Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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METHODS FOR THE PREVENTION AND TREATMENT OF DISEASES CAUSED BY AN INFLAMMATORY
RESPONSE MEDIATED BY ENDOGENOUS SUBSTANCE P BY USING ANTI-SUBSTANCE P
ANTIBODIES
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
The present invention relates to methods for preventing and/or treating
diseases caused by an inflammatory response mediated by endogenous substance
P.
l0 The methods comprise the administration to a subject of a pharmaceutically-
effective
amount of anti-substance P antibodies, or anti-substance P antibody fragments,
thereby inhibiting the activity of endogenous substance P. Such inhibition
reduces the
levels of cytokines produced by T lymphocytes, alters the signals which direct
the
inflammatory response to the infection, and reduces cytokine-induced
inflammation.
I5 The present invention especially relates to methods for preventing and
treating
diseases caused by an inflammatory response to viral or bacterial infections.
Moreover, the present invention especially relates to methods for preventing
and
treating diseases caused by an inflammatory response to respiratory syncytial
virus.
2o BACKGROUND
Inflammation. Inflammation is a localized, protective response of the
immune system which is produced to protect a subject from disease. It is
elicited by
destruction of tissues, foreign substances such as allergens, infectious
agents and cells
such as viruses and bacteria. The inflammatory response destroys, dilutes or
25 sequesters both the injurious agent and the injured tissue. It is
characterized in the
acute form by the classical signs of pain, heat, redness, swelling and loss of
function.
Histologically, it involves a complex series of events, including dilatation
of
arterioles, capillaries and venules, with increased permeability and blood
flow,
exudation of fluids, including plasma proteins, and leukocytic migration into
the
3o inflammatory focus.
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T lymphocytes, which are products of lymphoid tissue, and their soluble
protein products, known as cytokines, participate in humoral and cell-mediated
immunity, and mediate the inflammatory response. T lymphocytes can suppress or
assist the stimulation of antibody production in the presence of antigen, and
can kill
tumor and transplant tissue cells. Cytokines made by T lymphocytes, such as
the
intracellular cytokines interleukin-2 (IL-2), interleukin-4 (IL-4),
interleukin-6 (IL-6)
and interferon-y (IFN~), function to augment or suppress the immune response,
or to
change the type of immune response.
The immune-system-mediated inflammatory response, however, can also
1 o cause, or contribute to, disease, rather than prevent it. Some diseases,
such as those
caused by infection by respiratory syncytial virus appear to be caused in part
by the
inflammatory response. Small molecule anti-inflammatory agents which are
currently
employed to treat inflammation, disadvantageously have adverse side effects,
such as
gastrointestinal discomfort and decreased blood clotting efficiency. Steroid-
based
anti-inflammatory drugs also have adverse side effects, such as reduced
adrenal gland
function and generalized immune system inhibition.
Substance P. Substance P is a naturally-occurring, endogenously-released,
undecapeptide which has multiple sites of action as a major pro-inflammatory
neuromediator or neuromodulator, both in the central and peripheral nervous
systems.
Substance P is known to have regulatory effects upon several cell types
present in the
immune system, and to regulate the inflammatory response to foreign substances
or
pathogens by altering the expression of proinflammatory cytokines, which have
been
implicated in the pathogenesis of different neuropathologies. High levels of
substance
P have been implicated in the pathogenesis of diseases associated with
inflammation.
Substance P is also known to modulate the activity of cells affiliated with
respiratory
inflammation (neutrophils, mast cells and alveolar macrophages). Elevated
concentrations of substance P and cytokines have been associated in the
pathogenesis
of a wide variety of diseases, including arthritis, experimental allergic
encephalomyelitis, altered immunoregulation during human immunodeficiency
virus
3o infection, irritable bowel syndrome and airway hyperactivity.
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Substance P has the following amino acid sequence (SEQ ID NO: l in the
Sequence Listing):
H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NHZ ,
and belongs to a family of closely-related peptides known as the tachykinins
(neurokinins), along with two other related, endogenously-released peptides,
neurokinin A (NKA) and neurokinin B (NKB). Neurokinins are a group of
structurally-related peptides characterized by a similar C-terminal
pentapeptide that
1 o interacts with different receptor subtypes. The amidated C-terminal
portion of the
substance P neuropeptide is responsible for receptor binding (Otsuka et al.,
"Neurotransmitter Functions of Mammalian-Tachykinins," Physiological Reviews
73:229-307 (1993)). Substance P and neurokinin A are widely distributed in the
airways and lungs of several species, including humans and guinea pigs.
15 Substance P is known to participate in inflammatory processes, and in the
immune response, and appears to be involved in the process of neurogenic
inflammation, pain transmission, regulation of blood pressure, inflammation
processes
and endocrine and exocrine secretion. Substance P has been strongly implicated
in
the transmission of pain, being coreleased alongside other transmitters and
peptides,
2o from small-diameter primary afferents. The primary role of substance P in
neurogenic
inflammation, smooth muscle contraction, and vasodilation is well established.
In the
central nervous system, where substance P and its receptors are widely
distributed, its
functional roles are less clear. Given parenterally, substance P and
neurokinin A
induce a variety of responses, including contraction of bronchial smooth
muscle,
25 mucus secretion, vasodilation, extravasation of plasma proteins and
recruitment of
inflammatory cells. The principal biological activity of these tachykinins
resides in
their structurally similar carboxyl sequence, ...Phe-X-Gly-Leu-Met-COOH (SEQ
ID
NO. 2). Several binding experiments have shown that intestinal and vascular
substance P receptors bind to the C-terminal pentapeptide of substance P. It
has been
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suggested that the release of endogenous substance P and neurokinin A from
pulmonary afferent C-fibers contributes to bronchial obstruction in asthma.
Specific membrane receptors for substance P have been found in neurons, and
in muscular, glandular and immunocompetent cells, which helps to explain the
s diversity and importance of the physiological roles of substance P.
Substance P
effects its biological activity by ligation with these receptors, and the
binding of
substance P to its receptors enhances the incorporation of phosphate or
inositol into
phosphatidylinositol. Substance P, neurokinin A and neurokinin B appear to
exert
their biological effects primarily through interactions with three distinct
receptors of
the neurokinin family, named neurokinin receptor-1 (NK 1 ), neurokinin
receptor-2
(NK2) and neurokinin receptor-3 (NK3), respectively. Substance P affects
antibody
synthesis, or promotes cytokine production, through NK1 receptors which are
well-
characterized on T- and B-lymphocytes, monocytes and astrocytes, and possibly
also
via nonneurokinin receptors. While substance P preferentially binds to the NK1
receptor, its binding is not limited to this receptor. Further, the cationic,
amphiphilic
nature of the substance P molecule allows it to bypass the NK1 receptor and
insert
itself into the cell membrane, where it can directly interact with proteins.
Thus, any
inflammation-reduction therapy which targets the NK1 receptors (i.e., the use
of
compound or antibody NK1 receptor antagonists) will not fully inhibit the
substance P
2o pathway of inflammation, or be effective in preventing the pro-inflammatory
actions
of substance P.
The functional roles of substance P in the mammalian central nervous system
and peripheral nervous system are currently being investigated by a variety of
experimental approaches, including the use of antibodies and pharmacological
agonists and antagonists. However, a continuous delivery of pharmaceutical
drugs to
the nervous tissue poses several practical problems, including limited
diffusion and
half life of the drugs, secondary, non-specific effects of the drugs (because
the
endogenous neurokinins are preferential, but not selective agonists) and side-
effects of
the drugs. While studies have been performed with pharmaceutical antagonists
of
3o substance P receptors, studies in this area have been limited by the lack
of efficacious
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antagonists specific for the NK1 receptors, and by the difficulty of using
these
antagonists in vivo, due to short half lives, and the resulting lack of
effect, and non-
specific effects. Thus, the use of antibodies and antibody fragments which
bind
directly with substance P, as is done in the methods of the present invention,
rather
than using substance P receptor antagonists, is believed to be a preferable
approach
for inhibiting the action of endogenous substance P and, consequently for
reducing the
levels of cytokine production and cytokine-induced inflammation.
