Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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SMALL PEPTIDES AND METHODS FOR DOWNREGULATION OF IgE
FIELD OF THE INVENTION
This invention relates to small peptides, particularly to N-formyl-
methionyl peptides, having downregulating activity of IgE and to methods
for treating indications resulting from IgE-mediated responses. More
particularly, the peptides can be used to replace corticosteroids in any
application in which corticosteroids are used.
BACKGROUND OF THE INVENTION
Immunoglobulin E (IgE) is one of five classes of antibody occurring in
man and has been known for over three decades that it is the
immunoglobulin responsible for allergic reactions. IgE is produced and
secreted by B cells upon allergen invasion. However, IgE constitutes only a
small fraction of the total antibody in human serum (50-300ng/ml
compared to lOmg/ml of IgG) and thus, is not present in sufficient amount
to directly neutralize antigens. Instead, its action is amplified through
target cellular receptors and elicits a wide range of cellular responses to
antigens, culminating in inflammation, itching, coughing, lacrimation,
bronchoconstriction, mucus secretion, vomiting and diarrhea, all symptoms
commonly associated with allergic disorders.
Immediate hypersensitivity reactions are triggered through the high-
affinity IgE receptors (FceRI) found on mast cells and basophils. Allergen
binding to the FcERI-bound IgE causes cross-linking of receptor molecules
on the cell membrane, which triggers degranulation of the cell and
subsequent release of histamines and other mediators associated with the
immediate phase of the allergic response. These products of mast cell
degranulation cause activation of inflammatory cells and further induces a
low-affinity IgE receptor, FceRII, also known as CD23. FcERII can be found
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on activated B cells, various inflammatory cells (macrophages, eosinophils,
platelets, natural killer cells), T cells, follicular dendritic cells (FDC),
Langerhans cells and epithelial cells of the bone marrow and thymus
(Delespesse et al., Adv. Immun. 49:149-190, 1991; Delespesse et al.,
Immunol. Rev. 125:78-97, 1992). On the surface of B cells, FcERII plays a
role in IgE-dependent antigen presentation to T cells and also in the cross-
linking of B cells. On FDCs, FcERII is expressed in large amounts and is
therefore implicated in the recruitment of B cells to the germinal centers of
secondary follicles in the lymph nodes and spleen. When expressed on
inflammatory cells, it is thought to be responsible for IgE-dependent
cytotoxic activities, such as phagocytosis of immune complexes by
monocytes. Soluble FcERII (sFcERII) can also initiate humoral and cell-
mediated immune responses by triggering the growth and differentiation of
precursors of plasma cells, T cells and basophils.
Differentiation of B cells into IgE-secreting plasma cells involves a
complex signaling cascade of cytokines and surface molecules, thought to
take place mainly in the germinal centers of secondary follicles in the lymph
nodes and spleen. Surface molecules are essential in order to provide the
physical interaction of B cells with T cells and mast cells that is required
for
triggering IgE production. These surface molecules are CD40 ligand (CD40-
L) and FcERII. When T helper type 2 cells (Thz) are activated upon exposure
to antigen-presenting cells (APCs), they transiently express CD40L. CD40L
interacts with CD40 on B cells, resulting in B cell activation. The activated
Thz cells secrete various cytokines, such as IL-4 and IL-13, which act on
the activated B cells to switch to IgE production. IL-4 in addition
upregulates Fc~RII expression on B cells and inflammatory cells, providing a
further source of contact stimulation and soluble growth factor.
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Mast cells and basophils also secrete IL-4 and express CD40L and
can thus induce IgE synthesis by B cells upon physical interaction with B
cells in the presence of IL-4, in a similar manner as Tha cells. It is likely
that IgE synthesis can also take place in the skin, lungs and gut, in view of
the tissue distribution of the various types of cells involved in IgE
production.
Upregulation of IgE synthesis and rescue of germinal center B cells
from apoptosis is mediated by the cross-linking of B cell membrane-bound
IgE and complement receptor 2 (CR2), also called CD21, by sFcERII. CR2 is
a highly glycosylated membrane protein found on B cells, FDCs, and some
T cells and basophils. sFcERII can participate in the positive feedback
control of IgE synthesis by triggering CR2 on B cells to enhance IgE
synthesis while also promoting the survival of IgE-committed B cells.
Activation of IgE production can lead to two different situations.
Acute inflammation due to allergen exposure begins with an early phase
reaction involving rapid activation of mast cells, airway macrophages, and
bronchial epithelial cells which release proinflammatory mediators
including histamines, eicosanoids, platelet-activating factor, oxygen free
radicals, neuropeptides, and cytokines. These can induce constriction of the
airway smooth muscle, mucous secretion, and vasodilation. Inflammation
of the airways causes increased microvascular leakage, leading to plasma
exudation into the airways. Thickening of airway walls and narrowing of the
airway lumen result.
In the late-phase reaction, peripheral blood cells are recruited into
the airways to establish a chronic-type of inflammation. Such cells include
eosinophils, lymphocvtes, and monocytes, and recruitment is dependent on
cytokines such as IL-5 and granulocvte-macrohage colony-stimulating
factor (GMC-SF). Chemokines such as RANTES and eotaxin also appear to
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enhance recruitment of eosinophils. At the site of inflammation, these cells
are activated and their survival is increased by reduced apoptosis, mediated
by factors such as GMC-SF.
Treatments for asthma have traditionally been based on the severity
and persistence of the disorder. For acute, intermittent symptoms,
treatments have generally involved bronchodilators. Bronchodilators
include (3-adrenergic agonists, methylxanthines, and anticholinergic drugs.
These agents can improve airway obstruction in asthma patients but they
do not appear to be effective in reducing airway inflammation or bronchial
hyperreactivity. In more recent years, leukotriene inhibitors have become
available for treatment of mild to moderate asthma. Leukotrines are
generated from arachidonic acid through the 5-lipoxygenase metabolic
pathway and have long been known to possess powerful
bronchoconstrictive properties. These so-called slow-reacting substance of
anaphylaxis ("SRS-A") also induce migration, adhesion and aggregation of
various white blood cells to blood vessels and increase capillary
permeability, culininating in interstitial edema, leukocyte chemotaxis,
mucus production, mucocilliary dysfunction, and bronchospasm in the
lungs. Leukotriene D4 (LTD4) > in particular, appears to be primarily
responsible for this activity in airways and acts through a specific receptor
on airway smooth muscle cells. Leukotrines, including cysteinyl
leukotrienes, are released during IgE-mediated mast cell degranulation.
Leukotriene inhibitors consist of two types: one that blocks the
synthesis of leukotrienes by inhibiting the activity of 5-lipoxygenase (5-LO),
which is required for the synthesis of leukotriene, and another that
competitively blocks the LTD4 receptor on smooth muscle cells. Zileutin is
the first of the 5-LO inhibitors that have become available. Zafirlukast is
the
first LTD receptor antagonist to be approved, while others such as
monelukast and pranlukast are currently undergoing clinical trials. These
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leukotriene inhibitors have so far been used for treatments of mild
persistent asthma but have not yet been proved effective for more severe
forms of asthma.
