JP2001245973A - Blood purifying adsorbent formed of titanium oxide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an excellently plasma adaptable titanium oxide used for efficiently adsorbing and eliminating bilirubin in plasma. SOLUTION: When this titanium oxide for adsorbing bilirubin in plasma is brought into contact with human fresh plasma, a partial thromboplastin time PTT, a prothrombin time PT, and a fibrin deposition amount Fib serving as the plasma coagulation factors are substantially equal to those of intact human fresh plasma. The titanium oxide for adsorbing bilirubin in plasma is provided with physical characteristics such as a specific surface of 20-80 m2/g, a mean pore diameter of 150-200 Å, and a pH of 6-7.5 and selectively adsorbs total bilirubin in plasma when it is brought into direct contact with the plasma.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to the present invention, particularly to hyperbilirubinemia (hyperbilirubinemia) associated with liver diseases and the like.
The present invention relates to titanium oxide having excellent blood compatibility, which is used for efficiently adsorbing and removing bilirubin in plasma after separating the constituents from blood of a patient with inemia. Here, “excellent in blood compatibility” means that the coagulation characteristics upon contact with fresh human plasma are substantially the same as those without contact (blank). More specifically, partial thromboplastin time (second) (PTT) and proton bin time (second), which are pseudo-solid factors of plasma when brought into contact with human fresh plasma
(PT), the amount of deposited fibrin (Fib), etc., do not substantially differ from the value of the blank (uncontacted plasma).

[0002]

2. Description of the Related Art Erythrocytes aged in vivo are destroyed by reticulocytes in the liver and spleen, and hemoglobin in blood cells is changed to free bilirubin (hemobilirubin). This free bilirubin is fat-soluble, and as it increases in the blood,
It deposits while dissolving in lipids in the nuclei of brain cells and exerts a neurotoxin to cause nuclear jaundice. In humans, to detoxify this free bilirubin, albumin and some α2-
Globulin binds and is carried to the liver by blood. Then, free albumin is converted from uridine diphosphate glucuronic acid (UDPGA) to glucuronic acid (G) by the action of glucuronic acid convertase, one of the liver enzymes in the liver.
A), it undergoes glucuronidation by bilirubin UDP-glucuronyltransferase contained in the smooth endoplasmic reticulum undergoing microsome division in liver cells, and becomes bilirubin glucuronide (conjugated bilirubin: direct bilirubin) and becomes water-soluble (detoxification) ). Generally, two molecules of glucuronic acid are bonded to one molecule of bilirubin to form bilirubin diglucuronide (conjugated bilirubin: direct bilirubin).
This is further excreted from hepatocytes into the bile duct and duodenal tract, and the total bilirubin concentration in the blood of healthy subjects (sum of indirect bilirubin and direct bilirubin) is almost constant even though it fluctuates due to diet, muscle work, etc. Kept in range.

[0003] However, if the excretion into the duodenal tract is inhibited due to hepatic parenchyma or bile duct obstruction, the total bilirubin concentration in the blood rises, causing jaundice and hyperbilirubinemia, resulting in impaired consciousness. It is not unusual to have somnolence and even coma and renal impairment progressing from oliguria to anuria.

Therefore, as an initial blood purification method for such a symptom, a direct blood adsorption method using activated carbon (Direct Blood Adsorption Method)
Hemoperfusion, hereinafter, may be expressed as DHP. ) Was performed, but free bilirubin (C ₃₃ H ₃₆ N ₄
O ₆ , molecular weight: 564: fat-soluble) was removed, but the removal of conjugated bilirubin (direct bilirubin) was insufficient, and the therapeutic effect could not be sufficiently improved. In the direct blood adsorption method, there was only a proposal to use activated carbon as an adsorbent.

