TITLE

Therapeutic compositions and methods for treating colon

diseases.

Background of the invention

Inflammatory bowel disease (IBD) is the general name for diseases that cause inflammation in the small intestine and colon. Ulcerative colitis is the most common inflammatory bowel disease and it can be difficult to diagnose because its symptoms are similar to other intestinal disorders and to another type of IBD called Crohn's disease. Crohn's disease differs from ulcerative colitis because it causes inflammation deeper within the intestinal wall. Also, Crohn's disease usually occurs in the small intestine, although it can also occur in the mouth, oesophagus, stomach, duodenum, large intestine, appendix, and anus .

Ulcerative colitis may occur in people of any age, but most often it starts between ages 15 and 30, or less frequently between ages 50 and 70. Children and

adolescents sometimes develop the disease. Ulcerative colitis affects men and women equally and appears to run in some families.

It is also important to consider that about 5

percent of people with ulcerative colitis develop colon cancer. The risk of cancer increases with the duration

and the extent of involvement of the colon. For example, if only the lower colon and rectum are involved, the risk of cancer is no higher than normal. However, if the entire colon is involved, the risk of cancer may be as

much as 32 times the normal rate.

Treatment for ulcerative colitis depends on the seriousness of the disease . Most people are treated with medication. In severe cases, a patient may need surgery to remove the diseased colon.

Irritable bowel syndrome (IBS) is a common but poorly understood disorder that causes a variety of bowel symptoms including abdominal pain, diarrhoea and/or constipation, bloating, gassiness and cramping. While these symptoms may be caused by a number of different bowel diseases, IBS is usually diagnosed only after

exclusion of a more serious problem.

The goal of therapy is to induce and maintain remission, and to improve the quality of life for people

with IBD/IBS.

Irritable bowel syndrome (IBS) and inflammatory bowel diseases (IBD) are intestinal disorders

characterized by low to high grade of gut inflammation.

In many patients affected by one of these pathologies,

these conditions are often associated with one or more of the following symptomatic disturbances at various degree of severity, that depends also on the status (early, mature or advanced; active or quiescent, etc.) of the disease process: gut hypermotility, gut hypersensitivity, abdominal cramp, visceral pain, diarrhea, etc.

Aminosalicylates, drugs that contain 5- aminosalicylic acid (5-ASA) , help control inflammation. Sulfasalazine is a combination of sulfapyridine and 5-ASA and is used to induce and maintain remission.

Other 5-ASA agents such as olsalazine, ipsalazide and balsalazide, have a different carrier, offer fewer side effects than sulphasalazine, and may be used by people who cannot take sulphasalazine. 5-ASAs are given orally, through an enema, or in a suppository, depending on the location of the inflammation in the colon. Most people with mild or moderate ulcerative colitis are treated with this group of drugs first. However, in general therapy cannot be considered optimal, mainly

because of the poor potency of the drug that causes also a poor compliance for the patient.

Moreover aminosalicylate derivatives are reported to exacerbate colitis (and thereby abdominal cramps and pain

episodes, diarrhea, etc) in a significant number of

patients and is often limited in its regular use for this reason.

Among several possible drugs commonly indicated to treat gut disorders, dicyclomine is sometimes used systemically to reduce gut disorders, but the incidence

of classic antimuscarinic side-effect (dryness of mouth, blurred vision, tachycardia and costipation) often limits its regular use.

Other agents, such as 5-HT. sub.3 receptor antagonists, 5-HT.sub.4 receptor agonists or antagonists, anxiolytics, benzodiazepine compounds, anti- spasmodic/anti-muscarinic agents, selective serotonin reuptake inhibitors ("SSRIs"), tricyclic antidepressants, selegeline, belladonna alkaloids, muscarinic, metoclopramide, anti-inflammatory compounds, antacids, GI relaxants, loperamide, diphenoxylate, anti-gas compounds, bismuth-containing preparations , are effective for the treatment of gastrointestinal altered motility, sensitivity and secretion and abdominal viscera disorders, including both functional and organic diseases .

