Note: Descriptions are shown in the official language in which they were submitted.
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GREEN PORPHYRINS AS IIrIIr1UNOMODULATORS
Technical Field
The invention is in the field of modulating immune
responses by administering green porphyrins in the absence of
light. Antigen-specific immune responses are modulated when
the green porphyrins are administered during the course of the
response to a specific antigen. In addition, green porphyrins
in the absence of light interfere with intercellular
communication, thus permitting prevention or treatment of, for
example, restenosis.
Background Art
A group of compounds useful in photodynamic therapy,
collectively designated green porphyrins, is disclosed in a
series of patents including U.S. 5,283,255; 4,883,790;
4,920,143; 5,095,030; and 5,171,749.
These green porphyrins
are prepared using a Diels-Alder reaction with hematoporphyrin
and optional rearrangement or reduction of the resulting
product. A particularly preferred form of these green
porphyrins, as outlined in the above-referenced patents, is
designated a benzoporphyrin derivative in the monoacid form,
or "BPD-MA". This drug is currently in clinical trials with
respect to photodynamic treatment of various tumors and other
conditions.
Photodynamic therapy rests on the assumption that
the photoactive compounds administered, in this case the green
porphyrins, are without physiological effect in the absence of
light. However, when irradiated, the excited forms of the
compounds exert local toxic effects. Therefore, for the
treatment of tumors, for example, advantage has been taken of
the tendency of these photoactive compounds to be retained in
tumor tissue after clearance from normal tissue has been
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effected. It has also been found that local irradiation to
areas of neovasculature is effective even before the compounds
have been cleared from normal tissue.
It has now been found that the green porphyrins have
immunomodulating activity and interfere with intercellular
communication independent of their ability to behave as
photoactive agents in PDT. The present invention takes
advantage of this property, hitherto unexpected, of the green
porphyrins.
Disclosure of the Invention
The invention takes advantage of the ability of
green porphyrins, without irradiation, to participate in an
immune response generated with respect to a particular
antigen. The resulting immunomodulation is antigen-specific,
as is illustrated hereinbelow using results obtained in the
delayed-type hypersensitivity reaction. Therefore, the
subject can be protected against unwanted responses with
respect to autoantigens, allergens and the like without the
disadvantage of being immunocompromised generally. In
addition, the green porphyrins interfere with intercellular
communication such as that which results in thrombosis as
illustrated hereinbelow. The green porphyrins are also shown
to have conformational similarity to known integrins
characterized by the sequence RGD. This permits use of the
green porphyrin to prevent or treat conditions characterized
by unwanted intercellular communication.
Thus, in one aspect, the invention is directed to a
method to modulate an antigen-specific immune response, which
method comprises administering to a subject who is
experiencing an activated immune response to an antigen, an
amount of green porphyrin effective to modulate said immune
response to the antigen, in the absence of light absorbed by
the green porphyrin, wherein said administering is during the
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active phase of the immune response to the specific antigen
per se.
In another aspect, the invention is directed to a
method to modulate intercellular communication in a subject,
which comprises administering to a subject in need of such
modulation, an amount of green porphyrin effective to
modulate the intercellular communication in the absence of
light absorbed by the green porphyrin, said administering
being performed during the course of the unwanted
intercellular communication.
In other aspects, the invention includes
formulations of green porphyrins useful in the method of the
invention.
This invention provides the use of green porphyrin
for modulating, under ambient light conditions, an antigen
specific immune response in a subject.
This invention also provides the use of green
porphyrin for the manufacture of a medicament for use in
modulating, under ambient light conditions, an antigen
specific immune response in a subject.
This invention also provides the use of a green
porphyrin for modulating, under ambient light conditions,
inappropriate intercellular communication.
This invention also provides the use of a green
porphyrin for the manufacture of a medicament for use in
modulating, under ambient light conditions, inappropriate
intercellular communication.
Various embodiments of this invention provide the
use of green porphyrin for treating under ambient light
conditions, a condition which activates an antigen specific
immune response selected from allergy, autoimmune disorder,
rejection of a transplant, rejection of an allograft or
xenograft, rheumatoid arthritis, multiple sclerosis, lupus
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erythematosus, psoriasis, diabetes, and inflammation in a
subject.