[0005] Thereafter, a method of easily separating blood into solid matter and plasma was established by a blood centrifugation method (Hemonetic) and a plasma separation method using a membrane, for example, a hollow membrane. As a result, even if the platelets are irritating (procoagulant action), bilirubin can be efficiently removed from the separated plasma, and the adsorbent does not significantly affect the coagulation-fibrinolysis system of the plasma. The search for is continuing. In such a situation, based on the concept that anionic adsorbents would be effective for two carboxylic acids in the molecule of free bilirubin, styrene / divinylbenzene copolymers now have amino groups as active sites. Anionic ion exchange resins having (NH ₂ ), quaternary ammonium groups (N ^+. (CH ₃ ) ₃ .Cl ⁻ ) and the like have been clinically used. However, such ion exchange resins are also poorly compatible with plasma,
In order to improve the above problem, a method has been adopted in which the above-mentioned inconvenience is improved by coating with a blood-compatible polymer such as polyhydroxyethyl methacrylate (PHEMA) (JP-A-63-275351, Shuhei Nakaji et al.). ). However, the treatment causes a disadvantage of reducing the pore volume and the surface area of the ion exchange resin which contribute to the removal of bilirubin. Further, the ion exchange resin in water does not have sufficient mechanical strength, and may be disadvantageously crushed during use. Furthermore, it cannot be used for sterilization, which is an essential condition for clinical use, except for high-pressure steam sterilization.
Moreover, the resistance to the sterilization conditions (130 ° C., 10 minutes) is not sufficient.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide an adsorbent which overcomes the above-mentioned drawbacks of the ion exchange resin and has improved properties even in the treatment by removing bilirubin using the activated carbon. It is in. In examining the adsorbability of bilirubin for various materials in order to solve the above-mentioned problems, unexpectedly, titanium oxide, which is not so well known as an adsorbent, is subjected to a specific calcination treatment, whereby bilirubin, especially Having high adsorption performance for conjugated bilirubin and finding compatibility with plasma,
This has solved the above-mentioned problem. Titanium oxide gradually changes its crystal form from brookite type and anatase type to rutile type at 650 ° C. and 9155 ± 15 ° C. by baking, and at the same time, changes from acidic to neutral and further to basic. It is generally known that the surface area also decreases (Satoru Kiyono, "Titanium Oxide-Physical Properties and Applied Technology", published by Gihodo Publishing, May 10, 1997, 1st edition, 3rd printing, 53)
Page, see). Also, regarding the safety of titanium oxide as a medical material, it has already been used as a bone substitute material in the field of orthopedic surgery, and its mechanical strength and safety for living tissues have been confirmed. Therefore, the present inventors have studied to find the optimum calcination temperature for obtaining titanium oxide having high adsorption performance not only for free bilirubin but also for conjugated bilirubin, and have found that titanium oxide meeting the above-mentioned problem can be synthesized.

[0007]

DISCLOSURE OF THE INVENTION The present invention provides human fresh plasma.
Partial thrombo, a pseudo-solid factor of plasma when exposed to
Plastin time (PTT), proton bin time (P
T), the blood that does not come into contact with the amount of deposited fibrin (Fib)
No substantial difference from plasma, and specific surface area 20 to 80 m ^Two/
g, average pore diameter 150-200 °, pH value 6-7.
5. Direct contact with plasma characterized by having physical properties such as 5.
To selectively adsorb total bilirubin in the plasma
Is a titanium oxide for bilirubin adsorption. Preferably, warm
It is prepared by firing at a temperature range of 300 to 600 ° C.
Contacting the plasma directly, characterized in that the plasma
Bilirubin absorption in plasma to selectively adsorb total bilirubin
Wearing titanium oxide, more preferably firing temperature
The blood has a range of 400 to 550 ° C.
Plasma is brought into direct contact to selectively absorb total bilirubin in the plasma.
This is a titanium oxide for adsorbing bilirubin in plasma to be deposited.
In addition, the present invention provides a method for firing a titanium alkoxide compound during firing.
In water where volatile acidic or alkaline compounds are present
The precipitate obtained by stirring is filtered, dried and then
Temperature rate 2 ° C to 10 ° C / min, temperature range 300 to 600 ° C
After heating for 2 to 5 hours in the above temperature range,
The ratio of anatase type to rutile type obtained by cooling is 90 to
55:10 to 45.
Blood to selectively adsorb total bilirubin in the plasma
It is a titanium oxide for adsorbing bilirubin in plasma. Preferably
Is prepared by firing at a temperature in the range of 300 to 600 ° C.
Wherein said plasma is brought into direct contact with said blood
Bilirs in plasma selectively adsorb total bilirubin in plasma
Titanium oxide for bottle adsorption, and more preferably
Temperature range from 400 to 550 ° C.
The plasma is brought into direct contact to select total bilirubin in the plasma.
With titanium oxide for bilirubin adsorption in plasma
is there.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in more detail. A. Preparation method of titanium oxide of the present invention Preparation of raw titanium oxide. Here, a method for preparing titanium oxide, which is particularly preferable for adsorbing bilirubin in plasma, will be described. However, it goes without saying that the titanium oxide of the present invention is not limited to one obtained by such a preparation method.
A small amount of 0.2N monohydrochloric acid as a catalyst was added to 50 times the volume of distilled water, and tetraethyl orthotitanate (Tokyo Kasei Co., Ltd.) was stirred with a peristaltic pump (peristaltic pump). )
Mixed. This mixture was left and aged for 12 hours. The resulting precipitate was collected by filtration and placed in a 40 ° C constant temperature bath.
Let dry for days. The obtained powder was fractionated by sieving to obtain a sample having a particle size of 150 to 212 µm. 105, 245, 48 at a heating rate of 3 ° C./min using a muffle furnace.
After firing at 5, 550, and 705 ° C. and holding at that temperature for 3 hours, the product was naturally cooled. 2. As the catalyst, an alkaline substance can be used, and the catalyst is preferably one which is volatilized and removed from titanium oxide at an optimum firing temperature or lower. Specific examples of the catalyst include mineral acids such as nitric acid and sulfuric acid in addition to the above-mentioned hydrochloric acid, and ammonium hydroxide as a basic catalyst. The catalyst may be added in an amount of about 320 mL of a 0.1 N to 0.3 N aqueous solution of each acid or base per 100 g of orthotitanate. 3. The rate of temperature rise, firing time, etc., differ depending on the scale of the titanium oxide manufacturing equipment, as the heat transfer situation varies.
Basically, some contrivance is required to satisfy the characteristics of the titanium oxide. The heating rate during firing is in the range of 2 ° C./min to 10 ° C./min.
Preferably, the temperature is maintained at 400 to 550 ° C. for 2 to 5 hours.

