U.S. patent application number 10/083803 was filed with the patent office on 2002-07-18 for screening methods for presqualene diphosphate analogs.
Invention is credited to Levy, Bruce D., Serhan, Charles N..
Application Number | 20020094549 10/083803 |
Document ID | / |
Family ID | 25263032 |
Filed Date | 2002-07-18 |
United States Patent
Application |
20020094549 |
Kind Code |
A1 |
Serhan, Charles N. ; et
al. |
July 18, 2002 |
Screening methods for presqualene diphosphate analogs
Abstract
The present invention is directed to presqualene diphosphate
(PSDP) analogs having an active region of natural PSDP and a
metabolic transformation region resistant to rapid intracellular
inactivation in vivo. For example, PSDP and its stable analogs can
inhibit neutrophil signal transduction events in cellular
activation that result in the generation of active oxygen species,
regulation of leukocyte adherence, both homotypic
(leukocyte-leukocyte) or heterotypic adherence with leukocytes and
epithelial cells of mucosal origin or endothelial cells of vascular
origin. These analogs can also be used to regulate
leukocyte-dependent tissue injury.
Inventors: |
Serhan, Charles N.;
(Needham, MA) ; Levy, Bruce D.; (West Roxbury,
MA) |
Correspondence
Address: |
Scott D. Rothenberger
DORSEY & WHITNEY LLP
Suite 1500
50 South Sixth Street
Minneapolis
MN
55402-1498
US
|
Family ID: |
25263032 |
Appl. No.: |
10/083803 |
Filed: |
February 27, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10083803 |
Feb 27, 2002 |
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09793005 |
Feb 26, 2001 |
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09793005 |
Feb 26, 2001 |
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09539591 |
Mar 31, 2000 |
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6251622 |
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09539591 |
Mar 31, 2000 |
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09055592 |
Apr 6, 1998 |
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6066466 |
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09055592 |
Apr 6, 1998 |
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08832952 |
Apr 4, 1997 |
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6008205 |
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Current U.S.
Class: |
435/19 ;
435/25 |
Current CPC
Class: |
C12Q 1/34 20130101; C07F
9/117 20130101; G01N 33/68 20130101; G01N 33/5047 20130101; G01N
2333/916 20130101 |
Class at
Publication: |
435/19 ;
435/25 |
International
Class: |
C12Q 001/44; C12Q
001/26 |
Claims
What is claimed is:
1. A method of screening for a compound which modulates
phospholipase D (PLD) activity, comprising the steps of: a)
combining a compound with phospholipase D (PLD), thereby forming a
mixture; b) treating said mixture with phosphatidylcholine, such
that a cleavage reaction between phosphatidylcholine and PLD can
occur, resulting in generation of phosphatidic acid and choline ;
and c) evaluating the amount of choline produced, such that a
compound which modulates PLD activity is determined.
2. The method of claim 1, wherein said compound inhibits PLD from
cleaving phosphatidylcholine.
3. The method of claim 1, wherein said compound increases cleavage
of phosphatidylcholine by PLD.
4. The method of claim 1, wherein step c) includes an oxidation
step of choline.
5. The method of claim 4, wherein said oxidation step includes
peroxidase, 4-aminoantipyrine, phenol and choline oxidase.
6. The method of claim 1, wherein said compound is PSDP.
7. The method of claim 1, wherein said compound is a PSDP
analog.
8. A method of screening for a compound which modulates
intracellular signaling, comprising the steps of: a) combining a
compound with phospholipase D (PLD), thereby forming a mixture; b)
treating said mixture with phosphatidylcholine, such that a
cleavage reaction between phosphatidylcholine and PLD occurs,
resulting in generation of phosphatidic acid and choline; and c)
evaluating the amount of choline produced, such that a compound
which modulates intracellular signaling is determined.
9. The method of claim 8, wherein said compound inhibits PLD from
cleaving phosphatidylcholine.
10. The method of claim 8, wherein said compound increases cleavage
of phosphatidylcholine by PLD.
11. The method of claim 8, wherein step c) includes an oxidation
step of choline.
12. The method of claim 11, wherein said oxidation step includes
peroxidase, 4-aminoantipyrine, phenol and choline oxidase.
13. The method of claim 8, wherein said compound is PSDP.
14. The method of claim 8, wherein said compound is a PSDP
analog.
15. A method of screening for a compound which associates with
protein phosphate-sensing domains, comprising the steps of: a)
contacting a Gst-Grb2 fusion protein complexed to a support with a
labeled lipid compound with; and b) evaluating the amount of
labeled compound associated with said protein.
16. The method of claim 15, further comprising the step of treating
said labeled lipid compound associated with a competing compound
and evaluating the amount of labeled compound removed from said
Gst-Grb2 fusion protein.
17. The method of claim 15, wherein said support is agarose.
18. The method of claim 15, wherein said labeled lipid compound is
radiolabeled.
19. The method of claim 18, wherein said radiolabeled compound is
radiolabeled PSDP or a radiolabeled PSDP analog.
20. The method of claim 16, wherein said competing compound is
unlabeled PSDP, a PSDP analog, or delipidated albumin.
21. A method of screening for a compound which modulates the
production of inositol trisphosphate, comprising the steps of: a)
treating polymorphonuclear leukocytes with a stimulation agent,
causing activation of cell, thereby producing inositol
trisphosphate; b) treating said activated cells with a modulating
compound; and c) measuring the effect of said modulating compound
on production of said inositol trisphosphate.
22. The method of claim 21, wherein said stimulation agent is FMLP,
a leukotriene, a cytokine, gm-csf, a lip opolysaccharid or CFA.
23. The method of claim 21, wherein said modulating compound is
PSDP.
24. The method of claim 21, wherein said modulating compound is a
PSDP analog.
25. The method of claim 21, further including the step of
contacting a radiolabel to said inositol trisphosphate of step
c).
26. The method of claim 21, wherein production of phospholipase C
activity is determined.
27. A method of screening for a compound which modulates neutrophil
activation, comprising the steps of: a) treating neutrophils with a
stimulation agent, causing activation of said neutrophils, thereby
producing inositol phosphate; b) treating said activated
neutrophius with a modulating compound; and c) measuring the effect
of said modulating compound on production of said inositol
phosphate.
28. The method of claim 27, wherein said stimulation agent is FMLP,
a leukotriene, a cytokine, gm-csf, a lipopolysaccharid or CFA.
29. The method of claim 27, wherein said modulating compound is
PSDP.
30. The method of claim 27, wherein said modulating compound is a
PSDP analog.
31. The method of claim 27, further including the step of
contacting a radiolabel to said inositol phosphate of step c).
32. The method of claim 27, wherein production of phospholipase C
activity is determined.
Description
RELATED APPLICATION
[0001] This application is a continuation-in-part of U.S. Ser. No.
08/832,952, entitled "Novel Polyisoprenyl Phosphate Stable Analogs
For Regulation of Neutrophil Responses", filed on Apr. 4, 1997, the
contents of which are hereby expressly incorporated by
reference.
BACKGROUND OF THE INVENTION
[0002] The environment contains a variety of infectious microbial
agents, such as viruses, bacteria, fungi and parasites, any one of
which can cause pathological damage to the host organism.
Consequently, most organisms, such as mammals, i.e. humans, have
developed an immune system. The immune system is divided into two
functional divisions, the innate immune system and the adaptive
immune system.
[0003] The innate and adaptive immune systems consists of a variety
of molecules and cells distributed throughout the body. The most
important cells are leukocytes. Leukocytes are categorized as
phagocytes, including polymorphonuclear neutrophils (PMNs),
monocytes and macrophages, and lymphocytes, which mediate adaptive
immunity.
[0004] Inflammation is the body's response to invasion or an
injury, such as an invasion by an infectious microbial agent and
includes three broad actions. First, the blood supply is increased
to the area. Second, capillary permeability is increased, thereby
permitting larger molecules to reach the site of infection. Third,
leukocytes, particularly PMNs, migrate out of the capillaries and
into the surrounding tissue. Once in the tissue, the PMNs migrate
to the site of infection or injury by chemotaxis. These events
manifest themselves as inflammation. Examples of conditions which
cause these reactions to occur include clamping or tourniquet
vessel-induced ischemia reperfusion injury, chronic inflammatory
conditions such as asthma, rheumatoid arthritis, and inflammatory
bowel disease, as well as autoimmune diseases.
[0005] Aberrant activation of phagocytic cells, in particular
neutrophils, leads to the generation of superoxide anion, which
when released to the extracellular milieu can evoke damage to
surrounding tissues. Reactive oxygen species derived from
neutrophil oxygen burst can play a deleterious role in generating
secondary products that lead to loss of function. During surgery,
in particular clamping of vessels, there is clear evidence that
reperfusion following the release of the clamp involves
neutrophil-derived mediators. The neutrophil-derived oxygen
radicals and other toxic products that are normally intended for
killing of microbial agents once they spill over into the
surrounding tissue can lead to second organ injury, most notably in
the lung and cardiac tissues, sequelae which are observed following
ischemia reperfusion injury (Welbourn et al., Brit. J. Surg. 1991;
78:651-655).
[0006] Once at the site of infection, PMNs perform phagocytic and
degradative functions to combat the infectious agent. As part of
the response to the infectious agent, PMNs generate superoxide
anions, reactive oxygen species (ROS) to kill infested material and
adhere to epithelial cells of mucosal surfaces or vascular
endothelial cells of the blood vessels. As a consequence, the host
can experience undesirable side effects during the elimination of
the infectious agent such as, pain, swelling about the site, and
nausea.
SUMMARY OF THE INVENTION
[0007] The present invention relates to novel presqualene
diphosphate (PSDP) analogs and their use.
[0008] In one embodiment, the present invention is directed to a
PSDP analog having an active region of natural PSDP and a metabolic
transformation region resistant to in vivo metabolism. For example,
the analog can inhibit leukocyte activation, leukocyte generation
of active oxygen species, adhesion between a leukocyte cell and an
epithelial cell or an endothelial cell or leukocyte generation of
reactive oxygen species (ROS).
[0009] In another embodiment, the present invention is directed to
a method for treating or preventing inflammation and/or an
inflammatory response in the subject. The method includes
administering to a subject an anti-inflammatory amount of a PSDP
analog having an active region of natural PSDP and a metabolic
transformation region resistant to in vivo metabolism.
[0010] In yet another embodiment, the present invention is directed
to a compound represented by one of the formulae (Formulae I-IV):
1
[0011] R.sub.1, R.sub.2 and R.sub.3 are each independently,
selected from the group consisting of hydrogen, F, Cl, Br, I,
CH.sub.3 and substituted or unsubstituted, linear or branched
alkyl, alkoxy, aryl, aralkyl or heteroaryl groups. Y.sub.1,
Y.sub.2, Y.sub.3, Y.sub.4, and Y.sub.5 are each independently
selected from hydrogen atoms or lower alkyl groups. X.sub.1 is an
oxygen atom, a sulfur atom, an N.dbd.N group, a methylene or,
NR.sub.5, wherein R.sub.5 is a hydrogen atom or a substituted or
unsubstituted, linear or branched alkyl, aryl, aralkyl or
heteroaryl group. X.sub.2 is an OH group, SH, CH.sub.3, or
NR.sub.6R.sub.7, wherein R.sub.6 and R.sub.7 are each
independently, a hydrogen atom or a substituted or unsubstituted,
linear or branched alkyl, aryl, aralkyl or heteroaryl group.
A.sub.1, A.sub.2, A.sub.3, and A.sub.4 are each independently, a
substituted or unsubstituted aromatic or nonaromatic carbocyclic or
heterocyclic group. Preferably, carbon-carbon bonds are not formed
between one or more of C.sub.1 and C.sub.4, C.sub.2 and C.sub.5,
and C.sub.3 and C.sub.6 carbon atoms. Salts of Formulae I-IV are
also included in the present invention.
[0012] In yet another embodiment, the present invention is directed
to a pharmaceutical composition comprising an effective amount of a
compound represented by one or more of the formulae (Formulae
I-IV): 2
[0013] R.sub.1, R.sub.2 and R.sub.3 are each independently,
selected from the group consisting of hydrogen, F, Cl, Br, I,
CH.sub.3 and substituted or unsubstituted, linear or branched
alkyl, alkoxy, aryl, aralkyl or heteroaryl groups. Y.sub.1,
Y.sub.2, Y.sub.3, Y.sub.4, and Y.sub.5 are each independently
selected from hydrogen atoms or lower alkyl groups. X.sub.1 is an
oxygen atom, a sulfur atom, an N.dbd.N group, a methylene or,
NR.sub.5, wherein R.sub.5 is a hydrogen atom or a substituted or
unsubstituted, linear or branched alkyl, aryl, aralkyl or
heteroaryl group. X.sub.2 is an OH group, SH, CH.sub.3, or
NR.sub.6R.sub.7, wherein R.sub.6 and R.sub.7 are each
independently, a hydrogen atom or a substituted or unsubstituted,
linear or branched alkyl, aryl, aralkyl or heteroaryl group.
A.sub.1, A.sub.2, A.sub.3, and A.sub.4 are each independently, a
substituted or unsubstituted aromatic or nonaromatic carbocyclic or
heterocyclic group. Preferably, carbon-carbon bonds are not formed
between one or more of C.sub.1 and C.sub.4, C.sub.2 and C.sub.5,
and C.sub.3 and C.sub.6 carbon atoms. The present invention also
includes pharmaceutically acceptable salts of Formulae I-IV.
