Methods and compositions for improving health

Abstract

Methods and compositions for improving gastrointestinal, respiratory, and musculoskeletal health in human and animal subjects are provided. Methods and compositions for improving feed conversion, reducing the rate of occurrence of mortality in a population of subjects, reducing the rate of occurrence of morbidity in a population of subjects, and reducing the severity of morbidity are also provided. Parasitic, amebic, bacterial, and protozoal infections and related conditions, disorders, or diseases are also treated with or prevented by the methods and compositions herein.

Description

METHODS AND COMPOSITIONS FOR IMPROVING HEALTHFIELD OF THE INVENTION

[0001] The present invention concerns methods and compositions for improving health in human and animal subjects. The invention provides improved gastrointestinal (GI), respiratory, and musculoskeletal health. It also provides improved feed conversion, reduced occurrence of mortality in a population of subjects, reduced severity of morbidity, and reduced occurrence of morbidity in a population of subjects. Parasitic, amebic, bacterial, and protozoal infections and related conditions, disorders, or diseases are also treated or prevented by administration of a cardiac glycoside-containing composition.BACKGROUND OF THE INVENTION

[0002] Humans and animals suffer from deteriorating GI, respiratory, and musculoskeletal health. Such deterioration may or may not be related to disease states. Commercial and companion animals are particularly susceptible to such health issues especially if they reside or are processed through commercial facilities housing populations of animals. Factors such as the occurrence of mortality and morbidity in a population of animals as well as inefficient feed conversion are problematic in the commercial animal industry.

[0003] In terms of gastrointestinal health, parasitic, amebic, bacterial, and protozoal infections are common in both humans and animals. They are particularly common in third- world countries and in animal breeding or boarding facilities, especially for dogs, cats, horses, cows, chickens, turkeys, and pigs.

[0004] Giardiasis is a common parasitic infection that can cause a range of symptoms in mammals and birds, e.g. diarrhea, gas, foul-smelling greasy feces, stomach cramps or pain, intestinal cramps or pain, upset stomach, nausea, and / or dehydration. Other less common symptoms include fatigue, fever, itchy skin, hives, and swelling of the eyes and joints. Even so, some giardia seropositive subjects exhibit no related symptoms. Giardiasis is caused by an intestinal parasite called Giardia duodenalis (also known as Giardia intestinalis, Giardia lamblia, Giardia muris, Giardia ardeae, or Giardia psiUaci phylum: metamonada, class: trepomonadea, order: diplomonadida; domain: eukaryote; family: hexamitidae), a protozoan flagellate which can be found in feces-contaminated soil, food and water. Giardia is passed along by ingesting feces-contaminated water, soil, food or objects. For example, giardiasismay occur in subjects that have are in close contact with someone who has the disease, in subjects that travel within areas that have poor sanitation, in subjects that have contact with poop (feces) during sexual activity, in backpackers or campers who drink untreated water from springs, lakes, or rivers, in swimmers who swallow water from swimming pools, hot tubs, splash pads, or untreated recreational water from springs, lakes, or rivers, in people who get their household water from a shallow well, in subjects with weakened immune systems, or in subjects who have contact with infected animals or animal environments contaminated with poop.

[0005] Giardia has two forms: trophozoites, which live in the intestines of infected animals, and cysts, which are trophozoites protected by an outer shell and that get shed in the animal’s stool. Cysts are hardy and can survive in the surrounding environment for months. They are instantly infectious, and just a few ingested cysts can cause infection. A dog can easily get reinfected by grooming themselves, for example, if cysts remain on their fur, paw pads or hind end.

[0006] Giardia canis (Assemblage C / D) is the host-specific species infecting dogs; however, G. duodenalis (certain subtypes of Assemblage A) and G. enterica (Assemblage B) have also been found in dogs. Mixed infections are possible. The prevalence of Giardia in dogs varies with age, environment and diagnostic technique used, but in North America is usually less than 15% in pet dogs. Giardia was detected in 8% of dogs in Calgary using a zinc sulfate flotation method. In a recent national study using a centrifugal flotation method, Giardia was detected in 3.5% of dogs nationally and in western Canada (AB, SK, and MB), and in 2.2% of dogs in BC. Prevalence was higher (5.5%) in dogs less than a year of age. Given that most infections are likely subclinical, and that flotation methods are relatively insensitive for detection of protozoans, true prevalence, especially in young dogs, is likely higher.

[0007] Giardiasis is typically treated by administration of ronidazole, tinidazole, metronidazole, nimorazole, flunidazole, dimetridazole, ipronazole, fenbendazole, albendazole, antidiarrheic, pyrantel, ponazuril, a 5-nitroimidazole antibiotic, or combinations thereof.

[0008] Coccidiosis is an intestinal tract infection caused by a single-celled organism (protozoa) called coccidia (also known as canine Cystoisospora spp ). Coccidiosis typically refers to gastrointestinal infections with the Isospora species (sometimes called Cystoisospora) of coccidia, though other species can be found. Cats have speciesof Hammondia, Isospora, Besnoilia, Toxoplasma, and Sarcocystis. Two species infect cats: I fells and / rivolta,' both can be identified easily by oocyst size and shape. Dogs have species of Hammondia, Isospora, and Sarcocystis. Four species infect dogs: I burrowsi, I canis, I neorivolta, and I ohioensis. In dogs, only I canis can be identified by the oocyst structure; the other three Isospora spp overlap in dimensions and can be differentiated only by endogenous developmental characteristics. In puppies with severe cases with high oocyst counts, associated signs can occur. Both I canis and I ohioensis may cause bloody diarrhea and poor growth. Isospora burrowsi may not cause clinical disease. Isospora neorivolta occasionally causes diarrhea.

[0009] Coccidiosis may also be caused by Eimeria sp. parasite such as Gallus gallus domesticus (in chickens; especially E. tenella, E. acervuline, E. necatrix, and E. maxima), Meleagris gallopavo (in turkeys), Phasianus colchicus (in pheasants), Capra hircus (in goats), Ovis aries (in sheep), Bos taurus (in cattle; especially, E. bovis, E. zuernii, E. ellipsoidalis, andE. aiiburnensis), or Oryctolagus cuniculus (in rabbits).

[0010] The most common clinical sign of coccidiosis is diarrhea, but most dogs infected with coccidia do not have any clinical signs. However, in puppies and debilitated adult dogs, coccidiosis may cause severe watery diarrhea, dehydration, abdominal distress, and vomiting. In severe cases, death may occur.

[0011] Coccidiosis is typically treated by administration of sulfa-type antibiotic (e.g. sulfadimethoxine), amprolium (Albac / Amprol Hi-E [+ Bacitracin, + Ethopabate]), ponazuril, toltrazuril, metronidazole, ionophore(s) (e.g. rumensin), sulfaquinoxaline, sulfamethazine, lasalocid, decoquinate, monensin, or combinations thereof.

[0012] Cryptosporidium is a microscopic parasite that causes the diarrheal disease cryptosporidiosis. Symptoms of cryptosporidiosis include diarrhea, loose or watery stool, vomiting, weight loss, stomach cramps, and / or fever. Cryptosporidium is a leading cause of waterborne disease among humans in the United States. One can get this infection after eating food or drinking water that is contaminated with stool. Nitazoxanide has been FDA- approved for treatment of diarrhea caused by Cryptosporidium in people with healthy immune systems and is available by prescription. However, the effectiveness of nitazoxanide in immunosuppressed subjects is unclear. Cryptosporidiosis is normally not a serious disease in healthy people, but it can lead to a life-threatening illness for people with a weak immune system. Those at risk are subjects with HIV / AIDS, cancer and transplantpatients who are taking certain medicines that suppress the immune system, and subjects with inherited diseases that affect the immune system.

[0013] Campylobacteriosis is a diarrheal disease caused by Campylobacter sp. bacteria. Every year almost 1 in 10 people fall ill and 33 million healthy life years are lost. Campylobacter is 1 of the 4 key global causes of diarrheal diseases. Currently, there are 17 species and 6 subspecies assigned to the genus Campylobacter, of which the most frequently reported in human diseases are C. jejuni (subspecies jejuni) and C. coli. Other species such as C. lari and C. upsaliensis have also been isolated from patients with diarrhoeal disease, but are reported less frequently. Symptoms include diarrhea (often bloody), abdominal pain, fever, headache, nausea, and / or vomiting, and the symptoms typically last several days. Complications such as bacteraemia (presence of bacteria in the blood), hepatitis, pancreatitis (infections of liver and pancreas, respectively), and miscarriage have been reported with various degrees of frequency. Post-infection complications may include reactive arthritis (painful inflammation of the joints which can last for several months) and neurological disorders such as Guillain-Barre syndrome, a polio-like form of paralysis that can result in respiratory and severe neurological dysfunction in a small number of cases.

[0014] Johne’s disease is s a contagious, chronic and usually fatal infection that affects primarily the small intestine of ruminants. All ruminants are susceptible to Johne's disease. Johne's disease is caused by Mycobacterium avium subspecies paratuberculosis bacteria. A national study of US dairies, Dairy NAHMS 96, found that approximately 22 percent of US dairy farms have at least 10% of the herd infected with Johne's disease. The study determined that infected herds experience an average loss of $40 per cow in herds with a low Johne's disease clinical cull rate while herds with a high Johne's disease clinical cull rate lost on average of $227. This loss was due to reduced milk production, early culling, and poor conditioning at culling. Signs of Johne's disease include weight loss and diarrhea with a normal appetite. Several weeks after the onset of diarrhea, a soft swelling may occur under the jaw (bottle jaw). Bottle jaw or intermandibular edema is due to protein loss from the bloodstream into the digestive tract. Animals at this stage of the disease will not live very long, perhaps a few weeks at most. Animals are most susceptible to the infection in the first year of life. Newborns most often become infected by swallowing small amounts of infected manure from the birthing environment or udder of the mother. In addition, newborns may become infected while in the uterus or by swallowing bacteria passed in milk and colostrum.

[0015] Dysentery is an infection of the intestines that causes diarrhea, which may contain blood and / or mucus. Dysentery may be caused by parasitic, protozoal (as described herein and others), amebic, or bacterial (e.g. Campylobacter sp., Salmonella sp., Escherichia sp. or Shigella sp.) infection.

[0016] In terms of deterioration of respiratory health, such deterioration commonly occurs when humans or animals are exposed to unhealthy subjects or to unhealthy living conditions. This is particularly problematic for immune-compromised humans and animals. Deterioration of respiratory health may or may not be disease related; however, symptoms of respiratory health deterioration often include pulmonary congestion, nasal congestion, coughing, wheezing, hacking, or difficulty breathing.

[0017] The occurrence of mortality and / or morbidity in a population of animals (commercial or domestic) is particularly problematic and producers are continually seeking news ways to reduce the rate of occurrence of mortality, reduce the rate of occurrence of morbidity, and reduce the severity of morbidity in animals. For example, cattle sale bams handle healthy as well as at-risk or high-risk calves exhibiting anywhere from 5-40% death loss per lot. The sick calves, termed “no-bids”, are not purchased by buyers and so represent a significant loss to rancher s / producers. The challenge for ranchers is that the death loss needs to be less than 10% for the rancher to profit.

[0018] Nerium oleander, a member of the Nerium species, is an ornamental plant widely distributed in subtropical Asia, the southwestern United States, and the Mediterranean. Its medical and toxicological properties have long been recognized. In humans, it has been proposed for use, for example, in the treatment of hemorrhoids, ulcers, leprosy, snake bites, cancers, tumors, neurological disorders, warts, and cell -proliferative diseases.

[0019] Oleandrin is a cardiac glycoside obtained by extraction from Nerium species (Nerium oleander, Nerium odorum) plant. It is widely recognized in the animal industry that consumption of the plant material is toxic to animals and on occasion may result in fatal poisoning. (Rubini et al., “A probable fatal case of oleander (Nerium oleander) poisoning on a cattle farm: a new method of detection and quantitation of the oleandrin toxin in rumen” in Toxins (2019), 11, 442; Ceci et al., “Outbreak of oleander (Nerium oleander) poisoning in dairy cattle: clinical and food safety implications” in Toxins (2020), 12, 471; Aslani et al., “Clinical and pathological aspects of experimental oleander (Nerium oleander) toxicosis in sheep” in Vet. Res. Commun. (2004), 28, 609-616; Barbosa et al., “Toxicity in goats caused by oleander (Nerium oleander)” in Res. Vet. Sci. (2008), 85, 279-281; Soto-Blancoet al., “Acute cattle intoxication from Nerium oleander pods” in Trop. Anim. Health Prod. (2006), 38, 451-454).

[0020] Oleander is considered the most important cause of livestock poisoning in South Africa. Accidental intoxications have been reported in horses, donkeys, cattle, camelids (alpaca and llama), dogs, cats and pet birds. Mydriasis in animals, after oleander ingestion, is also observed in relation to the increased sympathetic tone. Moreover, oleandrin or oleander toxicity is well known to cause diarrhea in humans and animals. For fear of these side effects, no therapeutic products derived from the plant have been developed for use in animals such as domestic animals, commercial animals, or livestock, e.g. dogs, cats, horses, cows, pigs, goats, sheep, poultry, turkeys, etc. Therefore, it would be extremely unexpected that Nerium oleander extract or oleandrin would provide any clinical benefit in treating or preventing diarrhea, improving GI health, improving respiratory health, improving musculoskeletal health, increasing feed conversion, reducing the rate of occurrence of mortality, reducing the severity of morbidity, or reducing the rate of occurrence of morbidity.

[0021] Extraction of components from plants of Nerium species has traditionally been carried out using boiling water, cold water, supercritical fluid, or organic solvent.

[0022] ANVIRZEL™ (US 5,135,745 to Ozel) contains the concentrated form or powdered form of the hot- water extract of Nerium oleander. Muller et al. (Pharmazie. (1991) Sept. 46(9), 657-663) disclose the results regarding the analysis of a water extract of Nerium oleander. They report that the polysaccharide present is primarily galacturonic acid. Other saccharides include rhamnose, arabinose and galactose. Polysaccharide content and individual sugar composition of polysaccharides within the hot water extract of Nerium oleander have also been reported by Newman et al. (J. Herbal Pharmacotherapy, (2001) vol 1, pp.1-16). Compositional analysis of ANVIRZEL™, the hot water extract, was described by Newman et al. (Anal. Chem. (2000), 72(15), 3547-3552). U.S. Patent No. 5,869,060 to Selvaraj et al. pertains to extracts o Nerium species and methods of production. To prepare the extract, plant material is placed in water and boiled. The crude extract is then separated from the plant matter and sterilized by filtration. The resultant extract can then be lyophilized to produce a powder. U.S. Patent No. 6,565,897 (U.S. Pregrant Publication No. 20020114852 and PCT International Publication No. WO 2000 / 016793 to Selvaraj et al.) discloses a hot-water extraction process for the preparation of a substantially sterile water extract. Ishikawa et al. (J. Nutr. Sci. Vitaminol. (2007), 53, 166-173) disclosesa hot water extract of Nerium oleander and fractionation thereof by liquid chromatography using mixtures of chloroform, methanol, and water. They also report that extracts of the leaves of N. oleander have been used to treat Type II diabetes. US20060188585 published Aug. 24, 2006 to Panyosan discloses a hot water extract of Nerium oleander. US 10323055 issued June 18, 2019 to Smothers discloses a method of extracting plant material with aloe and water to provide an extract comprising aloe and cardiac glycoside. US20070154573 published July 5, 2007 to Rashan et al. discloses a cold-water extract of Nerium oleander and its use.

[0023] Erdemoglu et al. (J. Ethnopharmacol . (2003) Nov. 89(1), 123-129) discloses results for the comparison of aqueous and ethanolic extracts of plants, including Nerium oleander, based upon their anti-nociceptive and anti-inflammatory activities. Fartyal et al. (J. Sci. Innov. Res. (2014), 3(4), 426-432) discloses results for the comparison of methanol, aqueous, and petroleum ether extracts of Nerium oleander based upon their antibacterial activity.

[0024] Organic solvent extracts of Nerium oleander are also disclosed by Adome et al. (Afr. Health Sci. (2003) Aug. 3(2), 77-86; ethanolic extract), el-Shazly et al. (J. Egypt Soc. ParasitoL (1996), Aug. 26(2), 461-473; ethanolic extract), Begum et al. (Phytochemistry (1999) Feb. 50(3), 435-438; methanolic extract), Zia et al. (J. EthnolpharmacoL (1995) Nov. 49(1), 33-39; methanolic extract), and Vlasenko et al. (Farmatsiia. (1972) Sept.-Oct. 21(5), 46-47; alcoholic extract). Turkmen et al. (J. Planar Chroma. (2013), 26(3), 279-283) discloses an aqueous ethanol extract of Nerium oleander leaves and stems. US 3833472 issued Sept. 3, 1974 to Yamauchi discloses extraction of Nerium odorum SOL (Nerium oleander Linn) leaves with water, organic solvent, or aqueous organic solvent, wherein the leaves are heated to 60°-170°C and then extracted, and the organic solvent is methanol, ethanol, propyl ether or chloroform.

[0025] A supercritical fluid extract (PBI-05204) of Nerium species is known (US 8394434, US 8187644, US 7402325) and has demonstrated efficacy in treating neurological disorders (US 8481086, US 9220778, US 9358293, US 20160243143 Al, US 9877979, US 10383886) and cell-proliferative disorders (US 8367363, US 9494589, US 9846156), and some viral infections (US 10596186, WO 2018053123A1, W02019055119A1).

[0026] Triterpenes are known to possess a wide variety of therapeutic activities. Some of the known triterpenes include oleanolic acid, ursolic acid, betulinic acid, bardoxolone,maslinic acid, and others. The therapeutic activity of the triterpenes has primarily been evaluated individually rather than as combinations of triterpenes.

[0027] Triterpenes are found in extracts of Nerium oleander. Addington et al. (US 8481086, US 9220778, US 9358293, US 20160243143 Al) disclose a supercritical fluid extract (SCF; PBI-05204) of Nerium oleander containing oleandrin and triterpenes for the treatment of neurological conditions. Addington et al. (US 9011937, US 20150283191 Al) disclose a triterpene-containing fraction (PB 1-04711) of the SCF extract of Nerium oleander containing oleandrin and triterpenes for the treatment of neurological conditions.

[0028] Oleanolic acid (O or OA), ursolic acid (U or UA) and betulinic acid (B or BA) are the three major triterpene components found in PBI-05204 (PBI-23; a supercritical fluid extract of Nerium oleander) and PBI-04711 (a triterpene-containing fraction 0-4 of PBI- 05204). Van Kanegan et al. previously reported (Nature Scientific Reports (May 2016), 6:25626. doi: 10.1038 / srep25626) on the contribution of the triterpenes toward efficacy by comparing their neuroprotective activity in a brain slice oxygen glucose deprivation (OGD) model assay at similar concentrations. PBI-05204 (PBI) and PBI-04711 (Fraction 0-4) were found to provide neuroprotective activity.

[0029] Extracts of Nerium species are known to contain many different classes of compounds: cardiac glycosides, glycones, steroids, triterpenes, polysaccharides and others. Specific compounds include oleandrin, neritaloside, odoroside, oleanolic acid, ursolic acid, betulinic acid, oleandrigenin, desacetyloleandrin, gitoxigenin, oleaside A, betulin (urs-12- ene-30,28-diol), 28-norurs-12-en-30-ol, urs-12-en-30-ol, 30,3 P-hydroxy-12-oleanen -28- oic acid, 30,2Oa-dihydroxyurs-21-en-28-oic acid, 30,27-dihydroxy-12-ursen-28-oic acid, 30,130-dihydroxyurs-l l-en-28-oic acid, 30,12a-dihydroxyoleanan-28, 130-olide, 30,27- dihydroxy-12-oleanan-28-oic acid, and other components.

[0030] WO 2023 / 022866 Al of Phoenix Biotechnology, Inc. was published on February 23, 2023 and discloses methods and compositions for treating viral infections in animals. WO 2021 / 201903 Al of Phoenix Biotechnology, Inc. was published on Oct. 7, 2021 and discloses methods and compositions for treating viral infections in humans. WO 2021 / 202103 A2 of Phoenix Biotechnology, Inc. was published on Oct. 7, 2021 and discloses methods and compositions for treating viral infections in humans. WO 2018 / 053123 Al of Phoenix Biotechnology, Inc. was published on March 22, 2018 and discloses methods and compositions for treating viral infections in humans. The entire disclosure of each cited publication is hereby incorporated by reference in its entirety.

[0031] Transfer factors, which are produced by leucocytes and lymphocytes, are small water soluble polypeptides of about 44 amino acids that stimulate or transfer cell mediated immunity from one individual to another and across species but do not create an allergic response. Since transfer factors are smaller than antibodies, they do not transfer antibody mediated responses nor do they induce antibody production. The properties of, characteristics of, and processes for obtaining transfer factor or transfer factors are discussed in U.S. Pat. Nos. 4,816,563; 5,080,895; 5,840,700, 5,883,224 and 6,468,534, the entire disclosures of which are hereby incorporated by reference into the present application.

[0032] Transfer factor has been described as an effective therapeutic for Herpes simplex virus (Viza, et al.), a treatment for acne blemishes, U.S. Pat. No. 4,435,384 and as a treatment against C. albicans (Khan et al.). Transfer factor has also been used to treat intestinal cryptosporidiosis in recipients treated with specific transfer factor (McMeeking, et al.). Still, et al. also showed that chicken pox infections were prevented by pretreatment of children treated with transfer factor from individuals that had chicken pox or who in other words had been sensitized to the varicella antigen. The antigen specific transfer factors are the most well studied and have been demonstrated to be able to convey the antigen recognition ability of the experienced donor to the naive recipient. It may be assumed that the individual or animal that is the source of the transfer factor has been sensitized to the antigen of interest. However, transfer factor as found in commercial bovine colostrum extract coming from a pool of animals (e.g., cows) contains the acquired immunity from all of the pool and therefore provides a type of generalized adoptive transfer of immunity. Transfer factors or transfer factor can be obtained from a dialyzable extract of the lysed cells or from an extract of extracellular fluid containing transfer factor. Common sources of transfer factors are colostrum and ova. It is common practice to refer to preparations that contain transfer factor by the name of the active component (i.e., transfer factor or TF). Transfer factor extract containing transfer factors is also herein referred to as transfer factor. Transfer factor from bovine colostrum extract is defined as defatted water soluble material from colostrum that will pass through a nominal 10,000 molecular weight filter. The colostral derived transfer factor has been prepared with activity against various organisms including infectious bovine rhinotracheitis virus. One of the specific effects of transfer factor is a significantly increased natural killer (NK) cell activity. Natural killer cells provide protection against viruses as part of the innate immune defense system.

[0033] Administration of transfer factor, such as bovine colostrum or avian colostrum transfer factor, glucans, and probiotics to animals is known: US 6506413 Bl, issued Jan. 14, 2003, US 6962718 B2, issued Nov. 8, 2005, US 9463218 B2, issued Oct. 11, 2016, US 9610347, issued April 4, 2017, US 9999667, issued June 19, 2018, US 9125874 B2, issued Sept. 8, 2015, US 8357663 B2, issued Jan. 22, 2013, US 9125874 B2, issued Sep. 8, 2015, US 2013 / 0122075 Al, published May 16, 2013, US 2013 / 0142816 Al, published Jun. 6, 2013, US 2013 / 0243829 Al, published Sep. 19, 2013, US 2013 / 0302412 Al, published Nov. 14, 2013, US 2014 / 0205618 Al, published Jul. 24, 2014, US 2015 / 0104425 Al, published Apr. 16, 2015, US 2015 / 0147298 Al, published May 28, 2015, US 2015 / 0174241 Al, published June 25, 2015, US 2015 / 0231188 Al, published Aug. 20, 2015, US 2015 / 0366941 Al, published Dec. 24, 2015, US 2017 / 0224746 Al, published Aug. 10, 2017, and US 2018 / 0000874 Al, published Jan. 14, 2018, the entire disclosures of which are hereby incorporated by reference. Transfer factor has been used for immunotherapy in many different animal species. Even with the availability of transfer factor, glucans, and probiotics, their use in treating at-risk or high-risk sick (no-bid) calves has met with some but limited success. For this reason, it would be very important to develop compositions and methods for improving the effectiveness of therapies based upon transfer factor, glucans, and probiotics.

