Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
  • A61K31/593—9,10-Secocholestane derivatives, e.g. cholecalciferol, i.e. vitamin D3
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

• the present invention relates to therapeutics for the prevention and treatment of inflammatory bowel disease, and in particular the prevention and treatment of inflammatory bowel disease in humans as well as other animals through the use of biologically active vitamin D compounds.
• IBD Inflammatory bowel diseases
• UC ulcerative colitis
• Both diseases appear to involve either a dysregulated immune response to GI tract antigens, a mucosal barrier breach, and/or an adverse inflammatory reaction to a persistent intestinal infection.
• the GI tract luminal contents and bacteria constantly stimulate the mucosal immune system, and a delicate balance of proinflammatory and anti-inflammatory cells and molecules maintains the integrity of the GI tract, without eliciting severe and damaging inflammation [MacDermott, R.P., J. Gastroenterology, 31 :907:-916 (1996)].
• IBD ulcers and inflammation of the inner lining of the intestines lead to symptoms of abdominal pain, diarrhea, and rectal bleeding. Ulcerative colitis occurs in the large intestine, while in Crohn's, the disease can involve the entire GI tract as well as the small and large intestines.
• IBD is a chronic condition with symptoms lasting for months to years. It is most common in young adults, but can occur at any age. It is found worldwide, but is most common in industrialized countries such as the United States, England, and northern Europe. It is especially common in people of Jewish descent and has racial differences in incidence as well.
• the clinical symptoms of IBD are intermittent rectal bleeding, crampy abdominal pain, weight loss and diarrhea.
• IBD Intradilation Diagnosis of IBD is based on the clinical symptoms, the use of a barium enema, but direct visualization (sigmoidoscopy or colonoscopy) is the most accurate test. Protracted IBD is a risk factor for colon cancer. The risk for cancer begins to rise significantly after eight to ten years of IBD.
• UC ulcerative colitis
• prognosis for patients with disease limited to the rectum (proctitis) or UC limited to the end of the left colon (proctosigmoiditis) is better then that of full colon UC.
• Brief periodic treatments using oral medications or enemas may be sufficient.
• blood loss from the inflamed intestines can lead to anemia, and may require treatment with iron supplements or even blood transfusions.
• Crohn's disease can occur in all regions of the gastrointestinal tract. With this disease intestinal obstruction due to inflammation and fibrosis occurs in a large number of patients. Granulomas and fistula formation are frequent complications of Crohn's disease. Disease progression consequences include intravenous feeding, surgery and colostomy.
• the most commonly used medications to treat IBD are anti-inflammatory drugs such as the salicylates.
• the salicylate preparations have been effective in treating mild to moderate disease. They can also decrease the frequency of disease flares when the medications are taken on a prolonged basis.
• Examples of salicylates include sulfasalazine, olsalazine, and mesalamine. All of these medications are given orally in high doses for maximal therapeutic benefit. These medicines are not without side effects.
• Azulfidine can cause upset stomach when taken in high doses, and rare cases of mild kidney inflammation have been reported with some salicylate preparations.
• Corticosteroids are more potent and faster-acting than salicylates in the treatment of IBD, but potentially serious side effects limit the use of corticosteroids to patients with more severe disease. Side effects of corticosteroids usually occur with long term use. They include thinning of the bone and skin, infections, diabetes, muscle wasting, rounding of faces, psychiatric disturbances, and, on rare occasions, destruction of hip joints.
• immunosuppressants include azathioprine and 6-mercaptopurine. Immunosuppressants used in this situation help to control IBD and allow gradual reduction or elimination of corticosteroids. However, immunosuppressants cause increased risk of infection, renal insufficiency, and the need for hospitalization.
• vitamin D compounds include, but are not limited to compounds which have at least one of the following features: the C-ring, D-ring and 3 ⁇ - hydroxycyclohexane A-ring of vitamin D interconnected by the 5,7 diene double bond system of vitamin D together with any side chain attached to the D-ring (i.e. compounds with a 'vitamin D nucleus' and substituted or unsubstituted A-, C-, and D-rings interconnected by a 5,7 diene double bond system typical of vitamin D together with a side chain attached to the D-ring).
• nonsecosteroidal vitamin D mimics is defined as nonsecosteroid compounds which are capable of mimicking various activities of the secosteroid calcitriol.
• biologically active vitamin D compound is defined as encompassing vitamin D compounds and nonsecosteroidal vitamin D mimics which are biologically active in vivo, or are acted upon in a subject (i.e. host) such that the compound becomes active in vivo.
• examples of such compounds include, but are not limited to: vitamin D, 1,25 dihydroxyvitamin D 3 (l,25(OH) 2 D 3 ) [a.k.a. calcitriol], and analogs thereof [e.g.
• symptoms of IBD is herein defined to include symptoms, including, but not limited to, abdominal pain, diarrhea, rectal bleeding, weight loss, fever, loss of appetite, and other more serious complications, such as dehydration, anemia and malnutrition. A number of such symptoms are subject to quantitative analysis (e.g. weight loss, fever, anemia, etc.). Some symptoms are readily determined from a blood test (e.g. anemia) or a test that detects the presence of blood (e.g. rectal bleeding).
• calcemic response is herein defined as the biological response caused by many biologically active vitamin D compounds (e.g. calcitriol) when administered to a subject.
• the response includes, but is not limited to, elevated calcium concentrations in serum, increased intestinal absorption of dietary calcium, increased urinary calcium excretion, and increased bone calcium mobilization.
• symptoms of hypercalcemia is herein defined to detected symptoms including, but not limited to, calcium deposition in the kidneys (nephrocalcmosis), kidney stones (nephrolithiasis), uremia, manifestations of muscle weakness, lethargy, coma, constipation, anorexia, nausea, vomiting, shortening of the QT interval, hypotension, and arrhythmias.
• the phrase "serious hypercalcemia” is herein defined as the condition where a subject is suffering from symptoms of hypercalcemia which require immediate medical attention to prevent life threatening illness or death. Examples include, but are not limited to, nephrocalcmosis, nephrolithiasis, uremia, coma, and anorexia.
• mild hypercalcemia is herein defined as the condition where a subject is suffering from symptoms of hypercalcemia which do not require immediate medical attention to prevent life threatening illness or death. Examples include, but are not limited to, manifestations of muscle weakness, lethargy, constipation, nausea, vomiting, shortening of the QT interval, hypotension, and arrhythmia.
• a therapeutically effective amount of a biologically active vitamin D compound is herein defined as the dosage level required for a patient such that the symptoms of IBD are reduced.
• the phrase "under conditions such that the symptoms are reduced” refers to any degree of qualitative or quantitative reduction in detectable symptoms of IBD, including but not limited to, a detectable impact on the rate of recovery from disease (e.g. rate of weight gain), or the reduction of at least one of the following symptoms: abdominal pain, diarrhea, rectal bleeding, weight loss, fever, loss of appetite, dehydration, anemia, distention, fibrosis, inflamed intestines and malnutrition.
• the phrase "at risk for IBD” is herein defined as encompassing the segment of the world population that has an increased risk (i.e. over the average person) for IBD. IBD is most commonly found in young adults, but can occur at any age. It occurs worldwide, but is most common in the United States, England, and northern Europe. It is especially common in people of Jewish descent. An increased frequency of this condition has been recently observed in developing nations. Increased risk is also present in people with family members who suffer from inflammatory bowel disease.
• compositions containing the biologically active vitamin D compounds of the present invention refers to compositions containing the biologically active vitamin D compounds of the present invention, or the biologically active vitamin D compounds of the present invention provided together with one or more other compounds or agents including, but not limited to, other biologically active vitamin D compounds, physiologically tolerable liquids, gels, carriers, diluents, adjuvants, excipients, salicylates, steroids, immunosuppressants, antibodies, cytokines, antibiotics, binders, fillers, preservatives, stabilizing agents, emulsifiers, and buffers.
• other biologically active vitamin D compounds include, but not limited to, other biologically active vitamin D compounds, physiologically tolerable liquids, gels, carriers, diluents, adjuvants, excipients, salicylates, steroids, immunosuppressants, antibodies, cytokines, antibiotics, binders, fillers, preservatives, stabilizing agents, emulsifiers, and buffers
• continuous manner when used in reference to the method of delivery or administration of the biologically active vitamin D compounds of the present invention, is defined as meaning a substantially uninterrupted administration of the compounds of the present invention, such that a therapeutic dosage is stretched over a period of time and avoids a dosage 'spike' which is common among other modes of administration (e.g. oral administration or intravenous administration).
