WO2014036379A2 - Bile acids prevent or abolish progression of parkinson's and related neurodegenerative diseases - Google Patents

Description

BILE ACIDS PREVENT OR ABOLISH PROGRESSION OF PARKINSON'S AND RELATED NEURODEGENERATIVE DISEASES

BACKGROUND OF THE INVENTION

Parkinson's Disease:

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the selective loss of dopaminergic neurons in the substantia nigra of brain. Although there are multiple pathogenic mechanisms in PD, the most common postulated pathogenic mechanism in PD is a vicious cycle of oxidative stress. Postmortem studies showed that oxidative damage and decrease in anti-oxidative glutathione in PD brain tissues, and multiple signs of apoptosis, such as mitochondrial dysfunction, chromatin condensation, and caspase activation in dying cells. For these reasons, much interest has focused on the antioxidant and anti-apoptotic defenses that may be promising therapeutics for PD.

Unknown at this juncture are the underlying causes of PD, although it is believed to result from a combination of genetic predisposition and a possible external stimulus. The general symptoms of PD are triggered by a severe loss of dopamine production in the substantia nigra. Along these lines, it has been shown that in a small number of clinical patients with PD who are also recipients of transplanted human embryonic dopamine neurons, return to a normal life. A minimum of 80,000 dopamine-producing neurons is required for benefits from any clinical intervention, greatly accentuating the need for enhanced life of transplanted neurons.

SUMMARY OF THE FNVENTION

The present invention describes a method of preventing or delaying the onset of or abolishing Parkinson's and related diseases by preventing cell death of neurological tissue. The patient is a human patient, while the administering step involves administering, through various means, an amount of UDCA or TUDCA, in any formulation in any combination that is effective in providing the necessary pharmacological benefit.

One feature of the present invention involves the administering of an effective amount of UDCA or TUDCA or any of their analogs or formulations or any combination thereof. The mode of administering UDCA or TUDCA includes, but is not limited to, intravenously, parenterally, orally or intramuscularly or any combination of these methods thereof.

Another feature of the invention involves the administering of an effective amount of UDCA or TUDCA or any of their analogs or derivatives conjugated with pro-drugs of DA.

Herein, a "patient" includes a human or any mammal.

BRIEF DESCRIPTION OF THE FIGURES

Shows the effects of UDCA AND YS on the SNP -induced cell death in human dopaminergic neurons. Cell viability was estimated by MTT reduction assay. SH-SY5Y cells were treated with the identical concentrations of SNP (A), and UDCA or YS (B) for 24 h. (C) Cells were pretreated with different concentrations of UDCA or YS for 1 h, and exposed to 1 mM SNP for 24 h. (D) SH-SY5Y cells were treated for 24 h with SNP (1 mM), potassium ferricyanide (0.5, 1 mM), sodium cyanide (0,5, 1 mM), or 5-day light- degraded SNP (SNP_EXP, 1 mM), and analyzed for viability).

Shows that UDCA and YS reduce SNP-induced apoptosis in SH-SY5Y cells.

(A) Morphological analysis visualized nuclear chromatin in SH-SY5 Y cells stained with Hoechst 33342 (blue color) and propidium iodide (pink or red color). Apoptotic cells were showed as condensed chromatin and fragmented nuclei, and necrotic cells were observed as propidium idodide-stained cells, (a-f) Representative fluorescence photomicrographs show the nuclei morphology of SH-SY5Y cells, (a) Untreated control cells, (b) UDCA (200 μΜ; 24 h) treated cells, (c) YS (200 μΜ; 24 h) treated cells, (d) SNP (ImM; 24h) treated cells, (e) Treaded with UDCA (200 μΜ; 1 h) followed by SNP (ImM; 24h). (f) Treated with YS (200 μΜ; 1 h) followed by SNP (1 mM; 24 h). Scale bar indicates 20 μιη. (B) Apoptosis and necrosis rates were calculated as ratio of apoptotic nuclei or necrotic nuclei to total nuclei. (C) Activities of caspase-3/7 and caspase-9 were assayed using specific fluorogenic substrates.

Shows the effects of UDCA and YS on SNP-induced nitric oxide (NO) and reactive oxygen species (ROS). (A) The amount of NO production from cells was evaluated by the Triess reaction after SNP treatment for 24 h with our without UDCA or YS, and quantitatively assessed using NaN0₂ as a standard.

(B) SH-SY5Y cells were exposed to 1 mM SNP with or without different concentrations of UDCA or YS for 12 h. Total intracellular ROS and ONOO production was determined by fluorogenic assay. FIG. 4 Shows changes of intracellular GSH level and mitochondrial membrane potential in SH-SY5Y cells. After exposing the cells to 1 mM SNP for 12 h with or without various concentration of UDCA or YS pretreatment, GSH level was determined using monochlorobimane (MCB) (A), and mitochondrial membrane potential was evaluated using the fluorescent JC-1 dye. (B).

Shows the effects of UDCA on the protein levels of Bcl-2, Bax, and cytochrome c in SNP -treated SH-SY5Y cells. (A) Cells were pretreated with UDCA (200 μΜ) for 1 h and then exposed to SNP (1 mM) for 12 h. The change in expression levels of mitochondria-related programmed cell death markers, Bcl-2, Bax and cytochrome c was determined by Western blot analysis. Anti-actin and anti-cox4 antibodies were used for normalization. (B) The intensity of gands were quantified by densitometric analysis and assessed as the ratio against the value of untreated control.

