WO2001058496A1 - System for the extracorporeal treatment of blood - Google Patents

Abstract

A method is provided for treating blood so as to remove protein-bound toxins (PBT) which comprises: (a) separating plasma from the blood; (b) subjecting separated plasma from step (a) to a first dialysis procedure in which separated plasma and a first dialysis medium are maintained on opposite sides of a first semi-permeable membrane; (c) transferring separated plasma from step (b) to a second dialysis procedure wherein treated separated plasma and a second dialysis medium are maintained on opposite sides of a second semi-permeable membrane; and (d) recovering separated plasma depleted in PBT from step (c). The method is characterised in that (i) the first dialysis medium comprises a low molecular weight component that is capable of passing through the first semi-permeable membrane in step (b) into the separated plasma whereby interaction between said component(s) and PBT occurs resulting in toxin release; and (ii) the composition of the second dialysis medium is such that transfer of said low molecular weight component(s) from the plasma to the second dialysis medium occurs.

Description

SYSTEM FOR THE EXTRACORPOREAL TREATMENT OF BLOOD

Background to the invention

The present invention relates to an extracorporeal blood treating system which is particularly effective for removing protein-bound toxins from patients' blood The invention more specifically provides a novel method for treating patients' blood in an extracorporeal system, a novel dialysis fluid for use in this method and novel haemodialysis apparatus

The invention is based, in a preferred aspect, upon use of what we have referred to as a "crystalloid chelator"-based dialysate which may be used in plasmapheresis and/or haemodialysis apparatus for the therapeutic field of acute liver failure, acute and chronic renal failure, rheumatoid arthritis, and drug and metal intoxication The invention can also be applied to procedures for recycling or purifying a protein solution in vitro The term "crystalloid chelator" is particularly used to refer to a class of low molecular weight nitrogen-containing compounds

Extracorporeal blood purification is a technology that includes haemodialysis, and plasmapheresis and may utilise so-called "bioartificial-based" cells to treat a wide variety of metabolic and immunological diseases by treating the circulating blood The number of therapeutic applications has increased remarkably in recent years, together with an increase in acquired knowledge of the association between diseases and corresponding endogenous toxins accumulated in the body and in the blood circulation (Therapeutic haemopheresis A reference of diseases American Red Cross 1994) The link between the nature and properties of blood health and disease has been known since before the development of modern medicine Thus, studies have focused on the composition of blood and its relationship with disease from a time which predates the clinical introduction of extracorporeal devices to treat the blood (Thackrah CT, 1819) In general, methods utilized in the therapeutical extracorporeal treatment of the blood consist of a plasma separation, and/or haemodialyser, and/or adsorbent devices, and/or a bioreactor to achieve the purification and the amelioration of the metabolic or immunologic function of the patient

The main metabolic and immunologic diseases for which abnormal toxins are known and should be removed, are the rheumatological diseases, neurological diseases, liver disease, haematological disease, renal disease, malignant diseases, hyper pidemia, poison and drug intoxications Therapeutical methods that seek to purify albumin are important because albumin bound toxins are related to many diseases In this respect, various protein bound toxins have been recognized to be involved in many diseases The most important known protein bound toxins are idols, phenols, mercaptans, beπzodiazepines, ammonia, Indoxyl sulfate, hippuπc acid, polyamme, uforanic acids, chloramine These toxins are mainly bound to albumin, since albumin is the most important toxin carrier in the blood circulation This is due to the fact that albumin has a high binding affinity to several substances and it is the protein carrier with the highest concentration in the blood circulation. If the liver and/or the kidney are in failure, their metabolic functions are disturbed with the consequences that several albumin bound toxins increase dramatically in the blood circulation In this situation the toxin-carrying albumin is extremely overloaded and this leads to an increased fraction of unbound toxins The albumin toxin overloading induces a lack of binding capacities for several enzymes such as endothehal derived enzymes involving the alteration of the physiological control system of the blood pressure Albumin-bound toxins are mainly related to liver failure, which remains a problem since hvertransplantations, if available, need a bridging time Despite recent advances in the treatment of liver diseases, especially fulminant hepaticfailure, there continues to be as high as a 90% mortality and this accounts for greater than 27 thousand deaths annually (Konstantine et al Artificial liver Artif organs 16 235-42, 1992) The current storage of donor organs has prompted the development of liver support systems as a bridge to transplantation A variety of artificial liver support systems have been developed, which include haemodiafiltration, haemoperfusion over charcoal, plasmapheresis, hybrid bioartificial cell based membranes, and ex vivo xenotransplantation, but none has proven to be entirely effective

The need for an improved therapeutic system for deintoxication is heightened by a dramatic clinical situation which is associated with a poor economical climate, as documented by the US National Institute of Health (Digestive Diseases Statistics, NIH Publication NO 95-3873, February 1995)

It is well known that beside the production of important enzymes the liver has an important function in cleaning albumin by metabolizing the albumin bound toxins To date, several methods have been proposed to perform albumin purification for artificial liver support systems used as a temporary artificial liver The use of membrane filters, of solid phase matrices with an adsorbent capacity of the albumin bound and unbound toxins, and of so called bioreactors based on animal hepatocyte systems which stimulate the liver function, have been proposed

In the field of blood purification, haemodialysis and haemodiafiltration using diffusion and convective transport mechanisms are not effective in removing albumin bound toxins With the introduction of highly permeable asymmetrical synthetic membranes with a cut off up to 60 000 MW (which is the molecular weight of albumin), dialysis and haemodiafiltrations have become very effective in removing unbound small and middle molecules, but are limited in the extraction of protein bound toxins

Plasma elimination by the way of plasma filtration with the infusion of fresh plasma containing albumin has been applied to overcome the problem of hepatic failure The method is still clinically applied in acute liver failure for the removal of hepatic toxins Therefore, the survival rates of the patients remain low, which is due to the fact that the efficiency in the removal of significant protein bound toxins needs a large volume of plasma exchange for a long period of time In this respect therapy with large volumes of plasma exchange is impossible to apply because of the costs The use of charcoal as an adsorbent has improved the removal of toxins from albumin in combination with plasma exchange However, because of an insufficient removal of toxins, charcoal adsorption continues to be used in association with other procedures, for example those using cell based hybrid membranes, (so-called bioreactors)

Cell based devices have been seen for a long time as the ideal solution since they combine elimination of toxins and production of important vital enzymes Thus, a large number of patent applications have been filed in this field and much research has been carried out which has generated new knowledge concerning liver function The application of porcine or human cell line hepatocyte based bioreactors are by far the most popular and feature most frequently in publications Bioreactors are in general devices made with human or animal cells The cells which are grown on a permeable surface, the principal element of bioreactors, are mounted in an extracorporeal system operating in a similar way as haemodialysis The pore size of the membrane determines the transport of molecules between the patients' blood and the hepatocytes The technical problems, therefore, involved in these techniques were from the beginning the limited number of cells resulting in a reduced transport of toxins across the cell membrane A number of new inventions focused on these problems in order to make the system more effective by way of combining the biorectors with haemodialysis systems and in building a 3-dιmensιonal cell chamber to increase the number of cells

In US Patent No 1995000376095 Cerra Frank B et al propose a bioreactor apparatus comprising a feed and waste chamber and a cell chamber separated by a selectively permeable membrane Within the cell chamber, a biocompatible three- dimensional matrix entraps animal cells or genetic modifications

