CA1316820C

CA1316820C - Biocompatible, stable and concentrated fluorocarbon emulsions for contrast enhancement and oxygen transport in internal animal use

Info

Publication number: CA1316820C
Application number: CA000574015A
Authority: CA
Inventors: David M. Long, Jr.
Original assignee: Alliance Pharmaceutical Corp
Current assignee: Alliance Pharmaceutical Corp
Priority date: 1987-08-05
Filing date: 1988-08-05
Publication date: 1993-04-27
Anticipated expiration: 2010-04-27
Also published as: NO883456L; NO179162C; ES2056932T3; DE3850012T2; NO883456D0; EP0307087A1; IE64245B1; NO179162B; IE882416L; US4987154A; DE3850012D1; ZA885796B; ATE106722T1; EP0307087B1

Abstract

ABSTRACT OF THE DISCLOSURE

An up to 125% fluorocarbon emulsion for use in or with animal bodies and organs thereof, maintains emulsion stability through normal sterilization procedures with selective osmotic and buffering agents, maintains the emulsion at within predetermined osmolarity levels and, when desired, free of excessive calcium precipitation, reduces in vivo and in vitro red blood cell injury, reduces adverse anemia effects, reduces viscosity and reduces the rate of oxidation, and tends to equilibrate its distribution in major body organs thereby reducing toxicity. The osmotic agents may buffer and may provide nutrient in the form of sugars. The osmotic and buffering agents can comprise, selectively, hexahydric alcohols, namely mannitol and sorbitol; certain sugars, namely glucose, mannose and fructose; along with buffering agents that will affect osmolarity including imidazole, tris(hydroxy-methyl)aminomethane, sodium chloride, sodium bicarbonate, monobasic potassium phosphate, dibasic potassium phosphate, calcium chloride, magnesium sulfate, monobasic sodium phosphate, dibasic sodium phosphate or combinations of them.
The emulsion may include tocopherol. A method of emulsifying the fluorocarbon includes forced flow impingement under pressure after mixing the fluorocarbon into the discontinuous phase. The fluorocarbon emulsion can be used to deliver drugs and medicines soluble in, or transportable by the emulsion.

Description

1 31 6~2a B7~CKGI~OUND OF T~IE INVENTION

1. Field of the Invention:
The present invention relates to the art of non-toxic oxygen transpor-t and contras-t enhancement agents for . in-ternal and external animal use, and more particularly -to stable high concentration fluorocarbon emulsions capable of s-terilization and wl-ich are selectively free of calcium precipitation, reduce in vivo and in vitro red blood cell, or erythrocyte, injury, reduce anemia effects, and have reduced viscosity and reduced rate of oxidation or free radical damage, ~articularly of components of the emulsion and of contacted body tissue.

2. Description of tlle Prior ~rt:

In the past, efforts to use emulsified fluorocarbons as an oxygen transport or carrier, as in a blood substitute, and as a contrast enhancement agent, as for X-ray, ul-trasowld and magnetic resonance imaging, have encountered certain difficulties. Purity, non-toxicity, chemical and biological ::
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131~,20 1 iner-tness and ability to excrete are desirable objectives. The 2 emulsified fluorocarbon must be capable of sterilization, ,~ preferably by hea-t, have long-term size and function stability preferably in the fluid sta-te, be industrially r) feasible, persist for sufficiently long or e~fective times in the blood stream when used intravascularly and be elimina-ted I sufficien-tly rapidly from the body.
~ For intravenous use, it is considered important to () have small particle size. ~lowever, long term storage for extended periods of time for a month or longer, of blood 11 substitutes has heretofore resulted in conglomeration or 12 coalescence of the fluorocarbon particles in the emulsion into 13 larger particles, especially after heat sterilization. For a 1~ general discussion of~the objectives and a review of the efforts and problems in achieving these objec~ives in 1~; fluorocarbon blood substitutes, see "Reassessment of Criteria 17 for the Selection of Perfluoro Chemicals for Second-Generation 18 Blood Substitutes: Analysis of Structure/Property 1~ Relationship" by Jean G. Riess, 8 Artificial Organs, 34-56, (1984).
21 Larger particle sizes are dangerous in intravenous 22 use in that they tend to collect in the lung, liver, spleen 23 and some other organs, enlarging them and endangering their 2~ functioning. On the other hand, it is desired *o have sufficient particle size in the fluorocarbon particles for 2~ them to collect in tumors and other areas when fluorocarbons 27 are used as a contrast enhancement medium. Larger particle 2~ sizes within reasonable limits, also, are unobjectionable when 2~ used in other, non-venous systems in the body, such as, for example, the cerebrospinal fluid ventricles and cavities.
31 In the past, it has been observed that fluorocarbon 32 emulsio~s used intravascularly accumulate disproportionately ., . .,, , , 1316~320 I more in the spleen, as opposed to other organs such as the 2 liver. This concentration in the spleen sometimes causes a 3 transient hypersplenism, a condition characterized by an l enlarged and over-active spleen from which a transient anemia !) resul-ts. A fluorocarbon emulsion having the above-indicated G characteristics but also having a more even distribu-tion among / the major body organs is desired.
8 Glyeerol is normally a good osmotie ayent for fluorocarbon emulsions, but in concen-trations has been observed to hemolize the red blood cells. The glyeerol 11 apparently swells the red blood eells, damaging the eells, 12 promoting the egress of hemoglobin and thus causing damage to 13 the eells. Certain other additives, notably sugars have similar red blood eell damaging effeets. It has long been desired to avoid or to limit the amount of sueh hemolytic 1G agents in the emulsion.
1~ It is known that leeithin and other phospholipids lS are subject to oxidation in the vascular system. Sueh lD oxidation of leeithin phospholipids is also observable in respeet to the lecithin phospholipid emulsifier eomponents of 21 stored or paekaged fluoroearbon emulsions. It is desired to 22 have effeetive, stable and non-toxie fluorocarbon emulsions 23 having phospholipid emulsifying agents or other oxidizable 2-~ components wherein oxidation is inhibited.
It is frequently desired to have high eoncentration 2G fluorocarbon emulsions, but they unfortunately tend to have 27 high viseosity. It may also be desired to have emulsions 2~ containing nutrients, sueh as glueose and like sugars.
29 Glucose, however, has been known to make fluoroearbon emulsions more viseous. It is desired to have fluoroearbon 31 emulslons that are less viseous and more fluidie, to :~

