“BILE ACID SEQUESTERING COMPOSITION OF RESIN CHELATE AND PROCESS FOR PREPARATION THEREOF”

Technical field of the Invention:

The present invention relates to bile acid sequestering composition comprising resin chelate with improved affmity/binding activity towards bile acid(s) and to the process for preparation thereof.

Background and Prior art of the Invention:

The bile acid sequestrants are a group of polymeric compounds used to bind certain bile acid components in the gastrointestinal tract. The components of the bile acid sequestrant disrupt the enterohepatic circulation of bile acids by combining with bile constituents and preventing their reabsorption from the gut. They are classified as hypolipidemic agents, although they may be used for purposes other than lowering cholesterol such as in the treatment of chronic diarrhea due to bile acid malabsorption.

Bile acid sequestrants are polymeric compounds that serve as ion-exchange resins. Bile acid sequestrants exchange anions such as chloride ions for bile acids. By doing so, they bind bile acids and sequester them from the enterohepatic circulation. The liver then produces more bile acids to replace those that have been lost. Because the body uses cholesterol to make bile acids, this reduces the amount of LDL cholesterol circulating in the blood.

Bile acid sequestrants are large polymeric structures and they are not significantly absorbed from the gut into the bloodstream. Thus, bile acid sequestrants, along with any bile acids bound to it are excreted via the feces after passage through the gastrointestinal tract.

There are Patents/Patent Publications that describes the compositions comprising polymers for binding the bile acids. ETS4205064 discloses bile acid sequestering composition containing poly[{alkyl- (3-ammoniopropyl)imino}-trimethylenedihalides. The polymers have a linear backbone which is free from both branching and cross-linking and comprises of quaternized nitrogen atoms linked to each other through trimethylene groups. The polymers are obtained by the polymerization of dihydro-oxazine, reductive alkylation of the resulting polymer, followed by quaternization.

EP0162388 discloses resins useful to sequester non-absorbed bile acids from the intestinal tract to form a complex excreted in the feces with the consequent effect of lowering blood cholesterol level. The resins Quaternized vinylimidazole- ethylene glycol dimethacrylate copolymers comprising 1% to 25% by weight of ethylene glycol dimethacrylate moieties, the remainder being polyvinylimidazolium moieties having the structural formula

wherein the variables are as described in EP0162388.

ETS5453429 relates to bile acid sequestrant polymer compositions wherein the polymer particles are prepared by crosslinking an amine-containing polymer with an amount of a poly-functional amine-reactive compound sufficient to crosslink the polymer so that it is essentially water insoluble and has bile acid sequestering efficacy greater than that of cholestyramine. A need in the art still exists for new bile acid sequesters that effectively binds the bile acid. The inventors have surprisingly found chelating resins which can effectively bind to the bile acids.

It is therefore the object of present invention to provide the bile acid sequestering compositions comprising resin chelates for effective binding of the bile acids in the gastrointestinal track.

Summary of the Invention:

In an aspect, the present invention provides bile acid sequestering composition comprising resin chelate of Formula (I);

R-Y -MT-X wherein R-Y represents the resin/resin acid/diterpene/ triterpene resin acid, either purified or from resin containing components such as plant/animal extract, oleoresins, oleo-gum-resin, gum resin, resins and rosins having (un) substituted or substituted Ce-Cn linear, branched or cyclicalkyls optionally containing heteroatoms, (un) substituted or substituted aryls, (un)substituted or substituted alkylaryls, (un)substituted or substituted heteroaryls; (un)substituted or substituted spiroalkyls, (un) substituted or substituted naphthalenes, phenanthrenes, azulenes, furans, anthracenes, anthocyanins, chromenes picenes; or its salts, stereoisomers, enantiomers, optical isomers or derivatives thereof;

‘M ⁺ ’ is a divalent metal ion; and

‘C is a monovalent counter ion(s).

The bile acid sequestering composition of the present invention may further comprise pharmaceutically acceptable excipients.

