CA2078589A1

CA2078589A1 - Alkylated polyethyleneimine derivatives, a process for the preparation thereof, the use thereof as pharmaceuticals, and pharmaceutical products

Info

Publication number: CA2078589A1
Application number: CA002078589A
Authority: CA
Inventors: Stefan Muellner; Heiner Glombik
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: JPH0616809A; EP0534304A1

Abstract

Abstract of the disclosure Alkylated polyethyleneimine derivatives, a process for the preparation thereof, the use thereof as pharmaceuti-cals, and pharmaceutical products.

Uncrosslinked alkylated polyethyleneimines which, because of their bile acid-binding properties, can be used as hypolipidemics and additives to foodstuffs and fruit juices are described.

Description

HOECHST AKTIENGESELLSCHAFT ~OE 91/F 304 Dr.D~p785 ~9 Description Alkylated polyethyleneimine derivatives, a process for the preparation thereof, the use thereof as pharmaceuti-cals, and pharmaceutical products.

The invention relates to alkylated polyethyleneimine derivatives, to a process for the preparation thereof, to a pharmaceutical composition based on these compounds, and the use thereof as pharmaceuticals, especially for lowering elevated lipid levels.

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 is covered by the circulating LDL (low 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 athersgenic cholesterol content in the blood.

The ion exchangers used as pharmaceuticals have as active groups either quaternary ammonium groups (such as colestyramine) or secondary or tertiary amino groups (such as colestipol). The daily dose of colestyramine is expediently 12 -24 g, the recommended maximum daily dose is 32 g. 15 - 30 g is the recommended daily dose of colestipol. The taste, odor and high dosage make patient compliance difficult. The side effects derive from lack of selectivity (for example avitaminoses) and must also .
- : . ' ~ ~ ~ ' ' ' ;
.

- 2 - 2~785 89 be taken into 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 sig-nificance for both products has been described owing toa combination with other drugs with hypolipidemic acti-vity, such as fibrates, HMG-CoA reductase inhibitors, probucol tcf, for example, M.N. CAYEN, Pharmac. Ther. 29, 187 (1985) and 8th International Symposium on Atherosclerosis, Rome, Oct. 9-13, 1988, Abstracts pp 544, 608, 710), the effects achieved also making therapy of severe hyperlipidemias possible. This is why it appears important with the given principle of action to find suitable substances without the disadvantages of the lS 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 relatively low binding rate at neutral pH in isotonic 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 cholelithiasis.

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

2~78589 5. A further improvement on the presentation can be achieved.

Elimination of the listed deficiencies is possible, surprisingly, by the use of h:igh molecular weight alkyl-ated polyethyleneimines. The macromolecules, which arenot absorbable, display their action both in soluble 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 crosslinking is brought about, inter alia, via alkylene groups with 2 to 8 carbon atoms, and the molecular weight of the initial polymers is between 800 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 is 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 stright-chain or branched C1-C30-alkyl radical and, in the case of the crosslinked alkylated polyethylene-imines, the crosslinker is an .
' ~

2078~89 alpha,omega-dihalogenoalkane w:ith 2-10 carbon atoms or a more highly functional halogenoalkane with 2-10 carbon atoms.

The object of the invention was now to provide polyethy-leneimine-based bile acid adsorbers which are able, in an improved manner, to lower elevated cholesterol levels and undergo increased and more selective binding with bile acid.

It has now been found, surprisingly, that uncrosslinked cycloalkylated polyethyleneimines with a molecular weight of 10,000 to 10,000,000 meet this object in an excellent manner.

The invention therefore relates to uncrosslinked cyclo-alkylated polyethyleneimines which can be prepared from an initial polyethyleneimine with a molecular weight of 10,000 to 10,000,000 and a cycloalkylating agent of the formula I
R-X (I) wherein R is a cycloalkyl radical with 5-30 carbon atoms which can be monocyclic, bicyclic, tricyclic or polycyclic and/or bridged and X is chlorine, bromine, iodine, CH3-S02-0- or : CH3 ~ 02-0-R in particularly preferred uncrosslinked polyethylene-imines is cyclopentyl, cyclohexyl, cycloheptyl, cyclo-octyl, cyclononyl, cyclodecyl, bicyclic systems such as decalyl, hydrindanyl, bridged systems such as norbornyl, or polycyclic systems such as, for example, cyclopentano-perhydrophenanthrene and ring systems derived therefrom or derivatives.

