Note: Descriptions are shown in the official language in which they were submitted.
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MODULATION OF VITAMIN STORAGE
Field of the invention
The invention relates to modulating vitamin levels in animals, particularly
levels of
endogenous storage forms of tocopherol (Vitamin E), retinol (Vitamin A) or
Vitamin
K1 by administration of phosphate derivatives.
Background of the invention
In this specification, where a document, act or item of knowledge is
referred~to or
discussed, this reference or discussion is not an admission that the document,
act
or item of knowledge or any combination thereof was at the priority date:
(a) part of common general knowledge; or
(b) known to be relevant to an attempt to solve any problem with which
this specification is concerned.
Whilst the following discussion concerns tocopherol and tocopheryl phosphate
(TP), it is ~o be understood that this is merely illustrative and that the
invention is
not limited to tocopherol or TP but that the invention also similarly relates
to retinol
and vitamin K1 and their storage and transport forms.
Vitamin E is widely recognised as an anti-pxidant of considerable biological
importance. It is a potent free radical scavenger with a vital role in the
maintenance of cellular integrity through its capacity to protect the
polyunsaturated
2o fatty acyl moieties of phospholipids in biological membranes and plasma
lipoproteins. Vitamin E consists of the group of isoprenoids know as
'tocopherols'
which provide benefits to the health and well-being of humans and animals.
Several different tocopherols having Vitamin E activity are known, the most
active
and abundant being a-tocopherol.
2s There have been many attempts to supplement the body's dietary intake of
vitamin
E. In order for supplements of biologically active species and nutrients to be
valuable in clinical practice, they should exhibit certain properties. For
example,
the supplements should have adequate stability, solubility, perrrieability and
bioavailability.
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2
Free tocopherol is not stable and therefore not suitable as a bioavailable
delivery
form of Vitamin E. For this reason, derivatisation has long been recognised as
an
important means of increasing stability and bioavailability of tocopherol, but
it is
generally accepted that absorption of these derivatives has still been low.
Tocopherol organic esters, such as tocopheryl acetate or tocopheryl succinate
are
examples of such derivatised species which are currently in widespread
commercial use. These esters are cheap to produce and more stable than free
tocopherol.
In order to be useful, tocopherol derivatives should be capable of converting
to the
1o biologically active species after they have been absorbed into the systemic
circulation of a human or animal subject. Formulators believed that the free
form
of tocopherol was biologically active and thus believed that when the
pancreatic
esterase and lipase activity released free tocopherol from the organic esters
of
tocopherol in the small intestine lumen, the body's levels of Vitamin E were
increased.
When investigating the effectiveness of various tocopherol derivatives as
sources .
of tocopherol as dietary supplements, analytical techniques of the prior art
have
concentrated on measuring how efficiently these supplements increase levels of
free tocopherol in plasma and tissue. Tocopherol acetate has been favoured as
a
2o Vitamin E dietary supplement of the prior art because it reportedly causes
an
immediate, readily measurable increase in plasma free tocopherol levels.
Formulators who have been attempting to increase the water solubility of
tocopherol derivatives have prepared formulations containing TP as it is much
more water soluble than organic esters of tocopherol. While TP has been used
as
a water soluble source of Vitamin E in vitro, it has not previously been
commonly .
used as a suitable in vivo source of Vitamin E because the phosphate group is
a
substrate of phosphorylases, is resistant to passive transport, and therefore
considered to interfere with absorption.
To date, Vitamin E supplements containing either tocopheryl acetate or
tocopheryl
ao phosphate have aimed to provide a recommended daily intake of 7 - 15 mg/day
per person of free tocopherol in the body. Supplements have contained 200 -
600
mg as this amount is considered to be innocuous in adult humans, although
there
is limited evidence of this.
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Paradoxically, as well as being a potent free radical scavenger a-tocopherol
is
also a pro-oxidant and a potential source of free radicals which would cause
damage to the body. Free tocopherol is.pro-oxidant at high concentrations so
it is
unlikely hat this is the biological storage or transport form within the body.