[0034] The combined use of oleandrin (ov Nerium sp. extract containing oleandrin) with transfer factor, glucan(s), and / or probiotics is not disclosed or suggested in the art. Given the continuing occurrence of deterioration of health in animals and humans, a need remains for compositions suitable as effective treatments and prevention thereof.SUMMARY OF THE INVENTION

[0035] It is an object of the invention to provide compositions and methods for improving GI, respiratory, and / or musculoskeletal health in animals and humans. The invention employs oleandrin-containing compositions, optionally in combination with a) at least one transfer factor; b) at least one glucan; c) a combination of at least one transfer factor and at least one glucan; d) or a combination of at least one transfer factor, at least one glucan, and at least one probiotic. The invention provides at least additive, and preferably synergistic, improvement in performance as compared to either agent alone.

[0036] It is also an object of the invention to provide compositions and methods for increasing feed conversion, reducing the rate of occurrence of mortality and / or morbidity,and reducing the severity morbidity in subjects treated according to the invention. The invention thus provides compositions and methods for increasing feed conversion, reducing the rate of occurrence of mortality and morbidity, and reducing the severity of morbidity in a population of subjects, the invention comprising administering to subjects of said population one or more compositions of the invention in amounts sufficient and at a frequency sufficient to increase feed conversion, reduce the rate of occurrence of mortality and / or morbidity, and / or reduce the severity of morbidity of said subjects as compared to a population of similar subjects not treated according to the invention. The invention is particularly suitable for production animals, e.g. cattle, pigs, sheep, goats, chickens, turkeys, pheasants, quail, etc.

[0037] The inventors have succeeded in preparing compositions that exhibit sufficient health-improving activity to justify their use in treating and preventing health deterioration- related disorders, while at the same time being administered at safe doses that are nontoxic to the animals or humans.

[0038] The inventors have developed corresponding treatment and prevention methods employing particular dosing regimens. The inventors have also developed a cotherapeutic regimen for treating and preventing health deterioration, said cotherapeutic regimen including treating a subject with at least oleandrin-containing composition and another health-improving composition, wherein the compositions can be administered at the same time or separately.

[0039] An aspect of the invention provides a combination composition comprising oleandrin-containing composition in combination with at least one transfer factor, with at least one glucan, with at least one transfer factor and at least one glucan, or with at least one transfer factor, at least one glucan, and at least one probiotic.

[0040] Another aspect of the invention provides a method of improving GI, respiratory, and / or musculoskeletal health in an animal or human subject, the method comprising administering to said subject an oleandrin-containing composition. Another embodiment of the invention provides a method of improving GI, respiratory, and / or musculoskeletal health in an animal or human subject, the method comprising administering to said subject oleandrin-containing composition and at least one other health-improving composition. The other health-improving composition preferably comprises at least one transfer factor, at least one glucan, or a combination thereof.

[0041] In some embodiments, the transfer factor is mammalian colostrum transfer factor. In particular embodiments, the transfer factor is selected from the group consisting of bovine colostrum transfer factor, avian transfer factor, ovine colostrum transfer factor, human colostrum transfer factor, caprine colostrum transfer factor, and a combination of any two or more thereof. In some embodiments, the transfer factor is combination of bovine colostrum transfer factor and avian transfer factor.

[0042] In some embodiments, the bovine colostrum transfer factor is a fraction of colostrum comprising material that will pass through a nominal 10,000 Da filter.

[0043] In some embodiments, the avian transfer factor is present in the composition as powdered whole egg yolks comprising avian transfer factor.

[0044] In some embodiments, the at least one glucan is present in at least one whole fungal organism.

[0045] In other particular embodiments, the concentration of at least one transfer factor in the composition is between about 10 milligrams per ounce and about 12,000 milligrams per ounce, and the concentration of the at least one glucan is between about 10 milligrams of whole organism per ounce and about 18,000 milligrams of whole organism per ounce.

[0046] In some embodiments, the at least one transfer factor is encapsulated with a hydrophobic or lipid coating that is between about 25 and about 150 wt % of the at least one transfer factor.

[0047] Another aspect of the invention provides a method of reducing the occurrence or extent of deterioration of GI, respiratory, and / or musculoskeletal health in a population of animal or human subjects, the method comprising administering to subjects of said population an oleandrin-containing composition in an amount sufficient and for a period sufficient to reduce said occurrence or extent of said deterioration. In some embodiments, said subjects are also administered at least one other health-improving composition.

[0048] In some embodiments, the deterioration of GI health is coincident or synonymous with the occurrence of diarrhea in the subject. The cause of diarrhea may or may not be associated with a disease or infection.

[0049] In some embodiments, said deterioration in health is caused by a parasitic, amebic, bacterial or protozoal infection.

[0050] The invention also provides a method of treating and / or preventing parasitic, amebic, bacterial, or protozoal infection in animals or humans. The invention also provides compositions and methods for treating and / or preventing parasitic, amebic, bacterial, orprotozoal infection-related disorders in animals or humans. The invention also provides a method of treating and / or preventing parasitic, amebic, bacterial, or protozoa infection- related disorder in animals and humans by non-systemic or systemic administration of the composition.

[0051] In some embodiments, the parasitic or protozoal infection is caused by Giardia sp., Coccidia sp., Sarcocystis sp., Eimeria sp., or Cryptosporidium sp.

[0052] In some embodiments, the bacterial infection is caused by Campylobacter sp., Salmonella sp., Escherichia sp., Spirochaeta sp., Treponema sp., Borrelia sp., Leptospira sp., Mycobacterium sp., or Shigella sp.

[0053] The methods of the invention comprise chronically administering to a subject in need thereof one or more doses of oleandrin-containing composition (OCC) on a recurring basis over a treatment period, thereby treating and / or preventing the health deterioration- related disorder in the subject. In some embodiments, the disorder is diarrhea. In production animals, practice of this method provides increased feed conversion, reduced rate of occurrence of mortality and / or morbidity, and / or reduced severity of morbidity of said subjects as compared to a population of similar subjects not treated according to the invention.

[0054] In every embodiment of the invention, the OCC may also be referred to as a cardiac glycoside-containing composition (CGCC). Oleandrin and digoxin are both cardiac glycosides. Unless otherwise specified herein, the CGCC comprises a) oleandrin (excluding digoxin); b) digoxin (excluding oleandrin); or c) a combination of oleandrin and digoxin. DCC refers to a digoxin-containing composition. In preferred embodiments, the OCC of the invention comprises at least oleandrin.

[0055] Administration of the OCC can be a) one or more times daily; b) at least three days per week; c) at least five days per week; d) every day of the week; or e) five days on and two days off. A treatment period can last at least three days, at least five days, at least one week, at least 10 days, at least two weeks, at least three weeks, at least one month, at least two months, or longer. One or more doses are administered on a daily, weekly, and / or monthly basis. One or more doses per day can be administered. One, two, three, four, five, six or more doses can be administered per day. The composition can be administered chronically, i.e. on a recurring basis, such as daily, every other day, every second day, every third day, every fourth day, every fifth day, every sixth day, weekly, every other week, every second week, every third week, monthly, bimonthly, semi-monthly, every other monthevery second month, quarterly, every other quarter, trimesterly, seasonally, semi-annually and / or annually. The treatment period may be for one or more weeks, one or more months, one or more quarters and / or one or more years. An effective dose of OCC can be administered one or more times a day.

[0056] Chronic administration can be achieved by repeated administration of dosage form(s), feed, feed supplement s), liquid(s), solution(s), suspension(s), paste(s), or other composition(s). Suitable oral dosage forms include rapid or immediate release dosage form(s), delayed release, and / or extended (controlled, sustained, prolonged) release dosage form(s). The composition is preferably administered systemically, and even more preferably perorally, orally, buccally, anally, vaginally, perianally, and / or sublingually.

[0057] The subject can be an animal or human. The animal can be a domestic or commercial animal, e.g. dog, cat, cow, horse, sheep, goat, pig, llama, alpaca, buffalo, deer, elk, giraffe, camel, chicken, turkey, or other feather-bearing, hair-bearing, or fur-bearing animal. In preferred embodiments, the subject is a human, dog, cat, horse, cow, pig, sheep, goat, chicken, or turkey.

[0058] An aspect of the invention provides a health-improving composition comprising a) oleandrin; and b) one or more excipients. Another aspect of the invention provides a health-improving combination composition comprising oleandrin, one or more excipients, and at least one other health-improving agent. Another aspect of the invention provides a health-improving combination composition comprising oleandrin, one or more excipients, and at least one transfer factor. Another aspect of the invention provides a health-improving combination composition comprising oleandrin, one or more excipients, and at least one glucan. Another aspect of the invention provides a health-improving combination composition comprising oleandrin, one or more excipients, at least one transfer factor, and at least one glucan. The excipients are acceptable for animal and / or human use.

[0059] In some embodiments, the at least one health-improving agent is selected from the group consisting of at least one transfer factor, at least one glucan, and at least one probiotic.

[0060] An aspect of the invention provides a method of preventing or treating a protozoa infection in a subject, the method comprising administering to a subject one or more doses of OCC, wherein the protozoa is selected from the group consisting of Giardia sp., Cryptosporidium sp., Sarcocystis sp., and Coccidia sp.

[0061] Another aspect of the invention provides a method of preventing or treating a protozoa infection related disorder in a subject, the method comprising administering to a subject one or more doses of OCC, wherein the protozoa is selected from the group consisting of Giardia sp., Cryptosporidium sp., Sarcocystis sp., and Coccidia sp.

[0062] In some embodiments, the Giardia sp. is selected from the group consisting of Giardia duodenalis, Giardia canis, Giardia enterica, Giardia intestinalis, Giardia lamblia, Giardia muris, Giardia ardeae, and Giardia psittaci.

[0063] In some embodiments, the Coccidia sp. is selected from the group consisting of Isospora species (sometimes called Cystoisospora) Hammondia sp., Besnoitia sp., Toxoplasma sp., Sarcocystis sp., Isospora felis, Isospora rivolta, Sarcocystis sp, Isospora burrowsi, Isospora canis, Isospora neorivolta, and Isospora ohioensis.

[0064] In some embodiments, the Eimeria sp. is selected from the group consisting of Gallus gallus domesticus (in chickens; especially E. tenella, E. acervuline, E. necatrix, and E. maxima), Meleagris gallopavo (in turkeys), Phasianus colchicus (in pheasants), Capra hircus (in goats), Ovis aries (in sheep), Bos taurus (in cattle; especially, E. bovis, E. zuernii, E. ellipsoidalis, andE. auburnensis), or Oryctolagus cuniculus (in rabbits).

[0065] In some embodiments, the health deterioration-related disorder is selected from the group consisting of diarrhea, gas, foul-smelling greasy feces, stomach cramps or pain, intestinal cramps or pain, abdominal distress, vomiting, upset stomach, nausea, fatigue, fever, itchy skin, hives, and swelling of the eyes and joints. In some embodiments, the health deterioration-related disorder is not associated with a disease.

[0066] In some embodiments, the subject may be treated with one or more of the following in addition to the OCC: a) one or more compounds extracted from Nerium oleander plant material; b) one or more triterpenoid acids, e.g. one or more of oleanolic acid, ursolic acid, or betulinic acid; c) one or more deworming medications; d) one or more antibiotics; e) one or more antioxidants; f) one or more solvents; g) one or more oils; h) one or more compounds selected from the group consisting of ronidazole, tinidazole, metronidazole, nimorazole, flunidazole, dimetridazole, ipronazole, fenbendazole, albendazole, antidiarrheic, pyrantel, ponazuril, a 5-nitroimidazole antibiotic, sulfa-type antibiotic (e.g. sulfadimethoxine), amprolium (Albac / Amprol Hi-E [+ Bacitracin, + Ethopabate]), ponazuril, toltrazuril, ionophore(s) (e.g. rumensin), sulfaquinoxaline, sulfamethazine, lasalocid, decoquinate, rumensin (monensin as salt or free acid), and nitazoxanide; i) one or more antidiarrheic compounds; j) one or more preservatives; k) oneor more feed materials; 1) one or more nutritional materials; m) one or more surfactants; n) one or more anti-foaming agents; o) one or more lubricants; p) one or more corticosteroids (glucocorticoids); q) collagen, e.g. hydrolyzed collagen; r) one or more carboxylic acids; s) one or more amino acids; t) one or more humectants; u) one or more buffering agents; v) one or more proteins; or w) one or more vaccines. The above may or may not be included in the same composition as the OCC. The above may be administered simultaneously, sequentially, or in an overlapping manner with the OCC. The dosing regimen of the above may be the same as or different than the dosing regimen for the OCC.

[0067] Embodiments of the invention include those wherein one or more compounds in the composition is / are present in free base form, free acid form, and / or salt form. Some of the above components also possess anti-protozoa activity.

[0068] In some embodiments, a) the one or more oils is selected from the group consisting of bitter orange oil, chamomile oil, clove oil, silicone oil (dimethicone; polydimethylsiloxane), coconut oil, medium chain triglyceride (MCT), fractionated MCT, and others; b) the one or more solvents is selected from the group consisting of water, caprylyl glycol, hexylene glycol, phenoxyethanol, ethanol, glycerin (glycerol), propylene glycol, and others; c) the one or more antioxidants is selected from the group consisting of tocopherol (vitamin E), and others; and / or d) the one or more carboxylic acids is selected from the group consisting of pyrrolidine carboxylic acid (PCA), ascorbic acid, citric acid, acetic acid, salicylic acid, and others.

[0069] Another aspect of the invention provides a cotherapeutic method of treating or preventing diarrhea, the method comprising administering to a subject one or more doses of OCC and one or more doses of one or more of the following agents: a) one or more deworming agents; b) one or more antidiarrheic agents; c) one or more anti-protozoa agents; d) one or more antibiotics; e) one or more probiotics; f) one or more transfer factors; g) one or more glucans; h) one or more antibacterials; or i) one or more antimicrobials. The established doses of the agents are suitable for use according to the invention in combination with the OCC. In some embodiments, the dose of the agents may be reduced because of the additive or synergistic anti-protozoa activity of cardiac glycoside, in particular oleandrin.

[0070] The oleandrin and other agents (compositions) may be administered a) in the same composition or in separate compositions; b) via the same route of administration or via different routes of administration; c) simultaneously, sequentially, or in an overlapping manner; and / or d) with overlapping dosing periods or separate dosing periods.

[0071] Recommended dosages for metronidazole in dogs and cats are indication specific: giardiasis: 25 mg / kg, PO (peroral), every 12 hours for 5 days; inflammatory GI conditions or inflammatory bowel disease (IBD): 10-15 mg / kg, PO, every 12 hours; hepatic encephalopathy: 7.5 mg / kg, PO, every 8-12 hours. Small animals with hepatic dysfunction require lower dose rates of 15-20 mg / kg, PO, every 24 hours. In horses, dosages of 15-25 mg / kg, PO, every 6-8 hours are recommended. Neonatal foals (< 2 weeks of age) require lower doses of 10 mg / kg, PO, every 12 hours.

[0072] Trimethoprim-sulfonamide (30-60 mg / kg per day for 6 days; halve dose for small dogs) can be used in clinically infected subjects. Sulfonamides, such as sulfadimethoxine (50 mg / kg the first day and 25 mg / kg per day for 1-3 weeks thereafter or 50 mg / kg until 2- 3 days after clinical signs resolve), can be used in severe cases. Triazine antiprotozoal s, including diclazuril and toltrazuril, may be effective. Diclazuril (5 mg / kg, PO, once) is effective in cats. Ponazuril (20 to 50 mg / kg, PO, for 2 to 5 days) is effective in dogs and cats.

[0073] The recommended dose for a) ronidazole is about 30 mg / kg once or twice daily in cats and 10 mg / kg / day in dogs; b) tinidazole is about 10-30 mg / Kg once or twice daily; c) nimorazole is about 10-30 mg / Kg once or twice daily; d) flunidazole is about 5-10 mg / kg peroral once or twice daily; e) dimetridazole is about 20-25 mg / kg peroral once or twice daily; f) fenbendazole is about 50 mg / kg once or twice daily; g) for albendazole is about 25 mg / kg peroral once or twice daily; h) pyrantel is about 5-10 mg / kg once or twice daily; and i) ponazuril is about 7.5-50, about 20-50, or about 7.5-15 mg / kg peroral once or twice daily.

[0074] In some embodiments, a) the OCC and other composition are both administered systemically; b) the OCC is administered non-systemically, and the other composition is administered systemically; c) the OCC is administered systemically, and the other composition is administered non-systemically; or d) the OCC is administered orally, buccally, anally, vaginally, perianally, and / or sublingually, and the other composition is administered orally, buccally, anally, vaginally, perianally, and / or sublingually.

[0075] One or more excipients or other compounds as described herein can be further included in the compositions of the invention.

[0076] Another aspect of the invention provides a method of preventing an animal from exhibiting one or more symptoms associated with protozoal infection, amebic infection, bacterial infection, or parasitic infection, the method comprising administering to said animal one or more effective doses of oleandrin-containing composition (OCC). Themethod optionally further comprises administering one or more other anti-protozoa compositions. The composition(s) may be administered to prevent occurrence or recurrence of protozoa infection related disease or disorder in the animal. For example, the composition(s) may be administered prior to the animal exhibiting symptoms of the disease (prevention of occurrence) or as a maintenance therapy after disease in the animal has already been resolved (prevention of recurrence). In some embodiments, the one or more symptoms is at least diarrhea.

[0077] A subject infected with protozoa may or may not exhibit related symptoms, meaning the subject may be symptomatic or asymptomatic. The invention is useful for treating symptomatic and asymptomatic subjects and for preventing infections in subjects.

[0078] Administration of one or more effective doses of an oleandrin-containing composition to a human or animal subject may help a) maintain healthy skin; b) maintain healthy coat in an animal; c) support coat health in an animal; d) support skin health in an animal; e) support a healthy response to seasonal skin allergy; f) support the immune system; g) assist the immune system; h) enhance the immune response; i) provide immune enhancing effects; j) provide nutritional support for healthy immune system; k) support a healthy immune system; 1) support immune system function; m) support and promote longterm health; n) promote the body’s innate resistance to pathogens; o) reduce oxidative stress; p) provide antioxidant support; q) help maintain a normal inflammatory response; r) maintain a normal inflammatory cytokine cascade; s) maintain or improve GI health; t) maintain or improve respiratory health; u) maintain or improve musculoskeletal health; or v) support a healthy immune response.

[0079] The equivalent of plural daily doses of oleandrin can be achieved by administering to said animal one or more extended-release dosage forms that release therapeutically effective daily doses of oleandrin throughout a treatment period. Additional means of administering effective daily doses may be achieved through use of dosage forms suitable for use in water, milk, liquid feed, milk substitute, colostrum, colostrum substitute, edible treat, paste, suppository, implant, bolus, or solid feed.

[0080] The invention also provides a method of treating or preventing protozoa infection-related disease in an animal, the method comprising administering to the animal 1-10 doses of oleandrin-containing composition per day for a treatment period of at least 2 days up to about 9 months. One to eight, two to six, or two to four doses can be administered daily during the treatment period. Doses can be administered for 2 days to about 60 days,2 days to about 45 days, 2 days to about 30 days, 2 days to about 21 days, or 2 days to about 14 days. Said administering can be through any of the modes of administration discussed herein.

[0081] The invention also provides a method of reducing fetal mortality in a population of animals, the method comprising administering OCC of the invention to a pregnant animal (according to any of the dosing protocols or regimens described herein), thereby reducing the occurrence of fetal mortality in the pregnant animal. The reduction of fetal mortality can be as determined by comparison to a population of untreated pregnant animals. In particular embodiments, the reduction in fetal mortality is a reduction in aborted fetuses, stillborn fetuses, and / or mummified fetuses. In particular embodiments, before said administering, the pregnant animal is seropositive for a viral infection during at least the latter part of the animal’s gestation period. In some embodiments, the method of invention excludes embodiments wherein, before said administering, the animal is seropositive for a viral infection during at least the latter part of the animal’s gestation period. In some embodiments of the invention, the animal is seronegative for viral infection prior to said administering. In particular embodiments, the animal is a pig, cow, horse, sheep, or goat.

[0082] In some embodiments, the OCC is administered to one or more puppies according to the following schedule, wherein the number of days is relative to the number of days after birth of the pup: a) starting a day-2 and for 2 weeks: 2 doses per week (doses spaced apart by 3-4 days); b) starting at about 3 weeks of age: 1-7 doses per week; and c) starting at about 4 weeks of age and continuing as needed, e.g. for up to 9 weeks of age, up to 12 weeks of age, or up to 16 weeks of age: 2-7 doses per week.

[0083] An oleandrin-containing composition comprises oleandrin, which may be provided in pure form, impure form, or as part of an extract of Nerium sp. plant material(s), wherein the extract further comprises one or more components extracted from said plant material(s), said one or more components selected from those described herein.

[0084] In some embodiments, an oral composition further comprises at least one cardiac glycoside-metabolism inhibitor, at least one cardiac glycoside-digestion inhibitor, at least one enzyme inhibitor, or a combination thereof.

[0085] A veterinary clinician will be able to use known dose escalation or de-escalation protocols to determine a safe and effective dose of oleandrin to be administered to a subject. The dose or dosing regimen can be adjusted as needed until the subject reaches the desiredclinical endpoint(s) such as a reduction, alleviation, or elimination of specific health deterioration-related symptoms.

[0086] The oral maximum tolerated dose (MTD) of oleandrin may vary according to animal species. In some embodiments, a) said animal is a cow and the dose of composition provides a maximum plasma concentration of oleandrin of no more than 5 or no more than 10 ng / mL; b) said animal is a pig and the dose of composition provides a maximum plasma concentration of oleandrin of no more than 5 or no more than 10 ng / mL; c) said animal is a horse and the dose of composition provides a maximum plasma concentration of or oleandrin of no more than 5 or no more than 10 ng / mL; d) said animal is a sheep and the dose of composition provides a maximum plasma concentration of oleandrin of no more than 5 or no more than 10 ng / mL; e) said animal is a goat and the dose of composition provides a maximum plasma concentration of oleandrin of no more than 5 or no more than 10 ng / mL; or f) said animal is a dog and the dose of composition provides a maximum plasma concentration of oleandrin of no more than 5 or no more than 10 ng / mL.

[0087] Suitable target oral doses (one to four times daily) for oleandrin in animals are as follows: a) in dogs- less than about 100 microg / kg bodyweight, about 0.5-60 microg / kg bodyweight, about 0.1-10 microg / kg bodyweight, or about 0.1-5 microg / kg bodyweight; b) in cows, pigs, goats, or sheep- less than about 100 micro / kg bodyweight, about 0.1-50 microg / kg bodyweight, about 0.1-25 microg / kg bodyweight, about 0.1-10 microg / kg bodyweight, or about 0.1-5 microg / kg bodyweight; c) in cats- less than about 100 microg / kg bodyweight, about 0.5-50 microg / kg bodyweight, about 0.1-10 microg / kg bodyweight, or about 0.1-5 microg / kg bodyweight; d) in horses- less than about 100 microg / kg bodyweight or 0.5-50 microg / kg body weight; e) in poultry (chickens, turkeys, etc.)- less than about 1000 micro / kg bodyweight, about 0.5-500 microg / Kg bodyweight, about 0.5-250 microg / Kg bodyweight, about 0.5-100 microg / Kg bodyweight, about 0.5-50 microg / Kg bodyweight, about 0.5-25 microg / Kg bodyweight, about 0.5-10 microg / Kg bodyweight, or about 0.5-5 microg / Kg bodyweight.

