Methods useful in endotoxin based therapy

Treatment and prophylaxis of endotoxin caused toxicity is disclosed. This is accomplished by administering phospholipid containing compositions to the subject. The compositions are protein and peptide free, and may contain triglycerides, or other polar or neutral lipids.

FIELD OF THE INVENTION 
This invention relates to the treatment of endotoxin related endotoxemia. 
More particularly, it relates to the treatment of such poisoning via 
administration of various compositions which act to neutralize and/or to 
remove endotoxins from the organism, as well as prophylaxis utilizing 
these compositions. 
BACKGROUND AND PRIOR ART 
Normal serum contains a number of lipoprotein particles which are 
characterized according to their density, namely, chylomicrons, VLDLS, 
LDLS and HDLS. They are composed of free and esterified cholesterol, 
triglycerides, phospholipids, several other minor lipid components, and 
protein. Very low density lipoprotein (VLDL) transports energy, in the 
form of triglycerides, to the cells of the body for storage and use. As 
triglycerides are delivered, VLDL is converted to low density lipoprotein 
(LDL). Low density lipoprotein (LDL) transports cholesterol and other 
lipid soluble materials to the cells in the body, while high density 
lipoprotein (HDL) transports excess or unusable cholesterol to the liver 
for elimination. Normally, these lipoproteins are in balance, 
ensuring/proper delivery and removal of lipid soluble materials. 
Abnormally low HDL can cause a number of diseased states as well as 
constitute a secondary complication in others. 
Under normal conditions, a natural HDL is a solid particle with its surface 
covered by a phospholipid monolayer that encloses a hydrophobic core. 
Apolipoprotein A-I and A-II attach to the surface by interaction of the 
hydrophobic face of their alpha helical domains. In its nascent or newly 
secreted form the particle is disk-shaped and accepts free cholesterol 
into its bilayer. Cholesterol is esterified by the action of 
lecithin:cholesterol acyltransferase (LCAT) and is moved into the center 
of the disk. The movement of cholesterol ester to the center is the result 
of space and solubility limitations within the bilayer. The HDL particle 
"inflates" to a spheroidal particle as more and more cholesterol is 
esterified and moved to the center. Cholesterol ester and other water 
insoluble lipids which collect in the "inflated core" of the HDL are then 
cleared by the liver. 
Anantharamaiah, in Segrest et al., Meth. Enzymol. 128: 627-647 (1986) 
describes a series of peptides which form "helical wheels", as a result of 
the interaction of the amino acids in the peptide with each other. Such 
helical wheels present a nonpolar face, and a polar face in their 
configuration. The reference shows, generally, that peptides can replace 
aproproteins in these particles. 
Jonas et al., Meth. Enzym. 128A: 553-582 (1986) have produced a wide 
variety of reconstituted particles resembling HDL. The technique involves 
the isolation and delipidation of HDL by standard methods (Hatch et al., 
Adv. Lip. Res. 6: 1-68 (1968); Scanu et al., Anal. Biochem. 44:576-588 
(1971) to obtain apo-HDL proteins. The apoproteins are fractionated and 
reconstituted with phospholipid and with or without cholesterol using 
detergent dialysis. 
Matz et al., J. Biol. Chem. 257(8): 4535-4540 (1982) describe a micelle of 
phosphatidylcholine, with apoliprotein A1. Various ratios of the two 
components are described, and it is suggested that the described method 
can be used to make other micelies. It is suggested as well to use the 
micelles as an enzyme substrate, or as a model for the HDL molecule. This 
paper does not, however discuss application of the micelles to cholesterol 
removal, nor does it give any suggestions as to diagnostic or therapeutic 
use. 
Williams et al., Biochem. & Biophys. Acta 875:183-194 (1986) teach 
phospholipid liposomes introduced to plasma which pick up apoproteins and 
cholesterol. Liposomes are disclosed, which pick up apoprotein in vivo, as 
well as cholesterol, and it is suggested that the uptake of cholesterol is 
enhanced in phospholipid liposomes which have interacted with, and picked 
up apoproteins. 