Other embodiments of this invention provide the
use of green porphyrin for the manufacture of a medicament
for use in treating, under ambient light conditions, a
condition which activates an antigen specific immune
response selected from allergy, autoimmune disorder,
rejection of a transplant, rejection of an allograft or
xenograft, rheumatoid arthritis, multiple sclerosis, lupus
erythematosus, psoriasis, diabetes, and inflammation in a
subject.
Other embodiments of this invention provide a
composition comprising a pharmaceutically acceptable
excipient and a green porphyrin, for use in treating, under
ambient light conditions, a condition which activates an
antigen specific immune response selected from allergy,
autoimmurie disorder, rejection of a transplant, rejection of
an allograft or xenograft, rheumatoid arthritis, multiple
sclerosis, lupus erythematosus, psoriasis, diabetes and
inflammation.
Medicaments and compositions manufactured in
accordance with this invention may be formulated as a
liposomal composition suitable for systemic administration.
Medicaments for treatment of psoriasis may be formulated for
topical administration.
Inappropriate intercellular communication that may
be modulated in accordance with this invention may be
conditions which result in restenosis or thrombosis.
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Brief Description of the Drawings
Figure 1 shows representative structures for the
green porphyrins useful in the methods of the invention.
Figure 2 shows the superimposed conformation of the
family of conformations represented by RGDA.
Figure 3 shows the superimposed conformation of the
family of conformations represented by RGDB.
Figure 4 shows the superimposed conformation of the
family of conformations represented by RGDC.
Figure 5 shows the superimposition of the three-
dimensional structure of BPD-MPA with a representative
conformation of RGDA.
Figure 6 shows the superimposition of the three-
dimensional structure of BPD-MA with a representative
conformation of RGDB.
Figure 7 shows the superimposition of the three-
dimensional structure of BPD-MA with a representative
conformation of RGDC.
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Figure 8 shows a comparison of the influence of four
BPD analogueues on the DTH response in naive, unshaven Balb/c
mice. =
Modes of Carrying Out the Invention
There are numerous instances in which an immune
response to a particular antigen is undesirable. Prominent
among these situations are allergic responses, autoimmune
responses, and immune rejections of organ transplants, skin
grafts and the like. The green porphyrins of the invention
are effective when administered in the active phase of
eliciting an immune response, and are thus effective when
administered in the time period following exposure to the
antigen, or if the immune response in its active form is
continuous, during this continuous phase.
The green porphyrins exert their effects in the
absence of radiation absorbed by them. By this phrase is
meant that no deliberate radiation of the target is employed.
Minimal background light may still be present. As described
in the Background section above, green porphyrins have been
used extensively in photodynamic therapy protocols. In these
protocols, a group of cells or a tissue modified to contain
substantial amounts of the green porphyrin is deliberately
irradiated'with light including wavelengths absorbed by the
green porphyrin compounds. The absorption of these
wavelengths by the green porphyrins results in excitation of
the molecules in such a way that surrounding materials are
damaged. It is assumed that singlet oxygen is generated in
the course of decay of the excited compounds which accounts
for the toxicity. The photoactive agent itself, is presumed to be nontoxic.
In the methods of the present invention, irradiation
with light to effect excitation is not included in the
protocol. There is no necessity, however, to block out all
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light from the subject being treated. Thus, as used herein,
the phrase "in the absence of radiation absorbed by the green
porphyrin" refers to typical ambient conditions rather than
total darkness. It simply indicates that the known
= 5 photodynamic effects of the green porphyrins are not employed
in the methods of the invention.
Green Porphyrins
The nature of the green porphyrins is described in
the patents referenced in the Background section hereinabove.
Briefly, these are derivatives of protoporphyrin IX that are
obtainable using a Diels-Alder reaction with a substituted
acetylene, optionally followed by rearrangement and/or
reduction. Typical formulas for the compounds thus obtained
are shown in Figure 1. Preferred embodiments of the formulas
shown in Figure 1 are those wherein the ring system has the
formulas shown in Figures 1-3 or 1-4; and/or wherein each of R1
and R 2 is independently selected from the group consisting of
carbalkoxyl (2-6C), alkyl (1-6C), arylsulfonyl ( 6-10C) , cyano;
and -CONR5CO wherein R5 is aryl (6-10C) or alkyl (1-6C); each
R3 is independently carboxyl, carboxyalkyl (2-6C) or a salt,
amide, ester or acylhydragone or is alkyl (1-6C); R4 is CH=CH2
or -CH (OR4' ) CH3 wherein R4' is H, or alkyl (1-6C) optionally
substituted with a hydrophilic substituent. Of course,
mixtures can be used.