In US Pat No 19960006636591 Naughton et al a bioreactor is disclosed for growing three-dimensional tissue Cells are seeded onto a mesh and provided with two media flows, each contacting a different side of the cells The bioreactor can be used to grow liver tissue, and is designed as an extracorporeal liver assistance device in which blood or plasma is exposed to the three-dimensional livertissue The blood or plasma from patients are directed to flow against the liver tissue The liver tissue is further exposed on its opposite side to media providing nutrients and gases

In US Patent No 1997000861503 Amiots and Coon Rapids disclosed a bioreactor containing living animal cells at a density approaching that of normal animal tissue High cell loading is achieved by providing a flow restriction which controls fluid flow through the bioreactor during cell loading

In WO 99/32171 , Shell and Wang disclosed a cell based bioreactor with two exchanging sides The initial motivation for the development of extracorporeal livers is based on animal hepatocytes as the imitation of the natural liver for unknown metabolism that occurs in the organ

The potential clinical use of hepatocyte-based liver support is still growing However problems remain because of high costs to build a bioreactor and the problems involved with the use of animals (Jauregui HO, Gann KL Mammalian hepatocytes as foundation for treatment in human verfailure J Cell Biochem 1991 45 359-65) and human hepatoma ( Nyberg SL, Remmel RP, Mann HJM, Peshwa MV, Hu WS, Cerra FB Primary hepatocytes outperform HepG2 cells as the source of biotransformation functions in a bioartificial liver Ann Surg 1994, 220(1 ) 59-67) its application is the subject of discussions In this respect, problems which may issue from the application of animal hepatocytes can be very similar to those involved in xenotransplantation in terms of viral infection (which has @ FDA cπticicism This danger is due to the use of permeable membranes with the adhesion of the cells on the outer side next to the patients' blood in the inner side, and this may enable entry of virus or DNA particles to the patients' blood

In view of the problems involved with animal or genetically transformed cell based bioreactors, traditionally safe methods such as carrier molecules or adsorbent combined with dialysis systems, have been proposed Implementation of features based on albumin carriers or an adsorbent suspension make the therapy less expensive and conform with the clinical needs The proposed methods involve the extraction of protein bound toxins by physical processes The extraction of protein bound liver failure toxins has been for instance reported by using haemodiafiltration with a dialysate containing albumin (Awad et al Characteristics of an albumin dialysate haemofiltration system for the clearance of unconjugated bilirubin) The clearance of protein bound toxins using haemodiafiltration with albumin in the dialysate involves the interaction between the protein bound toxin, the carrier protein (albumin in the dialysate), and the filter membrane polymer that binds the carrier protein The clearance of protein bound toxins can be enhanced by increasing the concentration of the carrier protein and the pH

A commercially available device has been based on the system proposed by Award et al In this device, a molecular adsorbent system has been proposed for the elimination of albumin bound toxins In the Strange et Mitzner publication (A carrier- mediated transport of toxins in a hybrid membrane Safety barrier between a patients blood and a bioartifical liver Int J Artif organs 1996, 19 677-691 ) a combination of a membrane filter, a dialysate solution and a recycling system based on charcoal and resin adsorbent are used in an extracorporeal system in order to eliminate albumin bound toxins In this system, the dialysate solution containing albumin adsorbed the albumin bound toxins through a highly permeable membrane In the dialysate compartment, albumin molecules are recycled using the charcoal adsorbent As an intermediate step toward a bioartificial liver, the regeneration of the toxin loaded carrier proteins on the dialysate side is carried out by albumin carrier agents instead of a bioreactor based on cells This method was shown to be efficient in removing protein bound as well as water soluble toxins from blood However this method uses a dialysate solution with human albumin which remains a contamination risk (similar to blood transfusion) for which may in the future be expensive due to the testing controls related to hepatitis and the HIV-virus In spite of the well known association between unknown viruses and contamination with human biological product, today no devices eliminate all existing risks Thus, regulations for the future will limit the use of human albumin derived from donors and this will be replaced by genetically engineered albumin, making the clinical applications expensive

SUBSTITUTE SHEET (RULE 26) An alternative to albumin detoxification based on haemodiafiltration has been the use of suspensions of adsorbent The principle of such methods is to transport the protein bound toxins from the inner side to the outer side of a membrane, whereas the adsorbent are exchanging the toxins from one side to the other side In US Patent No 5919369, Stephen combines plasmafiltration and dialysis devices, designed for the circulation of an adsorbent suspension or capsuled hepatocytes Since the albumin content in the body is very high when compared with other precursor proteins, adsorbent technologies can only be effectively applied, if the adsorbents are continuously recycled In this respect, this proposal claims the use of an adsorbent suspension system which is applied in a haemodiafiltration system, and the recycling of the adsorbent suspensions

In US patent No 5078885, Matsmura claims a method for removing protein bound toxins using a so called albumin-dimensioned bottle-neck pore membrane In this procedure, the body fluid to be treated is brought into contact with one side of a semi-permeable membrane and an adsorbent is placed on the opposite side of the membrane having narrow bottle-neck pores The membrane is not permeable to the adsorbent

In US patent No 5211850, Shettiger and McRen claim a system based on the circulation of the blood through a filter device The devices comprises a bundle of "U" shaped hollow fibers, which are immersed in a chamber containing adsorbent which are not permeable to the membrane

Chemical procedures using adsorbent and membranes have been described in the prior art

The present invention has been designed in an attempt to overcome the aforementioned problems Further advantages of the invention include 1 ) an effective system for removing protein bound toxins, 2) a safe procedure, wherein hazardous contamination or genetic alteration is not a problem since the system does not require the use of human or animal substances such as albumin or cells, 3) the implementation of a system which should make therapeutic methods cost effective, i e. the cost should be much lower than cell based hybrid membrane, and should successfully compete with any other methods of plasmapheresis

Summary of the invention

According to an aspect of the present invention there is provided an effective plasma separation followed by a dialysis system based on a new formulated crystalloid ionic detergent for example a mixture of a chelating compound and a high concentration of sodium chloride

Specifically, according to one aspect of the invention, there is provided a method for treating blood so as to remove protein-bound toxins (PBT) which comprises (a) separating plasma from the blood,

(b) subjecting separated plasma from step (a) to a first dialysis procedure in which separated plasma and a first dialysis medium are maintained on opposite sides of a first semi-permeable membrane,

(c) transferring separated plasma from step (b) to a second dialysis procedure wherein treated separated plasma and a second dialysis medium are maintained on opposite sides of a second semi-permeable membrane, and

(d) recovering separated plasma depleted in PBT from step (c), characterised in that (i) the first dialysis medium comprises a low molecular weight component (herein also referred to as a "detoxicant") that is capable of passing through the first semi-permeable membrane in step (b) into the separated plasma whereby interaction between said component(s) and PBT occurs resulting in toxin release, and (n) the composition of the second dialysis medium is such that transfer of said low molecular weight component(s) from the plasma to the second dialysis medium occurs The process of the invention may be applied to blood that is continuously being removed from a patient, treated and then returned to the patient's circulation

In carrying out this procedure, plasma and cellular components are separated in step (a) and the separated cellular components may be reconstituted with the treated plasma after step (d) above (or step (g) below)

Alternatively, recovered plasma may be reintroduced into the patient without adding back cellular components

Steps (b) and (c) may be repeated Thus according to a preferred method of operation the recovered plasma from step (d) is subjected to at least one further cycle of purification comprising

(e) subjecting separated plasma from step (d) to a third dialysis procedure in which separated plasma and a third dialysis medium are maintained on opposite sides of a third semi-permeable membrane,

