U.S. patent application number 14/130020 was filed with the patent office on 2014-05-22 for fullerene and its use to maintain good health and to prolong the expected lifespan of mammals.
The applicant listed for this patent is Manef Abderrabba, Fathi Moussa. Invention is credited to Manef Abderrabba, Fathi Moussa.
Application Number | 20140140985 14/130020 |
Document ID | / |
Family ID | 54696184 |
Filed Date | 2014-05-22 |
United States Patent
Application |
20140140985 |
Kind Code |
A1 |
Moussa; Fathi ; et
al. |
May 22, 2014 |
FULLERENE AND ITS USE TO MAINTAIN GOOD HEALTH AND TO PROLONG THE
EXPECTED LIFESPAN OF MAMMALS
Abstract
A water-insoluble fullerene is at least partially dissolved in a
biocompatible lipid carrier, especially a fat or an oil such as
butter, olive oil, and liquid paraffin. When administered to
mammals, the fullerene, most preferably [60]fullerene dissolved in
olive oil, scavenges free radicals and prolongs life span in
rats.
Inventors: |
Moussa; Fathi; (Ben Arous,
TN) ; Abderrabba; Manef; (Sidi Rezig-Megrine,
TN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Moussa; Fathi
Abderrabba; Manef |
Ben Arous
Sidi Rezig-Megrine |
|
TN
TN |
|
|
Family ID: |
54696184 |
Appl. No.: |
14/130020 |
Filed: |
June 28, 2012 |
PCT Filed: |
June 28, 2012 |
PCT NO: |
PCT/TN2012/000003 |
371 Date: |
December 30, 2013 |
Current U.S.
Class: |
424/125 ;
426/541 |
Current CPC
Class: |
A61K 33/44 20130101;
A61P 39/06 20180101; A23D 9/007 20130101; A23L 3/358 20130101; A23L
33/10 20160801; A23D 7/0053 20130101 |
Class at
Publication: |
424/125 ;
426/541 |
International
Class: |
A61K 33/44 20060101
A61K033/44; A23L 3/358 20060101 A23L003/358 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 30, 2011 |
TN |
TN2011/0327 |
Claims
1. A stable biocompatible composition comprising: (a) a carrier
selected from the group consisting of fats and oils; and (b) one
compound selected from the group consisting of water-insoluble
fullerenes, wherein said fullerenes are mostly dissolved in said
earner.
2. The stable biocompatible composition of claim 1, wherein the
carrier is selected from the group consisting of butter, cocoa
butter, peanut butter, olive oil, soybean oil, cod-liver oil,
liquid paraffin, and mixtures thereof.
3. The stable biocompatible composition of claim 1, wherein said
water-insoluble fullerenes are present in an amount ranging from
0.01 to 0.12% by weight relative to the total weight of the
composition, preferably from 0.01 to 0.0.08% by weight.
4. The stable biocompatible composition of claim 1, wherein the
fullerene core of said water-insoluble fullerenes is Ceo.
5. The stable biocompatible composition of claim 1, wherein said at
least one water-insoluble fullerene is comprised in a
therapeutically effective amount.
6. The stable biocompatible composition of claim 5, wherein said at
least one water-insoluble fullerene can eliminate biologically
reactive radical species.
7. The stable biocompatible composition of claim 6, wherein the
biologically reactive radical species are initially generated from
O2 or H2O2.
8. A method to maintain good health and/or to prolong the expected
lifespan of a mammal, which comprises a step of administering to
said mammal a stable biocompatible composition according to any
claim 1, wherein said composition is administered intravenously,
intramuscularly, subcutaneously, intradermally, intrathecal ,
intraperitoneally, rectally by suppositories, sublingually, orally,
or by inhalation.
9. The method of claim 8, wherein said composition is administered
in a pure form or in the form of an emulsion in water.
10. The method of claim 8, wherein the at least one water-insoluble
fullerene is administered in an amount of at least 0.1 mg/kg of
body weight per day.
11. The method of claim 8, wherein said mammal is a human.
12. The method of claim 8, wherein it maintains the health and
prolong the longevity of said mammal.
13. A method to preserve a substance or mixture of substances
sensitive to damages caused by free radicals, which comprises a
step of adding to said substance or mixture of substances a stable
composition according to claim 1.
14. The method of claim 12, wherein said substance or mixture of
substances is food or a nutritional composition.
Description
[0001] This application claims benefit of Tunisian Provisional
Application No. TN 2011/327 filed Jun. 30, 2011 the contents of
which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention concerns [60]fullerene and stable
biocompatible compositions comprising [60]fullerene dissolved in a
carrier. The present invention also relates to a method for
increasing the expected lifespan (longevity) of a mammal, which
comprises a step of administering [60]fullerene or a composition
comprising a therapeutically effective amount of [60]fullerene. The
present invention further concerns a method for preserving a mammal
to damages caused by free radicals using said [60]fullerene or said
composition.