Description of the ^■ invention

This invention is related to pharmaceutical

compositions that combine mesalamine, or its derivatives, with a co-agent effective for the treatment of

gastrointestinal altered motility, sensitivity and

secretion and abdominal viscera disorders including both functional and organic diseases.

The invention aims at the treatment of intestinal diseases by combining two agents with two different characteristics . One agent (mesalamine or mesalazine and its derivatives) has anti-inflammatory activity and the other agent is active on intestinal smooth muscle dysfunctions. The two agents are delivered locally to the target site. Therefore there is a unit of invention related to the targeted colon delivery for the treatment (relief of motility disorders and mitigation of inflammatory processes) by combining an anti-inflammatory agent and a gut motility regulator.

Mesalamine, or 5-ASA, is the 5-amino-2-

hydroxybenzoic acid. Its derivatives are considered sulfasalazine, olsalazine, balsalazide, ipsalazide.

Co-agents considered in the present application are

anti-spasmodic/anti-muscarinic agents, (such as trimebutine, dicyclomine, hyoscyamine, darifenacin , cimetropium, octilonium) , 5-HT.sub.4 receptor agonists

or antagonists, (such as tegaserod, cisapride, nor-

cisapride, renzapride, zacopride, mosapride, itopride) , 5-HT.sub.3 receptor antagonists, (such as ondansetron,

cilansetron, alosetron, ramosetron, azasetron, granisetron and tropisetron) , anxiolytics, (such as chlordiazepoxide) , benzodiazepine compounds, selective serotonin reuptake inhibitors, ("SSRIs" such as fluvoxamine, fluoxetine, paroxetine, sertraline, citalopram, venlafaxine, duloxetine, etc.) _/ tricyclic antidepressants, (such as amitriptyline, buproprione, etc.), selegeline, belladonna alkaloids, (such as atropine and scopolamine) , muscarinic, metoclopramide , anti-inflammatory compounds, antacids, GI relaxants, loperamide, diphenoxylate, anti-gas compounds, bismuth- containing preparations .

Oxidative stress plays an important pathogenetic role in intestinal pathologies, and an anti-oxidant therapy (vitamin E, N-acetylcysteine, lipoic acid, hydroxyphenylacetic acid, cinnamic acid, caffeic acid, ascorbic acid, carnosine, glutamine, taurine, tromethamine, etc.) consequently has been indicated or at least suggested. According to the present invention, additionally to the mesalamine and to the co-agent, also an antioxidant can be added.

A preferred combination is 5-ASA and trimebutine.

The actions of trimebutine [3 , 4, 5-trimethoxybenzoic acid

2- (dimethylamino) -2-phenylbutylester] on the

gastrointestinal tract are mediated via (i) an agonist effect on peripheral mu, kappa and delta opiate receptors and (ii) release of gastrointestinal peptides such as

motilin and modulation of the release of other peptides, including vasoactive intestinal peptide, gastrin and glucagon (Delvaux M, Wingate D., J. Int Med Res 1997 25 (5) 225-246.). Trimebutine accelerates gastric emptying, induces premature phase III of the migrating motor complex in the intestine and modulates the contractile activity of the colon. Recently, trimebutine has also been shown to decrease reflexes induced by distension of the gut lumen in animals and it may therefore modulate visceral sensitivity. Clinically, trimebutine has proved to be effective in the treatment of both acute and chronic abdominal pain in patients with functional bowel

disorders, especially irritable bowel syndrome, at doses ranging from 300 to 600 mg/day. It is also effective in children presenting with abdominal pain.

It has been discovered that the combination of these two components (a mesalamine compound and a co-agent active on the GI tract) improves significantly the

therapy of colonic diseases.

