DESC:Form 2

THE PATENT ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)

“HYDROCOLLOID COMPOSITION MIMICKING MUCUS- A PHARMACEUTICAL BASE FOR OTHER THERAPEUTIC AGENTS”

in the name of PONDICHERRY UNIVERSITY an Indian National having address at, R. VENKATARAMAN NAGAR, KALAPET, PUDUCHERRY, PUDUCHERRY– 605014, PUDUCHERRY, INDIA.

The following specification particularly describes the invention and the manner in which it is to be performed.

FIELD OF THE INVENTION:

The present invention relates to the field of pharmaceutical biotechnology. Specifically, the present invention relates to a composition similar to mucus. More particularly the present invention relates to a hydrocolloid composition mimicking mucus, a pharmaceutical base for therapeutic agents and process thereof.

BACKGROUND OF THE INVENTION:

The human body is a complex system, and its intricate design often inspires innovations in various scientific fields, including medicine. One such area of exploration involves the development of a novel composition that mimics natural substances in the human body for the delivery of therapeutic agents. In recent years, researchers have turned their attention to mucus – a viscous, gel-like hydrocolloid that lines the mucosal surfaces of various organs, including the respiratory, gastrointestinal, and reproductive tracts. Mucus is not merely a passive barrier; it plays a crucial role in protecting and maintaining the health of these mucosal surfaces. It acts as a physical barrier, preventing pathogens and foreign particles from entering the body, while also serving as a habitat for beneficial bacteria. Furthermore, mucus is a dynamic environment that responds to changes in the body, such as infection or inflammation. The unique properties of mucus, such as its viscoelasticity, adhesive nature, and responsiveness to stimuli, make it an attractive candidate for the design of drug delivery systems. Scientists are exploring the possibility of creating synthetic compositions that mimic the characteristics of mucus to enhance the targeted delivery of therapeutic agents to specific tissues or organs. This innovative approach could revolutionize drug delivery, improving the efficacy and reducing side effects of various treatments. The potential applications of a mucus-mimicking drug delivery system are vast. For example, respiratory diseases such as asthma or chronic obstructive pulmonary disease (COPD) could benefit from inhalable medications designed to adhere to the mucus lining the airways. Similarly, gastrointestinal disorders may be targeted more effectively through oral formulations that interact intelligently with the mucus lining of the digestive tract.

There are some compositions available in the prior art related to mucus-mimicking composition.

US10736854B2 describes about nanocrystals, compositions that aid particle transport through mucus. Various polymers like PEG, HPMC, PVP etc. were used in minimal quantity. Various surfactants viz. Tween 20, tween 80, triton X, span 20, span 80 etc. were screened for novel composition. Synthetic and natural antimicrobial agents were incorporated and validated for its application through mucus of various organs.

KR101811917B1 is a patent on a polymeric nanoparticle, wherein the nanoparticle comprises of a core polymer and a surface-permeable reinforcing coating on the surface, wherein the nanoparticle comprises a therapeutic, prophylactic, diagnostic or nutritional supplement for administration to mucosal tissues.

JP2007537169A5 explains about a medium for oral administration of one or more active substances, comprising a hydrophilic agent selected from the group consisting of electrolytes, organic acids and osmotic agents, and mixtures thereof that improves the swelling of gellan gum.

ES2517244T3 is a patent about inflatable pharmaceutical form comprising gellan gum. The differentiated unit in the form of a tablet, a capsule, a seal or an envelope for oral administration of one or more active ingredients, the differentiated unit comprising a vehicle comprising i) a gellan gum having an acetylation degree of a glycerate by repetition and one acetate for every two repetitions, and ii) a hydrophilic agent selected from the group consisting of electrolytes, organic acids and osmotic agents, and mixtures thereof, to improve swelling of the gellan gum.

CN107847604B describes an in situ gel, wherein the in situ gel is an aqueous preparation, and the components of the in situ gel comprise an anti-infective agent, a biocompatible polysaccharide, an osmotic pressure regulator, a pH regulator and water; the anti-infective agent is povidone iodine, and the concentration of the povidone iodine in the aqueous preparation is as follows: a mass/mass ratio or a mass/volume ratio of 0.1% to 5.0%; the biocompatible polysaccharide comprises deacetylated gellan gum at a concentration of 0.1% to 0.5% mass/mass or mass/volume in an aqueous formulation; the osmotic pressure regulator comprises sodium chloride, glycerol, polyethylene glycol 400, mannitol or boric acid or any mixture thereof.

