Note: Descriptions are shown in the official language in which they were submitted.
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THIENYLHYDRAZON WITH DIGTTALIS-LIKE PROPERTIES (POSITIVE INU1ROPIC EE~FECTS)
Field of the Invention
The invention relates to chemical compounds exhibiting digitalis-like
properties
and activity, and to methods of making and using this compound. The invention
further
includes use of these compounds in the treatment of cardiac disease and muscle
fatigue.
Thus, tt~e invention relates to pharmaceutical compositions containing these
compounds.
The invention further relates to methods of synthesis of these compounds.
Technology Review
Several drugs in common use, such as digetoxin and digoxin, are derived from
digitalis. The common chemical structure of these drugs is a steroid nucleus
containing an
1 S unsaturated lactone ring and one or more glycoside residues. The mechanism
of action of
digitalis-derived drugs, or digitalis glycosides, is to selectively inhibit
active transport of
K+ and Na+ , in cardiac muscle, which increases the rate of Ca2+ cycling. This
has the
effect of increasing the velocity and extent of shortening of cardiac muscle
by increasing
the availability of Ca2+ to interact with contractile proteins. Digitalis
glycosides also
affect the sympathetic nervous system and reduce neurohumeral activation.
Examples of
currently marketed digitalis glycosides are Lanoxin~ and Lanoxicaps~ made by
Glaxo-
Wellcome.
Digitalis glycosides share the property of being toxic immediately above their
therapeutic range. Toxic effects of these drugs include: arrhythmias, ECG
effects such as
increased blood pressure and heart rate, pulmonary congestion, delirium,
fatigue,
disturbance of color vision, anorexia, nausea, and vomiting. The drugs are
cardiotoxic and
neurotoxic because of their effect on the sympathetic nervous system. There is
significant
infra-patient variability in the therapeutic and toxic levels, so that each
patient must be
individualized to achieve safe therapeutic drug levels. This problem is
exacerbated by the
fact that many co-administered drugs, such as verapamil, guanidine, and
amiodamne, can
shift the therapeutic and toxic ranges, requiring additional modification of
the dosing
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regimen. Digoxin is also metabolized by intestinal flora and antibiotic
treatment can
increase drug bioavailability, causing an overdose.
Thus, the risks due to incorrect dosing of digitalis glycosides are great; and
potential effects of overdosing are serious. The treatment for overdose is to
bind
circulating drug with antibodies to digitalis glycosides. The cost of this
treatment can be
in excess of $3,000 per incident. An example of such an antibody is Digibind~
made by
Glaxo-Wellcome. Digibind~ is a protein derived from sheep and carries its own
risks.
Summary of the Invention
There is a great medical need for drugs to treat congestive heart failure.
Congestive heart failure is an important cause of mortality and morbidity in
the U.S.: over
2.5 million patients are currently diagnosed. Compounds derived from
digitalis, which are
in the class of cardiac glycosides, are the primary drugs used in the
treatment of
congestive heart failure, particularly systolic dysfunction. However,
digitalis ameliorates
congestive heart failure by producing a positive inotropic effect. A positive
inotropic
agent strengthens the contractility of muscular tissue. Digitalis-derived
drugs have the
defect of being cardio-toxic and neuro-toxic at doses just above their
therapeutic range
(Hardman, J.G. and Limbird, L.E., The Pharmaceutical Basis of Therapeutics,
9"' ed.
McGraw-Hill New York, 1996, chapter 34). There is a need for drugs that can
treat
congestive heart failure and are less toxic near their therapeutic range.
The invention includes the novel chemical compound having the formula (I)
(I)
0
N~N\ S
o \ ~ Ri R2
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In formula (I), each of R1 and RZ is hydrogen,
alkyl of 1 to 6 carbon atoms, phenyl and substituted phenyl.
Preferably at least one of R1 or R2 is hydrogen.
The invention further includes the synthesis of a
novel compound that, like digitalis, produces a positive
isotropic effect on cardiac and skeletal muscle. Like
digitalis, it has utility in the treatment of congestive
heart failure and systolic dysfunction. Unlike digitalis,
it does not have toxic properties near its effective
therapeutic range and therefor the invention has a medical
advantage over that class of drugs. The invention has
further utility in the treatment of muscle fatigue in
pathological states.
The invention compounds) acts) as a calcium
sensitizer in heart and skeletal muscle. It delays and
shortens fatigue of skeletal muscle and thus also has
utility in the treatment of muscle fatigue. Muscle fatigue
is a symptom of certain pathological states such as: major
injury, cancer, HIV infection, sepsis, Crohn's disease,
ulcerative colitis, and athletic over-training.
According to one aspect of the present invention,
there is provided a chemical compound having the formula (I)
O
O / N.N w S/
O \ I Ri R
2 (I)
wherein, R1 is selected from the group consisting of
hydrogen, alkyl of 1 to 6 carbon atoms, unsubstituted
phenyl, and substituted phenyl; RZ is selected from the group
consisting of H, alkene, un-substituted phenol, and
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substituted phenyl; or a pharmaceutically acceptable salt
thereof.
According to another aspect of the present
invention, there is provided a use of a therapeutically
effective amount of LASSBio-294 for treating a patient in
need of treatment with a calcium sensitizer.
According to still another aspect of the present
invention, there is provided a use of a therapeutically
effective amount of LASSBio-294 in manufacture of a
medicament for treating a patient in need of treatment with
a calcium sensitizer.
According to yet another aspect of the present
invention, there is provided a pharmaceutical composition
comprising LASSBio-294 or a pharmaceutically acceptable salt
thereof, and a pharmaceutically acceptable carrier, diluent
or excipient.
According to a further aspect of the present
invention, there is provided a pharmaceutical composition
comprising LASSBio-249 and a dipeptide or a tripeptide,
wherein the composition products at least 200 oral
bioavailability of LASSBio-294 when taken orally by a
patient.
According to yet a further aspect of the present
invention, there is provided a pharmaceutical composition,
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comprising a compound of formula (III)
R6 R7
R3 O ~ ~ Rs
O / N~Nw S~
\ ~ R
O ~ 1 RZ
Rs
and a pharmaceutically acceptable carrier, wherein each of R1
and RZ is selected from hydrogen, C1-C6 alkyl, phenyl and
substituted phenyl and each of R3, R9, R5, R6, R~ and R8 is
selected from hydrogen; C1-C6 alkyl; substituted or
unsubstituted phenyl; secondary, tertiary or quaternary
amino; vitro; an ester; -COOH; -OH and an ether.
Brief Description of the Figures.
Figure 1. Table 1. Histopathological Study of
Tissues and Organs of Rats Injected with BASSBio-294.
Table 2. Toxicity Study of LASSBio-294 Injected in Mice.
Weight of mice in grams. Table 3. Blood Cell Analysis in
Mice Treated with LASSBio-294. Table 4. Blood Biochemistry
Analysis in Mice Exposed to LASSBio-294. Table 5.
Measurements of the time parameters described in Fig. 24.
The first column shows the numbers done in control
experiments (DMSO) or in 25 uM of compound 294. The last
line is the ratio between the values obtained in
compound 294 and the control experiments in Ringer plus
DMSO.
Figure 2. The papillary muscle, and bundles of
atrial and ventricular cells obtained from rat hearts were
dissected and set in an aerated chamber to enable recording
of isometric tension. LASSBio-294 was added to the chamber
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in a cumulative manner, first 10 uM and increasing to 50 uM
and recordings made after allowing 5 min. for equilibration.
Figure 3. LASSBio-294 was added to the
physiologic solution at increasing concentrations. The
isometric tension was expressed as a percent of control, as
measured prior to addition of test solution. N=9 for each
concentration.
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Figure 4. Hearts of adult rats were quickly removed and placed in an aorta
retrograde perfusion system (modified Langendorrff) for measurement of
electrocardiogram (ECG). .
Figure 5. Recordings of twitch tension in isolated hearts. Control (top),
LASSBio-.