5 Antiinflammatory agents are currently employed for treating more
severe and persistent forms of asthma. Agents categorized as
antiinflammatory agents include theophylline, corticosteroids, cromolyn
sodium, and nedocromil sodium. Corticosteroids, in particular, appear to be
more effective in decreasing bronchial hyperreactivity and severe
exacerbations. They act by suppressing eosinophil recruitment by
inhibiting cytokine and chemokine production, as well as by inducing
apoptosis of eosinophils. They also act to abrogate airway edema and
bronchorrhea and therefore, inhaled corticosteroids are the most common
treatment for patients with chronic asthma. Inhaled corticosteroids include
beclomethasone, flunisolide, triamcinolone, fluticasone, and budesonide.
For chronic asthma, (3a-agonists are ineffective, except in that they can
temporarily improve bronchial obstruction. Thus, optimal treatment may be
to combine both inhaled corticosteroids and long-acting ~i2-agonists.
However, potential side effects of corticosteroids include oropharyngeal
candidiasis, dysphonia, adrenal suppression, growth retardation in
children, thinning of skin, osteoporosis, glaucoma, and cataracts. In
addition, it is unclear at the present time, the relationship between
"effective" versus "toxic" doses of these corticosteroids.
In addition to targeting the downstream events of the IgE signaling
pathway, some new therapeutic strategies are being developed to directly
intervene with IgE and its synthesis. The central position IgE plays in the
complex network leading to allergic reactions suggests that therapy targeted
to eliminate IgE or to block IgE binding to receptors would in effect, prevent
allergic responses altogether. Although still in its early stages, some
success has been shown by the use of monoclonal antibodies directed
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against IgE. Fahy et al. (Am. J. Respir. Crit. Care Med., 155:1828-1834,
1997) have reported that a humanized murine monoclonal antibody
developed against IgE reduced free IgE and was successful in blocking both
the early and late phase responses to allergen stimulation. Anti-IgE
antibodies that target a region of IgE necessary for binding to FcERI not only
blocks binding of IgE to its receptor, but also prevents mast cell
degranulation and anaphylaxis induced by the cross-linking of IgE bound
to FcERI on basophils and mast cells. Two such anti-IgE antibodies are
currently being tested (MacGlashan et al., Jlmmunol. 158:1438-1445,
1997; Corne et al., J. Clin. Invest. 99:879-887, 1997). So far, they appear to
reduce IgE concentrations in serum and also lower the levels of FcERI on
basophils, suggesting that IgE-dependent responses may be altered by
modulating the levels of circulating IgE.
The treatments to date typically have focused on downstream events,
which result from IgE activation. It would therefore be desirable to develop
treatments that modulate IgE levels in order to treat IgE-mediated
responses. Chemotactic peptides such as N-formyl-methionyl-leucyl
phenylalanine and pepstatin have been reported to inhibit mast cell
degranulation (Inflammation, Vol. 5, No. 1, pp. 13-16, 1981). The peptides
of the present invention downregulate IgE levels and therefore can be used
to modulate a variety of IgE-mediated responses.
SUMMARY OF THE INVENTION
The present invention provides methods for treating a variety of
indications resulting from IgE-mediated responses using pharmaceutical
compositions containing in a suitable pharmacological carrier a N-formyl-
methionyl-leucyl ("f Met-Leu") peptide having IgE-downregulation activity.
Particularly useful peptides are those having the formula f Met-Leu-X where
X is selected from the group consisting of Tyr, Tyr-Phe, Phe-Phe and Phe-
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Tvr. The peptides of the present invention can be used to replace
corticosteroids in any application in which corticosteroids are used.
In accord with the present invention, a method for treating an IgE-
mediated response in a mammal comprises administering to the mammal
an IgE downregulating effective amount of a peptide having the formula f-
Met-Leu-X where X is selected from the group consisting of Tyr, Tyr-Phe,
Phe-Phe and Phe-Tyr.
The invention also provides a method for downregulating membrane-
bound and soluble receptors for IgE. The method comprises administering
to the patient a IgE receptor downregulating effective amount of a peptide
having the formula f Met-Leu-X where X is selected from the group
consisting of Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr.
The invention further provides a method for inhibiting IgE secretion
by plasma cells. The method comprises administering to the patient an IgE
secretion inhibiting effective amount of a peptide having the formula f Met-
Leu-X where X is selected from the group consisting of Tyr, Tyr-Phe, Phe-
Phe and Phe-Tyr.
In accord with another embodiment, the invention provides a method
for downregulating CD40L expression. The method comprises administering
to a patient a CD40L downregulating effective amount of a peptide having
the formula f Met-Leu-X where X is selected from the group consisting of
Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr.
In certain preferred embodiments of the present invention, patients
can benefit by administering the peptide of the present invention in
combination with a second active ingredient. Particularly useful other
active ingredients for such combination in accord with the present
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invention are, for example, antileukotrienes, beta2 agonists, corticosteroids,
and the like.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a log dose response curve illustrating the effects of various
dosages of HK-X on OVA-specific serum IgE levels in acute asthmatic mice.
FIG. 2 shows lung sections from acute asthmatic mice administered
with 50 ~g of HK-X. Limited cellular infiltrates were present in (A) and (B)
and limited mucus accumulation in (C).
FIG. 3 shows lung sections from acute asthmatic mice administered
with 10 ug of HK-X. Very few cells were associated with the airway (A) and
(B) and mucus was limited to the surface of airway epithelial cell layer (C).
FIG. 4 shows lung sections from acute asthmatic mice administered
with 1 ~,g of HK-X. Therapeutic effect dimished, with an increase in cellular
infiltrates (A), and increase in mucus secretion into airways (B) and (C).
FIG. 5 shows lung sections from OVA-immunized mice challenged
with either saline (A) or vehicle (0.05% DMSO) (B). No mucus secretion was
detected in the airways (C).
FIG. 6 is a schematic illustration of the immunization and treatment
regime used in establishing a chronic asthma mouse model.
FIG. 7 is a histogram illustrating the granuloma number in lungs of
chronic asthmatic mice.
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FIG. 8 shows the histology of chronic asthmatic lung tissues from
mice immunized weekly with OVA for 6 months and treated with either HK-
X or saline. (A) shows lung histology of control mice, (B) shows histology of
HK-X-treated mice, and (C) shows histology of OVA-challenged but
untreated mice.
FIG. 9 shows light micrographs of chronic asthmatic mouse lung
tissue accumulation of collagen fibrils. (A) shows a lung section of a control
mouse administered with saline, (B) shows a lung section of a mouse
treated with HK-X, and (C) shows a lung section of an OVA-immunized but
untreated mouse.
FIG. 10 shows lung sections of mice chronically OVA-immunized and
treated with saline.
FIG. 11 shows lung sections of mice chronically OVA-immunized and
treated with vehicle (0.5% DMSO).
FIG. 12 is a histogram illustrating the histomorphometry in chronic
asthma.
FIG. 13 is a histogram illustrating the frequency of mucus containing
cells in the airways of chronic asthmatic mice after various treatments.
FIG. 14 is a histogram illustrating the effects of various treatments
on eosinophil and neutrophil infiltrates in the lungs of chronic asthmatic
mice.
FIG. 15 is a schematic illustration of the immunization and
treatment protocol with HK-X and dexamethasone in an acute asthmatic
mouse model.
