CN1179719A - Azathioprine compositions for colonic administration - Google Patents

Abstract

A method is provided to treat inflammatory bowel disease by topically administering to the colon an effective amount of azathioprine or a pharmaceutically acceptable salt thereof, preferably via formulations adapted for delayed-release oral or rectal administration.

Description

Azathioprine compositions for colonic administration

Inflammatory bowel disorders (IBD) comprise a series of intertwined clinical conditions that seem to lack a common etiology. But inflammatory bowel disease is characterized by chronic inflammation in different parts of the gastrointestinal tract. Examples of inflammatory bowel diseases are Crohn's disease, idiopathic ulcerative colitis, idiopathic colorectum and infectious colitis. Most hypotheses regarding the pathogenesis of inflammatory bowel disease involve immunological, infectious, and dietary factors.

6-Mercaptopurine and its prodrugs have been used in the treatment of inflammatory bowel disease for more than 25 years. Multiple controlled trials and recent transition analyzes support the efficacy of 6-mercaptopurine and azathioprine in the treatment of Crohn's disease. See: J.M.T. Willoughby et al., Lancet, ii, 944 (1971); J.L. Rosenberg et al., Dig Dis., 20, 721 (1975). Several controlled trials support the use of azathioprine in the treatment of ulcerative colitis, most recently reported by Hawthorne and colleagues in Brit. Med. J. 1992, Vol. 305, p. 20. However, the use of mercaptopurine and azathioprine is limited due to concerns about their toxicity. Dose-related leukopenia was detected in 2-5% of patients with inflammatory bowel disease taking 6-mercaptopurine and azathioprine for a long time, see for example: D.H.Present et al., Am.lnt.Med., 111, 641 (1989); W. R. Connell et al., Gut, 34, 1081 (1993).

Therefore, there is a need for effective and non-toxic treatments for inflammatory bowel diseases.

The present invention provides a therapy for treating inflammatory bowel disease comprising administering locally to the colon of a patient in need of such treatment an amount of azathioprine effective to alleviate the symptoms of said IBD. The azathioprine is preferably orally administered, and the unit dose is enterically coated to selectively release the azathioprine in the patient's distal ileum and/or colon. Azathioprine can also be effectively administered to the colon by administering an enema formulation containing azathioprine through the rectum. Because azathioprine is poorly absorbed into the blood from the colon, relatively high doses of azathioprine can be given to damaged tissue, i.e. Crohn's disease or ulcerative colitis, without Cause systemic toxicity such as leukopenia. Thus an effective dose of about 150-1000 mg of azathioprine can be administered to adult patients 1-4 times daily (150 mg once daily to 1000 mg 4 times daily) without excessive toxicity (azathioprine The dosage is about 2-20 mg/kg body weight).

The term "azathioprine" used in this specification includes its pharmacologically acceptable salts thereof, as well as functionally equivalent analogs, derivatives and metabolites such as 6-mercaptopurine and the like. See, eg, US Patent No. 3,056,785 and W.P. Wilson et al., Anal. Profiles of Drug Substances, 10, 29-53 (1981).

Colonic administration has been used in patients to reduce the toxicity associated with oral or intravenous corticosteroids and with oral 5-aminosalicylate. This reduction in toxicity is thought to be due to reduced systemic bioabsorption, and several types of colonic drug delivery systems are currently in use, including enemas (see L.R. Sutherland et al., Med. Clin. North Amer., 74, 119 (1990)), rectal foam method (Drug.Ther.Bull., 29, 66 (1991)), and the oral delayed-release formulation of the acrylic emulsion-coated capsule dissolved in the distal ileum pH7 environment, (See K.W.Schroeder et al., New Engl, J.Med., 317, 1625 (1987)). An effective amount of azathioprine may be administered orally ingested topically to the colon of a patient in a unit dosage form comprising an effective amount of azathioprine coated with an enteric coating so that it is released from the unit dose in the lower alimentary tract. out, for example in the distal portion of the ileum and in the patient's colon. Microparticles of azathioprine can be individually coated and administered as a suspension in a liquid carrier, can be encapsulated as a powder, or can be compressed into pellets or tablets for swallowing. Alternatively, azathioprine may be combined with adjuvants, such as fillers and binders, used in solid unit dosage forms, such as pills or tablets, which may be compressed into Treated so that an enteric coating of appropriate thickness is applied on top.