[0014] In still another embodiment, the invention is directed to a
method for treating or preventing inflammation and/or an
inflammatory response in the subject, comprising: administering to
a subject an anti-inflammatory amount of more or more compounds
having the formulae (Formulae I-IV): 3
[0015] R.sub.1, R.sub.2 and R.sub.3 are each independently,
selected from the group consisting of hydrogen, F, Cl, Br, I,
CH.sub.3 and substituted or unsubstituted, linear or branched
alkyl, alkoxy, aryl, aralkyl or heteroaryl groups. Y.sub.1,
Y.sub.2, Y.sub.3, Y.sub.4, and Y.sub.5 are each independently
selected from hydrogen atoms or lower alkyl groups. X.sub.1 is an
oxygen atom, a sulfur atom, an N.dbd.N group, a methylene or,
NR.sub.5, wherein R.sub.5 is a hydrogen atom or a substituted or
unsubstituted, linear or branched alkyl, aryl, aralkyl or
heteroaryl group. X.sub.2 is an OH group, SH, CH.sub.3, or
NR.sub.6R.sub.7, wherein R.sub.6 and R.sub.7 are each
independently, a hydrogen atom or a substituted or unsubstituted,
linear or branched alkyl, aryl, aralkyl or heteroaryl group.
A.sub.1, A.sub.2, A.sub.3, and A.sub.4 are each independently, a
substituted or unsubstituted aromatic or nonaromatic carbocyclic or
heterocyclic group. Preferably, carbon-carbon bonds are not formed
between one or more of C.sub.1 and C.sub.4, C.sub.2 and C.sub.5,
and C.sub.3 and C.sub.6 carbon atoms. The present invention also
includes pharmaceutically acceptable salts of Formulae I-IV.
[0016] In still another embodiment, the present invention is
directed to a method of screening for a compound which modulates
phospholipase D (PLD) activity. The method includes combining the
compound with phospholipase D (PLD), thereby forming a mixture and
treating the mixture with phosphatidylcholine, such that a cleavage
reaction between phosphatidylcholine and PLD can occur, resulting
in generation of phosphatidic acid and choline. The amount of
choline produced is evaluated such that a compound which modulates
PLD activity is determined. In a preferred embodiment, the compound
increases cleavage of phosphatidylcholine by PLD.
[0017] In still yet another embodiment, the present invention is
directed to a method of screening for a compound which modulates
intracellular signaling. The method includes combining a compound
with phospholipase D (PLD), thereby forming a mixture and treating
the mixture with phosphatidylcholine, such that a cleavage reaction
between phosphatidylcholine and PLD occurs, resulting in generation
of phosphatidic acid and choline. The amount of choline produced is
evaluated, such that a compound which modulates intracellular
signaling is determined.
[0018] In another embodiment, the invention is directed to a method
of screening for a compound which associates with protein
phosphate-sensing domains. The method includes contacting a
Gst-Grb2 fusion protein complexed to a support with a labeled lipid
compound with and evaluating the amount of labeled compound
associated with the protein. The method can further include
treating the labeled lipid compound associated with a competing
compound and evaluating the amount of labeled compound removed from
the Gst-Grb2 fusion protein.
[0019] In still another embodiment, the invention is directed to a
method of screening for a compound which modulates the production
of inositol triphosphate. The method includes treating
polymorphoneutrophils with a stimulation agent, causing activation
of cell, thereby producing inositol phosphate and treating the
activated cells with a modulating compound. The effect of the
modulating compound is measured on production of the inositol
phosphate. A preferred stimulation agent is fMLP. A preferred
modulating compound is PSDP, e.g., an inhibititory compound.
[0020] In yet another embodiment, the invention is directed to a
method of screening for a compound which modulates neutrophil
activation. The method includes treating neutrophils with a
stimulation agent, causing activation of the neutrophils, thereby
producing inositol phosphate and treating the activated neutrophils
with a modulating compound. The effect of the modulating compound
is measured on production of the inositol phosphate. A preferred
stimulation agent is fMLP. A preferred modulating compound is PSDP,
e.g., an inhibititory compound.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1a depicts superoxide anion generation in the presence
of neutrophil lipid extracts.
[0022] FIG. 1b is a representative time course for neutrophil
Fraction B's inhibition of phosphatitic acid-triggered
O.sub.2.sup.- production.
[0023] FIG. 1c shows Fraction B phosphorus content and bioactivity
in sequential TLC segments.
[0024] FIG. 1d is a representative densitometric analysis of
Fraction B nonsaponifiable phosphorus containing lipids
[0025] FIG. 1 e is a comparison of compound V-VIII's Rf value with
available standards.
[0026] FIG. 1f shows a representative time course for Neutrophil
Fraction B's inhibition of arachiodonic acid-stimulated superoxide
anion generation.
[0027] FIG. 1g shows the ratio of mass determination by phosphorus
quantitation compared to scanning densitometry.
[0028] FIG. 2a is a mass spectrum of compound VI, consistent with
presqualene diphosphate.
[0029] FIG. 2b is a limited ion chromatogram for m/z 136 (i.e., 2
internal isoprene units) after direct injection.
[0030] FIG. 2c shows acid-treated compound VI, having shifted to
8.70 min and the resultant mass spectrum now consistent with
squalene.
[0031] FIG. 2d shows preliminary GC/MS analysis of compound VI.
[0032] FIG. 2e shows the physical chemical properties of compounds
V-VIII.
[0033] FIG. 3a is a representative phosphoimager profile of
.sup.32P-content after TLC of resting neutrophil extracts from
cells incubated with .gamma.-.sup.32PO.sub.4-ATP and (inset), time
course for FMLP-stimulated changes in phosphorus content of
compounds VI and VIII.
[0034] FIG. 3b depicts the impact of GM-CSF on .sup.32PO.sub.4
incorporation into compounds VI and VIII.
[0035] FIG. 3c depicts superoxide anion generation by
electroporated neutrophils.
[0036] FIG. 4a is a graph of FMLP-stimulated superoxide anion
generation with electroporated neutrophils in the presence of
compounds VI and VIII.
[0037] FIG. 4b is a graph of PMA-stimulated superoxide anion
generation with electroporated neutrophils in the presence of
compound VI and related isoprenoids and dose response (b inset) of
compound VI's inhibition of O.sub.2.sup.-- production.
[0038] FIG. 4c is a representative tracing of neutrophil homotypic
adhesion.
[0039] FIG. 4d shows the % inhibition of FMLP-stimulated homotypic
adhesion.
[0040] FIG. 4e, shows the % inhibition of FMLP-stimulated IP.sub.3
formation.
[0041] FIG. 4f, is a proposed scheme for polyisoprenyl phosphate
regulation of neutrophil responses.
[0042] FIG. 5 shows the inhibition of phospholipase D by PSDP at
several concentrations.
[0043] FIG. 6 shows the inhibition of fMLP-stimulated [Ca.sup.+2]
mobilization.
[0044] FIG. 7 shows the concentration dependence of inhibition of
fMLP-stimulated [Ca.sup.+2] mobilization.
[0045] FIG. 8 demonstrates that presqualene diphosphate associates
with Grb2 SH2 domains.
DETAILED DESCRIPTION OF THE INVENTION
[0046] The features and other details of the invention will now be
more particularly described and pointed out in the claims. It will
be understood that the particular embodiments of the invention are
shown by way of illustration and not as limitations of the
invention. The principle features of this invention can be employed
in various embodiments without departing from the scope of the
invention.
[0047] As used herein, the following phrases and terms are defined
as follows:
[0048] The term "precursor" is intended to refer to chemical
intermediates that can be converted in vivo, ex vivo and/or in
vitro to form the PSDP analogs of the invention. The term
"precursor" also contemplates prodrugs which are converted in vivo
to the analogs of the invention (see, e.g., R. B. Silverman, 1992,
"The Organic Chemistry of Drug Design and Drug Action", Academic
Press, Chp. 8). Examples of such prodrugs include, but are not
limited to esters of hydroxyls and/or carboxyl groups and/or
compounds which can be hydrolyzed or otherwise converted in vivo
or, ex vivo and/or in vitro into the analogs of the present
invention.
[0049] The term "epithelial cells" includes epithelial cells of
mucosal surfaces and/or endothelial cells of vascular origin.
[0050] The term "active region" shall mean the region of a natural
PSDP or PSDP analog, which is associated with in vivo cellular
interactions, e.g. inflammation. The active region may bind the
"recognition site" of a cellular PSDP receptor or a macromolecule
or complex of macromolecules, including an enzyme and its cofactor.
Preferred PSDP analogs have an active region comprising the
diphosphate moieties of natural PSDP.
[0051] The term "recognition site" or receptor is art-recognized
and is intended to refer generally to a functional macromolecule or
complex of macromolecules with which certain groups of cellular
messengers, such as hormones, leukotrienes, PMNs and PSDP
derivatives, must first interact before the biochemical and
physiological responses to those messengers are initiated. As used
in this application, a receptor may be isolated, on an intact or
permeabilized cell, or in tissue, including an organ. A receptor
may be from or in a living subject, or it may be cloned. A receptor
may normally exist or it may be induced by a disease state, by an
injury, or by artificial means. A compound of this invention may
bind reversibly, irreversibly, competitively, noncompetitively, or
uncompetitively with respect to the natural substrate of a
recognition site.
[0052] The term "detectable label molecule" is meant to include
fluorescent, phosphorescent, radiolabeled molecules, and other such
labels as are conventional in the art used to trace, track, or
identify the compound or receptor recognition site to which the
detectable label molecule is bound. The label molecule may be
detected by any of the several methods known in the art.
[0053] The term PSDP analog refers to analogs and derivatives of
natural PSDP including structural or functional analogs which have
the same or greater activity in vivo or in vitro as PSDP. Suitable
examples of PSDP analogs include Formulae I-IV, supra.
[0054] The term "labeled PSDP analog" is further understood to
encompass compounds which are labeled with radioactive isotopes,
such as but not limited to tritium (.sup.3H), deuterium (.sup.2H),
carbon (.sup.14C), (.sup.31P) or otherwise labeled (e.g.
fluorescently). The compounds of this invention may be labeled or
derivatized, for example, for kinetic binding experiments, for
further elucidating metabolic pathways and enzymatic mechanisms, or
for characterization by methods known in the art of analytical
chemistry.
[0055] The term "inhibits" means the blocking or reduction of
activity of a leukocyte, leukocyte generation of active oxygen
species, or adhesion between a leukocyte cell and endothelial cell
or an epithelial cell. The blockage or reduction can occur by
covalent bonding, by irreversible binding, by reversible binding,
e.g. which can have the practical effect of irreversible binding,
or by any other means which prevents the leukocyte from operating
in its usual manner.
[0056] The term "tissue" is intended to include intact cells,
blood, blood preparations such as plasma and serum, bones, joints,
muscles, smooth muscles, and organs, both in vivo and in vitro.
[0057] The term "halogen" is meant to include fluorine, chlorine,
bromine and iodine, or fluoro, chloro, bromo, and iodo.
[0058] The term "pharmaceutically acceptable salt" is intended to
include art-recognized pharmaceutically acceptable salts. These
non-toxic salts are usually hydrolyzed under physiological
conditions, and include organic and inorganic bases. Examples of
suitable salts include sodium, potassium, calcium, ammonium,
copper, and aluminum as well as primary, secondary, and tertiary
amines, basic ion exchange resins, purines, piperazine, and the
like. The term is further intended to include esters of lower
hydrocarbon groups, such as methyl, ethyl, and propyl.
[0059] The term "pharmaceutical composition" comprises one or more
PSDP analogs as active ingredient(s), or a pharmaceutically
acceptable salt(s) thereof, and may also contain a pharmaceutically
acceptable carrier and optionally other therapeutic ingredients.
The compositions include compositions suitable for oral, rectal,
ophthalmic, pulmonary, nasal, dermal, topical, parenteral
(including subcutaneous, intramuscular and intravenous) or
inhalation administration. The most suitable route in any
particular case will depend on the nature and severity of the
conditions being treated and the nature of the active
ingredient(s). The compositions may be presented in unit dosage
form and prepared by any of the methods well-known in the art of
pharmacy. Dosage regimes may be adjusted for the purpose to
improving the therapeutic response. For example, several divided
dosages may be administered daily or the dose may be proportionally
reduced over time. A person skilled in the art normally may
determine the effective dosage amount and the appropriate regime. A
PSDP analog pharmaceutic composition can also refer to a
combination comprising PSDPs,-PSt)P analogs, and/or PSDP
metabolites, including metabolites of PSDP analogs. A nonlimiting
example of a combination is a mixture comprising a PSDP analog x
which inhibits one enzyme which metabolizes PSDPs and which
optionally has specific activity with a PSDP receptor recognition
site, and a second PSDP analogy which has specific activity with a
PSDP receptor recognition site and which optionally inhibits or
resists PSDP metabolism. This combination results in a longer
tissue half-life for at least y since x inhibits one of the enzymes
which metabolize PSDPs. Thus, the PSDP action mediated or
antagonized by y is enhanced.
[0060] The term "subject" is intended to include living organisms
susceptible to conditions or diseases caused or contributed to by
inflammation, inflammatory responses, vasoconstriction, myeloid
suppression and/or undesired cell proliferation. Examples of
subjects include warm blooded aminals, more preferably mammals such
as humans, dogs, cats, cows, goats, and mice. The term subject is
further intended to include transgenic species.
[0061] The term "ameliorate" is intended to include treatment for,
prevention of, limiting of and/or inhibition of undesired leukocyte
activation, leukocyte generation of oxygen active species,
leukocyte generation of ROS and/or adhesion between a leukocyte
cell and a epithelial cell or endothelial cells.
[0062] Active compounds are administered at a therapeutically
effective dosage sufficient to inhibit leukocyte mediated
responses, such as, leukocyte activation, leukocyte generation of
oxygen species, leukocyte generation of ROS and/or adhesion between
a leukocyte cell and an epithelial cell or endothelial cells in a
subject. A "therapeutically effective dosage" preferably reduces
the degree of leukocyte mediated responses in the subject by at
least about 20%, more preferably by at least about 40%, even more
preferably by at least about 60%, and still more preferably by at
least about 80% relative to untreated subjects. The ability of a
compound to inhibit or ameliorate leukocyte mediated responses can
be evaluated in an animal model system that may be predictive of
efficacy in treating said responses.
