U.S. patent application number 09/733932 was filed with the patent office on 2002-05-02 for mixtures which can be isolated from eugenia jambolana lamarck seeds, their preparation and the use of these mixtures and some of their constituents as medicaments.
This patent application is currently assigned to RHONE-POULENC RORER S.A. and INSTITUT MALGACHE DE RECHERCHES APPLIQUEES. Invention is credited to Leboul, Jean, Provost, Jean, Rakoto Ratsimamanga, Suzanne, Rasoanaivo, Philippe, Ratsimamanga, Albert Rakoto, Reisdorf, Daniel.
Application Number | 20020051828 09/733932 |
Document ID | / |
Family ID | 9488865 |
Filed Date | 2002-05-02 |
United States Patent
Application |
20020051828 |
Kind Code |
A1 |
Ratsimamanga, Albert Rakoto ;
et al. |
May 2, 2002 |
Mixtures which can be isolated from eugenia jambolana lamarck
seeds, their preparation and the use of these mixtures and some of
their constituents as medicaments
Abstract
The present invention relates to mixtures which can be isolated
from grains of Eugenis Jambolana Lamarck, the preparation of such
mixtures, the medicaments containing said mixtures or constituents
of said mixtures, and the use of these mixtures and constituents
for the preparation of a medicament.
Inventors: |
Ratsimamanga, Albert Rakoto;
(Antananarivo, MG) ; Rakoto Ratsimamanga, Suzanne;
(Antananarivo, MG) ; Rasoanaivo, Philippe;
(Antananarivo, MG) ; Leboul, Jean; (Gometz La
Ville, FR) ; Provost, Jean; (Monts, FR) ;
Reisdorf, Daniel; (Thiais, FR) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW,
GARRETT and DUNNER, L.L.P.
1300 I Street, N.W.
Washington
DC
20005-3315
US
|
Assignee: |
RHONE-POULENC RORER S.A. and
INSTITUT MALGACHE DE RECHERCHES APPLIQUEES
|
Family ID: |
9488865 |
Appl. No.: |
09/733932 |
Filed: |
December 12, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09733932 |
Dec 12, 2000 |
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09328421 |
Jun 9, 1999 |
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6194412 |
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09328421 |
Jun 9, 1999 |
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09117843 |
Aug 6, 1998 |
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5972342 |
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09117843 |
Aug 6, 1998 |
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PCT/FR97/00207 |
Feb 3, 1997 |
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Current U.S.
Class: |
424/776 ;
514/100 |
Current CPC
Class: |
A61K 36/185 20130101;
C07D 241/12 20130101; A61K 31/495 20130101; A61P 3/10 20180101;
A61K 36/61 20130101; A61P 5/50 20180101; A61K 31/195 20130101; A61K
36/185 20130101; A61K 2300/00 20130101; A61K 36/61 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
424/776 ;
514/100 |
International
Class: |
A61K 035/78 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 6, 1996 |
FR |
96/01389 |
Claims
1. Mixtures free of polyphenol and sterol derivatives and which can
be isolated by grinding Eugenia Jambolana Lamarck seeds, maceration
of the powder with a lower aliphatic alcohol with the use of heat,
filtration, recovery of the insoluble part no longer containing
polyphenol and sterol compounds, treatment of the insoluble part
with an ammoniacal solution, treatment of the ammoniacal mixture
with a lower aliphatic alcohol with the use of heat, filtration,
recovery of the insoluble matter and drying this insoluble matter
which constitutes the mixture I and optionally treatment of the
mixture I with a water-lower aliphatic alcohol solution,
filtration, partial concentration of the filtrate, purification on
non-polar adsorbent resins, partial concentration, centrifugation,
ultrafiltration and isolation of the mixture II.
2. Process for preparing the mixture I according to claim 1,
characterized in that Eugenia Jambolana Lamarck seeds are dried,
finely ground, screened and the powder obtained is subjected to the
following treatment: a--maceration, with stirring, in a lower
aliphatic alcohol, at a temperature of between 40 and 70.degree.
C., b--filtration under vacuum and recovery of the insoluble
matter, c--maceration, with stirring, of the insoluble matter with
a lower aliphatic alcohol at a temperature of between 40 and
70.degree. C., d--filtration under vacuum and removal of the
alcoholic phases containing mainly the undesirable polyphenols and
sterols, e--taking up the insoluble matter in an ammoniacal
solution at a temperature of between 10 and 30.degree. C.,
f--taking up the whole wet ammoniacal mass in a water-lower
aliphatic alcohol solution, at a temperature of between 40 and
70.degree. C., g--filtration and removal of the alcoholic solution,
h--washing of the insoluble matter with a lower aliphatic alcohol,
filtration and removal of the alcoholic solution, i--recovery of
the insoluble matter and drying the mixture I.
3. Process according to claim 2, for which, in step a, the
procedure is carried out by means of 2 to 10 liters of a lower
aliphatic alcohol per 1 kg of screened powder.
4. Process according to either of claims 2 and 3, for which, in
step a, 5 liters of ethanol with a titre of 93-95.degree. Gay
Lussac are used at 60.degree. C. for 1 hour.
5. Process according to one of claims 2 to 4, for which the
filtration of steps b and d are carried out under a vacuum of 40
kPa.
6. Process according to one of claims 2 to 5, for which, in step c,
the procedure is carried out by means of 2 to 10 liters of a lower
aliphatic alcohol per 1 kg of starting screened powder.
7. Process according to one of claims 2 to 6, for which, in step c,
4 liters of ethanol with a titre of 93-95.degree. Gay Lussac are
used at a temperature of 60.degree. C. for 1 hour.
8. Process according to one of claims 2 to 7, for which, in step e,
750 to 1250 ml of an aqueous ammoniacal solution are used per 1 kg
of starting screened powder.
9. process according to one of claims 2 to 8, for which, in step e,
per 1 kg of starting screened powder, 1 liter of aqueous ammoniacal
solution containing 35 ml of 28% ammonium hydroxide is used and the
procedure is carried out at a temperature close to 20.degree. C.
for 10 to 30 hours hours.