Enteric coatings are those that remain intact in the stomach but dissolve and release the contents of the dosage form once it reaches the small intestine. The purpose of the enteric coating is to delay the release of azathioprine until it reaches its target site of action in the colon. Since azathioprine is locally administered to the colon tissue in this way, only about 10% is absorbed into the blood, and systemic side effects of azathioprine are avoided or minimized.

Thus, an effective enteric coating is one that remains in a non-dissociated state in the low pH environment of the stomach and only readily ionizes when the pH rises to about 4 or 5. The most effective enteric coating polymers are Polyacids with pHa 3 to 5.

The most widely used polymer is cellulose acetate phthalate (CAP), which functions effectively as an enteric coating. However, a pH greater than 6 is usually required for dissolution, thus allowing delayed release of the drug. Another useful polymer is polyvinyl acetate phthalate (PVAP), which is less permeable to moisture and gastric juices, is hydrolytically stable, and ionizes at lower pH, resulting in active It is released earlier in the duodenum.

A more recently available polymer is hydroxypropylmethylcellulose phthalate, which has similar stability and decomposes in the same pH range as polyvinyl acetate phthalate. Further examples of polymers in use today are those based on methacrylic acid, such as methacrylic acid copolymers, which carry acid ion groups, such as Eudragit S-100 (methacrylic acid copolymer). Various systems exist to make these enteric coating polymers suitable for aqueous diffusion, thus facilitating the enteric coating of pharmaceutical dosage forms using aqueous film coating techniques.

Another preferred dosage form for topical administration of azathioprine to the colon is an enema acid, which is administered from the rectum to the descending colon. The formulations used include an effective amount of azathioprine dissolved or dispersed in a suitable flowable carrier such as water, ethanol or hydro-alcoholic fluid, preferably a natural or synthetic thickener such as gums, acrylates or Denatured cellulose is thickened. The formulation may also include an effective amount of lubricants, such as natural or synthetic oils, ie, triglycerides or lecithin. Nontoxic, nonionic surfactants can also be included as wetting and spreading agents. Unit doses of enema formulations may be administered in pre-filled sachets or syringes. The carrier may also contain an effective amount of a foaming agent, such as n-butane, propane or isoalkane. Such a formulation can be delivered from a pressurized container such that the carrier is delivered to the colon in a foamy form, thereby preventing its escape from the target site.

The present invention is further illustrated by citing the following examples.

Example 1 Preparation of hydrophilic rectal azathioprine foam.

Add 2.0 grams of KELTROL TF (xanthan gum suspension) to 948.4 grams of pure distilled water, which contains 1.4 grams and 0.14 grams of methyl paraben and propyl paraben as preservatives respectively. Spread it out. Then 2.0 grams of soybean lecithin emulsifier (primary color) and 5.0 grams of water-soluble vinyl polymer (carbomer, CARBOPOL974PNF) were also diffused. Thereafter, the water-soluble vinyl polymer was neutralized with 2 g of sodium hydroxide dissolved in 20 ml of purified water (pH 7.1). Next, 10.0 grams of POLYSORBATE 80 (surfactant) was added to this mixture along with 0.25 grams of CITRAL (perfume) and the two components were mixed well. Finally, azathioprine 2.366 grams was also added and stirred well. The theoretical content of azathioprine in this formula is 50mg/21g of mixed solution. The viscosity of the concentrated solution measured by a Broodfield viscometer DV-II at 20° C. and 12 rpm (with a 64 measuring axis) is: 29,700 CPS.