[0063] The term "alkyl" refers to the radical of saturated
aliphatic groups, including straight-chain alkyl groups,
branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl
substituted cycloalkyl groups, and cycloalkyl substituted alkyl
groups. In preferred embodiments, a straight chain or branched
chain alkyt has 30 or fewer carbon atoms in its backbone (e.g.,
C.sub.1-C.sub.30 for straight chain, C.sub.3-C.sub.30 for branched
chain), and more preferably 20 or fewer. Likewise, preferred
cycloalkyls have from 4-10 carbon atoms in their ring structure,
and more preferably have 5, 6 or 7 carbons in the ring
structure.
[0064] Moreover, the term alkyl as used throughout the
specification and claims is intended to include both "unsubstituted
alkyls" and "substituted alkyls", the latter of which refers to
alkyl moieties having substituents replacing a hydrogen on one or
more carbons of the hydrocarbon backbone. Such substituents can
include, for example, halogen, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxy alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phQsphinato,
cyano, amino (including alkyl amino, dialkylamino, arylamino,
diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, aralkyl, or an
aromatic or heteroaromatic moiety. It will be understood by those
skilled in the art that the moieties substituted on the hydrocarbon
chain can themselves be substituted, if appropriate. Cycloalkyls
can be further substituted, e.g., with the substituents described
above. An "aralkyl" moiety is an alkyl substituted with an aryl
(e.g., phenylmethyl (benzyl)).
[0065] The term "aryl" as used herein includes 5- and 6-membered
single-ring aromatic groups that may include from zero to four
heteroatoms, for example, benzene, pyrrole, furan, thiophene,
imidazole, oxazole, thiazole, triazole, pyrazole, pyridine,
pyrazine, pyridazine and pyrimidine, and the like. Aryl groups also
include polycyclic fused aromatic groups such as naphthyl,
quinolyl, indolyl, and the like. Those aryl groups having
heteroatoms in the ring structure may also be referred to as "aryl
heterocycles", "heteroaryls" or "heteroaromatics". The aromatic
ring can be substituted at one or more ring positions with such
substituents as described above, as for example, halogen, hydroxyl,
alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,
phosphinato, cyano, amino (including alkyl amino, dialkylamino,
arylamino, diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, sulfona to, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, aralkyl, or an
aromatic or heteroaromatic moiety. Aryl groups can also be fused or
bridged with alicyclic or heterocyclic rings which are not aromatic
so as to form a polycycle (e.g., tetralin).
[0066] Unless the number of carbons is otherwise specified, "lower
alkyl" as used herein means an alkyl group, as defined above, but
having from one to ten carbons, more preferably from one to six
carbon atoms in its backbone structure. Preferred alkyl groups are
lower alkyls.
[0067] The terms "heterocyclyl" or "heterocyclic group" refer to 3-
to 10-membered ring structures, more preferably 4- to 7-membered
rings, which ring structures include one to four heteroatoms.
Heterocyclyl groups include pyrrolidine, oxolane, thiolane,
oxazole, piperidine, piperazine, morpholine, lactones, lactams such
as azetidinones and pyrrolidinones, lactones, sultams, sultones,
and the like. The heterocyclic ring can be substituted at one or
more positions with such substituents as described above, as for
example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl,
phosphate, phosphonato, phosphinato, cyano, amino (including alkyl
amino, dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl,
sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,
aralkyl, or an aromatic or heteroaromatic moiety. A beteroalkyl
moiety is an alkyl substituted with a heteroaromatic group.
[0068] The terms "polycyclyl" or "polycyclic group" refer to two or
more cyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls,
aryls and/or heterocyclyls) in which two or more carbons are common
to two adjoining rings, e.g., the rings are "fused rings". Rings
that are joined through non-adjacent atoms are tenned "bridged"
rings. Each of the rings of the polycycle can be substituted with
such substituents as described above, as for example, halogen,
hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,
phosphinato, cyano, amino (including alkyl amino, dialkylamino,
arylamino, diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkyl, aralkyl, or an
aromatic or heteroaromatic moiety.
[0069] The term "heteroatom" as used herein means an atom of any
element other than carbon or hydrogen. Preferred heteroatoms are
nitrogen, oxygen, sulfur and phosphorus.
[0070] The term "substantially pure," as used herein, refers to a
compound which is substantially free of impurities, including (but
not limited to) starting materials, side products, and the like. A
compound is "substantially pure" if it comprises at least about
80%, more preferably 90%, still more preferably at least about 95%
of the composition. If a single isomer of a compound is desired
(e.g., a single diastereomer, enantiomer, or regioisomer), the
compound is preferably substantially free of any undesired isomers
(e.g., the unwanted enantiomer, diastereomers, or regioisomers),
i.e., the desired isomer comprises at least about 80%, more
preferably 90%, still more preferably at least about 95% of the
weight of the isomers present in the composition.
[0071] It will be noted that the structure of some of the compounds
of this invention includes asymmetric carbon atoms. It is to be
understood accordingly that the isomers arising from such asymmetry
(e.g., all enantiomers and diastereomers) are included within the
scope of this invention, unless indicated otherwise. Such isomers
can be obtained in substantially pure form by classical separation
techniques and by stereochemically controlled synthesis.
[0072] I. PSDP Analogs
[0073] The instant invention is based on the surprising finding
that PSDP ameliorates potentionally undesirable leukocyte mediated
responses during neutrophil activation, such as inhibition of the
superoxide generating system. Applicants have discovered that PSDP
inhibits key neutrophil responses, which are important in
leukocyte-mediated tissue injury as observed in reperfusion injury
or overt acute inflammatory resonses. Based upon these findings, a
series of PSDP analogs that resist rapid inactivation of PSDP have
been designed in order to prevent or control the unwanted release
of noxious agents from activated neutrophils. The PSDP analogs of
the present invention include, compounds which can be represented
by Formulae I-IV: 4
[0074] wherein R.sub.1, R.sub.2 and R.sub.3 are each independently,
selected from the group consisting of hydrogen, F, Cl, Br, I,
CH.sub.3 and substituted or unsubstituted, linear or branched
alkyl, alkoxy, aryl, aralkyl or heteroaryl groups;
[0075] wherein Y.sub.1, Y.sub.2, Y.sub.3, Y.sub.4, and Y.sub.5 are
each independently selected from hydrogen atoms or lower alkyl
groups;
[0076] wherein X.sub.1 is an oxygen atom, a sulfur atom, an N.dbd.N
group, a methylene or, NR.sub.5, wherein R.sub.5 is a hydrogen atom
or a substituted or unsubstituted, linear or branched alkyl, aryl,
aralkyl or heteroaryl group;
[0077] wherein X.sub.2 is an OH group, SH, CH.sub.3, or
NR.sub.6.sub.7, wherein R.sub.6 and R.sub.7 are each independently,
a hydrogen atom or a substituted or unsubstituted, linear or
branched alkyl, aryl, aralkyl or heteroaryl group; and
[0078] wherein A.sub.1, A.sub.2, A.sub.3, and A.sub.4 are each
independently, a substituted or unsubstituted aromatic or
nonaromatic carbocyclic or heterocyclic group or a salt
thereof.
[0079] In preferred embodiments of the compound of Formulae I and
II, Y.sub.1-5 are F or CH.sub.3, X is N.dbd.N or methylene and
X.sub.2 is OH.
[0080] It has now been unexpectedly found that the compounds of the
invention inhibit leukocyte mediated responses. For example, as
described in FIG. 1, infra, certain compounds of the invention have
activity against superoxide anion. Moreover, the compounds of the
invention can have a variety of closely spaced functionalities and
may serve as interesting molecular scaffolds. The
leukocyte-mediated inflammation or inflammatory responses cause or
contribute to a wide variety of diseases and conditions including
various forms of asthma and arthritis. Included within the present
invention are inflammatory responses to physical injury, such as
physical trauma, radiation exposure, and otherwise. The compounds
of the invention can be used to treat inflammatory related
disorders, such as rheumatoid arthritis, asthma, psoriasis, related
leukocyte dependent reperfusion injury and adult respiratory
distress syndrome (ARDS).
[0081] II. Preparation of PSDP Analogs
[0082] Derivatives of farnesyl diphosphate (1) can be incorporated
into analogs of presqualene diphosphate (2) (PSDP) by enzymatic
synthesis using squalene synthetase in the absence of reducing
conditions (as in Jarstfer, M. B. et al., J. Am. Chem. Soc. 1996,
118, 13089). 5
[0083] Only minor variations in the farnesyl diphosphate structure
are recognized by squalene synthetase (Ortiz de Montellano, P. R et
a., Biochemistry 1977, 16, 2680), therefore, synthesis of complex
analogs of presqualene diphosphate require that structural
similarity is retained. Analogs of PSDP can be prepared by chemical
syntheses as described below and can be prepared by standard
techniques including Wittig-type coupling, Grignard synthesis,
carbene addition, selective hydrogenation, epoxidation and
asymmetric reductions. For example, presqualene derivatives have
been prepared by Jarstfer et al., J. Am. Chem. Soc. 1996, 118,
13089; Cohen L. H. et al., Biochem. Pharmacol. 1995, 49(6), 839;
Poulter C. D., Rilling H. C. in Biosynthesis of Isoprenoid
Compounds 1981, Vol. 1, Chap. 8, 413; Corey E. J., Volante R. P.,
J. Am. Chem. Soc. 1975,98, 1291; Coates R. M., Robinson W. H., J.
Am. Chem. Soc. 1971, 93, 1785; Good R. S., Eckstein F., J. Am.
Chem. Soc. 1971, 93, 6252; Kornforth R, Popjak G., Methods Enzymol.
1969, 15,382), the teachings of which are incorporated herein by
reference.
[0084] In one exemplary synthesis, PSDP analogs of the invention
can be prepared in three major fragments (A, B and C). These
fragments can then be combined, under appropriate conditions, to
form (3). R.sub.1, R.sub.2, R.sub.3, Y.sub.1, Y.sub.2, Y.sub.3,
Y.sub.4, Y.sub.5, X.sub.1 and X.sub.2 are as described above. 6
[0085] Polyisoprenyl alcohol precursors (4a) and (4b) can be
prepared with substituents R.sub.1 or R.sub.2, independently
selected from halogen (e.g., F, Cl), hydrogen, methyl and
substituted or unsubstituted linear or branched alkyl, alkoxy,
aryl, aralkyl or heteroaryl groups, as described previously. Each Y
group can be independently selected from hydrogen or substituted or
unsubstituted alkyl groups, preferably methyl, ethyl or propyl
groups. R.sub.1, R.sub.2, R.sub.3 and the Y groups help stabilize
the PSDP analog to prevent inactivation by P.sub.450 oxidation and
slow first pass metabolism in the liver, kidneys and lungs. 7
[0086] The method of Coates and Robinson (J. Am. Chem. Soc. 1971,
93, 1785) can be modified to couple polyisoprenyl alcohol (4b)
(fragment B) to cyclopropylcarbinyl ring (fragment A).
Polyisoprenyl alcohol (4b) (fragment B) can be transformed into
diazoacetate derivative (5) by treatment with glyoxalyl chloride
tosylhydrazone and triethylamine in methylene chloride (House H.
O., Blankley C. J., J. Org. Chem. 1963, 33, 53). 8
[0087] Transformation of (5) by copper-catalyzed decomposition with
cupric acetylacetonate in refluxing toluene can result in formation
of cyclopropyl lactone (6) having a cis-formed lactone ring. 9
[0088] Cyclopropyl lactone (6) can be hydrolyzed to hydroxy acid
(7) which can be further oxidized to cis-aldehyde acid (8) with
chromium trioxide-dipyridine complex in methylene chloride. 10
[0089] Aldehyde ester (8) can then be esterfied with diazomethane
to yield ester (9). 11
[0090] A second polyisoprenyl side chain (4a) can be converted to
phosphorane derivative (13). For example, polyisoprenyl alcohol
(4a) can be treated with tosyl 12
[0091] chloride, sodium iodide-acetone and triphenylphosphine in
benzene, to generate primary phosphonium iodide (10). Deprotonation
of (10) with n-butyllithium in ether followed 13
[0092] by alkylation with an excess of an alkyl halide, such as,
for example methyl iodide, can yield monosubstituted ylide (11),
thereby producing second phosphonium iodide (12). 14
[0093] Addition of a second equivalent of n-butyllithium to (12) in
tetrahydrofuran generates the disubstituted ylide (13).
[0094] A Wittig reaction between ester (9) and ylide (13) in
tetrahydrofuran can produce an isomeric mixture of esters (14).
Reduction of esters (14) with lithium aluminum hydride affords
corresponding alcohols (15) which can be separated by
chromatography. 15
[0095] To generate diphosphorylated analog (16), one mmole of
alcohol (15) can be added to trichloroacetonitrile (6 mmoles)
followed by addition of di-triethylammonium phosphate (2.4 mmoles)
dissolved in acetonitrile over 3-4 h at room temperature.
Diphosphate (16) can be extracted with 0.1 N aqueous ammonia and
ether and isolated by techniques known to those skilled in the art
(as in Kornforth R., Popjak G., Methods Enzymol. 1969, 15, 382).
16
[0096] In another embodiment, modification of the synthesis of
GTP.gamma.S (Goody R. S., Eckstein F., J. Am. Chem. Soc. 1971, 93,
6252) provides presqualene diphosphate analogs with sulfur in the
X.sub.1 and X.sub.2 positions. For example, the lithium salt of
S-2-carbamoylethyl thiophosphate (17) (R=2 carbamoylethyl) can be
converted to a pyridinium salt-via ion exchange column and then to
a mono(tri-n-octylammonium) salt by addition of tri-n-octylamine in
methanol. The solution is concentrated under reduced pressure to
afford a residue which is suspended in dixoane and combined with
diphenyl phosphorochloridate and tri-n-butylamine to yield (18).
17
[0097] Conversion of the polyisoprenyl alcohol (15) to a
monophosphorylated derivative can be accomplished by adding (15) in
pyridine and ether drop-wise over 30 min to freshly distilled
POC1.sub.3/ ether at -10.degree. C. with stirring. Pyridine HC1 is
removed by filtration. Lithium hydroxide can then be added to the
filtrate, followed by aqueous ammonia until a pH of 12 is reached.
