CA2078588A1

CA2078588A1 - Use of alkylated polyethyleneimine derivatives for concentrating bile acids

Info

Publication number: CA2078588A1
Application number: CA 2078588
Authority: CA
Inventors: Stefan Muellner
Original assignee: Individual
Current assignee: Hoechst AG
Priority date: 1991-09-21
Filing date: 1992-09-18
Publication date: 1993-03-22
Also published as: EP0534316A1; JPH069686A

Abstract

Abstract of the disclosure:

The use of alkylated polyethyleneimine derivatives for concentrating bile acids.

The use of uncrosslinked cycloalkylated polyethylene-imines for concentrating bile acids and their derivatives from biological fluids or extracts is described.

Description

~OECHS~ AKTIENGESELLSCHAFT HOE 91/F 305 Dr.D~rh - Description The use of alkylated polyethyleneimine derivatives for concentrating bile acids.

The invention relates to the use of alkylated poly-ethyleneimine derivatives.

Insoluble basic crosslinked polymers have been used for some time for binding bile acid and utilized therapeutic-ally on the basis of these properties. The object of therapy is the causal treatment of chologenic diarrhea (for example following resection of the ileum) and elevated cholesterol levels in the blood. The latter case comprises intervention in the enterohepatic circulation, where in place of the bile acid content taken out of the circulation the corresponding neosynthesis from chol-esterol is provoked in the liver. The cholesterolrequirement in the liver i8 covered by the circulating LDL tlow density lipoprotein) cholesterol, with action by an increased number of hepatic LDL receptors. The increase brought about in this way in the rate of LDL
catabolism has the effect of reducing the atherogenic chole~terol content in the blood.

The ion exchangers used as pharmaceutical~ have as active groups either quaternary ammonium groups (such as coles-tyramine) or secondary or tertiary amino groups (such as colestipol). The daily dose of cole~tyramine i9 expedien-tly 12 - 24 g, the recommended maximum daily dose is 32 g. 15 - 30 g i8 the recommended daily do~e o~ colestipol.
The taste, odor and high dosage make patient compliance difficult. The side effects derive from lack of selec-tivity (for example avitaminoses) and must also be takeninto account in the dosage of medicaments given concurrently, but also from bile acid depletion and cause various gastrointestinal disturbances (constipation, steatorrhea) of various degrees. The therapeutic significance for both products has been described owing .,~
, . .: ; . :
.~ ~ . . -to a combination with other drugs with hypolipidemic activity, such as fibrates, ~MG-CoA reductase inhibitors, p;robucol (cf, for example, M.N. CAYEN, Pharmac. Ther. 29, 1137 (1985) and 8th International Symposium on Athero-sclerosis, Rome, Oct. 9-13, 1988, Abstracts pp 544, 608, 710), the effects achieved also making therapy of severe hyperlipidemia~ possible. This is why it appears import-ant with the given principle of action to find suitable substances without the disadvantages of the products used at present.

The following features of the said products and, in particular, of colestipol are to be regarded as worthy of improvement:

1. The high daily doses, which are attributable to a xelatively low binding rate at neutral pH in iso-tonic medium and the (partial) release of the adsorbed bile acid again.

2. The qualitative shift in the bile acid composition of the bile with a decreasing trend for chenodeoxy-cholic acid and the increasing risk, associated therewith, of cholelithiasi~.

3. The lack of a suppressant action on cholesterol metabolism of intestinal bacteria.
5 4. The excessively high binding rate of vitamins and drugs makes replacement of these substances neces-sary and checks on blood levels possibly necessary.

5. A further improvement in the presentation can be achieved.

Elimination of the listed deficiencie~ is possible, surprisingly, by the use of high molecular weight alkyl-ated polyethyleneimines. The macromolecules, which are .
.
: :, . .
-.

_ 3 _ 2078S88 not absorbable, display their action both in ~oluble form and pH-dependently in insoluble form, corresponding to the uncrosslinked structure, and in insoluble state as crosslinked polymers.

Crosslinked polyethyleneimines are described in US Patent 3,332,841. The cros~linking is brought about, inter alia, via alkylene groups with 2 to 8 carbon atoms, and the molecular weight of the initial polymers is between 80~
and 100,000. For treatment of transient gastric hyper-acidity, 0.25 to 5 g per dosage unit are administered.Neither the binding of bile acid nor a lipid-lowering activity, associated therewith, of the crosslinked polyethyleneimines is described, since the binding capacity of the polyethyleneimines without alkylation for bile acids is insignificant or zero, depending on type.
Owing to the large potential charge density, it iæ
possible by alkylation to ensure an adequate binding capacity and by the choice of the substituents of appro-priately hydrophilic/hydrophobic nature to ensure the affinity and binding specificity.

