U.S. patent application number 13/475751 was filed with the patent office on 2013-03-07 for treatment of diseases modulated by a h4 receptor agonist.
The applicant listed for this patent is Emile Loria, Michalis Nicolaou, Gaetan Terrasse, Yves Trehin. Invention is credited to Emile Loria, Michalis Nicolaou, Gaetan Terrasse, Yves Trehin.
Application Number | 20130059878 13/475751 |
Document ID | / |
Family ID | 42231773 |
Filed Date | 2013-03-07 |
United States Patent
Application |
20130059878 |
Kind Code |
A1 |
Loria; Emile ; et
al. |
March 7, 2013 |
TREATMENT OF DISEASES MODULATED BY A H4 RECEPTOR AGONIST
Abstract
The invention provides a method for the treatment of H4R
modulated diseases and/or conditions comprising administering to
the subject an effective amount of a H4R agonist. The invention
also provides a method for treating COPD comprising administering
to the subject an effective amount of a H4R agonist, a H1R
antagonist and an anticholinergic drug. Further, the invention
provides a pharmaceutical formulation comprising a H4R agonist, a
second active agent and a pharmaceutically acceptable carrier.
Inventors: |
Loria; Emile; (La Jolla,
CA) ; Nicolaou; Michalis; (San Diego, CA) ;
Terrasse; Gaetan; (Saint-Valier, FR) ; Trehin;
Yves; (Toulouse, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Loria; Emile
Nicolaou; Michalis
Terrasse; Gaetan
Trehin; Yves |
La Jolla
San Diego
Saint-Valier
Toulouse |
CA
CA |
US
US
FR
FR |
|
|
Family ID: |
42231773 |
Appl. No.: |
13/475751 |
Filed: |
May 18, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12486725 |
Jun 17, 2009 |
8207188 |
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13475751 |
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11784992 |
Apr 9, 2007 |
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12486725 |
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12069775 |
Feb 12, 2008 |
8207292 |
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12486725 |
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60790490 |
Apr 7, 2006 |
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60816754 |
Jun 26, 2006 |
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60889423 |
Feb 12, 2007 |
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60892325 |
Mar 1, 2007 |
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60974685 |
Sep 24, 2007 |
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61073288 |
Jun 17, 2008 |
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Current U.S.
Class: |
514/291 |
Current CPC
Class: |
C07D 491/056 20130101;
A61P 1/04 20180101; A61P 27/02 20180101; C07D 491/04 20130101; A61P
17/00 20180101; A61P 37/00 20180101; A61P 29/00 20180101; A61P 1/00
20180101; A61P 35/00 20180101 |
Class at
Publication: |
514/291 |
International
Class: |
A61K 31/4355 20060101
A61K031/4355; A61P 1/04 20060101 A61P001/04; A61P 29/00 20060101
A61P029/00; A61P 35/00 20060101 A61P035/00; A61K 31/4545 20060101
A61K031/4545; A61K 31/4439 20060101 A61K031/4439; A61P 37/00
20060101 A61P037/00; A61P 17/00 20060101 A61P017/00 |
Claims
1.-45. (canceled)
46. A method for the treatment of a disease modulated by a H4R
agonist comprising administering to a subject an effective amount
of a H4R agonist to treat the disease.
47. The method of claim 46, wherein the disease modulated by a H4R
agonist is an immune system disease.
48. The method of claim 47, wherein the immune system disease is
selected from eczema, Crohn's disease, ulcerative colitis, celiac
disease, autoimmune gastritis, uveitis and disease conditions in
cancer.
49. The method of claim 46, wherein the H4R agonist is selected
from a group consisting of Tritoqualine (TRQ), Tritoqualine isomer,
4-methylhistmime (4MeHA) and clobenprobit (CB).
50. The method of claim 47, wherein the Tritoqualine isomer is E1
or E2.
51. The method of claim 46, wherein the effective amount of the H4R
agonist is about 200 mg/day, about 1 g/day, about 2 g/day, about 3
g/day, about 1-5 mg/day, about 5-10 mg/day, about 10-15 mg/day,
about 15-20 mg/day, about 20-25 mg/day, about 25-30 mg/day, about
30-35 mg/day, about 35-40 mg/day, about 40-45 mg/day, about 45-50
mg/day, about 50-55 mg/day, about 55-60 mg/day, about 60-65 mg/day,
about 65-70 mg/day, about 70-75 mg/day, about 75-80 mg/day, about
80-85 mg/day, about 85-90 mg/day, about 90-95 mg/day, about 95-100
mg day, about 100-105 mg/day, about 105-110 mg/day, about 110-115
mg/day, about 115-120 mg/day, about 120-125 mg/day, about 125-130
mg/day, about 130-135 mg/day, about 135-140 mg/day, about 140-145
mg/day, about 145-150 mg/day, about 150-155 mg/day, about 155-160
mg/day, about 160-165 mg/day, about 165-170 mg/day, about 170-175
mg/day, about 175-180 mg/day, about 180-185 mg/day, about 185-190
mg/day, about 190-195 mg/day, about 195-200 mg/day, about 200-205
mg/day, about 205-210 mg/day, about 210-215 mg/day, about 215-220
mg/day, about 220-225 mg/day, about 225-230 mg/day, about 230-235
mg/day, about 235-240 mg/day, about 240-245 mg/day, about 245-250
mg/day, about 250-255 mg/day, about 255-260 mg/day, about 260-265
mg/day, about 265-270 mg/day, about 270-275 mg/day, about 275-280
mg/day, about 280-285 mg/day, about 285-290 mg/day, about 290-295
mg/day, about 295-300 mg/day, about 300-305 mg/day, about 305-310
mg/day, about 310-315 mg/day, about 315-320 mg/day, about 320-325
mg/day, about 325-330 mg/day, about 330-335 mg/day, about 335-340
mg/day, about 340-345 mg/day, about 345-350 mg/day, about 350-355
mg/day, about 355-360 mg/day, about 360-365 mg/day, about 365-370
mg/day, about 370-375 mg/day, about 375-380 mg/day, about 380-385
mg/day, about 385-390 mg/day, about 390-395 mg/day, about 395-400
mg/day, about 400-405 mg/day, about 405-410 mg/day, about 410-415
mg/day, about 415-420 mg/day, about 420-425 mg/day, about 425-430
mg/day, about 430-435 mg/day, about 435-440 mg/day, about 440-445
mg/day, about 445-450 mg/day, about 1 mg/day-1 g/day, about 1
mg/day-2 g/day or about 1 mg/day-3 g/day.
52. The method of claim 46, further comprising administering to the
subject an effective amount of one or more of an anti-H1, anti-H2
and anti-H3 drug.
53. The method of claim 46, further comprising administering to the
subject an effective amount of an anti-H1 drug.
54. The method of claim 46, further comprising administering to the
subject an effective amount of an anti-H2 drug.
55. The method of claim 46, further comprising administering to the
subject an effective amount of an anti-H3 drug.
56. The method of claim 46, further comprising administering to the
subject an effective amount of an anti-H1, anti-H2 and anti-H3
drug.
57. The method of claim 52, wherein the anti-H1 drug is selected
from a group consisting of brompheniramine, cetirizine,
fexofenadine, cyproheptadine, dexchlorpheniramine, hydroxizine,
ketotifen, loratadine, mequitazine, oxotomide, mizolastine,
ebastine, astemizole, carbinoxamide, alimemazine, buclizine,
cyclizine hydrochloride, doxylamine, mepyramine, antazoline,
diphenhydramine, carbinoxamine, clemastine, dimenhydrinate,
pheniramine, chlorphenamine, triprolidine, chlorcyclizine,
hydroxyzine, meclizine, promethazine, and azatadine.
58. The method of claim 57, wherein the effective amount of anti-H1
drug is about 10.0 mg/day, about 120.0 mg/day, about 0.1 to 1.0
mg/day, about 1.0 to 2.0 mg/day, about 2.0 to 3.0 mg/day, about 3.0
to 4.0 mg/day, about 4.0 to 5.0 mg/day, about 5.0 to 6.0 mg/day,
about 6.0 to 7.0 mg/day, about 7.0 to 8.0 mg/day, about 8.0 to 9.0
mg/day, about 9.0 to 10.0 mg/day, about 10.0 to 11.0 mg/day, about
11.0 to 12.0 mg/day, about 12.0 to 13.0 mg/day, about 13.0 to 14.0
mg/day, about 14.0 to 15.0 mg/day, about 15.0 to 16.0 mg/day, about
16.0 to 17.0 mg/day, about 17.0 to 18.0 mg/day, about 18.0 to 19.0
mg/day, about 19.0 to 20.0 mg/day, about 20.0 to 21.0 mg/day, about
21.0 to 22.0 mg/day, about 22.0 to 23.0 mg/day, about 23.0 to 24.0
mg/day, about 24.0 to 25.0 mg/day, about 25.0 to 26.0 mg/day, about
26.0 to 27.0 mg/day, about 27.0 to 28.0 mg/day, about 28.0 to 29.0
mg/day, about 29.0 to 30.0 mg/day, about 30.0 to 31.0 mg/day, about
31.0 to 32.0 mg/day, about 32.0 to 33.0 mg/day, about 33.0 to 34.0
mg/day, about 34.0 to 35.0 mg/day, about 1 mg/day to about 35
mg/day, 1.0 to 30.0 mg/day, about 30.0 to 50.0 mg/day, about 50.0
to about 70.0 mg/day, about 70.0 to 90.0 mg/day, about 90.0 to
110.0 mg/day, about 110.0 to 130.0 mg/day, about 130.0 to 150.0
mg/day, about 150.0 to 170.0 mg/day, about 170.0 to 190.0 mg/day,
about 190.0 to 210.0 mg/day, about 210.0 to 230.0 mg/day, about
230.0 to 250.0 mg/day, about 250.0 to 270.0 mg/day, about 270.0 to
290.0 mg/day, about 290.0 to 310.0 mg/day, about 310.0 to 330.0
mg/day, about 330.0 to 350.0 mg/day, about 350.0 to 370.0 mg/day,
about 370.0 to 390.0 mg/day, about 390.0 to 410.0 mg/day, about
410.0 to 430.0 mg/day, about 430.0 to 450.0 mg/day, about 450.0 to
470.0 mg/day, about 470.0 to 490.0 mg/day, about 490.0 to 510.0
mg/day or about 1 mg/day to 510 mg/day.
59. (canceled)
60. The method of claim 52, wherein the effective amount of anti-H2
drug is about 150.0 mg/day, about 1.0 to 20.0 mg/ml, about 20.0 to
40.0 mg/ml, about 40.0 to 60.0 mg/ml, about 60.0 to 80.0 mg/ml,
about 80.0 to 100.0 mg/ml, about 100.0 to 120.0 mg/ml, about 120.0
to 140.0 mg/ml, about 140.0 to 160.0 mg/ml, about 160.0 to 180.0
mg/ml, about 180.0 to 200.0 mg/ml, about 200.0 to 220.0 mg/ml,
about 220.0 to 240.0 mg/ml, about 240.0 to 260.0 mg/ml, about 260.0
to 280.0 mg/ml, about 280.0 to 300.0 mg/ml, about 300.0 to 320.0
mg/ml, about 320.0 to 340.0 mg/ml, about 340.0 to 360.0 mg/ml,
about 360.0 to 380.0 mg/ml, about 380.0 to 400.0 mg/ml, about 400.0
to 420.0 mg/ml, about 420.0 to 440.0 mg/ml, about 440.0 to 460.0
mg/ml, about 460.0 to 480.0 mg/ml, about 480.0 to 500.0 mg/ml,
about 500.0 to 520.0 ing/ml, about 520.0 to 540.0 mg/ml, about
540.0 to 560.0 mg/ml, about 560.0 to 580.0 mg/ml, about 580.0 to
600.0 mg/ml, about 600.0 to 620.0 mg/ml, about 620.0 to 640.0
mg/ml, about 640.0 to 660.0 mg/ml, about 660.0 to 680.0 mg/ml,
about 680.0 to 700.0 mg/ml, about 700.0 to 720.0 mg/ml, about 720.0
to 740.0 mg/ml, about 740.0 to 760.0 mg/ml, about 760.0 to 780.0
mg/ml, about 780.0 to 800.0 mg/ml, about 800.0 to 820.0 mg/ml,
about 820.0 to 840.0 mg/ml, about 840.0 to 860.0 mg/ml, about 860.0
to 880.0 mg/ml, about 880.0 to 900.0 mg/ml or about 1 mg/ml to
about 900 mg/ml.
61. The method of claim 52, wherein the anti-H3 drug is selected
from a group consisting of betahistine
(N-methyl-2-pyridin-2-ylethanamine), ABT-239
(4-(2-{2-[(2R)-2-Methylpyrrolidin-1-yl]ethyl}-benzofuran-5-yl)ben-
zonitrile),
Cipralisant(1R,2R)-4-(2-(5,5-dimethylhex-1-ynyl)cyclopropyl)imidazole,
Ciproxifan cyclopropyl 4-(3-(1H-imidazol-4-yl)propyloxy)phenyl
ketone, Clobenpropit
N'-[(4-chlorophenyl)methyl]-1-[3-(3H-imidazol-4-yl)propylthio]formamidine-
, Thioperamide
N-Cyclohexyl-4-(1H-imidazol-4-yl)-1-piperidinecarbothioamide.
62. The method of claim 61, wherein the effective amount of anti-H3
drug is about 0.1 to 1.0 mg/day, about 1.0 to 2.0 mg/day, about 2.0
to 3.0 mg/day, about 3.0 to 4.0 mg/day, about 4.0 to 5.0 mg/day,
about 5.0 to 6.0 mg/day, about 6.0 to 7.0 mg/day, about 7.0 to 8.0
mg/day, about 8.0 to 9.0 mg/day, about 9.0 to 10.0 mg/day, about
10.0 to 15.0 mg/day, about 15.0 to 20.0 mg/day, about 20.0 to 25.0
mg/day, about 25.0 to 30.0 mg/day, about 30.0 to 35.0 mg/day, about
35.0 to 40.0 mg/day, about 45.0 to 50.0 mg/day, about 50.0 to 60.0
mg/day, about 60.0 to 70.0 mg/day, about 70.0 to 80.0 mg/day, about
80.0 to 90.0 mg/day, about 90.0 to 100.0 mg/day, about 100.0 to
150.0 mg/day, about 150.0 to 200.0 mg/day, about 200.0 to 250.0
mg/day, about 250.0 to 300.0 mg/day, about 300.0 to 350.0 mg/day,
about 350.0 to 400.0 mg/day, about 400.0 to 450.0 mg/day, about
450.0 to 500.0 mg/day, about 500.0 to 550.0 mg/day, about 550.0 to
600.0 mg/day, about 600.0 to 650.0 mg/day, about 650.0 to 700.0
mg/day, about 700.0 to 750.0 mg/day, about 750.0 to 800.0 mg/day,
about 800.0 to 850.0 mg/day, about 850.0 to 900.0 mg/day, about
900.0 to 950.0 mg/day, about 950.0 to 1000.0 mg/day, about 1000.0
to 1100.0 mg/day, about 1100.0 to 1200.0 mg/day, about 1200.0 to
1300.0 mg/day, about 1300.0 to 1400.0 mg/day, about 1400.0 to
1500.0 mg/day, about 1500.0 to 1600.0 mg/day, about 1600.0 to
1700.0 mg/day, about 1800.0 to 1900.0 mg/day or about 1900.0 to
2000.0 mg/day.
63. The method of claim 46, wherein the subject is suffering from
eczema, Crohn's disease, ulcerative colitis, celiac disease,
autoimmune gastritis, uveitis and disease conditions in cancer.
64. The method of claim 46, wherein the subject is a mammal.
65.-69. (canceled)
70. A pharmaceutical formulation comprising a combination of (a) a
H4R agonist, (b) one or more of an anti-H1, anti-H2 and anti-H3
drug, and (c) a pharmaceutically acceptable carrier.
71.-100. (canceled)
101. The method of claim 46, wherein the H4R agonist does not
inhibit Organic Cation Transporter-3 (OCT-3) and/or Serotonin
Transporter (SERT).
Description
[0001] This application claims the priority of U.S. Ser. No.
61/073,288, filed Jun. 17, 2008 and is a continuation-in-part of
U.S. Ser. No. 11/784,992, filed Apr. 9, 2007 (which claims the
priorities of U.S. Ser. No. 60/790,490, filed Apr. 7, 2006 and U.S.
Ser. No. 60/816,754, filed Jun. 26, 2006); and U.S. Ser. No.
12/069,775, filed Feb. 12, 2008 (which claims the priorities of
U.S. Ser. No. 60/889,423, filed Feb. 12, 2007, U.S. Ser. No.
60/892,325, filed Mar. 1, 2007 and U.S. Ser. No. 60/974,685, filed
Sep. 24, 2007), the contents of all of which are hereby
incorporated by reference, in their entirety, into this
application, and from which priority is hereby claimed.
[0002] Throughout this application various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this invention pertains.
BACKGROUND OF THE INVENTION
[0003] The H4R is the most recently identified and characterized
histamine receptor (for reviews, see de Esch J. P., et al., Trends
Pharmacol. Sci. 2005, 26(9), 462-469). The receptor is found in the
bone marrow and spleen and is expressed on eosinophils; basophils,
mast cells (Liu, C., et al., Mol. Pharmacol. 2001, 59(3), 420-426;
dendritic cells, and human synovial cells from rheumatoid arthritis
patients (Ikawa, Y., et al., Biol. Pham. Bull. 2005, 28(10),
2016-2018). More recently new variants of H4 receptors were
described by Richard M. van Rijn, et al. (Biochem. J. (2008)
Immediate Publication, doi:10.1042/BJ20071583).
[0004] In contrast to the other histamine receptors, H4R has a
distinct expression profile on immune and other cells and modulates
their function (immune-modulatory role). Such cells include: mast
cells, eosinophils, dendritic cells, T cells, monocytes and
macrophages and antigen presenting cells in general. It is also
present in endothelial and epithelial cells. The H4R appears to
play a role in multiple functions of these cells, such as,
activation, migration, differentiation, and cytokine and chemokine
production. While the H4R has been identified and characterized,
its functions and involvement in disease is still under study.
[0005] Currently, treatment of histamine related diseases generally
focuses on the design of antagonists for H1R and H2R for the
treatment of such diseases. For example, for allergies, antagonists
of H1R such as loratadine, fexofenadine, diphenyl-hydramine,
cetirizine, brompheniramine, cyproheptadine, dexchlorpheniramine,
hydroxizine, ketotifen, mequitazine, oxotomide, mizolastine,
ebastine, astemizole, carbinoxamide, alimemazine, buclizine,
cyclizine hydrochloride, and doxylamine and others were developed.
For stomach conditions exacerbated by gastric acid, antagonists of
H2R such as cimetidine, ranitidine, famotidine, and nizatidine and
others, were developed.
[0006] H4R antagonists have been proposed to have therapeutic
potential in a number of inflammatory diseases including
Inflammatory Bowel Disease (IBD), Systemic Lupus Erythematosus
(SLE), atherosclerosis, allergy and asthma and others. (Zhang M,
Thurmond R L, Dunford P J, The histamine H(4) receptor: a novel
modulator of inflammatory and, immune disorders. Pharmacol Ther.
2007 March; 113(3):594-606).
[0007] There are very few examples of compounds, much less of
marketed therapeutics, that have H1R, H2R or H3R agonist activity.
An example of a compound that has some H1R agonist activity is
betahistine, N-methyl-2-pyridin-2-ylethanamine (betahistine), a H3
antagonist low H1 agonist. Betahistine is not a full H1 agonist. It
is a potent H3 antagonist with a low H1 agonist activity.
Betahistine has a very strong affinity for histamine H3 receptors
and a weak affinity for histamine H1 receptors. Betahistine seems
to dilate the blood vessels within the middle ear which can relieve
pressure from excess fluid and act on the smooth muscle. It is used
for the treatment of Meniere syndrome (Review Article. CNS Drugs
2001: 15(11) 855-870). Examples of H3 agonists include: immepip
4-(3H-imidazol-4-ylmethyl)piperidine, imetit
S-[2-(4-imidazolyl)ethyl]isothiourea. Immepip and Immetit, although
not marketed drugs, are H3 receptor agonists and are currently used
in studies in animals with aim to elucidate the H3 receptor
function and derive, possible therapeutic utility, for brain
disorders. Examples of H2 agonists include: Betazole
2-(2H-Pyrazol-3-yl)ethanamine, impromidine,
N-[3-(imidazol-4-yl)-propyl]-N'-{2-[(5-methylimidazol-4-yl)methylthio]eth-
yl}-guanidine. Betazole and impromidine are histamine H2 agonists,
used clinically as diagnostic tools to test gastric secretory
function.
[0008] All examples of therapeutic molecules presented above do not
utilize the H-receptor agonist activity as the major therapeutic
function. In the case of Betahistine, the main therapeutic activity
may not be predominantly due to the H1 agonist activity, but due to
the H3 antagonist activity. The remainder agonist cases have a
diagnostic utility or basic research and investigative use.
[0009] There is currently no commercially available drug to treat
H4R modulated diseases that is a H4R agonist. Thus, there is an
unmet and unperceived need to develop H4R agonists to treat H4R
modulated diseases.
Anticholinergics
[0010] Anticholinergics are used in the treatment of COPD because
they widen the airways by relaxing smooth muscle. They do this by
blocking acetylcholine receptors. Acetylcholine is a chemical
produced by the brain that causes muscle contraction, which in tarn
constricts airways. Anticholinergics are considered first-line
therapy for COPD.
[0011] Examples of anticholinergics include, but are not limited
to: tiotropium bromide (Spiriva.RTM.) and ipratropium bromide
(Atrovent.RTM.). Atrovent is the only inhaled anticholinergic agent
available in the United States.
Combination Inhalers
[0012] Recently, a new product called Advair.RTM. was FDA approved
for asthma but it may also be beneficial in the treatment of COPD.
It combines two medications that have been on the market,
salmeterol (a longer acting beta2-agonist) and fluticasone (a
steroid). Many patients require both medications to help prevent
asthma or COPD symptoms from worsening, but until now were only
available as separate inhalers. Advair.RTM. cannot be used to
quickly relieve asthma or COPD symptoms, it is to be taken on a
scheduled basis without regard for the symptoms the patient is
having at that particular moment.
[0013] Another combination inhaler is Combivent.RTM.. It contains
two medications: albuterol and ipratropium. Albuterol is an inhaled
beta-agonist that works in the lungs to open airways and allow for
easier breathing. It does this by stimulating the beta-receptors,
which are a certain type of receptor located in the lungs, which
help regulate constriction and dilation of the airways. Ipratropium
is an anticholinergic used in the treatment of COPD to widen the
airways by relaxing and opening air passages to the lungs, making
it easier to breathe.
Corticosteroids
[0014] Corticosteroids are used to treat many health conditions.
This drug class is mainly used for treating asthma, but it has been
used for treating COPD. Oral corticosteroids decrease inflammation
in the lungs that is associated with COPD. They may take longer to
work than inhaled corticosteroids, since they have to travel
through the bloodstream before they get to the lungs to work.
Corticosteroids are only used in COPD patients who do not respond
well to other standard therapies.
Inhaled Beta-2 Agonists
[0015] Beta2-agonists work in a manner similar to adrenaline,
opening airways and easing breathing. They work by binding with,
and thus stimulating, "beta2-receptors" that line the cell walls of
the lungs and the bronchioles. The effect of this stimulation is to
relax smooth muscles and widen the airways. In COPD, beta2-agonists
should be scheduled instead of taken on as needed basis. Possible
side effects to the beta2-agonists include shakiness, rapid
heartbeat, and upset stomach.
[0016] Until recently, all available beta2-agonists were ones that
worked quickly but lasted for a relatively short time--about 4-6
hours. Longer-acting beta2-agonists have since been introduced.
They cannot be used to quickly relieve symptoms, because there is a
delay before they start working. Currently there are two on the
market: salmeterol (Serevent.RTM.) and formoterol (Foradil.RTM.),
Longer-acting beta2-agonists are prescribed as maintenance
medications which are to be taken on a scheduled basis without
regard for the symptoms the patient is having at that particular
moment. A short-acting beta2-agonist is best to treat acute
symptoms of shortness of breath.
Inhaled Corticosteroids
[0017] Corticosteroids suppress the body's production of substances
that trigger inflammation and reduce the production of substances
that maintain inflammation. This drug class is mainly used for
treating asthma, but it has been used for treating COPD.
Corticosteroids are only used in COPD patients who do not respond
well to other standard therapies.
Mucolytics
[0018] This class of drugs is used to thin the mucus associated
with cough caused by thick mucus. Mucolytics make it easier to
clear the mucus, which can be irritating and cause a cough.
Oral Beta-2 Agonists
[0019] Oral beta2-agonists works in a similar fashion to inhaled
beta2-agonists, but they may take longer to work than the inhaled
formulation. Oral beta-agonists must be absorbed in the digestive
tract and travel through the circulatory system before they begin
working in the lungs, whereas the inhaled formulations go straight
to the lungs.
Theophyllines
[0020] Theophyllines appear to widen airways by relaxing the smooth
muscles surrounding the airways. Theophylline is also used as a
long-acting bronchodilator to prevent COPD symptoms. Taken orally
as tablets, capsules, or liquids, theophylline is available in
immediate-release and controlled-release formulations as well as
injection (aminophylline).
Tritoqualine
[0021]
7-Amino-4,5,6-triethoxy-3-(5,6,7,8-tetrahydro-4-methoxy-6-methyl-1,-
3-dioxolo[4,5-g]isoquinolin-5-yl)phthalide or Tritoqualine (TRQ) is
a drug, currently formulated in 100 mg tablets and sold in
pharmacies in Europe for the treatment of allergy.
