Note: Descriptions are shown in the official language in which they were submitted.
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Hypericin and Hypericum extract: Specific T ,type calcium channel blocker,
and their use as T-type calcium channel targeted therapeutics
Field of the Invention
This invention relates to Hypericum perfor atum, extracts of Hypericum
perforatum, compounds found in Hypericum perforatum, e.g. hypericin, and
the derivatives and analogs of hypericin. One aspect of the present invention
is the discovery that Hypericum perforatum (referred to as Hypericum herein
after unless otherwise indicated), Hypericum extracts, certain compounds in
Hypericum, including hypericin, pseudohypericin, hyperforin, ashyperforin,
quercerin,, quercitrin, isoquercitrin, hyperoside, rutin, amentoflavone and
hyperin, hypericin derivatives and hypericin analogs can be used as
therapeutics targeted at T-type calcium channels in various biological
systems, such as cardiovascular system, central nervous system and
endocrine system, to treat diseases treatable with T-type calcium blocking
agents. The diseases treatable with T-type calcium blocking agents include
depression, chronic heart failure, congestive heart failure, ischaemc
condition, arrhythmia, angina pectoris, hypertension, hypo- and
hyperinsulinemia, diabete mellitus, hyperaldosteronemia, epilepsy, migraine
headache, brain aging or neurodegenerative related diseases, such as
Alzheimer's disease, and preterm labor.
Background of the Invention.
Hypericin is one of the chemical constituents from a perennial
herbaceous plant, Hypericum perforatum or St. John's Wort. Hypericum is
known to have medicinal properties since ancient times and it is widely
used in phytotheraphy. Hypericum has been widely researched for its
antidepressant and anti-viral properties. In addition to these properties,
Hypericum has historically been used for a variety of neurological
conditions, including anxiety, insomnia due to restlessness, irritability,
neuralgia, trigeminal neuralgia, neuroses, migraine headaches, fibrositis,
dyspepsia, and sciatica. Hypericum contains several compounds of
biological interest, including naphthodianthrones, e.g. hypericin and
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pseudohypericin, phloroglucinols, e.g. hyperforin and ashyperforin, and a
broad spectrum of flavonoids which are considered to be primarily
responsible for Hypericum's activity. However, the lack of a clearly
definable pharmacologic mechanism of Hypericum and its chemical
components cause the failure of identifying the constituents most
responsible for Hypericum's activity.
Clinical studies demonstrated that Hypericum is effective in treating
mild depression. Animal studies also showed that Hypericum extract
relieved depressant symptoms. It was reported that Hypericum extract
resulted in a down-regulation of adrenergic receptors in the rat frontal
cortex after subchronic treatment. Some reported that hypericin inhibited
monoamine oxidase (MAO) activity in vitro, but others have failed to
confirm this effect. Other proposed mechanisms involve effects on
serotonin. At very high doses, Hypericum extract inhibited seretonin
re-uptake although it is not known which chemical in the extract is
responsible. Studies have shown that both hypericin and pseudohypericin
inhibited a variety of virus. Hypericin has been reported to inhibit the
growth of glioma cell lines in vitro and to be a potent inducer of glioma cell
death due to inhibition of protein kinase C(PKC). Receptor tyrosine kinase
activity of epidermal growth factor has also been reported to be inhibited
by hypericin. These later effects have been linked to both the antiviral and
antineoplastic activity.
It is known (F.R. Buhler, J. Hypertension supplement 15(5):s3-7,
1997; B. Cremers et al., J. Cardiovascular Pharmacology, vol. 29(5), pp.
692-6, 1997) that T-type channels are involved in pacemaker activity,
low-threshold calcium spikes, neuronal oscillations and resonance, and
rebound burst firing. It was reported that Mibefradil, a selective T-channel
blocker, induces peripheral and coranary vasodilation. There is no reflex
sympathetic activation and no negative inotropic effect. It increases
coronary blood flow without increasing oxygen consumption and causes a
slight slowing of the heart rate, thereby inducing diastolic relaxation. The
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latter improves subendocardial and small artery perfusion. Ventricular
ectopic activity is reduced with mibefradil. The
renin-angiotensin-aldosterone system and endothelin effects are blunted
by T-channel inhibition. It is believed that mibefradil could lead to a
greater
therapeutic index and greater safety over conventional non-selective or
L-type calcium channel blockers in the treatment of cardiovascular
diseases. Mibefradil has been used to treat hypertension and angina
clinically. It was reported that Zonisamide, a antiepileptic drug reduces
T-type calcium current (M. Kito et al., Seizure, vol. 5(2), pp. 115-9, 1996).