[0088] Where oleandrin is administered in the form of Nerium species (Nerium sp.), e.g. Nerium oleander or Nerium indicum. leaf material, the amount of dried leaf material will preferably be a) less than 100 mg / Kg bodyweight or less than 50 mg / Kg bodyweight for a cow; b) less than 110 mg / Kg body weight for a goat; c) less than 110 mg / Kg body weight or less than 250 mg / Kg bodyweight for a sheep. The amount of leaf material may be adjusted further according to the oleandrin content of the leaf material on a dry weight basis. Thehigher the oleandrin content of the leaf material, the lower the amount of leaf material required on a dry weight basis. Depending upon the variety of Nerium sp., the content of oleandrin in the leaf material can be in the range of about 250-6500 microg OLE / g, about 250-5500 microg OLE / g, about 250-4500 microg OLE / g, about 250-3500 microg OLE / g, about 250-2500 microg OLE / g, or about 250-1500 microg OLE / g, on a dry weight basis (DWB). The content of OLE can be determined by conducting exhaustive solvent extraction, e.g. according to Example 21, optionally with a Soxhlet extractor and then analyzing the extract by HPLC, e.g. according to Example 8.

[0089] In some embodiments, following oral administration, the concentration of oleandrin in the plasma of a treated subject is about 10 ng / mL or less, about 5 ng / mL or less, about 2.5 ng / mL or less, about 2 ng / mL or less, about 1 ng / mL, or about 0.5 ng / mL or less. In some embodiments, following oral administration, the concentration of oleandrin in the plasma of a treated animal is about 0.0001 ng / mL or more, about 0.0005 ng / mL or more, about 0.001 ng / mL or more, about 0.0015 ng / mL or more, about 0.01 ng / mL or more, about 0.015 ng / mL or more, about 0.1 ng / mL or more, about 0.15 ng / mL or more, about 0.05 ng / mL or more, or about 0.075 ng / mL or more. The daily oral dose of composition administered will be sufficient to provide a plasma concentration of oleandrin within at least one of the ranges set forth herein. The invention includes all combinations and selections of the plasma concentration ranges set forth herein.

[0090] Depending upon the subj ect’ s bodyweight, the dose of oleandrin can also be about 0.5 to about 500 microg / day or less, about 0.5 to about 400 microg / day or less, about 0.5 to about 300 microg / day or less, about 0.5 to about 200 microg / day or less, about 0.5 to about 100 microg / day or less, about 1 to about 80 microg / day, about 1.5 to about 60 microg / day, about 1.8 to about 60 microg / day, about 1.8 to about 40 microg / day.

[0091] All dosing regimens, dosing schedules, and doses described herein are contemplated as being suitable; however, some dosing regimens, dosing schedules, and doses may be more suitable for some subjects than for others. The target clinical endpoints are used to guide said dosing.

[0092] The composition can be administered systemically. Modes of systemic administration include parenteral, buccal, enteral, intramuscular, subdermal, sublingual, peroral, pulmonary, or oral. The composition can also be administered via injection or intravenously. The composition may also be administered by two or more routes to the same subject. In some embodiments, the composition is administered by a combination ofany two or more modes of administration selected from the group consisting of parenteral, buccal, enteral, intramuscular, subdermal, sublingual, peroral, pulmonary, intranasal, anal, rectal, vaginal, perianal, and oral.

[0093] When administered to production animals, it may be preferable to administer the composition in its food and / or water supply, i.e. where the composition is added as a supplement to the supply. The oleandrin may be included in a feed and / or a liquid and administered orally to the animal. The solid feed may comprise oleandrin and at least one feedstuff. The liquid feed may comprise oleandrin, at least one liquid, and at least one nutrient. Oleandrin may also be administered in a milk substitute product or in water. The oleandrin may also be administered to the animal by feeding the animal plant material, e.g. leaves and / or stems, from the Nerium sp. plant. The plant material may be dried or undried. In preferred embodiments, the plant material has been reduced in size to less than 1” in greatest length.

[0094] The invention also provides a sublingual / buccal dosage form comprising oleandrin and liquid carrier. The invention also provides a method of treating protozoal infection related disease or disorder comprising sublingually administering plural doses of an oleandrin-containing composition to an animal having said disease or disorder. The invention also provides a method of preventing protozoal infection related disease or disorder comprising sublingually administering plural doses of an oleandrin-containing composition to an animal prone to exhibiting symptoms of said infection. One or more doses can be administered per day for two or more days per week and for one or more weeks per month, optionally for one or months per year. The liquid carrier can comprise water, oil, liquid feed, or a combination of any thereof.

[0095] In some embodiments, the composition comprises oleandrin and oil. The oil can comprise medium chain triglycerides (MCT). The composition can comprise one, two or more oleandrin-containing extracts and one or more pharmaceutical excipients.

[0096] In some embodiments, the oleandrin-containing composition comprises an extract of Nerium sp., said extract comprising a) at least oleandrin; b) at least oleandrin, oleanolic acid, ursolic acid, and betulinic acid; c) at least oleandrin, oleanolic acid, ursolic acid, betulinic acid, oleandrigenin, desacetyl-oleandrin, and gitoxigenin; d) at least oleandrin, oleanolic acid, ursolic acid, betulinic acid, oleandrigenin, desacetyl-oleandrin, gitoxigenin, kanerocin, and kanerodione; or e) at least at least oleandrin, oleanolic acid,ursolic acid, betulinic acid, oleandrigenin, gitoxigenin, desacetyl-oleandrin, kanerocin, kanerodione, Nerium F, neritaloside, odoroside, adynerin, and odoroside-G-acetate.

[0097] The oleandrin-containing composition (or the extract) may further comprise polyphenol(s), carbohydrate(s), flavonoid(s), amino acid(s), soluble protein(s), cellulose, starch, alkaloid(s), saponin(s), tannin(s), and any combination thereof.

[0098] The amino acid can be selected from the group consisting of aspartic acid, glutamic acid, asparagine, serine, glutamine, glycine, histidine, arginine, threonine, alanine, proline, tyrosine, valine, methionine, cysteine, isoleucine, leucine, phenylalanine, tryptophan, and lysine. In some embodiments, the amino is selected from the group consisting of asparagine, arginine, threonine, alanine, proline, tyrosine, valine, isoleucine, leucine, phenylalanine, tryptophan, and lysine.

[0099] The aglycone oleandrigenin can also be further included. In some embodiments, the composition further comprises a) one or more triterpenes; b) one or more steroids; c) one or more triterpene derivatives; d) one or more steroid derivatives; or e) a combination thereof. In some embodiments, the composition comprises oleandrin and a) two or three triterpenes; b) two or three triterpene derivatives; c) two or three triterpene salts; or d) a combination thereof. In some embodiments, the triterpene is selected from the group consisting of oleanolic acid, ursolic acid, betulinic acid, and salts or derivatives thereof. As used herein, the generic term triterpene also encompasses salts and derivatives thereof, unless otherwise specified.

[0100] The oleandrin can be present in a pharmaceutical composition in pure form or as part of an extract containing oleandrin or as a plant material containing oleandrin. In some embodiments, the oleandrin is present as the primary therapeutic component, meaning the component primarily responsible for antipruritic activity of the composition.

[0101] In some embodiments, an oleandrin-containing extract is obtained by extraction of plant material. The extract can comprise a hot-water extract, cold-water extract, supercritical fluid (SCF) extract, subcritical liquid (SbCL) extract, organic solvent extract, aqueous organic solvent extract, or combination thereof of the plant material. In some embodiments, the extract has been (biomass) prepared by subcritical liquid extraction of Nerium plant mass (biomass) using, as the extraction fluid, subcritical liquid carbon dioxide, optionally comprising alcohol. In some embodiments, the oleandrin-containing composition comprises two or more different types of oleandrin-containing extracts.

[0102] Embodiments of the invention include those wherein the oleandrin-containing biomass (plant material) is Nerium sp., e.g. Nerium oleander, Nerium oleander L (Apocynaceae), Nerium odouriim, Nerium indicum Mill, white oleander, pink oleander, red oleander, any variant of Nerium sp., Agrobacterium lumefaciens, cell culture (cellular mass) of any of said species, or a combination thereof. In some embodiments, the plant material (biomass) comprises leaves, stems, flowers, bark, fruits, seeds, sap, and / or pods. In preferred embodiments, the plant material comprises a) leaves; b) stems; c) flowers; d) buds; e) seeds; f) leaves and stems; g) leaves, stems, and flowers; h) leaves, stems, and buds; or i) a combination of any two or more of the above.

[0103] In some embodiments, the molar ratio of total triterpene content (OA + UA + BA) to oleandrin ranges from about 15: 1 to about 5: 1, or about 12: 1 to about 8: 1, or about 100: 1 to about 15: 1, or about 100: 1 to about 50: 1, or about 100: 1 to about 75: 1, or about 100: 1 to about 80: 1, or about 100: 1 to about 90: 1, or about 10: 1.

[0104] In some embodiments, the molar ratios of the individual triterpenes to oleandrin range as follows: about 2-8 (OA) : about 2-8 (UA) : about 0.1-1 (BA) : about 0.5-1.5 (OL); or about 3-6 (OA) : about 3-6 (UA) : about 0.3-8 (BA) : about 0.7-1.2 (OL); or about 4-5 (OA) : about 4-5 (UA) : about 0.4-0.7 (BA) : about 0.9-1.1 (OL); about 4.6 (OA) : about 4.4 (UA) : about 0.6 (BA) : about 1 (OL); about 9-12 : up to about 2 : up to about 2, or about 10 : about 1 : about 1, or about 9-12 : about 0.1-2 : about 0.1-2, or about 9-11 : about 0.5- 1.5 : about 0.5-1.5, or about 9.5-10.5 : about 0.75-1.25 : about 0.75-1.25, or about 9.5-10.5 : about 0.8-1.2 : about 0.8-1.2, or about 9.75-10.5 : about 0.9-1.1 : about 0.9-1.1.

[0105] In some embodiments, the other therapeutic agent, such as that obtained by extraction of Nerium sp. plant material, is not a polysaccharide obtained during preparation of the extract, meaning it is not an acidic homopolygalacturonan or arabinogalaturonan. In some embodiments, the extract excludes another therapeutic agent and / or excludes an acidic homopolygalacturonan or arabinogalaturonan obtained during preparation of the extract.

[0106] In some embodiments, the method includes administration of an aloe extract of Nerium sp., esp. Nerium oleander, and the composition includes an aloe extract of Nerium oleander, wherein the extract has been prepared by extraction of Nerium oleander biomass with aloe vera gel, e.g. has been prepared according to the process described in US 10323055 B2, which issued June 18, 2019 and which was filed July 17, 2013 as USSN 13 / 944720, US 8524286 B2, which issued Sept. 3, 2013 and which was filed Oct. 13, 2009 as USSN 12 / 578436, and WO 2010045243 Al, which published April 22, 2010. In someembodiments, the method includes administration of egg membrane and of an aloe extract of Nerium oleander, and the composition includes egg membrane and includes the aloe extract of Nerium oleander, wherein the extract has been prepared by extraction of Nerium oleander biomass with aloe vera gel.

[0107] In some embodiments, the other therapeutic agent, such as that obtained by extraction of Nerium sp. plant material, is a polysaccharide obtained during preparation of the extract, e.g. an acidic homopolygalacturonan or arabinogalaturonan. In some embodiments, the extract comprises another therapeutic agent and / or comprises an acidic homopolygalacturonan or arabinogalaturonan obtained during preparation of the extract from said plant material.

[0108] In some embodiments, the extract comprises oleandrin and at least one other compound selected from the group consisting of glycone, aglycone, steroid, triterpene, polysaccharide, saccharide, alkaloid, fat, protein, neritaloside, odoroside, oleanolic acid, ursolic acid, betulinic acid, oleandrigenin, gitoxigenin, desacetyl-oleandrin, oleaside A, betulin (urs-12-ene-30,28-diol), 28-norurs-12-en-30-ol, urs-12-en-30-ol, 30,30-hydroxy- 12-oleanen-28-oic acid, 3 P,20a-dihydroxyurs-21-en -28-oic acid, 30,27-dihydroxy-12- ursen-28-oic acid, 30,130-dihydroxyurs-l l-en-28-oic acid, 30,12a-dihydroxyoleanan- 28, 130-olide, 30,27-dihydroxy-12-oleanan-28-oic acid, homopolygalacturonan, arabinogalaturonan, chlorogenic acid, caffeic acid, L-quinic acid, 4-coumaroyl-CoA, 3-O- caffeoylquinic acid, 5- O-caffeoylquinic acid, cardenolide B-l, cardenolide B-2, oleagenin, neridiginoside, nerizoside, odoroside-H, 3-beta-O-(D-diginosyl)-5-beta, 14 beta-dihydroxy - card-20(22)-enolide pectic polysaccharide composed of galacturonic acid, rhamnose, arabinose, xylose, and galactose, polysaccharide with MW in the range of 17000-120000 D, or MW about 35000 D, about 3000 D, about 5500 D, or about 12000 D, cardenolide monoglycoside, cardenolide N-l, cardenolide N-2, cardenolide N-3, cardenolide N-4, pregnane, 4,6-diene- 3,12,20-trione, 20R-hydroxypregna-4,6-diene-3, 12-dione, 16beta,17beta-epoxy-12beta-hydroxypregna-4,6-diene-3, 20-dione, 12beta-hydroxypregna- 4,6, 16-triene-3, 20-dione (neridienone A), 20S,21-dihydroxypregna-4,6-diene-3, 12-dione (neridienone B), neriucoumaric acid, isoneriucoumaric acid, oleanderoic acid, oleanderen, 8alpha-methoxylabdan- 18-oic acid, 12-ursene, kaneroside, neriumoside, 30-( -(D- diginosyl)-2a- hydroxy-8, 140-epoxy-5P-carda-16: 17, 20: 22- dienolide, 3P-( -(D- diginosyl)-2a,14P- dihydroxy-5P- carda-16:17,20:22-dienolide, 3p,27-dihydroxy-urs-18- en-13, 28-olide, 3p,22a,28-trihydroxy-25-nor-lup-l(10),20(29)-dien-2-one, c / .s-karenin (3P-hydroxy-28-Z-p-coumaroyloxy-urs-12-en-27-oic acid), / ra / z.s-karenin (3-P-hydroxy-28-E- p-coumaroyloxy-urs-12-en-27-oic acid), 3beta-hydroxy-5alpha-carda- 14(15), 20(22)- di enolide (beta- anhydroepidigitoxigenin), 3 beta-O-(D-digitalosyl)-21-hydroxy-5beta- carda-8, 14,16,20(22)-tetraenolide (neriumogenin- A-3beta-D-digitaloside), proceragenin, neridienone A, 3beta,27-dihydroxy-12-ursen -28-oic acid, 3beta,13beta-dihydroxyurs-l 1- en-28-oic acid, 3beta-hydroxyurs-12-en-28-aldehyde, 28- orurs-12-en-3beta-ol, urs-12-en- 3beta-ol, urs-12-ene-3beta,28-diol, 3beta,27-dihydroxy-12-oleanen-28-oic acid, (20S, 24R)-epoxydammarane-3beta,25-diol, 20beta,28-epoxy-28alpha-methoxytaraxasteran- 3beta-ol, 20beta,28-epoxytaraxaster-21-en-3beta-ol, 28-nor-urs-12-ene-3beta,17 beta-diol, 3beta-hydroxyurs-12-en-28-aldehyde, alpha-neriursate, beta-neriursate, 3 alphaacetophenoxy -urs-12-en-28-oic acid, 3beta-acetophenoxy-urs-12-en-28-oic acid, oleanderolic acid, kanerodione, 3 P- / ?-hydroxyphenoxy- l la-m ethoxy- 12a-hydroxy-20- ursen-28-oic acid, 28-hydroxy-20(29)-lupen-3, 7-dione, kanerocin, 3 alpha-hydroxy -urs- 18,20-dien-28-oic acid, D-sarmentose, D-diginose, neridiginoside, nerizoside, isoricinoleic acid, gentiobiosylnerigoside, gentiobiosylbeaumontoside, gentiobiosyloleandrin, folinerin, 12P-hydroxy-5P-carda-8, 14, 16,20(22)-tetraenolide, 8P-hydroxy-digitoxigenin, A16-8P- hydroxy-digitoxigenin, A16-neriagenin, uvaol, ursolic aldehyde, 27(p- coumaroyloxy)ursolic acid, oleanderol, 16-anhydro-deacteyl-nerigoside, 9-D-hydroxy-cis- 12-octadecanoic acid, adigoside, adynerin, alpha-amyrin, beta-sitosterol, campestrol, caoutchouc, capric acid, caprylic acid, choline, cornerin, cortenerin, deacetyloleandrin, diacetyl-nerigoside, foliandrin, pseudocuramine, quercetin, quercetin-3 -rhamnoglucoside, quercitrin, rosaginin, rutin, stearic acid, stigmasterol, strospeside, urehitoxin, and uzarigenin. Additional components that may be present in the extract are disclosed by Gupta et al. (IJPSR (2010(, 1(3), 21-27, the entire disclosure of which is hereby incorporated by reference).

[0109] Oleandrin may also be obtained from extracts of suspension cultures derived from Agrobacterium tumefaci ens -transformed calli (Ibrahim et al., “Stimulation of oleandrin production by combined Agrobacterium tumefaciens mediated transformation and fungal elicitation in Nerium oleander cell cultures” in Enz. Microbial Techno. (2007), 41(3), 331- 336, the entire disclosure of which is hereby incorporated by reference). Hot water, organic solvent, aqueous organic solvent, subcritical liquid extract, or supercritical fluid extract of agrobacterium may be used according to the invention.

[0110] Oleandrin may also be obtained from extracts of Nerium sp. microculture in vitro, whereby shoot cultures can be initiated from seedlings and / or from shoot apices of the Nerium sp. cultivars, e.g. Splendens Giganteum, Revanche or Alsace, or other cultivars (Vila et al., “Micropropagation of Oleander (Nerium oleander L.)” in HortScience (2010), 45(1), 98-102, the entire disclosure of which is hereby incorporated by reference). Hot water, organic solvent, aqueous organic solvent, or supercritical fluid extracts of microcultured Nerium sp. may be used according to the invention.

[0111] The extract may also be obtained by extraction of cellular mass (such as is present in cell culture) of any of said Nerium species.

[0112] The invention also provides use of OCC in the manufacture of a medicament for the treatment of protozoal, amebic, parasitic, or bacterial infection related disease or disorder or for improvement of GI health, musculoskeletal health, and / or respiratory health in an animal or human. The invention also provides use of OCC in the manufacture of a medicament for increasing feed conversion, reducing the rate of occurrence of mortality and morbidity, and reducing the severity of morbidity in a population of subjects, in particular animals. In some embodiments, the manufacture of such a medicament comprises: providing one or more compositions of the invention; including a dose of said composition in a pharmaceutical dosage form; and packaging the pharmaceutical dosage form. In some embodiments, the manufacture can be conducted as described in PCT International Application No. PCT / US06 / 29061. The manufacture can also include one or more additional steps such as: delivering the packaged dosage form to a vendor (retailer, wholesaler and / or distributor); selling or otherwise providing the packaged dosage form to an animal having protozoal, amebic, parasitic, or bacterial infection related disease or disorder or poor GI health, musculoskeletal health, and / or respiratory health; including with the medicament a label and a package insert, which provides instructions on use, dosing regimen, administration, content and toxicology profile of the dosage form. In some embodiments, the treatment of protozoal, amebic, parasitic, or bacterial infection related disease or disorder and improvement of GI health, musculoskeletal health, and / or respiratory health comprises: determining that a subject has protozoal, amebic, parasitic, or bacterial infection related disease or disorder or poor GI health, musculoskeletal health, or respiratory health; indicating administration of pharmaceutical dosage form to the subject according to a dosing regimen; administering to the subject one or more pharmaceuticaldosage forms, wherein the one or more pharmaceutical dosage forms is administered according to the dosing regimen.

[0113] The pharmaceutical composition can further comprise a combination of at least one material selected from the group consisting of a water soluble (miscible) co-solvent, a water insoluble (immiscible) co-solvent, a surfactant, an antioxidant, a chelating agent, and an absorption enhancer.

[0114] The solubilizer is at least a single surfactant, but it can also be a combination of materials such as a combination of: a) surfactant and water miscible solvent; b) surfactant and water immiscible solvent; c) surfactant, antioxidant; d) surfactant, antioxidant, and water miscible solvent; e) surfactant, antioxidant, and water immiscible solvent; f) surfactant, water miscible solvent, and water immiscible solvent; or g) surfactant, antioxidant, water miscible solvent, and water immiscible solvent.

[0115] The composition optionally further comprises a) at least one liquid carrier; b) at least one emulsifying agent; c) at least one solubilizing agent; d) at least one dispersing agent; e) at least one other excipient; f) at least one filler; or g) a combination of any two or more of the above.

[0116] In some embodiments, the water miscible solvent is low molecular weight (less than 6000) PEG, glycol, or alcohol. In some embodiments, the surfactant is a pegylated surfactant, meaning a surfactant comprising a polyethylene glycol) functional group.

[0117] The invention includes all combinations of the aspects, embodiments and sub-embodiments of the invention disclosed herein.DETAILED DESCRIPTION OF THE INVENTION

[0118] The invention provides a method of improving GI, respiratory, and / or musculoskeletal health in an animal or human by chronic or acute administration of one or more effective doses of oleandrin-containing composition (OCC). The composition is administered according to a dosing regimen best suited for the subject, the suitability of the dose and dosing regimen to be determined clinically according to conventional clinical practices and clinical treatment endpoints for said disease or disorder. The invention is particularly suitable for a subject suffering from respiratory, GI, and / or musculoskeletal health deterioration. The invention is even more particularly suitable for treating or preventing health deterioration-related symptom(s), in particular symptom(s), as described herein, associated with GI health deterioration. The invention is also particularly suitablefor increasing feed conversion, reducing the rate of occurrence of mortality and morbidity, and reducing the severity of morbidity in a population of subjects, in particular animals.

[0119] As used herein, the term “subject” is taken to mean humans and warm blooded animals such as mammals, for example, dog, cat, cow, horse, sheep, goat, pig, llama, alpaca, buffalo, deer, elk, giraffe, camel, chicken, turkey, or other hair-bearing or fur-bearing animal susceptible to suffering from deteriorating GI health, deteriorating respiratory health, deteriorating musculoskeletal health, protozoal infection, bacterial infection, amebic infection, and / or parasitic infection. Said deteriorating health may or may not be disease related.

[0120] A subject treated according to the invention will exhibit a therapeutic (clinically beneficial) response. According to a specific example, by “therapeutic response” is meant that a subject presenting with a condition, e.g. diarrhea or other as described herein, will, as a result of said treatment, a) have diarrhea less often; or b) will stop having diarrhea. The therapeutic response can be a full or partial therapeutic response. In a population of subjects, a therapeutic response is deemed to be a reduction in the rate of occurrence of or severity of said condition in the treated population as compared to the occurrence of said condition in an equivalent (similar) but untreated population.

[0121] A dosing regimen includes a therapeutically relevant dose (or effective dose) of OCC administered according to a dosing schedule. A therapeutically relevant dose, therefore, is a therapeutic dose at which a therapeutic response to treatment with OCC is observed and at which a subject can be administered the OCC without an excessive amount of unwanted or deleterious side effects. A therapeutically relevant dose is non-lethal to a subject, even though it may cause some side effects. It is a dose at which the level of clinical benefit to the subject being administered the OCC exceeds the level of deleterious side effects experienced by the subject due to administration of the OCC or component(s) thereof. This same definition of therapeutically relevant dose applies to other compounds and health improving agents described herein.