Additional information concerning substance P is present in the following
publications: U.S. Patent No. 4,680,283; Mantyh, "Substance P and the
Inflammatory
1 o and Immune Response," Annals of the New York Academy of Sciences 632:263-
271
( 1991 ); Ho et al., "Substance P Modulates Human Immunodeficiency Virus
Replication in Human Peripheral Blood Monocyte-Derived Macrophages," AIDS
Research & Human Retroviruses 12:195 (1996); Kudlacz et al., "Parainfluenza
Virus
Type 3 Induced Alterations in Tachykinin NK1 Receptors, Substance P Levels and
Respiratory Functions in Guinea Pig Airways," European Journal of Pharmacology
270:291 (1994); Yamawaki et al., "Viral Infection Potentiates the Increase in
Airway
Blood Flow Produced by Substance P," Journal ofApplied Physiology 79:398
(1995);
Ben-Jebria et al., "Effect of Passive Sensitization on the Mechanical Activity
of
Human Isolated Bronchial Smooth Muscle Induced by Substance P, Neurokinin A
and
2o VIP," British Journal of Pharmacology 109:131 ( 1993); Boichot et al.,
"Inhaled
substance P Induces Activation of Alveolar Macrophages and Increases Airway
Responses in the Guinea-Pig," Neuropeptides 25:307 (1993); Cheung et al.,
"Effects
of Inhaled Substance P on Airway Responsiveness to Methacholine in Asthmatic
Subjects In Vivo," Journal ofApplied Physiology 77:1325 (1994); Heaney et al.,
"Substance P induces Histamine Release from Human Pulmonary Mast Cells,"
Clinical & Experimental Allergy 25:179 (1995); Murris-Espin et al., "Substance
P and
Alveolar Macrophages: Effects on Oxidative Metabolism and Eicosanoid
Production,"
Allergy 50:334 (1995); Tomaki et al., "Elevated Substance P Content in Induced
Sputum from Patients with Asthma and Patients with Chronic Bronchitis,"
American
Journal ofRespiratory & Critical Care Medicine 151:613 (1995); Yoshihara et
al.,
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"Involvement of Substance P in the Paroxysmal Cough of Pertussis," Regulatory
Peptides 46:238 (1993); Ahmed et al., "Capsaicin Effects on Substance P and
CGRP
in Rat Adjuvant Arthritis," Regulatory Peptides 55:85 (1995); Jarrah et al.,
"Cholecystokinin-Octapeptide (CCK-OP) and Substance P (SP) Influence Immune
Response to Cholera Toxin in Live Animals," Advances in Experimental Medicine
&
Biology 371A:563 (1995); Kincy-Cain et al., "Substance P-Induced IL-12
Production
by Murine Macrophages," Journal oflmmunology 158:2334 (1997); Palma et al.,
"Interleukin-6 Production by U373 MG, a Human Astrocytoma Cell Line; Different
Pathways Involved in Substance P and Lipopolysaccharide Activation," Journal
of
to Immunology 59:155 (1995); and Shadiack et al., "Lipopolysaccharide Induces
Substance P in Sympathetic Ganglia Via Ganglionic Interleukin-1 Production,"
Journal of Neuroimmunology 49:51 ( 1994).
Respiratory Syncytial Virus . Respiratory syncytial virus is one example of
an agent which causes an infection which, in turn, often results in a disease
produced
15 by an inflammatory response to the infection that may be mediated by
endogenous
substance P. The immune response to respiratory syncytial virus infection is
characterized by the enhanced production of cytokines, increased levels of
substance
P, and bronchial inflammation. Respiratory syncytial virus is the most
important
cause of pneumonia and bronchiolitis in infants, and can result in death.
2o The only available treatment for respiratory syncytial virus is ribavirin,
a
purine nucleoside analog which inhibits the replication of a wide range of RNA
and
DNA viruses, and which has many disadvantages. The administration of ribavirin
to
patients in the form of an aerosol is costly and difficult, and limits its
therapeutic
efficacy. Furthermore, ribavirin antagonizes the activity of the antiviral
agent
25 zidovudine against human immunodeficiency virus type 1 (HIV-1), the
causative
agent of AIDS. When administered orally or intravenously, ribavirin causes
anemia
due to extravascular hemolysis and suppression of the bone marrow. Further,
ribavirin is mutagenic in small animals, and the oral bioavailability of
ribavirin is only
about 4~ percent. The long-term oral therapy of ribavirin is associated with
both
30 gastrointestinal and adverse symptoms in the central nervous system.
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A need presently exists for an efficacious and convenient method for treating
and preventing diseases which are caused by an inflammatory response mediated
by
substance P. Accordingly, the present invention provides an effective and
convenient
method for preventing and treating diseases caused by an inflammatory response
to a
variety of conditions that is mediated by substance P comprising inhibiting
the
activity of endogenous substance P with anti-substance P antibodies and/or
anti-
substance P antibody fragments. The data presented in the examples set forth
below
show that the inhibition of the biological activity of substance P with anti-
substance P
F(ab)2 antibody fragments reduces the production of cytokines by T
lymphocytes,
thereby altering the signals which direct the inflammatory response to an
infection
caused by respiratory syncytial virus, and reducing potential cytokine-induced
inflammation.
In accordance with the methods of the present invention, blocking the activity
of endogenous substance P can potentially be employed to prevent or treat a
wide
variety of diseases or syndromes caused in whole or part by an inflammatory
response
mediated by substance P. Such diseases or syndromes include, by way of
nonlimiting
example, diseases ascribable to viral or bacterial infection such as
infections
associated with inflammatory bowel disease, virus-mediated bronchiolitis
including
that mediated by respiratory syncytial virus, bacterial colitis, inflammation
associated
2o with chlamydial diseases, lung injury associated with staphylococcal
enterotoxin B,
inflammation due to cytomegalovirus or hepatitis B virus, and sepsis, allergic
diseases
such as asthma, autoimmune diseases such as rheumatoid arthritis,
pancreatitis, and
inflammation associated with multiple sclerosis, and rejection of allografts
and other
transplanted tissues or organs. The prevention, reduction or elimination of
the
substance P-mediated inflammatory response can also be used to prevent adverse
effects which may otherwise result from tissue or organ transplantation.
The administration of anti-substance P antibodies, or anti-substance P
antibody fragments, to a subject to inhibit the activity of endogenous
substance P is
preferential to the administration of substance P NK1 receptor antagonists to
the
3o subject because, unlike substance P NK1 receptor antagonists, anti-
substance P
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antibodies and anti-substance P antibody fragments inhibit the activity of
substance P
without receptor ligation, and before the substance P can cross the cell
membrane in a
receptor-independent manner.
DESCRIPTION OF THE RELATED ART
Cytokine Networks, Inc. and IDEC Pharmaceuticals are apparently developing
products based on anti-macrophage inhibitory factor antibody technology. The
inhibition of the cytokine macrophage inhibitory factor may represent a novel
approach to the management of a variety of acute and chronic inflammatory
diseases
to (Printout of company press release dated September 9, 1997, provided and
copyrighted by Business Wire Health Wire as downloaded from
http://biz.yahoo.com/bw/97/09/09/idph~000_1.html).
U.S. Patent No. 5,688,806 describes spirocyclic piperidine derivatives which
are stated to be substance P receptor antagonists. Such compounds were used in
the
treatment and prevention of inflammatory and central nervous system disorders.
Jafarian et al., "Passive Immunization with an Anti-Substance P Antibody
Prevents Substance P- and Neurokinin A-Induced Bronchospasm in Anesthetized
Guinea Pigs," Life Sciences 57:143-153 (1995), describe experiments to
determine
whether the intravenous administration of a monoclonal IgG-type anti-substance
P
2o antibody derived from the rat-mouse hybridoma NC1/34 to guinea pigs would
alter
pulmonary responses to endogenous substance P and neurokinin A. The passive
immunization experiments were stated to confirm earlier reports which
indicated that,
in a guinea-pig model of asthma, active immunization against substance P
reduced the
bronchiospastic response to substance P, and to show that passive immunization
of
guinea pigs with a monoclonal anti-substance P antibody prevents the
bronchiospastic
effects of exogenous substance P and neurokinin A in vivo.
