DESC:TITLE OF THE INVENTION
A conjugate compound for regulation of MBP expression and novel in-situ process for preparation thereof.

FIELD OF THE INVENTION
The present invention relates to the conjugate compound for treating the disorders related to dysregulation of myelin basic protein (MBP) expression. Particularly, it pertains to the conjugate of magnesium with acetylated aminosulfonic acid which regulates the myelin basic protein (MBP) expression, thereby facilitating myelin sheath formation and maintenance.

Further, the present invention relates to novel in-situ process for preparation of the conjugate of magnesium with acetylated aminosulfonic acid with high purity and high yield. Particularly, the process comprising steps of in-situ catalytic acetylation of aminosulfonic acid followed by conjugation of magnesium in presence of base.

More particularly, the present invention relates to magnesium conjugate or conjugate of magnesium that easily penetrates the blood-brain barrier and effectively influence MBP expression which ensures targeted action, improve bioavailability, minimizes systemic side effects, and provides prolonged effect.

The present magnesium conjugate is useful in the treatment of disorders related to dysregulation of myelin basic protein (MBP) expression such as leukodystrophies, dementia, traumatic brain injury (TBI), adrenoleukodystrophy (ALD), Neuromyelitis Optica (NMO), schizophrenia, meningitis, age-related macular degeneration, multiple sclerosis (MS), Huntington's disease, Parkinson's disease (PD), bipolar disorder and hypomyelination disorder.

BACKGROUND OF THE INVENTION
Myelin Basic Protein (MBP) is a crucial protein in the central nervous system (CNS) involved in the formation and maintenance of the myelin sheath, which insulates axons and enhances the speed of electrical signal transmission. MBP maintains the correct structure of myelin, interacting with the lipids in the myelin membrane.

MBP belongs to a family of proteins that are highly conserved across species. MBP is rich in basic amino acids, giving it a positive charge, which is essential for its interaction with the negatively charged lipid components of the myelin membrane. MBP is critical for the compaction of the myelin sheath. It brings together the layers of the myelin membrane by binding to the lipid bilayers. It helps maintain the structural integrity of the myelin sheath, ensuring proper insulation of axons. By insulating axons, MBP aids in the rapid transmission of electrical signals in the nervous system. MBP plays major role in cell adhesion processes within the CNS, contributing to the stability of the myelin sheath.

MBP is a target in autoimmune reactions in MS, where the immune system attacks myelin, leading to demyelination and neurological impairment. Genetic mutations affecting MBP which leads to leukodystrophies, a group of disorders characterized by defective myelin formation. Abnormal MBP expression has been observed in various neurological disorders, suggesting a potential role in their pathogenesis. MBP levels in cerebrospinal fluid (CSF) can serve as a biomarker for demyelinating diseases like MS.

Ongoing research focuses on understanding the regulation of MBP expression, its role in myelin dynamics, and developing therapeutic approaches to modulate its function in neurological diseases. Understanding the regulation of MBP expression sheds light on the underlying mechanisms governing CNS myelination. MBP expression is regulated at multiple levels such as alternative promoter utilization, alternative splicing events, mRNA translocation, translational control, posttranslational modifications of the proteins. [Cell Biology and Pathology of Myelin. edited by Juurlink et at. Plenum Press. New Yark. 1997].

MBP has broader role in myelination beyond being a structural component. MBP may be more actively involved in myelination not only as a main building block but also as a self-regulating element [Evgeniya V. Smirnova et.al., Biomolecules 2021, 11, 1628].

As on date, MBP antibodies and peptides offer benefits in both diagnostic and therapeutic contexts. They are valuable tools in research, providing insights into myelin biology and disease mechanisms, and hold potential for developing targeted therapies for demyelinating diseases such as multiple sclerosis. MBP antibodies can help to detect the presence and levels of MBP in cerebrospinal fluid, aiding in the diagnosis and monitoring of MS. MBP peptides can modulate immune responses and have neuroprotective effects, promoting remyelination and repair of damaged nerves in animal models of demyelination.

WO2011046462 describes a method for diagnosing and treating multiple sclerosis by detecting and modulating MBP. It includes techniques for identifying specific MBP epitopes that may play a role in the disease's progression and potential therapeutic interventions targeting these epitopes.

WO2024130249 describes use of MBP peptides for the treatment of autoimmune neural diseases. It proposes that certain MBP peptides can modulate immune responses and may be useful in treating conditions like neuroinflammation-related disorder which includes multiple sclerosis.

