Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
SYNTHESIS OF DIHYDROHONOKIOL COMPOSITIONS
BACKGROUND OF THE INVENTION
1.1 Field of the Invention
The present invention relates to the synthesis of dihydrohonokiol, its
derivatives, analogs and homologs, and to methods of use for the
dihydrohonokiol
compounds. Also included are compositions particularly useful for treatment of
anxiety disorders.
1.2 Description of Related Art
Anxiety and anxiety-related disorders are extremely common. Anxiety-related
conditions can be relatively mild or can be sufficiently severe as to be
disabling. Also
noteworthy is that anxiety, while infrequently a "disease" in itself, is an
almost
inevitable and often exacerbating consequence of many other medical and
surgical
conditions. Estimates of the number of patients suffering from various anxiety
disorders range between 12 and 35 million persons in seven major
industrialized
nations.
Saiboku-to, a Chinese herbal medicine, has long been used to treat anxiety and
neurotic disorders (Hosaya and Yamamura, 1988; Nartia, 1990). A disadvantage
of
Saiboku-to is that it requires approximately seven days or more of daily
administration
before an anxiolytic effect is observed (Maruyama et al., 1998). Fractionation
of
Saiboku-to has identified the two putative principal active anxiolytic
components as
magnolol (5,5'-di-(2-propenyl)-1,1'-biphenyl-2,2'-diol) and its positional
isomer,
honokiol (3,5'-di-(2-propenyl)-1,1'-biphenyl-2',4diol) (Maruyama et al.,
1998).
Behavioral tests indicate that honokiol is at least 5000 times more potent
than
Saiboku-to. But honokiol still requires several days of administration to
elicit an
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anxiolytic effect (Maruyama et al., 1998). The delay in the onset of
anxiolytic activity
of honokiol is thought to be due to either changes in receptors or a slow
build-up of
honokiol metabolites within the body.
The metabolic pathway of honokiol has not been fully established. The
positional isomer magnolol is known to be metabolized to a number of compounds
including the hydrogenated products dihydromagnolol (5-(2-propenyl)-5'-n-
propyl-
l,l'biphenyl-2,2'diol) and tetrahydromagnolol (5,5'-8v-n-propyl-1,1'biphenyl-
2,2'-
diol) (Hattori et al., 1984a). The excretion of the reduced metabolites in
feces and
urine increased significantly in amount after repeated daily administration of
magnolol. This pattern was possibly the result of induction of the enzymes
responsible for the metabolism.
Benzodiazapines, such as diazepam and alprazolam, represent the most
commonly used class of anxiolytic agents administered for the treatment of
anxiety.
Benzodiazapines can act to counteract anxiety by depressing the electrical
afterdischarge in the limbic system, and appear to potentiate neural
inhibition that is
mediated by gamma-aminobutyrate (GABA) (Baldessarini, 1990). These compounds
have proven to be effective at reducing anxiety, but they also have
significant side
effects including sedation, ataxia, amnesia, dependence, tolerance, and
behavioral
disturbances and act as skeletal muscle relaxers. These side effects can
render these
compounds unsuitable for many patients, particularly those whose anxiety is
coupled
to another form of illness.
In addition to benzodiazepines, other drugs used to treat anxiety include
barbiturates, certain anticholinergic agents, antihistamines, and
azaspirodecanediones
(Baldessarini, 1990). These drugs are sedatives, or at least have many
properties in
common with traditional sedatives. The barbiturates are general neuronal
depressants.
The use of certain anticholinergic agents and antihistamines for treating
anxiety
appears to be based on their sedative properties. The azaspirodecanediones,
buspirone
in particular, are used in treating anxiety but require weeks of
administration before
anxiolytic activity is noted. The mechanism of action of the
azaspirodecanediones is
unknown.
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2.0 SUMMARY OF THE INVENTION
The present invention addresses problems associated with commonly-used
anxiolytic drugs, with Saiboku-to therapy in general, and with honokiol in
particular,
for treatment of anxiety. The present invention provides compounds which
exhibit
anxiolytic activity within hours without many of the negative side effects
associated
with well-known anxiolytic compounds, such as the benzodiazepines. The
invention
also provides a method of treating anxiety disorders. Further, the invention
provides
methods of synthesis of novel anxiolytic compounds.
2.1 Novel Anxiolytic Compounds
The present invention provides new anxiolytic compounds that do not have the
adverse side effects of the benzodiazepines. In particular embodiments, the
invention
relates to the use of dihydrohonokiol, its derivatives, analogs and homologs,
as
anxiolytic agents. The inventors have demonstrated that dihydrohonokiol, for
example, exerts an anxiolytic effect more rapidly than honokiol and exhibits
greater
potency and fewer side effects than diazepam, a benzodiazepine anxiolytic.
An aspect of the present invention encompasses novel compositions of matter
comprising compounds of the formula:
OH
R R
In a preferred embodiment, R is CH2-CH2-CH3 and X is 5'-CH2-CH=CH2 and
X' is 3-CHZ-CH=CH2.
In other embodiments, the group R represents -CH2-CH=CH2, CH=CH-CH3,
or -CH2-CH2CH3. The group X represents from one to two substituents in any of
the
3-, 4-, 5-, or 6-positions and is separately and independently fluorine,
hydroxy,
methoxy, (1-adamantyl), CI-CS alkyl, C2-C3 alkenyl, C2-C3 alkylcarbonyl or C1 -
C4
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carboxyalkyl. The C1-CS alkyl group may be substituted with one or more
fluorine or
hydroxyl. Excepted from this list are the compounds where R is -CHZ-CH=CHZ and
X is 5'-CH2-CH3, 5'-CH2-CH=CH2, 5'-OH, 3'-OH, 5'-CH2-CH-CH3, and 3'-OCH3,
5'-CH2-CH=CH2, and where R is -CHZ-CHZ-CH3. and X is 3-CHZ-CHI-CH3.
In particular, X may represent: 5'-CH=CH2; 5'-CH2-CHI; 5'-CH=CH2, 3'-OH;
5'-CHZ-CH=CH2; 5'-CH2-CH=CH2, 3'-OH; 5'-CHZ-CHZ-CH3, 3'-OH; 5'-CH2-
CH=CH2, 3'-OCH3; 5'-CHI-CHZ-CH3, 3'-OCH3; 5'-CH=CH-CH3, 3'-OH; 5'-CH=CH-
CH3, 3'-OCH3; 5'-CH3; 5'-CH(CH3)2; S'-CH2CH(CH3)2; 5'-C(CH3)3; 5'-
CH(CH3)ZC2H5; 5'-(1-adamantyl); 5'-CH(CH3)2, 6 -CH3; 5'-CH(CH3)2, 4 -CH3; 5'-
C(=O)-CH3; 5'-CH(-OH)-CH3; 5'-CH2-C(=O)-CH3; 5'-CHZ_CH(-OH)-CH3; 5'-CHZ-
COOH; 5'-CH2CH2-COOH; 5'-CHZCH2CH2-COON; 5'-CHZ-COOH, 3-OH; 5'-OH;
5'-OCH3; 3'-F; 4'-F; 5'-F; 3'-F, 5'-CH3; 3'-F, 5'-CH2-CH3; 3'-F, 5'-CH2-OH; 3'-
F, 5'-
CH2-CHZ-OH; 3'-F, 5'-COOH; 3'-F, 5'-CHZ-COON; 3'-F, 5'-CH=CH-CH3; 3'-F, 5'-
CHZ-CH2-CH3; 3'-F, 5'-CH2-CH=CHZ; 3'-F, 5'-CHZ-CHF-CH3; 3'-F, 5'-CH2-CHF-
CHZF; 4'-F, 5'-CH3; 4'-F, 5'-CHZ-OH; 3'-F, 5'-CHZ-CH3; 6'-F, 5'-CH2-CH3; 6'-F,
5'-
CH2-OH; and 6'-F, 5'-COON.
In other embodiments, the group X' represents from one to two substituents in
any of the 3-, 4-, 5-, or 6-positions and is separately and independently
fluorine,
hydroxy, methoxy, C1-C4 alkyl, C3 alkenyl, Cl-C3 alkylcarbonyl, C1 -C3
carboxyalkyl
or C3 carboxyalkenyl. The alkyl group may be substituted with one or more
fluorine
or hydroxyl. Excepted from this list are where R = -CH2-CH=CH2 and X' = 3-CHZ-
CH3, 3'-CH2-CH=CHZ, and where R = -CH2-CH2-CH3 and X' = 3-CH2-CHZ-CH3.
In particular, X' may represent: 3-CH3; 3-CH2-CH3; 3-CH2-CH=CH2; 3-CH2-
CH=CH2, 5-OH; 3-CHZ-CH2-CH3, 5-OH; 3-CHZ-CH=CH2, 5-OCH3; 3-CH2CHZCH3,
5-OCH3; 3-CH=CH-CH3; 3-CH3, 6-CH(CH3)2; 3,5 di-CH3; 2,6 di-CH3; 3-CH(CH3)2;
3-CH(CH3)2, 6-CH3; 3-CHZCH(CH3)2; 3-C(=O)-CH3; 3-CH(-OH)-CH3; 3-CHZ-C(=O)-
CH3; 3-CHZ-CH(-OH)-CH3; 3-COOH, 6-OH; 2-COOH; 3-OCH3; 3-CHZCOOH;
3-CHZCH2COOH; 3-CH=CHCOOH; 3-CHO, 2-OH; 3-CHZOH, 2-OH; 3-CHO, 5-
OCH3; 3-CHZOH, 5=OCH3; 3-CHO, 5-CH3; 3-CH20H, 5-CH3, 3-F; 2F; 2-F, 3-CH3;
6-F, 3-CH3; 5-F, 3-CH=CH-CH3; 5-F,3-CHZ-CHI-CH3; 5-F, 3-CHZ-CH=CHZ; 5-F, 3-
CH2-CHF-CH3; or 5-F, 3-CHZ-CHF-CHZF.
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2.1.2 Pharmaceutical Compositions
Another aspect of the present invention includes novel compositions
comprising dihydrohonokiol, its derivatives, analogs and homologs. It will, of
course,
5 be understood that one or more dihydrohonokiol, dihydrohonokiol derivative,
analogs
and homologs may be used in the methods and compositions of the invention. The
maximum number of novel anxiolytic compounds of the present invention that may
be
administered is limited only by practical considerations, such as the
possibility of
eliciting an adverse effect.
Compositions employing the novel anxiolytic compounds will contain a
biologically effective amount of the anxiolytic compound. As used herein, a
"biologically effective" amount of an anxiolytic compound refers to an amount
effective to alter, modulate, or reduce anxiety or related conditions. As
disclosed
herein, different amounts of the novel anxiolytic compounds may be effective.
Clinical doses will of course be determined by the nutritional status, age,
weight and health of the patient. The quantity and volume of the composition
administered will depend on the subject and the route of administration. The
precise
amounts of active anxiolytic compounds required will depend on the judgment of
the
practitioner and may be peculiar to each individual. However, in light of the
data
presented here, the determination of a suitable dosage range for use in
different
mammals will be straightforward.
Pharmaceutical compositions prepared in accordance with the present
invention find use in several applications, including inhibition or reduction
of anxiety.
Such methods generally involve administering to a mammal a pharmaceutical
composition comprising an anxiolytically effective amount of dihydrohonokiol,
its
derivatives, analogs or homologs.
Therapeutic kits comprising dihydrohonokiol, its derivatives, analogs and
homologs comprise another aspect of the present invention. Such kits will
generally
contain, in suitable container means, a pharmaceutically acceptable
formulation of
dihydrohonokiol, its derivatives, analogs and homologs. The kit may have a
single
container means that contains the novel compositions) or it may have distinct
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container means for the novel compositions) and other reagents which may be
included within such kits.
2.2 Method of Treating Anxiety Disorders
The invention also includes a method of treating anxiety disorders with
dihydrohonokiol, its derivatives, analogs and homologs. This method comprises
administration of an anxiolytic amount of a suitable composition containing
dihydrohonokiol, its derivatives, analogs and homologs, to a subject in need
thereof.