Piccioli et al., "Neuroantibodies: Ectopic Expression of A Recombinant Anti-
Substance P Antibody in the Central Nervous System of Transgenic Mice," Neuron
15:373-384 (1995), describe the generation of transgenic mice that express
3o recombinant antibodies against substance P under the control of the
promoter of the
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neuronal vgf gene. The anti-substance P transgenic antibodies were stated to
be
effective in competing with endogenous substance P, as demonstrated by a
marked
inhibition of neurogenic inflammation by the antibodies in mice after
neurogenic
inflammation in the mice was produced by the application of mustard oil to the
skin of
the mice.
Swenberg et al., "Development of an Anti-Idiotypic Antibody that Blocks
Substance P Primary Antibodies and Substance P Membrane Binding," Brain
Research 417:131-138 (1987), describe the preparation of antibodies raised
against
substance P coupled to bovine albumin, and of substance P anti-idiotypic
antibodies
l0 (antibodies raised against anti-substance P antibodies). This reference
also describes
the effects of substance P anti-idiotypic antibodies on substance P binding to
both
anti-substance P primary antibodies, and the substance P receptor in membrane
preparations from rat duodenum, and the biological effect on substance P-
induced
spasmogemc responses.
Couraud et al., "Anti-substance P Anti-Idiotypic Antibodies, Characterization
and Biological Activities," Journal of Biological Chemistry 260:9461-9469 (
1985),
describe the production of anti-substance P antibodies, and the production of
anti-
idiotypic antibodies which bind with substance P receptors, and which either
mimic or
block the physiological actions of substance P, depending upon the biological
2o preparation.
Maillet et al., "Anti-substance P Anti-idiotypic Antibodies Modulate the
Secretory Process in the Rat Parotid Gland in Vitro," European Journal of
Pharmacology 187:357-367 (1990), describe the preparation of anti-idiotypic
antibodies from rabbits in response to immunization with polyclonal anti-
substance P
antibodies, and the comparison of the effects of both anti-idiotypic
antibodies and
substance P on the rat parotid gland (a tissue in which substance P receptors
have been
well established).
None of the references cited above teaches or suggests the prevention or
treatment of diseases caused by an inflammatory response to a viral or
bacterial agent
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mediated by endogenous substance P by the administration of anti-substance P
antibodies, or anti-substance P antibody fragments, to subjects.
SUMMARY OF THE INVENTION
The present invention provides effective and convenient methods for
preventing or treating diseases in a subject caused by an inflammatory
response
mediated by endogenous substance P, especially a response to a viral or
bacterial
infection. The methods comprise administering to a subject a pharmaceutically-
effective amount of anti-substance P antibodies, or anti-substance P antibody
1o fragments, thereby inhibiting the activity of endogenous substance P in the
subject.
By inhibiting the activity of endogenous substance P in the subject, the
levels of
cytokines produced by T lymphocytes in the subject are reduced, and the
signals
which direct the inflammatory response to the viral or bacterial infection
become
altered, thereby reducing cytokine-induced inflammation. Generally, from about
0.5
15 mg to about 2 g of anti-substance P antibodies, or anti-substance P
antibody
fragments, per kilogram of body weight per day are administered to a mammalian
subject, with from about 1 mg to about 1 g of anti-substance P antibodies, or
anti-
substance P antibody fragments, per kilogram of body weight per day being
preferred.
The methods of the present invention are particularly useful for preventing
and
20 treating diseases caused by an inflammatory response to an infection by
respiratory
syncytial virus .
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph for the amount of substance P present in bronchoalveolar
25 lavage fluid obtained from the lungs of BALB/c mice which had been infected
with
respiratory syncytial virus versus the number of days post-infection. This
graph is
derived from the data from Example 1.
FIG. 2 shows a graph which plots the stimulation index in mean counts per
minute (cpm) of stimulated cells over the mean counts per minute of
unstimulated
3o cultures for intact, CD4+ and CD8+T lymphocytes which were stimulated with
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concanavalin, with or without the addition of various concentrations of
substance P
(in concentrations ranging from 10'5 M to 10'9 M), or which were left
unstimulated in
tissue culture medium, on the vertical axis, and the type of treatment on the
horizontal
axis, for the experiments described in Example 2. In FIG. 2, TCM indicates
cells
which were left unstimulated in tissue culture medium, CA indicates
stimulation with
concanavalin, open bars indicate intact T lymphocytes, black/white bars
indicate
CD4+ T lymphocytes, and solid bars indicate CD8+ T lymphocytes.
FIG. 3 shows a graph which plots the stimulation index of intact, CD4+ and
CD8+ T lymphocytes which were stimulated with lipopolysaccharide, with or
without
1 o the addition of various concentrations of substance P (in concentrations
ranging from
10'5 M to 10'9 M), or were left unstimulated in tissue culture medium, on the
vertical
axis, and the type of treatment on the horizontal axis, for the experiments
described in
Example 2. In FIG. 3, TCM indicates cells which were left unstimulated in
tissue
culture medium, LPS indicates stimulation with lipopolysaccharide, open bars
indicate intact T lymphocytes, black/white bars indicate CD4+ T lymphocytes,
and
solid bars indicate CD8+ T lymphocytes.
FIG. 4 shows a graph which plots the percent of intracellular (IC) cytokines
produced by T lymphocytes in the bronchoalveolar lavage (BAL) fluid of BALB/c
mice infected with respiratory syncytial virus collected 18 hours after the
mice were
2o treated with rabbit anti-substance P F(ab)z antibody fragments on the
vertical axis, and
the type of intracellular cytokine examined (CD3+ /IL-2, CD3+ /IL-4, CD3+ /IL-
5,
CD3+/IL-6 and CD3+/IFNy) on the horizontal axis. This graph is based on the
experiments described in Example 3. In FIG. 4, BAL indicates bronchoalveolar
lavage fluid, diagonally stripped bars indicate 2 ~g of anti-substance P
antibody
fragment, open bars indicate 20 p,g of anti-substance P antibody fragment,
open with
black horizontal cross-hatching bars indicate 200 ~g of anti-substance P
antibody
fragment, solid bars indicate treatment with 200 ~,g of normal rabbit Ig (nIg)
antibody,
and solid with white horizontal cross-hatching bars indicate no treatment
(SHAM).
FIG. 5 shows a graph which plots the percent of intracellular cytokines
3o produced by T lymphocytes in the bronchoalveolar lavage fluid of BALB/c
mice
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infected with respiratory syncytial virus collected 36 hours after the mice
were treated
with rabbit anti-substance P F(ab)z antibody fragments on the vertical axis,
and the
type of intracellular cytokine examined on the horizontal axis, for the
experiments
described in Example 3. The abbreviations and symbols appearing in FIG. 5 are
defined in the same manner as for FIG. 4.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Definitions. For purposes of clarity, terms and phrases used herein are
defined in the manner set forth directly below.
1 o As used herein, "anti-substance P antibody" means any polyclonal or
monoclonal antibody which inhibits the action of substance P. Such antibodies
may
be obtained from any species. They include, for example, monoclonal anti-
substance
P antibodies obtained from a mouse B cell hybridoma. In general, any class and
subclass of antibody is effective in the present invention.
1 s As used herein, "anti-substance P antibody fragment" means a portion of an
anti-substance P antibody, as defined hereinabove, which is of a sufficient
size and
conformation to inhibit the action of substance P, and includes F(ab)Z
fragments of the
antibody, wherein Fab represents the antigen binding Fragment. F(ab)z
fragments are
obtained by digestion of an immunoglobulin with pepsin. Fab fragments,
conversely,
2o are obtained by digestion of an immunoglobulin with papain. Both F(ab)2
fragments
and Fab fragments may be used to inhibit substance P. An advantage of using
Fab
and (Fab)~ fragments is that they do not have the Fc fragment of the
immunoglobulin
molecule, and therefore will not bind to the Fc receptor present on some
cells.
As used herein, the term "bind" means the well-understood interaction or other
25 nonrandom association between antibodies, or antibody fragments, and
antigens.
