U.S. patent application number 14/889512 was filed with the patent office on 2016-04-07 for oral composition comprisng (+)-catechin.
This patent application is currently assigned to VALORE. The applicant listed for this patent is VALORE. Invention is credited to Julien Estager, Bronislav Henric May, Paul Niebes, Said Rachidi, Bruno Schoentjes.
Application Number | 20160095923 14/889512 |
Document ID | / |
Family ID | 48951281 |
Filed Date | 2016-04-07 |
United States Patent
Application |
20160095923 |
Kind Code |
A1 |
Niebes; Paul ; et
al. |
April 7, 2016 |
ORAL COMPOSITION COMPRISNG (+)-CATECHIN
Abstract
The invention relates to a gastroenteric composition comprising
a compound of monomeric (+)-catechin and at least one basic amino
acid or at least one derivative of a basic amino acid, said
compound being in the form of a complex having a molar equivalence
ratio of said monomeric (+)-catechin to said at least one basic
amino acid or its derivative of between 1:1 and 1:2.5, preferably
between 1:1 and 1:2.
Inventors: |
Niebes; Paul; (Grez-Doiceau,
BE) ; May; Bronislav Henric; (Overijse, BE) ;
Rachidi; Said; (Hyon, BE) ; Estager; Julien;
(Kontich, BE) ; Schoentjes; Bruno; (Sevres,
FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
VALORE |
Seneffe |
|
BE |
|
|
Assignee: |
VALORE
Seneffe
BE
|
Family ID: |
48951281 |
Appl. No.: |
14/889512 |
Filed: |
May 13, 2014 |
PCT Filed: |
May 13, 2014 |
PCT NO: |
PCT/EP2014/059781 |
371 Date: |
November 6, 2015 |
Current U.S.
Class: |
514/456 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 9/0053 20130101; A61K 9/08 20130101; A61K 31/353 20130101;
A61K 31/194 20130101; A61P 29/00 20180101; A61P 37/08 20180101;
A61P 37/06 20180101; A61P 17/00 20180101; A61P 1/16 20180101; A61K
31/375 20130101; A61K 9/0095 20130101; A61P 1/04 20180101; A61P
35/02 20180101; A61K 31/19 20130101; A61P 19/02 20180101; A61K
31/198 20130101; A61K 31/375 20130101; A61K 2300/00 20130101; A61K
31/198 20130101; A61K 2300/00 20130101; A61K 31/353 20130101; A61K
2300/00 20130101; A61K 31/194 20130101; A61K 2300/00 20130101; A61K
31/19 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 47/18 20060101
A61K047/18; A61K 31/353 20060101 A61K031/353; A61K 9/00 20060101
A61K009/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 17, 2013 |
BE |
2013/0350 |
Claims
1. A gastroenteric composition for oral administration, comprising
a compound of monomeric (+)-catechin and at least one basic amino
acid, said composition being characterized in that said compound is
in the form of a complex and has a molar equivalence ratio of said
monomeric (+)-catechin to said at least one basic amino acid or to
said at least one basic amino acid derivative of between 1:1 and
1:2.5.
2. The gastroenteric composition for oral administration as claimed
in claim 1, wherein said molar equivalence ratio is greater than or
equal to 1:1, in particular greater than 1:1.
3. The gastroenteric composition for oral administration as claimed
in claim 1, wherein said ratio is less than or equal to 1:2.5, in
particular less than 1:2.5.
4. The gastroenteric composition for oral administration as claimed
in claim 1, wherein said molar equivalence ratio is less than or
equal to 1:2, more particularly less than 1:2.
5. The gastroenteric composition for oral administration as claimed
claim 1, wherein said at least one basic amino acid is lysine or
arginine of natural or synthetic origin, or a mixture of the
two.
6. The gastroenteric composition for oral administration as claimed
in claim 1, wherein said at least one basic amino acid is
lysine.
7. The gastroenteric composition for oral administration as claimed
in claim 1, characterized in that said complex is in the form of a
salt of said complex, said salt comprising said complex, said
complex comprising at least one proton derived from at least one
acid and at least one anion derived from said at least one acid,
said salt exhibiting said proton in equimolar amount relative to
the amount of basic amino acid or basic amino acid derivative.
8. The gastroenteric composition for oral administration as claimed
in claim 7, wherein said acid is chosen from ascorbic acid, acetic
acid, citric acid and hydrochloric acid.
9. The gastroenteric composition for oral administration as claimed
in claim 7, wherein said acid is ascorbic acid.
10. The gastroenteric composition for oral administration as
claimed in claim 1, characterized in that it also comprises one or
more biocompatible excipients.
11. The gastroenteric composition for oral administration as
claimed in claim 1, wherein the content of complex of (+) catechin
with said at least one basic amino acid or said at least derivative
of a basic amino acid is between 15% and 95% by weight relative to
the total weight of said composition, preferably between 60% and
90%, advantageously from 65% to 85%.
12. The gastroenteric composition for oral administration as
claimed in claim 1, in solid form, in particular in the form of a
powder, a tablet or a lozenge.
13. The gastroenteric composition for oral administration as
claimed in claim 9, comprising monomeric (+)-catechin and said at
least one basic amino acid or said at least one basic amino acid
derivative, and optionally at least one acid, as precursors of said
complex or of said salt of the complex as a combined preparation
for simultaneous use, said complex forming post-oral
administration, said monomeric (+)-catechin and said at least one
basic amino acid or said at least one basic amino acid derivative
being present in a molar equivalent ratio of between 1:1 and 1:2.5,
preferably greater than or equal to 1:1, in particular greater than
1:1, preferably less than or equal to 1:2.5, in particular less
than 1:2.5, more particularly less than or equal to 1:2, more
particularly less than 1:2.
14. The gastroenteric composition for oral administration as
claimed in claim 9, comprising monomeric (+)-catechin and said at
least one basic amino acid or said at least one basic amino acid
derivative, and optionally at least one acid, as precursors of said
complex or of said salt of the complex, as a combined preparation
for simultaneous use in solution in an aqueous phase, said complex
forming pre-oral administration, said monomeric (+)-catechin and
said at least one basic amino acid or said at least one basic amino
acid derivative being present in a molar equivalent ratio of
between 1:1 and 1:2.5, preferably greater than or equal to 1:1, in
particular greater than 1:1, preferably less than or equal to
1:2.5, in particular less than 1:2.5, more particularly less than
or equal to 1:2, more particularly less than 1:2.
15. The gastroenteric composition for oral administration as
claimed in claim 1, in liquid form.
16. The gastroenteric composition for oral administration as
claimed in claim 10, characterized, in 0.01 molar solution at
25.degree. C., by a pH greater than or equal to 3, preferably of
between 4 and 11, advantageously between 4.5 and 9.
17. The gastroenteric composition for oral administration as
claimed in claim 1, for the preventive and/or curative treatment of
a disease selected from the group consisting of cancer,
hepatocellular cancer, leukemia, myeloma, lymphoma, liver cancer,
prostate cancer, breast cancer, uterine cancer, testicular cancer,
bladder cancer, kidney cancer, lung cancer, bronchial cancer, bone
cancer, mouth cancer, esophageal cancer, stomach cancer, pancreatic
cancer and colorectal cancer.
18-21. (canceled)
22. The gastroenteric composition for oral administration as
claimed in claim 1, for the preventive and/or curative treatment of
a disease selected from the group consisting of an
anti-inflammatory disease, arthrosis, a collagen deficiency, a
connective tissue genetic disease, osteogenesis imperfecta,
Marfan's disease, Ehlers-Danlos disease, Cutis Laxa disease,
systemic scleroderma, and a connective tissue disease where
collagen and/or elastin.
23-25. (canceled)
26. The gastroenteric composition for oral administration as
claimed in claim 1, for the preventive and/or curative treatment of
peptic ulcers and also gastrointestinal allergies.
27. The gastroenteric composition for oral administration as
claimed in claim 1, for the preventive and/or curative treatment of
cell aging.
Description
[0001] The present invention relates to a gastroenteric composition
for oral administration, comprising a compound of monomeric
(+)-catechin and at least one basic amino acid, intended for
mammals, and in particular for human beings.
[0002] Polyphenols constitute a family of organic molecules widely
present in the plant kingdom. They are characterized, as the name
indicates, by the presence of several phenolic groups combined in
structures generally of high molecular weight. These compounds are
the products of secondary metabolism in plants.
[0003] In particular, (+)-catechin in its monomeric form is
directly obtained from Uncaria gambir extract.
[0004] Such a composition is already known, for example, from
document U.S. Pat. No. 4,285,964 in the context of the treatment of
chronic diseases of degenerative type of connected tissues. Among
these degenerative diseases, including the most well known and most
widespread in human beings, are for example arthrosis,
chondromalacia and parodontosis.
[0005] According to document U.S. Pat. No. 4,285,964, the
composition comprising a compound of monomeric (+)-catechin and at
least one basic amino acid is a medicament in which the active
substance is the monomeric (+)-catechin.
[0006] This document discloses moreover that, in order to be able
to treat these diseases, it is important to be able to administer
this compound on the site where the active substance must act, i.e.
in the joints affected by the disease.
[0007] The principal objective of U.S. Pat. No. 4,285,964 is
therefore to form an injectable composition which can be directly
injected into the body, for example into the joints.
[0008] This objective is achieved by the formation of a double salt
of monomeric (+)-catechin which, in addition, is not exactly
characterized since it is an equimolar mixture of (+)-catechin, of
lysine and of hydrochloric acid, in order to obtain solubilization
of the (+)-catechin in an aqueous medium, at physiological pH, for
the purpose of having an injectable form, the addition of the acid
making it possible to modulate the solubility of this double salt
and to adjust the pH of the latter to the physiological value
(pH=7.2-7.4).
[0009] Indeed, this document demonstrates that the solubility of
the (+)-catechin in an aqueous solution (such as water at
physiological pH) is high, up to 400 WI, when said catechin forms a
double salt in the presence of a basic amino acid such as
L-arginine or L-lysine and of an acid, for instance HCl.
[0010] This improved solubility in an aqueous medium is more
advantageous for an administration of parenteral type than the use
of the pure monomeric (+)-catechin which has a solubility in water
limited to 1 WI.
[0011] Document U.S. Pat. No. 4,285,964 extrapolates in passing the
abovementioned injectable composition in order to obtain an oral
formulation by drying the injectable composition.
[0012] However, this document does not give any indication as to
the stability and the capacities of assimilation by the organism in
vivo of the oral composition described above.
[0013] Consequently, in the light of the current prior art, there
is a need for a gastroenteric composition for oral administration
based on monomeric (+)-catechin which is assimilable after oral
administration and which remains stable once ingested.
[0014] For this purpose, the present invention provides a
composition characterized in that said compound is in the form of a
complex and has a molar equivalence ratio of said monomeric
(+)-catechin to said at least one basic amino acid or to said at
least one basic amino acid derivative or precursor of between 1:1
and 1:2.5 (hereinafter referred to as [C:AA/1:1-1:2.5] complex, C
for monomeric (+)-catechin and AA for basic amino acid or basic
amino acid derivative or precursor).
