DE4136322A1 - New pectin deriv. bile acid adsorption agents - useful for treating hyperlipidaemia and can be administered at lower doses than colestyramine or colestipol resins - Google Patents

Abstract

Pectin derivs. of formula (I) are new. In formula, R = OH, 6-30C opt. unsatd. natural or synthetic fatty acid bonded via carboxy, 6-30C alcohol function bonded via the alcohol gp., a natural or synthetic aminoacid bonded via carboxy, a bile acid bonded through a 2-12C alkyl or alkoxy gp. in position 3 and/or -XANR3R4; X = O or -OC(=O)-; A = 2-15C (pref. 2-8C and pref. branched) alkylene, alkenylene or alkynylene; R3, R4 = H or 1-6C alkyl pref. 1-3C alkyl; R2 = CO2H or -C(=O)Y-A-B; Y = O or NH; B = H, NH2 or a bile acid bonded through a mono or di(1-6C)alkyl gp. in position 3; and n = 10-500, pref. 30-200, esp. 40-100. Also claimed is a conjugate comprising (I) and at least one bile acid. USE/ADVANTAGE - (I) inhibit bile acid resorption and are useful as hypolipidaemic agents or for concentating bile acids in biological fluids used in food or fruit juice. (I) are prepd. in high yields under mild conditions. Dosage is 0.5-10g/Kg/day which is lower than that of known colestyramine and colestipol.

Description

The invention relates to water-soluble and insoluble pectin derivatives, pectin derivatives, which are loaded with bile acids, a process for their preparation and their Application as a medicinal product.

Bile acids have an important physiological function in fat digestion, z. B. as cofactors of pancreatic lipases. As end product of Cholesterol metabolism they are synthesized in the liver, in the gallbladder stored and released from this by contraction in the small intestine, where they develop their physiological effect.

Most of the secreted bile acids are released via the enterohepatic Recovered cycle through the mesenteric veins of the small intestine and The portal vein system returns them to the liver. In the reabsorption Both active and passive transport processes play a role in the intestine. in the enterohepatic circulation, the bile acids occur both as free acids, however also in the form of glycine and taurine conjugates in appearance.

So far, bile acid is known to non-absorbable, insoluble, basic crosslinked polymers (ion exchange resins = "Resins") to bind. When Therapy object are all diseases in which inhibition of Bile acid reabsorption in the intestine, especially in the small intestine, desirable appears, viewed. For example, the chologene diarrhea becomes weaker Ileal resection, or increased blood cholesterol levels in this way treated. In the case of elevated blood cholesterol levels may be due to the intervention  in the enterohepatic circulation a reduction of this level can be achieved. By lowering the bile acid pool in the enterohepatic circulation is the corresponding new synthesis of bile acids from cholesterol in the Forced liver. To cover the cholesterol requirement in the liver, is on the circulating LDL cholesterol (low density lipoprotein) recourse to the hepatic LDL receptors in increased numbers to Effect come. The resulting acceleration of LDL catabolism has an effect by lowering the atherogenic cholesterol content in the blood. So far These mentioned polymeric, insoluble ion exchange resins made the only way to increase the enterohepatic circulation Bile acid excretion and consequent lowering of cholesterol levels to influence.

It now turned out that the drugs that crosslinked on Ion exchange resins based, have various disadvantages.

In particular, for the "resins" in use as drugs to keep a very high daily dose. It is z. B. for colestyramine (contains quaternary ammonium groups) 12-24 g, maximum dose 32 g, for colestipol (contains secondary or tertiary amino groups) 15-30 g.

Another disadvantage is that taste, smell and said high Dosage complicates patient compliance.

Furthermore, it is known that conventional "Resins" show side effects. These Side effects are due to a lack of selectivity (eg avitaminosis), which be taken into account when dosing simultaneously given drugs but also on bile acid depletion, the various gastrointestinal Cause disorders (constipation, steatorrhea) of varying degrees.  

For both preparations was a therapeutic significance by combination with other hypolipidemic drugs such as fibrates, HMG-CoA reductase Inhibitors, probucol (see, for example, M.N. Cayen, Pharmac. Ther., 29, 187 (1985) and 8th International Symposium on Atherosclerosis, Rome, Oct. 9-13, 1988, abstracts P. 544, 608, 710), whereby the effects achieved also the therapy of enable severe hyperlipidemia. That's why it seems significant suitable substances without the disadvantages of to find currently used preparations. Following features of the mentioned Preparations and in particular colestipol are in need of improvement to watch:

• 1. The high daily doses that are necessary because only a relatively small one Binding rate at neutral pH in isotonic medium, as well as the (partial) Releasing the adsorbed bile acids.
• 2. The qualitative shift of bile acid composition of bile with decreasing tendency for chenodeoxycholic acid and its associated increasing danger for cholelithiasis.
• 3. The absence of a depressant effect on cholesterol metabolism Intestinal bacteria.
• 4. The too high binding rate of vitamins and pharmaceuticals makes one Substitution requirements for these substances and blood level controls possibly necessary.
• 5. The dosage form is currently considered inadequate.

