U.S. patent application number 13/048587 was filed with the patent office on 2011-09-15 for combinations for the treatment of immunoinflammatory disorders.
Invention is credited to Alexis Borisy, Michael A. Foley, Nicole W. Hurst, Edward Roydon Jost-Price, Curtis Keith, Palaniyandi Manivasakam, Grant Zimmermann.
Application Number | 20110223621 13/048587 |
Document ID | / |
Family ID | 23277540 |
Filed Date | 2011-09-15 |
United States Patent
Application |
20110223621 |
Kind Code |
A1 |
Keith; Curtis ; et
al. |
September 15, 2011 |
COMBINATIONS FOR THE TREATMENT OF IMMUNOINFLAMMATORY DISORDERS
Abstract
The invention features pharmaceutical compositions that include
dipyridamole and a corticosteroid.
Inventors: |
Keith; Curtis; (Boston,
MA) ; Borisy; Alexis; (Arlington, MA) ;
Zimmermann; Grant; (Somerville, MA) ; Jost-Price;
Edward Roydon; (West Roxbury, MA) ; Manivasakam;
Palaniyandi; (West Roxbury, MA) ; Hurst; Nicole
W.; (Boston, MA) ; Foley; Michael A.;
(Chestnut Hill, MA) |
Family ID: |
23277540 |
Appl. No.: |
13/048587 |
Filed: |
March 15, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11354552 |
Feb 15, 2006 |
7915265 |
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13048587 |
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10264991 |
Oct 4, 2002 |
7253155 |
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11354552 |
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60327674 |
Oct 5, 2001 |
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Current U.S.
Class: |
435/7.92 ;
435/375 |
Current CPC
Class: |
A61P 1/00 20180101; A61K
45/06 20130101; A61P 3/10 20180101; A61P 19/02 20180101; A61K 31/56
20130101; A61K 31/573 20130101; A61P 37/02 20180101; A61P 11/06
20180101; A61P 17/00 20180101; A61P 1/04 20180101; A61P 37/00
20180101; A61P 37/06 20180101; A61P 17/06 20180101; A61K 31/675
20130101; A61P 29/00 20180101; A61P 21/00 20180101; A61K 31/519
20130101; A61P 25/00 20180101; A61P 43/00 20180101; A61K 31/519
20130101; A61K 2300/00 20130101; A61K 31/56 20130101; A61K 2300/00
20130101; A61K 31/573 20130101; A61K 2300/00 20130101; A61K 31/675
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
435/7.92 ;
435/375 |
International
Class: |
G01N 33/566 20060101
G01N033/566; C12N 5/078 20100101 C12N005/078 |
Claims
1. A method of reducing the levels of TNF.alpha. in mammalian
blood, comprising contacting cells in the blood with a composition
consisting of a corticosteroid, dipyridamole, and excipients.
2. The method of claim 1, wherein the method suppresses the
secretion of TNF.alpha..
3. The method of claim 1, wherein the cells have been stimulated to
secrete TNF.alpha..
4. The method of claim 3 wherein the cells comprise white blood
cells.
5. The method of claim 1, further comprising the step of confirming
the reduction in TNF.alpha. levels.
6. The method of claim 5, wherein the confirming step comprises the
use of an antibody or an antibody-based assay.
7. The method of claim 6 wherein the antibody-based assay is an
ELISA.
8. The method of claim 1, wherein said corticosteroid is algestone,
6-alpha-fluoroprednisolone, 6-alpha-methylprednisolone,
6-alpha-methylprednisolone 21-acetate, 6-alpha-methylprednisolone
21-hemisuccinate sodium salt, 6-alpha,9-alpha-difluoroprednisolone
21-acetate 17-butyrate, amcinafal, beclomethasone, beclomethasone
dipropionate, beclomethasone dipropionate monohydrate,
6-beta-hydroxycortisol, betamethasone, betamethasone-17-valerate,
budesonide, clobetasol, clobetasol propionate, clobetasone,
clocortolone, clocortolone pivalate, cortisone, cortisone acetate,
cortodoxone, deflazacort, 21-deoxycortisol, deprodone, descinolone,
desonide, desoximethasone, dexamethasone, dexamethasone-21-acetate,
dichlorisone, diflorasone, diflorasone diacetate, diflucortolone,
doxibetasol, fludrocortisone, flumethasone, flumethasone pivalate,
flumoxonide, flunisolide, fluocinonide, fluocinolone acetonide,
9-fluorocortisone, fluorohydroxyandrostenedione, fluorometholone,
fluorometholone acetate, fluoxymesterone, flupredidene,
fluprednisolone, flurandrenolide, formocortal, halcinonide,
halometasone, halopredone, hyrcanoside, hydrocortisone,
hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone
cypionate, hydrocortisone sodium phosphate, hydrocortisone sodium
succinate, hydrocortisone probutate, hydrocortisone valerate,
6-hydroxydexamethasone, isoflupredone, isoflupredone acetate,
isoprednidene, meclorisone, methylprednisolone, methylprednisolone
acetate, methylprednisolone sodium succinate, paramethasone,
paramethasone acetate, prednisolone, prednisolone acetate,
prednisolone metasulphobenzoate, prednisolone sodium phosphate,
prednisolone tebutate, prednisolone-21-hemisuccinate free acid,
prednisolone-21-acetate, prednisolone-21(beta-D-glucuronide),
prednisone, prednylidene, procinonide, tralonide, triamcinolone,
triamcinolone acetonide, triamcinolone acetonide 21-palmitate,
triamcinolone diacetate, or triamcinolone hexacetonide.
9. The method of claim 8, wherein said corticosteroid is prednisone
or prednisolone.
10. The method of claim 9, wherein said composition comprises 0.5
to 10 mg of prednisone or prednisolone.
11. The method of claim 9, wherein said composition comprises 100
to 400 mg of dipyridamole.
12. The method of claim 1, wherein said composition comprises 100
to 400 mg of dipyridamole.
13. The method of claim 1, wherein said composition comprises 0.5
to 10 mg corticosteroid.
14. The method of claim 13, wherein said composition comprises 0.5
to 10 mg corticosteroid and 100 to 400 mg of dipyridamole.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of and claims priority
from U.S. patent application Ser. No. 11/354,552, filed Feb. 15,
2006, which is a divisional application of U.S. patent application
Ser. No. 10/264,991, filed Oct. 4, 2002, which claims benefit of
the filing date of U.S. Provisional Application No. 60/327,674,
filed Oct. 5, 2001, each of which is hereby incorporated by
reference.
FIELD OF THE INVENTION
[0002] The invention relates to the treatment of immunoinflammatory
disorders.
BACKGROUND OF THE INVENTION
[0003] Immunoinflammatory disorders (e.g., rheumatoid arthritis,
psoriasis, ulcerative colitis, Crohn's disease, stroke-induced
brain cell death, ankylosing spondylitis, fibromyalgia, and
inflammatory dermatoses, asthma, multiple sclerosis, type I
diabetes, systemic lupus erythematosus, scleroderma, systemic
sclerosis, and Sjogren's syndrome) are characterized by
dysregulation of the immune system and inappropriate activation of
the body's defenses, resulting in damage to healthy tissue.
