DE102010012223A1 - Pharmaceutical preparation, useful for the treatment of autoimmune disease, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, Crohn's disease or ulcerative colitis, comprises 12-imidazolyl-1-dodecanol - Google Patents

Abstract

Pharmaceutical preparation comprises 12-imidazolyl-1-dodecanol or its salt. ACTIVITY : Immunosuppressive; Antiinflammatory; Hepatotropic; Gastrointestinal-Gen.; Antiulcer. MECHANISM OF ACTION : Cytochrome P450 2E1 (CYP2E1) inhibitor. The CYP2E1 inhibiting activity of the preparation was tested in microsomes of rat using para-nitrophenol hydroxylation assay. The results showed that imidazolyl-dodecanol exhibited an IC 50value of 727.06 nM.

Description

The invention relates to a pharmaceutical preparation containing 12-imidazolyl-1-dodecanol for the treatment of an autoimmune disease; It also means here and below with 12-imidazolyl-1-dodecanol its pharmaceutically acceptable salts.

Autoimmune disease refers to diseases whose cause is an excessive reaction of the immune system against the body's own tissue. By mistake, the immune system recognizes the body's own tissue as a foreign body to be controlled. This leads to severe inflammatory reactions that lead to damage to the affected organs. The exact cause of autoimmune diseases remains unclear despite intensive research. Accepted hypotheses assume that autoimmune diseases are acquired through congenital "susceptibility" (genetic disposition, such as the presence of certain MHC molecule variants) in combination with external influences. If there are such genetic factors in the body of the person affected and unfavorable environmental factors such as high levels of stress, infections, pregnancy are added, the onset of autoimmune diseases can occur. The target structures of the autoimmune reaction can be confined to a specific organ (from the hair root to the liver) or invade the whole body (multiple organs and vascular systems). Mixed forms with several autoimmune diseases are not rare.

Because the exact molecular pathomechanisms of autoimmune diseases are unknown, no causal therapy is possible. Autoimmune diseases can therefore only be treated symptomatically (anti-inflammatory or immunosuppressive). The basic principle of symptomatic therapy here is to suppress the activity of the immune system by administering immunosuppressants, such as corticosteroids or azathioprine. Due to the manifold systemic side effects and interactions of these substances, attempts have been made to develop new drugs that specifically affect the mechanisms involved in the disease process. Examples include natalizumab and infliximab, which are used to treat multiple sclerosis or rheumatoid arthritis. Although these newer substances are specific and tolerated by most patients, severe side effects may occur (such as progressive multifocal leukoencephalopathy (PML) with natalizumab).

The object of the invention was to help improve the treatment of autoimmune diseases with a pharmaceutical preparation that does not have the disadvantages of the prior art, or at least not to the extent that weaken esp. The inflammation processes caused by the autoimmune reaction.

This is achieved by a pharmaceutical preparation containing 12-imidazolyl-1-dodecanol for the treatment of an autoimmune disease.

It has surprisingly been found that the use of 12-imidazolyl-1-dodecanol can significantly reduce the inflammation processes caused by an autoimmune reaction. This leads to a decrease in the intracellular formation of highly reactive oxygen radicals, such as hydrogen peroxide, superoxide radical anions or hydroxyl radicals as well as the autoimmune disease typical, excessive cytokine production. As a result, the pathological tissue changes and remodeling processes can be mitigated or even stopped. In addition, a beneficial effect of simultaneously administered immunosuppressive drugs is achieved. A particularly pronounced effect of 12-imidazolyl-1-dodecanol can be achieved due to the relatively high expression of cytochrome P450 isoforms in autoimmune diseases of the gastrointestinal tract. Examples include autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, and Crohn's disease and ulcerative colitis.

It has been found that surprisingly low doses of 12-imidazolyl-1-dodecanol are sufficient to achieve the desired purpose. Thus, for an adult with an autoimmune disease, 1 mg of 12-imidazolyl-1-dodecanol may be sufficient as a daily dose; and even a lower dose is sufficient for a child or even an infant.

