U.S. patent application number 10/374003 was filed with the patent office on 2003-08-14 for methods for reducing intraocular pressure.
Invention is credited to Civan, Mortimer M., Jacobson, Kenneth A., Mitchell, Claire H., Stone, Richard A..
Application Number | 20030153626 10/374003 |
Document ID | / |
Family ID | 26787065 |
Filed Date | 2003-08-14 |
United States Patent
Application |
20030153626 |
Kind Code |
A1 |
Civan, Mortimer M. ; et
al. |
August 14, 2003 |
Methods for reducing intraocular pressure
Abstract
A method for decreasing intraocular pressure by administering an
A.sub.3 subtype adenosine receptor antagonist, a calmodulin
antagonist or an antiestrogen such as tamoxifen. These agents, by
inhibiting influx or promoting efflux of aqueous humor, can be used
to treat glaucoma.
Inventors: |
Civan, Mortimer M.;
(Wynnewood, PA) ; Stone, Richard A.; (Havertown,
PA) ; Mitchell, Claire H.; (Philadelphia, PA)
; Jacobson, Kenneth A.; (Silver Springs, MD) |
Correspondence
Address: |
Evelyn H. McConathy, Esquire
Dilworth Paxson LLP
3200 Mellon Bank Center
1735 Market Street
Philadelphia
PA
19103
US
|
Family ID: |
26787065 |
Appl. No.: |
10/374003 |
Filed: |
February 25, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10374003 |
Feb 25, 2003 |
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09743744 |
May 4, 2001 |
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6528516 |
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09743744 |
May 4, 2001 |
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PCT/US99/16211 |
Jul 15, 1999 |
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60122965 |
Mar 3, 1999 |
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60093097 |
Jul 16, 1998 |
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Current U.S.
Class: |
514/651 |
Current CPC
Class: |
A61K 31/455 20130101;
A61K 31/4425 20130101; A61K 31/517 20130101; A61K 31/138 20130101;
A61P 27/06 20180101; A61K 31/435 20130101; A61K 31/00 20130101;
Y10S 514/913 20130101; A61K 31/5415 20130101; A61K 31/4422
20130101; A61K 31/498 20130101 |
Class at
Publication: |
514/651 |
International
Class: |
A61K 031/137 |
Claims
1. A method for reducing intraocular pressure in an individual with
an ocular disorder, comprising the step of administering to said
individual an effective intraocular pressure-reducing amount of a
pharmaceutical composition comprising an A.sub.3 subtype adenosine
receptor antagonist.
2. The method of claim 1, wherein said A.sub.3 subtype receptor
antagonist is a dihydropyridine, pyridine, pyridinium salt or
triazoloquinazoline.
3. The method of claim 1, wherein said A.sub.3 subtype receptor
antagonist is selected from the group consisting of MRS-1097,
MRS-1191, MRS-1220, MRS-1523 and MRS-1649.
4. The method of claim 1, wherein said pharmaceutical composition
is administered topically, systemically or orally.
5. The method of claim 1, wherein said pharmaceutical composition
is an ointment, gel or eye drops.
6. The method of claim 1, wherein said ocular disorder is
glaucoma.
7. A method for reducing intraocular pressure in an individual with
an ocular disorder, comprising the step of administering to said
individual an effective intraocular pressure-reducing amount of a
pharmaceutical composition comprising an antiestrogen.
8. The method of claim 7, wherein said antiestrogen is
tamoxifen.
9. The method of claim 7, wherein said pharmaceutical composition
is administered topically, systemically or orally.
10. The method of claim 7, wherein said pharmaceutical composition
is ointment, gel or eye drops.
11. The method of claim 7, wherein said ocular disorder is
glaucoma.
12. A method for reducing intraocular pressure in an individual
with an ocular disorder, comprising the step of administering to
said individual an effective intraocular pressure-reducing amount
of a pharmaceutical composition comprising a calmodulin
antagonist.
13. The method of claim 12, wherein said calmodulin antagonist is
trifluoperazine.
14. The method of claim 12, wherein said pharmaceutical composition
is administered topically, systemically or orally.
15. The method of claim 12, wherein said pharmaceutical composition
is ointment, gel or eye drops.
16. The method of claim 12, wherein said ocular disorder is
glaucoma.
17. A method for reducing intraocular pressure in an individual
with an ocular disorder, comprising the step of administering to
said individual an effective intraocular pressure-reducing amount
of a pharmaceutical composition comprising a prodrug which is
converted into a A.sub.3 subtype adenosine receptor antagonist
after said administering step.
18. An A.sub.3 subtype adenosine receptor antagonist for use in
reduction of intraocular pressure.
19. The A.sub.3 subtype adenosine receptor antagonist of claim 18,
wherein said receptor antagonist is selected from the group
consisting of MRS-1097, MRS-1191, MRS-1523 and MRS-1649.
20. Use of an antiestrogen in the preparation of a medicament for
the reduction of intraocular pressure.
21. The use of claim 20, wherein said antiestrogen is
tamoxifen.
22. A calmodulin antagonist for use in reduction of intraocular
pressure.
23. The calmodulin antagonist of claim 22, wherein said calmodulin
antagonist is trifluoperazine.
24. A prodrug which is converted into an A.sub.3 receptor
antagonist in vivo for use in reduction of intraocular pressure.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the use of A3 subtype
adenosine receptor antagonists, calmodulin antagonists and
antiestrogens for reduction of intraocular pressure.
BACKGROUND OF THE INVENTION
[0002] The aqueous humor of the eye is formed by the ciliary
epithelium, which comprises two cell layers: the outer pigmented
epithelial (PE) cells facing the stroma and the inner nonpigmented
epithelial (NPE) cells in contact with the aqueous humor. Secretion
is generally thought to reflect a primary transfer of solute,
largely NaCl, from the stroma to the aqueous humour, providing an
osmotic driving force for the secondary osmotic transfer of water
down its chemical gradient (Cole,. Exp. Eye Res. 25 (Suppl.),
161-176, 1977), although a more direct coupling between water and
solute may also proceed across epithelia (Meinild et al. J.
Physiol. 508: 15-21, 1998). One major factor governing the rate of
secretion is the rate of chloride ion (Cl.sup.-)release from the
NPE cells into the aqueous humor (Civan, News Physiol. Sci.
12:158-162, 1997). The activity of Cl.sup.- channels is likely to
be a rate-limiting factor in aqueous humour secretion, given the
low baseline level of channel activity and the predominance of the
chloride anion in the fluid transferred (Coca-Prados et al., Am. J.
Physiol. 268: C572-C579, 1995). The secretion of aqueous humor into
the eye is believed to result as a consequence of two opposing
physiological processes: fluid secretion into the eye by the NPE
cells and fluid reabsorption (secretion out of the eye) by the PE
cells. Thus, both release of Cl.sup.- by the nonpigmented ciliary
epithelial (NPE) cells into the adjacent aqueous humour would
enhance secretion, and Cl.sup.- release by the pigmented ciliary
epithelial (PE) cells into the neighboring stroma would reduce net
secretion (Civan, Current Topics in Membranes 45: 1-24, 1998).
[0003] Recently, adenosine has been found to activate NPE Cl.sup.-
channels which subserve this release (Carre et al., Am. J. Physiol.
(Cell Physiol. 42) 273:C1354-C1361, 1997). Purines, a class of
chemical compounds which includes adenosine, ATP and related
compounds, may regulate aqueous humour secretion, in part through
modifying Cl.sup.--channel activity. Both NPE and PE cells have
been reported to release ATP to the extracellular surface, where
ATP can be metabolized to adenosine by ecto-enzymes (Mitchell et
al. Proc. Natl. Acad. Sci U.S.A. 95: 7174-7178, 1998), and both
cell types possess adenosine receptors (Wax et al., Exp. Eye Res.
57:89-95, 1993; Wax et al, Invest. Ophthalmol. Vis. Sci.,
35:3057-3063, 1994; Kvanta et al., Exp. Eye Res. 65:595-602, 1997)
and ATP receptors (Wax et al., supra., 1993; Shahidullah et al.,
Curr. Eye Res. 16:1006-1016, 1997). Furthermore, in vitro studies
of rabbits have associated A.sub.2-adenosine receptors with
increased secretion and elevated intraocular pressure (Crosson et
al., Invest. Ophthalmol. Vis. Sci. 37:1833-1839, 1996) and
A.sub.1-adenosine receptors with the converse (Crosson, J.
Pharmacol. Exp. Ther. 273:320-326, 1995). Qualitatively similar
associations with intraocular pressure, but not with secretion,
have been observed in cynomologus monkeys (Tain et al., Exp. Eye
Res., 64: 979-989, 1997). A particular role for Cl.sup.- channels
has been suggested by the observations that adenosine agonists
stimulate Cl.sup.- channels of immortalized human and
freshly-dissected bovine NPE cells and of aqueous-oriented Cl.sup.-
channels of the intact rabbit iris-ciliary body (Carr et al.,
supra., 1997). Adenosine triggered isotonic shrinkage of cultured
human cells from the HCE cell line. The contribution of Cl.sup.-
channels to this shrinkage was identified by performing the
experiments in the presence of the cation ionophore gramicidin. In
addition, adenosine produced a Cl.sup.- dependent increase in
short-circuit current across rabbit iris-ciliary body while the
non-metabolizable adenosine analogue 2-Cl-adenosine was shown to
activate Cl.sup.- currents in HCE cells using the whole cell
patch-clamp technique. Although this study clearly established that
adenosine could activate Cl.sup.- channels on NPE cells, the
concentrations of agonist used were capable of stimulating all four
known adenosine receptor sub-types: A.sub.1, A.sub.2A, A.sub.2B and
A.sub.3 (Fredholm et al., Pharmacol. Rev., 46:143-156, 1994;
Fredholm et al., Trends Pharmacol. Sci, 18:79-82, 1997; Klotz et
al., Naunyn Schmiedebergs Arch. Pharmacol., 357:1-9., 1998).
Ciliary epithelial cells are known to possess A.sub.1 A.sub.2A and
A.sub.2B adenosine receptors (Kohno et al., Blood, 88:3569-3574,
1996, Stambaugh et al., Am. J. Physiol. 273 (Heart Circ. Physiol.
42):H501-H505, 1997; Wax et al., supra., 1994). Although
stimulation of these receptors can be associated with specific
changes in the levels of second messengers cAMP (Crosson, supra.;
Stambaugh et al., supra.; Wax et al., supra., 1994) and Ca.sup.2+
(Farahbakhsh et al., Exp. Eye Res., 64:173-179, 1997), the effect
of these receptors upon Cl.sup.- channels of NPE cells was
unknown.
[0004] Alternatively, the intraocular pressure could be reduced by
stimulating reabsorption of aqueous humor. In principle, this could
be achieved by activating chloride channels on the basolateral
surface of the pigmented cell layer. This would release chloride
back into the stroma. One way to accomplish this with the PE cells
has been identified using the antiestrogen tamoxifen. Tamoxifen is
known to exert multiple actions on biological cells. However,
recently, Mitchell et al. (Invest Ophthalmol. Vis. Sci.
38(Suppl.):S1042, 1997) have noted that the only known action of
tamoxifen which could account for the phenomenon is its
antiestrogenic activity, probably on the plasma membrane.
[0005] Glaucoma is a disorder characterized by increased
intraocular pressure that may cause impaired vision, ranging from
slight loss to absolute blindness. The increased intraocular
pressure is related to an imbalance between production and outflow
of the aqueous humor. Current drugs prescribed for glaucoma, in the
form of eyedrops, include pilocarpine, timolol, betaxolol,
levobunolol, metipranolol, epinephrine, dipivefrin, latanoprost,
carbachol, and potent cholinesterase inhibitors such as
echothiophate and carbonic anhydrase inhibitors such as
dorzolamidet. Many of these effective approaches to medical therapy
of glaucoma involve a reduction in the rate of flow into the eye.
However, none of these drugs are satisfactory, in part due to side
effects.
[0006] Because of side effects of available agents and inconvenient
dosing schedule, there is an ongoing need for compounds capable of
reducing intraocular pressure for the treatment of glaucoma with
improved efficacy, prolonged action and reduced side effects. The
present invention addresses this need.
SUMMARY OF THE INVENTION
[0007] One embodiment of the present invention is a method for
reducing intraocular pressure in an individual, comprising the step
of administering to the individual an effective intraocular
pressure-reducing amount of a pharmaceutical composition comprising
an A.sub.3 subtype adenosine receptor antagonist. In one aspect of
this preferred embodiment, the A.sub.3 receptor antagonist is a
dihydropyridine, pyridine, pyridinium salt or triazoloquinazoline.
Preferably, the A.sub.3 subtype receptor antagonist is selected
from the group consisting of MRS-1097, MRS-1191, MRS-1220 and
MRS-1523. Advantageously, the pharmaceutical composition is
administered topically, systemically or orally. Preferably, the
pharmaceutical composition is an ointment, gel or eye drops.
[0008] Another embodiment of the present invention is a method for
reducing intraocular pressure in an individual, comprising the step
of administering to the individual an effective intraocular
pressure-reducing amount of a pharmaceutical composition comprising
an antiestrogen. Preferably, the antiestrogen is tamoxifen.
Advantageously, the pharmaceutical composition is administered
topically, systemically or orally. Preferably, the pharmaceutical
composition is ointment, gel or eye drops.
[0009] The present invention also provides a method for reducing
intraocular pressure in an individual, comprising the step of
administering to the individual an effective intraocular
pressure-reducing amount of a pharmaceutical composition comprising
a calmodulin antagonist. Preferably, the calmodulin antagonist is
trifluoperazine. Advantageously, the pharmaceutical composition is
administered topically, systemically or orally. Preferably, the
pharmaceutical composition is ointment, gel or eye drops.
[0010] Another embodiment of the present invention is an A.sub.3
subtype adenosine receptor antagonist for use in reduction of
intraocular pressure. Preferably, the A.sub.3 subtype adenosine
receptor antagonist is MRS-1097, MRS-1191 or MRS-1523.
[0011] The present invention also provides the use of an
antiestrogen in the preparation of a medicament for the reduction
of intraocular pressure. Preferably, the antiestrogen is
tamoxifen.
[0012] The present invention also provides a calmodulin antagonist
for use in reduction of intraocular pressure. Preferably, the
calmodulin antagonist is trifluoperazine
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1 is a schematic diagram showing the ocular
nonpigmented epithelial (NPE) and pigmented epithelial (PE) cells,
and the effects of ATP, adenosine (Ado) and tamoxifen (TMX) on the
movement of aqueous humor. Ecto=ecto-enzymes; A.sub.3=A.sub.3
subtype adenosine receptor.
[0014] FIGS. 2-7 relate to the effects of A.sub.3 receptors on NPE
cells, while FIGS. 8-17 relate primarily to the actions of ATP on
PE cells.
[0015] FIGS. 2A-D show the concentration-response relationship for
the A.sub.3-selective agonist
N.sup.6-(3-iodobenzyl)-adenosine-5'-N-methyluro- namide
(IB-MECA)-stimulated isotonic shrinkage of NPE cells in the
presence of 5 .mu.M gramicidin. In FIGS. 2-5, solid trajectories
are least-square fits with monoexponentials, whereas data sets
displaying no significant shrinkage are connected by dotted
lines.
[0016] FIG. 2A:--least-squares fits yielded the following estimated
values for data obtained in parallel at concentrations of 30 nM-1
.upsilon.M IB-MECA (n=4 experiments): 30 nM [steady-state cell
volume (V.infin.)=98.5.+-.0.1%, .tau.=3.3.+-.1.5 min), and 1 .mu.M
(V.infin.=95.9.+-.0.3%, .tau.=2.4.+-.0.7 min). Control and 3 sets
of experimental results were significantly different (P<0.01,
F-test).
[0017] FIG. 2B--data obtained over concentration range of 1-10
.mu.M IB-MECA (n=4): 1 .mu.M (V.infin.=97.5.+-.0.1%,
.tau.=7.2.+-.1.0 min), 3 .mu.M (V.infin.=96.7.+-.0.3%,
.tau.=10.0.+-.2.3 min), and 10 .mu.M (V.infin.=97.9.+-.0.2%,
.tau.=3.2.+-.1.1 min). Data obtained at 1 .mu.M and 3 .mu.M did not
significantly deviate from the fit obtained with 10 .mu.M
IB-MECA.
