WO2006016349A1

WO2006016349A1 - Compositions of cholera-adsorbing insoluble or poorly soluble polysaccharide particles

Info

Publication number: WO2006016349A1
Application number: PCT/IL2004/000734
Authority: WO
Inventors: Eyal Shimoni; Yechezkel Kashi; Hanan Gancz; Orly Meir
Original assignee: Technion Research And Development Foundation Ltd.
Priority date: 2004-08-08
Filing date: 2004-08-08
Publication date: 2006-02-16

Abstract

The present invention relates to compositions and kits for preparing suspensions of insoluble or poorly soluble starch or cellulose particles and use of these suspensions in pharmaceutical formulations for the treatment of cholera. Furthermore, the present invention provides kits, apparatus and methods for isolating or identifying the Vibrio cholerae bacteria in a liquid sample.

Description

COMPOSITIONS OF CHOLERA-ADSORBING INSOLUBLE OR POORLY SOLUBLE POLYSACCHARIDE PARTICLES

FIELD OF THE INVENTION The present invention relates to compositions and kits for preparing suspensions of insoluble or poorly soluble starch or cellulose particles and use of these suspensions in pharmaceutical formulations for the treatment of cholera. Furthermore, the present invention provides kits, apparatus and methods for isolating or identifying the Vibrio cholerae bacteria in a liquid sample.

BACKGROUND OF THE INVENTION

Cholera is a severe gastrointestinal disease caused by the pathogen Vibrio cholerae that results in diarrhea and severe dehydration. The disease is caused after the ingestion of V. cholerae infested drinking water or food, or unprotected contact with excretions from cholera infected individuals. The mortality rate from V. cholerae induced infection can reach up to 50%, yet when medical help is at hand, antibiotics and rehydration therapy reduce this rate. The inaccessibility to medical care in areas where cholera outbreaks usually occur led to the development of the Oral Rehydration Solutions (ORS) by the WHO in the late 70' s. Oral rehydration solutions (ORS) are now routinely utilized throughout the world to correct the fluid and electrolyte losses associated with diarrhea. They have significantly decreased the mortality rate in third world children.

Traditionally, ORS contains, at a minimum, water, glucose, and sodium. The principle underlying oral rehydration is the phenomenon of coupled transport. The presence of glucose in the ORS increases the absorption of sodium by the body. Every glucose molecule that crosses the intestinal epithelium brings a sodium ion with it, raising the concentration of ions in the blood stream and pulling water out of the gut. The exact concentration of glucose in the oral fluid is very important. Sodium absorption improves as the glucose concentration of the oral fluid is increased up to about 2.5% w/w. At higher concentrations, the glucose can no longer be efficiently absorbed leading to a net reduction in sodium and water absorption. In fact, higher concentrations of glucose increase the osmotic load in the gut, which pulls water out of the blood stream. This leads to a net loss of fluids and electrolytes farther exacerbating dehydration.

The World Health Organization has recommended that an ORS contain 90 mEq of sodium per liter, 20 mEq of potassium per liter, 30 mEq carbonate per liter and 111 mM of glucose per liter. ORS is a simple-to-use and easily distributed therapy for moderate cholera, yet some drawbacks inhibit its general usage and have led many to attempt to develop improved oral rehydration solutions.

For example, United States Patents 5,096,894 and RE 36,032 disclose an ORS comprising a mixture of rice dextrin and required electrolytes where the ORS rice dextrin purportedly functions better than ORS glucose in infants with chronic diarrhea. United States Patent 5,164,192 relates to an effervescent product for the preparation of an ORS, where the product comprises oligosaccharides and/or disaccharides, and/or monosaccharides and/or amino acids as energy carriers, and (bi)carbonate and a bicarbonate precursor as alkalizing substances. United States Patent 5,733,579 discloses an ORS including indigestible oligosaccharides. United States Patent Application publication number 2003/0077333 discloses a zinc supplemented ORS and its use in the treatment of diarrhea. United States Patent Application publication number 2003/0119755 discloses methods for alleviating mucositis, associated with radiation and chemotherapy, via the administration of an oral rehydration solution. United States Patent Application publication number 2003/01493293 discloses a anti-diarrhea pharmaceutical composition containing various chemical agents, including tormentil root extract, antisecretory agents, and antibiotics. United States Patent Application publication number 2003/0194448 discloses an ORS including a low-fiber colloidal hydrolyzed rice suspension. United States Patent Application 2004/0071793 discloses an improved oral rehydration solution comprising standard formulae of sugars, mineral salts and bicarbonates in combination with Croton species materials with or without the inclusion of material from the Uncaria species.

None of the aforementioned patents and patent applications discloses or suggests an ORS that includes suspensions of insoluble or poorly soluble polysaccharide particles.

Ramakrishna et al 2000 (New Engl. J. Med, 342:5 308-313, 2000) reported a study where adolescent or adult patients with cholera were administered 5O g per liter of uncooked high-amylose maize starch in standard ORS containing glucose in combination with electrolytes. The authors concluded that the addition of a specific amylase resistant high amylose starch to oral rehydration solution reduces fecal fluid loss and shortens diarrhea in adolescents and adults with cholera. Ramakrishna et al. 2000 attributed the effect of the high amylose starch to purportedly enhancing the production of short chain fatty acids in the colon and thus enhancing water absorption.

Insoluble or poorly soluble polysaccharides are widely used as pharmaceutical excipients rather than as active pharmaceutical ingredients (see Handbook of pharmaceutical Excipients, 2nd ed., Wade, A. and Weller, P. J., eds., American Pharmaceutical Association (1994)). WO 00/28974 entitled "Local Delivery of Drugs to the Colon for Local Treatment of Colonic Diseases" discloses that specific configurations of insoluble polysaccharides and modified polysaccharides may be used as excipients to deliver pharmaceutical active ingredients to the colon.

Recently, WHO recommended a revised oral rehydration solution containing less sodium and glucose for all acute diarrheas, including cholera. This change was proposed to reduce gross stool volume and use of intravenous therapy by lowering solution osmolarity. There is an ongoing need for oral rehydration pharmaceutical compositions for treating cholera that include functional ingredients that do not significantly contribute to solution osmolarity.

Recently, the inventors of the present invention presented their preliminary findings concerning the attachment of Vibrio cholerae to starch granules (H. Gancz, O.

Meyer, Y. Kashi, and E. Shimoni, "Vibrio cholerae and Starch - an Improved Oral

Rehydration Solution at Sight?" Annual meeting of the Israel Society for Microbiology,

Haifa, Israel, February 9-10, 2004).

It would be advantageous to have a more effective Oral Rehydration Therapy (ORT) for treating cholera. Furthermore, there is an unmet need for compositions for treating cholera that are effective in the absence of or independently of antibiotics or other drugs. In addition, it would be advantageous to have an ORT that works even with reduced glucose levels, or in the absence of glucose.

SUMMARY OF THE INVENTION The present invention relates generally to compositions and methods for adsorbing Vibrio cholerae to insoluble or poorly soluble polysaccharide particles. In particular, the present invention provides kits and improved Oral Rehydration compositions useful for treating cholera. Furthermore, the present invention provides kits, apparatus and methods for isolating or identifying Vibrio cholerae bacteria in a liquid sample.

The present invention is based, in part, on the surprising discovery that bacteria of the species Vibrio cholerae adsorb to the surface of certain types of insoluble or poorly soluble starch or cellulose particles. Unexpectedly, this adsorption is effective even at physiological temperatures and may be used for improved oral rehydration therapy in cholera patients. This absorption is comparatively specific to Vibrio cholerae, with dramatically lower adhesion efficiencies observed for E CoIi, Listeria monocytogenes, and Salmonella typhimurium, and also highly efficient, with measured adhesion efficiencies of over 90% for certain types of starch. Thus, ingested non- gelatinized starch or cellulose particles, traveling through the alimentary canal of a human or other animal subject, adsorb Vibrio cholerae bacteria located in the digestive track and facilitate excretion of the virulent bacteria, ameliorating the cholera in the subject. Thus, the present invention minimizes the need for antibiotics and other antimicrobial compounds when treating cholera.

It is noted that the insoluble or poorly soluble polysaccharide particles of this invention do not substantially dissolve in electrolyte solutions, and thus do not substantially contribute to osmotic pressure. Thus, one advantage of the present invention is that the insoluble or poorly soluble starch or cellulose particles may be suspended in any previously disclosed ORS and imbue this ORS with additional therapeutic qualities without concomitantly raising the osmotic pressure above desirable levels. Thus, unlike many soluble functional agents that can be added to ORS, use of the insoluble or poorly soluble starch or cellulose particles of the present invention does not require concomitantly lowering the concentration of other therapeutic agents dissolved in the composition.