[0004] 2. Description of Related Art
[0005] Free radicals, such as oxygen radicals and other reactive
oxygen/nitrogen/chlorine species (hydroxyl, nitric oxide radicals),
are constantly formed in vivo. Some of these molecules are
physiologically useful, but they can also result in pathological
oxidative stress to cells and tissues. Endogenous defences include
both antioxidants and repairing systems. However, excess production
of free radicals, their production in inappropriate relative
amounts or deficiencies in endogenous defences can have deleterious
effects. Free radicals can cause oxidative damage to lipids, DNA,
bio molecules, rises in the concentration of intracellular calcium,
as well as activation of proteases, nucleases and protein kinases.
Considerable evidence supports the view that oxidative damage
involving free radicals occurs in most, if not all, human diseases.
Oxidative stress is now recognized as an important contributor to
the development of many human diseases including liver fibrosis,
ischemia-reperfusion, atherosclerosis, neurodegenerative disease
and age-related cancer as well as to process of ageing. Thus
antioxidants and systems that can protect against oxidative stress
are needed to maintain health. A large body of scientific evidence
supports that oxidative stress is directly responsible for aging
(Aging Cell. 2009, 8(3):258-69) and an array of neuropathology
conditions (Nutrition 2010, 26:595-603. Neurochem Res. 2007,
32:757-73). The free radical theory of aging proposes that the
organism is unable to repair all of them and that, with time,
unrepaired damages accumulate and put the organism at risk: in
other words, free radicals provoke aging and death (FEBS Letters
2009, 498: 183-186. J. Neurochem. 2009, 108:1251-65). Antioxidants
are the substances able to react with free radicals and to protect
the body from the damage caused by these molecules (Ital J Biochem.
2006, 55:263-282). In particular, consumption in excess of some
foods which are rich sources of antioxidants is considered to
promote good health and longevity. It is now believed that the
maintenance of redox balance within the body can forestall aging
and promote good health and longevity.
[0006] Due to its 30 carbon double bonds, [60]fullerene
(Buckminsterfullerene, Ceo) is a powerful free radical scavenger
which characterize it as a radical sponge (Science 1991, 254, 1
83-1185). Biological applications of fullerenes and derivatives, in
particular as antioxidants, have been extensively reviewed (Bioorg.
Med. Chem. 1996, 4: 767-779. Eur. J. Med. Chem. 2003, 38: 913-923.
Biomedicine & Pharmacotherapy, 2005, 59: 351-358). Ceo is only
soluble in a limited number of organic solvents, such as toluene,
benzene, chloronaphtalene and dichlorobenzene. Availability of
biocompatible aqueous solutions of Ceo and its derivatives that are
insoluble in water have been major obstacles to toxicity and in
vivo studies of this new family of compounds. Biological properties
of water-insoluble fullerenes are still misunderstood and to our
knowledge there are no certified toxicology data about them. Most
of the fullerenes studied until now are water-soluble derivatives,
since study of water-insoluble fullerenes, such as pristine Ceo, in
biological medium proves difficult. It is a common practice to
derivatize the fullerene core with substituants such as OH, COOH,
NH2 to increase hydrophilicity (Bioorg. Med. Chem. 1996, 4:
767-779. Eur. J. Med. Chem. 2003, 38: 913-923. Biomedicine &
Pharmacotherapy, 2005, 59: 351-358). Water-soluble Ceo derivatives
have been found to retain in vitro the free radical scavenger
properties of their parent fullerene molecule, allowing these
properties to be exploited in biological systems. Many patents
already exist for a broad range of biomedical applications and
other commercial applications of water-soluble fullerenes,
including anticancer and anti-HIV therapies, drugs for
neurodegenerative diseases, drug delivery systems, and preparations
that retard aging. In particular, a group of hydrophilic Ceo
derivatives, carboxyfullerenes, were proposed to increase
metazoan's lifespan (U.S. Patent Application 2003/0162837).
However, water-soluble fullerenes are difficult to synthesize and
to purify. Besides, in contrast to pristine Ceo, which is
non-toxic, some C6o-derivatives can be highly toxic (Adv Exp Med
Biol, 2007, 620, 168-80).
[0007] Pristine C6o has been shown to be more effective as an
antioxidant than certain carboxyfullerenes in Wang, I. et al., J.
