JPS6025925A - Application of fructose-1,6-diphosphate to blood medicine - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides the use of fructose 1, which has the effect of normalizing red blood cell metabolism and resistance to hemolysis in stored blood for transfusion to patients or for addition to extracorporeal circulation circuits (C:EC). , 6-diphosphate.

It is known empirically that red blood cells are damaged mechanically, chemically, and miraculously by the extracorporeal circulation (CEC) of blood required for cardiac surgery (Bern-stei et al.
n, F+F+ et al., Am, J. Surg., 114.1
26 (1967)), which manifests as postperfusion anemia due to intravascular hemolysis and the short average lifespan of red blood cells. Its delayed effect is greater than the direct effect, is strictly dependent on intravascular hemolysis, and can be measured by extravascular hemolysis, which is completely absent in the absence of intravascular hemolysis (Bcrn- 5tCIn+ F I F
, et al., C1rcu/1ation 45 (5upp
1.1) +226, (1967) and H, W, and Coburn.

R, F +, J, Thorac, Cardio
vasc, Surg, 68 +792, (1974
)).

The degeneration of red blood cell membranes caused by CEC is accompanied by changes in red blood cell metabolism and function, particularly modulation of oxygen affinity that impairs oxygen delivery to tissues (Glynn.

M., F., X. and Cornhill, F., C.
an, J., Surg.

18.73. (1975)).

The metabolic and hemolytic damage caused by CEC, in particular, is similar to that caused by blood storage that interferes with oxygen affinity function (Valtis D, J and Kennedy
A, C,, LanceL266.119. (1954)
).

For these reasons, the altered substitution and increased vulnerability of red blood cells due to both CEC and blood storage makes it difficult for patients who receive CEC during cardiac surgery and who simultaneously receive large amounts of stored blood to be transfused. It is clear that there will be serious negative consequences.

The present invention significantly inhibits damage to red blood cell membranes and red blood cell metabolism caused by blood storage and CE C, thereby protecting patients from subsequent intravascular hemolysis, post-laminar anemia and worsened oxygen affinity. , fructose-1,6
- Diphosphate compounds, especially diphosphate compounds) It was discovered that IJium salt was effective and was completed.

Fructose-1,6-diphosphinate sodium salt is a substance known to have activity in the fields of heart disease, hepatitis, cirrhosis, toxemia, diabetes and metabolic disorders, and in the present invention The substance is added to the blood transfused directly to the patient or contained in the filling medium of an extracorporeal circulation circuit. fructose-1
, 6-diphosphate compounds can be added directly to stored blood for transfusion or to blood contained in an extracorporeal circulation circuit.

In fact, this method is much more efficient than administering compounds to patients to improve metabolism and resist hemolysis of red blood cells.

Hemolytic and hypostatic insults that affect the oxygen affinity of red blood cells occur primarily after blood storage or during blood flow through extracorporeal circulation circuits.

Therefore, the method of the present invention focuses therapy on damaged red blood cells and prevents them from dispersing throughout the organism. This direct therapeutic effect is due to the fact that fructose-
This is achieved by adding 1,6-diphosphate to stored blood for transfusion or blood contained in an extracorporeal circulation circuit, thereby normalizing the red blood cell metabolism and resistance to hemolysis of the treated blood. Furthermore, in this method, fructose-1,6-diphosphate may be added, for example, a) during the blood storage period if the storage period is extended beyond a certain limit, b) at the end of the blood storage, and C) in an extracorporeal circulation circuit. It can be administered to blood that has already been added.

A great advantage of the method according to the invention is therefore that when stored blood has to be transfused or when extracorporeal circulation (CEC) is required, i.e. these techniques are routine operations in cardiac surgery, first aid and resuscitation. Hail appears on the ground, causing serious damage.

It is therefore an object of the present invention to provide fructose-1,6-in the form of microcrystalline powder or pyrodiene-free double-distilled aqueous solution.
To add the sodium salt of the diphosphate to stored blood at a rate of 0.2-2.5 g of fructose-1,6-diphosphneeto sodium salt per 100 ml of blood (or 3-4 g per II2) or in extracorporeal circulation. Add 2.30 g of fructose-1,6-diphosphneeto sodium salt (or 10 to 3
fructose-1,6-diphosphneeto sodium salt in the form of a microcrystalline powder or a pyrodiene-free redistilled aqueous solution for addition in a proportion of preferably 15 to 20 g).

