DK152256B - PROCEDURE FOR THE PREPARATION OF A STABLE BLOOD REMOVAL - Google Patents

Description

The invention relates to a process for producing a stable blood substitute in which homogeneously mixed physiologically acceptable water, oxygen-transmitting perfluorocarbon compounds and emulsifiers are obtained to obtain a coarse emulsion, after which it is emulsified by injecting it at a temperature of up to 55 ° C through a gap under a pressure of from about 10 to about 50 MPa, thereby undergoing shear forces and mixing action based on a strong velocity gradient until the particle size of the perfluorocarbon compounds in the resulting emulsion reaches 0.05-0.3 µm.

It has already been reported by a number of researchers that fluorocarbon compound emulsions may optionally be used as artificial blood substitutes for mammals and as perfusion fluid for preservation of internal organs to be transplanted, especially as an infusion fluid replacement capable of carrying oxygen [Leland C. Clark, Jr., F. Becattini, and S. Kaplan: The physiology of syn-thetic biodiversity, Journal of Thoracic Cardiovascular Surgery, 60, 757-773 (1970); R. P. Geyer: Fluorocarbon-polyol artificial biodegraders, New England Journal of Medicine, 289 »1077-1082 (1973) J ·

However, these emulsions may not be considered sufficient for practical use in terms of their pharmaceutical stability and safety for the living organism. In order for fluorocarbon compound emulsions to be qualified for practical use as a germ-free blood substitute, it is necessary to develop a composition which is sufficiently stable to be stored for a long time without change in particle size.

In fluorocarbon compound emulsions, particle size plays an important role in the emulsion's toxicity and efficiency [k. Yokoyama, K. Yamanouchi, M. Watanabe, R. Murashima, T. Matsumoto, T. Hamano, H. Ikamoto, T. Suyama, R. Watanabe, and R. Naito: Preparation of perfluorodecalin emulsion, an approach to the red cells substitute, Federation Proceedings, 34, 1478-1483 (May, 1975)] · A larger particle size emulsion is more toxic and has shorter residence time of the particles in the blood stream. Therefore, when the fluorocarbon compound emulsion is intended for use as artificial blood replacement to maintain the life of a patient suffering from massive bleeding, its average particle size must be 0.3 µm or less, preferably 0.2 µm or less, No. 849 412].

In addition to the particle size, it is necessary for the fluorocarbon compound emulsion to be useful as an artificial blood substitute that the intravenously administered fluorocarbon compound, after being eliminated from the blood stream, is excreted from the body as soon as possible. Previous studies on the rate of excretion and toxicity of several types of fluorocarbon compound emulsions have found that perfluorocarbon compounds of 9-11 carbon atoms are useful as artificial blood substitute materials, the best compound being perfluorodecalin. K. Yokoyama, K. Yamanouchi, and R. Murashima: Excretion of perfluorochemicals after intravenous injection of their emulsion, Chemical Pharmaceutical Bulletin, 23, 1368-1373 (June 1975)].

Furthermore, it has been found that fine and stable fluorocarbon compound emulsions can be prepared from these selected fluorocarbon compounds of 9-11 carbon atoms by emulsifying the fluorocarbon compounds with a mixture of egg yolk phospholipids or soybean phospholipids and a small amount. fatty acids having 8-22 carbon atoms or salts or monoglycerides thereof {Japanese Patent No. 954,944 and corresponding DE Publication No. 24 04 564].

Compared to a perfluorotributylamine emulsion stabilized with a high molecular weight polyoxyethylene-polyoxypropylene copolymer (R.P.

Geyer, loc.cit.), The aforementioned emulsion is stabilized with both phospholipids and fatty acids better in terms of excretion rate, but inferior in stability of circulating blood flow after intravenous injection, with the half-life being about 2/3 of the former.

Further, a fluorocarbon compound emulsion prepared with a high molecular weight nonionic surfactant such as perfluorotributylamine emulsion can be used as a mixture in any ratio with the commercial plasma pre-emitting agents such as dextran or hydroxyethyl starch or modified gelatin solution and the perfluorodecaline emulsion described in DE 24 04 564 cannot be used in combination with the aforementioned plasma enhancers due to precipitation formation when mixed with the latter. It appears that the precipitation is due to the degradation of emulsified particles caused by mutual interaction between the high concentration phospholipids contained in the emulsion and the plasma propagating agent such as dextran or hydroxyethyl starch which is a high molecular weight colloidal substance.

