DE2522821A1 - Permselective membrane for artificial kidney - contg. support modified on blood-side with polyelectrolyte and or enzyme dissociating toxins - Google Patents

Abstract

Permselective dialysis and diafiltration membranes, for use as artificial kidney, consists of a membrane support material provided, on blood-side, with a surface-layer of polyelectrolytes and bonded to support either covalently or adsorptively. The surface layer is pref. additionally modified with enzymes, which dissociate partic. those toxic materials present in blood which find difficulty in passing through membrane on account of their mol. wt. and/or charge. The elimination of toxic metabolites is selectively increased without enlarging the membrane pores. Membrane allows an improved discrimination of toxic metabolites as against water, urea and aminoacids.

Description

Permselective Membrane for Artificial Kidneys The invention relates on a permselective membrane for dialysis or diafiltration, which for the Use as an artificial kidney is provided. The membrane consists essentially made of a membranous carrier material with a blood-side surface layer or with a blood-side modification.

In the human kidney, metabolic products are first made through Ultrafiltration (glomerular filtrate) is extracted. Due to the high molecular weight exclusion limit The natural nephron membrane also permeates low-molecular body essentials Substances (e.g. amino acids, glucose), but these can through active transport are partially resorbed via the proximal and distal tubules, a possibility which is not the case with artificial systems.

The prior art for this can be outlined as follows.

Numerous membrane materials for hemodialysis, e.g.

Collagen, polymethacrylates, cellulose derivatives, cross-linked polyvinylpyrrolidone and nylon / epoxy blends have already been tested, primarily in the laboratory; most of the commercial systems are equipped with regenerated cellulose membranes, since their Dialysance of urinary substances has been clinically proven (cf. DT-OS 2 116 067; DT-AS 2 236 226 and DT-OS 2 009 515).

The development of membranes with increased dialysance or filtration rate for toxic metabolites focuses solely on increasing the molecular weight cutoff. This will in fact be exemplary mean molecular weights eliminated more quickly, partly with a relatively small increase in the losses of water and Urea. But to selectively eliminate toxic substances from the blood without that at the same time the loss e.g.

Increases in essential amino acids are in the way of pore enlargement narrow limits set. Deficiency syndromes occur when these limits are exceeded; replacement solutions or infusions are required to avoid them. Also goes with the increase in the molecular weight exclusion limit, an unexpectedly rapid elimination of water or urea.

On the other hand, the advantage of diafiltration is often mentioned in publications the faster elimination of toxic substances of medium molecular weight (approx 300-5000), the necessary dialysance for hemodialysis units of the Square Meter Hour Hypothesis (Trans.Amer. Soc.

Art. Int. Organs 17 (1971) 81) was used. Indeed are already conventional ultrafiltration units made of capillary modules with polysulfone, PVC / PAN (Amicon Corp.) or polyamide membranes (Berghof Institute) as well as for example newly developed membranes based on Polycarbonate or polyacrylonitrile (Zlône-Poulenc) able to remove larger molecules from the blood better than the standard Cuprophan 150 PM membrane. This has been proven in most cases using the B12 (MW 1355) or inulin test (MW 5200). The principal ones listed above However, there are difficulties here too.

Since the uremic also binds, for example, amino acids to If serum proteins are reduced, amino acid losses can already occur during hemodialysis up to 30g per dialysis occur. This in turn leads to pathological symptoms such as Hypoproteinemia, loss of transferin or complement factors (decreased resistance to infection) and, at the same time, increased levels of serum nitrogen as a result of impaired nitrogen utilization. These symptoms are to be expected and can be increased with diafiltration of blood cannot be compensated by dieting. The use of amino acid replacement fluid or infusion solution (e.g.

-Ketocarboxylic acid), as already stated above, required.

The development of modules for hemodialysis is state-of-the-art mainly through the optimization of plate kidneys (e.g. Kiil, Gambro, Rh & ne-Poulenc), Coiled kidneys (e.g. Goro-Tex, Extracorporeal) or hollow fibers (e.g. Dow-Cordis) with regard to flow conditions, "priming volume" or "rest volume" characterized. In addition, individual systems are designed for home dialysis or as disposable units (see DT-OS 2 156 734, DT-OS 2 252 341s DT-AS 1 546 657).