Administration is preferably by oral dosage, but any route of administration
capable
of delivering an effective dose my be used. The choice of the composition to
be used
for treatment, the amount of the composition to be administered, and the
duration of
treatment will depend on the judgment of the practitioner and may be peculiar
to each
individual, as disclosed above. The treatment may be maintained as long as
necessary
and may be used in conjunction with other forms of treatment.
2.3 Methods of Synthesis of Anxiolytic Compounds
In yet another aspect, the invention relates to synthetic methods for
producing
dihydrohonokiol and its various derivatives, analogs and homologs. The
compounds
may be synthesized from honokiol derived from plant sources. This reaction is
described herein as a hemi-synthesis. Alternatively, the total synthesis may
be
accomplished from the asymmetric coupling of 4-allylphenylalkyl ether with 4-
alkoxy
haloaryls, followed by dealkylation of the dialkoxyl biaryl formed.
In the hemi-synthesis of dihydrohonokiol, honokiol is partially hydrogenated
by reacting hydrogen atoms in the presence of a tris-(triphenylphosphine)
rhodium(I)
chloride catalyst with honokiol dissolved in a non-polar solvent. In preferred
embodiments, the non-polar solvent is selected from toluene, benzene or
hexane.
Under conditions of a limited supply of hydrogen, the predominant reaction
product
has only one of the two similar allylic groups of the biphenyl molecule
reduced.
Tetrahydrohonkiol is formed as a minor component, thus enabling unreacted
honokiol
to be recovered and reused. The partially hydrogenated honokiol products are
collected and the two dihydrohonokiol isomers are isolated and purified.
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The total synthesis of 3-n-propyl-5'-(2-propenyl)-l,l'-biphenyl-2',4-diol, one
of the two isomers of dihydrohonokiol, designated herein as I, utilizes 4-
allylanisole
as the starting chemical compound. This starting chemical compound is reacted
with
tert-butyllithium and anhydrous zinc chloride. The intermediate compound so
formed
is coupled with 2-propyl-4-iodo-anisole in the presence of a catalyst,
resulting in
2',4-dimethoxy-3-n-propyl-5'-(2-propenyl)-1,1'-biphenyl, which after
demethylation
generates 3-n-propyl-5'-(2-propenyl)-1,1'-biphenyl-2',4-diol (I) as one of the
reaction
products. The synthesis of the other isomer of dihydrohonokiol, 5'-n-propyl-3-
(2-
propenyl)-l,l'-biphenyl-2',4-diol, hereinafter designated as II, can be
accomplished in
a similar manner, using 4-propylanisole as the starting chemical compound and
reacting it with tent-butyllithium and anhydrous zinc chloride. This
intermediate
compound is coupled with 2-allyl-4-iodo-anisole in the presence of a catalyst,
resulting in 2',4-dimethoxy-5'-n-propyl-3-(2-propenyl)-1,1'-biphenyl, which
after
demethylation generates 5'-n-propyl-3-(2-propenyl)-l,l'-biphenyl-2',4-diol
(II) as one
of the reaction products. The two structural isomers of dihydrohonokiol can be
isolated from the reaction mixtures by procedures known in the art, such as
preparative reverse phase high pressure liquid chromatography or preparative
thin
layer chromatography.
Analogs and homologs of 3-n-propyl-5'-(2-propenyl)-l,l'-biphenyl-2',4-diol
(I) and 5'-n-propyl-3-(2-propenyl)-1,1'-biphenyl-2',4-diol (II) are
synthesized from
substituted 4-allyl or alkylphenyl alkyl ethers which are converted to the
corresponding 2-alkoxy-5-allylphenyl metal halide or 2-alkoxy-5-alkylphenyl
metal
halide by a directed ortho metalation reaction. The products of such reactions
are, in
turn, reacted with various 4-alkoxyhaloaryls in-situ to generate the required
homologs
or analogs of compounds I and II. Carboxylic groups may be protected as esters
(e.g.,
ethyl ester) during the synthetic process. The dihydrohonokiol analog formed
after the
coupling reaction and demethylation can be deprotected by base catalyzed
hydrolysis
of the ester group. Alcoholic groups may be protected as acyl esters, e.g., by
base
catalyzed acetylation with acetic anhydride. The dihydrohonokiol analog formed
after
the coupling and demethylation can be deprotected by base catalyzed removal of
acyl
groups.
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3.0 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The anxiolytic compounds of the present invention, including
dihydrohonokiol, its derivatives, analogs and homologs, in contrast to the
benzodiazepines, can be administered without significant side effects. The
benzodiazepines have side effects including sedation, ataxia, amnesia,
dependence,
tolerance, and behavioral disturbances. These side effects have not been
observed
with therapeutic doses of the dihydrohonokiol isomers I and II.
In tests in mice maximum anxiolytic activity of dihydrohonokiol was observed
three hours after oral administration. This contrasts with up to at least
seven days for
anxiolytic activity to manifest in mice treated with honokiol.
The novel dihydrohonokiols shown below have been synthesized:
(I> 4H (y OH
3'~~~' -'~~15 3'
/~ 4'
OH Y, ~~ OH
The nomenclature adopted for the dihydrohonokiols, 3-h-propyl-5'-(2-
propenyl)-1,1'-biphenyl-2',4-diol (I) and 5'-n-propyl-3-(2-propenyl)-1,1'-
biphenyl-
2',4-diol (II), is based on the original nomenclature for honokiol and
tetrahydrohonokiol as used by Takeya et al, ( 1986).
The dihydrohonokiol compounds I and II can be synthesized by partial
hydrogenation of honokiol as described in Example 1, ~ 5.1.2. This methods
allows
for the hydrogenation of only one of the two very similar allylic groups on
the
biphenyl moiety. In a typical reaction of the exemplified synthesis, 55 % of
the
reaction products was dihydrohonokiol, 35.5% unreacted honokiol, and 9.5%
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tetrahydrohonokiol. Thus dihydrohonkiol represents almost 86% of the consumed
honokiol. This method allows for the recovery of unreacted honokiol, the reuse
of
which in the synthesis of the dihydrohonokiols isomers markedly increases the
potential efficiency of this synthesis.
Limiting the supply of hydrogen was important in preventing conversion to
tetrahydrohonkiol. Thus increasing the total amount of hydrogen in the
reaction
mixture, e.g., by increasing the volume of solvent and associated dissolved
hydrogen,
or increasing the time hydrogen is passed through the reaction mixture after
the
addition of honokiol, increases the amount of tetrahydrohonokiol produced.
Preferred
reaction conditions were those which resulted in the reaction of 0.6 to 1.2
mol of
hydrogen per mol of honokiol. Despite use of a variety of conditions,
dihydrohonkiol
could not be prepared by hydrogenation using either 5% or 10% palladium on
activated carbon as catalyst. With palladium/carbon, either unreacted honokiol
or
fully reduced tetrahydrohonokiol was recovered from the reaction mixture.
Alternatively, compounds I and II can be synthesized by a total synthesis,
details of which are given in Example 1, ~ 5.1.3. Briefly, 4-allyl or alkyl
phenyl
ethers are converted to the respective 2-alkoxy 5-allyl or 5-alkyl phenyl
metal halides
by a directed ortho metalation reaction. Such intermediates are reacted with 4-
alkoxy
haloaryls in the presence of a palladium catalyst to yield unsymetrical
biphenyl
coupling products. The removal of alkyl protecting groups by boron tribromide
under
mild conditions, followed by preparative high pressure liquid chromatographic
purification, produces purified compounds I and II.
In like fashion, homologs and analogs of 3-n-propyl-5'-(2-propenyl)-1,1'-
biphenyl-2',4-diol (I) may be generated by converting 4-allylphenyl alkyl
ethers to
2-alkoxy-5-allylphenyl metal halides by a directed ortho metalation reaction,
which in
turn will be reacted with various 4-alkoxyhaloaryls in-situ. Similarly,
various
substituted-phenyl alkyl ethers will be converted to 2-alkoxy-substituted
phenyl metal
halides and be reacted in-situ with various 3-allyl-4-alkoxyhalophenyls to
generate the
homologs and analogs of 5'-n-propyl-3-(2-propenyl)-l,l'-biphenyl-2',4-diol
(II).
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3.1 Anxiety
As used herein, the term "anxiety" is intended to refer to a condition of
apprehension, uncertainty, dread, or fear unattached to a clearly defined
stimulus
accompanied by numerous physiological and psychological symptoms such as
5 tachycardia, dyspnia, tension, restlessness, inattentiveness, and loss of
appetite,
skeletal motor function, initiative, cognitive logic, short- and long-term
memory, and
the like. Practice of the method of the present invention can combat, i.e.,
reduce or
alleviate, some, most, or all of these physiological symptoms.
A suitable subject to be treated by the present method is an animal, such as a
10 human or other mammal (e.g., house pets such as dogs and cats, or other
commercially valuable or domestic animals), which experience anxiety-related
symptoms due to some external or internal stimulus that are desirably
combated.
Preferably, the subject is human.
As used herein the term "treating" includes prophylaxis of a physical and/or
mental condition or amelioration or elimination of the developed physical
and/or
mental condition once it has been established or alleviation of the
characteristic
symptoms of such condition.
The term "antianxiety dose", as used herein, represents an amount of
compound necessary to prevent or treat a human susceptible to or suffering
from
anxiety following administration to such human. The active compounds are
effective
over a wide dosage range. For example, dosages per day will normally fall
within the
range of about 0.005 to about 500 mg/kg of body weight. In the treatment of
adult
humans, the range of about 0.05 to about 100 mg/kg, in single or divided
doses, is
preferred. However, it will be understood that the amount of the compound
actually
administered will be determined by a physician, in the light of the relevant
circumstances including the condition to be treated, the choice of compound to
be
administered, the age, weight, and response of the individual patient, the
severity of
the patient's symptoms, and the chosen route of administration, and therefore
the
above dosage ranges are not intended to limit the scope of the invention in
any way.
While the present compounds are preferably administered orally to humans
susceptible to or suffering from anxiety, the compounds may also be
administered by
a variety of other routes such as the transdermal, parenteraly, subcutaneous,
intranasal,
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intramuscular and intravenous routes. Such formulations may be designed to
provide
delayed or controlled release using formulation techniques which are known in
the art.
Examples of anxiety disorders which may preferably be treated using an
effective amount of a named compound or pharmaceutically acceptable salt
thereof
include, but are not limited to: Panic Attack; Agoraphobia; Acute Stress
Disorder;
Specific Phobia; Panic Disorder; Psychoactive Substance Anxiety Disorder;
Organic
Anxiety Disorder; Obsessive-Compulsive Anxiety Disorder; Posttraumatic Stress
Disorder; Generalized Anxiety Disorder; and Anxiety Disorder NOS.
The named anxiety disorders have been characterized in the DSM-IV-R.
Diagnostic and Statistical Manual of Mental Disorders, Revised, 4th Ed.
(1994). The
DSM-1V-R was prepared by the Task Force on Nomenclature and Statistics of the
American Psychiatric Association, and provides clear descriptions of
diagnostic
categories. The skilled artisan will recognize that there are alternative
nomenclatures,
nosologies, and classification systems for pathologic psychological conditions
and that
these systems evolve with medical scientific progress. Social anxiety disorder
may
also be preferably treated using an effective amount of this compound or
pharmacologically acceptable salt thereof.
3.2 Antidepressant Activity
The compounds of the present invention are also contemplated to be useful for
the treatment of depression. There is considerable support the involvement of
GABA
in mood disorders. Somatic treatment for depression and mania upregulate the
GAGA
B receptor similar to the effect of GABA agonists. Decreased GABA function
accompanies depressed or manic mood states. Low GABA levels are found in
brain,
cerebrospinal fluid and plasma in patients with depression and in plasma of
patients
with mania. Low GABA function is proposed to be an inherited biological marker
of
vulnerability for development of mood disorders.