10. Process according to one of claims 2 to 9, for which, in step
f, the wet ammoniacal mass obtained from 1 kg of starting screened
powder is taken up in 2 to 10 liters of a water-lower aliphatic
alcohol solution.
11. Process according to one of claims 2 to 10, for which, in step
f, the procedure is carried out in 5 liters of an ethanol-water
mixture (75/25 by volume), at 60.degree. C. for 1 hour.
12. Process according to one of claims 2 to 11, for which, in step
h, the washing is carried out with 500 to 1500 ml of a lower
aliphatic alcohol per 1 kg of starting screened powder.
13. Process according to one of claims 2 to 12, for which, in step
h, the washing is carried out with 1 liter of ethanol and the
filtration is carried out on a cotton cloth and under a vacuum of
about 80 kPa.
14. Process according to one of claims 2 to 12, for which, in step
i, the drying is carried out in the open air and protected from
light.
15. Process for preparing the mixture II according to claim 1, for
which the mixture I according to claim 1 is subjected to the
following operations: j--treatment of the mixture I according to
claim 1 by means of a water-lower aliphatic alcohol solution,
k--decantation and then, on the one hand, drawing off the top phase
which is filtered to give the filtrate 1 and, on the other hand,
treating the bottom phase with water and filtration to give the
filtrate 2, pooling of the filtrates 1 and 2 and concentration to
aqueous phase, l--treatment with a nonpolar adsorbent resin and
then filtration, m--concentration of the filtrate, filtration and
then ultrafiltration, n--freeze-drying and isolation of the extract
II.
16. Process according to claim 15, for which, in step j, 10 to 25
liters of the water-lower aliphatic alcohol solution (95/5 to 90/10
by volume) are used per 1 kg of the mixture I per 1 kg of the
mixture I.
17. Process according to either of claims 15 and 16, for which, in
step j, 18 liters of a water-ethanol solution (17.7-0.93 by volume)
are used.
18. Process according to one of claims 15 to 17, for which, in step
k, the top phase is filtered on a cotton cloth and 10 to 25 liters
of water are added to the bottom phase per 1 kg of the mixture I
and the mixture is filtered on sintered glass.
19. Process according to one of claims 15 to 18, for which, in step
k, the concentration is generally carried out in a thermosiphon
concentrator at a temperature of 35.degree. C. under a vacuum of
0.4 kPa.
20. Process according to one of claims 15 to 19, for which, in step
1, S861 resin or XAD-type resins marketed by Rhom and Hass are used
and the mixture is filtered on sintered glass.
21. Process according to one of claims 15 to 20, for which, in step
m, the concentration is carried out in a thermosiphon concentrator
at a temperature of 35.degree. C. under a vacuum of 0.4 kPa.
22. Process according to one of claims 15 to 21, for which, in step
m, 3 successive ultrafiltrations are carried out on 10 kd, 3 kd and
1 kd cartridges.
23. Process for preparing sodium oxamate and the compounds of
formula: 2in which either R.sub.1 represents a hydrogen atom and
R.sub.2 represents a chain of formula:
--CH.sub.2--CHOH--CHOH--CH.sub.2OH (A)
--CHOH--CHOH--CHOH--CH.sub.2OH (B) or R.sub.1 represents a chain of
formula (A) and R.sub.2 represents a hydrogen atom for which the
mixture II according to claim 1 is subjected to the following
operations: o--chromatography of the mixture II according to the
invention on an infusorial earth column, recovery of the fractions
containing the 4 products and pooling of these fractions into a
single fraction, p--chromatography of the fraction previously
obtained on a Sephadex.RTM. column in order to obtain sodium
oxamate, the compound of formula (I) for which R.sub.1 represents a
hydrogen atom and R.sub.2 represents a residue (B) and a mixture of
the compound of formula (I) for which R.sub.1 represents a hydrogen
atom and R.sub.2 represents a residue (A) and of the compound of
formula (I) for which R.sub.1 represents a residue (A) and R.sub.2
represents a hydrogen atom, q--optionally, chromatography of the
mixture of the compound of formula (I) for which R.sub.1 represents
a hydrogen atom and R.sub.2 represents a residue (A) and of the
compound of formula (I) for which R.sub.1 represents a residue (A)
and R.sub.2 represents a hydrogen atom by HPLC.
24. Process according to claim 23, for which the chromatography of
step o is carried out on a CHEM ELUT.RTM. column marketed by
Prolabo, saturated with water and then eluted successively with
heptane, a heptane-ethyl acetate mixture (50/50 by volume), an
ethyl acetate-n-butanol mixture (95/5; 90/10; 80/20; 50/50; 20/80
and then 0/100 by volume) and an n-butanol-water mixture (98/2 and
then 95/5 by volume).
25. Process according to claim 24, for which the chromatography of
step p is carried out by means of a water-ethanol mixture (50/50 by
volume).
26. Process according to claim 24, for which the chromatography of
step q is carried out on a YMC 180DS-AQ column marketed by AIT
with, as eluent, a water containing 0.1% of formic acid
mixture.
27. Medicaments characterized in that they contain, as active
ingredient, a mixture I according to claim 1 or a mixture II
according to claim 1 or sodium oxamate or one or more compounds of
formula: 3in which either R.sub.1 represents a hydrogen atom and
R.sub.2 represents a chain of formula:
--CH.sub.2--CHOH--CHOH--CH.sub.2OH (A) --CHOH--CHOH--CHOH--CH.s-
ub.2OH (B) or R.sub.1 represents a chain of formula (A) and R.sub.2
represents a hydrogen atom and one or more excipients.
28. Use of a mixture I according to claim 1 or a mixture II
according to claim 1 or sodium oxamate or one or more compounds of
formula: 4in which either R.sub.1 represents a hydrogen atom and
R.sub.2 represents a chain of formula:
--CH.sub.2--HOH--CHOH--CH.sub.2OH (A)
--CHOH--CHOH--CHOH--CH.sub.2OH (B) or R.sub.1 represents a chain of
formula (A) and R.sub.2 represents a hydrogen atom for the
preparation of a medicament for the prevention and treatment of
diabetes and of the complications of diabetes.