When a fluorocarbon compound emulsion is to be used as an infusion liquid or as an artificial blood substitute to save a patient's life in case of massive bleeding, the combination with a plasma diluent becomes important to produce isotonicity, i.e. to equalize the oncotic pressures of the two colloidal solutions, namely the emulsion and the blood. The fluorocarbon compound emulsion delivers oxygen, while the plasma expanding agent allows the circulating blood volume to be maintained at an appropriate level. Therefore, it is preferred to use a high molecular weight non-ionic surfactant which is inactive against the plasma pre-emitting agent to prepare a fluorocarbon compound emulsion for use as artificial blood. Although these high molecular weight non-ionic surfactants are effective as emulsifiers for some fluorocarbon compounds such as perfluorotributylamine and other amine type fluorocarbons, they are not suitable for fluorocarbon compounds having 9-11 carbon atoms such as perfluorodecline having a high excretion rate.

In these circumstances, the inventors have conducted extensive studies to find a process for emulsifying perfluorodecaline without using a phospholipid as the main emulsifier and as a result have found that when perfluorodecaline is emulsified as a mixture with perfluorotripropylamine, the desired emulsion can be prepared using a nonionic surfactant as the main emulsifier. Furthermore, using a nonionic surfactant, it was possible to add a plasma pre-emitting agent which renders the emulsion's oncotic pressure isotonic. As a result, the hemolytic effect otherwise produced by an emulsion is reduced so that the animal's survival becomes possible when the emulsion is used as artificial blood substitute. Thus, it has been the selection of particular perfluorocarbon compounds and nonionic surfactant in association with a plasma pre-emitting agent. it is possible to produce an excellent fluorocarbon emulsion that is stable in the circulating bloodstream and has a low toxicity in the living body and desirable properties so that it has been used for the first time in the world as an artificial blood substitute in the clinical field.

Thus, the peculiarity of the method according to the invention lies in the selection of the optimum components and their use in the optimum ratio, thereby obtaining a surprisingly advantageous result.

Accordingly, the process of the invention is peculiar to the characterizing part of the claim.

The symbol "% (w / v) n means the amount ratio of a material calculated as weight (grams) per 100 ml of the resulting emulsion.

For the rest of the description, this ratio is simply referred to as

The polyoxyethylene-polyoxypropylene copolymers used as emulsifiers have a molecular weight of 2000-20000. The concentration of this high molecular weight nonionic surfactant in the emulsion is from about 2.0 to about 5.0%, preferably 3.0-3.5%.

The phospholipids used as the emulsifier in the emulsion are those commonly used in the art and those comprising egg yolk phospholipid or soybean phospholipid are preferred. The amount present in the emulsion may range from about 0.1 to 1.0%, and preferably about 0.4 to about 0.6%.

The fatty acid compound used as an emulsifier is a fatty acid having 8-22 carbon atoms, a physiologically acceptable salt thereof, such as the sodium or potassium salt, or a monoglyceride thereof. Such fatty acid compounds include, e.g. caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, palmitic acid, oleic acid, linoleic acid and arachidonic acid, and the sodium and potassium salts thereof and monoglycerides thereof. These fatty acid compounds can be used alone or as a mixture of two or more kinds thereof in an amount as low as 0.004-0.1%, and preferably from about 0.02 to about 0.04%. Of these fatty acid compounds, those with 14-20 carbon atoms and their physiologically acceptable salts are preferred, and the most preferred are potassium palmitate and potassium oleate, given their good solubility and ease of preparation of the emulsion.

The process of the invention is carried out by homogeneously mixing prescribed amounts of the aforementioned components in any order in a physiologically acceptable aqueous medium, such as distilled water, or an isotonic solution, to obtain a crude emulsion and then emulsifying the crude emulsion by inject it at a temperature of up to 55 ° C through a gap under a pressure of from about 10 to about 50 MPa, subjecting it to shear forces and mixing action based on a strong velocity gradient until the aforementioned desired particle size is achieved.

The homogeneous mixing of the materials is carried out using a conventional mixer such as a homo mixer or propeller stirrer.

The emulsion of the crude emulsion is obtained by a high pressure homogenizer, which is a high pressure pump which homogenizes a mixture of two immiscible liquids by injecting it through a high pressure slit at a very high speed to produce a shear and mixing. of the fluids. The typical homogenizer on the market is of the Manton-Gaulin type (trade name for a homogenizer sold by Manton-Gaulin Manufacturing Co., Inc., USA), which has a multi-stage valve in combination of two or more valves, each containing a spring, from which the slits are formed.

The mixture is circulated in this type of homogenizer several times under a total pressure of about 50 MPa, thus obtaining a stable emulsion. The operating temperature is maintained in the range up to 55 ° C, and preferably at 25-40 ° C.