FIG. 6 Shows the effects of protein kinase inhibitors on UDCA-mediated protection in SH-SY5Y cells. Cells were pretreated with either 1 μΜ triciribine (TR, Akt/PKB inhibitor), 2 μΜ LY294002 (LY, P13K inhibitor), 1 μΜ PKI (PKA inhibitor), or 2 μΜ Go6983 (GO, PKC inhibitor) 1 h before adding UDCA (200 μΜ) and SNP (1 mM). Cell viability was estimated by MTT assay 24 h after treatment. The protective effect of UDCA on SNP -induced cell death was reversed by AKP/PKB inhibitor (TR) and P13K inhibitor (LY).

FIG. 7 Shows inhibition of Bax translocation by UDC A via P 13K and Akt/PKB pathways. SH-SY5Y cells were pre-incubated for 1 h with either 2 μΜ LY294002. (LY, P13 kinase inhibitor) or 1 μΜ triciribine (TR, Akt/PKB inhibitor) and then exposed to UDC A (200 μΜ) and SNP 12 h. Mitochondrial fraction was prepared and subjected to Western blotting with specific antibody for Bax. The specific mitochondrial protein Cox-4 expression was determined for normalization. The photographs are representative of three separate experiments. The intensity of each band was quantified by densitometric analysis, and the fold changes of Bax expression wee plotted as a column graph. P13K inhibitor and Akt/PKB inhibitor reversed the inhibitory effect of UDCA on the Bax translocation to mitochondria.

FIG. 8 Illustrates downregulation of p53 by UDCA.

FIG 9 Shows a cluster analysis of untreated and UDCA-treated primary rat

hepatocytes.

FIG 10 Shows dopamine (1) and L-Dopa (2) conjugates with UDCA. FIG 11 Shows glutamate receptor antagonists.

FIG 12 Shows thiol antioxidants for conjugation or non-covalent combination with bile acids and their precursors and derivatives.

DETAILED DESCRIPTION OF THE INVENTION

The current invention describes a method of treating a patient exhibiting symptoms of several neurodegenerative diseases including Parkinson's disease. Currently there is no effective therapy that would either prevent or cure Parkinson's disease or several other neurodegenerative diseases.

Patients with neurodegenerative diseases such as Parkinson's disease and

Alzheimer's disease; Huntington's disease; multiple sclerosis; amyotrophic lateral sclerosis; cerebellar ataxia; lysosomal storage disorders; can greatly benefit from the neuroprotective properties of bile acids either alone or in combination with pro-drugs.

Along these lines, antioxidants such as the bile acids, ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA), and their analogues and derivatives are novel agents for the reduction of risk of neurodegenerative diseases. UDCA is a hydrophilic tertiary bile acid that is normally produced endogenously in the liver. Although hydrophilic bile acids, such as glycochenodeoxycholic acid and

taurochenodeoxycholic acid, are toxic and induce programmed cell death, UDCA and TUDCA are non-toxic. TUDCA can not only prevent hepatic cell death but also block oxygen radical production and programmed cell death in non-hepatic cells including neuronal cells.

In one embodiment, bile acids and all derivatives and precursors thereof with or without pro-drugs slow or reverse or completely abolish the progression of Parkinson's disease.

In one embodiment, bile acids and all derivatives and precursors thereof with or without pro-drugs protect neurons and brain tissue from degeneration or toxicity.

In one embodiment, bile acids and all derivatives and precursors thereof with or without pro-drugs protect neurons and brain tissue from apoptosis

In one embodiment, bile acids and all derivatives and precursors thereof with or without pro-drugs protect neurons and brain tissue from reactive oxidative damage.

In one embodiment, bile acids and all derivatives and precursors thereof with or without pro-drugs protect neurons and brain tissue from mitochondrial dysfunction or destruction.

In one embodiment, bile acids and all derivatives and precursors thereof with or without pro-drugs prevents or abolishes apoptosis in neurons and brain tissues.

In another embodiment of this invention, bile acids and all derivatives and precursors thereof can be conjugated to any anti-neurodegenerative pro-drug molecules involved in modulating neuronal apoptosis.

In another embodiment of this invention, bile acids and all derivatives and precursors thereof can be conjugated to pro-drugs of DA neurons such as L-DOPA and any analog of L-DOPA. In another embodiment of this invention, bile acids and all derivatives and precursors thereof are conjugated to glutamate receptor antagonists.

In another embodiment of this invention, bile acids and all derivatives and precursors thereof are conjugated to antioxidants.

In another embodiment of this invention, bile acids and all derivatives and precursors thereof can be combined, without conjugation, to any anti-neurodegenerative pro-drug molecules involved in modulating neuronal apoptosis.

In another embodiment of this invention, bile acids and all derivatives and precursors thereof can be combined, without conjugation, to pro-drugs of DA neurons such as L-DOPA and any analog of L-DOPA.

In another embodiment of this invention, bile acids and all derivatives and precursors thereof are combined, without conjugation, to glutamate receptor antagonists.

In another embodiment of this invention, bile acids and all derivatives and precursors thereof are combined, without conjugation, to antioxidants.

In another embodiment of this invention, the bile acid pro-drug in all its forms preserves the integrity of any aspect of the nervous system.

The term "effective amount" as used herein includes useful dosage levels of the compound of the present invention that will be effective to prevent or mitigate or completely cure the patients of any neurodegenerative disease. Useful dosages of the desired compound described herein can be determined by comparing its in vitro activity and its in vivo activity in animal models. Methods for extrapolation of effective dosages in mice, and other animals, to humans are known in the art.

It will be understood, however, that the specific "effective amount" for any particular subject will depend upon a variety of factors including the activity of the specific compound employed; the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the medical condition for the subject being treated.