However, the development of Myelin Basic Protein (MBP) antibodies and peptides for therapeutic purposes has faced several challenges and leads to limited success. The reasons such as immunogenicity [peptides or antibodies] can potentially exacerbate the autoimmune response rather than ameliorate it. The immune system might recognize these therapeutic peptides or antibodies as foreign, leading to an immune attack on myelin, worsening demyelination, potential toxicity [long-term administration of MBP antibodies or peptides can lead to toxicity or adverse effects, such as increased inflammation or unintended immune reactions against other CNS components] and technical and manufacturing issues including ensuring the stability of MBP peptides or antibodies during manufacturing, storage, and delivery is challenging. Unstable products can lose their efficacy or become immunogenic. Further developing and producing these therapies can be expensive, limiting their widespread use and accessibility.

The main reason for failure of MBP antibodies and peptides is blood brain barrier (BBB). The BBB is a major obstacle for delivering therapeutic agents to the central nervous system (CNS). Many MBP-targeting antibodies and peptides struggle to cross the BBB effectively, reducing their therapeutic efficacy. Ensuring that MBP peptides or antibodies reach the specific sites of demyelination without affecting other parts of the CNS is difficult. Non-specific targeting can lead to unintended side effects.

However, the BBB is highly selective, allowing only certain molecules to pass through. Many potential therapeutics may be unable to cross the BBB due to their size, polarity, or other chemical properties. The blood-brain barrier (BBB) separates the circulating blood from the brain and extracellular fluid in the central nervous system (CNS). It is crucial for maintaining the brain's microenvironment, which is essential for proper neuronal function. The BBB is formed by endothelial cells of the brain's capillaries, which are tightly joined together with tight junctions, along with astrocytic end-feet and pericytes.

Therefore, the penetration of the BBB is important for the regulation of the Myelin Basic Protein (MBP). Effective regulation of the MBP requires the delivery of therapeutic agents directly to the oligodendrocytes and other cells in the CNS. Therapeutic agents must reach the CNS and influence MBP expression directly in oligodendrocytes. The therapeutic agents that can cross the BBB can help create a more favourable environment for myelination and MBP regulation. By ensuring that therapeutic agents cross the BBB, the treatment can be localized to the CNS, reducing the risk of systemic side effects that might occur if the agents acted peripherally. Effective penetration of the BBB can allow for lower dosages of therapeutics, as the agents will be delivered directly to the site of action.

To overcome the challenge of BBB penetration, new strategy can be employed such as designing therapeutic agent that can passively diffuse through the BBB. Current therapeutic approaches are limited in efficacy and often associated with significant side effects. Therefore, there is a need for novel therapeutic agents that can easily penetrate BBB and effectively regulate MBP expression and promote remyelination.

Interestingly, the inventors of present invention have developed novel conjugate compound which can easily penetrate blood brain barrier (BBB) to influence MBP expression and it is generally safe for administration with improved bioavailability and reduced side effects.

OBJECTIVE OF THE INVENTION
The primary object of the invention is to provide a novel therapeutical approach for the disorder related to dysregulation of myelin basic protein (MBP) expression.

Yet another object of the invention is to provide therapeutically active conjugate which ensures targeted action and improve bioavailability.

Yet another object of the invention is to provide a conjugate which minimizes systemic side effects, and provides prolonged effect.

Another object of the invention is to provide cost-effective, industrially viable process for the preparation of the conjugate.

Another object of the invention is to provide present conjugate based medicinal composition for enhancing neurological health.

SUMMARY OF THE INVENTION
To meet the above objects, the inventors of the instant invention carried out thorough experiments to establish significant effects of the conjugate compound that ameliorate therapeutic efficacy in the treatment of neurological disorders and disabilities by regulation of myelin basic protein (MBP) expression.

In another aspect, the present invention provides a conjugate of magnesium with acetylated aminosulfonic acid which is prepared by commercially viable and non-hazardous process.
In an aspect, the present invention provides a conjugate compound which promote the formation and maintenance of the myelin sheath.

In another aspect, the invention provides a conjugate compound with potent and targeted approach to promote myelination and repair demyelinated neurons.

In yet another aspect, the invention provides a conjugate compound which easily penetrates the blood-brain barrier and effectively influence MBP expression and ensures targeted action with prolonged effect.

In another aspect, the invention provides therapeutically effective, non-toxic and safe conjugate compound with no major side effects.

In a further aspect, the present invention provides pharmaceutical composition comprising conjugate compound along with pharmaceutically acceptable excipients/carriers useful for treating disorders related to dysregulation of myelin basic protein (MBP) expression such as leukodystrophies, dementia, traumatic brain injury (TBI), adrenoleukodystrophy (ALD), Neuromyelitis Optica (NMO), schizophrenia, meningitis, age-related macular degeneration, multiple sclerosis (MS), Huntington's disease, Parkinson's disease (PD), bipolar disorder and hypomyelination disorder.