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1 facilitate packaging, injectability and avoidance of blood 2 vessel blockage.
3 It has been desired, furt~er, to provide a vehicle 4 carrier for delivering fat or oil soluble and fluorocarbon soluble medicines through the intravascular, intraperitoneal, oral, respiratory, cerebrospinal and other internal animal / body tissue or systems, including human tissue, as well as for ~ delivering such medicines externally such as cutaneously (3 through the skin. "Tissue" in this specification will be used to include blood.
11 It is often desirable to have some emulsions which 12 contain or deliver calcium, and which do not have calcium 13 precipitating components. Many buffers, however, are 14 phosphates or carbonates and form excessive calcium precipitates which not only reduce the amount of calcium lG available for therapeutic use, but dangerously deposit calcium 17 compounds in the tissue.
18 The present invention is directed toward 19 improvements in the formulation and use of fluorocarbon emulsions to meet these and other objectives while providing a 21 stable, non-toxic and efficacious fluorocarbon emulsion.

In brief, in accordance with one aspec~ of the 2~ invention, fluorocarbon emuIsions having a concentration in 27 the continuous phase of from 20% to 125% weight per volume is 28 described whose mean particle size and particle distribution 29 is maintained substantially stable through normal sterilization and storage procedures.
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The continuous phase of the emulsion shall be used herein to 2 refer to the aqueous phase of the emulsion. In particular, 3 for example, the term "weight per volume" or "w/v" will be 4 used and should be understood to mean the ratio of the weight in grams per 100 cubic centimeters or 100 milliliters, or equivalent expressions or mathematical identities thereof.
7 The fluorocarbon in emulsion may be mono-brominated 8, perfluorocarbons, such as l-bromoseptadecafluoroctane 9 (C8F17Br, sometimes designated perfluoroctylbromide or l~ "PFOB"), l-bromopentadecafluoroseptane (C7F15Br), and 1l l-bromotridecafluorohexane (C6F13Br, sometimes known as li perfluorohexylbromide or "PFHB"), C~F9CH-C~C4F9 ("F-~4E"), 1_ i-C3F7CH-C~C6F13 ("F-i36E"), C6F13CH=CHC6F13 ("F-66E"), 14 F-adamantane t"FA"), F-1,3-dimethyladamantane ("FDMA"), c F-declin ("FDC"), F-4-methyloctahydroquinolidizine ~"FMOQ"), 1~ F-~-methyldecahydroquinoline ("FHQ"), F-~-cyclohexylpyrroli-17 dine ("FC~P"), F-2- butyltetrahydrofuran ("FC-75"), 18 (CF3)2CFO(CF2CF2)20CF~CF3)2, (CF3)2CFO(CF2CF2)30CF(CF3)2, 19 (CF3)2CFO(CF2CF2)2F, (CF3)2CFO(CF2CF2)3F, (C6F13)20 and FtCF(CF3)CF20]2CHFCF3.
21 ~he emulsion has for an emulsifying agent a 22 phospholipid, an anionic surfactant, a fluorosurfactant or ~3 combinations thereof.`
~4 Osmolarity is maintained by an osmotic agent which ~5 has benefit independent of osmolarity, such as the hexahydric ~6 alcohols, namely mannitol and sorbitol which also are used to 27 control viscosity and stabilize particle membrane structure.
28 other osmotic agents, such as certain sugars, namely glucose, 2 mannose and fructose may be used which provide nutrition.
3 Osmolarity is also affected by buffers, which are selected ., ~

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1 from imida~ole or tris(hydroxymethyl)aminomethane, which do 2 not precipi-tate calcium, or may be selected from such 3 bu~fering agents as sodium chloride, sodium bicarbonate, 4 magnesium chloride, monobasic potassium phosphate, dibasic potassium phosphate, calcium chloride, magnesium sulfate, monobasic sodium phosphate and dibasic sodium phosphate.
~ Certain biocompatible combinations of these osmotic agents 8 provide variously or inclusively for reduction of red blood 9 cell injury in vivo and in vitro, for reduction of viscosity, for reduction in the rate of oxidation, for 11 nutrition and for buffering the acidity or pH le~el.
12 Tocopherol, mannitol, ascorbyl palmitate and imida~ole may be 13 added or increased to further reduce the rate of oxidation of 14 the emulsion components in vitro, and also are believed to have similar effects in vivo -to reduce t~e rate of oxidation lG of the body tissue or organ to which the emulsion may be 17 applied.
13 A buffering agent maintains the pH at predetermined 19 levels, and may provide osmotic pressure to maintain osmolarity. The buffering agents may include the non-calcium 21 precipitating buffers imidazole, tris(hydroxymethyl)amino-22 methane and other buffering agents such as sodium bicarbonate, 23 monobasic potassium phosphate, dibasic potassium phosphate, 24 monobasic sodium phosphate and dibasic sodium phosphate.
Tris(hydroxy- methyl)aminomethane is sometimes called TH~M, or 2G by several of its trade names, such as, for example, Trizma by 27 Sigma Chemical Company of St. Louis, Missouri.
~8 The fluorocarbon emulsions are prepared, . . ,. . - . ;
29 first by mixing in the aqueous or continuous phase the "vehicle" by adding osmotic agent(s), 31 buffering agent(s), electrolytes if desired, emulsifying , : ' ' ' : `

1316~,~?0 ~ l 1 agent(s) and additional anti-oxidant(s) if desired. The 2 fluorocarbon is mixed into the vehicle at a tempered rate so 3 that the emulsion is tempered or homogeneous. The emulsion is 4 then divided into separate flows which are impinged at high velocities upon each other in sheets in a cavity under relatively high pressure. The emulsions are then filtered, 7 packaged, sterilized and otherwise processed for storage and 8 use.
~ Other novel features which are believed to be characteristic of the invention, both as to organization and 11 methods of operation, together with further objects and 12 advantages thereof, will be better understood from the 13 following description in which preferred embodiments of the 14 invention are described by way of example.

lG DESCRIPTION OF THE PREFERRED EMBODIMENTS

18 A fluorocarbon emulsion comprises a continuous, i.e.
19 aqueous phase and a discontinuous phase. The discontinuous phase comprises the fluorocarbon with an emulsifying agent.
21 Osmotic agents and biological p~ buffers are included 22 generally in the continuous phase to maintain osmolarity and 23 P~.
24 The emulsifying agent generally surrounds and forms ~5 a layer around the discontinuous phase creating essentially 2G fluorocarbon particles suspended within the continuous phase.
2~ Lecithin is used frequently as the emulsifying agent ~: . ' ~ .
2~ Other emulsifying agents may be used with good 29 effect, such as fluorinated surfactants, also known as fluorosurfactants and anionic surfactants. Fluorosurfactants 3/ which will pro-ide stable emuls ons include tripcrfluoroalkyl-, .. .. .