The object of the invention was to provide a way bile acids in to bind in a concentration-dependent manner, the bile acids-binding Compounds themselves can not be reabsorbed with and thus not in the enterohepatic circulation. Likewise, the compounds should have a high  Binding rate for bile acids at neutral pH and simultaneously ensure that these do not recur under physiological conditions be released and thus can not be absorbed.

In addition, these compounds are said to be the existing disadvantages of known "Resins" not or no longer have the known extent.

The solution of the problem consists in the provision of pectin derivatives of general formula I

wherein
R¹ OH, a bonded via their carboxy group saturated or unsaturated natural or synthetic fatty acid having 6-30 carbon atoms, bound via its alcohol alcohol having 6-30 carbon atoms, bound via their carboxy natural amino acid, bound via their carboxy group synthetic amino acid, a bile acid bound via an alkyl or alkoxy group having 2-12 C atoms at position 3 and / or an aminoalkyl radical of the general formula II

-X-A-N (R₃) R₄ (II)

means, in which
X -O- or

wherein the carbonyl functions are adjacent to the pectin residue,
A is an alkylene, alkenylene or alkynylene radical having 2 to 15, preferably 2 to 8 C atoms, which is branched or straight-chain, preferably straight-chain,
R₃, R₄ are identical or different and are H or (C₁-C₆) -alkyl, preferably (C₁-C₃) -alkyl,
R₂ is a carboxy group or derived from it derivative of general formula III

means, in which
Y is -O- or -NH-,
A has the meaning already mentioned and
BH, NH₂ or a (C₁-C₆) alkyl mono- or -dialkylated amino group or a bonded via position 3 bile acid, and
n is 10-500, preferably 30-200, especially 40-100.

The radicals R mentioned can be side by side in the polysaccharide chain of Pectins are present.

However, it is preferred that only one type of substituent is preferred, e.g. B. only amino acids, including the radicals of the formula II or the fatty acids  and / or the alcohols. It is particularly preferred that the amino acids and the radicals of the formula II are not present side by side in the pectin polymer.

The compounds of the invention generally have a molecular weight from 3000 to 100,000, preferably from 10,000 to 70,000.

The degree of substitution to be achieved of the radicals R, based on a pectin unit in the Pectin polymer, is dependent on the radical R used and of the selected Reaction conditions. Finally, it is set in such a way that one to Achieving pharmacological activity of the compounds of the invention is optimally achieved.

In general, the degree of substitution DS is between 0.2 and 1.0, preferably between 0.3 and 1.0.

Derivatization of pectin with natural or synthetic amino acids or the reaction to compounds with the radicals of the formula II leads to a Cationization of the polymeric pectin. Of the amino acids are here especially the basic amino acids are preferred.

If derivatized with fatty acids or alcohols, one reaches one Lipophilization.

In both cases, the derivatization leads to increased bile acids and especially at a physiological pH, fixed to the pectin polymer be adsorptively bound. This is a bond that out an ionic and a structural interaction between the Bile acid molecule and the pectin derivative.

Bile acids are ionized or protonated at a physiological pH in front. In this respect, there is an ionic interaction. The structural  Interaction is due to the fact that bile acids from the lipophilic pectin spatially be surrounded. They are therefore inclusion compounds.

In principle, it is therefore possible to ionize both the pectins (amino acids, Aminoalkoxyreste) as to lipophilisieren (fatty acids, alcohols), whereby the supplement adsorptive properties respectively. It is also possible bile acids to be used as derivatizing reagents for pectins, and the derivatives as Adsorber for bile acids to use.

In this case, the degree of substitution is for the covalently bound bile acids 0.01 to 0.3, preferably 0.05 to 0.1.

As natural amino acids are called: Glycine, L-alanine, L-valine, L-leucine, L-serine, L-threonine, L-lysine, L-arginine, L- Asparagine, L-glutamine, L-phenylalanine, L-tyrosine, L-proline, L-tryptophan.

Synthetic amino acids are enantiomeric mixtures of the natural Amino acids, homoamino acids, isoamino acids, γ-aminobutyric acid, α-aminobutyric acid, etc. are preferred.

