U.S. patent application number 11/352275 was filed with the patent office on 2006-06-29 for tnf receptor and steroid hormone in a combined therapy.
This patent application is currently assigned to Applied Research Systems ARS Holding N.V.. Invention is credited to Alessandra Boe, Francesco Borrelli.
Application Number | 20060142197 11/352275 |
Document ID | / |
Family ID | 35998746 |
Filed Date | 2006-06-29 |
United States Patent
Application |
20060142197 |
Kind Code |
A1 |
Boe; Alessandra ; et
al. |
June 29, 2006 |
TNF receptor and steroid hormone in a combined therapy
Abstract
The present invention relates to the use of a TNF Receptor
together with a steroid hormone to produce a pharmaceutical
composition for the treatment of lethal bacterial and viral
infections as well as autoimmune and inflammatory diseases. It also
relates to said pharmaceutical compositions for the simultaneous
separate or sequential use of its active ingredients for the above
specified treatment. In particular, it relates to the use of TBP-1,
together with dehydroepiandrosterone (DHEA) or its metabolites to
produce a pharmaceutical composition for the treatment of septic
shock.
Inventors: |
Boe; Alessandra; (Rome,
IT) ; Borrelli; Francesco; (Rome, IT) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.;624 NINTH STREET, NW
SUITE 300
WASHINGTON
DC
20001-5303
US
|
Assignee: |
Applied Research Systems ARS
Holding N.V.
Curacao
NL
|
Family ID: |
35998746 |
Appl. No.: |
11/352275 |
Filed: |
February 13, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09687122 |
Oct 13, 2000 |
7012060 |
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11352275 |
Feb 13, 2006 |
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08983223 |
Feb 27, 1998 |
6225300 |
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PCT/EP95/02767 |
Jul 14, 1995 |
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09687122 |
Oct 13, 2000 |
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Current U.S.
Class: |
514/21.2 ;
514/1.4; 514/171; 514/3.7 |
Current CPC
Class: |
A61K 38/1793 20130101;
A61K 31/56 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 31/56 20130101; A61K 38/1793 20130101 |
Class at
Publication: |
514/012 ;
514/171 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61K 31/56 20060101 A61K031/56 |
Claims
1. A method for treating lethal virus infections in a patient which
comprises administering to a patient a tumor necrosis factor (TNF)
receptor in combination with dehydroepiandrosterone (DHEA)
simultaneously, separately or sequentially.
2. The method of claim 18, wherein the TNF receptor is TNF Binding
Protein-2.
3. The method of claim 18, wherein the TNF receptor is TNF Binding
Protein-1.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the use of a TNF Receptor
together with a steroid hormone to produce a pharmaceutical
composition for the treatment of lethal bacterial and viral
infections as well as autoimmune and inflammatory diseases. It also
relates to said pharmaceutical compositions for the simultaneous,
separate or sequential use of its active ingredients for the above
specified treatment.
[0002] In particular, it relates to the use of TBP-1 together with
dehydroepiandrosterone (DHEA) or its metabolites to produce a
pharmaceutical composition for the treatment of septic shock.
BACKGROUND OF THE INVENTION
[0003] Septic shock as a consequence of Gram-negative bacteremia or
endotoxemia remains a critical clinical condition in spite of
adequate antibiotic therapy.
[0004] It is now known that the lethal consequences of septic shock
results from an exaggerated host response, mediated by protein
factors such as TNF and interleukin 1 (IL-1), rather than from the
pathogen directly.
[0005] Tumor necrosis factor (TNF) is a cytokine produced mainly by
activated macrophages, which elicits a wide range of biological
effects. These include an important role in endotoxic shock and in
inflammatory, immunoregulatory, proliferative, cytotoxic and
anti-viral activities.
[0006] The induction of the various cellular responses mediated by
TNF is initiated by its interaction with two distinct cell surface
receptors of approximately 55 kDa (also called TNF-R1) and 75 kDa
(also called TNF-R2). The extracellular soluble portions of these
receptors, called respectively TBP-1 (TNF Binding Protein-1) and
TBP-2 (TNF Binding Protein-2), have been isolated and cloned (see
EP Patents 308 378, 398 327 and U.S. Pat. No. 5,811,261).