T-type calcium channels also facilitate insulin secretion by enhancing the
general excitability of these cells. Therefore, T-type calcium channels may
be therapeutic targets in hypo- and hyperinsulinemia (A. Bhattacharjee et
al., Endocrinology, vol. 138(9), pp. 3735-40, 1997). A direct link between
T-type calcium channel activity and steroidogenesis has been suggested
(M. F. Rossier et al., 1996).
Summary of the Invention
Consequently, T-type calcium channel blockers, such as
Hypericum, Hypericum extracts, Hypericum constituents, including
hypericin, pseudohypericin, hyperforin, ashyperforin, quercetin, quercitrin,
isoquercitrin, hyperoside, rutin, amentoflavone and hyperin, hypericin
derivatives and hypericin analogs of the present invention are effective in
treating disorders characterized by an insufficiency of a steroid hormone.
Specific T-Channel inhibitory effect of hypericin and' Hypericum
extract containing 0.3% of hypericin have been found by the present
inventors. The present inventors have unexpectedly found that hypericin
and Hypericum extracts act as specific T-calcium channel blockers. In view
of this, Hypericum, Hypericum extracts, extracts of species of the
Hypericum genus other than Hypericum perforatum, extracts of other
plants containing hypericin, constituents of Hypericum, including hypericin,
hypericin derivatives and hypericin analogs according to the present
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invention are expected to be useful for treating T-calcium channel targeted
diseases such as arrthymia, coronary diseases, angina, hypertension,
migraine, diabetes and preterm labor, etc.
Within the scope of the present invention are processes of using
Hypericum; Hypericum extracts; Hypericum constituents, including
hypericin, pseudohypericin, hyperforin, ashyperforin, quercetin, quercitrin,
isoquercitrin, hyperoside, rutin, amentoflavone and hyperin; hypericin
derivatives; or hypericin analogs to treat health disorders related to T-type
calcium channels or treatable with T-calcium channel blockers in animals,
including humans. The health disorders related to T-type calcium
channels or treatable with T-type calcium blocking agents include
depression, chronic heart failure, congestive heart failure, ischaemc
condition, arrhythmia, angina pectoris, hypertension, hypo- and
hyperinsulinemia, diabete mellitus, hyperaldosteronemia, epilepsy,
migraine headache, brain aging or neurodegenerative related diseases,
such as Alzheimer's disease, and preterm labor.
Brief Description of Drawings
Figure 1 shows the effect of hypericin on L-type calcium current activity in
cultured N1 E-115 cells. At doses of 0.1, 1 and 10 uM, hypericin causes a
slight but not significant increase in L-type calcium currents.
Figure 2. Shows the effect of nifedipine on L-type calcium current activity
in cultured N1 E-1 15 cells. The cells were added into nifedipine after not
responding to hypericin (1OuM). 1 uM of nifedipine significantly inhibited
the currents, indicating the existence of L-type calcium channels.
Figure 3 shows the effect of hypericin on T-type calcium current in
N1 E-1 15 cells. Hypericin causes a dose-dependent inhibitory effect on
T-type calcium current from 0.1 to 10 uM.
Figure 4 shows the effect of solvent control for hypericin on T-type calcium
channel activity. Since hypericin is dissolved in a solvent of ethanol
(50%)+DMSO (50%). This solvent is tested for its effect on T-type current.
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In the amount equivalent to dissolving 0.1 to 10 uM of hypericin, this
control solvent does not affect T-type calcium currents.
Figure 5 shows the effect of Hypericum extract on T-type calcium current.
Hypericum extract contains about 0.3% of hypericin and is dissolved in
5 external solution. At 50ug/ml, Hypericum extract significantly inhibited
T-type calcium channel currents.
Figure 6 shows the effect of hypericin on L-type calcium current in vascular
smooth muscle cells.
Figure 7 shows the effect of hypericin on L-type calcium current in
ventricular cells from baby rats.