The novel use of this fructose-1,6-diphosphneeto sodium salt in the modulated red blood cell production and vulnerability resulting from CEC and blood storage was proposed by Bjomeclica.
It was discovered through in vitro tests carried out using human blood at the Foscama Institute.

This is based on previously unknown activity confirmed in subsequent clinical trials. Illustrated below are the results of a number of in vitro studies and clinical studies conducted on patients undergoing CEC.

1) Materials and Methods From 4 healthy subjects and 11 patients without hematological diseases, 20 to 3 Oynl was administered using a syringe containing heparin.
Blood was collected a total of 96 times. In one test, a portion of the collected blood was centrifuged (3000 rpm for 10 minutes) and
Plasma was separated. Blood and plasma samples were combined in treatment sets for each test, and the treated samples were incubated at 37°C using a stirrer and a thermostat. In some cases, incubations were performed at 22°C or 4°C.

Treatments 1) FDP = amount of fructose-1,6-diphosphneeto sodium salt determined for each test. The sodium content is F ]) Pll of a 10% aqueous solution of P is 5.5
The amount is such that

2) F+P = molar amount of fructose equivalent to that of the FDP used in the same test, thorium (an amount equal to the sodium of the corresponding FDP) phosphate (an amount equivalent to the sodium of the corresponding FDP)
2 molar amount of treatment agent).

3) SAL = amount of physiological solution used to dilute the corresponding FDP treatment. FDP and F-1-P treatment agents are added to 1/100 volume of the planned amount of blood (or plasma) and added to double distilled water and/or pyrogen-free water.
or diluted with physiological solution (when this is necessary to avoid hypotonicity of the solution). In some studies, the volume in which the treatment is diluted is 1/20.1/10.115 or 1/2 of the intended blood volume.

In some tests, solutions containing FDP and F+P treatment gave a pI of 7.4 at Na 01-1.

In preliminary tests, various incubation times r&I
Changes in red blood cell azone triphosphate and 2,3-diphosphoglycerate, and red blood cell and plasma fructose-1,6-diphosphate were checked. Preliminary tests have shown that, under the experimental conditions employed, the metabolic state and hemolytic resistance of red blood cells are best expressed by changes in the fructose-1,6-diphosphate content.

Therefore, in these studies, fructose-
The concentration of 1,6 diphosphate was measured (Mich
al G,, Beutler I-1,0゜In B
ergmeyer HoU, -Ed,” Metbod
s of Enzymatic Analysis”
Vol, 3.1314 pages, Academic Press
s Inc. New York I 974).

Additionally, FDP-incubated plasma and FDP, F+
The concentration of inorganic phosphate (Mar
tin J, B., Doty O, M.

(1949), Anal, Cbem, 21*9
65 ) and hemoglobin (to calculate the degree of hemolysis) were measured.

Results: Even if no treatment is performed, the FDP value of red blood cells is
There is a marked increase after blood collection, and it is due not only to the influence of the substance with which the blood was treated, but also to other factors, namely: 1) incubation time and temperature; 2) treatment agents added to the blood being incubated; It was found that it varies depending on the effects of pH and volume, 3) % hemolysis in the incubated blood, etc. Furthermore, F in plasma
The DP% shows a gradual decrease as the incubation time progresses, which is due to factors present in the plasma (e.g. the activity of plasma phosphates) as well as factors related to the presence of red blood cells and other blood cells in the plasma. I also understood.

Regarding this effect, we will report the results for 8 types of collected blood. A portion of each blood draw was used to separate plasma. In this way, 8 samples of whole blood and 8 samples of plasma
A sample was obtained. These 16 samples were treated with F ]) P;
FDP plasma concentration was 1.3 milJ mol/β. 2.2
After incubation for 0 and 80 min, directly FD
FDP (
FDP present in plasma obtained by centrifugation of blood incubated with plasma a) and FDP (plasma b)
) I did some research. As a result of four incubation tests, plasma FDP changed as shown below (D%F I) P
) showed: 2 20 40 '80 min plasma a) -5-13-20-35 plasma b) -20-31-140-60 Change in FDPa degree in plasma a) (D FDP/a, μL
4 nt) is significantly correlated with time 0 min).

D FDP/a=+3.22-1.74 min 1°=0.9
6 (p(0,001).