Williams et al., Persp. Biol. & Med. 27(3): 417-431 (1984) discuss lecithin 
liposomes as removing cholesterol. The paper summarizes earlier work 
showing that liposomes which contain apoproteins remove cholesterol from 
cells in vitro more effectively than liposomes which do not contain it. 
They do not discuss in vivo use of apoprotein containing liposomes or 
micelies, and counsel caution in any in vivo work with liposomes. 
It is important to note that there is a clear and significant difference 
between the particles of the present invention, and the liposomes and 
micelles described in the prior art. The latter involve a bilayer 
structure of lipid-containing molecules, surrounding an internal aqueous 
core space. The structure of liposomes precludes filling the internal 
space with a lipid soluble component, however, and any molecular uptake of 
lipid soluble components is limited to the space defined between the two 
lipid layers. As a result, there is much less volume available for pick up 
and discharge of materials such as cholesterol and other lipid soluble 
materials than there is for the particles of this invention, which expand 
in a fashion similar to a balloon, with interior space filling with the 
material of choice. 
Endotoxic shock is a condition, often fatal, provoked by the release of 
lipopolysaccharide (LPS) from the outer membrane of most gram negative 
bacteria (e.g., Escherichia coli; Salmonella tymphimurium). The structure 
of the bacterial LPS has been fairly well elucidated, and a unique 
molecule, referred to as lipid A, which is linked to acyl chains via lipid 
A molecule's glucosamine backbone is a component of LPS. See Raetz, Ann. 
Rev. Biochem. 59:129-170 (1990) in this regard. 
The lipid A molecule serves as membrane anchor of a lipopolysaccharide 
structure ("LPS") and it is the LPS which is implicated in the development 
of endotoxic shock. It should be pointed out that LPS molecules are 
characterized by a lipid A type structure and a polysaccharide portion. 
This latter moiety may vary in molecular details in different LPS 
molecules, but it will retain the general structural motifs characteristic 
of endotoxins. It would be incorrect to say that the LPS molecule is the 
same from bacteria to bacteria (see Raetz, supra). It is common in the art 
to refer to the various LPS molecules as "endotoxins", and this term will 
be used hereafter to refer to LPS molecules collectively. 
In U.S. Pat. No. 5,128,318 the disclosure of which is incorporated by 
reference, it was taught that reconstituted particles containing both an 
HDL associated apolipoprotein and a lipid capable of binding an endotoxin 
to inactivate it could be used as effective materials for alleviating 
endotoxin caused toxicity. 
In the parent and grandparent applications cited in the Related Application 
section and incorporated by reference herein, it was disclosed that 
various other materials may be used to treat endotoxin caused toxicity. 
Specifically, it was found that apolipoproteins are not required in 
reconstituted particles, and that the reconstituted particle may contain a 
peptide and a lipid wherein the peptide is not an apolipoprotein. 
It was also found by the inventors that endotoxin caused toxicity may be 
treated via sequential administration of either an apolipoprotein or a 
peptide followed by a lipid. Following sequential administration, the 
components assemble as a reconstituted particle and then act to remove 
endotoxin. 
It was also found that at least some individuals possess native levels of 
apoliprotein which are higher than normal levels such that effective 
endotoxemia therapy may be effectuated by administering reconstituted 
particles containing no apolipoprotein or peptide, but containing the 
lipid of the disclosure. 
In addition, the invention disclosed in these applications involved the use 
of the reconstituted particles and the components discussed herein for 
prophylaxis against endotoxin caused toxicity, by administering 
prophylactically effective amounts to subjects in need of prophylaxis. 
Such subjects include patients suffering from infections or recovering 
from surgery. These patients sometimes have very low plasma HDL levels, 
sometimes as little as 20% of normal levels. It is highly desirable, in 
these cases, for early prophylaxis with HDL, so as to compensate for these 
drops. 