[0007] Several studies in animal models of TNF-mediated endotoxic
shock indicated that both anti-TNF antibodies and soluble
TNF-receptor are able to counteract the lethal effects induced (see
for example: Bentler, B et al., Science, 229:869 (1985), Lesslauer,
W. et al., Eur. J. Immunol., 21:2883 (1991), Evans, T. J. et al, J.
Exp. Med., 180:2173 (1994) and Mohler K. M. et al., J. Immunol.
151:1548 (1993)).
[0008] The in vivo protective effects of urinary as well as
recombinant TBP-1 (derived from both CHO and E. Coli cells) in
experimental models of septic shock have already been demonstrated
(see Bertini, R. et al., Eur. Cytokine Netw. 4(1):39 (1993) and
Ythier A., et al., Cytokines, 5:459 (1993)).
[0009] DHEA (INN: Prasterone) is an adrenocortical steroid hormone
which is an intermediate in the biosynthesis of other hormones
including testosterone and estradiol-17.beta..
[0010] The precise biological functions of DHEA are still unclear.
Experimental and epidemiological data suggest an inverse
relationship between low levels of DHEA in serum and morbidity from
atherosclerotic cardiovascular disease (see Barret-Connor, D. et
al., N. Engl. J. Med. 315:1519 (1986)), cancer (see Gordon, G. B.,
et al., Cancer Res. 51:1366 (1991)) and immunodeficiency virus
(HIV) infection (Villette, J. M. et al. J. Clin. Endocrinol. Metab.
70:572 (1990)).
[0011] An immunomodulating activity of this drug has also been
reported; in particular, DHEA has been shown to prevent the
development of systemic lupus erythematosus in a mouse model
(Lucas, J. et al. J. Clin. Invest. 75:2091(1985)).
[0012] It has been demonstrated that DHEA regulates the systemic
resistance against lethal viral infections induced by different
viruses: coxsackievirus B4 (as reported in Loria, R. M. et al.,
Ann. N.Y. Acad. Sci, 293), herpes virus type 2 (see Loria, R M. et
al., J. Med. Virol., 26:301 (1988)), West Nile virus (a
neurovirulent Sindbis virus) and Semliki Forest virus (as reported
in Ben-Nathan, D. et al. Arch. Virol. 20:263 (1989)).
[0013] It has also been reported that DHEA has similar protective
effects against a lethal bacterial infection induced by
Enterococcus faecalis (see Loria, R. M. et al. in Symposium
Pharmaco-Clinique, Roussel-Uclaf 9:24 (1989)).
[0014] Danenberg, H. D. et al. (Antimicrob. Agents Chemother.
36:2275 (1992)) reported that DHEA has the capability of protecting
mice from septic shock induced by lipopolysaccharides (LPS) alone
or by Tumor Necrosis Factor alpha (TNF-.alpha.) in combination with
D-Galactosamine. LPS administration resulted in high levels of
TNF-.alpha., a response that was significantly blocked by DHEA,
both in vivo and in vitro.
DISCLOSURE OF THE INVENTION
[0015] The main object of the present invention is the use of a TNF
Receptor in combination with a steroid hormone to produce a
pharmaceutical composition for the treatment of lethal bacterial
and viral infections as well as autoimmune and inflammatory
diseases. The TNF Receptor and the steroid hormone can be
administered simultaneously, separately or sequentially.
[0016] The Applicants have, in fact, found that in such treatment
there is a synergic effect between the two active ingredients.
[0017] Another object of the present invention is, therefore, the
method for treating lethal bacterial and viral infections as well
as autoimmune and inflammatory diseases by administering
simultaneously, separately or sequentially an effective amount of
TNF Receptor and an effective amount of a steroid hormone, together
with a pharmaceutically acceptable excipient.
[0018] A further object of the present invention are the
pharmaceutical compositions containing a TNF Receptor and a steroid
hormone, in the presence of one or more pharmaceutically acceptable
excipients, for the simultaneous, separate or sequential
administration of its active ingredients in the treatment of lethal
bacterial and viral infections as well as autoimmune and
inflammatory diseases.
[0019] In case of separate or sequential use of the two active
ingredients, the pharmaceutical compositions of the invention will
consist of two different formulations, each comprising one of the
two active ingredients together with one or more pharmaceutically
acceptable excipients.
