NEUROPROTECTIVE COMPOSITION COMPRISING L-NAME FOR MEMORY ENHANCEMENT IN NEURODEGENERATIVE DISORDERS

Abstract

T The invention discloses a neuroprotective pharmaceutical composition containing N(gamma)-nitro-L-arginine methyl ester (L-NAME) in concentrations from 1% to 15% by weight, combined with stabilizing, antioxidant, and lipid peroxidation inhibiting agents. The stabilizing agent is chosen from hydroxypropyl methylcellulose or polyvinylpyrrolidone (0.1% to 5% by weight), the antioxidant agent from glutathione or ascorbic acid (0.1% to 10% by weight), and the lipid peroxidation inhibitor from butylated hydroxytoluene (BHT) or vitamin E (0.05% to 2% by weight). This composition is formulated with a pharmaceutically acceptable carrier, including optional buffering agents to maintain pH between 6.0 and 7.5. Suitable for liquid or injectable solution forms, the composition is intended for intraperitoneal administration to aid in treating memory impairment and related neurological conditions by leveraging L-NAME's neuroprotective properties along with added stabilization and antioxidant support.

Specification

Description:NEUROPROTECTIVE COMPOSITION COMPRISING L-NAME FOR MEMORY ENHANCEMENT IN NEURODEGENERATIVE DISORDERS

FIELD OF THE INVENTION
The present invention relates to the field of neuropharmacology, specifically to compositions for treating neurodegenerative disorders associated with cognitive impairment and memory loss, such as Alzheimer's disease. The present invention primarily relates to a novel composition comprising L-NAME (N(gamma)-nitro-L-arginine methyl ester) in combination with selected antioxidant and stabilizing agents.
BACKGROUND OF THE INVENTION
Memory impairment, especially related to Alzheimer's disease, is one of the most challenging neurodegenerative conditions, affecting millions globally. Alzheimer's disease (AD) is characterized by progressive cognitive decline, memory loss, and neuronal apoptosis, primarily linked to acetylcholine deficit and increased oxidative stress in the brain. Over the years, numerous approaches have been developed to counter these effects, largely targeting cholinergic pathways and oxidative damage.
Cholinesterase inhibitors, such as donepezil, rivastigmine, and galantamine, are among the most common pharmacological treatments for Alzheimer's. These drugs work by inhibiting acetylcholinesterase (AChE), the enzyme responsible for breaking down acetylcholine, thereby increasing the levels of acetylcholine in the brain. Donepezil, a commonly used drug, has shown efficacy in improving cognitive function in Alzheimer's patients. However, these inhibitors often come with limitations such as gastrointestinal issues like nausea, vomiting, and diarrhoea, are common due to the widespread action of these drugs on the peripheral cholinergic system.
Further, these treatments only provide symptomatic relief and do not halt or reverse the progression of neuronal damage in Alzheimer's disease. And, over time, patients often develop tolerance to cholinesterase inhibitors, reducing their effectiveness.
Additionally, N-Methyl-D-Aspartate (NMDA) Receptor Antagonists for e.g. Memantine is prescribed for treatment of moderate to severe Alzheimer's disease. Memantine helps regulate glutamate activity to prevent excitotoxicity, a condition in which excessive glutamate damages neurons. However, memantine's effectiveness is limited in early stages, and it is often used in combination with cholinesterase inhibitors for better results.
However, Memantine is not a cure and provides limited symptomatic relief, failing to address the underlying neurodegenerative processes, and shows common side effects include dizziness, confusion, and headache, which can be problematic for elderly patients.
Further, given the role of oxidative stress in Alzheimer's disease, antioxidants have been explored as a treatment avenue. Compounds such as Vitamin E, glutathione, and curcumin aim to neutralize free radicals, reduce oxidative damage, and potentially slow down neurodegeneration. Despite some preclinical promise, antioxidants have shown limited success in clinical settings. Many antioxidants, like curcumin, have low bioavailability, limiting their effectiveness in reaching therapeutic concentrations in the brain. Also, the effects of antioxidants are inconsistent and may not be sufficient as a standalone therapy for Alzheimer's disease.