[0004] One percent of humans world-wide are afflicted with
rheumatoid arthritis (RA), a relentless, progressive disease
causing severe swelling, pain, and eventual deformity and
destruction of joints. According to the Arthritis Foundation,
rheumatoid arthritis currently affects over two million Americans,
of which women are three times more likely to be afflicted.
Rheumatoid arthritis is characterized by inflammation of the lining
of the joints and/or other internal organs, and the presence of
elevated numbers of lymphocytes and high levels of proinflammatory
cytokines.
[0005] Diagnosis of RA generally includes: (i) morning stiffness in
joints lasting at least one hour before improvement, (ii) arthritis
of three or more joint areas having simultaneously soft tissue
swelling or fluid; (iii) arthritis of at least one hand joint; (iv)
symmetric arthritis, i.e., simultaneous involvement of the same
joint area on both sides of the body; (v) rheumatoid nodules; (vi)
abnormal serum rheumatoid factor; and (vii) radiographic changes
typical of rheumatoid arthritis on posteroanterior hand and wrist
radiographs, which include erosions or unequivocal bony
decalcification localized in or most marked adjacent to the
involved joints. Patients are classified as having RA if at least
four of these seven criteria, and (i) through (iv) must have been
present for at least six weeks. (American College of Rheumatology,
1987 Criteria for the Classification of Acute Arthritis of
Rheumatoid Arthritis, based on Arnett F C et al., Arthritis Rheum.
1988; 31:315-324). Pain per se is not required for the diagnosis of
RA.
[0006] Treatment of RA generally includes anti-inflammatory
strategies directed at suppressing the clinical manifestations of
joint inflammation, including synovial thickening, joint
tenderness, and joint stiffness. Drugs used to address these signs
and symptoms generally include (i) non-steroidal anti-inflammatory
drugs (NSAIDs; e.g., detoprofen, diclofenac, diflunisal, etodolac,
fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen,
meclofenameate, mefenamic acid, meloxicam, nabumeone, naproxen
sodium, oxaprozin, piroxicam, sulindac, tolmetin, celecoxib,
rofecoxib, aspirin, choline salicylate, salsalte, and sodium and
magnesium salicylate)--these drugs may be adequate for mild RA, but
do not appear to alter the longterm course of the disease; and (ii)
steroids (e.g., cortisone, dexamethasone, hydrocortisone,
methylprednisolone, prednisolone, prednisone, triamcinolone).
[0007] Treatment for RA may also include strategies directed at
limiting the long term joint damage and deformity caused by the
inflammation in the joints. Such treatments are generally described
as DMARDs, i.e., disease modifying antirheumatic drugs (e.g.,
cyclosporine, azathioprine, methotrexate, leflunomide,
cyclophosphamide, hydroxychloroquine, sulfasalazine,
D-penicillamine, minocycline, gold, etanercept (soluble TNF
receptor) and infliximab (a chimeric monoclonal anti-TNF
antibody)).
[0008] There is a need to develop new regimens for the treatment of
immunoinflammatory disorders.
SUMMARY OF THE INVENTION
[0009] We have discovered that the combination of tetra-substituted
pyrimidopyrimidines, such as dipyridamole (also known as
2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido(5,4-d)pyrimidine),
and corticosteroids, such as fludrocortisone (as known as
9-alpha-fluoro-11-beta, 17-alpha,
21-trihydroxy-4-pregnene-3,20-dione acetate), and prednisolone
(also known as 1-dehydrocortisol; 1-dehydrohydrocortisone; or
1,4-pregnadiene-11beta,17alpha,21-triol-3,20-dione; or
11beta,17alpha,21-trihydroxy-1,4-pregnadiene-3,20-dione), brings
about substantial suppression of TNF.alpha. levels induced in
peripheral blood mononuclear cells (PBMCs).
[0010] Accordingly, the invention features, in one aspect, a method
for treating a patient who has, or who is at risk for developing,
an immunoinflammatory disorder. The method includes administering
(i) a corticosteroid; and (ii) a tetra-substituted
pyrimidopyrimidine having the formula (I):
##STR00001##
wherein each Z and each Z' is, independently, N, O, C,
##STR00002##
When Z or Z' is O or
##STR00003##
[0011] then p=1, when Z or Z' is N,
##STR00004##
then p=2, and when Z or Z' is C, then p=3. In formula (I), each
R.sub.1 is, independently, X, OH, N-alkyl (wherein the alkyl group
has 1 to 20, more preferably 1-5, carbon atoms); a branched or
unbranched alkyl group having 1 to 20, more preferably 1-5, carbon
atoms; or a heterocycle, preferably as defined in formula (Y),
below. Alternatively, when p>1, two R.sub.1 groups from a common
Z or Z' atom, in combination with each other, may represent
--(CY.sub.2).sub.k-- in which k is an integer between 4 and 6,
inclusive. Each X is, independently, Y, CY.sub.3,
C(CY.sub.3).sub.3, CY.sub.2CY.sub.3, (CY.sub.2).sub.1-5OY,
substituted or unsubstituted cycloalkane of the structure
C.sub.nY.sub.2n-1, wherein n=3-7, inclusive. Each Y is,
independently, H, F, Cl, Br, or I. In one embodiment, each Z is the
same moiety, each Z' is the same moiety, and Z and Z' are different
moieties. The two compounds are each administered in an amount
that, when combined with the second compound, is sufficient to
treat or prevent the immunoinflammatory disorder.
[0012] In a related aspect, the invention features a method for
suppressing the production of one or more proinflammatory cytokines
in a patient in need thereof by administering to the patient (i) a
corticosteroid; and (ii) a tetra-substituted pyrimidopyrimidine
having formula (I).
[0013] In particularly useful tetra-substituted pyrimidopyrimidines
in both aspects of the invention, R.sub.1 is a substituted or
unsubstituted furan, purine, or pyrimidine, (CH.sub.2CH.sub.2OY),
(CH.sub.2CH(OH)CH.sub.2OY), (HCH.sub.2CH(OH)CX.sub.3),
((CH.sub.2).sub.nOY), where n=2-5,
##STR00005##
In other useful tetra-substituted pyrimidopyrimidines, each Z is N
and the two associated R.sub.1 groups combine in the form of
--(CH.sub.2).sub.5--, and each Z' is N and each Z'-- associated
R.sub.1 group is --CH.sub.2CH.sub.2OH.
[0014] The tetra-substituted pyrimidopyrimidine and the
corticosteroid may be present in pharmaceutical compositions that
contain a pharmaceutically acceptable carrier, diluent, or
excipient, and are administered at dosages and frequencies
sufficient to suppress TNF.alpha. levels enough to produce a
therapeutic benefit to the patient. The tetra-substituted
pyrimidopyrimidine and the corticosteroid can be administered
within 14 days of each other (e.g., within 10 days, within five
days, twenty-four hours, or one hour of each other, or even
simultaneously). Administration of each compound can occur, e.g., 1
to 5 times each day, or as necessary to alleviate symptoms.
