Note: Descriptions are shown in the official language in which they were submitted.
CA 02305294 2000-04-10
WO 99/48483 PCT/US99/02023
(-)-PHENYLPROPANOLAMINE AS A SYMPATHOMIMETIC DRUG
FIELD OF THE INVENTION:
The present application provides pharmaceutical compositions and methods of
using
s the sympathomimetic composition of (-)-phenylpropanolamine as a
decongestant,
physiological antagonist of histamine, mydriatic agent, and for treating other
conditions
typically treated with sympathomimetic drugs. The present compositions of (-)-
phenylpropanolamine are substantially-free of (+)-phenylpropanolamine. As a
result, the
present compositions and methods have increased potency and reduced adverse
side effects.
Adverse side effects avoided by the present invention include drug
interactions, central
nervous system stimulation and depression. According to the present invention,
the potency
of the present (-)-phenylpropanolamine compositions is greater than (+)-
phenylpropanolamine alone, or a racemic mixture of (+)- and (-)-
phenylpropanolamine, and
the (-) isomer of phenylpropanolamine can be used at lower doses, for example,
to provide
improved decongestion therapy.
BACKGROUND OF THE INVENTION:
Sympathomimetic drugs are structurally and pharmacologically related to
amphetamine. They act by binding to or activating a- and ~3-adrenergic
receptors, resulting
in vascular constriction, reduced blood flow and/or reduced secretion of
fluids into the
surrounding tissues. Such receptor binding generally decreases the amount of
mucous
secreted into nasal passages. Sympathomimetic drugs are therefore used to
treat nasal
congestion, allergies and colds. In addition, because they can influence the
activity of the
central nervous system, sympathomimetic drugs are also used as appetite
suppressants.
They are known as mydriatic agents, meaning that they dilate the eye.
At the present time, sympathomimetic drugs often are sold as racemic mixtures.
However, one stereoisomer may interact more selectively than the other with
the receptors
involved in sympathomimetic action. Isolation and use of the more selective
stereoisomer
may therefore reduce not only the required dosage, but many unwanted side
effects.
Many organic compounds exist in optically active forms. This means that they
have
the ability to rotate the plane of plane-polarized light. An optically active
compound is
often described as a chiral compound. Such a chiral compound has at least one
asymmetric
carbon atom which can exist in two different, mirror-image configurations.
Compounds
which have the same composition but are mirror images of each other are called
enantiomers. The prefixes d and l, or (+) and (-), identify the direction in
which an
enantiomer rotates light. The d or (+) enantiomer is dextrorotatory. In
contrast, the 1 or (-)
enantiomer is levorotatory. A mixture of (+) and (-) enantiomers is called a
racemic
mixture. An alternative classification system for stereoisomers uses prefixes
(S) and (R).
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WO 99/48483 PCT/US99/02023
This classification system is based on the structure of the compound rather
than on the
optical activity of the compound.
Alpha-(1-aminoethyl)benzenemethanol is a molecule with two chiral, or
optically
active, centers. As such, this compound has four stereoisomers, occurring as
two pairs of
enantiomers. One pair of enantiomers is called "phenylpropanolamine" or
"norephedrine."
The other pair is called "norpseudoephedrine," "nordefrin" or
"pseudonorephrine."
The subject matter of this application relates to phenylpropanolamine
(norephedrine), which has two enantiomers, (+)-phenylpropanolamine and (-)-
phenylpropanolamine. The structures of the enantiomers of phenylpropanolamine
t 0 and noipseudoephedrine are depicted below.
According to the present invention, and the rules of structural chemistry, (-)-
phenylpropanolamine is (1R, 2S)-(-)-phenylpropanolamine.
The racemic mixture of (+)- and (-)-phenylpropanolamine, is known as a
sympathomimetic amine which can be used as a decongestant or anoretic.
However, the
15 racemic mixture has undesirable side effects -- it may be contraindicated
in patients having
glaucoma and is known to stimulate the central nervous system. 95 AMERICAN
Hosprr~.
FORMULATORY SERVICE 846. Moreover, according to the present invention, the (-)-
isomer
of phenylpropanolamine does not interact with other drugs, for example, with
antihistamines. Hence, a need exists for a composition having the beneficial
decongestant
2o and mydriatic activities of the racemic mixture of (+)- and (-)-
phenylpropanolamine,
without its undesirable side effects.
SUMMARY OF THE INVENTION:
According to the present invention, the (-) enantiomer of phenylpropanol-amine
is a
25 much more potent decongestant, and a better antagonist of histamine than is
(+)-
phenylpropanolamine but does not cause central nervous system stimulation, and
does not
interact with other drugs. Hence, the present compositions of (-}-
phenylpropanolamine can
be provided in lower dosages than the racemic mixture, and will, surprisingly,
counteract
the physiological effects of histamine as well as act as a decongestant,
without causing the
3o adverse side effects of the racemic mixture or the (+}-enantiomer.
The present invention is directed to a pharmaceutical composition containing (-
)-
phenylpropanolamine and a pharmaceutically acceptable carrier, wherein the
pharmaceutical composition is substantially-free of (+)-phenylpropanolamine.
The
pharmaceutical composition has (-)-phenylpropanolamine in a therapeutic dosage
suitable
35 for treating nasal or bronchial congestion, counteracting the physiological
effects of
histamine, dilating the pupil, treating attention deficit hyperactivity
disorder (ADHD),
suppressing the appetite and for treating other conditions typically treated
with
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sympathomimetic drugs. Upon administration to a mammal in a therapeutically
effective
amount, the present compositions have reduced side effects relative to
administration of
(+)-phenylpropanolamine, or a racemic phenylephrine mixture of (+)- and {-)-
phenylpropanolamine. Side effects caused by administration of (+)-
phenylpropanolamine,
or the racemic mixture of (+)- and (-)-phenylpropanolamine, include increased
central
nervous system stimulation and depression, and increased intraocular pressure
and drug
interactions, for example, with antihistamines.
The present invention is also directed to a method of relieving nasal and
bronchial
congestion which includes administering a therapeutically effective amount of
(-}-
to phenylpropanolamine to a mammal, wherein such (-)-phenylpropanolamine is
substantially-free of (+}-phenylpropanolamine. This method permits
administration of less
drug than a method which includes administration of a racemic mixture of (+)-
and (+)-
phenylpmpanolamine, or a composition of (+)-phenylpropanolamine alone. In this
embodiment, a therapeutically effective amount of (-)-phenylpropanolamine is a
dosage
suitable for treating nasal and/or bronchial congestion.
The present invention is also directed to a method of antagonizing the
physiological
effects of histamine which includes administering a therapeutically effective
amount of (-)-
phenylpropanolamine to a mammal, wherein such (-)-phenylpropanolamine is
substantially-free of (+)-phenylpropanolamine. According to the present
invention, (-)-
2o phenylpropanolamine is surprisingly a more potent physiological antagonist
of histamine
than is (+)-phenylpropanolamine. This method has less side effects than a
method which
includes administration of a racemic mixture of (+)- and (+)-
phenylpropanolamine, or a
composition of (+)-phenylpropanolamine alone. In this embodiment, a
therapeutically
effective amount of (-)-phenylpropanolamine is a dosage suitable for relieving
the
physiological effects of histamine, for example, nasal congestion,
inflammation and allergic
responses.
The present invention is further directed to a method of dilating the pupil
which
includes administering a therapeutically effective amount of (-)-
phenylpropanolamine to a
mammal, wherein the (-)-phenylpropanolamine is substantially-free of (+)-
3o phenylpropanolamine. The (-)-phenylpropanolamine is preferably administered
topically.