~ 5 ~ 2078~8~
The invention likewise relates to a process for the preparation of uncrosslinked polyethyleneimines, which comprises alkylation by methods customary in polymer chemistry of a polyethyleneimine with a molecular weight between 10,000 and 10,000,000 with an alkylating agent of the formula R-X in which X and R have the stated mean-lngs .

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 some of the secondary amino groups in the chain being converted into tertiary and quaternary structures. The formation of tertiary amino groups i9 preferred.

The invention likewise relates to a pharmaceutical product which contains one or more of the polyethylene-imines according to the invention or the physiologically tolerated salts thereof with an acid.

The invention furthermore relates to the use of the compounds according to the invention for preparing a pharmaceutical. The compounds can be used dissolved or suspended in pharmacologically acceptable organic sol-vents such as monohydric or polyhydric alcohols, for example ethanol or glycerol, in triacetin, oils, for example sunflower oil, fish liver oil, ethers, for example diethylene glycol dimethyl ether, or else poly-ethers, for example polyethylene glycol, or else in the presence of other pharmacologically acceptable polymeric - 6 - 2078~89 vehicles, such as, for example, polyvinylpyrrolidone, or other pharmaceutically acceptable additives, such as lactose, starch, gelatin, cyclodextrin, talc or polysac-charides. Active substances resulting in solid form are initially converted to a required particle size, for example by fine milling. It is furthermore possible for the compounds according to the invention to be given in combination with other pharmaceutical substances, for example with HMG-CoA reductase inhibitors, vitamins, geriatric agents and antidiabetics.

The compounds according to the invention are administered in various dosage forms, preferably orally in the form of tablets, emulsifiable formulations, capsules or liquids such as, for example, also foodstuffs or fruit juices.

The compounds according to the invention can be used as pharmaceuticals, in particular for inhibiting bile acid readsorption in the intestine and as hypolipidemic and for all disorders in which elevated bile acid concentra-tions in the intestine or serum occur.

It is likewise possible for compounds according to the invention to be used as additives in foodstuffs, espe-cially in fruit juices, bread etc. or to be taken to-gether with the latter.

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.

~ 7 ~ 2n78s~9 The alkylated polyethyleneimines according to the inven-tion adsorb endogenous acids, especially bile acids.
Because of these properties, they are able to lower elevated cholesterol levels. The alkylated polyethylene-imines according to the invention have, by comparisonwith colestipol, considerably more beneficial bile acid-adsorbing properties, as is evident from the experiments described hereinafter.

Concerning the tolerability and the selectivity of bile acid binding, as well as the simultaneous cholesterol binding, both in vitro and in vivo experiments in Wistar rats show a distinct superiority (soft gel characteris-tics).

Figure 6 shows the pH dependency of bile acid binding, which remains virtually constant over the physiological pH range and diminishes only above pH 8.

These properties of the uncrosslinked alkylated polymer ensure good to optimal bile acid binding both in the stomach (reflux of bile in liver disorders) and the slightly basic medium of the small intestine, as well as in the slightly acid environment in the rectum.

Example 4.3 g (0.1 mol) of polyethyleneimine (in 50~ aqueous solution) are diluted with 100 ml of water and heated 25 with 23.7 g (0.2 mol) of chlorocyclohexane to reflux while stirring vigorously for 24 h. After brief cooling, 100 ml of 2N NaOH are added the mixture is again heated to reflux for 24 h.
. .
The 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.
~ .

- ': .: .

. ' ': : '' - 8 - 2~78589 Removal of water (rotary evaporator, oil pump vacuum, freeze-drying) results in the cyclohexane-substituted polyethyleneimine as pale yellow powder (characteri2ation by elemental analysis).

1. In vitro experiments The in vitro experiments to examine the binding proper-ties of the compounds according to the invention were carried out using an ox bile assay developed especially for this purpose. This is because it was found that the binding affinity and capacity of bile acid adsorbents may differ greatly owing to ionic effects and pH (Figure 6) and are also dependent on the particular incubation medium. Ox bile is very similar in general composition to human bile and can be obtained in reproducible quality and in sufficient quantity. Because of the closeness to the physiological conditions prevailing in the duodenum, and of the better possibility of extrapolation of the in vitro data to the data from animal experiments and clinical data to be expected, therefore in place of the otherwise customary binding test with commercial pure bile acid in pure physiological buffer, a binding assay based on ox bile was developed. Figures 1 to 5 show the evaluation of the adsorber test in ox bile with corresponding W HPLC, fluorescence HPLC and TLC
evaluation. Adsorbers from DE-A 3,901,527 (Example 2) are compared with the substance according to the invention ~` from Example 1.

