DE10135694A1 - New amphiphilic conjugate of starch or hydroxyethylstarch, useful as drug carrier, contain e.g. fatty acyl residues, are not taken up by the reticuloendothelial system - Google Patents

Abstract

Amphiphilic (hydroxyethyl)starch conjugates (I) are new. Amphiphilic (hydroxyethyl)starch conjugates of formula (I) are new. B = (hydroxyethyl)starch, or its individual components, amylopectin or amylose, in soluble or hydroxyethylated form, attached through alpha -glycosidic bond; R2 = hydrogen , 2-hydroxyethyl or B; R<3> and R<4> = hydrogen or 2-hydroxyethyl; R' = -C(A)-R''; R'' = -NH((CH2)x-NH)y-R'''; x = 0, 1 or 2; y = 0 or 1; A = oxygen or two hydrogens; R''' = -C(O)O-cholesteryl, fatty acid acyl or, where y = 0, phosphatidylethanolaminyl.

Description

The present invention relates to amphiphilic conjugates of starch or hydroxyethyl starch. Derivatives and their use for the production of parenterally applicable colloidal Dosage forms, such as liposomes, fat emulsions or mixed micelles.

Liposomes are made of phospholipids and various other amphiphilic excipients such as z. B. Cholesterol-built vesicles, which are replaced by one (unilamellar) or more (multilamellar) double-layer membranes exist.

In recent years, liposomes have been developed as drug delivery systems, whereby the drugs either because of their lipophilic properties in the membranes themselves be installed, or in the case of hydrophilic substances in the aqueous compartment the vesicles are loosened.

However, such liposomal pharmaceutical preparations often have the disadvantage of being very short Half-life in the blood, as they enter the reticuloendothelial relatively quickly as colloidal systems System (RES).

For the targeted targeting of drug systems with a controlled release profile such liposomes are therefore often unsuitable.

It has therefore not been lacking in the past to develop liposomes that have these disadvantages did not have and which were aimed at a target organ by targeted modification (Targeting).

One of the best known modifications in this regard is the development of the so-called Stealth liposomes. Such camouflage caps are made using liposomes Modified polyethylene glycol modified cholesterol or phosphatidylethanolamine (PE) (so-called pegylated lipids). But it is also possible to use conventional liposomes according to their Manufacture to chemically conjugate with polyethylene glycol derivatives and in this way too Arrange camouflage caps liposomes.

With these camouflage caps liposomes, the outward-facing polyethylene glycol Chain the liposomes against interaction with plasma proteins or corpuscular Components of blood. Such camouflage caps become less liposomes from the RES system recognized more effectively than conventional liposomes and therefore less in scope strongly absorbed.

However, camouflage caps liposomes based on pegylated lipids have disadvantages that so far have not been eliminated. It is known that polyethylene glycol is not metabolizable and can only be excreted via the kidney. With regard to their molecular weight or hydrodynamic volume in solution, these polymers must be so-called Kidney threshold remain. This is around 70,000 D for polyethylene glycols. Restricted patients are also polyethylene glycol fractions below the Kidney threshold problematic, since these patients have an insufficient Eliminate such substances. Furthermore, such will not excreted polyethylene glycol fractions stored in the tissue and can Side effects such as B. cause itching.

There are also occasional immunological interferences derivatized with polyethylene glycol Liposomes or proteins have become known.

Bendele et. al describe in Toxicological Sciences 42, 152-157 (1998) vacuolizations of the Kidney tubules in rats after injection of pegylated protein drugs, if that Molecular weight of the PEG was less than 70,000 D.

Another disadvantage of polyethylene glycol liposomes is that the relative proportion of the pegylated phospholipids on the total lipids must be relatively high in order to be sufficient To achieve coverage of the entire vesicle surface with polyethylene glycol groups. It are therefore more recent developments with branched-chain polyethylene glycol systems have been described in which the proportion of pegylated molecules or the pegylation itself could be reduced because such branched chain polyethylene glycols a relative can shield larger areas and thus their molar use can be reduced in Trap of liposomes. The disadvantage of such branched chain liposomes is that their Metabolism or pharmacokinetics as well as their toxicological profile is so far unknown.

O / w emulsions are also known parenteral dosage forms, which are used as Drug carriers for lipophilic active substances are now established in the market. It is known for. B. a parenteral fat emulsion as a carrier for propofol, an injectable anesthetic (Disoprivan®, Astra Zeneka). Another well-known example is the product Etomidat Lipuro® B. Braun, which is a carrier emulsion for the anesthetic etomidate. Numerous parenteral o / w emulsions are according to the scientific literature in the Development.

Parenteral o / w emulsions also tend to accumulate in the RES system due to their Larger particle size compared to liposomes even much more pronounced than this. It has already been described in the literature that liposomes or parenteral emulsions in their pharmacokinetic properties and tissue distribution after injection by wrapping with amylopectin derivatized with palmitoyl or cholesteryl groups was influenced. The fatty acid residues or cholesterol residues served as "anchors" for the Polysaccharide amylopectin in the outer, lipophilic phosphatide layer of the liposomes or Fat emulsions (Sunamoto, J .; Iwamoto, K., CRC Critical Review in Therapeutic Drug Carrier Systems 1986, 2, 117-136).