Med. Chem. 1999, 42, 4614-4620. However, Ceo has not been employed
as an active ingredient to develop an in vivo treating method in
this publication. Aqueous suspensions of Ceo are well known in the
art. They are stable for long periods and can be delivered to
cells. A study of 14C-labeled C-60 reported that it is possible to
form suspensions of C6o in water that are stable for long periods
(J. Am. Chem. Soc. 1994, 116, 4517-4518). However, the authors
failed to detect the fullerene inside the cells and these
suspensions containing very low concentrations of fullerene
(typically 0.1 mg per ml) were inadequate to perform in vivo
studies, especially toxicity studies. and metabolic fate
investigations ((J. Am. Chem. Soc. 1994, 116, 4517-4518). Other
vectorisation methods include the formation of inclusion complexes
with cyclodextrins, calixarenes, tween-20, micelles, liposomes, and
vesicles; however the Ceo concentrations reached by such methods
are still very low (1 mg/mL at most) and inadequate to perform in
vivo toxicity studies. Further, these methods present another
drawback because they generally necessitate a preliminary
dissolution step of the fullerene in an organic solvent. Other
studies proposed the use of Ceo nanoparticles suspended in aqueous
media to form a colloidal solution so-called nC60, however such
solutions prpved to be highly toxic because they contain impurities
linked to the oxidation byproducts of the solvents used during
their preparation (Adv Exp Med Biol, 2007, 620, 168-80. Journal of
Nanoscience Letters 2011, 1: 62-63). Another method, disclosed in
J. Med. Chem. 2000, 43, 3186-3188 uses polyvinyl-pyrrolidone to
solubilize C6o; however this vehicle can react with fullerene and
the formed complex may cause harmful effects on mice embryos.
[0008] Moussa et al. described in Fullerene Science &
Technology 1995, 3, 333-342 that partially micronized Ceo particles
can be incorporated into living human phagocyte cells. C6o was
directly suspended in the culture media and did not exhibit acute
toxicity. Moussa et al. also described in Fullerene Science &
Technology 1996, 4, 21-29 that micronized particles of
water-insoluble fullerenes may be administered to mice on the form
of a biocompatible aqueous suspension comprising a surfactant
(tween 80) and a suspending agent (carboxym ethyl cellulose) which
stabilizes the suspension. The authors disclosed that C6o is
non-toxic, can cross cellular membranes and accumulates in liver
and spleen (Fullerene Science & Technology 1996, 4:21-29). The
same group headed by F. Moussa have already used Micronized Ceo
suspensions as free radical scavenger in vivo (Nano Letters 2005,
5: 2578-2585). However, the effective doses were very high (i.e.
>1 g/kg of body-weight) and intra peritoneal (i. p.)
administration was the only route of administration for such
suspensions. The authors also disclosed that Ceo can solubilize in
vivo inside lipid droplets (Nano Letters 2005, 5: 2578-2585). This
result has been confirmed in vitro by other authors whom studied
O.beta.o solubility in vegetable oils (Fullerenes, Nanotubes, and
Carbon Nanostructures, 2007, 15: 311-314. Fullerenes, Nanotubes,
and Carbon Nanostructures, 2007, 15: 331-339). Stable biocompatible
compositions comprising water insoluble fullerenes dispersed and/or
dissolved in a carrier selected from the group consisting of fats
and oils in an amount ranging from 0.2 to 10% by weight relative to
the total weight of the composition, preferably from 0.1 to 2% by
weight, were already proposed by N Gharbi and F Moussa for
preventing damages caused by free radicals (2005/International
Application No, PCT/EP2005/004963). However, in such compositions
the water-insoluble fullerene is not fully dissolved and their oral
absorption was unknown. Further, large aggregates of the
administered fullerene can be filtered by liver and spleen and
confined in their reticulo-endothelial system (RES) thus altering
the diffusion and biodistribution of Ceo in the whole body. Thus,
the in vivo use of water-insoluble fullerenes as free radical
scavengers through delivery thanks to a non-aqueous carrier is
still not satisfactory.
[0009] The inventors of the instant invention have now discovered a
surprising use of [60]fullerene as agent that promotes an increases
in the overall length of the expected lifespan of mammals.
[0010] Compositions comprising [60]fullerene and their use for
preventing damages caused to metazons by free radicals are
disclosed in TN Patent No.TN 2011/327 issued Jun. 30, 2011 to
Moussa et al. which is incorporated herein by reference in its
entirety.
SUMMARY OF THE INVENTION
[0011] It is in view of the above problems that the present
invention was developed. The main objective of the invention is to
provide a process or method for extending the longevity of a
mammal, which comprises a step of administering to said mammal a
composition comprising an effective amount of [60]fullerene, which
avoids the drawbacks of the prior art processes, and in particular:
1--avoids the use of charge transfer complexes, 2--avoids the use
of organic solvents, and 3--avoids in situ aggregation of the
administered fullerene. It has now been discovered by the inventors
that the compositions comprising [.alpha.]fullerene dissolved in a
suitable carrier selected from the group consisting of oils and
fats proved suitable to achieve the aforementioned objectives. In
particular, not only they allow [60]fullerene to be administered
orally or intramuscularly or intra peritoneally to prolong the
longevity of mammals but they are at least several times more
active than previous compositions. Thus, a first embodiment of the
instant invention comprises a stable biocompatible composition
comprising (a) a carrier selected from the group consisting of fats
and oils; and (b) [60]fullerene, wherein [60]fullerene is almost
dissolved in said carrier. The embodiment is further drawn to
compositions, in which [60]fullerene is dissolved in the carrier.