Another preferred combination is 5-ASA with

dicyclomine. Surprisingly and unexpectedly we discovered that by combining mesalamine with an anti-muscarinic

agent such as dicyclomine in a proper delivery system releasing the active principles in the intestinal situ it could be possible to reduce intestinal inflammation process, to mitigate the above described intestinal inflammation related symptomatic disturbances, to improve mesalamine tolerability, by limiting its colitis exacerbation associated adverse effects, and to improve dicyclomine tolerability, by reducing classical anti- muscarinic side-effects.

Another preferred combination is 5-ASA with itopride as it combines the antinflammatory pharmacological activity of mesalamine with the gastroprokinetic activity of itopride. The ratio between 5-ASA or its derivative and co-agent is of from 5:95 to 95:5%. The antioxidant compound, if present, is in an amount of from 0,1 to 20% by weight of the composition.

It is also further part of the present invention the delivery of the combination to the colon through specific

local delivery systems. Preferred ones are coated tablets that releases the active ingredients to the colon and the suppositories. The targeted delivery to the colon, via

coated tablets that release the active ingredients in the

colon area, is advantageous to reduce substantially the

side effects of the spasmolitic compound (s) and to enhance the anti-inflammatory activity of 5-ASA at the

site of action, namely the colon.

Pharmaceutical acceptable salts, such as for example, salts with alkaline metals and alkaline earth metals, non-toxic amines and amino acids, such as carnosine, are also part of the present invention. Preferred salts are the salts with arginine, agmatine of mesalamine or its derivatives in combination with a co-agent.

The L-arginine and agmatine (its decarboxylated analog) salts are precursor for nitric oxide, an endogenous messenger molecule involved in a variety of endothelium-mediated physiological effects in the vascular system.

Depending on the specific condition or disease state to be treated, subjects may be administered compounds of the present invention at any suitable therapeutically effective and safe dosage, as may be readily determined

within the skill of the art. For example, compounds of the present invention may be administered at a dosage between about 0.5 and 100 mg/kg, preferably between about 5 and 50 mg/kg.

The compounds of the invention are preferably

administered in combination orally or intrarectally.

The present invention is also directed to pharmaceutical compositions which include mesalamine and a co-agent compound as described above, in association

with one or more pharmaceutically acceptable diluents, excipients or carriers therefor.

In making the pharmaceutical compositions of the present invention, the compounds will usually be mixed with, diluted by or enclosed within a carrier which may be in the form of a capsule, sachet, or other container. When the carrier serves as a diluent, it may be a solid, semi-solid or liquid material which acts as a vehicle, excipient or medium for the active ingredient. Thus, the compositions can be in the form of tablets, coated tablets, pills, powders, lozenges, sachets, cachets, suspensions, emulsions, solutions, foams, soft and hard gelatine capsules, suppositories.

Some examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate,

alginates, tragacanth, gelatin, calcium silicate, microcrystalline ^• cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl- and

propyl-hydroxybenzoates, talc, magnesium stearate and mineral oil .

The formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, propellents. The compositions of the invention may be formulated so as to provide rapid, sustained or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art .

Preferred formulations are enteric coated tablets and most preferred film coated tablets that at acid pH (<6) remain intact (no release of active ingredients) and that at basic pH (>7) release the medicaments. The coatings therefore only begin to dissolve when they have left the stomach and entered the small intestine. A thick layer of coating is provided which will dissolve in about 3-4 hours thereby allowing the tablets to disintegrate only when it has reached the terminal ileum or the colon.

Such a coating can be made from a variety of polymers such as cellulose acetate trimellitate (CAT) ,

hydroxypropylmethyl cellulose phthalate (HPMCP) , polyvinyl acetate phthalate (PVAP) , cellulose acetate

phthalate (CAP) and shellac as described by Healy in his article "Enteric Coatings and Delayed Release" Chapter 7

in Drug Delivery to the Gastrointestinal Tract, editors Hardy et al . , Ellis Horwood, Chichester, 1989. For

coatings of cellulose esters, a thickness of 200-250 μm

would be suitable.