Description
[0001] The present invention relates to mixtures which can be
isolated from Eugenia Jambolana Lamarck seeds (Myrtaceae family),
medicaments containing these mixtures or some of their
constituents, the use of these mixtures and constituents for the
preparation of an antidiabetic medicament and their
preparations.
[0002] A plant extract prepared from Eugenia Jambolana seeds or
bark containing a polyphenol and sterol mixed complex is described
in patent FR 2,465,484.
[0003] New mixtures which can be isolated from Eugenia Jambolana
Lamarck seeds, and which are free of polyphenol and sterol complex,
as well as certain constituents of these mixtures endowed with
hypoglycaemic properties, have now been found.
[0004] These mixtures are characterized in that they are free of
polyphenol and sterol derivatives and can be isolated by grinding
Eugenia Jambolana Lamarck seeds, maceration of the powder with a
lower aliphatic alcohol with the use of heat, filtration, recovery
of the insoluble part no longer containing polyphenol and sterol
compounds, treatment of the insoluble part with an ammoniacal
solution, treatment of the ammoniacal mixture with a lower
aliphatic alcohol with the use of heat, filtration, recovery of the
insoluble matter and drying this insoluble matter which constitutes
the mixture I, then optionally treatment of the mixture I with a
water-lower aliphatic alcohol solution, filtration, partial
concentration of the filtrate, purification on nonpolar adsorbent
resins, partial concentration, centrifugation, ultrafiltration and
isolation of the mixture II.
[0005] From the mixture II, there may also be separated sodium
oxamate and the compounds of formula: 1
[0006] in which either R.sub.1 represents a hydrogen atom and
R.sub.2 represents a chain of formula:
--CH.sub.2--CHOH--CHOH--CH.sub.2OH (A)
--CHOH--CHOH--CHOH--CH.sub.2OH (B)
[0007] or R.sub.1 represents a chain of formula (A) and R.sub.2
represents a hydrogen atom.
[0008] Sodium oxamate has already been described by TOUSSAINT,
Ann., 120, 237 (1861).
[0009] The compounds of formula (I) have already been described by
KUHN et al., Ann., 644, 122-127 (1961); TSUCHIDA et al., Agr. Biol.
Chem., 39 (5), 1143-1148 (1975); TSUCHIDA et al., Agr. Biol. Chem.,
40 (5), 921-925 (1976); TSUCHIDA et al., Nippon Shokuhin Kogyo
Gakkaishi, 37, 154-161 (1990) and AVALOS et al., tetrahedron, 49,
2655-2675 (1993).
[0010] The present invention also relates to the process for
preparing the mixture I from dried and finely ground Eugenia
Jambolana Lamarck seeds.
[0011] The powder is screened, preferably with the aid of a
normalized screen with holes 0.5 .mu.m in diameter and then
subjected to the following treatment:
[0012] a--maceration, with stirring, in a lower aliphatic alcohol,
at a temperature of between 40 and 70.degree. C.,
[0013] b--filtration under vacuum and recovery of the insoluble
matter,
[0014] c--maceration, with stirring, of the insoluble matter with a
lower aliphatic alcohol at a temperature of between 40 and
70.degree. C.,
[0015] d--filtration under vacuum and removal of the alcoholic
phases containing mainly the undesirable polyphenols and
sterols,
[0016] e--taking up the insoluble matter in an ammoniacal solution
at a temperature of between 10 and 30.degree. C.,
[0017] f--taking up the whole wet ammoniacal mass in a water-lower
aliphatic alcohol solution, at a temperature of between 40 and
70.degree. C.,
[0018] g--filtration and removal of the alcoholic solution,
[0019] h--washing of the insoluble matter with a lower aliphatic
alcohol, filtration and removal of the alcoholic solution;
[0020] i--recovery of the insoluble matter and drying.
[0021] In step a, the procedure is generally carried out by means
of 2 to 10 liters of a lower aliphatic alcohol such as methanol or
ethanol per 1 kg of screened powder. Preferably, 5 liters of
ethanol with a titre of 93-95.degree. Gay Lussac are used at
60.degree. C. for 1 hour.
[0022] The filtration of step b is preferably carried out under a
vacuum of 40 kPa.
[0023] In step c, the procedure is generally carried out by means
of 2 to 10 liters of a lower aliphatic alcohol such as methanol or
ethanol per 1 kg of starting screened powder. Preferably, 4 liters
of ethanol with a titre of 93-95.degree. Gay Lussac are used at a
temperature of 60.degree. C. for 1 hour.
[0024] The filtration of step d is preferably carried out under a
vacuum of 40 kPa.
[0025] In step e, per 1 kg of starting screened powder, 750 to 1250
ml of an aqueous ammoniacal solution preferably containing 350 ml
of 28% ammonium hydroxide per 1000 ml are generally used. It is
particularly advantageous to use 1 liter of the aqueous ammoniacal
solution and to carry out the procedure for 10 to 30 hours and,
preferably, 20 hours at a temperature close to 20.degree. C.
[0026] In step f, the wet ammoniacal mass obtained from 1 kg of
starting screened powder is generally taken up, with stirring, in 2
to 10 liters of a lower aliphatic alcohol-water mixture (methanol
or ethanol for example) (70/30 to 80/20 by volume) and, preferably,
in 5 liters of an ethanol-water mixture (75/25 by volume), at
60.degree. C., for 1 hour.
[0027] In step g, the filtration is preferably carried out on a
cotton cloth and under a vacuum of about 80 kPa.
[0028] In step h, the washing is generally carried out with 500 to
1500 ml of a lower aliphatic alcohol (methanol or ethanol for
example) per 1 kg of starting screened powder and, preferably, with
1 liter of ethanol and the filtration is carried out on a cotton
cloth and under a vacuum of about 80 kPa.
[0029] In step i, the drying is preferably carried out in the open
air and protected from light.
[0030] The present invention also relates to the process for
preparing the mixture II.
