Therapeutic use of multilamellar liposomal prostaglandin formulations

This invention provides a multilamellar liposome containing an arachidonic acid metabolite, two or more lipid-containing bilayers and two or more aqueous compartments containing a release-inhibiting buffer. Preferred arachidonic acid metabolites are the prostaglandins, particularly PGE.sub.1. The liposomal formulations can be used to treat animals, particularly humans, for diseases, disorders or conditions which can be ameliorated by prostaglandins, e.g., disorders characterized by cellular activation and adhesion, inflammation and/or toxemia.

This application is directed to multilamellar liposomal formulations of 
arachidonic acid metabolites; such formulations can be used 
therapeutically in diseases, disorders or conditions such as cell 
activation and adhesion disorders, inflammatory disorders or toxemic 
disorders. 
Arachidonic acid, and other twenty carbon "essential" fatty acids having at 
least three double bonds, can be used to make prostaglandins (for a 
review, see, e.g., Goodman and Gilman's The Pharmacoloaical Basis of 
Therapeutics (A. Goodman Gilman et al., eds.), Pergamon Press, New York 
(1990), pp. 600-611); L. Stryer, Biochemistry (2nd edition), W. H. Freeman 
and Co,, New York (1981), pp. 853-854)). The various prostaglandins are 
grouped into several categories (A-I), which are distinguished by varying 
substituents on the five-carbon ring introduced into the twenty-carbon 
fatty acid precursor during prostaglandin synthesis. These groups can be 
further subdivided based upon the number, and position, of double bonds in 
the prostaglandins' carbon chains. Prostaglandins are believed to act on 
their target cells by way of cellular surface receptors; these receptors 
are believed to be coupled to second messenger systems by which 
prostaglandin action is mediated. Prostaglandins can have a broad spectrum 
of biological activities. They can act on smooth vascular muscle and 
thereby be potent vasodilators; prostaglandins can also affect the 
functioning of blood cells, particularly neutrophils and platelets. 
Uterine contractions can be affected by prostaglandin action, which can 
also affect renal, central nervous system and afferent nerve function, 
Various endocrine tissues can respond to prostaglandins. Furthermore, 
prostaglandins can modulate inflammatory conditions in animals. 
Enzymes in the body can rapidly deactivate prostaglandins. This typically 
necessitates frequent administrations of high doses of the compounds to 
maintain therapeutically effective levels in the serum, thereby increasing 
the expense of prostaglandin treatment and leading to the possibility of 
unwanted side effects. Furthermore, as prostaglandin deactivation occurs 
primarily as blood passes through the lungs, the compounds are generally 
administered intra-arterially. 
Liposomal formulations can prolong the circulatory half-lives of 
arachidonic acid metabolites, e.g., prostaglandins, and can help avoid 
their deactivation in the lungs. Accordingly, such liposomal formulations 
can useful provide therapeutic alternatives. Mizishuma et al. (J. 
Rheumatol. 14:97 (1987)) and Hoshi et al. (Drugs. Exptl. Clin. Res. 
12(8):681 (1986)) describe lipid microspheres containing prostaglandin 
E.sub.1 (PGE.sub.1). However, as disclosed in Mizishuma et al. (U.S. Pat. 
No, 4,493,847) and Imagawa et al. (U.S. Pat. No. 4,684,633), these 
"microspheres" are actually prostaglandin-containing fat emulsions, which 
are not liposomes, and have neither the same properties, nor the same 
advantages, as the liposomal prostaglandins provided herein. Shell and See 
(U.S. Pat. Nos. 4,820,732 and 4,955,878) disclose treatments for reducing 
dysfunction during angioplasty procedures which involve administering 
prostaglandin-containing compositions to patients. These compositions also 
contain a carrier. However, the liquid carriers disclosed, e.g., 
dehydrated alcohols and saline solutions, generally cannot provide 
sustained release of a prostaglandin. The fat-laden microsphere carriers 
disclosed are taught to be at least as large as a red blood cell, i.e, at 
least 7microns in diameter, and can be much larger. Administration of 
particles of such large size to animals can cause difficulties because the 
microspheres can become stuck in, and clog, small blood vessels, e.g., 
lung capillaries. 
