Claims:We Claim:
1. A pharmaceutical composition of silver nitrate comprises an acrylamide polymer containing residual acrylamide monomer content less than 0.2mcg/gm, wherein the acrylamide content is significantly reduced by treating with sodium sulphite solution compared to treatment of water.
2. A pharmaceutical composition of silver nitrate according to claim 1, wherein the treatment solution of sodium sulphite is in the concentration of 0.01 to 0.2% (w/v).
3. A pharmaceutical composition of silver nitrate according to claim 1, wherein the treatment solution of sodium sulphite is particularly in the concentration of 0.2% (w/v).
4. A pharmaceutical composition of silver nitrate wherein the residual Acrylamide monomer forms a complex of lower toxicity with Sodium sulphite during washing of the polymer with the same.
5. A pharmaceutical composition of silver nitrate according to claim 3, wherein washing with sodium sulphite imparts sixty times less free acrylamide monomer content than polymer without washing.
6. A pharmaceutical composition of silver nitrate according to claim 3, wherein washing with sodium sulphite imparts five times less free acrylamide monomer content than polymer washed with water.
7. A pharmaceutical composition of silver nitrate according to claim 3, wherein the treatment with sodium sulphite solution the polymer to sodium sulphite solution is taken in the ratio of 1:3 to 1:6.
8. A pharmaceutical composition of silver nitrate according to claim 3, wherein the treatment with sodium sulphite solution the polymer to sodium sulphite solution is taken particularly in the ratio of 1:3.

Date: Day 5th, Feb, 2018
, Description:FORM 2

THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003

Complete
Specification
(See section 10 and rule 13)

