WO2011024049A2

WO2011024049A2 - Method for producing proanthocyanidin polymer compositions for pharmaceutical formulations

Info

Publication number: WO2011024049A2
Application number: PCT/IB2010/002060
Authority: WO
Inventors: John Chow; Nitin Pradhan; Suresh Mahadev Kadam; Yingzhong Chen; Jonathan Tsang; Mubeen Ahmed Khan; Vipin Kumar Pandey
Original assignee: Glenmark Pharmaceuticals Ltd; Napo Pharmaceuticals Inc
Priority date: 2009-08-26
Filing date: 2010-08-24
Publication date: 2011-03-03
Also published as: EA023814B1; WO2011024049A9; WO2011024049A3; ZA201202160B; EA201290098A1

Abstract

Processes for producing pharmaceutical compositions containing a proanthocyanidin polymer useful for the treatment and prevention of various conditions are provided. The invention specifically relates to processes for producing pharmaceutical formulations of a proanthocyanidin polymer composition which has been isolated from a Croton spp. or a Calophyllum spp. In particular, the invention relates to a process for producing proanthocyanidin polymer compositions which achieve concentrations and purities useful for addition to pharmaceutically effective formulations.

Description

METHOD FOR PRODUCING PROANTHOCYANIDIN POLYMER COMPOSITIONS FOR PHARMACEUTICAL FORMULATIONS

RELATED APPLICATIONS

This application claims the benefit of Indian Provisional Application 1962/MUM/2009 filed on August 26, 2009 which is hereby incorporated by reference in its entirety.

BACKGROUND

Secretory Diarrhea

Secretory diarrhea, also called watery diarrhea, is a major source of illness and mortality in developing countries, particularly in infants and young children. Secretory diarrhea also affects a significant proportion of visitors from developed to developing countries, but can affect any person visiting a foreign country (called "traveler's diarrhea"). Secretory diarrhea is characterized by the loss of both fluid and electrolytes through the intestinal tract, leading to serious and sometimes life- threatening dehydration. Secretory diarrhea is caused by a variety of bacterial, viral and protozoal pathogens and also results from other non-infectious etiologies such as cancers and neoplasias of the gastrointestinal tract.

Two major bacterial sources of secretory diarrhea are Vibrio cholerae (V. cholerae) and Escherichia coli (E. coli). The enterotoxigenic types of E. coli represent a source of secretory diarrhea in developing countries and are the major cause of traveler's diarrhea. Other strains of E. coli which cause diarrhea include enterohemorrhagic, enteroinvasive, and enteropathogenic and other strains. Other bacterial agents which cause secretory diarrhea include other Vibrio spp., Campylobacter spp., Salmonella spp., Aeromonas spp., Plesiomonas spp., Shigella spp., Klebsiella spp., Citrobacter spp., Yersinia spp., Clostridium spp., Bacteriodes spp., Staphylococcus spp., and Bacillus spp, as well as other enteric bacteria. Secretory diarrhea can also be caused by protozoal pathogens such as Cryptosporidium spp, for example, Cryptosporidium parvum. See generally, Holland, 1990, Clin. Microbiol. Rev. 3:345; Harris, 1988, Ann. Clin. Lab. Sci. 18:102; Gracey, 1986, Clin, in Gastroent., 15:21; Ooms and Degryse, 1986, Veterinary Res. Comm. 10:355; Black, 1982, Med. Clin. Nor. Am., 66:611.

V. cholerae, the enterotoxigenic strains of E. coli, and a variety of other enteric bacteria elicit secretory diarrhea via similar mechanisms. These pathogens produce a toxin which binds a specific receptor on the apical membrane of the intestinal epithelium. Binding of the receptor triggers an adenylate cyclase- or guanylate cyclase-mediated signal transduction leading to an increase in cAMP or cGMP. This regulatory cascade, apparently acting through phosphorylation of specific apical membrane proteins, stimulates chloride efflux into the gut from the intestinal epithelial crypt cells and inhibits normal resorption of sodium and chloride ions by the intestinal epithelial villus cells. The increased chloride and sodium ion concentration osmotically draws water into the intestinal lumen, resulting in both dehydration and loss of electrolytes. Agents which reduce chloride ion secretion will, therefore, prevent the fluid movement into the intestine and resulting net fluid elimination. Thus, such agents are particularly useful for treating and preventing the dangerous dehydration and electrolyte loss associated with secretory diarrhea.

Secretory diarrhea is also a significant problem in non-human animals, particularly in farm animals, such as bovine animals, swine, sheep (ovine animals), poultry (such as chickens), and equine animals, and other domesticated animals such as canine animals and feline animals. Diarrheal disease is particularly common in young and recently weaned farm animals. Diarrheal disease in farm animals, particularly food animals such as cattle, sheep and swine, is often caused by bacterial pathogens such as enterotoxigenic, enterohemorrhagic and other E. coli, Salmonella spp., Clostridium perfringens, Bacteriodes fragilis, Campylobacter spp., and Yersinia enterocolitica. Additionally, protozoal pathogens, particularly Cryptosporidium parvum, and viral agents, particularly rotaviruses and coronaviruses, are significant causes of diarrhea in farm animals. Other viral agents which have been implicated in diarrhea of farm animals include togavirus, parvovirus, calicivirus, adenoviruses, bredaviruses, and astroviruses. See generally Holland, 1990, Clin. Microbiology Rev. 3:345; see also Gutzwiller and Blum, 1996, AJVR 57:560; Strombeck, 1995, Veterinary Quarterly 17(Suppl. 1):S12; Vermunt, 1994, Austral. Veterinary J. 71:33; Driesen et al., 1993, Austral. Veterinary J. 70:259; Mouricout, 1991, Eur. J. Epidemiol. 7:588; Ooms and Degryse, 1986, Veterinary Res. Comm. 10:355.

Proanthocyanidin and proanthocyanidin polymers are phenolic substances found in a wide variety of plants, particularly those with a woody habit of growth (e.g., Croton spp. and Calophyllum spp.). The general chemical structure of a polymeric proanthocyanidin consists of linear chains of 5, 7, 3', 4' tetrahydroxy or 5, 7, 3', 5' pentahydroxy flavonoid 3-ol units linked together through common C(4)-(6) and/or C(4)-C(8) bonds.

Biosynthetic studies have indicated that proanthocyanidin polymers consist of monomer units. See Fletcher et al., 1977, J.C.S. Perkin, 1:1628. The monomer unit (generally termed "leucoanthocyanidin") of the polymer chain may be based on either of two stereochemistries of the C-ring, at a 2 and/or 4 position designated cis (called epicatechins) or trans (called catechin). Therefore, the polymer chains are based on different structural units, which create a wide variation of polymeric proanthocyanidins and a large number of possible isomers (Hemingway et al., 1982, J.C.S. Perkin, 1:1217). ¹³C NMR has been useful to identify the structures of polymeric proanthocyanidins and recent work has elucidated the chemistry of di-, tri- and tetra-meric proanthocyanidins. Larger polymers of the flavonoid 3-ol units are predominant in most plants, and are found with average molecular weights above 2,000 daltons, containing 6 or more units (Newman et al., 1987, Mag. Res. Chem., 25:118).

SUMMARY

Provided herein are processes for the production of proanthocyanidin polymeric compositions for use in pharmaceutically effective formulations. In particular, provided herein are processes for the production of pharmaceutical formulations of a proanthocyanidin polymeric composition, which has been isolated from a Croton spp. or Calophyllum spp., which formulations can be effective, for example, for the treatment of secretory diarrhea, particularly for the reduction of the fluid loss and resulting dehydration associated with secretory diarrheas. Processes are provided for the production of proanthocyanidin polymer suitable for pharmaceutical formulations having a level of purity and concentration which is therapeutically effective. The present invention relates to processes for producing a proanthocyanidin polymer composition at a purity and concentration to be used for therapeutically effective pharmaceutical compositions. The proanthocyanidin polymer composition may be, for example, prepared from a latex from Croton spp, preferably Croton lechleri. The proanthocyanidin polymer in the composition produced according to the methods disclosed herein is crofelemer (USAN). Also described herein are novel pharmaceutical compositions made according to process for producing a proanthocyanidin polymer composition. Processes disclosed herein provide proanthocyanidin polymer compositions comprising greater than 90% by weight proanthocyanidin polymer (by chromatographic purity which, may be for example determined by the analysis of detectable components compared to a reference standard using chromatography), of greater than about 95% by weight proanthocyanidin polymer, and or, for example, from between about 95 and about 100% proanthocyanidin polymer. Also disclosed herein are proanthocyanidin polymer compositions with less than about 0.2% taspine (by chromatographic purity which is determined by the analysis of detectable components compared to a reference standard using chromatography). In addition, also disclosed herein are proanthocyanidin polymer compositions with increased homogeneity. For example, the proanthocyanidin polymer compositions disclosed herein have polydispersity between about 1.2 and about 1.8, or for example, between about 0.9 and about 1.2, or from between about 0.5 to about 1.5, or from between about 0.8 and bout 1.3.

In one embodiment, disclosed herein are method of producing a proanthocyanidin polymer composition comprising: (i) maintaining a latex from a Croton spp. below room temperature for at least 48 hours to permit sediment to settle; (ii) adjusting the pH of the latex to between about 6.5 to about 8.5; (iii) filtering the latex to produce a filtrate; (iv) adjusting the pH of the filtrate to between about 3.5 to about 5.5; (v) performing a solid phase extraction of the filtrate to obtain an eluate containing the proanthocyanidin polymer; (vi) performing a solid phase extraction with a solid phase that differs from the solid phase in step (v) to obtain an eluate containing the proanthocyanidin polymer; and (vii) processing the eluate to yield a proanthocyanidin polymer composition. The method may include an additional optional step that is performing an additional processing step similar to step (vii) but placed between steps (v) and (vi).

Alternatively, the method of producing a proanthocyanidin polymer composition may comprise providing or obtaining a partially-purified proanthocyanidin polymer composition; performing an extraction (e.g., a solid phase extraction) of the partially- purified proanthocyanidin polymer composition to obtain an eluate containing proanthocyanidin polymer; and processing the eluate to yield a purified proanthocyanidin composition. In one embodiment the extraction is performed with a solid phase extraction resin, such as ion exchange resins, adsorption resins, partition resins, size exclusion resins, resins with mixtures of activity, and mixtures of more than one type of resin.

Another embodiment, is a process for preparing proanthocyanidin polymer comprising:

a), providing a solution of plant latex (e.g., crude plant latex, partially purified plant latex, concentrated crude plant latex, or concentrated partially purified plant latex) comprising the mud;

b). adding an organic solvent to the solution of plant latex and mud;

c). separating the organic solvent and concentrating aqueous layer to obtain a solid;

Alternatively

d). separating the aqueous layer and concentrating organic solvent to obtain a solid;

e). dissolving the solid in an aqueous solvent;

f). subjecting the solution to chromatography; and

g). isolating Crofelemer.

As used herein, mud refers to sediment formed on storage. Crude plant latex can be obtained from the bark of Croton lecheri. This latex is collected and stored in barrels at 0-2°C. On storage, sediment deposited is referred as "Mud". This mud is generally discarded.

Disclosed herein, are methods of producing a proanthocyanidin polymer composition, which method, in certain embodiments, may include subprocessing by co- precipitation of impurities from a polar solvent further comprising filtering material (e.g., a methanolic solution with phyllosilicate, (e.g., one or more of bentonite, prehnite, clay minerals, palygorskite, muscovite, clintonite and the like and diatomaceous earth such as available under the Celatom® or Celite® names, cellulose-type mesh filter aids, perlite, charcoal, glass filtration media, sand, filter paper, dowex, salt, or the like) as a filtering material, and an acetate as a solvent (e.g., methyl acetate, ethyl acetate) in extraction and/or washing. For example, filtering materials such as a phyllosilicate may assist in removal of impurities, for example, taspine, protein, and other substances. The acetate may assist in dissolving and removing later eluting materials, for example, lower molecular weight related substances or lower molecular weight phenolic compounds. The subprocess may occur before, after, or in place of solid phase extraction. Alternatively, the method of producing a proanthocyanidin polymer composition may involve purification processes known in the art, but also includes partially purified latex dissolved in alcohols (for example, methanol, ethanol, propanol butanol and other alcohols) and extracting the dissolved compound using an alcohol followed by charcoal treatment and filtering through bentonite. The filterate, in certain embodiments, may be further treated with the acetate solvent followed by filtering through a solid filter (for example, paper, e.g., Whatman paper) to obtain the desired compound which is free of taspine and other impurities.

In addition, methods disclosed herein include methods suitable for commercial scale production. For example, methods disclosed herein can be used to minimize the need for disposal of toxic materials, solvents, and/or can be used to reduce the volume of liquid at a particular step in the process on an industrial scale of production.

In another embodiment, there is provided a pharmaceutical formulation comprising proanthocyanidin polymer composition. Preferably, the pharmaceutical formulation is intended for oral administration.

According to one aspect, provided herein are methods of producing a pharmaceutically acceptable proanthocyanidin polymer composition, comprising: (i) allowing sediment to settle from latex of a Croton Spp.; (ii) adjusting the pH of the latex to between about 6.5 to about 8.5; (iii) filtering the latex to produce a filtrate; (iv) adjusting the pH of the filtrate to between about 3.5 to about 5.5; (v) performing a first extraction of the filtrate to obtain an eluate comprising the proanthocyanidin polymer; and (vi) collecting the eluate comprising the proanthocyanidin polymer composition.

In one embodiment, the first extraction comprises a solid phase extraction.

In one embodiment, the method further comprises performing a second extraction, wherein the second extraction comprises a solid phase extraction.

In one embodiment, the second solid phase is distinct from the first solid phase.

In one embodiment, the proanthocyanidin polymer composition comprises greater than 90% proanthocyanidin polymer.

In one embodiment, the proanthocyanidin polymer composition comprises greater than 95% proanthocyanidin polymer.

In one embodiment, the proanthocyanidin polymer composition consists essentially of proanthocyanidin polymer and water.

In one embodiment, the sediment is allowed to settle for at least about 48 hours.

In one embodiment, the period of time that the sediment is allowed to settle from the latex comprises between about 48 hours and about 48 months.

In one embodiment, the sediment is allowed to settle at a temperature of less than about 20⁰C; at a temperature of between about 20⁰C and about 0⁰C; between about 30°C.and about 20⁰C; between about 2O⁰C and about -20⁰C; or between about 0⁰C and 15°C.

In one embodiment, the sediment is allowed to settle at a temperature between 0⁰C and 15°C for at least 2 hours. In one embodiment, the pH is adjusted in step (ii) to pH 8. In one embodiment, step (iii) is performed with a filtering aid.

In one embodiment, step (iii) further comprises one or more aqueous washes of the filtering material.

In one embodiment, step (iii) further comprises centrifuging the latex instead of filtering.

In one embodiment, the filtering material comprises one or more of diatomaceous earth, charcoal, bentonite, cellulose, glass, sand, or filter paper.

In one embodiment, the filtrate of step (iii) and any optional washes are combined and processed by ultrafiltration.

In one embodiment, the ultrafiltration is performed with a semipermeable membrane.

In one embodiment, the semipermeable membrane permits passage of solutes up to a molecular weight selected from the group consisting of 500 Da, 1 kDa, 5 kDa, 10 kDa, 20 kDa, and 30 kDa.

In one embodiment, the pH of step (iv) comprises 4.

In one embodiment, the solid phase extraction of step (v) is performed with solid phase extraction resin.

In one embodiment, the solid phase extraction resin is selected from the group consisting of ion exchange resins, affinity resins, adsorption resins, partition resins, and mixtures thereof.

In one embodiment, the solid phase extraction resin comprises a carboxymethyl- modified agarose resin. In one embodiment, step (v) is performed in a batch-wise fashion.

In one embodiment, the method further comprises processing the eluate of the first solid phase extraction to yield a composition for the second solid phase extraction.

In one embodiment, the eluate of the first extraction is produced by elution of the solid phase with a solvent system selected from the group consisting of water, acetone, methanol, ethanol, glycol and mixtures thereof.

In one embodiment, the second solid phase extraction is performed with a solid phase extraction resin.

In one embodiment, the solid phase extraction resin comprises one or more of size exclusion resins, ion exchange resins, affinity resins, adsorption resins, partition resins, or mixtures thereof.

In one embodiment, the solid phase extraction resin comprises a modified polysaccharide.

In one embodiment, the modified polysaccharide comprises a hydroxypropylated cross-linked dextran.

