U.S. patent application number 12/654779 was filed with the patent office on 2010-05-06 for treatment of neurodegenerative conditions.
This patent application is currently assigned to BTG International Limited. Invention is credited to Paul Barraclough, Lawrence S. Harbige, Michael J. Leach, Mohammed Sharief.
Application Number | 20100113595 12/654779 |
Document ID | / |
Family ID | 34219616 |
Filed Date | 2010-05-06 |
United States Patent
Application |
20100113595 |
Kind Code |
A1 |
Harbige; Lawrence S. ; et
al. |
May 6, 2010 |
Treatment of neurodegenerative conditions
Abstract
A method is provided for treating a patient in need of therapy
for a neurodegenerative disease comprising administering to that
patient a therapeutically effective dose of a lipid glyceride
comprising a glycerol moiety and a fatty acid moiety, the fatty
acid moiety being selected from the group consisting of
.gamma.-linolenic acid, dihomo-.gamma.-linolenic acid and
arachidonic acid characterised in that the selected fatty acid
moiety is attached to the glycerol moiety at its sn-2 position.
Preferably the method is that wherein the lipid is administered for
a duration and at a dose sufficient to maintain or elevate
TGF-.beta.1 levels in the patient to therapeutic levels.
Inventors: |
Harbige; Lawrence S.;
(London, GB) ; Leach; Michael J.; (London, GB)
; Sharief; Mohammed; (London, GB) ; Barraclough;
Paul; (Surrey, GB) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Assignee: |
BTG International Limited
London
GB
|
Family ID: |
34219616 |
Appl. No.: |
12/654779 |
Filed: |
December 31, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10567778 |
Feb 9, 2007 |
|
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PCT/GB2004/003524 |
Aug 13, 2004 |
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12654779 |
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Current U.S.
Class: |
514/547 ;
554/150; 554/224 |
Current CPC
Class: |
A61P 21/02 20180101;
C07C 67/29 20130101; C07C 69/65 20130101; A61P 25/00 20180101; A61P
25/04 20180101; A61K 31/232 20130101; A61P 25/28 20180101; C07C
69/587 20130101; C07C 67/14 20130101; A61P 25/16 20180101; A61P
43/00 20180101; C07C 67/14 20130101; C07C 69/65 20130101; C07C
67/14 20130101; C07C 69/28 20130101; C07C 67/14 20130101; C07C
69/587 20130101; C07C 67/29 20130101; C07C 69/30 20130101 |
Class at
Publication: |
514/547 ;
554/224; 554/150 |
International
Class: |
A61K 31/232 20060101
A61K031/232; C07C 57/02 20060101 C07C057/02; C07C 51/00 20060101
C07C051/00; A61P 25/00 20060101 A61P025/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 18, 2003 |
GB |
0319358.8 |
May 14, 2004 |
GB |
0410846.0 |
Claims
1-38. (canceled)
39. A method of treating a patient in need of therapy for a
neurodegenerative disease comprising administering to that patient
a therapeutically effective dose of a defined structure lipid
glyceride comprising a glycerol moiety esterified with one or more
fatty acid moieties, characterised in that the lipid has a fatty
acid moiety at the sn-2 position selected from the group consisting
of .gamma.-linolenic acid, dihomo-.gamma.-linolenic acid and
arachidonic acid.
40. A method as claimed in claim 39 wherein the neurodegenerative
disease involves demyelination.
41. A method as claimed in claim 39 wherein the treatment
specifically arrests underlying neurodegeneration and restores
neuronal function.
42. A method as claimed in claim 39 which normalizes neuronal
membrane composition with respect to .gamma.-linolenic acid,
dihomo-.gamma.-linolenic acid and arachidonic acid lipid
content.
43. A method as claimed in claim 39 which restores healthy
TGF-.beta.1/TNF.alpha. ratios as measured from spontaneous release
from peripheral blood mononuclear cell release.
44. A method as claimed in claim 39 wherein the disease is multiple
sclerosis.
45. A method as claimed in claim 39 wherein the disease is
relapsing remitting multiple sclerosis, primary progressive
multiple sclerosis or chronic progressive multiple sclerosis.
