JPS5835485B2 - oxygen delivery infusion - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an injection and a perfusion emulsion having oxygen-carrying ability, which are used to save patients suffering from massive bleeding and to preserve organs following organ transplantation.

Many researchers have reported that fluorocarbon emulsions have the potential to be used as red blood cell substitutes for borehole animals, as organ preservation perfusion fluids for organ transplants, and in particular as infusion fluids for the purpose of substituting oxygen-carrying ability. [Leland C. Clark Jr. (Leland C, C1
ark Jr.), Becateini, F.
cattini, F.), Kablan, Varnish (K.
aplan, S,) F Physiology of synthetic blood J (Th
ePhysiology of 5ynthetic
Blood) Journal of Thoracic Surgery
c Cardiovascular Surg,) No. 60
Volume No. 757-773, 1970, Gaii.

Geyer, R.P. Fluorocarbon-Polyol Artificial Blood J (Fluorocar
bon-polyol artificial bl
oodsubstitutes, ) =-i--English Journal of Medicine (New E
ngl, J. Med, ) Vol. 289 No. 1077~
1082 pages, 1973].

However, these emulsions cannot be said to be sufficiently practical in terms of their pharmaceutical stability, and in order to put fluorocarbon emulsions into practical use as artificial red blood cells, stable particles with no change in particle size over a long period of time are required. It is necessary to develop a formulation.

In fluorocarbon emulsions, the particle size plays an important role in the toxicity and efficacy of the emulsion [Yokoyama, K1, Yamanouchi, K0, Watanabe, M1, Murashima, R, Matsumoto, T8, Hamano, T 0,
Okamoto, H9, Suya Ma, T1, Watanabe,
R0, Naito, R, "Preparation of perfluorodecalin emulsion - approach to artificial red blood cells" (Preparation of perfluoro
Decal emulsion.

an approach to the red ce
lls 5ubstitut) federation,
Federation Proceedings Vol. 34, pp. 1478-1483, May 1975.
].

In other words, emulsions with small particle sizes are highly toxic and the particles remain for a short time in the bloodstream.

Therefore, when a fluorocarbon emulsion is used as an artificial red blood cell as a lifesaving infusion for patients with massive bleeding, the average particle diameter of the emulsion particles must be 0.3μ or less, preferably 0.2μ or less (Japanese Patent Application Laid-Open No. 48-1979 22612).

In addition, in order to use fluorocarbon for the purpose of artificial red blood cells, regardless of the particle size, it is necessary that the fluorocarbon administered intravenously be excreted from the body promptly after completing its original purpose of transporting oxygen.

The present inventors previously used several types of fluorocarbon emulsions and determined their excretion rates [Yokoyama, K9, Yamanouchi, K0, Murashima, R, [Excretion of perfluorochemicals after intravenous injection J]
orochemicals after 1ntrav
enousinjection of their e
Mulsion) Chemical Pharmaceutical Bulletin (Chem, Pharm.

Bull, ) Volume 23, pages 1368-1373,
June 1975] and toxicity, it was found that fluorocarbons having 9 to 11 carbon atoms can be used as artificial red blood cells, and perfluorodecalin is particularly the best.

Furthermore, the present inventors conducted extensive studies on these selected fluorocarbon emulsifiers having 9 to 11 carbon atoms, and
In addition to these, egg yolk or soybean phospholipid contains trace amounts of 8 to 8 carbon atoms.
It has been discovered that by adding 22 fatty acids or physiologically acceptable salts or monoglycerides of the fatty acids, it is possible to produce fine grain emulsions that are stable for a long period of time [JP-A-50-69219, (JP-A-48]). -112047)].

However, when these emulsions are used as artificial red blood cells, there are some drawbacks compared to perfluorotributylamine emulsions emulsified with a high molecular weight polyoxyethylene-polyoxypropylene copolymer.

In other words, in terms of long-term storage stability, an emulsion emulsified with phospholipids shows approximately the same stability as a perfluorotributylamine emulsion emulsified with a polymeric nonionic surfactant; In terms of stability in the bloodstream, the former is inferior to the latter, and therefore, the half-life in the blood of the former is approximately % of that of the latter.