[0015] Accordingly, this invention also features pharmaceutical
compositions, pharmaceutical packs, and kits containing one or more
tetra-substituted pyrimidopyrimidine and one or more
corticosteroid. The methods and compositions (pharmaceutical
compositions and pharmaceutical packs) of the invention may feature
higher order combinations of tetra-substituted pyrimidopyrimidines
and corticosteroids. Specifically, one, two, three, or more
tetra-substituted pyrimidopyrimidines may be combined with one,
two, three, or more corticosteroids. In preferred embodiments, the
tetra-substituted pyrimidopyrimidine, the corticosteroid, or both
are approved by the United States Food and Drug Administration
(USFDA) for administration to a human.
[0016] Exemplary tetra-substituted pyrimidopyrimidines that are
useful in the methods and compositions of this invention include
2,6-disubstituted 4,8-dibenzylaminopyrimido[5,4-d]pyrimidines.
Particularly useful tetra-substituted pyrimidopyrimidines include
dipyridamole (also known as
2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido(5,4-d)pyrimidine),
mopidamole, dipyridamole monoacetate, NU3026
(2,6-di-(2,2-dimethyl-1,3-dioxolan-4-yl)-methoxy-4,8-dipiperidinopyrimido-
pyrimidine), NU3059
(2,6-bis-(2,3-dimethyoxypropoxy)-4,8-dipiperidinopyrimidopyrimidine),
NU3060
(2,6-bis[N,N-di(2-methoxy)ethyl]-4,6-dipiperidinopyrimidopyrimidin-
e), and NU3076
(2,6-bis(diethanolamino)-4,8-di-4-methoxybenzylaminopyrimidopyrimidine).
[0017] The invention described herein has been exemplified using
the corticosteroids fludrocortisone; however, a skilled artisan
will recognize that structural and functional analogs of these
corticosteroids can also be used in combination with the
tetra-substituted pyrimidopyrimidines in the methods and
compositions of the present invention. Other useful corticosteroids
may be identified based on the shared structural features and
mechanism of action among the corticosteroid family.
[0018] The tetra-substituted pyrimidopyrimidine and the
corticosteroid may be administered in the same or different
pharmaceutical formulations. Pharmaceutical compositions or
components of the pharmaceutical pack may be administered by the
same or different routes and include oral, rectal, intravenous,
intramuscular, subcutaneous, intra-articular, inhalation, topical
or transdermal, vaginal, and ophthalmic administration.
[0019] Dosages of the tetra-substituted pyrimidopyrimidine and the
corticosteroid may be determined individually. In prior art
therapeutic regimines, tetra-substituted pyrimidopyrimidine are
typically administered to human patients at about 0.5-800 mg/day,
18-600 mg/day, or 50-400 mg/day. Corticosteroids are typically
administered at about 0.1-1500 mg/day, 0.5-30 mg/day, or 0.5-10
mg/day. Low doses of corticosteroids (e.g., 10 mg/day or less of
prednisolone, or its equivalent) are preferred. In the methods and
compositions of the invention, both components typically will be
used in lower dosages than those given above, because the two drugs
operate together to treat or suppress the subject disorder. Thus,
the pyrimidopyrimidine can be used, according to the invention, at
a dosage of 0.5-50 mg/day, and the corticosteroid can be used at a
dosage of 0.1 to 10 mg/day. The total daily dosage of the
tetra-substituted pyrimidopyrimidine and the corticosteroid may be
administered in one, two, three, four, or more dosages. It is not
necessary for the tetra-substituted pyrimidopyrimidine and the
corticosteroid to be administered in the same number of daily
doses. Further, there is no need for the tetra-substituted
pyrimidopyrimidine and/or the corticosteroid to be administered
every day or by the same route of administration. For example, the
tetra-substituted pyrimidopyrimidine may be administered by
intravenous injection every second day and the corticosteroid
administered by topical application twice every day. Accordingly,
when administered in different compositions, pharmaceutical
formulations, packs, and kits are prepared in form and dosage
suitable for achieving the desired treatment regimen.
[0020] The diseases or disorders treated using the methods and
compositions of this invention are immunoinflammatory disorders
including, for example, rheumatoid arthritis, psoriasis, ulcerative
colitis, Crohn's disease, stroke-induced brain cell death,
ankylosing spondylitis, fibromyalgia, asthma, multiple sclerosis,
type I diabetes, systemic lupus erythematosus, scleroderma,
systemic sclerosis, inflammatory dermatoses, or Sjogren's
syndrome.
[0021] The invention also features a method for identifying
compounds useful for treating a patient having an
immunoinflammatory disorder. The method includes the steps of:
contacting immune cells in vitro with (i) an immunomodulatory
compound selected from the group of a tetra-substituted
pyrimidopyrimidine having formula (I) or a corticosteroid; and (ii)
a candidate compound, and determining whether the immune response
is modulated relative to (a) immune cells contacted with the
immunomodulatory compound but not contacted with the candidate
compound, and (b) immune cells contacted with the candidate
compound but not with the immunomodulatory compound. A candidate
compound that, when combined with an immunomodulatory compound,
modulates the immune response to a greater degree than controls, is
a compound that is potentially useful for treating a patient having
an immunoinflammatory disorder.
[0022] Compounds useful in the invention include those described
herein in any of their pharmaceutically acceptable forms, including
isomers such as diastereomers and enantiomers, salts, solvates, and
polymorphs thereof, as well as racemic mixtures of the compounds
described herein.
[0023] By "heterocycle" is meant any cyclic molecule, wherein one
or more of the ring atoms is an atom other than carbon. Preferable
heterocycles consist of one or two ring structures. Preferable
heteroatoms are N, O, and S. Each ring structure of the heterocycle
consists of 3-10 atoms, preferably 4-8 atoms, and most preferably
5-7 atoms. Each ring structure need not contain a heteroatom,
provided that a heteroatom is present in at least one ring
structure. Preferred heterocycles are, for example, beta-lactams,
furans, tetrahydrofurans, pyrroles, pyrrolidines, thiophenes,
tetrahydrothiophenes, oxazoles, imidazolidine, indole, guanine, and
phenothiazine.
[0024] By "patient" is meant any animal (e.g., a human).
[0025] The term "immunoinflammatory disorder" encompasses a variety
of conditions, including autoimmune diseases. Immunoinflammatory
disorders result in the destruction of healthy tissue by an
inflammatory process. Examples of immunoinflammatory disorders
include, rheumatoid arthritis, psoriasis, ulcerative colitis,
Crohn's disease, stroke-induced brain cell death, ankylosing
spondylitis, fibromyalgia, asthma, multiple sclerosis, type I
diabetes, systemic lupus erythematosus, scleroderma, systemic
sclerosis, inflammatory dermatoses, myasthenia gravis, and
Sjogren's syndrome.
[0026] By "corticosteroid" is meant any naturally occurring or
synthetic steroid hormone which can be derived from cholesterol and
is characterized by a hydrogenated cyclopentanoperhydrophenanthrene
ring system. Naturally occurring corticosteroids are generally
produced by the adrenal cortex. Synthetic corticosteroids may be
halogenated. Functional groups required for activity include a
double bond at .DELTA.4, a C3 ketone, and a C20 ketone.
Corticosteroids may have glucocorticoid and/or mineralocorticoid
activity. In preferred embodiments, the corticosteroid is either
fludrocortisone or prednisolone.
