CA1072446A - Oxygen-transferable fluorocarbon emulsion - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Fluorocarbon-polyols have been suggested as an "artificial blood".
Emulsion of these compounds lack the stability required for pharmaceutical utility. The present invention seeks to overcome this drawback by providing a stable oxygen transferable fluorocarbon containing emulsion.

Description

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1 This invention relates to an oxygen-transferable fluorocarbon compound emulsion for injection and per-fusion, which is used in the lifesaving of a patient suffering from massive hemorrhage and in the preserva~tion of internal organs in transplantation and process for preparing same.
It has already been reported by a number o-f research people that fluorocarbon compound emulsions may possibly be used as an artifical blood substitute for mammals and as a perfusion fluid for preservation of internal organs to be transplanted 9 particularly as a substitute infusion fluid capable of transporting oxygen ~eland C. Clark, Jr., ~. Becattini, and S.
Kaplan: The physiology of synthetic blood., Journal of Thoracic Cardiovascular Surgery, 60, 757-773 (1970);
R. P. Geyer: ~luorocarbon-polyol artificial blood substitutes., ~ew ~ngland Journal of Medicine, 289, 1077 - 1082 (1973)~.
These emulsions, however, cannot be assumed as satisfactory enough for practical use in view of their pharmaceutical stability and of safety of living animal body. In order that fluorocarbon compound emulsions be qualified for practical use as an artificial blood substitute, it is necessary to develop a preparation which is sufficiently stable to be kept for a long period of time without change in particle size.
In fluorocarbon compound emulsions, the size of particle plays an important role in the to~icity and efficacy of the ernulsion ~K. Yokoyama, K. Yamanouchi, M. Watanabe, R. Murilshirna, 'L'. Matsurnoto, T. Hamnno, ~'7Z~

1 H. Okamoto, T. Suyama, R. Watanabe 7 and R. ~aito:
Preparation of perfluorodecalin emulsion, an approach to the red cells substitute., Federation Proceedings, 34, 1478-1483 (May, 1975) ~. An emulsion of larger 5 particle size is more toxic and shorter in retention time of the particles in the blood stream. According-ly, when the fluorocarbon compound emulsion is intended for use as an artificial blood substitute for saving the life of a patient suffering from massive hemorrhage, its average particle size should be 0. 3~ or less in diameter, preferably 0.2~ or less [Japanese Patent Application Kokai (~aid Open) ~o. 22612/73].
Besides the particle size, in order that the fluorocarbon compound emulsion be usable as artificial 15 blood substitute, it is necessary that after being eliminated from the blood stream, the intravenously administered fluorocarbon compound must be excre-ted from the body as rapidly as possible. Some of the present inventors had previously studied on~the excretion rate and toxicity of several sorts of fluorocarbon compound emulsions and, as a result, found that perfluorocarbon compounds of 9 to 11 carbon atoms are usable as the material of artificial blood substitute, particularly perfluorodecalin being the best of all [K. Yokoyama, K. Yamanouchi, and R.
Murashima: ~xcretion of perfluorochemicals after intravenous injection of their emulsion., Chemical Pharmaceutical Bulletin, 23, 1368-1373 ( June, 1975) ~.
In addition, some of the present inventors 3~ had also found tha-t the fine and stable fluorocarbon c ~L~'7Z~

1 compound emulsion can be prepared from these selected fluorocarbon compounds of 9 to 11 carbon atoms by emulsifying said fluorocarbon compounds with the mixture of egg yolk phospholipids or soybean phos-pholipids and a small amount of fatty acids of ~to 22 carbon atoms or salts thereof or monoglycerides thereof [Japanese Patent Application Kokai (Laid Open), No. 69,219/75].
However, compared with a perfluorotributylamine emulsion stabilized with a high-molecular-weight polyoxyethylene-polyoxypropylene copolymer emulsifier (R. P. Geyer, loc. cit.)~ the above-noted emulsion stabilized with both phospholipids and fatty acids is superior in the rate of excretion, bu-t inferior in 15 the stability in circulating blood stream after intra- ::
venous injection, the half life being about two-thirds of that of the former.
Further, a fluorocarbon compound emulsion prepared with a high-molecular-weight nonionic surface 20 active agent such as perfluorotributylamine emulsion, can be used as a mixture of any proportion with the commercial plasma expanders such as dextran, or hydroxyethylstarch, or modified gelatin solution, whereas the perfluorodecalin emulsion described in Japanese Patent Application Kokai (~aid Open) No.
69,219/75 cannot be used in combination with said plasma expander because of formation of precipitates when mixed with the latter. It seems that the precipitation is due to the destruction of emulsified particles caused by interaction between the phospholipid~