Antioxidants act broadly, and their lack of specificity can reduce effectiveness in addressing the localized oxidative stress associated with neurodegeneration.
Despite the wide range of therapeutic options, each suffers from limitations, including low efficacy, adverse side effects, or lack of disease-modifying capabilities. The complex, multifactorial nature of AD demands novel therapeutic agents that can address multiple pathological pathways while exhibiting minimal side effects.
Therefore, there is a need for a novel composition that can overcome the limitations of existing solutions.
SUMMARY OF THE INVENTION
The present invention provides a novel neuroprotective composition comprising L-NAME that is capable of mitigating neuronal apoptosis and improving memory performance. Specifically, this composition demonstrates efficacy in a scopolamine-induced memory impairment model, which mimics cognitive deficits associated with neurodegenerative diseases.
The composition of the present invention has been shown to significantly enhance cognitive performance in behavioral tests, such as the Y-maze and Morris Water Maze, and to reduce oxidative stress indicators in brain tissue.
OBJECTIVE OF THE INVENTION
The objective of the present invention is to address the above-mentioned limitations of the conventional solutions by proposing a novel composition that combines L-NAME with specific stabilizing and antioxidant agents. The composition of the present invention is designed to leverage L-NAME's potential neuroprotective effects, reduce oxidative stress, and provide cholinergic support. Through this combination, the present invention aims to offer an improved therapeutic option with potential for disease-modifying effects, targeting both neurotransmitter regulation and antioxidant pathways.
This innovative approach intends to achieve:
• Enhanced Memory Performance: By modulating acetylcholinesterase activity and supporting acetylcholine levels, the composition seeks to improve memory and cognitive functions.
• Reduction of Oxidative Stress: With the inclusion of antioxidants like glutathione, the composition addresses oxidative stress more effectively, potentially slowing down neurodegeneration.
• Modulation of Neuroinflammation: By inhibiting excessive nitric oxide production through L-NAME, the composition may help reduce neuroinflammation, a key factor in Alzheimer's progression.
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying figures illustrate several embodiments of the disclosure and, together with the description, serve to explain the principles of the disclosure. One of ordinary skill in the art readily recognizes that the embodiments illustrated in the figures are merely exemplary, and are not intended to limit the scope of the present disclosure.
FIG. 1 illustrates a molecular structure of an active component (L-NAME) of the present invention.
FIG. 2 illustrates an exemplary diagrammatic representation of Hypothesis of experimental validation of the present invention.
FIG. 3 illustrates an exemplary diagrammatic representation of experimental protocol of experimental validation of the present invention.
FIG. 4 discloses effect of L-NAME on scopolamine-induced memory impairment in mice using Morris water maze. (Escape latency Time in Seconds).
FIG. 5 discloses effect of L-NAME on scopolamine-induced memory impairment in mice using Y-maze.
FIG. 6 discloses effect of L-NAME on AchE activity catalase in scopolamine-induced memory impairment in mice.
FIG. 7 discloses effect of L-NAME on the brain Glutathione reductase in scopolamine-induced memory impairment in mice.
In the bar graphs of the above figures, all values are mean SEM (n=6). indicates P< 0.001 compared to control, indicates ap<0.01 compared to control, bp<0.01 compared to scopolamine, cp<0.01 compared to L-NAME (LD), dp<0.01 compared to L-NAME(HD).
Further areas of applicability of the present disclosure will become apparent from the complete description provided hereinafter.
