Therapeutic sepsis treatment using antagonists to PTHrP

Methods and compositions are provided for the treatment or prophylaxis of systematic inflammatory response syndrome by administering an antagonist to parathyroid hormone-related protein, such as antibodies to PTHrP.

FIELD OF THE INVENTION 
This invention relates generally to a method of treating severe 
inflammatory conditions, such as systemic inflammatory response syndrome, 
which results from e.g., sepsis, and more particularly to administering an 
antagonist to parathyroid hormone-related protein. 
BACKGROUND OF THE INVENTION 
Systematic inflammatory response syndrome is the designation recently 
established by a group of researchers to describe related conditions 
resulting from, for example, sepsis, pancreatitis, multiple trauma such as 
injury to the brain, and tissue injury, such as laceration of the 
musculature, brain surgery, hemorrhagic shock, and immune-mediated organ 
injuries. A variety of different approaches have been suggested for 
treating inflammatory conditions or septic (endotoxin) shock. 
U.S. Pat. No. 5,308,834, issued May 3, 1994, inventors Scott et al., 
discloses a method said to prevent endotoxemia in a subject by 
administering an amount of a leukocyte protein (BPI) effective to prevent 
endotoxemia in the subject. 
Among the therapeutic approaches, antibodies directed against endotoxin or 
its components have been evaluated for their utility in immunotherapy of 
sepsis. Murine and human monoclonal antibodies directed against the core 
lipopolysaccharide of the endotoxin have been reported to exert protection 
during Gram-negative bacterial sepsis in animals. Dunn, Transplantation, 
45, 424-429 (1988). Antibodies directed against lipid A also have been 
reported to have a protective effect in humans. Jaspers et al., Infection, 
15 Supp. 2, S89-95 (1987). Antibodies to the J5 mutant of E. coli are 
reported to be protective against septic shock in animals and humans. 
Cohen et al., Lancet, 1, 8-11 (1987); Law and Marks, J. Infect. Dis., 151, 
988-994 (1985). Antibodies to endotoxin core glycolipid have been reported 
to prevent the serious consequences of Gram-negative infections in 
surgical patients. Baumgartner et al., Lancet, 2, 59-63 (1985). In 
addition, human monoclonal antibodies to P. aeruzinosa exotoxin A and 
exoenzyme S have been described as useful for this purpose. U.S. Pat. No. 
4,677,070, issued Jun. 30, 1987. 
Many of the toxic effects of endotoxin are mediated by cytokines, hormones, 
and other small molecules. Blockade of these various mediators has been 
used to treat sepsis. In animal models prior administration of antibody 
directed against TNF was reported to protect from the lethal effects of 
endotoxin. Beutler et al., Science, 229, 869 (1985). Also, antibody 
blockade experiments were reported showing that various cytokines, such as 
TNF, are mediators of the lethal effects of endotoxin. Tracey et al., 
Nature, 330, 662 (1987); Ohlsson et al., Nature, 348, 550 (1990); Heinzel, 
J. Immunol., 145, 2920 (1990); Doherty et al., J. Immunol., 149, 1666 
(1992); Bernhagen et al., Nature, 365, 756 (1993). More recently, either 
polyclonal or monoclonal antibodies to a human tumor necrosis factor 
binding protein (TBP-I) have been described for application in modulating 
the response to tumor necrosis factor, such as to suppress deleterious 
effects of this cytokine. U.S. Pat. No. 5,359,037, issued Oct. 25, 1994, 
inventors Wallach et al. 
The treatment of endotoxemia or sepsis by passive immunization with 
endotoxin neutralizing antibodies or cytokine antibodies is a relatively 
new approach. However, to date many of the approaches suggested for sepsis 
treatment have not proven very efficacious. It is likely that treatment 
for septicemia in the future will combine a plurality of approaches, in 
view of the large cascade of pro-inflammatory cytokines unleashed during 
the host response to infection. 
Since morbidity and mortality associated with endotoxemia remains high, new 
adjunct therapies are being sought because septicemia remains the leading 
case of death in intensive care units in the United States. 
SUMMARY OF THE INVENTION 
In one aspect of the present invention, a method of treating a patient for 
a systemic inflammatory response syndrome comprises administering a 
pharmaceutically effective amount of a parathyroid hormone-related protein 
blocker to the patient. Thus, patients are treated for a systemic 
inflammatory response syndrome resulting from conditions such as sepsis. 
