WO2018022475A1

WO2018022475A1 - Methods of treating porcine epidemic diarrheal virus in piglets

Info

Publication number: WO2018022475A1
Application number: PCT/US2017/043435
Authority: WO
Inventors: Michael Hauser
Original assignee: Jaguar Health, Inc
Priority date: 2016-07-25
Filing date: 2017-07-24
Publication date: 2018-02-01

Abstract

Methods of treating neonatal and young non-human animals, particularly piglets, suffering from and/or having been exposed to Porcine Epidemic Diarrhea virus, and/or symptoms thereof, by administering to an animal in need thereof a proanthocyanidin polymer composition isolated from a Croton spp. or a Calophyllum spp. are provided. The composition, either enteric or non-enteric, can be in aqueous soluble form and orally administered to the affected neonatal and young animals.

Description

METHODS OF TREATING PORCINE EPIDEMIC DIARRHEAL VIRUS IN PIGLETS

FIELD OF THE INVENTION

[0001] The invention relates to the treatment of diarrhea caused by Porcine Epidemic

Diarrheal Virus in neonatal, unweaned and young non-human animals, particularly, piglets, with a composition comprising a proanthocyandin polymer isolated from the plant Croton spp. or Calophyllum spp., or with a latex, extract, or food supplement derived therefrom.

BACKGROUND OF THE INVENTION

[0002] According to the U.S. Department of Agriculture's (USD A) National Veterinary

Services Laboratories, Porcine Epidemic Diarrhea virus (PEDv) pathogenic agent is a coronavirus, and PEDv is most serious in neonatal piglets where morbidity and mortality can be 80 to 100 percent. The coronavirus infects the cells lining the small intestine of a pig, causing porcine epidemic diarrhea (PED), a condition of severe diarrhea and dehydration. The USD A confirmed the first PED diagnosis in the United States on May 17, 2013 in Iowa.

[0003] PED can lead to significant economic loss in the swine industry because of the high morbidity and mortality that occurs in immunologically naive neonatal piglets, according to the USDA. Vaccination and increased biosecurity needs related to PED further increase production costs, and, per the USDA, there is currently no effective treatment against PED other than control of secondary infections.

[0004] Accordingly, there is a need for an effective treatment for PED in piglets, particularly neonatal piglets in which the mortality is greatest. The present invention addresses such a need.

SUMMARY OF THE INVENTION

[0005] The present invention relates to methods of treating, ameliorating or managing

Porcine Epidemic Diarrhea Virus (PEDv) in neonatal, young, or non-adult animals, particularly porcines, in need thereof by administering a polymeric proanthocyanidin, i.e., a proanthocyanidin polymer, from a Croton species or Calophyllum species. In an embodiment, a pharmaceutically or physiologically acceptable formulation or composition comprising a proanthocyanidin polymer from a Croton species or Calophyllum species is administered. In particular embodiments, a proanthocyanidin polymer from Croton lechleri, or pharmaceutically acceptable formulation or composition comprising a proanthocyanidin polymer from Croton lechleri is administered.

[0006] In an embodiment, the proanthocyanidin polymer composition is a latex or extract from a Croton species or Calophyllum species, in particular, Croton lechleri. In another embodiment, the composition is a botanical extract of Croton lechleri containing a proanthocyanidin oligomer, or a food supplement formulation of the botanical extract of Croton lechleri. Such Croton species or Calophyllum species latex or extract compositions can be more highly purified as described herein. In an embodiment, the methods involve the administration of a pharmaceutically acceptable composition comprising a proanthocyanidin polymer from Croton lechleri to a non-human animal in need thereof. In an embodiment, the methods involve the administration of a proanthocyanidin polymer from Croton lechleri, or a pharmaceutically acceptable composition comprising a proanthocyanidin polymer from Croton lechleri, wherein the proanthocyanidin polymer or oligomer from C lechleri is also known as crofelemer (a purified proanthocyanidin oligomer), SP 303, or SB 300, as further described herein. In certain embodiments, the C lechleri proanthocyanidin polymer, or composition thereof, is in an enteric coated form that protects the proanthocyanidin polymer from the stomach environment of the non-human animal. In other preferred embodiments, the C lechleri proanthocyanidin polymer, or composition thereof, is in a non-enteric coated form.

[0007] PEDv is a porcine coronavirus that infects cells of the lining of the small intestine, causing severe diarrhea and dehydration. PEDv has high morbidity and mortality, particularly in unweaned piglets. Mortality can be close to 100% in newborn piglets in the first 2 to 3 weeks of life. Surprisingly, administration of proanthocyanidin polymer compositions isolated from C lechleri to newborn piglets with PEDv reduced mortality significantly. Accordingly, the invention provides a method of treating and preventing the debilitating effects of diarrhea caused by PEDV in neonatal and young non-human animals, particularly neonatal piglets. In particular, the methods treat and prevent dehydration associated with water, fluid and electrolyte losses in animals afflicted with diarrhea. The methods of the invention further prevent or reduce the incidence of intestinal lesions, weakness and death in the neonatal and young non-human animals, particularly neonatal and unweaned piglets. [0008] Accordingly, provided are methods of treating a neonatal, unweaned or young porcine, suffering from or exposed to PEDv infection, comprising orally administering to an animal in need thereof a pharmaceutically acceptable composition comprising an aqueous soluble proanthocyanidin polymer from Croton lechleri, preferably as a standardized botanical extract, Neonorm™ The methods provided ameliorate diarrhea in the animals, including reducing the amount or frequency of bowel movements or to improve stoof formation, to ameliorate or reduce vomiting, reduce, ameliorate, prevent or treat lesions in the small intesting or colon in the animals infected and/or exposed to PEDv. In addition, the proanthocyanidin polymer can be SB 300, SP 303, crofelemer and pharmaceutically acceptable compositions thereof. The composition is preferably not enteric coated. In certain embodiments, the composition is enteric coated. In embodiments of the method, the proanthocyanidin polymer composition or botanical extract derived from C. lechleri is administered as a powder reconstituted with oral electrolytes, milk or a milk substitute, physiological saline, or water; or as a bolus; or as a paste or gel; or in animal feed. The treated animals, particularly piglets, can be less than two weeks of age, including as young as 1 day old, 1 to 3 days of age, 5 to 10 days of age, or 10 to 15 days of age, or two to four weeks of age. In embodiments of the method, the composition is administered to the animal in an amount of 8 mg to 100 mg, or in amount of 10 mg, 20 mg, 30 mg or 40 mg either one time per day or twice per day. In other embodiments, the neonatal piglet is approximately 1 to 4 kg in weight. In a particular embodiment, the proanthocyanidin polymer composition or botanical extract derived from C. lechleri is administered in a liquid formulation at a dose of 2 mg/kg, 5 mg/kg, 10 mg/kg or 20 mg/kg either daily or BID. The composition is administered for 1 day, 2 days, 3 days, 4 days, 5 days or longer. Preferably, the composition is administered when piglets exhibits symptoms of PEDv, such as diarrhea and vomiting, particularly, when PEDv has been confirmed by standard diagnostic methods for identifying PEDv, or have been exposed to PEDv, i.e., another pig in proximity has been diagnosed with PEDv.

[0009] The disclosed methods and C. lechleri-derived proanthocyanidin polymer and botanical extract products used in the methods provide several advantages in the treatment of PEDv and associated diarrhea in neonatal, pre-weaned non-human animals, e.g., piglets. Such advantages include significantly reduced mortality of piglets, spread of disease and reduction in the economic impact and loss of swine. DESCRIPTION OF THE INVENTION

[0010] The invention provides treatment methods effective for treating PEDv in piglets, including reducing the mortality associated with the disease in neonates and unweaned piglets, and reducing and/or alleviating diarrhea in neonatal, unweaned non-human animals suffering from or having been exposed to PEDv. The invention further provides formulations and compositions suitable for treating diarrhea in neonatal and young animals, particularly piglets. Unless otherwise noted herein, use of the term "animal" herein denotes non-human, warmblooded mammals of a number of different species. In addition, the terms "young", non- adult", "immature" and "juvenile" are used synonymously herein and generally refer to animals under one year of age.

