DE19814814C2 - Water-soluble pharmaceutical compositions containing iminophenazine derivatives, their preparation and use - Google Patents

Description

The present invention relates to pharmaceutical compositions containing at least one Iminophenazine derivative contained in aqueous or water-soluble formulation. Further The present invention relates to a method for producing such pharma zeutischer compositions, and the use of the invention Compositions for the manufacture of a medicament.

Iminophenazine derivatives have anti-inflammatory and immunosuppressive Properties also antimycobacterial properties. Most famous representative of this Compound class is clofazimine (2-p-chloroanilino-5-p-chlorophenyl-3,5-dihydro-3- isopropyliminophenazine), which u. a. against Mycobacterium leprae and Mycobacterium avium-Mycobacterium intracellurare complex (hereinafter referred to as MAC) acts. Mycobacterium leprae is the causative agent of leprosy, while MAC is the most common bacterial opportunistic infection in AIDS. But also other mycobacterial ones Infections such as B. tuberculosis can be treated with iminophenazine derivatives.

However, iminophenazine derivatives mostly have the disadvantage of a poor one Solubility in water, which limits their use or is also responsible for the fact that Patients must be given high doses to obtain effective serum concentrations to reach. So z. B. clofazimine a solubility in water of about 0.5 to 1 mg / l, which means that a single dose of 200 mg of clofazimine is necessary to People to achieve an average maximum serum concentration of 0.47 mg / l (Garrelts in DICP, The Annals of Pharmacotherapy 25 (1991) 525-531).

Various approaches have been taken to increase the bioavailability of these substances already undertaken. So z. B. as a commercial formulation of clofazimine encapsulated suspension of the microcrystalline substance in an oil-wax base for the oral application available.

O'Reilly et al. (International Journal of Pharmaceutics 109 (1994) 137-154) the effect of simple and mixed micellar systems made of sodium cholate or Sodium cholate / fatty acid mixtures on the solubility and intestinal absorption of  Clofazimin in anesthetized rats. Increases in solubility of clofazimin in the context of the 16- to 63-fold compared to the solubility in water were for various aqueous Bile acid salt-fatty acid combinations described. A 63-fold solubility The increase was B. achieved in an 80 mM aqueous sodium cholate solution. The of O'Reilly et al. found increased gastrointestinal absorption became the solubility increase and better membrane permeability attributed.

Other approaches to increasing water solubility, that is to say the hydrophilicity of the formulations, led to considerations to modify the iminophenazines as such by introducing salt-forming groups. These approaches can best be described using the following general formula (I) of the iminophenazine derivatives:

So z. B. introduced for R ² different salt-forming groups, but this greatly reduced the activity of the iminophenazine derivatives. Nonetheless, many iminophenazine derivatives in the R ² group contain primary, secondary or tertiary amino groups, which makes them more hydrophilic than clofazimine (R ² = isopropyl). The increased hydrophilicity, however, must mostly by an additional introduction of aliphatic radicals with z. B. 6-8 carbon atoms (also in R ² ) can be counteracted to avoid the impending loss of activity.

Cyclodextrins are already suitable for solubilizing hydrophobic substances known for a long time. Cyclodextrins are another starting point Increase in solubility. Usually, the geometric increase in solubility Responsible for the structure of the cyclodextrins. They have a hydrophobic, in Molecular region in which various hydrophobic substances can be included. The outer area is hydrophilic and conveys the  Solution, these so-called inclusion complexes in aqueous media. Usually the unsubstituted cyclodextrins are used. Beyond that, too some substituted cyclodextrins such as hydroxypropylated or permethylated Cyclodextrins found their use in this area. Not every substance class is however suitable to form complexes with the above cyclodextrin derivatives. On the other hand, it is Increase in solubility does not necessarily result in the formation of inclusion compounds (Complexes), other types of association are also conceivable, see e.g. B. DE-38 09 808-A1.

In the Garrelts (DICP, The Annals of Pharmacotherapy 25 (1991) 525-531) was suggested to include clofazimine consider formulations. Nevertheless, this food for thought did not lead to Success reports in this area. About the formation of inclusion compounds between iminophenazines and the known cyclodextrin derivatives has so far not been possible be reported.