Comparison between groups of inventively treated and control animals showed 
that the inventive treatment provided a significant protective effect from 
an induced inflammatory response syndrome mortality, although the 
treatment delayed but, ultimately, did not prevent death. Thus, therapy in 
accordance with the invention preferably includes an additional (or a 
plurality of) approach(es) to block the cascade of pro-inflammatory 
cytokines.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Parathyroid hormone-related protein (PTHrP) was isolated and identified in 
the late 1980s. The amino acid sequence of human, rat, mouse, and chicken 
PTHrPs are known to the art and are discussed by Martin et al., Crit. Rev. 
Biochem. Mol. Biol., 26, pp. 377-395 (1991). PTHrP and PTH, one of the 
primary hormones responsible for calcium homeostasis, appear to be derived 
from a common ancestral gene. However, PTHrP is more highly conserved than 
PTH. Although PTHrP shares little primary sequence homology with PTH, 
N-terminal segments of both peptides bind with similar affinity to PTH 
receptors present in kidney and bone, the classic target tissues for PTH 
action. The 1-13 region of PTHrP is 70% homologous with the corresponding 
region of PTH. The 14-34 region of PTHrP shares no homology with the 14-34 
region of PTH, although there is apparently a similar tertiary, 
3-dimensional or steric homology between PTHrP and PTH in the 14-34 
region. 
In malignancy, high circulating levels of tumor-derived PTHrP cause 
hypercalcemia by inappropriately stimulating bone resorption and renal 
calcium reabsorption via interaction with PTH/PTHrP receptors at these 
sites. In contrast, PTHrP is barely detectable in the circulation of 
normal individuals. However, PTHrP is widely expressed in normal tissues, 
thus leading to the hypothesis that this peptide normally acts at its site 
of production in a paracrine or autocrine fashion. Consistent with this 
hypothesis, the recently cloned PTH/PTHrP receptor, in addition to being 
present in bone and kidney, has also been found to be expressed in many of 
the same tissues that produce PTHrP. 
While PTHrP has been defined by its relation to PTH, a hormone that follows 
the normal endocrine paradigm of localized production and distant action, 
we believe that PTHrP may in fact act more like a cytokine than a classic 
hormone and that its effect on bone and calcium metabolism may be but one 
of many important biological functions. We have found a therapeutic role 
for antibodies to PTHrP in significantly protecting from sepsis lethality. 
Although the precise cause of death from sepsis is unknown, hypotension is 
a hallmark of lethal endotoxemia. Alterations in vascular hemodynamics are 
also thought to contribute to the multisystem organ failure that 
accompanies sepsis. In septic shock, systemic vascular resistance is low 
due to vasodilation and, although cardiac output increases in an attempt 
to maintain blood pressure, cardiac contractility is decreased. 
The present invention is the therapeutic use of a PTHrP antagonist. By 
"PTHrP antagonist" is meant to include compounds that block PTHrP activity 
at the PTHrP receptor, and which include PTH or PTHrP fragments (e.g. 
fragments with the 3-34 or 7-34 amino acid residues of PTH or PTHrP and 
fragments in which one or more amino acid residues have been replaced with 
analogues), monoclonal or polyclonal antibodies to PTHrP, and non-peptide 
analogs that can be designed to mimic the effects of peptides such as the 
3-34 or 7-34 amino acid residue fragments. The polyclonal or monoclonal 
antibodies may be raised in rabbits, mice, or other animals or tissue 
cultured cells or can be products of cells of human origin. They may also 
be produced of recombinant DNA technology either in a form identical to 
that of the native antibody or as chimeric molecules, constructed by 
recombination of antibody molecules of man and animal origins or in other 
forms chosen to make the antibodies most suitable for use in therapy. The 
replacement of amino acid residues and the amide forms (at the C terminus) 
for analogues are known. Illustrative suitable analogues in accordance 
with the invention are where Nle is at the 8 and 18 positions and Tyr at 
the 34 of an amidated 3-34 parathyroid (bovine or human) fragment, where 
Tyr is at the 34 position of an amidated 7-34 (bovine) fragment, the 
amidated 7-34 fragment of PTHrP (human), where Leu is at 11 and D-Trp is 
at 12 of an amidated 7-34 PTHrP (human) fragment, where Asn is at 10, Leu 
at 11, and D-Trp at 12 of the 7-34 PTHrP fragment, and where D-Trp is at 
12 and Tyr at 34 of an amidated 7-34 PTHrP (bovine) fragment. 