[0018] FIG. 2C shows a Lineweaver-Burk plot generated from
nonlinear least-square fits of FIGS. 1A and 1B. Change in volume
was calculated as (VO-V.infin.). Variation with passage number was
noted for .tau. and (V0-V.infin.). For this reason, both
experimental sets (FIGS. 1A and 1B) included measurements with one
concentration (1 .mu.M) in common. Ratio (in V0-V.infin.) obtained
at 1 .mu.M in B to A was used as a scaling factor to accommodate
results obtained in B with 3 and 10 .mu.M. Using this approach,
linear least-squares analysis (r.sup.2=0.95) led to an estimated
value for the apparent K.sub.d of 55.+-.10 nM.
[0019] FIG. 2D shows isotonic cell shrinkage stimulated by 100 nM
Cl-IB-MECA. Fits for Cl-IB-MECA (V.infin.=97.9.+-.0.2%,
.tau.=2.5.+-.1.3 min) and IB-MECA (V.infin.=96.8.+-.0.4%,
.tau.=6.3.+-.2.2 min) are not significantly different
(P>0.05).
[0020] FIGS. 3A-3B show the effect of A.sub.3 antagonists on the
IB-MECA-stimulated isotonic shrinkage of NPE cells. FIG. 3A shows
that the A.sub.3-selective antagonist MRS-1097 (300 nM) prevented
shrinkage triggered by IB-MECA (P<0.01, F-distribution). FIG. 3B
shows that the A.sub.3-selective antagonist MRS-1191 (100 nM)
prevented characteristic shrinkage triggered by IB-MECA (n=4,
P<0.01 by F-distribution). MRS did not affect cell volume in the
absence of IB-MECA, confirming the specificity of the interaction
(n=4).
[0021] FIGS. 4A-4C shows the effects of selective A.sub.3-receptor
antagonists on adenosine-stimulated isotonic shrinkage of NPE
cells. Application of 300 nM MRS-1097 (FIG. 3A; n=4), 100 nM
MRS-1191 (FIG. 3B; n=3), and 100 nM MRS-1523 (FIG. 3C; n=3) all
prevented the characteristic shrinkage triggered by nonselective
activation of adenosine receptors with 10 .mu.M adenosine
(P<0.01, F-distribution).
[0022] FIGS. 5A-5C show the effects of adenosine-receptor agonists
on isoosmotic volume of NPE cells. In FIG. 5A, the
A.sub.3-selective agonist IB-MECA produced prompt shrinkage at 100
nM (n=4, V.infin.=95.6.+-.0.2%, .tau.4.5.+-.0.6 min, P<0.01 by
F-distribution). In contrast, the A.sub.1-selective agonist
N.sup.6-cyclopentyladenosine (CPA) had little effect at 100 nM, and
none at all at 3 .mu.M (n=4). In FIG. 5B, at 100 nM, the
A.sub.2-selective agonist CGS-21680 exerted no effect, but the
A.sub.3-selective agonist IB-MECA again produced shrinkage (n=4,
P<0.01 by F-distribution). In FIG. 5C, at high concentration (3
.mu.M), the A.sub.2-selective agonist CGS-21680 also triggered
isoosmotic shrinkage. However, preincubation of the cells with the
selective A.sub.3 receptor antagonist MRS-1191 (100 nM) abolished
this effect (n=4, P<0.01, F-distribution).
[0023] FIG. 6 shows the effects of IB-MECA on the level of free
intracellular calcium of NPE cells. Concentration of intracellular
Ca.sup.2+ increased steadily after application of 100 nM IB-MECA
and returned to baseline levels once IB-MECA was removed. Data were
obtained at a sampling rate of 1 Hz and smoothed by 21 points. The
box indicates the duration of the IB-MECA application.
[0024] FIG. 7 shows the effect of IB-MECA on short-circuit current
(I.sub.SC) across intact rabbit ciliary epithelium. As an initial
step in data analysis, 20-min period of baseline current just
before addition of any agent was fit by linear least-squares
analysis. The line generated by that analysis was extrapolated to a
point 45 min beyond introduction of that agent. Each current
response was subtracted from its respective extrapolated baseline
to yield a common initial baseline approximating constant zero
current. All recordings were placed in register relative to time of
agent introduction (time 0). Records of control (solvent), IB-MECA
with solvent, and B-MECA corrected for solvent were separately
averaged. IB-MECA was always added in the presence of 5 nM
Ba.sup.2+ to isolate contribution of Cl.sup.- to the response.
[0025] FIGS. 2-7 show that A.sub.3-selective adenosine receptors
increase chloride channel activity of NPE cells, and that blocking
of these receptors reduces chloride channel activity and secretion
by the NPE cells into the aqueous humor.
[0026] FIGS. 8-17 provide data to support an alternative approach
to reducing net aqueous humor secretion and intraocular pressure by
enhancing reabsorption by the PE cells.
[0027] FIGS. 8A-8C show the dependence of PE cell volume on ATP
concentration. In all figures, the volumes are normalized to the
initial values. The nonlinear least square fits are presented as
uninterrupted or interrupted curves, and data points displaying no
significant shrinkage are connected by dotted lines.
[0028] FIG. 8A shows that in .about.15% of the preparations, a
concentration-dependent shrinkage was observed with ATP alone (N=4,
P<0.01). The values generated by the fits were:
v.sub..infin.=94.3.+-.0.3% and .tau.=2.8.+-.0.7 min (10 .mu.M ATP),
and v.sub..infin.=94.3.+-.0.3%, .tau.=2.8.+-.0.7 min (100 .mu.M
ATP).
[0029] FIG. 8B shows the combined effect of tamoxifen (TMX) and ATP
on suspensions responding to ATP alone. The presence of 10 .mu.M
tamoxifen enhanced the response to both 3 .mu.M (P<0.01) and 10
.mu.M ATP (P<0.05). The values of the fits were:
v.sub..infin.=98.0.+-.0.4%, .tau.=10.8.+-.4.9 min (3 .mu.M ATP
alone); v.sub..infin.=96.7.+-.0.4%, .tau.=5.8.+-.2.1 min (3 .mu.M
ATP+10 .mu.M TMX); v.sub..infin.=97.2.+-.0.- 3%, .tau.=1.6.+-.1.0
min (10 .mu.M ATP alone); v.sub..infin.=95.7.+-.0.3%,
.tau.=1.4.+-.0.7 min (10 .mu.M ATP+10 .mu.M TMX).
[0030] FIG. 8C shows the dependence of synergistic shrinkage on ATP
concentration. Tamoxifen (6 .mu.M) was present throughout (N=5).
Shrinkage was observed in the simultaneous presence of 10 .mu.M or
1 mM ATP (P<0.01), but not at 1 .mu.M ATP. The data obtained at
10 .mu.M and 1 mM ATP were not significantly different from each
another. The fit obtained at 10 .mu.M ATP was characterized by
v.sub..infin.=95.7.+-.0.7% and .tau.=6.6.+-.3.2 min.
[0031] FIGS. 9A-9C show the effects of ATP and NPPB on whole-cell
currents of PE cells. The solid bars above show the time period of
drug application. In FIG. 9A, at a holding potential of -60 mV, 1
mM ATP produced reversible and reproducible increases in inward
current in the cultured bovine PE cell of the panel. In FIG. 9B,
the negatively-charged chloride-channel blocker NPPB partially
inhibited the ATP-stimulated current of the cell of the cell
patched, even at -60 mV. In FIG. 9C, in the freshly-dissociated
cell of the panel, increasing concentrations of ATP (10 .mu.M, 100
.mu.M and 1 mM) elicited increasing large stimulations, partially
reversible at 1 mM before losing the seal. These effects of ATP
alone were observed only in half of the total cultured and
freshly-dissociated cells studied.
[0032] FIGS. 10A-10B. show the synergism between tamoxifen and ATP
on isosmotic cell volume of PE cells. In FIG. 10A, neither ATP (10
mM) nor tamoxifen (6 .mu.M) separately produced substantial
shrinkage, whereas even 10 .mu.M ATP added together with 6 .mu.M
tamoxifen substantially enhanced the baseline shrinkage. The values
of the fits were: v.sub..infin.=96.2.+-.0.6%, .tau.=15.9.+-.5.2 min
(Control), 97.7.+-.0.2%, .tau.=2.2.+-.0.8 min (10 mM ATP alone),
v.sub..infin.=97.2.+-.0.3%, .tau.=1.6.+-.1.0 min (10 .mu.M ATP
alone); and v.sub..infin.=92.8.+-.0.8%, .tau.=11.4.+-.0.3 min (10
.mu.M ATP+6 .mu.M TMX) (N=6, P<0.01). In contrast, in FIG. 10B,
no such synergism was noted between tamoxifen (6 .mu.M) and
adenosine (10 .mu.M) (N=4).
[0033] FIG. 11 shows the effect of tamoxifen on the regulatory
volume decrease (RVD) of NPE cells. Gramicidin D (5 .mu.M) was
present in all suspensions to provide an exit pathway for K.sup.+.
In the absence of tamoxifen (TMX), the NPE cells displayed a
regulatory volume response to hypotonic swelling with a half-time
of .about.5 min (N=5). Adding TMX at the conclusion of the RVD
(t=10 min) had no effect on cell volume, adding TMX at t=5 min
partially inhibited the steady-state response, and adding TMX at
the same time as applying the hypotonic stress both slowed the rate
of initial slowing and abolished the RVD. This confirms the concept
that tamoxifen can inhibit swelling-activated chloride channels of
the NPE cells, but selectively enhances the stimulatory effect of
ATP on chloride channels by the PE cells.
[0034] FIGS. 12A-12B show the dependence on Cl.sup.- of the
synergistic shrinkage triggered by tamoxifen and ATP in PE cells.
Gramicidin D (5 .mu.M) was present in all suspensions to provide an
exit pathway for K.sup.+ As shown in FIG. 12A, the simultaneous
application of 6 .mu.M tamoxifen and 10 .mu.M ATP produced
isosmotic shrinkage in the presence of Cl.sup.- to a steady-state
value (v.sub..infin.) of 90.6%.+-.1.1% with a time constant (.tau.)
of 13.2.+-.3.2 min (N=4, P<0.01), but not in its absence. FIG.
12B, shows the effect of Cl.sup.--channel blockers on ATP,
tamoxifen-activated shrinkage. In the absence of inhibitors, the
shrinkage was fit with v.sub..infin.=94.2%.+-.0.3% and
.tau.=4.4.+-.0.8 min. The Cl.sup.- channel blockers DIDS (500
.mu.M) reduced and NPPB (100 .mu.M) each abolished the synergistic
shrinkage (N=4, P<0.01).
[0035] FIGS. 13A-13C. show the potential roles of histamine and
muscarinic receptors in PE cells As shown in FIG. 13A, in the
presence of 100 .mu.M ATP, either 10 .mu.M carbachol or 10 .mu.M
tamoxifen, but not 10 .mu.M histamine, enhanced shrinkage (N=4).
The value of the fits were: v.sub..infin.=94.9.+-.1.0%,
.tau.=45.2.+-.14.0 min (100 .mu.M ATP alone);
v.sub..infin.=94.1.+-.0.6%, .tau.=6.5.+-.2.3 min (ATP+10 .mu.M
TMX); v.sub..infin.=97.4.+-.0.5%, .tau.=21.0.+-.8.0 min (ATP+10
.mu.M histamine); v.sub..infin.94.2.+-.0.4%, .tau.=5.6.+-.1.2 min
(ATP+10 .mu.M carbachol).
[0036] In FIG. 13B, carbachol (10 .mu.M) produced nearly the same
degree of shrinkage in the presence or absence of 100 .mu.M ATP
(N=3). This effect was entirely abolished by preincubating for 2
min with 10 .mu.M atropine and retaining atropine in the test
suspension. The fits generated the following values:
v.sub..infin.=87.5.+-.1.8%, .tau.=45.2.+-.14.0 min (ATP alone);
v.sub..infin.=88.4.+-.0.5%, .tau.=7.2.+-.0.9 min (ATP+TMX);
v.sub..infin.=87.4.+-.0.5%, .tau.=6.3.+-.0.8 min
(ATP+TMX+atropine); v.sub..infin.=91.2.+-.1.6%, .tau.=13.4.+-.5.2
min (ATP+atropine).
[0037] In FIG. 13C, atropine (10 .mu.M) had no significant effect
on the volumetric response to the combined application of 100 .mu.M
ATP and 10 .mu.M tamoxifen (N=4). The values of the fits were:
v.sub..infin.=93.9.+-.1.7%, .tau.=34.8.+-.13.8 min (ATP alone);
v.sub..infin.=89.6.+-.1.9%, .tau.=11.3.+-.4.7 min (ATP+TMX); and
v.sub..infin.=87.8.+-.1.5%, .tau.=15.1.+-.3.6 min
(ATP+TMX+atropine). The cells exposed to ATP and atropine did not
display statistically significant shrinkage.
[0038] FIGS. 14A-14B show the potential role of calcium/calmodulin
in ATP/tamoxifen-mediated cell shrinkage of PE cells. FIG. 14A
shows the interactions of the calcium/calmodulin inhibitor
trifluoperazine (10 .mu.M) with ATP (100 .mu.M) and tamoxifen (10
.mu.M) on cell volume (N=6). The presence of trifluoperazine
reduced the response to the combined application of ATP and
tamoxifen. The trajectories displaying significant shrinkage were
fit with: v.sub..infin.=94.8.+-.0.4%, .tau.=2.2.+-.10.8 min
(ATP+TMX); v.sub..infin.=96.2.+-.0.3%, .tau.=6.8.+-.1.4 min
(ATP+trifluoperazine); v.sub..infin.=95.7.+-.0.4%, .tau.=3.9.+-.1.2
min (ATP+TMX+trifluoperazine). The trifluoperazine enhanced the
shrinkage produced by ATP alone (P<0.01), but also significantly
reduced the response produced by ATP+TMX (P<0.05).
[0039] FIG. 14B shows the effects of trifluoperazine (10 .mu.M) and
ATP (100 .mu.M) on PE cell volume in the absence of tamoxifen
(N=5). The shrinkage triggered by trifluoperazine was the same,
whether or not ATP was present (P>0.05). The values of the fits
were: v.sub..infin.=99.5.+-.0.002%, .tau.=0.5.+-.0.06 min
(Control); v.sub..infin.=96.8.+-.0.5%, .tau.=8.3.+-.3.7 min (ATP
alone); v.sub..infin.=96.8.+-.0.2%, .tau.=0.9.+-.0.6 min
(trifluoperazine alone); v.sub..infin.=96.7.+-.0.3%,
.tau.=0.5.+-.1.5 min (ATP+trifluoperazine).
[0040] FIG. 15 shows the potential role of protein kinase C on ATP
and tamoxifen-mediated PE cell shrinkage. The effect of 250 .mu.M
DiC.sub.8 and 0.3 .mu.M staurosporine with 10 .mu.M tamoxifen and
100 .mu.M ATP on cell volume (N=4) was determined. Only the
aliquots exposed to tamoxifen and ATP displayed shrinkage
(v.sub..infin.=89.7.+-.6.1%; .tau.=22.9.+-.19.0 min). The analyses
were conducted only with the first 6 time points because of the
unusually large shrinkage triggered by ATP+tamoxifen at 30 min.
[0041] FIGS. 16A-16B show the potential role of estrogen receptors
on tamoxifen and ATP-mediated PE cell shrinkage. FIG. 16A shows the
interactions of 17.beta.-estradiol (100 nM) with ATP (100 .mu.M)and
tamoxifen (10 .mu.M) on cell volume (N=4). Estradiol together with
ATP initiated a small and slow shrinkage, different from the
baseline null response to ATP, itself (P<0.05). The maximal
response obtained with tamoxifen and ATP
(v.sub..infin.=93.9.+-.1.0%, .tau.=15.6.+-.5.0 min) was
significantly inhibited (P<0.05) by adding estradiol 2 min
before initiating the measurements.
[0042] FIG. 16B shows the interactions of estradiol and tamoxifen
on PE cell volume. At the same 100-nM concentration, the active
(17.beta.-estradiol) and inactive (17.alpha.-estradiol) forms of
the estrogen exerted very small and opposite effects on the time
course of cell shrinkage.
[0043] FIGS. 17A-17B show the effects of tamoxifen and ATP on
intracellular Ca.sup.2+ of PE cells. In FIG. 17A, 100 .mu.M ATP or
100 .mu.M ATP+tamoxifen was applied for 20 sec as indicated by the
short black bars. The drugs were washed off for 5 min between
applications. Although the response attenuates, the presence of
tamoxifen does not alter the magnitude of the response. Tamoxifen
alone had no significant effect on Ca.sup.2+.sub.i (not shown)
[0044] In FIG. 17B, mean results from 7 experiments in which 20-sec
applications of 100 .mu.M ATP were alternated with 20-sec
applications of 100 .mu.M ATP+10 .mu.M TMX. In order to adjust for
the attenuation, two sets of experiments were performed. In the
first set illustrated in A, the order of drugs was 1) ATP+TMX, 2)
ATP, 3) ATP+TMX and 4) ATP. In the second type of experiment the
order was inverted. The magnitude of the Ca.sup.2+ response when
the first application was ATP alone (A1) was compared with
experiments in which the first application was with ATP+TMX (T1).