294 (middle) and wash (bottom). ..
Figure 6. Recording of left intraventricular pressure and arterial pressure in
dogs
anesthetized and ventilated normally.
Figure 7. Bundles of left ventricular fibers were treated with saponin and
exposed
to 0.5 mM CaCl2 to enable maximal tension development (Po). The contracture
initiated
by caffeine (20 mM) was measured after treatment of the sarcoplasmic reticulum
(SR)
with a solution of pCa 6.6 in the absence or presence of LASSBio-294 (100 p.MO
for 0.5,
1.0 and 5.0 min. N=6. Calibration: Vertical, 10 mm = 20 mg load; Horizontal,
10 mm =
40 sec.
Figure 8. Ccntracture induced by caffeine in relation to loading of the
sarcoplasmic reticulum with a solution of pCa 6.6.
Figure 9. The contractual responses were obtained during exposure to 20 mM
caffeine after loading of the sarcoplasmic reticulum with pCA 6.6 in the
absence and the
presence of LASSBio-294.
Figure 10. Calibration: Vertical, 10 mm=20 mg; Horizontal, 10 mm=40 sec.
Figure 11. Concentration-response relationship for contracture induced by
caffeine
in the presence of 100 pM LASSBio-294. * p < 0.01.
TM
Figure 12. Muscle bundles were treated with 1 % Triton X-100 for 60 min and
exposed to solutions containing increasing concentrations of Ca2+ in the
presence or
absence of LASSBio-294 (100 ~M). Calibration: Vertical, 10 mm = 20 mg;
Horizontal,
10 mm = 40 sec.
Figure 13. Bundles of left ventricular muscles were treated with Triton X-100
and
exposed to increasing concentrations of Ca2+. Muscle tension expressed as % of
the
maximal response induced by 0.5 mM CaCl2 is graphed as a function of pCa. - _
Figure 14. Isolated human skeletal muscle fiber from the vastus lateralis
(sarcolemmal membrane-free) was exposed to 0.5 mM of CaCl2 to enable maximal
muscle
tension (Po). The histogram show the effect of LASSBio-294 at concentrations
of 25, 50
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and 100 N.M on induced tension in human fibers. N=6 Calibration: Vertical; 10
mm = 20
mg; Horizontal, 10 mm = 40 s.
Figure 15. Effect of 12.5 pM of compound 294 on force development in a single
muscle fiber stimulated at different frequencies. The frequency of stimulation
in Hz is
indicated under each trace. Top panel, stimulating cycle done in Ringer
without
compound 294. Middle panel, stimulating cycle done 17 min after the fiber was
bathed
with compound 294. Bottom panel, second cycle of stimulation done 17 min after
compound 294 was washed out with Ringer. Force calibration bars are 50 mg for
twitches
and 10 Hz simulations and 100 mg for 30, 60, and 90 Hz.
Figure 16. Histogram showing the effect of 12.5 ~M compound 294 and after
washing out LASSBio-294 on fractional twitch tension (Tx/To). TX represents
the twitch
tension obtained when the fiber was bathed with the solutions indicated above
each
column. The order of the columns are the order in which the simulating cycles
were
performed.
Figure 17. Time course of fatigue development. Fatigue was produced by 60 Hz,
0.8 sec tetanic stimulations repeated every 4.75 sec with a twitch elicited
2.2 sec after each
tetanic stimulation. a) single tetanus. b) repeated tetanic stimulations.
Notice the time
calibration. A) In SOpM of DMSO only B) Same as in A, but the fiber was bathed
with 50
~M of compound LASSBio-294.
Figure 18. Same as Fig. 17, but with 25 p.M of LASSBio-294. The force
calibration bar is the same for all the stimulation frequencies.
Figure 19. Same as Fig. 17, but with 50 ~M of LASSBio-294. The force
calibration bar is the same for all the stimulation frequencies.
Figure 20. Effect of 12.5 pM LASSBio-294 on peak twitch tension and maximal
10 Hz tetanic tension. The measurements were taken after the fibers had been
in each
solution for 40 min without being stimulated. Each column represents the ratio
of the
corresponding tension (TX) divided by the corresponding control tension (To)
which was
taken as 1.
Figure 21A. Effect of 12.5 (circles) and 25 (dots) ~M of LASSBio-294 on
fractional tension potentiation at different frequencies of stimulation.
Fractional tension is
expressed as the ratio of the maximal force obtained at each different
frequency (T#294)
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divided by the maximal force obtained in Ringer plus DMSO at each
corresponding
different .frequency (TDMSO).
Figure 21B. Histogram showing the effect of 12.5 pM LASSBio-294 and wash
out of the compound on twitch tension ratio (Tx/To). Twitch tension obtained
during the
first cycle of stimulation in Ringer and DMSO was taken as one (To). Tx
represents the
twitch tension obtained when the fiber was bathed with the solutions indicated
above each
column. The order of the columns is the order in which the stimulating cycles
were done.
Figure 22. Comparison of the time course of twitch tension obtained in Ringer
plus DMSO (dots) with the time course of twitch tension elicited in Ringer
plus 25 pM of
LASSBio-294 (squares).
Figure 23. Relationship between fractional twitch tension and LASSBio-294
concentration. Peak twitch tensions (T~) were measured during the second
stimulation
cycle after the cells were in different LASSBio-294 concentrations and ratio
against peak
twitch tension (T°) in Ringer without LASSBio-294 which was taken as 1.
Figure 24. Time parameters measured during the twitch tension time course. Tp
is
the time to reach peak force. To,s is the relaxation time it takes for tension
to decline from
peak tension to 50% of the peak tension. To,g is the relaxation time it takes
for tension to
80% of the peak tension.
Figure 25. Effect of LASSBio-294 on the time course of fatigue development.
Top trace control experiment, the fiber was bathed with Ringer plus DMSO.
Bottom
trace, the fiber was bathed with Ringer plus 12.5 pM of LASSBio-294. The
fibers were
stimulated with the parameters described in methods. In both panels, the first
trace is the
tension elicited with a 10 Hz stimulation frequency. Force and time
calibration bars are SO
mg and 1 sec for the 10 Hz stimulation frequencies and 100 mg and 1 min for
the
repetitive stimulation.
Figure 26. Summary of the time course of the index of fatigue development
elicited in fibers bathed with Ringer only (green dots), Ringer plus DMSO
(squares) and
Ringer plus 12.5 pM of LASSBio-294 (red dots). tNR tDMSO ~d tF,M indicate the
times it
takes for tetanic tension to start declining (fatigue) after the beginning of
the repetitive
stimulation when the fibers were bathed with Ringer only, Ringer plus DMSO and
Ringer
plus 12.5 pM respectively. Abscissas are expressed either as the number of
tetanic
stimulations or the time of stimulation.
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Figure 27. Time course of the index of fatigue development with different
concentrations of compound 294 as indicated. The control curve, done in Ringer
only, is
the average of three experiments; each of the other correspond to only one
fiber.
Figure 28. Time (in min) for tetanic force to decrease (fatigue) to 50% of the
S original tetanic force during repetitive stimulations as in Fig. 17. Left
column in SO~M of
DMSO only (~ 6 in). Right column in SO~M of LASSBio-294 (~ 9 min).
Figure 29. Time of recovery to 80% of the pre-fatigue tetanic force. Left
column
in SO~M of DMSO only (~ 90 min). Right column in 50 ~M of LASSBio-294 (~ 10
min).
Figure 30. Records of contractions induced by stimulation of the sciatic nerve
of
equilibrated rat gastrocnemius muscles and of arterial pressure. In control,
contractions
were induced by intravenous injection of DMSO (- 175 ml). LASSBio-294 was
administered in DMSO. Calibration: Horizontal, 1 cm = 24 s; Vertical 2 g = 100
mm Hg.