• modes of administration which employ a continuous manner of delivery include, but are not limited to, a transdermal patch, a suppository, or a slow release oral formulation.
• subject refers to a patient which is administered the therapeutic composition comprising biologically active vitamin D compounds of the present invention.
• IL-10 deficient subject refers to a patient that is known (or determined) to express less IL-10 (interleukin-lO) than a wild- type subject. Examples include, but are not limited to, subjects that express 90%, 50%, or 10% of wild-type levels of IL-10, or subjects that do not express any IL-10.
• the present invention relates to therapeutics for the prevention and treatment of IBD. Specifically, the present invention contemplates the prevention and treatment of IBD in humans as well as other animals through the use of biologically active vitamin D compounds.
• the present invention provides a method of treatment, comprising; providing a subject and a therapeutic composition comprising a biologically active vitamin D compound; and administering the therapeutic composition to the subject. It is not intended that the present invention be limited to any particular subject. Indeed, a variety of subjects are contemplated.
• the subject is a mammal.
• the subject is a mammal selected from the group of a human, horse, non- human primate, dog, and cat.
• the subject is a human.
• the subject is on a low calcium diet.
• the subject is screened for the presence of interleukin- 10 prior to the administration of the composition comprising a biologically active vitamin D compound.
• the subject is IL-10 deficient and the therapeutic composition administered to the subject comprises a biologically active vitamin D compound and IL-10.
• the subject is not IL-10 deficient and the therapeutic composition administered to the subjected comprises a biologically active vitamin D compound and IL-10.
• the subject is suffering from symptoms of inflammatory bowel disease.
• the subject is suffering from ulcerative colitis.
• the subject is suffering from Crohn's disease.
• a therapeutic composition comprising a biologically active vitamin D compound reduces the symptoms of disease (e reduces the symptoms of inflammatory bowel disease, ulcerative colitis, or Crohn's disease)
• the biologically active vitamin D compounds are administered under conditions such that the symptoms of IBD are reduced
• the subject is at risk for inflammatory bowel disease, and the therapeutic composition is administered prophylactically
• a therapeutically effective amount of a biologically active vitamin D compound is administered to the subject
• the biologically active vitamin D compound is selected from vitamin D, 1,25 dihydroxyvitamin D 3 , l ⁇ -hydroxyvitamm D 3 , 1,25-d ⁇ hydroxyv ⁇ tam ⁇ n D 2 , l ⁇ -hydroxyvitamin D 2 , l ⁇ ,25-(OH) 2 -16-ene-D 3 , l ⁇ ,25-(OH) 2 - 24-oxo-16-ene-D 3 , l ⁇ ,24R(OH) 2 -D 3 , l ⁇ ,25(OH) 2 -22-oxa-D 3 , 20-ep ⁇ -22-oxa-24a,24b,- dihomo- 1 ⁇ ,25(OH) 2 -D 3 , 20-ep ⁇ -22-oxa-24a,26a,27a,-t ⁇ homo- 1 ⁇ ,25(OH) 2 -D
• the biologically active compound is 1,25-d ⁇ hydroxyv ⁇ tam ⁇ n D 3
• the biologically active vitamin D compound is selected from the analogs represented by the following formula
• X' and X 2 are each selected from the group consisting of hydrogen and acyl, wherein Y' and Y 2 can be H, or one can be O-aryl or O-alkyl while the other is hydrogen and can have a ⁇ or ⁇ configuration, Z 1 and Z 2 are both H, or Z 1 and Z 2 taken together are CH 2 , and wherein R is an alkyl, hydroxyalkyl or fluoroalkyl group, or R may represent the following side chain'
• (a) may have an S or R configuration and wherein R 1 represents hydrogen, hydroxy or O-acyl, R 2 and R 3 are each selected from the group consisting of alkyl, hydroxyalkyl and fluoroalkyl, or, when taken together represent the group— (CH 2 ) m — where m is an integer ha ⁇ ⁇ ng a value of from 2 to 5, R 4 is selected from the group consisting of hydrogen, hydroxy, fluorine, O-acyl, alkyl, hydroxyalkyl and fluoroalkyl, R 5 is selected from the group consisting of hydrogen, hydroxy, fluorine, alkyl, hydroxyalkyl and fluoroalkyl, or, R 4 and R 5 taken together represent double-bonded oxygen, R 6 and R 7 taken together form a carbon— carbon double bond and R 8 may be H or CH 3 , and wherein n is an integer having a value of from 1 to 5, and wherein the carbon at any one of positions 20, 22, or 23 in the side chain may be replaced by an O
• the biologically active vitamin D compounds of the present ent ⁇ on do not cause symptoms of hypercalcemia when administered to a subject
• the biologically active vitamin D compounds of the present invention do not generate as much (t e a lesser degree) of a calcemic response as compared to calcitriol when administered to a subject
• the biologically active vitamin D compounds have low calcemic response characteristics, inhibit cell proliferation and promote cell differentiation as compared to calcitriol.
• these compounds are selected from l ,25-(OH) 2 -24- epi-D 2 , l ⁇ ,25-(OH) 2 -24a-Homo-D 3 , l ⁇ ,25-(OH) 2 -24a-Dihomo-D 3 , l ⁇ ,25-(OH) 2 -19-nor- D 3 , and 20-epi-24-homo-l ⁇ ,25-(OH) 2 -D 3 .
• the present invention also contemplates the administration of a therapeutic composition comprising more than one of the biologically active compounds of the present invention. In other embodiments, the biologically active compounds of the present invention are administered in therapeutically effective amounts.
• a preferred dose of the biologically active vitamin D compound for the present invention is the maximum that a patient can tolerate and not develop serious hypercalcemia.
• a daily dose between 1.0 and 100 ⁇ g per day per 160 pound patient is administered, while a particularly advantageous daily dose is between 5.0 and 50 ⁇ g per day per 160 pound patient.
• a daily dose of between 0.1 and 20 ⁇ g per day per 160 pound patient is administered, while a preferred dose is between 0.5 and 10 ⁇ g per day per 160 pound patient.
• the dose is between 3-10 ⁇ g per day.
• the therapeutic administration of the biologically active vitamin D compounds does not cause serious hypercalcemia.
• the therapeutic administration of the biologically active vitamin D compounds only causes mild hypercalcemia.
• the biologically active vitamm D compounds do not cause symptoms of hypercalcemia.
• the present invention be limited to a particular mode of administration.
• modes of administration are contemplated, including intravenously, intra-muscularly, subcutaneously, intradermally, intraperitoneally, intrapleurally, intrathecally, orally, rectally and topically.
• the therapeutic compositions are administered via suppository, or in tablet or capsule formulations for oral delivery.
• administration of the therapeutic compositions occurs at night.
• multiple doses e.g. 3 or 4
• the administration of the therapeutic composition is by pulse intravenous therapy.
• the therapeutic compositions are administered via a transdermal patch (skin patch).
• the present invention also provides a method of treatment, comprising, providing a subject with symptoms of inflammatory bowel disease and a therapeutic composition comprising a biologically active vitamin D compound, and administering the therapeutic compound to the subject.
• the biologically active vitamin D compounds are administered to a patient after the surgical removal of damaged tissue.
• the present invention provides a method of treatment, comprising, providing a human patient with symptoms of inflammatory bowel disease, a therapeutic composition comprising a biologically active vitamin D compound, and administering the therapeutic composition to the patient under conditions such that said symptoms are reduced.
• the subject is screened for the presence of interleukin-10 prior to the administration of the composition comprising a biologically active vitamin D compound.
• the subject is administered IL-10 in addition to a biologically active vitamin D compound.
• the subject is an IL-10 deficient subject.
• the present invention also provides a method of treatment, comprising, providing a subject at risk for inflammatory bowel disease and a therapeutic composition comprising a biologically active vitamin D compound, and prophylactically administering the therapeutic compound to the subject.
• the prophylactic administration of the biologically active vitamin D compounds delays the onset of the symptoms of inflammatory bowel disease.
• the prophylactic administration of the biologically active vitamin D compounds prevents the onset of one or more symptoms of inflammatory bowel disease (e.g. prevents the onset of abdominal pain, diarrhea, rectal bleeding, weight loss, fever, loss of appetite, dehydration, anemia, or malnutrition, or any combination thereof).