Trials in which the second application was with ATP (A2) were
compared with trials in which the second application was ATP+TMX
(T2), and likewise for the third and fourth applications. There was
no significant difference between any of the four pairs (p<0.1,
n=3-4)
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0045] The ciliary epithelium of the eye is a bilayer, comprising a
deeper layer of pigmented epithelial (PE) cells and a superficial
layer of nonpigmented epithelial (NPE) cells (FIG. 1). The proposed
mechanism of action of ATP, tamoxifen (TMX) and A3 receptor
agonists, such as adenosine (Ado), in influencing aqueous humor
secretion is shown in FIG. 1. ATP is released from PE and/or NPE
cells. ATP is then converted to adenosine by ecto-enzymes (ecto).
The adenosine then binds to A.sub.3 receptors on NPE cells,
resulting in opening of Cl.sup.- channels. This results in an
increase in aqueous humor production and increased intraocular
pressure. In addition, simultaneous stimulation by ATP and
tamoxifen activates Cl.sup.- efflux from PE cells, leading to a net
decrease in aqueous humour formation. ATP acts on P.sub.2 receptors
of PE cells, promotes opening of Cl.sup.- channels, and a decrease
in aqueous humor production resulting in decreased intraocular
pressure.
[0046] The present invention includes the observation that the
A.sub.3 subtype adenosine receptor antagonists (referred to herein
as A.sub.3 antagonists) inhibit shrinkage of NPE cells as
determined by measurements of cell volume in isoosmotic solution.
This inhibition of cell shrinkage implies a net reduction of
secretion of aqueous humor through the NPE cell membrane which
would result in a reduction of intraocular pressure (FIG. 1). These
A.sub.3 receptors are present on human and rabbit NPE cells and
underlie the activation of NPE chloride (Cl.sup.-) channels by
adenosine. In addition, it was found that the antiestrogen
tamoxifen, the calcium/calmodulin inhibitor trifluoperazine and the
muscarinic agonist carbachol all promoted cell shrinkage in PE
cells. The shrinkage of PE cells implies a stimulation of a net
reabsorption of aqueous humor through the PE cell membrane towards
the stroma, which would result in a net reduction in aqueous humor
formation and a reduction in intraocular pressure (FIG. 1). Thus,
these compounds, or related compounds, can be used to lower
intraocular pressure as a treatment for glaucoma and other ocular
conditions in which it is desirable to lower intraocular
pressure.
[0047] Measurements of short-circuit current across intact rabbit
ciliary epithelium, of cell volume in suspended cultured human NPE
cells, and of whole-cell currents from patch-clamped cultured human
and fresh bovine NPE cells have indicated that adenosine-receptor
occupancy stimulates Cl.sup.- secretion in mammalian NPE cells
(Carre et al., supra., 1997). As evidenced by the data presented in
the examples below, these effects are mediated by A.sub.3
receptors. A.sub.3 receptors are present in both human HCE cells (a
cell line of human NPE cells) and rabbit ciliary body. The
A.sub.3-selective agonist IB-MECA increased the short circuit
current across rabbit iris-ciliary body in the presence of
Ba.sup.2+, a change consistent with an increased efflux of Cl.sup.-
from NPE cells. In the presence of gramicidin to isolate the
Cl.sup.- conductance, IB-MECA caused human HCE cells to shrink in a
dose-dependent manner; the K.sub.d of .sup..about.55 nM is
consistent with a maximal stimulation of A.sub.3 receptors in
cardiac myocytes at 100 nM IB-MECA (Shahidullah et al., Curr. Eye
Res., 16:1006-1016, 1997). The highly specific A.sub.3 agonist
Cl-IB-MECA also produced shrinkage of HCE cells in the presence of
gramicidin. Gramicidin readily partitions into plasma membranes to
form a cation-selective pore and is widely used for studying volume
regulation (Hoffmann et al., Interaction of Cell Volume and Cell
Function, Lang et al., eds., Springer, Heidelberg, Germany, p.
188-248, ACEP Series 14). Under these conditions, release of cell
Cl.sup.- becomes the rate-limiting factor in both hypo-(Civan et
al., Invest. Ophthalmol. Vis. Sci., 35:2876-2886, 1994) and
isosmotic cell shrinkage (Carre et al., supra., 1997).
[0048] The A.sub.3 antagonists MRS 1097 and MRS 1191 were able to
prevent the shrinkage induced by IB-MECA at concentrations far
below their K.sub.i for A.sub.1 and A.sub.2A receptors. The A.sub.1
agonist CPA did not have a consistent effect upon cell volume. The
A.sub.2A agonist CGS-21680 had no effect at low concentrations. The
effect of CGS-21680 on shrinkage was only detected at a
concentration 500 fold higher than the K.sub.i values for the
A.sub.3 receptor, and this effect was blocked by the A.sub.3
antagonist MRS-1191. The A.sub.3 antagonists MRS 1097, MRS 1191 and
MRS 1523 blocked the shrinkage produced by 10 .mu.M adenosine; at
the concentrations used, <20% of the A.sub.1 and A.sub.2A
receptors could have been occupied by MRS 1097 and <1% of those
receptors could have been blocked by MRS 1191 and MRS 1523.
Together, these observations indicate that the adenosine-stimulated
activation of Cl.sup.- release by the HCE line of human NPE cells
is primarily mediated by occupancy of an A.sub.3-subtype adenosine
receptor.
[0049] Adenosine but not ATP shrinks nonpigmented ciliary
epithelial (NPE) cells by activating Cl.sup.- channels. Although
adenosine had no effect on PE cells, PE cell volume was
occasionally reduced by ATP, and was always reduced by simultaneous
application of ATP with the antiestrogen tamoxifen. Cultured bovine
PE cells were studied volumetrically by electronic cell sorting.
ATP alone (.gtoreq.3 .mu.M) shrank .about.15% of the suspensions,
but had little/no effect in most suspensions. Whole cell patch
clamping indicated that this baseline response reflected activation
of Cl.sup.- permeant channels in a heterogeneous population of
cells. The antiestrogen tamoxifen (6-10 .mu.M) enhanced the
ATP-triggered shrinkage, whether or not a baseline response to ATP
was detected. This was unexpected since swelling activated Cl.sup.-
channels are either blocked (in NPE cells) or unaffected (in PE
cells) by tamoxifen. Tamoxifen in itself exerted no consistent
effect on PE-cell volume. The tamoxifen, ATP-activated shrinkage
required Cl.sup.- release since the response was blocked by
removing Cl.sup.- and was inhibited by Cl.sup.- channel blockers
(NPPB and DIDS). The modulating effect of tamoxifen could have
reflected .gtoreq.5 actions of tamoxifen. Our data argue against
actions of tamoxifen to inhibit PKC or calcium/calmodulin and on
histamine or carbachol receptors. The cooperative interaction
between tamoxifen and ATP was not mediated by an enhanced rise in
Ca.sup.2+. The results indicate that tamoxifen interacts
synergistically with ATP to activate Cl.sup.- release by the PE
cells. Tamoxifen may act in part by occupying plasma-membrane
estrogen-binding sites.
[0050] The present results demonstrate that tamoxifen markedly
enhanced the effects of extracellular ATP on the transport
properties of cultured bovine pigmented ciliary epithelial cells.
The synergism was particularly clear in those preparations with
little or no baseline response to ATP alone and was detected at ATP
and tamoxifen concentrations likely to be physiologically and
clinically relevant. In the presence of tamoxifen, an approximately
half-maximal response was elicited by 3 .mu.M ATP, a concentration
likely reached physiologically by ATP release into the constrained
space between the PE cells and the underlying basement membrane
(Mitchell et al., supra., 1998). Tamoxifen is used clinically as an
antiestrogen by occupying nuclear estrogen-receptor sites (Klinge
et al., Oncology Research, 4,:145-150, 1992), and the
concentrations applied here (6-10 .mu.M) also appear to be
clinically relevant (Stuart et al., Br. J. Cancer, 66, 833-839,
1992).
[0051] The precise signaling pathway involved in the modulating
action of tamoxifen is presently unknown. In addition to binding to
nuclear estrogen receptors (Klinge et al., supra.), and blocking
plasma-membrane swelling-activated Cl.sup.- channels (Valverde et
al., Pflugers Archiv. 425: 552-554, 1993; Zhang et al., J. Clin.
Investi. 94:1690-1697, 1994; Nilius et al., Pflugers Archiv.
428:364-371, 1994; Wu et al., J. Physiol., 491.3: 743-755, 1996),
tamoxifen has been observed to affect: histamine receptors (Brandes
et al. Biochem. Biophys. Res. Commun., 126: 905-910, 1986),
muscarinic receptors (Ben-Baruch et al. Mol. Pharmacol., 21:
287-293, 1981), activation by calcium/calmodulin (Lam, Biochem.
Biophys. Res. Commun. 118: 27-32,1984), plasma-membrane estrogen
receptors (Hardy et al., FASEB J. 8: 760-765. 1994), and protein
kinase C activity (O'Brien et al., Cancer Res. 45:2462-2465, 1985).
The current data indicate that four of these five latter effects
play no role in the synergism between tamoxifen and ATP: (1)
because histamine does not alter the response of cell volume to
ATP, histamine receptors appear to be irrelevant in the present
context. (2) Although carbachol itself shrinks cell volume, the
response is not synergistic with ATP, and atropine does not affect
the tamoxifen/ATP synergistic effect. Thus, muscarinic receptors
are not involved. (3) Protein kinase C (PKC) activity cannot be
playing a major role since both activation and inhibition of
enzymatic activity produced similar small reductions in volume, 1-2
orders of magnitude smaller than that triggered by tamoxifen and
ATP together. At the same concentrations used here, the PKC
activator DiC.sub.8 and the PKC inhibitor staurosporine exert large
and opposing actions on swelling-activated Cl.sup.- channels of
nonpigmented ciliary epithelial cells (Civan et al., Invest.
Ophthalmol. Vis. Sci. 35: 2876-2886, 1994). (4) Finally,
calcium/camodulin antagonism is unlikely to mediate the synergistic
effect since the shrinkage produced by another such antagonist
(trifluoperazine) was independent of the presence of ATP.
[0052] It should be emphasized that although tamoxifen is not
acting like carbachol or trifluoperazine, both of these compounds
did reduce PE cell volume. Both carbachol and trifluoperazine
reduced cell volume on their own, in the absence of ATP. Thus,
compounds like trifluoperazine, which inhibit calcium/calmodulin,
or substances like carbachol, which stimulate muscarinic receptors,
could be used to reduce the production of aqueous humor.
[0053] The potential role of binding to plasma-membrane estrogen
receptors is less clear. Hardy et al. (supra.) have reported that
that tamoxifen activates a large-conductance Cl.sup.- channel with
an EC.sub.50.about.15 .mu.M applied to NIH 3T3 fibroblasts, but
only after stable transfection with MDR1 and after growth in
colchicine for >24 hrs. In contrast, no consistent effect of
tamoxifen on cell volume was observed in the absence of ATP. Both
the active (17-.beta.-estradiol) and inactive
(17-.alpha.-estradiol) forms of the estrogen had only slight
effects on volume, but 17-.beta.-estradiol did enhance the
ATP-activated shrinkage slightly. Interestingly, the active (but
not the inactive) form of the estrogen also reduced the synergistic
effect elicited by tamoxifen and ATP. This indicates that tamoxifen
and estrogen compete for binding sites and that tamoxifen is more
effective than 17-.beta.-estradiol in activating these sites. Since
the response to tamoxifen was relatively rapid (within 2 min), it
is likely that these receptors are located at the plasma
membrane.
[0054] ATP produced an elevation in the levels on intracellular
Ca.sup.2+ which attenuated with repeated application, but the
response was not affected by the inclusion of tamoxifen. This
implies that: 1) the cell shrinkage produced synergistically by ATP
and tamoxifen is not mediated by a synergistic elevation in
Ca.sup.2+, and 2) the presence of TMX does not affect the
attenuation of the Ca.sup.2+ response to ATP. This attenuation has
been reported previously in ciliary epithelial cells, and it has
been suggested that the attenuation is mediated by the inhibition
of IP.sub.3 production by increasingly elevated levels of PKC
(Shahidulla et al., 1997). The inability of tamoxifen to modify the
rate of attenuation supports the interpretation that tamoxifen does
not act by modifying PKC in these cells.
[0055] Thus, cells derived from the pigmented ciliary epithelial
cell layer can respond to extracellular ATP by releasing Cl.sup.-,
and this release is strongly modulated by tamoxifen. Using the
fluorescent probe quinacrine, intracellular stores of ATP have been
identified in NPE and PE cells in the intact epithelium and in
culture (Mitchell et al., supra, 1998). The ATP can be released by
both cell types (Mitchell et al., supra., 1998) and metabolized to
adenosine by ectoenzymes (Mitchell et al., supra., 1998). Adenosine
is known to activate Cl.sup.- channels NPE cells (Carr et al.,
supra., 1997). Taken together with these previous observations, the
current information suggests that ATP can enhance fluid movement in
both directions across the ciliary epithelium: increasing secretion
by stimulating NPE cells (indirectly through adenosine formation)
to release Cl.sup.- into the aqueous humor, and increasing
reabsorption by directly stimulating PE cells to release Cl.sup.-
into the stroma of the ciliary processes. It is likely that one or
more additional factors is necessary to coordinate these opposing
purinergic actions to permit ATP to regulate net aqueous humor
formation.
[0056] This putative coordination could be provided in at least two
ways. In principle, ATP could be released heterogeneously
throughout the epithelium. Little information is as yet available
on this point, but ATP release triggered by anisosmotic swelling
appears to be comparable for NPE and PE cells (Mitchell et al.,
supra., 1998). An alternative possible mechanism would be for a
regulator, like tamoxifen, to modify the effects of purines on
Cl.sup.- release at the contralateral surfaces of the ciliary
epithelium. In the absence of tamoxifen, physiologic concentrations
(.about.3 .mu.M) of adenosine stimulate Cl.sup.- release by NPE
cells (Carr et al., supra., 1997), whereas even higher
concentrations of ATP usually had little effect on release by PE
cells. Under these conditions; ATP release would favor secretion.
In contrast, in the presence of 6-10 .mu.M tamoxifen, at least some
of the Cl.sup.- channels of the NPE cells are blocked (Wu et al.,
supra.; FIG. 4), and ATP is far more effective in stimulating
Cl.sup.- release from PE cells. Under these latter conditions, ATP
release is expected to favor reabsorption.
[0057] The data presented below demonstrates the ability of various
agents to block shrinkage of NPE cells and to promote shrinkage of
PE cells. The net effect of these agents would be to reduce
intraocular pressure in vivo. The use of four chemical classes of
A.sub.3 receptor antagonists for reduction of intraocular pressure
is also contemplated: dihydropyridines, pyridines, pyridinium salts
and triazoloquinazolines. These generic compounds are shown in
Appendix A, along with the possible substituents at each variable
position of the compound. These classes of compounds are also
described in PCT/W097/27177.
[0058] In addition to the particular A.sub.3 receptor antagonists
discussed in the examples below: MRS-1097 (3-ethyl
5-benzyl-2-methyl-6-phenyl-4-styryl-1,4-(.+-.)-dihydropyridine-3,5-dicarb-
oxylate), MRS-1191 (3-ethyl
5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1,4-
-(.+-.)-dihydropyridine-3,5-dicarboxylate) (Jiang et al., J. Med.
Chem. 40:2596-2608, 1997), and MRS-1523 (Li et al., J. Med. Chem.
42:706-721, 1999), the use of any A.sub.3 receptor antagonist or
analog thereof to reduce intraocular pressure is within the scope
of the invention. Other A.sub.3 receptor antagonists for use in the
present invention are described by Jacobson (Trends Pharmacol. Sci.