Thereafter the concentrate is mixed with the foaming agent n-butane using standard aerosol-making equipment. The content of n-butane in the aerosol mixture is 2.5%. Then pour this mixture into the sleeve (bag-in-can) of the Sepro type metal casing. In the aluminum tank, the propellant nitrogen is equipped with a nozzle and a sleeve in this integral tank. At a temperature of 37°C, the discharge volume of each metal tank concentrate is 21 ± 1 g, resulting in 200-220 ml Foam.

Example 2 Hydrophobic rectal azathioprine foam

Add 2.0 grams of KELTROL TF (xanthan gum suspension), 2.0 grams of primary color soybean lecithin (emulsifier) and 5.0 grams of water-soluble vinyl polymer (carbomer, CARBOPOL 974NF) into 948.45 grams of pure distilled water, respectively Contains 1.43 g and 0.14 g of methylparaben and propylparaben, which are homogenized with a SILVERSON homogenizer. The water-soluble vinyl polymer (carbomer) was neutralized with 2.0 g of sodium hydroxide dissolved in 20 ml of pure water (pH 7.05).

Then add 47.40 grams of hydrophobic agent WITEPSOL H 15 (Hard fat NF) and use a SILVERSON homogenizer to stir the mixture efficiently. A creamy concentrate with a hydrophobic feel is obtained.

Add 10.50 grams of surfactant POLYSORBATE 80 (polysorbate) and 0.25 grams of CITRAL (perfume), and also stir well with a homogenizer. In order to stabilize this emulsification, especially the corresponding foam, add 20.80 g of POLAWAX NF (as non-ionic emulsifying wax) to the mixture and stir well. Finally 1.93 grams of azathioprine were added and mixed well. The theoretical content of azathioprine in the formula is 50 mg in 27.5 g of the mixed solution. The measured viscosity of Azathioprine (AZA) concentrate AZA-1: 41.000CPS. Thereafter the concentrate for the AZA-1 sample was mixed with the blowing agent n-butane, the n-butane content being 2.5%. This mixture is then poured into the metal cans of the Sepro-type metal sheath, the discharge of each can of concentrate is 27.5 grams ± 1 gram, and at a temperature of 37° C., 200-220 milliliters of foam is produced. Hydrophobic foams have a lower diffusivity than hydrophilic foams.

Example 3 Preparation of azathioprine enteric-coated capsules

One azathioprine (CIMURAN) tablet (Azathioprine 50mg Tablets Containing 50mg Azathioprine, Burroughs Wellcone) was crushed in a mortar and the powder was filled into 2 equal-sized capsules, which were manually Sealed and each capsule is individually weighed.

The capsules are capped with a 50% by weight aqueous solution of gelatin (1% polysorbate) on a laboratory capping machine. Capsules were finally coated in a laboratory coater by spraying Eudragit S-100 (methacrylic acid copolymer 1N _NH4OH ). Capsules made (12 wt% methacrylic acid copolymer (12wt-% Eudragit)) were able to withstand simulated gastric fluid (pH 1.2) for 2 hours, but less than 60% in simulated intestinal fluid (pH 7.2). Minutes are broken down.

Example 4 Local administration of azathioprine in the colon

A. Subjects

24 healthy volunteers were recruited for physical screening and laboratory tests (full blood count, chemical examination, urinalysis). Before entering the study, all patients and subjects were tested for erythrocyte thiopurine methyltransferase (TPMT) activity in order to exclude homozygotes or heterozygotes with low TPMT activity from the study, as previously reported Severe neutropenia associated with the use of azathioprine or 6-mercaptopurine occurred in this population. Subjects were screened for medical conditions and surgical history that may affect the absorption or metabolism of azathioprine. The demographics of the subject cohorts are summarized in Table 1, indicating that there were no statistically significant differences between the cohorts for the parameters.