As used herein, the abbreviation "i.n." means that anti-substance P
antibodies,
or anti-substance P antibody fragments, were administered intranasally. As
used
herein, the abbreviation "i.p." means that anti-substance P antibodies. or
anti-
substance P antibody fragments, were administered intraperitoneally. As used
herein,
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the abbreviation "i.v." means that anti-substance P antibodies, or anti-
substance P
antibody fragments, were administered intravenously.
The phrases "parenteral administration" and "administered parenterally" as
used herein mean modes of administration other than enteral administration,
usually
by injection, and includes, without limitation, intraarterial,
intraarticulare,
intracapsular, intracardiac, intradermal, intramuscular, intraorbital,
intraperitoneal,
intraspinal, intrasternal, intrathecal, intravenous, subcutaneous,
subcuticular,
subarachnoid, subcapsular, and transtracheal injection and infusion.
As used herein, the phrase "inflammation-related diseases" means a disease or
Io disorder which is caused in part or entirely by an inflammatory response in
a subject
due to a variety of diseases or syndromes, wherein the inflammatory response
may be
produced in part or entirely by the activity of substance P, and wherein the
disease or
disorder may be prevented or treated by inhibiting in part of whole the
activity of
substance P. Examples of viral or bacterial pathogenic agents, or conditions,
that
15 induce an inflammatory response that is the subject of this invention
include, by way
of nonlimiting example, respiratory syncytial virus, cytomegalovirus-
associated
allograft rejection, hepatitis B virus and chronic hepatitis, viruses that
induce T cell
activation, staphylococcal enterotoxin B, bacterial superantigens, bacterial
endotoxin,
bacterial sepsis, cardiopulmonary bypass, and multiple sclerosis lesions.
20 The phrase "pharmaceutically acceptable" is employed herein to refer to
those
compounds, materials, compositions, and/or dosage forms which are, within the
scope
of sound medical judgement, suitable for use in contact with the fluids and
tissues of
subjects without excessive toxicity, irritation, allergic response, or other
problem,
complication, or undesirable biological effect, and without interacting in a
deleterious
25 manner with other components which may be present in a pharmaceutical
composition containing such components, materials, compositions and/or dosage
forms commensurate with a reasonable benefit/risk ratio.
The phrase "pharmaceutical carrier" as used herein means a pharmaceutically-
acceptable material, composition or vehicle, such as a liquid or solid filler,
diluent,
3o excipient, solvent or encapsulating material, involved in carrying or
transporting an
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active agent, such as anti-substance P antibodies, or anti-substance P
antibody
fragments, from one organ, or portion of a subject's body, to another organ,
or portion
of a subject's body. Some examples of materials which can serve as
pharmaceutical
carriers include: ( 1 ) sugars, such as lactose, glucose and sucrose; (2)
starches, such as
corn starch and potato starch; (3) cellulose, and its derivatives, such as
sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered
tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa
butter and
suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower
oil, sesame
oil, olive oil, corn oil, and soybean oil; ( 10) glycols, such as propylene
glycol; ( 11 )
polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12)
esters,
such as ethyl oleate and ethyl laurate; ( 13) agar; ( 14) buffering agents,
such as
magnesium hydroxide and aluminum hydroxide; ( 15) alginic acid; ( 16) pyrogen-
free
water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)
phosphate
buffer solutions; and (21 ) other non-toxic compatible substances employed in
pharmaceutical formulations.
As used herein, the term "purified" in relation to anti-substance P antibodies
and anti-substance P antibody fragments means that the anti-substance P
antibodies
and anti-substance P antibody fragments are of sufficient purity so that they
may be
employed, and will function properly, in the methods of the present invention,
as well
2o as in clinical, diagnostic, experimental or other procedures. Many
procedures are
known by those of ordinary skill in the art for purifying antibodies and
antibody
fragments prior to their use in other procedures. The term "purified" as used
herein in
relation to substance P means that the substance P is of sufficient purity so
that it may
bind with antibodies, or antibody fragments, which have the ability to bind
with
substance P.
As used herein, the term "subject" includes humans and animals, particularly
mammals.
The phrase "pharmaceutically-effective amount" as used herein means that
amount of anti-substance P antibodies, or anti-substance P antibody fragments,
which
is effective for inhibiting the activity of at least some endogenous substance
P, and
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which is effective for preventing an inflammation-related disease, as defined
hereinabove, for treating an inflammation-related disease, as defined
hereinabove (i.e.,
for eliminating the disease from the subject, or for reducing the effects of
the disease
in the subject), or for producing some other desired therapeutic effect in a
subject, at a
reasonable benefit/risk ratio applicable to any medical treatment.
Antibodies and Antibody Fragments. The anti-substance P antibodies, and
anti-substance P antibody fragments, may be employed in the methods of the
invention in pharmaceutically-effective amounts to prevent, or treat,
inflammation-
related diseases in a subject. The anti-substance P antibodies, and anti-
substance P
1 o antibody fragments, which may be employed in the methods of the present
invention,
have the ability to inhibit the action of endogenous substance P, for example,
by
binding to an epitope present on substance P and blocking substance P binding
to
cellular receptors for substance P, thereby preventing substance P from
binding to
such receptors, and exhibiting biological activity (i.e., neutralization of
the substance
P peptide by the antibodies, or antibody fragments).
The antibodies which may be employed in the methods of the invention may
be polyclonal or monoclonal, and the antibody fragments which may be employed
in
these methods may be derived from polyclonal or monoclonal antibodies.
Polyclonal
and monoclonal antibodies may be prepared by methods which are well-known by
2o those of skill in the art. See, for example, Harlow and Lane, Antibodies; A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
(1988). For the production of polyclonal antibodies, purified substance P can
be
injected into an animal in an amount, and in intervals, sufficient to elicit
an immune
response (i.e., the production of antibodies against the substance P). Such
polyclonal
antibodies can be obtained from the animal by standard methods, and purified
directly
by well-known methods.
Monoclonal antibodies are generally preferred, however, due to their highly
specific nature for a particular epitope on an antigen. For.the production of
monoclonal antibodies, spleen cells can be removed from an animal which has
been
3o injected several times over a period of time with an amount of substance P
which is
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sufficient to elicit an immune response in the animal, fused with an immortal
cell line
(rapidly-dividing myeloma cells), placed into wells, grown, and screened for
the
production of monoclonal antibodies to a particular epitope present on
substance P.
For additional information concerning the preparation of monoclonal antibodies
against substance P, see Couraud et al., "Monoclonal Antibodies to Substance
P;
Production, Characterization of their Fine Specificities, and Use in
Immunocytochemistry," Journal ofNeurochemistry 49:1708 (1987).
Because substance P and neurokinin A have a structurally similar carboxyl
sequence, in order to increase the specificity of antibodies generated against
substance
I o P (the ability of the antibodies to bind to substance P, but not to
neurokinin A), and
antibody fragments thereof, it is preferable to generate the antibodies
against the N-
terminal portion of the substance P amino acid sequence, rather than against
the C-
terminal portion of the sequence. It is preferable that the antibodies, and
antibody
fragments, employed in the methods of the invention do not cross react with
the
related peptides neurokinin A and neurokinin B, so that their binding occurs
with
epitopes present on endogenous or exogenous substance P, and not with epitopes
present on neurokinin A or neurokinin B.
Many procedures are known by those of ordinary skill in the art for purifying
antibodies and antibody fragments from other proteins, contaminants, and
materials
2o with which they may normally be associated prior to their use in various
procedures.
For example, Jafarian et al., "Passive Immunization with an Anti-Substance P
Antibody Prevents Substance P- and Neurokinin A-Induced Bronchospasm in
Anesthetized Guinea Pigs," supra., describes the purification of anti-
substance P
monoclonal antibodies from culture supernatant using three chromatographic
methods: protein A-sepharose, protein G-sepharose and thiophilic gel.