Accordingly, a pharmaceutical preparation according to the invention preferably contains 1 to 30 mg of 12-imidazolyl-1-dodecanol for the treatment of an autoimmune disease, more preferably 5 to 20 mg, especially 10 to 15 mg.

For the treatment of, inter alia, drug intoxications recommended WO 2007/124734 A2 12-imidazolyl-1-dodecanol. On the other hand, it has been shown that surprisingly low doses of 12-imidazolyl-1-dodecanol allow the successful treatment of autoimmune disease.

The present invention thus allows a considerable advance over the current possibilities of treating an autoimmune disease; and this with surprisingly low doses of the drug 12-imidazolyl-1-dodecanol.

The preparation according to the invention may contain other pharmaceutical active ingredients, auxiliaries and / or additives in a manner known per se, especially preservatives, solubilizers and extenders.

The advantageous properties of the preparation according to the invention are confirmed by the following investigations.

For 12-imidazolyl-1-dodecanol, the following kinetic parameters were determined, which prove the low dosage according to the invention with a simultaneously high inhibition potential.

para-nitrophenol hydroxylation Assay

The para-nitrophenol hydroxylation assay serves to determine the activity of cytochrome P450 2E1 ( Chang et al. 1998 ) and was performed with rat microsomes and human CYP2E1 supersomes. The human supersomes contain CYP2E1, reductase and cytochrome b5. The reaction buffer of the assay was HEPES (0.1 M, pH 7.6). For the p-NP hydroxylation assay, HEPES, p-Np, MgCl ₂ (0.1 M), microsomes / supersomes and optionally inhibitor were pipetted into an Eppendorf tube and then incubated for 5 min at 37 ° C in a water bath. The addition of NADPH (20 mM) started the enzymatic reaction. The samples were incubated for 30 min at 37 ° C in a water bath. Subsequently, the enzymatic reaction was stopped by addition of trichloroacetic acid (20%). The samples were centrifuged off for 5 min at 10,000 rpm at RT. The supernatant was pipetted into a cuvette to 2 M sodium hydroxide solution (NaOH). The absorbance was measured on the photometer at 535 nm against distilled water. The measurements were performed in triplicate. The calculation of the 4-nitrocatechol concentration and thus the enzymatic activity was carried out by means of a 4-nitrocatechol calibration line.

Determination of the K _m value

The determination of the K _m value serves to determine the enzymatic activity of CYP2E1 without inhibitors. The K _m value provides the substrate concentration at which the half-maximal rate of the enzymatic reaction is reached. The p-Np concentration used was between 30 and 600 μM.

Determination of the IC ₅₀ value

The IC ₅₀ value provides the inhibitor concentration at which the residual enzymatic activity has fallen to 50% of the initial value without inhibitor. The IC ₅₀ value was determined at the substrate concentration corresponding to the Km value of the microsomes. The inhibitor used to inhibit CYP2E1 was imidazolyldodecanol. The inhibitor solution was dissolved in HEPES with the addition of 0.1% DMSO (Stock). The required dilution steps of the inhibitor were prepared by serial dilution in pure HEPES (0.1 M, pH 7.6). Furthermore, the determinations of the inhibitory effect of imidazolyl-dodecanol were also carried out dissolved in cyclodextrin.

The inhibitor concentration used for 12-imidazolyl-1-dodecanol in 0.1% DMSO / HEPES (0.1 M, pH 7.6) was 0-3000 nM in the microsome and 0-2000 nM in the supersome fraction. For imidazolyl-dodecanol dissolved in cyclodextrin, concentration values were found in the microsome fraction of 0-2000 nM and in the supersome fraction 0-2000 nM.

Determination of the K _i value

The K _i value provides the dissociation constant between an enzyme complex and an inhibitor molecule. The K _i value was determined at a constant inhibitor concentration and a variable substrate concentration. The inhibitor concentration used corresponded to the determined IC ₅₀ value of the inhibitor used. The substrate concentrations used were between 50 and 300 μM for the supersomes and between 40 and 400 μM for the microsomes.