19:184-191, 1998) and include MRS-1334 (3-ethyl 5-(4-nitrobenzyl)
2-methyl-6-phenyl-4-phenyleth-
ynyl-1,4-(.+-.)-dihydropyridine-3,5-dicarboxylate)), MRS1067
(3,6-dichloro-2'-(isopropoxy)-4'-methylflavone), MRS-1220
(9-chloro-2-(2-furyl)-5-phenylacetylamino[1,2,4]triazolo[1,5-c]quinazolin-
e), L249313
(6-carboxymethyl-5,9-dihydro-9-methyl-2-phenyl-[1,2,4]-triazol-
o[5,1-a][2,7]naphthyridine) and L268605
(3-(4-methoxyphenyl)-5-amino-7-oxo- -thiazolo[3,2]pyrimidine),
VUF8504 (4-methoxy-N-[2-(2-pyridinyl)quinazdin-- 4-yl]benzamide)
and the like.
[0059] In a particularly preferred embodiment, the A.sub.3
antagonist
2,4-diethyl-1-methyl-3-(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl-6-pheny-
lpyridium iodide (MRS 1649, 11) is used to reduce intraocular
pressure. The synthesis of this compound is described in Example
15. This 3,5-diacyl-1,2,4-trialkyl-6-phenylpyridinium derivative
displays a uniquely high water solubility (43 mM) and can be
extracted readily into ether. In addition, the prodrug form of this
compound, the corresponding 1-methyl-1,4-dihydropyridine, can be
oxidized to form compound 11 in vitro in the presence of a tissue
homogenate. Thus, it is contemplated that prodrug forms of A.sub.3
receptor antagonists (e.g., compounds 24 and 25) can be
administered to the eye which will then be converted to the active
antagonists which will reduce intraocular pressure.
[0060] The determination of whether a compound can act as an
A.sub.3 receptor antagonist can be determined using standard
pharmacological binding assays. Similarly, although the
antiestrogen tamoxifen is exemplified herein, other antiestrogens
are also contemplated, including, but not limited to, 4-hydroxy
tamoxifen, toremifine, icosifene, droloxifene, LY117018, ICI
164,384, ICI 182,780, RU 58,668, EM-139, EM-800. EM-652, GW 5638,
and the like. Lowering of intraocular pressure with a combination
of an antiestrogen and ATP, or any compound capable of promoting
ATP release from NPE cells, is also contemplated. Finally, although
the calcium/calmodulin antagonist trifluoperazine is exemplified
herein, the use of any calmodulin antagonist for lowering
intraocular pressure is also within the scope of the invention
including, but not limited to calmidazolium chloride, calmodulin
binding domain, chlorpromazine HCl, melittin, phenoxybenzamine HCl,
trifluoperazine dimaleate, W-5, W-7, W-12 and W-13. These compounds
are available from Calbiochem, San Diego, Calif. The use of analogs
of the above-identified compounds for the reduction of intraocular
pressure is also within the scope of the present invention.
[0061] These agents can be used to treat ocular disorders resulting
associated with or caused by an increase in intraocular pressure,
such as glaucoma. The agents can be processed in accordance with
conventional methods to produce medicinal agents for administration
to mammals, preferably humans. The agents can be employed in
admixture with conventional excipients, i.e. pharmaceutically
acceptable organic or inorganic carrier substances suitable for
parenteral, enteral (e.g. oral) or topical application which do not
deleteriously react with the agents. Suitable pharmaceutically
acceptable carriers include, but are not limited to, water, salt
solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols,
polyethylene glycols, gelatin, carbohydrates such as lactose,
amylose or starch, magnesium stearate, talc, silicic acid, viscous
paraffin, perfume oil, fatty acid monoglycerides and diglycerides,
pentaerythritol fatty acid esters, hydroxy methylcellulose,
polyvinyl pyrollidone, etc. The pharmaceutical preparations can be
sterilized and, if desired, mixed with auxiliary agents, e.g.,
lubricants, preservatives, stabilizers, wetting agents,
emulsifiers, salts for influencing osmotic pressure, buffers,
coloring, flavoring and/or aromatic substances and the like which
do not deleteriously react with the active compounds. They can also
be combined where desired with other active agents, e.g.,
vitamins.
[0062] For parenteral application, particularly suitable are
injectable, sterile solutions, preferably oily or aqueous
solutions, as well as suspensions emulsions, or implants, including
suppositories. Ampules are convenient unit dosages. For enteral
application, particularly suitable are tablets, liquids, drops,
suppositories or capsules. A syrup, elixir or the like can be used
when a sweetened vehicle is employed. Sustained or directed release
compositions can be formulated, e.g. liposomes or those wherein the
active compound is protected with differentially degradable
coatings, e.g. by microencapsulation, multiple coatings, etc. It is
also possible to lyophilize the agents for use in the preparation
of products for injection.
[0063] Topical administration is preferred. For topical
application, there are employed nonsprayable forms, viscous to
semi-solid or solid forms comprising a carrier compatible with
topical application and having a dynamic viscosity preferably
greater than water. Suitable formulations include, but are not
limited to, solutions, suspensions, emulsions, creams, ointments,
powders, liniments, salves, aerosols, etc., which are, if desired,
sterilized or mixed with auxiliary agents, e.g., preservatives,
stabilizers, wetting agents, buffers or salts for influencing
osmotic pressure, ocular permeability, etc. For topical
application, also suitable are sprayable aerosol preparations
wherein the active ingredient, preferably in combination with a
solid or liquid inert carrier material, is packaged in a squeeze
bottle or in admixture with a pressurized volatile, normally
gaseous propellant, e.g. a freon. In a particularly preferred
embodiment, the agent is formulated into a pharmaceutical
formulation appropriate for administration to the eye, including
eye drops, gels and ointments.
[0064] For systemic administration, the dosage of the agents
according to this invention generally is between about 0.1 .mu.g/kg
and 10 mg/kg, preferably between about 10 .mu.g/kg and 1 mg/kg. For
topical administration, dosages of between about 0.000001% and 10%
of the active ingredient are contemplated., preferably between
about 0.1% and 4%. It will be appreciated that the actual preferred
amounts of agent will vary according to the specific agent being
used, the severity of the disorder, the particular compositions
being formulated, the mode of application and the species being
treated. Dosages for a given host can be determined using
conventional considerations, e.g.,by customary comparison of the
differential activities of the subject compounds and of a known
agent, e.g. by means of an appropriate, conventional pharmacologic
protocol. The agents are administered from less than once per day
(e.g., every other day) to four times per day.
[0065] Gramicidin, adenosine, 2-chloroadenosine, tamoxifen, ATP,
17.alpha.- and .beta.-estradiol, DiC.sub.8, carbachol, atropine,
histamine, and trifluoperazine were obtained from the Sigma
Chemical Co. (St. Louis, Mo.). CPA (N.sup.6-cyclopentyl-adenosine),
CGS-21680, IB-MECA, Cl-IB-MECA and MRS-1191 (3-ethyl 5-benzyl
2-methyl-6-phenyl-4-phenylethynyl-1,4-(.+-.)-dihydropyridine-3,5-dicarbox-
ylate) were obtained from Research Biochemicals International
(Natick, Mass.). Fura-2 AM was bought from Molecular Probes
(Eugene, Oreg.). MRS 1097, and MRS 1523 were provided by Drs.
Kenneth A. Jacobson (National Institutes of Health) and Bruce L.
Liang (University of Pennsylvania). The compound Cl-IB-MECA
(MH-C-7-08; Lot No. CMVIII-12) was provided by Research
Biochemicals International as part of the Chemical Synthesis
Program of the National Institute of Mental Health, Contract
N01MH30003. DIDS [4,4'-diisothiocyano-2,2'-disulfonic acid] and
fura-2 AM were obtained from Molecular Probes, Inc. (Eugene,
Oreg.). NPPB [5-nitro-2-(3-phenylpropylamino)benzoate] and
staurosporine were obtained from Biomol Research Laboratories, Inc.
(Plymouth Meeting, Pa.).
[0066] Values are presented as the means.+-.1 SE. The number of
experiments is indicated by the symbol N. The null hypothesis, that
the experimental and baseline measurements shared the same mean and
distribution, was tested with Student's t-test and by the upper
significance limits of the F-distribution, as indicated. The t-test
was applied to compare the significance between single means or
single fit parameters. The F-distribution was applied to test
whether the time course of volume measurements in different
suspensions could reflect a single population of data points.
EXAMPLE 1
Cell Culture
[0067] The HCE (human ciliary epithelial) cell line (Carre et al.,
supra.) is an immortalized NPE cell line obtained from primary
cultures of adult human epithelium. Cells were grown in Dulbecco's
modified Eagle's medium (DMEM, #11965-027, Gibco BRL, Grand Island,
N.Y.) with 10% fetal bovine serum (FBS, A-1115-L, HyClone
Laboratories, Inc., Logan, Utah) and 50 .mu.g/ml gentamycin
(#15750-011, Gibco BRL), at 37.degree. C. in 5% CO.sub.2 (Wax et
al., Exp. Eye Res. 57:3057-3063, 1993). The growth medium had an
osmolality of 328 mOsm. Cells were passaged every 6-7 days and were
studied 8-13 days after passage, after reaching confluence.
[0068] For the tamoxifen experiments, The cells used were an
immortalized PE-cell line from a primary culture of bovine
pigmented ciliary epithelium. Cells were grown in Dulbecco's
modified Eagle's medium (DMEM, #11965-027, Gibco BRL, Grand Island,
N.Y.; and 51-43150, JRH Biosciences, Lenexa, Kans.) with 10% fetal
bovine serum (FBS, A-1151-L, HyClone Laboratories, Inc., Logan,
Utah) and 50 .mu.g/ml gentamycin (#15750-011, Gibco BRL), at
37.degree. C. in 5% CO.sub.2 (Yantorno et al., Exp. Eye Res.
49:423-437, 1989). The medium had an osmolality of 328 mOsm. Cells
were passaged every 6-7 days and, after reaching confluence, were
suspended in solution for study within 6-10 days after passage.
EXAMPLE 2
Measurement of Cell Volume in Isosmotic Solution
[0069] The volume of NPE cells was measured as the movement of
fluid that underlies a change in NPE cell volume, this is thought
to be the same as the movement of fluid which underlines the
secretion of aqueous humor (FIG. 1).
[0070] A 0.5-ml aliquot of the HCE cell suspension in DMEM was
added to 20 ml of each test solution, which contained (in mM):
110.0 NaCl, 15.0 HEPES
[4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], 2.5
CaCl.sub.2, 1.2 MgCl.sub.2, 4.7 KCl, 1.2 KH.sub.2PO.sub.4, 30.0
NaHCO.sub.3, and 10.0 glucose, at a pH of 7.4 and osmolality of
298-305 mOsm. Parallel aliquots of cells were studied on the same
day. One aliquot usually served as a control, and the others were
exposed to different experimental conditions at the time of
suspension. The same amount of solvent vehicle (dimethylformamide,
DMSO or ethanol) was always added to the control and experimental
aliquots. The sequence of studying the suspensions was varied to
preclude systematic time-dependent artifacts (Civan et al., Exp.
Eye Res. 54:181-191, 1992).
[0071] Cell volumes of isosmotic suspensions were measured with a
Coulter Counter (model ZBI-Channelyzer II), using a 100 .mu.m
aperture (Civan et al., supra., 1994). As previously described (Wax
et al., supra., 1993), the cell volume (v.sub.C) of the suspension
was taken as the peak of the distribution function. Cell shrinkage
was fit as a function of time (t) to a monoexpenential
function:
v.sub.C=v.sub.28+(v.sub.0-v.sub..infin.)-[e.sup.-(t-t.sub..sup.0.sup.)/.ta-
u.] {1}
[0072] where v.sub..infin. is the steady-state cell volume, v.sub.0
is the cell volume at the first point (t.sub.0) of the time course
to be fit, and .tau. is the time constant of the shrinkage. For
purposes of data reduction, the data were normalized to the first
time point, taken to be 100% isotonic volume. Fits were obtained by
nonlinear least-squares regression analysis, permitting both
v.sub..infin. and .tau. to be variables.
[0073] In previous studies demonstrating that adenosine causes
isotonic cell shrinkage by activating Cl.sup.- channels in NPE
cells (Carre et al., supra., 1997), the levels of adenosine used
were sufficiently high to activate A.sub.1, A.sub.2A, A.sub.2B or
A.sub.3 adenosine receptor subtypes (Fredholm, et al., Pharmacol.
Rev. 46:143-156, 1994, Fredholm et al., Trends Pharmacol. Sci.
18:79-82, 1997; Klotz et al., Naunyn Schmiedebergs Arch. Pharmacol.
357:1-9, 1998). In order to differentiate among these receptors,
the experiments were repeated in the present study using a series
of agonists and antagonists selective for these receptors. As we
wished to identify the effects of these receptors specifically on
Cl.sup.- channels, 5 .mu.M gramicidin D was included in all
solutions to eliminate any potential contribution from K.sup.+
channels. This ionophore readily partitions into plasma membranes
to form a cation-selective pore, and is widely used for studying
volume regulation (Hoffmann et al., in Interaction of Cell Volume
and Cell Function, Lang et al., eds., Springer, Heidelberg,
Germany, pp. 188-248, (ACEP Series 14), 1993)). Under these
conditions, release of cell Cl.sup.- becomes the rate-limiting
factor in both hypo-(Civan et al., Exp. Eye Res. 54:181-191, 1992)
and isoosmotic cell shrinkage (Carre et al., supra., 1997).
[0074] In the presence of gramicidin, the A.sub.3 agonist IB-MECA
caused the cells to shrink in a concentration-dependent manner
(FIGS. 2A-2B). Least-squares analysis of the linearized
Lineweaver-Burk plot generated from monoexponential fits of these
data indicates that the apparent K.sub.d for the IB-MECA-induced
shrinkage was 55.+-.10 nM (FIG. 2C). IB-MECA is a highly selective
agonist for the A.sub.3 receptor; the K.sub.i for the A.sub.3
receptor is 50 times lower than it is for A.sub.1 or A.sub.2A
receptor (Gallo-Rodrigez et al, J. Med. Chem. 37:636-646, 1994;
Jacobson et al., supra., 1995; Jacobson et al., FEBS Lett.
336:57-60, 1993)). Cl-IB-MECA is even more specific for A.sub.3
receptors, with a K.sub.i for A.sub.3 receptors 2500 times lower
than for A.sub.1 receptors and 1400 times lower than for A.sub.2A
receptors. The ability of Cl-IB-MECA to induce cell shrinkage (FIG.
2D) further strengthens the hypothesis that stimulation of A.sub.3
receptors stimulates Cl.sup.- channels.
[0075] It was also determined whether A.sub.3-selective antagonists
could prevent the putative A.sub.3-mediated shrinkage produced by
IB-MECA. Parallel aliquots of suspensions were preincubated with
MRS 1097, a selective A.sub.3-selective antagonist with K.sub.i
values for the binding (in nM) to human A.sub.1/A.sub.2/A.sub.3
receptors of 5,930/4,770/108.(Jacobson et al., Neuropharmacol.
36:1157-1165, 1997). Preincubation for 2 min with 300 nM MRS 1097
blocked the isoomotic shrinkage characteristically triggered by 100
nM IB-MECA (FIG. 3A). A second highly selective A.sub.3 antagonist,
MRS 1191, (Jlang et al., J. Med. Chem. 39:4667-4675, 1996), with
K.sub.i values for the binding (in nM) to human
A.sub.1/A.sub.2/A.sub.3 receptors of 40,100/>100,000/31.4
(Jacobson et al., supra.) was also used. Preincubation for 2 min
with 100 nM MRS 1097 also prevented the subsequent response to 100
nM IB-MECA (FIG. 3B). There was an indication in the results of
FIG. 3B that MRS 1191 might actually produce a small amount of cell
swelling. This was not a constant finding (FIG. 4B, and may have
reflected variations in the background level of A.sub.3-receptor
occupancy.
[0076] The physiologic agonist reaching the adenosine receptors is
likely to be the nucleoside adenosine itself, arising from release
of ATP by the ciliary epithelial cells and ecto-enzyme activity
(Mitchell et al., 1998, supra.). Adenosine triggers isoosmotic
shrinkage of cultured human NPE cells with an EC.sub.50 of 3-10
.mu.M (Civan et al., supra., 1997). In this concentration range,
adenosine acts as a nonselective agonist of all four subtypes of
the adenosine receptor (Fredholm et al., supra., 1994; Fredholm et
al., supra., 1997). As illustrated in FIG. 4, a 2 min preincubation
with either 100 nM of the A.sub.3-selective antagonist MRS 1191
(FIG. 4B) or 300 nM of the A.sub.3-selective antagonist MRS 1097
(FIG. 4A) blocked the shrinkage characteristically produced by 10
.mu.M adenosine. MRS 1523, an A.sub.3 antagonist with K.sub.i
values for the binding (in nM) to human A.sub.1/A.sub.2/A.sub.3
receptors of 15,600/2,050/19 (Li et al., J. Med. Chem.