Benzodiazepines are GABA A receptor agonists and augment the activity of
GABA while generally not affecting biogenic amine uptake or metabolism. A
metaanalysis of the use of benzodiazepines in the treatment of depression
found that
alprazolam had activity comparable to standard antidepressants.
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The anxiolytic activity of the mixture of compound I and II and the
benzodiazepine diazepam in the elevated plus maize model was blocked by the
GABA antagonist bicuculline. But the anxiolytic activity of diazepam but not
the I/II
mixture was blocked by flumazenil, a benzodiazepine GABA site antagonist.
While
these results differentiate the mechanism of action of compounds I and II from
that of
benzodiazepines, they also indicate a possible augmenting action of
dihydrohonokiol
compounds on the GABA system, an action indicative of antidepressant activity.
4.2 Pharmaceutical Compositions
Another aspect of the present invention includes novel compositions
comprising the novel dihydrohonokiol, its derivatives, analogs and homologs.
In a
preferred embodiment the composition will comprise dihydrohonokiol, either 3-n-
propyl-5'-(2-propenyl)-1,1'-biphenyl-2',4-diol (I) or 5'-n-propyl-3-(2-
propenyl)-1,1'-
biphenyl-2',4-diol (II). Alternatively, compositions may comprise any ratio of
the two
isomers such as 10-90% of I and 90-10% of II. The composition may further
comprise derivatives, analogs or homologs of dihydrohonokiol, or one or more
other
pharmacologically-active compounds, and particularly one or more anxiety-
altering
compounds. The methods of the invention may thus entail the administration of
one,
two, three, or more, of the new dihydrohonokiol, its derivatives, analogs and
homologs. The maximum number of species that may be administered is limited
only
by practical considerations, such as the particular effects of each compound.
Of
course, a composition of derivatives of I or II may be preferable, depending
on their
particular physical properties and physiological effects.
The compositions of the invention may include a dihydrohonokiol, its
derivatives, analogs and homologs, modified to render it biologically
protected.
Biologically protected compounds have certain advantages over unprotected
compounds when administered to human subjects and, may exhibit increased
pharmacological activity.
Compositions employing the novel compounds will contain a biologically
effective amount of the compounds. As used herein a "biologically effective
amount"
of a compound or composition refers to an amount effective to alter, modulate
or
reduce anxiety or related conditions. For oral administration, a satisfactory
result may
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be obtained employing the compounds in an amount within the range of from
about
0.001 mg/kg to about 50 mg/kg, preferably from about 0.005 mg/kg to about 35
mg/kg and more preferably from about 0.01 mg/kg to about 20 mg/kg alone or in
combination with one or more additional anti-anxiety compounds in an amount
within
the range from about 0.001 mg/kg to about 50 mg/kg, preferably from about
0.005
mg/kg to about 35 mg/kg and more preferably from about 0.01 mg/kg to about 20
mg/kg both being employed together in the same oral dosage form or in separate
oral
dosage forms taken at the same time. For parenteral administration, e.g.,
subcutaneously or intramuscularly, the dihydrohonokiol compounds) will be
employed in an amount within the range of from about 0.005 mg/kg to about 10
mg/kg and preferably from about 0.01 mg/kg to about 1 mg/kg, alone or with the
additional anti-anxiety compounds in an amount within the range of from about
0.005
mg/kg to about 20 mg/kg and preferably from about 0.01 mg/kg to about 2 mg/kg.
It will also be understood that, if desired, the disclosed dihydrohonokiol,
its
derivatives, analogs and homologs, may be administered in combination with
additional agents, such as, e.g., proteins or polypeptides or various
pharmaceutically
active agents. So long as the composition comprises a dihydrohonokiol, its
derivatives, analogs and homologs, there is virtually no limit to other
components
which may also be included, given that the additional agents do not cause a
significant
adverse effect upon contact with the target cells or host tissues. The
dihydrohonokiol,
its derivatives, analogs and homologs, may thus be delivered along with
various other
agents as required in the particular instance.
In carrying out the method of the present invention, the pharmaceutical
compounds) alone or in combination with one or more anxiety-modulating
compounds may be administered to mammalian species, such as monkeys, dogs,
cats,
rats and humans, and as such may be incorporated in a conventional systemic
dosage
form, such as a tablet, capsule, elixir or injectable. The above dosage forms
will also
include the necessary carrier material, excipient, lubricant, buffer,
antibacterial,
bulking agent (such as mannitol), anti-oxidants (ascorbic acid of sodium
bisulfite) or
the like. Oral dosage forms are preferred, although parenteral forms such as
intramuscular, intraperitoneal, or intravenous are quite satisfactory as well.
The dose
administered must be carefully adjusted according to age, weight and condition
of the
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patient, as well as the route of administration, dosage form and regimen and
the
desired result.
The pharmaceutical compositions disclosed herein may be orally administered,
for example, with an inert diluent or with an assimilable edible carrier, or
they may be
enclosed in hard or soft shell gelatin capsule, or they may be compressed into
tablets,
or they may be incorporated directly with the food of the diet. For oral
therapeutic
administration, the active compounds may be incorporated with excipients and
used in
the form of solutions, suspensions, elixirs, troches, tablets, pills,
capsules, sustained
release formulations, powders, syrups, wafers and the like, and contain about
0.1 % to
about 95% of active ingredient, preferably about 1 % to about 70%. Normally
employed
excipients may include, for example, pharmaceutical grades of mannitol,
lactose, starch,
magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the
like.
Such compositions and preparations should contain at least 0.1 % of active
compound.
The percentage of the compositions and preparations may, of course, be varied
and
may conveniently be between about 2 to about 60% of the weight of the unit.
The
amount of active compounds in such therapeutically useful compositions is such
that a
suitable dosage will be obtained.
The tablets, troches, pills, capsules and the like may also contain the
following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin;
excipients, such
as dicalcium phosphate; a disintegrating agent, such as corn starch, potato
starch,
alginic acid and the like; a lubricant, such as magnesium stearate; and a
sweetening
agent, such as sucrose, lactose or saccharin may be added or a flavoring
agent, such as
peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form
is a
capsule, it may contain, in addition to materials of the above type, a liquid
carrier.
Various other materials may be present as coatings or to otherwise modify the
physical form of the dosage unit. For instance, tablets, pills, or capsules
may be
coated with shellac, sugar or both. A syrup of elixir may contain the active
compounds sucrose as a sweetening agent methyl and propylparabens as
preservatives, a dye and flavoring, such as cherry or orange flavor. Of
course, any
material used in preparing any dosage unit form should be pharmaceutically
pure and
substantially non-toxic in the amounts employed. In addition, the active
compounds
may be incorporated into sustained-release preparation and formulations.
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Tablets of various sizes can be prepared, e.g., of about 30 to 900 mg in total
weight, containing one or more of the active substances in the ranges
described above,
with the remainder being a physiologically acceptable carrier of other
materials
according to accepted pharmaceutical practice. These tablets can, of course,
be scored
5 to provide for fractional doses. Gelatin capsules can be similarly
formulated. Liquid
formulations can also be prepared by dissolving or suspending one or the
combination
of active substances in a conventional liquid vehicle acceptable for
pharmaceutical
administration so as to provide the desired dosage in one to four
teaspoonfuls. Such
dosage forms can be administered to the patient on a regimen of one to four
doses per
10 day.
According to another modification, in order to more finely regulate the dosage
schedule, the active substances may be administered separately in individual
dosage
units at the same time or carefully coordinated times. Since blood levels are
built up
and maintained by a regulated schedule of administration, the same result is
achieved
15 by the simultaneous presence of the two substances. The respective
substances can be
individually formulated in separate unit dosage forms in a manner similar to
that
described above.
In formulating the compositions, the active substances, in the amounts
described above, are compounded according to accepted pharmaceutical practice
with
a physiologically acceptable vehicle, carrier, excipient, binder,
preservative, stabilizer,
flavor, etc., in the particular type of unit dosage form.
The active compounds may also be administered parenterally or
intraperitoneally. Solutions of the active compounds as free base or
pharmacologically acceptable salts can be prepared in water suitably mixed
with a
surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared
in
glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under
ordinary
conditions of storage and use, these preparations contain a preservative to
prevent the
growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of
sterile injectable solutions or dispersions. In all cases the form must be
sterile and
must be fluid to the extent that easy syringability exists. It must be stable
under the
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conditions of manufacture and storage and must be preserved against the
contaminating action of microorganisms, such as bacteria and fungi. The
carrier can
be a solvent or dispersion medium containing, for example, water, ethanol,
polyol (for
example, glycerol, propylene glycol, and liquid polyethylene glycol, and the
like),
suitable mixtures thereof, and vegetable oils. The proper fluidity can be
maintained,
for example, by the use of a coating, such as lecithin, by the maintenance of
the
required particle size in the case of dispersion and by the use of
surfactants. The
prevention of the action of microorganisms can be brought about by various
antibacterial and antifungal agents, for example, parabens, chlorobutanol,
phenol,
sorbic acid, thimerosal, and the like. In many cases, it will be preferable to
include
isotonic agents, for example, sugars or sodium chloride. Prolonged absorption
of the
injectable compositions can be brought about by the use in the compositions of
agents
delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active
compounds
in the required amount in the appropriate solvent with various of the other
ingredients
enumerated above, as required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized active
ingredients into
a sterile vehicle which contains the basic dispersion medium and the required
other
ingredients from those enumerated above. In the case of sterile powders for
the
preparation of sterile injectable solutions, the preferred methods of
preparation are
vacuum-drying and freeze-drying techniques which yield a powder of the active
ingredient plus any additional desired ingredient from a previously sterile-
filtered
solution thereof.
The dihydrohonokiol compounds) described above may be administered in
the dosage forms as described above in single or divided doses of one to four
times
daily. It may be advisable to start a patient on a low dose combination and
work up
gradually to a high dose combination.
The formulations as described above may be administered for a prolonged
period, that is, for as long as the potential for onset of anxiety remains or
the
symptoms of anxiety continue. Sustained release forms of such formulations
which
may provide such amounts biweekly, weekly, monthly and the like may also be
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employed. A dosing period of at least one to two weeks are required to achieve
minimal benefit.
Formulations suitable for rectal administration are preferably presented as
unit
dose suppositories. These may be prepared by admixing the active compound with
one or more conventional solid carriers, for example, polyalkylene glycols or
triglycerides; such suppositories may be formed from mixtures containing the
active
ingredient in the range of about 0.5% to about 10%, preferably about 1 to
about 2%.
Formulations suitable for transdermal administration may be presented as
discrete patches adapted to remain in intimate contact with the epidermis of
the
recipient for a prolonged period of time. Such patches suitably contain the
active
compound as an optionally buffered aqueous solution of, for example, 0.1 to
0.2M
concentration with respect to the said active compound.
Formulations suitable for transdermal administration may also be delivered by
iontophoresis (see, for example, Pharmaceutical Research, 3(6):318, 1986) and
typically take the form of an optionally buffered aqueous solution of the
active
compound. Suitable formulations comprise citrate or bis tris buffer (pH 6) or
ethanol/water and contain from 0.1 to 0.2M active ingredient.
The compositions are administered in a manner compatible with the dosage
formulation, and in such amount as will be therapeutically effective. The
quantity to be
administered depends on the subject to be treated, including, e.g., age,
physical
condition and degree of symptoms presented. Precise amounts of active
ingredient
required to be administered depend on the judgment of the practitioner.
However,
suitable dosage ranges are of the order of several hundred micrograms active
ingredient
per dose. Suitable regimes for initial administration and booster doses are
also variable,
but are typified by an initial administration followed by subsequent
administrations.
Clinical doses will of course be determined by the nutritional status, age,
weight and health of the patient. The quantity and volume of the composition
administered will depend on the subject and the route of administration. The
precise
amounts of active compound required will depend on the judgment of the
practitioner
and may be peculiar to each individual. However, in light of the data
presented
herein, the determination of a suitable dosage range for use in humans will be
straightforward.
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As used herein, "pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal agents,
isotonic and
absorption delaying agents and the like. 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. Supplementary active ingredients can
also
be incorporated into the compositions.