[0011] Porcine Epidemic Diarrhea virus (PEDv) is a coronavirus that is transmissible among swine and causes severe diarrhea and vomiting in infected animals. Infection has the greatest impact on neonatal piglets for which morbidity and mortality can be 80 to 100 percent. The virus was first detected in the United Kingdom in 1971, has since spread through Asia and Europe, and was detected in the United States in 2013. The incubation period is generally 3 to 4 days, initial symptoms include watery feces, vomiting, with dehydration and metabolic acidosis as the disease develops. Lesions in the small intestine and colon may occur. Morbidity may reach 100% and mortality depends upon the age and susceptibility of the swine. Piglets under 7 days old may have a mortality rate of 50% or greater. According to the USD A, there is no treatment for PEDv other than to ameliorate secondary symptoms. See, Technical Note: Porcine Epidemic Diarrhea (USDA) at (https ://www. aasv. org/aasv%20web site/ Resources/Diseases/ PED/usda ped tech note.pdf). The inventors have found that standardized botanical extracts of C. lechleri administered to piglets infected with PEDv treat the symptoms of PEDv, including diarrhea and dehydration, and reduce mortality to 0%, to 10%), to 20%), to 30 %>, to 40%) or to 50%. Accordingly, the methods provided herein are a solution to a significant need for the swine industry faced with an increased incidence in PEDv, which results in loss of livestock and economic losses.

[0012] The methods and treatments of the invention are particularly suitable for treating piglets having or exposed to PEDv of a young age. In an embodiment, the animals, particularly, piglets, are neonatal (or newborn), unweaned, non-adult animals that are born, bred, raised and/or maintained in a domesticated and/or agricultural setting, e.g., as livestock and farm animals. Without wishing to be limiting, "young" animals are generally under one year of age. "Neonatal" animals are generally two weeks of age or less.

[0013] The present invention relates to treating PEDv, and diarrhea associated therewith, in neonatal, unweaned and young animals, particularly piglets, with physiologically and pharmaceutically acceptable formulations and compositions comprising a therapeutically effective amount of a proanthocyanidin polymer obtained from a Croton spp., preferably Croton lechleri. The proanthocyanidin polymer composition can also be obtained from a Calophyllum spp., in particular Calophyllum inophylum. In a specific embodiment, the pharmaceutically acceptable composition comprises a proanthocyanidin polymer from Croton lechleri. In a specific embodiment, the pharmaceutically acceptable composition comprises a botanical extract derived from Croton lechleri, such as a standardized botanical extract, such as Neonorm™.

[0014] In general terms, "treating" an animal according to the present methods refers to achieving or obtaining a desired physiologic and/or pharmacologic effect, whether prophylactic, therapeutic, or both. As used herein "treating" or "treatment" can refer to ameliorating, preventing, inhibiting, reversing, attenuating, alleviating, abrogating, minimizing, suppressing, reducing, decreasing, diminishing, stabilizing, eradicating, curing, or eliminating the deleterious effects of a disease or condition, or the progression or worsening of the disease or condition. For example, successful treatment may involve alleviating one or more symptoms of a disease or condition, although not necessarily all of the symptoms, of the disease or condition, or attenuating the symptoms or progression of the disease or condition. Curing or eliminating the disease or condition from the animal is an optimal outcome of the practice of the methods of the invention.

[0015] According to the invention, treatment of an animal in need thereof typically involves the use or administration of an effective amount or a therapeutically effective amount of a proanthocyanidin polymer or a proanthocyanidin polymer composition, or a botanical extract, preferably from a Croton spp., particularly C lechleri, provided as either an enteric or non-enteric formulation. In preferred embodiments, the composition is not enterically protected or coated. Effective amount refers to the quantity (amount) of the composition, and the like, that induces a desired response in the animal subject upon administration or delivery to the animal. Optimally, an effective amount produces a therapeutic effect in the absence of, or with little or virtually no, adverse effects or cytotoxicity in the animal. Alternatively, any adverse effects associated with an effective amount are optimally outweighed by the therapeutic benefit achieved.

[0016] PEDv infects the lining of the small intestine, disrupting the epithelium.

Typically, the epithelium of the digestive tube is protected from insult by a number of mechanisms that constitute the gastrointestinal barrier. However, the gastrointestinal barrier can be breached and result in diarrhea. Destruction of the epithelium results not only in leaking of serum and blood into the lumen but also is often associated with significant destruction of adsorptive epithelium. When this occurs, the absorption of water becomes highly inefficient and diarrhea results. In addition, the response of the immune system to inflammatory conditions in the bowel contributes greatly to the development of diarrhea. Activated white blood cells are stimulated to produce and secrete inflammatory mediators and cytokines that stimulate secretion. A secretory component is thus imposed upon and exacerbates an inflammatory diarrhea. Moreover, reactive oxygen species produced by leukocytes can damage or destroy intestinal epithelial cells, which are replaced with immature cells that are generally lacking in the brush border enzymes and transporters necessary for the absorption of nutrients and water. Thus, components of an osmotic (malabsorption) diarrhea provide additional pathology and problems for an afflicted animal

[0017] As a consequence of the infection, the absorption of fluids from the intestine is altered and life-threatening electrolyte imbalances can occur. The affected animals lose fluids, rapidly dehydrate and suffer from electrolyte loss and acidosis. Although infectious agents may cause an initial damage to the animal's intestine, actual death from diarrhea (serious diarrhea) in animals usually is a consequence of dehydration, acidosis and loss of electrolytes, which may be difficult to replenish in adequate amount and time. Accordingly, the methods and formulations of the invention are suitable for treating diarrhea and the symptoms of diarrhea, such as dehydration, weight loss, and electrolyte loss, in an effort to prevent more severe dehydration and animal death. The methods also treat, ameliorate or prevent lesions in the small intestine or colon. Proanthocyanidins and Tannins Obtained from Plant Extracts

[0018] Proanthocyanidins are types of condensed tannins, which are found in a large number of plants and are classified as hydrolyzable or condensed. Tannins and, in particular, proanthocyanidins are contained in many plants used in traditional medicine as treatment or prophylaxis for diarrhea {See, e.g., Yoshida et al., 1993, Phytochemistry, 32: 1033; Yoshida et al., 1992, Chem. Pharm. Bull., 40: 1997; Tamaka et al., 1992, Chem. Pharm. Bull, 40:2092).

[0019] Proanthocyanidins are comprised of at least two or more monomer units that may be of the same or different monomeric structure. The monomer units (generally termed "leucoanthocyanidins") are generally monomeric flavonoids which include catechins, epicatechins, gallocatechins, galloepicatechins, flavanols, flavonols, flavan-3,4-diols, leucocyanidins and anthocyanidins. The polymer chains are thus based on different structural units, creating a wide variation of polymeric proanthocyanidins and a large number of possible isomers (Hemingway et al., 1982, J. C. S. Perkin, 1 : 1217). Larger polymers of the flavonoid 3-ol units are predominant in most plants and often have average molecular weights above 2,000 daltons (Da), containing 6 or more units (Newman et al., 1987, Mag. Res. Chem., 25: 118).

