WO1995028146A1

WO1995028146A1 - Fluorochemical emulsions containing 1,3-dialkylglycerophosphoryl choline surfactants and methods of use

Info

Publication number: WO1995028146A1
Application number: PCT/US1995/004415
Authority: WO
Inventors: Robert J. Kaufman; Thomas J. Richard
Original assignee: Hemagen/Pfc
Priority date: 1994-04-15
Filing date: 1995-04-11
Publication date: 1995-10-26
Also published as: AU2245295A

Abstract

Fluorochemical emulsions containing 1,3-dialkylglycerophosphoryl choline surfactants and methods of use are disclosed. These emulsions have general utility for many industrial uses and are especially useful as oxygen transport agents, artificial bloods or red blood cell substitutes and as contrast agents for biological imaging.

Description

FLUOROCHEMICAL EMULSIONS CONTAINING 1,3-DIALKYLGLYCEROPHOSFHORYL CHOLINE SURFACTANTS AND METHODS OF USE

RELATED APPLICATION

This application is related to application

Ser. No. 07/791,420, filed November 13, 1991, now U.S.

Patent No. 5,304,325, and the disclosure thereof is incorporated herein by reference in its entirety.

TECHNICAL FIELD OF THE INVENTION

This invention relates to emulsions of fluorochemicals and water, and processes of making and using them. More particularly, this invention relates to novel emulsions that contain a perfluorochemical, water and a novel surfactant that may be generally identified as a 1, 3-dialkylglycerophosphoryl choline.

Such emulsions have general utility for many industrial uses and are especially useful as oxygen transport agents, artificial bloods or red blood cell substitutes and as contrast agents for biological imaging. BACKGROUND OF THE INVENTION

One class of emulsions that has developed over a number of years is fluorocarbon emulsions as oxygen transport agents or artificial bloods. U.S. Patent No. 3,911,138 which issued to Clark is an early example from the patent art which discloses perfluorocarbon emulsions as artificial bloods. As developed in this patent, neat fluorocarbon liquids cannot be injected into the blood stream because their hydrophobic character makes them immiscible in the blood and, as a result, when they are transported in small blood vessels, they may cause vascular obstruction and death. As a consequence, for medical uses that require intravascular injection, highly fluorinated organic compounds or fluorochemicals must be dispersed as physiologically accepted emulsions as developed in the above Clark patent and U.S. Patent Nos. 4,110,474; 4,178,252 and 4,443,480.

There have been various attempts to make emulsions that are both stable and incorporate relatively large amounts of fluorocarbons that are required in clinical practice where the total volume of the emulsion that can be administered is limited, e.g., as in artificial bloods. An objective in the preparation of such emulsions is the employment, of an acceptable fluorocarbon that may be excreted from the body within a clinically acceptable time period. Furthermore, compositions are required that are sterilizable without destruction of their stability. A fluorocarbon emulsion that has been approved by the FDA is FLUOSOL DA which is an emulsion of perfluorocarbon and perfluorotripropylamine in a mixture of two surfactants, namely, egg yolk phospholipid and Pluronic F-68. This product, however, is not stable in the liquid state and must be stored frozen.

Furthermore, the required presence of the perfluorotripropylamine in this emulsion to help stabilize it disadvantages the emulsion's medical usefulness because the half life of the perfluorotripropylamine in the liver and other body tissues is longer than desirable (see K. Yokoyama et al., "A Perfluorochemical Emulsion as an Oxygen Carrier"

Artif. Organs (Cleve), 8 (1) pp. 34-40 (1984)). Finally, this emulsion contains only about 12% fluorocarbon by volume and thus it is much less therapeutically effective than desired because of its low oxygen content capacity.

Various surfactants have also been investigated in an attempt to produce useful and stable emulsions of fluorocarbons as oxygen transport agents in artificial bloods. For example, fluorocarbon emulsions containing a hydrogenated phospholipid, a nonionic polymeric surfactant and a surfactant selected from C6- 22 fatty acids, their salts and monoglycerides, must also be stored at 4ºC. See, e.g., Japanese Patent Application 59-067, 229; U.S. Patent No. 4,252,827 and German Offen. DE 2630506. European Patent Application 87300454.3 of Clark and Shaw describes novel emulsions of highly fluorinated organic compounds for use as oxygen transport agents and artificial bloods. This Clark and Shaw application discloses emulsions that are stable even when they contain higher levels of perfluorocarbons of up to about 75% by volume. The fluorocarbons of these emulsions display acceptable rapid excretion times from the liver and other body tissues, as well as being easily sterilized. These emulsions contain an oil as an emulsifying adjuvant in a composition containing the fluorochemical, surfactant and water. While improvements disclosed in this Clark and Shaw application are significant and alleviate many of the difficulties in the long search for effective transport agents in artificial bloods, there is a continuing need for further development.

In brief, emulsions of fluorochemicals and water provide a very important role in many industries and numerous patents have been granted covering them. Research continues with efforts toward developing new emulsifying agents that provide emulsions having greater stability and broader utility in many industries including medical and non-medical fields.