In another aspect, the present invention provides a process for preparation of said resin(s) chelate composition. Brief description of Figure(s):

Figure 1 depict the comparative data showing binding of resin chelate of Examples 3 and standard drug to bile acid.

Figure 2 depict the bile acid binding of resin chelate of Example 11 and the positive control

Source of the Plant Material:

Detailed description of the Invention:

The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated. The term resin chelates/ chelating resins used in the entire specification means and relates to the resins, rosin, resinoids, oleoresins, oleo-gum-resin, gum resin, resins of plant extracts as bile acid sequesterants.

In an embodiment, the present invention discloses bile acid sequestering composition comprising resin chelate of the Formula (I);

R-Y--M ⁺ -X wherein R-Y represents the resin/resin acid/diterpene/triterpene resin acid, either purified or from resin containing components such as plant extract, oleoresins, oleo- gum-resin, gum resin, resins and rosins having (un) substituted or substituted C6- C22 linear, branched or cyclic alkyls optionally substituted with heteroatom, (un) substituted or substituted aryls, (un) substituted or substituted alkylaryls, (un) substituted or substituted heteroaryls; (un)substituted or substituted spiroalkyls, (un)substituted or substituted naphthalenes, phenanthrenes, azulenes, furans, anthracenes, anthocyanins, chromenes, picenes; or its salts, stereoisomers, enantiomers, optical isomers or derivatives thereof in an amount of 15-98%;

‘M ⁺ ’ is a divalent metal ion in an amount of 5-50%; and

‘C is a monovalent counter ion in an amount of 4-5% of the total composition.

The metal ion(s) comprises hydroxides, oxides of divalent metal ion such as Mg ²⁺ , Ca ²⁺ , Fe ²⁺ , Co ²⁺ , Zn ²⁺ , Mn ²⁺ , Cr ²⁺ , Cu ²⁺ or Se ²⁺ . The monovalent counter ion(s) is selected from inorganic acid chlorides or nitrates.

In yet another embodiment, the resin chelate has the particle size in the range of 50 to 500 microns.

In yet another embodiment, the bile acid sequestering composition of Formula (I) and Formula 1(a) further comprise pharmaceutically acceptable excipients such as wetting agents, dispersing agents, glidants, preservatives, stabilizer, anti-oxidants, pH modifiers and the like alone or in combination in an amount ranging from 0.01% to 20%; preferably in the range of 1.0 to 10% of the composition.

The stabilizer or pH modifier is selected from organic or inorganic acids such as propionic acids, formic acids, acetic acids, citric acid, butyric acids, succinic acid, valeric acids, caproic acids, oxalic acid, lactic acid, malic acid, benzoic acid and carbonic acid, hydrochloric acid, sulphuric acid, phosphoric acid either alone or in combination.

In an embodiment, the resins of the composition of Formula (I) and Formula (la) is obtained from the extracts of plants selected from Ginger root, Turemeric rhizome, Artichoke ( Cynara cardunculus ), Boswellia, Commiphora mukul (Myrrh, Guggul), Curcuma longa , Boswellia serrata , Bacopa monnieri , Marigold, Ginger, Glycyrrhiza glabra , Cinnamon species, Terminalia chebula , Scutellaria baicalensis, Pinus pinaster (Maritime pine bark), Euterpe oleracea and Acacia catechu , Silybum marianum , Viscum album , Punica granatum , Camellia sinensis (Green Tea), Green coffee bean, Cassia Fistula , Carica papaya , Centella asiatica , Cinnamomum zeylanicum , Cissus quadrangularis , Chlorophytum tuberosum , Curcuma zedoaria , Curcuma xanthorrhiza , Emblica officinalis , Eugenia jambolana , Eurycoma longifolia Root, Garcinia cambogia , Garcinia mangostana , Gymnema sylvestre , Indigofer a Tinctoria , Momordica charantia Fruit (Chamomile), Morinda citrifolia , Moringa oleifera , Mucuna pruriens , Piper nigrum Fruit, Phyllanthus niruri , Salacia oblonga , Salacia reticulata , Sphaeranthus indicus , Sida cordifolia , Tagetes erecta Flower, Tamarindus indica , Cannabis , Berberin , Terminalia arjuna , Terminalia chebula , Tribulus terrestris , Trigonella foenum-graecum , Triphala , Ashwagandha, resverarol, hupericin, Guaiacum officinale, G. sanctum Linn , Garlic, Allium cepa, Paprika, colophony resin, Tetraclinis articulate (Sandarac resin), Coleus forskohlii (frankincense/ Olibanum), Shellac, Rosin (s), Dandelion, alfalfa seeds, Milk Thistle, Fenugreek, Achillea wilhelmsii, Silybum marianum, Psyllium, Cayenne, Konjac, Basil, Flaxseeds, Hawthorn , Celery, sugar cane, asafetida, Justicia wynaadensis, Agathosma betulina, Annona muricata, Apium graveolens, Camellia sinensis, Cassia absus, Cassia occidentalis, Castanospermum austral, Crinum glaucum, Hibiscus sabdarijfa, Linum usitatissimum, Lycopersicon esculentum, Ocimum basilicum, Pinus pinaster, Punica granatum, Rauwolfia serpentine and Uncaria rhynchophylla.