[0022] Tritoqualine is an inhibitor of the enzyme histidine
decarboxylase (HDC), which catalyzes histidine decarboxylation in
vivo to produce histamine, an endogenous biogenic amine, plus
carbon dioxide. Inhibiting histamine production in the body is
proposed to ameliorate symptoms of allergy.
Leukotriene Receptor Antagonists
[0023] Leukotriene Receptor Antagonists (LRAs), e.g.,
Montelukast.RTM. and Zafirlukast.RTM.) have been traditionally used
for the treatment of asthma.
SUMMARY OF THE INVENTION
[0024] The invention disclosed herein relates to the surprising
discovery that H4R agonists can be used for the treatment of H4R
related diseases modulated by H4R. The invention relates generally
to the treatment or amelioration of H4R modulated diseases with H4R
agonists.
[0025] The invention further relates to methods of using H4R
agonists, alone, or in combination with one or more other active
agents to achieve desirable therapeutic effects for H4R modulated
diseases. Agents that can be used in combination with H4R agonists
are, for example, other H4R agonists, H1R antagonists (e.g. anti-H1
drug), H2R antagonists (e.g. anti-H2 drug), H3R antagonists (e.g.
anti-H3 drug), LRA and NSAIDS.
[0026] The invention also relates to methods of providing a plasma
concentration of H4R and/or the other compound(s) with a
peak-to-trough ratio of less than 3.5, less than 3.0, less than 2.5
or less than 2.0, over a time period spanning from about 1 hour to
about 6 hours after administration to a subject.
[0027] The invention further relates to pharmaceutical formulations
comprising a H4R agonist, one or more of a H1R antagonist, H2R
antagonist, H3R antagonist, LRA and NSAID, and a pharmaceutically
acceptable carrier.
[0028] The invention further provides pharmaceutical formulations
comprising therapeutically effective amounts of an H1R antagonist,
H2R antagonist, H3R antagonist, LRA and NSAID, and a
pharmaceutically acceptable carrier useful to treat a H4R modulated
disease.
[0029] The active compounds (e.g., H4R agonist, H1R antagonist, H2R
antagonist, H3R antagonist, LRA, NSAID) in the pharmaceutical
formulation may be combined in a single dosage form or for
unit-dose or multi-dose administration. Pharmaceutical carriers
suitable for administration of the compounds include any such
carriers known to those skilled in the art to be suitable for the
particular route of administration and/or suitable for a desired
release rate (e.g., immediate or controlled release) of the active
compounds. The multiple active ingredients can be formulated into
suitable pharmaceutical preparations such as solutions,
suspensions, tablets, dispersible tablets, capsules, powders,
sustained release, for oral administration or in sterile solutions
or suspensions for parenteral administration, as well as
transdermal patch preparation, nasal formulation and dry powder
inhalers et al. The compositions are formulated into pharmaceutical
compositions using techniques and procedures well known in the
art.
[0030] The pharmaceutical formulations of the invention provide a
plasma concentration of the active compounds with a peak-to-trough
ratio of less than 3.5, less than 3.0, less than 2.5 or less than
2.0 over a time period spanning from about 1 hour to about 6 hours
after administration to a subject.
[0031] The invention further relates to treatment or prevention of
COPD using drug combinations comprising a H4R agonist, an anti-H1
drug and an anticholinergic drug. The drug combination can further
comprise any one or more of existing COPD therapies including but
not limited to combination inhaler, corticosteroids, inhaled beta-2
agonists, inhaled corticosteroids, mucolytics, oral beta-2 agonists
and theophyllines.
BRIEF DESCRIPTION OF THE FIGURES
[0032] FIG. 1 illustrates the chemical formula of Tritoqualine
(7-Amino-4,5,6-triethoxy-3-(5,6,7,8-tetrahydro-4-methoxy-6-methyl-1,3-dio-
xolo[4,5-g]isoquinolin-5-yl)phthalide), as described in Example 3,
below.
[0033] FIG. 2 illustrates the sterical structure of the
Tritoqualine diastereomer D1, as described in Example 3, below.
[0034] FIG. 3 illustrates the sterical structure of the
Tritoqualine diastereomer D2, as described in Example 3, below.
[0035] FIG. 4 shows a chromatogram of the separation of
Tritoqualine stereoisomers via a chiral column. In the bottom part,
the UV absorbance at 190 nm has been detected, while the top part
depicts polarimetric detection at an averaged absorption in the
range of 200-800 nm, as described in Example 3, below.
[0036] FIG. 5 shows a UV spectrum of each of the peaks of FIG. 4,
as described in Example 3, below.
[0037] FIG. 6 illustrates the 3D-structures of the two
stereoisomers (enantiomers) of FIGS. 4 and 5 as determined by X-Ray
crystallography, as described in Example 3, below.
[0038] FIG. 7 shows the % improvement for each patient from the
visit T0 to visit T1 as described in Example 12. Statistical
analysis using a paired t-test on the values generated on visits T0
and T1 showed a statistically significant improvement due to
Tritoqualine treatment. P-value for the paired t-test P-0.0012
calculated using GraphPad Prism.RTM., as described in Example 3,
below.
[0039] FIG. 8 shows the results of a cell proliferation assay using
human TF1 cells that express H4R. When 10 .mu.M TRQ and 10 .mu.M CB
were compared in three separate assays (n=3), TRQ was found to be
superior, more potent inhibitor of cell proliferation than CB
(approximately double the potency of CB). Average inhibition of
cell proliferation by CB and TRQ was found to be 23% and 49%,
respectively, as described in Example 13.
[0040] FIG. 9 shows a comparison between TRQ alone and, in
association with Clobenpropit (TRQ+CB) on the inhibition of human
TF1 cell proliferation assay; both TRQ and CB were used each, at 10
.mu.M concentration. TRQ and Clobenpropit in 50:50-mixture produced
a more potent inhibition of cell proliferation compared to
Tritoqualine alone. This suggests that TRQ and CB demonstrate an
additive effect on the inhibition of cell proliferation on TF1
cells, as described in Example 14.
[0041] FIG. 10 shows the effects of TRQ or CB on a cell assay using
mouse medullary cells C57B1/6 expressing H4R. CB inhibits the
proliferation of CFC at a dose of 10.sup.-5M (10 .mu.M) and was
compared to TRQ at the dose of 10.sup.-5M (10 .mu.M). TRQ is a more
potent inhibitor of cell proliferation than Clobenpropit, as
described in Example 16.
[0042] FIG. 11 shows the effects of E2 enantiomer on a cell assay
using mouse medullary cells C57B1/6 expressing H4R. Approximately
60% of haematopoietic progenitor cells are blocked by E2 enantiomer
of Tritoqualine. This G0/G1 blocking of the cell cycle is reversed
if a known H4R antagonist such as JNJ7777120 is used, suggesting
that the inhibition of cell proliferation is due to activation of
the H4R, as described in Example 15.
[0043] FIG. 12 shows the effects of E1 enantiomer on a cell assay
using mouse medullary cells C57B1/6 expressing H4R. Approximately
48% of haematopoietic progenitor cells are blocked by the E1
enantiomer of Tritoqualine. This G0/G1 blocking of the cell cycle
is reversed if a known H4R antagonist such as JNJ7777120 is used
suggesting that the inhibition of cell proliferation is due to
activation of the H4R, as described in Example 15.
[0044] FIG. 13 is a graph showing that enantiomer E2 is a more
effective inhibitor of cell growth than TRQ and E1 (approximately
50% inhibition of G0/G1 phase was observed), whereas E2 inhibited
G0/G1 by 57%, as described in Example 17.
[0045] FIG. 14 is a bar graph showing that tritoqualine (TRQ) is an
1-14 agonist. CB-5 is clobenpropit at 1.times.10.sup.-5 M (10 uM);
AT-5 is a mixture of tritoqualine isomer 1 (also referred to herein
as E1 or D1) and 2 (also referred to herein as E2 or D2) at
1.times.10.sup.-5 M (10 uM); AT-6 is a mixture of tritoqualine
isomer 1 and 2 at 1.times.10.sup.-6 M (1 uM); E1-5 is tritoqualine
isomer 1 at 1.times.10.sup.-5 M (10 uM); E2-5 is tritoqualine
isomer 2 at 1.times.10.sup.-5 M (10 uM).
[0046] FIG. 15A is a scatter plot and FIG. 15B is a bar graph both
showing that TRQ inhibits progenitor cell cycling. CB-5 is
clobenpropit-5; AT-5 is a mixture of tritoqualine isomer 1 and 2 at
1.times.10.sup.-5M (10 uM); AT-6 is a mixture of tritoqualine
isomer 1 and 2 at 1.times.10.sup.-6M (1 uM); E1-5 is tritoqualine
isomer 1 at 1.times.10.sup.-5 M (10 uM); E1-6 is tritoqualine
isomer 1 at 1.times.10.sup.-6 M (1 uM); E2-5 is tritoqualine isomer
2 at 1.times.10.sup.-5M (10 uM); E2-6 is tritoqualine isomer 2 at
1.times.10.sup.-6M (1 uM).
[0047] FIG. 16 is a bar graph that shows that TRQ inhibits ckit
positive hematopoietic progenitor cell proliferation. The reference
to CB-5, E1-5, and E2-5 is the same as FIG. 15.
[0048] FIG. 17 includes three bar graphs (FIGS. 17A, B and C)
showing that TRQ promotes cells to enter and remain in the G0
and/or G1 phase of the cell cycle.
[0049] FIG. 18 is a bar graph that shows that pertussis toxin (PTX)
reverses TRQ's effects by decreasing number of ckit positive bone
marrow cells in the G0 and/or G1 phase of the cell cycle.
[0050] FIG. 19 is a bar graph that shows that TRQ maintains the
hematopoietic progenitor cells in the G0 and/or G1 phase of the
cell cycle so that even in the presence of Ara-C the cell CFU
remains high.
[0051] FIG. 20 includes two bar graphs (FIGS. 20A and B) that shows
that TRQ is an H4 agonist but does not affect organic cation
transporter-3 (OCT-3) or serotonin transporter (SERT).
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0052] As used herein "allergy" is an abnormal or altered
immunologic reaction induced by an allergen in a subject who
suffers from hypersensitivity to that allergen including
antibody-antigen reactions that include immediate type
hypersensitivity reactions such that when IgE molecules are
crosslinked with an allergen(s), mast cells and basophils release
mediators such as histamines. Examples of allergy symptoms include
sinusitis, rhinitis, hives, headaches, post-nasal drip, coughing,
sneezing, respiratory difficulties, sore throats, allergic asthma,
allergic conjunctivitis, allergic rhinitis, tightness in throat and
chest, and loss of voice.
[0053] For purposes of the present invention the term "controlled
release" refers to a pharmaceutical dosage form which releases one
or more active pharmaceutical agents over a prolonged period of
time, e.g. over a period of more than 1 hour. Controlled release
(CR) components can also be referred to as sustained release (BR),
prolonged release (PR), or extended release (ER). When used in
association with the dissolution profiles discussed herein, the
term "controlled release" refers to that portion of a dosage form
made according to the present invention which delivers the
compositions of the invention over a period of time e.g. greater
than 1 hour. The term "modified release" and "controlled release"
is used interchangeably herein.
[0054] The term "immediate release" refers to a dosage form which
releases the compositions of the invention substantially
immediately upon contact with gastric juices and will result in
substantially complete dissolution within about 1 hour. Immediate
release (IR) components can also be referred to as instant release.
When used in association with the dissolution profiles discussed
herein, the term "immediate release" refers to that portion of a
dosage form made according to the present invention which delivers
the compositions of the invention over a period of time less than 1
hour.
[0055] The term "derivative" means a chemically modified compound
wherein the modification is considered routine by the ordinary
skilled chemist, such as an ester or an amide of an acid,
protecting groups, such as a benzyl group for an alcohol or thiol,
and tert-butoxycarbonyl group for an amine.
[0056] The term "effective amount" means an amount of a compound or
composition according to the present invention effective in
producing the desired therapeutic effect.
[0057] The term "analog" means a compound which comprises a
chemically modified form of a specific compound or class thereof,
and which maintains the pharmaceutical and/or pharmacological
activities characteristic of said compound.
[0058] As used herein, "pharmaceutically acceptable salts" refer to
derivatives of the disclosed compounds wherein the parent compound
is modified by making acid or base salts thereof. Examples of
pharmaceutically acceptable salts include, but are not limited to,
mineral or organic acid salts of basic residues such as amines;
alkali or organic salts of acidic residues such as carboxylic
acids; and the like. The pharmaceutically acceptable salts include
the conventional non-toxic salts or the quaternary ammonium salts
of the parent compound formed, for example, from non-toxic
inorganic or organic acids. For example, such conventional
non-toxic salts include those derived from inorganic acids such as
hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric
and the like; and the salts prepared from organic acids such as
acetic, propionic, succinic, glycolic, stearic, lactic, malic,
tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic,
phenylacetic, glutamic, benzoic, salicylic, sulfanilic,
2-acetoxybenzoic, fumaric, toluensulfonic, methanesulfonic, ethane
disulfonic, oxalic, isethionic, and the like.
[0059] The phrase "pharmaceutically acceptable" is employed herein
to refer to those compounds, materials, compositions; and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication commensurate with a reasonable
benefit/risk ratio.
[0060] The term "about" when used in connection with percentages
means.+-.1-5%.
[0061] The term "H4R agonists" means any molecule that is an
agonist to the H4 receptor. Examples of H4 agonists include, but
are not limited to, Tritoqualine (TRQ) or derivative thereof (e.g.
racemic versions thereof), Tritoqualine isomers (e.g., E1 and E2
enantiomers), 4-methylhistmime (4MeHA) and clobenprobit (CB).
[0062] The term "anti-H1" refers to any drag that is an antagonist
to the H1 receptor. Examples of H1R antagonists include, but are
not limited to, brompheniramine, cetirizine, fexofenadine,
cyproheptadine, dexchlorpheniramine, hydroxyzine, ketotifen,
loratadine, mequitazine, oxotomide, mizolastine, ebastine,
astemizole, carbinoxamide, alimemazine, buclizine, cyclizine
hydrochloride, doxylamine, mepyramine, antazoline, diphenhydramine,
carbinoxamine, clemastine, dimenhydrinate, pheniramine,
chlorphenamine, triprolidine, chlorcyclizine, hydroxyzine,
meclizine, promethazine, and azatadine.
[0063] The term "anti-H2" refers to any drug that is an antagonist
to the H2 receptor. Examples of H2R antagonists include, but are
not limited to, ranitidine, cimetidine, famotidine, and
nizatidine.
[0064] The term "anti-H3" refers to any drug that is an antagonist
to the H3 receptor. Examples of H3R antagonists include, but are
not limited to, betahistine (N-methyl-2-pyridin-2-ylethanamine),
ABT-239
(4-(2-{2-[(2R)-2-Methylpyrrolidin-1-yl]ethyl}-benzofuran-5-yl)benzonitril-
e),
Cipralisant(1R,2R)-4-(2-(5,5-dimethylhex-1-ynyl)cyclopropyl)imidazole,
Ciproxifan cyclopropyl 4-(3-(1H-imidazol-4-yl)propyloxy)phenyl
ketone, Clobenpropit
N'-[(4-chlorophenyl)methyl]-1-[3-(3H-imidazol-4-yl)propylthio]formamidine-
, Thioperamide
N-Cyclohexyl-4-(1H-imidazol-4-yl)-1-piperidinecarbothioamide.
[0065] The term "anti-H4" refers to any drag that is an antagonist
to the H4 receptor. Examples of H4R antagonists include, but are
not limited to, thioperamide, JNJ7777120 and JNJ10191584 (Zhang et
al Pharmacology and therapeutics, 113 (2007) 594),
N-Cyclohexyl-4-(1H-imidazol-4-yl)-1-piperidinecarbothioamide,
R-.alpha.-methylhistamine. A number of other H4R antagonists are
described in a review by Venable et al, (Anti Inflamm. Anti Allergy
Agents Med. Chem, 2006, 5 307). None of the antagonists described
in the literature are currently used as therapeutic agents.
[0066] The term H4R modulated disease includes, but is not limited
to immune system diseases, and gastroinstestinal conditions
ameliorated by proper histamine management. Examples include GERD;
food allergies; Zollinger-Ellison Syndrome; peptic ulcer;
dyspepsia; allergic eosinophilic gastroenteritis; mastocytosis with
gastrointestinal symptoms; diseases provoked by CD4 Th2 lymphocytes
(asthma, rhinitis conjunctivitis eczema, gastro-esophageal reflux
disease and other gastric diseases exacerbated by gastric acid),
CD4 Th1 lymphocytes (IBD, Crohn's disease, ulcerative colitis and
celiac disease and autoimmune gastritis, chronic bronchitis etc.)
and/or CD4 Th17 lymphocytes (Uveitis and inflammatory digestive
diseases); Chronic Obstructive Pulmonary Disorder (COPD) and other
pulmonary types of inflammation including asthma; disease
conditions in cancer; and other diseases where histamine or the H4R
is involved. H4R agonists may also be used in vaccines as adjuvant
to improve immune responses to the inoculated antigens.
[0067] The gastrointestinal disease condition may be any of GERD, a
food allergy, Zollinger-Ellison Syndrome, peptic ulcer, dyspepsia,
allergic eosinophilic gastroenteritis, and mastocytosis with
gastrointestinal symptoms.
[0068] The term "desirable therapeutic effect" means to treat a
subject with the active agents of the invention in order to prevent
or ameliorate a disease and/or disease condition.
[0069] The terms "therapeutic agent", "drug" and "active agent" are
used interchangeably herein and refer to the compounds disclosed
herein that affect a H4R modulated disease and/or disease
condition. For example, active agents of the invention include, but
are not limited to, H4R agonists, H1R antagonists, H2R antagonists,
H3R antagonists, LRAs, steroids, NSAIDs.
[0070] The terms "pharmaceutical formulations", "pharmaceutical
compositions" and "dosage forms" are used interchangeably herein
and refer to a composition containing the active ingredient(s) of
the invention in a form suitable for administration to a
subject.
[0071] The term "anticholinergics" or "anticholinergic drugs" refer
to compounds that block acetylcholine receptors. Acetylcholine is a
chemical produced by the brain that causes muscle contraction,
which in turn can constrict airways. Anticholinergics can be use
for treatment of COPD.
[0072] The term "peak-to-trough ratio" refers to a comparison of
the values for a peak (e.g., a high point) plasma level and a
trough (e.g., a low point) plasma level of an active agent over a
set amount of time. For example, a line graph with plasma levels of
a drug with values ranging from 400 ng/ml (peak) to 200 ng/ml
(trough) over a four hour period, gives a peak-to-trough ratio of 2
for that time. More than one peak-to-trough ratio can be
illustrated in a graph.
METHODS OF THE INVENTION
[0073] The invention relates to the use of a Tritoqualine (TRQ)
(including derivatives or isomers thereof (e.g. Tritoqualine
isomers (E1 and E2)) as histamine H4 receptor (H4R) agonists. The
invention further relates to the use of H4R agonists to treat
diseases and/or disease conditions modulated by H4R agonists.
[0074] The invention relates generally to the treatment or
amelioration of H4R modulated diseases or conditions with one or
more compositions of the invention (e.g., H4R agonists).
[0075] In one embodiment, the invention provides methods for the
treatment or amelioration of H4R modulated diseases and/or
conditions in a subject, comprising administering to the subject an
effective amount of a H4R agonist. In a further embodiment, the H4R
agonist may not inhibit (or at least not significantly inhibit)
Organic Cation Transporter-3 (OCT-3) and/or Serotonin Transporter
(SERT).
[0076] The invention further provides methods for the treatment or
amelioration of H4R modulated diseases or conditions in a subject,
comprising administering to the subject an effective amount of a
H4R agonist, in combination with one, or more other compounds to
reach desirable therapeutic effects for H4R modulated diseases.
Compounds that can be used in combination with H4R agonists are,
for example, other H4R agonists, H1R antagonists (e.g. anti-H1
compounds or drugs), H2R antagonists (e.g. anti-H2 compounds or
drugs), H3R antagonists (e.g. anti-H3 compounds or drugs), LRAs and
NSAIDs. The method of the invention provides the administration of
a single or a combination of H4R agonist drugs.
[0077] In one embodiment, the subject is given an effective amount
of a H4R agonist(s) and an anti-H1 drug(s). In another embodiment,
the subject is given an effective amount of a H4R agonist(s) and an
anti-H2 drug(s). In another embodiment, the subject is given an
effective amount of a H4R agonists) and an anti-H3 drug(s). In yet
another embodiment, the subject is given an effective amount of a
H4R agonist(s) and one or more of an anti-H1 drug, an anti-H2 drug
and an anti-H3 drug. In a further embodiment, the subject may be
given an effective amount of a combination of a H4R agonist(s), and
any one or more of an anti-H1 drug, an anti-H2 drug and an anti-H3
drug, a LRA, a steroid and NSAID.
[0078] The H4R modulated disease and/or condition may be any of
GERD, a food allergy, Zollinger-Ellison Syndrome, peptic ulcer,
dyspepsia, allergic eosinophilic gastroenteritis, and mastocytosis
with gastrointestinal symptoms, diseases provoked by CD4 Th2
lymphocytes (e.g., asthma, rhinitis conjunctivitis eczema,
gastro-esophageal reflux disease and other gastric diseases
exacerbated by gastric acid), diseases provoked CD4 Th1 lymphocytes
(e.g., IBD, Crohn's disease, ulcerative colitis and celiac disease
and autoimmune gastritis, chronic bronchitis etc.), diseases
provoked CD4 Th17 lymphocytes (e.g., uveitis and inflammatory
digestive diseases); Chronic Obstructive Pulmonary Disorder (COPD)
and other pulmonary types of inflammation including asthma; disease
conditions in cancer; and other diseases where histamine or the H4R
is involved. H4R agonists may also be used in vaccines as adjuvant
to improve immune responses to inoculated antigens. In a preferred
embodiment, the H4R modulated disease or condition is COPD and/or
asthma. To treat COPD, a preferred pharmaceutical composition
comprises a tritoqualine and an anti-H4 drug such as loratadine. To
treat GERD, a preferred pharmaceutical composition comprises a
tritoqualine and an anti-H2 drag such as ranitidine. To treat food
allergy, a preferred pharmaceutical composition comprises a
tritoqualine and an antileukotriene such as Montelukast.RTM..
However other compositions of the invention may be used.
[0079] The invention also relates to methods of providing a plasma
concentration of H4R agonist and/or the other active agent(s) with
specific peak-to-trough ratios in a subject. In one embodiment, the
peak-to-trough ratio is less than 3.5, over a time period spanning
from about 1 hour to about 6, hours after administration to a
subject. In another embodiment, the peak-to-trough ratio is less
than 3.0, over a time period spanning from about 1 hour to about 6
hours, after administration to a subject. In yet another
embodiment, the peak-to-trough ratio is less than 2.5, over a time
period spanning from about 1 hour to about 6 hours, after
administration, to a subject in a further embodiment, the
peak-to-trough ratio is less than 2.0 over a time period spanning
from about 1 hour to about 6 hours after administration to a
subject.
[0080] A dose of a H4R agonist (e.g., Tritoqualine, or an isomer or
derivative thereof) administered to a subject may be about 200
mg/day. In another embodiment, the dose of a H4R agonist
administered to a subject may be about 1 g/day. In an additional
embodiment, the dose of a H4R agonist administered to a subject may
be about 2 g/day. In yet another embodiment, the dose of a H4R
agonist administered to a subject may be about 3 g/day. In a
further embodiment, the dose of a H4R agonist administered to a
subject may be about 1-5 mg/day, about 5-10 mg/day, about 10-15
mg/day, about 15-20 mg/day, about 20-25 mg/day, about 25-30 mg/day,
about 30-35 mg/day, about 35-40 mg/day, about 40-45 mg/day, about
45-50 mg/day, about 50-55 mg/day, about 55-60 mg/day, about 60-65
mg/day, about 65-70 mg/day, about 70-75 mg/day, about 75-80 mg/day,
about 80-85 mg/day, about 85-90 mg/day, about 90-95 mg/day, about
95-100 mg day, about 100-105 mg/day, about 105-110 mg/day, about
110-115 mg/day, about 115-120 mg/day, about 120425 mg/clay, about
125-130 mg/day, about 130-135 mg/day, about 135-140 mg/day, about
140-145 mg/day, about 145-150 mg/day, about 150-155 mg/day, about
155-160 mg/day, about 160-165 mg/day, about 165-170 mg/day, about
170-175 mg/day, about 175-180 mg/day, about 180-185 mg/day, about
185-190 mg/day, about 190-195 mg/day, about 195-200 mg/day, about
200-205 mg/day, about 205-210 mg/day, about 210-215 mg/day, about
215-220 mg/day, about 220-225 mg/day, about 225-230 mg/day, about
230-235 mg/day, about 235-240 mg/day, about 240-245 mg/day, about
245-250 mg/day, about 250-255 mg/day, about 255-260 mg/day, about
260-265 mg/day, about 265-270 mg/day, about 270-275 mg/day, about
275-280 mg/day, about 280-285 mg/day, about 285-290 mg/day, about
290-295 mg/day, about 295-300 mg/day, about 300-305 mg/day, about
305-310 mg/day, about 310-315 mg/day, about 315-320 mg/day, about
320-325 mg/day, about 325-330 mg/day, about 330-335 mg/day, about
335-340 mg/day, about 340-345 mg/day, about 345-350 mg/day, about
350-355 mg/day, about 355-360 mg/day, about 360-365 mg/day, about
365-370 mg/day, about 370-375 mg/day, about 375-380 mg/day, about
380-385 mg/day, about 385-390 mg/day, about 390-395 mg/day, about
395-400 mg/day, about 400-405 mg/day, about 405-410 mg/day, about
410-415 mg/day, about 415-420 mg/day, about 420-425 mg/day, about
425-430 mg/day, about 430-435 mg/day, about 435-440 mg/day, about
440-445 mg/day, about 445-450 mg/day, about 1 mg/day-1 g/day, about
1 mg/day-2 g/day or about 1 mg/day-3 g/day.