It has now been found, quite surprisingly, that phospholipids may be used 
alone, or in combination with neutral lipids, as effective agents to 
alleviate and/or prevent endotoxemia. It is especially preferred to use 
phosphatidylcholines ("PC" hereafter), either alone, or in combination 
with other phospholipids, such as sphingolipids, in compositions which are 
essentially free of peptides and proteins, such as apolipoproteins or 
peptides derived therefrom. Neutral lipids such as mono-, di-, and 
triglycerides may be combined with the phospholipids, as long as the total 
amount of neutral lipids is below certain weight percents when the 
compositions are used in the form of an intravenous bolus. When used in 
other forms of administration, such as intravenously for example, by 
continuous infusion, the weight percents are not so critical, but are 
desirable. 
Particularly preferred embodiments of the invention are those compositions 
where the neutral lipid is cholesterol ester, or a mixture of cholesterol 
ester and triglycerides. 
The invention is described in greater detail in the disclosure which 
follows.

DETAILED DESCRIPTION OF THE EMBODIMENTS 
EXAMPLE 1 
Studies were carried out to determine the survival rate of mice challenged 
with S. tymphimurium endotoxin. Outbred male, Swiss-Webster mice received 
either saline solution (20 mice), reconstituted HDL particles (40 mice), 
or reconstituted peptide 18A (20 mice), via injection through the tail 
vein. The particulars of the injection materials are as follows: 
a. HDL particles 
Particles were prepared from apo-Hu-HDL (85%-AI; 15% AII and apo C), 
reconstituted with 95% pure egg phosphatidylcholine (2:1 W/W), using 
detergent dialysis, in accordance with Matz et al., J. Biol. Chem. 
257:4535-4540 (1982), and U.S. Pat. No. 5,128,318, the disclosure of which 
is incorporated by reference. 
b. peptide particles 
The peptide 18A has the amino acids sequence: 
Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-GlY-LYs-Leu-LYs-Glu-Ala-Phe 
SEQ ID NO: 1 
Samples of peptide were also mixed and reconstituted with 95% pure eggs 
phosphatidylcholine as per Matz et al., supra (2:1 w/w), and U.S. Pat. No. 
5,128,318 also using detergent dialysis. The resulting particles are 
identical to those disclosed in U.S. Pat. No. 5,128,318 except that a 
peptide component was present, rather than the apo-HDL of the Matz and 
patent references. 
Within fifteen minutes of administration of the reconstituted material, the 
mice were administered, intraperitoneally, 10 mg/kg body weight of 
Salmonella LPS. The criterion for evaluation was survival. FIG. 3 presents 
these results, and indicates nearly 4 fold superiority over the saline 
control. The synthetic peptide is almost as effective as the reconstituted 
apo-HDL containing particles. 
EXAMPLE 2 
Factors which affect the LPS-mediated stimulation of TNF-.alpha. while 
preserving the integrity of interaction between plasma proteins, and 
cellular elements of blood, can be appropriately studied in an in vitro, 
human whole blood system. Such a system was used to determine which of the 
components of lipoproteins is important in neutralizing LPS. 
Materials tested were reconstituted high density lipoprotein (R-HDL), 
natural plasma lipoproteins (VLDL, LDL, HDL), lipoprotein deficient serum 
(LPDS), and the triglyceride rich emulsion 20% INTRALIPID.RTM. (a mixture 
of triglycerides and phospholipids). 
All particles described herein were made via the same protocol, which 
involved mixing a phospholipid, sphingomyelin or phosphatidylcholine, 
triolein, and/or unesterified cholesterol ester, dissolved in chloroform, 
and weighing it into a flask. Vitamin E (0.02% w/v) was added as 
antioxidant. A dry lipid film was then prepared by blowing nitrogen or 
argon gas over the sample. A volume of non pyrogenic saline was then added 
to the flask, followed by mixing on a vortex mixer until all lipid was 
suspended. The solution was then homogenized in a high pressure 
homogenizer. Samples containing phosphatidylcholine (PC), with or without 
triolein, were cycled through the homogenizer 10 times at 20,000 psi. 
