U.S. patent application number 11/401469 was filed with the patent office on 2007-04-19 for asthma associated factors as targets for treating atopic allergies including asthma and related disorders.
This patent application is currently assigned to Genaera Corporation. Invention is credited to Steve Jones, William A. Kinney, Roy C. Levitt, Nicholas C. Nicolaides.
Application Number | 20070088003 11/401469 |
Document ID | / |
Family ID | 22617916 |
Filed Date | 2007-04-19 |
United States Patent
Application |
20070088003 |
Kind Code |
A1 |
Levitt; Roy C. ; et
al. |
April 19, 2007 |
Asthma associated factors as targets for treating atopic allergies
including asthma and related disorders
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, R.sup.1, R.sup.2, R.sup.3, and R.sup.4 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.
Inventors: |
Levitt; Roy C.; (Plymouth
Meeting, PA) ; Nicolaides; Nicholas C.; (Plymouth
Meeting, PA) ; Kinney; William A.; (Plymouth Meeting,
PA) ; Jones; Steve; (Plymouth Meeting, PA) |
Correspondence
Address: |
MORGAN LEWIS & BOCKIUS LLP
1111 PENNSYLVANIA AVENUE NW
WASHINGTON
DC
20004
US
|
Assignee: |
Genaera Corporation
|
Family ID: |
22617916 |
Appl. No.: |
11/401469 |
Filed: |
April 11, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10148553 |
Oct 9, 2002 |
7074778 |
|
|
PCT/US00/33526 |
Dec 11, 2000 |
|
|
|
11401469 |
Apr 11, 2006 |
|
|
|
60169959 |
Dec 9, 1999 |
|
|
|
Current U.S.
Class: |
514/114 ;
514/169; 514/175; 514/176 |
Current CPC
Class: |
A61K 31/66 20130101;
Y10S 514/826 20130101; A61P 29/00 20180101; C07J 17/00 20130101;
A61K 31/58 20130101; G01N 33/5047 20130101; A61K 31/56 20130101;
A61P 11/00 20180101; C07J 41/0005 20130101; A61P 11/02 20180101;
A61P 11/06 20180101; A61P 37/08 20180101; A61P 17/04 20180101; A61P
27/16 20180101; C07J 43/003 20130101; G01N 2333/54 20130101; A61P
43/00 20180101; C07J 41/0061 20130101; A61P 1/04 20180101; C07J
41/0055 20130101; C07J 51/00 20130101 |
Class at
Publication: |
514/114 ;
514/176; 514/169; 514/175 |
International
Class: |
A61K 31/585 20060101
A61K031/585; A61K 31/58 20060101 A61K031/58; A61K 31/66 20060101
A61K031/66; A61K 31/56 20060101 A61K031/56 |
Claims
1-20. (canceled)
21. A method of treating atopic allergy and asthma in a mammal,
comprising administering an effective amount of a compound of the
following chemical formula: ##STR24## wherein X is selected from
the group consisting of --CH.sub.2--PO(OR.sup.5).sub.2,
--NH--SO.sub.2--R.sup.5, --NH--CO--OR.sup.5,
--CH.sub.2--CO--NH.sub.2, --CH.sub.2--CO--NH--R.sub.8,
--CH.sub.2--CO.sub.2--R.sup.5, ##STR25## R.sup.1 is selected from
the group consisting of R.sup.6--NH--, ##STR26## R.sup.2, R.sup.3
and R.sup.4 are each independently selected from the group
consisting of H, --OH, --OAc and ##STR27## R.sup.5 is a C.sub.1-12
alkyl; and R.sup.6, R.sup.7 and R.sup.8 are each independently
selected from the group consisting of H, C.sub.1-6 alkyl and
phenyl.
22. A compound having the formula: ##STR28## wherein X is selected
from the group consisting of --CH.sub.2--PO(OR.sup.5).sub.2,
--NH--SO.sub.2--R.sup.5, --NH--CO--OR.sup.5,
--CH.sub.2--CO--NH.sub.2, --CH.sub.2--CO--NH--R.sub.8,
--CH.sub.2--CO.sub.2--R.sup.5, ##STR29## R.sup.1 is selected from
the group consisting of R.sup.6--NH--, ##STR30## R.sup.2, R.sup.3
and R.sup.4 are each independently selected from the group
consisting of H, --OH, --OAc and ##STR31## R.sup.5 is a C.sub.1-12
alkyl; and R.sup.6, R.sup.7 and R.sup.8 are each independently
selected from the group consisting of H, C.sub.1-6 alkyl and
phenyl.
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. application
Ser. No. 08/478,763 (filed Jun. 7, 1995), now U.S. Pat. No.
5,721,226 all of which are herein incorporated by reference in
their entirety.
FIELD OF THE INVENTION
[0002] This invention relates to methods for treating atopic
allergies and related disorders, such as asthma, in a mammal. More
particularly, the invention relates to methods for regulating IL-9
activity in a mammal by administering a 3-aminosteroid compound
that down regulates the IL-9 pathway and asthmatic responses to
allergen. The invention also relates to certain novel
3-aminosteroid compounds. In addition, the invention also provides
methods for identifying immunomodulatory 3-aminosteroid
compounds.
BACKGROUND OF THE INVENTION
[0003] Inflammation is a complex process in which the body's
defense system combats foreign entities. 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.
[0004] Atopic allergy or atopy, is an ecogenetic disorder, where
genetic background dictates the response to environmental stimuli,
such as pollen, food, dander and insect venoms. The disorder is
generally characterized by an increased ability of lymphocytes to
produce IgE antibodies in response to ubiquitous antigens.
Activation of the immune system by these antigens leads to allergic
inflammation and may occur after ingestion, penetration through the
skin or after inhalation. When this immune activation occurs and is
accompanied by pulmonary inflammation and bronchial
hyperresponsiveness, this disorder is broadly characterized as
asthma. Many cell types are involved in this inflammatory reaction
and they include T cells and antigen-presenting cells, B cells that
produce IgE and basophils and eosinophils that bind IgE. These
inflammatory cells accumulate at the site of allergic inflammation
and the toxic products they release contribute to tissue
destruction related to these disorders.
[0005] While asthma is generally defined as an inflammatory
disorder of the airways, clinical symptoms arise from intermittent
air flow obstruction. It is a chronic, disabling disorder that
appears to be increasing in prevalence and severity (Gergen et al.,
(1992) Am. Rev. Respir. Dis. 146, 823-824). It is estimated that
30-40% of the population suffer with atopic allergy and 15% of
children and 5% of adults in the population suffer from asthma
(Gergen et al., (1992) Am. Rev. Respir. Dis. 146, 823-824). Thus,
an enormous burden is placed on our health-care resources.
[0006] Interestingly, while most individuals experience similar
environmental exposures, only certain individuals develop atopic
allergy and asthma. This hypersensitivity to environmental
allergens known as atopy, is often indicated by elevated serum IgE
levels or abnormally intense skin test response to allergens in
atopic individuals as compared to non-atopics (Marsh et al., (1982)
New Eng. J. Med. 305, 1551-1559). Strong evidence for a close
relationship between atopic allergy and asthma is derived from the
fact that most asthmatics have clinical and serologic evidence of
atopy (Clifford et al., (1987) Arch. Dis. Childhood 62, 66-73;
Gergen, (1991) Arch. Intern. Med. 151, 487-492; Burrows et al.,
(1992) J. Allergy Clin. Immunol. 90, 376-385; Johannson et al.,
(1972) Prog. Clin. Immunol. 1, 1-25; Sears et al., (1991) New Engl.
J. Med. 325, 1067-1071; Halonen et al., (1992) Am. Rev. Respir.
Dis. 146, 666-670). In particular, younger asthmatics have a high
incidence of atopy (Marsh et al., (1982) New Eng. J. Med. 305,
1551-1559). In addition, immunologic factors associated with an
increase in total serum IgE levels are very closely related to
impaired pulmonary function (Burrows et al., (1989) New Eng. J.
Med. 320, 271-277).
[0007] Both the diagnosis and treatment of these disorders are
problematic (Gergen et al., (1992) Am. Rev. Respir. Dis. 146,
823-824). The assessment of inflamed lung tissue is often difficult
and frequently the source of the inflammation cannot be determined.
Without knowledge of the source of the airway inflammation and
protection from the inciting foreign environmental agent or agents,
the inflammatory process cannot be interrupted. It is now generally
accepted that failure to control pulmonary inflammation leads to
significant loss of lung function over time.