Figure 31. Records of contractions of rat gastrocnemius muscles induced by
stimulation of the sciatic nerve and of arterial pressure. For control,
contraction was
induced by intravenous injection of DMSA (0.04 ml). LASSBio-294 was
administered in
DMSO. As a test of the functionality of the model, the depression of muscle
contraction
induced by alloferine was recorded.
Figure 32. Effect of LASSBio-294 on 1 ~.M noradrenaline-indicated rat aorta
contraction. The drugs were added after 1 ~,M Nor maximal effect. * p=0.023
versus
control, p=0.000 versus DMSO 0.06% ** p=D.001 versus control, p=0.002 versus
DMSO
0.12%
Figure 33. Effect of LASSBio-294 on endothelium-denuded rat aorta. The drugs
were added at 1 ~.M Nor maximal effect. * p = 0. OS versus control
Figure 34. Independence of NO for the LASSBio 294-induced rat aorta
relaxation.
The drugs were added at 1 ~.M Nor maximal effect. * p=0. 01 versus DMSO 0.12%
Figure 35. LASSBio-294 effect on rat aorta pretreated with 1-NAME and
indomethacin. The drugs were added at 1 ~M Nor maximal effect. * p=0.01 versus
DMSO 0.12%
Figure 36. Reversal of LASSBio 294-induced rat aorta relaxation by 30 ~M
methylene blue. The drugs were added at 1~M Nor maximal effect. *, # p= 0.001
versus
Nor 1 fiM and Ach 1 fcM, respectively. ** p=0. 002 versusLB294100fcM.
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Figure 37. Pre-treatment with 30 ~M methylene blue abolishes LASSBio-294-
induced rat aorta relaxation. The drugs were added at 1 ~M, the Nor maximal
effect. *,
p = 0. 000 versus LB294100 lcM and Ach I,ccM, respectively.
Detailed Description of the Invention
A compound of the invention, like digitalis, produces a positive isotropic
effect on cardiac and skeletal muscle. Like digitalis, it has utility in the
treatment
of congestive heart failure. Unlike digitalis, it does not have toxic
properties at its
effective therapeutic dosage levels; and, therefor, the invention provides a
medical
advantage over that class of drugs. A compound of the invention has further
utility
in the treatment of muscle fatigue in pathological states. The invention acts
as a
calcium sensitizer in heart and skeletal muscle and it delays and shortens
fatigue of
skeletal muscle and thus also has utility in the treatment of muscle fatigue.
Muscle
fatigue is a symptom of certain pathological states such as: major injury,
cancer,
HIV infection, sepsis, Crohn's disease, ulcerative colitis, and athletic over-
training.
The invention includes the novel chemical compound having the formula
(I)
(I)
0
N~N\ S
o \ Ri R2
Each of R1 and R2 is a substituent selected from the following in any
combination:
hydrogen, alkyl of 1 to 6 carbon atoms phenyl, and substituted phenyl.
Preferably, at least
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one of Rl or R2 is hydrogen. In a preferred embodiment, each of R~ and R2 I
hydrogen;
and then the compound has formula (II):
(II)
O /N
~N \ S
\ H H
O
The compound of formula (II) is 3, 4-methylenedioxybenzoyl-2-thienylhydrazone
and has
been designated LASSBio-294.
The invention further includes pharmaceutically acceptable salts of the
compounds
of formula (I) and (II). Such salts can include the acetate, citrate,
phosphate, fumarate,
benzoate, tartrate, succinate, chlorate, sulfate, butyrate, stearate,
palmitate, lactate,
methylate, and carbonate salts. These salt forms can be prepared by reacting
the
compound with appropriate acids under standard conditions.
In accordance with the present invention, a compound of formula I, and
particularly of formula II, can be a potent, positive inotropic agent for
cardiac and skeletal
muscle. It is within the scope of the invention to use the invention
compounds) to
increase the strength of heart muscle contraction. A compound of formula II
can be used,
like cardiac glycosides, to treat congestive heart failure. In another aspect,
the invention
encompasses the effects of the compound on skeletal muscle; and its use in the
therapeutic
treatment of muscle fatigue. Muscle fatigue is a serious complication of
certain
pathological states.
The synthesis of compounds of formulas (I) and (II) from safrole is described
in
the following steps. Although the techniques used and some of the
intermediates in the
synthesis are known, the use of these techniques to produce this novel
compound is itself
novel in the art. The synthesis uses as the starting material, safrole, (4-
allyl-1, 2-
methyldioxybenzene). Safrole is the principal constituent of sassafras oil,
from which it is
readily isolated. It is also available from commercial sources.
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The compound of formula (I) and (II) is synthesized from the starting material
safrole by means of the following scheme; technical details of the synthetic
method are
provided in CHEMICAL EXAMPLE 1.
O
I I C ~ I \ cH3 0
H
O \ -~ O ~ <O
O)
i
O
N~RyN~R2)H ~ ~ ~ I OMe
O \ O \
C5) C4)
i
O ~ N~N~ S
w I RI R2
In the formula (II) compound each of RI and RZ is hydrogen.
The invention includes the novel chemical compound having the formula (I) .
Compounds of formula (I) may contain substitution on the 2 and/or 5 and/or 6
position of
the benzoyl moiety and/or on the thienyl moiety in the 2 andlor on the 3
and/or 4
positions) of the thienyl moiety. Thus, R3, R4, R5, R6, R~, and Rg may be
hydrogen,
alkyl of 1 to 6 carbon atoms, phenyl [unsubsituted or substituted], amino
[secondary,
tertiary or quaternary amino] , nitro-, ester [RCOO-], acid [-COOH , alcohol [-
OH] or
ether [F O-], to form compounds of the following structure (III):
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(III)
R6 R~
R3 0
I >-Rs
N~N\ S
o \ Ri R2
Rs
wherein each of R3-R8 is as defined above.
Pharmaceutical compositions of the invention comprise a compound of formula
(I), formula (II) and/or of formula (III). The dose range for the compound of
formula (II),
based on in vitro and in vivo tests, is one that produces between levels of
100 nM and 500
~.M in plasma, a more effective dose produces between levels of 1 ~,M and 100
~M, the
most effective dose produces between levels of 20-50 ~,M in plasma. This
plasma
concentration can be achieved in several ways depending on the therapeutic
requirements
of the patient.
Dosage forms that target alternative routes of administration are within the
scope
of the invention. The exact form of the dosage of pharmaceutical compositions
of the
invention can be established by such experimentation as one of skill in the
art would
normally undertake and by the requirements of the clinical situation to be
treated. A
parenteral form can be used for intravenous and intramuscular infusion. This
can be
supplied as either a powder or a concentrate to be used as a solution at the
time of dosing
or as an injectable, sterile solution. The diluent could be water, saline, or
a lipid based
diluent containing ethanol and buffers such as citrate to stabilize pH, and
preservatives
such as sodium benzoate and methylparaben, as required. The diluent could
further
contain such other excipients and could contain pharmaceutically acceptable
Garners as
may be desirable such as a protein carrier, including serum albumen. The
invention also
encompasses the use of solvents such as dimethyl sulfoxide (DMSO), alcohol,
ethylene
glycol or polyethylene glycol. Such solvents can be used alone or in
combination. A
nasal spray is also encompassed by the invention and could readily be
compounded by one
of skill in the art using such diluents and inactive ingredients as are
commonly used.
Solutions can be emulsions or micro-emulsions containing an oily phase, an
aqueous
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phase, and optionally a surfactant. The oily phase can contain one or more of
the
following: carboxylic acid esters, fatty acids, fatty esters, glyceryl
derivatives such as
glyceryl behenate, short, medium and long chain triglycerides, and others.
Surfactants that
may be used with the invention to produce a pharmaceutically acceptable
formulation
include polyoxyethylene sorbitan esters, ethyleneoxide propylene oxide block
co-
polymers, polyglycolized glycerides, sucrose esters, polyoxyethylene laurel
esters, and
others.