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~ cholate [C7Fl5C(=0)0]3, perfluoroalkylcholestanol 2 [C7Fl5C(=0)0], perfluoroalkyloxymethylcholate, ~M0-lO and 3 fluorinated polyhydroxylated surfaetants, such as, for examples, those discussed in "Design, Synthesis and Evaluation _ of Fluorocarbons and Surfactants for In Vivo Applications New Perfluoroallcylated Polyhydroxylated Surfaetants" by J. ~.
~iess, et al. Such fluorosurfactants discussed therein include a fluorophilic tail, a hydrocarbon prolongator, a ~J junetion unit eomprised of an ether, an ester or an amide, and a hydrophilic head. Fluorophilie tails include, for example, 11 C3(CF2)n, where n equals from ~ to lO. XM0-lO is a 12 fluorinated surfaetant having a formula 13 C3F70(CF2)3C(=O)NH(CH2)3N(=O)(C~13)2. To be an non-toxie 1~ fluorosurfactant, the fluorinated surfaetant and the lS fluorocarbon should have an elimination rate from the animal lG body or organ such that the fluorocarbon and the fluorinated 17 cosurfactant are eliminated from the body or organ before 1g carcinosis, teratogenesis or embryotoxieity oeeurs. Suitable 1~ anionie surfaetant whieh will provide a stable, non-toxie and bioeompatible emulsion are polyoxyethylene-polyoxypropylene 21 eopolymers. ' 22 ~he osmolarity of normal, for example human tissue 23 is approximately from 290 milliosmols to 300 milliosmols.
2~ Maintaining this osmolarity is important in preventing injury 26 to eells, sueh as red blood eells and endothelial eells which 2~ line the blood vessels into whieh, for example, the emulsion 27 may be injeeted. When the osmolarity is less than 290 2~ milliosmols, down to 200 milliosmols, water tends to diffuse 2~ into the eells eausing them to swell and sometimes burst.
~0 When the osmolarity is too high, on the order of greater than ~1 700 milliosmols, the eells lose water and may shrink.
~2 Injeetion of hyperosmotie medieines often are painful and ' 1 31 ~ r!
1 burn, and further may also cause clotting and obstruction of 2 the veins. These complications may be prevented by 3 controlling the osmolarity of the emulsion prior to 1 administration.
r) Fluorocarbon emulsions with low osmolarity tend to ~) show instability in coalescense of the discontinuous 7 particles, especially when subjected to stress shelf life studies such as freeze and thaw cycles. Normally when the ~ osmolarity is too high, on the order of greater than 650 milliosmols, the fluorocarbon emulsion particles tend to 11 aggregate, which can lead to coalescence and separation of the 12 emulsion. It has been found, however, that in formulating 13 fluorocarbon emulsions, slight hyperosmolarity, in the range 11 of from 300 milliosmols to approximately ~50 milliosmols is ~avored in order (l) to protect more against ~reezing and thus ~) -to obtain more stability, and (2) to accommodate increased 17 amounts of the osmotic and other active agents, especially 18 where the osmotic agent has therapeutic and other beneficial 19 effects, as will be explained more below.
In the preferred embodiment of the present 21 invention, mannitol is added to the emulsion. It has been 22 found that mannitol provides a means for maintaining 23 osmolarity, for reducing red blood cell injury, for reducing 2-1 viscosity, for providing anti-oxidant effects in the emulsion and for stabilizing the fluorocarbon particles. Because 2~ mannitol has such beneficial effects, greater amounts of 27 mannitol can be tolerated in the body's tissues. When using 2~ mannitol as the osmotic agent, for example, the stability of 29 the emulsion can be maintained at the desired osmolarity range of from 2~0 milliosmols to 650 milliosmols with from 0.25%
31 weight per volume to l.5% weight per volume. The body's 32 tissues can tolerate substantially more mannltol for obtaining .~
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1 anti-oxida~ion effects, for emulsion stabilizing effects, for ~) viscosity reducing effects and for red blood cell protection ,~ effects.
l It is believed, further, that mannitol is _ responsible for an observed improvement in the distribution of (; the fluorocarbon emulsion particles among the major organs when applied within the animal body. The effects of mannitol are believed to reduce organ toxicity, which in turn is !) believed to largely account for the reduction of adverse anemia effects when using the emulsion.
11 It is believed that mannitol is incorporated into or 12 interacts in some way with the lecithin or other emulsifier 13 membrane of the fluorocarbon particle in emulsion, to form a 1~ more protective membrane. For lecithin, this interaction is believed to be a more competent cell barrier structure that is 1~ more renitent in the membrane. It is believed, further, that 17 the mannitol does not adversely affect the stability of the 18 particle size in -the fluorocarbon emulsion, as will be 1~ discussed in greater detail below.
Additionally, the mannitol, it is believed, assists 21 in forming a more competent and renitent cell barrier in the 22 somewhat similar lecithin membrane barriers of red blood 23 cells, thus protecting against injury to the red blood cell, 2~1 which injury allows hemoglobin to escape. Reduction of red blood cell injury has been observed with mannitol added to the 2~ emulsion in both in vivo and in vitro experiments.
27 Glycerol has been used as an osmotic agent, but 2~ ~lycerol readily penetrates the red blood cell walls. This 2() penetration causes swelling of the red blood cells allowing their hemoglobin to escape. The escape of hemoglobin results 31 in red blood cell ~hosts which cannot transport oxygen. This 32 condition may contribute to observed transient anemia effects ; ' .... ~ , - :