[0008] Current treatments suffer from their own set of
disadvantages. The main therapeutic agents, beta receptor agonists,
reduce the symptoms thereby transiently improving pulmonary
function, but do not affect the underlying inflammation so that
lung tissue remains in jeopardy. 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). The
agents that can diminish the underlying inflammation, the
anti-inflammatory steroids, have their own list of disadvantages
that range from immunosuppression to bone loss (Molinoff et al.,
(1995) Goodman and Gilman's The Pharmacologic Basis of
Therapeutics, MacMillan Publishing).
[0009] Because of the problems associated with conventional
therapies, alternative treatment strategies have been evaluated.
Glycophorin A (Chu et al., (1992) Cell. Immunol. 145, 223-239),
cyclosporin (Alexander et al., (1992) Lancet 339, 324-328; Morely,
(1992) J. Autoimmun. 5 Suppl A, 265-269) and a nonapeptide fragment
of interleukin 2 (IL-2) (Zavyalov et al., (1992) Immunol. Lett. 31,
285-288) all inhibit potentially critical immune functions
associated with homeostasis. What is needed in the art is a
treatment for asthma that addresses the underlying pathogenesis.
Moreover, these therapies should address the episodic nature of the
disorder and the close association with allergy and intervene at a
point downstream from critical immune functions.
[0010] In the related patent applications mentioned above,
applicants have demonstrated that interleukin 9 (IL-9), its
receptor and activities effected by IL-9 are the appropriate
targets for therapeutic intervention in atopic allergy, asthma and
related disorders.
[0011] Mediator release from mast cells by allergen has long been
considered a critical initiating event in allergy. IL-9 was
originally identified as a mast cell growth factor and it has been
demonstrated that IL-9 up-regulates the expression of mast cell
proteases including MCP-1, MCP-2, MCP-4 (Eklund et al., (1993) J.
Immunol. 151, 4266-4273) and granzyme B (Louahed et al., (1995) J.
Immunol. 154, 5061-5070). Thus, IL-9 appears to serve a role in the
proliferation and differentiation of mast cells. Moreover, IL-9
up-regulates the expression of the alpha chain of the high affinity
IgE receptor (Dugas et al., (1993) Eur. J. Immunol. 23, 1687-1692).
Elevated IgE levels are considered to be a hallmark of atopic
allergy and a risk factor for asthma. Furthermore, both in vitro
and in vivo studies have shown IL-9 to potentiate the release of
IgE from primed B cells (Petit-Frere et al., (1993) Immunology 79,
146-151).
[0012] There is substantial support for the role of IL-9 gene in
asthma. First, linkage homology between humans and mice suggests
that the same gene is responsible for producing biologic
variability in response to antigen in both species. Second,
differences in expression of the murine IL-9 candidate gene are
associated with biologic variability in bronchial responsiveness.
In particular, reduced expression of IL-9 is associated with a
lower baseline bronchial response in B6 mice (Nicolaides et al.,
(1997) Proc. Natl. Acad. Sci. USA 94, 13175-13180). Third, recent
evidence for linkage disequilibrium in data from humans suggests
IL-9 may be associated with atopy and bronchial hyperresponsiveness
consistent with a role for this gene in both species (Doull et al.,
(1996) Am. J. Respir. Crit. Care Med. 153, 1280-1284). Moreover, a
genetic alteration in the human gene appears to be associated with
loss of cytokine function and lower IgE levels. Fourth, the
pleiotropic functions of this cytokine and its receptor in the
allergic immune response strongly support a role for the IL-9
pathway in the complex pathogenesis of asthma. Fifth, in humans,
biologic variability in the IL-9 receptor also appears to be
associated with atopic allergy and asthma. Finally, despite the
inherited loss of IL-9 receptor function, these individuals appear
to be otherwise healthy. Thus, nature has demonstrated in atopic
individuals that the therapeutic down-regulation of IL-9 and IL-9
receptor genes or genes activated by IL-9 and its receptor is
likely to be safe.
[0013] Airway hyperresponsiveness is found in virtually all
asthmatics and in some strains of inbred mice (DBA2) (Levitt et
al., (1995) Clin. Exp. Allergy 25, 61-63). Airway
hyperresponsiveness is a risk factor for the development of asthma
in humans and is used in animal models of asthma as a physiologic
measure to assess the efficacy of treatment for asthma. This data
along with human (Postma et al., (1995) New Engl. J. Med. 333,
894-900) and murine genetic mapping results (U.S. Pat. No.
5,908,839) suggests a critical role for the murine IL-9 gene
product in the airway response of the mouse. In particular, the
bronchial hyperresponsive DBA2 mice differ from the C57BL6
hyporesponsive mice (Nicolaides et al., (1997) Proc. Natl. Acad.
Sci. USA 94, 13175-13180) in their expression of steady state
levels of IL-9 (U.S. Pat. No. 5,908,839). Furthermore, pretreatment
with blocking antibodies to IL-9 and its receptor can optionally
provide complete protection from antigen induced airway
hyperresponsiveness and inflammation in mice demonstrating a
critical regulatory role for IL-9 in these immune responses. This
data demonstrates that although different molecular changes produce
biologic variability in airway responsiveness in humans and mice,
these changes arise in the same gene(s) (IL-9 and its receptor)
that regulate this pathway. Taken together, these observations
confirm the critical role of IL-9 and its receptor in airway
hyperresponsiveness, asthma and atopic allergy. Moreover, this data
demonstrates that agents of the invention, which block IL-9
action(s), protect against an antigen induced response such as
those detailed above.
[0014] While the role of the IL-9 gene, its receptor and their
functions in atopic allergy, asthma and related disorders has been
elucidated, a specific need in the art exists for elucidation of
the role of genes which are regulated by IL-9 in the etiology of
these disorders. Furthermore, most significantly, based on this
knowledge, there is a need for the identification of agents that
are capable of regulating the activity of these genes, their gene
products and their subsequent biological activities for treating
these disorders.
SUMMARY OF THE INVENTION
[0015] This invention relates to methods of treating atopic allergy
and asthma in a mammal comprising administering an effective amount
of a 3-aminosteroid compound. In a preferred embodiment, the
3-aminosteroid compound down regulates the IL-9 pathway and
asthmatic responses to allergen.
[0016] Exemplary 3-aminosteroid compounds used in the methods of
the invention include compounds having the chemical formula (I)
below: ##STR1##
[0017] In Formula (I), the X group is selected from the group
consisting of --CH.sub.2--PO(OR.sup.5).sub.2,
--NH--SO.sub.2--R.sup.5, --NH--CO--OR.sup.5,
--CH.sub.2--CO--NH.sub.2, --CH.sub.2--CO--NH--R.sup.8,
--CH.sub.2--CO.sub.2--R.sup.5, ##STR2## The R.sup.1 group is
selected from the group consisting of R.sup.6--NH--, ##STR3##
[0018] The R.sup.2, R.sup.3, and R.sup.4 groups are each
independently selected from the group consisting of H, --OH, --OAc,
and ##STR4## The R.sup.5 group is a C.sub.1-12 alkyl, and the
R.sup.6, R.sup.7 and R.sup.8 are each independently selected from
the group consisting of H, C.sub.1-6 alkyl, and phenyl. The
invention also relates to certain novel 3-aminosteroid compounds
having the formula (I).
[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.
[0020] In an another embodiment, this invention also encompasses 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; and determining the
level of IL-9 mRNA wherein an immunomodulatory 3-aminosteroid
compound reduces the level of IL-9 mRNA when compared to peripheral
blood lymphocytes cultured in the absence of the 3-aminosteroid
compound. In a preferred embodiment the peripheral blood
lymphocytes are cultured in the presence of mitogen for about
twelve hours.
[0021] In yet another embodiment, the invention includes a method
for identifying a immunomodulatory 3-aminosteroid compound
comprising culturing peripheral blood lymphocytes isolated from
antigen-stimulated mammal in the presence of a 3-aminosteroid
compound and an antigen to form cell aggregates; and determining
the number of cell aggregates wherein an immunomodulatory
3-aminosteroid compound reduces the number of cell aggregates when
compared to peripheral blood lymphocytes cultured in the absence of
the 3-aminosteroid compound. In a preferred embodiment, the
peripheral blood lymphocytes are cultured in the presence of
antigen for three days and the antigen-stimulated mammal is a
mouse.