[0015] Indeed, in the context of the present invention, it has been
noted, entirely surprisingly, that the complex of monomeric
(+)-catechin with at least one basic amino acid or at least one
basic amino acid derivative or precursor in a specific molar
equivalence ratio of between 1:1 and 1:2.5 is stable once ingested
and has a high bioavailability after oral administration. This high
bioavailability after oral administration is made possible by the
in vivo stability of the complex according to the invention.
[0016] It is in fact thought that the presence of said at least one
basic amino acid linked to the monomeric (+)-catechin allows the
complex to easily cross the intestinal wall; this therefore results
in a higher proportion of free monomeric (+)-catechin in the
blood.
[0017] On the basis of the results presented in the context of the
present invention, it is observed that the high bioavailability,
when administered orally, of one gram of monomeric (+)-catechin in
the form of a complex reaches a value of at least 900 ng h/ml (54
000 ng min/ml) in humans. In light of the results of the present
invention, the term "high bioavailability in humans" is therefore
intended to mean a bioavailability at least equal to 900 ng h/ml
(54 000 ng min/ml) for an oral administration of one gram.
[0018] The results obtained by the inventors of the present patent
application show that the bioavailability measured in humans for
one gram of (+)-catechin ingested in the form of a complex of
monomeric (+)-catechin and of at least one basic amino acid in a
molar equivalence ratio of between 1:1 and 1:2.5 is at least 900 ng
h/ml (54 000 ng min/ml).
[0019] By way of illustration, it is observed in the context of
this invention that the bioavailability, when administered orally
in human beings, of the (+)-catechin ingested in the form of a
complex of monomeric (+)-catechin is much higher [1263.+-.88 ng
h/ml (75 780 ng min/ml) for 0.920 g of monomeric (+)-catechin
ingested in the form of 1.5 g of complex] than that of the
monomeric (+)-catechin ingested alone[832.+-.150 ng h/ml (49 920 ng
min/ml) for an equivalent amount of 0.920 g of an oral intake of
pure monomeric (+)-catechin], i.e. +52% increase in bioavailability
by virtue of the complex.
[0020] This characteristic is all the more unexpected since it has
been demonstrated in the context of the present invention that the
pure monomeric (+)-catechin has a solubility in water which is 400
times lower than that measured when it is solubilized in the form
of a complex formed with at least one basic amino acid or at least
one derivative or precursor of a basic amino acid.
[0021] Indeed, the solubility of the lysine salts of the
(+)-catechin reaches, at 20.degree. C., 400 g per liter of water,
whereas the (+)-catechin itself exhibits, under the same
conditions, a solubility of 0.9 g per liter.
[0022] Given that the composition according to the invention has a
high bioavailability after oral administration, the latter then
allows an assimilation at reduced doses, which constitutes, on the
one hand, a perspective of gain in terms of treatment costs, since
a lower dose has to be absorbed, and, on the other hand, a decrease
in the side effects associated with this treatment, in particular
in the context of a long-term treatment, given that it is known
that the monomeric (+)-catechin, even though it is generally
well-tolerated in the body.
[0023] In addition, it has been observed that: [0024] the use of
(+)-catechin administered orally in the form of a [C:AA/1:1-1:2.5]
complex gives a maximum plasma concentration of free monomeric
(+)-catechin, c (max), that is more than doubled compared with that
obtained with the same dose of pure monomeric (+)-catechin ingested
under the same conditions: c (max) of 571 ng/ml for 1 g of
(+)-catechin ingested in the form of a complex, compared with 280
ng/ml for 1 g of monomeric (+)-catechin ingested as it is, i.e. an
increase of +103%; and that [0025] the more than doubled maximum
plasma concentrations are reached [T (max)] more than two times
faster when the (+)-catechin is ingested in the form of a
[C:AA/1:1-1:2.5] complex rather than in the form of monomeric
(+)-catechin alone: T (max) of 30 min compared with 80 min, i.e. an
increase of +166%.
[0026] Thus, in addition to the high bioavailability per os of the
complex according to the invention, it is also demonstrated in the
present application that, in the case of absorption after oral
administration, a proportion of free monomeric (+)-catechin in the
blood is maintained in a delayed manner, thereby making it possible
to envision an improved curative and/or preventive action of the
monomeric (+)-catechin when it is administered per os.
[0027] Preferably, said molar equivalence ratio is greater than or
equal to 1:1, in particular greater than 1:1.
[0028] Advantageously, said molar equivalence ratio is less than or
equal to 1:2.5.
[0029] In particular, said molar equivalence ratio is less than
1:2.5, more particularly less than or equal to 1:2.
[0030] Preferentially, said molar equivalence ratio is less than
1:2.
[0031] Alternatively, the molar equivalence ratio is between 1:1.5
and 1:2.5, preferably between 1:1.5 and 1:2.
[0032] Advantageously, said molar equivalence ratio is greater than
or equal to 1.00:1.00 and less than or equal to 1.00:2.50.
[0033] Very advantageously, said molar equivalence ratio is greater
than or equal to 1.00:1.00 and less than or equal to 1.00:1.50.
[0034] Preferably, said molar equivalence ratio is greater than or
equal to 1.00:1.50 and less than or equal to 1.00:2.00.
[0035] Preferably, said molar equivalence ratio is greater than or
equal to 1.00:1.50 and less than or equal to 1.00:2.50.
[0036] Preferably, said at least one basic amino acid is
lysine.
[0037] In one preferential embodiment of the present invention,
said complex is a complex comprising one molecule of lysine for one
molecule of monomeric (+)-catechin.
[0038] In one advantageous embodiment of the present invention,
said complex is a complex comprising one molecule of arginine for
one molecule of monomeric (+)-catechin.
[0039] In one particular embodiment of the present invention, said
complex is a complex comprising two molecules of lysine for one
molecule of monomeric (+)-catechin.
[0040] The complex of (+)-catechin with two lysines is by far, in
all the experiments, the best combination for improving the
bioavailability of the monomeric (+)-catechin when administered
orally.
[0041] In another particular embodiment of the present invention,
said complex is a complex comprising two molecules of arginine for
one molecule of monomeric (+)-catechin.
[0042] In another preferential form, said complex is a complex
comprising one molecule of lysine and one molecule of arginine for
one molecule of monomeric (+)-catechin.
[0043] Preferably, the composition according to the invention is
characterized in that said complex is in the form of a salt of said
complex, said salt comprising said complex, said complex comprising
at least one proton derived from at least one acid and at least one
anion derived from said at least one acid, said salt exhibiting
said proton in equimolar amount relative to the amount of basic
amino acid or of basic amino acid derivative or precursor.
[0044] In this way, the complex salt is defined by the following
formulae: [0045] [C:AA:H.sup.+:A.sup.-/1:x:x:x], in the case of a
monofunctional acid such as HCl; or [0046]
[C:xAA:xH.sup.+:x/yA.sup.y-], in the case of an acid, [0047]
H.sup.+ representing the proton of the acid, [0048] A.sup.-
representing the anion of the acid, [0049] x representing the
number of molar equivalents of AA, [0050] y representing the charge
carried by the anion.
[0051] Advantageously, said at least one acid is preferably chosen
from ascorbic acid, acetic acid, citric acid and hydrochloric acid.
Very advantageously, said acid is ascorbic acid.
[0052] The role of ascorbic acid is that of a vitamin supplement.
Furthermore, since this acid is also an antioxidant, it therefore
plays a synergistic antioxidant role with respect to that of the
(+)-catechin.
[0053] This addition of an acid also makes it possible to improve
the bioavailability of the (+)-catechin when administered orally.
This observation is explained by the more stable state of the
(+)-catechin in an acidic medium.
[0054] In one particular embodiment, said composition is
characterized in that it also comprises one or more biocompatible
excipients.
[0055] Preferably, the content of complex of (+)-catechin with said
basic amino acid or said at least one derivative or precursor of a
basic amino acid is between 15% and 95% by weight relative to the
total weight of said composition, preferably between 60% and 90%,
advantageously from 65% to 85%.
[0056] Preferably, the composition is in liquid form or in solid
form, preferably in water-soluble solid form, in particular in the
form of a powder, a tablet or a lozenge.
[0057] Advantageously, the composition in liquid form has, in a
0.01 molar solution at 25.degree. C., a pH greater than or equal to
3 (theoretical pH of a C:L salt in a ratio of 1:0.5), preferably of
between 4 and 11, advantageously between 4.5 and 9.
[0058] Optionally, the composition according to the invention is a
solid composition, with a pH greater than or equal to 3, preferably
of between 4 and 11, advantageously between 4.5 and 9, when it is
dissolved at 0.01 M at 25.degree. C., comprising monomeric
(+)-catechin and said at least one basic amino acid or said at
least one basic amino acid derivative, and optionally at least one
acid, as precursors of said complex or of said salt of the complex,
as a combined preparation for simultaneous oral use, said complex
forming post-oral administration.
[0059] The solid composition according to the invention comprises
the monomeric (+)-catechin and said at least one basic amino acid
or said at least one basic amino acid derivative or precursor in a
molar equivalence ratio of between 1:1 and 1:2.5.
[0060] Preferably, said molar equivalence ratio is greater than or
equal to 1:1, in particular greater than 1:1.
[0061] Advantageously, said molar equivalence ratio is less than or
equal to 1:2.5.
[0062] In particular, said molar equivalence ratio is less than
1:2.5, more particularly less than or equal to 1:2.
[0063] Preferentially, said molar equivalence ratio is less than
1:2.
[0064] Alternatively, the molar equivalence ratio is between 1:1.5
and 1:2.5, preferably between 1:1.5 and 1:2.
[0065] Advantageously, said molar equivalence ratio is greater than
or equal to 1.00:1.00 and less than or equal to 1.00:2.50.
[0066] Very advantageously, said molar equivalence ratio is greater
than or equal to 1.00:1.00 and less than or equal to 1.00:1.50.
[0067] Preferably, said molar equivalence ratio is greater than or
equal to 1.00:1.50 and less than or equal to 1.00:2.00.
[0068] Preferably, said molar equivalence ratio is greater than or
equal to 1.00:1.50 and less than or equal to 1.00:2.50.
[0069] The monomeric (+)-catechin and said at least one basic amino
acid, or said at least one basic amino acid derivative, are
precursors which participate, in an aqueous medium, in a
complexation reaction so as to form the complex according to the
invention.
[0070] In this case, the complex is formed in vivo in the
gastrointestinal tract of contact with the water of the saliva or
with the water contained in stomach bolus.
[0071] Alternatively, the composition according to the invention is
a solid composition comprising monomeric (+)-catechin and said at
least one basic amino acid or said at least one basic amino acid
derivative, and optionally at least one acid, as precursors of said
complex or of said salt of the complex, as a combined preparation
for simultaneous oral use in solution in an aqueous phase, said
complex forming pre-oral administration, said monomeric
(+)-catechin and said at least one basic amino acid or said at
least one basic amino acid derivative being present in a molar
equivalence ratio of between 1:1 and 1:2.5.