[0031] The mixture I obtained above is subjected to the following
operations:
[0032] j--treatment of the mixture I by means of a water-lower
aliphatic alcohol solution,
[0033] k--decantation and then, on the one hand, drawing off the
top phase which is filtered to give the filtrate 1 and, on the
other hand, treating the bottom phase with water and filtration to
give the filtrate 2, pooling of the filtrates 1 and 2 and
concentration to aqueous phase,
[0034] l--treatment with a nonpolar adsorbent resin and then
filtration,
[0035] m--concentration of the filtrate, filtration and then
ultrafiltration,
[0036] n--freeze-drying and isolation of the extract II.
[0037] In step j, 10 to 25 liters of the water-lower aliphatic
alcohol solution (methanol or ethanol for example) (95/5 to 90/10
by volume) are generally used per 1 kg of the mixture I. It is
preferable to carry out the procedure in 18 liters of a
water-ethanol solution (17.7-0.93 by volume).
[0038] In step k, it is preferable to filter the top phase on a
cotton cloth. It is advantageous to add, per 1 kg of the mixture I,
10 to 25 liters of water to the bottom phase and in particular 10
liters and to filter on sintered glass.
[0039] In step k, the concentration is generally carried out in a
thermosiphon concentrator at a temperature of 35.degree. C. under a
vacuum of 0.4 kPa.
[0040] In step l, S861 resin or XAD-type resins marketed by Rhom
and Hass are preferably used and the mixture is filtered on
sintered glass.
[0041] In step m, the concentration is generally carried out in a
thermosiphon concentrator at a temperature of 35.degree. C. under a
vacuum of 0.4 kPa. It is also advantageous to carry out 3
successive ultrafiltrations on 10 kd, 3 kd and 1 kd cartridges.
[0042] The present invention also relates to the process for
preparing sodium oxamate and the compounds of formula (I).
[0043] The said process consists in subjecting the mixture II to
the following operations:
[0044] o--chromatography of the mixture II on an infusorial earth
column, recovery of the fractions containing the 4 products and
pooling of these fractions into a single fraction,
[0045] p--chromatography of the fraction previously obtained on a
Sephadex.RTM. column in order to obtain sodium oxamate, the
compound of formula (I) for which R.sub.1 represents a hydrogen
atom and R.sub.2 represents a residue (B) and a mixture of the
compound of formula (I) for which R.sub.1 represents a hydrogen
atom and R.sub.2 represents a residue (A) and of the compound of
formula (I) for which R.sub.1 represents a residue (A) and R.sub.2
represents a hydrogen atom,
[0046] q--optionally, chromatography of the mixture of the compound
of formula (I) for which R.sub.1 represents a hydrogen atom and
R.sub.2 represents a residue (A) and of the compound of formula (I)
for which R.sub.1 represents a residue (B) and R.sub.2 represents a
hydrogen atom by HPLC.
[0047] The chromatography of step o is carried out by means of an
organic solvent such as heptane, ethyl acetate or a lower aliphatic
alcohol. Preferably, a CHEM ELUT.RTM. column marketed by Prolabo is
used, saturated with water and then eluted successively with
heptane, a heptane-ethyl acetate mixture (50/50 by volume), ethyl
acetate, an ethyl acetate-n-butanol mixture (95/5; 90/10; 80/20;
50/50; 20/80), n-butanol, and an n-butanol-water mixture (98/2 and
then 95/5 by volume).
[0048] The chromatography of step p is preferably carried out by
means of a water-ethanol mixture (50/50 by volume).
[0049] The chromatography of step q is generally carried out on a
YMC 18ODS-AQ column marketed by AIT with, as eluent, a water
containing 0.1% of formic acid mixture.
[0050] In the preceding definitions and those which follow, the
lower aliphatic alcohols preferably contain 1 to 4 carbon
atoms.
[0051] The medicaments containing the mixtures I or II or sodium
oxamate or one or more compounds of formula (I) generally form part
of the invention.
[0052] The present invention also relates to the use of the
mixtures I and II, of sodium oxamate and of the compounds of
formula (I) or a mixture of these with the preparation of
medicaments for the treatment or prevention of diabetes and of the
complications of diabetes.
[0053] The following examples illustrate the invention.
EXAMPLE 1
Preparation of the Mixture I
[0054] Eugenia Jambolana Lamarck seeds are dried in the open air,
protected from light, and then finely ground. The powder thus
obtained is screened with the aid of a screen of mesh 0.5 .mu.m. 1
kg of screened powder is macerated, with mechanical stirring, in 5
liters of ethanol with a titre of 93-95.degree. Gay Lussac, for one
hour at 60.degree. C. After filtration under vacuum, the insoluble
matter is further treated under the same conditions with 4 liters
of ethanol of the same titre at 60.degree. C. for a further one
hour. The two ethanolic extracts containing mainly the undesirable
polyphenol and sterol complex are removed. After complete
filtration, the insoluble matter is taken up in 1 liter of
ammoniacal solution (28% NH.sub.4OH 350 ml and distilled H.sub.2O
in sufficient quantity to obtain 1 liter of solution) and the
mixture is left in contact for about 20 hours at a temperature
close to 20.degree. C. The wet ammoniacal mass is allowed to
macerate again in 5 liters of the ethanol of titre 93 to 95.degree.
Gay Lussac-water mixture (75/25 by volume), with mechanical
stirring, for 1 hour at 60.degree. C. The insoluble matter is
filtered and it is washed with 1 liter of ethanol of the same
titre; the filtrate and the washings are removed. The final
insoluble matter is dried in the open air and protected from light.
The mixture I is thus obtained in the form of a powder which is
free of polyphenol and sterol derivatives. The yield from the
pulverized and screened seeds is 80%.
