DE19637890A1 - Bisphosphonate liposomes - Google Patents

Abstract

A liposomal medicament (A) comprises a biphosphonic acid salt (I) as active agent, contained in a liposomal shell consisting of cholesterol (Chol), 1,2-dialkoylglycero-3-phosphocholine and 1,2-dialkoylglycerophosphoglycerol in a ratio of 1-2:2-6:1-3. The alkoyl groups contain 14-22C.

Description

The invention relates to a drug with a bisphosphone acid salt as active ingredient, packed in liposomes.

Clodon acid is dichloromethylenediphosphonic acid and is in Form of their physiologically acceptable salts as Calciumregu lator known in metabolism. Surprisingly, now More found that clodronate packed in liposomes the Ab Repulsion of transplanted kidneys in animal experiments (rats) prevented. In addition to clodronic acid have due to first Untersu also other active pharmaceutical ingredients from Biphos phonsäurederivatgruppe appropriate usability.

The invention therefore relates to a liposomal drug tel, which is characterized in that it as active ingredient a Biphosphonsäuresalz inside a liposome shell ent holds.

Clodon acid is preferred as the active ingredient. Other suitable Biphosphonsäurederivate that in the invention etidronate (1-hydroxyethylidene-bi phosphonic acid), aleudronate (4-amino-1-hydroxy-butylidene-bi phosphonic acid), pamidronate (3-amino-1-hydroxy-propylidene-bi phosphonic acid), BM 21.0955 (1-hydroxy-3 (methylpentylamino) propylidene-bisphosphonic acid).

According to the invention, preference is given to such a medicament, its liposome shell of cholesterol, 1,2-dialkoylglycero-3 phosphocholine and 1,2-dialkoylglycerophosphoglycerol, wherein the alkoyl groups are the same or different and 14 to 22 C atoms, in the molar ratio of 1: 1 to 2.5: 0.5 to 1 consists. Particularly preferred is as Phosphocholinbestandteil the 1,2-dipalmitoylglycero-3-phosphocholine and as phospho Glycerinbestandteil the 1,2-Dipalmitoylglycerophosphoglycerin.  

Another object of the invention is a method for Preparation of a drug to prevent rejection of implants, which is characterized in that a physiologically acceptable biphosphonic acid salt in one Packed liposome shell. As Biphosphonsäuresalz is how already mentioned above, Clodronsäuresalz preferred. Likewise suitable are the other mentioned Biphosphonsäuresalze and structurally analogous connections.

The preparation is carried out so that the liposome shell a Diameter of less than 200 nm. Preference is given here worked under such conditions that one with liposomes a mean diameter below 100 nm. The liposome is preferably cholesterol, 1,2-dialkoylglycero-3-phospho choline and 1,2-dialkoylglycerophosphoglycerol in a molar ratio of preferably 1: 1 to 2.5: 0.5 to 1. The alkoyl groups in the constituents mentioned can may be the same or different and preferably contains 14 to 22 C atoms.

The preparation of the medicament according to the invention can according to known to those skilled methods from a physiolo gisch compatible biphosphonate and the components of Liposome shell done. Appropriately, the lipids, from which the liposomes are composed in a suitable dissolved organic solvent and the solution of the Lipidgemi dried and then in an aqueous Clodronate solution dispersed and finally by high pressure Homogenization converted into liposomes. The not-included active substance is removed by a suitable method (eg Cross-flow filtration) separated.

Another object of the invention is a method for Prevention of rejection of transplants, which thereby characterized in that at least one biphosphonic acid salt administered in liposomes. The administration takes place by injection, preferably intravenously.  

The experiments on the effect of intravenously administered Clodronate on the rat allograft kidney have one almost complete prevention of rejection in renal vessels and an improvement in tissue rejection. It is Thus, clodronate can generally be expected in allogeneic trans implanted organs (eg heart, liver, small intestine) a vasku can inhibit rejection. Clodronate could be called "rescue in therapy with conventional immunosuppressive drugs Baren vessel rejection processes in humans who used the. The prevention of repulsion is after the received histological picture on a vascular elimination of Monozy returned.

The following example explains the production of the inventions Clodronate liposomes according to the invention.

example Preparation of clodronate liposomes material

1,2-dipalmitoyl-sn-glycero-3-phosphocholine, synthetic (DPPC)
1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, synthetic (DPPA)
Cholesterol (Chol)
Clodronic acid di-sodium 4H2O po (clo)
Sucrose.

Preparation of the 200 mM clodronate solution

14.44 g clodronate are dissolved in just under 200 ml of water, with Sodium carbonate (about 4 g) adjusted to pH and up the exact final volume of 200 ml was added to form a 200 mM clodronate solution with a pH of 7.2.