(f) transferring separated plasma from step (e) to a fourth dialysis procedure wherein treated separated plasma and a fourth dialysis medium are maintained on opposite sides of a fourth semi-permeable membrane, and

(g) recovering separated plasma depleted in PBT from step (f),

characterised in that (i) the third dialysis medium comprises a low molecular weight component that is capable of passing through the third semi-permeable membrane in step (e) into the separated plasma whereby interaction between said component(s) and PBT occurs resulting in toxin release, and (n) the composition of the fourth dialysis medium is such that transfer of said low molecular weight component(s) from the plasma to the fourth dialysis medium occurs, and the first and third dialysis media optionally have different compositions

Preferably, the low molecular weight component has a molecular weight less than 5000, more preferably less than 1000 and most preferably less than 500 The molecular weight of the low molecular weight component is preferably greater than 50 The low molecular weight component desirably has one or more of the following characteristics (a) it is water-soluble, (b) it is basic or amphoteπc, (c) it has at least one amine functionality, preferably provided by a primary, secondary or tertiary amine group, which may comprise a ring nitrogen, (d) the nitrogen lone pair of the amine group interact with the PBT, (e) it may possess one or more hydroxy groups and (f) it may possess one or more -COOH groups

Specific preferred examples of the low molecular weight component in the first dialysis medium are selected from the group consisting of ethanolamine, diethanolamine, tnethanolamine, pyrrolidine, N-(2-hydroxyethyl)pyrrolιdιne, N-(2- hydroxyethyl)pιperιdιne, phenanthrohne, ethylenediammetetraacetic acid (EDTA) and diethyleπetπamiπepentaacetic acid (DPTA)

The concentration of the low molecular weight component in the first dialysis medium is preferably from 1 mM to 200 mM, more preferably from 10 mM to 100 mM and most preferably from 40 mM to 60 mM The pH is preferably mildly alkaline, but reasonably close to physiologically acceptable pH values, for example from 8 to 11 5, preferably from 9 to 10 Typically the pH of the first dialysis medium is 9 0

Desirably, the first dialysis medium additionally comprises an ionic salt, which provides a source of physiologically acceptable cations and anions, which generally may be selected from sodium, potassium, calcium, magnesium, ammonium, chloride, bicarbonate and phosphate

The ionic salt is preferably sodium chloride, optionally at a concentration between 1 mM and 200 mM, more preferably between 50 mM and 150 mM, most preferably between 80 mM and 120 mM

Similarly, the second dialysis medium would normally comprise a source of physiologically acceptable cations and anions, such as cations selected from sodium, potassium, magnesium, calcium and ammonium and anions selected from chloride, bicarbonate and phosphate The concentration of the ions in the second dialysis medium is preferably from 0 01 mM to 20 mM, and more preferably from 0 01 mM to 5 mM

A preferred optional component of the second dialysis medium is glucose

In carrying out the method of the invention it is mainly the low molecular weight component(s) in the first dialysis medium that are instrumental in removing the toxin in the PBT from the protein by binding to the toxin In certain instances, this is achieved by inducing a conformational change in the protein of the PBT which causes the toxin to be released The conformation change induced in the protein may be reversible

In carrying out the invention, the released toxin may be removed from the plasma in a number of different ways For example, toxin released during the second orfourth dialysis procedure may pass through the dialysis membrane into the second or fourth dialysis medium This is a practical possibility when the toxin has a low enough molecular weight to pass through the membrane Alternatively, the released toxin may become bound to the dialysis membranes, i e in steps (b) and (c) and/or (e) and (f)

Preferably in carrying out the method of the invention, the first and/or third dialysis media are circulated between devices in which they are contacted with the first and/or third semi-permeable membrane and a respective regenerative device Similarly, the second and/or fourth dialysis media may be circulated between devices in which they are contacted with the second and/or fourth semi-permeable membranes and a respective regenerative device

The dialysis membranes are desirably synthetic asymmetrical semi-permeable membranes, preferably ones wherein the pores of the semi-permeable membranes are narrower on one side of the membrane When such membranes are used, the treated separated plasma in step (c) and/or step (e) would contact the side of the asymmetric semi-permeable membrane having the larger surface area

SUBSI H UTE SHEET (RULE 26) Preferably, the present invention, as currently envisaged, may be applied to the extracorporeal removal of protein bound toxins for the application of a liver support system, for acute and chronic renal failure, and for drug and metal deintoxification It is designed to provide a method of haemodialysis or haemodiafiltration which has been extended to a unique application of a chemical process involving the desorption of the protein bound toxins which occurs directly in the fluid phase of cell free plasma medium This process enables the desorption of protein bound toxins and their removals by way of diffusion and filtration through a membrane

The present invention according to one preferred aspect thereof provides a new method involving the application of what we term a "crystalloid-based chelator solution" This "crystalloid" dialysate may be used in a haemodialysis or haemodiafiltration extracorporeal apparatus and enables the removable of protein bound toxins in a competitive mode between proteins and the chelators The "crystalloid" and "crystalloid-toxin" complex may be removed from the body fluid before entering the patient So far, as the inventors of this invention are aware, no methods using soluble "crystalloid chemicals" which enable the release of protein bound toxin in the fluid phase by crystalloid chemicals have been described hither to

The invention may involve the use of a crystalloid solution which is a mixture of a sufficient concentration of sodium chloride and a chelating agent A chelating agent or chelator is defined as a compound which is able to form complexes by binding ions and metal ions The use of a mixture of sodium chloride and diethanolamine (ethylendiamine) as the chelating agent is preferred since diethanolamine is a phosphate acceptor and a detergent Other chelators may also be used, if they satisfy the properties of chelating agents A second characteristic of the invention is also an efficient adsorption of hydrophobic large molecules into a membrane, as defined above The invention provides apparatus for carrying out the procedure of any of the invention comprising

(i) conduit means for receiving blood withdrawn from a patient

(n) means for separating plasma from blood delivered via conduit means (i) and for recovering separated plasma and cellular blood components (in) a first dialysis device for dialysing plasma separated in means

(II) against a first dialysis medium (iv) a second dialysis device for subjecting plasma from the first dialysis device to dialysis against a second dialysis medium

(v) conduit means for transferring plasma from dialysis device (in) to a second dialysis device (vi) means for recombining plasma from the second dialysis device

(iv) with cellular blood compounds obtained from separation means (n) and

(VII) means for receiving recombined plasma and cellular blood components for re-infusion into the patient

The invention further provides apparatus for carrying out the procedure of any invention wherein steps (b) and (c) are repeated comprising

(i) conduit means for receiving blood withdrawn from a patient

(u) means for separating plasma from blood delivered via conduit means (i) and for recovering separated plasma and cellular blood components ιιι(a) a first dialysis device for dialysing plasma separated in means

(II) against a first dialysis medium ιv(a) a second dialysis device for subjecting plasma from the first dialysis device to dialysis against a second dialysis medium v(a) conduit means for transferring plasma from dialysis device ιιι(a) to the second dialysis device ιιι(b) a third dialysis device for dialysing plasma from the second dialysis device ιv(a) ιv(b) a fourth dialysis device for subjecting plasma from the third dialysis device to dialysis against a fourth dialysis medium v(b) conduit means for transferring plasma from dialysis device ιιι(b) to the fourth dialysis device (vi) means for recombining plasma from the fourth dialysis device

(iv) with cellular blood compounds obtained from separation means (n) and (VII) means for receiving recombined plasma and cellular blood components for re-infusion into the patient

The apparatus may include respective reservoirs for the dialysis media as defined above, which are preferably charged with said dialysis media

