U.S. patent application number 10/614702 was filed with the patent office on 2004-07-15 for method of identifying partial agonists of the a2a receptor.
This patent application is currently assigned to CV Therapeutics, Inc.. Invention is credited to Belardinelli, Luiz, Blackburn, Brent K., Gao, Zhenhai.
Application Number | 20040137533 10/614702 |
Document ID | / |
Family ID | 26880004 |
Filed Date | 2004-07-15 |
United States Patent
Application |
20040137533 |
Kind Code |
A1 |
Belardinelli, Luiz ; et
al. |
July 15, 2004 |
Method of identifying partial agonists of the A2A receptor
Abstract
The present invention provides a method for identifying and
using partial adenosine A.sub.2A receptor agonists that are useful
as adjuncts in myocardiological perfusion imaging.
Inventors: |
Belardinelli, Luiz; (Menlo
Park, CA) ; Blackburn, Brent K.; (Los Altos, CA)
; Gao, Zhenhai; (San Jose, CA) |
Correspondence
Address: |
A. Blair Hughes
McDonnell Boehnen Hulbert & Berghoff
32nd Floor
300 S. Wacker Drive
Chicago
IL
60606
US
|
Assignee: |
CV Therapeutics, Inc.
|
Family ID: |
26880004 |
Appl. No.: |
10/614702 |
Filed: |
July 2, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10614702 |
Jul 2, 2003 |
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09792617 |
Feb 23, 2001 |
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60219876 |
Jul 21, 2000 |
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60184296 |
Feb 23, 2000 |
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Current U.S.
Class: |
435/7.2 |
Current CPC
Class: |
C07H 19/16 20130101;
G01N 33/566 20130101; G01N 33/502 20130101; G01N 33/5088 20130101;
G01N 2500/04 20130101; A61K 51/00 20130101; A61P 9/08 20180101;
G01N 33/5082 20130101; G01N 33/5008 20130101 |
Class at
Publication: |
435/007.2 |
International
Class: |
G01N 033/53; G01N
033/567 |
Claims
We claim:
1. A method for identifying compounds that are selective partial
A.sub.2A adenosine receptor agonists with a short duration of
action, comprising: a. measuring the intrinsic efficacy of a test
compound in a cell line that express adenosine A.sub.2A receptors.
b. measuring the intrinsic efficacy of a full agonist in said cell
line; and c. selecting those compounds that have a lower intrinsic
efficacy than said fall agonist;
2. The method of claim 1, comprising the additional steps of
measuring the binding affinity (K.sub.i) of the selected compounds;
and selecting a compound with a K.sub.i>1 .mu.M.
3. The method of claim 1, wherein said test compound is a selective
A.sub.2A adenosine receptor agonist.
4. The method of claim 1, wherein said cell line is rat
pheochromocytoma PC12 cells, HEK-293 cells or porcine striatal
cells.
5. The method of claim 4, wherein said cell line is rat
pheochromocytoma PC12 cells.
6. The method of claim 1, wherein said full agonist is CGS21680 or
WRC 0470.
7. The method of claim 6, wherein said full agonist is
CGS21680.
8. The method of claim 7, wherein the intrinsic efficacy of the
compound is 10%-95% of the intrinsic efficacy of CGS21680.
9. The method of claim 7, wherein the intrinsic efficacy of the
compound is 50%-85% of the intrinsic efficacy of CGS21680.
10. The method of claim 1, wherein said binding affinity of the
selected, compound is greater than or equal to 1 .mu.M.
11. A method of myocardial perfusion imaging of a mammal,
comprising administering a radionuclide and a compound identified
by the method of claim 1 to a mammal in need thereof, and
determining areas of insufficient blood flow.
12. The method of claim 11, wherein the compound is CVT-3033 or
CVT-3146.
13. The method of claim 12, wherein the dose of CVT-3033 or
CVT-3146 is from 0.20 .mu.g/kg to 9 .mu.g/kg.
14. The method of claim 12, wherein the dose of CVT-3033 or
CVT-3146 is 0.25 to 5.0 .mu.g/kg.
Description
[0001] This application is entitled to claim the benefit of
priority to Provisional Patent Applications Serial Nos. 60/184,296,
filed Feb. 23, 2000, and 60/219876, filed Jul. 21, 2000 the
specifications of each of which are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] This invention relates to a method of identifying compounds
that are selective partial A.sub.2A-adenosine receptor agonists,
preferably With a short duration of action. Such compounds provide
coronary dilation in mammals without causing corresponding
significant peripheral vasodilation. The invention also relates to
a method of using such compounds as adjuncts in cardiac
imaging.
BACKGROUND
[0003] Myocardial perfusion imaging (MPI) is a diagnostic technique
useful for the detection and characterization of coronary artery
disease. Perfusion imaging uses materials such as radionuclucides
to identify areas of insufficient blood flow. In MPI, blood flow is
measured at rest, and the result compared with the blood flow
measured during exercise on a treadmill (cardiac stress testing),
such exertion being necessary to stimulate blood flow.
Unfortunately, many patients are unable to exercise at levels
necessary to provide sufficient blood flow, due to medical
conditions such as peripheral vascular disease, arthritis, and the
like.
[0004] Therefore, a pharmacological agent that increases CBF for a
short period of time would be of great benefit, particularly one
that did not cause peripheral vasodilation. Vasodilators, for
example dipyridamole, have been used for this purpose in patients
prior to imaging with radionuclide. Dipyridamole is an effective
vasodilator, but side effects such as pain and nausea limit the
usefulness of treatment with this compound.
[0005] Adenosine, a naturally occurring nucleoside, also is useful
as a vasodilator. Adenosine exerts its biological effects by
interacting with a family of adenosine receptors characterized as
subtypes A.sub.1, A.sub.2A, A.sub.2B, and A.sub.3. AdenoScan.RTM.
(Fujisawa Healthcare Inc.) is a formulation of a naturally
occurring adenosine. AdenoScan has been marketed as an adjuvant in
perfusion studies using radioactive thallium-201. However, its use
is limited due to side effects such as flushing, chest discomfort,
the urge to breathe deeply, headache, throat, neck, and jaw pain.
These adverse effects of adenosine are due to the activation of
other adenosine receptor subtypes in addition to A.sub.2A, which
mediates the vasodilatory effects of adenosine. Additionally, the
short half-life of adenosine necessitates multiple treatments
during the procedure, further limiting its use. AdenoScan is
contraindicated in many patients including those with second-or
third-degree block, sinus node disease, bronchoconstructive or
bronchospastic lung disease, and in patients with known
hypersensitivity to the drug.
[0006] Other potent and selective agonists for the A.sub.2A
adenosine receptor are known. For example, MRE-0470 (Medco) is an
adenosine A.sub.2A receptor agonist that is a potent and selective
derivative of adenosine. VWRC-0470 (Medco) is an adenosine A.sub.2A
agonist used as an adjuvant in imaging. These compounds, which have
a high affinity for the A.sub.2A receptor, and, consequently, a
long duration of action, which is undesirable in imaging.
[0007] Thus, there is still a need for a method of producing rapid
and maximal coronary vasodilation in mammals without causing
corresponding peripheral vasodilation, which would be useful for
myocardial imaging with radionuclide agents.. Preferred compounds
would be selective for the A.sub.2A adenosine receptor and have a
short duration of action (although longer acting than compounds
such as adenosine), thus obviating the need for multiple
dosing.
[0008] Selective A.sub.2A receptor agonists are well known; for
example, see Provisional Patent Applications Serial Nos. 60/1844296
and 60/21987. The compounds disclosed therein have a high
specificity for the adenosine A.sub.2A receptor subtype but are not
necessarily selective to heart. We have discovered a method for
identifying A.sub.2A receptor agonists that produce the desired
vasodilation in the heart but do not significantly affect the
peripheral vasculature and have a short duration of action.
[0009] In addition to discovering a method to identify A.sub.2a
agonists that are selective coronary vasodilators, we have
discovered that compounds that meet our criteria that would be
superior as adjuncts to MPI techniques.
[0010] Additionally, in the above-identified Provisional Patent
Applications Serial Nos. 60/1844296 and 60/219876 the effective
dose of the compounds of is disclosed to be in the range of
0.01-100 mg/kg/day. Surprisingly, we have discovered that the
compounds are active at much lower doses (0.0002-0.009 mg/kg) than
those disclosed as effective in Accordingly, a novel and effective
method of using the compounds is provided, which is virtually free
of undesirable side effects.
SUMMARY OF THE INVENTION
[0011] In a first aspect, the invention relates to a method for
identifying compounds useful as adjuncts in MPI, comprising the
steps;
[0012] a. measuring the intrinsic efficacy of test compounds in a
cell line that stabily express adenosine A.sub.2A receptors.
[0013] b. measuring the intrinsic efficacy of a full agonist in
said cell line.
[0014] c. selecting those compounds that have a lower intrinsic
efficacy than said full agonist;
[0015] d. measuring the binding affinity (K,) of the selected
compounds; and
[0016] e. selecting a compound with a K.sub.i>1 uM.
[0017] Such compounds are selective partial A.sub.2A-adenosine
receptor agonists, which produce coronary dilation in mammals
without causing corresponding peripheral vasodilation at
significant levels. They are low affinity compounds having a short
duration of action.
[0018] In a, second aspect, the invention relates to a method of
measuring coronary blood flow (CBF) in a mammal, comprising
administering to a a mammal low doses of an A.sub.2A agonist
referred to as CVT-3033, or CVT-3146.