• the subject is screened for the presence of interleukin-10 prior to the administration of the composition comprising a biologically active vitamin D compound.
• the subject is administered both a biologically active vitamin D compound and IL-10.
• the subject is an IL-10 deficient subject and is administered both IL-10 and a biologically active vitamin D compound.
• the present invention also provides a composition of matter comprising a transdermal patch, wherein said transdermal patch comprises a therapeutic composition comprising biologically active vitamin D compounds.
• the transdermal patch comprises a therapeutically effective amount of a biologically active vitamin D compound.
• the transdermal patch further comprises IL-10.
• the transdermal patch further comprises a single polymer.
• the transdermal patch further comprises multiple polymers.
• the transdermal patch further comprises a polyurethane acrylic copolymer. In another embodiment, the transdermal patch further comprises silicone or polyisobutylene or both. In a preferred embodiment, the transdermal patch is worn by a subject at risk for Inflammatory Bowel Disease. In another preferred embodiment, the transdermal patch is worn by a subject with symptoms of Inflammatory Bowel Disease.
• the transdermal patch delivers biologically active vitamin D compounds to a subject in a continuous manner under conditions such that symptoms of IBD are reduced.
• the present invention also provides a method of treatment, comprising, providing an IL-10 deficient subject with symptoms of inflammatory bowel disease and a therapeutic composition comprising a biologically active vitamin D compound and IL-10, and administering the therapeutic compound to the subject.
• the present invention also provides a method of treatment, comprising, providing an IL- 10 deficient subject at risk for inflammatory bowel disease and a therapeutic composition comprising a biologically active vitamin D compound and IL-10, and prophylactically administering the therapeutic compound to the subject.
• the present invention provides therapeutic compositions comprising a biologically active vitamin D compound and IL-10 (e.g. a therapeutic amount of a biologically active vitamin D compound and a therapeutic amount of IL-10).
• the present invention provides a transdermal patch comprising a biologically active vitamin D compound and IL-10.
• the present invention provides a suppository comprising a biologically active vitamin D compound and IL-10.
• the present invention provides kits comprising a biologically active vitamin D compound, and IL-10 (e.g. in separate containers, or separate pills or as part of separate devices).
• the kit further comprises instructions for employing the biologically active vitamin D compound and IL- 10 to treat or prevent disease (e.g. a printed insert describing the use of these compounds).
• the instructions describe the treatment of IBD with a biologically active vitamin D compound and IL-10.
• Figure 2 depicts five nonsecosteroidal vitamin D compounds (mimics) useful in the present invention.
• the present invention relates to therapeutic compositions and methods for the prevention treatment of IBD, and in particular the prevention and treatment of IBD in humans as well as other animals through the use of biologically active vitamin D compounds.
• IBD Intestinal Bowel Disease
• CD Crohn's Disease
• UC Ulcerative Colitis
• CD and UC are distinct, but related polygenic disorders. Familial clustering of IBD strongly suggests that IBD susceptibility genes contribute to disease development. The relative risk of IBD among siblings of CD patients was 36.5 and among siblings of UC patients was 16.6, compared to the general population. Possible IBD susceptibility genes have been mapped to chromosomes 2, 3, 6, 7, 12 and 16. The identity and function of these susceptibility genes is unknown [See, Ecuadorgi et al, Clin. Sci. (Colch), May;94(5):473-8 (1998)].
• Vitamin D stimulates bone mineralization, and there is evidence for poor bone mineralization in IBD patients [Andreassen et al, Scand. J. GastroenteroL, 32:1247-1255 (1997)]. Many reports found osteoporosis in greater than 40% of adult CD patients, while pediatric CD patients had significantly decreased bone length and mineral density compared to controls [Boot et al, Gut, 42:188-94 (1998)]. Neither malabsorption, nor steroid use explained the poor bone mineralization. Recently, an IBD susceptibility locus was mapped to chromosome 12 [Satangi et al, Clin. Sci. (Colch), May;94(5):473-8 (1998), and Duerr et al, Am. J. Hu.
• This IBD susceptibility locus is in the same region as the vitamin D receptor and the 25-hydroxyvitamin D 3 -l- ⁇ - hydroxylase genes [Labuda et al, J. Bone Min. Res. 7: 1447-53 (1992)].
• IBD prevalence studies show a striking latitude gradient in the United States [Sonnenber et al, GastroenteroL 102:1827 (1992)], and Europe [Shivananda et al, Gut, 39:690-7 (1996)].
• the IBD incidence is high at northern latitudes and low at southern latitudes.
• Vitamin D 3 is biologically inert, and must be activated by 25-hydroxylation in the liver and l ⁇ - hydroxylation in the kidney to produce the vitamin D hormone l ⁇ ,25-dihyroxyvitamin D 3 (calcitriol) [Haussler et al, J. Endocrinol. 154:557-573 (1997)].
• Vitamin D deficiency is common at northern latitudes. In one study, at 42°N, vitamin D deficiency affected 57% of a random population sample [Thomas et al, N. Eng. J. Med., 338:777-783 (1998)]. It is not intended that the present invention be limited to a particular mechanism of action. Indeed, an understanding of the mechanism is not necessary to make and use the present invention.
• insufficient sunlight for vitamin D 3 biosynthesis may be a disease-determining environmental risk factor for IBD.
• the therapeutic affect of the administration of biologically active vitamm D compounds to patients may be achieved by compensating for insufficient vitamin D biosynthesis in certain patients.
• T lymphocyte infiltration of the mucosa is characteristic of IBD; these infiltrating cells display activation markers [Pallone et al.Gut, 28:745-753 (1987], and broad specificity [Kaulfersch et al, Gastroenterology, 95:364-370 (1988)].
• mucosal T cells display upregulated IL-2 receptors, and spontaneously produce IL-2 and
• TCR T cell receptor
• MHC major histocompatibility complex
• calcitriol is a potent and selective inhibitor of autoimmune disease in mice.
• calcitriol administered to mice blocked disease induction for EAE [Cantorna et al, Proc. Natl Acad. Sci. USA, 93:7861-7864 (1996)], Lyme arthritis and collagen induced arthritis [Cantorna et al, J. Nutr. 128:68-72 (1998)].
• the present invention contemplates the prevention and treatment of Inflammatory Bowel Disease in a subject through the use of biologically active vitamin D compounds.
• Various forms of treatment and dosages are contemplated, as well as the avoidance of the development of the symptoms of hypercalcemia.
• the present invention contemplates both the treatment and prevention of IBD in humans as well as other animals (e.g. mammals) with biologically active vitamin D compounds in therapeutic amounts. It is not intended that the present invention be limited to particular dosages. A broad range of dosages for the therapeutic administration of the biologically active vitamin D compounds are contemplated.
• a preferred dose of the biologically active vitamin D compound for the present invention is the maximum that a patient can tolerate and not develop serious hypercalcemia. If the biologically active vitamin D compound is not a l ⁇ -hydroxy compound, a daily dose between 1.0 and 100 ⁇ g per day per 160 pound patient is administered, while a particularly advantageous daily dose is between 5.0 and 50 ⁇ g per day per 160 pound patient.
• a daily dose of between 0.1 and 20 ⁇ g per day per 160 patient is administered, while a preferred dose is between 0.5 and 10 ⁇ g per day per 160 pound patient. In a particularly preferred embodiment, the dose is between 3-10 ⁇ g per day. In general, a preferred dose is the highest amount of the biologically active vitamin D compound the patient can tolerate. The dose is preferably divided between two, three, four or five treatments within a 24 hour period.
• the accepted safe dose of l,25(OH 2 )D 3 and 19-nor-21-epi- l,25(OH 2 )D 3 in patients having normal calcium intakes is between 0.5 and 15 ⁇ g per day for l,25(OH 2 )D 3 , and is 10-20 ⁇ g/day for 19-nor- l,25-(OH) 2 D 2 . Therefore, a preferred dose for patients with normal calcium intakes is between 0.5 and 0.75 ⁇ g per day for a
• treatment with biologically active vitamin D compounds is administered in as high a dose as the patient can tolerate without developing symptoms of hypercalcemia.
• high doses e.g. 3-10 ⁇ g per day
• the safe dosage range is 10-20 ⁇ g per day per 160 pound patient.
