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| United States Patent | 4,748,187 |
| Kakimoto , et al. | May 31, 1988 |
Opioid peptide-degrading enzyme inhibitor with a germanium
compound
Abstract
The present invention provides (1) a new organogermanium compound of the following general formula: ##STR1## wherein A represents a hydrogen atom, a lower alkyl group such as a methyl or ethyl group or a phenyl group, B represents a hydrogen atom or a lower alkyl group as mentioned above and Z represents a hydroxyl or amino group and (2) an opioid peptide-degrading enzyme inhibitor containing the compound (1) as a principal ingredient.
| Inventors: | Kakimoto; Norihiro (Tokyo, JP); Katayama; Takashi (Tokyo, JP); Hazato; Tadahiko (Saitama, JP); Ohnishi; Tsutomu (Tokyo, JP) |
| Assignee: | Asai Germanium Research Institute (Tokyo, JP) |
| Appl. No.: | 930561 |
| Filed: | November 14, 1986 |
Foreign Application Priority Data
| Jul 01, 1983[JP] | 58-119856 | |
| Jul 11, 1983[JP] | 58-125725 |
| Current U.S. Class: | 514/492 |
| Intern'l Class: | A61K 031/28 |
| Field of Search: | 514/492 |
References Cited [Referenced By]
U.S. Patent Documents
| 4271084 | Jun., 1981 | Ishikawa et al. | 260/429. |
| 4508654 | Apr., 1985 | Chang et al. | 260/429. |
| Foreign Patent Documents | |||
| 55-105696 | Aug., 1980 | JP. | |
| 57-203090 | Dec., 1982 | JP. | |
Primary Examiner: Friedman; Stanley J.
Attorney, Agent or Firm: Burns, Doane, Swecker & Mathis
Parent Case Text
This application is a division of application Ser. No. 626,787, filed July 2,
1984, now U.S. Pat. No. 4,681,960.
Claims
What is claimed is:
1. A method of inhibiting the action of an opioid peptide-degrading enzyme
comprising administering to an organism in need of such treatment an effective
opioid peptide-degrading enzyme amount of a compound having the formula:
##STR63## wherein A represents a hydrogen atom, a lower alkyl group or a phenyl
group, B represents a hydrogen atom or a lower alkyl group and Z represents a
hydroxyl or amino group; wherein said lower alkyl group is selected from the
group consisting of methyl, ethyl and propyl.
2. A method as recited in claim 1 wherein A represents a hydrogen atom, B
represents a hydrogen atom and Z represents a hydroxyl group.
3. A method as recited in claim 1 wherein A represents a hydrogen atom, B
represents a lower alkyl group and Z represents a hydroxyl group.
4. A method as recited in claim 1 wherein A represents a hydrogen atom, B
represents a hydrogen atom and Z represents an amino group.
5. A method as recited in claim 1 wherein A represents a hydrogen atom, B
represents a lower alkyl group and Z represents an amino group.
6. A method as recited in claim 1 wherein A represents a lower alkyl group, B
represents a lower alkyl group and Z represents a hydroxy group.
7. A method as recited in claim 1 wherein A represents a lower alkyl group, B
represents a lower alkyl group and Z represents an amino group.
8. A method as recited in claim 1 wherein A represents a phenyl group, B
represents a hydrogen atom and Z represents a hydroxy group.
9. A method as recited in claim 1 wherein A represents a phenyl group, B
represents a hydrogen atom and Z represents an amino group.
10. A method as recited in claim 1 wherein A represents a phenyl group, B
represents a lower alkyl group and Z represents a hydroxy group.
11. A method as recited in claim 1 wherein A represents a phenyl group, B
represents a lower alkyl group and Z represents an amino group.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to organogermanium compounds having new structures
and a strong opioid peptide-degrading enzyme inhibitor containing the same as a
principal ingredient.
2. Description of the Prior Art
Germanium (Ge), known as a homologue of carbon, has semiconductive effect like
silicon (Si) as a special property and, in addition, it has been studied in this
aspect for a long time. Recently, the studies of organogermanium compounds have
been advanced and the results thereof have been reported and they have attracted
public attention in various technical fields.