46. A method as claimed in claim 39 wherein the disease is multiple
sclerosis and the treatment restores, in part or completely,
neuronal function or neuronal integrity as measured by one or more
of MRI scan, CAT scan or by EDSS score.
47. A method as claimed in claim 39 wherein the treatment is of
cerebral impairment after stroke, head trauma and intracranial
bleeding, Alzheimer's disease or Parkinson's disease where there is
demyelination or neuronal damage.
48. A method as claimed in claim 39 wherein the lipid is
administered for a duration and at a dose sufficient to maintain or
elevate TGF-.beta.1 levels in the patient to therapeutic
levels.
49. A method as claimed in claim 39 wherein the lipid is
administered for a duration and at a dose sufficient to maintain or
elevate TGF-.beta.1 levels in the patient to a
TGF-.beta.1/TNF-.alpha. ratio released spontaneously from
peripheral blood mononuclear cells isolated from the blood of a
patient, after 18 months of daily dosing, of 0.4 to 3.0, at least
0.5, more preferably at least 0.75 and most preferably at least
1.
50. A method as claimed in claim 49 wherein the dose is such as to
produce a TGF-.beta.1/IL-1.beta. ratio in PBMCs isolated from blood
of a patient, after 18 months of daily dosing, of at least of at
least 0.75.
51. A method as claimed in claim 39 wherein the amount of lipid
administered is between 0.5 and 30 grams, typically 3 to 5 grams,
per day.
52. A method as claimed in claim 39 wherein the lipid is a
monoglyceride, diglyceride or triglyceride containing the at least
one sn-2 .gamma.-linolenic acid, dihomo-.gamma.-linolenic acid or
arachidonic acid moiety, the lipid being of general Formula I
##STR00005## wherein R.sup.1 and R.sup.2 are independently selected
from hydrogen and acyl groups, and R.sup.2 is selected from the
group consisting of .gamma.-linolenic acid,
dihomo-.gamma.-linolenic acid and arachidonic acid residues having
their carbonyl carbon attached to the oxygen of the glycerol
moiety.
53. A method as claimed in claim 39 wherein R.sup.1 and R.sup.3 are
saturated fatty acid moieties of formula
--CO--(CH.sub.2).sub.n--CH.sub.3, wherein n is an integer selected
from 1 to 22.
54. A method as claimed in claim 53 wherein R.sup.1 and R.sup.3 are
the same and n is an integer of from 5 to 12.
55. A method as claimed in claim 54 wherein n is an integer of from
6 to 10.
56. A method as claimed in claim 52 wherein R.sup.1 and R.sup.3 are
selected from the group consisting of essential fatty acids or
physiologically acceptable fatty acids metabolisable by the human
body.
57. A method as claimed in claim 52 wherein R.sup.1, R.sup.2 and
R.sup.3 are all the same and are selected from the group consisting
of .gamma.-linolenic acid, dihomo-.gamma.-linolenic acid and
arachidonic acid residues.
58. A pharmaceutical composition characterised in that it comprises
a defined structure lipid glyceride comprising a glycerol moiety
esterified with one or more fatty acid moieties, characterised in
that the lipid has a fatty acid moiety at the sn-2 position
selected from the group consisting of .gamma.-linolenic acid,
dihomo-.gamma.-linolenic acid.
59. A pharmaceutical composition for treating neurodegeneration
characterised in that it comprises a defined structure lipid
glyceride comprising a glycerol moiety esterified with one or more
fatty acid moieties, characterised in that the lipid has a fatty
acid moiety at the sn-2 position selected from the group consisting
of .gamma.-linolenic acid, dihomo-.gamma.-linolenic acid and
arachidonic acid.
60. A pharmaceutical composition for treating demyelinating disease
comprising a glycerol moiety esterified with one or more fatty acid
moieties, characterised in that the lipid has a fatty acid moiety
at the sn-2 position selected from the group consisting of
.gamma.-linolenic acid, dihomo-.gamma.-linolenic acid and
arachidonic acid.
