Claims:
We claim:

1. A composition for eliminating or reducing the concentration of metal ions, the composition comprising of
sodium polystyrene sulphonate (SPS) in an predetermined amount with at least one excipient to rapidly remove the metal ions from a solution,
wherein said SPS is present in the range of 1g to 12g/100 ml of 1 ppm metal solution.
said composition being formulated as powder, solution, in suspension form or in lyophilized form.

2. The composition as claimed in claim 1 wherein said excipients are selected from thermoreversible polymers, viscosity modifiers and other adjuvants.

3. The composition as claimed in claim 1, wherein said composition comprises of 6g/dl of SPS.

4. The composition as claimed in claim 2 wherein said thermoreversible polymers are selected from poloxamers in combination with mucoadhesive polymers.

5. The composition as claimed in claim 2 wherein said viscosity modifiers are selected from cellulose derivatives, clays, natural gums, synthetic polymers and miscellaneous compounds.

6. The composition as claimed in claim 5 wherein
- said cellulose derivatives are selected from methylcellulose, microcrystalline cellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose and hydroxypropyl methylcellulose,
- said clays are selected from hectorite, bentonite, aluminium and/or magnesium silicate,
- said natural gums are selected from acacia, guar gum, tragacanth, xanthan gum, alginates, carrageenan and locust bean gum,
- said synthetic polymers are selected from carbomers, polyvinyl pyrrolidone, polyvinyl alcohol and poloxamer, and
- said miscellaneous compounds are selected from colloidal silicon dioxide and silicates.

7. The composition as claimed in claim 2 wherein said adjuvants are selected from Benzoic acid-0.01- 0.1%, Butyl paraben 0.006–0.05%, Glyceryl palmitostearate 1.0–3.0%, Guar gum-1 -2.5%, Hydroxyethylmethyl cellulose 1-2%, Lactic acid-0.015–6.6, Lecithin- 0.25–10.0%, Maltitol- 1-10%, Methyl cellulose 1-2%, Polycarbophil 0.1 -1%, Potassium bicarbonate:25 -50%, Povidone- 0.1 to 5%, Sodium alginate-1- 5%, Sorbitol- 10-50%, Thaumatin- 0.5 to 3 ppm.

8. The composition as claimed in claim 1, wherein the metal ions is present in as is or as an aqueous matrix,
- wherein the matrix is a solution of metal ions from ordinary solutions, herbal extracts or soil samples or a porous substrate, or a non-porous Substrate.

9. A method for producing the composition as claimed in claim 1wherein said method comprises the steps of
- combining SPS with said excipients,
wherein said SPS is in the range of in the range of 1g to 12g/100 ml of 1 ppm metal solution;
wherein said excipients are in the range of 0.01% to 5%.
to provide a composition capable of reducing the concentration of metal ions wherein
- said composition is delivered into a metal ion solution at pH 4, 7 or 9
? said metal ion solution is incubated at temperatures 2°C to 40°C, preferably at ambient temperature

10. The method as claimed in claim 9, wherein said metal ions are mercury, lead, cadmium, or all types of divalent heavy metal ions.

11. The composition as claimed in claim 1 wherein:
a) said composition is administered to a subject with chronic kidney disease, such that a single dose of 15g is administered once orally per day to children (1g/kg oral for children), or every 6hr for adults. Through rectal route, it is 30-50 g every 6hr in adults and 1 g/kg every 2-6hr for children
b) at least 4 doses equivalent to 15g doses administered to the subject, including wherein at least two of the holin-modified bacteriophage have specificity for different bacterial host cells, or
c) said subject has a mixed bacterial infection due to metal toxicity

12. The composition as claimed in claim 1 wherein SPS may be substituted by sodium zirconium cyclosilicate (SCZ), Patiromer or calcium polystyrene sulphonate.

13. The composition as claimed in claim 1 wherein said composition capable of reducing metal toxicity in soil samples, herbal extracts, herbal raw materials, in fishes or other animals in water bodies, soil used in green house vegetables, in drinking water and all other water bodies.
Dated this the 8th day of October 2021

________________________
Sunita K. Sreedharan
IN/PA-310
of SKS Law Associates
Attorney for the Applicant

To
The Controller of Patents, The Patent Office, Mumbai
, Description:FIELD OF THE INVENTION
The present invention relates to composition comprising of sodium polystyrene sulphonate to eliminate/reduce metal ions concentration from the solutions and the method thereof. More preferably, the present invention relates to provide composition and method for preparing SPS in a pharmaceutical acceptable form for reducing the concentration of metals such as mercury, lead, cadmium or all types of divalent heavy metal ions in solutions at different temperatures and pH’s.

BACKGROUND OF THE INVENTION
Metals are found in the earth's crust naturally and there are spatial variations in their compositions among different localities. Various sources of heavy metals include soil erosion, natural weathering of the earth's crust, mining, industrial effluents, urban runoff, sewage discharge, insect or disease control agents applied to crops, and many others (Morais et al., 2012). Heavy metals are generally referred to as those metals which possess a specific density of more than 5 g/cm3 and adversely affect the environment and living organisms (Järup, 2003). Heavy metals are significant environmental pollutants, and their toxicity is a problem of increasing significance for ecological, evolutionary, nutritional and environmental reasons (Jaishankar et, 2014a; Nagajyoti et al., 2010).

Heavy metal poisoning is the accumulation of heavy metals, in toxic amounts, in the soft tissues of the body. Such an accumulation might occur as a result of industrial exposure, air or water pollution, foods, medicines, improperly coated food containers, or the ingestion of lead-based paints. The most commonly heavy metals that are associated with poisoning of humans are lead, mercury, arsenic and cadmium. Metal toxicity depends upon the absorbed dose, the route of exposure and duration of exposure, i.e. acute or chronic. This can lead to various disorders and can also result in excessive damage due to oxidative stress induced by free radical formation.

Greenhouse vegetables are also highly contaminated with heavy metals such as Copper, Zinc, Manganese, Lead and Cadmium (Li et al., 2017). Additional metals that may cause poisoning include antimony, aluminum, barium, bismuth, copper, gold, iron, lithium, platinum, silver, tin, and zinc. Common symptoms of poisoning from these metals may include gastrointestinal, renal, and neurological symptoms, such as headaches, irritability, psychosis, stupor, coma, and convulsions.

These heavy metals are deployed in common everyday use. For instance, Cadmium is used for electroplating, storage batteries, vapor lamps and in some solders. Overexposure to cadmium causes fatigue, headaches, nausea, vomiting, abdominal cramps, diarrhea, and fever. Certain individuals exposed to cadmium renal tubular dysfunction causing proteinuria, changes in liver function, and osteomalacia is disclosed by Jaishankar et al., 2014b. Similarly, exposure to lead makes children dull, clumsier, irritable, and lethargic. At high levels of exposure, lead attacks the brain and central nervous system and results in coma, convulsions, impaired consciousness and even death. Headaches, vomiting, abdominal pain are common symptoms of lead poisoning (Wani et al., 2015). Chronic lead exposure was found to reduce fertility in males (Sokol & Berman, 1991). Long-time exposure to lead has been reported to cause anemia, increase in blood pressure, damage to the brain and kidneys, both in adults and children. In pregnant women, high exposure to lead may cause miscarriage (Wani et al., 2015).

Mercury is used by dental assistants and hygienists, and chemical workers. Mercury can affect the lungs, kidneys, brain, and/or skin. Symptoms of mercury poisoning include fatigue, depression, sluggishness, irritability, and headaches. There may be behavioral and neurological changes associated with overexposure to mercury poisoning, such as excitability and quick-tempered behavior, lack of concentration, and loss of memory. Some individuals may experience skin changes such as painful swelling and pink coloration of the fingers and toes. Mercury is mainly excreted through the urine and feces.

Thus, these heavy metals, such as Cd, Pb and Hg are considered toxic not only to humans but also to animals, fishes and environment in general (Govind and Madhuri., 2014). The toxic effects include mutagenicity, carcinogenicity, teratogenicity, immunosuppression, poor body condition and impaired reproduction.

In order to eliminate or minimize such toxicity, scientists have been working to find the best way to attend to these situations.

Three common drugs for treatment of metal poisoning are: BA. (Dimercaprol), Calcium EDTA (Calcium Disodium Versenate) and Penicillamine. Each of these agents work by binding actions that permit the metals to be eliminated from the body through the urine. Treatment of subjects affected by metal toxicity should be symptomatic and supportive. Occupational exposure to heavy metals requires prevention through the use of masks and protective clothing. In cases of swelling of the brain (cerebral edema), treatment with a diuretic called Mannitol, and corticosteroid drugs, along with intracranial monitoring, is required. Hemodialysis is preferred for patients with kidney damage due to metal ion toxicity. In 1991 the FDA approved the drug succimer (Chemet) for the treatment of children with severe lead poisoning. Chemet is manufactured by Johnson & Johnson Co. There is no proven effective therapy for the treatment of cadmium poisoning.

While clinical protocols exist for the use of EDTA, DMPS, DMSA and British Anti-Lewisite (BAL) for treatment of cadmium toxicity, EDTA is the most widely accepted treatment (Bernhoft, 1999) for clinical use. The efficacy of EDTA is apparently improved with concomitant use of glutathione [125] which also protects against nephrotoxicity; efficacy may also be improved with concomitant use of antioxidants. While DMPS has been widely used in Germany for the past fifty years and is available over the counter, published absorption of ingested DMPS varies from 39% to 60% (FDA document, 1999). The antidotal potential of the chelating agents dimercaptosuccinic acid (DMSA) and sodium dimercaptopropanesulfonate (DMPS) is discussed (Bernhoft, 2011).