41:3186-3201, 1998) also eliminated the actions of adenosine.
[0077] The ability of specific A.sub.3 antagonists to inhibit the
response to the nonspecific adenosine suggests that the
contribution of the other receptors to Cl.sup.- channel activation
was minimal. To test this further, the effect of A.sub.1 and
A.sub.2A agonists were tested. CPA is an A.sub.1-selective agonist
with a K.sub.i for the A.sub.1-receptor of 0.6 nM (31). However,
CPA produced no significant shrinkage at 30 nM and 1 .mu.M (data
not shown, N=3) and 3 .mu.M (FIG. 5A). A small slow effect of
uncertain significance was detected at the intermediate
concentration of 100 nM (FIG. 5A). Some cross-reactivity with
A.sub.3 receptors might be expected, given the K.sub.i of CPA for
the A.sub.3-subtype of 43 nM (Klotz et al., supra.). CGS-21680 is a
widely used A.sub.2A agonist with an IC.sub.50 value of 22 nM for
the A.sub.2A-receptor (Hutchison et al., J. Pharmacol. Exp. Ther.
251:47-55, 1989, Jarvis et al., J. Pharmacol. Exp. Ther.
253:888-893, 1989). CGS-21680 had no detectable effect at 100-nM
concentration (FIG. 5B), but did trigger isoosmotic shrinkage at a
30-fold higher concentration (3 .mu.M) (FIG. 5C). However, the
K.sub.i for the CGS-21680 at the A.sub.3 receptor is 67 nM (Klotz
et al., supra.) and thus CGS-21680 could have been acting though
either A.sub.2A receptors or A.sub.3 receptors at the higher
concentration. To distinguish between these possibilities, we
preincubated parallel aliquots of suspensions with the antagonist
100 nM MRS 1191. MRS 1191 prevented the shrinkage produced by the
high concentration of CGS-21680 (FIG. 5C, P<0.01, F-test),
indicating that the shrinkage observed was mediated by
cross-reactivity with A.sub.3 receptors. As there are presently no
high-affinity A.sub.2B agonists (Klotz et al., supra.), the
contribution of A.sub.2B receptor stimulation was not pursued,
although the ability of A.sub.3 antagonists to inhibit the response
to 10 .mu.M adenosine (FIG. 5) argues against a role for the
A.sub.2B receptor. For example, MRS1191 at 10 .mu.M did not
displace radioligand binding to recombinant human A.sub.2B
receptors, thus it is a truly selective A.sub.3 antagonist.
EXAMPLE 3
Effects of IB-MECA on Free Intracellular Calcium Levels
[0078] In other cells, stimulation of the A.sub.3-receptor can lead
to an elevation of intracellular Ca.sup.2+ (Kohno et al., supra.),
so intracellular Ca.sup.2+ was monitored in HCE cells to provide an
additional physiologic assay for the presence of A.sub.3 receptors.
HCE cells grown on coverslips for 24-48 hrs were loaded with 1-5
.mu.M fura-2 AM for 30-45 min at room temperature. The cells were
subject to a post-incubation interval of 20-40 minutes at room
temperature before recording began. The coverslips were mounted on
a Nikon Diaphot microscope and visualized with a .times.40
oil-immersion fluorescence objective. The emitted fluorescence (510
nm) from 10-12 confluent cells was acquired at a sampling frequency
of 1 Hz following excitation at 340 nm and 380 nm, and the ratio
was determined with a Delta-Ram system and Felix software (Photon
Technology International Inc., Princeton, N.J.).Cells were perfused
with an isotonic solution consisting of (in mM) 105 NaCl, 6 HEPES
(acid), 4 HEPES (Na.sup.+), 2 CaCl.sub.2, 1 MgCl.sub.2, 4 KCl, 5
glucose and 90 mannitol, at an osmolality of 327 mOsm, pH 7.4. The
ratio of light excited at 380 nm vs. 340 nm was converted into
Ca.sup.2+ concentration using the following equation (Grynkiewicz
et al., J. Biol Chem. 260:3440-3550, 1985): 1 [ Ca 2 + ] = K d * (
( R - R min ) ( R max - R ) ) ( S 2 f S 2 b ) { 2 }
[0079] where R.sub.min and R.sub.max are the ratio of fluorescence
at 340 nM vs. 380 nM in the absence of Ca.sup.2+ and in the
presence of saturating Ca.sup.2+, respectively. R is the ratio
measured experimentally. The S.sub.2f and S.sub.2b are the
fluorescence emitted at 380 nM in the Ca.sup.2+ free and Ca.sup.2+
bound states respectively. An in situ K.sub.d value for fura-2 of
350 nM was used (32). R.sub.min was obtained by bathing cells in a
Ca.sup.2+ free isotonic solution containing 10 mM EGTA and 10 .mu.M
ionomycin. R.sub.max was obtained by bathing the cells in isotonic
solution with 10 mM Ca.sup.2+ and 10 .mu.M ionomycin. Both
calibration solutions were maintained at pH 8.0 to facilitate
Ca.sup.2+ exchange through ionomycin. Background fluorescence
obtained from confluent HCE cells in the absence of Fura-2 was
subtracted from all traces. Mean values of R.sub.min and R.sub.max
were used to obtain the mean responses for a set of experiments.
Data were analyzed using a one-sided unpaired t-test.
[0080] Superfusion of HCE cells with 100 nM IB-MECA produced a
sustained, repeatable and frequently reversible increase in the
intracellular Ca.sup.2+ concentration (FIG. 6). The increase in
Ca.sup.2+ was dependent upon concentration, with 100 nM IB-MECA
leading to a mean rise of 17.+-.5 nM Ca.sup.2+ ((p<0.01, N=8)
while 1 .mu.M IB-MECA intracellular Ca.sup.2+ by 22.+-.6 nM
(p<0.05, N=3). Although these changes were relatively small,
they were sustained, suggesting that these increases in Ca.sup.2+
could be responsible for physiologic effects occurring on a time
scale of minutes to hours.
EXAMPLE 4
Reverse Transcriptase (RT)-PCR Assays
[0081] RT-PCR amplifications of RNA from the human and rabbit NPE
cells were conducted using primers for the human A.sub.3-type
adenosine receptor. RNA was isolated from the HCE human NPE cell
line using Trizol Reagent (Gibco BRL). Template was synthesized in
vitro from the total RNA using an RNA-PCR kit (Gene AMP, Perkin
Elmer, Emeryville, Calif.). The reaction mixture contained MuLV
reverse transcriptase, an antisense primer specific for the A.sub.3
subtype of adenosine receptor, and 1-5 .mu.g of total RNA. Primers
for the human A.sub.3 receptor (Accession No. X76981) were selected
according to the Primer Select program (DNASTAR Inc., Madison,
Wis.). The forward (sense) primer (nucleotides 914-937) was:
5'-GCGCCATCATCTTGACATCTTTT-3' (SEQ ID NO: 1). The reverse
(antisense) primer (nucleotides 1373-1355) was:
5'-CTTGGCCCAGGCATACAGG-3' (SEQ ID NO: 2). The cDNA was amplified by
annealing the set of oligonucleotide primers (0.2 .mu.M) in a final
volume reaction of 100 .mu.l in an Omnigene Thermal Cycler (#480,
HYBAID, Franklin, Mass.). The PCR reaction was conducted for 35
cycles, each cycle comprising 1 min at 95.degree. C., 1 min at
55.degree. C., and 1 min at 72.degree. C. The final extension was
prolonged by 7 min at 72.degree. C. The PCR product was reamplified
using the touchdown PCR method with fresh primers and TAQ
polymerase, using an annealing temperature ranging from 58.degree.
C. to 48.degree. C. The resulting PCR product was size-fractionated
by electrophoresis on 1% agarose gel. To sequence the PCR product,
a band of the expected size (462 bp) was extracted from low
melting-point agarose gel using a Qiaex II Agarose Gel Extraction
kit (Qiaex, Calif.). The purified reaction product was directly
sequenced on an ABI100 sequencer by the DNA Sequencing Facility at
the Cell Center of the University of Pennsylvania and compared with
the predicted sequence using a DNASTAR program.
[0082] The RT-PCR assay of rabbit A3 message was conducted in the
same way with the following changes. RNA was obtained from the tips
of New Zealand White rabbit ciliary processes using Trizol Reagent,
and was reverse transcribed using 3-6 .mu.g total RNA, MuLV reverse
transcriptase and oligo-dT primers. The reaction was carried out at
42.degree. C. for 30 minutes, followed by 5 minutes at 95.degree.
C. The PCR reaction and reamplification steps were performed using
Amplitaq Gold (Perkin-Elmer, Foster City, Calif.) and 10% glycerol
was included in the reamplification step. Specific primers for the
rabbit A3 receptor were selected from the rabbit A3 sequence
(Accession No. U90718); the forward primer (nucleotides 147-167)
was 5'-CAACCCCAGCCTGAAGACCAC-3' (SEQ ID NO: 3) while the reverse
primer (nucleotides 608-587) was 5'-TGAGAAGCAGGGGGATGAGAAT-3' (SEQ
ID NO: 4). Both PCR amplification and reamplification were
performed for 35 cycles, each cycle consisting of 1 min at
95.degree. C., 1 min at 58.5.degree. C. and 1 min at 72.degree. C.
A final extension cycle of 7 minutes at 72.degree. C. minutes
completed the reaction.
[0083] The product of the PCR reamplification of rabbit tissue was
cloned into the PCR-TOPO vector using the TOPO TA cloning kit
(Invitrogen Corporation, Carlsbad, Calif.) following the
manufacturer's directions. After transformation, plasmids were
isolated using the Wizard Plus Miniprep DNA Purification System
(Promega Corporation, Madison, Wis.). The cloned plasmid was cut
with EcoR I restriction nuclease, and a band of approximately the
expected size (479 bp) was identified by running the cut product on
an agarose gel. The plasmid was sequenced from the Sp6 promoter
site 80 base pairs proximal to the PCR product. The sequence was
compared to the expected rabbit A.sub.3 sequence using a DNASTAR
program.
[0084] From the RT-PCR amplifications of human NPE cells using
primers for the human A.sub.3 receptor, a fragment of the expected
462-bp size was obtained, and was enhanced by direct PCR
amplification of the product. The sequence obtained from the
reamplified product was compared to the sequences of known human
adenosine receptors using the DNASTAR program. The results
displayed a 97.4% similarity to the published base sequence for the
A.sub.3 receptor, whereas the similarity indices for the other
known adenosine-receptor subtypes were all <40% [37.9% for
A.sub.1 (Accession No. 68485), 35.0% for A.sub.2A (Accession No.
68486), and 36.7% for A.sub.2B (Accession No. 68487)]. No product
was detected when reverse-transcriptase was excluded from the
initial reaction mixture.
[0085] RT-PCR amplification was also conducted with rabbit ciliary
processes, using primers for the rabbit A.sub.3-type adenosine
receptor. The RT-PCR product was reamplified, cloned and sequenced.
The sequence displayed a 97.4% similarity with the published base
sequence for the rabbit A.sub.3 receptor There was only 27.9%
homology between rabbit A.sub.1 (Accession No. L01700) and A.sub.3
receptors. Sequences are not yet available for the remaining
A.sub.2A- and A.sub.2B-subtypes of adenosine receptors in the
rabbit. Our rabbit product also displayed 75.1% similarity to the
human A.sub.3 receptor but only <30% similarity indices for the
other human adenosine-receptor subtypes (28.2% for A.sub.1, 27.7%
for A.sub.2A and 29.5% for A.sub.2B). No product was detected when
reverse-transcriptase was excluded from the reaction mixture.
EXAMPLE 5
Transepithelial Measurements
[0086] Adult male Dutch belted rabbits weighing 1.8-2.4 kg (Ace
Animals, Boyertown, Pa.) were anesthetized with pentobarbital and
sacrificed (Carre et al., J. Membr. Biol. 146:293-305, 1995). After
enucleation, the iris-ciliary body (I-CB) was isolated as
previously described (Carre et al., 1995, supra.). The experiments
were in accordance with the Resolution on the Use of Animals in
Research of the Association for Research in Vision and
Ophthalmology.
[0087] The pupil and central iris were occluded with a Lucite disc,
and the iris-ciliary body was mounted between the two halves of a
Lucite chamber (1). The annulus of exposed tissue provided a
projected surface area of 0.93 cm.sup.2. Preparations were
continuously bubbled with 95%O.sub.2-5%CO.sub.2 for maintenance of
pH 7.4 in a Ringer's solution comprising (in mM): 110.0 NaCl, 10.0
HEPES (acid), 5.0 HEPES (Na.sup.+), 30.0 NaHCO.sub.3, 2.5
CaCl.sub.2, 1.2 MgCl.sub.2, 5.9 KCl, and 10.0 glucose, at an
osmolality of 305 mOsm. BaCl.sub.2 (5 mM) was added to the solution
to block K.sup.+ currents. The transepithelial potential was fixed
at 0 mV, corrected for solution series resistance, and the
short-circuit current was monitored on a chart recorder. Data were
digitally acquired at 10 Hz via a DigiData 1200A converter and
AxoScope 1.1 software (Axon Instruments, Foster City, Calif.).
Automatic averaging was performed with a reduction factor of 100 to
achieve a final sampling rate of 6/min.
[0088] Adenosine in high concentration (100 .mu.M) has been found
to increase the short-circuit current across the rabbit ciliary
body (Carre et al., supra., 1997). We therefore tested whether a
high concentration (30 .mu.M) of the A.sub.3 agonist IB-MECA also
affected short-circuit current. At this concentration, the vehicle
(dimethylformamide) itself exerts significant effects (FIG. 7,
lowest trajectory). We corrected for the solvent effect in the
following way. Solvent alone was initially introduced (to 0.1%),
followed by the same volume of solvent (to 0.2%) containing
agonist, and ending with addition of a third identical volume of
solvent alone (to a final concentration of 0.3%). The reduction in
short-circuit current following the first addition of solvent was
always greater than the third. In each of four experiments, we
averaged the time courses of the first and third additions to
estimate the effect of raising the solvent concentration without
agonist from 0.1% to 0.2% during the experimental period. FIG. 7
presents the mean trajectory for the averaged solvent effect, the
uncorrected mean time course following exposure to IB-MECA, and the
mean trajectory.+-.1 SE for the solvent-corrected response. The
experiments were performed in the presence of 5 mM Ba.sup.2+ to
minimize the contribution of K.sup.+ currents. IB-MECA produced a
significant increase in the short-circuit current; an increase in
short-circuit current in the presence of Ba.sup.2+ suggests that
the effect is mediated by activating a Cl.sup.- conductance on the
basolateral membrane of the NPE cells. The sustained nature of the
stimulation is consistent with the time course of the cell
shrinkage in response to A.sub.3 stimulation.
EXAMPLE 6
Volumetric Measurements and Analysis
[0089] The volume of PE cells was measured as the movement of fluid
that underlies a change in PE cell volume, this is thought to be
the same as the movement of fluid which underlies the reabsorption
of aqueous humor (FIG. 1).
[0090] After harvesting a single T-75 flask by trypsinization
(Yantorno et al., supra.), a 0.5-ml aliquot of the bovine cell
suspension in DMEM (or in Cl.sup.--free medium, where appropriate),
described in Example 1 was added to 20 ml of each test solution.
The standard test solution contained (in mM) 110.0 NaCl, 15.0 HEPES
[4-(2-hydroxyethyl)-1-piperazine- ethanesulfonic acid], 2.5
CaCl.sub.2, 1.2 MgCl.sub.2, 4.7 KCl, 1.2 KH.sub.2PO.sub.4, 30.0
NaHCO.sub.3, and 10.0 glucose, at a pH of 7.4 and osmolality of
298-305 mOsm. The Cl.sup.- free solution comprised: 110.0 sodium
methanesulfonate, 15.0 HEPES, 2.5 calcium methanesulfonate, 1.2
MgSO.sub.4, 4.7 potassium methanesulfonate, 1.2 KH.sub.2PO.sub.4,
30.0 NaHCO.sub.3, and 10.0 glucose, at a pH of 7.4 and osmolality
of 294-304 mOsm. Parallel aliquots of cells were studied on the
same day. One aliquot usually served as a control, and the others
were exposed to different experimental conditions at the time of
suspension. The same amount of solvent vehicle (dimethylformamide,
DMSO or ethanol) was always added to the control and experimental
aliquots. The sequence of studying the suspensions was varied to
preclude systematic time-dependent artifacts (Civan et al.,
1994).