~0724~L6 1 contained in a high concentration in the emulsion and the plasma expander, such as dextran or hydroxyethyl-starch, which is a high-molecular-weight colloidal substance.
~hen it is intended to use a fluorocarbon eompound emulsion as an infusion fluid or an artificial blood substitute to save the life of patient in the ease of massive hemorrhage, the combined used with a plasma expander becomes important in order to make isotonicity, that is, to equalize oncotic pressures of both colloidal solutions, i.e. the emulsion and the blood; the fluorocarbon compound emulsion supplies oxygen, while the plasma expander makes it possible to maintain the circulating blood volume at a proper level. Therefore, it is preferable to use a high-moleeular-weight nonionic surface active agent which is inactive with the plasma expnader, in preparing a fluorocarbon emulsion intended for use as an artificial blood. Although these high-molecular-weight nonionic surface active agents are effeetive for some fluoro-carbon compounds such as perfluorotributylamine and other amine-type fluorocarbons as an emulsifier, they are not suitable for fluorocarbon compounds of 9 to 11 carbon atoms, such as perfluorodecalin, which have a high rate of excretion.
Under the circumstances, the present in-ventors eonducted extensive pharmaceutical investigations on preparing the emulsions of those fluorocarbons of 9 to 11 carbon atoms represented by perfluorodecalin, which have a high rate of excretion, which emulsions 1~72~

1 are stable in the circulating blood strcam and a.re able to mix with the plasma expander without any destruction of emulsified particles. As a result, the present invention has now been accomplished.
According to this invention, there is pro-vided a stable emulsion in a physiologically acceptable aqueous medium of an oxygen-transferable perfluorocarbon compound having a particle size of about 0.05 to 0.3,~ , which comprises (A) at least one perfluorocarbon com-pound having 9 - 11 carbon atoms selected *rom the group consisting of perfluorodecalin, perfluoromethyl-decalin, perfluoro alkylcyclohexanes having 3 to 5 carbon atoms in the alkyl, perfluoro alkyltetrahydro-furans having 5 to 7 carbon atoms in the alkyl, perfluoro alkyltetrahydropyrans having 4 to 6 carbon atoms in the alkyl, perfluoroalkanes having 9 to 11 carbon atoms; ~B) at least one perfluoro tert-amine having 9 to 11 carbon atoms selected from the group consisting of perfluoro tert-alkylamines having 9 to 11 carbon atoms, perfluoro N-alkylpiperidines having 4 to 6 carbon atoms in -the alkyl, and perfluoro N-alkylmorpholines having 5 to 7 carbon atoms in the alkyl; a high-molecular-weight nonionic surfactant having a molecular weight of about 2,000 to 207000; a phospholipid; and at least one fatty acid compound selected from the group consisting of fatty acids having ~ to 22 carbon atoms 9 physiologically acceptable salts and monoglycerides thereof; the ratio of the said perfluorocarbon compound and the said ~erfluoro-tert-~0 amine being 95 - 50 to 5 - 50 by weight.
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the sum of the concentration of the perfluorocarbon compound (A) and the perfluoro tert-amine (B) belng 10 to 50% (W/V), the concentration of the high molecular weight nonionic surfactant being 2.1 to 5.0%
(~/V), the concentration of the phospholipid being 0.1 to 1.0% (W/V) and the concentration of the fatty acid compound being 0.004 to 0.1% (W/V).

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l The "high-molecular-weight nonionic surfactant", as herein referred to, has a molecular weight of 2,000 to 20,000 and includes polyoxyethylene-polyoxypropylene copolymers, polyoxyethylene alkyl ethers, and poly-oxyethylene alkyl aryl ethers. The concentration of said surfactant in the emulsion is about 2.0 to about 5.0, preferably 3.0 to 3.5, ~o (W/V).
The symbol "% (W/V)" referred to in the specification and claim of this application means the amount proportion of a material by weight (gram) based on lO0 ml of the resulting emulsion.
Examples of the perfluorocarbons (A) having 9 to ll carbon atoms are a perfluorocycloalkane or perfluoro alkylcycloalkane which includes, for example, perfluoro C3_s-alkylcyclohexanes such as perfluoro-methylpropylcyclohexane, perfluorobutylcyclohexane, perfluorotrimethylcyclohexane, perfluoroethylpropyl-cyclohexane, perfluorodecalin and perfluoromethyl-decalin; a perfluoro C4_6-alkyltetrahydropyran such as perfluorohexyltetrahydropyran; a perfluoro Cs_7-alkyltetrahydrofuran such as perfluoro pentyltetra-hydrofuran, perfluoro hexyltetrahydrofuran and per-fluoro heptyltetrahydrofuran; and a perfluoroalkane having 9 - ll carbon atoms such as perfluorononane and perfluorodecane.
Examples of the perfluoro tert-amine (B) having 9 to ll carbon atoms are a perfluoro tert-alkylamine having 9 to ll carbon atoms which includes, for example, perfluorotrialkylamines such as perfluoro ~0 N,N-dibutylmonomethylamine, perfluoro N,N-diethyl-~C~72~