Abbreviations:
MBP: Myelin basic protein
TBI: Traumatic brain injury
ALD: Adrenoleukodystrophy
NMO: Neuromyelitis Optica
PD: Parkinson's disease
MS: Multiple sclerosis
BBB: Blood brain barrier
CNS: Central nervous system
CSF: Cerebrospinal fluid
SCO: Scopolamine

BRIEF DESCRIPTION OF FIGURES
Figure1: illustrates MPB expression of Brain Homogenate of Rats for magnesium salt i.e., MgSO4, MgOH, Mg Taurate, Taurine, Mg Conjugate with acetylated 2-aminoethanesulfonic acid.
DESCRIPTION OF THE INVENTION
It is further to be understood that all terminology used herein is for the purpose of describing particular embodiment only and is not intended to be limiting in any manner or scope. Unless
defined otherwise, all technical and scientific expressions used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention pertain.

In describing and claiming the embodiments of the present invention, the following terminology will be used in accordance with the definitions set out below which are known in the state of art.

The singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Also, the term ‘composition’ does not limit the scope of the invention for multiple compositions that can be illustrated for best mode of the invention.

The term “pharmaceutically/nutraceutically acceptable salt,” as used herein, represents those salts which are within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit/risk ratio.

Particularly the term “pharmaceutically-acceptable salts” refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds, amino acid salt, sugar-based salt, alkali or alkaline earth metal salts, as well as solvates, co-crystals, polymorphs and the like of the salts.

All modifications and substitutions that come within the meaning of the description and the range of their legal equivalents are to be embraced within their scope. A description using the transition “comprising” allows the inclusion of other elements to be within the scope of the invention.

The term "conjugate" in this context refer to "conjugate of magnesium”. A conjugate refers to a compound formed by the joining of two or more chemical compounds. Particularly it refers to type of magnesium-containing species, especially in coordination compounds or biological molecules.

The inventors of the present invention have developed therapeutically significant novel conjugate of magnesium with acetylated aminosulfonic acid in stable form.

In a preferred embodiment, the present invention provides the conjugate compound of acetylated aminosulfonic acid and magnesium for disorder related to dysregulation of myelin basic protein (MBP) expression.

In another embodiment, the present invention provides cost-effective, industrially viable process for the preparation of conjugate compound of magnesium with acetylated aminosulfonic acid compounds.
In one preferred embodiment, the present invention provides novel in-situ process for preparation of conjugate of magnesium with acetylated aminosulfonic acid, comprising steps of;
a) catalytic acetylation of aminosulfonic acid compounds in presence of a catalyst and solvents to obtain acetyl aminosulfonic acid compounds,
b) conjugation of acetyl aminosulfonic acid compounds with magnesium salts in presence of base to obtain the conjugate of magnesium with high yield and purity.

In another embodiment, the present invention provides cost-effective, viable process for the preparation of conjugate of magnesium with acetylated aminosulfonic acid compounds.

In another preferred embodiment, the invention provides feasible process for the preparation of stable conjugate compound, the process comprising steps of; catalytic acetylation of aminosulfonic acid compounds followed by conjugation with magnesium.

In another embodiment, the aminosulfonic acid compound is selected from the group consisting of sulfamic acid, m-aminobenzenesulfonic acid, sulfinic acid, p-aminobenzenesulfonic acid, 2-aminoethanesulfonic acid, a-aminomethanesulfonic acid and like thereof.

In yet another embodiment, the magnesium salt is selected from the group consisting of in magnesium acetate, magnesium hydroxide, magnesium chloride, magnesium sulfate, magnesium lactate and the magnesium nitrate.

In yet another embodiment, wherein the solvents used in the reaction are green solvents selected from the group consisting of water, ethanol, methanol, propanol, ethyl acetate, 2- propanol, butanol or combination thereof.

In some embodiment, the invention provides acetylation of aminosulfonic acid, wherein the substrate and acetylating agent is present in the molar ratio ranges from 1: 0.5 to 1:2.

In another embodiment, the invention provides catalytic acetylation in presence of the combination of ethyl acetate with DMAP which offers selective acetylation of aminosulfonic acid compounds.

Ethyl acetate is generally safer to handle. It has a lower toxicity compared to other acetylating agents such as acetic anhydride. Other acetylating agents such as acetic anhydride, acetic acid are quite hazardous. They are corrosive and can cause severe burns upon contact with skin or eyes, and its vapours are irritating to the respiratory tract.