The invention is based on the object of those described above known dialyzers or diafiltration apparatus encountered difficulties to overcome and create a permselective membrane that allows improved discrimination of toxic metabolites to water, urea and amino acids.

According to the invention it has been found that this task with a Membrane can loosen if this has a surface layer of polyelectrolytes on the blood side possesses, which are either covalently or adsorptively bound to the carrier material.

According to an advantageous embodiment of the invention, the surface layer is additionally modified with enzymes, primarily those in the split existing toxic substances for which the membrane is difficult to pass due to their molecular weight and / or their charge.

According to a further embodiment, the polyelectrolyte consists of Invention predominantly composed of strongly anionic and / or strongly cationic groups.

In some cases it should be sufficient if the permselective membrane according to the invention does not have a layer of polyelectrolytes on the blood side, but is only modified on the blood side with enzymes which are either covalently or adsorptively bound to the carrier material; these are again enzymes, which are primarily responsible for the im split existing toxic substances which otherwise, due to their molecular weight and / or their charge, would not be able to pass the membrane or would only be able to pass it with difficulty.

With the measures according to the invention is therefore without increasing the Membrane pores selectively increases the elimination of toxic metabolites. It was namely recognized that in surface modification, i.e. surface coverage of effective hemodialysis and diafiltration membranes with polyelectrolyte molecules there is a feasible way of solving the problems described. For this, besides commercially available (b, c) laboratory scale synthesized membrane materials used (see appendix table 1). The prerequisite is that these membranes have a ' have a dense and regular sequence of functional groups for surface coverage (e.g. alternating copolymers) or that such groups on the blood-side surface can be generated.

The grafting of the membrane surface was completely dissociated with - (i.e. "strong") - rigid polyelectrolytes (see appendix: Table 2) of high Molecular weight carried out, suppressed their adsorption on the membrane surface is.

The mentioned polyelectrolytes were with specifically reacting end groups made to suppress multiple bonding to the membrane surface. Appropriate End groups (-COOH, -NH2) are in poly (-amino acids and polyamides already given. In the case of polyvinyl electrolytes, for example, they were specified by default dilute solutions of preferably radical-forming monomers and addition of the polyelectrolyte-forming Monomers generated. End groups suitable for the purposes of the invention are -CH = CH2 e.g. Butadiene -CH2) 2-OH e.g. hydroxyethyl methacrylate -CH2-CO-NH- e.g. vinylpyrrolidone -COCl e.g. acrylic chloride -NH2 e.g. p-aminostyrene The addition or condensation of the Polyelectrolytes took place after additional activation of the membrane surface e.g. thionyl chloride, acrylic chloride, N-carbonylsulfamic acid chloride, aryl diisocyanate or heterobifunctional reagents such as 4-fluoro-3-nitrophenyl azide. Even the direct one Polymerization of polyelectrolytes on the membrane is after a radical Activation possible.

Toxic metabolites in the blood of uraemic patients can be from Due to their structure, they can be assigned to some classes of substances that affect the pH of the blood protonated or as Anions are present, e.g. indolecarboxylic acid, phenolcarboxylic acid, Amines or mono- or disubstituted guanidyl derivatives, but also phosphates. on the other hand are all essential, semi-essential and non-essential amino acids (with the exception of the non-essential amino acids arginine, aspartic acid, glutamic acid) at pH 7.4 it is still close to its isoelectric point (see appendix: table 3). They are therefore still partially in the betaine structure, i.e. without a net charge before.

According to the current state of knowledge, membranes are charged toxic metabolites with net charges or

allow relevant model compounds to permeate faster than neutral amino acids, also in the same sense between other neutral molecules discriminate like urea or water and do not increase their clearance.

The separation of essential amino acids was based on simple model systems simulated by a possible toxin.

An ultrafiltration experiment was carried out with a cuprophane membrane, for example carried out with an aqueous solution, each 0.1 wt .-% phenylalanine and 2,5-dihydroxybenzoic acid. The experiment was also carried out with a by Polyacrylic acid covalently grafted cuprophane membrane and as a comparison with addition made free polyacrylic acid.

As the results in Table 4 (see Appendix) show, the retention capacity was the membrane compared to the phenol carboxylic acid is selectively reduced, while the amino acid remained unaffected. The drastic effect of adding free polyacrylic acid to the solution finally shows the achievable extent of the reduction in retention capacity charged toxins, with betaines or neutral molecules remaining unaffected.