SUMMARY OF THE INVENTION

According to this invention, novel surfactants have been found to form surprisingly stable fluorochemica1-in-water emulsions. More particularly. fluorochemical (hereinafter sometimes simply "PFC") emulsions have been made and found to significantly increase the circulatory blood residence time of the PFC and favorably alter the tissue distribution of the PFC in critical organs, such as the liver and spleen. Furthermore, the novel surfactants of this invention have been found to significantly ameliorate the adverse drop of hematocrit, or red blood cell count, after intravenous infusion, normally associated with most lecithin based PFC emulsions.

The fluorochemical emulsions of this invention contain novel surfactants that may generally be identified as 1,3-dialkylglycerophosphoryl choline surfactants or related 1,3-diaklylglycerophosphoryl group-containing surfactants. These surfactants have the following general structure:

More specifically, R₁ and R₂ are a C₁₂-C₂₂ saturated or unsaturated aliphatic group. For physiologically acceptable emulsions, the total carbon content of R₁ and R₂ is preferably at least 28. In general, these R groups are the residues of aliphatic alcohol reactants when synthesized by methods of this invention. PC in the above structures is the phosphoryl choline or similar group and salts thereof represented by the structure:

where R₄ is hydrogen or lower alkyl such as methyl, ethyl and propyl. The hydrogen or methyl group is preferred in medical PFC emulsions of this invention.

This invention also includes methods of making these surfactants, emulsions containing them and methods of using them as oxygen transport agents, artificial bloods or red blood cell substitutes. Other objectives of this invention and advantages will become apparent from the following detailed description.

DETAILED DESCRIPTION

The fluorochemical emulsions of this invention comprise a fluorochemical, water and a surfactant of the above identified type. More specifically, for instance, in medical applications for intravenous use, about 60% v/v (115 w/v%) of fluorochemical is a practical limit because of viscosity limitations for an intravenous product. Higher amounts may be employed for other applications. The surfactant may be contained in amounts from about 0.5 to about 10% by weight, usually about 1-2% by weight of the emulsion. These components are identified with greater particularity as follows. A. Fluorochemical

In this description, "fluorochemical" or "PFC" is used to describe either a highly fluorinated organic compound of perfluorocarbon or fluorinated chemical. Further, these terms are used interchangeably. The term

"perfluorocarbon" includes a "cyclic" or "acyclic" compound of carbon. Substituted derivatives thereof are also included where fluorocarbons have other elements within their structures such as oxygen, nitrogen and bromine, etc. It should also be noted that the term

"perfluorocarbon" is meant to include partially or substantially fluorinated compounds. This is permissible providing that the lack of complete replacement of all hydrogens does not affect the essential non-toxic characteristics of the preferred medical fluorocarbons of this invention. Among the perfluorocarbon compounds which may be employed are perfluorotributylamine (FC47), perfluorodecalin (PP5), perfluoromethyldecalin (PP9), perfluorooctylbromide, perfluorotetrahydrofuran (FC80), perfluoroether (PID)

[(CF₃)₂CFOCF₂(CF₂)₂CF₂OCF(CF₃)₂] perfluoroether (PIID)

[CF₃)₂CFOCF₂(CF₂) ₆CF₂OCF(CF₃)₂] , perfluoropolymer (E3)

, perfluoropolymer (E4)

perfluoroetherpolymer (Fomblin Y/01), perfluorododecane, perfluorobicyclo [ 4 . 3 . 0 . ] nonane , perfluorotritrimethylbl cyclohexane, perfluorotripropylamine, perfluoroisopropyl cyclohexane, perfluoroendotetrahydrodicyclopentadiene, perfluoroadamantane, perfluoroexot e t r a h y dr o d i cy c l o p e n t a d i e n e , per f luorbi cycl o [ 5 . 3 . 0 . ] decane , perfluorotetramethylcyclohexane, perfluoro-1-methyl-4╌ isopropylcyclohexane, perfluoro-n-butyleyclohexane, perfluorodimethylbicyclo[3.3.1.]nonane, perfluoro-1-methyl adamantane, perfluoro-1-methyl-4-t butylcyclohexane, perfluorodecahydroacenapthane, perfluorotrimethylbicyclo[3.3.1.]nonane, perfluoro-1-methyl adamantane, perfluoro-1-methyl-4-t butylcyclohexane, perfluorodecahydroacenaphthene, perfluorotrimethylbicyclo[3.3.1.]nonane, perfluoro-n-undecane, perfluorotetradecahydrophenanthrene, perfluoro-1,3,5,7-tetramethyladamantane, perfluorododecahydrofluorene, perfluoro-1-3- dimethyladamantane, perfluoro-n-octylcyclohexane, perfluoro-7-methylbicyclo[4.3.0.] nonane, perfluoro-p- diisopropylcyclohexane, and perfluoro-m- diisopropylcyclohexane. Chlorinated perfluorocarbons, such as chloroadamantane and chloromethyladamantane as described in U.S. Patent No. 4,686,024 may be used. Such compounds are described, for example in U.S. Patent

Nos. 3,962,439; 3,493,581, 4,110,474, 4,186,253; 4,187,252; 4,252,824; 4,423,077; 4,443,480; 4,534,978 and 4,542,147, European Patent Application Nos. 80710 and 158,996, British Patent specification 1,549,018 and German Offen. 2,650,586. Of course, it should be understood that mixtures of any of these highly fluorinated organic compounds may also be used in the emulsions and processes of this invention.