BRIEF DESCRIPTION OF FIGURES
[0019] FIG. 1. Competititive radiolabeling binding assays of of
adenosine receptor, agonists for A.sub.2A and A.sub.1 binding sites
Membranes prepared from HEK-293 expressing human A.sub.2A adenosine
receptors were incubated with [.sup.3H]ZM241385 (1.5-5 M) and from
10.sup.-9M-10.sup.-5 M of the various agonists (FIG. 1A). Membranes
from CHO-K1 cells expressing human A.sub.1 adenosine receptors were
incubated with [.sup.3H]CPX (2.5-3.0 M) and from
10.sup.-9M-10.sup.-5 M of the various agonists (FIG. 1B). The cells
were incubated for two hours at room temperature in 50 mmol/L
Tris-HCl buffer (pH 7.4) containing ADA (1U/mL) and 100 .mu.M
Gpp(NH)p. Non-specific binding of [.sup.3H]ZM241385 or [.sup.3H]CPX
was determined in the presence of either 100 .mu.mol/L NECA or 1
.mu.mol/L CPX, respectively. Each point represents the mean.+-.SEM
of triplicates pooled from at least three experiments. Values of
K.sub.i and pK.sub.i are given in Table 2.
[0020] FIG. 2. Competititive radiolabeling binding assays of of
adenosine receptor agonists for A.sub.2band A.sub.3 binding site.
Membranes suspensions from HEK-293 cells expressing A.sub.2b
adenosine receptors were incubated with [.sup.3H]DPCPX (1.5-5 M)
and from 10.sup.-8M-10.sup.-5 M of the various agonists (FIG. 2A).
Membranes from CHO-K1 cells expressing A.sub.3 adenosine receptors
were incubated with [.sup.125I]ABMECA (2.5-3.0 M) and from
10.sup.-8M-10.sup.-5 M of the various agonists (FIG. 2B). The cells
were incubated for two hours at room temperature in 50 mmol/L
Tris-HCl buffer (pH 7.4) containing ADA (1U/mL). Each point
represents the mean.+-.SEM of triplicates pooled from three
experiments.
[0021] FIG. 3. Effects of adenosine receptor agonists on cAMP
content in intact PC12 cells. (FIG. 3A) PC12 cells were incubated
for 10 minutes with various concentrations of adenosine receptor
agonists in the presence of 50 .mu.mol/L rolipram. Cyclic AMP
levels were determined as described under "Methods". Values
represent mean.+-.SEM of results of triplicate samples from three
experiments. (FIG. 3B) Effect of CVT-3033 (1 .mu.M) on CGS21680
stimulated increase cAMP accumulation in PC12 cells. PC12 cells
were stimulated for 10 minutes with various concentrations of
CGS21680 in the absence or presence of the partial agonist CVT-3033
(1 .mu.M). Values represent mean.+-.SEM of triplicate determinants
from one representative experiment.
[0022] FIG. 4. Effect of CVT-3146 and CVT-3033 on cAMP content in
HEK-293 cells. HEK-293 cells were incubated for 10 minutes with
various concentrations of CVT-3146 or CVT-3033 in the presence of
50 .mu.mol/L rolipram. Cyclic AMP levels were determined as
described under "Methods". Values represent mean.+-.SEM of
triplicate samples from three experiments.
[0023] FIG. 5. Time course of the decline in agonist stimulated
cAMP following addition of an A.sub.2A adenosine receptor
antagonist SCH58261. (FIG. 5A) PC12 cells were stimulated with
various adenosine receptor agonists in the presence of 50 .mu.mol/L
rolipram at 37.degree. C. After a 10 minute incubation, SCH58261
(20 .mu.mol/L) was added and cAMP content was determined at the
times indicated. (FIG. 5B) Linear regression analysis of the
relationship between the T.sub.1/2 (min) and pK.sub.i for the
agonists. Binding affinities were determined by measurement of the
displacement of specific binding of [.sup.3H] ZM241385 from
membranes prepared from PC12 cells using the data from Table 2
[0024] FIG. 6. Effects of adenosine receptor agonists on coronary
conductance in isolated rat hearts. Concentration-dependent
stimulation by CGS21680, CVT-2995, WRCO470, CVT-3146, CVT-3033,
CVT-3032 and CVT-3100 of coronary conductance of isolated rat
hearts. Symbols and error bars represent the mean.+-.SEM of single
determination from 4 to 6 hearts per agonist. Hearts were paced at
a cycle length of 250 msec. Coronary conductance in the absence of
drug was 0.17.+-.0.01 ml/min/mmHg (mean.+-.SEM, n=26).
[0025] FIG. 7A. Effect of CPX and ZM241385 on CVT-3146 (10 nM) on
CPP by isolated rat hearts. FIG. 7B. Effect of concentration of
ZM241385 on CVT-3146 stimulation of CC by isolated rat hearts.
[0026] FIG. 8. Functional selectivity of CVT-3146 and CVT-3033 for
adenosine receptor subtypes. (FIG. 8A) Effect of concentration on
AV conduction time and coronary conductance in isolated rat hearts.
Symbols and error bars represent means.+-.SEM of single
determinations from each of three hearts. (FIG. 8B)
[0027] FIG. 9. Effect of adenosine receptor agonists on coronary
perfusion pressure (CCP) in isolated rat hearts. Decreases in CPP
caused by infusion of CVT-3146 (10 nM, 4 min) or CGS21680 (100 nM,
4 min) (FIG. 9A and FIG. 9B). Decrease in CPP caused by the
infusion of CVT-3146 (10 nM) with or without additional infusion of
CGS21680 (100 nM).
[0028] FIG. 10. Reversal of effect of agonist stimulation on CC.
CVT-3033, CGS21680 and adenosine were given as boluses by iv
infusion to isolated rat hearts and then. CC was measured at 8, 16
and 24 minutes after administration (FIG. 10A). Also, CVT-3146, WRC
0470 and adenosine were given as boluses by iv infusion to isolated
rat hearts and then CC was measured at 8, 16 and 24 minutes after
administration (FIG. 10B). Linear regression analysis of the
relationship between the pK.sub.i (data from Table 2) and the
reversal time (t.sub.0.9) of coronary vasodilation are given in
FIG. 10C and D. Each data point represents the mean.+-.SEM of
pK.sub.i and T.sub.0.9 values. R and N are the correlation
coefficient and number of agonists, respectively.
[0029] FIG. 11. Increases in CBF caused by CVT-3146 and adenosine
in conscious dogs. Each data point is mean.+-.SEM of the peak
effect in CBF from 6 dogs. *: p<0.05,.dagger.: p<0.01 and
.dagger-dbl.: p<0.001. Adenosine: (6) and CVT-3146 (O).
[0030] FIG. 12. Time course of changes in average peak coronary (O)
and peripheral (O) artery blood flow velocity after an IV bolus
injection ofCVT-3146 (1 ug/kg) (A) and adenosine (200-300
.mu.g/kg).(B). Each point represents the changes in average peak
flow velocity (.DELTA.APV) in comparison with the baseline values
and represent the mean.+-.SEM of single determinations.
DETAILED DESCRIPTION
[0031] Definitions and General Parameters
[0032] This invention provides methods for identifying partial
adenosine A.sub.2A receptor agonists, which are particularly useful
in MPI.
[0033] The interaction of an adenosine A.sub.2A agonist with the
extracellular domain of its receptor causes a cascade of
intracellular responses that culminate with vasodilation. However,
there are quantitative differences in the ability of various
agonists to achieve this effect. With respect to drug design, the
ideal agent for imaging would have a duration of action that is
brief enough not to cause serious side effects, but long enough to
obviate the necessity for multiple treatments. It would be
selective for the A.sub.2A receptor, thus avoiding side effects due
to interaction with other adenosine subtypes, and it would produce
coronary vasodilation without causing corresponding peripheral
vasodilation.
[0034] Compounds that act as A.sub.2A agonists produce a variety of
effects that depend on both the characteristics of the agonist, its
receptor, and the tissue bearing A.sub.2A receptors. Factors relate
to agonist properties are the intrinsic efficacy (E) and the
equilibrium dissociation constant of the agonist-receptor complex
(K.sub.d).
[0035] Intrinsic efficacy (maximal efficacy) is the maximum effect
that an agonist can produce if the dose is taken to its maximum.
Efficacy is determined mainly by the nature of the receptor and its
associated effector system. By definition, partial agonists have a
lower maximal efficacy than full agonists.
[0036] The K.sub.d of a drug is obtained from data generated from a
saturation experiment analyzed according to a Scatchard plot (B/F
versus F), which leads to a linear curve. The K.sub.d is estimated
as the negative reciprocal of the slope of the line of best fit,
and B.sub.max by the abscissa intercept of the line. The reciprocal
of K.sub.d measures the affinity constant (K.sub.a) of the
radioligand for the receptor. Thus, for a given ligand-receptor
pair, the smaller the K.sub.d (0.1-10 nM) the higher its affinity.
B.sub.max is expressed as pmol or fmol per mg tissue or
protein.
[0037] When the saturation experiment is performed in the presence
of a displacer (competitor), the line of best fit of the Scatchard
plot can be modified in a manner that depends on the type of
receptor interaction exhibited by the displacer. Two main cases
exist: (1) decreased slope and unchanged B.sub.max, the
displacement is of the competitive type; (2) unchanged slope and
unchanged displacement of the non-competitive type. Intermediate
cases where both the slope and B.sub.max are modified also
exist.
[0038] Data generated from a displacement experiment are generally
fitted by a sigmoidal curve termed the displacement or inhibition
curve, that is the percentage radiolabeled ligand specifically
bound versus log [displacer] in M). The abscissa of the inflexion
point of the curve gives the IC.sub.50 value, the concentration of
dsplacer that displaces or inhibitor 50% of the radioactive ligand
specifically bound. IC.sub.50 is a measure of the inhibitor or
affinity constant (Ki) of the displacer for the receptor. IC.sub.50
and K.sub.i are linked as follows if the displacement is of the
competitive type then
K.sub.i=IC.sub.50/(1+[C*]/K.sub.d*
[0039] This is the Cheng-Prusoff equation (Biochem. Pharmacol,
22:3099 (1973)). [C*] is the concentration of radioligand and
K.sub.d* is its dissociation constant. The duration of the
biological effect of an agonist is directly related to the binding
affinity of a compound. It is desirable that compounds that act as
adjuncts in imaging have an effect that is long enough to produce a
response without repeated administration but short enough to avoid
adverse side effects. Consequently, the, preferred compounds of the
invention will have a relatively low binding affinity and a
relatively short duration of action.
