Asthma associated factors as targets for treating atopic allergies including asthma and related disorders

a technology of asthma and associated factors, applied in the field of methods for treating atopic allergies and related disorders, can solve the problems of allergic inflammation, huge burden on our health-care resources, and damage to surrounding tissues

Inactive Publication Date: 2007-04-19
GENAERA CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0019] This invention includes a method for identifying a immunomodulatory 3-aminosteroid compound comprising culturing peripheral blood lymphocytes in the presence of a 3-aminosteroid compound and a mitogen to form cell aggregates; and determining the number of cell aggregates wherein an immunomodulatory 3-aminosteroid compound reduces the number of cell aggregates when compound to peripheral blood lymphocytes cultured in the absence of the 3-aminosteroid compound.

Problems solved by technology

While the battle against foreign entities may be necessary for the body's survival, some defense systems respond to foreign entities, even innocuous ones, as dangerous and thereby damage surrounding tissue in the ensuing conflict.
Activation of the immune system by these antigens leads to allergic inflammation and may occur after ingestion, penetration through the skin or after inhalation.
Thus, an enormous burden is placed on our health-care resources.
The assessment of inflamed lung tissue is often difficult and frequently the source of the inflammation cannot be determined.
It is now generally accepted that failure to control pulmonary inflammation leads to significant loss of lung function over time.
Current treatments suffer from their own set of disadvantages.
In addition, constant use of beta receptor agonists results in desensitization which reduces their efficacy and safety (Molinoff et al., (1995) Goodman and Gilman's The Pharmacologic Basis of Therapeutics, MacMillan Publishing).
Second, differences in expression of the murine IL-9 candidate gene are associated with biologic variability in bronchial responsiveness.

Method used

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  • Asthma associated factors as targets for treating atopic allergies including asthma and related disorders
  • Asthma associated factors as targets for treating atopic allergies including asthma and related disorders
  • Asthma associated factors as targets for treating atopic allergies including asthma and related disorders

Examples

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example 1

RNA Isolations, RT-PCR, Cloning and Sequencing of RT-PCR Products

[0065] Total cellular RNA was extracted after 24 hours from cultured PBMC, murine spleen cells and M07e cells using RNA PCR corekit (Perkin-Elmer, Foster City Calif.) according to manufacturer's instructions. One microgram of RNA from each source was denatured for five minutes at 65° C. and then reverse transcribed into cDNA using a 20 μl reaction mixture containing 50 units of MLV Reverse Transcriptase, one unit per μl RNAse inhibitor, 2.5 mM oligo d(T)16 primer, 1 mM each dATP, dCTP, dGTP, dTTP, 50 mM KCl, 10 mM Tris-HCl, pH 7.0, 25 mM MgCl2. The reaction mixture was pipetted into thermocycler tubes, placed in a PCR thermal cycler and subjected to one cycle (fifteen minutes at 42° C., five minutes at 99° C. and five minutes at 4° C.). A mock reverse transcription reaction was used as a negative control.

[0066] This mixture was then added to a second tube containing 2 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl, pH 7.0, 65.5...

example 2

IL-9 Biological Assay in M07e Cells

[0068] The M07e line is a human megakaryoblastic cell line, cultured in RPMI-1640, 20% fetal bovine serum and 10 ng / ml IL-3 (R&D Systems). The cell line responds to cytokines including IL-9. The cells were fed and split at 2×105 cells per milliliter every 72 hours.

[0069] The cells were centrifuged for ten minutes at 2000 rpm and resuspended in RPMI-1640 with 0.5% bovine serum albumin and insulin-transferrin-selenium (ITS) cofactors (Gibco-BRL). Cells were counted using a hemocytometer and diluted to a concentration of 1×105 cells / ml and plated in a 96-well microtiter plate. Each well contained 2×104 cells per well. The cells were stimulated with 50 ng / ml Stem Cell Factor (SCF) alone, 50 ng / ml SCF plus 50 ng / ml IL-3 (R&D Systems) or 50 ng / ml SCF plus 50 ng / ml IL-9. A control was included which contained cells and basal media only. Serial dilutions of test compounds were added to each test condition in triplicate. Cultures were incubated for 72-96 ...

example 3

Identification of Immunomodulatory 3-Aminosteroids in vitro

[0072] Immunomodulatory 3-aminosteroids (FIGS. 3-7 and Table I) were identified in vitro based on their ability to inhibit homo- or hetero-typic aggregation and subsequent proliferation of mitogen or antigen stimulated murine or human lymphocytes. Human or mouse lymphocytes were isolated from peripheral blood by Ficoll-Hypaque as described (Stoeckert et al., (1990) Exp. Hematol. 18, 1164-1170). For mitogen stimulation, 1×105 cells per well were plated in varying amounts of 3-aminosteroid compounds and assayed for aggregation and proliferation after twelve hours of stimulation by PHA-PMA mitogens. Wells were microscopically counted for aggregates of greater than 100 cells to assess aggregation and proliferation was determined using tritiated thymidine incorporation and analysis on a Packard Top Count as suggested by the manufacture. For antigen stimulation, lymphocytes were isolated from BALBc mice which had been sensitized ...

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Abstract

This invention relates to methods for treating asthma or allergy in a mammal by administering a 3-aminosteroid compound to a mammal in need thereof. The 3-aminosteroid compound being capable of down regulating the IL-9 pathway and alleviating asthmatic responses to allergen. Exemplary 3-aminosteroid compounds used in the methods of the invention include compounds having the chemical formula (I), wherein X, R1, R2, R3, and R4 groups are as defined herein. The invention also relates to certain novel compounds of formula (I). Moreover, the invention also provides methods for identifying an immunomodulatory 3-aminosteroid compound.

Description

CROSS REFERENCE TO RELATED APPLICATION [0001] The present application is a divisional application of U.S. application Ser. No. 10 / 148,553 (filed Oct. 9, 2002), which is a U.S. National Phase Application of International Application PCT / US2000 / 33526 (filed Dec. 11, 2000), which claims the benefit of U.S. Provisional Application 60 / 169,959 (filed Dec. 9, 1999), both of which are herein incorporated by reference in their entirety. This application is related to U.S. application Ser. No. 09 / 325,571 (filed Jun. 4, 1999), now U.S. Pat. No. 6,261,559, which is a continuation of U.S. application Ser. No. 08 / 874,503 (filed Jun. 13, 1997), now abandoned, which claims the benefit of U.S. Provisional 60 / 002,765 (filed Aug. 24, 1995), all of which are herein incorporated by reference in their entirety. This application is also related to U.S. application Ser. No. 09 / 198,486 (filed Nov. 24, 1998); U.S. application Ser. No. 08 / 769,689 (filed Dec. 18, 1996), now U.S. Pat. No. 5,856,535; and U.S. ap...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K31/585A61K31/58A61K31/66A61K31/56G01N33/50A61K31/575A61K31/662A61P1/04A61P11/00A61P11/02A61P11/06A61P17/04A61P27/16A61P29/00A61P37/08A61P43/00C07J17/00C07J41/00C07J43/00C07J51/00C12Q1/06C12Q1/68G01N33/15
CPCA61K31/56A61K31/58A61K31/66C07J17/00C07J41/0005C07J41/0055C07J41/0061C07J43/003C07J51/00G01N33/5047G01N2333/54Y10S514/826A61P1/04A61P11/00A61P11/02A61P11/06A61P17/04A61P27/16A61P29/00A61P37/08A61P43/00
Inventor LEVITT, ROY C.NICOLAIDES, NICHOLAS C.KINNEY, WILLIAM A.JONES, STEVE
Owner GENAERA CORP
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