[0081] Suitable examples of H4R agonists include, but are not
limited to, any of Tritoqualine includes an isomer or derivative
thereof, 4-methylhistmime (4MeHA) and clobenprobit (CB).
[0082] Tritoqualine maybe used successfully alone and in
combinatorial therapy to treat allergy, GERD, food allergy, and
COPD. The drug has two chiral centers and therefore, it exists in
four isomeric forms. The commercial product includes a mixture of
two isomers, one with the chiral centers at the SS configuration
and the other one at the RR configuration. Examples of Tritoqualine
isomers include, but are not limited to, E1 and E2 enantiomers.
[0083] Surprisingly, Tritoqualine and each of its isomers may have
different activity as an H4R agonist i.e., various H4R agonists
will have varying effects on. H4R. Thus, binding of various H4R
agonists to the H4R can provide varying therapeutic profiles (e.g.,
cytokine activation profiles) in a subject.
[0084] In accordance with the practice of the invention, suitable
examples of Leukotriene Receptor Antagonists (LRA) include, but are
not limited to, Montelukast.RTM. (Singulair.RTM.), Pranlukast.RTM.
and Zafirlukas.RTM.. The method provides the administration of a
single or a combination of LRA drugs.
[0085] The dose of LRAs (e.g. Montelukast.RTM. (Singulair.RTM.))
administered to a subject may be about 10.0 mg/day. In another
embodiment, the dose of a LRA(s) (e.g. Montelukast.RTM.)
administered to a subject may be about 0.1 to 1.0 mg/day, about 1.0
to 2.0 mg/day, about 2.0 to 3.0 mg/day, about 3.0 to 4.0 mg/day,
about 4.0 to 5.0 mg/day, about 5.0 to 6.0 mg/day, about 6.0 to 7.0
mg/day, about 7.0 to 8.0 mg/day, about 8.0 to 9.0 mg/day, about 9.0
to 10.0 mg/day, about 10.0 to 11.0 mg/day, about 11.0 to 12.0
mg/day, about 12.0 to 13.0 mg/day, about 13.0 to 14.0 mg/day, about
14.0 to 15.0 mg/day, about 15.0 to 16.0 mg/day, about 16.0 to 17.0
mg/day, about 17.0 to 18.0 mg/day, about 18.0 to 19.0 mg/day, about
19.0 to 20.0 mg/day, about 20.0 to 21.0 mg/day, about 21.0 to 22.0
mg/day, about 22.0 to 23.0 mg/day, about 23.0 to 24.0 mg/day, about
24.0 to 25.0 mg/day, about 25.0 to 26.0 mg/day, about 26.0 to 27.0
mg/day, about 27.0 to 28.0 mg/day, about 28.0 to 29.0 mg/day, about
29.0 to 30.0 mg/day, about 30.0 to 31.0 mg/day, about 31.0 to 32.0
mg/day, about 32.0 to 33.0 mg/day, about 33.0 to 34.0 mg/day, about
34.0 to 35.0 mg/day or about 1 mg/day to 35 mg/day.
[0086] In accordance with the practice of the invention, suitable
examples of anti-H1 drugs includes, but are limited to, any of
brompheniramine, cetirizine, fexofenadine, cyproheptadine,
dexchlorpheniramine, hydroxyzine, ketotifen, loratadine,
mequitazine, oxotomide, mizolastine, ebastine, astemizole,
carbinoxamide, alimemazine, buclizine, cyclizine hydrochloride,
doxylamine, mepyramine, antazoline, diphenhydramine, carbinoxamine,
clemastine, dimenhydrinate, pheniramine, chlorphenamine,
triprolidine, chlorcyclizine, hydroxyzine, meclizine, promethazine,
and azatadine or analogs, equivalents, isomers, pharmaceutically
acceptable salts, and solvate fauns thereof. The method provides
the administration of a single or a combination of anti-H1
drugs.
[0087] The dose of an anti-H1 drug (e.g., loratadine) administered
to a subject may be about 10.0 mg/day. In another embodiment, the
dose of the anti-H1 drag(s) (e.g. loratadine) administered to a
subject may be about 0.1 to 1.0 mg/day, about 1.0 to 2.0 mg/day,
about 2.0 to 3.0 mg/day, about 3.0 to 4.0 mg/day, about 4.0 to 5.0
mg/day, about 5.0 to 6.0 mg/day, about 6.0 to 7.0 mg/day, about 7.0
to 8.0 mg/day, about 8.0 to 9.0 mg/day, about 9.0 to 10.0 mg/day,
about 10.0 to 11.0 mg/day, about 11.0 to 12.0 mg/day, about 12.0 to
13.0 mg/day, about 13.0 to 14.0 mg/day, about 14.0 to 15.0 mg/day,
about 15.0 to 16.0 mg/day, about 16.0 to 17.0 mg/day, about 17.0 to
18.0 mg/day, about 18.0 to 19.0 mg/day, about 19.0 to 20.0 mg/day,
about 20.0 to 21.0 mg/day, about 21.0 to 22.0 mg/day, about 22.0 to
23.0 mg/day, about 23.0 to 24.0 mg/day, about 24.0 to 25.0 mg/day,
about 25.0 to 26.0 mg/day, about 26.0 to 27.0 mg/day, about 27.0 to
28.0 mg/day, about 28.0 to 29.0 mg/day, about 29.0 to 30.0 mg/day,
about 30.0 to 31.0 mg/clay, about 31.0 to 32.0 mg/day, about 32.0
to 33.0 mg/day, about 33.0 to 34.0 mg/day, about 34.0 to 35.0
mg/day or about 1 mg/day to about 35 mg/day.
[0088] Also, the dose of an anti-H1 drug (e.g., cetirizine)
administered to a subject may be about 10 mg/day. In another
embodiment, the dose of the anti-H1 drug(s) (e.g. cetirizine)
administered to a subject may be about 0.1 to 0.5 mg/day, about 0.5
to 1.0 mg/day, about 1.0 to 1.5 mg/day, about 1.5 to 2.0 mg/day,
about 2.0 to 2.5 mg/day, about 2.5 to 3.0 mg/day, about 3.0 to 3.5
mg/day, about 3.5 to 4.0 mg/day, about 4.0 to 4.5 mg/day, about 4.5
to 5.0 mg/day, about 5.0 to 5.5 mg/day, about 5.5 to 6.0 mg/day,
about 6.0 to 6.5 mg/day, about 6.5 to 7.0 mg/day, about 7.0 to 7.5
mg/day, about 7.5 to 8.0 mg/day, about 8.5 to 9.0 mg/day, about 9.0
to 9.5 mg/day, about 9.5 to 10.0 mg/day, about 10.5 to 11.0 mg/day,
about 11.0 to 11.5 mg/day, about 11.5 to 12.0 mg/day, about 12.0 to
12.5 mg/day, about 12.5 to 13.0 mg/day, about 13.0 to 13.5 mg/day,
about 13.5 to 14.0 mg/day, about 14.0 to 14.5 mg/day, about 14.5 to
15.0 mg/day, about 15.0 to 15.5 mg/day, about 15.5 to 16.0 mg/day,
about 16.0 to 16.5 mg/day, about 16.5 to 17.0 mg/day, about 17.0 to
17.5 mg/day, about 17.5 to 18.0 mg/day, about 18.5 to 19.0 mg/day,
about 19.0 to 19.5 mg/day, about 19.5 to 20.0 mg/day, about 20.5 to
21.0 mg/day, about 21.0 to 21.5 mg/day, about 21.5 to 22.0 mg/day,
about 22.0 to 22.5 mg/day, about 22.5 to 23.0 mg/day, about 23.0 to
23.5 mg/day, about 23.5 to 24.0 mg/day, about 24.0 to 24.5 mg/day,
about 24.5 to 25.0 mg/day, about 25.0 to 25.5 mg/day, about 25.5 to
26.0 mg/day, about 26.0 to 26.5 mg/day, about 26.5 to 27.0 mg/day,
about 27.0 to 27.5 mg/day, about 27.5 to 28.0 mg/day, about 28.5 to
29.0 mg/day, about 29.0 to 29.5 mg/day, about 29.5 to 30.0 mg/day
or about 0.5 mg/day to 30.0 mg/day.
[0089] Further, the dose of an anti-H1 drug (e.g., fexofenadine)
administered to a subject may be about 120.0 mg/day. In another
embodiment, the dose of the anti-H1 drug(s) (e.g. fexofenadine)
administered to a subject may be about 1.0 to 30.0 mg/day, about
30.0 to 50.0 mg/day, about 50.0 to about 70.0 mg/day, about 70.0 to
90.0 mg/day, about 90.0 to 110.0 mg/day, about 110.0 to 130.0
mg/day, about 130.0 to 150.0 mg/day, about 150.0 to 170.0 mg/day,
about 170.0 to 190.0 mg/day, about 190.0 to 210.0 mg/day, about
210.0 to 230.0 mg/day, about 230.0 to 250.0 mg/day, about 250.0 to
270.0 mg/day, about 270.0 to 290.0 mg/day, about 290.0 to 310.0
mg/day, about 310.0 to 330.0 mg/day, about 330.0 to 350.0 mg/day,
about 350.0 to 370.0 mg/day, about 370.0 to 390.0 mg/day, about
390.0 to 410.0 mg/day, about 410.0 to 430.0 mg/day, about 430.0 to
450.0 mg/day, about 450.0 to 470.0 mg/day, about 470.0 to 490.0
mg/day, about 490.0 to 510.0 mg/day or about 1 mg/day to 510
mg/day.
[0090] Suitable examples of anti-H2 drugs include, but are not
limited to, any of ranitidine, cimetidine, famotidine, and
nizatidine and/or analogs, equivalents, isomers, pharmaceutically
acceptable salts, and solvate forms thereof. The method provides
the administration of a single or combination of anti-H2 drags.
[0091] The dose of an anti-H2 drug (e.g., ranitidine) administered
to a subject may be about 150 mg/day. In another embodiment, the
dose of the anti-H2 drug(s) (e.g. ranitidine) administered to a
subject may be about 1.0 to 20.0 mg/ml, about 20.0 to 40.0 mg/ml,
about 40.0 to 60.0 mg/ml, about 60.0 to 80.0 mg/ml, about 80.0 to
100.0 mg/ml, about 100.0 to 120.0 mg/ml, about 120.0 to 140.0
mg/ml, about 140.0 to 160.0 mg/ml, about 160.0 to 180.0 mg/ml,
about 180.0 to 200.0 mg/ml, about 200.0 to 220.0 mg/ml, about 220.0
to 240.0 mg/ml, about 240.0 to 260.0 mg/ml, about 260.0 to 280.0
mg/ml, about 280.0 to 300.0 mg/ml, about 300.0 to 320.0 mg/ml,
about 320.0 to 340.0 mg/ml, about 340.0 to 360.0 mg/ml, about 360.0
to 380.0 mg/ml, about 380.0 to 400.0 mg/ml, about 400.0 to 420.0
mg/ml, about 420.0 to 440.0 mg/ml, about 440.0 to 460.0 mg/ml,
about 460.0 to 480.0 mg/ml, about 480.0 to 500.0 mg/ml, about 500.0
to 520.0 mg/ml, about 520.0 to 540.0 mg/ml, about 540.0 to 560.0
mg/ml, about 560.0 to 580.0 mg/ml, about 580.0 to 600.0 mg/ml,
about 600.0 to 620.0 mg/ml, about 620.0 to 640.0 mg/ml, about 640.0
to 660.0 mg/ml, about 660.0 to 680.0 mg/ml, about 680.0 to 700.0
mg/ml, about 700.0 to 720.0 mg/ml, about 720.0 to 740.0 mg/ml,
about 740.0 to 760.0 mg/ml, about 760.0 to 780.0 mg/ml, about 780.0
to 800.0 mg/ml, about 800.0 to 820.0 mg/ml, about 820.0 to 840.0
mg/ml, about 840.0 to 860.0 mg/ml, about 860.0 to 880.0 mg/ml,
about 880.0 to 900.0 mg/ml or about 1 mg/ml to about 900 mg/ml.
[0092] Suitable examples of anti-H3 drugs include, but are not
limited to, any of betahistine (N-methyl-2-pyridin-2-ylethanamine),
ABT-239
(4-(2-{2-[(2R)-2-Methylpyrrolidin-1-yl]ethyl}-benzofuran-5-yl)benzonitril-
e),
Cipralisant(1R,2R)-4-(2-(5,5-dimethylhex-1-ynyl)cyclopropyl)imidazole,
Ciproxifan cyclopropyl 4-(3-(1H-imidazol-4-yl)propyloxy)phenyl
ketone, Clobenpropit
N'-[(4-chlorophenyl)methyl]-1-[3-(3H-imidazol-4-yl)propylthio]formamidine-
, Thioperamide
N-Cyclohexyl-4-(1H-imidazol-4-yl)-1-piperidinecarbothioamide. The
method provides the administration of a single or combination of
anti-H3 drugs.
[0093] The dose of an anti-H3 drug administered to a subject may be
about 0.1 to 1.0 mg/day, about 1.0 to 2.0 mg/day, about 2.0 to 3.0
mg/day, about 3.0 to 4.0 mg/day, about 4.0 to 5.0 mg/day, about 5.0
to 6.0 mg/day, about 6.0 to 7.0 mg/day, about 7.0 to 8.0 mg/day,
about 8.0 to 9.0 mg/day, about 9.0 to 10.0 mg/day, about 10.0 to
15.0 mg/day, about 15.0 to 20.0 mg/day, about 20.0 to 25.0 mg/day,
about 25.0 to 30.0 mg/day, about 30.0 to 35.0 mg/day, about 35.0 to
40.0 mg/day, about 45.0 to 50.0 mg/day, about 50.0 to 60.0 mg/day,
about 60.0 to 70.0 mg/day, about 70.0 to 80.0 mg/day, about 80.0 to
90.0 mg/day, about 90.0 to 100.0 mg/day, about 100.0 to 150.0
mg/day, about 150.0 to 200.0 mg/day, about 200.0 to 250.0 mg/day,
about 250.0 to 300.0 mg/day, about 300.0 to 350.0 mg/day, about
350.0 to 400.0 mg/day, about 400.0 to 450.0 mg/day, about 450.0 to
500.0 mg/day, about 500.0 to 550.0 mg/day, about 550.0 to 600.0
mg/day, about 600.0 to 650.0 mg/day, about 650.0 to 700.0 mg/clay,
about 700.0 to 750.0 mg/day, about 750.0 to 800.0 mg/day, about
800.0 to 850.0 mg/day, about 850.0 to 900.0 mg/day, about 900.0 to
950.0 mg/day, about 950.0 to 1000.0 mg/day, about 1000.0 to 1100.0
mg/day, about 1100.0 to 1200.0 mg/day, about 1200.0 to 1300.0
mg/day, about 1300.0 to 1400.0 mg/day, about 1400.0 to 1500.0
mg/day, about 1500.0 to 1600.0 mg/day, about 1600.0 to 1700.0
mg/day, about 1800.0 to 1900.0 mg/day or about 1900.0 to 2000.0
mg/day.
[0094] The invention further relates to the treatment or prevention
of COPD using drug combinations comprising a H4R agonist, an
anti-H1 drug and an anticholinergic drug.
[0095] In one embodiment of the invention, the invention provides
methods for the treatment of COPD comprising administering to a
subject an effective amount of a H4R agonist, an anti-H1 drug and
an anticholinergic drug. Suitable H4R agonists and anti-H1 drugs
are described above. Suitable examples of anticholinergics include,
but are not limited to: tiotropium bromide (Spiriva.RTM.) and
ipratropium bromide (Atrovent.RTM.).
[0096] The dose of an anticholinergic drug administered to a
subject may be about 0.1 to 1.0 mg/clay, about 1.0 to 2.0 mg/day,
about 2.0 to 3.0 mg/day, about 3.0 to 4.0 mg/day, about 4.0 to 5.0
mg/day, about 5.0 to 6.0 mg/day, about 6.0 to 7.0 mg/day, about 7.0
to 8.0 mg/day, about 8.0 to 9.0 mg/clay, about 9.0 to 10.0 mg/day,
about 10.0 to 15.0 mg/day, about 15.0 to 20.0 mg/day, about 20.0 to
25.0 mg/day, about 25.0 to 30.0 mg/day, about 30.0 to 35.0 mg/day,
about 35.0 to 40.0 mg/day, about 45.0 to 50.0 mg/day, about 50.0 to
60.0 mg/day, about 60.0 to 70.0 mg/day, about 70.0 to 80.0 mg/day,
about 80.0 to 90.0 mg/day, about 90.0 to 100.0 mg/day, about 100.0
to 150.0 mg/day, about 150.0 to 200.0 mg/day, about 200.0 to 250.0
mg/day, about 250.0 to 300.0 mg/day, about 300.0 to 350.0 mg/day,
about 350.0 to 400.0 mg/day, about 400.0 to 450.0 mg/day, about
450.0 to 500.0 mg/day, about 500.0 to 550.0 mg/day, about 550.0 to
600.0 mg/day, about 600.0 to 650.0 mg/day, about 650.0 to 700.0
mg/day, about 700.0 to 750.0 mg/day, about 750.0 to 800.0 mg/day,
about 800.0 to 850.0 mg/day, about 850.0 to 900.0 mg/day, about
900.0 to 950.0 mg/day, about 950.0 to 1000.0 mg/day or about
1000.0. The dose of an anticholinergic drug dispensed by an inhaler
to a subject may be about 0.1 mg per puff, about 0.5 mg per puff,
about 1.0 mg per puff, about 2.5 mg per puff, about 5.0 mg per pug
about 10.0 mg per puff, about 15.0 mg per puff, about 18.0 mg per
puff, about 20.0 mg per puff, about 25.0 mg per puff, about 30.0 mg
per puff, about 35.0 mg per puff, about 40.0 mg per pug about 45.0
mg per puff or about 50.0 mg per puff.
[0097] In a specific embodiment of the invention, the H4R agonist
is tritoqualine, the anti-H1 drug is loratadine and the
anticholinergic drug is Spiriva.RTM..
[0098] In a further embodiment of the invention, the drug
combination can further comprise any one or more of existing COPD
therapies including but not limited to combination inhaler,
steroids, inhaled beta-2 agonists, inhaled corticosteroids,
mucolytics, oral beta-2 agonists and theophyllines.
[0099] According to the practice of the invention, suitable
examples of steroids include, but are not limited to,
corticosteroids, Cortisone, Hydrocortisone/cortisol, Desoxycortone,
Alclometasone, Aldosterone, Amcinonide, Beclometasone,
Betamethasone, Budesonide, Ciclesonide, Clobetasol, Clobetasone,
Clocortolone, Cloprednol, Cortivazol, Deflazacort,
Deoxycorticosterone, Desonide, Desoximetasone, Dexamethasone,
Diflorasone, Diflucortolone, Difluprednate, Fluclorolone,
Fludrocortisone, Fludroxycortide, Flumetasone, Flunisolide,
Fluocinolone acetonide, Fluocinonide, Fluocortin, Fluocortolone,
Fluorometholone, Fluperolone, Fluprednidene, Fluticasone,
Formocortal, Halcinonide, Halometasone, Hydrocortisone aceponate,
Hydrocortisone buteprate, Hydrocortisone butyrate, Loteprednol,
Medrysone, Meprednisone, Methylprednisolone, Methylprednisolone
aceponate, Mometasone furoate, Paramethasone, Prednicarbate,
Prednisone, Prednisolone, Prednylidene, Rimexolone, Tixocortol,
Triamcinolone and Ulobetasol. The method provides the
administration of a single or combination of steroid drags.
[0100] Suitable examples of NSAIDs include, but are not limited to,
acetyl salicylic acid (Aspirin), Amoxiprin, Benorilate, choline
magnesium salicylate, diflunisal, Faislamine, Methyl salicylate,
magnesium salicylate, salsalate, sodium salicylate, diclofenac,
etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen,
ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone,
piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2
inhibitors, meloxicam, tramadol, Aceclofenac, Acemetacin,
Bromfenac, Etodolac, Indometacin, Nabumetone, Sulindac, Tolmetin,
Ibuprofen, Carprofen, Fenbufen, Loxoprofen, Oxaprozin, Tiaprofenic
acid, Suprofen, Mefenamic acid, Meclofenamicacid, Phenylbutazone,
Azapropazone, Metamizole, Oxyphenbutazone, Sulfinpyrazone,
Meloxicam, Piroxicam, Lornoxicam and Tenoxicam. In a preferred
embodiment, NSAIDs include Aspirin, Meloxicam, Ibuprofen, Naproxen.
The method provides the administration of a single or combination
of NSAIDS.
[0101] In accordance with the practice of the invention, for each
given daily dose of a drug, or combination of drags, listed above,
the given dose may be administered once a day or multiple times a
day. For example, 1/2 of the given dose may be administered twice a
day. In another embodiment of the invention, 1/3 of the given dose
may be administered 3 times a day. In a further embodiment of the
invention, 1/4 of the given dose may be administered 4 times a day.
In yet another embodiment of the invention, 1/5 of the given dose
may be administered 5 times a day. In yet another embodiment of the
invention, 1/6 of the given dose may be administered 6 times a day.
In yet another embodiment of the invention, 1/7 of the given, dose
may be administered times a day. In yet another embodiment of the
invention, 1/8 of the given dose may be is administered 8 times a
day. In yet another embodiment of the invention, 1/9 of the given
dose may be administered 9 times a day. In yet another embodiment
of the invention, 1/10 of the given dose may be administered 10
times a day.
[0102] Additionally, for each given daily dose of a drug listed
above, various fractions of the given dose may be administered to
the subject at multiple times during the day, with the sum of the
various fractions adding up to the given dose. For example, the
amount of the fraction of the given dose administered to the
subject at a given time may be any of 1/2 of the given dose, 1/3 of
the given dose, 1/4 of the given dose, 1/5 of the given dose, 1/6
of the given dose, 1/7 of the given dose, 1/8 of the given dose,
1/9 of the given dose, or 1/10 of the given dose. In another
embodiment of the invention, the fraction of the given dose that is
administered, and the total number of times the drug is
administered can vary by day. In a further embodiment of the
invention, the time of day the given dose or fraction of the given
dose is administered can vary by day.
[0103] For example, a daily dose of a drug (e.g., H4R agonist) can
total about 200 mg per clay. The drug can be given once a day at a
dose of about 200 mg, twice a day with each dose about 100 mg,
three times a day with each dose about 66.6 mg or four times a day
with each dose about 50 mg.
[0104] In accordance with the practice of the invention, the drug
can be administered one or more times a day, daily, weekly, monthly
or yearly.
[0105] Dosage of the therapeutic agent(s) of the invention is
dependant upon many factors including, but not limited to, the type
of tissue affected, the type of disease being treated, the severity
of the disease, a subject's health and response to the treatment
with the agents. Accordingly, dosages of the agents can vary
depending on each subject and the mode of administration.
[0106] In accordance with the practice of the invention, the
subject may be a mammal. In other embodiments of the invention, the
subject may be any of human, monkey, ape, dog, cat, cow, horse,
sheep, rabbit, mouse, or rats.
[0107] In accordance with the practice of the invention, the
administration of a given drug may be effected locally or
systemically. Additionally, the route of administration of a given
drug may be any of topical, enteral or parenteral. In other
embodiments of the invention, the route of administration of a
given drug may be any of rectal, intercisternal, bucal,
intramuscular, intrasternal, intracutaneous, intrasynovial,
intravenous, intraperitoneal, intraocular, periostal,
intra-articular injection, infusion, oral, inhalation,
subcutaneous, implantable pump, continuous infusion, gene therapy,
intranasal, intrathecal, intracerebroventricular, transdermal, or
by spray, patch or injection.
[0108] In accord with the practice of the invention, the route of
administration of a given drug can vary during a course of
treatment, or during a given day. For example, if a given drug is
administered in conjunction with one or more additional drugs, each
additional drug may be administered by identical or different
routes compared to the other drugs.
[0109] The combination of a H4R agonist alone, or in combination
with one or more of an anti-H1 drug, anti-H2 drug, anti-H3 drug, a
LRA drug, a steroid and a NSAID, can be prepared in a single or
multiple dosage form for administration to a subject.
[0110] The administration of a given drug to a subject can be
performed daily, weekly, monthly, every other month, quarterly, or
any other schedule of administration as a single dose
administration, in multiple doses, or in continuous dose form.
Additionally, a given drug can be administered to a subject
intermittently, or at a gradual, continuous, constant, immediate or
controlled rate to a subject.
[0111] In accord with the practice of the invention, if other drugs
are being administered in addition to the agents of the invention,
the timing of administration of each drug may be identical to or
different from the timing of the other drugs. The administration of
the drugs of the invention can be concurrent or at different
times.
[0112] The active agents of the invention may be administered alone
or in combination with other therapeutic agents. Components of the
combinations may be administered either concomitantly, (e.g., as an
admixture), separately but simultaneously or concurrently or
sequentially. This includes presentations in which the combined
active agents are administered together as a therapeutic mixture,
and also procedures in which the combined active agents are
administered separately but simultaneously, e.g., as through
separate intravenous lines into the same individual. Administration
"in combination" further includes the separate administration of
one of the active agents given first, followed by the one or more
sequential active agent(s).