EXAMPLE 2
Preparation of the Mixture II
[0055] 1 kg of the mixture I obtained in Example 1, in a solution
containing 17.7 liters of water and 0.93 liter of ethanol, is
stirred for 3 hours. After decantation overnight, on the one hand,
the top phase (13.5 liters) is drawn off and then filtered on a
cotton cloth in a 7-liter filter (Schott) to give the filtrate 1
(13.5 liters) and, on the other hand, the bottom phase is stirred
for 1 hour with 20 liters of water, filtered on No. 3 sintered
glass to give the filtrate 2 (24 liters). The filtrates 1 and 2 are
pooled and then concentrated in aqueous phase (33.5 liters) in a
thermosiphon concentrator (Schott) at 35.degree. C. under a vacuum
of 0.4 kPa. The concentrate is stirred for one hour with 2.5 liters
of S861 resin (Rhom and Haas) and then filtered on No. 3 sintered
glass. The filtrate is concentrated in a thermosiphon concentrator
(Schott) at 35.degree. C. and then under a vacuum of 0.4 kPa to 5.5
liters. The concentrate is centrifuged in a tubular centrifuge
(centrifugal force 62000 g) and filtered on a 0.22 .mu.m filter
(Gelman suporcap 100 type) pump. After 3 successive
ultrafiltrations on 10 Kd, 3 Kd and 1 Kd cartridges and
freeze-drying, 25 g of the mixture II are obtained in the form of a
dark brown hygroscopic powder.
EXAMPLE 3
Constituents Obtained from the Mixture II
[0056] 525 mg of the mixture II obtained in Example 2 in solution
in 1.1 ml of milli Q-filtered water are chromatographed on a CHEM
ELUT.RTM. column marketed by PROLABO, 50 cm in height and with a
diameter of 1 cm, saturated with milli Q-filtered water. The
elution is carried out with heptane using increasing gradients of
ethyl acetate and then of n-butanol, of n-butanol/hydrochloric acid
and of water (fraction 1 heptane; fraction 2 heptane/ethyl acetate
(50/50 by volume); fractions 3-4: ethyl acetate; fractions 5-6
ethyl acetate/n-butanol (95/5 by volume), fractions 7-8: ethyl
acetate/n-butanol (90/10 by volume), fractions 9-10: ethyl
acetate/n-butanol (80/20 by volume); fractions 11-12: ethyl
acetate/n-butanol (50/50 by volume), fractions 13-14: ethyl
acetate/n-butanol (20/80 by volume), fractions 15-16: n-butanol,
fractions 17-18: n-butanol/water (98/2 by volume), fractions 19-21:
n-butanol/water (95/5 by volume). 50 ml fractions are collected.
Fractions 19 to 21 are combined and concentrated to dryness under
reduced pressure. 108 mg of a fraction is thus obtained which is
chromatographed on a Sephadex.RTM. LH20 column marketed by
Pharmacia (height 100 cm, diameter 1 cm) produced with a
methanol-water mixture (50-50 by volume). The elution is carried
out with the same eluent mixture; 1 ml fractions are collected.
[0057] 1--Fractions 88 to 91 are combined and concentrated to
dryness. 14.4 mg of a product are obtained, which product gives,
upon crystallization from 0.5 ml of ethanol, 2 mg of
2,5-di-(tetrahydroxybutyl- )pyrazine in the form of white crystals
whose characteristics are the following:
[0058] optical rotation [.alpha.].sup.20 (Na
589)=-137.degree..+-.2.0 (dimethyl sulphoxide; c=0.5),
[0059] infrared spectrum produced on a Nicolet 60SX-R apparatus in
solution in KBr, main characteristic absorption bands: 3281
cm.sup.-1 (.nu. bound OH groups including H.sub.2O),
2972+2940+2901+2880 cm.sup.-1 (.nu. CH of the CH.sub.2 and CHOH
groups), 2733 cm.sup.-1 (v bound OH groups), 1635 cm.sup.-1
(deformations of the OH groups, including H.sub.2O), 1491 cm.sup.-1
(.nu. C.dbd.C and C.dbd.N of the pyrazine nucleus), 1449 cm.sup.-1
(.nu. C.dbd.C and C.dbd.N of the pyrazine nucleus+deformations of
the OH groups), 1413 cm.sup.-1 (deformations of the OH groups),
1343 cm.sup.-1 (.nu. C.dbd.C and C.dbd.N of the pyrazine nucleus),
1309+1290+1251+1215+1181+1161+1123 cm.sup.-1 (deformations of the
CH groups), 1092 cm.sup.-1 (.nu. CO of the secondary alcohols),
1048+1035 cm.sup.-1 (.nu. CO of the primary alcohols+pyrazine
nucleus), 947+899+854 cm.sup.-1 (secondary alcohols), 877 cm.sup.-1
(.nu. CH of the pyrazine nucleus), 727 cm.sup.-1 (pyrazine
nucleus+deformations of the OH groups), 639 cm.sup.-1 (pyrazine
nucleus), 607+531 cm.sup.-1 broad (deformations of the OH groups),
451+411 cm.sup.-1 (pyrazine nucleus),
[0060] mass spectrum performed on a FINNIGAN TSQ46 apparatus, mass
ionization mode M/z=321 (MH)+,
[0061] .sup.1H NMR spectrum (600 MHz, DMSO, chemical shift in ppm):
3.40 and 3.61 (2 mts, 2H each: CH.sub.2 at position 4', 4"); 3.58
(2 mts, 2H each: CH at position 2', 2", 3', 3"); 4.36 (broad t, 2H:
OH at position 4', 4"); 4.40 (d, J=7.2 Hz, 2H: OH at position 2',
2"); 4.63 (d, J=4.8 Hz, 2H: OH at position 3', 3"); 4.95 (dd J=6.0
and 0.6 Hz, 2H: CH at position 1', 1"); 5.30 (d, J=6.0 Hz, 2H: OH
at position 1', 1"); 8.61 (s, 2H, CH at position 3 and 6),
[0062] ultraviolet spectrum: .lambda. max=275 nm (.epsilon.=8260);
206 nm (.epsilon.=10220) (c=19 mg/ml; water), .lambda. max=276 nm
(.epsilon.=7960); 206 nm (.epsilon.=9920) (c=19 mg/ml; HCl 0.1N),
.lambda. max=275 nm (.epsilon.=7690); (c=19 mg/ml; KOH 0.1N),
[0063] HPLC on Y.M.C. 180DS-AQ column of 150.times.4.6 mm (batch
AIT/DE940377) marketed by AIT, isocratic elution H.sub.2O+0.1%
formic acid with a flow rate of 1 ml/min, UV detection at 270 nm,
retention time: 2 min 32 s.