The apparatus may also include a regeneration device for the first and/or third dialysis media and means for circulating said first and/or third dialysis media between the first and third dialysis devices respectively and the respective regeneration devices

Similarly, the apparatus may include a regeneration device for the second and/or fourth dialysis media and means for circulating said second and/or fourth dialysis media between the second and fourth dialysis devices respectively and the respective regeneration devices

The dialysis fluids used in the first and/or third stage of the method of the invention include ones which are novel in their own right

Thus, also provided according to the invention are dialysis fluids comprising water, at least one ionic salt, and a low molecular weight, water-soluble, nitrogen- containing detoxicant having the formula /

HO (CH₂)_n N I

\

R²

R³ R⁵

\ /

N- — X— -N

/ \ _R

R⁴ R⁶

wherein either R¹ and R² are independently selected from hydrogen or lower alkyl groups, and said lower alkyl groups are optionally substituted by one or more of hydroxy, -COOH, -COOR, nitro or ammo, wherein R represents C,_₄ alkyl, or R¹ and R² together form a ring having from four to seven carbon atoms, said ring being optionally substituted by one or more of hydroxy, -COOH, -COOR, nitro or ammo, and R represents C^ alkyl, wherein R³, R⁴, R⁵ and R⁶ are independently selected from hydrogen, -CH₂COOH, -CH₂COOR, -CH₂COO^'Na⁺, or -CH₂CH₂NH₂, and R represents C^ alkyl, X represents a covalent bond, or a group -(CH₂)_p-Y-(CH₂)_q-, wherein Y represents a covalent bond or N-R⁷, and R⁷ is selected from hydrogen, -CH₂COOH, -CH₂COOR, -CH₂COO^"Na⁺, and R represents C^ alkyl, and wherein n, p and q are integers from 1 to 6

Preferably, the components thereof and their concentrations are as defined above in relation to the process of the invention

The invention further provides concentrate compositions which may be reconstituted to form dialysis media as defined above Such concentrations preferably have a pH in the range 9 5 to 1 1 , at which pH the low molecular weight component is stable

A further embodiment of the invention is the storage of the contents of the first and/or third dialysis media in a two component pack Such a pack preferably contains a concentrate composition in one component comprising components in aqueous solution suitable for reconstitution to form a dialysis fluid suitable for use as the first or third dialysis media as defined above Another component of the pack would comprise a diluent for reconstituting the dialysis solution

The contents of the pack may thus be divided into two separate aqueous solutions, one of which comprises a concentrate composition of the low molecular weight component, of the type described above, maintained at a pH range of 9 5 to 11 , but preferably 10 The other component of the pack comprises an aqueous diluent composition of physiologically acceptable salt and acid, preferably HCI and NaCI, with a pH less than 7 Thus, when the aqueous diluent composition and the concentrate composition are mixed, the resulting fluid has a pH from 8 to 1 1 5, but preferably 9 5 and a composition as defined above for the first or third dialysis fluids of this invention Such a means of storage is desirable as the premixed dialysis fluid may be unstable at the pH of use and as the pH of the dialysis media may be critical to the method of the invention, fluctuations in pH may produce adverse affects

The method of the invention preferably includes the steps of circulating patient blood extracorporaly through a plasma filter, filtrating a sufficient amount of a cell fraction prior circulating the cell free plasma fraction through the inner side of a hollow fibre membrane device, circulating the sodium chloπde/cheiator solution through the outer side of the membrane As further steps, after the passage of the cell free plasma fraction, the treated plasma is recirculated through a dialyser in a reverse mode in a way that the plasma is pumped through the outer side of a hollow fiber membrane Meanwhile, physiological bicarbonate solution at physiological pH and containing electrolytes at physiological concentration, circulates at the inner side of the membrane Thus, the treated plasma mixes in with the untreated blood cell fraction prior the back infusion of the patients

The preferred embodiment of the invention includes as principal elements

1 A device for separating blood plasma by a way of a plasma filter This first device is used for separating the blood withdrawn from the patient's arterial or venous blood with a plasma filter into a concentrated blood fraction containing blood cells and cell free plasma fraction The concentrated cell blood fraction is pumped back into the patient after been diluted with the finally treated plasma fraction in the dilution chamber The plasma cell free fraction is pumped into the device No 2 This second device comprises a haemodialyser for the circulation of the cell free plasma fraction in the inner side of the membrane and the circulation of a desorb g crystalloid dialysate solution which may contain the sodium chloride and the chelating agent in the outer side of the membrane into the inner side of the membrane According to the invention, the said solution permeates across the membrane by the way of diffusion and enables the desorption of albumin bound compounds in patient plasma by forming toxin-chelator complexes Simultaneously, the newly formed toxin-chelator complexes are removed through the membrane The desorbmg crystalloid dialysate containing the chelating agent is preferentially based on the formulation of an alkali osmotic salt based agent based on sodium chloπde/diethanoiamine Both chemicals react synergically for the effective desorption of protein bound toxins in the plasma compartment prior elimination through the membrane The device is preferably a hollow fiber dialyser from the type low-permeable (with a nominal cut off below 5000 KD) or a high permeable with a nominal cut off below 60 000 KD A device comprising a second dialyser perfused through the inner side of the membrane with a so-called "crystalloid" physiological solution such as conventional bicarbonate dialysate solution, whereas the outer side is perfused with the cell free plasma through the outer side of the membrane The bicarbonate solution enables removal by the way of diffusion and adsorption unbound toxins and chemical compounds used in the membrane Preferably the said "crystalloid" solution contains 50 mM diethanolamine (HOCH₂CH₂)₂NH and 100mM NaCI, and a pH of 9 0 (preferably adjusted by hydrochloric acid) This solution may be referred to as a "sodium chloride/ diethanolamine/hydrochloπde liquid" The proportions of the compounds can vary, but normally would be in the range of about 1 to 500 mM for diethanolamine and 1 to 200 mM for NaCI The pH of the solution may varies between 8 and 11 5 The term "desorbmg crystalloid dialysate" is used herein to refer to the proposed dialysate containing sodium chloride and diethanolamine which is used as a dialysate to exchange molecules and has the ability to desorb protein bound toxins In accordance with preferred aspects of this invention, the formulated dialysate is circulated through the dialysate compartment of a haemodialyser, whereas the patient cell free plasma is circulated in the blood compartment of the haemodialyser