[0040] The potency is the dose or concentration required to bring
about some fraction of a compound's maximal effect (i.e., the
amount of compound needed to produce a given effect). In graded
dose-response measurements, the effect usually chosen is 50% of the
maximum effect and the dose causing the effect is called the
EC.sub.50. Dose-response ratios using EC.sub.50 values for an
agonist for a given receptor in the absence and presence of various
concentrations of an antagonist for the same receptor are
determined and used to construct a Schild plot from which the
K.sub.b and .sub.PA.sub.2 (-log.sub.10K.sub.b) values are
determined.
[0041] The concentration of antagonist that causes 50% inhibition
is known as the IC.sub.50. IC.sub.50 is used to determine the
K.sub.b, the equilibriumdissociation constant for the
antagonist-receptor complex. Thus,
K.sub.b=[IC.sub.50]/1+[A]/K.sub.A
[0042] Wherein K.sub.A=equilibrium dissociation constant for an
agonist binding to a receptor (concentration of agonist that causes
occupancy of 50% of the receptors) and [A] is the concentration of
agonist.
[0043] A compound may be potent but have less intrinsic activity
than another compound. Relatively potent therapeutic compounds are
preferable to weak ones in that lower concentrations produce the
desired effect while circumventing the effect of concentration
dependent side effects.
[0044] The tissue specific factors that determine the effect of an
agonist are the number of viable specific receptors in a particular
tissue [RT] and the efficiency of the mechanisms that convert a
stimulus (S) into an effector response. Thus, there exists for a
given tissue, a complex function f(S) that determines the magnitude
of the response: 1 Response = f ( S ) = f ( [ A ] E [ RT ] ) ( [ A
] + K d )
[0045] Therefore, a response to a drug is a function of both the
stimulus produced by agonist interaction with the receptor and the
efficiency of the transduction of that stimulus by the tissue.
Stimulus is proportional to the intrinsic efficacy of the agonist
and the number of receptors. Consequently, variation in receptor
density in different tissues can affect the stimulus for response.
Furthermore, some tissues have very efficiently coupled receptors
and other relatively inefficient coupled receptors. This has been
termed `receptor reserve` (or spare receptor) since in the first
case, a maximum effect can be achieved when a relatively small
fraction of the receptor is apparently occupied and, further
receptor occupancy can produce no additional effect. The magnitude
of the response will thus depend on the intrinsic efficacy value so
that, by classical definition, full agonists (E=1) produce the
maximum response for a given tissue, partial agonists produce a
maximum response that is below that induced by the full agonist
(0.ltoreq.E.ltoreq.1), and antagonists produce no visible response
and block the effect of agonists (E=0). These activities can be
completely dependent upon the tissue, i.e., upon the efficiency
coupling. Therefore, low-efficacy adenosine agonists may be partial
agonists in a given tissue and yet full agonists in peripheral
arteries with respect to a function such as vasodilation.
[0046] The presence of spare receptors in a tissue increases
sensitivity to an agonist. An important biologic consequence of
spare receptors is that they allow agonists with low efficacy for
receptors to produce full responses at low concentrations and
therefore elicit a selective tissue response. Thus, a drug may be
designed to elicit a maximal effect in a desired tissue but elicit
a less than maximal effect in other tissues when such action of a
drug would lead to undesirable side effects.
[0047] Thus, the invention provides a method of identifying drugs
by first determining their efficacy compared to a known full
agonist. Then, the binding affinity of the compound is determined.
Compounds identified by this method will demonstrate partial
agonist effects in the cAMP assays and a low IC as determined by
affinity binding assays.
[0048] One preferred compound of the invention that is a selective
partial A.sub.2A-adenosine receptor agonist with a short duration
of action is a compound of the formula: 1
[0049] CVT-3033 is particularly useful as an adjuvant in
cardiological imaging. The preparation of CVT-3033 and related
compounds is described in U.S. patent application Ser. No.
09/338185, filed on Jun. 22, 1999, the specification of which is
incorprated herein by reference.
[0050] Another preferred compound of the invention that is a
selective partial A.sub.2A-adenosine receptor agonist with a short
duration of action is a compound of the formula: 2
[0051] The preparation of CVT-3146 and related compounds is set
forth in U.S. patent application Ser. No. 09/338327 filed on Jun.
22, 1999, the specification of which is incorporated herein by
reference.
[0052] Compounds identified by the method of the invention are
partial A.sub.2A agonists that increase CBF but do not
significantly increase peripheral blood flow. That is, the
stimulation of blood flow in the periphery is less than 50% of the
increase of that in the heart.
[0053] Preferred compounds identified by the method of the
invention have a duration of less than 5 seconds but longer than
the effect produced by adenosine.
[0054] The compounds identified by the method of the invention are
useful A.sub.2A agonists that may be used as adjuncts in cardiac
imaging when added: either prior to dosing with an imaging agent or
simultaneously with an imaging agent.
[0055] Suitable imaging agents are .sup.201Thallium or
.sup.99mTechnetium-Sestamibi, .sup.99mTcteboroxime, and
.sup.99mtc(III). :
[0056] The term "optional" or "optionally" means that the
subsequently described event or circumstance may or may not occur,
and that the description includes instances in which said event or
circumstance occurs and instances in which it does not.
[0057] The compositions may be administered orally, intravenously,
through the epidermis or by any other means known in the art for
administering therapeutic agents.
[0058] The method of treatment of comprises the administration of
an effective quantity of the chosen compound, preferably dispersed
in a pharmaceutical carrier. Dosage units of the active ingredient
are generally selected from the range of 0.3 to 103 .mu.g/kg, but
will be readily determined by one skilled in the art depending upon
the route of administration, age and condition of the patient.
These dosage units may be administered one to ten times daily for
acute or chronic disorders. No unacceptable toxicological effects
are expected when compounds of the invention are administered in
accordance with the present invention.
[0059] Pharmaceutical compositions including the compounds of this
invention, and/or derivatives thereof, may be formulated as
solutions or lyophilized powders for parenteral administration.
Powders may be reconstituted by addition of a suitable diluent or
other pharmaceutically acceptable carrier prior to use. If used in
liquid form the compositions of this invention are preferably
incorporated into a buffered, isotonic, aqueous solution. Examples
of suitable diluents are normal isotonic saline solution, standard
5% dextrose in water and buffered sodium or ammonium acetate
solution. Such liquid formulations are suitable for parenteral a
administration, but may also be used for oral administration. It
may be desirable to add excipients such as polyvinylpyrrolidinone,
gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol,
sodium chloride, sodium citrate or any other excipient known to one
of skill in the art to pharmaceutical compositions including
compounds of this invention. Alteratively, the pharmaceutical
compounds may be encapsulated, tableted or prepared in an emulsion
or syrup for oral administration. Pharmaceutically acceptable solid
or liquid carriers may be added to enhance or stabilize the
composition,%or to facilitate preparation of the composition.
Liquid carriers include syrup, peanut oil, olive oil, glycerin,
saline, alcohols and water. Solid carriers include starch, lactose,
calcium sulfate, dihydrate, teffa alba, magnesium stearate or
stearic acid, talc, pectin, acacia, agar or gelatin. The carrier
may also include a sustained release material such as glycerol
monostearate or glycerol distearate, alone or with a wax. The
amount of solid carrier varies but, preferably, will be between
about 20 mg to about 1 gram per dosage unit. The pharmaceutical
dosages are made using conventional techniques such as milling,
mixing, granulation, and compressing, when necessary, for tablet
forms; or milling, mixing and filling for hard gelatin capsule
forms. When a liquid carrier is used, the preparation will be in
the form of a syrup, elixir, emulsion or an aqueous or non-aqueous
suspension. Such a liquid formulation may be administered directly
or filled into a soft gelatin capsule.
[0060] The Examples that follow serve to illustrate this invention.
The Examples are intended to in no way limit the scope of this
invention, but are provided to show how to make and use the
compounds of this invention.
EXAMPLES
[0061] The following abbreviations are used in the Examples:
1TABLE 1 List of Abbreviations and Activities of Experimental
Compounds Chemical Compound Abbreviation Receptor/Activity
4-aminobenzyl-5'-N- ABMECA A.sub.3 agonist methylcarbox-
amidoadenosine adenosine deaminase ADA 2-p-(2-carboxy-ethyl)
CGS21680: High affinity A.sub.2A agonist phenethyl-
amino-5'-N-ethyl- carboxamido- adenosine 8-cyclopentyl- CPX A.sub.1
antagonist 1,3-dipropylxanthine 5'-guanylyl-imididodiphosph- ate
Gpp(NH)p Stabilizes GPCR 2-hexynyladenosine- HENECA 5'-N-ethyl-
N-ethylcarboxamido-adenosine NECA Non-selective adenosine receptor
agonist Phenylisopropyl- R-PIA A.sub.1 receptor agonist adenosine
Rolipram phosphodiesterase inhibitor SCH58261: A.sub.2A antagonist
2-cyclohexylmethyliden- WRC-0470: High affinity 2A agonist
ehydrazinoadenosine 4-(2-[7-amino-2- ZM241385: 2A antagonist
(2furyl)[1,2,4]- triazolo[2,33-a][1, 3,5]triazin-5-yl
amino]ethyl)phenol
[0062] Other abbreviations include cAMP (cyclic adenosine
monophosphate), APV (average peak velocity), CBF (coronary blood
flow), CHO-Ki (Chinese hamster ovary cell line), HEK-293 (human
cell line),CPP (coronary perfusion pressure), CR (coronary
resistence), HR (heart rate), im (intramuscular), iv (intravenous)
LVSP (left ventricle systolic pressure), MAP (mean arterial
pressure). PBF (peripheral blood flow).