Liposomes are self-assembling structures comprising one or more bilayers of 
amphipathic lipid molecules, each of which encloses an internal aqueous 
volume. Unilamellar liposomes have a single lipid bilayer. Multilamellar 
liposomes have two or more lipid bilayers. Liposomes can be produced by a 
variety of methods (for a review, see, e.g., Cullis et al., in: Liposomes, 
From Biophysics to Therapeutics (M. J. Ostro, ed.), Marcel Dekker, pp. 
39-72 (1987), the contents of which are incorporated herein by reference). 
Liposomal formulations of drugs can have an enhanced therapeutic index by 
reducing the drug's toxicity, increasing its efficacy, or both. 
Furthermore, liposomes, like other particulate matter in the circulation, 
are typically taken up by phagocytic cells of the reticuloendothelial 
system in tissues having sinusoidal capillaries, and are thereby often 
directed to sites of intracellular infections. 
Maximizing the efficiency with which drugs are entrapped in liposomes can 
minimize the lipid load presented to treated subjects and can also 
minimize the waste of valuable drug products. The release of compounds 
which tend to leak from liposomes should also be inhibited to derive the 
maximum benefit from their encapsulation. Furthermore, the provision of 
liposomal formulations which can be stably stored will increase the 
therapeutic benefits derived therefrom. 
The liposomal arachidonic acid metabolite formulations of this invention 
are useful in ameliorating or preventing diseases, disorders or conditions 
which can be treated with a prostaglandin. Disorders which can be treated 
with these formulations include cell activation and adhesion disorders, 
toxemic disorders and inflammatory disorders. 
SUMMARY OF THE INVENTION 
This invention provides a multilamellar liposome which comprises an 
arachidonic acid metabolite, two or more lipid bilayers comprising a lipid 
and two or more aqueous compartments comprising a release-inhibiting 
buffer. 
Preferably, the multilamellar liposome comprises a solute entrapped in its 
aqueous compartments, wherein the concentration of the solute in each of 
the aqueous compartments of the multilamellar liposome is substantially 
equal; i.e., the multilamellar liposome of this invention preferably has 
substantially equal interlamellar solute distribution. 
Preferably, the arachidonic acid metabolite is a prostaglandin. Preferred 
prostaglandins are prostaglandins of the E series or prostaglandins of the 
I series. Most preferably, the metabolite is prostaglandin E1 (PGE.sub.1). 
Preferably, the lipid has saturated acyl chains. In one embodiment of this 
invention, the saturated acyl chain lipid is dipalmitoyl 
phosphatidylcholine (DPPC). Preferably, the release-inhibiting buffer is a 
citric acid buffer, more preferably, a citric acid buffer having a pH of 
about 4.5. 
The multilamellar liposome can comprise a drying protectant. Preferably, 
the drying protectant is a sugar, e.g., maltose, dextrose, galactose, 
lactose, raffinose or trehalose. Preferably, the sugar is maltose. 
Accordingly, in a preferred embodiment of the invention, the multilamellar 
liposome comprises prostaglandin E.sub.1, two or more lipid bilayers 
comprising DPPC and two or more aqueous compartments comprising a citric 
acid buffer having a pH of about 4.5. This preferred multilamellar 
liposome comprises a solute entrapped in its aqueous compartments, wherein 
the concentration of the solute in each of the aqueous compartments of the 
multilamellar liposome is substantially equal, and can comprise a drying 
protectant. 
The multilamellar liposome of this invention can comprise an additional 
bioactive agent, i.e., a bioactive agent in addition to the arachidonic 
acid metabolite. The multilamellar liposome can further comprise a 
headgroup-modified lipid. 
Also provided herein is a pharmaceutical composition comprising a 
pharmaceutically acceptable carrier and the multilamellar liposome of this 
invention. Further provided is a dehydrated multilamellar liposome 
comprising an arachidonic acid metabolite and two or more lipid bilayers 
comprising a lipid. Still further provided is a two-component system 
comprising an aqueous solution and such a dehydrated liposome, wherein the 
dehydrated multilamellar liposome and the aqueous solution are combined so 
as to rehydrate the dehydrated liposome. 
This invention provides a method of administering an arachidonic acid 
metabolite to an animal which comprises administering to the animal a 
multilamellar liposome comprising the metabolite, two or more lipid 
bilayers comprising a lipid, and two or more compartments comprising an 
aqueous release-inhibiting aqueous buffer. Preferably, the administration 
comprises intravenous administration. Preferably, the animal is a human, 
Preferably, the arachidonic acid metabolite is prostaglandin E1. 