[0091] Cell volumes of isoosmotic suspensions were measured with a
Coulter Counter (model ZBI-Channelyzer II), using a 100-.mu.m
aperture (Civan et al., 1992). As previously described (Yantorno et
al., supra.), the cell volume (v.sub.C) of the suspension was taken
as the peak of the distribution function. Cell shrinkage was fit as
a function of time (t) to the simple exponential function:
v.sub.C=(v.sub.0-v.sub..infin.).multidot.(e.sup.-t/.tau.)+v.sub..infin.
(1)
[0092] where v.sub..infin. is the steady-state cell volume, v.sub.0
is cell volume at t=0, and .tau. is the time constant of of the
shrinkage. For purposes of data reduction; the data were normalized
to the first time point, taken to be 100% isotonic volume. The
baseline isotonic value was 2488.+-.203 fl (mean.+-.SE, N=15). Fits
were obtained by nonlinear least-squares regression analysis,
permitting both v.sub..infin. and .tau. to be variables (Carr et
al., supra., 1997).
[0093] In approximately 15% of the volumetric studies, ATP produced
shrinkage of the PE cells in suspension. As shown in FIG. 8A, the
shrinkage was faster and larger after exposure to 100 .mu.M than to
10 .mu.M ATP. In contrast to the results displayed in FIGS. 8A and
9A-C, ATP alone exerted very little effect on cell volume in
.about.85% of the PE cell-suspensions studied over the
concentration range 100 .mu.M-10 mM (FIGS. 10A, 13A, 13C,
14A-C).
[0094] The clinically important, nonsteroidal antiestrogen
tamoxifen triggered no consistent response in cell volume over a
30-min period of observation (FIGS. 10A-B). However, in the
presence of tamoxifen, the response to ATP was strongly enhanced.
The effect was most striking in those preparations which displayed
little or no shrinkage when exposed to 100 .mu.M ATP (FIGS. 13A,
13C, 14A-C) or to 10 mM ATP (FIG. 10A). However, the volumetric
response to ATP was also enhanced in cell preparations responsive
to ATP alone (FIG. 8B). No such interactive response was observed
between the corresponding nucleoside adenosine and tamoxifen (FIG.
10B).
[0095] At a constant tamoxifen concentration, ATP triggered
detectable shrinkage at 10 .mu.M, but not at 1 .mu.M (FIG. 1C). At
3 .mu.M ATP, the shrinkage was about half that noted with 10 .mu.M
ATP (both in the presence of tamoxifen, FIG. 8B). The effects of
ATP were comparable at 10 .mu.M and 1 mM (FIG. 8C). A concentration
of 100 .mu.M ATP was used in all of the subsequent experiments to
ensure that the concentration of ATP was not limiting the rate of
shrinkage. The choice of a tamoxifen concentration of 6-10 .mu.M
was based on two considerations: 10 .mu.M is the concentration used
in probing Cl.sup.- channels in other cells (Wu et al., supra.,
1996), and the concentration needed to produce a minimum detectable
effect on the response to swelling the current bovine PE cells is
>2 .mu.M and .ltoreq.6 .mu.M (Mitchell et al., Invest.
Ophthalmol. Vis. Sci.38 (Suppl.):S1042, 1997).
[0096] Several observations suggest that the ATP,
tamoxifen-activated shrinkage involved Cl.sup.- release. First, the
synergism occurred even when gramicidin was present to provide a
constant pathway for K.sup.+ release (Civan et al., supra., 1994)
(FIG. 11A), suggesting that the reduction in volume prompted by ATP
and tamoxifen was due to the activation of an anionic conductance.
Second, removal of Cl.sup.- from the preincubation and test
solutions abolished the synergistic response (FIG. 11A). Third, the
Cl.sup.--channel blockers NPPB (100 .mu.M) (Wangemann et al.,
supra., 1986) and DIDS (500 .mu.M) (Cabantchik et al., Am. J.
Physiol., 262: C803-C827, 1992) both inhibited the volume reduction
(FIG. 11B).
[0097] The synergistic stimulators effect of tamoxifen in shrinking
PE cells was unexpected, given its inhibition of swelling-activated
Cl.sup.- channels in many cells (Valverde et al., supra.; Zhang et
al., supra.; Nilius et al., supra.), including NPE cells (Wu et
al., supra, 1996), and the absence of any effect on
swelling-activated Cl.sup.- channels in PE cells (Mitchell et al.,
supra., 1997). Therefore, we reexamined the effect of tamoxifen on
the swelling-activated Cl.sup.- channels of the immortalized human
NPE cells we have previously characterized (FIG. 12). After
hypotonic swelling, the cell volume spontaneously fell (the
regulatory volume decrease, RVD), reflecting the release of KCl and
secondarily water (Civan, et al., supra., 1994). Addition of
tamoxifen 10 min later, after the conclusion of the RVD, did not
affect cell volume, but addition 5 min after hypotonic suspension
reduced the magnitude of the RVD. Inclusion of tamoxifen at the
time of the initial hypotonic suspension completely abolished the
RVD, consistent with the earlier report (Wu et al., supra., 1996)
that tamoxifen blocks swelling-activated Cl.sup.- channels of NPE
cells. Tamoxifen also markedly slowed the rate of hypotonic
swelling (FIG. 12), raising the possibility that the antiestrogen
also blocks the aquaporin-1 (AQP1) water channels of the NPE cells
(Stamer et al., Invest. Ophthalmol. Vis. Sci. 35: 3867-3872, 1994;
Lee et al., Current Topics in Membranes 45:105-134, 1998).
[0098] The block of RVD in NPE cells by tamoxifen suggested that
tamoxifen would reduce efflux of aqueous humor from NPE cells in
addition to its action of stimulating reabsorption from PE cells.
Thus, tamoxifen could reduce aqueous humor production by two
separate mechanisms.
EXAMPLE 7
Ruptured-Patch Whole-Cell Recording
[0099] Harvested cultured PE cells were resuspended and permitted
to settle and attach to glass coverslips, which were then
transferred to a perfusion chamber (Carr et al., supra., 1997).
Solutions were designed to isolate any C.sup.- current activated by
ATP. Thus both internal and external solutions were devoid of
K.sup.+, and the cation reversal potential was >95 mV. The
perfusate contained (in mM): 105.0 NaCl, 6.0 HEPES acid, 4.0 HEPES
Na.sup.+, 1.3 CaCl.sub.2, 0.5 MgCl.sub.2 and 90.0 mannitol (pH 7.4,
316 mOsm) and the micropipette-filling solution contained (in mM):
40.0 NCl, 135.0 NMDG-OH, 95.0 CH.sub.3SO.sub.4 acid, 2.0 MgATP,
0.05 GTP, 1.1 EGTA and 0.55 CaCl.sub.2 (pH 7.2, 275 mOsm).
Supplementary experiments were also conducted with
freshly-dissected bovine PE cells prepared by the method of Jacob
et al. (Am. J. Physiol., 261:C1055-C1062, 1991), as previously
described (Carr et al., supra., 1997). Cells were patched while
still round, usually one day after dissociation. The perfusate
contained (in mM): 105.0 NaCl, 6.0 HEPES acid, 4.0 HEPES Na.sup.+,
1.3 CaCl.sub.2, 0.5 MgCl.sub.2, 8.0 sucrose and 70.0 mannitol (pH
7.4, 305 mOsm) and the micropipette-filling solution contained (in
mM): 105.0 NMDG-Cl, 10.0 HEPES acid, 70.0 mannitol, 2.0 MgATP, 0.01
GTP, 1.1 EGTA and 0.55 CaCl.sub.2 (pH 7.2, 286 mOsm). Cells were
perfused with Na.sub.2ATP dissolved directly into isotonic solution
(1.0) or serially diluted (100 .mu.M and 10 .mu.M), while NPPB was
diluted 1000:1 from a 100 mM stock in DMSO.
[0100] Data were acquired at 1 kHz using Axopatch-1B electronics
and associated headstage (Axon Instruments, Foster City, Calif.)
and filtered at 500 Hz with a Bessel filter. The micropipettes were
double-pulled from Corning No. 7052 glass, coated with Sylgard and
fire polished. The membrane potential was held without
series-resistance compensation at 0 mV and stepped to voltages over
the range from -100 to +100 mV in 20 mV increments for 200 msec
periods. The mean current measured between 150-200 msec was used to
obtain the patch-clamp data used here. The data presented in FIG. 2
was obtained at -60 mV [the approximate membrane potential (Green
et al., Invest. Ophthalmol. Vis. Sci., 26:371-381, 1985)] to
facilitate comparison with the volumetric data.
[0101] ATP increased the current (FIG. 9A). The activated currents
were likely carried by Cl.sup.- as solutions were chosen to
minimize cationic currents, and the Cl.sup.--channel blocker NPPB
(100 .mu.M) (Wangemann et al., Pflugers Archiv. 407 (Suppl.
2):S128-S141, 1986) reduced the ATP-enhanced currents at -60 mV by
56.+-.11% (FIG. 9B, N=3). The activation of Cl.sup.- channels was
not restricted to the PE cell line, for ATP also stimulated
currents in fresh bovine PE cells (FIG. 9C) cells. Although current
was activated in both fresh and cultured cells, both cell types
contained a heterogeneous population with cultured cells responding
in 10/17 trials and fresh cells activated in 3/9 trials. Thus, ATP
alone can activate Cl.sup.- curents directly, but only in half the
cells.
EXAMPLE 8
Measurements of Intracellular Ca.sup.2+
[0102] Bovine PE cells grown on coverslips for 2-4 days were loaded
with 5 .mu.M fura-2 AM for 30-45 min at room temperature, and then
rinsed and maintained in fura-free solution before beginning data
acquisition. The coverslips were mounted on a Nikon Diaphot
microscope and visualized with a .times.40 oil-immersion
fluorescence objective. The emitted fluorescence (510 nm) from
10-12 confluent cells was sampled at 1 Hz following excitation at
340 nm and 380 nm, and the ratio determined with a Delta-Ram system
and Felix software (Photon Technology International Inc.,
Princeton, N.J.). The ratio of light excited at 340 nm to that at
380 nm was converted into Ca.sup.2+ concentration using the method
of Grynkiewicz et al. (J. Biol. Chem. 260:3440-3450, 1985). An in
situ K.sub.d value for fura-2 of 350 nM was used (Negulescu et al.,
Meth. Enzymol. 192:38-81, 1990). R.sub.min was obtained by bathing
cells in a Ca.sup.2+ free isotonic solution of pH 8.0 containing 10
mM EGTA and 5 .mu.M ionomycin. R.sub.max was obtained by bathing
the cells in isotonic solution with 1.3 mM Ca.sup.2+ and 5 .mu.M
ionomycin. Calibration was performed separately for each
experiment. Baseline levels from PE cells in the absence of fura-2
were subtracted from records to control for autofluorescence.
Experiments were performed predominantly at room temperature, but
several trials were performed at 37.degree. C. using a temperature
control unit from Warner Instrument Corp., (Hamden, Conn.). Cells
were perfused with an isotonic solution containing (in mM) 105
NaCl, 6 HEPES (acid), 4 HEPES (Na.sup.+), 1.3 CaCl.sub.2, 1
MgCl.sub.2, 4 KCl, 5 glucose and 90 mannitol, at an osmolarity of
317 mOsm, pH 7.4. Tamoxifen was stored as a 10 mM stock in ethanol
for 2 days. In comparing the effects of ATP and of ATP+tamoxifen,
0.1% ethanol was also added to the solutions containing ATP
alone.
EXAMPLE 9
Interaction with Histamine and Muscarinic Receptors
[0103] In addition to its actions on nuclear estrogen receptors
(Klinge et al., supra.) and swelling-activated Cl.sup.- channels
(Valverde et al., supra.; Zhang et al., supra.; Nilius et al.,
supra.; Wu et al., supra.), tamoxifen has been reported to produce
multiple other effects. Experiments were conducted in order to
address the possibility that one or more of the following known
actions of tamoxifen may be involved in ATP, tamoxifen-activated
shrinkage: interaction with histamine and muscarinic receptors,
antagonism of calcium/calmodulin, inhibition of protein kinase C,
and antagonism of plasma- or nuclear membrane estrogen
receptors.
[0104] The antiestrogens are known to interact with histamine
(Brandeset al., supra.) and muscarinic receptors (Ben-Baruch et
al., supra.) in other preparations. FIG. 13A indicates that 10
.mu.M histamine did not enhance the volumetric response to 100
.mu.M ATP (N=4). The nonmetabolizable muscarinic agonist carbachol
did trigger a prompt shrinkage in the presence of 100 .mu.M ATP
(FIG. 13A, N=4). However, carbachol triggered approximately the
same response whether or not ATP was present, and 10 .mu.M atropine
abolished that response (FIG. 13B, N=3). In contrast, tamoxifen had
little effect in the absence of ATP (FIGS. 8C, 10A and 10C), and 10
.mu.M atropine did not alter the response to the combined presence
of tamoxifen and ATP (FIG. 13C, N=4). FIG. 13 indicates that the
volumetric actions of tamoxifen cannot be mediated by either
histamine or muscarinic receptors.
[0105] However, the results do show that carbachol can act alone to
reduce PE cell volume. This suggests that carbachol, or similar
agents, can stimulate fluid reabsorption by the PE cells and thus
reduce the net production of aqueous humor.
EXAMPLE 10
Antagonism of Calcium/Calmodulin
[0106] Tamoxifen can inhibit calcium/calmodulin at the same
concentration (10 .mu.M) typically used to block Cl.sup.- channels
in NPE cells (Lam, supra.; Wu et al., supra., 1996). In PE cells,
trifluoperazine triggered a partial shrinkage of the bovine PE
cells in the presence of ATP but this effect was not synergistic as
similar effects were observed in the absence of ATP (FIG. 14). This
suggests that although calcium/calmodulin can modulate cell volume,
it does not mediate the synergistic action of tamoxifen. Thus,
inhibitors of calcium/calmodulin can provide a separate additional
route by which fluid efflux from the PE cells can be stimulated and
the net production of aqueous humor reduced.
EXAMPLE 11
Protein Kinase C Inhibition
[0107] Tamoxifen can also inhibit protein kinase C (PKC), with a
K.sub.i of 5-100 .mu.M depending on the assay system (O'Brien et
al., supra.). However, inhibiting PKC activity with the PKC
inhibitor staurosporine produced a small, insignificant shrinkage
in the presence of 100 .mu.M ATP, substantially less that that
produced by tamoxifen (FIG. 15). Activating PKC with DiC.sub.8 in
the presence of 100 .mu.M ATP also had no significant effect on
cells volume. Thus, the synergistic effect of tamoxifen cannot be
mediated by its inhibition of baseline PKC activity.
EXAMPLE 12
Antagonism of Estrogen Receptors
[0108] In the presence of 100 nM 17.beta.-estradiol, the response
to the combined application of ATP and tamoxifen was reduced,
consistent with the known antiestrogen action of tamoxifen (FIG.
16A, N=4, P<0.05, F-distribution). The 17.beta.-estradiol also
reduced the synergistic shrinkage produced by ATP and tamoxifen in
another series of four experiments (P<0.01, F-distribution, data
not shown), whereas the inactive estrogenic isomer
17.alpha.-estradiol had no significant effect (P>0.05,
F-distribution). In the absence of tamoxifen, the 17.alpha.- and
17.beta.-estradiols exerted very small effects on cell volume (FIG.
16B, N=4). The data are consistent with the possibility that
tamoxifen and estrogen compete for occupancy of the same population
of receptors.
EXAMPLE 13
Potential Role of Ca.sup.2+
[0109] Non-pigmented ciliary epithelial cells show a synergistic
elevation in free intracellular Ca.sup.2+ concentration
(Ca.sup.2+.sub.i) upon simultaneous presentation of certain drug
pairs, and this synergism may involve the activation of the G.sub.i
G-protein (Farahbakhsh et al., Exp. eye Res. 64:173-179, 1997). As
ATP can activate G.sub.i in a variety of tissues (Murthy et al., J.
Biol. Chem. 273:4695-4704, 1998), it was determined whether the
signaling cascade for the synergistic shrinkage produced by
tamoxifen and ATP could reflect a synergistic change in
Ca.sup.2+.sub.i. Tamoxifen (10 .mu.M) itself triggered no
significant change (.DELTA.) in Ca.sup.2+.sub.i (.DELTA.=7.+-.6 nM,
N=4). Although Ca.sup.2+.sub.i increased in response to both ATP
and ATP+TMX, the comparison of the response was complicated by the
attenuation of the Ca.sup.2+ spike with repeated exposure, and the
variation between preparations. A 3-min application of either 100
.mu.M ATP or 100 .mu.M ATP+10 .mu.M TMX usually produced an
elevation in Ca.sup.2+but it was difficult to elicit a response of
similar magnitude to a second application 5 min later. Elevating
the temperature to 37.degree. C. did not eliminate the
attenuation.