4.2.1 Liposomes and Nanocapsules
In certain embodiments, the inventors contemplate the use of liposomes and/or
nanocapsules for the introduction of one or more of the disclosed
pharmaceutical
composition into a host cell. Such formulations may be preferred for the
introduction
of pharmaceutically-acceptable formulations of the honokiol derivatives and/or
analogs disclosed herein.
The formation and use of liposomes is generally known to those of skill in the
art (see for example, Couvreur et al., 1977 which describes the use of
liposomes and
nanocapsules in the targeted antibiotic therapy of intracellular bacterial
infections and
diseases). More recently, liposomes were developed with improved serum
stability
and circulation half-times (Gabizon and Papahadjopoulos, 1988; Allen and
Choun,
1987).
In one instance, the disclosed composition may be entrapped in a liposome.
Liposomes are vesicular structures characterized by a phospholipid bilayer
membrane
and an inner aqueous medium. Multilamellar liposomes have multiple lipid
layers
separated by aqueous medium. The term "liposome" is intended to mean a
composition arising spontaneously when phospholipids are suspended in an
excess of
aqueous solution. The lipid components undergo self-rearrangement before the
formation of closed structures and entrap water and dissolved solutes between
the
lipid bilayers (Ghosh and Bachhawat, 1991).
Nanocapsules can generally entrap compounds in a stable and reproducible
way (Henry-Michelland et al., 1987). To avoid side effects due to
intracellular
polymeric overloading, such ultrafine particles (sized around 0.1 pm) should
be
designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-
cyano-
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acrylate nanoparticles that meet these requirements are contemplated for use
in the
present invention, and such particles may be are easily made, as described
(Couvreur
et al., 1977; 1988). Methods of preparing polyalkyl-cyano-acrylate
nanoparticles
containing biologically active substances and their use are described in U.S.
Patent
4,329,332, U.S. Patent 4,489,055, and U.S. Patent 4,913,908.
Pharmaceutical compositions containing nanocapsules for the oral delivery of
active agents are described in U.S. Patent 5,500,224 and U.S. Patent
5,620,708.
U.S. Patent 5,500,224 describes a pharmaceutical composition in the form of a
colloidal suspension of nanocapsules comprising an oily phase consisting
essentially
of an oil containing dissolved therein a surfactant and suspended therein a
plurality of
nanocapsules having a diameter of less than 500 nanometers. U.S. Patent
5,620,708
describes compositions and methods for the oral administration of drugs and
other
active agents. The compositions comprise an active agent carrier particle
attached to a
binding moiety which binds specifically to a target molecule present on the
surface of
a mammalian enterocyte. The binding moiety binds to the target molecule with a
binding affinity or avidity sufficient to initiate endocytosis or phagocytosis
of the
particulate active agent carrier so that the carrier will be absorbed by the
enterocyte.
The active agent will then be released from the carrier to the host's systemic
circulation. In this way, degradation of the disclosed pharmaceutical
compounds in
the intestines can be avoided while absorption of compound form the intestinal
tract is
increased.
An excellent review of nanoparticles and nanocapsular carriers is provided by
Arshady 1996. Arshady notes that one of the major obstacles to the targeted
delivery
of colloidal carriers, or nanocapsules, is the body's own defense mechanism in
capturing foreign particles by the reticuloendothelial system (RES). This
means that
following intravenous administration, practically all nanometer size particles
are
captured by the RES (mainly the liver). The review describes recent
initiatives on the
design of macromolecular homing devices which seem to disguise nanoparticles
from
the RES and, hence, are of potential interest to the targeted delivery of
nanocapsular
carriers. The idea is based on a graft copolymer model embodying a link site
for
attachment to the carrier, a floating pad for maintaining the particles afloat
in the
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blood stream, an affinity ligand for site-specific delivery and a structural
tune for
balancing the overall structure of the homing device.
Yu and Chang, 1996 describe the use of nanocapsules containing hemoglobin
as potential blood substitutes. They use different polymers including
polylactic acid
5 and polyisobutyl-cyanoacrylate and modify the surface of the nanocapsules
with
polyethylene glycol (PEG) or with PEG 2000 PE. The surface modified
nanocapsules
containing hemoglobin survive longer in the circulation.
U.S. Patent 5,451,410 describes the use of modified amino acid for the
encapsulation of active agents. Modified amino acids and methods for the
preparation
10 and used as oral delivery systems for pharmaceutical agents are described.
The
modified amino acids are preparable by reacting single amino acids or mixtures
of two
or more kinds of amino acids with an amino modifying agent such as benzene
sulfonyl
chloride, benzoyl chloride, and hippuryl chloride. The modified amino acids
form
encapsulating microspheres in the presence of the active agent under sphere-
forming
15 conditions. Alternatively, the modified amino acids may be used as a
carrier by
simply mixing the amino acids with the active agent. The modified amino acids
are
particularly useful in delivering peptides or other agents which are sensitive
to the
denaturing conditions of the gastrointestinal tract.
20 4.3 Diagnostic and Therapeutic Kits
Therapeutic kits comprising dihydrohonokiol, its derivatives, analogs and
homologs, comprise another aspect of the present invention. Such kits will
generally
contain, in suitable container means, a therapeutically-effective amount of a
pharmaceutically acceptable composition of dihydrohonokiol, its derivatives,
analogs
or homologs, and a pharmaceutically acceptable excipient. The
diagnostic/therapeutic
kits comprising the pharmaceutical compositions disclosed herein will
generally
contain, in suitable container means, a therapeutically-effective amount of
dihydrohonokiol, its derivatives, analogs and homologs, in a pharmaceutically
acceptable excipient. The kit may have a single container means that contains
the
dihydrohonokiol, its derivatives, analogs and homologs, and a suitable
excipient or it
may have distinct container means for each compound.
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The components of the kit may be provided as liquid solution(s), or as dried
powder(s). When the components are provided in a liquid solution, the liquid
solution
is an aqueous solution, with a sterile aqueous solution being particularly
preferred.
When reagents or components are provided as a dry powder, the powder can be
reconstituted by the addition of a suitable solvent. It is envisioned that the
solvent
may also be provided in another container means.
When the components of the kit are provided in one or more liquid solutions,
the liquid solution is an aqueous solution, with a sterile aqueous solution
being
particularly preferred. The dihydrohonokiol, its derivatives, analogs and
homologs,
may also be formulated into a syringeable composition. In which case, the
container
means may itself be a syringe, or other such like apparatus, from which the
formulation may be administered into the body, preferably by injection or even
mixed
with the other components of the kit prior to injection. The dihydrohonokiol,
its
derivatives, analogs and homologs, to be administered may be a single
compound, or
a composition comprising two or more of dihydrohonokiol, its derivatives,
analogs
and homologs, in a single or multiple dose for administration. Alternatively,
one or
more dihydrohonokiol, its derivatives, analogs and homologs, may be
administered
consecutively or concurrently with other agents as deemed appropriate by the
clinician. Dosage of each of the compositions will vary from subject to
subject
depending upon severity of conditions, size, body weight, etc. The calculation
and
adjustment of dosages of pharmaceutical compositions is well-known to those of
skill
in the art.
In an alternate embodiment, components of the kit may be provided as dried
powder(s). When reagents or components are provided as a dry powder, the
powder
can be reconstituted by the addition of a suitable solvent. It is envisioned
that the
solvent may also be provided in another container means.
The container means will generally include at least one vial, test tube,
flask,
bottle, syringe or other container means, into which the dihydrohonokiol, its
derivatives, analogs and homologs, may be placed, preferably, suitably
allocated.
Where two or more of dihydrohonokiol, its derivatives, analogs and homologs,
are
provided, the kit will also generally contain a second vial or other container
into
which this additional compound may be formulated. The kits may also comprise a
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second/third container means for containing a sterile, pharmaceutically
acceptable
buffer or other diluent.
The kits of the present invention will also typically include a means for
containing the vials in close confinement for commercial sale, such as, e.g.,
injection
or blow-molded plastic containers into which the desired vials are retained.
Alternatively, the vials may be prepared in such a way as to permit direct
introduction
of the composition into an intravenous drug delivery system.
Irrespective of the number or type of containers, the kits of the invention
may
also comprise, or be packaged with, an instrument for assisting with the
injection/administration or placement of the ultimate dihydrohonokiol, its
derivatives,
analogs and homologs, composition within the body of an animal. Such an
instrument
may be a syringe, pipette, forceps, measured spoon, eye dropper or any such
medically
approved delivery vehicle.
5.0 EXAMPLES
5.1 Example 1 ~ Synthesis of Dihydrohonokiols
5.1.1 Materials and Methods:
Materials
4-Allyl anisole, t-butyllithium ( 1.7 M solution in anhydrous pentane),
anhydrous tetrahydrofuran, anhydrous dichloromethane, anhydrous zinc chloride
( 1.0 M solution in anhydrous diethyl ether), bis(triphenylphosphine)
palladium(II)chloride, diisobutylaluminum hydride ( 1.0 M solution in hexane),
hexane, tris-(tiphenylphosphine) rhodium(I) chloride, toluene, deuterated
chloroform,
tetramethylsilane and florisil were purchased from Aldrich Chemical Co.
(Milwaukee,
Wisconsin).
Honokiol was obtained from Wako Pure Chemical Industries Ltd. (Osaka,
Japan). Alternatively, honokiol may be extracted by supercritical carbon
dioxide
extraction from suitable portions of Magnolia trees (Chandra and Nair, 1995).
Tetrahydrohonokiol was prepared by the hydrogenation in the presence of
5°70
Palladium on active carbon catalyst as described by Fujita et al. (1972).
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2-Propyl-4-iodo anisole was obtained from Rann Research Laboratory (San
Antonio, Texas).
Ammonium chloride, sodium chloride, anhydrous diethyl ether and anhydrous
sodium sulfate were purchased from Fisher Scientific (Fair Lawn, New Jersey).
HPLC grade Ethyl acetate, acetone, water and acetonitrile were obtained from
Burdick
and Jackson Inc. (Muskegon, Michigan).
Analytical and preparative TLC plates (20 x 20 cm glass plates coated with
250 micron and 1 mm thickness of silica gel (G) were purchased from Analtech
Inc.
(Newark, Delaware).
HPLC
The analytical and preparative high pressure liquid chromatography were
performed on a Milton Roy HPLC equipment consisting of a CM 4000 solvent
delivery system, the autoinjector A1000 and the spectro-monitor 3100 variable
wavelength detector from LCD Analytical (Riviera Beach, Florida). A reverse
phase
HPLC column (Econosil C 18, l Op, 250 x 10 mm, Catalog #C231 ) was purchased
from Alltech Associates, Inc. (Deerfield, Illinois). The solvent for elution
consisted of
a mixture of acetonitrile and water (6:4). The elution of the honokiol and its
analogs
was detected at a wavelength of 280 nm. The chart speed was 0.5 cm/min,
solvent
flow rate was 5 ml/min and the detection was performed at the sensitivity of
0.05 and
2.0 absorbance units full scale (for analytical and preparative use,
respectively). The
proton magnetic resonance spectra were recorded on a 300 MHz GE QE 300 NMR
spectrometer. The samples were dissolved in deuterated chloroform and
tetramethylsilane was used as the internal standard.
5.1.2 Synthesis of 3-n-Propyl-5'-(2-propenyl)-1,1'-biphenyl-2',4-diol (I) and
5'-
n-Propyl-3-(2-propenyl)-1,1'-biphenyl-2',4-diol (II) by the Partial
Hydrogenation of Honokiol:
Hydrogen gas was passed for two hours with stirring in a solution of
tris-(triphenylphosphine) rhodium(I) chloride ( 10 mg) in toluene (4 ml) at
room
temperature. Then, honokiol (20 mg) dissolved in toluene (1 ml) was added
slowly
into the reaction flask with stirring and the hydrogen gas was passed for
another 10
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min after the complete addition of honokiol solution. The reaction mixture was
stirred overnight at room temperature. The reaction mixture was passed through
a
column of florisil (5 gm), which was finally washed with 50 ml of dry diethyl
ether.