Preparation of 200 ml liposomes

The weight is chosen so that the lipid concentration bezo to the volume of the sucrose solution is 100 mM. The  Molar composition of the lipid components Chol / DPPC / DPPG is 3: 5: 2.

1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) | 7.34g 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPA) 2.98 g Cholesterol (Chol) 2.32 g.

To produce the liposomes, the lipids in a Ge mixed with chloroform / methanol under rotation with a water Bath temperature of + 35 ° C to a maximum of + 40 ° C dissolved. The lipid concentration is 200mM. Subsequently, the solvent completely removed under vacuum (T 40 ° C, 240 RPM) and at Room temperature under vacuum for 2 hours (P 15 mbar).

The lipids are dissolved in the clodronate solution (200 mM, pH 7.2). predispersed. The lipid concentration is 100 mM. The lipid approach is under rotation for 1 hour at a Wasserbadtempe temperature of + 50 ° C tempered. The resulting suspension looks milky and should contain no lumps. Without rotation A sediment forms, which shake up before refilling is.

Subsequently, a high-pressure homogenization is carried out. The pre-annealed lipid suspension becomes + 30 ° C to + 40 ° C cooled, filled and the homogenization ge in circulation starts. During homogenization, the lipid approach touched. 10 passes at 900 to 1000 bar are sufficient. After homogenization, the lipid approach is less milky and has a slightly bluish tinge.

For the separation of possibly occurring abrasion (due to high-pressure homogenization), a refrigerated centrifuge is suitable with corresponding rotors. The centrifugation is at 5 ° C  and takes 30 minutes. Subsequently, the Supernatant decanted through a pleated filter.

The separation of the clodro not enclosed in liposomes nates takes place at room temperature by diafiltration (Cross-flow filtration). The Liposomendispersion is with it the 15-fold volume of a 5% sucrose solution gewa rule.

Remarkably, the liposomes according to the invention are without Quality loss freezable.

The liposome suspension thus prepared has the following characteristics:
pH value: 7.3 ± 0.5
av. Diameter: <100 nm (num. Weighting)
Liposomal agent: 11 μmol / ml
Phospholipid: 42.4 μmol / ml (DPPC + DPPA)
Phospholipid degradation: <3% (mol)
Total lipid: 61.2 μmol / ml (DPPC + DPPA + Chol)
Lipid ratio: DPPC / DPPA / Chol (4.9 / 2.0 / 3.1, mol / mol / mol)
Appearance: opalescent dispersion
Sterile filtration: possible
Transport: as frozen goods (-80 ° C) on dry ice
Storage: at -80 ° C
Shelf life: as frozen goods (-80 ° C) at least 12 months after production, after thawing for a maximum of 2 days at + 4 ° C to + 10 ° C.

Claims (8)

1. Liposomal medicament, characterized in that it contains as active ingredient a biphosphonic acid salt in a liposome shell of cholesterol, 1,2-dialkoylglycero-3-phosphocholine and 1,2-dialkoylglycerophosphoglycerol in a ratio of 1 to 2: 2 to 6: 1 to 3, wherein the alkoyl groups are the same or different and have 14 to 22 carbon atoms. 2. Medicament according to claim 1, characterized, that the liposome shell of cholesterol, 1,2-Dialkoylgly cero-3-phosphocholine and 1,2-dialkoylglycerophosphoglyce in which the alkoyl groups are identical or different and Containing 14 to 22 carbon atoms, in a quantitative ratio 1 to 2: 2 to 6: 1 to 3. 3. Medicament according to claim 2, characterized, that it contains 1,2-dipalmitoylglycero-3-phosphocholine and 1,2- Dipalmitoylglycerophosphoglycerin contains. 4. Medicament according to one of claims 1 to 3, characterized, that it contains clodronate as biphosphonic acid salt. 5. Medicament according to one of claims 1 to 3, characterized, that it is at least one active substance as biphosphonic acid salt from the group 1-hydroxyethylene-bisphosphonic acid, 4-amino 1-hydroxy-butylidene-bisphosphonic acid, 3-amino-1-hydroxy propylidene bisphosphonic acid and 1-hydroxy-3 (methylpentyl amino) -propylidene-bisphosphonic acid).   6. A process for the preparation of a medicament for verhin the rejection of implants, characterized, that is a physiologically acceptable biphosphonic acid salt packed in a liposome casing. 7. The method according to claim 6, characterized, that one works under such conditions, which one average diameter of the liposomes below 100 nm. 8. A method of preventing graft rejection characterized, that is a medicament according to one of claims 1 to 5 administered.