In addition, fluorocarbon emulsions emulsified with polymeric nonionic surfactants can be mixed with commercially available plasma expanders such as Textran, hydroxyethyl starch, modified ceratin preparations, etc. in any ratio; however, fluorocarbon emulsions emulsified with phospholipids When these fluorocarbon emulsions are mixed with these plasma expanders, a precipitate is deposited, and the two cannot be mixed and used.

This is because the emulsion is destroyed by the interaction between the high concentration of phospholipids in the emulsion and polymeric colloid substances such as dextran and hydroxyethyl starch.

The combination with a plasma expander is important when using a fluorocarbon emulsion as a life-saving infusion during massive bleeding, that is, as an artificial red blood cell.During massive bleeding, the fluorocarbon emulsion supplies oxygen while the plasma expander maintains circulating blood volume. is necessary.

Therefore, a fluorocarbon emulsion intended for use as an artificial red blood cell is preferably prepared using a polymeric nonionic surfactant.

However, with the exception of some fluorocarbons such as amine-based fluorocarbons, nonionic surface active polymers
Most fluorocarbons that are effective as fluorocarbons for artificial red blood cells, ie, fluorocarbons having 9 to 11 carbon atoms, such as perfluorodecalin, which has a good excretion rate, are not suitable as emulsifiers.

Therefore, the present inventors used a polymeric nonionic surfactant to store fluorocarbons with a carbon number of 9 to 11, which have a fast excretion rate, as represented by perfluorodecalin, and made it possible to store them stably for long periods of time and to ensure that they are stable in the circulating bloodstream. Extensive pharmaceutical and pharmacological studies were carried out with the aim of preparing emulsions that could be mixed with plasma expanders without any disintegration of the emulsified particles.

As a result, the present inventors have succeeded in providing a stable emulsion consisting of a physiologically acceptable aqueous solution of a fluorocarbon compound with an oxygen-carrying ability having a particle size of 0.05 to 0.3 microns.

That is, its composition is (N component is perfluorodecalin, perfluoromethyldecalin, perfluorocycloalkane or perfluoroalkylcycloalkane such as perfluoroalkylcyclohexane having an alkyl group having 3 to 5 carbon atoms, 5 carbon atoms) A perfluorocarbon compound having 9 to 11 carbon atoms selected from the group of perfluoroalkyltetrahydrofuran having an alkyl group of ~7, perfluoroalkyltetrahydropyran having an alkyl group having 4 to 6 carbon atoms, and perfluoroalkane. At least one: perfluoro-tertiary alkylamine as component (B), perfluoro-N-furkylpiperidine having an alkyl group having 4 to 6 carbon atoms, perfluoro-N having an alkyl group having 5 to 7 carbon atoms - at least one perfluorocarbon compound of tertiary amine having 9 to 11 carbon atoms selected from the group of furkylmorpholines; polymeric nonionic surfactant with a molecular weight of approximately 2,000 to 20,000; phospholipid; carbon number 8 ~22 fatty acids and their physiologically acceptable salts, at least one fatty acid compound selected from the group of monoglycerides; and a physiological aqueous solution, in which (N
The weight ratio of component and component (B) is 95 to 50:5 to 50
Then, an emulsion was provided which solved the above object.

The polymer nonionic surfactant mentioned here has a molecular weight of 20
00 to 20,000, for example, polyoxyethylene-
Polyoxypropylene copolymer polyoxyethylene alkyl ether, polyoxyethylene alkyl allyl ether, etc.

The concentration of this surfactant in the emulsion is from about 2.0 to about 5
．． 0 w/v%, preferably 3.0-3.5
It is w/v%.

The perfluorocarbon belonging to component (A) is perfluorocycloalkane or perfluoroalkylcycloalkane, and examples thereof include perfluoromethylpropylcyclohexane, perfluorobutylcyclohexane, perfluorotrimethylcyclohexane,
Perfluorocyclohexane having an alkyl group having 3 to 5 carbon atoms, such as perfluoroethylfurobylcyclohexane, perfluorodecalin, and perfluoromethyldecalin.