Inorganic trilithium phosphate salt can be removed from the
solution by centrifugation and contaminates are removed by
extraction with ethanol/water. The pH of the supernatant can be
adjusted to 8 with HCl. Lyophilization of the remaining solution
yields monophosphorylate derivative (19). 18
[0098] Compound (18) can be treated with ether and added to (19) in
pyridine at room temperature to produce diphosphorylated analog
(20). Similarly, reaction of (15) with (18) can produce
monophosphorylated analog (21) with sulfur in the X.sub.1 position.
Additionally (21) can also be a substrate for a second
phosphorylation by (18) to yield sulfur in both the X.sub.1 and
X.sub.2 positions. Therefore, when R.dbd.H in compound (21),
further reaction with (18) can yield (22). 19
[0099] In yet another embodiment, the present invention provides
analogs of PSDP, such as (23), which incorporate at least one ring
system, A.sub.1, into the molecule. These PSDP analogs can also be
prepared from fragments A, B and C (shown in (23)), which can be
coupled to form (23). As described above, A.sub.1 can be a
substituted or unsubstituted aromatic or non-aromatic carbocyclic
or heterocyclic ring. The presence of A.sub.1 can help stabilize
the conformation of (23). 20
[0100] For example, fragment B can be coupled to fragment A,
thereby forming a compound which can be phosphorylated as described
above. In one embodiment, fragment B can be prepared by
dehalogenating 1,4-dichlorocyclohexane (24) to conjugated diene
(25). This dehalogenation provides a substrate suitable for
addition of chlorocarbene to form cyclopropylcarbinyl ring (26).
Compound (26) can then undergo syn addition epoxidation in the
presence of performic acid to form (27). Hydrolysis of (27) can
then form diol (28). Alternatively, exposure of cyclopropyl
chloride (26) 21
[0101] to osmium tetroxide can produce diol (28). Oxidation of the
hydroxyl groups of (28), followed by Grignard addition of suitable
alkyl groups and subsequent reduction of resultant alcohols
provides (29). Conversion of the chloride to a hydroxyl group leads
to (30) which can be derivatized as described above. 22 23
[0102] Each of the PSDP analogs and their intermediates can be
isolated by chromatography (TLC, RP-HPLC) for homogeneity.
[0103] III. Pharmaceutical Compositions
[0104] In another aspect, the present invention provides
pharmaceutically acceptable compositions which comprise a
therapeutically-effective amount of one or more of the compounds
described above, formulated together with one or more
pharmaceutically acceptable carriers (additives) and/or diluents.
As described in detail below, the pharmaceutical compositions of
the present invention may be specially formulated for
administration in solid or liquid form, including those adapted for
the following: (1) oral administration, for example, drenches
(aqueous or non-aqueous solutions or suspensions), tablets,
boluses, powders, granules, pastes for application to the tongue;
(2) parenteral administration, for example, by subcutaneous,
intramuscular or intravenous injection as, for example, a sterile
solution or suspension; (3) topical application, for example, as a
cream, ointment or spray applied to the skin; or (4) intravaginally
or intrarectally, for example, as a pessary, cream or foam.
[0105] The phrase "therapeutically-effective amount" as used herein
means that amount of a compound, material, or composition
comprising a compound of the present invention which is effective
for producing some desired therapeutic effect by treating (i.e.,
inhibiting, preventing or ameliorating) a bacterial infection or a
leukocyte mediated response in a subject, at a reasonable
benefit/risk ratio applicable to any medical treatment.
[0106] 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.
[0107] The phrase "pharmaceutically-acceptable carrier" as used
herein means a pharmaceutically-acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient,
solvent or encapsulating material, involved in carrying or
transporting the subject peptidomimetic agent from one organ, or
portion of the body, to another organ, or portion of the body. Each
carrier must be "acceptable" in the sense of being compatible with
the other ingredients of the formulation and not injurious to the
patient. Some examples of materials which can serve as
pharmaceutically-acceptable carriers include: (1) sugars, such as
lactose, glucose and sucrose; (2) starches, such as corn starch and
potato starch; (3) cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)
powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)
excipients, such as cocoa butter and suppository waxes; (9) oils,
such as peanut oil, cottonseed oil, safflower oil, sesame oil,
olive oil, corn oil and soybean oil; (10) glycols, such as
propylene glycol; (11) polyols, such as glycerin, sorbitol,
mannitol and polyethylene glycol; (12) esters, such as ethyl oleate
and ethyl laurate; (13) agar; (14) buffering agents, such as
magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)
pyrogen-free water; (17) isotonic saline; (18) Ringer's solution;
(19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other
nontoxic compatible substances employed in pharmaceutical
formulations.
[0108] As set out above, certain embodiments of the present
compounds can contain a basic functional group, such as amino or
alkylamino, and are, thus, capable of forming
pharmaceutically-acceptable salts with pharmaceutically-acceptable
acids. The term "pharmaceutically-acceptable salts" in this
respect, refers to the relatively non-toxic, inorganic and organic
acid addition salts of compounds of the present invention. These
salts can be prepared in situ during the final isolation and
purification of the compounds of the invention, or by separately
reacting a purified compound of the invention in its free base form
with a suitable organic or inorganic acid, and isolating the salt
thus formed. Representative salts include the hydrobromide,
hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,
valerate, oleate, palmitate, stearate, laurate, benzoate, lactate,
phosphate, tosylate, citrate, maleate, fumarate, succinate,
tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and
laurylsulphonate salts and the like. (See, e.g., Berge et al.
(1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19)
[0109] In other cases, the compounds of the present invention may
contain one or more acidic functional groups and, thus, are capable
of forming pharmaceutically-acceptable salts with
pharmaceutically-acceptable bases. The term
"pharmaceutically-acceptable salts" in these instances refers to
the relatively non-toxic, inorganic and organic base addition salts
of compounds of the present invention. These salts can likewise be
prepared in situ during the final isolation and purification of the
compounds, or by separately reacting the purified compound in its
free acid form with a suitable base, such as the hydroxide,
carbonate or bicarbonate of a pharmaceutically-acceptable metal
action, with ammonia, or with a pharmaceutically-acceptable organic
primary, secondary or tertiary amine. Representative alkali or
alkaline earth salts include the lithium, sodium, potassium,
calcium, magnesium, and aluminum salts and the like. Representative
organic amines useful for the formation of base addition salts
include ethylamine, diethylamine, ethylenediamine, ethanolamine,
diethanolamine, piperazine and the like. (See, for example, Berge
et al., supra)
[0110] Wetting agents, emulsifiers and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, coating agents, sweetening, flavoring and perfuming
agents, preservatives and antioxidants can also be present in the
compositions.
[0111] Examples of pharmaceutically-acceptable antioxidants
include: (1) water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; (2) oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and (3) metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0112] Formulations of the present invention include those suitable
for oral, nasal, topical (including buccal and sublingual), rectal
and/or parenteral administration. The formulations may conveniently
be presented in unit dosage form and may be prepared by any methods
well known in the art of pharmacy. The amount of active ingredient
which can be combined with a carrier material to produce a single
dosage form will vary depending upon the host being treated, the
particular mode of administration. The amount of active ingredient
which can be combined with a carrier material to produce a single
dosage form will generally be that amount of the compound which
produces a therapeutic effect. Generally, out of one hundred
percent, this amount will range from about 1 percent to about
ninety-nine percent of active ingredient, preferably from about 5
percent to about 70 per cent, most preferably from about 10 percent
to about 30 per cent.
[0113] Methods of preparing these formulations or compositions
include the step of bringing into association a compound of the
present invention with the carrier and, optionally, one or more
accessory ingredients. In general, the formulations are prepared by
uniformly and intimately bringing into association a compound of
the present invention with liquid carriers, or finely divided solid
carriers, or both, and then, if necessary, shaping the product.
[0114] Formulations of the invention suitable for oral
administration may be in the form of capsules, cachets, pills,
tablets, lozenges (using a flavored basis, usually sucrose and
acacia or tragacanth), powders, granules, or as a solution or a
suspension in an aqueous or non-aqueous liquid, or as an
oil-in-water or water-in-oil liquid emulsion, or as an elixir or
syrup, or as pastilles (using an inert base, such as gelatin and
glycerin, or sucrose and acacia) and/or as bronchoaveolar lavages
for intended delivery systems to the lung and the like, each
containing a predetermined amount of a compound of the present
invention as an active ingredient. A compound of the present
invention may also be administered as a bolus, electuary or
paste.
[0115] In solid dosage forms of the invention for oral
administration (capsules, tablets, pills, dragees, powders,
granules and the like), the active ingredient is mixed with one or
more pharmaceutically-acceptable carriers, such as sodium citrate
or dicalcium phosphate, and/or any of the following: (1) fillers or
extenders, such as starches, lactose, sucrose, glucose, mannitol,
and/or silicic acid; (2) binders, such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
sucrose and/or acacia; (3) humectants, such as glycerol; (4)
disintegrating agents, such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates, and sodium
carbonate; (5) solution retarding agents, such as paraffin; (6)
absorption accelerators, such as quaternary ammonium compounds; (7)
wetting agents, such as, for example, cetyl alcohol and glycerol
monostearate; (8) absorbents, such as kaolin and bentonite clay;
(9) lubricants, such a talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof; and (10) coloring agents. In the case of capsules, tablets
and pills, the pharmaceutical compositions may also comprise
buffering agents. Solid compositions of a similar type may also be
employed as fillers in soft and bard-filled gelatin capsules using
such excipients as lactose or milk sugars, as well as high
molecular weight polyethylene glycols and the like.
[0116] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (for example, gelatin or hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent.
Molded tablets may be made by molding in a suitable machine a
mixture of the powdered compound moistened with an inert liquid
diluent.
[0117] The tablets, and other solid dosage forms of the
pharmaceutical compositions of the present invention, such as
dragees, capsules, pills and granules, may optionally be scored or
prepared with coatings and shells, such as enteric coatings and
other coatings well known in the pharmaceutical-formulating art.
They may also be formulated so as to provide slow or controlled
release of the active ingredient therein using, for example,
hydroxypropylmethyl cellulose in varying proportions to provide the
desired release profile, other polymer matrices, liposomes and/or
microspheres. They may be sterilized by, for example, filtration
through a bacteria-retaining filter, or by incorporating
sterilizing agents in the form of sterile solid compositions which
can be dissolved in sterile water, or some other sterile injectable
medium immediately before use. These compositions may also
optionally contain opacifying agents and may be of a composition
that they release the active ingredient(s) only, or preferentially,
in a certain portion of the gastrointestinal tract, optionally, in
a delayed manner. Examples of embedding compositions which can be
used include polymeric substances and waxes. The active ingredient
can also be in micro-encapsulated form, if appropriate, with one or
more of the above-described excipients.
[0118] Liquid dosage forms for oral administration of the compounds
of the invention include pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active ingredient, the liquid dosage forms may
contain inert diluents commonly used in the art, such as, for
example, water or other solvents, solubilizing agents and
emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, oils (in particular,
cottonseed, groundnut, corn, germ, olive, castor and sesame oils),
glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty
acid esters of sorbitan, and mixtures thereof
[0119] Besides inert diluents, the oral compositions can also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, coloring, perfuming and
preservative agents.
[0120] Suspensions, in addition to the active compounds, may
contain suspending agents as, for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth, and mixtures thereof
[0121] Formulations of the pharmaceutical compositions of the
invention for rectal or vaginal administration may be presented as
a suppository, which may be prepared by mixing one or more
compounds of the invention with one or more suitable nonirritating
excipients or carriers comprising, for example, cocoa butter,
polyethylene glycol, a suppository wax or a salicylate, and which
is solid at room temperature, but liquid at body temperature and,
therefore, will melt in the rectum and release the active
compound.
[0122] Formulations of the present invention which are suitable for
vaginal administration also include pessaries, creams, gels,
pastes, foams or spray formulations containing such carriers as are
known in the art to be appropriate.
[0123] Dosage forms for the topical or transdermal administration
of a compound of this invention include powders, sprays, ointments,
pastes, creams, lotions, gels, solutions, patches and inhalants.
The active compound may be mixed under sterile conditions with a
pharmaceutically-acceptable carrier, and with any preservatives,
buffers, or propellants which may be required.
[0124] The ointments, pastes, creams and gels may contain, in
addition to an active compound of this invention, excipients, such
as animal and vegetable fats, oils, waxes, paraffins, starch,
tragacanth, cellulose derivatives, polyethylene glycols, silicones,
bentonites, silicic acid, talc and zinc oxide, or mixtures
thereof.
[0125] Powders and sprays can contain, in addition to a compound of
this invention, excipients such as lactose, talc, silicic acid,
aluminum hydroxide, calcium silicates and polyamide powder, or
mixtures of these substances. Sprays can additionally contain
customary propellants, such as chlorofluorohydrocarbons and
volatile unsubstituted hydrocarbons, such as butane and
propane.
[0126] Transdermal patches have the added advantage of providing
controlled delivery of a compound of the present invention to the
body. Such dosage forms can be made by dissolving or dispersing the
peptidomimetic in the proper medium. Absorption enhancers can also
be used to increase the flux of the peptidomimetic across the skin.
The rate of such flux can be controlled by either providing a rate
controlling membrane or dispersing the peptidomimetic in a polymer
matrix or gel.
[0127] Ophthalmic formulations, eye ointments, powders, solutions
and the like, are also contemplated as being within the scope of
this invention.
[0128] Pharmaceutical compositions of this invention suitable for
parenteral administration comprise one or more compounds of the
invention in combination with one or more
pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous
solutions, dispersions, suspensions or emulsions, or sterile
powders which may be reconstituted into sterile injectable
solutions or dispersions just prior to use, which may contain
antioxidants, buffers, solutes which render the formulation
isotonic with the blood of the intended recipient or suspending or
thickening agents.