[0113] In accordance with the practice of the invention, the
subject being administered an active agent may have any one or more
of the following diseases and/or conditions: GERD; food allergies;
Zollinger-Ellison Syndrome; peptic ulcer; dyspepsia; allergic
eosinophilic gastroenteritis; mastocytosis with gastrointestinal
symptoms; those provoked by CD4 Th2 lymphocytes (asthma, rhinitis
conjunctivitis eczema, gastro-esophageal reflux disease and other
gastric diseases exacerbated by gastric acid), CD4 Th1 lymphocytes
(IBD, Crohn's disease, ulcerative colitis and celiac disease and
autoimmune gastritis, chronic bronchitis etc.), CD4 Th17
lymphocytes (Uveitis and inflammatory digestive diseases); COPD and
other pulmonary types of inflammation including asthma; disease
conditions in cancer; and other diseases where histamine or the H4R
is involved. H4R agonists may also be used in vaccines as adjuvant
to improve immune responses to the inoculated antigens.
[0114] The invention provides methods for the treatment or
prevention of gastrointestinal disease conditions ameliorated by
histamine management in a subject, comprising administering to the
subject an effective amount of a histidine decarboxylase inhibitor.
In one embodiment, the subject is given an effective amount of a
histamine decarboxylase inhibitor and an anti-E11 drug. In another
embodiment, the subject is given an effective amount of a histidine
decarboxylase inhibitor and an anti-H2 drug. In yet another
embodiment, the subject is given an effective amount of a histidine
decarboxylase inhibitor, an anti-H1 drug and an anti-H2 drug. In a
further embodiment, the subject may be given an effective amount of
a combination of a histidine decarboxylase inhibitor, and any one
or both an anti-H1 drug and an anti-H2 drug together with an
NSAID.
[0115] An embodiment of the invention also provides methods for the
treatment or prevention of gastrointestinal disease conditions
ameliorated by histamine management in a subject comprising
administering to the subject an effective amount of a HDC inhibitor
in combination with an effective amount a LRA. In a further
embodiment, the subject is given an effective amount of a HDC
inhibitor in combination with an effective amount of a LRA and an
effective amount of an anti-H1 drug. In another embodiment of the
invention, the subject is given an effective amount of a HDC
inhibitor in combination with an effective amount of a LRA and an
effective amount of an anti-H2 drug. In a further embodiment of the
invention, the subject is given an effective amount of a HDC
inhibitor in combination with an effective amount of a LRA, an
effective amount of an anti-H1 drug and an effective amount of an
anti-H2 drug.
[0116] The invention further provides methods for treatment or
prevention of COPD comprising administering to the subject an
effective amount of HDC inhibitor. In one embodiment, the patient
is administered an effective amount of a HDC inhibitor in
combination with an effective amount of an anti-H1 drug. Histidine
decarboxylase inhibitors in general, as well as histamine receptor
antagonists e.g. Anti-H1 drugs have never been used for the
treatment of COPD. Tritoqualine (an HDC inhibitor) and Loratadine
(an anti-H1 drug) were traditionally used for the treatment of
allergy and primarily allergic rhinitis. The combination of
Tritoqualine and Loratadine showed statistically significant
effectiveness in the management of COPD when compared with the
standard treatment of beta agonists, bronchodilators, steroids and
oxygen. The presumed action of combining Tritoqualine and an
anti-H1 drug may be by increasing the TH1 cells thus balancing the
equilibrium between TH1 and TH2 cells to the detriment of TH2
cells. As a result, secretion of inflammatory cytokines such as
IL4, IL5, and IL10 is reduced causing less pulmonary
inflammation.
[0117] In another embodiment of the invention the patient is given
HDC inhibitor in combination with any one or more of COPD therapies
including but not limited to Anticholinergics, Combination Inhaler,
Corticosteroids, Inhaled Beta-2 Agonists, Inhaled Corticosteroids,
Mucolytics, Oral Beta-2 Agonists, Bronchodilators and
Theophyllines. In a further embodiment, the patient is given HDC
inhibitor and anti-H1 drug in combination with any one or more of
COPD therapies including but not limited to Anticholinergics,
Combination Inhaler, Corticosteroids, Inhaled Beta-2 Agonists,
Inhaled Corticosteroids, Mucolytics, Oral Beta-2 Agonists,
Bronchodilators and Theophyllines. In yet another embodiment of the
invention, the patient is administered an effective amount of a HDC
inhibitor, an anti-H1 drug and a NSAID.
[0118] Suitable examples of histidine decarboxylase inhibitors
include, but are not limited to, any of Tritoqualine or an isomer
thereof, alpha-fluoromethylhistidine,
3-methoxy-5,7,3',4'-tetrahydroxyflavan, naringenin,
(+)-cyanidanol-3, the dipeptide His-Phe, and
4-imidazolyl-3-amino-2-butanone, polyphenols such as catechins and
related structures; these include, but are not limited to:
(-)-epigallocatechin gallate, (-)-epicatechin gallate,
(-)-epicatechin, (-)-epigallocatechin, and is composed of
(-)-epicatechin, (-)-epigallocatechin; and other flavonoids such as
O-methyl-3(+)catechin; or analogs, equivalents, isomers,
pharmaceutically acceptable salts, and solvate forms of any of the
above. The Tritoqualine isomer may be an SS isomer of Tritoqualine
or an RR isomer of Tritoqualine. The method provides the
administration of single or a combination of histidine
decarboxylase inhibitors.
[0119] In accordance with the practice of the invention, the
subject may have any one or more of the following: 1) a history of
GERD; 2) a history of allergies (for example food allergies); 3) a
history of COPD; 4) previous unsatisfactory treatment with an
anti-H1 and/or anti-H2 drug; 5) previously unsatisfactory treatment
with existing COPD therapies; 6) previous unsatisfactory treatment
with proton pump inhibitors (PPI); 7) previous diagnosis of GERD
and concurrent symptoms of allergy; and/or 8) previous
unsatisfactory treatment with cromoglycate.
[0120] In one embodiment, the subject may suffer from COPD or GERD
(or any of the gastrointestinal disorders or diseases disclosed
herein) but does not exhibit allergy symptoms which include any one
or more of allergic asthma, allergic conjunctivitis, and allergic
rhinitis.
[0121] For example, the allergy may be confirmed with two positive
prick tests for the same allergen or set of allergens. The
allergens may be any of Dermatophagoid Pternzonysisnus,
Demathophagoid Farinae, cat dander, dog dander, food allergens,
fungal proteins, and pollen proteins. Additionally, the food
allergen may be any of wheat, egg, soy, potato, peanut, and/or
tomato proteins. The fungal protein may be any of a protein from a
species in the alternaria genus, a protein from a species in the
mucor genus, and a protein from a species in the aspergillus genus.
The pollen protein may be any of pollen of birch tree, pollen of
cypress tree, pollen of Quercus tree, pollen of lolium perenne, and
pollen of ray grass.
COMPOSITIONS OF THE INVENTION
[0122] The present invention provides pharmaceutical formulations
(also known as pharmaceutical compositions or dosage forms)
comprising a first active agent (e.g., a H4R agonist), one or more
additional active agent (e.g., a H1R antagonist, a H2R antagonist,
a H3R antagonist, a LRA, a steroid, a NSAID or other active agent),
and a pharmaceutically acceptable carrier or vehicle.
[0123] The present invention also provides pharmaceutical
formulations for the treatment of COPD comprising a first active
agent (e.g., a H4R agonist), a second active agent (e.g., a H1R
antagonist), a third active agent (e.g., an anticholinergic drug)
and a pharmaceutically acceptable carrier or vehicle.
[0124] Pharmaceutically acceptable carrier or vehicle refers to a
non-toxic solid, semisolid (also referred to herein as softgel) or
liquid filler, diluent, encapsulating material or formulation
auxiliary of any type. The invention also provides methods for
treating or ameliorating H4R modulated diseases using said
pharmaceutical formulations.
[0125] H4R agonists include, but are not limited to, Tritoqualine
or an isomer thereof, 4-methylhistmime (4MeHA) and clobenprobit
(CB). The pharmaceutical formulation of the invention provides one
or more H4R agonist drugs. Examples of Tritoqualine isomers
include, but are not limited to, the E1 and E2 enantiomer.
[0126] H1 antagonists (anti-H1 drugs) include, but are not limited
to, brompheniramine, cetirizine, levocetirizine, fexofenadine,
cyproheptadine, dexchlorpheniramine, hydroxizine, ketotifen,
loratadine, mequitazine, oxotomide, mizolastine, ebastine,
astemizole, carbinoxamide, alimemazine, buclizine, cyclizine
hydrochloride, doxylamine, mepyramine, antazoline, diphenhydramine,
carbinoxamine, clemastine, dimenhydrinate, pheniramine,
chlorphenamine, triprolidine, chlorcyclizine, hydroxyzine,
meclizine, promethazine and azatadine and/or analogs, equivalents,
isomers, salts, and solvate forms thereof. The pharmaceutical
formulation of the invention provides one or more anti-H1
drugs.
[0127] H2 antagonists (anti-H2 drugs) include, but are not limited
to, ranitidine, cimetidine, famotidine, and nizatidine and/or
analogs, equivalents, isomers, salts, and solvate forms thereof.
The pharmaceutical formulation of the invention provides one or
more anti-H2 drugs.
[0128] H3 antagonists (anti-H3 drugs) include, but are not limited
to, betahistine (N-methyl-2-pyridin-2-ylethanamine), ABT-239
(4-(2-{2-[(2R)-2-Methylpyrrolidin-1-yl]ethyl}-benzofuran-5-yl)benzonitril-
e),
Cipralisant(1R,2R)-4-(2-(5,5-dimethylhex-1-ynyl)cyclopropyl)imidazole,
Ciproxifan cyclopropyl 4-(3-(1H-imidazol-4-yl)propyloxy)phenyl
ketone, Clobenpropit
N'-[(4-chlorophenyl)methyl]-1-[3-(3H-imidazol-4-yl)propylthio]formamidine-
, Thioperamide
N-Cyclohexyl-4-(1H-imidazol-4-yl)-1-piperidinecarbothioamide. The
pharmaceutical formulation of the invention provides one or more
anti-H3 drugs.
[0129] Anticholinergic drugs include, but are not limited to:
tiotropium bromide (Spiriva.RTM.) and ipratropium bromide
(Atrovent.RTM.).
[0130] LRAs include, but are not limited to, Montelukast.RTM.
(Singulair.RTM.), Pranlukast.RTM. and Zafirlukast.RTM.. The method
provides the administration of a single or a combination of
LRAs.
[0131] Steroids include, but are not limited to, corticosteroids,
Aldosterone, Cortisone, Hydrocortisone/cortisol, Desoxycortone,
Alclometasone, Amcinonide, Beclometasone, Betamethasone,
Budesonide, Ciclesonide, Clobetasol, Clobetasone, Clocortolone,
Cloprednol, Cortivazol, Deflazacort, Deoxycorticosterone, Desonide,
Desoximetasone, Dexamethasone, Diflorasone, Diflucortolone,
Difluprednate, Fluclorolone, Fludrocortisone, Fludroxycortide,
Flumetasone, Flunisolide, Fluocinolone acetonide, Fluocinonide,
Fluocortin, Fluocortolone, Fluorometholone, Fluperolone,
Fluprednidene, Fluticasone, Formocortal, Halcinonide, Halometasone,
Hydrocortisone aceponate, Hydrocortisone buteprate, Hydrocortisone
butyrate, Loteprednol, Medrysone, Meprednisone, Methylprednisolone,
Methylprednisolone aceponate, Mometasone furoate, Paramethasone,
Prednicarbate, Prednisone, Prednisolone, Prednylidene, Rimexolone,
Tixocortol, Triamcinolone and Ulobetasol. The method provides the
administration of a single or combination of steroid drugs.
[0132] NSAIDs include, but are not limited to, acetyl salicylic
acid (Aspirin), Amoxiprin, Benorilate, choline magnesium
salicylate, diflunisal, Faislamine, Methyl salicylate, magnesium
salicylate, salsalate, sodium salicylate, diclofenac, etodolac,
fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac,
meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam,
sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors,
meloxicam, tramadol, Aceclofenac, Acemetacin, Bromfenac, Etodolac,
Indometacin, Nabumetone, Sulindac, Tolmetin, Ibuprofen, Carprofen,
Fenbufen, Loxoprofen, Oxaprozin, Tiaprofenic acid, Suprofen,
Mefenamic acid, Meclofenamicacid, Phenylbutazone, Azapropazone,
Metamizole, Oxyphenbutazone, Sulfinpyrazone, Meloxicam, Piroxicam,
Lornoxicam and Tenoxicam. In a preferred embodiment, NSAIDs include
Aspirin, Meloxicam, ibuprofen, Naproxen, Phosphodiesterase (PDE4)
inhibitors Prostaglandin E4. The method provides the administration
of a single or combination of NSAIDS.
[0133] In one embodiment of the invention, the pharmaceutical
formulation comprises the H4R agonist and a pharmaceutically
acceptable vehicle. In a particular embodiment, the H4R agonist is
Tritoqualine or an isomer thereof.
[0134] In another embodiment of the invention, the pharmaceutical
formulation comprises the H4R agonist, one or more other active
agent and a pharmaceutically acceptable vehicle. In a particular
embodiment, the H4R agonist is Tritoqualine or an isomer thereof.
The active agent can include, but is not limited to, H1R
antagonists, H2R antagonists, H3R antagonists, H4R agonist, LRAs,
steroids and NSAIDs.
[0135] In yet another embodiment, the pharmaceutical composition
comprises a tritoqualine and an anti-H2 drag.
[0136] The present invention also provides pharmaceutical
formulations comprising a solid or liquid dosage form of a H4R
agonist (e.g., Tritoqualine, E1 or E2) and a plurality of particles
which permit the formulation of solid or liquid dosage form of the
H4R agonist. The particles comprise, but are not limited to,
excipients disclosed herein below, e.g., typical excipients for
softgels.
[0137] In one embodiment, the solid or liquid dosage form of the
H4R agonist formulation further comprises an H1R antagonist. In
another embodiment, the solid or liquid dosage form of the H4R
agonist formulation further comprises an H2R antagonist. In another
embodiment, the solid or liquid dosage form of the H4R agonist
formulation further comprises an H3 antagonist. In yet another
embodiment, the solid or liquid dosage form of the H4R agonist
formulation further comprises one or more of an H1R, an H2R
antagonist, an H3 antagonist, a LRA drug and an NSAID.
[0138] In one aspect, the present invention provides a
pharmaceutical composition for the treatment of H4R modulated
diseases or conditions comprising a solid or liquid dosage form of
the H4R agonist (e.g., Tritoqualine, E1 or E2), wherein the
composition is an administrable formulation that allows resorption
of the H4R agonist into a subject. In one embodiment, the
administrable formulation can be, e.g., an inhalant or a topically
administrable formulation such as an ointment or cream.
[0139] Anti-H1, anti-H2 and anti-H3 drugs are present in the
various compositions of the invention in a proportion of the order
of 0.1 to 2000 mg.
[0140] In the case of a pharmaceutical composition according to the
invention containing an antihistamine compound (for example,
anti-H1, anti-H2 or anti-H3) and a H4R agonist, these compounds are
present in a proportion of the order of: [0141] 0.1 to 2000 mg of
anti H2 compound (when used), [0142] 0.1 to 2000 mg of anti-H1
compound (when used), [0143] 0.1 to 2000 mg of anti-H3 compound
(when used), and [0144] 0.10 to 3000 mg of a H4R agonist such as
Tritoqualine or its isomers.
[0145] In the case of a composition according to the invention
containing H4R agonist and one or more of an antihistamine compound
(for example, anti-H1, anti-H2 or anti-H3), an LRA drug, a steroid
drug and a NSAID, these compounds are present in a proportion of
the order of: [0146] 0.1 to 2000 mg of anti H2 compound (when
used), [0147] 0.1 to 2000 mg of anti-H1 compound (when used) [0148]
0.1 to 2000 mg of anti-H3 compound (when used), [0149] 0.1 to 2000
mg of a LRA drug (when used) [0150] 0.01 to 2000 mg of steroid
compound (when used), [0151] 0.1 to 5000 mg of NSAID compound (when
used), and [0152] 0.10 to 3000 mg of a H4R agonist such as
Tritoqualine or its isomers.
[0153] Further, the active agents of the invention can be
pegylated, phosphorylated, esterified, derivatized with amino acids
and/or peptides, to improve solubility for both formulation and
bioavailability. Additionally, lipid derivatization and other
lipophile derivatization can be used to improve mucosal
permeability, absorption and formulation of the active agents of
the invention in oily vehicles.
Dosage Forms
[0154] Dosage forms can be made according to well known methods in
the art. Some preferred methods are described below.
[0155] The pharmaceutical compositions of the invention may be
formulated as solid dosage forms, such as capsules, pills,
softgels, tablets, caplets, troches, wafer, sprinkle, chewing gum
or the like, for oral administration. The pharmaceutical
compositions of the invention may also be formulated as liquid
dosage forms such as elixir, suspension or syrup.
[0156] The pharmaceutical compositions of the invention may also be
presented in a dosage form for transdermal application, for example
an ointment for children, a form for oral administration, for
example a slow release product, or in gastro-resistant tablet form
or gum form. They may also be in spray, bronchial form or eye
lotion form, or other galenic forms with programmed mucosal and
secondarily per os disintegration.
[0157] Therefore the different pharmaceutical compositions of the
invention can be administered by several routes chosen in
accordance with the patient's pathological profile and age. For
children, the patch form, syrup form or tablets to be dissolved in
the mouth. The other forms, eye lotion or injection may also be
used. In adults all galenic forms (also known as dosage forms) can
be contemplated.
[0158] The advantage of a coupled or combined galenic form also
provides simplicity of treatment, patient compliance with the
simplified treatment and therefore a more successful outcome.
[0159] The pharmaceutical compositions of the present invention may
be mixed with pharmaceutically acceptable carriers, binders,
diluents, adjuvants, excipients, or vehicles, such as preserving
agents, fillers, polymers, disintegrating agents, glidants, wetting
agents, emulsifying agents, suspending agents, sweetening agents,
flavoring agents, perfuming agents, lubricating agents, acidifying
agents, coloring agent, dyes, preservatives and dispensing agents,
or compounds of a similar nature depending on the nature of the
mode of administration and dosage forms. Such ingredients,
including pharmaceutically acceptable carriers and excipients that
may be used to formulate oral dosage forms, are described in the
Handbook of Pharmaceutical Excipients, American Pharmaceutical
Association (1986), incorporated herein by reference in its
entirety.
[0160] Pharmaceutically acceptable carriers are generally non-toxic
to recipients at the dosages and concentrations employed and are
compatible with other ingredients of the formulation. Examples of
pharmaceutically acceptable carriers include water, saline,
Ringer's solution, dextrose solution, ethanol, polyols, vegetable
oils, fats, ethyl oleate, liposomes, waxes polymers, including gel
forming and non-gel forming polymers, and suitable mixtures
thereof. The carrier may contain minor amounts of additives such as
substances that enhance isotonicity and chemical stability. Such
materials are non-toxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, succinate, acetic acid, and other organic acids or their
salts; antioxidants such as, ascorbic acid; low molecular weight
(less than about ten residues) polypeptides, e.g., polyarginine or
tripeptides; proteins, such as serum albumin, gelatin, or
immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids, such as glycine, glutamic acid, aspartic acid, or
arginine; monosaccharides, disaccharides, and other carbohydrates
including cellulose or its derivatives, glucose, mannose, or
dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol or sorbitol; counterions such as sodium; and/or nonionic
surfactants such as polysorbates, poloxamers, or PEG. Preferably
the carrier is a parenteral carrier, more preferably a solution
that is isotonic with the blood of the recipient.
[0161] Examples of binders include, but are not limited to,
microcrystalline cellulose and cellulose derivatives, gum
tragacanth, glucose solution, acacia mucilage, gelatin solution,
molasses, polyinylpyrrolidine, povidone, crospovidones, sucrose and
starch paste.
[0162] Examples of diluents include, but are not limited to,
lactose, sucrose, starch, kaolin, salt, mannitol and dicalcium
phosphate.
[0163] Examples of excipients include, but are not limited to,
starch, surfactants, lipophilic vehicles, hydrophobic vehicles,
pregelatinized starch, Avicel, lactose, milk sugar, sodium citrate,
calcium carbonate, dicalcium phosphate, and lake blend purple.
Typical excipients for dosage forms such as a softgel include
gelatin for the capsule and oils such as soy oil, rice bran oil,
canola oil, olive oil, corn oil, and other similar oils; glycerol,
polyethylene glycol liquids, vitamin E TPGS as a surfactant and
absorption enhancer (Softgels: Manufacturing Considerations;
Wilkinson P, Foo Sog Hom, Special Drug Delivery Systems; Drugs and
the Pharmaceutical Sciences Vol 41 Praveen Tyle Editor, Marcel
Dekker 1990, 409-449; Pharmaceutical Dosage Forms and Drug Delivery
by Ansel, Popovich and Allen 1995, Williams and Wilkins, Chapter 5
pp 155-225). Tritoqualine and anti H1 may form either a solution in
a selected oil vehicle or a suspension of fine particles
(comprising any of the excipients disclosed herein, e.g., typical
excipients for softgels).
[0164] Examples of disintegrating agents include, but are not
limited to, complex silicates, croscarmellose sodium, sodium starch
glycolate, alginic acid, corn starch, potato starch, bentonite,
methylcellulose, agar and carboxymethylcellulose.
[0165] Examples of glidants include, but are not limited to,
colloidal silicon dioxide, talc, corn starch.
[0166] Examples of wetting agents include, but are not limited to,
propylene glycol monostearate, sorbitan monooleate, diethylene
glycol monolaurate and polyoxyethylene laural ether.
[0167] Examples of sweetening agents include, but are not limited
to, sucrose, lactose, mannitol and artificial sweetening agents
such as saccharin, and any number of spray dried flavors.
[0168] Examples of flavoring agents include, but are not limited
to, natural flavors extracted from plants such as fruits and
synthetic blends of compounds which produce a pleasant sensation,
such as, but not limited to peppermint and methyl salicylate.
[0169] Examples of lubricants include magnesium or calcium
stearate, sodium lauryl sulphate, talc, starch, lycopodium and
stearic acid as well as high molecular weight polyethylene
glycols.
[0170] Examples of coloring agents include, but are not limited to,
any of the approved certified water soluble FD and C dyes, mixtures
thereof; and water insoluble FD and C dyes suspended on alumina
hydrate.
[0171] The artisan of ordinary skill in the art will recognize that
many different ingredients can be used in formulations according to
the present invention, in addition to the active agents, while
maintaining effectiveness of the formulations in treating the H4R
modulated diseases. The list provided herein is not exhaustive.
Matrix Based Dosage Forms
[0172] Dosage forms according to one embodiment of the present
invention may be in the form of coated or uncoated matrices. The
term matrix, as used herein, is given its well known meaning in the
pharmaceutical arts as a solid material having an active agent
(e.g., the components of the compositions of the invention) of the
invention incorporated therein. Upon exposure to a dissolution
media, channels are formed in the solid material so that the active
agent can escape.
[0173] The skilled artisan will appreciate that the matrix material
can be chosen from a wide variety of materials which can provide
the desired dissolution profiles. Materials can include, for
example, one or more gel forming polymers such as polyvinyl
alcohol, cellulose ethers including, for example,
hydroxypropylalkyl celluloses such as hydroxypropyl cellulose,
hypromellose, prop-2-enoic acid, hydroxypropyl methyl cellulose,
hydroxyalkyl celluloses such as hydroxypropyl cellulose, natural or
synthetic gums such as guar gum, xanthum gum, and alginates, as
well as ethyl cellulose, polyvinyl pyrrolidone, fats, waxes,
polycarboxylic acids or esters such as the Carbopol R series of
polymers, meth acrylic acid copolymers, and methacrylate
polymers.
[0174] In addition to the above-mentioned ingredients, a controlled
release matrix may also contain suitable quantities of other
materials, for example, diluents, lubricants, binders, granulating
aids, colorants, flavorants, and glidants that are conventional in
the pharmaceutical arts. The quantities of these additional
materials should be sufficient to provide the desired effect to the
desired formulation. A controlled release matrix incorporating
particles may also contain suitable quantities of these other
materials such as diluents, lubricants, binders, granulating aids,
colorants, flavorants, and glidants that are conventional in the
pharmaceutical arts in amounts up to about 75% by weight of the
particulate, if desired.
[0175] Methods of making matrix dosages are well known in the art
and any known method of making such dosages which yields the
desired immediate release and controlled release dissolution
profiles can be used. One such method involves the mixture of the
compositions of the invention with a solid polymeric material and
one or more pharmaceutically acceptable excipients which can then
be blended and compressed in controlled release tablet cores. Such
tablet cores, can be used for further processing as bi-layer or
multilayer tablets, press coated tablets, or film coated
tablets.
[0176] In addition, the formulation of respective release
components can occur by appropriate granulation methods as is well
known in the art. In wet granulation, solutions of the binding
agent can be added with stirring to the mixed powders. The powder
mass can be wetted with the binding solution until the mass has the
consistency of damp snow or brown sugar. The wet granulated
material can be forced through a sieving device. Moist material
from the milling step can be dried by placing it in a temperature
controlled container. After drying, the granulated material can be
reduced in particle size by passing it through a sieving device.
Lubricant can be added, and the final blend can then be compressed
into a matrix dosage form such as a matrix tablet.
[0177] In fluid-bed granulation, particles of inert material and/or
active agent (e.g., the components of the compositions of the
invention) can be suspended in a vertical column with a rising air
stream. While the particles are suspended, a common granulating
material in solution can be sprayed into the column. There will be
a gradual particle buildup under a controlled set of conditions
resulting in tablet granulation. Following drying and the addition
of lubricant, the granulated material will be ready for
compression.
[0178] In dry-granulation, the active agent (e.g., the components
of the compositions of the invention), binder, diluent, and
lubricant can be blended and compressed into tablets. The
compressed large tablets can be comminuted through the desirable
mesh screen by sieving equipment. Additional lubricant can be added
to the granulated material and blended gently. The material can
then be compressed into tablets.