Examples of perfluoroalkyltetrahydropyran having an alkyl group having 4 to 6 carbon atoms include perfluorohexyltetrahydropyran, and examples of perfluoroalkyltetrahydrofuscine having an alkyl group having 5 to 7 carbon atoms include perfluoro Pentyltetrahydrofuran, perfluorohexyltetrahydrofuran, perfluorohexyltetrahydrofuran; carbon? 9-1
Examples of the perfluoroalkane 1 include perfluorononane and perfluorodecane.

Examples of the perfluorocarbon of the tertiary amine having 9 to 11 carbon atoms as component (B) include perfluoro N-N-diptyl monomethylamine, perfluoro N-N-diethylpentyl amines, perfluorotrialkylamines such as perfluoroN-N-dipropylbutylamine, perfluorotrialkylamines, and perfluoroN-N
- perfluoro N-N-dialkylcyclohexylamine such as diethylcyclohexylamine.

Examples of the perfluoroalkylpiperidine having an alkyl group having 4 to 6 carbon atoms include perfluoroN-pentylpiperidine, perfluoroN-hexylpiperidine, and perfluoroN-butylpiperidine.

Examples of the perfluoroalkylmorpholine having an alkyl group having 5 to 7 carbon atoms include perfluoro N-pentylmorpholine, perfluoro N-hexylmorpholine, and perfluoro N-hebutylmorpholine.

The composition weight ratio of component (4) and component (B) of perfluorocarbon is 95 to 50:5 to 50, and the ratio of perfluorocarbon in the emulsion is about 10 to 50 w/w/.
v%.

Any phospholipids known to those skilled in the art can be used as the emulsification aid, but egg yolk phospholipids and soybean phospholipids are preferred.

The composition ratio in the emulsion is about 0.1 to about 1. Ow/v
%, preferably about 0.4 to about 0.6 w/v%.

Fatty acid compounds used as emulsification aids include fatty acids having 8 to 22 carbon atoms and physiologically acceptable salts or monoglycerides of these fatty acids, such as sodium and potassium.

These include, for example, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, haloleic acid, oleic acid, linoleic acid, arachidonic acid and their sodium or potassium salts. These monoglycerides.

These fatty acids can be used alone or in a mixture of two or more, and the composition in the emulsion is 0.004 to 0.1 w
/v% preferably about 0.02 to 0.04 w/v%.

Among these fatty acids, particularly preferred ones have 14 carbon atoms.
~20 fatty acids and their physiologically acceptable salts, such as potassium palmitate, potassium oleate, chosen for their emulsion solubility and stability.

The emulsion of the fluorocarbon compound of this invention can be mixed and coarsely emulsified in any order in a physiologically acceptable aqueous medium according to the above composition.

The coarsely emulsified liquid is reduced to a particle size of 0.0 by an effective emulsifying machine.
By homogenizing it so that it becomes 05 to 0.3μ,
The preparation of emulsions of the invention is accomplished.

Since the emulsion of the present invention has ultrafine particles and does not coarsen during long-term storage, a high degree of safety is ensured for the animals to be administered without any problems associated with coarsening of the particles.

Furthermore, the emulsion of the present invention has a long residence time in the circulating bloodstream,
Oxygen transport function is maintained for a long time.

For example, when a fluorocarbon emulsion emulsified with phospholipid as the main emulsifier and having the same particle size as the emulsion of the present invention with an average particle diameter of 0.1 μm was intravenously administered as fluorocarbon to domestic rabbits at 8 f/9 body weight, the fluorocarbon blood The intermediate half-lives were 72 hours and 44 hours, respectively, indicating that the emulsion of the present invention had a high ability to be maintained in blood.

When the emulsion of the present invention is used as an infusion,
It is used as a physiologically isotonic solution.

Furthermore, the emulsion of the present invention can be mixed with commercially available plasma expanders such as dextran, hydroxyethyl starch, modified gelatin preparations in any ratio.

In addition, this emulsion can be used as a blood substitute for mammals.
It can also be used as a perfusate for organ preservation.

Next, the present invention will be explained in more detail with reference to Examples.