• a determination of whether a patient is benefiting from treatment is performed by monitoring the qualitative and quantitative symptoms of IBD.
• Qualitative symptoms which may monitored include, but are not limited to, abdominal pain, diarrhea, rectal bleeding, weight loss, fever, loss of appetite, dehydration, anemia, and malnutrition.
• Quantitative symptoms which may be monitored include, but are not limited to, weight loss, fever, and anemia (using a blood test).
• a successful treatment is indicated wherein the symptoms of IBD are reduced. Preferably, treatment should continue as long as IBD symptoms are suspected or observed.
• a determination of whether a subject would benefit from prophylactic treatment of IBD is determined by assessing various risk factors. In other words, a determination of whether a subject is at risk for IBD is made. It is not intended that the present invention be limited to particular risk factors. Indeed, a variety of risk factors may be monitored, including, but not limited to; genetic predisposition, amount of sunlight the patient normally receives, the age of the patient (common in young people), nationality (common in U.S, England, and Northern Europe), and ancestry/race (common in people of Jewish decent). Patients at risk are prophylactically administered the therapeutic compositions of the present invention to delay or prevent the onset of symptoms of IBD.
• calcitriol biologically active vitamin D compounds
• the major physiological function of vitamin D is to maintain extracellular calcium levels within a very limited normal range for normal cellular and metabolic processes (including neuromuscular function and bone mineralization).
• calcitriol primarily increases intestinal absorption of dietary calcium and phosphate, and when required, mobilizes bone calcium.
• calcitriol has a potent calcemic effect (i.e. generates a calcemic response in a subject).
• the primary concern associated with administering calcitriol or its analogues to subjects is elevated serum calcium (hypercalcemia) and phosphate levels, a condition accompanied by a corresponding increase in urinary calcium excretion (hypercalcuria).
• vitamin D compounds can have serious consequences for renal function; prolonged hypercalcemia can result in calcium deposition in the kidneys (nephrocalcmosis), kidney stones (nephrolithiasis), and ultimately in renal dysfunction leading to uremia. Vitamin D intoxication may also have serious consequences for neurological functions.
• severe hypercalcemia the threshold for excitation of nerve and muscles is increased, resulting in clinical manifestations of muscle weakness, lethargy, and even coma.
• Gastrointestinal manifestations of vitamin D intoxication include constipation, anorexia, nausea, and vomiting, with subsequent fluid loss which exacerbates the hypercalcemic crisis.
• Hypercalcemia can also affect cardiovascular functioning, including shortening of the QT interval, hypotension, and arrhythmias. Therefore, it is important to monitor the development of hypercalcemia in patients receiving biologically active vitamin D compounds. Hypercalcemia may be monitored in a patient by measuring the terminal serum calcium levels.
• TGF-B1, TCR, MHC, or IL-10 knockout mice employing a chosen biologically active vitamin D compound.
• these studies involve assaying the level of the biologically active vitamin D compound in the serum and correlating dietary dose of this compound to biologically efficacy and the symptoms of hypercalcemia such that the minimum effective dose is determined.
• Another method of minimizing the risks of hypercalcemia involves administering the biologically active vitamin D compound using timed drug release methods (e.g. suppository or transdermal patch) or "slow release" biologically active vitamin D derivatives (See, US Pat. 5,952,317, hereby incorporated by reference).
• transdermal patch reduces the risk of hypercalcemia (caused by mobilization of calcium across the intestinal wall) by preventing a delivery 'spike' of the biologically active vitamin D compound.
• a transdermal patch is believed to deliver a continuous, lower dosage stream of the biologically active vitamin D compounds such that a spike (total dosage all at once) which could cause a severe increase in the mobilization of calcium across the intestine wall, is avoided.
• biologically active vitamin D compounds of the present invention encompass vitamin D compounds which are biologically active in vivo, or are acted upon in a subject (i.e. host) such that the compound becomes active in vivo.
• examples of such compounds include: vitamin D, l,25(OH 2 )D 3 and analogs thereof (e.g.
• the present invention also contemplates other biologically active vitamin D compounds which may be represented by various functional classes.
• the first functional class are vitamin D compounds which exhibit significant activity in vivo as inhibitors of autoimmunity (e.g. multiple sclerosis or experimental autoimmune encephalomyelitis, type one diabetes, arthritis or lyme arthritis or collagen-induced arthritis, glomerulonephritis, thyroidits, systemic lupus erythematosis), and which exhibit calcemic activity in vivo that is less than or equal to but not more than calcitriol (i.e. l,25(OH) 2 D 3 .
• autoimmunity e.g. multiple sclerosis or experimental autoimmune encephalomyelitis, type one diabetes, arthritis or lyme arthritis or collagen-induced arthritis, glomerulonephritis, thyroidits, systemic lupus erythematosis
• calcemic activity in vivo that is less than or equal to but not more than calcitriol (i
• Examples of this class include, but are not limited to, l ⁇ ,25-dihydroxy-16ene-vitamin D 3 and l ⁇ ,25-dihydroxy-24-oxo-16ene-vitamin D 3 [See, Lemire et al, Endocrinology, 135:2818-2821, (1994)]; l ⁇ ,24R-dihydroxy-vitamin D 3 [See, Koizumi et al, Int. Arch.
• the second functional class are vitamin D compounds which exhibit significant activity in vivo as an inhibitor of transplanted cells, tissue, or organ rejection (e.g. skin graft, heart graft, islet graft, etc.), and exhibit calcemic activity in vivo that is less than or equal to calcitriol.
• this class include, but are not limited to, 1,25-dihydroxy-
• the third functional class are vitamin D compounds which exhibit significant activity in an in vitro cell differentiation assay (e.g. HL-60, U-937, NB4, etc.) and exhibit in vivo calcemic activity that is less than or equal to calcitriol.
• this class include, but are not limited to l,25-dihydroxy-16,23E-diene-26-trifluoro-19-nor- cholecalciferol [See, Asou et al. Blood, 92:2441-2449 (1998)]; 1 l ⁇ -vinyl-l ⁇ ,25- dihydroxy-vitamin D 3 [See, Bouillon et al, J.
• the fourth functional class are vitamin D compounds (mimics) which exhibit significant activity as an activator of the nuclear vitamin D receptor in an in vitro transcriptional activation assay, while also exhibiting a binding affinity for the serum vitamin D binding protein that is less than or equal to calcitriol.
• An example of this class includes, but is not limited to LG190090, LG1901 19, LG190155, LG190176, and LG1900178 [See, Boehm et al, Chemistry & Biology 6:265-275 (1999)].
• vitamin D compounds are contemplated for use in the present invention, including, but not limited to, compounds described in: US Pat No. 5,936,105 (incorporated herein by reference), US Pat. No. 5,932,565 (incorporated herein by reference), US Pat. No. 5,929,056 (incorporated herein by reference), US Pat. No. 5,905,074 (incorporated herein by reference), 5,902,806 (incorporated herein by reference), US Pat. No. 5,883,271 (incorporated herein by reference), US Pat. No. 5,877,168 (incorporated herein by reference), US Pat. No. 5,872,140 (incorporated herein by reference), US Pat. No. 5,81 1,562 (incorporated herein by reference), US Pat. No.
• X 1 and X 2 are each selected from the group consisting of hydrogen and acyl; wherein Y 1 and Y 2 can be H, or one can be O-aryl or O-alkyl while the other is hydrogen and can have a ⁇ or ⁇ configuration, Z' and Z 2 are both H, or Z 1 and Z 2 taken together are CH 2 , and wherein R is an alkyl, hydroxyalkyl or fluoroalkyl group, or R may represent the following side chain
• (a) may have an S or R configuration and wherein R 1 represents hydrogen, hydroxy or O-acyl, R 2 and R 3 are each selected from the group consisting of alkyl, hydroxyalkyl and fluoroalkyl, or, when taken together represent the group— (CH 2 ) m — where m is an integer having a value of from 2 to 5, R 4 is selected from the group consisting of hydrogen, hydroxy, fluorine, O-acyl, alkyl, hydroxyalkyl and fluoroalkyl, R "5 is selected from the group consisting of hydrogen, fluorine, alkyl, hydroxyalkyl and fluoroalkyl, or, R 4 and R" taken together represent double-bonded oxygen, R° and R 7 taken together form a carbon—carbon double bond and R s may be H or CH 3 , and wherein n is an integer having a value of from 1 to 5, and wherein the carbon at any one of positions 20, 22, or 23 in the side chain may be replaced by an O, S, or N
• the present invention also contemplates "slow release" de ⁇ vatives of the biologically active vitamin D compounds of the present invention (See US Pat No 5,952,317, inco ⁇ orated herein by reference). The following formula describes these various derivatives:
• R 5 and R 6 each represent hydrogen, or taken together R 5 and R 6 represent a methylene group.
