FI65912C - PROCEDURE FOR FRAMING PROCESSING OF PHARMACEUTICAL COMPOSITION INKLUSIONSKOMPLEXER AV PROSTACYKLIN OCH PROSTACYKLIN-C1-4-ESTRAR MED BETA-CYKLODEXTRIN - Google Patents

Description

6591 2

In the first stage of prostaglandin biosynthesis, starting from Araki-donic acid, endo-peroxides, denoted by PGG® and PG1, are formed in the enzymatic process. endoperoxide compounds are highly unstable and have a half-life of a few seconds. When platelets collide with the walls of blood vessels, these endoperoxides are released from the platelets, which are then converted to prostacyclin by an enzyme formed by the walls of the blood vessels. If a blood vessel is damaged and does not produce an enzyme that converts endoperoxides to prostacyclin, they are converted to thromboxane, which accelerates platelet aggregation and leads to thrombosis.

Thrombosis is the number one cause of death among young and middle-aged people worldwide. There is currently no truly effective method available for breaking up blood clots that have already formed and preventing the formation of a fatal blood clot. In the case of heart attacks, infarctions and cardiac thromboses, an effective anticoagulant could prevent the further development of the blood clots that have already formed and break them down, which could save lives. Such drugs could also be used, for example, to prevent pre- and post-operative thrombosis.

Prostacycline (PGI ~) is chemically (5Z) -9-deoxy-6,9 5β-epoxy-Δ-prostaglandin. This compound has been found to be very effective in preventing platelet aggregation and removing already formed blood clots. However, its use has so far seemed impossible due to its very short biological half-life (about two minutes). In aqueous solution, the compound decomposes mainly in about four minutes. The highly labile nature of this compound is due to its chemical structure. It would be highly advantageous to provide a stabilized and thus a prostacyclin or prostacyclin derivative suitable for therapeutic use (Science, December 20, 1976, page 17).

Cyclodextrins are cyclic molecules that contain 6, 7 or 8 glucopyranose units to form rh-1,4-glucoside units. Structurally, they are characterized by a special arrangement of hydroxyl groups. All secondary hydroxyls are located on one side of the ring, while all primary hydroxyls are located on the other side of this ring. Thus, the outer surface of the ring is substantially hydrophilic, resulting in the water solubility of cyclodextrins. On the other hand, the inner surface of the rings is hydrophobic in nature because only hydrogen atoms and glucosidic oxygen bridges are detectable in this part of the molecule. Thus, if molecules with a lower polarity than water and whose shape and size allow them to penetrate the cavity inside the cyclodextrin are added to the aqueous solution of cyclodextrins, cyclodextrin barrier complexes are already formed in the aqueous solution. This method has proven useful for stabilizing several compounds. This method is also useful for lowering the stress of several compounds and protecting various materials from the oxidizing effect of air. A ring containing 6 glucopyranose units is called -S-cyclodextrin, a ring containing 7 units is called -cyclodextrin, and a ring containing 8 glucopyranose units is called T-cyclodextrin.

Prostacycline and its derivatives can be prepared by the method described in Tetr. Let. 30 (1977) 26-27 and J. Am. Chem. Soc. 99 (1977) 2006.

The inclusion complexes of prostacyclin and prostacyclin C 1-4 alkyl esters with β-cyclodextrin prepared by the process of this invention are considerably more stable and have a much longer duration of action than prostacyclin and its derivatives and can be used as active ingredients in pharmaceutical compositions. These compositions can be administered by intravenous injection or sublingual tablets, etc. for the treatment and prevention of the aforementioned thrombosis. Because these complexes act very quickly, their use can save lives in many cases.

The process according to the invention for the preparation of inclusion complexes is characterized in that an organic solution of prostacyclin or prostacyclin C 1-4 alkyl ester is added to an aqueous solution of β-cyclodextrin, optionally also containing organic or inorganic salts; the organic solvent is removed from the resulting mixture; and the closure complex of prostacyclin or prostacyclin-C 1-4 alkyl ester with β-cyclodextrin is isolated from the remaining aqueous solution with lyophil.

4 6591 2

The inclusion complexes prepared according to the invention can be converted into conventional pharmaceutical compositions in ways known per se. These compositions consist of inclusion complexes as well as conventional excipients, diluents, ionic strength regulators and / or other additives and may be administered as injections and infusion solutions, tablets for oral or sublingual administration, capsules, beads or powder vials.

Prostacycline or a derivative thereof can be dissolved in various water-miscible, water-immiscible or other solvents which have limited miscibility with water, preferably ether is used as the solvent. Aqueous solutions of β-cyclodextrin preferably also contain various inorganic or organic salts or buffers which stabilize the solutions and which affect the pH and ionic strength of the solution.