An object of the present invention was to provide compositions pose the problem of low water solubility and / or water dispersibility of iminophenazine derivatives. The poorly water-soluble iminophenazine derivatives (hereinafter also referred to as clofazimine and its analogues) moreover, be made available in a wording that is in addition to a Higher gastrointestinal uptake also opens up other application options. So sets z. B. parenteral application of a water-soluble formulation of Iminophenazine derivatives ahead. Other forms of administration can also be of the Providing highly concentrated water soluble formulations benefit. So are z. B. sprays conceivable, but also the admixture of such formulations to ointments and aqueous-based gels, the peroral optionally encapsulated or rectal Administration is also an option. Also encapsulated in pharmaceutical relevant liposomes can be made. Even unencapsulated aqueous iminophenazine derivative Formulations which, if appropriate, in a strongly acidic or strongly basic medium extremely microcrystalline precipitation of the iminophenazine derivative capable are advantageous can be used, because by providing such formulations a finest Distribution of the active substance in a large amount of liquid, which causes the absorption of the Active ingredient is facilitated.  

Another object was to use in the compositions anti-inflammatory, immunosuppressive, antimycobacterial and / or antiviral To provide treatment.

The invention was surprisingly achieved by the provision of aqueous or water-soluble pharmaceutical compositions which

• a) at least one iminophenazine derivative of the general formula (I):
  wherein
  R ¹ = an optionally halogenated phenyl radical,
  R ² = a branched or unbranched alkyl radical having 1 to 6 carbon atoms,
  and
  R ³ = an optionally halogenated phenyl radical,
  and
• b) at least one water-soluble cyclodextrin, which as polysulfate or Polyphosphate is present and covalent with a sterol or sterol derivative is linked

contain.  

Z. B. that of Quigley et al. (Int. J. Pharm. 58 (1990) 107-113) described iminophenazine derivatives, which also fall under the general formula (I) and known in the literature as B628, B633, B749, B826, B3779, B3785, B3770 and B3640 are used.

R ¹ = p-chlorophenyl radical, R ² = isopropyl radical and R ³ = p-chlorophenyl radical, ie chlofazimine (B663), are particularly preferred.

As sterols covalently bonded to the anionic cyclodextrin, compounds are to be understand which the framework of a fully or partially hydrogenated cyclopenta [a] phen anthrens and which have a hydroxyl group in the 3β position. These sterols can also further hydroxyl groups or in the branched at position C-17 or straight-chain alkyl or alkenyl side chain containing 1 to 10 carbon atoms carry a carboxyl group.

Such sterols come e.g. B. cholesterol, lanosterol, ergosterol, stigmasterol or β- Sitosterol, but also bile acids such as cholic acid.

Is particularly preferred, not least because of its inexpensive and simple Availability, cholesterol.

There is a carboxy function in the side chain of position C-17 Sterins, this can, if necessary after activation with z. B. N-hydroxysuccinimide can be selectively linked to a monoaminocyclodextrin to form an amide.  

If the only functionality of sterol is the mandatory hydroxyl group in the 3β position, so the sterol is preferably via difunctional "bridge molecules" with the Cyclodextrin linked. These molecules include, for example, diacids such as Succinic acid, glutaric acid, adipic acid, or corresponding compounds in which instead of the carboxyl groups, other hydroxy or amino reactive groups are, such as B. isocyanate, acid anhydride or N-hydroxysuccinimide groups. A functional group can be linked to the hydroxyl group of sterol accordingly are used for binding to a hydroxyl group of the cyclodextrin or the other Amino group of an aminocyl codextrin.

A sterol modified in this way with bridge molecules is in the sense of claim 1 as To understand sterol derivative. Preferred is the sterol derivative Succinic acid monocholesteryl ester, in which a carboxyl group of succinic acid associated with cholesterol. The other can, if necessary after activation with z. B. N-hydroxysuccinimide, for selective binding to the preferred mono-6 Deoxy-6-amino-β-cyclodextrin serve.

Wear the cyclodextrins so lipophilically linked to a sterol or sterol derivative at least as much anionic groups in the form of sulfate or phosphate groups as are necessary to this a high water solubility, for. B. <10 mg / ml.

As mentioned, cyclodextrins are particularly good and selective via bridge molecules link to the sterol if you convert them to monoaminocyclodextrins beforehand. The Monoaminocyclodextrins can be produced in various ways. Petter et al. Describes one possibility. (J. Am. Chem. Soc. 112 (1990) 3860-3868), further methods were described by Croft and Bartsch in Tetrahedron 39 (1983) 1417-1474 summarized. Should z. B. the succinic acid monocholesteryl ester with a amino-modified cyclodextrin are linked in this way Succinic monocholesteryl ester preferably initially, such as. B. from Bellanger and Perly in J. Mol. Struct. 273 (1992) 215-226 for fatty acids, with N- Hydroxysuccinimide in a suitable solvent, such as. B. ethyl acetate in the activated succinic acid monocholesteryl ester and sets this with a Monoaminocyclodextrin to cholesterol-linked cyclodextrin. Below is z. B. in solvents such as dimethylformamide or pyridine sulfation or  Phosphating with commercially available sulfating or phosphating reagents carried out. Trimethylamine is particularly suitable for gentle sulfation. Sulfur trioxide complex. Other possible sulfation and phosphation cracks ions and reagents are also from Croft and Bartsch in Tetrahedron 39 (1983) 1417-1474 or in Otake et al. (Antiviral Chem. Chemother. 5 (1994) 155-161) described.