Particle Based Dosage Forms
Immediate Release and Controlled Release Particles
[0179] Dosage forms according to another embodiment of the present
invention may be in the form of coated or uncoated immediate
release/controlled release dosage forms. The immediate
release/controlled release dosage forms of the present invention
can take the form of pharmaceutical particles. The dosage forms can
include immediate release particles in combination with controlled
release particles in a ratio sufficient to deliver the desired
dosages of active agents (e.g., the components of the compositions
of the invention). The controlled release particles can be produced
by coating the immediate release particles with an enteric
coat.
[0180] The particles can be produced according to any of a number
of well known methods for making particles. The immediate release
particles can comprise the active agent combination (the
compositions of the invention) and a disintegrant. Suitable
disintegrants can include, for example, starch, low-substitution
hydroxypropyl cellulose, croscarmellose sodium, calcium
carboxymethyl cellulose, hydroxypropyl starch, and microcrystalline
cellulose.
[0181] In addition to the above-mentioned ingredients, a controlled
release matrix may also contain suitable quantities of other
materials, for example, diluents, lubricants, binders, granulating
aids, colorants, flavorants, and glidants that are conventional in
the pharmaceutical arts. The quantities of these additional
materials should be sufficient to provide the desired effect to the
desired formulation. A controlled release matrix incorporating
particles may also contain suitable quantities of these other
materials such as diluents, lubricants, binders, granulating aids,
colorants, flavorants, and glidants that are conventional in the
pharmaceutical arts in amounts up to about 75% by weight of the
particulate, if desired.
[0182] Particles can assume any standard structure known in the
pharmaceutical arts. Such structures can include, for example,
matrix particles, non-pareil cores having a drug layer and active
or inactive cores having multiple layers thereon. A controlled
release coating can be added to any of these structures to create a
controlled release particle.
[0183] The term particle as used herein means a granule having a
diameter of between about 0.01 mm and about 5.0 mm, preferably
between about 0.1 mm and about 2.5 mm, and more preferably between
about 0.5 mm and about 2 mm. The skilled artisan will appreciate
that particles according to the present invention can be any
geometrical shape within this size range and so long as the mean
for a statistical distribution of particles falls within the
particle sizes enumerated above, they will be considered to fall
within the contemplated scope of the present invention.
[0184] The release of the therapeutically active agent (e.g., the
components of the compositions of the invention) from the
controlled release formulation of the present invention can be
further influenced, i.e., adjusted to a desired rate, by the
addition of one or more release-modifying agents. The
release-modifying agent may be organic or inorganic and include
materials that can be dissolved, extracted, or leached from the
coating in the environment of use. The pore-formers may comprise
one or more hydrophilic materials such as hydroxypropyl
methylcellulose. The release-modifying agent may also comprise a
semi-permeable polymer. In certain preferred embodiments, the
release-modifying agent is selected from hydroxypropyl
methylcellulose, lactose, metal stearates, and mixtures
thereof.
[0185] The controlled release particles of the present invention
can slowly release the compositions of the invention when ingested.
The controlled release profile of the formulations of the present
invention can be altered, for example, by increasing or decreasing
the thickness of a retardant coating, i.e., by varying the amount
of overcoating. The resultant solid controlled release particles
may thereafter be placed in a gelatin capsule in an amount
sufficient to provide an effective controlled release dose when
ingested and contacted by an environmental fluid, e.g., gastric
fluid, intestinal fluid or dissolution media.
[0186] The dosage forms of the invention may be coated (e.g., film
coated or enterically coated) as known by those of skill in the
art. For example, the composition can be formulated in an enteric
coating that maintains its integrity in the stomach and releases
the active compound in the intestine.
[0187] Examples of enteric-coatings include, but are not limited
to, phenylsalicylate, fatty acids, fats, waxes, shellac, ammoniated
shellac and cellulose acetate phthalates. Film coatings include,
but are not limited to, hydroxyethylcellulose, sodium
carboxymethylcellulose, polyethylene glycol 4000 and cellulose
acetate phthalate.
[0188] In one example, the dosage forms e.g., particles of the
invention as described above, may be overcoated with an aqueous
dispersion of a hydrophobic or hydrophilic material to modify the
release profile. The aqueous dispersion of hydrophobic material
preferably further includes an effective amount of plasticizer,
e.g. triethyl citrate. Preformulated aqueous dispersions of
ethylcellulose, such as AQUACOAT.TM. or SURELEASE.TM. products, may
be used. If a SURELEASE.TM. product is used, it is not necessary to
separately add a plasticizer.
[0189] The hydrophobic material may be selected from the group
consisting of alkylcellulose, acrylic and methacrylic acid polymers
and copolymers, shellac, zein, fatty oils, hydrogenated castor oil,
hydrogenated vegetable oil, or mixtures thereof. In certain
preferred embodiments, the hydrophobic material can be a
pharmaceutically acceptable acrylic polymer including, but not
limited to, acrylic acid and methacrylic acid copolymers, methyl
methacrylate, methyl methacrylate copolymers, ethoxyethyl
methacrylates, cyanoethyl methacrylate, aminoalkyl methacrylate
copolymer, poly(acrylic acid), poly(methacrylic acid), methacrylic
acid alkylamine copolymer, poly(methyl methacrylate),
poly(methacrylic acid anhydride), polymethacrylate, polyacrylamide,
poly(methacrylic acid anhydride), and glycidyl methacrylate
copolymers. In alternate embodiments, the hydrophobic material can
be selected from materials such as one or more hydroxyalkyl
celluloses such as hydroxypropyl methylcellulose. The hydroxyalkyl
cellulose can preferably be a hydroxy (C.sub.1 to C.sub.6) alkyl
cellulose, such as hydroxypropylcellulose,
hydroxypropylmethylcellulose, or preferably hydroxyethylcellulose.
The amount of the hydroxyalkyl cellulose in the present oral dosage
form can be determined, in part, by the precise rate of active
agents (e.g., the components of the compositions of the invention)
desired and may vary from about 1% to about 80%.
[0190] In embodiments of the present invention where the coating
comprises an aqueous dispersion of a hydrophobic polymer, the
inclusion of an effective amount of a plasticizer in the aqueous
dispersion of hydrophobic polymer can further improve the physical
properties of the film. For example, because ethylcellulose has a
relatively high glass transition temperature and does not form
flexible films under normal coating conditions, it may be necessary
to plasticize the ethylcellulose before using it as a coating
material. Generally, the amount of plasticizer included in a
coating solution can be based on the concentration of the
film-former, e.g., most often from about 1 percent to about 50
percent by weight of the film-former. Concentration of the
plasticizer, however, can be preferably determined after careful
experimentation with the particular coating solution and method of
application.
[0191] Examples of suitable plasticizers for ethylcellulose include
water-insoluble plasticizers such as dibutyl sebacate, diethyl
phthalate, triethyl citrate, tributyl citrate, and triacetin,
although other water-insoluble plasticizers (such as acetylated
monoglycerides, phthalate esters, castor oil, etc.) may be used.
Triethyl citrate may be an especially preferred plasticizer for the
aqueous dispersions of ethyl cellulose of the present
invention.
[0192] Examples of suitable plasticizers for the acrylic polymers
of the present invention include, but are not limited to, citric
acid esters such as triethyl citrate NF XVI, tributyl citrate,
dibutyl phthalate, and possibly 1,2-propylene glycol. Other
plasticizers which have proved to be suitable for enhancing the
elasticity of the films formed from acrylic films such as
EUDRAGIT.TM. RL/RS lacquer solutions include polyethylene glycols,
propylene glycol, diethyl phthalate, castor oil, and triacetin.
Triethyl citrate may be an especially preferred plasticizer for
aqueous dispersions of ethyl cellulose. It has further been found
that addition of a small amount of talc may reduce the tendency of
the aqueous dispersion to stick during processing and acts a
polishing agent.
[0193] One commercially available aqueous dispersion of
ethylcellulose is the AQUACOAT.TM. product which is prepared by
dissolving the ethylcellulose in a water-immiscible organic solvent
and then emulsifying the ethylcellulose in water in the presence of
a surfactant and a stabilizer. After homogenization to generate
submicron droplets, the organic solvent can be evaporated under
vacuum to form a pseudolatex. The plasticizer will not be
incorporated into the pseudolatex during the manufacturing phase.
Thus, prior to using the pseudolatex as a coating, the AQUACOAT.TM.
product can be mixed with a suitable plasticizer.
[0194] Another aqueous dispersion of ethylcellulose is commercially
available as SURELEASE.TM. product (Colorcon, Inc., West Point,
Pa., U.S.A.). This product can be prepared by incorporating
plasticizer into the dispersion during the manufacturing process. A
hot melt of a polymer, plasticizer (dibutyl sebacate), and
stabilizer (oleic acid) can be prepared as a homogeneous mixture
which can then be diluted with an alkaline solution to obtain an
aqueous dispersion which can be applied directly onto
substrates.
[0195] In one embodiment, the acrylic coating can be an acrylic
resin lacquer used in the form of an aqueous dispersion, such as
that which is commercially available from Rohm Pharma under the
trade name EUDRAGIT.TM.. In additional embodiments, the acrylic
coating can comprise a mixture of two acrylic resin lacquers
commercially available from Rohm Pharma under the trade names
EUDRAGIT.TM. RL 30 D and EUDRAGIT.TM.RS 30 D. EUDRAGIT.TM. RL 30 D
and EUDRAGIT.TM. RS 30 are copolymers of acrylic and methacrylic
esters with a low content of quaternary ammonium groups, the molar
ratio of ammonium groups to the remaining neutral (meth)acrylic
esters being 1:20 in EUDRAGIT.TM. RL 30 and 1:40 in EUDRAGIT.TM. RS
30 D. The mean molecular weight is about 150,000 Daltons. The code
designations RL (high permeability) and RS (low permeability) refer
to the permeability properties of these agents. EUDRAGIT.TM. RL/RS
mixtures are insoluble in water and in digestive fluids; however,
coatings formed from them are swellable and permeable in aqueous
solutions and digestive fluids.
[0196] The EUDRAGIT.TM. RL/RS dispersions may be mixed together in
any desired ratio in order to ultimately obtain a
controlled-release formulation having a desirable dissolution
profile. Desirable controlled release formulations may be obtained,
for instance, from a retardant coating derived from one of a
variety of coating combinations, such as 100% EUDRAGIT.TM. RL; 50%
EUDRAGIT.TM. RL and 50% EUDRAGIT.TM. RS; or 10% EUDRAGIT.TM. RL and
EUDRAGIT.TM. 90% RS. Of course, one skilled in the art will
recognize that other acrylic polymers may also be used, for
example, others under the EUDRAGIT.TM. brand. In addition to
modifying the dissolution profile by altering the relative amounts
of different acrylic resin lacquers, the dissolution profile of the
ultimate product may also be modified, for example, by increasing
or decreasing the thickness of the retardant coating.
[0197] The stabilized product may be obtained by subjecting the
coated substrate to oven curing at a temperature above the Tg
(glass transition temperature) of the plasticized acrylic polymer
for the required time period, the optimum values for temperature
and time for the particular formulation being determined
experimentally. In certain embodiments of the present invention,
the stabilized product is obtained via an oven curing conducted at
a temperature of about 45.degree. C. for a time period from about 1
to about 48 hours. It is also contemplated that certain products
coated with the controlled-release coating of the present invention
may require a curing time longer than 24 to 48 hours, e.g., from
about 48 to about 60 hours or more.
[0198] The coating solutions preferably contain, in addition to the
film-former, plasticizer, and solvent system (i.e., water), a
colorant to provide elegance and product distinction. Color may be
added to the solution of the compositions of the invention instead
of, or in addition to the aqueous dispersion of hydrophobic
material. For example, color may be added to an AQUACOAT.TM.
product via the use of alcohol or propylene glycol based color
dispersions, milled aluminum lakes and opacifiers such as titanium
dioxide by adding color with shear to the water soluble polymer
solution and then using low shear to the plasticized AQUACOAT.TM.
product.
[0199] Alternatively, any suitable method of providing color to the
formulations of the present invention may be used. Suitable
ingredients for providing color to the formulation when an aqueous
dispersion of an acrylic polymer is used include titanium dioxide
and color pigments, such as iron oxide pigments. The incorporation
of pigments, may, however, increase the retardant effect of the
coating.
[0200] Spheroids or beads coated with the compositions of the
invention can be prepared, for example, by dissolving the
compositions of the invention in water and then spraying the
solution onto a substrate, for example, non pareil 18/20 beads,
using a Wuster insert. Optionally, additional ingredients can also
be added prior to coating the beads in order to assist the binding
of the compositions of the invention to the beads, and/or to color
the solution, etc. For example, a product which includes
hydroxypropyl methylcellulose with or without colorant (e.g.,
OPADRY.TM. product, commercially available from Coloron, Inc.) may
be added to the solution and the solution mixed (e.g., for about 1
hour) prior to application onto the beads. The resultant coated
substrate, beads in this example, may then be optionally overcoated
with a bather agent to separate the compositions of the invention
from the hydrophobic controlled release coating. An example of a
suitable barrier agent is one which comprises hydroxypropyl
cellulose. However, any film-former known in the art may be used.
It is preferred that the barrier agent does not affect the
dissolution rate of the final product.
[0201] Immediate release particles according to the present
invention may be coated with a controlled release coating in order
to change the release rate to obtain the dissolution rates
according to the present invention.
Press Coated, Pulsatile Dosage Form
[0202] In another embodiment of the present invention, the
compositions of the invention can be administered via a press
coated pulsatile drug delivery system suitable for oral
administration with a controlled release component, which contains
a compressed blend of an active agent (e.g., the components of the
compositions of the invention) and one or more polymers,
substantially enveloped by an immediate release component, which
contains a compressed blend of the active agent and hydrophilic and
hydrophobic polymers. The immediate-release component preferably
comprises a compressed blend of active agent and one or more
polymers with disintegration characteristics such that the polymers
disintegrate rapidly upon exposure to the aqueous medium.
[0203] The controlled-release component preferably can comprise a
combination of hydrophilic and hydrophobic polymers. In this
embodiment, once administered, the hydrophilic polymer will
dissolve away to weaken the structure of the controlled-release
component, and the hydrophobic polymer will retard the water
penetration and help to maintain the shape of the drug delivery
system.
[0204] In accordance with the present invention, the term "polymer"
includes single or multiple polymeric substances, which can swell,
gel, degrade or erode on contact with an aqueous environment (e.g.,
water). Examples include alginic acid, carboxymethylcellulose
calcium, carboxymethylcellulose sodium, colloidal silicon dioxide,
croscarmellose sodium, crospovidone, guar gum, magnesium aluminum
silicate, methylcellulose, microcrystalline cellulose, polacrilin
potassium, powdered cellulose, pregelatinized starch, sodium
alginate, sodium starch glycolate, starch, ethylcellulose, gelatin,
hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl
methylcellulose, polymethacrylates, povidone, pregelatinized
starch, shellac, and zein, and combinations thereof.
[0205] The term "hydrophilic polymers" as used herein includes one
or more of carboxymethylcellulose, natural gums such as guar gum or
gum acacia, gum tragacanth, or gum xanthan, hydroxyethyl cellulose,
hydroxypropyl cellulose, hydroxypropyl methylcellulose,
methylcellulose, and povidone, of which hydroxypropyl
methylcellulose is further preferred. The term "hydrophilic
polymers" can also include sodium carboxymethylcellulose,
hydroxymethyl cellulose, polyethelene oxide, hydroxyethyl methyl
cellulose, carboxypolymethylene, polyethelene glycol, alginic acid,
gelatin, polyvinyl alcohol, polyvinylpyrrolidones, polyacrylamides,
polymethacrylamides, polyphosphazines, polyoxazolidines,
poly(hydroxyalkylcarboxylic acids), an alkali metal or alkaline
earth metal, carageenate alginates, ammonium alginate, sodium
alganate, or mixtures thereof.
[0206] The hydrophobic polymer of the drug delivery system can be
any hydrophobic polymer which will achieve the goals of the present
invention including, but not limited to, one or more polymers
selected from carbomer, carnauba wax, ethylcellulose, glyceryl
palmitostearate, hydrogenated castor oil, hydrogenated vegetable
oil type 1, microcrystalline wax, polacrilin potassium,
polymethacrylates, or stearic acid, of which hydrogenated vegetable
oil type 1 is preferred. Hydrophobic polymers can include, for
example, a pharmaceutically acceptable acrylic polymer, including,
but not limited to, acrylic acid and methacrylic acid copolymers,
methyl methacrylate copolymers, ethoxyethyl methacrylates,
cyanoethyl methacrylate, aminoalkyl methacrylate copolymer,
poly(acrylic acid), poly(methacrylic acid), methacrylic acid
alkylamide copolymer, poly(methyl methacrylate), poly(methyl
methacrylate) copolymer, polyacrylamide, aminoalkyl methacrylate
copolymer, poly(methacrylic acid anhydride), and glycidyl
methacrylate copolymers. Additionally, the acrylic polymers may be
cationic, anionic, or non-ionic polymers and may be acrylates,
methacrylates, formed of methacrylic acid or methacrylic acid
esters. The polymers may also be pH dependent.
[0207] The present invention also provides a method for preparing a
press coated, pulsatile drug delivery system comprising the
compositions of the invention suitable for oral administration.
This method can include the steps of combining an effective amount
of the components of the compositions of the invention, or a
pharmaceutically acceptable salt thereof, and a polymer to form an
immediate-release component; combining an effective amount of an
active agent (e.g., the components of the compositions of the
invention), or a pharmaceutically acceptable salt thereof, and a
combination of hydrophilic and hydrophobic polymers to form a
controlled release component; and press coating the
controlled-release component to substantially envelop the immediate
release component.
[0208] A preferred embodiment further can include the steps of
combining an effective amount of an active agent (e.g., the
components of the compositions of the invention), or a
pharmaceutically acceptable salt thereof, and a polymer to form an
immediate release component, and press coating the immediate
release component to substantially envelop the controlled release
component. In another preferred embodiment, the combining steps can
be done by blending, wet granulation, fluid-bed granulation, or dry
granulation according to methods recognized in the art.
[0209] The active agents of the invention can also be mixed with
other active materials which do not impair the desired action, or
with materials that supplement the desired action, such as
antacids, H2 blockers and anti-inflammatory agents. Higher
concentrations, up to about 98% by weight of the components of the
compositions of the invention may be included.
[0210] The dosage form of the invention may be administered to
mammalian subjects, including: humans, monkeys, apes, dogs, cats,
cows, horses, rabbits, pigs, mice and rats.
[0211] The dosage form of the invention may be administered orally
(e.g., in liquid form within a solvent such as an aqueous or
non-aqueous liquid, or within a solid carrier), rectally,
parenterally, intracistemally, intravaginally, intraperitoneally,
topically (as by powders, ointments, lotion, gels, drops,
transdermal patch or transcutaneous patch), bucally, in bronchial
form or as an oral or nasal spray. The term "parenteral" as used
herein refers to modes of administration which include intravenous
(e.g., within a dextrose or saline solution), intramuscular,
intrasternal, subcutaneous, intracutaneous, intrasynovial,
intrathecal, periostal, intracerebroventricularly, intra-articular
injection and/or infusion. Alternative methods include
administration by pump or continuous infusion, injection (e.g., as
a suspension or as an emulsion in a pharmaceutically acceptable
liquid or mixture of liquids), or liposomes. Administration can be
performed daily, weekly, monthly, every other month, quarterly or
any other schedule of administration as a single dose injection or
infusion, multiple doses, or in continuous dose form. The
administration of the pharmaceutical compositions of the present
invention can be intermittent or at a gradual, continuous, constant
or controlled rate to a subject. In addition, the time of day and
the number of times per day that dosage form(s) is administered can
vary.
[0212] For parenteral administration, in one embodiment, the agents
of the invention can be formulated generally by mixing it at the
desired degree of purity, in a unit dosage injectable form
(solution, suspension, or emulsion), with a pharmaceutically
acceptable carrier(s) described above.
[0213] Any dosage form used for therapeutic administration should
be sterile. Sterility can readily be accomplished by filtration
through sterile filtration membranes (e.g., 0.2 micron membranes).
Therapeutics generally are placed into a container having a sterile
access port, for example, an intravenous solution bag or vial
having a stopper pierceable by a hypodermic injection needle.
[0214] The appropriate dose of the compound will be that amount
effective to prevent occurrence of the symptoms of the food
allergy, COPD or GERD and/or other gastrointestinal conditions
ameliorated by proper histamine management or to treat some
symptoms of the food allergy, COPD or GERD and/or other
gastrointestinal conditions ameliorated by proper histamine
management from which the patient suffers. By "effective amount",
"therapeutic amount" or "effective dose" is meant that amount
sufficient to elicit the desired pharmacological or therapeutic
effects, thus resulting in effective prevention or treatment of the
disorder or condition. Prevention of the food allergy, COPD, GERD
and/or other gastrointestinal conditions ameliorated by proper
histamine management can be manifested by delaying the onset of the
symptoms of food allergy, COPD or GERD (e.g., acid reflux,
heartburn, a burning sensation in the chest, occasionally a bitter
taste in the mouth, cough, back pain) and/or other gastrointestinal
conditions ameliorated by proper histamine management. Treatment of
the disorder can be manifested by a decrease in the symptoms
associated with food allergies, COPD, GERD and/or other
gastrointestinal conditions ameliorated by proper histamine
management or an amelioration of the recurrence of the symptoms of
the food allergies, COPD, GERD and/or other gastrointestinal
conditions ameliorated by proper histamine management.
KITS OF THE INVENTION
[0215] In a further embodiment, the present invention provides kits
(i.e., a packaged combination of reagents with instructions)
containing the active agents of the invention useful for treating
H4R modulated diseases and/or conditions.
[0216] The kit can contain a pharmaceutical composition that
includes one or more agents of the invention effective for treating
H4R modulated diseases and an acceptable carrier or adjuvant, e.g.,
pharmaceutically acceptable buffer, such as phosphate-buffered
saline, Ringer's solution or dextrose solution. It may further
include other materials desirable from a commercial and user
standpoint, including other buffers, diluents, filters, needles,
syringes, and package inserts with instructions for use.
[0217] The agents may be provided as dry powders, usually
lyophilized, including excipients that upon dissolving will provide
a reagent solution having the appropriate concentration.
[0218] The kit comprises one or more containers with a label and/or
instructions. The label can provide directions for carrying out the
preparation of the agents for example, dissolving of the dry
powders, and/or treatment for H4R modulated diseases and/or
conditions.
[0219] The label and/or the instructions can indicate directions
for in viva, use of the pharmaceutical composition. The label
and/or the instructions can indicate that the pharmaceutical
composition is used alone, or in combination with another agent to
treat H4R modulated diseases or conditions.
[0220] The label can indicate appropriate dosages for the agents of
the invention as described supra.
[0221] Suitable containers include, for example, bottles, vials,
and test tubes. The containers can be formed from a variety of
materials such as glass or plastic. The container can have a
sterile access port (for example the container can be an
intravenous solution bag or a vial having a stopper pierceable by a
needle such as a hypodermic injection needle).
ADVANTAGES OF THE INVENTION
[0222] The invention disclosed herein relates to the surprising
discovery that H4R agonists can be used for the treatment of H4R
modulated diseases.
[0223] As discussed supra, current treatment of histamine related
diseases generally focuses on the use of antagonists for H1R and
H2R for the treatment of such diseases. Based on the current state
in the art of the therapeutic utility of histamine receptors, those
skilled in the art have proposed using H4R antagonists as the main
therapeutic approach to exploit the H4R functions.
[0224] There is currently no commercially available drug to treat
H4R modulated diseases that is a H4R agonist. Thus, there is an
unmet and unperceived need to develop H4R agonists to treat H4R
modulated diseases.
EXAMPLES
Example 1
TRQ+Anti-H1 or TRQ+Anti-H1+Anti-H2 Treatment of GERD Patients
[0225] The effectiveness of the combination of Tritoqualine with an
anti-H1 and combination of Tritoqualine with an anti-H1 drug and an
anti-H2 drug was demonstrated in human studies.
Patient Cohort
[0226] Patients at baseline had at least a one year history of GERD
for whom treatment with proton pump inhibitors (PPI) did not
satisfactorily manage GERD symptoms. Baseline patients were
diagnosed with GERD and Respiratory symptoms of allergy such as
Allergic Rhinitis and Asthma.
[0227] Two groups of patients were treated in this study. The first
group (Group A) was treated with an H4R agonist, Tritoqualine at
200 mg daily and an anti-H1 drug, Loratadine at 10 mg daily. The
second group (Group B) was treated with a combination of a H4R
agonist, Tritoqualine at 200 mg daily; an anti-H1 drug, Loratadine
at 10 mg daily, and an anti-H2 drag, Ranitidine at 150 mg daily.
The demographic characteristics of the patients are shown on Table
1.
[0228] Patients were recruited based upon the aforementioned
criteria, and were examined by a physician at the initial visit
(T0) and following visit (T1) set after 6-8 weeks of treatment with
the relevant drug combination.
Results
[0229] Each patient was scored in both the initial visit (T0) and
the visit 6-8 weeks later (T1) by the physician. Scoring of the
symptoms is shown in Table 2. Both groups showed improvement using
either combination of two drugs (Group A: H4R agonist and anti-H1
drug) or three drugs (Group B: H4R agonist and anti-H1 drug and
anti-H2 drag). Overall, Group B showed superior score compared to
Group A.
[0230] Based on the data presented above, patients diagnosed with
GERD were inadequately treated with PPI and AntiH1 drug alone prior
to the treatments described in this invention (patients were taking
AntiH1 to control allergic symptoms and not to control GERD). The
average score of 6.4 which represents GERD symptoms such as
heartburn, regurgitation, and cough was indicative of the
discomfort and inadequate treatment.
