WO2009035387A1

WO2009035387A1 - A medicament prepared using methotrexate and suitable for topical administration, for the treatment of a lower gastrointestinal tract inflammatory disease

Info

Publication number: WO2009035387A1
Application number: PCT/SE2007/050651
Authority: WO
Inventors: Anders Hallberg; Claes Post; Per Hellström; Sofie Lundberg
Original assignee: Softcure Pharmaceuticals Ab
Priority date: 2007-09-14
Filing date: 2007-09-14
Publication date: 2009-03-19

Abstract

Use of methotrexate in the preparation of a medicament for the treatment of a lower gastrointestinal tract inflammatory disease in a mammal by topical administration to the lower gastrointestinal tract of said mammal. A method of treatment of a lower gastrointestinal tract inflammatory disease by topical administration of a therapeutically effect ive dose of methotrexate to the lower gastrointestinal tract of a mammal in need of such treatment. A medicament for topical administration containing methotrexate.

Description

A medicament prepared using methotrexate and suitable for topical administration, for the treatment of a lower gastrointestinal tract inflammatory disease

Field of the invention

The present invention relates to the use of methotrexate in the treatment of a lower gastrointestinal tract inflammatory disease. In particular, the invention relates to the topical administration of methotrexate in the treatment of such disease.

Background to the invention

Inflammatory bowel disease (IBD) is a general term that includes both ulcerative colitis and Crohn's disease, disorders of largely unknown ethiology that result in inflammation in the gastrointestinal tract. Ulcerative colitis is an inflammatory disease of the large intestine. In this disease, ulcers develop in the inner lining, or mucosa, of the colon or rectum, often resulting in diarrhea, blood, and pus.

Crohn's disease is an inflammation that extends into the deeper layers of the intestinal wall. It is found most often in the ileum and the first part of the large intestine, known as the ileocecal region.

Ulcerative colitis and Crohn's disease share many symptoms, although they also differ in important ways. Both are chronic diseases characterized by frequent relapses and remissions, and symptoms usually appear in young adults. The most common symptom of both ulcerative colitis and Crohn's disease is diarrhea. Constipation may develop during active flare-ups of both Crohn's disease and ulcerative colitis. Cramps can occur from intestinal contractions caused by inflammation. Fever, fatigue and loss of appetite are often present. Neurological or psychiatric symptoms may be early signs of Crohn's disease when accompanied by gastrointestinal problems.

Drugs presently on the market cannot cure IBD, but some are effective in reducing the inflammation and accompanying symptoms in up to 80% of patients. Many such drugs are available, including corticosteroids, aspirin-like medications, and drugs that suppress the immune system. The primary goal of drug therapy is to put acute flares into remission and/or prevent relapse. Mesalazine is the common name of the compound 5-aminosalicylic acid or S- ASA, which inhibits substances in the immune system that cause inflammation. For very active IBD that does not respond to standard treatments, immunosuppressant drugs are now being used for long-term treatment All of these drugs suppress actions of the immune system and thereby its inflammatory response that causes ulcerative colitis and Crohn's disease. The two most common immunosuppressants used for IBD are azathioprine and mercaptopurine. Other immunosuppressants that are investigated for IBD and showing promising results in promoting remission include cyclosporine and methotrexate.

Methotrexate is an inhibitor of dihydro folate reductase, and is cytotoxic at high doses. The mechanism of its action in Crohn's disease is unknown, but may involve inhibiting inflammatory cell activation and cytokine release. Response to therapy is seen over several months.

In the treatment of lower gastrointestinal tract inflammatory diseases, methotrexate according to the state of the art is given as a tablet, infusion or injection. As an example, for Crohn's disease, methotrexate is commonly given by weekly injection in a physician's office or hospital.

The clinical use of methotrexate is limited due to iis side-effects. Dose-limiting toxicity occurs in 10% to 15% of patients. The most common adverse effects are nausea and mouth ulcers, which occur in about 5% of individuals, but may decrease with continued use. The adverse effects can be limited by a dose reduction, co-administration of folic acid, and parenteral delivery of the methotrexate.

