NO145370B - PROCEDURE FOR THE PREPARATION OF A HEAT AND STORAGE STABLE BLOOD REPLACEMENT CONSISTING OF A MIXTURE OF OXYGEN TRANSFERING PERFLUOR CARBON COMPOUNDS - Google Patents

Description

The present invention relates to a method

for the preparation of an emulsion of an oxygen-transmitting fluorocarbon compound for injection and perfusion, which is used in the resuscitation of patients suffering from severe bleeding and for the maintenance of internal organs during transplantation.

It has already been indicated by a number of researchers that fluorocarbon compound emulsions can possibly be used as artificial blood substitutes in mammals and as perfusion fluid for the maintenance of internal organs to be transplanted, particularly as a suitable replacement for infusion fluid that can transport oxygen (Leland, C. Clark, Jr. F. Becattini and S. Kaplan: "The physiology of synthetic blood", Journal of Thoracic Cardiovascular Surgery, 60, 757-773 (1970); R. P. Geyer; "Fluorocarbon-polyol artificial blood substitutes",

New England Journal of medicine, 289, 1077-1082 (1973).

However, these emulsions cannot be assumed to be sufficient for practical use with regard to pharmaceutical stability and patient safety. In order for emulsions of fluorocarbon compounds to be suitable for practical use as an artificial blood substitute, a preparation must be prepared that is sufficiently stable to be stored over a long period of time, without change in particle size.

In emulsions of fluorocarbon compounds, the particle size plays a significant role in the toxicity and effect of these emulsions (K. Yokoyama,

K. Yamanouchi, M. Watanabe, R. Murashima, T. Matsumoto,

T. Hamano, H. Okamoto, T. Suyama, R. Watanabe, of R. Naito: "Preparation of perfluorodecalin emulsion, an approachto the red cells substitute", Federation Proceedings, 34, 1478-1483, (May 1975). An emulsion containing larger particles is more toxic and has a shorter retention time for the particles in the bloodstream. When fluorocarbon compounds as emulsion there-

for is to be used as an artificial blood substitute to save the life of a patient suffering from severe bleeding, the average particle size should be 0.3 y or less in diameter, preferably 0.2 y or less (DE off.skrift 2 144 094).

In order for the fluorocarbon compound emulsion to be able to

used as a blood substitute must the compound, next to

have a suitable particle size, could be removed or flushed out of the body as quickly as possible after the intravenously administered fluorocarbon compound has been eliminated from the blood stream, cf. DE Off.skrift 2 409 598. The excretion rate and toxicity of several types of fluorocarbon compound have previously been studied emulsions and found that perfluorocarbon compounds with 9 to 11 carbon atoms are suitable as artificial blood substitutes, with perfluorodecalin in particular proving to be the best compound (K. Yokoyama, K. Yamanouchi and R. Murashima: "Excretipn of perfluorochemicals after intravenous injection of their emulsion ", Chemical Pharmaceutical Bulletin, 23, 1368-1373 (June 1975).

Furthermore, it has been found that finely divided and stable fluorocarbon compound emulsions can be prepared from certain selected 9 to 11 C fluorocarbon compounds by emulsifying them with a mixture of egg yolk phospholipids or soybean phospholipids and a small amount of 8 to 22 C fatty acids. atoms or their salts or monoglycerides (DE Off.skrift 2 404 564).

Compared to a perfluorotributylamine emulsion stabilized with high molecular weight emulsifiers of polyoxyethylene-polyoxypropylene copolymer (R.P. Geyer, loe. eit.) the above-mentioned emulsion stabilized with both phospholipids and fatty acids is superior in terms of excretion rate, but inferior in terms of stability in the circulating blood stream after intravenous injection, the half-life being about two-thirds of the former.

Furthermore, an emulsion of fluorocarbon compounds such as perfluorotributylamine, in admixture with high molecular weight non-ionic surfactants, can be used as admixture in all proportions with marketed plasma thickening agents such as dextran or hydroxyethyl starch or modified gelatin solution, while the perfluorodecalin emulsion as described in DE off .skrift 2 404 564 cannot be used in combination with plasma thickeners due to precipitation that forms with the latter. It appears that these precipitations are due to breakdown of the emulsified particles due to reaction between the phospholipids contained in high concentration in the emulsion and the plasma thickener such as dextran or hydroxyethyl starch, which are high molecular weight colloidal substances.

When one wants to use a fluorocarbon emulsion as an infusion fluid or artificial blood substitute for saving the life of patients during severe bleeding, it is important that the emulsion can be used with plasma thickeners to ensure isotonic quality, i.e. to equalize the oncotic pressures for both colloidal solutions, i.e. , the emulsion and the blood, the fluorocarbon compound supplies oxygen while the plasma replacement makes it possible to keep the circulating blood volume at a suitable level. It is therefore advantageous to use a non-ionic surfactant with a high molecular weight which is inactive in the plasma substitute when producing a fluorocarbon emulsion to be used as artificial blood. Although these high molecular weight nonionic surfactants are effective in conjunction with perfluorotributylamine as an emulsifier, they are not suitable for perfluorodecalin, which has a high shedding rate.