Another embodiment of the instant invention is a method to prolong
the longevity of mammals, which comprises a step of administering
to said mammal a stable biocompatible composition comprising an
effective amount of [60]fullerene dissolved in a carrier selected
from the group consisting of fats and oils. In a preferred
embodiment, the invention is drawn to a method of prolonging the
longevity of mammals, which comprises a step of adding to food or
any nutritional composition a stable composition comprising an
effective amount of [60]fullerene dispersed in a carrier selected
from the group consisting of fats and oils.
[0012] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating specific
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] The foregoing and other objects, features and advantages of
the present invention will become readily apparent to those skilled
in the art from a reading of the detailed description hereafter
when considered in conjunction with the accompanying drawings
wherein:
[0014] FIG. 1 is a representation of the growth rate of rats as a
function of time, which were treated or not with a composition
according to the present invention.
[0015] FIG. 2 shows whole blood Ceo concentrations-time plot (mean
.+-.S.E . . . ) following single dose oral administration (4 mg/kg,
n=3) or single dose intra-peritoneal (ip) bolus injection of the
same dose (n=3) of Ceo dissolved in olive oil (0.8 mg/ml)
(n=3),
[0016] FIGS. 3 and 4 show the results of some biochemical tests for
Ceo pre-treated and control rats before CCl4 administration,
and
[0017] FIG. 5 represents the survival percentage of rats (n=6 per
group) orally treated with Ceo at the age of 10 months (1 ml/kg of
body-weight, weekly until the end of the second month then every
two weeks until the end of the 7th month, with water, olive oil or
Ceo dissolved in olive oil (0.8 mg/ml)). Table 1 summarizes the
mean pharmacokinetic parameters obtained in rats after oral (n=3)
or intra-peritoneal (n=3) administration of Ceo dissolved in olive
oil and table 2 summarizes Ceo concentrations in whole blood (WB),
liver, spleen and brain of rats daily treated with a single dose of
Ceo dissolved in olive oil (4 mg/kg body weight) by oral (n=3) or
ip route (n=3).
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
[0018] Free radicals are constantly formed in vivo. Some of these
molecules are physiologically useful, but they can also result in
pathological oxidative stress to cells and tissues. Endogenous
defences include both antioxidants and repairing systems. However,
excess production of free radicals, their production in
inappropriate relative amounts or deficiencies in endogenous
defences can have deleterious effects. Free radicals can cause
oxidative damage to lipids, DNA, bio molecules, rises in the
concentration of intracellular calcium, as well as activation of
proteases, nucleases and protein kinases. Considerable evidence
supports the view that oxidative damage involving free radicals
occurs in most, if not all, human diseases. Oxidative stress is now
recognized as an important contributor to the development of many
human diseases including liver fibrosis, ischemia-reperfusion,
atherosclerosis, several neurological disorders and age-related
cancer as well as to process of ageing. Thus antioxidants and
systems that can protect against oxidative stress are needed to
maintain health and prolong the expected lifespan in metazoans.
This has led to attempts to develop additional antioxidants to
supplement the antioxidant defences of cells as potential
therapeutic agents. Diet-derived antioxidants and a number of small
molecules that can scavenge free radicals as well as super oxide
dismutase-mimetics and chelators of transition metal ions were
proposed as potential therapeutic agents against oxidative stress.
Compositions according to the invention comprising [60]fullerene
have been found to exhibit highly efficient antioxidant properties
in vivo. The fullerene core, i.e. the fullerene skeleton without
lateral substituants, used in the practice of this invention
comprise clustered carbon structures generally spherical in shape
and having a carbon content of 60 carbon atoms. Typically,
[60]fullerene according to the invention is present in an amount
ranging from 0.01 to 0.0.08% by weight relative to the total weight
of the composition, preferably 0.08% by weight. [60]fullerene is
preferably dissolved in the carrier, i.e. the composition can be
filtered through a 0.2 pm filter. The stable, biocompatible
compositions according to the invention comprise a carrier selected
from the group consisting of fats and oils; and [60]fullerene,
wherein said fullerene is mostly dissolved in said carrier. The
carrier used in the present invention is a pharmaceutically
acceptable and biocompatible carrier, selected from the group
consisting of fats and oils. The fat or oil may be any natural or
synthetic fat or oil suitable for administration to a mammal. They
are not particularly restricted inasmuch as they are components
which can be used in pharmaceutical preparations or in foods. Oils
and fats can be hydrogenated or partially hydrogenated. They are
used at a solid, a semisolid, or a liquid state. Vegetable and
animal fats and oils are preferred, vegetable fats and oils are
most preferred. Oils and fats include, without limitation fatty
acid esters, fatty acids, fatty alcohols and fatty alcohol esters.