[0020] The administration of such active ingredients may be by
intravenous, intramuscular or subcutaneous route. Other routes of
administration, which may establish the desired blood levels of the
respective ingredients, are comprised by the present invention.
[0021] Non-limitative examples of pathologies, in which the above
active ingredients are indicated, are the following: septic shock,
AIDS, rheumatoid arthritis, lupus erythematosus and multiple
sclerosis. Particularly preferred is the treatment of septic
shock.
[0022] The TNF Receptor is preferably selected between the
extracellular soluble domain of TNF-R1, i.e. TBP-1, and the
extracellular soluble domain of TNF-R2, i.e. TBP-2. TBP-1 is
particularly preferred. Recombinant human TBP-1 (r-hTBP-1) is
advantageously used according to the invention.
[0023] The steroid hormone can be both a corticosteroid or an
androgen. Preferably, it is an androgen. More preferably, it is
DHEA or one of its metabolites, dispersed in a suitable medium, for
example a phospholipid emulsion or carboxymethyl cellulose or
polyvinylpirrolidone.
[0024] Therefore, a preferred embodiment of the invention consists
in the combined use of r-hTBP-1 and DHEA in the treatment of septic
shock. In this case, the Applicants have found that it is possible
to reduce at least four times the effective dose of r-hTBP-1.
[0025] The above effect has been showed with in vivo experiments in
mice.
[0026] In particular, a murine model has been used in the present
invention, according to which septic shock is induced by
administering lipopolysaccharides (LPS) in combination with.
D-Galactosamine.
[0027] For the human therapy, the preferred doses of the active
ingredient are 20 mg of r-hTBP-1 and 80 mg of DHEA, preferably
divided into four administrations in a 24-hours period.
[0028] The invention will now be described by means of the
following Examples, which should not be construed as in any way
limiting the present invention. The Examples will refer to the
Figures specified here below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1: Protective effects of CHO r-hTBP-1 (62.5
.mu.g/mouse) in combination with single (250 .mu.g/mouse) or
repeated injections (4.times.62.5 .mu.g/mouse) of DHEA in the
experimental marine septic shock are shown.
[0030] FIG. 2: Protective effects of different doses of CHO
r-hTBP-1 in the experimental murine septic shock induced by
LPS+D-Galactosamine are shown for comparative purposes. The number
of mice in the r-hTBP-1 groups is the same as that reported for the
corresponding control groups. In FIG. 2a and 2b are shown the
results observed after 24 and 48 hours (respectively) after the
inoculum of LPS+D-Galactosamine.
EXAMPLE 1
LPS+D-Galactosamine-Induced Septic Shock Model
[0031] The experiments were carried out in vivo on mice. The septic
shock was induced by an i.p. administration of a mixture of LPS
(0.1 .mu.g/mouse) and D-Galactosamine (18 mg/mouse).
[0032] The treatment schedule for r-hTBP-1 based on repeated
administrations (the total dose was divided into 4 administrations
given at time 0 and 3, 6 and 24 hours after), was adopted. CHO
r-hTBP-1- was tested, alone or in combination with DHEA, at dose
levels of 62.5 and 125 .mu.g/mouse (total dose).
Assessment of the Effects of DHEA in the LPS+D-Galactosamine
Model
[0033] DHEA was tested both in a single administration injected
i.p. one hour before and contemporaneously to the septic
shock-inducing agent at doses encompassed between 100 and 2,000
.mu.g/mouse and in repeated treatment with the total dose (250
.mu.g/mouse) divided into 4 injections given within 24 hours.
Assessment of the Effects of DHEA in Combination with r-hTBP-1
[0034] The above treatment schedules of DHEA (doses from 250 to
1,500 .mu.g/mouse) were followed also in combination with r-hTBP-1
which was tested at the total dose varying from 62.5 to 125
.mu.g/mouse (divided into 4 administrations).
Materials
Animals
[0035] SPF female BALB/c inbred mice, 6-8 weeks (about 18-20 g
b.w.), from Charles River were used throughout the whole study
after an acclimatization period of at least 7 days.
[0036] Unless otherwise specified, in all the experiments the mice
were randomly divided into groups of 10 animals each.