Thus, according to one aspect, the present invention provides a method for generating an improved oral rehydration therapy without significantly elevating osmolarity comprising adding insoluble or poorly soluble polysaccharide particles to an Oral Rehydration Solution and suspending the particles in the solution. According to preferred embodiments, the polysaccharide is selected from the group consisting of non- gelatinized starches and celluloses. Insoluble and poorly soluble polysaccharides are widely used as pharmaceutical excipients and as thickening agents. The present invention for the first time reveals that insoluble and poorly soluble are useful for removing cholera from the alimentary canal of a subject and may be used as active ingredients of a pharmaceutical composition. According to one aspect, the present invention provides a kit for extemporaneous reconstitution to produce a pharmaceutical composition for treating cholera, the kit comprising: a) a plurality of insoluble or poorly soluble polysaccharide particles; and b) an oral rehydration solution. According to preferred embodiments, the polysaccharide is selected from the group consisting of non-gelatinized starches and celluloses.

According to certain embodiments, the polysaccharide is a non-gelatinized starch selected from the group consisting of corn starch, waxy maize starch, rice starch and wheat starch. In alternative embodiments, the polysaccharide is a cellulose. In one particular embodiment, the cellulose is microcrystalline cellulose.

According to some embodiments, the insoluble or poorly soluble polysaccharide particles range in size from 5 to 45 microns. Not wishing to be bound by theory, it is understood that when the particles are larger than 45 microns, the suspension may assume a sandy texture, making it difficult and unpleasant to ingest. Not wishing to be bound by theory, it is understood that particles smaller than 5 microns will have a surface area on the same order of magnitude as the surface area of the bacteria Vibrio cholerae, and thus it is more difficult for bacteria to adsorb to these smaller insoluble or poorly soluble polysaccharide particles. In specific embodiments, the polysaccharide particles range in size from 10 to 30 microns. According to some embodiments, the kit contains 40 to 120 grams of insoluble or poorly soluble polysaccharide particles per liter of electrolyte solution.

The oral rehydration solution (ORS) contains at a minimum sodium and chloride. Optionally, the ORS electrolyte solution further contains other electrolytes, which may include but are not limited to potassium, zinc, citrates, carbonates and bicarbonates. In a preferred embodiment, the ORS may be provided as a prepared solution. Alternatively, one or more ingredients of the ORS may be provided as dry, particulate matter for extemporaneous reconstitution, in an appropriate aqueous solution.

According to some embodiments, the kit further comprises an additional functional ingredient selected from the group consisting of carbohydrates, thickening agents, preservatives, amino acids, drugs, mineral salts, vitamins, nutraceuticals, probiotics, flavors, and sweeteners.

In certain embodiments, the functional ingredient is dry, particulate matter to be added to the electrolyte solution upon reconstitution. In yet other embodiments, this functional ingredient is part of the electrolyte solution.

In one embodiment, the kit further contains glucose. Other appropriate soluble carbohydrates include, but are not limited to glucose, fructose, sucrose, and maltodextrin.

Thus, according to another aspect the present invention provides a kit comprising dry, particulate matter for reconstitution to produce a pharmaceutical composition for treating cholera. Kits comprising dry, particulate matter are lighter and more compact than kits containing solutions. These kits may be reconstituted extemporaneously, though in many situations special care must be taken to reconstitute using potable cholera-free water, preferably sterilized water. The present invention provides a kit for extemporaneous reconstitution of an oral rehydration therapy comprising: a) a plurality of insoluble or poorly soluble polysaccharide particles; and b) at least one dry electrolyte.

According to preferred embodiments, the polysaccharide is selected from the group consisting of non-gelatinized starches and celluloses.

According to another aspect, the invention provides a pharmaceutical composition for treatment of cholera comprising insoluble or poorly soluble polysaccharide particles suspended in an electrolyte solution, wherein the polysaccharide is other than a high-amylose starch. According to preferred embodiments, the polysaccharide is selected from the group consisting of non-gelatinized starches other than a high-amylose starches and celluloses.

According to some embodiments, the polysaccharide is a non-gelatinized starch. selected from the group consisting of corn starch with an amylose content of less than

50%, waxy maize starch, rice starch and wheat starch. In alternative embodiments, the polysaccharide is a cellulose. In one particular embodiment, the cellulose is microcrystalline cellulose.

According to some embodiments, the insoluble or poorly soluble polysaccharide particles range in size from 5 to 45 microns. In specific embodiments, the polysaccharide particles range in size from 10 to 30 microns.

According to some embodiments, the insoluble or poorly soluble polysaccharide particles are present in the quantity from about 40 grams/L to about 120 grams/L.

According to some embodiments, the viscosity of the pharmaceutical composition does not differ substantially from the viscosity of water, with 0.9 centipoises at 25° C a typical value. In other embodiments, a thickening agent may be added to the composition, in order to impede settling of the suspending particles. This may be especially important when the suspension is not immediately consumed, but is packaged for later use. Suitable thickening agents include but are not limited to agar, alginic acid and salts, gum arabic, gum acacia, gum talha, cellulose derivatives, curdlan, fermentation gums, furcellaran, gellan gum, gum ghatti, guar gum, iota carrageenan, irish moss, kappa carrageenan, konjac flour, gum karaya, lambda carrageenan, larch gum/arabinogalactan, locust bean gum, pectin, tamarind seed gum, tara gum, gum tragacanth, xanthan gum and mixtures thereof. The skilled practitioner is able to select the appropriate concentrations for each particular thickening agent.

In particular embodiments, the composition contains all the necessary electrolytes and levels thereof required by the Food and Drug Administration for oral rehydration formulations sold in the United States. According to these embodiments, in addition to sodium (Na+), potassium (K+), chloride (Cl-) and citrate ions, the composition contains a source of simple carbohydrate, such as glucose, fructose, or dextrose. Typically, the composition for these embodiments comprises water, simple carbohydrates, sodium ions, potassium ions, chloride ions, and citrate ions. The quantity of sodium ions used in various embodiments can vary widely, as is known to those skilled in the art. Typically, the composition will contain from about 30 mEq/L to about 95 niEq/L of sodium, hi certain embodiments, sodium content can vary from about 30 mEq/L to about 110 mEq/L, most preferably from about 50 mEq/L to about 95 mEq/L. Suitable sodium sources include but are not limited to sodium chloride, sodium citrate, sodium bicarbonate, sodium carbonate, and mixtures thereof.

In various embodiments, the composition will also contain a source of potassium ions. The quantity of potassium can vary widely. However, as a general guideline, the composition will typically contain from about 10 mEq/L to about 30 mEq/L of potassium. In a further embodiment, it may contain from about 15 mEq/L to about 25 mEq/L of potassium. Suitable potassium sources include but are not limited to, potassium citrate, potassium chloride, potassium bicarbonate, potassium carbonate, potassium hydroxide, and mixtures thereof.

According to certain embodiments, the composition will also contain a source of soluble carbohydrate. Quantities ranging from about 1.5% w/w to about 3.0% w/w are suitable. Not wishing to be bound to any particular theory, excessive soluble carbohydrate may exacerbate the fluid and electrolyte losses associated with diarrhea.

Any soluble carbohydrate used in previously disclosed oral rehydration solutions may be used to practice the present invention. Suitable carbohydrates include but are not limited to glucose, fructose, sucrose, and maltodextrin.

According to various embodiments, the composition will also typically include a source of buffer. The type and quantity of buffer can vary as is known in the art. Typically, a citrate buffer is used, the citrate content ranges from about 10 mEq/L to about 40 mEq/L, more preferably from about 20 mEq/L to about 40 mEq/L, and most preferably from about 25 mEq/L to about 35 mEq/L. Suitable citrate sources include, but are not limited to, potassium citrate, sodium citrate, citric acid, and mixtures thereof.

In various embodiments, the composition will also typically contain a source of chloride. The quantity of chloride can vary as is known in the art. Typically the composition will contain chloride in the amount of from about 30 mEq/L to about 80 mEq/L, more preferably from about 30 mEq/L to about 75 mEq/L, and most preferably from about 30 mEq/L to about 70 mEq/L. Suitable chloride sources include but are not limited to, sodium chloride, potassium chloride and mixtures thereof. In various embodiments, the composition will also contain a source of zinc ions. The quantity of zinc used in the composition of this invention can vary widely. Zinc ingested in the composition can replace the zinc lost due to the underlying diarrhea and/or vomiting. Concentrations at from about 0.3 mEq to about 10 mEq of zinc per liter of composition will typically accomplish this result. Typically, the composition will contain from about 0.6 mEq to about 3 mEq of zinc per liter. Alternatively, the composition may contain from about 0.6 mEq to about 1.2 mEq of zinc per liter. The source of zinc ions is not critical. Any zinc salt suitable for human consumption may be used in the composition of this invention. Examples of suitable zinc sources include zinc gluconate, zinc sulfate, zinc chloride, zinc citrate, zinc bicarbonate, zinc carbonate, zinc hydroxide, zinc lactate, zinc acetate, zinc fluoride, zinc bromide, and zinc sulfonate.