It is well known from reports of numerous scientific meetings and literature
that a carboxyethylgermanium sesquioxide (GeCH.sub.2 CH.sub.2 COOH).sub.2
O.sub.3, as a macromolecular compound (a propionic acid derivative of germanium)
containing a 12-membered ring as a unit structure in which germanium atoms and
oxygen atoms are arranged alternately, has quite excellent physiological effects
such as strong hypotensive and antineoplastic effects, and it is free from
toxicity or adverse reaction.
It has also been reported that when the above mentioned organogermanium compound
is administered to a patient who complains of pain such as a cancerous pain, the
growth of the tumor is inhibited and the dose of a narcotic analgesic such as
morphine required for relieving the pain can be reduced. For this fact, the
following hypothesis has been given.
Namely, when morphine or the like is administered, peptides generally called "opioid
peptides" are liberated in vivo. This opioid peptide and morphine share the same
receptor to control the autoanalgesic activity in vivo. A reason why the dose of
morphine or the like can be reduced by the administration of the organogermanium
compound is that the organogermanium compound inhibits the action of opioid
peptide-degrading enzyme which inactivate the opioid peptide by decomposition in
vivo to improve the efficiency of the opioid peptide in vivo.
However, the mechanism of the physiological activity of the organogermanium
compound has not fully been known. As for the antineoplastic effects, some
researchers reported that the effect is realized based on a germanium-oxygen
bond in the structure. If an organogermanium compound containing an analogous
atom in place of the oxygen atom can be synthesized, the use of the resulting
compound for a purpose different from that of the known organogermanium compound
can be expected.
SUMMARY OF THE INVENTION
The present invention has been completed under these circumstances. It is an
object of the present invention to provide organogermanium compounds having the
following general formula: ##STR2## wherein A represents a hydrogen atom, a
lower alkyl group such as a methyl or ethyl group or a phenyl group, B
represents a hydrogen atom or a lower alkyl group as mentioned above and Z
represents a hydroxyl or amino group.
Another object of the present invention is to provide an opioid
peptide-degrading enzyme inhibitor which comprises as a principal ingredient an
organogermanium compound of the following general formula: ##STR3## wherin A
represents a hydrogen atom, a lower alkyl group such as a methyl or ethyl group
or phenyl group, B represents a hydrogen atom or a lower alkyl group as
mentioned above and Z represents a hydroxyl or amino group.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
In the organogermanium compound of the present invention, a germanium atom is
bonded with propionic acid derivative (when Z is OH) or its amide (when Z is
NH.sub.2), in which a substituent A is placed in an .alpha.-position and
substituent(s) B is (are) placed in .alpha.-position and/or .beta.-position to
the germanium atom on this propionic acid skeleton to form a germylpropionic
acid as a base construction (in which carbon atoms on the propionic acid
skeleton not bonded with the substituent B are bonded with hydrogen atoms), and
germanium atoms of this base construction and the sulfur atoms are bonded in a
ratio of 2/3 to form a ethylgermanium sesquisulfide.
The substituent A is a hydrogen atom, a lower alkyl group such as a methyl,
ethyl or propyl group or a substituted or unsubstituted phenyl group. The
substituent B is a hydrogen atom or an alkyl group as mentioned in the
substituent A. Therefore, the organogermanium compounds of the present invention
include the following compounds: ##STR4##
The compounds in the present invention are represented as above, since the ratio
of the germylpropionic acid to the sulfur atom is 2/3 in these compounds. The
compounds of the present invention may be represented also as follows: ##STR5##
The compounds of the present invention having the above mentioned structures may
be prepared by various processes.