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1 wi-th high doses of fluorocarbon emulsions. Mannitol is 2 preferred as the osmotic agent to glycerol where injury to red 3 blood cells may be a problem.
Mannitol establishes an osmotic pressure in the continuous phase of the emulsion, and is preferred in the G present invention as an osmotic àgent. Mannitol, unlike other 7 osmotic agents, such as, for examples, glucose, glycerol and 8 saline, generally does not penetrate the red blood cell, and ~ generally does not cause the red blood cells to swell and be damaged. Swollen and damaged red blood cells allow hemoglobin ll to be released from the red blood cell, thus possibly 12 contributing to the observed anemia effects.
13 The use of manni*ol in the fluorocarbon emulsion, it 1~ is believed, reduces the temporary anemia effects sometimes observed during discrete time periods in animals after lG receiving exaggerated doses of perfluorocarbon emulsion. It 17 is believed that the highly desired and long sought reduction 1~ in anemia effects is due to distribution equilibration of the 1J fluorocarbon emulsion among the body organs by mannitol, and to reduction of red blood cell injury . This reduction in 21 anemia effects has been observed in adolescent Sprague Dawley 22 rats, as may be better seen in the following Examples I and 23 II.
2~
EXAMPLE I
2G Two grams per kilogram of body weight of a 100%
27 weight per volume emulsion of perfluoroctylbromide were 2~ infused intravenously into twenty-two Sprague Dawley rats, 2J some (ten) of the rats getting an emulsion having 0.6% weight per volume of mannitol while other rats ttwelve) received an 31 emulsion having no mannitol but having a saline concentration 32 providing equivalent osmotic pressure. There were ten other - .
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I control rats which received a placebo injection of physiologic 2 saline in a dose of two milliliters per kilogram of body 3 weigh-t. The emulsion was further comprised of 6% weight per ~1 volume of lecithin, 0.0252% weight per volume of T~I~M. The r) emulsion was prepared in accordance with the process and procedure given in my co-pending application referenced / hereinabove. At two weeks, the rats receiving the emulsion including mannitol had in their red blood cells an average of ~J 97% of hemoglobin (measured in grams/deciliter) as found in 0 the control rats. The rats receiving the emulsion having no mannitol had at two weeks an average of 91% blood hemoglobin 12 as compared to the control rats. The hemoglobin was measured 13 by hemolyzing the red blood cells in the blood and measuring the amount of hemoglobin released.

lG EXAMPLE II
17 Rats of the same type as used in Example I were used in further tests, into which rats ten grams per kilogram of 1~ body weight of the emulsions as described for Example I above, were injected intravenously. At two weeks, the rats receiving 21 the emulsion containing mannitol averaged 87% hemoglobin as 22 compared with the control rats. The rats receiving the 23 emulsion not having the mannitol averaged 70% hemoglobin at 24 two weeks.
Mannitol thus was successful in reducing anemïa 2G effects even in rats receiving very high doses o~ 1uorocarbon 27 emulsions.
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2~ More significantly affecting these reductions in anemia effects, it is believed, is the observed difference in 31 major body organ distribution resulting from using mannitol as 32 an osmotic agent and as an emulsion stabilizer over other , . . :

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13168~'`i 1 osmotic agents. As noted, it has been observed in the past 2 that fluorocarbon emulsions accumulate more in the spleen, on 3 the order of lO to 15 times more than in other organs such as '1 the liver. It is believed tha-t -this high concentration of _ fluorocarbon emulsion particles in the spleen is caused by the macrophages engulfing the particles and trapping them in the / spleen. This large accumulation is unnecessary for effective imaging and sometimes causes hypersplenism, a condition ~ characterized by an enlarged and over-active spleen from which 1() anemia may result. When using mannitol as the osmotic agent, 11 this accumulation is significantly reduced, on the order of 12 approximately forty-eight percent (48%) as may be appreciated 13 from the following Example III. Thus, the risk of 1~ hypersplenism and accompanying anemia is believed to be significantly reduced. This more equilibrated distribution 1B can be seen be-tter from the following experiment given by way 17 of example:

1~ EX~MPLE III
A dose of the 100% weight per volume 21 perfluoroctylbromide emulsion having 0.6% weight per volume of 22 mannitol comprising one gram per kilogram of body weight was 23 injected intravenously into adolescent Sprague Dawley rats, 2-1 and the level of concentration of the perfluoroctylbromide in the spleen was measured at twenty-four hours. The 2B concentration was measured at 30.l + 1.5 milliyrams per gram 27 of spleen tissue. A substantially comparable 100% emulsion 28 not having mannitol has typically in the past resulted in, for 2~ example, 57.61 ~ 2.345 milligrams per gram of spleen tissue for the same dose.
31 Other organs, such as the liver showed a slight 32 increase in perfluoroctylbromide concentration when using the - .-,:

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1 same mannitol containing emulsion. In the rats receiving the 2 emulsion with mannitol, a concentration of 5.6 ~ 0.14 mg/gm.
3 liver tissue was observed, as compared with ~.605 + 0.533 mg./gm. liver tissue in a typical 100% emulsion not containing mannitol.
~) ~ The anemia is very significantly and substantially 8 reduced if not virtually eliminated altogether when mannitolis incorporated into the fluorocarbon emulsion.
0 Mannitol is, further, an anti-oxidant interacting with the free radicals in the body's systems generally, as 12 well as with free radicals in stored emulsion. Further, it 13 has been found that mannitol reduces the viscosity of the 1~ emulsion. With mannitol, reduced viscosity is observed in high concentration fluorocarbon emulsions and in fluorocarbon 1~ emulsions in which glucose or other nutrients have been added.
17 ~s noted, glucose has been found to make fluorocarbon 1~ emulsions more viscous, but it has been observed that adding l~ mannitol to such an emulsion restores viscosity to even less than the viscosity of an emulsion without glucose.
~1 The anti-oxidation characteristics of the emulsion 2~ are improved dramatically by adding tocopherols, such as 23 alpha tocopherol acetate, as may be seen from the results of 2~ experiments given in the following Example IV.

2~ EXAMPLE IV
27 Fluorocarbon emulsions were prepared without 28 mannitol or tocopherol (B~tch I in the table 1 below), with 2~ mannitol but without tocopherol (Batch II in the table 1 below), with tocopherol but without mannitol (Batch III in the 31 table 1 below) and with mannitol and tocopherol together 32 (Batch IV in the table 1 below); In Batch II, mannitol was ....