• the side chain group R in the above-shown structure represents a steroid side chain of the structure below:
• m and n independently, represent the integers from 0 to 5, where R 1 is selected from hydrogen, OX 4 , fluoro, trifluoromethyl, and C,. 5 -alkyl, which may be straight chain or branched and, optionally, bear a hydroxy substituent, and where R 2 is selected from hydrogen, fluoro, trifluoromethyl and C,. 5 alkyl, which may be straight-chain or branched, and optionally, bear a hydroxy substituent, and where R 3 and
• X 1 , X 2 and X 4 independently represent hydrogen, an acyl group or a hydrocarbyloxycarbonyl group, and X 3 represents an acyl group or a hydrocarbyloxycarbonyl group, as previously defined herein.
• an animal model of IBD e.g. DS-induced mice, carageenan-induced guinea pigs, or IL-2, TGF- ⁇ l, TCR, MHC, IL-10 knockout mice, HLA-B27/ ⁇ 2m transgenic rat, trinitrobenzene sulfonic acid induced colitis in rodents, or spontaneous IBD in cotton-top tamarin colonies held in temperate climates
• IBD an animal model of IBD (e.g. DS-induced mice, carageenan-induced guinea pigs, or IL-2, TGF- ⁇ l, TCR, MHC, IL-10 knockout mice, HLA-B27/ ⁇ 2m transgenic rat, trinitrobenzene sulfonic acid induced colitis in rodents, or spontaneous IBD in cotton-top tamarin colonies held in temperate climates) may be employed [See also, Bouillon et al, Endocr Rev. Apr;16(2):200-57 (1995)]. The inhibition
• Useful biologically active vitamin D compounds reduce the symptoms of IBD. Especially useful biologically active vitamin D compounds reduce the symptoms of IBD, and do not cause substantial hypercalcemia at therapeutic dosages.
• the present invention contemplates using therapeutic compositions of biologically active vitamin D compounds. It is not intended that the present invention be limited by the particular nature of the therapeutic composition. For example, such compositions can be provided together with physiologically tolerable liquids, gels, solid carriers, diluents, adjuvants and excipients (and combinations thereof).
• biologically active vitamin D compounds may be used together with other therapeutic agents, including, but not limited to, salicylates, steroids, immunosuppressants, antibodies or antibiotics.
• biologically active vitamin D compounds are used together with IL-10.
• Particular therapeutic agents which may be used with the biologically active vitamin D compounds of the present invention include, but are not limited to, the following agents: azobenzene compounds (US Pat.
• the therapeutic compositions of the present invention can be administered to mammals for veterinary use, such as with domestic animals and non-human primates, and clinical use in humans in a manner similar to other therapeutic agents.
• the dosage required for therapeutic efficacy varies according to the type of use and mode of administration, as well as the particularized requirements of individual hosts.
• a preferred dose of the biologically active vitamin D compounds of the present invention is the maximum that a patient can tolerate and not develop serious hypercalcemia.
• the attending medical professional is capable of determining the therapeutically effective dosage based on the characteristics of the subject (e.g., gender, age, weight, amount of calcium in diet, etc.).
• the biologically active vitamin D compounds are administered intravenously, intra-muscularly, subcutaneously, intradermally, intraperitoneally, intrapleurally, intrathecally, orally, rectally or topically.
• formulations for such administrations may comprise an effective amount of the biologically active vitamin D compound in sterile water or physiological saline.
• formulations for such administrations may comprise an effective amount of the biologically active vitamin D compound in an organic solvent (e.g. ethanol, vegetable oil, or glycerol).
• compositions may contain such normally employed additives as binders, fillers, carriers, preservatives, stabilizing agents, emulsifiers, buffers and excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.
• binders such normally employed additives as binders, fillers, carriers, preservatives, stabilizing agents, emulsifiers, buffers and excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.
• binders such normally employed additives as binders, fillers, carriers, preservatives, stabilizing agents, emulsifiers, buffers and excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbon
• the biologically active vitamin D compounds of the present invention can also be mixed with diluents or excipients which are compatible and physiologically tolerable. Suitable diluents and excipients are, for example, water, saline, dextrose, glycerol, or the like, and combinations thereof.
• the compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, stabilizing or pH buffering agents.
• the therapeutic compositions of the present invention are prepared either as liquid solutions or suspensions, as sprays, or in solid forms.
• Oral formulations usually include such normally employed additives such as binders, fillers, carriers, preservatives, stabilizing agents, emulsifiers, buffers and excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.
• These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, and typically contain l%-95% of active ingredient, preferably 2%-70%.
• an oral composition useful for delivering the therapeutic compositions of the present invention is described in US Pat.
• the therapeutic composition comprising biologically active vitamin D compounds is administered via a transdermal patch.
• a transdermal patch optimally includes a therapeutically effective amount of the biologically active vitamin D compounds of the present invention. While not limited to any mechanism, it is believed that transdermal delivery would provide a continuous supply of the vitamin D compound, maintaining the vitamin D receptor occupancy at a stable, optimal level, to achieve the desired biological effect. This is in contrast to other modes of delivery (e.g. oral or intravenous) which could provide a peak hormone concentration (spike) shortly after delivery, which would subsequently decline (leading to cyclical hormone occupancy and peak concentrations stimulating calcium abso ⁇ tion, bone reso ⁇ tion, or soft tissue calcification).
• Transdermal delivery may also bypass delivery of the vitamin D compounds to the intestine, decreasing binding to the vitamin D receptors in the intestinal epithelial cells. This in turn may decrease stimulation of intestinal calcium abso ⁇ tion, and decrease the calcemic effect of the vitamin D compound. Transdermal delivery may also be preferred because intestinal physiology is disturbed in patients with IBD, which may alter uptake of the vitamin D compound in a patient-specific, non-predictable manner (making dose determination difficult for other modes of administration such as oral delivery). Transdermal delivery may be more convenient than other modes of delivery (especially for children), and could increase patient compliance.
• transdermal patch for delivering therapeutics employs a polyurethane acrylic copolymer (U.S. Pat. No. 4,638,043, inco ⁇ orated herein by reference).
• Another example of a transdermal patch employs polymers and vitamin E (U.S. Pat. No. 5,830,505, inco ⁇ orated herein by reference).
• a third example of a transdermal patch employs an adhesive matrix of silicone or polyisobutylene or both
• enteric formulations are employed.
• the covering may comprise an enteric coating or a capsule.
• enteric coating or “enteric film” are used interchangeably and refer to a material or compound which is resistant to acid pH (i.e., an acid-resistant compound), such as that found in the stomach.
• acid pH i.e., an acid-resistant compound
• Standard techniques are known to the art for the encapsulation of solid compositions. These techniques include microencapsulation of a solid composition wherein an enteric coating is applied to the solid composition.
• the coated material may be delivered orally to a subject by suspending the microencapsulated particles in pharmaceutical suspension solutions known to the art.
• the capsule would preferably have the characteristic of being resistant to dissolution in the stomach and being capable of dissolving in the intestines.
• Numerous suitable capsule formulations are available to the art; in addition standard techniques are available for the filling of capsules including the use of inert filler materials to provide sufficient bulk of the filling of a capsule with a therapeutic composition in a solid form.
• the biologically active vitamin D compounds may be delivered orally in tablet or pill form.
• the biologically active vitamin D compounds may be combined with inert materials to provide sufficient bulk for the pressing of the tablet or pill. Once formed, the tablet or pill may then be coated with an enteric film to prevent dissolution in the stomach and to enhance dissolution in the intestines.
• the present invention provides methods of treating subjects with biologically active vitamin D compounds and IL-10.
• subject that are known (or determined to be) IL-10 deficient are treated for IBD with therapeutic compositions comprising a biologically active vitamin D compound and IL- 10.
• IL-10 deficient subjects are treated with combination therapy of IL-10 and a biologically active vitamin D compound (e.g. separate administration of IL-10 and a biologically active vitamin D therapy).