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FIG. 16 is a histogram comparing the effects of intranasal
administration of dexamethasone and HK-X on OVA-specific IgE levels.
5 FIG. 17 is a schematic illustration of the immunization and
treatment protocol with HK-X and a control peptide in an acute asthmatic
mouse model.
10 DETAILED DESCRIPTION OF THE INVENTION
In accord with the present invention, certain small peptides having
the formula f Met-Leu-X where X is selected from the group consisting of
Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr have been found to have surprising
activity for downregulating the levels of IgE. As a result, such peptides are
useful for treatment of a variety of indications resulting from IgE mediated
responses. The peptides of the present invention can be used to replace
corticosteroids in any application in which corticosteroids are used.
Preferred peptides, in accord with the present invention, reduce
blood IgE levels and block IgE activation of lymphocytes such as, for
example, macrophages, monocytes, eosinophils, neutrophils, TNF, and the
like.
Continued mast cell degranulation and its release of leukotrienes,
histamines, and other cvtokines also decrease, or cease entirely in preferred
embodiments, following treatment with peptides of the present invention.
In accord with preferred embodiments of the present invention, the peptides
also can reduce the infiltration of eosinophils, basophils and neutrophils
into inflammatory tissues. Lymphocytes, eosinophils, and neutrophils do
not exhibit chemotaxis in response to preferred peptides of the present
invention. As a consequence, the chemotactic adhesion, migration and
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aggregation of lymphocytes, eosinophils and neutrophils to the site of
inflammation is significantly reduced, as is vascular permeability at the
inflammation site. Further, preferred compounds of the present invention
exhibit no toxicity to vital organs such as heart, liver and lungs.
The peptides of this invention can be prepared by conventional small
peptide chemistry techniques. The peptides when used for administration
are prepared under aseptic conditions with a pharmaceutically acceptable
carrier or diluent.
The pharmaceutical compositions may conveniently be presented in
unit dosage form and prepared for each type of indication resulting from
IgE-mediated responses that is to be treated. The compositions may be
prepared by any of the methods well known in the art of pharmacy.
1 S Methods typically include the step of bringing the active ingredients of
the
invention into association with a Garner that constitutes one or more
accessory ingredients.
For example, doses of the pharmaceutical compositions will vary-
depending upon the subject, type of indication to be treated, and upon the
particular route of administration used. Dosages of active peptide when
treating acute IgE-mediated responses can range from 0.1 to 100,000 ~g/kg
a day, more preferably 1 to 10,000 ~g/kg. Most preferred dosages range
from about 1 to 100 ~g/kg of body weight, more preferably from about 1 to
20 ~g/kg and most preferably 10 to 20 ~g/kg. Dosages of active peptide
when treating chronic IgE-mediated responses can range from 0.1 to
100,000 ~g/kg a day, more preferably 1 to 10,000 ~g/kg. Most preferred
dosages range from about 1 to 1000 ~g/kg of body weight, more preferably
from about 1 to 100 ~g/kg and most preferably 50-70 ug/kg. Doses are
typically administered from once a day to every 4-6 hours depending on the
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severity of the condition. For acute conditions, it is preferred to administer
the peptide every 4-6 hours. For maintenance, it may be preferred to
administer only once or twice a day. Preferably, from about 0.18 to about
16 mg of peptide are administered per day, depending upon the route of
administration and the severity of the condition. Desired time intervals for
delivery of multiple doses of a particular composition can be determined by
one of ordinary skill in the art employing no more than routine
experimentation.
Routes of administration include oral, parenteral, rectal.
intravaginal, topical, nasal, ophthalmic, direct injection, etc. In a
preferred
embodiment, the peptides of this invention are administered to the patient
in an IgE downregulating effective amount. An exemplary pharmaceutical
composition is an IgE modulating effective amount of a peptide in accord
with the present invention that provides an IgE downregulating effect,
typically included in a pharmaceutically acceptable carrier.
The term "pharmaceutically acceptable carrier" as used herein, and
described more fully below, includes one or more compatible solid or liquid
filler diluents or encapsulating substances that are suitable for
administration to a human or other animal. In the present invention, the
term "carner" thus denotes an organic or inorganic ingredient, natural or
synthetic, with which the molecules of the invention are combined to
facilitate application. The term "IgE modulating-effective amount" is that
amount of the present pharmaceutical compositions, which produces an
IgE downregulating effect on the particular condition being treated. Various
concentrations may be used in preparing compositions incorporating the
same ingredient to provide for variations in the age of the patient to be
treated, the severity of the condition, the duration of the treatment and the
mode of administration.
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The carrier must also be compatible. The term "compatible", as used
herein, means that the components of the pharmaceutical compositions are
capable of being commingled with a small peptides of the present invention,
and with each other, in a mariner such that does not substantially impair
the desired pharmaceutical efficacy.
The small peptides of the invention are typically administered per se
(neat). However, they may be administered in the form of a
pharmaceutically acceptable salt. Such pharmaceutically acceptable salts
include, but are not limited to, those prepared from the following acids:
hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, malefic, acetic,
salicylic, p-toluene-sulfonic, tartaric, citric, methanesulphonic, formic,
malonic, succinic, naphthalene-2-sulfonic, and benzenesulphonic. Also,
pharmaceutically acceptable salts can be prepared as alkaline metal or
alkaline earth salts, such as sodium, potassium or calcium salts of the
carboxylic acid group. Thus, the present invention provides pharmaceutical
compositions, for medical use, which comprise peptides of the invention
together with one or more pharmaceutically acceptable carriers thereof and
optionally any other therapeutic ingredients.
The compositions include those suitable for oral, rectal, intravaginal,
topical, nasal, ophthalmic or parenteral administration, all of which may be
used as routes of administration using the materials of the present
invention. Pharmaceutical compositions containing peptides of the present
invention may also contain one or more pharmaceutically acceptable
carriers, which may include excipients such as stabilizers (to promote long
term storage), emulsifiers, binding agents. thickening agents, salts,
preservatives, solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the Like.
The use of such media and agents for pharmaceutical active substances is
well known in the art. Except insofar as any conventional media or agent is
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incompatible with the peptide of this invention, its use in pharmaceutical
preparations is contemplated herein. Supplementary active ingredients can
also be incorporated into the compositions of the present invention.
S Compositions suitable for oral administration are typically prepared
as an inhalation aerosol, nebule, syrup or tablet. Compositions suitable for
topical administration typically are prepared as a cream, an ointment, or a
solution. For treating an acute IgE-mediated response, the concentrations
of the peptide active ingredient in such compositions is typically less than
1000 ~g/ml, more preferable less than S00 ~g/ml, and most preferably
from about 200 to 400 ~g/ml. For treating a chronic IgE mediated
response, the concentrations of the peptide active ingredient in such
compositions is typically less than 3 mg/ml, more preferable less than 2
mg/ml, and most preferably from about 1 to 1.5 mg/ml.