However several challenges exist in designing a mucus-mimicking composition. Achieving the right balance of viscosity, adhesiveness, and responsiveness to physiological cues is crucial for replicating the dynamic nature of natural mucus. Additionally, ensuring biocompatibility and stability of the composition within the human body is a paramount concern. Therefore, a model that similar to that of natural mucus is proposed. Therapeutic agents of choice such as Antimicrobial peptides, liposomes etc, are incorporated to study the therapeutic effectiveness also.

OBJECT OF THE INVENTION

The main object of the present invention relates to a hydrocolloid composition mimicking mucus, a pharmaceutical base for therapeutic agents.

Another object of the present invention is to develop process of preparation of hydrocolloid composition mimicking mucus, a pharmaceutical base for therapeutic agents.
Yet another object of the present invention relates to therapeutic agents incorporated hydrocolloid composition mimicking mucus.

Yet another object of the present invention is to develop a process of preparation of therapeutic agents incorporated hydrocolloid composition mimicking mucus.

Another object of the present invention is to study the biological activities of therapeutic agents incorporated hydrocolloid composition mimicking mucus.

Further object of the present invention is to utilize the developed hydrocolloid composition mimicking mucus and therapeutic agents incorporated hydrocolloid composition mimicking mucus for targeted drug delivery

SUMMARY OF THE INVENTION

The present invention discloses a hydrocolloid composition mimicking mucus, a pharmaceutical base for therapeutic agents, a therapeutic agents incorporated hydrocolloid composition mimicking mucus and process thereof. The present invention discloses a composition that mimics the composition and physicochemical properties of mucus of various tissues using a polysaccharide, osmotic regulating agent, surfactant and water. In particular low acyl gellan gum, sodium chloride, polysorbate 40 and water are utilized for the composition. Physicochemical characterization of the prepared composition is evaluated using various analytical techniques which confirmed the similar characteristics as that of mucus. The composition exhibited a non-Newtonian behaviour with gel-sol /sol-gel transition, surface tension, and morphology similar to mucus, making it suitable to enhance mucociliary clearance. Therapeutic agents of choice (natural and synthetic), dosage forms and drug delivery systems are loaded to this pharmaceutical base and characterized for its structural stabilization and its biological activity which is observed to be noteworthy. Further, the composition is in nanosize of approximately 188 nm which makes it suitable for targeted drug delivery to the organ like the lung in the respiratory system.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 depicts Rheology of hydrocolloid composition of the present invention.

Figure 2 depicts Scanning microscopy images of (a) hydrocolloid composition and (b) Lysozyme loaded Hydrocolloid composition of the present invention.

Figure 3 depicts Comparative CD spectra of model therapeutic agent lysozyme and hydrocolloid composition of the present invention.

Figure 4 depicts SEM images of liposome loaded hydrocolloid composition of the present invention.

Figure 5 depicts Cell viability of therapeutic agent loaded hydrocolloid composition of the present invention on (a)Lung cancer cell line A549 and in (b)normal cell line L132.

Figure 6 depicts anti-inflammatory activity of therapeutic agent loaded hydrocolloid composition of the present invention in the Zebra fish model.
DETAILED DESCRIPTION OF THE PRESENT INVENTION

The present invention discloses a hydrocolloid composition mimicking mucus, a pharmaceutical base for therapeutic agents, a therapeutic agents incorporated hydrocolloid composition mimicking mucus and process thereof.

The hydrocolloid composition mimicking mucus, a pharmaceutical base for therapeutic agents comprises of a polysaccharide, osmotic regulating agent, surfactant and water.

In an embodiment, the present invention provides hydrocolloid composition mimicking mucus which contains therapeutic agents of choice and will be important in the development of therapeutic medicines for various abnormalities related to lung, eyes, intestine etc.

In an embodiment of the present invention discloses hydrocolloid composition mimicking mucus, a pharmaceutical base for other therapeutic agents, its excipients optimization using design of expert.

In yet another embodiments the polysaccharide is in the range of 0.25%w/v to 1.0 % w/v, osmatic agents ranges from 75mM to 223 mM, the surfactant is in the range of 0.1% w/v to 0.5% w/v.

In yet one of the embodiments, present invention provides a hydrocolloid composition mimicking mucus, wherein the polysaccharide is selected from the group which form gels consisting of Agar, Pectin, levan, gellan gum, alginates, dextran, and chitosan, wherein the polysaccharide is gellan gum, wherein the gellan gum selected is low acyl.
In yet one of the embodiments, present invention provides a hydrocolloid composition mimicking mucus wherein the osmotic regulating agent is selected from the group consisting of salts, sugar or amino acid wherein the osmotic regulating agent is salt, wherein the salt is selected from the group consisting of trivalent, divalent or monovalent wherein the osmolytic agent is monovalent particularly sodium chloride.