Alternatively the said crystalloid desorbmg solution may also be a mixture of sodium chloride and a sufficient quantity of a chelator or a mixture of chelators The chelating agent may be a ethanolamine or diethanolamine or diethanoiamine-EDTA (ethylendiaminetetraacedic acid ) or tnethanolamine or pyrrolidine or N-(2- hydroxyethyl) pyrrolodme or (N-(2-hydroxyethyl)pιpeπdιne or diethanooamme tertraacetate, or phenanthrolin and Chelex-100 Other chelating agent which may be used are the group of substituted dithiocarbamates used in mobilizing lead, such as morpholme dithiocarbamate, tetraammonium ethylendiamiπe diacetic dithiocarbamate, or ammonium diethanolamine dithiocarbamate, or sodium diethyldithiocarbamate, or N-benzyl-D-glucoamine dithiocarbamate, ordimercaptosuccinic acid Other acidic chelators which are clinically used pharmacologically as a drug to remove endogenous metals such as dιethylentπamιnepentaacetιcacιd (DTPA), or ethylendiaminetetraacedic acid (EDTA), or 2,3-dιmermaycaptosuccιnιc acid (NTA), or 2,3-dιmercaptopropanol (BAL), can also be used The proportion of the chelating agents can vary, but normally would be in the range of about 1 mM to 1 M for the chelator and 1 to 200 mM for NaCI, the pH of the solution vanes between 5 and 11 5 according to the chelator used Another specific aspect relates to the application of desorbmg crystalloid dialysate solution which contain one or a mixture of several chelating agents which provide physico-chemical forces in cell free plasma in terms that it has the ability to bind toxins and desorb the protein bound toxins from the target proteins when it is dialyzed against cell free plasma solution These preferred aspects are based upon specific choices of the desorbmg crystalloid dialysate solution which contains one or several chelating agents and which exercises hydrophobic and ionic forces on protein solutions In accordance with the preferred aspects of the invention, the desorbmg crystalloid dialysate solution contains the chelating agent, which is able to pass from the outer side to the inner side of a membrane, and is able to change plasma physico-chemical condition in terms that the pH changes, the ionic strength increases and the binding of the toxins to proteins are in competition with the chelating agents which have stronger binding affinity than proteins Thereby when the toxins are desorbed, their elimination occurs rapidly by diffusion

Still another preferred embodiment is the use of a desorbmg crystalloid dialysate solution which does contain the chelating agent and which does not irreversibly damage the proteins in terms of their function and structure The use of chelators, which have hither to been used in therapeutic applications and which have limited toxic effects are preferred Although, the chelators are removed by dialysis residual amounts should be non-toxic for the patients

A further specific aspect of the invention involves the use of a second hemodialyser, to remove the chemicals of the desorbmg crystalloid dialysate solution which contains the chelating agent In accordance with the preferred embodiment it is important that after elimination of the protein bound toxins in the first dialyser device, the used sodium chloride and the toxin bound and the toxin unbound chelating agents are removed and plasma physiological conditions are restored Another important aspect is to return important metals back to the patients, if they have a clinical significance in a vital enzyme activity In addition the preferences are closely related with the specificity of mode and the characterisations of the dialysate and the device ln accordance with the preferred embodiment, a specific aspect of the invention is to exchange the sodium chloride and the chelating agents treated cell free plasma fraction in the way that initial physiological electrolytes conditions and pH are reached

Still preferred embodiment of the present invention provides a method for circulating the cell free plasma processed with the desorbmg crystalloid dialysate solution which contains the chelating agent through the outer side of the second dialyser membrane, and for circulating the dialysate through the inner side of the membrane These preferred aspects are termed "reverse dialysis mode" In accordance with the invention the reverse dialysis mode is preferably applied during the exchanging process between the cell free plasma and the dialysate During this process, two mechanisms may be involved In the first PBT may be removed by diffusion, and in the second, PBT e g hydrophobic toxins are removed by adsorption on the membrane In this respect, the advantage of the invention is based on the fact that the process of the reverse dialysis involves an increase in the hydrophobic interaction between plasma and the surface of a synthetic membrane

Preferably a dialysate solution is chosen which contains typical physiological inorganic salts which are commonly used in haemodialysis solutions e g sources of Na⁺, K⁺, Ca²⁺ and Cl ions Buffers to be used in the solution preferably include bicarbonate The dialysate may also contain important metals which were lost in the first membrane In accordance with the invention and according to the clinical application important enzymes may also be mixed with the dialysate Preferably, the dialyser to be used is a synthetic asymmetrical permeable membrane The membrane is preferably made with hollow fibers

In accordance with the invention, a preferred embodiment provides a plasmaseparation / haemodialysis or a plasmaseparation / haemodiafiltration device performing the methods described, which is effective for removing protein bound toxins and unbound toxins from blood The device includes a supply for processing of the blood and a supplyfor processing the dialysate The preferred device includes a plasma filter from hollow fibre type, and a pump in fluid connection at the blood inlet of the plasma filter The plasma filter is composed of two compartments, the blood compartment with at the inlet and outlet connection and the filtrated plasma compartment with an outlet connection The outlet of the blood side of the plasma filter is in fluid connection to a mixing chamber The outlet of the filtrated plasma is in fluid connection with a pump to the inlet of the membrane inner side of the first dialyser The hollowfibre dialyser has two compartments the inner compartment with two connections (inlet and outlet) and the outlet compartment with two connections (inlet and outlet) and the outlet Thus, the outlet of the membrane inner side of the first dialyser is in fluid connection to the inlet of the membrane outer side of a second dialyser Whereas the outlet of the membrane outer side of the membrane is in fluid connection with a pump to the mixing chamber to mix the concentrated blood with the processed cell free plasma before entering the patient The second hollow fibre dialyser also has two compartments the inner compartment with two connections (inlet and outlet) and the outlet compartment with two connections (inlet and outlet) and the outlet The devices include a pressure monitoring of each dialyser in order to control the transmembrane pressure The device further includes a haemodialysis or a haemodiafiltration supply for the circulation of the crystalloid desorption dialysate with a pump which is adapted to circulate the dialysate through the exterior surface of the first dialyser Furthermore the device also includes a haemodialysis or a haemodiafiltration supply with a pump which is adapted to circulate the bicarbonate dialysate through the interior of the second dialyser

In accordance with the invention, a further preferred embodiment provides a device for processing the blood The preferred device includes a plasma filter from hollow fibre type, and a pump in fluid connection at the blood inlet of the plasma filter The plasma filter is composed with two compartments, the blood compartment with at the inlet and outlet connection and the filtrated plasma compartment with an outlet connection The outlet connection of the blood compartment of the plasma filter is in fluid connection to a mixing chamber The outlet of the filtrated plasma is in fluid connection with a pump to the inlet of membrane inner side of the first dialyser The hollow fibre dialyser has two compartments the inner compartment with two connections (inlet and outlet) and the outlet compartment with two connections (inlet and outlet) and the outlet Thus, the outlet of the membrane inner side of the first dialyser is in fluid connection to a the inlet of the membrane outer side of a dialyser Whereas the outlet of the membrane outer side of the membrane is in fluid connection with a pump to the mixing chamber to mix the concentrated blood with the processed cell free plasma before entering the patient The hollow fibre dialyser has two compartments the inner compartment with two connections (inlet and outlet) and the outlet compartment with two connections (inlet and outlet) and the outlet

In accordance with the invention, a device includes a supply for processing the dialysate The device includes a haemodialysis or a haemodiafiltration supply for the circulation of the desorbmg crystalloid dialysate solution which contains the chelating agent with a pump which is adapted to circulate the dialysate through the exterior surface of the first dialyser Furthermore, the device also includes a haemodialysis or an haemodiafiltration supply with a pump which is adapted to circulate the bicarbonate dialysate through the interior of the second dialyser

Thus in accordance with a further aspect of the invention there are provided compositions for use in preparing a desorbmg crystalloid dialysate solution which contains a chelating agent as defined herein, reconstitution by addition of sterile, pyrogen free water, said composition comprising the specified components in dry form or in the form of an aqueous concentrate

The invention further provides a method of performing plasma separation and plasma dialysis or plasmaseparation and plasmadiafiltration which comprises 1 ) separating plasma, 2) dialysing or diafiltratmg the plasma with a desorbmg crystalloid dialysate, 3) and dialysing or diafiltratmg the processed plasma Features and advantages of the present invention will be apparent from the descriptions which follows Description of the drawing

Additional reference will be to the accompanying drawing of which Figure 1 illustrates diagrammatically the preferred extracorporeal system for the removal of protein bound toxins

