WO1993016720A1

WO1993016720A1 - Inhibition of bacterial endotoxin

Info

Publication number: WO1993016720A1
Application number: PCT/US1992/001296
Authority: WO
Inventors: Thomas A. Lane; Edward V. Wancewicz; Rhonda Funk; Dan J. Smith
Original assignee: The Regents Of The University Of California; Alliance Pharmaceutical Corporation
Priority date: 1992-02-20
Filing date: 1992-02-20
Publication date: 1993-09-02
Also published as: AU2198592A

Abstract

A method for inhibiting the biological effect of bacterial endotoxin on mammalian cells in a system containing such endotoxin, comprising the step of administering to the cells an effective endotoxin-inhibiting amount of a fluorocarbon emulsion. Although the method may be used to treat cells in vitro, the invention is preferably used to treat whole organisms. In a preferred aspect of the invention, the emulsion is administered to a mammal having a bacterial infection in a dosage effective to prevent or reduce endotoxic or septic shock in the mammal.

Description

INHIBITION OP BACTERIAL ENDOTOXIN

FIELD OF THE INVENTION This invention relates to inhibition of bacterial endotoxin through administration of fluorocarbon emulsions.

BACKGROUND OF THE INVENTION It has been estimated that up to 300,000 people develop bacteremia annually. Bacteremic shock or septic shock is a complication of this condition in approximately one-half of. these patients. The mortality rate for bacteremic shock is 40-60%.

The prime mediator of the mortality and morbidity associated with bacteremic shock is bacterial endotoxin. Endotoxin consists of a core polysaccharide with oligosaccharide side chains linked to a lipid called lipid-A.

Classic septic shock syndrome results primarily from activation by bacterial endotoxin of the complement, coagulation, kinin, and ACTH/endorphin systems. This activation results in a series of metabolic events that ultimately progress to a state of shock.

Septic shock related to bacteremic infections advances in two hemodynamic stages. First, patients exhibit vasomotor dysfunction resulting from ACTH/endorphin release, kallikrein- kinin system activation, and histamine release. Next, complement-mediated leukoagglutination and capillary damage cause a sever capillary leak syndrome. A dramatic decrease in intravascular blood volume, a decline in cardiac output, and disseminated intravascular coagulation follow, with high morality rates.

Prevention and treatment of bacteremic shock is of major importance. Numerous efforts are underway to address this problem. It has been demonstrated, for example, that immune plasma from volunteers who have been vaccinated with strains of gram negative bacteria may ameliorate the effects of septic shock to some extent. (Ziegler, et al., K. Engl. J. Med. 307:1225-1230 (1982).) More recently, it has been demonstrated that monoclonal antibodies to lipid-A are capable of improving the outcome of septic shock. (See, e.g., Greenman, et al. , JAMA 266:1097-1102 (1991).) Furthermore, very low density lipoprotεin, chylomicrons, and a synthetic lipid emulsion have been shown to prevent endotoxin-induced death in mice. (Harris, et al., J. Clin. Invest. 86:696-702 (1990).)

Although antibody therapy shows great promise in treating septic shock, it is not an altogether satisfactory solution. Such therapy has proved only partially successful in controlling the morbidity and mortality associated with endotoxin shock. It is believed that such therapy may suffer from disadvantages such as unfavorable kinetics, biological half-life, an immune response to the therapeutic antibodies, or possible generation of anti-idiotypic antibody that would neutralize the therapeutic antibody.

This invention provides an alternative therapy for prevention and treatment of disease associated with bacterial endotoxin.

SUMMARY OF THE INVENTION

The present invention utilizes fluorocarbon emulsions to inhibit bacterial endotoxin. It has been demonstrated that even relatively low quantities of fluorocarbon emulsion are effective, through mechanisms not fully understood, to inhibit the activity of bacterial endotoxin.

One embodiment of the present invention comprises a method for inhibiting the biological effect ^' of bacterial endotoxin on mammalian cells in a system containing such endotoxin, comprising the step of administering to the cells an effective endotoxin-inhibiting amount of a fluorocarbon emulsion. Although the method may be used to treat cells in vitro , the invention is most valuable in treating cells in vivo; e.g., treating whole organisms. In a preferred aspect of the invention, the emulsion is administered to a mammal having a bacterial infection in an amount effective to prevent or reduce endotoxic shock in the mammal.