B. Surfactant

According to this invention, novel surfactants have been found to form surprisingly stable oi1-in-water emulsions. More particularly, stable PFC emulsions have been made and found to significantly increase the circulatory blood residence time of the PFC and favorably alter the tissue distribution of the PFC in critical organs, such as the liver and spleen. Furthermore, surfactants of this invention have been found to significantly ameliorate the adverse drop in hematocrit, or red blood cell count, after intravenous infusion, normally associated with most lecithin based PFC emulsions.

The emulsions of this invention contain 1,3- dialkylglycerophosphoryl choline or similar 1,3- dialkylglycerophosphoryl group-containing surfactants.

These surfactants have the following general structure:

More specifically, R₁ and R₂ are a C₁₂-C₂₂ saturated or unsaturated aliphatic group. In general, these R groups are the residues of aliphatic alcohol reactant when synthesized by methods of this invention.

PC in the above structure is the phosphoryl choline or similar group and salts thereof represented by the structure:

Specific examples of the surfactants under the general structures are selected from the group consisting of 1-dodecyl-3-hexadecylglycero-2-phosphoryl choline, 1-dodecyl-3-octadecylglycero-2-phosphoryl choline, 1-dodecyl-3-oleylglycero-2-phosphoryl choline, 1,3-ditetradecylglycero-2-phosphoryl choline, 1-tetradecyl-3-hexadecylglycero-2-phosphoryl choline, 1-tetradecyl-3-octadecylglycero-2-phosphoryl choline, 1-tetradecyl-3-oleylglycero-2-phosphoryl choline, 1,3-dihexadecylglycero-2-phosphoryl choline, 1-hexadecyl-3- (2-hexyldecyl)glycero-2-phosphoryl choline, 1,3-di(2-hexyldecyl)glycero-2-phosphoryl choline, 1-hexdecyl-3-octadecylglycero-2-phosphoryl choline, 1-hexadecyl-3-oleylglycero-2-phosphoryl choline, 1,3-dioleylglycero-2-phosphoryl choline, 1,3-dioctadecylglycero-2-phosphoryl choline, 1-octadecyl-3-oleylglycero-2-phosphoryl choline, and mixtures of these novel surfactants with other known surfactants may be employed hereinafter.

C. Emulsion Characteristics

The emulsions of this invention are made by dispersing PFC in water in the presence of the above identified surfactants. The surfactant enhances the dispersion and stabilization of the liquid phases. While dispersons may be generally referred to herein as emulsions, it should be understood that they may be considered solutions, micellar solutions, microemulsions, vesicular suspensions, or mixtures of all of these physical states. Accordingly, the term "emulsion" as used herein covers all these states and the novel surfactant or solubilizing agent is employed to enhance stable mixtures of these physical states of the PFC and water phases. For example, where a fluorochemical is emulsified in water, another oil may serve as an emulsifying adjuvant as described in the aforementioned Clark and Shaw European Patent Application 87300454.3. We wish to emphasize that, according to this invention, this emulsifying adjuvant is optional. Successful emulsions have been prepared with no such adjuvant. When an adjuvant is employed, a liquid fatty oil such as mono-, di-, or triglyceride or mixtures thereof are the preferred agents. Where such oil and oleophilic PFC combinations are emulsified in water, as provided hereinafter, complex phases and interfaces may form. Preferably, for artificial blood, the emulsions of this invention contain a PFC or mixture of PFCs, and most preferably contain a fluorocarbon selected from the group consisting of perfluorodecalin, perfluormethyldecalin, perfluorodimethyladamantane, perfluorooctylbromide, perfluoro-4-methyloctahydroquinolidizine, perfluoro-N-methyldecahydroquinoline, F-methyl-1-oxadecalin, perfluorobicylo(5.3.0) decane, perfluorooctahydroquinolidizine, perfluoro-5, 6-dihydro-5-decene perfluoro-4 , 5-dihydro-4-octene, perfluorodichlorooctane, perfluorobischlorobutyl ether, and chlorinated perfluorocarbons. For use as a contrast agent for bioligical imaging perfluorooctylbromide is one of the preferred PFCs according to this invention.

While the PFCs or mixture of PFCs may comprise 10% up to about 75% by volume, or more, of the emulsions, preferably they comprise at least 40% by volume. When the emulsions are to be used as "artificial bloods" or red blood cell substitutes, the PFC is present in as high a volume concentration as possible, e.g., 40% by volume is often preferred because that concentration matches the approximate oxygen content capacity of whole blood. PFC/oil emulsions may also be used as stated above. For example, when used as an artificial blood, PFC is present in an acceptable amount along with an oil emulsifying adjuvant. The actual oil concentration to produce an acceptable emulsion for any given set of components is determined by preparing and testing the stabilities of emulsions at various oil concentrations. Within this teaching for PFC artificial bloods, for instance, between 0 and 30% by weight oil adjuvant and 10-70% by volume PFC oil are used as described in the above European Patent Application 87300454.3 of Clark and Shaw.