In accordance with the inventive method, the PTHrP antagonist is 
administered prophylactically or therapeutically (that is, before, 
simultaneously with, or after) infection has set in. For example, when 
administering prophylactically, one particularly considers patients at 
risk such as those suffering from severe thermal burns, receiving 
immunosuppressive therapy, undergoing extensive surgical procedures, organ 
transplantation, or suffering other serious injuries or disease. 
The PTHrP antagonist as therapeutic agent is administered to the patient by 
any suitable technique, preferably parenteral and, if desired 
intralesional. The specific method of administration will depend, e.g., on 
whether the administration is therapeutic or prophylactic. Thus, in view 
of the therapeutic urgency usually attending shock, the PTHrP antagonist 
may be intravenously infused at the same time as solutions used for 
initial volume expansion. Continuous infusion is preferred for 
administering peptides while bolus infusion may be used when administering 
antibodies. Prophylaxis is generally accomplished, e.g., by intramuscular 
or subcutaneous administration or other parenteral administration, 
including intraarterial and intraperitoneal administration, preferably 
intravenous or intraperitoneal. 
The PTHrP antagonist compositions to be used in the inventive therapy will 
be formulated and dosed in a fashion consistent with good medical practice 
taking into account the clinical condition of the individual patient, the 
cause of the septic shock, whether the PTHrP antagonist is used for 
therapy of shock or prophylaxis of incipient septic shock, the site of 
delivery of the PTHrP antagonist, the method of administration, the 
scheduling of administration, and other factors known to practitioners. 
The "effective amount" for purposes herein is thus determined by such 
considerations. 
As a general proposition, the total pharmaceutically effective amount of 
the PTHrP antagonist administered parenterally per dose will be in the 
range of approximately 1 .mu.g/kg to 10 mg/kg of patient body weight once 
per day, although, as noted above, this will be subject to a great deal of 
therapeutic discretion. As earlier noted, where the PTHrP antagonist is a 
peptide or an analog of a peptide, then administration is preferably by 
continuous infusion. The key factor is selecting an appropriate dose and 
scheduling is the result obtained. Relatively higher doses may be needed 
initially for the treatment of profound shock, i.e., for patients in acute 
renal failure or respiratory distress, or having severely depressed blood 
pressure (mean arterial pressure below about 60 mm Hg). 
For parental administration, the PTHrP antagonist is formulated generally 
by mixing it at the desired degree of purity, in a unit dosage injectable 
form (solution, suspension, or emulsion), with a physiologically 
acceptable carrier, i.e., one that is non-toxic to recipients at the 
dosages and concentrations employed. Preferably the carrier is a 
parenteral carrier. Examples of such carrier vehicles include water, 
saline, Ringer's solution, dextrose solution, and 5% human serum albumin. 
Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful 
herein, as well as liposomes. Generally, the carrier can contain minor 
amounts of additives such as substances that enhance isotonicity and 
chemical stability, e.g., buffers and preservatives, as well as low 
molecular weight (less than about 10 residues) polypeptides, proteins, 
amino acids, carbohydrates including glucose or dextrans, chelating agents 
such as EDTA, or other excipients. The PTHrP antagonist is typically 
formulated in such vehicles at a concentration of about 0.1 mg/ml to 100 
mg/ml at pH range 4 to 7. 
A PTHrP antagonist for use in therapeutic administration must be sterile. 
This is readily accomplished by sterile filtration through (0.2 micron) 
membranes. The PTHrP antagonist selected ordinarily will be stored as an 
aqueous solution or can be lyophilized. 
PTHrP antagonist therapy or prophylaxis is suitably combined (indeed, 
preferably combined) with other proposed or conventional therapies or 
prophylactic treatment for septic shock. For example, for treatment of 
burns, the PTHrP antagonist therapy may be delivered by separate means, 
simultaneously with and by the same administration route as other 
substances such as antibiotics, or anti-microbial agents that inhibit 
bacterial colonization of the burn wound surface. Other therapies that can 
be combined with PTHrP antagonist therapy include other cytokine 
antagonists, such as anti-TNF, antagonists of IL-1, and inhibitors of 
platelet activating factor. For example, Ohlsson et al., supra, have 
reported that a specific interleukin-1 receptor agonist reduces mortality 
from endotoxin shock while Doherty et al. and Heinzel, supra, discuss the 
use of anti-IFN-.gamma. to reduce mortality from endotoxic shock. Further, 
the inventive method can be practiced in conjunction with primary 
therapeutic agents, for example, potent anti-microbial agents such as 
aminoglycosides (such as amikacin, tobramycin netilmicin, and gentamicin), 
cephalosporin, related beta lactam agents such as moxalactam, carbopenems 
such as imipenem, monobactam agents such as aztreonam, ampicillin and 
broad-spectrum penicillins (e.g., penicillinase-resistant penicillins, 
ureidopenicillins, or anti-pseudomonal penicillins). 