Samples containing cholesterol ester with one or more other lipids were 
cycled through 15-20 times at 30,000 psi. Homogenized solutions were 
filtered through 0.45 .mu.m syringe filters, and the filtrate was stored 
at room temperature until used (within three days). 
Blood was collected in a heparinized tube, diluted with Hank's Balanced 
Salt Solution ("HBSS" hereafter), or the material to be tested, dissolved 
in HBSS. The resulting material was transferred to Starstedt tubes (250 
ul/tube). LPS was dissolved in pyrogen free saline containing 10 mM HEPES, 
and added (2.5 ul) to a final concentration of 10 ng/ml. After incubation 
for four hours at 37.degree. C., tubes were chilled to 4.degree. C., 
followed by centrifugation at 10,000.times. g for 5 minutes. Supernatant 
was collected, and assayed for determination of TNF-.alpha., using a 
commercially available ELISA. 
Table 1, which follows, compares the compositions of the materials tested. 
FIGS. 5A and 5B present the results. Data are plotted as amount of 
TNF-.alpha. produced, plotted against concentration of added protein (FIG. 
5A), and phospholipid (FIG. 5B). Logarithmic scales were used, in order to 
display the wide range of concentrations used, with 10.degree. equal to 1 
mg/ml. All whole blood incubations contained 10 ng/ml of E. coli 0111:B4 
LPS, supplemented with one of the compositions, as the key for FIGS. 5A 
and 5B show. 
The fact that the materials differ in effectiveness when protein content is 
plotted (FIG. 5A), while being very similar when phospholipid content is 
plotted (FIG. 5B) suggest that the phospholipid is the important 
component. This is confirmed by the finding that a protein free lipid 
emulsion is more effective than is natural HDL, but less effective than 
R-HDL. Protein does not appear important to the neutralization. 
______________________________________ 
Composition of natural lipoproteins and reconstituted HDL 
Lipoprotein TC TG PC Protein 
Class Density (g//ml) 
Weight % 
______________________________________ 
VLDL &lt;1.006 22 53 18 7 
LDL 1.007-1.063 
48 11 22 20.9 
HDL 1.063-1.21 18 8 22 52 
R-HDL 1.063-1.21 -- -- 79 21 
LPDS &gt;1.21 0 0 2 98 
Intralipid 
-- 1 93 6 0 
______________________________________ 
EXAMPLE 3 
As the next step, protein free lipid emulsions, containing different 
amounts of neutral lipid, were tested in human whole blood. The same in 
vitro human whole blood assay as set forth in example 2 was used. FIGS. 6A 
and 6B present these results. In these studies, LPS-dependent, TNF-.alpha. 
production is plotted against concentration of added triglyceride (FIG. 
6A), or phospholipid (FIG. 6B). The compositions, as indicated by the key, 
contained (by weight) 7% triglyceride ("TG"), 45% TG, 89% TG, 94% TG, 
R-HDL, or phospholipid without TG, (shown in FIG. 6B only). An 89% TG 
composition is a 10% INTRALIPID.RTM. formulation, while 94% TG refers to 
20% INTRALIPID. In all other tests, egg phosphatidylcholine (PC), and 
triolein were used. 
These results show that the protein free compositions, when compared via 
triglyceride content, are very different. They are very similar when 
tested via phospholipid (PC) content. This confirms the role of 
phospholipid, especially since phospholipid alone is effective, but less 
so than emulsions containing up to 45% TG. 
EXAMPLE 4 
The work then proceeded to in vivo experiments in a mouse model, which is 
accepted as a reliable system for predicting human efficacy. 
In these experiments, mice were injected, in bolus form, with sufficient 
amounts of the formulations described in example 3 as well as others (pure 
phosphatidylcholine, 7% TG, 45% TG, 71% TG, 80% TG, 89% TG, 94% TG), or a 
saline control, to provide doses of phospholipid (either 200 mg/kg or 400 
mg/kg), together with 25 mg/kg of E. coli 0111:B4 LPS. The control group 
received intravenous physiological saline in a volume sufficient to match 
the volume of emulsion. Survival after 72 hours is presented in FIG. 7. 