[0129] Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol; and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0130] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of the action of microorganisms may be ensured
by the inclusion of various antibacterial and antifungal agents,
for example, paraben, chlorobutanol, phenol sorbic acid, and the
like. It may also be desirable to include isotonic agents, such as
sugars, sodium chloride, and the like into the compositions. In
addition, prolonged absorption of the injectable pharmaceutical
form may be brought about by the inclusion of agents which delay
absorption such as aluminum monostearate and gelatin.
[0131] In some cases, in order to prolong the effect of a drug, it
is desirable to slow the absorption of the drug from subcutaneous
or intramuscular injection. This may be accomplished by the use of
a liquid suspension of crystalline or amorphous material having
poor water solubility. The rate of absorption of the drug then
depends upon its rate of dissolution which, in turn, may depend
upon crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally-administered drug form is accomplished
by dissolving or suspending the drug in an oil vehicle.
[0132] Injectable depot forms are made by forming microencapsule
matrices of the subject compounds in biodegradable polymers such as
polylactide-polyglycolide. Depending on the ratio of drug to
polymer, and the nature of the particular polymer employed, the
rate of drug release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the drug in liposomes or microemulsions which are
compatible with body tissue.
[0133] When the compounds of the present invention are administered
as pharmaceuticals, to humans and animals, they can be given per se
or as a pharmaceutical composition containing, for example, 0.1 to
99.5% (more preferably, 0.5 to 90%) of active ingredient in
combination with a pharmaceutically acceptable carrier.
[0134] The preparations of the present invention may be given
orally, parenterally, topically, or rectally. They are of course
given by forms suitable for each administration route. For example,
they are administered in tablets or capsule form, by injection,
inhalation, eye lotion, ointment, suppository, etc. administration
by injection, infusion or inhalation; topical by lotion or
ointment; and rectal by suppositories. Oral administration is
preferred.
[0135] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticulare, subcapsular,
subarachnoid, intraspinal and intrasternal injection and
infusion.
[0136] The phrases "systemic administration," "administered
systemically," "peripheral administration" and "administered
peripherally" as used herein mean the administration of a compound,
drug or other material other than directly into the central nervous
system, such that it enters the patient's system and, thus, is
subject to metabolism and other like processes, for example,
subcutaneous administration.
[0137] These compounds may be administered to humans and other
animals for therapy by any suitable route of administration,
including orally, nasally, as by, for example, a spray, rectally,
intravaginally, parenterally, intracisternally and topically, as by
powders, ointments or drops, including buccally and
sublingually.
[0138] Regardless of the route of administration selected, the
compounds of the present invention, which may be used in a suitable
hydrated form, and/or the pharmaceutical compositions of the
present invention, are formulated into pharmaceutically-acceptable
dosage forms by conventional methods known to those of skill in the
art.
[0139] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of this invention may be varied so as
to obtain an amount of the active ingredient which is effective to
achieve the desired therapeutic response for a particular patient,
composition, and mode of administration, without being toxic to the
patient.
[0140] The selected dosage level will depend upon a variety of
factors including the activity of the particular compound of the
present invention employed, or the ester, salt or amide thereof,
the route of administration, the time of administration, the rate
of excretion of the particular compound being employed, the
duration of the treatment, other drugs, compounds and/or materials
used in combination with the particular compound employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the
medical arts.
[0141] A physician or veterinarian having ordinary skill in the art
can readily determine and prescribe the effective amount of the
pharmaceutical composition required. For example, the physician or
veterinarian could start doses of the compounds of the invention
employed in the pharmaceutical composition at levels lower than
that required in order to achieve the desired therapeutic effect
and gradually increase the dosage until the desired effect is
achieved.
[0142] In general, a suitable daily dose of a compound of the
invention will be that amount of the compound which is the lowest
dose effective to produce a therapeutic effect. Such an effective
dose will generally depend upon the factors described above.
Generally, intravenous, intracerebroventricular and subcutaneous
doses of the compounds of this invention for a patient, when used
for the indicated analgesic effects, will range from about 0.0001
to about 100 mg per kilogram of body weight per day, more
preferably from about 0.01 to about 50 mg per kg per day, and still
more preferably from about 0.1 to about 40 mg per kg per day.
[0143] If desired, the effective daily dose of the active compound
may be administered as two, three, four, five, six or more
sub-doses administered separately at appropriate intervals
throughout the day, optionally, in unit dosage forms.
[0144] While it is possible for a compound of the present invention
to be administered alone, it is preferable to administer the
compound as a pharmaceutical composition.
[0145] IV. Exemplification
[0146] Receptor-ligand recognition initiates membrane remodeling
events that generate bioactive lipids which serve as both inter-
and extracellular mediators crucial to coordinate leukocyte
responses (Serhan, C. N., Haeggstrom, J. Z. & Leslie, C. C.
FASEB J. 10:1147-1158 (1996), the teachings of which are
incorporated herein by reference.). While several classes of
prophlogistic lipids have been described (Serhan, C. N.,
Haeggstrom, J. Z. & Leslie, C. C. FASEB J. 10:1147-1158
(1996)), the present invention is directed to novel intracellular
lipid signals that down-regulate neutrophil responses.
Neutrophil-derived extracts rich in phosphorylated,
non-saponifiable lipids potently inhibit superoxide anion
generation. Structural analysis of these bioactive fractions
revealed four major phosphorylated lipids: (V) farnesyl
diphosphate, (VI) presqualene diphosphate (PSDP), (VII) famesyl
monophosphate and (VIII) presqualene monophosphate (PSMP).
Following receptor activation, compounds VI and VIII underwent
rapid phosphate turnover. Compound VI but not VIII, in nM-.mu.M
amounts, inhibited neutrophil superoxide anion generation,
homotypic adhesion and 1,4,5-inositol-triphosphate formation. These
results indicate that PSDP, present in immune effector cells, is a
potent receptor-activated mediator which regulates cellular
responses of interest in inflammation and tissue injury.
[0147] Receptor-ligand interaction results in the elaboration of
both positive and negative signals, the timing and balance of which
determines cellular responses (Serhan, C. N., Haeggstrom, J. Z.
& Leslie, C. C. FASEB J. 10:1147-1158 (1996)). This phenomenon
is exemplified by formyl-methionyl4eucyl-phenylalanine (FMLP)
binding to its receptor on neutrophils with subsequent generation
of intracellular signals that stimulate (e.g., intracellular
calcium influx) as well as inhibit (e.g. formation of cyclic AMP)
neutrophil function (Weismann, G., Smolen, J. E. & Korchak, H.
M. N. Engl. J. Med. 303, 27-34 (1980), the teachings of which are
incorporated herein by reference). Although several classes of
intracellular lipids are known to activate leukocytes during
inflammation and reperfilsion injury, relatively few
counterregulatory lipid-derived mediators have been identified
(Serhan, C. N., Haeggstrom, J.Z. & Leslie, C. C. FASEB J.
10:1147-1158 (1996)). To this end, the capacity of neutrophil
extracts to regulate superoxide anion generation was screened with
cell sonicates.
[0148] Neutrophil-derived lipid extracts triggered generation of
substantial amounts of superoxide anions (2.71 nmoles
O.sub.2.sup.-/mg protein/min) (FIG. 1a). To identify regulatory
signals, phospholipids with ester or amide bonds were hydrolyzed by
saponification prior to extraction and CC4 silica chromatography
(generally, as in Adair, W. L. & Keller, R. K. Methods Enzymol.
111, 201-215 (1985); Van Dessel, G. A. F., Lagrou, A. R. Hilderson,
H. J. J. & Dierick, W. S. H. In: CRC Handbook of Chromatography
(eds Mukherjee, K. D., Weber, N. & Sherma, J.) 321-337 (CRC
Press, Boca Raton, 1993), the teachings of which are incorporated
herein by reference). The initial chloroform:methanol (2:1, v/v)
elutions (Fraction "A") contained a mixture of fatty acids and
neutral lipids that gave similar rates of superoxide anion
generation (2.31 nmoles/mg protein/min). O.sub.2 -- production was
comparable to that observed with arachidonic acid (C20:4 (75
.mu.M), 1.38 nmoles/mg protein/min) and phosphatitic acid (C10:0
(100 .mu.M), 1.34 nmoles/mg protein/min), known activators in this
system, and consistent with reported values (FIG. 1a) (as in
references: McPhail, L. C., Shirley, P. S., Clayton, C. C. &
Snydennan, R. J. Clin Invest. 75, 1735-1739 (1985) and Agwu, D. E.,
McPhail, L. C., Sozzani, S., Bass, D. A. & McCall, C. E. J
Clin. Invest. 88,531-539 (1991), the teachings of which are
incorporated herein by reference). In contrast, subsequent elutions
with chloroform: methanol:water (10:10:3, v/v) (Fraction "B"),
which contained non-saponifiable, phosphorylated lipids, stimulated
much less superoxide anion generation (0.62 nmoles/mg protein/min).
Furthermore, the lipids present in Fraction B potently inhibited
O.sub.2 -- production stimulated by either phosphatitic acid (52%,
FIG. 1b) or arachidonic acid (56%, see FIG. 1f). These results
indicate that materials present in Fraction B can counteract
intracellular lipid signals relevant in early neutrophil
activation.
[0149] Characterization of PSDP analogs and metabolites include
standard techniques such as extraction, chromatography, and
quantitative HPLC followed by trimethyl silyl derivatization,
O-methoxime derivatization and gas chromatography/mass spectroscopy
analysis. The experimental details of this embodiment are described
above.
[0150] Neutrophil lipids were saponified (10% KOH in methanol,
37.degree. C., 30 min), extracted (Van Dessel, G. A. F., Lagrou, A.
R. Hilderson, H. J. J. & Dierick, W. S. H. In: CRC Handbook of
Chromatography (eds Mukherjee, K. D., Weber, N. & Sherma, J.)
321-337 (CRC Press, Boca Raton, 1993)) and separated by CC4 silica
(1 gm/10.sup.8 PMN) column chromatography as in FIG. 1f. To
determine O.sub.2 -- production, post-nuclear supernatants from
neutrophils after sonication (as in McPhail, L. C., Shirley, P. S.,
Clayton, C. C. & Snyderman, R. J. Clin Invest. 75, 1735-1739
(1985)), 75 .mu.M arachiodonic acid(C20:4, NuCheck Prep) and
Fraction B (0.1% ethanol) were added to cytochrome c (60 .mu.M),
sucrose (170 mM), EGTA (ethylene glycol-bis (.beta.-aminoethyl
ether)-N,N,N',N'-tetraacetic acid). (1 mM), FAD (flavin adenine
dinucleotide) (10 .mu.M), NaN.sub.3 (2 mM) and either superoxide
dismutase (30 .mu.g/ml) or H.sub.2O at O.degree. (as in 20). After
2 min (37.degree. C.) in a thermal-jacketed cuvette, p-NAPH (200
.mu.M) was added to initiate reactions and absorbance (550 nm) was
monitored at 15 second intervals. Representative results are
depicted from n=6. The inset shows % inhibition of arachidonic
acid-stimulated superoxide anion generation by Fraction B lipids.
Values represent mean.+-.Standard Error of the Mean.
[0151] To identify the inhibitory compounds in Fraction B,
extracts, isolated from stimulated neutrophils, were examined after
thin layer chromatography (TLC). Lipids were eluted from sequential
5 mm fractions from origin to solvent front and assessed for
phosphorus content and bioactivity. Regions 10-15 mm from the
origin carried phosphorus (11.3 nmoles phosphorus/10.sup.6 PMN) and
resulted in a 52% inhibition of phosphatitic acid stimulated
superoxide anion generation (FIG. 1c). To determine the Rf value of
Fraction B's bioactive, non-saponifiable phosphorus-containing
lipids, materials chromatographed in parallel were stained with
iodine to identify double bonds and treated with molybdenum blue to
visualize phosphorus. Four major compounds were observed (FIG. 1d).
In preliminary experiments to elucidate the structures, material
analyzed by gas chromatography/mass spectrometry (GC/MS), gave base
peaks at (m/z 69) with a fragmentation pattern of repeating
C.sub.5H.sub.8 (m/z 68) units, characteristic of isoprenoids (FIG.
2d) (see, for example, Farnsworth, C. C., Gelb, M. H. &
Glomset, J. A. Science 247, 320-322 (1990), the teachings of which
are incorporated herein by reference).
[0152] After TLC (as in FIG. 1C-c), compound VI was eluted with 1
ml mobile phase (chloroform:methanol:water 65:25:4, v/v) .times.4,
brought to dryness under N.sub.2, concentrated in bexane (2 .mu.l)
and taken to GC/MS (as in FIG. 2c under methods). Representative
results (n=7) indicate a base peak of 69
[(CH.sub.3).sub.2CCHCH.sub.2] with prominent ions at repeating
intervals of 68 [CH.sub.2C(CH.sub.3)CHCH.sub.2] or 69: 136, 203,
273, 341, 410. The absence of a clear molecularion (or M+-15) was
noted. This fragmentation pattern is characteristic of isoprenoids
(Farnsworth, C. C., Gelb, M. H. & Glomset, J. A. Science 247,
320-322 (1990); Popjk, G., Edmond, J. Clifford, K. & Williams,
V. J. Biol. Chem. 244, 1897-1918 (1969); Epstein, W. W. &
Rilling, H. C. J. Biol. Chem. 245, 4597-4605 (1970), the teachings
of which are incorporated herein by reference).
[0153] For control purposes, Rf values were determined for
available isoprenoids: isopentenyl diphosphate, geranyl
diphosphate, geranylgeranyl diphosphate, dolichyl monophosphate,
isoprenyl alcohols, squalene or cholesterol (FIG. 1e). Compounds V
and VII (Rf values of 0.05.+-.0.01 and 0.17.+-.0.01) comigrated
with farnesyl diphosphate (FDP) and farnesyl monophosphate (FMP),
respectively. Compounds VI and VIII gave Rf values (0.10.+-.0.01
and 0.25.+-.0.02) identical to the TLC fractions with the most
phosphorus/cell but different from available standards; Based on Rf
values and the relationship between phosphorus content and mass
(measured by scanning densitometry of charred TLC plates, Table
1g), compound VI contained two phosphates and compounds VII and
VIII one phosphate. Together the results indicated the presence of
four major non-saponifiable, phosphorylated lipids in neutrophils
with chromatographic ehavior consistent with polyisoprenyl
phosphates.