61. A lipid of formula II ##STR00006## wherein R.sup.1 and R.sup.3
are the same and are --C(O)(CH.sub.2).sub.nCH.sub.3 wherein n is
selected from 4 to 14, more preferably 6 to 10 and most preferably
7, 8 or 9 and R.sup.2 is selected from .gamma.-linolenyl,
dihomo-.gamma.-linolenyl and arachidonyl residues.
62. Use of a defined structure lipid glyceride comprising a
glycerol moiety esterified with one or more fatty acid moieties,
characterised in that the lipid has a fatty acid moiety at the sn-2
position selected from the group consisting of .gamma.-linolenic
acid, dihomo-.gamma.-linolenic acid and arachidonic acid, for the
manufacture of a medicament for the treatment of neurodegenerative
disease.
63. Use as claimed in claim 62 wherein the degenerative disease is
a demyelinating disease.
64. Use as claimed in claim 62 wherein the disease is multiple
sclerosis.
65. Use as claimed in claim 62 wherein the medicament normalises
neuronal membrane composition with respect to lipid
.gamma.-linolenic acid, dihomo-.gamma.-linolenic acid and
arachidonic acid levels.
66. Use as claimed in claim 62 wherein the medicament restores
TGF-.beta.1/TNF.alpha. ratios spontaneously released from
peripheral blood mononuclear cells of a patient to healthy
levels.
67. Use as claimed in claim 62 wherein treatment is for multiple
sclerosis or the degenerative sequelae associated with head trauma,
stroke and intracranial bleeds or neuronal damage caused by
Alzheimer's or Parkinson.sup.1s disease.
68. Use as claimed in claim 62 wherein the medicament repairs CNS
lesions.
69. Use as claimed in claim 62 wherein the medicament relieves
muscle spasticity and/or pain.
70. Use as claimed in claim 62 wherein the medicament eliminates
relapses.
71. Use as claimed in claim 62 wherein the medicament improves EDSS
score by at least 1 unit over a period of 1 years treatment.
72. Use as claimed in claim 62 wherein the medicament is sufficient
to restore EDSS of a patient with EDSS above 2.5 to below 2 over a
period of 1 years treatment.
73. Use as claimed in claim 62 wherein there is improvement in
bladder control.
74. A method for synthesis of a compound of general formula III
##STR00007## wherein R.sup.1 and R.sup.3 are the same and are
--C(O)(CH.sub.2).sub.nCH.sub.3 wherein n is selected from 4 to 14,
more preferably 6 to 10 and most preferably 7, 8 or 9 and R.sup.2
is .gamma.-linolenyl residue, dihomo-.gamma.-linolenyl residue or
arachidonyl residue comprising reacting 1,3-dihydroxyacetone with a
compound of formula X--C(O)(CH.sub.2).sub.nCH.sub.3 wherein X is
selected from Cl, Br and I, to give the corresponding
1,3-di-(C(O)(CH.sub.2).sub.nCH.sub.3) 2-keto compound reducing the
keto group to the corresponding
1,3-di-(C(O)(CH.sub.2).sub.nCH.sub.3) 2-ol and reacting that with
.gamma.-linolenyl halide or dihomo-.gamma.-linolenyl halide or
arachidonyl halide, wherein halide is chloride, bromide or
iodide.
75. A method for synthesis of a compound of general formula IV
##STR00008## wherein R.sup.1 to R.sup.3 are the same and selected
from .gamma.-linolenyl residue, dihomo-.gamma.-linolenyl residue or
arachidonyl residue comprising reacting the corresponding
.gamma.-linolenyl halide, dihomo-.gamma.-linolenyl halide or
arachidonyl halide, wherein halide is chloride, bromide or iodide,
with glycerol.
76. A lipid selected from the group consisting of Glycerol
1,3-didecanoate-2-octadecatri(6-Z,9-Z,12-Z)enoate Glycerol
1,3-didecanoate-2-eicosa-(8Z,11Z,14Z)-trienoate Glycerol
trieicosotetra5-Z,8-Z,11-Z,14Z-eneoate.