[0027] Exemplary corticosteroids include algestone,
6-alpha-fluoroprednisolone, 6-alpha-methylprednisolone,
6-alpha-methylprednisolone 21-acetate, 6-alpha-methylprednisolone
21-hemisuccinate sodium salt, 6-alpha,9-alpha-difluoroprednisolone
21-acetate 17-butyrate, amcinafal, beclomethasone, beclomethasone
dipropionate, beclomethasone dipropionate monohydrate,
6-beta-hydroxycortisol, betamethasone, betamethasone-17-valerate,
budesonide, clobetasol, clobetasol propionate, clobetasone,
clocortolone, clocortolone pivalate, cortisone, cortisone acetate,
cortodoxone, deflazacort, 21-deoxycortisol, deprodone, descinolone,
desonide, desoximethasone, dexamethasone, dexamethasone-21-acetate,
dichlorisone, diflorasone, diflorasone diacetate, diflucortolone,
doxibetasol, fludrocortisone, flumethasone, flumethasone pivalate,
flumoxonide, flunisolide, fluocinonide, fluocinolone acetonide,
9-fluorocortisone, fluorohydroxyandrostenedione, fluorometholone,
fluorometholone acetate, fluoxymesterone, flupredidene,
fluprednisolone, flurandrenolide, formocortal, halcinonide,
halometasone, halopredone, hyrcanoside, hydrocortisone,
hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone
cypionate, hydrocortisone sodium phosphate, hydrocortisone sodium
succinate, hydrocortisone probutate, hydrocortisone valerate,
6-hydroxydexamethasone, isoflupredone, isoflupredone acetate,
isoprednidene, meclorisone, methylprednisolone, methylprednisolone
acetate, methylprednisolone sodium succinate, paramethasone,
paramethasone acetate, prednisolone, prednisolone acetate,
prednisolone metasulphobenzoate, prednisolone sodium phosphate,
prednisolone tebutate, prednisolone-21-hemisuccinate free acid,
prednisolone-21-acetate, prednisolone-21(beta-D-glucuronide),
prednisone, prednylidene, procinonide, tralonide, triamcinolone,
triamcinolone acetonide, triamcinolone acetonide 21-palmitate,
triamcinolone diacetate, triamcinolone hexacetonide, and
wortmannin. Desirably, the corticosteroid is fludrocortisone or
prednisolone.
[0028] By "an effective amount" is meant the amount of a compound,
in a combination of the invention, required to treat or prevent an
immunoinflammatory disease. The effective amount of active
compound(s) used to practice the present invention for therapeutic
treatment of conditions caused by or contributing to an
inflammatory disease varies depending upon the manner of
administration, the age, body weight, and general health of the
patient. Ultimately, the attending physician or veterinarian will
decide the appropriate amount and dosage regimen. Such amount is
referred to as an effective amount.
[0029] By the term "cytokine suppressing amount" is meant an amount
of the combination which will cause a decrease in the vivo presence
or level of the proinflammatory cytokine, when given to a patient
for the prophylaxis or therapeutic treatment of an
immunoinflammatory disorder which is exacerbated or caused by
excessive or unregulated proinflammatory cytokine production.
[0030] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
DETAILED DESCRIPTION
[0031] We have discovered that the combination of a
tetra-substituted pyrimidopyrimidine with a corticosteroid
substantially has substantial TNF.alpha. suppressing activity
against stimulated white blood cells. The combinations of
dipyridamole with fludrocortisone, and dipyridamole with
prednisolone were particularly effective. Thus, the combination of
a tetra-substituted pyrimidopyrimidine with a corticosteroid is
useful for the treatment of immunoinflammatory disorders.
Dipyridamole
[0032] Dipyridamole
(2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido(5,4-d)pyrimidine)
is a tetra-substituted pyrimidopyrimidine that is used as a
platelet inhibitor, e.g., to prevent blood clot formation following
heart valve surgery and to reduced the moribundity associated with
clotting disorders, including myocardial and cerebral infarction.
Typically, anticoagulation therapy (prophylaxis or treatment) is
effected by administering dipyridamole at about 75-200 mg b.i.d,
t.i.d., or q.i.d. either alone or in combination with aspirin. In
the invention, lower doses generally can be used, e.g., 20-80 mg,
administered by any of the prior art routes.
Tetra-Substituted Pyrimidopyrimidines
[0033] Tetra-substituted pyrimidopyrimidines are structural analogs
that can replace dipyridamole in the methods and compositions of
this invention. Tetra-substituted pyrimidopyrimidines generally are
of formula (I), above.
Therapy
[0034] Combination therapy according to the invention may be
performed alone or in conjunction with another therapy and may be
provided at home, the doctor's office, a clinic, a hospital's
outpatient department, or a hospital. Treatment generally begins at
a hospital so that the doctor can observe the therapy's effects
closely and make any adjustments that are needed. The duration of
the combination therapy depends on the type of immunoinflammatory
disorder being treated, the age and condition of the patient, the
stage and type of the patient's disease, and how the patient
responds to the treatment. Additionally, a person having a greater
risk of developing an immunoinflammatory disorder (e.g., a person
who is genetically predisposed or previously had an
immunoinflammatory disorder) may receive prophylactic treatment to
inhibit or delay an inflammatory response.
[0035] The dosage, frequency and mode of administration of each
component of the combination can be controlled independently. For
example, one compound may be administered orally three times per
day, while the second compound may be administered intramuscularly
once per day. Combination therapy may be given in on-and-off cycles
that include rest periods so that the patient's body has a chance
to recovery from any as yet unforeseen side-effects. The compounds
may also be formulated together such that one administration
delivers both compounds.
Formulation of Pharmaceutical Compositions
[0036] The administration of each compound of the combination may
be by any suitable means that results in a concentration of the
compound that, combined with the other component, is
anti-inflammatory upon reaching the target region. The compound may
be contained in any appropriate amount in any suitable carrier
substance, and is generally present in an amount of 1-95% by weight
of the total weight of the composition. The composition may be
provided in a dosage form that is suitable for the oral, parenteral
(e.g., intravenously, intramuscularly), rectal, cutaneous, nasal,
vaginal, inhalant, skin (patch), or ocular administration route.
Thus, the composition may be in the form of, e.g., tablets,
capsules, pills, powders, granulates, suspensions, emulsions,
solutions, gels including hydrogels, pastes, ointments, creams,
plasters, drenches, osmotic delivery devices, suppositories,
enemas, injectables, implants, sprays, or aerosols. The
pharmaceutical compositions may be formulated according to
conventional pharmaceutical practice (see, e.g., Remington: The
Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro,
Lippincott Williams & Wilkins, 2000 and Encyclopedia of
Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan,
1988-1999, Marcel Dekker, New York).
[0037] Pharmaceutical compositions according to the invention may
be formulated to release the active compound substantially
immediately upon administration or at any predetermined time or
time period after administration. The latter types of compositions
are generally known as controlled release formulations, which
include (i) formulations that create a substantially constant
concentration of the drug within the body over an extended period
of time; (ii) formulations that after a predetermined lag time
create a substantially constant concentration of the drug within
the body over an extended period of time; (iii) formulations that
sustain drug action during a predetermined time period by
maintaining a relatively, constant, effective drug level in the
body with concomitant minimization of undesirable side effects
associated with fluctuations in the plasma level of the active drug
substance (sawtooth kinetic pattern); (iv) formulations that
localize drug action by, e.g., spatial placement of a controlled
release composition adjacent to or in the diseased tissue or organ;
and (v) formulations that target drug action by using carriers or
chemical derivatives to deliver the drug to a particular target
cell type.