[0122] A therapeutically relevant dose will vary from subject to subject according to a variety of established pharmacologic, pharmacodynamic and pharmacokinetic principles. A therapeutically relevant daily dose of oleandrin will typically be in the range of about 0.1 micrograms to 100 micrograms, about 0.1 microg to about 500 microg, about 0.1 to about 100 microg / kg of body weight, about 15 to about 25 microg / kg of body weight, about 25 to about 50 microg / kg of bodyweight, about 50 to about 100 microg / kg of bodyweight, about100 to about 200 microg / kg of bodyweight, about 200 to about 500 microg / kg of bodyweight, about 10 to about 750 microg / kg of bodyweight, about 16 to about 640 microg / kg of bodyweight, about 15 to about 750 microg / kg of bodyweight, about 15 to about 700 microg / kg of bodyweight, about 15 to about 650 microg / kg of bodyweight, about 0.05 to about 10 microg / Kg of bodyweight, about 0.1 to about 7.5 microg / Kg of bodyweight, about 0.1 to about 5 microg / Kg of body weight, about 0.5 to about 5 microg / Kg body weight, about 0.1 to about 50 microg / Kg bodyweight, about 0.1 to about 25 microg / Kg bodyweight, about 0.1 to about 15 microg / Kg bodyweight, or about 0.1 to about 10 microg / Kg bodyweight. Such dose can be administered one to five times daily.

[0123] Vaccines that can be administered to dogs or other subjects as part of a treatment protocol include, for example, bordetella intranasal or oral vaccine (BDTV), NOBIVAC PUPPY DPV subcutaneous vaccine (NOBV; canine distemper virus, adenovirus type 1 and 2, canine parainfluenza virus, and canine parvovirus), NEOPAR subcutaneous parvo virus vaccine (NEOP), NEOTECH DA2 subcutaneous canine distemper adenovirus type 2 vaccine (NEOT), 7-way with coronavirus subcutaneous vaccine (7XCV), and others.

[0124] Oleandrin may be administered to ruminant animals including cattle, sheep, goats, buffalo, deer, elk, giraffes, and camels. For ruminant animals, the young animals have a different digestive tract than adult animals, meaning the young animals are nonruminants. Accordingly, the dose of oleandrin (microg of oleandrin per Kg of body weight) may be different in a young animal as compared to an adult animal of the same species. For example, a calf may require a different (lower) dose than an adult cow in order to benefit from oleandrin therapy. Likewise, a puppy may require a different (lower) dose than an adult dog, or a kitten may require a different (lower) dose than an adult cat. A veterinary clinician will be able to use known dose escalation or de-escalation protocols to determine a safe and effective dose to be administered.

[0125] It is known that young ruminants are initially non-ruminating and become ruminating after they reach a certain age. Oleandrin may undergo an excessive degree of metabolism or degradation in the rumen. Accordingly, for ruminating animals, the OCC is preferably administered in a coated, time-release, delayed release, sustained release, controlled release, or prolonged release formulation. For subjects with acidic stomachs, oleandrin may undergo excessive acid-catalyzed degradation, so for such subjects, it may be necessary to increase the oral dose to account for gastric degradation while still providing a therapeutic dose.

[0126] A therapeutically relevant dose can be administered according to any dosing regimen typically used in the treatment of protozoal, amebic, bacterial, or parasitic infection related disease or disorder. A therapeutically relevant dose can be administered once, twice, thrice, or more, or continuously daily. It can be administered every other day, every third day, every fourth day, every fifth day, semiweekly, weekly, biweekly, every three weeks, every four weeks, monthly, bimonthly, semimonthly, every three months, every four months, semiannually, annually, or according to a combination of any of the above to arrive at a suitable dosing schedule. For example, a therapeutically relevant dose can be administered one or more times daily (up to 10 times daily for the highest dose) for one or more weeks.

[0127] Oleandrin may be included in feed and / or liquid administered to an animal. Oleandrin may be included in any feed format including solid feed, liquid feed, or gel feed. The solid feed may be loose granules, pellets, foodstuff, salt block, or other such feed used to feed animals.

[0128] The solid feed may comprise oleandrin and at least one feedstuff. Suitable feedstuffs include Whole cottonseed, cottonseed hulls, cottonseed meal, soybean meal, meat, protein, meat substitute, blood, bloodmeal, soybean hulls, corn gluten feed, hominy feed, dried distiller’s grains, and rice mill feed are examples of commodity feedstuffs. Additional ingredients that may be included are selected from the group consisting of silage, nutritious supplement, vitamin, mineral, salt, grain (wheat, barley, oat, corn), fiber, hay, alfalfa, rye grass, beet, molasses, blood meal, bone meal, yeast, brome grass, canary grass, tomato, carrot, peas, pea vine hay, safflower, sage brush, sorghum, cheatgrass, clover, fat, grape, hominy, hops, meadow hay, sundan grass, sunflower, timothy hay, meat meal, milo, orange, orchard grass, potato, navy beans, peanut, prairie hay, rape meal, soybean, protein, meat, animal protein, plant protein, dextrin, sugar, maltodextrin, molasses, and combinations of any thereof.

[0129] The liquid feed comprises oleandrin, at least one liquid, and at least one nutrient. The liquid can be water, fermentation broth, milk, or milk substitute or other such liquid suitable for administration to an animal.

[0130] Given the bitter taste of oleandrin and oleander extracts, an oral, buccal, sublingual, or orodispersible composition administered to an animal can include one or more taste-masking agents. One or more sweeteners is / are advantageously included in a feed or water supply. Suitable sweeteners include nutritive and nonnutritive sweeteners, such assucram (combination comprising 98% sodium saccharin, 1% neohesperidin dihydrocalcone, and 1% maltol; Pancosma SA, Geneva, Switzerland), sucrose, dextrose, maltose, lactose, stevia, sucralose, maltose, corn syrup, molasses, sugar cane syrup, maple syrup, saccharin, stevioside, rebaudioside, neotame, steviol glycoside, NHDC (neohesperidin dihydrochalcone), aspartame, cyclamate, maltodextrin, glycerin, acesulfame, alitame, dulcin, monellin, maltol, and combinations of two or more thereof. For some animals, a meat-flavoring agent is included. Sweeteners that are obtained from plant materials made be provided as pure compounds, extracts of said plant materials, or as said plant materials.

[0131] Oleandrin can also be included in water or other liquid given to the subject.

[0132] A composition can also include one or more additives suitable for administration to animals. For example, ammonium sulfate, calcium carbonate, sodium chloride, defluorinated phosphate, diammonium phosphate, dicalcium phosphate, limestone, monoammonium phosphate, monocalcium phosphate, sodium tripolyphos, urea, or any combination thereof may be used as additive.

[0133] According to certain aspects of the invention, compositions and formulations are provided comprising transfer factor. According to certain embodiments of the invention, various forms of transfer factor may be used. They include, without limitation, excreted transfer factor released from transfer factor containing cells such as lymphocytes, leukocytes, and ova, and collected from extracellular fluids such as colostrum and blood. Another form includes pre-excreted transfer factor found within the cell or on the cell surface. In certain embodiments of the invention, substantially purified transfer factor originating from leukocytes, colostrum, or ova and having a molecular weight of less than 10,000 daltons and a specific activity of at least 5000 units per absorbance unit at 214 nanometers, may also be used. The transfer factor used in the Examples of this invention and referred to in the following Tables and further referred to in the rest of the detailed description is generally extracted from colostrum collected from a general pool of lactating cows; although, in some cases, it is derived from eggs.

[0134] Alternative sources of transfer factor include, but are not limited to, avian transfer factor, ova transfer factor, and transfer factor isolated from colostrum collected from non- bovine animals such as goats, pigs, horses and humans. In addition, combinations of transfer factors from any number of sources may be used in the formulations of the instant invention. Transfer factor may also be derived from recombinant cells that are genetically engineered to express one or more transfer factors or by clonal expansion of leukocytes.

[0135] In certain embodiments of the invention, transfer factor may be obtained from colostrum. In a preferred embodiment, transfer factor is obtained from bovine colostrum. The fraction of colostrum comprising material having a molecular weight of approximately 10,000 daltons (Da) and below is designated as transfer factor. A fraction obtained that is approximately 10,000 to approximately 150,000 Da is designated an antibody fraction, also known as an antibody-colostrum fraction. In certain embodiments, a colostral fraction having a molecular weight of about 10,000 to about 400,000 Da may be used as an antibody fraction. The fraction comprising material having a molecular weight of approximately 10,000 Da and above is designated as the growth factor fraction. The growth factor fraction may include high molecular weight proteins.

[0136] In certain embodiments, an antibody fraction comprises antibodies from about 1% to about 99%, about 5% to about 95%, about 10% to about 90%, about 15% to about 85%, about 20% to about 80%, about 25% to about 75%, about 30% to about 70%, about 35% to about 65%, about 40% to about 60%, about 45% to about 55%, or about 50% by weight, the remainder comprising other colostrum components.

[0137] According to certain embodiments of the invention, transfer factor, as used in the formulations described in the Tables, particularly when not defined as obtained from an avian source, may be further defined as defatted water soluble material from bovine colostrum that will pass through a nominal 10,000 molecular weight filter.

[0138] In other embodiments, the transfer factor may be obtained from an avian source. In one embodiment, chickens are given a feed mixture containing excrement from an animal, including without limitation, at least one selected from the group consisting of a human, a fish, a goat, a llama, an alpaca, a pig, a sheep, a cow, and a horse. The excrement will contain a large variety of pathogens and upon administration in a feed to an animal, the animal will develop transfer factor and / or antibodies to such pathogens. Avian transfer factor can then be obtained from the eggs produced by the above-treated chickens. In certain embodiments of the invention, transfer factor may be found in whole egg yolks. As a nonlimiting example, the transfer factor of avian source (which is believed to also contain antibodies) is supplied as powdered whole egg yolks.

[0139] Alternative kinds of transfer factor include, but are not limited to, targeted transfer factors. Target transfer factors include transfer factor collected from sources which have been exposed to (1) one or more viral or otherwise infectious organisms; (2) one or more antigens that produce an immune response; or (3) a combination of organisms andantigens. The term antigen is defined herein is anything that will initiate the cell mediated immune response. Examples of such viral or other infectious organisms include Herpes Simplex Virus 1, Herpes Simplex Virus 2, H. Pylori, Campylobacter and Chlamydia, Bovine Rhinotracheitis Virus, Parainfluenza, Respiratory Syncytial Virus Vaccine, modified live virus, Campylobacter Fetus, Leptospira Canicola, Grippotyphosa, Hardjo, Leterohaemorrhagiae, Pomona Bacterin, Bovine Rota-Coronavirus, Escherichia Coli Bacterin, Clostridium Chauvoei, Septicum, Haemolyticum, Novy, Sordellii, Perfringens Types C & D, Bacterin, Toxoid, Haemophilus Somnus, Pasteurella Haemolytica, Multocida Bacterin. However, one of skill in the art would readily recognize that a wide variety of other viral and otherwise infectious organisms can find use in the instant invention.

[0140] Additionally, transfer factor and antibodies may be derived from any suitable source, as described, for example, in U.S. Pat. Nos. 4,816,563; 5,080,895; 5,840,700; 5,883,224; and 6,468,534; and U.S. patent application Ser. No. 11 / 762,727, the entire disclosures of which are hereby incorporated by reference herein.

[0141] In certain embodiments, the component of a given formulation that is referred to as the "transfer factor" may optionally include a colostral component of higher molecular weight; for example, a portion of the fraction referred to above as an antibody or antibodycolostrum fraction. In certain embodiments, the mammalian "transfer factor" component of a formulation comprises both transfer factor fraction and antibody fraction. In certain preferred embodiments, "mammalian transfer factor" comprises about 70% transfer factor fraction from colostrum (i.e., 10,000 Da or below colostrum fraction) and about 30% antibody colostrum fraction. In other preferred embodiments, "mammalian transfer factor" comprises about 80% transfer factor fraction from colostrum and about 20% antibody colostrum fraction. (The foregoing are in weight percents of the composition). In certain embodiments, the "transfer factor" component of the composition or formulation may include one or both of mammalian and avian transfer factor.

[0142] In preferred embodiments, the transfer factor is encapsulated by mixing with a hydrophobic substance or a lipid to form a coating around the growth factor(s). In additional embodiments, one or more additional components such as antibody, antibody fraction, and / or glucans may be encapsulated. Other optional components of compositions and formulations of the invention may be encapsulated, such as, without limitation, inositol hexaphosphate, olive leaf extract, mannans, phytosterol, vitamin C and mixtures thereof. The transfer factor, antibody or antibody fraction and / or additional optional componentsmay each be individually encapsulated or encapsulated as a mixture. Alternatively, the entire formulation can be encapsulated. The encapsulated component(s) and / or formulation can be produced in a variety of ways. In a preferred embodiment, each of the transfer factor, glucans, antibody or antibody fraction and / or additional labile component(s) in the formulation may be encapsulated as described in U.S. Pat. Nos. 5,190,775, 6,013,286 and U.S. Application 2003 / 0129295, the entire disclosures of which are incorporated herein by reference.

[0143] The transfer factor may be encapsulated with a hydrophobic or lipid coating that is preferably between about 25% and about 150 wt / % of the transfer factor, about 50-150 wt / % and about 75-125 wt / %, with an equal weight being most preferred.

[0144] In additional embodiments of the invention, additional components may be used in the formulation administered. Particular components may be encapsulated. For example, IP6, beta-sitosterol, olive leaf extract, aloe extract matter and / or vitamin C may be used; in certain embodiments, one or more of these components may be encapsulated. In preferred embodiments, IP6 is present at between 10 mg and 3 gm / oz, or one preferably between 100 mg and 2 gm / oz, and most preferably between 100 mg and 1 gm / oz. The .beta. -sitosterol is preferable in the amount of between 10 mg and 3 gm / oz, or preferably between 100 mg and 2 gm / oz, and most preferably between 100 mg and 1 gm / oz. Olive leaf extract is preferably present in the amount of 2 mg to 2 gm / oz, more preferably between 5 mg and 1 gm / oz, and most preferably between 5 mg and 500 gm / oz. Aloe extract is preferably present at between 2 mg and 1000 mg, more preferably between 5 and 500 mg / oz, and most preferably between 5 and 250 mg / oz. Vitamin C may be present at between 10 mg / oz and 10 gm / oz, or preferably between 100 mg and 8 gm / oz, and most preferably between 100 mg and 5 gm / oz.

[0145] Glucans are polysaccharides found in mushrooms. In some conversations, glucans and mushrooms are used interchangeably. Glucans may be derived from multiple sources, including, but not limited to, fungi, oats, and yeast. Preferably, glucans are present in or derived from fungi. In certain embodiments, the glucans which may be included in the formulations are present in whole fungi.

[0146] In certain embodiments, glucans are derived from hybrid strains of fungi. In a preferred embodiment the hybrid glucans used in the invention are present in, or derived from, hybrid strains of Cordyceps and in particular Cordyceps sinensis. One technique to induce the hybridization of Cordyceps involves plating two different strains or species on a single agar plate which has been inoculated with rattlesnake venom as described in, forexample, U.S. Patent Application Publication No. 2006 / 0073197, published Apr. 6, 2006, and U.S. Patent Application Publication No. 2007 / 0128253, published Jun. 7, 2007, each of which is incorporated herein by reference. In a preferred embodiment, the hybrid strain producing the hybrid glucans that may be used in compositions and formulations of the invention is Cordyceps sinensis Alohaensis, which is available from Pacific Myco Products, Santa Cruz, Calif.

[0147] In addition to Cordyceps sinensis hybrids, suitable sources of glucans may include, but are not limited to, Agaricus blazeii, Coriolus, Poira Cocos, Inonotus obliquus, Maitake Mushroom, Shiitake Mushroom, and combinations thereof.

[0148] In certain embodiments, glucans of the formulation may be encapsulated, preferably with a hydrophobic or lipid coating. It is preferred that the amount of hydrophobic or lipid coating be between about 25% and 150 wt / % of the glucan, about 50-150 wt %, or about 75-125 wt / %, with an equal weight being most preferred.

[0149] In certain preferred embodiments, compositions may further comprise one or more of inositol hexaphosphate (Ip6), mannans, olive leaf extract, and phytosterols. In certain preferred embodiments, mannans are derived from Aloe vera. In certain preferred embodiments, phytosterols may be derived from soya (soy) bean.

[0150] In certain embodiments, compositions may further comprise one or more of lactic acid producing bacteria, ascorbic acid, Vitamin A, Vitamin D, Vitamin E, Vitamin B 1, Vitamin B2, Vitamin B12, dipotassium phosphate, potassium chloride, magnesium salts or chelates, and calcium pantothenate.

[0151] In certain embodiments, compositions and formulations comprising transfer factor may be combined with minerals, antioxidants, amino acids, and other nutraceuticals.

[0152] In certain preferred embodiments, the formulation comprises OCC, at least one transfer factor, lactic acid generating bacteria, and at least one glucan. In some embodiments, one or more additional ingredients are included. In certain embodiments, the transfer factor fraction may be lyophilized. In certain embodiments, the optional growth factor colostral fraction, other colostral fraction, antibody or antibody fraction may be lyophilized.

[0153] In certain embodiments, a transfer factor formulation includes at least encapsulated transfer factor derived from bovine and / or avian sources, and / or one or more of hybrid glucans. It is preferred that the glucan portion of this formulation also be encapsulated. Other components include zinc proteinate, targeted avian transfer factors,.beta. -sitosterol, inositol hexaphosphate (IP6), olive leaf extract, aloe extract powder, probiotics, B. subtlis, B. longum, B. thermophilium, L. acidophilus, E. faecium, and S. cerevisiae. In a preferred embodiment, all of the foregoing are included in this transfer factor formulation.

[0154] In another embodiment, the formulation includes a probiotic. Exemplary probiotics include, but are not limited to B. subtlis, B. longum, B. thermophilium, B. coagulans, L. acidophilus, E. faecium, and S. cerevisia, L. casei, L. plantarum, Pediococccus acidilacticii, Kluyveromyces marxianus fragillis and combinations thereof.

[0155] In another preferred embodiment, the component listed as "Transfer factor (mammal source)" is substituted with a composition containing 80% bovine colostrum transfer factor as described herein, combined with 20% bovine colostrum antibody fraction as described herein (both weight percents of the composition). In certain embodiments, the mammalian transfer factor and the colostrum antibody fraction are both lyophilized. In a preferred embodiment, the component listed as "Transfer factor (avian source)" is present in the formulation in an amount of 3000.0 mg / oz. This component may be supplied as powdered whole egg yolk obtained from hyperimmunized chickens, i.e., chickens that had been exposed to pathogens prior to laying the eggs which serve as a source of transfer factor. In various embodiments, the avian transfer factor may be obtained from commercial sources (for example, 4Life® Research; Labelle, Inc., Bellingham, Wash.; Troue; and Ghen Corporation, Japan).

[0156] The amount of transfer factor and / or antibody or antibody fraction used in the formulation or the amount of formulation administered will vary depending upon the severity of the clinical manifestations presented. In addition, the amount of transfer factor administered to a recipient will vary depending upon the species from the transfer factor is derived as compared to the species of the recipient. It has been observed that transfer factor derived from bovine species administered to cattle is more efficacious than transfer factor from another species such as avian species. Accordingly, when the source of the transfer factor and recipient are different species, it is preferred that the amount of transfer factor be increased.

[0157] In certain embodiments, oral or subcutaneous administration of a composition comprising OCC, transfer factor, glucan, and lactic acid generating bacteria may be achieved by the use of a time-release or controlled-release implantable dosage form. Examples of suitable implantable dosage forms have been described in U.S. Pat. Nos.5,665,363, 6,290,980, and RE 39,014 (a reissue of U.S. Pat. No. 6,290,980). In certain embodiments, the invention relates to formulations that provide controlled (delayed) release of active agents. In certain embodiments, these formulations may be administered orally. In certain embodiments, the formulation may include a combination of water soluble and water insoluble polymers.

[0158] In certain embodiments, OCC, transfer factor, lactic acid generating bacteria, probiotic, and glucans may be consumed at different times within a one week period.

[0159] A reasonable weight range for transfer factor is 0.05-50 mg per pound of body weight. A reasonable weight range for lactic acid generating bacteria is 0.47-10 mg per pound of body weight, based on a nominal live count of 15-20 billion CFU / ounce (colony- forming-units-per-ounce).

[0160] Proof of the anti -protozoan infection related activity, in particular anti-giardia activity, of oleandrin was established in vivo according to Example 13. Puppies (280) living in a large scale dog-breeding kennel were administered OCC in the form of MCT-based buccal / sublingual supplement (PBI-06150) comprising oleander extract and fractionated medium chain triglyceride. The supplement contains 12-15 (usually 13-14) microg of oleandrin (OLE) per mL. Administration was started when the puppies were 2 days of age and administered orally according to the dosing regimen of Example 13. The PBI-06150 was mixed with transfer factor and glucan mix (as described below) to make the paste.

[0161] Two kennels were selected for treatment: Kennel A (population of puppies with moderate problem of Giardia infection (diarrhea; -20% positive Imagyst fecals) and respiratory distress; and Kennel B (population of puppies with no Giardia infection- diarrhea and no respiratory distress). After completion of treatment, only one litter of the puppies of Kennel A exhibited diarrhea, and none of the litters of Kennel B exhibited diarrhea. Moreover, the puppies of Kennel B exhibited better food consumption, improved overall health, improved stool characteristics. None of the puppies of either kennel exhibited respiratory distress.

[0162] A separate study was conducted to determine whether the non-oleandrin composition components on their own would provide adequate treatment of diarrhea. A large population of puppies were administered substantially the same formulations as those ofExample 13, except that oleandrin and oleander extract were excluded. The non-oleandrin components on their own were not effective at reducing the rate of occurrence of diarrhea(-40%) in the puppies. This means that oleandrin (and oleander extract) worked synergistically with the other components to provide substantially improved GI health.

[0163] Accordingly, the invention provides a method of treating diarrhea in a subject, the method comprising administering one or more effective doses of oleandrin (OCC) to a subject having diarrhea. The invention also provides a method of preventing recurrence of diarrhea (perhaps associated with giardiasis), the method comprising administering one or more effective doses of OCC to a subject having already experienced resolution of a prior occurrence of diarrhea, thereby preventing recurrence of diarrhea in the subject. The administration can be according to any of the dosing regimens described herein.

[0164] The invention also provides a method of reducing the rate of occurrence of diarrhea in a population of subjects, the method comprising administering to subjects of said population OCC, at least one transfer factor, at least one glucan, and at least one probiotic, thereby reducing the rate of occurrence of diarrhea in treated subjects as compared to untreated subjects.

[0165] The concentrations of oleandrin evaluated in the assays are clinically relevant in terms of dosing and plasma concentration.

[0166] Proof of the in vivo safety of the oleandrin-containing composition was provided by clinical evaluation of the above-mentioned puppies. At the doses administered, no oleandrin-related toxicity over control vehicle was observed.

[0167] A safety study on the administration of a Nerium oleander hot water extract (ANVIRZEL) was conducted on beagle dogs according to Example 6. At doses ranging from 0.14 to 0.56 microg of oleandrin per Kg of bodyweight per day, no drug-related side effects, no morbidity, and no mortalities were observed.

[0168] A safety study on the administration of a Nerium oleander supercritical fluid extract (PBI-05204) was conducted in dogs according to Example 23. The results indicate that a daily dose of up to 4.6 microg OLE / Kg of body weight were well tolerated.