Figure 2 illustrates diagrammatically the process of the removal of protein bound toxins in the first hollow dialyser membrane by exchanging the crystalloid desorbmg dialysate at the level of the first membrane The diagram illustrate the process of the diffusion of the chelators/sodium chloride from the dialysate compartment to the plasma compartment After the diffusion of the chelator/sodium chloride from the dialysate compartment to the plasma compartment, the hydrophobic and the ionic binding of the proteins with the toxins is challenged with chelators The use of a chelator which has the capability to be a solvent like the diethanolamine has the capability to challenge the toxins by hydrophobic and by charge interaction Thus, the formed chelator-toxin complex is easily removed by diffusion from the plasma compartment to the dialysate compartment according to the gradient of the concentration of the chelator-toxins complex The use of an internal filtration in the hollow fibre membrane will increase the removal of the chelator-toxins complex by the way of convection

Figure 3 illustrate concentration gradients of the different chemicals involved in the first dialyser The primary phase in the exchange process is attributed to concentration gradients of the chelators and sodium chloride which take place between both compartments, which represent the driving forces of the diffusion of the chelators and sodium chloride from the dialysate to the plasma compartment The secondary phase during the exchanging process is attributed to a concentration gradients between the chelator-toxins complex formed in the plasma compartment, taking place between the compartments, which represents the driving force of the diffusion of the chelator-toxin complex from the plasma to the dialysate compartment

Figure 4 illustrates diagrammatically the process of the removal of the crystalloid chelators and sodium chloride and the process of adsorbing large toxins in a reverse dialysis at the level of the second membrane This process is important for removing the free chelator and the excess of sodium chloride, as well as the correction of the pH During this process which is called the process of equilibrium the adsorptive capacity of the synthetic membrane is increased In this respect the dialyzer is used in a reversed form in the way that plasma is in the side of membrane presenting the large surface area During this process, large molecule toxins can be removed by adsorption when using an asymmetrical synthetic membrane

Figure 5 illustrate concentration gradients of the different chemicals involved in the second dialyser In the first phase of the exchange process, a reversed concentration gradient of the chelators and sodium chloride takes place between the compartments, which represents the driving forces of the diffusion of the chelators and sodium chloride from the plasma to the dialysate compartment In addition to the exchange process, a concentration gradient of the physiological electrlytes takes place between the compartments, which represents the driving forces of the diffusion of electrolytes from the dialysate to the plasma compartment

Figure 6 illustrates graphically the results of the experimental procedure described in the example

The invention will now be described, by ways of illustration without limitations, in the following example and in vitro experimental procedures

Example 1

First dialysate solution A desorbmg crystalloid dialysate solution which contains a mixture of 50 mM diethanolamine (HOCH₂CH₂)₂NH and 100mM sodium chloride, and a pH of 9 0 adjusted by hydrochlonde The dialysate solution is prepared in a concentrated form x 35 concentrated which are reconstituted with the dialysate device

Second dialysate solution A reversed dialysis solution based on bicarbonate dialysis fluid supplied by BRAUN SHIWA (Glansdorf Germany) with the following final composition 140 mM Na⁺ , 2 mM K⁺, 1 5 mM Ca⁺⁺, 0 5 mM Mg⁺⁺,

111 mM Cl^" 32,5 mM HC0₃ , 2 5mM CH₃COO- , 1 g/L glucose The dialysate is supphed in 1/35 concentrated form in two bags (acidic and alkaloid part) which are reconstituted with the dialysate device

Example 2 A two compartment dialysis bag was formed from polyethylene.

The compartment reservoirs were made by ultrasonically welding two thin rectangular polymer films around their periphery to make a bag with an additional transverse welded strip dividing the bag into two compartments. The contents of the bag can be mixed by peeling apart the transverse weld so as to create a single large bag.

2.1 10X Concentrate

The two compartments contained the following :

Compartment 1

2.5 litres deionized water containing 262.75 g diethanolamine.

Compartment 2 2.5 litres deionized water containing 438.6 g NaCI and 1 00 ml of a

36.8% HCI solution.

On mixing, the contents form a 10X concentrate solution that can be diluted with deionized water for use to form a working solution with a pH of 9.00 ± 0.05.

2.2 20X Concentrate

The two compartments contained the following :

Compartment 1

2.5 litres deionized water containing 525.5 g diethanolamine. Compartment 2

2.5 litres deionized water containing 876.6 g NaCI and 200 ml of a 36.8% HCI solution.

On mixing, the contents form a 20X concentrate solution that can be diluted with deionized water for use to form a working solution with a pH of 9.00 ± 0.05.

Apparatus: A device which includes a plasma separation supply, a haemodialysis supply and haemodiafiltration supply The device is build like a haemodiafiltration device which comprises 3 blood pumps and a double dialysis fluid supply The device has been constructed with the components of the Nikkiso machine (Model DBB-03, Nikkiso Tokyo) according to the instructions and permission delivered by Nikkiso Medical GmbH (Hamburg Germany) A plasma filter (Asahi Plasmaflow AP-05H), is in fluid connection to the first pump of the device using blood tubing The inlet of the plasma filter is in fluid connection with the first pump to the patient by a tubing set The outlet of the blood side the plasma filter is in fluid connection to a mixing chamber

The outlet of the filtrated plasma is in fluid connection with a tubing set with the second pump to the inlet of the membrane inner side of the first dialyser (Nikkiso FLX-15GWS) The hollow fibre dialyser has two compartments the inner compartment with two connections (inlet and outlet) and the outlet compartment with two connections (inlet and outlet) and the outlet The dialysate compartment is connected to the supply of the crystalloid desorption dialysate, which is adapted to circulate the dialysate through the exterior surface of the first dialyser

The outlet of the membrane inner side of the first dialyser is in fluid connection to the inlet of the membrane outer side of a second dialyser (Nikkiso FLX-18GWS) Whereas the outlet of the membrane outer side of the membrane is in fluid connection with the third pump to the mixing chamber to mix the concentrated blood with the processed cell free plasma before entering the patient The dialysate compartment is connected to the supply of the bicarbonate dialysate, which is adapted to circulate the dialysate through the exterior surface of the first dialyser Experimental procedure

The purpose of this experiment was to evaluate the ability of the method described in the example to eliminate protein bound toxins in vitro

A volume of 3 liter of fresh human plasma which was obtained by pooling exchanges plasma from acute liver patients from the Medical School Hannover (Prof R Brunkhorst) has been proceeded with the method described in the example The plasma reservoir was mounted in a closed fashion in the device described in the example except that the plasma filtration was not used The plasma flow was regulated by 10 ml/mm, 25 ml/mm and 50 ml/mm, the total ultrafiltration rate was 0 ml/mm and the dialysate flow for both dialysate was respectively 25 ml/mm, 72 5 ml/mm and 125 ml/mm

After starting the in vitro plasma treating procedure, plasma samples were taken at the inlet of the first dialyser and at the outlet of the second dialyser The duration of test was 300 minutes The protein bound toxins were measured by high performance chromatography using a C18 column (Waters) The extraction of the protein bound toxins were first extracted by acidic precipitation using tπfluoroacedic acid (TFA) A volume of 20 μl of the supernatant which represents the protein toxins contained in 50 ml plasma was loaded in the column using 0 1 % TFA in water Finally, the toxins were eluded from the column using as eludaπt, isopropanol- diethaπolamine-EDTA Concentrations of the substance were measured by optical detection using a standard for each substance The concentration values were further to determine the clearance using the following formula Clearance =( C , - C₀)x