The bile acids and their derivatives or precursors with or without pro-drugs are used in amounts effective to treat Parkinson's disease or any other neurodegenerative disease by either or both prophylactic or therapeutic treatments. Treatment involves prevention of onset or retardation or complete reversal of any or all symptoms or pharmacological or physiological or neurological or biochemical indications associated with Parkinson's disease or other neurodegenerative disease. Treatment can begin wither with the earliest detectable symptoms or established symptoms of Parkinson's disease or other neurodegenerative disease.

The "effective" amount of the compound thereof is the dosage that will prevent or retard or completely abolish any or all pathophysiological features associated with various stages (late or end) Parkinson's disease (sporadic or familial) or other neurodegenerative disease.

The bile acids and their derivatives or precursors with or without pro-drugs can be combined with a formulation that includes a suitable carrier. Preferably, the compounds utilized in the formulation are of pharmaceutical grade. This formulation can be administered to the patent, which includes any mammal, in various ways which are, but not limited to, oral, intravenous, intramuscular, nasal, or parental (including, and not limited to, subcutaneous, intramuscular, intraperitoneal, intravenous, intrathecal, intraventricular, direct injection into the brain or spinal tissue).

Formulations may be presented to the patient may be prepared by any of the methods in the realm of the art of pharmacy. These formulations are prepared by mixing the biologically-active bile acid and its derivative or precursor with or without pro-drugs into association with compounds that comprise the carrier. The carrier can be liquid, granulate, solid (coarse or finely broken), liposomes (including liposomes prepared in combination with any non-lipid small or large molecule), or any combination thereof.

The formulation in the current invention can be furnished in distinct units including, but not limited to, tablets, capsules, caplets, lozenges, wafers, troches with each unit containing specific amounts of the active molecule for treating Parkinson's or other neurodegenerative disease. The active molecule can be incorporated either in a powder, encapsulated in liposomes, in granular form, in a solution, in a suspension, in a syrup, in any emulsified form, a drought or an elixir.

Tablets, capsules, caplets, pills, troches, etc. that contain the biologically-active bile acid and its derivatives or precursors with or without pro-drugs can contain binder (including, but not limited to, corn starch, gelatin, acacia, bum tragacanth), an excipient agent (including but not limited to dicalcium phosphate), a disintegrating agent (including but not limited to corn starch, potato starch, alginic acid) a lubricant (including but not limited to magnesium stearate), a sweetening agent (including but not limited to sucrose, fructose, lactose, aspartame), a natural or artificial flavoring agent. A capsule may additionally contain a liquid carrier. Formulations can be of quick or sustained or extended-release type.

Syrups or elixirs can contain one or several sweetening agents, preservatives, crystallization-retarding agents, solubility-enhancing agents, etc.

Any or all formulations containing the biologically-active bile acids and their precursors or derivatives with or without pro-drugs can be included into the food (liquid or solid or any combination thereof) of the patient. This inclusion can either be an additive or supplement or similar or a combination thereof.

Parenteral formulations are sterile preparations of the desired biologically-active bile acid and its precursor or derivative with or without pro-drugs can be aqueous solutions, dispersions of sterile powders, etc., that are isotonic with the blood physiology of the patient. Examples of isotonic agents include, but are not limited to, sugars, buffers (example saline), or any salts.

Formulations for nasal spray are sterile aqueous solutions containing the biologically-active bile acid and its precursors or derivatives with or without pro- drugsalong with preservatives and isotonic agents. The sterile formulations are compatible with the nasal mucous membranes.

The formulation can also include a dermal patch containing the appropriate sterile formulation with the active agent. The formulation would release the active agent into the blood stream either in sustained or extended or accelerated or decelerated manner.

The formulation can also consist of a combination of compounds, in any of the afore mentioned formulations designed to traverse the blood-brain barrier. Examples

In the following examples, the role of biologically-active bile acid in the protection of neurons from destruction or dysfunction is described. In a dose- dependent manner, UDCA prevented sodium nitroprusside (SNP)-induced cytotoxicity in human dopaminergic SH-SY5Y cells. UDCA effectively attenuated the production of total reactive oxygen species (ROS), peroxynitrite (ONOO ) and nitric oxide (NO), and markedly inhibited the mitochondrial membrane potential (MMP) loss and intracellular reduced glutathione (GSH) depletion. In another example, SNP-induced programmed cell death or apoptotic events, such as nuclear fragmentation, caspase-3/7 and -9 activation, Bcl-2/Bax ratio decrease, and cytochrome c release, were significantly attenuated by UDCA.

In another example, the selective inhibitor of phosphatidylinositol-3 -kinase (P13K), LY294002, and Akt/PKB inhibitor, triciribine, reversed the preventive effects of UDCA on the SNP-induced cytotoxicity and Bax translocation. These results indicate that UDCA can protect SH-SY5 Y cells under programmed cell death process by regulating P13K-Akl/PKB pathways.