The solvents were evaporated to obtain crude residue which was resuspended in
aforesaid HPLC eluant to be injected in the HPLC system for analysis. The HPLC
analysis indicated the presence of three peaks:
(a) peak 1: Retention time- 9 min, honokiol.
(b) peak 2 (86.4% based on honokiol consumed): Retention time- 11 min.
(c) peak 3 ( 13.6% based on honokiol consumed): Retention time- 14.2
min, m/z 270, confirmed to be tetrahydrohonokiol when compared
with standard tetrahydrohonokiol.
The compounds eluting as peak 2 were identified as the two isomers of
dihydrohonokiol. The first component of the peak (up to the begining of the
shoulder)
was 92% of the total peak, [m/z 268; 1H NMR(CDC13): 8: 0.994 (t,3H), 1.663
(dt,3H), 2.629 (t,2H), 3.347 (d,2H), 5.03-5.11 (m,2H), 5.90-6.04 (m,lH), 6.88-
7.20
(m, 6H) with a small shoulder (approximately 8% of total peak 2) [1H
NMR(CDC13):
8: 0.994 (t,3H), 1.663 (dt,3H), 2.55(t,2H), 3.45(d,2H), 5.13(m,2H), 5.90-6.04
(m,lH),
6.88-7.20 (m,6H). The dihydrohonokiol isomers in peak 2 were isolated by
preparative HPLC purification. The major component (approximately 92%) was
assigned the structure as 3-n-propyl-5'-(2-propenyl)-1,1'-biphenyl-2',4-diol
(I), while
the minor component (approximately 8%). was assigned the structure as
5'-n-propyl-3-(2-propenyl)-l,l'-biphenyl-2',4-diol, (II). The HPLC retention
times for
the purified isomers was 11 min for isomer I and 12 min for isomer Ii. A
typical
reaction resulted in a product mixture consisting of 55% dihydrohonokiols-,
35.5%
unreacted honokiol and 9.5% tetrahydrohonokiol. The dihydrohonokiol isomers
represented approximately 86% of the dihydrohonokiol consumed in the reaction.
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HZ O O O O
OH ~ ~OH
OH (ph3p)sRhCl
t n
Synthesis of 3-h-propyl-5'-(2-propenyl)-1,1'-biphenyl-2',4-diol (I) and
5 5'-n-propyl-3-(2-propenyl)-1,1'-biphenyl-2',4-diol (II) from Honokiol by
Partial
Hydrogenation
5.1.3 Total Synthesis of 3-n-Propyl-5'-(2-propenyl)-1,1'-biphenyl-2',4-diol
(I)
and 5'-n-Propyl-3-(2-propenyl)-1,1'biphenyl-2',4-diol (II)
10 5.1.3.13-n-Propyl-5'-(2-propenyl)-1,1'-biphenyl-2',4-diol (I):
To a solution of 4-allylanisole (148 mg, 1.0 mmol) in 1 ml anhydrous
tetrahydrofuran was added a solution of tert-butyllithium in pentane ( 1.5
mmol; as
0.88 ml of 1.7 M solution in anhydrous pentane) with stirring under the
nitrogen
atmosphere at -78°C. After two hours, the mixture was warmed to -
10°C and the
15 solution was reacted with anhydrous zinc chloride ( 1 ml, 1.0 mmol; as 1.0
M solution
in anhydrous diethyl ether). Then, the reaction mixture was stirred at room
temperature for 1 h. The palladium catalyst was prepared separately. Thus, an
anhydrous hexane solution of diisolbutyl aluminum hydride (0.066 ml, 0.066
mmol;
1.0 M solution in hexane) was added to a solution of
20 bis-(triphenylphosphine)palladium(II) chloride (22 mg; 0.033 mmol) in 1 ml
anhydrous tetrahydrofuran with stirring under nitrogen atmosphere. This
organic
catalyst solution was then reacted with 2-propyl-4-iodoanisole ( 193 mg, 0.70
mmol)
in 2 ml of anhydrous tetrahydrofuran and a solution of the aryl zinc chloride,
prepared
as above. The mixture was stirred for 2 h at room temperature and then
quenched
25 with 5 ml saturated ammonium chloride solution. The aqueous layer was
extracted
with three 10 ml portions of ethyl acetate. The combined extract was washed
once
with brine and dried over anhydrous sodium sulphate. After the removal of
solvent in
vacuo, the crude product was chromatographed on a 1 mm thickness 20 x 20 cm
silica
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gel plate (silica gel G; eluent: hexane, ethyl acetate; 9:1) to get 2',4-
dimethoxy-
3-n-propyl-5'-(2-propenyl)-1,1'-biphenyl (95 mg, 45.9%; based on the amount of
2-propyl-4-iodoanisole): m/z: 296; 'H NMR (CDCl3): 8: 0.90 (t, 3H,
3-CH2.CHZ.CH3), 1.45 (qt,3H, 3-CH2.CHZ.CH3), 2.53 (t,2H, 3-CH2.CH~.CH3), 3.38
(d,2H, 5'-CH?.HC=CHI), 3.71 (s, 3H, 4-OCH3), 3.77 (s, 3H, 2'-OCH3), 4.99
(dd,2H,
5'-CH2.HC=CH2), 5.85-5.98 (m, 1H, 5'-CHZ.HC=CHZ), 6.79-7.28 (m, 6H, aromatic
H).
To a solution of 2',4-dimethoxy-3-n-propyl-5'-(2-propenyl)-1,1'-biphenyl
( 10 mg: 0.03 mmol) in 1 ml of anhydrous dichloromethane was slowly added a
dichloromethane solution of boron tribromide (75 microliters, 1M solution in
dichlormethane) at -78°C with magnetic stirring for one h. Then, the
reaction mixture
was stirred at room temperature overnight. Next day, the reaction was cooled
and
subjected to an aqueous quenching. The aqueous phase was extracted three times
with 2 ml portions of dichloromethane. The combined organic layer was washed
once
with brine ( 1 ml) and dried over anhydrous sodium sulfate. The solid was
filtered off
and the solvent was evaporated under nitrogen. The crude product was
chromatographed on a 250 micro thickness 20 x 20 cm silica gel plate (silica
gel G;
eluent: hexane, ethylacetate, 80:20) to get 3-n-
propyl-5'-(2-propenyl)-1,1'-biphenyl-2',4-diol (6.5 mg; 72%). In a HPLC
analysis, the
retention time of total synthetic 3-n-propyl-5'-(2-propenyl)-l,l'- biphenyl-
2',4-diol
was comparable to that of the hemi-synthetic sample made earlier; and in a
mixed
co-injection both co-eluted together. The structure was further confirmed by
the
proton magnetic resonance analysis on a HPLC pure sample. 'H NMR (CDC13): 8:
0.994 (t, 3H, 3-CH2.CHZ.CH3), 1.663 (qt,3H, 3-CH2.CHZ.CH3), 2.629 (t,2H,
3-CH2.CH2.CH3), 3.347 (d,2H, 5'-CH2.HC=CHZ), 5.03-5.11 (dd,2H,
5'CH~.HC=CHZ), 5.90-6.04 (m,lH, 5'-CH2.HC=CH2), 6.88-7.20 (m, 6H, aromatic H).
5.1.3.2 5'-n-Propyl-3-(2-propenyl)-l,l'biphenyl-2',4-diol (II):
The same method as described above in ~ 5.1.3.1 for the synthesis
2',4-dimethoxy-3-n-propyl-5'-(2-propenyl)-1,1'-biphenyl can be used to
synthesize
2'4-dimethoxy-5'-n-propyl-3-(2-propenyl)-1,1'-biphenyl. Thus, 4-propylanisole
is
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reacted with tert-butyllithium and anhydrous zinc chloride to form an aryl
zinc
chloride which is then coupled with 2-allyl-4-iodo-anisole, in the presence of
the
palladium catalyst made in a separate reaction of bis-
(triphenylphosphine)palladium(II) chloride and diisolbutyl aluminum hydride.
The
product, 2'4-dimethoxy-5'-n-propyl-3-(2-propenyl)-1,1'-biphenyl, is purified
by
extraction and thin layer chromatography as described above.
The methyl protecting groups are removed from 2'4-dimethoxy-5'-n-propyl-3-
(2-propenyl)-l,l'-biphenyl as described above in ~ 5.1.3.1 for
3-n-propyl-5'-(2-propenyl)-1,1'- biphenyl-2',4-diol. The product,
5'-n-propyl-3-(2-propenyl)-1,1'biphenyl-2',4-diol (II), is purified by
extraction and
thin layer chromatography, as described above.
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OCH3
OCH3 OCH3 OCH3
ZnCI
t.bu.
ZnCl2 _
I OCH3
(Ph3P)2PdC12
DIBALH I BBr3
OH
OCH3
OCH3 OCH3 OCH3
ZnCI
t.b~
ZnCl2 _
I OCH3
(Ph3P)ZPdCl2
DIBALH BBr3
OH
Total synthesis of 3-h-propyl-5'-(2-propenyl)-1,1'-biphenyl-2',4-diol (I) and
5'-
n-propyl-3-(2-propenyl)-1,1'-biphenyl-2',4-diol (II)
5.2 Example 2 ~ Synthesis of Analogs and Homologs of Dihydrohonokiol
For the synthesis of analogs and homologs of dihydrohonokiol, 4-allylphenyl
alkyl ethers can be converted to 2-alkoxy, 5-allylphenyl metal halides by a
directed
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ortho metalation reaction, which in turn is reacted with various 4-
alkoxyhaloaryls in
situ to generate the homologs and analogs of 3-n-propyl, 5'-(2-propenyl)-1,1'-
biphenyl-2',4-diol. Similarly, various substituted-phenyl metal halides are
reacted in
situ with various 5'-n-propyl, 3-(2-propenyl)-1,1'-biphenyl-2',4-diol.
Carboxylic groups may be protected as esters (e.g., ethyl ester) during the
synthetic process. The dihydrohonokiol analog formed after the coupling
reaction and
demethylation can be deprotected by based catalyzed hydrolysis of the ester
group.
Thus, for example, for the synthesis of 3-n-propyl-5'-(3-carboxy propyl)-1,1'-
biphenyl-2',4-diol, the starting compound 3-(4-methoxyphenyl)-propionic acid,
available from Aldrich (Milwaukee, WI) can be converted to its acid chloride
and then
to the ethyl ester, i.e. 3-(4-methoxyphenyl)-propionate. This compound may be
treated with tert-butyllithium followed by anhydrous zinc chloride to form the
intermediate 2-methoxy, 5-(3-ethoxy carbonyl propyl)-phenyl zinc chloride.
This
intermediate can be coupled with 2-n-propyl-4-iodo anisole in the presence of
the
palladium catalyst (prepared in situ by the reaction of bis-(triphenyl
phosphine)
palladium (II) chloride and diisobutyl aluminum hydride) to synthesize 2',4-
dimethoxy-3-n-propyl-5'-(3-ethoxycarbonyl propyl)-1,1'-biphenyl. The
demethylation
of this compound with borontribromide will give 3-n-propyl-5'-(3-
ethoxycarbonyl
propyl)-1,1'-biphenyl-2',4-diol. Finally, the base catalyzed hydrolysis of the
ester
group, e.g., with potassium hydroxide, will generate 3-n-propyl-5'-(3-carboxy
propyl)-
1,1'-biphenyl-2',4-diol.
Alcoholic groups may be protected as acyl esters, e.g., by base catalyzed
acetylation with acetic anhydride. The dihydrohonokiol analog formed after the
coupling and demethylation can be deprotected by base catalyzed removal of
acyl
groups. Thus, for example, for the synthesis of 3-n-propyl-5'-(2-hydroxy
propyl)-l,l'-
biphenyl-2',4-diol, the intermediate 1-(4-methoxyphenyl)-2-propanol is made by
sodium borohydride reduction of 4-methoxyphenyl acetone, available from
Aldrich
(Milwaukee, WI). The 1-(4-methoxyphenyl)-2-propanol can be reacted with acetic
anhydride in basic medium to get 1-(4-methoxyphenyl)-2-propyl acetate. This
compound may be treated with tert-butyllithium followed by anhydrous zinc
chloride
to get 2-methoxy, 5-(2-acetoxypropyl)-phenyl zinc chloride as an intermediate.