It is within the scope of the invention to formulate the pharmaceutical
compositions of the invention in tablet form. One of skill in the art can
readily formulate
such a tablet using inactive ingredients such as cellulose, microcrystalline
cellulose, corn
starch, lactose, starch, silica, dextrose and stearic acid, and such
additional ingredients as
dis-integrants, including carboxy methyl cellulose, soy polysaccharides, pre-
gelatinized
starches, and polyethylene glycol and lubricants to achieve a pharmaceutical
preparation
that can be readily manufactured. Such lubricants can include polyethylene
glycol,
leucine, glycerol behenate, magnesium stearate, or calcium stearate.
Similarly, the
pharmaceutical composition of the invention can be used as a hard or soft
gelatin capsule
in combination with suitable inactive ingredients such as lactose, cornstarch,
microcrystalline cellulose, soy polysaccharides, calcium phosphate dihydrate,
calcium
sulfate, lactose, sucrose, sorbitol, or suitable liquids or gels. The tablet
or capsule could
readily be coated. Such a coating could be an enteric coating to provide for
intestinal
release of the drug, or a neutral coating to improve stability of the tablet
or capsule.
As a drug pharmaceutical compositions of the invention can also be provided as
an
elixir for pediatric and geriatric dosing. Such an elixir could readily by
formulated by one
of skill in the art and could contain water, ethanol, solvents and surfactants
as well as a
preservative, such as methyl paraben, citric acid, and coloring and flavoring
ingredients, as
desired. A rectal suppository is also with in the scope of the invention and
could be
readily compounded using standard methods. Such a suppository could contain
waxes,
oils, lipids or gelling agent to produce a stable formulation which melts at
body
temperature. It could contain such solubilizers, surfactants, and stabilizers
as might be
required.
Other routes of administration such as buccal, sublingual, transdermal, and
subcutaneous are within the scope of the invention. Such form of
administration might be
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preferred when a treatment was needed with rapid effectiveness or when the
patient has
difficulty swallowing. Any of the dosage forms described could be formulated
as an
immediate, sustained or delayed release form. The compound could be
administered as a
single dose, multiple doses, or a continuos dose over a time period depending
on the
therapeutic need.
If the therapeutic situation requires it, it is within the scope of the
present invention
to create a pro-drug of LASSBio294. Such a molecule would combine LASSBio-294
with
a carrier molecule, for example a dipeptide, tripeptide, or any molecule
absorbed in the
intestine via transporter-mediated transport., so as to increase the
bioavailability of the
I 0 drug in oral formulation.
Various tests on whole animals and in isolated hearts and muscle fibers have
been
carried out, which show that the invention produces reactions that can be
correlated with
positive inotropic activity in humans. These tests indicate that LASSBio-294
will have
therapeutic utility in the treatment of disease states. Whole animal and in
vitro models are
known in the art to predict of the behavior of drugs in humans, including
drugs used in the
treatment of congestive heart failure.( Hoffmeister, H.M., Beyer, M.E., and
Oeipel, L.
(1997) Am. J. Cardiol. 80, 25G; Remy-Jouet, L, Cartier, F., Lesouhaitier, O.,
Kuhn, J.M.,
Fournier, A., Vaudry, H., Delaure, C., (1998) Horm. Metab. Res. 30, 341).
One such predictive test is the test of isometric tension in rat cardiac
muscle.
LASSBio-294 has a positive effect on contractility of cardiac muscle. It
increases the
isometric tension achieved by isolated bundles of muscle. Ventricular,
papillary and atrial
cardiac muscle bundles all achieved increased tension up to a 2-fold over
control, when
treated with LASSBio-294 at concentrations of up to 200uM. This test predicts
that the
invention will have positive inotropic effects in human cardiac muscle.
Predictive tests can also be performed on isolated hearts of animals. Testing
intraventricular and arterial pressure in isolated dog hearts, under pre-load,
is an animal
model for human congestive heart disease, as is well known in the art. This is
called
Langendorff's method. It allows examination of therapeutic strategies to treat
congestive
heart disease. When dog hearts were so tested, LASSBio-294 treated hearts
achieved a 50
increa.~e in intraventricular and aortic pressure compared to control or wash
out after
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67880-~~ CA 02384525 2005-02-02
treatment. The electro-cardiogram (ECG) of isolated rat heart, without pre-
load, showed
no change after treatment with LASSBio-294. When LASSBio-294 was tested on
intact,
anesthetized dogs with normal cardiac function and no pre-load, there was no
change in
pressure after injection of LASSBio-294. These tests predict that the
invention has utility -
as a medicament in the treatment of congestive heart failure (Curtis, M.J.,
(1998)
Cardiovasc. Res. 39, 194-21 ).
Another predictive test can be performed on isolated cardiac muscle fibers.
The
mechanism of action of LASSBio-294 was determined by examining the
sarcoplasmic
reticulum of cardiac muscle fibers. Tests showed that treatment with LASSBio-
294
increases uptake of Ca2+. Further tests demonstrated that LASSBio-294
increases the
storage of Ca2+ by the sarcoplasmic reticulum. Thus, the mechanism of action
of
LASSBio-294 is different from cardiac glycosides, which act by inhibiting
active transport
of Na+ and K+. LASSBio-294 increases bioavailability by increasing the storage
of
calcium in the sarcoplasmic reticulum (SR); it does not change the sensitivity
of muscle
fibers to calcium. The tests were performed on human skeletal as well as rat
cardiac
muscle fibers. These tests predict how LASSBio-294 will achieve its
therapeutic effects
in human cardiac and skeletal muscle (Weir, W.G. and Hess, P., (1984 )J. Gen.
Physiol.,
83, 395). LASSBio-294 treated skeletal muscle fibers showed increased
resistance to
muscle fatigue compared to control fibers. Neuromuscular transmission was
unaffected
by LA3SBio-294. Supportive tests were also successfully performed in
amphibians.
These tests predict a second therapeutic use of LASSBio-294 in the treatment
of muscle
fatigue (Albuquerque, E.X., Daly, J.W., Wannick, J.E. (1988) Ion Channels, 1,
95; Gallant,
E.M., Godt, R.E., and Gronert, G.A.,. ( 1980) J. Pharmacol. Exp. Ther. 213, 91
).
LASSBio-294 has very low toxic effects. This was tested in whole mouse, rat,
and
dog models (Greaves, P. (1998) Exp. Toxicol. Pathol. 50, 283). These models
are
commonly used to predict toxicity in humans (Chou, W.L., Robbie G., Chung,
S.M., Wu,
T.C., and Ma, C. (1998) Pharm. Res. 15, 1474). Major tissue types and organs
were
unaffected by LASSBio-294 given in doses greater than those which showed
isotropic
activity, and animal weights and blood cell counts were unaffected. The LD 50
in dogs is
l.Sg/kg, an amount more then 1000 fold greater than the effective dose. These
tests
predict LASSBio-294 can be dosed in humans to achieve therapeutic plasma
levels
v~rithout significant risk of toxicity.
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Definitions
Calcium sensitizer: An agent that increases the Ca2+ and/or the amount of Ca2+
available.
Congestive heart failure: Heart failure in which the heart is unable to
maintain an adequate
circulation of blood in the bodily tissues or to pump out the venous blood
returned to it by
the veins.
Langendorff's method: The experimental method using perfusion of the isolated
mammalian heart by carrying fluid under pressure into the sectioned aorta, and
thus into
the coronary system.
Muscle fatigue: Temporary loss of power to respond induced in a muscle by
continued
stimulation. This symptom is found in patients with HIV infection, cancer,
major injuries,
sepsis, Crohn's disease, ulcerative colitis, chronic fatigue syndrome, and to
some extent in
over-trained athletes.
Positive inotropic a ent: an agent that strengthens the contractility of
muscular tissue.
Pharmaceutically or therapeutically acceptable Garner: a carrier medium which
does not
1 S interfere with the effectiveness of the biological activity of the active
ingredients and
which is not toxic to the host or patient.