In this embodiment, a therapeutically effective amount of (-)-
phenylpropanolamine is a
dosage suitable for dilating the eye pupil. This method has less side effects
than does a
method which includes administration of a racemic mixture of (+)- and (-)-
phenylpropanolamine, or (+)-phenylpropanolamine alone, in that it avoids
substantial
increases in intraocular pressure.
The present invention is further directed to a method of treating attention
deficit
hyperactivity disorder which includes administering a therapeutically
effective amount of (-
-phenylpropanolamine to a mammal, wherein the (-)-phenylpropanolamine is
substantially-
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WO 99/48483 PGT/US99/02023
free of (+)-phenylpropanolamine. In this embodiment, a therapeutically
effective amount of
(-)-phenylpropanolamine is a dosage suitable for relieving the symptoms of
attention deficit
disorder. This method has less side effects than does a method which includes
administration of a racemic mixture of (+)- and (-)-phenylpropanolamine, or
(+)-
phenylpropanolamine alone.
The present invention is further directed to a method of suppressing the
appetite
which includes administering a therapeutically effective amount of
(-)-phenylpropanolamine to a mammal, wherein the (-)-phenylpropanolamine is
substantially-free of (+)-phenylpropanolamine. In this embodiment, a
therapeutically
effective amount of (-)-phenylpropanolamine is a dosage suitable for
suppressing or
depressing the appetite. This method requires a smaller dosage and has less
side effects
than does a method which includes administration of a racemic mixture of (+)-
and (-)-
phenylpropanolamine, or (+)-phenylpropanolamine alone.
The present invention is also directed to a method of treating conditions
typically
t 5 treated with sympathomimetic drugs, which includes administering a
therapeutically
effective amount of (-)-phenylpropanolamine to a mammal, wherein such (-)-
phenylpropanolamine is substantially-free of (+)-phenylpropanolamine. This
method
requires administration of less drug than does a method which includes
administration of a
composition of (+)- and (-)-phenylpropanolamine, or of (+)-phenylpropanolamine
alone.
In this embodiment, a therapeutically effective amount of (-)-
phenylpropanolamine is a
dosage suitable for treating the condition typically treated with a
sympathomimetic drug.
BRIEF DESCRIPTIONS OF THE DRAWINGS:
Figure 1 depicts the mean arterial blood pressure after administration of a
"75%
dose" of the indicated enantiomers, with (stippled bar) and without (solid
bar) histamine
treatment. The "75% dose" is 75% of the dosage required to produce a 10%
increase in
mean arterial blood pressure. As indicated, (-)-phenylpropanolamine causes a
lesser
increase in blood pressure at this 75% dose than is observed for several
enantiomers which
are commercially available as decongestants.
Figure 2 provides the observed percent nasal airway pressure after a 75% dose
of the
different enantiomers, with (stippled bar) and without (solid bar) histamine
treatment. The
"75% dose" is 75% of the dosage required to produce a 10% increase in mean
arterial
blood pressure. Even at this reduced dosage, the (-) enantiomer of
phenylpropanolamine is
more effective than the (+) enantiomer.
DETAILED DESCRIPTION OF THE INVENTION:
The present invention provides pharmaceutical compositions of (-)-
phenylpropanolamine and a pharmaceutically acceptable carrier that are
substantially free of
4
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WO 99/48483 PCT/US99/02023
(+)-phenylpropanolamine. The present invention also provides methods of using
such (-)-
phenylpropanolamine compositions for treating colds, nasal congestion,
bronchial
congestion, histamine-related inflammations, allergies, attention deficit
disorder, and for
other conditions typically treated with sympathomimetic drugs. The present
invention also
provides methods for dilating the pupil. Accordingly, the present compositions
of (-)-
phenylpropanolamine can be used as decongestants, bronchodilators,
physiological
antagonists of histamine, agents for treating attention deficit disorder,
mydriatric agents,
and the like.
The structures of the free amines of (+)-phenylpropanolamine and (-)-
phenylpropanolamine are: CAS #: 37577-28-9, [S-(R*, S*)]-alpha-CAS #: 492-41-
1, (i-
aminoethyl)benzenemethanol, [R-(R*, S*)]-alpha-(1-aminoethyl)benzenemethanol.
As used herein, the term "substantially free of (+)-phenylpropanolamine" means
that
the composition contains at least 90% (-)-phenylpropanolamine and 10% or less
(+)-
phenylpropanolamine. In a more preferred embodiment, "substantially free of
(+)-
phenylpropanolamine" means that the composition contains at least 95% (-)-
phenylpropanolamine and 5% or less (+)-phenylpropanolamine: Still more
preferred is an
embodiment wherein the pharmaceutical composition contains 99% or
more (-)-phenylpropanolamine and 1 % or less (+)-phenylpropanolamine.
According to the present invention, compositions of (-)-phenylpropanolamine
which
2o are substantially free of (+)-phenylpropanolamine are also substantially
free of the adverse
side effects related to administration of (+)-phenylpropanolamine. Such
adverse side
effects include but are not limited to: central nervous system stimulation,
central nervous
system depression and drug interactions. As a result, administration of the
present
compositions of (-)-phenylpropanolamine produces reduced side effects relative
to the
administration of the racemic phenylpropanolamine mixture or the (+)-
stereoisomer of
phenylpmpanolamine.
The (-)-phenylpropanolamine of this invention may be prepared by known
procedures. Methods for separating the stereoisomers in a racemic mixture are
well-known
to the skilled artisan.
3o The present invention also provides pharmaceutically acceptable salts of (-
)-
phenylpropanolamine. For example, (-)-phenylpropanolamine can be provided as a
hydrochloride, bitartrate, tannate, sulfate, stearate, citrate or other
pharmaceutically
acceptable salt. Methods of making such pharmaceutical salts of (-)-
phenylpropanolamine
are readily available to one of ordinary skill in the art.
As used herein, "pharmaceutically acceptable carrier" includes any and all
solvents,
dispersion media, coatings, isotonic and absorption delaying agents,
sweeteners and the like.
The pharmaceutically acceptable carriers may be prepared from a wide range of
materials
including, but not limited to, diluents, binders and adhesives, lubricants,
disintegrants,
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WO 99/48483 PGT/US99/02023
coloring agents, bulking agents, flavoring agents, sweetening agents and
miscellaneous
materials such as buffers and adsorbents that may be needed in order to
prepare a particular
therapeutic composition. The use of such media and agents for pharmaceutical
active
substances is well known in the art. Except insofar as any conventional media
or agent is
incompatible with the active ingredient, its use in the therapeutic
compositions is
contemplated.
According to the present invention, (-}-phenylpropanolamine does not interact
with
other drugs, whereas the (+) enantiomer of phenylpropanolamine does interact
with drugs.
For example, when (-)-phenylpropanolamine is administered with an
antihistamine like
to triprolidine, no effect is observed. However, when (+)-phenyl-
propanolamine is administered with triprolidine, significant changes in
central nervous
system activity (stimulation or depression) are observed.
Due to the lack of drug interaction which (-)-phenylpropanolamine exhibits,
supplementary active ingredients, such as additional antihistamines and
decongestants, can
15 be incorporated into the present compositions. The amount of the added
antihistamine or
decongestant present in the pharmaceutical composition will depend upon the
particular
drug used. Typical antihistamines include: diphenhydramine; chlorpheniramine;
astemizole; terfenadine; terfenadine carboxylate; brompheniramine;
triprolidine; acrivastine;
and loratadine
2o The present invention further contemplates a method of relieving nasal and
bronchial congestion which comprises administering a therapeutically effective
amount of
(-)-phenylpropanolamine, which is substantially free of (+)-
phenylpropanolamine.