1 penty]amine, perfluoro N,N-dlethylhexylamine, per-fluoro N,N-dipropylbutylamine and per~luorotripropyl-amine; a perfluoro N,N-dialkylcyclohexylamlne having 9 - 11 carbon atoms such as perfluoro N9N-die-thyl-cyclohexylamine; a perfluoro N-C~_6~alkylpiperidine such as perfluoro N-pentylpiperidine, perfluoro ~-hexylpiperidine and perfluoro N-butylpiperidine; and ~a perfluoro ~-C5_7-alkylmorpholine such as perfluoro N-pentylmorpholine, perfluoro ~-hexylmorpholine and perfluoro N-heptylmorpholine.
The ratio of the perfluorocarbon compound (A) to the perfluoro tert-amine (B) to be used is 50 - 95 to 50 - 5 by weight and the total amount of (A) and (B) contained in the emulsion is about 10 to about 50 % (W/V).
The phospholipids used as emulsifier adjuvant in the invention are ones commonly used in the art, and those comprising yolk phospholipid or soybean phospholipid are preferable. The amount present in the emulsion ranges from about 0.1 to about 1.0 ~0 (W/V), and preferabIy about 0.~ to about 0.6 % (W/V).
The fatty acid compound used as emulsifying adjuvant is a fa-tty acid having 8 to 22 carbon atoms, a physiologically acceptable salt such as sodium or potassium salt or a monoglyceride thereof, ~hich in-cludes, for example, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, palmitoleic acid, oleic acid, linoleic acid, arachidonic acid and sodium or potassiurn salt and monoglyceride thereof. These ~atty acid compounds 7Z~

1 may be used alone or as a mixture of two or more kinds thereof in such a minor amount of 0.004 to 0.1 % (W/V), and preferably about 0.02 to 0.04 ~o (W/V). Among these fa.tty acid compounds the preferable ones are those having 14 to 20 carbon atoms and their physiological.ly acceptable salts, and the most preferable are potassium palmitate and potassium olea-te, taking into consideration of their good solubility and ease of the preparation of the emulsion.
The fluorocarbon compound emulsion of this invention is prepared by homogeneously mixing prescribed amounts of the aforesaid components in any order in a physiologically acceptable aqueous medium, such as distilled water 9 or isotonic solution to obtain a crude emulsion, and then emulsifying ~he crude emulsion by injecting it at a temperature of up to 55C through a slit under a pressure of about 100 kg/cm2 to 500 kg/cm2 thereby subjecting it to shearing force and mixing action based on a strong velocity gradient, until the desired .particle size previously mentioned is obtained The homogeneously mixing of the materials used is carried out by the use of a conventional mixer such as homoblender or propeller stirrerO
The emulsification of the crude emulsion is attained by means of a high pressure homogenizer, which is a high pressure pump which homogenizes a mixture of two immiscible liquids by injecting through a slit under a high pressure at a very high velocity to give a shear and mixing to the liquids. The typical homogenizer on market is Manton-Gaulin type homogenizer (Trldema:rk of this type of homogenizer sold by Manton-Gaulin .

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1 Manufacturing Co., Inc., U.S.A.) which has a multiple-stage valve in combination of -two or more valves each having a spring therein by which the slits are formed.
The mlxture is circulated in this type of homogenizer several times under the total pressure of about 500 kg/cm2 thereby to obtain the stable emulsion of the invention. The operating temperature is kept in a range of up to 55C, and preferably 25 to 40C.
The present emulsion has a dispersed phase of ultrafine par-ticles whose diameter is less than 0.2~4 or at most less than 0.3 ~ . Moreover, it is stable, showing no growth in particle size, even when heated or stored for a long period of time. Therefore, the present emulsion thus secures the administered animal to a high degree against harmful effect due to agglomeration of the emulsion particles.
The present emulsion, moreover, has a long retention time in the circulating blood stream, so that the oxygen-carrying capacity is maintained for a long period.
For instance 7 as compared with a fluorocarbon compound emulsion prepared by use of phospholipids as emulsifier according to Japanese Patent Application Kokai (~aid Open) No. 69219/75, the present emulsion retains for much longer time in the blood stream of animal. The excretion of the present emulsion from the body is far faster than that of a perfluorotri-bu-tylamine emulsion.
The present emulsion may be used as infusion fluid, after having been made physiologically isotonic.