It is known that the pathophysiologic consequences of Gram-negative sepsis 
are primarily mediated by the release of bacterial endotoxin. Thus, 
endotoxin treatment of mice is used as an experimental model of gram 
negative sepsis. For example, in U.S. Pat. No. 5,308,834, a group of rats 
was given a single, bolus injection of 0.5 mg/kg body weight bacterial 
endotoxin in studying the effects of leukocyte protein ("BPI"), with the 
results said to support the use of BPI to reduce mortality due to sepsis. 
Similarly, U.S. Pat. No. 5,055,447, issued Oct. 8, 1991, inventors 
Palladino et al., used mice as a model in endotoxin studies. 
We have also used the injection of a near-lethal dose of endotoxin as a 
model for septic shock (a state of profound hypotension and multi-organ 
failure resulting from a systemic inflammatory response to overwhelming 
infection by Gram-negative or other bacteria) in mice and rats as models 
of sepsis. 
Thus, mice were passively immunized with antibody generated against the 
1-34 fragment of PTHrP, which is a peptide fragment that is active at the 
PTH/PTHrP receptor, prior to the administration of a lethal dose of 
endotoxin. Anti-PTHrP antibodies were raised both in goats and in rabbits 
by immunizing the animals with synthetic human PTHrP. 
The immunoglobulin fraction of immune sera (PTHrP antibody) or naive sera 
(control antibody) was partially purified by ammonium sulfate 
precipitation (33% saturation) using previously described techniques to 
avoid the introduction of endotoxin contamination. The endotoxin content 
of PTHrP and control antibody solutions, determined by Limulus assay 
(sensitivity, 10 pg/ml), was below the levels required to alter 
sensitivity to subsequent (6 h) LPS challenge in mice. 
Administration of goat PTHrP antibody 6 h prior to the injection of the 
endotoxin ("LPS") significantly protected mice from death when compared to 
the mortality rate (LD.sub.90) seen in mice treated with goat control 
antibody (p&lt;0.03 by log rank analysis of Kaplan Meier curves) (FIG. 1). 
The protective effect of PTHrP antibody was confirmed in other studies 
utilizing antiserum raised in a different species (rabbit) against the 
same antigen. Passive immunization of mice with rabbit PTHrP antibody 
similarly protected mice from death caused by administration of LPS when 
compared to control mice treated with rabbit control antibody (p&lt;0.004 by 
log rank test of Kaplan Meier survival curves) (FIG. 2). The degree of 
protection from LPS lethality seen here with passive immunization against 
PTHrP is similar to that reported for passive immunization of mice against 
TNF, a major mediator of sepsis. 
Comparison of survival curves for PTHrP antibody- versus control 
antibody-treated groups showed that PTHrP antibody provided a significant 
protective effect from LPS-induced death over the 3 to 4 day course of the 
experiments (FIGS. 1 and 2), although the relative survival rates at the 
end of the observation periods were no different (as determined by 
Fisher's exact test). Thus, the PTHrP antibodies administered delayed but, 
ultimately, did not prevent death. Compilation of data from multiple 
experiments (n=90 mice) examining the ability of rabbit PTHrP antibody to 
protect from LPS-induced death confirms this conclusion. While overall 
survival was improved by pretreatment with rabbit PTHrP antibody 
(p&lt;0.00005 by log rank analysis), comparison of lethality rates at 12 h 
intervals following LPS administration only showed a significant 
protective effect during the first 48 hours following LPS administration 
(FIG. 3). 
In summary, these data show that PTHrP is effective in mediating at least 
some of the toxic effects of endotoxin, as evidenced by the ability of 
antibody directed against PTHrP to delay LPS-induced death. 
Antibody blockade experiments, similar to those presented here, have 
previously shown that numerous other cytokines, such as TNF, IL-1, 
macrophage inhibitory factor (MIF) and IFN-.gamma., are also mediators of 
the lethal effects of endotoxin. The fact that passive immunization 
against PTHrP delays, but does not ultimately prevent, lethality from LPS 
is consistent with the hypothesis that PTHrP is one member of a larger 
cascade of pro-inflammatory cytokines that is unleashed during the host 
response to infection, and therapy in accordance with the invention will 
preferably include one or more approaches to block the cascade. 