Overall survival in the control group was 29%.+-.8% (mean: 63 animals in 8 
experiments). Each preparation was tested in a minimum of 3 experiments on 
18 or more animals. 
PC alone had a modest protective effect, not statistically significant at 
the 95% confidence level, while 7%, 45% and 71% TG compositions 
significantly improved survival. The 80% and 89% TG compositions were 
marginally effective, while the 94% TG decreased survival. 
When the dose was increased to provide 400 mg/kg of PC both the 89% and 94% 
TG emulsions significantly decreased survival time, probably due to TG 
poisoning, as explained infra. 
EXAMPLE 5 
The work described in examples 2-4 established that phospholipids are an 
active agent useful in inhibiting endotoxemia. The fact that non-polar 
lipids other than triglycerides may form emulsions with phospholipids 
other than PC suggested that others may be tried. Exemplary are 
sphingomyelin (another phospholipid), and unesterified cholesterol (a 
polar neutral lipid), and mixtures of these. So, too, esterified 
cholesterol (a nonpolar ester), squalene (a hydrocarbon), and vitamin E (a 
nonpolar antioxidant) may be used. A series of experiments were designed 
to test these, using the human whole blood assay of example 2, supra, and 
the mouse survival assay of example 4. 
Emulsions were prepared, in the manner described supra, using pure 
phosphatidylcholine, phosphatidylcholine with 10% (wt/wt) unesterified 
cholesterol, 10% (wt/wt) sphingomyelin, or 10% total of a mix of both. 
Emulsions were added to whole blood, at a concentration of 100 mg/dl, with 
reference to PC, and 10 ng/ml of LPS. The mixture was incubated, and 
TNF-.alpha. release measured. 
The results are shown in FIG. 8. TNF-.alpha. production was substantially 
reduced with PC alone. Emulsions containing unesterified cholesterol, 
sphingomyelin, or the mix of both, were also suppressive of TNF-.alpha. 
release. 
EXAMPLE 6 
The whole blood assay was also used to determine the effect of unesterified 
cholesterol and/or sphingomyelin to neutral lipid containing emulsions. 
Again, the emulsions were added at 100 mg/dl PC. The various compositions 
(wt/wt) are set forth in the following table. 
______________________________________ 
Emulsion Composition 
______________________________________ 
PC with 45% TG 55:45 
PC + TG + C 54.4:45.3:0.3 
PC + TG + SP 51.6:43.0:5.4 
PC + TG + C + SP 51.4:42.9:0.3:5.4 
PC + CE 54.5:45.5 
PC + CE + C 54.4:45.3:0.3 
PC + CE + SP 51.6:43.0:5.4 
PC + CE + C + SP 51.5:42.9:0.3:5.4 
______________________________________ 
FIGS. 9A and 9B display the results. PC emulsions made with either neutral 
lipid, with or without additional polar lipids, demonstrated inhibition. 
Again, the LPS concentration used is a 70% lethal dose. The cholesterol 
ester containing emulsions are less effective than are TG containing 
emulsions, while those emulsions containing unesterified cholesterol did 
not suppress TNF-.alpha. as well as those emulsions which did not contain 
it. Adding sphingomyelin to the emulsions appeared to improve suppression 
of TNF-.alpha. production. 
EXAMPLE 7 
Cholesterol ester containing emulsions were tested in an in vivo model 
(i.e., that used in example 4), with a lethal dose of endotoxin. Emulsions 
were prepared with PC and TG, or PC and cholesterol ester (CE), and were 
administered to provide a single bolus dose of 200 mg/kg of PC, together 
with 25 mg/kg of E. coli 0111:B4 LPS (a lethal dose), through the tail 
vein. Control groups received intravenous physiological saline in a volume 
to match the volume of emulsion. 
In FIG. 10, the data compare the results from the CE and TG containing 
emulsions. Each emulsion was tested in a minimum of two experiments, using 
a total of 16 or more animals. 