The absorbance values obtained were divided by the slope of the 4-nitrocatechol calibration line and the concentration of 4-nitrocatechol calculated. Using the calculated rates of enzymatic reaction, the software program SigmaPlot 10 (Systat Software Inc. (SSI), San Jose (USA)) the IC ₅₀ value determined. The K _m and K _i values were determined using the Enzyme Kinetics 1.3 module.

Results: Microsomes of rat liver.

Determinations of the enzymatic activity of CYP2E1 were made with the acetone-induced microsomal extracts from the rat liver. The amount of protein used in CYP2E1 was unknown. A test for different amounts of microsomes was used to determine the volume of 20 μL microsome extract used. The calculations were based on the total protein concentration of the extracts and a theoretically determined proportion of CYP2E1 on this total protein extract.

Determination of the K _m value

The K _m value was carried out with p-Np concentrations between 20 and 400 μM. The measurement values were plotted using the Enzyme Kinetics 1.3 module for the SigmaPlot 10 program. The module was used to generate a Michaelis quantity diagram. The resulting K _m value was calculated independently by the module.

The calculated K _m value for the rat liver microsomes is 111.5 μM. This means that at a p-Np concentration of 111.5 μM the enzymatic reaction has reached its half-maximal velocity (v _max / 2). The maximum enzymatic activity (v _max ) is 1.6 moles of 4-nitrocatechol per minute and per mmol of CYP2E1.

Determination of IC ₅₀ values

The IC ₅₀ value was determined at a constant p-Np concentration and a variable inhibitor concentration. The p-Np concentration used corresponded to the K _m value (110 μM). The obtained values for the enzymatic activity were plotted against the inhibitor concentration by means of the SigmaPlot 10 program. The residual enzymatic activity of 50% was calculated. The resulting IC ₅₀ value was determined by the SigmaPlot 10 program.

12-Imidazolyl-1-dodecanol in HEPES / 0.1% DMSO

The determination of the IC ₅₀ value for imidazolyl-dodecanol was carried out for concentration of imidazolyl-dodecanol between 0 and 2500 nM. The solution of imidazolyl-dodecanol was in HEPES and 0.1% DMSO.

The obtained IC ₅₀ value for imidazolyldodecanol in HEPES with 0.1% DMSO is 727.06 nM. Thus, at an imidazolyl-dodecanol concentration of 727.06 nM, the residual enzymatic activity of CYP2E1 is 50% of the baseline without inhibitor.

12-imidazolyl-1-dodecanol in cyclodextrin

The inhibitory effect of imidazolyl-dodecanol was additionally investigated by means of a cyclodextrin complex solution of imidazolyldodecanol. The imidazolyl-dodecanol concentrations used were between 0 and 2000 nM. The determined IC ₅₀ value for imidazolyl dodecanol in cyclodextrin is 402.73 nM. The residual enzymatic activity of 50% of the initial value without inhibitor is thus reached at an imidazolyl-dodecanol concentration of 402.73 nM.

Determination of K _i values

The inhibitor concentration used corresponded to the IC ₅₀ value of the inhibitor used. Thus, the residual enzymatic activity of CYP2E1 is 50% of the baseline without inhibitor. The substrate concentration was varied as in the determination of the K _m value.

The calculated enzymatic activities were evaluated by means of the module Enzyme Kinetics 1.3 for the program SigmaPlot 10. For the calculation of the K _i value, the measured values for the K _m value were used as reference value. The plots were plotted as a Lineweaver-Burk diagram. In the Lineweaver-Burk plot, the reciprocal of the substrate concentration (1 / [p-Np]) is plotted against the reciprocal of the enzymatic activity (1 / v). The resulting graph represents a straight line.