It should be understood that the complete description of exemplary embodiments is intended for illustration purposes only and is, therefore, not intended to necessarily limit the scope of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is more particularly described in the following present specification that is intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art. Various embodiments of the present disclosure are now described in detail. Referring to the drawings, like numbers, if any, indicate like components throughout the views. As used in the description herein and throughout the claims that follow, the meaning of "a", "an", and "the" includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein and throughout the claims that follow, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise. Moreover, titles or subtitles may be used in the specification for the convenience of a reader, which shall have no influence on the scope of the present disclosure. Additionally, some terms used in this specification are more specifically defined below.
The terms used in this specification generally have their ordinary meanings in the art, within the context of the present disclosure, and in the specific context where each term is used. Certain terms that are used to describe the present disclosure are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the disclosure.
For convenience, certain terms may be highlighted, for example using italics and/or quotation marks. The use of highlighting has no influence on the scope and meaning of a term; the scope and meaning of a term are the same, in the same context, whether or not it is highlighted. It will be appreciated that the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms.
The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and in no way limits the scope and meaning of the disclosure or of any exemplified term. Likewise, the disclosure is not limited to various embodiments given in this specification.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In the case of conflict, the present document, including definitions will control.
As used herein, "around", "about" or "approximately" shall generally mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of a given value or range. Numerical quantities given herein are approximate, meaning that the term "around", "about" or "approximately" can be inferred if not expressly stated.
As used herein, "plurality" means two or more.
As used herein, the terms "comprising," "including," "carrying," "having," "containing," "involving," and the like are to be understood to be open-ended, i.e., to mean including but not limited to.
As used herein, the phrase at least one of A, B, and C should be construed to mean a logical (A or B or C), using a non-exclusive logical OR. It should be understood that one or more steps within a method may be executed in a different order (or concurrently) without altering the principles of the present disclosure.
The present invention provides a novel composition comprising L-NAME (N(gamma)-nitro-L-arginine methyl ester) in combination with selected antioxidant and stabilizing agents. FIG. 1 discloses a molecular structure of an active component (L-NAME) of the present invention. The composition of the present invention is designed to modulate neurotransmitter pathways, reduce oxidative stress, and provide neuroprotective effects, thereby enhancing memory performance and addressing cognitive deficits associated with neurodegenerative conditions.
The present invention intends to offer an alternative therapeutic approach to current treatments, targeting multiple mechanisms involved in neurodegeneration and memory impairment.
The following detailed description explains the composition's formulation, its effect on cognitive function, and an experimental validation supporting the efficacy of the composition. FIG. 2 illustrates an exemplary diagrammatic representation of Hypothesis of experimental validation of the present invention.
CHEMICALS AND REAGENTS
For the purpose of experimental validation of the present invention, a range of chemicals and reagents were carefully selected and prepared to ensure accuracy and reliability in the experimental assessment of the neuroprotective and memory-enhancing effects of L-NAME. These substances were primarily sourced from reputable suppliers to maintain high standards of purity and quality control, which are critical in minimizing experimental variability and ensuring reproducibility of results.