The emulsion prepared by the process of the invention has a dispersion phase of ultrafine particles whose diameter is less than 0.2 µm or at least less than 0.3 µm. Furthermore, it is stable in that it shows no particle size growth even when heated or stored for a long time. Therefore, the emulsion greatly protects the animal to which it is administered against deleterious effect due to agglomeration of the emulsion particles.

Furthermore, the emulsion has a long retention time in the circulating blood stream, so that the oxygen carrying capacity is maintained for a long time.

Eg. For example, the emulsion prepared according to the invention remains much longer in the animal's blood stream than a fluorocarbon compound emulsion prepared using phospholipids as emulsifier according to JP 954 944 and DE 24 04 564. emulsion.

The present emulsion can be used as an infusion liquid as it is made physiologically isotonic with commercial plasma extenders such as dextran and hydroxyethyl starch. Furthermore, it can be used as a blood substitute for mammals and as a perfusate for preserving internal organs.

The invention is further illustrated by the following examples in which the particle size was measured by the centrifugal sedimentation method proposed by K. Yokoyama, A. Suzuki, I. Utsumi and R. Naito. Chem. Pharm. Buil. 22 (12), 2966-2971 (1974).

Example 1 181 distilled water was dissolved 300 g of a polyoxyethylene-polyoxy-propylene copolymer (molecular weight 10800). To the solution was added 40 g of soybean phospholipids, 2 g of potassium oleate and a mixture of 3 kg of perfluorodecaline and 300 g of perfluorotripropylamine.

The resulting mixture was stirred in a mixer to form a coarse emulsion. The resulting coarse emulsion was charged into the liquid tank of a jet emulsifier (manufactured by Manton-Gaulin Co.) and emulsified by passage 12 times through a valve at a high pressure of 20-49 MPa while maintaining the liquid temperature at 35-5 ° C. to produce emulsification. The resulting emulsion contained 30.5% perfluorodecaline and 2.9% perfluorotripropylamine. The average particle diameter was 0.09 - 0.1 µm, as measured by the centrifugal settling method. The emulsion showed essentially no particle size growth when enclosed in a vial for injection and subjected to thermal sterilization at 115 ° C for 12 minutes in the specially designed rotary sterilizer. Table 1 lists the particle size distribution of this emulsion and of an emulsion of perfluorodecaline alone, prepared without the use of perfluorotripropylamine.

As can be seen from Table 1, the present emulsion when stored at 4 ° C for 6 months showed no agglomeration, the mean particle diameter being essentially unchanged.

Table 1. Particle size distribution of different emulsions

(The table continues from here on the next page.

* = polyoxyethylene-polyoxypropylene copolymer, average molecular weight 10,800.

Table 1 (continued)

(continued from page 9)

To prepare a blood substitute by the method of the invention, the emulsion is added to a plasma preening agent to fill the lack of oncotic pressure. When this emulsion was mixed with a plasma pre-emulsifier, the reversible precipitation, which may be caused by mutual interaction between colloidal solutions, was not observed, demonstrating that one of the difficulties to be expected using the blood substitute prepared as an infusion liquid, is eliminated.

Perfluorodecaline / perfluorotripropylamine (10: 1) emulsions of various concentrations prepared in the same manner as above and by reference perfluorodecalin emulsions of different concentrations prepared according to DE Publication No. 24 04 564 [egg yolk phospholipid 4%; potassium oleate 0.02% 1 was individually made isotonic with lactate-containing Ringer's solution or Krebs-Ringer hydrogen carbonate solution, and then mixed with a plasma substitute so that the final concentration of the plasma excipient was 1-6%, whereupon the formation of precipitate was obtained. visually observed within 6 hours of mixing at room temperature. The plasma replacements used are hydroxyethyl starch (average molecular weight 200,000, 20% in physiological saline; supplied by Ajinomoto Co., Ltd.) and "Dextran 40" average molecular weight 40,000, 10% in physiological saline; provided by The Green Cross Corp.J.

By "plasma replacement" is meant a solution of the high molecular weight compounds, the "plasma amplifiers", in a physiologically acceptable aqueous medium at such a concentration that they can be used to replace plasma.

The results are given in Tables 2 and 3.

Table 2

Table 3

Note: no precipitate +: formation of precipitate

From the above results, it can be seen that the blood substitute produced by the process of the invention is much less affected by the presence of a plasma pre-emitting agent than the emulsion of DE Publication No. 24 04 564, which shows that the emulsion used in the process of the invention can be mixed with "Dextran". 40 "and hydroxyethyl starch separator replacement in any ratio to provide a blood substitute with the physiological colloidal isotonicity obtained by the addition of" Dextran 40 "and hydroxyethyl starch at a final concentration up to 2% and 4% respectively.