Ethyl acetate is also more environmentally friendly. It is biodegradable and less harmful to the environment. It has a lower boiling point (77°C), making it easier to remove from reaction mixtures by simple distillation. More particularly, ethyl acetate generally less expensive and more readily available.

In another preferred embodiment the invention provides feasible process for the preparation of stable conjugate compound, the process comprising first step of catalytic acetylation of aminosulfonic acid compounds using 4-Dimethylaminopyridine (DMAP) as a catalyst, wherein the catalyst 4-dimethylaminopyridine is present in 1 to 10% molar equivalent to substrate.

DMAP is an efficient catalyst that can significantly speed up the reaction and increase the yield by activating the ester toward nucleophilic attack.

Particularly the present process is carried out under milder conditions, which is beneficial for sensitive substrates like aminosulfonic acid. The acetylation required low catalyst loading where small amount of catalyst is used.

Further the DMAP catalyst is recyclable catalyst which can be recovered by simple filtration and reused several times.

In another preferred embodiment the invention provides in-situ, process for the preparation of stable conjugate compound, the process comprising second step of conjugation of acetylated aminosulfonic acid compounds with magnesium compound.

In another embodiment, the conjugation of magnesium salt with acetylated aminosulfonic acid compounds carried out at temperature ranged from 25°C– 35°C. The reaction is neutralized in presence of base like NaOH, trimethyl amine, KOH, ammonia, ethylamine and like thereof.

In one more embodiment, the invention provides novel in-situ process for preparation of conjugate of magnesium with acetylated 2-aminoethanesulfonic acid with high purity.
comprising steps of;
a) acetylation of 2-aminoethanesulfonic acid in presence of 4-5 % molar equivalent of 4 dimethylaminopyridine at temperature 60-80°C to obtain acetylated 2-aminoethanesulfonic acid;
b) in situ conjugation of magnesium chloride to the acetylated 2-aminoethanesulfonic acid with stirring in presence of triethylamine followed by crystallization to obtain conjugate of magnesium with acetylated 2-aminoethanesulfonic acid with high purity.

In another embodiment, the present invention provides novel conjugate of magnesium with acetylated aminosulfonic acid compounds that facilitate myelin sheath formation and maintenance in a subject in need thereof, wherein the subject is human.

In another embodiment the present novel in-situ process affords the final magnesium conjugate product or conjugate of magnesium, with the purity in the range of 90 to 99% and w/w yield in the range of 75%-95%.

In another embodiment, the present invention provides novel conjugate compound of magnesium with acetylated aminosulfonic acid compounds that easily penetrates the blood-brain barrier and effectively influence MBP expression which ensures targeted action, improve bioavailability, minimizes systemic side effects, and provides prolonged effect.

Further the present novel conjugate compounds are useful in the treatment of disorders related to dysregulation of myelin basic protein (MBP) expression such as leukodystrophies, dementia, traumatic brain injury (TBI), adrenoleukodystrophy (ALD), Neuromyelitis Optica (NMO), schizophrenia, meningitis, age-related macular degeneration, multiple sclerosis (MS), Huntington's disease, Parkinson's disease (PD), bipolar disorder and hypomyelination disorder.

The term ‘subject in need thereof’ pertains to a subject preferably mammal, more preferably a human suffering or suspected with nervous system related disorder.

In the context of the present invention, the term “treatment” refers to alleviate, mitigate, prophylaxis, attenuate, manage, regulate, modulate, control, minimize, lessen, decrease, down regulate, up regulate, moderate, inhibit, restore, suppress, limit, block, decrease, prevent, inhibit, stabilize, ameliorate, cure, heal metabolic or nervous system related disorders observed in the patient.

Notably, the present novel conjugate moieties are non-hazardous, non-toxic, and safe for human consumption without any severe adverse effects, therefore the present medicinal composition can also be used as preventive therapy, adjuvant therapy, add-on therapy, combination, adjunctive therapy in a subject in need thereof.

In some embodiment, the present conjugate compounds or pharmaceutically acceptable salts thereof, are formulated for medicaments, which preferably take the form of therapeutically effective individual doses adjusted to the form of administration.

Certain compounds of the present invention exist in unsolvated forms as well as solvated forms, including hydrated forms. Further, some compounds of the present invention exist in multiple crystalline or amorphous forms (“polymorphs”). Compounds of the invention are formulated in geometric or, enantiomeric or stereoisomeric forms.

In general, all physical forms are of use in the methods contemplated by the present invention and are intended to be within the scope of the invention.

Compound or pharmaceutically acceptable salts includes, hydrates, polymorphs, solvates, enantiomers or racemates. Some of the crystalline forms of the compound exist as polymorphs and as such are intended to be included in the present disclosure. In addition, some of the compounds may form solvates with water (i.e., hydrates) or common organic solvents, and such solvates are intended to be encompassed by some embodiments.