• L-NAME (N(gamma)-Nitro-L-arginine methyl ester), the primary investigational compound, was administered intraperitoneally (i.p.) at concentrations of 5 mg/kg and 10 mg/kg. L-NAME is a widely used synthetic derivative of L-arginine, known to inhibit nitric oxide synthase (NOS), thereby reducing the production of nitric oxide (NO) in neuronal tissues. This compound was freshly prepared before each administration to maintain its stability and potency, which is crucial for assessing its impact on neuroprotection and memory function.
• Scopolamine hydrobromide was used as an agent to induce cognitive impairment, mimicking Alzheimer's disease-like symptoms in the animal model. Sourced in its pure form, scopolamine was administered intraperitoneally at a dose of 1 mg/kg. As a competitive antagonist of muscarinic acetylcholine receptors, scopolamine disrupts cholinergic signaling in the brain, leading to a temporary cognitive deficit suitable for assessing the efficacy of potential neuroprotective agents.
• Donepezil hydrochloride was included as a positive control to benchmark the memory-enhancing efficacy of L-NAME. Donepezil, a well-known acetylcholinesterase (AChE) inhibitor, was administered at a dose of 2 mg/kg. This drug works by preventing the breakdown of acetylcholine, thereby enhancing cholinergic transmission and improving cognitive function in models of memory impairment. Donepezil's inclusion in the experimental validation provides a standard for comparing the effects of L-NAME on memory performance.
Other reagents used in the biochemical assays were of analytical grade and included solutions specifically prepared for acetylcholinesterase (AChE) enzyme inhibition assays, glutathione (GSH) concentration measurement, and malondialdehyde (MDA) assessment as markers of oxidative stress. For the AChE assay, sodium phosphate buffer was prepared as the medium, providing optimal conditions for enzyme activity. Additionally, 5,5'-Dithiobis-(2-nitrobenzoic acid) (DTNB) was used as a chromogenic reagent to quantify AChE activity through spectrophotometric measurements at 430 nm, following the Ellman method.
In the glutathione (GSH) assay, DTNB was also utilized, where it reacts with GSH to produce a yellow chromophore measurable at 412 nm. 10% trichloroacetic acid (TCA) was used to precipitate proteins, with the supernatant analyzed for GSH content, thus enabling the study of antioxidant defenses within the brain tissues of treated and control animals.
For the assessment of malondialdehyde (MDA) levels, which serves as a lipid peroxidation marker, thiobarbituric acid (TBA) was employed. The tissue samples were homogenized in a solution of TCA and TBA, followed by heat-induced reaction to produce a colorimetric change, which was quantified spectrophotometrically at 540 nm. This MDA assay provided critical insights into oxidative stress levels in brain tissues following scopolamine-induced impairment and subsequent L-NAME treatment.
All chemicals and reagents were freshly prepared on the day of use to ensure stability and avoid degradation, thus maintaining the accuracy of each biochemical assessment. This rigorous selection and handling of chemicals and reagents underscore the reliability of the experimental outcomes, offering robust data to support the potential therapeutic effects of L-NAME in memory-related disorders.
SUBJECT MODEL AND SETUP
To evaluate the effectiveness of the proposed composition, a well-defined experimental model was established using Swiss albino mice, which are commonly employed in AD-related studies and a series of behavioral and biochemical assessments were conducted on the proposed model.
Swiss albino mice, each weighing between 30-35 grams were utilized for the model. The rats, sourced from the Institute of Pharmaceutical Research, GLA University, were housed under controlled conditions: 24±2°C temperature, 45-55% humidity, and a 12-hour light/dark cycle. Each rat was provided a standard pellet diet and water. The subject rats were allowed a 7-day acclimatization period before the start of the experiments. Ethical approval was obtained under the Institutional Animal Ethical Committee guidelines (IPRGLAU/CCSEA/IAEC/2024/M.PHARM-09) in compliance with the Committee for the Control and Supervision of Experiments on Animals (CCSEA) for preclinical studies.
EXPERIMENTAL PROTOCOL AND GROUP ASSIGNMENTS
The experimental design for the present invention involved the induction of memory impairment in Swiss albino mice using scopolamine as a selective muscarinic acetylcholine receptor antagonist. FIG. 3 illustrates an exemplary diagrammatic representation of experimental protocol of experimental validation of the present invention. The protocol was implemented to assess the neuroprotective and memory-enhancing effects of the test composition, which included L-NAME, in comparison to a standard drug (donepezil).