The mechanism of action of the tested inhibitor is competitive inhibition. In a competitive inhibition v _max with inhibitor is identical to v _max without inhibitor. In the Lineweaver-Burk plot, the intersection of the line with the enzymatic activity axis (1 / v axis) gives the reciprocal of maximum enzymatic activity (1 / v _max ). This intersection point is identical for the measured values without inhibitor and the measured values with inhibitor. The intersection of the straight line without inhibitor with the 1 / [p-Np] axis gives the value for -1 / K _m . The calculation of the K _m / K _i value was carried out independently by the module Enzyme Kinetics 1.3.

12-Imidazolyl-1-dodecanol in HEPES / 0.1% DMSO

The inhibitor concentration used corresponded to the determined IC ₅₀ value for imidazolyl-dodecanol in HEPES / 0.1% DMSO (727.06 nM). The p-Np concentrations used were between 40 and 200 μM.

The calculated K _m value and the v _max value without inhibitor are 111.5 μM and 1.6 moles of 4-nitrocatechol per minute and per mmol of CYP2E1.

The determined K _i value in the presence of imidazolyl-dodecanol in HEPES / 0.1% DMSO is 300.3 nM. The v _max value with inhibitor is identical to the v _max value without inhibitor.

12-imidazolyl-1-dodecanol in cyclodextrin

The inhibitor concentration of imidazolyl-dodecanol in cyclodextrin was 402.73 nM (IC ₅₀ value). The p-Np concentrations used were between 40 and 200 μM.

The K _m value and v _max value without inhibitor are 109.3 μM and 1.6 mol 4-nitro-catechol / min / mmol CYP2E1. The calculated K _i value at an imidazolyl-dodecanol concentration of 402.73 nM is 252.8 nM. The _vmax value with inhibitor remains identical to the value without inhibitor.

Human CYP2E1 supersomes

The determination of the enzymatic activity was carried out with human CYP2E1 supersomes. The supersome concentration used was 50 nM in the total incubation approach. The resulting enzymatic activities were calculated by means of Excel.

Determination of the K _m value

The K _m value was determined with p-Np concentrations of 50 to 600 μM. The evaluation was carried out by means of a Michaelis-Menten diagram. Calculations of the K _m and v _max values were performed using the Enzyme Kinetics 1.3 module. The determined K _m value is 86.4 μM. The v _max value for the human CYP2E1 supersomes is 7.5 moles of 4-nitrocatechol per minute and per mmol CYP2E1.

Determination of IC ₅₀ values

The IC ₅₀ values were determined at a constant p-Np concentration. The p-Np concentration used corresponded to the K _m value of the acetone-induced microsomes 110 μM. The inhibitor concentration was varied. The evaluation was carried out by means of the SigmaPlot 10 program. The resulting IC ₅₀ value was determined graphically.

12-Imidazolyl-1-dodecanol in HEPES / 0.1% DMSO

The inhibitor concentrations used were between 0 and 2000 nM. The imidazolyl-dodecanol was dissolved in HEPES and 0.1% DMSO. The determined IC ₅₀ value is at an imidazolyl-dodecanol concentration of 267.25 nM.

12-imidazolyl-1-dodecanol in cyclodextrin

The inhibitory effect of imidazolyl-dodecanol was also investigated in cyclodextrin dissolved. The used imidazolyl-dodecanol concentrations were varied between 0 and 2000 nM. The resulting IC ₅₀ value for imidazolyldodecanol in cyclodextrin is 602.74 nM. Thus, CYP2E1 has one Imidazolyldodecanol concentration of 602.74 nM a residual activity of 50% from baseline without inhibitor.

Determination of K _i values

The K _i values were determined at a constant inhibitor concentration and a variable substrate concentration. The inhibitor concentration used corresponded to the determined IC ₅₀ value for the inhibitor used.

The obtained values for the enzymatic activity were evaluated with the module Enzyme Kinetics 1.3. As a comparison value, the measured values from the determination of the K _m value for the supersomes were used. The values were plotted as a Lineweaver-Burk diagram. The module Enzyme Kinetics 1.3 calculated the K _m , K _i and v _max values.