Numerous well-known assay techniques based upon immunological reactions
between antigens and antibodies may be performed using substance P as the
antigen
to determine whether or not a particular antibody, or antibody fragment, has
the
ability to bind to an epitope present on the substance P, and to potentially
inhibit the
3o activity of endogenous substance P. These techniques include the enzyme-
linked
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immunosorbent assays (ELISA), immunofluorescence assays (IFA), radioimmuno
assays, immunoelectrophoreses, immunoblotting and the like. For example,
Jafarian
et al., supra., describe an inhibition assay which was employed to estimate
the relative
affinities of an anti-substance P monoclonal antibody derived from the rat-
mouse
heterohybridoma NCl/34 for substance P, neurokinin A and calcitonin gene-
related
peptide. Using any of the known assay techniques which are based upon
immunological reactions, the ability of particular antibodies, or antibody
fragments, to
bind with substance P may be determined by contacting a fluid, solid or other
medium
which contains substance P with purified antibodies, or antibody fragments,
and
to detecting the presence of binding between the antibodies, or antibody
fragments, and
the substance P. The presence of binding indicates the ability of the
antibodies, or
antibody fragments, to bind with the substance P. Preferably, the antibodies,
or
antibody fragments, employed in the methods of the invention will be specific
for
substance P (i.e., they will bind to an epitope present on substance P which
is not
common to other related proteins, or to other molecules, and will bind with a
higher
affinity to substance P than to other antigens).
The various epitopes which may be present on substance P, and with which an
antibody, or antibody fragment, may bind, can be readily determined using the
well-
known techniques of epitope mapping and conformational dependency analysis.
The
2o activity of a particular antibody or antibody fragment produced may be
determined by
its ability to inhibit the action of substance P. In making such a
determination, one
first needs to associate substance P with the inflammatory disease by assaying
for
systemic or local increases in substance P in a subject. At least two methods
are
available for determining inhibitory effectiveness of an antibody or antibody
fragment. In one method, anti-substance P antibodies or fragments may be
tested in
vitro for their ability to inhibit substance P by examining the dose-response
effect of
adding the antibody or fragment to cultures of lymphocytes, such as peripheral
blood
lymphocytes. Using a flow cytometer, one could then readily assess whether
there
was an inhibition of intracellular cytokines at various time-points after
treatment. In a
3o second method, one may administer one or more pharmacological doses of an
anti-
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substance P antibody in vivo in a mouse model, either locally or systemically,
to
inhibit the actions of substance P. Inhibition of substance P would result in
an
inhibition or change in the type of inflammatory response. Quantitating levels
of
proinflammatory cytokines in inflammatory cells could assess a change in the
type of
inflammatory response. Furthermore, additional assays for substance P or the
cytokines whose levels it modulates may be performed by methods such as
Western
blot analysis, ELISA, affinity chromatography, immunoprecipitation,
competitive
inhibition analysis, and the like.
To determine whether a particular antibody, or antibody fragment, has the
to ability to prevent or treat an inflammation-related disease, the mouse
model discussed
in the preceding paragraph may be studied. In such determinations, a mouse may
be
infected with an inflammation-producing pathogen. The procedures outlined
above
may be applied to evaluate the ability to prevent (if administered before
infection) or
treat (if administered after infection) the inflammation associated with the
pathogen.
Antibody fragments, such as F(ab)2 fragments, may be prepared from
polyclonal or monoclonal antibodies by standard methods known by those of
skill in
the art. For example and in brief, an antibody is incubated with pepsin in a
buffer of
100 mM sodium citrate, pH 3.5, at 37°C for 24 h. The digestion is
terminated by
adding 3 M Tris buffer pH 8.8. The F(ab)2 fragments are resolved from the Fc
2o fragments by chromatography over a protein A column, which selectively
binds Fc
fragments. The F(ab)2 fragments are collected in the flow through fractions.
Conventional techniques of molecular biology which may be employed in
producing anti-substance P antibodies, and anti-substance P antibody
fragments, and
in generally carrying out the methods of the present invention, are fully
explained in
the literature. See, for example, Rose et al., Manual of Clinical Laboratory
Immunolo~v, 5th ed., Eds. American Society for Microbiology, Washington (
1996);
Harlow and Lane, Antibodies; A Laboratory Manual, supra.; Roitt et al.,
Immunolo~v,
The C.V. Mosby Company, St. Louis, MO (1985); Kuby, Immunolo~y, W.H.
Freeman and Co., New York ( 1992); Ausubel et al., Current Protocols in
Molecular
3o Bioloav, Green Publishing Associates and Wiley-Interscience, John Wiley and
Sons,
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New York ( 1987; updated quarterly); Sambrook et al., Molecular Cloning - A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
(1985);
Davis et al., Basic Methods in Molecular Biolo y, Elsevier Science Publishing
Co.,
Inc., New York (1986); Rodriguez et al., Recombinant DNA Techniques: An
Introduction, The Benjamin/Cummings Publishing Company, Inc., Menlo Park, CA
(1983); and Maniatis et al., Molecular Cloning: A Laboratory Manual Cold
Spring
Harbor, New York ( 1982).
Dosage and Mode of Administration. The methods of the present invention
are useful for preventing and/or treating inflammation-related diseases in a
subject. A
1 o physician or veterinarian of ordinary skill in the art can readily
determine whether or
not a particular subject has an inflammation-related disease, or is
particularly
susceptible to an inflammation-related disease.
While anti-substance P antibodies, and anti-substance P antibody fragments,
may be employed in the methods of the invention alone, they will preferably be
15 employed in the form of a pharmaceutical composition or formulation. For
human
subjects or patients, these antibodies, fragments and pharmaceutical
compositions
should be used under the guidance of a physician.
The pharmaceutical compositions which may be employed in the methods of
the invention will typically comprise one or more types of anti-substance P
antibodies,
2o and/or anti-substance P antibody fragments, as an active ingredient in
admixture with
one or more pharmaceutically-acceptable carriers and, optionally, with one or
more
other types of antibodies, antibody fragments, compounds, drugs, therapeutic
agents
or other materials. The appropriate dosage and mode of administration of the
anti-
substance P antibodies, anti-substance P antibody fragments and pharmaceutical
25 compositions which may be employed in the methods of the invention should
be
suitably selected by methods which are consistent with conventional
pharmaceutical
practices.
The anti-substance P antibodies, anti-substance P antibody fragments and
pharmaceutical compositions which may be employed in the methods of the
invention
3o may be specifically formulated for oral administration in solid or liquid
form, for
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parenteral injection, for rectal or vaginal administration or for topical
administration.
They may be administered to humans and other subjects for the prevention or
therapy
of inflammation-related diseases by any suitable route of administration,
including
orally, nasally, as by, for example, a spray, rectally, intravaginally,
parenterally,
intracisternally or topically, as by powders, ointments and drops, including
bucally
and sublingually. Preferably the antibodies are administered via a parenteral
route.
Regardless of the route of administration, the anti-substance P antibodies,
anti-
substance P antibody fragments and pharmaceutical compositions which may be
employed in the methods of the invention are formulated into pharmaceutically-
to acceptable dosage forms by conventional methods known by those of skill in
the art.
Actual dosage levels of the active ingredients employed in the methods of the
invention may be varied so as to obtain an amount of the active ingredient
which is
effective to achieve the desired preventative effect, or therapeutic response,
for a
particular subject, composition, and mode of administration, without being
toxic to
the subject. The selected dosage level will depend upon a variety of factors,
including
the activity of the particular anti-substance P antibodies or anti-substance P
fragments
being employed, the route of administration, the time of administration, the
rate of
excretion of the particular antibodies or fragments being employed, the
severity of the
disease, the duration of the treatment, other drugs, therapeutic agents or
materials
2o which are being used in combination with the particular antibodies or
fragments being
employed, the species, age, sex, weight, condition, general health and prior
medical
history of the subject being treated, and like factors which are well known in
the
medical arts. A physician or veterinarian having ordinary skill in the art can
readily
determine and prescribe the effective amount of anti-substance P antibodies,
anti-
substance P antibody fragments or a pharmaceutical formulation containing such
antibodies or antibody fragments required to prevent or treat an inflammation-
related
disease. For example, in order to treat a particular inflammation-related
disease, the
physician or veterinarian could start doses of the anti-substance P
antibodies, anti-
substance P antibody fragments or pharmaceutical composition containing the
antibodies or antibody fragments at levels which are lower than that required
in order
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to achieve the desired therapeutic effect, and gradually increase the dosage
until the
desired therapeutic effect in achieved.