Being
fundamentally important to membrane stability, the natural form must be
transported efficiently and mobilized on demand to act as an electron transfer
agent. To date only free and protein-bound or associated a-tocopherol have
been
detected in biological tissue. The protein-bound and associated forms are
thought
to represent transport forms. Although it would appear to be a vital piece of
information, no one has pursued the concept that although free tocopherol is
the
biologically active form there must be another form which is stored or
transported
through the body. All attempts at supplementation of Vitamin E have focussed
on
increasing the levels of free tocopherol within tissue or plasma. There have
been
no attempts to increase the storage or transport form of Vitamin E.
15 These negative effects of high levels of free tocopherol limit the daily
recommended dose. There is also ~ concern that a high intake of fat soluble
nutrients can lead to toxicity. As a result, the recommended daily intake has
limited the amount of tocopherol provided by supplements.
Summary of the Invention
2o It has now been found by the presenf inventors that the storage form of
tocopherol, retinol and vitamin K1 is their respective phosphate derivatives.
These
phosphate derivatives are not typically a source of free radicals.
Surprisingly, the
present inventors have found that administering phosphate derivatives of
tocopherol, retinol or vitamin K1 to an animal can provide therapeutically
useful
25 levels of tocopherol, retinol or vitamin K1 in storage and transport forms.
Accordingly, in a first aspect, the present invention provides a method for
increasing levels of a storage form of a vitamin selected from the group
consisting
of tocopherol, retinol, vitamin K1 and mixtures thereof in a target tissue of
a
subject, the method comprising administering to the subject an effective
amount of
so a phosphate derivative of the vitamin so as to cause an accumulation of
stored
vitamin in the target tissue.
Preferably, the vitamin is tocopherol.
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Preferably, the subject is an animal, more preferably the animal is a human.
Preferably, the target tissue is liver because it is the storage organ of
preference
and the generation site of lipoprotein cholesterol. It is also known that
there is
Vitamin E activity in the skin and TP is important in skin metabolism. Vitamin
E
may also be important for adipose tissue where it may protect against
degradation
of unsaturated fats. Vitamin E may also be important in the brain.
Preferably, the increased levels of the stored vitamins) result in potentially
therapeutic levels of the vitamins) in the animal.
The present invention is particularly suitable for increasing the storage
levels of
the vitamins to be of therapeutic value. Increased levels of vitamins,
particularly
tocopherol, can have uses in the treatment of inflammatory diseases such as,
but
not limited to, coronary diseases, atherosclerosis and diabetes and a number
of
diseases affected by tocopherol, such as cancer where tocopherol affects cell
adhesion, and foetal development. In the brain, vitamin levels may assist
treatment of alzheimers.
Therapeutic levels of the vitamins would typically be in the order of at least
about
50% increase of the levels usually stored in healthy or normal tissue.
Preferably,
the levels are increased by at least about 100%, more preferably at least
about
200%, that is, about 2 to 3.times normal plasma or tissue levels. By 'loading
up
2o the body' with the vitamin using the present invention, higher levels of
vitamins can
then be released in a number of ways to provide potentially higher doses of
biologically available vitamin to organs or tissue in the body as required.
The present invention may also be suitable for treating or overcoming problems
with subjects unable to acquire or utilise vitamins by normal dietary uptake
or
processing. The ability to increase storage levels may also be useful to
overcome
consequences of periods of low or poor dietary intake.
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Tocopherol is used in three forms in the body, being
Form Typical levels in normal
of
tocopherol
adult human
(a) 'free' as described by the currenttotal plasma tocopherols
range
analytical methods where it from about 5 to 12 ~,g/ml
is readily
able to be oxidized to the quinone
form
or take part in free radical
reactions;
(b) present as TP in unique places This amount has not been
such as
the boundary between the epidermisquantified in the literature
and dermis where tocopherol
acts in the
initiator of the inflammatory
response
and other cell mediation;
(c) storage of tocopherol in an human adipose free tocopherol
inactive
form. - 259 ~g/gm, TP - Adipose
249 ~,g/gm~ & Liver 322
wg/gm
Typically, the increased storage forms acts as a source of tocopherol, retinol
or
vitamin K1 to tissue, organs or cells of the subject.