Preferred emulsions include perfluorocarbon emulsions, such as perflubron emulsions. Any effective concentration may be used. Suitable emulsions, for example, may have a perfluorocarbon concentration between about 5% and about 125%, weight per volume. An effective dose will generally be between about 1 and 20 grams fluorocarbon per kilogram of patient weight, although other dosage ranges are contemplated.

The invention further comprises the use of a systemically-administered fluorocarbon emulsion for binding circulating endotoxin in a mammal, as well as the use of a fluorocarbon emulsion in the preparation of a medicament for treatment or prevention of disease associated with bacterial endotoxin.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph showing the inhibition of LPS-induced ICAM-1 production by human umbilical vein endothelial cells as a function of perflubron emulsion concentration.

Figure 2 is a graph showing the inhibitory effect of perflubron emulsion on LPS-induced ICAM-1 production as a function of LPS concentration.

Figure 3 is a graph showing the inhibitory effect of perflubron emulsion on LPS-induced production of TNF by RAW cells.

DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention, a fluorocarbon emulsion is administered to a patient for treatment of endotoxin shock or septic shock. The treatment may be prophylactic (in a patient with a bacterial infection but with no endotoxin shock) or therapeutic (in patients suffering from endotoxin shock) . The fluorocarbon emulsion may be selected from a wide range of suitable emulsions. Advantageously, it is a fluorocarbon-in-water emulsion, having a preferred fluorocarbon concentration of about 5% to about 125%, w/v.

Fluorocarbons are fluorine substituted hydrocarbons that have been used in medical applications as imaging agents and as blood substitutes. U.S. Patent No. 3,975,512 to Long uses fluorocarbons, including brominated perfluorocarbons, as a contrast enhancement medium in radiological imaging. Brominated fluorocarbons and other fluorocarbons are known to be safe, biocompatible substances when appropriately used in medical applications. It is additionally known that oxygen, and gases in general, are highly soluble in some fluorocarbons. This characteristic has permitted investigators to develop emulsified fluorocarbons as blood substitutes. For a general discussion of the objectives of fluorocarbons as blood substitutes and a review of the efforts and problems in achieving these objectives see "Reassessment of Criteria for the Selection of Perfluorochemicals for Second-Generation Blood Substitutes: Analysis of Structure/Property Relationship" by Jean G. Reiss, Artificial Orσans 8:34-56, 1984.

The fluorocarbon, in one^" preferred embodiment, is a perfluorocarbon or substituted perfluorocarbon. Fluorocarbon molecules used in these emulsions may have various structures, including straight or branched chain or cyclic structures, as described in Riess, J. , Artificial Organs 8(l):44-56 (1984). These molecules may also have some degree of unsaturation, and may also contain bromine or hydrogen atoms, or they may be amine derivatives. The fluorocarbons may be present in the emulsion in useful concentration ranging from about 5% to 125% weight per volume (w/v) . As used throughout, concentrations defined as weight/volume are understood to represent grams/ml and % weight per volume to represent grams/100 ml.

Although concentrations as low as 5%, w/v are contemplated, in a preferred embodiment the concentrations are at least 25% or 30%, preferably at least 40%, 50%, 55%, 60%, 75% or 80% W/v. Preferred fluorocarbon emulsion formulations are those disclosed in U.S. Patent Nos. 4,865,836; 4,987,154; and 4,927,623, which are hereby incorporated by reference.