In another embodiment, the present invention discloses the bile acid sequestering composition comprising resin chelate of Formula (la);

R-Y--M ⁺ -X wherein R-Y represents turmeric resins, ginger resins, artichoke resins, coleus forskohlii resins, garlic resins, olibanum resinoid, silybum marium resinoid, myrrh resinoid, acacia catechu resins, bacopa monnieri resins alone or combination thereof in an amount of 15-98%;

‘M ⁺ ’ is a divalent metal ion such as Mg ²⁺ or Ca ²⁺ in an amount of 5-50%; and ‘C is a monovalent Cl counter ion in an amount of 4-5% of the total composition.

The counter ion is sourced from hydrochloric acid.

The metal ion(s) comprises hydroxides, oxides of magnesium and calcium in an amount of 5-50% of the total composition.

The bile acid displaces the counter ion bound to metal ion forming resin-metal ion- bile acid complex which is excreted via the feces after passage through the gastrointestinal tract.

In yet another preferred embodiment, the present invention discloses the process for preparation of bile acid sequestering composition of Formula (I) or Formula (la) comprising,

a) Heating the extracted resin or resin containing components; b) adding the hydrochloric acid;

c) adding metal hydroxide/oxides/ to step (a) and mixing vigorously followed by adding organic/inorganic acid;

d) optionally adding pharmaceutically acceptable excipients to the reaction mass of step (b) and mixing;

e) vaccuum drying the mixture of step (d) followed by milling the product to obtain free flowing powder; and

f) optionally adding excipients to free flowing powder of step (d) to obtain the composition.

According to the process, the extracted resin(s) or resin containing component(s) were added into liquid reactor and heated to the temperature between 25°C to 180°C to convert it into liquid form or to reduce the viscosity. This was followed by adding HC1. The metal hydroxide/oxides, organic/inorganic acid and other excipients were added to the mixture in sequential order. The mixture was mixed vigorously and was transferred to stainless steel tray and vacuum dried at temperature 25 to 80°C. The product was milled using milling machine to obtain free flowing powder. The excipients were further optionally added to this free flowing powder to obtain the final composition.

The excipients are selected from wetting agents, dispersing agents, glidants, preservatives, stabilizer, anti-oxidants, pH modifiers and the like alone or in combinations in an amount ranging from 0.01% to 20%; preferably in the range of 1.0 to 10% of the composition.

The stabilizers or pH modifiers include but is not limited to organic or inorganic acid(s) selected from propionic acids, formic acids, acetic acids, citric acid, butyric acids, valeric acids, caproic acids, oxalic acid, lactic acid, malic acid, benzoic acid, adipic acid, chlorine gas and carbonic acid, hydrochloric acid, sulphuric acid, phosphoric acid either alone or in combination in an amount of 3.0-7.0% of the composition. The said metal ion(s) is selected from the hydroxides, oxides of divalent ion such as Mg ²⁺ , Ca ²⁺ , Fe ²⁺ , Co ²⁺ , Zn ²⁺ , Mn ²⁺ , Cr ²⁺ , Cu ²⁺ or Se ²⁺ .