In one of the embodiments, the present invention provides medicinal composition comprising novel conjugate of magnesium with acetylated aminosulfonic acid which is present in therapeutically effective amount along with pharmaceutically acceptable excipients.

As used herein, the term “pharmaceutically acceptable carriers, diluents or excipients” is purported to mean, without limitation, any adjuvant, carrier, excipient, sweetening agent, diluents, preservative, dye/colorant, flavour enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, emulsifier, or encapsulating agent, encapsulating polymeric delivery systems or polyethylene glycol matrix, which is acceptable for use in the subject, preferably humans. Excipients also include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, fragrances, glidants (flow enhancers), lubricants, preservatives, sorbents, suspending or dispersing agents, sweeteners, surfactant, anticaking agent, food additives, or waters of hydration, salts.

In another embodiment, the present invention relates to medicinal composition of the conjugate of magnesium with acetylated aminosulfonic acid and prepared in a manner well known in the pharmaceutical art and administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated.

The preferable route of administration includes but is not limited to sublingual, rectal, topical,
parenteral, nasal, or oral.

In some embodiment, the medicinal compositions of the conjugate of acetylated aminosulfonic acid and magnesium are administered to a subject in need thereof, in the form which is suitable for oral use, such as a tablet, capsule (in the form of delayed release, extended release, sustained release, enteric coated release); hard gelatin capsules, soft gelatin capsules in an oily vehicle, veg capsule, hard or soft cellulose capsule, granulate for sublingual use, effervescent or carbon tablets, aqueous or oily solution, suspension or emulsion, encapsulate, matrix, coat, beadlets, nanoparticles, caplet, granule, particulate, agglomerate, spansule, chewable tablet, lozenge, troche, solution, suspension, rapidly dissolving film, elixir, gel, tablets, pellets, granules, capsules, lozenges, aqueous or oily solutions, suspensions, emulsions, sprays or reconstituted dry powdered form with a liquid medium or syrup; for topical use including transmucosal and transdermal use, such as a cream, ointment, gel, aqueous or oil solution or suspension, salve, parch or plaster; for nasal use, such as a snuff nasal spray or nasal drops; for vaginal or rectal use, such as a suppository; for administration by inhalation, such as a finely divided powder or a liquid aerosol; for sub-lingual or buccal use, such as a tablet, capsule, film, spray.

In a further embodiment, the present conjugate can be useful for medicinal composition which can be formulated in the form of age-appropriate pediatric oral dosage forms such as syrup, inhalation, spray minitablets, chewable formulations, orodispersible films and orodispersible tablets.
The magnitude of a prophylactic or therapeutic dose typically varies with the nature and severity of the condition to be treated and the route of administration. The dose, and perhaps the dose frequency, will also vary according to the age, body weight and response of the individual patient.

In general, the total daily dose (in single or divided doses) ranges from about 0.1 mg per day to about 5000 mg per day, preferably about 1mg per day to about 1000 mg per day.

In additional embodiment, the present conjugate of magnesium with acetylated aminosulfonic acid obtained from the novel in situ process is formulated in oral dosage form with effective dose ranges from 0.1 mg to 3000 mg; preferably 1 mg to 1000 mg.

In another embodiment, the conjugate compounds of the present invention are non-toxic, cost effective, enriched with nutrients or biomolecules and provide safeguard against problems associated with neurodevelopment without any adverse effect.

In some embodiments, the present magnesium conjugate compounds or conjugates of magnesium are useful for the formulation of medicaments by using pharmaceutically acceptable excipients such as diluents, binders, lubricants, solubilizing agents, surfactants, stabilizers, colors, flavoring agents, sweeteners, glidants, plasticizers, and other additives.

In yet another embodiment, the invention provides a medicinal composition comprising present magnesium conjugate compounds along with pharmaceutical excipients, wherein the pharmaceutical excipients are selected from a diluent, a binder, a lubricant, a glidant, an additive, a surfactant, a stabilizer or mixtures thereof.

In another embodiment, the present invention provides a method for treating neurological disorders in a subject in need thereof. The method comprises administering an oral dose of a therapeutically effective amount of a medicinal composition comprising present magnesium conjugate compounds with pharmaceutically acceptable excipients.

It is further recommended that children, patients over 60 years old, initially receive low doses and that the dosage be titrated based on individual physiological responses and/or pharmacokinetics. It can be necessary to use dosages outside these ranges in some cases, as will be apparent to those in the art. The present composition can be used as infant formula as well as adult formula by varying the concentration of active ingredients. Further, it is noted that the dietician or nutritionist or certified physician knows how and when to interrupt, adjust or terminate therapy in conjunction with an individual patient's response.