In yet one of the embodiments, present invention provides a hydrocolloid composition mimicking mucus wherein the surfactant is selected from a group consisting of anionic, cationic or non-ionic, wherein surfactant is non-ionic particularly polysorbate 40.

In yet one of the embodiments, present invention provides a hydrocolloid composition mimicking mucus wherein the medium is a buffer or water, wherein the medium is selected from the group consisting of miliQ water, deionized water or double distilled water.

The therapeutic agents are selected from the group either proteins, peptides, phytomolecules like terpenes, alkaloids, polyphenols, or synthetic APIs and drug delivery system like microparticles, nanoparticles, liposomes, dendrimers, niosomes, nanoemusiions etc.

In yet one of the embodiments, present invention provides a hydrocolloid composition mimicking mucus, wherein the hydrocolloid composition to therapeutic agent in the ratio is in w/v of 2:1 to 4:1 wherein the composition further contains hydrocolloid composition to therapeutic agent in the ratio in (w/v) used is 2:1 (w/v).

In yet one of the embodiments, present invention provides a process of preparing a hydrocolloid composition mimicking mucus containing the steps;

i. Stirring the polysaccharide, gellan gum in water under magnetic stirring for 30 min at 500 RPM at room temperature to obtain a first solution;

ii. Adding an osmotic regulating agent, sodium chloride to the first solution of step (i) and enabling mixing at a room temperature for 30 min at 500 rpm under magnetic stirring to obtain a second solution;
iii. Adding a surfactant, polysorbate 40 to the second solution of step (ii) under magnetic stirring which continued for 2h at 700 RPM at room temperature to obtain a final gel.

In yet one of the embodiments, present invention provides a process of preparing a hydrocolloid composition mimicking mucus, which can be made into any dosage forms like inhaler, in situ or mucoadhesive gel instilled into body cavities, oral solid dosage forms, semisolid dosage forms, transdermal patches etc.

Various analytical techniques viz. UV-Visible, Fluorescence spectroscopy FT-IR, DSC, XRD, CD, Rheology, Surface tension measurement, TEM, SEM, AFM, DLS, Surface charge etc., confirmed similar physicochemical characters between the hydrocolloid composition and natural mucus. The therapeutic agent loaded hydrocolloid retained the safety profile, anti-oxidant, anti-microbial and anti-inflammatory activity of active ingredient loaded which warrant its application as a pharmaceutical base for different therapeutic agents.

The present invention is illustrated by examples. The examples are mere embodiments of the present invention and cannot be construed as limiting.

EXAMPLE 1: PHARMACEUTICAL BASE COMPOSITION OF THE PRESENT INVENTION
S.No. Chemicals Composition
1. Gellan gum 0.25% w/v
2. Sodium chloride 75mM
3. Tween 40 0.1% w/v

EXAMPLE 2: PHARMACEUTICAL BASE COMPOSITION OF THE PRESENT INVENTION
S.No. Chemicals Composition
1. Gellan gum 0.5% w/v
2. Sodium chloride 124 mM
3. Tween 40 0.5% w/v

EXAMPLE 3: COMPOSITION WITH INCORPORATION OF THERAPEUTIC AGENT
S.No. Chemicals Composition
1. Gellan gum 0.5% w/v
2. Sodium chloride 124 mM
3. Tween 40 0.5% w/v
4. Lysozyme (Therapeutic agent) 300 µg/ml

EXAMPLe 4 COMPOSITION WITH INCORPORATION OF THERAPEUTIC AGENT
S.No. Chemicals Ratio
1. Base composition 2
2. API loaded Liposomes 1

EXAMPLES 5 RHEOLOGY STUDIES

Viscosity Measurement
This invention provides valuable insights into the viscosity behavior of gellan gum and sodium chloride-added gellan gum, its modifications with non-ionic surfactants and therapeutic agent of choice, lysozyme and the impact of shear rate on the viscosity of these compositions. When the viscosity of pure gellan gum (1200 mPa.s) was compared with sodium chloride-added gellan gum, it exhibited a higher viscosity of 3250 mPa.s. Furthermore, the addition of polysorbate 40 to GG-S further reduced viscosity, with a measured value of 700 mPa.s. The invention also examined the effect of introducing lysozyme into the system. Interestingly, the viscosity decreased even further to 300 mPa.s upon lysozyme addition. The results represented the influence of shear rate on the viscosity of gellan gum along with other components, and the formed gel confirmed to be a shear thinning effect, where an increase in shear rate probably led to a decrease in viscosity for all the samples.