The present inventors have found that at least about 3 mg/kg (at least about
240
s mg in an 80 kg adult), typically from about 3 to 50 mg/kg of the phosphate
derivative of the vitamin is required to increase storage levels to a
beneficial
amount. More preferably, 3 to 30 mg/kg is provided. More preferably, at least
10
mg/kg body mass of tocopherol provided orally as tocopheryl phosphate has been
found suitable to achieve a level of 68% absolute bioavailability. This is
significantly more than the recommended daily intake (RDI) of 7-15 mg/day for
a
normal person.
The dosage may be achieved by administering the phosphate derivative of the
vitamins) in one dose or may be administered over a period of minutes, hours
or
days to achieve the required stored amount of the vitamin(s).
The phosphate derivative of the vitamin can be given by any suitable route
such
as orally or parenterally.
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In a second aspect,~the present invention provides a method for alleviating or
treating a subject suffering a condition responsive to a vitamin treatment,
the
method comprising administering to a subject in need of such treatment an
amount of a phosphate derivative of the vitamin selected from the group
consisting
s of tocopherol, retinol, vitamin K1 and mixtures thereof effective to cause
an
accumulation of a therapeutic amount of tocopherol, retinol vitamin K1 or a
mixture
thereof in a tissue of the subject.
Preferably, the conditions expected to be responsive to a vitamin treatment
are
inflammatory diseases such as, but not limited to, coronary diseases!
artherosclerosis and diabetes and a number of diseases affected by tocopherol,
such as cancer where tocopherol affects cell adhesion, and foetal development.
Preferably, the vitamin is tocopherol.
Preferably, the subject is an animal, more preferably the animal is a human.
Preferably, the target tissue is liver as this tissue can be used to indicate
that there
are adequate stores for the various ubiquitous metabolic activities in
different
tissues.
The phosphate derivative of the vitamin can be given by any suitable route
such
as orally or parenterally. .
The present inventors have found that at least about 3 mg/kg (at least about
240
2o mg in an 80 kg adult), typically from about 3 to 30 mg/kg of the phosphate
derivative of the vitamin is required to increase storage levels to a
beneficial
amount which can result in therapeutic levels of the vitamin(s). More
preferably,
about 10 mg/kg body mass of tocopherol provided orally as tocopheryl phosphate
has been found suitable to achieve a level of 68% absolute bioavailability.
This is .
25 significantly more than the recommended daily intake (RDI) of 7-10 mg/day
for a
normal person.
In a third aspect, the present invention provides use of an effective amount
of a
phosphate derivative of a vitamin selected from the group consisting of
tocopherol,
retinol, vitamin K1 and mixtures thereof in the manufacture of a supplement
for
3o causing an accumulation of stored vitamin in the target tissue of an
animal.
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In a fourth aspect, the present invention provides use of an effective amount
of a
phosphate derivative of a vitamin selected from the group consisting of
tocopherol,
retinol, vitamin K1 and mixtures thereof in the manufacture of a medicament
for
alleviating or treating a subject suffering a condition responsive to a
vitamin
treatment.
The term "phosphate derivatives of the vitamin" is used herein to refer to the
acid
forms of phosphorylated tocopherol, retinol or vitamin K1, salts of the
phosphates
including metal salts such as sodium, magnesium, potassium and calcium and any
other derivative where the phosphate proton is replaced by other substituents
such
as ethyl or methyl groups or phosphatidyl groups.
In some situations, it may be necessary to use a phosphate derivative such as
a
phosphatide where additional properties such as increased water solubility are
preferred. Phosphatidyl derivatives are amino alkyl derivatives of organic
phosphates. These derivatives may be prepared from amines having a structure
of R~R2N(CH2)"OH wherein n is an integer between 1 and 6 and R~ and R2 may be
either H or short alkyl chains with 3 or less carbons. R~ and R2 may be the
same
or different. The phosphatidyl derivatives are prepared by displacing the
hydroxyl
proton of the electron transfer agent with a phosphate entity that is then
reacted
with an amine, such as ethanolamine or N,N' dimethylethanolamine, to generate
2o the phosphatidyl derivative of the electron transfer agent. One method of
preparation of the phosphatidyl derivatives uses a basic solvent such as
pyridine
or triethylamine with phosphorous oxychloride to prepare the intermediate
which is
then reacted with the hydroxy group of the amine to produce the corresponding
phosphatidyl derivative, such as P cholyl P tocopheryl dihydrogen phosphate.