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I added in the amount of 0.6% weight per volume in tha emulsion.
2 satch III had 0.05% weight per volume in alpha tocopherol 3 acetate added. Batch IV comprised 0.6% weight per volume of ~1 mannitol and 0.05% weight per volume of alpha tocopherol acetate. The emulsions were 100~ weight per volume G perfluoroctylbromide emulsions having as the emulsifying agent ~ ~.5% weight per volume lecithin, and further having 0.0252%
8 weight per volume of THAM as a buffer to maintain the pEI
~ before the experiment and storage at 7.6, having 0.2% weight per volume of glucose for osmolarity, having 0.025% weight per ~ volume of calcium chloride ~CaCl), having 0.005% ~eight per 12 volume of magnesium sulfate (MgS04), and having w~ter (H20) 13 quantity suficient to form the remainder of the emulsion.
~ All emulsions were saturated with oxygen at the time of preparation. Oxygenation was accomplished by sparging with lG 100% oxygen during the formulation of the emulsion.
17 Additionally, twenty milliliters (ml) of the emulsion were 18 placed in a 30 ml bottle having the head space filled with 1~ 100% oxygen. The bottle was sealed.
Thereafter, the oxygenated emulsions were then 21 sterilized at 121 degrees Centigrade for eight mlnutes by 22 autoclaving. Measurements of the partial pressure of oxygen 23 (pO2), partial pressure of carbon dioxide tPCo2)~ and hydrogen 2~ ion concentration (pH) were taken at ten days and thirty days, where the atmospheric pressure varied during the measurements 2G from 7~1 mm of mercury (Hg) to 7~6 mm Hg. Measurements were 27 taken at 38 degrees Centigrade. The results are given in 28 table 1 below, where in the first column are given the partial 29 pressures of oxygen ~pO2), in the second column are given the partial pressures of carbon dioxide (pC02) and in the third 31 column are given the resultant pH. The tocopherol used was 32 al h~ tocoph ~ol ~ceta-e a c-nce~tratlon of 0.05 grams per , .

'~ ' ' : ' 1 3 1 61Q,'''-., I 100 millili-ters of emulsion. The mannitol was 0.6 grams per 2 milliliter of emulsion. Readings were taken at ten (10) days .~ and thirty (30) daye: after preparation of the emulsion, and l the emulsion was stored at 10 degrees Centigrade. All r) measurements except for pH are given in millimeters of Hg.
TAsLE 1 ~ 10 days 30 days 9 Ba-tch ~ pO2pC02 ~
10 I 550.0 10.2 3.3 2~2.412.8 3.2 11II 650.1 0.7 7.171 643.41.2 7.072 12III 627.3 0.5 7.361 656.41.4 7.098 13IV 738.3 0.25 7.436 664.60.94 7.191 1~
Since the emulsion was saturated with water, lG approximately 47 mm Hg of the total 7~1 to 746 mm Hg pressure 17 should be attributed to H20 vapor. The emulsion having no 18 mannitol, tocopherol or any other effective anti-o~idant shows 1~ a significant reduction in oxygen content occurring, and an increase in C02 content with a pronounced acidity. No such 21 deleterious effect occurs with the addition of mannitol, 22 tocopherol or both. It can be observed that with mannitol and 23 tocopherol used together, the emulsion becomes super-saturated 24 with oxygen at ten days. At other times, the saturation of oxygen remains very high, close to full saturation at ten and 2~ at thirty days for emulsions with mannitol and/or tocopherol 27 added, with time having some effect.

29 As noted hereinabove, mannitol does not decrease the stability of the particle sizes in the emulsion. It is 31 believed tha~ mannitol actually improves the particle size stability by forming a protective interaction with the . ~:
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l lecithin membrane to protect the fluorocarbon particles and 2 prevent the particles from coalescing.
3 It has also been found that glucose is an effective l osmotic agen-t and works well in fluorocarbon emulsions. The 5 particle size characteristics of the emulsion are no-t ~; degraded with glucose being used as an osmotic agent, it has 7 also been found. Other sugars, such as mannose and fructose 8 are effective osmotic agents, and are also metabolized in (3 cells of the body to provide sources of energy. It is often desired, further, to have glucose in the emulsion as a 11 nutrient.
12 It is believed that glucose, like mannitol, 13 interacts with, or is incorporated in the lecithin membrane of 1~ the fluorocarbon particle to protect or stabilize the fluorocarbon particle membrane. This protection is IG particularly effective in freeze - thaw cycle accelerated 17 shelf life studies. In such studies, it has been found that 18 the particle size means remained substantially the 1~ same through as many as five rapid freezes to minus 2~ degrees Centigrade, each followed by thawing at room temperatures.
21 The most common bu~fering agents normally include 22 phosphate compounds. It is frequently desired, however, to 23 include calcium containing compounds in the emulsion as an 2~ additional electrolyte and as a nutrient, in particular when perfusing the heart and the cerebro-ventricular systems.
2G Calcium is essential, for example, for the heart muscle to 27 contract. Calcium containing compounds, however, such as 28 calcium chloride (CaCl~ will form calcium precipitates with 29 phosphate and carbonate buffers. Excessive amounts of such precipitates are harmful in the vascular and some other body 31 systems, in that calcium precipitates block vessels. In this 32 specification, the term "non-calcium precipitating" will be .:

1 3 t 6 ~J ! ~' 1 used to designate a mixture or solution which haY
.~ substantially no calcium precipitates or has calcium 3 precipitates in such small quantity so as not to result in ~i undesired or harmful body reactions.
~r) The hydrogen ion concentration (pH) of fluorocarbon / emulsions is related to the emulsion stability and biological 8 -tolerance. Acidic pH reduces the electronegativity of the 9 particles, which encourages aggregation and sedimentation.
Alkaline pH tends to stabilize the emulsion by increasing 11 electronegativity. Alkaline emulsions with a pH of up to 8.2 12 are well tolerated when injected into the coronary arteries.
When the pH is less than 7.0, the emulsion may cause decreased 1~ contractility of the heart muscle and ventricular ~ibrillation. For intracoronary use, the pH should be from lG 7.0 -to 7.8. An emulsion with a pH of between 4.0 and 8.4 can 17 be used intravenously and in certain other arteries such as 18 the femoral artery depending upon the purpose of the use.
19 Tris(hydroxymethyl)aminomethane, sometimes called THAM, is an effective buffering agent for fluorocarbon 21 emulsions to maintain the pH at predetermined levels. THAM, 22 also, is non-calcium precipitating; that is to say, THAM does ~, 23 not precipitate calcium salts.
2~ It has also been found that imidazole is a very -effective buffering agent for use in fluorocarbon emulsions.
2~ Imidazole is, also, non-calcium precipitating.
27 ~oth T~AM and im~dazole have an effect on the 2~ osmolarity of the emulsion. Use of imidazole or THaM
29 increases the alkalinity of the emulsion, and normally would be used ln conjunction with other osmotic agents to maintain 31 ~he osmolarity without causing the pH to vary beyond desired 32 levels.