The surfactant(s) are selected from phospholipids (plant or animal source) or polysorbate 80 or polysorbate 20.

The composition prepared by the aforesaid process can be formulated into different solid/semisolid and liquid compositions such as powder, tablet, capsule, liquid, gel, solution, suspension, granules, gummies and the like.

In yet another embodiment, the present invention discloses total bile acid assay kit for estimating bile acid binding capacity using bile acid sequestering composition of the present invention. Accordingly, the kit comprises (i) the bile sequestering composition containing the resin chelate of Formula (I); (ii) other additional components such as 3 a-hydroxy steroid dehydrogenase (3a-HSD) enzyme, NADH, and thio-NAD+; Cholestyramine (lOpg) as positive control, blank solution, bile acid control (5mM), buffer, colorimeter and (iii) instructions for estimating the unbound (free) bile acid.

In an embodiment, the present invention discloses a method of sequestering bile acids, comprising administering the bile acid sequestering composition of Formula (I) and/or Formula (la) in therapeutic effective amount. The“therapeutic effective amount” refers to an amount of the composition which is effective, upon single or multiple dose administration to a subject in treating or improving a subject with a disorder beyond that expected in the absence of such treatment. The term” subject” is intended to include human and animals.

The method comprises treating Hypercholesterolemia, Hyperlipidemia, Bile acid- induced diarrhea, Obesity, Gall stone, Pruritus, hyperthyroidism, high cholesterol associated diseases such coronary heart disease, stroke, high blood pressure, peripheral arterial disease, Type 2 diabetes, adsorption of toxin A and toxin B, Myxotoxins comprising administering the bile acid sequestering composition of Formula (I) and/or Formula (la) in therapeutic amount of 200mg to 5000mg.

In another embodiment, the present invention discloses use of bile acid sequestering composition of Formula (I) and/or Formula (la) for sequestering bile acids for treating Hypercholesterolemia, Hyperlipidemia, Bile acid-induced diarrhea, Obesity, Gall stone, Pruritus, hyperthyroidism, high cholesterol associated diseases such coronary heart disease, stroke, high blood pressure, peripheral arterial disease, Type 2 diabetes, adsorption of toxin A and toxin B, Myxotoxins.

In yet another embodiment, comparative data of the bile sequestering composition containing the resin chelate of the present invention against the standard drug (Colesevelam hydrochloride) is provided. Accordingly, the Turmeric resin chelate of Example 3 showed O.D. (Optical Density) value of 0.15 and standard drug showed O.D. value of 0.25. The lower OD shows the higher binding to cholesterol (Figure 1).

In yet another embodiment, the bile acid sequestering composition of the present invention described in Example 11 demonstrates effective binding of the bile acid over the positive control Cholestyramine in Figure 2.

Examples:

Some typical examples illustrating the embodiments of the present invention are provided; however, these are exemplary only and should not be regarded as limiting the elements of the present invention.

Example 1: Composition of Turmeric resin Chelate

Example 2: Composition of Olibanum resin chelate

Example 3: Composition of Turmeric resin chelate

Example 4: Composition of Ginger resin chelate

Example 5: Composition of Artichoke resin Chelate

Example 6: Composition of Coleus forskohlii resin chelate

Example 7: Composition of Gralic resin chelate

Example 8: Process for preparation of Composition of Example 1

a) Added Abietic acid resin (crude or purified) into liquid reactor;

b) heated the resin of step (a) at a temperature between at 25°C to l80°C to convert resin into liquid form or to reduce the viscosity;

c) added inorganic (hydrochloric acid) and /organic acid to step (b) and mixing for 5 minutes;

d) added magnesium hydroxide (Metal ion) to step (c) and mixed for 2 to 5 minutes followed by addition of polysorbate 80;

e) transferred the mixture of step (d) into stainless steel tray and vacuum dried (lmBar to 200mBars) and temperature (30°C to 80°C) to obtained hard cake;

f) product of step (e) was milled using milling machine to obtained free flowing powder, and

g) mixed the optional excipients to free flowing powder of step (f) and prepared final composition.