[0154] FIG. 1g shows that after TLC of materials in Fraction B,
plates were sprayed with molybdenum blue:4.2M H.sub.2SO.sub.4 (1:1,
v/v). Compounds containing phosphorus were quantitated first by
scanning densitometry (Model PD and integration software, Molecular
Dynamics, Inc.) and then by inorganic phosphorus determination (as
in Chen, P. S. et al. Anal. Chem. 28, 1756-1758 (1956), the
teachings of which are incorporated herein by reference) after
scraping, elution and concentration. Representative results are
reported for compound VI, VII and VIII.
[0155] For further analysis of compounds VI and VIII and to
substantiate the identification of compounds V and VII, each was
isolated and subjected to GC/MS by direct injection or after
conversion to Me.sub.3Si derivatives. All four lipids possessed the
characteristic fragmentation pattern of isoprenoids (Farnsworth, C.
C., Gelb, M. H. & Glomset, J. A. Science 247, 320-322 (1990),
Table 2e). Compounds V and VII gave retention times and prominent
ions also consistent with farnesyl diphosphate and farnesyl
monophosphate which were confirmed by direct comparison to
authentic samples (Table 2e). Compound VI gave a fragmentation
pattern resembling that of squalene (molecular ion (M.sup.+)=410),
yet anions m/z>410 were present and its retention was 0.10-0.12
min greater than that of squalene (FIGS. 2a & 2b). It is known
that acid hydrolysis liberates 80% of the phosphate from
polyisoprenyl diphosphates without substantial destruction of
polyisoprenyl monophosphates (Van Dessel, G. A. F., Lagrou, A. R.
Hilderson, H. J. J. & Dierick, W. S. H. In: CRC Handbook of
Chromatography (eds Mukherjee, K. D., Weber, N. & Sherna, J.)
321-337 (CRC Press, Boca Raton, 1993)) and likewise led to the
conversion of compound VI into a material with the retention and
mass spectrum of squalene (FIGS. 2b & 2c). Compound VIII's
retention and fragmentation pattern by GC/MS were very similar to
that seen with compound VI; however compound VIII was not
susceptible to acid hydrolysis. Molecular ions were not readily
apparent for either compound VI or VIII; a trait consistent with
GC/MS analysis of polyisoprenyl phosphates (Popjk, G., Edmond, J.
Clifford, K. & Williams, V. J. Biol. Chem. 244, 1897-1918
(1969) and Epstein, W. W. & Rilling, H. C. J. Biol. Chem. 245,
4597-4605 (1970)). Based on these physical properties, the proposed
structures for compound VI and VIII were presqualene diphosphate
(PSDP) (FIG. 2a) and presqualene monophosphate (PSMP),
respectively.
[0156] In FIG. 2e, the structures of the four major phosphorylated,
non-saponifiable neutrophil lipids were elucidated using several
analytical techniques: (Serhan, C. N., Haeggstrom, J. Z. &
Leslie, C. C. FASEB J. 10:1147-1158 (1996)) TLC (FIGS. 1d & e,
mean.+-.SEM, n.gtoreq.15) to determine the compounds' Rf values
relative to authentic dolichyl monophosphate (internal standard);
(Weismann, G., Smolen, J. E. & Korchak, H. M. N. Engl. J. Med.
303, 27-34 (1980)) GC/MS for structural identification after direct
injection, reaction (1:2 equivalents) with trimethylbromosilane in
CH.sub.2C1.sub.2 (RT, 2 hours) to generate trimethylsilyl (OTMS)
derivatives ("NP"=not performed) or acid hydrolysis to monitor for
the generation of squalene (FIGS. (2b)) and (3) quantitation by
inorganic phosphorus determination (mean.+-.SEM, n=3-5, d.gtoreq.6)
(19). Proposed structures were determined by comparison with
authentic material when available.
[0157] In yeast, plants and mammalian liver, squalene synthetase
condenses two molecules of farnesyl diphosphate to form the
cyclopropylcarbinyl intermediate, presqualene diphosphate (PSDP),
and in the presence of NADPH, squalene (Goldstein, J. L. &
Brown, M. S. Nature 343, 425-430 (1990); Corey, E. J. &
Volante, R. P. J. Amer. Chem. Soc. 98, 1291-1293 (1976); Mookhtiar,
K. A., Kalinowski, S. S., Zhang, D. & Poulter, C. D. J. Biol.
Chem. 269, 11201-11207 (1994), the teachings of which are
incorporated herein by reference). Alternatively, when reducing
equivalents are depleted, yeast phosphates convert PSDP into
presqualene monophosphate (PSMP) (Popjk, G., Edmond, J. Clifford,
K. & Williams, V. J. Biol. Chem. 244, 1897-1918 (1969)).
Although human leukocytes biosynthesize both FDP and squalene,
neither PSDP nor PSMP has been reported in neutrophils, which
uniquely lack the mixed-function oxidases required to convert
squalene to cholesterol (Shechter. I., Fogelman, A. M. &
Popjak, G. J. Lipid Res. 21, 277-283 (1980), the teachings of which
are incorporated herein by reference). Compounds V, VI, VII and
VIII represented 0.1, 1.8, 0.4 and 0.5%, respectively, of the total
phosphorylated lipid fraction (92.4) nmoles phosphorus/10.sup.7
unstimulated PMN, n=3). Taken together, the results indicate that
neutrophil non-saponifiable lipids include a series of
biosynthetically-related polyisoprenyl phosphates.
[0158] Resting neutrophils, exposed to .gamma..sup.32PO.sub.4-ATP
to enable mass quantitation and identify phosphate turnover,
selectively incorporated radiolabel into compounds VI and VIII
(FIG. 3a). Exposure to the chemotactic peptide, FMLP, resulted in
changes in compound VI and VIII's phosphorus content which were
rapid (within 60 seconds), reciprocal (decrements in compound VI
paralleled increments in compound VIII) and transient (FIG. 3a
inset). FMLP stimulates neutrophil superoxide anion generation and
increases carboxyl methylation of prenylated proteins via
interaction with a G-protein-coupled, seven transmembrane-spanning
receptor (Philips, M. R., Pillinger, M. H., Volker, C., Rosenfield,
M. G., Weissmann, G. & Stock, J. B. Science 259,977-980 (1992);
Baggiolini, M., Boulay, F., Badwey, J. A. & Curnutte, J. T.
FASEB J. 7, 10040-1010 (1993), the teachings of which are
incorporated herein by reference). Decrements in the mass of
compound VI paralleled the early increased rate of O.sub.2 --
production with FMLP stimulation, which after approximately 5
minutes terminates as levels of compound VI return to baseline.
These findings were consistent with a regulatory role for compound
VI in NADPH assembly.
[0159] Cytokines prime leukocytes for inflammatory responses,
including superoxide anion generation, by interacting with
receptors whose signaling mechanisms remain to be fully elucidated
(Gomez-Cambronero, J. & Sha'afi, R. I. in Cell-Cell
Interactions In The Release of Inflammatory Mediators. (eds Wong,
P. Y-K. & Serhan, C. N.) 35-71 (Plenum Press, New York, (1991),
the teachings of which are incorporated herein by reference).
Cytokine stimulation also regulates formation of compounds VI and
VIII, as neutrophils are exposed to granulocyte/monocyte-colony
stimulating factor (GM-CSF) which significantly increases
.sup.32PO.sub.4 incorporation into both compounds VI (45.0%) and
VIII (84.1%) (FIG. 3b) without statistically significant changes in
mass by inorganic phosphorus determination (n=3). Together these
results indicate that compounds VI and VIII are biosynthetically
related, and that neutrophil activation by different classes of
receptors stimulates the incorporation of .sup.32PO.sub.4 into
these lipids.
[0160] To test the impact of compounds VI and VIII on neutrophil
responses, the isolated lipids were introduced into neutrophils by
electroporation prior to determining NADPH oxidase activity. In
nM-.mu.M quantities, compound VI resulted in 60% inhibition of the
FMLP-triggered generation of superoxide anion, while O.sub.2 --
production in the presence of compound VIII was unaltered (FIG.
4a). At these concentrations, neither compound VI nor VIII alone
stimulated O.sub.2 -- production (FIG. 3c). Prenyl cysteine
analogues can disrupt G-protein interaction with activated FMLP
receptors (Scheer, A. & Gierschik, P. Biochemistry.
34,4952-4961 (1995), the teachings of which are incorporated herein
by reference); therefore to determine compound VI's level of
action, neutrophil superoxide anion generation was examined in the
presence of phorbol 12-myristate 13-acetate (PMA), a potent trigger
for O.sub.2 -- production which bypasses cell surface receptors to
stimulate protein kinase C and subsequent NADPH assembly (see for
example, Baggiolini, M., Boulay, F., Badwey, J. A. & Curnutte,
J. T. FASEB J. 7, 10040-1010 (1993); FIG. 3c). Compound VI (1
.mu.M) inhibited PMA (100 .mu.M) activated superoxide anion
generation by 44% in a concentration-dependent fashion with
significant inhibition still present at 10 nM (FIG. 4b, inset).
PMA-stimulated O.sub.2 -- production was not significantly
influenced by either compound VIII or other related eicosanoids
(FIG. 4b). These results indicate that compound VI selectively
inhibits O.sub.2 -- production at a site down-stream from
G-protein-receptor interactions.
[0161] FIG. 3c shows neutrophils (10.sup.7/500 .mu.l) that were
electroporated (as in FIGS. 4, 4a and 4b) in the presence of 0.2%
ethanol. After electroporation, cells in cytochrome c (0.7 mg/ml)
were exposed (10 min, 37.degree. C.) to PMA (10.sup.-7 M), FMLP
(10.sup.-7 M) plus cytochalasin b (15 .mu.g/ml), compound VI (1
.mu.M) or compound VIII (1 .mu.M) and supernatants monitored at 550
nm. Values represent the mean.+-.SEM for n.gtoreq.4.
[0162] Since leukocyte adhesion and diapedesis are also critical
early events in inflammation (reviewed in Serhan, C. N.,
Haeggstrom, J. Z. & Leslie, C. C. FASEB J. 10: 1147-1158 (1996)
and Baggiolini, M., Boulay, F., Badwey, J. A. & Cumutte, J. T.
FASEB J. 7, 10040-1010 (1993), the impact of polyisoprenyl
phosphates on neutrophil homotypic adhesion was evaluated with
intact, freshly isolated cells. Here too, exposure to .mu.M levels
of compound VI but not compound VIII resulted in significant
inhibition (63%, p<0.001) of the aggregtory response to FMLP
(FIGS. 4c and d), while neither compound VI nor VIII alone
stimulated adhesion. In addition, calcium mobilization by
1,4,5-inositol triphosphate (IP.sub.3) is important in neutrophil
activation and cytokine signal transduction (Weismann, G., Smolen,
J. E. & Korchak, H. M. N. Engl. J. Med. 303,27-34 (1980) and
Gomez-Cambronero, J. & Sha'afi, R.I. in Cell-Cell Interactions
In The Release of Inflammatory Mediators. (eds Wong, P. Y-K. &
Serhan, C. N.) 35-71 (Plenum Press, New York, (1991)). Compound VI
(1 .mu.M) also inhibited FMLP-triggered IP.sub.3 formation (56%,
FIG. 4e). These findings indicated that compound VI inhibits
agonist-induced neutrophil responses that are relevant during host
defense.
[0163] Isoprenoids are pivotal intermediates in the generation of
diverse classes of compounds including serols, retinoids,
dolichols, ubiquinone and prenylated proteins (Adair, W. L. &
Keller, R. K. Methods Enzymol. 111, 201-215 (1985); Goldstein, J.
L. & Brown, M. S. Nature 343,425-430 (1990); Quinn, M. T. J.
Leukoc. Biol. 58,263-276 (1995), the teachings of which are
incorporated herein by reference). Here, the major polyisoprenyl
phosphates in neutrophils have been indentified. In response to
receptor activation, presqualene diphosphate and presqualene
monophosphate rapidly remodel, and presqualene diphosphate
regulates relevant cellular bioactions (FIG. 4f). Together our
results indicate that these compounds represent potent, novel
lipid-derived signals and provide evidence for an intracellular
signaling role for isoprenoid remodeling.
[0164] Neutrophil lipids (isolated from human neutrophils from
purified periperal blood of healthy donors) (FIG. 1) were
saponified (10% KOH in methanol, 37.degree. C., 30 min), extracted
(as described generally in Van Dessel, G. A. F., Lagrou, A. R.
Hilderson, H. J. J. & Dierick, W. S. H. In: CRC Handbook of
Chromatography (eds Mukherjee, K. D., Weber, N. & Sherma, J.)
321-337 (CRC Press, Boca Raton, 1993)) and separated by CC4 silica
(1 gm/10.sup.8 PMN) chromatography with sequential elutions of
chloroform:methanol (2:1, v/v) (Fraction A) and
chloroform:methanol:water (10:10:3, vfv) (Fraction B).
[0165] FIGS. 1a-e represent neutrophil-derived non-saponifiable
phospholipids which regulate the generation of superoxide anion by
neutrophil sonicates. Eluents were evaporated under vacuum,
concentrated in ethanol and either (FIGS. 1a-c) added to
incubations for superoxide anion generation or (FIGS. 1c-e) spotted
on TLC plates for development (50 min 25.degree. C.) in an
environment saturated with chloroform:methanol:water (65:25:4,
v/v).
[0166] To determine O.sub.2 -- production (FIGS. 1a-c),
post-nuclear supernatants from neutrophils after sonication
(0.1-0.5 mg protein/ml) (as generally described in Bromberg, Y.