Viscosity Vs. Temperature
The present invention provides the impact of temperature on the viscosity of gellan gum with addition of other components- salt, surfactant and lysozyme. It was found that pure gellan gum displayed a gel point at 30°C, and a decrease in viscosity was observed leading up to this gel point. Once the gel point was reached, the viscosity curve levelled off and reached a plateau. By adding sodium chloride to the samples, it was identified that it significantly influenced viscosity when the temperature was varied. The multiple transitions between sol-gel and gel-sol states were noticed, indicating the variations in viscosity with changing temperatures. However, the introduction of polysorbate 40 disrupted this trend and resulted in the attainment of a steady state viscosity. Furthermore, the study revealed a gradual decrease in viscosity as the temperature increased for all the samples. Adding lysozyme did not substantially impact the viscosity compared to Hydrocolloid composition, and a similar viscosity pattern was observed for the gel. These findings shed light on the relationship between temperature and viscosity for gellan gum and its modified samples, the influence of sodium chloride and polysorbate 40 on viscosity transitions, and the minimal impact of protein addition on viscosity.

EXAMPLE 6
Antimicrobial Activity of Nanogel
The antimicrobial activities of the therapeutic agent loaded hydrocolloid were tested in comparison to the positive drug control Piperacillin (10µg/disc) using the zone of inhibition method. It was observed that it exhibited noteworthy antimicrobial activity against Staphylococcus aureus followed by Acinetobacter baumannii and E.coli both in low and high doses. 1mg of therapeutic agent loaded hydrocolloid consists of 320.7±0.12 µg of active ingredient, lysozyme and it is an anti-microbial protein effective against the gram positive bacteria than the gram negative species. Hence, the antibacterial activity of the therapeutic agent loaded hydrocolloid is attributed to the presence of lysozyme.

EXAMPLE 7
Anti-inflammatory activity of Nanogel
The nanogel formulation demonstrated anti-inflammatory activity, as evidenced by the downregulation of the inflammatory markers in the experimental Zebra model. However, its effect was diminished in comparison to that of the standard drug diclofenac and statistically not significant. The nanogel demonstrated a noticeable trend toward reducing inflammation, indicating potential for therapeutic activity as the antimicrobial peptide lysozyme is an innate immune defense factor. However, the antimicrobial peptide used in the formulation is a naturalcompound, and its anti-inflammatory effect was milder compared to the synthetic drug diclofenac. Although the nanogel’s activity was not as extensive as diclofenac, its performance still supports its potential as a safer, biocompatible alternative for managing inflammation, especially in cases where long-term use of synthetic drugs may pose risks.

In one of the preferred embodiment, the present invention shall discloses a hydrocolloid composition for use as a pharmaceutical base. The hydrocolloid composition comprising
a. a polysaccharide present at a concentration ranging from 0.25% w/v to 1.0% w/v;
b. an osmotic regulating agent present at a concentration ranging from 75 mM to about 223 mM; and
c. a surfactant present at a concentration ranging from 0.1% w/v to about 0.5% w/v;
in which the hydrocolloid composition mimics mucus and is adapted for loading with one or more therapeutic agents in a ratio of w/v of 2:1 to 4:1.

As per the invention, in the hydrocolloid composition the polysaccharide is gellan gum.

In accordance with the invention, in the hydrocolloid composition, the osmotic regulating agent is sodium chloride.

According to the invention, in the hydrocolloid composition, the surfactant is polysorbate 40.

In another preferred embodiment, the present invention shall discloses a method for preparing a hydrocolloid composition for use as a pharmaceutical base, comprising:
a. preparing a first solution by stirring a polysaccharide, in which the polysaccharide is of concentration ranging from 0.25% w/v to 1.0% w/v, in water under magnetic stirring at 500 RPM at room temperature for 30 minutes;
b. forming a second solution by adding an osmotic regulating agent, of concentration ranging from 75 mM to 223mM, to the first solution and mixing at room temperature under magnetic stirring at 500 RPM for 30 minutes;
c. generating a final gel by adding a surfactant, of concentration ranging from 0.1% w/v to about 0.5% w/v, to the said second solution and stirring magnetically at 700 RPM at room temperature for 2 hours to obtain a final hydrocolloid composition that mimics mucus.
As per the invention in the method, the polysaccharide is gellan gum, the osmotic regulating agent is sodium chloride and the surfactant is polysorbate 40.