There are a number of fluorocarbons that are contemplated for use in the present invention. These fluorocarbons include bis(F-alkyl) ethanes such as C₄F₉CH=CH_/(CF₉ (sometimes designated "F-44E") , i-C₃F₉CH=CHC₆F₁₃ ("F-i36E") , and C₆F₁₃CH=CHC₆F₁₃ ("F-66E") ;cyclic fluorocarbons, such as C10F18 ("F-decalin", "perfluorodecalin" or "FDC") , F-adamantane ("FA"), F-methyladamantane ("FMA") , F-l,3-dimethyladamantane ("FDMA"), F-di-or F-trimethylbicyclo[3,3,l]nonane ("nonane") ; perfluorinated amines, such as F-tripropylamine("FTPA") and F- tri-butylamine ("FTBA"), F-4-methyloctahydroquinolizine ("FMOQ"), F-n-methyl-decahydroisoquinoline ("FMIQ") , F-n- methyldecahydroquinoline ("FHQ") , F-n-cyclohexylpurrolidine ("FCHP") and F-2-butyltetrahydrofuran ("FC-75"or "RM101") . Other suitable fluorocarbons may be selected from brominated perfluorocarbons, such as 1-bromo-heptadecafluoro- octane (C₈F₁₇Br, sometimes designated perfluorooctylbro ide or "PFOB", now known by the U.S. Adopted Name "perflubron"), 1- bromopenta-decafluoroheptane (C₇F₁₅Br) , and 1- bro otridecafluorohexane (C₆F₁₃Br, sometimes known as perfluorohexylbromide or "PFHB") . Other brominated fluorocarbons are disclosed in US Patent No. 3,975,512 to Long. Also contemplated are fluorocarbons having nonfluorine substituents, such as perfluorooctyl chloride, perfluorooctyl hydride, and similar compounds having different numbers of carbon atoms, e.g., 6-12 carbon atoms.

Additional fluorocarbons contemplated in accordance with this invention include perfluoroalkylated ethers or polyetherε, such as (CF₃)₂CFO(CF₂CF₂)₂OCF(CF₃)₂, (CF₃)₂CF0- (CF₂CF₂)₃OCF(CF₃) , (CF₃)CFO(CF₂CF₂)F, (CF₃)₂CFO(CF₂CF₂)-,F, (C₆F₁₃)₂0. Further, fluorocarbon-hydrocarbon compounds, such as, for example compounds having the general formula C_nF_2n+1-

^Cn'^F2n.₊i'

^{or c}n^P2r».^CFβCHCn*^F2n-₊.' ^where " and n^» are the same Or different and are from about 1 to about 10 (so long as the compound is a liquid at room temperature) . Such compounds, for example, include C₈F₁₇C₂H₅ and C₆F₁₃ CH=CHC₆H₁₃. It will be appreciated that esters, thioethers, and other variously modified mixed fluorocarbon-hydrocarbon compounds are also encompassed within the broad definition of "fluorocarbon" materials suitable for use in the present invention. Mixtures of fluorocarbons are also contemplated. Additional "fluorocarbons" not listed here, but having those properties described in this disclosure that would lend themselves to use in vivo in accordance with the present invention are also contemplated.

Emulsifying agents used in the emulsions of this invention may be anionic, cationic or non-ionic surfactants or combinations thereof as are well known to those in the chemical arts or they may be mixtures of synthetic compounds such as Pluronic F-68, a condensate of ethylene oxide with propylene. glycol, as used in U.S. Patent No. 4,073,879 to Long. Fluorosurfactants, such as those described by J. Riess et al. Int'l Symposium on Blood Substitutes, Montreal, May, 1987, are particularly suitable can also be used. Emulsifying agents may also be mixtures of the above agents". Particularly suitable emulsifiers may include natural amphipathic compounds such as phospholipids, particularly phosphatidylcholine, wherein combined hydrophilic and hydrophobic properties enable the molecule to interface with both aqueous and fluorocarbon systems, thereby forming the emulsion droplets. There are various species of each class of phospholipids, such as the phospholipid cholines, comprising various pairings of saturated and unsaturated fatty acids in the glycerol structures. Phosphatidylcholine is an abundant natural material (lecithin) which may be purified from egg yolk, or may be produced synthetically (Avanti Polar Lipids, Pelham, ALA) . Phospholipid emulsifiers, particularly egg yolk phospholipid and lecithin, are particularly preferred.

The phospholipid emulsifying agent should be included in the range of from 2 to 14% w/v, increasing the phospholipid concentration with increasing fluorocarbon concentration. The preferred amount for an emulsion comprising 75% w/v bromofluorocarbon is 2.5 to 5% w/v and 3.5 to 10% w/v of phospholipid for an emulsion with 100% w/v bromofluorocarbon. In a preferred embodiment, the phospholipid comprises at least 2% w/v of the emulsion.