The amount of a particular surfactant used in the emulsions of this invention depends upon the amounts and properties of other components of the emulsion as indicated above. Generally about 0.5-10% by weight of surfactant, preferably about 1-4% by weight, is used.

The surfactant of this invention may be used with other surfactants as indicated above. Among other surfactants useful in the emulsions of this invention are any of the known anionic, cationic, non-ionic and zwitter-ionic surfactants. These include, for example, anionic surfactants, such as alkyl or aryl sulfates, sulfonates, carboxylates or phosphates, cationic surfactants such as mono-, di-, tri- and tetraalkyl or aryl ammonium salts, non-ionic surfactants, such as alkyl or aryl compounds, whose hydrophilic part consists of polyoxyethylene chains, sugar molecules, polyalcohol derivatives or other hydrophilic groups and zwitter-ionic surfactants that may be combination so the above anionic or cationic groups, and whose hydrophobic part consists of any other polymer, such as polysobutylene or polypropylene oxides. When the emulsions of this invention are to be used in artificial bloods or red blood cell substitutes, the surfactant, or combinations of them, must be physiologically acceptable. For example, in artificial bloods the 1, 3-dialkylglycerophosphoryl choline is used where the alkyl group is about C₁₂-C₂₂ as exemplified above.

The emulsions may be prepared using any order of mixing the main components of PFC, surfactant and water. However, for optimal PFC emulsions the PFC is first miked with the adjuvant oil in the presence of a combination of all or part of the surfactant and some water. Then the final emulsion is prepared by emulsifying this first emulsion in the remaining water and any remaining surfactant as described in the above Clark and Shaw European Application 87300454.3 which is incorporated herein by reference.

The mixing and emulsification of components may be done using any of the conventional mixers and emulsifiers. For example. Fisher brand touch mixers,

Microfluidizers, Gaulin and Rannie Homogenizers may be employed.

The following non-limiting examples illustrate various embodiments of this invention. DETAILED EXAMPLES

Surfactant Syntheses

The surfactants of this invention were synthesized from the corresponding dialkylglycerols and the synthetic sequences are represented by the following schemes or methods.

In connection with the above schemes and methods, the term "symmetrical" is employed to designate dialkyl compounds where both alkyl groups are identical and "unsymmetrical" where the alkyl groups differ. Employing the schemes and methods, the following illustrate specific examples of making 1,3-dialkylglycerols as precursers of the 1,3-dialkylglycerophosphoryl choline surfactants of this invention.

This invention also involves a novel method for the preparation of 1,3-dialkylglycerols. Alkyl glycidyl ethers (where the alkyl group contained from 12 to 22 carbons, saturated and unsaturated) were prepared from epichlorhydrin and subsequently treated with monohydric alcohols of 12 to 22 carbons (saturated and unsaturated) to produce 1,3-dialkylglycerols (symmetrical and unsymmetrical). This procedure allowed for catalytic use of an inexpensive reagent (KOH) in a recoverable solvent (toluene) according to the above scheme of preparation. Specific examples of preparation follow.

Preoaration of Alkyl Glycidyl Ethers

Hexadecyl glycidyl ether

Into a 22 L flask was added 40% NaOH (4.0 L) and n-tetrabutylammonium hydrogen sulfate (135 g, 0.4. mol). Hexadecanol (2314 g, 9.5 mol), epichlorohydrin

(4.0 L, 51.0 mol) and 40% NaOH (2.5 L) were added in portions during the course of 45 minutes. The temperature of the reaction mixture warmed to 45ºC after

3 hours. An ice-water bath was used to maintain a temperature of 45-50ºC. The reaction was complete after 3.5 hours. Upon cooling to room temperature, the mixture was poured over ice-water and NaCl (7 × 1500 mL). The organic layers were separated and concentrated. Approximately 2 L of epichlorohydrin was recovered. The aqueous portions were extracted with Et₂O and EtOAc (2 × 750 mL), organic layers were pooled, dried (Na₂SO₄ and MgSO₄) and concentrated. A honey colored syrup was recovered which was then recrystallized from methanol to yield the product as a white solid 2638 g, 93%.

Oleyl glycidyl ether

Reaction procedures and workup performed in the same manner as that above for hexadecyl glycidyl ether. The residue was distilled on a kugelrohr apparatus at 140-160ºC and 200 mT to yield the title compound as a translucent yellow oil 109 g, 91%. Preparation of Dialkylglycerols

Any of the above alkyl glyeidyl ethers, or similar ethers, may be converted to 1,3 dialkylglycerol, exemplified by the following conversions.

1-octadecyl-3-hexadecyl-glycerol

Octadecanol (70.0g, 0.26 mol) and toluene (30 mL) was warmed to form a solution. Hexadecyl glycidyl ether (15.4 g, 0.05 mol) and KOH (0.4 g, 0.01 mol) were added and the solution was stirred under N₂ at 110ºC for 2.5 hours. Toluene was distilled by rotary evaporation at 70ºC aind excess octadecanol was removed by kugelrohr distillation. A honey colored syrup was obtained and this was recrystallized from EtOAc to give the title compound as an off white solid, 21.7 g, 73%.