In the Examples, the particle size was measured using the centrifugal sedimentation method proposed by Yokoyama Gu et al.
.

Bull, 22(12), 2966-2971(1
974)).

Example 1 300P of polyoxyethylene polyoxypropylene copolymer (molecular weight: 8350) was dissolved in 81 parts of distilled water, and 3 parts of perfluorodecalin, 9 parts, and 300 parts of perfluorotripropylamine were added to this solution. A mixed fluorocarbon containing 40 P of soybean oil phospholipid and 21 P of potassium oleate was added and stirred with a mixer to prepare a rough emulsion.

A spray emulsifying machine (manufactured by Manton-Gorlin)
The mixture was placed in a liquid tank and circulated, and emulsification was carried out under high pressure of 200 to 500 kg/- while maintaining the liquid temperature at 35±5°C.

Perfluorodecalin M in the obtained emulsion was 30.5w
/v%, perfluorotripropylamine concentration is 2.9
W/V%.

The average particle diameter measured by centrifugal sedimentation is 0.09~
The particle size was 0.1μ, and no increase in particle size was observed even when the particles were dispensed into injection vials, capped, placed in a rotary sterilizer, and heat sterilized at 115° C. for 12 minutes.

The particle size distribution of the emulsion after sterilization is shown in Table 1 along with a control of perfluorodecalin alone.

Further, even when this emulsion was stored at 4°C for 6 months, no coarsening of the grains was observed, and the average grain size hardly changed.

The average particle size after storage at 4° C. for 6 months is shown in Table 1 along with the control emulsion containing perfluorodecalin alone.

Example 2 Polyoxyethylene octyl ether 3301 with an average molecular weight of 3500 was dissolved in 81% distilled water, and 40P was added to this solution.
Soybean phospholipid, 2F? Add potassium oleate and mix with a mixer to prepare a suspension. Add mixed fluorocarbons of perfluoromethyldecalin 3-dopa-fluoro N-pentylpiperidine 6001 to this solution and mix with a mixer to make a rough emulsion. Manufactured.

This rough emulsion was emulsified in the same manner as in Example 1,
After dispensing into vials, heat sterilization was performed at 115° C. for 12 minutes in a rotary sterilizer.

The perfluorodecalin concentration in the obtained emulsion was 29.7
w/v%, perfluoro N-pentylpiperidine concentration was 5.8 w/v%.

The particle size distribution of the emulsion after sterilization is shown in Table 1 along with the control.

Furthermore, no coarsening of the grains was observed in this emulsion even after storage at 4°C for 6 months.

Table 1 shows the average particle diameter after storage at 4°C for 6 months.

Example 3 100P of polyoxyethylene polyoxypropylene copolymer having an average molecular weight of 10,800 was dissolved in 21 parts of distilled water, and 20 parts of egg yolk phospholipid was added to this liquid. Add 0.5 fI of oleic acid and mix with a mixer to make a suspension. To this solution, add a mixed fluorocarbon prepared by mixing perfluorodecalin 6401 with 250 g of perfluorodibutyl monomethylamine, and mix with a mixer to make a rough emulsion. was manufactured.

This rough emulsion was emulsified in the same manner as in Example 1,
The emulsion was sterilized by heating at 115° C. for 12 minutes in a rotary sterilizer.

The concentration of perfluorodecalin in the emulsion is 25.3 w/v
%, the concentration of perfluorodiptyl monomethylamine is 9
．． It was 8 w/v%.

The particle size distribution of the particles after sterilization is shown in Table 1 along with a control emulsion of perfluorodecalin.

Furthermore, Table 1 shows the average particle diameter when this emulsion was stored at 4°C for 6 months.

Example 4 Polyoxyethylene polyoxypropylene copolymer 35r having an average molecular weight of 15,800 was dissolved in 800 ml of distilled water, 41 ml of egg yolk phospholipid and 0.11 lauric acid monoglyceride were added thereto, and a suspension was prepared by stirring with a mixer. To this, perfluorohexyltetrahydropyran 3
Add a mixed fluorocarbon prepared by mixing 40 g of perfluoro-N-N-diethylcyclohexylamine to No. 501, and mix with a □ mixer to prepare a rough emulsion.