Especially preferred materials are methylmethacrylates or copolymers of methacrylic acid and methylmethacrylate. Such materials are available as Eudragit polymers (trademark) (Rohm Pharma, Darmstadt, Germany) . Eudragits are copolymers of methacrylic acid and methylmethacrylate. Preferred compositions are based on Eudragit LlOO and Eudragit SlOO. Eudragit LlOO dissolves at pH 6 and upwards and comprises 48.3% methacrylic acid units per g dry substance; Eudragit SlOO dissolves at pH 7 and upwards and comprises 29.2% methacrylic acid units per g dry substance.

Preferred coating compositions are based on Eudragit LlOO and Eudragit SlOO in the range of 100 parts SlOO :0

parts LlOO to 20 parts SlOO: 80 parts LlOO. As the pH at which the coating begins to dissolve increases, the thickness necessary to achieve colon specific delivery

decreases. For formulations where the ratio of Eudragit LlOO: SlOO is high, a coat thickness of the order 150-200 μm is preferable. For coatings where the ratio Eudragit LlOO: SlOO is low, a coat thickness of the order 80-120

μra is preferable.

The following non-limitative examples further

describe and enable an ordinary skilled in the art to

make and use the invention.

EXAMPLES

Example 1. - Tablet containing 5ASA and trimebutine

Tablets of the combination are prepared according to the following formula:

Mesalazine 400.00 mg

Trimebutine base 150.00 mg Lactose monohydrate 96.00 mg Sodium starch glycolate 28.00 mg Povidone 9.20 mg

Magnesium stearate 6.20 mg Talc 10.40 mg

Preparation:

4 kg of mesalazine are mixed with 1.5 kg of trimebutine, 0.960 kg of lactose and 0.14 kg of sodium starch glycolate (Explotab) . The mixture is granulated with a 10% w/w solution of povidone (PVP K30) dissolved in water. After granulation the mixture is dried in a thermostatic oven at 42°C until a residual humidity of < 2% is reached. The remaining part, 0.14 kg (50%) of the disintegrant sodium starch glycolate, and the lubricant, 0.062 kg magnesium stearate and 0.10 kg talc are added.

The final mixture is compressed using a rotary-punch

machine to an average weight/tablet of 700 mg+5% and an hardness > 5.0 Kp.

Example 2.- Coated tablet with Eudragit SlOO

5 kg of tablets prepared according to Example 1 are film coated with a film having the following composition: Methacrylic Acid-Methyl Methacrylate copolymer (1:2) 23.90 mg Triethyl citrate 12.20 mg

Talc 12.20 mg

Ferric oxide red 2.70 mg

Titanium dioxide 4.00 mg

Preparation:

In a stainless steel container, disperse the Eudragit SlOO (Methacrylic acid-Methyl methacrylate copolymer) in water and maintaining stirring, add slowly 1.7% ammonia solution mixing for 60 minutes. Add triethyl citrate very slowly under stirring and mix for 60 minutes. In another stainless steel container disperse the other components of the film, talc, iron oxide and titanium dioxide until an homogeneous suspension is obtained. Mix together the two suspensions and maintain stirring during the coating process.

Transfer the tablets prepared in example 1 to a

rotary coating pan and spray the tablets, maintaining the

temperature of the tablet bed at about 40 ⁰ C, with the

film-forming suspension. Example 3. - Release in vitro

The dissolution performance of the tablet coated with Eudragit SlOO as described in example 2 was tested using the USP Method I (baskets rotating at 50 rpm) . For the first 2 h of the test, 0.1M HCl was used as the test medium. After 2 h, the test medium was changed to 0.05M phosphate buffer, pH 7.4. Samples were withdrawn at regular intervals from the dissolution vessels and the appearance of mesalazine and trimebutine was monitored spectrophotometrically.