[0020] Proanthocyanidin polymers and proanthocyanidin are found in a wide variety of plants, especially those having a woody habit of growth {e.g., Croton spp.. and Calophyllum spp.). A number of different Croton tree species, including Croton sakutaris, Croton gossypifolius, Croton palanostima, Croton lechleri, Croton erythrochilus and Croton draconoides, which are endemic to South America, produce a red viscous latex sap called Sangre de Drago or "Dragon's Blood". The red viscous latex is known for its medicinal properties. For example, U.S. Patent No. 5,211,944 describes the isolation of an aqueous soluble proanthocyanidin polymer composition from Croton spp. See also, Ubillas et al., 1994, Phytomedicine, 1 :77. The isolation of an aqueous soluble proanthocyanidin polymer composition from Calophyllum inophylum and the use of this composition as an antiviral agent are also described in U.S. Patent No. 5,211,944.

[0021] In an embodiment, a proanthocyanidin polymer from C. lechleri, or a composition thereof, is crofelemer. Crofelemer (CAS 148465-45-6) is an oligomeric proanthocyanidin of varying chain lengths derived from the Dragon's Blood of Croton lechleri, a tree of the family Euphorbiaceae, which is sustainably harvested under fair trade work practices in the Amazon. It has an average molecular weight of approximately 1900 Da to approximately 2700 Da. The monomers comprising crofelemer comprise catechin, epicatechin, gallocatechin, and epigallocatechin. The chain length of crofelemer ranges from about 3 to about 30 units with an average chain length of about 8 units. Crofelemer has the chemical formula: (Ci50_6j7Hi2)_n and a molecular mass of 860-9100 g/mol. The antisecretory mechanism of action of crofelemer involves the targeting and inhibition of two, distinct intestinal chloride channels, namely, the cystic fibrosis transmembrane regulator conductance (CFTR) channel, which is a cAMP-stimulated CI^" channel, and the calcium-activated chloride channel (CaCC), as reported, for example, by Tradtrantip, L et al., 2010, "Crofelemer, an Antisecretory Antidiarrheal Proanthocyanidin Oligomer Extracted from Croton lechleri, Targets Two Distinct Intestinal Chloride Channels", Mol. Pharmacol., 77(l):69-78). A general structure of crofelemer is shown below. In the structure, an H at the R position of the structure signifies procyanidin; an OH at the R position of the structure signifies prodelphinidin.

[0022] In accordance with an embodiment of the invention, crofelemer, or a pharmaceutically acceptable formulation or composition comprising crofelemer, is employed in the treatment methods as the proanthocyanidin polymer from Croton lechleri.

[0023] In an embodiment, SP 303, an oligomeric proanthocyanidin from Croton lechleri, (also known as crofelemer) is the proanthocyanidin polymer from Croton lechleri, or a pharmaceutically acceptable formulation or composition comprising SP 303, which is suitable for use in the treatment methods of the invention. SP-303 (R. Ubillas et al., 1994, Phytomedicine, 1 :77-106) is largely composed of purified proanthocyanidin oligomers (-)- galloepicatechin and (+)-gallocatechin,(-)-epicatechin and (+)-catechin and is suitable for use in the enteric and non-enteric formulations and compositions for administration in the treatment methods described herein. The C lechleri proanthocyanidin may also be isolated according to example 2 of patent application publication US2007/0254050 or in patent application publication US2005/0019389, which are both incorporated by reference herein in their entirety.

[0024] In another embodiment, SB 300 is the proanthocyanidin polymer from Croton lechleri, or a pharmaceutically acceptable formulation or composition comprising SB 300, which is suitable for use in the treatment methods of the invention. SB 300, as described, for example, by Fischer, H. et al., (2004, J. Ethnopharmacol. , 93(2-3):351-357) provides a natural product extract that is particularly amenable for both enteric and non-enteric formulations and compositions, and is highly functional and cost-effective in the treatment methods described herein.

[0025] A pharmaceutically acceptable composition comprising a proanthocyanidin polymer from Croton lechleri and employed in the treatment methods of the invention can be obtained from C lechleri, e.g., as described in WO 00/47062 to Shaman Pharmaceuticals, Inc., the contents of which are incorporated by reference herein, and formulated as a food or dietary supplement or nutraceutical formulation. A standardized botanical extract is Neonorm™.

[0026] In other embodiments, compositions useful in the methods of the invention comprise a raw latex obtained from a Croton species or a Calophyllum species, or an extract obtained from a Croton species or a Calophyllum species, which are not specifically polymeric proanthocyanidin polymer compositions. Exemplary extracts are described in Persinos et al., 1979, J. Pharma. Sci., 68: 124 and Sethi, 1977, Canadian J. Pharm. Sci., 12:7 ^'.

[0027] In an embodiment, the proanthocyanidin polymer from Croton lechleri is formulated with an enteric coating or matrix in a variety of dosage formats known in the art (See, e.g., WO 00/47062 and U.S. Patent Nos. 7,441,744 and 7,323,195, the contents of which are incorporated herein, and as briefly described below). In another embodiment, the proanthocyanidin polymer is formulation without an enteric coating or matrix. Both enteric and non-enteric forms of the proanthocyanidin polymer from Croton lechleri, for example, SB 300, are intended for use in the methods of the present invention.

Preparation of Proanthocyanidin Polymer Compositions and Formulations

[0028] The proanthocyanidin polymer composition, effective for treating secretory diarrhea according to the invention, is comprised of monomeric units of leucoanthocyanidins. More particularly, the composition is comprised of proanthocyanidin polymers of 2 to 30 flavonoid units, preferably 2 to 15 flavonoid units, more preferably 2 to 11 flavonoid units and most preferably an average of 7 to 8 flavonoid units with a number average molecular weight of approximately 2500 Da. The proanthocyanidin polymer composition is preferably soluble in an aqueous solution. Preferred for use in the methods according to the invention is a proanthocyanidin polymer from C. lechleri; such a C. lechleri proanthocyanidin polymer may be in the form of a pharmaceutically acceptable composition.

[0029] Examples of proanthocyanidin polymeric compositions useful in the present invention are preferably isolated or purified from a Croton spp., namely, Croton lechleri, or Calophyllum spp. by any method known in the art. For example, the proanthocyanidin polymer composition may be isolated from a Croton spp. or Calophylum spp. by the method disclosed in U.S. Pat. No. 5,211,944 or in Ubillas et al. (1994, Phytomedicine, 1 :77-106, called SP 303 therein), both of which are incorporated herein by reference. Other isolation methods are described in U.S. Patent Nos. 7,556,831 and 8,067,041 (Example 2 therein), the contents of which are incorporated by reference herein. PCT application PCT/USOO/02687, published as WO 00/47062, the contents of which are incorporated by reference herein, also discloses a method of manufacturing a proanthocyanidin polymeric composition isolated from Croton spp. or Calophyllum spp., and enteric formulations of proanthocyanidin polymer dietary supplements, as well as methods of their preparation. Another illustrative method for isolating proanthocyanidin polymer from C. lechleri (such as crofelemer) is found in U.S. Patent Nos. 7,341,744 and 7,323, 195, the contents of which are expressly incorporated herein. As described above, the SP 303 and SB 300 purified forms of oligomeric proanthocyanidin polymer from Croton lechleri are suitable for use in the treatment methods of the invention.

[0030] In an embodiment, the proanthocyanidin polymer composition may be generally isolated by the following process, such as provided in U.S. Patent No. 7,341,744. Latex collected from Croton lechleri plants is mixed with purified water (preferably one part latex to two parts purified water). Any insoluble material in the latex solution is allowed to settle, e.g., by leaving the mixture at 4°C overnight (12 hours). The supernatant is pumped away from the residue and is extracted with a short chain alcohol, such as n-butanol. The extraction is preferably performed multiple times, such as three times. After each extraction, the alcohol phase is discarded and the aqueous phase is retained. The aqueous phase is concentrated, for example, using an ultrafiltration device with a 1 kD cut-off membrane. This membrane can be a low protein binding cellulose membrane, or, alternatively, a polypropylene, teflon or nylon membrane can be used. The membrane used should be compatible with acetone. The purpose of the ultrafiltration is to remove the water from the material.