Preferably, the lipid is a saturated acyl chain lipid. Preferably the 
buffer is a citric acid buffer having a pH of about 4.5. The liposome 
preferably comprises a solute entrapped in its aqueous compartments, 
wherein the concentration of the solute in each of the compartments is 
substantially equal. 
The animal can be afflicted with a disorder characterized by cell 
activation and adhesion, inflammation and/or toxemia, that is one or more 
of these phenomena are occurring in the animal and are the object of 
treatment with the liposomal formulations of this invention. This method 
comprises administering to the animal an amount of the liposome which 
comprises an anti-disorder effective amount of the arachidonic acid 
metabolite. 
Disorders which can be treated with the formulations of this invention 
include, without limitation: reperfusion injury, systemic inflammatory 
response syndrome (SIRS), myocardial infarction, adult respiratory 
distress syndrome (ARDS), vasculitis, post-traumatic shock, burn injuries, 
vaso-occlusive disorders, arthritic disorders, such as rheumatoid and 
filary arthritis and gout, and auto-immune disorders, for example, 
systemic lupus erythematosus, juvenile diabetes, multiple sclerosis or 
Hashimoto's thyroiditis. Particularly preferred indications are ARDS and 
SIRS. 
Generally, the anti-disorder effective amount of the arachidonic acid 
metabolite is at least about 10.sup.-12 g of the metabolite per kg of body 
weight of the animal. Typically, the effective amount is from about 
10.sup.-12 g of the metabolite per kg of body weight of the animal to 
about 10.sup.-3 g per kg. Preferably, the effective amount is from about 
10.sup.-18 g of the metabolite per kg of body weight of the animal to 
about 10.sup.-4 g per kg. More preferably, the effective amount is about 
10.sup.-6 g of the metabolite per kg of body weight. 
The method of this invention can comprise administering to the animal an 
additional bioactive agent, for example, an antimicrobial or 
anti-inflammation agent; this additional agent is typically selected by 
means, and for reasons, well understood by ordinarily skilled artisans 
given the teachings of this invention. The additional bioactive agent can 
be an additional arachidonic acid metabolite.

EXAMPLES 
Example 1 
Preparation of Multilamellar Liposomes (MLVs) Containing PGE.sub.1 
Preparation of EPC-Containing PGE.sub.1 MLVs 
An egg phosphatidylcholine (EPC) stock solution (20 mg/ml in ethanol) was 
prepared as follows: 1 g of dried EPC was dissolved in 50 ml of absolute 
ethanol, with gentle swirling, in a 50-ml brown bottle with a Teflon-lined 
lid. The resulting solution was stored at minus 20 degrees Celsius. A 
PGE.sub.1 stock solution (1 mg/ml in ethanol) was prepared as follows: 20 
mg of dried PGE.sub.1 was transferred to a 20-ml vial, to which 20 ml of 
absolute ethanol was added. The PGE.sub.1 was dissolved in the ethanol 
with gentle swirling; the resulting solution was stored at minus 20 
degrees Celsius. 
An aliquot of the EPC stock solution (9.75 ml), and an aliquot of the 
PGE.sub.1 stock solution (0.5 ml), were combined in a 500-ml round-bottom 
flask; the ethanol was removed by rotoevaporation at about 30 degrees C. 
for at least two hours. The dried EPC/PGE.sub.1 was resuspended in a pH 
4.5 buffer (e.g., 50 mM acetate, 150 mM NaCl, pH brought to 4.5 with 10N 
NaOH; glass beads aided in resuspension of the dried EPC/PGE.sub.1) so as 
to form a liposome suspension. This suspension was stored at 4 degrees C. 
Preparation of DPPC-Containing PGE.sub.1 MLVs 
A DPPC stock solution was prepared as described above, using 1.035 g of 
dipalmitoyl phosphatidylcholine (DPPC) dissolved in methylene chloride. 
Rehydration of the dried DPPC/PGE.sub.1 mixture required heating in a 
water bath, with swirling, at about 52 degrees C. for about 3-5 minutes. 