Detailed Description of the Invention
The present inventors have found that a commercially available
standardized Hypericum extract containing the primary compounds, such
as hypericin, pseudohypericin, flavonol glycosides, and phloroglucinols,
etc, and pure hypericin inhibited T-type calcium channel activity in cultured
neuroblastoma cells.
Hypericum extracts can be obtained either through commercially
available sources or extracted from original whole fresh or dried
Hypericum plant containing not less than 0.04% naphthodianthrones of the
hypericin group calculated as hypericin. Hypericum extracts can be easily
prepared by organic solvent extraction or supercritical fluid extraction by
carbon dioxide (E. Bombardelli and P. Morazzoni, Fitoterapia, vol. 66, pp.
43-68, 1995; S.S. Chatterjee et al, Pharmacopsychiat., vol. 31 (Suppl.), pp.
7-15, 1998; W. Dimpfel et al, Pharmacopsychiat., vol. 31 (Suppl.), pp. 30-
35, 1998). As an example, 1 kg of finely ground dried Hypericum
perforatum was stirred with 8 I of 80% ethanol under nitrogen at 55 C for 1
hour. The mixture was centrifuged under nitrogen and the supernatant
was collected. Ascorbic acid (0.1 %) was added and the extract was dried
under reduced pressure to give a Hypericum extract. It is noted that other
organic solvents, e.g. alkyl alcohols other than ethanol, acetone, methyl n-
butyl ketone, n-hexane, DMSO and toluene, can be used to extract'
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Hypericum perforatum to make Hypericum extracts. The plant, Hypericum
perforatum, can be obtained worldwide such as England, China, Holland,
French, German, Italy, Russia, Spain and Sweden.
Another embodiment of the present invention includes extracts from
species of the Hypericum genus other than St. John's Wort or Hypericum
perforatum, and extracts from other plants containing hypericin, as well as
methods of using these extracts in treating health disorders related to T-
type calcium channels or treatable with T-calcium channel blockers. Also
within the scope of the present invention are methods of using species of
the Hypericum genus, other than St. John's Wort (Hypericum perforatum),
or other plants containing hypericin to treat health disorders related to T-
type calcium channels or treatable with T-calcium channel blockers.
Hypericum extracts usually contain hypericin and various amounts
of other chemicals, including pseudohypericin; a broad range of
flavonoids, such as quercetin, quercitrin, isoquercitrin, hyperoside, rutin,
amentoflavone and hyperin; phloroglucinols, such as hyperforin and
ashyperforin; the essential oil; and xanthones. In the present invention,
Hypericum constituents include hypericin, pseudohypericin, flavonoids,
such as quercetin, quercitrin, isoquercitrin, hyperoside, rutin,
amentoflavone and hyperin, phloroglucinols, such as hyperforin and
ashyperforin, the essential oil from Hypericumn perforatum, and xanthones.
Other embodiments of the present invention are Hypericum extract
constituents, which include hypericin, pseudohypericin, flavonoids, such as
quercetin, quercitrin, isoquercitrin, hyperoside, rutin, amentoflavone and
hyperin, phloroglucinols, such as hyperforin and ashyperforin, the
essential oil from Hypericum perforatum, and xanthones. Also within the
scope of the present invention are methods of using Hypericum
constituents or Hypericum extract constituents in treating health disorders
related to T-type calcium channels or treatable with T-calcium channel
blockers.
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A further embodiment of the present invention includes hypericin
derivatives and hypericin analogs. Chemicals other than hypericin in
Hypercum extract may potentiate the biological effect of hypericin as
proved by their synergistic effects of inhibiting T-type calcium channel
5, activity. Also within the scope of the present invention are methods of
using hypericin derivatives or hypericin analogs in treating health
disorders related to T-type calcium channels or treatable with T-calcium
channel blockers.
The term "hypericin analog" refers to compounds having a chemical
structure similar to hypericin and having T-type calcium channel blocking
activities like hypericin, but "hypericin analog" excludes Mibefradil.
Examples of hypericin analogs include emodin and a compound of formula
I shown below.
HO 0 on HO 0 OH
HO CH3 HO C 19H38
0
Ernodin
I
In the present application, hypericin derivatives are compounds
modified from hypericin. Pseudohypericin can be considered as a
hypericin derivative. Hyperin and hypericin derivatives of the present
invention include compounds of formula II shown below.