Administration of (-)-phenylpropanolamine avoids many of the side effects
related to
administering (+)-phenylpropanolamine. Side effects which can be reduced or
avoided by
25 using the present methods include central nervous system stimulation,
central nervous
system depression and drug interactions.
According to the present invention, (-)-phenylpropanolamine is surprisingly
more
effective as a decongestant and as a physiological antagonist of histamine
than is (+)-
phenylpropanolamine. As a physiological antagonist of histamine, (-)-
phenylpropanolamine
3o counteracts the physiological effects of histamine. Histamine can cause
physiological
effects like nasal congestion, bronchial congestion, inflammation and the
like. This present
invention contemplates (-)-phenylpropanolamine to counteract all of these
histamine-related
physiological responses.
The present invention also contemplates a method of treating histamine-
related
35 inflammation and/or sinus congestion which comprises administering a
therapeutically
effective amount of (-)-phenylpropanolamine. The pharmaceutical compositions
of (-)-
phenylpropanolamine used for this method are substantially-free of (+)-
phenylpropanolamine and induce less side effects than does administration of a
composition
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WO 99/48483 PCT/US99/02023
containing (+)-phenylpropanolamine, or the racemic mixture of
phenylpropanolamine.
The present invention further contemplates a method of dilating the pupil
which
comprises administering a therapeutically effective amount of (-)-
phenylpropanolamine to
the eye. According to the present invention, administration of (-)-
phenylpropanolamine
induces less intraocular pressure than does administration of (+)-
phenylpropanolamine or of
the racemic mixture of phenylpropanolamine. The pharmaceutical compositions of
(-)-
phenylpropanolamine used for this method are substantially-free of (+)-
phenylpropanolamine.
According to the present invention, a therapeutically effective amount of (-)-
to phenylpropanolamine is an amount sufficient to relieve the symptoms of a
condition which
can be treated by a sympathomimetic drug. In one embodiment, an amount
sufficient to
reduce the symptoms of a condition which can be treated by a sympathomimetic
drug is an
amount of (-)-phenylpropanolamine sufficient to bind or activate an adrenergic
receptor, for
example, an a- or a (3-adrenergic receptor. When the condition is nasal
congestion, the
therapeutically effective amount is the amount needed to reduce nasal
congestion. When
bronchial congestion is the condition, the therapeutically effective amount is
the amount
needed to reduce bronchial congestion or provide bronchodilation. When
inflammation
and/or allergic reaction is the condition, the therapeutically effective
amount is the amount
needed to counteract the physiological effects of histamine. When eye pupil
dilation is the
2o desired, such a therapeutically effective amount of (-)-phenylpropanolamine
is an amount
of (-)-phenylpropanolamine sufficient to dilate the pupil. Preferably, such a
pharmaceu-
tically effective amount also produces less side effects than are observed
upon
administration of (+)-phenylpropanolamine, or a racemic mixture of (+)- and
(-)-phenylpropanolamine. The skilled artisan can readily determine the
necessary
therapeutically effective amounts for treating these conditions, particularly
in light of the
teachings provided herein.
The pharmaceutical compositions of the present invention contain (-)-
phenylpropanolamine in a therapeutically effective amount that is sufficient
to provide
decongestion, bronchodilation, antagonize the effects of histamine, produce a
mydriatic
response or treat attention deficit hyperactivity disorder, while having less
side effects than
would similar doses of (+)-phenylpropanolamine or the racemic mixture of (+)-
and (-)-
phenylpropanolamine. Such a therapeutically effective amount would be about
0.01
micrograms (p,g) to about 50 milligrams (mg) per kilogram of body weight, and
preferably
of about 0.1 p,g to about 10 mg per kg of body weight. More preferably, the
dosage can
range from about 1.0 p,g to about 1 mg per kg of body weight. Even more
preferably, the
dosage can range from about 5.0 ~,g to about 50 pg per kg of body weight.
Dosages can be
readily determined by one of ordinary skill in the art and can be readily
formulated into the
subject pharmaceutical compositions.
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The subject (-)-phenylpmpanolamine may be administered by any convenient
route.
For example, (-)-phenylpropanolamine may be inhaled, ingested, topically
applied or
parenterally injected. The subject (-}-phenylpropanolamine may be incorporated
into a
cream, solution or suspension for topical administration. (-)-
Phenylpropanolamine is
preferably inhaled or administered orally or topically. The skilled artisan
can readily
determine the route for a specific use.
The following examples fiuther illustrate the invention.
FXAMPLF 1
10. oc -Adrenergic and (3-Adrenergic Receptor Binding Studies
Many physiological processes are mediated by the binding of chemical compounds
to 1, 2 and receptors. For example, many compounds which reduce nasal
congestion
bind to , and 2 receptors and some reduce bronchial congestion by binding to
receptors.
Accordingly, a compound that binds to ,, 2 and/or 2 receptors may be an
effective nasal or
bronchial decongestant.
More specifically, 2 adrenergic receptors, concentrated on precapillary
arterioles in
the nasal mucosa, induce arteriolar vasoconstriction when activated by a
sympathomimetic
compound. Such vasoconstriction decrease blood flow through those vessels and
reduces
excess extracellular fluid associated with nasal congestion and a runny nose.
On the other
2o hand, , adrenergic receptors are concentrated on postcapillary venules in
the nasal mucosa.
Binding to t receptors induces venoconstriction, which also reduces nasal
congestion.
Compounds that bind to 2 receptors may also help relieve the symptoms of nasal
congestion because 2 receptor binding is related to increased bronchodilation
and reduced
airway resistance.
The binding of (-)-phenylpropanolamine to ,, 2 and 2 various receptors was
compared to the receptor binding of (+}pseudoephedrine, (-)ephedrine, (-)-
phenylephrine
and (+)-phenylpropanolamine. The (+) isomer of pseudoephedrine is a known
decongestant, sold under the trade name SUDAFED~. (-)-Phenylephrine (Neo-
Synephrine~ and (-)-ephedrine are also known to be effective decongestants.
Methods:
Membrane Preparations.
PULMONARY ALPHA-1 AND BETA-2 RECEPTORS: The lungs of mongrel dogs were
separated from cartilaginous airways and major blood vessels, weighed, chopped
and placed
into 10 volumes of ice-cold buffered sucrose (SOmM Tris-HCl pH 7.4, 1mM EGTA,
0.32M
Sucrose). The tissue was then homogenized in a Polytron tissue homogenizes.
The
homogenate was filtered through two layers of cheesecloth, and the filtrate
was dounced
three times using a Con-Turque Potter homogenizes. The dounced filtrate was
centrifuged
8
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at 1000 x g for 15 min at 4 C. The supernatant was recentrifuged at 30,000 x g
for 30 min at
4 C and the resulting pellet was washed and resuspended in 10 volumes of Tris
buffer
(SOmM Tris HC1, pH 7.4, 1mM EGTA) and incubated at 37 C for 30 min in a
shaking
water bath. The suspension was centrifuged at 4 C at 30,000 x g for 30 min and
the
resulting pellet washed in 10 volumes of Tris buffer: The final pellet was
resuspended in
0.5 volume of SOmM Tris HCI, pH 7.4, 1mM EGTA, 25mM MgCl2. Protein
concentration
was then determined by the Lowry method and the final suspension was adjusted
to 10 mg
of protein/ml, aliquoted and stored at 90 C. Particulates were also prepared
for 2 receptors
using the identical procedure except the final protein concentration was
adjusted to 0.1 g/ml.