In one embodiment, in the eluate from the first extraction is produced by elution of the solid phase with a solvent selected from the group consisting of water, acetone, methanol, ethanol, glycol and mixtures thereof.

In one embodiment, the eluate from the second extraction is mixed with a solvent selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol, hexanol, ethylene glycol, propylene glycol, ethyl acetate, dichloromethane, trichloromethane, tetrachloromethane, dichloroethane, and mixtures thereof.

In one embodiment, the processing of step (vi) is selected from the group consisting of ultrafiltration, freeze drying, evaporation with heat, evaporation without heat, evaporation with vacuum, evaporation without vacuum, spray drying, and combinations thereof.

In one embodiment, the level of residual taspine in the proanthocyanidin polymer composition comprises less than 0.2% of the composition.

In one embodiment, the level of residual taspine in the proanthocyanidin polymer composition comprises less than 0.1% of the composition by chromatographic purity.

In one embodiment, the level of taspine is determined by chromatography.

In one embodiment, the polydispersity of the proanthocyanidin comprises between about 1.2 and about 1.8.

According to one aspect, provided herein are proanthocyanidin polymer compositions comprising proanthocyanidin polymer obtained according to the process described herein in the description and the claims.

In one embodiment, the proanthocyanidin polymer composition comprises greater than about 95% proanthocyanidin polymer.

In one embodiment, the proanthocyanidin polymer composition comprises proanthocyanidin polymer and water.

According to one aspect, provided herein are pharmaceutical formulations comprising the proanthocyanidin polymer composition wherein the proanthocyanidin polymer composition comprises greater than 90% proanthocyanidin polymer and a pharmaceutically acceptable carrier.

In one embodiment, the level of residual taspine in the proanthocyanidin polymer composition comprises less than 0.2% of the composition. In one embodiment, the level of residual taspine in the proanthocyanidin polymer composition comprises less than 0.1% of the composition.

According to one aspect, provided herein are methods of producing a proanthocyanidin polymer composition, comprising: (i) obtaining a partially-purified proanthocyanidin polymer composition, (ii) performing a solid phase extraction of the partially-purified proanthocyanidin polymer composition to obtain an eluate containing proanthocyanidin polymer, and (iii) processing the eluate to yield a proanthocyanidin composition; wherein step (ii) is performed with a solid phase extraction resin as the solid phase.

In one embodiment, the partially-purified proanthocyanidin polymer composition is partially purified by a method comprising: obtaining a latex from a Croton spp. and performing a solid phase extraction with solid phase extraction resin that is not hydroxypropylated cross-linked dextran.

In one embodiment, the solid phase extraction resin comprises a carboxymethyl- modifϊed agarose resin.

In one embodiment, the partially-purified proanthocyanidin polymer composition is partially purified by a method comprising obtaining a latex from a Croton spp. and performing a solid phase extraction with solid phase extraction resin that is not carboxymethyl-modified agarose resin.

In one embodiment, the solid phase extraction resin comprises a hydroxypropylated cross-linked dextran resin.

In one embodiment, the partially-purified proanthocyanidin polymer composition comprises between about 35 to about 90% proanthocyanidin polymer.

In one embodiment, the partially-purified proanthocyanidin polymer composition comprises SB-300. In one embodiment, the solid phase extraction resin of step (ii) is a modified polysaccharide.

In one embodiment, the solid phase extraction resin of step (ii) comprises carboxymethyl-modified agarose resin.

In one embodiment, the purified proanthocyanidin polymer composition comprises greater than about 90% proanthocyanidin polymer.

In one embodiment, the purified proanthocyanidin polymer composition comprises greater than about 95% proanthocyanidin polymer.

In one embodiment, the purified proanthocyanidin polymer composition comprises proanthocyanidin polymer and water.

In one embodiment, the eluate is mixed with a solvent selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol, hexanol, ethylene glycol, propylene glycol, ethyl acetate, dichloromethane, trichloromethane, tetrachloromethane, dichloroethane, and mixtures thereof.

In one embodiment, the processing is selected from the group consisting of ultrafiltration, freeze drying, evaporation with heat, evaporation without heat, evaporation with vacuum, evaporation without vacuum, spray drying, and combinations thereof.

In one embodiment, the level of residual taspine in the purified proanthocyanidin polymer composition comrpises less than 0.1% of the composition. In one embodiment, the polydispersity of the proanthocyanidin polymer in the purified proanthocyanidin polymer composition comprises between about 1.2 and about 1.8.

According to one aspect, provided herein are proanthocyanidin polymer compositions comprising proanthocyanidin polymer obtained according to the process comprising(i) obtaining a partially-purified proanthocyanidin polymer composition, (ii) performing a solid phase extraction of the partially-purified proanthocyanidin polymer composition to obtain an eluate containing proanthocyanidin polymer, and (iii) processing the eluate to yield a proanthocyanidin composition; wherein step (ii) is performed with a solid phase extraction resin as the solid phase.

In one embodiment, the proanthocyanidin polymer composition is of a purity and concentration sufficient for incorporation into a therapeutically effective pharmaceutical composition.

In one embodiment, the purified proanthocyanidin polymer composition comprises greater than 90% proanthocyanidin polymer.

In one embodiment, the purified proanthocyanidin polymer composition comprises greater than 95% proanthocyanidin polymer.

In one embodiment, wherein the proanthocyanidin polymer composition consists essentially of proanthocyanidin polymer and water.

According to one aspect, provided herein are pharmaceutical formulations comprising the purified proanthocyanidin polymer composition comprising: (i) obtaining a partially-purified proanthocyanidin polymer composition, (ii) performing a solid phase extraction of the partially-purified proanthocyanidin polymer composition to obtain an eluate containing proanthocyanidin polymer, and (iii) processing the eluate to yield a proanthocyanidin composition; wherein step (ii) is performed with a solid phase extraction resin as the solid phase.and a pharmaceutically acceptable carrier. In one embodiment, the level of residual taspine in the purified proanthocyanidin polymer composition comprises less than 0.2% of the composition.

In one embodiment, the level of residual taspine in the purified proanthocyanidin polymer composition comprises less than 0.1% of the composition.

According to one aspect, provided herein are methods of producing a proanthocyanidin polymer composition, comprising (i) maintaining a latex from a Croton spp. below room temperature for at least 48 hours to permit sediment to settle; and either step (ii) (a), or step (ii) (b), or both steps (ii) (a) and (ii) (b) as follows: (ii) (a) dissolving latex in water or methanol, adding bentonite to solution with agitation and optional adjustment of pH to between 5.0 to 7.0, and filtering to produce a filtrate; and/or (ii) (b) addition of ethyl acetate and optionally addition of water and/or adjustment of pH to between 6.5 to 8.5, agitation, and removal of the ethyl acetate layer to result in an aqueous or methanolic phase or solid precipitate; thereby producing a proanthocyanidin polymer composition.

In one embodiment, the method further comprises purifying aqueous or methanolic phase the phase extractions to yield a product.

In one embodiment, the purification comprises by solid phase extraction. In one embodiment, step (ii) (a) occurs in methanol. In one embodiment, step (ii) (a) is repeated between one and twenty times. In one embodiment, step (ii) (b) is repeated between one and twenty times.

In one embodiment, step (ii) (a) removes about 10% or more of protein existing in sample prior to step (ii) (a).

In one embodiment, taspine levels above 250 ppm in the latex are reduced to less than about 250 ppm. According to one aspect, provided herein are methods of producing a pharmaceutically acceptable proanthocyanidin polymer composition, comprising: (i) allowing sediment to settle from latex of a Croton Spp.; (ii) adjusting the pH of the latex to between about 6.5 to about 8.5; (iii) filtering the latex to produce a filtrate; (iv) adjusting the pH of the filtrate to between about 3.5 to about 5.5; (v) performing a first solid phase extraction of the filtrate to obtain an eluate comprising the proanthocyanidin polymer; and (vi) collecting the eluate . comprising the proanthocyanidin polymer composition.

According to one aspect, provided herein are methods of producing a proanthocyanidin polymer composition, comprising: a), providing a solution of plant latex comprising the mud; b). adding an organic solvent to the solution of plant latex and mud; c). separating the organic layer and concentrating aqueous layer to obtain a solid.

In one embodiment, the method further comprises, separating the aqueous layer and concentrating organic layer to obtain a solid; d). dissolving the solid in an aqueous solvent; e). subjecting the solution to chromatography; and f). isolating Crofelemer; thereby producing a proanthocyanidin polymer composition.

According to one aspect, provided herein are methods of producing a proanthocyanidin polymer composition, comprising: a), providing a solution of plant latex comprising the mud; b). adding an organic solvent to the solution of plant latex and mud; c). separating the organic layer and concentrating aqueous layer to obtain a solid; Alternatively; Separating the aqueous layer and concentrating organic layer to obtain a solid; d). dissolving the solid in an aqueous solvent; e). subjecting the solution to chromatography; and f). isolating Crofelemer; thereby producing a proanthocyanidin polymer composition.

In one embodiment, the organic solvent in step (b) is selected from one or more of alcohols, ketones, esters, ethers or mixtures therof.

In one embodiment, the ketone comprises methyl ethyl ketone. In one embodiment, the organic solvent in step (b) selected from mixture of methanol and ethyl acetate.

In one embodiment, the column used in chromatography in step (e) comprises a single column or two set column selected from CM-Sepharose Fast Flow Column and Sephadex LH-20.

Other aspects and embodiment are disclosed infra.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 -shows an amino acid analysis of a partially purified polymer composition sample before bentonite treatment in aqueous solution.

FIG. 2 shows an amino acid analysis of a partially purified polymer composition sample after bentonite treatment in aqueous solution.

FIG. 3 shows a chromatogram of scaled-up product after bentonite treatment, ethyl acetate co-precipitation, and first filtration.

FIG. 4 shows an amino acid analysis of a partially purified polymer composition sample before bentonite treatment in methanol solution.

FIG. 5 shows an amino acid analysis of a partially purified polymer composition sample after bentonite treatment in methanol solution.

FIG. 6 shows a flowchart of the process disclosed herein.

FIG. 7 is a reverse-phase HPLC chromatogram of product produced disclosed herein using LH-20 resin.

DETAILED DESCRIPTION

Described herein are processes of producing proanthocyanidin polymer composition, and the composition produced by the process. The starting material is plant latex, for example, a latex from a Croton spp. or Calophyllum spp. In one embodiment, the proanthocyanidin polymer composition is from Croton lechleri. In another embodiment, the proanthocyanidin polymer composition is from Calophyllum inophylum. In one embodiment, the proanthocyanidin polymer composition produced as disclosed herein has a lower concentration of taspine than the concentration of taspine in the latex starting material. In the composition produced according to the methods disclosed herein, the proanthocyanidin polymer comprise monomeric units of leucoanthocyanidins. Leucoanthocyanidins, include, for example, monomeric flavonoids which include catechins, epicatechins, gallocatechins, galloepicatechins, flavanols, flavonols, and flavan-3,4-diols, leucocyanidins and anthocyanidins. In one embodiment, the proanthocyanidin polymer comprises polymers of between about 2 to about 30 flavonoid units, between about 2 to about 15 flavonoid units, between about 2 to about 11 flavonoid units or an average of between about 7 to about 8 flavonoid units with a number average molecular weight of between about 2000 to about 3000 Da, or for example a molecular weigh of between about 1100 daltons to about 2900 daltons; or for example a molecular weigh of between about 1500 Da to about 3000 Da. The proanthocyanidin polymer may be soluble in an aqueous solution, the proanthocyanidin polymer may also be soluble in other solutions and mixtures of aqueous and non-aqueous solutions.

In the composition produced according to one embodiment, the proanthocyanidin polymer comprises crofelemer (USAN). For additional information on proanthocyanidin polymers and/or crofelemer, see, for example, CAS Registry Number 148465-45-6, and/or US Pat. No. 5,211,944 (Tempesta).

The proanthocyanidin polymer compositions produced as disclosed herein can be analyzed by any methods known in the art. For example, proanthocyanidin polymers can be detected by ultraviolet absorbance (lambda-max). Certain proanthocyanidin monomers and polymers, for example, have broad peaks around 200 to about 300 nm, for example between about 190 and about 215 nm (e.g., about 205 - 210 nm) and between about 260 and about 295 nm (e.g., about 275 - 280 nm). Fractions containing proanthocyanidin polymers can have additional major UV absorption maxima from about 400 nm to about 500 nm, from between 425 and 475 nm, and about 460 nm.

Preparation of the Proanthocvanidin Polymer Compositions:

Disclosed herein are methods of producing a proanthocyanidin polymer composition having a purity and concentration for use in a therapeutically effective pharmaceutical composition. The starting material comprises a latex isolated, for example, from a Croton spp. or Calophyllum spp.. The latex may be obtained by any method known in the art, e.g., scoring a Croton tree and collecting the latex accumulating within or exuded by the scores.

The method comprises, for example, one or more of the following steps: producing or obtaining a latex comprising proanthocyanidin polymer; allowing the latex (e.g., from a Croton spp.) to be below room temperature (e.g., from between about 0 to about 20 degrees C, either constantly or for intervals) for at least 48 hours (from between about 1 hour and about 30 days) to permit sediment to settle; adjusting the pH of the latex to between about 6.5 to about 8.5; filtering the latex to produce a filtrate; adjusting the pH of the filtrate to between about 3.5 to about 5.5; performing a first extraction using a first extraction phase (e.g., a solid phase extraction) of the filtrate to obtain an eluate containing the proanthocyanidin polymer; performing a second extraction using a second extraction phase (e.g., solid phase) to obtain an eluate containing the proanthocyanidin polymer; and processing the eluate to yield a proanthocyanidin polymer composition. The method may include an additional optional step such as performing an additional processing step, for example, an additional processing step between the first extraction and the second extraction.

In certain embodiments the maintaining at below room temperature comprises one or more of maintaining the material for the entire time below room temperature, maintaining the material for a majority of the time below room temperature, maintaining the material for intervals of time below room temperature, cycling the material through temperature including below room temperature. Maintaining may also include, for example, the time it takes to reduce the temperature to below room temperature and the time it is warming up to room temperature or above.

In one embodiment, the second extraction phase differs from the first extraction phase.

Alternatively, the method of producing a proanthocyanidin polymer composition may comprise obtaining or providing a partially-purified proanthocyanidin polymer composition; performing a first extraction using a first extraction phase of the partially-purified proanthocyanidin polymer composition to obtain an eluate containing proanthocyanidin polymer, and processing the eluate to yield a purified proanthocyanidin composition; wherein the first extraction is performed with a solid phase extraction resin.

As used herein and unless otherwise indicated, the term "purified" or "pure" means a composition that comprises proanthocyanidin polymer composition that is substantially free of impurities. For example, a pure proanthocyanidin polymer composition comprises greater than about 80% by weight proanthocyanidin polymer composition and less than about 20% by weight of impurities, more preferably greater than about 90% by weight of proanthocyanidin polymer composition and less than about 10% by weight of impurities, even more preferably greater than about 95% by weight of proanthocyanidin polymer composition and less than about 5% by weight of impurities. The degree of purity can be determined by methods known to the chemist or pharmacist. Preferably the compounds are greater than 99% pure (w/w), while purities of greater than 95%, 90% or 85% can be employed if necessary.

Alternatively, in addition to or instead of solid phase extraction, a method of producing a proanthocyanidin polymer composition may comprise a filtering step using a filtering material (e.g., bentonite, charcoal, or one or more filtering materials as indicated herein) wherein latex or a partially-purified proanthocyanidin polymer composition is mixed with a solvent such as an alcohol (e.g., methanol), water, an aqueous/organic mixture, or the like, and then washed or precipitated with another solvent, for example, ethyl acetate, methanol, ethanol, propanol, butanol, pentanol, hexanol, ethylene glycol, propylene glycol, dichloromethane, trichloromethane, tetrachloromethane, dichloroethane, diethyl ether, acetone, dimethylformamide, dimethylsulfoxide, ether combinations thereof, and the like.