TABLE 1 
______________________________________ 
PGE.sub.1 FORMULATIONS 
% Bound and/ 
or Entrapped at 
Lipid pH 4.6 pH 7.1 
______________________________________ 
EPC 90 54 (60)* 
90 61 (68) 
89 56 (63) 
DPPC 60 42 (68) 
59 38 (64) 
53 35 (67) 
______________________________________ 
*The numbers in parentheses represent the percentage of prostaglandin 
bound and/or entrapped at pH 4.6 which remains in the pellet at pH 7.1. 
The data (see Table 1, above, and Table 2, below) show that about 90% of 
the available prostaglandin was associated with EPC multilamellar vesicles 
when a prostaglandin-containing citrate buffer, pH 4.6 was used to 
rehydrate dried lipids so as to form liposomes. When these same liposomes 
were transferred to a pH 7.1 buffer, about 54-61% of the available 
prostaglandin remained associated with the liposomes after one half hour. 
When DPPC was used to make the multilamellar vesicles, about 53-60% of the 
available prostaglandin was entrapped in liposomes comprising the pH 4.6 
buffer. When these liposomes were transferred to the pH 7.1 buffer, about 
35-42% of the available prostaglandin remained within the liposomes after 
one half hour. 
TABLE 2 
______________________________________ 
PGE.sub.1 FORMULATIONS 
% Bound and/ 
or Entrapped at 
pH 7.12 
Lipid 0.5 Hours 4.5 Hours 
______________________________________ 
EPC 54 (60) 31 (35)* 
61 (68) 35 (39) 
56 (63) 31 (35) 
DPPC 42 (68) 31 (51) 
38 (64) 26 (44) 
35 (67) 25 (47) 
______________________________________ 
*The numbers in parentheses represent the percentage of prostaglandin 
bound and/or entrapped at pH 4.6 which remains in the pellet at pH 7.1. 
Example 2 
Rat Air Pouch Studies 
The rat subcutaneous air pouch, a model for acute inflammation and 
leukocyte extravasation from the peripheral vasculature to sites of 
inflammation (Tate, et al., Laboratory Investigation 59:192 (1988), the 
contents of which are incorporated herein by reference), was used to study 
the effect of systemic PGE.sub.1 liposomes in mediating fMLP induced fMLP 
inflammation. 
Male Sprague-Dawley rats, weighing 126-150 g, were obtained from Charles 
River Laboratories. Upon receipt, the rats were acclimated in the animal 
facility for 2 days. Throughout the experiments, the rats were watered and 
fed ad libitum. For air pouch formation, the rats were anesthetized via 
inhalent, their backs shaved, and swabbed with ethyl alcohol. Twenty cc of 
ambient air was injected subcutaneously into the animal's back to form an 
air pouch, and the animal was returned to it's cage. The air pouches were 
monitored to determine integrity, and additional air was injected, if 
warranted. At six days following air pouch formation, intra-air pouch 
inflammation was induced by direct injection into the air pouch of 2.15 
.mu.g fMLP. Free prostaglandin E.sub.1, or PGE.sub.1 liposome 
formulations, were simultaneously injected i.v. via the tail vein, and the 
animals returned to their cages. Six hours after stimulation, the rats 
were sacrificed by CO.sub.2 inhalation, and the total exudate fluid 
recovered from the air pouch via syringe. The results of these experiments 
are presented in FIGS. 2-6. 
Visual examination of the post-stimulation air pouch lining indicated that 
fMLP effected a thickening of the lining and a large number of invasive 
leukocytes, as compared to control animals, in which saline alone was 
injected into the air pouch. Treatment with free PGE.sub.1 resulted in a 
reduction in vascular reactivity and a concurrent reduction in the number 
of leukocytes invading the pouch lining. The neutrophil population evident 
in the lining was transient, i.e., the leukocytes were in the process of 
extravasation from the vasculature to the lumen/exudate fluid of the air 
pouch. Since the leukocytes were transiently crossing the air pouch 
lining, the subsequent analysis of liposomal prostaglandin formulations in 
ameliorating leukocyte invasion was confined to those cells present in the 
aspirated exudate fluid. 
Experiments were performed to evaluate the effect of PGE.sub.1 liposomes in 
mediating cellular influx to the air pouch. These experiments compared 
C-53 (unilamellar liposomal PGE.sub.1) and MLV-PGE.sub.1 (multilamellar 
liposomal PGE.sub.1) with free PGE.sub.1. The free stable prostaglandin 
analog 15-methyl-PGE.sub.1 was included in these experiments due to its 
longer bioavailability of.gtoreq.8 hours, as compared to the&lt;15 min. 
bioavailability of free PGE.sub.1. 