Compositions of the present invention suitable for inhalation
administration may be presented, for example, as aerosols or inhalation
solutions. An example of a typical aerosol composition for treating acute
IgE-mediated responses consists of about 0.1 to 100 ug of microcrystalline
peptide suspended in a mixture of trichloro-monofluoromethane and
dichlorodifluoromethane plus oleic acid, per dose. A more preferable
amount of microcrystalline peptide in the composition is 1 to 50 fig, and
most preferable is 10 to 20 ~g per dose of the aerosol composition. An
example of a typical aerosol composition for treating chronic IgE-mediated
responses consists of about 0.1 to 1000 ~g of microcrystalline peptide
suspended in a mixture of trichloro-monofluoromethane and
dichlorodifluoromethane plus oleic acid, per dose. A more preferable
amount of microcrvstalline peptide in the composition is 1 to 100 fig, and
most preferable is 50 to 70 ~g per dose of the aerosol composition . An
example of a typical solution consists of the desired quantity of peptide
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dissolved or suspended in sterile saline (optionally about 5 % v/v
dimethylsulfoxide ("DMSO") for solubility), benzalkonium chloride, and
sulfuric acid (to adjust pH).
5 Compositions of the present invention suitable for oral
administration also may be presented as discrete units such as capsules,
cachets, tablets or lozenges, each containing a predetermined amount of the
peptide of the invention depending on the type of IgE mediated response to
be treated, or which may be contained in liposomes or as a suspension in
10 an aqueous liquor or non-aqueous liquid such as a syrup, an elixir, or an
emulsion. An example of a tablet formulation base includes corn starch,
lactose and magnesium stearate as inactive ingredients. An example of a
syrup formulation base includes citric acid, coloring dye, flavoring agent,
hydroxypropylmethylcellulose, saccharin, sodium benzoate, sodium citrate
15 and purified water.
Compositions suitable for parenteral administration conveniently
comprise a sterile aqueous preparation of the molecule of the invention,
which is preferably isotonic with the blood of the recipient. This aqueous
preparation may be formulated according to known methods using those
suitable dispersing or wetting agents and suspending agents. The sterile
injectable preparation may also be a sterile injectable solution or
suspension in a non-toxic parenterally-acceptable diluent or solvent, for
example as a solution in 1,3-butane diol. Among the acceptable vehicles
and solvents that may be employed are water, Ringer's solution and isotonic
sodium chloride solution. In aqueous solutions, up to about 10 % v/v
DMSO or Trappsol can be used to maintain solubility of some peptides.
Also, sterile, fixed oils may be conventionally employed as a solvent or
suspending medium. For this purpose, a number of fixed oils can be
employed including synthetic mono- or diglycerides. In addition, fatty acids
(such as oleic acid or neutral fatty acids) can be used in the preparation of
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Missing upon filing
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A protocol for administration of ovalbumin (OVA) as a model allergen
has been developed to induce acute allergen-specific pulmonary disease in
normal Balb/C mice. The protocol involves intraperitoneal (i.p.)
immunization of mice with 100 ~g of ovalbumin (OVA) in alum adjuvant on
days 1 and 14, and single intranasal (i.n.) doses of 50 to 100 ~g of OVA in
normal saline on days 14, 25, 26, and 27. Control mice receive alum alone
by i.p. injections, and normal saline alone by i.n. administrations. On day
28, OVA-immunized mice display a disease strikingly similar to
allergen-induced human asthma. This animal model has been used for the
evaluation of drug efficacy in allergic acute pulmonary disease.
The Mouse Model for Late-Phase Chronic Allergen-Specif c Pulmonary Disease
The protocol for administration of ovalbumin (OVA) as a model
allergen to induce late-phase chronic allergen-specific pulmonary disease in
normal Balb/C mice includes intraperitoneal (i.p.) immunization of mice
with 100 ~.g of ovalbumin (OVA) in alum adjuvant on days I and 14, and
single intranasal (i.n.) doses of 50 to 100 ug of OVA in normal saline on
days 14, 25. 26, and 27 and then weekly thereafter for up to 6 months.
Control mice receive alum alone by i.p. injections, and normal saline alone
by i.n. administrations. On day 28, OVA-immunized mice display a disease
strikingly similar to allergen-induced human asthma. This animal model is
also useful for the evaluation of drug efficacy in chronic allergic pulmonary
disease.
Materials and Methods
Special Reagents: Crystalline OVA was obtained from Pierce Chem.
Co. (Rockford, IL) and aluminum potassium sulfate (alum) from Sigma
Chemical, St. Louis, MO. The OVA (500ug/ml) was mixed with equal
volumes of 10% (~~/vol.) alum in distilled water. The mixture was adjusted
to pH 6.5 with 10 N NaOH and incubated for 60 min at room temperature.
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The material was centrifuged at 750 g for 5 min; the pellet was resuspended
to the original volume in distilled water and used within 1 hr.
Immunization Protocol: the immunization protocol consisted of
intraperitoneal administration of 100 ~g OVA in alum on day 1 followed by
intraperitoneal administration of 100 ~g OVA in alum combined with
intranasal administration of 100 ~g OVA in saline on day 14. On days 25,
26, and 27 the mice were challenged with intranasal OVA (100 ug in saline).
For acute asthmatic studies, the animals were euthanized on day 28 and
lungs removed. For chronic asthmatic studies, mice were immunized
weekly thereafter for up to 6 months.
Analyses
ELISA protocol for serum IgE: Immulon 2 Microtiter plates (Dynex
Technologies) were coated with OVA solution in 50 mM carb-bicarbonate
buffer, pH 9.6 at 4 C overnight and blocked with 0.1% casein for 2 hr at
room temperature (RT). All test sera were diluted 1:100 in Tris-NaCL buffer,
pH 8.0 containing 0.1 % casein prior to incubation with OVA coated plates.
A positive serum sample known to contain IgE antibodies to OVA and
normal serum samples from unimmunized mice were included in each
assay as controls. The serum samples were incubated on the plates at room
temperature for 2 hours and washed 6X with PBS. Appropriately diluted
secondary antibody (sheep, antimouse IgE Biotin (Binding Site cat # PB
284, lot # 026917) was added for 2 hr at RT an d the plates were washed 6X
with PBS. OPD, Urea and peroxide solution were added for 30 min at RT.
The reaction was stopped with 2.5 M sulfuric acid. OD was read at 490/630
nm. All samples were run in duplicate. Inter and intra sample variation of
positive controls was less than 10% of the means.
Lung Histology: The lung and trachea were removed and fixed in
10°~0
neutral buffered formalin. The tissues were embedded in paraffin and cut
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into 7 ~m sections. After deparaffinization and hydration, the sections were
stained with eosinophil staining solution and counterstained with
methylene blue. Alcian blue, toluidine blue, and periodic acid Schiff stains
identified mucus within the airway. Tissues were examined by light
microscopy.
Bronchoalveolar Lavage (BAL): The left lung was tied off at the
mainstem bronchus. The right lung was ravaged with 0.4 ml of normal
saline three times, and the fluid pooled. The total cell number was
determined using a hemocytometer. The remaining cells were pelleted by
centrifugation and the cells placed into a 10% BSA solution and
resuspended. The cells were placed on a microscope slide and stained with
an eosinophil staining solution (eosin with methylene blue counterstain).
Histomorphometric Analysis of Lung: The following parameters of
allergic pulmonary disease were measured in the experiments reported
here:
1. Airway plug scores were scored as previously reported (Henderson
et al. J. Exp. Med. 184:1483-1494, 1996). A scoring system from + to ++++
was used, reflecting the degree of severity of mucus secretion.