Upon completion of the process, the proanthocyanidin polymer composition comprises proanthocyanidin polymer and residual or trace amounts of, for example, water, other solvents used in the process, monomeric units of the proanthocyanidin polymer, other naturally-occurring components from the original latex, and the like. In one embodiment, the proanthocyanidin polymer composition disclosed herein comprises greater than about 90% pure (for example, by chromatographic purity), proanthocyanidin polymer. Alternatively, the proanthocyanidin polymer composition obtained according to the process comprises greater than about 95% pure, proanthocyanidin polymer. In another embodiment, the proanthocyanidin polymer composition comprises proanthocyanidin polymer and a residual amount of water. In another embodiment, the proanthocyanidin polymer composition consists essentially of proanthocyanidin polymer and a residual amount of water. As used herein, "consists essentially" includes the proanthocyanidin polymer being greater than 95% pure proanthocyanidin polymer composition and wherein other components are less than 5%, and any other single component is individually less than 1.0%. Weight percentages of proanthocyanidin polymer can be obtained by methods known in the art, for example by HPLC analysis with a UV detector and measurement of the area under the curve of the resulting data. Weight percent refers, for example, to the detectable components compared to a reference standard and is independent of moisture content.

The lower limit for purity of the compositions produced according to the methods disclosed herein may be, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% proanthocyanidin polymer as measured, for example, by chromatographic purity, or any amount in-between the listed amounts. The purity of the compositions produced as disclosed herein may range up to and include 100% pure, proanthocyanidin polymer, where 100% indicates that any additional components are not present in detectable levels. Alternatively, the upper limit for purity may be 99.99%, 99.9%, and 99% of the proanthocyanidin polymer, or any amount in-between.

Provided herein, are methods of producing a proanthocyanidin polymer composition from latex containing proanthocyanidin polymer. The latex is kept below room temperature (for example, below 20 ⁰C) for a period of time to allow sediment to settle (for example, from about 1 hour to about 30 days). More time may be allowed for sediment to settle, for example, from between about 30 days to about 4 years. In one embodiment, the latex may be maintained at approximately 0°- 15⁰C for 48 hours to 48 months, or at a temperature or period of time in-between, such as 13, 14, or 15 days at 0°- 10⁰C, 2°- 8°C, or 5°C. In one embodiment, the latex is allowed to settle for 14 days at 2°- 8°C. Alternatively, any number within the above ranges may be used. Sufficient settling time, as used herein, is a time that will allow the sediment to settle at or near the bottom of the container containing the polymer. This may be measure by, for example, 1 hour or 30 days. Sufficient time may also be an average time determined to permit adequate settlement.

After sufficient settling time at below room temperature, the pH of the latex is adjusted to between about 6.5 and about 8.5. The pH may be adjusted with any pH- adjusting agent known to one of skill in the art, for example, sodium carbonate, sodium bicarbonate, sodium hydroxide, calcium carbonate, and the like. In one embodiment, the pH is adjusted to about 8. Upon adjustment of the pH, the latex is allowed to settle. For example, in one embodiment, the pH-adjusted latex is refrigerated for 2 hours to allow particulates or sediment to form and settle out from the latex. Refrigeration may range from below room temperature to the freezing point of the latex. For example, from about 0° to about 15°C.

Following adjustment of the pH and a period of settlement, the reaction mixture is filtered through a filtering material. Suitable filtering materials include diatomaceous earth (available as Celatom® or Celite®). Other filtering materials may include cellulose-type mesh filter aids, for example, perlite, charcoal, bentonite, glass filtration media, sand, filter paper, dowex, salt, or the like. Upon completion of filtration, the residue on the filtering material may be washed with additional aliquots of aqueous solution or water. Optionally, the filtering step may include centrifugation to assist in the filtering step.

In one embodiment, bentonite and/or one or more other filtering materials as listed is used as a filtering material in an aqueous, methanolic, aqueous/organic mixture, or other solvent system to reduce protein and/or taspine and/or related substances and/or impurity levels after filtration, either as a sole filtering material or in combination with one or more other filtering materials. Bentonite is commercially-available clay. For example, bentonite may be obtained from Aldrich Chemicals. Other filtering materials may include cellulose-type mesh filter aids, perlite, charcoal, bentonite, glass filtration media, sand, filter paper, dowex, salt, or the like. While not wishing to be bound by theory, it is believed that at certain pH, protein and/or taspine and/or related substances and/or impurities that may be present in the latex starting material or partially purified proanthocyanidin polymer compositions may exist as positively charged particles which interact with a negative charge on bentonite or other filtering material, resulting in removal of the protein and/or taspine and/or related substances and/or impurities with the bentonite or other filtering material upon filtration. In one embodiment, bentonite is added to an aqueous or methanol ic or aqueous/organic system containing partially-purified proanthocyanidin polymer composition and mixed, followed by filtration to remove the bentonite and any materials or precipitates that associate with the bentonite. The amount of protein removed in this manner may be greater than about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, and about 95%, and about any integer in-between these values, compared to the amount of protein in the original latex. For example, using bentonite in an aqueous system may remove greater than about 50% of the protein compared to the latex. Use of bentonite or other filtering material may be used alone or in combination with other steps mention herein, e.g., solid phase extractions, salting, etc.

The resulting filtrate can be combined with aqueous washes of the filtering material, and the combined aqueous layers can be processed to remove excess water and/or prepare the solution for further processing. Processing and/or concentration can be achieved in vacuo or, for example, by ultrafiltration (for example, with a semipermeable membrane). In one embodiment, concentration is by ultrafiltration. While not wishing to be bound by any particular theory, it is believed that ultrafiltration allows suspended solids and solutes of high molecular weight to be retained on one side of a semipermeable membrane, while water and low molecular weight solutes pass through the membrane. Appropriate semipermeable membranes may have a pass profile of 500 Da, 1 kDa, 5 kDa, 10 kDa, 20 kDa, 3OkDa, from 500 Da to about 750 Da, from 900 Da to about 5 kDa, or from 1 kDa to about 4OkDa. Optionally, ultrafiltration may occur more than once, or more than one type of semipermeable membrane may be used together or in sequence to isolate a portion with upper and lower cutoffs. By use of one or more types of semipermeable membranes, the chemical profile (e.g., number and amount of naturally-occurring components from the original latex apart from the proanthocyanidin polymer) and the polydispersity index of the proanthocyanidin polymer (e.g., the distribution of molecular weights of the polymer) in the resulting composition may be modified. In one embodiment, the semipermeable membrane permits passage of low molecular weight solutes up to 1 kDa in molecular weight. Alternatively, the semipermeable membrane will retain solutes greater than 5 kDa.

Following processing of the filtrate and optional washes, the pH of the resulting solution can be adjusted to between 3.5 to 5.5 using for example, a strong or weak acid solution, including hydrochloric acid, citric acid, and the like. In one embodiment, the pH is adjusted to about 4. As an alternative, the pH can be adjusted prior to ultrafiltration as described above.

In one embodiment, after treatment with bentonite or other filtering material, the latex or an aqueous solution or a methanolic solution or an aqueous/organic system containing the latex is treated with ethyl acetate one or more times. Suitable solvents include, for example, methanol, ethanol, propanol, butanol, pentanol, hexanol, ethylene glycol, propylene glycol, ethyl acetate, dichloromethane, trichloromethane, tetrachloromethane, dichloroethane, diethyl ether, acetone, dimethylformamide, dimethylsulfoxide, ether, combinations thereof, and the like. While not wishing to be bound by any particular theory, it is believed that ethyl acetate removes lower molecular weight related substances preferentially while leaving the desired proanthocyanidin polymers. Lower molecular weight related substances may include without limitation later eluting materials, possibly lower molecular weight phenolic compounds. Treatment with ethyl acetate may occur one or more times. For example, two, three, four, five, six, seven, eight, nine, ten or more washings with ethyl acetate may be performed. The pH may be adjusted to between about 6.5 to about 8.5, for example, with 0.5N NaOH. In one embodiment, six washings with ethyl acetate are performed. Where an aqueous or methanolic or aqueous/organic layer is present after washing with ethyl acetate, the desired proanthocyanidin polymer may remain in the aqueous layer and can, thus, be separated from the ethyl acetate layer to achieve purification. Alternatively, the ethyl acetate can be viewed as a co-solvent, which, upon addition by itself or with other precipitating agents such as salt, causes precipitation from the solution. In other words, where insufficient water or methanol or aqueous/organic system is present to maintain the proanthocyanidin polymer composition in solution due to addition of ethyl acetate, a precipitate may form, which may be separated from the liquid layers by filtration. Aqueous back-extractions of the ethyl acetate washings may also be performed. In one embodiment, the resulting proanthocyanidin polymer composition has an average molecular weight of between about 2000 to about 3000 Da, or for example a molecular weigh of between about 1100 daltons to about 2900 daltons; or for example a molecular weigh of between about 1500 Da to about 3000 Da. and a polydispersity between about 1.2 and about 1.8, or for example, between about 0.9 and about 1.2, or from between about 0.5 to about 1.5, or from between about 0.8 and bout 1.3.

In one embodiment, both bentonite and ethyl acetate treatments are performed. In an alternate embodiment, the bentonite and ethyl acetate treatments occur before, after, or in place of any solid phase extraction steps. Advantageously, the amount of proanthocyanidin polymer composition recovered following bentonite and/or ethyl acetate treatment is over 30% by weight compared to starting material. Alternatively, yields may be over 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% or more.

The solid phase extraction of the pH-adjusted solution provides additional purification of the proanthocyanidin polymer. Solid phase extraction may be performed with a variety of solid phases, such as crosslinked polystyrene, modified agarose, or modified dextran. The type of solid phase is not limited, and may include resins such as ion exchange resins, adsorption resins, partition resins, size exclusion resins, resins with mixtures of activity, and mixtures of more than one type of resin. In one embodiment, solid phase extraction is performed with a resin with some ion exchange properties such as carboxymethyl-modified agarose. Alternatively, the solid phase resin may be a modified polysaccharide such as hydroxypropylated cross-linked dextran. Commercially available supports for solid phase extraction include CM- Sepharose®, which is a carboxy-methyl modified agaroseand Sephadex LH20®, which is a hydroxypropylated cross-linked dextran. The solid phase extraction may be performed in a batch process or a continuous process. In one embodiment, the solid phase extraction is performed in a batch-wise fashion to improve efficiency. Following extraction, the solid phase is eluted to remove the proanthocyanidin polymer from the solid phase. Elution can be performed with any solvent or mixture of solvents that removes the proanthocyanidin from the solid phase. For example, solvents for elution include water, acetone, methanol, ethanol, glycol, and mixtures thereof. In one embodiment, the elution is performed with water and water/acetone mixtures.

The eluate in the methods described above can be used for additional rounds of solid phase extraction using a different solid phase from the solid phase used prior. The type of solid phase is not limited, and may include, for example, resins such as ion exchange resins, adsorption resins, partition resins, size exclusion resins, resins with mixtures of activity, and mixtures of more than one type of resin. For example, where CM-Sepharose® is used for the first solid phase extraction and a second solid phase extraction may be performed with Sephadex LH-20® as the solid support or vice versa.

Following the solid phase extractions or in the alternative, following use of a filtering material such as bentonite, the eluate containing proanthocyanidin polymer can be mixed with a solvent selected from one or more of methanol, ethanol, propanol, butanol, pentanol, hexanol, ethylene glycol, propylene glycol, ethyl acetate, dichloromethane, trichloromethane, tetrachloromethane, dichloroethane, diethyl ether, acetone, dimethylformamide, dimethylsulfoxide, ether, combinations thereof, and the like. While not wishing to be bound by any particular theory, mixing with an additional solvent may be useful in promoting additional precipitation and/or additional washing of the layer containing proanthocyanidin polymer. In one embodiment, the solvent for treating the aqueous concentrate is butanol. Following optional mixing with an additional solvent, the layer containing proanthocyanidin polymer may be processed by ultrafiltration, evaporation with or without heat, evaporation with or without vacuum, freeze drying, spray drying, and the like, including combinations of processing techniques, to yield a proanthocyanidin polymer composition.

Proanthocyanidin polymer compositions purified according to the processes described herein have concentrations and purities that are suitable for use in pharmaceutical compositions. The lower limit for purity of the compositions produced as disclosed herein may be about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% by chromatographic purity proanthocyanidin polymer, or any amount in-between the listed amounts. The purity of the compositions produced as disclosed herein may range up to and include 100% by chromatographic purity, where 100% indicates that any additional components are not present in detectable levels. Alternatively, the upper limit for purity may be about 99.99%, about 99.9%, and about 99% by chromatographic purity, or any amount in-between. For example, the amount of taspine present in the original latex can be reduced through the processes disclosed herein. Taspine levels in the proanthocyanidin polymer composition produced as disclosed herein may range from 1% by chromatographic purity down to below detectable limits. For example, the upper level for the amount of taspine in the proanthocyanidin polymer composition may be about 1.0%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, about 0.1%, or about 0.05% by chromatographic purity, or any amount in-between or below the listed amounts down to the limit of detectability. In one embodiment, taspine levels are below about 0.2% by chromatographic purity (e.g., 2000 ppm) in the proanthocyanidin polymer composition. Proanthocyanidin polymer compositions are advantageously between about 0.1% by chromatographic purity of taspine and about 0% by chromatographic purity of taspine (e.g., no detectable amount of taspine) when produced according to the processes described herein.

In one embodiment, the polydispersity index may range from approximately 1.2 to 1.8. Depending on the desired pharmaceutical formulation, a narrow band of molecular weights may be selected for a low polydispersity index, or a wider band of molecular weights, or various upper or lower cutoffs, may be selected. Polydispersity is not solely determined by this ultrafiltration step, but may also be affected by other process parameters.

In an alternative to the above method, the methods disclosed herein also encompasses producing a purified proanthocyanidin polymer composition in a purity and concentration for incorporation into a therapeutically effective pharmaceutical composition, comprising: obtaining a partially-purified proanthocyanidin polymer composition; performing an extraction (e.g., a solid phase extraction) of the partially- purified proanthocyanidin polymer composition to obtain an eluate containing proanthocyanidin polymer; and processing the eluate to yield a purified proanthocyanidin composition. In one embodiment, the the extraction is performed with a solid phase extraction resin as the solid phase. In one embodiment, the partially-purified proanthocyanidin polymer composition is partially purified by a method comprising obtaining a latex from a Croton spp. and performing a solid phase extraction with solid phase extraction resin that is not hydroxypropylated cross-linked dextran. In one embodiment, the solid phase extraction resin used to accomplish the partially purified starting material is carboxymethyl-modified agarose resin. The partially-purified proanthocyanidin polymer composition may comprise, for example, from between about 35 to about 90% by chromatographic purity of proanthocyanidin polymer. Within the range of from between about 35 to about 90%, the partially- purified proanthocyaidin polymer composition may have any sub-range, for example 40-80%, 45-75%, 50-70%, 55-65%, or about 60%. Alternatively, the partially- purified proanthocyanidin polymer composition may have any numerical amount within 35-90%, for example 36%, 37%, 38%, and the like, up to 87%, 88%, 89%, and 90%, or any value in-between. In one embodiment, the partially-purified proanthocyanidin polymer composition may be SB-300. In addition, the partially- purified proanthocyanidin polymer composition may have taspine levels of about 0.2% or higher, for example, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0% or higher, or any amount in- between the recited levels up to the naturally-occurring level in the latex.

Performing the solid phase extraction may be accomplished with one or more solid phase extraction resins. For example, such resins may include a modified polysaccharide or the like such as carboxymethyl-modified agarose. Modified polysaccharides may include hydroxypropylated cross-linked dextran. Commercially available supports for solid phase extraction include CM-Sepharose®, which is a carboxy-methyl modified agarose, and Sephadex LH20®, which is a hydroxypropylated cross-linked dextran.

Following the solid phase extractions, the eluate containing proanthocyanidin polymer can be mixed with a solvent selected from one or more of methanol, ethanol, propanol, butanol, pentanol, hexanol, ethylene glycol, propylene glycol, ethyl acetate, dichloromethane, trichloromethane, tetrachloromethane, dichloroethane, diethyl ether, acetone, dimethylformamide, dimethylsulfoxide, ether, mixtures thereof, and the like. In one embodiment, the solvent for washing the aqueous concentrate is butanol. Following optional mixing with an additional solvent, the layer containing proanthocyanidin polymer may be processed by ultrafiltration, evaporation with or without heat, evaporation with or without vacuum, freeze drying, spray drying, and the like, including combinations of processing techniques, to yield a final proanthocyanidin polymer composition.

In the process for preparing proanthocyanidin, organic solvents are selected from one or more of alcohols, ketones, esters, and ethers. Ketones are selected from, for example, methyl ethyl ketone and isobutyl ketone. Esters are selected from, for example, ethyl acetate, methyl acetate, propyl acetate, and butyl acetate. Ethers are selected from, for example, diethylether, methyl ethyl ether, dimethyl ether, etc. Organic solvents can also be mixtures of alcohols, ketones, esters, and ethers. For example mixture can be a combination of alcohol and esters, such as methanol and ethyl acetate.