Synthetic lipids can also be used. Fatty acids, as defined herein,
are intended to mean aliphatic monocarboxylic acids having a chain
of 4 to 40 carbon atoms, which may be branched or unbranched,
saturated or unsaturated, cyclic or acyclic. Fatty acids may be
natural or synthetic, polyunsaturated, mono-unsaturated or
saturated. Natural fatty acids, which are usually unbranched and
C4-C28 even-numbered, are preferred. Examples of fatty acids
include, but are not limited to, linoleic acid, arachidonic acid,
linolenic acid, gamma-linolenic acid, caprylic acid, stearic acid,
myristic acid, a palmitic acid, behenic acid, undecylenic acid,
oleic acid, an decosahexaenoic acid (DHA), eicosapentaenoic acid
(EPA), isostearic acid, 12-hydroxy-stearic acid. Salts thereof
[e.g. alkali metal salts (sodium salts, potassium salts, etc.),
alkaline earth metal salts (calcium, magnesium salts etc.)] can
also be employed. Fatty acid esters are preferably esters of fatty
acid as defined hereinabove with C-1-C40 aliphatic or aromatic
alcohols, preferably aliphatic, saturated or unsaturated,
straight-chain or branched-chain, cyclic or acyclic. Alcohols can
be polyols, having preferably up to five hydroxyl groups. Examples
of fatty acid esters include, but are not limited to, triglycerides
i.e. tri-esters of glycerol with fatty acids cited above, sterids
i.e. esters of sterols with fatty acids cited above, the group
consisting of the lower alkyl esters thereof (preferably methyl,
propyl, butyl, isopropyl and hexyl), 1,2- or 1,3-diglycerides, 1-
or 2-monoglycerides, polyglycolysed glycerides such as sucrose
fatty acid esters, polyglyceryl fatty acid esters, propylene glycol
fatty acid esters. Specific examples of fatty acid esters are
octyldodecyl behenate; isocetyl behenate; isocetyl lactate;
isostearyl lactate; linoleyl lactate; oleyl lactate; isostearyl
octanoate, isocetyl octanoate, decyl oleate, isocetyl isotearate,
isocetyl laurate; isocetyl stearate; isodecyl octanoate; isodecyl
oleate; isononyl isononanoate; isostearyl palmitate; myristyl
isostearate; octyl isononanoate; 2-ethylhexyl isononanoate; octyl
isostearate; octyldodecyl erucate; isopropyl palmitates,
2-ethylhexyl palmitate, 2-octyldecyl palmitate, branched alkyl
myristates such as isopropyl myristate, t-butyl myristate,
2-octyldodecyl myristate, hexyl isostearate, butyl isostearate,
isobutyl stearate, hexyl laurate, 2-hexyldecyl laurate, propylene
glycol monostearate and distearate. Examples of giycerides (fatty
acid esters) include, without limitation, triolein, trilinolein,
tripalmitin, tristearin, trimyristin, and triarachidonin. Examples
of sterids (fatty acid esters) include, without limitation,
cholesteryl oleate, cholesteryl linoleate, cholesteryl myristate,
cholesteryl palmitate, cholesteryl arachidate. Examples of fatty
alcohols include, without limitation, cetyl alcohol, stearyl
alcohol, lauryl alcohol, myristyl alcohol, palmityl alcohol,
behenyl alcohol, hexadecyl alcohol, oleic alcohol, isostearyl
alcohol, cetostearyl alcohol. They can be used as esters with
C4-C40 dicarboxylic, tricarboxylic or tetracarboxylic acids. Oils
may be natural oils such as vegetable oils and animal oils
(composed predominantly of triglycerides), or mineral oils such as
silicon oils, fluorinated oils. Liquid paraffin can also be used.
Examples of natural oil include, but are not limited to, oils from
plant sources, such as corn oil, wheat germ oil, soybean oil, rice
bran oil, rapeseed oil, canola oil, sesame oil, palm (kernel) oil,
olive oil, camellia oil, peanut oil, coconut oil, sunflower oil,
peanut oil, orange oil, evening primrose oil, borage oil,
blackcurrant seed oil, cottonseed oil, beaver oil, pineapple oil,
safflower oil, copra oil, oils found in coffee, and animal oils
such as turtle oil, fish oil, cod-liver oil. Fats may be mineral
fats or natural fats such as vegetable fats and animal fats.