EXAMPLES:
CHEMICAL EXAMPLES
EXAMPLE 1
The synthesis of LASSBio-294 from safrole is described in the following steps.
Although the techniques used and some of the intermediates in the synthesis
are known,
the use of these techniques to produce this novel compound is itself novel in
the art.
The synthesis uses safrole, (4-allyl-1, 2-methyldioxybenzene) as starting
material
or reagent. Safrole is the principal constituent of sassafras oil, from which
it is readily
isolated. It is also available from commercial sources.
LASSBio-294 is synthesized from safrole following the following scheme.
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CH3 O /
H
O \ -~ O \ <O
C1) C2) C3)
i
~NHNH2 ~ /O / I OMe
\ \O \
~5) ~4)
/O / N N~ S
\O \ I H H
LASSBio-294
Numbers below each structure refer to the number before each step in the
following synthesis. The molecule numbered is the starting material for that
step and is
reacted to form the next molecule. Clearly the invention is not limited to the
following
synthesis scheme but also encompasses all such variations and modifications as
will be
clear to one of skill in the art to produce similar results.
(1) Synthesis of isosafrale To 80 g (0.49 mmol) of safrole was added 100 ml of
a 3N
solution of potassium hydroxide (KOH) in n-butyl alcohol and the reaction
mixture was
stirred at room temperature for 3 h. The mixture was poured into a solution of
12 ml of
concentrated hydrochloric acid (HCl), and 52 ml of ice water. After
neutralization with
additional concentrated HCI, the organic layer was extracted with three 35-ml
portions of
ethyl acetate. The organic extracts were treated with brine, dried over
anhydrous sodium
sulfate and concentrated under reduced pressure, furnishing a crude oily
residue.
Distillation of this residue, under reduced pressure, furnished 78.4 g (97%)
of isosafrole,
as a colorless oil.
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(2) Synthesis of Piperonal A solution of isoafrole (2 g, 12.1 mmol) in acetic
acid (10 ml) was saturated with ozone at 0°C, until the formation of a
slight blue color.
After removal of the excess of ozone by bubbling NZ through for some time, the
ozonide
was decomposed by stirring in the presence of zinc (Zn) (5 g, 0.076 atg) at
0°C for 2 h.
After filtration, ethyl acetate was added, the reaction mixture was washed
with water, and
the solvent was evaporated, following which it was dried over anhydrous sodium
sulfate,
affording 1.42 g (77%) of piperonal, as a white solid, m.p. 36-37°C.
(3) Synthesis of Methyl 3,4-Methylenedioxybenzoate To a solution of piperonal
(0.30 mmol 0.045 g) in absolute methanol (4 ml) cooled at 0°C, were
successively added
methanolic solutions (each 3 ml) of iodine (0.100 g, 0.39 mmol) and KOH (0.440
g, 7.85
mmol) at 0°C. After stirring for 1.5 hour at 0°C, small amounts
of saturated NaHS03
solution were added until the disappearance of the brown color. Next, the
methanol was
almost totally evaporated under reduced pressure. To the residue was added
water and the
desired methyl 3,4-methylenedioxybenzoate was obtained by filtration, in 90%
yield, as a
white solid m.p. 53°C.
1NMR (200 MHz) CDC13/TMS (8-ppm): 7.63 (dd, H6, Jax-8.2 Hz, Jbx=1.7 Hz); 7.44
(d,
H2, Jax=1.6 Hz); 6.82 (d, H5, Jax=8.2 Hz); 6.02 (s, O-CH2-O); 3.87 (s, O-CH3);
i3C NMR
(50 MHz) CDC13/TMS (8-ppm); 166.0 (C=O); 151.4 (C4); 147.5 (C3); 125.1 (C6);
124.0
(C~); 109.3 (C2-AR); 107.7 (CS); 101.6 (O-CH2-O); 51.9 (OCH3); M.S. (70 eV)
m/z
(relative abundance); 180 (50%); 149 (100%), 121 (20%); 91 (8%), 65 (18%); IR
(KBr)
cm ~ : 1'723 (C=O); 1289 (C-O).
(4) Synthesis of 3,4-Methylenedioxybenzoylhydrazine To a solution of 2.67 g
(14.85 mmol) of methyl 3,4-methylenedioxybenzoate in 10 ml of ethanol, was
added 15
ml of 80% hydrazine monohydrate. The reaction mixture was maintained under
reflux for
3.5 hours, when thin layer chromatography indicated the end of the reaction.
Then, the
media was poured on ice, and the resulting precipitate was filtered out,
affording the 3,4-
methylenedioxybenzoylhydrazine derivative in 70% yield, as a white solid, m.p.
170-171
oC
1H NMR (200 MHz) DMSO/TMS (8-ppm): 10.74 (s, -CONH-); 7.44 (dd, H6, Jax=8.2
Hz,
Jbx=1.6 Hz); 7.36 (s, H2) 7.17 (d, HS,J=8.2 Hz); 6.10 (s, O-CH2-O); 4.45 (s, -
NH2); iaC
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NMR (50 MHz) DMSO/TMS (8-ppm); 165.2 (C=O); 149.6 (C4); 147.3 (C3); 127.2
(C1);
121.9 (Cg); 107.9 (CZ); 107.1 (CS); 101.6 (O-CH2-O); M.S. (70 eV) m/z
(relative
abundance); 180 (17%); 149 (100%), 121 (25%); 91 (8%); 65 (19%); IR (KBr) cm
1:
3303.9 NH2); 3220 (NH); 1605 (C=O); 1262 (C-O).
(5) Synthesis of 3,4-Methylenedioxybenzoyl-2-thienylhydrazone (LASSBio-294)
To a solution of 0.150 g (0.83 mmol) of 3,4-methylenedioxybenzoylhydrazine in
absolute
ethanol (7 ml) containing two drops of 37% hydrochloric acid, was added 0.098
g (0.87
mmol) of thiophene-2-carboxaldehyde. The mixture was stirred at room
temperature for
30 minutes, after which extensive precipitation was visualized. Next, the
mixture was
poured into cold water., and the precipitate formed was filtered out and
dried. The 3,4-
methylenedioxybenzoyl-2-thienylhydrazone was obtained in 85% yield, after
recrystallization in ethanol, as yellow needle crystals, m.p. 204-205
°C.
~NMR (200 MHz) DMSO/TMS (8-ppm): 11.53 (s, -CO-NH-); 8.62 (s, =CH-); 7.65 (d,
H5,
J=5.0 Hz); 7.53 (d, H4, J=5.0 Hz); 7.50 (dd, H6, Jax=8.2 Hz, Jax=8.2 Hz,
Jbx=0.8 Hz);
7.41 (s, H2); 7.12 (dd, H3', Jax=4.8 Hz, Jbx=3.9 Hz); 6.12 (s~ O-CH2-O); 13C
NMR (50
MHz) DMSO/TMS (-ppm); 162.1 (C=O); 150.2 (C4); 147.4 (C3); 142.6(=Ch-); 139.2
(C1~); 130.8 (C3~); 128.8 (C2~); 127.9 (C4'); 127.1 (C1); 122.8 (C8); 108.0
(C2); 107.6 (CS);
101.8 (O-CH2-O); IR (KBr) cm ~: 3075 (NH); 2798 (N=CH), 1610 (C=O); 1540
(C=N);
1275 (C-O).
Detailed technical methods for performing the described steps can be found in
the
following references (Barreiro, E.J. and Lima, M.E.F (1992) J. Pharm. Sci. 81,
1219);
(Barreiro, E.J., Costa, P.R., Coelho, F.A.S. and Farias, F.M.C. (1985) J.
Chem. Res., (M)
2301.); (Yamada, S., Morizono, D., Yamamoto, K. (1992) Tetrahedron Lett., 33,
4329);
(Dias, M.L.R., Alvim, J.J.F., Freitas, A.C.C., Barreiro, E.J., Miranda, A.L.P.