Methods

Cell culture and treatments

Human dopaminergic neuronal cell line, SH-SY5Y, was cultured in DMEM/F12 medium supplemented with 10% FBS (v/v), penicillin (100 U/ml)-streptomycin (100 μg/ml) in 5% C0₂ at 37 °C. SH-SY5Y cells were cultured at a seeding density of 3 x

10⁵ cells/ml. Usually, the culture medium was changed to DMEM/F12 medium with 0.5% FBS before any treatment to reduce the serum effect. In order to prevent the direct interaction between the treated chemicals, the culture medium was changed to fresh low- serum medium at the ent of pretreatment. UDCA was dissolved in ethanol as a lOOx stock solution and diluted to the desired final concentrations. To estimate cell viability, 3-(4,5- dimetnylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay was performed. After cells were treated ad culture medium was removed, MTT solution (50 μg, 1 mg/ml in phosphate buffered saline, PBS) was added to each well in 96-well plate and incubated for 4 h at 37 °C. The medium was carefully removed, 100 μΐ DMSO added to each well, and the plate agitated on an orbital shaker for 15 min to dissolve the formazan. The absorbance was measured at 540 nm using a microplate reader

(SepctraMax M2, Molecular Devices).

Nuclear staining for detecting apoptosis and necrosis

For the fluorescent detection of apoptotic and necrotic cells, nuclear staining with Hoechst dye 33342 and propidium iodide (PI) was performed. SH-SY5Y cells were exposed to SNP (1 mM) for 24 h with or without pretreatment with UDCA or YS. After fixation with 1% paraformaldehyde (PBS) for 30 min at room temperature, cells were washed with PBS and then stained with Hoechst 33342 (10 μΜ) for 10 min. Cells were washed with PBS and further stained with PI (10 μΜ) for 10 min. Stained cells were washed with PBS and ovserved under a fluorescent microscopy. The apoptotic cells were determined as bright condensed and fragmented nuclei. PI positive cells stained with pink to red color were counted as necrotic cells.

Analysis of caspase activity

Caspase-3/7 and caspase-9 activities were measured using the fluorogenic substrates. The assay was performed according to the manufacturer's protocol (Sensolyte Homogenous AMC Caspase Assay Kit, Anaspec Inc.). Briefly, cells were seeded at 3xl0⁴ cells/well in 96-well black wall and clear bottom culture plates. After 1 day, cells were pretreated for 1 h with UDCA (50, 100, 200 μΜ) or YS (100, 200 μΜ) then treated with SNP (1 mM) for 12 h. The fluorogenic peptide substrates Ac-DEVD-AMC and Ac-LEHD-AMC were used for caspase-3/7 and caspase-9, respectively. The reaction buffer containing 40 mM DTT and 100 μΜ substrate peptide was added into each well (50 μΐ of reaction buffer/well) and mixed completely by shaking and then incubated for 1 h. Fluorescende was read at 354 excitation and 442 emission on a fluorescence microplate reader

(SpectraMax M2, Molecular Devices).

Detection of total ROS, ONOO , and NO levels

The production of total ROS was measured using 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA, Sigma- Aldrich) and the formation of peroxynitrite ONOO^") was determined using dihydrorhodamine 123 (DHR 123, Molecular Probes). SH-SY5Y cells were treated with SNP (1 mM) with or without various concentrations of UDCA or YS for 12 h. After washing with Hank's balanced salt solution (HBSS), cells were incubated with 20 μΜ H₂DCFDA or 50 μΜ DHR at 37 °C for 30 min, and then rinsed with HBSS. The fluorescence intensity was measured using an automatic fluorescence microplate reader (SpectraMax M2, Molecular Devices) at an excitation wavelength of 485 nm and an emission of 535 nm. The values were expressed as a percentage of fluorescence intensity to the untreated control group. The production of NO was determined by measuring nitrite, a stable oxidation product of NO in the culture medium. After treatment of SNP (1 nM) with or without various concentrations of UDCA or YS for 24 h, cell culture medium was mixed with an equal volume of Griess reagent (Sigma- Aldrich). After a 10-min reaction, the absorbance at 550 nm was measured in a microplate reader (VersaMax, Molecular Devices). Sodium nitrite (NaN0₂) was used as a standard to calculate nitrite concentrate and the values were expressed in micromoles. Measurement of mitochondrial membrane potential (MMP)

MMP (ΔΨιη) was measured using the mitochondria-specific lipophilic cationic fluorescent dye 5,5',6,6'-tetrachloro-l,l ',3,3'-tetraethybenzimidazolocarbocyanine iodide (JC-1; Anaspec Inc.). JC-1 preferentially accumulates in mitochondria as red aggregates in normal conditions but it exists as green monomers in the cytosol when MMP collapsed during apoptosis. The ratio of red/green fluorescence correlates with MMP. SH-SY5Y cells were pretreated with various concentrations of UDCA or YS for 1 h and then treated with 1 mM SNP for additional 12 h. Next, 5 μg/ml JC-1 was added and incubated at 37 °C for 15 min in dark. After wash three times with PBS, MMP was measured at 535/590 nm (Ex/Em) for red fluorescence and 485/535 (Ex/Em) for green fluorescence using a fluorescence multimode microplate reader (Infinite 200; Tecan). Results were calculated as the ratio of red-to-green fluorescence and the values were expressed as the percentage over control. Measurement of cellular reduced glutathione (GSH) content

The intracellular GSH levels were analyzed using the fluorescent dye monochlorobimane (MCB, Sigma- Aldrich). Briefly, following treatments, SH-SY5Y cells in black 96-well culture plates were washed with HNSS and then incubated with 40 μΜ MCB for 20 min in dark. After washing twice with HBSS, fluorescence intensity was determined at 355/460 nm (Ex/Em) in a fluorescence microplate reader (SpectraMax M2, Molecular Devices). GSH content was determined from a standard curve constructed using known amounts of glutathione (Sigma- Aldrich). Values were expressed as a relative content of untreated group.