The composition of the present invention will also typically include a flavor to enhance its palatability, especially in a pediatric population or for embodiments wherein the glucose level is lower than that used in typical ORS formulations. The flavor should mask the salty notes of the composition. Useful artificial sweeteners include saccharin, aspartame, sucralose, acesulfane-K (ace-K), and the like.

Preservatives may be added to help extend shelf life. Persons knowledgeable in the art would be able to select the appropriate preservative, in the proper amount, to accomplish this result. Typical preservatives include, but are not limited to, potassium sorbate and sodium benzoate.

In accordance with the present invention, it has been discovered that Vibrio cholerae adsorb to insoluble or poorly soluble polysaccharide particles even in the absence of glucose. The present invention also provides pharmaceutical composition wherein the concentration of glucose is lower than that traditionally found in ORS formulations.

According to another aspect, the present invention provides pharmaceutical compositions for treatment of cholera comprising insoluble or poorly soluble polysaccharide particles suspended in an electrolyte solution, wherein the composition contains less than 2.0 w/w% glucose, preferably less than 1.5 w/w% glucose, and more preferably less than 1.0 w/w% glucose. According to preferred embodiments, the polysaccharide is selected from the group consisting of non-gelatinized starches and celluloses.

According to some embodiments, the polysaccharide is a non-gelatinized starch selected from the group consisting of corn starch, waxy maize starch, rice starch and wheat starch. In alternative embodiments, the polysaccharide is a cellulose. In one particular embodiment, the cellulose is microcrystalline cellulose.

In accordance with the present invention, it has been discovered that the Vibrio cholerae adsorb to granular insoluble or poorly soluble starch or cellulose particles, and thus these particles are useful also in apparatus and methods for isolating Vibrio cholerae from a fluid. Relevant applications include water purification and tools for detecting the presence of Vibrio cholerae in a fluid sample.

According to another aspect, the present invention provides apparatus for isolating V Cholerae from a fluid comprising a vessel and a plurality of insoluble or poorly soluble polysaccharide particles capable of adsorbing V Cholerae, the particles located inside the vessel.

According to preferred embodiments, the polysaccharide is selected from the group consisting of non-gelatinized starch and cellulose.

In various embodiments, the insoluble or poorly soluble polysaccharide particles range in size from 5 to 70 microns. According to some embodiments, the polysaccharide is a non-gelatinized starch selected from the group consisting of corn starch, waxy maize starch, rice starch and wheat starch. In alternative embodiments, the polysaccharide is a cellulose. In one particular embodiment, the cellulose is microcrystalline cellulose.

In one specific embodiment, the vessel has a fluid inlet and a fluid outlet. In specific embodiments, the plurality of insoluble or poorly soluble polysaccharide particles are further defined as a bed of particulate matter.

According to another aspect, the present invention provides methods for isolating V Cholerae from a fluid comprising contacting the fluid with a plurality of insoluble or poorly soluble polysaccharide particles thereby isolating V Cholerae from the fluid. In preferred embodiments, the polysaccharide is selected from the group consisting of non-gelatinized starch and cellulose. According to another aspect, the present invention provides a kit for detecting the presence of V Cholerae in a fluid sample said kit comprising: a) a plurality of insoluble or poorly soluble polysaccharide particles; and b) a plurality of reporting species, the reporting species capable of adhering to V Cholerae and capable of being detected, wherein detection of the reporter species is indicative of the presence of V Cholerae in a fluid sample.

According to preferred embodiments, the polysaccharide particles in the kits of the invention are selected from the group consisting of non-gelatinized starches and celluloses. According to preferred embodiments, the reporting species comprises a targeting species bound to a labeling species capable of being detected, the targeting species being capable of adhering to V Cholerae bacteria.

According to some embodiments, the targeting species is selected from antigens, haptens, monoclonal antibodies, polyclonal antibodies, oligonucleotides, polynucleotides, oligosaccharides, polysaccharides, lectins, avidin, streptavidin, biotin, growth factors, hormones, and receptor molecules.

According to other embodiments, the targeting species is insoluble or poorly soluble polysaccharide particles wherein the polysaccharide is selected from the group consisting of non-gelatinized starches and celluloses. According to some embodiments, labeling species is selected from proteins, enzymes, toxins, drugs, dyes, fluorescent, luminescent, phosphorescent and other light- emitting substances cells, metal-chelating substances, substances labeled with a radioactive isotope, and substances labeled with a heavy atom.

According to some embodiments, the kit further comprises a solid support, wherein the polysaccharide particles are bound to the solid support.

In various embodiments, the support is in the form of sheets, plates, test tubes, test sticks, ELISA plates, microwell plates, or any other support suitable for diagnostic reagents, as are well known in the art.

According to another aspect, the present invention provides a method of assaying a test sample for the presence of a V Cholerae, the method comprising: a) contacting the test sample with a plurality of insoluble or poorly soluble polysaccharide particles; b) contacting the insoluble or poorly soluble polysaccharide particles with a reporting species, the reporting species capable of adhering to V Cholerae and capable of being detected; c) detecting the presence of reporting species bound to the polysaccharide particles, the presence of the reporting species being positively correlated with the presence of V Cholerae in the test sample.

The aforementioned kits and methods for detecting the presence of cholera in a fluid sample are useful for ascertaining the presence of cholera in a drinking water sample, as well as for diagnosing cholera in a subject, wherein the presence of V

Cholerae in the fluid sample derived from the subject is indicative that the subject has cholera.

In embodiments where the reporting species comprises a targeting species comprising insoluble or poorly soluble polysaccharide particles, the aforementioned kits and methods for detecting cholera provide tools for detecting the presence of cholera in a fluid subject without the need for antibodies, which need refrigeration. Thus, in these embodiment, the aforementioned kits and methods for detecting cholera are particularly suitable for field use.

These and other embodiments of the present invention will become apparent in conjunction with the figures, description and claims that follow.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 provides a photomicrograph of insoluble starch granules washed with ORS containing fluorescently labeled V. cholera and then viewed under visible light and fluorescence microscope (XlOOO magnification). Images were captured by a digital camera. Fig. 2 presents a graph which illustrates the adhesion efficiency of V. cholera to several types of polysaccharide particles, where a low Residual CFU indicates a high adhesion efficiency.

Fig. 3 depicts a schematic diagram of V. cholera bacteria adsorbed to immobilized insoluble or poorly soluble polysaccharide particles, with labeled antibodies adhering to the bacteria.

Fig. 4 shows a schematic diagram of V. cholera bacteria adsorbed to immobilized insoluble or poorly soluble particles, with labeled insoluble or poorly soluble polysaccharide particles also adhering to the bacteria.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, it has been discovered that Vibrio cholerae adsorb to certain insoluble or poorly soluble polysaccharide particles with measured adhesion efficiencies of over 90%. The present invention provides kits and pharmaceutical compositions for treating cholera which facilitate excretion of the virulent Vibrio cholerae and thereby ameliorate the cholera condition. The present invention also provides kits, apparatus and methods for isolating Vibrio cholerae from a fluid, enabling purification of the fluid or detection of the presence of Vibrio cholerae in the fluid.

The pharmaceutical compositions and kits of the present invention reduce or minimize the need for antibiotics when treating cholera. Furthermore, one particular advantage of the non-gelatinized the present invention provides is that the non- gelatinized starch does not substantially dissolve in electrolyte solutions, and does not substantially contribute to osmotic pressure. Thus, the granular, insoluble or poorly soluble polysaccharide particles may be suspended in any previously disclosed ORS and imbue this ORS with additional therapeutic qualities without concomitantly raising the osmotic pressure above desirable levels. For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

Definitions and Terminology As used herein and in the claims, the term "non-gelatinized starch" refers to a native granular starch. This is in contrast with the term gelatinized starch which refers to starch that has been heated or cooked. Non-gelatinized starch is also known to exhibit some degree of resistance to digestion by amylase enzymes. The recited ranges of particle sizes of the particulate polysaccharides used refer to the longest dimension of the particles used. The particles may be spherical or elongated and are not intended to be limited to any particular shape or geometry.