In some situations complexes of phosphate derivatives of the vitamins may also
be utilized where additional properties such as improved stability or
deliverability
may be useful. The term "complexes of phosphate derivatives of the vitamin"
refers to the reaction product of one or more phosphate derivatives of
tocopherol,
retinol or vitamin K1 and mixtures thereof with one or more complexing agents
3o selected from the group consisting of amphoteric surfactants, cationic
surfactants,
amino acids having nitrogen functional groups and proteins rich in these amino
acids as disclosed in international patent application no PCT/AU01/01476.
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The preferred complexing agents are selected from the group consisting of
arginine, lysine and tertiary substituted amines, such as those according to
the
following formula:
N R~ R2R3
wherein R~ is chosen from the group comprising straight or branched chain
mixed
alkyl radicals from C6 to C22 and carbonyl derivatives thereof;
R2 and R3 are chosen independently from the group comprising H, CH2COOX,
CH2CHOHCH2S03X, CH2CHOHCH20P03X, CH2CH2COOX, CH2COOX,
CH2CH2CHOHCH2S03X or CH2CH2CHOHCH20P03X and X is H, Na, K or
alkanolamine provided R2 and R3 are not both H; and
wherein when R~ is RCO then R2 may be CH3 and R3 may be
(CH2CH2)N(C2H40H)-H2CHOP03 or R2 and R3 together may be
N(CH2)2N(C2H40H)CH2C00-.
Preferred complexing agents include arginine, lysine or lauryliminodipropionic
acid
15 'where complexation occurs between the alkaline nitrogen center and the
phosphoric acid ester to form a stable complex.
The'administration of phosphate derivatives of the vitamin to provide a
'therapeutic
effect' includes administration to achieve a curative, preventative or other
beneficial health effect. For example, administration to a subject may be
2o undertaken to treat a deficiency-of the vitamin, to ameliorate or cure a
disease or
disorder, to ameliorate or remove the symptoms of the disease or disorder, or
to
increase the subject's plasma and tissue level of the vitamin to provide a
beneficial
effect.
The phosphate derivative of the vitamin may be administered to humans or
25 animals through a variety of dose forms such as supplements, enteral feeds,
parenteral dose forms, suppositories, nasal delivery forms, dermal delivery
including patches, creams, and any other delivery system capable of
supplementing the natural storage and transport form of the electron transfer
agent.
so For example the phosphate derivative of the vitamin may be administered by
an
orally or parenterally administered dose form. These include, tablets,
powders,
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chewable tablets, capsules, oral suspensions, suspensions, emulsions or
fluids,
children"s formulations, enteral feeds, nutraceuticals, and functional foods.
The dose form may further include any additives routinely used in preparation
of
that dose form such as starch or polymeric binders, sweeteners, coloring
agents,
s emulsifiers, coatings and the like. Other suitable additives will be readily
apparent
to those skilled in the art.
In one embodiment, the dose form has an enteric coating as disclosed in
international patent application PCT/AU01 /01206.
In another embodiment, the dose form is a topical formulation as disclosed in
international patent application PCT/AU02/01003.
The term "endogenous" refers to a vitamin occurring in the body as the result
of
ingestion from a diet with no supplementation of the vitamin or its
derivatives, and
where only typical metabolic processes transform the dietary vitamin.
In order that the present invention may be more clearlyunderstood, preferred
15 forms will be described with reference to the following drawings and
examples.
Brief Description of Drawings
The following figures are referred to in the examples.
Figure 1: Calibration curve for TP vs T2P using ESMS.
Figure 2: GCMS of methylated liver extract and TP standard.
2o Figure 3: Example of TP ESMS spectra.
Figure 4: Example of liver extract ESMS spectra.
Modes for Carrying Out the Invention
It has now been demonstrated that the monophosphate ester of a-tocopherol, a-
tocopheryl phosphate, is present in significant quantities (8-147 p,g/g) in a
range of
2s biological tissues. a-Tocopheryl phosphate is resistant to oxidation and is
thus
inactive as an anti-oxidant; it is also resistant to acid and alkaline
hydrolysis so
that it cannot be detected by the standard assays for Vitamin E. The discovery
of
this storage form of Vitamin E heralds the need for a reassessment of the role
of
this essential vitamin in the body.