[0022] In a further embodiment, the invention includes a method for
identifying a immunomodulatory 3-aminosteroid compound comprising
culturing cells which proliferate in response to IL-9 in the
presence of IL-9 and a 3-aminosteroid compound; and measuring the
level of cell proliferation wherein an immunomodulatory
3-aminosteroid compound reduces the level of cell proliferation
induced by IL-9 when compared to cells cultured in the absence of
the 3-aminosteroid compound. In a preferred embodiment, the cells
which proliferate in response to IL-9 are Mo7e cells.
[0023] The accompanying figures, which are incorporated in and
constitute a part of this specification, illustrate several
embodiments of the invention and, together with the description,
serve to explain the principle of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1: Inhibition by various 3-aminosteroids of IL-9
mediated M07e proliferation.
[0025] FIG. 2: Aminosterols derived from the dogfish shark.
[0026] FIG. 3: Analogues of 3-aminosteroids derived from the
dogfish shark.
[0027] FIG. 4: 3-aminosteroids prepared from acylated or
sulfonylated 22-amines.
[0028] FIG. 5: 3-aminosteroid analogues prepared via the
22-aldehyde.
[0029] FIG. 6: 3-aminosteroids prepared via the Mitsunobu
reaction.
[0030] FIG. 7: 24-Amide 3-aminosteroid analogue.
[0031] FIG. 8: Novel 3-aminosteroids with ester-isosteres.
[0032] FIG. 9: Novel 3-aminosteroid esters with modified
polyamines.
[0033] FIG. 10: Novel acylated 3-aminosteroid esters.
[0034] FIG. 11: Effect of 3-aminosteroids on human lymphocyte
aggregation.
[0035] FIG. 12: Effect of 3-aminosteroids on human lymphocyte
proliferation.
[0036] FIG. 13: Effect of 3-aminosteroid on mitogen-induced
induction of IL-9
[0037] FIG. 14: Inhibition of bronchial hyperresponsiveness in mice
by 3-aminosteroids.
[0038] FIG. 15: Inhibition of bronchial hyperresponsiveness in
DBA2J mice by dexamethasone and Compound-A.
[0039] FIG. 16: Inhibition of eosinophilia in mice by dexamethasone
and Compound-A.
[0040] FIG. 17: Inhibition of IgG1 production in sensitized DBA2J
mice by dexamethasone and Compound-A.
[0041] FIG. 18: Inhibition of IgE production in sensitized DBA2J
mice by dexamethasone and Compound-A.
[0042] FIG. 19: Inhibition of bronchial hyperresponsiveness in
BALBc mice by Compound-A.
[0043] FIG. 20: Effect of dexamethasone or Compound-B on plasma
corticosterone in Sprague-Dawley rats.
[0044] FIG. 21: Effect of dexamethasone or Compound-B on spleen
weight in Sprague-Dawley rats.
[0045] FIG. 22: Effect of Compounds A, B or Dexamethasone on spleen
weight in mice.
DETAILED DESCRIPTION OF THE INVENTION
[0046] Applicant has resolved the needs in the art by elucidating
an IL-9 pathway and compositions that affect that pathway that may
be used in the diagnosis, prevention or treatment of atopic allergy
including asthma and related disorders. Asthma encompasses
inflammatory disorders of the airways with reversible airflow
obstruction. Atopic allergy refers to atopy and related disorders
including asthma, bronchial hyperresponsiveness, rhinitis,
urticaria, allergic inflammatory disorders of the bowel and various
forms of eczema. Atopy is a hypersensitivity to environmental
allergens expressed as the elevation of serum IgE or abnormal skin
test responses to allergens as compared to controls.
[0047] Further evidence defining the role of IL-9 in the
pathogenesis of atopic allergy, bronchial hyperresponsiveness,
asthma and related disorders derives directly from the applicants
observation that IL-9 is critical to a number of antigen induced
responses in mice. When the functions of IL-9 are down-regulated by
antibody or 3-aminosteroid, the animals can be completely protected
from the antigen induced responses. These responses include:
bronchial hyperresponsiveness, eosinophilia and elevated cell
counts in bronchial lavage, histologic changes in the lung
associated with inflammation and elevated serum IgE. The treatment
of such responses, which are critical to the pathogenesis of atopic
allergy and which characterize the allergic inflammation associated
with asthma, by the down-regulation of the functions of IL-9, are
within the scope of this invention.
[0048] Applicants have found that 3-aminosteroid compounds are also
useful in the inhibition of signal transduction due to IL-9
stimulation. 3-aminosteroid compounds which are useful in this
invention are described in U.S. Pat. No. 5,637,691 and related U.S.
Pat. Nos. 5,733,899 and 5,721,226 as well as in 5,840,740 and its
related U.S. Pat. Nos. 5,795,885; 5,763,430; 5,840,936; 5,874,597;
05,792,635; 5,994,336 and 5,847,172 which are specifically
incorporated herein by reference. In a preferred embodiment of the
invention, 3-aminosteroid compounds A, B, D, J and L and derived
analogues are useful for the treatment of atopic allergy and
asthma. Any compounds derived from compounds A, B, D, J and L
including alterations to the core sterol molecule, which are useful
for the treatment of atopic allergy and asthma is encompassed in
the invention.
[0049] Applicant also provides for a method to screen for the
compounds that down-regulate the expression of IL-9 or the
functions controlled by IL-9. One may determine whether the
functions expressed by IL-9 are down-regulated using techniques
standard in the art (Miyazawa et al., (1992) Blood 80, 1685-1692;
Yin et al., (1994) J. Biol. Chem. 269, 26614-26617; Renauld et al.,
(1992) Proc. Natl. Acad. Sci. USA 89, 5690-5694; Chang et al.,
(1994) Blood 83, 3199-3205). In one embodiment, serum IgE may be
measured using techniques well known in the art (Meyers et al.,
(1994) Genomics 23, 464-470) to assess the efficacy of a compound
in down-regulating the functions of IL-9 in vivo. In another in
vivo assay, bronchial hyperresponsiveness and eosinophilia in
bronchoalveolar lavage may be measured using techniques well known
in the art (Meyers et al., (1994) Genomics 23, 464-470).
[0050] In yet another embodiment, the functions of IL-9 may be
assessed in vitro. Specific assays may be based on regulation, in
part, of the proliferation of T lymphocytes, IgE synthesis and
release from mast cells by IL-9 (Renauld et al., (1990) J. Immunol.
144, 4235-4241; Kelleher et al., (1991) Blood 77, 1436-1441;
Houssiau et al., (1995) J. Immunol. 154, 2624-2630; Miyazawa et
al., (1992) Blood 80, 1685-1692; Yin et al., (1994) J. Biol. Chem.
269, 26614-26617; Renauld et al., (1992) Proc. Natl. Acad. Sci. USA
89, 5690-5694; Chang et al., (1994) Blood 83, 3199-3205). Another
assay involves the ability of human IL-9 to specifically induce the
rapid and transient tyrosine phosphorylation of multiple proteins
in M07e cells (Miyazawa et al., (1992) Blood 80, 1685-1692).
Because this response is dependent on the expression and activation
of the IL-9 receptor, it represents a simple method or assay for
the characterization of potentially valuable compounds. The
tyrosine phosphorylation of Stat3 transcriptional factor appears to
be specifically related to the actions of IL-9 (Yin et al., (1994)
J. Biol. Chem. 269, 26614-26617) and this response represents a
simple method for the characterization of compounds within the
invention. Still another method to characterize the function of
IL-9 and similar molecules involves the well known murine TS1 clone
and the D10 clone available from ATCC which is used to assess human
IL-9 function with a cellular proliferation assay (Renauld et al.,
(1992) Proc. Natl. Acad. Sci. USA 89, 5690-5694). Still another
method to monitor the effect of pharmacologic compounds is by
measuring IL-9 expression in mitogen-stimulated primary
lymphocytes, where the suppression of IL-9 prevents the activation
of the lymphocytes.
[0051] Exemplary 3-aminosteroid compounds for use in the invention
have chemical formula (I), shown below: ##STR5##
[0052] In Formula (I), the X group is selected from the group
consisting of --CH.sub.2--PO(OR.sup.5).sub.2,
--NH--SO.sub.2--R.sup.5, --NH--CO--OR.sup.5,
--CH.sub.2--CO--NH.sub.2, --CH.sub.2--CO--NH--R.sup.8, --CH.sub.2,
--CO.sub.2--R.sup.5, ##STR6## The R.sup.1 group is selected from
the group consisting of R.sup.6--NH--, ##STR7## The R.sup.2,
R.sup.3, and R.sup.4 groups are each independently selected from
the group consisting of H, --OH, --OAc, and ##STR8## The R.sup.5
group is a C.sub.1-12 alkyl, and the R.sup.6, R.sup.7 and R.sup.8
are each independently selected from the group consisting of H,
C.sub.1-6 alkyl, and phenyl.