Emulsification requires large amounts of energy to convert a two-phase immiscible system into a suspension of discontinuous small droplets of hydrophobic fluid in an aqueous continuous phase. Fluorocarbon emulsification may be carried out generally by either of two general processes which provide energy to the system to break up the fluorocarbon volume into small droplets. In sonication emulsification, a probe is inserted into the mixture of fluorocarbon, emulsifier, and aqueous phase, and bursts of energy are released from the tip of the probe. In a mechanical emulsification process, such as performed by a Microfluidizer™ apparatus (Microfluidics, Newton, MA 02164), streams of the mixed emulsion components are directed through the apparatus at high velocity and under high pressure (e.g. 15,000 psi) , and the high shear forces or cavitation resulting from the mechanical stress applied to the fluid produce the emulsion. The aqueous phase of the emulsion may have components dissolved therein which give the emulsion desirable properties. For example, it may comprise an osmotic agent to bring the emulsion to physiological isotonicity. The osmotic agent may be sodium chloride, or it may be a polyhydroxyl compound, such as a sugar or mannitol. The aqueous phase will also contain soluble buffering agents.

The lipid phase of the emulsion may also have components dissolved therein. For example, a phosphatidyl choline emulsifier may have glycerol, phosphatidyl glycerol, other phospholipids or cholesterol admixed, and further contain an antioxidant substance, such as a tocopherol, to protect against lipid oxidation.

Several fluorocarbon emulsions have been produced commercially for use as intravascular oxygen carriers. These include a mixed decalin emulsion sold by Alpha Therapeutics Corp. under the trademark FLUOSOL and perflubron emulsions produced by Alliance Pharmaceutical Corp. of San Diego, California.

The suitability of any combination of fluorocarbon and emulsifier in an emulsion for use in the present invention can be determined through the in vitro screening procedure outlined in Example l.

The emulsions may be used to protect cells in vitro against the action of endotoxin by administering the perfluorocarbon emulsion to the cells in cell growth medium. When a 90% or 100% w/v emulsion is used, for example, a sufficient quantity of emulsion may be added so that the growth medium contains from about 0.1% to about 80%, v/v, of the emulsion; preferably from about 5% to about 50%, v/v, of the emulsion. Although the emulsion may be administered after contact between the cells and the endotoxin, the greatest protection is achieved by administering the emulsion prior to or simultaneously with the endotoxin.

The more important use of the invention, of course, is in treating cells in vivo ; i.e., protecting a whole organism against deleterious effects of bacterial endotoxin, such as endotoxin shock or septic shock. In a preferred embodiment, this involves the prophylactic or therapeutic administration of fluorocarbon emulsion to a mammal, such as a human. Due to the -oxygen carrying properties of the emulsions, they not only provide protection against endotoxin, they also prevent or treat tissue ischemia associated with endotoxin shock.

In one preferred embodiment, a patient suffering from endotoxin shock receives from about 0.5 to 10 ml/kg, preferably 1 to 5 ml/kg of a 90% or 100% w/v biocompatible fluorocarbon emulsion at periodic intervals, e.g., every 4 to 8 hours. For an adult human, that dosage would be about 50- 400 ml of emulsion. Similar levels will be used for prophylactic administration of emulsion. The therapy will usually be continued for 24 to 48 hours, or as long as bacterial infection continues to produce harmful levels of endotoxin.

Typically, the emulsion will be administered intravenously. However, localized administration is also contemplated. Thus, for example, where intraperitoneal bacterial infection exists, the emulsion may be administered intraperitoneally. Similarly, administration into other body cavities, including such externally-connected cavities as the bronchiopulmonary tree, are also within the scope of the present invention.

Although it is contemplated that the invention can be practiced as broadly as it is disclosed and claimed, the following nonlimiting examples illustrate the efficacy of the emulsions in inhibiting the effect of endotoxins and suggest how the invention can be used in a clinical setting.

EXAMPLE 1 Effect of Perflubron on Endotoxin-Induced Expression of ICAM-1 Leukocyte Adhesion Molecules by Endothelial Cells

The E. coli lipopolysaccharide endotoxin (LPS) , which is implicated in endotoxic shock in humans and other animals, is a potent activator of mammalian endothelial cells. LPS activated endothelial cells produce leukocyte adhesion molecules, including intercellular adhesion molecule-1 (ICAM- 1) . Expression of ICAM-1 by human umbilical vein endothelial cells (HUVEC) to which LPS has been added can be used as a measurement of the biological activity of the LPS.