~(:77;~4~6 1 It may be used also as mixtures with commercial plasma expanders such as Dextran, hydroxyethylstarch, and modified gelatin. ~urther, it can be used as a blood substitute for mammals and as a perfusate for preserving internal organs.
The present invention is further illustrated by the following Examples which should not be construed to limit the invention thereto.
In ~xamples the particle size was measured by the centrifugal sedimentation method proposed by K. Yokoyama, A. Suzuki, I. Utsumi and R. ~aito.
~hem. Pharm. ~ull. 22 (12), 2966 - 2971 (1974)~

Example 1 In 8 liters of distilled water, was dissolved 300 g of a polyoxyethylene polyoxypropylene copolymer Q~ lO~ ~o~
(molecular weight: 8,35~). To the solution were added 40 g of soybean phospholipids, 2 g of potassium oleate, and a mixture comprising 3 kg of perfluorodecalin and 300 g of perfluorotripropylamine. The resulting mixture was stirred in a mixer to form a crude emulsion. The resulting crude emulsion was charged into the liquor tank of a jet emulsifier (made by Manton-Gaulin Co.) and emulsified by passing it twelve times through a valve at a high pressure of 200 to 500 kg/cm2, while maintaining the liquor temperature at 35 ~ 5C, to effect emulsification. The resulting emulsion contained 30,5 ~0 (W/V) of perfluorodecalin and 2.9 ~0 (W/V) of perfluorotripropylamine. The average particle diameter was 0.09 to 0.1 ~, as measured by the centrifugal - 10 - `, ''.' ~ :

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1 sedlmentation method. The emulsion showed substantially no growth in particle size, when enclosed in a vial for injection and subjected to thermal sterilization at 115C
for 12 minutes in the special designed rotary sterilizer.
In Table 1 are shown the particle size dis-tribution of this emulsion and -that of an emulsion of perfluorodecalin alone prepared without using perfluorotripropylamine.
As seen from Table 1, when stored at 4C for 6 months, the present emulsion did not show any agglomeration, the mean particle diameter having been substantially unchanged.
The above procedure was repeated, excep-t that perfluoropentyltetrahydrofuran was used in place of the perfluorodecalin, and similar results to those shown above were obtained.

Example 2 In 8 liters of distilled water, was dissolved 330 g of a polyoxyethylene octyl ether (average molecular weight: 3,500). To the solution were added 40 g of soybean phospholipid and 2 g of potassium oleate, and the resulting mixture was stirred in a mixer to prepare a dispersion. To the dispersion was added a mixture comprising 3 kg of perfluoromethyldecalin and 600 g of perfluoro-~-pentylpiperidine and the resulting mixture was stirred in a mixer to prepare a crude emulsion. The crude emulsion was thoroughly emulsified in the same manner as in Example 1, and the resulting emulsion was filled in small vials~ The vial containing the emulsion was subjected to thermal steriliæation at 1Ci~7~

1 115C for 12 minutes in the rotary sterilizer. The emulsion contained 29.7 ~ (W/V) of perfluoromethyl-decalin and 5.8 % (W/V) of perfluoro-N-pentylpiperidine.
The particle size distribution after sterilization and the average particle diameter after storage at 4C ~or 6 months, as well as those of` a reference emulsion of perfluorodecalin alone were as shown in Table 1.

Example 3 In 2 liters of distilled water, was dissolved 100 g of a polyoxyethylene-polyoxypropylene having an A~ average molecular weight of ~84~. To the resulting solution were added 20 g of yol~ phospholipids and 0.5 g of oleic acid, and the mixture was stirred in a mixer to prepare a dispersion. To the dispersion was added a mixture comprising 6~0 g of perfluorodecalin and 250 g of perfluorodibutylmonomethylamine, and the resulting mixture was stirred in a mixer to obtain a crude emulsion. The crude emulsion was emulsified in the same manner as in Example 1 and sterilized by heating at 115C for 12 min. in the rotary sterilizer. The emulsion contained 25.3 % (W/V) of perfluorodecalin and 9.8 % (W/V) of perfluorodibutylmonomethylamine.
The avsrage particle diameter and -the particle size distribution of the present emulsion and those of a reference sample of an emulsion prepared by use of perfluorodecalin alone were as shown in Table 1. In Table 1 is also shown the average particle diameter of the present emulsion after storage at ~C f`or 6 months.