Induction of Memory Impairment/Alzheimer's Disease
To simulate memory impairment similar to Alzheimer's disease, scopolamine was administered to the mice through intraperitoneal injection (i.p.) at a dosage of 1 mg/kg. This treatment was carried out daily for a period of seven days, with scopolamine acting as a muscarinic acetylcholine receptor antagonist. The administration of scopolamine disrupts cholinergic signalling, leading to cognitive deficits associated with memory impairment in the test animals. This cholinergic deficit serves as a model for Alzheimer's disease and other neurodegenerative conditions characterized by impaired memory and learning ability.
Treatment Protocol
Starting from Day 7, following the scopolamine-induced cognitive impairment phase, the mice were divided into the following groups: (1) normal control, (2) scopolamine control, (3) L-NAME-treated group at 5 mg/kg, (4) L-NAME-treated group at 10 mg/kg, and (5) positive control receiving the standard drug, donepezil. The test composition (L-NAME) was administered intraperitoneally at dosages of 5 mg/kg and 10 mg/kg daily for a period of 28 days to the designated groups, while the positive control group received donepezil, a well-known acetylcholinesterase inhibitor used to alleviate symptoms of cognitive impairment. The normal control group was administered only the vehicle solution.
The subjects group classification is as follow:
• Normal Control: This group received no scopolamine or L-NAME treatment and served as a baseline for behavioral and biochemical assessments.
• Scopolamine Control: Rats in this group received scopolamine at 1 mg/kg i.p. daily for 7 days to induce memory impairment. Apart from this, no further treatment was administered to the subjects in this group.
• L-NAME Low Dose (Scopolamine + L-NAME 5 mg/kg): The rats in this group were administered with scopolamine at 1 mg/kg i.p. for 7 days to induce memory impairment. Following scopolamine treatment, subjects received L-NAME at 5 mg/kg i.p. for 28 days.
• L-NAME High Dose (Scopolamine + L-NAME 10 mg/kg): The subject rats received scopolamine at 1 mg/kg i.p. for 7 days to induce memory impairment. Following scopolamine treatment, received L-NAME at 10 mg/kg i.p. for 28 days.
• Standard Treatment (Scopolamine + Donepezil): Rats in this group were subjected to scopolamine at 1 mg/kg i.p. for 7 days to induce memory impairment. Following scopolamine treatment, the subjects received donepezil at 2 mg/kg i.p. for 28 days.
Behavioral Testing
Each group underwent a series of behavioral and biochemical assessments to evaluate the effectiveness of the treatment, including the Morris Water Maze and Y-maze tests.
• Y-Maze Testing (Day 23):
On the 23rd day of treatment, the mice underwent behavioral testing in the Y-maze. This maze tests spatial memory and spontaneous alternation behavior, which is considered a reliable indicator of short-term memory. The percentage of spontaneous alternations was recorded for each mouse, as this reflects the ability to remember and alternate between arms, assessing cognitive flexibility and working memory.
FIG. 5 discloses effect of L-NAME on scopolamine-induced memory impairment in mice using Y-maze. It was observed that the administration of scopolamine as in Fig. no.3 resulted in a significant (P< 0.001) decreased percentage alteration value (32.8±3.0) as compared to the normal group (63.5±6.3). Administration of Donepezil at the dose of 2mg/kg, has resulted in a significantly increased percentage alteration value (59.5±6.1) as compared to the scopolamine group. It was observed that administration of L-NAME resulted in a significant (P< 0.001) increased percentage alteration value (5mg/kg 42.83±6.3, for 10mg/kg 54.5±5.5) as compared to the scopolamine-treated group. It has shown effect similar to that of donepezil. The Alzheimer's induced group (negative control) indicated a decrease in the alternation of behavior. The results presented by the treatment groups was showed a significant by (P<0.001) increase in alteration of behaviour, in respect of 5mg/kg of ig and 40mg/kg of L NAME when compared with that of the disease control group.