[0038] Administration of compounds in the form of a controlled
release formulation is especially preferred in cases in which the
compound, either alone or in combination, has (i) a narrow
therapeutic index (i.e., the difference between the plasma
concentration leading to harmful side effects or toxic reactions
and the plasma concentration leading to a therapeutic effect is
small; in general, the therapeutic index, TI, is defined as the
ratio of median lethal dose (LD50) to median effective dose
(ED50)); (ii) a narrow absorption window in the gastro-intestinal
tract; or (iii) a very short biological half-life so that frequent
dosing during a day is required in order to sustain the plasma
level at a therapeutic level.
[0039] Any of a number of strategies can be pursued in order to
obtain controlled release in which the rate of release outweighs
the rate of metabolism of the compound in question. In one example,
controlled release is obtained by appropriate selection of various
formulation parameters and ingredients, including, e.g., various
types of controlled release compositions and coatings. Thus, the
drug is formulated with appropriate excipients into a
pharmaceutical composition that, upon administration, releases the
drug in a controlled manner. Examples include single or multiple
unit tablet or capsule compositions, oil solutions, suspensions,
emulsions, microcapsules, microspheres, nanoparticles, patches, and
liposomes.
Solid Dosage Forms for Oral Use
[0040] Formulations for oral use include tablets containing the
active ingredient(s) in a mixture with non-toxic pharmaceutically
acceptable excipients. These excipients may be, for example, inert
diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol,
microcrystalline cellulose, starches including potato starch,
calcium carbonate, sodium chloride, lactose, calcium phosphate,
calcium sulfate, or sodium phosphate); granulating and
disintegrating agents (e.g., cellulose derivatives including
microcrystalline cellulose, starches including potato starch,
croscarmellose sodium, alginates, or alginic acid); binding agents
(e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium
alginate, gelatin, starch, pregelatinized starch, microcrystalline
cellulose, magnesium aluminum silicate, carboxymethylcellulose
sodium, methylcellulose, hydroxypropyl methylcellulose,
ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and
lubricating agents, glidants, and antiadhesives (e.g., magnesium
stearate, zinc stearate, stearic acid, silicas, hydrogenated
vegetable oils, or talc). Other pharmaceutically acceptable
excipients can be colorants, flavoring agents, plasticizers,
humectants, buffering agents, and the like.
[0041] The tablets may be uncoated or they may be coated by known
techniques, optionally to delay disintegration and absorption in
the gastrointestinal tract and thereby providing a sustained action
over a longer period. The coating may be adapted to release the
active drug substance in a predetermined pattern (e.g., in order to
achieve a controlled release formulation) or it may be adapted not
to release the active drug substance until after passage of the
stomach (enteric coating). The coating may be a sugar coating, a
film coating (e.g., based on hydroxypropyl methylcellulose,
methylcellulose, methyl hydroxyethylcellulose,
hydroxypropylcellulose, carboxymethylcellulose, acrylate
copolymers, polyethylene glycols and/or polyvinylpyrrolidone), or
an enteric coating (e.g., based on methacrylic acid copolymer,
cellulose acetate phthalate, hydroxypropyl methylcellulose
phthalate, hydroxypropyl methylcellulose acetate succinate,
polyvinyl acetate phthalate, shellac, and/or ethylcellulose).
Furthermore, a time delay material such as, e.g., glyceryl
monostearate or glyceryl distearate may be employed.
[0042] The solid tablet compositions may include a coating adapted
to protect the composition from unwanted chemical changes, (e.g.,
chemical degradation prior to the release of the active drug
substance). The coating may be applied on the solid dosage form in
a similar manner as that described in Encyclopedia of
Pharmaceutical Technology, supra.
[0043] The two drugs may be mixed together in the tablet, or may be
partitioned. In one example, the first drug is contained on the
inside of the tablet, and the second drug is on the outside, such
that a substantial portion of the second drug is released prior to
the release of the first drug.
[0044] Formulations for oral use may also be presented as chewable
tablets, or as hard gelatin capsules wherein the active ingredient
is mixed with an inert solid diluent (e.g., potato starch, lactose,
microcrystalline cellulose, calcium carbonate, calcium phosphate or
kaolin), or as soft gelatin capsules wherein the active ingredient
is mixed with water or an oil medium, for example, peanut oil,
liquid paraffin, or olive oil. Powders and granulates may be
prepared using the ingredients mentioned above under tablets and
capsules in a conventional manner using, e.g., a mixer, a fluid bed
apparatus or a spray drying equipment.
Controlled Release Oral Dosage Forms
[0045] Controlled release compositions for oral use may, e.g., be
constructed to release the active drug by controlling the
dissolution and/or the diffusion of the active drug substance.
[0046] Dissolution or diffusion controlled release can be achieved
by appropriate coating of a tablet, capsule, pellet, or granulate
formulation of compounds, or by incorporating the compound into an
appropriate matrix. A controlled release coating may include one or
more of the coating substances mentioned above and/or, e.g.,
shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl
alcohol, glyceryl monostearate, glyceryl distearate, glycerol
palmitostearate, ethylcellulose, acrylic resins, dl-polylactic
acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl
acetate, vinyl pyrrolidone, polyethylene, polymethacrylate,
methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydrogels,
1,3 butylene glycol, ethylene glycol methacrylate, and/or
polyethylene glycols. In a controlled release matrix formulation,
the matrix material may also include, e.g., hydrated
methylcellulose, carnauba wax and stearyl alcohol, carbopol 934,
silicone, glyceryl tristearate, methyl acrylate-methyl
methacrylate, polyvinyl chloride, polyethylene, and/or halogenated
fluorocarbon.
[0047] A controlled release composition containing one or more of
the compounds of the claimed combinations may also be in the form
of a buoyant tablet or capsule (i.e., a tablet or capsule that,
upon oral administration, floats on top of the gastric content for
a certain period of time). A buoyant tablet formulation of the
compound(s) can be prepared by granulating a mixture of the drug(s)
with excipients and 20-75% w/w of hydrocolloids, such as
hydroxyethylcellulose, hydroxypropylcellulose, or
hydroxypropylmethylcellulose. The obtained granules can then be
compressed into tablets. On contact with the gastric juice, the
tablet forms a substantially water-impermeable gel barrier around
its surface. This gel barrier takes part in maintaining a density
of less than one, thereby allowing the tablet to remain buoyant in
the gastric juice.
Liquids for Oral Administration
[0048] Powders, dispersible powders, or granules suitable for
preparation of an aqueous suspension by addition of water are
convenient dosage forms for oral administration. Formulation as a
suspension provides the active ingredient in a mixture with a
dispersing or wetting agent, suspending agent, and one or more
preservatives. Suitable dispersing or wetting agents are, for
example, naturally-occurring phosphatides (e.g., lecithin or
condensation products of ethylene oxide with a fatty acid, a long
chain aliphatic alcohol, or a partial ester derived from fatty
acids) and a hexitol or a hexitol anhydride (e.g., polyoxyethylene
stearate, polyoxyethylene sorbitol monooleate, polyoxyethylene
sorbitan monooleate, and the like). Suitable suspending agents are,
for example, sodium carboxymethylcellulose, methylcellulose, sodium
alginate, and the like.