Title: Acrylamide free Silver Containing Compositions and methods for making

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

Acrylamide free Silver Containing Compositions and methods for making
Field of Invention
The present invention comprises methods and compositions for silver containing antimicrobial hydrophilic material comprising a polymer network and an active agent. The present invention also relates to the removal of acrylamide content within the acceptance criteria to overcome the label warning associated with acrylamide content and provide a safer alternative in the field of wound healing. More particularly, the present invention illustrates a silver containing antimicrobial hydrophilic gel composition that is free or almost free of toxic acrylamide monomer content to provide a safer alternative in the field of wound healing.
Background of the Invention
Wounds to skin and the underlying tissues of humans and animals may be caused by external insult such as friction, abrasion, laceration, burning or chemical irritation. Damage to such tissues may also result from internal metabolic or physical dysfunction, including but not limited to bone protrudence, diabetes, circulatory insufficiencies, or inflammatory processes. Normally tissue damage initiates physiological processes of regeneration and repair. In broad terms, this process is referred to as the wound healing process.
The wound healing process usually progresses through distinct stages leading to the eventual closure, and restoration of the natural function of the tissues. Injury to the skin initiates an immediate vascular response characterized by a transient period of vasoconstriction, followed by a more prolonged period of vasodilation. Blood components infiltrate the wound site, endothelial cells are released, exposing fibrillar collagen, and platelets attach to exposed sites. As platelets become activated, components are released which initiate events of the intrinsic coagulation pathway. At the same time, a complex series of events trigger the inflammatory pathways generating soluble mediators to direct subsequent stages of the healing process.
Importantly, none of the presently available devices significantly contribute to or support the autolytic debridement phase, which is the natural removal process of necrotic tissue and debris from the wound. Autolytic debridement is a key early stage event that precedes repair phases of healing. When wound conditions are not optimal for supporting autolytic debridement, then clinical procedures such as surgical removal, irrigation, scrubbing, and enzymatic or chemical methods must be used to remove the necrotic tissue and escar that can inhibit wound healing.
One common method employed for the treatment of wounds is the topical application of a salve or ointment. Yet many times, topical application to a wound can be painful and short-lived. Additionally, in the case of a deeply cavitated wound in particular, an excess of active agent may be required because the agent must diffuse through layers of necrotic tissue and newly forming epidermal tissues. This difficulty in delivering the agent may require the application of an excessive amount of the agent and preclude an accurate determination of the effective amount of active agent to be added.
One active heavy metal, in particular silver, has long been recognized for its broad spectrum anti-microbial activity and compatibility with mammalian tissues. Although silver has been used in a large range of medical devices, its incorporation, as a prophylactic anti-infective agent, in primary wound contact products has been restricted due to silver's adverse properties. These properties include a short half-life, the rapid inactivation of silver by protein, and light-mediated discoloration of the product containing silver and any body parts touching the product, such as skin. Recently, manufacturers have tried methods to overcome some of the limitations to broaden the utility of silver in wound care. The currently available silver-containing wound care dressing materials have been unsuccessful in adequately overcoming the problems inherent in using silver.
The mode of action of silver is due to the reactivity of the ionic form with a variety of electron donating functional groups that contain reactive entities such as oxygen, sulfur or nitrogen. Electron donating functional groups in biological systems are many and varied, including groups such as phosphates, hydroxyl, carboxylates, thiol, imidizoles, amines, and indoles. Microbial macromolecules are richly endowed with these functional groups that, when bound by silver ion, may become inactivated and disfunctional resulting in the death of the microorganism. Ionic silver is known to disrupt microbial cell wall, cell membrane, electron transport, metabolic and anabolic enzymes, and nucleic acid function (A. D. Russel, W. B. Hugo, “Antimicrobial activity and action of silver” In Progress in Medicinal Chemistry. Vol. 3, G. P. Ellis & D. K. Luscombe, ed., Elsevier Science B. V., (1994)).
Attempts at overcoming the limitations of silver addition included applying silver or silver salts onto dry substrates where little or no ionization of silver could occur or the use of substrates containing few reactive functional groups that would react with ionic silver. However, this is impractical for applications where moisture abounds such as in moist devices such as soft contact lenses, hydrated plastic implants, or in moist wound dressing cover such as a hydrogels, hydrocolloids, or biologics, or in medical devices that contain reactive functional groups such as in a collagen matrix.
To overcome these problems, inventions describing stabilization of silver have been described. U.S. Pat. No. 5,709,870 describes a process for producing a light and heat stable silver complex with carboxymethyl cellulose for use in coating fibers. Similarly U.S. Pat. No. 5,744,151 describes a process for rendering silver photo-stable and antimicrobial for use as an adjunct to pharmaceuticals by forming an acyclic polyether polymer stabilized by ratios of cation and anions in the process.
These inventions have provided some solutions to the problems of stability, and half-life for silver for several silver antimicrobial applications. However they are cumbersome, may contain toxic accessory agents that support function, or are prohibitively expensive for application to commodity medical devices such as wound dressings.
Alternately, there are products that deliver the antimicrobial actives to the wound site more slowly over time. In such case, the bio-burden decrease takes place slowly but without interference with the body's natural healing process. Additionally, these products are compounded with agents that aid healing and provide for moisture management. Examples of such products include antimicrobial hydrogels (SilvaSorb®, SilverSept®, Normlgel Ag® and Elta®) and antimicrobial sheet dressings (SilvaSorb® and Covalon®)
U. S. Pat. No. 6,605,751 discloses a silver-containing antimicrobial hydrophilic material comprising a matrix comprising a polymer network and an active agent. Acrylamide has been used as starting monomer that has been cross linked with the use of Bisacrylamide to form the desired polymeric matrix. The marketed product “SilvaSorb®” is also using the same technology to provide antimicrobial activity in medical devices, and particularly for silver incorporation into medical devices such as moisture-containing wound dressings, skin contact devices, such as monitor leads, wound dressings and hydrated plastic implants.
Although these devices have provided certain solutions to combining antimicrobial activity with medical devices, these inventions have identified a number of limitations associated with Acrylamide monomer content that remain as free substance within the polymer matrix and induces certain toxicities including Cancer and Birth defects raising a serious concern regarding the maximum acceptance limit of free acrylamide within the mammals.
To reduce the levels of free acrylamide monomer less than prescribed limits and overcome label warning was a matter of primary concern. Several attempts had been taken to reduce the acrylamide content by washing with water but it was a cumbersome task to perform and even the results were not satisfactory. Inventors of present invention surprisingly found out that if the polymer in test is washed with Sulfite solution, any residual amounts of acrylamide monomer remaining in the polymer are substantially reduced or in some way converted to derivatives of low toxicity thus putting an end to an age old problem.
Summary of Invention
The present invention relates to compositions and methods for the incorporation and stabilization of silver onto and within the hydrophilic fibers of cross-linked and non-cross-linked celluloses such as carboxymethy cellulose and hydroxymethyl cellulose, cotton, rayon, and of fibers made from polyacrylates and other synthetic and natural polymers, and fibers of calcium alginates that may be used as a primary contact sustained-release silver antimicrobial materials.
The present invention also relates to washing of polymer, specifically polyacrylamide to remove the free acrylamide monomer to pharmacologically acceptable limits with a predetermined concentration of sulphite salt, specifically Sodium sulphite salt solution to avoid the label warning and provide a safer alternative in the field of wound healing.
One of the embodiments of present invention relates to the washing of polymer employing predetermined concentration of sodium sulphite wherein the residual acrylamide monomer forms a complex of low toxicity with the sodium sulphite and might get removed during filtration or induces no toxicity if remains within the polymer.
The present invention also discloses washing of polymer, specifically polyacrylamide with Sulfite salt solution to remove the free acrylamide monomer content almost ten times more than washing with water and almost sixty times more than non washed polymer.
The present invention also discloses washing of polymer, specifically polyacrylamide with Sulfite salt solution to bring down the free acrylamide monomer content within the prescribed limit.
These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments and the appended claims.
Brief Description of the Figures
Figure 1 depicts the chromatographic representation of non washed polymer having a significant amount of free acrylamide content.
Figure 2 illustrates the chromatographic representation of water washed polymer having a smaller peak of free acrylamide content almost ten times less than that of present in non washed polymer.
Figure 3 illustrates the chromatographic representation of Sulphite washed polymer having a negligibly smaller peak of free acrylamide content almost 5 times less than that of present in water washed polymer and sixty times less than that of present in non washed polymer.
Detailed Description of the invention
The present invention now is described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, this embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
Like numbers refers to like elements throughout. In the figures, the thickness of certain lines, layers, components, elements or features may be exaggerated for clarity. Broken lines illustrate optional features or operations unless specified otherwise. One or more features shown and discussed with respect to one embodiment may be included in another embodiment even if not explicitly described or shown with another embodiment.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. As used herein, phrases such as "between X and Y" and "between about X and Y" should be interpreted to include X and Y. As used herein, phrases such as "between about X and Y" mean "between about X and about Y." As used herein, phrases such as "from about X to Y" mean "from about X to about Y."
The term “almost” signifies the free acrylamide monomer content to be in the range of zero i.e., free of acrylamide content to 0.2 µg/g of final gel formulation.
The matrix of the preferred embodiment preferably comprises polymerized chains of acrylamide monomer, wherein the acrylamide monomers are cross-linked with a cross-linking agent and an active agent or pharmaceutical directly encapsulated into micro-cavities therein.
Acrylamide (or acrylic amide) is a chemical compound with the chemical formula C3H5NO. Its IUPAC name is prop-2-enamide.