As shown, emulsions containing 7% or 45% CE (wt %) significantly improved 
survival. These results, taken with those of example 6, show that CE can 
be substituted for TG to create emulsions that neutralize endotoxin. 
EXAMPLE 8 
Protein-free emulsions of phospholipid with triglyceride effectively block 
TNF-.alpha. production in whole blood stimulated with LPS. In theory, 
these emulsions might also be effective in vivo if they can be 
administered safely in doses that provide protective concentrations of 
phospholipid in plasma. Our previous experiments with R-HDL suggest that 
the minimum dose of phospholipid is approximately 200 mg/kg. Using this 
dose and a plasma volume of 4.5% of body weight, one can calculate the 
concentration of triglyceride expected in plasma following administration 
of a series of emulsions with increasing triglyceride content. The result 
is shown in FIG. 11 as a smooth line curving upward with increasing weight 
percent TG. Plasma TG concentrations rarely rise above 1000 mg/dl in 
healthy adults even after a fatty meal. Pancreatitis is reported in 
patients with plasma TG above 2000 mg/dl (Farmer, et al., Amer. J. Med. 
54: 161-164 (1973); Krauss, et al., Amer. J. Med. 62: 144-149 (1977); 
Glueck, et al., J. Lab. Clin. Med. 123: 59-61). Plasma TG above 4000 mg/dl 
is extremely rare and cause for serious concern. The last two thresholds 
are shown by horizontal lines in the figure above. Administration of 
either 10% or 20% INTRALIPID.RTM. in a dose to provide 200 mg/kg 
phospholipid is expected to raise plasma TG concentrations (see the two 
open circles) well above the safe limits. By contrast, administration of 
emulsions containing 7%, 45%, 71% or 78% (solid squares left to right) 
raises plasma TG to 136, 477, 1300 or 2000 mg/dl respectively. Emulsions 
with TG content up to .about.50% are expected to be free of toxicity from 
TG. 
The foregoing examples detail the invention which involves, in one aspect, 
the alleviation or prevention of endotoxemia in a subject via 
administering an effective amount of a phospholipid with which an 
endotoxin associates. The association of phospholipid and endotoxin is 
then removed from the subject via standard biological processes well known 
to anyone familiar with processes via which lipoprotein particles are 
removed. Association of the endotoxin with the phospholipid inactivates 
it. 
The subject being treated is preferably a human, but the practice of the 
invention is equally applicable in a veterinary context as well. 
"Alleviation" as used herein refers to treatment to ease the burden of 
endotoxemia caused by any of the various endotoxins produced by, e.g., 
gram negative bacteria (S. tymphimurium, E. coli, etc.). Prophylaxis may 
be accomplished by administering the agent at a point where the subject is 
in or about to be in, a situation where endotoxin exposure may result. 
Classically, this occurs during surgery. Thus, a subject who is about to 
experience a surgical procedure may have the active ingredient 
administered preparatory to the procedure. 
The effective amount of phospholipid necessary for treatment of the subject 
can vary. In general, a dose up to about 200 mg of phospholipid per 
kilogram of body weight of the subject is preferred, although the amount 
may drop, or increase, depending upon the severity of the endotoxemia or 
the degree of risk in the context of the prophylaxis. 
It is desirable to administer the phospholipids in compositions which also 
contain neutral lipids, but this is not necessary, as neutral lipid free 
emulsions of phospholipids are also envisioned. The desirability of the 
administration with phospholipids results from the fact that the neutral 
lipids and phospholipids associate into particles which resemble the 
lipoproteins, but differ therefrom in that they contain no protein of 
peptide components, which are of course, always present in the 
lipoproteins. 
Especially desirable forms of treatment are those where the phospholipid is 
a phosphatidylcholine, such as egg yolk phosphatidylcholine, soy based 
phosphatidylcholine or a sphingolipid. With respect to the neutral lipids, 
it is preferred to use cholesterol ester or triglyceride, but other 
neutral lipids, such as squalene or other hydrocarbon oils, di- and 
mono-glycerides and antioxidants such as vitamin E may also be used. 