Rt 0 R12 R
R2 u
R3 Rte
R4 R9
RS RS
R6 0 R7
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wherein
R, is H, OH, OR or OCOR;
R2 is H, R, F, Cl, Br, I or S03H;
R3 is H, R, OH, OR, OCOR, CH2OH, CH2OR, CH2OCOR, COOH or
COOR;
R4 is H, R, OH, OR, OCOR, CH2OH, CH2OR, CH2OCOR, COOH or
COOR;
R5 is H, R, F, Cl, Br, I or S03H;
R6 is H, OH, OR or OCOR;
R7 is H, OH, OR or OCOR;
R. is H, R, F, Cl, Br, I or SO3H;
R9 is H, R, OH, OR, OCOR, CH2OH, CH2OR, CH2OCOR, COOH or
COOR;
R10 is H, R, OH, OR, OCOR, CH2OH, CH2OR, CH2OCOR, COOH or
COOR;
Rõ is H, R, F, Cl, Br, i or S03H;
R12 is H, OH, OR or OCOR; and
R is an alkyl or substituted alkyl group.
It is noted that the compound of formula II wherein R,, R3, R4, R6, R7
and R12 are OH, R2, R5, R8 and Rõ are H, and R9 and R10 are methyl is
hypericin itself. It is also noted that the compound of formula II wherein R,,
R3, R4, R6, R7 and R12 are OH, R2, R5, R. and Rõ are H, R9 is methyl and
R10 is hydroxymethyl is pseudohypericin.
In the present invention, "alkyl" represents a C1-C30 linear or
branched saturated or unsaturated hydrocarbyl group. Preferably, "alkyl"
is a C,-C6 linear or branched saturated or unsaturated hydrocarbyl group.
The alkyl group of R can be optionally substituted with one to three
substituents independently selected from hydroxy, alkoxy, acyloxy,
carboxy, akoxycarbonyl, amino, alkylamino, dialkylamino, nitro or phenyl
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group or fluorine, chlorine, bromine or iodine atom.
Further preferred are compounds of formula II, wherein
R, is H, OH, OR or OCOR;
R2 is H or R;
R3 is H, OH, OR, OCOR, CH2OH, CH2OR or CH2OCOR;
R4 is H, OH, OR, OCOR, CH2OH, CH2OR or CH2OCOR;
R5 isHorR;
R6 is H, OH, OR or OCOR;
R7 is H, OH, OR or OCOR;
R8isHorR;
R. is H, OH, OR, OCOR, CH2OH, CH2C)R or CH2OCOR;
R10 is H, OH, OR, OCOR, CH2OH, CH2OR or CH2OCOR;
Rõ is H or R;
R12 is H, OH, OR or OCOR; and
R is an optionally substituted C1-C6 alkyl group, as well as methods
of using these compounds to treat health disorders treatable with T-
calcium channel blockers.
It is also preferred that R is an optionally substituted methyl or ethyl
group.
The hypericin derivatives of the present invention with alkyl or
substituted alkyl group(s), i.e. compounds of formula 11 with alkyl or
substituted alkyl group(s) at R2, R3, R4, R5, R8õ R9, R10, and/or R1t
position,
can be synthesized from appropriately substituted emodin anthones by
dimerization (see Y. Mazur et al, CA 2,029,993; H. Falk et al, Monatsh.
Chem., vol. 126, pp. 993-1000, 1995; H. Falk and T.N.H. Tran, Monatsh.
Chem., vol. 127, pp. 717-723, 1996; R. Altmann et al, Monatsh. Chem.,
vol. 129, pp. 235-244, 1998; G.A. Kraus and W. Zhang, Bioorg. Med.
Chem., vol. 5, pp. 2633-2636, 1995). For example, a hypericin derivative,
wherein R,, R3, R4, R6, R7, and R12 are OH, R;2, R5, R8, and Rõ are H, and
R9 and Rt0 are C19H39, was synthesized from anthone of formula I by
dimerization in the presence of pyridine N-oxide, piperidine, and ferrous
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sulfate in pyridine.
O-Substituted hypericin derivatives or hypericin analogs can be
synthesized by direct etherification and esterification of the phenolic
hydroxyl group of hypericin and/or hypericin analogs (see H. Falk and
5 T.N.H. Tran, Monatsh. Chem., vol. 127, pp. 717-723, 1996;
G.A. Kraus and W. Zhang, Bioorg. Med. Chem., vol. 5, pp. 2633-2636,
1995). For example, a hypericin derivative, wherein R,, R3, R4, R6, R7, and
R12 are OMe, R2, R5, R8, and Rõ are H, and R9 and Rio are methyl, was
prepared by methylation of hypericin with dimethyl sulfate under basic
10 condition.