[0072] Preferably, said molar equivalence ratio is greater than or
equal to 1:1, in particular greater than 1:1.
[0073] Advantageously, said molar equivalence ratio is less than or
equal to 1:2.5.
[0074] In particular, said molar equivalence ratio is less than
1:2.5, more particularly less than or equal to 1:2.
[0075] Preferentially, said molar equivalence ratio is less than
1:2.
[0076] Alternatively, the molar equivalence ratio is between 1:1.5
and 1:2.5, preferably between 1:1.5 and 1:2.
[0077] Advantageously, said molar equivalence ratio is greater than
or equal to 1.00:1.00 and less than or equal to 1.00:2.50.
[0078] Very advantageously, said molar equivalence ratio is greater
than or equal to 1.00:1.00 and less than or equal to 1.00:1.50.
[0079] Preferably, said molar equivalence ratio is greater than or
equal to 1.00:1.50 and less than or equal to 1.00:2.00.
[0080] Preferably, said molar equivalence ratio is greater than or
equal to 1.00:1.50 and less than or equal to 1.00:2.50.
[0081] In this way, the complex is formed as soon as the
composition is placed in aqueous solution, i.e. before oral
administration.
[0082] In particular, the present invention relates to a curative
or prophylactic, therapeutic or food composition.
[0083] Other embodiments of the composition according to the
invention are indicated in the appended claims.
[0084] The present invention also relates to the composition
according to the invention, for use for the preventive and/or
curative treatment, by oral administration, of cancer, preferably
of hepatocellular cancer.
[0085] Advantageously, the present invention also relates to the
composition according to the invention, for use for the preventive
or curative treatment, by oral administration, of leukemias.
[0086] Very advantageously, the present invention also relates to
the composition according to the invention, for use, by oral
administration, for the preventive and/or curative treatment of
myelomas.
[0087] Optionally, the present invention also relates to the
composition according to the invention, for use, by oral
administration, for the preventive and/or curative treatment of
lymphomas.
[0088] Optionally, the present invention also relates to the
composition according to the invention, for use in the context of
the preventive and/or curative treatment of liver cancer, prostate
cancer, breast cancer, uterine cancer, testicular cancer, bladder
cancer, kidney cancer, lung cancer, bronchial cancer, bone cancer,
mouth cancer, esophageal cancer, stomach cancer, pancreatic cancer
and colorectal cancer.
[0089] Other embodiments of the composition according to the
invention for the preventive and/or curative treatment, by oral
administration, of cancer are indicated in the appended claims.
[0090] The present invention also relates to the composition
according to the invention, for use, by oral administration, in the
context of the preventive and/or curative treatment of inflammatory
and/or degenerative diseases, in particular arthrosis.
[0091] This use is made possible by virtue of the recognized
anti-inflammatory action of the monomeric (+)-catechin.
[0092] Other embodiments of the composition according to the
invention for the preventive and curative treatment, by oral
administration, of inflammatory diseases are indicated in the
appended claims.
[0093] The present invention also relates to the composition
according to the invention, for use, by oral administration, in the
context of the preventive and/or curative treatment of cell
aging.
[0094] This use is made possible by virtue of the recognized
antioxidant action of the monomeric (+)-catechin.
[0095] Other embodiments of the composition according to the
invention for the preventive and/or curative treatment, by oral
administration, of cell aging are indicated in the appended
claims.
[0096] The present invention also relates to the composition
according to the invention, for use, by oral administration, in the
context of the preventive and/or curative treatment of collagen
deficiencies.
[0097] This use is made possible by virtue of the action which
stimulates collagen production and the protection of these fibers
by the monomeric (+)-catechin and salts thereof, as demonstrated in
the present application.
[0098] In particular, the present composition according to the
invention targets the preventive and/or curative treatment of
genetic connective tissue diseases, such as osteogenesis
imperfecta, Marfan's disease, Ehlers-Danlos disease, Cutis Laxa
disease, systemic scleroderma, and other connective tissue diseases
where collagen and/or elastin appear to be abnormally soluble, as
is also the case in parodontosis-parodontitis, scleroderma, stretch
marks, or problems of tissue repair or of lesions of the connective
tissue, caused by irradiation, radiotherapy or prolonged exposure
to sunlight.
[0099] Other embodiments of the composition according to the
invention for the preventive and/or curative treatment, by oral
administration, of collagen deficiencies are indicated in the
appended claims.
[0100] The present invention also relates to the composition
according to the invention, for use, by oral administration, in the
context of the preventive and/or curative treatment of peptic ulcer
and gastrointestinal allergies.
[0101] This use is made possible by virtue of the protective action
of the composition according to the invention against gastric ulcer
and its effect on decreasing histamine levels at the level of the
gastric mucosa, as demonstrated in the present application.
[0102] Other embodiments of the composition according to the
invention for the preventive and/or curative treatment of peptic
ulcer and gastrointestinal allergies are indicated in the appended
claims.
[0103] Other characteristics and advantages of the invention will
emerge from the description given hereinafter, in a nonlimiting
manner and with reference to the examples (and in particular to the
comparative examples) described below.
[0104] FIG. 1 illustrates the NMR spectra of the complex of
monomeric (+)-catechin and of lysine for a molar equivalence ratio
of 1:2 (a) and a molar equivalence ratio of 1:1 (b).
[0105] FIG. 2 illustrates the NMR spectra of the complex of
monomeric (+)-catechin and of arginine for a molar equivalence
ratio of 1:2 (a) and a molar equivalence ratio of 1:1 (b).
[0106] FIG. 3 illustrates the evolution of the plasma
concentrations (cc) of free (+)-catechin measured in human beings
in ng/ml over time T (hours) after the assimilation of (+)-catechin
hydrochlorolysine (rich in monomeric (+)-catechin in an amount of
61% by weight, group A) and of one gram of pure monomeric
(+)-catechin (group B).
[0107] FIG. 4 illustrates the (powder) X-ray diffraction spectrum
of the complex of monomeric (+)-catechin and of lysine for a molar
equivalence ratio of 1:2.
[0108] FIG. 5 illustrates the evolution of the concentration of
free monomeric (+)-catechin in the plasma as a function of the
molar equivalence ratio between the monomeric (+)-catechin and the
lysine: 1:0, 1:1, 1:2, 1:2.5, 1:3, and 1:5, for a [C:Lys:HCl]
complex salt (see table 14).
[0109] In the description, examples 1 to 6 and also comparative
examples 1 to 5 relate to the results of bioavailability per os and
of anticancer activity obtained in mammals (rats and human beings)
for the composition based on the [C:AA/1:1-1:2.5] complex according
to the invention.
[0110] Comparative examples 6 to 10 describe the results of
bioavailability per os obtained in rats for the composition based
on the mixture of monomeric (+)-catechin and of at least one basic
amino acid or of at least one derivative of a basic amino acid in a
molar equivalence ratio of between 1:1 and 1:2.5, preferably
between 1:1 and 1:2.
[0111] In the context of the present invention, the equivalence
between the results of the tests obtained in rats and in human
beings has also been demonstrated. It is therefore proven that the
results obtained in rats are actually reproduced in human
beings.
[0112] In the examples which follow, with the exception of the
results of table 7b, the contents of (+)-catechins in the blood
correspond to the contents of monomeric free (+)-catechin.
Materials and Methods: Bioavailability Measurement
[0113] The bioavailability corresponds to the proportion of free
monomeric (+)-catechin which is found in the organism compared with
the amount initially administered.
[0114] In the context of the present invention, and in a
nonlimiting manner, the (+)-catechin can be absorbed in the form of
the [C:AA] complex or in the form of pure monomeric (+)-catechin,
i.e. which is not linked to at least one basic amino acid or at
least one basic amino acid derivative.
[0115] In the context of the present invention, it is first of all
necessary to distinguish, the (+)-catechin having been absorbed,
the presence in the blood of free monomeric (+)-catechin, i.e. in a
non-conjugated form, from the conjugated (+)-catechin, i.e. which
has been derivatized by the mammal's metabolism.
[0116] The derivatized monomeric (+)-catechin results from the
cycle of elimination of the free monomeric (+)-catechin in the
blood by the metabolism (for example, and in a nonlimiting manner,
by the enterohepatic cycle).
[0117] The elimination of active substances (or molecules) foreign
to the organism results from the joint action of several processes.
It comprises the metabolic capacity of various organs, first and
foremost the liver, and excretion in all its forms, in particular
renal (urine), but also hepatic (bile).
[0118] This elimination metabolism provides the derivation of the
monomeric (+)-catechin by means of a well-known bioconversion cycle
which involves two metabolic phases according to the enzymatic
conversion processes. The phase-I reactions and the phase-II
reactions are therefore distinguished: [0119] the phase-I reactions
comprise the oxidation reactions which are predominantly located in
the hepatic microsomes; the reduction reactions which are much less
frequent and have been less well examined; and the hydrolysis
reactions which constitute a banal metabolic pathway, which occurs
in the liver, in various tissues and even in the plasma; and then
[0120] the derivatives resulting from the phase-I reactions are
then conjugated. It is this conjugation which constitutes the
phase-II reactions, including glucuronidation which involves the
conjugation of these derivatives with glucuronic acid.
[0121] In this context, a given bioavailability corresponds to a
given proportion (or content) of free monomeric (+)-catechin in the
blood after administration per os of monomeric (+)-catechin.
[0122] Procedure:
[0123] The experimental results described below were obtained on
the basis of a study carried out in healthy human beings and in
healthy Wistar rats weighing approximately 250 grams.
[0124] With regard to the studies carried out on rats, the
individuals were housed in groups of a maximum of 4 individuals in
a cage with sawdust litter at an ambient temperature of between
20.degree. C. and 25.degree. C., with illumination for 12 h out of
24 h. An acclimation period was observed before beginning the
experiments.
[0125] During the experimental study, the rats had access to a
standard commercial feed and to water ad libitum. The rats were
given nothing to eat through the night preceding the administration
of the source of active product, it being understood that the
active product is monomeric (+)-catechin (or other polyphenols:
quercetin and EGCG).
[0126] The rats received an administration per os of a volume of 5
ml of a solution of active product.
[0127] With regard to taking the blood samples from the rats and
from the human beings, the blood was collected in tubes internally
coated with EDTA and was then centrifuged at 3000 rpm (revolutions
per minute) at ambient temperature for 45 min, and then for 15 min
at a temperature of 4.degree. C. The plasma was then collected and
stored at -70.degree. C. before analysis.
[0128] The analytical technique (LC-MS/MS for Liquid Chromatography
coupled to tandem Mass Spectrometry) for measuring the active
substance in the blood was based on the method described by
Mata-Bilbao et al., published in 2007 in the Journal of
Agricultural and Food Chemistry, volume 55, page 8857.
[0129] With regard to the methodological development, the reference
plasmas were collected internally, from rats and from cows not
treated with the active substance, and then stored at -20.degree.