Test Drugs
[0037] CHO recombinant human TBP-1 produced according to the known
methods in the field of biotechnology.
[0038] The protein mass of the r-hTBP-1 bulk was determined by the
Lowry method before starting the experiments.
[0039] E. Coli lipopolysaccharides 055:B5 supplied by Sigma.
[0040] D-(+)-Galactosamine hydrochloride, 99% supplied by
Janssen.
[0041] Dehydroepiandrosterone supplied by Sigma.
Reagents
[0042] Sodium chloride 0.9% (Saline), manufactured by Baxter.
[0043] Phospholipid emulsion (PLE) composed of phosphatidyl choline
(71%), phosphatidyl ethanolamine (16%), lysophosphatidyl choline
(7.5%), sphingomyelin (5%) and phosphatidic acid (0.3%).
Methods
Septic Shock Model (LPS+D-Galactosamine Model)
[0044] Both substances were dissolved in saline. The mixture of LPS
(0.1 .mu.g/mouse) and D-Galactosamine (18 mg/mouse) was prepared by
mixing equal volumes of these substances. The mixture was
subsequently injected to animals by the i.p. route. This dose was
selected under previous experiments as that inducing around 80%
mortality calculated as the number of dead vs. total number of mice
for each group. The lethality was monitored after 24, 48, 72 and 96
hours and at day 7 post-treatment.
Treatment Schedule for r-hTBP-1
[0045] The drug was diluted in saline and injected by repeated
administrations, i.e. i.v. immediately before the inoculum of LPS
plus D-Galactosamine and s.c. 3, 6 and 24 hours later.
Treatment Schedules for DHEA
[0046] The effects of DHEA, alone or in combination with r-hTBP-1,
were assayed after single i.p. administrations at different times
in respect to LPS+D-Galactosamine injection; in particular, it was
administered 1 hour before and contemporaneously to the septic
shock-inducing agent.
[0047] A repeated treatment schedule was also investigated in which
DHEA was administered at time 0 (septic shock induction) and 3, 6
and 24 hours later. DHEA was initially vehicled in a mixture of
2.5% alcohol and 10% rabbit serum in saline. To improve the
solubility and standardise the preparation, the steroid was
successively dispersed in a mixture of 2.5% alcohol and 10%
phospholipid emulsion in saline.
Data Evaluation
[0048] The primary end point was the survival rate at 48 hours
after induction of septic shock. The comparison of the effects of
each treatment group vs. controls and among treatment groups was
effected by Fisher's exact test, one-tailed. The same comparison
was also performed at the other time points. The results in each
treatment group were expressed as percent survival.
Results
[0049] DHEA Administered 1 hour before LPS+D-Galactosamine
[0050] The results of the DHEA dose-range finding experiments are
reported in Table 1. No protective effects were exerted by this
substance up to 1,000 .mu.g/mouse; by contrast, statistically
significant protective effects were obtained at higher doses with a
complete protection at 2,000 .mu.g/mouse at all time points
considered.
[0051] When a suboptimal dose of CHO r-hTBP-1 (125 .mu.g/mouse),
inducing about 50% survival, was administered in combination with
two suboptimal doses of DHEA (1,000 and 1,500 .mu.g/mouse), an
additive effect between the two substances was observed (Table 2).
The result of the combination with the highest DHEA dose, where a
100% survival rate was obtained, is significantly different from
that of the r-hTBP-1 group (50%) and DHEA group (40%).
DHEA Administered Contemporaneously with LPS+D-Galactosamine
[0052] The results obtained in the DHEA dose-range finding test,
where the substance, diluted in saline 10% rabbit serum, was
administered contemporaneously to septic shock induction are
reported in Table 3.
[0053] Although a clear dose-effect relationship has not been
found, a higher protection was produced by 1,000 .mu.g DHEA in
comparison to what observed with the same dose by the previous
treatment schedule (DHEA administered 1 hour before).
[0054] The combination of 62.5 .mu.g/mouse of r-hTBP-1 with 250
.mu.g/mouse of DHEA induced a slightly higher protection (60%) in
respect to the single treatments (20% and 30% for r-hTBP-1 and
DHEA, respectively, see Table 4). When the same combination was
vehicled in a 10% phospholipid emulsion, to improve the solubility
of DHEA, a significantly higher survival was obtained compared to
DHEA and r-hTBP-1 alone (Table 4) at 24 hours which, however,
successively decreased.