2. Total mucous cells were estimated by randomly counting the number
of epithelial cells containing mucus per 100 epithelial cells in medium to
large airways (600 ~m to 1,000 ~m diameters). Ten fields were counted in
different lung lobes.
3. Cell density of infiltrating cells located either in the perivascular
compartment or in the areas adjacent to airways (neutrophils, eosinophils,
monocytes and lymphocytes) was approximated by using a scoring system
ranging from o to ++++, A score of + indicates an inflammatory cell layer of
3 but less than 5 cells; ++ indicates an inflammatory density of 5 cells to 10
cells; +++ indicates an inflammatory density of 10 to 20 cells; and ++++
indicates an inflammatory density of 20 to 40 cells.
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4. Numbers of various cell types were quantified by counting the
numbers per high power held (1 OX by 40X).
5. Degree of edema was calculated by using a scoring system wherein
the degree of accumulation of fluid surrounding blood vessels was
5 estimated.
Statistical Analyses of Histomorohometric Data: SigmaStat version
2.0 was used to perform statistical analyses. Differences were analyzed for
significance (p<0.05) by ANOVA using the appropriate posthoc tests for
10 independent means. SigmaPlot version 4.0 or GraphPad Prism was
employed for the construction of graphical representations of the data.
EXAMPLE 1: Therapeutic Dose Response of Acute Asthmatic Mice to HK-X
15 Ideally, an experiment which demonstrates that therapeutic efficacy
correlates with drug dosage will show three distinct regions of behavior:
1) At low doses, there will be no therapeutic effect; 2) At higher dosage,
therapeutic efficacy will be dose dependent; 3) The third range of doses
(highest) will not demonstrate therapeutic efficacy greater than that
20 observed at the highest middle range dose.
Dose response curves are an important source of information on
dosages safe for human use. Occasionally, when doses of drugs that
exceed the optimal therapeutic dosages are administered, toxic responses
can be observed. This is particularly true if the drug is administered in
situ,
such as intranasally.
To establish therapeutic effectiveness of a range of doses of f-met-
Leu-Phe-Phe (HK-X) during the acute effector phase of bronchial asthma at
days 25, 26 and 27 induced by repeated immunization with OVA, doses of
0.1, 1.0, 10 and 50 dug of intranasal HK-X were chosen. HK-X was
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administered 30 min before OVA challenge. Control groups consisted of
OVA-immunized and OVA-challenged mice as well as animals immunized
with Alum in saline and challenged with saline alone. All animals were
sacrificed one day after (day 28) the final OVA challenge. Serum IgE levels
were determined and serum and lung tissues were collected for further
analysis.
To first establish the optimal dose that will effectively downregulate
serum IgE levels in an acute asthma model, 0.1, 1.0, 10 and 50 ~g doses of
HK-X (in 40 ~L of saline) were infused into the lung 15-30 min prior to
antigenic challenge on days 25, 26, and 27. A dose response curve of
serum IgE levels is depicted in Figure 1.
The effects of varying doses of HK-X on the response of lung tissue to
acute allergic challenge are depicted in Figures 2A to 4C. Fifty micrograms
of HK-X administered intranasally to acute asthmatic mice provided some
degree of protection against the effects of acute asthma (Figs. 2A-2C). There
was limited perivascular and peri-bronchial accumulation of inflammatory
cells (Figs. 2A and 2B). Figure 2C demonstrates that mucus accumulation
was present but limited.
Ten micrograms of HK-X appeared to be the most efficacious dose
(Figs. 3A-3C). Figures 3A and 3B show minimal inflammatory infiltrate
surrounding vessels and airways. The degree of mucus secretion in airways
is illustrated in Figure 3C. The mucus is confined to the surface of the
airway epithelial cells.
As the dose of HK-X decreased 10 fold to 1 dug, therapeutic effect
diminished. The amount of perivascular and airway inflammation increased
(Figure 4A). There was a corresponding increase in mucus secretion by
airway epithelial cells (Figure 4 B and 4C).
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For the purposes of contrast and control, Figures 5A through C
illustrate the benign response of immunized mice to administration of
saline or the HK-X vehicle (0.05% DMSO). As shown in Figures 5A and 5B,
there was little detectable inflammatory infiltrate in the perivascular or
periairway zones of the lung. Correspondingly, there was no accumulation
of mucus in the airway lumen or on the airway epithelial cell surfaces
(Figure 5C).
Of the key histological measurements of the severity of acute asthma,
mucus plug, the numbers of eosinophils and the fraction of airway cells
secreting mucus showed a dose dependent improvement after treatment
with 0.1 dug to 10 ~g HK-X. 10 dug of HK-X provided a 70% reduction in
mucus plug score (p<0.05). Interestingly, 50 ~g of HK-X provided
significantly less reduction in mucus plug (p<0.05). This same pattern of
responsiveness was observed for the numbers of eosinophils and fraction of
airway cells secreting mucus. The 10 ~g dose of HK-X showed a 57%
decline in the number of interstitial eosinophils, which was significantly
greater than the 0.1 ug dose effect of (p<0.05). The reduction in eosinophils
by the 50 dug dose was less than one-half that provided by 10 ~g HK-X
(p<0.05).
The fraction of airway cells secreting mucus was also inhibited in a
dose dependent manner from 0.1 ~g to 10 ~g (37% reduction, p<0.05). The
50 ~g dose provided a small amount of reduction (11%) which was not
significantly less than the 0.1 ~g dose of HK-X. The effect of HK-X on the
accumulation of fluid surrounding vessels showed a modest decline at 10
and 50 ~g doses. However, none of the doses was different from the 0.1 ~g
dose of HK-X. The dose of HK-X showing the greatest reduction in the
inflammatory cell score or accumulation of inflammatory cells was 10 fig.
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These data demonstrate that the following parameters showed a dose
response effect: serum IgE levels and histopathological features (cellular
infiltration, mucus plug formation, and total eosinophlis in interstitium).
Ten micrograms of HK-X administered intranasally was the most effective
dosage compared to lower doses and a higher dose, 50 fig. Compared to
controls, animals treated with 10 ~g of HK-X demonstrated a 60% reduction
in serum IgE levels, 50% reduction in cellular infiltration of the lung, 70%
reduction in mucus plug formation and 67% reduction in eosinophil
number.
EXAMPLE 2: Chronic Asthma With and Without HK-X Intervention
The animal model is also useful for the evaluation of drug efficacy in
chronic allergic pulinonary disease. In this study, an immunization period
of 6 months induced a persistent inflammation that was maintained by
weekly intranasal challenges with OVA. The mice were treated with saline 8
times over a 20 day period to assess changes which occurred in the lungs.
HK-X treatments of mice with chronic asthma were performed as indicated
in Figure 6. ~0 ~.g of HK-X (in 50 ~L of saline containing less than
2.5°~0
DMSO) was administered i.n. for a total of 8 dosages delivered over a period
of 16 days. The animals were sacrificed 4 days after the last saline or HK-X
dose. The experimental results were compared between HK-X treated and
HK-X untreated mice.