The compounds of the general formula (I) wherein Z represents OH [i.e. compounds
(I')] may be prepared by reacting a corresponding trichlorogermanium compound
(II) with dry hydrogen sulfide gas (H.sub.2 S) in the presence of a base such as
pyridine in an organic solvent as shown by the following reaction scheme (I):
##STR6##
The compounds of the general formula (I) wherein Z is NH.sub.2 [i.e. compounds
(I")] may be prepared by first converting the same trichlorogermanium compound
(II) as above into a corresponding acid chloride (III), then reacting the same
with ammonia (NH.sub.3) to form an amide (IV) and reacting the product with dry
hydrogen sulfide gas in the presence of a base in an organic solvent in the same
manner as above, as shown by the following reaction scheme (2): ##STR7##
In the above reaction scheme (1) and (2), a mercapto compound of the formula:
##STR8## is formed by the reaction with hydrogen sulfide. This mercapto compound
may be either isolated or not. When this compound is isoloated, intermolecular
hydrogen sulfide elimination occurs to form a structure of the general formula
(I).
The trichlorogermanium compound (II) being used in the above mentioned reaction
may be prepared by a process disclosed in the specification of Japanese Patent
Publication No. 2964/1971 as follows: ##STR9## Alternatively, the compound (II)
may be prepared by directly reacting the same starting material as above with an
acrylic acid derivative as follows: ##STR10##
The thus-obtained compounds of the present invention including the above
mentioned compounds (1) to (14) are colorless, transparent crystals having a
melting poing (or decomposition point) of generally around 200.degree. C. The
results of elementary analyses coincide with values calculated from the
respective molecular formulae, differences between them being within the range
of measuremental error. The results of infrared (IR) absorption spectrum and
nuclear magnetic resonance (NMR) absorption spectrum prove that the compounds of
the present invention are those shown by the above general formula (I).
The compounds of the present invention are characterized in that they are
slightly soluble in water and highly soluble in an organic solvent miscible with
water, such as acetone or alcohol, namely they are oil-soluble, while the above
mentioned carboxyethylgermanium sesquioxide is slightly soluble in water and
insoluble in an organic solvent at all.
The organogermanium compounds of the present invention have the germanium-sulfur
bonds very close to the germanium-oxygen bonds in the known
carboxyethylgermanium sesquioxide. It is expected, therefore, when the compound
of the present invention is administered to a living body, similar
antineoplastic effect, etc., are obtained. In this connection, it is to be noted
that the effect of the organogermanium compounds of the present invention
resides in a strong inhibition of the opioid peptide-degrading actions of the
above mentioned opioid peptide-degrading enzyme.
Namely, as described above, the substances generally called "opioid peptides"
which are peptides found in the living bodies are quite important compounds
managing the autoanalgesic activity in vivo. The opioid peptides includes
several compounds such as enkephalin isolated from swine or bovine brains by
Hughes et al. in 1975 and having the following structure:
H.sub.2 N-Tyr-GlY-Gly-Phe-Met-OH
As the enzymes which degrade the opioid peptides such as enkephalin, there have
been found numerous enzymes such as dipeptidylaminopeptidase and aminopeptidase
which can be separated from various living tissues and purified. It has been
found that when these enzymes are reacted on the opioid peptides or their model
compounds in the presence of the compound of the present invention, the compound
of the invention strongly inhibit the action of the enzymes.
The effects of the compounds of the present invention are quite strong. For
example, the compound (4) has 97.0% inhibition against the effect of
aminopeptidase (derived from bovine longitudinal muscle) on enkephalin, i.e. one
of the opioid peptide. Thus, when an opioid peptide-degrading enzyme inhibitor
in the form of a solid preparation such as tablets, powder, granules or capsules
or a liquid preparation such as an injection, containing the organogermanium
compound of the present invention as the principal ingredient is administered to
a living body, the effects of the opioid peptide-degrading enzyme is remarkably
inhibited and the effective utilization of the opioid peptide is improved.
Consequently, the medical effects of a narcotic substance such as morphine
become remarkable and the dose of the narcotic substance to be used for
obtaining a given medical effect can be reduced. Thus, side effects brought
about by the continuous use of the narcotic substance such as habituation and
addiction can be relieved.
Dipeptidylcarboxypeptidase which is one of the opioid peptide-degrading enzymes
acts also as an converting enzyme for angiotensin I which is a precursor of
angiotensin II (an enzyme having a quite strong hypertensive effect). Therefore,
when this effect of the enzyme is inhibited, the inhibitor also acts on a renin/angiotensin/aldosterone
system to exert preferred influences on the living body, particularly blood
pressure maintenance mechanism.