[0063] Adenosine deaminase was purchased from Boehringer Mannheim
Biochemicals Indianapolis, Ind.). [.sup.3H] ZM241385 was purchased
from Tocris Cookson Ltd (Langford, Bristol, UK). [.sup.3H] CPX was
from New England Nuclear (Boston, Mass.). HENECA CGS21680,
adenosine, NECA, R-PIA, phenylephrine, DMSO, rolipram and
HEK-hA.sub.2AAR membranes were obtained from Sigma-RBI (Natick,
Mass.). Nitroglycerin was obtained from Parke-Davis, Morris Plains,
N.J. Aminophylline was obtained from Abbott Laboratories, Chicago,
Ill.
[0064] HENECA was a gift from Professor Gloria Cristalli of the
University of Camerino, Italy. Drug stock solutions (10 mmol/L)
were prepared in DMSO. Sprague Dawley rats were purchased from
Simonsen Laboratories (Gilroy, Calif.). Ketamine was purchased from
Fort Dodge AnimalHealth (Fort Dodge, Iowa) and xylazine from Bayer
(Shawnee Mission, Kans.). Succinyl cAMP-tyrosyl methyl ester
(ScAMP-TME) was purchased from Sigma and iodinated in the presence
of chloramine T. CVT-2995,
CVT-3003-((4S,2R,3R,5R)-2-(6-amino-2-(2-thienyl)pruin-9-yl)-5-(hydroxymet-
hyl)oxolane-3,4-diol),
CVT-3006-((4S,2R,3R,5R)-2-(6-amino-2-{3-[2-benzylph-
enoxy]prop-1-ynyl} purin-9-yl)-5-(hydroxymethyl)oxolane-3,4-diol),
CVT-3032, CVT-3033,
CVT-3100-((4S,2R,3R,5R)-2-[6-amino-2,-(5-methyl(2-thi-
enyl))purin-9-yl]-5-(hydroxymethyl)oxolane-3,4-dion),
CVT-3101-(4-(3-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2--
yl]-6-aminopurin-2-yl} prop-2-ynyloxy)benzenecarbonitrile),
CVT-3126-(4S,2R,3R,5R)-2-{6-amino-2-[1-(3-phenylpropyl)pyrazol-4-yl]purin-
-9-yl}-5-(hydroxymethyl)oxolane-3,4-diol), CVT-3127 -(ethyl
1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydromethyl)oxolan-2-yl]-6-aminopuri-
n-2-yl}pyrazole-4-carboxylate),
CVT-3141-((4S,2R,3R,5R)-2-{6-amino-2-[4-(4-
-chlorophenyl)pyrazole]purin-9-yl}-5-(hydroxymethyl)oxolane-3,4-diol),
CVT-3144-((4S,2R,3R,5R)-2-{6-amino-2-[4-(4-methylphenyl)pyrazolyl]purin-9-
-yl}-5-(hydroxymethyl)oxolane-3,4-diol), CVT-3 146, YT-146 and
WRC0470 were synthesized by CV Therapeutics, Department of
Medicinal and Bioorganic Chemistry, Palo Alto, Calif. The
structures of several of these compounds are set forth on the
following page. 3
EXAMPLES 1 AND 2
[0065] Examples 1 and 2 demonstrate the selectivity and binding
affinity of the compounds of the invention to adenosine receptors
on cells obtained as follows. Rat pheochromocytoma PC12 cells were
obtained from the American Type Culture Collection and grown in
DMEM with 5% fetal bovine serum, 10% horse serum, 0.5 mmol/L
L-glutamine, 100U/mL penicillin, 0.1 mg/mL streptomycin, and 2.5
.mu.g/mL amphotericin.
[0066] HEK-293 cells stabily expressing recombinant human A.sub.2B
adenosine receptors (HEK-hA.sub.2B adenosine receptors) were grown
in DMEM supplemented with 10% fetal bovine serum and 0.5 mg/mL
G-418.
[0067] CHO-K1 cells stabily expressing the recombinant human
A.sub.1 adenosine receptors (CHO-hA.sub.1 adenosine receptors) or
A.sub.3 adenosine receptors (CHO-hA.sub.3 adenosine receptors) were
grown as monolayers on 150-mm plastic culture dishes in Ham's F-12
media supplemented with 10% fetal bovine serum in the presence of
0.5 mg/mL G-418. Cells were cultured in an atmosphere of 5%
CO.sub.2/95% air and maintained at 37.degree. C.
[0068] Cell membranes were harvested from the cell lines by
detaching cells from the culture plates into ice-cold 50 mmol/L
Tris-HCl buffer (pH 7.4). The cell suspensions were homogenized and
centrifuged at 48,000 g for 15 minutes. The pellets were washed
three times by re-suspension in ice-cold Tris-HCl buffer and
centrifugation. The final pellet was re-suspended in Tris-HCl,
aliquoted and frozen at -80.degree. C. until used for receptor
binding assays. The protein concentration of membrane suspensions
was determined using the Bradford (Bradford, M. M. (1976. Anal.
Biochem. 72, 248) with bovine serum albumin as standard.
[0069] Membranes were also obtained from porcine striatial cells as
follows. Porcine striatum was obtained from Pel Freeze Inc.
Striatum was minced and homogenized in 10 volumes of ice-cold 50
mmol/L Tris HCl buffer (pH7.4). The homogenate was filtered through
cotton gauze and centrifuged at 48,000 g for 15 minutes at
4.degree. C. The supernatant was discarded, and the membrane pellet
was suspended in 10 volumes of 50 mmol/L Tris-HCl buffer (pH 7.4)
and washed three times by centrifugation and resuspended in fresh
buffer. The final pellet was frozen at -80.degree. C. until used
for receptor binding assays.
[0070] Competitive radiolabeling binding assays were performed to
determine the binding affinities of the compounds of the invention
for the adenosine receptor subtypes, A.sub.1, A.sub.2A, A.sub.2B
and A.sub.3. Briefly, membrane suspensions, obtained from cells
expressing either adenosine receptor subtypes A.sub.1, A.sub.2A,
A.sub.2B or A.sub.3 were incubated for 2 hours at room temperature
in 50 mmol/L Tris-HCl buffer (pH 7.4) containing ADA (1U/mL).
[.sup.3H]-ZM241385 (.about.1.5 to 5 mmol/L) was added to membranes
from cells expressing A.sub.2A, [.sup.3H]-CPX (.about.2.5 to 3.0
mmol/L) was added to membranes from cells expressing A.sub.1,
[.sup.3H]-CPX (30 nM) was added to membranes from cells expressing
A.sub.2B and [.sup.125I]ABMECA (1 nM) was added to membranes from
cells expressing A.sub.3. The competing agents, that is the
agonists (10.sup.-9-10.sup.-4M) were also added along Gpp (NH) p
(100 .mu.g) which stabilizes the receptor in the low affinity state
thereby obviating the complication of multiple affinity states
(Gao., Z. et al. (1999). Biochem J 338(3):729). At the end of the
incubation, free radioligand was separated from membrane-bound
radioligand by filtration through Whatman GF/C glass fiber filters
using a Brandel tissue harvester (Gaithersburg, Md.). Triplicate
determinations were performed for each concentration of unlabelled
compound.
[0071] The results of radioligand binding assays, presented in
Table 2, show that binding affinities of CVT-3033 (1417 nM) and
CVT-3146 (1095 nM) to A.sub.2A receptors in pig striatum were lower
(i.e. K.sub.i higher) than a full A.sub.2A agonist, CGS21680 (157
nM).
[0072] The binding affinities of CVT-3033 (3623 nM) and CVT-3146
(1734 nM) to A.sub.2A were also lower than CGS21680 (210 nM) in
PC12 cells. Binding affinities of CVT-3033 (2895 nM) and CVT-3146
(1269 nM) were also lower than CGS21680 (609 nM)in HEK cells
expressing human A.sub.2A receptors. CGS-21680, CVT-3033, and
CVT-3146 had relatively low affinities for the A.sub.1 receptor
expressed by CHO-K1 cells. Binding to the A.sub.2A receptor was
relatively greater than binding affinities to A.sub.1, A.sub.2b or
A.sub.3 (FIGS. 1A and B and FIGS. 2A and B).
2TABLE 2 Binding affinity of adenosine receptor agonists for
A.sub.2A and A.sub.1 AdoRs K.sub.1, nmol/L (pK.sub.1 .+-. SEM) Pig
Striatum(A.sub.2A) PC12 cells(A.sub.2A) HEK-hA.sub.2AAdoR
CHO-hA.sub.1AdoR Compounds Affinity N Affinity N Affinity N
Affinity N NECA ND 297 (6.54 .+-. 0.06) 4 360 (6.45 .+-. 0.06) 3
328 (6.49 .+-. 0.06) 3 R-PIA 1106 (5.96 .+-. 0.06) 4 3476 (5.48
.+-. 0.10) 3 1656 (5.78 .+-. 0.02) 3 477 (6.45 .+-. 0.11) 3
CGS21680 157 (6.85 .+-. 0.23) 5 210 (6.74 .+-. 0.09) 3 609 (6.22
.+-. 0.06) 3 3540 (5.47 .+-. 0.20) 3 CVT-2995 14.4 (7.86 .+-. 0.12)
4 53 (7.28 .+-. 0.05) 4 305 (6.52 .+-. 0.04) 6 866 (6.07 .+-. 0.05)
3 WRC0470 20.7 (7.68 .+-. 0.04) 3 18 (7.77 .+-. 0.04) 8 272 (6.55
.+-. 0.04) 6 7278 (5.16 .+-. 0.09) 3 CVT-3032 1594 (5.88 .+-. 0.10)
3 5516 (5.26 .+-. 0.05) 3 13651 (4.87 .+-. 0.02) 3 6350 (5.22 .+-.