Q_p c, C, is the concentration of the toxin at the inlet of the first dialyser

C₀ is the concentration at the outlet of the second dialyser Q_p is the plasma flow

Figure 6 shows the clearances of the different relevant toxins as a function of the plasma flow As it can be seem from the figure all toxins are significantly eliminated Increasing the plasma flow resulted in an increase of the clearances of the protein bound toxins, which suggests that there is no saturation in the elimination of protein bound toxins According to the results, the process eliminates more than 60% of the protein bound toxins after a single passage through the device Thus, by a second passage, the total protein bound toxins may be eliminated, so the washed proteins which enter the body have free binding sides forfurther toxins contained in the body

References

NIH Publication, Digestive Diseases Statistics, NIH Publication NO 95-3873, February 1995

American red cross Therapeutic hemopheresis A reference of diseases American red cross 1994 Thackrah CT, 1819 Cox London As a inquiry into the nature and properties of the blood, as existent in health and disease

Konstantme et al Artificial liver Artif organs 16 235-42, 1992 Jauregui HO, Gann KL Mammalian hepatocytes as foundation for treatment in human liver failure J Cell Biochem 1991 45 359-65

Nyberg SL, Remmel RP, Mann HJM, Peshwa MV, Hu WS, Cerra FB Primary hepatocytes outperform HepG2 cells as the source of biotransformation functions in a bioartificial liver Ann Surg 1994, 220(1 ) 59-67

Strange et Mitzner A carrier-mediated transport of toxins in a hybrid membrane Safety barrier between a patients blood and a bioartifical liver Int J Artif organs 1996, 19 677-691

Cerra Frank B et al US Patent No 1995000376095

Amiots and Coon Rapids US Patent No 1997000861503 Sheil and Wang , WO patent 9932171

Stephen, US patent NO 5919369

Matsmura , US patent NO 5078885

Shettiger and McRen , US patent No 5211850

Claims

CLAIMS:

1 A method for treating blood so as to remove protein-bound toxins (PBT) which comprises (a) separating plasma from the blood,

(b) subjecting separated plasma from step (a) to a first dialysis procedure in which separated plasma and a first dialysis medium are maintained on opposite sides of a first semi-permeable membrane,

(c) transferring separated plasma from step (b) to a second dialysis procedure wherein treated separated plasma and a second dialysis medium are maintained on opposite sides of a second semi-permeable membrane, and

(d) recovering separated plasma depleted in PBT from step (c), characterised in that (i) the first dialysis medium comprises a low molecular weight component that is capable of passing through the first semi-permeable membrane in step (b) into the separated plasma whereby interaction between said component(s) and PBT occurs resulting in toxin release, and (n) the composition of the second dialysis medium is such that transfer of said low molecular weight component(s) from the plasma to the second dialysis medium occurs

2 A method according to Claim 1 wherein the recovered plasma from step (d) is subjected to at least one further cycle of purification comprising

(e) subjecting separated plasma from step (d) to a third dialysis procedure in which separated plasma and a third dialysis medium are maintained on opposite sides of a third semi-permeable membrane,

(f) transferring separated plasma from step (e) to a fourth dialysis procedure wherein treated separated plasma and a fourth dialysis medium are maintained on opposite sides of a fourth semi-permeable membrane, and (g) recovering separated plasma depleted in PBT from step (f), characteπsed in that (i) the third dialysis medium comprises a low molecular weight component that is capable of passing through the third semi-permeable membrane in step (e) into the separated plasma whereby interaction between said component(s) and PBT occurs resulting in toxin release, and (u) the composition of the fourth dialysis medium is such that transfer of said low molecular weight component(s) from the plasma to the fourth dialysis medium occurs, and the first and third dialysis media optionally have different compositions

3 A method according to Claim 2 wherein the first and third dialyasis media contain different low molecular weight components

4 A method according to Claim 2 or Claim 3 wherein the first and third dialyasis media have different pHs

5 The method according to any preceding claim wherein the low molecular weight component has a molecular weight less than 5000, preferably less than 1000, most preferably less than 500

6 The method according to any preceding claim wherein the low molecular weight component has a molecular weight greater than 50

7 The method according to any preceding claim wherein the low molecular weight component is water-soluble

8 The method according to any preceding claim wherein the low molecular weight component is basic or amphoteπc

9 The method according to any preceding claim wherein the low molecular weight component has at least one amine functionality

10 The method according to any preceding claim wherein low molecular weight component additionally possesses one or more hydroxy groups l l The method according to any preceding claim wherein the low molecular weight component additionally possesses one or more -COOH groups

12 The method according to any preceding claim wherein the low molecular weight component is selected from the group consisting of ethanolamme diethanolamine, tnethanolamine, pyrrolidine, N-(2-hydroxyethyl)pyrrolιdιne N-(2- hydroxyethyl)pιperιdιne, phenanthrol e, ethyleπediaminetetraaceticacid (EDTA)and diethylenetπaminepentaacetic acid (DPTA)

13 The method according to Claim 12 wherein the low molecular weight component is selected from ethanolamme, diethanolamine, tnethanolamine, and ethylenediaminetetraacetic acid (EDTA)

14 The method according to Claim 12 wherein the low molecular weight component is diethanolamine

15 The method according to any preceding claim wherein the concentration of the low molecular weight component in the first and/or third dialysis medium is from 1 mM to 200 mM, preferably from 10 mM to 100 mM, most preferably from 40 mM to 60 mM

16 The method according to any preceding claim wherein the pH of the first and/or third dialysis medium is from 8 to 11 5

17 The method according to Claim 16 wherein the pH of the first and/or third dialysis medium is from 8 5 to 9 5

18 The method according to Claim 17 wherein the pH of the first and/or third dialysis medium is from 8 7 to 9 3

19 The method according to Claim 17 wherein the pH of the first and/or third dialysis medium is 9 0

20 The method according to any preceding claim wherein the pH of the second and/or fourth dialysis medium is from 6 9 to 7 4, preferably about pH 7 2

21 The method according to any preceding claim wherein the first and/or third dialysis medium additionally comprises an ionic salt, which provides a source of physiologically acceptable cations and anions, preferably cations are selected from sodium, potassium, calcium, magnesium and ammonium and anions are selected from chloride, bicarbonate and phosphate

22 The method according to Claims 21 wherein the ionic salt is sodium chloride

23 The method according to Claim 21 or Claim 22 wherein the concentration of the ionic salt is between 1 mM and 200 mM, preferably between 50 mM and 150 mM and most preferably 80 mM and 120 mM

24 The method according to any preceding claim wherein the second and/or fourth dialysis medium comprises a source of physiologically acceptable cations and anions, preferably cations selected from sodium, potassium, magnesium, calcium and ammonium and anions selected from chloride, bicarbonate and phosphate

25 The method according to Claim 24 wherein the concentration of the ions in the second and/orfourth dialysis medium is from 0 01 mM to 20 mM, preferably from 0 01 mM to 5 mM

26 The method according to Claim 24 or Claim 25 wherein the second dialysis medium additionally comprises glucose

27 The method according to any preceding claim wherein the low molecular weight component(s) in the first dialysis medium remove(s) the toxin in the PBT from the protein by binding to the toxin

SUBS! M UTE SHEET (RULE 26)

28 The method according to any of Claims 1 to 26 wherein the low molecular weight component(s) in the first dialysis medium ιnduce(s) a conformational change in the protein of the PBT which causes the toxin to be released which conformation change induced is preferably reversible.