[0064] 2--Fractions 92 and 93 are combined and concentrated to
dryness. 9 mg of a fraction are obtained, which fraction gives,
upon crystallization from 0.5 ml of ethanol, 1.2 mg of sodium
oxamate having the same characteristics as those described by
TOUSSAINT, Ann., 120, 237 (1861).
[0065] 3--Fractions 94 to 97 are combined and concentrated to
dryness. 25.9 mg of a fraction are obtained, which fraction gives,
upon crystallization from 0.5 ml of ethanol, 6.2 mg of a mixture of
2-(tetrahydroxybutyl)-5-(2',3',4'-trihydroxybutyl)pyrazine and
2-(tetrahydroxybutyl)-6-(2',3',4'-trihydroxybutyl)pyrazine which is
further separated by HPLC on a Y.M.C. 180DS-AQ column of
150.times.4.6 mm (batch AIT/DE940377); isocratic elution
H.sub.2O+0.1% formic acid with a flow rate of 1 ml/min, UV
detection at 270 nm.
[0066] 5.2 mg of
2-(tetrahydroxybutyl)-5-(2',3',4'-trihydroxybutyl)pyrazin- e are
thus obtained, which has the following characteristics:
[0067] optical rotation [.alpha.].sup.20 (Na
589)=-116.3.degree..+-.1.7 (dimethyl sulphoxide; c=0.5),
[0068] infrared spectrum performed on a Nicolet 60SX-R apparatus in
solution in KBr, main characteristic absorption bands at 3398
cm.sup.-1 (.nu. bound OH groups including H.sub.2O), 2951+2922+2891
cm.sup.-1 (.nu. CH of the CHOH groups), 2761 cm.sup.-1 (.nu. bound
OH groups), 1636 cm.sup.-1 (deformations of the OH groups,
including H.sub.2O), 1483+1463 cm.sup.-1 (.nu. C.dbd.C and C.dbd.N
of the pyrazine nucleus), 1411 cm.sup.-1 (deformation of the OH
groups), 1367+1328+1270+1227+1191 cm.sup.-1 (deformation of the CH
groups), 1071 cm.sup.-1 (.nu. CO of the secondary alcohols), 1041
cm.sup.-1 (.nu. CO of the primary alcohols+pyrazine nucleus),
943+897 cm.sup.-1 (secondary alcohols), 869 cm.sup.-1 (.nu. CH of
the pyrazine nucleus), 645 cm.sup.-1 (CH of the pyrazine nucleus),
607 cm.sup.-1 broad (deformations of the OH groups), 446+409
cm.sup.-1 (pyrazine nucleus),
[0069] mass spectrum performed on a FINNIGAN TSQ46 apparatus, mass
ionization mode M/z=305 (MH)+,
[0070] .sup.1H NMR spectrum (600 MHz, DMSO, chemical shift in ppm):
2.70 and 3.04 (2 dd, J=9.0 and 15.0 Hz and J=3.0 and 15.0 Hz, 1H
each: CH.sub.2 at position 1"); between 3.30 and 3.45 (mts, CH at
position 3", 4', 4"); between 3.53 and 3.65 (mts, 4H: CH at
position 2', 3', 4", 4"), 3.73 (mt, 1H: CH at position 2"); 4.37
(broad t, 1H OH at position 4'); 4.42 (mt, 2H: OH at position 2'
and 4"); 4.60 (d, J=6 Hz, 1H: OH at position 2"); 4.63 (d, J=6 Hz,
1H: OH at 3'); 4.67 (d, J=6 Hz, 1H: OH at position 3"); 4.92 (dd
J=6.0 and 0.6 Hz, 1H: CH at position 1'); 5.30 (d, J=6.0 Hz, 1H: OH
at position 1'); 8.39 (s, 1H: CH at position 6); 8.61 (s, 1H: CH at
position 3),
[0071] ultraviolet spectrum: .lambda. max=276 nm (.epsilon.=7756);
206 nm (.epsilon.=8738) (c=19 mg/ml; water), .lambda. max=277 nm
(.epsilon.=7218); 208 nm (.epsilon.=7171) (c=19 mg/ml; HCl 0.1 N),
.lambda. max=276 nm (.epsilon.=7467) (c=19 mg/ml; KOH 0.1N),
[0072] HPLC on Y.M.C. 180DS-AQ column of 150.times.4.6 mm (batch
AIT/DE940377) marketed by AIT, isocratic elution H.sub.2O+0.1%
formic acid with a flow rate of 1 ml/min, UV detection at 270 nm,
retention time: 3 min 75 s and 1 mg of
2-(tetrahydroxybutyl)-6-(2',3',4'-trihydroxy- butyl)pyrazine which
has the following characteristics:
[0073] .sup.1H NMR spectrum (600 MHz, DMSO, chemical shift in ppm):
2.70 and 3.04 (2 dd, J=9.0 and 15.0 Hz and J=3.0 and 15.0 Hz, 1H
each: CH.sub.2 at position 1"); between 3.30 and 3.45 (mts, CH at
position 3", 4', 4"); between 3.53 and 3.65 (mts, 4H: CH at
position 2', 3', 4', 4"), 3.73 (mt, 1H: CH at position 2"); 4.37
(broad t, 1H: OH at position 4'); 4.42 (mt, 2H: OH at position 2'
and 4"); 4.60 (d, J=6 Hz, 1H: OH at position 2"); 4.63 (d, J=6 Hz,
1H: OH at 3'); 4.67 (d, J=6 Hz, 1H: OH at position 3"); 4.92 (dd
J=6.0 and 0.6 Hz, 1H: CH at position 1'); 5.30 (d, J=6.0 Hz: 1H, OH
at position 1'); 8.31 (s, 1H: CH at position 5); 8.53 (s, 1H: CH at
position 3),
[0074] HPLC on Y.M.C. 180DS-AQ column of 150.times.4.6 mm (batch
AIT/DE940377) marketed by AIT, isocratic elution H.sub.2O+0.1%
formic acid with a flow rate of 1 ml/min, UV detection at 270 nm,
retention time: 3 min 61 s.
