U.S. patent application number 12/447566 was filed with the patent office on 2009-12-31 for analgesic agent comprising cyclic phosphatidic acid derivative.
This patent application is currently assigned to OCHANOMIZU UNIVERSITY. Invention is credited to Harumi Kanashiki, Kimiko Murofushi.
Application Number | 20090326256 12/447566 |
Document ID | / |
Family ID | 39588269 |
Filed Date | 2009-12-31 |
United States Patent
Application |
20090326256 |
Kind Code |
A1 |
Murofushi; Kimiko ; et
al. |
December 31, 2009 |
ANALGESIC AGENT COMPRISING CYCLIC PHOSPHATIDIC ACID DERIVATIVE
Abstract
An object of the present invention is to elucidate an analgesic
effect as one of new cPA bioactivities and thus to provide a novel
analgesic agent. The present invention provides an analgesic agent
which comprises a cyclic phosphatidic acid derivative that is one
type of phospholipid.
Inventors: |
Murofushi; Kimiko; (Tokyo,
JP) ; Kanashiki; Harumi; (Tokyo, JP) |
Correspondence
Address: |
GREENBLUM & BERNSTEIN, P.L.C.
1950 ROLAND CLARKE PLACE
RESTON
VA
20191
US
|
Assignee: |
OCHANOMIZU UNIVERSITY
Tokyo
JP
|
Family ID: |
39588269 |
Appl. No.: |
12/447566 |
Filed: |
December 25, 2007 |
PCT Filed: |
December 25, 2007 |
PCT NO: |
PCT/JP2007/001463 |
371 Date: |
August 18, 2009 |
Current U.S.
Class: |
558/83 |
Current CPC
Class: |
A61P 29/00 20180101;
A61K 31/665 20130101; A61P 25/04 20180101 |
Class at
Publication: |
558/83 |
International
Class: |
C07F 9/02 20060101
C07F009/02 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 28, 2006 |
JP |
2006-353642 |
Claims
1. An analgesic agent, which comprises a compound represented by
the formula (1) as an active ingredient: ##STR00004## wherein R
represents C1-30 linear or branched alkyl, C2-30 linear or branched
alkenyl, or C2-30 linear or branched alkynyl, provided that such
groups may optionally contain a cycloalkane ring or an aromatic
ring; X and Y each independently represents --O-- or --CH.sub.2--,
provided that X and Y do not simultaneously represent --CH.sub.2--;
and M represents a hydrogen atom or a counter cation.
2. The analgesic agent according to claim 1, wherein in the formula
(1), X and Y represent --O--.
3. The analgesic agent according to claim 1, wherein in the formula
(1), X or Y represents --CH.sub.2--.
4. The analgesic agent according to claim 1, wherein the compound
represented by the formula (1) is 1-oleoyl cyclic phosphatidic acid
or 1-palmitoleoyl cyclic phosphatidic acid.
5. The analgesic agent according to claim 2, wherein the compound
represented by the formula (1) is 1-oleoyl cyclic phosphatidic acid
or 1-palmitoleoyl cyclic phosphatidic acid.
6. The analgesic agent according to claim 3, wherein the compound
represented by the formula (1) is 1-oleoyl cyclic phosphatidic acid
or 1-palmitoleoyl cyclic phosphatidic acid.
Description
TECHNICAL FIELD
[0001] The present invention relates to an analgesic agent
comprising a cyclic phosphatidic acid derivative that is one type
of phospholipid.
BACKGROUND ART
[0002] "Pain" treatment in Japan is said to be at a level lower
than that in Europe and the United States. The environment in Japan
for basic research concerning "pain" is greatly inferior to that in
Europe and the United States. Low social or governmental
understanding concerning "pain" currently does not lead to "pain"
treatment.
[0003] To improve medical care for "pain" by overcoming the current
situation, activities should be promoted in Japan with cooperation
among doctors in clinical practice, researchers involved in basic
research, people involved in the government, and the like. There
are many things that remain to be done including, in addition to an
actual condition survey of "pain" treatment in Japan, efforts to
examine effects on the social economy and the like, elucidation of
the mechanism of "pain," development of forms of treatment,
formulation of treatment guidelines, enhanced "pain" education in
the field of education, and educational activity for general people
and patients, for example. Also, in preparation for the aging
society of the near future, establishment of medical care for
"pain" is thought to be an urgent need. This will improve the
quality of life of patients suffering from "pain" and reduce the
burden of social cost for medical care. It will also suppress
social labor productivity loss and improve the Japanese
economy.
[0004] Known major analgesic agents are largely classified as
narcotic analgesics or antipyretic analgesics. Narcotic analgesics
mainly act on the central nervous system and have strong analgesic
effects. Narcotic analgesics act on opioid receptors. Examples of
such narcotic analgesics include weak opioids (e.g., codeine)
having relatively weak analgesic effects and strong opioids (e.g.,
morphine) having strong analgesic effects, but having chemical
resistance. On the other hand, aspirin, acetaminophen, ibuprofen,
indomethacin, and the like are known as antipyretic analgesics,
which act mainly on the peripheral nervous system, and have
moderate analgesic effects. Antipyretic analgesics generally have
anti-inflammatory effects and analgesic antipyretic effects via COX
inhibition. Analgesic antipyretic drugs, such as acetaminophen,
lacking COX inhibitory action also exist.