0.11) 3 CVT-3033 1417 (5.67 .+-. 0.16) 3 3623 (5.45 .+-. 0.03) 5
2895 (5.54 .+-. 0.03) 3 5836 (5.24 .+-. 0.04) 3 CVT-3146 1095 (5.95
.+-. 0.11) 5 1734 (5.76 .+-. 0.01) 3 1269 (5.90 .+-. 0.03) 7
>16460 (4.59 .+-. 0.35) 3 YT-146 16.3 (7.86 .+-. 0.33) 4 ND ND
ND CVT-3003 1007 (6.00 .+-. 0.07) 3 ND ND ND CVT-3006 64 (7.24 .+-.
0.26) 3 ND ND ND CVT-3100 697 (6.16 .+-. 0.06) 5 ND ND ND CVT-3101
94 (7.03 .+-. 0.06) 3 ND ND ND CVT-3126 1667 (5.78 .+-. 0.09) 3 ND
ND ND CVT-3127 64 (7.22 .+-. 0.17) 3 ND ND ND CVT-3141 1138 (5.95
.+-. 0.07) 3 ND ND ND CVT-3144 502 (6.31 .+-. 0.12) 3 ND ND ND
HENECA 7.96 (8.10 .+-. 0.08) 10 ND ND ND The binding affinities of
adenosine receptor agonists for A.sub.2AAdoRS and A.sub.1AdoRs were
determined by their effect to compete with specific binding of
[.sup.3H]ZM241385 or [.sup.3H]CPX, respectively, to membranes from
the indicated tissue/cells. Values are means .+-. SEM # of results
of at least three experiments (n) performed in triplicate. Numbers
in parentheses are means of pK.sub.1 .+-. SEM. Values of the
equilibrium dissociation constant (K.sub.d) for [.sup.3H]ZM241385
binding that were used in calclations of K.sub.1 values were 0.5,
0.5 and 0.8 nM for pig # striatum, PC12 and HEK-hA.sub.2AAdoR
cells, respectively. A K.sub.d value of 1 nM for [.sup.3H]CPX
binding to CHO-hA.sub.1AdoR membrane was used in calculation of
K.sub.1 values is 1.
EXAMPLE 3
[0073] Example 3 demonstrates the ability of the compounds of the
invention to stimulate cAMP levels, a measure of the intrinsic
efficacy of the agonists. Briefly, PC12 cells were rinsed three
times with Hanks' balanced saline solution (HBSS), detached using a
cell lifter, and pelleted by centrifugation at 500 g for 5 minutes.
Aliquots of the cell suspension (0.1 to 0.2 mg protein) were placed
in microfuge tubes with 250 .mu.L of HBSS containing rolipram (50
.mu.mol/L) to inhibit phosphodiesterases that degrade cAMP and
warmed to 37.degree. C. Appropriate drugs were added to the cell
suspensions, and incubations were allowed to continue for 10
minutes. Tubes were placed in a boiling water bath for 5 minutes to
terminate the incubation. The samples were then cooled to room
temperature, diluted by the addition of 1 mL of 10 mmol/L Tris-HCl
buffer at pH 7.4, and then centrifuged for 2 minutes at 13000
g.
[0074] The cAMP content of the supernatant was determined by
modification of a radioimmunoassay method described by Harper and
Brooker (1975. J. Cyclic nucleotide Res 1:207). Briefly, an aliquot
of the supernatant (0.01 ML) was mixed with 0.04 mL of HBSS, 0.05
mL of 50 mmol/L sodium acetate buffer (pH 6.2) containing 10 mmol/L
CaCl.sub.2, [.sup.125]ScAMP-TME (12500 dpm), and 0.05 mL of
anti-cAMP antibody (1:2000 dilution with 0. 1% bovine serum albumin
in distilled water). The samples were then incubated at 4.degree.
C. for 16 hours. At the end of the incubation, 70 .mu.L of a 50%
(wt/vol) hydroxyapatite suspension was added to each tube. The
suspensions were gently agitated and then incubated for 10 minutes
at 4.degree. C. Antibody-bound radioactivity adsorbed to
hydroxyapatite was collected onto glass fiber filters by vacuum
filtration using a Brandel cell harvester. Radioactivity retained
by the filter was counted in a gamma counter. Nonspecific binding
of [.sup.125I]ScAMP-TME was defined as radioactivity bound in the
presence of 3 .mu.mol/L unlabeled cAMP and was subtracted from
total binding. The: amount of cAMP present in samples was
calculated based on a standard curve using known amounts of
cAMP.
[0075] As illustrated in FIG. 3A, all compounds increased the
cellular content of cAMP in a concentration-dependent manner. The
low affinity A.sub.2A agonists CVT-3032, CVT-3033 and CVT-3146,
were not only less potent (10-15 fold), but also less effective in
stimulating cAMP accumulation compared to CGS21680. The maximal
responses induced by CVT-3146, CVT-3033 and CVT-3032 were 85%, 63%
and 65% of that induced by CGS21680, respectively. These data
demonstrate that CVT-3146, CVT-3033 and CVT-3032 behave as partial
A.sub.2A agonists in PC12 cells. Additionally, CVT-3033 (1 .mu.M)
inhibit the ability of CGS21680 to stimulate cAMP levels (FIG. 3B)
causing an approximate 5-fold shift to the right of the CGS21680
concentration-response curve
[0076] It is notable that the stimulation of cAMP levels in PC12
cells was related to the binding affinity of the compounds to
A.sub.2A receptors (Table 3).
3TABLE 3 Rate of decline (t.sub.0.5) of cAMP accumulated after
exposure to the A.sub.2A agonists in PC12 cells Agonists t.sub.0.5
(min) n WRC0470 5.9 .+-. 0.8 6 CGS21680 5.3 .+-. 0.5 5 CVT-2995 3.9
.+-. 0.6 5 CVT-3146 2.6 .+-. 0.2 6 CVT-3033 1.9 .+-. 0.1 4 R-PIA
1.6 .+-. 0.2 6 Values are mean .+-. SEM of the least three
experiments performed in triplicate. The pK.sub.1 (-logK.sub.1)
values were obtained from the results of competition binding assays
under [.sup.3H]ZM241385 as the radioligand for A.sub.2A AdoRs. The
pEC50 (-logEC50) values were # determined from concentration
response relationships for agonist-induced cAMP accumulation in
PC12 cells. The t.sub.0.5 values were calculated from the rate of
decline of cAMP accumulated during exposure to each agonist (1
.mu.M) following the addition of the A.sub.2A AdoR antagonists #
SCH58261 (20 .mu.M)>
[0077] The selectivity of the effects of CVT-3033 and CVT-3146 on
cAMP accumulation in HEK-293 cells expressing A.sub.2B adenosine
receptors is also shown. NECA, a non-selective adenosine receptors
agonist, caused a concentration-dependent increase of cellular cAMP
content whereas neither CVT-3033 nor CVT-3146 had any detectable
effects even at a high concentration of 100 .mu.M (FIG. 4). These
results indicate that CVT-3033 and CVT-3146 have very weak, if any,
interaction with A.sub.2B receptors.
[0078] The effect of an A.sub.2a antagonist on agonist-mediated
cAMP accumulation in PC12 was also demonstrated. PC12 cells
cultured in DMEM at 37.degree. C. were treated with WRC0470,
CGS21680, CVT-2995, CVT-3146, CVT-3033 and R-PIA each at a
concentration of 1 .mu.M in the presence of rolipram (50 .mu.M) for
10 minutes. Then, an A.sub.2A antagonist, SCH58261 (20 .mu.M), was
added and cAMP content was determined at various periods. The time
for cAMP levels to decrease to half maximal (t.sub.0.5) was
calculated and plotted against the affinity (pK.sub.i) of each
agonist for the A.sub.2A adenosine receptor, as determined by
competition radioligand binding assays (above).
[0079] FIG. 5A shows the time-course of the decline of
agonist-stimulated cAMP accumulation following the addition of
SCH58261when compared to the control cultures (CGS2160 incubated
without SCH58261). The calculated values of t.sub.0.5 from these
experiments are presented in Table 3. The apparent t.sub.0.5 values
of agonists were inversely related to their affinities for A.sub.2A
adenosine receptors, that is, the greater the agonist affinity, the
lower the rate of decline of cAMP content upon application of the
A.sub.2A adenosine receptors antagonist SCH58261 (FIG. 5A). As
depicted in FIG. 5B, the relationship between the apparent
t.sub.0.5 and pK.sub.i for the agonists was best fit by linear
regression with a correlation coefficient (r value) of 0.84.
EXAMPLE 4
[0080] The effect of the compounds of the invention on coronary
conductance (CC), an estimate of vasodilation, was demonstrated ex
vivo using perfused rat hearts. Briefly, rats of either sex
weighing 230-260 grams were anesthetized by intraperitoneal
injection of a mixture of ketamine (100 mg/ml) and xylazine (20
mg/ml). The chest of each rat was opened and the heart removed, and
rinsed in ice-cold modified Krebs-Henseleit (K-H) solution
containing NaCl 117.9, KCl 4.5, CaCl.sub.2 2.5, MgSO.sub.4 1.18,
KH.sub.2PO.sub.4 1.18, pyruvate 2.0 mmo/L. The aorta was cannulated
and the heart was perfused at a flow rate of 10 ml/min with
modified K-H solution. The K-H solution (pH 7.4) was gassed
continuously with 95% O.sub.2 and 5% CO.sub.2 and warmed to
35.+-.0.50 C. The heart was electrically paced at a fixed cycle
length of 240 ms (250 beats/min) using a bipolar electrode placed
in the left atrium. The electrical stimuli were generated by a
Grass stimulator (Model S48, W. Warwick, R.I.) and delivered
through a Stimuli Isolation Unit (Model SIU5, Astro-Med, Inc., NY)
as square-wave pulses of 3-msec in duration and with an amplitude
of at least twice the threshold intensity.