Suitable fatty acids are saturated and unsaturated, straight-chain or branched carboxylic acids such as caproic, heptane, octane, decane, dodecane, Tetradecane, hexadecane, octadecanoic, oleic, linoleic, linolenic, 2-methylhexanoic acid, u. ä.

Also compatible are aromatic carboxylic acid esters, such as. B. cinnamic acid or Di-hydrocinnamic acid used.

Of the alcohols, the following are used for lipophilization: saturated and unsaturated linear or branched alcohols, such as hexanol, octanol, Decanol, dodecanol, tetradecanol, hexadecanol, octadecanol, and the like. a. Likewise are  Also to understand those that can carry more OH groups, such as about 1,2-hexanediol, 1,2-dodecanediol or 1,2-hexadecanediol.

The following aminoalkyl radicals of the formula X-A-N (R₃) R₄ are used: 2-aminoethanol, 3-aminopropanol, N, N-dimethyl- or -diethyl-2-aminoethanol, N, N-dimethyl- or -diethyl-3-aminopropanol, 4-aminobutanol, 6-aminohexanol, 4- Aminobutyric acid, 6-aminocaproic acid, and the like; a.

The following bile acids can be bound to the dextrans according to the invention cholic acid, deoxycholic acid, lithocholic acid, ursodeoxycholic acid, Chenodeoxycholic acid, as well as their corresponding taurine and Glycinkonjugate.

By using the compounds of the invention, the described deficiencies of the market in enterohepatic Circulatory "Resins" are completely eliminated. By inhibition of Bile acid reabsorption using the compounds of the invention in Small intestine is in a much more effective way in enterohepatic circulation decreased bile acid concentration, so that a reduction in the Cholesterol level in the serum. Avitaminoses are when using the Compounds according to the invention then just as little present as the Influence on the absorption of other drugs or even the negative effect on the intestinal flora, since the bile acid binding to the inventive Compounds is extremely stable.

By using the compounds of the invention, the usual Dosage of the Resins can be significantly lowered, the recommended dose is 0.5-10 g / kg / day. The known side effects (constipation, Steratorrhoe) are therefore not observed, d. H. the fat digestion by the natural Origin of pectins on the one hand and the known positive influence from so-called fiber to digestion on the other hand will not negatively influenced.  

Because of the high affinity of the compounds of the invention for bile acids The problem arises in comparison to the high daily dose of "Resins" Dosing and therefore also that of compliance no longer. In addition, the Improves compliance characterized in that the pectins according to the invention are water-soluble are and thus for the formulation no surcharges such. B. Taste enhancers, emulsifiers, sweeteners, etc. are needed.

Compounds of general formula I can be substantially after produce known methods. For example, the esterification of Polysaccharides with fatty acids or N-protected amino acids after activation the carboxylic acid with carbonyldiimidazole (C.H.BAMFORD, I.P. MIDDLETON, K.G. AL-LAMEE, Polymer 27, 1981 (1986)) in good yields under mild conditions Conditions. For alkylation, the reaction of the polysaccharide in DMSO solution with alkyl bromides and iodides under the action of strong bases such as Sodium hydride or potassium hydroxide (J.S. BRIMACOMBE Meth. Carbohydr. Chem. Vol. VI, 376 (1972)). Analogously, the implementation of the corresponding Cholic acid tosylates in which the tosyl group is attached via position 3 to a spacer bound as a leaving group, to covalently linked cholic acid pectin Conjugates.

Amino groups can be prepared by reaction of N-containing alkyl halides such as N-2. Chloroethyl-N, N-diethylamine with dextranes (R.W. HOLLEY, J. Am. Chem. Soc. 83, 4861 (1961)), reaction with amino epoxides such as 1,2-epoxypropyldiethylamine, Addition of acrylonitrile to polysaccharides and subsequent reduction (J. COMPTON Meth. Carbohydr. Chem. Vol. III, 317 (1963) or after cleavage of Release N-protecting group of amino acid esterified pectins.

Compounds having the following R radicals have been prepared according to the above Regulation made:

• 1) natural amino acids: L-serine, L-alanine, L-arginine, L-lysine, L-valine.  
• 2) synthetic amino acids: D, L mixtures of all (natural) amino acids, Ornithine, homo-serine, homo-alanine, iso-serine, γ-aminobutyric acid, α-aminobutyric acid
• 3) fatty acids: caproic acid, capric acid, caprylic acid, lauric acid, Stearic acid, oleic acid, linoleic acid, linolenic acid
• 4) alcohols: butanol, hexanol, octanol, decanol, dodecanol, hexadecanol, 2-hydroxyhexanol, 2-hydroxydodecanol
• 5) Aminoalkyl radicals of the formula II in which the following applies:

The degree of substitution is 0.2-1.0, preferably 0.3-1.0.