The compounds obtained are preferably in physiologically acceptable salts, such as z. B. sodium salts, what z. B. by adding a concentrated aqueous Sodium acetate solution and subsequent precipitation with ethanol can happen.

Liquid Secondary Ion Mass Spectrometry (LSIMS) shows that the mean number is anionic Groups strongly on the ratio of the lipophilically modified Cycdextrinderivat to "Anionizing agent" and that the reaction in any case leads to compounds, which have a distribution around a medium number of anionic groups. As long as it is ensured that the resulting compounds are sufficiently water-soluble, preferably about <10 mg / ml, plays the number of anionic groups per Cyclodextrin molecule regarding the water solubility of the iminiphenazine derivatives mediating properties a subordinate role.

The inventive method for producing the water-soluble according to the invention Formulations include mechanical agitation, e.g. B. stirring or shaking one Suspension of the corresponding iminophenazine in an aqueous solution of the Cyclodextrin derivative. Here, the maintenance of a pH value in slightly acidic, i.e. H. between 6 and 7 advantageous. This can be done by buffering the solution with commercially available Buffers can be achieved. Volatile buffers such as e.g. B. Ammonium acetate, which occurs when freeze-drying aqueous solutions of the Compositions according to the invention volatilize, so that compositions result, which only the iminophenazine derivative and the cyclodextrin derivative contain. Stirring or shaking at 5 to 95 ° C, but preferred because gentle and energy saving, at room temperature instead to achieve the desired solution effect achieve. Depending on the duration of the stirring or shaking process and its intensity, as well as in. Depending on the type of the corresponding iminophenazine derivatives and cyclodextrin derivatives, and the concentration of the components are different amounts of  Iminophenazine derivative solved. Preferably corresponding to a cyclodextrin Iminophenazine ratio of 20: 1 to 0.8: 1 (here, as in the following, it is always that Molar ratio meant), particularly preferably according to a ratio of 10: 1 to 1: 1. Preferably, under an inert gas atmosphere, such as. B. a nitrogen atmosphere worked to oxidize oxidation-sensitive iminophenazine derivatives prevent. Any iminophenazine derivative which remains undissolved is filtered off. The solutions obtained are optically clear and can, for. B. by filtration through 400 nm Sterilize the sterile filter. Followed by freeze drying gives powdery products that can be taken up again in aqueous medium.

That already mentioned above linked with the succinic acid monocholesteryl ester Sodium salt of β-cyclodextrin sulfate was, for. B. for Solubilization of clofazimine used. In this way, solutions could be obtained are in addition to 10 mg / ml of the cyclodextrin derivative z. B. 1.7 mg / ml clofazimine. This corresponds to a solubility of 0.5 µg / ml clofazimine in water, a 3400-fold increase in solubility. Taking into account that from such Solutions by freeze-drying solid compositions are available, which are also in a smaller amount of aqueous volume can be absorbed Solubility increases in the range of 10,000 times and higher can be achieved.

As already mentioned above, another task was to make it available an antiviral composition. This task is also carried out by the compositions according to the invention solved. That through the sterol linkage Lipophilized anionic cyclodextrin derivatives have solubility in addition to their ability of iminophenazines also increase an anti-HIV effect, which is even stronger is pronounced, the higher the degree of sulfation of the compounds. The already mentioned with the sodium salt of monoamino-β- linked to the succinic acid monocholesteryl ester cyclodextrin sulfate (on average 14.5 sulfate groups) has e.g. B. the ability that Fusion of HIV fusion protein (gp160) -containing cells with uninfected cells, who have a receptor that carries the HIV gp160 at concentrations of less than 8 µg / ml completely in the cell culture assay.

The higher the number of anionic groups on the cyclodextrin, the more active they are Cyclodextrin derivatives in the syncytium formation assay. Preferably contain the  Cyclodextrin derivatives on average more than 10, particularly preferably more than 14 anionic Groups, especially sulfate groups.