Column purification involves, for example, a single column or sets of columns selected from, for example, CM-Sepharose Fast Flow Column and Sephadex LH-20. When using multiple columns, they may be used jointly in series or separately. Eluents are selected from, for example, aqueous solvents, such as water and water miscible solvents.

In another embodiment, the method of producing a Proanthocyanidin polymer composition may involve purification processes and may include partially purified latex being dissolved in alcohols, (for example, methanol) and extracting the dissolved compound with the alcohol followed by filter (e.g., charcoal) treatment and filtered through another filter bed, e.g., Bentonite Bed. The filtrate may then be treated with an acetate (e.g., ethylacetate) followed by filtering through a filter (e.g., Whatman paper) to obtain the Proanthocyanidin free of Taspine impurity, e.g., wherein Taspine is below a detectable level.

Therapeutic Uses Described herein are uses of compositions according to the production methods set forth herein for treating and/or preventing one or more symptoms associated with diarrhea-predominant irritable bowel syndrome (dIBS), in warm blooded animals, including male and female humans, which symptoms include, but are not limited to, pain, abdominal discomfort, diarrhea, presence of urgency, abnormal stool frequency and abnormal stool consistency. The methods described herein generally comprise administering to a subject in need of d-IBS treatment a proanthocyanidin polymer composition. In one embodiment, the proanthocyanidin polymer composition is orally administered and is not systemically absorbed. In one embodiment, the subject comprises an animal. In one embodiment, the subject comprises a primate. In another embodiment the subject comprises a human. In another embodiment the subject comprises a female and/or a male.

In one embodiment, provideded herein are methods of treating pain and diarrhea associated with d-IBS comprising administering to a subject in need of such treatment, an amount of a proanthocyanidin polymer composition effective to treat pain and diarrhea associated with d-IBS. In another embodiment, presented herein are methods of treating abdominal discomfort and diarrhea associated with d-IBS comprising administering to a subject in need of such treatment, an amount of a proanthocyanidin polymer composition effective to treat abdominal discomfort and diarrhea associated with d-IBS. In certain embodiments, the amount of the proanthocyanidin polymer composition is co-administered with an analgesic and/or anti-inflammatory compound, for example, one that inhibits COX-2 and preferably inhibits COX-2 over COX-I.

In other embodiments, the proanthocyanidin polymer composition is co-administered, either simultaneously, prior to, during or after administration with another composition.

In exemplary embodiments, stool frequency is decreased by at least 10%, 20%, 30%, 40% 50%, 60%, 70% 80% or more compared to the stool frequency of a subject prior to treatment with a proanthocyanidin polymer composition. In other embodiments, stool frequency is decreased by at least one bowel movement per day compared to before treatment with a proanthocyanidin polymer composition. In other embodiments, stool consistency is increased, e.g., there is a decrease in the amount of water in the stool, by at least 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70% 80% or more compared to before treatment. In yet other embodiments, presence of urgency of the subject is decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70% 80% or more compared to before treatment with a proanthocyanidin polymer composition.

In another embodiment, a method of treating diarrhea associated with d-IBS is provided and comprises orally administering to a patient in need of such treatment, an amount of a proanthocyanidin polymer composition, for example, enterically- protected crofelemer (CAS 148465-45-6), effective to treat the diarrhea associated with d-IBS, in which said amount is between about 50 mg per day and about 750 mg per day, wherein the crofelemer is produced in a method disclosed herein. In yet another embodiment, provided herein is a method of treating abnormal stool frequency, abnormal stool consistency or presence of urgency associated with d-IBS comprising orally administering to a patient in need of such treatment, an amount of enterically-protected crofelemer effective to treat the abnormal stool frequency, abnormal stool consistency or presence of urgency associated with d-IBS, in which said amount is between about 50 mg per day and about 750 mg per day, wherein the crofelemer is produced in a method disclosed herein.

The pharmaceutical formulations described herein can also be used to treat diarrhea- predominant irritable bowel syndrome (d-IBS) in non-human animals, particularly in farm animals, such as but not limited to, bovine animals, swine, ovine animals, poultry (such as chickens), and equine animals, and other domesticated animals such as canine animals and feline animals. In particular, the pharmaceutical formulations described herein can be used to treat d-IBS disease in nonhuman animals, particularly food animals such as cattle, sheep and swine by incorporating the proanthocyanidin polymer composition or pharmaceutical compositions thereof into the animal's feed.

In determining whether a subject has diarrhea-predominant IBS, any art-recognized method can be used to diagnose the subject including, but not limited to, the Rome II criteria for diagnosis of irritable bowel syndrome (Thompson et al., 1999, Gut 45 (Suppl II):Ih43-l 147). Briefly, the Rome II diagnostic criteria state that for at least 12 weeks, which need not be consecutive, in the preceding 12 months of abdominal discomfort or pain that the subject has two of following three features: (1) relief with defecation, (2) onset associated with a change in frequency of stool; and (3) onset associated with a change in form (appearance) of stool. The following symptoms cumulatively support the diagnosis of BIBS: (i) abnormal stool frequency, e.g., greater than 3 times per day; (ii) abnormal stool form, e.g., loose/watery stool; and (iii) the presence of urgency (having to rush to have a bowel movement).

Pain and discomfort can be measured by any method known in the art. For example, a pain or discomfort scale in which a patient assigns the level of pain or discomfort on a scale of 0 to 5, with 0 being no pain or discomfort and 5 being assigned the highest level of pain or discomfort can be employed. In certain embodiments, the alleviation of pain or discomfort is measured by a lowering of the average level of pain or discomfort, and/or an increase in the number of pain- or discomfort-free days. In certain embodiments, the number of pain- or discomfort-free days is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or more compared to before treatment with a proanithocyanidin polymer composition. In other embodiments, the level of pain or discomfort decreased by at least 0.1, 0.2, 0.3, 0.4, 0.5, .75, 1.0, 1.5, or 2.0 units or more compared to before treatment.

In another embodiment, provided herein aremethods of treating constipation- predominant irritable bowel syndrome (c-IBS). The methods comprise administering to a subject in need of c-IBS treatment a proanthocyanidin polymer composition. In one embodiment, the proanthocyanidin polymer composition is orally administered and is not systemically absorbed. In one embodiment, the patient is a human female. In another embodiment provided herein are methods of treating pain or any other symptom or combination of symptoms associated with c-IBS comprising orally administering to a patient in need of such treatment, an amount of enterically- protected crofelemer (CAS 148465-45-6) effective to treat the pain associated with c- IBS, in which said amount is between about 500 mg per day and about 3 grams per day, wherein the crofelemer is produced by a method disclosed herein.

In another embodiment, provided herein are methods of treating secretory diarrhea with a composition disclosed herein. The pharmaceutical compositions are useful for treatment of traveler's diarrhea and non-specific diarrhea. In another embodiment, pharmaceutical compositions produced according to the methods presented herein are useful treatment of secretory diarrheas associated with viral infections, such as, diarrheas which accompany Human Immunodeficiency Virus (HIV) infection and Acquired Immuno Deficiency Syndrome (AIDS), and rotavirus infection. Almost all AIDS patients suffer from diarrhea at some point during the course of the disease, and 30% of AIDS patients suffer from chronic diarrhea. The diarrhea that accompanies AIDS and HIV infection has been termed "HIV-Associated Chronic Diarrhea." This diarrheal component of HIV disease is thought to be caused by secondary infections that include protozoal pathogens, for example, Cryptosporidium spp., by the HIV virus itself, or by the therapies used to control viral load. Pharmaceutical compositions are useful for the above HIV and AIDS related-secretory diarrheas in adults and in children. In addition, rotavirus infection is a substantial cause of diarrhea particularly in infants and young children in developing countries for which the pharmaceutical compositions disclosed herein are useful.

Described herein are uses of compositions according to the production methods set forth herein for treating and/or preventing of colon cancer in animals, including male and female humans.- Specifically, provided herein are methods of treating or preventing colon cancer comprising administering to a patient in need of such prevention an amount of a polymeric proanthocyanidin composition produced by the methods disclosed herein. For example, a patient in need of such treatment or prevention includes those patients who have been identified to have colon polyps, or those patients who have been diagnosed with colon cancer (for example, colon cancer at any of stages 0, 1, II, III, or IV), or those patients who have been treated previously for colon cancer (e.g., to prevent recurrence), or those patients who have a familial or genetic predisposition to colon cancer, or those patients living in areas that have higher than average rates of colon cancer. Other patients in need of such treatment or prevention are those who have had a colon biopsy indicating pre-cancerous changes.

The methods provided herein comprise administering to a subject in need of colon cancer treatment a proanthocyanidin polymer composition. In one embodiment, the proanthocyanidin polymer composition is orally administered and is not systemically absorbed. In one embodiment, the subject comprises an animal. In one embodiment, the subject comprises a primate. In another embodiment the subject comprises a human. In another embodiment the subject comprises a female and/or a male.

In certain embodiments, the amount of the proanthocyanidin polymer composition is co-administered to treat colon cancer with an analgesic and/or anti-inflammatory compound, for example, one that inhibits COX-2 and preferably inhibits COX-2 over COX-I.

In determining whether a subject has colon cancer or is at increased risk of developing colon cancer, any art-recognized method can be used.

In certain embodiments, the treatment of colon cancer is measured by a decrease in the size of a colon tumor. In certain embodiments, the size of a colon cancer tumor is decreased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or more compared to the size before treatment with a proanthocyanidin polymer composition. In other embodiments, the treatment of colon cancer is measured by the lack of an increase in growth of a colon cancer tumor.

In one embodiment, the patient is a human, e.g., a male or female human. In another embodiment, provided is a method of treating colon cancer comprising orally administering to a patient in need of such treatment, an amount of enterically- protected crofelemer (CAS 148465-45-6) effective to treat colon cancer, in which said amount is between about 500 mg per day and about 3 grams per day, wherein the crofelemer is produced by a method disclosed herein.

In certain embodiments, the polymeric proanthocyanidin compositions can be administered as adjuvant therapy with other known therapies for treating or preventing colon cancer. For example, the compositions can be administered before, concurrently with or after surgery, radiation therapy, chemotherapy or biologic therapy (biologic therapy includes immunologic therapy with engineered antibodies such as ERBITUX^®, AVASTIN^®, or a therapy to boost the immune response to the cancer).

Pharmaceutical Dosages When used according to the formulations and methods as a treatment for the conditions, diseases and disorders disclosed herein, effective dosage ranges of the pharmaceutical formulations of the proanthocyanidin polymer composition for oral administration are in the range of 0.1 to 100 mg/kg per day, for example, about 0.1 to about 40 mg/kg per day, about 0.1 to about 25 mg/kg per day, or about 0.1 to about 10 mg/kg per day. It should be appreciated that the appropriate dose will depend upon the type and severity of the condition or disease. It has been found that human subjects can tolerate at least up to 2 grams of the proanthocyanidin polymer composition per day (25-30 mg/kg/day) for up to 27 days.

In another embodiment, the pharmaceutical composition comprises a proanthocyanidin polymer composition prepared from a Croton spp, the dosage of which does not exceed 750 mg per day, preferably less than 250 mg/day. In one embodiment, the proanthocyanidin polymer composition is crofelemer (CAS 148465- 45-6).

In other embodiments, crofelemer is orally administered in an enteric protected form (enteric coated) in a total amount of not more than about 750 mg/day. As used herein, about means within the margin of error. In specific embodiments, the enteric coated crofelemer is orally administered to a subject in an amount of from about 50 mg/day to 750 mg/day. In another embodiment, the enteric coated crofelemer is orally administered to a subject in a total amount of not more than about 500 mg/day. In specific embodiments, the enteric coated crofelemer is orally administered to a subject in an amount of from about 50 mg/day to 500 mg/day. In other embodiments, the enteric coated crofelemer is orally administered to a subject at not more than about 700 mg/day, about 650 mg/day, about 600 mg/day, about 550 mg/day, about 500 mg/day, about 450 mg/day, about 400 mg/day, about 350 mg/day, about 300 mg/day, about 250 mg/day, about 200 mg/day, about 150 mg/day or about 100 mg/day of enteric coated crofelemer. In yet another embodiment, the enteric coated crofelemer is orally administered to a subject in an amount from about 100 mg/day to 750 mg/day. In other embodiments, the enteric coated crofelemer is orally administered to a subject in an amount of from about 125 mg/day to about 500 mg/day, from about 250 mg/day to about 500 mg/day, from about 250 mg/day to about 450 mg/day, from about 250 mg/day to about 400 mg/day, from about 250 mg/day to about 350 mg/day, or from about 250 mg/day to about 300 mg/day. In other particular embodiments, the total dosage of the enteric coated crofelemer orally administered to a subject is about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, about 150 mg, about 155 mg, about 160 mg, about 165 mg, about 170 mg, about 175 mg, about 180 mg, about 185 mg, about 190 mg, about 195 mg, about 200 mg, about 205 mg, about 210 mg, about 215 mg, about 220 mg, about 225 mg, about 230 mg, about 235 mg, about 240 mg, about 245 mg, about 250 mg, about 255 mg, about 260 mg, about 265 mg, about 270 mg, about 275 mg, about 280 mg, about 285 mg, about 290 mg, about 295 mg, about 300 mg, about 305 mg, about 310 mg, about 315 mg, about 320 mg, about 325 mg, about 330 mg, about 335 mg, about 340 mg, about 345 mg, about 350 mg, about 355 mg, about 360 mg, about 365 mg, about 370 mg, about 375 mg, about 380 mg, about 385 mg, about 390 mg, about 395 mg, about 400 mg, about 405 mg, about 410 mg, about 415 mg, about 420 mg, about 425 mg, about 430 mg, about 435 mg, about 440 mg, about 445 mg, about 450 mg, about 455 mg, about 460 mg, about 465 mg, about 470 mg, about 475 mg, about 480 mg, about 485 mg, about 490 mg, about 495 mg, or about 500 mg once, twice, or three-times per day.

According to the methods disclosed herein for treating farm animals for d-IBS, the pharmaceutical compositions of comprising proanthocyanidin polymer composition are administered to a subject in a total amount that is bioequivalent to not more than 750 mg/day of orally administered enteric protected proanthocyanidin polymer composition. In specific embodiments, pharmaceutical compositions comprising proanthocyanidin polymer composition are administered to a subject in an amount of between about 50 mg per day and about 250 mg/day. In specific embodiments, ranges may be between about 50 to about 450 mg per day, or about 50 to about 400 mg per day, or about 50 to about 350 mg per day, or about 50 to about 300 mg per day, or about 50 to about 250 mg per day, or about 50 to about 200 mg per day, or about 50 to about 150 mg per day or about 50 to about 100 mg per day.

The proanthocyanidin polymer compositions can be incorporated into a pharmaceutical dosage to be administered in a single or a divided dosage from one, two, three or four times per day. In a particular embodiment, the pharmaceutical dosage is administered twice daily. In yet another embodiment, the pharmaceutical dosage is administered twice daily for at least two consecutive days. In yet another embodiment, the pharmaceutical dosage is administered for at least a period of time selected from the group consisting of 24 hours, 48 hours, 72 hours, 96 hours, one week, two weeks, one month, two months, and three months. In certain embodiments, the pharmaceutical dosage is taken indefinitely.

Pharmaceutical Formulations

In another embodiment, there is provided a pharmaceutical formulation comprising proanthocyanidin polymer composition.

Methods of administering a proanthocyanidin polymer composition disclosed herein include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous) and mucosal (e.g., intranasal and oral routes). In a specific embodiment, a proanthocyanidin polymer composition is administered intramuscularly, intravenously, or subcutaneously. A proanthocyanidin polymer composition may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. Preferably, the proanthocyanidin polymer composition is orally administered.