Petrolatum, paraffin can also be used. Examples of natural fat
include, but are not limited to, butter, cocoa butter, theobroma,
peanut butter, lard, beef fat, chicken fat, horse fat, lanolin and
lanolin derivatives. Oils and fats can be polyunsaturated such as
corn, soybean, safflower oils, or saturated, such as palm, coconut
oils and butter, or mono-unsaturated, such as olive oil and canola
oil. Other suitable carriers according to the invention are
diisopropyl sebacate; diisopropyl adipate; diisostearyl adipate;
octyldodecyl stearoyl stearate; pentaerythrityl tetra-isononanoate;
pentaerythrityl tetraisostearate; triisopropyl citrate;
triisostearyl citrate; and trioctyldodecyl citrate. Preferred
carriers according to the invention are butter, cocoa butter,
peanut butter, olive oil, soybean oil, cod-liver oil and liquid
paraffin. As defined above, carriers may be used each alone or in a
combination of two or more species. [60]fullerene is dissolved in
the carrier, depending on the nature of the carrier. Some carriers
are able to dissolve substantial amounts of water-insoluble
fullerenes (several mg/g of carrier). In one embodiment, at least
0.8 mg of fullerene is dissolved per ml of the carrier (the carrier
being a liquid). As an example, it is possible to dissolve a total
weight up to 1 mg of C60 per g of olive or soybean oil in less than
one week. The compositions according to the invention may be
pharmaceutical compositions comprising the fullerene in a
therapeutically effective amount. Preferably, said fullerene can
protect against biologically reactive radical species, which means
chemicals that are free radicals or contribute to the generation of
free radicals. Generally, the biologically reactive radical species
are generated from O2 or H2O2. Thus, the invention also concerns a
method to prolong the longevity of a mammal, which comprises a step
of administering to said mammal a stable biocompatible composition
as defined hereinabove. [60]fullerene, when dissolved in the
carriers of the present invention, can be administered to mammals
and this compound is well absorbed by said mammals. Generally, the
said fullerene is administered in an amount of at least 0.1 mg/kg
of body weight per day. According to the method of the invention,
the inventive compositions may be administered orally,
intramuscularly, subcutaneously, intra dermally or intra
peritoneally, rectally by suppositories or sublingually. For oral
ingestion by a mammal to be treated, the carrier is preferably an
edible carrier. In at least one embodiment, said composition is
administered in a pure form. In another embodiment, it is
administered in the form of an emulsion in water. In another
embodiment the fullerene is administered as a labile C6o-derivative
derivative that can deliver Ceo after administration. The
compositions of the instant invention can be in any liquid or solid
conventional pharmaceutical formulation. The carrier enables the
fullerene to be formulated as tablets, pills, dragees, capsules,
liposome, pomade, ointment, cream , lotion, emulsions, gels,
syrups, slurries and the like. The compositions of the present
invention are preferably presented for oral administration to
mammals in unit dosage forms, such as tablets, capsules, and oral
solutions, containing suitable quantities of [60]fullerene. The
compositions may be sterilized and/or may contain some adjuvants
such as preservatives, stabilizers, acidity regulators, natural or
synthetic flavour, anti-foaming agents, viscosity-control agents,
emulsifiers, salts for varying the osmotic pressure and/or other
buffers. In addition, compositions may contain other
pharmaceutically active agents. The level of free radicals and
reactive oxygen species in mammal cells decreases following
treatment as compared to the level of reactive oxygen species in a
cell that has not been contacted with a composition according to
the invention. Indeed, [60]fullerene according to the invention can
act as antioxidant and supplement the antioxidant defences of
cells. That means they inhibit oxidation or inhibit reactions
promoted by reactive oxygen species. Physiologically relevant
reactive oxygen species, which contribute to the generation of free
radicals, include hydrogen peroxide, super oxide anion, and the
like. The protective method of the invention reduces cell damage
and death, and thus generally maintains the health of treated
mammals. Further, the inventors discovered that [60]fullerene
administered as biocompatible composition as described herein--1)
can be absorbed after oral administration (FIG. 2);--2) they can
react inside the liver with vitamin A (retinol) and esters thereof
following a Diels-Alder-like reaction without any toxic effect (New
J Chem, 1998, 989-992);--3) they are eliminated through the bile
ducts (Nano Letters 2005, 5 (12): 2578-2585); and--4) despite the
large amounts administered, no acute or sub-acute toxicity could be
observed in mice and rats. No behaviour or growth disorder could be
observed in treated animals either, which can be seen on FIG. 1.
The latter shows growth rate of three groups (n=6) of rats which
received weekly per os 1 ml of olive oil containing 0.8 mg of Ceo
or 1 ml of olive oil only or 1 ml of water only. The in vivo new
properties of [60]fullerene are due to fullerenes themselves and/or
to the fullerene-retinol and fullerene-retinyl ester adducts formed
after administration inside the liver. Also disclosed herein is a
method for preparing a composition according to the present
invention, comprising a carrier and particles of [60]fullerene.
Said method comprises the steps of:--(a) Charging a milling vessel
with the fullerene, the carrier and balls, said milling vessel and
balls being made out of any biocompatible metal or polymer;--(b)
Agitating the mixture resulting from step (a) until a homogeneous
dissolution is obtained; and--(c) Sterilizing the composition
resulting from step (b) by filtration. Direct mechanical milling in
the carrier presents the advantages to accelerate the dissolution.