& Pick, E. Cell. Immunol. 88,213-221 (1984), the teachings of
which are incorporated herein by reference), 100 .mu.M phosphatitic
acid (C10:0, Avanti Polar Lipids, Inc.) and lipid extracts (0.1%
ethanol) were added to cytochrome c (60 .mu.M), sucrose (170 mM),
EGTA (1 mM), FAD (10 .mu.M), NaN.sub.3 (2 mM) and either superoxide
dismutase (30 ug/ml) or H.sub.2O at 0.degree. C. (as in 20). After
2 min (37.degree. C.) in a thermal-jacketed cuvette, B-NADPH (200
.mu.M) was added to initiate reactions and absorbance (550 nm) was
monitored at 15 second intervals or following termination (10
minutes, 0.degree. C.).
[0167] For screening of Fraction B by TLC, sequential 5 mm segments
of each lane were scraped, eluted with 1 ml.times.4 of
chloroform:methanol (2:1) and brought to dryness under N.sub.2.
Inorganic phosphorus levels were quantitated as in described in
Chen, P. S. et al. Anal. Chem. 28, 1756-1758 (1956).
[0168] Prior to densitometry, TLC plates with material run in
parallel were either stained with iodine, sprayed with 10%
CuSO.sub.4 in 8% phosphoric acid and charred (100.degree. C., 20
min followed by 120.degree. C., 10 min) (as described generally in
Tou, J. & Dola, T. Lipids 30, 373-381 (1995), the teachings of
which are incorporated herein by reference) or sprayed directly
with molybdenum blue: 4.2 M sulfuric acid (1:1, v/v).
[0169] Rf values for authentic FDP, geranylgeranyl diphosphate
(GGDP), dolichyl monophosphate (DOL-MP), cholesterol, farnesol,
squalene (Sigma Chemical Co.) and FMP (American Radiolabeled
Chemicals, Inc.) are indicated for comparison. Compounds VI, VII
and VIII frequently ran as doublets suggesting the presence of
isomers or closely related compounds in each major peak.
[0170] The isolation and elucidation of phosphorylated
non-saponifiable neutrophil lipids are depicted by FIGS. 2(a-c).
Specifically, GC/MS of compound VI before and after acid hydrolysis
shows a mass spectrum of compound VI consistent with presqualene
diphosphate with a limited ion chromatogram for m/z 136 (i.e., 2
internal isoprene units) after direct injection. Acid-treated
compound VI was found to shift to 8.70 min and had a mass spectrum
now consistent with squalene.
[0171] After quantitation by phosphorus determination, .about.500
ng/compound in hexane (2 .mu.l) was injected into GCIMS
(Hewlett-Packard Co., Model 5890 GC and 5781A MS). To enhance
detection, Me.sub.3Si-derivatives were produced by treatment with
BSTFA (N,O-bis(trimethylsiyl) tri-fluoroacetamide), or compounds
were subjected to acid (0.01 N HCl in 50% methanol, pH
2.0@100.degree. C., 20 min) to liberate pyrophosphate when present.
Diagnostic ions in (FIG. 2a), namely m/z 489 (M-96), 447
M-[96-C(CH.sub.3).sub.2], 410 M-(HP.sub.2O.sub.7), 341
[(HP.sub.2O.sub.7)--(C(CH.sub.3).sub.2CHCH.sub.2)], 273
M-[(HP.sub.2O.sub.7)-69-(CH.sub.2C(CH.sub.3)CHCH.sub.2)], 205
M-[(HP.sub.2O.sub.7)-69-(CH.sub.2C(CH.sub.3)CHCH.sub.2.times.2)],
136 (CH.sub.2C(CH.sub.3)CHCH.sub.2.times.21, 95
[C(CH.sub.3).sub.2CH(CH.sub.2- ).sub.2C], 81
[(CH.sub.2C(CH.sub.3)CH(CH.sub.2).sub.2)-H.sup.+], and 69 [base
peak; (CH.sub.3).sub.2CCHCH.sub.2] were consistent with presqualene
diphosphate, and in (FIG. 2c), squalene (Popjk, G., Edmond, J.
Clifford, K. & Williams, V. J. Biol. Chem. 244, 1897-1918
(1969), Epstein, W. W. & Rilling. H. C. J. Biol. Chem.
245,45974605 (1970)).
[0172] PMN (20.times.10.sup.6/ml in PO.sub.4-free HBSS (Hanks
buffered saline solution) were labeled with
.gamma.-.sup.32PO.sub.4-ATP (40 .mu.Ci/ml, 90 min, 37.degree. C.),
and saponified (10% KOH in methanol, 30 min, 37.degree. C.).
Non-saponifiable lipids were extracted and separated by
one-dimensional TLC (chloroform:methanol:water (65:25:4 (v/v), 50
min, RT). .sup.32P content was determined by phosphoimager (Model
425E and integration software, Molecular Dynamics, Inc.)
(O=origin). Because destruction of the TLC plate was not required
for .sup.32P analysis, the same lane was later exposed to iodine
vapor and the corresponding Rf values for compounds V-VIII are
demonstrated for the purposes of comparison (representative of n=6,
d=12). To determine FMLP (10-.sup.7 M)-induced changes in compound
VI and VIII mass (inset), radioactive compounds were eluted and
inorganic phosphorus levels were measured. In addition, PMN labeled
with .gamma.-.sup.32PO.sub.4 ATP were incubated in the presence or
absence of GM-CSF (25 ng/ml, 60 min, 37.degree. C.)(b) with
.sup.32P content quantitated as above. Values represent the
mean.+-.SEM for duplicate determinations from at least two separate
PMN. The percent increase after exposure to GM-CSF is relative to
parallel incubations with cells in the absence of agonist.
**p<0.0005 and * p<0.03 by Student's paired t-test.
[0173] FIGS. 3a-e represent neutrophils which incorporate 32P04
into non-saponifiable lipids; receptor activation triggers
incorporation and turnover in compounds VI and VIII. For example,
FIG. 3a depicts a representative phosphoimager profile of
.sup.32P-content after TLC of resting neutrophil extracts from
cells incubated with .gamma..sup.32PO.sub.4-ATP and (inset), time
course for FMLP-stimulated changes in phosphorus content of
compounds VI and VIII. FIG. 3b shows the impact of GM-CSF (b) on
PO.sub.4 incorporation into compounds VI and VIII.
[0174] FIGS. 4a-f demonstrate how compound VI selectively inhibits
neutrophil responses.
[0175] Impact of compounds VI and VIII and related isoprenoids on
(FIG. 4a) FMLP and (FIG. 4b) PMA-stimulated superoxide anion
generation with electroporated neutrophils and dose response (b.
inset) of compound VI's inhibition of O.sub.2 -- production. FIG.
4c is a representative tracing of neutrophil homotypic adhesion.
FIG. 4d shows the % inhibition of FMLP-stimulated homotypic
adhesion and FIG. 4e, IP.sub.3 formation. FIG. 4f is a proposed
scheme for polyisopryenyl phosphate regulation of neutrophil
responses.
[0176] Neutrophils (10.sup.7/500 .mu.l) were electroporated (1.65
kV/cm, 250 .mu.F,.OMEGA..infin., Invitrogen Electroporator VI) at
0.degree. C. in the presence of 10.sup.-9-10-.sup.6 M compound VI,
VIII, FDP, FMP, squalene or vehicle (ethanol 0.2%). After
electroporation, cells, in cytochrome c (0.7 mg/ml), were exposed
(10 minutes, 37.degree. C.) to either cytochalasin b (5 .mu.g/mL)
and FMLP (10.sup.-7 M)(a) or PMA ((10.sup.-7 M)(b) and supernatants
monitored at 550 mm. Electroporation gave a 49.9% reduction in
FMLP-stimulated NADPH oxidase compared to naive cells. Values
(FIGS. 4a and 4b) represent the mean.+-.SEM for n.gtoreq.3,
d.gtoreq.6. For adhesion (FIGS. 4c and 4d), changes in light
transmittance were monitored (as in Fiore, S., Nicolaou, K. C.,
Caulfield, T., Kataoka, H. & Serhan, C. N. Biochem. J. 266,
25-31 (1990), the teachings of which are incorporated herein by
reference) after addition of compounds VI, VIII (1.6 .mu.M) or
vehicle (0.5 min) and FMLP (0.5-5 .mu.M) (2.5 min). Cell viability
remained >99%. Values represent the mean.+-.SEM for n=3, d=9.
1,4,5-inositol-triphosphate levels were determined (Amersham) in
fresh PMN (10.sup.7 cells) exposed (5 minutes, 37.degree. C.) to
compound VI, compound VIII (1 .mu.M) or vehicle and stimulated with
FMLP (10-.sup.7M, 37.degree. C.) (as in FIG. 4e). % Inhibition
after 10 second exposure to FMLP is reported as mean.+-.SEM for
n=4, d.gtoreq.8. *p<0.05 in Student's paired t-test compared
with control, while "NS" indicates no significant change compared
to incubations in the presence of vehicle (ethanol 0.2%) alone.
Stereochemistry of PSDP and PSMP (FIG. 4f) were assigned by routine
methods (Poulter, C. D. and Rilling, H. C. Acc. Chem. Res. 11,
307-313, (1978), the teachings of which are hereby incorporated by
reference).
[0177] Phospholipase D (PLD) plays a central role in cellular
activation in diverse cell types via the release of the bioactive
fatty acid, phosphatidate. Several agonists operating through
different classes of receptors have been defined that stimulate
cellular PLD activity. No known endogenous regulators of PLD have
been identified. Activation of neutrophils leads to an increase in
intracellular phosphatidic acid that is generated predominantly via
the hydrolysis of phosphatidyl choline by phospholipase D (Agwu, D
E, et al., Journal of Clinical Investigation 1991; 88:531). Because
PSDP inhibited phosphatidate mediated superoxide anion generation
in human neutrophil sonicates (Levy, B D, et al., Nature 1997;
389:985), its impact on phospholipase D was determined. A purified
enzyme was utilized to directly evaluate the action of PSDP with
PLD. Lineweaver-Burk plots for PLD (3 units/125 .mu.L; EC 3.1.4.4,
Sigma Chemical Co.) in the presence of EtOH (0.2%) or increasing
concentrations of PSDP are reported in FIG. 5. After addition of
PSDP to PLD (5 min, 30.degree. C.), phosphatidyl choline (lecithin)
in 60 mM Tris/HCl+10 mM CaC1.sub.2, pH 7.5 was added (30.degree.
C.) and reactions were terminated at 30 second intervals (t0-90sec)
with Tris-HCl (1 M) plus EDTA (50 mM). Choline release was
quantitated as in Abousalham A. et al. Biochimica et Biophysica
Acta 1;1158 (1993). For PLD, the Vmax was 2.49 nmoles choline/sec
and the Km was 3.62 mM (n.gtoreq.3, d.gtoreq.2). PSDP inhibited
this enzyme and gave a Ki<100 nM. These results indicate that
PSDP is a potent inhibitor of PLD and suggest that the mechanism
for the inhibition, based on the L-B analysis, is competition for
the enzyme's substrate. In addition, this assay is ideally suited
to screen novel polyisoprenyl phosphate analogs for
bioactivity.
[0178] In addition to PLD, phospholipase C plays a critical role in
intracellular signaling via the hydrolysis of inositol lipids to
diacyl glyceride (DAG) and inositol trisphosphate (IP.sub.3) to
effect a cascade of cellular events in response to pro-inflammatory
stimuli. DAG activates protein kinases and is converted, in part,
to phosphatidic acid by DAG kinase. Therefore, PLC together with
DAG kinase provides a second major source of phosphatidic acid in
human neutrophils (Agwu, D E, et al., Journal of Clinical
Investigation 1991; 88:531). Because of PLC's participation in
phosphatidate formation, PSDP was analyzed to determine whether it
also inhibited this enzyme's activity. To this end, the impact of
PSDP on fMLP-stimulated IP.sub.3 formation as a surrogate for
phospholipase C activity was examined (FIG. 4e). Incubation (5 min.
37.degree. C.) of freshly isolated human neutrophils
(10.times.10.sup.6) with PSDP (1 .mu.M) just prior to exposure to
fMLP (100 nM) resulted in >50% inhibition of IP.sub.3 levels 10
seconds after exposure to an agonist (time to peak formation in
these cells) as determined using a D-myo-inositol
1,4,5-trisphosphate[.sup.3H]-based assay (Biotrak radioimmunoassay
system, Amersham Life Science #TRK 1000).
[0179] Inositol trisphosphate is known to be an important
intracellular mediator of calcium mobilization from intracellular
stores, a critical event in cellular activation (Exton, J H, Annual
Review of Pharmacology and Toxicology 1996; 36:481). In order to
determine the functional importance of the PSDP-mediated reduction
in IP.sub.3 levels, fMLP-stimulated intracellular calcium
mobilization in the presence of polyisoprenyl phosphates was
measured. FURA-2 (1 .mu.M) loaded neutrophils (10.times.10.sup.6/2
ml) in the presence of EDTA (5 mM) were exposed (5 min, 37.degree.
C.) to PSDP (10.sup.-5-10.sup.-6 M) or EtOH (0.1%), followed by 1
nM FMLP. FIG. 6 shows a representative incubation demonstrating
inhibition by PSDP (1 .mu.M) of fMLP-stimulated [Ca.sup.2+].sub.i
mobilization. This inhibition was concentration dependent FIG. 7
and significant (n=3, mean.+-.SEM, *P<0.05 by Student's t-test).
Together these results indicate that in addition to phospholipase
D, PSDP also inhibits PLC activity and that this assay can be
utilized to screen novel polyisoprenyl phosphate analogues for
bioactivity.