Abnormal liver function tests are common in patients treated with methotrexate, particularly in those with diabetes, obesity, abnormal renal function, or excessive alcohol intake. Hepatotoxicity correlates with total cumulative methotrexate dose, and manifests itself as fibrosis of increasing severity, culminating in cirrhosis. While hepatotoxicity is rare in appropriately selected patients in the absence of abnormalities in albumin or transaminases, methotrexate should be used cautiously in patients with pre-existing liver disease, or alcohol abuse.

Methotrexate can also cause leucopenia, thrombocytopenia and anemia. The frequency of these haemalological events is low, but is higher in the elderly, those with reduced renal function, and in patients with acute illness (e.g. viral infection, dehydration). Treatment should be ceased temporarily in patients who are acutely unwell. Dose reduction is required when using methotrexate in patients with mild renal impairment, and methotrexate is contraindicated in moderate to severe renal impairment.

Methotrexate has also been associated with pneumonitis, rash, menorrhagia, fatigue, alopecia and depression. The compound also is teratogenic. It therefore should be avoided in pregnancy, and ceased 3 months before conception if possible.

The combination of methotrexate with other drugs that inhibit folic acid synthesis, such as trimethoprim and sulfamethoxazole (colrimoxazole), trimethoprim alone, or triamterene, should be avoided because of increased risk of haemalological toxicity (e.g. pancytopenia, megaloblastic anaemia).

From the above it is obvious that, while in many cases offering a means of treating various inflammatory conditions in the lower gastrointestinal tract, methotrexate as presently used nonetheless is associated with a number of serious side effects and limitations that substantially reduce its clinical use.

It thus appears that there still exists an unfulfilled need for an effective therapy directed to patients suffering from an inflammatory disease in the lower gastrointestinal tract. Jn particular there is still a need for such a therapy that is associated with less or less serious drug-related side effects than with present methotrexate usage.

More particularly, it would be desirable to be able to use methotrexate with fewer and less harmful side effects in the treatment of inflammatory disorders of the lower gastrointestinal tract.

Likewise, it would be an advantage to be able to use methotrexate in wider group of patients, advantageously comprising also subjects that are especially sensitive to the negative side effects of methotrexate such as those referred to herein above or for which the use of methotrexate by administration according to the state of the art is for some reason contraindicated.

It is an object of the present invention to fulfill such needs and achieve such advantages. Summary of the invention

Thus, one object of the present invention is to provide the use of methotrexate for the treatment of various inflammatory conditions in the lower gastrointestinal tract at a substantially reduced risk of the adverse effects that are commonly associated with the treatment by use of methotrexate.

Another object of the invention is to render possible the use of methotrexate in the treatment of various inflammatory conditions in the lower gastrointestinal tract also in patients that hitherto has been precluded from such treatment, due e.g. to the side effects associated with the use of methotrexate according to the prior art.

Furthermore, one object of the invention is to provide a method of treatment of a lower gastrointestinal tract inflammatory disease wherein the systemic distribution of the methotrexate in the treated subject is substantially reduced.

The present inventors surprisingly have found that the above objects may be achieved by topical administration of methotrexate to the lower gastrointestinal tract of a mammal suffering from a lower gastrointestinal tract inflammatory disease. Indeed, by use of a formulation allowing for the topical delivery of methotrexate to the site of inflammation, a beneficial therapeutic effect on the disease is obtainable at a substantially lower risk of negative side effects. While the topically delivered methotrexate still acts positively on the inflammation locally, the systemic toxic effect of the compound is reduced due to the limited and unpredictable absorption of the drug into the bloodstream.

Consequently, according to a first aspect, the present invention provides the use of methotrexate in the preparation of a medicament for the treatment of a lower gastrointestinal tract inflammatory disease in a mammal by topical administration of said medicament to the lower gastrointestinal tract of said mammal.