[0075] The hypoglycaemic activity of the mixtures I and II, of
sodium oxamate and of the compounds of formula (I) have been
determined in mice made diabetic with streptozocin and in normal
mice with post-prandial hyperglycaemia according to the following
procedures:
[0076] I--Mice Made Diabetic with Streptozotocin
[0077] Swiss albino mice weighing between 22 and 25 g are made
diabetic with streptozotocin administered by intraperitoneal
injection at a dose of 210 or 265 mg/Kg of mouse, diluted in a
citrate buffer at a concentration such that each mouse receives 0.2
ml of the solution on day 1 (D1). 3 to 4 days later, a check is
made on 2 to 3 mice to see if the diabetes is established
(glycaemia greater than 7.2 mmol/liter (130 mg per 100 ml). The
glycaemia is then measured after starving for 4 hours. If the mice
have become diabetic, they are divided into batches of 5 to 7 mice.
Each of the batches receives, from D1 and daily, a selected dose of
product. The administration is made once a day and at a fixed time,
by gastric intubation and in a volume of 0.4 ml of distilled water
as vehicle. Two batches of controls are made up:
[0078] one batch of untreated diabetic mice
[0079] one batch or normal mice
[0080] These two batches of controls receive 0.4 ml of vehicle by
gastric intubation and simultaneously with the treated mice.
[0081] The treatment lasts for 4 days. On the 5th day (D5), there
is no administration of product. After fasting for 4 hours, the
final glycaemias are measured.
[0082] II--Normal Mice with Post-prandial Hyperglycaemia
[0083] Swiss albino mice weighing between 22 and 25 g are made to
have post-prandial hyperglycaemia by the following procedure:
[0084] fasting 2 hours
[0085] food in excess for 1 hour
[0086] fasting 2 hours
[0087] The glycaemia is measured at the end of the last two hours
of fasting, which constitutes the glycaemia at time T0. The mice
are then divided into homogeneous batches according to the measured
glycaemias. The products to be evaluated are administered without
delay by gastric intubation in 0.4 ml of distilled water. The
control batch receives the excipient (0.4 ml of distilled water).
After one hour, the final glycaemias are measured which constitute
the glycaemia at time T60.
1 Results obtained on the glycaemia of mice made diabetic with
streptozotocin GLY- GLY- NUMBER CAEMIA CAEMIA % PRODUCTS OF AT D1
AT D5 inhibi- mg/mouse/day MICE mg/100 ml mg/100 ml tion Sodium
oxamate (0.5 mg) 5 153.40 .+-. 89.90 .+-. -41.46 11.53 13.94
Mixture II (0.5 mg) 5 270.50 .+-. 117.50 .+-. -56.56 58.72 17.35
2-(tetrahydroxybutyl)-5- 5 304.25 .+-. 164.50 .+-. -45.93
(2',3',4'-trihydroxybutyl)- 99.35 95.13 pyrazine (0.5 mg)
2-(tetrahydroxybutyl)-5- 6 320.83 .+-. 174.00 .+-. -45.77
(2',3',4'-trihydroxybutyl)- 130.30 97.74 pyrazine and 2-
(tetrahydroxy- butyl)-6-(2',3',4'- trihydroxybutyl)pyrazine (50/50)
(0.25 mg) Controls 5 221.66 .+-. 210.33 .+-. -5.11 50.17 20.07
[0088]
2 Results obtained on the glycaemia of normal mice with
post-prandial hyperglycaemia GLY- GLY- NUMBER CAEMIA CAEMIA %
PRODUCTS OF AT T0 AT T60 inhibi- mg/mouse/day MICE mg/100 ml mg/100
ml tion Mixture I (5 mg) 5 129.50 .+-. 87.60 .+-. -32.35 29.06
14.16 Sodium oxamate (0.5 mg) 15 136.46 .+-. 102.93 .+-. -24.57
23.69 20.95 Mixture II (0.5 mg) 15 133.93 .+-. 105.60 .+-. -21.15
17.50 16.91 2,5-di(tetrahydroxybutyl) 10 136.25 .+-. 102.41 .+-.
-24.83 pyrazine (0.5 mg) 16.92 14.12 2-(tetrahydroxybutyl)-5- 10
128.75 .+-. 90.50 .+-. -29.70 (2',3',4'-trihydroxy- 21.23 19.55
butyl)pyrazine (0.5 mg) 2-(tetrahydroxybutyl-5- 5 126.60 .+-.
100.60 .+-. -20.53 (2',3',4'-trihydroxy- 17.03 16.88 butyl)pyrazine
and 2- (tetrahydroxybutyl)-6- (2',3',4'-trihydroxybutyl) pyrazine
(50/50) (0.25 mg) Controls 10 137.83 .+-. 130.50 .+-. -5.32 13.99
20.23
[0089] The mixtures according to the invention, the sodium oxamate
and the compounds of formula (I) have a low toxicity. Their
LD.sub.50 is greater than 2000 mg/kg orally in mice.
[0090] The mixtures according to the invention, the sodium oxamate
and the compounds of formula (I) reduce the glycaemia in a diabetic
subject and are therefore useful in the treatment of diabetes and
the complications of diabetes.
[0091] When these products are used in monotherapy in the treatment
of diabetes, there is no risk of hypoglycaemia. They are true
antidiabetic agents. It appears that this effect results from a
peripheral increase in glucose. The products do not significantly
stimulate the secretion of insulin but the presence of small
quantities of insulin is necessary for their action.
[0092] In sand rats (Psammomys obesus) which are spontaneously
diabetic in captivity, the products decrease hyperglycaemia,
prevent or decrease cataract and restore a degree of fertility.