The same is also reported for parenteral emulsions for cholesteryl pullulan, Cholesteriyl-Mannan and Cholesterol Amylopectin (Iwamoto, K. et. All Journal of Pharmaceutical Sciences 1991, 80, No. 3, 219 ff).

However, the polysaccharide derivatives used in the work cited above are considerable Disadvantages associated with the use described.

So no clear molecular structure is realized in that synthesis-related lipophilic "anchors are statistically distributed over the polysaccharide chains.

Therefore z. B. for cholesterol-derivatized amylopectin an average degree of substitution of 1.8 cholesterol groups per 100 anhydroglucose units in the polysaccharide chain.

However, such multiple anchor groups per polysaccharide molecule can also be negative affect the stability of liposomes or fat emulsions, e.g. B. not necessarily each cholesterol group of an amylopectin molecule is bound to a specific fat droplet must be, but such cholesterol groups also exist within a macromolecule, that are free or unstable and thus interact with a second Fat droplets can occur. Such "bridge connections" lead to aggression and Instability of liposomes and fat emulsions with those described above Polysaccharide derivatives.

Furthermore, the pullulans described above are not physiological or sufficiently rapid degradable by the serum amylase due to the α-1,6 glycosidic linkage of the Maltotriose repeating units. The same applies to the Mannan polysaccharide cited above.

Amylopectin on the other hand is degraded extremely quickly by serum amylase, so that the stealth effect is lost very quickly, a fact that in many cases is not is acceptable. Dextrans, which are considered as polysaccharides also as conjugation partners come, have the disadvantage that they are known to run the risk of anaphylactic Initiate reaction after injection in patient. This has resulted in it nowadays The prior art is a low molecular weight one before application of a dextran plasma expander Pre-inject variant of dextrans with an average molecular weight Mw of 1,000 D.

Since dextrans can only be broken down by the tissue-resistant dextranase, theirs is Pharmacokinetics are also relatively slow and also more difficult to control than is the case with Hydroxyethyl starch, also a plasma expander, which is now due to considerable Benefits in terms of side effects are widely used worldwide.

The present invention is therefore based on the object based on polysaccharide based compounds for use in the production of parenteral fat emulsions or To provide liposomes which do not have the disadvantages mentioned above and enable a targeted change in pharmacokinetics and tissue targeting. It has now surprisingly been found that starch or hydroxyethyl starch, which via the reducing end group which occurs only once in the molecules, if appropriate after chemical modification, contained selectively bound lipophilic anchor groups, as Camouflage cap polysaccharides advantageous for liposomes and parenteral o / w emulsions could be used.

By varying the molecular weights of the degree of substitution of the hydroxyethyl groups as well of the substitution pattern of the hydroxyethyl groups on the various carbons of the Anhydroglucose basic units can change the properties in a manner adapted to requirements become.

By varying the molecular weight of the HES component used, the So-called HLB value (hydrophilic lipophilic balance) can be set specifically. The Starch and HES derivatives according to the invention can thus also be used to generate w / o or multiple emulsions w / o / w can be used advantageously. The same applies to the Production of so-called mixed micelles.

The compounds according to the invention are preferably used in a manner known per se produced by the (only) reducing end group of the starch or hydroxyethyl starch is specifically oxidized to the lactone group.

The lipophilic group is then coupled to the oxidized polysaccharide in one possible variant directly via an NH ₂ group present in the lipophilic molecule via an amide bond. This coupling can be carried out either in the anhydrous system with DMSO or in dimethylformamide / DMSO mixtures. However, it can also be carried out in aqueous solution in a second variant in a manner known per se by means of a water-soluble carbodiimide, provided that the lipophilic component has a sufficiently high water solubility or critical micelle concentration.

Furthermore, lipophilic compounds with free NH ₂ groups can also be prepared in a manner known per se via shipbase formation with simultaneous reduction (so-called reductive armination) or also reduction in a second step with borohydrides.

If necessary, so-called spacers can also be introduced, such as. B. diamines of the structure NH ₂ - (CH ₂ ) _n -NH ₂ , which are reacted in a further step with a lipophilic component. For example, the cholesterol residue can be introduced by reacting the amino-functionalized starch or hydroxyethyl starch with cholesterol chloroformate in dry DMF / DMSO mixtures analogous to Iwamoto et al.

The introduction of a fatty acid acyl group on the amino-functionalized polysaccharides can be carried out by carbodiimide-catalyzed reaction with the corresponding Fatty acid.

In the following examples, the manufacturing processes of the invention Compounds described as well as the preparation of a polysaccharide coated Fat emulsion.

example 1 Production of hydroxyethyl starch oxidized to lactone at the reducing end group (HES lactone)

To 30 ml of a 30% HES solution with a molecular weight Mw of 45,000 D. added two ml of a 0.1 N iodine solution and about 3 ml of a 0.1 NaOH solution. The mixture is stirred until the color of the iodine has disappeared. The process is repeated repeated until a total of about 30 ml of iodine solution and 20 ml of the sodium hydroxide solution are used are.