This
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intermediate can be coupled with 2-n-propyl-4-iodo anisole in the presence of
the
palladium catalyst (prepared in situ by the reaction of bis-(triphenyl
phosphine)
palladium (II) chloride and diisobutyl aluminum hydride) to synthesize 2',4-
dimethoxy-3-n-propyl-5'-(2-acetoxypropyl)-1,1'-biphenyl. The demethylation of
this
5 compound with borontribromide will yield 2',4-dimethoxy-3-n-propyl-5'-(2-
acetoxypropyl)-1,1'-biphenyl-2',4-diol. Finally the base catalyzed hydrolysis
of the
acetyl group will generate 3-n-propyl-5-(2-hydroxy propyl)-1,1-biphenyl-2,4-
diol.
5.3 Example 3 ~ Anxiolytic Effect of a Mixture of Dihydrohonokiol Isomers in
10 the Elevated Plus-Maze Test in Mice
5.3.1 Materials and Methods
5.3.1.1 Animals
Male mice of the Balb/C strain (Harlan, Indianapolis, 1N) were used at about
6 wk of age and weighing of 22-27 g. Groups of 5 mice each were housed in
standard
15 polycarbonate cages ( 15W x 25Lx 12H cm) with woodchip bedding with free
access
to a standard solid diet and tap water. The environment of the animal room was
well
controlled with a temperature of 23~1°C, humidity 40-55% and a 12:12-h
light-dark
cycle; lights on between 0600-1800h).
All the experimental procedures were carried out according to "The Guide for
20 the Care and Use of Laboratory Animals and Animal Welfare Act, and the
experimental protocol (97031-34-O1-A) was approved by the Institutional Animal
Care and Use Committee of the University of Texas Health Science Center at San
Antonio.
25 5.3.1.2 Drugs
For oral administration, the mixture of dihydrohonokiol isomers I and II
prepared by the partial hydrogenation of honokiol (designated I/11 mixture) as
described in ~ 5.1.2, was first dissolved in a very small amount of ethanol,
and the
solution was diluted with physiological saline containing Tween-80 (0.1 %)
(vehicle).
30 Diazepam was suspended in the Tween-80/physiological saline. The
concentration of
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each drug solution or suspension was adjusted so that each volume administered
was
constant at 0.1 ml/10 g body weight of the mouse.
5.3.1.3 Apparatus and Experimental Procedures
The elevated plus-maze test: The elevated plus-maze used was the same as
that used in previous studies (Kuribara et al., 1996; Maruyama et al., 1997),
and was
an improvement of the original apparatus for rats (Pellow et al., 1985) and
for mice
(Lister, 1987). Briefly, the plus-maze consisted of two closed-arms and two
open-arms (6W x 30L cm). The arms extended from a center platform (8 x 8 cm).
The closed arms had side-walls ( l OH cm), and they were made of non-
transparent
polyvinyl chloride of gray color. The open arms were made of transparent
acrylic
fiber, and had no side-walls. The platform was made of non-transparent
polyvinyl
chloride of gray color. This plus-maze was set 40 cm above the base. Each
mouse
was placed at the center platform facing one of the closed arms, and the
cumulative
time of entering into the open arms during the 5 min observation period was
recorded.
The criterion of the mouse's entering into the open arms was crossing with all
four
paws the borderline separating the open arm and the center platform.
Activity test: Mouse activity was measured with a tilting-type ambulometer
which had a bucket-like Plexiglas activity cage 20 cm in diameter (SMA-1:
O'Hara
and Co., Tokyo). Only a horizontal movement (ambulation) of the mouse caused a
slight tilt of the activity cage, and was detected by microswitches attached
to the cage.
The duration of measurement was 5 min for each mouse. This activity test was
carried out immediately after the end of the plus-maze test.
Traction test: The traction test was carried out to assess muscle strength.
This
experimental procedure was described elsewhere (Kuribara et al., 1977).
Briefly, a
wire ( 1.6 mm in diameter, and 30 cm in length) was set horizontally 30 cm
above the
base. The mouse was first allowed to grip the wire with the fore paws, and the
duration of clinging to the wire was measured up to 60 sec. The trial was held
twice
for each mouse, and the longer duration of clinging was used in the
calculation of
mean value. When the duration of clinging was over 60 sec, the mouse was
released
from the wire, and the clinging time was recorded as 60 sec. The traction test
was
carried out immediately after the end of the activity test.
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5.3.1.4 Statistical Analysis
The time in open arms in the elevated plus-maze test, the activity counts in
the
activity test, and the durations of clinging in the traction test were
compared by the
Fisher's protected least significant difference statistical test. Values of P
less than
0.05 were considered significant.
5.3.2 Dose Response of Dihydrohonokiol Isomers I/II Mixture
TABLE 1
Dose Effect in the Mouse of I/II Mixture Assessed Using the Elevated
Plus-Maze and Motor Activity
Treatment, P.O.Time (sec) Motor Activity
spent
in open
arm
mg/kg 1 h post 3 h post adm. 1 h post 3 h post
adm. adm. adm.
Vehicle 13.2 13.1 4.7 30.6 1.7 31.2
2.7 1.5
I/II 0.1 26.0 8.4 30.5
1.5
1/>I 0.2 27.66.2 37.9 6.2* 36.12.3 21.93.3~
I/11 0.5 33.84.9 87.312.8* 32.72.7 26.01.8
1/II 1.0 53.87.7 90.0 9.2* 32.01.7 30.81.5
I/II 2.0 50.25.7 95.3 8.7* 34.62.0 27.01.7
The data for the plus-maze are mean times ~ SEM spent in the open arm
during the observation period of 5 min. The data for the activity test are
mean counts
~ SEM during the observation period of 5 min. The group size was 10. *p<0.002.
Increasing doses of the 1/II isomer mixture cause an increasing anxiolytic
effect- a
dose/effect relationship. A 0.2 mg/kg oral dose of the I/II mixture shows
significant
anxiolytic effects at 3 h post administration, while a higher dose of 1 mg/kg
is
significantly anxiolytic at only one hour. Since the inventors used the 0.2
mg/kg dose
orally in this series the tests were performed 3 h after dosing of the I/II
mixture.
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Motor activity was unchanged by dose or time except for the 0.2 mg/kg dose at
3 h
post administration.
5.3.3 Duration of I/II Mixture Induced Anxiolytic Activity
TABLE 2
Duration of Pharmacological Action Following 0.5 mg/kg I/II Mixture Given
Orally
Treatment Hours post treatmentTime spent in Motor activity
mg/kg open arm, sec
Vehicl 0 12.9 3.8 26.9 2.1
a
I/II 0.5 1/2 15.5 3.7 28.6 1.7
1/ll 0.5 1 33.84.9 32.72.7
1/B 0.5 2 43.0 7.5 * 25.8 1.1
I/11 0.5 3 87.3 12.8* 26.0 1.8
I/II 0.5 4 77.7 13.2* 26.92.0
I/>I 0.5 6 37.4 11.0 22.3 1.8
I/11 0.5 8 46.2 9.1 * 27.5 2.1
I/lZ 0.5 12 47.0 13.1 * 24.0 2.0
1/II 0.5 24 14.0 5.0 17.3 3.1
I/1Z 0.5 48 11.5 4.5 27.3 1.5
The data for the plus-maze test are mean times ~ SEM spent in the open arm
during the observation period of 5 min. The data for the activity test are the
mean
counts ~ SEM during the test period of 5 min. The group size was 10 mice.
*p<0.02
vs control vehicle.
The duration of anxiolytic activity of the I/11 mixture following an oral dose
of
0.5 mg/kg extends for at least 12 h post administration, ending between 12 h
and 24 h
after oral administration. The anxiolytic activity was not measured between 12
and
24 h. This long duration of action indicates the possibility of a one single
dose per
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day schedule. Single dosage greatly improves patient compliance. The motor
activity
was unchanged.
5.3.4 Duration of Diazepam Anxiolytic Activity
TABLE 3
Duration of Pharmacological Action Following 1.0 mg/kg Diazepam Given
Orally
Treatment, Hours postTime spent Motor Traction
dose in mg/kgtreatment in activity
open arm,
sec.
Vehicle 0 12.3 2.6 24.9 1.7 60.0 0.0
Diazepam 0.17 43.5 6.1* 38.9 3.2* 42.6 6.3*
1.0
Diazepam 0.5 41.2 5.2* 23.2 3.8 53.3 3.7
1.0
Diazepam 1 29.7 6.9* 32.1 2.3 60.0 0.0
1.0
Diazepam 2 27.0 8.0 32.2 4.1 60.0 0.0
1.0
Diazepam 4 21.7 6.3 31.6 3.1 60.0 0.0
1.0
Diazepam 8 13.2 5.9 27.0 2.4 60.0 0.0
1.0
Diazepam 24 12.2 2.7 29.5 3.4 60.0 0.0
1.0
The data for the plus-maze test are mean times ~ SEM spent in the open arm
during the observation period of 5 min. The data for the activity test are the
mean
counts ~ SEM during the test period of 5 min. The group size was 10 mice.
*p<0.04
vs control.
The benzodiazepine compound diazepam was used as a positive control for
anxiolytic activity in this series of tests because of wide use and its
acceptance as
representative of the benzodiazepine series of compounds. The time of peak
anxiolytic action of diazepam is 0.17 h (10 min.). The anxiolytic tests in the
subsequent series were done on diazepam 10 min after oral dosing to obtain the
peak
response. The duration of diazepam following the effective dose of 1 mg/kg
orally is
shown to be 1 h for its anxiolytic activity and 0.17 h for its effect on motor
activity
and its detrimental affect on traction (ataxia production).
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5.3.5 Effect of Multiple Doses of I/II Mixture and Diazepam
TABLE 4
5 Effect of seven days treatment with 0.2 mg/kg po I/II Mixture and
1.0 mg/kg po diazepam
Treatment Time in open arm Motor activityTraction
(sec)
vehicle 15.65.0 25.93.8 60.00.0
44.97.7 * 31.94.1 60.00.0
vehicle 12. 32.6 24.9 1.7 60.00.0
Diazepam 43.56.1 * 38.93.2* 42.66.2*
*p<0.016 vs vehicle control
The data for the plus-maze are mean times ~ SEM spent in the open arm
10 during the observation period of 5 min. The data for the activity test are
mean counts
~ SEM during the test period of 5 min. Traction was evaluated by the ability
of the
mouse to hold on to a bar for 60 sec. The group size was 10 mice.
Tolerance following chronic use, that is diminished anxiolytic activity
requiring increasing doses in long term use, is evaluated in this data. The
1/II mixture
15 and diazepam were given orally each day for 7 days and then the anxiolytic
activity
was tested. There was no decrease in the effectiveness of either agent. The
dose of
each compound produced the same anxiolytic activity after 7 days of dosing as
occurred after one dose. The I/II mixture still did not change motor activity.
Diazepam still retained its change of motor activity and diminution of
traction.
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5.3.6 Synergistic Effect of Combination of I/II Mixture and Diazepam
TABLE 5
Synergy in the anxiolytic activity of I/II Mixture and Diazepam
Treatment mg/kg Time in open Motor ActivityTraction
arm,
po in sec
Vehicle 13.1 4.7 31.21.5 60.00.0
1/1T 0.2 37.9 6.2* 21.93.1 60.00.0
Diazepam 1.0 43.5 6.1 * 38.93.2# 42.66.3
I/II + Diazepam 137.0 11.0** 27.14.5 44.04.8*
*p<0.02 vs vehicle control.
**p<0.0001 vs I/II mixture or diazepam alone.
#p<0.02 vs I/II mixture + diazepam.