(1994)
Pharm. Acta Helvetiae, 69, 163.).
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PHARMACEUTICAL EXAMPLES
EXAMPLE 1
Toxicity
a. Table 1 shows a histo-pathological table of rat tissues and organs examined
after
the animal had been treated with LASSB-294. The table shows that there were no
pathological changes in the tissues and organs examined after LASSBio-294
injection.
b. Rats were weighed before and after treatment with LASSBio 204. Table 2
shows
weights of mice in grams after injection with LASSBio294, compared to saline
and
solvent vehicle controls. Six rats were examined in each group, measurements
were made
after 1-15 days of treatment. The data show that there is no change in weight
in any group
during the test period.
c. Mice were injected with LASSBio-294 and their blood examined for cell
changes.
Experimental groups received 2 or 10 mg/kg of drug. Table shows no significant
change
in blood cell values for any group. Hematocrit levels, leucocytes and
hemoglobin were not
significantly changed by LASSBio-294 (Table 3).
d. Table 4 lists blood chemistry analysis of mice treated with LASSBio-294.
Experimental groups received 2 or 10 mg/kg of drug. Controls were saline and
solvent.
The data show no significant change in blood chemistry values for any group.
The low
values of glucose found could be due to an artifact of the technique, glucose
being a
byproduct of hemolysis. In male and female mice, subjected to long-term
treatment with
the compound, tests of hemoglobin, erythrocytes and blood biochemistry again
showed
values similar to control.
General Method
To test for possible systemic toxicological changes induced by LASSBio-294,
animals were divided into four groups, control (saline), solvent
(dimethylsulphoxide
(DMSO)/polyethylene glycol (PEG), LASSBio-294 2 mg/kg, and LSSBio-294 10
mg/kg.
Animals were injected daily for more than 14 days. The results show that
LASSBio-294
at concentrations that produce significant positive inotropic effect on the
heart muscle
produces no significant histopathological changes in the organ and tissues
studied.
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EXAMPLE 2
Isometric Tension in Cardiac Muscle
a. The test shown in Figure 2 measured the effect of LASSBio-294 on the
isometric
tension of papillary, atrial, and ventricular bundles of rat cardiac muscle.
Chart traces are
of muscle tension for each muscle group. All muscle groups show an increase in
tension
after 10 ~M and 50 p.M of LASSBio-294.
b. Figure 3 contains a graphic representation of the effect of LASSBio-294 on
isometric tension of papillary, atrial, and ventricular bundles of rat cardiac
muscle.
LASSBio-294 at concentrations of 0 ~.M, 25 ~M, 50 ~,M, 100 ~M, 200 ~,M, and
S00 pM
was used. Isometric tension is expressed as a percent of control that was
measured prior
to addition of the test solution. Isometric tension increased for each
concentration up to
200 gM. Figure 3 shows the accumulated results of nine tests for each
concentration.
General Method
The papillary muscle, and bundles of atrial and ventricular cells obtained
from rat
hearts were dissected and set in an aerated verticle chamber to enable
recording of
isometric tension. LASSBio-294 was added to the chamber in a cumulative
manner.
Recordings made after allowing 5 min. for equilibration.
EXAMPLE 3
Isolated Hearts
a. Recordings of electrocardiogram (ECG) of isolated rat heart are shown in
Figure 4
which compare control, LASSBio-294 treatment, 10 uM, 50 ~,M and 100 pM and
post-
treatment wash. No treatment had any effect on ECG. These data indicates that
LASSBio-294 does not cause abnormal ECG even at dose levels that show
increased
isometric tension.
b. Figure 5 illustrates a test of change in isometric tension induced by
LASSBio-294
in isolated hearts under preload. Concentration of LASSBio-294 in the bathing
solution is
50 ~,M, which is half of the highest concentration tested in the ECG tests
described in
Figure 4. Hearts were treated under a pre-load of 1 g; control was compared to
LASSBio-
294 and wash. LASSBio-294 treated hearts demonstrated an increase in isometric
tension
compared to control. This effect was lost in the wash out period. The pre-load
tension
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test is considered to be an animal model of congestive heart disease.
Comparing the effect
of drugs with this test is predictive of therapeutic effect of treatment in
human congestive
heart disease.
General Method
S Hearts were quickly removed and placed in an aorta retrograde perfusion
system
for measurement of ECG. Increasing concentrations of LASSBio-294 were added to
the
bathing solution. The system equilibrated for 5 min. before measurement of
isometric
tension after each addition.
EXAMPLE 4
Whole Animal Testing
Figure 6 illustrates a test that measured pressure from the hearts of intact
dogs
during LASSBio-294 application. Recordings were made of left intraventricular
pressure
and arterial pressure. These measures show no change with LASSBio-294
application
indicating that, in dogs without congestive heart failure, the compound had no
significant
inotropic effect.
General Method
Dogs were anesthetized and normally ventilated. Recordings were made before
and after application of LASSBio-294, lmg kg 1 miri 1 for 13 min.
EXAMPLE 5
Isolated Cardiac Muscle
a. Figure 7 illustrates a test that demonstrates increased uptake of Ca2+ by
the
sarcoplasmic reticulum (SR) of LASSBio-294 treated fibers after caffeine
induced
contracticn.
b. Graphic representation of the tests in Figure 7 showing the effect of
LASSBio-294
on uptake of Ca2+ by sarcoplasmic reticulum in isolated cardiac muscle with
sarcolemma
removed. Uptake follows contracture induced by caffeine measured as a function
of
loading time, shown in Figure 8.
c. Figure 9 illustrates the effect of LASSBio-294 on tension in isolated
cardiac
muscle with sarcolemma removed was measured. Measurement of contraction
induced by
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caffeine, as a function concentration of LASSBio-294 in bathing solution.
Figure 9 is a
graphic representation of the effect of increasing concentrations of LASSBio-
294 between
0 and 20 pM. Response to caffeine increases until 10~.M LASSBio-294 level.
d. This test demonstrates the effect of increasing concentrations of caffeine
from 0.05
to 10 mM on contractile tension. Figure 10 shows data comparing control to 100
~M
LASSBio-294.
e. Figure 11 shows a graphic representation of data in Figure 10 showing
isometric
tension elicited by caffeine. Contraction of LASSBio-294 treated fibers is
increased over
control at all concentrations.
f. Figure 12 illustrates the effect of LASSBio-294 on sensitivity of cardiac
fibers to
calcium. Calcium levels can induce isometric twitch. At each level of pCa the
isometric
twitch response of fibers was greater in the presence of LASSBio-294 than in
control.
g. Figure 13 provides a graphic representation of data in Figure 12.
General Method
Bundles of left ventricular muscle (130 ~.m diameter and 1-2 mm in thickness)
from Wistar rats were dissected out and mounted for measurement of isometric
tension.
The muscles were treated with saponin solution (0.5%) for 5 minutes to block
the
permeability of the sarcolemma. The membranes of the SR were then fixed with
Triton
X-100 (1%v/v) with the objective of investigating the effects of LASSBio-294
on the
sensitivity of the contractile system Ca2+. The treatment does not interfere
with the
maximal tension developed by cardiac muscle induced by CaCl2 (0.05). Caffeine
is used
to elicit contraction in muscle. Comparison of caffeine alone to caffeine in
the presence of
LASSBio-294 was measured.
EXAMPLE 6
Isolated Human skeletal muscle
Figure 14 illustrates that LASSBio-294 induces the release of Ca2+ from the
sarcoplasmic reticulum of isolated human skeletal muscle fibers with
sarcolemmal
membrane removed. The histogram shows effect of LASSBio-294 at concentrations
of 25
~M, 50 gM, and 100 ~.M on induced tension in human muscle fibers.
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General method
Isolated muscle fiber from the vastus lateralis was exposed to 0.5 mM of CaCl2
to
enable maximal muscle tension. The contractile response to caffeine was
observed after
loading of the sarcoplasmic reticulum (SR) with a solution of pCa of 7.0 for 3
min.,
LASSBio-294 evoked a contraction in the human by liberating Ca2+ from the SR.