The spectrum of toxic substances to be discharged can also be biotransformed Metabolites are expanded without thereby affecting the membrane with contrary requirements to be loaded if, according to the invention, enzymes on the blood-side membrane surface e.g. ß-glucuronidase is grafted on.

Enzymes are biocatalysts that make the activation energy more specific significantly lower chemical reactions. This results in the possibility with certain enzymes certain substances at precisely defined Reaction direction to be implemented quickly.

The detoxification of toxic and no longer required substances in the body takes place through various chemical reactions and is called biotransformation.

An important reaction here is the turnover of the toxic substances to glucuronides, whereby an increase in water solubility is usually achieved. In addition to the formation of glucuronides, glucosides and ribosides, conjugation with amino acids especially glycine, cysteine, ornithine, glutamine, the formation of sulfuric acid esters and methylation and acetylation play a role.

The use of certain enzymes now offers the possibility of being preferred by hydrolytic cleavage on the one hand the molecular weight of the biotransformed Substances, especially the glucuronides, to reduce and on the other hand the net charge to change.

Ss-Glucuronidase (EC 3.2.1.31), which is Gluduronide, is particularly suitable for this to glucuronic acid and the corresponding Alcohol or phenolic components splits.

The enzyme can be obtained cheaply and in good quality from microorganisms. If aryl sulfates are to be split at the same time, the mixture of ß-glucuronidase / Aryl sulfatase are used.

While the glucuronides due to their chemical-physical properties (e.g. molecular weight etc.) cannot pass through the membranes discussed here Fission products permeate. For this purpose, depending on the structure of the above-mentioned membranes either ß-glucuronidase immobilized directly on the membrane surface or else fixed to the polyelectrolytes bound to the membrane. For this were known Procedure used; hydroxyl-containing membranes were with BrCN, carboxyl-bearing Membranes activated with carbodiimides or azides (see e.g. Immobilized Enzymes 1973, CRC Press, Cleveland, Ohio; Ang. Chemie 34, (1972) 319).

Dialysis membranes according to the invention can be flat or tubular can be produced according to the usual methods, but in any case is irrelevant on the applied Procedure to ensure that the blood side in In the sense of the description given, a surface modification has been carried out. as well can, under the same precondition, those known from the prior art Modules are produced with the membrane according to the invention.

Further details, advantages and possible uses of the invention are evident from the specific examples below.

Example 1: In a vessel equipped with a stirrer, a reflux condenser, two feed devices and a thermometer were fitted 3, there were 550 cm of water filled. The water was heated to about 80 ° C. and from the dropping funnels on the one hand 300 g of acrylic acid and on the other hand 6 g of potassium peroxydisulfate dissolved in 150 cm3 of water added dropwise with stirring. Duration approx. 3 1/2 hours. It fell a clear, syrupy Polyacralic acid solution.

Viscosity = 0.045 poise dry residue = 30%; 2 h; 120 0C Cuprophan (see Developments in the fields of the 7th Work Session of Working Group for clinical nephrology in Hamburg on June 14, 1974) was initially 20 h in dimethylformanide pretreated and finally with 5% hexamethylene diisocyanate at 60 ° C for 5 minutes implemented. The cuprophane membrane activated in this way was finally applied for 30 min. polyacrylic acid grafted on at room temperature, 5% based on polyacrylic acid on the membrane weight were used. The modified membrane thus obtained became watered overnight.

In an ultrafiltration cell, a aqueous test solution consisting of 0.1% by weight each of PhyJalanin and 2,5-dihydroxybenzoic acid existed, at pH 7.4 and a working pressure of 1 atm, the permselectivity of the modified Cuprophane membrane tested; The following retention capacities were determined. Retention evermögen fiu Cuprophan unfashionable. Phe 1.7 2.5 - DHB 11.0 Cuprophan mod. Phe 1.6 2.5 - DHB 4.2 Phe = phenylalanine; 2.5 DHB = 2.5 - dihydroxybenzoic acid example 2: About 1% by weight of polyacrylic acid, prepared according to Example 1, became the test solution added and an unmodified cuprophane membrane in the ultrafiltration cell built-in.

Thereafter, under the same conditions as in Example 1 the retention capacity was determined.