B. Preparation of Albumin Containing Conjugated Bilirubin for Adsorption Test a. Reagent: Bilirubin (Wako Pure Chemical Industries, Ltd.), 0.12 g
Was dissolved in 50 mL of 0.1N-sodium hydroxide aqueous solution. b. Bovine serum albumin (Wako Pure Chemical Industries, Ltd., purity, 98-
99%) and 7.50 g were dissolved in 50 mL of distilled water. a. The solution and the solution b were mixed, and 50 mL of a 0.1 N aqueous hydrochloric acid solution was added to the mixed solution. A 5N aqueous solution of sodium hydroxide was added thereto to adjust the pH to 7.2, and 1.06 g of sodium chloride was added to obtain a total bilirubin standard solution.

C. Measurement of adsorption and removal ability of titanium oxide for bilirubin. Three kinds of titanium oxide powders (particle size: 213 to 30) sintered at 245 ° C. (1), 485 ° C. (2), and 705 ° C. (3)
0 μm) and as control samples, activated carbon for blood purification (4) [(Kuraray Co., Ltd., PHEMA coating-AC)] currently used clinically, anionic ion exchange resin for bilirubin adsorption (5) [Kuraray Co., Ltd.] , Active point: quaternary ammonium salt type, PHEMA coating] and its raw material, PHEMA uncoated resin (6), were used to measure the total bilirubin adsorption ability. 0.1 mL of each of the above-mentioned six types of adsorbents was collected in a disposable polyethylene test tube having a volume of 5.0 mL, and a physiological saline 1.0
mL, and degassed for 1 hour in a vacuum desiccator. The previously prepared total bilirubin standard solution, 3
mL was added, and the mixture was shaken in a constant temperature bath at 37 ° C. for a predetermined time (0.5, 1.3, 5 hours) in a dark place. After shaking, the samples were filtered through a disposable filter having a pore size of 0.45 μm, and the total bilirubin (T, Bi) in the solution was filtered.
l) and the free (direct) bilirubin (D.Bil) concentration were determined by the clinical test Kid [Wako Pure Chemical Industries, Ltd., bilirubin BII
-Test Wako]. Conjugated (indirect) bilirubin (I.Bil) concentration was calculated from the difference between total bilirubin concentration and free bilirubin concentration. For each sound absorbing agent, direct bilirubin (D.Bil) and indirect bilirubin (In. Bi)
l), the total bilirubin (T.Bil) sucker capacity [(ml) per unit volume capacity] is shown in Fig. 1-a, b and c.