1-oleyl-3-hexadecyl-glycerol

Reaction and workup procedure same as above. The title compound was obtained by recrystallized from MeOH: EtOAc (6:1) as an off white waxy solid 82 g, 87%.

1,3-dioleyl-glycerol

Reaction and workup procedure same as above.

Chromatography on 250 g silica gel (100-200 mesh) with CHCl₃ as eluant yielded 82 g, 90% of the title compound as a translucent yellow oil. Preparation of 1,3 Dialkylglycerophosphoryl choline

Any of the above dialkylglycerols, or similar compounds, may then be converted to the 1,3-dialkylglycerophosphoryl choline surfactants by either of the above Methods A and B, and the following specific examples illustrate such conversions.

1,3-Dihexadecylglycero-2-phosphoryl choline (Method B)

Into a dry, nitrogen purged 1 liter 3-necked round bottom flask equipped with an overhead stirrer and heating mantel was added 1,3-dihexadecyl glycerol (51.1g, 0.093 mol). Anhydrous ether (500 ml) was added via a dry cannula. The mixture was heated to 30°C with stirring, until a clear solution of glycerol in ether was obtained. At this point, a solution of 2-chloro-2-oxo-1,3,2-dioxaphospholane¹ (13.44 g, 0.093 mol) in anhydrous ether (10 ml) was added, followed shortly thereafter by triethylamine (14.5 ml, 0.102 mol). A precitipate quickly formed, and the mixture was allowed to stir at 30°C overnight. The precipitate (triethylamine hydrochloride) was filtered, and the filtrate was rotary evaporated to yield the intermediate dihexadecylglycero-2-phospholane (55.7 g).

This phospholane was placed in a 500 ml serum bottle and dimethylformamide (400 ml) was added via cannula. The bottle was placed in a dry ice bath, and trimethylamine (~16 g, 0.28 mol) was condensed into the bottle. The bottle was then sealed and immersed in a 60ºC water bath for 3 days. During this time, a precipitate formed. The bottle was removed from the water bath, allowed to cool, and the preciiptate was collected. This solid was purified by column chromatorgraphy (chloroform: methanol:water - 75:22:3) 1R. S. Edmundson, Chemistry and Industry, 1828 (1962) to yield the desired 1,3-dihexadecylglycero-2-phosphoryl choline ( 40 g, 61% from starting glycerol).

It was later found that certain of these products could be purified by either crystallization or trituration from 4:1 ether:hexane, withour performing column chromatography. It is expected that other crystallization solvents such as acetone may be employed as well.

1-dodecyl-3-hexadecylglycero-2-phosphoryl choline

(Method B)

Into a dry, nitrogen purged 1 liter 3-necked round bottom flask equipped with an overhead stirrer and heating mantel was added 1-dodecyl-3-hexadecylglycerol

(10.0 g, 0.21 mol). Anydrous ether (100.0 ml) was added via a dry cannula. The mixture was heated to 30ºC with stirring, until a clear solution of glycerol in ether was obtained. At this point, a solution of 2-chloro-2-oxo-1,3,2-dioxaphospholane (3.00g, 0.021 mol) in anhydrous ether (2 ml) was added, followed shortly thereafter by triethylamine (3.2 ml, 0.023 mol). A precipitate quickly formed, and the mixture was allowed to stir at 30ºC overnight. The precipitate (triethylamine hydrochloride) was filtered, and the filtrate was rotary evaporated to yield the intermediate dihexadecylglycerophospholane (12.4 g).

This phospholane was placed in a 100 ml serum bottle and dimethylformamide (80 ml) was added via cannula. The bottle was placed in a dry ice bath, and trimethylamine (~4.1 g, 0.069 mol) was condensed into the bottle. The bottle was then sealed and immersed in a 60ºC water bath for 3 days. During this time, a precipitate formed. The bottle was removed from the water bath, allowed to cool, and the precipitate was collected. This solid (7.5 g, 55% crude yield) was further purified by trituration with 4:1 ether;hexane to yield pure 1-dodecyl-3-hexadecylglycerophosphoryl-2- choline (4.8 g, 35% from starting glycerol). Employing the above methods, a large variety of 1,3 dialkylglycero-2-phosphoryl cholines were prepared according to the above general formula and these novel compounds are set forth in Table I.

PERFLUOROCARBON EMULSIONS AND THEIR PROPERTIES

The surfactants of Table I were used to make 40 volume % (v/v) perfluorochemical emulsions. In the above described European Patent Application 87300454.3, it was reported that certain oils, i.e., trigiycerides of fatty acids as co-additives greatly improved the stability of lecithin containing emulsions. Therefore, those perfluorochemical emulsions were used as a control for comparison with the perfluorochemical emulsions of this invention. The control emulsion was a 40v/v% perfluorodichlorooctane emulsion prepared according to the technique of the mentioned European patent application and containing 2 w/v% egg yolk lecithin as the surfactant and 2w/v% safflower oil. The surfactants of the invention were substituted for lecithin and the results of the emulsion's properties are reported in Table II. In Table II, the emulsion characteristics for most of the Table I compounds are reported, namely viscosity and particle size. All data is for emulsions sterilized for 15 minutes at 121ºC in a rotating basket autoclave, the industry standard for large volume parenterals.