This rough emulsion was emulsified in the same manner as in Example 1,
After dispensing the obtained emulsion into vials, it was heated to 115°C in a rotary sterilizer.
, heat sterilized for 12 minutes.

The concentration of perfluorohexylpyran in the emulsion is 35.7
The concentration of perfluorinated N-N-diethylcyclohexylamine was 4.1 w/v%.

The particle size of this emulsion after sterilization is shown in Table 1 along with a control emulsion containing perfluorohexylpyran alone.

Furthermore, no coarsening of the grains was observed in this emulsion even after storage at 4°C for 6 months.

Table 1 shows the average grain diameters of this emulsion and the control emulsion after storage at 4°C for 6 months.

Test Example 1 Combination experiment with plasma expander When FC preparation is used as an infusion, colloid osmotic pressure is low, so plasma expander (plasma expander) is not recommended for clinical use.
expander) is preferred.

In the preparation of the present invention, even when mixed with a plasma expander, there is no interaction with the plasma expander.
No reversible precipitate was observed, which solved one of the problems when used as an infusion.

The FC formulations used in the experiment were emulsions of various concentrations obtained by the same method as in Example 3 and JP-A-50-69219.
Comparative tests were conducted using fluorinated carbon emulsions of various concentrations using 0.02 w/v% of potassium oleate as an emulsifier.

After each solution was made isotonic with lactated Ringer's solution, a plasma expander was mixed to a final concentration of 1 to 6 w/v%, and the presence or absence of precipitate formation was visually observed at room temperature for 6 hours.

As a plasma expander, 20 w/v% HES (hydroxyethyl starch) (average molecular weight 200000)
formulation and 10 w/v% Dextran 40 (Dex
tran40) (average molecular weight 40,000) formulation was used.

The test results are shown in Tables 2 and 3.

-Bon formulation is commercially available Gixtran 40. HES preparations can be mixed at any ratio up to a final concentration of 2 w/v% or 3 w/v%.

The preparations obtained in Example 1.2.4.5 of the present application also obtained similar effects.

Test Example 2 In order to examine the safety of the preparation in circulating blood after intravenous administration from the survival time of rats, Wistar rats (male 200 to 250 f) were treated with an emulsifier of the composition shown in the table. A blood exchange transfusion experiment was performed.

As test samples, the formulation provided in Example 3 as a representative of the main rule and the formulation provided in JP-A-50-69219 as a control were used, and these formulations were made isotonic with an electrolyte.

The composition was as follows, and the FC formulation and electrolyte were mixed at a ratio of 9 volumes: 1 volume.

The test results are shown in Table 4. At the same time, since the FC preparation alone has a low colloid osmotic pressure and has little effect on maintaining circulating blood volume, plasma and a plasma expander such as hydroxyethyl starch (HES) were used in combination.

The HES infusion is Kyorin Hespander ■ (6w/v%
HES preparation) was used, and plasma obtained from Wistar rats was used.

The mixing ratio of the FC preparation and the plasma expander was 1:3.

Blood was removed from the carotid artery, and the FC preparation was injected from the tail vein.

After setting the final hematocrit value to 7.0.4.0.1.0 (total blood red blood cell content 7 w/v%), the survival time of the rats was measured.

The results are shown in Table 5. This result indicates that the present preparation is extremely safe in circulating blood compared to the control FC preparation.

Test Example 3 Table 4 of Experiment Example 2 shows the acute toxicity test for the formulation of the present invention.

Tests were carried out using physiologically isotonic formulations of the present invention and ☆☆ control formulations.

The experimental animals were Wistar male rats (body weight 100-120
? ) was used.

The emulsion was administered intravenously and observed for one week after administration.

The results are shown in Table 6.

The amphoteric agent had an LD5o of approximately 130 m1/body weight of 9, and had very low toxicity.

Test Example 4 In order to investigate the hemolytic effect of FC preparations in the extracorporeal circulation system, rabbit red blood cells were used to conduct in vitro experiments.
o) experiments were conducted.