Parenteral Compositions
[0049] The pharmaceutical composition may also be administered
parenterally by injection, infusion or implantation (intravenous,
intramuscular, subcutaneous, or the like) in dosage forms,
formulations, or via suitable delivery devices or implants
containing conventional, non-toxic pharmaceutically acceptable
carriers and adjuvants. The formulation and preparation of such
compositions are well known to those skilled in the art of
pharmaceutical formulation. Formulations can be found in Remington:
The Science and Practice of Pharmacy, supra.
[0050] Compositions for parenteral use may be provided in unit
dosage forms (e.g., in single-dose ampoules), or in vials
containing several doses and in which a suitable preservative may
be added (see below). The composition may be in form of a solution,
a suspension, an emulsion, an infusion device, or a delivery device
for implantation, or it may be presented as a dry powder to be
reconstituted with water or another suitable vehicle before use.
Apart from the active drug(s), the composition may include suitable
parenterally acceptable carriers and/or excipients. The active
drug(s) may be incorporated into microspheres, microcapsules,
nanoparticles, liposomes, or the like for controlled release.
Furthermore, the composition may include suspending, solubilizing,
stabilizing, pH-adjusting agents, and/or dispersing agents.
[0051] As indicated above, the pharmaceutical compositions
according to the invention may be in the form suitable for sterile
injection. To prepare such a composition, the suitable active
drug(s) are dissolved or suspended in a parenterally acceptable
liquid vehicle. Among acceptable vehicles and solvents that may be
employed are water, water adjusted to a suitable pH by addition of
an appropriate amount of hydrochloric acid, sodium hydroxide or a
suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic
sodium chloride solution. The aqueous formulation may also contain
one or more preservatives (e.g., methyl, ethyl or n-propyl
p-hydroxybenzoate). In cases where one of the compounds is only
sparingly or slightly soluble in water, a dissolution enhancing or
solubilizing agent can be added, or the solvent may include 10-60%
w/w of propylene glycol or the like.
Controlled Release Parenteral Compositions
[0052] Controlled release parenteral compositions may be in form of
aqueous suspensions, microspheres, microcapsules, magnetic
microspheres, oil solutions, oil suspensions, or emulsions.
Alternatively, the active drug(s) may be incorporated in
biocompatible carriers, liposomes, nanoparticles, implants, or
infusion devices. Materials for use in the preparation of
microspheres and/or microcapsules are, e.g.,
biodegradable/bioerodible polymers such as polygalactin,
poly-(isobutyl cyanoacrylate), poly(2-hydroxyethyl-L-glutamine)
and, poly(lactic acid). Biocompatible carriers that may be used
when formulating a controlled release parenteral formulation are
carbohydrates (e.g., dextrans), proteins (e.g., albumin),
lipoproteins, or antibodies. Materials for use in implants can be
non-biodegradable (e.g., polydimethyl siloxane) or biodegradable
(e.g., poly(caprolactone), poly(lactic acid), poly(glycolic acid)
or poly(ortho esters)).
Rectal Compositions
[0053] For rectal application, suitable dosage forms for a
composition include suppositories (emulsion or suspension type),
and rectal gelatin capsules (solutions or suspensions). In a
typical suppository formulation, the active drug(s) are combined
with an appropriate pharmaceutically acceptable suppository base
such as cocoa butter, esterified fatty acids, glycerinated gelatin,
and various water-soluble or dispersible bases like polyethylene
glycols and polyoxyethylene sorbitan fatty acid esters. Various
additives, enhancers, or surfactants may be incorporated.
Compositions for Inhalation
[0054] For administration by inhalation, typical dosage forms
include nasal sprays and aerosols. In a typically nasal
formulation, the active ingredient(s) are dissolved or dispersed in
a suitable vehicle. The pharmaceutically acceptable vehicles and
excipients (as well as other pharmaceutically acceptable materials
present in the composition such as diluents, enhancers, flavoring
agents, and preservatives) are selected in accordance with
conventional pharmaceutical practice in a manner understood by the
persons skilled in the art of formulating pharmaceuticals.
Percutaneous and Topical Compositions
[0055] The pharmaceutical compositions may also be administered
topically on the skin for percutaneous absorption in dosage forms
or formulations containing conventionally non-toxic pharmaceutical
acceptable carriers and excipients including microspheres and
liposomes. The formulations include creams, ointments, lotions,
liniments, gels, hydrogels, solutions, suspensions, sticks, sprays,
pastes, plasters, and other kinds of transdermal drug delivery
systems. The pharmaceutically acceptable carriers or excipients may
include emulsifying agents, antioxidants, buffering agents,
preservatives, humectants, penetration enhancers, chelating agents,
gel-forming agents, ointment bases, perfumes, and skin protective
agents.
[0056] Examples of emulsifying agents are naturally occurring gums
(e.g., gum acacia or gum tragacanth) and naturally occurring
phosphatides (e.g., soybean lecithin and sorbitan monooleate
derivatives). Examples of antioxidants are butylated hydroxy
anisole (BHA), ascorbic acid and derivatives thereof, tocopherol
and derivatives thereof, butylated hydroxy anisole, and cysteine.
Examples of preservatives are parabens, such as methyl or propyl
p-hydroxybenzoate, and benzalkonium chloride. Examples of
humectants are glycerin, propylene glycol, sorbitol, and urea.
Examples of penetration enhancers are propylene glycol, DMSO,
triethanolamine, N,N-dimethylacetamide, N,N-dimethylformamide,
2-pyrrolidone and derivatives thereof, tetrahydrofurfuryl alcohol,
and AZONE.TM.. Examples of chelating agents are sodium EDTA, citric
acid, and phosphoric acid. Examples of gel forming agents are
CARBOPOL.TM., cellulose derivatives, bentonite, alginates, gelatin
and polyvinylpyrrolidone. Examples of ointment bases are beeswax,
paraffin, cetyl palmitate, vegetable oils, sorbitan esters of fatty
acids (Span), polyethylene glycols, and condensation products
between sorbitan esters of fatty acids and ethylene oxide (e.g.,
polyoxyethylene sorbitan monooleate (TWEEN.TM.)).
[0057] The pharmaceutical compositions described above for topical
administration on the skin may also be used in connection with
topical administration onto or close to the part of the body that
is to be treated. The compositions may be adapted for direct
application or for introduction into relevant orifice(s) of the
body (e.g., rectal, urethral, vaginal or oral orifices). The
composition may be applied by means of special drug delivery
devices such as dressings or alternatively plasters, pads, sponges,
strips, or other forms of suitable flexible material.
Controlled Release Percutaneous and Topical Compositions
[0058] There are several approaches for providing rate control over
the release and transdermal permeation of a drug, including:
membrane-moderated systems, adhesive diffusion-controlled systems,
matrix dispersion-type systems, and microreservoir systems. A
controlled release percutaneous and/or topical composition may be
obtained by using a suitable mixture of the above-mentioned
approaches.