According to another aspect, the invention provides methotrexate for use as a medicament in the treatment of a lower gastrointestinal tract inflammatory disease in a mammal by topical administration of the medicament to the lower gastrointestinal tract of said mammal. According to another aspect, the invention provides a medicament comprising a therapeutic amount of methotrexate for the treatment of a lower gastrointestinal tract inflammatory disease in a mammal, said medicament being formulated so as to allow for the topical administration of methotrexate to the lower gastrointestinal tract of said mammal.

According to still a further aspect, the invention provides a method of treatment of a lower gastrointestinal tract inflammatory disease by topical administration of a therapeutically effective dose of methotrexate to the lower gastrointestinal tract of a mammal in need of such treatment.

In one embodiment, the lower gastrointestinal tract inflammatory disease is inflammatory bowel disease (IBD), e.g. ulcerative colitis or Crohn's disease.

The mammal preferably is a human, but also may be a non-human mammal, such as a laboratory, pel or farm animal, e.g. a dog, a cat or a horse.

Brief description of the drawings

Fig. 1 : The catheter, employed for rectal administration of methotrexate and vehicle to mice.

Fig. 2: Damage of the mucosa. Expressed as percent 0=no damage.

Fig. 3: Acute inflammation, graded from 0=no inflammation to 4=severe.

Fig. 4: Body weight from day 13 compared with healthy controls. All animals received DSS from day 1. Only rectal administration of methotrexate completely prevented weight loss (2.7

± 1.5% decrease in weight, (p< 0.05 vs. DSS alone).

Fig. 5: Colon length in healthy animals and in DSS-treated animals with different treatments.

Box plot with median values.

Fig. 6: Hematoxylin-eosin staining showing circular section of the distal colon from healthy controls (A), DSS alone (19 days) (B), and methotrexate, high dose (G). The images are representative for each treatment group.

Detailed description of the invention

Methotrexate, or N-[4-[[(2,4-diammo-6-pteridmyl)methyl]methy-amino]benzoyl]-L-glutamic acid, has the structural formula

Since methotrexate contains a number of basic and acid groups, it may be provided as a salt, e.g. with one or several cations, e.g. metal cations such as Na⁺, or as hydrochloride salt. For the purpose of the invention, and if the contrary is not apparent from the context, the reference to methotrexate is understood also to include any such pharmaceutically acceptable salt.

By "topical administration", as used herein, is meant administration to a localized surface of the body of a mammal subject.

By "topical administration to the lower gastrointestinal tract", as used herein, is meant administration to the inner wall, i.e. the epithelial lining, of the lower gastrointestinal tract of a mammal subject.

By a "medicament in a form suitable for topical administration to the lower gastrointestinal tract" is meant a medicament formulated so as to reach said site before the active ingredient is allowed to come into contact in any essential degree with and exert any therapeutic effect on the surface of the inner wall of the tract.

By "lower gastrointestinal tract" is meant the intestines, including the large intestine (cecum, colon and rectum) and the small intestine (duodenum, jejunum and ileum), and the anus.

"Lower gastrointestinal tract inflammatory disease" refers to an inflammatory disease affecting one or more of the parts of the lower gastrointestinal tract, e.g. an inflammatory bowel disease, such as ulcerative colitis and Crohn's disease. The disease may be chronic or not.

By "therapeutic amount" or "therapeutically effective dose" or similar expression as used herein is meant an amount or dose sufficient to procure a desired therapeutic effect in a mammal on administration to said mammal. It is well within the capability of the skilled person to determine the therapeutic amount of methotrexate to be included in a formulation, by suitable experiments and/or taking guidance from the examples given herein below. While the examples refer to mice, the effective dosage may easily be converted to the human equivalent dose (HED) based on the body surface area, as is known to the person of skill in the art. By HED is meant the dose which, when administered to humans, produces an effect equal to that produced by the dose in an animal.