In one embodiment, crofelemer comprises an enteric coating to protect it from degradation by the acidic conditions of the stomach and/or from interactions with proteins, such as pepsin, present in the stomach, e.g, an enteric protected formulation. In a specific embodiment, crofelemer is in tablet form. In yet another specific embodiment, the tablet is enteric coated, e.g., EUDRAGIT®. In one embodiment, crofelemer is formulated as an enteric coated bead or granule in an enteric coated capsule shell. In another embodiment, crofelemer is formulated in a delayed release composition, e.g., Merck GEM, Alza OROS, wax matrix (release is delayed primarily until the formulation passes out of the stomach and into the intestine). In certain embodiments, the proanthocyanidin polymer composition is formulated with a compound or compounds which neutralize stomach acid. Alternatively, the pharmaceutical composition containing the inhibitor molecule is administered either concurrently with or subsequent to or after administration of a pharmaceutical composition which neutralize stomach acid. Compounds, such as antacids, which are useful for neutralizing stomach acid include, for example, aluminum carbonate, aluminum hydroxide, bismuth subnitrate, bismuth subsalicylate, calcium carbonate, dihydroxyaluminum sodium carbonate, magaldrate, magnesium carbonate, magnesium hydroxide, magnesium oxide, and mixtures thereof. Compounds that are able to reduce the secretion of stomach acid and/or are able to reduce the acidity of stomach fluid are known in the art and include, for example, antacids (aluminum hydroxide, aluminum carbonate, aluminum glycinate, magnesium oxide, magnesium hydroxide, magnesium carbonate, calcium carbonate, sodium bicarbonate), stomach acid blockers (cimetidine (Tagamet™), famotidine (Mylanta™, Pepcid™), nizatidine (Axid™), ranitidine (Zantac™), omeprazole (Zegerid™)) and a combination of any of the foregoing. In general, any drug that has been approved for sale by the relevant government agency and is able to reduce the production of stomach acid and/or reduce the acidity of stomach fluid can be administered in combination with a proanthocyanidin polymer composition, such as crofelemer.

In other embodiments, the proanthocyanidin polymer compositions disclosed herein are administered with other compounds which are useful in treating diarrhea, pain or cancer. Such compounds include, but are not limited to, COX-2 inhibitors such as 5- ASA, sulfasalazine, mesalamine, APAZA, as well as other commercially available COX-2 inhibitors such as celecoxib and rofecoxib. Compounds for the treatment of cancer include small molecule and biologies that specifically target cancerous cells. Preferably, such compounds are not systemically absorbed or are modified so as to not be systemically absorbed.

In a particular embodiment where crofelemer is not enteric coated, crofelemer is formulated with one or more compounds that are able to reduce the secretion of stomach acid and/or able to reduce the acidity of stomach fluid. Preferably, the dosage of crofelemer to be given in this formulation is a dosage that is bioequivalent to orally administered enteric coated crofelemer at a dosage of about 50 mg per day to about 750 mg per day. In an exemplary embodiment, crofelemer is formulated in a controlled release (delayed release) composition.

In other embodiments, the proanthocyanidin polymer composition can be administered in combination with analgesic or anti-inflammatory agents. In one embodiment, the analgesic or anti-inflammatory agent is formulated or modified such that it is not substantially systemically absorbed, e.g., only 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% or 0.5% absorbed of the dosage given.

The proanthocyanidin polymer composition disclosed herein can be provided in any therapeutically acceptable pharmaceutical form. The pharmaceutical composition can be formulated for oral administration as, for example but not limited to, drug powders, crystals, granules, small particles (which include particles sized on the order of micrometers, such as microspheres and microcapsules), particles (which include particles sized on the order of millimeters), beads, microbeads, pellets, pills, microtablets, compressed tablets or tablet triturates, molded tablets or tablet triturates, and in capsules, which are either hard or soft and contain the composition as a powder, particle, bead, solution or suspension. The pharmaceutical composition can also be formulated for oral administration as a solution or suspension in an aqueous liquid, as a liquid incorporated into a gel capsule or as any other convenient formulation for administration, or for rectal administration, as a suppository, enema or other convenient form. The inhibitor molecule or anti-cancer agents can also be provided as a controlled release system (see, e.g., Langer, 1990, Science 249: 1527- 1533).

The pharmaceutical formulation can also include any type of pharmaceutically acceptable excipients, additives or vehicles. For example, diluents or fillers, such as dextrates, dicalcium phosphate, calcium sulfate, lactose, cellulose, kaolin, mannitol, sodium chloride, dry starch, sorbitol, sucrose, inositol, powdered sugar, bentonite, microcrystalline cellulose, or hydroxypropylmethylcellulose may be added to the formulation to increase the bulk of the composition. Also, binders, such as but not limited to, starch, gelatin, sucrose, glucose, dextrose, molasses, lactose, acacia gum, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapgol husks, carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone, Veegum and starch arabogalactan, polyethylene glycol, ethylcellulose, and waxes, may be added to the formulation to increase its cohesive qualities. Additionally, lubricants, such as but not limited to, talc, magnesium stearate, calcium stearate, stearic acid, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, carbowax, sodium Iauryl sulfate, and magnesium lauryl sulfate may be added to the formulation. Also, glidants, such as but not limited to, colloidal silicon dioxide or talc may be added to improve the flow characteristics of a powdered formulation. Finally, disintegrants, such as but not limited to, starches, clays, celluloses, algins, gums, crosslinked polymers (e.g., croscarmelose, crospovidone, and sodium starch glycolate), Veegum, methylcellulose, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, alginic acid, guar gum, citrus pulp, carboxymethylcellulose, or sodium lauryl sulfate with starch may also be added to facilitate disintegration of the formulation in the intestine.

In one aspect, the proanthocyanidin polymer compositions produced as disclosed herein are formulated for oral administration. In other aspects, the pharmaceutical dosage form is formulated to protect the proanthocyanidin polymer composition from degradation by the acidic conditions of the stomach and from interactions with proteins, such as pepsin, present in the stomach. Thus, in a preferred aspect, the formulation is enteric coated. For example, the enteric coated formulation is enteric coated tablets, beads or granules, which optionally contain a lubricant such as, but not limited to, magnesium stearate. The enteric coated formulations include enteric coated beads in a capsule, enteric coated microspheres in a capsule, enteric coated microspheres provided in a suspension or mixed with food, which suspensions are particularly convenient for pediatric administration, and enteric coated compressed tablets. The capsule can be a hard-shell gelatin capsule or a cellulose capsule. In particular, the pharmaceutical composition is formulated as an enteric coated capsule. In one specific aspect, a proanthocyanidin polymer composition is administered in tablet form, which tablet is backfilled with microcrystalline cellulose.

In one embodiment, the proanthocyanidin polymer composition is directly compressed, that is, the proanthocyanidin polymer composition, with or without any excipients, can be compressed into a tablet, or other pharmaceutical formulation, that has a pharmaceutically acceptable hardness and friability. Preferably, the directly compressible pharmaceutical composition can be compressed into tablets having a hardness of greater than 4 kp (kiloponds), preferably a hardness of 8 to 14 kp, more preferably a hardness of 10 to 13 kp. A directly compressible composition can be compressed into a tablet that has a friability of not more than 1% loss in weight, preferably less than 0.8% loss in weight, more preferably less than 0.5% loss in weight.

In one embodiment, the directly compressible formulations consist of 99.93 % proanthocyanidin polymer composition and 0.07 % magnesium stearate and is coated with a methacrylic acid copolymer. In another embodiment, the pharmaceutical formulation contains a directly compressible proanthocyanidin polymer composition but no excipients, additives or vehicles other than an enteric coating; however, the formulation may contain a lubricanf, such as but not limited to, magnesium stearate. Preferably, a directly compressed proanthocyanidin polymer composition formulation is formulated as a tablet of pharmaceutically acceptable hardness (greater than 4 kp, preferably 8-14 kp, and more preferably 10-13 kp) and friability (not more than 1% loss in weight, preferably less than 0.8% loss in weight, and more preferably less than 0.5% loss in weight).

In another embodiment, the proanthocyanidin polymer compositions are enteric coated. Enteric coatings are those coatings that remain intact in the stomach, but will dissolve and release the contents of the dosage form once it reaches the small intestine. A large number of enteric coatings are prepared with ingredients that have acidic groups such that, at the very low pH present in the stomach, e.g., pH 1.5 to 2.5, the acidic groups are not ionized and the coating remains in an undissociated, insoluble form. At higher pH levels, such as in the environment of the intestine, the enteric coating is converted to an ionized form, which can be dissolved to release the inhibitor molecule. Other enteric coatings remain intact until they are degraded by enzymes in the small intestine, and others break apart after a defined exposure to moisture, such that the coatings remain intact until after passage into the small intestines.

Polymers which are useful for the preparation of enteric coatings include, but are not limited to, shellac, starch and amylose acetate phthalates, styrene-malefic acid copolymers, cellulose acetate succinate, cellulose acetate phthalate (CAP), polyvinylacetate phthalate (PVAP), hydroxypropylmethylcellulose phthalate (grades HP-50 and HP-55), ethylcellulose, fats, butyl stearate, and methacrylic acid- methacrylic acid ester copolymers with acid ionizable groups (including "ACRYLEZE®" and "EUDRAGIT®"), such as "EUDRAGIT® L 3OD", "EUDRAGIT® RL 30D", "EUDRAGIT® RS 30D", "EUDRAGIT® L 100-55", and "EUDRAGIT® L 30D-55". In one embodiment, the pharmaceutical compositions contain a proanthocyanidin polymer composition and the enteric coating polymer "EUDRAGIT® L 30D", an anionic copolymer of methacrylic acid and methyl acrylate with a mean molecular weight of 250,000 Daltons. In another preferred embodiment, the enteric coating polymer is "EUDRAGIT® L 30D-55".

The disintegration of the enteric coating occurs either by hydrolysis by intestinal enzymes or by emulsification and dispersion by bile salts, depending upon the type of coating used. For example, esterases hydrolyze esterbutyl stearate to butanol and stearic acid and, as the butanol dissolves, the stearic acid flakes off of the medicament. Additionally, bile salts emulsify and disperse ethylcellulose, hydroxypropylmethylcellulose, fats and fatty derivatives. Other types of coatings are removed depending on the time of contact with moisture, for example coatings prepared from powdered carnauba wax, stearic acid, and vegetable fibers of agar and elm bark rupture after the vegetable fibers absorb moisture and swell. The time required for disintegration depends upon the thickness of the coating and the ratio of vegetable fibers to wax.

Application of the enteric coating to the proanthocyanidin polymer can be accomplished by any method known in the art for applying enteric coatings. For example, but not by way of limitation, the enteric polymers can be applied using organic solvent based solutions containing from 5 to 10% w/w polymer for spray applications and up to 30% w/w polymer for pan coatings. Solvents that are commonly in use include, but are not limited to, acetone, acetone/ethyl acetate mixtures, methylene chloride/methanol mixtures, and tertiary mixtures containing these solvents. Some enteric polymers, such as methacrylic acid-methacrylic acid ester copolymers can be applied using water as a dispersant. The volatility of the solvent system must be tailored to prevent sticking due to tackiness and to prevent high porosity of the coating due to premature spray drying or precipitation of the polymer as the solvent evaporates.

Furthermore, plastic izers can be added to the enteric coating to prevent cracking of the coating film. Suitable plasticizers include the low molecular weight phthalate esters, such as diethyl phthalate, acetylated monoglycerides, triethyl citrate, polyethyl glycoltributyl citrate and triacetin. Generally, plasticizers are added at a concentration of 10% by chromatographic purity of enteric coating polymer weight. Other additives such as emulsifiers, for example detergents and simethicone, and powders, for example talc, may be added to the coating to improve the strength and smoothness of the coating. Additionally, pigments may be added to the coating to add color to the pharmaceutical formulation.

In specific embodiments, pharmaceutical compositions comprising proanthocyanidin polymer compositions are provided as enteric coated beads in hard-shell gelatin capsules. Proanthocyanidin polymer beads are prepared by mixing a proanthocyanidin polymer composition with hydroxypropylmethylcellulose and layering the mixture onto nonpareil seeds (sugar spheres). In a more preferred embodiment, crofelemer, which is directly compressible, without any excipients, additives or vehicles other than an enteric coating, is milled and fractionated into beads (e.g., as beads that do not contain the nonpareil sugar seeds). The beads may be covered with a seal coat of Opadry Clear (mixed with water). A preferred enteric coating of the beads is "EUDRAGIT™ L 30D" or "EUDRAGIT™ L 30D-55" applied as an aqueous dispersion containing 20%-30% w/w dry polymer substance, which is supplied with 0.7% sodium lauryl sulfate NF (SLS) and 2.3% polysorbate 80 NF (Tween™ 20) as emulsifiers, to which plasticizers, such as polyethylene glycol and/or citric acid esters, are added to improve the elasticity of the coating, and talc can be added to reduce the tendency of the enteric coating polymer to agglutinate during the application process and to increase the smoothness of the film coating.

In a specific formulation, the final composition of enteric coated proanthocyanidin polymer composition beads containing the nonpareil seeds is 17.3% w/w nonpareil seeds, 64.5% w/w proanthocyanidin polymer composition, 1.5% w/w hydroxypropylmethylcellulose, 0.5% w/w Opadry clear, 14.5% w/w "EUDRAGIT™ L 30D", 1.45% w/w triethyl citrate, and 0.25% w/w glyceryl monostearate. This pharmaceutical formulation may be prepared by any method known in the art.

A specific formulation of the proanthocyanidin polymer composition beads not containing the nonpareil seeds is 78% w/w directly compressible proanthocyanidin polymer composition, 0.76% w/w Opadry Clear, 19% w/w "EUDRAGIT™ L 30D- 55", 1.9% triethyl citrate, and 0.34% w/w glyceryl monostearate. This pharmaceutical formulation may be prepared by any method known in the art.

Another formulation contains 54.58% w/w proanthocyanidin polymer composition beads (without non-pareil seeds and made of a directly compressible proanthocyanidin polymer composition), 1.78% wlw Opadry Clear, 39% w/w "EUDRAGIT™ L 30D-55", 3.9% triethylcitrate, and 0.74% wlw glyceryl monostearate.

In another embodiment, the pharmaceutical composition comprising the proanthocyanidin polymer composition is formulated as enteric coated granules or powder (microspheres with a diameter of 300-500 p.) provided in either hard shell gelatin capsules or suspended in an oral solution for pediatric administration. The enteric coated powder or granules may also be mixed with food, particularly for pediatric administration. This preparation may be prepared using techniques well known in the art.

In general, the granules and powder can be prepared using any method known in the art, such as but not limited to, crystallization, spray-drying or any method of comminution, preferably using a high speed mixer/granulator. Examples of high speed mixer/granulators include the "LITTLEFORD LODIGE™" mixer, the "LITTLEFORD LODIGE™" MGT mixer/granulator, and the "GRAL™" mixer/granulator. During the high-shear powder mixing, solutions of granulating agents, called binders, are sprayed onto the powder to cause the powder particles to agglomerate, thus forming larger particles or granules. Granulating agents which are useful for preparing the granules, include but are not limited to, cellulose derivatives (including carboxymethylcellulose, methylcellulose, and ethylcellulose), gelatin, glucose, polyvinylpyrrolidone (PVP), starch paste, sorbitol, sucrose, dextrose, molasses, lactose, acacia gum, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, Veegum and larch arabogalactan, polyethylene glycol, and waxes. Granulating agents may be added in concentrations ranging from 1 to 30% of the mass of the particles or granules.

The powder or granules are preferably coated using the fluidized bed equipment. The granules or powder may then be covered with a seal coat of Opadry Clear (mixed with water). A preferred enteric coating is "EUDRAGIT™ L 30D" applied as an aqueous dispersion containing 30% w/w dry polymer substance, which is supplied with 0.7% sodium lauryl sulfate NF (SLS) and 2.3% polysorbate 80 NF (Tween™ 20) as emulsifiers, to which the plasticizers, polyethylene glycol and citric acid esters, are added to improve the elasticity of the coating, and talc is added to reduce the tendency of the enteric coating polymer to agglutinate during the application process and to increase the smoothness of the film coating. In one embodiment, the final composition of an enteric coated powder is 81.8% w/w proanthocyanidin polymer composition, 1.5% w/w hydroxypropylmethylcellulose, 0.5% w/w Opadry clear, 14.5% w/w "EUDRAGIT™ L 3OD", 1.45% w/w triethyl citrate, and 0.25% w/w glyceryl monostearate. The final composition of the enteric coated granules is 81.8% w/w proanthocyanidin polymer composition, 10% polyvinylpyrrolidone, 1.5% w/w hydroxypropylmethylcellulose, 0.5% w/w Opadry clear, 14.5% w/w "EUDRAGIT™ L 30D", 1.45% wlw triethyl citrate, and 0.25% w/w glyceryl monostearate.