Repeated Administrations of DHEA after LPS+D-Galactosamine
[0055] Comparable results (30 and 40% survival rate at 48 hours)
were obtained when a dose of 250 .mu.g/mouse of DHEA was
administered as a single injection (Table 5, group D) or divided
into 4 inocula (given at 0, 3, 6 and 24 hours from septic shock
induction, group E), respectively. Similarly, no statistically
significant differences were found when the animals were injected
the combination of r-hTBP-1 and DHEA with the latter administered
either as a single inoculum (Table 5, group F) or divided into 4
injections (Group G).
[0056] Due to the high survival rate obtained in the r-hTBP-1
group, an additive effect is not evident in these tests after 24
and 48 hours where the combination treated groups were
significantly different only from control and DHEA groups.
[0057] However, it has to be stressed that the combined therapy
induced a more prolonged survival in respect to the r-hTBP-1 group
alone since a statistically significant difference was found after
72 and 96 hours.
[0058] FIG. 1 reports the data obtained combining the results of
the three experiments reported in Tables 4 and 5 in which the dose
of 250 .mu.g/mouse of DHEA was injected as a single inoculum or
divided into 4 inocula (62.5 .mu.g each).
[0059] FIG. 2 reports, for comparative purposes, the effects of
different doses of CHO r-hTBP-1 alone in the experimental marine
septic shock induced by LPS+D-Galactosamine. The number of mice in
the r-hTBP-1 groups is the same as that reported for the
corresponding control groups. In FIGS. 2a and 2b are shown the
results observed after 24 and 48 hours (respectively) after the
inoculum of LPS+D-Galactosamine.
[0060] In all the experiments, percent survival values were
calculated using the survival Tables for the follow up studies
(Armitage P., Cap. XIV, Tavole disopravvivenza in Statistica
Medica, Feltrinelli, Milano). The X.sup.2-test was applied to each
observation time by comparing the DHEA+r-hTBP-1 groups to the other
groups.
[0061] In addition to showing that at all time points the combined
therapy is significantly different from control and DHEA groups,
this analysis confirmed that more prolonged survival times were
obtained with the combined treatment in respect to r-hTBP-1 alone.
Although no statistical difference between the two treatment
combinations has been found, a trend to a more sustained protection
can be observed when both drugs were divided into four injections,
which is reflected by the different levels of significance at
different time-points (Table 6).
Conclusions
[0062] A significant protection from death (70% survival) was found
with at least 1,500 .mu.g/mouse of DHEA, injected 1 hour before
LPS+D-Galactosamine, which confirm literature data.
[0063] When suboptimal doses of DHEA (1,000 and 1,500 .mu.g/mouse)
and of r-hTBP-1 (125 .mu.g/mouse, total dose) were combined,
additive effects (90-100% survival) were produced, compared to
0-40% and 50% with the two doses of DHEA and of r-hTBP-1,
respectively.
[0064] Similar results were obtained even with lower doses of DHEA
(250 .mu.g/mouse) in combination with r-hTBP-1 (62.5 .mu.g/mouse)
(90-100% survival), which also provided a more prolonged protection
(up to 96 hours) in respect to r-hTBP-1 alone, both when DHEA was
injected contemporaneously to LPS+D-Galactosamine in a single
inoculum (250 .mu.g/mouse) or when the same dose was divided into 4
inocula (62.5 .mu.g/each) injected at times 0, 3, 6 and 24 hours
after septic shock induction.
[0065] The present findings are of particular relevance in showing
that a combined treatment of r-hTBP-1 with DHEA allows one to
reduce the dose of r-hTBP-1 by at least four times.
EXAMPLE 2
Example of Pharmaceutical Manufacturing
A) r-h-TBP-1
Materials
[0066] Pure saccharose Ph EurBP, Ph Nord, NF, (Merck);
H.sub.3PO.sub.4 Suprapur (Merck); NaOH for analysis (Merck); water
for injectable.