[0005] Meanwhile, the present inventors have conducted various
cellular biochemical analyses using the true slime mold Physarum
polycephalum as an experimental material. The true slime mold has
been revealed to undergo morphological changes depending on changes
in exterior environment and exert significant changes in the
composition and metabolism of biomembrane lipids along with its
proliferation and differentiation. As a result of the structural
analysis of a novel lipid component isolated and identified from
haploid myxoamoeba in 1992, the lipid component was confirmed to be
a substance containing cyclopropane-containing hexadecanoic acid at
position sn-1 of the glycerol backbone and phosphoric acids
circularly ester-linked at positions sn-2 and 3
(Murakami-Murofushi, K., et al.: J. Biol. Chem., 267, 21512-21517
(1992)). This substance was named PHYLPA since it is an LPA analog
derived from Physarum. PHYLPA suppresses the activity of DNA
polymerase a of eukaryotic cells and is obtained from a lipid
fraction in which the proliferation of cultured animal cells has
been suppressed. PHYLPA has been confirmed to exhibit such
bioactivities. PHYLPA has characteristic fatty acids. As a result
of examining the bioactivities of structural analogs prepared via
organic synthesis involving substitution of the fatty acid portions
with other general fatty acids, physiological effects similar to
those of PHYLPA were confirmed (Murakami-Murofushi, K., et al.:
Biochem. Biophys. Acta, 1258, 57-60 (1995)). Accordingly, an
important structure for these physiological effects is inferred to
be a cyclic phosphate structure at glycerol positions sn-2 and 3.
Lipids having such a structure are collectively referred to as
cyclic phosphatidic acids (cPAs). Moreover, it was demonstrated
that cyclic phosphatidic acids (cPAs) are bioactive lipids
universally existing not only in human sera, but also in various
organisms (Kobayashi, T., et al.: Life Sci., 65, 2185-2191
(1999)).
Non-Patent Document 1: Murakami-Murofushi, K., et al.: J. Biol.
Chem., 267, 21512-21517 (1992) Non-Patent Document 2:
Murakami-Murofushi, K., et al.: Biochem. Biophys. Acta, 1258, 57-60
(1995)
Non-Patent Document 3: Kobayashi, T., et al.: Life Sci., 65,
2185-2191 (1999)
DISCLOSURE OF THE INVENTION
Object to be Achieved by the Invention
[0006] An object of the present invention is to elucidate an
analgesic effect as one of new cPA bioactivities and thus to
provide a novel analgesic agent.
Means for Attaining the Object
[0007] The present inventors have discovered cyclic phosphatidic
acids (cPAs), which are bioactive phospholipids, from various
eukaryotes and have studied for over dozen years concerning the
physiological effects. Thus, the present inventors have discovered
that cPAs have significant analgesic effects. Cyclic phosphatidic
acids (cPAs) have analgesic effects equivalent to that of morphine
as demonstrated by employed assays. Furthermore, cPAs are
originally contained in living body, so that cPAs exhibit no
toxicity or addiction when administered to living bodies from the
outside, as revealed by animal experiments. The summary of the
results of experiments concerning the effects of cPAs on the
suppression of pain is as follows.
[0008] Specifically, the present inventors have examined the
possibility of the intravenous administration of cPAs to induce
analgesic effects using anesthetized rats with uninjured central
nervous systems and two indicators: (1) heart rate increase
reaction and (2) reflex potential in sympathetic cardiac branch,
which are induced by electrical stimulation of hind limb afferent
nerves. As a result, it could have been demonstrated by the two
methods that cPAs clearly have analgesic effects and the effects
may be exerted via an opioid receptor. In particular, first, the
present inventors have applied electric pulse at a level at which
rats feel pain, so as to induce rises in heart rate and examined
possibility of suppression of rises in heart rate by cPA
administration. Furthermore, the present inventors have exposed
cardiac nerves and examined possibility of reduction (by cPA
administration) in discharge levels induced by electric pulse using
an experimental system for measurement of the discharge levels of
nerve fibers. As a result, it has been demonstrated that cPAs
suppress the discharge level in a dose-dependent manner and have
high potential as an analgesic agent. The present invention has
been completed based these findings.
[0009] The present invention provides an analgesic agent, which
comprises a compound represented by the formula (1) as an active
ingredient:
##STR00001##
wherein R represents C1-30 linear or branched alkyl, C2-30 linear
or branched alkenyl, or C2-30 linear or branched alkynyl, provided
that such groups may optionally contain a cycloalkane ring or an
aromatic ring; X and Y each independently represents --O-- or
--CH.sub.2--, provided that X and Y do not simultaneously represent
--CH.sub.2--; and M represents a hydrogen atom or a counter
cation.
[0010] Preferably, in the formula (1), X and Y represent --O--.
[0011] More preferably, in the formula (1), X or Y represents
--CH.sub.2--. The former is 2 carba cPA (abbreviated as 2 ccPA),
and the latter is 3 carba cPA (abbreviated as 3 ccPA). Particularly
preferably, X is --CH.sub.2-- and Y is --O-- (2 carba cPA).