C.
[0130] The free (+)-catechin was extracted from the plasma with a
solution of phosphoric acid, EDTA and ascorbic acid and was then
purified by SPE (solid phase extraction); in particular, it is an
Oasis.TM. HLB extraction which is analyzed by LC-MS/MS. The
quantification was carried out according to a standard calibration
procedure with (+/-)-catechin-2,3,4-.sup.13C3 as internal standard
(SI).
[0131] In this context, the content of free (+)-catechin summarizes
not only the content of monomeric free (+)-catechin isolated from
the plasma, but also its isomeric forms which result from the
isomerization of the monomeric free (+)-catechin pertaining to the
conditions for extraction of the free (+)-catechin from said
plasma.
[0132] The procedure is identical with regard to the extraction and
the quantification of free quercetin or ECGC in the blood.
[0133] With regard to the extraction and quantification of the
total (+)-catechin, the procedure is the same as that applied to
the free (+)-catechin, with the exception of the fact that an
additional step prior to the purification by SPE is carried out.
This additional step consists in treating the sample with a
digestive solution of arylsulfates and glucoronidase, in order to
extract the active substance from the plasma cells.
[0134] The total concentration (ct) of (+)-catechin (free or
derivatized) is calculated by measuring, for example using the
trapezium method, the area under the curve of evolution of the
plasma concentrations (cc) measured in ng/ml over time after oral
administration of the source of monomeric (+)-catechin.
[0135] Standards
[0136] A solution of internal standard of 1 mg/ml (methanol) of
(+)-catechin was prepared from stock (+)-catechin and was then
stored at -20.degree. C.
[0137] The catechin-C13 (reference: 719579, purity of 99.3% by
weight) produced by Sigma Aldrich was used as SI.
[0138] The SI solution for the quantification of the (free and
total) (+)-catechin was prepared by diluting 5 ml of the stock
solution in methanol in a final volume of 10 ml. This SI solution
was then stored at -20.degree. C.
[0139] Methods [0140] a) Extraction Protocol for Analysis of the
Free Monomeric (+)-Catechin in the Plasma [0141] transfer of 500
.mu.l of homogenized plasma into a 15 ml flask; [0142] addition of
50 .mu.l of SI (10 ppm); [0143] addition of 180 .mu.l of an
antioxidant solution (20 mg/ml of ascorbic acid and 1 mg/ml of
EDTA) and 10 .mu.l of ortho-phosphoric acid; [0144] vortex mixing
for 2 min; [0145] addition of 1.5 ml of water for dilution in order
to obtain an extraction mixture; [0146] solid-phase extraction
applied to the extraction mixture using Water Oasis.TM. HLB
according to the standard protocol well known to those skilled in
the art and adaptable to the present procedure; [0147] drying of
the eluent by evaporation at 45.degree. C. under an inert
atmosphere; [0148] solubilization of the dry extract in
acetonitrile, followed by centrifugation at 3000 rpm for 10 min;
and [0149] HPLC (for high performance liquid chromatography)
analysis.
[0150] b) Extraction Protocol for Analysis of the Total Monomeric
(+)-Catechin in the Plasma [0151] transfer of 500 .mu.l of
homogenized plasma into a 15 ml flask; [0152] addition of 50 .mu.l
of SI (10 ppm); [0153] addition of 180 .mu.l of an antioxidant
solution (20 mg/ml of ascorbic acid in 1 mg/ml of EDTA) and 10
.mu.l of ortho-phosphoric acid; [0154] vortex mixing for 2 min;
[0155] addition of 750 .mu.l of a 0.2 M acetate buffer solution (pH
of 4.8); [0156] addition of 5 .mu.l of a mixture of Helix pomatia
which follows a step of digestion of the solution for 2 hours at
55.degree. C.; [0157] centrifugation at 4000 rpm for 10 min in
order to obtain an extraction mixture; [0158] solid-phase
extraction applied to the extraction mixture using Water Oasis.TM.
HLB according to the standard protocol well known to those skilled
in the art and adaptable to the present procedure; [0159] drying of
the eluent by evaporation at 45.degree. C. under inert atmosphere;
[0160] solubilization of the dry extract in acetonitrile, followed
by centrifugation at 3000 rpm for 10 min; and [0161] HPLC (for high
performance liquid chromatography) analysis.
[0162] c) Extraction--Water Oasis.TM. HLB
[0163] Activation Solution: [0164] 1 ml of methanol; [0165] 1 ml of
water; and [0166] 1 ml of a solution of DMF (70% by volume)
containing 0.1% (by volume) of formic acid.
[0167] Washing Solution: [0168] E ml of water; [0169] 1 ml of a
methanol solution (30% by volume); [0170] 1 ml of ethyl
acetate.
[0171] Elution Solution [0172] 5 ml of a mixture of ethyl acetate
and methanol in a molar equivalence ratio of 1:2.
[0173] d) LC-MS/MS Analysis
[0174] Liquid Chromatography (LC) [0175] column: Alltima C18 5 u;
[0176] HPLC aqueous mobile phase A: formic acid (concentrated in an
amount of 0.1% by volume); [0177] HPLC mobile phase B: solution of
acetonitrile containing formic acid in an amount of 0.1% by volume;
[0178] flow rate: 0.6 ml/min; [0179] oven temperature: 30.degree.
C.; and [0180] injection volume: 50 .mu.l; [0181] elution
program:
TABLE-US-00001 [0181] Time A B (min) (% by volume) (% by volume)
0.00 95 5 0.50 95 5 4.00 100 0 4.50 100 0 5.50 95 5 8.00 95 5
[0182] Mass Spectroscopy (MS) [0183] negative-mode ESI ionization
with a desolvation temperature of 400.degree. C.; [0184] source
temperature: 120.degree. C.; [0185] cone gas flow: 150 I/h; [0186]
desolvation gas flow: 1000 I/h.
Synthesis of the Complex According to the Invention
[0187] The complex corresponds to a molecular structure in which
the monomeric (+)-catechin is bonded to said at least one basic
amino acid, or to said at least one basic amino acid derivative, by
bonds of the hydrogen bridge type.
[0188] This means that, when the complex does not dissociate in
aqueous solution.
[0189] The monomeric (+)-catechin corresponds to a polyphenol
present in the form of a monomer.
[0190] The basic amino acid has a radical which is positively
charged at neutral pH.
[0191] The basic amino acid derivative, for its part, is defined in
the context of the present invention as a molecule originating from
a basic amino acid, and which results from one or more chemical
conversions performed on this amino acid.
[0192] Preferentially, the complex (optionally in salt form)
comprises monomeric (+)-catechin and at least one amino acid
precursor which is a molecule intended to be converted into an
amino acid or into an amino acid derivative after oral
administration.
[0193] For example, and in a nonlimiting manner, the amino acid
precursor may be a glucuronide or a glucuronoside of an amino acid,
which, once absorbed, is converted by the organism into an amino
acid, by virtue of the action of .beta.-glucuronidase enzymes.
EXAMPLE 1
Preparation of a Complex of Monomeric (+)-Catechin and of
Lysine
[0194] 1a. Complex of Monomeric (+)-Catechin and of Lysine in a
Molar Equivalence Ratio of 1:2
[0195] Monomeric (+)-catechin, extracted from Uncaria gambir
according to one of the methods well known to those skilled in the
art, is milled and then dried at 50.degree. C. under vacuum for 30
minutes.
[0196] After this drying step, the (+)-catechin extracted comprises
water at least in trace amounts.
[0197] Two equivalents of lysine are then added successively to the
(+)-catechin with stirring until dissolution in distilled water
into which helium is bubbled. The solution is then heated up to a
temperature between 40.degree. C. and 45.degree. C. and stirred for
approximately one hour until complete dissolution of the two
equivalents of lysine and is then placed in a fridge for 16 h.
[0198] Alternatively, the (+)-catechin can be added to a solution
of two equivalents of lysine, brought to a temperature between
40.degree. C. and 45.degree. C. with stirring.
[0199] The solution is then filtered through a buchner funnel and
washed with 50 ml of distilled water and then dried with
evaporation of the water first in a rotary evaporator and then
using a vacuum line (for 4 h at a temperature of 50.degree.
C.).
[0200] The product obtained is not soluble in deuterated methanol
but is soluble in deuterated water (D.sub.2O, solvent used for the
NMR analysis). The NMR spectrum obtained is illustrated in FIG.
1a.
[0201] On reading the NMR spectrum, it is observed that the complex
is made up of two mol of lysine for one mol of monomeric
(+)-catechin.
[0202] Moreover, the presence of the three peaks A, B and C on the
spectrum shows that the lysine is indeed bonded to the catechin. A
strong modification of the aromatic peaks of the (+)-catechin, with
disappearance of the peaks around 5.95-5.87 ppm and poor
integration of the peaks at 6.50-7.00 ppm, is in fact observed.
This indicates that there is an interaction between the lysine and
the aromatic rings of the (+)-catechin, at the level of the
phenolic groups, these interactions involving bonds of the hydrogen
bridge type.
[0203] X-ray diffraction spectra collected using a Siemens D5000
powder diffractometer with a Cu K.alpha. radiation (.lamda.=1.542
.ANG.) were taken for the following compounds: the monomeric
(+)-catechin (JE143), a mixture (JECatLysH) of lysine
hydrochloride, of monomeric (+)-catechin and of protonated lysine
(mixture C:Lys:HCl/1:2:2), and three mixtures of monomeric
(+)-catechin and of non-protonated lysine (JE149b, JE150, JE151).
The data were recorded in a range of between 5.degree. and
60.degree. in steps of 0.0167.degree..
[0204] The spectra are illustrated in FIG. 4. As shown in FIG. 4a,
associated with the JECatLysH sample is an XRD spectrum which does
not appear to be an overlap of the spectra of the monomeric
(+)-catechin and of the lysine hydrochloride, thus showing the
formation of a complex and not a mixture of the two components. In
particular, the disappearance of the peaks at the angles of 31.5
and 36 degrees of the lysine in the spectrum of the complex is,
inter alia, noted.
[0205] The spectra for the three mixtures of monomeric (+)-catechin
and of non-protonated lysine (JE149b, JE150, JE151) do not show any
indication of crystallinity for these compounds, thereby indicating
that, for these references JE149b, JE150 and JE151, there is
formation of a composition in the amorphous crystalline state,
contrary to what is observed for the JECatLysH mixture for which a
crystalline network was identified, which does not correspond to
the superposition of the spectra of the lysine hydrochloride and of
the monomeric (+)-catechin.
1b. Complex of Monomeric (+)-Catechin and of Lysine in a Molar
Equivalence Ratio of 1:1
[0206] The complex of monomeric (+)-catechin with lysine in a molar
equivalence ratio of 1:1 was synthesized according to the same
protocol as that used for the synthesis of the lysine complex of
example 1a, but with the amount of lysine being reduced by a factor
of 2 so that there is an excess of (+)-catechin which promotes the
formation of said complex in a molar equivalence ratio of 1:1 by
virtue of the lysine deficit.