• IL-10 is administered to a subject in a protein form (e.g. intravenous injection).
• the gene expressing IL-10 is administered to the subject such that IL-10 is expressed in the subject (e.g.
• IL-10 preparation, production, and subject dosages are provided in U.S. Patent 5,945,097 to Cutler and U.S. Patent 5,776,451 to Hsu et al, both of which are hereby inco ⁇ orated by reference.
• daily IL-10 dosages for a subject include; 1-100 micrograms/kg/day, 1-15 micrograms/kg/day, or approximately 8 micrograms/kg/day.
• the present invention contemplates screening subjects for the presence of IL-10 prior to the administration of biologically active vitamin D compounds (e.g. prior to administration of biologically active vitamin D compounds to subjects with IBD or at risk for IBD, or any other disease where biologically active vitamin D compounds are administered for treatment).
• Screening subjects for the presence of IL-10 may be performed, for example, by obtaining a biological sample (e.g. blood sample, tissue sample, or other bodily fluid) from the subject and contacting the sample with an IL-10 binding ligand.
• a biological sample e.g. blood sample, tissue sample, or other bodily fluid
• binding ligands includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, nucleic acid sequences, and IL-
• U.S. patent 6,028,186 to Tasset et al provides nucleic acid molecules (isolated by the SELEX procedure) capable of binding IL-10.
• U.S. patent 5,231,012 to Mosmann et al. describes antibodies to IL-10 (e.g. polyclonal and monoclonal antibodies).
• IL-10 detection kits are also available commercially (e.g.
• PromoCell which supplies a human IL-10 ELISA kit, www.promocell). Any method of determining the IL-10 status of a subject is useful in the present invention.
• subjects identified as IL-10 deficient may be administered both IL-10 and a biologically active vitamin D compound in order to treat (or prevent) IBD.
• N normal; M (molar); mM (millimolar); ⁇ M (micromolar); mol (moles); mmol (millimoles); ⁇ mol (micromoles); nmol (nanomoles); pmol (picomoles); g (grams); mg (milligrams); ⁇ g (micrograms); ng (nanograms); 1 or L (liters); ml (milliliters); ⁇ l (microliters); cm (centimeters); mm (millimeters); ⁇ m (micrometers); run (nanometers); DS (dextran sulfate); °C (degrees Centigrade); Sigma (Sigma Chemical Co., St. Louis,
• the fist group is the widely used DS-induced murine model, which reflects the involvement of enteric microbes, innate immunity, and non-specific inflammation in IBD.
• Mouse strains vary in their susceptibility to DS- induced IBD [Mahler et al, Am. J. Physiol- Gastro. & Liver Physiol., 37 G: 544-G 551, (1998)], so the highly susceptible strains C3H/HeJ and C57BL/6J are employed.
• the second group of IBD models involves mice with targeted disruptions in immunologically relevant genes.
• the widely used IL-10 knockout on the C57BL/6J background is employed (i.e. C57BL/6J-IL-10 ko).
• mice with a homozygous IL-10 ko mutation on the mixed 129/Ola x C57BL/6 genetic background are employed as described below to demonstrate the effect of calcitriol on the inhibition of IBD induction (See Table 1, overview of Experimental Groups). Table 1. Experimental Groups.
• mice 1-8 The DS-induced IBD study (groups 1-8; Table 1) are treated as follows. Beginning on day 0 and continuing, groups of 10 mice are fed a purified diet without or with calcitriol (50 ng/day females; 200 ng/day males); this dose is based on dose-response studies in EAE [Cantorna et al, J. Immunol, 160:5314-5319 (1998)]. The prednisolone- treated group is fed a purified diet with prednisolone (20 ng/day). These experimental diets have been published [Cantorna et al, Proc. Natl Acad. Sci. USA, 97:7861-7864 (1996)], and are replenished each 2-3 days.
• mice On day 2, the mice are weighed and DS (3.5% wt/vol) is given in the drinking water on days 2-6 (Okayasu, et al, Gastro., 98:694-702 (1990)].
• the mice receive acidified drinking water without DS for days 7- 22.
• days 7, 11, 15, and 19 mice are weighed and stool samples are collected.
• a blood sample is collected on day 11.
• mice On day 22, mice are weighed, euthanized, and stool, blood, and colon samples are collected. The samples are analyzed as described below.
• the mock-treated control mice in this Example are expected to show severe weight loss, bloody diarrhea (as evidenced by fecal hemoglobin), shortening of the colon and thickening of the colonic wall, mucosal ulceration, goblet cell loss, and crypt elongation and loss (as evidenced by histopathologic score), and inflammatory infiltration by lymphocytes, macrophages, neutrophils, and granulocytes (as evidenced by fecal lactoferrin and colonic myeloperoxidase activity) during IBD induction and progression.
• the calcitriol-treated mice exhibit significantly reduced incidence or severity of disease as compared to the mock-treated controls. With respect to severity, the calcitriol-treated mice exhibit significantly reduced weight loss, bloody diarrhea, shortening and thickening of the colon, histopathologic score, and inflammatory infiltration as compared to the mock-treated controls.
• mice 9-11 The spontaneous IBD study (groups 9-11; Table 1) are treated the same as the DS- induced mice, except purified diet feeding begins when the IL-10-ko mice are weaned at age 3 weeks, and no DS is administered. Mice are weighed and stool samples are collected twice weekly for four weeks. A blood sample is collected at age 5 weeks.
• mice After four weeks, mice are weighed, euthanized, and stool, blood, and colon samples are collected. These samples are analyzed as described below in Example 2.
• the mock- treated control mice carrying a null-mutation of the IL-10 gene described in this Example are expected to show growth retardation by age 4 weeks (as evidenced by low blood hemoglobin levels), and a significant mortality incidence by 10 weeks.
• mice are also expected to show clinical signs of IBD, specifically bloody fecal lactoferrin and colonic myeloperoxidase activity, perianal ulceration, and occasional rectal prolapse.
• these mice are expected to show histopathological signs of IBD, specifically inflammatory infiltration with lymphocytes, plasma cells, marcophages, and neutrophils, ulceration, abnormal enlarged and branched crypts, branched and fused villus structures, and marked hype ⁇ lasia of the mucosa leading to thickening of the intestinal wall.
• the calcitriol-treated mice exhibit significantly reduced growth retardation, anemia, mortality, bloody diarrhea, intestinal inflammation, perianal ulceration and rectal prolapse, and histopathologic score as compared to the mock-treated controls.
• EXAMPLE 2 Analysis of Samples From Treated Mice This Example describes the analysis of stool, serum, and colon samples from both groups of mice described in Example 1. Stool extracts are analyzed individually for protein, hemoglobin, and lactoferrin. Serum samples are analyzed for Ca, IFN- ⁇ , and
• TGF- ⁇ l Colon samples are analyzed individually for myeloperoxidase (5/group), IFN- ⁇ and TGF- ⁇ l immunohistochemistry, and for histopathology (5/group). Fecal extracts and intestinal tissue extracts are assayed for protein content by a micro-Bradford assay [Bradford, M.M., Anal. Biochem., 72:248-254 (1976)]. Results of the hemoglobin, lactoferrin, and myeloperoxidase assays are then determined per mg of protein in the sample. Fecal hemoglobin and fecal lactoferrin (as well as weight) are plotted as a function of time.
• Histopathology scores for disease incidence and severity, myeloperoxidase activity, and IFN- ⁇ and TGF- ⁇ l values are tabulated, and differences between groups are compared by the nonparametric Wilcoxon test (Schefler, W.C., Statistics for the Biological Sciences, 2nd Edition, Addison- Wesley Pub. Co., Reading, MA, USA, 1979).
• Intestinal bleeding in a breached intestinal barrier is measured in live animals by measuring fecal hemoglobin levels, which have been shown to be elevated in UC patients [Saitoh, et al, Digestion 56:67-75 (1995)]. Stool samples, collected over a 24 hour period are weighed, homogenized in a small amount of water, and centrifuged in order to detect the relative hemoglobin concentration of each sample (ng/mg of protein).