The term "water-soluble" as used herein refers to a compound that typically has solubility in water in the range of 1 gr/ml to 1 gr/30 ml at room temperature. The term "poorly water-soluble" as used herein refers to a compound that typically has solubility in water in the range of 1 gr/30 ml to 1 gr/10,000 ml at room temperature. The term "water-insoluble" of polysaccharide particles as used herein refers to a compound that typically has solubility in water of less that 1 gr/10,000 ml at room temperature. The present invention encompasses insoluble or poorly soluble polysaccharide particles selected from the group consisting of non-gelatinized starch and cellulose.

As used herein, one milliequivalent (mEq) refers to the number of ions in solution as determined by their concentration in a given volume. This measure is expressed as the number of milliequivalents per liter (mEq/L). Milliequivalents may be converted to milligrams by multiplying mEq by the atomic weight of the mineral and then dividing that number by the valence of the mineral.

Starch is a carbohydrate polymer made up of anhydroglucose units linked primarily through alpha- 1,4 glucosidic bonds. Diversity stems from a starch's mixture of two types of polymers - amylose and amylopectin. As used herein, high amylose starch refers to genetic varieties of starch containing over 50% amylose. Current commercial varieties contain 55% and 70% amylose.

As used in, low amylose starch refers to genetic varieties of starch containing 50% amylose or less. Both low amylose as well as high amylose non-gelatinized starch are readily available for purchase from suppliers such as National Starch and Chemical (Bridgewater, New Jersey, USA) as well as Starch Australasia (Sydney, Australia). The term "electrolyte" means any compound that conducts electricity while in solution and is decomposed (electrolyzed) by it. Such compounds are ionizable in solution. Examples of an electrolyte that may be used in the present invention include, for example, inorganic acids, bases, and salts. Additional examples of electrolytes and electrolyte solutions that may be used in the present invention are described in Remington's The Science and Practice of Pharmacy, Meade Publishing Co., and United States Pharmacopeia/National Formulary. The term "polysaccharide" refers to any complex carbohydrate, or derivative thereof, from any natural or synthetic source.

Any insoluble or poorly soluble polysaccharide may be used for the presently provided kits, apparatus, methods and pharmaceutical compositions including modified polysaccharides, insoluble metal salts, and cross-linked derivatives. In specific embodiments, these modified polysaccharides are not necessarily poorly soluble or insoluble in native form but have been modified so as to render them poorly soluble or insoluble.

In various embodiments, appropriate polysaccharides include but are not limited to corn starch, waxy maize starch, rice starch, wheat starch and microcrystalline cellulose.

In other embodiments, the polysaccharides may be modified polysaccharides. In specific embodiments, these modifies polysaccharides are not necessarily poorly soluble or insoluble in native form but have been modified so as to render them poorly soluble or insoluble. Examples of such modified polysaccharides are disclosed in WO 00/28974, entitled "Local Delivery of Drugs to the Colon for Local Treatment of Colonic Diseases."

Modified insoluble or poorly soluble polysaccharides are known in the art as pharmaceutical excipients (see Handbook of pharmaceutical Excipients, 2nd ed., Wade, A. and Weller, P. J., eds., American Pharmaceutical Association (1994)). Insoluble or poorly soluble polysaccharides previously disclosed as pharmaceutical excipients are appropriate active ingredients for the pharmaceutical compositions and kits of the present invention.

In various embodiments, the poorly soluble or insoluble polysaccharides include, but are not limited to, water insoluble cross linked polysaccharides, water insoluble polysaccharide metal salts, water insoluble protein:polysaccharide complexes, water insoluble protein:polysaccharide complexes, and polysaccharides rendered insoluble by interaction with a poly-cation or poly-anion. Appropriate polysaccharides to be modified so as to make them poorly soluble or insoluble include but are not limited to insoluble or poorly soluble metal salts of pectin, xanthan gum, carrageenan, tragacanth gum, locust bean gum, and alginic acid; insoluble crosslinked derivatives of xanthan gum, guar gum, dextran, carrageenan. tragacanth gum, locust bean gum, pectin, hydroxypropylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose and alginic acid, and cellulose.

Appropriate insoluble metal salts of alginic acid include but are not limited to calcium alginate, zinc alginate, aluminum alginate, ferric alginate, and ferrous alginate. Appropriate insoluble metal salts of pectin include but are not limited to calcium pectinate, zinc pectinate, aluminum pectinate, ferric pectinate, and ferrous pectinate.

Appropriate crosslinking agents include, but are not limited to formaldehyde, glutaraldehyde, epichlorhydrin, diacid chlorides, diacid anhydrides, diisocyanates, and diamines. Pharmaceutical Compositions for Treatment of Cholera

According to one aspect, the present invention provides packaged kits for reconstitution to produce a pharmaceutical composition for treating cholera, the kit comprising a plurality of insoluble or poorly soluble polysaccharide particles and an oral rehydration solution. According to preferred embodiments, the polysaccharide is selected from the group consisting of non-gelatinized starch and cellulose.

According to another aspect, the present invention provides packaged kits for reconstitution to produce a pharmaceutical composition for treating cholera, the kit comprising a plurality of insoluble or poorly soluble polysaccharide particles and at least one dry electrolyte.

In various embodiments, both the polysaccharide particles and electrolytes are dry particulate matter, and the packaged kit provides a container (e.g., a vial, a bottle, a pouch, an envelope, a can, etc) and instructions for carrying out drug administration in a effective manner. The instructions will typically be written instructions on a package insert, a label, and/or on other components of the kit. hi certain embodiments, the kit will also contain a container, preferably glass, metal, or a plastic known not to adsorb hydrophobic compounds, in which to reconstitute the pharmaceutical composition.

The present invention additionally provides compositions for treating cholera. The composition of this invention can be manufactured using techniques well known to those skilled in the art. As a general guideline, all the water-soluble ingredients may be dry blended together; dispersed in water with agitation; and optionally heated to the appropriate temperature to dissolve all the constituents.

Subsequently, the granular, insoluble or poorly soluble polysaccharide particles may be added to the electrolyte solution to obtain a disperse suspension. Thus, in certain embodiments, it is recommended that the granular, insoluble or poorly soluble polysaccharide particles be mixed into water of a temperature less than 20° C to avoid caking. Furthermore, in various embodiments the suspension is obtained by adding the granular, insoluble or poorly soluble polysaccharide particles to the electrolyte solution in small quantities, rather than all at once, in order to avoid caking. Once the starch is completely suspended in the electrolyte solution, the composition may be mixed with gentle shaking.

The composition is then optionally packaged and sterilized to food grade standards as is known in the art. In the event that the starch is suspended into an aqueous solution derived from locally available water, it is recommended that potable cholera free water be used. If this is not possible, it is recommended to first boil the water in order to eliminate any pathogens such as V Cholerae.

The oral rehydration solution (ORS) contains at a minimum sodium and chloride. Optionally, the ORS electrolyte solution further contains other electrolytes, which may include but are not limited to potassium, zinc, citrates, carbonates and bicarbonates.

In a preferred embodiment, the ORS may be provided as a prepared solution. Alternatively, one or more ingredients of the ORS may be provided as dry, particulate matter for extemporaneous reconstitution, in an appropriate aqueous solution. According to some embodiments, the kit further comprises an additional functional ingredient selected from the group consisting of carbohydrates, thickening agents, preservatives, amino acids, drugs, mineral salts, vitamins, nutraceuticals, probiotics, flavors, and sweeteners.

In one embodiment, the kit further contains glucose. Other appropriate soluble carbohydrates include, but are not limited to glucose, fructose, sucrose, and maltodextrin. Thus, according to another aspect the present invention provides a kit comprising dry, particulate matter for reconstitution to produce a pharmaceutical composition for treating cholera. Kits comprising dry, particulate matter are lighter and more compact than kits containing solutions. These kits may be reconstituted extemporaneously, though in many situations special care must be taken to reconstitute using potable cholera-free water, preferably sterilized water.

The present invention provides a kit for extemporaneous reconstitution of an oral rehydration therapy comprising: a) a plurality of insoluble or poorly soluble polysaccharide particles; and b) at least one dry electrolyte. According to preferred embodiments, the polysaccharide is selected from the group consisting of non-gelatinized starches and celluloses. According to another aspect, the invention provides a pharmaceutical composition for treatment of cholera comprising insoluble or poorly soluble polysaccharide particles suspended in an electrolyte solution, wherein the polysaccharide is other than a high-amylose starch. According to preferred embodiments, the polysaccharide is selected from the group consisting of non-gelatinized starches other than a high-amylose starches and celluloses.

According to some embodiments, the polysaccharide is a non-gelatinized starch. selected from the group consisting of corn starch with an amylose content of less than 50%, waxy maize starch, rice starch and wheat starch. In alternative embodiments, the polysaccharide is a cellulose. In one particular embodiment, the cellulose is microcrystalline cellulose.