Immunoblot analysis

SH-SY5Y cells were pretreated for 1 h with UDCA (200 μΜ) and then treated with SNP (1 mM) for fixed time according to our pretests (12 h for the analysis of Bcl-2, Bax, and cytochrome c). Whole cell proteins were extracted using RIPA buffer (PBS, 1% NP-40, 0.5% Na deoxycholate, 0.1% SDS, 0.1 mg/ml PMSF, 30 mg/ml aprotinin, 1 mM Na₃V0₄). Cells were washed twice with PBS, lysed with RIPA buffer for 30 min on ice, and then centrifuged at 14,000xg for 10 min at 4 °C. The supernatants were used as the total cell lysates. In some experiments, mitochondrial fraction was prepared from SH-Sy5Y cells using mitochondrial/cytosolic fraction kit (Biovision, Inc., Mountain View) according to the manufacturer's protocol. Protein concentration was determined by BCA protein assay kit (BioRad, Hercules, CA) using bovine serum albumin as a standard. Protein samples (40 μg) were separated on a 10 - 15%> SDS-polyacrylamide gel and transferred onto PVDF membrane. The membrane was flocked in fresh blocking buffer (5% nonfat dry milk in Tris-buffered saline, pH 7.4, and containing 0.1% Tween 20) for 2 h at room temperature and rinsed in TBST buffer (0.1% Tween 20 in Tris-buffered saline, pH 7.4). The membrane was incubated at 4 °C with the following primary antibodies at dilutions of 1/1000: Bax, cytochrome c, Cox -4 or 1/4000: Bcl-2, actin. After three times washing with TBST buffer, membranes were incubated with horse radish peroxidase (HRP)-conjugated secondary antibodies (1 :2000 dilutions) for 2 h at room temperature. Subsequently, the membrane was washed in TBST and the immunoreactive bands were detected by ECL chemiluminescence kit (GE Healthcare, USA). Protein bands were quantified by densitometric analysis.

Statistical Analysis

All experiments were performed at least three times, and results were expressed as the mean +SEM. The data were analyzed using the SPSS 12.0 software package (SPSS Inc., Chicago, IL). Differences were analyzed using one-way factorial analysis of variance (ANOVA), and the Duncan's post hoc test. Results

Protective effect of UDCA and YS against SNP-induced neurotoxicity

Initial studies were performed to examine the cytotoxic response of SH-SY5Y cells to various concentrations (100 μΜ - 2 mM) of SNP. The loss of viability occurred by SNP in a dose-dependent manner, and 1 mM SNP induced approximately 56% cell loss after 24 hr of treatment (Fig. 1A). Thus, we did subsequent experiments using 1 mM SNP.

Treatnemt with UDCA alone or YS alone for 24 h at doses of 50 - 200 μΜ showed no obvious change in the viability compared with the control group (Fig. IB). To investigate the effect of UDCA and YS on SNP-induced human dopaminergic cell death, SH-SY5Y cells were pretreated with 50-200 μΜ UDCA or 100-200 mM YS for 1 h, followd by 1 mM SNP treatment for 24 h. As shown in Fig. 2C, SNP-induced loss of cell viability was significantly attenuated by UDCA or YS pretreatment dose-dependently.

Although SNP acts as a NO donor, the molecular structure of SNP shows a complex of NO with ferrous ion and five cyanides. Therefore, SNP not only produces NO but also generates cyanides and free iron. To distinguish the role of NO, cyanides, and free iron in the SNP-induced dopaminergic cell death, SH-SY5Y cells were treated with potassium ferricyanide or sodium cyanide. However, treatment with potassium

ferricyanide (0.5, 1 mM) or sodium cyanide (0.5 or 1 mM) did not change the cell viability obviously (Fig. ID). Also, to confirm a causative role of NO moiety in SNP, we treated SH-SY5Y cells with the 50day light exposed SNP (SNP_EXP), which corresponds to its NO- exhausted SNP. As shown in Fig ID, SNP_EXp did not effect the cell viability of SH-SY5Y cells. Thus, we can speculate that NO may be a cytotoxic mediator involved in SNP- induced dopaminergic cell death. UDCA and YS ameliorated SNP-induced apoptosis and caspase activation

We investigated the effect of UDCA and SNP-induced programmed cell death characteristics, such as nuclear morphology changes, caspase-3/7 activation and caspase-9 activation in SH-SY5Y cells. A significant proportion of SNP-induced cell death was apoptotic, based on Hoechst 33342-stained nuclear changes in morphology and PI staining. We observed a significant increase in condensed, fragmented nuclei after 24 h treatment with SNP (1 mM). However, a low percentage of nuclei were stained red by the necrotic marker dye PI (Fig 2A). The number of those hallmarks of apoptotic or necrotic nuclei was similar to untreated control cells and both UDCA and YS treated cells.

Moreover, we found that both UDCA and YS effectively inhibited SNP -mediated apoptotic nuclear damages (Fig2A). As quantified in Fig 2B, although SNP increased the apoptotic rate to 30.59+3.38%, UDCA or YS pretreatment prior to SNP treatment caused a statistically significant reduced apoptotic rate (8.45+2.01%) and 11.67+1.75%,

respectively).

Next, we examined caspase-3/7 and caspase-9 activity as another marker of programmed cell death. A shown in Fig. 2C, the exposure of SH-SY5Y cells to 1 mM SNP for 12 h increased caspase-3/7 and -9 activities by 2.43 and 4.21 -fold respectively. Either UDCA (50-200 μΜ) or YS (100-200 μΜ) pretreatment strongly attenuated the effects of SNP on caspase-3/7 and caspase-9 activity. These results suggest that the protective effects of UDCA and YS are mediated by anti-apoptotic pathway.