• the fecal hemoglobin two-site ELISA is performed on these samples as described [Saitoh, et al, Digestion, 56:67-75 (1995)] with modifications. All ELISA steps are separated by washing four times (TRIS-buffered saline with 0.05% Tween 20). ELISA plates (Immulon, Dynatech; 96-well) are coated overnight in the cold with rabbit polyclonal antibodies (pAb) to mouse hemoglobin (ICN Biomedical Research Products; 5 ⁇ g/ml in 0.1 M sodium carbonate buffer pH 8.2), and blocked with 0.1 M TRIS-buffered saline (pH 8) containing 1% bovine serum albumin.
• pAb rabbit polyclonal antibodies
• the samples and the hemoglobin standard are serially diluted in buffer (0.1 M TRIS-buffered saline, pH 7.5, with 0.1% bovine serum albumin) and aliquots are incubated 1 hour at 37°C in duplicate assay wells; assay blank wells receive buffer only.
• An optimal amount of biotinylated rabbit pAb to mouse hemoglobin (ICN) is added to each well and incubated 1 hour at 37°C.
• the assay is developed with tetramethylbenzidine (TMB) plus hydrogen peroxide substrate solution (ICN), the reaction is stopped by adding 100 ⁇ l of IM phosphoric acid, and the color is measured on an ELISA plate reader.
• TMB tetramethylbenzidine
• ICN hydrogen peroxide substrate solution
• the hemoglobin in each sample is determined from the linear portion of a log-linear plot of A-450 nm versus hemoglobin standard concentration, and results are expressed in ng mg protein.
• the calcitriol-treated mice exhibit less hemoglobin protein in stool extracts than the mock-treated control mice.
• GI tract inflammation in live animals is indicated by measuring fecal lactoferrin concentrations. High levels of fecal lactoferrin have been demonstrated in patients with colorectal diseases [Sugi et al, Am. J. GastroenteroL 91 :927 (1996)].
• the fecal lactoferrin two-site ELISA is performed in the same manner as the hemoglobin assay above, except that rabbit pAb to human lactoferrin (ICN Biomedical Research Products) replaces rabbit pAb to mouse hemoglobin as the coating Ab, and biotinylated rabbit pAb to human lactoferrin replaces rabbit pAb to mouse hemoglobin as the detecting Ab.
• the lactoferrin in each sample is determined from the linear portion of a log-linear plot of A- 450 nm versus lactoferrin standard concentration. Results are expressed as lactoferrin ng/mg protein.
• the calcitriol-treated mice show less lactoferrin protein in stool extracts than the mock-treated control mice.
• Myeloperoxidase assays have been used as objective and quantitative measures of inflammation in humans [e.g. Dwarakanath et al, Clin. Sci. 92:307-13 (1997)] and animals [e.g. Hogaboam et al, J. Clin. Invest., 100:2766-76 (1997)] with IBD. Accordingly, intestinal inflammation is measured in the mouse intestinal tissue samples by measuring myeloperoxidase activity levels. The myeloperoxidase assay is performed as in Krawisz et al, GastroenteroL 87:1344-50 (1984), as modified by Schneider and Issekutz, J. Immunol. Methods, 198:1-14 (1996).
• washed intestinal tissue samples (3/mouse; 200 mg each) are minced and homogenized in hexadecyltrimethylammonium bromide (HTAB) buffer (1 ml; 0.5% HTAB in 50mM phosphate, pH 6) to release the enzyme.
• the homogenate is frozen and thawed four times, then centrifuged.
• the supernatant (10 ⁇ l) is added to a well of a 96-well plate containing 0.29 ml assay mix (TMB plus hydrogen peroxide substrate solution; ICN), and the A-450 nm is measured on an ELISA plate reader as a function of time.
• An assay blank is prepared with a heat-inactivated supernatant and subtracted.
• a unit of enzyme activity is defined as the amount catalyzing the oxidation of 1 ⁇ mole substrate/min under these conditions. Results are expressed as myeloperoxidase U/mg protein. The calcitriol-treated mice show less myeloperoxidase protein in the intestinal tissue extracts than the mock-treated control mice.
• Intestinal tissue is also analyzed for the presence of IFN- ⁇ and TGF- ⁇ l (cytokines thought to regulate mucosal inflammation, see below).
• Intestinal tissue specimens are snap frozen in OCT embedding compound (Miles Laboratories). Longitudinal 10 ⁇ m sections are applied to coated glass slides, air dried, and fixed briefly in acetone. Sections are then re-hydrated in PBS with 0.05% Tween 20, and washed in this buffer between each subsequent reaction step. All reactants are dissolved in PBS-Tween buffer.
• Sections are reacted 30 minutes with 3% hydrogen peroxide, then 10 minutes with 3% goat serum, then overnight in the cold with polyclonal rabbit antibodies to IFN- ⁇ and TGF- ⁇ l or control rabbit serum, and finally with biotinylated goat antibodies to rabbit IgG. Color is developed with the Vector Elite ABC kit.
• the intestinal tissue sections from the calcitriol-treated mice exhibit qualitatively greater immunohistochemical staining with polyclonal rabbit antibodies to TGF- ⁇ l and qualitatively less immunohistochemical staining with the polyclonal rabbit antibodies to IFN- ⁇ than the intestinal tissue sections from the mock-treated control mice.
• Anemia can be a sign of IBD due to blood loss in the stool.
• 0.5 ml of blood is obtained on days 11 and 22 (prevention of DS-induced mouse IBD models) and at age 5 and 7 weeks (prevention of spontaneous mouse IBD model).
• a blood hemoglobin determination is performed.
• a small blood aliquot is dispensed into lysis buffer [3 mM K 3 (FeCN) 6 , 1.5 mM KCN, 5mM Na 2 BO 4 , 0.1% Nonidet P-40] and the absorbance at 546 nm is measured using lysis buffer as a blank. This absorbance is compared to a reference curve produced using purified moused hemoglobin dissolved in lysis buffer.
• the remaining blood is allowed to clot overnight in the cold, centrifuged, and the serum is collected for further analysis.
• Serum calcium is measured by a colormetric reaction (Sigma Diagnostics). Calcium ions form a pu ⁇ le complex with o- cresolphthalein complexone at alkaline pH (0.5 M 2-amino-2-methyl-l,3-propanediol buffer); 8-hydroxyquinoline (0.25%) is included to prevent magnesium ion interference. Sample, standard, or buffer blank (lO ⁇ l) is added to duplicate tubes of a working solution (1 ml) of equal parts Ca binding reagent and Ca buffer.
• EXAMPLE 3 Treating Established IBD In Mice This example describes the treatment of established IBD in mice using calcitriol.
• Example 2 The procedure is the same as Example 1 above, except treatment is not started until the mice show signs of IBD. This is accomplished by administering DS in the drinking water of the mice listed in groups 1-4 in Table 1, followed by acidified water without DS thereafter. The mice are also weighed at the beginning of this procedure. At two- day intervals, stool samples are tested as in Example 1 for hemoglobin and lactoferrin. Treatment with calcitriol is begun when these test indicate the mice are suffering from IBD. Thereafter, weights and stool samples are obtained at 4-day intervals. On day 22, the mice are weighed, euthanized, and stool, blood, and colon samples are collected. Stool extracts, blood, serum, and colon samples are analyzed as described in above in Example 2.
• Weight, fecal and blood hemoglobin, and fecal lactoferrin are plotted as a function of time. Disease incidence, severity (as evidenced by histopathologic score), myeloperoxidase activity, and IFN- ⁇ and TGF- ⁇ l values are then tabulated. The results of this experiment indicate that calcitriol treatment of mice exhibiting symptoms of IBD exhibit reduced symptoms of disease compared to controls.
• Therapeutic formulations of calcitriol are used prophylactically and therapeutically to treat IBD in humans.
• Individuals at risk of contracting IBD, particularly young adults, or those or Jewish descent are administered an effective amount of calcitriol in a therapeutic formulation to prevent or reduce the severity of the disease.
• a patient with symptoms of IBD is administered an effective dose of calcitriol daily until symptoms of
• EXAMPLE 5 Therapeutic and Prophylactic Use of Vitamin D to Treat and Prevent IBD
• Therapeutic formulations of vitamin D are used prophylactically and therapeutically to treat IBD in humans. Individuals at risk of contracting IBD, particularly young adults, or those or Jewish descent are administered an effective amount of vitamin D in a therapeutic formulation to prevent or reduce the severity of the disease. A patient with symptoms of IBD is administered an effective dose of vitamin D daily until symptoms of IBD are reduced.