As is clear from FIG. 1 (a, b, c),
The total bilirubin adsorption activity of these six adsorbents is
It was as follows. Titanium oxide (485 ° C)> PHEMA coating exchange resin ≒ Uncoated exchange resin> Titanium oxide (745 ° C) ≧ P
HEMA coated activated carbon> titanium oxide (245 ° C.)
At ℃, the pores occupying most of the pores, which had a diameter of 3 nm, disappeared by sintering, and at 485 ℃, the relatively large pores of 15 to 25 nm became the majority (FIG. 2). At the same time, the surface area also decreased, and the firing temperature: 245
What was 120 m ² / g at ℃, 485, 55
At 0 and 705 ° C., they were 25, 15, and 5 m ² / g, respectively (FIG. 3). Uncoated exchange resin of control sample, PHE
22, ²⁰ m ² for MA coating exchange resin respectively
/ G, which is almost the same surface area as titanium oxide fired at 485 ° C (Fig. 3). As shown in FIG. 1, direct bilirubin is a biomolecule having a molecular weight of 546 and is adsorbed on titanium oxide (245 ° C.) having a large surface area even if it is weakly acidic. The surface of titanium oxide (485 ° C.), which is larger in size than direct bilirubin and has a larger pore diameter, has a weakly acidic or weakly basic property (zwitterionic) on the surface, and this property functions to improve the suction performance. It is presumed that it is. The weight per unit volume (mL) is 1.30 g for titanium oxide.
However, in the case of an exchange resin, about 1/4 is 0.35 g, and at the same filling rate, the surface area of titanium oxide is 4 times that of titanium oxide.
Although the weight increases, the total amount of bilirubin removed by one adsorption column is more efficient when filled with titanium oxide for clinical use. In this case, a powdery titanium oxide of 213 to 300 μm was examined. However, actually, a method of processing titanium oxide into beads having a particle size of about 1 mm and filling the beads as they are, or a sheet in which activated carbon is conventionally fixed is used. For example, a sheet (preferably having a large surface area) made of cellulose, cellulose diacetate, cellulose triacetate, ethylene-vinyl alcohol copolymer (ethylene content: about 30 mol%), polysulfone, polyacrylonitrile, etc. Immobilize the beads, stack the sheets or roll the sheet into a coil and fill the housing (wound into a coil and pass through the plasma between layers (so as to contact the surface to which the beads are fixed) There is a method of using it in a state where it is left.

Hemocompatibility and Safety of Titanium Oxide Titanium oxide has been confirmed to exhibit high adsorption activity to bilirubin in plasma, but when it is used clinically, it has compatibility with plasma, In particular, for use in blood purification therapy by extracorporeal circulation, it is an essential requirement that the anticoagulant property is sufficient. Thus, when the above six samples were brought into contact with human fresh plasma (mixed plasma of 10 healthy subjects),
The coagulation parameters (PTT, PT, Fib) of the plasma were measured (manufactured by Ortho Diagnostics, Inc.-KoaguL).
abo.MJ. use). Table 1 shows the coagulation parameters (PTT, PT, Fib) of the plasma before and after contact with the adsorbent.

[0013]

[Table 1]

As is clear from Table 1, when the firing temperature of the titanium oxide was 105 ° C., only a measurement result regarded as a slightly abnormal value was obtained. The reason is considered to be the effect of residual hydrochloric acid added as a contact butterfly when synthesizing titanium oxide from tetraethyl orthotitanate. When calcined at a higher temperature, the value of the coagulation parameter of any of the titanium oxide samples was not much different from that of the plasma-only negative blank, confirming that titanium oxide had excellent plasma compatibility. From this measurement result, if the correlation between the surface area of titanium oxide, the pore size, the weak basicity of the surface, and the blood compatibility is considered, the optimum firing temperature range is in the range of 400 to 550 ° C. FIG. 4 shows the calcination temperature of titanium oxide and the solidification parameters (other conventional treatment agents and blanks are also shown).