Compared with traditional chelating agents (dimercaprol, penicillamine, and CaNa2EDTA), DMSA and DMPS have relatively low toxicity, allowing chelation therapy to be administered for extended time periods. Other workers have reported that cerebellar damage characterizing methylmercury-poisoned animals could be prevented by DMSA treatment (Magos et al., 1978). Oral treatment with DMSA or DMPS lowered the kidney mercury level substantially in mice injected with Mercury chloride (Aaseth et al., 1982).

Oxalic and acetic acids commonly are known as moderate and week chelating agents (Oustan et al., 2011). There are limits set for presence of heavy metals in herbal products. This is listed in Annexure 1.

The Food and Drug Administration (FDA) has also approved Sodium polystyrene sulfonate (SPS) in the late 1950s (Hunt et al., 2019) for the treatment of hyperkalemia. It is typically administered to patients suffering from hyperkalemia as an oral solution or in an enema. As the resin passes along the intestine after oral administration or is retained in the colon by rectal administration, the sodium ions are partially released and replaced by potassium ions. For the most part, this action occurs in the large intestine, which excretes potassium ions to a greater extent than does the small intestine. It is also an effective topical microbicide and spermicide, inhibiting the genital transfection of, among others, HIV. Poly (sodium 4-styrene sulfonate) (T-PSS) is reported for its antimicrobial activity in primary culture systems and in a genital herpes murine model. Results obtained indicate that T-PSS is highly effective against herpes simplex viruses, Neisseria gonorrhoeae, and Chlamydia trachomatis in vitro, inactivates virus at higher concentrations, and exhibits a broad spectrum of antimicrobial activity (Herold et al., 2000).

Further, polystyrene sulfonate polymer has also been prescribed for the treatment of various medical conditions, including antibiotic-associated diarrhea, caused by toxins expressed from pathogenic bacteria, such as Clostridium difficile. A published patent application no. WO2008030512A2 by Ho et al discloses preparation of SPS tablets containing at least about 70% of polystyrene sulfonate polymer, binder and moisture for treating medical conditions including antibiotic-associated diarrhea.

The binding of â-lactoglobulin to a synthetic polyelectrolyte, polystyrene sulfonate, displays a pH dependence where low pH was found to make the binding stronger as compared to binding at alkaline pH’s (Hallberg and Dubin, 1998). In 2004, Mikrut and Brockmiller-Sell studied the dose response between SPS and serum potassium concentration. Their data demonstrated an average reduction in potassium concentration of approximately 1 mmol/L and 1.48 mmol/L following a 30 gm and a 60 gm dose of SPS, respectively. Serum potassium levels were reduced by 0.39, 0.69, 0.91, and 0.22 mEq/L following 15-, 30-, and 60-g oral doses and a 30-g rectal dose of SPS, respectively (Mistry et al., 2016).
Also, ponds and the surroundings are one of the most important protectors of biodiversity. Freshwater is significantly important for all living creatures on earth and human interferences like discharge of large quantities of untreated effluents from industrial and domestic sector have enhanced the rate of contaminating the pond and lake ecosystems.
In India, there are temples by the side of these water bodies and the practice of immersion of idols and waste generated from fairs organized on the fields next to these water bodies contaminate the natural ecosystem. Currently naturally growing plants are used in disturbed aquatic environs to clean up the unwanted toxic pollutants. These include sunflower, ragweed, cabbage, geranium etc. (Lasat 2002). The roots of the plants serve as sieves, trapping algae, and other suspended particles in dirty water (Qin et al. 2016).
Phytoremediants scavenge the harmful substances such as nutrients and heavy metals from disturbed surface waters. Some of the examples are aquatic macrophytes such as Limnocharis flava for the removal of low cadmium (Cd) levels from water. Ipomea aquatica showed good Cr (VI) uptake ability in wastewater effluent (Bhat et al. 2005) etc. Table A gives details of other metal ions and plants used to remove them. These take up heavy metals mainly through the root, although uptake through the leaves may also be of significance.
Water pollution has become one of the major issues faced by the world because most of the rivers have been contaminated by pollution from industrial areas and chemical use for agricultural purposes (Kusin et al., 2016a). The presence of heavy metals such as Cu, Zn, Pb, Mn and Fe in aquatic environments could bring adverse effects on human health, aquatic life as well as environment (Kusin et al. 2016b).
Since the lifecycle of these plants is generally very short; after their death and decomposition, chemical elements return in part to the water or are accumulated in bottom sediments, hence alternate methods of metal ion removal that is cost-effective and not harmful to environment will be always useful.

The uses of SPS are numerous. While softening of water by percolating the hard water through a bed of the sodium form of cross-linked polystyrene sulfonate is reported, where the Ca2+ and Mg2+ ions adhere to the sulfonate groups and displaces sodium ions resulting in softened water. Sodium polystyrene sulfonate is also used as a superplastifier in cement, as a dye improving agent for cotton, and as proton exchange membranes in fuel cell applications. In its acid form, the resin is used as a solid acid catalyst in organic synthesis.

There is another potassium binder approved for the treatment of hyperkalemia in adults, sodium zirconium cyclosilicate (SZC), which is a crystal (rather than a resin) that binds with high affinity and high specificity to potassium throughout the gastrointestinal tract (Spinowitz et al., 2019). Potassium is exchanged for hydrogen and sodium, which elicits a rapid and sustained reduction in serum potassium. The safety and efficacy of SZC has been demonstrated in a number of clinical trials, with recently published data extending to 52 weeks of treatment (Packham et al., 2015; Kosiborod et al., 2014)

Patiromer has similar property of binding potassium ions (Sabir, 2019). Patiromer is a non-absorbed polymer that binds potassium is under investigation for the treatment of hyperkalemia. Patiromer consists of smooth, spherical beads ~100 µm in diameter that are free-flowing and do not swell appreciably when placed in liquids is disclosed by Bushinsky et al.,2015.

High molecular weight insoluble polymers which contain functional anionic groups that are capable of undergoing exchange reactions with cations. Each gram of resin binds approximately 0.65 mmol of K in vivo, although the effect is highly variable and unpredictable.

Commercially available resins for removal of metal ions such as Diaion CR11 (Mitsubishi Corporation, Japan), ion-exchange resins (Wastech, USA), chelating ion-exchange resins (Purolite, USA).

In view of above there arises a need for a stable pharmaceutically acceptable composition which is able to remove or eliminate the metal ions toxicity from humans, marine animals. Also, present invention provides a composition which is able to remove or eliminate the metal ions toxicity from soil and other water bodies for better growth and development of plants. There is an urgent requirement for a composition which act rapidly, is industrially scalable and therefore, cost effective.

In view of the aforesaid drawbacks of various chelating agents used in the art to remove metal ion toxicity, present invention discloses the composition which eliminate or reduce the metal ion toxicity efficiently and a method thereof which is rapid and effective in removing the toxicity of metal ions in soil, herbal extracts, herbal raw materials, toxicity in fishes in lakes, ponds and other animals etc.

OBJECTOF THE INVENTION
In order to obviate the drawbacks in the existing state of art, the main object of the present invention is to provide an acceptable pharmaceutical composition of sodium polystyrene sulphonate (SPS) with an excipient to eliminate or to minimize the concentration of heavy metals such as mercury, lead, cadmium or all types of divalent heavy metal ions.

Yet another object of the invention is to provide method for preparing said composition.

Yet another object of the preset invention is to provide said composition in appropriate dose and dosage form capable of reducing the concentration of metals such as mercury, lead, cadmium or all types of divalent heavy metal ions in solutions of different temperatures ranging from ambient temperature to 40°C preferably at ambient temperature.

Yet another object of the present invention is to provide said composition of SPS in reducing the concentration of metals as a function of different concentrations of SPS ranging from 1g/100 ml to 12g/100 ml, preferably 6g/dl.

Yet another object of the invention is to provide a composition of SPS in reducing the concentration of metals at various pH’s.

Yet another object of the invention is to provide composition of SPS capable of rapidly reducing the concentration of metals and provides a method which is industrially scalable and cost effective.

Yet another object of the invention is to provide a dosage regimen, as a single dose or in divided doses.

Yet another object of the present invention is to provide a pharmaceutical acceptable composition comprising of calcium polystyrene sulphonate (CPS) along with excipients in appropriate dose and dosage form to eliminate/reduce metal ions concentration from the solutions and the method thereof.

Yet another object of the invention to provide a composition comprising of sodium zirconium cyclosilicate (SZC) in appropriate dose and dosage form for removing toxicity due to metals such as mercury, lead and cadmium or all types of divalent heavy metal ions.

Yet another object of the invention to provide a composition comprising of alternate potassium binder such as calcium polystyrene sulphonate etc. in appropriate dosage for removing toxicity due to metals such as mercury, lead and cadmium or all types of divalent heavy metal ions.

SUMMARY OF THE INVENTION:
Accordingly, present invention discloses a pharmaceutically acceptable composition comprising of Sodium Polystyrene Sulfonate (SPS) and method of preparing the same. The said composition has been found effective in eliminating or minimizing the concentration of metals in a solution, including soil samples, herbal extracts and herbal raw materials which have high amounts of metal ions.

Sodium polystyrene sulfonate (SPS) is a benzene, diethenyl-, polymer with ethenylbenzene, sulfonated, sodium salt and has the following structural formula. The sodium polystyrene sulfonate exists as a cation exchange resin.

Structure of SPS

Experimental data described below establish that said composition comprising of sodium polystyrene sulphonate (SPS) is effective in for eliminating or reducing metal induced poisoning or toxicity in humans. The present invention also discloses the effective dosage range of the said composition as well as optimal conditions for maximal removal of metal ions involved in toxicity.

Present invention discloses a composition of SPS for removing metal ion toxicity wherein SPS is added in various ratios from 1 g to 12 g/100 ml of 1 ppm metal solution preferably 6g/dl. Various viscosity modifiers used in liquid pharmaceutical dosage forms include cellulose derivatives, clays, natural gums, synthetic polymers and miscellaneous compounds such as colloidal silicon dioxide and silicates.