[0110] Nevertheless, it did prove possible to compare the
magnitudes of successive Ca.sup.2+ responses when each drug
application was limited to periods of 20 sec (FIG. 17). Experiments
were performed by alternating 20-sec exposures to 100 .mu.M ATP+10
.mu.M TMX with 20 sec exposures to 100 .mu.M ATP alone (including
0.1% ethanol as a vehicle control for the TMX). Cells were washed
in isotonic solution for 5 min between drug applications, and 4-5
applications were possible per trial (FIG. 17A). The order of drug
application shown in FIG. 10A, beginning first with 100 .mu.M
ATP+10 .mu.M TMX, is termed the T series. A parallel set of
experiments termed the A series was performed which began with the
application of ATP alone followed 5 min later by ATP+TMX.
[0111] To check for synergism while compensating for the
attenuation, the responses to each application were compared for
those experiments where ATP was first added (A series) with where
ATP+TMX was first added (T series). Comparing the responses to
successive applications of drugs, it is clear that there was no
significant difference between the two series, whether ATP alone or
ATP+TMX was added at a given point in time (FIG. 17B). The presence
of TMX did not affect the size of the Ca.sup.2+ response to ATP
regardless of whether it was included in the first, second, third
or fourth application (P>0.05 for applications 1-4, No=3.4). We
conclude that ATP and tamoxifen did not produce a synergistic
elevation in the level of intracellular Ca.sup.2+.
EXAMPLE 14
In vivo Animal Model
[0112] A rabbit model is used to determine the ability of A.sub.3
subtype adenosine receptor antagonists, antiestrogens and
calmodulin antagonists to reduce intraocular pressure. Ten normal
New Zealand White rabbits are used for the study, sedated, and
their intraocular pressure is measured by standard optometric
methods for several days at various times of the day to account for
normal pressure variations. Once the average baseline pressure of
each obtained, five rabbits are assigned to one of two groups. In
the first group, one eye is administered vehicle (e. g., corn oil
or dimethyl sulfoxide (DMSO)) in the form of liquid drops and the
other eye is left as an untreated control. In the second group, one
eye is administered vehicle plus test compound in the form of
liquid drops, and the other eye is left as an untreated control.
The test compound is administered at a concentration ranging from
about 1 nM to 100 mM to see a dose response relationship of
intraocular pressure reduction. Intraocular pressure is then
measured several hours after the administration to determine
reduction of intraocular pressure by the test compound.
EXAMPLE 15
Synthesis of MRS-1649 and Related A.sub.3 Receptor Antagonists
[0113] Materials. lodomethane was purchased from Fluka (Buchs,
Switzerland). Iodoethane and 1-iodopropane were purchased from
Aldrich (Milwaukee, Wis.). PBS (1.times. pH 7.4) was purchased from
Biofluids, Inc. (Rockville, Md.). Starting
3,5-diacyl-2,4-dialkylpyridine and dihydropyridine derivatives were
described previously (Li et al., J. Med. Chem. 41:3186-3201, 1998;
Li et al., J. Med. Chem. 42:706-721, 1999). All other materials
were obtained from commercial sources.
[0114] Proton nuclear magnetic resonance spectroscopy was performed
on a Varian GEMINI-300 spectrometer, and all spectra were obtained
in CDCl.sub.3. Chemical shifts (.delta.) relative to
tetramethylsilane are given. Chemical-ionization (CI) mass
spectrometry was performed with a Finnigan 4600 mass spectrometer,
and electron-impact (EI) mass spectrometry with a VG7070F mass
spectrometer at 6 kV. Elemental analysis was performed by Galbraith
Laboratories, Inc. (Knoxville, Tenn.) and/or Atlantic Microlab,
Inc. (Norcross, Ga.).
[0115] General Procedure for Preparation of Pyridinium Salt (10,
11, 14-23) by Quaternary Amination of
3,5-Diacyl-2,4-Dialkylpyridine Derivatives with Iodomethane: A
mixture of a 3,5-diacyl-2,4-dialkylpyridi- ne derivative (14 mg,
0.038 mmol) and iodomethane (59 mg, 0.38 mmol) in 2 mL of anhydrous
nitromethane was sealed in a Pyrex tube and was heated at
80.degree. C. for 2 days. After the mixture cooled to room
temperature, the solvent and excess Mel were removed under reduced
pressure to leave a yellow oil. It was applied to TLC separation
[ethyl acetate:petroleum ether=1:4 (v/v) for the first development;
methanol:chloroform=1:5 (v/v) for a second development and ethyl
acetate:petroleum ether=1:1 (v/v) for a third development] and 9.5
mg of the desired product (Pyridinium Salt, such as MRS 1649, 11)
was afforded as a yellow solid (yield: 49%). If methanol or acetone
was used as the solvent, the yield was much lower than with
nitromethane.
[0116] HPLC results showed that 11 is free of the starting 2
(2,4-diethyl-3-(ethylsulfanylcarbonyl)-5-ethyloxy-carbonyl-6phenylpyridin-
e). The mobile phase used for the analysis consisted of methanol,
acetonitrile and water (45:45:10). At a flow rate of 1.0 mL/min
with a 4.6.times.250 mm (internal diameter) reverse-phase 300 .ANG.
C-18 column operated at ambient temperature, 11 had a retention
time of 2.3 min (purity>99%). CHN analysis of 11: Calcd for
C.sub.22H.sub.28INO.sub.3S C: 51.47%, H: 5.50%, N: 2.73%. Found: C:
51.42%, H: 5.14%, N: 2.43%. HR-MS (FAB, m-b): Calcd for
C.sub.22H.sub.28NO.sub.3S (M.sup.+-I): 386.1790. Found: 386.1776.
UV spectra was measured using a Beckman DU 640 Spectrophotometer.
In methanol at ambient temperature, 11 had a .lambda..sub.max=203
nm, .epsilon..sub.max=6.65.times.10.sup.4 lmol.sup.-1cm.sup.-1;
.lambda..sub.max=224 nm, .epsilon..sub.max=3.30.tim- es.10.sup.4
lmol.sup.-1cm.sup.-1; .lambda..sub.max=288 nm,
.epsilon..sub.max=9.62.times.10.sup.3 lmol.sup.-1cm.sup.-1.
[0117] The water solubility of 11 was measured by the following
method. 100 .mu.L of de-ionized water was saturated with 5 mg 11
with heating. After cooling to room temperature and the
disappearance of turbidity, 50 .mu.L of the clear supernatant was
withdrawn and lyophilized to give 1.1 mg of 11. The water
solubility of 11 was calculated to be 42.8 mM at room
temperature.
1-Methyl-2-methyl-4-ethyl-3-(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl-6-p-
henylpyridinium Iodide (10)
[0118] .sup.1H-NMR .delta.: 0.88 (t, J=6.9 Hz, 3 H), 1.26 (t, J=7.5
Hz, 3 H), 1.33 (t, J=7.5 Hz, 3 H), 2.69 (s, 3H), 2.79 (q, J=7.5 Hz,
2 H), 3.24 (q, J=7.5 Hz, 2 H), 4.01 (q, J=6.9 Hz, 2 H), 4.17 (s, 3
H), 7.49-7.53 (m, 3 H), 7.65-7.68 (m, 2 H). MS (Cl/NH.sub.3): m/z
500 (MH.sup.+), 358 (MH.sup.+-Me--I), 297 (MH.sup.+-Me--I--SEt).
K.sub.i(hA.sub.3)=379 nM.
1-methyl-2,4-Diethyl-3-(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl-6-phenyl-
pyridinium Iodide (MRS 1649, 11)
[0119] .sup.1H-NMR .delta.: 0.86 (t, J=7.2 Hz, 3 H), 1.32 (t, J=7.8
Hz, 3 H), 1.41 (t, J=7.8 Hz, 3 H), 1.44 (t, J=7.8 Hz, 3 H), 2.84
(q, J=7.8 Hz, 2 H), 3.22 (q, J=7.8 Hz, 2 H), 3.44 (q, J=7.8 Hz, 2
H), 3.98 (q, J=7.2 Hz, 2 H), 4.22 (s, 3 H), 7.56-7.62 (m, 3 H),
7.72-7.75 (m, 2 H). MS (Cl): m/z 514 (MH.sup.+), 372
(MH.sup.+-Me--I). K.sub.i(hA.sub.3)=219 nM.
1-methyl-2,4-Diethyl-3-(ethylsulfanylcarbonyl)-5(2-fluoroethyloxycarbonyl)-
-6-phenyl pyridinium Iodide (14)
[0120] .sup.1H-NMR .delta.: 1.20 (t, J=7.5 Hz, 3 H), 1.39 (t, J=7.5
Hz, 3 H), 1.46 (t, J=7.5 Hz, 3 H), 2.79 (q, J=7.5 Hz, 2 H), 2.99
(q, J=7.5 Hz, 2 H), 3.27 (q, J=7.5 Hz, 2 H), 4.20 (s, 3 H), 4.28
(m, 2 H), 4.30-4.40 (m, 2 H), 7.47-7.50 (m, 3 H), 7.64-7.67 (m, 2
H). MS (Cl/NH.sub.3): m/z 390 (MH.sup.+-Me--I).
K.sub.i(hA.sub.3)=364 nM.
1-Methyl-2-ethyl-4-ethyl-3-(ethylsulfanylcarbonyl)-5-propyloxycarbonyl-6-p-
henylpyridinium Iodide (15)
[0121] .sup.1H-NMR .delta.: 0.68 (t, J=7.8 Hz, 3 H), 1.25 (t, J=7.8
Hz, 3 H), 1.39 (m, 2 H), 1.49 (t, J=7.8 Hz, 3 H), 1.57 (t, J=7.8
Hz, 3 H), 2.97 (q, J=7.8 Hz, 2 H), 3.28 (q, J=7.8 Hz, 2 H), 3.38
(q, J=7.8 Hz, 2 H), 4.07 (t, J=6.9 Hz, 2 H), 4.21 (s, 3 H),
7.69-7.76 (m, 5 H). MS (Cl/NH.sub.3): m/z 386 (MH.sup.+-Me--I).
K.sub.i(hA.sub.3)=483 nM.
1-Methyl-2-ethyl-4-propyl-3-(ethylsulfanylcarbonyl)-5-propyloxycarbonyl-6--
phenylpyridinium Iodide (16)
[0122] .sup.1H-NMR .delta.: 0.67 (t, J=7.8 Hz, 3 H), 1.04 (t, J=7.8
Hz, 3 H), 1.40 (m, 2 H), 1.48 (t, J=7.8 Hz, 3 H), 1.55 (t, J=7.8
Hz, 3 H), 1.74 (m, 2 H), 2.87 (t, J=7.8 Hz, 2 H), 3.27 (q, J=7.8
Hz, 2 H), 3.42 (q, J=7.8 Hz, 2 H), 4.05 (t, J=7.8 Hz, 2 H), 4.20
(s, 3 H), 7.62-7.74 (m, 5 H). MS (Cl/NH.sub.3): m/z 558
(M.sup.++NH.sub.4), 525 (M.sup.+-1-Me), 414 (M.sup.+-I), 369
(M.sup.+-1-I--Me--Et). K.sub.i(hA.sub.3)=2.02 .mu.M.
1-Methyl-2-ethyl-4-propyl-3-(3-fluoropropylsulfanylcarbonyl)-5-propyloxyca-
rbonyl-6-phenylpyridinium Iodide (17)
[0123] .sup.1H-NMR .delta.: 0.68 (t, J=7.8 Hz, 3 H), 1.04 (t, J=7.8
Hz, 3 H), 1.41 (m, 2 H), 1.55 (t, J=7.8 Hz, 3 H), 1.73 (m, 2 H),
2.16 (m, 2 H), 2.88 (m, 2 H), 3.35 (q, J=7.8 Hz, 2 H), 3.41 (t,
J=6.9 Hz, 2 H), 4.06 (t, J=6.9 Hz, 2 H), 4.22 (s, 3 H), 4.55 (t,
J=6.0 Hz, 1 H), 4.71 (t, J=6.0 Hz, 1 H), 7.68-7.75 (m, 5 H).
[0124] MS (Cl/NH.sub.3): m/z 432 (MH.sup.+-Me--I).
K.sub.i(hA.sub.3)=465 nM.
1-Methyl-2-ethyl-4(2-acetylthioethyl)-3-(ethylsulfanylcarbonyl)-5-propylox-
ycarbonyl-6-phenylpyridinium Iodide (18)
[0125] .sup.1H-NMR .delta.: 0.66 (t, J=7.5 Hz, 3 H), 1.37 (m, 2 H),
1.39 (t, J=7.8 Hz, 3 H), 1.45 (t, J=7.2 Hz, 3 H), 2.35 (s, 3 H),
2.89-3.02 (m, 4 H), 3.11 (m, 2 H), 3.28 (q, J=7.2 Hz, 2 H), 4.00
(t, J=6.9 Hz, 2 H), 4.23 (s, 3 H), 7.53-7.55 (m, 3 H), 7.67-7.70
(m, 2 H). MS (Cl/NH.sub.3): m/z 460 (MH.sup.+-Me--I).
K.sub.i(hA.sub.3)=538 nM.
1-Methyl-2-ethyl-4-(2-phthalimidoethyl)-3-(ethylsulfanylcarbonyl)-5-ethylo-
xycarbonyl-6-phenylpyridinium Iodide (19)
[0126] .sup.1H-NMR .delta.: 0.94 (t, J=6.9 Hz, 3 H), 1.34 (t, J=7.2
Hz, 3 H), 1.48 (t, J=7.2 Hz, 3 H), 2.94 (q, J=6.9 Hz, 2 H), 3.15
(t, J=7.8 Hz, 2 H), 3.24 (q, J=7.2 Hz, 2 H), 4.01 (t, J=7.8 Hz, 2
H), 4.21 (s, 3 H), 4.25 (q, J=7.2 Hz, 2 H), 7.51 (m, 3 H), 7.65 (m,
2 H), 7.78 (m, 2 H), 7.89 (m, 2 ). MS (Cl/NH.sub.3): m/z 517
(MH.sup.+-Me--I). K.sub.i(hA.sub.3)=1.25 .mu.M.
1-Methyl-2-butyl-4-ethyl-3-(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl-6-ph-
enylpyridinium Iodide (20)
[0127] .sup.1H-NMR .delta.: 0.90 (t, J=7.5 Hz, 3 H), 1.04 (t, J=7.5
Hz, 3 H), 1.29 (t, J=7.5 Hz, 3 H), 1.34-1.43 (m, 2 H), 1.46 (t,
J=7.5 Hz, 3 H), 1.84 (m, 2 H), 2.76 (q, J=7.5 Hz, 2 H), 2.88 (t,
J=7.5 Hz, 2 H), 3.16 (q, J=7.5 Hz, 2 H), 4.05 (q, J=7.5 Hz, 2 H)
4.26 (s, 3 H), 7.52-7.55 (m, 3 H), 7.69-7.72 (m, 2 H). MS
(Cl/NH.sub.3): m/z 400 (MH.sup.+-Me--I). K.sub.i(hA.sub.3)=436
nM.
1-Methyl-2-cyclobutyl-4-ethyl-3-(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl-
-6-phenyl pyridinium Iodide (21)
[0128] .sup.1H-NMR .delta.: 0.99 (t, J=7.5 Hz, 3 H), 1.29 (t, J=7.5
Hz, 3 H), 1.45 (t, J=7.5 Hz, 3 H), 1.88-1.97 (m, 1 H), 1.97-2.07
(m, 1 H), 2.18-2.32 (m, 2 H), 2.53-2.65 (m, 2 H), 2.71 (q, J=7.5
Hz, 2 H), 3.13 (q, J=7.5 Hz, 2 H), 3.81 (m, 1 H), 4.01 (q, J=7.5
Hz, 2 H), 4.23 (s, 3 H), 7.54-7.56 (m, 3 H), 7.73-7.75 (m, 2 H).
MS(Cl/NH.sub.3): m/z 398 (MH.sup.+-Me--I). K.sub.i(hA.sub.3)=1.41
.mu.M.