Acrylamide is considered a potential occupational carcinogen by U.S. government agencies and classified as a Group 2A carcinogen by the IARC. The different toxicities and safe level of free acrylamide content are explained in California proposition 65. The details are mentioned in below table.
Table-1
S. No Type of Toxicity
NSRL or MADL
1 Cancer 0.2 µg/day*
2 Developmental male 140 µg/day**

*NSRL: “No Significant Risk Level” for cancer-causing chemicals.
**MADL: “Maximum Allowable Dose Level” for reproductive toxins.
If the polyacrylamide obtained contains free acrylamide more than mentioned in the table-1, as per California proposition 65 a warning label shall be kept on the gel under test which states.
‘‘This product contains a chemical known to the state of California to cause cancer and birth defects or other reproductive harm.’’
To reduce the levels of free acrylamide monomer less than prescribed limits and overcome label warning was a matter of primary concern. Several attempts had been taken to reduce the acrylamide content by washing with water but it was a cumbersome task to perform and even the results were not satisfactory. Inventors of present invention surprisingly found out that if the polymer in test is washed with Sulfite solution, any residual amounts of acrylamide monomer remaining in the polymer are substantially reduced or in some way converted to derivatives of low toxicity. The residual acrylamide monomer forms a complex of lower toxicity with sodium sulfite and might get removed during filtration or induces no toxicity if remains within the polymer.