1o
BRAIN ALPHA-2 RECEPTORS: Membranes of mongrel dogs were harvested from the
canine frontal cortex and prepared as described for lung except that the final
membrane
protein concentration was adjusted to 0.5 mg/ml.
15 Binding Assays.
ALPHA-1 BINDING, 3H-PRAZOCIN: Canine lung membrane preparations (SOOug
protein/100 ul) were incubated with 3H-Prazocin (77.9Ci/mmol) for 60 min at 25
C in a
final volume of 0.25 ml of buffer (50 mM Tris-HCl/1mM EGTA, pH 7.4). Each
experimental point was determined in triplicate. Nonspecific binding was
determined
2o separately for each concentration point using 10 pM phentolamine. The final
concentration
of 3H-Prazocin was 0.7-1.1 nM in competition studies and between 0.1 and IOnM
in
saturation experiments. All binding assay incubations were terminated by rapid
dilution
with 2 ml of ice-cold wash buffer (SOnM Tris-HCI, pH 7.4) and filtration
through Whatman
GFB filters using a Brandel receptor-binding harvester. The filters were
washed twice
25 more with 4 ml of wash buffer and then added to 6 ml Cytoscint (ICN, Costa
Mesa CA) for
liquid scintillation counting (Barnes et al., 1983). In all experiments less
than 17% of the
added radio ligand was bound, and specific binding was about 65-70% of total
binding.
ALPHA-2 BINDING P-1~IODOCLONIDINE. Canine brain membranes (50 ug
3o protein/100 ul) were incubated with p-iodoclonidine (2200 Ci/mmol) for 120
min at 25 C in
a final volume of 0.25 ml. Nonspecific binding was determined in separate
incubations in
the presence of 10 uM phentolamine. The final concentration of p-iodoclonidine
was 44-45
pM in competition studies and between 50 pM and 10 nM in saturation
experiments. Bound
and free were separated and the bound quantitated as described above for the
ICYP assays.
35 An average of 6% of radioligand was bound, and specific binding was about
91 % of total
binding.
BETA-2 BINDING, ~25IODOCYANOPINDOLOL (~25ICYP). Canine lung membranes (10
9
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ug protein/ 100 ul) were incubated with ~25ICYP (2200 Cilmmol) for 110 min at
30 C in a
final volume of 0.25 ml. Nonspecific binding was determined in separate
incubations in the
presence of 2 pM dipropranolol. Each experimental point was determined in
triplicate. The
final concentration of ~ZSICYP was 8-12 pM in competition studies and between
2 and 200
pM in saturation experiments. Incubations were terminated as described above
for the 1
assays. Filters were placed into polyethylene tubes and the bound ligand was
determined by
gamma spectrometry (Sano et al., 1993). An average of 27% of radio ligand was
bound,
and specific binding was about 90% of total binding.
All data were analyzed with the aid of microcomputer nonlinear curve fitting
1o programs (PRISM 2.0, Graphpad Software, San Diego CA).
Results:
The receptors resident in each of the three membrane preparations were
evaluated by
standard saturation analysis following the addition of increasing
concentrations of the
15 appropriate radioligand. In the case of the a ,- and (3 2- assays the
mathematical analysis
was consistent with a one site fit. The a 2-receptor analysis was best fit as
two sites, one
high and one iow affinity.
The radio ligand added for subsequent a 2-displacement assays was adjusted to
evaluate only the high affinity receptor. Contributions from p-iodoclonidine
binding to
2o imidazoline receptors in the a 2- displacement assay were evaluated with
epinephrine.
Epinephrine easily displaced all bound p-iodoclonidine which indicates that at
the
concentrations employed, p-iodoclonidine labeled few if any imidazoline
receptors.
Similarly, with the ~i 2-assay, contributions from the binding of ICYP to (3 ,
sites was
evaluated with the ~i ,-selective antagonist, atenolol. Atenolol was largely
ineffective in
25 displacing ICYP from pulmonary membranes indicating little if any [3 ,
binding within the
assay. All subsequent analyses with displacement by individual test compounds
used the
Kd determined from the saturation analysis since it is generally considered a
more reliable
estimate of the true equilibrium dissociation constant.
3o Table 1 provides the binding characteristics of the a ,-receptors in the
membrane
preparation for prazocin. The Kd is the apparent equilibrium dissociation
constant for
prazocin. The BMAX is the number of a ,-receptor binding sites for prazocin in
this
membrane preparation expressed as femtomoles per mg protein.
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TABLE 1
a ,-Receptor
Binding Characteristics
(canine lung
membranes)
Measure Summary
Scatchard Analysis
Kd 0.84 nM
BMAX 55
Saturation Analysis
Kd 0.73 nM
BMAX 53
Table 2 provides the binding characteristics of the a 2-receptors in the
membrane
preparation for p-iodoclonidine. The Kd is the apparent equilibrium
dissociation constant
for p-iodoclonidine. The BMAX is the number of a 2-receptor binding sites for
p-
iodoclonidine in this membrane preparation expressed as femtomoles per mg
protein. Note
to that the two site data from the Saturation Analysis is more reliable than
the Scatchard
Analysis because the Scatchard Analysis assumes only one site. In order to
obtain both
values from the Scatchard plots, the points in the transition zone were
arbitrarily divided and
assigned to high and low affinity plots.
t1
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WO 99/48483 PCT/US99/02023
TABLE 2
a 2-Receptor Binding
Characteristics
(canine cerebral
cortex membranes)
Measure Summary
Scatchard Analysis
Kd, (high affinity) 0.15 nM
Kd2 (low affinity) 0.87 nM
BMAX, (high affinity)67
BMAX2 (low affinity)120
Saturation Analysis
Kd, (high affinity) 0.15 nM
Kd2 (low affinity) 3.01 nM
BMAX, (high affinity)57
BMAX2 (low affinity]73
Table 3 provides the binding characteristics of the (3 2-receptors in the
membrane
preparation for ~25iodocyanopindolol (ICYP). The Kd is the apparent
equilibrium
dissociation constant for ICYP. The Bl~tvc is the number of [3 2-receptor
binding sites for
ICYP in this membrane preparation expressed as femtomoles per mg protein.
to
TABLE 3
~i2-Receptor
Binding
characteristics
(canine
lung membranes)
Measure Run 1 Run 2 Summary
Scatchard
Analysis
Kd 9.9 pM 7.8 pM 8.9 pM
BMAX 150 139 145
Saturation
Analysis
Kd 9.6 pM 9.3 pM 9.5 pM
BMAX 149 142 146
The concentration of test drug required to inhibit 50% of specific prazocin, p-
15 iodoiclonidine or ICYP binding (ICsa) is provided in Table 4. The Ki values
of a ,, a 2 and
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~i2-receptors for each drug are also provided in Table 4, where the Ki is ICso
= ( 1 + I/Kd).
The variable, I, is the concentration of tracer added and the variable, Kd, is
the equilibrium
dissociation constant empirically determined for this receptor population. In
general, lower
ICso and/or K; values mean that less drug need be administered to effectively
bind to these
a~, a 2 and (32-receptors.