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1 ~xample 4 In 800 ml of distilled water, was dissolved 35 g of a polyoxyethylene-polyoxypropylene copolymer havi.ng an averag~e molecular weight of 15,800. To the solution were added 4 g of yolk phospholipids and 0.1 g of monoglyceride of lauric acid, and the resulting mixture was stirred in a mixer to prepare a dispersion.
To the dispersion was added a mixture comprising 350 g of perfluorohexyltetrahydropyran and 40 g of per1uoro-N,N-diethylcyclohexylamine, and the resulting mixture was stirred in a mixer to prepare a crude emulsion.
The crude emulsion was further emulsified in the same manner as in Example 1, and the resulting emulsion was subdivided into small portions which were enclosed in vials. The vial containing the emulsion was subjected to thermal sterilization at 115C for 12 min. in a rotary sterilizer. The emulsion contained 35.7 ~ (W/V) of perfluorohexyltetrahydropyran and 4.1 % (W/V) of perfluoro-N,N-diethylcyclohexylamine.
The average particle diameter of the present emulsion and that of a reference sample of emulsion prepared by use of perfluorohyxyltetrahydropyran alone after sterilization is shown in Table 1. The present emulsion showed no change of particle size after storage at 4C for 6 months.

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1 Experimental Example 1 Test for mixing with plasma expander For the clinical use as infusion fluid, the present emulsion is used preferably in combination with a plasma expander to replenish the defficiency in oncotic pressure. When the present emulsion was mixed with a plasma expander, the reversible precipitation which migh-t be caused by interaction between both colloidal solutions was not detected, indicating that one of the difficulties which might be encountered in using the present emulsion as an infusion fluid has been eliminated.
The emulsions used in the experiment were perfluorodecalin-perfluoro-~,~-dibutylmethylamine ;~
(5 : 2) emulsions of various concen-trations prepared in the same manner as in Example 3 [polyoxyethylene-polyoxypropylene copolymer, 3.4 % (W/V); yolk phos-pholipids, 0.6 % (W/V); potassium oleate, 0.04 ~0 (W/V)]
and, as a reference, perfluorodecalin emulsions of various concentrations prepared according to Japanese Patent Application Kokai (Laid Open) ~o. 69219/75 [yolk phospholipid, 4 % (W/V); potassium oleate, 0.02 % (W/V)]. Each emulsion was made isotonic with the lactated Ringer's solution or Krebs-Ringer bi-carbonate solution, then admixed with a plasma expanderso that the final concentration of the latter may become 1 to 6 ~ (W/V), and forma-tion of precipitates was visually observed during 6 hrs. after mixing at room temperature. Plasma expanders used are hydroxy-~0 ethyistarch (HE~) (average molecular weight: 200,000 ~7;~44~

1 20 ~ (W/V) in saline, supplied by Ajinomoto Co., IJtd.) and Dextran 40 [Dextran (average molecular weight, 40,000) 10 ~ (W/V) in saline in water; supplied by The Green Cross Corp.]~
The results are shown in Tables 2 and 3.

Table 2 _ \ Final fluoro- Emulsion, Jap.
\ carbon Emulsion of the Pat. Appl. ~aid \ conten-t, invention Open No.
Einal \ ~0 (W/V) 69219/ 75 Dextran 40 con~

Table \ Final fluoro- _ ~ Emulsion, Jap.
\ carbon content Emulsion of the Pat. Appl. I,aid \ % (W/V) invention Open No.
Final \ 69219/75 ~ 1~

~ 19 -~ ~ 7 ~

1 Note: -: non precipita-tes +: formation of precipitates Form the results obtained above, it was made clear that the p-resent emulsion is much less affected by the presence of a plasma expander compared wlth the emulsion according to Japanese Patent Application Kokai (~aid Open) ~o. 69219/75, indicating that -the present emulsion can be mixed wlth the Dextran 40 and HES
porf/on preparation in any~ to make it the physiologically ~: .
colloidal isotonicity which obtained by addition of Dextran 40 and HES as a final concentration of

2 % (W/V) and 3 ~0 (W/V), respectively.
Similar results to those mentioned above were also found in the emulsions prepared in Examples 1, 2, and 4.

Experimental ~xample 2 In order to evaluate the efficacy of the present emulsion, the study on the exchange-transfusion in rats was done.

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1 Two kinds of fluorocarbon compound emulsion which were the present emulsion, the mixture of per-fluorodecalin and per-fluorodibutylmonome-thylamine prepared in Example 3 and per~luorodecalin emulsion stabilized with yolk phospholipids according to Japanese Patent Application Kokai (Laid Open) No.
69219/75 were used in this experiment.
The ingredients of both emulsion were shown in Table 4.
To make the emulsion electrolyte and colloidal isotonification, one volume of hypertonic electrolytes solution shown Table 4 is added to 9 volume of the emulsion, and then9 1 volume of resultant emulsion containing electrolytes were mixed with 3 volume f 6 % hydroxyethyl starch (molecular weight 40 000 -50,000) in Ringer's lactated solution or rat plasma as reference, prior to use.
sJ~er The rats (~ri~ strain weighing 200 to 250 g) were exchange transfused with the emulsion containing electrolytes and hydroxyethyl starch or plasma by repeated bleeding from carotid artery and replacement transfusion through tail vein alternately up to hematocrit 1 %, 4 % and 7 %, respectively~ under the 100 % oxygen atomosphere.
Then, the survival time of rats exchange~
transfused were determined.
The results are shown in Table 5. As is evident in Table 5, the present emulsion was far more effective in saving the life o-f animal hemorraged massively in comparing with the perfluorodecalin ~7~

1 emulsion st~bilized ~lith yolk phospholipids.