[0231] Removal of the PPI and introduction of the H4R agonist such
as Tritoqualine with anti-H1 drugs in one group and Tritoqualine
plus anti-H2 drug such as Ranitidine on the other group clearly
improved the symptoms of GERD, by 82.9% (100*(6.4-3.5)/3.5) and
209.5% (100*(6.5-2.1)/2.1), respectively.
TABLE-US-00001 TABLE 1 Summary of the population demographics Age
Average Age Interval Female Male Group A 20-60 44 15 31 14 Group B
20-60 46 13 13 10 Total # of patients 44 24
TABLE-US-00002 TABLE 2 Scoring guide for the magnitude of symptoms
for GERD patients Scores Symptoms Symptoms No Moderate occurring
occurring each Symptoms symptoms Symptoms each day day and night
Heartburn 0 1 2 3 Regurgitation 0 1 2 3 Cough 0 1 2 3 Global Score
0 3 6 9
TABLE-US-00003 TABLE 3 Results of the study Global Score Global
Score To (initial visit) T1 (visit after 6-8 weeks) p-value Group A
6.4 3.5 0.005 Group B 6.5 2.1 0.002 p-value NS 0.01 NS = Not
significant
Example 2
TRQ+Anti-H1, TRQ+Anti-H2 or TRQ+Anti-H1+Anti-H2 Treatment of GERD
Patients
[0232] The effectiveness of the combination of Tritoqualine with an
anti-H1, combination of Tritoqualine with an anti-H2 and
combination of Tritoqualine with both anti-H1 and anti-H2 was
demonstrated in human studies.
Patient Cohort
[0233] Patients at baseline had at least one year history of GERD,
for whom treatment with proton-pump inhibitors, anti-H1, anti-H2
and combination of anti-H1 and anti-H2 yielded unsatisfactory
management of GERD symptoms. Baseline patients had received anti-H2
in the dosages range of 400-800 mg of Ranitidine per day, anti-H1
in the dosage range of about 10 mg Loratadine per day, and proton
pump inhibitors (PPI) in the dosage range of about 40-60 mg of
Omeprazole or Esomeprazole per day. The baseline patients were
diagnosed with GERD and respiratory symptoms of allergy such as
allergic rhinitis and asthma.
[0234] The one year history of allergies was confirmed with two
positive prick tests. The list of prick tested allergens included
Dermatophagoid Pteronysinus, Dermatophagoid Farinae, and cat and
dog dander; food allergen such as wheat egg, soy, potato, peanut
and tomato proteins; fungal proteins such as alternaria, mucor, and
apergillus; pollen proteins such as birch tree, cypress free,
Quercus tree, lolium perenne and ray grass pollen.
[0235] Three groups of patients were treated in this study. The
first group, Group A, was treated with an H4R agonist,
Tritoqualine, 200 mg daily and an Anti-H1 drug, Loratadine, 10 mg
daily. The second group, Group B, was treated with a combination of
a H4R agonist Tritoqualine, 200 mg daily; an Anti-H1 drug,
loratadine 10 mg daily, and an Anti-H2 drug, Ranitidine, 150 mg
daily. The third group, Group C, was treated with an H4R agonist,
Tritoqualine, 200 mg daily, and an Anti-H2 drug, Ranitidine, 150 mg
daily.
[0236] The study population characteristics of the patients are
shown on Table 4.
[0237] Patients were recruited based upon the aforementioned
criteria, and were examined by an allergist physician at the
initial visit (T0) and another doctor visitation (T1) set 6-8 weeks
after treatment using the relevant drug combination (three drug
combinations used described above).
[0238] Each patient was examined by the allergist both by a
physical exam and answering a standard questionnaire. The
questionnaire included questions such as whether the patient took
proton-pump inhibitors and whether and the frequency at which the
patient experienced heartburn, regurgitation and cough. Each
patient was then assigned a score by the allergist for each of the
GERD symptoms pursued in this study. Scores were assigned both
during the initial visit (T0) and the visit (T1) (scheduled within
6-8 weeks from T(0)). The severity of the following symptoms was
assessed: heartburn, regurgitation, and cough. If the patient
presented no symptoms of any of the specific attribute (heartburn,
regurgitation, and cough) was assigned a 0 score for that
attribute. Moderate symptoms (when the symptom occurs in the
frequency of less than once per day) were given the score of 1.
Symptoms occurring each day were given a score of 2 and symptoms
occurring each day and night the score of 3. The scores of each
symptom were added together to result in the global GERD score for
each patient. Global scores were then compared from visits T(0) and
T(1). Significant reduction in global scores indicated that the
therapy improved the global GERD condition for each patient. A
summary of the scoring system is shown on Table 5. The average T(0)
and T1 scores for all male and female patients was calculated (need
standard deviation) and reported in Table 6. Conducting a t-test,
the p-value for each group was calculated. P-Values<0.05 would
indicate that symptoms at visit T(0) and T(1) are significantly
different from each other. If the total score at visit T(1) is
significantly lower than at visit T(0) it will be concluded that
the effect of the drug combination was positive and therefore the
treatment of the relevant combination of drugs, efficacious.
Results and Discussion
[0239] All groups, A, B and C, showed an improvement using all
three combinations of drugs (Group A: H4R agonist and AntiH1 drug);
(Group B: H4R agonist and AntiH1 drug and Anti H2 drug) and (Group
C: H4R agonist and AntiH2 drug). Overall, groups B and C showed
superior score compared to group A. It is therefore concluded that
combinations of and H4R agonist such as Tritoqualine plus AntiH2
drugs such as Ranitidine at low dosages is an effective way to
control the symptoms of GERD of allergic patients that are not
responding to proton pump inhibitors or Anti H2 drugs alone. The
treatment in group A is also effective in treating GERD. The
results however of groups B and C are superior to group A.
TABLE-US-00004 TABLE 4 Summary of the patient details in the study
Age Average Age Interval Female Male Group A 20-60 44 15 31 14
Group B 20-60 46 13 13 10 Group C 20-60 48 12 1 2 Total 45 26
Percentage 63 37
TABLE-US-00005 TABLE 5 Scoring Symptoms Symptoms No Moderate
occurring occurring each Symptoms symptoms Symptoms each day day
and night Heartburn 0 1 2 3 Regurgitation 0 1 2 3 Cough 0 1 2 3
Global Score 0 3 6 9
TABLE-US-00006 TABLE 6 Global Score GERD T(o) Global Score GERD T1
(initial visit) (visit after 6-8 weeks) P value Group A 6.4 3.5
0.005 Group B 6.5 2.1 0.002 Group C 6.5 2.0 0.01 P value NS
0.01
Example 3
Characterization of TRQ, E1 and E2
[0240] The known chemical structure of Tritoqualine, illustrated in
FIG. 1, is characterized by, amongst other structural features, the
presence of two asymmetric carbons, A and B (marked with asterisk).
Thus, depending on the method of synthesis, Tritoqualine active
pharmaceutical ingredient can be produced as either one or two
diastereomeric structures each one comprising of its corresponding
two mirror images, enantiomers. Thus, Tritoqualine can exist as
either two or four possible isomeric structures. Using the
convention of R and S designation in each asymmetric carbon, one of
the two possible diastereomeric structures will be comprised of the
RR and SS enantiomers, and the other of RS and SR enantiomers.
Embodiments of the two enantiomers include an isolated stereoisomer
of Tritoqualine having the structure D1 of FIG. 2 and an isolated
stereoisomer of Tritoqualine having the structure D2 of FIG. 3 and
pharmaceutical compositions thereof
Materials and Methods
[0241] Extraction of Tritoqualine from tablets: Forty 100 mg
Tritoqualine tablets were crushed using mortar and pestle and the
white powder was transferred to an Erlenmeyer flask. Addition of
400 mL ethyl acetate resulted in the formation of a fine white
suspension. The suspension was allowed to stir for 1 hour under
ambient conditions. Filtration of all insoluble matter, removal of
solvent by rotary evaporation afforded a white crystalline solid.
This solid was then dissolved in approximately 100 mL of
dichloromethane. Hexane was added to the above solution until it
became cloudy. After overnight storage at room temperature,
Tritoqualine crystalline material formed at the bottom of the glass
affording 3.5 g of pure Tritoqualine.
Analytical Separation and Isolation of Tritoqualine
Stereoisomers:
[0242] Thin layer chromatography: various proportions of ethyl
acetate/hexane, dichloromethane/hexane, and ethyl acetate
dichloromethane were used in conjunction with silica-based thin
layer chromatography to identify the number of compounds available
in the mixture. In all cases of mobile phase mixtures, there was
only one single spot observed (seen under UV light) indicating the
presence of only one diastereomer. The two enantiomers comprising
the diastereomer could not be resolved using silica-based thin
layer chromatography.
[0243] HPLC separation of Tritoqualine enantiomers: HPLC separation
was conducted using an Agilent 1100 HPLC system equipped with a
quaternary pump, injector, diode array detector and a Jasco OR-990
polarimetric detector. The successful chromatographic separation
utilized the chiral HPLC column CHIRALPAK.RTM.IA (250 mm, 4.6 mm, 5
.mu.m) with the following conditions: mobile phase:
n-heptane/dichloromethane 60:40; flow rate 1 ml/min; temp
25.degree. C.; Tritoqualine concentration injected was 8 g/l in
mobile phase; injection volume 1 .mu.l; UV detection:290 nm. UV
spectra for each enantiomer were obtained using the diode array
detector and absorption of polarized light using a polarimetric
detector.
[0244] HPLC purification of Tritoqualine enantiomers: Purification
of each Tritoqualine enantiomer was conducted using a similar
Agilent HPLC with a preparatory chiral column CHIRALPAK.RTM.IA (250
mm, 4.6 mm, 5 .mu.m). Mobile phase: n-heptane dichloromethane
60:40; flow rate 20 mL/min; temp 25.degree. C., UV detection 250
nm. Each enantiomer was collected as was eluted from the column. To
ensure purity of each enantiomer HPLC analysis using the analytical
column CHIRALPAK.RTM.IA (250 mm, 4.6 mm, 5 .mu.m), mobile phase:
n-heptane/dichloromethane 60:40; flow rate 1 ml/min; temp
25.degree. C.; UV detection:250 nm. Enantiomer A eluted at
retention time of -5.95 min and enantiomer B at retention time of
7.19 mins. Chemical purities for each isolated compound exceeded
the 99.5%. Enantiomeric excess for enantiomer A was 99.5% and
enantiomer B was 99.0%. Solvent removal afforded each isolated
isomer as an amorphous white powder.
Characterization of the Commercial Mixture of Tritoqualine and of
Each Isolated Enantiomer by NMR.
[0245] .sup.1H NMR spectra were recorded on a Brucker AMX 500 (500
MHz). Chemical shifts are expressed in parts per million (.delta.)
relative to residual solvents as internal standards.
[0246] .sup.1H NMR characterization of the commercial Tritoqualine
product isolated from tablets: .sup.1H NMR (CDCl.sub.3) .delta.
6.36 (1H, s), 5.88 (2H, m), 5.59 (1H, d, J=1.71 Hz), 5.03 (2H, s),
4.54 (1H, s), 4.08 (9H, m), [3.08 (1H, m), 2.76 (1H, m), 2.56 (1H,
m), 2.43 (1H, m), 2.14 (3H, s)], 1.39-1.45 (9H, m).
[0247] .sup.1H NMR characterization of isolated enantiomer A:
.sup.1H NMR (CDCl.sub.3) .delta. Ar 6.35 (1H, s), O--, 5.87 (2H,
m), 5.58 (1H, s), 5.02 (2H, s), 4.54 (1H, s), 4.08 (9H, m), [3.04
(1H, m), 2.79 (1H, m), 2.55 (1H, m), 2.41 (1H, m)], 2.13 (3H, s),
1.37-1.45 (9H, m).
[0248] .sup.1H NMR characterization of isolated enantiomer B:
.sup.1H NMR (CDCl.sub.3) .delta. 6.36 (1H, s), 5.88 (2H, m), 5.58
(1H, d, J=1.71 Hz), 5.02 (2H, s), 4.54 (1H, s), OCH.sub.3 4.07 (9H,
m), 3.04 (1H, m), 2.77 (1H, m), 2.55 (1H, m), 2.41 (1H, m), 2.13
(3H, s), 1.37-1.45 (9H,
Crystallography
[0249] A crystal of Tritoqualine, afforded by the recrystallization
procedure described above, was chosen for X-Ray crystallography.
The crystal structure of commercial Tritoqualine was determined by
an expert crystallographer. The data is reported in Tables S1-S5
and a picture of the existing structures is illustrated in FIG. 6
below.
Crystal Structure Determination C.sub.26H.sub.32N.sub.2O.sub.8
[0250] The Brucker X8-APEX X-ray diffraction instrument with
Mo-radiation was used for data collection. All data frames were
collected at low temperatures (T=90 K), using an .omega.,
.phi.-scan mode (0.3.degree. .omega.-scan width, hemisphere of
reflections), and integrated using a Brucker SAINTPLUS software
package. The intensity data were corrected for Lorentzian
polarization. Absorption corrections were performed using the
SADABS program. The SIR97 was used for direct methods of phase
determination, and Brucker SHELXTL software for structure
refinement and difference Fourier maps. Atomic coordinates,
isotropic and anisotropic displacement parameters, of all the
non-hydrogen atoms were refined, by means of a full matrix
least-squares procedure on F.sup.2. All H-atoms were included in
the refinement, in calculated positions riding on the C atoms, with
U[iso] fixed at 20% higher, than isotropic parameters of carbons
atoms which they were attached. Drawing of molecule was performed
using Ortep 3.
[0251] Crystal and structure parameters: size
0.38.times.0.20.times.0.10 mm.sup.3, monoclinic, space group
P2(1)/n, a=16.7348(6) .ANG., b=7.8819(3) .ANG., c=18.5117(6) .ANG.,
.alpha.=90.0.degree. .beta.=985090(10).degree.
.gamma.=90.0.degree., V=2414.85(15) .ANG..sup.3,
.rho..sub.calcd=1.377 g/cm.sup.3, 2.theta.=65.26.degree.,
Mo-radiation (.lamda.=0.71073 .ANG.), low temperature=90(2) K,
reflections collected=33322, independent reflections=8434
(R.sub.int=0.0372, R.sub.sig=0.0382), 6524 (77.4%) reflections were
greater than 2.sigma.(I), index ranges 25<=h<=24,
-11<=k<=10, -27<=l<=25, absorption coefficient
.mu.=0.102 min.sup.-1; max/min transmission=0.9898 and 0.9621, 399
parameters were refined and converged at R1=0.0493, wR2=0.1210,
with intensity I>2.sigma.(I), the final difference map was 0.431
and -0.272 e.ANG..sup.-3.
Mass Spectrometry
[0252] Mass spectrometry results showed molecular ion peaks for
each enantiomer to be 500. The mass spectrometry data was recorded
on Applied Biosystems PI 100 electrospray mass spectrometer. The
samples were run in positive mode and (M.sup.++1) values are
reported 501.6 for enantiomer A and 501.5 for enantiomer B.
Results and Discussion
Separation, Characterization and Isolation of Tritoqualine
Enantiomers:
[0253] Silica-based thin layer chromatography was not able to
separate and resolve any Tritoqualine diastereomers. In general,
diastereomeric compounds can be separated in silica-based thin
layer chromatography. Enantiomers, on the other hand cannot be
separated by silica-based chromatography. A chiral solid phase is
necessary to separate and resolve enantiomers. Therefore, it was
postulated that the commercial Tritoqualine material was a mixture
of enantiomers.
[0254] Chiral chromatography was employed in order to test
commercial Tritoqualine (two chiral centers) for the presence of
enantiomers. FIG. 4 (bottom part) illustrates a representative
chromatogram of Tritoqualine chromatographed on a chiral column.
Clearly, two distinct and well resolved peaks of approximately the
same area could be identified, at 5.95 and 7.19 minutes
respectively. Polarimetric detection (FIG. 4, top part) indicates
that each peak on the chromatogram absorbs polarized light
suggesting that each molecule eluting from the chiral column is an
optically active compound. However, the polarimetric detector, in
contrast to the standard polarimeters, does not measure the sign of
the rotatory power at a given wavelength, but only gives an average
response over a range of wavelengths (200-800 nm). As the sign of
the rotatory power may change depending on the wavelength for the
same isomer (for certain compounds), especially for compounds
having UV absorption at high wavelengths (>300 nm) which is the
case of Tritoqualine (FIG. 5), it was not possible to draw
conclusions by this technique beyond the notion that each peak
represents an optical isomer. From the diode array detector
available on the HPLC setup the UV spectrum of each peak was
obtained as shown on FIG. 5. Both compounds show almost identical
UV spectra, which is the case of enantiomers. To further confirm
the presence of enantiomers, .sup.1HNMR spectra of the mixture and
of the individual components are identical. If two optically active
diastereomers were present in the mixture, then two sets of peaks
for each diastereomer would have been expected.
Diastereomer Identification in Commercial Tritoqualine:
[0255] The Tritoqualine structure contains two chiral centers (FIG.
1). Thus, there could only be two possible diastereomeric
structures. One comprised of the enantiomers RR and SS and a second
comprised of the enantiomers RS and SR.
[0256] Based on the data generated above, the only reasonable
conclusion was that commercial Tritoqualine is a single
diastereomeric structure. The challenge to find whether commercial
Tritoqualine is the RR/SS or the RS/SR remains.
[0257] To solve this issue, a single crystal from the
recrystallized Tritoqualine was identified and the crystal
structure was determined by an expert crystallographer. The
crystallography data indicates that on the single Tritoqualine
crystal there are two molecules present that are enantiomers of a
single diastereomer. The two enantiomers bear the RR and the SS
configuration.
[0258] All relevant information is shown on Tables 8-12 and the
molecular structures of the two enantiomers are illustrated on FIG.
6.
Isolation of Tritoqualine Enantiomers for the Purposes of
Biological Activity Determination:
[0259] Using the preparatory chiral column CHIRALPAK.RTM.IA (250
mm, 4.6 mm, 5 .mu.m) and the HPLC system described above, the two
enantiomers, enantiomer A and B have been successfully isolated as
amorphous white powders.
Purification of Human Histidine Decarboxylase
[0260] The DNA encoding for residues 1-512 of human HDC was
subcloned in the pGEX-6P-1 vector (GE-Healthcare). The recombinant
plasmid transformed into the Escherichia coli BL21(DE3)pLysS
strain. Transformed cultures were induced to express the HDC 1/512,
which was purified by affinity chromatography using Glutathione
sepharose (GE-Healthcare). 1/512 HDC was released from the fusion
protein bound to the affinity chromatography support by digestion
with the Pre-Scission.TM. protease (GE-Healthcare). The final
preparations were dissolved in 50 mM potassium phosphate, 0.1 mM
PLP, pH 7.0. Purity of the HDC 1/512 construct was checked by
Coomassie blue staining and Western blotting, and was higher than
95% in the final preparations.
Human HDC Activity Determination
[0261] HDC activity was assayed, as described in Engel at al.
(1996) Biochem J. 320: 365-368, by measuring the production of
.sup.14CO2 from L-[U-.sup.14C]histidine (GE-Healthcare) in a
mixture containing 0.2 mM dithiothreitol, 10 .mu.M PLP, 10 mg/ml
poly(ethylene glycol)-300, 100 mM potassium phosphate, pH 6.8, and
purified protein in a total volume of 100 .mu.L. When recombinant
HDC was used, the concentration of L-[U-.sup.14C]histidine was 13.3
.mu.M (with 1/3 isotopic dilution). The released .sup.14CO2 was
measured as previously described for HDC activity determinations
(Urdiales et al. (1992) FEBS Lett. 305, 260-264).
Assessment of Inhibitory Activity of Each Isomeric Component,
Versus the Mixture:
[0262] 10 .mu.M concentration of each isomer, A and B (A
corresponds to the isolated pure isomer eluting at 5.9 minutes, B
corresponds to the isolated pure isomer eluting at 7.1 minutes of
the chromatogram shown in FIG. 4 (bottom)) and their corresponding
racemic mixture (starting material prior to separating the
individual isomers, indicated as A+B) along with 4 us of
recombinant human HDC were used to assess the inhibitory effect of
each isomer and the mixture on the enzymatic conversion of
histidine to histamine Table 7 summarizes results obtained. Results
are presented as means of duplicates samples. As shown in Table 7,
the pure isomers (isomer A and isomer B) have more activity
compared to the racemic mix (A+B).
TABLE-US-00007 TABLE 7 Effect of compound A, B and A + B on
activity of recombinant HDC at micromolar concentration. Specific
activity Activity (.mu.mole/h % of % of Sample DPM (.mu.mole/h) mg
prot) control inhibition Control 12340 0.35 87.00 100.00 Isomer A
8895 0.25 62.71 72.08 27.92 (10 .mu.M final) Isomer B 7831 0.22
55.21 63.46 36.54 (10 .mu.M final) Racemic 10176 0.29 71.74 82.46
17.54 mix A + B (10 .mu.M final)
TABLE-US-00008 TABLE 8 Crystal data and structure refinement
Empirical formula C.sub.26H.sub.32N.sub.2O.sub.8 Formula weight
500.54 Temperature 90(2) K Wavelength 0.71073 .ANG. Crystal system
Monoclinic Space group P2(1)/n Unit cell dimensions a = 16.7348(6)
.ANG. .alpha. = 90.degree. b = 7.8819(3) .ANG. .beta. =
98.5090(10).degree. c = 18.5117(6) .ANG. .gamma. = 90.degree.
Volume 2414.85(15) .ANG..sup.3 Z 4 Density (calculated) 1.377
Mg/m.sup.3 Absorption coefficient 0.102 mm.sup.-1 F(000) 1064
Crystal size 0.38 .times. 0.20 .times. 0.10 mm.sup.3 Theta range
for data 2.22 to 32.63.degree. collection Index ranges -25 <= h
<= 24, -11 <= k <= 10, -27 <= 1 <= 25 Reflections
collected 33322 Independent 8434 [R(int) = 0.0372] reflections
Completeness to 95.7% theta = 32.63.degree. Absorption correction
Sadabs Max. and min. 0.9898 and 0.9621 transmission Refinement
method Full-matrix least-squares on F.sup.2 Data/restraints/
8434/0/399 parameters Goodness-of-fit on F.sup.2 1.021 Final R
indices R1 = 0.0493, wR2 = 0.1210 [I > 2sigma(I)] R indices (all
data) R1= 0.0677, wR2 = 0.1309 Largest diff. peak 0.431 and -0.272
e..ANG..sup.-3 and hole
TABLE-US-00009 TABLE 9 Atomic coordinates (.times.10.sup.4) and
equivalent isotropic displacement parameters (.ANG..sup.2 .times.
10.sup.3) U(eq) is defined as one third of the trace of the
orthogonalized U.sup.ij tensor. x y z U(eq) N(1) 4187(1) 505(1)
7064(1) 15(1) N(2) 2469(1) 6526(1) 7408(1) 22(1) O(1) 4787(1)
3926(1) 7526(1) 17(1) O(2) 8074(1) 270(1) 7914(1) 30(1) O(3)
7817(1) 1237(1) 6723(1) 25(1) O(4) 4067(1) 5361(1) 8264(1) 22(1)
O(5) 6084(1) 1991(1) 5982(1) 19(1) O(6) 3737(1) 2797(1) 5250(1)
20(1) C(1) 4114(1) 4785(1) 7666(1) 16(1) C(2) 3536(1) 4844(1)
6994(1) 16(1) C(3) 2767(1) 5590(1) 6881(1) 17(1) C(4) 2330(1)
5387(2) 6178(1) 20(1) C(5) 2649(1) 4475(2) 5638(1) 20(1) C(6)
3433(1) 3771(2) 5762(1) 18(1) C(7) 3863(1) 3974(1) 6453(1) 15(1)
C(8) 4689(1) 3349(1) 6771(1) 15(1) C(9) 4825(1) 1417(1) 6743(1)
14(1) C(10) 5687(1) 1028(1) 7087(1) 15(1) C(11) 6312(1) 1350(1)
6668(1) 16(1) C(12) 7096(1) 1016(2) 6998(1) 19(1) C(13A) 8410(5)
438(8) 7244(5) 32(1) C(13B) 8434(13) 880(20) 7355(13) 43(4) C(14)
7249(1) 432(2) 7708(1) 22(1) C(15) 6658(1) 112(2) 8126(1) 23(1)
C(16) 5857(1) 420(2) 7800(1) 18(1) C(17) 5150(1) 4(2) 8183(1) 22(1)
C(18) 4479(1) -737(2) 7633(1) 20(1) C(19) 6648(1) 1882(2) 5474(1)
24(1) C(20A) 3999(3) 3784(5) 4676(2) 27(1) C(20B) 3831(7) 3420(13)
4531(6) 34(2) C(21) 4431(1) 2557(2) 4224(1) 28(1) O(7A) 2204(2)
4414(3) 4960(2) 18(1) C(22A) 1509(1) 3252(2) 4911(1) 18(1) C(23A)
1771(2) 1542(3) 4685(2) 44(1) O(7B) 2188(7) 3888(8) 4956(7) 28(2)
C(22B) 1726(6) 2260(20) 5010(5) 93(5) C(23B) 1508(4) 1574(8)
4304(4) 36(1) O(8A) 1549(3) 6094(11) 6050(4) 20(1) C(24A) 1469(6)
7557(10) 5574(5) 22(1) O(8B) 1575(10) 5820(30) 6002(10) 28(4)
C(24B) 1476(16) 7230(30) 5472(14) 28(3) C(25) 578(1) 7703(2)
5283(1) 29(1) C(26) 3628(1) -354(2) 6496(1) 20(1)
TABLE-US-00010 TABLE 10 Bond lengths [.ANG.] and angles [.degree.].