EXAMPLE 7
Amphibian Muscle
a. Figure 15 illustrates the measurement of the effect of LASSBio-294 on
force development in a single muscle fiber stimulated at different
frequencies. Control is
Ringer's solution without compound; following is stimulation after wash with
plain
Ringer's solution.
b. The histogram in Figure 16 shows effect of 12.5 ~M LASSBio-294 on
fractional twitch tension with 10 Hz stimulation. TX/To reflects twitch
tension after
bathing, TX, divided by initial tension in Ringer's solution alone, To. Bars
are labeled and
1 S are placed right to left in the histogram, in the order performed.
c. A time course of fatigue development, in Figure 17, in single muscle fibers
comparing SOuM LASSBio-294 to solvent control demonstrated that fatigue is
produced
by 60 Hz, 0.8 sec tetanic stimulations, repeated every 4.75 seconds with a
twitch elicited
every 2.2 sec after tetanic stimulation. A is SO~,M DMSO in Ringer's solution,
B is SO~,M
LASSBio-294. The time to fatigue in LASSBio-294-treated is approximately 50 %
longer
than control.
d. Figure 18, same conditions as 7c, with 25 ~M of LASSBio-294.
e. Figure 19, same conditions as 7c, with 50 ~M of LASSBio-294.
~ Figure 20 is a histogram of TX/To, 12.5 uM LASSBio-294 compared to
DMSO.
g. Figure 21A shows the effect of two different doses of LASSBio-294 on
fractional tension potentiation. Fig 21A.
h. Figure 21B shows a histogram effect of 12.S~M LASSBio-294 on twitch
tension ratio, at 10 Hz, 30 Hz, 60 Hz, and 90 Hz.
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i. Figure 22 illustrates a comparison of time course of twitch tension in
control v~. LASSBio-294.
j. Figure 23 is a graphic representation of the relationship between
fractional
twitch tension and LASSBio-294 concentration from 0 to 100 ~M.
k. Figure 24 shows time parameters measured during twitch tension time
course. Table 5 gives the time parameters experimentally measured in vehicle
control and
LASSBio-294 12.5 ~,M.
1. Figure 25 is a graphic representation of effect of LASSBio-294 on time
course of fatigue development, comparing Ringer's solution, vehicle control
and
LASSBio-294 12.5 ~M.
m. Figure 26 shows data giving the time course of the index of fatigue
development.
n. Figure 27 is a graphic representation of the time for tetanic force to
decrease to 50% of original tetanic force comparing Ringer's solution to 12.5,
25, and 50
pM of LASSBio-294.
o. Figure 28 is a graphic representation of the time required tetanic force to
decrease (fatigue) to 50% of the pre-fatigue tetanic force.
p. Figure 29 is a graphic representation of the time to recovery to 80% of the
pre-fatigue tetanic force.
General Method:
The tests in amphibians were performed in isolated single muscle cells freshly
isolated from either the semitendinous or the tibialis anterior muscles of the
frog, rang
pipiens. The isolated muscle cells were left resting for at least half an hour
in Ringer's
solution. The fibers were then stimulated with single, low-voltage, electric
shocks. If
fibers gave brisk twitches and had no signs of membrane damage, they were
used.
Otherwise, they were discarded. Healthy fibers were transferred to the
experimental
chamber, which consisted of a 0.3 ml narrow channel where the solutions could
be
changed several times within five seconds. One tendon of the fiber was gripped
with a
small clamp and the other tendon was attached to a hook of an Ekhart;s type
force
transducer. The stimulating electrode consisted of platinum wires placed to
each side of
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the fiber. The muscle fibers were stretched 1.3 times their slack length to
reach
approximately an average sarcomere length of 1.6 Vim.
T1e fibers were stimulated with single electric pulses of 0.5 msec duration
and
variable voltage. The voltage was increased until the threshold for
contraction was
reached. This voltage was then increased by 50% and the experimental protocol
started.
To find out if LASSBio-294 has an effect on contractility, a stimulating
protocol was used
consisting of a series of single twitches elicited every 3 sec. followed by
different
frequencies of tetanic stimulation of 10 Hz, 30 Hz, and 60 Hz. The fibers
rested for three
minutes between each tetanic stimulation. When the whole series of
stimulations were
repeated, the fibers rested for 10 min. between each series of twitches and
tetanic
stimulations. Fatigue was induced by repetitive cycles of electrical
stimulation. Each
cycle consisted of a train of electric shocks delivered at 60 Hz for 0.8 sec
followed by a
single twitch after 2.2 sec and repeated every 4.15 sec. To compare different
curves of
fatigue development from different preparations, we have used a fatigue index
at different
times of stimulation by taking the ratio of maximum tetanic tension produced
during every
3rd tetanrs to the tension output in the 1st tetanus i.e. T"/T1.
The first series of tests were done with LASSBio-294 in normal Ringer and then
in
Ringer's solution plus LASSBio-294.
Solutions:
The Ringer's solution contained, in mM: NaCI, 115; KCI, 2.5; CaCl2, 1.8;
MgCl2,
0.2. pH was adjusted with phosphates to 7.2.
A stock solution of 50 mM LASSBio-294 was dissolved in Ringer's solution to
produce a 100 ~M solution by diluting 40 p,l of the original 50 mM LASSBio-294
in 20 ml
of Ringer's solution. From this 100 gM 294 compound solution final dilutions
were
prepared; e.g. for a 12.5 wM LASSBio-294 solution, 1.25 ml of the 100 ~M
solution was
dissolved in 10 ml of normal Ringer's solution.
Single twitches. As shown in Figures 15 and 16, three minutes after the fiber
was
bathed with 12.5 ~.M of LASSBio-294, twitch peak tension increased by
approximately
25% when compared with control values. However, when a second cycle of
different
frequencies of stimulation was repeated 17 min. after the fiber had been in
LASSBio-294,
twitch tension had increased by approximated 50% of the control value. Washing
away
LASSBio-294 with normal Ringer induced five min later a further twitch
potentiation
(Fig. 16). During the second stimulating cycle and 17 min. after the fiber was
in Ringer
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without LASSBio-294, twitch tension was further potentated by approximately
77% of the
control value. Afterwards, the fiber was exposed again to 12.5 g,M of LASSBio-
294.
This caused a decrease in twitch force. However, twitch tension was still
potentated above
the control valve.
EXAMPLE 8
Isolated Skeletal Muscle
a. Figure 30 illustrates a test to measure muscle tension stimulation,
comparing
LASSBio-294 treated with vehicle control. LASSBio-294 has no effect on the
neuromuscular junction.
b. Figure 31 illustrates a test to measure muscle tension after neural
stimulation,
comparing LASSBio-294 to vehicle control. LASSBio-294 has no effect on
neuromuscular transmission.
General Method
Small bundles of fibers were dissected from the extensor digitorum longus
(EDL)
or the soleus (SOL) muscles of young and adult rats. Rats were anesthetized
with ether,
and the EDL or SOL muscles were removed quickly and placed in a bicarbonate
buffer:
Krebs' solution of the following composition in mM: NaCI, 118 ~M; KCI, 4.7 ~M;
KH2PO4, 1.2 ~,M; MgCl2, 0.6 ~,M; NaHC03, 25 ~M; glucose, 11 ~,M, and CaCl2,
2.5;
equilibrated with 95 % 02-S% C02 to a pH of 7.4 at 22°C. Bundles of 100
to150 fibers
were carefully dissected under a stereo microscope. A new chamber has been
designed
which allows for bubbling the solution with the OZ, C02 gas mixture directly
in the
chamber instead of in a separate container. This avoids a continuous flow of
bathing
solution that would cause the use of a very large amount of LASSBio-294. Prior
experimentation had determined the degree of gas flow necessary for the
mammalian
preparations to survive in a healthy state during a long period of time.