[0053] Exemplary 3-aminosteroid compounds used in the methods of
the invention, include the following: ##STR9##
[0054] In addition, the invention also relates to certain novel
3-aminosteroid compounds of formula (I). Such compounds are also
useful in the practice of the methods of the invention. These
include compounds of formula (II), below: ##STR10## The X group is
selected from the group consisting of
--CH.sub.2--PO(OR.sup.5).sub.2, --NH--SO.sub.2--R.sup.5,
--NH--CO--OR.sup.5, --CH.sub.2--CO--NH.sub.2,
--CH.sub.2--CO--NH--R.sup.8, --CH.sub.2--CO.sub.2--R.sup.5,
##STR11## The R.sup.1 is selected from the group consisting of
R.sup.6--NH.sub.2--, ##STR12## The R.sup.2, R.sup.3, and R.sup.4
groups are each independently selected from the group consisting of
H, --OH, --OAc, and ##STR13## The R.sup.5 group is a C.sub.1-12
alkyl, and the R.sup.6, R.sup.7 and R.sup.8 groups are each
independently selected from the group consisting of H, C.sub.1-6
alkyl, and phenyl.
[0055] An embodiment of the invention relates to compounds of
formula (II), where the X is selected from the group consisting of
--CH.sub.2--PO(OR.sup.5).sub.2, --NH--SO.sub.2--R.sup.5,
--NH--CO--OR.sup.5, --CH.sub.2--CO--NH.sub.2,
--CH.sub.2--CO--NH--R.sup.8, ##STR14##
[0056] Another embodiment of the invention relates to compounds of
formula (II), where the X group is --CH.sub.2--CO.sub.2--R.sup.5,
and the R.sup.1 group is selected from the group consisting of:
R.sup.6--NH.sub.2--, ##STR15## In this embodiment, the R.sup.2,
R.sup.3, and R.sup.4 groups are independently selected from the
group consisting of H, --OH, --OAc, and ##STR16## the R.sup.5 group
is a C.sub.1-12 alkyl, and the R.sup.6 and R.sup.7 groups are each
independently selected from the group consisting of H, C.sub.1-6
alkyl, and phenyl.
[0057] In yet another embodiment, the invention also relates to
compounds of formula (II), where the X group is
--CH.sub.2--CO.sub.2--R.sup.5, the R.sup.1 group is ##STR17## and
the R.sup.2, R.sup.3, and R.sup.4 groups are each independently
selected from the group consisting of H, --OH, --OAc, and ##STR18##
with the proviso that at least one of R.sup.2, R.sup.3, and R.sup.4
is ##STR19## In this embodiment, the R.sup.5 group is a C.sub.1-12
alkyl, and R.sup.7 is selected from the group consisting of H,
C.sub.1-6 alkyl, and phenyl.
[0058] It is to be understood in the above discussion that the
alkyl groups may be straight or branched. The alkyl and phenyl
groups may be optionally substituted with halogen, alkoxy, or a
water-solubilizing group. A "water-solubilizing group" is a
substituent that increases the solubility of a compound in aqueous
solution. Exemplary water-solubilizing groups include, but are not
limited to, quaternary amine, sulfate, sulfonate, carboxylate,
phosphate, phosphonate, polyether, polyhydroxyl, boronate, and
amide groups such as--CONH.sub.2 and CONHCH.sub.3. The water
solubilizing groups may also include sulfo, sulfonamido,
carbonamido, sulfamoyl, carbamoyl, hydroxyl, and salts thereof.
[0059] In addition, the invention includes pharmaceutical
compositions comprising the compounds of the invention or their
salts together with a pharmaceutically acceptable carrier.
Pharmaceutically acceptable carriers can be sterile liquids, such
as water and oils, including those of petroleum, animal, vegetable
or synthetic origin, such as peanut oil, soybean oil, mineral oil,
sesame oil and the like. Water is a preferred carrier when the
pharmaceutical composition is administered intravenously. Saline
solutions and aqueous dextrose and glycerol solutions can also be
employed as liquid carriers, particularly for injectable solutions.
Suitable pharmaceutical carriers are described in Remington's
Pharmaceutical Sciences, Mack Publishing Company, 1995,
specifically incorporated herein by reference.
[0060] In another embodiment, a pharmaceutical compound or
composition of the invention is provided as a packaged formulation.
The packaged formulation may include a pharmaceutical composition
of the invention in a container or inhalation device and printed
instructions for administration of the composition to a subject or
patient exhibiting the symptoms of asthma or allergy. The packaged
formulation may also contain instructions for the administration of
the composition to a subject or patient in combination with another
compound or composition having a known activity against asthma or
allergy. In another format, the packaged formulation may contain
the pharmaceutical composition with general written material
indicating or suggesting the use of the composition and any other
compounds or formulations contained therein for treating a patient
diagnosed with or exhibiting the symptoms of asthma or allergy.
[0061] The compounds used in the method of treatment of this
invention may be administered systemically or topically, depending
on such considerations as the condition to be treated, need for
site-specific treatment, quantity of drug to be administered and
similar considerations.
[0062] Topical administration may be used. Any common topical
formulation such as a solution, suspension, gel, ointment or salve
and the like may be employed. Preparation of such topical
formulations as are well described in the art of pharmaceutical
formulations as exemplified, for example, by Remington's
Pharmaceutical Sciences. For topical application, these compounds
could also be administered as a powder or spray, particularly in
aerosol form. The active ingredient may be administered in
pharmaceutical compositions adapted for systemic administration. As
is known, if a drug is to be administered systemically, it may be
confected as a powder, pill, tablets or the like or as a syrup or
elixir for oral administration. For intravenous, intra-peritoneal
or intra-lesional administration, the compound will be prepared as
a solution or suspension capable of being administered by
injection. In certain cases, it may be useful to formulate these
compounds in suppository form or as an extended release formulation
for deposit under the skin or intramuscular injection. In a
preferred embodiment, the compounds of this invention may be
administered by inhalation. For inhalation therapy the compound may
be in a solution useful for administration by metered dose inhalers
or in a form suitable for a dry powder inhaler.
[0063] An effective amount is that amount which will down-regulate
IL-9 activity. A given effective amount will vary from condition to
condition and in certain instances may vary with the severity of
the condition being treated and the patient's susceptibility to
treatment. Accordingly, a given effective amount will be best
determined at the time and place through routine experimentation.
However, it is anticipated that in the treatment of atopic allergy,
asthma and asthma-related disorders in accordance with the present
invention, a formulation containing between 0.001 and 5% by weight,
preferably about 0.01 to 1%, will usually constitute a
therapeutically effective amount. When administered systemically,
an amount between 0.01 and 100 mg per kg body weight per day, but
preferably about 0.1 to 10 mg/kg, will effect a therapeutic result
in most instances.
[0064] Other embodiments of the invention will be apparent to those
skilled in the art from consideration of the specification and
practice of the invention disclosed. It is intended that the
specifications and examples be considered exemplary only with a
true scope of the invention being indicated by the claims. Having
provided this background information, applicant now describes
preferred aspects of the invention.
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.degree. C. and then reverse
transcribed into cDNA using a 20 .mu.l reaction mixture containing
50 units of MLV Reverse Transcriptase, one unit per .mu.l 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 MgCl.sub.2. The
reaction mixture was pipetted into thermocycler tubes, placed in a
PCR thermal cycler and subjected to one cycle (fifteen minutes at
42.degree. C., five minutes at 99.degree. C. and five minutes at
4.degree. 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
MgCl.sub.2, 50 mM KCl, 10 mM Tris-HCl, pH 7.0, 65.5 .mu.l deionized
water, 2.5 units Amplitaq DNA polymerase and 1 .mu.l (20 .mu.M)
each of oligonucleotides representing human cDNA IL-9 exon 1
(forward) and exon 5 (reverse), for a final volume of 100 .mu.l.
The reaction mixture was subjected to the following PCR conditions:
two minutes at 98.degree. C., then 30 cycles at thirty seconds at
94.degree. C., forty seconds at 55.degree. C., forty seconds at
72.degree. C. Finally, the reaction mixture was cycled one time for
fifteen minutes at 72.degree. C. for extension.