[0081] As shown in FIG. 6A, adenosine, CGS21680, WRC0470, and the
CVT compounds caused concentration-dependent increases in coronary
conductance.
[0082] The potencies (EC.sub.50 values) of adenosine, CGS21680,
WRC0470 and the CVT compounds are summarized in Table 4. The low
affinity agonist CVT-3146 was found to be approximately 1 0-fold
more potent than adenosine but 1 0-fold less potent than the high
affinity agonists CGS21680 and WRC0470 with respect to increasing
coronary conductance. The results show that CVT 3146 was a potent
agonist of coronary conductance in heart but only a weak agonist in
PC 12 cells: (EC.sub.50=291 nM).
4TABLE 4 Potency of adenosine and A.sub.2A Adenosine receptor
agonists to increase cAMP accumulation in PC12 cells and coronary
conductance in rat isolated perfused heart EC.sub.50 (pEC.sub.50
.+-. SEM), nM cAMP Accumulation Coronary Conductance Agonist (PC12
cells) (Rat Isolated Heart) CGS21680 18 (7.75 .+-. 0.03) N = 3 0.54
(9.27 .+-. 0.03) N = 3 CVT-2995 6.6 (8.82 .+-. 0.25) N = 3 0.68
(9.17 .+-. 0.03) N = 5 CVT-3146 291 (6.54 .+-. 0.03) N = 3 6.40
(8.19 .+-. 0.04) N = 4 CVT-3032 613 (6.21 .+-. 0.02) N = 3 66.50
(7.18 .+-. 0.07) N = 4 CVT-3033 487 (6.31 .+-. 0.01) N = 3 67.95
(7.19 .+-. 0.08) N = 4 WRC0470 ND 0.62 (9.19 .+-. 0.6) N = 5
Adenosine ND 59.20 (7.24 .+-. 0.11) N = 4 Values are the mean
concentrations of agonists that caused 50% increase in cAMP
accumulation or coronary conductance (EC.sub.50 and pEC.sub.50).
ND; Not determined.
[0083] Coronary vasodilatory effect of CVT-3146 in the absence and
presence of adenosine receptor antagonists were also demonstrated.
The identity of the adenosine receptor subtype. (A.sub.1 or
A.sub.2A) mediating the coronary vasodilation was determined.
Hearts (n=6) were exposed to CVT-3 146 (10 nM), and after the
effect of this agonist reached steady-state, CPX (60 nM), an A1
antagonist and then ZM241385 (60 nM), an A2a antagonist were added
to, the perfusate and the changes in CPP were recorded. As depicted
in FIG. 7A, CVT-3146 significantly increased coronary conductance
to 0.22.+-.0.01 ml mm Hg.sup.-1 min.sup.-1 from a baseline value of
0.16.+-.0.02 ml mm Hg.sup.-1 min.sup.-1. This increase in coronary
conductance caused by CVT-3146 was not affected by 60 nM CPX but
was completely reversed by 60 nM ZM241385. Furthermore, the
inhibition by ZM241385 of an increase of coronary conductance
caused by CVT-3146 was concentration-dependent (FIG. 7B).
[0084] A.sub.1 adenosine receptor-mediated depression of A-V nodal
conduction time by CVT-3033 and CVT-3146 (negative dromotropic
effect) was measured using atrial and ventricular surface
electrograms as described by Jenkins and Belardinelli (Circ Res
63:97).
[0085] As shown in FIG. 8, CVT-3146 and CVT-3033 increased coronary
conductance in a concentration-dependent manner, but did not
prolong A-V nodal conduction time
[0086] Coronary perfusion pressure (CPP) was measured using a
pressure transducer that was connected to the aortic cannula via a
T-connector positioned approximately 3 cm above the heart. CPP was
monitored throughout an experiment and recorded either on a chart
recorder (Gould Recorder 2200S, Valley View, Ohio) or a
computerized recording system (PowerLab/4S, ADInstruments Pty Ltd,
Australia). Only hearts with CPP ranging from 60 to 85 mmHg (in the
absence of drugs) were used in the study. CC conductance (in
ml/min/mmHg) was calculated as the ratio between coronary perfusion
rate (10 ml/min) and CPP.
[0087] As shown in FIG. 9A the extent of the decrease in coronary
perfusion pressure (an index of the coronary vasodilation) caused
by CVT-3146 was similar to that caused by a supramaximal
concentration of CGS21680 (FIG. 9B). Both 10 nM CVT-3146 and 100 nM
CGS21680 decreased coronary perfusion pressure by 23 mmHg. In
addition, in the presence of 10 nM CVT-3146, CGS21680 (100 nM) did
not cause a further decrease of the coronary perfusion pressure
(FIG. 9C). Thus, CVT-3146 is a full agonist with respect to
coronary artery conductance.
[0088] The relationship between the affinity of agonists for the
A.sub.2A receptor and the rate of reversal of agonist-mediated
responses (coronary vasodilation in heart and cAMP accumulation in
PC12 cells) upon termination of drug administration were
determined. All compounds were given as boluses into the perfusion
line at their respective minimal concentrations that caused equally
or near-equally maximal increases in coronary conductance.
Likewise, the onset and time to peak effect: (i.e. maximal coronary
vasodilation) were similar for all agonists. Although adenosine and
the various agonists caused equal maximal increases in coronary
conductance, the durations of their effects were markedly
different. The duration of the effect of adenosine was the shortest
followed by those of CVT-3033 and CVT-3146. The effects of CGS21680
and WRC0470 had the longest duration (FIG. 10). The duration of the
coronary vasodilation in isolated rat hearts caused by adenosine,
the CVT compounds, and other agonists measured as the time to 50%
and 90% (t.sub.0.5 and t.sub.0.9, respectively) reversal of the
increases in coronary conductance after termination of drug
administration are summarized in Table 4. The reversal time of
coronary vasodilation correlated with the affinity of the adenosine
derivatives for the A.sub.2A receptors. As shown in FIG. 9C, there
was a significant (p<0.05) inverse relationship (r=0.87) between
the affinity (pK.sub.i) of the agonists for the A.sub.2A adenosine
receptors (Table 2) and the reversal time (t .sub.0.9) (Table 4) of
the coronary vasodilation caused by the same agonists in rat
isolated hearts.
EXAMPLE 5
[0089] The magnitude of the effect of A.sub.2A adenosine receptor
agonists on coronary dilation and the duration of the effect was
determined in pigs weighing 22-27 kg. All animals received humane
care according to the guidelines set forth in "The Principles of
Laboratory Animal Care" formulated by the National Society for
Medical research and the "Guide for the Care and Use of Laboratory
Animals" prepared by the Institute of Laboratory Animal Resources
and published by the National Institutes of Health (NIH Publication
No. 86-23, revised 1996). In addition, animals were used in
accordance with the guidelines of the University of Kentucky
Institutional Animal Care and Use Protocol.
[0090] Animals were anesthetized with ketamine (20 mg/kg, i.m.) and
sodium pentobarbital (15-18 mg/kg, i.v.). Anesthesia was maintained
with additional sodium pentobarbital (1.5-2 mg/kg, i.v.) every
15-20 minutes. Animals were ventilated via a tracheotomy tube using
a mixture of room air and 100 % O.sub.2 Tidal volume, respiratory
rate and fraction of O.sub.2in inspired air were adjusted to
maintain arterial blood gas (ABG) and pH values. Core body
temperature was monitored with an esophageal temperature probe and
maintained at 37.0-37.5.degree. C. by use of a heating pad. Lactate
Ringers solution was administered via an ear or femoral vein as an
initial bolus of 300-400 ml followed by a continuous infusion at a
rate of 5-7 ml/kg/hr. A catheter was inserted into the femoral
artery to monitor arterial blood pressure.
[0091] The heart was exposed through a median sternotomy and
suspended in a pericardial cradle. Left ventricular pressure (LVP)
was measured with a 5F high fidelity pressure sensitive tip
transducer (Millar Instruments, Houston, Tex.) placed in the left
ventricular cavity via an apical incision and secured with a purse
string suture. A segment of the left anterior descending coronary
artery (LAD), proximal to the origin of the first diagonal branch,
was dissected free of surrounding tissue. A transit time
perivascular flow probe (Transonic Systems Inc., Ithaca, N.Y.) was
placed around this segment to measure CBF. Proximal to the flow
probe, a 24-gauge modified angiocatheter was inserted for
intracoronary infusions. All hemodynamic data were continuously
displayed on a computer monitor and fed through a 32-bit analog to
digital converter into an online data acquisition computer with
customized software (Augury, Coyote Bay Instruments, Manchester,
N.H.). A.sub.2A adenosine receptors agonists were dissolved in DMSO
to produce stock concentrations of 1-5 mM, which were diluted in
0.9% saline and infused at rates of 1-1.5 ml/min via the catheter.
The A.sub.2A, adenosine receptors agonists were administered
intracoronary.
[0092] Relationship between affinity of various agonists for
A.sub.2A adenosine receptor and the reversal time of their effect
to increase coronary conductance Was determined in pigs. Each
experiment was preceded by a 30-minute stabilization period
following the completion of all instrumentation of the animal.
Baseline hemodynamic data were then recorded and an intracoronary
infusion of an A.sub.2A Adenosine receptors agonist was initiated.
Each infusion was maintained for 4-5 minutes to allow LAD.degree.
CBF to reach a steady-state, after which the infusion was
terminated. The times to recovery of CBF by 50% (t.sub.0.5) and 90%
(t.sub.0.9) of the difference from peak effect to baseline CBF were
recorded. Ten to 15 minutes after CBF returned to pre-drug values a
second infusion with a different agonist was started. In
preliminary studies it was found that the intracoronary infusion of
adenosine receptor agonists produced varying degrees of systemic
hypotension, and hence, in all subsequent experiments,
phenylephrine was administered intravenously at dose of .about.1
.mu.g/kg/min. Hemodynamic measurements were made prior to and
following the initiation of the phenylephrine infusion. The
phenylephrine infusion rate was adjusted during and following the
infusions of the adenosine receptor agonists to maintain arterial
blood pressure within 5 mmHg of pre-infusion values. The effect of
a maximum of three different agonists was determined in each
experiment.