[0169] The safe administration of oleandrin (in a hot water extract or an alcohol extract) was also established in chickens and turkeys. Birds were administered, via water or feed, doses ranging from 5 microg of OLE / Kg BW to over 500 microg of OLE / Kg BW with no side effects observed. Additional studies establishing safe administration of OLE to chicks and pullets of egg-laying hens according to Example 29. Doses ranged from about 40 to about 142 microg OLE / Kg. The OLE was administered in water or water and feed. All chicks and pullets fared well.

[0170] The safe administration of oleandrin (in an extract) was also established in nursing sows and piglets according to Example 28. Nursing sows and piglets were administered doses ranging from 0.125 microg of OLE / Kg BW to over 25 microg of OLE / Kg BW with no side effects observed. Tissues (organs and muscles) and biological fluids of piglets were analyzed for oleandrin content at different time points. Oleandrin was detected in heart and kidney tissue but not in muscle or lung tissue at 24 h after administration. No oleandrin was detected in heart, kidney, lung, or muscle tissue at 8 days after administration. For the nursing sow, no oleandrin was detected in the milk at five hours after administration of doses ranging from 0.125-1.0 microg OLE / Kg BW. In terms of the plasma clearance rate of oleandrin for the piglet, OLE content in the plasma peaked at about 1 h (Cmax) and fell to below the level of quantitation (BLQ = 2.5 ng / mL) at about 5-6 hours after administration.

[0171] The compositions of the invention were tested (according to Example 26) in roping calves to determine their impact upon the rate of occurrence of mortality, the rate of occurrence of morbidity, and the severity of morbidity. Calves treated with the immune primer paste (containing transfer factor, glucan, and probiotics) exhibited a morbidity rate of about 10-20% and a mortality rate of 5% or less. Calves treated with the immune primer paste and Nerium oleander extract (containing oleandrin) exhibited no illness, morbidity rate of 3%, or no mortality for at least 14 days after treatment. Importantly, the OCC provided these substantial improvements regardless of whether treatment of the calves began immediately upon arrival to the ranch / bam or five days after arrival to the ranch / barn. The treated cattle had improved overall health.

[0172] A health supplement was evaluated in pigs according to the placebo-controlled study detailed in Example 34. The purpose of the study was to determine whether an OCC of the invention would provide any improvement in the overall health of a production animal, e.g. sows and piglets. Pregnant sows, which tested seropositive for wild-type PRRSV (porcine reproductive respiratory syndrome virus), were divided into a treatment group and an untreated control group. The sows of the treatment group were given a health supplement mixed into their daily food ration. The supplement contained Nerium oleander powdered biomass (dried and ground leaves and stems), powdered corncob, and a mixture of sweeteners. For both groups, the rate of occurrence of fetal mortality (fetal abortions, fetal stillbirths, and mummified fetuses) and the average viral titer were determined periodically and at the end of a three-week (21 -day) treatment period. The treatment periodbegan at about day 78-80 of the gestation period for the sows. The feed consumption and feed conversion of the sows was also determined, as was the overall health of the sows and the farrowed piglets. The treatment group exhibited an improvement in the overall health (reduced morbidity) of the sows and their farrowed piglets and also exhibited an overall reduction in fetal mortality (reduction in aborted fetuses, stillbirth fetuses, and mummified fetuses). The supplement did not cause any adverse events. There did not appear to be a direct correlation between changes in viral titer, and the observed improvements (reduced fetal mortality).

[0173] In another placebo-controlled study with pregnant PRRSV seropositive sows, the composition (in this particular case, a powdered composition containing Nerium oleander extract adsorbed onto maltodextrin; prepared as described herein) was included as a supplement in the daily ration of feed for a period of three weeks. Oral administration begat at about day 78-80 of the gestation period. The sows became seronegative after one week of daily rations, and seronegativity was further confirmed after three weeks of daily rations. No fetal mortalities (stillbirths, abortions, and mummified fetuses) were observed in the treated sows. Moreover, the entire farrow of one of the treated sows was found to be seronegative, which is absolutely astounding and unexpected, because it is well-known in the porcine industry that seropositive sows give birth to seropositive piglets.

[0174] Accordingly, the invention also provides a method of reducing fetal mortality in a population of animals, the method comprising administering OCC of the invention to a pregnant animal (according to any of the dosing protocols or regimens described herein), thereby reducing the occurrence of fetal mortality in the pregnant animal. In particular embodiments, the reduction in fetal mortality is a reduction in aborted fetuses, stillborn fetuses, and / or mummified fetuses. In particular embodiments, before said administering, the pregnant animal is seropositive for a viral infection during at least the latter part of the animal’s gestation period. In some embodiments, the method of invention excludes embodiments wherein, before said administering, the animal is seropositive for a viral infection during at least the latter part of the animal’s gestation period. In some embodiments of the invention, the animal is seronegative for viral infection prior to said administering. In particular embodiments, the animal is a pig, cow, horse, sheep, or goat.

[0175] As used herein, the term feed conversion is synonymous with feed conversion ratio (FCR or feed conversion rate), and it is a ratio or rate measurement of the efficiency with which the bodies of livestock convert animal feed into the desired output.For dairy cows, for example, the output is milk, whereas in animals raised for meat (such as beef cows, pigs, chickens, and fish) the output is the flesh, that is, the body mass gained by the animal, represented either in the final mass of the animal or the mass of the dressed output. FCR is the mass of the input divided by the output (thus mass of feed per mass of milk or meat).

[0176] The impact of OCC upon feed conversion was evaluated in dairy cattle according to Example 27. Cattle exhibiting low feed conversion in terms of milk production were administered dried finely ground Nerium oleander biomass (primarily leaves and some stems). The study was conducted for only three weeks. Within each of those weeks, the average daily milk production went from 48 lbs of milk (considered a very low production baseline) to 53-55 lb, meaning the OCC resulted in almost a 20% increase in average daily milk production across the population of cows.

[0177] A study on the prevention of diarrhea in humans exposed to diarrhea-causing microbes was conducted on backpackers, campers, and hikers according to Example 24. For several days, the subjects drank untreated water from creeks in outdoors areas known to have diarrhea-causing microbes. At the same time, the subjects were dosed twice daily with oleandrin-containing extract. None of the subjects experienced diarrhea during or after completion of their outdoor activities.

[0178] In some embodiments, an OCC comprises a) oleandrin, or b) oleandrin and one or more other compounds extracted from Nerium species. The oleandrin may be present as part of an extract of Nerium species, which extract may be a a) supercritical fluid extract; b) hot-water extract; c) organic solvent extract; d) aqueous organic solvent extract; e) extract using supercritical fluid, optionally plus at least one organic solvent (extraction modifier); f) extract using subcritical liquid, optionally plus at least one organic solvent (extraction modifier); or g) any combination of any two or more of said extracts.

[0179] PB 1-05204 (as described herein and in US 8187644 B2 to Addington, which issued May 29, 2012, US 7402325 B2 to Addington, which issued July 22, 2008, US 8394434 B2 to Addington et al, which issued Mar. 12, 2013, the entire disclosures of which are hereby incorporated by reference) comprises cardiac glycoside (oleandrin, OL) and triterpenes (oleanolic acid (OA), ursolic acid (UA) and betulinic acid (BA)) as the primary pharmacologically active components. The molar ratio of OL to total triterpene is about l :(10-96). The molar ratio of OA:UA:BA is about 7.8:7.4: 1, about 3:2.2: 1, about 2-8 (0 A) : about 2-8 (UA) : about 0.1-1 (BA) : about 0.5-1.5 (OL); or about 3-6 (OA) : about 3-6(UA) : about 0.3-8 (BA) : about 0.7-1.2 (OL); or about 4-5 (OA) : about 4-5 (UA) : about 0.4-0.7 (BA) : about 0.9-1.1 (OL); about 4.6 (OA) : about 4.4 (UA) : about 0.6 (BA) : about 1 (OL); about 9-12 : up to about 2 : up to about 2, or about 10 : about 1 : about 1, or about 9-12 : about 0.1-2 : about 0.1-2, or about 9-11 : about 0.5-1.5 : about 0.5-1.5, or about 9.5- 10.5 : about 0.75-1.25 : about 0.75-1.25, or about 9.5-10.5 : about 0.8-1.2 : about 0.8-1.2, or about 9.75-10.5 : about 0.9-1.1 : about 0.9-1.1.

[0180] OCCs comprising oleandrin as the sole anti-giardia agent are within the scope of the invention. OCCs comprising oleandrin and plural triterpenes as the anti-giardia agents are within the scope of the invention. In some embodiments, the OCC comprises oleandrin, oleanolic acid (free acid, salt, derivative or prodrug thereof), ursolic acid (free acid, salt, derivative or prodrug thereof), and betulinic acid (free acid, salt, derivative or prodrug thereof). The molar ratios of the compounds are as described herein.

[0181] The OCCS of the invention can be administered as primary anti-giardia therapy, adjunct anti-giardia therapy, or co-anti-giardia therapy. Methods of the invention include separate administration or coadministration of the OCC with at least one other known anti- giardia composition, meaning the OCC of the invention can be administered before, during or after administration of a known anti-giardia composition (compound(s)) or of a composition for treating symptoms associated with giardia infection, e.g. diarrhea.

[0182] The one or more other therapeutic agents can be administered at doses and according to dosing regimens that are clinician-recognized as being therapeutically effective or at doses that are clinician-recognized as being sub-therapeutically effective. The clinical benefit and / or therapeutic effect provided by administration of a combination of OCC and one or more other therapeutic compositions can be additive or synergistic, such level of benefit or effect being determined by comparison of administration of the combination to administration of the individual anti-giardia composition component(s) and one or more other therapeutic agents. The one or more other therapeutic agents can be administered at doses and according to dosing regimens as suggested or described by the Food and Drug Administration (Center for Veterinary Medicine), World Health Organization, European Medicines Agency (Veterinary Medicines Division), Australian Pesticides and Veterinary Medicines Authority (APVMA), Pan American Health Organization (Veterinary Public Health Program), Agricultural Compounds and Veterinary Medicines Authority (New Zealand) or the various Ministries of Health worldwide.

[0183] Where a dose of the composition is intended to be administered in a single dosage form, the composition will contain amounts of the components sufficient to provide a unit dose of about 0.05-100 microg (or about 0.1-50 microg, about 0.1-25 microg, 0.1-15 microg, 0.1-10 microg, or 0.1-5 microg) of oleandrin. Such composition can be administered one to four times daily or two to four times daily, once daily, or twice daily.

[0184] One or more compound(s) present in the composition can be present in their unmodified form, salt form, derivative form or a combination thereof. As used herein, the term “derivative” is taken to mean: a) a chemical substance that is related structurally to a first chemical substance and theoretically derivable from it; b) a compound that is formed from a similar first compound or a compound that can be imagined to arise from another first compound, if one atom of the first compound is replaced with another atom or group of atoms; c) a compound derived or obtained from a parent compound and containing essential elements of the parent compound; or d) a chemical compound that may be produced from first compound of similar structure in one or more steps. For example, a derivative may include a deuterated form, oxidized form, dehydrated, unsaturated, polymer conjugated or glycosylated form thereof or may include an ester, amide, lactone, homolog, ether, thioether, cyano, amino, alkylamino, sulfhydryl, heterocyclic, heterocyclic ring- fused, polymerized, pegylated, benzylidenyl, triazolyl, piperazinyl or deuterated form thereof.

[0185] As used herein, the term “oleandrin” is taken to mean all known forms of oleandrin unless otherwise specified. Oleandrin can be present in racemic, optically pure or optically enriched form. Nerium sp. plant material can be obtained, for example, from commercial plant suppliers such as Aldridge Nursery, Atascosa, Texas.

[0186] The supercritical fluid (SCF) extract can be prepared as detailed in US 7,402,325, US 8394434, US 8187644, or PCT International Publication No. WP 2007 / 016176 A2, the entire disclosures of which are hereby incorporated by reference. Extraction can be conducted with supercritical carbon dioxide in the presence or absence of a modifier (organic solvent) such as ethanol.

[0187] A hot-water extract is available under the tradename ANVIRZEL™ (Nerium Biotechnology, Inc., San Antonio, TX; Salud Integral Medical Clinic, Tegucigalpa, Honduras; www.saludintegral.com; www.anvirzel.com) as a liquid dosage form. ANVIRZEL™ comprises oleandrin, oleandrigenin, polysaccharides extracted (hot water extraction) from Nerium oleander. Commercially available vials comprise about 150 mg ofoleander extract as a freeze-dried powder (prior to reconstitution with water before administration) which comprises about 200 to about 900 microg of oleandrin, about 500 to about 700 microg of oleandrigenin, and polysaccharides extracted from Nerium oleander. Said vials may also include pharmaceutical excipients such as at least one osmotic agent, e.g. mannitol, sodium chloride, at least one buffering agent, e.g. sodium ascorbate with ascorbic acid, at least one preservative, e.g. propylparaben, methylparaben.

[0188] Other extracts containing cardiac glycoside, especially oleandrin, can be prepared by various different processes. An extract can be prepared according to the process developed by Dr. Huseyin Ziya Ozel (U.S. Patent No. 5,135,745) describes a procedure for the preparation of a hot water extract. The aqueous extract reportedly contains several polysaccharides with molecular weights varying from 2KD to 30KD, oleandrin, oleandrigenin, odoroside and neritaloside. The polysaccharides reportedly include acidic homopolygalacturonans or arabinogalaturonans. U.S. Pregrant Patent Application Publication No. 20040247660 to Singh et al. discloses the preparation of a protein stabilized liposomal formulation of oleandrin for use in the treatment of cancer. U.S. Pregrant Patent Application Publication No. 20050026849 to Singh et al. discloses a water soluble formulation of oleandrin containing a cyclodextrin. U.S. Pregrant Patent Application Publication No. 20040082521 to Singh et al. discloses the preparation of protein stabilized nanoparticle formulations of oleandrin from the hot-water extract.

[0189] The extracts also differ in their polysaccharide and carbohydrate content. The hot water extract contains 407.3 glucose equivalent units of carbohydrate relative to a standard curve prepared with glucose while analysis of the SCF CO2 extract found carbohydrate levels that were found in very low levels that were below the limit of quantitation. The amount of carbohydrate in the hot water extract of Nerium oleander was, however, at least 100-fold greater than that in the SCF CO2 extract. The polysaccharide content of the SCF extract can be 0%, <0.5%, <0.1%, <0.05%, or <0.01% wt. In some embodiments, the SCF extract excludes polysaccharide obtained during extraction of the plant mass.

[0190] The partial compositions of the SCF CO2 extract and hot water extract were determined by DART TOF-MS (Direct Analysis in Real Time Time of Flight Mass Spectrometry) on a JEOL AccuTOF-DART mass spectrometer (JEOL USA, Peabody, MA, USA).

[0191] The SCF extract of Nerium species or Thevetia species is a mixture of pharmacologically active compounds, such as oleandrin and triterpenes. The extract obtained by the SCF process is a substantially water-insoluble, viscous semi-solid (after solvent is removed) at ambient temperature. The SCF extract comprises many different components possessing a variety of different ranges of water solubility. The extract from a supercritical fluid process contains by weight a theoretical range of 0.9% to 2.5% wt of oleandrin or 1.7% to 2.1% wt of oleandrin or 1.7% to 2.0% wt of oleandrin. SCF extracts comprising varying amount of oleandrin have been obtained. In one embodiment, the SCF extract comprises about 2% by wt. of oleandrin. The SCF extract contains a 3-10 fold higher concentration of oleandrin than the hot-water extract. This was confirmed by both HPLC as well as LC / MS / MS (tandem mass spectrometry) analyses.

[0192] The SCF extract comprises oleandrin and the triterpenes oleanolic acid, betulinic acid and ursolic acid and optionally other components as described herein. The content of oleandrin and the triterpenes can vary from batch to batch; however, the degree of variation is not excessive. For example, a batch of SCF extract (PB 1-05204) was analyzed for these four components and found to contain the following approximate amounts of each.WRT denotes “with respect to”.

[0193] The content of the individual components may vary by ±25%, ±20%, ±15%, ±10% or ±5% relative to the values indicated. Accordingly, the content of oleandrin in the SCF extract would be in the range of 20 mg ± 5 mg (which is ±25% of 20 mg) per mg of SCF extract.

[0194] Oleandrin, oleanolic acid, ursolic acid, betulinic acid and derivatives thereof can also be purchased from Sigma-Aldrich (www.sigmaaldrich.com; St. Louis, MO, USA). Digoxin is commercially available from HIKMA Pharmaceuticals International LTD (NDA N012648, elixir, 0.05 mg / mL; tablet, 0.125 mg, 0.25 mg), VistaPharm Inc. (NDA A213000, elixir, 0.05 mg / mL), Sandoz Inc. (NDA A040481, injectable, 0.25 mg / mL), West-Ward Pharmaceuticals International LTD (NDA A083391, injectable, 0.25 mg / mL), Covis Pharma BV (NDA N009330, 0.1 mg / mL, 0.25 mg / mL), Impax Laboratories (NDA A078556, tablet, 0.125 mg, 0.25 mg), Jerome Stevens Pharmaceuticals Inc. (NDA A076268, tablet, 0.125 mg, 0.25 mg), Mylan Pharmaceuticals Inc. (NDA A040282, tablet, 0.125 mg, 0.25 mg), Sun Pharmaceutical Industries Inc. (NDA A076363, tablet, 0.125 mg, 0.25 mg), Concordia Pharmaceuticals Inc. (NDA A020405, tablet, 0.0625, 0.125 mg, 0.1875 mg, 0.25 mg, 0.375 mg, 0.5 mg, LANOXIN), GlaxoSmithKline LLC (NDA 018118, capsule, 0.05 mg, 0.1 mg, 0.15 mg, 0.2 mg, LANOXICAPS).

[0195] As used herein, the individually named triterpenes can independently be selected upon each occurrence in their native (unmodified, free acid) form, in their salt form, in derivative form, prodrug form, or a combination thereof. Compositions containing and methods employing deuterated forms of the triterpenes are also within the scope of the invention.

[0196] The composition can be formulated in any suitable pharmaceutically acceptable dosage form. Parenteral, otic, ophthalmic, nasal, inhalable, buccal, sublingual, enteral,topical, oral, peroral, transdermal, and injectable dosage forms are particularly useful. Particular dosage forms include a solid or liquid dosage forms. Exemplary suitable dosage forms include tablet, capsule, paste, pill, caplet, troche, sache, solution, suspension, dispersion, vial, bag, bottle, injectable liquid, i.v. (intravenous), i.m. (intramuscular) or i.p. (intraperitoneal) administrable liquid and other such dosage forms known to the artisan of ordinary skill in the pharmaceutical sciences.

[0197] Suitable dosage forms for administering oleandrin (or digoxin) to an animal can be made according to known procedures wherein oleandrin (or digoxin) is used in place of another drug: Klink et al. (“Formulations of Veterinary Dosage Forms” in Development and Formulation of Veterinary Dosage Forms, 2nded., Eds. G.E. Hardee and J.D. Baggot, New York, CRC Press, 1998), Foster et al. (“Veterinary Dosage Forms” in Encyclopedia of Pharmaceutical Science and Technology, 4thed., Eds. J. Swarbrick, New York, CRC Press, 2015).

[0198] The desired dose for oral administration is up to 5 dosage forms although as few as one and as many as ten dosage forms may be administered as a single dose. Doses will be administered according to dosing regimens that may be predetermined and / or tailored to achieve specific therapeutic response or clinical benefit in an animal.

[0199] The cardiac glycoside can be present in a dosage form in an amount sufficient to provide an animal with an initial dose of oleandrin of about 20 to about 100 microg, about 12 microg to about 300 microg, or about 12 microg to about 120 microg. For example, a dosage form can comprise about 20 of oleandrin to about 100 microg, about 0.01 microg to about 100 mg or about 0.01 microg to about 100 microg oleandrin, oleandrin extract or extract of Nerium sp. containing oleandrin.

[0200] The composition can be included in an oral dosage form. Some embodiments of the dosage form are not enteric coated and release their charge of cardiac glycoside composition within a period of 0.5 to 1 hours or less. Some embodiments of the dosage form are enteric coated and release their charge of cardiac glycoside downstream of the stomach, such as from the jejunum, ileum, small intestine, and / or large intestine (colon). Enterically coated dosage forms will release cardiac glycoside into the systemic circulation within 1-10 hr after oral administration.

[0201] The composition can be included in a rapid release, immediate release, controlled release, sustained release, prolonged release, extended release, burst release, continuous release, slow release, or pulsed release dosage form or in a dosage form that exhibits two ormore of those types of release. The release profile of cardiac glycoside from the dosage form can be a zero order, pseudo-zero, first order, pseudo-first order or sigmoidal release profile. The plasma concentration profile for cardiac glycoside in an animal to which the CGCC is administered can exhibit one or more maxima.

[0202] The anticipated oleandrin plasma concentration (Cmax or Cavg as measure in a 24-h period) will be in the range of about 0.005 to about 5 ng / ml, about 0.005 to about 4 ng / mL, about 0.005 to about 3 ng / mL, about 0.005 to about 2 ng / mL, or about 0.005 to about 2 ng / mL. A veterinary clinician will use known dose escalation and de-escalation protocols to determine the appropriate dose of oleandrin or digoxin to be safely administered per day.

[0203] It should be noted that a compound herein might possess one or more functions in a composition or formulation of the invention. For example, a compound might serve as both a surfactant and a water miscible solvent or as both a surfactant and a water immiscible solvent.

[0204] A liquid composition can comprise one or more pharmaceutically acceptable liquid carriers. The liquid carrier can be an aqueous, non-aqueous, polar, non-polar, and / or organic carrier. Liquid carriers include, by way of example and without limitation, a water miscible solvent, water immiscible solvent, water, buffer and mixtures thereof.

[0205] As used herein, the terms “water soluble solvent” or “water miscible solvent”, which terms are used interchangeably, refer to an organic liquid which does not form a biphasic mixture with water or is sufficiently soluble in water to provide an aqueous solvent mixture containing at least five percent of solvent without separation of liquid phases. The solvent is suitable for administration to animals. Exemplary water soluble solvents include, by way of example and without limitation, PEG (polyethylene glycol)), PEG 400 (poly(ethylene glycol having an approximate molecular weight of about 400), ethanol, acetone, alkanol, alcohol, ether, propylene glycol, glycerin, triacetin, polypropylene glycol), PVP (poly(vinyl pyrrolidone)), dimethylsulfoxide, N,N-dimethylformamide, formamide, N,N-dimethylacetamide, pyridine, propanol, N-methylacetamide, butanol, soluphor (2-pyrrolidone), pharmasolve (N-methyl-2-pyrrolidone).

[0206] As used herein, the terms “water insoluble solvent” or “water immiscible solvent”, which terms are used interchangeably, refer to an organic liquid which forms a biphasic mixture with water or provides a phase separation when the concentration of solvent in water exceeds five percent. The solvent is suitable for administration to animals. Exemplary water insoluble solvents include, by way of example and without limitation,medium / long chain triglycerides, oil, castor oil, corn oil, vitamin E, vitamin E derivative, oleic acid, fatty acid, olive oil, softisan 645 (Diglyceryl Caprylate / Caprate / Stearate / Hydroxy stearate adipate), miglyol, captex (Captex 350: Glyceryl Tricaprylate / Caprate / Laurate triglyceride; Captex 355: Glyceryl Tricaprylate / Caprate triglyceride; Captex 355 EP / NF: Glyceryl Tricaprylate / Caprate medium chain triglyceride).