N(1)--C(26) 1.4661(14) N(1)--C(18) 1.4673(15) N(1)--C(9) 1.4828(14)
N(2)--C(3) 1.3728(16) N(2)--HN1 0.885(18) N(2)--HN2 0.96(2)
O(1)--C(1) 1.3714(13) O(1)--C(8) 1.4559(14) O(2)--C(13B) 1.36(2)
O(2)--C(14) 1.3841(13) O(2)--C(13A) 1.442(10) O(3)--C(12)
1.3866(14) O(3)--C(13A) 1.424(9) O(3)--C(13B) 1.47(2) O(4)--C(1)
1.2090(15) O(5)--C(11) 1.3678(14) O(5)--C(19) 1.4294(14) O(6)--C(6)
1.3759(14) O(6)--C(20A) 1.436(5) O(6)--C(20B) 1.448(12) C(1)--C(2)
1.4590(15) C(2)--C(7) 1.3898(16) C(2)--C(3) 1.4035(14) C(3)--C(4)
1.4036(17) C(4)--O(8B) 1.305(17) C(4)--C(5) 1.4000(18) C(4)--O(8A)
1.409(6) C(5)--O(7A) 1.361(4) C(5)--C(6) 1.4115(15) C(5)--O(7B)
1.454(11) C(6)--C(7) 1.3801(16) C(7)--C(8) 1.5043(14) C(8)--C(9)
1.5419(15) C(9)--C(10) 1.5197(14) C(10)--C(16) 1.3930(16)
C(10)--C(11) 1.4144(15) C(11)--C(12) 1.3880(14) C(12)--C(14)
1.3810(18) C(14)--C(15) 1.3662(19) C(15)--C(16) 1.4065(15)
C(15)--H(15) 0.9500 C(16)--C(17) 1.5032(17) C(17)--C(18) 1.5168(17)
C(20A)-C(21) 1.529(5) C(20B)-C(21) 1.402(13) O(7A)-C(22A) 1.473(4)
C(22A)-C(23A) 1.496(3) O(7B)-C(22B) 1.509(17) C(22B)-C(23B)
1.412(10) O(8A)-C(24A) 1.445(12) C(24A)-C(25) 1.512(10)
O(8B)-C(24B) 1.47(3) C(24B)-C(25) 1.54(3) C(26)--N(1)--C(9)
110.92(9) C(26)--N(1)--C(18) 108.46(9) C(3)--N(2)--HN1 115.2(12)
C(18)--N(1)--C(9) 115.39(8) HN1--N(2)--HN2 116.8(16)
C(3)--N(2)--HN2 114.0(11) C(13B)-O(2)--C(14) 107.1(9)
C(1)--O(1)--C(8) 110.92(8) C(12)--O(3)--C(13A) 104.7(4)
C(14)--O(2)--C(13A) 104.7(3) C(11)--O(5)--C(19) 117.87(9)
C(12)--O(3)--C(13B) 103.4(9) C(6)--O(6)--C(20B) 123.1(5)
C(6)--O(6)--C(20A) 113.09(18) O(4)--C(1)--C(2) 130.14(10)
O(4)--C(1)--O(1) 121.60(10) C(7)--C(2)--C(3) 123.33(10)
O(1)--C(1)--C(2) 108.26(10) C(3)--C(2)--C(1) 128.12(11)
C(7)--C(2)--C(1) 108.53(9) N(2)--C(3)--C(4) 121.72(10)
N(2)--C(3)--C(2) 122.73(11) O(8B)-C(4)--C(5) 114.4(10)
C(2)--C(3)--C(4) 115.48(11) C(5)--C(4)--C(3) 121.34(10)
O(8B)-C(4)--C(3) 123.7(9) C(3)--C(4)--O(8A) 117.0(3)
C(5)--C(4)--O(8A) 121.6(3) O(7A)-C(5)--C(6) 120.30(18)
O(7A)-C(5)--C(4) 117.59(17) C(4)--C(5)--O(7B) 125.1(5)
C(4)--C(5)--C(6) 121.85(11) O(6)--C(6)--C(7) 120.53(9)
C(6)--C(5)--O(7B) 112.2(4) C(7)--C(6)--C(5) 116.79(11)
O(6)--C(6)--C(5) 122.43(10) C(6)--C(7)--C(8) 130.40(10)
C(6)--C(7)--C(2) 121.17(9) O(1)--C(8)--C(7) 103.86(8)
C(2)--C(7)--C(8) 108.42(9) C(7)--C(8)--C(9) 116.21(9)
O(1)--C(8)--C(9) 110.19(9) N(1)--C(9)--C(8) 110.29(8)
N(1)--C(9)--C(10) 115.37(9) C(16)--C(10)--C(11) 121.12(9)
C(10)--C(9)--C(8) 108.67(8) C(11)--C(10)--C(9) 117.83(9)
C(16)--C(10)--C(9) 121.03(10) O(5)--C(11)--C(10) 116.61(9)
O(5)--C(11)--C(12) 126.46(10) C(14)--C(12)--O(3) 110.05(9)
C(12)--C(11)--C(10) 116.92(10) O(3)--C(12)--C(11) 129.17(11)
C(14)--C(12)--C(11) 120.74(11) O(2)--C(13B)-O(3) 109.6(14)
O(3)--C(13A)-O(2) 107.6(5) C(15)--C(14)--O(2) 127.12(12)
C(15)--C(14)--C(12) 123.63(10) C(14)--C(15)--C(16) 116.61(11)
C(12)--C(14)--O(2) 109.24(11) C(10)--C(16)--C(17) 117.20(9)
C(10)--C(16)--C(15) 120.97(11) C(16)--C(17)--C(18) 108.89(10)
C(15)--C(16)--C(17) 121.72(11) O(6)--C(20A)-C(21) 106.4(3)
N(1)--C(18)--C(17) 111.09(10) C(5)--O(7A)-C(22A) 113.4(3)
C(21)--C(20B)-O(6) 113.0(7) C(5)--O(7B)-C(22B) 115.0(8)
O(7A)-C(22A)-C(23A) 108.44(19) C(4)--O(8A)-C(24A) 114.7(6)
C(23B)-C(22B)-O(7B) 109.2(9) C(4)--O(8B)-C(24B) 112.0(16)
O(8A)-C(24A)-C(25) 106.0(6) O(8B)-C(24B)-C(25) 110.1(18)
TABLE-US-00011 TABLE 11 Anisotropic displacement parameters
(.ANG..sup.2 .times. 10.sup.3). The anisotropic displacement factor
exponent takes the form: -2.pi..sup.2[h.sup.2a*.sup.2U.sup.11 + . +
2 h k a* b* U.sup.12] U.sup.11 U.sup.22 U.sup.33 U.sup.23 U.sup.13
U.sup.12 N(1) 15(1) 14(1) 16(1) 1(1) 4(1) -2(1) N(2) 19(1) 20(1)
29(1) 1(1) 10(1) 3(1) O(1) 16(1) 16(1) 19(1) -2(1) 1(1) 1(1) O(2)
17(1) 36(1) 35(1) -1(1) -6(1) 8(1) O(3) 12(1) 27(1) 36(1) 3(1) 2(1)
2(1) O(4) 26(1) 20(1) 21(1) -3(1) 5(1) 0(1) O(5) 15(1) 23(1) 19(1)
5(1) 5(1) 3(1) O(6) 22(1) 22(1) 17(1) 1(1) 5(1) 2(1) C(1) 17(1)
13(1) 21(1) 0(1) 4(1) -1(1) C(2) 14(1) 14(1) 20(1) 2(1) 3(1) 0(1)
C(3) 15(1) 15(1) 23(1) 4(1) 7(1) 1(1) C(4) 13(1) 23(1) 24(1) 8(1)
5(1) 3(1) C(5) 14(1) 27(1) 19(1) 6(1) 2(1) 1(1) C(6) 15(1) 20(1)
18(1) 2(1) 3(1) 1(1) C(7) 13(1) 14(1) 18(1) 3(1) 3(1) 1(1) C(8)
13(1) 14(1) 17(1) 1(1) 2(1) 0(1) C(9) 13(1) 14(1) 15(1) 1(1) 2(1)
0(1) C(10) 14(1) 13(1) 17(1) -1(1) 1(1) 2(1) C(11) 15(1) 14(1)
19(1) 0(1) 1(1) 2(1) C(12) 14(1) 16(1) 27(1) 0(1) 2(1) 2(1) C(13A)
14(1) 36(2) 45(2) 5(2) -3(1) 5(1) C(13B) 16(3) 64(10) 45(8) 23(7)
-3(4) -2(7) C(14) 17(1) 19(1) 28(1) -3(1) -5(1) 5(1) C(15) 24(1)
23(1) 19(1) 1(1) -3(1) 6(1) C(16) 21(1) 17(1) 16(1) -1(1) 1(1) 4(1)
C(17) 25(1) 24(1) 16(1) 4(1) 3(1) 4(1) C(18) 24(1) 17(1) 19(1) 3(1)
7(1) 0(1) C(19) 22(1) 31(1) 23(1) 1(1) 10(1) 4(1) C(20A) 42(2)
18(1) 24(2) -5(1) 18(1) -4(1) C(20B) 46(5) 34(6) 23(5) 13(4) 10(3)
19(4) C(21) 34(1) 25(1) 28(1) -3(1) 14(1) 0(1) O(7A) 14(1) 21(1)
18(1) 3(1) 0(1) -2(1) C(22A) 11(1) 17(1) 24(1) 4(1) -1(1) 2(1)
C(23A) 35(1) 28(1) 67(2) -2(1) 3(1) -2(1) O(7B) 25(2) 32(4) 23(2)
6(4) -5(2) 2(3) C(22B) 55(5) 191(16) 36(5) -50(8) 13(4) -42(8)
C(23B) 30(3) 25(3) 54(4) 5(3) 9(3) 4(2) O(8A) 10(1) 24(2) 26(1)
7(1) 4(1) 8(1) C(24A) 19(1) 22(2) 25(2) 9(1) 8(1) 7(2) O(8B) 26(3)
28(7) 36(5) 24(5) 22(3) 18(3) C(24B) 16(3) 35(9) 36(8) 13(5) 10(5)
14(5) C(25) 20(1) 36(1) 29(1) 11(1) 3(1) 9(1) C(26) 19(1) 19(1)
20(1) -3(1) 4(1) -5(1)
TABLE-US-00012 TABLE 12 Hydrogen coordinates (.times.10.sup.4) and
isotropic displacement parameters (.ANG..sup.2 .times. 10.sup.3) x
y z U(eq) H(8) 5100 3931 6517 18 H(9) 4772 1086 6216 17 H(13A) 8908
1134 7326 39 H(13B) 8548 -692 7065 39 H(13C) 8822 31 7219 51 H(13D)
8735 1929 7510 51 H(15) 6781 -297 8613 27 H(17A) 4958 1043 8403 26
H(17B) 5315 -825 8579 26 H(18A) 4682 -1755 7405 24 H(18B) 4026
-1091 7888 24 H(19A) 7125 2571 5650 37 H(19B) 6395 2304 4997 37
H(19C) 6810 698 5428 37 H(20A) 4372 4694 4884 32 H(20B) 3530 4311
4369 32 H(20C) 3310 3304 4204 41 H(20D) 3968 4642 4567 41 H(21A)
4626 3178 3826 42 H(21B) 4054 1669 4020 42 H(21C) 4890 2039 4537 42
H(21D) 4421 1412 4429 42 H(21E) 4993 2921 4235 42 H(21F) 4157 2551
3718 42 H(22A) 1061 3682 4548 21 H(22B) 1317 3174 5391 21 H(23A)
1988 1642 4223 66 H(23B) 1306 771 4620 66 H(23C) 2189 1093 5063 66
H(22C) 2065 1440 5324 112 H(22D) 1234 2489 5234 112 H(23D) 1148
2365 4004 54 H(23E) 1229 490 4339 54 H(23F) 1995 1390 4079 54
H(24A) 1786 7400 5168 26 H(24B) 1662 8592 5849 26 H(24C) 1689 6883
5022 34 H(24D) 1788 8224 5681 34 H(25A) 489 8679 4953 43 H(25B) 273
7859 5691 43 H(25C) 396 6666 5017 43 H(25D) 283 6790 5488 43 H(25E)
499 7611 4750 43 H(25F) 376 8803 5424 43 H(26A) 3915 -1252 6274 29
H(26B) 3411 469 6121 29 H(26C) 3183 -852 6714 29 HN1 1937(11)
6530(20) 7378(10) 39(3) HN2 2775(11) 6410(20) 7888(11) 39(3)
TRQ or TRQ+Montelukast.RTM. Treatment of Food Allergies in
Patients
[0263] In the following examples (EXAMPLES 4-6) baseline patients
with food allergies treated with Tritoqualine alone or
Montelukast.RTM. alone (Singulair.RTM.) did not yield satisfactory
management of food allergy symptoms. The patients with food
allergies described below demonstrate a synergic and surprising
effect with the drug combination of Tritoqualine and
Montelukast.RTM.. Allergens tested came from either STALLERGENES,
or ALLERBIO.
Example 4
TRQ+Montelukast.RTM. Treatment of Nut Allergy in a Patient
[0264] A pediatric patient had a history of anaphylaxis due to nut
allergy (hazelnut/peanut) caused by ingestion of a hazelnut
flavored cocoa spread marketed as "Nutella.RTM.". The direct test
with the Nutella.RTM. was strongly positive for allergy. This
patient was hospitalized in two occasions for the same dramatic
allergic reaction to Nutella.RTM. spread. Furthermore, the patient
presented allergies to pneumoallergens.
[0265] The patient underwent anti-allergic treatment that did not
modify his reaction to nut allergens. The second hospitalization
occurred while the patient was under H1 antagonist and
cromoglycate.
[0266] After treatment with a composition of the present invention
containing the combination of Montelukast.RTM. at the dose of 10 mg
daily and Tritoqualine at the dose of 200 mg daily for a period of
six weeks, it was possible to re-introduce the causal agent
(Nutella.RTM. spread) to the patient without any major allergic
reaction. Further, continuing the aforementioned treatment for six
months, the patient was able to eat more than 5 spoons of
Nutella.RTM. spread without any noticeable allergic reaction.
Digestive symptoms associated with this patient's allergy (stomach
pain, diarrhea, constipation) completely disappeared after the
treatment with the combination of Tritoqualine and
Montelukast.RTM.. Continuation of the above treatment for over 8
months afforded reversal of allergic and digestive symptoms due to
food allergy.
Example 5
TRQ+Montelukast.RTM. Treatment of Shrimp Allergy in a Patient
[0267] The patient under study had survived two cases of
anaphylactic shock due to ingestion of shrimps. Each case of
anaphylaxis resulted in hospitalization. Treatment with the
composition of the invention containing a combination of
Tritoqualine 200 mg and Montelukast.RTM. 10 mg for six weeks
afforded gradual reintroduction of the allergenic food without
symptoms of allergy. The first shrimp did not generate any local
signs. Later the patient was able to ingest five shrimps without
any local signs and no global reaction.
Example 6
TRQ+Montelukast.RTM. Treatment of Food Allergies in Patients
[0268] Baseline patients had signs of food allergies and the food
allergy symptoms were not adequately managed with an H1 antagonist
and cromoglycate. A group of 15 patients presenting signs of food
allergies is tested with allergens such as the following: peanut,
milk, tomato, potato, flour of wheat, soybean, fish, and shrimp.
Allergens tested came from either STALLERGENES, or ALLERBIO.
[0269] All patients had history of hospitalization and reactions
such as Quincke's edema and asthma crisis. Most of the causal
allergens were peanut (six patients), egg (four patients) and
soybean (five patients) of them (with other several positive food
allergens at different degrees of reaction). The patients
presenting loco-regional reactions to food allergens after
ingestion were treated with a composition of the invention
containing the combination of Tritoqualine and
Montelukast.RTM..
[0270] Treatment with Tritoqualine 200 mg with 10 mg of
Montelukast.RTM. for 6 to 8 weeks afforded gradual reintroduction
of the causal allergen without major allergic symptoms. One patient
with a history of peanut allergy after 8 weeks of treatment
presented a mild local reaction (itching of the lips). At 8 months
of treatment the reintroduction of peanut did not cause local or
general symptoms.
Example 7
TRQ+Loratadine Treatment of COPD in a Patient
[0271] In this study, the baseline patient was a 64 year old male
with a COPD history. The COPD history of this patient extended 1
year of treatment prior to the initial visit (T0) and presented an
average of eight hospitalization days per month due to
complications of COPD.
[0272] At the initial visit to the physician (T0), the patient was
examined and the spirometry parameter FEV1 was recorded.
Tritoqualine 200 mg/day and Loratadine 10 mg/day were added to the
patient's existing drug therapy. The patient had undergone
physician examinations: 6 weeks, 6 months and 10 months after the
start of the Tritoqualine/Loratadine treatment (T0). During each
visit the patient's overall health and the FEV1 parameter was
assessed by spirometry. Results from each visit are illustrated in
Table 13.
TABLE-US-00013 TABLE 13 Spirometry T0 T2 T3 T4 Parameter (L/s)
(initial visit) (6 weeks) (6 months) (10 month) FEV1 0.4 0.6 0.8
0.9 FEV1 Normal 3.2 3.2 3.2 3.2 value* *Normal Value is a value
expected for healthy individuals based on age, and height
[0273] During the period of treatment the patient showed
improvement as evidenced by the fact that no hospitalization was
necessary during the treatment period. Thus hospitalization
decreased from an average of eight days per month to no
hospitalization days at all. Further, the spirometry parameter FEV1
increased by 125% (from 0.4-0.9) during the 10 month treatment
period, indicating improvement.
[0274] Therefore, the additional therapy, Tritoqualine 200 mg/day
and Loratadine 10 mg/day ameliorated the COPD symptoms of the
patient by significantly decreasing the frequency of
hospitalization and improving the spirometry parameter FEV1 by 125%
(0.5 L/s in 10 months).
Example 8
TRQ+Loratadine Treatment of COPD in a Patient
[0275] The baseline patient in this study was a 60 year old male
with a COPD history that extended 2 years of treatment prior to the
initial visit (T0).
[0276] At the initial visit to the physician (T0), the patient was
examined and the spirometry parameter FEV1 was recorded.
Tritoqualine 200 mg/day and Loratadine 10 mg/day were added to the
patient's existing drug therapy. The patient had undergone
physician examinations: 6 weeks, 6 months and 8 months after the
start of the Tritoqualine/Loratadine treatment (T0). During each
visit the patient's overall health and the FEV1 parameter was
assessed by spirometry. Results from each visit are illustrated in
Table 14.
TABLE-US-00014 TABLE 14 Spirometry T0 T2 T3 T4 Parameter (initial
visit) (6 weeks) (6 months) (8 month) FEV1 (L/s) 1.4 1.7 2.1 2.4
FEV1 Normal 3.8 3.8 3.8 3.8 value*(L/s) *Normal Value is a value
expected for healthy individuals based on age, and height.
[0277] During the period of treatment the patient showed
improvement as evidenced by an increase in the spirometry parameter
FEV1, which increased by 71.4% (from 1.4-2.4) during the 8 month
treatment period.
[0278] Therefore, the additional therapy, Tritoqualine 200 mg/day
and Loratadine 10 mg/day, ameliorated the COPD symptoms of the
patient by significantly improving the spirometry parameter FEV1 by
71.4% (1.0 L/s in 8 months).
Example 9
TRQ+Loratadine Treatment of COPD in a Patient
[0279] The baseline patient in this study was a 68 year old male
with a COPD history that extended 3 years of treatment prior to the
initial visit (T0).
[0280] At the initial visit to the physician (T0), the patient was
examined and the spirometry parameter FEV1 was recorded.
Tritoqualine 200 mg/day and Loratadine 10 mg/day were added to the
patient's existing drug therapy. The patient had undergone
physician examinations: 6 weeks, and 8 months after the start of
the Tritoqualine/Loratadine treatment (T0). During each visit the
patient's overall health and the FEV1 parameter was assessed by
spirometry. Results from each visit are illustrated in Table
15.
TABLE-US-00015 TABLE 15 Spirometry T0 T2 T3 Parameter (initial
visit) (6 weeks) (6 months) FEV1 (L/s) 1.2 1.8 1.9 FEV1 Normal 3.8
3.8 3.8 value*(L/s) *Normal Value is a value expected for healthy
individuals based on age, and height.
[0281] During the period of treatment the patient showed
improvement as evidenced by an increase in the spirometry parameter
FEV1 increased by 58.3% (from 1.2-1.9) during the 8 month treatment
period.
[0282] Therefore, the additional therapy, Tritoqualine 200 mg/day
and Loratadine 10 mg/day, ameliorated the COPD symptoms of the
patient by significantly improving the spirometry parameter FEV1 by
58.3% (0.7 L/s in 8 months).
Example 10
TRQ+Loratadine Treatment of COPD in a Patient
[0283] The baseline patient in this study was a 58 year old male
with a COPD history that extended 3 years of treatment prior to the
initial visit (T0).
[0284] At the initial visit to the physician (T0), the patient was
examined and the spirometry parameter FEV1 was recorded.
Tritoqualine 200 mg/day and Loratadine 10 mg/day were added to the
patient's existing drug therapy. The patient had undergone
physician examinations: 6 weeks, 6 months, and 12 months after the
start of the Tritoqualine/Loratadine treatment (T0). During each
visit the patient's overall health and the FEV1 parameter was
assessed by spirometry. Results from each visit are illustrated in
Table 16.
TABLE-US-00016 TABLE 16 Spirometry T0 T2 T3 T4 Parameter (initial
visit) (6 weeks) (6 months) (12 months) FEV1 (L/s) 1.8 2.1 2.1 2.6
FEV1 Normal 3.1 3.1 3.1 3.1 value*(L/s) *Normal Value is a value
expected for healthy individuals based on age, and height.
[0285] During the period of treatment the patient showed
improvement as evidenced by the increase in the spirometry
parameter FEV1, which increased by 100.0% (from 1.8-2.6) during the
12 month treatment period.
[0286] Therefore, the additional therapy Tritoqualine 200 mg/day
and Loratadine 10 mg/day ameliorated the COPD symptoms of the
patient by significantly improving the spirometry parameter FEV1 by
44.4% (0.8 L/s in 12 months).
Example 11
TRQ+Loratadine Treatment of COPD in a Patient
[0287] The baseline patient in this study was a 67 year old male
with a COPD history that extended 2 years of treatment prior to the
initial visit (T0).
[0288] At the initial visit to the physician (T0), the patient was
examined and the spirometry parameter FEV1 was recorded.
Tritoqualine 200 mg/day and Loratadine 10 mg/day were added to the
patient's existing drug therapy. The patient had undergone
physician examinations: 8 weeks, 4 months, and 8 months after the
start of the Tritoqualine/Loratadine treatment (T0). During each
visit the patient's overall health and the FEV1 parameter was
assessed by spirometry. Results from each visit are illustrated in
Table 17.
TABLE-US-00017 TABLE 17 Spirometry T0 T2 T3 T4 Parameter (initial
visit) (6 weeks) (6 months) (12 months) FEV1 (L/s) 1.5 1.7 1.9 2.0
FEV1 Normal 3.2 3.2 3.2 3.2 value*(L/s) *Normal Value is a value
expected for healthy individuals based on age, and height.
[0289] During the period of treatment the patient showed
improvement as evidenced by the increase in the spirometry
parameter FEV1, which increased by 100.0% (from 1.5-2.0) during the
8 month treatment period.
[0290] Therefore, the additional therapy, Tritoqualine 200 mg/day
and Loratadine 10 mg/day, ameliorated the COPD symptoms of the
patient by significantly improving the spirometry parameter FEV1 by
33.3% (0.5 L/s in 8 months).
[0291] The treatment with a combination of a histidine
decarboxylase inhibitor such as Tritoqualine 200 mg/day and an
anti-H1 drug such as Loratadine 10 mg/day dramatically ameliorated
symptoms of COPD as shown by the respiratory parameter FEV1 and the
decrease of the need of hospitalization.
Example 12
TRQ+Existing Treatment of COPD in Patients
[0292] 13 patients (38-84 years of age; nine male and four female)
were examined by a physician and their condition of COPD was
assessed. The doctor did not remove other medications previously
taken from the patient, even though the patient was not responding
well with those medications.
[0293] At the initial visit to the physician (T0), the patients
were examined and the spirometry parameter FEV1 was recorded.
Tritoqualine 200 mg/day were added to the patient's existing drug
therapy. The patients had undergone physician examination on a
second visit (T1) 6-8 weeks after the start of the Tritoqualine
treatment. During each visit the patient's overall health and the
FEV1 parameter was assessed by spirometry. Results from each visit
are illustrated in Table 18.
TABLE-US-00018 TABLE 18 % T2 (6-8 % Deviation weeks Deviation
Expected T0 from the follow-up from the Normal (initial expected
visit) expected FEV1 visit) normal FEV1 normal Patient # value*
FEV1 value. normalized value patient 1 1.8 1.30 72.22% 1.60 88.89%
patient 2 2.15 1.86 86.51% 2.26 105.12% patient 3 1.97 1.00 50.76%
1.00 50.76% patient 4 2.66 2.39 89.85% 2.60 97.74% patient 5 3.93
1.86 47.33% 3.36 85.50% patient 6 2.09 1.60 76.56% 1.50 71.77%
patient 7 2.13 1.53 71.83% 1.98 92.96% patient 8 2.63 1.64 62.36%
1.90 72.24% patient 9 1.35 1.00 74.07% 1.18 87.41% patient 10 2.45
1.32 53.88% 1.59 64.90% patient 11 1.52 0.82 53.95% 1.04 68.42%
patient 12 1.7 0.58 34.12% 0.74 43.53% patient 13 2.72 2.28 83.82%
2.40 88.24% *Normal Value is a value expected for healthy
individuals based on age, and height according to American thoracic
Society.