UDCA and YS inhibited SNP-induced NO, ΟΝΟΟ^', and total ROS production in SH- SY5Y cells

To determine the changes of RNS and ROS production in human dopaminergic cells during the SNP-induced cell death and UDCA- or YS-mediated protection, we measured NO, total ROS, and ONOO^" production in SH-SY5Y cells using Griess reagent, fluorescent dye H₂DCFDA, and DHR-123, respectively. As shown in Fig. 3 A, NO production after 24 h SNP treatment was increased to 527.74% that of the control group. Both UDCA and YS attenuated the SNP-induced NO production. UDCA pretreatment (50, 100, and 200 μΜ) dose-dependently reduced the NO production to 91.44%, 82.88%, and 77.26%), respectively, compared with the group treated with SNP alone. Next, we further investigated whether the protective effects of UDCA and YS were due to the decreased production of total ROS and peroxynitrite. Treatment with 1 mM SNP increased total ROS and ONOO^" generation up to 324.17% and 174.9%), respectively compared with the control group (Fig 3A). However, ROS generation was dose- dependency reduced to 79.68%, 72.59%, and 58.09% of SNP-treated group by UDCA pretreatment (50, 100 and 200 μΜ) and reduced to 76.74% and 66.57% by YS

pretreatment (100 and 200 μΜ), respectively. SNP-induced peroxynitrite generation was inhibited by UDCA (50, 100, and 200 μΜ) or YS (100 and 200 μΜ) dose-dependently. Interestingly, pretreatment of cells with high dose of UDCA (200 μΜ) or YS (200 μΜ) produced almost complete blocking of SNP-induced peroxynitrie generation (Fig 3 A). UDCA and YS restored the SNP-induced cellular GSH content depletion and mitochondrial dysfunction

To further evaluate the anti-oxidative effects of UDCA and YS, we determined the levels of intracellular GSH, a major cellular protective antioxidant. As shown in Fig 4A, cellular GSH level was significantly decreased after treatment with 1 mM SNP for 12 h (49.52+8.4% of control). However, pretreatment with UDCA (50, 100, and 200 μΜ) or YS (100 and 200 μΜ) markedly attenuated SNP-induced GSH depletion in SH-SY5Y cells (Fig 4A).

As shown in Fig 4B, the control cells and UDCA or YS treated cells did not show any alterations in MMP. Treatment of cells with 1 mM SNP for 12 h significantly decreased MMP to 47%> of control group. However, the SNP-induced MMP loss was relieved by UDCA (71%, 88%, and 87% of control group at 50, 100, 200 μΜ UDCA, respectively) or YS (71 > and 74%> of control group at 100 and 200 μΜ YS, respectively). UDCA restored the Bcl-2/Bax ratio and prevented the cytochrome c release

The mitochondrial dysfunction is accompanied by modulation of Bcl-2 family proteins and release of cytochrome c. To investigate the involvement of Bcl-2 family proteins in SNP-induced cell death and UDCA-mediated protection, we determined the expression of the programmed cell death suppressor protein Bcl-2 and programmed cell death inducer protein Bax by Western blot (Fig 5A). SNP treatment showed no alterations in Bcl-2 expression but an increase in Bax expression, which resulted in a decreased ratio of Bcl-2/Bax (0.63+0.05 fold of control). However, UDCA per se and pretreatment with UDCA prior to SNP treatment significantly increased the ratio of Bcl-2/Bax (2.52+0.16 fold and 2.21+0.09 fold of control, respectively) in SH-SY5Y cells (Fig 5B). In addition, SNP (1 mM) markedly induced cytochrome c release from the mitochondria into the cytosol (2.48+0.11 fold of control). However, the release of cytochrome c was significantly restored (1.41+0.06 fold of control) of pretreatment with UDCA (Fig 5).

UDCA-mediated neuroprotection is associated with P13K and Akt/PKB signal pathways To evaluate the signaling pathways in UDCA-mediated neuroprotection against the insult of SNP on SH-SY5Y cells, a pharmacological approach was used with specific inhibitors of various signaling molecules. Cells were pretreated with specific Akt/PKB inhibitor triciribine (1 μΜ), P13K inhibitor LY294002 (2 μΜ), PKA inhibitor PK1 (1 μΜ), or PKC inhibitor Go6983 ( 2 μΜ) for 1 h, and then treated with UDCA (200 μΜ) for 1 h and stimulated with SNP (1 mM) for 24 h. However, PKI (PKA inhibitor) and Go6983 (PKC inhibitor) did not have significant impact on the UDCA-mediated neuroprotection. All those inhibitors themselves had no effects on cell viability in SH- SY5Y cells. To further confirm the role of PI 3K- Akt/PKB pathways in UDCA-mediated neuroprotection, translocation of the programmed cell death inducer Bax was evaluated after pretreatment with specific inhibitors of P13K and Akt/PKB. As shown in Fig 7, Bax translocation to the mitochondria induced by SNP (ImM) treatment was almost completely blocked by UDCA (200 μΜ) pretreatment. However, the inhibitory effect of UDCA on SNP-induced Bax translocation was markedly reversed by LY294002 (P 13K inhibitor) and triciribine (Akt/PKB inhibitor). These results indicate that UDCA can exert a neuroprotective effect, at least in part, through the P13K- Akt/PKB pathways in SH- SY5Y cells. p53 is a key molecular target of UDCA in regulating apoptosis