Table 5 . Jointly withJointly with Flnal plasma ~ES
hematocrit _ _ value % ~urvival time 7> 72 hours> 72 hours The present 4 50 0O min. ~ 72 emulsion 1 29 02 61 oo min.
_ ~mulsion 7 23 hUrs 10 min. 5 hours 53 min-acc. to Jap.
Pat. Appl. 4 11 5 5 2 ~aid Open ~o. 69219/75 1 8 10 2 56 . _ ~ote: The values obtained from 5 rats in each group.

~xperimental Example 3 The present fluorocarbon compound emulsion obtained in Example 3 and the emulsion obtained accord-ing to Japanese Patent Application Kokai (~aid Open) No. 69219/75 were subjected to acute toxicity test.
In order -to make these emulsion isotonifica-tion, one volume of electrolytes is added to 9 volume f the emulsion prior to use The ingredients of these emulsions were shown in Table 4. As test animals were used wister strain male rats weighing 100 to 120 g.
The rats were intravenously injected with the emulsion and the survival rate was observed during one week after injection. The results obtained are shown in 4~

1 Table 6. As seen in Table 6, the ~Dso of both emul-sions was around 130 ml/kg body weight, indicating they were quite low-toxic.

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1 ~xperimental ~xample 4 In order to examine hemolytic effect of the fluorocarbon preparations in the c~tracorporea:L circula-tion system, experiments in vitro were carried out by using red blood cells of rabbit.
Two emulsion preparations used in Experi-mental Example 2 as shown in Table 4 were admixed with a lactated Ringer's solution so as to become substantially isotonic physiologically. The result-ing isotonic emulsion preparations were mixed withthe heperinized rabbit blood in a ratio of 3 : 1, 1 : 1, and 1 : 3 to prepare sample solutions for the tests. The hemolytic effect was evaluated by measuring the free hemoglobin content of 8 ml of the blood after having been kept at 37C for 6 hours. Determination of hemolyzed hemoglobin was determined by the cyanomethemo-globin method (Kampen, ~. J. and Ziilstram, W. J.
Clin. Chim. Acta. 6, 538, 1961).
The results obtained are shown in Table 7.

Table 7 ______ ~ _ ~ _ Free hemoglobin, mg . _ . _ Fluorocarbon : blood ratio 1 : 3 1 : 1 3 : 1 Fmulsion of this invention 36 81 180 Emulsion acc. to Jap. Pat.
Appl. ~aid Open No. 298.5> 4,000 3,380 ~actated Ringer's solution 36 128 141 (r~r~ ' _. _ ~7~4~

1 As seen from Table 7, the hemolytic effect of the present emulsion is far smaller than that of prior art and not much different from that of the lactated Ringer's solution used as reference.

Claims (39)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A stable emulsion in a physiologically acceptable aqueous medium of an oxygen-transferable perfluorocarbon compound having a particle size of about 0.05 to 0.3µ, which comprises (A) at least one perfluorocarbon com-pound having 9 - 11 carbon atoms selected from the group consisting of perflu-orodecalin, perfluoromethyldecalin, perfluoro alkylcyclohexane having 3 to 5 carbon atoms in the alkyl, perfluoro alkyltetrahydrofuran having 5 to 7 carbon atoms in the alkyl, and perfluoro alkyltetrahydropyran having 4 to 6 carbon atoms in the alkyl, perfluoroalkanes having 9 - 11 carbon atoms, (B) at least one perfluoro tert-amine having 9 - 11 carbon atoms selected from the group consisting of perfluoro tert-alkylamine having 9 - 11 carbon atoms, perfluoro N-alkylpiperidine having 4 to 6 carbon atoms in the alkyl, and perfluoro N-alkylmorpholine having 5 - 7 carbon atoms in the alkyl; a high molecular weight nonionic surfactant having a molecular weight of about 2,000 to about 20,000; a phospholipid; and at least one fatty acid compound selected from the group consisting of fatty acids having 8 to 22 carbon atoms, physiologi-cally acceptable salts and monoglycerides thereof; the proportion of the said perfluorocarbon compound (A) and the said perfluoro tert-amine (B) being 95 - 50 to 5 - 50 by weight, the sum of the concentration of the perfluoro-carbon compound (A) and the perfluoro tert-amine (B) being 10 to 50% (W/V), the concentration of the high molecular weight nonionic surfactant being 2.1 to 5.0% (W/V), the concentration of the phospholipid being 0.1 to 1.0% (W/V) and the concentration of the fatty acid compound being 0.004 to 0.1% (W/V).