[0067] Vials DIN 2R (borosilicate glass type I), rubber closures
(Pharmagummi W1816 V50) and Aluminium rings and Flip-off caps
(Pharma-Metal GmbH) are used as containers.
Preparation of r-hTBP-1 Solution Containing Saccharose (for 1,000
Vials each Containing 5 mg of r-hTBP-1)
[0068] Saccharose (30.0 g) and H.sub.3PO.sub.4 (1.96 g) are
dissolved into water for injectables (800 ml) in order to obtain
the starting saccharose solution.
[0069] The bulk of r-hTBP-1 (5 g) is added to the saccharose
solution that, after the pH has been adjusted at 7.0 by means of
2.5 M NaOH, is brought to the final volume of 1,000 ml. The
solution is filtered through a 0.22 .mu.m Duarapore sterile filter
(Millipore). During the process, the solution temperature is kept
between 40 and 8.degree. C.
Filling Up and Lyophilization
[0070] The vials are filled up with 1 ml of r-hTBP-1 sterile
solution, transferred to the freeze dryer and cooled at -45.degree.
C. for 6 hours. The lyophilization is started at the temperature of
-45.degree. C. with a vacuum of 0.07 mBar. The heating is performed
according to the following scheme: 10.degree. C. for 12 hours; then
+35.degree. C. until the end of the cycle.
B) DHEA
Materials
[0071] Pure Mannitol Ph Eur, BP, FU, USP, FCC, E241 (Merck):
H.sub.3PO.sub.4 Suprapur (Merck); NaOH for analysis (Merck); water
for injectable.
[0072] Vials DIN 2R (borosilicate glass type I), rubber closures
(Pharmagummi W1816 V50) and Aluminium rings and Flip-off caps
(Pharma-Metal GmbH) are used as containers.
Preparation of DHEA Solution Containing Mannitol (for 1,000 Vials
Containing 10 mg of DHEA)
[0073] Mannitol (45.0 g) and H.sub.3PO.sub.4 (1.96 g) are dissolved
into water for injectables (800 ml) in order to obtain the starting
saccharose solution.
[0074] The bulk of DHEA (20 g) is added to the saccharose solution
that, after the pH has been adjusted at 7.0 by means of 2.5 M NaOH,
is brought to the final volume of 1,000 ml. The solution is
filtered through a 0.22 .mu.m Durapore sterile filter (Millipore).
During the process, the solution temperature is kept between
4.degree. and 8.degree. C.
Filling Up and Lyophilization
[0075] The vials are filled up with 1 ml of DHEA sterile solution,
transferred to the freeze dryer and cooled at -45.degree. C. for 6
hours. The lyophilization is started at the temperature of
-45.degree. C. with a vacuum of 0.07 mBar. The heating is performed
according to the following scheme: +20.degree. C. for 12 hours;
then +35.degree. C. until the end of the cycle. TABLE-US-00001
TABLE 1 Effects of DHEA in the Septic Shock Induced by LPS +
D-Galactosamine, in Mice % Cumulative Survival No. 24 hrs 48 hrs 72
hrs 96 hrs Treatment, of 1.sup.st 2.sup.nd 1.sup.st 2.sup.nd
1.sup.st 2.sup.nd 1.sup.st 2.sup.nd dose/mouse and route mice Exp
Exp Exp Exp Exp Exp Exp Exp LPS 0.1 .mu.G None 10 20 20 20 10 20 10
20 10 i.p..sup.(1) + D- DHEA, i.p. 10 20 -- 20 -- 20 -- 20 -- Gal
18 mg 100 .mu.g.sup.(2) i.p..sup.(1) DHEA, i.p. 10 10 -- 10 -- 10
-- 10 -- 500 .mu.g.sup.(2) DHEA, i.p. 10 -- 20 -- 20 -- 10 -- 0
1000 .mu.g.sup.(2) DHEA, i.p. 10 -- .sup. 70.sup.a -- .sup.
70.sup.a -- 50 -- 50 1500 .mu.g.sup.(2) DHEA, i.p. 10 .sup.
100.sup.a .sup. 100.sup.a .sup. 100.sup.a .sup. 100.sup.a .sup.