[0207] The NMR spectrum taken in deuterated water is illustrated in
FIG. 1b. On reading the NMR spectrum, it is observed that the
complex is made up of one mol of lysine for one mol of monomeric
(+)-catechin.
EXAMPLE 2
Preparation of a Complex of Monomeric (+)-Catechin and of
Arginine
[0208] 2a. Complex of Monomeric (+)-Catechin and of Arginine in a
Molar Equivalence Ratio of 1:2
[0209] The complex of monomeric (+)-catechin with arginine was
synthesized according to the same protocol as that used in the
synthesis of the lysine complex of example 1a.
[0210] On reading the NMR spectrum illustrated in FIG. 2a, it is
observed that the complex is made up of two molecules of arginine
bonded, via bonds of hydrogen bridge type, to one molecule of
monomeric (+)-catechin.
2b. Complex of Monomeric (+)-Catechin and of Arginine in a Molar
Equivalence Ratio of 1:1
[0211] The complex of monomeric (+)-catechin with arginine was
synthesized with the same protocol as that used in the synthesis of
the lysine complex of example 1b.
[0212] On reading the NMR spectrum of said complex illustrated in
FIG. 2b, it is observed that the complex is made up of one molecule
of arginine bonded, via bonds of hydrogen bridge type, to one
molecule of monomeric (+)-catechin.
Bioavailability after Oral Administration of the Complex According
to the Invention
EXAMPLE 3
Measurement of the Bioavailability after Oral Administration of
Monomeric (+)-Catechin Hydrochlorolysinate (or [C:Lvs:HCl] Complex
Salt) in Human Beings
[0213] 1.5 g of a complex of monomeric (+)-catechin and of at least
one basic amino acid: monomeric (+)-catechin hydrochlorolysinate.
Starting from the [C:Lys] complex obtained according to protocol 1
b, the monomeric (+)-catechin is present in a proportion of 61% by
weight relative to the total weight of said complex (i.e. 0.92 g
for 1.5 g), the monomeric (+)-catechin hydrochlorolysinate
[C:Lys:HCl] complex salt) is synthesized by neutralization of said
[C:Lys] complex with HCl added in an amount equimolar to that of
said complex.
[0214] In this way, a complex salt in which the lysine is bonded to
the proton (Lys-H.sup.+) resulting from the HCl and in which the
chloride (Cl.sup.- anion) is free in solution and does not interact
with the [C:Lys-H.sup.+] complex.
[0215] This complex salt is administered to a group A of 5 healthy
volunteers.
[0216] The content of free monomeric (+)-catechin in the blood was
measured for the group A of volunteers, over time, after 1.5 g of
monomeric (+)-catechin hydrochlorolysinate containing 0.92 g of
monomeric (+)-catechin had been taken (table 1).
TABLE-US-00002 TABLE 1 plasma concentration (cc) of free monomeric
(+)-catechin (in ng/ml) for the group A as a function of time T
(hours) Group A T (h) cc (ng/ml) 0.5 524 .+-. 30 1 487 .+-. 47 2
328 .+-. 35 3 173 .+-. 38 4 78 .+-. 24 6 16 .+-. 2
TABLE-US-00003 TABLE 2 parameters for evaluating the
bioavailability after oral administration, calculated from the
curves of plasma concentrations (cc) of the free monomeric
(+)-catechin (in ng/ml) for the group A as a function of time T
(hours) Group A T (max) (h) 0.5 ct (ng h/ml) (ng min/ml) 1263 .+-.
88 (75780) c (max) (ng/ml) 525
[0217] FIG. 3 illustrates the evolution of the plasma
concentrations (cc) measured in ng/ml over time T (hours) after
oral administration of (+)-catechin hydrochlorolysinate (curve A);
this curve is extrapolated from the data listed in table 1.
[0218] The area measured, for example using the trapezium method
well known in the literature, under the curve indicates the total
concentration (ct) of free monomeric (+)-catechin assimilated in
the plasma, measured over a period of six hours starting from the
ingestion of the (+)-catechin in the form of a complex formed with
the hydrochlorolysinate. This ct parameter makes it possible to
calculate, from the data of table 1, the bioavailability of the
(+)-catechin expressed in ng h/ml. The ct is 1263.+-.88 ng h/ml for
the (+)-catechin hydrochlorolysinate.
[0219] From FIG. 3, the maximum concentration c (max) corresponds
to the peak of the curve. For the group having received the
[C:Lys:HCl] complex salt, the c (max) reaches 525 ng/ml. This
maximum concentration is reached at a time T (max) of 0.5 h for the
group A having received the monomeric (+)-catechin complex.
COMPARATIVE EXAMPLE 1
Measurement of the Bioavailability after Oral Administration of
Pure Monomeric (+)-Catechin in Human Beings
[0220] A dose of 1 g of pure monomeric (+)-catechin is administered
to a group B of 5 healthy volunteers.
[0221] The content of free monomeric (+)-catechin in the blood was
measured for the group of volunteers B over time after (+)-catechin
had been taken. The results are indicated in table 3.
TABLE-US-00004 TABLE 3 plasma concentration (cc) of free monomeric
(+)-catechin (in ng/ml) for the group B as a function of time T
(hours) Group B T (h) cc (ng/ml) 0.5 59 .+-. 61 1 266 .+-. 147 2
230 .+-. 87 3 226 .+-. 38 4 128 .+-. 51 6 27 .+-. 13
TABLE-US-00005 TABLE 4 parameters for evaluating the
bioavailability after oral administration, calculated from the
curves of plasma concentrations (cc) of the free monomeric
(+)-catechin (in ng/ml) for the group B as a function of time T
(hours) Group B T (max) (h) 1.20 ct (ng h/ml) (ng min/ml) 904 .+-.
163 (54240) c (max) (ng/ml) 280
[0222] FIG. 3 illustrates the evolution of the plasma
concentrations (cc) measured in ng/ml over time T (hours) after
oral administration of pure (+)-catechin (curve B). This curve is
extrapolated from the data listed in table 3.
[0223] The area measured under the curve indicates the total
concentration (ct) of free monomeric (+)-catechin assimilated in
the plasma, measured over a period of six hours starting from the
ingestion of the pure (+)-catechin. This ct parameter makes it
possible to calculate, from the data of tables 1 and 3, the
bioavailability of the (+)-catechin, expressed in ng h/ml. The ct
is calculated at 1263.+-.88 ng h/ml for 1.5 g of (+)-catechin
hydrochlorolysinate containing 0.92 g of pure (+)-catechin. It is
calculated at 904.+-.163 ng h/ml for 1 g of pure (+)-catechin, i.e.
at 832.+-.150 ng h/ml for 0.92 g of pure (+)-catechin,
corresponding to the dose ingested with the [C:Lys:HCl] complex
salt (see example 2 according to the invention), that is to say an
improvement in bioavailability of at least 52%.
[0224] In FIG. 3, the maximum concentrations c (max) correspond to
the peaks of the curves. For the group having received 1 g of pure
monomeric (+)-catechin, the c (max) is calculated at 280 ng/ml,
i.e. 258 ng/ml for 0.92 g of pure (+)-catechin corresponding to the
dose of (+)-catechin ingested with the [C:Lys:HCl] complex salt.
For the group having received the [C:Lys:HCl] complex salt, the c
(max) reaches 525 ng/ml, which corresponds to an increase of 103%
compared with the pure (+)-catechin. These maximum concentrations
are reached respectively at a time T (max) of 1.20 h for the group
B and of 0.5 h for the group having been treated with the monomeric
(+)-catechin complex.
[0225] From this first comparative example, it is shown that the
(+)-catechin complex, and in particular the (+)-catechin
hydrochlorolysinate, significantly improved (+52%) the
bioavailability after oral administration of the monomeric
(+)-catechin compared with the taking thereof in pure form. The
speed with which the monomeric (+)-catechin passes into the blood
(and is then in free form in the plasma), measured by the T (max)
parameter, and also the maximum concentration, c (max), are also
very significantly increased: respectively by +166% and by +103%,
compared with taking monomeric (+)-catechin in its pure form.
COMPARATIVE EXAMPLE 2
Measurement of the Bioavailability after Oral Administration of the
Pure Monomeric (+)-Catechin and of the Complex of (+)-Catechin and
of Lysine in Rats
[0226] A dose of 100 mg (per kg of body weight) of pure monomeric
(+)-catechin (CP) or a dose of (+)-catechin lysinate equivalent to
100 mg of (+)-catechin was administered to each individual of a
sample of 5 Wistar rats.
[0227] The parameters for evaluating the bioavailability after oral
administration are given in table 5 for various sources of
(+)-catechin: CP, or various lysine complexes.
TABLE-US-00006 TABLE 5 parameters for evaluating the
bioavailability after oral administration, calculated from the
curves of plasma concentrations (cc) of free monomeric (+)-catechin
(in ng/ml) for each source of (+)-catechin, as a function of time T
(minutes) Source of monomeric (+)-catechin [C:Lys:HCL/ CP
[C:Lys/1:1] 1:1:1] [C:Lys/1:2] T (max) (min) 60 60 60 120 c (max)
(ng/ml) 716 1127 955 1547 ct (ng min/ml) 96 238 112 422 132 349 186
348 .DELTA.ct (as %) 0 +17 +36 +94
[0228] In this table, the .DELTA.ct (expressed as %) corresponds to
the measurement of the difference between the ct obtained with each
of the complexes and the ct measured for the pure monomeric
(+)-catechin, related back to the bioavailability value for the CP.
By way of illustration, the .DELTA.ct calculated for the
[C:Lys/1:1] complex is obtained as follows:
100.times.[(112 422-96 238)/96 238]=17%
[0229] The best parameter values are achieved for the [C:Lys/1:2]
complex for which a value of bioavailability after oral
administration of close to 1.9.times.10.sup.5 ng min/ml is
achieved, which corresponds to an increase of 94% compared with the
(+)-catechin ingested in pure form.
[0230] Furthermore, the maximum concentration [C(max)] of catechin
having passed into the blood is increased by 116%; the peak which
gives this Cmax is located at 120 min [T (max)] compared with 60
min for the other sources, which also has the effect of favorably
increasing the blood levels of free monomeric (+)-catechin over
time, confirming the delayed action of the active substance and
therefore an improved anticancer action.
[0231] These data correspond to what we had found in human beings
by comparing the oral intake of pure (+)-catechin (table 4) to that
of the [C:Lys:HCL, 1:1:1] complex salt (table 2). The
bioavailabilities are in fact increased in the same proportions,
+52% in humans, +36% in rats.
[0232] In the present example, in rats, we show that the
[C:Lys/1:2] complex increases even more significantly the
bioavailability of the (+)-catechin after oral administration.