According to some embodiments, the viscosity of the pharmaceutical composition does not differ substantially from the viscosity of water, with 0.9 centipoises at 25° C a typical value. In other embodiments, a thickening agent may be added to the composition, in order to impede settling of the suspending particles. This may be especially important when the suspension is not immediately consumed, but is packaged for later use. Suitable thickening agents include but are not limited to agar, alginic acid and salts, gum arabic, gum acacia, gum talha, cellulose derivatives, curdlan, fermentation gums, furcellaran, gellan gum, gum ghatti, guar gum, iota carrageenan, irish moss, kappa carrageenan, konjac flour, gum karaya, lambda carrageenan, larch gum/arabinogalactan, locust bean gum, pectin, tamarind seed gum, tara gum, gum tragacanth, xanthan gum and mixtures thereof. The skilled practitioner is able to select the appropriate concentrations for each particular thickening agent. In particular embodiments, the composition contains all the necessary electrolytes and levels thereof required by the Food and Drug Administration for oral rehydration formulations sold in the United States. According to these embodiments, in addition to sodium (Na+), potassium (K+), chloride (Cl-) and citrate ions, the composition contains a source of simple carbohydrate, such as glucose, fructose, or dextrose. Typically, the composition for these embodiments comprises water, simple carbohydrates, sodium ions, potassium ions, chloride ions, and citrate ions. The quantity of sodium ions used in various embodiments can vary widely, as is known to those skilled in the art. Typically, the composition will contain from about 30 mEq/L to about 95 mEq/L of sodium. In certain embodiments, sodium content can vary from about 30 mEq/L to about 110 mEq/L, most preferably from about 50 mEq/L to about 95 mEq/L. Suitable sodium sources include but are not limited to sodium chloride, sodium citrate, sodium bicarbonate, sodium carbonate, and mixtures thereof.

Any soluble carbohydrate used in previously disclosed oral rehydration solutions may be used to practice the present invention. Suitable carbohydrates include but are not limited to glucose, fructose, sucrose, and maltodextrin. According to various embodiments, the composition will also typically include a source of buffer to replace diarrheal losses. The type and quantity of buffer can vary as is known in the art. Typically, citrate buffer is used, the citrate content ranges from about 10 mEq/L to about 40 mEq/L, more preferably from about 20 mEq/L to about 40 mEq/L, and most preferably from about 25 mEq/L to about 35 mEq/L. Suitable citrate sources include, but are not limited to, potassium citrate, sodium citrate, citric acid, and mixtures thereof. In various embodiments, the composition will also typically contain a source of chloride. The quantity of chloride can vary as is known in the art. Typically the composition will contain chloride in the amount of from about 30 mEq/L to about 80 mEq/L, more preferably from about 30 mEq/L to about 75 mEq/L, and most preferably from about 30 mEq/L to about 70 mEq/L. Suitable chloride sources include but are not limited to, sodium chloride, potassium chloride and mixtures thereof.

In various embodiments, the composition will also contain a source of zinc ions. The quantity of zinc used in the composition of this invention can vary widely. Zinc ingested in the composition can replace the zinc lost due to the underlying diarrhea and/or vomiting. Concentrations at from about 0.3 mEq to about 10 mEq of zinc per liter of composition will typically accomplish this result. Typically, the composition will contain from about 0.6 mEq to about 3 mEq of zinc per liter. Alternatively, the composition may contain from about 0.6 mEq to about 1.2 mEq of zinc per liter. The source of zinc ions is not critical. Any zinc salt suitable for human consumption may be used in the composition of this invention. Examples of suitable zinc sources include zinc gluconate, zinc sulfate, zinc chloride, zinc citrate, zinc bicarbonate, zinc carbonate, zinc hydroxide, zinc lactate, zinc acetate, zinc fluoride, zinc bromide, and zinc sulfonate.

In accordance with the present invention, it has been discovered that Vibrio cholerae adsorb to insoluble or poorly soluble polysaccharide particles even in the absence of glucose. The present invention also provides pharmaceutical composition wherein the concentration of glucose is lower than that traditionally found in ORS formulations. According to another aspect, the present invention provides pharmaceutical compositions for treatment of cholera comprising insoluble or poorly soluble polysaccharide particles suspended in an electrolyte solution, wherein the composition contains less than 2.0 w/w% glucose, preferably less than 1.5 w/w% glucose, and more preferably less than 1.0 w/w% glucose.

Any reference to a numerical range in this application should be considered as being modified by the adjective "about". Further, any numerical range should be considered to provide support for a claim directed to a subset of that range. For example, a disclosure of a range of from 1 to 10 should be considered to provide support in the specification and claims to any subset in that range (i.e. ranges of 2-9, 3- 6, 4-5, 2.2-3.6, 2.1-9.9, etc.). Any reference in the specification or claims to a quantity of an electrolyte should be construed as referring to the final concentration of the electrolyte in the composition. Tap water often contains residual sodium, chlorine, etc. A value of 40 mEq of sodium, in this application, means that the total sodium present in the ORS equals 40 mEq, taking into account both added sodium as well as the sodium present in the water used to manufacture the composition.

Besides being useful for human treatment, the compositions of the present invention are also useful for veterinary treatment of companion mammals, exotic animals and domesticated animals, including mammals, rodents, and the like. In one embodiment, the mammals include horses, dogs, and cats. In another embodiment of the present invention, the human is an adolescent or infant under the age of eighteen years of age.

Isolation of V. Cholera from a Fluid

In accordance with the present invention, it has been discovered that the Vibrio cholerae adsorb to insoluble or poorly soluble particles, and thus it is possible to construct apparatus for isolating Vibrio cholerae from a fluid. Relevant applications include water purification and tools for detecting the presence of Vibrio cholerae in a fluid sample.

According to preferred embodiments, the polysaccharide used in the device or apparatus of the invention is selected from the group consisting of non-gelatinized starch and cellulose.

Possible varieties of starch to be used include, but are not limited to corn starch, waxy maize starch, and rice starch, and wheat starch.

In various embodiments, the size of the insoluble or poorly soluble polysaccharide particles ranges in size from 5 to 70 microns. In one embodiment, the size of the insoluble or poorly soluble polysaccharide particles ranges in size from 15 to 45 microns.

It is noted that there are no limitations on the size, shape or material composition of the vessel. In one specific embodiment, the vessel is a cylindrically shaped with an axially length of about 50 centimeters and a radius of about 10 centimeters. As a non-limiting example, it is noted that to remove over 95% of the virulent

Vibrio cholerae bacteria from one liter of fluid containing 10 CFU/ml of the Vibrio cholerae, it is necessary to use 100 grams of insoluble or poorly soluble polysaccharide particles, and to expose the fluid to the polysaccharide for 2 minutes.

Tools for Detecting the Presence of Vibrio cholerae in a fluid sample

In recent years, there has been a recognized need for analytical techniques capable of fast, sensitive and reliable detection of pathogenic agents in general, and Vibrio cholerae in particular. Traditionally, Vibrio cholerae have been detected in samples by culturing the samples on TCBS agar and other selective and nonselective media. In the case of stool samples, one typical technique is to examine a wet mount of liquid stool microscopically. Other methodologies are based upon fluorescent antibody tests or genetically-based rapid techniques. Yet other methodologies detect the presence of a specific pathogen by sensing the presence of a relevant toxin. For example, United States Patent Application Publication Number 2004/0002064 discloses a method for detecting and identifying a toxin in a sample based upon contacting a microarray of biological membranes with a sample, and monitoring for binding activity.

United States Patent Application Publication Number 2004/0063160 relates to a method for rapid detection of a biological cell and/or a biological particle contained in a fluid sample. The 160 application discloses that the method may be used for rapidly diagnosing a condition in an individual resulting from an infection by a virus, a fungus or a bacteria. The disclosed method comprises the further steps of detecting a plurality of infection and/or inflammatory response agents, preferably cytokines, and performing a profile of such agents.

To date, there have been no published disclosures of using tagged and untagged non-gelatinized starch particles to identify the presence of Vibrio cholerae in aqueous samples. There is an ongoing need for simple, inexpensive tests for diagnosing cholera in individuals as well as for detecting the presence of Vibrio cholerae both in water supply samples, such as samples obtained in undeveloped countries or in areas where natural disaster has struck, and in clinically obtained samples.

The present invention also relates to kits and methods for detecting the presence of V cholerae in a fluid sample. According to another aspect, the invention provides techniques analogous to ELISA for detecting the presence of Vibrio cholerae, where insoluble or poorly soluble polysaccharide particles starch in some way substitute for specific antibodies used in traditional ELISA assays. The economics of using specific antibodies is often prohibitive, and the present invention provides inexpensive kits for the detection of Vibrio cholerae.