The term "treating" and "treatment" as used herein refers to administering to a subject a therapeutically effective dose of methotrexate so that the subject has an improvement in the parameters relating to the treated disease. The improvement is any observable or measurable improvement. Thus, the treatment may improve the patient condition, but may not be a complete cure of the disease.

"Pharmaceutically acceptable" as used herein denotes the lack of any significant interference with the effectiveness or the therapeutic activity of the active ingredient, viz. methotrexate and of any additional active ingredient as well as lack of any significant toxicity at the administered dosage.

The topical administration may be rectal and effected e.g. by enema or rectiol. The administration also may be effected by a suppository. It is well within the knowledge of the person of ordinary skill in the art to select suitable excipients, optionally referring to suitable handbooks, such e.g. Remington: The Science and Practice of Pharmacy. A. R. Gennaro, Editor. Lippincott, Williams and Wilkins, 20th edition (2000). Thus, in addition to methotrexate, the medicament in a form suitable for topical administration to the lower gastrointestinal tract may comprise any suitable, pharmaceutically acceptable excipient such as binders, fillers, solubilizing agents, preservatives, antioxidants, flavouring agents and the like. The medicament also may contain other active ingredients, such as antibiotics, analgesics, spasmolytics etc.

The topical administration to the lower gastrointestinal tract also may be achieved by the oral route using e.g. an enteric-coated formulation. In such a formulation the methotrexate, appropriately formulated, e.g. as a viscous liquid or semi-liquid composition, is coated with a material that permits transit through the stomach to the small intestine, where the active ingredient is released. The enteric coated formulation may be in the form of a capsule, or a tablet. One class of enteric coating materials are those that remain intact in the environment of the stomach but solubilise at the higher pH of the intestines. Materials of this type are known in the art and examples are disclosed e.g. in Eury et al., U.S. Pat. No. 5,316,774, issued May 31, 1994, the contents of which are hereby incorporated herein by reference.

In one suitable embodiment, topical, e.g. rectal, administration to the lower gastrointestinal tract is performed by use of a mucoadhesive delivery system, such as a hydrogel. By "mucoadhesive delivery system" is meant a system capable of adhering to an epithelial surface of the body covered by mucus, such as the inner wall of the lower gastrointestinal tract. This mucoadhesive system may be a viscous liquid or semi solid composition.

In one embodiment of the invention, methotrexate is administered by means of a rectiol, i.e. rectiol is a small container provided with a rectal catheter and filled with the methotrexate suitably formulated, e.g. as a viscous liquid having mucoadhesive properties.

Mucoadhesive hydrogels are a class of polymeric biomaterials that exhibit the basic characteristic of a hydrogel to swell by absorbing water and interact by means of adhesion with the mucus that covers epithelia. Other types of mucoadhesive systems that may be used according to the invention are e.g. those based on lectins.

In one embodiment, the mucoadhesive delivery system for topical administration of methotrexate to the lower gastrointestinal tract is rectally administered, e.g. as a capsule suppository or as an enema or rectiol. In another embodiment, the mucoadhesive delivery system for topical administration of methotrexate to the lower gastrointestinal tract is orally administered using a suitable enteric coating.

The medicament of the invention may be formulated so as to allow for prolonged (controlled, sustained) release of methotrexate in the lower gastrointestinal tract. The prolonged release may be achieved e.g. by use of a mucoadhesive system or an enteric coated tablet. A prolonged release tablet or capsule suitably contains methotrexate and optionally any additional active ingredient in a matrix of insoluble or only slowly dissolving substance, which preferably is mucoadhesive. The precise mode, frequency and dosage applied for the topical administration of methotrexate to the treated subject is to be decided by the attending physician. One skilled in the art will recognize that these parameters will depend upon a variety of factors including the age, condition and body weight of the patient, as well as the stage/severity of the disease.