[0012] Preferably, the compound represented by the formula (1) is
1-oleoyl cyclic phosphatidic acid or 1-palmitoleoyl cyclic
phosphatidic acid.
[0013] Another aspect of the present invention provides a method
for suppressing pain which comprises administering an effective
amount of the compound represented by the aforementioned formula
(1) to mammals including human beings.
[0014] Further another aspect of the present invention provides a
use of the compound represented by the aforementioned formula (1)
for the production of an analgesic agent.
EFFECTS OF THE INVENTION
[0015] According to the present invention, it has been confirmed
that a compound represented by the formula (1) has analgesic
effects. Therefore, it has been revealed that the compound to be
used in the present invention represented by the formula (1) is
useful as an analgesic agent. According to the present invention, a
novel analgesic agent is provided.
BEST MODES FOR CARRYING OUT THE INVENTION
[0016] The embodiments of the present invention will be described
in detail below.
[0017] An analgesic agent of the present invention comprises a
compound represented by the following formula (1) as an active
ingredient.
##STR00002##
(wherein R represents C1-30 linear or branched alkyl, C2-30 linear
or branched alkenyl, or C2-30 linear or branched alkynyl, provided
that such groups may optionally contain a cycloalkane ring or an
aromatic ring. X and Y each independently represents --O-- or
--CH.sub.2--, provided that X and Y do not simultaneously represent
--CH.sub.2--. M represents a hydrogen atom or a counter
cation.)
[0018] In the formula (1), specific examples of C1-30 linear or
branched alkyl represented by substituent R include methyl, ethyl,
propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl,
pentadecyl, and octadecyl.
[0019] Specific examples of C2-30 linear or branched alkenyl
represented by substituent R include allyl, butenyl, octenyl,
decenyl, dodecadienyl, and hexadecatrienyl and more specifically
include 8-decenyl, 8-undecenyl, 8-dodecenyl, 8-tridecenyl,
8-tetradecenyl, 8-pentadecenyl, 8-hexadecenyl, 8-heptadecenyl,
8-octadecenyl, 8-icosenyl, 8-docosenyl, heptadeca-8,11-dienyl,
heptadeca-8,11,14-trienyl, nonadeca-4,7,10,13-tetraenyl,
nonadeca-4,7,10,13,16-pentaenyl, and
henicosa-3,6,9,12,15,18-hexaenyl.
[0020] Specific examples of C2-30 linear or branched alkynyl
represented by substituent R include 8-decinyl, 8-undecinyl,
8-dodecinyl, 8-tridecinyl, 8-tetradecinyl, 8-pentadecinyl,
8-hexadecinyl, 8-heptadecinyl, 8-octadecinyl, 8-icocinyl,
8-dococinyl, and heptadeca-8,11-diinyl.
[0021] Specific examples of a cycloalkane ring that can be
contained in the above alkyl, alkenyl, or alkynyl include a
cyclopropane ring, a cyclobutane ring, a cyclopentane ring, a
cyclohexane ring, and a cyclooctane ring. A cycloalkane ring may
contain one or more heteroatoms and examples of such a ring include
an oxirane ring, an oxetane ring, a tetrahydrofuran ring, and a
N-methylprolidine ring.
[0022] Specific examples of an aromatic ring that can be contained
in the above alkyl, alkenyl, or alkynyl include a benzene ring, a
naphthalene ring, a pyridine ring, a furan ring, and a thiophene
ring.
[0023] Therefore, when substituent R represents an alkyl
substituted with a cycloalkane ring, specific examples thereof
include cyclopropylmethyl, cyclohexylethyl, and
8,9-methanopentadecyl.
[0024] When substituent R represents an alkyl substituted with an
aromatic ring, specific examples thereof include benzyl, phenethyl,
and p-pentylphenyloctyl.
[0025] X and Y in the compound represented by the formula (1) each
independently represents --O-- or --CH.sub.2--, however, X and Y do
not simultaneously represent --CH.sub.2--. Hence, the three
combinations of X and Y are as follows.
(1) X represents --O-- and Y represents --O--. (2) X represents
--CH.sub.2-- and Y represents --O--. (3) X represents --O-- and Y
represents --CH.sub.2--.
[0026] M in a compound represented by the formula (1) represents a
hydrogen atom or a counter cation. When M represents a counter
cation, examples thereof include an alkaline metal atom, an
alkaline-earth metal atom, a substituted or unsubstituted ammonium
group. Examples of an alkaline metal atom include lithium, sodium,
and potassium and examples of an alkaline-earth metal atom include
magnesium and calcium. Examples of a substituted ammonium group
include a butyl ammonium group, a triethyl ammonium group, and a
tetramethyl ammonium group.
[0027] An example of cPAs represented by the formula (1), which is
used in the present invention, is an oleoyl cPA (Ole-cPA) and
particularly preferable examples of the same include a palmitoleoyl
2 carba cPA (Pal-2 ccPA) and a palmitoleoyl 3 carba cPA (Pal-3
ccPA).
