WO2025006992A2

WO2025006992A2 - Compositions and methods for treating neurological disorders

Info

Publication number: WO2025006992A2
Application number: PCT/US2024/036166
Authority: WO
Inventors: Amal Alachkar; Olivier Civelli
Original assignee: University of California Berkeley; University of California San Diego UCSD
Current assignee: University of California Berkeley; University of California San Diego UCSD
Priority date: 2023-06-30
Filing date: 2024-06-28
Publication date: 2025-01-02
Anticipated expiration: 2025-12-30
Also published as: WO2025006992A3

Abstract

In alternative embodiments, provided are pharmaceutical compositions, including products of manufacture and kits, and methods, for treating or ameliorating a neurological disease, for example, a degenerative neurological disorder such as Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS), Friedreich ataxia, Lewy body disease, Spinal muscular atrophy or Motor neuron disease.

Description

COMPOSITIONS AND METHODS FOR TREATING NEUROLOGICAL DISORDERS

RELATED APPLICATIONS

This Patent Convention Treaty (PCT) International Application claims the benefit of priority to U.S. Provisional Patent Application Serial No. (USSN) 63/524.294, filed June 30, 2023. The aforementioned application is expressly incorporated herein by reference in their entirety and for all purposes.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under NS107671 awarded by the National Institutes of Health (NIH), National Institute of Neurological Disorders and Stroke. The government has certain rights in the invention.

TECHNICAL FIELD

This invention generally relates to biology and cancer treatments. In alternative embodiments, provided are pharmaceutical compositions, including products of manufacture and kits, and methods, for treating or ameliorating a neurological disease, for example, a degenerative neurological disorder such as Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS), Friedreich ataxia, Lewy body disease, Spinal muscular atrophy or Motor neuron disease.

BACKGROUND

The dopamine theory of movement control has been the prevailing model for explaining the symptoms and treatment responses seen in individuals with Parkinson's disease (PD) for over five decades. This widely accepted theory' suggests that dopamine neurons in the basal ganglia regions are involved in initiating and modulating voluntary’ movement, with dopamine acting as a 'go' signal that activates the neural pathways that control movement.

Individuals affected by PD often have low levels of dopamine in the basal ganglia as a result of degenerative damage to dopamine neurons in the substantia nigra pars compacta. This brings about tremors and difficulty initiating and controlling movement, as well as rigidity and slowed mobility’ (bradykinesia). Treatment with agents that raise dopamine levels can significantly improve movement-related symptoms in PD patients. Dopamine is unable to cross the bloodbrain barrier, so L-DOPA (also known as L-3. 4, hydroxypheylalanine, and levodopa), a dopamine precursor, is regarded as the gold standard treatment for PD.

L-DOPA is converted to dopamine by the enzyme aromatic amino acid decarboxylase (AADC). This enzy me is found in the brain and a number of peripheral organs. The conversion of L-DOPA to dopamine peripherally can lead to two main issues. First, the amount of L-DOPA that reaches the brain is reduced, where it is needed to replace dopamine in PD patients. This can diminish the therapeutic effects of L-DOPA in relieving PD movement symptoms. Second, the increased levels of dopamine can cause a range of side effects, such as nausea, vomiting, and arrhythmias. To reduce the peripheral metabolism of L-DOPA to dopamine, peripheral AADC inhibitors such as carbidopa (or LODOSYN™) can be used in combination with L-DOPA.

Despite being the most effective treatment for PD for over 50 years, the precise mechanisms of L-DOPA action remain uncertain. Initial treatments with L- DOPA can provide great relief from motor symptoms, but over time its therapeutic effects tend to diminish and dyskinesia, abnormal involuntary movements, can increase in PD patients. This phenomenon, known as L-DOPA-induced dyskinesia (LID), can be difficult to treat and may require a change in treatment approach. The mechanisms underlying LID are also not fully understood.

SUMMARY

In alternative embodiments, provided are compositions, including products of manufacture and kits, and methods, for treating or ameliorating a neurological disease, for example, a degenerative neurological disorder such as Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), Amy otrophic lateral sclerosis (ALS), Friedreich ataxia. Lewy body disease, Spinal muscular atrophy or Motor neuron disease.

In alternative embodiments, provided are compounds having a formula comprising:

(a) ophthalmic acid (ophthalmate, or “OA”). or

; or

(b) a deuterated form of a compound of (a), or an isomer, optical isomer or stereoisomer thereof, or a racemate or racemic mixture thereof, an enantiomer, or an individual diastereomer or a diastereomeric mixture thereof, or an analog thereof, or a crystalline product or cry stalline intermediate thereof, or a pharmaceutically acceptable salt thereof, or prodrug or a bioisostere thereof, of a compound of (a).

In alternative embodiments, provided are pharmaceutical compositions or formulations comprising:

(a) a deuterated form of ophthalmic acid (ophthalmate, or “OA”) or a deuterated form of an isomer, optical isomer or stereoisomer, a racemate or racemic mixture thereof, an enantiomer, an individual diastereomer or a diastereomeric mixture thereof, or an analog, thereof, or a crystalline product or crystalline intermediate thereof, or a pharmaceutically acceptable salt thereof, or prodrug or a bioisostere thereof, of ophthalmic acid (ophthalmate, OA);

(b) a compound having a formula comprising:

(i) ophthalmic acid (ophthalmate, or '‘OA”), or

³C _ CD₂

HO. .

NH₂ ; or o

(c) a deuterated form of a compound of (b), or an isomer or optical isomer or stereoisomer thereof, or a racemate or racemic mixture thereof, or an enantiomer, an individual diastereomer or a diastereomeric mixture thereof, or an analog thereof, or a crystalline product or cry stalline intermediate thereof, or a pharmaceutically acceptable salt thereof, or prodrug or a bioisostere thereof of a compound of (b).

In alternative embodiments of pharmaceutical compositions or formulations as provided herein;

- the pharmaceutical compositions or formulations further comprising a pharmaceutically acceptable excipient, or wherein the pharmaceutical composition is formulated in a sterile solution or liposome, wherein optionally the sterile solution comprises saline: or

- pharmaceutical composition or the formulation is a solid, liquid, aerosol, powder, lyophilized, gel or emulsion formulation. wherein optionally the pharmaceutical composition is formulated for enteral or parenteral administration, and optionally the compound is formulated for administration in vivo,' or for enteral or parenteral administration, or as a tablet, pill, capsule, lozenge, gel, geltab, liquid, lotion, aerosol, patch, spray, or implant, and optionally the compound is formulated as a liposome, a nanoparticle or a nanolipoparticle.

In alternative embodiments provided is: a kit, an implant, a pump, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, an infusion pen, a needles, a reservoir, an ampoules, a vial, a syringe, a cartridge, a pen, a disposable pen or jet injector, a prefdled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump, comprising a compound as provided herein, or a formulation or pharmaceutical composition as provided herein.

In alternative embodiments provided are methods for treating, ameliorating, slowing the progression of, decreasing the severity of symptoms of a neurological disease, optionally a degenerative neurological disorder, optionally Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS). Friedreich ataxia, Lewy body disease. Spinal muscular atrophy or Motor neuron disease, in an individual in need thereof, comprising administering: ophthalmic acid (ophthalmate, OA), or, a compound as provided herein, or a pharmaceutical composition as provided herein, or a kit, an implant, a pump, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, an infusion pen, a needles, a reservoir, an ampoules, a vial, a syringe, a cartridge, a pen, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multichambered pump as provided herein, to an individual in need thereof.

In alternative embodiments provided are uses of: ophthalmic acid (ophthalmate, OA), or a compound as provided herein, or a pharmaceutical composition as provided herein, or a kit, an implant, a pump, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, an infusion pen, a needles, a reservoir, an ampoules, a vial, a syringe, a cartridge, a pen. a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump as provided herein, for treating, ameliorating, slowing the progression of, decreasing the severity⁷ of symptoms of a neurological disease, optionally a degenerative neurological disorder, optionally Parkinson's disease (PD). Alzheimer's disease (AD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS), Friedreich ataxia, Lewy' body disease, Spinal muscular atrophy or Motor neuron disease, in an individual in need thereof.

In alternative embodiments provided are uses of ophthalmic acid (ophthalmate, OA), or a compound as provided herein in the preparation of a medicament, for example, preparation of a medicament for treating, ameliorating, slowing the progression of, decreasing the severity of symptoms of a neurological disease, optionally a degenerative neurological disorder, optionally Parkinson's disease (PD). Alzheimer's disease (AD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS). Friedreich ataxia, Lewy body disease. Spinal muscular atrophy or Motor neuron disease, in an individual in need thereof.

In alternative embodiments provided are compounds as provided herein, or a pharmaceutical composition comprising ophthalmic acid, or a pharmaceutical composition as provided herein, or a kit. an implant, a pump, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, an infusion pen, a needles, a reservoir, an ampoules, a vial, a syringe, a cartridge, a pen, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump as provided herein, for use in treating, ameliorating, slowing the progression of, decreasing the severity of symptoms of a neurological disease, optionally a degenerative neurological disorder, optionally Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS), Friedreich ataxia. Lewy body disease, Spinal muscular atrophy or Motor neuron disease, in an individual in need thereof.

The details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

All publications, patents, patent applications cited herein are hereby expressly incorporated by reference in their entireties for all purposes.

DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

The drawings set forth herein are illustrative of exemplary embodiments provided herein and are not meant to limit the scope of the invention as encompassed by the claims.

FIG. 1A-G illustrates motor response to L-DOPA (also known as levodopa and 1-3,4-dihydroxyphenylalanine) is enhanced by inhibition of DOPA decarboxylase in mouse model of Parkinson’s disease (PD):

FIG. 1A schematically illustrates the biosynthesis of dopamine from L-DOPA and its proposed inhibition by benserazide and NSD1015. a DOPA decarboxylase inhibitor (Sigma Aldrich), also known as a-Hydrazino-m-cresol dihydrochloride, 3- (Hydrazinomethyl)phenol dihydrochloride) in the periphery' and the central nervous system, respectively;

FIG. IB graphically illustrates the effects of L-DOPA and NSD1015 (Sigma Aldrich) on normal mice: mice were injected subcutaneously (s.c.) with a vehicle, and 18 hours later, injected intraperitoneally (i.p.) with either NSD1015 or saline, followed by an L-DOPA or saline injection 30 minutes after, and motor activity' was monitored for 20 hours, where data so generated is graphically represented in (left image) a time-course and (right image) the area under the curve (AUC) of the effects of L-DOPA, NSD1015, and combination of L-DOPA and NSD1015; and

FIG. 1C graphically illustrates the effects of L-DOPA and NSD1015 (Sigma Aldrich) on reserpinized mice, where data is graphically represented: (left image) time-course of the effect of L-DOPA alone and L-DOPA in conjunction with NSD1015, and (right image) the area under the curve,

FIG. 1D-E graphically illustrate the effect of varying doses of DOPA and NSD1015 (Sigma Aldrich) on motor activity', where mice were injected (i.p.) with reserpine 1 mg/kg and 18 hours later, mice were injected with varying doses of NSD1015 followed by varying doses of L-DOPA 30 minutes after, and locomotion was monitored for 20 hours, and the same data are presented in FIG. 1D-E; data represents (FIG. ld'-d"') the time-course and (FIG. IE) the area under the curve of the effect of varying doses of DOPA with fixed doses of NSD;

FIG. 1F-G graphically illustrate data representing (FIG. If'-f") the time-course and (FIG. 1G) the area under the curve of the effect of varying doses of NSD1015 (Sigma Aldrich) with fixed doses of DOPA;

FIG. 1H-I graphically illustrate data showing the motor effect of the combination of dopaminergic agents and NSD1015 (Sigma Aldrich), where the data represents the time-course of the effect of conjunction ofNSD1015 (Sigma Aldrich) with (FIG. 1H) amphetamine and (FIG. II) apomorphine; and

FIG. 1J graphically illustrates data showing the effect of haloperidol on the hyperactivity induced by DOPA and NSD1015 (Sigma Aldrich); data represents the time-course of the effect of haloperidol on DOPA alone and DOPA in conjunction with NSD1015 (Sigma Aldrich), as described in further detail in Example 1 , below.

FIG. 2A-I illustrate the motor Response to L-DOPA and AADC Inhibitor correlates with alterations in brain and striatum metabolites, with highest alteration in ophthalmate levels:

FIG. 2A illustrates the study's experimental design and timeline.

FIG. 2B graphically illustrates data of an unsupervised principle component analysis (PCA) of the metabolomics data of mouse brain and striatum from two treatment groups at the two time points FIG. 2C graphically illustrates data of an unsupervised hierarchical clustering analysis (HCA) of the differential metabolites of mouse brain and striatum from two treatment groups at two time points;

FIG. 2D illustrates a table showing the count of altered metabolites in the (left section table) whole brain and (right section table) striatum;

FIG. 2E graphically illustrates a box plot legend showing the range, median, and quartiles;

FIG. 2F graphically illustrates fold changes in the major components of dopamine synthesis pathway after L-DOPA/ NSD1015 (Sigma Aldrich) administration;

FIG. 2G graphically illustrates fold changes in the major L-DOPA metabolites that are not related to the dopamine pathway;

FIG. 2H graphically illustrates fold changes in the major metabolites of the metabolic pathways for the aromatic amino acids: L-tryptophan, phenylalanine, and tyrosine; and

FIG. 21 graphically illustrates fold changes in ophthalmate and the key metabolites of the proposed pathways leading to its synthesis, noting that the asterisk (*) is used to compare metabolite levels within the same group at the two different time points, whereas the hashtag (#) is used to compare metabolite levels between the two treatment groups at the same time point, as described in further detail in Example 1 , below.

FIG. 3A-J illustrate data showing how ophthalmic acid (OA) is synthesized in the brain and modulates motor function in mouse model of PD:

FIG. 3 A graphically illustrates data showing that peripheral OA injection did not induce motor activity in MPTP -treated mice, where the data represent the timecourse of the effect of OA injected i.p. at three different doses and are expressed as mean ± S.E;

FIG. 3B graphically illustrates data showing OA levels following peripheral administration as measured using Liquid chromatography-mass spectrometry (LC- MS), where data represent the interpolated concentrations of d5-OA in the blood and brain and are expressed as mean ± S.E;

FIG. 3C-D graphically illustrate data showing the effect of central administration of OA on the motor activity of normal mice: the data presented shows (FIG. 3C) the time-course and (FIG. 3D) the AUC of the effect of OA injected i.c.v. at three different doses effect; and

FIG. 3E-J graphically illustrate data showing the effect of central administration of OA on the motor activity of MPTP -treated mice: the data presented include (FIG. 3E) the time-course of the effect of MPTP, (FIG. 3F) the AUC of the effect of MPTP during the 20-hour experiment time; unpaired t-test, ns: not significant. (FIG. 3G) the time-course of the effect of OA injected i.c.v. at four different doses, (FIG. 3H) the AUC of the effect of different doses of OA during the 20-hour experiment time, (FIG. 31) the AUC of the effect of different doses of OA during the first 10 hours of the experiment time, and (FIG. 3 J) the AUC of the effect of different doses of OA during the 10-20 hours of the experiment time, as described in further detail in Example 1 , below.

FIG. 4A-L illustrates that OA acts as a neuromodulator on Calcium sensing receptors (CaSRs):

FIG. 4A-B graphically illustrate data showing Saturation curve of [3H]-0A binding to mouse brain sections in the presence of unlabeled OA. NPS2143 (a selective calcium-sensing receptor antagonist), Ca²⁺, and L-DOPA:

FIG. 4A graphically illustrates a representative plot of total, specific, and nonspecific binding of [3H]-OA to mouse brain sections;

FIG. 4B graphically illustrates a representative plot of specific binding of [3H]-OA binding to mouse brain sections, where non-specific bindings were defined as the levels of [3H]-OA binding in the presence of 10 pM NPS-2143, 100 pM calcium, and 100 pM L-DOPA;

FIG. 4C graphically illustrates a representative plot of specific binding of [3H]-OA binding to mouse brain sections, where non-specific binding was defined as the level of [3H]-0A binding in the presence of combinations of NPS-2143+ Ca²⁺, L- DOPA+ Ca2+, and NPS-2143 + L-DOPA;

FIG. 4D graphically illustrates data showing inhibition of [3H]-0A binding to mouse brain sections by Ca2+;

FIG. 4E-H graphically illustrate data showing forskolin- stimulated cAMP GLOSENSOR™ luminescence responses in HEK 293 cells transiently transfected with the GLOSENSOR™ cAMP biosensor and the CaSR plasmid: (FIG. 4E-H) representative dose-response curves of cAMP signal for (FIG. 4E) OA. (FIG. 4F) L- DOPA, and (FIG. 4G) Ca2+ with and without CaSR antagonist NPS-2143, and (FIG. 4H) different combinations of the three ligands OA. L-DOPA, and Ca²⁺;

FIG. 4I-L schematically illustrate exemplary docking models for ligand-bound states for CaSR binding site in domain A (named A in amino acid hereinafter):

FIG. 41 schematically illustrates a ligand-bound CaSR structure (domain A) in the closed-closed conformation (5 Angstroms);

FIG. 4J schematically illustrates an interface analysis of Tryptophan (Trp)- bound state in CSRS binding are shown with Serl70A, Serl47A, Ala298A, and Thr 145 A and Alai 68 A;

FIG. 4K schematically illustrates an interface analysis of L-dopa (LDP)-bound state in CaSR binding are shown with Tyr218A. Serl70A, Asp216A. and Alal68A; and

FIG. 4L schematically illustrates an interface analysis of Ophthalmate (OPT) bound state in CaSR binding are shown with Serl47A, Glyl48A, Tyr218A, Serl70A, Asp216A, and Alal68A. Vall49A, the green curve shows the transition state of Ala298A and Thr 145A in the Trp-bound state, Tyr218A in LDP-bound state, and Glyl46A in the OPT-bound state, as described in further detail in Example 1 , below⁷.