In general, a suitable daily dose of anti-substance P antibodies, anti-
substance
P antibody fragments or a pharmaceutical composition containing these
antibodies or
fragments will be at least that amount of the antibodies, antibody fragments
or
pharmaceutical composition which is the lowest dose which is effective to
produce a
therapeutic effect. Pharmaceutically-effective amounts of the anti-substance P
antibodies, anti-substance P antibody fragments and pharmaceutical
compositions
containing these antibodies and fragments for a particular subject may be
determined
l0 using standard procedures. Generally, dosage levels which range from about
0.5
mg/kg body weight to about 2 g/kg body weight, and more preferably range from
about 1 mg/kg body weight to about 1 g/kg body weight, and still more
preferably
range from about 5 mg/kg body weight to about 100 mg/kg body weight, of the
anti-
substance P antibodies, or anti-substance P antibody fragments, per day are
administered to a mammalian subject. However, the total daily usage of the
anti-
substance P antibodies, or anti-substance P antibody fragments, or of the
pharmaceutical compositions comprising such active agents, will be determined
by an
attending physician or veterinarian within the scope of sound medical
judgement.
If desired, the effective daily dose of the anti-substance P antibodies or
anti-
2o substance P antibody fragments may be administered as two, three, four,
five, six or
more sub-doses administered separately at appropriate intervals throughout the
day,
optionally, in unit dosage forms. Such a multiple dosage regime allows for the
observation of the patient after each dose and, if appropriate, modification
of the
dosage amount and/or regime.
The pharmaceutical compositions which may be employed in the methods of
the invention comprise anti-substance P antibodies, or anti-substance P
antibody
fragments, together with one or more pharmaceutically-acceptable carriers
thereof
and, optionally, with other therapeutic agents. Each carrier should be
"acceptable" in
the sense of being compatible with the other ingredients of the formulation,
and not
injurious to the patient. Wetting agents, emulsifiers and lubricants, such as
sodium
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lauryl sulfate and magnesium stearate, as well as coloring agents, release
agents,
coating agents, preservatives, antioxidants, and the like can also be employed
in the
pharmaceutical compositions used in the methods of the invention.
Formulations of the anti-substance P antibodies and anti-substance P antibody
fragments used in the methods of the invention may be conveniently prepared in
a unit
dosage form, and may be prepared by any of the numerous methods which are well
known in the art of pharmacy. The amount of active ingredient (anti-substance
P
antibodies or fragments) which can be combined with a carrier material to
produce a
single dosage form will vary depending upon the patient being treated, the
particular
1 o mode of administration and all of the other factors described above. The
amount of
active ingredient which can be combined with a carrier material to produce a
single
dosage form will generally be that amount of the active ingredient which is
the lowest
dose which is effective to produce a therapeutic effect. Generally, out of one
hundred
percent for the pharmaceutical formulation, this amount will range from about
one to
about ninety-nine percent of active ingredient, preferably from about five to
about
seventy percent of active ingredient, and most preferably from about ten to
about
thirty percent of active ingredient.
Methods for preparing the pharmaceutical compositions which may be
employed in the methods of the invention include the step of bringing into
association
2o the active ingredient with the carrier and, optionally, with one or more
accessory
ingredients, and then, if necessary, shaping the product. Formulations of the
active
ingredient which are suitable for oral administration may be in the form of
capsules,
cachets, pills, tablets, lozenges, powders, granules, solutions or suspensions
in an
aqueous or nonaqueous liquid, or oil-in-water or water-in-oil emulsions.
Formulations of the active ingredient for rectal or vaginal administration may
be in
the form of a gel, cream, foam or suppository, which may be prepared by mixing
the
active ingredient with one or more non-irntating excipients or carriers
comprising, for
example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate.
They
will preferably be solid at room temperature, but liquid at body temperature,
so that
3o they will melt in the rectum or vaginal cavity of the subject and release
the active
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ingredient. Dosage forms for the topical or transdermal administration of the
active
ingredient include powders, sprays, ointments, pastes, creams, lotions, gels,
solutions,
patches and inhalants.
Pharmaceutical compositions containing anti-substance P antibodies and/or
anti-substance P antibody fragments which may be employed in the methods of
the
invention, and which are suitable for parenteral administration, comprise one
or more
types of anti-substance P antibodies and/or anti-substance P antibody
fragments in
combination with one or more pharmaceutically-acceptable carriers. Such
Garners
may be sterile, isotonic, aqueous or nonaqueous solutions, dispersions,
suspensions or
Io emulsions, or sterile powders which may be reconstituted into sterile
injectable
solutions or dispersions just prior to use. These pharmaceutical formulations
may
contain antioxidants, buffers, bacteriostats, solutes that render the
formulation isotonic
with the blood of the intended recipient and/or suspending or thickening
agents.
Examples of suitable aqueous and nonaqueous carriers which may be employed in
these compositions include water, ethanol, polyols (glycerol, propylene
glycol,
polyethylene glycol, and the like, and suitable mixtures thereof), vegetable
oils, such
as olive oil, and injectable organic esters, such as ethyl oleate. Proper
fluidity can be
maintained, for example, by the use of coating materials, such as lecithin, by
the
maintenance of the required particle size in the case of dispersions, and by
the use of
2o surfactants. These compositions may also contain adjuvants, such as
preservatives,
wetting agents, emulsifying agents and dispersing agents. Prevention of the
action of
microorganisms may be ensured by the inclusion of various antibacterial and
antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid,
and the
like. It may also be desirable to include isotonic agents, such as sugars,
sodium
chloride, and the like into the compositions.
In some cases, in order to prolong the effect of an active ingredient, it is
desirable to slow the absorption of an active ingredient administered by, for
example,
subcutaneous or intramuscular injection. This may be accomplished by the use
of a
liquid suspension of a material having poor water solubility. The rate of
adsorption of
3o the active ingredient will then depend upon its rate of dissolution.
Delayed adsorption
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of a parenterally-administered active ingredient may also be accomplished by
dissolving or suspending the active ingredient in an oil vehicle. In addition,
prolonged absorption of the injectable pharmaceutical form may be brought
about by
the inclusion of agents which delay absorption, such as aluminum monostearate
and
gelatin.
Forming microencapsule matrices of the active ingredient in biodegradable
polymers, such as polylactide-polyglycolide, may make injectable depot forms
of the
active ingredient. Depending upon the ratio of the active ingredient to
polymer
employed, and the nature of the particular polymer employed, the rate of
release of the
l0 active ingredient can be controlled. Examples of other biodegradable
polymers
include poly(orthoesters) and poly(anhydrides). Depot injectable formulations
may
also be prepared by entrapping the active ingredient in liposomes or
microemulsions
which are compatible with body tissue.
Materials which may be used to prepare an injectable pharmaceutical
formulation of anti-substance P antibodies and anti-substance P antibody
fragments
can be sterilized, for example, by filtration through a bacterial-retaining
filter, or by
incorporating sterilizing agents in the form of sterile solid compositions
which can be
dissolved or dispersed in sterile water, or in other sterile injectable media,
into the
formulations just prior to use. Injectable pharmaceutical formulations
containing anti-
2o substance P antibodies or anti-substance P antibody fragments may be
present in unit-
dose or mufti-dose sealed containers, such as ampoules and vials, and may be
stored
in a lyophilized condition requiring only the addition of the sterile liquid
carrier, for
example, water, immediately prior to use. All of the pharmaceutical
formulations
discussed may be prepared using standard pharmaceutical techniques which are
well-
known to those skilled in the art.
The following examples describe and illustrate the methods of the invention.
These examples are intended to be merely illustrative of the present
invention, and not
limiting thereof in either scope or spirit. Those of skill in the art will
readily
understand that variations of the materials used in, and the conditions and
processes
of, the procedures described in these examples can be used. All materials and
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equipment employed in the examples, and generally employed to practice the
methods
of the invention, are commercially available. Sources for these materials and
equipment are set forth in the examples, or are known by those of skill in the
art.
EXAMPLE 1: Quantification of Substance P Levels in Mouse
Bronchoalveolar Lavage Specimens. In these experiments, the levels of
substance P
in cell-free bronchoalveolar lavage (BAL) fluid specimens collected from
BALB/c
mice i.n. infected with 1 O6 pfu of respiratory syncytial virus during the
acute immune
response to the virus at various timepoints post infection were quantitated by
to competitive enzyme immunoassay (ELISA).