Together! Iqningsforsøq

The blood replacement emulsions were prepared by the procedure of Example 1 and their constituents were as listed in the following Table 4.

The following compounds were used as starting materials:

Component (A): Perfluorodecaline Component (B): Perfluorotripropylamine

Emulsifier: Polyoxyethylene-polyoxypropylene copolymer ("PLURONIC F-68") in the samples prepared according to the invention; phospholipid (egg yolk) in the comparison samples.

Emulsifier: Phospholipid (egg yolk) and fatty acid in the samples prepared according to the invention; fatty acid in the comparison samples.

Physiologically acceptable carrier: Aqueous solution containing glycerol (0.8% w / v), NaCl (0.6% w / v), KCl (0.034% w / v), MgCl2 (0.02% w / v). %), CaCl 2 (0.028 w / v%), NaHCO 3 (0.21 w / v%) and glucose (0.18 w / v%).

Plasma prewetting agent: Hydroxyethyl starch.

The comparison experiments were performed on the following subjects: 1) Particle size of the emulsion:

The average particle size of the emulsion was determined after sterilization and after 6 months of storage at 4 ° C according to the centrifugal sedimentation method described by K. Yokoyama et al. Tchem. Pharm. Buil., 22 (12), 2966-2971 (1974) '].

2) Effect of the plasma pre-emitting agent:

Hydroxyethyl starch was used as a plasma pre-emulsifier in an amount of 3% w / v, and the occurrence of precipitation was observed visually after 6 hours storage at room temperature.

If a reversible precipitate is formed by the mutual action of the fluorocarbon (FC) emulsion and the plasma pre-emulsion, the emulsion cannot be used as a blood substitute.

3) Replacement transfusion in rats:

Comparative experiments with replacement transfusion were performed using rats (Wistar strain, males, weight 200-250 g) by repeated bleeding from the carotid artery and replacement transfusion through the tail vein alternately up to a hematocrit value of 4.0 under 100% oxygen atmosphere. Then, the survival time of the replacement transfused rats was determined.

4) Hemolytic effects:

Experiments with the haemolytic action of FC emulsion in the extracorporeal circulatory system were performed in vivo using rabbit red blood cells.

The FC emulsion was mixed with a lactate-containing Ringer's solution so that it became essentially physiologically isotonic, and the resulting isotonic emulsion preparation was mixed with 1: 1 heparinized rabbit blood to prepare a test solution for the test. 8 ml of the sample solution was kept at 37 ° C for 6 hours and then the content of free hemoglobin was determined by the cyanmethemoglobin method (Kampen, EJ and Ziilstram, WJ, Clin. Chim. Acta. 6, 538, 1961). lactated Ringer's solution was used alone as a control without the use of FC emulsion, the free hemoglobin content was 128 mg.

5) Exhalation rate of FC emulsion in vivo.

The rate of exhalation of the FC emulsion in vivo was determined by the method described in Chemical Pharmacological Buil.

26, (3) 956-966, 1978, (K. Yokoyama, "Fate of perfluorochemicals in aminals after intravenous injection or hemodilution with their emulsions").

The FC emulsion was administered intravenously to the rat at a rate of 4 g / kg body weight and the exhalation rate was shown by the number of days until half of the administered FC was consumed by exhalation.

The results obtained are listed in Table 4.

_________Table 4 _.______

NOTE: (A): Perfluorodecaline (B): Perfluorotripropylamine F: "PLURONIC F68" L: Phospholipid FA: Fatty Acid HES: Hydroxyethyl Starch

From the results shown, the following can be deduced.

In case of sample # 1, the resulting FC emulsion is insufficient. emulsion and has low stability so that an emulsion with an average particle size of less than 0.2 µm could not be obtained. Thus, the resulting emulsion is completely unsuitable as a drug.

In case of sample # 2, the resulting emulsion was excellent with respect to emulsification and stability, but it precipitated by mixing with hydroxyethyl starch, and additionally showed a remarkable hemolytic effect and rats could not survive the treatment by replacement transfusion. Thus, this emulsion also cannot be used as a drug.

In the case of sample # 3, the resulting emulsion has low stability, but it can be administered in vivo as a drug just after it is made.

Sample # 4 showed the same result as # 2.

Samples Nos. 5, 7 and 9 showed very good properties. Therefore, they can be used as a blood substitute.

Samples Nos. 6, 8, 10, 12 and 14 showed the same results as Nos. 2.

Sample # 11 has a low rate of exhalation of FC in a living body compared to samples # 5, 7, and 9, but could be used as a drug under adequate control.