The use of any and all examples, or exemplary language (e.g., such as) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

While in the foregoing specification this invention has been described in relation to certain embodiments thereof, and many details have been put forth for the purpose of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein can be varied considerably without departing from the basic principles of the invention.

The present invention is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the invention.
The invention may be further illustrated by the following examples, which are for illustrative purposes only and should not be construed as limiting the scope of the invention in anyway.

The present disclosure is therefore to be considered as in all respects illustrative and not restrictive, the scope of the invention being indicated by the appended claims and examples, and all changes or alterations which come within the ambit of equivalency are intended to be encompassed therein.

Examples:
Having described the basic aspects of the present invention, the following non-limiting examples illustrate specific embodiments thereof. Those skilled in the art will appreciate that many modifications may be made in the invention without changing the essence of invention.

Example 1: In-situ process for preparation of conjugate of magnesium with acetylated aminosulfonic acid group.
Step 1: Acetylation of 2-aminoethanesulfonic acid in the presence of 4-Dimethylaminopyridine (DMAP)
For the acetylation of 2-aminoethanesulfonic acid, 10 g (0.11 mol) of 2-aminoethanesulfonic acid weighted and dissolved in 100 mL of ethanol measured with a graduated cylinder. Complete dissolution was ensured by using an IKA magnetic stirrer. Then, 11.2 g (0.12 mol) of ethyl acetate and 1 g of DMAP was added to the solution. The reaction mixture was placed on hot plate with a magnetic stirrer and heat it to 70°C, monitored the temperature with a thermometer. The mixture was stirred for 4 hours to ensure complete acetylation.
H2N-CH2-CH2-SO3H + CH3COOCH2CH3 CH3-CO-HN-CH2-CH2-SO3H + CH3CH2OH
Step 2: In situ conjugation of magnesium with acetylated 2-aminoethanesulfonic acid
Once the acetylation reaction was completed, the reaction mixture was removed from the hot plate and allowed to cool to room temperature. Slowly added 9.5 g (0.1 mol) of magnesium chloride to the reaction mixture while stirring. To neutralize any acidic by-products, 10 mL of triethylamine added and stirred the reaction mixture for another 1-2 hours at room temperature.
2[CH3-CO-HN-CH2-CH2-SO3H] + MgCl2 Mg(CH3-CO-HN-CH2-CH2-SO3)2+ 2HCl
Purification, Yield and Purity:
The reaction mixture was filtered through vacuum filtration apparatus and remove any insoluble impurities. A hot filtration setup was used to prevent premature crystallization. This can be done using a Büchner funnel and filter paper or a sintered glass filter. The filtered solution was allowed to cool slowly to room temperature, and then placed it in an ice bath to promote crystallization of the conjugate compound. Once crystals have formed, filtered them out using vacuum filtration. The crystals were washed with a small amount of cold solvent like ethyl acetate to remove any adhering impurities. The purified magnesium conjugate crystals were dried under vacuum or in a drying oven at a suitable temperature (e.g., 50-60°C) to remove any residual solvent. The final product is formed by the acetylation of 2-aminoethanesulfonic acid and subsequent conjugation with magnesium. The purity of the purified magnesium conjugate with acetyl 2-aminoethanesulfonic acid verified using analytical techniques such as NMR spectroscopy, HPLC, or melting point determination. (Yield -88% and Purity 97%w/w)
1H NMR (in DMSO-d6) : d ~ 2.2 ppm (singlet, 3H, -CH3 group) ; d ~ 8.5 ppm (multiplet, NH); d ~ 3.0 ppm (triplet, CH2-SO3): d ~ 3.5 ppm (triplet, CH2-NH).
13C NMR (in DMSO-d6): d ~ 170 ppm (C=O); d ~25 ppm (CH3): d ~ 55 ppm (C-SO3); d ~ 45 ppm (CH-NH).
Molecular Formula: C8H16MgN2O8S2, Molecular Weight : 356.7 g/mol.

Example 2: Animal Study
“To measure the concentration of Myelin Basic Protein (MBP) in brain tissue of Swiss Albino Rats using ELISA”
Studying Myelin Basic Protein (MBP) levels in rats treated with scopolamine, a muscarinic receptor antagonist known to induce cognitive deficits and impairments in learning and memory, can provide insights into the effects of cholinergic dysfunction on myelination processes.