77. A lipid as claimed in claim 76 for use in therapy.
Description
[0001] The present invention relates to a method for treating
neurodegenerative conditions, particularly those in which increase
in transforming growth factor .beta. (TGF-.beta.) is beneficial,
particularly TGF-.beta.1. More particularly the present invention
provides treatment for neurodegenerative conditions, particularly
those such as demyelinating diseases, such as multiple sclerosis,
Alzheimer's and Parkinsons diseases and the degenerative sequelae
associated with head trauma, stroke and intracranial bleeds,
whereby neuronal function may be improved or restored from an
impaired condition, eg. by remyeleination.
[0002] Further provided are novel use of known and novel compounds
comprising unsaturated fatty acid moieties for the manufacture of
medicaments capable of effectively treating such conditions, more
particularly being capable of achieving previously unattained
levels of success with regard to recovery of neurological
function.
[0003] The inventor's copending unpublished patent application
PCT/GB04/002089, incorporated herein by reference, relates to the
use of plant and fungal oils for the treatment of neurodegenerative
diseases. These oils have high percentages of the essential fatty
acid .gamma.-linolenic acid (GLA) at the sn-2 position of their
lipids, typically being over 40% of the sn-2 fatty acid total of
the oil.
[0004] It is well reported in the literature that essential fatty
acids (EFAs) of the n-3 and n-6 unsaturation pattern have
beneficial effect in a wide variety of human physiological
disorders, including autoimmune disease (WO 02/02105). Harbige
(1998) Proc. Nut. Soc. 57, 555-562 reviewed the supplementation of
diet with n-3 and n-6 acids in autoimmune disease states, and
particularly noted evidence of benefit of .gamma.-linolenic (GLA)
and/or linoleic acid (LA) rich oils.
[0005] Bates et al noted that lipid oils comprising a mixture of
linoleic acid and .gamma.-linolenic acid residues had been
suggested back in 1957 to be possibly more efficacious in treating
inflammation and autoimmune diseases, but found that at 3 g oil per
day (Naudicelle Evening Primrose oil 7:1 LA:GLA) patients who had
relapses became more ill on the trial oil than on the control.
[0006] Although the aetiology of MS remains unknown studies have
shown that MS patients have higher than normal neuro-antigen
autoreactive T-cells levels. These T-cells react inter alia to
myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein
(MOG) and are in an increased state of activation compared with
healthy controls. The actual processes of axonal damage e.g.
chronic inflammation, demyelination and astrogliosis in MS is
complex but white matter inflammation and demyelination are
considered to determine disease severity, whilst recent studies
suggested that axonal damage in MS begins in the early stages of
the disease and contributes to disability (De Stefano et al,
2001).
[0007] Experimental autoimmune encephalomyelitis (EAE) is the most
frequently used animal model for immune mediated effects of MS.
Studies in the guinea-pig have shown that linoleic acid partially
suppresses the incidence and severity of EAE (Meade et al (1978)).
(Harbige et al (1995), 1997b) demonstrated disease modifying
effects of linoleic acid and .gamma.-linolenic acid on clinical and
histopathological manifestations of EAE. Depending on dose,
.gamma.-linolenic acid was fully protective in acute rat EAE
whereas linoleic acid had dose-dependent action on the clinical
severity but did not abolish it.
[0008] Despite these experimental findings, it is recognised that
the human disease, multiple sclerosis, is highly complex and can be
conversely exacerbated and ameliorated by the activity of T-cells
and other immune response factors. It is thought that the n-6 fatty
acids promote autoimmune and inflammatory disease based upon
results obtained with linoleic acid only. TGF-.beta.1 and PGE.sub.2
production has been shown to be increased non-specifically in
.gamma.-linolenic acid fed mice ex vivo. TGF-.beta.1 has been
reported to protect in acute and relapsing EAE ((Racke et al
(1993); Santambrogio et al (1993)), and PG inhibitors such as
indomethacin augment, and thus worsen, the disease (Ovadia &
Paterson (1982)).