Hypericin derivatives having halogen or sulfonate substitution, i.e.
compounds of formula II wherein R2, R5, R8, and/or R11 are halogen or
S03H, or halogenated or sulfonated hypericin analogs can be synthesized
by direct halogenation or sulfonation of hypericin or hypericin analog (H.
Falk and W. Schmitzberger, Monatsh. Chem., vol. 124, pp. 77-81, 1993; H.
Falk et al, Monatsh. Chem., vol. 129, pp. 309-318, 1998). For example, a
hypericin derivative, wherein R2, R5, R8, and/or R11=Br, was prepared by
bromination of hypericin in pyridine.
Other hypericin derivatives of formula 11 can be prepared by
derivatization of hypericin, pseudohypericin or the hypericin derivatives
described above using processes known in the art. For instance, one or
more of the hydroxy groups in hypericin, pseucohypericin or the hypericin
derivatives described above can be derivatized by converting the hydroxy
group(s) to a protected hydroxy group(s) according to the processes
described in T.W. Greene and P.G.M. Wuts, Protective Groups in Organic
Synthesis, John Wiley & Sons, Inc., New York, 1991, the disclosure of
which is incorporated by reference. Similarly, hypericin derivatives can be
prepared by converting hypericin or pseudohypericin into a prodrug of
hypericin or pseudohypericin using processes known in the art, e.g. the
processes described in H. Bundgaard, Design of Prodrugs, Elsevier
Science Publishers, Amsterdam, 1985, the disclosure of which is
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incorporated by reference. The present invention includes prodrug forms
of the agents disclosed above and methods of using the prodrug forms to
treat health disorders related to T-type calcium channels or treatable with
T-calcium channel blockers. For instance, hypericin prodrugs,
pseudohypericin prodrugs and methods of using hypericin prodrugs or
pseudohypericin prodrugs to treat health disorders related to T-type
calcium channels or treatable with T-calcium channel blockers are also
contemplated in the present invention.
The present invention also provides pharmaceutical compositions
comprising Hypericum, Hypericum extract, or a compound found in
Hypericum, i.e. Hypericum constituent, including hypericin,
pseudohypericin, hyperforin, ashyperforin, quercetin, quercitrin,
isoquercitrin, hyperoside, rutin, amentoflavone and hyperin, hypericin
derivatives or hypericin analogs, admixed with a pharmaceutically
acceptable carrier. Also within the scope of the present invention is a
pharmaceutical composition comprising at least two Hypericum
constituents, one of which is preferably hypericin, with or without a
pharmaceutically acceptable carrier. It is further preferred that the
pharmaceutical composition comprises hypericin and pseudohypericin. It
is also preferred that the pharmaceutical composition comprises hypericin
and hyperforin. A further aspect of the present invention is a
pharmaceutical composition comprising a Hypericum constituent and a
hypericin derivative or hypericin analog.
Numerous standardized extracts are available yielding from 0.4 to
2.7 mg of hypericin per daily dose. These are prepared in a variety of ways
according to the various manufacturers. The extracts are normally
standardized by containing 0.24-0.32% total hypericin. '
The primary compounds, hypericin and pseudohypericin
(naphthodianthrones), are naturally occurring pigments and characteristic
markers for Hypericum plant and can be extracted in methanol or ethanol.
Hypericin and pseudohypericin can also be obtained synthetically by
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processes known in the art. Synthetic hypericin is also available
commercially. Similarly, Hypericum extract constituents, such as hypericin,
pseudohypericin, flavonoids, phloroglucinols and xanthones, can be
obtained by synthetic processes known in the art or by high pressure liquid
chromatography of Hypericum extracts, followed by work-up procedures
known to one skilled in the art.