Platelet aggregation inhibitor

The antiplatelet assay was performed on a Born aggregometer with human blood. Accumulation was induced using 20 mmol of ADP (adenosine triphosphate) or 1.2 mmol of arachidonic acid. The β-cyclodextrin-prostacyclin complex was added to the platelet suspension before the addition of the active ingredient. The experiments were performed at 20 ° C.

Arachidonic acid-induced aggregation

Fresh aqueous solution of prostacyclin / 5-cyclodextrin occlusion complex containing prostacyclin 40 μg / mlf showed no inhibitory effect on platelet aggregation. If the same solution was administered after standing for 30 minutes at room temperature, a 20% inhibition was observed.

With a solution containing the complex in an amount corresponding to a prostacyclin concentration of 5 pg / ml, complete inhibition of platelet aggregation was observed after 30, 60 and 150 minutes of preparation of the solution.

If the aqueous solution of prostacyclin-β-cyclodextrin occlusion complex was allowed to stand at room temperature for two hours, its inhibition was complete (100%) even at concentrations of 1 μg / ml.

6591 2 ADP-induced aggregation

The prostacyclin / '-cyclodextrin occlusion complex was dissolved in water and the resulting solution was allowed to stand at room temperature for four hours. An inhibition of ADP-induced accumulation was then prescribed. The following results were obtained:

Prostacyclin concentration Inhibition 10 ng / ml 50% 100 ng / ml 100% 1 μg / ml 100% 10 pg / ml 100% Using solutions containing 20-400 μg / ml Prostacycline, it was possible to achieve the dispersion of blood clots formed ADP or ristocetin. The same results were obtained if the solutions were allowed to stand at room temperature for 120 minutes before dosing.

Given that prostacyclin alone disintegrates in aqueous solutions within four minutes, the above results indicate that the stability of prostacyclin is significantly improved in the preparation of its cyclodextrin complex. The observation that fresh solutions do not have an anti-aggregation effect is due to the fact that dissociation of inclusion complexes takes some time. For pharmacological reasons, this property is very advantageous because the concentration of PGI2 at the time of dosing is not detrimentally high and the rapid dissociation of the occlusion complex later ensures that the concentration of PGI2 remains at a biologically active level for a long time.

If only PGI2 is added to human blood, the disintegrating effect of the accumulations can be observed in about two minutes.

During this very short period, PGI2 is completely dispersed. Contrary to this finding, the duration of action of the prostacyclin occlusion complex is 10 minutes, i. its duration of action is five times that of PGI2.

The Mesenteris artheris preparation was treated with an aqueous solution of the PGI2-v3-cyclodextrin occlusion complex. A transient, prostacyclin-specific trigger effect was observed. A contraction was observed with the rat uterus preparation, which corresponded to 0.01-0.001 of that caused by PGR2j, if the preparation was treated with «6 6591 2 occlusion complex. Contractions in the arteries of the rabbit caused by electric shock could be prevented by a closure complex with an active ingredient concentration of 10-20 μg / ml. PGI2 ethyl ester showed the same potency at concentrations of 0.2 to 0.4 ug / ml.

The effect of salivary amylase on cyclodextrins and their complexes has been studied. Determining the mode of action of this reaction is necessary to determine whether the rate of PGI2 release is controlled by complex dissociation or enzymatic hydrolysis of cyclodextrin. According to our experiments, in starch phosphate pH 6, the decomposition of starch soluble at 35 ° C into reducing sugar is complete (100%). On the other hand, a measurable increase in the reduction capacity of β-cyclodextrin only occurs after five hours. Thus, it can be concluded that β-cyclodextrin is virtually able to completely resist the action of saliva. It follows from this finding that prostacyclin can be released from a sublingual tablet only by dissociation of the occlusion complex.

The complex prepared according to Example 2 was kept in a sealed ampoule for five days at room temperature. The vial was opened, then its contents were poured into a vial and 500 times the amount of ether was added. The flask was sealed and the mixture was stirred at room temperature for 30 hours. Samples from the reaction mixture were periodically subjected to thin layer chromatography (silica gel F254 'manufactured by Merck, using a 1: 2 mixture of ethyl acetate and acetone as solvent and chromium sulfuric acid as developer). Even after 30 hours, only a spot corresponding to prostacyclin methyl ester could be detected (Rf = 0.50). No signs of dispersion were observed, i. the complex was stable in aprotic solution at neutral pH.

Examining the stability of prostacyclin ethyl ester-β-cyclodextrin prepared essentially according to the procedure described in Example 2, the following results were obtained:

The decomposition half-life of the occlusion complex was 87 minutes at a concentration of 10 μg / ml in aqueous solution (pH = 6.8-7).

Under the same conditions, the half-life of prostacyclin ethyl ester itself is 11 minutes.

7 65912

When administered orally, cyclodextrins are non-toxic. Female GFY rats with an average weight of 250 g were administered 500 mg of β-cyclodextrin by gavage as a suspension in 0.01% methylcellulose solution. Experimental animals were observed for 34 days. It was found that the experimental animals developed in the same way as the control animals, no signs of intoxication were observed.