The competitive ELISA performed is based upon the competition between free
substance P (Biomol Research Laboratories, Plymouth Melting, PA) and a
substance
P tracer (Cayman Chemical, Ann Arbor, MI) for a limited number of substances P-
specific antibody binding sites. Dilutions of bronchoalveolar lavage fluid
were
15 analyzed against a substance P standard, and the results were calculated as
the percent
of sample or standard bound, and the maximum bound. Intra- and interassay
coefficients of variation (CV) were less than or equal to 10 percent.
Four- to six-week old specific pathogen-free female BALB/c mice (Harlan
Sprague Dawley, Indianapolis, IN) were anesthetized with avertin (2,2,2,-
2o tribromoethanol, Aldrich Chemical Co., Milwaukee, WI). The lungs were
washed via
a catheter inserted through a tracheal incision with 1 ml of phosphate
buffered saline
(GIBCO Laboratories, Grand Island, NY). The responses of three mice were
examined for each time-point, and the mean values reported.
The inflammatory response, in the lungs of the mice peaked between days 3
25 and 7 post-infection with the respiratory syncytial virus. Compared with
naive mice
(i.e., uninfected mice), the inflammatory response in the lungs of the BALB/c
mice
was characterized by an approximately 50-fold increase in cell numbers in the
lung, of
which macrophages constitute about 60%, lymphocytes about 30%, and
polymorphonuclear cells about 10%. CD4+ T lymphocytes produced peak levels of
30 intracellular cytokines IL-2, IL-4 and IL-6 at day 3 post infection, and IL-
5 and IFN~y
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levels peaked at day 5 post-infection. CD8+T lymphocytes also participated in
the
cytokine-mediated inflammatory response to respiratory syncytial virus, but at
a lesser
magnitude. All cytokines decreased to near baseline (levels at day 0) by day
12 post-
infection. The T lymphocyte response to respiratory syncytial virus was
predominantly characterized by a Thl-type mRNA cytokine pattern (IL-2, IFNy)
in
the BALB/c mice. That is, only Th 1-type cytokine mRNA (which may include
mRNA for IFNy, IL-2, IL-12, and lymphotoxin mRNAs) is found. This pattern is
the
same as occurs with humans. FIG. 1 shows that changes in levels of substance P
found in the cell-free bronchoalveolar lavage fluid follow a pattern very
similar to that
to for cell numbers and intracellular cytokine levels. FIG. 1 shows that
substance P
levels in the bronchoalveolar lavage fluid rise from a baseline of about 250
pg/mL at
day 0 (naive mice) to about 800 pg/ml by day 3 post infection, peak at about
1000
pg/mL on day 5 post infection, and then rapidly decline to about 350 pg/mL at
day 12
post infection.
EXAMPLE 2: Examination of the Impact of Exogenous Substance P on
CD4+ and CD8+ T Lymphocyte Proliferation. In order to better understand how
substance P directly affects the immune response, the effect of exogenous
substance P
on the proliferation of intact, or unfractionated, T lymphocytes, and of CD4+
and
2o CD8+ enriched T lymphocyte populations, induced with the mitogens
(substances
which induce mitosis and cell transformation, especially lymphocyte
transformation)
concanavalin and lipopolysaccharide in BALB/c mice was examined. T
lymphocytes,
isolated from three pooled spleens of BALB/c mice were enriched with CD4+ and
CD8+ T lymphocytes by standard methods using streptavidin-coated magnetic
beads
(Dynal A S., Oslo, Norway) coupled to biotin-anti-CDBa (53-6.7, PharMingen,
San
Diego, CA), or to biotin-anti-CD4 (RM4-5, PharMingen). A portion of the CD4+
and
CD8+ T lymphocytes were analyzed by flow cytometry using an FACScan (Becton-
Dickinson, Mountain View, CA) and found to be enriched to greater than 95
percent
using magnetic beads.
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Intact, and CD4+ and CD8+ enriched, T lymphocytes were stimulated with the
mitogen concanavalin (Sigma Chemical Co., St. Louis, MO, 2 ~g/mL; FIG. 2) or
lipopolysaccharide (Sigma Chemical Co., St. Louis, MO, 100 ~g/mL; FIG. 3),
with or
without the addition of concentrations of substance P (Biomol Research
Laboratories)
ranging from 10'5 M to 10'9 M, or were left unstimulated in tissue culture
medium
(GIBCO) containing 10% heat-inactivated FBS (Hyclone Laboratories, Logan, UT)
plus 1 % of antibiotic/antimycotic (GIBCO BRL, Grand Island, NY). Substance P
was
added to the cell cultures of intact, and CD4+ or CD8+ enriched, T lymphocytes
at
concentrations comparable to those found in bronchoalveolar lavage fluid (
10'5 M to
10'9 M). Cells were cultured at 37°C for 48 hours, and all of the wells
for the cells
were pulsed with 1 ~Ci of tritiated thymidine (Amersham, Arlington Heights,
IL) for
25 hours, and then harvested with a Matrix Cell Harvester (Packard
Instruments,
Meriden, CT).
The results of these experiments are shown in FIGS. 2 and 3. The stimulation
index is the mean counts per minute (cpm) of stimulated, or experimental,
cells
divided by the mean counts per minute of unstimulated, or control, cultures
(intact,
CD4+ or CD8+ T lymphocytes which were left unstimulated in tissue culture
medium).
A statistically significant value of the stimulation index above 1.0 indicates
a
significant effect of the experimental parameter above that of control.
2o The exogenous addition of substance P at concentrations from 10'5 M to 10'9
M to the intact T lymphocytes did not induce any detectable increase in cell
proliferation. In contrast, and as is shown in FIGS. 2 and 3, CD4+ T
lymphocytes
stimulated with concanavalin (FIG. 2), but not CD4+ T lymphocytes stimulated
with
lipopolysaccharide (FIG. 3), showed a 7- to 8-fold increase in proliferation
when
cocultured with substance P at concentrations ranging from 10'5 M to 10'' M.
CD8+T
lymphocytes stimulated with concanavalin A showed a 4- to 5-fold increase in
proliferation. These data suggest that activated T lymphocytes are more
sensitive to
substance P than are naive or quiescent lymphocytes, and that CD4+ T
lymphocytes
are more sensitive to substance P than are CD8+ T lymphocytes.
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To confirm the specificity of substance P-induced cell proliferation, rabbit
anti-substance P antibody (PharMingen) was added to a portion of the cell
cultures
containing concanavalin and substance P during incubation. Enhanced cell
proliferation caused by the addition of substance P was found to be inhibited
by the
addition of 100 ~g rabbit anti-substance P antibody to each culture. Further,
no effect
of rabbit anti-substance P antibody was observed on cell cultures only
stimulated with
mitogens (i.e., not with substance P).
EXAMPLE 3: Treatment of Mice Infected with Respiratory Syncytial
Io Virus with Anti-Substance P Antibod Fragments. In order to ascertain the
connection of substance P with cytokine-based inflammation caused by
respiratory
syncytial virus infection, BALB/c mice, which were acutely infected with
respiratory
syncytial virus' were i.n. treated with rabbit anti-substance P F(ab)z
antibody fragments
to inhibit the biological activity of substance P. An anti-substance P F(ab)2
antibody
fragment dose-response experiment was also performed in order to determine the
amount of the antibody fragments which was required to block the effects of
substance P on the intracellular generation of cytokines by CD3+ T
lymphocytes. The
effect of the rabbit anti-substance P F(ab)2 antibody fragments on
intracellular
cytokine production by T lymphocytes isolated from the lungs was also
examined.
Rabbit anti-substance P antibodies (PharMingen) were incubated overnight at
37°C in citrate buffer containing 5 ~g of pepsin (Sigma Chemical
Company) per 1 ~.g
of antibody. The antibodies were then centrifuged at 10,000 G for 30 minutes,
resuspended in phosphate buffered saline (GIBCO), and run over a protein A
column
(Sigma Chemical Company) to separate anti-substance P F(ab)Z antibody
fragments
2s from anti-substance P Fc antibody fragments.