The present invention also provides methods of using Hypericum;
Hypericum extracts; Hypericum constituents, e.g. hypericin,
pseudohypericin, flavonoids, such as quercetin, quercitrin, isoquercitrin,
hyperoside, rutin, amentoflavone and hyperin, phloroglucinols, such as
hyperforin and ashyperforin, the essential oil from Hypericum perforatum,
and xanthones; hypericin derivatives; hypericin analogs; or the
pharmaceutical compositions disclosed above for treating health disorders
related to T-type calcium channels or treatable with T-calcium channel
blocksers. In these methods, Hypericum, Hypericum extract, hypericin,
pseudohypericin, flavonoids, such as quercetin, quercitrin, isoquercitrin,
hyperoside, rutin, amentoflavone and hyperin, phloroglucinols, such as
hyperforin and ashyperforin, the essential oil from Hypericum perforatum,
xanthones, hypericin derivatives, hypericin analogs, or the pharmaceutical
compositions disclosed above may be administered to an animal, e.g. a
mammal such as a human, in need of such a treatment by a parenteral,
opthalmological, topical, oral or rectal route or by inhalation. Examples of
parenteral route are intravenous, subcutaneous and intramuscular routes.
Hypericum extract, hypericin, pseudohypericin, flavonoids, such as
quercetin, quercitrin, isoquercitrin, hyperoside, rutin, amentoflavone and
hyperin, phloroglucinols, such as hyperforin and ashyperforin, the
essential oil from Hypericum-perforatum, and xanthones, hypericin
derivatives, or hypericin analogs may be formulated for parenteral,
ophthalmological, topical, oral or rectal administration by compounding
these active agents with a conventional vehicle, excipient, binder,
preservative, stabilizer, dye, flavoring agent, or the like, as called for by
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accepted pharmaceutical practice.
The doses of the active agents used in the methods of the present
invention are described herein. Daily doses are in the range of 0.05 to
500 mg per kg of body weight, prefereably 0.5 to 50 mg per kg of body
weight, for Hypericum extract, an extract of a species of the genus
Hypericum other than Hypericum perforatum, or an extract of a plant
containing hypericin. Daily doses are in the range of 0.0001 to 10 mg per
kg of body weight, preferably 0.0015 to 0.15 mg per kg of body weight, for
hypericin and 0.001 to 5 mg per kg of body weight for a hypericin
derivative, such as pseudohypericin, or hypericin analog. For Hypericum
extract constituents or Hypericum constituents, other than hypericin and
pseudohypericin, the daily doses are in the range of 0.01 to 100 mg per kg
of body weight, preferably of 0.05 to 50 mg per kg of body weight.
Hypericum perforatum, fresh or dried, can be used in the methods
of treating health disorders related to T-type calcium channels or treatable
with T-calcium channel blockers. In the present invention, "fresh
Hypericum perforatum" includes the entire plant of Hypericum perforatum
or a portion of Hypericum perforatum plant in a fresh state. In the present
invention, "dried Hypericum perforatum" includes the entire plant of
Hypericum perforatum or a portion of Hypericum perforatum plant in a dry
state, as well as powder resulting from grinding a dried Hypericum
perforatum plant or a dried portion of a Hypericum perforatum plant. The
methods of treatment of the present invention can be performed by
administering fresh Hypericum perforatum or dried Hypericum perforatum.
For fresh Hypericum perforatum, the daily doses are in the range of 1 to
5000 mg per kg of body weight. For dried Hypericum perforaturn, the daily
doses range from 0.5 to 2000 mg per kg of body weight.
The actual dose of the active agent used in the method of the
present invention to treat a particular subject can be selected from the
"daily doses" disclosed above depending on the T-calcium channel activity
of the active agent, i.e. Hypericum perforatum, Hypericum extracts,
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Hypericum constituents, hypericin derivatives or hypericine analogs, used
in the treatment, the age, race, sex, species and health condition of the
subject to be treated and the type and severity of the health disorder to be
treated. If used for acute treatments, the above active agents can be
administered at the daily doses disclosed above for no more than one day.
If needed, the above active agents can be administered to the subject
being treated at the above daily doses repetitively day after day.
The extract or hypericin can also be combined with drugs or any
other natural substances known to be effective for treating the condition in
question.
The following examples illustrate, but are not intended to limit, the
present invention.
Example 1.
Effect of hypericin on L-type calcium current activity in cultured
neuroblastoma cells.