Particularly preferred are those compounds of
Figures 1-3 and 1-4 wherein each of R1 and R2 is independently
carbalkoxyl (2-6C); one R3 is carboxyalkyl (2-6C) and the other
R3 is the ester of a carboxyalkyl (2-6C) substituent; and R" is
' 30 CH=CH2 or -CH (OH) CH3.
Especially preferred is the compound shown as Figure
' 1-3 wherein R' and R2 are methoxycarbonyl; one R3 is
-CH2CHZCOOCH3 and the other R3 is CH2CH2COOH; and R' is CH=CHZ.
This latter compound is referred to as BPD-MA, an acronym for
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benzohydroporphyrin derivative monoacid wherein the Diels-
Alder addition occurs on ring A.
Nature of the Conditions Treated
The green porphyrins, when administered to a
vertebrate subject during the course of an activated immune
response to a particular antigen, modulate the response to the
specific antigen without resulting in any overall
immunosuppression. The timing of administration is
facilitated if the subject is naive with respect to the
immunogen/antigen administered. Unwanted immune responses to
deliberately administered antigens are found, for example, in
transplantation protocols using allografts, such as skin
transplants or organ transplants, individual cellular
transplants, such as those used in insulin replacement for
diabetes, and any other surgical procedure or procedure
involving injection wherein foreign cells or tissues are
deliberately introduced into the subject. Other instances
where an unwanted immune response may occur to a deliberately
administered compound include those wherein proteins of
species xenogeneic to those of the subject are used. A
typical example is the administration of monoclonal antibodies
in the treatment of tumors. In many cases, although attempts
at humanizing antibodies have been made, monoclonal antibodies
derived from mice are used in human therapy. An undesired
immune response to such antibodies can be modulated by the
methods of the invention. In these instances, a primary
immune response is activated and the administration of green
porphyrins contemporaneously with or immediately following the
administration of the antigen provides the desired effect.
Another instance wherein an immune response occurs =
to a foreign antigen, is illustrated by allergic reactions.
While these responses are generally secondary immune
responses, administration of the green porphyrins of the
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invention simultaneously with this secondary exposure, or in a
time period proximal thereafter, is also effective.
In both of the foregoing cases, the green porphyrin
will be administered at the same time that the antigen or
allergen is administered or within the time period required
for the active response to the antigen. In general, this time
period is within 24-48 hours of the administration of antigen.
The subjects for whom the methods of the invention
are appropriate are generally vertebrate subjects, preferably
mammalian subjects. However, vertebrate organisms generally
utilize similar intercellular communication systems and
assemble immune responses in analogueous ways. Particularly
preferred subjects are domesticated animals and avian
subjects, as well as human subjects. The appropriate
protocols, dosage, and formulation will depend, of course, on
the nature of the subject.
Administration of the Antigen
The administration of the antigen simultaneously
with or proximally before the administration of the green
porphyrin will vary depending on the nature of the allergin.
For deliberately administered antigens, such as drugs,
monoclonal antibodies or other foreign proteins which are used
for therapeutic or diagnostic purposes, the dosage level and
form of administration will be controlled by the purpose for
which the antigen is administered generally. The antigen is
typically already available in a suitably formulated
pharmaceutical composition and the dosage levels and expected
routes of administration are already known.
` 30 With respect to allografts, it is generally believed
that the cells that define tissues or organs from members of
the same species as the intended recipient, including
vascularized organs such as the heart, kidney, liver, lungs,
etc. and endocrine glands such as pituitary, thyroid, adrenal,
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parathyroid and pancreas and skin grafts, while immunogenic,
may not contain the major histocompatibility antigen that
trigger rejection of the transplant. Rather, these antigens
are believed to be carried by passenger cells such as
leukocytes that are included in the transplanted cells as
impurities.