FIG. 5A-F illustrate that OA induces motor function by acting on Calcium sensing receptors (CaSRs):

FIG. 5A-B graphically illustrate data showing CaSR antagonist NPS-2143 inhibition of motor response induced by L-DOPA/ NSD1015 (Sigma Aldrich) in MPTP -treated mice, and data represents (FIG. 5A) the time-course and (FIG. 5B) the AUC of the effect of NSD1015 (Sigma Aldrich) and L-DOPA with or without NPS2143;

FIG. 5C-F graphically illustrate data showing CaSR antagonist inhibits OA- induced motor response in MPTP -treated mice, were mice were subjected to surgery and MPTP treatment as in (FIG. 5A-B), and on the day following the final MPTP treatment, mice were administered, mice were injected i.c.v. with either saline or OA at (FIG. 5C-D) 5 pM and (FIG. 5E-F) 10 pM with or without NPS2143 (10 nM and 20 nM), and locomotion was monitored for 20 hours. Data represent (c,e) the timecourse and (FIG. 5D. FIG. 5F) the AUC of the effect of OA w ith and without NPS2143 on motor activity, and in (FIG. 5D) and (FIG. 5F) One way ANOVA, followed by Tukey post-test, as described in further detail in Example 1 , below.

FIG. 6A-K (or, Figure 1, Example 2) illustrate Motor response to L-DOPA is enhanced by inhibition of DOPA decarboxylase in mouse model of Parkinson’s disease (PD):

FIG. 6A schematically illustrates the biosynthesis of dopamine from L-DOPA and its proposed inhibition by benserazide and NSD1015 in the periphery and the central nervous system, respectively;

FIG. 6B and FIG. 6C graphically illustrate data showing the effects of L- DOPA and NSD1015 (NSD) on normal mice: data represent (FIG. 6B) the timecourse and (FIG. 6C) the Area Under the Curve (AUC) of the effects of L-DOPA, NSD 1015, and combination of L-DOPA and NSD1015;

FIG. 6D-E graphically illustrate data representing the (FIG. 6D) time-course of the effect of L-DOPA alone and L-DOPA in conjunction with NSD1015, and (FIG. 6E) the area under the curve;

FIG. 6F-H graphically illustrate data representing the time-course of the effect of varying doses of NSD1015 with fixed doses of L-DOPA (FIG. 6F: 50mg/kg, FIG. 6G: lOOmg/kg, FIG. 6H: 200mg/kg); and

FIG. 6I-K graphically illustrate data representing the time-course of the effect of varying doses of L-DOPA with fixed doses of NSD1015 (FIG. 61: 50mg/kg, FIG. 6H: lOOmg/kg. FIG. 6K: 200mg/kg), as described in further detail in Example 2, below.

FIG. 7A-J (or, Figure 2, Example 2) illustrate data showing that motor Response to L-DOPA and AADC inhibitor correlates with alterations in brain and striatum metabolites, with highest alteration in ophthalmate levels:

FIG. 7A schematically illustrates this exemplary study's experimental design and timeline;

FIG. 7B graphically illustrates data representing an unsupervised principal component analysis (PCA) of the metabolomics data of mouse whole brain and striatum from two the treatment groups at the two time points;

FIG. 7C graphically illustrates a Volcano plot illustrating the differential expression of metabolites in the brains of L-DOPA+NSD1015 treated mice compared to L-DOPA treated mice, with brain samples taken 2 hours post-DOPA injection; FIG. 7D graphically illustrates a Volcano plot showing the differential expression of metabolites in the brains of L-DOPA + NSD1015 treated mice versus L-DOPA treated mice, with brain samples obtained 7 hours after L-DOPA injection, and shows contrasts: significantly increased metabolites in red, and significantly decreased metabolites in blue;

FIG. 7E graphically illustrates a Volcano plot displaying the differential expression of metabolites in the brains of L-DOPA + NSD1015 treated mice at 7 hours compared to L-DOPA + NSD 1015 treated mice at 2 hours post-DOPA injection, and shows contrasts: significantly increased metabolites in red, and significantly decreased metabolites in blue;

FIG. 7F graphically illustrates a Volcano plot demonstrating the differential expression of metabolites in the striatum tissues of L-DOPA+NSD1015 treated mice in comparison to L-DOPA treated mice, with brain samples collected 7 hours following L-DOPA injection, and shows contrasts: significantly increased metabolites in red. and significantly decreased metabolites in blue;

FIG. 7G-J graphically illustrate lists of top altered metabolites in the brains of different treatment groups (> 2 -fold change, q< 0.05, X axis represents fold changes; L-DOPA+NSD1015 versus DOPA at (FIG. 7G) 2 hours (h), (FIG. 7H) 7 h, (FIG. 71) 2h versus 7 h, and (FIG. 7 J) 7h in striatum, as described in further detail in Example 2, below.

FIG. 8A-C (or, Figure 3 Example 2) illustrate data showing that the motor response to L-DOPA and AADC inhibitor is associated with alterations in dopamine and ophthalmate synthesis pathways:

FIG. 8A graphically illustrates a Box plot legend showing the range, median, and quartiles;

FIG. 8B graphically illustrates the fold changes in the major components of dopamine synthesis pathway after L-DOPA/ NSD 1015 administration; and

FIG. 8C graphically illustrates the fold changes in ophthalmate and the key metabolites of the proposed pathways leading to its synthesis, as described in further detail in Example 2, below.

FIG. 9A-I (or, Figure 4 Example 2) illustrate data showing that central but not peripheral ophthalmate (OA) rescues motor activity in MPTP 10 mouse model of PD: FIG. 9A illustrates images of representative immunostaining of tyrosine hydroxylase (TH, green) and DAPI (blue), showing the effect of MPTP (lower image) on dopamine neuronal loss in the substantia nigra (top image control);

FIG. 9B graphically illustrates the time-course of the effect of MPTP;

FIG. 9C graphically illustrates the AUC of the effect of MPTP during the 20- hour experiment time; unpaired t-test;

FIG. 9D graphically illustrates data showing that peripheral ophthalmate injection did not induce motor activity in MPTP-treated mice;

FIG. 9E-H graphically illustrate data representing the time-course of the effect of ophthalmate injected i.p. at three different doses and are expressed as mean±S.E. n=8 for each group:

FIG. 9E-H graphically illustrate the effect of central administration of ophthalmate on the motor activity of MPTP-treated mice, where the data presented include (FIG. 9E) the time-course of the effect of ophthalmate injected i.c.v. at four different doses. (FIG. 9F) the AUC of the effect of different doses of ophthalmate during the 20-hour experiment time. (FIG. 9G) the AUC of the effect of different doses of ophthalmate during the first 10 hours of the experiment time, and (FIG. 9H) the AUC of the effect of different doses of ophthalmate during the 11-20 hours of the experiment time; and

FIG. 91 illustrates a deuterated ophthalmate (d5-OA) structure, and also graphically illustrates deuterated ophthalmate (d5-OA) levels following peripheral administration, measured using liquid chromatography-mass spectrometry' (LC-MS), as described in further detail in Example 2, below.

FIG. 10A-G (or, Figure 5 Example 2) illustrate data showing that ophthalmate binds to and activates calcium sensing receptor (CaSR):

FIG. 10A-B graphically illustrate saturation curves of [³H]-0A binding to mouse brain sections in the presence of unlabeled ophthalmate, NPS2143, Ca²⁺, and L- DOPA;

FIG. 10A graphically illustrates a reprehensive plot of total, specific, and nonspecific binding of [³H]-OA to mouse brain sections;

FIG. 10B graphically illustrates a representative plot of specific binding of [³H]-0A binding to mouse brain sections, where non-specific bindings were defined as the levels of [³H]-0A binding in the presence of 10 pM NPS-2143, 100 pM calcium, and l O pM L-DOPA; FIG. 1 OC graphically illustrates a representative plot of specific binding of [³H]-OA binding to mouse brain sections, where non-specific binding was defined as the level of [³H]-OA binding in the presence of combinations of NPS-2143+ Ca²⁺, L-DOPA+ Ca²⁺, and NPS-2143 + L-DOPA;

FIG. 10D graphically illustrates data showing inhibition of [³H]-OA binding to mouse brain sections by Ca²⁺;

FIG. 10E-G graphically illustrate forskolin-stimulated cAMP GLOSENSOR™ luminescence responses in HEK 293 cells transiently transfected with the GLOSENSOR™ cAMP biosensor and the CaSR plasmid;.

FIG. 10E-E" graphically illustrate representative dose-response curves of cAMP signal for (E) Ca²⁺. (E') OA, and (E") L-DOPA, with and without CaSR antagonist NPS-2143;

FIG. 10F-F" graphically illustrate data showing the Emax of (10F) Ca²⁺, (10F') OA, and 10F" illustrates data showing L-DOPA at CaSR in the absence and presence of CaSR antagonist NPS-2143: and

FIG. 10G-G" graphically illustrate data showing EC50 of (FIG. 10G) Ca²⁺, (FIG. 10G') OA, and (FIG. 10G")L-DOPA at CaSR in the presence and absence of CaSR antagonist NPS-2143;

FIG. 10H-K schematically illustrate docking models and ligand-bound states in CaSR binding site in domain A (named A in amino acid):

FIG. 10H schematically illustrates a cartoon presentation of ligand-bound CaSR structure (domain A) in the closed-closed conformation (5 Angstroms);

FIG. 101 schematically illustrates an interface analysis of Try ptophan (Trp)- bound state in CaSR; binding is shown with Serl70A, Serl47A. Ala298A, and Thr 145A and Alal68A;

FIG. 10J schematically illustrates an interface analysis of Ophthalmate (OA) bound state in CaSR; binding is shown with Serl47A, Glyl48A, Tyr218A, Serl70A, Asp216A, and Alal68A, Vall49A; and

FIG. 10K schematically illustrates an interface analysis of L-DOPA bound state in CaSR; binding is shown with Tyr218A, Serl70A, Asp216A, and Alal68A. Green curve shows the transition state of Ala298A and Thr 145A in the Trp-bound state, Glyl46A in the OA-bound state, and Tyr218A in DOPA-bound state, as described in further detail in Example 2, below. FIG. 11 A-H illustrate data showing that CaSR mediates the motor-enhancing effects of L-DOPA/NSD1015 and ophthalmate in PD mice:

FIG. 11A-B illustrate data showing CaSR antagonist NPS-2143 inhibition of motor response induced by L-DOPA/NSD1015 in MPTP-treated mice: data represents (FIG. 11A) the time-course and (FIG. 1 IB) the AUC of the effect of NSD1015 and L-DOPA with or without NPS2143.

FIG. 11C-F illustrate data showing CaSR antagonist inhibits ophthalmate- induced motor response in MPTP-treated mice: data represent (FIG. 11 C, FIG. 1 IE) the time-course and (FIG. 1 ID, FIG. 1 IF) the AUC of the effect of ophthalmate with and without NPS2143 on motor activity;

FIG. 11G-H illustrate data showing CaSR antagonist NPS2143 inhibits ophthalmate-induced motor response in reserpine-treated mice; mice were injected (s.c.) with reserpine 1 mg/kg, and 18 hours later, mice were injected (i.c.v) with ophthalmate (10 pM) with or without NPS2143 (20 pM), and the (FIG. 11G) timecourse and (FIG. 11H) the AUC of the effect of ophthalmate with and without NPS2143 on motor activity, as described in further detail in Example 2, below.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

We discovered that ophthalmic acid (ophthalmate, OA) acts as a novel neurotransmitter to counteract the motor symptoms in art-accepted animal models of human Parkinson’s disease, with a long duration of action. We discovered that the ophthalmate system compounds, including the ophthalmate compound and its precursors and/or modified structures (for example, deuterated forms, isomers, optical isomers or stereoisomers, racemate or racemic mixtures, enantiomers, diastereomers or a diastereomeric mixtures, analogs, crystalline products or crystalline intermediates or a prodrug or a bioisostere thereof) can be used as novel drugs for the treatment of degenerative neurological disorders such as Parkinson's disease (PD). Alzheimer's disease (AD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS). Friedreich ataxia, Lewy body disease, Spinal muscular atrophy or Motor neuron disease, and other neurological disorders.

As discussed in Example 1, we used a standard animal model of PD where we administered L-DOPA the anti PD drug to restore locomotion. The L-DOPA effect is active for only a few hours. We discovered that hours after L-DOPA lost its activity. OA is dramatically increased. We injected OA in mice and found that it increases locomotion and a such carries anti PD activity. We discovered OA mechanism of action. We concluded that acting on the OA system is a novel way of treating PD. Inhibition of brain conversion of L-DOPA to dopamine delays, increases and prolongs 1-DOPA-induced motor activity in PD mice: We used a standad animal model of PD (reserpine-treated mice) where we administered Idopa the anti PD drug to restore locomotion. We tested the effects of L-DOPA in a condition of pharmacological inhibition of central aromatic amino acid decarboxylase (AADC), which converts L- DOPA to dopamine. We used NSD1015 as a central AADC inhibitor (27) (Goshima et al., 1990) (Fig. la).

We administered L-DOPA (lOOmg/kg) with and without NSD1015 (lOOmg/kg) in combination with the peripheral AADC inhibitor benserazide (25mg/kg) to reserpine-treated mice (reserpinized-mice). L-DOPA alone enhanced activity that lasted up to four hours. In the condition of inhibition of L-DOPA conversion to dopamine, mice exhibited a greater motor response to L-DOPA than those treated with L-DOPA alone. The motor activity onset in these mice was delayed by around 120 minutes, and the duration of the effect was also extended, compared with L-DOPA alone (Fig.1 b).

NSD1015/L-DQPA-provoked motor activity is accompanied by elevated ophthalmic acid: Analyzing the metabolites found in mice subjected to these conditions, we found that Ophthalmate is dramatically increased during the peak of motor activity in the AADC inhibition condition, hours after the anti PD drug L-DOPA has been administered (Fig. 1C).

OA rescues motor symptoms in PD model: We studied the direct action of OA on PD mice, and found that central administration of OA (at doses 1, 2.5, 5, 10 pM) can reverse the PD symptoms for prolonged duration. Further, OA precursor 2- aminobutyrate was able to increase and prolong motor activity when administered peripherally with L-DOPA/NSD1015.

OA acts through activating Calcium sensing receptors (CaSR): Screening literature, we found that OA can bind to CaSR. We reproduced the these data on mouse brain sections, and also discovered that OA activates CaSR. We then found that CaSR could block the motor action of OA in PD model.

Bioisosteres of Compounds

In alternative embodiments, provided are bioisosteres of compounds and compositions as provided herein, or a compound used to practice methods as provided herein. In alternative embodiments, bioisosteres as provided herein are compounds comprising one or more substituent and/or group replacements with a substituent and/or group having substantially similar physical or chemical properties which produce substantially similar biological properties to a compound as provided herein or a stereoisomer, racemate or isomer thereof. In one embodiment, the purpose of exchanging one bioisostere for another is to enhance the desired biological or physical properties of a compound without making significant changes in chemical structures.

For example, in one embodiment, bioisosteres of compounds and compositions as provided herein, or a compound used to practice methods as provided herein, are made by replacing one or more hydrogen atom(s) with one or more fluorine atom(s). for example, at a site of metabolic oxidation; this may prevent metabolism (catabolism) from taking place. Because the fluorine atom is only slightly larger than the hydrogen atom the overall topology of the molecule is not significantly affected, leaving the desired biological activity unaffected. However, with a blocked pathway for metabolism, the molecule may have a longer half-life or be less toxic, and the like.

In alternative embodiments, compounds as provided herein contain one or more asymmetric centers and can thus occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. Additional asymmetric centers may be present depending upon the nature of the various substituents on the molecule. Each such asymmetric center will independently produce two optical isomers, and all of the possible optical isomers and diastereomers in mixtures, as pure or partially purified compounds, are provided herein. In alternative embodiments, compounds provided herein encompass any and all existing isomers and mixtures thereof in any proportion. In alternative embodiments, compounds herein are provided as isomers in pure form or as part of a mixture with other isomers in any proportion.

In alternative embodiments, provided are independent syntheses of diastereomers or their chromatographic separations, and their absolute stereochemistry may be determined by the x-ray crystallography of crystalline products or crystalline intermediates which are derivatized, if necessary, with a reagent containing an asymmetric center of known absolute configuration.

In alternative embodiments, racemic mixtures are separated so that the individual enantiomers are isolated. The separation can be carried out by methods well known in the art, such as the coupling of a racemic mixture of compounds to an enantiomerically pure compound to form a diastereomeric mixture, followed by separation of the individual diastereomers by standard methods, such as fractional crystallization or chromatography. In alternative embodiments, a coupling reaction comprises formation of salts using an enantiomerically pure acid or base. The diastereomeric derivatives may then be converted to the pure enantiomers by cleavage of the added chiral residue. The racemic mixture of the compounds can also be separated directly by chromatographic methods utilizing chiral stationary phases, which methods are well known in the art.