[0031] The retentate from the ultrafiltration is then concentrated to dryness, for example using tray-dryers at approximately 37°C (± 2°C). The dried material is subsequently dissolved in water and is then chromatographed on a cation exchange column (e.g., a CM- Sepharose column) and a size exclusion column (e.g., an LH-20 column). In the preferred two column system, material is run over a CM-Sepharose and then an LH-20 column in a series. Specifically, the dissolved material is loaded onto the cation exchange column and is then washed with purified water. The proanthocyanidin polymer material is eluted from the cation exchange column with an aqueous acetone solution (preferably 30% acetone), thereby loading the proanthocyanidin polymer material onto the sizing column. The sizing column is disconnected from the cation exchange column and the material is then eluted off of the sizing column with an aqueous acetone solution (preferably 45% acetone). The fractions are collected and monitored with a UV detector, e.g., at a wavelength of 460 nm. Fractions containing the proanthocyanidin polymer material are combined and concentrated, for example, by ultrafiltration using, e.g., a 1 kD cut-off membrane (as described above for the ultrafiltration step prior to the chromatography steps). The retentate may then be concentrated to dryness using a suitable drying method, such as, but not limited to, a rotary evaporator, at a temperature of approximately 37°C (± 2°C). Other suitable drying methodologies include, but are not limited to, tray drying and spray drying. Example 10 of U.S. Patent No. 7,341,744 provides additional, non-limiting, methodology for preparing a composition comprising proanthocyanidin polymer, which can be used according to the invention. A detailed protocol for isolating an enriched proanthocyanidin polymer extract suitable for use in the methods of the invention is described in WO 00/47062 as noted herein above.

Methods of Treatment and Applications of Use

[0032] The invention is directed to methods of treating diarrhea and other sequelae associated with PEDv, particularly in neonatal and young animals, comprising administering to an animal in need of such treatment, a pharmaceutically acceptable composition comprising a proanthocyanidin polymer from a Croton species or Calophyllum species in an amount effective to treat the PEDv infection and/or the diarrhea associated with the infection. In preferred embodiments, the proanthocyanidin polymer is from a Croton species, namely, Croton lechleri. Treating the diarrhea can involve reducing the severity and duration of the diarrhea in the animal. Treating the diarrhea preferably involve increasing the survivability, vigor and weight of the animal, particularly a neonatal or young animal undergoing treatment.

[0033] The methods of the invention relate to the treatment of non-human animals, notably, but not limited to, the newborns and young of livestock, domestic and farm animals, particularly swine. In one embodiment, the immature animals to which treatment with the proanthocyanidin polymer from Croton lechleri is administered are neonatal (newborn) or infant animals, for example, one to ten hours after birth, one to fifteen hours after birth, twelve to twenty-four hours after birth, twenty-four to thirty-six hours after birth, one to three days after birth, one to four days after birth, one to six days after birth, or one to seven days after birth or up to two weeks after birth. Neonatal animals generally being those under two weeks of age. In an embodiment, the animals are treated between day one and day four after birth. In some embodiments, the neonatal or young animals are treated one to five days of age, less than one week of age, or only a few weeks of age. In an embodiment, treatment occurs during the first weeks of life, for example, one to six weeks of age. In an embodiment, the animals are from two to ten weeks of age, for example, less than one, two, three, four, five, six, seven, eight, nine, or ten weeks of age. The animals undergoing treatment may also be from one to four weeks of age, from one to six weeks of age, or from two to four weeks of age. In some embodiments, the animals are one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, or thirty days old. In other embodiments, the animals are thirty to forty days old. In other embodiments, the animals are young animals, generally up to one year in age. In many cases, the animals are not weaned (unweaned), i.e., they are still drinking milk. Pigs are weaned at 3 to 4 weeks of age. In some cases, the animals are newly weaned or weaned, but still juvenile, young, and non-adult.

[0034] According to the methods of the invention, the neonatal and young animals can be treated with a proanthocyanidin polymer from C. lechleri, preferably, a botanical extract derived from C. lechleri, for one, two, three, four, five, six, seven, eight, nine, or ten days, etc. The C. lechleri proanthocyanidin polymer can be administered to the animal on consecutive days or intermittently, such as every other day, every two days, every three days, every four days, and the like. In an embodiment, the C. lechleri proanthocyanidin polymer is administered to the animals for three consecutive days. The composition is preferably administered twice per day, or may be administered once per day, three times per day or four times per day. The methods provide for administering the compositions to pigs, particularly, neonatal or pre-weaned pigs, which have been diagnosed with PEDv or exposed to PEDv, i.e., other animals in the herd, yard, or otherwise in proximity, have been diagnosed with PEDv. The administration may be given to animals exposed but not necessarily diagnosed or exhibiting symptoms of PEDv as prophylaxis. In other embodiments, the animals are administered the compositions provided herein after exhibiting one or more symptoms, such as diarrhea, and/or having been diagnosed with PEDv by a diagnostic method known in the art. In an embodiment, the C. lechleri proanthocyanidin polymer is administered to neonatal animals between one and four days after birth for three consecutive days.

[0035] In a particular embodiment, a formulation or composition comprising a botanical extract derived from C. lechleri, SB 300, or SP 303, is provided in the form of a gel or paste formulation or a suspension of a powder formulation in water or other fluid that is orally administered to the neonatal or young animal, such as a piglet, twice daily for three days, preferably, three consecutive days. In a particular embodiment, the twice daily doses are administered to the animal twelve hours apart.

[0036] In accordance with the described methods, the C. lechleri proanthocyanidin polymer composition reduces chloride flux across intestinal epithelial cells and reduces fluid movement into the intestinal lumen, which results in fluid loss and dehydration associated with secretory diarrhea. Therefore, the pharmaceutically acceptable formulations and methods of the invention are useful in prophylactic and therapeutic applications in the treatment of diarrhea symptoms of PEDv, especially in preventing the dehydration and electrolyte loss that accompanies diarrhea.

[0037] In an embodiment, neonatal and young animals are treated prophylactically with a C. lechleri proanthocyanidin polymer composition, such as SB 300 or SP 303, in enterically protected or non-enterically protected form, to prevent or reduce the risk or severity of the debilitating effects of diarrheal disease and its associated symptoms, e.g., dehydration and weight loss, in neonatal and young animals. The methods can also reduce the incidence or severity of lesions of the small intestine or the colon. According to the treatment method, a C. lechleri proanthocyanidin polymer composition is administered to neonatal and young animals at a suitable time after birth to protect the animals from diarrhea outbreaks typically caused by PEDv. Administering a C. lechleri proanthocyanidin polymer composition to neonatal and young animals can also serve to ameliorate or reduce the risk of the animals' suffering from a more serious or severe form of diarrhea relative to animals that are not provided with the C. lechleri proanthocyanidin polymer composition prior to an outbreak of PEDv infection. The C. lechleri proanthocyanidin polymer composition can be enteric or non-enteric and can be, for example, SB 300 or SP 303, or a standardized botanical extract of C. lechleri. The dose and regimen of C. lechleri proanthocyanidin polymer composition administration are within the skill of the practitioner to determine and will depend on the environmental conditions and health of the neonatal and young animals to be treated. The animals can be prophylactically treated a with C. lechleri proanthocyanidin polymer composition according to the invention, for example and without limitation, one to seven days, one to six days, one to four days, one to three days, or one or two days after birth, particularly when PEDv infection has been detected in another animal in the herd or in proximity of the neonatal animal or the neonatal animal is thought to have been exposed to PEDv. The treatment regimen can involve one, two, three, four, five, six, seven or more days, of C. lechleri proanthocyanidin polymer composition administration to the animals, modified or adjusted as necessary or desired, once or multiple times, e.g., twice, three or four times, per day.