Thus, the administration of the green porphyrin can
either be performed at the time of, or approximately after,
the transplant itself carrying the MHC-bearing cells, or the
subject may be protected in advance by administering the
relevant histocompatibility antigens separately, either as
proteins per se, or included on the surfaces of cells bearing
them. The green porphyrin is then administered at a time
proximal to the preadministration of the cells or
histocompatibility antigens. For example, a major determinant
of histocompatibility in humans is designated HLA-DR in the
group of MHC Class II antigens. These have been
subclassified, and if the donor has been typed, the subtype of
DR antigen can be administered along with proximal
administration of the green porphyrin prior to the transplant
per se.
If the antigen is an allergen, the allergen can be
administered directly by injection or orally along with the
green porphyrin or immediately prior to administration of the
green porphyrin. Alternatively, the administration may mimic
natural exposure by placing the subject in proximity to, for
example, plants which contain pollen known to elicit an
allergic response in the subject. When the subject is placed
in this environment, green porphyrin is administered
simultaneously or very soon thereafter. If the antigen is an autoantigen, such
as believed
responsible for a number of conditions including rheumatoid
arthritis, multiple sclerosis, lupus erythematosus, certain
types of diabetes, or inflammatory reactions generally caused
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by an autoantigen, the administration of the antigen cannot
separately controlled. The efficacy of the method of the
invention depends on the continuing active immune response to
the autoantigen which is supplied internally. In this case,
the green porphyrin is administered to the subject afflicted
with the autoimmune disease, preferably during episodes when
the immune response is most evident.
An additional condition believed to be related to an
autoimmune response is psoriasis. The method of the invention
is suitable, especially using topical administration, for
treatment of this condition.
Intercellular Communication
In addition to its ability to modulate an immune
i5 response, the green porphyrins of the invention are able to
interfere with intercellular communication putatively by
virtue of their similarity in form to the integrins. As shown
hereinbelow, the three-dimensional structure of the green
porphyrins of the invention mimics the three-dimensional
structure of the favored conformations representative of
molecules containing the adhesion sequence RGD. Further, it
is demonstrated that the green porphyrins are able to
interrupt thrombosis, a known capacity of the RGD-based
compounds. Therefore, the methods of the invention are also
suitable for the treatment of conditions where intercellular
communication has negative effects on the subject.
One such instance is in the formation of restenoses
following surgery or other traumatic disturbance of the
vasculature. For this purpose, the method of the invention
= 30 can be employed proximal to surgical procedures representing
risks of formation of blood clots in response to this stress.
The administration will typically be systemic and timed so as
to effect interruption of the required cellular communication
responsible for thrombosis.
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Formulation and Administration
The green porphyrins of the invention may be
formulated and administered in a manner convenient generally
for small-molecule drugs as is known in the art, for example,
as set forth in Remington's Pharmaceutical Sciences, Mack
Publishing Company, Easton, PA, Latest Edition. The
composition will contain a quantity of the green porphyrin
effective to provide immunomodulation or interruption of
intercellular communication. The dosage level will vary
depending on the mode of administration, formulation,
condition to be treated, and the nature of the subject;
however, in general, the amount of green porphyrin for
systemic administration is of the order of 10 Tg/kg to 100
mg/kg, preferably 100 Tg/kg-10 mg/kg, and most preferably
around 1 mg/kg. If administration is topical, suitable
concentrations in the composition ranging from about 5% to
about 95% of the composition, preferably about 10$-50$ of the
composition are employed.
Routes of systemic administration can be by
injection, including intravenous, intramuscular,
intraperitoneal and the like; oral, transmucosal or
transdermal using appropriate excipients, and the like.
Localized administration can be achieved also by transdermal
or transmucosal means using suppositories or skin patches or
the green porphyrins may be applied topically in the form of
gels or salves.
When systemic administration is employed, liposomal
compositions are particularly preferred. Liposomes can be
prepared using standard methods; they typically are prepared
from negatively charged phospholipids such as phosphatidyl
glycerol, phosphatidyl serine or phosphatidyl inositol as well as lipids per
se and various stabilizing agents. The
liposomes may be multilamilar or monolaminar and are in a
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range of sizes. The concentration of green porphyrin in the
liposomal composition is typically on the order of 1-20%.
The following examples are intended to illustrate
but not to limit the invention.