As shown in FIG. 2, both C-53 and MLV-PGE.sub.1 inhibited the influx of 
cells to the air pouch more effectively than free PGE.sub.1. MLV-PGE.sub.1 
was more inhibitory than C-53. Both C-53, and MLV, placebo liposomes 
inhibited the cellular extravasation in the absence of PGE.sub.1 ; when 
this placebo inhibition was increased with the addition of free PGE.sub.1 
to the placebos. 
The leukocyte subset distribution in the air pouch exudate was also 
determined and this data is shown in FIG. 3, below. All leukocyte 
subpopulations were preferentially inhibited by PGE.sub.1 liposome 
formulations as compared to free PGE.sub.1. The predominate leukocyte 
subpopulation extravasating into the air pouch in response to fMLP is 
neutrophils. The greatest inhibition was seen for this neutrophil 
population. Monocyte influx to the air pouch was completely abolished by 
liposomal PGE.sub.1, but not by free PGE.sub.1. 
The dose responses of inhibition of leukocytes extravasation in response to 
free PGE.sub.1 and liposomal PGE.sub.1 are shown in FIG. 4. As shown, the 
inhibition of leukocyte influx to the rat air pouch is dose-dependently 
inhibited by PGE.sub.1 liposomes. Maximal inhibition is attained at 10.0 
.mu.g/kg, as greater concentrations have no additional effect. Leukocyte 
subpopulation was similar to that shown in FIG. 3, as all leukocyte 
subpopulations were preferentially inhibited by PGE.sub.1 liposome 
formulations, as compared to free PGE.sub.1. The greatest inhibition was 
seen for the neutrophil population, and the monocyte influx to the air 
pouch was completely abolished by liposomal PGE.sub.1, but not by free 
PGE.sub.1. The above data indicated that PGE.sub.1 -MLVs had a slightly 
greater inhibitory response on air pouch extravasation that C-53. To 
further separate the response to these two formulations, we assessed the 
air pouch leukocyte population at both 6 and 24 hours. This data is shown 
in FIG. 5, below. 
The leukocyte subpopulation distribution in these experiments was similar 
to that shown in FIGS. 3 and 4, as: a) the predominate leukocyte 
population infiltrating the air pouch was comprised of neutrophils; and b) 
all leukocyte subpopulations were preferentially inhibited by PGE.sub.1 
liposome formulations as compared to free PGE.sub.1. Monocytes were absent 
at 6 hours, but present in small numbers at 24 hours (&gt;4.times.10.sup.4 in 
all treatment groups as compared to 7.8.times.10.sup.4 for the 24 hour 
saline control). 
The efficacy of alternative PGE.sub.1 liposome formulations in mediating 
leukocyte extravasation to the rat air pouch was assessed. The specific 
formulations assessed, along with their characteristics, are listed below 
in Table 3 (see below), with the data from these experiments being 
presented in FIG. 6. 
TABLE 3 
______________________________________ 
Alternative PGE.sub.1 Liposome Formulations 
Formulation Characteristics 
______________________________________ 
EPC SPLV Multi-lamellar, and similar to 
MLVS as to PGE.sub.1 leak rate. 
EPC/Chol/POPE-GA/PGE.sub.1 
Surface modified with glutaric acid 
to effect circulation times of up to 24 
hours. PGE.sub.1 is membrane associated. 
EPC/Chol/POPE-GA Surface modified with glutaric acid 
to effect circulation times of up to 24 
hours. No PGE.sub.1. 
EPC/Chol/POPE-GA/DOPE-PGE.sub.1 
Surface modified with glutaric acid 
to effect circulation times of up to 24 
hours. PGE.sub.1 covalently linked to the 
membrane. 
EPC/Chol/DOPE-PGE.sub.1 
PGE.sub.1 is membrane associated. 
______________________________________ 
As these data indicate, all PGE.sub.1 liposome formulations are more 
efficacious than free PGE.sub.1 in inhibiting leukocyte extravasation to 
the rat air pouch. The leukocyte subpopulation distribution in these 
experiments was similar to that shown in FIGS. 4 and 5, as: a) the 
predominate leukocyte population infiltrating the air pouch was comprised 
of neutrophils, and b) all leukocyte subpopulations were preferentially 
inhibited by PGE.sub.1 liposome formulations as compared to free 
PGE.sub.1. Monocytes were absent in all liposomal PGE.sub.1 treatment 
groups. 