100.sup.a .sup. 100.sup.a .sup. 100.sup.a .sup. 100.sup.a 2000
.mu.g.sup.(2) .sup.(1)Administered as a mixture at time 0
.sup.(2)Administered 1 hour before LPS + D-Gal administration
.sup.aSignificantly different from LPS + D-Gal controls (Fisher's
exact test, one-tailed) N.B. DHEA was vehicled in saline 10% rabbit
serum
[0076] TABLE-US-00002 TABLE 2 Effects of CHO 4-h TBP-1 and DHEA in
Septic Shock Induced by LPS + D-Galactosamine, in Mice No.
Treatment, of % cumulative survival dose/mouse and route Mice 24 hr
48 hr 72 hr 96 hr LPS, i.p. None 10 10 25 25 25 0.1 .mu.g.sup.(1) +
D- r-hTBP-1 125 .mu.g.sup.(2) 10 50 10 0 0 Gal, i.p. DHEA 1000
.mu.g i.p..sup.(3) 10 0 30 30 30 18 mg.sup.(1) DHEA 1500 .mu.g
i.p..sup.(3) 10 40 50 50 50 DHEA 1000 .mu.g i.p..sup.(3) + r- 10
90.sup.a,b 90.sup.a,b 90.sup.a,b 90.sup.a,b hTBP-1 125
.mu.g.sup.(2) DHEA 1500 .mu.g i.p..sup.(3) + r- 10 100.sup.a,b,c
100.sup.a,b,c 100.sup.a,b,c 100.sup.a,b,c hTBP-1 125 .mu.g.sup.(2)
.sup.(1)Administered as a mixture at time 0 .sup.(2)Administered at
time 0 (i.v.), and at 3, 6 and 24 hours (s.c.) after LPS + D-Gal
injection .sup.(3)Administered 1 hour before LPS + D-Gal
administration .sup.aSignificantly different from LPS + D-Gal.
controls .sup.bSignificantly different from DHEA alone
.sup.cSignificantly different from rhTBP-1 alone (Fisher's exact
test, one-tailed) N.B. DHEA was vehicled in saline 10% rabbit
serum.
[0077] TABLE-US-00003 TABLE 3 Effects of DHEA in the Septic Shock
Induced by LPS and D-Galactosamine, in Mice % Cumulative Survival
No. 24 hrs 48 hrs 72 hrs 96 hrs Treatment, of 1.sup.st 2.sup.nd
1.sup.st 2.sup.nd 1.sup.st 2.sup.nd 1.sup.st 2.sup.nd dose/mouse
and route mice Exp Exp Exp Exp Exp Exp Exp Exp LPS 0.1 .mu.g None
10 10 40 10 40 10 40 10 40 i.p..sup.(1) + D- DHEA, i.p. 10 -- 20 --
20 -- 20 -- 20 Gal 18 mg 250 .mu.g.sup.(2) i.p..sup.(1) DHEA, i.p.
10 50 30 40 30 30 30 30 30 500 .mu.g.sup.(2) DHEA, i.p. 10 40 50 40
50 40 50 40 50 750 .mu.g.sup.(2) DHEA, i.p. 10 .sup. 80.sup.a -- 60
-- 60 -- 60 -- 1000 .mu.g.sup.(2) .sup.(1)Administered as a mixture
at time 0 .sup.(2)Administered at time 0 .sup.aSignificantly
different from LPS + D-Gal controls (Fisher's exact test,
one-tailed) N.B. DHEA was vehicled in saline 10% rabbit serum
[0078] TABLE-US-00004 TABLE 4 Effects of CHO r-hTBP-1 and DHEA in
the Septic Shock Induced by LPS and D-Galactosamine, in Mice No.
Treatment, of % cumulative survival dose/mouse and route Mice 24 hr
48 hr 72 hr 96 hr LPS 0.1 .mu.g none 20 25 25 25 25 i.p..sup.(1) -
D- r-hTBP-1 62.5 .mu.g.sup.(2) 10 20 10 0 0 Gal 18 mg DHEA 250
.mu.g.sup.(3) 10 30 30 30 30 i.p..sup.(1) (NaCl 10% RS) DHEA 250
.mu.g.sup.(3) 10 60 50 50 50 (NaCl 10% RS) - r- hTBP-1 62.5
.mu.g.sup.(2) DHEA 250 .mu.g.sup.(3) 10 40 30 30 30 (NaCl 10% PLE)
DHEA 250 .mu.g.sup.(3) 10 90.sup.a,b,c .sup. 60.sup.c .sup.