The compositions of Examples 2-7 were prepared similarly by the process described in Example 8.

Example 9: Efficacy data

The binding capacity to the bile acid of the Turmeric resin chelate of Example 3 (lOpg) was compared with standard drug (Colesevalam lOpg) and the control. The Optical density (OD) for the turmeric resin chelate of Example 3 was lowest (i.e. O.D 0.15) in comparison to the standard drug which showed O.D of 0.25. Lower the O.D. higher the binding capacity. Hence, the Turmeric resin chelate of Example 3 showed superior binding capacity to the bile acids as against the standard drug

(Figure 1).

Example 10: Composition of Turmeric resin chelate

Process:

a) Measured amount of resin extracted from turmeric rhizome was taken in the reaction vessel fitted with stirrer and heated upto 35°C temperature with continuous mixing;

b) hydrochloric acid and propionic acid were added and stired well;

c) magnesium hydroxide was then added slowly and mix well untill the product temperature reached between 53-58°C;

d) the content was then transferred to the trays and vacuum dried. The resulting product was milled in milling machine and shifted through 40 mesh sieves; and

e) the powder was further vacuum dried and the free flowing powder was stored in an air tight container.

Example 11: Composition of resin chelate from Turmeric rhizome extract

Process:

a) Measured amount of resin extracted from turmeric rhizome was taken in the reaction vessel fitted with stirrer and heated upto 35°C temperature with continuous mixing;

b) propionic acid and hydrochloric acid was added and stirred well;

c) weighed amount of polysorbate 80 / Sunflower lecithin was added and mixed well;

d) magnesium hydroxide was then added slowly and mix well untill the product temperature reached between 53-58°C;

e) the content was then transferred to the trays and vacuum dried. The resulting product was milled in milling machine and shifted through 40 mesh sieves; and

f) the powder was further vacuum dried and the free flowing powder was stored in an air tight container.

Example 12: Composition of resin chelate from Olibanum resinoid

Process:

a) Measured amount of Olibanum resinoid was taken in the reaction vessel fitted with stirrer and heated upto 80 °C temperature with continuous mixing;

b) hydrochloric acid was added and stirred well;

c) weighed amount Calcium oxide slowly and stir well till it get mixed properly;

d) the reaction mixture was then vacuum dried and then in milling machine and shifting through 40 mesh sieves; and

e) the powder was further vacuum dried and the free flowing powder was stored in an air tight container.

Example 13: Composition of resin chelate from Olibanum resinoid

Process:

a) Measured amount of Olibanum resinoid was taken in the reaction vessel fitted with stirrer and heated upto 80 °C temperature with continuous mixing;

b) hydrochloric acid was added and stirred well;

c) weighed amount Calcium oxide slowly and stir well till it get mixed properly;

d) the reaction mixture was then vacuum dried and then in milling machine and shifting through 40 mesh sieves; and e) the powder was further vacuum dried and the free flowing powder was stored in an air tight container.

Example 14: Composition of resin chelate from Myrrh resinoid

Process:

a) Measured amount of Myrrh resinoid was taken in the reaction vessel fitted with stirrer and heated upto 80°C temperature with continuous mixing; b) hydrochloric acid was added and stirred well;

c) weighed amount Calcium hydroxide slowly and stir well till it get mixed properly;

d) the reaction mixture was then vacuum dried and then milled in milling machine and shifting through 40 mesh sieves; and

e) the powder was further vacuum dried and the free flowing powder was stored in an air tight container.

Example 15: Composition of resin chelate from Ginger resin

Process: a) Measured amount of Ginger resin extracted from ginger root was taken in the reaction vessel fitted with stirrer and heated upto 80°C temperature with continuous mixing;

b) hydrochloric acid was added and stirred well;

c) weighed amount Calcium hydroxide slowly and stir well till it get mixed properly;

d) the reaction mixture was then vacuum dried and then milled in milling machine and shifting through 40 mesh sieves; and

e) the powder was further vacuum dried and the free flowing powder was stored in an air tight container.