Key to Figures 1 to 6 Figure 1 The diagram shows HPLC analysis (W detection) of the binding characteristics of the compound according to Example 1 of the invention and the compound according to Example 2 from DE-A 3,901,527 (comparison compound).

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'.

2078~89 The supernatant of the diluted ox bile (1:10 with 50 mM
NaHCO3, pH 6.5) was analyzed after incubation (at 37C) of 10 mg/ml bile acid sequestrant for 24 h.
The results are shown in the form of bound bile acid/g of adsorber.

Figure 2 The diagram shows HPLC analysis (W detection) of the binding characteristics of the compound according to Example 1 of the invention, the compound according to Example 2 from DE-A 3,901,527 and of commercially obtainable colestipol.
The supernatant of the diluted ox bile (1:10 with 50 mM
NaHCO3, pH 6.5) was analyzed after incubation (at 37C) of 10 mg/ml bile acid sequestrant for 24 h.
The results are shown in % bound bile acid (taurocholate and glycocholate).

Figure 3 The diagram shows TLC (thin layer chromatography) ana-lysis (fluorescence scan) of the binding characteristics of the compound according to Example 1 of the invention and the compound according to Example 2 from DE-A 3,901,527 (compariRon compound).
~he supernatant of the diluted ox bile (1:10 with 50 mM
NaHCO3, pH 6.5) was analyzed after incubation (at 37C) of -10 mg/ml bile acid sequestrant for 24 h.
The results are shown as % bound bile acid (cholate, taurocholate and glycocholate).

Figure 4 The diagram shows HPLC analysis (fluorescence detection) of the binding characteristics of the compound according to Example 1 of the invention and the compound according to Example 2 from DE-A 3,901,527 (comparison compound).
The supernatant of the diluted ox bile (1:10 with 50 mM
NaHCO3, pH 6.5) was analyzed after incubation (at 37C) of 10 mg/ml bile acid sequestrant for 24 h.

: : -: ~'' ' - ' -'' ' ' ' .
. -.

" ' - lO 2078~89 The results are shown as % bound bile acid (cholate and glycocholate).

Figure 5 The diagram shows the means for all the analyses (Figures 2, 3 and 4). The binding characteristics of the compound according to Example 1 of the invention are compared with those of the compound according to Example 2 from DE-A
3,901,527.

Figure 6 The diagram shows the pH-dependence of the bile acid binding of the compound according to Example 1 of the invention.
The supernatant of the diluted ox bile (1:10 with 50 mM
NaHCO3, pH 6.5) was analyzed after incubation (at 37C) of 10 mg~ml bile acid sequestrant for 24 h.
The results are shown as % bound bile acid (taurocholate and glycocholrate) and were examined by HPLC (W detec-tion).
. .~
Design of test with ox bile 20 - Ox bile (from a slaughterhouse, with addition of 0.02% sodium azide, stored in a refrigerator at +4C) is diluted 1:10 with sodium bicarbonate buffer 50 mM, pH 6.5. This is intended to achieve a substan-tial adjustment to the conditions prevailing in the small intestine (information from Prof. A.F.
Hoffman, University of California, San Diego, USA:
90~ of the substances bound by bile acid adsorbers are not bile acids but predominantly bicarbonate.).
The pH of 6.5 corresponds to that of undiluted ox bile.

- 100 mg samples of the adsorbers to be tested are weighed into an Erlenmeyer flask with ground joint.

.

.
, ~ ' - 11 - 2078~89 - After addition of 10 ml of the bile/buffer mixture, the mixtures are stoppered and incubated in a shaking water bath at 37C for 24 h.

- Each test includes two control mixtures without added product and two standard mixtures with colestipol (pure active substance). One duplicate determination is carried out for each product.

- After incubation, the samples are transferred into centrifuge tubes and centrifuged at 6,000 rpm for 10 min.
- The supernatant is removed and analyzed as follows:
a) Enzymatic determination of total bile acid - 900 ~1 portions of the following mixture are placed --in Eppendorf tubes:
6 ml of tetrasodium diphosphate buffer 0.1 M, pH 8.9 2 ml of NAD solution (4 mg/ml of watex) 20 ml of ~Millipore water - 30 ~1 of the sample and 30 ~1 of the enzyme solution are pipetted into this 20 - Enzyme solution: 3alpha-hydroxysteroid dehydrogenase 0.5 units/ml - The mixtures are mixed and incubated at room temper-ature for 2 h.
- Subsequent transfer into 1 ml disposable cuvettes and measurement in a photometer at 340 nm.
- Of limited suitability for bile samples because the green color interferes.

b) TLC with scanner evaluation and photography (single bile acids and cholesterol) TLC plates: precoated silica gel 60 TLC plates, 20 x cm, layer thickness 0.5 mm, with .. . . . .
- . ~ : . .
- . .