In alternative embodiments, a compound is made using stereoselective synthesis using optically pure starting materials or reagents of known configuration by methods well known in the art.

In alternative embodiments, a compound is isotopically labeled with one or more atoms having an atomic mass or mass number different from the atomic mass or mass number most abundant in nature. Examples of isotopes that can be incorporated into compounds provided herein include isotopes of hydrogen, carbon, nitrogen, oxygen and fluorine, for example, 2H (deuterium), 3H (tritium), 13C, 14C, 15N, 180 or 18F. In alternative embodiments, compounds provided herein may be substituted with an alternative isotope, for example, a 2H (deuterium) in place of a hydrogen, to. for example, increase metabolic stability and/or in vivo half-life. In alternative embodiments, a compound is selectively modified, for example, selectively deuterated, to modify all or only part of a reactive site, or a portion of the compound that is a site of chemical modification in vivo, for example, for the purpose of changing its solubility or pharmacokinetics, for example, metabolic profile or halflife. In alternative embodiments, compounds as provided herein, prodrugs thereof, and pharmaceutically acceptable salts of these compounds may contain the aforementioned isotopes and/or isotopes of other atoms.

Formulations and pharmaceutical compositions

In alternative embodiments, provided are compounds and compositions, including formulations and pharmaceutical compositions, for use in in vivo, in vitro or ex vivo methods for treating, ameliorating, preventing or reversing a neurological disease, for example, a degenerative neurological disorder such as Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS), Friedreich ataxia, Lewy body disease, Spinal muscular atrophy or Motor neuron disease.

In alternative embodiments, the pharmaceutical compositions as provided herein can be administered parenterally, topically, orally or by local administration, such as by aerosol or transdermally. In alternative embodiments, pharmaceutical compositions can be prepared in various forms, such as granules, tablets, pills, capsules, suspensions, taken orally, suppositories and salves, lotions and the like. Pharmaceutical formulations as provided herein may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, lozenges, gels, geltabs, on patches, in implants, etc. In practicing embodiments as provided herein, the pharmaceutical compounds can be delivered by transdermally, by atopical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols. Oral carriers can be elixirs, syrups, capsules, tablets, pills, geltabs and the like.

In alternative embodiments, provided are pharmaceutically acceptable salts of compounds as provided herein, including pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. In alternative embodiments, salts are derived from inorganic bases such as aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganese, potassium, sodium, zinc, and the like; or, salts can be in a solid form, or in a cry stal structure, or the form of hydrates. In alternative embodiments, salts are pharmaceutically acceptable organic non-toxic bases including salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2- diethylaminoethanol. 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N- ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like. In alternative embodiments, for example, if a compound provided herein is basic, salts are prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, carbonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like.

In alternative embodiments, pharmaceutically acceptable salts include hemisalts of non-toxic acids or bases, or hemihydrates.

In alternative embodiments, compounds and compositions as provided herein, or a compound used to practice methods as provided herein, are delivered orally, for example, as pharmaceutical formulations for oral administration, and can be formulated using pharmaceutically acceptable carriers well known in the art in appropriate and suitable dosages. Such carriers enable the pharmaceuticals to be formulated in unit dosage forms as tablets, pills, powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient. Pharmaceutical preparations for oral use can be formulated as a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired, to obtain tablets or dragee cores. Suitable solid excipients can be carbohydrate or protein fillers, for example, sugars, including lactose, sucrose, mannitol, or sorbitol; starch from com, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethyl cellulose; and gums including arabic and tragacanth; and proteins, for example, gelatin and collagen. Disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

In alternative embodiments, liquid carriers are used to manufacture or formulate compounds as provided herein, or a composition used to practice the methods as provided herein, including carriers for preparing solutions, suspensions. emulsions, syrups, elixirs and pressurized compounds. The active ingredient (for example, a composition as provided herein) can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid carrier can comprise other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers or osmo-regulators.

In alternative embodiments, solid carriers are used to manufacture or formulate compounds as provided herein, or a composition used to practice the methods as provided herein, including solid carriers comprising substances such as lactose, starch, glucose, methyl-cellulose, magnesium stearate, dicalcium phosphate, mannitol and the like. A solid earner can further include one or more substances acting as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet-disintegrating agents; it can also be an encapsulating material. In powders, the carrier can be a finely divided solid which is in admixture with the finely divided active compound. In tablets, the active compound is mixed with a carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free flowing form such as a powder or granules, optionally mixed with a binder (for example, povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose) surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropyl methylcellulose in vary ing proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach. In alternative embodiments, concentrations of therapeutically active compound in a formulation can be from between about 0. 1% to about 100% by weight.

In alternative embodiments, therapeutic formulations are prepared by any method well known in the art, for example, as described by Brunton et al., eds., Goodman and Gilman's: The Pharmacological Bases of Therapeutics , 12th ed., McGraw-Hill, 2011; Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th ed., 2000; Avis et al., eds., Pharmaceutical Dosage Forms: Parenteral Medications, published by Marcel Dekker, Inc., N.Y., 1993; Lieberman et al., eds., Pharmaceutical Dosage Forms: Tablets, published by Marcel Dekker, Inc., N.Y.. 1990; and Lieberman et al., eds., Pharmaceutical Dosage Forms: Disperse Systems, published by Marcel Dekker, Inc., N.Y., 1990.

In alternative embodiments, therapeutic formulations are delivered by any effective means appropriated for a particular treatment. For example, depending on the specific antitumor agent to be administered, the suitable means include oral, rectal, vaginal, nasal, pulmonary administration, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) infusion into the bloodstream. For parenteral administration, antitumor agents as provided herein may be formulated in a variety of ways. Aqueous solutions of the modulators can be encapsulated in polymeric beads, liposomes, nanoparticles or other injectable depot formulations known to those of skill in the art. In alternative embodiments, compounds and compositions as provided herein, or a compound used to practice methods as provided herein, are administered encapsulated in liposomes (see below). In alternative embodiments, depending upon solubility, compositions are present both in an aqueous layer and in a lipidic layer, for example, a liposomic suspension. In alternative embodiments, a hydrophobic layer comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surfactants such a diacetylphosphate, stearylamine, or phosphatidic acid, and/or other materials of a hydrophobic nature.

The pharmaceutical compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration are well described in the scientific and patent literature, see. /w example, the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co.. Easton PA ("Remington’s"). For example, in alternative embodiments, compounds and compositions as provided herein, or a compound used to practice methods as provided herein, are formulated in a buffer, in a saline solution, in a powder, an emulsion, in a vesicle, in a liposome, in a nanoparticle, in a nanolipoparticle and the like. In alternative embodiments, the compositions can be formulated in any way and can be applied in a variety of concentrations and forms depending on the desired in vivo, in vitro or ex vivo conditions, a desired in vivo, in vitro or ex vivo method of administration and the like. Details on techniques for in vivo, in vitro or ex vivo formulations and administrations are well described in the scientific and patent literature. Formulations and/or carriers used to practice embodiments as provided herein can be in forms such as tablets, pills, powders, capsules, liquids, gels, syrups, slurries, suspensions, etc., suitable for in vivo, in vitro or ex vivo applications.

In practicing embodiments as provided herein, the compounds (for example, formulations) as provided herein can comprise a solution of compositions disposed in or dissolved in a pharmaceutically acceptable carrier, for example, acceptable vehicles and solvents that can be employed include water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can be employed as a solvent or suspending medium. For this purpose any fixed oil can be employed including synthetic mono- or diglycerides, or fatty acids such as oleic acid. In one embodiment, solutions and formulations used to practice embodiments as provided herein are sterile and can be manufactured to be generally free of undesirable matter. In one embodiment, these solutions and formulations are sterilized by conventional, well known sterilization techniques.

The solutions and formulations used to practice methods as provided herein can comprise auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in these formulations can vary widely, and can be selected primarily based on fluid volumes, viscosities and the like, in accordance with the particular mode of in vivo, in vitro or ex vivo administration selected and the desired results. The compounds and compositions as provided herein, or a compound used to practice methods as provided herein, can be delivered by the use of liposomes. In alternative embodiments, by using liposomes, particularly where the liposome surface carries ligands specific for target cells or organs, or are otherwise preferentially directed to a specific tissue or organ type, one can focus the delivery⁷ of the active agent into a target cells in an in vivo, in vitro or ex vivo application.

The compounds and compositions as provided herein, or a compound used to practice methods as provided herein, can be directly administered, for example, under sterile conditions, to an individual (for example, a patient) to be treated. The modulators can be administered alone or as the active ingredient of a pharmaceutical composition. Compositions and formulations as provided herein can be combined with or used in association with other therapeutic agents. For example, an individual may be treated concurrently with conventional therapeutic agents. Nanoparticles. Nanolipoparticles and Liposomes

Provided are nanoparticles, nanolipoparticles, vesicles and liposomal membranes comprising compounds and compositions used to practice the methods and embodiments as provided herein. Provided are multilayered liposomes comprising compounds used to practice embodiments as provided herein, for example, as described in Park, et al., U.S. Pat. Pub. No. 20070082042. The multilayered liposomes can be prepared using a mixture of oil-phase components comprising squalane, sterols, ceramides, neutral lipids or oils, fatty acids and lecithins, to about 200 to 5000 nm in particle size, to entrap a composition used to practice embodiments as provided herein.

Liposomes can be made using any method, for example, as described in Park, et al., U.S. Pat. Pub. No. 20070042031, including the method of producing a liposome by encapsulating an active agent (for example, compounds and compositions as provided herein, or a compound used to practice methods as provided herein), the method comprising providing an aqueous solution in a first reservoir; providing an organic lipid solution in a second reservoir, and then mixing the aqueous solution with the organic lipid solution in a first mixing region to produce a liposome solution, where the organic lipid solution mixes with the aqueous solution to substantially instantaneously produce a liposome encapsulating the active agent; and immediately then mixing the liposome solution with a buffer solution to produce a diluted liposome solution. In one embodiment, liposome compositions used to practice embodiments as provided herein comprise a substituted ammonium and/or polyanions, for example, for targeting del i v ery of a compound as provided herein, or a compound used to practice methods as provided herein, to a desired cell type or organ, for example, brain, as described for example, in U.S. Pat. Pub. No. 20070110798.

Provided are nanoparticles comprising compounds as provided herein, for example, used to practice methods as provided herein in the form of active agentcontaining nanoparticles (for example, a secondary nanoparticle), as described, for example, in U.S. Pat. Pub. No. 20070077286. In one embodiment, provided are nanoparticles comprising a fat-soluble active agent used to practice embodiments as provided herein, or a fat-solubilized water-soluble active agent to act with a bivalent or trivalent metal salt.

In one embodiment, solid lipid suspensions can be used to formulate and to deliver compositions used to practice embodiments as provided herein to mammalian cells in vivo, in vitro or ex vivo, as described, for example, in U.S. Pat. Pub. No. 20050136121.

Delivery vehicles

In alternative embodiments, any delivery' vehicle can be used to practice the methods as provided herein, for example, to deliver compounds and compositions as provided herein, or a compound used to practice methods as provided herein, to mammalian cells, for example, in vivo, in vitro or ex vivo. For example, delivery vehicles comprising poly cations, cationic polymers and/or cationic peptides, such as polyethyleneimine derivatives, can be used for example as described, for example, in U.S. Pat. Pub. No. 20060083737.

In one embodiment, a dried polypeptide-surfactant complex is used to formulate compounds and compositions as provided herein, or a compound used to practice embodiments as provided herein, for example as described, for example, in U.S. Pat. Pub. No. 20040151766.

In one embodiment, compounds and compositions as provided herein, or a compound used to practice methods as provided herein, can be applied to cells using vehicles with cell membrane-permeant peptide conjugates, for example, as described in U.S. Patent Nos. 7,306,783; 6,589,503. In one aspect, the composition to be delivered is conjugated to a cell membrane-permeant peptide. In one embodiment, the composition to be delivered and/or the delivery vehicle are conjugated to a transport-mediating peptide, for example, as described in U.S. Patent No. 5,846,743, describing transport-mediating peptides that are highly basic and bind to polyphosphoinositides.

In one embodiment, electro-permeabilization is used as a primary or adjunctive means to deliver the composition to a cell, for example, using any electroporation system as described for example in U.S. Patent Nos. 7,109,034; 6,261,815; 5.874,268.

Dosaging

The pharmaceutical compositions and formulations as provided herein can be administered for prophylactic and/or therapeutic treatments, for example, for treating a neurological disease, for example, a degenerative neurological disorder such as Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS), Friedreich ataxia, Lewy body disease, Spinal muscular atrophy or Motor neuron disease. In therapeutic applications, compositions are administered to a subject, for example, a human in need thereof, in an amount of the agent sufficient to cure, alleviate or partially arrest the clinical manifestations and/or its complications (a “therapeutically effective amount”).

The amount of pharmaceutical composition adequate to accomplish this is defined as a "therapeutically effective dose." The dosage schedule and amounts effective for this use. i.e., the “dosing regimen,” will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient’s physical status, age and the like. Dosage levels may range from about 0.01 mg per kilogram to about 100 mg per kilogram of body weight. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration.

The dosage regimen also takes into consideration pharmacokinetics parameters well know n in the art, i.e., the active agents’ rate of absorption, bioavailability, metabolism, clearance, and the like (see, for example. Hidalgo- Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617; Groning (1996) Pharmazie 51 :337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24: 103-108; the latest Remington’s, supra). The state of the art allows the clinician to determine the dosage regimen for each individual patient, active agent and disease or condition treated. Guidelines provided for similar compositions used as pharmaceuticals can be used as guidance to determine the dosage regiment, i.e., dose schedule and dosage levels, administered practicing the methods as provided herein are correct and appropriate.

Products of manufacture and Kits

Provided are products of manufacture and kits for practicing methods as provided herein; and optionally, products of manufacture and kits can further comprise instructions for practicing methods as provided herein.

Any of the above aspects and embodiments can be combined with any other aspect or embodiment as disclosed here in the Summary, Figures and/or Detailed Description sections.

As used in this specification and the claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and”.

Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About (use of the term “about”) can be understood as within 20%. 19%. 18%. 17%. 16%. 15%. 14%. 13%. 12% 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”

Unless specifically stated or obvious from context, as used herein, the terms “substantially all”, “substantially most of’, “substantially all of’ or “majority of’ encompass at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, or more of a referenced amount of a composition.

The entirety of each patent, patent application, publication and document referenced herein hereby is incorporated by reference. Citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Incorporation by reference of these documents, standing alone, should not be construed as an assertion or admission that any portion of the contents of any document is considered to be essential material for satisfying any national or regional statutory disclosure requirement for patent applications. Notwithstanding, the right is reserved for relying upon any of such documents, where appropriate, for providing material deemed essential to the claimed subj ect matter by an examining authority or court.

Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the invention has been described in substantial detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, and yet these modifications and improvements are within the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of, and "consisting of may be replaced with either of the other two terms. Thus, the terms and expressions which have been employed are used as terms of description and not of limitation, equivalents of the features shown and described, or portions thereof, are not excluded, and it is recognized that various modifications are possible within the scope of the invention. Embodiments of the invention are set forth in the following claims.

The invention will be further described with reference to the examples described herein; however, it is to be understood that the invention is not limited to such examples.

EXAMPLES

Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols, for example, as described in Sambrook et al. (2012) Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). Standard materials and methods for polymerase chain reactions can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory⁷ Press, and in McPherson at al. (2000) PCR - Basics: From Background to Bench, First Edition, Springer Verlag, Germany.

Example 1 : Ophthalmic Acid Acts as a Neurotransmitter with Motor Functions

This example demonstrates that methods and compositions as provided herein using the exemplary embodiment comprising administration of ophthalmic acid (ophthalmate) to an individual in need thereof, for example, an individual having a degenerative neurological disorder such asParkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS), Friedreich ataxia, Lewy body disease, Spinal muscular atrophy or Motor neuron disease.

In this study, we challenged the traditional view that dopamine is the primary neurotransmitter involved in motor functions. Our investigations in Parkinson's disease mouse models revealed motor activity in the absence of dopamine, indicating the involvement of non-dopamine systems. Our further comprehensive investigations revealed that ophthalmic acid (OA) acted as a mediator of this activity' through the calcium-sensing receptor (CaSR) signaling. Our findings identify OA as a novel neurotransmitter in motor function regulation, acting at the CaSR receptors. The discovery of a new neurotransmitter has important implications for the understanding of the complex mechanisms underlying motor function and opens up new avenues for the development of effective treatments for movement disorders with fewer side effects than current treatments. The study highlights the therapeutic potential of targeting CaSR signaling and suggests that OA and its derivatives could be a promising option for the treatment of movement disorders.