DE-A 3,901,527 (corresponding to US Patent Application No. 07/466,923 or No. 07/762,177 and corresponding to EP-A-0,379,161) describes uncrosslinked and crosslinked alkylated polyethyleneimine derivatives in which the basic polymer has molecular weights of 10,000 to 10,000,000 and the alkylating agents correspond to the formula R-X where X is a halogen, pseudohalogen or halogen-like compound, R is a straight-chain or branched C,-C30-alkyl radical and, in the case o~ the crosslinked alkylated polyethyleneimines, the cros~linker is an alpha,omega-dihalogenoalkane with 2-10 carbon atoms or a more highly functional halogenoalkane with 2-10 carbon atoms.

German Patent Application P 41 31 507.3, filed at the same time, de~cribes uncro~slinked cycloalkylated ~ . . , -;,, . ..... , :
.. , , .~ ; : ~ . .
.: .. : , :, :. .. . . . ... .

., ;, ~, ' :,, : ':
. ~ . . , . ~ . .. .

2~78588 polyethyleneimines with a molecular weight of 10,000 to 10,000,000 which can be prepared from an initial poly-ethyleneimine with a molecular weight of 10,000 to 10,000,000 and a cycloalkylating agent of the formula I

R-X ~I) in which R is a cycloalkyl radical with 5-30 carbon atoms which can be monocyclic, bicyclic, tricyclic or polycyclic and/or bridged, and 0 X i8 chlorine, bromine, iodine, CH3-SO2-O- or C~3 4~ s02_o_.

These compounds are now used for concentrating bile acid from biological fluids and extracts.

Particularly preferred uncrosslinked polyethyleneimines are those in which R i8 cyclopentyl, cyclohexyl, cyclo-heptyl, cyclooctyl, cyclononyl, cyclodecyl, bicyclic systems such as decalyl, hydrindanyl, bridged systems such as norbornyl, or polycyclic systems such as, for example, cyclopentanoperhydrophenanthrene and ring systems derived therefrom or derivatives.

Polyethyleneimines with a molecular weight above 100,000 are preferably employed.

X in the alkylating agents R-X is preferably chlorine or bromine.

The ratio of the alkylating agent employed to the amino groups in the polyethyleneimine is 0.2 : 1 to 5 : 1, preferably 0.5 : 1 to 2 : 1.

.. . .
:, . , , -, , .. : , .
~ . . , . . . . ~.

The reaction with alkylating agent results in ~ome of the secondary amino groups in the chain being converted into tertiary and quaternary structures. The formation of tertiary amino groups is preferred.

The bile acids include natural bile acids, derivatives of natural bile acids and their alkali metal or alkaline earth metal salts. These particularly include: glyco-cholate, taurocholate, cholate, cholic acid, deoxy-, chenodeoxy-, ursodeoxy- and lithocholic acid, and the conjugates thereof with glycine or taurine. It is addi-tionally also possible for cholesterol to be adsorbed.

Serum means, in particular, human or animal serum.

The alkylated polyethyleneLmines used according to the invention adsorb endogenous acids, especially bile acids.
Because of these properties, they are able to lower elevated cholesterol levels. Increased concentrations of cholesterol, but also of cholic acid, chenodeoxycholic acid, deoxycholic acid and lithocholic acid occur, inter alia, in the following diseases:
primary biliary cirrhosis (PBC), cholestasis and biliary atresia. It is possible with the alkylated polyethylene-imines in an analytical method to normalize the con-centrations of the bile acids again.

The analytical method according to the invention can compri~e, in particular, an ion exchange chromatography in which the alkylated polyethyleneimines bound to the solid phase are used for concentrating the bile acid.

The described use of these compounds in analytical methods is very important particularly because detection of bile acid in serum is generally extremely difficult.

After passage through the liver, only a small proportion of the bile acid remains in the peripheral blood. The . . .
, - .~ . , ` . ; . . : ~

' - .' ~ -, - 6 - 207~88 concentrations of the individual bile acids in the serum are therefore usually low. In this, they are bound to albumin and lipoproteins which carry out the transport Eunction. The strength of bile acid binding increases with the hydrophobic nature of the bile acid (G. Salvioli et al., Bile Acid Binding in Plasma: the importance of lipoproteins. FEBS-Letters 187, 272-276 (1985~). The concentration of bile acids in the serum is influenced by bile acid synthesis, their secretion and reabsorption in the intestine and by renewed uptake in the liver. Impair-ment of these functions may result in alterations in the serum bile acid profile, ie. the total bile acid con-centration may be higher or lower than in healthy sub-jects. However, it is also pos~ible for the concentra-tions of only a few bile acids to change, for example ontreatment with bile acid products. Thus, in liver dis-orders the concentration of the bile acids in the serum is usually raised and the ratio of the endogenous bile acids to one another is often greatly changed (C.R. Pennington et al., Serum Bile Acids in the Diag-nosis of Hepatobiliary Disease. Gut 18, 903-908, 1977).