EXAMPLE 9
Kinetics
Measurement of plasma concentrations of LASBio-294 revealed a retention time
(RT) of 14.59 minutes. The plasma concentration of the drug reached a maximum
at that
time and afterwards tapered off to control levels within 2 hours.
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Effects on ATPases
The compound did not affect either Ca2+-ATPase or Na+/K+-ATPase extracted
from heart muscle or gastrocnemius muscle of the rat, as well as binding was
not affected
by the compounds. Determination of phosphodiesterase direct effect was also
negative.
EXAMPLE 10
Studies of LASSBio-294 using the rat thoracic aorta.
Introduction
In vascular smooth muscle, an increase in the content of the second messenger
cyclic GNP causes vasodilatation, either by reducing the cytoplasmic Ca2+
and/or Ca2+
sensitivity of the contractile machinery. Cyclic GMP, as well as cyclic AMP,
are
degraded by phosphodiesterase isoenzymes (PDE), which have been classified
into at least
seven isoenzymes families, according to the nucleotide preferentially
hydrolyzed and to
the regulatory properties of the enzyme: PDE 1 (Ca2+ -calmodulin dependent),
PDE 2
(cyclic GMP-stimulated), PDE 3 (cyclic GNP-inhibited), PDE 4 (cyclic AMP-
specific
PDE) and PDE 5 (cyclic GMP-specific PDE). The main objective of the present
work was
to investigate the vascular actions of the recently synthesized inotropic drug
namely
LASSBio-294.
Results
LASSBio-294 100 ~,M relaxed noradrenaline-precontracted aortic rings (Fig.
32),
although more slowly than the acetylcholine- and IBMX-induced relaxation.
However, in
endothelium-denuded aorta this relaxation was abolished, indicating an
endothelial
contribution to its effect (Fig. 33). In order to establish a possible
involvement of 1
arginine/nitric oxide (NO) pathway the aortic rings were treated with the
nitric oxide
synthase (NOS) inhibitor 1-NAME (100~M) during 30 min. This treatment caused
no
difference in the relaxation induced by LASSBio-294 (Fig. 34), suggesting no
direct role
for NO. The pharmacological inhibition of both NOS and cycloxigenase pathways
caused the same level of relaxation observed in the presence of only 1-NAME
(Fig. 35).
Finally, the relaxation induced by LASSBio-294 was reversed by the addition of
30~M
methylene blue, an inhibitor of soluble guanylate cyclase (Fig. 36, 36A).
Furthermore
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when methylene blue was added before LASSBio-294, the later was not able to
induce
relaxation (Fig. 37). In preliminary assays ODQ 10 p.M also prevented the
vasorelaxation
induced by LASSBio-294 100 N.M.
Discussion
LASSBio-294 relaxed intact aortic rings in a concentration-dependent manner,
however its effect was less pronounced than either endothelium dependent and
independent relaxation produced by acetylcholine and IBMX (a rion-specific
phosphodiesterase PDE inhibitor), respectively Furthermore its effect was
abolished by
the removal of the endothelial cells. It has been recently reported that
DMPPO, an inhibitor of PDE S, also lost its effect after the removal of
endothelium. As
this relaxant effect could be due to the activation of 1-argininelNO pathway
we
investigated the influence of the NOS inhibitor on LASSBio-294 effect. I-NAME
treatment caused no difference in the relaxation elicited by LASSBio-294
indicating that
1 S basal vascular NO was not directly involved in this response, despite the
endothelial
dependence for the vaso-relaxant effect. Furthermore when the tissue was
treated with
1-NAME plus indomethacin, the relaxant effect induced by LASSBio-294 was the
same
as observed in the presence of 1-NAME alone, discarding a direct contribution
of PGIZ.
The relaxant effect of LASSBio-294 was fully reversed by the addition of
methylene blue,
an inhibitor of soluble guanylate cyclase, and prevented by the pretreatment
of this tissue
with it, which might reflect an increase in the aortic cyclic GMP and/or
cyclic AMP
content induced by LASSBio-294. In preliminary results ODQ, a selective
inhibitor of
soluble guanylate cyclase, had the same effect of methylene blue. Delpy and
colleagues
(1996) showed that in vascular smooth muscle methylene blue also impaired
isoprenaline-
induced relaxation, due to a cross-talk between these two nucleotides, where
cyclic GMP
could enhance cyclic AMP mediated vascular relaxation through the inhibition
of PDE 3.
LASSBio-294 did not inhibit directly particulate PDE 3 and 4 iso-forms present
in rabbit
and rat heart, respectively. As a conclusion, the inotropic agent LASSBio-294,
has
vasodilator activity. A non-limiting explanation is that LASSBio 294 works by
increasing
cyclic GNP and/or cyclic AMP.
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General Method
Aortic rings: Male wistar rats (300-350 g) were anaesthetized with ether and
killed by
cervical dislocation. The thoracic aorta was quickly removed, placed in
physiological
solution, cleaned of fat and connective tissue, and cut into 3-mm rings. In
some
experiments endothelium was mechanically removed by gently rubbing inverted
rings on a
cotton surface moistened with physiological solution. The rings were fixed in
an organ
bath chamber filled with physiological solution (composition (mM): NaCI 122,
KCl 5,
NaHC03 15, glucose 11.5, MgCl2 1.25, CaCl2 1.25 and KH2P04 1.25) aerated with
OZ/COZ, maintained at 37°C, and left to equilibrate for 60 min during
which the
physiological solution was changed twice. All experiments were carned out
under an
initial tension of 20 mN, and the developed active tension was measured
isometrically
using a Grass Transducer (FT03). Data were acquired and analyzed by Chart
3.4/s
software (MacLab, USA). The contraction was induced by 1 pM Noradrenaline
(NOR)
and when it reached a plateau the relaxant drugs (or solvent - time-matched
control) were
added. The rings contracted with 1 ~M NOR that relaxed 40 - 50% in response to
1 ~,M
acetylcholine were considered with intact endothelium.
Drugs: LASSBio-294 and ODQ were dissolved in 100% dimethyl sulphoxide (DMSO).
Noradrenalin, 1-NAME, IBMX, methylene blue, acetylcholine and indomethacin
were
purchased from SIGMA (USA). All drugs but indomethacin were dissolved in water
in
the day of the experiment. Indomethacin was dissolved in 5% sodium carbonate.
Statistics: Data are presented as mean ~ sem. Statistical analysis was
performed using the
Primer software. Vasodilator responses are expressed in percentage of the
maximal
contraction induced by 1 ~M NOR. Differences were considered significant at p
< 0.05
(Student's t test).
EXAMPLE 11
Composition of a Tablet
0.01 mg LASSBio
90 mg Lactose anhydrate
9.45 mg Glycerol behenate
0.5 mg Magnesium stearate
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EXAMPLE 12
Composition of a Parentarel
0.01 mg LASSBio 294
100 mg Polysorbate 90
900 mg water
EXAMPLE 13
Composition of an Injectable
1 mg LASSBio
150 mg soybean oil
50 mg diacetylated monoglyceride
50 mg . water ,
sodium hydroxide to adjust pH
EXAMPLE 14
Composition of a Tablet
0.01 mg LASSBio
10 mg magnesium stearate
90 mg microcrystaline cellulose
EXAMPLE 15
Composition of a Sublingual Solution
1 mg LASSBio
mg Tweeri 20
60 mg Linoleic Acid
10 mg - ~ water
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EXAMPLE 16
Composition of a Suppository
0.1 mg LASSBio
50 mg glycerin
150 mg glyceryl mono stearate
700 mg hydrogenated coconut oil
100 mg hydrogenated fatty acids
EXAMPLE 17
Composition of a Liquid
mg Tween 20
10 mg glycerin
10 mg propylene glycol
0.1 mg sodium benzoate
0.1 mg citric acid
50 mg sucrose
800 mg ~ water
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