Table 1 Group Number Age Weight TPMT Level ^*

(Years old) (kg) (unit/ml, RBC) Orally taking 6 27.7 ± 7.5 80.3 ± 20.0 21.9 ± 2.3dro 6 36.7 ± 18.9 ± 44.0 21.2 ± 2.6hbf 6 29.2 ± 5.8 83.5 ± 4.0hpf 6 33.2 ± 8.2 71.7±10.0 19.5±1.8*average±standard deviation

B. Research Design

24 healthy human subjects were randomized to receive a dose of 50 mg azathioprine prescribed in 4 groups (n=6 in each group): Oral; Delayed Release Oral (DRO); Hydrophobic Rectal Foam (HBF); and Hydrophilic Rectal Foam (HPF). All subjects also received a 50 mg dose of intravenous (IV) azathioprine. The two azathioprine doses were separated by at least 3 weeks, and the order of administration (IV vs. non-IV) was randomized. Subjects fasted after midnight before the study. If colonic foam administration was to be administered, subjects were given a quick enema if their bowels were not moving on the morning of the study.

A single intravenous dose (see below) is administered as an injection diluted in 20 mL of normal saline, delivered by infusion over 5 minutes. Oral preparations were swallowed with water, and rectal prescriptions were administered by nurses, and subjects were required to fast for 3 hours after administration. A time zero blood sample was drawn prior to the study. Blood samples were drawn into 7 mL at the following intervals: 5, 10, 15, 30 minutes and 1, 1.5, 2, 3, 4, 5, 6, and 8 hours after one dose was administered or after the IV infusion was initiated In a vacuum tube containing EDTA ( _K3 ) (Sherwood Medical, St. Louis, Mo.).

Pharmacokinetic studies of azathioprine were performed by measuring the bioabsorption rate of 6-mercaptopurine rather than azathioprine because reliable techniques for measuring plasma 6-mercaptopurine levels are available. Furthermore, 6-mercaptopurine is a more biologically relevant molecule since azathioprine functions as a precursor of mercaptopurine. Azathioprine is rapidly converted to 6-mercaptopurine after absorption, which is accomplished by the non-enzymatic chemical action of the linkage between the imidazole ring and the 6-mercaptopurine molecule by a compound containing a sulfhydryl group such as glutathione. 6-Mercaptopurine is then metabolized to a compound with immunomodulatory activity, 6-mercaptoguanine nucleotide (6TGN).

C. Determination of 6-mercaptopurine concentration in plasma

Put the blood sample into water immediately after collection, and centrifuge the blood sample at 1000 g at 4°C for 10 minutes within 30 minutes. Plasma was then transferred to plastic cryogenic tubes (Nunc Inc., Naperbille, IL) and stored at -70°C until analysis. 6-Mercaptopurine concentrations were determined by high pressure liquid chromatography using a modified technique from Zimm (20). Solid-phase extraction cartridges (C18 Sep-Paks, Waters, Inc., Nillford, MA) were prerinsed sequentially with 2.5 mL methanol and 5 mL 0.2% acetic acid. One milliliter of plasma was placed on the Sep-Pak and 0.04 milliliters of saturated EDTA.2Na (Aldrich, Milwaukee, Wisconsin) solution was added to the plasma. Dithiothreitol (DTT) was not included because azathioprine was immediately converted to mercaptopurine in the presence of DTT. The extraction cartridge was rinsed with 2 ml of 0.2% acetic acid, and then centrifuged at 3200 rpm for 5 minutes to remove excess water. The sample was eluted from the cartridge with 2 mL of methanol and evaporated to dryness at 37°C under nitrogen flow. Samples were then reconstituted into the free phase in 200 μL, vortexed for 30 minutes, transferred to 1.5 mL microconical tubes and centrifuged in a microcentrifuge for 5 minutes. A portion of the supernatant (0.175 mL) was transferred to a vial for HPLC.