The following examples will further illustrate the present invention.
EXAMPLE 1
Preparation of compound (I') of the present invention
Synthesis of compound (1)
25.2 g (0.1 mol) of .beta.-trichlorogermylpropionic acid was dissolved in 200 ml
of anhydrous benzene. 24 g (0.1 mol) of anhydrous pyridine was added to the
solution and the mixture was stirred. Then, dry hydrogen sulfide gas was
introduced therein for 60 min. Benzene was removed carefully from the resulting
oily product and then the residue was dissolved in 100 ml of methanol. The
solution was added to 300 ml of purified water and crystals thus formed were
recrystallized from methanol to obtain 16.2 g of compound (1) of the present
invention in the form of colorless plate. Yield was 78%.
Compound (1)
melting point: 200.degree. C. (calcutated from the DTA spectrum; the same shall
apply hereinafter)
______________________________________
elementary analysis:
Ge C H S
______________________________________
found 37.44 18.61 2.62 24.83
calculated 37.41 18.58 2.62 24.88
______________________________________
IR (KBr, cm.sup.-1) 3420, 1710, 425
NMR (methanol d.sub.4 .sigma.) 1.97 (2H, t, Ge--CH.sub.2), 2.67 (2H, t, CH.sub.2
--CO)
Synthesis of compound (4)
20.02 g (0.2 mol) of (E)-2-methyl-2-butenoic acid was dissolved in 100 ml of dry
ethyl ether. 36.0 g (0.2 mol) of trichlorogermane was added to the solution and
stirred for 2 hrs. Crystals thus formed were recrystallized from n-hexane to
obtain 42.86 g (yield: 76.5%) of 2-methyl-3-(trichlorogermyl)butanoic acid in
the form of colorless plate.
Then, 5.6 g (0.02 mol) of 2-methyl-3-(trichlorogermyl)-butanoic acid prepared as
above was dissolved in 100 ml of anhydrous benzene. 5.2 g (0.066 mol) of
anhydrous pyridine was added to the solution and the mixture was stirred and dry
hydrogen sulfide gas was introduced therein for 60 min. A compound thus
precipitated was separated and then recrystallized from anhydrous acetone or
purified by isolating the same by means of a molecular sieve such as Sephadex
LH-20 (trade name) using methanol as a eluant to obtain 3.2 g of compound (4) of
the present invention. Yield was 72.1%.
Compound (4)
melting point: 235.degree. C.
______________________________________
elementary analysis:
Ge C H S
______________________________________
calculated: 32.73 27.07 4.09 21.68
found: 32.50 27.13 4.02 21.92
______________________________________
IR (KBr, cm.sup.-1) 3400, 2960, 1700, 1445, 1225, 820, 680, 600, 425
NMR (CD.sub.3 OD, .sigma.) 1.33 (3H, dd, Ge--CH--CH.sub.3), 1.40 (3H, dd,
CO--CH--CH.sub.3), 2.18 (1H, m, Ge--CH), 2.80 (1H, m, CO--CH)
Other compounds may also be prepared in the same manner as above. The physical
properties of the compounds (I') are shown in Tabel (1).
EXAMPLE 2
Preparation of compound (I") of the present invention
Synthesis of compound (11)
28.0 g (0.1 mol) of 2-methyl-3-(trichlorogermyl)butanoic acid was treated with
100 ml of thionyl chloride and then distilled under reduced pressure to obtain
27.0 g (yield: 90.4%) of 2-methyl-3-(trichlorogermyl)-butanoyl chloride as a
light yellow fraction having a boiling point of 99.degree. to 100.degree. C./6
mmHg.
5.8 g (0.02 mol) of this chloride was dissolved in 50 ml of anhydrous benzene.
Dry ammonia was introduced therein under cooling with ice for 1 h. Then, dry
hydrogen chloride gas was introduced therein for 1 h. 100 ml of methyl acetate
was added thereto and the mixture was stirred and filtered. The filtrate was
distilled and the residue was recrystallized from a liquid mixture of
acetone/benzene (1/2) to obtain 4.1 g (yield: 76.0%) of
2-methyl-3-(trichlorogermyl)butanamide.