Under these conditions, extensive pharmaceutical research has been carried out regarding the production of emulsions of perfluorodecalin, which have a high excretion rate and which are stable in the circulating blood stream and can be mixed with plasma replacement (plasma diluent),

without breakdown of the emulsified particles. The present invention is a result of these research works.

According to the present invention, a method has been provided for the production of a heat- and storage-stable blood substitute consisting of a mixture of oxygen-transferring perfluorocarbon compounds with particle sizes of 0.05-0.3 y in a physiological aqueous medium, and this method is characterized by homogeneously mixing 10-50% (w/v) together of (A) perfluorodecalin and (B) perfluorotripropylamine; 2-5% (w/v) of a high molecular weight non-ionic surfactant which is a polyoxyethylene-polyoxypropylene copolymer with a molecular weight of 2000-20,000, 0.1-1.0% (w/v) of a phospholipid , and 0.004-0.1% (w/v) of at least one fatty acid compound selected from fatty acids with 8-22 C atoms, their physiological salts or monoglycerides, where the quantity ratio between perfluorodecalin (A) and perfluorotripropylamine (B) is 95- 50 to 5-50 by weight, which components are mixed in said physiological aqueous medium to form a crude emulsion which is finely emulsified in a manner known per se by injection at a temperature of up to 55°C through a gap under a pressure of 100-500 kg/cm which subjects the emulsion to shear forces and mixing action based on a high velocity gradient, until the particle size of the perfluorocarbon compounds in the emulsion has reached down to 0.05-0.3 y.

This high molecular weight non-ionic surfactant has, as mentioned, a molecular weight of 2000 - 20,000, and includes polyoxyethylene-polyoxypropylene copolymers, polyoxyethylene alkyl ethers and polyoxyethylene alkyl aryl ethers. The concentration of surfactant in the emulsion is 2.0-5.0 and preferably 3.0-3.5% (w/v).

The symbol "% (w/v)" indicates the relative amount of a compound in grams based on 100 ml of emulsion formed.

The phospholipids used as emulsifier additives are of the usual type in the area, and those consisting of egg yolk phospholipid or soybean phospholipid are preferred. The amount in the emulsion is from 0.1-1.0% (w/v), and preferably 0.4-0.6% (w/v).

The fatty acid compound used as an emulsifying additive is a fatty acid with 8-22 C atoms, a physiological salt thereof such as the sodium or potassium salt or a mono-glyceride thereof, e.g. including caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, palmitoleic acid, oleic acid, linolenic acid, aracidic acid and sodium or potassium salts or monoglycerides thereof. Said fatty acid compounds can be used alone or as a mixture of two or more in a smaller amount of 0.004-0.1%

(w/v), preferably 0.02-0.04% (w/v). Among said fatty acid compounds, those with 14-20 C atoms and their physiological salts are preferred, and most preferably

potassium palmitate and potassium oleate are used because of their good solubility and ease of emulsification.

Emulsions of fluorocarbon compounds prepared

according to the invention is achieved as mentioned in a known manner by homogeneously mixing specific amounts of said components in the desired order in a physiologically applicable aqueous

medium such as distilled water or isotonic solution, e.g. for the production of a crude emulsion, and then emulsify the crude emulsion by injecting it at a temperature up to 55°C through a gap under a pressure of 100-500 kg/cm<2>

which thereby subjects the mixture to a shear force and mixing action based on a high velocity gradient until the desired particle size is reached.

Homogeneous mixing of the compounds is carried out in an ordinary mixing device such as a "homoblender" or propeller stirrer.

Emulsification of the crude emulsion is achieved with the help of a high-pressure homogenizer, which is a high-pressure pump that homogenizes a mixture of two immiscible liquids by pressing through a gap under high pressure and very high speed, which provides shearing force during mixing of the liquids.

A typical homogenizer on the market is a Manton-Gaulin homogenizer that has a multistage valve in combination with two or more additional valves that are spring-loaded to form slit openings.

The mixture is circulated through such a homogenizer several times under a total pressure of approx. 500 kg/cm until a stable emulsion according to the invention is formed. The operating temperature is kept in the range up to 55°C, preferably 25-40°C.

The emulsion has a dispersed phase of ultrafine particles with a diameter below 0.2 y or at most 0.3 y. Emul-

tion is stable and shows no growth in particle size even when heated or stored for longer periods.

The produced emulsion therefore secures the patient or the animal

against harmful effects caused by agglomeration of emulsified particles.