Said composition of present invention comprises of SPS and any one or more of the excipients selected from above. The resultant composition can be administered either in powder form or in solution form.

For suspension composition, the adjuvants used are selected from Benzoic acid-0.01- 0.1%, Butyl paraben 0.006–0.05%, Glyceryl palmitostearate 1.0–3.0%, Guar gum-1 -2.5%, Hydroxyethylmethyl cellulose 1-2%, Lactic acid-0.015–6.6, Lecithin- 0.25–10.0%, Maltitol- 1-10%, Methyl cellulose 1-2%, Polycarbophil 0.1 -1%, Potassium bicarbonate:25 -50%, Povidone- 0.1 to 5%, Sodium alginate-1- 5%, Sorbitol- 10-50%, Thaumatin- 0.5 to 3 ppm.

The invention also discloses a similar dose of sodium zirconium cyclosilicate (SZC), potassium absorbing polymer such as Patiromer, and calcium polystyrene sulphonate that also be used as substitute for removing toxicity due to metals.

The composition is also useful for reducing or removing the metal ions concentration in soil, herbal extracts and herbal raw materials. These samples could be pre-treated with the powder or a suspension of sodium polystyrene sulphonate or the substitutes as mentioned in above and reduce the concentration of metal ions.

The present invention also relates to a method which can be used to reduce/remove metal toxicity in fishes in lakes, ponds and other animals.

Following is the proposed methodology for removal of metal ions from ponds using SPS/CPS

1. SPS brushes can be attached to silica nanoparticles which will have high affinity towards metal ions. This could be similar to one described by Isahak et al (2016).
2. Tablets of SPS could be made and added to lakes and ponds. These will enable to adsorb metal ions and reduce the concentration of metal ions in the water. Since these particles are insoluble, they will settle down and hence would act like a bed to remove metal ions.
3. Sodium nanopowders are available commercially. These could be also uses in lakes and ponds and soil. For soil, these nanopowders could be added along with nutrients so that plants do not absorb metal ions and there is minimal contamination of ions in herbal plants used for drugs.
4. The nanoshapes could be sheets, spherical particles, nanofibers, nanosheets with enhanced surface area for better adsorption and activity.
5. The proportionality of ion-exchange capacity (IEC) to degree of sulfonation is primarily controlled by the SPS particle size. Larger IEC values were obtained for larger particles of SPS rather than smaller ones (Hazarika et al., 2012)

BRIEF DESCRIPTION OF DRAWINGS
Figure 1 displays the effect of test formulation on histology of liver tissue in lead-induced toxicity induced rats.
Figure 2 displays the effect of test formulation on histology of kidney tissue in lead-induced toxicity in rats.
Figure 3 displays the effect of test formulation on histology of testis in lead-induced toxicity in rats.
Figure 4 displays the effect of test formulation on histology of epididymis in lead-induced toxicity in rats.
Figure 5 displays the effect of test formulation on histology of ovary in lead-induced toxicity in rats.
Figure 6 displays the effect of test formulation on histology of uterus in lead-induced toxicity in rats.
Figure 7 displays a chart of plasma concentrations of Mercury in plasma following treatment with test items. The error bars represent mean ± SD.

DETAILED DESCRIPTION OF THE INVENTION ILLUSTRATIONS AND EXAMPLES
While the invention has been disclosed with reference to certain embodiments, it will be understood by those skilled in the art that various changes may be made, and equivalents may be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt to a particular situation or material to the teachings of the invention without departing from its scope.

Throughout the specification and claims, the following terms take the meanings explicitly associated herein unless the context clearly dictates otherwise. The meaning of “a”, “an”, and “the” include plural references. Additionally, a reference to the singular includes a reference to the plural unless otherwise stated or inconsistent with the disclosure herein.

Accordingly, present invention discloses a pharmaceutically acceptable composition comprising of Sodium polystyrene sulfonate (SPS) and method of preparing the same. The said composition has been found effective in eliminating or minimizing the concentration of metals in a solution, including soil samples, herbal extracts and herbal raw materials which have high amounts of metal ions.

Experimental data described below establish that said composition comprising of sodium polystyrene sulphonate (SPS) is effective in for eliminating or reducing metal induced poisoning or toxicity in humans. The present invention also discloses the effective dosage range of the said composition as well as optimal conditions for maximal removal of metal ions involved in toxicity.

Present invention discloses a composition of SPS for removing metal ion toxicity wherein SPS is added in various ratios from 1 g to 12 g/100 ml of 1 ppm metal solution preferably 6g/dl. Various viscosity modifiers used in liquid pharmaceutical dosage forms include cellulose derivatives selected from methylcellulose, microcrystalline cellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose and hydroxypropyl methylcellulose, clays selected from hectorite, bentonite, aluminium and/or magnesium silicate, natural gums selected from acacia, guar gum, tragacanth, xanthan gum, alginates, carrageenan and locust bean gum, synthetic polymers selected from carbomers, polyvinyl pyrrolidone, polyvinyl alcohol and poloxamer), and miscellaneous compounds such as colloidal silicon dioxide and silicates. Said composition of present invention comprises of SPS and any one or more of the excipients selected from above. The resultant composition can be administered either in powder form or in solution form.

For suspension composition, the adjuvants used are selected from Benzoic acid-0.01- 0.1%, Butyl paraben 0.006–0.05%, Glyceryl palmitostearate 1.0–3.0%, Guar gum-1 -2.5%, Hydroxyethylmethyl cellulose 1-2%, Lactic acid-0.015–6.6, Lecithin- 0.25–10.0%, Maltitol- 1-10%, Methyl cellulose 1-2%, Polycarbophil 0.1 -1%, Potassium bicarbonate:25 -50%, Povidone- 0.1 to 5%, Sodium alginate-1- 5%, Sorbitol- 10-50%, Thaumatin- 0.5 to 3 ppm.

Formulations applied through rectal route are formulated as a polymer solution consisting of thermoreversible polymers selected from poloxamers, in combination with mucoadhesive polymers that enable gel attachment to the rectal mucosa. These in situ thermoreversible liquid-gel formulations, also called thermoreversible “liquid suppositories”, are liquid at low temperatures (<10C) and turn to gel at body temperature. Poloxamer (thermoreversible polymer), sodium alginate (mucoadhesive polymer) and hydroxypropyl-beta-cyclodextrin (solubility enhancer). These are some of the excipients used for such formulations, hence can be used for SPS formulations for metal removal.

Present invention provides a composition and method for use of SPS in reducing the concentration of metals as a function of various incubation time in the range of 30 min to 720 minutes preferably 30 min.

Another advantage of the invention is that the use of SPS is already established in humans, although for a different indication. The recommended average daily dose is 15 gm to 60 gm given as a single dose or in divided doses for hyperkalemia.

The composition of SPS is administered to the humans with chronic kidney disease, a single dose of 15 g of said composition is given orally per day to children, (1g/kg oral for children), or every 6hr for adults. Through rectal route, it is 30-50 g every 6hr in adults, wherein the maximum dose is 50 g/day in adults and 1 g/kg every 2-6 hr for children; at least 4 doses equivalent to 15 g doses is administered to the subject, including wherein at least two of the holin-modified bacteriophage have specificity for different bacterial host cells, or the subject has a mixed bacterial infection.

The invention also discloses a similar dose of sodium zirconium cyclosilicate (SZC) that can be used for removing toxicity due to metals. This is due to the fact that sodium zirconium cyclosilicate (SZC), a potassium binder is approved for the treatment of hyperkalemia in adults. Present application discloses a method for reducing the concentration of metal ions in all sources using substitute of SPS like sodium zirconium cyclosilicate (SCZ). SCZ is a crystal that binds to potassium with high affinity and specificity throughout the gastrointestinal tract. The safety and efficacy of SZC has been demonstrated in a number of clinical trials, with recently published data extending to 52 weeks of treatment.

The present invention discloses other potassium absorbing polymer such as Patiromer has similar property of removing toxicity due to metal ions. Patiromer is a non-absorbed polymer that binds potassium is under investigation for the treatment of hyperkalemia. Patiromer consists of smooth, spherical beads ~100 µm in diameter that are free-flowing and do not swell appreciably when placed in liquids.

The composition is also useful for reducing or removing the metal ions concentration in soil, herbal extracts and herbal raw materials. These samples could be pre-treated with the powder, or a suspension of sodium polystyrene sulphonate and reduce the concentration of metal ions.

The present invention also relates to a method which can be used to reduce/remove metal toxicity in fishes in lakes, ponds and other animals. Said method can also be applied to removal of metal ions from the soil used in green house vegetables.

Aforementioned objective is accomplished by providing experimental conditions for maximum efficiency in removal of metals from solutions.