[0093] In human therapy, these products are therefore useful in the
prevention and treatment of diabetes and especially type II
diabetes (NID diabetes) not exhibiting acetonuria, obese diabetes,
diabetes at the age of about fifty, metaplethoric diabetes,
diabetes affecting the elderly and mild diabetes. They can be used
as a supplement to insulin therapy (because of their
insulin-potentiating activity) in insulin-dependent diabetes where
they make it possible to gradually reduce the dose of insulin,
unstable diabetes, insulin-resistant diabetes, and as a supplement
to hypoglycaemic sulphamides when these do not provide a sufficient
decrease in glycaemia. These products can also be used in the
complications of diabetes such as hyperlipaemias, lipid metabolism
disorders, dyslipaemias and obesity. They are also useful in the
prevention and treatment of the lesions of atherosclerosis and
their complications (coronopathies, myocardial infarction,
cardiomyopathies, progression of these three complications into
left ventricular insufficiency, various arteriopathies, arteritis
of the lower limbs with claudication and progression into ulcers
and gangrene, cerebral vascular insufficiency and its complications
and sexual impotence of vascular origin), diabetic retinopathy and
all its manifestations (increase in capillary permeability,
dilation and capillary thrombosis, microaneurysms, arteriovenous
shunt, venous dilation, punctiform and macular haemorrhages,
exudates, macular oedemas, manifestations of proliferative
retinopathy: neovessels, proliferative retinitis scars,
haemorrhages of the vitreous body, retinal detachment), diabetic
cataract, diabetic neuropathy in its various forms (peripheral
polyneuropathies and its manifestations such as paresthesias,
hyperesthesias and pain, mononeuropathies, radiculopathies,
autonomous neuropathies, diabetic amyotrophies), the manifestations
of diabetic foot (ulcers of the lower extremities and of the foot),
diabetic nephropathy in its two diffuse and nodular forms,
atheromatoses (rise in HDL lipoproteins promoting the elimination
of cholesterol from the atheroma plaques, decrease in the LDL
lipoproteins, decrease in the LDL/HDL ratio, inhibition of
oxidation of the LDLs, decrease in platelet adhesiveness),
hyperlipaemias and dyslipaemias (hypercholesterolaemias,
hypertriglyceridaemias, normalization of the fatty acid level,
normalization of the uricaemia, normalization of the A and B
apoproteins), cataracts, high blood pressure and its
consequences.
[0094] The medicaments according to the invention consist of a
mixture according to the invention, sodium oxamate, a compound of
formula (I) or a combination of these products, in the pure state
or in the form of a composition in which it is combined with any
other pharmaceutically compatible product which may be inert or
physiologically active. The medicaments according to the invention
can be used orally, parenterally, rectally or topically.
[0095] As solid compositions for oral administration, there may be
used tablets, pills, powders (gelatine capsules, cachets) or
granules. In these compositions, the active ingredient according to
the invention is mixed with one or more inert diluents, such as
starch, cellulose, sucrose, lactose or silica, under an argon
stream. These compositions may also comprise substances other than
the diluents, for example one or more lubricants such as magnesium
stearate or talc, a colorant, a coating (sugar-coated tablets) or a
glaze.
[0096] As liquid compositions for oral administration, there may be
used pharmaceutically acceptable solutions, suspensions, emulsions,
syrups and elixirs containing inert diluents such as water,
ethanol, glycerol, vegetable oils or paraffin oil. These
compositions may comprise substances other than the diluents, for
example wetting, sweetening, thickening, flavouring or stabilizing
products.
[0097] The sterile compositions for parenteral administration may
preferably be solutions in aqueous or nonaqueous form, suspensions
or emulsions. As solvent or vehicle, there may be used water,
propylene glycol, polyethylene glycol, vegetable oils, in
particular olive oil, injectable organic esters, for example ethyl
oleate or other suitable organic solvents. These compositions may
also contain adjuvants, in particular wetting, isotonizing,
emulsifying, dispersing and stabilizing agents. Sterilization can
be performed in several ways, for example by aseptizing filtration,
by incorporating sterilizing agents into the composition, by
irradiation or by heating. They can also be prepared in the form of
sterile solid compositions which can be dissolved at the time of
use in sterile water or any other injectable sterile medium.
[0098] The compositions for rectal administration are suppositories
or rectal capsules which contain, in addition to the active
product, excipients such as cocoa butter, semisynthetic glycerides
or polyethylene glycols.
[0099] The compositions for topical administration may be for
example creams, lotions, collyria, collutoria, nasal drops or
aerosols.
[0100] The doses depend on the desired effect, the duration of
treatment and the route of administration used; they are generally
between 150 mg and 600 mg per day via the oral route for an adult
with unit doses ranging from 50 mg to 200 mg of active
substance.
[0101] In general, the doctor will determine the appropriate dosage
according to the age, weight and all the other factors specific to
the subject to be treated.
[0102] The following examples illustrate compositions according to
the invention:
EXAMPLE A
[0103] Gelatine capsules in doses of 50 mg of active product having
the following composition are prepared according to the usual
technique:
3 Active product 50 mg Cellulose 18 mg Lactose 55 mg Colloidal
silica 1 mg Sodium carboxymethyl starch 10 mg Talc 10 mg Magnesium
stearate 1 mg
EXAMPLE B
[0104] Tablets in doses of 50 mg of active product having the
following composition are prepared according to the usual
technique:
4 Active product 50 mg Lactose 104 mg Cellulose 40 mg Polyvidone 10
mg Sodium carboxymethyl starch 22 mg Talc 10 mg Magnesium stearate
2 mg Colloidal silica 2 mg Hydroxymethylcellulose, glycerine,
titanium 245 mg oxide (72-3.5-24.5) qs 1 finished film-coated
tablet containing
EXAMPLE C
[0105] An injectable solution containing 50 mg of active product
having the following composition is prepared:
5 Active product 50 mg Benzoic acid 80 mg Benzyl alcohol 0.06 ml
Sodium benzoate 80 mg Ethanol at 95% 0.4 ml Sodium hydroxide 24 mg
Propylene glycol 1.6 ml Water qs 4 ml
* * * * *