[0207] Suitable solvents are listed in the “International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidance for industry Q3C Impurities: Residual Solvents” (1997), which makes recommendations as to what amounts of residual solvents are considered safe in pharmaceuticals. Exemplary solvents are listed as class 2 or class 3 solvents. Class 3 solvents include, for example, acetic acid, acetone, anisole, 1 -butanol, 2-butanol, butyl acetate, tert-butlymethyl ether, cumene, ethanol, ethyl ether, ethyl acetate, ethyl formate, formic acid, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, methyl- 1 -butanol, methylethyl ketone, methylisobutyl ketone, 2-methyl-l -propanol, pentane, 1 -pentanol, 1- propanol, 2-propanol, or propyl acetate.

[0208] Other materials that can be used as water immiscible solvents in the invention include: Captex 100: Propylene Glycol Dicaprate; Captex 200: Propylene Glycol Dicaprylate / Di caprate; Captex 200 P: Propylene Glycol Dicaprylate / Dicaprate; Propylene (jlycol Dicaprylocaprale: Captex 300: Glyceryl Tricaprylate / Caprate; Captex 300 EP / NF: Glyceryl Tricaprylate / Caprate Medium Chain Triglycerides; Captex 350: Glyceryl Tricaprylate / Caprate / Laurate; Captex 355: Glyceryl Tricaprylate / Caprate; Captex 355 EP / NF: Glyceryl Tricaprylate / Caprate Medium Chain Triglycerides; Captex 500: Triacetin; Captex 500 P: Triacetin (Pharmaceutical Grade); Captex 800: Propylene Glycol Di (2- Ethythexanoate); Captex 810 D: Glyceryl Tricaprylate / Caprate / Linoleate; Captex 1000: Glyceryl Tricaprate; Captex CA: Medium Chain Triglycerides; Captex MCT-170: Medium Chain Triglycerides; Capmul GMO: Glyceryl Monooleate; Capmul GMO-50 EP / NF: Glyceryl Monooleate; Capmul MCM: Medium Chain Mono- & Diglycerides; Capmul MCM C8: Glyceryl Monocaprylate; Capmul MCM CIO: Glyceryl Monocaprate; Capmul PG-8: Propylene Glycol Monocaprylate; Capmul PG-12: Propylene Glycol Monolaurate; Caprol 10G10O: Decaglycerol Decaoleate; Caprol 3 GO: Triglycerol Monooleate; Caprol ET: Polyglycerol Ester of Mixed Fatty Acids; Caprol MPGO: Hexaglycerol Dioleate; Caprol PGE 860: Decaglycerol Mono-, Dioleate.

[0209] As used herein, a “surfactant” refers to a compound that comprises polar or charged hydrophilic moieties as well as non-polar hydrophobic (lipophilic) moieties; i.e., a surfactant is amphiphilic. The term surfactant may refer to one or a mixture of compounds. A surfactant can be a solubilizing agent, an emulsifying agent or a dispersing agent. A surfactant can be hydrophilic or hydrophobic.

[0210] The hydrophilic surfactant can be any hydrophilic surfactant suitable for use in pharmaceutical compositions. Such surfactants can be anionic, cationic, zwitterionic or nonionic, although non-ionic hydrophilic surfactants are presently preferred. As discussed above, these non-ionic hydrophilic surfactants will generally have HLB values greater than about 10. Mixtures of hydrophilic surfactants are also within the scope of the invention.

[0211] Similarly, the hydrophobic surfactant can be any hydrophobic surfactant suitable for use in pharmaceutical compositions. In general, suitable hydrophobic surfactants will have an HLB value less than about 10. Mixtures of hydrophobic surfactants are also within the scope of the invention.

[0212] Examples of additional suitable solubilizer include: alcohols and polyols, such as ethanol, isopropanol, butanol, benzyl alcohol, ethylene glycol, propylene glycol, butanediols and isomers thereof, glycerol, pentaerythritol, sorbitol, mannitol, transcutol, dimethyl isosorbide, polyethylene glycol, polypropylene glycol, polyvinylalcohol, hydroxypropyl methylcellulose and other cellulose derivatives, cyclodextrins and cyclodextrin derivatives; ethers of polyethylene glycols having an average molecular weight of about 200 to about 6000, such as tetrahydrofurfuryl alcohol PEG ether (glycofurol, available commercially from BASF under the trade name Tetraglycol) or methoxy PEG (Union Carbide); amides, such as 2-pyrrolidone, 2-piperidone, caprolactam, N- alkylpyrrolidone, N-hydroxyalkylpyrrolidone, N-alkylpiperidone, N-alkylcaprolactam, dimethylacetamide, and polyvinypyrrolidone; esters, such as ethyl propionate, tributyl citrate, acetyl tri ethyl citrate, acetyl tributyl citrate, tri ethyl citrate, ethyl oleate, ethyl caprylate, ethyl butyrate, triacetin, propylene glycol monoacetate, propylene glycol diacetate, caprolactone and isomers thereof, valerolactone and isomers thereof, butyrolactone and isomers thereof; and other solubilizers known in the art, such as dimethyl acetamide, dimethyl isosorbide (Arlasolve DMI (ICI)), N-methyl pyrrolidones (Pharmasolve (ISP)), monooctanoin, diethylene glycol nonoethyl ether (available from Gattefosse under the trade name Transcutol), and water. Mixtures of solubilizers are also within the scope of the invention.

[0213] Except as indicated, compounds mentioned herein are readily available from standard commercial sources.

[0214] Although not necessary, the composition or formulation may further comprise one or more chelating agents, one or more preservatives, one or more antioxidants, one or more adsorbents, one or more acidifying agents, one or more alkalizing agents, one or more antifoaming agents, one or more buffering agents, one or more colorants, one or more electrolytes, one or more salts, one or more stabilizers, one or more tonicity modifiers, one or more diluents, or a combination thereof.

[0215] The composition of the invention can also include oils such as fixed oils, peanut oil, sesame oil, cottonseed oil, com oil, coconut oil, or olive oil; fatty acids such as oleic acid, stearic acid and isostearic acid; and fatty acid esters such as ethyl oleate, isopropyl myristate, fatty acid glycerides and acetylated fatty acid glycerides. The composition can also include alcohol such as ethanol, isopropanol, hexadecyl alcohol, glycerol and propylene glycol; glycerol ketals such as 2,2-dimethyl-l,3-dioxolane-4-methanol; ethers such as polyethylene glycol) 450; petroleum hydrocarbons such as mineral oil and petrolatum; water; a pharmaceutically suitable surfactant, suspending agent or emulsifying agent; or mixtures thereof.

[0216] One or more of the components of the formulation can be present in its free base, free acid or pharmaceutically or analytically acceptable salt form. As used herein, “pharmaceutically or analytically acceptable salt” refers to a compound that has been modified by reacting it with an acid as needed to form an ionically bound pair. Examples of acceptable salts include conventional non-toxic salts formed, for example, from non-toxic inorganic or organic acids. Suitable non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfonic, sulfamic, phosphoric, nitric and others known to those of ordinary skill in the art. The salts prepared from organic acids such as amino acids, acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and others known to those of ordinary skill in the art. On the other hand, where the pharmacologically active ingredient possesses an acid functional group, a pharmaceutically acceptable base is added to form the pharmaceutically acceptable salt. Lists of other suitable salts are found in Remington 's Pharmaceutical Sciences, 17th. ed.,Mack Publishing Company, Easton, PA, 1985, p. 1418, the relevant disclosure of which is hereby incorporated by reference.

[0217] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with tissues of animals and without excessive toxicity, irritation, allergic response, or any other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0218] A dosage form can be made by any conventional means known in the pharmaceutical industry. A liquid dosage form can be prepared by providing at least one liquid carrier and CGCC composition in a container. One or more other excipients can be included in the liquid dosage form. A solid dosage form can be prepared by providing at least one solid carrier and CGCC composition. One or more other excipients can be included in the solid dosage form.

[0219] A dosage form can be packaged using conventional packaging equipment and materials. It can be included in a pack, bottle, via, bag, syringe, envelope, packet, blister pack, box, ampoule, or other such container.

[0220] The composition of the invention can be included in any dosage form. Particular dosage forms include solid or liquid dosage forms. Exemplary suitable dosage forms include tablet, capsule, pill, caplet, troche, sache, and other such dosage forms known to the artisan of ordinary skill in the pharmaceutical sciences.

[0221] The CGCC can further comprise at least one cardiac glycoside-metabolism inhibitor, at least one cardiac glycoside-digestion inhibitor, at least one enzyme inhibitor, or a combination thereof. A cardiac glycoside-metabolism inhibitor is a compound that inhibits metabolism of a cardiac glycoside. A cardiac glycoside-digestion inhibitor is a compound that inhibits digestion of a cardiac glycoside. An enzyme inhibitor is a compound that inhibits an enzyme. The metabolism or digestion can be caused by the animal or one or more microbes in the animal. These categories of inhibitors are herein referred to together more broadly as inhibitors. The purpose of said inhibitors is to reduce the rate of metabolism or digestion of the cardiac glycoside, thereby increasing the plasma concentration half-life of the cardiac glycoside in the animal.

[0222] In view of the above description and the examples below, one of ordinary skill in the art will be able to practice the invention as claimed without undue experimentation. The foregoing will be better understood with reference to the following examples that detailcertain procedures for the preparation of embodiments of the present invention. All references made to these examples are for the purposes of illustration. The following examples should not be considered exhaustive, but merely illustrative of only a few of the many embodiments contemplated by the present invention.Example 1Supercritical fluid extraction of powdered oleander leavesMethod A. With carbon dioxide.

[0223] Powdered oleander leaves were prepared by harvesting, washing, and drying oleander leaf material, then passing the oleander leaf material through a comminuting and dehydrating apparatus such as those described in U.S. Patent Nos. 5,236,132, 5,598,979, 6,517,015, and 6,715,705. The weight of the starting material used was 3.94 kg.

[0224] The starting material was combined with pure CO2 at a pressure of 300 bar (30 MPa, 4351 psi) and a temperature of 50°C (122°F) in an extractor device. A total of 197 kg of CO2 was used, to give a solvent to raw material ratio of 50: 1. The mixture of CO2 and raw material was then passed through a separator device, which changed the pressure and temperature of the mixture and separated the extract from the carbon dioxide.

[0225] The extract (65 g) was obtained as a brownish, sticky, viscous material having a nice fragrance. The color was likely caused by chlorophyll and other residual chromophoric compounds. For an exact yield determination, the tubes and separator were rinsed out with acetone and the acetone was evaporated to give an addition 9 g of extract. The total extract amount was 74 g. Based on the weight of the starting material, the yield of the extract was 1.88%. The content of oleandrin in the extract was calculated using high pressure liquid chromatography and mass spectrometry to be 560.1 mg, or a yield of 0.76%.Method B. With mixture of carbon dioxide and ethanol

[0226] Powdered oleander leaves were prepared by harvesting, washing, and drying oleander leaf material, then passing the oleander leaf material through a comminuting and dehydrating apparatus such as those described in U.S. Patent Nos. 5,236,132, 5,598,979, 6,517,015, and 6,715,705. The weight of the starting material used was 3.85 kg.

[0227] The starting material was combined with pure CO2 and 5% ethanol as a modifier at a pressure of 280 bar (28 MPa, 4061 psi) and a temperature of 50°C (122°F) in an extractor device. A total of 160 kg of CO2 and 8 kg ethanol was used, to give a solvent to raw material ratio of 43.6 to 1. The mixture of CO2, ethanol, and raw material was then passed througha separator device, which changed the pressure and temperature of the mixture and separated the extract from the carbon dioxide.

[0228] The extract (207 g) was obtained after the removal of ethanol as a dark green, sticky, viscous mass obviously containing some chlorophyll. Based on the weight of the starting material, the yield of the extract was 5.38%. The content of oleandrin in the extract was calculated using high pressure liquid chromatography and mass spectrometry to be 1.89 g, or a yield of 0.91%.Example 2Water extraction of powdered oleander leaves.

[0229] Water extraction was used to extract oleandrin and other active components from oleander leaves. Examples of hot water extraction processes can be found in patent nos. US 5,135,745 and US 5,869,060. Examples of cold or ambient temperature water extraction processes can be found in publication No. US 2007 / 0154573 Al.Hot water extraction

[0230] A hot water extraction was carried out using 5 g of powdered oleander leaves. Ten volumes of boiling water (by weight of the oleander starting material) were added to the powdered oleander leaves and the mixture was stirred constantly for 6 hours. The mixture was then filtered and the leaf residue was collected and extracted again under the same conditions. The filtrates were combined and lyophilized. The appearance of the extract was brown. The dried extract material weighed about 1.44 g. 34.21 mg of the extract material was dissolved in water and subjected to oleandrin content analysis using high pressure liquid chromatography and mass spectrometry. The amount of oleandrin was determined to be 3.68 pg. The oleandrin yield, based on the amount of extract, was calculated to be 0.26%.Hot water extraction

[0231] Cold water extraction was conducted by placing 1 Kg of Nerium oleander plant material (ground leaves and stems) with 8 Kg of water and stirring the mixture for 1-12 hours. The mixture was then filtered and the filtrate was used as is or freeze-dried to a powder.OCC from water extract

[0232] A suitable aqueous liquid composition was prepared by dissolving 300 mg of dried extract material (containing about 350-400 pg of oleandrin (OLE) per g of driedextract) in 20 mL of water to provide, thereby resulting in a concentration of 15 mg of extract / mL (about 17.5-20 pg OLE / mL). Solutions having higher and lower concentrations of OLE were made.Example 3Preparation of veterinary compositions.Method A. Cremophor-based drug delivery system

[0233] The following ingredients were provided in the amounts indicated.

[0234] The excipients were dispensed into ajar and shook in a New Brunswick Scientific C24KC Refrigerated Incubator shaker for 24 hours at 60°C to ensure homogeneity. The samples were then pulled and visually inspected for solubilization. Both the excipients and OCC were totally dissolved for all formulations after 24 hours.Method B. GMO / Cremophor-based drug delivery system

[0235] The following ingredients were provided in the amounts indicated.

[0236] The procedure of Method A was followed.Method C. Labrasol-based drug delivery system

[0237] The following ingredients were provided in the amounts indicated.

[0238] The procedure of Method A was followed.Method D. Vitamin E-TPGS based micelle forming system

[0239] The following ingredients were provided in the amounts indicated.

[0240] The procedure of Method A was followed.Method E. Multi-component drug delivery system

[0241] The following ingredients were provided in the amounts indicated.

[0242] The procedure of Method A was followed.Method F. Multi-component drug delivery system

[0243] The following ingredients were provided in the amounts indicated and included in a capsule.

[0244] The procedure of Method A was followed.Example 4Preparation of enteric coated capsulesStep I: Preparation of liquid-fdled capsule

[0245] Hard gelatin capsules (50 counts, 00 size) were filled with a liquid composition of Example 3. These capsules were manually filled with 800 mg of the formulation and then sealed by hand with a 50% ethanol / 50% water solution. The capsules were then banded by hand with 22% gelatin solution containing the following ingredients in the amounts indicated.

[0246] The gelatin solution mixed thoroughly and allowed to swell for 1-2 hours. After the swelling period, the solution was covered tightly and placed in a 55 °C oven and allowed to liquefy. Once the entire gelatin solution was liquid, the banding was performed

[0247] Using a pointed round 3 / 0 artist brush, the gelatin solution was painted onto the capsules. Banding kit provided by Shionogi was used. After the banding, the capsules were kept at ambient conditions for 12 hours to allow the band to cure.Step II: Coating of liquid-filled capsule

[0248] A coating dispersion was prepared from the ingredients listed in the table below.

[0249] If banded capsules according to Step I were used, the dispersion was applied to the capsules to a 20.0 mg / cm2coating level. The following conditions were used to coat the capsules.* Spray nozzle was set such that both the nozzle and spray path were under the flow path of inlet air.Example 5Treatment of Giardiasis in an animal- all dosage forms

[0250] An animal presenting with giardiasis is chronically administered OCC according to a prescribed dosing regimen for a period of time. The animal’s level of response isdetermined periodically to determine if the target treatment goal (reduction or elimination of giardia infection or of diarrhea associated with giardia infection) has been achieved. If the level of response is too low at one dose, then the dose is escalated according to a predetermined dose escalation schedule until the desired level of therapeutic response in the animal is achieved. Treatment of the animal with OCC composition is continued as needed, and the dose or dosing regimen can be adjusted as needed until the animal reaches the target treatment goal. The animal is maintained on treatment as long as desired or as long as it is prone to exhibit symptoms associated with giardiasis.Example 6Intramuscular Administration of OCC to Dogs (Safety study)

[0251] A stock aqueous solution containing ANVIRZEL powder (300 mg; ANVIRZEL contained 350-400 microg oleandrin (OLE) per g of powder) dissolved in water for injection (20 mL) was prepared (resulting concentration: 15 mg ANVIRZEL / mL; 17.5-20 microg OLE / mL).

[0252] Beagle dogs (16 male, 16 female) ranging in weight from 9.4-11.5 kg (males) and 6.5-10.7 kg (females) were selected for the study. The age of the dogs ranged from 7-11 months on day-1 of administration. Each gender was divided into four cohorts according to the dose (amount of stock solution) administered once daily (for 28 days) via i.m. injection. Water for injection was used as the control.

[0253] No mortality was observed. No drug-related undesirable side effects were observed.Example 7Preparation of a tablet composition

[0254] An initial tabletting mixture of 3% Syloid 244FP and 97% microcrystalline cellulose (MCC) was mixed. Then, an existing batch of composition prepared according to Example 3 was incorporated into the Syloid / MCC mixture via wet granulation. This mixture is labeled "Initial Tabletting Mixture) in the table below. Additional MCC was added extra- granularly to increase compressibility. This addition to the Initial Tabletting Mixture was labeled as "Extra-granular Addition." The resultant mixture from the extra-granular addition was the same composition as the "Final Tabletting Mixture."Extragranular additionFinal Tabletting Mixture:AbbreviatedFinal Tabletting Mixture:Detailed

[0255] Syloid 244FP is a colloidal silicon dioxide manufactured by Grace Davison. Colloidal silicon dioxide is commonly used to provide several functions, such as an adsorbant, glidant, and tablet disintegrant. Syloid 244FP was chosen for its ability to adsorb 3 times its weight in oil and for its 5.5 micron particle size.Example 8HPLC analysis of solutions containing oleandrin

[0256] Samples (oleandrin standard, SCF extract and hot-water extract) were analyzed on HPLC (Waters) using the following conditions: Symmetry C18 column (5.0 pm, 150 x4.6 mm I.D.; Waters); Mobile phase of MeOH:water = 54: 46 (v / v) and flow rate at 1.0 ml / min. Detection wavelength was set at 217 nm. The samples were prepared by dissolving the compound or extract in a fixed amount of HPLC solvent to achieve an approximate target concentration of oleandrin. The retention time of oleandrin can be determined by using an internal standard. The concentration of oleandrin can be determined / calibrated by developing a signal response curve using the internal standard.Example 9Preparation of Veterinary Pharmaceutical Compositions

[0257] A pharmaceutical composition of the invention were prepared according to any of the following methods. Mixing was done under wet or dry conditions as needed. The pharmaceutical compositions could be compacted, ground, and / or dried during preparation. The pharmaceutical composition were portioned into dosage forms described in this disclosure.Method A.

[0258] At least one pharmaceutical excipient is mixed with at least one OCC disclosed herein. For example, maltodextrin was mixed with an organic liquid extract of Nerium oleander, and the mixture was then dried to form a powdered, which in some cases was ground. The oleandrin content of the final composition was determined by HPLC.Method B.

[0259] At least one pharmaceutical excipient is mixed with oleandrin. For example, maltodextrin was mixed with an organic solution (alcohol) in which oleandrin was dissolved. The mixture was then dried to form a powdered, which in some cases was ground. The oleandrin content of the final composition was determined by HPLC.Method C.

[0260] At least one pharmaceutical excipient is mixed with an extract containing oleandrin.Method D.

[0261] At least one pharmaceutical excipient is mixed with a combination of two or more extracts containing oleandrin.Method E.

[0262] At least one pharmaceutical excipient is mixed with any of the compositions of Methods A-D and at least one triterpene. The triterpenes are selected from the group consisting of oleanolic acid, ursolic acid, and betulinic acid.Method F.

[0263] At least one pharmaceutical excipient is mixed with any of the compositions of Methods A-E and at least one compound selected from the group consisting of oleandrigenin, desacetyl-oleandrin, gitoxigenin, kanerocin, kanerodione, Nerium F, neritaloside, odoroside, adynerin, and odoroside-G-acetate.Method G.

[0264] At least one filler is mixed with dried ground Nerium sp. plant material. For example, leaves and stems were dried and ground to a powder, which was then mixed to homogeneity with a powdered filler suitable for use in animal feed to form a food or health supplement. The powdered carriers evaluated include maltodextrin, cornmeal, wheat bran, rice bran, and corncob. In some cases, at least one sweetener or a combination of sweeteners was added to the supplement.

[0265] The veterinary composition of any one of methods A-G can further comprise polyphenol(s), carbohydrate(s), flavonoid(s), amino acid(s), soluble protein(s), cellulose, starch, alkaloid(s), saponin(s), tannin(s), or a combination thereof.Example 10Preparation of Triterpene Mixtures

[0266] The following compositions were made by mixing the specified triterpenes in the approximate molar ratios indicated.

[0267] For each composition, three different respective solutions were made, whereby the total concentration of triterpenes in each solution was approximately 9 pM, 18 pM, or 36 pM.Example 11Preparation of Topical Composition

[0268] Topical aqueous liquid compositions were prepared by mixing the following components in the approximate amounts indicated. The compositions were included in bottles or spray bottles intended for multi-use, e.g. an amount sufficient to provide doses for a treatment period of one week up to three months.Ingredient Content water 55-60% (q.s) glycerin 15-25% polysorbate 20 5-10% propylene glycol 4-6% hydrolyzed collagen 0.5-5% aloe vera 200x 0.5-5% tocopherol (vitamin e) 0.5-3% pram oxine 0.5-2% sodium PCA 0.5-5% caprylyl glycol, hexylene glycol, phenoxyethanol 0.5-2%0.01-10 pg / mL Oleandrin or 0.1-5 pg / mL allantoin 0.05-0.5% chamomile oil 0.01-0.05% clove essential oil 0.01-0.05%Dimethicone 0.05-2%

[0269] When included in a spray bottle, the amount of fluid per spray varied from 0.25 mL to 2.5 mL.Example 12Treatment of Respiratory distress (oral administration)

[0270] Cows, pigs, dogs or cats presenting with respiratory distress are orally administered oleandrin containing composition. The animals may also be administered at least one transfer factor, at least one glucan, at least one probiotic.

[0271] Animals are administered an oral OCC as described herein, in particular of the formulations containing any of the following extracts: supercritical fluid (SCF) extract, ethanolic (EtOH) extract, aqueous ethanolic (wEtOH) extract, methanolic (MeOH) extract, water (W) extract, subcritical fluid (SbCF) extract, and combinations thereof. Specific extracts can be selected from PB 1-05204 (SCF), PBI-01220 (EtOH), PBI-1X (aqueous EtOH), PBI-06150 (SbCF+EtOH), PBI-01207 (W). The extract can be combined with feedstuff, liquid (such as water, milk, or oil), or any edible excipient if needed.