TABLE 4
Alpha-1 Alpha-2 Beta-2 Ki-Ratio
Drugs ICso K; ICso K; ICso K; al/a al/(I ~i2/
2 2 a2
(+)- 691 299 28 21 502 220 14.23 1.35 10.48
Pseudoephedrin
a
(-)-Ephedrine109 47 0.77 0.59 12 5 79.67 9.40 8.47
(-)- 7 3 0.02 0.01510 5 200.00 0.60 333.33
Phenylephrine
(+)- 1464 612 15 12 486 215 51.00 2.85 17.92
phenylpropanola
-mine
(-)- 116 48 0.31 0.24 SO 22 200.00 2.18 91.67
phenylpropano
1-amine
i 0 These data show that (-)-phenylpropanolamine has significantly lower ICso
and/or K,
values, than does (+)-phenylpropanolamine and several known decongestants.
EXAMPLE 2
(-)-Phenylpropanoiamine Induces Pupil Dilation
Without Increasing Intraocular Pressure
The induction of pupil dilation or mydriasis by (-)-phenylpropanolamine was
2o compared to the pupil dilation caused by (+)pseudoephedrine, (-)ephedrine,
(-)-
phenylephrine and (+)-phenylpropanolamine. The (-) enantiomer of phenylephrine
is
known to be a mydriatic agent.
Methods:
Enantiomers (-)-phenylpropanolamine, (+)-pseudoephedrine, (-)-ephedrine,
(-)-phenylephrine and (+)-phenylpmpanolamine were evaluated for their efficacy
in
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producing mydriasis and for their effects on intraocular pressure (IOP). These
agents were
administered topically as either 1 and 2% solutions in buffered saline.
Pupillary diameter
and IOP were measured in all animals over a six how time period during the day
to
minimize diurnal variations in IOP and pupil diameter.
The experiments were performed on adult male New Zealand white rabbits
weighing
3.0-6.0 kg. All rabbits were caged individually and maintained on a l2hr/l2hr
light/dark
schedule with free access to food and water. All animal procedures were in
conformity
with the ARVO Resolution on the Use and Care of Animals in Research. All
treated rabbits
had served as controls by having received a saline treatment on a different
day.
1o Drug or saline-control solutions were applied to the superior aspect of the
globe in a
volume of 25 ~,1 and allowed to spread over the cornea and sclera, while a
conjunctival
trough was formed by retracting the lower eyelid for approximately 30 seconds.
Only one
eye received drug treatment. The contralateral eye served as a control. Saline
(or PBS) and
drug treated rabbits were treated and observed simultaneously. A single dose
was given at 0
time and IOP and pupil diameter measured at -1.0, -0.5, 0.5, l, 3 and S hrs
post-treatment.
IOP measurements were recorded with an Alcon Applanation Pneumotonograph
(Surgical Products Division, Alcon Laboratories, Inc., Ft. Worth, TX) in
rabbits placed in
Lucite restraining cages. Initial topical application of a two drop 0.5%
proparacaine HCl
(Ophthetic~, Allergen Pharmaceuticals, Inc.) was performed on each rabbit.
2o Pupil diameter was measured visually at the point of the greatest
horizontal diameter
with a transparent millimeter ruler. All measurements were made under the
identical
ambient lighting conditions.
Mean and Standard Error values were used to construct time-response and dose-
response curves for the treated and contralateral eye of research rabbits. The
data were
analyzed statistically by an analysis of variance and a Bonferoni's test for
significance.
P<0.05 was the accepted level of significance.
Results:
Although some variation in baseline IOP was noted among the total rabbits
tested,
there were no significant changes in IOP or pupil diameter (PD) in the saline
control groups
(Tables 5-7) during the six hour time period selected for drug testing.
The adrenergic agonist (+)-pseudoephedrine is known to be an active
sympathomimetic amine which has both a- and (i- agonist activity. In this
study, (+)
pseudoephedrine produced mydriasis in only the treated eye. A slight acute
elevation in
IOP in the treated eye was observed following 1 % and 2% topical application
of (+)-
pseudoephedrine. A delayed elevation in IOP was also observe in the
contralateral eye.
(-)-Ephedrine increased IOP but had no effect on pupil diameter.
(-~Phenylephrine, which is clinically available as a mydriatic agent, produces
both
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an elevation in IOP and an increase in pupil diameter.
At a 1 % topical dose, (-)-phenylpropanolamine caused significant mydriasis
and
only mild IOP. Even higher doses (-)-phenylpropanolamine resulted in only mild
IOP
elevation, while causing marked mydriasis. (+)-Phenylpropanolamine was less
effective
than (-)-phenylpropanolamine. (Tables 5-7).
TABLE 5 - I OP in
mmIig
Time in Hr. -1 -0.5 0.5 1 3 5
SALINE U 2710.925f0.4 2611.5 25f 2710.92611.2
1.4
( 15) T 26f0.92511.1 2611.1 25~ 27f0.826f0.8
1.3
SALINE U 200.7 19f0.9 2011.0 19f 20f 1911.1
I .0 1.0
(15) T 1910.81810.9 180.8 1710.7 1910.91811.1
DRUG (1%)
(+)-Pseudoephedrine1911.01811.6 18f2.0 1911.2 20f2.021f0.9
U 20+0.220f1.7 I9t2.3 22+1.4 2112.02312.3
T
(-)-Ephedrine 19f 17f 20f I 9~ 160.9 1 Sf I
U I 1.0 1.9 I .2 .0
.7
T 2212.02310.9 232.5 2611.0 2410.62310.9
(-)-Phenylephrine16+ 1 St2.118+ 19+ 20~ 1911.7
1.0 1.6 1.3 1.9
U 20+1.61611.5 240.6 24+1.0 231.5 2111.9
T
(+)- 2310.92211.3 21 t 221 2311.52211.0
1.2 I .1
Phenylpropanolamine2511.22211.7 2510.7 2411.2 22~ 2110.7
1.2
U
T
(-)- 2312.12112.3 2712.0 250.9 25~ 22f 1.3
1.9
Phenylpropanolamine25+0.22211.6 2512.7 2512.2 232.6 2413.4
U
T
DRUG (2%)
(+}-Pseudoephedrine20f 16~ 18f2.3 I 72.1 1811.222f 1.4
1.0 10
U 2411.118f 260.8 23~ 2212. 2312.4
1.4 1.3 I
T
(-)-Ephedrine 1711.219f0.9 1911.4 1911.9 180.7 1911.5
U
T 16f2.016f0.7 1611.0 1 St0.4170.7 1910.8
(-)-Phenylephrine17~ 1711.9 2012.0 202.4 I 4t 142.0
1.9 1.7
U 1811.615~ 1210.8 1412.0 160.8 14f 1.3
1.5
T
(+)- 2111.020f1.4 19+1.5 170.3 1711.015f0.7
Phenylpropanolamine2411.223f 240.9 270.9 2011.81 St 1.0
1.4
U
T
(-)- I 712.11711.4 1912.9 2011. 22f 1911.2
7 1.9
Phenylpropanolamine1812.016~ 21 t 21 t 21 20f2.1
1.3 I .9 1.9 t
I
.7
U
T
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Results are as mean t S.E. of five-six rabbits per drug.
