DE2725608A1 - METHOD AND DEVICE FOR CONTINUOUS REMOVAL OF BLOOD SUBSTANCES IN AN EXTRACORPORAL CIRCUIT - Google Patents

Description

Claimed Priority: June 22nd, 1997, Japan,

No. 72 827/1076

Applicant: MITSUI TOATSU CHEMICALS, INCORPORATED

No. 2-5 Kasumigaseki 3-chome, Chiyoda-ku, Tokyo, Japan

Method and device for the continuous removal of blood substances in an extracorporeal

Cycle.

The present invention relates, inter alia, to a device for the continuous removal of special substances in Blood. In particular, the present invention relates to a device having an inlet line, a filter, an adsorber and includes an outlet conduit, and the device is for designed to selectively remove specific substances in blood that is carried from the body through the apparatus and back to the blood Body circulates.

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In recent years, the noticeable progress in immunology has brought detailed information about various self-antibodies in self-immunizable diseases. For example, it has been thought that anti-nuclear factor (ANF) _1, especially anti-DNA antibodies, play etiological roles in patients with systemic lupus erythematosus (SLE) and rheumatoid factor (RF) in patients with rheumatoid arthritis (RA).

Dr. DS Terman et al. (USA) reported the selective removal of anti-DNA antibodies by immunological adsorbents in "Clinical and Experimental Immunology" (1976) 24 , 231-237. In their investigations, they used DNA-CeHulose, which was embedded in agar as an adsorbent. The blood prepared for experiments in vitro was serum from SLE patients and that for experiments in vivo was whole rabbit blood immunized with DNA and methylated bovine serum albumin mixed with Freund's complete adjuvant. The blood was passed through a column with a feed pump and sampled at constant intervals to analyze the anti-DNA antibodies.

The researchers concluded that the DNA CeHulose agar adsorbent used was effective at selectively circulating Remove anti-DNA antibodies in both the in vitro and in vivo experiments, and that is expected showed that the adsorbent is an effective therapeutic agent in clinical medicine. It was also found that the experimental duration of the investigations was rather short and that further investigations with a view to achieving it better results should be continued.

Under these circumstances provides the creation of a new apparatus by means of which continuously specific factors of blood can be removed, as proposed in the present invention, an essential contribution to the treatment of the respective diseases and to the elimination

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the causes of such diseases. It is assumed, that no effective compact apparatus has yet been developed that can use plasma in large quantities in vivo can be continuously separated and treated with a wide range of applications. Artificial kidneys made according to the whole blood process work can cause undesirable influx of adsorbents into the body and destruction of blood cells entail. If a suitable coating is applied to the adsorber to avoid such difficulties, the effectiveness is slightly reduced.

The present invention is the result of extensive studies made in order to develop satisfactory apparatus. It has now been found that, for example, the removal of specific factors from blood can be achieved with markedly increased effectiveness by providing a filter in front of an adsorber in an apparatus in which the plasma from the blood circulating externally from the body is passed through the filter is separated from the blood cells including red blood cells, white blood cells and platelets, and treated in the adsorber with an adsorbent comprising insolubilized substances such as enzymes, antigens and antibodies which adsorb the specific factors from the plasma.

The invention provides an apparatus adapted for use in continuous, exogenous exposure to blood circulating from the body to the apparatus and back to the body, and which apparatus comprises (a) means for continuously separating plasma from Blood cells, which include red blood cells, white blood cells and platelets; (b) means for removing specific factors from the separated plasma; (c) means for back-mixing the separated blood cells with the plasma from which the specific factors have been removed; (d) an inlet for introducing the blood into the partition; and (e) an outlet for returning the remixed blood to the body.

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The means for removing specific factors from plasma contain preferably an adsorber.

By properly selecting the insolubilized substances to be packed in the adsorber, the device can not just for removing anti-DNA antibodies from patients with systemic lupus erythematosus by insolubilization of DNA but also for purposes such as removing rheumatic factors from rheumatoid arthritis patients by insolubilizing accumulated, aggregated ^ - globulin, the removal of anti-insulin antibodies from patients with diabetes mellitus by making insulin insoluble, for the removal of reagents from allergic patients by insolubilization of allergy factors and for the removal of Self-antibodies can be used by patients with other self-immunizable diseases. Furthermore, the apparatus can also be used for plasma dialysis as an artificial kidney.