COMPARATIVE EXAMPLE 3
Measurement of the Bioavailability after Oral Administration of the
Pure Monomeric (+)-Catechin, of the Complex of (+)-Catechin and of
Arginine, and of the Complex of (+)-Catechin and of Lysine, in
Rats
[0233] A dose of 100 mg (per kg of body weight) of pure monomeric
(+)-catechin (CP) or a dose of (+)-catechin complex equivalent to
100 mg of (+)-catechin was administered to each individual of a
sample of 5 Wistar rats.
[0234] The parameters for evaluating the bioavailability after oral
administration are given in table 6 for various sources of
(+)-catechin: CP, or various lysine complexes or arginine
complexes.
TABLE-US-00007 TABLE 6 parameters for evaluating the
bioavailability after oral administration, calculated from the
curves of plasma concentrations (cc) of free monomeric (+)-catechin
(in ng/ml) for each source of (+)-catechin, as a function of time T
(minutes) Source of monomeric (+)-catechin CP [C:Lys/1:1]
[C:Arg/1:1] T (max) (min) 60 60 60 c (max) (ng/ml) 716 1127 1292 ct
(ng min/ml) 96 238 112 422 116 351 .DELTA.ct (as %) 0 +17 +21
[0235] From these results, it is deduced that any basic amino acid
(i.e. chosen from lysine, arginine and histidine) which bonds to
the (+)-catechin, and therefore which neutralizes the acidity of
the ortho-diphenol groups located on the nucleus of the
(+)-catechin, could have the same beneficial effect on the
bioavailability of said catechin after oral administration.
COMPARATIVE EXAMPLE 4
Measurement of the Bioavailability after Oral Administration of the
Pure Monomeric (+)-Catechin, and of the Complex of (+)-Catechin and
of Lysine, at a Molar Equivalence Ratio of 1:1 and 1:5 in Rats
[0236] A dose of 25 mg (per kg of body weight) of pure monomeric
(+)-catechin (CP) or a dose of (+)-catechin lysinate equivalent to
25 mg of (+)-catechin was administered to each individual of a
sample of 3 Wistar rats.
[0237] The parameters for evaluating the bioavailability after oral
administration are given in table 7a for various sources of
(+)-catechin: CP, or various lysine complexes.
[0238] The T (max), c (max) and ct parameters in terms of total
(+)-catechin present in the blood are given in table 7b. The total
(+)-catechin summarizes the free monomeric (+)-catechin and the
monomeric (+)-catechin which has been conjugated by the rat's
body.
TABLE-US-00008 TABLE 7a parameters for evaluating the
bioavailability after oral administration, calculated from the
curves of plasma concentrations (cc) of free monomeric (+)-catechin
(in ng/ml) for each source of (+)-catechin as a function of time T
(minutes) Source of monomeric (+)-catechin CP [C:Lys/1:2]
[C:Lys/1:5] T (max) (min) 30 60 60 c (max) (ng/ml) 154 173 74 ct
(ng min/ml) 21 145 24 845 13 770 .DELTA.ct (as %) 0 +17 -35
TABLE-US-00009 TABLE 7b T (max), c (max) and ct parameters in terms
of total (+)-catechin present in the blood, calculated from the
curves of plasma concentrations (cc) of total monomeric
(+)-catechin (in ng/ml) for each source of (+)-catechin as a
function of time T (minutes) Source of monomeric (+)-catechin CP
[C:Lys/1:2] [C:Lys/1:5] T (max) (min) 30 60 60 c (max) (ng/ml) 2200
2366 1100 ct (ng min/ml) 273 875 339 245 163 080 .DELTA.ct (as %) 0
+24 -40
[0239] The [C:Lys/1:2] complex allows an increase in the contents
of both free and total plasma (+)-catechin, while the [C:Lys/1:5]
complex clearly decreases these contents compared with the pure
(+)-catechin, i.e. the (+)-catechin ingested without lysine.
[0240] The results of this example, compared to the results
illustrated in table 5, show that the optimum in terms of
bioavailability after oral administration is actually achieved for
the [C:AA/1:2] complex, optionally in the presence of HCl. It
should be noted that the presence of an acid favorably influences
the parameters of bioavailability after oral administration (see
table 5); however, the presence of this acid is not essential to
obtain an increased bioavailability after oral administration.
Anticancer Action of the Complex According to the Invention
EXAMPLE 4
Measurement of the Action of Monomeric (+)-Catechin
Hydrochlorolysinate and of Monomeric (+)-Catechin Ascorbolysinate
on the Total Incorporation and Incorporation into Collagen of
3H-Proline in the Skin
[0241] The stabilization of lysosome membranes prevents the release
of the proteolytic enzymes responsible for the degradation of the
connective matrix, in particular of collagen. This degradation is
responsible for the propagation of metastatic cancer cells,
whatever the type of cancer (Cell Communication and Signaling 2010,
8:22).
[0242] The skin is a tissue particularly rich in connective matrix
and in collagen fibers, which gives it all its elasticity.
[0243] The effectiveness of the (+)-catechin hydrochlorolysinate
and of the monomeric (+)-catechin ascorbolysinate, administered
orally, on the one hand on the total incorporation (on the entire
skin) and on the other hand the incorporation into the collagen of
said skin, of 3H-proline is demonstrated here.
[0244] To do this, a study was carried out in vivo on a batch of 24
female Wistar rats weighing from 95 to 115 g, divided into two
groups C and D, each of 12 rats. For each group, the radioactive
3H-hydroxyproline, the specific activity of which is 13.6
Ci/10.sup.-3 mol, was administered to all the animals by
intraperitoneal injection of proline-L-(5-3H) at a dose, relative
to the weight of the individuals, of 1 mCi/kg, which represents an
activity in terms of radioactive 3H-proline of 100
.mu.Ci/individual.
[0245] 6 rats of each group received a treatment based on monomeric
(+)-catechin by gavage in a proportion of 100 mg/kg for 5 days
before sacrifice. The other 6 rats which did not receive treatment
based on monomeric (+)-catechin are a control subgroup (ctrl). The
group C received monomeric (+)-catechin hydrochlorolysinate, while
the group D received monomeric (+)-catechin ascorbolysinate.
TABLE-US-00010 TABLE 8 action of the monomeric (+)-catechin in its
complex forms (100 mg/kg) on the level (in dpm/mg) of total
incorporation (IT) and incorporation into collagen (IC) of
3H-hydroxyproline in rat skin Group C D Level ctrl treated ctrl
treated IT 5620 .+-. 186 6248 .+-. 124 4864 .+-. 231 5112 .+-. 259
IC 1178 .+-. 39 1343 .+-. 48 963 .+-. 42 1117 .+-. 49
[0246] In table 8, the level of incorporation is measured by the
number of disintegrations of the radioactive 3H-hydroxyproline per
minute (dpm) per mg of dry weight. The higher this number, the
higher the concentration of 3H-hydroxyproline.
[0247] The results of table 8 show that the catechin and the
complex of (+)-catechin hydrochlorolysinate stimulate connective
tissue biosynthesis and particularly collagen synthesis.
[0248] In addition, this example, according to the invention,
confirms the protection of the connective web in vivo by oral
absorption of the monomeric (+)-catechin complexes given orally
and, consequently, the protection of this web, in particular
against the intrusion of cancer cells.
COMPARATIVE EXAMPLE 5
Measurement of the Action of the Pure Monomeric (+)-Catechin on the
Total Incorporation and the Incorporation into Collagen of
3H-Proline in the Skin
[0249] In this comparative example, a group E of 12 Wistar rats
weighing from 95 to 115 g, in which radioactive 3H-hydroxyproline,
the specific activity of which is 13.6 Ci/10.sup.-3 mol, was
administered to all the individuals by intraperitoneal injection of
proline-L-(5-3H) at a dose, relative to the weight of the
individuals, of 1 mCi/kg, which represents an activity in terms of
radioactive 3H-proline of 100 .rho.Ci/individual.
[0250] 6 rats of each group received a treatment based on pure
monomeric (+)-catechin by gavage in a proportion of 100 mg/kg for 5
days before sacrifice. The other 6 rats which did not receive
treatment based on monomeric (+)-catechin are a control subgroup
(ctrl).
TABLE-US-00011 TABLE 9 action of the pure monomeric (+)-catechin
(100 mg/kg) on the level (in dpm/mg) of total incorporation (IT)
and incorporation into collagen (IC) of 3H-hydroxyproline in rat
skin Group E Level ctrl treated IT 4864 .+-. 231 5689 .+-. 410 IC
963 .+-. 42 1124 .+-. 51
[0251] In table 9, the level of incorporation is measured by the
number of disintegrations of the radioactive 3H-hydroxyproline per
minute (dpm) per mg of dry weight. The higher this number, the
higher the concentration of 3H-hydroxyproline.
[0252] The results of tables 8 and 9 show that the increase in the
level of incorporation for the complexes of monomeric (+)-catechin
hydrochlorolysinate (+15%) and of monomeric (+)-catechin
ascorbolysinate (+14%) is equivalent to that measured when the rats
are given pure monomeric (+)-catechin by gavage (+16%).
[0253] This comparative example confirms the protection of the
connective web in vivo by oral absorption of the monomeric
(+)-catechin complexes given orally and, consequently, the
protection of this web in particular against cancer cell intrusion,
this being at monomeric (+)-catechin contents which are de facto
lower (61 mg of (+)-catechin for 100 mg of complex) than that
encountered when pure (+)-catechin is taken (100 mg of
(+)-catechin).
EXAMPLE 5
Measurement of the Action of the Monomeric (+)-Catechin
Hydrochlorolysinate ([C:Lys:HCl] Complex Salt) on the Incidence of
Gastric Ulcers and on the Histamine Level in the Gastric Mucosa in
Mastomys Rats
[0254] In this example, the effectiveness of the monomeric
(+)-catechin hydrochlorolysinate in vivo in an African rat,
Mastomys, which is known to spontaneously generate gastric lesions
and in particular gastric carcinomas, is demonstrated.
[0255] Mastomys in fact has a special system of cells which store
histamine in the gastric mucosa; these cells can give rise to
carcinoid tumors.
[0256] In this example, a sample of n=48 Mastomys are sensitized a
first time by intraperitoneal injection of 3 .mu.g of ovalbumin
dissolved in 0.2 ml of saline solution containing 1 mg of aluminum
hydroxide the ovalbumin which brings about anti-immunoglobulin
E(IgE) and G(IgG) antibodies. After 7 days, a second injection of 1
mg of ovalbumin in 0.01 ml of saline solution is given in the
gastric mucosa at the level of the corpus of the anesthetized
animals, which causes an ulcerated lesion at the actual site of the
injection.
[0257] The 48 rats are divided up into three groups: one of 24 and
two of 12 individuals. The first group of 24 individuals (G1)
received a placebo (concentrated NaCl solution at 0.15 M).
[0258] The second group (G2) of 12 individuals was treated with the
monomeric (+)-catechin hydrochlorolysinate administered per os
twice a day (2.times.300 mg), two days before the injection and
three days after injection. The third group (G3) of 12 individuals
received, under the same conditions as those of the G2 group, the
monomeric (+)-catechin hydrochlorolysinate in a proportion of 100
mg, but intraperitoneally.