1 3 1 6 ! ', ' 1 If calcium is not desired or if moderate amounts of 2 calcium precipitates can be tolerated, phosphate and carbonate 3 buffers, including monobasic sodium phosphate, dibasic sodium phosphate, monobasic potassium phosphate, dibasic potassium phosphate, sodium bicarbonate and combinations including these G buffers will be suitable.
The osmotic a~ents and buffers discussed herein are ~ effective for formulating several stable, non-toxic and/or !) efficacious fluorocarbon emulsions. For a stable emulsion, the fluorocarbon in emulsion may be mono-brominated perfluoro-11 carbons, such as l-bromoseptadecafluoroctane (C8Fl7Br, some-12 times desi~nated perfluoroctylbromide or ''PFO~'I), l-bromo-13 pentadecafluoroseptane (C7Fl5Br), and l-bromotridecafluorohex-11 ane (C6Fl3~r, sometimes known as perfluorohexylbromide or "PFHB"). Other stable fluorocarbon emulsions are C4F~C}I-C~rC4F9 1~ (sometimes designated "F-44E"), i-C3F7CH-CHC6Fl3 ("F-i36E"), 1~ and C6Fl3CH=CHC6Fl3 ("F-66E"), ClOFl8 ("F-declin"), F-adaman-1~ tane ("FA"), F-methyladamantane ("FM~"), F-l,3-dimethylada-19 mantane ("FDMA"), F-declin ("FDC"), F-4-methyloctahydroquino-lidizine ("FMOQ"), F-4-methyldecahydroquinoline ("FHQ"), 2~ F-4-cyclohexylpyrrolidine ("FC~IP"), F-2-butyltetrahydrofuran 22 ("FC-75"). Addi-tional stable fluorocarbon emulsions that can 23 achieve small particle sizes and long shelf lives when made in 2~ accordance with this invention include 2~ (CF3)2CFO(CF2CF2)20CF(CF3)2, (CF3)2CFOtCF2CF2)30CF(CF3)2, 2~ (CF3)2CFO(CF2CF2)2F, (CF3)2CFO(CF2CF2)3F, (C6Fl3)20 and 27 F~CF(CF3)CF20]2CHFCF3. The present invention as it relates to 2~ the aspects of such fluorocarbon emulsion stability can be 29 further understood by reference to the follo~ing illustrative examples.

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ExaMpLl3V~ G ~
2l An emulsion of F-4gE, that is C4F9C~-CHC4F~, was prepared by first preparing an aqueous phase. The aqueous phase was in a solution containing 2.08% weight per volume of mannitol, 18.75% weight per volume of lecithin, and 0.10 G!! weight per volume of alpha tocopherol aceta-te.
The aqueous phase was buffered with 0.0515% weight ~¦1 per volume TH~M, resulting in a pH of approximately 7.8 after 9 the emulsion was prepared for further testing. In order to 0 arrive at this pH, the initial pH after adding the buffer was l1 approximately 8.2. This buffered, aqueous phase solution is 12 sometimes designated the vehicle. The vehicle is homogenized 3 or mixed.
1l The fluorocarbon F-44E was then metered in a predetermined, measured rate into the vehicle or aqueous phase 1~ to ultimately achieve ~6.1% weight per volume of the F~4~E in 1/ the emulsion. The resulting amounts of the emulsion 18 components were 9% weight per volume of lecithin, 1% weight 19 per volume of mannitol, 0.05% weight per volume of tocopherol, 0.0247% wei~ht per volume of T~I~M, and 100% weight per volume 21 of F-44E.
22 The resulting mixture was then placed into a flow 23 path which was divided into a plurality of flow paths. The 2~ flows were redirected to impinge upon each other at velocities in excess of 1500 feet per second in sheets of interaction in 2~ a cavity under 4,000 pounds per square inch or more of 27 pressure and subjected to an ice bath kept at from five 2~ degrees to ei~ht degrees Centigrade surrounding the chamber 29 containing the cavity. This flow procedure was repeated six times.
31 The emulsion was then sterilized by autoclave at 121 32 degrees Centigrade for eight minutes. The particle size ' 22 ' ' , ` " ~ ' ' .

1316~L'''A''' 1 distribution was analyzed in a Nicomp submicron par-ticle sizer 2 manufactured by Pacific Scientific Co. of Anaheim~ CaliEornia.
3 This analyzer determines relative quantities of va~ious sized particles by a method of dynamic light scattering. The 5 fluorocarbon particles in the emulsion had a size G characteristic of 188.1 nanometers mean diameter after this 7 initial heat step.
8 The emulsion was then alternately frozen to minus 20 9 degrees Centi~rade and thawed to room tempera-ture three times.
0 The mean fluorocarbon particle size measured after the third 1l thaw was 193.8 nanometers. The emulsion was then subjected to 12 three heat stress sessions of 121 degrees Centigrade for sixty 13 minutes each. The particle size was then analyzed and found ~4 to have a characteristic mean diameter of 601.2 nanometers.

I G EXAMPLE VI
17 An emulsion of F-declin, that is ClOF18, was 18 prepared by first preparing an aqueous phase. The aqueous 19 phase was in a solution containing 2.08% weight per volume of mannitol as an osmotic agent, 18.75% weight per volume of 21 lecithin, and 0.104 weight per volume of alpha tocopherol 22 acetate.
23 ~he aqueous phase was buffered with 0.0515% weight 2~1 per volume THAM, resulting in a pH of approximately 7.8 after the emulsion was prepared for further testing. In order to 2G arrive at this p~, the initial pH after adding the buffer was 27 approximately 8.2. This buffered, aqueous phase solution is 28 sometimes designated the vehicle. The vehicle is homogenized 29 or mixed.
The fluorocarbon F-declin was then metered at a 31 predetermined, measured rate into the vehicle or aqueous phase 32 to ultimatel chieve 99 53~ weight per volume of the F-declin : .
' :