[0028] A compound among compounds represented by the formula (1),
in which X and Y each represents --O--, can be chemically
synthesized according to the methods disclosed in JP Patent
Publication (Kokai) No. 5-230088 A (1993), JP Patent Publication
(Kokai) No. 7-149772 A (1995), JP Patent Publication (Kokai) No.
7-258278 A (1995), or JP Patent Publication (Kokai) No. 9-25235 A
(1997), for example.
[0029] Furthermore, a compound among compounds represented by the
formula (1), in which X and Y each represents --O--, can also be
synthesized by reacting phospholipase D with lysophospholipids
according to the method disclosed in JP Patent Publication (Kokai)
No. 2001-178489 A. Lysophospholipids to be used herein are not
particularly limited, as long as they are lysophospholipids that
can react with phospholipase D. Many types of lysophospholipid are
known, including those differing in fatty acid type and molecular
species having an ether or vinylether bond. They are available in
the market. As for phospholipase D, those derived from a higher
plant such as cabbage and peanut or from a microorganism such as
Streptomyces chromofuscus, Actinomadula sp. are available in the
market as commercial reagents. cPAs can be highly selectively
synthesized with the enzyme derived from Actinomadula sp. No. 362
(JP Patent Publication (Kokai) No. 11-367032 A (1999)). Conditions
for reaction between lysophospholipids and phospholipase D are not
specifically limited, as long as an enzyme can exert its activity.
The reaction of lysophospholipids with phospholipase D is carried
out, for example, in an acetate buffer (around pH 5-6) containing
calcium chloride at a temperature ranging from room temperature to
a warmed temperature (preferably approximately 37.degree. C.) for
approximately 1 to 5 hours. The thus generated cPA derivative can
be purified by extraction, column chromatography, thin-layer
chromatography (TLC) or the like according to a conventional
method.
[0030] Furthermore, a compound among compounds represented by the
formula (1), in which X represents --CH.sub.2-- and Y represents
--O--, can also be synthesized by the method disclosed in JP Patent
Publication (Kokai) No. 2004-010582 A.
[0031] Furthermore, a compound among compounds represented by the
formula (1), in which X represents --O-- and Y represents
--CH.sub.2--, can be synthesized according to the method disclosed
in the document (Kobayashi, S., et al., Tetrahedron Letters 34,
4047-4050 (1993); and "The Pharmaceutical Society of Japan, The
23.sup.rd Symposium of Advancement of Reaction and Synthesis, Nov.
17 and 18, 1997 (Kumamoto citizen hall), Synthesis and
Physiological Effects of Cyclic Phosphatidic Acid and Carba
Derivative, Summaries (pp. 101-104)) or the method disclosed in
International Publication WO2002/094286. A specific example of the
synthetic pathway is as follows.
##STR00003##
[0032] In the above synthetic pathway, first, commercially
available (R)-benzylglycidyl ether (1) is activated with
BF.sub.3.Et.sub.2O followed by reaction with a lithio derivative
obtained by reacting methyl phosphonic acid dimethyl ester with
n-BuLi, so that alcohol (2) is obtained.
[0033] The thus obtained alcohol is reacted with an excess of a
pyridinium salt of p-toluenesulfonic acid in toluene at 80.degree.
C., so that a cyclized product (3) is obtained. The cyclized
product is decomposed with hydrogenation under a hydrogen
atmosphere using 20% Pd(OH).sub.2--C, so that debenzylation is
carried out (4). 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide
hydrochloride as a condensation agent is reacted with fatty acid,
so that a coupling product (5) is obtained. Next, bromotrimethyl
silane is used as a nucleophile to position-selectively remove
methyl groups alone, so that cyclic phosphonic acid (6) is
obtained. The cyclic phosphonic acid (6) is transferred into a
separatory funnel using ether, a small amount of a 0.02N sodium
hydroxide aqueous solution is added dropwise, and then separation
is carried out, so that a target compound is extracted and purified
as a sodium salt (7).
[0034] Compounds represented by the formula (1) to be used as
active ingredients in the present invention have analgesic effects.
Therefore, according to the present invention, an analgesic agent
comprising such compound as an active ingredient is provided.
[0035] In the Example described below in the description, a method
for evaluation of analgesic effects was employed. Specifically,
possible exertion of analgesic effects due to intravenous
administration of cPAs was examined using anesthetized rats with
uninjured central nervous systems and indicators: (1) heart rate
increase reaction and (2) reflex potential in sympathetic cardiac
branch, which are induced by electrical stimulation of hind limb
afferent nerves. The two experimental systems used herein are
basically according to the method of Uchida et al., (Neurosci Lett
269: 161, 1999) who have examined the effects of morphine.
[0036] Many methods are employed for examining the effects of
analgesic substances, including a method for evaluation of the
analgesic effects in humans and a method using as an indicator
animals' escape behavior against various noxious stimuli such as
tail pinching and heat. However, application of noxious stimuli to
unanesthetized animals affects the emotion of the animals. Thus,
quantitative evaluation of analgesic effects is difficult and the
provision of agonies to animals raises an ethical issue.
[0037] Noxious stimuli can induce various reactions also in
anesthetized animals. Such noxious stimuli activate peripheral
nociceptive primary afferent nerve fibers, activate nociceptive
neurons of the central nervous system, and can cause further
various reflex reactions. Examples of well known reflex reactions
under anesthesis include somatic kinetic reflex such as noxious
flexion reflex, sympathetic reflex such as noxious pressor reflex,
and endocrine reflex such as a noxious increase in blood
catecholamine level.