[0067] PCR products representing human IL-9 or IL-9R cDNA were
subjected to gel electrophoresis through 1.5% agarose gels and
visualized using ethidium bromide staining. Products of a mock
reverse transcriptase reaction, in which water was substituted for
RNA was used as negative control amplification in all
experiments.
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.times.10.sup.5 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.times.10.sup.5 cells/ml and plated in a 96-well microtiter
plate. Each well contained 2.times.10.sup.4 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 hours at 37.degree. C. in 5% CO.sub.2.
[0070] Cell proliferation was assayed using the Abacus.RTM. Cell
Proliferation Kit (Clontech) which determines the amount of
intracellular acid phosphatase present as an indication of cell
number. The substrate p-nitrophenyl phosphate (pNPP), was converted
by acid phosphatase to p-nitrophenol, which was measured as an
indicator of enzyme concentration. pNPP was added to each well and
incubated at 37.degree. C. for one hour. Sodium hydroxide was then
added to stop the enzymatic reaction and the amount of
p-nitrophenol was quantified using a Dynatech.RTM. 2000 plate
reader at 410 nm wavelength. Standard curves that compare cell
number with optical absorbance were used to determine the linear
range of the assay. Assay results were only used when absorbance
measurements were within the linear range of the assay.
[0071] FIG. 1 illustrates the effect of aminosterols isolated from
the shark liver (FIG. 2) as set forth in U.S. Pat. Nos. 5,637,691;
5,733,899; 5,721,226 and 5,840,740 incorporated herein by
reference, on the IL-9 dependent growth of M07e cells in vitro.
Each 3-aminosteroid was incubated with M07e cells at 20 .mu.g/ml of
the culture media and inhibition of cellular growth induced by IL-9
was determined by comparison with control conditions (no
treatment). There was no evidence for cytotoxicity with any of the
treatments. 3-aminosteroids 3 and 6 (FIG. 2) consistently provided
the greatest inhibition of growth.
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.times.10.sup.5 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 to Aspergillus fumigatus
antigen for three weeks and plated at 1.times.10.sup.5 cells per
well with or without 100 units of Aspergillus fumigatus antigen.
Cells were grown for three days and then scored for cellular
aggregates and proliferation as described above in the presence or
absence of increasing amounts of Compound-B or analogue compounds.
Compound-B and Compound-A were able to suppress lymphocyte
aggregation (FIG. 11) and proliferation (FIG. 12) at lower doses
(IC.sub.50=2.5 .mu.g/ml & 0.5 .mu.g/ml, respectively) than the
highly similar compounds D (IC.sub.50=10 .mu.g/ml) and E
(IC.sub.50>10 .mu.g/ml). Moreover, the treatment of
mitogen-stimulated lymphocytes with compounds A or B was found to
block the expression of IL-9 in these cultures in contrast to the
effect of control compound E (FIG. 13). Similar results were
obtained in assays using antigen mediated response to compounds
(not shown). These data demonstrate that both the aggregation and
proliferation assays are useful for determining structure-activity
relationships for this family of 3-aminosteroids. Table I shows the
activity of other 3-aminosteroid compounds in the assays described
above. All of the active compounds shown in Table I as well as
rational modifications of these compounds as depicted in FIGS. 8-10
are embodiments of the invention
EXAMPLE 4
Efficacy of Immunomodulatory 3-Aminosteroids in Suppression of
Asthmatic Response
[0073] DBA2, C57BL6 or B6D2F1 mice, five to six weeks of age, were
obtained from the National Cancer Institute or Jackson
Laboratories. Animals were housed in high-efficiency particulate
filtered air laminar flow hoods in a virus and antigen free
facility and allowed free access to pelleted rodent chow and water
for three to seven days prior to experimental manipulation. The
animal facilities were maintained at 22.degree. C. and the
light:dark cycle is automatically controlled (10:14 hour
cycle).
[0074] Phenotyping and efficacy of pretreatment. Animals either
received no pretreatment or were sensitized by nasal aspiration of
Aspergillus fumigatus antigen to assess the effect on bronchial
hyperresponsiveness, bronchoalveolar lavage and serum IgE. Mice
were challenged with Aspergillus or saline intranasally (Monday,
Wednesday and Friday for three weeks) and phenotyped twenty-four
hours after the last dose. The effect of pretreatment by
aminosteroids was used to assess the effect of down-regulating the
IL-9 pathway in mice. To determine the bronchoconstrictor response,
respiratory system pressure was measured at the trachea and
recorded before and during exposure to the drug. Mice were
anesthetized and instrumented as previously described. (Kleeberger
et al., (1990) Am. J. Physiol. 258, L313-320; Levitt et al., (1995)
Clin. Exp. Allergy 25, 61-63; Ewart et al., (1995) J. Appl.
Physiol. 79, 560-566). Airway responsiveness was measured to one or
more of the following: 5-hydroxytryptamine, acetylcholine,
atracurium or a substance-P analogue. A simple and repeatable
measure of the change in peak inspiratory pressure following
bronchoconstrictor challenge was used which has been termed the
Airway Pressure Time Index (APTI). The APTI was assessed by the
change in peak respiratory pressure integrated from the time of
injection until the peak pressure returns to baseline or plateau.
The APTI was comparable to airway resistance, however, the APTI
includes an additional component related to the recovery from
bronchoconstriction.
[0075] Prior to sacrifice, whole blood was collected for serum Ig
measurements by needle puncture of the inferior vena cava in
anesthetized animals. Samples were centrifuged to separate cells
and serum was collected and used to measure total IgG.sub.1,
IgG.sub.2a and IgE levels. Samples not measured immediately were
frozen at -20.degree. C.
[0076] Serum Igs were measured using an ELISA antibody-sandwich
assay. Microtiter plates were coated, 50 .mu.l per well, with rat
anti-murine IgG.sub.1, IgG.sub.2a or IgE antibody (Southern
Biotechnology and PharMingen) at a concentration of 2.5 .mu.g/ml in
coating buffer of sodium carbonate-sodium bicarbonate with sodium
azide. Plates were covered with plastic wrap and incubated at
4.degree. C. for sixteen hours. The plates were washed three times
with a wash buffer of 0.05% Tween-20 in phosphate-buffered saline,
incubating for five minutes for each wash. Blocking of nonspecific
binding sites was accomplished by adding 200 .mu.l per well 5%
bovine serum albumin in phosphate-buffered saline, covering with
plastic wrap and incubating for two hours at 37.degree. C. After
washing three times with wash buffer, duplicate 50 .mu.l test
samples were added to the wells. Test samples were assayed after
being diluted 1:10, 1:50 and 1:100 with 5% bovine serum albumin in
wash buffer. In addition to the test samples, a set of Ig standards
(PharMingen) at various liner concentrations in 5% bovine serum
albumin in wash buffer, were assayed to generate a standard curve.
A blank of no sample or standard was used to zero the plate reader
(background). After adding samples and standards, the plate was
covered with plastic wrap and incubated for two hours at room
temperature. After washing three times with wash buffer, 50 .mu.l
of secondary antibody rat anti-murine IgG.sub.1, IgG.sub.2a or
IgE-horseradish peroxidase conjugate was added at a concentration
of 250 ng/ml in 5% bovine serum albumin in wash buffer. The plate
was covered with plastic wrap and incubated two hours at room
temperature. After washing three times with wash buffer, 100 .mu.l
of the substrate 0.5 mg/ml o-phenylenediamine in 0.1 M citrate
buffer was added to every well. After five to ten minutes the
reaction was stopped with 50 .mu.l of 12.5% sulfuric acid and
absorbance was measured at 490 nm on a Dynatech.RTM. MR5000 plate
reader. A standard curve was constructed from the standards with
antigen concentration on the x axis (log scale) and absorbance on
the y axis (linear scale). The concentration of IgG.sub.1,
IgG.sub.2a or IgE in the samples was interpolated from the standard
curve.
[0077] Bronchoalveolar lavage and cellular analysis were preformed
as previously described (Kleeberger et al., (1990) Am. J. Physiol.
258, L313-320). Lung histology was carried out after the lungs were
removed under anesthesia. Since prior instrumentation may introduce
artifact, separate animals were used for these studies. Thus, a
small group of animals was treated in parallel exactly the same as
the cohort undergoing various pretreatments except these animals
were not used for other tests aside from bronchial responsiveness
testing. After bronchial responsiveness testing, the lungs were
removed and submersed in liquid nitrogen. Cryosectioning and
histologic examination was carried out in a manner obvious to those
skilled in the art.