It is clear from the above data that compounds from Table I are generally useful as surfactants for aqueous emulsions of perfluorochemicals. These compounds were screened for toxicity. Groups of ten rats each were infused with 20 cc/kg of a given emulsion and sacrificed at fourteen days. The liver, lung and spleen were monitored for organ growth, typically against a standard emulsion (control) and saline. All of this data has been summarized in Table III as follows.

With reference to the above Table III, the following meanings of the headings are provided in Table IV.

Table IV

Parameter Meaning

Heading

Liver grams of Liver per 100 grams of body weight at sacrifice

Lung grams of Lung per 100 grams of body weight at sacrifice

Spleen grams of Spleen per 100 grams of body weight at sacrifice

Hct hematocrit as a volume percentage of red blood cells determined by centrifugation

Platelet platelets (count per cubic millimeter of whole blood)

Survival 100% (Number of Survivors) per (Number of animals used)

Lee The weight/weight percentage of lecithin in the emulsion

Oil The weight/weight percentage of safflower oil in the emulsion

Glyc The weight/weight percentage of glycerin in the emulsion. Glycerin is used to make the emulsion isotonic

%PFC The volume/volume percentage of perfluorodichlorooctane in the emulsion Amt CPD The amount of the 1,3 alkylglycerophosphoryl choline surfactant in the emulsion

pH The pH of the emulsion prepared

Osm The osmolarity of the emulsion as determined by a Wescor vapor pressure osmometer in the milliosmoles per liter

Viscosity The viscosity of the sample as determined by a Brookfield Cone Plate fiscometer in centipoise.

PSD The mean particle size, in nanometers, of the particles in the emulsin that are under

1 micron in size

μ The mean particle size, in microns, of the particles in the emulsion that are over 1 micron in size

Vol The volume percentage of particles in the emulsion that are over 1 micron in size.

The lower this number, the better the emulsion. Typically better emulsions have no more than 3% of the particles in this range. With reference to Table III, where no glycerin is listed, the emulsions were prepared in phosphate buffered saline prior to sterilization, with no adverse effect on the quality of the emulsions prepared. With respect to multiple emulsions of a single compound reported in Table III, data is included for the emulsion actually used in a given toxicity experiment. The physical properties are generally within experimental error of those listed in Table II, but not identical.

In summary, with reference to Table III, a number of observations may be made for each of the examples. For each of the compounds reported, hereinafter the letters "PCE" will refer to the glycerophosphoryl choline for the sake of brevity.

With reference to Example 1, 1-n-hexadecyl-3-octadecyl PCE (Compound 15) was compared to its 1,2 isomer. In both cases, a significant improvement in circulating platelets and hematocrit was noted relative to the emulsion control. There was no significant difference in the physical properties of the emulsions used. The differences in survival are not considered significant because there are many occasions when a control emulsion gives 70-90% survival, with reference, for example to Examples 3, 4 and 6.

In Example 2, the 1-n-hexadecy1-3-octadecyl

PCE (Compound 15) was again tested against a control emulsion to ensure the reproducibility and validity of the observations. Again, there was a marked increase in circulating platelets and hematocrit. Survival was 100% for both emulsions, supporting the conclusions above.

In Example 3, another repeat of Examples 1 and 2 was conducted, this time including saline control animals. The same conclusions were reached, especially since circulating platelets are the same in emulsion treated animals as in the saline controls. It is therefore inconceivable that the effects being observed are random occurrences.

In Example 4, additional experiments were performed with 1-n-hexadecy1-3-octadecyl PCE (Compound 15) in order to determine if the addition of lecithin would have any impact on the result. Lecithin does not adversely affect platelets or hematocrit when used in conjunction with the 1,3-dialkylglyceryophosphoryl cholines. By itself, however, it is not effective at maintaining high platelets or hematocrit, as demonstrated by the low response of these parameters to the control emulsion.

In Example 5, further evidence is obtained that the effects obtained in accordance with the principles of this invention are general to the genus of the surfactants. It was found that the 1,3-ditetradecyl PCE (Compound 8) and 1,3-dioleyl PCE (Compound 17) increase platelets and hematocrit against the control emulsion.

In Example 6, the mixture of fatty chains derived from safflower oil prove to be the first of this class of compounds that did not have a positive impact on platelets in the rodent model. Hematocrit was still somewhat improved relative to the control. In this study, it was surprising to find that the 1,2 isomer of Compound 8 was toxic, and had no positive effect on platelets. In this particular case, it is apparent that there is a marked advantage to the 1,3 substituted PCEs.

In Example 7, the effect for the ditetradecyl PCE (Compound 8) was verified. Furthermore, the same effects were observed without the toxicity observed for its 1,2 isomer. The earlier observations made for the 1,3 dioleyl PCE (Compound 17) were reproducible with this experiment. This experiment showed, however, that compounds of molecular weight lower than that of ditetradecyl PCE (Compound 8) may have a tendency to be toxic. Thus, for physiologically acceptable emulsions, the total carbon content of the dialkyl groups is preferably at least 28.