10.8 g (0.04 mol) of the obtained 2-methyl-3-(trichlorogermyl)butanamide was
dissolved in 200 ml of anhydrous benzene. 9.5 g (0.12 mol) of anhydrous pyridine
was added to the solution and the mixture was stirred. Dry hydrogen sulfide gas
was introduced therein for 60 min. A compound thus precipitated was separated
and then recrystallized from anhydrous acetone or purified by isolating the same
by means of a molecular sieve such as Sephadex LH-20 (trade name) using methanol
as a eluant to obtain 7.8 g of compound (11) of the present invention. Yield was
88.3%.
Compound (11)
melting point: 205.degree. C. (decomposition)
______________________________________
elementary analysis:
Ge C H N S
______________________________________
calculated:
32.87 27.20 4.56 6.34 21.87
found: 32.59 27.37 4.43 6.25 21.56
______________________________________
IR (KBr, cm.sup.-1) 3400, 3200, 2960, 1660, 1460, 1400, 780, 570, 420
NMR (CD.sub.3 OD, .sigma.) 1.30 (3H, d, Ge--CH--CH.sub.3), 1.38 (3H, d,
CO--CH--CH.sub.3), 2.14 (1H, m, Ge--CH),
2.71 (1H, m, CO--CH),
Other compounds were prepared in the same manner as above. The physical
properties of the compounds (I") are shown in Table (2).
TABLE (1)
__________________________________________________________________________
Physical Properties
Elementary
calculated
Melt-
Com-
analysis found ing Yield
pound
Ge C H S point
IR(KBr, cm.sup.-1)
(Solvent)
NMR (.delta.) (%)
__________________________________________________________________________
(2)
##STR11##
##STR12##
##STR13##
##STR14##
185 (dec)
3420, 1705, 425
CD.sub.3 OD
##STR15## 57.7
(3)
##STR16##
##STR17##
##STR18##
##STR19##
196 3410, 1705, 425
CD.sub.3 OD
##STR20## 93
(5)
##STR21##
##STR22##
##STR23##
##STR24##
205 (dec)
3450, 2960, 1700, 1460, 1380 1220, 1130, 680,
620, 425 CD.sub.3 OD
1.46(6H, s,
(CH.sub.3).sub.2), .sub.--
2.60(2H, s, CH.sub.2)
80.3.--
(6)
##STR25##
##STR26##
##STR27##
##STR28##
265 (dec)
3450, 3040, 2850, 1710, 1600 1410, 1230, 700,
425 CD.sub.3 OD
3.00(2H, d, C H.sub.2 CO),
.sub.-- 3.55(1H, t, GeCH),
.sub.-- 7.25(5H, m, C.sub.6
H.sub.5) .sub.--
94.1
(7)
##STR29##
##STR30##
##STR31##
##STR32##
215 (dec)
3450, 3030, 2980, 1705, 1455 1210, 820, 700,
680, 420 CD.sub.3 OD
1.43(3H, d, CH.sub.3),
.sub.-- 3.27(2 .sub.--H, m,
CHCH), .sub.-- 7.17(5H, m,
C.sub.6 H.sub.5) .sub.--
86.0
__________________________________________________________________________
TABLE (2)
__________________________________________________________________________
Elementary
calculated Melt-
Com-
analysis found ing IR(KBr, Yield
pound
Ge C H N S point
cm.sup.-1)
(solvent)
NMR (.delta.) (%)
__________________________________________________________________________
(8)
##STR33##
##STR34##
##STR35##
##STR36##
##STR37##
225.about. 226 (dec)
3340, 1665, 1620, 1200 1400,
DMFd.sub.7
1.98(2H, t, GeCH.sub.2)
.sub.-- 2.56(2H, t, COC
.sub.--H.sub.2)
60.2
(9)
##STR38##
##STR39##
##STR40##
##STR41##
##STR42##
248 (dec)
3300, 3200, 1600, 1400 430
CD.sub.3 OD
1.33(3H, d, GeCHCH.sub.2)
.sub.-- 2.17.about.2.77 (3H,
m, GeC .sub.--HC .sub.--H.sub.