29 A method according to any preceding claim wherein released toxin passes from the plasma to at least one of the first and second dialysis media in steps (b) and/or (c)

30 A method according to any preceding claim wherein released toxin passes from the plasma to at least one of the third and fourth dialysis media in steps (e) and/or (f)

31. A method for treating blood so as to remove protein-bound toxins (PBT) which comprises

(a) separating plasma from the blood,

(b) subjecting separated plasma from step (a) to a first dialysis procedure in which separated plasma and a first dialysis medium are maintained on opposite sides of a first semi-permeable membrane, (c) transferring separated plasma from step (b) to a second dialysis procedure wherein treated separated plasma and a second dialysis medium are maintained on opposite sides of a second semi-permeable membrane, and

(d) recovering separated plasma depleted in PBT from step (c), characterised in that (i) the first dialysis medium comprises a low molecular weight component that is capable of passing through the first semi-permeable membrane in step (b) into the separated plasma whereby interaction between said component(s) and PBT occurs resulting in toxin release, (n) the composition of the second dialysis medium is such that transfer of said low molecular weight component(s) from the plasma to the second dialysis medium occurs, (in) released toxin passes from the plasma to at least one of the first and second dialysis media in steps (b) and/or (c), and, if necessary (iv) the ionic balance of the plasma is restored to a physiologically acceptable level in step (c)

32 A method according to any preceding claim wherein released toxin binds to at least one of the first and second semipermeable membranes in steps (b) and (c)

33 A method according to any preceding claim wherein released toxin binds to at least one of the third and fourth semipermeable membranes in steps (e) and (f)

34 The method according to any preceding claim wherein at least one of the first and/or third dialysis media is circulated between a device in which it is contacted with the respective first or third semi-permeable membranes and one or more regenerative devices

35 The method according to any preceding claim wherein at least one of the second and/or fourth dialysis media is circulated between a device in which it is contacted with the respective second or fourth semi-permeable membranes and one or more regenerative devices

36 The method according to any preceding claim wherein the PBT is bound to albumin

37 The method according to any preceding claim wherein said semipermeable membranes permit passage of dissolved components with molecular weights below about 60,000 Daltons, and impede passage of dissolved components with molecular weights above about 60,000 Daltons

38 The method according to any preceding claim wherein at least one of the semi-permeable membranes are synthetic asymmetrical semi-permeable membranes

39. The method according to Claim 40 wherein the pores of the semi-permeable membranes are narrower on one side of the membrane.

40. The method according to Claim 37 or Claim 38 wherein the treated separated plasma in steps (c) and/or (f) contacts the side of the asymmetric semi-permeable membrane having the larger surface area.

41. The method of any preceding claim wherein the PBT is selected from paracetamol, phenolic compounds, mercaptans, benzodiazepines, ammonia, hippuπc acid, polyamines, and chloramine.

42. The method according to any preceding claim wherein the plasma is additionally subjected to haemoperfusion over charcoal.

43. The method according to any preceding claim wherein the separation of plasma from blood is effected by filtration or centrifugation.

44. Apparatus for carrying out the procedure of any of Claims 1 to 43 comprising: (i) conduit means for receiving blood withdrawn from a patient (ii) means for separating plasma from blood delivered via conduit means (i) and for recovering separated plasma and cellular blood components (iii) a first dialysis device for dialysing plasma separated in means

(ii) against a first dialysis medium (iv) a second dialysis device for subjecting plasma from the first dialysis device to dialysis against a second dialysis medium, (v) conduit means for transferring plasma from dialysis device (iii) to the second dialysis device (vi) means for recombining plasma from the second dialysis device (iv) with cellular blood compounds obtained from separation means (ii) and (VII) means for receiving recombined plasma and cellular blood components for re-infusion into the patient

45 Apparatus for carrying out the procedure of any of Claims 1 to 43 comprising (i) conduit means for receiving blood withdrawn from a patient

(II) means for separating plasma from blood delivered via conduit means (i) and for recovering separated plasma and cellular blood components ιιι(a) a first dialysis device for dialysing plasma separated in means (II) against a first dialysis medium ιv(a) a second dialysis device for subjecting plasma from the first dialysis device to dialysis against a second dialysis medium v(a) conduit means for transferring plasma from dialysis device ιιι(a) to the second dialysis device ιιι(b) a third dialysis device for dialysing plasma from the second dialysis device ιv(a) ιv(b) a fourth dialysis device for subjecting plasma from the third dialysis device to dialysis against a fourth dialysis medium v(b) conduit means for transferring plasma from dialysis device ιιι(b) to the fourth dialysis device

(vi) means for recombiniπg plasma from the fourth dialysis device

(iv) with cellular blood compounds obtained from separation means (n) and (vii) means for receiving recombined plasma and cellular blood components for re-infusion into the patient

46 Apparatus according to Claim 44 or Claim 45 including one or more reservoirs for dialysis media as defined in any of Claims 3 to 36

47 Apparatus accordmg to Claim 46 wherein said reservoirs are charged with of said dialysis media 48 Apparatus according to any of Claims 44 to 47 including a regeneration device for the first and/or third dialysis media and means for circulating said first and/or third dialysis media between the first and third dialysis devices respectively and the respective regeneration devices

49 Apparatus according to any of Claims 44 to 47 including a regeneration device for the second and/or fourth dialysis media and means for circulating said second and/or fourth dialysis media between the second and fourth dialysis devices respectively and the respective regeneration devices

50 A dialysis fluid comprising water, at least one ionic salt and a low molecular weight, water-soluble nitrogen-containing detoxicant having the formula

R^π

/

HO— (CH₂)_n-

\

R³ R⁵

\ /

N X— -N

/ \

R⁴ R⁶

wherein either R¹ and R² are independently selected from hydrogen or lower alkyl groups, and said lower alkyl groups are optionally substituted by one or more of hydroxy, -COOH, -COOR, nitro or ammo, wherein R represents C^ alkyl, or R¹ and R² together form a ring having from four to seven carbon atoms, said ring being optionally substituted by one or more of hydroxy, -COOH, -COOR, nitro or ammo, and R represents C_1-4 alkyl, wherein R³, R⁴, R⁵ and R⁶ are independently selected from hydrogen, -CH₂COOH, -CH₂COOR, -CH₂COO^"Na⁺, or -CH₂CH₂NH₂, and R represents

C ₄ alkyl, X represents a covalent bond, or a group -(CH₂)_p-Y-(CH₂)_q-, wherein Y represents a covalent bond or N-R⁷, and R⁷ is selected from hydrogen, -CH₂COOH, -CH₂COOR, -CH₂COO^"Na⁺, and R represents C_{1 4} alkyl, and wherein n, p and q are integers from 1 to 6

51 A dialysis fluid according to Claim 50 comprising components, concentrations and/or pHs as defined in any of Claims 3 to 36

52 A concentrate composition comprising components in aqueous solution suitable for reconstitution to form a dialysis fluid as defined in Claim 50 or 51

53 A concentrate composition according to Claim 52 which contains the low molecular weight component and which has a pH in the range of 9 5 to 1 1

54 A two component pack comprising separate aqueous solutions, one of which comprises a concentrate composition according to Claim 52 and the other of which comprises an aqueous diluent composition which, when mixed with said concentrate, will form a dialysis fluid as claimed in any of Claims 3 to 36

55 A two component pack according to Claim 53 wherein said aqueous diluent composition has a pH less than 7, whereby when the aqueous diluent composition and the concentrate composition are mixed, the pH of the resulting dialysis medium is as defined in any one of Claims 16 to 19