In one embodiment of the invention, in order to prepare a suitable pharmaceutical formulation, methotrexate is dissolved in a pharmaceutically acceptable solvent. Preferably the solvent has a high solubilizing power without being irritating to the tissues to be treated or causing any other adverse effects. Examples of solvents that can be used according to the present invention are organic solvents such as N-methylpyrrolidone, propylene glycol, polypropylene glycol and mixtures thereof.

In one preferable embodiment of the invention, methotrexate, optionally dissolved in a suitable organic solvent, is formulated with a hydrogel-forming material. Examples of hydrogel-forming materials that may be used according to the invention are synthetic polymers, such as polyvinyl alcohol, polyvinylpyrrolidon, polyacrylic acid, polyethylene glycol, poloxamer block copolymers and the like; semi-synthetic polymers, such as cellulose ethers, including carboxymethylcellulose (as the sodium salt), hydroxyethylcellulose, hydroxypropylcellulose, methylcellulose, hydroxypropylmethylcellulose/ methylhydroxypropylcellulose and ethylhydroxyethylcellulose, and the like; natural gums, such as acacia, carrageen, chitosan, pectin, starch, xanthan gum and the like.

It is advantageous to use a hydrogel that is mucoadhesive. In that respect it is particularly useful to use cross-linked polyacrylic acids of the carbomer and polycarbophil types, polymers that readily form gels, which are known to adhere strongly to mucous membranes.

It is also advantageous to use block copolymers of the poloxamer type, i.e., polymers consisting of polyethylene glycol and polypropylene glycol blocks. Certain poloxamers dispersed in water are thermoreversible: at room temperature they are low viscous but exhibit a marked viscosity increase at elevated temperatures, resulting in a gel formation at body temperature. Thereby the contact time of a pharmaceutical formulation administered to the relatively warm intestine/rectum may be prolonged and thus the efficacy of the incorporated substance may be improved.

The formulation may comprise optional, pharmaceutically acceptable additives known in the art for improving different aspects of the composition, such as preservatives, antioxidants, flavouring agents and the like.

The invention will be further illustrated by the following examples, which should not be construed as limiting the scope of the invention.

Examples

Example 1

Preparation of a viscous methotrexate solution or hydrogel

Methotrexate is weighed in a suitable container, preferably a clear glass vial, to which the desired amount of organic solvents) is added. The mixture is then alternately shaken by hand or on a vortex mixer, if necessary heated on a heating block or in a water bath, adjusted to about 40°C, for a few minutes, and then sonicated in a sonication bath for a few minutes. This treatment is repeated until a clear solution is obtained. Between the treatments the sample may be equilibrated at room temperature.

The resulting solution is a clear, yellow solution which should be stored at room temperature protected from light until use.

The hydrogelling material is weighed in a separate container to which the desired amount of water (water for injection or purified water) is added, normally during vigorous mixing. If non-ionic cellulose ether is used, the water may be heated above the cloud point of the resulting solution in order to facilitate the dissolution. Optionally, a few percent of glycerol may be added to the water in order to facilitate the swelling and gelling of the hydrogelling material. The mixture is left at room temperature or in a refrigerator until a clear viscous solution or hydrogel is formed, typically over night. If air bubbles have been trapped in the hydrogel during swelling, these may be removed by gentle centrifugation (for example, by transferring the hydrogel to a suitable container and centrifuging at 1500 rpm for 10 min in a bench centrifuge). Example 2

Preparation of methotrexate formulations for rectal administration in mouse

Table 1 Methotrexate formulations for rectal administration

Methotrexate (Sigma-Aldrich, M9929, lot no. 045K1337) was dissolved in N-methyl-2- pyrrolidone (NMP; Aldrich, Cat. 32,863-4, lot S29935-245) and then diluted with a polymer solution consisting of 2.0 % (w/w) hydroxypropylmethylcellulose (HPMC or Hypromellose; Sigma-Aldrich, H-3785, lot 013K0621) in 2.6 % (w/w) glycerol in water. The samples were prepared in 10 ml glass vials with Al caps. The total volume of each sample was about 5 ml. The samples were stored at room temperature and protected from light.