Because both MAC and HI viruses share a common host for both possess, namely macrophages, the compositions according to the invention are in the At the same time, their antimycobacterial or antiviral effects are in one for both Components to unfold effective common place of action. So not only that Solved the problem of the solubility of iminophenazine derivatives, but at the same time an HIV-effective composition provided.

The invention is explained in more detail below with the aid of examples.

example 1 (a) Preparation of Mono-6 linked with succinic acid monocholesteryl ester deoxy-6-amino-β-cyclodextrin and subsequent sulfation

The preparation of mono-6-deoxy-6-amino-β-cyclodextrin was described in detail by Henke et al. (Anal. Chem. 68 (1996) 3158-3165).

The commercially available succinic acid monocholesteryl ester was, as for fatty acids from Bellanger and Perly (J. Mol. Struct. 273 (1992) 215-226) in ethyl acetate Room temperature with equimolar amounts of N-hydroxysuccinimide and Dicyclohexylcarbodiimid reacted overnight. The precipitated dicyclohexyurea was filtered off. The solvent was removed in vacuo and the product from ethanol recrystallized. The yield was quantitative.

Equimolar amounts of the activated succinic acid monocholesteryl ester and mono-6 Deoxy-6-amino-β-cyclodextrins were used in just an adequate amount Dimethylformamide dissolved at 60 ° C and reacted with stirring. The The progress of the reaction was determined by means of thin layer chromatography (silica gel; eluent: Ethyl acetate / isopropanol / water / concentrated ammonium hydroxide solution 7: 7: 10: 0.5; development  checked with ethanol / sulfuric acid spray). After full implementation, that was Product obtained by precipitation with ethanol / water, washed with hot ethanol and dried in a vacuum drying cabinet. The yield was quantitative.

The thus obtained succinic acid monocholesteryl-linked β-cyclodextrin was mixed with Trimethylamine sulfur trioxide at 60 ° C in dimethylformamide with stirring overnight sulfated. Here, per mole of the cyclodextrin derivative (corresponding to 20 equivalents OH groups) about 60 moles of the sulfation agent. The product came with a sufficient ethanol precipitated, taken up in water and again in ethanol precipitated. Then the product became 30% aqueous in just a sufficient amount Sodium acetate solution dissolved, the solution was mixed with activated carbon and about 5 min on the Water bath heated to 80 ° C and filtered through diatomaceous earth. This solution became that Product precipitated with ethanol, taking care that the solubility of the Sodium acetate in the ethanol / water mixture is not undercut. You get in quantitative yield the sodium salt of the succinic acid monocholesteryl ester linked β-Cyclodextrinsulfats. This was in aqueous medium before use in Example 1 (b) recorded, sterile filtered (200 nm sterile filter) and freeze-dried.

The degree of sulfation corresponds to approx. 14.5 ± 3 sulfate groups per molecule (using LSIMS certainly).

(b) Preparation of an aqueous formulation containing clofazimine with the in (a) obtained sulfated, cholesterol-linked β-cyclodextrin derivative

100 mg of the sulfated, cholesterol-linked β-cyclodextrin derivative were in 10 ml of a 5 mM ammonium acetate buffer (pH 6.5) dissolved. In this solution, 20 mg of crystalline Clofazimin stirred in. The suspension was then added under nitrogen Room temperature stirred. Aliquots were taken by the hour and by means of a Undissolved clofazimine is removed from the sterile filter (400 nm pore diameter). The The clofazimine content of the remaining aliquoted solution was in 2N hydrochloric acid determined spectrophotometrically at 530 nm. With a cyclodextrin-to-clofazimine Ratio of approx. 3: 1, the experiment was terminated and clofazimine was not dissolved separated by means of a frit. The solution was then analyzed using a 400 nm  Sterile filter sterile filtered. The analog and parallel ones described below Comparative experiments 1 to 3 were also stopped at this time.

The solution was frozen in liquid nitrogen and freeze-dried. It became a buffer-free deep red powder, which can be found in aqueous medium or water and which had a cyclodextrin to clofazimine ratio of about 3: 1 receive.

Comparative Examples 1 to 3

The procedure was as in Example 1 (b), but instead of the one used there Cyclodextrin derivatives, unsubstituted β-cyclodextrin (Comparative Example 1), sulfated β-cyclodextrin (Comparative Example 2) and sulfated, with palmitic acid linked mono-6-deoxy-6-amino-β-cyclodextrin (Comparative Example 3) used. None of the three comparison substances, not even the palmitoylated, i.e. also lipophilic sulfated cyclodextrin derivative, the solubility of clofazimine was noticeable compared to pure water solubility.