Test system details:
Species: Rat
Strain: Wistar; Sex: Male / Female
No. of animals: 42 Animals (n=6 per group)
Body weight : 180-200gm
Animal House conditions:
Lighting: 12 / 12 hour light-dark cycle
Temperature: 22 ± 3 °C
Relative Humidity: 30 to 70%
Feed: Normal chow diet
Drinking water: Rats were provided with ad libitum drinking water passed through water filter
system in autoclaved poly propylene bottles.

Housing conditions:
Three rats /cage were housed in autoclaved polypropylene cages using autoclaved paddy husk as the bedding material. Each cage was fitted with top grill having provision for keeping rodent feed and water bottle.

Group, Designation and Dose Levels:
Table 1: Animal grouping and treatment details

Groups Dose No. of Animals
Normal Control Vehicle (PBS) 6
Disease Induced Scopolamine 6
MgSO4 250mg 6
MgOH 250mg 6
Mg Taurate 250mg 6
Taurine 250mg 6
Conjugate 250mg 6

Study Procedure:
Administration of scopolamine:
Administration of scopolamine via intraperitoneal (i.p.) injection. The systemic scopolamine treatment induces cognitive impairment along with a significant decrease of myelin basic protein (MBP) expression in the rat brain parts.

Scopolamine is a non-selective postsynaptic muscarinic receptor blocker and can cause myelin sheath impairments in rodents and humans via decreasing the effectiveness of MBP in animals and humans CNS.

Administration and Procedure -
All the animals were acclimatized for at least 7 days under controlled environmental condition. Total 42 animals were divided into seven groups consisting of 6 animals per group.

Normal control group receiving vehicle, disease control group receiving scopolamine intraperitoneal (i.p.) injection only, and treatment groups MgSO4, MgOH, Mg Taurate, Taurine, Mg Conjugate were treated with the test substance followed by the scopolamine injection.

Rats were intraperitoneally injected with 1 mg/kg of scopolamine (SCO), once daily, for 1, 2, 3 and 4 weeks (n = 36 at each point in time). The control rats (n = 6 at each point in time) were injected with the same volume of saline (pH 7.4). The rats were sacrificed after 4 weeks SCO or saline treatment.

After sacrification of animals, whole fresh brains were removed and weighed and further processed in PBS (Phosphate buffer saline) pH- 7.4. The brain parts cortex, cerebellum, striatum, cortex, midbrain were carefully isolated with the help of sharp blade and microspatula.

ELISA for MBP:
Sample Preparation and Analysis:
After successful isolation of all brain parts, all parts were immersed in 2-5 ml of PBS buffer solution and homogenized for 5 to 10 minutes with 5000 rpm by tissue homogenizer machine and allowed for centrifugation for 15 min at 8000 to 10,000 rpm. The resultant supernatant was collected and preserved at cold temperature in deep freeze. 100 microlitres of sample were added to the particular wells and incubation for 90 min at 37°C. Further 100 microlitres of anti-MBP antibody working solution was added to each well and incubated again for 1 hour at 37°C in the incubator. After the incubation completion washed the plate by washing buffer solution. 100 microlitres of horseradish peroxidase (HRP) conjugate working solution was added to the plate and incubated for 30 min at 37°C incubator then 90 microlitres of a substrate tetramethylbenzidine (TMB) reagent was added that reacted with the enzyme and incubated, subsequently 50 microlitres of Stop solution was added to stop the enzymatic reaction. The blue colour was converted to yellow colour just after the addition of Stop solution which indicates that the reaction has been stopped. The reading of the plate in ELISA plate reader at 450 nm and results were displayed with graph plots in units measurement (pg/mg protein).

The MBP concentration in each sample (expressed in picograms per milligram of total protein) was determined and plotted in bar charts. All results of the statistical analysis were summarized in separate tables.

Statistical analysis
The values were expressed in Mean±sem. The significance of in vivo data was analysed by one-way Anova followed by Dunnet test. P < 0.001 was considered as significant.

Results:
Changes in MBP levels was observed in the different parts of brain once treated with different salts of magnesium after chronic systemic treatment with 1 mg/kg SCO. MBP level at 1 week after SCO treatment was not significantly different compared to the control group. MBP level was increased after treatment with Magnesium salt.