NON-LIMITING EXAMPLES
EXAMPLE 1
Study 1: Effect of pH on metal removing property of SPS
Effect of pH on metal removal
sub batches (pH ) Results in %
Product Code Metal Ion pH SPS
1134-06 G Lead 4.0 Blank (1 ppm Pb) 100.00
1134-06 H 7.0 Blank (1 ppm Pb) 100.00
1134-06 I 9.0 Blank (1 ppm Pb) 100.00
1134-06 J 4.0 100 ml Blank + 6 gm SPS BLQ
1134-06 K 7.0 100 ml Blank + 6 gm SPS BLQ
1134-06 L 9.0 100 ml Blank + 6 gm SPS BLQ
1134-06 M Cadmium 4 Blank (1 ppm Cd) 100.00
1134-06 N 7 Blank (1 ppm Cd) 100.00
1134-06 O 9 Blank (1 ppm Cd) 100.00
1134-06 P 4 100 ml Blank + 6 gm SPS BLQ
1134-06 Q 7 100 ml Blank + 6 gm SPS BLQ
1134-06 R 9 100 ml Blank + 6 gm SPS BLQ

EXAMPLE 2
Study 2: Effect of incubation temperature on metal removing property of SPS
Effect of Temperature on metal removal Results (ppm)
Product Code Metal Ion Sub batches
1134-07A Mercury 40 °C Blank (1 ppm Hg) 0.87
1134-07 B 40 °C 1 h 100 ml Blank + 6 gm SPS 0.06
1134-07 C Lead 40°C Blank (1 ppm Pb) 0.49
1134-07 D 40 °C 1 h 100 ml Blank + 6 gm SPS BLQ
1134-07 E Cadmium 40 °C Blank (1 ppm Cd) 0.99
1134-07 F 40 °C 1 h 100 ml Blank + 6 gm SPS BLQ

From study 2, it is clear that the efficiency of metal ion reduction by SPS is not affected by temperature tested 40°C in comparison to the regular temperature of 25-30°C.

EXAMPLE 3
Study 3: Effect of Incubation Time of SPS and metal ions on metal removal from solutions
Effect of Incubation Time on metal removal Results (ppm) Results in %
Product Code Metal Ion Procedure Hr
1134-08 A Mercury As such Mercury 1.09 100
1134-08 B 200 ml Blank +12 gm SPS 1 0.18 16.51
1134-08 C 6 0.19 17.43
1134-08 D 12 0.24 22.01
1134-08 E Lead As such Lead 1.23 100
1134-08 F 200 ml Blank +12 gm SPS 1 BLQ BLQ
1134-08 G 6 BLQ BLQ
1134-08 H 12 BLQ BLQ
1134-08 I Cadmium As such Cadmium 0.94 100
1134-08 J 200 ml Blank +12 gm SPS 1 BLQ BLQ
1134-08 K 6 BLQ BLQ
1134-08 L 12 BLQ BLQ

From all the above experiment, it is evident that composition of SPS is more effective in removal of lead and cadmium over mercury. Also, removal of metal ions is not affected by incubation time and this reflects that the potency of SPS in adsorption of metal ions could be achieved in almost 1 hour of interaction, which will help to decide on the dose regime in animal experiments.

EXAMPLE 4
Study 4: Effect of various concentration of SPS in metal removal
Effect of Concentration of SPS on metal removal Results (ppm)
Product Code Metal Ion SPS
1134-09 A Mercury Blank (1 ppm Hg) 1.00
1134-09 B 100 ml Blank + 1 gm SPS 0.20
1134-09 C 100 ml Blank + 3 gm SPS 0.22
1134-09 D 100 ml Blank + 6 gm SPS 0.23
1134-09 E 100 ml Blank + 12 gm SPS 0.21
1134-09 F Lead Blank (1 ppm Pb) 0.94
1134-09 G 100 ml Blank + 1 gm SPS BLQ
1134-09 H 100 ml Blank + 3 gm SPS BLQ
1134-09 I 100 ml Blank + 6 gm SPS BLQ
1134-09 J 100 ml Blank + 12 gm SPS BLQ
1134-09 K Cadmium Blank (1 ppm Cd) 0.97
1134-09 L 100 ml Blank + 1 gm SPS BLQ
1134-09 M 100 ml Blank + 3 gm SPS BLQ
1134-09 N 100 ml Blank + 6 gm SPS BLQ
1134-09 O 100 ml Blank + 12 gm SPS BLQ

Conclusion of Study 4: Metal binding property of SPS is efficient in all concentration of SPS used.

Animal Study:
EXAMPLE 5:
DOSE PREPARATION:
Test and Standard formulations were suspended in distilled water. Lead solution was prepared by using lead chloride in distilled water with the help of sonicator. These suspensions and solution were prepared freshly prior to administration.
Below are the details of Standard and Test formulations

Innovator Product characterization SAVA Product Characterization
Sodium Polystyrene Sulfonate (Kayexalate) Sodium Polystyrene Sulfonate composition
General description
Brand Name Kayexalate SODIUM POLYSTYRENE
Generic name Sodium Polystyrene Sulfonate USP Sodium Polystyrene Sulfonate Powder
Label Claim Each gm contains
Sodium Polystyrene Sulfonate ………..1 gm Each gm contains
Sodium Polystyrene Sulfonate USP ………..1 gm
Excipients….. 0.01% to 5%.
Mfg. By Sanofi Aventis, Canada Inc. SAVA Healthcare Ltd., Pune
Fill Weight 454 gm 454 gm
Storage temp Store at 15-30°C Store at 15-30°C
Shelf Life 5 Years 5 Years
Physical Characterization
Description Light brown coloured finely ground free flowing powder Golden brown, odorless fine powder with a characteristic taste
Particle size Sieve analysis: 100 % Passes through 100 # Sieve analysis: 100 % Passes through 100 #
Container White opaque HDPE jar (Induction seal Present)/625 cc White opaque HDPE jar (Induction seal Present)/625 cc
Scoop Not given With Jar (Teaspoon to be used) Not given With Jar (Teaspoon to be used)
Adult dose Oral: 4 level teaspoon =15 gm; 1 to 4 times daily in water or syrup
Rectal: 8 level teaspoon= 30 gm once or twice daily in 150-200 ml aqueous vehicle Oral: 4 level teaspoon =15 gm; 1 to 4 times daily in water or syrup
Rectal: 8 level teaspoon= 30 gm once or twice daily in 150-200 ml aqueous vehicle
Flow Properties
Bulk density 0.714 gm/ml 0.689 gm/ml
Tapped density 0.830 gm/ml 0.833 gm/ml
Hausner’s Ratio 1.16 1.20
Carrs index 13.97 % 17.16 %
LOD 6.09% 6.75
Pack In white opaque HDPE jars of 454g. In white opaque HDPE jars of 454g.
Head space Calculation Total volume of the container: 700 ml
Volume occupied by powder: 635 ml
Head space: 700-635 = 65; = 9.28 % Total volume of the container: 700 ml
Volume occupied by powder: 659 ml
Head space: 700-657=200; = 5.85 %
After Reconstitution
Description Light brown coloured slurry, powder settles at the bottom of the container leaving clear to slightly turbid supernatant Light brown coloured slurry, powder settles at the bottom of the container leaving clear to slightly turbid supernatant
Sedimentation time 10-15 min 10-15 min
pH 7.5 5.82
Taste Bland Bland
Chemical Characterization
Assay: (Sodium Content) 10.42% (Limit: 9.4 % -11.5 %) 9.58 %
Water 5.72 (Limit: NMT 10.0%) 3.68 %
Potassium Exchange Capacity 122.0 mg (Limit: 110 to 135 mg) 119.0 mg/gm
Related Substances
Styrene content Less than 1 ppm Less than 1 ppm

TEST SYSTEM AND MANAGEMENT:
1. Species : Rat
2. Strain : Wistar
3. Source : National Institute of Biosciences, Pune-411051
4. Sex : Male and Female
5. Body weight range : 150 to 200 g at the beginning of the study
6. Identification : By unique identification number marked by writing on cage tag and by corresponding colour body markings.
7. No. of animals : 1. Evaluation of LD50 dose of lead: Three female Wistar rats per group
2. Evaluation of effect of test formulation on lead-induced toxicity: Eight animals per group (Male: female- 1:1).
8. Acclimatization : The Rats were housed individually in their cages for six days prior to start of dosing in the experimental room after veterinary examination.
9. Environmental
Conditions : Room temperature between 25±30 C, relative humidity 55 ± 5% and illumination cycle set to 12 hours light and 12 hours dark.
10. Accommodation : Rats was housed in polypropylene cages with stainless steel grill top, facilities for food and water bottle, and bedding of clean paddy husk.
11. Diet : Pelleted feed supplied by Nutrivet Life Sciences, Pune, was provided ad libitum during acclimatization and during the study, except for period specified below:

Rats were fasted overnight prior to administration of lead chloride solution and food was offered 30 min after dosing
12. Water : Potable water passed through ‘Aquaguard’ water filter
was provided ad libitum in polypropylene bottles with stainless steel sipper tubes.

STUDY DESIGN:
Evaluation of LD50 dose of lead:
Female Wistar rats were divided into groups containing three in each. Group-1 was vehicle treated group and remaining were lead chloride treated groups. Doses selected for LD50 estimation were 5, 50, and 100 mg lead/kg body weight. Starting dose was 50 mg lead/kg body weight. The lead solution was administered to overnight fasted rats once daily for 7 days by intraperitoneal route.

Table 1: Study Design and Treatment details
Groups No. of Animals Test Material Dose Route of administration
Group-I
(Normal Control) 3 Vehicle (water) 1 ml/100 g i.p.
Group-II
(Lead chloride-treated Group) 3 Lead chloride solution 5 mg lead/kg i.p.
Group-III
(Lead chloride-treated Group) 3 Lead chloride solution 50 mg lead/kg i.p.
Group-IV Confirmation group
(Lead chloride-treated Group) 3 Lead chloride solution 100 mg lead/kg i.p.
Confirmation group 3 Lead chloride solution - i.p.

Evaluation of effect of test formulation on lead-induced toxicity:
Wistar albino rats (male:female-1:1) were used to investigate effect of test formulation on lead-induced toxicity. Based on results of lead toxicity, 50 mg lead/kg dose was selected for the study. The animals were divided in 4 groups containing 8 animals (4 males and 4 females). Group-1 was normal control and received water (1 ml/100 g) which was used for preparation of lead solution. All remaining groups were given lead solution at the dose of 50 mg lead/kg intraperitoneally once daily for seven days. Group-2 was lead-treated group and did not received any other treatment. Group-3 and Group-4 were test formulation treated group and standard formulation treated group respectively. They received test formulation or standard formulation at the dose of 3250 mg/kg body weight twice daily, 6 h apart. On each day, first dose of test formulation and Standard formulation were given 2 minutes after lead solution injection and second dose were administered after 6 h of first dose. The animals were dosed using a stainless-steel intubation needle fitted onto a suitably graduated syringe. The dosage was administered to individual rat according to its most recently recorded body weight.