• EXAMPLE 6 DS-induced Animal Model of Ulcerative Colitis
• This Example describes the generation of a mouse model for dextran sulfate (DS) induced colitis.
• This DS-induced colitis model was adapted from Okayasu et al. (1990) for use with a synthetic diet.
• male C3H/HeJ mice were used, as these mice are highly susceptible to DS-induced colitis. Initially, mice were given 3.5% DS w/v in acidified water for 5 days, followed by acidified water without DS, and continuously fed a synthetic diet (synthetic diet described in Smith and Hayes, PNAS, USA, 84:5878-5882, 1987). These mice showed no signs of colitis (e.g., diarrhea, blood in the stool, weight loss).
• mice shunned the water containing DS, and met their hydration needs by consuming the synthetic diet (which is approximately 60% water by weight).
• a second group of mice were given 3.5% DS w/v in acidified water for 5 days, followed by acidified water without DS, and continuously fed laboratory chow (Purina mouse chow #5008) as described in Mahler et al, Gastrointest, Liver Physiol, 37:G544- G551 (1996).
• These mice consuming an average of 0.57g DS/mouse, lost weight
• mice were given graded amounts of DS in acidified water and a synthetic diet for 5 days, followed by acidified water and the synthetic diet without DS. These mice were weighed twice weekly and subjected to various other assays, including: fecal hemoglobin analysis, and histopathologic analysis. The results are presented below in Tables 2 and 3.
• Fecal hemoglobin was analyzed in the mice to determine bleeding from a breached intestinal barrier.
• Fresh stool samples were collected from individual mice, dried overnight, weighed, and suspended in 0.5 mL of TRIS-buffered saline (TBS; 25 mM TRIS, 0.15 M NaCl, pH 7.6). The stool samples were centrifuged (10,000 x g for TRIS-buffered saline (TBS; 25 mM TRIS, 0.15 M NaCl, pH 7.6). The stool samples were centrifuged (10,000 x g for TRIS-buffered saline (TBS; 25 mM TRIS, 0.15 M NaCl, pH 7.6). The stool samples were centrifuged (10,000 x g for TRIS-buffered saline (TBS; 25 mM TRIS, 0.15 M NaCl, pH 7.6). The stool samples were centrifuged (10,000 x g for TRIS-buffered saline (TBS; 25 m
• supematants were decanted into fresh tubes and stored frozen at -20°C. Hemoglobin in the supematants was analyzed by a method adapted from Saitoh et al, Digestion, 56:67-75, 1995, and Liem et al, Anal. Biochem., 98:388- 393, 1979, with rat hemoglobin as standard (Sigma- Aldrich). The supematants were diluted 1 :25 in TBS.
• the assay was done in round-bottom, polystyrene, 96 well microtite ⁇ lates (#3555, DYNEX Technologies, Chantily, VA).
• the samples, standards, and buffer blanks (20 ⁇ L each) were incubated in duplicate for 10 min with 100 ⁇ L of a substrate solution made with 9 volumes 3,3'5,5'-tetramethylbenzidine dihydrochloride peroxide solution (Turbo TMB-ELISA, Pierce Chemical Co., Rockford, IL) and 1 volume of hydrogen peroxide solution (Stable Peroxide Substrate Buffer, Pierce Chemical Co.).
• the reaction was stopped with 100 ⁇ L of 2 M phosphoric acid, and the absorbance at 450 nm was measured with an MRX Revelation Microplate Reader (DYNEX
• the fecal hemoglobin assay working range was 1 to 100 ⁇ g/mL sample.
• the hemoglobin mg/g stool was calculated and reported in Table 3.
• mice were euthanized with an i.p. injection of pentobarbital.
• the cecal and rectal GI tract segments were collected, cut longitudinally, flushed with 10% neutral- buffered formalin, arranged in a spiral pattern in a cassette, and fixed in formalin.
• the fixed tissue was embedded in paraffin, sectioned at 6 ⁇ m, stained with hematoxylin and eosin, and examined by light microscopy. Scoring was conducted by a veterinary pathologist in blinded fashion.
• Each colon segment (proximal, medial, and distal colon, and rectum) was given a score from 0 to 4, based on the criteria described by Berg et al, J. Clin. Invest., 98: 1010-1020, 1996.
• the scores were summed to provide a total colitis score for each mouse.
• the total colitis score could range from 0 (no pathology in any segment) to 16 (grade 4 lesions in all colon segments).
• the DS was in the acidified drinking water and in the synthetic diet.
• E the DS was in the drinking water and the mice were fed laboratory chow.
• This Example demonstrates the ability of calcitriol to prevent colitis in DS- induced mouse models of colitis.
• age-matched C3H/HeJ male mice were fed a synthetic diet (see Example 6) with or without calcitriol or prednisolone for one week.
• the drug treatments were continued, and IBD was induced with a 5 day administration of 1% DS in the food and water as described in Example 6.
• the mice were weighed every 3-4 days thereafter.
• Fecal Hg was analyzed daily from day 3 to day 7. Serum and colon tissue were collected on day 28. Most assays were performed as described in Example 6, and the terminal serum calcium assay was performed as described below. The results are reported in Tables 4 and 5.
• DS was fed on days 0 to 5 in the acidified drinking water and in the diet. The experiment was terminated on day 27.
• b Hormones were fed continuously in the diet beginning one week before DS treatment (day -7). TABLE 5 Calcitriol Prevention of IBD in Mice - Colon Histopathology
• the peak Hg in the stool of the calcitriol-treated group was 37% lower than that in the untreated group, and 65% lower than that in the prednisolone-treated group.
• the total colon histopathology score of the calcitriol-treated group C was 61% lower than the score of the untreated group B, and 51% lower than the score of the prednisolone-treated group D (Table 5).
• This Example demonstrates the ability of calcitriol to treat chronic colitis in DS-induced mouse models of colitis.
• age-matched adult C3H/HeJ male mice were subjected to three DS treatment cycles treatment separated by 6-8 weeks.
• Each DS treatment was a 5-day administration of 1% DS in acidified water and in synthetic diet. Between treatments, the mice were fed laboratory mouse chow. These treatments induced chronic IBD. Beginning 10 days after the last DS treatment was completed, the mice were divided into two groups. The groups were matched with respect to fecal Hg and weight (Table 6). Group A was fed synthetic diet and group B was fed calcitriol in synthetic diet. The experiment was terminated 21 days later. The colon histopathology results indicate that calcitriol treatment reduced the severity of chronic DS-induced IBD. In particular, the mice treated with calcitriol for 21 days had an approximately 45% reduction in colon histopathology (Table 7).
• mice made genetically deficient in IL-10 spontaneously develop chronic enterocolitis (Kuhn et al, Cell, 75:263-274, 1993) if resident enteric bacteria is present (Sellon et al, Infect. Immunity, 66:5224-5231, 1998).
• the effect of calcitriol on spontaneous colitis in 129-IL-10 " ' " mice is examined. Mice raised in gnotobiotic conditions were transferred to conventional conditions and fed synthetic diet with and without graded doses of calcitriol. The mice were age 7 weeks when they were exposed to soiled bedding from mice housed under conventional conditions. This was done to provide the enteric bacteria for development of colitis.
• mice The results (presented in Table 8 and Table 9) indicate that calcitriol did not prevent spontaneous colitis in the 129-IL-10 " " mice.
• the calcitriol-treated mice had a lower terminal weight than the mock-treated mice (Table 8).
• the calcitriol-treated mice developed hypercalcemia (Table 8), indicating that they are capable of responding to calcitriol treatment as regards Ca abso ⁇ tion.
• the calcitriol-treated mice had colitis histopathology scores equal to the mock-treated mice (Table 9), indicating that calcitriol treatment had no effect on spontaneous colitis in strain 129-IL-10 ⁇ mice.
• EXAMPLE 10 Treatment of IBD in an IL-10 Deficient Subject
• This Example describes the treatment of a subject that is IL-10 deficient and suffering from IBD.
• a human subject with symptoms of IBD is screened for the presence of IL-10 and determined to be IL-10 deficient.
• the subject is administered a therapeutic formulation (daily) that includes approximately 7 micrograms of calcitriol and approximately 8 micrograms/kg of IL-10 in order to prevent or reduce the severity of the symptoms of IBD.
• This therapeutic formulation is administered daily until symptoms of IBD are reduced or eliminated.

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