U = Untreated contralateral eye
T = Drug treated eye
TABLE 6 - Pupil
Diameter in
mm
Time is Hr. -1 -0.5 0.5 1 3 5
SALINE U 50.2 St0.2 S+0.2 Sf0.2 5+0.2 St0.2
( 15) T St0.2 5+0.2 50.2 St0.2 Sf0.2 St0.2
SALINE U St0.2 St0.2 St0.2 St0.2 Sf0.2 St4.2
( 15) T St0.2 St0.2 St0.2 St0.2 St0.2 5+0.2
DRUG (1 %)
(+)-Pseudoephedrine610.4 6+0.4 610.4 6f0.4 6f0.4 610.4
U 710.2 7f0.2 810.2 8f0.2 8f0.2 810.2
T
(-)-Ephedrine 610.4 610.4 610.4 610.4 6f0.4 6f0.4
U
T 6+0.4 6f0.4 6+0.4 6+0.4 610.4 610.4
(-)-Phenylephrine610.4 610.4 60.4 6+0.4 6f0.4 610.4
U 6+0.4 6+0.4 7f0.4 910.4 9f0.4 9f0.4
T
(+)- 7f0.3 7+0.3 7+0.3 7+0.4 710.4 7+0.3
Phenylpropanolamine7f0.3 7+0.3 810.3 810.4 8f0.4 7f0.5
U
T
(-)- 710.4 70.4 7t 7f 1.2 7t 1.2 7t 1.2
1.2
Phenylpropanolamine710.5 7f0.5 lOf0.21010.2 lOf0.2 10+0.2
U
T
DRUG (2%)
(+)-Pseudoephedrine610.4 610.4 610.4 6f0.4 610.4 610.4
U 610.4 610.4 7+0.4 710.4 710.4 710.4
T
(-)-Ephedrine St0.2 St0.2 9+0.2 9f0.2 90.2 910.2
U
T Sf0.2 St0.2 910.2 910.2 910.2 9f0.2
(-)-Phenylephrine610.2 6f0.2 1210.21210.2 1210.2 1210.2
U 610.2 6f0:2 12+0.21210.2 12+0.2 12f0.2
T
(+)- 610.4 6i-0.4 710.7 710.5 7f0.5 710.7
Phenylpropanolamine6+0.4 610.4 8i-0.48f0.4 8f0.4 8f0.4
U
T
(-)- St0.5 St0.5 Sf0.5 St0.5 St0.5 Sf0.5
Phenylpropanolamin5+0.5 Sf0.5 6f0.5 8f0.5 810.5 810.5
a U
T
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Results are as mean ~ S.E. of flue-sia rabbits per drug.
U = Untreated contralateral eye
T = Drug treated eye
TABLE 7 - Mydriatic
Responses
DRUGS TREATED EYE UNTREATED EYE
SALINE 0 0
(+}-Pseudoephedrine 1 + 1 0
% + % 0
2% 2%
(-)-Ephedrine 1 0 I 0
% +++ % +++
2% 2%
(+)-Phenylephrine 1 ++ 1 0
% ++-~- % +++
2% 2%
(+)-Phenylpropanolamine1 + I 0
% ++ % +
2% 2%
(-)-Phenylpropanolamine1 ++ 1 +
% ++ % 0
2% 2%
SCALE: 0 = No change; + = 0-2 mm change; ++ = 2-4 mm change; +++ _ >4 mm
change
FXAMPLF 3
(-rPhenylpropanolamine Does Not Stimulate the Central Nervous System
Many sympathomimetic compounds stimulate the central nervous system. This is
one reason that decongestants have side effects like insomnia. These tests
compare the
degree of central nervous system stimulation, in the presence and absence of
an
antihistamine, of (-)-phenylpropanolamine, (+)-pseudoephedrine, (-)-ephedrine,
(-)-
phenylephrine and (+)-phenylpropanolamine. Unlike many sympathomimetic
compounds,
(-)-phenylpropanolamine does not stimulate the central nervous system.
2o Moreover, decongestants are often sold in combination with other active
ingredients
(e.g. Claritin-D~ and Seldane-D~). In products containing two or more active
ingredients,
interactions between the active ingredients are undesirable. In these tests,
the extent of (-)-
phenylpropanolamine interaction with a known antihistamine triprolidine, was
observed and
compared to any such interaction between triprolidine and (+)-pseudoepdedrine,
(-)-
ephedrine and (-)-phenylephrine.
Methods:
Animals
Male Swiss-Webster mice (HSD ND4, Harlan Sprague Dawley, Houston, TX) aged
2-3 months were used in these studies. Each dose group consisted of 8 mice.
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The mice were housed 2 to 4 per cage in 30.4 x 22.9 x 15.2 cm clear
polycarbonate cages
with food and water available ad libitum for at least one week prior to
locomotor activity
testing. The colony was maintained at 2311 C, on a normal light-dark cycle
beginning at
0700 hr. All testing took place during the light portion of the light-dark
cycle.
Apparatus
Horizontal (forward movement) locomotor activity was measured using a
standardized, optical activity monitoring system [Model KXYZCM ( 16), Omnitech
Electronics, Columbus, OH]. Activity was monitored in forty 40.5 x 40.5 x 30.5
cm clear
acrylic chambers that where housed in sets of two within larger sound-
attenuating chambers.
1o A panel of 16 infrared beams and corresponding photodetectors were spaced 2
cm apart
along the sides and 2.4 cm above the floor of each activity chamber. A 7.5-W
incandescent
light above each chamber provided dim illumination via a rheostat set to 20%
of full scale.
Fans provided an 80-dB ambient noise level within the chamber.
Drugs.
(+)-Pseudoephedrine, (-)-ephedrine, (-)-phenylephrine, (+)-
phenylpropanolamine, (-
-phenylpropanolamine and (+)-amphetamine were obtained from Sigma Chemical Co.
Triprolidine HCl was obtained from Research Biochemicals International,
(Natick, MA).
All compounds were dissolved in 0.9% saline and injected i.p. in a volume of
10 ml/kg body
weight.
Procedure.
Locomotor stimulant ef,~'ects. In these studies, mice were placed in the
activity
testing chambers immediately following injection of saline or a dose of one of
the test
compounds ranging from 0.1 mg/kg to 250 mg/kg. (+)-Amphetamine was used as a
positive
control. The total horizontal distance traversed (cm) was recorded at 10
minute intervals for
a 2-hour session. Separate groups of 8 mice were assigned to each dose or
saline group, and
dose-effect testing continued for each compound until maximal stimulant or
depressant
effects could be estimated. A separate control group was tested along with
each compound.
For compounds with significant stimulant effects, the potency and efficacy
were
estimated for the 30-minute time period in which maximal stimulant effects
were observed
3o at the lowest dose. Using TableCurve 2D v2.03 (Jandel Scientific), the mean
average total
distance traversed (cm/10 min) for that period was fit to a 3-parameter
logistic peak function
of logio dose (with the constant set to the mean of the saline group), and the
maximum
effect estimated from the resulting curve. The IDso (dose producing 1/z
maximal stimulant
activity) was estimated from a linear regression against loglo dose of the
ascending portion
of the dose-effect curve. The stimulant efficacy was the peak effect of the
compound
(cm/10 min) as estimated from the logistic peak fimction, minus the mean
control distance
traveled (cm/10 min), and was expressed for each stimulant compound as a ratio
to the
stimulant efficacy determined for (+)-amphetamine.
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For compounds with significant depressant effects, the potency and efficacy
were
estimated for the 30-minute time period in which maximal depression occurred
at the lowest
dose. The mean average total distance traversed (cm/10 min) for that period
were fit to a
linear fimction of loglo dose of the descending portion of the dose-effect
curve. The IDso
was the dose producing %z maximal depressant activity, where maximal
depression = 0
cm/30 min. Efficacy was the ratio of maximal depressant effect to maximum
possible
depression for each compound (mean average total distance of the control group
minus the
lowest mean total distance, expressed as a ratio to the control group total
distance).