The invention also creates an artificial kidney, comprising an apparatus according to the present invention.

The device can also be used to collect human immunoglobulin in large amounts of blood for clinical use, such as human anti-D immunoglobulins given to Rh (-) women when they have their second child, or human Anti-tetanus immunoglobulin; In addition, the apparatus can also be used for collecting pharmacologically useful substances from non-human animal blood.

The invention also provides a method for the production of pharmacologically useful substances from blood which circulates from the body outside the body and back to the body, comprising the following method steps: (a) outside the body separating the plasma from blood cells which

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include red blood cells, white blood cells, and platelets; (b) obtaining the pharmacologically acceptable substances from the separated plasma; (c) Subsequent mixing back of the plasma, of which the pharmacologically useful ones Substances have been obtained with the separated blood cells and (d) returning the remixed blood to the body.

There now follows a description of an apparatus embodying an embodiment with reference to the accompanying drawings of the invention. This description, in which the principles of the method according to the invention are also described, however, it is only to be regarded as an example and is not intended to limit the invention in any way.

In the attached drawings show:

Figure 1 is a schematic view of an apparatus which is an embodiment of the invention;

FIG. 2 is a graph depicting a radio allergic adsorbent test, and FIG

Figure 3 is a graph showing dialysis results.

The device according to the invention is used for the continuous removal of specific substances from circulating blood. The device includes an inlet line that directs blood from the body to the device, a filter that communicates therewith Inlet conduit and capable of continuously separating plasma from blood cells, the blood cells including red blood cells, white blood cells and platelets, a Outlet line connected to an outlet for the blood cells in the filter and returning blood to the body can, an adsorber containing insolubilized substances such as enzymes, antigens and antibodies to adsorb specific factors in the plasma and to connect to the outlet for the Plasma in the filter is connected by a conduit, and a conduit that carries the plasma from the adsorber into the output

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line returns. To fully exploit the effect of the device In order to take advantage of this, various auxiliary devices can be developed and used. In some cases it is desirable to use such devices. Such bodies are e.g. a heparin injector to prevent the blood from coagulating, a blood pump with the plasma at a constant Flow rate can be allowed to flow, a thermostat, to keep the filter and the adsorber at a constant temperature, means for removing bubbles and a Monitoring device or a monitoring unit for checking whether or not the device works without interference.

The function of the device according to the invention is now further developed described in detail with reference to FIG.

In Figure 1, blood is drawn from the \ rtery of the body is conducted at a constant flow rate by means of a blood pump 2 through an inlet line 1 to a plasma filter 3. The filter, which has a filter membrane 3-1, a channel 3-2 and a membrane support 3-3, separates plasma from blood cells, and the latter are connected through an outlet line 7 to circulated back to the body. While the separated in the filter plasma to the adsorber 5 through a line is passed, heparin is added through a heparin injector 4 to prevent coagulation. The plasma from which in the adsorber 5 specific factors have been removed, is pumped through a filter pump 6, mixed with the blood cells and circulated back through the outlet line 7 to the body.

As the inlet pipe 1, the pipe for conveying the plasma and the outlet pipe 7, silicone pipes or -Hoses and pipes or hoses made of soft polyvinyl chloride are suitable. A small roller pump is suitable as the blood pump 2. As the filter 3, there are a Kiil type filter and a Hollow fiber type filter useful as it is used for hemodialysis and which is provided with a membrane that has one

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2 effective area of less than 1 m, what of the desired purpose. It is assumed that a filter membrane with a pore size between 0.15 μm and 0.8 μm is optimal for separating the blood cells and the plasma. The membranes can be those made of cellulose acetate, poly carbonate, polyvinyl chloride, epoxy resin, glass fibers, polyamide, regenerated cellulose, cellulose nitrate, Teflon, nylon, cellulose or the like. The membranes are well sterilized before practical use.