HUVEC were grown from primary cultures, and activation as measured by the expression of ICAM-1 was assessed by ELISA in accordance with the procedures described by Lane, et al., Biochem. Biophys. Res. Comm. 172:1273-1281 (1991).

A. Effect of Emulsion Concentration on ICAM-1 Production

A perflubron emulsion was obtained from Alliance Pharmaceutical Corp., San Diego, California, having the following composition: Raw Material % W/V

Perflubron (perfluorooctylbromide) Egg Yolk Phospholipid (EYP) Tromethamine, USP Mannitol, USP Sodium Chloride, USP Edetate Calcium Disodium, USP D-alpha-Tocopherol Acetate Water for Injection

HC1 to adjust pH to about 7

A vehicle having substantially the same composition as the emulsion, but without the fluorocarbon, was also obtained from Alliance Pharmaceutical Corp. for use as a control.

The emulsion was added to the HUVEC cultures in concentrations ranging from 1.25% to 20%, expressed as volume of emulsion per volume of culture medium. Immediately thereafter, LPS was added to the cultures in a concentration of 100 ng/ml. The experiment was repeated, using the emulsion vehicle without perflubron. Tumor necrosis factor (TNF) also induces expression of ICAM-1 by HUVEC. The above procedure was repeated substituting TNF at 100 U/ml for LPS to determine whether the inhibitory effect of the fluorocarbon emulsion related to its action on LPS or whether the emulsion generally inhibited cellular expression of ICAM-1.

The production of ICAM-1 was determined in each instance by ^"ELISA as discussed previously, and the results were expressed in terms of optical density. The results are presented in Table 1, and are graphically displayed in Figure 1.

TABLE 1: Effect of perflubron on activation of HUVEC

13. Refers to the concentration of Agent (100% w/v perflubron emulsion or Vehicle), as X v/v required to diminish the Activator-induced increase in expression of ICAH-1 on HUVEC by 50%.

2. Refers to the X inhibition of the increase in 1CAM-1 expression induced by the Activator, in the presence of a 10X v/v concentration of the Agent. 20

As shown in Figure 1 and Table 1, perflubron inhibited or prevented LPS-induced HUVEC activation in dose-dependent fashion. The ID50 concentration of perflubron emulsion (v/v) was 6.0% ± 0.6%. At a 10% concentration of perflubron 5 emulsion, ICAM-1 expression was inhibited by 95% ± 6%. Vehicle also inhibited LPS-induced activation, but with much lower potency and less reliably than perflubron emulsion, with only 7 ± 11% inhibition at 10% concentration. Thus, the emulsions of the invention are clearly more effecive than the 10 phospholipid component of the emulsion alone. TNF-induced production of ICAM-1 was not significantly inhibited by the emulsion, indicating that results with LPS are not due to general cellular inhibition of ICAM-1 production.

15 B. Effect of LPS Concentration on ICAM-1 Production

HUVEC cells were contacted with perflubron emulsion at a concentration of 10%, v/v, and were then contacted with LPS at concentrations ranging from 0 to 2500 ng/ l. ICAM-1 production was assayed as above. Perflubron emulsion 20 inhibited the action of LPS at all concentrations of the activating agent, but increasing concentrations of LPS gradually overcame the inhibitory effect, as shown in Figure 2. C. Duration of Inhibition

A time course study of the inhibition of ICAM-1 production by HUVEC after activation by LPS was conducted, using the general procedures outlined above. This study indicated that the inhibitory effect of the perflubron emulsion persisted for the duration of the study, 72 hours.

EXAMPLE 2 Inhibition of LPS-Induced TNF Production by Macrophaqes

Murine macrophage cells (RAW cells) produce tumor necrosis factor (TNF) in response to incubation with LPS. This model was used to verify the inhibitory effect of fluorocarbon emulsions on LPS.