LD, .q (i.v.) for cK cyclodextrin = 1 g / kg body weight; (I.v.) / '? for cyclodextrin = 0.788 g / kg body weight in Sprague-Dawlwy rats (Amer. J. Pathol. 83 (1976) 367).

From the results of the above experiments, it can be seen that the effective prostacyclin concentration of prostacyclin-cyclodextrin occlusion complexes is in the range of 0.1 to 1 μg / ml. Calculated per 5 liters of blood, 5 mg of PGI2 is required, which means 2-100 mg of PGI2 occlusion complex with a PGI2 content of 5%. Concentrations of the active ingredient in intravenous injections and sublingual tablets should be in this range.

The invention is further illustrated by the following examples.

Example 1

To one milliliter of a phosphate buffer solution having a pH of 8 was added 6 milliliters of distilled water. The solution was warmed to 30 ° C and 190 mg (water content 14%) was added. cyclodextrin. After dissolution was complete, 8 mg of PGI2 in 32 ml of ether was added to the solution over 1 hour with stirring. The ether vapor was removed by stirring under slightly reduced pressure. After complete removal of the ether vapor, the solution was allowed to cool to room temperature with constant stirring over about an hour. The solution was cooled and lyophilized in the usual manner. 182 mg of dry prostacyclin /? cyclodextrin complex as a white powder which was readily soluble in water.

Example 2 10 mg of prostacyclin methyl ester was dissolved in 2 ml of diethyl ether. Separately dissolved 200 mg of anhydrous / '? cyclodextrin to a mixture of 1 ml of a pH 8 buffer solution and 5.5 ml of distilled water at 30 ° C, and the above-mentioned prostacyclin methyl ester solution was added to the resulting solution. The mixture was stirred for one hour under slightly reduced pressure and then shaken on a shaker for a further two hours at room temperature. The solution was cooled and lyophilized. 198 mg of prostacyclin-methyl ester-N, N-cyclodextrin complex were obtained as an amorphous, water-soluble powder which decomposed on heating without melting.

Example 3

Capsule containing 2.5 mg of active ingredient per capsule.

Composition: PG ^ -, ^ -cyclodextrin complex (containing 5% of active ingredient) 50 mg

Colloidal silicic acid 9 mg

Talc 5 mg

Magnesium stearate 15 mg

Lactose 20 mg

40 mg of crystalline cellulose

Potato starch 86 mg

Example 4 Preparation of capsules

The powder mixture is prepared by dry granulating a mixture containing the ingredients shown in Example 3 in a given ratio. The powder mixture is homogenized and encapsulated in a standard apparatus to give 225 mg capsules.

Example 5

Tablet containing 2.5 mg of active ingredient per capsule.

Composition: PGJ ^ - · '' - cyclodextrin complex (containing 5% of active ingredient) 50 mg

Amylopectin 10 mg

Crystalline cellulose 60 mg

Stearic acid 2 mg

Talc 13 mg

280 mg of potato starch

Example 6 Preparation of tablets

Prostasykliini- /? cyclodextrin-barrier complex, amylopectin and microcrystalline cellulose together with an equivalent amount of potato starch are thoroughly mixed in a homogenisation medium.

Claims (4)

9,65912 in the ratios shown in Example 5. The required amount of stearic acid and talc is homogenized and sieved through a No. 100 sieve. The obtained fine powder is mixed with the above-mentioned homogenized powder mixture, and the resulting mixture is made into tablets each weighing 415 mg. If desired, the resulting tablets are converted into granules or coated tablets. Example 7 Injection (lyophilized) Ampoules are filled with an aqueous solution of the PGβ-cyclodextrin complex prepared according to Example 2 so that each ampoule contains 5 mg of complex with an active ingredient content of 5%. The ampoules are then lyophilized and sealed under nitrogen. Prior to use, the lyophilized powder is dissolved in physiological sodium chloride solution to the desired volume. A process for the preparation of barrier complexes of pharmaceutically acceptable prostacyclin and prostacyclin C 1-4 alkyl esters with β-cyclodextrin, characterized in that an organic solution of prostacyclin or prostacyclin C 1-4 alkyl ester is optionally added to the β-cyclodextrin organic solution. inorganic salts or buffers; the organic solvent is removed from the resulting mixture; and the closure complex of prostacyclin or Prosta-cyclin C 1-6 alkyl ester with β-cyclodextrin is isolated from the remaining aqueous solution by lyophilization. Process according to Claim 1, characterized in that the organic solution of prostacyclin methyl ester is reacted with an aqueous solution of β-cyclodextrin, which optionally also contains inorganic or organic salts or buffers. Process according to Claim 1 or 2, characterized in that the organic solvent is an ether. Process according to one of Claims 1 to 3, characterized in that an aqueous solution of β-cyclodextrin, which also contains a phosphate buffer, is used.