Composition and Manufacturing Process:
1) Preparation of Polyacrylamide Powder:
Material Description Quantity %w/w

Purified water (1) 2 - 3
Purified water (2) 60 - 70
Purified water (3) 10 - 15
Acrylamide 2 - 10

Purified water (4) 5 - 10

Bis acrylamide NMT 0.5

Purified water (5) 1 - 5

TEMED 0.05 – 0.1

Ammonium persulfate 0.01 – 0.1

Purified water (6) 1 - 5

Manufacturing of Polyacrylamide Powder:
Preparation of Bis acrylamide solution: Purified water (3) was added in a container; Bisacrylamide was added to it and mixed for 40min to dissolve completely.
Preparation of acrylamide solution: Purified water (4) was added in a container; acrylamide was added to it and mixed until it is dissolve completely.
Preparation of TEMED solution: Purified water (5) was added in a glass container; TEMED was added to it and mixed until it mix completely.
Preparation of Ammonium persulfate solution: Purified water (6) was added in a container; Ammonium persulfate was added to it and mixed until it is dissolved completely.
Preparation of Polyacrylamide base sheets:
• Purified water (2) was added in main vessel and the temperature of purified water was maintained in between 15-21°C. Then Bis-acrylamide solution was added to the main vessel and mix for 2-3 min and simultaneously Acrylamide, TEMED and Ammonium persulphate solution were added into the vessel and mixed properly.
• After completion of mixing the solution was poured into the mold boxes, covered with shrink wrap and kept aside for 24hr for polymerization.
• After 24hr the Polyacrylamide sheets were collected from mold box and stored for further use.
Analysis for Acrylamide Content
PROCEDURE:
Preparation of Mobile Phase: HPLC Grade water was transferred into a mobile phase bottle and sonicated for 5 minutes.
Preparation of Diluent: Mobile phase as the Diluent.
Chromatographic parameters
Column Phenomenox Luna C18, 5 µ, 250 x 4.6 mm ( Or ) Equivalent.
Wavelength 205 nm
Flow Rate 1.0 mL/min
Column Temperature 50°C
Sample Temperature 25°C
Injection Volume 80µL
Run time 15 min

Reagents:
S. No. Name of the Chemical / Reagent
1 Acrylamide standard
2 Acetone

Instrument: HPLC with UV or PDA detector.
Result: Figure 1 depicts the chromatographic illustration of free Acrylamide monomer content in the polymer without washing.
To get rid of this out of limit free acrylamide monomer content from the Polymer sheets several washing techniques had been introduced. Those washing techniques along with the results have been demonstrated in the following examples and figures attached herewith.
Example 1:
Washing with Water:
Experiment No.1:
• 250g of Polyacrylamide base sheet prepared as per the process mentioned above was mashed in to small pieces and transferred in to beaker containing 2.5L of water and mixed for 5hr by using overhead stirrer at 750RPM.
• The above mixture was filtered at 5min, 1hr & 5hr intervals and water was replaced with fresh 2.5L purified water every time.
• After 5hr mixing the Polyacrylamide base gel was transferred on to the white plates and loaded into hot air oven at 55°C for drying.
• The dried Polyacrylamide base gel was made into fine powder and given for analysis.
The results were mentioned below
S.No Parameter Results
1 Free Acrylamide monomer content (ppm) in Polyacrylamide powder 195.0

Observations: The washing of Polyacrylamide gel with water for 5 hour still needs to be optimized to achieve the level of free acrylamide monomer less than 0.2µg/g in final formulation.
Experiment No.2: (1: 10 ratio)
• Four sheets of Polyacrylamide base gel accurately weighed (940g) & mixed in 9.4L (1:10 ratio) of purified water for 24 hrs using overhead stirrer.
• Purified water was replaced with fresh 9.4L purified water at 4, 8, 12, 16 & 20hr intervals.
• After 24 hours the mixture was filtered and gel was kept in oven for drying at 55°C.
• The gel was dried and then given for analysis.

S.No Parameter Results
1 Free Acrylamide monomer content (ppm) in Polyacrylamide powder 23.97
Conclusion:
The washing of Polyacrylamide base gel with water still needs to be optimized to achieve the level of free acrylamide monomer less than 0.2µg/g in the final gel formulation.
Experiment No.3: (1: 6ratio)
In order to reduce the water quantity experiment has been taken by preparing the 18 sheets as per the manufacturing process mentioned above and reducing the proportion of Polyacrylamide base gel to Purified water to 1:6 instead of 1:10.
• 30kg of sheets were accurately weighed & mashed into small pieces. 180L of (1:6 ratios) of purified water was added in mixing vessel and mixed for 24hr by mixing with impeller present at the bottom.
• After every 4 hour the mixture was filtered by using muslin cloth (200#) and fresh Purified water was added in gel. The same process was repeated at 8, 12, 16 & 20hr intervals and filtrate was collected at each interval and given for analysis of free acrylamide content.
• After 24hr, gel was kept in oven at 55°C for drying.
• The dried Polyacrylamide powder was given for analysis.
S.No Parameter FD-BGM-095/17
1 Assay of free Acrylamide monomer content (ppm) 17.96