Example 1
Effect on MBP-Induced EAE
Experimental allergic encephalomyelitis (EAE) in PL
mice has been used as a model for multiple sclerosis in
humans. This condition can be induced by injecting
splenocytes from donor mice primed with a myelin basic protein
(MBP). Splenocytes were obtained as follows: PL mice were
administered 0.1 ml of a mixture prepared by emulsifying MBP
(4 mg/mi phosphate-buffered saline) in an equal volume of____
complete Freund's adjuvant (4 mg of Mycobacterium tuberculosis
H37RA in incomplete Freund's adjuvant). Mice were injected
subcutaneously at the tail base. Ten days following
immunization, spleens were removed and teased into single cell
suspensions. Cells were pelleted by centrifugation and
erythrocytes removed by lysis in 0.14 M NH4C1. Cells
(4x106/ml) were cultured in RPMI 1640 medium containing 5%
fetal calf serum, 5x10-5 M 2-mercaptoethanol, 1 mM sodium
pyruvate, 20 mM HEPES, 2 mM 1-glutamine, 100 U/ml penicillin,
100 Tg/ml streptomycin and 100 Tg/ml MBP in 75 cm2 flasks.
Following three days at 370C, 5% CO2 the cultures were pulsed
with human recombinant interleukin-2 (rIL-2, 50 U/ml) and
returned to the incubator for a further 48 hours. Cells were
then harvested, washed with serum-free medium and 5x10' cells
injected intravenously (iv) into naive, syngeneic animals.
Mice thus treated typically developed EAE with 18-25 days
post-cell transfer.
Four control mice were injected intravenously with
5x10' of the cells prepared and cultured as described in the
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preceding paragraph. After 19-30 days, three of the four mice
developed the characteristic paralytic disease associated with
EAE. This condition is induced when MBP-reactive T
lymphocytes invade the central nervous system. However, in
the experimental group, four mice, similarly injected with
MBP-primed and cultured cells, were administered 1.0 mg/kg
BPD-MA intravenously 24 hours later. None of these mice
developed the EAE condition. However, mice administered the
same dose of BPD 24 hours prior to the injection of the MBP-
primed and cultured cells, showed the same course of EAE as
did the controls.
Since BPD-injection was effective only when
administered within about 24 hours after administration of the
cultured cells, BPD is shown to act directly on the
transferred activated T cells, rather than on the endogenous
cells of the recipient animals.
Splenocytes prepared as described in paragraph 1 of
this example, when cultured with 100 Tg/ml of MBP, generate a
proliferative response by expansion of T cells specific for,
and activated by, MBP. This proliferative response is indexed
at 100% in comparison with cells cultured in the absence of
MBP which is indexed at 0%. When the cells are cultured in
the presence of BPD at concentrations of 1 ng/ml-1 Tg/ml, the
proliferation is inhibited. At 1 ng/ml of BPD the
proliferation is only 20%; the index falls to zero at 10
ng/ml. At over 100 ng/ml, the proliferative response is less
than that seen in the cells cultured in the absence of MBP.
It is known that BPD at the concentration range used
has no innate toxicity in the absence of light; therefore,
these results suggest that BPD induces anergy in MBP-specific T cells.
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Example 2
Effect of BPD on DTH
The well documented delayed-type hypersensitivity
(DTH) model was employed. Dinitrofluorobenzene (DNFB) was
painted onto the inguinal area of hairless strain mice on day
0. On day 5, the DNFB is painted onto the ear, resulting in
significant inflammatory response, including swelling of the
ear, during the following 24 hours orchestrated by antigen-
specific T cells.
Mice painted with DNFB on day 0 were injected
intravenously with liposomal BPD-MA at a dose of 1 mg/kg on
day -2, -1, 0, +1, +3 or +4. On day 5 the mice were
challenged by painting DNFB onto the ear and ear swelling was
measured 24 hours later. Minimal ear swelling was noted in
unprimed mice. DNFB-sensitized mice administered saline
rather than BPD showed a strong ear-swelling response
following challenge with DNFB. Swelling was recorded as
percent swelling, or
ear thickness post-challenge minus ear thickness pre-challenge X 100
ear thickness pre-challenge
Swelling was usually in the range of 60-90% above
control levels. However, when BPD was given on days -2, -1,
0, +1, or +3, ear swelling was less than 50% of that observed
in the control animals. Inhibition of the DTH response was
approximately 25% when BPD was given on day 4.