The molecular weight of the pectin used is 25,000 to 70,000 D, as well however, other molecular weight fractions can also be used.  

The invention further relates to a conjugate of the invention derivatized pectin derivative and at least one bile acid resulting from a structural interaction adsorptive on the derivatized pectin derivative is bound.

Furthermore, the invention relates to the use of the conjugates according to the invention for the manufacture of a medicament. The connections can be solved or suspended in pharmacologically acceptable organic solvents, such as monohydric or polyhydric alcohols such. As ethanol or glycerol, in triacetin, Oils, such as As sunflower oil, cod liver oil, ethers, such as. B. Diethylenglykoldimethylether or polyethers such. For example, polyethylene glycol, or even in the presence of other pharmacologically acceptable polymer carrier, such as. B. Polyvinylpyrrolidone, or other pharmaceutically acceptable additives such as starch, cyclodextrin or polysaccharides. Furthermore, the Compounds of the invention in combination with other drugs are given.

The conjugates of the invention are in various dosage forms administered, preferably orally in the form of tablets, capsules or liquids, such as As well as foods or fruit juices.

The conjugates of derivatized pectin and bile acids according to the invention can be used as pharmaceuticals, especially for the inhibition of Bile acid reabsorption in the intestine and as hypolipidemic.

The derivatized pectins according to the invention can also be used in analytical Method for the group selective enrichment of bile acids from biological Liquids such. As plasma, serum, urine, bile, etc. are used.  

Determination of adsorber capacity

For in vitro testing of the absorber capacity of the compounds of the invention the following bile acids: 15.1 mg cholic acid, 13.8 mg desoxycholic acid, 13.8 mg chenodeoxycholic acid, 13.2 mg lithocholic acid, 17.1 mg glycocholic acid, 15.8 mg glycodeoxycholic acid, 16.6 mg glycochenodeoxycholic acid and 16.0 mg Glycolithocholic acid dissolved in 700 μl methanol, with 0.92 ml phosphate buffered Saline (pH 7.2) and added together with 5 mg of the invention Compounds incubated for 24 h at 37 ° C in a shaking water bath. After that, the Mixture in a Visking dialysis tube filled and at 72 h Room temperature against phosphate-buffered saline (pH 7.2) dialysed. The Determination of bile acid binding is carried out by analysis of the external medium, for. B. by the methods described below.

1) HPLC with fluorescence detection

Equipment:
HPLC system from Kontron, consisting of three pumps and mixing chamber, autosampler, UV detector and evaluation unit with software MT2. Fluorescence detector from Merck and Hitachi. Since the samples are sensitive to light and temperature, the autosampler is cooled to approx. 5 ° C.
Mobile phase:
Eluent A: Millipore water (own plant)
Eluent B: acetonitrile / methanol 60:30
Pillar:
LiChrospher 100 RP-18, 25 mm, 5 μm, from Merck
Guard column:
LiChrospher 60 RP-select B, 4 mm, 5 μm, from Merck
Flow rate:
1.3 ml / min
detection:
Excitation: 340 nm
Emission: 410 nm
Gradient:
0.00 min 66% B
7.50 min 66% B
8.00 min 76% B
12.50 min 76% B
13.00 min 83% B
25.00 min 83% B
25,50 min 91% B
40.00 min 91% B

2) Enzymatic determination of total bile acid

• - Eppendorf tubes are each given 900 μl of the following mixture:
  6 ml Tetra-sodium diphosphate buffer 0.1 M, pH 8.9
  2 ml NAD solution (4 mg / ml water)
  20 ml Millipore water
• - For this purpose, 30 μl of the sample and 30 μl of enzyme solution are pipetted.
• - Enzyme solution: 3-alpha-hydroxysteroid dehydrogenase 0.5 units / ml
• - The mixtures are mixed and incubated for 2 h at room temperature.
• - Transfer to 1 ml disposable cuvettes and measure in the photometer at 340 nm.
• - Only conditionally suitable for bile acid samples as the green color interferes.