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For example, without being bound by theory, it is believed that TP as a
nascent
storage form is not a source of free radicals in marked contrast to free
tocopherol
which is an excellent source of undesirable free radicals or pro-oxidant at
high
concentrations. Oxidation with active phosphorylation of adenosine diphosphate
s for example, is known suggesting that a-tocopheryl phosphate may be a
reducing
agent capable of producing phosphorylated secondary messengers. This would
account for the non-antioxidant roles ascribed to tocopherol.
Again without wishing to be bound by theory, the following model is proposed
to
help understand the role of a-tocopheryl phosphate in tissue. a-Tocopheryl
~ o phosphate is stored in the lipoprotein environment in association with a
lipophilic
protein or as a glycerophosphate derivative. The proximity of a free radical
or the
generation of an oxidative environment stimulates the dephosphorylation of a-
tocopheryl phosphate and the release of free a-tocopherol. When the oxidative
challenge is neutralised, excess antioxidant, (a-tocopherol) is drawn back
into
~5 storage by phosphorylation. This conservative strategy avoids pro-
oxidation. This
proposed mode of action for a-tocopheryl phosphate may also apply to other
lipophilic bioactive compounds and provide a new explanation for its function
in
VIVO.
The invention will now be further illustrated and explained in the following
non-
limiting examples.
EXAMPLES
Example 1:
In this example, the storage form of Vitamin E was investigated in various
tissues.
The standard analytical methodology for the detection of a-tocopherol in
tissue
2s samples and foodstuffs normally includes a hydrolysis step as part of the
extraction process. The hydrolysis ensures that a-tocopheryl esters, such as
added a-tocopherol acetate, are converted to free a-tocopherol prior to
analysis.
There is scant literature on the analysis of a-tocopheryl phosphate. The
earliest
report appears to be a method for the separation of synthetic a-tocopheryl
so phosphate from other phosphate esters using paper chromatography. Although
the authors reported good sensitivity, they failed to detect natural levels of
a-
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tocopheryl phosphate in rat liver. It seems highly likely that the a-
tocopheryl
phosphate was lost at the step in their protocol at which protein was removed
from
the homogenate.
Surprisingly, a-tocopheryl phosphate is resistant to all of the alkaline and
acid
hydrolysis conditions encountered in these procedures and is consequently not
included with free a-tocopherol in typical analyses. Indeed, refluxing a-
tocopheryl
phosphate under strongly alkaline or acidic conditions for extended periods in
excess of 24 hours does not lead to any cleavage of the phosphate bond. We
have been unable to discover conditions under which a-tocopheryl phosphate can
be hydrolysed to yield free a-tocopherol. a-Tocopheryl phosphate has also
proven
to be resistant to oxidation and does not give a positive colour test under
conditions of the standard colorimetric oc-tocopherol analysis.
Although the stability of a-tocopheryl phosphate is such that vigorous
conditions
can be used to isolate it from tissue, but the detection of a-tocopheryl
phosphate is
~5 extremely difficult and most common instrumentation is inadequate. Gas
chromatography/mass spectrometry (GCMS) is the method of choice for a-
tocopherol analysis, but this technique is not particularly suitable for
analysis of a-
tocopheryl phosphate. We were eventually able to detect a-tocopheryl phosphate
by GCMS after derivatisation to dimethyl tocopheryl phosphate using
2o diazomethane. This method was used to confirm the presence of endogenous a-
tocopheryl phosphate in tissue samples, the endogenous material had the same
retention time, molecular ion (m/z 538) and base peak (m/z 273) as the
synthetic
a-tocopheryl phosphate (see Figure 2). This method was however found to be
r
unsuitable for routine quantitative analysis. We examined a number of
analytical
25 ~ techniques and determined that electrospray mass spectrometry (ESMS) was
clearly the best method for the analysis of a,-tocopheryl phosphate (see
Figure 3).
a-Tocopheryl phosphate is readily ionisable and is soluble in water at high pH
and
in organic solvents at low pH; these are properties that are ideally suited to
ESMS.
With the addition of a suitable internal standard, levels of a-tocopheryl
phosphate
3o in tissues were routinely determined by ESMS (see Figure 4).