[0009] Cytokines are implicated in the pathogenesis of MS, with
many studies showing an increase in myelinotoxic inflammatory
cytokines (TNF-.alpha., IL-1.beta. and IFN-.gamma.) coinciding with
the relapse phase of the disease. Conversely, levels of the
anti-inflammatory and immunosuppressive cytokine transforming
growth factor-beta1 (TGF-.beta.1) appear to be reduced during a
phase of relapse and increase as the patient enters remission. Thus
the balance between biologically active TGF-.beta.1 and the
pro-inflammatory TNF-.alpha., IL-1.beta. and IFN-.gamma. appears to
be dysregulated during MS relapse-remission.
[0010] During natural recovery phase from EAE,
TGF-.beta.1-secreting T-cells inhibit EAE effector cells,
TGF-.beta.1 is expressed in the CNS and, in oral-tolerance-induced
protection in EAE, TGF-.beta. and PGE.sub.2 are expressed in the
brain (Karpus & Swanborg (1991); Khoury et al (1992)). Harbige
((1998) concluded that dietary .gamma.-linolenic acid effects on
EAE are mediated through Th.sub.3-like mechanisms involving
TGF-.beta.1 and possibly through superoxide dismutase antioxidant
activity.
[0011] Borage oil (typically 20% to 23% .gamma.-linolenic acid and
34 to 40% linoleic acid per 100% fatty acid content) and Mucor
javanicus fungal oil (see FIG. 1) have been shown to be effective
in the EAE animal model used to identify MS candidates, whilst
never having been shown to be significantly effective in the human
disease. High levels of linoleic rich oil containing low levels of
.gamma.-linolenic acid (EP0: linoleic acid:.gamma.-linolenic acid
7:1) partially suppressed the incidence and severity of EAE in rat
(Mertin & Stackpoole, 1978) whereas the Bates' Naudicelle study
referred to above led to worsening of patients. In spite of the use
of Borage oil and other GLA/LA containing oils such as Evening
Primrose oil by multiple sclerosis sufferers over the past 30 years
or so, the vast majority of patients fail to recover from the
disease, showing no significant improvement, with the underlying
disease continuing to progress to death.
[0012] It has been suggested to use, inter alia, .gamma.-linolenic
acid and linoleic acid rich Borage oil as a means to provide
immuno-suppression in multiple sclerosis (U.S. Pat. No. 4,058,594).
Critically, the dose suggested is 2.4 grams of oil per day and no
actual evidence of efficacy is provided. This is much lower than
the low 5 g/day dose found to be ineffective in vivo in man in the
PCT/GB04/002089 study.
[0013] Other more dramatic immunosuppressant treatments, including
T cell depleters and modulators such as cyclophosphamide, are also
shown to be effective in the EAE model, but where these are
employed in the human multiple sclerosis disease symptoms improve,
but the underlying disease continues to progress. T-cells indeed
produce beneficial cytokines, such as TGF-.beta.1, as well as
deleterious ones in man. David Baker of Institute of Neurology, UK
summed up the disparity between what is effective in the EAE and in
MS with a paper entitled `Everything stops EAE, nothing stops MS`
at the 10 May 2004 UK MS Frontiers meeting of the UK MS
Society.
[0014] It is clear that immunosuppression alone cannot cure MS.
This is almost certainly due to a fundamental underlying metabolic
disorder in MS patients, in addition to the autoimmune disease,
that leads to membrane abnormality, cytokine dysregulation and
subsequent immune attack and lesioning. Although patients go into
remission in relapse-remitting disease, the underlying
demyelination proceeds.
[0015] The `gold standard` treatment for MS remains interferon,
such as with .beta.-Avonex.RTM., Rebif.RTM. and other interferon
preparations. This gold standard treatment only addresses needs of
some, eg 30%, of the patients and even in these symptom improvement
is restricted to reduced severity of relapses. Whilst symptoms may
be reduced in a proportion of patients, the disease tends to
progress to further disability and death due to underlying
degeneration.
[0016] In their as yet unpublished PCT/GB04/002089 study the
present inventors have surprisingly determined that with compliance
to a `high dose` treatment with triglyceride oil containing high
levels of sn-2 .gamma.-linolenic acid (>40% of residues at the
sn-2 being of .gamma.-linolenic acid) with suitable accompanying
fatty acid content, remarkable levels of improvement in almost all
symptoms of MS can be achieved, way surpassing that provided by the
current gold standard treatment. Such success is particularly
surprising in the light of the prior use of other .gamma.-linolenic
acid containing preparations without success, such as the
Naudicelle study.