H~ receptor antagonist interaction studies. The potential for each compound to
to interact with an H~ antihistamine was determined by testing whether a known
antihistamine,
triprolidine, produced a dosage shift in the observed stimulant or depressant
effects of each
sympathomimetic compound. Triprolidine was used as an example of the class of
Hi
receptor antagonists that are typically used as antihistaminic drugs. Twenty
minutes prior to
administering each test sympathomimetic compound, either triprolidine (at
0.01, 0.1, 1.0,
or 25 mg/kg) or saline was injected. The mice were then immediately placed in
the
apparatus for a two hour session. Doses of the test compound were selected
from the
ascending or descending time of the dose-effect curve determined from the
compound-alone
studies. Eight mice were tested for each triprolidine/ syrnpathomimetic
combination.
Statistical analysis. Time course data for each compound were considered in 2-
way
analyses of variance with dose as a between-group and time as a within-group
factor. The
dose-effect data were considered in 1-way analyses of variance, and planned
individual
comparisons were conduced between each dose and the saline control group.
Interaction
studies were considered in 2-way analyses of variance, with Pretreatment and
Test dose as
the factors.
Two-way analyses of variance were conducted on horizontal distance traveled
using
dose as a between-subject factor and time as a within-subject factor. Only (+)-
amphetamine
exhibited a significant dose- and time-effect, with an interaction of dose and
time (all
Fs>2.7; all p values <0.01 ).
Results:
The effects of sympathomimetic enantiomers on locomotor activity are
summarized
in Table 8.
Locomotor stimulant effects
Time course.
Mice injected with (+)amphetamine showed a dose- and time-dependent increase
in
the distance traversed within 10 minutes following injection. The peak
stimulant effects
occurred during the first 30 minutes following 2.5 mg/kg and continued for at
least 60
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minutes.
(-)-Ephedrine resulted in increased locomotion within 40 minutes following 50
to
100 mg/kg, with peak effects occurring 60 to 90 minutes following injection
and
diminishing thereafter.
Little or no stimulant effects were evident within two hours following
treatment with
(+)-phenylpropanolamine, (+)-pseudoephedrine or (-)-phenylpropanolamine (Table
8).
Locomotor depressant eJ~'ects
Time course. (+)-Amphetamine and (-)-ephedrine treatment did not cause
1 o locomotor depression. However, treatment with (+)-pseudoephedrine, (+)-
phenylpropanolamine and (-)-phenylpropanolamine did result in some locomotor
depression
within 10 to 20 minutes following injection. These effects lasted from 20
minutes to > 2
hours, depending upon dose and compound.
15 Depressant efficacylpotency.
Dose-response relationships for locomotor depressant effects of the
sympathomimetics are provided in Table 8, for the time period in which the
maximal
depressant effects were first observed as a function of dose. The maximal
depressant effect
was estimated as the difference between the control group mean and the mean of
the dose
2o group with lowest locomotor activity. The maximum possible effect was
assumed to be
equivalent to the mean of the control group. Depressant efficacy was the ratio
of maximal
depressant effect to the maximum possible effect, and was highest for (+)-
pseudoephedrine
(0.58}. The IDso for depressant effects was estimated from a linear regression
through the
descending portion of the dose-effect curve, assuming zero locomotor activity
(horizontal
25 distance) as the maximal effect. The order of potency for the depression
was:
(+)-phenylpropanolamine~(-)-phenylephrine > (-)-phenylpropanolamine
» (+)-pseudoephedrine
CA 02305294 2000-04-10
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TABLE 8
Stimulation ression
(+)- 1-100 0.21 12.6 40-70 0.58 72.4 10-40
Pseudoephedrine0.5-2500.80 38.2 50-80 0.45 7.4 10-40
(-)-Ephedrine
0.1-100 - 60-90 0.77 2.3 0-30
(-)-Phenylephrine2.5-250.19 7.9 50-80 0.76 2.6 90-120
(+)- 1-25 0 - 60-90 0.70 5.8 10-40
Phenylpropanolamin
a
(_)-
Phenylpropanolamin
1 Dose range tested in mg/kg.
2 The ratio of the maximal stimulant effect of the test compound to the
maximal effect of
(+)amphetamine.
3 The dose resulting in '/s the maximal stimulant effect (IDS) in mg/kg i.p.
4 The 30-min period following injection in which the maximal stimulant effect
occurred.
The ratio of the maximal depressant effect to the maximum possible effect
(zero locomotor
activity)
6 The dose resulting in'/~ the maximal depressant effect (IDS) in mg/kg i.p.
7 The 30-min minute period following injection in which the maximal depression
occurred.
8 "-" denotes absence of significant effect
Triprolidine interactions.
Triprolidine alone. When injected immediately prior to testing, doses of
triprolidine
from 0.25 to 25 mg/kg failed to affect horizontal distance during the 2-hour
test period.
Dose-dependent depression of locomotion was observed following 50 and 100
mg/kg,
beginning within 10-minutes following injection and lasting for 30 to 40
minutes. A
separate one-way analysis of variance on average distance/ 10 min for the
period 0-30
1o minutes following injection suggested a significant dose main effect where
F(8,102) = 7.7
and p<0.00 1, although individual comparisons of dose groups with control in
that analysis
verified that significant effects of triprolidine were
restricted to the 50 and 100 mg/kg doses (ps<.01 ).
Triprolidine interactions. When tested for dose-response in mice pretreated
with
0.01, 0.1, or 1.0 mg/kg triprolidine, (-)-phenylpropanolamine and (-)-
phenylephrine did not
show significant modification of stimulant or depressant effects. Significant
effects for
pretreatment with triprolidine were observed for (+)-pseudoephedrine, (-)-
ephedrine and
(+)-phenylpropanolamine.
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Conclusion.
(-)-Phenylpropanolamine causes no central nervous system stimulation and has
less
potency for depression than does (+)-phenylpropanolamine. Moreover, combining
triprolidine and (-)-phenylpropanolarnine did not give rise to any negative
drug interactions
such as central nervous system stimulation or depression. Hence,
(-)-phenylpropanolarnine does not appear to interact with H, histamine
receptors, and may
be used in combination with other drugs, such as H, antihistamines,
particularly,
1 o triprolidine.
EXAMPLE 4
Decongestant Activity of (-~phenylpropanolamine
The decongestant activity of (-)-phenylpropanolamine was compared to that of
15 (+)pseudoephedrine, (-)ephedrine, (-)-phenyiephrine and (+)-phenylpropanol-
amine, in
normal and histamine-challenged rats.
Experimental Protocol:
The method was based on one reported by Lung for the measurement of nasal
2o airway resistance. Sprague Dawley rats (weight range 247-365 gram) were
anesthetized
with sodium pentobarbital infra-peritoneally (50 mg/ka). Rats were placed on a
heating pad,
in a V trough, dorsal side down. A tracheotomy was performed and a tracheal
cannula was
positioned, tied and left open to room air. A cannula was placed into the
superior part of the
trachea and was advanced till lodged in the posterior nasal opening. Normal
saline (0.5 ml)
25 was injected into the nasal cannula to confirm position as well as to
moisten the nasal
mucosa. After nasal cannulation was confirmed the cannula was tied in place
with a suture
placed around the trachea. Excess fluid was expelled from the nasal airway
with a short (2-
3 second) air flow via the nasal cannula. Additionally, in studies correlating
blood pressure
changes to those in the nasal airway pressure, a cannula was positioned in the
internal
3o carotid artery (PE. 50) and connected to a multipen (Grass) recorder using
pressure
transducer (Isotec).