40.sup.c .sup. 40.sup.c (NaCl 10% PLE) - r- hTBP-1 62.5
.mu.g.sup.(2) .sup.(1)Administered as a mixture at time 0.
.sup.(2)Administered at time 0 (i.v.) and at 3, 6 and 24 hours
(s.c.) after LPS + D-Gal. injection. .sup.(3)Administered as a
single injection at time 0 i.p. .sup.aSignificantly different from
LPS + D-Gal. controls (Fisher's exact test, one-tailed).
.sup.bSignificantly different from DHEA alone (Fisher's exact test,
one-tailed). .sup.cSignificantly different from r-hTBP-1 alone
(Fisher's exact test, one-tailed). N.B. DHEA was vehicled in saline
10% rabbit serum (NaCl 10% RS) or in saline 10% phospholipid
emulsion(NaCl 10% PLE).
[0079] TABLE-US-00005 TABLE 5 Effects of CHO r-hTBP-1 and DHEA in
the Septic Shock Induced by LPS + Galactosamine, in Mice %
Cumulative Survival No. 24 hrs 48 hrs 72 hrs 96 hrs Treatment, of
1.sup.st 2.sup.nd 1.sup.st 2.sup.nd 1.sup.st 2.sup.nd 1.sup.st
2.sup.nd dose/mouse and route mice Exp Exp Exp Exp Exp Exp Exp Exp
LPS A None 10 10 10 10 10 10 10 10 10 0.1 .mu.g B NaCl 10% PLE 10
-- 11 -- 11 -- 11 -- 11 i.p..sup.(1) + D- (n = 9) (n = 9) (n = 9)
(n = 9) Gal 18 mg C r-hTBP-1 62.5 .mu.g.sup.(2) 10 90.sup.a
70.sup.a 80.sup.a 40 70.sup.a 30 70.sup.a 30 i.p..sup.(1) D DHEA
250 .mu.g.sup.(3) 10 40 -- 30 -- 30 -- 30 -- E DHEA 62.5 .mu.g
x4.sup.(4) 10 40 40 40 40 40 40 40 40 F DHEA 250 .mu.g.sup.(3) + r-
10 100.sup.a,b -- 100.sup.a,b -- 100.sup.a,b 100.sup.a,b -- hTBP-1
62.5 .mu.g.sup.(2) G DHEA 62.5 .mu.g.sup.(3) + r- 10 100.sup.a,b
90.sup.a,b 100.sup.a,b 90.sup.a,b 100.sup.a,b 80.sup.a,b
100.sup.a,b 80.sup.a,b hTBP-1 62.5 .mu.g.sup.(2)
.sup.(1)Administered as a mixture at time 0 .sup.(2)Administered at
time 0 i.v., and at time 3, 6 and 24 hours (s.c.)
.sup.(3)Administered as a single injection at time 0 i.p.
.sup.(4)Administered at time 0, 3, 6 and 24 hours i.p.
.sup.aSignificantly different from LPS + D-Gal. controls (Fisher's
exact test, one-tailed) .sup.bSignificantly different from DHEA
alone (Fisher's exact test, one-tailed) .sup.cSignificantly
different from r-hTBP-1 alone (Fisher's exact test, one-tailed N.B.
DHEA was vehicled in 10% phospholipid emulsion (NaCl 10% PLE)
[0080] TABLE-US-00006 TABLE 6 Statistical Significance of r-hTBP-1
Group vs. r-h TBP-1 + DHEA Groups (.chi..sup.2-Test) p Level
Observation Times r-hTBP-1 r-hTBP-1 (hours Post vs. vs. LPS + D-
r-hTBP-1 62.5 + DHEA r-hTBP-1 62.5 + DHEA Cal Injection)
250.sup.(1) 4 .times. 62.5.sup.(1) 24 0.015 0.015 48 0.022 0.0006
72 0.024 0.0002 96 0.024 0.0002 .sup.(1)Doses are expressed as
.mu.g/mouse
* * * * *