The adsorber 5 is generally a column packed with an insolubilized substance. The insoluble support must be resistant to serum enzymes, nonspecific adsorption allowed to own and must be stable at a temperature between 0 ⁰ C and 40 ° C. Preferred examples of the insolubilized carriers or fillers include crosslinked dextran, crosslinked polyacrylamide, polyaminopolystyrene, cellulose, cellulose derivatives, agarose and polymerized proteins. These insoluble carriers are usually modified according to the specific circulating factor that is to be removed. For example, DNA Sepharose, made by covalently attaching DNA to Sepharose 4B (pharmaceutical; agarose), is a suitable insolubilized substance for specifically binding anti-DNA antibodies.

The filter pump 6, which is of the same type as the blood pump 2, facilitates filtering by having one Exerts negative pressure on the filter 3.

A result of the animal experiments is described below, performed using the present apparatus.

Example 1 - Adsorption of Anti-Egg Albumin Antibody In Vivo.

A male rabbit weighing 3.3 kg was used in this experiment. The rabbit was through intradermal injection of 8 mg egg albumin mixed with

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Freund's complete adjuvant, immunized in the back. After 7 weeks, a flow tube and a recirculation cannula was inserted into the common carotid arteries at a distance of 5 mm, which were then connected to the device shown in FIG. 1 in order to circulate the blood of the to circulate the living animal through the apparatus. the

The filter membrane in the filter was a cellulose membrane with a

ο
effective area of 43 cm and pores with 0.2 µm diameter.

The adsorber was a column packed with 13 ml of Elalbumin-Sepharose 4B. The pumps were controlled so that the Flow rate of the total blood to 7 ml / min, and that of the Plasma at 1.6 ml / min. After The egg albumin-Sepharose 4B column was exchanged for 90 minutes and treatment was continued for an additional 90 minutes before all flushed blood was returned to the body became. During this treatment of plasma, 10000 units / kg of heparin was added to prevent coagulation. the Egg albumin Sepharose 4B on which the antibodies had been adsorbed was washed with 100 ml of a phosphate buffer containing was kept at pH 7.5. The adsorbed antibodies were then separated with 20 ml of a 0.1 M alanine buffer which was kept at pH 2.3 was held. The separated antibodies were neutralized with a 1.0 M alanine buffer maintained at pH 10.0, dialyzed against distilled water for 3 days and lyophilized (freeze-dried) to obtain 480 mg of a powder. A result of electrophoresis showed that the powder consists mainly of LT-globulin with a small Amount of oC -globulin impurity was composed. The amount of the antibodies in the powder became 450 mg by one Radioallergosorbent test (RAST) determined using the purified anti-egg albumin antibody as a standard.

0.1 ml aliquots of 5.1 and 0.2 pg / ml solutions of this antibody were administered intradermally to guinea pigs. After 4 hours, the guinea pigs were given 0.5 ml of 10% Evan's blue and 0.5 ml of 1% egg albumin are injected intravenously. After 30 minutes, the result of the PCA reaction was determined

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measure, which is given in Table 1.

Table 1

Concentration of the antibody obtained from egg albumin-Sepharose 4B (pg / ml)

5 1 0.2

Diameter of 13.8 7.9 2.5 Reaction plate

The antigen levels in the blood measured by the radioallergosorbent test are shown in FIG. As a result, was the amount of antibody removed from the plasma with the present device is calculated to be 3.3 40 χ (4.0-0.9) - (mg), provided that the amount of plasma is 40 ml / kg. The calculated value is almost equal to 450 mg, which is the amount of the antibody adsorbed on egg albumin Sepharose 4B in the present device.

Example 2 - Removal of Urea and Creatinine by Dialysis.

One hundredth of a milliliter of rabbit blood, that is 2OOO units Containing heparin to prevent coagulation was used for this test.

20 mg of urea and G mg of creatinine were added to the blood. The blood was then treated according to the same procedure, as described in Example 1, allowed to flow. The adsorber was made by a hollow fiber type dialyzer manufactured by Dow (surface area: 1,000 cm; nominal molecular weight cut off): 50,000; Model number: b / HFD-1) replaced. The blood was collected using a Ringer's solution as an external dialysis solution

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rinsed solution. The pumps were controlled so that the flow rate for the whole blood of 5 ml / min, for the plasma of 1.6 ml / min and for the external dialysis solution of 5 ml / min was discontinued.

The changes in the amounts of total protein, albumin, creatinine and urea are shown in FIG. urea and creatinine in the blood were quickly removed. In the figure, A stands for the entire protein, B for \ lbumin, C for creatinine and D for urea.