[0093] All CVT-compounds as well as CGS21680 and other A2a
adenosine receptors agonists (i.e., WRC-0470 and YT-146) caused
significant increases in CBF (Table 6). Selected doses of these
compounds given as continuous (4 to 5 min) intracoronary infusions
caused 3.1 to 3.8-fold increases in CBF. Once it was established
that all agonists caused comparable increases of CBF (i.e., "fold
increase") and caused little or no changes in heart rate and mean
arterial blood pressure (data not shown), the reversal time of
their coronary vasodilatory effects was determined. As summarized
in Table 5 the reversal times of the effect of the low affinity,
partial agonists, CVT-3146, CVT-3032 and CVT-3033, were shorter
than those of CGS21680, WRC-0470 or YT-146. More importantly, as
depicted in FIG. 9D, there was a significant (p<0.05) inverse
relationship (r=0.93) between the affinity (pK.sub.i) of the
A.sub.2A Adenosine receptors agonists for pig brain striatum
A.sub.2A receptors and the reversal time (t.sub.0.9) of coronary
vasodilation in pig heart Table 2).
5TABLE 5 Reversal time of coronary vasodilation by adenosine and
adenosine receptor agonists in rate isolated perfused Agonist
t.sub.0.5 (min) t.sub.0.9 (min) n R-PIA 7.9 .+-. 0.1 12.6 .+-. 0.8
3 CGS21680 14.5 .+-. 0.5 19.5 .+-. 09 3 CVT-2995 17.5 .+-. 1.2 23.2
.+-. 2.1 4 WRC0470 21.9 .+-. 0.9 27.9 .+-. 1.4 6 CVT-3032 4.1 .+-.
0.3 9.8 .+-. 1.4 4 CVT-3033 3.4 .+-. 0.5 8.4 .+-. 2.2 4 CVT-3146
5.2 .+-. 0.2 11.3 .+-. 1.1 5 YT-146 17.7 .+-. 1.0 25.8 .+-. 4.0 3
CVT-3003 3.4 .+-. 0.1 9.2 .+-. 2.2 4 CVT-3006 16.1 .+-. 0.1 21.8
.+-. 2.0 3 CVT-3100 5.1 .+-. 0.6 10.1 .+-. 0.2 4 CVT-3101 16.7 .+-.
0.5 25.6 .+-. 0.3 3 CVT-3126 8.3 .+-. 0.4 12.6 .+-. 0.3 4 CVT-3127
14.8 .+-. 2.1 15.0 .+-. 0.8 3 CVT-3141 14.4 .+-. 1.9 21.3 .+-. 3.9
4 CVT-3144 13.6 .+-. 1.3 18.9 .+-. 1.9 4 HENECA 28.6 .+-. 1.1 32.8
.+-. 3.1 3 Adenosine 1.6 .+-. 0.1 5.6 .+-. 0.8 11 Times (in
minutes) to 50% and 90% (t.sub.0.5 and t.sub.0.9, respectively)
reversal of the increases in coronary conductance caused by
adenosine and adenosine receptor agonists. Values are the mean .+-.
SEM of single determination in each of n preparation.
[0094]
6TABLE 6 Magnitude and reversal time of coronary dilation caused by
various adenosine receptor agonists in open-chest anesthetized pigs
CBF (fold- Agonist Dose increase) t.sub.0.5 (min) t.sub.0.9 (min) n
CVT-3146 10 ug/kg/min 3.40 .+-. 0.04 1.9 .+-. 0.2 10.1 .+-. 0.7 3
CVT-3146 30 ug/kg/min 3.38 .+-. 0.39 2.6 .+-. 0.5 12.3 .+-. 1.1 6
CVT-3032 30 ug/kg/min 3.78 .+-. 0.70 2.3 .+-. 0.6 9.6 .+-. 1.0 3
CVT-3033 50 ug/kg/min 3.33 .+-. 0.58 3.1 .+-. 0.9 12.0 .+-. 1.0 3
WRC0470 1 ug/kg/min 3.14 .+-. 0.24 9.5 .+-. 0.8 22.5 .+-. 1.6 6
CGS21680 2 ug/kg/min 3.54 .+-. 0.09 9.7 .+-. 0.8 21.4 .+-. 0.8 3
YT-146 1 ug/kg/min 3.44 .+-. 0.47 17.8 .+-. 3.4 32.9 .+-. 5.6 3
Maximal "fold-increase" and time (in minutes) to 50% and 90%
(t.sub.0.5 and t.sub.0.9, respectively) reversal of the increases
in coronary blood flow caused by various adenosine receptor
agonists. Data represent mean .+-. SEM of one or two measurements
in each of n pigs.
EXAMPLE 6
[0095] The following example demonstrate the effects of CVT-3146 on
the hemodynamic parameters in dogs.
[0096] Ten mongrel dogs (weighing 23-27 kg) were premedicated with
Acepromazine (0.3 mg/kg im) and anesthetized with sodium
pentobarbital (25 mg/kg) and then intubated and ventilated with
room air. A thoracotomy was performed in the left fifth intercostal
space using sterile surgical techniques. A Tygon catheter
(Cardiovascular Instruments, Inc.) was placed in the descending
thoracic aorta for the measurements of blood pressure. A
solid-state pressure gauge (P6.5, Konisberg Instrument, Inc) was
placed in the apex of the left ventricle for the measurement of the
left ventricular systolic pressure (LVSP) and calculation of first
derivative of left ventricular pressure (LV dP/dt). A Doppler
transducer (Craig Hartley) was placed on the left circumflex
coronary artery for measurement of CBF. An hydraulic coronary
occluder (In: Vivo Metric, Inc) was implanted in 4 dogs around the
left circumflex coronary artery. The chest was closed in layers and
the catheters and wires were run subcutaneously and exited in the
interscapular area. The dogs were allowed 10 to 14 days to recover
fully from the surgery and were trained to lie on a laboratory
table. The protocols were approved by the Institutional Animal Care
and Use Committee of New York Medical College and conform to the
"Guiding Principles for the use and Care of Laboratory Animals" of
the National Institute of Health and the American Physiology
Society.
[0097] Arterial pressure was measured by connecting the previously
implanted catheter to a strain-gauge transducer (P23ID, Statham)
and mean arterial pressure (MAP) was derived using 2-Hz low-pass
filter. LV pressure was measured from the solid-state pressure
gauge, and LV dP/dt was calculated using a microprocessor set as a
differentiator and having a frequency response flat to 700 Hz (LM
324, National Semiconductor). Left circumflex CBF was measured
using a pulsed Doppler flowmeter (System 6, Triton technology), and
mean CBF was derived using a 2-Hz low-pass filter. Mean CR was
calculated as the quotient of MAP and CBF. Heart rate was monitored
from the pressure pulse interval using a cardiotachometer (Beckman
Instruments). The lead-2 of the electrocardiogram was recorded
during the experiments in order to examine the alterations in the
AV nodal conduction (PR interval). All signals were recorded on a
direct-writing oscillograph (Gould 2800).
[0098] To determine the effects of adenosine and CVT 3146 on CBF
and CR in resting dogs baseline hemodynamics and CBF were recorded
and then, increasing doses of adenosine: 13, 27, 67, 134 and 267
.mu.g/kg and CVT-3146: 0.1, 0.175, 0.25, 0.5, 1.0, 2.5, 5 .mu.g/kg
in 10 ml volumes were administered iv for 10 minutes in 10 ml via a
catheter inserted into a peripheral vein. Hemodynamics were
measured before, during and after each dose. Following each dose
hemodynamics were allowed to return to baseline before the
administration of the next dose. Changes in heart rate, blood
pressure, CBF; and ECG were recorded
[0099] The duration of coronary vasodilation was determined using
two different injection protocols: 1) an iv infusion of 10 ml in 10
seconds and; 2) iv infusion of 10 ml in 30 seconds. The time to the
peak effect in mean CBF increase and the duration during which CBF
remained at least 2 fold above baseline.
[0100] To determine whether tachyphylaxis occurred, three
consecutive injections of 1 .mu.g/kg CVT-3146 were given as
intravenous injections (10 ml in 30 seconds) via a catheter
inserted into a peripheral vein. The hemodynamics were allowed to
return to baseline between doses. Hemodynamics were measured
before, during and after each dose.
[0101] Hemodynamic results are expressed as mean.+-.SEM. Data were
analyzed using one way repeated measures analysis of variance, with
Student-Neuman-Keuls post hoc analysis to identify which means were
significantly (p<0.05) different (Sigma Stat, Version 2.2,
Jandel Scientific, San Rafael, Calif.). To determine the agonist
potency from dose-response curves, doses producing 50% of maximum
effect (ED.sub.50) were calculated by fitting curves using the
Boltzmann equation. ED.sub.50 were compared using a Student's
t-test (p<0.05 being considered as significant). Because no
statistical differences were found among the baseline values,
between each dose for each parameter measure, the first baseline
registered during each experiment was used as the control value. A
Student's t-test was used to compare the changes in CBF and
hemodynamic parameters produced by 2.5 .mu.g/kg CVT-3146, 267
.mu.g/kg adenosine and 25 kg/kg nitroglycerin (p<0.05 being
considered significant).
[0102] A dose-response curve of the effect of CVT-3146 on CBF is
shown in FIG. 11. An IV bolus injection of CVT-3146 caused a
dose-dependent increase in mean CBF, with a ED.sub.50 of
0.34.+-.0.08 .mu.g/kg and a maximal increase of 154.+-.16 ml/min
from baseline (45.+-.3 ml/min). In comparison to CVT-3146,
adenosine was less potent having an ED.sub.50=51.+-.15 kg/kg
(p<0.05). The maximal increase in mean CBF stimulated by
CVT-3146 and adenosine were similar.