[0272] The animal is orally administered 1-4 doses per day of one or more of the above compositions according to a prescribed dosing regimen described in this specification for a period of time. The animal’s level of clinical response is determined periodically. The level of therapeutic response can be determined by determining the animal’s reduction or elimination of respiratory distress. If the level of clinical response is too low at one dose, then the dose is escalated according to a predetermined dose escalation schedule until the desired level of clinical response in the animal is achieved. Chronic daily treatment of the animal with the composition is continued as needed and the dose or dosing regimen can be adjusted as needed until the animal reaches the desired clinical endpoint.Example 13Treatment and Prevention of Diarrhea and Microbial Infection in a Population of Puppies

[0273] Products administered: a) PBI-06150 (PBI; about 12-15 (usually 13-14) microg of oleandrin / mL; Phoenix Biotechnology, Inc., San Antonio, TX) comprises a combination subcritical CO2 liquid extract and ethanol extract of Nerium oleander leaves diluted into fractionated medium chain triglyceride; b) Paste (PREMIER RPM; described below; provided by Ramaekers Nutrition; California, USA; containing transfer factor mix, glucan and other ingredients); c) fenbendazole (FEN; 100 mg / mL); d) ponazuril (PON; 50 mg / mL); e) bordetella intranasal or oral vaccine (BDTV); f) NOB IV AC PUPPY DPV subcutaneous vaccine (NOBV; canine distemper virus, adenovirus type 1 and 2, canine parainfluenza virus, and canine parvovirus); g) metronidazole benzoate (MET; 200 mg / mL); h) azithromycin (AZT; 50 mg / mL); i) CLEAN EARS otic solution (CE); j) NEOPAR subcutaneous parvo virus vaccine (NEOP); k) NEOTECH DA2 subcutaneous canine distemper adenovirus type 2 vaccine (NEOT); 1) 7-way with coronavirus subcutaneous vaccine (7XCV); m) pyrantel (PYR; 50 mg / mL).

[0274] Prior to administration, some of the PREMIER RPM powder was converted to a paste by dissolving it in water according to the following ratio: 1 teaspoon (4 g powder) ofpowder per 5 mL of water. The product was administered as a paste or powder depending upon the age of the puppy.

[0275] Puppies (280) in a large-scale breeding facility were administered the products listed above according to the following schedules. Administration began when the puppies were 2 days old.

[0276] Through the first 5 weeks of age the puppies were given the PREMIER PRM (described further herein) as a paste containing oleandrin (in the form of the PBI-06150 oilbased liquid). From the age of 2 days to 9 weeks of age, the puppies were given the paste twice weekly (once every 3-4 days). The corresponding doses of oleandrin, transfer factor, and glucan blend were as follows.BW denotes bodyweight.

[0277] The Premier RPM (Stress pack) formulation used in this study contained the following ingredients in the amounts specified.

[0278] Additional excipients included dextrose, sodium chloride, silicon dioxide, citric acid, glycine, flavor.

[0279] Paste was made by mixing the above ingredients with a sufficient amount of coconut oil and starch to form the paste. The paste was administered orally.

[0280] The treatment was effective at improving GI health and respiratory health. None of the treated puppies exhibited respiratory distress after treatment and only one litter out of several hundred puppies showed any sign of GI distress (diarrhea), both results of which are remarkable because in this kennel the use of transfer factor and glucan, absent oleandrin, still resulted in about 30% of the puppies exhibiting GI distress. The puppies also exhibited increased energy levels and firm stools.Example 14Treatment of Pruritus in an animal (buccal / sublingual)Method A. administration of oleandrin-containing aqueous solution

[0281] A dog presenting with pruritus was administered the aqueous liquid composition of Example 2. It contained 0.1-5 pg OLE / mL. The composition (0.25-1 mL) was administered (1-4 times daily) directly to the dog’s mouth, thereby resulting in a combination of buccal and sublingual administration. The dog exhibited relief from the itching. Both inflammation and itchiness were relieved. Daily administration 1-4 times per day was continued and the dog exhibited no return of the pruritus and no composition- related side effects.Method B. administration of oleandrin-containing oil-based solution

[0282] A dog presenting with pruritus was administered the MCT-based composition of Example 21. It contained 12-25 pg OLE / mL. A few drops of the composition (0.25-1 mL) is administered (1-4 times daily) directly to the dog’s mouth, thereby resulting in a combination of buccal and sublingual administration. The dog exhibited relief from the itching. Both inflammation and itchiness were relieved. Daily administration 1-4 times per day is continued and the dog exhibits no return of the pruritus and no composition-related side effects.Example 15Treatment of Pruritus in an animal (topical)

[0283] A dog presenting with pruritus was administered the aqueous liquid composition of Example 11. The composition was sprayed (1-4 pumps) directly onto the itchy area of the dog and rubbed into the skin and fur / hair. The dog exhibited almost immediate relief from the itching. Both inflammation and itchiness were relieved. Daily administration 2-4 times per day was continued, and the dog exhibited no return of the pruritus and no composition-related side effects.Example 16Statistical Analysis

[0284] The statistical significance of experimental data sets was determined using unpaired two-tailed Student’s / -tests (alpha =0.05) and calculated -values using the Shapiro-Wilk normality test and Graphpad Prism 7.03 software. The / 3-values were defined as: 0.1234 (ns), 0.0332 (*), 0.0021 (**), 0.0002 (***), <0.0001 (****). Unless otherwise noted, error bars represent the SEM from at least three independent experiments.Example 17Preparation of aqueous ethanolic extract of Nerium oleander

[0285] The purpose of this was to prepare an ethanolic extract by extraction of Nerium oleander biomass (plant material) with aqueous ethanol.

[0286] Ground dried leaves were repeatedly treated with aqueous ethanol (60-70% v / v ethanol; 40-35% v / v water). In some cases, the temperature was above ambient, e.g. hot (40°C to refluxing). The combined ethanolic supernatants were combined and filtered and then concentrated by evaporation in vacuo to reduce the amount of ethanol and water therein (by about 10-fold) and provide crude ethanolic extract comprising about 2-4 mg of oleandrin / mL of extract (which has about 50% v / v ethanol content).Example 18Preparation of Subcritical fluid extract of Nerium oleander

[0287] An improved process for the preparation of an oleandrin-containing extract was developed by employing subcritical liquid extraction rather than supercritical fluid extraction of Nerium oleander biomass.

[0288] Dried and powdered biomass was placed in an extraction chamber, which was then sealed. Carbon dioxide (about 95% wt) and alcohol (about 5% wt; methanol or ethanol) were injected into the chamber. The interior temperature and pressure of the chamber were such that the extraction medium was maintained in the subcritical liquid phase, rather than the supercritical fluid phase, for a majority or substantially all of the extraction time period: temperature in the range of about 2°C to about 16°C (about 7°C to about 8°C), and pressure in the range of about 115 to about 135 bar (about 124 bar). The extraction period was about 4 h to about 12 h (about 6 to about 10 h). The extraction milieu was then filtered and the supernatant collected. The carbon dioxide was vented from the supernatant, and the resulting crude extract was diluted into ethanol (about 9 parts ethanol : about 1 part extract)and frozen at about -50°C for at least 12 h. The solution was thawed and filtered (100 micron pore size filter). The filtrate was concentrated to about 10% of its original volume and then sterile filtered (0.2 micron pore size filter). The concentrated extract was then diluted with 50% aqueous ethanol to a concentration of about 1.5 mg of extract per mL of solution.

[0289] The resulting subcritical liquid (SbCL) extract comprised oleandrin and one or more other compounds extractable from Nerium oleander, said one or more other compounds being as defined herein.Example 19Preparation of ethanolic extract of Nerium oleander

[0290] The purpose of this was to prepare an ethanolic extract by extraction of Nerium oleander biomass with aqueous ethanol.

[0291] Ground dried leaves were repeatedly treated with ethanol. Loading ratios ranged from about 1 :2 to about 1 : 10 w / w parts of dried biomass to parts of ethanol. In some cases, the temperature was above ambient. The combined ethanolic supernatants were combined and filtered and then concentrated by evaporation in vacuo to reduce the amount of ethanol and water therein and provide crude ethanolic extract comprising about 25 mg of oleandrin / mL of extract (which has about 50% v / v ethanol content).Example 20Preparation of dosage form comprising a combination of extracts of Nerium oleander

[0292] The purpose of this was to prepare a dosage form (OCC, PBI-06150) containing a portion (1 wt %) of the ethanolic extract of Example 18 combined with a portion (1 wt %) of the SbCL extract of Example 19, medium chain triglyceride (95 wt %), and flavoring agent (3 wt %).Example 21Preparation of methanolic extract of Nerium oleander

[0293] The purpose of this was to prepare a methanolic extract by extraction of Nerium oleander biomass with methanol.

[0294] Ground dried leaves were repeatedly treated with methanol. In some cases, the temperature was above ambient, e.g. hot (>21°C to refluxing). The combined methanolic supernatants were combined and filtered and then concentrated by evaporation in vacuo tosubstantially to dryness and provide crude methanolic extract comprising about 2-4 mg of oleandrin / g of solid extract.Example 22Preparation of foodstuff containing oleandrin (oleander extract)

[0295] The purpose of this was to prepare an edible product comprising oleandrin or comprising an oleandrin-containing extract, in any suitable form.

[0296] An extract of Nerium oleander is prepared. Its solvent can be removed to either concentrate the extract or to remove the solvent completely. The resulting concentrate is mixed with one or more edible materials suitable for animals. The foodstuff can be any treat, food, food replacement, or liquid known to be suitable for consumption by animals, in particular dogs, cats, turkeys, pigs, cows, horses, sheep, and goats.

[0297] In particular examples, an organic liquid extract, aqueous extract, or aqueous organic liquid extract was mixed with an edible carrier, and the mixture was then dried to remove the liquid. The resulting mixture was then milled to a powder if needed. Plant based powdered edible carriers, as described herein, were used. The oleandrin content of the product was determined as described herein.Example 23Administration of oleandrin containing composition to dogs (oral) (Safety study)

[0298] The SCF extract (PBI-05204) was formulated as detailed above into a micelle forming delivery system, i.e. a composition that forms micelles when the composition is mixed with aqueous, such as water, buffer, saliva, gastric fluid, intestinal fluid, or colonic fluid. The content of OLE in the formulated composition was typically in the range of 0.05- 0.1%.

[0299] Formulated PBI-05204 composition or control vehicle were orally administered to beagle dogs at once daily doses of 0.1, 0.3, or 1.0 / 0.6 mg / kg oleander extract (equivalent to 2.3 pg, 6.9 pg, and 23 / 13.8 pg of oleandrin / kg, respectively) for 28 consecutive days followed by a 14-day recovery period. This resulted in a no-observable-adverse-effect-level (NOAEL) of 0.1 mg / kg. The adverse effects at 0.3 or 1.0 / 0.6 mg / kg were reversible following a 14-day recovery period.

[0300] In order to determine an approximate MTD, a dose-range finding (single dose) study of formulated PBI-05204 composition was conducted in beagle dogs. The relative drug toxicity in dogs treated with escalating oral doses was determined. One male and onefemale dog were provided the following escalating doses of 5, 10, and 20 mg / kg (equivalent to 115 pg, 230 pg, and 460 pg of oleandrin / Kg, respectively) followed by a 3-day washout / observation period between doses. The results in the male indicated a dosedependent increase in abnormal clinical observations consisting of vomiting, abnormal stool, increased salivation, labored breathing, decreased activity, and death. After the female dog received 20 mg / kg of formulated PBI-05204 (equivalent to 460 pg of oleandrin / Kg), a severe arrhythmia characterized as sinus arrest with ventricular escape rhythm.

[0301] A 28-day toxicity study of formulated PBI-05204 in beagle dogs followed by a 14-day recovery period was conducted. The objective of this study was to determine the potential systemic toxicity of test article administered once daily for 28 consecutive days via oral (gavage) administration followed by a 14-day recovery period. A total of 36 dogs were dosed with test article PBI-05204 containing 37.2 mg / mL (at concentrations of 0.1, 0.3, or 1.0 / 0.6 mg / kg) oleander extract (equivalent to 2.3, 6.9, 23 / 13.8 ug oleandrin / kg, respectively) or control vehicle PBI-05204 Control Vehicle (Placebo) via oral gavage once daily for 28 consecutive days (except for Group 4 animals). There were no early deaths during the study. All animals survived until their scheduled necropsy date. Test article- related clinical findings include the following: abnormal stool primarily in Groups 3 and 4 males, thin body only in Group 4 animals with a delayed onset in females when compared to males, decreased activity in Group 4 males only, inappetence in Group 4 males only, defecation decreased in Group 4 males only, predominant post-dose vomiting in Group 4 (males > females), predominant increased salivation in Group 4 (males > females), bloodshot eyes in Group 4 males only, predominant excessive lacrimation in Group 4 animals, and ptosis in Group 4 males only. Compared to Study Day 1, Study Day 29 bodyweights for Groups 3 and 4 males were decreased. There were no apparent test articleaffects on feed consumption with the exception of decreased feed intake in Group 4 males on Study Days 7 through 13. There were no apparent test article-affects on individual body temperatures with the exception of decreased body temperatures in Group 4 males on Study Day 8. Based on the measured hematology parameters, there were no apparent biological or toxicological findings. On Study Day 29, Group 4 male heart weight trends (absolute and relative) were decreased in comparison to concurrent controls. This finding appears to be test article-related. The administration of test article did not cause a discernable change in QRS duration, or QT interval, or QTcf or QTcv throughout this study. However, in dogs receiving 1.0 / 0.6 mg / kg / day, the heart rate was decreased. In dogs receiving 0.3 or 1.0 / 0.6mg / kg / day, atrioventricular conduction was slowed and indicated by prolongation of the PR interval and the development of secondary degree atrioventricular block. Also atrial premature depolarization developed in dogs receiving over 0.3 or 1.0 / 0.6mg / kg / day. The presence of these findings both before and after test article-administration on Study Week 4 suggests at least a 23 hour duration of the test article-induced changes. In conclusion, the administration of O. l; 0.3; or 1.0 / 0.6 mg / kg) oleander extract, or control vehicle PBI-05204 Control Vehicle (Placebo) via oral gavage once daily for 28 consecutive days (except for Group 4 animals) followed by a 14 day recovery period resulted in a no-observable-adverse- effect-level (NOAEL) of 0.1 mg / kg (equivalent to 2.3 ug oleandrin / kg). The adverse effects at 0.3 or 1.0 / 0.6 mg / kg were reversible following a 14-day recovery period.Example 24Treatment of amebic, protozoal, bacterial, or parasitic infection in a human (oral)

[0302] Campers, hikers, backpackers and other subjects participating in outdoor sports (wherein they may be subject to exposure to amebic, protozoal, bacterial, or parasitic infection) can be administered OCC (with or without other agents / compounds described in this specification) to prevent or treat giardiasis.

[0303] The subject is typically administered 0.2-100 microg of OLE in an OCC one or more times daily. The administration can be initiated prior to, during, or after exposure to the microbe causing the infection.

[0304] In particular cases, subjects were administered 2-25 microg of OLE twice daily during a backpacking, hiking, and camping trip that lasted several days. The trip was conducted in terrain having creeks known to possess diarrhea-inducing microbes, such as giardia. The subjects drank from untreated (meaning the water was not treated with an antimicrobial prior to drinking) creek water for several days. The subjects did not contract any corresponding infection or suffer from any symptoms associated with amebic, protozoal, bacterial, or parasitic infection, e.g. diarrhea or gastrointestinal pain.Example 25Formulations containing transfer factor, glucans, probiotics, nutrients and OCC

[0305] The following are exemplary formulations that are administered to improve health.

[0306] Transfer Factor Formula (No Encapsulation) (Amounts are in mg / lb of body weight unless otherwise stated)

[0307] * These amounts are calculated for livestock animals weighing about 450 to 1,000 pounds, goats weighing about 150 pounds, and dogs and cats weighing from about 8 to about 15 pounds.

[0308] Equine premix (Amounts in mg / lb of body weight unless otherwise stated). This formulation can be used for many different species. A sufficient amount of OCC is mixed with the premix such that a 0.5-2.0 oz dose of the final mixture provides the target dose of oleandrin on a microg OLE / Kg BW basis. The OLE dose is about 0.5-5.0 microg OLE / Kg BW or about 225-2250 microg OLE per dose.(*) Lactic acid generating bacteria is two-thirds of component and yeast is one-third; lactic acid generating bacteria is 500,000,000 CFU / gm, yeast (e.g., "Saccharomyces") 250,000,000 CFU / gm.

[0309] Canine preMix (Amounts in mg / lb of body weight unless otherwise stated). This formulation can be used for puppies and adult dogs. A sufficient amount of OCC is mixed with the premix such that a 0.5-2.0 oz dose of the final mixture provides the target dose of oleandrin on a microg OLE / Kg BW basis. The OLE dose is about 0.5-5.0 microg OLE / Kg BW or about 5-50 microg OLE per dose.(*) Lactic acid generating bacteria is two-thirds of component and yeast is one-third; lactic acid generating bacteria is 500,000,000 CFU / gm, yeast (e.g., "Saccharomyces") 250,000,000 CFU / gm.

[0310] Feline premix (Amounts in mg / lb of body weight unless otherwise stated). This formulation can be used for puppies and adult dogs. A sufficient amount of OCC is mixed with the premix such that a 0.5-2.0 oz dose of the final mixture provides the target dose ofoleandrin on a microg OLE / Kg BW basis. The OLE dose is about 0.5-5.0 microg OLE / Kg BW or about 2.0-20 microg OLE per dose.(*) Lactic acid generating bacteria is two-thirds of component and yeast is one-third; lactic acid generating bacteria is 500,000,000 CFU / gm, yeast (e.g., "Saccharomyces") 250,000,000 CFU / gm.

[0311] Livestock Stress Rumen By-Pass (Amounts in mg / lb of body weight unless otherwise stated)*These amounts are calculated for livestock animals weighing about 450 to 1,000 pounds, goats weighing about 150 pounds, and dogs and cats weighing from about 8 to about 15 pounds. Stabilized active ingredients are included in a formulation of 50% soybean oil and 50% active ingredient.

[0312] Additional exemplary formulations that can be mixed with OCC to provide improved health.

[0313] A 2000 mg per dose:*The colostrum blend indicated above contains higher molecular weight components including antibodies, praline-rich peptides, lactoferrin, growth factors in addition to the transfer factor filtrate.

[0314] Electrolyte, vitamin and probiotic blend: serving of 2000 mg.

[0315] Transfer factor controlled release formulation. OCC was added to this formulation.

[0316] Transfer factor rumen bypass (Stress pack) formulation. OCC was added to this formulation. Amounts expressed in mg / lb of body weight unless otherwise stated* Stabilized active ingredients are included in a formulation of 50% soybean oil and 50% active ingredient.

[0317] The following Active Blend is used in the formulations of the invention. The amounts indicated are per serving.

[0318] The following is a time release oral formulation. Once Active blend is coated with formulation below, the composition is formed into pellets. These pellets are placed inside gelatin capsules (size 7) for oral administration.

[0319] The following stress formulation is useful for many species of animals. It does not require encapsulation. Amounts are in mg / lb of body weight unless otherwise stated.

[0320] The above formulations are mixed with the appropriate amount of OCC or administered separately from the OCC.Example 26Reducing morbidity and mortality in a population of nonruminating calves

[0321] The following method and compositions were used to reduce the rate of occurrence of mortality, reduce the rate of occurrence of morbidity, and reduce the severity of morbidity in a population of nonruminating calves.

[0322] Subjects: high-risk nonruminating roping calves (averaging 100 Kg bodyweight (BW)) exhibiting a morbidity rate of about 40% and a mortality rate of 10-20% when untreated.

[0323] Products used: a) oil-based liquid containing Nerium oleander extract dissolved in MCT oil to provide an oleandrin content of about 13.5 microg / mL; b) immune primer mix paste (50:50 mix of actives (transfer factor, glucans, and other ingredients as set forth in Example 25 or otherwise herein) and soybean oil carrier; Ramaekers Nutrition, California).

[0324] Method A- comparative treatment without Nerium oleander extract. Calves were repeatedly administered 35 mL of immune primer mix paste. Only a minor reduction in mortality and morbidity was observed.

[0325] Method Bl- combination treatment. Upon arrival to the barn, calves (10) were administered Nerium oleander extract to provide a dose of 5.4 microg of oleandrin / Kg BW and 35 mL of immune primer mix paste for two consecutive days. No illness, morbidity or mortality were observed for at least 14 days after treatment.

[0326] Method B2- combination treatment. For five days after arrival to the barn, untreated calves (10) were monitored and found to be sluggish (unhealthy). After the five days, they were administered Nerium oleander extract to provide a dose of 5.4 microg of oleandrin / Kg BW and 35 mL of immune primer mix paste for two consecutive days. The calves recovered from their sluggishness, and no illness, morbidity or mortality were observed for at least 14 days after treatment.

[0327] Method B3- combination treatment. Upon arrival to the barn, calves (20) were administered Nerium oleander extract to provide a dose of 2.7 microg of oleandrin / Kg BW b.i.d. and 35 mL of immune primer mix paste for two consecutive days. No illness, morbidity or mortality were observed for at least 14 days after treatment.Example 27Increasing milk production in ruminating dairy cattle

[0328] The following method and compositions were used to improve the health of ruminating dairy cattle exhibiting morbidity and low milk production.

[0329] Subjects: Holstein dairy cows (mature, 1200-1500 lbs BW) exhibiting low milk production (about 45-50 Ibs / day). They did not have any disease; however, their poor overall health prior to treatment resulted in low daily milk production.

[0330] Product: powdered mineral mix supplement containing dried powdered Nerium oleander biomass (0.0025 lbs; containing 1000-1500 microg of oleandrin / Kg dry weight basis; primarily leaves and some stems) per pound of commercial mineral mix. The supplement was mixed in with feed prior to administration.

[0331] The cows were divided into two groups: a) untreated control (105 head); and b) treated (56 head). The cows were administered 4 oz of the product (equivalent to 0.4-0.6 microg of oleandrin) per head per day for a period of three weeks. Prior to administration, the baseline average milk production per head was determined to be about 50 lbs per day.

[0332] During the treatment period, the daily average milk production per head was calculated and expressed as the average increase in daily pounds of milk produced above the average baseline. During the first week, the cows exhibited an increase of 6.87 lbs of milk per head as compared to baseline. During the second week, the cows exhibited an increase of 6.47 lbs of milk per head as compared to baseline. During the first week, the cows exhibited an increase of 5.27 lbs of milk per head as compared to baseline.Example 28Administration of Nerium oleander extract to pigs

[0333] The following method was used to evaluate the safe administration of the MCT oil-based product (PBI-06150) described herein containing a combination of Nerium oleander subcritical extract and alcohol extract dissolved in the MCT oil to provide a concentration of about 13.5 microg of oleandrin / mL.

[0334] A nursing sow (weighing about 400 lbs or about 182 Kg) was administered product according to the following schedule and the content of oleandrin in plasma and milk were determined according to the method of Tor et al., who used HPLC-MSMS analysis.

[0335] A weaned piglet (about 23 days of age and weighing about 12.7 lbs) was administered a sufficient amount of PBI-06150 to provide 160 microg of OLE (a dose equivalent to 27.7 microg of OLE / Kg BW). Blood samples were drawn periodically, and their content of OLE determined. The following results were obtained.BLQ denotes below level of quantitation (below 2.5 ng / mL)

[0336] OLE tissue distribution data was obtained for four of the above piglets after euthanization: subjects 1 and 2 were euthanized 1 day after administration; and subjects 3 and 4 were euthanized 8 days after administration. Muscle tissue and organs were collected after euthanization and tested for OLE content by HPLC-MSMS. The following results were obtained.Example 29Administration of Nerium oleander extract to chickens

[0337] The following method was used to evaluate the safe administration of the hot water extract-based product (PBI-01207) described herein. The lyophilized dry extract contained approximately 1200 microg OLE / g. The lyophilized extract is water soluble. It was dissolved in drinking water which was provided ad libitum. The concentration of oleandrin (based upon the extract added) in the water was varied and increased over time as follows.

[0338] Twelve chicks (weighing about 30 g each) were started on drinking water containing such that average water consumption would provide a dose of about 5 microg OLE / Kg BW in a 24-h period. The chicks fared well, and no product-related side effects were observed.