A plethora of data suggest that L-DOPA has more functions than merely being a precursor for dopamine creation in the brain. Our earlier research showed that L- DOPA produces hyperkinesia in the presence of a central AADC inhibitor (NSD1015) in the reserpine-treated rat model of PD. We speculated, therein, that this hyperkinesia is mediated through the direct action of a metabolite(s) - other than dopamine- on non-dopamine receptors. We based our speculation on the following observations: 1) the onset of motor activity induced by the administration of L-DOPA and a potent central AADC inhibitor (3-hydroxybenzyl hydrazine, NSD1015 (Sigma Aldrich), also known as a-Hydrazino-m-cresol dihydrochloride, 3- (Hydrazinomethyl)phenol dihydrochloride) was delayed (110 minutes) compared to that with L-DOPA alone (45 minutes), suggesting that NSD1015, at least initially, inhibited central AADC activity and prevented the enzymatic conversion of L-DOPA to dopamine or any other effective metabolites [8-10]; 2) 110 minutes following L- DOPA administration, motor activity was greatly increased and hyperkinesia occurred for a prolonged duration of time. We argued that L-DOPA alone could not be responsible for this hyporeactivity solely through its conversion to dopamine, as one would expect that the motor behavior would be abolished subsequent to the inhibition by AADC. These observations provoked our inquiry into the potential molecule(s) behind such a remarkable boost and extension of motor activity. We hypothesized that other neuromodulators are related to motor activity.

To understand the specific mechanisms underlying the observed motor activity when L-DOPA conversion to dopamine is inhibited, we conducted a series of behavioral, metabolomics, binding, and functional studies, and identified the tripeptide ophthalmic acid as a potential neuromodulator, with motor functions that act through activating calcium sensing receptors (CaSR).

Results and Discussion

Brain AADC inhibition delays, increases and prolongs L-DOPA-induced motor activity in PD mice

We sought to examine whether pharmacological inhibition of central AADC, by NSD1015, produces similar phenotypes in mice as those we previously observed in rats. We employed two mouse models of PD: reserpine- and MPTP treatment. The AADC inhibitor NSD1015 acts through binding to the catalytic site of the enzy me and forms (3-hydroxybenzyl)hydrazone of pyridoxal-5 '-phosphate (PLP), which is used by AADC as a cofactor [11, 12] (Fig. la).

We first administered L-DOPA (lOOmg/kg, equivalent to 0.5mmole/kg, labelled 1XL-DOPA) with and without NSD1015 (lOOmg/kg, equivalent to 0.47mmole/kg, labelled 1XNSD1015) in combination with the peripheral AADC inhibitor benserazide (25mg/kg) to reserpine-treated (reserpinized-mice) and control mice (non-reserpinized mice). No alteration of motor activity was found in control animals (Fig. lb). As expected, L-DOPA-treated reserpinized-mice showed a period of enhanced activity that lasted up to four hours (Fig. 1c). Reserpinized mice pretreated with NSD1015 exhibited a greater motor response to L-DOPA than those treated with L-DOPA alone (Fig. Id). The motor activity onset was delayed by around 120 minutes, and the duration of the effect was also extended, compared with L- DOPA alone (Fig. lc,d). While Although the motor response to L-DOPA was dependent on its doses, the impact of NSD1015 doses on the motor response was less clear (Fig. ld'-d"',e). Specifically, the effects of ‘AX and IX ofNSD1015 on L-DOPA- induced motor activity were equivalent, whereas 2X NSD1015 caused an additional increase in hyperactivity induced by ‘AX L-DOPA and IX L-DOPA, but not by 2X L-DOPA (Fig. lf-f".g). This suggests a complex relationship between NSD1015 doses and their effect on motor response to L-DOPA.

We next sought to evaluate whether the hyperactivity provoked by the combination of L-DOPA and NSD1015 was exclusive to L-DOPA or if it could be activated by other dopamine agonists. To answer this question, we administered in two separate experiments a dopamine releasing agent (amphetamine) and a D1/D2 agonist (apomorphine), in combination with NSD1015 to the reserpine-treated mice. Our findings revealed that both drugs produced effects on motor activity similar to their effects when administered alone, indicating that the hyperactivity⁷ provoked by NSD1015 is specific to L-DOPA (Fig. Ih.i). We further assessed the role of dopamine receptors in the L-DOPA/NSD1015-induced hyperactivity in reserpine-treated mice, by pre-treating these mice with a DI receptor antagonist (SCH23390) or a D2 receptor antagonist (haloperidol), and found that neither treatment blocked the hyperactivity induced by L-DOPA/NSD1015 (Fig. Ij). These results indicate that the hyperactivity likely does not involve either DI or D2 dopamine receptors, and imply that the NSD 1015 - L-DOPA combination has a distinct effect on motor activity in these mice.

NSD1015/L-DQPA-treatment is associated with elevated ophthalmic acid levels

In order to acquire a comprehensive understanding of the neurochemical effects of NSD1015-L-DOPA administration in PD mice, we employed a combination of targeted and untargeted metabolomics approaches on the whole brain and striatum tissues (Fig. 2a-e). We found that the primary metabolic consequences of administering L-DOPA in conjunction with AADC disruption were remarkable changes in the levels of L-DOPA and its metabolites in the brain and striatum (Fig. 2f-h). Dopamine levels in L-DOPA-treated mice correlated well with the motor activity, as they were significantly lower at 7 hours than at 2 hours. In the NSD1015/L-DOPA group, dopamine levels did not vary⁷ between 2 hours and 7 hours, suggesting that dopamine was not likely the compound responsible for the hyperactivity. This implies that non-dopamine pathways are involved in the hyperactivity caused by NSD1015/L-DOPA at 7 hours (Fig. 2g). We found substantial changes in a number of phenylalanine/tyrosine metabolites and other metabolites derived from aromatic amino acids in the brain and striatum, noticeably elevations in their methylated, deaminated, and hydroxylated metabolites (Fig. 2g, h). Some of these changes are characteristic of AADC deficiency, including high 5- hydroxytryptophan and 3-0- methyldopa, and low homovanillic acid (HVA) and 5- hydroxyindoleacetic acid (Fig. 2g.h) [13, 14],

The notable (13-fold) increase in 3-methoxytyramine (3-MT, 3- methyldopamine) between L-DOPA and the NSD1015/L-DOPA groups at the peak of hyperactivity (at 7 hours) suggests that 3-MT may be involved in the hyperactivity. However, the fact that these levels were still lower at 7 hours than at 2 hours, when the NSD1015/L-DOPA mice were hypoactive, suggests that 3-MT is unlikely to be the sole compound responsible for the observed hyperactivity, though it might be one of the contributing factors.

Ophthalmic acid (OA; L-Y-Glutamyl-L-2-Aminobutyryl-Glycine, y-Glu-2AB- Gly) exhibited the largest change in response to NSD1015 pretreatment. Specifically, OA increased during the hyperactivity peak (7 hours) by 20-fold in the whole brain and 8-fold in the striatum of L-DOPA/NSD1015- compared to L-DOPA-treated mice (Fig. 2i). OA was initially isolated from the calf lens [15], and thus the name, and has subsequently been detected in the lenses and other tissues of higher animals, and in microorganisms [16-19],

Structurally, OA is analogous to glutathione (GSH; L-y-Glutamyl-L- Cysteinyl -Glycine, y-Glu-Cys-Gly), which functions as an essential antioxidant and detoxifying agent in the biological systems [15], OA is generated through the sequential reactions of glutamate-cysteine synthase (GCS) and glutathione synthase (GS), which are also involved in GSH synthesis (Fig. 2i) [20, 21], The notable increase in OA levels in the brain and striatum of L-DOPA/NSD1015-treated mice suggests that these two drugs have an effect on glutathione metabolism and OA production. GSH and OA synthesis is intricately linked to methionine (Met) metabolism via the transsulfuration pathway, which is facilitated by cystathionine [>- synthase (CBS) and cystathionine y-lyase (CSE) (Fig. 2k) [22], CSE catalyzes the cleavage of cystathionine to generate equal amounts of cysteine (Cys) and 2- ketobutyric acid (2KB), which is subsequently converted to 2-aminobutryate (2AB) through a transamination reaction with glutamate and other keto-carboxyl compounds as the donor substrate and AST aminotransferase as the catalytic enzy me [23-25], Based on these findings, the elevations of OA levels observed in the NSD/L-DOPA group may be related to the alteration of methylation pathways and the increase in methylated and hydroxyl-carboxyl metabolites observed in this group.

The scarcity' of research on OA implies that its physiological importance is yet to be established. In the literature, two contexts of OA production have been identified: 1) by E. coli that lack PLP-dependent proteins [19. 26], and 2) in conditions where GSH synthesis is required, thus being viewed as a biomarker for oxidative stress and cysteine or GSH depletion [27, 28], GSH depletion conditions associated with elevated OA levels include acetaminophen-overdose-induced liver toxicity, starvation and extreme stress [28-32], This is due to the fact that, under reducing conditions, GSH inhibits GCS and blocks the production of OA. However, during oxidative stress, the depletion of GSH activates GCS, leading to the biosynthesis of OA [28, 31, 32], By examining these two contexts in which OA has been identified, it can be inferred that the rise in OA levels observed in our study may be linked to the effects of the combination of L-DOPA and NSD1015 on AADC. oxidative stress, and GSH metabolism. First, NSD1015 inhibits AADC, which is a PLP-dependent enzy me [11, 12], mimicking the production of OA in E. coli lacking PLP-dependent proteins. Second, L-DOPA is a highly reactive molecule that can cause oxidative stress, and excessive L-DOPA, in the absence of its conversion to dopamine, is removed through reacting and being conjugated with GSH, leading to GSH depletion [33, 34], The observations of altered PLP and GSH levels in the NSD1015/L-DOPA group supports these notions (Fig. 2i). These findings suggest that the rise in OA levels observed in our study may be a result of a combination of factors. These factors include changes in PLP and GSH metabolism due to the effects of NSD1015 on AADC and the presence of L-DOPA, which can cause oxidative stress, in addition to the augmented methylation pathways, which facilitate the synthesis of both OA and GSH (Fig. 2i). This conclusion is further substantiated by the observed rise in the stress hormone corticosterone and the changes in energy pathways, specifically, the glycolysis and mitochondrial tricarboxylic acid (TCA) cycle metabolism pathways.

OA is synthesized in the brain and regulates motor function

We investigated whether the increase in OA levels is related to the hyperactivity induced by L-DOPA/NSD1015 treatment in mouse models of PD. Initially, we examined the direct effect of OA on PD mice and found that when administered peripherally (i. p. ), it did not produce a motor response (Fig. 3a). However, due to insufficient data on OA's pharmacokinetics, it was unclear whether the lack of response was due to its inactive nature or inability to cross the blood-brain barrier (BBB). To address this, we administered deuterated OA (D-OA) peripherally (i.p.) in mice and used liquid chromatography -mass spectrometry (LC-MS) to track its presence in various tissues, including the brain. The results indicated that D-OA was undetectable in the brain after peripheral injection, although it was present in the plasma and other peripheral organs (Fig. 3b). This suggests that OA cannot cross the BBB and, more importantly, provides evidence that the increase in OA levels following NSD1015/L-DOPA treatment is due to its synthesis within the brain.

Consequently, we infused OA and L-DOPA directly into the brain (intracerebroventricular injection, i.c.v.) and found that while OA did not produce any motor activity' in normal animals at doses of 2.5pM, 5pM and lOpM (Fig. 3c, d), it increased motor activity in MPTP- mice in a dose-dependent manner (Fig. 3e-h). Moreover, the duration of OA motor effects was prolonged, lasting for approximately 24 hours, with the highest effects observed during the first 10 hours (Fig. 3i-j). OA induces motor function by acting as a neuromodulator on CaSR

We then sought to elucidate the mechanism through which OA induces motor activity. We first investigated the specific interactions of OA with receptor sites on brain sections, using a radioligand binding saturation experiment with increasing concentrations of labeled OA ([3H]-OA). A saturating concentration of unlabeled OA was used to assess non-specific binding. Our analysis indicated that [3H]-OA binds reversibly to saturable sites in the brain sections, with a single population of moderate-affinity sites (Kd 2.61± 0.36 pM) (representative binding curves are shown in Fig. 4a).

The similarity' between OA and GSH and the prior discoveries that GSH and its other gamma-glutamyl-tri-peptides analogs modulate calcium sensing receptors (CaSR) [35, 36] led us to explore whether OA binds to and modulates CaSR activity. CaSR is a class C G protein-coupled receptor (GPCR) that is activated by extracellular calcium (Ca2+) [37-40], and can activate multiple signaling pathways through Gq/11, Gi/o, G12/13, and Gs proteins [37, 41-43], Aromatic L-amino acids (AAAs) such as L-phenylalanine, L-tyrosine, and L-tryptophan increase the sensitivity of CaSR to Ca2+ and, thus, are considered positive allosteric modulators of the receptor [44-49], Ca2+ ions and aromatic L-amino acids have also been proposed to act as co-agonists of the receptor [45, 48, 50],

We, therefore, aimed to determine whether the saturable sites, to which OA binds in brain sections, are CaSRs. We conducted a similar radioligand binding saturation experiment in the presence of a saturating concentration of an unlabeled CaSR antagonist (NPS-2143). At low concentrations of [3H]-OA (0. 1-0.5 pM), 80- 85% of the total binding of the labeled peptide to brain sites was inhibited by NPS- 2143 (Fig. 4b). The specific component of binding was saturable, while the nonspecific binding was linearly dependent on OA concentration. The calculated Kd of OA binding to CaSR was 1.62± 0.22 pM. [3H]-0A binding was also inhibited by a saturating concentration of Ca2+ and L-DOPA, rendering a Kd for [3H]-OA binding of 4.06±1.47 pM and 5.55± 0.85 pM, respectively (Fig. 4b). While this finding does not conclusively prove that L-DOPA binds to CaSR, it indicates that OA and L- DOPA bind to the same target in the brain. [3H]-0A binding was also inhibited by combinations of saturating concentrations of L-DOPA/NPS-2143, Ca2+/L-DOPA, Ca2+/NPS-2143. rendering a kd for [3HJ-OA binding of 3.99±0.05, 5.28±0.57. and 5.99±0.1 respectively (Fig. 4c). Finally, using a radioligand competitive (displacement) experiment, with a fixed concentration of [3H]-0A (2.5 pM), we found that Ca2+ inhibited the binding of [3H]-0A in a dose-dependent manner, with Ki of 0.98±0.13 pM (Fig. 4d), thereby verifying the binding of OA to calcium binding sites in the brain. Neither SKF-82958, a DI dopamine receptor ligand, nor haloperidol, a D2 dopamine receptor ligand, displaced [3H]-0A binding.

To investigate whether OA acts as an agonist or antagonist at CaSR, we conducted a cAMP -luminescence assay, using HEK cells expressing CaSR, and forskolin to enhance cAMP production. We found that OA activated Gi-coupled CaSR (decreased forskolin-induced cAMP) in a dose-dependent manner with an EC50 of 1.56 pM (Fig. 4e). Under the same conditions, L-DOPA also activated the CaSR at an EC50 comparable to that of the EC50 of Ca2+, the orthosteric agonist for CaSR (Fig. 4f,g). L-DOPA agonistic action on CaSR is noteworthy given that other aromatic amino acids, such as L-tryptophan and phenylalanine, are known to act as allosteric agonists at CaSR. In the presence of NPS-2143, a negative allosteric modulator (NAM), the CaSR displayed a decrease in maximal signaling capacity (Emax) and rightward shift of the dose-response curves of OA, L-DOPA. and Ca2+ (Fig. 4e-g). Lastly, we found that the response curve exhibited an increase in Emax when all three ligands, OA, Ca2+, and L-DOPA, were present together, as opposed to when each ligand was present alone (Fig. 4h). Our results provide evidence that both OA and L-DOPA bind to and activate CaSR at an EC50 comparable to that of the orthosteric agonist (Ca2+), and that L-DOPA, like other aromatic amino acids, enhances the activation of CaSR by OA. Further, our study showed that L-DOPA displayed increased Emax of the dose-response curves of OA.

Since these experiments did not identify the specific binding sites of OA and L-DOPA to CaSR, we constructed a model for their binding to CaSR using computational modeling and structure-based docking. Our docking model predicted binding of OA to CaSR, similar to TRP, to the L-aromatic acid binding pocket (Fig. 4i-l).

We lastly examined the involvement of CaSR in motor activity induced by L- DOPA/NSD1015 or OA. We found that the CaSR antagonist NPS-2143 inhibited the motor activity induced by L-DOPA/NSD1015 in MPTP-treated mice (Fig. 5a, b), indicating that activating CaSR plays a role in the observed hyperactivity. Additionally, we found that pretreatment with NPS-2143 inhibited OA-induced motor activity in a dose-dependent manner in MPTP model of PD (Fig. 5c-f), suggesting that the effect of OA on motor activity' is mediated through its action on CaSR.

In conclusion, our study provides evidence for the role of ophthalmic acid (OA) as a neurotransmitter that regulates motor function through its action at CaSR. The fact that OA regulates motor function through CaSR highlights the importance of this pathway in the development of novel therapies for movement disorders such as Parkinson’s disease. The results open up new avenues for the development of therapeutic interventions that target CaSR signaling, and suggest that OA and/or its derivatives may be a promising therapeutic agent for the treatment of these conditions. Additionally, OA can act as a therapeutic agent for neurological and movement disorders beyond PD.

Figure Legends Example 1

Figure 1. Motor response to L-DOPA is enhanced by inhibition of DOPA decarboxylase in mouse model of Parkinson’s disease (PD).