[0103] CVT-3146 produced a maximal decrease in CR of 73.+-.2% and
75.+-.2% at 2.5 .mu./kg at 5 .mu.g/kg, respectively. Adenosine
produced a maximal decrease of 73.+-.1% at 267 .mu.g/kg (data not
shown).
[0104] The effects of CVT-3146 and adenosine on left ventricular
systolic pressure and dP/dt were compared. Increasing doses of
CVT-3 146 did not cause significant changes in LVSP (data not
shown). In comparison, adenosine increased LVSP at 67 .mu.g/kg, 134
.mu.g/kg and 267 .mu.g/kg, by 12.+-.3%, 12.+-.3% and 18.+-.6%,
respectively. Both adenosine (267 .mu.g/kg) and CVT-3146 (2.5
.mu.g/kg) increased the dP/dt by 29.+-.7% and 39.+-.7%
respectively.
EXAMPLE7
[0105] Example 7 demonstrates the differential effects of CVT-3146
on blood flow velocity in coronary and peripheral arteries,
systemic arterial blood pressure and heart rate in anesthetized
dogs.
[0106] Mongrel dogs (either sex, 17-21 kg; n=6) were obtained from
a local vendor (Barton, Oxford, N.J.). Blood flow velocity in the
coronary and cranial circumflex arteries was measured using Doppler
transducer-tipped guide wires 0.014" in diameter (FloWire.RTM.,
model 1400J) purchased from Cardiometrics, Inc., Mountain View,
Calif. For the positioning of the FloWire, a Judkins left coronary
guiding catheter (JL3.5, 8F; Cordis) was used. Vascular arterial
angiography was performed using Hypaque-76 contrast fluid (Bracco
Diagnostics, Inc., Princeton, N.J.; Lot #9H28899) and a mobile
fluoroscopic unit (Philips, BV 29). Systemic arterial blood
pressure was measured using an electronic transducer-tipped
catheter (Millar). The following pharmacologic agents were used:
CVT-3 146 (CV Therapeutics; Lot #315-53), heparin (Solopak
Laboratories, Inc., Elk Grove Village, Ill.; Lot # 960211) and
sodium pentobarbital (lot # 9700), and acepromazine (lot #
3960960), obtained from J. A. Webster (Fort Dodge, IW).
[0107] Dogs were sedated with acepromazine (0.25 mg/kg),
anesthetized with sodium pentobarbital (30 mg/kg+additional doses
(1 mg/kg) given as necessary to maintain the level of anesthesia),
intubated with endotracheal tube and artificially ventilated with
room air using a respirator. Following the administration of
heparin (20 U/kg+100 U/hr), the pressure transducer-tipped catheter
was introduced through the left femoral artery and positioned in
the descending aorta. The Doppler FloWire was introduced through
the right femoral artery. A peripheral vein was cannulated for the
administration of all drugs. Doses of 1 .mu.g/kg of CVT-3146 and
300 or 200 .mu.g/kg adenosine (Adenosine) were given multiple
times, once or twice when the Doppler catheter was positioned in a
coronary artery and again when the catheter was positioned in the
cranial circumflex humeral artery. The sequence of positioning of
the Doppler catheter was reversed in consecutive experiments. In
each dog, baseline values of measured parameters were recorded
following a stabilization period of 20 minutes, and CVT-3146 and
Adenosine were given as intravenous bolus injections (<0.5 ml)
followed by a physiologic saline solution flush (10 ml); the time
required for both injections of each drug was <15 sec. All
parameters were allowed to recover (>30 min) to their respective
baseline values between two consecutive drug administrations. In
all six dogs studied, the effect of CVT-3146 on coronary artery APV
was determined. The effect of CVT-3146 on peripheral artery APV was
determined in five of the six dogs. The effect of adenosine on both
coronary and peripheral artery APV was studied in five of the six
dogs.
[0108] Systemic arterial blood pressure (BP) and electrocardiograms
(ECG) were monitored and recorded using Gould Data Acquisition
System (model 13-4615-65A), a video cassette recorder (Teac, XR
5000) and a chart recorder (Astromed, 9600). The following
parameters were monitored and recorded: Average peak coronary and
peripheral artery blood flow velocity (APV), mean arterial blood
pressure (MAP) (mmHg), and sinus cycle length (SCL; msec).
Differences in measure parameters were tested for statistical
significance using ANOVA and Student's t test corrected for
multiple measurements. Data are expressed as the mean.+-.SEM.
[0109] CVT-3146 increased APV in the coronary vasculature by
2.6.+-.0.2-fold while its increase of APV in the peripheral
arteries was only 1.1.+-.0.1 -fold (Table 7). In contrast the
vasodilatory action of adenosine was similar in the two vascular
beds: specifically, adenosine increased APV in the coronary
vasculature by 2.5.+-.0.3-fold while its increase of APV in the
periopheral arteries was 2.0.+-.0.4-fold.
7TABLE 7 Magnitude of Increases in Coronary and Peripheral Blood
Flow Velocity of CVT-3146 and Adenosine in Anesthetized Close-chest
Dogs Agonist Coronary N Peripheral N CVT-3146 2.6 .+-. 0.2 6 1.1
.+-. 0.1 5 Adenosine 2.5 .+-. 0.3 5 2.0 .+-. 0.4 5 Data are the
maximal "folds increase" in average peak velocity (APV) above
baseline (APV.sub.max/APV.sub.baseline) after intravenous injection
of CVT-3146 (1 .mu.g/kg) and adenosine (200 or 300 .mu.g/kg)
[0110] The time course of the changes in CBF, PBF, HR and MAP and
heart rate caused by CVT-3146 and adenosine are depicted in FIG. 12
and summarized in Table 8. The duration of >2-fold increase in
coronary APV caused by CVT-3146 and adenosine was <120 sec and
<20 sec, respectively; all parameters returned to baseline
within 10 min and 5 min post injection of adenosine and CVT-3 146,
respectively. Based on the differential selectivity of the
vasodilatory effects of CVT-3146 and adenosine in the coronary and
the peripheral arteries, and the dosage used, CVT-3146 is
approximately 600 times more selective than adenosine in
vasodilating the coronary vs. the peripheral arterial
vasculature.
8TABLE 8 Differential effects of CVT-3146 and Ado on blood flow
velocity, heart rate and mean arterial blood pressure (MAP)
CVT-3146 (1 ug/kg) Ado (200 or 300 ug/kg) Coronary (n = 6)
Peripheral (n = 5) Coronary (n = 5) Peripheral (n = 5) Time HR MAP
APV HR MAP APV HR MAP APV HR MAP APV Baseline 171 .+-. 8 128 .+-. 7
49 .+-. 7 162 .+-. 9 116 .+-. 8 34 .+-. 8 170 .+-. 10 126 .+-. 6 42
.+-. 5 165 .+-. 10 118 .+-. 8 33 .+-. 3 10" 177 .+-. 8 126 .+-. 9
84 .+-. 4 167 .+-. 6 114 .+-. 9 34 .+-. 4 174 .+-. 7 126 .+-. 8 90
.+-. 10 171 .+-. 6 107 .+-. 9 62 .+-. 9 20" 186 .+-. 9 116 .+-. 9
106 .+-. 8 177 .+-. 7 107 .+-. 8 38 .+-. 4 166 .+-. 11 90 .+-. 9 85
.+-. 10 162 .+-. 15 85 .+-. 9 76 .+-. 10 30" 188 .+-. 7 117 .+-. 7
109 .+-. 10 183 .+-. 8 111 .+-. 6 41 .+-. 4 173 .+-. 6 107 .+-. 9
75 .+-. 9 168 .+-. 9 87 .+-. 9 59 .+-. 11 1' 186 .+-. 7 112 .+-. 7
100 .+-. 12 182 .+-. 7 102 .+-. 6 42 .+-. 4 175 .+-. 5 113 .+-. 6
39 .+-. 8 171 .+-. 6 105 .+-. 6 44 .+-. 7 1.5' 186 .+-. 7 110 .+-.
7 97 .+-. 11 184 .+-. 7 99 .+-. 7 42 .+-. 4 171 .+-. 5 117 .+-. 6
55 .+-. 8 171 .+-. 6 106 .+-. 7 38 .+-. 4 2' 185 .+-. 7 110 .+-. 7
88 .+-. 11 187 .+-. 8 98 .+-. 7 40 .+-. 4 170 .+-. 5 120 .+-. 6 52
.+-. 7 169 .+-. 6 110 .+-. 6 35 .+-. 3 3' 185 .+-. 7 110 .+-. 6 81
.+-. 11 187 .+-. 8 97 .+-. 7 40 .+-. 4 168 .+-. 8 125 .+-. 6 49
.+-. 6 165 .+-. 9 115 .+-. 7 33 .+-. 3 4' 183 .+-. 8 110 .+-. 6 75
.+-. 11 186 .+-. 8 100 .+-. 6 39 .+-. 4 164 .+-. 10 126 .+-. 6 47
.+-. 6 160 .+-. 9 119 .+-. 8 31 .+-. 3 5' 183 .+-. 8 112 .+-. 6 72
.+-. 11 185 .+-. 8 101 .+-. 6 37 .+-. 5 163 .+-. 10 128 .+-. 6 47
.+-. 6 158 .+-. 10 121 .+-. 7 30 .+-. 2 6' 182 .+-. 8 113 .+-. 6 65
.+-. 11 7' 181 .+-. 8 115 .+-. 6 63 .+-. 12 8' 180 .+-. 8 117 .+-.
6 55 .+-. 11 9' 177 .+-. 9 119 .+-. 6 55 .+-. 11 10' 176 .+-. 9 120
.+-. 6 53 .+-. 11 Values are the mean .+-. SEM of single
determinations in each of the preparations (n).
* * * * *