BALB/c mice (Harlan Sprague Dawley) were i.n. infected with 106 pfu of
respiratory syncytial virus (Long, A strain), and then rested for three days.
The mice
were then anesthetized with avertin (Aldrich Chemical Co.), and i.n. treated
with 2, 20
or 200 ~.g per mouse of rabbit anti-substance P F(ab), antibody fragments or
with 200
~g per mouse of normal rabbit Ig (nIg) antibodies. Controls were not treated.
This
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treatment was given to the mice during the peak of intracellular cytokine
production
by T lymphocytes, which occurs on day 4 post-infection with the respiratory
syncytial
virus.
T lymphocytes were collected from bronchoalveolar lavage specimens
obtained from the mice either 18 hours (FIG.4) or 36 hours (FIG.S) after
treatment
with the rabbit anti-substance P F(ab)2 antibody fragments, or with normal
rabbit Ig
antibodies, and were stained for the observation of intracellular cytokines in
order to
examine the levels of intracellular cytokines produced by the T lymphocytes 18
hours
and 36 hours post-treatment. The procedure used for immunofluorescence
1 o intracellular cytokine staining was modified from the protocol described
by
PharMingen. Briefly, the intracellular transport of cytokines in the T
lymphocytes
was inhibited using 1 ~g/mL brefeldin (Sigma Chemical Company). The T
lymphocyte cells were washed in phosphate buffered saline (GIBCO), and the
cell
surface antigen was stained with either anti-CD4+ (RM4-5) or anti-CD8+ (53-
6.7)
antibody, and subsequently fixed with 4% paraformaldehyde (Ted Pella Inc.,
Redding,
CA) in D-phosphate buffered saline containing 1 % bovine serum albumin. The
cells
were washed in phosphate buffered saline, and the membranes were permeabilized
using saponin (Sigma Chemical Company). All intracellular antibodies were
labeled
with phycoerythrin, and were purchased from PharMingen. Anti-IL-2 (JES6-SH4),
2o anti-IL-4 (BVD4-1D11), anti-IL-5 (TRFKS), anti-IL-6 (MPS-20F3), and anti-
IFN~y
(XMG 1.2) antibodies were diluted in D-phosphate buffered saline containing 1
bovine serum albumin and 0.1 % saponin. The cells were stained on ice using an
appropriate amount of the intracellular antibody (equivalent to 1 ~g/mL),
washed
phosphate buffered saline, resuspended in D-phosphate buffered saline
containing 1
bovine serum albumin, and analyzed by flow cytometry using an FACScan (Becton-
Dickinson).
FIG. 4 shows that, at 18 hours post-treatment, a single i.n. treatment of 200
~,g
of the rabbit anti-substance P F(ab)~ antibody fragments per mouse induced
approximately a 3-fold reduction in the intracellular cytokines IL-2, IL-4, IL-
6 and
3o IFNy made by T lymphocytes in the bronchoalveolar lavage fluid of the mice.
The
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inhibitory effect of the rabbit anti-substance P F(ab)z antibody fragments on
the
intracellular cytokines was found to be dose dependent, and specific for T
lymphocytes in the bronchoalveolar lavage fluid of the mice. (No effect of the
rabbit
anti-substance P F(ab)Z antibody fragments on the levels of intracellular
cytokines was
observed in spleens obtained from the mice.) FIG. 4 also shows that less of an
inhibitory effect of the rabbit anti-substance P F(ab)2 antibody fragments on
the levels
of intracellular cytokines present in the bronchoalveolar lavage fluid of the
mice was
observed when 20 pg of the rabbit anti-substance P F(ab)2 antibody fragments
were
administered. No inhibitory effect was observed when 2 pg of the rabbit anti-
l0 substance P F(ab)z antibody fragments were administered. FIG. 5 shows that,
at 36
hours post treatment, the only detectable and significant inhibitory effect of
the rabbit
anti-substance P F(ab)2 antibody fragments occurred for the production of the
IL-2
cytokine by the T lymphocytes in the bronchoalveolar lavage fluid of the mice.
T lymphocytes were obtained from the bronchoalveolar lavage fluid of the
infected BALB/c mice by inserting a catheter through a tracheal incision, and
washing
the lungs. T lymphocytes were analyzed by flow cytometry after binding
labelled
anti-CD3 MAb. Phenotypic analysis of the T lymphocytes was performed by cell
surface staining the lymphocytes with phycoerythrin-conjugated MAbs specific
for
cell activation molecules such as CD49d or CD54, and assaying by flow
cytometry.
The results showed a reduction in the expression of inflammatory molecules,
such as
CD49d and CD54, on the surface of T lymphocytes following treatment of the
mice
with rabbit anti-substance P F(ab)z antibody fragments, as well as a decrease
in the
numbers of granular cells.
The data resulting from these experiments show that inhibiting the biological
activity of endogenous substance P found in bronchoalveolar lavage fluid
obtained
from mice infected with respiratory syncytial virus, with anti-substance P
antibody
fragments, can reduce the levels of intracellular cytokine production by T
lymphocytes present in the lungs, and beneficially modify the immune response
to
infection by respiratory syncytial virus. The magnitude of the inhibition of
intracellular cytokine production by T lymphocytes in the bronchoalveolar
lavage
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fluid of the BALB/c mice by administration to the mice of rabbit anti-
substance P
F(ab)Z antibody fragments suggests that this treatment may have broad effects
on the
immune and inflammatory response to respiratory syncytial virus infection.
Similar experiments were performed to those described hereinabove, with the
exception that the anti-substance P F(ab)Z antibody fragments were
administered to
the mice either by i.v. or i.p. routes. The data resulting from these
experiments
demonstrated that treatment of the mice with the anti-substance P F(ab)2
antibody
fragments inhibited the production of intracellular cytokines. The results of
the anti-
substance P treatment were found to be rapid (i.e., occurring within eighteen
hours), to
1 o be persistent (i.e., having an effect up to seventy-two hours post-
treatment for IL-2
and IL-4), and to operate in a dose-dependent manner. These data suggest that
the
results observed are specific, and are attributable to the anti-substance P
treatment. In
addition, it was observed that the i.p. administration of the anti-substance P
F(ab)Z
antibody fragments appeared to have a slightly stronger effect at inhibiting
the
production of intracellular cytokines when compared to either i.n. or i.v.
routes of
administration. Consistent with these observations are results which were
obtained
from the histological examination of bronchoalveolar fluid from the lungs of
BALB/c
mice obtained at various time points post treatment with the anti-substance P
F(ab)z
antibody fragments. These data showed that i.p. treatment with anti-substance
P
2o antibody fragments induced the least accumulation of inflammatory cells
(e.g.,
eosinophils and polymorphonuclear cells) in the bronchoalveolar fluid, when
compared to i.n. or i.v. treatment.
The foregoing examples are provided to enable one of ordinary skill in the art
to practice the present invention. These examples are merely illustrative,
however,
and should not be read as limiting the scope of the invention as it is claimed
in the
appended claims. While the present invention has been described herein with
some
specificity, and with reference to certain preferred embodiments thereof,
those of
ordinary skill in the art will recognize numerous variations, modifications
and
substitutions of that which has been described which can be made, and which
are
within the scope and spirit of the invention. It is intended that all of these
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modifications and variations be within the scope of the present invention as
described
and claimed herein, and that the invention be limited only by the scope of the
claims
which follow. Specific anti-substance P antibodies and anti-substance P
antibody
fragments, and methods, within the scope of the invention include, but are not
limited
to, the anti-substance P antibodies and anti-substance P antibody fragments,
and
methods, described herein. Contemplated equivalents of the anti-substance P
antibodies and anti-substance P antibody fragments, and methods, described
herein
include anti-substance P antibodies and anti-substance P antibody fragments,
and
methods, which otherwise correspond thereto, and which have the same general
1 o properties thereof, wherein one or more simple variations are made which
do not
adversely affect the function of the anti-substance P antibodies and anti-
substance P
antibody fragments, and methods, described herein.
Throughout this application, various patents, publications, books, and amino
acid sequences have been cited. The entireties of each of these patents,
publications,
I5 books, and amino acid sequences are hereby incorporated by reference into
this
application.
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