Nasal airway pressure was measured using a validyne pressure transducer (with
a
2.25 cm H20 range membrane) connected to a multipen recorder (Grass). Air was
passed
thmugh an in-line direct measure flow meter (Gilmont instruments) connected to
the nasal
35 opening cannula. Pressure was measured in the line with a constant flow
rate (150 mUmin)
of air. Enantiomeric drugs were directly injected into the jugular vein using
a 30 gauge
needle. All injections were of a constant 0.1 ml volume. In the congestion
challenged
groups congestion was achieved by an intranasal administration of histamine
(50 mM,
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0.02m1/nostril}. The histamine was expelled after 2 min with a short nasal
cannula airflow
and subsequent enantiomeric drug doses were directly injected into the jugular
vein. The
doses of injection for each of the enantiomers tested were determined from a
previous study
in our laboratory in which each of the dose of drug we chose resulted in an
increase in mean
arterial pressure (MAP) of 10% (Table 9). The dose causing a 10% increase in
MAP served
as our "100%" dose for the initial nasal airway studies.
TABLE 9
Dosage of Enantiomer which raised mean arterial pressure 10%
Drug Name Dog (ug/kg) Rat (ug)
(+)-pseudoephedrine 200 60
(-)-Ephedrine 100 30
(-)-Phenylephrine 10 3-5
(+)-Phenylpropanolamine 400 120
(-)-Phenylpropanolamine 20 6
io
Three investigations were performed as follows:
Investigation 1: A comparison was made of the effect of the different
enantiomers on
nasal airway resistance prior to and following histamine congestion. The
amount of drug
required to raise the mean arterial pressure by 10% was chosen as the " 100%
dose" for
these decongestant studies (see Table 9}. Control changes in nasal airway
resistance were
obtained by recording nasal airway resistance prior to and following this dose
100% dose. In
a test group of rats the 100% dose was injected into the jugular vein two
minutes after nasal
airway congestion was produced by introduction of 0.02 ml/nostril of 50 mM
histamine
2o into the nasal airway. Nasal airway resistance was thus increased by
histamine challenge.
To assess the effect of administering an enantiomer changes in airway
resistance were then
observed.
Inves~tgation 2: A comparison of the effect on nasal airway resistance of
various doses of
each enantiomer was made to determine an effective dose of the enantiomer.
Dosages
tested were 50%, 25%, 10% and 5% of the "100%" enantiomer dosage required to
increase
the mean arterial pressure 10%. Changes in nasal airway resistance were
observed by
comparing pre-enantiomer injection nasal airway resistance with decreases in
nasal airway
resistance following jugular vein injection of the enantiomer. Five rats were
tested at each
3o dose for each of the enantiomers.
23
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PCT/US99/02023
Investigation 3: A 75% dose of enantiomer was tested following 0.02 ml/nostril
of 50mM
histamine. As before, this "75% dose" was 75% of the dose required to increase
the mean
arterial pressure 10%. The 75% dosages employed were as provided in Table 10.
Tabte 10
Drug 75% Dosage (~g/kg i.v.)
(+)-pseudoephedrine 150
(-)-Ephedrine 75
(-)-Phenylephrine 7.5
(+)-Phenylpropanolamine 300
(-)-Phenylpropanolamine 15
Blood pressure was monitored. Effects on airway resistance and blood pressure
of each of
~o the eight enantiomeric were evaluated at the 75% dose prior to and
following histamine in
five rats for each enantiomer and each histamine condition.
Results
Investigation 1:
Each drug gave rise to a significant decrease in nasal airway pressure,
relative to
control, in non-histamine-challenged rats (Table 11 ). While the control for
the
(-)-phenylephrine was significantly different from the other controls, this
difference in
control level did not translate into a difference caused by administration of
the drug.
2o TABLE 11
Drug Control Post Drug % Change Paired
t test
(mm H20) (mm H20) pValue
(+)- 910.5 7f0.9 -21.3f7.6 0.015
Pseudoephedrine
(-)-Ephedrine7 t 0.8 6 t 0.5 -14 + 3.4 0.034
(-)-Phenylephrine5 f 0.2 4 f 0.2 -20.3 f 0.010
4.3
(+)- 7 ~ 0.2 610.2 -10.5 + 0.001
1.4
Phenylpropanol-
amine
(-)- 80.6 70.7 -8.81 t2.6 0.028
Phenylpropanol-
amine
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* mm water.
In the histamine-challenged rats, administration of each drug again showed a
significant decrease in nasal passage pressure (Table 12). However, after
treatment with
histamine, rats treated with (-)-phenylpropanolamine showed a significantly
greater
reduction in airway passage resistance than did rats treated with (+)-
phenylpropanolamine
(Table 12). These data indicate that (-)-phenylpropanolamine is a stronger
antagonist of the
physiological effects of histamine than is (+)-phenylpropanolamine.
TABLE
12
Drug Control*t test Post* t test Post* %Change
p Value HistaminepValue Drug
(+)- 6.6 10.60.1 9.3 ~ 0.001 6.1 t -3 6.7f2.7
1. 8 1.5
Pseudo-
ephedrine
(-)- 6.7 f0.40.06 8.2 X0.7 0.007 6.4 f0.6-21.8f3.5
Ephedrine
(-)- 7.5 X0.50.04 13.2 X2.10.05 10.5 -22.3f8.5
12.1
Phenyl-
ephrine
(+)- 0.5 10.90.004 12.2 10.80.04 10.6 -13.6
t 1.0 X4.4
Phenyl-
pmpanola
mine
(-)- 7.8 f0.20.07 10.5 t 0.04 7.6 f0.4-25.215.6
1.0
Phenyl-
propanola
mine
* mm water.
The results indicate that the antihistamine and/or decongestant activity of
(-)-phenylpropanolamine was superior to (+)-phenylpropanolamine.
Investigation #2:
Table 13 summarizes the mean nasal airway pressure of different enantiomer
dosages ranging from 5%, 10%, 25% and 50% of the dose that produced a 10%
change in
2o resting mean arterial pressure (the " 100%" dose). The standard error of
the mean is also
provided. In general, the 50% dose was an approximate threshold dose at which
nasal
airway pressure was reduced.
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WO 99/48483 PCT/US99/02023
TABLE 13
Mean Decrease in Nasal Airway Pressure
With Variable Enantiomer Dosages
Drug 5% 10% 25% 50%
(+)-pseudoephedrine-3.8 t -6.8 t 3.6 -13.5 t 4.3 -16.1 ~
2.8 2.4
(-)-Ephedrine -1.0 t -2.5 f 1.5 -3.6 t 1.9 -1.9 t 2.0
0.8
(-)-Phenylephrine-1.6 f -4.8 t 0.8 -12.1 t 2.3 -5.2 f 1.6
0.9
(+)-phenyl- -0.03 t -0.7 t 1.2 -0.8 f 1.0 -0.07 t
0.3 1.5
propanolamine
(-)-Phenyl- 0.1 t 0.7 -1.4 t 0.5 -1.4 t 2.1 -1.9 f 0.6
propanolamine
*percent enantiomer dose that increased the dog resting mean arterial pressure
10%
Investigation #3:
Figure 1 summarizes the percent change in mean arterial blood pressure after a
75%
dose of various enantiomers. As indicated, (-)-phenylpropanolamine causes a
lesser
increase in blood pressure than is observed for several enantiomers which are
commercially
available as decongestants.
Figure 2 provides the observed percent nasal airway pressure after a 75% dose
of the
i5 different enantiomers.
26