Biocompatibility Biocompatibility is as important as plasma compatibility. It is always pointed out as one of the major problems of blood purification therapy using non-specific physical or chemical adsorbents. It is. There are a huge variety of components in human plasma, and the concentrations of these components vary from 0.1 ppm to the order of%.
Therefore, albumin, which is the highest concentration in plasma components and is indispensable for human life support, is used as an indicator of useful components.
The albumin adsorption ratio of the titanium oxide of the present invention and the exchange resin was compared using 4.0 g / dl PBS. Table 2 shows the results.

[0016]

[Table 2]

Measurement of albumin concentration: manufactured by Wako Pure Chemical Industries, Ltd.
Albumin Kit Wako (Bromcresol Green Method) Bath ratio (volume ratio): albumin PBS 10: adsorbent 1 As shown in Table 2, titanium oxide, especially titanium oxide having high adsorption characteristics to total bilirubin, also has albumin adsorption ability Is not so high, and plasma adsorption therapy (Plasma p
When performing erfusion, replacement of a small amount of fresh frozen plasma is usually performed, and it is considered that there is no particular problem. FIG. 5 shows the calcination temperature of titanium oxide and the albumin adsorption characteristics [albumin adsorption% (a): ▲ indicates the amount of albumin adsorbed on the anion exchange resin, and the amount of albumin adsorbed (g) per unit area (1 m ² ) (b). ]

FIG. 7 shows the relationship between the firing temperature of titanium oxide and the surface polarity. Incidentally, the characteristics of the treating agent are also described.
Titanium oxide calcined at the optimal calcining temperature is almost neutral in polarity, but has both polarities and is presumed to be neutralized.

[0019]

As described above, the blood purification adsorbent comprising titanium oxide of the present invention has sufficient practical characteristics in terms of adsorption characteristics and blood compatibility, and selects total bilirubin in plasma. This provides an excellent effect of being able to be used as a practical adsorbent for adsorbing on a surface.

[Brief description of the drawings]

FIG. 1 shows the total bilirubin adsorption activity of the titanium oxide of the present invention and a conventional treating agent:

Fig. 2 Pore distribution of calcined titanium oxide and conventional treating agent

FIG. 3 Specific surface area of calcined titanium oxide and conventional treating agent

FIG. 4 Solidification parameters of calcined titanium oxide and conventional treating agents

FIG. 5: Calcination temperature and albumin adsorption characteristics of titanium oxide

FIG. 6: Surface polarity of calcined titanium oxide and conventional treating agents

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Claims (6)

[Claims] The present invention is characterized in that there is substantially no difference in partial thromboplastin time, proton bin time, and fibrin deposition amount, which are pseudo-solid factors of plasma, when brought into contact with human fresh plasma, and the specific surface area is 20%. ８０80 m ² / g, average pore size of 150-200 °, pH value of 6-7.5, characterized in that the plasma is brought into direct contact with plasma to selectively adsorb total bilirubin in the plasma. Titanium oxide for bilirubin adsorption. 2. The plasma according to claim 1, which is prepared by calcining at a temperature in the range of 300 to 600 ° C., wherein the plasma is brought into direct contact with the plasma to selectively adsorb the total bilirubin in the plasma. Titanium oxide for bilirubin adsorption. 3. The method for adsorbing bilirubin in blood plasma according to claim 2, wherein the temperature range for baking is 400 to 550 ° C., wherein the plasma is brought into direct contact and the total bilirubin in the blood plasma is selectively adsorbed. Titanium oxide. 4. A precipitate obtained by stirring the titanium alkoxide compound in water in which an acidic or alkaline compound which volatilizes at the time of calcination is present, and then the resulting precipitate is separated by filtration, dried and heated at a rate of 2 ° C to 10 ° C / min. After heating to a temperature range of 300 to 600 ° C. and maintaining the temperature range for 2 to 5 hours, the ratio of anatase type to rutile type obtained by natural cooling is 90 to 5;
5:10 to 45, a titanium oxide for adsorbing bilirubin in plasma, which is brought into direct contact with plasma to selectively adsorb total bilirubin in the plasma. 5. The plasma according to claim 4, which is prepared by calcining at a temperature in the range of 300 to 600 ° C., wherein plasma is brought into direct contact with the plasma to selectively adsorb total bilirubin in the plasma. Titanium oxide for bilirubin adsorption. 6. The method for adsorbing bilirubin in plasma, wherein the temperature range for baking is 400 to 550 ° C., and the total bilirubin in the plasma is selectively adsorbed by directly contacting the plasma. Titanium oxide.