Example 3 
Adjuvant Arthritis 
Male Lewis rats, weighing 126-150 g each, were obtained from Charles River 
Laboratories. Upon receipt, the rats were acclimated in the animal 
facility for 2 days. Throughout the experiments the rats were watered and 
fed ad libitum. Chronic bilateral arthritis was induced by the i.d. 
(intra-dural) injection of complete Freund's adjuvant at the base of the 
tail. The onset of arthritis was abrupt, occurring between days 10 and 14 
in Freund's induced animals. The symptoms exhibited by untreated control 
animals were tenderness upon palpation in most active joints, symmetric 
edema involving the joints of the paws, ankles and knees, flexation 
contractures of the forepaws, malaise, and weight loss attributable to 
both primary disease as well as inability or disinclination to access food 
supplies, due to pain and decreased mobility. 
Experiments were conducted to assess the efficacy of free PGE.sub.1 in 
mediating the progression of adjuvant arthritis. The parameters assessed 
in these experiments were changes in joint size measured at the rear knee, 
changes in body weight, and a subjective scoring of general health, vigor 
and motility. The results from these experiments are shown in FIGS. 7-12. 
The data in FIGS. 7, 8 and 9 indicates that free PGE.sub.1 attenuated the 
progression of adjuvant arthritis, as objectively determined by 
maintaining weight gain and inhibiting joint edema. Subjective scoring 
indicated a maintenance of general health and mobility in PGE.sub.1 
-treated animals. The rats could be treated as late as 10 days 
post-adjuvant administration and still receive protection from disease 
progression, although the inhibition of arthritis progression was not as 
profound as that seen in animals treated with PGE.sub.1 beginning at day 
0. 
Because of the protective effect of free PGE.sub.1 in ameliorating 
arthritis, we next addressed the question of whether liposomal PGE.sub.1 
was as effective as free PGE.sub.1. The experiments included the same 
objective parameters and subjective scoring as in the previous 
experiments, and compared C-53, PGE.sub.1 30 containing MLVs and free 
PGE.sub.1. The data from these experiments are shown in FIGS. 10, 11 and 
12, and indicate a greater efficacy for liposomal than free PGE.sub.1 in 
decreasing the progression of rat adjuvant induced arthritis. 
The optimal formulation thus far tested is PGE.sub.1 -containing MLVs, 
presumably because of longer bioavailability of PGE.sub.1 due to slower 
leak rate. MLV formulations effected an almost total inhibition of disease 
manifestation and progression. 
Example 4 
Rat Endotoxemia 
Fever, hypotension, changes in leukocyte counts and diarrhea are symptoms 
of gram-negative bacterial infections. These infections may lead to 
disseminated intravascular coagulation and irreversible shock. A large 
volume of literature indicates the involvement of leukocyte derived IL-1, 
IL-6 and TNF.alpha. in mediating the progression of endotoxic shock. 
Because our in vitro data indicated an inhibition of these cytokines from 
cultured monocytes, we developed an in vivo model of rat endotoxemia, 
using mortality as an end point, to assess the effectiveness of PGE.sub.1 
liposome formulations in attenuating LPS-induced death. 
Experiments were designed to establish an LD.sub.50 for E. coli LPS 
(lipopolysaccharide) in Sprague-Dawley rats. The data from these 
experiments are shown in FIG. 13, and indicate that the LD.sub.50 is at 50 
.mu.g/kg. This LPS dosage was used in subsequent experiments, unless 
otherwise indicated. 
Experiments was designed to assess the efficacy of free and liposomal 
PGE.sub.1 in mediating LPS-induced mortality. The results from these 
experiments is shown in FIG. 14, and indicate that free PGE.sub.1 
increased both the rate and magnitude of LPS induced mortality. In 
contrast, C-53 afforded almost complete protection against LPS induced 
death. PGE.sub.1 -containing MLVs imparted also protection. All animals 
receiving LPS exhibited non purulent conjunctivitis, profuse watery 
diarrhea and profound lethargy. These symptoms were manifested within the 
first two hours post-LPS administration, and persisted throughout the time 
course of the experiment. Dying animals exhibited syncope and shock.