1-Methyl-2-(2-benzyloxyethyl)-4-propyl-3-(ethylsulfanylcarbonyl)-5-propylo-
xycarbonyl-6-phenylpyridinium Iodide (22)
[0129] .sup.1H-NMR .delta.: 0.69 (t, J=7.2 Hz, 3 H), 1.02 (t, J=7.2
Hz, 3 H), 1.44 (t, J=7.2 Hz, 3 H), 1.45 (m, 2 H), 1.68 (m, 2 H),
2.71 (m, 2 H), 3.17 (q, J=7.2 Hz, 2 H), 3.24 (t, J=7.2 Hz, 2 H),
3.99 (t, J=7.2 Hz, 2 H), 4.02 (q, J=7.2 Hz, 2 H), 4.25 (s, 3 H),
4.58 (s, 2 H), 7.29-7.35 (m, 5 H), 7.53-7.56 (m, 3 H), 7.68-7.71
(m, 2 H). MS (Cl/NH.sub.3): m/z 648 (MH.sup.+), 506
(MH.sup.+-Me--I), 445 (MH.sup.+-Me--I--SEt). K.sub.i(hA.sub.3)=348
nM.
1-Methyl-2,4-diethyl-3(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl)-6-cyclop-
entylpyridinium Iodide (23)
[0130] .sup.1H-NMR .delta.: 1.10 (t, J=7.5 Hz, 3 H), 1.32 (t, J=7.5
Hz, 3 H), 1.41 (t, J=7.5 Hz, 3 H), 1.44 (t, J=7.5 Hz, 3 H), 1.66
(m, 2 H), 1.95 (m, 7 H), 2.62 (q, J=7.5 Hz, 2 H), 2.81 (q, J=7.5
Hz, 2 H), 3.97 (q, J=7.5 Hz, 2 H), 4.28 (s, 3 H), 4.40 (q, J=7.5
Hz, 2 H). MS(Cl/NH.sub.3): m/z 364 (MH.sup.+-Me--I).
K.sub.i(hA.sub.3)=695 nM.
1-Ethyl-2,4-diethyl-3-(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl-6-phenylp-
yridinium Iodide (12)
[0131] .sup.1H-NMR .delta.: 0.89 (t, J=7.5 Hz, 3 H), 1.28 (t, J=7.5
Hz, 3 H), 1.39 (t, J=7.5 Hz, 3 H), 1.44 (t, J=7.2 Hz, 3 H), 2.79
(q, J=7.5 Hz, 2 H), 2.94 (t, J=7.5 Hz, 3 H), 3.11 (q, J=7.5 Hz, 2
H), 3.34 (q, J=7.5 Hz, 2 H). 4.02 (q, J=7.2 Hz, 2 H), 5.20 (q,
J=7.5 Hz, 2 H), 7.52-7.58 (m, 3 H), 7.68-7.71 (m, 2 H). MS
(Cl/NH.sub.3): m/z 372 (MH.sup.+-Et--I). K.sub.i(hA.sub.3)=577
nM.
1-Propyl-2,4-diethyl-3-(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl-6-phenyl-
pyridinium Iodide (13)
[0132] .sup.1H-NMR .delta.: 0.91 (m, J=7.2 Hz, 6 H), 1.29 (t, J=7.5
Hz, 3 H), 1.39 (t, J=7.5 Hz, 3 H), 1.43 (t, J=7.2 Hz, 3 H), 2.44
(t, J=7.2 Hz, 3 H), 2.79 (q, J=7.2 Hz, 2 H), 3.07 (q, J=7.5 Hz, 2
H), 3.38 (q, J=7.2 Hz, 2 H), 4.01 (q, J=7.2 Hz, 2 H), 4.29 (q,
J=7.2 Hz, 2 H), 5.22 (q, J=7.2 Hz, 2 H), 7.51-7.54 (m, 3 H),
7.68-7.71 (m, 2 ). MS (Cl/NH.sub.3): m/z 372 (MH.sup.+-Pr--I).
K.sub.i(hA.sub.3)=1.35 .mu.M.
[0133] General Procedure for Preparation of 1-Methyl
Dihydropyridines (24 and 25) by Alkylation of
3,5-Diacyl-2,4Dialkyl-1,4-Dihydropyridine Derivatives with
Iodomethane (Scheme 1): A solution of the appropriate DHP
(2,4-diethyl-3(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl-6-phenyl-1,4-
-dihydropyridine, 15 mg 0.04 mmol) in 2 mL of anhydrous THF was
treated with NaH (60%, 2 mg, 0.08 mmol) at room temperature under
stirring for 5 min. Then iodomethane (28 mg, 0.2 mmol) was added,
and the reaction mixture was stirred for another 10 min (monitor by
TLC). At completion the reaction mixture was applied to TLC
separation (ethyl acetate:petroleum ether=1:19 v/v), and 11 mg of
24 was obtained (yield: 71%).
1-Methyl-2,4-diethyl-3-(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl-6-phenyl-
-1,4-dihydropyridine (24)
[0134] .sup.1H-NMR .delta.: 0.85 (t, J=7.2 Hz, 3 H), 0.93 (t, J=7.2
Hz, 3 H), 1.20 (t, J=7.2 Hz, 3 H), 1.29 (t, J=7.2 Hz, 3 H), 2.74
(q, J=7.2 Hz, 2 H), 2.85 (s, 3 H), 2.92 (q, J=7.2 Hz, 2 H), 3.07
(q, J=7.2 Hz, 2 H), 3.87 (q, J=7.2 Hz, 2 H), 4.02 (t, J=7.2 Hz, 1
H), 7.20 (m, 2 H), 7.38-7.41 (m, 3 H). MS (Cl/NH.sub.3): m/z 388
(MH.sup.+).
1-Methyl-2,4-diethyl-3-(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl-6-cyclop-
entyl-1,4-1,4-dihydropyridine (25)
[0135] .sup.1H-NMR .delta.: 0.78 (t, J=7.8 Hz, 3 H), 1.11 (t, J=7.8
Hz, 3 H), 1.28 (t, J=7.8 Hz, 3 H), 1.32 (t, J=7.8 Hz, 3 H), 1.46
(m, 2H), 1.60-1.78 (m, 7H), 2.10 (m, 2 H), 2.60-2.67 (m, 1 H), 2.89
(q, J=7. Hz, 2 H), 3.01-3.13 (m, 1 H), 3.15 (s, 3 H), 4.10 (t,
J=6.0 Hz, 1 H), 4.13-4.26 (m, 2 H). MS (Cl/NH.sub.3): m/z 380
(MH.sup.+), 318 (M.sup.+-SEt, base).
[0136] Chemical Transformation of 24 to 11 through Oxidation with
Iodine (Scheme 1): A solution of 24 (5 mg, 0.013 mmol) in 0.5 mL of
dry nitromethane was treated with iodine (10 mg, 0.040 mmol) at
room temperature with stirring for 1 day (monitor by TLC). At
completion the reaction mixture was applied to TLC separation
(ethyl acetate:petroleum ether=1:4 v/v for the first development
then 1:1 for a second development), and 2 mg of a yellow solid was
obtained (yield: 30%), with .sup.1H-NMR and MS data consistent with
those of compound 11.
[0137] Oxidation of a 1-Methyl-1,4-Dihydropyridine Derivative (24)
in the Presence of Rat Brain Homogenate (Bodor et at., J. Med.
Chem. 26:528-534, 1983; Wu et al., J. Med. Chem. 32:1782-1788,
1999).
[0138] The rat brain homogenate was prepared by the following
method. One rat (1 kg) was killed and the brain (weighing 2.26 g)
was removed, and homogenized in 12 mL PBS (Biofluids, Inc.,
1.times. pH 7.4). The homogenate was centrifuged at 12,000.times.
rpm, and the supernatant was used. 5 mg of 24 dissolved in 0.2 mL
of DMSO was mixed with 10 mL of brain homogenate (initial
concentration of 1.27 mM), which was previously equilibrated to
37.degree. C. in a water bath incubator, and shaking was continued
at that temperature. Aliquots of 500 .mu.L were withdrawn at 2, 4,
8, 16, 32, 64, 128, 256, 768 min (2.times. each) from the test
medium, added immediately to 3 mL of ice-cold ethyl ether, shaken
vigorously, and placed in a freezer. When all samples had been
collected, and the ether layer of each sample was separated. After
evaporation of the ether, each residue was dissolved in 200 .mu.L
methanol, filtered through Whatman 1 filter paper and analyzed by
HPLC.
[0139] Pyridinium salt 11 could be generated through oxidation of
the corresponding reduced precursor,
1-methyl-2,4-diethyl-3-(ethylsulfanylcar-
bonyl)-5-ethyloxycarbonyl-6-phenyl-1,4-dihydropyridine, 24. The
conversion of 24 to 11 by chemical means (iodine in nitromethane)
and during incubation at 37C with rat brain membranes, to simulate
in vivo conditions, was studied. The chemical conversion occurred
readily, and the time course of the biochemical oxidation was
recorded. At regular time points, aliquots were removed from the
incubation mixture, extracted with ether, and both the precursor
and the pyridinium salt were assayed in the evaporated organic
phase using HPLC. The oxidation occurred cleanly with a t.sub.1/2
of approximately 47 min.
[0140] The 1-methyl dihydropyridine 24 (precursor of MRS 1649) was
found to bind selectively to human A.sub.3 adenosine receptors. At
human A.sub.3 receptors, the K.sub.i value was 379.+-.122 nM (n=4).
The K.sub.i value at rat A.sub.1 receptors was 28.4.+-.2.5 .mu.M
(n=3). At rat A.sub.2A receptors, the percent displacement of
specific radioligand binding was 48.+-.7% at 100 .mu.M. Therefore
to demonstrate the prodrug principle, i.e. an inactive prodrug that
could be converted to a selective adenosine antagonist, the
corresponding 6-cyclopentyl dihydropyridine 25 was prepared. The
6-cyclopentyl analogue,
1-methyl-2,4-diethyl-3-(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl6-cyclop-
entyl-1,4-dihydro pyridine (25), was shown to increase affinity at
human A.sub.3 receptors upon oxidation to the corresponding
pyridinium salt (23). Compound 25 had a K.sub.i value at human
A.sub.3 receptors of 6.40.+-.0.78 .mu.M, while 23 was an order of
magnitude more potent (K.sub.i of 695 nM), suggesting a prodrug
scheme. At rat A.sub.1 receptors, both the precursor, compound 25
(34.+-.1% displacement at 100 .mu.M) and the oxidized product, 23
(K.sub.i value 11.5 .mu.M) bound only weakly.
[0141] Thus, in the present study, one reduced precursor, 25, was
found to be clearly less potent than the corresponding pyridinium
salt, 23, in binding to adenosine receptors, especially the A.sub.3
subtype, and another precursor, 24, was found to have the identical
human A.sub.3 receptor affinity before and after oxidation. Thus,
depending on the structure of the pyridine substitutents, one can
determine the degree to which the antagonist requires preactivation
of a prodrug form in order to acheive antagonism of A.sub.3
receptors.
EXAMPLE 16
Pharmacology
[0142] Adenylate cyclase assay. Adenylate cyclase assays were
performed with membranes prepared from Chinese hamster ovary (CHO)
cells stably expressing either the human A.sub.1 receptor or human
A.sub.3 receptor by the method of Salomon et al.., (Anal. Biochem.
58:541-548, 1974) as described previously with the following
modifications (Jacobson et al., Neuropharmacol., 36:1157-1165,
1997). 4-(3-Butoxy-4-methoxybenzyl)-2-imid- azolidinone (Ro
20-1724, 20 .mu.M, Calbiochem, San Diego, Calif.) was employed to
inhibit phosphodiesterases rather than papaverine, and the NaCl
concentration in the assay was 25 mM. Membranes were pretreated
with 2 units/mL adenosine deaminase, and the antagonist 11 (10
.mu.M) at 30 C for 5 min prior to initiation of the adenylate
cyclase assay. Adenylate cyclase was stimulated with forskolin (1
.mu.M). Concentration-response data for the inhibition of adenylate
cyclase activity by IB-MECA (human A.sub.3 receptor) were obtained.
Maximal inhibition of adenylate cyclase by IB-MECA at the human
A.sub.3 receptor correlated to .sup..about.60% of total
stimulation, respectively. IC.sub.50 values were calculated using
InPlot (GraphPad, San Diego, Calif.). K.sub.B values were
calculated as described. (Aluniakshana et al., Brit. J. Pharmacol
Chemother. 14:48, 1959).
[0143] Responses for agonist alone (0) or in combination with the
A.sub.3 adenosine antagonist 11 (10 .mu.M) were measured. IC.sub.50
values were 41.4.+-.14.9 nM (IB-MECA alone), 1.08.+-.0.19 .mu.M
(+11). Compound 11 effectively antagonized the effects of an
agonist in a functional A.sub.3 receptor assay, i.e. inhibition of
adenylate cyclase in CHO cells expressing cloned human A.sub.3
receptors..sup.8,14 In this functional assay, IB-MECA inhibited
adenylate cyclase via human A.sub.3 receptors with an IC.sub.50 of
41.4.+-.14.9 nM (n=3). In the presence of 10 .mu.M of 11, the
concentration response curve was shifted 26-fold to the right, with
an IC.sub.50 of 1.08.+-.0.19 .mu.M (n=3). From a Schild analysis
(Alumakshana et al., supra.), a K.sub.B value obtained for
antagonism by 11 was 399 nM, i.e. approximately 1.8-times the
K.sub.i value obtained in binding to human A.sub.3 receptors.
[0144] Radioligand binding studies. Binding of
[.sup.3H]R-N.sup.6-phenylis- opropyladenosine ([.sup.3H]R-PIA) to
A.sub.1 receptors from rat cerebral cortex membranes and of
[.sup.3H]-2-[4-[(2-carboxyethyl)phenyl]ethylamino-
]-5'-N-ethylcarbamoyladenosine ([.sup.3H]CGS 21680) to A.sub.2A
receptors from rat striatal membranes was performed as described
previously (Schwabe et al., Naunyn Schmiedeberg Arch. Pharmacol.
313:179-187, 1980; Jarvis et al., J. Pharmacol. Exp. Ther.
251:888-893, 1989) Adenosine deaminase (3 units/mL) was present
during the preparation of the brain membranes, in a pre-incubation
of 30 min at 30.degree. C., and during the incubation with the
radioligands. Nonspecific binding was determined in the presence of
10 .mu.M (A.sub.1 receptors) or 20 .mu.M (A.sub.2A receptors)
2-chloroadenosine.
[0145] Binding of
[.sup.125I]N.sup.6-(4-amino-3-iodobenzyl)-5'-N-methylcar-
bamoyladenosine ([.sup.125I]AB-MECA).sup.24 to membranes prepared
from human embryonic kidney (HEK-293) cells stably expressing the
human A.sub.3 receptor (Salvatore et al., Proc. Natl Acad. Sci.
U.S.A. 90:10365-10369, 1993), clone HS-21a (Receptor Biology, Inc.,
Beltsville, Md.) or to membranes prepared from Chinese hamster
ovary (CHO) cells stably expressing the rat A.sub.3 receptor (Zhou
et al., Proc. Natl. Acad. Sci. U.S.A. 89:7432-7436, 1992) was
performed as described at 4.degree. C. (Olah et al., Mol.
Pharmacol.i 45:978-982, 1994). The assay medium consisted of a
buffer containing 10 mM Mg.sup.2+, 50 mM Tris, 3 units/mL adenosine
deaminase, and 1 mM EDTA, at pH 8.0 (4 C). The glass incubation
tubes contained 100 .mu.L of the membrane suspension (0.3 mg
protein/mL, stored at -80.degree. C. in the same buffer), 50 .mu.L
of [.sup.125I]AB-MECA (final concentration 0.3 nM), and 50 .mu.L of
a solution of the proposed antagonist. Nonspecific binding was
determined in the presence of 100 .mu.M
N.sup.6-phenylisopropyladenosine (NECA).
[0146] All non-radioactive compounds were initially dissolved in
DMSO, and diluted with buffer to the final concentration, where the
amount of DMSO never exceeded 1%. Incubations were terminated by
rapid filtration over Whatman GF/B filters, using a Brandell cell
harvester (Brandell, Gaithersburg, Md.). The tubes were rinsed
three times with 3 mL buffer each.
[0147] At least five different concentrations of competitor,
spanning 3 orders of magnitude adjusted appropriately for the
IC.sub.50 of each compound, were used. IC.sub.50 values, calculated
with the nonlinear regression method implemented in the InPlot
program (Graph-PAD, San Diego, Calif.), were converted to apparent
K.sub.i values using the Cheng-Prusoff equation (Cheng et al.,
Biochem. Pharmacol. 22:3099-3108, 1973) and K.sub.d values of 1.0
nM ([.sup.3H]R-PIA); 15.5 nM ([.sup.3H]CGS 21680); 0.59 nM and 1.46
nM ([.sup.125I]AB-MECA at human and rat A.sub.3 receptors,
respectively).
* * * * *