2. The emulsion according to Claim 1 wherein the perfluorocarbon com-pound (A) is perfluoro alkylcyclohexane having 3 to 5 carbon atoms in the alkyl, perfluorodecalin or perfluoro methyldecalin.

3. The emulsion according to Claim 1 wherein the perfluorocarbon com-pound (A) is a perfluoro alkyltetrahydropyran having 4 to 6 carbon atoms in the alkyl, a perfluoro alkyltetrahydrofuran having 5 to 7 carbon atoms in the alkyl.

4. The emulsion according to Claim 1, wherein the perfluoro tert-amine is a perfluoro tert-alkylamine.

5. The emulsion according to Claim 4, wherein the perfluoro tert-amine is perfluoro N,N-dibutylmethylamine, perfluoro N,N-diethylpentylamine, per-fluoro N,N-diethylhexylamine, perfluoro N,N-dipropylbutylamine, perfluoro tripropylamine, or perfluoro N,N-diethylcyclohexylamine.

6. The emulsion according to Claim 4, wherein the perfluoro tert-amine is a N-alkyl heterocyclic amine selected from the group of a perfluoro N-alkylpiperidine having 4 to 6 carbon atoms in the alkyl, and a perfluoro N-alkylmorpholine having 5 to 7 carbon atoms in the alkyl.

7. The emulsion according to Claim 1, wherein the physiologically acceptable aqueous medium is water or an isotonic solution.

8. The emulsion according to Claim 7, wherein the isotonic solution is a lactated Ringer's solution or a Ringer's solution containing glucose.

9. The emulsion according to Claim 1, wherein the fatty acid compound is caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, palmitoleic acid, oleic acid, linoleic acid or arachidonic acid.

10. The emulsion according to Claim 1, wherein the fatty acid is an alkali metal salt of a fatty acid selected from caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, palmitoleic acid, oleic acid, linoleic acid and arachidonic acid.

11. The emulsion according to Claim 10, wherein the alkali metal salt of the fatty acid is potassium oleate or sodium oleate.

12. The emulsion according to Claim 10, wherein the alkali metal salt of the fatty acid is potassium palmitate or sodium palmitate.

13. The emulsion according to Claim 1, wherein the fatty acid compound is monoglyceride of a fatty acid selected from caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, palmitoleic acid, oleic acid, linoleic acid and arachidonic acid.

14. The emulsion according to claim 1, wherein the phospholipid is egg yolk phospholipids or soy-bean phospholipids.

15. The emulsion according to Claim 1, wherein the emulsion is made iso-tonicity in its oncotic pressure with blood by the addition of plasma or plas-ma expander.

16. The emulsion according to Claim 15, wherein the plasma expander is hydroxyethyl starch modified gelatin or dextran.

17. The emulsion according to Claim 1, wherein the high molecular weight nonionic surfactant is a polyoxyethylene-polyoxypropylene copolymer having a molecular weight of 2,000 to 20,000, or a polyoxyethylene alkyl ether having a molecular weight of 2,000 to 20,000, or a polyoxyethylene alkyl aryl ether having a molecular weight of 2,000 to 20,000.

18. The emulsion according to Claim 17, wherein the polyoxethylene-polyoxypropylene copolymer has a molecular weight of 8,350 to 15,800.

19. The emulsion according to Claim 17, wherein the polyoxyethylene alkyl ether is polyoxyethylene octyl ether having a molecular weight of 3,500.

20. A process for preparing a stable emulsion in a physiologically acceptable aqueous medium of an oxygen-transferable perfluorocarbon compound having a particle size of about 0.05 to 0.3µ , which comprises homogeneously mixing (A) at least one perfluorocarbon compound having 9 - 11 carbon atoms selected from the group consisting of perfluorodecalin, perfluoromethyl-decalin, perfluoro alkylcyclohexane having 3 to 5 carbon atoms in the alkyl, perfluoro alkyltetrahydrofuran having 5 to 7 carbon atoms in the alkyl, and perfluoro alkyltetrahydropyran having 4 to 6 carbon atoms in the alkyl, perfluoroalkanes having 9 - 11 carbon atoms, (B) at least one perfluoro tert-amine having 9 - 11 carbon atoms selected from the group consisting of per-fluoro tert-alkylamine having 9 - 11 carbon atoms, perfluoro N-alkylpiperi-dine having 4 to 6 carbon atoms in the alkyl, and perfluoro N-alkylmorpho-line having 5 - 7 carbon atoms in the alkyl; a high molecular weight non-ionic surfactant having a molecular weight of about 2,000 to about 20,000;