The 1/II mixture was given 3 h before testing and diazepam was given 10 min
before testing. The data for the plus-maze are mean times ~ SEM spent in the
open
arm during the observation period of 5 min. The data for the motor activity
are mean
counts ~ SEM during the test period of 5 min. Traction was evaluated by the
ability
of the mouse to hold on to a bar for 60 sec. The group size was 10 mice.
To establish whether the I/11 mixture and diazepam interact, an effective dose
of the I/II mixture and diazepam were given to separate mice to show the
anxiolytic
activity of each compound when given alone. The effective dose of each
compound
given using appropriate timing was then combined mice. The combination of 1/II
mixture and diazepam resulted in a 3 fold increase in anxiolytic activity over
either
agent given separately. The compounds act synergistically as anxiolytics. The
effect
of diazepam on motor activity is abolished by combining with the I/II mixture
UT2 but
the I/II mixture has no effect on the ataxia produced by diazepam.
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5.3.7 Effect of Coadministration of Flumazenil
TABLE 6
The Effect of the Benzodiazepine Receptor Antagonist Flumazenil
on the Anxiolytic Effect of the I/II Mixture and Diazepam
Treatment Challenge Time in Open Arms
Vehicle saline 13.0 3.4
Vehicle flumazenil 6.7 2.9
I/11 saline 37.9 6.2
lII flumazenil 31.2 3.8
Diazepam saline 43.5 6.1
Diazepam flumazenil 13.5 3.8*
*p<0.0001 vs the mice pretreated only with diazepam.
The time in the open arm of the elevated plus maze was observed during a 5
min period. The 1/II mixture (0.2 mg/kg po) was given 3 h before testing.
Diazepam
( 1 mg/kg po) was given 10 min before testing. Flumazenil (0.3 mg/kg sc) was
given
10 min before testing.
Flumazenil is a benzodiazepine receptor blocking agent which when given
blocks the anxiolytic effects of benzodiazepines. Flumazenil blocks the
anxiolytic
activity of diazepam but does not block the anxiolytic activity of the 1/II
mixture. This
indicates that the I/II mixture does not physiologically act at the same site
as the
benzodiazepine anxiolytics.
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TABLE 7
The Effects of the Benzodiazepine Receptor Antagonist Flumazenil
on Motor Activity and Traction Measured Following
Administration of Diazepam or I/II mixture
Treatment Challenge Motor Activity Traction
Vehicle saline 29.0 2.4 60.0 0.0
Vehicle flumazenil 25.6 3.0 60.0 0.0
I/>I saline 21.9 3.1 60.0 0.0
I/11 flumazenil 28.6 2.7 60.0 0.0
Diazepam saline 38.9 3.2* 42.6
6.3#
Diazepam flumazenil 27.9 1.8** 54.5
2.9##
*p< 0.01 vs control
**p< 0.007 vs diazepam alone
#p<0.0001 vs control
##p<0.004 vs diazepam alone
The data for the activity test are for an observation period of 5 min. The
data
for the traction test are the mean duration of clinging. The I/II mixture (0.2
mg/kg po)
was given three hours before testing. Diazepam ( 1 mg/kg po) was given ten min
before testing as was flumazenil (0.3 mg/kg sc).
Motor activity and traction are not changed by the I/II mixture or flumazenil.
Flumazenil blocks the motor activity effect of diazepam and significantly
decreases
the diazepam alteration of traction. These serious side effects of the
benzodiazepine
anxiolytics appear to reside in that receptor interaction.
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5.3.8 Effect of Coadministration of Bicuculline
TABLE 8
The Effect of the GABA Antagonist Bicuculline Administered
with the I/II mixture and Diazepam
Treatment Bicuculline,Time spent
mg/kg po mg/kg in Motor Traction
open arm, Activity
sec.
Vehicle saline 13.03.5 29.02.4 60.00.0
Vehicle 0.1 7.12.8 21.52.7 60.00.0
I/II 0.2 saline 37.96.2* 21.93.1 60.00.0
I/ll 0.2 0.1 16.75.3 36.12.7 60.00.0
Diazepam saline 43.56.1* 38.93.2* 42.66.3*
1.0
Diazepam 0.1 17.44.9 37.92.3* 46.66.9*
1.0
*p<0.5 vs control vehicle.
The 1/II mixture was given 3 h before testing and diazepam and bicucuIline
were given 10 min before testing. The data for the plus-maze are mean times ~
SEM
spent in the open arms during the observation period of 5 min. The data for
the motor
activity are mean counts ~ SEM during the test period of 5 min. Traction was
evaluated by the ability of the mouse to hold on to a bar for 60 sec. The
group size
was 10 mice.
The GABAergic antagonist bicuculline abolished the anxiolytic activity of
both the I/II mixture and diazepam identifying a common effect on the
GABAergic
system. Bicuculline did not block the effect of diazepam on motor activity or
traction.
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5.3.9 Effect of Coadministration of Caffeine
TABLE 9
Effect of Caffeine on the Pharmacological Activity of Diazepam
5 and I/II Mixture
Treatment Challenge Time in Motor Traction
Open Activity
Vehicle saline 14.03.8 28.92.4 60.00.0
Vehicle Caffeine 13.99.0 60.57.4*** 60.00.0
I/11 saline 37.96.2 21.93.1 60.00.0
I/>I Caffeine 6.12.2* 61.15.0# 60.00.0
Diazepam saline 43.56.1 38.93.2 42.66.3+
Diazepam Caffeine 93.811.3** 55.17.1## 50.45.1+
*p<0.003 vs I/II mixture
* *p<0.0001 vs diazepam
***p<0.0001 vs control
10 #p<0.0001 vs I/>I mixture
##p<0.03 vs diazepam
+ p<0.05 vs vehicle control
I/11 mixture, 0.2 mg/kg, was given po 3 hr before testing. Diazepam 1.0 mg/kg
was given po 10 min before testing. Caffeine 30 mg/kg was given ip 15 min
before
15 testing. The time in sec in the open arms is reported as means ~ SEM during
a 5 min
observation period. The motor activity is reported as mean ~ SEM for the five
min
test period. Traction reports the ability of the mouse to hold to a bar for 60
sec mean
~ SEM. Group size was 10 mice.
Caffeine is an axiogenic agent in high doses and increases motor activity.
20 Given at a high dose with the I/II mixture, caffeine abolished the
anxiolytic activity of
the I/II mixture. A series of doses would be needed to establish a dose effect
curve.
Diazepam combined with caffeine resulted in increased anxiolytic activity and
motor
activity but did not change the ataxia production.
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5.3.10 Effect of Coadministration of CCK
TABLE 10
The Effect of CCK on the Pharmacological Activity of the
I/II Mixture and Diazepam
Treatment Challenge Time in Open Motor Traction
Activity
Vehicle saline 13.7 4.9 27.1 1.9 60.0 0.0
Vehicle CCK 2.2 1.1 29.2 1.9 60.0 0.0
I/11 saline 37.9 6.2 21.9 3.1 60.0 0.0
I/II CCK 11.13.6* 20.23.7 60.00.0
Diazepam saline 43.5 6.1 38.9 3.2 42.6 6.3
Diazepam CCK 57.3 13.0 34.1 5.1 41.3 7.7
*p<0.008 vs I/II mixture alone.
The I/II mixture was given 0.2 mg/kg po 3 h before the test. Diazepam 1.0
mg/kg po and CCK 50 ug/kg ip were given 10 min before the test. The data for
the
plus-maze test are mean ~ SEM time spent in the open arms during the 5 min
observation period. The data for the motor activity are mean counts ~ SEM
during the
5 min test period. Traction was evaluated by the ability of the mouse to hold
on to a
bar for 60 sec. The group size was 10 mice. CCK is cholecystokinin Ac-fragment
26-
29 amide non-sulfated.
Cholecystokinin (CCK) is anxiogenic and its administration is used as a screen
for discovery of new anxiolytic agents. CCK in this initial test shows it
anxiogenic
effect and effectively abolished the anxiolytic effect of I/II mixture. It had
no effect
on any of the three effects of diazepam.
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TABLE 11
The Effect of a Constant Dose of CCK on Three Doses of the I/II Mixture
Treatment Dose mg/kg Challenge Time in Open Motor
Activity
Vehicle 0 saline 13.7 4.9 27.1 1.9
Saline 0 CCK 2.2 1.1 29.2 2.2
I/II 0.2 saline 37.9 6.3 21.9 3.1
I/II 0.2 CCK 11.1 3.5 20.23.7
I/II 0.5 saline 87.3 12.8 26.0 1.8
I/II 0.5 CCK 28.5 5.4* 35.5
4.7#
I/II 2.0 saline 95.3 8.7 27.0 1.7
I/II 2.0 CCK 48.3 15.0*# 32.3 2.1
*p<0.05 vs CCK
# p<0.05 vs control vehicle.
The I/II mixture was given orally 3 h before the test. CCK, 50 ug/kg, was
given ip 10 min before the test. The data for the plus-maze are mean times ~
SEM
spent in the open arms during the 5 min observation period. The data for the
motor
activity are mean counts ~ SEM during the 5 min test interval. The group size
was 10
mice. CCK is cholecystokinin Ac-fragment 26-29 amide non-sulfated.
CCK at a constant dose of 50 micrograms/kg abolishes the anxiolytic activity
of the I/II mixture at a dose of 0.2 mg/kg po. CCK is antagonized in a dose
dependent
manner by 0.5 mg/kg and 2 mg/kg doses of the I/II mixture.
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5.4 Example 4 ~ Evaluation of Dependence Liability of the Dihydrohonokiol
Isomer Mixture
TABLE 12
Number of Mice in Groups of Ten Showing Symptoms When Treated with
Flumazenil Following 12 Days Administration of Diazepam or I/II Mixture
Drug Flumaz. .~ Hr Tr Cc Tc Tf Rf
Saline 0 1 0 0 0 0 0
S aline 10 mg/kg3 0 0 0 1 0
Diazepam 10 7 0 0 0 0 0
0.5 mg/kg
1.0 10 10 0 0 0 0 3
2.0 10 10 5 0 0 2 5
5.0 10 10 7 0 0 1 8
10.0 10 10 5 3 1 1 10
I/11 10 4 0 0 0 0 0
0.2 mg/kg
0.5 10 0 0 0 0 0 0
2.0 10 3 0 0 0 0 0
Saline and Tween 80, I/II mixture and Diazepam were given orally once a day
for twelve days. The challenge with 10 mg/kg I p Flumazenil followed 24 h
after the
last treatment. The number represents the number of mice of the ten treated
mice
showing symptoms during the 20 min observation period following Flumazenil
treatment.
Hr: hyper-reactivity indicated by vocalization caused by light pressure on the
back
Tr: tremor
Cc: clonic convulsions
Tc: tonic convulsions
Tf: tail flick
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Rf: running fit evoked by auditory stimulus
To evaluate dependence liability, I/II mixture and diazepam were given for
12 days. This was followed by administration of flumazenil the benzodiazepine
antagonist. No dihydrohonokiol (I/II mixture) antagonist is known. Six signs
of
withdrawal following flumazenil were observed and recorded. Diazepam showed
extensive withdrawal signs following all doses 0.5 mg/kg to 10 mg/kg. The I/II
mixture showed no withdrawal response at the tested doses of 0.2 mg/Kg, 0.5
mg/Kg
or 2.0 mg/Kg. The 2.0 mg/Kg dose is 10 times the effective dose. The hyper-
reactivity response of the I/II mixture given flumazenil was no different from
flumazenil given alone. The 0.5 mg/kg dose of the I/II mixture followed by
flumazenil produced no hyper-reactivity response at all.
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5.5 Example 5 ~ Evaluation of Sedative Potential of the Dihydrohonokiol
Isomer Mixture
TABLE 13
5 Effect of I/II Mixture, Diazepam and I/II mixture plus
Diazepam on the Duration of Hexobarbital Induced Sleep
Treatment, Additional Duration of
dose mg/kg drug, sleep
po dose in sec
following mg/kg po
hexobarbital
Vehicle 2552 209
I/II 0.1 2822 138
1/II 0.2 2580 255
I/II 0.5 2753 296
I/II 1.0 2440 37
I/II 2.0 2870 191
Vehicle 2902 84
Diazepam 1.0 4476 336*
Diazepam 1.0 I/II 0.1 4342 195*
Diazepam 1.0 I/II 0.2 4248 66*
Diazepam 1.0 I/II 0.5 4526 467*
Diazepam 1.0 I/B 1.0 4052 217*
Diazepam 1.0 I/II 2.0 4758 299*
*p<0.0003 vs vehicle containing hexobarbital control.