Mouse neuroblastoma cells (N1 E115) were cultured in Dulbecco's
modified Eagle's medium (GIBCO) containing 110% fetal bovine serum at 37 C
in a humidified atmosphere of 5% CO2 in air. The medium was changed
every 3-4 days. After mechanical agitation, 3 x 1104 cells were replanted in
35
mm tissue culture dishes containing 4 ml of bath solution. After cell
attachment, the dish was mounted on the stage of an inverted phase-contrast
microscope (Nikon) for Cat+channel current recording. These cells expressed
predominately T channel currents. In experiments where L channels were
specifically sought, the cells were grown and maintained at confluence for 3-4
weeks under the same culture conditions with the addition of 2% (vol/vol)
dimethyl sulfoxide. Three to five days before use, the cells were replanted
with the same medium. These cells expressed predominately L channel
currents. A small number of these cells also expressed T channel currents.
Hence, cells were selected so that at a holding potential of -40 mV, the T
channel component was very small and the inward current measured was
conducted predominantly by L channels.
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The whole-cell version of the patch-clamp technique was used. The
pipettes had resistance of 2-15 M. Membrane current recordings were made
with an Axopatch-1 C (Axon Instruments) patch-clamp amplifier. All signals
were filtered at 1 kHz and stored in the computer. Since the peak currents
5 measured with 20 mM Ba2+ as the charge carrier were usually small (about
200 pA), the series resistance compensation was not usually employed. If the
capacitive transient overlapped with the onset of the inward current, or if
the
spatial voltage control was inadequate (i.e., NIE-115 cells with long neural
outgrowths), the experimental data were rejected. Unless otherwise specified
10 the current-voltage plots were constructed by using the peak values
(corrected for leakage) from the original records for both T and L channel
currents. The holding membrane potential was fixed at -80 mV when the T
channels were under investigation or at -40 mV when the L channels were
studied. Ba2+ currents through Cat} channels were elicited by 200 msec
15 depolarization at intervals of 5 sec. For event single-cell recording,
stable
readings were first obtained for 5 min; the drug was then added to the bath
solution. Experiments were performed at room temperature(21-22 C) to
prolong cell survival and channel recording time. The bath solution contained
110 mM Tris, 5 mM KCL, 5 mM CsCL, 20 mM Hepes, 30 mM glucose, 20 mM
BaCL2, and 0.5 M tetrodotoxin. The pipette (internal) solution contained 70
mM Cs2 -aspartame, EGTA 10, 2 mM ATP-Na2, 5 mM K-pyruvate, 5 mM
K-succinate, 5 mM Phosphocreatine-Na2,15 units/ml Creatine kinase, Hepes
and 5 mM glucose. The osmolarity of all solutions was adjusted to 310-320
mOsm and pH to 7.4 using HCL or CsOH as required.
Figure 1 shows that hypericin does not significantly affect L-type
calcium current. Figure 2 shows that Nifedipine, a known L-type calcium
channel blocker significantly inhibits L-type calcium currents.
Example 2.
Effect of Hypericin on T-type Calcium Currents in N1 E-1 15 cells.
The method is described as above (Example 1). Figure 3 shows that
hypericin inhibited T-type calcium currents in a dose-dependent manner.
CA 02336781 2001-01-08
WO 00/02455 PCT/US99/14132
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Figure 4 shows that the solvent controls did not affect the T-type calcium
currents.
Example 3.
Effect of St. John's Wort extract (Hypericum extract) on T-type calcium
current in N1 E-115 Cells.
The method is described as above (Example 1). Hypericum extract
is standardized with about 0.3% of hypericin. As shown in Figure 5, at, 50
ug/mI, Hypericum extract containing about 0.15 ug/ml of hypericin
significantly inhibited T-type calcium current by more than 60%. However,
0.15 ug/ml of pure hypericin, as-shown in Figure 3 produces less than 10%
of inhibition on T-type calcium current. This results suggested that the
chemicals other than hypericin in Hypericum extract cause a synergistic
effect to hypericin on T-type calcium channel activity.
Example 4
Effect of Hypericin on L-type calcium current in VSMC
The method is similar to the method described above for Example 1,
except that vascular smooth muscle cells (VSMMC) were used. As shown in
Fig. 6, hypericin did not affect the L-type calcium current in vascular
smooth muscle cells.
Example 5
Effect of Hyericin on L-type calcium current in Ventricular Cells
The method is similar to the method described above for Example 1,
except that ventricular cells from baby rats were used. As shown in Fig. 7,
hypericin did not affect the L-type calcium current in ventricular cells.