Sample # 13 has a very low exhalation rate so that it can cause unpredictable disadvantages in the living body. In this case, rats died after 69 hours when subjected to replacement transfusion.

As can be seen from these comparative experiments, the blood substitute produced by the method of the invention (samples Nos. 3, 5, 7, 9 and 11) shows significant effect on the stability of the particles, the concomitant use of plasma diluent, replacement transfusion, and safety in circulatory systems without for the body as compared to the FC emulsions of the aforementioned patents (Sample # 1 is composed according to DE Publication No. 21 44 094 and samples Nos. 2, 4, 6, 8, 10, 12 and 14 are composed according to DE disclosure no.

24 04 564). The above difference in effect between the blood substitute prepared according to the invention and the known FC emulsions is due to the difference in the constituents of the FC emulsions which led to such a significant advance as the toxicity of the FC emulsion almost disappeared and it was possible for the first time to use such a FC emulsion for clinical treatment.

Clinical Testing Based on the previously reported test results and extensive additional studies on toxicity, pharmacology, pharmacodynamics and efficacy in animals, the blood substitute manufactured according to the invention known as "Fluosol-DA" has been shown to be safe and effective such as artificial blood replacement. The studies have now reached the clinical stage.

The composition in w / v of "Fluosol-DA (20%)" is as follows:

Perfluorodecaline 14.0

Per fluorotripropylamine 6.0 "Pluronic F-68" 2.7 Egg yolk phospholipids 0.4

Potassium oleate 0.04

Glycerol 0.8

NaCl 0.600 KCl 0.034

MgCl2 0.020

CaCl2 0.028

NaHCO3 0.210

Glucose 0.180

Hydroxyethyl starch 3.0 March 1, 1979, the Phase I study of "Fluosol-DA" in humans has been conducted in Japan by Professor Mitsuno and Dr. Ohyanagi at Kobe University Medical School. Ten normal adult male volunteers, including 7 physicians, were infused with 20 to 500 ml of "Fluosol-DA", the three individuals receiving 500 ml being previously tapped for 200 ml of blood.

Blood samples were taken before the infusion and at 3, 24 and 48 hours and 1, 2 and 4 weeks after the infusion. The subjects who received either 200 ml or 500 ml were further sampled 8 and 14 weeks after the infusion. The following parameters were examined in the samples: Hematology

Complete blood count Reticulocyte count B1odp1ad counting Count and differential count of white blood cells Serum protein electrophoresis

Chemistry

Thymol Turbidity Test Amylase

Bilirubin, Total and Direct Lactate Dehydrogenase (LDH)

Cobalt reaction Cholinesterase

Serum glutamate oxaloacetate transaminase (SGOT) Blood urine nitrogen (BUN)

Serum glutamate pyruvate transaminase (SGPT) Total cholesterol

Alkaline phosphatase Creatinine

Total protein

In addition, a thromboelastogram was performed and the complement activity was determined on the blood samples taken 3 and 24 hours after the infusion.

No changes in blood pressure, heart rate, respiratory rate, body temperature or ECE were observed. No other symptoms were observed. No adverse reactions or complaints were reported by any of the 10 individuals. Throughout the investigation

Claims (1)

The tests were the tests for all individuals within the normal ranges of the above tests. It shows that "Fluosol-DA" is safe and can be used on patients. Patent claim: A process for preparing a stable blood substitute in which homogeneously mixed physiologically acceptable water, oxygen-transferring perfluorocarbon compounds and emulsifiers are obtained to obtain a coarse emulsion and then emulsified by injecting it at a temperature of up to 55 ° C. a gap under a pressure of from about 10 to about 50 MPa, thereby undergoing shear forces and mixing action based on a strong gradient of velocity until the particle size of the perfluorocarbon compounds in the resulting emulsion reaches 0.05 - 0.3 µm, characterized in that as perfluorocarbon compounds uses 10 - 50% (w / v) of (A) perfluorodecaline and (B) perfluorotripropylamine, where the weight ratio A: B is between 95: 5 and 50:50 and emulsifiers use 2.0 - 5.0 % (w / v) of a polyoxyethylene / polyoxypropylene copolymer having a molecular weight of 2000-20,000, 0.1 - 1.0% (w / v) of a phospholipid, and 0.004 -0.1% ( weight / vol.) of at least one fat acid compound selected from fatty acids having 8-22 carbon atoms and physiologically acceptable salts and monoglycerides thereof, and after preparation of the fine emulsion, it is mixed with an amount of plasma substitute supplying 1-2% (w / v) Dextran 40 or 1- 4% (w / v) hydroxyethyl starch as a plasma pre-emulsifier.