12-Imidazolyl-1-dodecanol in HEPES / 0.1% DMSO

The imidazolyl-dodecanol concentration used corresponded to the determined IC ₅₀ value for imidazolyldodecanol and was 267.25 nM. The p-Np concentrations used were 50, 100, 150, 200 and 300 μM. The determined K _m and v _max values are 86.3 μM and 7.5 moles of 4-nitrocatechol per minute and per mmol CYP2E1. The calculated K _i value for imidazolyl-dodecanol in HEPES / 0.1% DMSO is 225.3 nM.

The v _max value with inhibitor is identical to the v _max value without inhibitor and is 7.5 mol formed 4-nitrocatechol per min and per mmol CYP2E1.

12-imidazolyl-1-dodecanol in cyclodextrin

Furthermore, the competitive inhibition of imidazolyl-dodecanol dissolved in cyclodextrin was investigated. The used imidazolyl-dodecanol concentration was 602.74 nM corresponding to the determined IC ₅₀ value for imidazolyl-dodecanol in cyclodextrin. As p-Np concentrations 50, 100, 150, 200 and 300 μM were used. The calculated K _m value without inhibitor is 83.9 μM. The value of v _max without inhibitor was than 7.4 mol of 4-nitrocatechol per min and per determined mmol CYP2E1. The v _max value with inhibitor is identical to the v _max value without inhibitor. The determined K _i value for imidazolyl-dodecanol in cyclodextrin is 634.6 nM.

Summary of the measurement results

A summary of the measurement results for the measurement of inhibitory enzymatic activity by means of the p-Np hydroxylation assay is given in the following table. inhibitor microsomes Super omen without inhibitor K _m 111.5 μM 86.9 μM v _max 1.6 mol / min / mmol 7.4 mol / min / mmol Imidazolyl dodecanol in HEPES / 0.1% DMSO IC ₅₀ 727.06 nM 267.25 nM K _i 300.3 nM 225.3 nM Imidazolyl dodecanol in cyclodextrin IC ₅₀ 402.73 nM 602.74 nM K _i 252.8 nM 634.6 nM

The K _m value and the v _max value without inhibitor were constant during the determination of the K _i values for the inhibitors. The V _max values with inhibitors is identical to the v _max value without inhibitor.

In the following, the invention will be explained by way of example without wishing to restrict it unnecessarily.

Example 1: Film-coated tablet

On the basis of magnesium stearate, polysorbate 80, povidone K 25, microcrystalline cellulose, fumed silica, crospovidone (type A), talc, hypromellose and Macrogol 6000 film tablets are prepared containing 12.5 mg of 12-imidazolyl-1-dodecanol per tablet ,

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• WO 2007/124734 A2 [0009]

Cited non-patent literature

• Chang et al. 1998 [0014]

Claims (9)

A pharmaceutical preparation containing 12-imidazolyl-1-dodecanol or a pharmaceutically acceptable salt thereof for the treatment of an autoimmune disease. A pharmaceutical preparation according to claim 1, containing 1 to 30 mg of 12-imidazolyl-1-dodecanol or a pharmaceutically acceptable salt thereof. A pharmaceutical preparation according to claim 1, containing 5 to 20 mg of 12-imidazolyl-1-dodecanol or a pharmaceutically acceptable salt thereof. A pharmaceutical preparation according to claim 1, containing 10 to 15 mg of 12-imidazolyl-1-dodecanol or a pharmaceutically acceptable salt thereof. A pharmaceutical preparation according to any one of claims 1-4 for the treatment of autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, Crohn's disease or ulcerative colitis. A pharmaceutical preparation according to any one of claims 1-5, which additionally contains an immunosuppressive agent. A pharmaceutical preparation according to any one of claims 1-5, additionally containing a corticosteroid. Use of a pharmaceutical preparation according to any one of claims 1-7 for the treatment of an autoimmune disease. A process for the preparation of a pharmaceutical preparation according to any one of claims 1-7 by mixing the components.