According to one aspect, the present invention provides a kit for detecting the presence of V Cholerae in a fluid sample, said kit comprising: i) a plurality of insoluble or poorly soluble polysaccharide particles; and ii) a plurality of reporting species, the reporting species capable of adhering to V Cholerae and capable of being detected, wherein detection of the reporter species is indicative of the presence of V Cholerae in a fluid sample. In preferred embodiments, the polysaccharide used in the kits according to the present invention is selected from the group consisting of non-gelatinized starches and celluloses.

Possible varieties of starch to be used include, but are not limited to high corn starch, waxy maize starch, and rice starch, and wheat starch. In one particular embodiment, the cellulose is microcrystalline cellulose.

In various embodiments, the insoluble or poorly soluble polysaccharide particles range in size from 5 to 70 microns. In one embodiment, the insoluble or poorly soluble polysaccharide particles range in size from 15 to 45 microns. In some embodiments, the reporting species comprises a targeting species capable of adhering to V Choϊerae bacteria bound to a labeling species capable of being detected.

Appropriate targeting species include but are not limited to antigens, haptens, monoclonal antibodies, polyclonal antibodies, oligonucleotides, polynucleotides, oligosaccharides, polysaccharides, lectins, avidin, streptavidin, biotin, growth factors, hormones, and receptor molecules.

In yet other embodiments, the targeting species is insoluble or poorly soluble polysaccharide particles. In specific embodiments, the polysaccharide is selected from the group consisting of non-gelatinized starches and celluloses. In some embodiments, the kit further comprises a solid support, and the polysaccharide bound to the solid support.

By "immobilized on a solid support " or "attached to a solid support" or "bound to a solid support", it is meant that the insoluble or poorly soluble polysaccharide is attached to a solid surface in such a manner that the system containing the immobilized polysaccharide may be subjected to washing or other physical or chemical manipulation without being dislodged from that location.

In one particular embodiment, the targeting species is insoluble or poorly soluble polysaccharide particles, wherein the insoluble or poorly soluble polysaccharide is selected from the group consisting of non-gelatinous starch and cellulose. In one particular embodiment, the targeting insoluble or poorly soluble polysaccharide is fluorescently labeled by FITC-concanavalin A (McHaIe, R. H. Starch/Staerke (1986), 38(12), 413-17).

As a non-limiting example, it is noted that Vibrio cholerae concentrations on the order of magnitude of 10⁴ cells/ml may presently be detected. Nevertheless, no theoretical minimum concentration exists. More advanced detection technologies, like single photon cameras capable of single fluorophore detection, may theoretically be employed to identify the presence of even a single cell in a sample.

It is noted that the labeling species, which is bound to the target species, is detectable by methods understood to those schooled in the art. Thus, the presence of the labeling species would be indicative of the presence of V Cholerae in the sample. Appropriate labeling species include, but are not limited to proteins, enzymes, toxins, drugs, dyes, fluorescent, luminescent, phosphorescent and other light-emitting substances cells, metal-chelating substances, substances labeled with a radioactive isotope, and substances labeled with a heavy atom. The kit of the present invention can detect the presence of V cholerae in any fluid sample. In one embodiment, the fluid sample is an aqueous sample, and the invention provides tools for detecting the presence of V cholerae in a body of possibly contaminated water. In another embodiment, the fluid sample is a body fluid sample.

Other applications relate to the testing of water supplies. Although potable water supplies in developed country have been cholera-free for years, there are a plethora of situations where there is a need for fast, sensitive, inexpensive, portable and reliable analytical tools for detecting Vibrio cholerae. For example, in regions that have been visited by natural disasters the water supply is often compromised.

Furthermore, it is possible that a terrorist element could contaminate a local water supply with virulent biological agents such as Vibrio cholerae. Similarly, a military team operating in unfriendly territory would prefer kits that are light, portable, and need no or minimal refrigeration.

For the embodiments where the insoluble or poorly soluble polysaccharides particles are not immobilized on a solid surface, it is preferred that the insoluble or poorly soluble polysaccharides particles be placed in a vessel. Suitable vessels include test tubes, columns, microwell plates, and the like. After the insoluble or poorly soluble polysaccharides particles are placed in the vessel, the particles are contacted with the sample, and then with the reporter species. Subsequently, the insoluble or poorly soluble polysaccharide particles are subjected to washing to remove reporter species that do not bind to the insoluble or poorly soluble polysaccharide particles. In the event that the sample does indeed contain Vibrio cholerae, the Vibrio cholera adsorbs to the insoluble or poorly soluble polysaccharides particles as well as to the reporter species, and the presence of the detectable reporter species adsorbed to the insoluble or poorly soluble polysaccharides particles after washing indicates the presence of Vibrio cholerae in the sample. In the event that there is no Vibrio cholerae in the samples, the detectable reporter species are simply washed away. Figures 3 and 4 schematically portray different embodiments of the present invention as related to detecting the presence of Vibrio cholerae in a fluid sample.

In the embodiment illustrated in Figure 3, non-gelatinized starch particles (2) are first adsorbed to a solid support (1). Subsequently, the immobilized insoluble or poorly soluble polysaccharide particles (2) are contacted with a fluid sample, and some of the Vibrio cholerae bacteria (3) present in the sample adsorb to the immobilized insoluble or poorly soluble polysaccharide particles (2). Subsequently, labeled antibodies (4) are introduced into the system, some of which adhere to the immobilized bacteria (3). It is noted that the labeled antibodies(4) are not depicted to scale in Figure 3, but in reality are much smaller than both Vibrio cholerae bacteria as well as the insoluble or poorly soluble polysaccharide particles. The labeled antibodies (4) may be detected using well known techniques. Unlike a traditional ELISA assay, there is no need for an immobilized layer of labeled antibodies, and thus the embodiment described in Figure 3 obviates, in part, the need for potentially costly antibodies when detecting V cholerae bacteria. In the embodiment illustrated in Figure 4, insoluble or poorly soluble polysaccharide particles (2) are first adsorbed to a solid support (1). Subsequently, the immobilized insoluble or poorly soluble polysaccharide particles (2) are contacted with a fluid sample, and some of the Vibrio cholerae bacteria (3) present in the sample adsorb to the immobilized insoluble or poorly soluble polysaccharide particles (2). Subsequently, labeled insoluble or poorly soluble polysaccharide particles (5) are introduced into the system, some of which adhere to the immobilized bacteria (3). The labeled insoluble or poorly soluble polysaccharide particles (5) may be detected using well known techniques such as fluorescent labeling by FITC-concanavalin A (McHaIe 1986). It is noted that the embodiment described in Figure 4 does not require any antibodies which themselves require refrigeration. Thus, this embodiment could be quite advantageous for mobile teams that would prefer kits that do not require any refrigeration.

The Solid Support Element

The extended solid phase used in the present invention may be employed in a variety of forms or structures. The solid phase has a location where a targeting species, such as a insoluble or poorly soluble polysaccharide particle or an antibody; can bind or associate, and the formation of such a solid phase with said targeting species, such as a non-gelatinized particle or an antibody, enables contacting a specimen and other materials used in the method of the invention.

The extended solid phase is best formed in a way that enables simple manipulation for easy contact with the specimen and other reagents. For this purpose, the extended solid phase may form at least part of a dipstick, syringe, tube or container.

The fluid sample and other reagents can be drawn in and ejected from a syringe, caused to flow through a tube, or deposited in a container such as a test tube shaped container. In such devices, the extended solid phase can form the whole of the device, or part of it, where, in the case of a syringe, tube or container, the part formed of the extended solid phase will at least be exposed at the inside of the device to permit contact with fluid sample and reagents. Targeting species, such as a non-gelatinized particle or an antibody, is preferably concentrated at one location of the extended phase, to be exposed to the fluid sample.

One more preferred form of the extended solid phase is a dipstick. In such a dipstick, it is further preferred that the extended solid phase should be included at least one end, and that the targeting species, such as a non-gelatinized particle or an antibody, conjugated on the extended solid phase should be concentrated at the end of the dipstick. The extended solid phase can however comprise the entire dipstick, with the targeting species, such as a non-gelatinized particle or an antibody, concentrated at one end, or in more than one location.

The dipstick may be entirely formed from the extended solid phase, at one end of which has been conjugated a coating of targeting species, such as a non-gelatinized particle or an antibody. In another embodiment the dipstick has an extended solid phase one end of which is adhered to a body portion. A coating of targeting species, such as a non-gelatinized particle or an antibody, is conjugated to the extended solid phase. In yet another embodiment the extended solid phase entirely forms a tubular container into which a fluid sample can be placed. Coatings of targeting species, such as a non- gelatinized particle or an antibody, are located near the bottom of the container and are concentrated in one or more locations.