- 12 - 2078~9 concentration zone from Merck, Cat. No.

Mobile phase: chloroform : methanol : glacial acetic acid 85:20:8 Coloring reagent: manganese(II) chloride~sulfuric acid Coloring time: about 20 min in an oven at 120C
Sample volume: 5 ~1 Evaluation: a. "optically~' under W lamp b. with scanner supplied by Camag and evaluation software c. photography c) HPLC with W detection (conjugated bile acids) Apparatus: HPLC system supplied by Kontron, compris-ing three pumps and mixing chamber, autosampler, W detector and evaluation unit with MT2 software.
Mobile phase: mobile phase A: ammonium carbamate buffer 0.019 M, adjusted to pH 4.0 with phosphor-ic acid.
mobile phase B: acetonitrile Column: ~LiChrospher 100 RP-8, 25 mm, 5 ~m from Merck Precol D : LiChrospher 60 RP-select B, 4 mm, 5~m from Merck Flow rate: Gradient: 0.00 min 0.8 ml/min 20.00 min 0.8 ml/min 23.00 min 1.3 ml/min 51.00 min 1.3 ml/min Detection: 200 nm (additionally at 254 nm for products) 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
-:~ ~

:

- 13 - 2078~9 30.00 min 50% B
45.00 min 60% B

d) Derivatization, HPLC with fluorescence detection tunconjugated and glycine-conjugated bile acids) Apparatus: HPLC system supplied by Kontron, compris-ing three pumps and mixing chamber, autosampler, W detector and evaluation unit with MT2 software. Fluorescence detector from Merck-Hitachi. 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 phaæe B: 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% B
40,00 min 91% B
e) HPLC (cholesterol) Apparatus: HPLC system supplied by Kontron, compris-ing three pumps and mixing chamber, autosampler, W detector and evaluation unit with MT2 software.
; Mobile phase: acetonitrile : isopropanol 50:50 Column: LiChrospher 100 RP-18, 25 mm, 5 ~m from Merck 5 Precolumn: LiChrospher 60 RP-select B, 4 mm, 5 ~m from Merck Flow rate: 1.0 ml/min Detection: 207 nm f) ~optically" (bile pigment) 1.2 Reversibility test The adsorbate removed from the equilibrium in a mixture as described above was subsequently incubated with fresh medium containing no bile acid, for example water or phosphate buffer, and determined as above.

Claims

1. Uncrosslinked alkylated polyethyleneimines which can be prepared from an initial polyethyleneimine 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, CH3SO2-O- or

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

3. Polyethyleneimines as claimed in claim 1 or 2, wherein R is cyclopentyl, cyclohexyl, cycloheptyl, cyclo-octyl, cyclononyl, cyclodecyl, decalyl, hydrin-danyl, norbornyl or cyclopentanoperhydrophenan-threne and derivatives thereof.

4. Polyethyleneimines as claimed in claims 1 to 3, wherein the initial polyethyleneimine has a molecu-lar weight above 100,000.

5. A process for the preparation of uncrosslinked alkylated polyethyleneimine derivatives, which comprises alkylation by methods customary in polymer chemistry of an initial polyethyleneimine with a molecular weight of 10,000 to 10,000,000 with an alkylating agent of the formula I as claimed in claim 1.

6. The process as claimed in claim 5, wherein one or more of the following measures are observed:
a) polyethyleneimines with a molecular weight range above 100,000 are employed, b) X in the alkylating agents R-X is chlorine or bromine, c) R in the alkylating agents is a cyclic alkyl radical, d) the ratio of the alkylating agent employed to the amino groups in the polyethyleneimine is 0.5 : 1 to 2 : 1.

7. A pharmaceutical product which contains a compound as claimed in claims 1 to 4 or the physiologically tolerated salt thereof with an acid.

8. A process for the preparation of a pharmaceutical product, which comprises converting a compound as claimed in claims 1 to 4 into a suitable presenta-tion.

9. The use of a compound as claimed in claims 1 to 4 as hypolipidemic.

10. The use of compounds as claimed in claims 1 to 4 as additives in foodstuffs and fruit juices.