1 31 6~i2 l 1 in the emulsion. The resulting amounts of the emulsion 2 components were 9~ weight per volume of lecithin, 1% weight 3 per volume of mannitol, 0.05% weight per volume of tocopherol, .~ 0.0247% weight per volume of T~IAM, and 100% weight per volume 5 of F-declin.
The resulting mixturs was then placed into a flow / path which was divided into a plurality oF flow paths. The g flows were redirected to impinge upon each other at velocities D in excess of 1500 feet per second in sheets of interaction in ~ a cavity under ~,000 pounds per square inch or more of 11 pressure and subjected to an ice bath as described for Example 12 V above. This flow procedure was repeated six times.
13 The emulsion was then sterilized by autoclave at 121 1~ degrees Cen-tigrade for eight minutes. The particle size distribution was analyzed in the same Nicomp submicron lG particle sizer described above in Example V. The fluorocarbon 17 particles in the emulsion had a size characteristic of 1~5.7 18 nanometers mean diameter after this initial heat step.
1~ The emulsion was then alternately frozen to minus 20 degrees Centigrade and thawed to room temperature three times.
21 The mean fluorocarbon particle size measured after the third 22 thaw was 145.1 nanometers. The emulsion was then subjected to 23 three heat stress sessions of 121 degrees Centigrade for sixty 2~ minutes each. The particle size was then analyzed and found to have a characteristic mean diameter of 86.9 nanometers.
2~
27 It has been found that, in general, it is desirable 28 to repeat the flow and impingement steps for four times, and 2~ sometimes five and six times in order to maximize stability of the emulsion. Sometimes the heat generated by the impingemen-t 31 has a tendency to hydrolyze lecithin. This hydrolysis can be 32 reduced or eliminated by mainta ning the cavity in which the .
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, ' 1316~2fl 1 impingement takes places in an ice bath at approximately five 2 to ten degrees Centigrade. It should be unnecessary to cool 3 or otherwise remove heat from the impin~ement cavity when an ~1 emulsifying agent which is not heat sensitive is used. Many of the fluorinated surfactants are not heat sensitive, such as triperfluoralkylcholate and perfluoroalkylcholestanol for ~ examples.

!) Fluorocarbon emulsions can be used effectively for delivery of therapeutic agents, medicines and drugs throughout 11 the body, tissue and organs. The particles comprising the 12 discontinuous fluorocarbon phase of the emulsion comprise two 13 principal components, the fluorocarbon and the encasing 1~ membrane. The stability of this discontinuous fluorocarbon phase allows at least two modes of carrying the therapeutic IG agent, medicine or drug, namely solution of the agent, 17 medicine or drug within the fluorocarbon phase, and complexing 18 of the agent, medicine or drug with the membrane. ~xamples of 19 medicines, drugs and therapeutic agents which dissolve in the fluorocarbon are diazepam, cyclosporin, rifampin, clindamycin, 21 isoflurane, halothane and enflurane. Examples of medicines, 22 therapeutic agents and drugs which do not dissolve in 23 fluorocarbon, but which complex with, for example, a lecithin 2~ membrane include mannitol, tocopherol, streptokinase, dexamethasone, prostaglandin E, Interleukin II, gentamycin and 2B cefoxitin. ~ntibiotics may be delivered transcutaneously 27 through the skin when added to a fluorocarbon emulsion.
~28 Thrombolytic agents, such as streptokinase and other 29 enzymes have been transported and delivered by fluorocarbon emulsions. It is believed that the low surface tension of the 31 fluorocarbons, and of the fluorocarbon emulsions having S2 lecithin or fluorosurfactants as the emulsifylng agent, ~:
: ' 1 31 6~, ) provide a very effective wetting fluid that permeates capillaries and vascular channels, as well as other narrow channels within the body. Transport of thrombolytic agents carried by such a fluorocarbon emulsion is demonstrated by the following Example VII:
EXAMPLE VII
A 40% weight per volume perfluoroctylbromide emulsion was prepared using the method described hereinabove in Example V, having 6% weight per volume lecithin as the emulsifying agent, 0.01% weight per volume dexamethasone, 0.01% weight per volume tocopherol, 1.5% weight per volume glycerol, and having as a buffer monobasic sodium phosphate at 0.012% w/v and dibasic sodium phosphate at 0.0563% w/v. The emulsion was formulated in accordance with the procedure described hereinabove and in my U.S. Patent No. ~,865,836, with the dexamethasone added during the vehicle formation. Streptokinase was added before the impingement flow steps, and three flow steps were performed.
The emulsion was placed in test tubes having clotted human blood. From 80% to 90% of the clots lysed in less than twenty minutes. Streptokinase alone, not in the presence of the fluorocarbon emulsion lyses the clots at substantially the same rate. Fluorocarbon emulsions, therefore, do not inhibit the action of the streptokinase.
The foregoing detailed description of my invention and of preferred embodiments, as to products, compositions and processes, is illustrative of specific embodiments only. It is to be understood, however, that additional embodiments may be perceived by those skilled in the art. The embodiments described herein, together with those additional embodiments, JJ ~ 26 ~r i,~

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Claims

1. A fluorocarbon emulsion, prepared by:
combining an aqueous phase with an effective amount of emulsifying agent and a fluorocarbon to form a mixture having from greater than 50% to about 125% weight per volume of said fluorocarbon; and passing the fluorocarbon-containing mixture through a mechanical emulsification apparatus in which said mixture is subjected to sufficiently high flow rates and pressures to form a stable, heat sterilizable fluorocarbon-in-water emulsion;
wherein said emulsion is biocompatible and exhibits substantial particle size stability in the non-frozen state following heat sterilization.

2. The emulsion of claim 1, wherein said emulsion further comprises an effective amount of an osmotic agent for adjusting and maintaining the osmolarity of the emulsion.

3. The emulsion of claim 1, which has been heat sterilized.

4. A storage stable, heat sterilizable fluorocarbon emulsion, comprising:
a continuous aqueous phase, a discontinuous fluorocarbon phase, and an effective amount of emulsifying agent, wherein the concentration of said fluorocarbon phase in said emulsion is greater than 75% and no more than 125%, weight per volume, and wherein said emulsion exhibits substantial particle size stability on storage in the non-frozen state following heat sterilization and is biocompatible.

5. The emulsion of claim 4, wherein the concentration of said fluorocarbon phase in said emulsion is at least about 80%, weight per volume.

6. The emulsion of claim 4, wherein the concentration of said fluorocarbon phase in said emulsion is at least about 100%, weight per volume.