In an additional experiment, mice that had been
previously sensitized with DNFB were treated with a different
skin contact sensitizer, oxazolone. The mice were
subsequently challenged with DNFB and oxazolone. Mice treated
with BPD showed suppression of the response to DNFB. However,
no diminution in the ear swelling in response to oxazolone was
found in mice administered BPD. Thus, only the antigen-
specific T cells (reactive to DNFB) were affected by BPD. The
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mice retained their ability to respond to the second antigen,
oxazolone, despite the treatment with BPD in conjunction with
DNFB sensitization.
Example 3
Effect of BPD on Fibrinogen Binding
Platelets were activated by thrombin according to a
modification of the method of Shattil et al. Blood (1987)
70:307. Briefly, after a 15-minute preincubation at 220C with
100 Tg/ml liposomal BPD or control, citrated whole blood was
treated with I-thrombin (10 U/ml final concentration) in the
presence of the fibrin polymerization inhibitor Gly-Pro-Arg-
Pro (1.25 mM final concentration) for 30 minutes at 220C.
Platelet activation was measured by assessing bound fibrinogen
using fluorescein-labeled antifibrinogen polyclonal antibodies
in a flow cytometric analysis. The results are shown in Table
1 as the percentage of platelets positive for antifibrinogen
(determined as the mean of three experiments plus or minus one
standard deviation).
TABLE 1
PERCENTAGE PLATELETS POSITIVE FOR ANTI-FIBRINOGEN
Mean of 3 (1 S.D.)
BPD-Liposomes Liposomes No
Treatment
No agonist 2.9 2.1 2.2
(0.8) (2.5) (2.2)
Thrombin 28.9 86.7 86
(2.0) (1.3) (2.2)
As seen in Table 1, when treated with thrombin the
percentage of platelets positive for antifibrinogen rises from
about 2% to about 86-87% when BPD is absent. However, when
100 Tg/ml of liposomal BPD is present, the level of activation
is diminished by about two-thirds.
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Example 4
Effect of BPD on Skin Allograft Rejections
The skin allograft rejection assay involving skin
grafts between MHC-incompatible mice was conducted accordinq
to the method of Medewar and Billingham (1951) J. of
Experimental Biology, 28:385. Control mice (n=16) reject
the grafts at day 11.1 1.9 following allograft.
In the experimental group, (n=6) mice received a
single intravenous injection of 0.25 mg/kg BPD-MA liposomal
preparation 3-4 hours after allografting and showed prolonged
engraftment of 20.7 0.9 days to rejection. Another group of
mice who received an additional 0.25 mg/kg injection of BPD on
day 8 showed a median reduction time of 23.3 1.9 days.
Thus, BPD without irradiation appears to suppress
rejection of allografts.
Example 5
Effect of BPD on Adhesion Molecules in Rheumatoid Arthritis
The cell surface adhesion molecule ICAM-1 is
upregulated in activated cells. Therefore, appearance of this
molecule is considered an indicator of cellular activation.
Synovial fluids from patients with rheumatoid
arthritis were incubated in tissue culture medium for 18 hours
with varying concentrations of BPD, treated with anti-ICAM-1
and subjected to flow cytometry. The results of this assay
are shown in Table 2.
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TABLE 2
(BPD) Ungated Cell Lymphocytes Monocytes PMNs
ng/ml Population
MED +VE MED +VE MED +VE MED +VE
0 24.4 (85.7%) 14.2 (23.7%) 478 (85.9%) 20.4 (99.3%)
15 26.7 (87.7%) 13.6 (30.7%) 509 (95.9%) 22.0 (99.3%)
25 26.7 (87.0%) 14.6 (29.1%) 496 (93.8%) 21.9 (99.5%)
50 23.9 (84.5%) 12.9 (23.5%) 443 (94.2%) 20.0 (99.2%)
100 14.9 (66.9%) 13.7 (22.7%) 309 (46.6%) 12.5 (69.0%)
+VE: Percentage of cells positive for ICAM-1.
MED: Median value of fluorescence intensity of cells
expressing ICAM-1.