[0017] The PCT/GB04/002089 study shows that over an 18-month
period, patients taking high dose (15 g/day) selected high sn-2
.gamma.-linolenic acid borage oil showed significant (p<0.001)
and marked improvements in EDSS score, a reduced rate of relapse,
symptomatic relief of muscle spasticity and painful sensory
symptoms, and improved objective measures of cognitive functions.
Low doses of 5 g/day of this borage oil were without effect.
[0018] Patients taking the highest dose of this borage oil
maintained their level of peripheral blood mononuclear cell
production (PBMC) of TGF-.beta.1 during the trial period, their
pro-inflammatory cytokines TNF-.alpha. and IL-1.beta. were
significantly and markedly (<70%) reduced and they either
maintained or increased the PBMC membrane long chain omega-6 fatty
acids dihomo-'.gamma.-linolenic acid (DHLA) and arachidonic acid
(AA) in contrast to patients taking placebo who demonstrated loss
of these fatty acids over the course of the trial period.
[0019] This whilst immuno-suppression would be expected to reduce
increase of active lesioning and neurodegeneration, the high sn-2
GLA oil treatment apparently targeted maintenance and/or increase
of key membrane lipid components that are otherwise specifically
lost in MS, being consistent with a correction of a metabolic
defect not otherwise effectively treated by current therapies. The
fact that the low dose (5 grams/day) had no effect on this supports
such determination.
[0020] .gamma.-Linolenic acid (18:3n-6, or GLA) is known to be
rapidly converted to longer-chain omega-6 polyunsaturated fatty
acids dihomo-.gamma.-linolenic acid and arachidonic acid in vivo
(Phylactos et al 1994, Harbige et al 1995, 2000). Therefore to
determine how to increase the level of membrane long chain omega-6
fatty acids in MS the inventors have reviewed their results
obtained with several GLA-containing oils:--both fungal (from Mucor
javanicus) and plant (Borago officianalis), Evening primrose
Oenothera spp. or Blackcurrant Ribes spp) as well as a synthetic
tri-GLA oil as GLA delivery systems in an in vivo experimental
animal model of MS known as chronic relapsing experimental
autoimmune encephalomyelitis (CREAE).
[0021] Induction of EAE in rats does not produce histological
features of demyelination (Brosnan et al 1988) but induces an acute
mono-phasic disease pattern, unlike MS which is characterised by
CNS demyelination and is in the majority of cases clinically
relapsing-remitting. Chronic relapsing and demyelinating EAE models
(CREAE) however are characterised by demyelination and relapse
phases. With the demonstration that myelin oligodendrocyte
glycoprotein (MOG) is an important neuroantigenic target in MS
(Genain et al 1999) and the demonstration of far greater responses
of peripheral blood auto-reactive lymphocytes to this neuroantigen,
compared with MBP, in MS (Kerlero de Rosbo et al 1993, 1997) MOG
induced CREAE has become the animal model of choice with features
closely resembling those observed in MS (Fazakerely et al 1997,
Genain et al 1999, Amor et al 1994).
[0022] Evidence from the inventor's CREAE and rat EAE feeding
studies indicates that an enriched blackcurrant seed oil (72% w/w
18:3n-6, GLA) did not protect against EAE (see Table 3).
Importantly blackcurrant seed oil has a low sn-2 GLA with most of
the GLA in the sn-1 and sn-3 positions (Lawson and Hughes 1988).
Furthermore a structured triacylgcerol containing three GLA
moieties (TG-GLA) provided protective effects similar to that of
the borage oil used in CREAE (Table 2). This would also be
consistent with the sn-2 GLA being important i.e. the outer pair
sn-1 and sn-3 GLA being enzymatically removed in vivo and probably
undergoing oxidation leaving the sn-2 GLA only. This selective
hydrolysis arises from the known ability of specific lipases to
remove the sn-1 and sn-3 fatty acids from triacylgycerol molecules
but an apparent protection of the sn-2 position in vivo (Lawson and
Hughes 1988, Kyle 1990).