7. The fluorocarbon emulsion of claim 1 wherein the emulsifying agent is a phospholipid.

8. The fluorocarbon emulsion of claim 7 wherein the phospholipid is lecithin.

9. The fluorocarbon emulsion of claim 1 wherein the emulsifying agent is an anionic surfactant.

10. The fluorocarbon emulsion of claim 1 wherein the fluorocarbon is a mono-brominated perfluorocarbon.

11. The fluorocarbon emulsion of claim 10 wherein the mono-brominated perfluorocarbon is 1-bromosep-tadecafluoroctane.

12. The fluorocarbon emulsion of claim 10 wherein the mono-brominated perfluorocarbon is 1-bromo-tridecafluorohexane.

13. The fluorocarbon emulsion of claim 10 wherein the mono-brominated perfluorocarbon is 1-bromopen-tadecafluoroseptane.

14. The fluorocarbon emulsion of claim 1 wherein the fluorocarbon is C4F9CH=CHC4F9.

15. The fluorocarbon emulsion of claim 1, wherein the fluorocarbon is F-decalin.

16. The fluorocarbon emulsion of claim 1 wherein the emulsifying agent comprises a biocompatible fluorinated surfactant.

17. The fluorocarbon emulsion of claim 16 wherein the fluorocarbon emulsion and fluorinated surfactant have a sufficient elimination rate that the fluorocarbon emulsion and fluorinated cosurfactant are substantially eliminated from the animal body or organ before carcinosis occurs.

18. The fluorocarbon emulsion of claim 16 wherein the fluorocarbon emulsion and fluorinated surfactant have a sufficient elimination rate that the fluorocarbon emulsion and fluorinated cosurfactant are substantially eliminated from the animal body or organ before teratogenesis occurs.

19. The fluorocarbon emulsion of claim 16 wherein the fluorocarbon emulsion and fluorinated surfactant have a sufficient elimination rate that the fluorocarbon emulsion and fluorinated cosurfactant are substantially eliminated from the animal body or organ before embryotoxicity occurs.

20. The fluorocarbon emulsion of claim 16 wherein the fluorinated cosurfactant comprises a fluorinated polyhydroxylated surfactant.

21. The fluorocarbon emulsion of claim 1 further comprising a buffering agent selected from the group consisting of imidazole, tris(hydroxymethyl)aminomethane, and combinations thereof.

22. The fluorocarbon emulsion of claim 21 wherein said buffering agent group further consists of sodium bicarbonate, monobasic sodium phosphate, dibasic sodium phosphate, magnesium sulfate, magnesium chloride, sodium chloride, potassium chloride, monobasic potassium phosphate, dibasic potassium phosphate and non-calcium precipitating combinations thereof.

23. The fluorocarbon emulsion of claim 21 wherein the pH of the emulsion is maintained at from approximately 4.0 to approximately 8.4 after sterilization and before use intravenously.

24. The fluorocarbon emulsion of claim 21 for use wherein the pH of the emulsion is maintained at from approximately 7.0 to approximately 7.8 before use in the coronary arteries.

25. The fluorocarbon emulsion of claim 21 wherein said buffering agent is imidazole.

26. The fluorocarbon emulsion of claim 1 further comprising an anti-oxidant.

27. The fluorocarbon emulsion of claim 26 wherein said anti-oxidant comprises mannitol.

28. The fluorocarbon emulsion of claim 26 wherein the anti-oxidant comprises a tocopherol.

29. The fluorocarbon emulsion of claim 26 wherein the anti-oxidant comprises mannitol and tocopherol.

30. The fluorocarbon emulsion of claim 28 wherein the tocopherol comprises alpha-tocopherol acetate.

31. The fluorocarbon emulsion of claim 26 including an anti-oxidant for reduction of oxidation of components of said emulsion comprising an effective amount of an anti-oxidant selected from the group consisting of ascorbyl palmitate, mannitol, tocopherol, imidazole and combinations thereof.

32. The fluorocarbon emulsion of claim 26 including an anti-oxidant for reduction of oxidation of tissues of animal bodies and organs thereof comprising an effective amount of an anti-oxidant selected from the group consisting of ascorbyl palmitate, mannitol, tocopherol, imidazole and combinations thereof.

33. The fluorocarbon emulsion of claim 1 for application to tissue of animal bodies and organs thereof, further comprising mannitol and tocopherol in an effective amount for reduction of oxidation in said emulsion.

34. The fluorocarbon emulsion of claim 1 for application to tissue of animal bodies and organs thereof, further comprising mannitol and tocopherolan in an effective amount for reduction of oxidation in said tissue of animal bodies and organs thereof.

35. The fluorocarbon emulsion of claim 1 for application to tissue of animal bodies and organs thereof, further comprising mannitol in an effective amount for reduction of oxidation in said emulsion.

36. The fluorocarbon emulsion of claim 1 for application to tissue of animal bodies and organs thereof, further comprising mannitol in an effective amount for reduction of oxidation in said tissue of animal bodies and organs thereof.

37. The fluorocarbon emulsion of claim 31 for application to tissue of animal bodies and organs thereof for reduction of oxidation therein wherein said anti-oxidant group further includes ascorbic acid, salts and complexes thereof and non-calcium precipitating combinations thereof.

38. The fluorocarbon emulsion of claim 1 wherein the fluorocarbon in emulsion is in an amount of from 80%
weight per volume to 125% weight per volume.

39. The fluorocarbon emulsion of claim 2, wherein the osmolarity of the emulsion is maintained between from about 240 milliosmols to about 650 milliosmols.

40. The fluorocarbon emulsion of claim 2, wherein the osmolarity of the emulsion is maintained between from about 400 milliosmols to about 450 milliosmols.

41. The fluorocarbon emulsion of claim 39 wherein the osmolarity is maintained, at least in part, by a hexahydric alcohol.

42. The fluorocarbon emulsion of claim 41, wherein the hexahydric alcohol is selected from the group consisting of mannitol and sorbitol.

43. The fluorocarbon emulsion of claim 42, wherein the hexahydric alcohol is mannitol, present at a concentration of from about 0.25% weight per volume to about 1.5% weight per volume.

44. The fluorocarbon emulsion of claim 39 wherein the osmolarity is maintained, at least in part, by a sugar.

45. The fluorocarbon emulsions of claim 44, wherein the sugar is selected from the group consisting of glucose, mannose and fructose, or combinations thereof.

46. The fluorocarbon emulsion of claim 39 or 40, wherein the osmolarity is maintained at least in part, by a buffering agent.

47. The fluorocarbon emulsion of claim 39 or 40, wherein the osmolarity is maintained, at least in part, by chloride or sulfate salts.