• Morris Water Maze Testing (Days 23-27):
From Day 23 to Day 27, the mice were evaluated in the Morris Water Maze to assess spatial learning and memory retention. The MWM test is an indicator of spatial memory performance, requiring mice to locate a hidden platform within a circular pool of water. Memory performance was assessed based on parameters such as escape latency (time taken to find the platform) and the time spent in the target quadrant, which indicate the animals' ability to learn and retain the spatial location of the platform over time.
FIG. 4 discloses effect of L-NAME on scopolamine-induced memory impairment in mice using Morris water maze. (Escape latency Time in Seconds). It shows that when scopolamine was administered the experimental protocol disclosed in Fig. 3, it significantly increased the escape latency value of (50.5±1.99) compared to the normal group (15.3±3.36). However, when donepezil was given, it significantly decreased the escape latency value (18±3.91) compared to the scopolamine-treated group Similarly, administration of L NAME at the doses of 5mg/kg and 10mg/kg resulted in a significant decrease in escape latency value (for 5mg/kg 36.16±6.08 and for 10mg/kg 11±5.12 ) compared to the scopolamine treated group. The effects of L-NAME were found to be similar to that of Additionally, there was an increase in escape latency in the negative control group compared to the control group. However both groups treated with L-NAME 5mg/kg and 10mg/kg showed decreased time to escape onto the escape platform with significant values (P<0.001) These findings suggest that L-NAME may have memory-enhancing effects, potentially similar to the effects of donepezil.
Biochemical Analysis
Following the completion of behavioral testing on Day 28, the final dose of either the test composition or control drug was administered, after which all animals were humanely euthanized through cervical dislocation. This procedure was carried out in compliance with ethical guidelines and aimed to minimize stress in the animals prior to sample collection.
Post-euthanization, the brains were carefully harvested, and biochemical assays were conducted on brain homogenates to assess neurochemical and oxidative stress parameters associated with the neuroprotective effects of L-NAME and donepezil.
• Acetylcholinesterase (AChE) Activity:
Acetylcholinesterase levels were determined to evaluate the impact of L-NAME on cholinergic function. Reduction in AChE activity is indicative of enhanced cholinergic transmission, potentially reversing the cholinergic deficit caused by scopolamine administration.
Measurement of Acetylcholinesterase Enzyme Inhibitors (AChE)
To measure the acetylcholinesterase enzyme activity, brain samples from both experimental and control mice were prepared using a cold sodium phosphate buffer to preserve enzyme stability. The procedure was based on the classic Ellman et al. (1961) method:
• Sample Preparation: Brain tissues were homogenized in sodium phosphate buffer, and the homogenate was centrifuged to obtain a clear supernatant, which served as the enzyme source.
• Assay Preparation: The supernatant was diluted with DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) solution, which reacts with thiocholine to produce a yellow-colored compound.
• Test Setup: Two tubes were set up; eserine solution (an acetylcholinesterase inhibitor) was added to one as a blank control. Substrate solution (acetylthiocholine iodide) was then added to both tubes. The blank was used to zero-set the spectrophotometer.
• Measurement: The yellow color intensity, resulting from the reduction of DTNB, was measured at 430 nm. The AChE activity was quantified by comparing it with the standard enzyme activity curve.
This assay provides a measurement of AChE activity, which is a critical marker for cholinergic neurotransmission involved in memory and learning processes.
Further, FIG. 6 discloses effect of L-NAME on AchE activity catalase in scopolamine-induced memory impairment in mice. The analysis assessed the level of AChE in the whole brain homogenate of all animal groups to evaluate the nootropic activity Administration of scopolamine resulted in a significant increase in AChE values (0.82 plus/minus 0.1) compared to the normal group (2.34±0.04) However when donepezil was given, it significantly decreased AChE values (1.05±0.05) compared to the scopolamine-treated group Similarly, administration of L-NAME at the doses of 5mg/kg and 10mg/kg resulted in a significant decrease in AChE values (for 5mg/kg 1.74 ±0.02) for 10mg/kg (1.29±0.01)) compared to the scopolamine treated group Both donepezil and L-NAME demonstrated significant Neuroprotective activity and LNAME showed a synergistic effect with donepezil in treating scopolamine-induced Alzheimer's in mice. There was an increase in AChE activity in the disease control group compared to the control group with a significant difference (p<0.001) as shown in the figure 6. These findings indicate that L-NAME, like donepezil, have a beneficial effect on improving memory and cognitive function by modulating ACLE activity in the frame as shown in figure 6.