Following on from the development of a suitable assay technique for a-
tocopheryl
phosphate, it was also necessary to develop an effective extraction protocol
since
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a-tocopheryl phosphate could not be detected using the extraction methodology
commonly employed for vitamin E determination. In the procedure we have
developed, the tissue was homogenized in dichloromethane containing the
internal
standard, di-a-tocopheryl phosphate. The mixture was centrifuged and the
dichloromethane layer was removed, leaving the majority of protein behind. The
residue remaining after evaporation of the dichloromethane was then hydrolyzed
with potassium hydroxide for 1 hour at room temperature. If the sample was not
hydrolyzed then a-tocopheryl phosphate could not be detected, which suggests
that it is associated with a protein or other some other compound; for
example, the
a-tocopheryl phosphate may be present as a glycerophosphate or similar ester,
and that hydrolysis is required to liberate free a-tocopheryl phosphate. The
hydrolysate was then washed with hexane to remove all of the organic soluble
material. Free a-tocopherol was removed at this stage and could be readily
analysed using standard techniques; a-tocopheryl phosphate was present as the
potassium salt, and being water soluble was not extracted. Acidification of
the
aqueous layer converted the a-tocopheryl phosphate to the free acid, which was
readily extracted into hexane.
Our findings that a-tocopheryl phosphate is resistant to hydrolysis and
oxidation,
coupled with the requirement to liberate free a-tocopheryl phosphate by
alkaline
2o hydrolysis during the extraction process, explains why the a-tocopherol
present as
a-tocopheryl phosphate has not previously been detected using the standard
assays for vitamin E. Reported amounts of "total a-tocopherol" present in
samples
will consequently have always been underestimated.
Using this extraction protocol and ESMS analysis, the a-tocopheryl phosphate
content of a range of animal and human tissues was measured. Table 1 presents
the results of these analyses.
Naturally occurring a-tocopheryl phosphate has been detected in pig, guinea
pig,
chicken and rat liver, and in human, pig and guinea pig adipose tissue. Liver
and
adipose have been reported to be the two main storage sites for a-tocopherol
and
3o it is therefore not surprising that a-tocopheryl phosphate is also present
in these
tissues. Our results indicate that the a-tocopheryl phosphate makes up a
significant proportion of the total a-tocopherol present.
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TP has hitherto been considered to be unable to be deposited directly in
tissues
where it can remain as a storage form of tocopherol. However, the following
example illustrates the detection of ingested TP in human and animal tissue.
The level of TP was determined using ditocopheryl phosphate (T2P) as the
internal
standard. T2P is not a naturally occurring substance. A calibration curve was
determined for TP vs T2P (Figure 1 ) using ES/MS. As can be seen from Figure 1
the response is virtually linear.
An extraction method was developed which gave the maximum recovery of TP.
The stability of TP to the extraction methodology was tested by subjecting
pure TP
1o to the extraction protocol, virtually 100% recovery was obtained. The
extraction
protocol is outlined below:
1. Weigh 1 g and homogenize in 10 ml dichloromethane.
2. Add 0.1 mg T2P (1 mg/ml in 50% tetrahydrofuran) as internal standard.
3. Mix using homogenizer and centrifuge sample.
4. Remove and evaporate the dichloromethane.
5. Add 9 ml 2 M KOH (2M) and stir for 1 h at room temperature.
6. Add 10 ml hexane, shake and remove hexane (upper) layer.
7. Add 10 ml 2M HCI (2M) to the 9 ml KOH (2M) solution.
8. Add 10 ml hexane, shake and remove hexane layer.
9. Evaporate to dryness.
10.Analyse on ESMS, ESI (-ve mode)
ESMS conditions
Sample was dissolved in 1 ml THF containing 1 % NH3, 20 p,1 was injected into
the
sample loop. The sample was eluted with THF:H20 (9:1 ) 20p,1/min. MS analysis
.
2s was conducted in ve ion mode with cone voltage of 40V.
Diazomethane reaction with liver extract and TP standard.
A solution of potassium hydroxide (5 g) in water (8 ml) and ethanol (10 ml)
was
preheated to 65°C in a water bath. A solution of Diazald~ (3.0 g, 13.8
mmol) in
ether (40 ml) was slowly added to the potassium hydroxide solution and a
distillate
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14
was distilled across. When the Diazald~ addition was completed, 10 ml of ether
was added and also distilled across. The final ether distillate contained
approximately 420 mg (9.95 mmol) of diazomethane.