Physiologically and pharmaceutically acceptable formulations

[0038] The proanthocyanidin polymer from C. lechleri, or a composition thereof, can be provided in any physiologically, pharmaceutically, or therapeutically acceptable form. The pharmaceutically acceptable composition can be formulated for oral administration as, illustratively, but without limitation, powders; crystals; granules; small particles, including microparticles; particles sized on the order of micrometers, e.g., microspheres and microcapsules; particles sized on the order of millimeters, particles sized on the order of nanometers, e.g., nanoparticles; beads; microbeads; pellets; pills; tablets; 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 pharmaceutically acceptable 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, as a gel, as a paste or gel paste, or as any other convenient formulation for administration. The composition can be formulated for rectal administration, as a suppository, enema or other convenient form. The proanthocyanidin polymeric composition can also be provided as a controlled release system (See, e.g., Langer, 1990, Science 249: 1527-1533). The composition can be formulated as a dietary supplement or food supplement, e.g., as described in WO 00/47062, for administration to an animal in need thereof according to the present invention.

[0039] In a particular embodiment, a formulation or composition comprising a botanical extract derived from C. lechleri, SB 300, or SP 303, is provided in the form of a powder suspended in a liquid, preferably water, but may also be saline, milk or milk substitute, or other physiologically acceptable fluid, formulation that is orally administered to the animal, in need, twice daily for three days, preferably, three consecutive days. In a particular embodiment, the twice daily doses are administered to the animal twelve hours apart. The formulation is preferably a standardized botanical extract of C. lechleri, preferably Neonorm™, and is a powder that does not have an enteric coating or enteric protection.

[0040] The pharmaceutical formulation can also include any type of pharmaceutically acceptable excipients, additives, carriers, or vehicles. By way of nonlimiting 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 can be added to the proanthocyanidin polymer composition to increase the bulk of the composition. In addition, 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, can be added to the formulation to increase its cohesive qualities. Further, 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 lauryl sulfate and magnesium lauryl sulfate can be added to the formulation. Also, glidants, such as, but not limited to, colloidal silicon dioxide or talc can be added to improve the flow characteristics of a powdered formulation. 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 can also be added to facilitate disintegration of the formulation in the intestine.

[0041] In some other embodiments, the pharmaceutically acceptable formulations contain the proanthocyanidin polymer composition with an enteric coating, in addition to another pharmaceutically acceptable vehicle. In another embodiment, the pharmaceutically acceptable compositions containing the proanthocyanidin polymer composition alternatively include one or more substances that either neutralize stomach acid and/or enzymes or are active to prevent secretion of stomach acid. These formulations can be prepared by methods known in the art {see, e.g., methods described in Remington's "The Science and Practice of Pharmacy," 22nd Edition, Editor-in-Chief: Lloyd V. Allen, Jr., Pharmaceutically acceptable Press, Royal Pharmaceutically acceptable Society, London, UK, 2013; and U.S. Patent No. 7,323, 195).

[0042] In an embodiment, the proanthocyanidin polymer composition is formulated with a substance that protects the proanthocyanidin polymer and/or the polymer composition from the stomach environment. For such protection, the proanthocyanidin polymer composition can be 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, i.e. 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 proanthocyanidin polymer composition. 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. A variety of polymers are useful for the preparation of enteric coatings, and the application of an enteric coating to the proanthocyanidin polymer composition can be accomplished by any method known in the art for applying enteric coatings, as may be found, for example, and without limitation, in U.S. Patent Nos. 7,323, 195 and 7,341,744, incorporated herein by reference.

[0043] In another embodiment, the pharmaceutically acceptable composition of the proanthocyanidin polymer composition is formulated as enteric coated granules or powder (microspheres with a diameter of 300-500 microns) provided in either hard shell gelatin capsules or suspended in an oral solution for pediatric administration. The enteric coated proanthocyanidin polymer composition powder or granules can also be mixed with food, particularly for administration to neonatal or young animals. Such preparations may be prepared using techniques well known in the art. In addition, the proanthocyanidin polymer composition 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, as described, for example and without limitation, in U.S. Patent No. 7,323,195, incorporated herein by reference. [0044] In other embodiments, the proanthocyanidin polymer composition is in the form of an aqueous suspension in admixture with suitable excipients. Non-limiting examples of excipients that are suitable for the manufacture of aqueous suspension include suspending agents, for example, methylcellulose, sodium carboxymethylcellulose, hydroxy- propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents, which may be a naturally-occurring phosphatide, e.g., lecithin, or condensation products of an alkylene oxide with fatty acids, e.g., polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, e.g., heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol, for example, polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, such as polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, e.g., sucrose, saccharin or aspartame.

[0045] Dispersible powders and granules suitable for the preparation of an aqueous suspension by the addition of water provide the proanthocyanidin polymer composition in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those stated above. Additional excipients, for example, sweetening, flavoring and coloring agents, may also be present.

[0046] In an embodiment, the proanthocyanidin polymer composition is a gel or gel formulation. In an embodiment, the proanthocyanidin polymer composition is a paste formulation. In an embodiment, the paste formulation contains a purified botanical extract derived from C. lechleri that is preferably not enteric coated. In another embodiment, the paste formulation contains enterically coated beads comprising SB 300 or SP 303. In an embodiment, the paste formulation contains enteric protected SB 300 beads. In an embodiment, the gel or paste is contained or preloaded in a delivery device, such as a syringe, e.g., a needle-less syringe, or other type of applicator or delivery system, especially for oral delivery In an embodiment, the gel or paste is not contained in a delivery device, but is administered to the roof of the mouth of the animal, particularly one that is too incapacitated or ill to eat or drink, thereby eschewing an oral or other mode of administration. In an embodiment, the gel or paste comprises pH-sensitive polymeric particles, such as microparticles or nanoparticles, to allow for pH-dependent uptake of the active compound into cells and/or the pH-dependent release of the active compound in different pH environments in an animal. Processes for generating granules and particles comprising the C. lechleri botanical extract, proanthocyanidin polymer composition, or a compressible form thereof are as known and practiced in the art, and as provided, for example, in U.S. Patent No. 7,341,744, the contents of which are incorporated by reference herein. In an embodiment, gels are prepared for oral delivery and contain copolymers, such as poloxamers and Pluronics of different types, e.g., Pluronic R

[0047] In another embodiment, the proanthocyanidin polymer composition is in a paste formulation, preferably for oral administration. For example, an oral paste may comprise, without limitation, an oily vehicle or excipient, such as a hydrophobic oily vehicle, a basifying agent, a flavoring agent and a coloring agent. Illustrative and nonlimiting examples of hydrophobic oily vehicles include vegetable oil, triglyceride or polypropylene glycol, as well as a thickening agent, e.g., aluminum stearate. Flavoring agents can include, for example, fruit flavors, mint flavors, honey flavor, and other natural and organic flavorings known to those skilled in the art. Coloring agents can include, for example, iron oxide or titanium dioxide. Alternatively, the oily vehicle can be liquid paraffin or other suitable waxes, including a thickening agent.

[0048] Oily suspensions may be formulated by suspending the C. lechleri proanthocyanidin polymer as active ingredient in a vegetable oil, e.g., arachis oil, olive oil, sesame oil or coconut oil, or in mineral oil, such as liquid paraffin. The oily suspensions may contain a thickening agent, e.g., beeswax, hard paraffin or cetyl alcohol. Oral preparations can include sweetening agents as mentioned above and flavoring agents to improve palatability. Pharmaceutically acceptable preservatives, for example, an anti-oxidant such as ascorbic acid, can also be added to such compositions.

[0049] The C. lechleri proanthocyanidin polymer pharmaceutical compositions used in the methods of the invention may also be in the form of an oil-in-water emulsions. The oily phase may be a vegetable oil such as olive oil or arachis oil, or a mineral oil such as liquid paraffin or mixtures of these oils. Examples of emulsifying agents include, without limitation, naturally-occurring phosphatides, e.g., soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, e.g., sorbitan monooleate, and condensation products of partial esters with ethylene oxide, e.g., polyoxyethylene sorbitan monooleate. Sweetening, coloring and flavoring agents can be included in the emulsions.