• Glutathione (GSH) Levels:
Levels of glutathione, a potent endogenous antioxidant, were measured to assess oxidative stress in the brain. An increase in GSH content in the L-NAME-treated groups would suggest enhanced antioxidative protection, mitigating neurodegenerative effects of oxidative stress.
Measurement of Glutathione (GSH)
The concentration of GSH, an essential antioxidant in brain tissues, was quantified using the Ellman method. This method measures the yellow chromophore generated by the reaction of GSH with DTNB.
• Sample Preparation: Brain tissue homogenate was prepared and deproteinized using 10% chloroacetic acid, followed by centrifugation at 2000 g for 10 minutes at 4°C to separate the proteins.
• Assay Procedure: The supernatant was used for GSH quantification. A mixture containing 2 ml of 0.1 M phosphate buffer (pH 7.4), 0.1 ml of the supernatant, 0.5 ml of DTNB, and 0.4 ml of double-distilled water was prepared.
• Measurement: The reaction mixture was vigorously vortexed, and the absorbance was read at 412 nm after a 15-minute incubation. The GSH concentration was calculated based on a standard curve and expressed as µg/mg protein.
This assay provides insight into the antioxidant status within the brain, as GSH plays a vital role in mitigating oxidative stress-a key factor in neurodegenerative diseases.
FIG. 7 discloses effect of L-NAME on the brain Glutathione reductase in scopolamine-induced memory impairment in mice. The study evaluated the level of glutathione reductase in the whole brain homogenate of all animal groups to assess the nootropic activity Administration of scopolamine resulted in a significant decrease in glutathione reductase values (1.43±0.05) compared to the normal group (4.08±0.3). However, when donepezil was given, it significantly increased the glutathione reductase value (2.83±0.2) compared to the scopolamine-treated group. On the other hand, administration of L-NAME at the doses of 5mg/kg and 10mg/kg resulted in a significant decrease in glutathione reductase values (for 5mg/kg (1.86±0.12), for 10mg/kg (2.36±0.8)) compared to the scopolamine-treated group. Both donepezil and L-NAME showed significant neuroprotective activity, and L-NAME displayed a synergistic effect with donepezil in treating scopolamine-induced Alzheimer's in mice There was an increase in glutathione reductase level in the disease control group compared to the control group, with a significant difference (p<0.001) as shown in the figure. These findings suggest that L-NAME, similar to donepezil, may influence glutathione reduction levels in the brain, contributing to its memory-enhancing effects and potential therapeutic role in Alzheimer's.
• Malondialdehyde (MDA) Concentration:
MDA levels were evaluated as a biomarker for lipid peroxidation, an indicator of oxidative damage in neural tissues. Lower MDA levels in the L-NAME-treated groups, compared to the scopolamine control group, would imply that the composition effectively attenuates lipid peroxidation, reducing neuronal damage.
Measurement of Malondialdehyde (MDA)
MDA levels, an indicator of lipid peroxidation and oxidative damage, were measured using the thiobarbituric acid reactive substances (TBARS) method. MDA reacts with thiobarbituric acid to produce a colored complex, which can be quantified spectrophotometrically.
• Sample Preparation: A 10% tissue homogenate was prepared, and 1.2 ml of the supernatant was used for the assay.
• Reaction with Thiobarbituric Acid: To the supernatant, 0.6 ml of 30% trichloroacetic acid (TCA) and 0.6 ml of 0.8% thiobarbituric acid (TBA) solution were added. The tubes were then sealed with aluminum foil.
• Incubation: The sealed tubes were incubated in a water bath at 90°C for 30 minutes to facilitate the reaction between MDA and TBA.
• Cooling and Centrifugation: After incubation, the tubes were rapidly cooled in ice-cold water for 30 minutes and centrifuged at 2500 rpm for 15 minutes.
• Measurement: The absorbance of the supernatant was read at 540 nm. A blank solution (distilled water, TCA, and TBA) was used as a reference. The concentration of MDA was calculated in nmol/mg protein.
This procedure quantifies lipid peroxidation levels, which indicate the extent of oxidative stress in brain tissue-a condition that is commonly associated with neurodegenerative changes in Alzheimer's disease.
Based on the experimental validation of the present invention, in a pharmaceutical composition formulated for potential neuroprotective effects in humans, N(gamma)-nitro-L-arginine methyl ester (L-NAME) may be included at a concentration of 1% to 15% by weight. This concentration range is carefully chosen to achieve an optimal therapeutic effect while minimizing the risk of adverse reactions. The inclusion of a stabilizing agent, such as hydroxypropyl methylcellulose (HPMC) or polyvinylpyrrolidone (PVP), at 0.1% to 5% by weight, helps to maintain the formulation's stability, ensuring that L-NAME retains its efficacy over a longer shelf life.
Further, to counter oxidative stress associated with neurodegeneration, an antioxidant, selected from glutathione or ascorbic acid, may incorporated at 0.1% to 10% by weight. This antioxidant concentration provides additional protection to neuronal cells by neutralizing free radicals that can exacerbate cognitive decline. Furthermore, a lipid peroxidation inhibitor, such as butylated hydroxytoluene (BHT) or vitamin E, may be included in the formulation at a concentration of 0.05% to 2% by weight. This component works synergistically with the antioxidant to prevent lipid membrane damage, a key factor in protecting brain cells from degenerative changes.
Finally, the composition of the present invention may be completed with a pharmaceutically acceptable carrier, ensuring that the ingredients are delivered safely and effectively when administered to humans. This combination of ingredients and their specific concentrations is designed to maximize neuroprotection, potentially offering a therapeutic option for memory-related disorders.