Said method comprises the steps of:--(a) Charging a milling vessel
with the fullerene, the fat or oil and balls, said milling vessel
and balls being made out of any biocompatible metal or
polymer;--(b) Agitating the mixture resulting from step (a) until
complete homogenization of the solution;--(c) Agitating the
composition resulting from step (b) until complete dissolution of
the fullerene; and--(d) Sterilizing by filtration the composition
resulting from step (c). Other than in the operating examples, or
where otherwise indicated, all numbers expressing quantities of
ingredients, reaction conditions, and so forth used in the
specification and claims are to be understood as being modified in
all instances by the term "about." Accordingly, unless indicated to
the contrary, the numerical parameters set forth in the following
specification and attached claims are approximations that may vary
depending upon the desired properties sought to be obtained by the
present disclosure. At the very least, and not as an attempt to
limit the application of the doctrine of equivalents to the scope
of the claims, each numerical parameter should be construed in
light of the number of significant digits and ordinary rounding
approaches. Notwithstanding that the numerical ranges and
parameters setting forth the broad scope of the disclosure are
approximations, the numerical values set forth in the specific
examples are reported as precisely as possible. Any numerical
value, however, inherently contain certain errors necessarily
resulting from the standard deviation found in their respective
testing measurements. The invention is further illustrated by the
examples described below. These examples are meant to illustrate
the i nvention and are not to be interpreted as limiting the scope
of the invention.
EXAMPLES
General Considerations
[0019] C60 (Purity: 99.98%) was purchased from Term USA (Fort
Bragg, Calif., USA). Its characterization and its purity were
assessed by HPLC, UV, C-NMR, and Mass Spectrometry. No impurity
could be observed. It was used without further purification as well
as after sublimation. All the other reagents were analytical grade
and were purchased from Sigma (St Louis, Mo.). Animals received
human care and the study protocols complied with general guidelines
for the care and use of laboratory animals. Male Wistar rats
(Charles River, France) were housed by groups of 3 in polypropylene
cages at constant temperature (22.degree. C.) and humidity (60%)
and with a 12 h light/dark cycle, and fed a standard diet ad
libitum. All rats were allowed to acclimate to this facility for at
least one week before being used in the experiments. At the end of
the experiment, body weights were determined and the animals were
sacrificed under the same conditions by bleeding through the
thoracic aorta after sodium pentobarbital (1.0 mL/kg of body
weight) anaesthesia.
[0020] Biochemical tests, Cso determinations and statistics were
processed as previously described in Nano Letters 2005, 5 (12):
2578-2585.
Example 1
Direct Dissolution of [60]Fullerene in a Vegetable Oil
[0021] In the stainless steel milling vessels of a Pulverisette 7
(Fritsch, Idar-Oberstein, Germany) or a similar device, add 8 mg of
[60]fullerene and 10 mL of olive oil or 10 g of butter and 6
stainless steel balls (8 mm of diameter) (the milling vessels and
the balls can be made out of any biocompatible metal or polymer
such as stainless steel, tempered chrome steel, silicon nitride,
corundum, tungsten carbide, agate, oxide of zirconium etc). Agitate
the mixture during several hours (at 600 rpm for instance) until
complete dissolution. The resulting homogenous solution or paste is
then ready for use for oral administration or by any route of
administration after appropriate sterilization. Sterilization may
be achieved by filtration under vacuum (pore size: 0.2 .mu..eta.L).
The sterilized composition is stable for at least 1 month. It is
also possible to dissolve water-insoluble fullerenes in natural or
mineral oils without stirring however the dissolution may be time
consuming (up to several days at room temperature). Therefore, the
former protocol is preferred. The fullerene concentration in
compositions according to the invention can be determined by HPLC
after adequate dilution in mobile phase as described previously (J.
Chromatogr. B 1997, 696: 153-159).
Example 2
Pharmacokinetics and Biodistribution of an Oily Solution after Oral
and Intra-Peritoneal Administration in Rats
[0022] Pharmacokinetic studies were carried out with male Wistar
rats (weighing 200-220 g). Rats were housed in individual cages and
maintained in an air-conditioned room (22-25.degree. C.) on a 12 h
light/dark cycle with water and food available. The rats were
acclimated for 7 days and they were fasted overnight but with
access to water, before treatment.
[0023] Under general anesthesia, a catheter was introduced into the
rat right jugular vein, positioned subcutaneously with the tip in
the inter-scapular region. The prepared rats were then allowed to
recover for 24 h, and the blood catheters were flushed with 0.9%
NaCl solution containing 20 I U/ml of heparin to avoid possible
clot obstruction. Before Ceo administration, the rats were fasted
overnight but with access to water. The same single dose of Ceo (4
mg/kg) was delivered orally, by a gavages needle, or
intra-peritoneally to two groups of three rats. Blood (0.20 ml) was
withdrawn via the canular prior to dosing (f=0) and at 15, 30, 60
min and then at 2, 4, 8, 10, 12, 24 and 48 h post-dosing.
Antithrombin heparin (20 IU/m1) was added in each blood sample.
After each blood collection 0.20 ml of sterile 0.9% NaCl solution
were injected to the animal, to avoid hypovolemia. The rats were
sacrificed 48 h after eo administration for organ collection
(livers, spleens, and brains).