At this time, 6-mercaptopurine was determined by high-pressure liquid chromatography, and the analytical column was Hewlett-Packard (Rockville, Maryland) octadecasilane (ODS) Hypersil, 200×4.6 mm, 5 μm particle size. It was protected by a Zorbax (Mac-mod Analytical, Chadds Ford, Pennsylvania) ODS 4 x 12.5 mm guard column. The mobile phase was 0.8% acetonitrile in 1 mM ( _C2H5 )3N ( _{triethylamine} ), pH adjusted to 3.2 with phosphoric acid. Absorbance was monitored at 340nm. The injection volume is 80 μL. Unknowns were determined by comparing them to a standard curve established on the same day by spiking known amounts of 6-mercaptopurine into blank plasma. The lower limit of quantitation for 6-mercaptopurine was 2 ng/ml. The coefficients of variation for the average calculated concentration ± 2 ng/ml and the 50 ng/ml standard were: 2.0 ng/ml ± 18% and 50.2 ng/ml ± 3.4%, respectively.

D. Pharmaceutical Research

(1) Intravenous administration of azathioprine, sodium salt lyophilized formulation (Burroughs Wellcome, Research Triangle Park, North Carolina).

(2) Standard 50 mg oral tablet (Burroughs Wellcome., Research Triangle Park, North Carolina).

(3) 50 mg delayed release oral tablet (DRO).

(4) Hydrophobic rectal foam (HBF), 50 mg of azathioprine (manufactured by Fernion/Orion, Espoo, Finland) is administered rectally through a pressurized foam cartridge, which is dissolved in an oil-containing base (Witepsol H 15) in the foam agent to make it hydrophobic.

(5) Hydrophilic rectal foam (HPF) for rectal administration of 50 mg of azathioprine (manufactured by Fernion/Orion, Espoo, Finland) through a pressurized foam cartridge dissolved in a foam. Both the azathioprine delayed-release oral formulation and the rectal foam are manufactured by a Swiss company (Tillotts Pharma.A.G., Ziefen, Switzerland).

E. Results

1. Pharmacokinetic parameters for intravenous (IV) azathioprine agents

The indicators of AUC, CL, V _ds , T _1/2 (mean ± standard deviation) of all 24 subjects after intravenous administration of 50 mg of azathioprine are listed in Table 2. Table 2 shows the pharmacokinetic parameters of 6-mercaptopurine after intravenous administration of 50 mg of azathioprine to 24 healthy volunteers ^* . Table 2 AUC CL V _ds T _1/2 (ng h/ml) (L/kg h) (L/kg) (h) 100.7±30.5 3.8±1.2 6.7±3.1 1.2±0.37 ^* mean±standard deviation

2. Pharmacokinetic parameters of colonic release

AUC, F, T _max and C _max (mean ± standard error) for each non-intravenous route of administration are listed in Table 3. The average bioabsorption rate of the oral formulation was significantly greater than that of other formulations, and the average T _max of the delayed-release oral formulation formulation was significantly greater than that of other formulations. None of the other differences were statistically significant. Table 3 shows the pharmacokinetic parameters ^* of 6-mercaptopurine after oral administration, DRO, HBF, and HPF of 50 mg of azathioprine. table 3

Group AUC F Tmax Cmax

(n＝6) (ng h/ml) (%) (h) (ng/ml)

Oral 55±27 52±26 1.8±0.8 21±13

DRO 11±9 13±15 5.4± ^0.2C 5± ^5C

HBF 5±4 5±4 2.1± ^0.3d 2± ^1d

HPF 3±4 2±3 2.0 ^± 0.0e 1± ^1e

P-values 0.01 ^a 0.01 ^a 0.01 ^b 0.01 ^{a a} P-values were derived from analysis of covariance. Post-test results (Duncan's): a) oral administration>DRO=HRF=HPF; b) DRO>oral administration=HBF=HPF; C) mean ± standard error (6-mercaptopurine concentration of 5 subjects); d) mean ± standard error (4 subjects detected 6-mercaptopurine concentration); e) mean ± standard error (3 subjects detected 6-mercaptopurine concentration).