[0050] Syrups and elixirs containing the C. lechleri proanthocyanidin polymer may also be formulated with sweetening agents, for example, glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions may be in the form of a sterile, orally deliverable or administrable aqueous or oleagenous suspension. This suspension may be formulated according to methods known in the art using suitable dispersing or wetting agents and suspending agents, such as those mentioned above. The sterile pharmaceutical preparation may also be a sterile solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, a solution in 1,3 -butane diol. Illustrative, acceptable vehicles and solvents that may be used in the preparations include water, Ringer's solution and isotonic sodium chloride solution. Co-solvents, e.g., ethanol, propylene glycol or polyethylene glycols, may also be included. In addition, sterile, fixed oils, e.g., any bland, fixed oil such as synthetic mono- or diglycerides, are conventionally employed as solvents or suspending media and may be used. In addition, fatty acids, such as oleic acid and the like, may be used in injectable preparations.

Dosage forms and administration

[0051] In a particular embodiment for treating diarrhea in neonatal animals, e.g., without limitation, bovine and camel calves, foals, kids, lambs, etc., but preferably piglets, the proanthocyanidin polymer composition is in powder, e.g., reconstitutable powder, form. The composition may be not enterically coated or alternatively may be enterically coated. In an embodiment, the neonates are less than one week in age. In an embodiment, the neonatal animals are piglets diagnosed with or having been exposed to PEDv.

[0052] In an embodiment, the powder form of the proanthocyanidin polymer composition used for treatment is reconstituted or mixed with liquid, such as oral electrolytes, milk or a milk replacer, water, physiological saline, to produce a liquid form or suspension. Milk replacer is generally a source of protein from different origins (for example, milk from a different species, soy, or eggs) and energy (lactose and fat) given to the calf or other animals to replace milk from the mother. The proanthocyanidin polymer composition, preferably a standardized botanical extract from C. lechleri, Neonorm™, is reconstituted at a concentration of 5 mg/ml, 8 mg/ml, 10 mg/ml or 20 mg/ml. In embodiments, piglets are administered 1-2 ml of the resuspended powder formulation per dose (including per day or twice per day) for a dose of 8 to 40 mg per day and/or per dose. In an embodiment, the powder form of the proanthocyanidin polymer composition is provided in the form of individual dosages in packets, e.g., packaged dosage forms, wherein some number of individual packets are provided for use in a treatment regimen. In certain embodiments, the packaged dosage form contain 10 to 100 mg of the proanthocyanidin polymer composition, preferably, 20 to 40 mg of the proanthocyanidin polymer composition. The number of individual doses that can be packaged and provided together is not intended to be limiting, and can include, for example, one to twenty packaged doses; one to ten packaged doses; two, four, six, eight, ten, or more packaged doses, as well as numbers of packaged doses in-between the foregoing, for efficiency of use, handling and for commercial efficacy. Those skilled in the art will appreciate that due to the higher purity of compositions such as SP-303 or crofelemer and SB 300, more by weight of SB 300 than SP-303 will need to be used in formulations to achieve the same amount of the active ingredient of the proanthocyanidin polymer composition. SB 300 generally has about 67% by weight of the proanthocyanidin polymer composition while SP-303 has higher purity, for example 99-100%.

[0053] In an embodiment, the proanthocyanidin polymer composition is administered or delivered to a neonatal animal afflicted with diarrhea and in need thereof by providing the compound as a bolus. In an embodiment, the proanthocyanidin polymer composition formulated as bolus, i.e., a pill, capsule, or tablet, is orally administered to the neonatal animals afflicted with diarrhea or symptoms thereof, e.g., piglets, directly in the mouth. In an particular embodiment, the treatment regimen comprises administering a dose of 10, 20 or 40 mg of the product, e.g., as embraced by one bolus per sick animal for a determined time period, for example, for one, two, or three or more days. The product can be provided to an animal in need thereof in portions of the complete dose, in which the portions are administered one or two or more times per day. Alternatively, the complete dose can be administered to an animal in need thereof one or two or more times per day. In a particular embodiment, the treatment encompasses a dose of 10 mg or 20 mg given two times a day. In another embodiment, the treatment encompasses an oral bolus dose of 10 mg or 20 mg given two times a day for 3 days. In an embodiment, the dose is the botanical extract of Croton lechleri, Neonorm™ that is not non-enteric protected, e.g., a reconstituted powder form.

[0054] In an embodiment, the proanthocyanidin polymer composition is in a gel or gel formulation. In an embodiment, the gel is contained or preloaded in a delivery device, such as a syringe or other type of injector or delivery system, especially for oral delivery. In an embodiment, the gel comprises pH-sensitive polymeric particles, such as microparticles or nanoparticles, to allow for pH-dependent uptake of the active compound into cells and/or the pH-dependent release of the active compound in different pH environments in an animal. In an embodiment, the gel is not contained in a delivery device, but is administered to the roof of the mouth of the animal, particularly one that is too incapacitated or ill to eat or drink, thereby eschewing an oral or other mode of administration. In an embodiment, gels are prepared for oral delivery and contain copolymers, such as poloxamers and Pluronics of different types, e.g., Pluronic F. Processes for generating granules and particles comprising the proanthocyanidin polymer composition or a compressible form thereof are as known and practiced in the art, and as provided, for example, in U.S. Patent No. 7,341,744, the contents of which are incorporated by reference herein.

[0055] The routes of administration of the C. lechleri proanthocyanidin polymer product to afflicted animals are not intended to be limiting. Illustratively, administration can be via any suitable, convenient or preferred route of administration including oral, buccal, dental, periodontal, via food source (animal feed), nutrition source, or libation source, otic, inhalation, endocervical, intramuscular, subcutaneous, intradermal, intracranial, intralymphatic, intraocular, intraperitoneal, intrapleural, intrathecal, intratracheal, intrauterine, intravascular, intravenous, intravesical, intranasal, ophthalmic, biliary perfusion, cardiac perfusion, spinal, sublingual, topical, transdermal, intravaginal, rectal, ureteral, or urethral. In certain embodiments, oral, buccal, and food and/or drink supplement are particularly suitable routes. In an embodiment, the product is an aqueous formulation and is provided to the animal as a drench or directly from a ready-to-use (RTU) bottle directed to the esophageal cavity so as to more effectively reach the animal's intestine/gut for optimal activity. In a related embodiment, administration can also be by inclusion in the regular or special diet of the animal, such as in a functional food for the animals or companion animals.

[0056] Dosage forms can include, without limitation, oral, injectable, transdermal, aerosol including metered aerosol, chewable products or pellets, capsules, capsule containing coated particles, nanoparticles, or pellets, capsule containing delayed release particles, capsule containing extended release particles, concentrates, creams and augmented creams, suppository creams, discs, dressings, elixirs, emulsions, enemas, extended release films or fibers, gases, gels, metered gels, granules, delayed release granules, effervescent granules, implants, inhalants, injectable lipid complexes, injectable liposomes, inserts or devices, extended release inserts, intrauterine devices, jelly s, liquids, extended release liquids, lotions, augmented lotions, oils, ointments, augmented ointments, pastes, pastilles, pellets, powders, reconstituted powders, extended release powders, metered powders, solutions, drops, concentrated solutions, gel forming solutions/drops, sponges, sprays, metered sprays, suppositories, suspensions, suspensions/drops, extended release suspensions, syrups, tablets/pills, chewable tablets/pills, tablets/pills containing coated particles, delayed release tablets/pills, dispersible tablets/pills, effervescent tablets/pills, extended release tablets/pills, orally disintegrating tablets/pills, tapes, or troches/lozenges. The dosages can be provided as formulations, compositions, pharmaceutically acceptable formulations and compositions, physiologically acceptable formulations and compositions, including pharmaceutically and physiologically acceptable carrier, excipients, diluents, or vehicles as known and used in the art.