p53 plays an important role in regulating expression of genes that mediate cell cycle progression and/or apoptosis. We have previously shown UDCA prevents TGF-βΙ- induced p53 stabilization and apoptosis in primary rat hepatocytes. We therefore hypothesized that p53 may represent an important target in bile acid-induced modulation of apoptosis and cell survival. Functional studies revealed that UDCA reduced both transcriptional and DNA binding activity of p53 tumor suppressor, while promoting its nuclear export in primary rat hepatocytes. These effects led to abrogation of all apoptotic hallmarks induced by p53 overexpression, such as Bax mitochondrial translocation, cytochrome c release and caspase-3 activation. We have also evaluated whether UDCA inhibited p53 via its major repressor, the Mdm-2 protein. Indeed, increased association between p53 and Mdm-2 was detected in hepatocytes preincubated with UDCA. We suggested that by inducing Mdm-2/p53 complex formation, UDCA reduced p53 activity by simultaneously blocking its transactivation domain and enhancing its export to the cytosol. Target knockdown of the mdm-2 gene by posttranscriptional silencing resulted in increased accumulation of p53 in the nucleus, even in the presence of UDCA, thus confirming the specific role of Mdm-2 in the anti-apoptotic function of UDCA.

We have further extended these studies to explore the role of UDCA in downregulating p53 by Mdm-2. The results showed that the bile acid increases cellular proteasomal activity, thereby decreasing p53 half-life (Fig 8). Importantly, after proteasomal inhibition, UDCA pre-treatment resulted in accumulation of Mdm-2- dependent ubiquitinated p53. Finally, the protective effect of UDCA against p53 -induced apoptosis was abolished after inhibition of proteasome activity. In conclusion, these findings suggest that UDCA protects cells from p53 -induced apoptosis by promoting its degradation via the Mdm-2-ubiquitin-proteasome pathway.

The fact that proteasomal degradation has been described as the main mechanism by which Mdm-2 inhibits p53 prompted us to investigate the role of UDCA in this pathway. Our data indicated that UDCA stimulated Mdm-2-dependent ubiquitination of p53; further increased proteasome activity triggered by wild-type p53. After proteasomal inhibition, UDCA pre-treatment resulted in accumulation of Mdm-2-dependent ubiquitinated p53. Of note, the protective function of UDCA was abolished by inhibiting proteasome activity.

These data suggest that UDCA protects hepatocytes from p53 -induced apoptosis by enhancing complex formation between p53 and its inhibitor Mdm-2. Furthermore, by acting as a chaperone-like molecule, UDCA modulate specific and diverse regulatory events such as transcription, subcellular localization, and degradation of precise apoptosis- related molecular targets. Genomic Profiling of Rat Hepatocytes after Incubation with UDCA by Microarray Analysis

We have investigated the effects of UDCA on gene expression in primary rat hepatocytes by microarray analysis of the rat genome. We determined the global profile of genes regulated by UDCA by using Affymetrix GeneChip® Rat Expression Array 23 OA, consisting of approximately 16,000 transcripts and variants. cRNA prepared from vehicle- treated cells was used for comparative analysis. The relative levels of gene expression after 24 h treatment of hepatocytes with 100 μΜ UDCA were compared by plotting the average difference between cells, and determining the fold change in gene expression. Approximately 441 genes (2.76%) exhibited alterations in expression following UDCA treatment, with a greater than 1.5-fold change in genes expression. Among these, approximately 25% fulfilled the filtering criteria for detection in at least one of the arrays. Of these 96 genes, 28 were up-regulated and 68 were down-regulated. These genes fall into several broad categories, although some of the most prominent are involved in cell cycle/proliferation and apoptosis. For example, the array analysis indicated that Apaf-1 is robustly down-regulated in rat hepatocytes in response to UDCA. We also assessed the specificity and sensitivity of the microarray analysis. Hierarchical clustering was performed using specific gene subsets. As expected, all three controls clustered with remarkable identity and separated from the three UDCA treated samples on the

dendrogram (Fig. 9).

Our data indicate that UDCA and TUDCA have markedly anti-apoptotic properties. Characterization of the molecular basis for their anti-apoptotic effects will provide significant new information about the events involved in cell death and the potential check points that may promote cell survival. The toxicity of MPTP and 3-NP are closely related. MPTP toxicity is mediated by inhibition of complex I of the electron transport chain, and is preferentially taken up by dopaminergic cells. 3-NP acts by irreversibly inhibiting complex II of the electron transport chain. By impairing mitochondrial function, MPTP and 3-NP both cause depressed oxidative phosphorylation leading to decreased ATP production and mitochondrial stress. We have previously generated extensive data using 3-NP as the primary toxin. However, the similarities between MPTP and 3-NP suggests that TUDCA will affect MPTP toxicity in a manner similar to that of 3-NP. Design and Synthesis of UDCA Pro-drugs

Figure 10 depicts the structures of UDCA conjugates of DA and L-Dopa. Included here are alkyl derivatives of L-dopa, monoamine oxidase inhibitors (MAO), catechol-O- methyl transferase (COMT) and the monoamine re -uptake inhibitors. Converting these molecules and their analogs to pro-drugs by conjugating them with UDCA (and its derivatives and analogs) would greatly enhance the transport through the blood brain barrier which currently is a huge challenge.