Bile acids can he determined by spectroscopic methods (S.J. Levin et al., Spectrofluorom~etric determination of total bile acids in bile. Anal. Chem. 33, 856-860, 1961), radioimmunoassays (J.D. Palombo et al., Assessment of the effects of above-normal bilirubin on radioimmunoassay of conjugated bile acids in serum. Clin. Chem. 32, 2204-2205, 1986), thin-layer chromatography (S.S. Ali et al., Quantitative estLmation of bile salts in serum. Can.
J. Biochem 48, 1054-1057, 1970), HPLC (G.R. Cambell et al., Modified sample preparation and chromatography for the separation of human bile acid conjugates. Anal.
Proceed. 23, 33-34, 1986) or gas chromatography (T. Laatikainen et al., Determination of serum bile acids by glass capillary gas-liquid chromatography. Clin. Chim.
Acta 64, 63-68, 1975). HPLC and gas chromatography are, because of the high separation efficiency, good methods ,~.., .. ~ , :

i, ~
, _ 7 _ 2078588 of determination but in gas chromatographic methods it is impossible to dispense with derivatization, for example silylation of the OH groups and eQterification or methyl-ation of the carboxyl group. Thi~ is why GC takes considerably longer and moreover requires high purity of the samples, which in turn makes an elaborate sample preparation process necessary.

The sample preparation method of K.D.R.Setchell et al. (A
rapid method for the quantitative extraction of bile acids and their conjugates from serum using commercially available reversed-phase octadecylsilane bonded silica cartridges. Clin. Chim. Acta 125, 135-144, 1982) which has been most commonly used to date proves in practice not to be reproducible and has the disadvantage that large sample volumes are required in order to concentrate sufficient amounts for further analysis (R. Nuber et al., J. Lipid Research 31, 1517-1522, 1990).

~he method of Nuber et al. in turn now ha~ the di~-advantage that a high sample throughput is impossible owing to the elaborate pretreatment of the serum samples, and it is thus advisable only for selected sample~.

In the search for an alternative to the methods described hitherto, it has been found, surprisingly, that the compound according to the Example is particularly suitable for the selective and quantitative adsorption of bile acids in serum and allows equally quantitative desorption so that it is possible for the bile acid~ to be separated efficiently from their serum carrier proteins, to be derivatized after desorption, and to be subjected to a highly sensitive fluorescence ~PLC method.

Example 4.3 g (0.1 mol) of polyethyleneimine (in 50% aqueous solution) are diluted with 100 ml of water and heated : , . . . ~ ; .

'' ' . :. . , - 8 - 2~78588 with 23.7 g (0.2 mol) of chlorocyclohexane to reflux while stirring vigorously for 24 h. Atar br~ef cooling, 100 ml of 2N NaOH are added and the mixture is again heated to reflux for 24 h.

~he organic phase is separated off. The aqueous phase is washed with dichloromethane and concentrated in a rotary evaporator and then under oil pump vacuum. The residue is dialyzed from aqueous solution.

Removal of water (rotary evaporator, oil pump vacuum, freeze-drying) results in the cyclohexane-substituted polyethyleneimine as pale yellow powder (characterization by elemental analysis).

The u~e according to the invention in an analytical method is shown by the following outline of the method:

Analytical method for concentrating bile acids from serum using cycloalkylated polyethyleneimines:

Step 1: Deproteinization - Incubate 1 ml of serum + 100 ~1 of NaOH (lM) at 96C
for 20 min.
- Add 100 ~1 of HCl (2M), mix (gives pH between 3 and 4), centrifuge.
- Dissolve pellet in 500 ~1 of NaOH, see above, incubate at 96C for 20 min.
- Add 300 ~1 of HCl, see above, centrifuge.

Step 2: Bile acid binding - Place the two supernatants from step 1 together in an Eppendorf tube containing 150 mg of compound from the Example.
- Incubate in an Eppendorf heated shaker at 37C for 4 hours.
- Centrifuge, discard supernatant.

. :
.: ~
,, . :.

:

.

2078~88 Step 3: Remove adsorbex - Add 600 ~l of lM ~aOH, incubate in an Eppendorf heater at 96C for 20 min (adsorber dissolves).
- Add 300 ~1 of approx. 5M methanolic HCl in order to precipitate adsorber and residual protein.
- Centrifuge, collect supernatant.
- Dissolve in 600 ~1 of NaOH, see above, incubate at 96C for 20 min.
- Add 300 ~1 of meth. HCl, ~ee above, centrifuge.

Step 4: Concentration - Evaporate supernatants and take up in 0.5 ml of MeOH. Shake the sample for some time in an Eppendorf shaker to do this.