[0059] In a membrane-moderated system, the active drug is present
in a reservoir which is totally encapsulated in a shallow
compartment molded from a drug-impermeable laminate, such as a
metallic plastic laminate, and a rate-controlling polymeric
membrane such as a microporous or a non-porous polymeric membrane
(e.g., ethylene-vinyl acetate copolymer). The active compound is
only released through the rate-controlling polymeric membrane. In
the drug reservoir, the active drug substance may either be
dispersed in a solid polymer matrix or suspended in a viscous
liquid medium such as silicone fluid. On the external surface of
the polymeric membrane, a thin layer of an adhesive polymer is
applied to achieve an intimate contact of the transdermal system
with the skin surface. The adhesive polymer is preferably a
hypoallergenic polymer that is compatible with the active drug.
[0060] In an adhesive diffusion-controlled system, a reservoir of
the active drug is formed by directly dispersing the active drug in
an adhesive polymer and then spreading the adhesive containing the
active drug onto a flat sheet of substantially drug-impermeable
metallic plastic backing to form a thin drug reservoir layer. A
matrix dispersion-type system is characterized in that a reservoir
of the active drug substance is formed by substantially
homogeneously dispersing the active drug substance in a hydrophilic
or lipophilic polymer matrix and then molding the drug-containing
polymer into a disc with a substantially well-defined surface area
and thickness. The adhesive polymer is spread along the
circumference to form a strip of adhesive around the disc.
[0061] In a microreservoir system, the reservoir of the active
substance is formed by first suspending the drug solids in an
aqueous solution of water-soluble polymer, and then dispersing the
drug suspension in a lipophilic polymer to form a plurality of
microscopic spheres of drug reservoirs.
Dosages
[0062] The dosage of each compound of the claimed combinations
depends on several factors, including: the administration method,
the condition to be treated, the severity of the condition, whether
the condition is to be treated or prevented, and the age, weight,
and health of the person to be treated. Additionally,
pharmacogenomic (the effect of genotype on the pharmacokinetic,
pharmacodynamic or efficacy profile of a therapeutic) information
about a particular patient may affect the dosage used.
[0063] As described above, the compound in question may be
administered orally in the form of tablets, capsules, elixirs or
syrups, or rectally in the form of suppositories. Parenteral
administration of a compound is suitably performed, for example, in
the form of saline solutions or with the compound incorporated into
liposomes. In cases where the compound in itself is not
sufficiently soluble to be dissolved, a solubilizer such as ethanol
can be applied. Below, for illustrative purposes, the dosages for
dipyridamole and fludrocortisone are described.
Routes of Administration
[0064] For oral, intramuscular, subcutaneous, topical, inhalation,
rectal, vaginal and ophthalmic administration of the
tetra-substituted pyrimidopyrimidine, the dosage used according to
the invention is about 0.5-800 mg/day, preferably about 5-600
mg/day, 10-100 mg/day, and more preferably 0.5-50 mg/day.
Administration can be one to four times daily for one day to one
year, and may even be for the life of the patient. Chronic,
long-term administration will be indicated in many cases. In some
cases of serious illness, up to 1600 mg/day may be necessary. For
intravenous administration of the tetra-substituted
pyrimidopyrimidine, the dosage used is about 0.1-200 mg/day,
preferably about 0.5-150 mg/day, 1-100 mg/day, and more preferably
about 0.5-50 mg/day. Administration can be one to four times daily.
Systemic dosing will result in steady-state plasma concentrations
preferably of 0.1-7.0 .mu.M, more preferably, 0.5-5.0 .mu.M, and
most preferably, 1.0-2.0 .mu.M.
[0065] The dosage of the corticosteroid for use in combination with
the tetra-substituted pyrimidopyrimidine is about 0.1-1500 mg/day,
preferably about 0.5-30 mg/day, and more preferably about 0.1-10
mg/day. Administration can be one to four times daily for one day
to one year, and may even be for the life of the patient. Chronic,
long-term administration will be indicated in many cases. In cases
of serious illness, dosages up to 3000 mg/day may be necessary.
[0066] The following examples are to illustrate the invention. They
are not meant to limit the invention in any way.
Example 1
Preparation of Pairwise Compound Mixed Combination Serial Dilution
Matrix
[0067] Stock solutions at 16 mg/ml of dipyridamole, and 1.6 mg/ml
of fludrocortisone acetate (Sigma-Aldrich, St. Louis, Mo.; catalog
numbers D9766 and F6127, respectively) were made in
dimethylsulfoxide (DMSO). The dipyridamole master plates were made
by adding 25 .mu.l of the concentrated stock solution to columns 3,
9, and 15 (rows C through N) of a polypropylene 384-well storage
plate that had been pre-filled with 75 .mu.l of anhydrous DMSO.
Using a TomTec Quadra Plus liquid handler, the 25 .mu.l of
dipyridamole stock solution was serially diluted four times into
the adjacent columns (columns 4-7, 10-13, 16-19). The sixth column
(8, 14, and 20) did not receive any compound and served as a
vehicle control. The fludrocortisone master plates were made by
adding 25 .mu.l of the concentrated stock solution to the
appropriate wells (row C, columns 3-8; row C, columns 9-14; row C,
columns 15-20; row I, columns 3-8; row I, columns 9-14; row I,
columns 15-20) of the appropriate master polypropylene 384-well
storage plate. These master plates had been pre-filled with 75
.mu.l of anhydrous DMSO. Using the TomTec Quadra Plus liquid
handler, the 25 .mu.l was serially diluted four times in the
adjacent rows (rows D-G, and J-M). The sixth row (H and N) did not
receive any compound to serve as a vehicle control. Master plates
were sealed and stored at -20.degree. C. until ready for use.
[0068] The final dipyridamole/fludrocortisone combination plates
were generated by transferring 1 .mu.l from each of the
dipyridamole and fludrocortisone master plates to a dilution plate
containing 100 .mu.l of media (RPMI; Gibco BRL, #11875-085), 10%
Fetal Bovine Serum (Gibco BRL, #25140-097), 2%
Penicillin/Streptomycin (Gibco BRL, #15140-122)) using the TomTec
Quadra Plus liquid handler. This dilution plate was then mixed and
a 10 .mu.l aliquot transferred to the final assay plate, which had
been pre-filled with 40 .mu.l/well RPMI media containing the
appropriate stimulant to activate TNF.alpha. secretion (see
below).
Example 2
Assay for TNF.alpha. Suppressing Activity by the Combination of
Dipyridamole and Fludrocortisone
[0069] The compound dilution matrix was assayed using a TNF.alpha.