3) HPLC with UV detection

Equipment:
HPLC system from Kontron, consisting of three pumps and mixing chamber, autosampler, UV detector and evaluation unit with software MT2.
Mobile phase:
Eluent A: ammonium carbamate buffer 0.019 M, adjusted to pH 4.0 with phosphoric acid.
Eluent B: acetonitrile
Pillar:
LiChrospher 100 RP-8, 25 mm, 5 μm, from Merck
Guard column:
LiChrospher 60 RP-select B, 4 mm, 5 μm, from Merck
Flow rate:
Gradient:
0.00 min 0.8 ml / min
20.00 min 0.8 ml / min
23.00 minutes 1.3 ml / min
51.00 minutes 1.3 ml / min
detection:
200 nm (for preparations additionally at 254 nm)
Gradient:
0.00 min 32% B
8,00 min 35% B
17.00 min 38% B
20.00 min 40% B
24.00 min 40% B
30.00 min 50% B
45.00 min 60% B

The following results could be obtained. They are in Table 1 summarized. Table 1 shows the adsorption properties of Pectin derivatives according to the invention with regard to:

GC glycocholic GCDC glycochenodeoxycholic GDC glycodeoxycholic LC lithocholic

Example 1 (3-aminopropyl) pektinamid)

5 g (26.3 mmol) of pectin C (from Roth, Karlsruhe) are mixed with 10 ml (8.8 g, 119 mmol). Dissolved 1,3-diaminopropane in 50 ml of DMSO with 0.1 g of dimethylaminopyridine and 8 h stirred at 100 ° C. The solution is then concentrated, the residue in  Water taken over a membrane with the exclusion limit 3000 D ultrafiltered and freeze-dried.

Yield: 1.3 g
Elemental analysis: 42.3% C; 6.5% H; 8.8% N.

example 2 (3-dimethylaminopropyl) pektinamid

5 g (26.3 mmol) of pectin C (Roth, Karlsruhe) in 50 ml (41.1 g, 409 mmol) 3-dimethylaminopropylamine is stirred with 0.1 g of DMAP for 6 h at 100 ° C. To Concentrate, ultrafiltration over a 3000 D membrane and freeze-drying 6.0 g of the product isolated.

Elemental analysis: 43.0% C; 7.0% H; 6.3% N.

Claims (13)

1. pectin derivatives of general formula I. wherein
R¹ OH, a bonded via their carboxy group saturated or unsaturated natural or synthetic fatty acid having 6-30 carbon atoms, bound via its alcohol alcohol having 6-30 carbon atoms, bound via their carboxy natural amino acid, bound via their carboxy group synthetic amino acid, a bonded via an alkyl or alkoxy group having 2-12 C-atoms at position 3 bile acid and / or an aminoalkyl radical of the general formula II-XAN (R₃) R₄ (II), wherein
X -O- or wherein the carbonyl function is adjacent to the pectin residue,
A is an alkylene, alkenylene or alkynylene radical having 2 to 15, preferably 2 to 8 C atoms, which is branched or straight-chain, preferably straight-chain,
R₃, R₄ are identical or different and are H or (C₁-C₆) -alkyl, preferably (C₁-C₃) -alkyl,
R₂ is a carboxy group or derived from it derivative of general formula III means, in which
Y is -O- or -NH-,
A has the meaning already mentioned and
BH, NH₂ or a (C₁-C₆) alkyl mono- or -dialkylated amino group or a bonded via position 3 bile acid, and
n is 10-500, preferably 30-200, especially 40-100. 2. Compounds according to claim 1, characterized in that only one type a substituent, in addition the radicals of the formula II and / or an alcohol, is included. 3. Compounds according to claims 1 or 2, characterized in that this type corresponds to the amino acids. 4. Compounds according to claims 1 or 2, characterized in that the amino acids and the radicals of the formula II not next to each other in the pectin available.   5. Compounds according to claims 1 to 4, characterized in that they have a molecular weight of 3000 to 10,000. 6. Compounds according to claims 1 to 5, characterized in that they have a degree of substitution DS between 0.1 and 1.0, preferably between 0.2 and 0.7. 7. conjugates consisting of a compound according to claims 1 to 6 and at least one bile acid. 8. conjugates according to claim 7, characterized in that the Substitution degree DS is 0.01 to 0.3, preferably 0.05 to 0.1. 9. A pharmaceutical composition containing a conjugate according to claims 7 and 8 and a carrier. 10. A process for the preparation of a medicament, characterized in that to convert a pharmaceutical composition according to claim 9 into a suitable dosage form. 11. Use of the conjugates according to claim 7 or 8 as hypolipidemic. 12. Use of the conjugates according to claim 7 or 8 in foods and Fruit juices. 13. Use of the compounds according to claims 1 to 6 for Group selective enrichment of bile acids from biological fluids.