IgE levels of antibody to OVA in the blood of mice challenged with or
without OVA are shown in Table 1. It is important to note that all animals
were OVA immunized for the first 6 months, however, the group denoted as
"saline" were administered saline intranasally but not challenged with OVA
during the terminal 20 day period. Therefore, these IgE levels were carried
over from the immunization period and were used as background values
from which all comparisons were corrected. For example, animals treated
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with either saline or DMSO and OVA challenged had a 36% increase in IgE
levels compared to a 14% increase in IgE levels in the animals treated with
HK-X and OVA challenged. The amount of suppression of IgE levels by
HK-X was calculated to be -60%.
TABLE 1: IgE Values in the Sera of Chronic Asthmatic Mice
TREATMENT MEAN OD b'SE P VALUE
DMSO/OVA 1.115 40.017 @0.111
SALINE/OVA 1.113 40.093 @0.111
HK-X/OVA 0.929 b'0.033 0.049
@ 0.111
SALINE 0.814 b'0.079 @0.111
Note: These values represent relative IgE levels as OD values from
the ELISA test.
One of the important characteristics of chronic asthma in the murine
model is the appearance of granulomatous structures in the lung. The
effective IgE downregulating dose of 50 ~g of HK-X significantly (p<0.05)
reduced the numbers and sizes of these structures in the lungs of treated
animals compared to animals permitted to spontaneously reduce collagen
deposition (Saline or DMSOJ (Fig. 7).
Furthermore, during the immunization of mice with OVA and the
subsequent treatment with HK-X at a dosage of 50 ~g for a total of 8 times
over a 20 day period, no adverse reactions or signs of sickness were
observed. The mice were active during the experimental period.
Examination of the lung tissues from groups of animals immunized with
only OVA revealed severe pulmonary pathological changes consistent with
characteristics of chronic asthma observed in humans. There was a
significant infiltration of inflammatory cells in association with outer
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boundaries of the airway basal lamina (interstitial regions) and blood
vessels (Fig. 8C). When animals were treated with 8 doses of 50 ~g of HK-X
per treatment over a 20-day period, the number of inflammatory cells were
clearly reduced around airways and blood vessels (Fig. 8B). Saline
5 inhalation by saline sham or control immunized mice resulted in patent
airways and blood vessels with normal appearance (Fig. 8A). OVA
immunized mice contained increased accumulations of collagen (blue color)
around vessels and airways (Figure 9C). However, lungs treated with HK-X
demonstrated a reduced level of collagen deposition (Figure 9B). In control
10 mice (administered HK-X in saline) the pulmonary tissue was free of
inflammatory cells and fibrotic collagen deposits (Figure 9A). In contrast, in
OVA sensitized mice and treated only with either saline (Figures 10A- l OC)
or 0.5% solution DMSO (Figures 11A-11C), airways contained mucus and
had mucus secreting cells when Alcian blue at pH 2.3 was used to visualize
15 mucus. The amount of perivascular and airway inflammatory cell
infiltration was similar. Greater than 60 percent of the airways of these 6
month-old chronic asthmatic mice were plugged with mucus (Figs. l0A-lOB
and 1 1A-11 B, respectively) .
20 The pulmonary tissue changes in chronic asthma were also assessed
by morphometry methods to quantify the degree of persistent inflammation,
deposition of fibrotic collagen and airway narrowing with structural
changes. In Figures 12 through 14, the responses of animals immunized
with OVA for 6 months and treated with various agents are shown in the
25 left panels of the figures, whereas animals sham immunized and treated
with saline are shown in the right panel. There were significant differences
(p< 0.05) between the degree of mucus plug formation in HK-X treated
animals versus animals given only saline for the 20 day period (Fig. 12).
The animals insumated with the vehicle (0.5 % DMSO) for H K-X compared
to HK-X also did not demonstrate any improvement in the mucus plug
score. The results were the same for saline treatments. The patterns of
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responses for inflammatory cell accumulation in and about the airways
paralleled the mucus plug data for the various experimental treatments
(Fig. 12). When given 8 doses of 50 ~.g each of HK-X in 40 ~uL vehicle
intranasally over a 1 6-day period, there was a significant reduction in
mucus accumulation and mucus cells occurrence in the airway (Fig. 13).
Again the saline treatment alone demonstrated no therapeutic effects but
HK-X did significantly reduce the number of mucus containing cells within
the airways (p<0.05). This observation further substantiates that there was
no spontaneous repair after establishment of OVA-induced chronic asthma.
In the analyses of the numbers of infiltrating inflammatory cells in
association with airway, the data showed that eosinophils and neutrophils
per unit area was also reduced (Fig. 14). Intranasal saline infused animals
maintained high levels of eosinophils as did DMSO treated animals
(p>0.05); however, HK-X treatment reduced the numbers of eosinophils
significantly compared to the two control groups (p<0.05).
These studies show that there was very little or no spontaneous
reduction in airway inflammation or of mucus cell secretion in this model of
allergen induced chronic asthma unless HK-X was administered. In this
model, mice were sensitized to OVA and exposed to OVA via intranasal
route weekly for 5 months and were treated with HK-X intranasally 8 times
over a 20 day period. This allergen immunization and challenge regimen led
to a chronic airway infiltration of eosinophils and other types of
inflammatory cells, accumulation of mucus in the airways and hyperplasia
of mucus secreting cells. Administration of an effective IgE-downregulating
dose of 50 ~g of HK-X reduced airway hypersecretion, hyperplasia of mucus
cells, and recruitment of eosinophils and neutrophils. These results indicate
that by administering an efffective amount of HK-X and downregulating IgE
levels, HK-X can also downregulate IgE-mediated responses such as airway
hypersecretion of mucus and the deposition of collagen that occur in this
allergen-induced model of asthma.
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EXAMPLE 3: Effects of HK-X and Dexamethasone in Acute Murine Asthma
Glucocorticoids are potent inhibitors of inflammatory mediators
produced by a variety of cell types, including T cells, mast cells, monocytes,
dendritic cells and eosinophils. Glucocorticoids are effective in the
treatment of human asthma when inhaled or used systemically. They
suppress inflammatory cell infiltration and have been demonstrated to
decrease mucus secretion and pulmonary edema. These responses relate to
direct effects of glucocorticoids on bronchial epithelial cells. Equally
important, steroids reduce bronchial hyperresponsiveness. Because of their
demonstrated efficacy, the glucocorticosteroids represent a mainline
therapeutic armamentarium in the treatment of asthma and could be used
without reservation but for well documented cumulative toxicity that limits
their value over time. Because of their value as the current standard of
efficacy for asthma, new compounds fcr asthma treatment should be
evaluated in comparison to glucocorticoids.
This experiment compared the effectiveness of HK-X to
dexamethasone, a widely used glucocorticoid, to modulate mucus release,
eosinophil numbers, edema and allergen specific IgE levels in this mouse
model. Comparable dosages of 10 ~g and 50 ~g of HK-X and
dexamethasone were used in this study. Intranasal administration was
used for both drugs at all doses. Fifty micrograms of HK-X was selected as
the high dose based on the previous results from the chronic asthma
model. An outline of the immunization and treatment protocol is shown in
Figure 15.