As shown in Table 2, the percentage of ungated cells
positive for ICAM-1 decreases from 85.7% in the absence of BPD
to 66.9% in the presence of 100 ng/ml BPD. The percentage of
lymphocytes displaying this molecule was relatively unaffected
by BPD; however, for monocytes and polymorphonuclear cells,
100 ng/ml BPD significantly reduced the expression of ICAM-1.
Without wishing to be bound by any theory,
applicants believe that the antigen-specific immunomudulation
exerted by BPD in the dark results from its interaction with
integrins or adhesion molecules expressed on activated
hematopoietic cells so as to disrupt cellular communication.
Example 6
Relation of BPD-MA to Conformation of Integrin-Binding
Compounds
Two known integrin antagonists, RGDS and
acetylated/amidated CNPRGDYC (wherein tyrosine is present as
the methyl ether) were compared with respect to three-
dimensional structures to the three-dimensional structure of
BPD-MA. This comparison was done by the Alberta Peptide
Institute.
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Three-dimensional structure predictions for RGDS
showed 1584 possible conformations which could be grouped into
seven families according to their energies. Predictions with
respect to the acetylated, amidated peptide CNPRGDYC provided
1347 conformations grouped into five families according to
energies.
A subset of the major family groups in the
predictions for both peptides are similar to three groups of
conformations that are found experimentally, designated RGDA,
RGDB and RGDC. These three groups of conformations were used
as representative conformations for both peptides.
The conformations included within these families are
shown superimposed in Figures 2-4: Figure 2 shows RGDA;
Figure 3 shows RGDB; Figure 4 shows RGDC. All conformations
have a carboxylic acid (Asp) to nitrogen (Arg) functional
group distance of 12-13/. Figures 5-7 show superimposed
representative conformations of each of these three groups
with BPD-MA. Figure 5 represents BPD superimposed onto RGDA;
Figure 6 represents BPD-MA superimposed onto RGDB Figure 7
shows BPD-MA superimposed onto RGDC.
In all of these superimpositions, the superimposed
structures are flat and the functional groups of aspartic and
arginine are superimposed onto the corresponding carboxylic
acid and nitrogen functional groups of BPD-MA.
The results show that BPD appears to have several
arrangements of functional groups that would mimic the
conformations of integrin binding compounds.
Example 7
Experimental Testing of BPD Analogues on the DTH Response
A comparison of the influence of BPD-MA, BPD-MB,
BPD-DA, and BPD-DB on the DTH response to the topically
applied hapten DNFB, in the absence of direct light was
performed using the method outlined in Example 2 above. The
.CA 02210152 1997-07-10
WO 96/22090 PCT/CA96/00020
- 18 -
compounds were evaluated in naive, unshaved Balb/c mice. Each
treatment group consisted of 4-5 animals. BPD analogues were
reconstituted in DMSO and administered 24 hours following DNFB
application. Mice were ear challenged on day 5 of the
experiment and the response measured one day later. The final
DMSO concentration was 2%. Control mice received the
appropriate, matched solvent.
The results are provided in Tables 3 and 4 and
summarized in Figure 8. The mono-BPD analogues strongly
inhibited the DTH response. The di-BPD analogues did not
inhibit the DTH response.
Table 3
Effect of different BPD derivatives on the CHS response
BPD derivative AEarR (mm x 10- ) SD AWeight (g)
MA 0.1 4.02 1.54 0.2
MA 1.0 1.8 1.28 0.4
MB 0.1 2.32 0.69 0.3
MB 0.5 2.4 0.49 0.1
DA 0.1 6.46 3.6 0.8
DA 1.0 4.16 1.44 -0.1
DB 0.1 10.33 2.8 -0.4
DB 1.0 5.48 2.79 -0.7
(+) Control 6.53 1.87 -0.7
Irritant 1.78 0.59 0.5
Irrit-MB 1.0 1.5 0.23 0.3
Irrit-DA 1.0 1.78 0.29 0.6
Irrit-DB 1.0 0.85 0.47 0.4
Table 4
Compound Dose (mg/kg) Relative Inhibition
of the DTH Response
BPD-MA 1.0 ++++
BPD-MA 0.1 +++
BPD-MB 0.5 +++
BPD-MB 0.5 +++
BPD-DA 1.0 -
BPD-DA 0.1 -
BPD-DB 1.0 -
BPD-DB 0.1 -