[0259] As shown by the results of table 10, the oral administration
of the (+)-catechin hydrochlorolysinate gives a very significant
decrease (-72%) in the number of animals exhibiting a gastric ulcer
(NU) and also a significant decrease (-18%) in the histamine level
(HL), this level being expressed as 10.sup.-6 mg/kg of protein, in
the gastric mucosa. The histamine level is measured for the G1, G2
and G3 groups on the basis of a sample of n=12 individuals.
[0260] The decrease in the level of histamine accumulated in the
gastric mucosa results directly from the action of the monomeric
(+)-catechin on the wall of the cells which store histamine. By
contributing to the reinforcement of the wall of these cells, the
monomeric (+)-catechin makes it possible to decrease the level of
histamine assimilated in the gastric mucosa and therefore to reduce
the number of ulcers.
[0261] It is interesting to note, with respect to this experiment,
that in particular in many types of chronic or acute leukemias, an
increase in mast cells is found, with a concomitant increase in
histamine levels and also an increase in immunoglobulin levels
(Blood Cells Mol. Dis. 35 (3), 370-383, 2005).
TABLE-US-00012 TABLE 10 action of the (+)-catechin
hydrochlorolysinate on the incidence of gastric ulcers and on the
histamine level in the gastric mucosa in Mastomys Group n NU HL (n
= 12) G1 24 23 46.17 .+-. 2.30 G2 12 5 37.91 .+-. 3.73 G3 12 6
34.98 .+-. 3.52
EXAMPLE 6
Measurement of the Action of the Monomeric (+)-Catechin
Hydrochlorolysinate ([C:Lys:HCl] Complex Salt) in the Treatment of
Patients Suffering from Cancer
[0262] In this example, the results relating to the test of the
effect of the monomeric (+)-catechin hydrochlorolysinate in two
cancer patients treated for more than a year with a tablet of 500
mg of [C:Lys:HCl] complex salt per day are presented.
[0263] In the first patient, a 67-year-old man, a lymphoplasmocytic
lymphoma (of Waldenstrom macroglobulinemia type) was detected
during a post-operative blood test on day D.sub.0. The diagnosis is
based on the electrophoretic detection of a monoclonal peak of
immunoglobulins (IgM). This diagnosis was reconfirmed and the peak
was assayed during a second analysis using the same detection by
electrophoresis.
[0264] Immunoglobulins play an essential role in the organism's
defense against attacks and are normally secreted by B
lymphocytes.
[0265] In multiple myeloma or, for example, in the case of
Waldenstrom's disease, secretion of a single type of
immunoglobulin, or monoclonal immunoglobulin, by plasma cells
present in the bone marrow and which proliferate in an uncontrolled
manner, is observed.
[0266] These immunoglobulins are found at a high concentration in
the blood and in the urine. They therefore constitute real tumor
markers. The assaying thereof gives an account of the number of
sick cells and the extent of the disease and, consequently, makes
it possible to monitor its progression under treatment.
[0267] Successive blood tests gave the following data for the IgM
assay (in mg per 100 ml of plasma):
TABLE-US-00013 TABLE 11 evolution of the IgM level (in mg/100 ml)
in the blood over time Sampling period IgM D.sub.0 1910 D.sub.0 + 1
month 2010 D.sub.0 + 5 months 1966 D.sub.0 + 8 months 2467 D.sub.0
+ 12 months 2280 D.sub.0 + 15 months 2748 D.sub.T 2931 D.sub.T + 4
months 2417 D.sub.T + 9 months 2410 D.sub.T + 15 months 2412
[0268] The results of table 11 show that, since the taking of
monomeric (+)-catechin hydrochlorolysinate for a year and a half,
there has been a stabilization below the maximum detected on day
D.sub.T starting from which the treatment was administered.
[0269] The level of infiltration remained low, despite IgM values
greater than 2000 mg/100 ml. In addition, the patient did not
undergo chemotherapy treatment. It is therefore considered that he
is stabilized.
[0270] In the second patient, a left subclavicular cervical
adenopathy was first of all detected on day D.sub.0. Next, 2 weeks
later, a left renal adenocarcinoma was detected for this patient,
which was followed by a nephrectomy of the left kidney 3 days after
the detection of the adenocarcinoma in this kidney.
[0271] Thereafter, on day D.sub.0+3 months, a left cervical lymph
node dissection is performed on the patient. This procedure was
followed by a series of 15 radiotherapy sessions.
[0272] Starting from day D.sub.0+15 months (that is to say on day
D.sub.T), the patient was orally administered a tablet of 500 mg of
(+)-catechin hydrochlorolysinate per day.
[0273] On day D.sub.T+15 months, the appearance of a pathological
mediastinal adenopathy (45 cm.sup.3 in volume) was observed and
required a mediastinal lymph node dissection followed by a
pulmonary examination on day D.sub.T+17 months. This examination
demonstrated the involution of a small micronodular opacity of 5 mm
in the left lung and the absence of secondary lesion at the level
of the pulmonary parenchyma and of the abdominal stage.
[0274] On D.sub.T+19 months, the consultation report concluded that
the clear cell renal carcinoma was in complete remission. This
diagnosis was reconfirmed during controlled examinations carried
out on D.sub.T+23 months. Clearly, it appears that taking
(+)-catechin hydrochlorolysinate for one year stabilized the
patient's condition.
Synthesis of the Composition of Precursors of the Complex or of the
Complex Salt According to the Invention
[0275] The method for producing the composition comprising said
monomeric (+)-catechin and said at least one basic amino acid as
precursor of said complex comprises the following steps: [0276]
providing a first amount of monomeric (+)-catechin; [0277]
providing a second amount of at least one basic amino acid, or of
at least one basic amino acid derivative, so as to obtain a molar
equivalence ratio between said (+)-catechin and said at least one
basic amino acid or said at least one basic amino acid derivative
of between 1:1 and 1:2.5, preferably greater than or equal to 1:1,
in particular greater than 1:1, preferably less than or equal to
1:2.5, in particular less than 2.5, more particularly less than or
equal to 1:2, more particularly less than 2; and [0278] bringing
said monomeric (+)-catechin into contact with said at least one
basic amino acid, or with said basic amino acid derivative, so as
to obtain said mixture of monomeric (+)-catechin and of at least
one basic amino acid or of at least one derivative of a basic amino
acid.
[0279] Preferentially, the method comprises an additional step
which consists in providing an acid, preferably an ascorbic
acid.
[0280] Alternatively, the method comprises an additional step which
consists in providing an aqueous phase and in solubilizing said
mixture in said aqueous phase so as to form a complex of
(+)-catechin and of at least one basic amino acid, said complex
being solubilized in said aqueous phase.
[0281] Alternatively, the method also comprises a step of adding a
biocompatibility excipient to said mixture according to the
invention.
[0282] Preferably, said at least one amino acid provided is
selected from the group consisting of lysine and arginine, of
natural or synthetic origin, and of a mixture thereof.
[0283] In comparative examples 6 to 9 which follow, the orally
administered composition was prepared based on a mixture of
hydrochlorolysinate (Lys:HCl) in pulverulent form with pure
monomeric (+)-catechin at the molar equivalent ratios of 1:1, 1:2,
1:3 and 1:5.
[0284] A composition which comprises the monomeric (+)-catechin and
at least one basic amino acid as precursor of said [C:Lys:HCl]
complex salt is obtained.
[0285] Each of the mixtures is then solubilized in water. During
the solubilization of the mixture, formation of the complex of
(+)-catechin and of hydrochlorolysinate in the aqueous phase
occurs. The solution thus obtained is a solution of (+)-catechin
hydrochlorolysinate in the form of a complex: the complex obtained
in this solution is a complex of monomeric (+)-catechin and of at
least one basic amino acid (or derivative thereof) with a molar
equivalence ratio identical to that of the mixture according to the
invention. Thus, a mixture of monomeric (+)-catechin and of an
amino acid in a molar equivalence ratio of 1:1 will give, once
solubilized in the aqueous phase, a solution of complex of
monomeric (+)-catechin and of at least one basic amino acid (or
derivative thereof) in a molar equivalence ratio equal to 1:1.
[0286] This solution is then administered per os (PO) or by
intravenous (IV) injection to each individual of a group of 5
Wistar rats, at a dose of 25 mg (per kilogram of body weight) of
pure monomeric (+)-catechin or a dose of (+)-catechin lysinate
equivalent to 25 mg of (+)-catechin.
Bioavailability after Oral Administration of the Composition of
Precursors of the Complex or of the Complex Salt According to the
Invention
COMPARATIVE EXAMPLE 6
Measurement of the Bioavailability after Oral Administration and
after Administration by Injection of the Complex of (+)-Catechin
and of Lysine, at a Molar Equivalence Ratio of 1:1 and 1:2, in
Rats
TABLE-US-00014 [0287] TABLE 12a plasma concentration (cc) of the
total monomeric (+)-catechin (in ng/ml) as a function of time T
(min) IV PO [C:Lys:HCl/1:2:2] [C:Lys:HCl/1:1:1] [C:Lys:HCl/1:2:2] T
(min) cc (ng/ml) 10 8066 6406 1839 30 2639 2259 3780 60 1191 963
3190 120 338 270 1990 240 0 0 475
TABLE-US-00015 TABLE 12b plasma concentration (cc) of the free
monomeric (+)-catechin (in ng/ml) as a function of time T (min) IV
PO [C:Lys:HCl/1:2:2] [C:Lys:HCl/1:2:2] T (min) cc (ng/ml) 10 3340
428 30 991 1027 60 291 1188 120 0 767 240 0 179
TABLE-US-00016 TABLE 12c comparison of the total plasma
concentrations (ct) in ng min/ml of the free monomeric (+)-catechin
and of the total monomeric (+)-catechin after intravenous and oral
administration of 25 mg/kg of C:Lys:HCl/1:2:2 in rats IV PO Free 87
970 165 325 (.DELTA.ct*: +88%) Total 277 040 479 585 (.DELTA.ct*:
+73%) *.DELTA.ct measured between the IV and PO data.
[0288] After intravenous injection of the [C:Lys:HCl/1:2:2] complex
salt, the plasma levels (cc) of (+)-catechin, naturally very high
at the start, very rapidly come back down to very low levels, 340
ng/ml after 2 hours for the total (+)-catechin found in the plasma
and 0 ng/ml for the free (+)-catechin, whereas the total blood
levels of the same product taken orally under the same conditions
are maintained after 2 hours at 1990 ng/ml and the amount of free
(+)-catechin at 767 ng/ml, as indicated in the results summarized
in tables 12a and 12b.
[0289] This intravenous injection (made possible thanks to the
hydrochlorolysinate making the (+)-catechin soluble) shows, whether
there is or are one or two molecules of lysine linked to the
monomeric (+)-catechin, that this (+)-catechin is rapidly
eliminated from the blood stream.