Half of this diazomethane solution was immediately added to a liver sample
that
s ~ had been extracted from 10 g of rat liver and pre-dissolved in THF and the
other
half of this diazomethane solution was added to 100 pg of TP (TP) standard
that
had been dissolved in ether: The two reaction mixtures were sealed and placed
in
a dark cupboard for approximately 30 minute, after which the solvent and
diazomethane were removed from the liver extract and TP standard under
reduced pressure. The methylated liver extract sample was dissolved in 100 p1
of
chloroform, as was the TP standard; 30 p1 of this TP solution was then taken
and
re-dissolved in 1.5 ml of chloroform in order to mimic the concentration of TP
in the
liver extract. Both the liver extract and TP standard were then immediately
analysed using gas chromatograph mass spectrometry (GC-MS).
15 GCMS conditions
The methylated liver extract and TP standard were analysed using a Shimadsu
GCMS-QP5000 Gas Chromatograph Mass Spectrometer incorporating a
Shimadsu AOC-20i Auto Injector. One p1 of sample was injected onto a 15m
SGE.BP1 capillary column, with a thickness of 0.25pm and an internal diameter
of
20 0.25 mm utilising a temperature program from 260°C up to
300°C tamping at 3°C
per minute. The injector and interface temperatures were both set at
300°C. The
carrier gas used was helium, the column inlet pressure was set at 156 kPa and
the
total flow was 144 ml/min, thus presenting a column flow of 2.4 ml/min with a
linear
velocity of 86.5 cm/sec. The samples were run in split mode with a split ratio
of
25 56:1.
The acquisition mode for the mass spectrometer was SIM (single ion monitoring)
mode, with Channel 1-m/z = 538.00 and Channel 2-m/z = 273.00. The detector
gain for this acquisition was set at 1.5 kVolts.
TP in Rat Liver
so Non-treated rat livers were used to extract, detect and quantitate the
amount of
naturally occurring TP. Fresh livers from Sprague-dawley and Wistar rats (aged
between 10 to 11 weeks old, weighing approximately 120-180 grams) were used
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for each extraction. The amount of naturally occurring TP in the rat livers
was
found to be 12.5 ~.g/g of liver (n=15).
TP has also been detected in human abdominal fat, Guinea Pig and rat adipose
tissue, porcine and guinea pig liver of humans and animals which have 'not
been
s administered TP. Table 1 outlines the quantity of TP detected in each organ.
The results demonstrate that the levels of tissue TP are of the same order of
magnitude across the species. Interestingly, the phosphate form seems to be
used in the same way, but not surprisingly, at different levels in different
tissues
depending upon the life span of the animal. .
Table 1
Species and Organ Amount (p,g/g tissue) TP detected
using ESMS
Rat (male) Liver, n=15 124
Rat (male) Adipose tissue, 185
n=5
Guinea Pig (male) Liver, n=4 83
Guinea Pig (male) Adipose tissue,104
n=4
Pig (7 month old-male) Liver, 186
n=3
Human Liver, n=2 322
Human Adipose tissue, n=3 259
Chicken (2 month old male) 3617
n=14
Example 2:
This example investigates whether tocopheryl phosphate levels in storage sites
are increased after tocopheryl acetate and tocopheryl phosphate were
~s administered.
The procedure used is summarized as follows:
(a) Administer a single dose of the compounds to male Sprague-Dawley
rats (see table; oral gavage with an 18g gavage needle and 1 ml
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16
syringe, or intravenously with a 26g hypodermic needle and 1 ml
syringe).
(b) Twenty-four hours after administrations the rat will be anaesthetized
with Nembutal (60 mg/kg i.p.).
s (c) Once the rats were under deep anaesthesia a sample of blood was
taken from the tail vein, and the femoral vein exposed and injected
with 500 units of heparin. The abdominal cavity will be opened and
the rat perfused with saline. The liver, heart and epidydimal fat pad
removed and frozen in liquid nitrogen. Hind-leg muscle and brain will
also be collected and frozen.