Table 2: Study Design and Treatment details
Groups No. of Animals Test Material Dose Route of administration
Group-I
(Normal Control) 8
(4 males and 4 females) Vehicle (water) 1 ml/100 g i.p.
Group-II
(Lead-treated Group) 8
(4 males and 4 females) Lead solution 50 mg lead/kg i.p.
Group-III
(Test formulation-treated Group) 8
(4 males and 4 females) Lead solution 50 mg lead/kg i.p.
Test formulation
3250 mg/kg body weight twice daily, 6 h apart p.o.
Group-IV
(Standard formulation-treated Group) 8
(4 males and 4 females) Lead solution 50 mg lead/kg i.p.
Standard
formulation 3250 mg/kg body weight twice daily, 6 h apart p.o.

OBSERVATIONS
Evaluation of LD50 dose of lead:
The number of survivors was noted during the dosing period and these animals were then maintained for a further 14 days with a once daily observation.

Evaluation of effect of test formulation on lead-induced toxicity:
One hour after last dose of treatment, blood samples (3 ml) were collected in presence and absence of anticoagulant. Blood with anticoagulant was used for measurement of hematological parameters. Blood with anticoagulant was used for preparation of serum. Serum was analysed for estimation of BUN, creatinine, uric acid, total serum protein, albumin, total bilirubin, serum glutamate oxalate transaminase/aspartate aminotransferase (SGOT/AST), serum glutamate pyruvate transferase/alanine aminotransferase (SGPT/ALT), alkaline phosphatase (ALP), amylase (AMY), lactate dehydrogenase (LDH), calcium (Ca), inorganic phosphorus (P), magnesium (Mg) and chloride (Cl). After blood collection, animals were sacrificed to isolate liver, kidney, testis, ovary, epididymis and uterus for histopathological examination. Two representative samples of organs from each group will studied for histopathological evaluation.

INTERPRETATION OF RESULTS
The results were expressed as the mean±SEM. The statistical significance was assessed by Student’s t-test and One way ANOVA followed by Dunnett’s test. Graphpad prism 9.1. software (Free version) was used for data analysis.

RESULTS
Evaluation of LD50 dose of lead:
First dose level of lead i.e. 50 mg/kg lead caused no mortality during seven days treatment as well as fourteen days observation period. However, peritoneal cavity was found inflamed and filled with blood containing fluid. At higher dose of lead i.e. 100 mg/kg of lead two animals out of three died during experimental period. Confirmation group also produced no mortality of animals at 50 mg/kg lead during seven days treatment and fourteen days observation period.

Evaluation of effect of test formulation on lead-induced toxicity:
A. Survival rate:
Number of animals survived during experimental period is given in Table 3. There was morality of 1 animal in lead induced and test formulation treated group. In standard formulation treated group, two animals out of eight were died during experimental period.

Table 3: Animals survived during experimental period
Groups No. of Animals Used No. of Animals survived
Group-I
(Normal Control) 8 8
Group-II
(Lead-treated Group) 8 7
Group-III
(Test formulation-treated Group) 8 7
Group-IV
(Standard formulation-treated Group) 8 6

B. Effect on hematology parameters:
Effect of test formulation on hematology is summarized in Table 4. Lead administration caused significant decrease in hemoglobin, HCT, TEC and MCH levels. The decreased hemoglobin levels were significantly improved by both test and standard formulations. HCT level was significantly increased by standard formulation and no difference in observed in HCT level of lead treated group and test formulation treated group. Both test as well as standard formulations did not produce any change in MCH level. The decreased MCHC level was significantly improved by test formulation, whereas standard formulation did not cause improvement in MCHC level.

Table 4: Effect of test formulation on haematology parameters
Parameter Normal Control Lead treated Lead + Test Formulation treated Lead + Standard Formulation treated
Hemoglobin 15.21±0.61 11.31±0.36a*** 13.56±0.61b* 13.31±0.56b*
HCT 40.44±1.91 32.24±1.03a** 35.06±1.36 37.21±1.84b*
TEC 5.53±0.29 4.72±0.18a* 5.37±0.38 5.49±0.29
TLC 6.18±0.71 5.27±0.88 7.84±0.70 7.19±1.14
MCV 73.15±2.16 68.74±3.05 66.40±3.07 68.35±3.04
MCH 27.62±1.08 24.08±0.90a* 25.74±1.49 24.46±0.98
MCHC 37.76±0.93 35.17±0.98 38.69±0.95b* 35.90±0.88
Platelet Count 5.04±0.37 4.77±0.57 4.69±0.49 5.51±0.41
MPV 8.34±0.77 7.14±0.80 7.16±1.01 7.73±0.80

Values are expressed as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001. aCompared with normal control, bCompared with disease control group. Values of normal control group and lead treated group were compared by Student’s t-test. One-way analysis of variance (ANOVA) followed by Dunnett’s comparison test was used for comparison between drug-treated (Test formulation and standard’s formulation) groups and lead treated group or normal control group.
p < 0.05 was considered significant.

Effect on biochemical parameters:
Effect of test formulation on biochemical parameters in lead toxicity induced rats is summarized in Table 5. Lead administration caused significant decrease in serum albumin level. This decreased serum albumin level was increased significantly by both test as well as standard formulations.

There was significant increase in serum levels of SGPT, ALP, creatinine, BUN, LDH and SGOT in lead toxicity induced animals. The elevated serum levels of SGPT and creatinine were significantly decreased by both test as well as standard formulations. Test formulation was found to be effective in decreasing serum levels of BUN and SGOT. There was no significant change in serum levels of BUN and SGOT of lead toxicity induced animals and standard formulation treated animals. Serum levels of ALP and LDH were found unchanged by the treatment with test formulation and standard formulation.

There was significant increase in serum levels of SGPT, ALP, creatinine, BUN, LDH and SGOT in lead toxicity induced animals. The elevated serum levels of SGPT and creatinine were significantly decreased by both test as well as standard formulations. Test formulation was found to be effective in decreasing serum levels of BUN and SGOT. There was no significant change in serum levels of BUN and SGOT of lead toxicity induced animals and standard formulation treated animals. Serum levels of ALP and LDH were found unchanged by the treatment with test formulation and standard formulation.

Table 5: Effect of test formulation on biochemical parameters
Parameter Normal Control Lead treated Lead + Test Formulation treated Lead + Standards’ Formulation treated
Total Protein 6.28±0.24 5.83±0.13 6.18±0.18 6.04±0.14
Albumin 3.54±0.21 2.64±0.18a** 3.48±0.3b* 3.55±0.2b*
Globulin 2.73±0.36 3.2±0.23 2.7±0.23 2.5±0.24
SGPT 33.73±5.8 106.2±12.86a*** 67.14±8.6a**, b* 69.6±8.25a** b*
ALP 109.09±8.38 148.4±15.4a* 124.1±17.38 112±7.75
Calcium 9.36±0.45 7.89±0.53 9.25±0.25b* 8.97±0.35
Phosphorus 3.33±0.38 3.46±0.27 3.58±0.38 3.41±0.24
Magnesium 3.24±0.34 2.74±0.32 3.02±0.2 2.92±0.35
Creatinine 0.27±0.05 0.98±0.07a*** 0.68±0.04a***, b** 0.5±0.07a*, b***
BUN 13.33±1.24 20.59±1.84a** 17.76±1.37b* 17.69±1.26
Uric acid 2.63±0.38 3.65±0.32 2.91±0.25 2.88±0.42
LDH 88.44±9.20 123.9±13.17a* 111.1±10.88 98.71±6.81
Total Bilirubin 0.31±0.06 0.48±0.6 0.42±0.04 0.4±0.05
SGOT 57.46±10.64 106.6±9.62a** 97.36±6.02b** 92.54±8.46a*
Amylase 686.8±74.13 570.3±76.47 640.2±63.72 691±92.02
Chloride 104.08±1.10 104±1.22 105.3±1.31 106.05±1.19
Values are expressed as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001. aCompared with normal control, bCompared with disease control group. Values of normal control group and lead treated group were compared by Student’s t-test. One-way analysis of variance (ANOVA) followed by Dunnett’s comparison test was used for comparison between drug-treated (Test formulation and standard’s formulation) groups and lead treated group or normal control group.
p < 0.05 was considered significant.

Effect on Tissue Histopathology:
It was observed that abdominal cavities of animals of lead-treated group, test formulation treated group and standard formulation treated group were filled with fluid indicating severe inflammation of abdominal cavities. There was deposition of white-colored materials on all organs of abdominal cavities. In some animals, these abdominal organs were tightly attached to each other.

1. Effect on Liver Tissue:
Effect of test formulation on histology of liver tissue in lead-induced toxicity in rats is shown in Figure 1. Following changes were observed:
a) Group-1: No Abnormality Detected
b) Group-2: Minimal (+1) to Mild (+2) • Vascular changes-congestion/hemorrhage in hepatic parenchyma.
• Cellular changes or degenerative and granular cytoplasmic changes and vacuolar changes.
• Loss of nuclei and necrotic changes of hepatocytes.
• Inflammatory changes in hepatic tissue, MNC infiltration in hepatic parenchyma.

c) Group-3: No Abnormality Detected
d) Group-4: Minimal (+1)

It is concluded that lead administration to rats caused histological changes in liver tissue of mild to moderate grade. These changes were prevented by both test as well as standard formulation. Test formulation was found to be more effective than standard formulation in this regard.

Figure 1 shows the effect of test formulation on histology of liver tissue in lead-induced toxicity induced rats.