Techniques for immobilizing insoluble or poorly soluble polysaccharide particles are well known in the art. For example, covalent immobilization of polysaccharides such as in starch granules can be performed by using a reductive animation reaction. Furthermore, gas plasma (r.f. glow discharge) methods adapt the surfaces of normally unreactive polymeric substrates with chemical groups capable of reacting with polysaccharides (starch granules). The polysaccharide particles may be activated for covalent immobilization by periodate oxidation. The resulting Schiff base linkages are stabilized by reduction to secondary amine linkages using sodium cyanoborohydride (Dai LM, StJohn HAW, Bi JJ, Zientek P, Chatelier RC, Griesser HJ, Surface and Interface Analysis, 29 (1): 46-55 JAN 2000)

The extended solid phase is composed of any material onto which a non- gelatinized starch particle can be effectively bound. The kit according to the invention may also be applied in a micro system, such as a micro flow system described in WO 98/10267, one such system being marketed by Torsana Biosensor AJS, Denmark.

In addition to micro systems, the kit according to the invention may also form part of a conventional macro system such as e.g. a lateral flow device. Examples of such devices are listed disclosed in U.S. Pat. No. 5,610,077.

The Labeling Species

Detection of the labeling species in question preferably indicates both the localization and the amount of Vibrio cholerae. The detection may be performed by visual inspection, by light microscopic examination in the case of enzyme labels, by light or electron microscopic examination in the case of heavy metal labels, by fluorescence microscopic examination, using irradiated light of a suitable wavelength in the case of fluorescent labels, and by autoradiography in the case of an isotope label. Methods of detecting labeled antibodies are well known in the art.

In one particular embodiment, the targeting species is insoluble or poorly soluble polysaccharide particles that are fluorescently labeled by FITC-concanavalin A (McHaIe, R. H. Starch/Staerke (1986), 38(12), 413-17).

In particular embodiments, Vibrio cholerae concentrations on the order of magnitude of 10⁴ cells/ml may presently be detected. Nevertheless, it is noted that no theoretical minimum concentration exists. More advanced detection technologies, like single photon cameras capable of single fluorophore detection, may theoretically be employed to identify the presence of even a single cell in a sample.

In one embodiment, the visual detection is based on a cut-off point above which one color indicates the presence of the biological cell above a certain minimum amount (cut-off point), and below which cut-off point another color indicates that the biological cell is present in an amount of less than that indicated by the cut-off point. When fluorescent markers are used the amounts of the biological cell detected is directly correlatable with the fluorescence measured by a detection unit.

It is to be understood that while the invention has been described in conjunction with the preferred specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications will be apparent to those skilled in the art to which the invention pertains. Furthermore, the practice of the present invention will employ, unless otherwise indicated, conventional techniques of drug formulation that are within the skill of the art. Such techniques are fully explained in the literature. See Remington: The Science and Practice of Pharmacy, cited supra, as well as Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed. (New York: McGraw-Hill, 1996). EXAMPLES

EXAMPLE 1: Particle Size Distribution of Polysaccharide Particles

In this Example and in Example 2 seven different types of particulate polysaccharide were used: five different types of non-gelatinized starches, one soluble starch, and one cellulose. These included non-gelatinized Corn starch (corn) containing 30% amylose; non-gelatinized high amylose corn starch (HYLON®) containing 70% amylose; non-gelatinized amylose-free corn maize starch; non-gelatinized rice starch (-17% amylose content); non-gelatinized wheat starch (~17% amylose content); microcrystalline cellulose, and soluble instant starch. These starches differ in their plant source, chemical composition, mean particle size and other physico-chemical properties. Table 1 provides Chemo-physical characteristics of the polysaccharides used in Example 1. Particle size was measured with a laser particle size analyzer.

Table 1: Chemo-physical characteristics of the polysaccharides used in Example 1.

to

EXAMPLE 2: Measured Adsorption of Vibrio Cholera to Polysaccharide Particles in the Absence of Glucose

Materials and Methods

Vibrio cholerae Ol (a kind gift of Dr ARAKAWA NIID, Tokyo, Japan) were grown in 50 ml of Luria-Broth liquid medium in a 250 ml flask with agitation for 16-18 hours at 37° C. The bacteria were harvested by centrifugation (16,000 g, 5 minutes) and washed with modified glucose-free polysaccharide-free ORS. The bacteria were then diluted to a final concentration of ~10⁶ CFU/ml.

The polysaccharide containing ORS (pre-equilibrated) was mixed in a 9:1 ratio as to give a final concentration of 10% polysaccharide and approximately 10⁵ CFU/ml (10⁶ CFU/gram polysaccharide). The mixture was rotated on an axial spinning agitator and samples were drawn after 5 minutes. The samples were spin-centrifuged for 30 seconds at 38O g. A 100 μl sample of the supernatant was serially diluted and plated on Luria Agar plates, and after 24 hours incubation at 37° C CFU were recorded. Controls, containing modified ORS without polysaccharide were used to normalize the results (p<0.05).

Results

Fig. 1 provides micrographs of insoluble starch granules washed with ORS containing fluorescently labeled V. cholera for three times and then viewed under light and fluorescence microscope (100OX magnification). Images were captured by a digital camera. Based upon the images, it is evident that the labeled V. cholera adhere to the surface of the insoluble starch granules.

Fig. 2 illustrates the adhesion efficiency of V. cholera to several types of polysaccharide particles. A Residual CFU of 100% indicates that no bacteria is adsorbed, while a Residual CFU of 0% in indicative of perfect adhesion efficiency. From Figure 2 it is evident that starch source and composition play a critical role in determining the fate of the bacteria. EXAMPLE 3: Measured Adsorption of Other Pathogens to Suspended Insoluble or Poorly Soluble Polysaccharides The above procedure was repeated for bacteria other than V Cholerae. Table 2 contains adhesion efficiencies for bacteria other than V Cholerae.

Table 2: Adhesion efficiencies for bacteria other than V Cholerae

Starch E.coli 0157 Listeria Salmonella monocytogenes typhimurium

Wheat ~0%-l l% -0% -0%

Corn 14%-29% -0% -0%

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

Claims

WHAT IS CLAIMED IS:

1. A kit for reconstitution to produce a pharmaceutical composition for treating cholera, the kit comprising: a) a plurality of insoluble or poorly soluble polysaccharide particles; and b) an oral rehydration solution.

2. The kit of claim 1 wherein the polysaccharide is selected from the group consisting of non-gelatinized starches and celluloses.

3. The kit of claim 2 wherein the non-gelatinized starch is selected from the group consisting of corn starch, waxy maize starch, rice starch, and wheat starch.

4. The kit of claim 2 wherein the cellulose is microcrystalline cellulose.

5. The kit of claim 1 wherein the insoluble or poorly soluble polysaccharide particles range in size from 5 to 45 microns.

6. The kit of claim 1 wherein the insoluble or poorly soluble polysaccharide particles range in size from 10 to 30 microns.

7. The kit of claim 1 wherein the kit contains 40 to 120 grams of insoluble or poorly soluble polysaccharide particles per liter of oral rehydration solution.

8. The kit of claim 1 wherein the oral rehydration solution comprises at least one electrolyte selected from the group consisting of sodium, potassium, chloride, zinc, citrates, and bicarbonates.

9. The kit of claim 1 further comprising an additional functional ingredient selected from the group consisting of soluble carbohydrates, thickening agents, preservatives, amino acids, drugs, mineral salts, vitamins, nutraceuticals, probiotics, flavors, and sweeteners.

10. The kit of claim 9 wherein the soluble carbohydrate is selected from the group consisting of glucose, fructose, sucrose, and maltodextrin.

11. A kit for reconstitution to produce a pharmaceutical composition for treating cholera, the kit comprising: a) a plurality of insoluble or poorly soluble polysaccharide particles; and b) at least one dry electrolyte.

12. The kit of claim 11 wherein the polysaccharide is selected from the group consisting of non-gelatinized starches and celluloses.

13. The kit of claim 12 wherein the non-gelatinized starch is selected from the group consisting of corn starch, waxy maize starch, rice starch, and wheat starch.

14. The kit of claim 12 wherein the cellulose is microcrystalline cellulose.

15. The kit of claim 11 wherein the insoluble or poorly soluble polysaccharide particles range in size from 5 to 45 microns.

16. The kit of claim 11 wherein the insoluble or poorly soluble polysaccharide particles range in size from 10 to 30 microns.

17. The kit of claim 11 wherein the electrolyte is selected from the group consisting of sodium, potassium, chloride, zinc, citrates, and bicarbonates.