[0078] Active compounds identified in vitro were tested in vivo for
their ability to suppress airway hyperresponsiveness, lung
eosinophilia and serum Ig levels using assays described above.
Animals were either unsensitized or sensitized to antigen and dosed
with a 3-aminosteroid compound 1p at either 1 or 10 mg/kg/day for
up to three weeks or 2.5 mg/kg twice a week for four weeks. The
ability of these compounds to suppress asthmatic type responses is
demonstrated by the data in FIGS. 14-19. FIG. 14 demonstrates that
both Compounds-B and -D are effective in suppressing bronchial
hyperresponsiveness in D2 naive mice, which are hyperresponsive due
to elevated IL-9 levels in their lungs (Nicolaides et al., (1997)
Proc. Natl. Acad. Sci. USA 94, 13175-13180) and Compound-B is
effective at a lower dose than Compound-D. FIG. 15 indicates that
the 3-aminosteroid Compound-A is able to block airway
hyperresponsiveness in mice sensitized to antigen and that it was
more efficacious than the commonly used corticosteroid,
dexamethasone (Dex). FIG. 16 demonstrates that eosinophils were the
particular cell type affected by Compound-A, suggesting that this
compound acts on cell types associated with a TH2-allergic
response. The inhibition of a TH2-allergic response was also
indicated in FIGS. 17 and 18 where total serum IgG1 and IgE was
suppressed but not the TH1 associated immunoglobulin IgG2a. FIG. 19
indicates that the 3-aminosteroid Compound-A is able to block
airway hyperresponsiveness at a dose as low as 2.5 mg/kg
administered twice per week. In summary, these data suggest that
3-aminosteroid compounds and derived analogues have the potential
to inhibit asthmatic responses at very low drug concentrations and
therefore will be useful for the treatment of asthma with a low
incident of side effects in human patients.
EXAMPLE 5
Mechanism of Action in vivo of Immunomodulatory 3-Aminosteroids
[0079] To determine the mechanism by which 3-aminosteroid compounds
function to suppress the asthmatic response to allergen,
comparative physiologic assays were carried out utilizing the
extensively studied Sprague-Dawley rat model (Scaccianoce et al.,
(1995) Neuroendocrinology 62, 32-38; Hatzinger et al., (1996)
Neuroendocrinology 64, 349-356). Animals were administered 1
mg/kg/day of either the corticosteroid Dexamethasone or Compound-B
and analyzed at ten hours for corticosterone and ACTH. As shown in
FIG. 20, dexamethasone significantly suppressed absolute levels of
plasma corticosterone (>450 fold) eight to ten hours after
treatment while Compound-B had no significant effect. Similar
results were found for ACTH levels (>100 fold) supporting the
finding that Compound-B is acting by a different mechanism from
dexamethasone. Longer studies, where drug was administered for 28
days, demonstrated that animals tolerated the 3-aminosteroid
compounds better than dexamethasone where weight loss and splenic
atrophy were observed (FIG. 21). Similar data was obtained in mice
treated with effective doses of either Compounds A or B (10 mg/kg
per day for 22 days) where no splenic atrophy was observed in
contrast to dexamethasone treatment (FIG. 22). This data combined
with the data from Examples 2, 3 and 4 demonstrates that
3-aminosteroid compounds are novel anti-inflammatory and
anti-asthma compounds which appear to function by a mechanism
unlike that of the dexamethasone class of steroids to suppress the
biological response to allergen sensitization.
EXAMPLE 6
3-Aminosteroidal Esters
[0080] The majority of the analogues prepared were accessible from
the methods described previously (Jones et al., (1996) Steroids 61,
565-571) using the appropriate polyamine and steroidal ester (FIG.
3). In addition, general methods for preparing 3-aminosteroids have
also been described previously (Zasloff et al., (1999) U.S. Pat.
No. 5,856,535) this reference herein incorporated by reference in
its entirety.
[0081] A number of 3-aminosteroid analogues were prepared via
acylation or sulfonylation of the C22-amine 5, which was prepared
as outlined below. The preparation of compounds 1 and 2 has been
described previously (Rao et al., (1997) J. Org. Chem. 62,
4541-4545).
Analogues Prepared via Acylation or Sulfonylation of 22-Amine
[0082] ##STR20## Preparation of Compound 3
[0083] Compound 2 (5.5 g, 14.6 mmol) was added to THF (100 mL)
containing TEA (5 mL), and methanesulfonylchloride (2.0 g, 17.5
mmol) was added dropwise with ice bath cooling. The reaction was
judged complete by TLC on silica gel (elution with 60/40
hexane/ethyl acetate) after thirty minutes. The reaction mixture
was worked-up by addition of toluene (100 mL) and saturated sodium
bicarbonate. The organic layer was washed alternately with sodium
bicarbonate and 0.1 M HCl solution. The pooled organic layers were
then dried over sodium sulfate to afford crude compound 3 as an off
white solid (5.4 g, 11.9 mmol, 81%). The crude mesylate (3) was
carried on without further purification.
Preparation of Compound 4
[0084] The crude mesylate (5.0 g, 1.0 mmol) was added to DMSO (150
mL). Sodium azide (2.5 g, 38.5 mmol) was added and the reaction was
warmed to 60.degree. C. until judged complete by TLC on silica gel
(approximately four hours). The reaction was worked-up by the
addition of hexane/ethyl acetate 50/50 (.about.200 mL). The organic
layer was repeatedly washed with water to remove the DMSO. The
organic layer was dried over sodium sulfate and the solvent removed
in vacuo to give the crude azide 4 (4.2 g, 10.4 mmol, 94%) which
was carried on without further purification.
Preparation of Compound 5
[0085] The crude azide 4 (4.2 g, 10.4 mmol) was dissolved in THF
(100 mL) in a 250 mL Parr flask. The catalyst (400 mg, 5% Pd on
carbon) was added wet under a stream of N.sub.2. The flask was
purged with N.sub.2 before introduction of H.sub.2 at 50 psi. The
reaction was hydrogenated at room temperature for eight hours. The
reaction was worked-up by filtration through Celite.RTM. to give
the C22-amine 5 (3.9 g, 10.3 mmol, 99%), which was of satisfactory
purity.
General Procedure for Acylation of Compound 5
[0086] The acylations were carried out via a similar procedure.
Compound 5 (.about.500 mg) was dissolved in THF (10 mL) and TEA (2
mL) was added. The flask was chilled in an ice water bath, and the
acylating agent (approximately two equivalents) was added dropwise.
The reaction was followed by TLC on silica gel (elution with 20/1
chloroform/methanol). The acylating agents used were
methanesulfonyl chloride, methyl chloroformate and
pyrolidinecarbonyl chloride. After the reactions were judged to be
complete, 20% TFA in water (.about.20 mL) was added to the flask.
If precipitation occurred, acetone was added until the sterol
redissolved. The acid solutions were stirred for approximately two
hours before being worked-up by extracting into 50/50 toluene/ethyl
acetate. The organic layer was washed with water and saturated
sodium bicarbonate, dried over sodium sulfate, and evaporated in
vacuo. This provided crude 3-oxo-22-sulfonamide (compound 6, 437
mg,1.1 mmole, 73%), carbamate (compound 7, 477 mg, 1.2 mmole, 81%)
and urea (compound 8, 544 mg, 1.3 mmole, 86%) derivatives of
satisfactory purity.
EXAMPLE 7
3-Aminosteroids Prepared from Acylated or Sulfonylated
22-Amines
Compounds F, G, H and I (FIG. 4) were prepared from acylated or
sulfonylated 22-amines.
Reductive Aminations of Compounds 6, 7 and 8
[0087] The reductive aminations were accomplished by similar
procedures. The sterol (500 mg for, compound 6.2 g for compounds 7
and 8) was dissolved in 2-propanol (25 mL). Ethylenediamine (1 mL)
was added to the flask. The 3-oxo sterol and ethylenediamine were
allowed to stir at room temperature for approximately four hours.
Sodium cyanoborohydride (250 mg for compound 6 and 100 mg for
compounds 7 and 8) was dissolved in 2-propanol (2 mL) and acetic
acid (1 mL). The sodium cyanoborohydride solution was added to the
reaction flask after evolution of gas had almost ceased
(approximately five minutes). In all cases the reaction was
complete before the first TLC was taken (less than five minutes).