[0180] SH2 and SH3 domains on intracellular proteins are thought to
be critical sites of phosphorylation by specific kinases in the
regulation of signal transduction (Pawson, T., Schlessinger, J.,
Current Biology 1993; 3:434). Of special interest during
inflammation and wound repair, these domains appear to play pivotal
roles during cytokine (e.g., growth factors) signaling via tyrosine
kinases. Granulocyte/macrophage-colony stimulating factor
stimulates phosphate turnover in presqualene diphosphate and
presqualene monophosphate (FIG. 3b) indicating a potential role for
these compounds in cytokine signaling. Furthermore, SH2 or SH3
domains are present on important regulatory intracellular enzymes
(e.g., PLC.gamma.) and have been reported to play pivotal roles in
neutrophil responses (e.g., NADPH oxidase activation) (Ree, S G, et
al., Journal of Biological Chemistry, 1997; 272:15045) (McPhail, L
C, Journal of Experimental Medicine, 1994; 180:201 1) Therefore, it
was determined whether polyisoprenyl phosphates could associate
with these important intracellular phosphate-sensing domains.
Equilibrium binding was determined for .sup.32PO.sub.4-PSDP (1
.mu.M) to the adaptor protein, Grb2, which is known to carry two
SH3 domains and one SH2 domain (Lowenstein, E J, et al., Cell,
1992; 70:43 1). Radiolabeled PSDP bound to a Gst-Grb2 fusion
protein complexed to agarose beads (Upstate Biotechnology #14-128)
after incubation for 20 min at 37.degree. C. Bound material was
separated from unbound by spin filtration (0.2 .mu.m) and
quantitated by scintillation counting. The radioligand could be
competed off Gst-Grb2 by excess unlabeled PSDP or delipidated
albumin (50 .mu.g), but not by the SH3 containing peptide, Sos (50
.mu.g) (FIG. 8; n=3, mean.+-.SEM, *P<0.01). These results
indicate that PSDP can associate with SH2 domains and suggests
another potential intracellular target for this novel messenger. In
addition, these studies define a novel screening assay for
association of phosphorylated lipids with protein phosphate-sensing
domains in lipid-protein interactions.
[0181] V. Utilities
[0182] The compounds of this invention have the biological activity
of natural PSDPs, but are more resistant to degradation or
alternatively inhibit the degradation of natural PSDPs. The
disclosed compounds therefore have utility as pharmaceuticals for
treating or preventing a number of diseases or conditions
associated with inadequate or inappropriate inflammatory mediated
cellular response in a subject.
[0183] Also encompassed by this invention is a method of screening
PSDP analogs or other compounds to identify those having a longer
tissue half-life than the corresponding natural PSDP. This method
can be used to determine whether the compound inhibits, resists, or
more slowly undergoes metabolism compared to the natural PSDP. This
method is performed by preparing at least one enzyme which
metabolizes PSDPs, contacting the compound with the enzyme
preparation, and determining whether the compound inhibits,
resists, or more slowly undergoes metabolism by the enzyme. Cells
having a PSDP recognition site, such as polymorphonuclear
neutrophils, peripheral blood monocytes, and differentiated HL-60
cells are among the appropriate sources for the enzyme preparation.
The PSDP recognition site may exist naturally, or be induced
artificially, by a disease state, or by an injury. A non-limiting
example of artificially-induced PSDP recognition sites is the
induction of such sites in differentiated HL-60 cells in
culture.
[0184] PSDP analogs can also be screened for binding activity with
a PSDP receptor recognition site, for example by contacting the
compound with a receptor recognition site and determining whether
and to what degree the compound binds. Examples of kinetic binding
assays include homologous displacement, competitive binding,
isotherm, and equilibrium binding assays.
[0185] The receptor recognition site may normally exist or it may
be induced by a disease state, by an injury, or by artificial
means. For example, retinoic acid, PMA, or DMSO may be used to
induce differentiation in HL-60 cells. Differentiated HL-60 cells
express PSDP-specific receptor recognition sites. Examples of other
cells which may be screened for PSDP specificity include PMN,
epithelial cells, and peripheral blood monocytes or vascular
endothelial cells.
[0186] Selection of competitive ligands will depend upon the nature
of the recognition site, the structure of the natural substrate,
any structural or functional analogs of the natural substrate known
in the art, and other factors considered by a skilled artisan in
making such a determination. Such ligands also include known
receptor antagonists. The compounds of this invention may be
radiolabelled with isotopes including .sup.2H, .sup.3H, .sup.13C,
and .sup.10C. by standard techniques known in the art of
radiochemistry or synthetic chemistry.
[0187] In one embodiment, the present invention pertains to a
method of screening for a compound which modulates phospholipase D
(PLD) activity. The method includes combining the compound with
phospholipase D (PLD), thereby forming a mixture and treating the
mixture with phosphatidylcholine, such that a cleavage reaction
between phosphatidylcholine and PLD can occur, resulting in
generation of phosphatidic acid and choline. The amount of choline
produced is evaluated such that a compound which modulates PLD
activity is determined. Preferably, the compound is PSDP or a PSDP
analog which decreases cleavage of phosphatidylcholine by PLD.
Alternatively, the compound increases cleavage of phosphatidyl
choline by PLD. includes an oxidation step of choline. The method
can further include an oxidation step of choline, e.g., the
oxidation mixture can be peroxidase, 4-aminoantipyrine, phenol and
choline oxidase.
[0188] The terms "modulates" and "modulating" are intended to
include inhibition, eradication, or prevention of an activity,
e.g., intracellular signaling or production of an undesired
chemical species. In some instances the terms "modulates" and
"modulating" also includes the increase of an activity where
beneficial to the physiology of the cell or tissue environment.
[0189] In another embodiment, the present invention pertains to a
method of screening for a compound which modulates intracellular
signaling. The method includes combining a compound with
phospholipase D (PLD), thereby forming a mixture and treating the
mixture with phosphatidylcholine, such that a cleavage reaction
between phosphatidylcholine and PLD occurs, resulting in generation
of phosphatidic acid and choline. The amount of choline produced is
evaluated, such that a compound which modulates intracellular
signaling is determined. Preferably, the compound is PSDP or a PSDP
analog which decreases cleavage of phosphatidylcholine by PLD.
Alternatively, the compound increases cleavage of phosphatidyl
choline by PLD. The method can include an oxidation step of
choline. The method can further include an oxidation step of
choline, e.g., the oxidation mixture can be peroxidase,
4-aminoantipyrine, phenol and choline oxidase. Alternatively,
methods of detection include those recognized by those skilled in
the art and include, for example, TLC, HPLC, MS, GC, etc.
[0190] In still another embodiment, the invention pertains to a
method of screening for a compound which associates with protein
phosphate-sensing domains. The method includes contacting a
Gst-Grb2 fusion protein complexed to a support with a labeled lipid
compound, e.g., a radiolabeled PSDP or PSDP analog, with and
evaluating the amount of labeled compound associated with the
protein. The method can further include treating the labeled lipid
compound associated with a competing compound and evaluating the
amount of labeled compound removed from the Gst-Grb2 fusion
protein. Preferably, the support is agarose. Competing compounds
are unlabeled PSDP, PSDP analogs, or delipidated albumin.
[0191] The term "competing compound" is intended to include those
compounds which can displace labeled compounds, such as
radiolabeled PSDP, which are associated with the Gst-Grb2 fusion
protein.
[0192] The terms "associates" and "associated with" are intended to
include those physical interactions between a lipid and a protein,
e.g., hydrophobic, hydrophilic, ionic, hydrogen bonding, dipole
interactions, etc. which cause the lipid and protein to bind to
each other, e.g., absorption or adsorption.
[0193] In yet another embodiment, the invention pertains to a
method of screening for a compound which modulates the production
of inositol triphosphate. The method includes treating
polymorphoneutrophils with a stimulation agent, e.g., fMLP, causing
activation of cell, thereby producing inositol phosphate and
treating the activated cells with a modulating compound. The effect
of the modulating compound, e.g., PSDP or a PSDP analog, is
measured on production of the inositol phosphate. A preferred
stimulation agent is FMLP. Other stimulating agents include
leukotrienes, cytokines, granulocyte monocyte colony stimulating
factors (gm-csf), lipopolysaccharides and CFA. A preferred
modulating compound is PSDP, e.g., an inhibititory compound.
[0194] In still another embodiment, the invention pertains to a
method of screening for a compound which modulates neutrophil
activation. The method includes treating neutrophils with a
stimulation agent, e.g., FMLP, causing activation of the
neutrophils, thereby producing inositol phosphate and treating the
activated neutrophils with a modulating compound. The effect of the
modulating compound, e.g., PSDP or a PSDP anlog, is measured on
production of the inositol phosphate. A preferred stimulation agent
is fMLP. A preferred modulating compound is PSDP, e.g., an
inhibititory compound.
[0195] VI. Experimentals
[0196] Inositol Trisplosphate Assay
[0197] Amersham's D-myo-inositol 1,4,5-trisphosphate (IP.sub.3)
[.sup.3H] assay was used for this procedure (Amersham code TRK
1000, including product assay, the contents of which are
incorporated herein by reference). The assay is based on procedures
developed by Takata S. et al. J. Clin. Invest. 93:499 (1994) and
Dillon S. B. et al. J. Biol. Chem. 262:11546 (1987), the contents
of which are incorporated herein by reference in their entirety.
24
[0198] Solutions of 250 .mu.l of PMN (10.times.10.sup.6) in
phosphate buffered saline with 1 mM CaCl.sub.2 and MgCl.sub.2
(physiological pH) were placed into 1.5 ml Eppendorf tubes and
warmed at 37.degree. C. for 3 minutes. To the solutions were added
25 .mu.l of 0.25% aqueous ethanol or a test compound in 0.25%
aqueous ethanol. (Test compounds included PSDP and PSMP in
concentrations between 10.sup.-8 and 10.sup.-6 M) The mixture was
incubated for 5 minutes at 37.degree. C. To this solution was added
25 .mu.l of 0.25% aqueous ethanol or FMLP (10.sup.-7 M) in 0.25
aqueous ethanol for a total solution of 300 .mu.l. The solution was
incubated for 0 to 60 seconds and the reaction was stopped with 100
.mu.l of 10% perchloric acid (PCA) with vortexing.
[0199] The cell extract was centrifuged at 14,000 rpm for 5 minutes
at 4.degree. C. The supernatant was transferred to new tubes, to
which was added {fraction (1/40)} volume (10 .mu.l) of 1 M HEPES
(pH 7.4) and {fraction (1/20)} volume (20 .mu.l) of pH indictor
(Fisher). The color was adjusted from pink to green with 10 N KOH
for a pH between 7 and 8 (14-18 .mu.l KOH generally). (If pH>8
5% PCA was added until pH was between 7-8). The mixture was chilled
at O.degree. C. for 20 minutes.
[0200] The supernatants were centrifuged at 14,000 rpm for 5
minutes at 4.degree. C. To the supernatants were added 50 .mu.l of
buffer (0.1 M Tris buffer (pH 9.0) containing 4 mM EDTA and 4 mg/ml
BSA) and 50 .mu.l tracer radiolabel (D-myo-[.sup.3H]inositol
1,4,5-trisphosphate, 3 nmol in water) per tube. To this was added
50 .mu.l standards (D-myo-inositol 1,4,5-trisphosphate, 3 nmol in
water) or experimental samples (compounds) per tube. To this was
added 50 .mu.l binding protein (IP.sub.3 binding protein). The
solution was chilled at 0.degree. C. for 15 minutes.
[0201] The samples were centrifuged at 10,000 rpm for 5 minutes at
4.degree. C. to separate bound from unbound IP.sub.3. The
supernatant was discarded by gentle vacuum and the resultant pellet
was resuspended in 100 .mu.l water with vortexing. The dissolved
pellet was subjected to scintillation counting and rates of
reaction were determined (See FIGS. 6 and 7).
[0202] PSDP Equilibrium Binding Assay to GST-GRB2
[0203] GST-GRB2 complexed to agarose beads was purchased from
Upstate Biotechnology, Inc. Microcentrifuge spin nylon filters (0.2
.mu.m) were purchased from PGC Scientific).
[0204] To 100 .mu.l of 0.9% NaCl was added 2 .mu.g GST-GRB2 with
0-100 .mu.g compound (e.g., delipidated human serum albumin,
SH3-bearing SOS peptide or unlabeled PSDP) to be tested. The
samples were prepared directly in the top of PGC spin filter
tubes.
[0205] To the solution was added radiolabeled-PSDP in 1 .mu.l
ethanol. The mixture was vortexed and incubated at 37.degree. C.
for 30 minutes. After the incubation period, the sample was
centrifuged at 2000 rpm at 25.degree. C. for 3 minutes. The top of
the tube was rinsed with 200 .mu.l of chloroform:methanol 2:1 (v/v)
(the sample should not be vortexed at this stage). This sample was
centrifuged at 2000 rpm at 25.degree. C. for 3 minutes. The filter
was removed, placed in a scintillation vial and the amount of
radioactivity bound to the trapped agarose complex (i.e., bound
material) was determined.
[0206] 1 ml of CHCl.sub.3:MeOH (2:1) was added to the filtrate (a
clear interface should be evident). The upper phase was removed and
discarded and the lower phase was brought to dryness under a gentle
stream of nitrogen. The concentrate was resuspended in 40 .mu.l
CHC1.sub.3:MeOH (2:1 v/v). At this stage, dependent upon the amount
of radioactivity present, the radioactivity of the sample was
determined either in scintillation fluid or by phosphoimage after
TLC. When necessary, TLC was performed in CHCl.sub.3:MeOH:water
65:25:4 (v/v/v) at 25.degree. C..times.50 minutes. The phosphoimage
was analyzed by densitometry (FIG. 8).
[0207] Bound material is reflected in the filtrate by loss of
radioactivity when radiolabeled material is incubated in the
presence of the GRB2 complex compared to the absence of the agarose
complex. The difference is defined as 100% binding so that
comparisons can be made to unknown compounds tested in
parallel.
[0208] VII. Equivalents
[0209] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to specific embodiments of the invention described
specifically herein. Such equivalents are intended to be
encompassed in the scope of the following claims. The contents of
all references and issued patents cited throughout all portions of
this application including the background are expressly
incorporated by reference.
* * * * *