Enteric coated granules or powder particles can further be suspended in a solution for oral administration, particularly for pediatric administration. The suspension can be prepared from aqueous solutions to which thickeners and protective colloids are added to increase the viscosity of the solution to prevent rapid sedimentation of the coated powder particles or granules. Any material which increases the strength of the hydration layer formed around suspended particles through molecular interactions and which is pharmaceutically compatible with the proanthocyanidin polymer composition can be used as a thickener, such as but not limited to, gelatin, natural gums (e.g., tragacanth, xanthan, guar, acacia, panwar, ghatti, etc.), and cellulose derivatives (e.g., sodium carboxymethylcellulose, hydroxypropylcellulose, and fiydroxypropylmethylcellulose, etc.). Optionally, a surfactant such as Tween™ may be added to improve the action of the thickening agent. A preferred suspension solution is a 2% w/w hydroxypropylmethylcellulose solution in water containing

0.2% Tween™.

The proanthocyanidin polymer composition can also be formulated as enteric coated tablets. In one preferred embodiment, a proanthocyanidin polymer composition is granulated with any pharmaceutically acceptable diluent (such as those listed above) by the methods described above for preparing the granules. Then, the granules are compressed into tablets using any method well known in the art, for example but not limited to, the wet granulation method, the dry granulation method or the direct compression method. Preferred diluents include, but are not limited to, microcrystalline cellulose ("AVICEL™ PH 200/300") and dextrates ("EMDEX™"). Additionally, disintegrants, such as those described above, and lubricants, such those described above, may also be added to the tablet formulation. A preferred tablet formulation contains 250 mg proanthocyanidin polymer composition, 7 mg of the disintegrant "AC-DI-SOL™" (cross linked sodium carboxymethylcellulose), 1.75 mg of the lubricant magnesium stearate and the weight of "AVICEL™ PH 200/300" necessary to bring the mixture up to 350 mg. The tablets are coated with an enteric coating mixture prepared from 250 grams "EUDRAGIT™ L 30 D-55", 7.5 grams triethyl citrate, 37.5 grams talc and 205 grams water. This formulation may be prepared by any method well known in the art.

In one embodiment, a directly compressible proanthocyanidin polymer composition is made into granules by size reduction (e.g., as described above) and mixed with a lubricant, preferably, magnesium stearate. Then, the lubricated granules are compressed into tablets using any method well-known in the art, for example but not limited to, the direct compression method. Preferably, each tablet is 125 mg containing 99.6% w/w directly compressible proanthocyanidin polymer composition and 0.40% w/w magnesium stearate. The tablets are then preferably coated with an enteric coating mixture of a 30% suspension (6.66 g in 22.22 g) of "EUDRAGIT™ L 30D-55 ", 0.67 g triethyl citrate, 1.67 g talc and 20.44 g purified water, per 100 grams of tablet. The tablets can be prepared by any method known in the art.

In a more preferred embodiment, a directly compressible proanthocyanidin polymer composition is formulated into core tablets of either 125 mg, 250 mg or 500 mg containing 99.6% w/w directly compressible proanthocyanidin polymer composition and 0.40% w/w magnesium stearate. The tablets are then preferably coated with an enteric coating mixture. The final composition of the tablets is 86.6% w/w directly compressible proanthocyanidin polymer composition, 0.4% magnesium stearate, 6.5% "EUDRAGIT™ L30D-55", 0.9% triethyl citrate, 2.87% talc, and 2.74% white dispersion. The tablets can be prepared by any method known in the art, for example but not limited to the method described infra.

The compositions formed into small particles (which include particles sized on the order of micrometers, such as microspheres and microcapsules), particles (which include particles sized on the order of millimeters), drug crystals, pellets, pills and microbeads can be coated using a fluidized-bed process. This process uses fiuidized- bed equipment, such as that supplied by "GLATT™", "AEROMATIC™", "WURSTER™", or others, by which the composition cores are whirled up in a closed cylindrical vessel by a stream of air, introduced from below, and the enteric coat is formed by spray drying it onto the cores during the fluidization time. To coat tablets or capsules, Accela-Cota coating equipment ("MANESTY™") can be used. By this process, the tablets or capsules are placed in a rotating cylindrical coating pan with a perforated jacket and spraying units are installed within the pan and the dry air is drawn in through the rotating tablets or capsules. Any other type of coating pan, such as the "COMPU-LAB™" pan, Hi-coates "GLATT™" immersion sword process, the "DRIAM™" Dricoater, " STEINBERG™" equipment, "PELLEGRINI™" equipment, or "WALTHER™" equipment can also be used.

In another embodiment, the proanthocyanidin polymer composition is provided as a suppository for rectal administration. Suppositories can be formulated with any base substance which is pharmaceutically acceptable for the preparation of suppositories and which is compatible with the proanthocyanidin polymer composition. Because rectal administration does not expose the proanthocyanidin polymer composition to the stomach environment, the pharmaceutical formulations for rectal administration need not be formulated to protect the composition from the stomach environment. Suppository bases which may be used to prepare suppositories with the proanthocyanidin polymer composition include, but are not limited to, cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols or fatty acids of polyethylene glycols or glycol-surfactant combinations or nonionic surfactant materials (such as polyoxyethylene sorbitan fatty acid esters (Tweens), polyoxyethylene stearates, and mixtures of sorbitan fatty acid esters (Span and Arlacel)). However, because of the hydrophilic nature of the proanthocyanidin polymer composition, a hydrophilic base for the suppository is suggested. A preferred suppository formulation for the proanthocyanidin polymer composition is prepared from 91 grams glycerin, 9 grams sodium stearate, 5 grams purified water and can be 5% to 50% w/w proanthocyanidin polymer composition. Alternatively, the suppository may contain 10 grams proanthocyanidin polymer composition, 20 grams gelatin, and 70 grams of glycerin. Suppositories prepared from the proanthocyanidin polymer composition can be shaped by any method known in the art, including but not limited to, compression molding, fusion, or, preferably, melt molding.

In another embodiment, the proanthocyanidin polymer composition is formulated with a compound or compounds which neutralize stomach acid. Alternatively, the pharmaceutical composition containing the proanthocyanidin polymer composition is administered either concurrent with or subsequent to administration of a pharmaceutical composition which neutralize stomach acid. Compounds, such as antacids, which are useful for neutralizing stomach acid include, but are not limited to, aluminum carbonate, aluminum hydroxide, bismuth subnitrate, bismuth subsalicylate, calcium carbonate, dihydroxyaluminum sodium carbonate, magaldrate, magnesium carbonate, magnesium hydroxide, magnesium oxide, and mixtures thereof.

In another embodiment, the proanthocyanidin polymer composition is administered with a substance that inactivates or inhibits the action of stomach enzymes, such as pepsin. Alternatively, the pharmaceutical composition containing the proanthocyanidin polymer composition is administered either concurrent with or subsequent to administration of a pharmaceutical composition active to inactivate or inhibit the action of stomach enzymes. For example, but not by way of limitation, protease inhibitors, such as aprotin, can be used to inactivate stomach enzymes.

In another embodiment, the proanthocyanidin polymer composition is formulated with a compound or compounds which inhibit the secretion of stomach acid. Alternatively, the pharmaceutical composition containing the proanthocyanidin polymer composition is administered either concurrent with or subsequent to administration of a pharmaceutical composition active to inhibit the secretion of stomach acid. Compounds which are useful for inhibiting the secretion of stomach acid include, but are not limited to, omeprazole, esomeprazole, lansoprazole, rabeprazole, pantaprazole, ranitidine, nizatidine, famotidine, cimetidine, and misoprostol.

The following series of Examples are presented for purposes of illustration and not by way of limitation on the scope of the invention.

Experimental Section

Comparative example:

Isolation of Partially Purified Proanthocyanidin Polymer Composition

A directly compressible proanthocyanidin polymer composition was isolated from the latex of the Croton lechleri plant as follows: 460 liters of Croton lechleri latex was mixed with 940 liters purified water for ten minutes and then allowed to stand overnight (12 hours) at 4⁰C. The red supernatant was pumped into a holding tank and the residue discarded. The supernatant was then extracted with 200 liters n-butanol by mixing for ten minutes and then allowing the phases to separate. The n-butanol phase was discarded, and the aqueous phase was extracted two more times with 200 liters n- butanol each time. After extraction, the aqueous phase was concentrated by ultrafiltration using a 1 IeD cut-off membrane (a low protein binding cellulose membrane), and then the retentate was dried in a tray dryer at approximately 37⁰C ( + 2⁰C).

For purification by column chromatography, 6 kg of the dried extract was dissolved in 75 liters of purified water and stirred for 90 minutes. The dissolved material was chromatographed on a two column chromatography system consisting of a 35 liter CM-Sepharose column (a weak cation exchange resin) and a 70 liter LH-20 column (a size-exclusion resin) connected in series. The material was loaded onto the CM- Sepharose column, washed with 140 liters purified water, and then eluted onto the LH-20 column with 375 liters of 30% acetone. At this point, the two columns were disconnected, and the proanthocyanidin polymer composition was eluted from the LH-20 column with 250 liters of 45% acetone. Fractions were collected into 10 liter bottles and monitored with a UV detector at 460 nm. Fractions containing material having detectable absorbance at 460 nm were pooled and concentrated by ultrafiltration using a 1 IcD cut-off membrane (a low protein binding cellulose membrane). The retentate was dried using a rotary evaporator in a waterbath at approximately 37⁰C ( + 2⁰C) to yield a partially-purified proanthocyanidin polymer composition.

The partially-purified proanthocyanidin polymer composition was tested for direct compressibility. 250 mg portions of the proanthocyanidin polymer composition, in the absence of any binders or excipients, was placed into a tableting machine and then pressed into tablets of varying thicknesses (e.g., the greater the pressure on the composition to form it into a tablet, the thinner the resulting tablet). The hardness of the tablets was then determined in a hardness tester.

The friability of tablets having a hardness of 8-15 kp was determined as described in USP 23 (1216). The friability was less than 0.5% loss in weight.

Example 1 :

Preparation of a Proanthocyanidin Polymer Composition:

A partially-purified proanthocyanidin polymer composition according to comparative example was weighed and ground up in a glass jar using a glass rod. The ground polymer composition was dissolved and titrated to pH 8-8.1 with 0.5N NaOH and treated six times with dichloromethane. The aqueous layer was exposed to a 6O⁰C bath without vacuum to remove any remaining dichloromethane. The residue was titrated to pH 4 using IN HCl. Sufficient acetone to yield a 10% acetone solution was added. LH-20 resin was equilibrated in pH 4 distilled water, placed in a Buchner funnel, and excess liquid was removed. The aqueous/acetone residue containing proanthocyanidin polymer was mixed evenly with the semi-dried LH-20 resin and permitted to settle for 30 minutes. An amount of LH-20 equal to the initial aliquot of LH-20 and pre-conditioned in the same way was filtered on a Buchner funnel over Whatman 42 filter paper to remove excess liquid. The LH-20/proanthocyanidin polymer combination was layered on top of the layer of LH-20 in the Buchner funnel and allowed to set for 30 minutes. After the settling, 140 mL of 10% (w/w) Acetone in pH 4 DIW was added to the top of the Buchner funnel. The vacuum was pulled slightly to start collection. This was repeated 5 more times for a total of 6 fractions of 14OmL each. A total of 840 mL of solvent was used for the 10% (w/w) elution. Additional elutions were performed with 25% (w/w) acetone (1 OxHOmL), 32% (w/w) acetone (6x14OmL), 39% (w/w) acetone (6x14OmL), 45% (w/w) acetone (6x14OmL), and 54% (w/w) acetone (6x14OmL). Fractions containing proanthocyanidin polymer were combined and dried in a 6O⁰C vacuum oven to yield proanthocyanidin polymer composition in 16.59% yield. A comparison of the purity of Example 1 with Comparative Example is shown in the following table:

Table: Comparative Example and Example 1

Alternatively, the columns could be separated and not run in series. An example of columns run separated follows. For example, the columns may be run as two or more separate entities. For example, 6 kilos of dissolved material are loaded on to the first column (e.g., CM-Sepharose), and Solution "A" is collected. As the column is eluted with water, Solution "B" is collected. Then as the column is eluted with about 30% acetone, Solution "C" is collected.

These solutions (Solutions A, B, and C) are then loaded on to the second column (e.g., LH-20) consecutively for example, "Solution A" followed by "Solution B" followed by "Solution C"). Crofelemer is then eluted from the column (e.g., LH- 20) with about 45% acetone solution.

Example 2:

Step 1: Equal volume of methyl ethyl ketone was added to crude plant latex solution (500ml) along with mud stirred gently and kept undisturbed for 2 hours. Organic layer was separated and the process was repeated for two times. Aqueous layer was concentrated to obtain 60 gm Stage-A.

Step 2: 1Og obtained from Stage A was dissolved in 120 ml of water and stirred for 1 hour at 30-35⁰C and kept to room temperature for another 1 hour. The mass was filtered through a whatman filter paper under vacuum and filtrate was subjected to Sepharose column (11cm, LD. 3 cm) joined in series to Sephadex column (22cm, LD. 3 cm). The elution was carried out with 250ml water followed by 30% acetone water mixture (600ml). After 30% acetone elution Sepharose column was .removed and Sephadex column was eluted with 45% acetone water mixture. Initial 110 ml fraction was discarded and then 100 ml dark colored band was taken and concentrated to furnish 1.3-1.5g of Crofelemer.

*BDL = Below Detection Limit; *Mn = Molecular Weight, *PD = Polydispersity

Example 3:

Stage- 1 : Two volume of methyl ethyl ketone was added to crude plant latex solution (500ml) along with mud stirred gently and kept undisturbed for 2 hours. Organic layer was separated and the process was repeated for three more times. Combined organic layers were concentrated to obtain 50gm Stage-A.

Stage-2: 50 gms obtained from Stage -A was dissolved in 1Ov of distilled water and stir for 1 hour at 30-35⁰C. The solution obtained is kept at room temperature for another 1 hour. This solution was subjected to Sepharose column (6 volumes). Elution was carried out with 3Ov of water followed by 30 volumes of acetone water mixture. The aqueous portion eluted was discarded and 8 volumes of organic mixture (30% acetone) was discarded followed by elution of a dark band, the rich cut (15 volumes). The rich cut was taken and concentrated to furnish a yield of 7.5-8.0 gms of Crofelemer.

Example 4:

Stage- 1: Two volume of methanol and four volume of Ethyl acetate added to crude plant latex solution (500ml) along with mud stirred for 4 hours and kept undisturbed for 2 hours. Organic layer was separated and filtered through Whatman filter paper. The filtrate was concentrated to obtain 90 gm Stage-A.

Stage-2: 50 gms obtained from Stage -A was dissolved in 12 volumes of distilled water and stir for 1 hour at 30-35⁰C. The solution obtained is kept at room temperature for another 1 hour. This solution was subjected to Sepharose column (6 volumes). Elution was carried out with 30 volumes of water followed by 25 volumes of acetone water mixture. The aqueous portion eluted was discarded and 8 volumes of organic mixture (30% acetone) was discarded followed by elution of a dark band, the rich cut (15 volumes). The rich cut was taken and concentrated to furnish a yield of 12.0-13.0 gms of Crofelemer.

Example 5:

Process for the purification of Crofelemer with Charcoal treatment in Methanol/Ethyl acetate by precipitation method:

The crude Proanthocyanidin compound (lOOgms) is dissolved in methanol (1000ml) and filtered through sintered glass funnel. The undissolved solid is again extracted with methanol (200ml) followed by combining the two filtrates and treated with 30 grams of Charcoal stirred for 60 minutes. The compound is filtered through Bentonite

Bed two times and the residue is discarded. The filtrate is treated with 6.2ml of ethyl acetate and stirred for 60 minutes and filter through 2mm Whatman paper. 52 gms of

Brown coloured solid obtained having a purity of 99.81% and the content of Taspine impurity is below detection limit.

Using Bentonite to remove protein in aqueous solution:

3.3520 g partially purified proanthocyanidin polymer composition was weighted out and put into a 50 mL plastic tube with cap, 30 mL DI-water (pre-adjusted to pH = 8.8) was added to dissolve all partially purified polymer composition with shaking. The pH of the resulting solution was 6.12 and it was adjusted to 5.5 by IN HCl. The solution was then equally split into two tubes (some foam remaining in the original tube was discarded).

Tube one was shaken for lhr and then stored at room temperature for 22 hrs. The resulting solution was filtered through fine size filter funnel. A 100 μL sample of the filtrate was taken out, dried to get 9.7mg solid and tested by amino acid analysis to serve as AAA sample without bentonite treatment. The filtrate was adjusted to pH = 8.0 by 0.5N NaOH. A 50 μL sample was taken out, dried and tested for HPLC and molecular weight profile for sample without bentonite treatment.