[0024] It was now discovered by the inventors that:--1) Soluble Ceo
is absorbed and eliminated after either intraperitoneal or oral
administrations. Table 1 represents the main pharmacokinetic
parameters; 2--the maximal concentrations (Cmax) are reached 4 and
8 hours after i.p. and oral administrations, respectively (FIG. 2);
3--the maximal concentration after i.p. administration
(1.47.+-.0.15 pg/ml) is higher than that after oral administration
(0.52.+-.0.16 Mg/ml). Consequently, the area under the curve (AUC)
is about 5 times larger when Ceo is administered by i.p. route as
compared to oral route. Although these results do not allow
determining the bioavailability of Ceo, they clearly show that a
non-negligible % of the orally administered dose is absorbed as
compared to the i.p. administered one (FIGS. 2); and--3) the
fullerene is well distributed in the whole body, in particular it
can cross the brain barrier (table 2).
Example 3
Ceo-Induced Protection of the Liver against Acute Toxicity of
Carbon Tetrachloride (CCU) in Rats
[0025] Carbon tetrachloride is a classical hepatotoxicant that
causes rapid liver damage progressing from steatosis to
centrilobular necrosis. CCU intoxication in rodents is an important
model for elucidation of the mechanism of action of hepatotoxic
effects such as fatty degeneration, fibrosis, hepatocellular death,
and carcinogenicity. These effects are consistent with the known
induced metabolic activation of CCl4 to reactive intermediates,
including CCl3'' and CCl02' free radicals, and mobilization of
intracellular calcium. Kupffer cells (liver resident macrophages)
participate in the mechanism of toxicity of CCl4 in vivo by release
of chemoattractants for neutrophils and a series of chemical
mediators (cytokines). Both expression and synthesis of these
cytokines are mainly modulated through redox-sensitive reactions.
Further, involvement of reactive oxygen species and lipid
peroxydation products can be demonstrated in other fundamental
events of hepatic fibrogenosis, like activation of hepatic stellate
cells (HSC: liver resident nonparenchymal cells also referred to as
fat-storing or perisinusoidal cells, lipocytes and Ito cells). In a
previous work, the effects of C60-pretreatments on acute carbon
tetrachloride intoxication in rats, a classical model for studying
free-radical-mediated liver injury was reported. The results
obtained by the authors leaded by F Moussa (Nano Letters 2005, 5
(12), 2578-2585) showed that aqueous C6o suspensions not only have
no acute or subacute toxicity in rodents but they also protect
their livers in a dose-dependent manner against free-radical
damage. The most effective dose of C6o reported in the latter paper
was about 2.5 g/kg of body-weight and was administered
intra-peritoneally and the better protection was obtained at day 14
after administration. It was now discovered by the inventors that
the fullerene is about 100 times more active it is administered in
solution than in suspension, and the effect is more rapid (24 hours
after administration) as compared to the results published
previously in the same experimental model (Nano Letters 2005, 5:
2578-2585).
Example 4
Ceo Prolongs the Longevity in Rats without Chronic Toxicity
[0026] The rats were housed one per cage and acclimated for 14
days, before dosing. Three groups of 10 rats (10 months old,
weighing 495.+-.31 g) were administered daily for one week, then
weekly until the end of the second month and then every two weeks
until the end of the 7th month, by gavages with 1 ml of water or
olive oil or Ceo dissolved in olive oil (0.8 mg/ml), respectively.
The rats were weighed before each dosing. Routine observations
following official recommendations (EC Commission Directive
2004/73/EC of 29 Apr. 2004 Adapting to Technical Progress for the
Twenty-Ninth Time Council Directive 67/548/EEC on the Approximation
of the Laws, Regulations and Administrative Provisions Relating to
the Classification, Packaging and Labeling of Dangerous Substances.
O.J. No. L1522004) were made on all animals inside and outside the
cage once a day throughout the study for signs of departure from
normal activity, morbidity and mortality.
[0027] This experiment was initiated after observing that Ceo is
absorbed through oral administration. To study the chronic toxicity
of Ceo we designed a protocol according to the general guidelines
of US food and drug administration (Chronic Toxicity Studies with
Rodents in Toxicological Principles for the Safety Assessment of
Food Ingredients. Redbook 2000, revised July 2007, Chapter
IV.C.5.a. IV.C.5.a.) with some modifications. It was now discovered
by the inventors that oral administration of Ceo increases
significantly the longevity of rats (FIG. 5). At 25 months after
the beginning of the treatment, the % of survival is equal to 25%,
67% and 100% for the rats treated with water, olive oil or
C6o-dissolved in olive oil, respectively (FIG. 5). At 37 months,
after the beginning of the treatment, this percentage is always
equal to 100% for the rats treated with C6o-dissolved in olive oil,
17% for the rats treated with olive oil, and 0% for the rats
treated with water (FIG. 5). The increase of the expected lifespan
reported herein has never been reported for any other substance, to
our knowledge.
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