[0038] Among (1) "heart rate increase reaction induced by frequent
electrical stimulation of hindlimb afferent nerves" employed herein
is not induced by weak stimulation intensity (0.2 V) that excites
only thick medullated A.cndot.fibers, but induced by stimulation
intensity (1 V) that is the same or higher than the excitation
threshold of thin medullated A.cndot.fibers. When the stimulation
intensity is further increased (10 V) so as to cause excitation of
nonmedullated C fibers, the amplification of the heart rate
increase reaction is increased to reach the maximum level (Sato et
al, J Auton Nerv Syst 4: 231, 1981; Uchida et al, Neurosci Lett
269: 161, 1999). Noxious information is transmitted by
A.cndot.fibers and C fibers. Therefore, such heart rate increase
reaction induced by excitation of somatic afferent A.cndot.fibers
and C fibers can be an indicator for evaluation of analgesic
effects.
[0039] Among (2) "reflex potential induced by single electrical
stimulation of hindlimb afferent nerves in sympathetic cardiac
branch" employed herein, short latency A-reflex potential is
induced and increased mainly by excitation of A.cndot.fibers. When
the stimulation intensity is increased and nonmedullated C fibers
are excited, delayed C-reflex potential is induced after induction
of the above A-reflex potential (Ito et al, Neurosci Lett 39: 169,
1983; Adachi et al, Neurosci Res 15: 281, 1992).
[0040] C-reflex potential is specifically induced by the excitation
of nonmedullated C fibers involved in transmission of noxious
information, particularly burning delayed pain. Hence, a method
using C-reflex potential as an indicator is excellent for
evaluation of the action of analgesic agent. Among various types of
pain, delayed pain that is transmitted by C fibers causes strong
discomfort and thus is particularly important clinically. Sato et
al., have demonstrated using this model for anesthetized cats that
morphine selectively suppresses C-reflex potential alone and thus
have reported that C-reflex potential induced by the excitation of
somatic afferent C fibers can be an indicator for research
concerning analgesic effects (see Pain Clinic 10 (5) 605, 1989).
This method enables long-term stable measurement and enables
continuous examination of the effects by minutes, so that this
method is also excellent in evaluation of the time courses of drug
effects.
[0041] Meanwhile, regarding A-reflex potential, reflex due to
stimulation A.cndot.fibers (among medullated nerve fibers) involved
in transmission of noxious information overlaps reflex due to
A.cndot.fibers. Hence, examination of the effects of drugs on
reflexes due to only A.cndot.fibers involved in sharp and rapid
pain is difficult.
[0042] Anesthetized animals are used in both methods employed
herein. Accordingly, the methods are advantageous such that noxious
stimuli can be applied without causing pain and specific reflexes
induced by noxious stimuli can be examined. Thus, the methods can
create models for evaluation of analgesic effects as objective
measurement methods.
[0043] The analgesic agent of the present invention is preferably
provided in the form of a pharmaceutical composition containing 1,
2, or more pharmaceutically acceptable pharmaceutical additives and
a compound represented by the formula (1), which is an active
ingredient.
[0044] The analgesic agent of the present invention can be
administered in various forms. An appropriate form of
administration may be either oral administration or parenteral
administration (e.g., intravenous, intramuscular, subcutaneous, or
intradermal injection, intrarectal administration, and transmucosal
administration). Examples of pharmaceutical compositions
appropriate for oral administration include tablets, fine granules,
capsules, powders, solutions, suspensions, and syrups. Examples of
pharmaceutical compositions appropriate for parenteral
administration include injections, infusions, suppositories, and
transdermal absorption systems. However, the dosage forms of the
analgesic agent of the present invention are not limited to these
examples. Moreover, the analgesic agent of the present invention
can be produced in the form of prolonged action preparation by
known technology.
[0045] Types of pharmaceutical additives to be used for production
of the analgesic agent of the present invention are not
particularly limited and can be adequately selected by persons
skilled in the art. For example, excipients, disintegrators or
disintegration adjuvants, binders, lubricants, coating agents,
bases, solubilizing agents or solubilization adjuvants,
dispersants, suspensions, emulsifiers, buffers, antioxidants,
antiseptics, isotonizing agents, pH-adjusting agents, lytic agents,
and stabilizers can be used. Specific individual ingredients to be
used for these purposes are known by persons skilled in the
art.
[0046] Examples of pharmaceutical additives that can be used for
preparation of pharmaceutical preparations for oral administration
include: excipients such as glucose, lactose, D-mannitol, starch,
and crystalline cellulose; disintegrators or disintegration
adjuvants such as carboxymethyl cellulose, starch, and
carboxymethyl cellulose calcium; binders such as hydroxy propyl
cellulose, hydroxy propyl methyl cellulose, polyvinylpyrrolidone,
and gelatin; lubricants such as magnesium stearate and talc;
coating agents such as hydroxy propyl methyl cellulose, saccharose,
polyethylene glycol, and titanium oxide; and bases such as
Vaseline, liquid paraffin, polyethylene glycol, gelatin, kaoline,
glycerin, purified water, and hard fat.