[0023] This review has led the inventors to postulate that
glycerides having sn-2-.gamma.-linolenic acid,
dihomo-.gamma.-linolenic acid or arachidonic acid residues will be
superior in correcting MS metabolism even to the high
sn-2-.gamma.-linolenic acid Borage oil of their earlier trial. This
would allow lower doses of lipid to be taken and/or possibly
decrease the time of treatment which would result in beneficial
effect.
[0024] Table 3 of EP 0520624 (Efamol Holdings) compares the
triglyceride content of Evening Primrose and Borage Oils, the
former being taught to be more therapeutically effective than the
latter for a variety of GLA responsive disorders. This document
indicates Borage oil to have twenty seven different trigyceride
components, only 20% of which have sn-2 GLA. Page 3, lines 40-42
notes that biological testing has shown that equal amounts of GLA
may indeed have very different effects when that GLA is supplied as
different oil sources. Crucially, it then directs the reader to one
particular fraction present in Evening Primrose Oil (EPO), but not
Borage Oil, as being responsible for the former's superior effect
in raising PGE1 (see EP 0520624 Chart page 4 and Table 2) and thus
anti-inflammatory effect: that fraction being identified as
di-linoeoyl-mono-gamma-linolenyl-glycerol (DLMG) which it states to
be 18 to 19% of the total triglyceride in EPO. Critically, page 6
clearly teaches that the position of the GLA, in sn-1, 2 or 3, is
not important to this effect.
[0025] Dines et al (1994) Proceedings of the Physiological Society,
Aberdeen Meeting 14-16 Sep. 1994 report on studies of treatment of
diabetic neuropathy neuronal damage with .gamma.-linolenic acid
containing oils of the type advocated by EP 0520624 and again note
that Borage Oil was not very effective in treating this
neurodegeneration whereas Evening primrose oil was. The paper
concludes that Borage Oil contains other constituents that
interfere with GLA activity.
[0026] The present inventors now set out, in view of their results
for high sn-2-.gamma.-linolenic acid Borage Oil, to demonstrate
that it is indeed the presence of an sn-2-.gamma.-linolenic acid,
dihomo-.gamma.-linolenic acid or arachidonic acid residue in a
glyceride, particularly a triglyceride, that gives it efficacy in
treating EAE, CREAE and the human disease MS.
[0027] In a first aspect the present invention provides a method of
treating a patient in need of therapy for a neurodegenerative
disease comprising administering to that patient a therapeutically
effective dose of a defined structure lipid glyceride comprising a
glycerol moiety esterifed with one or more fatty acid moieties,
characterised in that the lipid has a fatty acid moiety at the sn-2
position selected from the group of residues consisting of residues
of .gamma.-linolenic acid, dihomo-.gamma.-linolenic acid and
arachidonic acid.
[0028] Particularly advantageously treated neurodegenerative
diseases are those involving demyelination. The present method
specifically arrests underlying neurodegeneration and restores
neuronal function. Particularly the method normalises neuronal
membrane composition, and restores healthy PBMC spontaneuosly
released TGF-.beta.1/TNF.alpha. ratios and the ratios of
TGF-.beta.1 with other PBMC released cytokines. Most advantageously
the method arrests neurodegeneration in multiple sclerosis of all
types but particularly relapsing remitting, primary progressive and
chronic progressive MS and the restoration, in part or completely,
of neuronal function such as measured, eg. By MRI or CAT scan or by
EDSS score. Such method may also be used in treatment of cerebral
impairment after stroke, head trauma and intracranial bleeding
where there is demyelination or neuronal damage. Further
application is provided in treating other chronic demyelination
such as in Alzheimer's and Parkinson's disease.