At administration the formulation was slowly transferred to a syringe through a coarse cannula. The cannula was then replaced by a suitable tip or catheter for rectal administration. The syringe was weighed before and after injection to the rectum.

Example 3

Preparation of methotrexate formulations for rectal administration in mouse

Table 2 Methotrexate formulations for rectal administration

The formulations were prepared in the same manner as in Example 1. All ingredients were weighed. MTX was dissolved in NMP and was then diluted with a polymer solution consisting of 2.0 % (w/w) HPMC in 2.0 % (w/w) glycerol in water. The final concentration of NMP was 10 % (w/w). The samples were prepared in 20 ml glass vials with Al caps. The total of each sample was about 10 g. The samples were stored at room temperature and protected from light.

Biological tests Material and Methods

Mice. The study was approved by the regional Ethics committee for the humane use of research animals in northern Stockholm. Thirty female BALB/c mice, weighing 19-21 g were purchased from B&K Universal AB (SoUentuna, Sweden). The mice were housed under standard conditions. Food and drinking water were available ad libitum.

Experimental design. The experiment was conducted using nine week-old female BALB/c mice. AU treatment groups, four mice in each group, had comparable body weights of 20.1 ± 0.3 g. In this report we use the dextran sodium sulfate (DSS) colitis model in mice. Clinical as well as histological features of the DSS model, originally reported by Okayasu et al (I) resemble IBD in humans. The immunopathogenesis of the model involves infiltration of neutrophils, monocytes and lymphocytes (2-7). Colitis was induced by 2.5% DSS (mol. Wt 40 kD; TdB Consultancy, Uppsala, Sweden), dissolved in purified drinking water, for 19 days. After 13 days, when the animals had established clinical signs of colitis, one group of animals was killed and their colons examined histologically. The other animals received daily rectal treatment with high, low dose methotrexate or vehicle from day 13 until the end of the experiment (day 19). Thus treatment with the compounds studied started first after onset of signs of colitis. This protocol enabled us to monitor potential degree of clinical remission. Clinical signs of disease such as diarrhea, weight loss and rectal bleeding were monitored daily. After 19 days all animals were killed, their colons removed and analyzed histologically. The different treatment groups were compared with an untreated control group and animals killed at an earlier time to disclose potential signs of disease remission.

Treatments. Two doses of methotrexate, 100 μM, and 30 μM, 0.1 ml/day were both administered rectally, Fig 1. Vehicle (MediGelium AB, Stockholm, Sweden) administered in the same way as the methotrexate. The used vehicle contains 10.0 % NMP (N-metyl-2-pyrrolidon), 1.8 % Hypromellos (hydroxipropylmetylcellulosa; HPMC), 1.8 % glycerol and 86.4% water.

Other treatments used in this study: Azathioprine, 5-ASA, and anti-integrin antibodies.

Assessments

Daily assessment of colitis. Daily assessment of animals included measurement of drinking water volume, body weight and evaluation of blood in feces and diarrhea. When the animals were killed, fecal samples were collected for analysis of blood content by Hemocult IVD (TRIOLAB AB, Mδlndal, Sweden).

Histopathological analysis.

The removed mice colons were stained with haematoxylin-eosin. The degree of inflammation on microscopic sections of the colon was graded in a blinded fashion by a GI pathologist. A subjective grading system of acute inflammatory activity in three steps was adopted. Normal mucosa was graded as 0, sporadic scattered segmented granulocytes in the lamina propria was defined as grade 1, few granulocytes in smaller foci was defined as grade 2. If larger quantities of granulocytes were present in lamina propria or if granulocytes were seen in crypts, the inflammation was defined as grade 3. Lymphocytes and plasma cells in the mucosa were not included in the grading system. The surface extension of the mucosal damage was also quantified in the circular microscopic sections of the distal colon and expressed as percentage of the total circular section.