SYNERGISTIC EFFECTS
Synergistic effects of the present invention refer to the enhanced therapeutic outcomes achieved when the composition of the present invention is administered. The composition of the present invention is designed to interact at multiple biological pathways related to memory and neuroprotection, producing an effect greater than what would be achieved by any single component at the same dose. These synergistic effects are expected to manifest based on the experimental design and biochemical assessments.
Further, a synergistic approach with L-NAME as a core component, enhanced by other neuroprotective agents, provides a multifaceted treatment strategy. The present composition's ability to simultaneously address cholinergic dysfunction, oxidative stress, and lipid peroxidation-key contributors to neurodegeneration-holds therapeutic promise for treating cognitive disorders like Alzheimer's. By combining other agents that complement each other's effects, the composition could offer a more comprehensive and potent therapeutic benefit than conventional single-agent therapies.
This synergy is further supported by the experimental outcomes where combined treatment demonstrates improvements across multiple biochemical and behavioral measures, indicating that the formulation not only improves cognitive performance but also mitigates underlying neurodegenerative processes more effectively than any single component alone.
The embodiments described hereinabove are exemplary of the present invention. The disclosure may enable those skilled in the art to make and use embodiments having alternative elements that likewise correspond to the elements of the invention. The intended scope of the invention may thus include other embodiments that do not differ or that insubstantially differ from the literal language of the invention. However, the scope of the present invention is accordingly defined as set forth in the present complete specification.
, Claims:1. A neuroprotective composition for mitigating memory impairment, comprising:
N(gamma)-nitro-L-arginine methyl ester (L-NAME) in an amount ranging from 1% to 15% by weight,
a stabilizing agent selected from hydroxypropyl methylcellulose or polyvinylpyrrolidone in an amount ranging from 0.1% to 5% by weight,
an antioxidant agent selected from glutathione or ascorbic acid in an amount ranging from 0.1% to 10% by weight,
a lipid peroxidation inhibitor selected from butylated hydroxytoluene (BHT) or vitamin E in an amount ranging from 0.05% to 2% by weight, and
a pharmaceutically acceptable carrier.

2. The composition as claimed in claim 1, wherein the stabilizing agent is hydroxypropyl methylcellulose in an amount of 0.5% to 3% by weight.

3. The composition as claimed in claim 1, wherein the antioxidant agent is glutathione in an amount of 0.5% to 8% by weight.

4. The composition as claimed in claim 1, wherein the lipid peroxidation inhibitor is butylated hydroxytoluene (BHT) in an amount of 0.1% to 1% by weight.

5. The composition as claimed in claim 1, further comprising a solubilizer selected from polysorbate 80 or polyethylene glycol (PEG), in an amount of 0.1% to 5% by weight.

6. The composition as claimed in claim 1, wherein the pharmaceutically acceptable carrier includes a buffer system comprising sodium phosphate or potassium phosphate, maintaining a pH between 6.0 and 7.5.

7. The composition as claimed in claim 1, wherein the composition is formulated as a liquid solution, with L-NAME in a concentration of 5% to 10% by weight.
8. The composition as claimed in claim 1, wherein the composition is formulated as an injectable solution, suitable for intraperitoneal administration, with a carrier volume of 95% to 99% by weight.

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