[0047] Examples of pharmaceutical additives that can be used for
preparation of pharmaceutical preparations for injection or drip
infusion include: solubilizing agents or solubilization adjuvants
that can compose aqueous injections or injections that are
dissolved upon use, such as distilled water for injection, a
physiological saline solution, and propylene glycol; isotonizing
agents such as glucose, sodium chloride, D-mannitol, and glycerine;
pH adjusting agents such as inorganic acid, organic acid, inorganic
base, and organic base.
[0048] The analgesic agent of the present invention can be
administered to mammals including humans.
[0049] The dose of the analgesic agent of the present invention
should be adequately increased or decreased depending on conditions
of a patient's age, sex, body weight, and symptoms, and the route
of administration, for example. Generally the amount of an active
ingredient per day for an adult ranges from approximately 1
.mu.g/kg to 1,000 mg/kg and preferably ranges from approximately 10
.mu.g/kg to 100 mg/kg. The above dose of the analgesic agent may be
administered once a day or at several separate times (e.g.,
approximately 2 to 4 times).
[0050] The analgesic agent of the present invention can also be
used in combination with other analgesic agent and the like.
[0051] In addition, cPAs themselves that are active ingredients of
the present invention are substances existing in the mammalian
sera, brains, or the like. Hence, cPAs are thought to be safe for
organisms.
[0052] The present invention is described more specifically with
reference to the following Examples, but is not limited to these
Examples.
EXAMPLE
(1) Experimental Methods
Biological Materials
[0053] Wister male rats (body weight: 300 g-375 g) were used.
Anesthesia
[0054] Urethane (1.1 g/Kg) was initially administered
intraperitoneally to rats and then the rats were anesthetized.
Anesthetic depth was observed based on changes in blood pressure
and heart rate, and then urethane was subcutaneously administered
every 1 to 2 hours in an amount 1/20 to 1/10 the initial dose, so
that anesthetic depth was maintained.
Maintenance of Respiration
[0055] The trachea of each rat was cut open, a tracheal cannula was
inserted, and then respiration was maintained using an artificial
respirator (SN-480-7, Shinano, Tokyo). The carbon dioxide
concentration in expired air was monitored using an expired gas
monitor (1H26, NEC San-ei, Tokyo), and the respiratory volume was
regulated to be approximately 3% during the experiment.
Maintenance of Body Temperature
[0056] A thermister was inserted rectally. The core temperature was
constantly monitored using a body temperature control device
(ATB-1100, Nihon kohden) so that the body temperature was
maintained at 37.5.degree. C.
Measurement of Blood Pressure Fluid Replacement
[0057] The right femoral artery was cannulated. Arterial blood
pressure was continuously recorded from the intra-arterial cannula
using a pressure transducer (TP-400T, Nihon Kohden, Tokyo).
Measurement of Heart Rate
[0058] Blood pressure waveform was collected using a pulse rate
tachometer (AT601-G. Nihon Kohden) and the heart rate was recorded
continuously.
Record of Sympathetic Cardiac Branch Activity
[0059] Left second rib was removed in the supine position. Left
sympathetic cardiac branch running from the stellate ganglion to
the heart was separated and dissected at a position as close as
possible to the heart. Nerves were immersed in paraffin oil so as
not to be dried. Separated nerve center ends were placed on a
platinum iridium electrode. Efferent nerve activity of the
sympathetic cardiac branch was derived and then amplified using an
amplifier (S-0476, Nihon Kohden, time constant 0.33 s). The result
of reflex reaction induced by electrical stimulation of hindlimb
afferent nerves was added 50 times with an adder (ATAC3700, Nihon
Kohden). The averaged reaction results were displayed on the screen
and then recorded on a mini writer. The size of reflex reaction was
evaluated via measurement of the area of the thus induced reaction
and the result was expressed with % with respect to the size of the
control (before cPA administration). At the time of recording
sympathetic nerve activity, a muscle relaxant, gallamine
triethiodide (20 mg/kg, i.v.), was administered to avoid body
motion that could block stable recording of nerve activity.
Electrical Stimulation of Hindlimb Afferent Nerves
[0060] Left tibial nerves were dissected from the peripheral
tissue. The dissected center end was placed on a stimulating
electrode and then electrically stimulated by a supramaximal
square-wave pulse (pulse width: 0.5 ms and intensity: 10-20V) using
an electric stimulator (SEN-7103, Nihon Kohden). In an experiment
for examining heart rate increase reaction, frequent electrical
stimulation (10 Hz) was applied for 5 seconds. In an experiment of
reflex potentials, single electrical stimulation was applied every
3 seconds.
Preparation and Administration of cPAs
[0061] Products chemically synthesized according to the method
disclosed in Kobayashi, S., et al.: Tetrahedron Lett., 34,
4047-4050 (1993) or products enzymatically synthesized according to
the method disclosed in JP Patent Publication (Kokai) No.
2001-178489 A were used as Pal-cPA (16:0) (palmitic acid is bound
at position sn-1), Pal-cPA (16:1) (palmitoleic acid is bound at
position sn-1) and Ole-cPA (18:1) (oleic acid is bound at position
sn-1). There are no differences in terms of effects between
purified chemically-synthesized products and
enzymatically-synthesized products.