[0057] For oral administration, the C. lechleri proanthocyanidin polymer product, or a composition thereof, is preferably a liquid or gel formulation, but may also be encapsulated and formulated with suitable carriers, and the like, in solid dosage forms. Nonlimiting examples of suitable carriers, excipients, diluents and vehicles include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, gelatin, syrup, methyl cellulose, methyl- and propylhydroxybenzoates, talc, magnesium, stearate, water, mineral oil, edible oils, and the like. The formulations can also include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents. The compositions can be formulated to provide rapid, sustained, extended, or delayed release of the active ingredient after administration to the animal by employing protocols and methods well known in the art. The formulations can also include compounds or substances that reduce proteolytic degradation and promote absorption such as, for example, surface active agents.

[0058] As will be appreciated by those having skill in the art, the specific dose can be calculated according to the approximate body weight, body mass, or body surface area of the animal, or the volume of body space or mass to be occupied. The dose also depends on the particular route of administration selected by the practitioner. Further refinement of the calculations necessary to determine an appropriate dosage for treatment is routinely made by those of ordinary skill in the art, for example, using appropriate assays and analytical procedures, such as has been described for certain compounds (e.g., Howitz et al., Nature, 425: 191-196, 2003). Exact dosages can be determined based on standard dose-response studies. Therapeutically effective doses for treatment of afflicted animals can be determined, by titrating the amount of the active product given to the animal to arrive at the desired therapeutic effect, while minimizing side effects.

[0059] For use in treating diarrhea associated with PEDv infection and its symptoms in neonatal and young animals, preferably piglets, in accordance with the methods of the invention, a therapeutically acceptable form of the C. lechleri proanthocyanidin polymer composition, including a C. lechleri botanical extract, is administered, particularly orally administered, in an amount ranging from 2 to 100 mg/kg per day, once, twice or more daily. In other embodiments, the amount can range from about 1 to about 10 mg/kg/day, once, twice or more daily; or from about 2 to about 20 mg/kg/day, once, twice or more daily; or from about 1 to about 40 mg/kg/day, once, twice or more daily; or from about 1 to about 40 mg/kg/day, once, twice or more daily. In other embodiments, the dose can be 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, etc., as well as incremental dose amounts in between. In still other embodiments, the amount can range from about 5 to about 40 mg/kg/day once, twice or more daily; or from about 5 to about 20 mg/kg/day, from about 1 to about 20 mg/kg/day, from about 1 to about 10 mg/kg/day, or from about 2 to about 4 mg/kg/day once, twice or more daily. In other embodiments, the foregoing amounts of the C. lechleri proanthocyanidin polymer composition are administered, for example, twice daily, three times daily, four times daily, or more than four times daily, rather than once per day. Higher doses, e.g., 50 mg/kg or 100 mg/kg per day or twice or more daily, may be required, as necessary, to treat diarrhea and accompanying dehydration in the neonatal and young animals. [0060] In other embodiments, for the treatment methods, a suitable dose for the C. lechleri proanthocyanidin polymer product, or the C. lechleri proanthocyanidin polymer composition, such as SP 303 or SB 300 or a C. lechleri botanical extract, may range from about 1 mg to about 100 mg, either daily or multiple times per day. In an embodiment, a suitable dose may range from about 5 mg to about 60 mg, either daily or multiple times per day. In an embodiment, a suitable dose may range from about 10 mg to about 100 mg, either daily or multiple times per day. In an embodiment, a suitable dose may range from about 10 mg to about 40 mg, either daily or multiple times per day. In an embodiment, a suitable dose may range from about 10 mg to about 20 mg, either daily or multiple times per day. It will be understood that the ranges include the lower and higher amounts specified, as well as amounts in between. The doses administered multiple times per day can be given for consecutive days, e.g., two days, three days, four days, five days, six, days, seven days, or more, in some embodiments. A dose administered multiple times per day may embrace two, three, four, five, six, or more times per day. Other dosing schedules, such as every other day, or every third day, every fourth day, etc. are embraced by the invention. In an embodiment a standardized botanical extract of C. lechleri, i.e., Neonorm™, which is not enterically coated is administered. In an embodiment the C. lechleri proanthocyanidin polymer is enterically coated SB 300. In an embodiment the C. lechleri proanthocyanidin polymer is non-enterically coated SB 300.

[0061] In some embodiments, daily doses, including multiple daily doses, e.g., twice or three times a day, of the C. lechleri proanthocyanidin polymer product may be 2 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 50 mg., 100 mg, or 150 mg (or there between) per animal. Administration schedules may also be altered to achieve a therapeutically effective concentration of the C. lechleri proanthocyanidin polymer to treat the PEDv infection and diarrhea and its symptoms as described herein. By way of specific, yet nonlimiting example, a suitable dosage amount for use in the methods according to the invention is 10 or 20 mg administered once or twice daily. In some embodiments, the compound may be administered once per day, twice per day, thrice per day, 4 times per day, 5 times per day, 7 times per day or 10 times per day. Often the dosage is divided into equal parts administered throughout the day, however in some embodiments related to treating more severe or entrenched symptoms, it may be useful to tailor the dosage administration schedule so that most of the daily treatment is administered at a predetermined time of the day, e.g., the beginning half of the day. In some embodiments, about 50% 60%>, 70% or 80%> of the dosage is administered in the first half of the day. In other embodiments, it may be more appropriate to administer most of the dosage in the latter half of the day so that about 50%, 60%>, 70% or 80%> of the dosage is administered in the latter half of the day.

[0062] It will be understood that the dose amount actually administered can be determined by the practitioner, in the light of the relevant circumstances, including the severity of the disease, condition, or symptoms thereof being treated, the form of the product to be administered, the age, weight, and response of the individual animal receiving treatment, as well as the chosen route of administration.

[0063] The methods of the invention further embrace the administration of pharmaceutically acceptable formulations of the proanthocyanidin polymer composition either alone or in combination with other supplements or agents for treatment or amelioration of the symptoms of secretory diarrhea, such as rehydration agents, electrolytes (e.g., sodium, potassium, magnesium, chloride and formulations thereof), antibiotics, gut-lining protectants, such as kaolin, pectin, or bismuth liquid, and fluid adsorbents, such as attapulgite. Other agents may include anti-motility agents, although because many of the microorganisms and pathogens that are associated with diarrhea induction in neonatal and young animals concomitantly decrease gut motility, the use of anti-motility drugs may be contraindicated. Natural biological products, e.g., Lactobacillus, Bifidobacterium, or Streptococcus faecium, other bacteria and yeast microorganisms, or probiotics, may also be employed as additives to restore the natural balance of intestinal flora in the affected neonatal animals. Such natural biological products, e.g., probiotics as known in the art, may be administered in conjunction with the C. lechleri proanthocyanidin polymer or composition thereof, for example, prior to, at the same time as, or after the administration of the proanthocyanidin polymer or composition to a non-human animal. In addition, a reconstituted C. lechleri proanthocyanidin polymer or composition thereof may include probiotics in accordance with the present invention.

Exemplary Specific Embodiments Encompassed by the Invention

[0064] The present invention is further directed to uses and methods encompassed by the following embodiments. [0065] In an embodiment, the invention provides the use of a standardized botanical extract from Croton lechleri in treating PEDv and diarrhea associated with PEDv infection in a neonatal or young piglet, wherein the composition is formulated as a pharmaceutically acceptable reconstituted powder and is orally administered to the neonatal or young piglet animal in an amount of at least 10 mg to 60 mg per day for two or more consecutive days. In specific embodiments, 1 to 2 milliliters (ml) of a 10 mg/ml or 20 mg/ml suspension of powdered Neonorm™ are administered once or two times per day, for 1, 2, 3 4, or 5 or more days, to neonatal piglets (approximately 1 to 14 days after birth) to treat or ameliorate the symptoms of PEDv infection or prevent PEDv infection in piglets exposed to PEDv. In an embodiment, the Neonorm™ formulation is administered orally. In particular, the administration of Neonorm™reduced mortality in the piglets by 50%, 60%, 70%, 80% or 100%). The administration reduces the severity of diarrhea and dehydration and may also reduce the number and/or severity of lesions in the small intestine or colon of the animal.