2. Effect on kidney tissue:
Effect of test formulation on histology of kidney tissue in lead-induced toxicity in rats is shown in Figure 2. Following changes were observed:
a) Group-1: No Abnormality Detected
b) Group-2: Minimal (+1) to Mild (+2)
• Vascular changes-congestion/hemorrhage in renal tissue.
• Cellular swelling, degenerative changes and necrotic changes of renal tubules and glomerular changes.
• Presence of tubular cast and degenerative changes of renal tubules.
• Inflammatory changes in renal tissue (cortex and medulla).

c) Group-3: No Abnormality Detected
d) Group-4: No Abnormality Detected

It is concluded that Lead administration to rats caused mild to moderate grade histological changes in renal tissue. These changes were prevented by both test as well as standard formulation. Both formulations were found equipotent in preventing histological changes in renal tissues.

Figure 2 shows the effect of test formulation on histology of kidney tissue in lead-induced toxicity in rats.

3. Effect on testis:
Effect of test formulation on histology of testis in lead toxicity induced rats is shown in Figure 3. Following changes were observed:
a) Group-1: No Abnormality Detected
b) Group-2: No Abnormality Detected
c) Group-3: No Abnormality Detected
d) Group-4: No Abnormality Detected

It is concluded that lead administration to rats caused no any histopathological changes in testes.
Figure 3 shows the effect of test formulation on histology of testis in lead-induced toxicity in rats.

4. Effect on epididymis:
Effect of test formulation on histology of epididymis in lead-induced toxicity in rats is shown in Figure 4. Following changes were observed:
a) Group-1: No Abnormality Detected
b) Group-2: No Abnormality Detected
c) Group-3: No Abnormality Detected
d) Group-4: No Abnormality Detected
It is concluded that lead administration to rats caused no any histopathological changes in epididymis.

Figure 4 shows the effect of test formulation on histology of epididymis in lead-induced toxicity in rats.

5. Effect on ovary:
Effect of test formulation on histology of ovary in lead-induced toxicity in rats is shown in Figure 5. Following changes were observed:
a) Group-1: No Abnormality Detected
b) Group-2: No Abnormality Detected
c) Group-3: No Abnormality Detected
d) Group-4: No Abnormality Detected

It is concluded that lead administration to rats caused no histopathological changes in ovary.

Figure 5 shows the effect of test formulation on histology of ovary in lead-induced toxicity in rats.

6. Effect on Uterus:
Effect of test formulation on histology of uterus in lead-induced toxicity in rats is shown in Figure 6. Following changes were observed:
a) Group-1: No Abnormality Detected
b) Group-2: No Abnormality Detected
c) Group-3: No Abnormality Detected
d) Group-4: No Abnormality Detected

It is concluded that lead administration to rats caused no any histopathological changes in uterus.

Figure 6 shows the effect of test formulation on histology of uterus in lead-induced toxicity in rats.

Therefore, from the above experimental data, it is inferred that, Intraperitoneal administration of lead at the dose of 50 mg/kg once daily for seven days did not cause mortality in the female Wistar rats during seven days of lead administration and fourteen days of observation period. Seven days intraperitoneal administration of lead once daily at the dose of 50 mg/kg produced histological changes in liver and kidney tissues. This was supported by serum biochemical estimations. These histological changes in liver and kidney tissues were prevented by both test as well as standard formulation. Test formulation was found to be more effective than standard formulation in preventing histological changes in liver tissues. Both formulations i.e., test and standard formulations were found equipotent in preventing histological changes in renal tissues. There were no changes in the tissues of testis, epididymis, uterus and ovary.

It is concluded that the “Test formulation” is having protective effect on liver and kidney damage caused by lead induced toxicity in rats.

Example 6:
Maximum Tolerated Dose of Mercury and Efficacy of N3S0 Powder (Batch No.: GTE0067 & 8108952) in a Mercury Induced Toxicity Rat Model.

PURPOSE
The purpose of this study was to evaluate
a) the maximum tolerated dose (MTD) of Mercury in rats and,
b) Evaluate the efficacy of test compounds in a Mercury induced toxicity rat model.

MTD: Male Wistar rats were dosed with vehicle, Mercury chloride (2, 4, 8 and 16 mg/kg single dose, p.o.,) orally as single doses. Animals were observed for general clinical signs. In all the tested doses, animals were apparently normal and were comparable with vehicle control group. Body weight increase was observed in vehicle and in all mercury chloride treated groups. At end of the experiment animals were subjected for gross pathological observations. No abnormalities were detected in all the groups. The MTD of Mercury Chloride was 16 mg/kg.

Efficacy: Male Wistar rats were dosed orally, once daily, with Mercury chloride (8 mg/kg) followed by N3SO (Batch No.: GTE0067, 3250 mg/kg) and N3SO (Batch No.810895270, 3250 mg/kg) for a period of seven consecutive days.

Animals were sacrificed on day 8 after the last dose. The vehicle control group showed increase of ~ 7 % in body weight whereas the mercury chloride group showed a mean decrease of 8% in body weight. However, the differences in mean body weight were not statistically significant (p>0.05). The animals treated with the test items showed a mean increase of 3-5% in body weight but were not significantly different when compared to the vehicle group.

In hematology, N3SO (Batch No. GTE0067) showed significantly lower HCT% when compared to Mercuric chloride alone (p<0.05). There were no significant differences for the other parameters when compared to Mercuric chloride group (p>0.05). N3SO (Batch No.: 81089527) did not show significant activity when compared to the Mercuric chloride group (p>0.05).

In clinical chemistry, N3SO (Batch No. GTE0067) showed significantly lower ALP and AST levels when compared to Mercuric chloride alone (p<0.05). There were no significant differences for the other parameters when compared to Mercuric chloride group (p>0.05). N3SO (Batch No.81089527) showed significantly lower AST levels when compared to the Mercuric chloride group (p< 0.05). There were no significant differences for the other parameters when compared to Mercuric chloride group (p>0.05).

Histopathology analysis showed that Mercury alone did not cause microscopic changes in liver when compared to vehicle (G1). A higher incidence of microscopic changes (minimal, multifocal) was observed in kidneys when compared to vehicle (G1). G3, N3SO (Batch No. GTE0067) showed higher incidence of microscopic changes in liver when compared to G2 (Mercury alone); changes in kidney were similar to G2. G4, N3SO (Batch No. 81089527) showed microscopic changes in liver which was similar to G2 (Mercury alone); changes in kidney were similar to G2.

In conclusion, the histological observations in kidney and liver of mice treated with N3SO (Batch No. GTE0067) and N3SO (Batch No. 81089527) were minimal in nature and equivocal when compared to the Mercury alone group (G1).

Taken together N3SO (Batch No. 81089527) and N3SO (Batch No. 81089527) showed significant activity for lowering liver enzymes ALT and AST in the Mercury induced toxicity model in Wistar rats. However, no significant efficacy was observed in hematology and histopathology of liver and kidneys. Further studies are required to characterize the efficacy of the test items.

MATERIALS AND METHODS
TEST SYSTEM
Species: Rats
Strain: Wistar,
Age: 6 weeks
Sex: Male

HUSBANDRY
Conditions: Animal room environment was monitored for temperature and relative humidity twice a day. Temperature range was 22+2 °C and humidity range was between 45 and 60%. The animals were provided with 12 hours light and 12 hours dark artificial photoperiod.
Housing: Group of 3 animals in IVC Cages
Diet : SDS (Special Diet Services) Lab Diet was provided ad libitum.
Water : Autoclaved water was provided ad libitum.
Identification: By unique cage number and corresponding tail marking

TEST ITEM PREPARATION
Mercuric Chloride [15 gm; Batch No. QH5Q652296], N3SO Powder [20 gm; Batch no. GTE0067], and N3SO Powder [20 gm; Batch no. 8108952] were provided by the sponsor.

STEP 1: Maximum Tolerated Dose (MTD):
G2, 2 mg/kg (Concentration: 0.2 mg/ml): 2.0 mg of Mercuric chloride was weighed accurately and dissolved in 10 ml of sterile water, vortexed for 2 minutes to achieve 0.2 mg/ml. The final appearance of the solution was clear colorless.
G3, 4 mg/kg (Concentration: 0.4 mg/ml): 4.0 mg of Mercury chloride was weighed accurately and dissolved in 10 ml of sterile water, vortexed for 2 minutes to achieve 0.4 mg/ml. The final appearance of the solution was clear colorless.
G4, 8 mg/kg (Concentration: 0.8 mg/ml): 8.0 mg of Mercury chloride was weighed accurately and dissolved in 10 ml of sterile water, vortexed for 2 minutes to achieve 0.8 mg/ml. The final appearance of the solution was clear colorless.
G5, 16 mg/kg (Concentration: 1.6 mg/ml): 16 mg of Mercury chloride was weighed accurately and dissolved in 10 ml of sterile water, vortexed for 2 minutes to achieve 1.6 mg/ml. The final appearance of the solution was clear colorless.