[0062] 2 carba and 3 carba derivatives were synthesized according
to the method disclosed in JP Patent Publication (Kokai) No.
2004-010582 and the method disclosed in International Publication
WO2002/094286, respectively.
[0063] Crystals were dissolved in ethanol and then evaporated to
dryness in the form of film using a nitrogen gas. The resultant was
dissolved in a physiological saline solution (0.9% NaCl)
immediately before administration, so that a 2.5 mg/mL or 0.5 mg/mL
solution was prepared.
[0064] Each cPA was administered intravenously via the cannula
inserted in the right femoral vein. When the dose was less than 1
mg/Kg body weight, a 0.5 mg/mL solution was injected and when the
dose was 1 mg/Kg body weight or more, a 2.5 mg/mL solution was
injected. Injection rate was 150 mL/minute in both cases.
(2) Results and Discussion
[0065] Effects of cPAs on Heart Rate Increase
[0066] The suppressive effect of Ole-cPA administration on heart
rate increase was verified using two 2 rats. The suppressive effect
was observed when the dose was 1 mg/Kg body weight or more and
observed to increase in a concentration-dependent manner (FIG. 1).
When the dose was 5 mg/Kg body weight, the heart rate increase was
found to be a half of or lower than that of the control. Regarding
the duration of the suppressive effect, the suppressive effect was
confirmed even at 20 minutes after administration when the dose was
higher than 1 mg/Kg body weight. The suppressive effect was then
observed to increase in a concentration-dependent manner. In the
case of 5 mg/Kg, the significant suppressive effect was observed
even after 20 minutes. As described above, it was demonstrated that
heart rate increase that is induced by electrical stimulation of
lower extremity nerves is significantly suppressed by
administration of Ole-cPA.
[0067] On the other hand, in the case of administration of Pal-cPA,
the suppressive effect on heart rate increase could be confirmed on
the whole, however, no consistent tendency could be confirmed.
Effects of cPA Administration on Blood Pressure
[0068] The effects of cPA administration on blood pressure were
examined using 2 rats simultaneously with the examination of the
suppressive effect on heart rate increase. When the dose of Ole-cPA
was 2 mg/Kg body weight or less, the blood pressure almost remained
unchanged at 90 mmHg and the effects due to administration was not
observed. On the other hand, when the dose of Pal-cPA was 300
.mu.g/Kg body weight, the blood pressure was found to briefly
increase to around 100 mmHg; and after administration of the dose
of 2 mg/Kg body weight, the blood pressure was found to increase to
130 mmHg. Then the blood pressure remained unchanged at around 130
mmHg and no decrease in blood pressure was confirmed even at 30
minutes after administration.
Changes in Discharge Levels of Cardiac Nerves Due to cPA
Administration
[0069] Measurement of the discharge levels of cardiac nerves was
verified using 4 rats. The suppressive effect of cPAs on heart rate
increase due to stimulation was significantly observed in the case
of Ole-cPA, but was not significant in the case of Pal-cPA. Hence,
changes in the discharge levels of cardiac nerves due to
stimulation were examined only in the case of Ole-cPA. FIG. 2 shows
typical electrograms obtained before cPA administration and after
administration. Particularly the level of C reflex decreased by cPA
administration.
[0070] FIG. 3 shows changes with time in discharge levels after cPA
administration. Here, discharge levels (peak areas in FIG. 2) are
expressed with relative values obtained via normalization with the
use of the level before cPA administration. At any concentrations,
the discharge level of C reflex was found to decrease by up to 40%
(approximately 1 mV) at 10 to 15 minutes after administration. The
discharge level was observed to increase again after 20 to 25
minutes. However, when the dose was 2 mg/Kg, a state of a decrease
by approximately 20% (compared with that before administration) was
maintained even at 30 minutes after administration. The time course
of the suppressed discharge level after cPA administration was
almost in agreement with that observed upon suppression of heart
rate increase. Furthermore, dose dependency similar to that
observed in the case of the suppressive effect on heart rate
increase was observed in the case of the suppression level.
[0071] Meanwhile, in the case of A.delta. reflex, changes were
observed in the discharge level after cPA administration, however,
the changes were observed to be inconsistent compared with C
reflex. Furthermore, a consistent change in the case of C reflex
was observed for 4 rats used in the experiment, whereas changes in
the discharge level of A.delta. reflex were thought to have
properties easily affected by individual differences or
physiological conditions.
[0072] The effects of 2 carba cPAs (Ole-, Pal-, and Pal-) and 3
carba cPAs (Ole-, Pal-, and Pal-) on heart rate, blood pressure,
the discharge level of cardiac nerve were examined by a method
similar to that employed for a natural cPA.
[0073] Regarding the effects on heart rate, the suppressive effect
of Ole-cPA and the same of Pal-carba cPA on heart rate increase
were observed at levels almost the same as that of a natural
Ole-cPA.
[0074] Regarding the effects on blood pressure, similar to Ole-cPA,
no effects due to administration were observed in all cases.