[0066] In an embodiment related to the above uses and methods, the composition comprising the C. lechleri proanthocyanidin polymer is administered to the animal as an enteric coated pharmaceutical composition or as a non-enteric coated pharmaceutical composition. In other embodiments, the composition comprising the C. lechleri proanthocyanidin polymer is a botanical extract of C. lechleri, SB 300, SP 303, or crofelemer.

[0067] The following examples describe the invention in its various aspects and are not intended to be limiting.

EXAMPLES

Example 1

[0068] A preliminary, multi-site, 3 -month study was conducted to evaluate the safety and effectiveness of a botanical extract comprising a proanthocyanidin polymer composition, as described herein, derived from Croton lechleri, in piglets. The study was conducted at pig farms in China, which have been known to have outbreaks of diarrhea related disease in facilities in which piglets are born, raised and maintained. The C. lechleri extract administered to the piglets was a non-enterically protected powder form of SB-300, known as Neonorm^™, dissolved in water, with no added excipients. [0069] The study took place from March to May of 2016 and involved 433 piglets ranging in age from 1-15 days. The piglets in the study were from two age groups: Group A: 1-3 days of age and Group B: 10-15 days of age) and had a history of diarrhea. The piglets in Groups A and B in this study were placed either in a treatment group in which the piglets were individually treated with 10-20 mg of the Neonorm^™ powder- in-water formulation twice daily (BID) for three days, or in a control group that was not treated with Neonorm^™.

[0070] Following treatment with Neonorm^™, 99% of the piglets in the 1-3 days of age treatment group achieved resolution of diarrhea, while no resolution of diarrhea was found in the control group. A 3% mortality rate was observed among piglets in the Neonorm^™- treatment group, while a 100% mortality rate was observed in the piglets in the control group. In the group of piglets aged 10-15 days, the rate of diarrhea resolution was 70%, versus no resolution of diarrhea in the control group. The mortality rate of piglets in the 10-15 days of age group was 30%, while the mortality rate of piglets in the control group was 100%. Comparisons of cure and mortality rates between the Neonorm^™-treated groups and the control groups of piglets generated p-values less than 0.01. The results of this study showed that Neonorm^™ treatment of diarrhea in piglets ranging from 1 to 15 days of age resolved the diarrhea, increased survival and decreased mortality in a significant percentage of the animals that were treated with Neonorm^™ during the treatment period compared with controls not treated with Neonorm^™.

Example 2

[0071] A second preliminary study to evaluate Neonorm^™'s safety and effectiveness was carried out on a farm in China that had experienced an outbreak of Porcine Epidemic Diarrhea Virus (PEDv), a specific coronavirus. As noted supra, PEDv is most serious in neonatal piglets where morbidity and mortality can be 80 to 100 percent. PEDv infects the cells lining the pig's small intestine, causing porcine epidemic diarrhea (PED), a condition of severe diarrhea and dehydration. The piglets in this second study were divided into three treatment groups with no control in which each treatment group piglet received 8-20 mg of Neonorm^™.

[0072] Treatment group A contained two hundred (200) piglets that suffered from diarrhea caused by PEDv. The animals in Treatment Group A received 10-20 mg of Neonorm^™ administered over 2 days. Treatment group B contained two hundred (200) piglets that had recently contracted PEDv. The piglets in Treatment group B received a lower dose of Neonorm^™ (8-16 mg) over 3 days. Treatment group C contained one hundred (100) piglets that were healthy and were treated prophylactically with 8-16 mg of Neonorm^™ for three days.

[0073] The results of the second study showed a 95% cure rate among the piglets in

Treatment group A that had received 10-20 mg of Neonorm^™ administered over two days. The cure rate among piglets in Treatment group B that had received 8-16 mg of Neonorm^™ for 3 days was 60%. Of the piglets in Treatment group C that were treated prophylactically with 8- 16 mg of Neonorm^™ for 3 days, 15% developed diarrheal symptoms, which is similar to the usual frequency of diarrheal disease on this farm in the absence of prophylactic treatment. The results of this study suggested a high resolution and survival rate following Neonorm^™ treatment of diarrhea in piglets.

[0074] The study results related to successful treatment of diarrhea associated with

PEDv infection in piglets are surprising, as PEDv is known to infect the cells lining the small intestine of piglets causing porcine epidemic diarrhea (PED), a condition of severe diarrhea and dehydration, with concomitant lesions in the small intestine or colon of the animal. It was therefore unexpected to find that treatment of piglets with Neonorm^™ resolved diarrhea and increased survival in piglets experiencing the symptoms of PED, and whose intestinal cells were severely compromised and/or had possibly undergone cytolysis resulting from their PEDv infection.

[0075] All patents, patent applications and publications referred to or cited herein are hereby incorporated by reference in their entireties for all purposes.

[0076] It is understood that the embodiments and examples described herein are for illustrative purposes and that various modifications or changes in light thereof will be suggested to persons skilled in the pertinent art and are to be included within the spirit and purview of this application and scope of the appended claims. It is to be understood that suitable methods and materials are described herein for the practice of the embodiments; however, methods and materials that are similar or equivalent to those described herein can be used in the practice or testing of the invention and described embodiments.

Claims

What is claimed is:

1. A method of treating a neonatal or young piglet suffering from or having been exposed to Porcine Epidemic Diarrhea virus (PEDv), the method comprising orally administering to the neonatal or young piglet in need thereof a pharmaceutical composition comprising an aqueous soluble proanthocyanidin polymer from Croton lechleri.

2. The method according to claim 2, wherein the piglet is neonatal.

3. The method according to claims 1 or 2, wherein the piglet is unweaned.

4. The method according to any one of claims 1 to 3 wherein the piglet is 1 to 5 days old.

5. The method according to any one of claims 1 to 4 wherein the piglet is 5 to 15 days old.

6. The method according to any one of claims 1 to 5, wherein the proanthocyanidin polymer composition is administered twice daily for one, two or three consecutive days.

7. The method according to any one of claims 1 to 6, wherein the proanthocyanidin polymer composition is a standardized botanical extract of C. lechleri.

8. The method according to any one of claims 1 to 6 wherein the proanthocyanidin polymer composition is SB300 or SP303.

9. The method according to any one of claims 1 to 8, wherein the proanthocyanidin polymer composition is administered as a powder reconstituted with water, oral electrolytes, milk or a milk substitute, or physiological saline.

10. The method according to any one of claims 1 to 9 wherein the proanthocyanidin polymer composition does not have an enteric coating.

11. The method according to any one of claims 1 to 10, wherein the composition is administered to the neonatal piglet in an amount of at least 8 mg to 20 mg daily.

12. The method according to any one of claims 1 to 10 wherein the composition is administered to the neonatal piglet in an amount of at least 8 mg to 20 mg twice a day.

13. The method according to any one of claims 1 to 12 wherein the piglet has been diagnosed with PEDv.

14. The method according to any one of claims 1 to 13 wherein the piglet has been exposed to an animal infected with PEDv.

15. The method according to any one of claims 1 to 14, wherein said administration ameliorates diarrhea in the piglet.

16. The method according to any one of claims 1 to 15, wherein said administration reduces the incidence of mortality by at least 50%

17. The method according to any one of claims 1 to 16 wherein said administration reduces the incidence of mortality by at least 70%.