STEP 2: Efficacy
G2, Mercury chloride alone, 8 mg/kg (Concentration: 0.8 mg/ml): 24.0 mg of Mercury chloride was weighed accurately and dissolved in 30 ml of sterile water, vortexed for 2 minutes to achieve 0.8 mg/ml. The final appearance of the solution was clear colorless.
G3, Mercuric Chloride + Test item N3SO (Batch No.: GTE0067, 8mg/kg+ 3250 mg/kg (Concentration: 0.8 +325 mg/ml): 325 mg of N3SO Powder was weighed accurately and dissolved in 10 ml of sterile water, vortexed for 2 minutes to achieve 325 mg/ml. The final appearance of the solution was uniform suspension.
G4, Mercuric Chloride + Test item N3SO (Batch No.: GTE0067, 8mg/kg+ 3250 mg/kg (Concentration: 0.8 +325 mg/ml): 325 mg of N3SO Powder was weighed accurately and dissolved in 10 ml of sterile water, vortexed for 2 minutes to achieve 325 mg/ml. The final appearance of the solution was uniform suspension

STUDY DEISGN
MTD
Group Test Item Dose
(mg/kg) Route of Administration Dose Volume (ml/kg) Total Number of animals
G1 Vehicle 0, single dose Oral (gavage) 10 5
G2 Mercuric Chloride 2 mg/kg, single dose Oral (gavage) 10 5
G3 Mercuric Chloride 4 mg/kg, single dose Oral (gavage) 10 5
G4 Mercuric Chloride 8 mg/kg, single dose Oral (gavage) 10 5
G5 Mercuric Chloride 16 mg/kg, single dose Oral (gavage) 10 5

The MTD was defined as the dose level which does not cause serious adverse effect in terms of clinical signs and body weight loss.
The MTD of Mercuric chloride was found to be at least 16 mg/kg following single dose administrations.

STEP 2: Efficacy (Mercuric Chloride dose was fixed based on MTD Results)
Group Test Item Dose Route of Administration Treatment Duration Total Number of animals
G1 Vehicle (PBS) NA, once daily Oral (gavage) 7 days 5
G2 Mercuric Chloride alone 8 mg/kg&, once daily Oral (gavage) 7 days 5
G3 Mercuric Chloride + Test item N3SO (Batch No.: GTE0067 * Mercuric Chloride (8 mg/kg, once daily) + N3SO (Batch No.: GTE0067) 3250 mg/kg, once daily) #@ Oral (gavage) 7 days 5
G4 Mercuric Chloride + Test item N3SO (Batch No.: 81089527) * Mercuric Chloride (8 mg/kg, once daily) + N3SO (Batch No.: 81089527) 3250 mg/kg, once daily)#@ Oral (gavage) 7 days 5

*Mercuric chloride was administered first; ten minutes later the test items were administered
# 3250 mg/kg is equivalent to 813 mg of test item per rat (weighing 0.25 kg) as per sponsor’s requirement; @The dose of 3250 mg/kg in rat is equivalent of 500 mg/kg in humans (was estimated using Allometric principles based on the human dose of 1000 mg/kg given by the sponsor); & MTD of Mercuric chloride from stage 1

Treatment
On each day, animals were first dosed orally with Mercuric Chloride solution at 8 mg/kg body weight. Ten minutes post dosing of Mercuric Chloride, animals were treated orally, once daily for 7 days, with test compounds (3250 mg/kg body weight which is equal to 813 mg per rat) as shown in experimental design for a period of seven days.

Clinical Signs
MTD: The results for clinical signs, body weights and gross pathology are shown in tables 1, 2 and 3, respectively.
Table 1: Clinical observations following single oral doses of Mercuric chloride in rats
Group Daily Clinical Signs on Days
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7
G1, Vehicle Control N N N N N N N
G2, Mercury Chloride, 2 mg/kg, Oral, Single Dose N N N N N N N
G3, Mercury Chloride, 4 mg/kg, Oral, Single Dose N N N N N N N
G3, Mercury Chloride, 8 mg/kg, Oral, Single Dose N N N N N N N
G4, Mercury Chloride, 16 mg/kg, Oral, Single Dose N N N N N N N
N: Apparently Normal

Table 2: Body weights of rats following single oral doses of Mercuric chloride
Group Body weight(g) (Mean ± SD)
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7
G1, Vehicle Control 133.0±6.3 138.2±7.3 142.8±6.6 146.6±7.5 149.4±6.9 152.4±6.4 154.8±6.3
G2, Mercury Chloride, 2 mg/kg, Oral, Single Dose 136.4±6.8 141.6±5.3 143.8±6.4 147.0±8.3 149.4±8.2 150.0±7.6 151.4±7.5
G3, Mercury Chloride, 4 mg/kg, Oral, Single Dose 137.0±5.6 141.0±4.8 143.4±4.7 146.2±4.1 147.4±3.6 148.2±4.3 148.6±5.1
G3, Mercury Chloride, 8 mg/kg, Oral, Single Dose 135.2±4.8 139.2±5.1 141.4±5.9 143.8±5.5 145.2±6.0 145.6±5.6 146.8±6.1
G4, Mercury Chloride, 16 mg/kg, Oral, Single Dose 136.6±5.0 139.4±4.8 141.2±4.8 144.0±5.4 145.0±5.3 145.0±5.4 145.0±6.6

Table 3: Gross pathology observations in rats following single oral doses of Mercuric chloride
Group Gross pathology Observations
External Internal
G1, Vehicle Control NAD NAD
G2, Mercury Chloride, 2 mg/kg, Oral, Single Dose NAD NAD
G3, Mercury Chloride, 4 mg/kg, Oral, Single Dose NAD NAD
G3, Mercury Chloride, 8 mg/kg, Oral, Single Dose NAD NAD
G4, Mercury Chloride, 16 mg/kg, Oral, Single Dose NAD NAD
NAD=No abnormality detected

Animals in the vehicle and test item dosed groups were found to be apparently normal throughout the study. Body weight gain was observed across all the groups throughout the study. No internal and external abnormalities in gross pathology were detected in all the tested doses. The NOAEL of Mercury chloride in rats was at least 16 mg/kg.

Efficacy:
The results of clinical signs, body weights and gross pathology are shown in tables 4, 5 and 6, respectively.

Table 4: Clinical observations in rats following treatment with test items
Group Daily Clinical Signs on Days
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8
G1, Vehicle Control N N N N N N N N
G2, Mercuric Chloride alone N N N N N N N N
G3, Mercuric Chloride + Test item N3SO (Batch No.: GTE0067) N N N N N N N N
G4, Mercuric Chloride + Test item N3SO (Batch No.: 81089527) N N N N N N N N
N: Apparently Normal

Table 5: Body weights of rats following treatment with test items

Group Body weight(g) (Mean ± SD)
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8
G1, Vehicle Control 166.2±18.2 166.8±17.8 168.2±17.1 170.6±17.3 174.2±18.2 178.0±17.8 177.4±15.8 178.0±14.1
G2, Mercuric Chloride alone 166.2±15.8 166.4±12.1 157.0±11.0 152.2±13.0 151.8±12.8 154.4±13.8 153.2±13.9 155.4±13.9
G3, Mercuric Chloride + Test item N3SO (Batch No.: GTE0067) 167.4±18.7 162.6±14.7 165.2±15.6 164.6±17.9 169.2±18.1 171.8±19.4 175.6±17.7 176.2±19.0
G4, Mercuric Chloride + Test item N3SO (Batch No.: 81089527) 168.2±18.8 171.0±14.4 171.4±14.4 173.2±14.1 170.2±16.2 170.4±14.9 172.6±14.2 171.0±14.1

Animals in all the groups were apparently normal throughout the study. The vehicle control group showed ~ 7 % increase in body weight whereas the mercury chloride group showed a mean decrease of 8% in body weight. However, the differences in mean body weight were not statistically significant (p>0.05). The animals treated with the test items showed a mean increase of 3-5% in body weight but were not significantly different (p>0.05) when compared to the vehicle group.

No abnormalities were observed following gross pathology observations in groups 1, 2, 3 and4.

Table 6: Gross pathology of rats following treatment with test items
Group Animal No. Gross Pathological Observations
Liver Spleen Kidneys Adrenals Heart Thymus Lungs Brain Testes
G1, Vehicle Control
1 NAD NAD NAD NAD NAD NAD NAD NAD NAD
2 NAD NAD NAD NAD NAD NAD NAD NAD NAD
3 NAD NAD NAD NAD NAD NAD NAD NAD NAD
4 NAD NAD NAD NAD NAD NAD NAD NAD NAD
5 NAD NAD NAD NAD NAD NAD NAD NAD NAD
G2, Mercuric Chloride alone
6 NAD NAD NAD NAD NAD NAD NAD NAD NAD
7 NAD NAD NAD NAD NAD NAD NAD NAD NAD
8 NAD NAD NAD NAD NAD NAD NAD NAD NAD
9 NAD NAD NAD NAD NAD NAD NAD NAD NAD
10 NAD NAD NAD NAD NAD NAD NAD NAD NAD
G3, Mercuric Chloride + Test item N3SO (Batch No.: GTE0067)
) 11 Mild
Congestion NAD NAD NAD NAD NAD NAD NAD NAD
12 NAD NAD NAD NAD NAD NAD NAD NAD NAD
13 NAD NAD NAD NAD NAD NAD NAD NAD NAD
14 Mild Congestion NAD NAD NAD NAD NAD NAD NAD NAD
15 NAD NAD NAD NAD NAD NAD NAD NAD NAD
G4, Mercuric Chloride + Test item N3SO (Batch No.: 81089527) 16 NAD NAD NAD NAD NAD NAD NAD NAD NAD
17 NAD NAD NAD NAD NAD NAD NAD NAD NAD
18 NAD NAD NAD NAD NAD NAD NAD NAD NAD
19 NAD NAD NAD NAD NAD NAD NAD NAD NAD
20 NAD NAD NAD NAD NAD NAD NAD NAD NAD

The results of hematology and clinical chemistry are shown in tables 7 and 8, respectively.