[0075] In the case of administration of palmitoleoyl 2 carba cPA
(Pal-2 ccPA) among 2 carba cPAs, a significant decrease was
observed in the depolarization level of cardiac nerves and
particularly of C reflex.
[0076] The effect of Pal-2 ccPA with a concentration ranging from
50 .mu.g/Kg to 500 .mu.g/Kg on the depolarization level of C reflex
was analyzed. As a result, at any concentration, the depolarization
level of C reflex was found to decrease at 10 to 15 minutes after
administration. Specifically, an approximately 30% or more decrease
was observed when the concentration was 50 .mu.g/Kg ( 1/40 of the
concentration (2 mg/Kg) at which natural Ole-cPA causes an
approximately 50% decrease), an approximately 60% decrease was
observed when the concentration was 100 .mu.g/Kg ( 1/20 of the
same), an approximately 70% decrease was observed when the
concentration was 200 .mu.g/Kg ( 1/10 of the same), and an
approximately 80% decrease was observed when the concentration was
500 .mu.g/Kg (1/4 of the same). The depolarization level was
observed to increase again after 20 to 25 minutes, and then
observed to gradually return to the level at the time of
administration.
[0077] In the case of administration of palmitoleoyl 3 carba cPA
(Pal-3 ccPA) among 3 carba cPAs, a significant decrease was
observed in the depolarization level of cardiac nerves and
particularly of C reflex, similarly to 2 carba derivatives. The
degree of the decrease was somewhat lower than those of 2 carba
derivatives. The depolarization level of C reflex was found to
decrease at 10 to 15 minutes after administration of Pal-3 ccPA
with a concentration ranging from 50 .mu.g/Kg to 200 .mu.g/Kg. An
approximately 30% decrease was observed when the concentration was
50 .mu.g/Kg ( 1/40 of the concentration (2 mg/Kg) at which natural
Ole-cPA causes an approximately 50% decrease), an approximately 50%
decrease was observed when the concentration was 100 .mu.g/Kg (
1/20 of the same), and an approximately 60% decrease was observed
when the concentration was 200 .mu.g/Kg ( 1/10 of the same). Also
in this case, the depolarization level was observed to increase
again after 20 to 25 minutes and then gradually return to the level
at the time of administration.
[0078] FIG. 4 shows comparison of Ole-cPA, Pal-2 ccPA, and Pal-3
ccPA.
[0079] The results are as described above such that heart rate
increase and depolarization of cardiac nerves due to electrical
stimulation were lowered by administration of a cPA, 2 ccPA, or 3
ccPA. The depolarization level of C reflex is thought to reflect
chronic pain. The results strongly suggest that cPAs and
derivatives thereof, 2 ccPA and 3 ccPA, have significant analgesic
effects.
Expected Mechanisms of Analgesic Effects Exerted by cPAs
[0080] The following action mechanisms of the analgesic effects
exerted by cPAs are expected.
(1) Evaluation is made using reactions due to electrical
stimulation of afferent nerve fibers on the central side from the
nociceptor, so as to eliminate the possibility that the effects are
peripheral effects on the periphery of the nociceptor. (2)
Transmission of excitation in primary afferent C fibers may be
blocked. (3) Nociceptive transmitter release within the central
nerve, such as glutamate or substance P release from primary
afferent C fiber ends in the posterior horn of the spinal cord, may
be suppressed. (4) Within the central nerve, such as within the
posterior horn of the spinal cord, an opioid analgesic system may
be activated by the effect of accelerating endogenous opioid
release or the effect of increasing opioid receptor sensitivity.
(5) A nonopioid analgesic system such as a serotoninergic
descending inhibitory system, a noradrenergic descending inhibitory
system, or a GABA-mediated analgesic system may be activated. (6)
The analgesic effects may be antagonistic to the pain generation
effects of LPA.
BRIEF DESCRIPTION OF THE DRAWINGS
[0081] FIG. 1 shows the effects of the administration of cPAs on
changes in heart rate after stimulation of lower extremity
nerves.
[0082] FIG. 2 shows representative examples of the electrogram
obtained before administration of a cPA (left) and the same
obtained after administration (right). Peaks on the left and the
right represent A.delta. reflex and C reflex, respectively.
[0083] FIG. 3 shows changes in A.delta. and C reflex levels of
cardiac nerves after stimulation of lower extremity nerves. Each
data point represents an average value calculated every 5 minutes.
Response levels obtained with 2 to 3 instances of administration to
each rat were averaged. The thus obtained average response levels
were further averaged for 4 rats. The bar denotes the standard
error.
[0084] FIG. 4 shows the results of comparing the effects of natural
Ole-cPA with the effects (changes in C reflex levels of cardiac
nerves after stimulation of lower extremity nerves by
administration of Pal-2 ccPA or Pal-3 ccPA having varied
concentrations (.mu.g/Kg)) of Pal-2 ccPA or Pal-3 ccPA. The peak
area of C reflex was measured and the result was used for the
depolarization level for each reflex. In each administration, the
depolarization level immediately before administration (0 minute)
was used as the standard and then a relative depolarization level
(%) was calculated. The graph shows average values of relative
depolarization levels measured at 10 to 15 minutes after
administration.
* * * * *