In Example 8, the conclusions for 1-n-dodecyl- 3-oleyl PCE (compound 7), were reaffirmed, although the effect on platelets is somewhat attenuated. The saturated 1-n-dodecy1-3-octadecyl PCE (Compound 6) retains the full advantages of the invention.

It is noteworthy that in all of the studies reported above in Table III, the liver, lung and spleen weights of the treatment animals trended toward lower values (in most cases statistically significant at the 0.05 confidence level) than those of the control emulsion animals, although they remained above saline control values.

In summary, emulsions made with the surfactants of this invention show increased abilities in the circulatory blood system as measured by the amount of perfluorochemical remaining at 48 hours. Therefore, the surfactants are considered to significantly increase the circulatory blood residence time of the PFC. Furthermore, it is clear from the data that the surfactant compounds of this invention greatly ameliorate the effects of perfluorochemical on the toxic responses of hepatomegally splenomegally, and lung enlargement. Furthermore, the surfactants did not cause nearly as large a drop in hematocrit (the v/v% of red blood cells (Hct) after infusion. Thus, the 1,3-dialkylglyceryophosphoryl cholines behave in very similar fashion to the 1,2 isomers reported in our U.S. Patent No. 5,304,325, but there are further advantages to be gained by their use. For example, the surfactants of this invention may be made by a significantly improved process that results in significant cost savings over all from previous reported syntheses and allows for greater functionality in the ether side chains. In addition, as noted above, there are further improved and unexpected effects noted for the emulsions containing surfactants of this invention in comparison to the earlier 1,2 isomer. The surfactants of this invention are also resistant to oxidation and degradation normally associated with egg yolk phospholipid lecithin emulsifying agents. Accordingly, emulsions containing the novel surfactants may be oxygenated during sterilization and through storage for extended period without degradation due to these oxygen resistant surfactants.

In view of the above detailed description, other variations and embodiments of this inventions will be understood by a person of ordinary skill in this art and such are within the scope and spirit of this description.

What is claimed is:

Claims

CLAIMS:

1. An emulsion comprising a fluorochemical, water and a surfactant having a general structure of

where R₁ and R₂ are a C₁₂-C₂₂ saturated or unsaturated aliphatic group and PC is the group or salt thereof represented by the structure

where R₄ is hydrogen or lower alkyl from the group consisting of methyl, ethyl and propyl.

2. The emulsion of claim 1 where R₄ is methyl.

3. The emulsion of claim 1 wherein said surfactant is selected from the group consisting of 1-dodecyl-3-hexadecylglycero-2-phosphoryl choline, 1-dodecyl-3-octadecylglycero-2-phosphoryl choline, 1-dodecyl-3-oleylglycero-2-phosphoryl choline, 1,3-ditetradecylglycero-2-phosphoryl choline, 1-tetradecyl- 3-hexadecylglycero-2-phosphoryl choline, 1-tetradecy1-3-octadecylglycero-2-phosphoryl choline, 1-tetradecy1-3-oleylglycero-2-phosphoryl choline, 1,3-dihexadecylglycero-2-phosphoryl choline, 1-hexadecyl-3- (2-hexyldecyl)glycero-2-phosphoryl choline, 1,3-di(2-hexyldecyl)glycero-2-phosphoryl choline, 1-hexdecyl-3-octadecylglycero-2-phosphoryl choline, 1-hexadecyl-3-oleylglycero-2-phosphorylcholine, 1,3-dioleylglycero-2-phosphoryl choline, 1,3-dioctadecylglycero-2-phosphoryl choline, 1-octadecyl-3-oleylglycero-2-phosphoryl choline, and mixtures thereof.

4. The emulsion of claim 1 wherein the fluorochemical is selected from the group consisting of perfluorodecalin, perfluoromethyldecalin, perfluorodimethyladamantane, perfluorooctylbromide, perfluoro-4-methyloctahydroquinolidizine, perfluoro-N-methyl-decahdroquinoline, F-methyl-1-oxadecalin, perfluorobicyclo(5.3.)decane,perfluorooctahydroquinolidizine, perfluoro 5,6-dihydro-5-decene, and perfluoro-4,5-dihydro-4-octene, perfluorodichlorooctane, perfluorobischlorobutyl ether, chlorinated perfluorocarbons, and mixtures thereof.

5. The emulsion of claim 4 that is stable after heat sterilization.

6. The emulsion of claim 4 wherein a liquid fatty oil is present as an emulsifying adjuvant in an amount between about 0.5 and about 30% by weight of the emulsion.

7. The emulsion of claim 6 wherein the oil is selected from the group consisting of mono-, di- and trigiycerides, and mixtures thereof.

8. The emulsion of claim 1 wherein the surfactant is present in an amount from about 0.5 to about 10% by weight of the emulsion.

9. The emulsion of claim 1 wherein the surfactant is present in an amount of from about 1 to about 4% by weight of the emulsion.

10. The emulsion of claim 1 wherein the fluorochemical is present in an amount of from about 10 to about 75% by volume of the emulsion.