Statistical analysis

Statistical significance was evaluated employing ANOVA or Student's /-test when appropriate. Values are expressed as mean and standard error of the mean (SEM) unless otherwise stated. P< 0.05 was considered statistically significant.

Results

Histological analysis of the distal colon revealed sign of remission, (fig. 2 and less mucosal damage, (fig. 3) in animals treated with high and low doses of methotrexate compared to animals treated with other treatments. Of the comparative treatments given, high dose methotrexate was most effective in ameliorating weight loss due to colitis. Clinical evaluation ofDSS-induced colitis

Clinical evaluation of DSS colitis included daily monitoring of the animals body weight, stool consistency, occult and gross rectal bleeding. All mice that received regular water were negative for clinical signs of colitis throughout the entire experiment. As shown in figure 4 animals that received DSS, substantially lost body weight during the test period. At day 19, when the experiment was ended, DSS-treated animals without treatment had lost 11.2 ± 1.1% body weight compared with healthy controls. Only rectal administration of high dose methotrexate completely prevented weight loss (2.7 ± 1.5% decrease in weight, p< 0.05 vs. DSS alone).

Histological evaluation ofDSS-induced colitis

Histological examination of the distal colon revealed mucosal lesions, edema, crypt damage, and inflammatory infiltrates. A subjective grading system of acute inflammatory activity in three steps was adopted. Normal mucosa was graded as 0 and defined as responder if the animal had received DSS, sporadic scattered segmented granulocytes in the lamina propria was defined as grade 1, few granulocytes in smaller foci was defined as grade 2. Larger quantities of granulocytes in lamina propria or if granulocytes were seen in crypts, the inflammation was defined as grade 3. The surface extension of the mucosal damage was quantified in the circular microscopic sections of the distal colon and expressed as percentage of the total circular section. Both severity and extension of mucosal damage are displayed in Table 3.

Table 3 Severity and extension of mucosal damage in mice

From the results shown in Table 3, it appears that high dose methotrexate could ameliorate both severity and the extension of mucosal damage. Interestingly, in all treatment groups that received vehicle, high or low dose methotrexate, one animal had a completely undamaged mucosa (expressed as percentage 'responders')-

Colonic shortening is a prominent indicator of IBD. As shown in figure S, colon length in healthy animals was 9.5 ± 0.4 cm and 6.0 ± 0.1 cm in animals receiving DSS alone (19 days). Methotrexate could significantly not prevent colonic shortening vs. DSS alone (19 days)). Figure 6 displays representative histological sections of the distal colon of all treatment groups. The most striking histopathological change in response to DSS was epithelial damage and mucosal crypt destruction. This pathological change was seen in all treatment groups receiving DSS, however at different levels of severity as previously illustrated in Table 3.

Discussion

Treatment of animals started first when clinical signs of colitis such as weight reduction and fecal blood were obvious. The therapeutic compounds tested included topically (here rectally) administered methotrexate. High dose methotrexate was most effective in reducing the severity of inflammation, as defined by standard histological grading including depth of mucosal damage and infiltration of inflammatory cells.

References

1. Okayasu, I., Hatakeyama, S., Yamada, M., Ohkusa, T., Inagaki, Y., and Nakaya, R. 1990. A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice. Gastroenterology 98:694-702.

2. Abdelbaqi, M., Chidlow, J.H., Matthews, K.M., Pavlick, K.P., Barlow, S.C., Linscott, A.J., Grisham, M.B., Fowler, M.R., and Kevil, CG. 2006. Regulation of dextran sodium sulfate induced colitis by leukocyte beta 2 integrins. Lab Invest 86:380-390.

3. Lundberg, S., Lindholm, J., Lindbom, L., Hellstrom, P.M., and Werr, J. 2006. Integrin alpha2betal regulates neutrophil recruitment and inflammatory activity in experimental colitis in mice. Inflamm Bowel Dis 12:172-177.