(a) The biosynthesis of dopamine from L-DOPA and its proposed inhibition by benserazide and NSD1015 in the periphery and the central nervous system, respectively. (b) Effects of L-DOPA and NSD on normal mice: The mice were injected subcutaneously (s.c.) with a vehicle, and 18 hours later, injected intraperitoneally (i.p.) with either NSD1015 or saline, followed by an L-DOPA or saline injection 30 minutes after. Motor activity was monitored for 20 hours. Data represent (left) the time-course and (right) the area under the curve (AUC) of the effects of L-DOPA, NSD1015, and combination of L-DOPA and NSD1015. Values are expressed as mean±S.E. n=8 for each group.

(c) Effects of L-DOPA and NSD on reserpinized mice: Mice were injected (s.c.) with reserpine 1 mg/kg, and 18 hours later, mice were injected with NSD or saline, followed by an DOPA injection 30 minutes after. Locomotion was monitored for 20 hours. Data represent the (left) time-course of the effect of L-DOPA alone and L-DOPA in conjunction with NSD1015, and (right) the area under the curve. One way ANOVA, followed by Tukey post-test: ***P < 0.001, ****p < 0.0001, ns, not significant. Values are expressed as mean±S.E. n=8 for each group.

(d-g) Effect of varying doses of DOPA and NSD on motor activity. Mice were injected (i.p.) with reserpine 1 mg/kg and 18 hours later, mice were injected with varying doses of NSD1015 followed by varying doses of L-DOPA 30 minutes after. Locomotion was monitored for 20 hours. Same data are presented in two figures. (d,e) Data represents (d'-d'") the time-course and (e) the area under the curve of the effect of varying doses of DOPA with fixed doses of NSD. Values are expressed as mean±S.E. n=6 for each group. (f,g) Data represents (F-f ") the time-course and (g) the area under the curve of the effect of varying doses of NSD w ith fixed doses of DOPA.

(h,i) Motor effect of the combination of dopaminergic agents and NSD. Mice were injected (s.c.) with reserpine 1 mg/kg and 18 hours later, mice were injected with NSD or saline, follow ed by apomorphine or amphetamine. Data represents the time-course of the effect of conjunction of NSD with (h) amphetamine and (i) apomorphine.

(j) Effect of haloperidol on the hyperactivity induced by DOPA and NSD. Mice were injected (s.c.) with reserpine 1 mg/kg and 18 hours later, mice were injected with NSD1015, followed by DOPA and haloperidol injection 30 minutes after. Locomotion w as monitored for 20 hours. Data represents the time-course of the effect of haloperidol on DOPA alone and DOPA in conjunction with NSD. Data are expressed as mean±S.E. n=8 for each group. Figure 2, Motor Response to L-DOPA and AADC Inhibitor correlates with alterations in brain and striatum metabolites, with highest alteration in ophthalmate levels

(a) The study's experimental design and timeline. Mice were injected (s.c.) with reserpine 1 mg/kg. After 18 hours, the mice were treated with NSD1015 or saline, followed by L-DOPA administration 30 minutes later. Brain tissues were collected from the two treatment groups at two time points (2 hours and 7 hours after L-DOPA administration). The brains were divided into two hemispheres, with one hemisphere homogenized entirely, and the other hemisphere's striatum was used to extract the striatum.

(b) Unsupervised principle component analysis (PCA) of the metabolomics data of mouse brain and striatum from two treatment groups at the two time points. n=6 for each group.

(c) Unsupervised hierarchical clustering analysis (HCA) of the differential metabolites of mouse brain and striatum from two treatment groups at two time points.

(d) Table showing the count of altered metabolites in the (left) whole brain and (right) striatum.

(e) Box plot legend showing the range, median, and quartiles.

(f) The fold changes in the major components of dopamine synthesis pathway- after L-DOPA/NSD1015 administration.

(g) The fold changes in the major L-DOPA metabolites that are not related to the dopamine pathway.

(h) The fold changes in the major metabolites of the metabolic pathways for the aromatic amino acids: L-tryptophan, phenylalanine, and tyrosine.

(i) The fold changes in ophthalmate and the key metabolites of the proposed pathways leading to its synthesis.

The asterisk (*) is used to compare metabolite levels within the same group at the two different time points, whereas the hashtag (#) is used to compare metabolite levels between the two treatment groups at the same time point. * and # P< 0.05. ** and ## P < 0.01, *** and ### P < 0.001, and **** and #### P < 0.0001.

Figure 3, QA is synthesized in the brain and modulates motor function in mouse model of PD

(a) Peripheral OA injection did not induce motor activity in MPTP -treated mice. Mice were injected with MPTP (i.p.) 20 mg/kg for consecutive three days, followed by a single injection of saline or OA at three different doses. Motor activity was monitored for 20 hours (12 hours shown). Data represent the time-course of the effect of OA injected i.p. at three different doses and are expressed as mean±S.E. n=8 for each group.

(b) OA levels following peripheral administration were measured using Liquid chromatography-mass spectrometry (LC-MS). Mice were injected (i.p.) with deuterated OA (d5-OA) and the brain and blood samples were collected 10 minutes after injection. Data represent the interpolated concentrations of d5-OA in the blood and brain and are expressed as mean±S.E. n=4 for each group.

(c-d) The effect of central administration of OA on the motor activity of normal mice. Anesthetized mice underwent surgery, during which a cannula was implanted for future intracerebroventricular (i.c.v.) injections. Following recovery, mice were injected i.c.v. with either saline or OA at three different doses, and their motor activity' was monitored for 20 hours. The data presented show (c) the timecourse and (d) the AUC of the effect of OA injected i.c.v. at three different doses effect. One way ANOVA, followed by Tukey post-test, ns: not significant. Data are expressed as mean±S.E. n=8 for each group.

(e-j) The effect of central administration of OA on the motor activity' of MPTP-treated mice. Mice underwent surgery under anesthesia to implant a cannula for future intracerebroventricular (i.c.v.) injections. After recovery, mice were injected (i.p.) with MPTP 20 mg/kg for three days. The following day, mice were injected i.c.v. with either saline or OA at four different doses, and their motor activity' was monitored for 20 hours. The data presented include (e) the time-course of the effect of MPTP, (f) the AUC of the effect of MPTP during the 20-hour experiment time; unpaired t-test, ns: not significant, (g) the time-course of the effect of OA injected i.c.v. at four different doses, (h) the AUC of the effect of different doses of OA during the 20-hour experiment time, (i) the AUC of the effect of different doses of OA during the first 10 hours of the experiment time, and (j) the AUC of the effect of different doses of OA during the 10-20 hours of the experiment time. In (h). (i), and (j) One way ANOVA, followed by Tukey post-test: **P < 0.01, ****P < 0.0001, ns: not significant. Data are expressed as mean±S.E. n=8 for each group.

Figure 4, OA acts as a neuromodulator on CaSR

(a-b) Saturation curve of [3H]-OA binding to mouse brain sections in the presence of unlabeled OA, NPS2143 (Chemical Abstracts Service (CAS) Number 284035-33-2) (a selective calcium-sensing receptor antagonist), Ca2+, and L-DOPA. [3H]-OA binding was carried out as described in the methods section, (a) Representative plot of total, specific, and nonspecific binding of [3H]-0A to mouse brain sections. Non-specific binding was determined as the levels of [3H]-0A binding in the presence of 100 pM unlabeled OA. (b) Representative plot of specific binding of [3H]-0A binding to mouse brain sections, where non-specific bindings were defined as the levels of [3H]-0A binding in the presence of 10 pM NPS-2143, 100 pM calcium, and 100 pM L-DOPA. (c) Representative plot of specific binding of [3H]-OA binding to mouse brain sections, where non-specific binding was defined as the level of [3H]-0A binding in the presence of combinations of NPS-2143+ Ca2+, L-DOPA+ Ca2+. and NPS-2143 + L-DOPA. In all binding experiments, the degree of binding is expressed in disintegrations per minute (dpm). Data represent binding from three experiments for non-specific binding for each point (total 36 sections repeats for each point).

(d) Inhibition of [3H]-0A binding to mouse brain sections by Ca2+. Competition experiments were carried out as described in the methods. Radioligand binding assay was performed in the presence of 2.5 nM [3H]-OA and increasing concentrations of Ca2+. Each point represents the mean ±S.E of at least 3 measurements from three experiments.

(e-h) Forskolin- stimulated cAMP GLOSENSOR™ luminescence responses in HEK 293 cells transiently transfected with the GLOSENSOR™ cAMP biosensor and the CaSR plasmid, (e-g) Representative dose-response curves of cAMP signal for (e) OA. (f) L-DOPA, and (g) Ca2+ with and without CaSR antagonist NPS-2143, and (h) different combinations of the three ligands OA, L-DOPA, and Ca2+. Data are represented as relative light units (R.L.U.), and are expressed as mean±S.E. of at least three experiments, each with at least triplicate measurements.

(i-1) Docking models Ligand-bound states in CaSR binding site in domain A (named A in amino acid hereinafter), (i) Cartoon presentation of ligand-bound CaSR structure (domain A) in the closed-closed conformation (5 Angstroms), (j) Interface analysis of Tryptophan (Trp)-bound state in CSRS binding are shown with Serl70A, Serl47A, Ala298A, and Thr 145A and Alal68A. (k) Interface analysis of L-dopa (LDP)-bound state in CaSR binding are shown with Tyr218A, Serl70A, Asp216A, and Alal68A. (1) Interface analysis of Ophthalmate (OPT) bound state in CaSR binding are shown with Serl47A, Glyl48A, Tyr218A, Serl70A, Asp216A, and Alal68A, Vall49A. Green curve shows the transition state of Ala298A and Thr 145A in the Trp-bound state, Tyr218A in LDP-bound state, and Gly 146A in the OPT-bound state.

Figure 5, QA induces motor function by acting on CaSR

(a,b) CaSR antagonist NPS-2143 inhibition of motor response induced by L- DOPA/NSD1015 in MPTP -treated mice. Mice were anesthetized and underwent surgery where a cannula was implanted for future intracerebroventricular (ICV) injections. Following recovery, the mice were injected (i.p.) with MPTP 20 mg/kg for three days. The following day, mice were injected with NSD1015 or saline, followed by a saline or L-DOPA injection 30 minutes after with or without NPS2143. Locomotion was monitored for 20 hours. Data represents (a) the time-course and (b) the AUC of the effect of NSD1015 and L-DOPA with or without NPS2143. One way ANOVA, followed by Tukey post-test: ****P < 0.0001, ns: not significant. Data are expressed as mean±S.E. n=8 for each group.

(c-f) CaSR antagonist inhibits OA-induced motor response in MPTP -treated mice. Mice were subjected to surgery and MPTP treatment as in (a-b). On the day following the final MPTP treatment, mice were administered, mice were injected i.c.v. with either saline or OA at (c,d) 5 pM and (e,f) 10 pM with or without NPS2143 (10 nM and 20 nM). Locomotion was monitored for 20 hours. Data represent (c,e) the time-course and (d,f) the AUC of the effect of OA with and without NPS2143 on motor activity. In (d) and (f) One way ANOVA, followed by Tukey post-test: **P < 0.01, ****P < 0.0001, ns: not significant. Values are expressed as mean±S.E. n=8 for each group.

Methods Example 1

Mouse Models of Parkinson’s disease

Swiss Webster mice (8-10 weeks old) were obtained from Charles River Laboratories. Animals were group housed with a maximum of four animals per cage and acclimated to the vivarium for a week prior to treatments. Animals were kept in a normal 12: 12 hour light/dark cycle with free access to food and water. For the reserpine treatment, animals were lightly anesthetized and then injected subcutaneously with either reserpine (1 mg/kg, dissolved in 1% (v/v) glacial acetic acid) or vehicle (1% (v/v) glacial acetic acid). For the MPTP treatment, mice were injected intraperitoneally for 3 days with MPTP (20 mg/kg) or saline once per day. Brain Tissue Harvesting: Adult male Swiss Webster mice, weighing approximately 20-30g, were sacrificed by asphyxiation. The brains were rapidly removed on a cold surface, frozen in isopentane at -40°C and then stored at -80°C.

Perfusion: Adult male Swiss Webster mice were anesthetized using isoflurane and using sterile PBS, the blood from the tissues and brain was flushed out. A 4% paraformaldehyde (PF A) solution was then flushed through the animal until the limbs became rigid. The brain was removed and kept in a 4% PFA solution for 24-48 hours at 4°C. The brain was then transferred to a solution of 30% sucrose where it was kept until it was ready to be sectioned.

Immunohistochemistry : Coronal sections were cut at 20 pm thickness. Sections were blocked with 4% goat serum in PBS with 0.3% triton x-100 for 1 hour and were then incubated with the primary antibody diluted in the blocking buffer overnight. The sections were then washed three times with PBS. The sections were then incubated with the secondary antibody for one hour, washed three times again, and then mounted onto gelatin-coated glass slides. Sections were then imaged using a BZ-9000 fluorescence microscope (Keyence, Osaka, Japan), looking for levels of TH and only data from animals with sufficient deletion were used.

Cannulation Surgery : Swiss Webster mice were anesthetized with isoflurane (5% for induction; 1-3% to sustain). Once anesthetized, each mouse w as placed on the stereotaxic device and implanted with a stainless-steel cannula (20G, 2.5 mm length) into the brain ventricle (0.2 mm posterior and 1 mm lateral to bregma and 2.3 mm below the surface of the skull). Mice were allowed to recover for 7 days.

Assessment of Locomotor Activity : Mice were placed in a locomotor test chamber (40 x 40 x 38 cm3) and the horizontal locomotor activity was monitored with a 16 x 16 photobeam array (San Diego Instruments, San Diego, CA) located 1.25 cm above the floor of the enclosure. Mice were transported to the activity chamber room at least 30 minutes prior to placement into the activity chambers. All mice were given time to acclimatize to the chamber prior to inj ections, and locomotor activity was recorded for 20-24 hours following drug administration. Mice were randomly distributed to each treatment group with all proper controls being run in parallel. cAMP Assay: Human embry onic kidney 203 (HEK293T) cells were grown in DMEM adjusted to contain 10% fetal bovine serum (FBS), 1% penicillin and streptomycin at 5-10% CO2 and 37°C HEK293T cells were transiently transfected to express both the PGLOSENSOR-22F™ cAMP plasmid and a CaSR plasmid. Cells were transfected using the JETPRIME™ transfection reagent using the standard conditions of 5 pg of each plasmid and 20 pl of the reagent. After an overnight incubation in 37°C with 5-10% CO2, the cells were then ready to be used. On the day of the experiment, cells were washed with PBS and detached using 0.05% trypsin. The cells are then pelleted and resuspended in CO2-independent media, 10% FBS, and the GLOSENSOR™ cAMP reagent. The cells are incubated at room temperature for 2 hours and are gently mixed every 15 minutes to prevent the cells from settling. The cells are then transferred to a 96-well plate and luminescence was measured using the MicroBeta2 2450 Microplate counter. Basal luminescence level is recorded prior to assay.

Radioligand Binding Assay: Sections were pre-incubated in buffer (50mM Tris, 1.5mM EDTA), PH 7.4 for 30 minutes at 4°C. Total binding was determined by incubating sections in buffer containing varying concentrations of [³H]-OA for 60 minutes at room temperature. Non-specific binding of [³H]-0A was defined by that seen in the presence of lOpM NPS-2143. The incubation was terminated by washing the sections in an ice-cold (4°C) incubation buffer for 10 minutes. The sections were then dipped in ice-cold distilled water and dried in a stream of cold air. Sections w ere scraped off of the slides and placed into vials containing 4.5 ml of scintillation fluid and were subsequently measured at a scintillation counter.

Example 2: Ophthalmate is a new regulator of motor functions via CaSR: implications for movement disorders

In this study we sought to determine the metabolites associated with the pronounced hyperactivity observed, using comprehensive metabolomics analysis. Our results revealed that the peak in motor activity induced by NSD1015, a DOPA decarboxylase inhibitor (Sigma Aldrich)/L-DOPA in PD mice is associated with a surge (20-fold) in brain levels of the tripeptide ophthalmic acid (OA, also know n as ophthalmate in its anionic form). Interestingly, we found that administering ophthalmate directly to the brain rescued motor deficits in PD mice in a dosedependent manner. We investigated the molecular mechanisms underlying ophthalmate’s action and discovered, through radioligand binding and cAMP- luminescence assays, that ophthalmate binds to and activates the calcium-sensing receptor (CaSR). Additionally, our findings demonstrated that a CaSR antagonist inhibits the motor-enhancing effects of ophthalmate, further solidifying the evidence that ophthalmate modulates motor functions through the activation of the CaSR. The discovery- of ophthalmate as a novel regulator of motor function presents significant potential to transform our understanding of brain mechanisms of movement control and the therapeutic management of related disorders.

Described in this Example are a series of behavioral, metabolomic, binding, and functional studies, where we identified the tripeptide ophthalmic acid (OA. also known as ophthalmate in its anionic form) as a modulator of motor functions through activating calcium sensing receptors (CaSR).

Materials and methods Example 2

1. Mouse models of Parkinson’s disease

Swiss Webster mice (8-10 weeks old) were obtained from Charles River Laboratories. Animals were group housed with a maximum of 4-5 animals per cage and acclimated to the vivarium for a week prior to treatments. Animals were kept in a normal 12: 12 hour light/dark cycle with free access to food and water. For the reserpine treatment, animals were lightly anesthetized and then injected subcutaneously (s.c.) with either reserpine (1 mg/kg, dissolved in 1% (v/v) glacial acetic acid) or vehicle (1% (v/v) glacial acetic acid), as we previously described. ⁸ For the MPTP treatment, mice were injected intraperitoneally (i.p.) for 3 days with MPTP (l-methyl-4-phenyl-l, 2,3,6- tetrahydropyridine) (20 mg/kg) or saline per day, based on previous studies, with slight modifications.^12,13 All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, Irvine, and were conducted in accordance with national and institutional guidelines for the care and use of laboratory animals.