The experimental parameters showed that 10 dug of intranasal HK-X
was more effective than either 10 ~g or 50 dug of dexamethasone in reducing
serum IgE levels (Figure 16). Ten ~g of HK-X reduced serum IgE levels by
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28%. HK-X was also more effective than dexamethasone at improving two
histopathological features: cellular infiltrate and total number of
eosinophils
in interstitium. Both 10 ~g and 50 ,ug doses of HK-X significantly reduced
inflammatory infiltrate by 54% compared to the OVA control (p<0.05) and
were significantly more effective than 10 ~g ( 13%) and 50 ~g ( 13%) of
dexamethasone (p<0.05). Both the 10 ~g and 50 dug doses of HK-X were
more effective than 10 ug of dexamethasone (p<0.05). Ten ug of HK-X
decreased the eosinophil cell count by 57%, while the same dose of
dexamethasone decreased the eosinophil cell count by 13%.
A high dose of 50 micrograms of HK-X administered intranasally was
as effective as either dosage of dexamethasone in reducing the following
histopathological features: number of eosinophils in Bronchoalveolar
Lavage (BAL), mucus plug formation, percentage of airway mucus secreting
cells, number of interstitial eosinophils, and edema. These results establish
the comparative efficacy of HK-X and a glucocorticoid, dexamethasone, in
the murine asthma model.
EXAMPLE 4: Effects of HK-X and a Related Control Peptide in Acute
Murine Asthma
This experiment compared the effectiveness of HK-X to a related
member of the peptide family, f Met-Met (referred to as the control peptide)
in relation to the following measures: mucus release, cellular inf'lltration,
eosinophil numbers, edema and allergen specific IgE levels in the acute
asthma mouse model. Comparable dosages of 50 ug of HK-X and the
control peptide were used in this study. Intranasal administration was used
for both compounds. The immunization and treatment regime is outlined
in Figure 17.
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While belonging to the same family of chemical compounds and
being closely related in molecular size, the control peptide did not exhibit
any of the therapeutic properties of HK-X. Most significantly, 50 ~g of HK-X
caused a 7% decrease in the serum IgE levels in the sera to the allergen,
OVA (p>0.05). 50 ~g of the control peptide did not affect the serum IgE
levels (p>0.05). Furthermore, the control peptide delivered in vehicle and
administered to control animals promoted clear-cut pro-inflammatory
increases in the following parameters: mucus plug formation, number of
airway cells secreting mucus, and the degree of interstitial inflammatory
cells. In previous experiments, HK-X did not demonstrate any
pro-inflammatory changes in the histological parameters measured.
Therefore, the unique composition of HK-X appears responsible for its
efficacy in downregulating IgE levels and IgE-mediated responses.
EXAMPLE 5: Pulmonary Tissue Response to Long Term Dosing of High
Therapeutic Levels of HK-X
To determine whether there are potential toxic effects of long-term
intranasal exposure to HK-X at the higher end of the therapeutic dose
range, mice were exposed to weekly doses of 20 ~g of intranasal HK-X for 3
months. During the last two weeks, the intranasal dose of HK-X was
increased to 50 fig. Lung tissue was collected 24 hr after the last H K-X
administration for histological analysis.
Weekly administration of 20 ug of HK-X intranasally for 3 months
followed by 2 weeks of administration of 50 ~g did not cause pathologic
alterations of lung tissue. There was no difference (p>0.05) between saline
and HK-X administration regarding mucus plug formation and
inflammatory infiltrate. Secretion of mucus by airway cells was elevated
after administration of HK-X but this is not judged to be biologically
significant. A similar phenomenon was observed regarding the number of
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interstitial eosinophils. While HK-X treated animals had approximately 2
eosinophils per 2,200 ~2, the saline treated animals demonstrated less than
1 per unit area (p<0.05). Livers, spleens, and kidneys were examined for
pathological changes. Except for occasional foci of inflammatory cells in the
5 livers of animals from control and treated groups, no pathologic changes
were observed. These data establish the tolerability of supra-therapeutic
doses of HK-X in mice.
EXAMPLE 6: Immunogenicity and Antigenicity of HK-X
The objective of this study is to determine whether HK-X, when
administered to mice via several different routes, will produce an immune
response as assessed by antibody production. Thus, immunogenicity and
antigenicity were both evaluated in relation to HK-X in this study.
HK-X is a small tetrapeptide. In most cases, such small molecules
are poorly immunogenic; however, in vivo, small molecules may conjugate
or absorb to (become haptens) larger proteins or to blood cells (carriers).
Penicillin, quinidine and a-methyl dopa allergic responses are examples of
such haptenic responses. Antibodies to the haptens can produce anemia
and immune complex diseases because of the destruction of red cells
(Garners).
A number of haptens such as dinitrophenol (DNP) or trinitrophenol
(TNP) used experimentally are covalently linked to carrier molecules. The
more antigenic the Garner molecule, the more likely that an immune
response to the hapten will be elicited. Keyhole Limpet hemocyanin (KLH) is
a widely used Garner and generally supports potent antibody responses to
haptens like DNP or TNP.
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The use of adjuvants greatly increases the likelihood that a potential
immunogen will elicit an immune response. Complete Freunds Adjuvant
(CFA) or bacterial peptidoglycans have been widely used to stimulate
immune responses to poorly immunogenic haptens.
Therefore, after first determining availability of antibodies from
normal drug exposure routes (with no anti- HK-X reactivity), the potential
immunogenicity of HK-X was examined when it was coupled to KLH and
administered in bacterial adjuvant. These extreme conditions determined
whether HK-X could be immunogenic.
Materials and Methods
Immunogenic Conjugates of HK-X: HK-X was conjugated to KLH via a
12 to 20 carbon spacer added at the carboxy terminus. The linkage was
completed through lysine residues on the KLH. United Biochemical, Seattle,
WA, prepared the conjugates.
Preparation of Immunogen: HK-X-KLH conjugate suspended in PBS
at 0.1 mg/ml was emulsified in complete Freund's adjuvant (CFA)
containing 1.0 mg/ml bovine Mycobacterium tuberculosis at a 1:1 ratio.
Adjuvant Immunization Protocol: Balb/C female mice were
immunized intradermally with 0.1 ml emulsion, boosted 4 weeks later and
bled at 6 weeks.
Soluble Immunization Protocol: Balb/C female mice were injected
intraperitoneally with 100 ~g of the conjugate without adjuvant in a volume
of 0.1 ml to 0.2 ml. The mice were bled after 21 days.
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Normal Drug Exposure Routes: Sera were collected from animals
administered HK-X via the intranasal route in therapeutic asthma studies.
Determination of antibodies: ELISA analyzed antibodies to
conjugated and unconjugated HK-X. Immulon 2 Microtiter Plates (Dynex
Technologies cat. # 3455) were coated overnight at 4°C with the
following
HK-X or HK-X conjugates at 10 ~g/ml in PBS:
HK-X- peptide alone
HK-X - KLH - peptide conjugated to KLH
~ HK-X LISA- peptide conjugated to BSA
HK-X - Spacer- peptide with 12 carbon linear spacer.