The FC formulations were the physiologically isotonic formulation of the present invention shown in Table 4 of Test Example 2 and the FC formulation prepared in JP-A-50-6921 as a control.
No. 9 formulation was used.

In the experiment, FC emulsion and heparinized rabbit blood were mixed in a volume ratio of 3:1.1:1.1:3, respectively.

8 ml of the sample solution was kept at 37°C, and the amount of hemolysis was measured 6 hours later.

Measurement is done using the cyanomethemogmobin method [Reference: Clinic
Acta (CIin, Chim, Acta,
) 6.538.1961).

The results are as follows.

As a result, the hemolytic effect of the preparation of the present invention is
It was much smaller than that of Preparation No. 9, and there was no significant difference from the control lactic acid Ringer's solution.

Claims (1)

[Scope of Claims] 1. As an essential composition component, (A) as an ff component, a perfluorocycloalkane such as perfluorodecalin, perfluoromethyldecalin, perfluoroalkylcyclohexane having an alkyl group having 3 to 5 carbon atoms; Also selected from the group of hahafluoroalkylcycloalkane, perfluoroalkyltetrahydrofuran having an alkyl group having 5 to 7 carbon atoms, perfluoroalkyltetrahydropyran having an alkyl group having 4 to 6 carbon atoms, and perfluoroalkane. At least 1 perfluorocarbon compound having 9 to 11 carbon atoms
As component (B), perfluoro-tertiary alkylamine, perfluoro-N-alkylpiperidine having an alkyl group having 4 to 6 carbon atoms, perfluoro-N-fluoro-N-fluorinated fluoride having an alkyl group having 5 to 7 carbon atoms; At least one perfluorocarbon compound of a tertiary amine having 9 to 11 carbon atoms selected from the group of chirmorpholines; a polymeric nonionic surfactant having a molecular weight of about 2,000 to 20,000; niphospholipids having 8 to 22 carbon atoms; at least one fatty acid compound selected from the group of fatty acids and their physiologically acceptable salts, monoglycerides; and a physiologically acceptable medium;
An emulsion preparation of a medical fluorocarbon compound having a particle size of 0.05 to 0.3μ and having an oxygen carrying ability, characterized in that the weight ratio of component () to component (B) is 95 to 50:5 to 50. 2. Claim 1, wherein the medium of the emulsion of the fluorocarbon compound is water or an isotonic solution that is physiologically acceptable.
formulation. 3 Fluorocarbon compound (A component + B component) is 10
~50 w/v%, polymeric nonionic surfactant at 2.0-5.0 w/v%, and phospholipid at 0.1%.
~1.0 w/v% and 0.004 to 0 fatty acids
．． 1 w/v%. 4. As an essential composition component, (4) As a component, perfluorocycloalkane or perfluoroalkylcycloalkane such as perfluorodecalin, perfluoromethyldecalin, perfluoroalkylcyclohexane having an alkyl group having 3 to 5 carbon atoms, Perfluoroalkyltetrahydrofuran having an alkyl group having 5 to 7 carbon atoms, perfluoroalkyltetrahydropyran having an alkyl group having 4 to 6 carbon atoms,
9 carbon atoms selected from the perfluoroalkane group
At least one of the perfluorocarbon compounds of ~II; as component (B), perfluoro tertiary alkylamine, perfluoro N-alkylpiperidine having an alkyl group having 4 to 6 carbon atoms, alkyl having 5 to 7 carbon atoms; At least one perfluorocarbon compound of a tertiary amine having carbons a9 to 11 selected from the group of perfluoro-N-furkylmorpholines having a molecular weight of about 2
000 to 20,000 polymeric nonionic surfactants; phospholipids; at least one fatty acid compound selected from the group of fatty acids having 8 to 22 carbon atoms, physiologically acceptable salts of the fatty acids, and monoglycerides; and a physiologically acceptable medium;
The weight ratio of component to component (B) is 95 to 50:5 to 5
0 and the addition of plasma or plasma expanders,
An emulsion preparation of a medical fluorocarbon compound having a particle size of 0.05 to 0.3μ and having an oxygen carrying ability, which is characterized by being isotonic with blood in terms of colloid osmotic pressure.