After 2 hours the release for each of the 2 active ingredients (mesalazine and trimebutine) is less than 5%. In the second medium, at pH 7.4 with sodium phosphate buffer aqueous solution in the same apparatus, after 1 hour the release of the 2 active ingredients is more than 80%.

Example 4. - Tablets of balasalazide and itopride

Tablets of the combination are prepared according to the following formula:

Balsalazide disodium 600.00 mg Itopride HCl 50.00 mg

Lactose monohydrate 100.00 mg Sodium starch glycolate 25.00 mg Povidone 9.00 mg

Magnesium stearate 6.00 mg Talc 10.00 mg

Preparation.-

6 kg of balsalazide disodium are mixed with 0.5 kg of itopride HCl, 1.00 kg of lactose and 0.125 kg of sodium starch glycolate (50%) (Explotab) . The mixture is granulated with a 10% w/w solution of povidone (PVP K30) dissolved in water. After granulation the mixture is dried in a thermostatic oven at 42°C until a residual humidity of < 2% is reached. The remaining part, 0.125 kg (50%) of the disintegrant sodium starch glycolate, and the lubricant, 0.060 kg magnesium stearate and 0.10 kg talc are added. The final mixture is compressed using a rotary-punch machine to an average weight/tablet of 800 mg ± 0,5% and an hardness > 5.0 Kp.

Example 5.- Suppository with mesalazine and trimebutine

The suppositories were prepared according to the following formula: Mesalazine 400 mg

Trimebutine 150 mg

Hard fat 2450 mg

Weigh in a suitable beaker Suppocire AM ™ (hard fat) and melt by heating at 60 ⁰ C. Weigh trimebutine, pass through a

1 mm sieve, and under stirring slowly pour it in the melted fatty base. Weigh mesalazine, pass through a 1 mm sieve and slowly add to the melted base containing also trimebutine.

Maintaining the temperature at 40 ⁰ C transfer the suspension to the filling-molding machine where it is poured into the shells at a weight of 3.0 g/suppository.

Allow the molded suppository to cool in the refrigerator at a constant temperature of 5°C. Example 6.- Suppository balsalazide, ondansetron, Vitamin E

The suppositories were prepared according to the following formula: Balsalazide 600 mg Ondansetron 15 mg Vitamin E 300 UI Hard fat 1385 mg

Weigh in a suitable beaker Suppocire AM ™ (hard fat) and melt by heating at 60 ⁰ C. Weigh ondansetron, pass through a 1 mm sieve, and under stirring slowly pour it

in the melted fatty base. Weigh balsalazide, pass through a 1 mm sieve and slowly add to the melted base containing

also ondansetron. Add Vitamin E (d-alpha tocopheryl

acetate) and mix carefully for 30 minutes.

Maintaining the temperature at 40 ⁰ C transfer the suspension to the filling-molding machine where it is poured into the shells at a weight of 2.0 g/suppository.

Allow the molded suppository to cool in the

refrigerator at a constant temperature of 5°C. Example 7. - Rectal foam

(% Composition of foam or pressurized liquid) Mesalazine 20.0

Citalopram HCl 2.0

Carnosine HCl 0.5

Xanthan gum 0.2

Potassium metabisulphite 0.2 EDTA bisodium salt 0.3 Polysorbate 20 4.0

Polyglycol 300 isostearate 4.0 Purified water 58.8

Freon 12 (propellant) 6.5 Freon 114 (propellant) 3.5 Preparation:

The components citalopram HCl, potassium metabisulphide, EDTA, carnosine HCl, and xanthan gum in

water in the stated order in a stainless steel dissolving

vessel of suitable capacity fitted with a propeller

stirrer and turboemulsifier.

Polysorbate 20, polyglycol 300 isostearate and mesalazine are added while stirring, and the

turboemulsifier is then operated for 15 minutes. Using a metering pump, the suspension is metered in the volume corresponding to the theoretical weight into aerosol cans while stirring.