Wells were washed the following day with PBS and then blocked for 30
minutes at room temperature with sample dilution buffer consisting of 0.1
M Tris - 0.15M NaCI buffer, pH 8.0, and 0.1% casein (ICN cat # 902896, lot
99333). Mouse sera samples were diluted either 1:100 or 1:200 with the
same buffer, added to the wells and incubated 2 hours at room temp. Wells
were then washed with PBS and incubated with goat anti-mouse IgG
peroxidase conjugated secondary antibody (Cappel cat # 55554, lot #
39714) for 2 hours at room temp. After washing with PBS, wells were
reacted with OPD chromagen (SIGMA cat # P-9187, lot 18H82111) for 30
minutes at room temp. The reaction was stopped with 50 ~l of 2.5 M
sulfuric acid. The ODs were then determined using a BIO-TEK EL800
reader at 490/630.
Results
Determination of HK X from normal, drug exposure route: Sera from
the following groups of mice were tested for anti-HK-X reactivity: OVA-
induced asthma and HK-X treated, OVA-induced asthma and DMSO
(vehicle) treated control, saline-immunized and DMSO (vehicle) treated.
Mice were treated intranasally every other day with 50 dug of HK-X or vehicle
for 16 days.
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No IgG reactivity was observed to HK-X conjugated to either the 12-C
spacer (HK-X+Spacer), KLH (KLH-HK-X) or BSA (BSA-HK-X). IgG reactivity
to OVA was observed in all OVA-immunized mice and one saline-immunized
control mouse and served as a control for the ELISA. IgG reactivity to
KLH-HK-X and BSA-HK-X was observed in sera from animals immunized
with KLH-HK-X in adjuvant and served as a control for the coating of these
antigens onto the ELISA plate.
Soluble-immunized HK X coupled to a carrier. Mice were immunized
with soluble KLH-HK-X and bled after 21 days. The results of the ELISA
show that 4/5 serum samples reacted to KLH and KLH-HK-X but no
reactivity to BSA-HK-X or HK-X +spacer was observed indicating that
antibodies were not generated against the HK-X that was coupled to KLH
after immunization with soluble Garner coupled to HK-X.
Adjuvant-immunized HKX coupled to a carrier. To force the
generation of antibodies to HK-X, mice were immunized with KLH or
KLH-HK-X in complete Freund's adjuvant, boosted once and bled after 6
weeks. The results of the ELISA show that antibodies were generated
against KLH. Antibodies were also generated to HK-X. This was supported
by the following: 1 ) antibody reactivity to KLH-HK-X from KLH-HK-X sera
was 2 fold higher than from KLH only immune sera and 2) sera from
KLH-HK-X immunized HK-X immunized mice reacted to BSA-HK-X but not
to BSA alone. However, no antibody reactivity was observed to H K-X
coupled to the 12C spacer.
From the results of these studies, several conclusions about the
immunogenicity and antigenicity of the HK-X peptide can be made. First,
mice did not generate antibodies to HK-X after therapeutic intranasal
administration of the peptide for 16 days. Second, mice did not generate
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antibodies when immunized with soluble peptide conjugated to the
immunogenic carrier KLH. Third, mice can be forced under extreme
conditions to generate antibodies to HK-X when coupled to KLH and
immunized with complete adjuvant. However, even in this case, antibody
reactivity is probably generated to neo-epitopes created by the conjugation
of HK-X and KLH since no antibody reactivity could be detected to HK-X
conjugated to the 12C spacer. This conclusion is supported by the
observation that addition of free HK-X to the antiserum for at least 30 min
prior to incubation with the test antigen, HK-X-KLH, did not reduce
antibody reactivity to HK-X-KLH.
Thus, it appears unlikely that clinically relevant antibody or other
immune responses to HK-X will be elicited in the clinical environment.
There are five observations supporting such a notion. These observations
5 are:
1) HK-X is only four amino acids in size (less than 600 Dalton), which
makes it unlikely to become immunogenic;
2) All of the amino acids in HK-X are hydrophobic, whose property is
not associated with immunogenicity;
3) To become immunogenic, HK-X would have to become covalently or
electrostatically associated with a larger and immunogenic carrier in vivo;
4) Antibodies produced to HK-X are likely to be directed towards an
epitope formed by the combination of the carrier and HK-X (neo-antigen);
5) Antibodies directed towards the neo-antigen react only weakly (low
affinity) if at all to free HK-X.
EXAMPLE 7: Primate Toxicology Study of HK-X
This study was conducted at BIOSUPPORT, an animal research
facility in Redmond, Washington, according to GLP standards. Six adult
male and female macaque monkeys obtained from Charles River were
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studied. Group A, considered a control group, consisted of two animals
given vehicle (buffered saline with 3% DMSO) IV daily for five days. Blood
sampling for CBC and chemistries was performed on days 0-4 and 7. Group
B consisted of three animals dosed with 20 ~.g/kg of HK-X in vehicle
5 (buffered saline with 3% DMSO) IV daily for five days. Blood sampling for
CBC and chemistries was performed on days 0 - 4 and 7. Group C
consisted of the three additional animals dosed with 150 ~.g/kg IV daily in
an identical regimen. Group D consisted of all six animals from Groups B
and C, dosed with 1000 ~.g/kg IV daily using the same regimen, five days
10 after Group C animals completed their regimen. All animals were observed
daily throughout the study for recording of weight and general health and
behavior. At the end of the Group D regimen, all animals were euthanized,
underwent necropsy, and had representative tissue samples from the
following organs collected for histological analysis: liver, kidney, spleen,
15 lung, heart, lymph node, and brain. Histopathological evaluation was
performed by a board certified veterinary pathologist associated with
BIOSUPPORT and independently by a histopathologist associated with
Histatek.
20 These dosages of HK-X were selected based on the effective
therapeutic dosages of HK-X of 10 and 50 ~g/kg in the mouse asthma
model.
No significant abnormalities of white blood cell,
25 hematocrit/hemoglobin, or platelet counts were noted on any day or at any
dose level. Similarly, no significant abnormalities of chemistry values were
noted at any of the three dosing levels. No histological abnormality was
noted in representative tissue samples of spleen, and lymph nodes obtained
from animals who were exposed to either 20 or 150 ~g/kg of HK-X followed
30 by 1000 ~.g/kg daily, or in the vehicle control group. Minimal multifocal
CA 02379323 2002-O1-15
WO 01/05420 PCT/US00/19496
36
Iymphocytic infiltrate was noted in liver, kidney, heart, and lung tissue
samples from both treated and control animals and was therefore judged
unrelated to treatment. Mild glomerular lesions, common in aging
macaques, did not segregate according to treatment and were thus also
considered unrelated to treatment. Other minor histological changes were
not considered significant.
There was no discernible toxicity observed in blood counts or
chemistries obtained from six macaque monkeys exposed to dosage levels of
HK-X significantly higher than dosages considered therapeutic. Minor
histopathological changes noted in liver, kidney, spleen, lymph nodes,
heart, and lung did not segregate according to treatment and were
considered manifestations of background pathology or artifactual change
related to euthanasia.
This primate study suggests that therapeutic amounts of HK-X can
be useful in human treatment without apparent toxicity or side effects.
The invention has been described in detail with reference to preferred
embodiments thereof. However, it will be appreciated that, upon
consideration of the present specification and drawings, those skilled in the
art may make modifications and improvements within the spirit and scope
of this invention as defined by the claims.