Retention capacity% Cuprophan unmod. Phe. 1.7 2.5-DHB 11.0 cuprophan + free polyacrylic acid Phe. 0.7 40.9 (1% by weight) Example 3: A cuprophane membrane was in the ultrafiltration chamber for 10 minutes with a solution of BrCN (25 mg / ml) activated at 200C. The BrCN solution pumped around became continuous Maintained at pH 11.0 with 2N NaOH. After washing with water, w-aminocarboxylic acid was used implemented.

The COOH function was activated with soluble carbodiimide; then the membrane activated with carbodiimide was in a buffer of pH 4.5 with a solution of ß-glucuronidase implemented. The membrane contained about 25 mg enzyme per m2. As a test solution for determining the retention capacity an aqueous solution of 0.1 wt. o each phenylalanine and the sodium salt of Phenolphthalein glucuronide used, furthermore, as in Example 2, 1 wt .-% Polyacrylic acid used freely for surface modification.

Retention capacity% Cuprophan + 1% by weight polyacrylic acid Phe. 1.6 Phenolphthalin 23.5 Cuprophan + Enzyme + 1% Phe. 0.9 20.0 polyacrylic acid Tabel 1 Table 1: Membrane materials suitable for surface modification. Membrane material functional remarks a) poly-d-amino acids -NH2 good blood tolerance, high (e.g. from lysine, serine) -OH mechanical strength, suitability for ultrafiltration of blood b) regenerated cellulose -OH comparison materials c) cellulose acetate -OH (deacetylated) d) copolymers of butadiene / vinylpyridine C = C easily modifiable, simple Processing, partly very good blood compatibility hydroxyethyl methacrylate / ethyl methacrylate - (CH2) 2OH "vinylsulfonic acid / acrylic acid / acrylonitrile -COOH (-COCl)" O e) polyamides -C-NH- activation by: a) partial hydrolysis b) dimethyl sulfate Tabel 2: Polyelectrolytes SBukne bases suitable for covering the membrane surface Disulfonated, aromatic polyvinylpyridine (quater polyamide nated) polyvinylbenzylal polyacrylic acid kylammonchloride poly-α-L-crystalline (oxid.) poly-α-L-lysine polystyrene sulfonate Ionene polymer sulfoethyl cellulose guanidinoethyl cellulose Tabel 3: Molecular weight and isoelectric point of the essential +) and some not essential amino acids amino acid molecular weight isoelectric point (pH) Alanine 89.09 6.00 histidine 155.16 7.59 glycine 75.07 5.97 tyrosine 181.19 5.66 proline 115.13 6.30 Serine 105.09 5.68 Valine +) 117.15 5.96 Isoleucine +) 131.17 5.94 Leucine +) 131.17 5.94 lysine +) 146.19 9.59 methionine +) 149.29 5.74 pheylalanine +) 165.19 5.48 TryptXhan +) 204.22 5.89 Threonine +) 5 119.12 5.64 Table 4: Retention a modified and an unmodified cuprophane membrane in ultrafiltration (p = 1 atm) of phenylalanine (Phe) and 2,5-dihydroxybenzoic acid (2,5-DHB) at pH 7.4 cuprophane membrane retention capacity in% Phe (MW 165) 2,5-DHB (MW 154) unmodified 1.7 11.0 modified (grafted) 1.6 4.2 free polyacrylic acid (1% by weight) 0.7 - 40.9

Claims (5)

Claims Permselective membrane for dialysis or diafiltration for use as an artificial kidney, consisting essentially of a membranous Carrier material with a blood-side surface layer, characterized in that that the surface layer consists of polyelectrolytes and on the carrier material is bound either covalently or adsorptively. 2. Membrane according to claim 1, characterized in that the surface layer is additionally modified with enzymes that are primarily those in the split existing toxic substances for which the membrane is difficult to pass due to their molecular weight and / or their charge. 3. Membrane according to one of claims 1 or 2, characterized in that that the polyelectrolyte consists predominantly of strongly anionic and / or of strongly cationic Groups. 4. Permselective membrane for dialysis or diafiltration for use as an artificial kidney, consisting essentially of a membranous carrier material modified on the blood side, characterized in that the modification consists of enzymes bound to the carrier material either covalently or adsorptively, mainly those in the split existing toxic substances for which the membrane is difficult to pass due to their molecular weight and / or their charge. 5. Use of the membrane according to one of claims 1 to 4 for production of flat, spiral or tubular modules.