Table 2: Treatments
Compound Dose (mg/kg) Number of
Rats
TP (IV) 10 3
TP (IV) 30 3
TP 10 3
TP 30 3
Tocopheryl acetate 10 3
Tocopheryl acetate 30 3
Tocopheryl acetate 100 ' 3
Control (0.3 ml water)(IV)0 3
Control (0.3 rnl corn~ 0 3
oil)
Livers were be extracted according to the method below. The extracts will be
analyzed and quantitated for TP (p,g) content by ESMS. Any tissue samples left
~s over at the end of the study was kept frozen at -80 °C.
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Liver extraction
17
One g liver was homogenized in 10 ml dichloromethane (analytical grade). Add
0.1 mg tocopheryl diphosphate (internal standard). Homogenize sample for 2
min,
centrifuge and remove upper layer and evaporate under nitrogen. Add 9 ml KOH
(2M) and stir for 1 hr at room temperature (or 20 min at 80 C). Add 10 ml
hexane,
shake and remove upper layer. Add 10 ml HCI (2M) and shake. Add 10 ml
hexane to the solution and shake and remove upper layer. Evaporate top layer
to
dryness.
Electrospray analysis
~o Add 1 ml tetrahydrofuran (THF) and 20 u1 of 25% Ammonia to sample and
analyse.
Results
Table 3 Treatment dose of TP (mg/kg) vs. liver TP (ug/gm)
TreatmentControl 3 10 30 100
(Oral & mg~kg mg/kg mg/kg mg/kg
LV.
TP i.v. 11.95 ug 17 ug 24 ug 28 ug 27 ug
(61 (86%) (100%)
%)
TP oral 19.18 18.35
ug ug
(68%) (65%)
TA oral 8.0 ug 12.81 13.71 14 ug
ug ug (50%)
(46%) (49%)
TP was administered by IV to provide a value for absolute bioavailability. The
amounts in brackets are percentages when compared with the IV value.
Conclusion
The above results in table 3 clearly demonstrate that:
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(a) absorption of a tocopherol analogue can improve tissue levels of TP
within a 24 hour period; from which it can be inferred that the acetate
group has been biologically removed and replaced by a phosphate
group
s (b) TP has higher bioavailability than tocopheryl acetate because the
transformation to phosphate is not required
This means that the higher bioavailability of TP can quickly increase tissue
concentrations of the electron transfer agent to~therapeutically efficacious
levels.
Example 3
~o This example investigates whether tocopheryl phosphate exists in commonly
consumed food products in a normal diet using analytical methods outlined in
example 1.
Table 4 shows the results of TP analysis in chicken fat, muscle and eggs.
Table 4: Concentration of TP in chicken fat, muscle and eggs as determined by
~5 ESMS
Chicken Number Ratio of TPIT2P [TP] ~.g/g tissue Mean
& (SIR SD
Tissue plots)
1, muscle 0.3193 13 9 5
2. muscle 0.1325 3
3. muscle 0.2560 10
Eggwhite black-x 0.0633 Below limit of detection
Eggwhite white-x 0.0419 Below limit of detection
Eggwhite white-x 0.0512 Below limit of detection
Eggwhite brown-x 0.0361 Below limit of detection
Eggwhite brown-x 0.0633 Below limit of detection
Fat 5.724 301 310 35
Fat 5.333 280
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Chicken Number Ratio of TP/T2P [TP] p.g/g tissue Mean
& (SIR SD
Tissue plots)
Fat 6.64 349
Yolk black-x 0.9578 47 40 9
Yolk white-x 0.6747 32
Yolk white-x 0.6145 29
Yolk brown-x 0.9548 47
Yolk brown-x 0.9608 47
Clearly large amounts of TP were detected in a variety of commonly consumed
chicken products. This means that TP is consumed in normal diets containing
poultry meats, fat and eggs, suggesting the compound is associated with a long
history of safe use. Importantly however, the levels required for disease
intervention would likely be inadequate from these sources and additional
doses of
TP are required.
The word 'comprising' and forms of the word 'comprising' as used in this
description and in the claims does not limit the invention claimed to exclude
any
variants or additions.
Modifications and improvements to the invention will be readily apparent to
those
skilled in the art. Such modifications and improvements are intended to be
within
the scope of this invention.