18. The kit of claim 11 further comprising an additional functional ingredient selected from the group consisting of soluble carbohydrates, thickening agents, preservatives, amino acids, drugs, mineral salts, vitamins, nutraceuticals, probiotics, flavors, and sweeteners.

19. The kit of claim 18 wherein the carbohydrate is selected from the group consisting of glucose, fructose, sucrose, and maltodextrin.

20. A pharmaceutical composition for treatment of cholera comprising insoluble or poorly soluble polysaccharide particles suspended in an electrolyte solution, wherein the polysaccharide is other than a high-amylose starch.

21. The pharmaceutical composition of claim 20 wherein the polysaccharide is selected from the group consisting of non-gelatinized starches other than high-amylose starches and celluloses.

22. The pharmaceutical composition of claim 21 wherein the starch is selected from the group consisting of corn starch with an amylose content of less than 50%, waxy maize starch, rice starch, and wheat starch.

23. The pharmaceutical composition of claim 21 wherein the cellulose is microcrystalline cellulose.

24. The pharmaceutical composition of claim 20 wherein the insoluble or poorly soluble polysaccharide particles range in size from 5 to 45 microns.

25. The pharmaceutical composition of claim 24 wherein the insoluble or poorly soluble polysaccharide particles range in size from 10 to 30 microns.

26. The pharmaceutical composition of claim 20 wherein the insoluble or poorly soluble polysaccharide particles are present in the quantity from about 40 grams/L to about 120 grams/L.

27. The pharmaceutical composition of claim 20 wherein the electrolyte solution comprises at least one electrolyte selected from the group consisting of sodium, potassium, chloride, zinc, citrates, and bicarbonates.

28. The pharmaceutical composition of claim 20 further comprising an additional functional ingredient selected from the group consisting of carbohydrates, thickening agents, preservatives, amino acids, drugs, mineral salts, vitamins, nutraceuticals, probiotics, flavors, and sweeteners.

29. The pharmaceutical composition of claim 28 wherein the carbohydrate is selected from the group consisting of glucose, fructose, sucrose, and maltodextrin.

30. A pharmaceutical composition for treatment of cholera comprising insoluble or poorly soluble polysaccharide particles suspended in an electrolyte solution, wherein the composition contains less than 2.0 wt./wt% glucose.

31. The pharmaceutical composition of claim 30 wherein the polysaccharide is selected from the group consisting of non-gelatinized starches and celluloses.

32. The pharmaceutical composition of claim 31 wherein the starch is selected from the group consisting of corn starch, waxy maize starch, rice starch, and wheat starch.

33. The pharmaceutical composition of claim 31 wherein the cellulose is microcrystalline cellulose.

34. The pharmaceutical composition of claim 30 wherein the insoluble or poorly soluble polysaccharide particles range in size from 5 to 45 microns.

35. The pharmaceutical composition of claim 30 wherein the insoluble or poorly soluble polysaccharide particles range in size from 10 to 30 microns.

36. The pharmaceutical composition of claim 30 wherein the insoluble or poorly soluble polysaccharide particles are present in the quantity from about 40 grams/L to about 120 grams/L.

37. The pharmaceutical composition of claim 30 wherein the electrolyte solution comprises at least one electrolyte selected from the group consisting of sodium, potassium, chloride, zinc, citrates, and bicarbonates.

38. The pharmaceutical composition of claim 30 further comprising an additional functional ingredient selected from the group consisting of soluble carbohydrates, thickening agents, preservatives, amino acids, drugs, mineral salts, vitamins, nutraceuticals, probiotics, flavors, and sweeteners.

39. The pharmaceutical composition of claim 38 wherein the carbohydrate is selected from the group consisting of glucose, fructose, sucrose, and maltodextrin.

40. A method for treating cholera comprising administering to a subject in need thereof a pharmaceutical composition according to any one of claims 20-39.

41. Use of any of the pharmaceutical compositions of claims 20-39 for treatment of cholera.

42. Apparatus for isolating V Cholerae from a fluid comprising a vessel and a plurality of insoluble or poorly soluble polysaccharide particles capable of adsorbing V Cholerae, the particles located inside the vessel.

43. Apparatus of claim 42 wherein the polysaccharide is selected from the group consisting of non-gelatinized starches and celluloses.

44. The apparatus of claim 42 wherein the insoluble or poorly soluble polysaccharide particles range in size from 5 to 70 microns.

45. The apparatus of claim 42 wherein the vessel has a fluid inlet and a fluid outlet.

46. The apparatus of claim 42 where the plurality of insoluble or poorly soluble polysaccharide particles are further defined as a bed of particulate matter.

47. A method for isolating V Cholerae from a fluid comprising contacting the fluid with a plurality of insoluble or poorly soluble polysaccharide particles thereby isolating V Cholerae from the fluid.

48. The method of claim 47 wherein the polysaccharide is selected from the group consisting of non-gelatinized starches and celluloses.

49. A kit for detecting the presence of V Cholerae in a fluid sample said kit comprising: a) a plurality of insoluble or poorly soluble polysaccharide particles; and b) a plurality of reporting species, the reporting species capable of adhering to V

Cholerae and capable of being detected, wherein detection of the reporter species is indicative of the presence of V Cholerae in a fluid sample.

50. The kit of claim 49 wherein the polysaccharide is selected from the group consisting of non-gelatinized starches and celluloses.

51. The kit of claim 49 wherein the reporting species comprises a targeting species capable of adhering to V Cholerae bacteria bound to a labeling species capable of being detected.

52. The kit according to claim 51 wherein the targeting species is selected from the group consisting of antigens, haptens, monoclonal antibodies, polyclonal antibodies, oligonucleotides, polynucleotides, oligosaccharides, polysaccharides, lectins, avidin, streptavidin, biotin, growth factors, hormones, and receptor molecules.

53. The kit according to claim 51 wherein the targeting species is insoluble or poorly soluble polysaccharide particles wherein the polysaccharide is selected from the group consisting of non-gelatinized starches and celluloses.

54. The kit according to claim 51 wherein the labeling species is selected from proteins, enzymes, toxins, drugs, dyes, fluorescent, luminescent, phosphorescent and other light-emitting substances cells, metal-chelating substances, substances labeled with a radioactive isotope, and substances labeled with a heavy atom.

55. The kit of claim 49, further comprising a solid support, the polysaccharide bound to the solid support.

56. The kit of claim 55, wherein the support is in the form of sheets, plates, test tubes, test sticks, ELISA plate or microwell plate.

57. A method of assaying a test sample for the presence of a V Cholerae, the method comprising: a) contacting the test sample with a plurality of insoluble or poorly soluble polysaccharide particles; b) contacting the insoluble or poorly soluble polysaccharide particles with a reporting species, the reporting species capable of adhering to V Cholerae and capable of being detected; c) detecting the presence of reporting species bound to the polysaccharide particles, the presence of the reporting species being positively correlated with the presence of V Cholerae in the test sample.

58. The method of claim 57 wherein the polysaccharide is selected from the group consisting of non-gelatinized starches and celluloses.

59. The kit of claim 57 wherein the reporting species comprises a targeting species bound to a labeling species capable of being detected, the targeting species capable of adhering to V Cholerae bacteria.

60. The kit according to claim 59 wherein the targeting species is selected from the group consisting of antigens, haptens, monoclonal antibodies, polyclonal antibodies, oligonucleotides, polynucleotides, oligosaccharides, polysaccharides, lectins, avidin, streptavidin, biotin, growth factors, hormones, and receptor molecules.

61. The kit according to claim 59 wherein the targeting species is insoluble or poorly soluble polysaccharide particles wherein the polysaccharide is selected from the group consisting of non-gelatinized starches and celluloses.

62. The kit according to claim 59 wherein the labeling species are selected from proteins, enzymes, toxins, drugs, dyes, fluorescent, luminescent, phosphorescent and other light-emitting substances cells, metal-chelating substances, substances labeled with a radioactive isotope, and substances labeled with a heavy atom.

63. The kit of claim 57, further comprising a solid support, the polysaccharide bound to the solid support.

64. The kit of claim 63, wherein the support is in a form selected from the group consisting of sheets, plates, test tubes, test sticks, ELISA plate or microwell plate.

65. The method of claim 57 wherein the sample is a body fluid sample.

66. The method of claim 57 wherein the sample is a water sample.

67. A method for generating an improved oral rehydration therapy without significantly elevating osmolarity comprising: a) adding insoluble or poorly soluble polysaccharide particles to an Oral Rehydration Solution; b) suspending the particles in the solution.

68. The method of claim 67, wherein the polysaccharide is selected from the group consisting of non-gelatinized starches and celluloses.