2, Global Metabolomics Profiling

Metabolomic analysis was performed by Metabolon (Durham, NC).

Brain tissue harvesting: Mice were injected (s.c.) with reserpine 1 mg/kg. After 18 hours, the mice were treated with NSD1015 or saline, followed by L-DOPA administration 30 minutes later. Brain tissues were collected from the two treatment groups at two time points (2 hours and 7 hours after L-DOPA administration). The brains were divided into two hemispheres, with one hemisphere homogenized entirely, and the other hemisphere used to extract the striatum.

Sample Preparation: Samples were prepared using the automated MICROLAB STAR® system from Hamilton Company. Several recovery standards were added prior to the first step in the extraction process for QC purposes. To remove protein. dissociate small molecules bound to protein or trapped in the precipitated protein matrix, and to recover chemically diverse metabolites, proteins were precipitated with methanol under vigorous shaking for 2 min (GLEN MILLS GENOGRINDER 2000™) followed by centrifugation. The resulting extract was divided into five fractions: two for analysis by two separate reverse phase (RP)/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI), one for analysis by RP/UPLC-MS/MS with negative ion mode ESI. one for analysis by HILIC/UPLC- MS/MS with negative ion mode ESI, and one sample was reserved for backup. Samples were placed briefly on a TURBOVAP® (Zymark) to remove the organic solvent. The sample extracts were stored overnight under nitrogen before preparation for analysis.

QA QC: Several types of controls were analyzed in concert with the experimental samples: a pooled matrix sample generated by taking a small volume of each experimental sample served as a technical replicate throughout the data set; extracted water samples served as process blanks; and a cocktail of QC standards that were carefully chosen not to interfere with the measurement of endogenous compounds were spiked into every analyzed sample, allowed instrument performance monitoring and aided chromatographic alignment. Instrument variability' was determined by calculating the median relative standard deviation (RSD) for the standards that were added to each sample prior to injection into the mass spectrometers. Overall process variability was determined by calculating the median RSD for all endogenous metabolites (i.e., non-instrument standards) present in 100% of the pooled matrix samples. Experimental samples were randomized across the platform run with QC samples spaced evenly among the injections.

Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC- MS/MS): All methods utilized a Waters ACQUITY ultra-performance liquid chromatography (UPLC) and a Thermo Scientific Q-Exactive high resolution/ accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and ORBITRAP™ mass analyzer operated at 35,000 mass resolution. The sample extract was dried then reconstituted in solvents compatible to each of the four methods. Each reconstitution solvent contained a series of standards at fixed concentrations to ensure injection and chromatographic consistency. One aliquot was analyzed using acidic positive ion conditions, chromatographically optimized for more hydrophilic compounds. In this method, the extract was gradient eluted from a C18 column (WATERS UPLC BEH™ C18-2.1x100 mm. 1.7 pm) using water and methanol, containing 0.05% perfluoropentanoic acid (PFPA) and 0.1% formic acid (FA). Another aliquot was also analyzed using acidic positive ion conditions; however, it was chromatographically optimized for more hydrophobic compounds. In this method, the extract was gradient eluted from the same afore mentioned C 18 column using methanol, acetonitrile, water, 0.05% PFPA and 0.01% FA and was operated at an overall higher organic content. Another aliquot was analyzed using basic negative ion optimized conditions using a separate dedicated C18 column. The basic extracts were gradient eluted from the column using methanol and water, however with 6.5mM Ammonium Bicarbonate at pH 8. The fourth aliquot was analyzed via negative ionization following elution from a HILIC column (Waters UPLC BEH Amide 2.1x150 mm, 1.7 pm) using a gradient consisting of water and acetonitrile with lOmM Ammonium Formate, pH 10.8. The MS analysis alternated between MS and data-dependent MSⁿ scans using dynamic exclusion. The scan range varied slighted between methods but covered 70-1000 m/z. Raw data files were archived and extracted as described below.

Bioinformatics: The informatics system consisted of four major components, the Laboratory' Information Management System (LIMS), the data extraction and peakidentification software, data processing tools for QC and compound identification, and a collection of information interpretation and visualization tools for use by data analysts. The hardware and software foundations for these informatics components were the LAN backbone, and a database server running ORACLE 10.2.0. 1™ (Enterprise Edition).

Data Extraction and Compound Identification: Raw data was extracted, peak- identified and QC processed using Metabolon’s hardware and software. These systems are built on a web-service platform utilizing MICROSOFT™’s .NET™ technologies, which run on high-performance application servers and fiber-channel storage arrays in clusters to provide active failover and load-balancing. Compounds were identified by comparison to library entries of purified standards or recurrent unknown entities. Metabolon maintains a library based on authenticated standards that contains the retention time/index (RI), mass to charge ratio (m/z), and chromatographic data (including MS/MS spectral data) on all molecules present in the library. Furthermore, biochemical identifications are based on three criteria: retention index within a narrow RI window of the proposed identification, accurate mass match to the library +/- 10 ppm, and the MS/MS forward and reverse scores between the experimental data and authentic standards. The MS/MS scores are based on a comparison of the ions present in the experimental spectrum to the ions present in the library spectrum. While there may be similarities between these molecules based on one of these factors, the use of all three data points can be utilized to distinguish and differentiate biochemicals. More than 3300 commercially available purified standard compounds have been acquired and registered into LIMS for analysis on all platforms for determination of their analytical characteristics. Additional mass spectral entries have been created for structurally unnamed biochemicals, which have been identified by virtue of their recurrent nature (both chromatographic and mass spectral). Metabolite Quantification and Data Normalization: Peaks were quantified using area-under-the-curve. For studies spanning multiple days, a data normalization step was performed to correct variation resulting from instrument inter-day tuning differences. Essentially, each compound was corrected in run-day blocks by registering the medians to equal one (1.00) and normalizing each data point proportionately (termed the “block correction’⁷).

3. Synthesis of d5-Qphthalmate

The detailed methods of d5-OA synthesis are described in a paper in preparation. Briefly, the synthesis of d5-ophthalmate as a mixture of isomers was accomplished with standard peptide coupling reagents. Glycine benzyl ester was first coupled with racemic tert-butyloxy carbonyl protected d5-2-aminobutyrate. This dipeptide was deprotected and N-carbobenzyloxy-L-glutamate 1 -methyl ester (Z-L-Glu-OMe) was added. Two deprotection steps, hydrolysis of the methyl ester, and hydrogenolysis of the benzyl ester and the N-carbobenzyloxy groups gave the acetate salt of d5-OA.

4, Measurement of d5-ophthalmate using tandem mass spectrometry (ms/ms) Adult male Swiss Webster mice, weighing approximately 20-30g, were sacrificed by asphyxiation. After decapitation, the blood was collected and the brains were rapidly- removed on a cold surface, frozen in isopentane at -40°C and then stored at -80°C. The protocol for metabolite extraction from the brain tissue and serum was adapted from a previously reported protocol ¹⁴ Frozen tissue was immediately plunged into methanol (1 ml) containing internal standards and homogenized for 1 minute to inactivate enzymes. 500 pl of deionized water was added and 300 pl of the solution was transferred to another tube and 200 pl of chloroform was added and mixed thoroughly. The solution was centrifuged at 12,000 x g for 15 minutes at 4°C. The upper aqueous layer was filtered and the filtrate was lyophilized and dissolved in 50 pl of methanol. For the serum, 200 pl of serum was plunged into 1.8 ml of methanol. 800 pl of deionized water was added and an additional 2 ml of chloroform was added as well. The solution was centrifuged at 2,500 x g for 5 minutes at 4°C. The upper aqueous layer was filtered, lyophilized and dissolved in 50 pl of methanol. Ophthalmate measurements were performed using a TSQ QUANTUM ULTRA MASS SPECTROMETER™ (Thermo Finnigan, San Jose, CA).

5. Perfusion and immunohistochemistry

Adult male Swiss Webster mice were anesthetized using isoflurane and using sterile PBS, the blood from the tissues and brain was flushed out. A 4% paraformaldehyde (PF A) solution was then flushed through the animal until the limbs became rigid. The brain was removed and kept in a 4% PFA solution for 24-48 hours at 4°C. The brain was then transferred to a solution of 30% sucrose where it was kept until it was ready to be sectioned. Coronal sections were cut at 20 pm thickness. Sections were blocked with 4% goat serum in PBS with 0.3% TRITON X-100™ for 1 hour and were then incubated with the tyrosine hydroxylase (TH) antibody overnight. The sections were then washed three times with PBS, and were then incubated with the secondary antibody for one hour, washed three times again, and then mounted onto gelatin- coated glass slides. Sections were then imaged using a BZ-9000™ fluorescence microscope (Keyence, Osaka, Japan), and TH-positive cells were counted.

6. Cannulation Surgery

Swiss Webster mice were anesthetized with isoflurane (5% for induction; 1-3% to sustain). Once anesthetized, each mouse was placed on the stereotaxic device and implanted with a stainless-steel cannula (20G. 2.5 mm length) into the brain ventricle (0.2 mm posterior and 1 mm lateral to bregma and 2.3 mm below the surface of the skull). Mice were allowed to recover for 7 days.

7. Assessment of Locomotor Activity⁷

Mice were placed in a locomotor test chamber (40 x 40 x 38 cm³) and the horizontal locomotor activity was monitored with a 16 x 16 photobeam array (San Diego Instruments, San Diego, CA) located 1.25 cm above the floor of the enclosure. Mice were transported to the activity⁷ chamber room at least 30 minutes prior to placement into the activity⁷ chambers. All mice were given time to acclimatize to the chamber prior to injections, and locomotor activity was recorded for 20-24 hours following drug administration. Mice were randomly distributed to each treatment group with all proper controls being run in parallel. OA peripheral doses were selected based on previous studies that established the pharmacokinetics of OA in the plasma and peripheral tissues.^15,16 For central administration, OA was initially delivered to the brain through intracerebroventricular (i.c.v.) injection at a high dose of 20pM, chosen empirically based on doses ty pically used for glutathione administration.¹⁷'¹⁹ Subsequent dose-response curve analysis was performed with OA doses below 20pM, specifically targeting those near the EC50 and the lowest effective dose. The doses of NPS2143 were selected based on previous studies, substantiating that these specific doses effectively block the CaSR in mice.²⁰'²²

8, cAMP assay

Human embryonic kidney 203 (HEK293T) cells were grown in DMEM adjusted to contain 10% fetal bovine serum (FBS), 1% penicillin and streptomycin at 5-10% CO2 and 37°C. HEK293T cells were transiently transfected to express both the PGLOSENSOR-22F™ cAMP plasmid and a CaSR plasmid. Cells were transfected using the JETPRIME™ transfection reagent using the standard conditions of 5 pg of each plasmid and 20 pl of the reagent. After an overnight incubation in 37°C with 5- 10% CO2, the cells were then ready to be used. On the day of the experiment, cells were washed with PBS and detached using 0.05% trypsin. The cells are then pelleted and resuspended in CO2-independent media, 10% FBS, and the GLOSENSOR™ cAMP reagent. The cells are incubated at room temperature for 2 hours and are gently mixed every 15 minutes to prevent the cells from settling. The cells are then transferred to a 96-well plate and luminescence was measured using the MICROBET A2 2450 MICROPLATE COUNTER™ (PerkinElmer). Basal luminescence level is recorded prior to assay.

9, Radioligand binding assay

Sections were pre-incubated in buffer (50mM Tris, 1.5mM EDTA), pH 7.4 for 30 minutes at 4°C. Total binding was determined by incubating sections in buffer containing varying concentrations of [³H]-OA for 60 minutes at room temperature. Non-specific binding of [³H]-OA was defined by that seen in the presence of lOpM NPS-2143. The incubation was terminated by washing the sections in an ice-cold (4°C) incubation buffer for 10 minutes. The sections were then dipped in ice-cold distilled water and dried in a stream of cold air. Sections were scraped off of the slides and placed into vials containing 4.5 ml of scintillation fluid and were subsequently measured at a scintillation counter. 10, Molecular Docking

To predict the experimental binding modes and affinities of L-DOPA and ophthalmate to the CaSR, the following steps were conducted. First, the crystal structure of the calcium sensing receptor was retrieved from a protein database (PBD ID #7M3F) ²³, and UCSF Chimera ²⁴²⁵ was used to prepare the structure by removing water molecules and other unwanted entities. The chemical structures of L-DOPA (PubChem SID 3648) and ophthalmate (PubChem SID 254741470) were obtained, protonated, and converted into PDBQT format using Open Babel. Autodock Vina ^26/27 was used for molecular docking, and simulations conducted under experimental conditions of pH = 4.5 (for X-ray) and in an aqueous buffer. The docking was performed with L-DOPA and Ophthalmate targeting chain A of 7M3F in the active state. The docking configurations are based on the Trp ligand binding site reported in the previous report ²³, with a grid box size of 40 x 40 x 40, coordinates of 190.442, 212.175, 134.085 for L-DOPA and 210.238, 186.017, 134.231 for ophthalmate and spacing of 0.375 A. Genetic algorithm was run 100 times for each ligand. After completing molecular docking, the predicted binding poses, interaction, and the ligand binding affinities were determined.

11. Statistical Analysis

Statistical analyses of data were carried out using GRAPHPAD PRISM™ (GraphPad Software, Inc.). Data were presented as means ± standard error means (S.E.M.). Results were analyzed by unpaired student t-test, one-way, and two-way ANOVA followed by the appropriate post hoc comparisons, and P < 0.05 was considered statistically significant.

Results

Brain AADC inhibition alters L-DOPA-induced motor activity in reserpine-treated PD mice

Building upon our prior observations in rats, we investigated the effects of central AADC inhibition, mediated by NSD1015, on L-DOPA-induced motor behaviors in reserpine-treated mouse models of PD. As expected, reserpine treatment led to hypoactivity in mice. We first administered L-DOPA (lOOmg/kg) with and without NSD1015 (lOOmg/kg), which prevents its conversion to dopamine inhibiting AADC ²⁸²⁹ (Fig. 1A). All treatments were administered in combination with the peripheral AADC inhibitor benserazide (25mg/kg). No alteration of motor activity was found in control animals (Fig. 1B,C). In the control group, motor activity remained largely unchanged (Fig. 1B,C). However, in the reserpine-treated mice, L-DOPA treatment noticeably increased activity, persisting up to four hours (Fig. 1C). Remarkably, reserpinized-mice pre-administered NSD 1015 showed a more pronounced motor response to L-DOPA than their counterparts treated with L-DOPA alone (Fig. 1D,E). This amplified response had an onset delay of approximately 120 minutes and was sustained for a longer duration than that induced by L-DOPA alone (Fig. ID). While the last-duration motor effects of L-DOPA remained dose-dependent, the modulating effects of varying NSD1015 doses were less distinct (Fig. 1F-K).

To determine whether the enhanced activity with the L-DOPA and NSD1015 combination was exclusive to L-DOPA or applicable to other dopamine agonists, we administered a dopamine-releasing agent (amphetamine) and a dopamine receptor D1/D2 agonist (apomorphine), along with NSD1015, to reserpine-treated mice. We found that both agents displayed motor activity patterns consistent with their expected individual effects, indicating NSD1015-associated hyperactivity is linked to L-DOPA. Additionally, to explore the potential role of dopamine receptors in this effect, mice were pre-treated with a D2 receptor antagonist (haloperidol). Notably, haloperidol failed to block the hyperactivity induced by the L-DOPA/NSD1015 combination, implying the possibility of alternative mechanisms or pathways being triggered by the L-DOPA and NSD 1015 combination.

NSD1015/L-DQPA-induced hyperactivity in PD mice is associated with a surge in ophthalmate levels

To elucidate the neurochemical consequences of combined NSD1015 and L-DOPA administration in PD mice, we employed both targeted and untargeted metabolomic analyses on whole brain and striatal tissues (Fig. 2A-G). Our observations revealed that L-DOPA administration, when combined with AADC disruption, caused remarkable changes in the levels of L-DOPA and its associated metabolites within the brain and striatum (Fig. 2C-I). Interestingly, dopamine levels in the L-DOPA-treated group correlated closely with motor activity patterns, notably dropping at the 7-hour mark compared to the levels observed at 2 hours (Fig. 21). In contrast, dopamine concentrations in the NSD1015/L-DOPA group remained consistent between these time points. This lends credence to the idea that dopamine may not be the primary agent inducing hyperactivity in these conditions, hinting at the possible involvement of alternative, non-dopamine pathways. Our data also showed that AADC inhibition viaNSD1015 shifts the metabolic processing of aromatic amino acids, prioritizing methylation, deamination, and hydroxylation over decarboxylation. This metabolic profile mirrors that observed in cases of AADC deficiency ^30,31. Though we noticed a 13-fold rise in 3-O-methyldopamine (3-MT) levels at the hyperactivity peak (7 hours) in the NSD1015/L-DOPA-treated mice, its reduced concentrations at the 7-hour mark (compared to the 2-hour mark) suggest that 3-MT is not the sole contributor to the heightened activity observed at 7 hours (Fig. 2C-G). An increase in the stress hormone corticosterone was observed at 7 hours in the NSD1015/L-DOPA-treated mice, accompanied by alterations in energy pathways, notably in glycolysis and the mitochondrial tricarboxylic acid (TCA) cycle metabolism pathways.