In further preferred embodiment, the present invention shall discloses a method for loading a therapeutic agent into the hydrocolloid composition comprising:
a. gradually adding a therapeutic agent, to the hydrocolloid composition while stirring continuously at a low speed to form a mixture;
b. maintaining the stirring under ice bath conditions such that temperature of the mixture remains within the range of 0°C to 4°C, thereby producing a hydrocolloid composition loaded with a therapeutic agent at a hydrocolloid composition to therapeutic agent weight-to-volume ratio of about 2:1.
As per the invention, in the method for loading a therapeutic agent into the hydrocolloid composition, the therapeutic agent is selected from a group comprising of natural or synthetic small or large molecules.

WORKING EXAMPLE 1:
The hydrocolloid composition for use as a pharmaceutical base comprising
a. gellan gum 0.25% w/v;
b. sodium chloride 75 mM; and
c. polysorbate 40 0.1% w/v.

WORKING EXAMPLE 2:
A method for preparing a hydrocolloid composition for use as a pharmaceutical base, comprising:
a. preparing a first solution by stirring gellan gum 0.25% w/v, in water under magnetic stirring at 500 RPM at room temperature for 30 minutes;
b. forming a second solution by adding sodium chloride 75 mM, to the first solution and mixing at room temperature under magnetic stirring at 500 RPM for 30 minutes;
c. generating a final gel by adding polysorbate 40 0.1% w/v, to the second solution and stirring magnetically at 700 RPM at room temperature for 2 hours to obtain a final hydrocolloid composition that mimics mucus.

WORKING EXAMPLE 3:
A method for loading a therapeutic agent into the hydrocolloid composition comprising:
a. gradually adding a 300µg/ml of lysozyme (therapeutic agent), to the 10 mg of hydrocolloid composition while stirring continuously at a low speed to form a mixture;
b. maintaining the stirring under ice bath conditions such that temperature of the mixture remains within the range of 0°C to 4°C, thereby producing a hydrocolloid composition loaded with a therapeutic agent.

While considerable emphasis has been placed herein on the various components of the preferred embodiment, it will be appreciated that many alterations can be made and that many modifications can be made in the preferred embodiment without departing from the principles of the invention. These and other changes in the preferred embodiment as well as other embodiments of the invention will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the invention and not as a limitation.
,CLAIMS:WE CLAIM:

1. A hydrocolloid composition for use as a pharmaceutical base, comprising
a. a polysaccharide present at a concentration ranging from 0.25% w/v to 1.0% w/v;
b. an osmotic regulating agent present at a concentration ranging from 75 mM to about 223 mM; and
c. a surfactant present at a concentration ranging from 0.1% w/v to about 0.5% w/v;
wherein the claimed composition mimics mucus and is adapted for loading with one or more therapeutic agents in a ratio of w/v of 2:1 to 4:1.

2. The hydrocolloid composition as claimed in claim 1, wherein the said polysaccharide is gellan gum.

3. The hydrocolloid composition as claimed in claim 1, wherein the said osmotic regulating agent is sodium chloride.

4. The hydrocolloid composition as claimed in claim 1, wherein the said surfactant is polysorbate 40.

5. A method for preparing a hydrocolloid composition for use as a pharmaceutical base, comprising:

a. preparing a first solution by stirring a polysaccharide, wherein the polysaccharide is of concentration ranging from 0.25% w/v to 1.0% w/v, in water under magnetic stirring at 500 RPM at room temperature for 30 minutes;
b. forming a second solution by adding an osmotic regulating agent, of concentration ranging from 75 mM to 223 mM, to the said first solution and mixing at room temperature under magnetic stirring at 500 RPM for 30 minutes;
c. generating a final gel by adding a surfactant, of concentration ranging from 0.1% w/v to about 0.5% w/v, to the said second solution and stirring magnetically at 700 RPM at room temperature for 2 hours to obtain a final hydrocolloid composition that mimics mucus.

6. The method as claimed in claim 1, wherein the said polysaccharide is gellan gum.

7. The method as claimed in claim 1, wherein the said osmotic regulating agent is sodium chloride.

8. The method as claimed in claim 1, wherein the said surfactant is polysorbate 40.

9. A method for loading a therapeutic agent into the hydrocolloid composition of claim 1 comprising:

a. gradually adding a therapeutic agent, to the said hydrocolloid composition while stirring continuously at a low speed to form a mixture;
b. maintaining the said stirring under ice bath conditions such that temperature of the mixture remains within the range of 0°C to 4°C, thereby producing a hydrocolloid composition loaded with a therapeutic agent at a hydrocolloid composition to therapeutic agent weight-to-volume ratio of about 2:1.

10. The method for loading a therapeutic agent into the hydrocolloid composition as claimed in claim 9 wherein the said therapeutic agent is selected from a group comprising of natural or synthetic small or large molecules.