The production of TNF was assayed using the L929 indicator cell method, as described by Hay, et al. , J. Clin. Lab. Immunol. 29:151-155 (1989). Specifically, LPS was. incubated with the 100% w/v perflubron emulsion described earlier. The resulting mixture was diluted to 20%, v/v prior to addition to RAW cells. After incubation for 18 hours at 37°C, the supernatant was collected and added to the L929 indicator cells. Supernates were incubated with the L929 cells for 18 hours, followed by assessment of Trypan blue dye exclusion (cell viability) as a measure of TNF activity. The results are shown in Figure 3, where high optical density reflects low TNF production. The emulsions clearly inhibited TNF production, whereas vehicle alone did not.

EXAMPLE 3

Treatment of Patient At Risk for Endotoxin Shock

This Example outlines one suitable treatment protocol for a patient suffering from bacteremia and at risk for endotoxin shock. Of course, other protocols, routes of administration, dosages, and emulsions can be used, as will be recognized by those skilled in the art.

An adult patient having a severe gram negative bacterial infection is treated by administering 3 ml of a 90% perflubron emulsion (fluorocarbon in water, egg yolk phospholipid emulsifier, Alliance Pharmaceutical Corp., San Diego, California) intravenously at 6 hour intervals. The treatment is continued for 24 hours. Conventional antibiotic therapy is simultaneously administered. This therapy is effective to prevent or reduce endotoxin shock.

Claims

WHAT IS CLAIMED IS:

1. A method for inhibiting the biological effect bacterial endotoxin on mammalian cells in a system containi such endotoxin, comprising the step of administering to sa cells an. effective endotoxin-inhibiting amount of fluorocarbon emulsion.

2. The method of Claim 1, wherein said cells are vitro.

The method of Claim 1, wherein said cells are vivo .

4. The method of Claim 3, wherein said emulsion administered to a mammal having a bacterial infection a said amount is a dosage effective to prevent or redu endotoxic shock in said mammal.

5. The method of any one of Claims 1-4, wherein sa emulsion is a perfluorocarbon emulsion.

6. The method of Claim 5, wherein said perfluorocarb is perflubron.

7. The method of Claim 5, wherein the concentration perfluorocarbon in said emulsion is between 5% and 125 weight per volume.

8. The method of Claim 4, wherein said effecti dosage is between about 1 and 20 grams fluorocarbon p kilogram.

9. Use of a systemically-administered fluorocarb emulsion for binding circulating endotoxin in a mammal.

10. Use of a fluorocarbon emulsion in the preparati of a medicament for treatment or prevention of disea associated with bacterial endotoxin.

11. The use according to Claim 10, wherein sa emulsion is a perfluorocarbon emulsion having perfluorocarbon concentration between about 5% and 125%, w/ 12. The use according to Claim 11, wherein t perfluorocarbon is perflubron. AMENDED CLAIMS

[received by the International Bureau on 20 January 1993 (20.01.93); original claims 1,9 and 10 amended; other claims unchanged (1 page)]

1. A method for inhibiting the biological effect of bacterial endotoxin on mammalian cells in a system containing such endotoxin, comprising the step of administering to said cells an effective endotoxin-inhibiting amount of an aqueous emulsion of a fluorocarbon liquid.

2. The method of Claim l, wherein said cells are in vitro . 3. The method of Claim 1, wherein said cells are in vivo .

4. The method of Claim 3, wherein said emulsion is administered to a mammal having a bacterial infection and said .amount is a dosage effective to prevent or reduce endotoxic shock in said mammal.

5. The method of any one of Claims 1-4, wherein said emulsion is a perfluorocarbon emulsion.

6. The method of Claim 5, wherein said perfluorocarbon is perflubron. 7. The method of Claim 5, wherein the concentration of perfluorocarbon in said emulsion is between 5% and 125%, weight per volume.

8. The method of Claim 4, wherein said effectiv dosage is between about 1 and 20 grams fluorocarbon pe kilogram.

9. Use of a systemically-administered aqueous emulsio of a fluorocarbon liquid for binding circulating endotoxin i a mammal.

10. Use of an aqueous emulsion of a fluorocarbon liqui in the preparation of a medicament for treatment o prevention of disease associated with bacterial endotoxin.

11. The use according to Claim 10, wherein sai emulsion is a perfluorocarbon emulsion having perfluorocarbon concentration between about 5% and 125%, w/v 12. The use according to Claim 11, wherein th perfluorocarbon is perflubron.