The work-up was the same for all analogues, solution was made basic
(pH 10-11) by the addition of carbonate buffer. The aqueous layer
was repeatedly extracted with chloroform. The chloroform was
removed in vacuo and the crude 3-aminosteroid was dissolved in 10%
acetonitrile/water, acidified with TFA. The solubility of compound
I was very poor. The solutions were passed through a 45.mu. Gelman
vacu-cap filter. The 3-aminosteroid isomers were separated by
reverse phase chromatography on C18 (Dynamax, 300, 8 .mu.M, 21.6 mm
ID, 25 cm/L) using a gradient of acetonitrile in water with 0.1%
TFA throughout. The separation of the sulfonamide -(G) and -(F)
isomers was accomplished relatively easily, while only the -isomer
of the methyl carbamate (H) was isolated cleanly. For the urea
analogue the isomers were not effectively separated by
chromatography on C18. The urea derivatives were submitted for
biological testing as the mixed isomers (I).
Analytical for Compounds F, G, H, and I
[0088] Compound-F: .sup.1H NMR (400 MHZ, CD.sub.3OD): 0.74 (s, 3H),
0.89 (s, 3H), 1.04 (d, 3H, J=6.7 Hz), 2.75 (m, 1H), 2.94 (s, 3H),
3.1-3.3 (m, buried in solvent signal); MS (ES) [M+H].sup.+: 454;
Anal. Calcd. for C.sub.25H.sub.47N.sub.3O.sub.2S-2TFA-0.8H.sub.2O:
C 50.03%, H 7.33%, N 6.04%. Found: C 49.98%, H 7.10%, N 6.00%.
[0089] Compound-G: .sup.1H NMR (400 MHZ, CD.sub.3OD): 0.74 (s, 3H),
0.88 (s, 3H), 1.02 (d, 3H, J=6.7 Hz), 2.75 (d of d, 1H, J.sub.1=10
Hz, J.sub.2=3 Hz), 2.94 (s, 3H), 3.15 (d of d, J.sub.1=10 Hz,
J.sub.2=2 Hz), 3.48 (sharp m, 1H); MS (ES) [M+H].sup.+: 454; Anal.
Calcd. for C.sub.25H.sub.47N.sub.3O.sub.2S-2TFA-0.8H.sub.2O: C
50.03%, H 7.33%, N 6.04%. Found: C 50.23%, H 7.38%, N 6.05%.
[0090] Compound-H: .sup.1H NMR (400 MHZ, CD.sub.3OD): 0.74 (s, 3H),
0.90 (s, 3H), 0.97 (d, 3H, J=6.7 Hz), 2.75 (d of d, 1H, J.sub.1=10
Hz, J.sub.2=3 Hz), 3.20 (m, 2H), 3.66 (s, 3H); MS (FAB)
[M+H].sup.+: 434; Anal. Calcd. for
C.sub.26H.sub.47N.sub.3O.sub.2-2TFA-1.5H.sub.2O: C 52.32%, H 7.61%,
N 6.10%. Found: C 52.10%, H 7.31%, N 5.78%.
[0091] Compound-I: .sup.1H NMR (400 MHZ, CD.sub.3OD): 0.69 (s, 3H),
0.84 (s, 3H, 2 signals from the mixed isomers), 0.93 (d, 3H, J=6.7
Hz), 2.75 (m, 1H), 3.10-3.30 (m, buried in solvent peak), 3.45
(sharp m); MS (FAB) [M+H].sup.+: 474; Anal. Calcd. for
C.sub.29H.sub.52N.sub.4O.sub.1-2TFA-2H.sub.2O: C 53.79%, H 7.93%, N
7.60%. Found: C 53.91%, H 7.35%, N 7.74%.
EXAMPLE 8
3-Aminosteroid Analogues Prepared Via the 22 Aldehyde
[0092] ##STR21## Preparation of Compound 13
[0093] Compound 1 (5.0 g, 15.2 mmol) was dissolved in
dichloromethane (100 mL) and pyridinium chlorochromate (6.5 g, 30.0
mmol) was added to the reaction in one portion. After the reaction
was magnetically stirred at room temperature for approximately six
hours, the reaction was worked up by the gradual addition of a 5%
sodium bisulfite solution. The organic layer was repeatedly washed
with sodium bicarbonate solution and finally with brine. The
organic layer was dried over sodium sulfate and the solvent removed
in vacuo to yield compound 13 (4.3 g, 13.1 mmol, 86%) as an off
white solid.
Preparation of Compound 16
[0094] Tetraethyl methylenediphosphonate (1.44 g, 5 mmol) was added
to 10 mL anhydrous THF. Sodium t-butoxide (480 mg, 5 mmol) was
added to the flask in one portion at rt. The solution was stirred
for approximately thirty minutes to ensure complete formation of
the phosphonate carbanion. Compound 13 (1.60 g, 4.8 mmol) was
dissolved in minimal THF, and added to the reaction vessel dropwise
via an addition funnel over five minutes. The reaction was allowed
to stir for thirty minutes at room temperature. The reaction was
worked-up by the addition of 50/50 toluene/ethyl acetate (50 mL).
The organic layer was repeatedly washed with 0.1 M NaOH solution
and then brine. The organic layer was dried over sodium sulfate and
the solvent removed in vacuo to yield compound 16 (1.48 g, 3.2
mmol, 67%) as a white crystalline solid.
Preparation of Compound 17
[0095] Compound 16 (1.48 g, 3.2 mmol) was dissolved ethyl acetate
(50 mL) and added to Parr flask. The flask was purged with nitrogen
and 5% palladium on carbon (280 mg) was added to the flask. The
flask was evacuated and filled with H.sub.2 at 50 psi. The flask
was shaken at room temperature overnight (approximately fourteen
hours). The reaction was worked-up by filtering through a bed of
Celite-7.RTM. and thoroughly washing the filter cake with ethyl
acetate. The filtrate was evaporated in vacuo to give compound 17
as a white crystalline solid (1.41 g, 3.1 mmol, 97%).
Preparation of Compound 19
[0096] The phosphonate carbanion was prepared by the addition of
2-(diethylphosphono)-butyrolactone (25 g, 120 mmol) (prepared by
heating a neat mixture of 2-bromobutyrolactone and
triethylphosphite) to THF (1.2 L). Sodium tert-butoxide (11.5 g,
120 mmol) was added with ice bath cooling. The reaction was allowed
to warm to room temperature over thirty minutes to insure complete
formation of the phosphonate carbanion. Compound 18 (25.0 g, 76.2
mmol) was dissolved in THF (200 mL) and added to the reaction
mixture, which was then warmed to reflux for approximately sixteen
hours. The reaction was worked-up by removal of some THF
(.about.700 mL) in vacuo. Toluene (500 mL) was then added and the
solution washed repeatedly with 0.1 M NaOH solution and then brine.
The organic layer was dried over sodium sulfate and the solvent
removed in vacuo. The resulting solid was recrystallized from
hexane/ethyl acetate to yield the mixed E,Z-isomers 19 as an off
white solid (25.7 g, 64.5 mmol, 85%).
Preparation of Compound 15
[0097] Compound 19 (23.0 g, 57.8 mmol) was dissolved in THF/toluene
3/1 (.about.1 L). The solution was then added to ammonia (1.2 L) at
-78.degree. C. Lithium wire was added to the reaction until a deep
blue color persisted. The reaction was warmed to reflux for thirty
minutes, chilled back to -78.degree. C., and then quenched by the
addition of ammonium chloride. The ammonia was allowed to boil off
overnight. The residue was acidified by the addition of 1.0 M HCl
solution (500 mL) with aggressive stirring and an additional
portion of toluene (500 mL) was added. There was a significant
amount of insoluble material at the organic aqueous interface so
the material was filtered through Celite-7.RTM.. The filtrate was
added to a separatory funnel and the organic layer was washed with
repeatedly with 0.1 M HCl, followed by sodium bicarbonate solution
and brine. The organic layer was dried over sodium sulfate and the
solvent removed in vacuo to yield compound 15 as a mixture of
isomers at C23 (14.3 g, 36.2 mmol, 63%).
Reductive Aminations of Compounds 15 and 17
[0098] The 3-aminosteroid analogues prepared from compounds 15 and
17, compounds J, K, L and M are depicted in FIG. 5. The reactions
and isolations for these compounds were all virtually identical to
those described previously for the preparation of compounds F, G, H
and I. The only changes being the scale at which various
preparations were run and the polyamine which was used;
ethylenediamine was replaced with homopiperazine in one case
(Compound-M).