2) 84.1
(10)
##STR43##
##STR44##
##STR45##
##STR46##
##STR47##
225 (dec)
3300, 3200, 1660, 1460 1400,
CD.sub.3 OD
1.23(3H, d, CH.sub.3) .sub.--
.67.about.2.25 (3H, m, GeC
.sub.--HCH.sub.2) .sub.--
83.2
(12)
##STR48##
##STR49##
##STR50##
##STR51##
##STR52##
230 (dec)
3400, 3200, 2960, 1660, 1460 1120,
CD.sub.3 OD
1.22(6H, s,
CH.sub.3CCH.sub.3) .sub.--
2.60(2H, s, CH.sub.2CO)
.sub.-- 76.5
(13)
##STR53##
##STR54##
##STR55##
##STR56##
##STR57##
210 (dec)
3450, 3350, 3200, 1660, 1600 1400, 765,
700, 420
CD.sub.3 OD
2.90(2H, m, CH.sub.2) .sub.--
.55(1H, m, CH) .sub.--
7.19(5H, s, -.sub.6 H.sub. 5)
.sub.-- 81.8
(14)
##STR58##
##STR59##
##STR60##
##STR61##
##STR62##
215 (dec)
3450, 3350, 3200, 1660, 1455 1400, 700,
420 CD.sub.3 OD
1.42(3H, m, CH.sub.3) .sub.--
.23(2H, m, CHCH) .sub.--
7.15(5H, S, C.sub.6 H.sub.5)
.sub.-- 82.3
__________________________________________________________________________
EXAMPLE 3
Pharmacological effects of the compound of the present invention
(1) As described above, the inhibitor of the present invention has a strong
effect of inhibiting the action of the opioid peptide-degrading enzyme. However,
it is difficult to prove the effects of the products of the present invention
unlike other general medicines, since problems are posed because the number of
cases in which narcotic drugs are used for the treatment of diseases is not so
large and the conditions of the patients in these cases are serious generally.
On the other hand, however, some opioid peptides released in vivo when such
narcotic substances are given and opioid peptide-degrading enzymes have been
known. Accordingly, the effects of the products of the present invention were
judged from inhibition rates realized when the products were allowed to act on
the opioid peptide-degrading enzyme in the presence of the opioid peptide in
vitro.
In the tests, the product of the invention was added to an opioid peptide such
as enkephalin or its model compound. After an incubation effected for a given
time, the inhibition rates of the product against the opioid peptide-degrading
enzyme were examined. Various opioid peptides were used. Generally, high
inhibition rates were exhibited as shown in Tables (3) and (4).
TABLE (3)
__________________________________________________________________________
Enzyme
Origin
Bovine longitudinal muscle
Name
Dipeptidyl- Dipeptidyl-
carboxy- Carboxy-
amino-
Amino-
Principal
peptidase
peptidase
peptidase
peptidase
ingredient
Substrate
(1 .mu.g/1 ml)
Hip--His--Leu
Hip--L-PheAla
Enkephalin
Enkephalin
__________________________________________________________________________
Compound (1)
76.4% - - -
Compound (2)
85.0% 6.2% - -
Compound (3)
80.0% - - -
Compound (4)
60% - - 58%
Compound (5)
78.8% - + 88.0%
Compound (6)
74.2% 89.8% + 97.0%
Compound (7)
76.8% - + -
Compound (8)
78.3% - - -
Compound (9)
68.4% - - -
Compound (10)
73.4% - - -
Compound (11)
+ - + -
Compound (12)
+ - + -
Compound (13)
+ - + -
Compound (14)
+ - + -
__________________________________________________________________________
TABLE (4)
______________________________________
Enzyme
Origin
Monkey brain
Name
Dipeptidylaminopeptidase
Aminopeptidase
Substrate
Compound Enkephalin
______________________________________
(1) 98.2% 87.8%
(2) 97.9% 87.6%
(3) 97.7% 85.6%
(8) - -
(9) - -
(10) - -
(4) 62% -
______________________________________
Further, to confirm the inhibition effects of the compounds of the present
invention, 50% inhibition coefficients (IC.sub.50) were determined to obtain the
results shown in Table (5). The effects of the compounds of the present
invention were thus clear.