4. Krieglstein, C.F., Cerwinka, W.H., Sprague, A.G., Laroux, F.S., Grisham, M.B., Koteliansky, V.E., Senninger, N., Granger, D.N., and de FougeroUes, A.R. 2002. Collagen- binding integrin alphalbetal regulates intestinal inflammation in experimental colitis. J Clin Invest 110:1773-1782. 5. Chen, Y., Chou, K., Fuchs, E., Havran, W.L., and Boismenu, R. 2002. Protection of the intestinal mucosa by intraepithelial gamma delta T cells. Proc Natl Acad Sci 1/5.4 99:14338-14343.

6. Domek, M.J., Iwata, F., Blackman, E.I., Kao, J., Baker, M., Vidrich, A., and Leung, F. W. 1995. Anti-neutrophil serum attenuates dextran sulfate sodium-induced colonic damage in the rat. ScandJ Gastroenterol 30: 1089-1094.

7. Tsuchiya, T., Fukuda, S., Hamada, H., Nakamura, A., Kohama, Y., Ishikawa, H., Tsujikawa, K., and Yamamoto, H. 2003. Role of gamma delta T cells in the inflammatory response of experimental colitis mice. J Immunol 171:5507-5513.

Claims

1. Use of methotrexate in the preparation of a medicament for the treatment of a tower gastrointestinal tract inflammatory disease in a mammal, which medicament is in a form suitable for topical administration to the lower gastrointestinal tract of said mammal.

2. Use according to claim 1, wherein the disease is an inflammatory bowel disease.

3. Use according to claim 1 or 2, wherein the disease is ulcerative colitis or Crohn's disease.

4. Use according to any one of the claims 1-3, wherein the medicament is formulated so as to allow for prolonged release of methotrexate.

5. Use according to any one of the claims 1-4, wherein the medicament comprises methotrexate in a mucoadhesive formulation.

6. Use according to any one of the claims 1-5, wherein the medicament is for rectal administration.

7. Use according to any one of the claims 1-5, wherein the medicament is for oral administration.

8. Methotrexate for the treatment of a lower gastrointestinal tract inflammatory disease in a mammal by topical administration thereof to the lower gastrointestinal tract of said mammal.

9. A medicament for the treatment of a lower gastrointestinal tract inflammatory disease in a mammal, comprising a therapeutic amount of methotrexate in a vehicle suitable for topical administration to the lower gastrointestinal tract of said mammal.

10. The medicament according to claim 9, wherein the disease is an inflammatory bowel disease.

11. The medicament according to claim 9 or 10, wherein the disease is ulcerative colitis or Crohn's disease.

12. The medicament according to any one of the claims 9-11, formulated so as to allow for prolonged release of methotrexate.

13. The medicament according to any one of the claims 9-12, comprising methotrexate in a mucoadhesive formulation.

14. The medicament according to any one of the claims 9-13, for rectal administration.

15. The medicament according to any one of the claims 9-13, for oral administration.

16. A method of treatment of a lower gastrointestinal tract inflammatory disease by topical administration of a therapeutically effective dose of methotrexate to the lower gastrointestinal tract of a mammal in need of such treatment.

17. The method according to claim 16, wherein (he disease is an inflammatory bowel disease.

18. The method according to claim 16 or 17, wherein the disease is ulcerative colitis or Crohn's disease.

19. The method according to according to any one of the claims 16-18, wherein the administration is performed using a pharmaceutical formulation that allows for prolonged release of methotrexate in the lower gastrointestinal tract of said mammal.

20. The method according to according to any one of the claims 16-19, wherein the administration is performed using methotrexate in a mucoadhesive formulation.

21. The method according to according to any one of the claims 16-20, wherein the administration is performed by rectal administration.

22. The method according to according to any one of the claims 16-20, wherein the administration is performed by oral administration.