Analytical for Compounds J, K, L and M
[0099] Compound-J: .sup.1H NMR (400 MHZ, CD.sub.3OD): 0.74 (s, 3H),
0.90 (s, 3H), 1.04 (d, 3H, J=6.7 Hz), 2.45 (m, 1H), 2.64 (m, 1H),
3.17 (m, 1H) 3.68 (m, 1H), 4.21 (m, 1H), 4.30 (m, 1H); MS (FAB)
[M+H].sup.+: 445; Anal. Calcd. for
C.sub.28H.sub.48N.sub.2O.sub.2-2TFA-1.0H.sub.2O: C 55.64%, H 7.59%,
N 4.06%. Found: C 55.75%, H 7.41%, N 4.17%.
[0100] Compound-K: .sup.1H NMR (400 MHZ, DMSO-d.sub.6): 0.74 (s,
3H), 0.90 (s, 3H), 1.04 (d, 3H, J=6.7 Hz), 2.35 (m, 1H), 2.60 (m,
1H), 3.17 (sharp m, 4H), 4.12 (m, 1H), 4.26 (m, 1H); MS (FAB)
[M+H].sup.+: 445; Anal. Calcd. for
C.sub.28H.sub.48N.sub.2O.sub.2-2TFA-1.0H.sub.2O: C 55.64%, H 7.59%,
N 4.06%. Found: C 55.43%, H 7.63%, N 4.10%.
[0101] Compound-L: .sup.1H NMR (400 MHZ, DMSO-d.sub.6): 0.72 (s,
3H), 0.80 (s, 3H), 0.94 (d, 3H, J=6.7 Hz), 1.21 (t, 6H, J=6.7 Hz),
3.10-3.25 (m, 4H), 3.95 (d of q, 4H, J.sub.1=6.7 Hz, J.sub.2=2 Hz);
MS (FAB) [M+H].sup.+: 511; Anal. Calcd. for
C.sub.29H.sub.55N.sub.2O.sub.3P-2TFA-1.0H.sub.2O: C 52.37%, H
7.86%, N 3.70%. Found: C 52.58%, H 7.82%, N 3.53%.
[0102] Compound-M: .sup.1H NMR (400 MHZ, DMSO-d.sub.6): 0.72 (s,
3H), 0.79 (s, 3H, 2 signals from mixed diastereomers), 0.94 (d, 3H,
J=6.7 Hz, 2 signals), 2.31 (m, 1H), 2.55 (m, 1H), 3.15-3.72 (m,
8H), 4.10 (m, 1H), 4.26 (m, 1H); MS (FAB) [M+H].sup.+: 485; Anal.
Calcd. for C.sub.31H.sub.52N.sub.2O.sub.2-2TFA-2.0H.sub.2O: C
56.14%, H 7.81%, N 3.74%. Found: C 56.34%, H 7.14%, N 3.79%.
EXAMPLE 9
3-Aminosteroids Prepared Via Mitsunobu Reaction with Compound 1
[0103] ##STR22## Preparation of Compound 24
[0104] Compound 1 (5.0 g, 15.0 mmol) was dissolved in anhydrous THF
(75 mL), and was treated with 2-hydroxypyridine (1.7 g, 18.0 mmol)
and triphenylphosphine (4.7 g, 18.0 mmol). Diethylazodicarboxylate,
DEAD, (3.1 g, 18.0 mmol) was added to the flask dropwise via an
addition funnel. The addition of the DEAD caused an exothermic
reaction. The reaction was allowed to stir at room temperature for
thirty minutes before the solution was reduced in volume and
applied directly to a 6.times.10 cm silica gel column (elution with
20% ethyl acetate in toluene). The fractions containing pure
compound 24 were pooled and the solvent removed in vacuo to yield a
white crystalline solid (3.7 g, 9.1 mmol, 61%).
EXAMPLE 10
[0105] Preparation of Compounds N, O, and P
[0106] Compounds N, O, and P (FIG. 6) were prepared by the same
reductive amination procedure described previously.
Analytical for Compounds N, O and P
[0107] Compound-N: .sup.1H NMR (400 MHZ, CD.sub.3OD): 0.69 (s, 3H),
0.83 (s, 3H), 1.05 (d, 3H, J=6.7 Hz), 3.14 (m, 4H), 3.96 (d of d,
1H, J.sub.1=10 Hz, J.sub.2=2 Hz), 4.24 (d of d, 1H, J.sub.1=10 Hz,
J.sub.2=3 Hz), 6.70 (m, 1H), 6.86 (m, 1H), 7.60 (m, 1H), 8.14 (m,
1H); MS(+FAB): [M+H].sup.+454; Anal. Calcd. for
C.sub.29H.sub.47N.sub.3O-2TFA-3H.sub.2O: C 53.87%, H 7.53%, N
5.71%. Found: C 53.72%, H 6.61%, N 5.85%.
[0108] Compound-0: .sup.1H NMR (400 MHZ, DMSO-d.sub.6): 0.70 (s,
3H), 0.81 (s, 3H), 1.07 (d, 3H, J=6.7 Hz), 3.15 (m, 4H), 3.94 (d of
d, 1H, J.sub.1=10 Hz, J.sub.2=2 Hz), 4.25 (d of d, 1H, J.sub.1=10
Hz, J.sub.2=3 Hz), 6.70 (m, 1H), 6.85 (m, 1H), 7.60 (m, 1H), 8.14
(m, 1H); MS(+FAB): [M+H].sup.+454; Anal. Calcd. for
C.sub.29H.sub.47N.sub.3O-2TFA-3H.sub.2O: C 55.22%, H 7.44%, N
5.85%. Found: C 55.12%, H 6.74%, N 5.95%.
[0109] Compound-P: .sup.1H NMR (400 MHZ, DMSO-d.sub.6): 0.70 (s,
3H), 0.81 (s, 3H), 1.14 (d, 3H, J=6.7 Hz), 2.90 (m, 4H), 4.04 (d of
d, 1H, J.sub.1=10 Hz, J.sub.2=2 Hz), 4.25 (d of d, 1H, J.sub.1=10
Hz, J.sub.2=3 Hz), 6.70 (m, 1H), 6.85 (m, 1H), 7.60 (m, 1H), 8.14
(m, 1H); MS(+FAB): [M+H].sup.+480; Anal. Calcd. for
C.sub.29H.sub.47N.sub.3O-3TFA-1H.sub.2O: C 52.92%, H 6.48%, N
5.00%. Found: C 53.18%, H 5.88%, N 4.22%.
EXAMPLE 11
Preparation of 24-Amide
[0110] ##STR23## Preparation of Compound 29
[0111] Compound 28 (1.0 g, 2.6 mmol) was dissolved in methanol (50
mL). The solution was chilled to 0.degree. C. and ammonia was
bubbled into the reaction vessel for thirty minutes. The reaction
was sealed and allowed to stir at room temperature for two weeks.
The reaction was worked-up by chilling the reaction to -20.degree.
C., opening the sealed tube, and then allowing the reaction to warm
to room temperature. After the excess ammonia had evaporated, the
remainder of the methanolic ammonia was removed in vacuo to yield
compound 29 (0.95 g, 2.5 mmol, 96%).
Preparation of Compound-O (FIG. 7)
[0112] The 3-aminosteroid analogue of compound 29 was prepared by
same methods described earlier for the preparation of the other
3-aminosteroid analogues.
[0113] Compound-Q: .sup.1H NMR (400 MHZ, CD.sub.3OD): 0.74 (s, 3H),
0.93 (s, 3H), 0.98 (d, 3H, J=6.7 Hz), other downfield signals
buried in the solvent peak; MS (+FAB): [M+H].sup.+418; Anal. Calcd.
for C.sub.29H.sub.47N.sub.3O-2TFA-0.7H.sub.2O: C 54.73%, H 7.72%, N
6.38%. Found: C 54.70%, H 7.51%, N 6.18%.
[0114] While the invention has been described and illustrated
herein by references to various specific materials, procedures and
examples, it is understood that the invention is not restricted to
the particular material combinations of material and procedures
selected for that purpose. Numerous variations of such details can
be implied as will be appreciated by those skilled in the art.
[0115] Other embodiments of the invention described above and will
be apparent to those skilled in the art from consideration of the
specification and practice of the invention disclosed within. All
cited patents and publications referred to in this application are
herein incorporated by reference in their entirety. It is intended
that the specification and examples considered as exemplary only,
with true scope and spirit of the invention being indicated by the
following
* * * * *