Furthermore, the produced emulsion has a long retention time in the circulating blood stream, so that the oxygen-carrying capacity or ability is maintained over a longer period of time.

Compared to e.g. with fluorocarbon emulsions prepared with phospholipids as emulsifier according to DE off. document 2 404 564, the emulsion prepared according to the invention will be kept longer, i.e. have greater retention in the blood stream. The excretion of this emulsion from the body takes place much faster than for the perfluorotributylamine emulsion.

The prepared emulsion can be used as an infusion liquid, after being physiologically isotonic.

It can be used as mixtures with marketed plasma expanders such as dextran, hydroxyethyl starch, and modified gelatin. Furthermore, the emulsions can be used as a blood substitute in mammals and perfusion fluid for the maintenance of internal organs.

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

Example_el_1_

300 g of polyoxyethylene-polyoxypropylene copolymer (molar weight: 10,800) are dissolved in 8 liters of distilled water. 40 g of soybean phospholipids, 2 g of potassium oleate and a mixture of 3 kg of perfluorodecalin and 300 g of perfluorotripropylamine are added to the solution. This mixture is stirred in a mixer to a crude emulsion. The raw emulsion is filled into the liquid tank in a spray emulsifier ("Manton-Gaulin") and emulsified by passing through the valve 12 times at a high pressure of 200 to 500 kg/cm and a liquid temperature of 35 - 5°C. The emulsion contains 30.5% (w/v) perfluorodecalin and 2.9% (w/v) perfluorotripropylamine. Average particle diameter is 0.09-0.1 m, measured by "centrifugal sedimentation". The emulsion showed no growth in particle size after filling the ampoule and temperature sterilization at 115°C for 12 minutes in a specially designed rotary sterilizer. Table 1 shows the particle size distribution for this emulsion and for an emulsion of perfluorodecalin alone prepared without perfluorotripropylamine.

As can be seen from table 1, the prepared emulsion stored at 4°C for 6 months shows no agglomeration and the average particle diameter is essentially unchanged.

?!S§E§£i2§£teksem£el_l

An emulsion was prepared according to the method in example 1 and the components used are listed in table 2 below. The following compounds were used as starting materials:

The comparison tests were carried out with a view to the following conditions:

1) Particle size of emulsion:

The mean particle size of the emulsion was determined after sterilization and after 6 months of storage at 4°C according to the centrifuge sedimentation method of K. Yokoyama,

et al. /Chem, Pharm. Bull., 22 (12), 2966-2971 (1974).7.

2) Co-use trials with a plasma substitute.

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

When the perfluorocarbon (FC) emulsion is used as a blood substitute, it is preferred to add a certain amount of plasma substitute to thereby set a colloid osmotic pressure. If, in this case, a reversible precipitate is formed upon impact between the FC emulsion and the plasma substitute, the emulsion cannot be used as a blood substitute. ' 3) Exchange-transfusion in the rat.

Comparative experiments with exchange transfusion were performed using rats (wistar strain weighing 200-250 g, male rats). Bleeding was performed from the carotid artery and replacement transfusion of FC emulsion through the tail vein up to a hematocrit value of 4.0. Then the survival time of the exchange-transfused amounts was determined.

4) Haemolytic effects.

Hemolytic effect of the FC emulsion in the extracorporeal circulatory system performed in vivo using rabbit red blood cells.

The FC emulsion was mixed with a lactated Ringer's solution to thereby render it substantially isotonic, and the resulting isotonic emulsion preparation was mixed with the heparinized rabbit blood in a 1:1 ratio to form a sample solution for the experiment. 8 ml of the sample solution was kept at 37°C for 6 hours, and then the free hemoglobin content was determined according to the cyanomethemoglobin method (Clin. Chim. Acta. 6, 538, 1961). In the case where a lactated Ringer's solution was used alone as a control without the use of FC emulsion, the free hemoglobin content was 128 mg. 5) Expiration rate of FC emulsion in vivo.

Expiration rate of FC emulsion in vivo was determined according to the method described in "Chemical Pharmacological Bull. 26, (3) 956-966, 1978", (K. Yokoyama, FATE OF PERFLUOROCHEMICALS IN ANIMALS AFTER INTRAVENOUS INJECTION OR HEMODILUTION WITH THEIR EMULSIONS). The FC emulsion was administered intravenously to rats in an amount of 4g/kg body weight, and the rate of expiration was noted as the number of days until half the administered amount of FC was used up by expiration.

The results obtained are shown in table 2 below.

An emulsion was prepared according to the method in example 1 and the components used are shown in table 3 below, and the starting materials were the same as used in experimental example 1.

The comparison tests were carried out as indicated in experimental example 1, and the results obtained are indicated in table 3 below.