The most striking finding was the pronounced surge in ophthalmic acid (OA; L-y- Glutamyl-L-2-Aminobutyryl-Glycine. y-Glu-2AB-Gly) following NSD1015 pretreatment. At the hyperactivity peak (7 hours), ophthalmate levels in the whole brain of L-DOPA/NSD1015-treated animals rose 20-fold compared to the 2-hour measurement and exhibited an 8-fold increase relative to L-DOPA-treated mice (see Fig. 2D-G,J).

Ophthalmate restores motor activity in MPTP PD mouse model

We investigated whether the observed hyperactivity' following L-DOPA/NSD1015 treatment in PD mouse models was directly caused by elevated OA levels. In our first set of experiments on the MPTP model of PD (Fig. 3A-C), peripheral administration (i.p.) of OA did not elicit any motor response in MPTP -treated mice (Fig. 3D). Given the limited data on OA’s pharmacokinetics, we were uncertain if the absence of a motor response was due to OA being intrinsically inactive or its inability to cross the blood-brain barrier (BBB). We, therefore, administered OA directly to the brain through i.c.v. in MPTP-treated mice. Remarkably, OA significantly enhanced motor activity in these mice in a dose-dependent manner (Fig. 3E-H). Notably, this enhanced motor activity⁷ persisted for around 24 hours, peaking during the initial 10 hours (Fig. 3G.H). To further explore ophthalmate's capability⁷ to cross the BBB, we injected deuterated ophthalmate (D5-OA) into mice and utilized mass spectrometry⁷ (MS) to monitor its distribution across various tissues, including blood, liver, kidney, and brain. Our findings show ed that, following peripheral administration, D5-OA w as detectable in the plasma and other organs but was absent from the brain (Fig. 31). This highlights that ophthalmate does not cross the BBB. suggesting that the surge in ophthalmate levels follow ing NSD1015/L-DOPA treatment w as likely a result of its synthesis in the brain.

Ophthalmate binds to and activates the CaSR

To elucidate the mechanism underlying ophthalmate's influence on motor activity, we probed ophthalmate's interactions with potential receptor sites in brain sections, using radioligand binding saturation experiment with increasing concentrations of labeled ophthalmate ([³H]-OA). A saturating concentration of unlabeled ophthalmate was used to assess non-specific binding. Our data revealed reversible binding of [3H]-0A to specific and saturable sites in the brain, with a single moderate-affinity population (Kd 2.61± 0.36 pM) (Fig. 4A). Given the structural similarities between ophthalmate and GSH, and past evidence suggesting modulation of CaSR by GSH and its gamma- glutamyl-tri-peptides analogs ^32,33, we sought to explore whether ophthalmate might interact with brain CaSR. CaSR is a class C G-protein-coupled receptor (GPCR) that is activated by extracellular calcium (Ca²⁺) ³⁴'³⁷, and can activate multiple signaling pathways through Gq/11, Gi/o, G12/13, and Gs proteins ³⁴’^38-40. Aromatic L-amino acids such as L-phenylalanine, L-tyrosine, and L-tryptophan increase the sensitivity of CaSR to Ca²⁺ and, thus, are considered positive allosteric modulators of the receptor ⁴¹'⁴⁶. Ca²⁺ ions and aromatic L-amino acids have also been proposed to act as coagonists of the receptor ⁴²⁴⁵-⁴⁷. We, therefore, sought to determine whether the saturable sites to which ophthalmate binds in brain sections are CaSR. We conducted a similar radioligand binding saturation experiment in the presence of a saturating concentration of an unlabeled CaSR antagonist (NPS-2143). At low concentrations of [³H]-0A (0.1-0.5 pM), 80-85% of the total binding of the labeled peptide to brain sites was inhibited by NPS-2143 (Fig. 4B). The specific component of binding was saturable, while the non-specific binding was linearly dependent on ophthalmate concentration. The calculated Kd of ophthalmate binding to CaSR was 1.62± 0.22 pM. [³H]-0A binding was also inhibited by a saturating concentration of Ca²⁺ and L- DOPA. rendering a Kd for [³H]-0A binding of 4.06±1.47 pM and 5.55± 0.85 pM, respectively (Fig. 4B). While this finding does not conclusively prove that L-DOPA binds to CaSR, it indicates that ophthalmate and L-DOPA can bind to the same target in the brain. [³H]-0A binding was also inhibited by combinations of saturating concentrations of L-DOPA/NPS-2143, Ca²⁺/L-DOPA, Ca²⁺/NPS-2143, rendering a Kd for [3H]-0A binding of 3.99±0.05. 5.28±0.57, and 5.99±0. 1 respectively (Fig. 4C). Lastly, using a radioligand competitive (displacement) experiment, with a fixed concentration of [³H]-OA (2.5 pM), we found that Ca²⁺ inhibited the binding of [³H]- OA in a dose-dependent manner, with Ki of 0.98±0.13 pM (Fig. 4D). thereby verifying the binding of ophthalmate to calcium binding sites in the brain. Neither SKF-82958, a DI dopamine receptor ligand, nor haloperidol, a D2 dopamine receptor ligand, displaced [³H]-OA binding.

To determine whether ophthalmate functions as an agonist or antagonist at CaSR, we employed a cAMP-luminescence assay using HEK cells that express CaSR and forskolin to amplify cAMP production. Our results demonstrated ophthalmate's dosedependent activation of the Gi-coupled CaSR, reducing forskolin-stimulated cAMP. The EC50 for ophthalmate was determined 1.55±0.1 pM, while for Ca² . an orthosteric agonist of CaSR, the ECso was 0.7±0.01 pM (Fig. 4E,E'). Under the same conditions, L-DOPA also activated the CaSR at an ECso of 2.44±0.45 pM (Fig. 4E"). L-DOPA agonistic action on CaSR is noteworthy given that other aromatic amino acids, such as L-tryptophan and phenylalanine, are known to act as allosteric agonists at CaSR ^41-46. In the presence of NPS-2143, a negative allosteric modulator (NAM), the CaSR displayed a decrease in maximal signaling capacity (Emax) (Fig. 4F-F''), and rightward shift of the dose-response curves (ECso) of ophthalmate, L-DOPA, and Ca²⁺ (Fig. 4G-G''). Our results provide evidence that both ophthalmate and L-DOPA bind to and activate CaSR at EC50 values comparable to that of the orthosteric agonist (Ca²¹ ).

While these experiments did not identify' the specific binding sites of ophthalmate and L-DOPA to CaSR, we utilized computational modeling and structure-based docking to propose a model for their interaction. Our model predicted that ophthalmate binds to the CaSR in a manner similar to L-tryptophan (TRP). specifically targeting the L- aromatic acid binding pocket (Fig. 4H-K).

CaSR mediates the motor-enhancing effects of L-DQPA/NSD1015 and ophthalmate in PD mice

We lastly examined the involvement of ophthalmate binding to the CaSR in motor activity. Our findings showed that when we used the CaSR antagonist NPS-2143, the motor-enhancing effects of L-DOPA/NSD1015 in MPTP -treated mice were suppressed (Fig. 5A,B). This suggests that CaSR activation contributes to the increased motor activity observ ed. Moreover, using NPS-2143 as a pretreatment revealed a dose-dependent decrease in ophthalmate-induced motor activity in both the MPTP (Fig. 5C-F) and reserpine (Fig. G,H) PD models. This substantiates that ophthalmate's effect on motor function is largely due to its interaction with the CaSR. Discussion

The central role of dopamine in governing motor functions has been deeply accepted in the scientific understanding for years. This view posits dopamine as the primary neurotransmitter mediating such functions. Our study introduces a paradigm shift in this understanding by highlighting the significant role of the tripeptide ophthalmic acid in mediating motor activity, particularly in conditions where the dopamine system is disrupted.

Dopamine-independent role of L-DOPA in movement

Earlier research proposed that L-DOPA may mediate its effects in part by acting directly, or via its metabolites, on alternative non-dopaminergic neurotransmitter systems involved in motor behaviour ⁴⁸'⁵⁰. For example, the presence has long been demonstrated of neurons containing L-DOPA that release it in a calcium-dependent fashion upon stimulation.⁵¹’⁵² Additionally, L-DOPA enhances the release of various neurotransmitters, including dopamine, noradrenaline, glutamate, and GABA,⁵³'⁵⁵ suggesting it may act as a neurotransmitter or neuromodulator in the CNS.^56,57 Additionally, L-DOPA undergoes nonenzymatic conversion into biologically active compounds like 2,4,5 -trihydroxy phenylalanine (TOP A), which can elicit neuronal responses in dopaminergic pathways independent of dopamine receptor stimulation, including neuronal firing and membrane depolarization.⁵⁸'⁶⁰

Our earlier research in a rat model of Parkinson’s disease demonstrated that L-DOPA, when its conversion to dopamine in the brain is inhibited, leads to delayed but significantly enhanced and prolonged motor activity [6], Our current study's observations in a mouse reserpine model of Parkinson’s Disease mirror those from earlier rat studies and are substantiated using another mouse Parkinson’s Disease model (MPTP), suggesting the generalizability of our results. While L-DOPA does not seem to be the molecule that directly induces the long-lasting enhanced activity - evidenced by its negligible levels during hyperactive periods - its presence is indispensable for activating the pathways responsible for this motor activity. This is demonstrated by our findings that both the intensity and duration of the motor activity strongly correlate with L-DOPA doses.

Interestingly, the long-duration response of L-DOPA has been recognized in early studies.⁶¹'⁶³ Researchers distinguished between the short-duration response, which lasts a few hours and correlates with plasma L-DOPA concentrations and the long- duration response, which persists after the elimination of L-DOPA, gradually declining back to baseline over a period of days to weeks.⁶⁴’⁶⁵ The long-duration response to L-DOPA has been largely overlooked for the past five decades, with only a handful of studies confirming early observations.⁶⁴'⁷⁰ However, the mechanisms underlying the long-duration response remained poorly understood.

Given the dominant hypothesis on dopamine replacement therapy in Parkinson's disease has focused on centrally formed dopamine, research into the effects of central AADC inhibitors has been limited. A study from the seventies showed that an AADC inhibitor with both peripheral and central effects, could potentiate the therapeutic effect of L-DOPA in Parkinson's disease patients.⁷¹ The study recommended extending the search for effective decarboxylase inhibitors to compounds that can cross the BBB, yet subsequent investigations have only focused on AADC inhibitors lacking this ability.⁷²

The neurobiological contexts of ophthalmate surge

Originally identified in the calf lens,⁷³ ophthalmate's presence has since been detected in various tissues in advanced animals and even microorganisms.⁷⁴'⁷⁷ Structurally, ophthalmate is analogous to glutathione (GSH; L-y-Glutamyl-L-Cysteinyl-Glycine, y- Glu-Cys-Gly), which functions as an essential antioxidant and detoxifying agent in biological systems.⁷³ The synthesis pathways of both GSH and ophthalmate share common enzymes: glutamate-cysteine synthase (GCS) and glutathione synthase (GS), which sequentially produce ophthalmate (Fig. 2J).^78,79 The notable increase in ophthalmate levels in the brain and striatum of L-DOPA/NSD1015-treated mice suggests that these two drugs have an effect on glutathione metabolism and ophthalmate’s synthesis. GSH and ophthalmate synthesis is closely linked to methionine metabolism via the transsulfuration pathway, which is facilitated by cystathionine -synthase (CBS) and cystathionine y-lyase (CSE).⁸⁰ CSE catalyzes the cleavage of cystathionine to generate equal amounts of cysteine (Cys) and 2- ketobutyric acid (2KB), which is subsequently converted to 2-aminobutryate (2 -AB) through a transamination reaction with glutamate and other keto-carboxyl compounds as the donor substrate and AST aminotransferase as the catalytic enzy me.⁸¹'⁸³ Our study revealed a pronounced elevation in ophthalmate levels under conditions where the conversion of L-DOPA to dopamine was inhibited. This unexpected surge in ophthalmate is indicative of alternative metabolic or signaling pathways being activated when traditional dopaminergic pathways are disrupted. This discovery' raises pertinent questions: What triggers the synthesis and release of ophthalmate under such conditions? Is there an intrinsic neural mechanism that compensates for reduced dopamine by increasing ophthalmate? Based on our findings, the elevations of ophthalmate levels observed in the NSD/L-DOPA group may be related to the alteration of methylation pathways, with a notable increase in methylated metabolites and hydroxyl-carboxyl metabolites observed within this group. The scarcity of research on ophthalmate implies that its physiological importance is yet to be established. Historically, two primary scenarios have been elucidated for ophthalmate synthesis: firstly, in the context of E. coli lacking PLP-dependent proteins,⁷⁷’⁸⁴ and secondly, under conditions necessitating GSH synthesis, positioning ophthalmate as a potential indicator for oxidative stress and the subsequent depletion of cysteine or GSH.^{14 15} It is noteworthy that conditions leading to GSH depletion, such as the hepatotoxic effects of acetaminophen overdose, or systemic responses to starvation and acute stress, have been associated with elevated ophthalmate levels.¹⁴’^85-88 This is due to the fact that, in environments favoring reductive reactions, GSH inhibits GCS, suppressing ophthalmate synthesis. In contrast, during oxidative stress, the depletion of GSH activates GCS, leading to the production of ophthalmate.¹⁴⁸⁷-⁸⁸

In light of these two contexts, it can be inferred that the rise in ophthalmate levels observed in our study may be linked to the effects of the combination of L-DOPA and NSD1015 on AADC, oxidative stress, and GSH metabolism. First, NSD1015 inhibits AADC, a PLP-dependent enzy me,²⁸²⁹ mimicking the production of ophthalmate observed in E. coli lacking PLP-dependent proteins. Second, L-DOPA, due to its highly reactive nature, can induce oxidative stress. When its conversion to dopamine is inhibited, the excess L-DOPA interacts and conjugates with GSH, resulting in GSH depletion ⁸⁹’⁹⁰. The observations of altered PLP and GSH levels in the NSD1015/L- DOPA group support these notions (Fig. 21, J). These findings suggest that the rise in ophthalmate levels observed in our study may be a result of a combination of factors. These include shifts in PLP and GSH metabolism influenced by NSD1015's impact on AADC and L-DOPA's propensity for oxidative stress, along with enhanced methylation pathways that facilitate the synthesis of both ophthalmate and GSH. Further supporting this conclusion is the notable rise in the stress hormone corticosterone, along with shifts in energy pathways, specifically, the glycolysis and mitochondrial TCA cycle metabolism pathways. Ophthalmate emergence in the regulation of motor function

Historically, dopaminergic pathways have been central to our comprehension of movement disorders, especially with dopamine's connection to conditions like PD. The effectiveness of dopaminergic drugs in treating motor symptoms of PD further reinforced this understanding. Yet, our study highlights a notable observation: pronounced motor activity persists even in dopamine-deprived states. This suggests alternative mechanisms and pathways influencing motor activity. Our findings point toward ophthalmate's significant role in influencing motor activity, specifically through its interaction with the CaSR signaling system. While CaSR has historically been recognized for its integral role in systemic calcium regulation,⁹¹'⁹⁴ its implications within the central nervous system and its potential influence on motor activities have largely remained underexplored. The direct connection between ophthalmate and CaSR, revealed by our study, presents a new perspective in understanding neural signaling beyond the conventional neurotransmitters. By elucidating the connection between ophthalmate and CaSR signaling, we have established a foundational basis for the development of potential new therapeutic strategies targeting this pathway. Leveraging this pathway holds the promise of refining therapeutic strategies for movement disorders, offering potential benefits with fewer complications compared to traditional dopaminergic approaches. If ophthalmate can indeed offer similar or superior therapeutic benefits with a reduced side-effect profile, it could revolutionize treatment approaches for movement disorders.

While our study has highlighted the significant role of ophthalmate in modulating motor functions, many questions remain. For example, are there other neurological conditions, beyond PD, where ophthalmate's role might be crucial? Additionally, are there specific neurons that synthesize and release ophthalmate in the brain? Lastly, are there other receptors or pathways influenced by ophthalmate that haven not been identified yet? Examining into these questions will extend our understanding of ophthalmate's role in the brain functions and open up avenues for therapeutic interventions. Future studies should also focus on confirming these results in primate models of Parkinson's disease to validate and broaden the applicability of our findings.

In summary, our study establishes the critical role of ophthalmate in regulating motor functions via its interaction with the CaSR. This discovery challenges the traditional understanding that primarily associates dopamine with motor functions. By identifying ophthalmate's connection with the CaSR pathway, we open up new therapeutic opportunities for movement disorders.