11. The emulsion ot claim 4 wherein said surfactant is selected from the group consisting of, 1-dodecyl-3-hexadecylglycero-2-phosphoryl choline, 1-dodecyl-3-octadecylglycero-2-phosphoryl choline, 1-dodecyl-3-oleylglycero-2-phosphoryl choline, 1,3-ditetradecylglycero-2-phosphoryl choline, 1-tetradecyl-3-hexadecylglycero-2-phosphoryl choline, 1-tetradecyl-3-octadecylglycero-2-phosphoryl choline, 1-tetradecyl-3-oleylglycero-2-phosphoryl choline, 1,3-dihexadecylglycero-2-phosphoryl choline, 1-hexadecyl-3- (2-hexyldecyl)glycero-2-phosphoryl choline, 1,3-di(2-hexyldecyl)glycero-2-phosphoryl choline, 1-hexdecyl-3-octadecylglycero-2-phosphoryl choline, 1-hexadecyl-3-oleylglycero-2-phosphoryl choline, 1,3-dioleylglycero-2-phosphoryl choline, 1,3-dioctadecylglycero-2-phosphoryl choline, 1-octadecyl-3-oleylglycero-2-phosphoryl choline, and mixtures thereof.

12. A physiologically acceptable emulsion comprising a fluorochemical, water and a surfactant having a general structure of

where R₁ and R₂ are C₁₄-C₂₂ saturated or unsaturated aliphatic group with the total carbon content of R₁ and R₂ being at least 28 and PC is the group or salt thereof represented by the structure

where R₄ is hydrogen or methyl.

13. The emulsion of claim 1 wherein said surfactant is selected from the group consisting of 1-dodecyl-3-hexadecylglycero-2-phosphoryl choline, 1-dodecyl-3-octadecylglycero-2-phosphoryl choline, 1-dodecyl-3-oleylglycero-2-phosphoryl choline, 1,3-ditetradecylglycero-2-phosphoryl choline, 1-tetradecyl-3-hexadecylglycero-2-phosphoryl choline, 1-tetradecyl-3-octadecylglycero-2-phosphoryl choline, 1-tetradecyl-3-oleylglycero-2-phosphoryl choline, 1,3-dihexadecylglycero-2-phosphoryl choline, 1-hexadecyl-3- (2-hexyldecyl)glycero-2-phosphoryl choline, 1,3-di(2-hexyldecyl)glycero-2-phosphoryl choline, 1-hexdecyl-3-octadecylglycero-2-phosphoryl choline, 1-hexadecyl-3-oleylglycero-2-phosphoryl choline, 1,3-dioleylglycero-2-phosphoryl choline, 1,3-dioctadecylglycero-2-phosphoryl choline, 1-octadecyl-3-oleylglycero-2-phosphoryl choline, and mixtures thereof.

14. The emulsion of claim 12 wherein said surfactant is contained in an amount of from about 0.5 to about 10% by weight of the emulsion.

15. The emulsion of claim 12 wherein a fluorochemical is contained in an amount of from about 10 to about 60% by volume of the emulsion.

16. The emulsion of claim 12 wherein said fluorochemical is contained in an amount of at least about 40% by volume of the emulsion.

17. The emulsion of claim 12 wherein said surfactant is 1-octadecyl-3-hexadecylglycero-2-phosphoryl choline.

18. The emulsion of claim 12 wherein the R₁ and R₂ groups are derived from a natural oil.

19. The emulsion of claim 18 wherein said oil is selected from the group of safflower, soybean, cottonseed, corn, coconut and olive, and mixtures thereof.

20. The emulsion of claim 12 wherein the R₁ and R₂ groups are selected from the group consisting of hexyldecyl, octadecyl, oleyl, linoleyl and other C₁₆-C₂₂ chains, and mixtures thereof.

21. The emulsion of claim 12 wherein the fluorochemical is selecting from a group consisting of perfluorodecalin, perfluoromethyldecalin, perfluorodimethyladamantane, perfluorooctylbromide, perfluoro-4-methyloctahydroquinolidizine, perfluoro-N-methyl-decahdroquinoline, F-methyl-1-oxadecalin, perfluorobicyclo(5.3.)decane,perfluorooctahydroquinolidizine, perfluoro 5,6-dihydro-5-decene, and perfluoro-4,5-dihydro-4-octene, perfluorodichlorooctane, perfluorobischlorobutyl ether, chlorinated perfluorocarbons, and mixtures thereof.

22. A red blood cell substitute comprising an amount of an emulsion of claim 12, said amount being therapeutically effective for oxygen carrying and transport in animals.

23. A contrast agent for biological imaging comprising an amount of an emulsion of claim 12, said amount being therapeutically effective for oxygen carrying and transport in animals.

24. A contrast agent for biological imaging comprising an amount of an emulsion of claim 12, said amount being clinically effective for imaging by modalities selected from the group consisting of nuclear magnetic resonance, X-ray and ultrasound.

25. The method of increasing the fluorochemical content of circulating blood in an animal for oxygen carrying and transport by administering the emulsion of claim 12.

26. The method of increasing the fluorochemical content of circulating blood in an animal for oxygen carrying and transport by administering the emulsion of claim 13.

27. The method of increasing the fluorochemical content of circulating blood in an animal for oxygen carrying and transport by administering the emulsion of claim 21.