From the results obtained above, the following can be concluded: In the case of Sample No. 1, the resulting FC emulsion is insufficient in terms of emulsification and it has low stability, so that an emulsion with an average particle size of less than 0.2 µ could not be achieved. The resulting emulsion is thus completely unsuitable as a medicine.

For sample No. 2, the resulting emulsion was excellent in terms of emulsification and stability, but it precipitated when mixed with hydroxyethyl starch and, in addition, it had a significant hemolytic effect and rats could not survive exchange-transfusion treatment. The emulsion cannot therefore be used as a medicine.

For Sample No. 3, the resulting emulsion has low stability, but can be administered in vivo as a drug only soon after it is prepared.

For Sample No. 4, the resulting emulsion showed the same result as for Sample No. 2.

For samples 5, 7 and 9, the resulting emulsions showed very excellent properties and can thus be used as a blood substitute.

For samples 6, 8, 10, 12 and 14, the resulting emulsions showed the same results as for sample 2.

For sample No. 11, the resulting emulsion showed

low expiration rate for FC in a living organism compared to what is the case for samples 5, 7 and 9, but the emulsion can be used as a medicine under satisfactory control.

For sample No. 13, the resulting emulsion showed a very low expiration rate, so that it may cause unexpected disadvantages in a living organism. In this case, the rat died after 69 hours when subjected to exchange transfusion.

From the above, it can be concluded that the FC emulsions that can be safely administered to a living organism are those according to samples 3, 5, 7, 9 and 11.

Comparison experiment

To further illustrate the advantage achieved with the present invention, a comparison was made with the emulsion used in Norwegian application no. 740641 (corresponding to US patent 3,911,188). An emulsion containing 10 to 30% w/v perfluorocarbon compound (e.g. perfluoromethylcyclohexane, perfluorodecalin, etc.) and 5 to 10% w/v polyoxyethylene-polyoxypropylene copolymer as emulsifier is described here. This reference teaches that said emulsion can be used as artificial blood, and the essential thing lies in the discovery of a fluorocarbon with a high excretion rate based on a number of fluorocarbons.

However, it appears from the comparison data below in Table 4 that the known emulsion is inferior to the one according to the present invention in terms of heat stability and storage stability.

The emulsions used were prepared using perfluorodecalin according to the method in the above example 1 and the storage stability and heat stability of the emulsions were compared. The composition of each emulsion is shown in the table. The stability of each emulsion was compared by measuring a particle diameter of the emulsion as shown by the average particle size (µm) according to the centrifugal sedimentation method described in Chem. Pharm. Bull., 22 (12), 2966-2971

(1974), (1974), K. Yokoyama et al.

As can be seen from table 4, the emulsion in the present invention is excellent in terms of storage stability. When the emulsion is prepared using egg yolk phospholipid or a nonionic surfactant or a combination of a nonionic surfactant, soybean phospholipid and K-oleate, the resulting emulsion is inferior in terms of storage and heat stability (experiments 3, 4 and 2) compared to the present invention, in which the emulsion is produced using perfluorotripropylamine,

a nonionic surfactant, soybean phospholipid and K-oleate in combination.

5ii2isj£§_studies

A perfluoro material {"Fluosol-DA" (20%)) as a whole blood substitute consisting of perfluorodecalin and perfluorotripropylamine was used in these studies. "Fluosol-DA" (20%) has the following composition:

"Fluosol-DA" (20%) material was infused into 10 normal, healthy, adult male volunteers. Three of them received a maximal amount of 500 ml h<y>er. No adverse reactions were noted and all haematological and biochemical values were within the normal range throughout the course of the infusion and subsequent follow-up. Based on these preliminary safe results, further safety and efficacy trials seem to be warranted.

Claims (1)

Process for the production of a heat- and storage-stable blood substitute consisting of a mixture of oxygen-transferring perfluorocarbon compounds with particle sizes of 0.05-0.3 y in a physiological aqueous medium, characterized by homogeneously mixing 10-50% (w/v) together of (A) perfluorodecalin and (B) perfluorotripropylamine > 2-5% (w/v) of a high molecular weight nonionic surfactant which is a polyoxyethylene-polyoxypropylene copolymer of molecular weight 2000-20,000; 0.1-1.0% (wt/vol.) of a phospholipid> and 0.004-0.1% (wt/vol.) of at least one fatty acid compound selected from fatty acids with 8-22 C atoms, their physiological salts or monoglycerides, ■ where the quantity ratio between perfluorodecalin (A) and perfluorotripropylamine (B) is 95-50 to 5-50 on a weight basis, which components are mixed in said physiological aqueous medium to form a crude emulsion which is finely emulsified in a manner known per se by injection at a temperature up to 55°C through a slit under a pressure of 100-500 kg/cm 2 which subjects the emulsion to shear forces and mixing action based on a high velocity gradient, until the particle size of the perfluorocarbon compounds in the emulsion reaches 0.05-0.3 y.