600mg Bentonite was added to tube two. The tube was capped and shaken for lhr and then stored at room temperature for 22 hrs. The resulting solution was filtered through fine size filter funnel. A 100 μL sample of the filtrate was taken out, dried to get 10.3 mg solid and tested by amino acid analysis to serve as AAA sample with bentonite treatment. The filtrate was adjusted to pH = 8.0 by 0.5N NaOH. A 50 μL sample was taken out, dried and tested for HPLC and molecular weight profile for sample with bentonite treatment.

Using Ethyl Acetate on a partially purified polymer composition:

1.6704 g partially purified polymer composition was weighed out and slowly added into a scintillation vial with stir bar and 15 mL DI-water (pre-adjusted to pH = 8.62). The vial was capped and kept stirring until all partially purified polymer composition was dissolved. The pH of the resulting solution was 6.49 and then it was adjusted to 8 by 0.5N NaOH. A 200 μL sample was taken out, dried and tested for HPLC and molecular weight profile for sample before EtOAc extraction.

The pH 8 partially purified polymer composition solution was extracted with 20 mL EtOAc 6 times. Inter-phase layer was observed during the extraction, same as dichloromethane extraction. 200 μL sample was taken out from the resulting aqueous phase, dried and tested for HPLC and molecular weight profile for sample after 6 times EtOAc extraction.

Using Bentonite and MeOH/EtOAc on partially purified polymer composition materials.

2.5 z Scale:

2.5g partially purified polymer composition was put in a 5OmL polypropylene tube with cap, 25g (~32mL) MeOH was added. The tube was capped and put on the shaker to shake for 10 minutes until all solids were dissolved. 130μL sample from this solution was dried in oven by vacuum at room temperature (RT) and tested.

750mg bentonite was added to the methanol solution of partially purified polymer composition and the tube was capped and shaken for lhr. The solution was filtered through a filter paper using Buchner funnel on an Erlenmeyer flask. Bentonite along with material adsorbed. on it was discarded. 130μL sample was taken out from the filtrate, dried by vacuum in oven at RT and then analyzed. The above filtrate (32mL) was put in a 25OmL polypropylene bottle, 5X volume of EtOAc (160ml) was added to it. Brown color precipitate was observed immediately. The bottle was capped and shaken for 10 minutes.

The mixture was filtered by filter paper using Buchner funnel. The solid was dried by vacuum at RT to obtain 930.2mg product, sample was analyzed. There were some particles passing through the filter paper to the filtrate. This filtrate was centrifuged and the precipitated solid was filtered and washed by EtOAc, dried under vacuum at RT to yield another 194.8mg solid. Sample was taken from this solid. The clear filtrate and EtOAc wash solution was combined and dried by rotary evaporation at 40 ⁰C to generate 538.7mg solid. Sample was also taken from this solid.

\2.5e Scale:

The process described above was scaled-up to 12.5g partially purified polymer composition. After adding EtOAc to precipitate out the desired products, the mixture was filtered through filter paper. The solid on top of the filter paper was dried by vacuum at RT to obtain 4.4896g solid. Sample from this solid was taken. Similar to the smaller scale process, the filtrate still contains some solids and it was left in refrigerator overnight. The resulting solution with precipitation was then slowly poured into a Buchner funnel with filter paper, this time no solid passed through the filter paper. The remaining solid was dried by vacuum at RT to produce 1.422 Ig solid. Sample was taken.

Protein removal study on 2.5g Scale:

Previously samples for amino acid analysis were not taken prior to and after bentonite treatment, experiments were repeated and the same volume of sample solutions were taken out and dried for amino acid analysis before and after bentonite treatment. After bentonite treatment, the solution was filtered before taking the sample. To eliminate the effect of protein removal just by filtration, the solution before bentonite treatment was also filtered prior to the sampling. Thus, the difference of the protein level in these two samples should be attributable to bentonite treatment.

Preparation of Pharmaceutical Formulations Described herein are illustrative methods for the manufacture and packaging of pharmaceutical formulations of the proanthocyanidin polymer composition from C. lechleri produced according to the methods disclosed herein.

ENCAPSULATED ENTERIC COATED BEADS

Descriptions of the methods used to prepare the encapsulated enteric coated proanthocyanidin polymer composition bead formulation based on sugar spheres are provided below. Each hard-shell gelatin capsule contained 250 mg proanthocyanidin polymer composition enteric coated beads. Capsules were packaged in HDPE bottles containing sixteen (16) 250 mg caps each. The formulation for enteric coated proanthocyanidin polymer composition beads contained 17.3% (w/w) of nonpareil seeds (sugar spheres 40/60 mesh, Paulaur, lot #60084060), 64.5% proanthocyanidin polymer composition from C. lechleri, 1.5% hydroxypropylmethylcellulose (Methocel E5 Premium,Dow Chemical Co., lot #MM9410162E), 0.5% Opadry Clear (Colorcon, lot #S83563), 14.5% "EUDRAGIT™ L 3OD" (Rohm Tech., lot #1250514132), 1.45% triethyl citrate (Morflex, lot #N5X291), glyceryl monostearate (Imwitor-900, Rohm Tech, lot #502-229), and purified water (USP).

The layering coating solution containing the proanthocyanidin polymer composition was prepared by adding hydroxypropylmethylcellulose and the proanthocyanidin polymer composition to purified water (USP) and mixing until dissolved. The nonpareil seeds were loaded into the product bowl of the fluid bed processor (NiorPrecision Coater). The polymer solution was then layered on the nonpareil seeds by spraying the solution onto the fluidized nonpareil seeds at a target bed temperature of 3035⁰C. Once the proanthocyanidin polymer layering had been completed, a seal coat using Opadry Clear (preparing by mixing the Opadry Clear with Purified Water, USP) was applied with a target bed temperature of 30-35⁰C. After the seal coat was applied, the pellets were discharged and screened through lOOOt and 425μ screens, and the layered spheres larger than 425g and smaller than lOOOμ were charged back into the fluid bed processor. Meanwhile, the enteric coating solution was prepared by mixing triethyl citrate and glyceryl monostearate to water that had been heated to 65°C and then mixing this solution with the "EUDRAGIT™ L 30D-55". The resulting enteric coating solution was then sprayed onto the layered spheres in the fluidized bed processor, at a bed temperature of 30-35⁰C, until all the enteric coating solution was layered on the beads. Enteric coated beads were hand filled into a Size #0 hard shell gelatin capsule to provide a 250mg dosage and then packaged into a suitable HDPE bottles with a heat induction lined cap.

TABLE 1: BATCH FORMULA

Product: Proanthocyanidin Polymer Enteric Coated Beads

Batch Size: 578.0 gm

Raw Material Amount Used Per Batch

Sugar Nonpareil Spheres, NF (40/60) 100.0 gm

Proanthocyanidin Polymer Composition 372.8 gm

Hydroxypropylmethylcellulose E5, USP 8.7 gm

(K29/32)

Opadry Clear (YS-1-19025A) 2.9 gm

"EUDRAGIT™ L 30D-55" 279.4 gm

(30% solids)

Triethyl Citrate, NF 8.4 gm

Glycerol Monostearate 1.4 gm

Water, USP (Removed during processing) 1284.8 gm

ENCAPSULATED ENTERIC COATED BEADS

Described below are methods for preparing encapsulated enteric coated bead formulations that do not contain nonpareil sugar spheres. One formulation contains 83.3% w/w proanthocyanidin polymer composition, 0.5% w/w Opadry clear, 14.5% w/w "EUDRAGIT™ L 30D-55" , 1.9% w/w triethyl citrate and a 0.34% glyceryl monostearate.

The beads were first seal coated with a 5% solution of Opadry clear in a 16 liter aeronatic MP-I fluidized bed processor with a 50mm Wurster column. The coating parameters for the application of the seal coating were an inlet temperature of 50⁰C to 60⁰C, an outlet temperature of 25°C to 40⁰C, an air volume of 30 to 40 CMH, a spray rate of 6 to 12 grams per minute, and an air pressure of 2.5 Bar. After the seal coat was applied, the beads were discharged and screened for beads larger than 425 μ and smaller than 100Ot. The beads of appropriate size were then charged back into the fluid bed processes for enteric coating. For each 1000 grams of proanthocyanidin polymer composition beads, an enteric coating suspension was prepared from 811.97 grams "EUDRAGIT™ L 30D-55", 24.36 grams triethyl citrate, 4.36 grams glyceryl monostearate and 248.55 grams purified water. This suspension was prepared by gently stirring the "EUDRAGIT™ L 30D-55" suspension continually and, in a separate container, suspending and homogenizing the triethyl citrate and talc in purified water. The triethyl citrate/talc mixture was then added to the "EUDRAGIT™ L 30D-55" suspension, and the resulting coating dispersion stirred during the spraying process to avoid settling. The beads were then coated in the fluidized bed processor under the following parameters: the inlet temperature was 42°C to 47°C; the outlet temperature was 28°C to 34°C; the air volume was 30-40 CMH; the spray rate was 612 grams/minute; and the air pressure was 2.5 Bars. The resulting enteric coated beads were then filled into a size #0 hard shell gelatin capsule.

ENTERIC COATED GRANULES AND POWDER PARTICLES

A method for formulating the proanthocyanidin polymer composition as enteric coated granules or powder in either hard shell gelatin capsules or suspended in an oral solution is set forth below. The proanthocyanidin polymer composition powder particles are prepared by high-shear powder mixing of the proanthocyanidin polymer composition and hydroxypropylmethylcellulose in a high speed mixer/granulator. The proanthocyanidin polymer composition granules are prepared by spraying polyvinylpyrrolidone on the powder in the high speed mixer/granulator so that the powder particles agglomerate to form larger granules. Using fluidized bed equipment, the granules or powder are then covered with a seal coat of Opadry Clear (mixed with water) and then coated with the enteric coating "EUDRAGIT™ L 30D" applied as an aqueous dispersion containing 30% w/w dry methacrylate polymer substance, which is supplied with 0.7% sodium lauryl sulfate NF (SLS) and 2.3% polysorbate 80 NF (Tween™ 20) as emulsifiers, to which the plasticizers, triethyl citrate and glyceryl monostearate, are added to improve the elasticity of the coating. The final composition of the enteric coated powder is 81.8% w/w proanthocyanidin polymer composition, 1.5% w/w hydroxypropylmethylcellulose, 0.5% w/w Opadry clear, 14.5% wlw "EUDRAGIT™ L 30D", 1.45% w/w triethyl citrate, and 0.25% w/w glyceryl monostearate. The final composition of the enteric coated granules is 81.8% w/w proanthocyanidin polymer composition, 10% polyvinylpyrrolidone, 1.5% w/w hydroxypropylmethylcellulose, 0.5% w/w Opadry clear, 14.5% w/w "EUDRAGIT™ L 30D", 1.45% w/w triethyl citrate, and 0.25% w/w glyceryl monostearate.

The enteric coated proanthocyanidin polymer composition granules or particles may be filled into a hard shell gelatin capsule in an amount which provides a suitable dosage. The enteric coated proanthocyanidin polymer composition granules or powder particles can also be suspended in a solution for oral administration, particularly for pediatric administration. The suspension solution is prepared by wetting 2 grams hydroxypropylmethylcellulose in 97.8 ml distilled water and 0.2 grams Tween™ 80; mixing this preparation to homogeneity by sonicating, heating the solution to 40⁰C and stirring for three hours; and then adding the enteric coated proanthocyanidin polymer composition powder particles or granules to the homogeneous solution.

ENTERIC COATED COMPRESSED TABLETS

A method for formulating the proanthocyanidin polymer composition with a diluent as enteric coated tablets is described below. For each 350 mg tablet, 250 mg proanthocyanidin polymer composition is granulated with 7 mg crosslinked sodium carboxymethylcellulose ("AC-DI-SOL™") and a sufficient mass of microcrystalline cellulose ("AVICEL™ PH 200/300") to bring the total mass to 350 mg. These ingredients are mixed for 20 to 30 minutes in a V blender. After the 20 to 30 minutes of mixing, 1.75 mg magnesium stearate is added and the mixture is blended for an additional 4 to 5 minutes. The resulting granules are compressed on a rotary tablet press using 5/16th inch standard concave punches. The tablets are coated with an enteric coating mixture prepared from 250 grams "EUDRAGIT™ L 30 D-55", 7.5 grams triethyl citrate, 37.5 grams talc and 205 grams water. The tablets are then placed in a perforated pan coater (e.g. the "ACCELACOTA™" system) and rotated at 15 rpm at 40⁰C. The enteric coating formulation is sprayed using the following conditions: inlet air temperature of 44°C-48°C, exhaust air temperature of 29°C-32°C, product temperature of 26°C-30°C, a 1 mm spray nozzle, a pan speed of 30 to 32 rpm, an airflow of 30-32 CFM, and a spray pressure of 20 PSI. The tablets are finally cured for 30 minutes as the pan is rotating at 15 rpm with an inlet air temperature of 60⁰C μnd then, after shutting off the heat, the tablets are rotated at 15 rpm until the tablets have cooled to room temperature.

ENTERIC COATED DIRECTLY COMPRESSED TABLETS

A method for formulating the proanthocyanidin polymer composition without a diluent as enteric coated tablets was carried out as described below. Directly compressible proanthocyanidin polymer composition was produced. 125 mg tablets were prepared by blending 99.6% w/w directly compressible proanthocyanidin polymer composition with 0.40% w/w magnesium stearate for two minutes and then directly compressing the material into 125 mg tablets on a rotary press using 1/4 inch diameter round standard concave punches to a tablet hardness of 4-10 Kp.

The core tablets were tested and found to have an average hardness (n=10) of 4-10 Kp, friability (n=20) of less than 0.7%, an average table weight (n=10) of 125 mg ± 7 mg, an average thickness (n=10) of 3.9 to 4.1 mm, and a disintegration time (n=6) of not more than 20 minutes.

The coating dispersion was prepared by mixing, per 100 grams of tablets, 22.22 grams of a 30% wlw " EUDRAGIT™ L 30D-55" suspension, kept gently stirred with a mixture of 0.67 grams triethyl citrate, 1.67 grams talc and 20.44 grams purified water which had been mixed until homogeneous. The coating dispersion was continually stirred to avoid settling.

The tablets (in batches of 100,000) were coated with the coating dispersion in a Compu-Lab 24 inch/30 L pan. The tablets were jogged in the pan at a speed of 3-5 rpm and pre-warmed to a temperature of 35°C to 40 ⁰C. The tablets were then coated with the enteric coating dispersion to a 6% to 8% weight gain with the following parameters: an inlet temperature of 45°C to 65°C; an exhaust air temperature of 27 ⁰C to 34°C; a product temperature of 28° C to 32⁰C; a pan speed of 8-14 rpm; an air flow of 180 to 240 CHM; an air spray pressure of 10-20 psi (pounds per square inch); an initial spray rate of 3 to 4 grams/min/kg; and a final spray rate of 4 to 8 grams/min/kg. The tablets were then cured for 30 minutes in the pan with an inlet temperature of 45°C to 50⁰C and a pan speed of 3 to 5 rpm. Finally, the tablets were allowed to cool

60 to room temperature in the pan at a pan speed of 3 to 5 rpm. Four of the 125 mg tablets were then filled into a size zero, opaque Swedish orange-colored gelatin capsule.

The enteric coated proanthocyanidin polymer composition tablets were tested for content uniformity, drug release, microbiological tests and stability, and some analytical in process tests were also performed. In stability studies, the proanthocyanidin polymer composition remained stable after six months of storage at room temperature as well as under accelerated temperature and humidity conditions. Finally, the core tablets were tested and found to have an average hardness (n=10) of 4-10 Kp, friability (n=20) of less than 0.7%, an average tablet weight (n=10) of 125 mg±7 mg, an average thickness (n=10) of 3.9 to 4.1 mm, and a disintegration time (n=6) of not more than 20 minutes.

ENTERIC COATED DIRECTLY COMPRESSED TABLETS

Formulations of the proanthocyanidin polymer composition descriebed herien, without a diluent, as enteric coated tablets were carried out as described below. The core tablets were prepared by milling 250 mg proanthocyanidin polymer composition per tablet (approximately 16 kg total) in a Quadro Comil with an 024R (30 mesh) screen and then blending the milled composition in a Patterson Kelley 2 cubic foot twin shell blender. 1 mg magnesium stearate (Spectrum Quality Products, Inc., New Brunswick, N.J) per tablet was then added to the composition in the blender and blended for 2 minutes. The blend was then compressed into 251 mg tablets (containing 250 mg proanthocyanidin polymer composition) on a rotary tablet press to a tablet hardness of 8-15 Kp and friability less than 0.5%.