3. Rectal administration

The rectal foam formulation has a very good hold. 22 subjects could keep the foam for more than 6 hours, only one subject said that the hydrophobic foam was discharged after only 1 hour, and the other said that the hydrophobic foam was discharged after 2 hours. No detectable (drug) absorption was found in the subject who reported foaming after 1 hour. The subject who reported foaming after 2 hours had a detectable Cmax of 2.35 ng/ml at 3 hours. There were no reports of adverse reactions to the foam product.

F. Discussion

This example shows that the systemic bioabsorption rate of 6-mercaptopurine after administration of azathioprine in delayed release oral formulations and rectal delivery formulations is significantly lower than the general oral azathioprine 6-mercaptopurine bioabsorption rate. There are several potential factors contributing to this observation. The most likely factor is less absorption of azathioprine across colonic mucus than through gastric and small intestinal mucus because there is no specific transport mechanism or different rates of passive absorption. A reduction in colonic absorption compared to jejunal absorption has been demonstrated by 5-aminosalicylate (5-ASA) (see S.Bondesen et al., Br.J.Clin.Pharm., 25, 269 (1988 )). Azathioprine and 6-mercaptopurine may be more completely metabolized in the colonic mucus closer to the gastroenteric tract. Also, some azathioprine may be lost in the stool. After administration of acrylic emulsion-coated tablets, approximately 25% of 5-ASA is excreted in feces (see B. Norlander et al., Aliment. Pharmacol. Therap., 4, 497 (1990)).

Azathioprine is rapidly converted to 6-mercaptopurine in plasma after absorption. This conversion is the result of the non-enzymatic chemical action of a thiol-containing compound such as glutathione on the linkage between the 6-mercaptopurine molecule and the imidazole ring of azathioprine (see L. Lennard et al., Clin. Pharmacol. Therap ., 46, 149 (1989)). Glutathione is present in every mammalian cell, including colonic epithelial cells and lymphocytes, and previous studies have shown that lymphocytes contain the enzyme 6TGNs required to convert 6-mercaptopurine into active metabolites (see B. Bostrom et al., Am. J. Ped. Hem./Onc., 15, 80 (1993)). Therefore, it is reasonable to expect that local release of azathioprine will result in a local immunosuppressive effect on colonic lymphocytes.

This example demonstrates that colonic release of azathioprine results in significantly lower bioabsorption of 6-mercaptopurine than conventional oral azathioprine. Colonic release of azathioprine is thought to reduce drug toxicity by reducing systemic exposure to 6-mercaptopurine. In addition, the topical delivery of azathioprine results in the release of higher locally concentrated doses to the intraepithelial lamina propria colonic lymphocytes.

The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that various changes and modifications may be made within the spirit and scope of the invention.

Claims (7)

1. A method for preparing a medicament for treating intestinal inflammatory diseases, comprising a certain amount of azathioprine suitable for local administration to the colon of patients who need such treatment, wherein said dose is effective for alleviating said intestinal inflammatory diseases symptoms are valid. 2. The method according to claim 1, characterized in that the intestinal inflammatory disease is ulcerative colitis. 3. The method according to claim 2, characterized in that the intestinal inflammatory disease is Crohn's disease. 4. The method of claim 1, wherein the agent is adapted to be administered to the patient's lower colon by a rectal enema. 5. The method of claim 1, wherein the medicament is in a unit dosage form suitable for oral administration, comprising an effective dose of enteric-coated azathioprine in the patient's terminal ileum and colon. Released from dosage form. 6. The method of claim 1, wherein the unit dosage form comprises an enteric coating that disintegrates at about pH 7. 7. The method of claim 1, wherein the dosage form releases a total daily dose of azathioprine of about 3-75 mg/kg.