Table 7: Hematology results in rats following treatment with test items
Group Treatment
WBC
(x 103/µL) RBC
(x 106/µL) Hb (g/dL) HCT (%) PLT
(103/µL) MCV
(fL) MCH
(pg) MCHC
(g/dL)
G1 G1, Vehicle Control 12.0±4.5ns 7.4±0.5ns 13.9±1.0ns 38.9±7.1ns 726.2±40.2ns 62.4±2.6ns 18.5±0.3ns 29.7±1.5ns

G2 G2, Mercuric Chloride alone 12.9± 2.5 7.7±0.5 14.3±1.0 48.0±2.8 773.8± 66.2 62.6±0.6 18.5± 0.3 30.2±1.0
G3
G3, Mercuric Chloride + Test item N3SO (Batch No. GTE0067) 12.0± 2.2 ns 7.2±0.6 ns 13.8±1.0 ns 37.4±5.9 * 723.2± 72.6 ns 63.0±3.3 ns 18.4± 0.4 ns 30.1±0.9 ns
G4 G4, Mercuric Chloride + Test item N3SO (Batch No.81089527) 12.3± 2.1ns 7.4±0.7 ns 13.8±1.2 ns 38.6±7.7 ns 731.2± 115.3 ns 62.9±1.7 ns 18.3± 0.5 ns 30.0±1.3 ns
ns: not statistically significant [p>0.05] from G2; * statistically significant [p<0.05] from G2

N3SO (Batch No. GTE0067) showed significantly lower HCT% when compared to Mercuric chloride alone (p<0.05). There were no significant differences for the other parameters when compared to Mercuric chloride group (p>0.05).
N3SO (Batch No.81089527) did not show significant activity when compared to the Mercuric Chloride group (p>0.05).

Table 8: Clinical chemistry results in rats following treatment with test items
Group Treatment
ALP
(IU/L) AST
(IU/L) ALT
(IU/L) Glucose
(mg/dl) Total protein (g/dl) Albumin
(g/dl) Triglycerides
(mg/dl)
G1 G1, Vehicle Control 51.09±9.29ns 49.88±7.72* 38.97±4.90ns 55.51±10.34ns 6.34±0.56ns 2.57±0.31ns 63.81±13.13ns

G2 G2, Mercuric Chloride alone 68.78± 13.13 69.94±10.24 42.75±7.48 56.79±16.59 7.00± 0.48 2.50±0.2 66.97± 21.85
G3
G3, Mercuric Chloride + Test item N3SO Batch No.GTE0067) 54.89± 7.72 * 51.55±7.22 * 39.56±4.05 ns 55.08±7.78 ns 6.46± 0.32 ns 2.54±0.25 ns 69.38± 15.82 ns
G4 G4, Mercuric Chloride + Test item N3SO (Batch No.81089527) 57.49± 9.58ns 50.40±6.40 * 39.02±2.57 ns 54.34±15.67 ns 6.64± 0.52 ns 2.44±0.60 ns 68.68± 5.85 ns
ns: not statistically significant [p>0.05] from G2; * statistically significant [p<0.05] from G2

N3SO (Batch No.: GTE0067) showed significantly lower ALP and AST levels when compared to Mercuric chloride alone (p<0.05). There were no significant differences for the other parameters when compared to Mercuric chloride group (p>0.05).
N3SO (Batch No.: 81089527) showed significantly lower AST levels when compared to the Mercuric Chloride group (p< 0.05). There were no significant differences for the other parameters when compared to Mercuric chloride group (p>0.05).

The results of histopathology are shown in table 9.
Table 9: Histopathology results in rats following treatment with test items
G1 (Vehicle Control)
Organ/Microscopic Finding Animal Number
1 2 3 4 5
Liver Sinusoidal Dilatation <1> NAD NAD NAD NAD
Mononuclear infiltration NAD NAD NAD NAD NAD
Kidneys Infiltration of inflammatory cell, Cortex <1> NAD NAD NAD NAD
Tubular necrosis, Cortex NAD NAD NAD NAD NAD
Hemorrhage, Cortex <1> NAD NAD NAD NAD

G2 (Mercury Alone)
Organ/Microscopic Finding Animal Number
6 7 8 9 10
Liver Sinusoidal Dilatation NAD NAD NAD NAD NAD
Mononuclear infiltration NAD NAD NAD NAD NAD
Kidneys Infiltration of inflammatory cell, Cortex NAD NAD <1> NAD (1)
Tubular necrosis, Cortex <1> NAD NAD NAD NAD
Hemorrhage, Cortex NAD NAD NAD <1> NAD

Note: - Minimal: - 1; Mild: - 2; Focal: - ( ); Multifocal: - < >, NAD:- No Abnormality Detected.

G3: N3SO (Batch No.GTE0067)
Organ/Microscopic Finding Animal Number
11 12 13 14 15
Liver Sinusoidal Dilatation (1) (1) NAD <1> NAD
Mononuclear infiltration NAD NAD NAD <1> <1>
Kidneys Infiltration of inflammatory cell, Cortex NAD <1> NAD NAD NAD
Tubular necrosis, Cortex NAD NAD NAD NAD NAD
Hemorrhage, Cortex <1> <1> NAD <1> NAD

Note: - Minimal: - 1; Mild: - 2; Focal: - ( ); Multifocal: - < >, NAD:- No Abnormality Detected.

G4 (N3SO (Batch No. 81089527))
Organ/Microscopic Finding Animal Number
16 17 18 19 20
Liver Sinusoidal Dilatation NAD NAD NAD NAD <1>
Mononuclear infiltration NAD NAD NAD NAD NAD
Kidneys Infiltration of inflammatory cell, Cortex NAD <1> NAD NAD NAD
Tubular necrosis, Cortex <1> NAD NAD NAD NAD
Hemorrhage, Cortex NAD <1> NAD <1> NAD

Note: - Minimal: - 1; Mild: - 2; Focal: - ( ); Multifocal: - < >, NAD:- No Abnormality Detected.

G2, Mercury alone: No significant microscopic changes were observed in liver when compared to vehicle (G1). A higher incidence of microscopic changes (minimal, multifocal) was observed in kidneys when compared to vehicle (G1).
G3, N3SO (Batch No. GTE0067): A higher incidence of microscopic changes was seen in liver when compared to G2; changes in kidney were similar to G2
G4, N3SO (Batch No. 81089527): The incidence of microscopic changes in liver was similar to G2; changes in kidney were similar to G2.
Plasma samples were sent to the Sponsor for bioanalysis. The results are shown in figure 1.

No significant differences were observed in plasma concentrations in the treatment groups when compared to mercury group alone (p>0.05). A possible reason could be that since Mercury was dosed first and then the test items were dose next, Mercury was absorbed in all the groups. However, the mechanism of action of the test items could be at the cellular level.

CONCLUSION
The MTD of Mercuric chloride was at least 16 mg/kg following single ascending oral doses in Wistar rats.

In the efficacy study, the vehicle control group showed increase of ~ 7 % in body weight whereas the mercury chloride group showed a mean decrease of 8% in body weight. However, the differences in mean body weight were not statistically significant (p>0.05). The animals treated with the test items showed a mean increase of 3-5% in body weight but were not significantly different when compared to the vehicle group.

In hematology, N3SO (Batch No. GTE0067) showed significantly lower HCT% when compared to Mercuric chloride alone (p<0.05). There were no significant differences for the other parameters when compared to Mercuric chloride group (p>0.05). N3SO (Batch No.: 81089527) did not show significant activity when compared to the Mercuric chloride group (p>0.05).

In clinical chemistry, N3SO (Batch No. GTE0067) showed significantly lower ALP and AST levels when compared to Mercuric chloride alone (p<0.05). There were no significant differences for the other parameters when compared to Mercuric chloride group (p>0.05). N3SO (Batch No.81089527) showed significantly lower AST levels when compared to the Mercuric chloride group (p< 0.05). There were no significant differences for the other parameters when compared to Mercuric chloride group (p>0.05).

Histopathology analysis showed that Mercury alone did not cause microscopic changes in liver when compared to vehicle (G1). A higher incidence of microscopic changes (minimal, multifocal) was observed in kidneys when compared to vehicle (G1). G3, N3SO (Batch No. GTE0067) showed higher incidence of microscopic changes in liver when compared to G2 (Mercury alone); changes in kidney were similar to G2. G4, N3SO (Batch No. 81089527) showed microscopic changes in liver which was similar to G2 (Mercury alone); changes in kidney were similar to G2.

In conclusion, the histological observations in kidney and liver of mice treated with N3SO (Batch No.GTE0067) and N3SO (Batch No. 81089527) were minimal in nature and equivocal when compared to the Mercury alone group (G1).

Taken together N3SO (Batch No. 81089527) and N3SO (Batch No. 81089527) showed significant activity for lowering liver enzymes ALT and AST in the Mercury induced toxicity model in Wistar rats. However, no significant efficacy was observed in hematology and histopathology of liver and kidneys. Further studies are required to characterize the efficacy of the test items.

Example 7:
Experimental data of metal absorption with Calcium polystyrene sulphonate (CPS) with lead, mercury and cadmium ions was also conducted and it can be seen from the results depicted in Table 10 that CPS is equally efficient for metal binding as sodium polystyrene sulphonate.

Accordingly, SPS and CPS have varied properties to binding to various metal ions, for instance, CPS is best to remove cadmium (as depicted from Table 10) while SPS is have shown unexpected results for removal of metal ion toxicity concerning lead and cadmium both.

Table 10:

Reproducible metal binding Study for calcium Polystyrene
Sava sample: Batch No ITE0070, Exp: 06/2023 Residual metal concentration in % Total metal binding, %
Date: 16.09.2021
Product Code Metal Ion CPS
1134-18 A Mercury Blank (1 ppm Hg) 100 NA
1134-18 B 50 ml Blank + 1.5 gm CPS 13.29 86.71
1134-18 C Lead Blank (1 ppm Pb) 100 NA
1134-18 D 50 ml Blank + 1.5 gm CPS 7.06 92.94
1134-18 E Cadmium Blank (1 ppm Pb) 100 NA
1134-18 F 50 ml Blank + 1.5 gm CPS 0.69 99.31
Blank: 1 ppm solution of each Metal Ion
Sample: 50 ml + each above qty of CPS
stirring time 30 min