Step 5: Solvolysi~ (Princen HMG., Meijer P. and Kuipers, F., One-Step solvolysi~ of 3-, 7- and 12-sulfated free and conjugated bile acids. Clin.
Chim. Acta. 192, 77-84, 1990).
- Add sample from Step 4 to 5 ml of dioxane/HCl, and incubate in test tubes in a ~haking water bath at 37C overnight.
- Evaporate and take up again in 0.5 ml of MeOH.

Step 6: Derivatization for HPLC with fluorescence detection - Employ complete sample from step 5, evaporate with 200 ~1 of methanolic KOH.
- Take up in 100 ~1 of dicyclohexano-18-crown ether (1 mg/ml of acetonitrile), and add 100 ~l of 4-bromomethyl-6,7-dimethoxycoumarin (1.5 mg/ml of acetonitrile).
- Incubate in a water bath at 37C for 40 min.
- Add 100 ~l of acetonitrile and ~tore in a deep-freeze overnight.
- HPLC analysis.

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. , -: :: . , . ..
-: : . - , . . ..

,: ., , -., ~ , : ~ . . ;
,:

2078~88 :
The derivati~ation, HPLC with fluorescence detection (unconjugated and glycine-conjugated bile acids), is carried out as indicated below:

Apparatus: HPLC system supplied by Kontron, compris-ing three pumps and mixing chamber, autosampler, W detector and evaluation unit with M~2 software. Fluorescence detector from Merck-~itachi. Since the samples are sensitive to light and heat, the autosampler is cooled to about 5C.
Mobile phase: mobile phase A: ~Millipore water (own system) mobile phase ~: acetonitrile/methanol 60:30 Column: ~LiChrospher 100 RP-18, 25 mm, 5~m from Merck Precolumn: LiChrospher 60 RP-select B, 4 mm, 5 ~m 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% 9 40.00 min 91% ~3 ~he following Table 1 shows the concentration rates ~%~
which can be achieved with the compounds used according to the invention:

. ~

.
:, ~ . - -,: ,, : :

2078~88 Table 1 Determination of the recovery rate using radioactively labeled bile acids (taurocholate TC, glycocholate GC and cholate C ) TC: 105~
Deproteinization GC: 106%
C: 102%

~ ~ TC: 79%
~ile acid binding GC: 78%
C: 88%

~ TC: 87% :
Remove adsorber GC: 99%
C: 73%

~ ~ TC: 43%
Concentration GC: 66%
C: 74%

~ ~ TC: 92%
Solvolysis GC: 77%
C: 53%

. ~ ~ ~ . . . :
:, , : ~

Claims

1. Use of uncrosslinked alkylated polyethyleneimines which can be prepared from an initial polyethylene-imine with a molecular weight of 10,000 to 10,000,000 and an alkylating agent, wherein the alkylating agent has the formula I

R-X (I) in which R is a cycloalkyl radical with 5-30 carbon atoms and X is chlorine, bromine, iodine, CH3-SO2-O- or CH3 - - SO2-O-for concentrating bile acids and their derivatives from biological fluids or extracts.

2. The use of polyethyleneimines as claimed in claim 1, wherein the cycloalkyl radical is mono-, bi-, tricyclic or polycyclic and/or bridged.

3. The use of polyethyleneimines as claimed in claims 1 or 2, wherein R is cyclopentyl, cyclohexyl, cyclo-heptyl, cyclooctyl, cyclononyl, cyclodecyl, decalyl, hydrindanyl, norbornyl or cyclopentanoperhydro-phenanthrene and derivatives thereof.

4. The use of polyethyleneimines as claimed in claim 1, wherein the initial polyethyleneimine has a molecu-lar weight above 100,000.

5. The use of polyethyleneimines as claimed in claim 1, wherein the cycloalkyl radical is mono-, bi-, tricyclic or polycyclic and/or bridged, and wherein the initial polyethyleneimine has a molecular weight above 100,000.

6. The use of polyethyleneimines as claimed in claim 1, wherein the initial polyethyleneimine has a molecu-lar weight above 100,000, and wherein R is cyclo-pentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclo-nonyl, cyclodecyl, decalyl, hydrindanyl, norbornyl, or cyclopentanoperhydrophenanthrene and derivatives thereof.

7. An analytical method for concentrating bile acids and their derivatives on a solid phase, containing uncrosslinked alkylated polyethyleneimines of claims 1 to 6, comprising the following steps:
- deproteinization of the serum and of the adsorber, - mixing of the serum and of the adsorber, - removal of the adsorber, - concentration of the supernatant, - solvolysis, - derivatization of the sample.

8. The method as claimed in claim 7, wherein the serum is human or animal serum.

9. The method as claimed in claims 7 and 8, which comprises an ion exchange chromatography.