The solution obtained is then passed through a sodium ion exchanger (Amberlite IR 120) given and then in a dialysis tube with an exclusion limit of approx. 3,500 daltons dialysed against distilled water over 24 hours. The product comes with Ethanol precipitated, filtered off, freed from the majority of residual ethanol under vacuum and then dried at approx. 80 ° C in a warming cabinet to constant weight.

Determination of the degree of oxidation

The content of the reducing end groups was used to check the successful oxidation according to Somogyi (Meth. Carbohydrate Chem., 1, 384-386, 1962).

No reducing end groups could be detected after the oxidation.

Example 2 Conversion of HES lactone according to Example 1 with phosphatidylethanolamine to HES PE

1.5 g of dried HES lactone according to Example 1 are placed in 10 ml in a reaction vessel dry DMSO with 5 mg of dried egg phosphatidylethanolamine at 80 ° C for approx. 24 hours long under argon as protective gas. The pH may be adjusted with pyridine or triethylamine set to about 6.

Then the DMSO is partially stripped off under vacuum and the product by ethanol precipitated and extracted with chloroform.

After resumption in DMSO, dialysis is carried out with a membrane with an exclusion limit of 3,500 Daltons against distilled water under argon for 50 hours at 4 ° C.

The freeze-dried product was almost colorless to very pale yellow. The yield based on egg PE was over 90%. The 1H NMR analysis showed the characteristic signals of the CH2 and CH protons of the fatty acid chains.

Example 3 Production of amino-functionalized HES

40 ml of a 25% aqueous solution of oxidized HES according to Example 1 becomes 0.75 g Added ethylenediamine dihydrochloride and 0.20 g 1 ethyl 3 (3-dimethylamino) propyl carbodiimide hydrochloride.

After adjusting the pH to about 4.8, the reaction solution reacts overnight. The The reaction solution was then dialyzed with a membrane with an exclusion limit of approx. 3,500 Daltons cleaned against water and the product isolated by freeze drying and dried.

Example 4 Manufacture of cholesteryl HES

1 g of the product from Example 3, dissolved in 50 ml of dry DMSO, was mixed with Cholesteryl chloroformate (0.23 g in 4 ml dry DMF) reacted at 80 ° C for 24 hours under argon. The DMSO was removed under reduced pressure and the product was precipitated with acetone. The Product taken up again in DMSO was by diafiltration against distilled water cleaned with a membrane with an exclusion limit of 3,500 daltons against distilled water. The product isolated by lyophilization showed the corresponding in the 1H NMR spectrum Signals for cholesterol.

Example 5 Production of an HES-coated parenteral fat emulsion

5 ml of a 10% parenteral fat emulsion (Lipovenös 10%) are added to 1 ml of a aqueous solution of cholesteryl HES prepared according to Example 4, the 12 mg of the substance contained, added. The mixture was sonicated for 2 minutes with 25 watts in an ice bath. The mean diameter, determined by dynamic light scattering, grew from 279 nm 285 nm and was determined after 28 months storage at 25 ° C with 284 nm.

The zeta potential dropped from -55 mvolt to -50 mvolt after treatment.

The HES-coated emulsion was compared to a salt and calcium chloride containing Electrolytic solution stable.

Claims (6)

1. Amphiphilic starch and hydroxyethyl starch conjugates of the general formula


in which mean
B = hydroxyethyl starch or starch, also its individual components amylopectin and amylose in soluble or hydroxyethylated form, in an α-glycosidic bond
R ₂ = H, - (CH ₂ ) ₂ -OH, B
R ₃ , R ₄ independently of one another H, or - (CH ₂ ) ₂ -OH


in which
R "= -NH [(CH ₂ ) _x - NH] _y R""where
x = 0 or an integer ≥ 2 means y = 0 or 1 and
A = O (oxygen) or 2 hydrogen atoms (2H)
Farther


or fatty acid acyl
or phosphatidylethanolaminyl (for y = 0) 2. Amphiphilic hydroxyethyl starch derivatives of the general formula 1. 3. Amphiphilic hydroxyethyl starch derivatives of the formula 1, the average molecular weight the proportion of hydroxyethyl starch is between 2,000 and 200,000 daltons, the degree of substitution MS of the hydroxyethyl groups is between 0.15 and 0.8 and the ratio of substitution at C2 and C6 of the anhydroglucose units between 3 and 10 lies. 4. Amphiphilic starch and hydroxyethyl starch derivatives according to claim 1 for the production of parenterally applicable liposomes, fat emulsions and mixed micelles as Excipient. 5. hydroxyethyl starch derivatives according to claim 2 for the production of parenteral liposomes, Fat emulsions and mixed micelles as drug carriers. 6. Hydroxyethyl starch derivatives according to claim 3 for the production of parenterally applied Liposomes, fat emulsions and mixed micelles as drug carriers.