[0290] On the other hand, the oral ingestion of the complexed form
of the monomeric (+)-catechin, on the one hand, increases the
amounts of free monomeric (+)-catechin in the blood and, on the
other hand, prolongs the maximum concentration peak.
[0291] This is a surprising result. Indeed, the plasma levels
obtained after direct injection into the blood give blood levels
that are much higher over the first hour after administration than
when the product is given orally, which is expected. On the other
hand, when the complex is administered per os, free monomeric
(+)-catechin ct values which are increased by 88% with regard to
the free (+)-catechin available in the plasma are observed (ct of
165 325 ng min/ml for an administration per os compared with only
87 970 ng min/ml for an IV administration); the same is true for
the total catechin found in the plasma; the ct increases in the
same manner by 73%, which is absolutely remarkable. Oral
administration therefore surprisingly proves to be superior to
intravenous administration for the overall bioavailability, whether
it is free or total, of the (+)-catechin in the plasma after
administration of the [C:Lys:HCl/1:2:2] complex salt.
[0292] Furthermore, while, after 120 min, there is virtually no
more (+)-catechin in the blood after IV injection, the maximum
levels of catechin in the blood are observed, at the same moment,
after oral intake of the [C:Lys:HCl/1:2:2] complex salt.
[0293] It is therefore demonstrated that the [C:Lys:HCl/1:2:2]
complex salt improves the bioavailability of the catechin in a
surprising manner; indeed, this blood bioavailability of the
(+)-catechin, whether it is free or conjugated, is surprisingly
better when administered orally than when administered by
intravenous injection.
[0294] This shows that the levels and the bioavailability data
after intravenous administration cannot be extrapolated to oral
administration and that the results of the present invention
clearly differ from document U.S. Pat. No. 4,285,964 in which no
bioavailability test for an intake per os was exemplified.
COMPARATIVE EXAMPLE 7
Measurement of the Effect of the Acid on the Bioavailability after
Oral Administration of the Complex of (+)-Catechin and of Lysine in
Rats
[0295] In order to verify the possible effect of the addition of an
acid to the [C:Lys/1:2] complex, we measured the levels of free
monomeric (+)-catechin in the plasma after oral ingestion of CP or
of the [C:Lys:HCl/1:2:2] complex salt. The results are given in the
table below:
TABLE-US-00017 TABLE 13 T (max), c (max) and ct parameters in terms
of free (+)-catechin present in the blood, calculated from the
curves of plasma concentrations (cc) of the free monomeric
(+)-catechin (in ng/ml) for each source of (+)-catechin as a
function of time T (minutes) Source of monomeric (+)-catechin CP
[C:Lys:HCl/1:2:2] T (max) (min) 60 60 c (max) (ng/ml) 580 1200 ct
(ng min/ml) 91 935 165 325 .DELTA.ct (as %) 0 +77
[0296] The positive effect of the 2 molecules of lysine on the
bioavailability of the monomeric (+)-catechin and on its maximum
concentration is again found; the addition of 2 molecules of HCl
influences the effect of the 2 molecules of lysine: .DELTA.ct of
+77% in the presence of HCl and of +17% without HCl (see table
7a).
COMPARATIVE EXAMPLE 8
Measurement, in Rats, of the Effect of the Molar Equivalence Ratio
Between the (+)-Catechin and the Basic Amino Acid on the
Bioavailability after Oral Administration of the Complex of
(+)-Catechin Hydrochlorolysinate According to the Invention
TABLE-US-00018 [0297] TABLE 14 T (max), c (max) and ct parameters
in terms of free (+)-catechin present in the blood, calculated from
the curves of plasma concentrations (cc) of the free monomeric
(+)-catechin (in ng/ml) for each source of (+)- catechin as a
function of time T (minutes) Source of monomeric (+)-catechin Ratio
CP 1:2:2 1:2.5:2.5 1:3:3 1:5:5 1:2:2.sup..sctn. T (max) 60 60 -- 60
60 60 (min) c (max) 580 1200 -- 280 265 226 (ng/ml) ct 91935 165325
105500* 38000 31136 44095 (ng min/ml) .DELTA.ct (as %) 0 +80 +15
-59 -66 -48 *ct value estimated from the profile of the curve of
evolution of ct as a function of the monomeric (+)-catechin: lysine
molar equivalence ratio (see FIG. 5). The other ct values estimated
at the start in FIG. 5 are those of the 1:1.5:1.5 ratio, which is
142000 ng min/ml, and of the 1:1:1 ratio, which is 127000 ng
min/ml. .sup..sctn.Separate per os intake of the
hydrochlorolysinate and of the pure (+)-catechin.
[0298] The value of the ct (ng min/ml) for the C:Lys:HCl/1:1.5:1.5
complex salt reported on the curve in FIG. 5 is 142 k ng min/ml,
that is to say better than for 1:1:1 (127 k, reported value) and
better than for 1:2.5:2.5 (105 k ng min/ml, reported value), but
not as good as for 1:2:2 (165 k, calculated value).
[0299] All the ct values obtained for (+)-catechin salts in
proportions less than or equal to 1:2.5:2.5 are much better than
the values obtained for these same salts in higher proportions,
1:3:3 (38 k, calculated value) and 1:5:5 (26 k, calculated
value).
[0300] These results confirm the results of comparative example 4:
the [C:Lys:HCl/1:2:2] complex salt is the only (+)-catechin-lysine
association which clearly improves the bioavailability of the
(+)-catechin taken orally.
[0301] The data of this example not only show that the
[C:Lys:HCl/1:2:2] complex salt gives an optimum bioavailability,
but also that, as soon as this proportion is exceeded, the effect
is clearly reversed, with a .DELTA.ct of -59% for the molar
equivalence ratio of 1:3:3 and of -66% for the molar equivalence
ratio of 1:5:5.
[0302] In addition, the separate ingestion (within a time period of
between 5 and 10 min) of the monomeric (+)-catechin and the
hydrochlorolysinate gives a negative effect (with a .DELTA.ct
of-48%) on the amount of free monomeric (+)-catechin in the plasma.
This demonstrates that the catechin and the lysine must be mixed
before ingestion (in a form solubilized optionally in water), or at
least ingested simultaneously.
COMPARATIVE EXAMPLE 9
Measurement, in Rats, of the Nature of the Therapeutic Composition
on the Bioavailability after Oral Administration
[0303] Whether the composition administered per os is based on the
[C:AA/1:1-1:2.5] complex prepared according to the method described
in example 1a (1 b) or 2a (2b) (see examples 1 and 2), or else the
composition administered per os is a mixture in a desired molar
equivalence ratio of a powder of (+)-catechin and of lysine
hydrochloride (also referred to as hydrochlorolysinate), the
results in terms of bioavailabilities after oral administration are
similar (see table 15 below).
[0304] Given the results of comparative example 2 in which the
[C:Lys/1:2] complex was prepared according to protocol 1a (see
example 1), and the results of comparative example 8, in which the
[C:Lys:HCl/1:2:2] complex salt was prepared by simply mixing one
mol of pulverulent (+)-catechin and two mol of pulverulent
hydrochlorolysinate and then dissolving before administration per
os, the results summarized in table 15 show that the improvement in
bioavailability after oral administration for these complexes
remains in the same order of magnitude.
TABLE-US-00019 TABLE 15 T (max), c (max) and ct parameters in terms
of free (+)-catechin present in the blood, calculated from the
curves of plasma concentrations (cc) of the free monomeric
(+)-catechin (in ng/ml) for each source of (+)-catechin as a
function of time T (minutes) Source of monomeric (+)-catechin
[C:Lys/1:2] [C:Lys:HCl/1:2:2] T (max) (min) 120 60 c (max) (ng/ml)
1547 1200 ct (ng min/ml) 186 348 165 325 .DELTA.ct (as %) +94
+80
[0305] Being able to obtain optimum bioavailability parameters on
the basis of a pulverulent mixture of (+)-catechin and of amino
acid represents an advantage in terms of production cost as long as
the preparation of this mixture is carried out in a single solid
phase and very easily, thereby making it possible to envision
large-scale production perspectives.
COMPARATIVE EXAMPLE 10
Measurement, in Rats, of the Bioavailability after Oral
Administration for Quercetin and Epigallocatechin Gallate
(EGCG)
[0306] In this example, it is demonstrated that the effect of
lysine is not significant, either on the bioavailability after oral
administration of quercetin, or on that of epigllocatechin gallate
(EGCG), as shown by the results of table 16 below:
TABLE-US-00020 TABLE 16 T (max), c (max) and ct parameters in terms
of free polyphenol present in the blood, calculated from the curves
of plasma concentrations (cc) of the polyphenol (in ng/ml) for each
polyphenol source as a function of time T (minutes) Source of
monomeric (+)-catechin QP [Q:Lys:HCl/1:2:2] .sup..sctn. T (max)
(min) 60 60 c (max) (ng/ml) 10 430 8777 ct (ng min/ml) 1 311 645 1
541 945 .DELTA.ct.sup.q (as %)** 0 +18 * QP: pure quercetin, i.e.
not complexed with at least one basic amino acid; .sup..sctn.
[Q:Lys:HCl/1:2:2]: complex of quercetin with two lysines.
**.DELTA.ct.sup.q: measurement of the difference between the ct
obtained with the complex and the ct measured for the QP, related
back to the bioavailability value for QP.
[0307] In this example, the orally administered composition was
prepared based on a mixture of hydrochlorolysinate (Lys:HCl) in
pulverulent form with pure monomeric quercetin or EGCG at the molar
equivalence ratios of 1:2 for quercetin, and of 1:1, 1:2, 1:3 and
1:5 with regard to EGCG.
[0308] Each of the mixtures is solubilized in water. This solution
is then administered per os (PO) to each individual of a group of 5
Wistar rats, in a proportion of one dose of 25 mg (per kg of body
weight) of pure monomeric quercetin or EGCG or a dose of quercetin
lysinate or EGCGC lysinate equivalent to 25 mg of polyphenol.
[0309] With regard to EGCG, the levels measured remain below the
reliable limits of detection (i.e. below 250 ng/ml) whether for
pure EGCG or for its complexed forms, whatever the proportion of
hydrochlorolysinate added to the epigallocatechin gallate (in a
molar equivalence ratio of 1:1:1, of 1:2:2, of 1:3:3, or else of
1:5:5).
[0310] In conclusion, in view of the results of optimum
bioavailability obtained for a complex between the (+)-catechin and
the basic amino acid at a molar equivalence ratio of 1:2, and given
the fact that the attempts at complexation of other acid
polyphenols such as quercetin or EGCG failed, it is clearly
demonstrated in the context of the present invention that the
[C:AA/1:1-1:2.5] complexes are complexes which have a specific
activity with regard to its passage through the gastrointestinal
tract. Clearly, it is not therefore a case of a simple acid-based
neutralization, which should, in the latter case, have also
promoted the bioavailability after oral administration of quercetin
and of EGCG.
[0311] It is also understood that the present invention is in no
way limited to the abovementioned particular embodiments and that
many modifications may be introduced therein without departing from
the context of the appended claims.
* * * * *