Turning to FIG. 1 and FIG. 2, the data shows the effect of goat (FIG. 1) or 
rabbit (FIG. 2) antiserum directed against PTHrP on LPS-induced lethality 
in mice. The details of the data are summarized by FIGS. 1-3 are given by 
Examples 1-3. 
EXAMPLE 1 
Male C57BL/6 5-6 week old mice (n=9/group) were injected intraperitoneally 
(ip) with 200 .mu.l ammonium sulfate-precipitated goat antisera (PTHrP 
antibody, open triangle) (titre 1:8,000 by ELISA; LPS, undetectable) or 
ammonium sulfate-precipitated naive goat sera (control antibody, open 
circle) (LPS, undetectable) 6 h prior to the administration of 700 .mu.g 
055:B5 LPS diluted in apyrogenic 0.9% saline. Animals, given access to 
chow and water ad libitum, were monitored for lethality for 96 h after LPS 
treatment. Statistical analysis of the Kaplan Meier survival curves for 
the two groups (Statistica 4.1, StatSoft) using either a log rank test 
which gives equal weight to all points or the Peto & Peto Wilcoxon Test 
which weights earlier time points more heavily showed that pretreatment 
with PTHrP antibody protected mice from LPS-induced lethality (p&lt;0.030 and 
p&lt;0.031, respectively). 
EXAMPLE 2 
Mice (n=8/group) were injected with 100 .mu.l ammonium sulfate precipitated 
rabbit antisera (open triangle) (titre 1:16,000; 6-fold concentrated by 
volume; 100 pg LPS/100 .mu.l ) or ammonium sulfate-precipitated naive 
rabbit serum (open circle) (5-fold concentrated by volume; 165 pg LPS/100 
.mu.l ) 6 h prior to the administration of 700 .mu.g LPS ip. Animals were 
then monitored as in (A). Statistical analysis of the Kaplan Meier 
survival curves for the two groups showed that pretreatment with PTHrP 
antibody protected mice from LPS-induced death (p&lt;0.004 by log rank test; 
p&lt;0.003 by Peto & Peto Wilcoxon Test). Results are representative of 5 
separate experiments. 
EXAMPLE 3 
Turning to FIG. 3, the data shows the effect of rabbit antisera directed 
against PTHrP on LPS-induced lethality. Lethality data from 5 separate 
experiments, performed as described in FIGS. 1 and 2, were combined for 
analysis. For a given experiment, C57BL/6 mice received an equal volume 
(100-500 .mu.l) of PTHrP or control rabbit antibody. Since PTHrP antibody 
was ammonium sulfate-precipitated from different rabbit bleeds for 
different experiments, the volume of ammonium sulfate precipitated-PTHrP 
antibody used was adjusted so that an equivalent titer of PTHrP antibody 
was administered in all experiments (e.g. 100 .mu.l of 1:16,000; 300 .mu.l 
of 1:5,500; or 500 .mu.l of 1:3,200). Control and PTHrP antibody solutions 
were matched for fold-concentration (4-6-fold concentrated relative to 
serum) and contained less than 165 pg LPS/volume injected. Six hours after 
antibody administration, mice from both treatment groups were injected 
with an equal amount of LPS (650-800 .mu.g LPS, corresponding to an 
LD.gtoreq.80 at t=48 h in control-antibody treated mice). Statistical 
analysis of Kaplan Meier survival curves (not shown here) for PTHrP (n=43) 
and control (n=47) antibody-treated groups showed that pretreatment with 
PTHrP antibody protected mice from LPS-induced death (p&lt;0.00005 by log 
rank analysis and p&lt;0.000001 by Peto & Peto Wilcoxon test). Comparison of 
lethality at 12 h intervals after LPS administration (from t=0-96 h) using 
Fisher's exact test (Instat 2.01, GraphPad Software, San Diego, Calif.) 
showed that PTHrP antibody significantly protected animals from 
LPS-induced lethality when compared to control antibody-treated mice at 
the early time points shown. 
It is to be understood that while the invention has been described above in 
conjunction with preferred specific embodiments, the description and 
examples are intended to illustrate and not limit the scope of the 
invention, which is defined by the scope of the appended claims.