a phospholipid; and at least one fatty acid compound selected from the group consisting of fatty acids having 8 to 22 carbon atoms, physiologically acceptable salts and monoglycerides thereof; the proportion of the said perfluorocarbon compound (A) and tile said perfluoro tert-amine (B) being 95 - 50 to 5 - 50 by weight, in said physiologically acceptable aqueous medium to obtain a crude emulsion, and further emulsifying the crude emul-sion by injecting it at a temperature of up to 55°C through a slit under a pressure of about 100 kg/cm2 to 500 kg/cm2 -thereby subjecting it to shearing force and mixing action based on a strong velocity gradient until the particle size of the perfluorocarbon compound in the resulting emulsion reaches 0.05 to 0.3 µ.

21. The process according to Claim 20, wherein the perfluorocarbon com-pound (A) is perfluoro alkylcyclohexane having 3 to 5 carbon atoms in the alkyl, perfluorodecalin or perfluoro methydecalin.

22. The process according to Claim 20, wherein the perfluorocarbon com-pound (A) is a perfluoro alkyltetrahydropyran having 4 to 6 carbon atoms in the alkyl, a perfluoro alkyltetrahydrofuran having 5 to 7 carbon atoms in the alkyl.

23. The process according to Claim 20, wherein the perfluoro tert-amine is a perfluoro tert-alkylamine.

24. The process according to Claim 23, wherein the perfluoro tert-amine is perfluoro N,N-dibutylmethylamine, perfluoro N,N-diethylpentylamine, perfluoro N,N-diethylhexylamine, perfluoro N,N-dipropylbutylamine, perfluoro tripropylamine or perfluoro N,N-diethylcyclohexylamine.

25. The emulsion according to Claim 24, wherein the perfluoro tert-amine is a N-alkyl heterocyclic amine selected from the group of a perfluoro N-alkylpiperidine having 4 to 6 carbon atoms in the alkyl, and a perfluoro N-alkylmorpholine having 5 to 7 carbon atoms in the alkyl.

26. The process according to Claim 21, wherein the physiologically acceptable aqueous medium is water or isotonic solution.

27. The process according to Claim 26, wherein the isotonic solution is a lactated Ringer's solution or a Ringer's solution containing glucose.

28. The process according to Claim 21, wherein the sum of the amount of the perfluorocarbon compound and the perfluoro tert-amine is 10 to 50%
(W/V), the amount of the high molecular weight nonionic surfactant is 2.0 to 5.0% (W/V), that of the phospholipid is 0.1 to 1.0% (W/V) and that of the fatty acid compound is 0.004 to 0.1% (W/V).

29. The process according to Claim 20, wherein the fatty acid compound is caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, palmitoleic acid, oleic acid, linoleic acid or arachidonic acid.

30. The process according to Claim 20, wherein the fatty acid is an alkali metal salt of a fatty acid selected from caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, palmitoleic acid, oleic acid, linoleic acid and arachidonic acid.

31. The process according to Claim 30, wherein the alkali metal salt of the fatty acid is potassium oleate or sodium oleate.

32. The process according to Claim 30, wherein the alkali metal salt of the fatty acid is potassium palmitate or sodium palmitate.

33. The process according to Claim 20, wherein the fatty acid compound is monoglyceride of a fatty acid selected from caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, palmitoleic acid, oleic acid, linoleic acid and arachidonic acid.

34. The process according to Claim 20, wherein the phospholipid is egg yolk phospholipids or soybean phospholipds.

35. The process according to Claim 20, wherein the emulsion is made isotonicity in its oncotic pressure with blood by the addition of plasma or plasma expander.

36. The process according to Claim 35, wherein the plasma expander is hydroxyethylstarch, modified gelatin or dextran.

37. The process according to Claim 20, wherein the high molecular weight nonionic surfactant is a polyoxyethylene-polyoxypropylene copolymer having a molecular weight of 2,000 to 20,000, a polyoxyethylene alkyl ether having a molecular weight of 2,000 to 20,000, or a polyoxyethylene alkyl aryl ether having a molecular weight of 2,000 to 20,000.

38. The process according to Claim 37, wherein the polyoxyethylene-polyoxypropylene copolymer has a molecular weight of 8,350 to 15,800.

39. The process according to Claim 37, wherein the polyoxyethylene alkyl ether is polyoxyethylene octyl ether having a molecular weight of 3,500.