The difference between the FDP change in plasma a and that in plasma is due to cellular factors (mainly red blood cells) ζ.
) shows the effect on the reduction of P.

This difference (DFDPb-a) does not change much over time.

D FDP ba = -62,70, 5 molecules = -Q,
46n, s: In other words, the ball is substantially constant.

Furthermore, treatment with FDP was a check-treatment aimed at improving red blood cell cost and hemolytic resistance at all incubation times examined.
s) was found to be more effective. In this regard, 37
Report the concentration of red blood cell fructose-1,6-diphosphate in 10 blood samples after incubation for 80 minutes at °C. These numbers are n, t,) without any treatment.
and FDP treatment (administering the amount necessary to make the plasma concentration 1.3 mmol/l) is effective against F+P treatment (administering the equivalent amount of FDP). . The treatment agent is a certain amount of distilled water (equal to 1/100 of the amount of blood to be incubated I, NaO■-IteP■)
7.4 Nishitamono).

n, n, 211±6.60 F T) P 283±9.85 F+P 225±7,69 Differences between treatments are: 1” D P vs. F+P=+58 (P<0.001)
F D 1.1 vs. n, n, = +72 (p<0.001)
F + P vs. n, n, = 10 l 4 n, s.

The average value of red blood cell FDP in 10 blood samples was 10.2 (±
0.6) μg/1 red blood cell t] and 2 for 80 minutes
The value for the same blood kept at 2°C was 68 (±4.4
6) μg/g/bulb (ml).

Clinical study ■) Materials and methods Thirty-five patients who underwent extracorporeal blood circulation and underwent surgery were randomly divided into the following two groups: 1) Test group (G-study) 17 patients;
2) Extracorporeal circulation circuit) Direct fructose-1,6
- Group treated with 15 g of diphosphneeto sodium salt (Fl)P), 18 cases (G-FDP).

In all cases, large amounts of Normosol R with pI-17,4 and Beucl filled with stored blood capable of giving a hematocrit of 25-28% were used.
An ey BO310 oxygenator was used. Filling of the extracorporeal circulation circuit was completed with heparin (257η/β) and hydryhydroxyaminomethane (2oo2Il) - as previously described, in G-Fl) I) 5 g of FDPI was added directly to the oxygenator. .

There were no significant differences in mean age, intraoperative blood volume and CEC time between the two patient groups. In all patients, preoperative red blood cell resistance and plasma free hemoglobin were in the normal range. Free I-1b
Blood was collected for measurement 2 hours before the end of CEC.

The results, expressed as mean error, were checked with Studecito's L test for unpaired theta.

■) Result The degree of hemolysis was evaluated using free hemoglobin (1-
1b) was calculated for two groups of patients by measuring the values. Hemolysis rate of CEC per minute (I-Ib
% min) was also measured. Since the greater the amount of blood circulating in the extracorporeal circulation circuit, the greater the mechanical damage, the absolute value was corrected per 14 discharged blood (1-1b% 1 x 103
).

The hemolysis rate (Hb%/1/min) per 4/min (which is a stress intensity factor independent of the stress period) was also calculated.

The F I) P treatment was able to significantly change all the coefficients of interest. i.e. stored blood and CEC
It showed the effect of improving red blood cell metabolism and lytic surface resistance of blood passing through the body.

The addition of fructose-1,6-diphosphate and/or substances containing it to blood to be added (i.e., stored blood for transfusion or extracorporeal circulation circuits) is not fully documented. It will be clear that the scope of the invention encompasses the following.

Patent applicant Biomezoica foscama indunu I
゛Ria Kimiko~Pharmaceutei Power Etsu Seripi!
A-1C

Claims (1)

[Claims] 1. Fructose-1,6-diphosphate compound 2
An agent that improves red blood cell metabolism and hemolysis resistance in the blood as an essential acid. 2. The improving agent according to item 1, wherein the lJl fluid is stored blood. 3. The improving agent according to item 1, wherein the blood is extracorporeally circulating blood. 4. The improving agent according to any one of items 1 to 3, wherein the amount of the fructose-1,6-diposphae 1 compound used is 3 to 4 y per 1 d of blood. 5. The improving agent according to item 3, wherein the amount of the fructose=1°6-diphosphate compound added to the extracorporeal circulation circuit is 10 to 30 g, preferably 15 to 20 g.