All mice received 100 mg/kg hexobarbital ip. The I/II mixture was given
10 orally 3 h prior to hexobarbital to obtain the maximum anxiolytic effect of
the I/II
mixture. Diazepam was given 10 min before hexobarbital to obtain the maximum
effect. The time from loss of righting reflex in each mouse to the return of
the
righting reflex was recorded as the sleep duration.
To evaluate the sedative effects of the I/11 mixture and diazepam all mice
15 received a sedative dose of hexobarbital. The duration of the loss of
righting reflex
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(ability of the mouse to change from lying on the side to upright posture) was
recorded. Doses of the I/II mixture up to 2 mg/kg which is 10 times the
effective
anxiolytic doses caused no increase in sleep time. Diazepam at the effective
anxiolytic dose of 1 mg/kg increased sleep time significantly. The I/II
mixture when
combined with diazepam, and hexobarbital, caused no increase in sleep time
over that
of diazepam plus hexobarbital.
5.6 Example 6~ Evaluation of Effect of Dihydrohonokiol Isomer Mixture on
Cognitive Function
TABLE 14
Effect of Diazepam and I/II Mixture on Learning and Memory
Treatment Dose, po mg/kg Training Retention Latency
Latency
Vehicle saline 59.1 10.1 32.4 9.4
Diazepam 1.0 41.8 9.3 109.2 16.9*
Vehicle saline 71.7 10.4 31.9 9.9
Diazepam 1.0 85.3 13.2 37.1 5.8
Vehicle saline 59.1 9.8 25.6 3.3
I/II 0.2 59.38.8 26.23.1
I/II 2.0 121.0 46.06.6*
12.7*
Vehicle saline 62.8 12.1 25.2 2.8
I/II 0.2 60.1 9.1 24.3 2.8
I/II 2.0 71.9 13.1 29.95.3
*p<0004 vs control vehicle and saline
The I/II mixture was given 3 h before testing and Diazepam was given 10 min
before testing. Group size was 10 mice.
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To test the cognitive effects of the 1111 mixture and diazepam, training
latency
and retention latency were evaluated using the elevated plus-maze. On one of
two
tests diazepam show significant retention latency at the effective anxiolytic
doses of 1
mg/kg po. In one of two tests the I/II mixture showed only at 10 times the
effective
anxiolytic dose a training and retention latency.
5.7 Example 7 ~ Evaluation of Effect of Dihydrohonokiol Isomer Mixture on
Conditioned Place Preference
TABLE 15
The Effect of the I/II mixture and Diazepam on Conditioned Place Preference
Treatment mg/kg orally for Time in sec in light compartment
six
days after conditioning
Saline 112.710.1 t
Diazepam 1.0 182.916.8*t
Saline 105.46.9t
I/II 0.2 105.210.81
*p<0.002 vs saline
tn=10 mice.
The elevated plus-maze test was used to evaluate the ability of the drugs to
engender a desire to continue the drug, time spent at the site of six days
prior
administration of the drug was observed. The time spent at the site of
administration
following diazepam was significantly increased over saline administration. The
time
spent at the site following the I/II mixture administration was no different
from that of
saline administration.
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5.8 Example 8 ~ Anxiolytic Activity of 3-n-Propyl-5'-(2-propenyl)-1,1'-
biphenyl-2',4-diol (I) and 5'-n-
Propyl-3-(2-propenyl)-1,1'biphenyl-2',4-diol (II)
The I/II mixture consists of the two dihydrohonokiol isomers in the ratio of
92:08. These isomers were separated by preparative HPLC as described in ~
5.1.1 and
their anxiolytic activity compared using a modified elevated plus-maze. The
major
modification change from the maze used in the previous studies was the use of
a
camcorder placed above and to the side of the maze so that no human observer
was
visible to the mouse running the maze. This change resulted in an increase in
the time
that the mouse remained on the transparent arm. The control time increased
from 13
sec to 41 sec and the treated times also increased. The small amount of the II
isomer
(5'-n-propyl-3-(2-propenyl)-1,1'-biphenyl-2',4-diol) separated limited the
inventors'
group size to 5 instead of 10 mice. The oral doses were diazepam 1 mg/kg and I
isomer (3-h-propyl-5'-(2-propenyl)-1,1'-biphenyl-2',4-diol) and II isomer each
at 0.2
mg/kg. As shown in Table 16 and Table 17, both isomers were active but the I
isomer
was more active than the II isomer.
TABLE 16
Anxiolytic Activity of Isomer I and Isomer II in the Modified Elevated
Plus Maze Test
Mean Std. Dev. Std. ErrorCount
Maze, Total 93.195 69.503 15.541 20
Maze, CONTROL 41.220 18.693 8.360 5
Maze, Group A 134.860 43.782 19.580 5
Maze, Group B 113.280 62.607 27.999 5
Maze, Group C 83.420 102.905 46.020 5
Group A: Diazepam
Group B: Isomer I
Group C: Isomer II
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TABLE 17
Statistical Analysis of Anxiolytic Activity of Isomer I and Isomer II in the
Modified Elevated Plus Maze Test
Mean Dif. Crit. Diff.P-Value
CONTROL, Group -93.6400 86.8267 0.0362
A
CONTROL, Group -72.0600 86.8267 0.0976
B
CONTROL, Group -42.2000 86.8267 0.3182
C
Group A, Group 21.5800 86.8267 0.6055
B
Group A, Group 51.4400 86.8267 0.2272
C
Group B, Group 29.8600 86.8267 0.4765
C
Fisher's PLSD for Maze. Descriptive statistics split by Group:
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5. 8.1 Further Studies on the Anxiolytic Activity of Isomer I
The I isomer was further tested as shown in Tables 17 and 18. The anxiolytic
activity of isomer I at an oral dose of 0.2 mg/kg was compared to diazepam at
an oral
dose of 1 mg/kg using the new elevated plus maze system with camcorder. Both
5 compounds showed significant anxiolytic effect.
TABLE 18
Anxiolytic Activity of Isomer I and Isomer II in the Modified
Elevated Plus Maze Test
Mean Std. Dev. Std. ErrorCount
Maze, Total 83.585 57.096 10.988 27
Maze, CONTROL 41.167 26.178 8.726 9
Maze, Group A 118.089 57.168 19.056 9
Maze, Group B 91.500 56.633 18.878 9
Group A: Diazepam
Group B: Isomer I
TABLE 19
Statistical Analysis of Anxiolytic Activity of Isomer I
in the Modified Elevated Plus Maize Test
Mean DifferenceCrit. DifferenceP-Value
CONTROL, Group -76.9222 47.5334 0.0027 S
A
CONTROL, Group -50.3333 47.5334 0.0388 S
B
Group A, Group 26.5889 47.5334 0.2597
B
Fisher's PLSD for Maze. Descriptive statistics split by Group.
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5.8.2 Evaluation of Effect of Isomer I on Ultrasonic Vocalization
TABLE 20
The Effect of Isomer I, Gepirone and the Anxiogenic Compound
WAY on Conditioned Ultrasonic Distress Vocalizations in Adult Male Rats
Treatment Number of Ultrasonic Vocalizations,
10 min.
Control 114.4 10.7
Vehicle 154.8 11.7
Vehicle 94.6 10.8
Isomer I 1.0 mg/kg 48.1 4.7*
po
Vehicle 62.5 8.4
Gepirone 0.5 mg/kg 20.0 14.6*
sc
Vehicle 30.0 7.4
WAY 0.25 mg/kg sc 107.1 19.2*
*p<0.0001 vs vehicle.
The rats were divided into four groups depending on their level of ultrasonic
vocalization. Each of these groups was divided into an ultrasonic vocalization
matched control and drug test group consisting of three rats each.
The conditioned ultrasonic distress vocalization test for anxiolytic activity
in
rats was used to compare isomer I to Gepirone, an anxiolytic of the azapirone
class of
compounds. The azapirone class of anxiolytics differ structurally and
pharmacologically from the benzodiazepines. Their exact mechanism is unknown.
The primary action appears to be binding to serotonin receptors in the brain.
Only
Buspirone is marketed and it is not prescribed, often the benzodiazepines
being
preferred. In this test, single pure isomer I reduced vocalization effectively
indicating
it is effective in aversive conditioning test for anxiolytic activity.
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5.9 Example 9- Methods for Assessing Antianxiety Activity of the Disclosed
Compounds
This example describes protocols to facilitate preclinical and clinical
studies of
the compounds of the present invention. The various elements of conducting
preclinical and clinical trials, including animal and patient treatment and
monitoring,
will be known to those of skill in the art in light of the present disclosure.
The
following information is being presented as a general guideline for use in
preclinical
and clinical antianxiety studies.
5.9.1 Punished Responding
This procedure has been used to establish antianxiety activity in clinically
established compounds. Response of rats or pigeons is maintained by a multiple
schedule of food presentation. In one component of the schedule, responding
produces
food pellet presentation only. In a second component, responding produces both
food
pellet presentation and is also punished by presentation of a brief electric
shock. Each
component of the multiple schedule is approximately 4 min in duration, and the
shock
duration is approximately 0.3 sec. The shock intensity is adjusted for each
individual
animal so that the rate of punished responding is approximately 15 to 30% of
the rate
in the unpunished component of the multiple schedule. Sessions are conducted
each
weekday and are approximately 60 min in duration. Vehicle or a dose of
compound
are administered 30 min to 6 hr before the start of the test session by the
subcutaneous
or oral route. Compound effects for each dose for each animal are calculated
as a
percent of the vehicle control data for that animal. The data are expressed as
the mean
+- the standard error of the mean.
5.9.2 Monkey Taming Model
The antianxiety activity of a compound may be established by demonstrating
that the compounds are effective in the monkey taming model. Plotnikoff,
(1973)
described the response of rhesus monkeys to pole prodding as a method of
evaluating
the antiaggressive activity of a test compound. In this method, the
antiaggressive
activity of a compound was considered to be indicative of its antianxiety
activity.
Hypoactivity and ataxia were considered to be indicative of a sedative
component of
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the compound. In one study, the pole prod response-inhibition induced by a
compound
of this invention may be analyzed and compared with that of a standard
antianxiety
compound such as diazepam as a measure of antiaggressive potential, and to
obtain an
indication of the duration of action of the compound.
Male and female rhesus, cynomolgus or squirrel monkeys, selected for their
aggressiveness toward a pole, are housed individually in a primate colony
room.
Compounds or appropriate vehicle are administered orally or subcutaneously and
the
animals are observed by a trained observer at varying times after drug
administration.
A minimum of three days (usually a week or more) elapses between treatments.
Treatments are assigned in random fashion except that no monkey receives the
same
compound two times consecutively. Aggressiveness and motor impairment are
graded by response to a pole being introduced into the cage. The individuals
responsible for grading the responses are unaware of the dose levels received
by the
monkeys.
5.9.3 Human Clinical Trials
Antianxiety activity may be demonstrated by human clinical trials. A study
may be designed as a double-blind, parallel, placebo-controlled multicenter
trial.
Patients are randomized into four groups, placebo and three different
appropriate
doses of the test compound either once, twice or three times per day depending
on the
pharmacokinetics of the drug. The dosages may be administered orally with
food.
Patients are then observed during four visits to provide baseline
measurements, and
then visits 5 and beyond may be used as the treatment phase for the study.
During the visits, patients and their caregivers may be questioned and
observed for signs of agitation, mood swings, vocal outbursts, suspiciousness,
and
fearfulness. Each of these behaviors are indicative of the effect of the test
compound
on an anxiety disorder.
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The following references, to the extent that they provide exemplary procedural
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