Conclusion:
Based on the above results, we conclude that washing of Polyacrylamide base sheets using purified water for 24hr is effectively reducing the free acrylamide monomer content, with no effect of lower proportion of purified water.
But to achieve objective of reduction of acrylamide content to safe level we need to reduce the monomer content to a level of 0.2µg/g in the final gel formulation.
The required result has been illustrated in Figure 2 (For Experiment 3 of Example 1) that clearly points out the reduction of Acrylamide content significantly but not within the prescribed limit.
Finally Inventors of present invention surprisingly found out a solution of this problem wherein the process comprises of washing of Polyacrylamide obtained with a predetermined concentration of Sodium Sulphite i.e., 0.2% (w/v) Sodium Sulphite thus any residual amounts of acrylamide monomer remaining in the polymer are substantially reduced or in some way converted to derivatives of low toxicity. The residual acrylamide monomer forms a complex of lower toxicity with sodium sulphite and might get removed during filtration or induces no toxicity if remains within the polymer thus putting an end to an age old problem.
Example 2:
Washing with 0.2% Sodium Sulphite:
Experiment No.1:
• Required quantity of Polyacrylamide base sheets prepared as per the process mentioned above were mashed into small pieces and transferred into a mixing vessel containing 0.2% sodium Sulphite solution 6 times more than the Polyacrylamide sheet weight (1:6 ratio)
• The above mixture was mixed for 2hr, filtered to remove the solution from the mixture. Then replaced with same quantity of 0.2% sodium Sulphite solution and mixed for next 2hr.
• At 4th hrs interval solution was filtered and replaced with same quantity of fresh purified water.
• Mixed for next 2hr, by replacing with fresh purified water at 1hr interval.
• After completion of 6hr mixing Polyacrylamide base gel was kept in hot air oven at 55°C for drying.

S.No Parameter
1 Assay of free Acrylamide monomer content (ppm) in Polyacrylamide sheets 4.57

Conclusion: Based on the above results, we conclude that the Sodium Sulphite solution method is ideal condition for the intended purpose where we could achieve the target level of acrylamide monomer content in Polyacrylamide sheets (NMT 10µg/g).
Experiment No.2:
In order to reduce the water quantity experiments has been taken by preparing the Polyacrylamide sheets as per the manufacturing process mentioned above and reducing the proportion of Polyacrylamide base sheets to Sodium Sulphite solution to 1:3 instead of 1:6.
• 1Kg of Polyacrylamide base sheets prepared as per the process mentioned above were mashed into small pieces and transferred into a mixing vessel containing 0.2% sodium Sulphite solution 3 times more than the Polyacrylamide sheet weight (1:3 ratio)
• The above mixture was mixed for 2hr, filtered to remove the solution from the mixture. Then replaced with same quantity of 0.2% sodium Sulphite solution and mixed for next 2hr.
• At 4th hrs interval solution was filtered and replaced with same quantity of fresh purified water.
• Mixed for next 2hr, by replacing with fresh purified water at 1hr interval.
• After completion of 6hr mixing Polyacrylamide base gel was kept in hot air oven at 55°C for drying.

Parameter
Assay of free Acrylamide monomer content (ppm) in Polyacrylamide sheets 0.71
Conclusions:
Basing on the above the experiments and the results obtained usage of 0.2%w/v sodium sulfite for washing the Polyacrylamide base sheets was finalized.
Results:
Figure 3 (Experiment 2 of Example 2) is an illustration of Chromatograph obtained from the assay of free acrylamide monomer content present in the polyacrylamide polymer after treating with 0.2% Sodium Sulphite. It clearly shows that the free acrylamide monomers have been removed to desired level.
The invention having been disclosed in connection with the foregoing embodiments, additional variations will now be apparent to persons skilled in the art. Various modifications and variations to the above described development of anti-snake venom of high potent can be made without departing from the scope of the invention.
From the foregoing it will be understood that the embodiments of the present invention described above are well suited to provide the advantages set forth, and since many possible embodiments may be made of the various features of this invention and as the process herein described may be varied in various parts, all without departing from the scope of the invention, it is to be understood that all matter hereinbefore set forth or shown in the accompanying process is to be interpreted as illustrative and that in certain instances some of the features may be used without a corresponding use of other features, all without departing from the scope of the invention.

Date: Day 5th, Feb, 2018