Example 2

The composition obtained in Example 1 (b) was evaluated for its antimycobacterial activity against MAC in a BACTEC experiment, based on Franzblau (Antimicrob. Agents Chemother. 33 (1989) 2115-2117). The anti-MAC Effect of the composition corresponds to its clofazimine content.

Example 3

The composition obtained in Example 1 (b) was anti-HIV effective tested in a syncytium formation assay. Syncytia are inoperable merged Cells that are formed when cells that have the HIV fusion protein gp160 on their Surface wear merge with uninfected receptor cells. Served as receptor cells in this experiment H9 cells. TF228.1.16 cells were used as gp160-carrying cells used. Then a syncytium formation assay analogous to Jonak et al. (Aids Res. Hum. Retrovir. 9 (1993) 23-32). However, as a medium  Commercial "Dulbecco's modified Eagles" (= DME) nutrient medium (containing 16% fetal bovine serum). Syncytia formation was studied after 18 hours. Already from a concentration of approx. 8 µg / ml (approx. 2.6 µM) based on the 14.5-fold sulfated, cholesterol-linked β-cyclodextrin derivative found none cell-killing gp160-induced syncytia formation takes place more. The invention The composition from Example 1 (b) was found to be highly anti-HIV active. in the considered concentration range, which is up to 50 times the necessary effective Concentration was sufficient, the composition according to the invention showed no measurable or visible cytotoxic influence on the cell cultures (Trypan Blue test and Alamar blue test).

Claims (15)

1. Aqueous or water-soluble pharmaceutical composition, which

• a) at least one iminophenazine derivative of the general formula (I):
  wherein
  R ¹ = an optionally halogenated phenyl radical,
  R ² = a branched or unbranched alkyl radical having 1 to 6 carbon atoms,
  and
  R ³ = an optionally halogenated phenyl radical,

and

• a) at least one water-soluble cyclodextrin, which is present as polysulfate or polyphosphate and is covalently linked to a sterol or sterol derivative

contains. 2. Composition according to claim 1, characterized in that the Iminophenazine derivative is clofazimine.   3. Composition according to one or more of claims 1 or 2, characterized characterized in that the water-soluble cyclodextrin is a β-cyclodextrin. 4. Composition according to one or more of claims 1 to 3, characterized characterized in that the cyclodextrin polysulfated as a physiologically acceptable salt is present. 5. Composition according to one or more of claims 1 to 4, characterized characterized in that the cyclodextrin is present as the sodium salt. 6. Composition according to one or more of claims 1 to 6, characterized characterized in that the sterol is a compound from the following group "cholesterol, Lanosterol, Ergosterol, Stigmasterol, β-sitosterol, cholic acid or deoxycholic acid ". 7. Composition according to one or more of claims 1 to 6, characterized characterized in that the sterol or sterol derivative is a cholesterol or a Succinic acid monocholesteryl ester. 8. The composition according to one or more of claims 1 to 7, characterized in that the sterol by means of a difunctional bridge molecule of the general formula
X- (C _m H _{m + 2} ) -Y
With
and with
m = 1 to 4 and Z = halogen, with which cyclodextrin is connected. 9. The composition according to claim 8, characterized in that the Bridge molecule is succinic acid or its N-hydroxysuccinimide. 10. The composition according to one or more of claims 1 to 9, characterized characterized in that the cyclodextrin is present as aminocyclodextrin and via its Amino group, optionally via a bridge molecule according to claim 8 or 9, with is linked to the sterol or sterol derivative. 11. The composition according to one or more of claims 1 to 10, characterized characterized in that the cyclodextrin to iminophenazine molar ratio is 20: 1 to 0.8: 1, is preferably 10: 1 to 1: 1. 12. A method for producing a composition according to any one of claims 1 to 11, wherein a suspension of the iminophenazine derivative (a) in an aqueous solution of the Cyclodextrin derivative (b), at a pH of preferably between 6 and 7, preferably under an inert gas atmosphere, stirred, shaken or otherwise is moved mechanically until the desired ratio of components (a) to (b) in the solution, and whereupon the solution is separated from undissolved constituents and freeze-dried if desired.   13. The method according to claim 12, wherein the pH between 6 and 7 by means of a Buffer, preferably a buffer that is volatile during freeze drying is checked. 14. Use of one or more compositions according to any one of claims 1 to 11 for antimycobacterial, immunosuppressive, anti-inflammatory and / or antiviral treatment. 15. Use according to claim 14, wherein in addition to the compositions Liposomes are used.