Each can is immediately sealed by clinching the dispenser valve and is then pressurized by means of the propellant, which is fed in under pressure in a suitable quantity by a pumping device .

Example 8.- Pharmacological activity in animals The activity of the different treatments was evaluated according to the method described by Chevalier et al . (Life Sciences 76,319-329,2004). Trimebutine (3 mg/kg) and mesalamine (10 mg/kg) were administered intrarectally alone or in combination 30 minutes before the administration of trinitrobenzene sulfonic acid (TNBS, 0,5 ml) (50 mg/kg dissolved in 30% ethanol) . Two groups

of male Sprague-Dawley rats (one healthy and one with colitis) of 10 animals each are also included. The

animals are killed 3 days after administration of TNBS and the intestine examined macroscopically for colitis.

Intestinal myeloperoxidase (MPO) was also measured as

marker of inflammation by a spectrophotometric method, as described in the above quoted reference. It was found

that the combination of the two drugs markedly reduced both the severity of colitis degree and MPO values, much more than the two drugs given separately. The tolerability was excellent for all the treatments. Example 9.- Clinical activity.

Fifteen patients, aged 43 to 68, with inflammatory bowel syndrome (IBS) were treated with 400 mg mesalazine and 150 mg trimebutine administered twice a day with a colon delivery tablet formulation prepared according to example 2.

Rapid clinical remission was obtained in these patients with cessation of symptoms.

After one month of treatment, five patients were examined colonoscopically. Four of these patients had normalised the colonic and terminal ileum mucosa while one continued to have patchy inflammatory changes and histological presence of minimal inflammatory infiltrate

with some eosinophils.

In this last patient, a combination of mesalazine, citalopram and cairnόsine dispensed as rectal foam

prepared as described in example 6 made up a preferred

therapy .

Seventy percent of the patients improved dramatically at 3 months with removal of all need for anti-inflammatory bowel disease medications.

Example 10.- Tablets of mesalazine and dicyclomine

Tablets of the combination are prepared according to the following formula:

Mesalazine 600.00 mg

Dicyclomine hydrochloride 20.00 mg

Lactose monohydrate 130.00 mg

Sodium starch glycolate 25.00 mg

Povidone 9.00 mg

Magnesium stearate 6.00 mg

Talc 10.00 mg

Preparation:

6 kg of mesalazine are mixed with 0.2 kg of dicyclomine

HCl, 1.30 kg of lactose and 0.125 kg of sodium starch glycolate (50%) (Explotab) . The mixture is granulated with a 10% w/w solution of povidone (PVP K30) dissolved in water. After granulation the mixture is dried in a thermostatic oven at 42°C until a residual humidity of <

2% is reached. The remaining part, 0.125 kg (50%) of the disintegrant sodium starch glycolate, and the lubricant,

0.060 kg magnesium stearate and 0.10 kg talc are added.

The final mixture is compressed using a rotary-punch

machine to an average weight/tablet of 800 mg ± 0,5% and an hardness > 5.0 Kp.

The tablets are film coated as described in example 2.

Example 11.- Suppository with mesalasine and dicyclomine

The suppositories were prepared according to the

following formula:

Mesalazine 800 mg

Dicyclomine hydrochloride 40 mg

Hard fat 2160 mg

Weigh in a suitable beaker Witepsol H15 ^® and melt by heating at 60 ⁰ C. Weigh dicyclomine, pass through a 1 mm sieve, and under stirring slowly pour it in the melted fatty base. Weigh mesalazine, pass through a 1 mm sieve and slowly add to the melted base containing also dicyclomine .

Maintaining the temperature at 40 ⁰ C transfer the suspension to the filling-molding machine where it is poured into the shells at a weight of 3.0 g/suppository.

Allow the molded suppository to cool in the refrigerator at a constant temperature of 5°C.