The form in which the compositions may be administered can vary, with a 
bolus or other intravenous forms being especially preferred. When a bolus 
form is used, and the composition contains triglyceride, e.g., some care 
must be given in dosing. It is fairly well known that triglycerides are 
toxic if administered in too large an amount. The artisan of ordinary 
skill, however, can easily formulate the compositions so that the risk of 
triglyceride poisoning is reduced, or eliminated. In general, when a bolus 
form is used, the compositions should contain no more than about 80 weight 
percent of triglyceride or other neutral lipid, preferably no more than 70 
weight percent. Most preferably, the compositions should contain no more 
than about 50 weight percent, of neutral lipid, when a bolus is 
administered. 
When non-bolus forms are employed, however, such as other intravenous 
forms, the risk of poisoning is decreased. Nonetheless, the ranges 
delineated supra are preferred for intravenous, or other forms of 
administration, although it must be understood that they are not required. 
Preferably, a dose of up to about 200 mg per kg of body weight of 
phospholipid is administered. Administration of up to about or even up to 
about 800 mg/kg is also feasible. Doses are general, however, and will 
vary depending upon the subject and the form of administration. 
As indicated, supra., the protein and peptide free formulations require 
that at least one phospholipid be present, preferably at least one neutral 
lipid is present. Optionally, these may include additional materials such 
as sterols (e.g., cholesterol, .beta.-sitosterol), esterified or 
unesterified lipids (e.g., cholesterol ester or unesterified cholesterol), 
hydrocarbon oils such as squalene, antioxidants such as vitamin E, but 
these are not required. Of course, more than one phospholipid, and/or more 
than one neutral lipid may be used in any formulation. 
Other aspects of the invention will be clear to the skilled artisan, and 
need not be repeated here. 
It will be understood that the specification and examples are illustrative 
but not limitative of the present invention and that other embodiments 
within the spirit and scope of the invention will suggest themselves to 
those skilled in the art. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 9 
(2) INFORMATION FOR SEQ ID NO: 1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: 
AspTrpLeuLysAlaPheTyr AspLysValAlaGluLysLeuLys 
51015 
GluAlaPhe 
(2) INFORMATION FOR SEQ ID NO: 2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 
LysTrpLeuAspAlaPheTyrLysAspValAlaLysGluLeuGlu 
51015 
LysAlaPhe 
(2) INFORMATION FOR SEQ ID NO: 3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: 
AspTrpLeuLysAlaPheTyrAspLysAlaGluLysLeuLysGlu 
51015 
AlaPhe 
(2) INFORMATION FOR SEQ ID NO: 4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 
ProLysLeuGluGluLeuLysGluLysLeuLysGluLeuLeuGlu 
510 15 
LysLeuLysGluLysLeuAla 
20 
(2) INFORMATION FOR SEQ ID NO: 5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: 
ValSerSerLeuLysGl uTyrTrpSerSerLeuLysGluSerPhe 
51015 
Ser 
(2) INFORMATION FOR SEQ ID NO: 6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
( xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 
ValSerSerLeuLeuSerSerLeuLysGluTyrTrpSerSerLeu 
51015 
LysGluSerLeuSer 
20 
(2) INFORMATION FOR SEQ ID NO: 7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: 
ValSerSerLeuLeuSerSerLeuLeuSerSerLeuLysGluTyr 
510 15 
TrpSerSerLeuLysGluSerGluSer 
20 
(2) INFORMATION FOR SEQ ID NO: 8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: 
ProValLeuAspGluPheA rgGluLysLeuAsnGluGluLeuGlu 
51015 
AlaLeuLysGlnLysMetLys 
20 
(2) INFORMATION FOR SEQ ID NO: 9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: 
ProLeuAlaGluAspLeuGlnThrLysLeuAsnGluAsnValGlu 
51015 
AspLeuArgLysGln LeuVal 
20