Figure legends - Example 2

FIG. 6 (or. Figure 1, Example 2): Motor response to L-DOPA is enhanced by inhibition of DOPA decarboxylase in mouse model of Parkinson’s disease (PD). (A) The biosynthesis of dopamine from L-DOPA and its proposed inhibition by benserazide and NSD1015 in the periphery and the central nervous system, respectively. (B,C) Effects of L-DOPA and NSD1015 (NSD) on normal mice: The mice were injected subcutaneously (s.c.) with a vehicle, and 18 hours later, injected intraperitoneally (i.p.) with either NSD 1015 or saline, followed by an L- DOPA/Benserazide (100/25mg/kg) or saline injection 30 minutes after. Motor activity was monitored for 20 hours. Data represent (B) the time-course and (C) the Area Under the Curve (AUC) of the effects of L-DOPA, NSD1015, and combination of L-DOPA and NSD1015. One-way ANOVA, followed by Tukey post-test: ns, not significant. Values are expressed as mean±S.E. n=8 for each group. (D,E) Effects of L-15 DOPA and NSD1015 on reserpinized mice: Mice were injected (s.c.) with reserpine 1 mg/kg, and 18 hours later, mice were injected with NSD1015 or saline, followed by an L-DOPA injection 30 minutes after. Locomotion was monitored for 20 hours. Data represent the (D) time-course of the effect of L-DOPA alone and L-DOPA in conjunction with NSD1015. and (E) the area under the curve. One-way ANOVA, followed by Tukey post-test: ****P < 0.0001 , ns, not significant. Values are expressed as mean±S.E. n=6-8 for each group. (F-K) Effect of varying doses of L-DOPA and NSD1015 on motor activity in reserpine- treated mice: Mice were injected (s.c.) with reserpine 1 mg/kg and 18 hours later, mice were injected (i.p.) with varying doses of NSD 1015 followed by varying doses of L-DOPA 30 minutes after. Locomotion was monitored for 20 hours. Same data are presented in two clusters of figures. (F-H) Data represents the time-course of the effect of varying doses of NSD1015 with fixed doses of L-DOPA (F: 50mg/kg. G: lOOmg/kg, H: 200mg/kg). (I-K) Data represents the time-course of the effect of varying doses of L-DOPA with fixed doses of NSD1015 (I: 50mg/kg, J: lOOmg/kg, K: 200mg/kg). Values are expressed as mean±S.E. n=6-8 for each group.

FIG. 7 (or. Figure 2, Example 2): Motor Response to L-DOPA and AADC inhibitor correlates with alterations in brain and striatum metabolites, with highest alteration in ophthalmate levels: (A) The study's experimental design and timeline. Mice were injected (s.c.) with reserpine 1 mg/kg. After 18 hours, the mice were treated with NSD1015 or saline, followed by L-DOPA/Benserazide administration 30 minutes later. Brain tissues were collected from the two treatment groups at two time points (2 hours and 7 hours after L-DOPA administration). The brains were divided into two hemispheres, with one hemisphere homogenized entirely, and the other hemisphere's striatum was used to extract the striatum, n=6 for each group. (B) Unsupervised principal component analysis (PCA) of the metabolomics data of mouse whole brain and striatum from two the treatment groups at the two time points. (C) Volcano plot illustrating the differential expression of metabolites in the brains of L-DOPA+NSD1015 treated mice compared to L- DOPA treated mice, with brain samples taken 2 hours post-DOPA inj ection. (D) Volcano plot showing the differential expression of metabolites in the brains of L-DOPA + NSD1015 treated mice versus L-DOPA treated mice, with brain samples obtained 7 hours after L-DOPA injection. One-Way ANO V A Contrasts: significantly increased metabolites in red, and significantly decreased metabolites in blue. (E) Volcano plot displaying the differential expression of metabolites in the brains of L-DOPA + NSD1015 treated mice at 7 hours compared to L-DOPA + NSD1015 treated mice at 2 hours post-DOPA injection. One-Way ANOVA Contrasts: Significantly increased metabolites in red, and significantly decreased metabolites in blue. (F) Volcano plot demonstrating the differential expression of metabolites in the striatum tissues of L-DOPA+NSD1015 treated mice in comparison to L-DOPA treated mice, with brain samples collected 7 hours following L-DOPA injection. One-Way ANOVA Contrasts: Significantly increased metabolites in red, and significantly decreased metabolites in blue. (G) List of top altered metabolites in the brains of different treatment groups (> 2 -fold change, q< 0.05, One-way ANOVA Contrasts), X axis represents fold changes.

FIG. 8 (or. Figure 3 Example 2): Motor response to L-DOPA and AADC inhibitor is associated with alterations in dopamine and ophthalmate synthesis pathways.

(A) Box plot legend showing the range, median, and quartiles. (B) The fold changes in the major components of dopamine synthesis pathway after L-DOPA/ NSD1015 administration. (C) The fold changes in ophthalmate and the key metabolites of the proposed pathways leading to its synthesis. The asterisk (*) is used to compare metabolite levels within the same group at the two different time points, whereas the hashtag (#) is used to compare metabolite levels between the two treatment groups at the same time point. * and # q< 0.05, ** and ## q < 0.01, *** and ### q < 0.001, **** and #### q < 0.0001. SAM: S-adenosylmethionine, SAH: S- adenosylhomocysteine, Hey: homocysteine, Glu: glutamate, a-KG: a-ketoglutarate, Gly: glycine, a-KB: a-ketobutyrate, GCS: glutamate-cysteine synthase, GS: glutathione synthase.

FIG. 9 (or. Figure 4 Example 2: Central but not peripheral ophthalmate (OA) rescues motor activity⁷ in MPTP 10 mouse model of PD.

Mice were given MPTP injections (i.p.) at 20 mg/kg over three days. Ophthalmate was administered 24 hours after the final MPTP injection, and motor activity was monitored for the following 20 hours. For the central ophthalmate experiments, mice underwent a surgical procedure to implant a cannula for intracerebroventricular (i.e.v.) injections a week prior to receiving the MPTP injection.

(A) Representative immunostaining of tyrosine hydroxylase (TH. green) and DAPI (blue), showing the effect of MPTP on dopamine neuronal loss in the substantia nigra;

(B) the time-course of the effect of MPTP,

(C) the AUC of the effect of MPTP during the 20-hour experiment time; unpaired t- test, ****p < 0.0001; (D) Peripheral ophthalmate injection did not induce motor activity in MPTP-treated mice; Data represent the time-course of the effect of ophthalmate injected i.p. at three different doses and are expressed as mean±S.E. n=8 for each group. (E-H) The effect of central administration of ophthalmate on the motor activity of MPTP-treated mice. The data presented include (E) the timecourse of the effect of ophthalmate injected i.e.v. at four different doses, (F) the AUC of the effect of different doses of ophthalmate during the 20-hour experiment time, (G) the AUC of the effect of different doses of ophthalmate during the first 10 hours of the experiment time, and (H) the AUC of the effect of different doses of ophthalmate during the 11-20 hours of the experiment time. In (F), (G). and (H).

One-way ANOVA, followed by Tukey post-test: *P < 0.05, ****P < 0.0001, ns: not significant. Data are expressed as mean±S.E. n=6-8 for each group.

(I) Deuterated ophthalmate (d5-OA) levels following peripheral administration measured using liquid chromatography-mass spectrometry (LC-MS). Mice were injected (i.p.) with deuterated ophthalmate (d5-OA) and the brain, blood, kidney, and liver samples were collected 10 minutes after injection. Data represent the nterpolated concentrations of d5-OA, using mass spectrometer (MS), and are expressed as mean±S.E. n=6 for each group.

FIG. 10 (or. Figure 5, Example 2): Ophthalmate binds to- and activates calcium sensing receptor (CaSR).

(A-B) Saturation curve of [³H]-0A binding to mouse brain sections in the presence of unlabeled ophthalmate. NPS2143, Ca²⁺, and L-DOPA. [³H]-0A binding was carried out as described in the methods section. (A) Reprehensive plot of total, specific, and nonspecific binding of [³H]-OA to mouse brain sections. Non-specific binding was determined as the levels of [³H]-0A binding in the presence of 100 pM unlabeled ophthalmate. (B) Representative plot of specific binding of [³H]-0A binding to mouse brain sections, where non-specific bindings were defined as the levels of [³H]- OA binding in the presence of 10 pM NPS-2143, 100 pM calcium, and l OO pM L-DOPA. (C) Representative plot of specific binding of [³H]-OA binding to mouse brain sections, where non-specific binding was defined as the level of [³H]-0A binding in the presence of combinations of NPS-2143+ Ca²⁺, L-DOPA+ Ca²⁺, and NPS-2143 + L-DOPA. In all binding experiments, the degree of binding is expressed in disintegrations per minute (dpm). Data represent binding from three experiments for each point (total 36 sections repeat for each point). (D) Inhibition of [³H]-0A binding to mouse brain sections by Ca²⁺. Competition experiments were carried out as described in the methods. Radioligand binding assay was performed in the presence of 2.5 pM [³H]-0A and increasing concentrations of Ca²⁺. Each point represents the mean ± S.E of at least 3 measurements from three experiments. (E-G) Forskolin-stimulated cAMP GLOSENSOR™ luminescence responses in HEK 293 cells transiently transfected with the GLOSENSOR™ cAMP biosensor and the CaSR plasmid. (E-E") Representative dose-response curves of cAMP signal for (E) Ca²⁺, (E') O A, and (E") L-DOPA, with and without CaSR antagonist NPS-2143. (F-F") Emax of (F) Ca²⁺, (F') OA, and (F") L-DOPA at CaSR in the absence and presence of CaSR antagonist NPS-2143. (G-G") EC50 of (G) Ca²¹ , (G') OA, and (G")L-DOPA at CaSR in the presence and absence of CaSR antagonist NPS-2143; unpaired t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001, ns: not significant.

(H-K) Docking models Ligand-bound states in CaSR binding site in domain A (named A in amino acid hereinafter). (H) Cartoon presentation of ligand-bound CaSR structure (domain A) in the closed-closed conformation (5 Angstroms).

(I) Interface analysis of Tryptophan (Trp)-bound state in CaSR; binding is shown with Serl70A, Serl47A, Ala298A, and Thr l45A and Alal68A.

(J) Interface analysis of Ophthalmate (OA) bound state in CaSR; binding is shown with Serl47A, Glyl48A, Tyr218A, Serl70A. Asp216A, and Alal68A, Vall49A.

(K) Interface analysis of L-DOPA bound state in CaSR; binding is shown with Tyr218A, Serl70A, Asp216A, and Alal68A. Green curve shows the transition state of Ala298A and Thr 145A in the Trp-bound state, Glyl46A in the OA-bound state, and Tyr218A in DOPA-bound state.

FIG. 11A-H (or. Figure 6, Example 2): CaSR mediates the motor-enhancing effects of L-DOPA/NSD1Q15 and ophthalmate in PD mice.

(A, B) CaSR antagonist NPS-2143 inhibition of motor response induced by L- DOPA/NSD1015 in MPTP-treated mice. Mice were injected (i.p.) with MPTP 20 mg/kg for three days. The following day, mice were injected (i.p.) with NSD1015 or saline, followed by a saline or L-DOPA injection 30 minutes after with or without NPS2143. Locomotion was monitored for 20 hours. Data represents (A) the time-course and (B) the AUC of the effect of NSD1015 and L-DOPA with or without NPS2143. One-way ANOV A. followed by Tukey post-test: ****P < 0.0001, ns: not significant. Data are expressed as mean±S.E. n=8 for each group. (C-F) CaSR antagonist inhibits ophthalmate-induced motor response in MPTP-treated mice. Mice w ere anesthetized and underwent surgery where a cannula w as implanted for future intra-cerebro-ventricular (i.c.v.) injections. Following recovery, the mice were injected (i.p.) with MPTP 20 mg/kg for three days. On the day following the final MPTP treatment, mice were injected i.c.v. with either saline or ophthalmate at (C,D) 5 pM and (E,F) 10 pM with or without NPS2143 (10 pM and 20 pM). Locomotion was monitored for 20 hours. Data represent (C,E) the time-course and (D,F) the AUC of the effect of ophthalmate with and without NPS2143 on motor activity’. In (D) and (F) One-way ANOV A, followed by Tukey post-test: **P < 0.01. ****P < 0.0001, ns: not significant. Values are expressed as mean±S.E. n=6-8 for each group. (G,H) CaSR antagonist NPS2143 inhibits ophthalmate-induced motor response in reserpine-treated mice. Mice were injected (s.c.) with reserpine 1 mg/kg, and 18 hours later, mice were injected (i.c.v) with ophthalmate (10 pM) with or without NPS2143 (20 pM). The (G) time-course and (H) the AUC of the effect of ophthalmate with and without NPS2143 on motor activity. In (H) One-way ANOVA, followed by Tukey post-test: **P < 0.01, ****p < 0.0001, ns: not significant.

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48. Zhang, C., et al., Structural basis for regulation of human calcium-sensing receptor by magnesium ions and an unexpected tryptophan derivative co-agonist. Sci Adv, 2016. 2(5): p. el 600241.

49. Wen, T., et al., Structural basis for activation and allosteric modulation of full- length calcium-sensing receptor. Sci Adv, 2021. 7(23). 50. Young, S.H. and E. Rozengurt, Amino acids and Ca2+ stimulate different patterns of Ca2+ oscillations through the Ca2+-sensing receptor. Am J Physiol Cell Physiol, 2002. 282(6): p. C1414-22.

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A number of embodiments of the invention have been described.

Nevertheless, it can be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A compound having a formula comprising:

(a) an ophthalmic acid (ophthalmate, or “OA”), or

; or

(b) a deuterated form of a compound of (b), or an isomer, optical isomer or stereoisomer thereof, or a racemate or racemic mixture thereof, or an enantiomer, an individual diastereomer or a diastereomeric mixture thereof, or an analog thereof, or a crystalline product or crystalline intermediate thereof, or a pharmaceutically acceptable salt thereof, or prodrug or a bioisostere thereof of a compound of (a).

2. A pharmaceutical composition or a formulation comprising:

(a) a deuterated form of ophthalmic acid (ophthalmate, OA) or a deuterated form of a an isomer, optical isomer or stereoisomer, a racemate or racemic mixture thereof, an enantiomer, an individual diastereomer or a diastereomeric mixture thereof, or an analog, thereof, or a crystalline product or cry stalline intermediate thereof or a pharmaceutically acceptable salt thereof, or prodrug or a bioisostere thereof of ophthalmic acid (ophthalmate, OA);

(b) a compound having a formula comprising:

(i) ophthalmic acid (ophthalmate, or “OA’'), or

3. The pharmaceutical composition or the formulation of claim 2, further comprising a pharmaceutically acceptable excipient, or wherein the pharmaceutical composition is formulated in a sterile solution or liposome, wherein optionally the sterile solution comprises saline.

4. The pharmaceutical composition of claim 2 or 3, wherein pharmaceutical composition or the formulation is a solid, liquid, aerosol, powder, lyophilized, gel or emulsion formulation. wherein optionally the pharmaceutical composition is formulated for enteral or parenteral administration, and optionally the compound is formulated for administration in vivo,' or for enteral or parenteral administration, or as a tablet, pill, capsule, lozenge, gel, geltab, liquid, lotion, aerosol, patch, spray, or implant, and optionally the compound is formulated as a liposome, a nanoparticle or a nanolipoparticle.

5. A kit, an implant, a pump, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, an infusion pen, a needles, a reservoir, an ampoules, a vial, a syringe, a cartridge, a pen, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump, comprising a compound as set forth in claim 1, or a formulation or pharmaceutical composition as set forth in any of claims 2 to 4.

6. A method for treating, ameliorating, slowing the progression of. decreasing the severity of symptoms of a neurological disease, optionally a degenerative neurological disorder, optionally Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS). Friedreich ataxia, Lewy body disease, Spinal muscular atrophy or Motor neuron disease, in an individual in need thereof, comprising administering, or use of: an ophthalmic acid (ophthalmate, OA), or, a compound of claim 1, or a pharmaceutical composition of any of claims 2 to 4, or a kit, an implant, a pump, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, an infusion pen, a needles, a reservoir, an ampoules, a vial, a syringe, a cartridge, a pen, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump of claim 5, to an individual in need thereof.

7. Use of: ophthalmic acid (ophthalmate, OA), or a compound of claim 1, or a pharmaceutical composition of any of claims 2 to 4, or a kit, an implant, a pump, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, an infusion pen, a needles, a reservoir, an ampoules, a vial, a syringe, a cartridge, a pen, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump of claim 5. for treating, ameliorating, slowing the progression of, decreasing the severity of symptoms of a neurological disease, optionally a degenerative neurological disorder, optionally Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS), Friedreich ataxia, Lewy body disease, Spinal muscular atrophy or Motor neuron disease, in an individual in need thereof.

8. Use of ophthalmic acid (ophthalmate, OA), or a compound of claim 1 in the preparation of a medicament for treating, ameliorating, slowing the progression of, decreasing the severity of symptoms of a neurological disease, optionally a degenerative neurological disorder, optionally Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS), Friedreich ataxia, Lewy body disease, Spinal muscular atrophy or Motor neuron disease, in an individual in need thereof.

9. A compound of claim 1, or a pharmaceutical composition comprising ophthalmic acid, or a pharmaceutical composition of any of claims 2 to 4, or a kit, an implant, a pump, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, an infusion pen, a needles, a reservoir, an ampoules, a vial, a syringe, a cartridge, a pen, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multichambered pump of claim 5, for use in treating, ameliorating, slowing the progression of, decreasing the severity of symptoms of a neurological disease, optionally a degenerative neurological disorder, optionally Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS). Friedreich ataxia, Lewy body disease, Spinal muscular atrophy or Motor neuron disease, in an individual in need thereof.