[0339] A week later, the concentration of OLE in the water was increased to provide a dose of about 256 microg OLE / Kg BW in a 24-h period daily for seven days. The chicks fared well, and no product-related side effects were observed.

[0340] At day-30, the dose was reduced to 95 microg OLE / Kg per day based upon the current weight of the chicks.

[0341] At the end of three months, three chickens were separated from the rest. Their average weight was about 2.9 lb, so their daily dose was about 72 microg OLE / Kg. These chicks were bled, and their blood tested for oleandrin content. No OLE was detected in the blood. The three chicks were taken off product, and eight days later, they were euthanized. Muscle tissue samples were obtained and analyzed for OLE content. No OLE was detected in the tissue samples.

[0342] OLE was then included in both feed and water. At about five months, three chickens were separated. Based upon their current weight their total daily dose was about 142 microg OLE / Kg. The remaining chickens were administered a daily dose of about 40 microg / Kg. All chickens fared well.Example 30Administration of Nerium oleander biomass in combination with transfer factor, and glucan (with and without monensin) to cattle

[0343] The following method was used to evaluate the safe administration of Nerium oleander biomass in combination transfer factor, and glucan to determine the impact of the therapy upon feed conversion. The biomass contained approximately 400 microg OLE / g. The biomass, transfer factor, and glucan encapsulated as described herein to provide rumen bypass delivery. The target dose for OLE was about 0.5 microg / Kg BW per day.

[0344] High risk cattle (80) were divided into two groups: control and treatment groups. The study lasted 240 days. The treatment group received the OLE, transfer factor and glucan daily via feed. The transfer factor and glucan were administered as otherwise described herein. The control group received RUMENSIN 90 (containing 90.7 g of monensin per pound). The target dose for monensin was about 0.14-0.42 or 0.14-1.0 mg / lbs BW per day (about 185-660 mg / head / day for lactating cows or about 115-40 mg / head / day for dry cows. Dosing schedules for monensin are known (ww drags. ) .Example 31Chew formulation for cats and dogs

[0345] A chew formulation was made as follows:

[0346] The above formulation containing was mixed with Nerium oleander biomass or powdered concentrate containing Nerium oleander extract to the target concentration of oleandrin.Example 32Adult stress pre-mix formula for cattle

[0347] A powdered formulation was made as follows:

[0348] The above formulation containing was mixed with Nerium oleander biomass or powdered concentrate containing Nerium oleander extract to the target concentration of oleandrin.Example 33Montmorillonite

[0349] The composition of montmorillonite (which is colloidal clay that contains a mixture of trace elements) is described in US 6506413 Bl.Example 34Reducing morbidity and mortality in a population of pregnant sows

[0350] The following method and composition were used to reduce the rate of occurrence of mortality, reduce the rate of occurrence of morbidity, and reduce the severity of morbidity in a population of pregnant sows infected with (seropositive for) wild-types PRRSV (porcine reproductive respiratory syndrome virus). A reduction in abortions, stillbirths, and mummified embryos was observed for the treated sows as compared to the untreated control sows.

[0351] Subjects: 120 pregnant sows divided into two groups: Treatment group (60 sows treated with a health supplement and control group (60 untreated). The average bodyweightwas about 225 Kg bodyweight. PRRSV wild-type infection in all sows was confirmed by PCR.

[0352] Product used: a health supplement containing a powdered mixture of Nerium oleander plant material (dried leaves and stems), rice bran, and sweeteners. The concentration of oleandrin the health supplement was determined as described herein and was determined to be 500-1500 microg oleandrin / g depending upon the batch.

[0353] Protocol: The sows were divided into two groups: treatment group and control group. Starting at about 78-80 days into their gestation period, the treatment group sows were given the health supplement by mixing it with their daily ration of feed. The corresponding dose of oleandrin was in the range of 5-20 microg oleandrin / kg of body weight. The sows received daily doses of the supplement for 21 days. The average PRRSV titers for the two groups were measured by drawing blood samples from random sows within each group. The total number of aborted fetuses, stillbirth fetuses, and mummified fetuses were determined periodically and at the end of the evaluation period.

[0354] Result: as compared to the control group, the treatment group exhibited an improvement in the overall health of the sows and their farrowed piglets and also exhibited an overall reduction in aborted fetuses, stillbirth fetuses, and mummified fetuses. When comparing the average viral titer of the treatment group and the control group, a direct correlation between viral titer reduction and the observed improvements was not found.

[0355] As used herein, the terms “about” or “approximately” are taken to mean ±10%, ±5%, ±2.5% or ±1% of a specified valued. As used herein, the term “substantially” is taken to mean “to a large degree” or “at least a majority of’ or “more than 50% of’.

[0356] The above is a detailed description of particular embodiments of the invention. It will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims. All of the embodiments disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure.

Claims

CLAIMS1) A method of increasing feed conversion in an animal, the method comprising administering to the animal one or more doses of a cardiac glycoside-containing composition (CGCC), thereby increasing the animal’s feed conversion as compared to the animal’s feed conversion prior to administration.2) A method of increasing the feed conversion rate in a population of subjects, the method comprising administering to subjects of said population one or more dose of CGCC in amounts sufficient and at a frequency sufficient to increase the feed conversion rate of said subjects as compared to a population of similar subjects not administered the CGCC.3) The method of claim 1 or 2 wherein the feed conversion rate is based upon milk production or meat production.4) A method of reducing the rate of occurrence of mortality and morbidity, and reducing the severity of morbidity in a population of subjects, the method comprising administering to subjects of said population one or more dose of CGCC in amounts sufficient and at a frequency sufficient to reduce the rate of occurrence of mortality and / or morbidity, and / or reduce the severity of morbidity of said subjects as compared to a population of similar subjects not administered the CGCC.5) A method of improving GI, respiratory, and / or musculoskeletal health in an animal or human subject suffering from deteriorating GI, respiratory, and / or musculoskeletal heath, respectively, the method comprising administering to said subject a cardiac glycosidecontaining composition (CGCC), thereby improving the subject’s GI, respiratory, and / or musculoskeletal health.6) The method according to any one of the above claims wherein said mortality, morbidity, deteriorating GI health, deteriorating respiratory health, or deteriorating musculoskeletal health is not associated with a disease state caused by microbial infection.7) A method of preventing a subject from exhibiting one or more symptoms associated with protozoal infection, amebic infection, bacterial infection, or parasitic infection, the method comprising administering to said subject one or more effective doses of cardiac glycoside-containing composition (CGCC).8) A method of treating parasitic, amebic, bacterial, or protozoal infection in a subject, the method comprising administering to said a subject in need thereof one or more effective doses of cardiac glycoside-containing composition (CGCC).9) A method of preventing parasitic, amebic, bacterial, or protozoal infection in a subject, the method comprising administering to said a subject at risk of contracting said infection one or more effective doses of cardiac glycoside-containing composition (CGCC).10) The method according to any one of claims 7, 8, or 9, wherein the infection is caused by Campylobacter sp., Salmonella sp., Escherichia sp., Spirochaeta sp., Treponema sp., Borrelia sp., Leptospira sp., Mycobacterium sp., Shigella sp., Giardia sp., Coccidia sp., Eimeria sp., Sarcocystis sp., or Cryptosporidium sp.11) The method according to any one of the above claims, wherein the subject is suffering from diarrhea, loose stool, bloating, and / or gastrointestinal pain or discomfort prior to treatment.12) The method according to any one of the above claims, wherein said subject is also administered at least one other health-improving composition.13) The method according to claim 12, wherein the at least one other health-improving composition comprises a) at least one transfer factor; b) at least one glucan; c) at least one probiotic; d) at least one transfer factor and at least one glucan; e) at least one transfer factor, at least one glucan, and at least one probiotic; or f) at least one transfer factor and at least one probiotic.14) The method of claim 13, wherein a) the at least one transfer factor is selected from the group consisting of bovine colostrum transfer factor, avian transfer factor, ovine colostrum transfer factor, human colostrum transfer factor, caprine colostrum transfer factor, and a combination of any two or more thereof; b) the at least one glucan is obtained from mushroom; and / or c) the at least one probiotic is lactic acid generating bacteria.15) The method of claim 14, wherein a) mushroom is selected from the group consisting of Cordyceps sp., Cordyceps sinensis Alohaensis, Agaricus blazeii, Coriolus, Poira Cocos, Inonotus obliquus, Maitake sp., Shiitake sp., and combinations thereof; and / or b) lactic acid generating bacteria is selected from the group consisting of B. subtlis, B. longum, B. thermophilium, B. coagulans, L. acidophilus, E. faecium, and S. cerevisia, L. casei, L. plantarum, Pediococccus acidilacticii, Kluyveromyces marxianus fragillis and combinations thereof.16) The method according to any one of the above claims, wherein the subject is administered any one or more of the following: a) one or more compounds extracted from Nerium oleander plant material; b) one or more triterpenoid acids, e.g. one or more of oleanolic acid, ursolic acid, or betulinic acid; c) one or more deworming medications; d) one or more antibiotics; e) one or more antioxidants; f) one or more solvents; g) one or more oils; h) one or more compounds selected from the group consisting of ronidazole, tinidazole, metronidazole, nimorazole, flunidazole, dimetridazole, ipronazole, fenbendazole, albendazole, antidiarrheic, pyrantel, ponazuril, a 5-nitroimidazole antibiotic, sulfa-type antibiotic (e.g. sulfadimethoxine), amprolium (Albac / Amprol Hi-E [+ Bacitracin, + Ethopabate]), ponazuril, toltrazuril, ionophore(s) (e.g. rumensin), sulfaquinoxaline, sulfamethazine, lasalocid, decoquinate, monensin, and nitazoxanide; i) one or more antidiarrheic compounds; j) one or more preservatives; k) one or more feed materials; 1) one or more nutritional materials; m) one or more surfactants; n) one or more anti-foaming agents; o) one or more lubricants; p) one or more corticosteroids (glucocorticoids); q) collagen, e.g. hydrolyzed collagen; r) one or more carboxylic acids; s) one or more amino acids; t) one or more humectants; u) one or more buffering agents; v) one or more proteins; or w) one or more vaccines.17) The method according to any one of the above claims, wherein the subject is administered any one or more of the following: a) one or more deworming agents; b) one or more antidiarrheic agents; c) one or more anti-protozoa agents; d) one or more antibiotics; e) one or more probiotics; f) one or more transfer factors; g) one or more glucans; h) one or more antibacterials; or i) one or more antimicrobials.18) The method according to any one of the above claims, wherein a) the CGCC and other composition are both administered systemically; b) the CGCC is administered non- systemically, and the other composition is administered systemically; c) the CGCC is administered systemically, and the other composition is administered non-systemically; or d) the CGCC is administered orally, buccally, anally, vaginally, perianally, rectally, and / or sublingually, and the other composition is administered orally, buccally, and / or sublingually.19) The method according to any one of the above claims, wherein cardiac glycosidecontaining composition (CGCC) and other composition are administered a) in the same composition or in separate compositions; b) via the same route of administration or viadifferent routes of administration; c) simultaneously, sequentially, or in an overlapping manner; and / or d) with overlapping dosing periods or separate dosing periods.20) The method of any one of the above claims, wherein the subject is a dog, cat, cow, pig, sheep, goat, horse, chicken, turkey, pheasant, deer, or human.21) The method of any one of the above claims, wherein a) a dose of said CGCC composition is selected from the group of about 0.05-0.5 microg / kg / day, about 0.05-0.35 microg / kg / day, about 0.05-0.22 microg / kg / day, about 0.05-0.4 microg / kg / day, about 0.05-0.3 microg / kg / day, about 0.5 to about 500 microg / day or less, about 0.5 to about 400 microg / day or less, about 0.5 to about 300 microg / day or less, about 0.5 to about 200 microg / day or less, about 0.5 to about 100 microg / day or less, about 1 to about 80 microg / day, about 1.5 to about 60 microg / day, about 1.8 to about 60 microg / day, about 1.8 to about 40 microg / day, 140 microg to 315 microg per day, 20 to 750 microg / day, 0.01 to 100 microg / day, 12 microg to 300 microg, or 12 microg to 120 microg; or b) said CGCC comprises oleandrin (present in isolated form, as part of an extract, or within plant material, and wherein oral doses (one to four times daily) for oleandrin in animals are as follows: i) in dogs- less than about 100 microg / kg bodyweight, about 0.5-60 microg / kg bodyweight, about 0.1-10 microg / kg bodyweight, or about 0.1-5 microg / kg bodyweight; ii) in cows, pigs, goats, or sheep- less than about 100 micro / kg bodyweight, about 0.1-50 microg / kg bodyweight, about 0.1-25 microg / kg bodyweight, about 0.1-10 microg / kg bodyweight, or about 0.1-5 microg / kg bodyweight; iii) in cats- less than about 100 microg / kg bodyweight, about 0.5-50 microg / kg bodyweight, about 0.1-10 microg / kg bodyweight, or about 0.1-5 microg / kg body weight; iv) in horses- less than about 100 microg / kg bodyweight or 0.5-50 microg / kg bodyweight; v) in poultry (chickens, turkeys, etc.)- less than about 1000 micro / kg bodyweight, about 0.5-500 microg / Kg bodyweight, about 0.5-250 microg / Kg bodyweight, about 0.5-100 microg / Kg bodyweight, about 0.5-50 microg / Kg bodyweight, about 0.5-25 microg / Kg bodyweight, about 0.5-10 microg / Kg bodyweight, or about 0.5-5 microg / Kg bodyweight.22) The method of any one of the above claims, wherein following administration of said one or more doses, the plasma concentration of cardiac glycoside in said subject is a) in the range of about 0.05 to about 2 ng / ml, about 0.005 to about 10 ng / mL, about 0.005 to about 8 ng / mL, about 0.01 to about 7 ng / mL, about 0.02 to about 7 ng / mL,about 0.03 to about 6 ng / mL, about 0.04 to about 5 ng / mL, or about 0.05 to about 2.5 ng / mL, in terms of the amount of cardiac glycoside per mL of plasma; b) the plasma concentration of oleandrin in said subj ect is about 10 ng / mL or less, about 5 ng / mL or less, about 2.5 ng / mL or less, about 2 ng / mL or less, about 1 ng / mL, or about 0.5 ng / mL or less; and / or c) the plasma concentration of oleandrin in said subject is about 0.0001 ng / mL or more, about 0.0005 ng / mL or more, about 0.001 ng / mL or more, about 0.0015 ng / mL or more, about 0.01 ng / mL or more, about 0.015 ng / mL or more, about 0.1 ng / mL or more, about 0.15 ng / mL or more, about 0.05 ng / mL or more, or about 0.075 ng / mL or more.23) The method of any one of the above claims, wherein a) plural doses are one or more doses administered per day for two or more days per week; b) 1-10 doses per day are administered for a treatment period of 2 days to about 2 months; or c) one or more doses are administered per day for plural days and plural weeks.24) The method of claim 23, wherein dosing is continued for one or more weeks per month.25) The method of claim 24, wherein dosing is continued for one or more months per year.26) The method according to any one of the above claims, wherein the cardiac glycoside is selected from the group consisting of oleandrin, digoxin, digitoxin, nerine, ouabain, and any combination of two or more thereof.27) The invention any one of the above claims wherein said CGCC further comprises one or more of the following: a) one or more compounds extracted from Nerium oleander plant material; b) one or more triterpenoic acids, e.g. one or more of oleanolic acid, ursolic acid, or betulinic acid; c) one or more antioxidants; d) one or more solvents; e) one or more oils; f) allantoin; g) one or more active polysaccharides from aloe vera, said polysaccharides being selected from the group consisting of glucomannan, polymannose, acemannan, and l-4)-acetylated polymannose; h) one or more preservatives; i) one or more surfactants; j) one or more anti-foaming agents; k) one or more lubricants; 1) one or more corticosteroids (glucocorticoids); m) collagen, e.g. hydrolyzed collagen; n) one or more carboxylic acids; o) one or more amino acids; p) one or more buffering agents; q) one or more antihistamines; and / or r) cyclosporine.28) The invention of any one of the above claims, wherein said CGCC composition further comprises polyphenol(s), carbohydrate(s), flavonoid(s), amino acid(s), soluble protein(s), cellulose, starch, alkaloid(s), saponin(s), tannin(s), or any combination thereof.29) The invention of any one of the above claims, wherein said CGCC composition comprises a) an extract of Nerium sp. biomass; b) oleandrin in isolated form; or c) Nerium sp. biomass.30) The invention of claim 29, wherein said extract is prepared by hot-water extraction, cold-water extraction, organic solvent extraction, supercritical fluid extraction, subcritical liquid extraction, or a combination or two or more thereof.31) The invention of claims 29 or 30, wherein said extract comprises a combination of oleandrin and one or more compounds extracted from said biomass.32) The invention of claim 31, wherein said extract further comprises one or more cardiac glycoside precursors, one or more glycone constituents of cardiac glycosides, or a combination thereof.33) The invention of claim 32, wherein said extract comprises oleandrin and one or more compounds selected from the group consisting of cardiac glycoside, glycone, aglycone, steroid, triterpene, polysaccharide, saccharide, alkaloid, fat, protein, neritaloside, odoroside, oleanolic acid, ursolic acid, betulinic acid, oleandrigenin, oleaside A, betulin (urs-12-ene- 30,28-diol), 28-norurs-12-en-30-ol, urs-12-en-30-ol, 30,30-hydroxy-12-oleanen -28-oic acid, 30,2Oa-dihydroxyurs-21-en-28-oic acid, 30,27-dihydroxy-12-ursen-28-oic acid, 30,130-dihydroxyurs-l l-en-28-oic acid, 30,12a-dihydroxyoleanan-28, 130-olide, 30,27- dihydroxy-12-oleanan-28-oic acid, homopolygalacturonan, arabinogalaturonan, chlorogenic acid, caffeic acid, L-quinic acid, 4-coumaroyl-CoA, 3-O-caffeoylquinic acid, 5- O-caffeoylquinic acid, cardenolide B-l, cardenolide B-2, oleagenin, neridiginoside, nerizoside, odoroside-H, 3-beta-O-(D-diginosyl)-5-beta, 14 beta-dihydroxy-card-20(22)- enolide pectic polysaccharide composed of galacturonic acid, rhamnose, arabinose, xylose, and galactose, polysaccharide with MW in the range of 17000-120000 D, or MW about 35000 D, about 3000 D, about 5500 D, or about 12000 D, cardenolide monoglycoside, cardenolide N-l, cardenolide N-2, cardenolide N-3, cardenolide N-4, pregnane, 4,6-diene- 3, 12,20-trione, 20R-hydroxypregna-4,6-diene-3, 12-dione, 16beta,17beta-epoxy-12beta- hydroxypregna-4,6-diene-3, 20-dione, 12beta-hydroxypregna-4,6,16-triene-3, 20-dione(neridienone A), 20S,21-dihydroxypregna-4,6-diene-3, 12-dione (neridienone B), neriucoumaric acid, isoneriucoumaric acid, oleanderoic acid, oleanderen, 8alpha-methoxylabdan- 18-oic acid, 12-ursene, kaneroside, neriumoside, 3P-O-(D-diginosyl)-2a- hydroxy-8,14P-epoxy-5P-carda-16: 17, 20: 22- dienolide, 3P-O-(D-diginosyl)-2a,14P- dihydroxy-5P- carda- 16: 17, 20:22-di enolide, 3p,27-dihydroxy-urs-18-en-13, 28-olide, 3p,22a,28-trihydroxy-25-nor-lup-l(10),20(29)-dien-2-one, c / .s-karenin (3P-hydroxy-28-Z- p-coumaroyloxy-urs-12-en-27-oic acid), / / zw / .s-karenin (3-P-hydroxy-28-E-p- coumaroyloxy-urs-12-en-27-oic acid), 3beta-hydroxy-5alpha-carda-14(15),20(22)- di enolide (beta- anhydroepidigitoxigenin), 3 beta-O-(D-digitalosyl)-21-hydroxy-5beta- carda-8, 14,16,20(22)-tetraenolide (neriumogenin- A-3beta-D-digitaloside), proceragenin, neridienone A, 3beta,27-dihydroxy-12-ursen -28-oic acid, 3beta,13beta-dihydroxyurs-l 1- en-28-oic acid, 3beta-hydroxyurs-12-en-28-aldehyde, 28- orurs-12-en-3beta-ol, urs-12-en- 3beta-ol, urs-12-ene-3beta,28-diol, 3beta,27-dihydroxy-12-oleanen-28-oic acid, (20S, 24R)-epoxydammarane-3beta,25-diol, 20beta,28-epoxy-28alpha-methoxytaraxasteran- 3beta-ol, 20beta,28-epoxytaraxaster-21-en-3beta-ol, 28-nor-urs-12-ene-3beta,17 beta-diol, 3beta-hydroxyurs-12-en-28-aldehyde, alpha-neriursate, beta-neriursate, 3 alphaacetophenoxy -urs-12-en-28-oic acid, 3beta-acetophenoxy-urs-12-en-28-oic acid, oleanderolic acid, kanerodione, 3 P- / ?-hydroxyphenoxy- l la-m ethoxy- 12a-hydroxy-20- ursen-28-oic acid, 28-hydroxy-20(29)-lupen-3, 7-dione, kanerocin, 3 alpha-hydroxy -urs- 18,20-dien-28-oic acid, D-sarmentose, D-diginose, neridiginoside, nerizoside, isoricinoleic acid, gentiobiosylnerigoside, gentiobiosylbeaumontoside, gentiobiosyloleandrin, folinerin, 12P-hydroxy-5P-carda-8, 14, 16,20(22)-tetraenolide, 8P-hydroxy-digitoxigenin, A16-8P- hydroxy-digitoxigenin, A16-neriagenin, uvaol, ursolic aldehyde, 27(p- coumaroyloxy)ursolic acid, oleanderol, 16-anhydro-deacteyl-nerigoside, 9-D-hydroxy-cis- 12-octadecanoic acid, adigoside, adynerin, alpha-amyrin, beta-sitosterol, campestrol, caoutchouc, capric acid, caprylic acid, choline, cornerin, cortenerin, deacetyloleandrin, diacetyl-nerigoside, foliandrin, pseudocuramine, quercetin, quercetin-3 -rhamnoglucoside, quercitrin, rosaginin, rutin, stearic acid, stigmasterol, strospeside, urehitoxin, and uzarigenin.34) The invention of any one of the above claims, wherein the CGCC further comprises at least one inhibitor that reduces the rate of metabolism or digestion of the cardiac glycoside, thereby increasing the plasma concentration half-life of the cardiac glycoside in the animal.35) The invention of claim 34, wherein said inhibitor inhibits metabolism or digestion of said cardiac glycoside.36) The invention of any one of the above claims, wherein said CGCC is included in a feed, paste, gel, and / or liquid administered orally to the subject.37) The invention according to any one of the above claims, wherein the method is a method of reducing fetal mortality in a pregnant animal or in a population of pregnant animals, the method comprising administering OCC of the invention to a pregnant animal according to any of the dosing protocols or regimens described herein, thereby reducing the occurrence of fetal mortality.38) The invention of claim 37, wherein a) the reduction in fetal mortality is a reduction in aborted fetuses, stillborn fetuses, and / or mummified fetuses; b) before said administering, the pregnant animal is seropositive for a viral infection during at least the latter part of the animal’s gestation period; c) the method of invention excludes embodiments wherein, before said administering, the animal is seropositive for a viral infection during at least the latter part of the animal’s gestation period; d) the animal is seronegative for viral infection prior to said administering.39) The invention of claim 37 or 38, wherein the animal is a pig, cow, horse, sheep, or goat.40) A composition as described herein.41) Use of a composition as described herein.42) Use of a composition as described herein for the preparation of a medicament for use as described herein in an animal or human.

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