Among the other magnesium salt present conjugate of magnesium with acetylated aminosulfonic acid showed significant results.
Table 1: The MBP expression in pg/mg in brain homogenate
Brain Parts Normal Diseased MgSO4 MgOH MgT Taurine Conjugate
Cortex 2.33 0.71 1.35 1.23 1.44 0.94 2.95
Hippocampus 1.23 0.5 0.92 1.01 1.03 0.66 2.02
Striatum 0.92 0.33 0.94 1.09 1.14 0.45 1.83
Mid brain 1.83 0.76 1.13 1.26 1.34 0.86 2.82
Cerebellum 1.14 0.42 0.77 0.83 0.96 0.63 1.86

Result Outcome Discussion:
Rodent models of MBP deficits have been established using SCO-induced amnesia. In the CNS, myelin is responsible for protection and insulation of axons as well as proper communication between neurons. Thus, the loss of myelin has marked negative effects on axonal function and neuronal connections. Many recent studies have demonstrated that myelin may play a crucial role in cognitive function, likely because of its involvement in information processing; it maintains connectivity between neuronal circuits and regulates conduction velocity.

In this study, it was first examined chronological changes in myelin in the rat brain after chronic systemic treatment with SCO using MBP immunohistochemistry. The density of MBP immunoreactive myelinated fibers was significantly decreased with SCO tretment. It is well known that the degradation of myelin is a key feature in the pathogenesis of neurological disorders such as stroke, Alzheimer’s disease, and Huntington’s disease, inducing cognitive dysfunction

After treatment with different magnesium salts like magnesium sulphate, magnesium hydroxide, magnesium taurate, taurine and the present magnesium conjugate. All magnesium salt except taurine showed certain action on different parts of brain that in turn ameliorate MBP expression, however among all salts present magnesium conjugate expressed significant results with substantial increase in MBP level, which was almost two times better that the known magnesium salts.

It is concluded that, the present conjugate of magnesium reduces hypomyelination, effectively prevents degradation of myelin. The MBP expression in brain homogenate shows significant increase in MBP level ranges from 1.8 to 2.9 pg/mg.
,CLAIMS:1. A novel in-situ process for preparation of conjugate of magnesium with acetylated aminosulfonic acid, the process comprising steps of;
a) catalytic acetylation of aminosulfonic acid compounds in presence of a catalyst and solvents to obtain acetyl aminosulfonic acid compounds;
b) conjugation of acetyl aminosulfonic acid compounds with magnesium salts in presence of base to obtain the conjugate of magnesium with high yield and purity.

2. The novel in-situ process as claimed in claim 1, wherein the aminosulfonic acid is selected from the group consisting of sulfamic acid, m-aminobenzenesulfonic acid, sulfinic acid, p-aminobenzenesulfonic acid, 2-aminoethanesulfonic acid, and a-aminomethanesulfonic acid.

3. The novel in-situ process as claimed in claim 1, wherein the magnesium salt is selected from the group consisting of magnesium acetate, magnesium hydroxide, magnesium chloride, magnesium sulfate, magnesium lactate, and magnesium nitrate.

4. The novel in-situ process as claimed in claim 1, wherein the catalyst is 4-dimethylaminopyridine and present in 1 to 10% molar equivalent.

5. The novel in-situ process as claimed in claim 1, wherein the solvents used in the reaction are green solvents selected from the group consisting of water, ethanol, methanol, propanol, ethyl acetate, 2- propanol, butanol or combination thereof.

6. The novel in-situ process as claimed in claim 1, wherein the purity of conjugate of magnesium is in the range of 90 to 99% and w/w yield is in the range of 75%-95%.

7. The novel in-situ process as claimed in claim 1, comprising steps of;
a) acetylation of 2-aminoethanesulfonic acid in presence of 4-5 % molar equivalent of 4-dimethylaminopyridine at temperature 60-80°C to obtain acetylated 2-aminoethanesulfonic acid;
b) in situ conjugation of magnesium chloride to the acetylated 2-aminoethanesulfonic acid with stirring in presence of triethylamine followed by crystallization to obtain conjugate of magnesium with acetylated 2-aminoethanesulfonic acid with high purity.

8. The conjugate of magnesium with acetylated aminosulfonic acid obtained from the process as claimed in claim 1, wherein the conjugate regulates the myelin basic protein (MBP) expression in a subject in need thereof, wherein the subject is human.

9. The conjugate of magnesium with acetylated aminosulfonic acid obtained from the process as claimed in claim 1, wherein the conjugate is useful in the treatment of disorders related to dysregulation of myelin basic protein (MBP) expression such as leukodystrophies, dementia, traumatic brain injury (TBI), adrenoleukodystrophy (ALD), Neuromyelitis Optica (NMO), schizophrenia, meningitis, age-related macular degeneration, multiple sclerosis (MS), Huntington's disease, Parkinson's disease (PD), bipolar disorder and hypomyelination disorder.

10. The conjugate of magnesium with acetylated aminosulfonic acid obtained from the process as claimed in claim 1, is formulated in oral dosage form with effective dose ranges from 1 mg to 1000 mg.