The coating dispersion was prepared by first mixing in a first container the 25 g (7.5 g solids) ΕUDRAGIT™ L 30 D-55" (HuIs America, Inc., Somerset, N.J.) (weight given per 115 grams coated tablets) dispersion. The pigment dispersion was prepared by adding sequentially with constant stirring in a second container 39.59 g purified water, 3.30 grams talc (Alphafil™ 500) (Whittaker, Clark & Daniels, Inc., South Plainfield, N. J.), 6.06 g (3.15 g solids) White Dispersion (pigment) (Warner- Jenkinson, Inc., St. Louis, Mo.), and then 1.05 g triethyl citrate (Morflex, Inc., Greensboro, N.C.). The mixture was then homogenized for 15 minutes or until homogenous. While slowly stirring, the pigment dispersion was added to the "EUDRAGIT™ L 30 D-55" dispersion and then stirred for 30 minutes before spraying. Stirring was also maintained during the spraying process to avoid settling.

The tablets were coated in batches of 50,000 in a Compu-Lab 24 inch/30 L pan with the following settings: 10-20 psi atomizing air pressure; 35°C-60°C pan inlet air temperature; 5 to 6 inches nozzle tip to tablet bed distance; and 4/2 baffles/nozzles. After adding the tablets to the pan, the pan was jogged at a speed of 3 to 5 rpm and heated to 40⁰C. The tablets were then sprayed to a weight gain of 11 to 13% with the following parameters: 27°-33°C target exhaust temperature (to be achieved within ten minutes of spraying); pan speed of 8 to 12 rpm; 180-240 CFM air flow; and a spray rate of 2-5 g/min/kg. After achieving the desired weight gain, the heat was shut off and the pan jogged at 3-5 rpm until the tablets were cooled to below 30⁰C. The tablets were encapsulated in size AA opaque Swedish orange colored DB gelatin capsules (Capsugel, Greenwood, S.C.). 500 mg tablets were also produced as described above, except that coating was done on batches of 25,000 tablets to a weight gain of 8 to 10%.

DRY POWDER FOR ORAL SUSPENSION

Proanthocyanidin polymer composition (1.6 g), sodium benzoate (0.06 g), Microcrystalline cellulose and carboxymethylcellulose sodium (AVICEL CL 611, 0.04 g), Colloidal silicon dioxide (0.01 g), Tutti Frutti flavor (0.03 g), and Pharma grade sugar (9.25 g) were individually sifted through ASTM # 40 sieve, mixed in a geometric proportion, and then blended together in a blender. The dry blend (total weight about 11 g) was light brown to brown color free flowing powder, and has bulk density 0.78 g/ml.

The dry blend, upon reconstitution with 23 ml of Purified water, yielded a brown colored suspension (approx. 30 ml), which has pH 5.2. Each 5 ml of the reconstituted suspension contains 250 mg of proanthocyanidin polymer composition.

The invention described and claimed herein is not to be limited in scope by the specific embodiments disclosed. Theembodiments are intended as illustrations of several aspects and embodiments. Any equivalent embodiments are intended to be within the scope. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. All publications cited herein are incorporated by reference in their entirety.

Claims

What is claimed is:

1. A method of producing a pharmaceutically acceptable proanthocyanidin polymer composition, comprising:

(i) allowing sediment to settle from latex of a Croton Spp.;

(ii) adjusting the pH of the latex to between about 6.5 to about 8.5;

(iii) filtering the latex to produce a filtrate;

(iv) adjusting the pH of the filtrate to between about 3.5 to about 5.5;

(v) performing a first extraction of the filtrate to obtain an eluate comprising the proanthocyanidin polymer;

and

(vi) collecting the eluate comprising the proanthocyanidin polymer composition.

2. The method of claim 1, wherein the first extraction comprises a solid phase extraction.

3. The method of claim 1, further comprising performing a second extraction, wherein the second extraction comprises a solid phase extraction.

4. The method of claim 2, wherein the second solid phase is distinct from the first solid phase.

5. The method according to claim 1, wherein the proanthocyanidin polymer composition comprises greater than 90% proanthocyanidin polymer.

6. The method according to claim 5, wherein the proanthocyanidin polymer composition comprises greater than 95% proanthocyanidin polymer.

7. The method according to claim 1, wherein the proanthocyanidin polymer composition consists essentially of proanthocyanidin polymer and water.

8. The method of claim 7, wherein the sediment is allowed to settle for at least about 48 hours.

9. The method according to claim 1, wherein the period of time that the sediment is allowed to settle from the latex comprises between about 48 hours and about 48 months.

10. The method of claim 1, wherein the sediment is allowed to settle at a temperature of less than about 20⁰C; at a temperature of between about 20⁰C and about 0⁰C; between about 30°C.and about 20⁰C; between about 20⁰C and about -20⁰C; or between about 0⁰C and 15°C.

1 1. The method according to claim 1, wherein the sediment is allowed to settle at a temperature between 0⁰C and 15°C for at least 2 hours.

12. The method according to claim 1, wherein the pH is adjusted in step (ii) to pH 8.

13. The method according to claim 1, wherein step (iii) is performed with a filtering aid.

14. The method of claim 13 wherein step (iii) ftirther comprises one or more aqueous washes of the filtering material.

15. The method of claim 1, wherein step (iii) further comprises centrifuging the latex instead of filtering.

16. The method of claim 13, wherein the filtering material comprises one or more of diatomaceous earth, charcoal, bentonite, cellulose, glass, sand, or filter paper.

17. The method of claim 1 or 16, wherein the filtrate of step (iii) and any optional washes are combined and processed by ultrafiltration.

18. The method according to claim 17, wherein the ultrafiltration is performed with a semipermeable membrane.

19. The method of claim 18, wherein the semipermeable membrane permits passage of solutes up to a molecular weight selected from the group consisting of 500 Da, 1 kDa, 5 kDa, 10 kDa, 20 kDa, and 30 kDa.

20. The method according to claim 1, wherein the pH of step (iv) comprises 4.

21. The method according to claim 1 or 2, wherein the solid phase extraction of step (v) is performed with solid phase extraction resin.

22. The method according to claim 21, wherein the solid phase extraction resin is selected from the group consisting of ion exchange resins, affinity resins, adsorption resins, partition resins, and mixtures thereof.

23. The method according to claim 21 wherein the solid phase extraction resin comprises a carboxymethyl-modifϊed agarose resin.

24. The method according to claim 21, wherein step (v) is performed in a batch-wise fashion.

25. The method according to claim 2, further comprises processing the eluate of the first solid phase extraction to yield a composition for the second solid phase extraction.

26. The method according to claim 1, wherein the eluate of the first extraction is produced by elution of the solid phase with a solvent system selected from the group consisting of water, acetone, methanol, ethanol, glycol and mixtures thereof.

27. The method according to claim 2, wherein the second solid phase extraction is performed with a solid phase extraction resin.

28. The method according to claim 27 wherein the solid phase extraction resin comprises one or more of size exclusion resins, ion exchange resins, affinity resins, adsorption resins, partition resins, or mixtures thereof.

29. The method according to claim 27, wherein the solid phase extraction resin comprises a modified polysaccharide.

30. The method according to claim 29, wherein the modified polysaccharide comprises a hydroxypropylated cross-linked dextran.

31. The method according to claim 1, wherein in the eluate from the first extraction is produced by elution of the solid phase with a solvent selected from the group consisting of water, acetone, methanol, ethanol, glycol and mixtures thereof.

32. The method according to claim 2, wherein the eluate from the second extraction is mixed with a solvent selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol, hexanol, ethylene glycol, propylene glycol, ethyl acetate, dichloromethane, trichloromethane, tetrachloromethane, dichloroethane, and mixtures thereof.

33. The method according to claim 1, wherein the processing of step (vi) is selected from the group consisting of ultrafiltration, freeze drying, evaporation with heat, evaporation without heat, evaporation with vacuum, evaporation without vacuum, spray drying, and combinations thereof.

34. The method according to claim 1, wherein the level of residual taspine in the proanthocyanidin polymer composition comprises less than 0.2% of the composition.

35. The method according to claim 34, wherein the level of residual taspine in the proanthocyanidin polymer composition comprises less than 0.1% of the composition by chromatographic purity.

36. The method of claim 34 or 35, wherein the level of taspine is determined by chromatography.

37. The method of claim 1, wherein the polydispersity of the proanthocyanidin comprises between about 1.2 and about 1.8.

38. A proanthocyanidin polymer composition comprising proanthocyanidin polymer obtained according to the process of any one of claims 1-37

39. The proanthocyanidin polymer composition according to claim 38, wherein the proanthocyanidin polymer composition comprises greater than 90% proanthocyanidin polymer.

40. The proanthocyanidin polymer composition according to claim 39, wherein the proanthocyanidin polymer composition comprises greater than about 95% proanthocyanidin polymer.

41. The proanthocyanidin polymer composition according to claim 40, wherein the proanthocyanidin polymer composition comprises proanthocyanidin polymer and water.

42. A pharmaceutical formulation comprising the proanthocyanidin polymer composition according to claim 39 and a pharmaceutically acceptable carrier.

43. The pharmaceutical formulation according to claim 42, wherein the level of residual taspine in the proanthocyanidin polymer composition comprises less than 0.2% of the composition.

44. The pharmaceutical formulation according to claim 43, wherein the level of residual taspine in the proanthocyanidin polymer composition comprises less than 0.1% of the composition.

45. A method of producing a proanthocyanidin polymer composition, comprises:

(i) obtaining a partially-purified proanthocyanidin polymer composition,

(ii) performing a solid phase extraction of the partially-purified proanthocyanidin polymer composition to obtain an eluate containing proanthocyanidin polymer, and

(iii) processing the eluate to yield a proanthocyanidin composition;

wherein step (ii) is performed with a solid phase extraction resin as the solid phase.

46. The method according to claim 45, wherein the partially-purified proanthocyanidin polymer composition is partially purified by a method comprising: obtaining a latex from a Croton spp. and performing a solid phase extraction with solid phase extraction resin that is not hydroxypropylated cross-linked dextran.

47. The method according to claim 46, wherein the solid phase extraction resin comprises a carboxymethyl-modified agarose resin.

48. The method according to claim 45, wherein the partially-purified proanthocyanidin polymer composition is partially purified by a method comprising obtaining a latex from a Croton spp. and performing a solid phase extraction with solid phase extraction resin that is not carboxymethyl-modified agarose resin.

49. The method according to claim 48, wherein the solid phase extraction resin comprises a hydroxypropylated cross-linked dextran resin.

50. The method according to claim 45, wherein the partially-purified proanthocyanidin polymer composition comprises between about 35 to about 90% proanthocyanidin polymer.

51. The method according to claim 45, wherein the partially-purified proanthocyanidin polymer composition comprises SB-300.

52. The method according to claim 45, wherein the solid phase extraction resin of step (ii) is a modified polysaccharide.

53. The method according to claim 52, wherein the modified polysaccharide comprises a hydroxypropylated cross-linked dextran.

54. The method according to claim 45, wherein the solid phase extraction resin of step (ii) comprises carboxymethyl-modifϊed agarose resin.

55. The method according to claim 45, wherein the purified proanthocyanidin polymer composition comprises greater than about 90% proanthocyanidin polymer.

56. The method according to claim 55, wherein the purified proanthocyanidin polymer composition comprises greater than about 95% proanthocyanidin polymer.

57. The method according to claim 56, wherein the purified proanthocyanidin polymer composition comprises proanthocyanidin polymer and water.

58. The method according to claim 45, wherein the eluate is mixed with a solvent selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol, hexanol, ethylene glycol, propylene glycol, ethyl acetate, dichloromethane, trichloromethane, tetrachloromethane, dichloroethane, and mixtures thereof.

59. The method according to claim 45, wherein the processing is selected from the group consisting of ultrafiltration, freeze drying, evaporation with heat, evaporation without heat, evaporation with vacuum, evaporation without vacuum, spray drying, and combinations thereof.

60. The method according to claim 45, wherein the level of residual taspine in the proanthocyanidin polymer composition comprises less than 0.2% of the composition.

61. The method according to claim 45, wherein the level of residual taspine in the purified proanthocyanidin polymer composition comrpises less than 0.1% of the composition.

62. The method according to claim 45, wherein the polydispersity of the proanthocyanidin polymer in the purified proanthocyanidin polymer composition comprises between about 1.2 and about 1.8.

63. A proanthocyanidin polymer composition comprising proanthocyanidin polymer obtained according to the process of claim 45.

64. The proanthocyanidin polymer composition of claim 63, wherein the proanthocyanidin polymer composition is of a purity and concentration sufficient for incorporation into a therapeutically effective pharmaceutical composition.

65. The proanthocyanidin polymer composition according to claim 45, wherein the purified proanthocyanidin polymer composition comprises greater than 90% proanthocyanidin polymer.

66. The proanthocyanidin polymer composition according to claim 56, wherein the purified proanthocyanidin polymer composition comprises greater than 95% proanthocyanidin polymer. ^'

67. The proanthocyanidin polymer composition according to claim 66, wherein the proanthocyanidin polymer composition consists essentially of proanthocyanidin polymer and water.

68. A pharmaceutical formulation comprising the purified proanthocyanidin polymer composition according to claim 45 and a pharmaceutically acceptable carrier.

69. The pharmaceutical formulation according to claim 68, wherein the level of residual taspine in the purified proanthocyanidin polymer composition comprises less than 0.2% of the composition.

70. The pharmaceutical formulation according to claim 69, wherein the level of residual taspine in the purified proanthocyanidin polymer composition comprises less than 0.1% of the composition.

71. A method of producing a proanthocyanidin polymer composition, comprising:

(i) maintaining a latex from a Croton spp. below room temperature for at least 48 hours to permit sediment to settle;

and either step (ii) (a), or step (ii) (b), or both steps (ii) (a) and (ii) (b) as follows:

(ii) (a) dissolving latex in water or methanol, adding bentonite to solution with agitation and optional adjustment of pH to between 5.0 to 7.0, and filtering to produce a filtrate; and/or (ii) (b) addition of ethyl acetate and optionally addition of water and/or adjustment of pH to between 6.5 to 8.5, agitation, and removal of the ethyl acetate layer to result in an aqueous or methanolic phase or solid precipitate;

thereby producing a proanthocyanidin polymer composition.

72. The method of claim 71, further comprising purifying aqueous or methanolic phase the phase extractions to yield a product.

73. The method of claim 72, wherein the purification comprises by solid phase extraction.

74. The method of claim 71, wherein step (ii) (a) occurs in methanol.

75. The method of either claim 71 or claim 74, wherein step (ii) (a) is repeated between one and twenty times.

76. The method of either claim 71 or claim 74, wherein step (ii) (b) is repeated between one and twenty times.

77. The method of claim 71, wherein step (ii) (a) removes about 10% or more of protein existing in sample prior to step (ii) (a).

78. The method of claim 71, wherein taspine levels above 250 ppm in the latex are reduced to less than about 250 ppm.

79. A method of producing a pharmaceutically acceptable proanthocyanidin polymer composition, comprising:

(i) allowing sediment to settle from latex of a Croton Spp.;

(ii) adjusting the pH of the latex to between about 6.5 to about 8.5;

(iii) filtering the latex to produce a filtrate;

(iv) adjusting the pH of the filtrate to between about 3.5 to about 5.5;

(v) performing a first solid phase extraction of the filtrate to obtain an eluate comprising the proanthocyanidin polymer;

and

(vi) collecting the eluate comprising the proanthocyanidin polymer composition.

80. A method of producing a proanthocyanidin polymer composition, comprising: a), providing a solution of plant latex comprising the mud;

b). adding an organic solvent to the solution of plant latex and mud;

c). separating the organic layer and concentrating aqueous layer to obtain a solid; Alternatively;

Separating the aqueous layer and concentrating organic layer to obtain a solid; d). dissolving the solid in an aqueous solvent;

e). subjecting the solution to chromatography; and

f). isolating Crofelemer;

thereby producing a proanthocyanidin polymer composition.

81. The method of claim 80, wherein organic solvent in step (b) is selected from one or more of alcohols, ketones, esters, ethers or mixtures therof.

82. The method of claim 81, wherein ketone comprises methyl ethyl ketone.

83. The method of claim 80, wherein organic solvent in step (b) selected from mixture of methanol and ethyl acetate.

84. The method of claim 80, wherein column used in chromatography in step (e) comprises a single column or two set column selected from CM-Sepharose Fast Flow Column and Sephadex LH-20.