Figure 11 depicts the thio-conjugates of UDCA and its analogs and derivatives. Glutamate plays a central role in the disruption of normal basal ganglia function, and it has been hypothesized that agents acting to restore normal glutamatergic function may provide therapeutic interventions that bypass the severe motor complications associated with current DA replacement strategies. Analysis of glutamate receptor ligands in the basal ganglia suggests that both ionotropic and metabotropic glutamate receptors could have anti-parkinsonian actions. Delivery of NMD A receptor antagonists that selectively target the NR2B subunit and antagonists of the metabotropic glutamate receptor mGluR5 also may hold promise. For example, amantadine releases DA from nerve endings of brain cells and stimulates norepinephrine response. Importantly, amantadine also relieves levodopa-induced dyskinesia. Conjugates of UDCA (and its analogs and derivatives) with amantadine (compound 4, Fig 11), kinurenic acid (compound 7) (metabolic product of L- tryptamine) and nipecotic acid (compound 6) isonipacotic acid (compound 5) will be used for their anti-parkinsonian activity.

Glutathione (GSH) is the most important antioxidant in biological systems. Several strategies have been used to increase GSH as a means to obtain protection against oxidants such as free radicals and reactive electrophiles such as quinones. Glutathione is present at up to 150mg/day in the human diet and can be absorbed intact in the intestine. Although cysteine that is released from protein degradation can be reutilized for the synthesis of GSH, cysteine is also used for production of taurine and needed for variety of biological functions including detoxification. Oxidative stress evoked by xenobiotics generally result in the depletion of cellular GSH. A current experimental therapy for Parkinson's disease involves intravenous infusion of GSH. The GSH conjugate of the metabolite of the anti- alcohol agent disulfiram (111) and metabolites of amphetamine and metamphetamine readily cross the BBB via a GSH transporter (112). The relevance to our drug design strategy is S-conjugated GSH with UDCA which is expected to be actively transported via GSH or bile acid transporters in the brain when administered intranasally. Therefore we propose to synthesize GSH -thio-conjugate of UDCA (Fig 12, compound 8).

In addition to lipoic acid's role as cofactor in the citrate synthase, it is a powerful antioxidant that is effective at scavenging both water and lipid soluble free radicals. It picks up some of the free radicals that vitamin C and E miss. Lipoic acid is emerging as one of the most promising agents for neuroprotection in neurodegenerative diseases. It acts as a metal chelator for ferrous iron, copper, cadmium and also participates in the regulation of endogenous antioxidants. UDCA (and its analogs and derivatives) conjugate of lipoic acid (Fig 12, compound 9) will be used for neuroprotection activity.

Acetyl-L-carnitine has been demonstrated to increase cellular ATP production. It was shown to prevent MPTP-induced neuronal injury in rats. Further, acetyl-L-carnitine reduces production of mitochondrial free radicals, helps maintain transmembrane mitochondrial potential, and enhances NAD/NADH electron transfer. Compound 10 (Fig 12, compound 10) as a conjugate of UDCA (and its analogs and derivatives) will be used for protection against neuronal injury.

While the invention has been described with reference to an exemplary

embodiment(s), it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment(s) disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method of treating or preventing the onset of Parkinson's or any

neurodegenerative disease in a patient through the administration of a bile acid, and its analogs and derivatives, conjugated to a pro-drug and a combination thereof to the patient.

2. The method of claim 1 wherein the bile acid in all its form is chemically

conjugated to a pro-drug of nigral DA neurons.

3. The method of claim 1 where the pro-drug is L-dopa or any of its analogs or precursors or derivatives with the general formulae:

4. The method of claim 1 wherein the pro-drug is monoamine oxidase inhibitor or any of its analogs or precursors or derivatives.

5. The method of claim 1 wherein the pro-drug is catechol-O-methyl transferase or any of its analogs or precursors or derivatives.

6. The method of claim 1 wherein the pro-drug is monoamine re-uptake inhibitor or any of its analogs or precursors or derivatives.

7. The method of claim 1 wherein the pro-drug is a glutamate receptor antagonist or any of its analogs or precursors or derivatives with the general formulae:

8. The method of claim 1 wherein the pro-drug is an antioxidant of any of its analogs or precursors or derivatives with the general formulae:

9. The method of claim 1 wherein the bile acid pro-drug in all its forms inhibits the onset of Parkinson's disease.

10. The method of claim 1 wherein the bile acid pro-drug in all its forms is an inhibitor of any process leading the loss of dopaminergic neurons in the brain or any nervous tissue.

11. A method for treating or preventing Parkinson's disease in a patient, the method

comprising: administering a combination of a bile acid in all its form in combination with pro-drugs of nigral DA neurons to the patient.

12. A method for treating a patient having Parkinson's or any neurodegenerative disease, the method comprising: administering to a patient an effective amount of the bile acid in all its forms, including pro-drug embodiments.

13. The method of claim 12 wherein the patient is a human patient.

14. The method of claim 12 wherein the compound is administered in combination with a pharmaceutical-grade carrier.

15. The method of claim 12 wherein the compound is administered by any at least one of oral, or parenterally or intramuscular, intravenous, or direct administration into any part of the brain.

16. The method of claim 12 wherein any foreign molecule elevates the physiological levels of the bile acid or any derivative, including pro-drug embodiments, or any analog or any precursor or salt in the patient.

17. The method of claim 12 further comprising a diet that would increase the levels of the bile acid or any of its pro-drug embodiments, or any derivative or any analog or any precursor or salt in the patient.

18. The method of claim 12 further comprising a drug therapy would alter the endogenous level of bile acid or any of its pro-drug embodiments or any derivative or any analog or any precursor or salt in the patient.

19. The method of claim 12 further comprising a gene therapy would enhance the

physiological levels bile acid or any of its pro-drug embodiments or any derivative or any analog or any precursor or salt in the patient.

20. A method of treating or preventing or delaying the onset of Parkinson's disease or any neurodegenerative disease comprising by administering the compound to a brain of a patient.