ELISA method. Briefly, a 100 .mu.l suspension of diluted human
white blood cells contained within each well of a polystyrene
384-well plate (NalgeNunc) was stimulated to secrete TNF.alpha. by
treatment with a final concentration of 10 ng/ml phorbol
12-myristate 13-acetate (Sigma) and 750 ionomycin (Sigma). Various
concentrations of each test compound were added at the time of
stimulation. After 16-18 hours of incubation at 37.degree. C. in a
humidified incubator, the plate was centrifuged and the supernatant
transferred to a white opaque polystyrene 384 well plate
(NalgeNunc, Maxisorb) coated with an anti-TNF antibody (PharMingen,
#18631D). After a two-hour incubation, the plate was washed (Tecan
PowerWasher 384) with phosphate buffered saline (PBS) containing
0.1% Tween 20 (polyoxyethylene sorbitan monolaurate) and incubated
for an additional one hour with another anti-TNF antibody that was
biotin labeled (PharMingen, 18642D) and horseradish peroxidase
(HRP) coupled to strepavidin (PharMingen, #13047E). After the plate
was washed with 0.1% Tween 20/PBS, the HRP substrate (which
contains luminol, hydrogen peroxide, and an enhancer such as
para-iodophenol) was added to each well and light intensity
measured using a LJL Analyst luminometer. Control wells contained a
final concentration of 1 .mu.g/ml cyclosporin A (Sigma).
[0070] Together, dipyridamole and fludrocortisone were able to
suppress TNF.alpha. secretion in blood stimulated with phorbol
12-myristate 13-acetate and ionomycin. As seen in Tables 1 and 2,
dipyridamole was able to enhance the potency of fludrocortisone by
60-fold. At a concentration of 947 nM, fludrocortisone alone
inhibited TNF.alpha. secretion by 39%. Addition of 124 nM
dipyridamole to a concentration of only 15 nM fludrocortisone
resulted in the inhibition of TNF.alpha. secretion by 39% (Table
1). Efficacy was maintained while reducing the total drug species
by over 80%, from 947 nM to 163 nM. In the presence of 2 .mu.M
dipyridamole, 50% TNF.alpha. inhibition is achieved by only 4 nM
fludrocortisone. This level of inhibition is not possible with
fludrocortisone alone at concentrations that would be expected to
cause serious mineralocorticoid-induced side effects. Dipyridamole
enhancement of fludrocortisone activity was observed in a secondary
screen (Table 2). Again, a low dose of 495 nM dipyridamole enhanced
the potency of fludrocortisone by over 135 fold. Specifically, 947
nM fludrocortisone alone was required to achieve a 52% reduction of
TNF.alpha. secretion. A similar reduction (49%) was measured for
the combination of 7 nM fludrocortisone and 495 nM dipyridamole.
Further, the addition of 248 nM dipyridamole resulted in a
supramaximal effect on the inhibition of TNF.alpha. secretion at
fludrocortisone concentrations as low as 59 nM.
TABLE-US-00001 TABLE 1 Primary Screen Data of Fludrocortisone vs
Dipyridamole Average Result of Plates (% TNF.alpha. suppression
from P/I-induced white blood cells) Dipyridamole [.mu.M] 7.927
1.982 0.495 0.124 0.031 0.000 Fludrocortisone 0.947 82.90 66.61
54.90 52.48 61.35 39.19 [.mu.M] 0.237 81.88 61.99 52.35 52.11 46.77
36.66 0.059 79.57 60.37 47.08 45.47 42.93 32.49 0.015 77.13 54.06
40.70 38.73 30.62 22.63 0.004 74.61 50.60 34.21 24.90 22.52 17.21
0.000 66.37 35.24 13.21 9.08 3.68 0.00
TABLE-US-00002 TABLE 2 Secondary Screen Data of Fludrocortisone vs
Dipyridamole Average Result of 2 Plates (% TNF.alpha. Suppression
from P/I-induced white blood cells) Dipyridamole [.mu.M] 7.927
3.964 1.982 0.991 0.495 0.248 0.124 0.062 0.031 0.000
Fludrocortisone 0.947 89.12 82.25 78.01 69.10 67.91 61.77 60.82
53.38 53.41 52.05 [.mu.M] 0.473 92.64 84.40 78.44 70.25 65.06 60.25
56.14 53.68 50.07 50.16 0.237 89.69 83.68 82.01 70.36 66.53 65.46
60.90 56.65 52.34 49.25 0.118 87.58 80.66 76.13 68.18 65.89 67.09
58.91 54.17 47.39 46.42 0.059 84.43 79.37 73.86 64.53 63.88 58.96
56.84 48.63 44.66 40.24 0.030 88.61 76.42 68.58 65.77 62.08 51.31
49.96 47.02 44.19 36.95 0.015 90.46 79.36 73.52 65.22 56.39 62.88
43.17 47.73 46.00 37.77 0.007 84.11 75.29 69.74 64.61 48.90 42.05
38.92 39.27 36.70 29.49 0.004 79.02 75.15 65.79 55.19 46.00 41.93
35.15 30.94 30.20 22.40 0.000 78.11 66.54 62.36 48.20 33.73 23.02
12.13 9.43 10.16 -3.50
Example 3
Preparation of Pairwise Compound Mixed Combination Serial Dilution
Matrix
[0071] A compound matrix of dipyridamole and prednisolone were
prepared according to the method of Example 1. The initial stock
solution of dipyridamole was 16 mg/ml, and prednisolone was 1.6
mg/ml.
Example 4
Assay for TNF.alpha. Suppressing Activity by the Combination of
Dipyridamole and Prednisolone
[0072] The compound dilution matrix of Example 3 was assayed using
the TNF.alpha. ELISA method of Example 2. The results are shown in
Table 3. Together, dipyridamole and prednisolone were able to
suppress TNF.alpha. secretion in blood stimulated with phorbol
12-myristate 13-acetate and ionomycin to a greater extent than
either compound alone. Specifically, dipyridamole greatly increased
the potency of prednisolone. Prednisolone alone, at a concentration
of 250 nM, can suppress TNF.alpha. secretion by 38%. The same level
of suppression (41%) can be achieved by only 1 nM prednisolone in
combination with 2 .mu.M dipyridamole. This represents a shift in
the potency of prednisolone of over 250-fold. Further, the addition
of 2 .mu.M dipyridamole to 250 nM prednisolone resulted in a
supramaximal effect (57%), compared to prednisolone alone (38%).
The combination of low doses of prednisolone and dipyridamole,
therefore, results in the inhibition TNF.alpha. to levels
previously unattainable without a high risk of
glucocorticoid-induced side effects.
TABLE-US-00003 TABLE 3 Primary Screen Data of Prednisolone vs
Dipyridamole Average Result of 2 Plates (% TNF.alpha. suppression
from P/I-induced white blood cells) Dipyridamole [.mu.M] 7.93 1.98
0.50 0.12 0.031 0.00 Prednisolone 0.250 70.30 56.72 48.90 50.82
46.08 38.25 [.mu.M] 0.063 68.53 57.68 51.61 47.24 37.57 33.00 0.016
66.48 45.20 40.12 40.99 32.42 37.84 0.004 61.06 47.25 34.66 33.48
32.42 19.99 0.001 57.35 40.84 32.10 25.47 20.64 4.86 0.000 47.51
27.21 18.30 12.63 11.24 0.00
Other Embodiments
[0073] Various modifications and variations of the described method
and system of the invention will be apparent to those skilled in
the art without departing from the scope and spirit of the
invention. Although the invention has been described in connection
with specific desired embodiments, it should be understood that the
invention as claimed should not be unduly limited to such specific
embodiments. Indeed, various modifications of the described modes
for carrying out the invention that are obvious to those skilled in
the fields of medicine, immunology, pharmacology, endocrinology, or
related fields are intended to be within the scope of the
invention.
[0074] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually incorporated by reference.
* * * * *