Example 3 - Effects on Blood Count and Blood Biochemistry.

A male dog weighing 14 kg was used for this experiment. In the femoral artery (Arteria femoralis) was a perfusion cannula and in the femoral vein (Vena femoralis) was a circulation cannula introduced, both of which were connected to the device shown in Figure 1, to circulate blood in vivo through the device allow. A filter membrane used in the filter was a cellulose membrane with an effective area of 300 cm and a pore diameter of 0.65 pm. The blood was from the femoral artery at a flow rate of 150 ml / min under a pressure of 50 mm Hg. The plasma separated from the filter membrane itself was with such pumped through at a flow rate that the plasma was separated in the filter (5 ml / min). The plasma was mixed with the blood cells near the outlet from the filter and circulated back into the body through the femoral vein calmly. After a 90 minute flush, the effects became apparent examined for blood count and blood biochemistry. The change in blood count is in Table 2 and that in blood biochemistry given in Table 3. The treatment had little effect on the two properties. It became that too Electrocardiogram examined without being influenced by treatment were found.

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Table 2 Haematological examinations

After circulation (h) before O 2 24

Ht (%) 40 , 4 39 , 5 39 , 0 39 , 7 Hb (g'dl) 14th , 3 13th .6 13th , 3 13th ,8th Red blood cells
(10 ⁴ xmm ³ ) 624 GOl 596 586 White blood cells
(1θ2χ mm ³ ) 96 87 138 158

Platelets (10 ⁴ x mm ³ )

38

Table 3 Blood chemistry results T.P. Albumin total urinary crea urinary

(g / dl) (g / dl) cholesterol tinine acid

sterol -N (mg / dl) (mg-'dl)

(mg / dl) (mg-'dl)

before 7, 3 3 . '* 268 26th , 5 O , 5O 0 , 40 later 6, 9 3 ,1 27Ο 27 , 0 0 , 48 0 , 35

ü ^:; λ .-, ι / π 7 η f;

Claims (11)

Claims Apparatus for use in continuous, exogenous exposure to blood flowing from the body to the apparatus and back to the body, having an inlet for the entry of the blood and an outlet for the return of the blood to the body, characterized by a filter (3) which can continuously separate plasma from blood cells, which include red blood cells, white blood cells and platelets, an inlet line (1) leading to the filter (3) leads, and means (5) for removing specific factors from the separated plasma as well as by that the separated blood cells with the plasma from which the specific factors have been removed are mixed and the remixed blood is returned to the body through the outlet line (7) will. 2. Device according to claim 1, characterized in that the means for removing specific factors from the separated plasma comprise an adsorber (5). 3. Apparatus according to claim 2, characterized in that the adsorber (5) contains insoluble substances which the specific Adsorb factors from the plasma. 4. Apparatus according to claim 2 or 3, since d u r c h marked that the adsorber is insoluble 7098R1 / 0706 ORfQiNAL INSPECTED - ² - 2775608 contains borrowed enzymes, antigens or antibodies. 5. The device according to claim 1, characterized in that the means for removing specific factors from the separated plasma include a dialyzer. 6. Device according to one of claims 1 to 5, characterized in that the filter has a filter membrane (3-1) with a pore size between 0.15 μπι and 0.8 μπι includes. 7. Device according to one of claims 1 to 6, characterized in that the Fi L- ter a filter membrane (3-1) with an effective area ο which is smaller than 1 m ". 8. Device according to one of claims 1 to 7, characterized in that it contains a heparin injector (4). 9. Process for the preparation of pharmacologically acceptable substances from blood drawn from the body externally and to circulates back to the body, being external to the body Plasma is separated from blood cells, which include red blood cells, white blood cells and platelets, the pharmacologically acceptable substances of the separated plasma are removed, then the plasma from which the pharmacologically useful substances have been removed are mixed again with the separated blood cells and the remixed blood is added is returned to the body. 10. The method according to claim 9, characterized in that the method step - ³ - 2775608 removal by adsorption from the plasma is performed. 11. The method according to claim 10, characterized in that the adsorption by
insolubilized substances is carried out , the pharmacologically useful substances of the
Adsorb plasma . 709BR1 / 07nß