Example 16: Composition of resin chelate from Artichoke resin

Process:

a) Measured amount of Artichoke resin extracted was taken in the reaction vessel fitted with stirrer and heated upto 80°C temperature with continuous mixing;

b) hydrochloric acid was added and stirred well;

c) weighed amount Magnesium hydroxide slowly and stirred well till it get mixed properly;

d) the reaction mixture was then vacuum dried and then milled in milling machine and shifting through 40 mesh sieves; and

e) the powder was further vacuum dried and the free flowing powder was stored in an air tight container. Example 17: Composition of resin chelate from Acacia catechu resin

Process:

a) Measured amount of Acacia catechu resin extract was taken in the reaction vessel fitted with stirrer and heated upto 80°C temperature with continuous mixing;

b) hydrochloric acid was added and stirred well;

c) weighed amount Magnesium hydroxide slowly and stirred well till it get mixed properly;

d) the reaction mixture was then vacuum dried and then milled in milling machine and shifting through 40 mesh sieves; and

e) the powder was further vacuum dried and the free flowing powder was stored in an air tight container.

Example 18: Composition of resin chelate from Bacopa monnieri resin

Process:

a) Measured amount of Bacopa monnieri resin extract was taken in the reaction vessel fitted with stirrer and heated upto 80°C temperature with continuous mixing;

b) hydrochloric acid was added and stirred well;

c) weighed amount Magnesium hydroxide slowly and stirred well till it get mixed properly;

d) the reaction mixture was then vacuum dried and then milled in milling machine and shifting through 40 mesh sieves; and

e) the powder was further vacuum dried and the free flowing powder was stored in an air tight container.

Example 19: Estimation of Bile acid binding of resin formulation using Total bile Acid Assay kit (cell Bio labs)

Principle: The assay is based on an enzyme driven reaction: when bile acids are incubated in the presence of enzyme 3 a-hydroxy steroid dehydrogenase (3a-HSD), NADH, and thio-NAD+, thio-NAD+ is converted into its reduced form Thio- NADH. Thio-NADH is then detected calorimetrically as an absorbance increase at 405 nm.

Assay Method : Microplate spectrophotometer (Primary absorbance 405nm, Secondary absorbance 630nm) method was used for the study . Phosphate buffer (pH 6.8) used as assay buffer. The bile acid standard used was Glycochenodeoxycholic acid @ 12.5 (mM) concentration. The positive control used for the study is Cholestyramine. The test sample and positive control sample was prepared using 40% DMSO. The assay content per well is given in below Table 1. Table 1:

Assay Procedure:

a) 40 pL of the mixed test sample, Positive control, Bile Acid control and Bile acid standard as per the Table 1 was added to the 96-well microtiter plate (in duplicates) and Incubated at 37°C for lhr.

b) 150 pL of Thio-NAD+ was added to each well and mixed thoroughly.

Incubated at 37°C for 5 minutes.

c) 50 pL of NADH Reagent was added to one half of the paired standard or sample wells and mixed the well thoroughly.

d) 50 pL of 3a-HSD was added to the another half of the paired wells and mixed thoroughly.

e) Incubated at room temperature for 30 minutes on an orbital shaker.

f) The plate was read at a primary wavelength of 405 nm and a secondary wavelength 630 nm using a microplate spectrophotometer

Calculation:

a) The 630 nm absorbance was subtracted from the 405 nm absorbance. b) The average absorbance values was determined for each sample, control, and standard. c) Subtracted the sample well values without 3a-HSD from the sample well values containing enzyme (3a-HSD ) to obtain the difference. The absorbance difference is due to the enzyme 3a-HSD activity:

DA = ARgtB - ANADH

Results: The Percentage Bile Acid Binding results are given in below Table 2 Table 2:

Conclusion:

From above Table 2, it is concluded that the Test Sample 2 (Composition from Example 11) exhibits higher Percentage Bile Acid Binding than Positive control (Cholestyramine).