[0294] Based upon the data illustrated on Table 18, there was a
significant improvement in the FEV1 parameter for at least 11
patients, one patient showed no improvement and one showed slight
deterioration. FIG. 7 and Table 19 show the % improvement for each
patient from the visit T0 to visit T1. Statistical analysis using a
paired t-test on the values generated on visits T0 and T1 showed a
statistically significant improvement due to Tritoqualine
treatment. The P-value for the paired t-test P=0.0012 was
calculated using GraphPad Prism.
TABLE-US-00019 TABLE 19 Visit T0 Visit T1 Mean 65.3 77.69 Standard
Deviation 16.81 18.27 Standard error of the mean 4.66 5.07 N 13
13
Example 13
TRQ Effect on H4R.sup.+ Human TF1 Cells, Demonstrating that TRQ has
Agonist Action on H4R
[0295] Tritoqualine was tested on a cell proliferation assay using
human TF1 cells that express H4R.
[0296] TF1 cell proliferation is promoted using
granulocyte/macrophage colony-stimulating factor (GM-CSF). In this
cell proliferation model H4R agonists inhibit cell proliferation.
Clobenpropit (CB), a known H4R agonist at a dose of 10.sup.-5M (10
.mu.M) was used as a known cell proliferation inhibition control.
Tritoqualine (racemic mixture: TRQ) was used at doses between
10.sup.-5 M (10 .mu.M) and 10.sup.-7 M (0.1 .mu.M) and the potency
of inhibition of cell proliferation was compared to CB 10.sup.-5M
(10 .mu.M). Each experiment used 100,000 TF1 cells/mL incubated for
three days with GM-CSF (10 ng/mL) After addition of TRQ in the
range of 10.sup.-5 M (10 .mu.M) to 10.sup.-7 M (0.1 .mu.M), and
incubation for 3 days, cell proliferation was assessed and compared
to that of 10 .mu.M CB.
[0297] Results and Discussion: When 10 .mu.M TRQ and 10 .mu.M CB
were compared in three separate assays (n=3), TRQ was found to be a
superior, more potent inhibitor of cell proliferation than CB
(approximately double the potency of CB). Average inhibition of
cell proliferation by CB and TRQ was found to be 23% and 49%,
respectively. Results are illustrated in FIG. 8. In addition to the
above experiment, TRQ, at concentration 10.sup.-6 M (1 .mu.M)
inhibited cell proliferation by 18%. TRQ at 10.sup.-7 (0.1 .mu.M)
demonstrated no inhibition of cell proliferation.
Example 14
TRQ or TRQ+CB Effect on H4R.sup.+ Human TF1 Cells Demonstrating
that TRQ has Agonist Action on H4R
[0298] Comparison between TRQ alone and, in association with
Clobenpropit (TRQ+CB) on the inhibition of human TF1 cell
proliferation assay; both TRQ and CB were used each, at 10 WI
concentration.
[0299] TRQ and Clobenpropit in 50:50-mixture produced a more potent
inhibition of cell proliferation compared to Tritoqualine alone.
This suggests that TRQ and CB demonstrate an additive effect on the
inhibition of cell proliferation on TF1 cells as shown in FIG.
9.
Example 15
Analysis of the Cell Cycle of Haematopoietic Progenitor Cells to
Test Tritoqualine Enantiomers Demonstrating that TRQ Enantiomers
have Agonist Action on H4R
[0300] The enantiomers of TRQ E1 and E2 M (10 .mu.M)) were tested
for inhibition of cell cycle according to the following procedure
on Colony Forming Cells (CFC, mouse medullary cells C57B1/6)
expressing H4R.
[0301] Haematopoietic progenitor cells were incubated for 1-3
day(s) with a cocktail of growth factors (IL-3, 10 ng/ml, IL-6 10
ng/ml), Fetal Calf Serum (FCS) (50 ng 1 ml) in the culture medium
StemSpan.RTM. (without serum) in the presence and absence of TRQ
and H4R antagonist (JNJ7777120, Sigma Aldrich).
[0302] Cells were later stained with propidium iodide (PI) for the
analysis of the cell cycle according to the following protocol:
[0303] 2.times.10.sup.6 cells/ml were labelled with CD1 17-FITC for
20 min in PBS, then washed in PBS and stained with PBS 1%
formaldehyde, without methanol, at 4.degree. C. for 5 min and again
washed in PBS.
[0304] The cells were then lysed with 1 ml of PVTritonX100 and
RNAse without DNAse (0.2 mcg/ml), PI (20 mcg/ml), and incubated in
PBS for 30 min at ambient temperature.
[0305] The lysed cells were analyzed with FACS scan (FL2-area
IFL2-width) for cell cycle analysis and FL1 for labelling of cell
Surface.
Staining with the CFSE
[0306] 10.sup.6 cells enriched in haematopoietic stem cells were
incubated in 2 ml of CFSE 5 mmol in PBS 10 min, 37.degree. C. The
reaction was stopped by the addition of cold PBS in excess
(5.times.) and centrifuged for 5 min at 1700 rpm.
[0307] Cells were resuspended in the culture medium StemSpan.RTM.
(without serum) and stimulated with the growth Factor cocktail, and
analyze in FACs scan (FL1) after 72 hours incubation.
[0308] The E2 enantiomer of TRQ inhibits the proliferation of the
Colony Forming Cells (CFC, mouse medullary cells C57B1/6)
expressing H4R by the blocking the cellular cycle in phase G0/G1
due to the activation of the H4 receptor.
[0309] Approximately 60% of haematopoietic progenitor cells were
blocked by the E2 enantiomer of Tritoqualine (FIG. 11). This G0/G1
blocking of the cell cycle is reversed if a known H4R antagonist
such as JNJ7777120 is used suggesting that the inhibition of cell
proliferation is due to activation of the H4R.
[0310] The E1 enantiomer of TRQ inhibits the proliferation of the
Colony Forming Cells (CFC, mouse medullary cells C57B1/6)
expressing H4R, by blocking the cellular cycle in phase G0/G1 to
the activation of the H4 receptor.
[0311] Approximately 48% of haematopoietic progenitor cells were
blocked by the E1 enantiomer of Tritoqualine (FIG. 12). This G0/G1
blocking of the cell cycle is reversed, if a known H4R antagonist
such as JNJ7777120 is used; suggesting that the inhibition of cell
proliferation is due to activation of the H4R.
[0312] These results demonstrate that the E1 enantiomer may be a
better inhibitor of cell cycle than the enantiomer E2.
Example 16
TRQ or CB Inhibition of Proliferation of Colony Forming Cells (CFC)
Demonstrating that TRQ has Agonist Action on H4R
[0313] Using the assay described in Example 15, above, the effect
of TRQ or CB on cells expressing H4R was tested. Mouse medullary
cells C57B1/6 expressing H4R (50,000 cells/Petri dish) were
incubated in a semi-solid media with haematopoietic growth factors
(IL-3: 10 ng/ml) for 7 days with TRQ or CB. In this test CB
inhibits the proliferation of CFC at the classic dose of 10.sup.-5M
(10 .mu.M) and was compared to TRQ at the dose of 10.sup.-5M (10
.mu.M).
[0314] TRQ was a more potent inhibitor of cell proliferation than
the Clobenpropit. This inhibition is due to the TRQ agonist
activity on the H4R as shown in FIG. 10.
Example 17
Effect of TRQ, E1 and E2 on Haematopoietic Progenitor Cells
[0315] Mouse C57B1/6 Haematopoietic stem cells were enriched with
bone marrow cells by magnetic sorting. Only cells expressing
surface tyrosine kinase were sorted. Cells were incubated for 2
hours with growth factors (IL-3 10 ng/ml, IL-6 10 ng/ml, SCF 50
ng/ml) and TRQ and its enantiomers, E1 and E2.
[0316] After 2 hours of incubation, cells were washed and incubated
with vibrating cell Dye cycle for 30 minutes read by FACS scan.
[0317] Enantiomer E2 was found to be a more effective inhibitor of
cell growth than TRQ and E1 (approximately 50% inhibition of G0/G1
phase was observed), whereas E2 inhibited G0/G1 by 57% (FIG. 13).
This G0/G1 blocking of the cell cycle is reversed if a known H4R
antagonist such as JNJ7777120 is used; therefore suggesting that
the inhibition of cell proliferation is due to activation of the
H4R. Thus, enantiomer E2 was a more potent inhibitor of cell
proliferation than E1.
Example 18
Effect of TRQ, Loratadine and Spiriva.RTM. to Treat COPD
[0318] Tritoqualine when combined with anti-cholinergic drugs with
and without loratadine ameliorates conditions of COPD. In this
example, the anticholinergic drug Tiotropium bromide (Spiriva.RTM.)
was combined with TRQ and/or Loratadine to treat the condition of
COPD.
[0319] The baseline patient was a 58 year old female (height 1.59
m, WEIGHT 70 Kg) with a COPD history.
The COPD History Prior to the Initial Visit (T0)
[0320] At the initial visit to the physician (T0), the patient was
examined, and the spirometry parameter FEV1 was recorded.
Tritoqualine 200 mg/day and Spiriva.RTM. (anticholinergic) 2
puffs/day (2.5 micrograms of drug per puff) were added to the
patient's existing drug therapy. The patient had undergone
physician examinations: 8 weeks, 6 months after the start of the
Tritoqualine/Spiriva.RTM. treatment (T0). During each visit, the
patient's overall health and the FEV1 parameter was assessed by
spirometry. Results from each visit are illustrated in Table
20.
TABLE-US-00020 TABLE 20 Spirometry T0 T2 T3 Parameter (L/s)
(initial visit) (8 weeks) (6 months) FEV1 1.39 1.99 2.02 FEV1
Normal 2.28 2.28 2.28 value* *Normal Value is a value expected for
healthy individuals based on age, and height
[0321] During the period of treatment the patient showed
improvement as the spirometry parameter FEV1 increased by 45.3%
(from 1.39-2.02) during the 6 month treatment period.
[0322] The results show that the additional therapy, Tritoqualine
200 mg/day and Spiriva.RTM. (2 puffs per day) ameliorated the COPD
symptoms of the patient by significantly improving the spirometry
parameter FEV1 by 45.3% (0.63 L/s in 6 months).
Example 19
Effect of TRQ, Loratadine and Spiriva.RTM. to Treat COPD
[0323] Tritoqualine when combined with anti-cholinergic drugs with
and without loratadine ameliorates conditions of COPD. In this
example, the anticholinergic drug Tiotropium bromide (Spiriva.RTM.)
was combined with TRQ and/or Loratadine to treat the condition of
COPD.
[0324] The baseline patient was a 62 year old male (height 1.70 m,
WEIGHT 85 Kg) with a COPD history.
The COPD History Prior to the Initial Visit (T0)
[0325] At the initial visit to the physician (T0), the patient was
examined and the spirometry parameter FEV1 was recorded.
Tritoqualine 200 mg/day, Loratidine 10 mg/day and Spiriva.RTM.
(anticholinergic) 2 puffs/day (2.5 micrograms of drug per puff)
were added to the patient's existing drag therapy. The patient had
undergone physician examinations: 6 weeks, 4 months and 10 months
after the start of the Tritoqualine/Loratadine/Spiriva.RTM.
treatment (T0). During each visit the patient's overall health and
the FEV1 parameter was assessed by spirometry. Results from each
visit are illustrated in Table 21.
TABLE-US-00021 TABLE 21 Spirometry T0 T2 T3 T3 Parameter (L/s)
(initial visit) (6 weeks) (4 months) (10 months) FEV1 1.21 1.3 1.70
1.86 FEV1 Normal 2.81 2.81 2.81 2.81 value* *Normal Value is a
value expected for healthy individuals based on age, and height
[0326] During the period of treatment the patient showed
improvement as the spirometry parameter FEV1 increased by 53.72.0%
(from 1.21-1.86) during the 10 month treatment period, indicating
improvement.
[0327] Therefore, the additional therapy, Tritoqualine 200 mg/day,
loratadine 10 mg/day and Spiriva.RTM. (2 puffs per day) ameliorated
the COPD symptoms of the patient by significantly improving the
spirometry parameter FEV1 by 53.72% (0.65 L/s in 6 months).
Example 20
[0328] TRQ racemic mixture (contains a mix of D1 and D2 (also known
as E1 and E2, respectively)) and purified isomers as well as CB
inhibit proliferation of Colony Forming Cells (CFC) and
demonstrates that TRQ exhibits agonist action on H4R similar to
CB.
[0329] Using the assay described in Example 15, above, the effect
of TRQ or CB on cells expressing H4R was tested. Mouse medullary
cells C57B1/6 expressing H4R (50,000 cells/Petri dish) were
incubated in a semi-solid media with haematopoietic growth factors
(IL-3: 10 ng/ml) for 7 days with a TRQ (racemic mixture of RR and
SS isomers at 1 .mu.M and 10 .mu.M concentration); TRQ isomer E1
(10 .mu.M concentration); TRQ isomer E2 (10 .mu.M concentration)
and CB (10 .mu.M concentration). In this test CB inhibits the
proliferation of CFC at the classic dose of 10.sup.-5M (10 .mu.M)
and was compared to TRQ at the doses of 10.sup.-5M and 10.sup.-6M
(10 .mu.M and 1 .mu.M).
[0330] Racemic TRQ exerted similar inhibitory activity of cell
proliferation compared to that of Clobenpropit (CB), both tested at
the 10 .mu.M concentration. At the 1 .mu.M level racemic TRQ showed
less inhibitory activity in comparison to 10 .mu.M CB. Purified TRQ
isomers E1 and E2 also exerted inhibitory activity of cell
proliferation at the 10 uM level with isomer E1 to be slightly more
potent than isomer E2. This inhibition of cell proliferation is due
to the TRQ agonist activity on the H4R. All results for this
example are shown in FIG. 14.
Example 21
[0331] Analysis of the Cell Cycle of Haematopoietic Progenitor
Cells to Test Tritoqualine racemate and purified enantiomers
demonstrating agonist action on H4R. This experiment was conducted
at two concentrations for all TRQ molecules (10 .mu.M and 1 .mu.M)
in the presence and absence of CB (10 .mu.M).
[0332] Using the assay described in Example 15, above, the effect
of TRQ or CB on cells expressing H4R was tested. Mouse medullary
cells C57B1/6 expressing H4R (50,000 cells/Petri dish) were
incubated in a semi-solid media with haematopoietic growth factors
such as IL-3 (10 ng/ml) for 7 days with a TRQ (racemic mixture of
RR and SS isomers at 1 .mu.M and 10 .mu.M concentration); purified
TRQ isomer E1 (10 .mu.M and 1 .mu.M concentration); purified TRQ
isomer E2 (10 .mu.M and 1 .mu.M concentration) and CB (10 .mu.M
concentration). Results from these experiments show that CB
increases the percentage of cells at the G0/G1 phase at the classic
dose of 10.sup.-5M (10 .mu.M); TRQ (racemic mixture) at the doses
of 10.sup.-5M and 10.sup.-6M (10 .mu.M and 1 .mu.M) also increased
the percentage of cells at the G0/G1 phase compared to the control
(GF only). When an equimolar quantity of CB was concomitantly
present with TRQ, the percentage of cells at the G0/G1 phase
further increased. Similar results were shown with the purified TRQ
isomers. In each experiment cells were incubated in the presence of
growth factors (GF, FIG. 15).
[0333] CB exerted slightly better inhibitory activity of cell
proliferation compared to that of Racemic TRQ, both tested at the
10 .mu.M concentration. At the 1 .mu.M level racemic TRQ presented
less inhibitory activity in comparison to 10 .mu.M CB. Purified TRQ
isomers E1 and E2 also exerted inhibitory activity of cell
proliferation at the 10 .mu.M level with isomer E1 to be slightly
more potent than isomer E2. Surprisingly, at the 1 .mu.M level the
purified isomers forced a higher percentage of cells at the G0/G1
phase than the racemic TRQ. Upon addition of 10 .mu.M CB the % of
cells at the G0/G1 phase further increased suggesting an additive
effect between the two molecules (TRQ and CB).
[0334] This experiment confirms that forcing cells in the G0/G1
phase, inhibiting the cell cycle, is due to agonist activity on the
H4R. Results from this experiment may also predict that H4R
agonists such as TRQ or its purified isomers can be used to protect
the vulnerable hematopoietic cells from the cytotoxicity of
anticancer drugs by forcing cells to stay in the G0/G1 phase
(inhibiting the cell cycle) whereby the chemotherapeutic agent is
less destructive. In general, anticancer agents affect dividing
cells; cells at the G0/G1 phase although metabolically active, are
not able to divide.
[0335] All results for this example are shown in FIG. 15 (two
graphs illustrating the same data).
Example 22
[0336] The data herein shows that TRQ inhibits ckit positive
hematopoietic progenitor cell proliferation.
[0337] In FIG. 16 the reference to CB-5, E1-5, and E2-5 is the same
as above. CKit is a receptor on the surface of hematopoetic cells.
Quantifying the ckit receptor is a measure of the differentiation
from the progenitor state to the hematopoetic cell state. Here we
show that H4 agonists have an inhibitory effect on the expression
of the ckit receptor.
[0338] CB inhibits of ckit expression on ckit positive
hematopoietic progenitor cells. The above graph shows that TRQ,
like CB, has a similar effect, e.g., at 10 .mu.M.
Example 23
[0339] This experiment shows that the results seen with CB and
Tritoqualine isomers E1 and E2 are due to action on the H4R. H4R
siRNA specifically inhibits the expression for the H4R. Therefore,
the activity of H4R agonists such as CB and Tritoqualine isomers E1
(10 .mu.M) and E2 (10 .mu.M) inhibiting the cell cycle at the G0/G1
phase is reversed.
Materials and Methods
[0340] H4R silencing was performed with freshly sorted c-kit.sup.+
cells. For transfection, 3 .mu.l of HiPerFect reagent (Qiagen;
Courtaboeuf, France) and 4 .mu.l (40 pmol) of human and 8 .mu.l (80
pmol) of murine H4R siRNA, scrambled FITC-labeled or unconjugated
control siRNAs (all from Santa Cruz Biotechnology) were incubated
in 50 .mu.l x-vivo medium (Cambrex; East Rutherford, N.J.) for 20
min at room temperature. The mixture was then added to 50 .mu.l of
cell suspension at a final concentration of 10.sup.6 cells/3 ml and
incubated for 20 h, when transfection efficiency was verified with
scrambled FITC-conjugated siRNA. Cells were then centrifuged and
resuspended at a concentration of 10.sup.5 cells/ml in StemSpan
medium with growth factor cocktail. The extinction of H4R protein
expression was ascertained by FACS analysis after 24 h. After 3
days of culture with or without tritoqualine (10 .mu.M) or
Clobenpropit (10 .mu.M) cells were counted and their viability was
assessed by trypan blue exclusion. The cell cycle status was
assessed after an overnight incubation.
Results and Discussion
[0341] As shown in figures below, the populations of cells treated
with SiRNA and H4R antagonists CB and Tritoqualine isomers were
significantly reduced compared to the control or non-specific,
scrambled, RNA (this scrambled RNA has no effect on the expression
of the H4R). Thus, H4R interacting with an H4R agonist will
increase the population of cells in the G0/G1 phase.
Example 24
[0342] This experiment shows that the increase of the percentage of
cells in the G0/G1 phase is reversed when effect of Bordetella
pertussis toxin (PTX), an inhibitor of G.sub.i/o coupling to the
H4R is used. This experiment confirms that tritoqualine isomers E1
and E2 have an H4R agonist activity similar to the one of CB.
[0343] Cell cultures. Bone marrow (BM) cells were prepared as
reported (Schneider, et al. J. Immunol. 139:3710-717) and adjusted
to a final concentration of 2.5.times.10.sup.6 per ml in culture
medium (MEM) supplemented with 10% horse serum. Various doses
(10.sup.-5-10.sup.-7 M) of Clobenpropit or Tritoqualine were added
shortly before the addition of IL-3 (1 ng/ml), followed by a 24-h
incubation at 37.degree. C., 5% CO.sub.2.
[0344] Progenitor-enriched murine c-kit.sup.+ BM or cord
blood-derived CD34.sup.+ cells were prepared by positive magnetic
selection, suspended at a final concentration of 10.sup.5 per ml in
serum-free StemSpan medium (StemCell Technologies) and cultured up
to 3 days in the presence of 10 ng of IL-3, 10 ng of IL-6 and 50 ng
of SCF (Stem Cell Factor) per ml, with or without Clobenpropit
(10.sup.-5M), Tritoqualine or both.
[0345] Murine CFU-GM (Colony-forming Unit-Granulocyte/Macrophage)
were quantified in MethoCult M3230 (StemCell Technologies)
supplemented with IL-3 (1 ng/ml). They were plated in a final
volume of 1 ml at a concentration of 5.times.104 total BM
cells/culture dish (Falcon 1008) with or without Clobenpropit,
Tritoqualine or both. Colonies were scored on day 7.
Evaluation of Cell Cycle Status
[0346] Magnetically sorted c-kit.sup.+ BM cells were incubated for
2 h in StemSpan medium with growth factor cocktail alone or in the
presence of 10.sup.-5M Clobenpropit, Tritoqualine or enantiomers.
The cell cycle status was then assessed using Vybrant DyeCycle
Violet (VDV) Stain (Invitrogen), according to the manufacturer's
instructions.
[0347] The effect of Bordetella pertussis toxin (PTX; 100 ng/ml),
an inhibitor of G.sub.i/o coupling to the H4R, on the cell cycle
arrest promoted by receptor activation was evaluated in the same
conditions. After incubation, cells were analyzed using FlowJo
software.
Results and Discussion
[0348] Tritoqualine Isomers (10 .mu.M) have a similar effect to CB
through the H4 receptor agonist activity manifested with the
percentage of cells at the G0/G1 phase.
Example 25
[0349] This data shows that TRQ inhibits the cell cycle and
maintains the hematopoietic progenitor cells in the G0 and/or G1
phase of the cell cycle so that even in the presence of Ara-C the
cell CFU remains high.
[0350] Ara-C is a chemotherapeutic agent that kills cancel cells
(or actively dividing cells). Since both CB and TRQ inhibit the
cell cycle and keeps the cells in the G0 and G1 cell phase, Ara-C
kills fewer cells (nondividing cells) and CB and TRQ provides
protection to the cells in the presence of chemotherapeutic agents
such as AraC.
Example 26
[0351] Organic Cation Transporter-3 (OCT-3) and Serotonin
Transporter (SERT) are important mechanisms of monoamine transport.
Many drugs and drug candidates may have significant activity on a
therapeutic target, are not therapeutically useful because they
significantly inhibit these transporters. It is therefore preferred
that tritoqualine isomers have significantly reduced activity on
the OCT-3 and SERT transporters.
[0352] This Example illustrates that TRQ and isomers do not
significantly alter biogenic monoamine update either by the Organic
Cation Transporter-3 (OCT-3) or by Serotonin Transporter
(SERT).
Materials and Methods
[0353] Cell cultures. Bone marrow (BM) cells were prepared as
reported (Schneider, et al. J. Immunol. 139:3710-717) and adjusted
to a final concentration of 2.5.times.10.sup.6 per ml in culture
medium (MEM) supplemented with 10% horse serum. Various doses
(10.sup.-5-10.sup.-7 M) of Clobenpropit or Tritoqualine were added
shortly before the addition of IL-3 (1 ng/ml), followed by a 24-h
incubation at 37.degree. C., 5% CO.sub.2.
[0354] Progenitor-enriched murine c-kit.sup.+ BM or cord
blood-derived CD34.sup.+ cells were prepared by positive magnetic
selection, suspended at a final concentration of 10.sup.5 per ml in
serum-free StemSpan medium (StemCell Technologies) and cultured up
to 3 days in the presence of 10 ng of IL-3, 10 ng of IL-6 and 50 ng
of SCF (Stem Cell Factor) per ml, with or without Clobenpropit
(10.sup.-5 M), Tritoqualine or both.
[0355] Murine CFU-GM (Colony-forming Unit-Granulocyte/Macrophage)
were quantified in MethoCult M3230 (StemCell Technologies)
supplemented with IL-3 (1 ng/ml). They were plated in a final
volume of 1 ml at a concentration of 5.times.10.sup.4 total BM
cells/culture dish (Falcon 1008) with or without Clobenpropit,
Tritoqualine or both. Colonies were scored on day 7.
[0356] In some experiments, enantiomers were used instead of the
racemic mixture of Tritoqualine.
[0357] Cytokine assays, measurement of histamine production, and
uptake. IL-6 and IL-4 production was measured in cell supernatants
recovered after a 24-h incubation. Histamine was quantified by an
automated continuous flow spectrofluorometric technique (Schneider,
et al. J. Immunol. 139:3710-717). For binding experiments, 10.sup.6
total BM cells were plated in round-bottomed 96-well polypropylene
plates (Costar). Unless stated otherwise, the cells were incubated
(37.degree. C., 5% CO.sub.2) for 3 h with 3 .mu.Ci/ml of
[.sup.3H]histamine dihydrochloride (2.5.times.10.sup.-7 M; 12
Ci/mmol) in a final volume of 100 .mu.l. Competition assays were
performed as previously described (Corbel, S., et al. 1995. Blood.
86:531-539; Corbel, S., et al. 1995, FEBS Lett. 404:289-293). Each
experiment was performed in triplicate, and histamine binding was
calculated from total cpm after subtraction of nonspecific binding
to filters.
Results and Discussion
[0358] It is evident that inhibition of histamine uptake and
serotonin uptake is greatly reduced in the presence of tritoqualine
isomers E1 and E2 at the 10 .mu.M level and negligible at the 1
.mu.M level. Thus, the fact that these transporters are not
affected by tritoqualine is a significant reason for tritoqualine's
low toxicity and great therapeutic utility as a drug.
* * * * *