TABLE (5)
______________________________________
Principal
ingredient
Enzyme/origin Substrate IC50
______________________________________
Compound
dipeptidyl-
bovine Hip--His--L-Lue
66 .mu.g/ml
(2) carboxy- longi-
peptidase tudinal
muscle
Compound
angiotensin
rat " 70 .mu.g/m.sup.
(2) converting
lung
enzyme
Compound
Angiotensin
mon- " 78 .mu.g/ml
(2) converting
key
enzyme brain
Compound
amino- bovine Enkephalin 110 .mu.g/ml
(5) peptidase longi-
tudinal
muscle
Compound
amino- bovine " 19 .mu.g/ml
(6) peptidase longi-
tudinal
muscle
Compound
carboxy- bovine Hip--L-PheAla
275 .mu.g/ml
(6) peptidase longi-
tudinal
muscle
Compound
dipeptidyl-
bovine Hip--His--Leu
100 .mu.g/ml
(6) carboxy- longi-
peptidase tudinal
muscle
______________________________________
The inhibition rate (IC.sub.50) of the compound of the present invention
containing the compound (6) as the principal ingredient on enkephalin (aminopeptidase
derived from bovine longitudinal muscle) was as high as 19 .mu.g/ml. This fact
suggests that the product can be used as an inhibitor against this enzyme.
The opioid peptide-degrading enzymes derived from bovine longitudinal muscle
used in the above examples were purified partially by a process of Goreustein
and Snyder S. H., ["Life Sci." 25, 2065 (1979)]. The inhibition effects of the
compounds of the present invention on the opioid peptide-degrading enzymes were
determined by a process of T. Hazato, M. Shimamura, T. Katayama and T. Yamamoto
[B.B.R.C. 105, 470-475 (1982)] (for dipeptidylaminopeptidase), a process of M.
Shimamura, T. Hazato and T. Katayama [B.B.A., 756, 223-229 (1983)] (for
aminopeptidase) and analogous processes.
(2) The effects of the compounds of the present invention on human bodies were
examined.
A human cerebrospinal fluid was dialyzed by using 25 mM of tris-HCl buffer
having a pH of 7.0 for 5 hrs. Enkephalin-degrading enzymes contained therein
were analyzed according to a radioautography or the like. In the cerebrospinal
fluid, the aminopeptidase activity was the strongest. Further,
dipeptidylaminopeptidase and dipeptidylcarboxypeptidase activites which were
non-selective for bestatin were also recognized.
The compound (3) and (6) of the present invention were allowed to act on the
respective enzymes. The compound (3) in a concentration of 2 mg/ml exhibited
inhibition effects on all the enzymes. The compound (6) in the same concentraton
as that of the compound (3) exhibited inhibition effects on aminopeptidase,
dipeptidylcarboxypeptidase and carboxypeptidase.
On the other hand, the aminopeptidase alone was eluted according to the
cellulose column chromatography using a NaCl solution as a eluant. IC.sub.50
values of the above two compounds on the aminopeptidase were determined
according to Porapak Q column process and high-performance liquid chromatography
using enkephalin as the substrate. The results were compared with those of
Arphamenin A and B the inhibitive actions of which on the enkephalin-degrading
enzyme have been known. As shown in Table (6), the compounds of the present
invention in concentrations lower than those of Arphamenin A and B exhibited the
inhibition activities.
TABLE (6)
______________________________________
Compound IC50 (.mu.g/m)
______________________________________
(3) 450
(6) 440
Arphamenine A 810
Arphamenine B 650
______________________________________
The external liquid used for the dialysis of the cerebrospinal fluid was
examined minutely to reveal that it contained an indogenous inhibitor against
the enkephalin-degrading enzymes.
Namely, it is considered that the human cerebrospinal fluid contains both the
enkephalin-degrading enzymes and the indogenous inhibitors which inhibit these
enzymes and they are well-balanced under normal conditions, a pain being caused
when the balance is broken. This fact suggests the usefulness of the compounds
of the present invention in vivo.
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