[0029] Preferably the lipid is administered for a duration and at a
dose sufficient to maintain or elevate TGF-.beta. levels, in the
patient to therapeutic levels. By therapeutic levels is meant
levels at least consistent with healthy subjects. Preferably the
dose is such as to produce a TGF-.beta.1/TNF-.alpha. ratio
spontaneously released from peripheral blood mononuclear cells
(PBMCs) isolated from blood of a patient, after 18 months of daily
dosing, of 0.4 to 3.0, at least 0.5, more preferably at least 0.75
and most preferably at least 1. Preferably the dose is such as to
produce a TGF-.beta.1/IL-1.beta. ratio in blood of a patient, after
18 months of daily dosing, of at least 0.5, more preferably at
least 0.75 and most preferably at least 1. Preferably said levels
are produced after 12 months and more preferably after 6
months.
[0030] Typically the amount of lipid administered daily will be
between 0.5 and 30 grams, orally dosed, still more preferably
between 1 and 20 grams and most preferably between 1 and 18 grams,
typically 3 to 5 grams.
[0031] Where the sn-2 moiety is that of a .gamma.-linolenic acid
residue, the dose may be toward the higher end of these ranges,
particularly where the sn-1 and sn-3 moieties are relatively inert,
eg. being metabolically utilised acids such as saturated fatty
acids. Where the sn-2 moiety is that of a dihomomlinolenic acid
residue, the dose may be less, whilst where the sn-2 moiety is that
of an aracidonic acid residue, efficacy is higher, but dosing
should be more cautious, due to possibilities of unwanted side
effects at higher levels.
[0032] More preferably the method is characterised in that the
lipid is a monoglyceride, diglyceride or triglyceride containing
the at least one sn-2 .gamma.-linolenic acid,
dihomo-.gamma.-linolenic acid or arachidonic acid moiety of general
Formula I below:
##STR00001##
[0033] wherein R.sup.1 and R.sup.3 are independently selected from
hydrogen and acyl groups,
[0034] and R.sup.2 is selected from the group consisting of
.gamma.-linolenic acid, dihomo-.gamma.-linolenic acid and
arachidonic acid residues.
[0035] For the purpose of the present invention acyl groups are
defined as comprising at least one carbonyl group on the end of an
optionally substituted hydrocarbyl chain selected from alkyl and
alkenyl chains, the carbonyl group being directly attached by its
carbon to the oxygen of the glycerol residue shown in Formula
1.
[0036] Preferred acyl groups R.sup.1 and R.sup.3 are saturated
fatty acid moities of formula .dbd.CO--(CH.sub.2).sub.n--CH.sub.3,
wherein n is an integer selected from 1 to 22, more preferably
being 4 to 16, still more preferably being from 5 to 12, most
preferably being from 6 to 10. Particularly preferred acyl groups
are those of caprylic and capric acids, particularly being
1,3-dicaprylic or 1,3-dicapric glycerols having the
.gamma.-linolenic acid, dihomo-.gamma.-linolenic acid or
arachidonic acid moiety at the sn-2 position.
[0037] Preferred glycerides for use in the invention are
triglycerides.
[0038] U.S. Pat. No. 4,701,469 describes some potential
triglycerides for nutraceutical use that the present inventors have
determined may be used in the method of the invention, although it
only specifically describes 1,3-dioctanyl triglycerides wherein the
sn-2 acid is of an EFA, only 1,3-dioctanoyl eicosapenta glycerol is
described as having been prepared. These are said to useful in
inter alia immunomodulation, but although a number of diseases are
specified, use in immunosuppresion in neurodegeneration and MS are
not listed.
[0039] Whilst most preferred groups R.sup.1 to R.sup.3 for
inclusion in the compound of formula I are simple saturated fatty
acids or naturally occurring fatty acids with structural or
metabolic function, such as medium chain or long chain fatty acids,
there are other possibilities. Particularly preferred fatty acids
are those that are utilised primarily by the metabolism for
producing energy. Where fatty acids are structural, that is
utilised in membranes, they are conveniently such as
.gamma.-linolenic acid, linoleic acid, dihomo-.gamma.-linolenic
acid and arachidonic acid residues. By residue is meant the moiety
that remains after the fatty acid carboxyl group esterifies to one
of the hydroxy groups of the glycerol molecule.
[0040] Other preferred acids for sn-1 and sn-3 are selected from
fatty acids that are metabolised in the human to yield energy as
opposed to a fatty acid that is primarily directed to the
structural membrane pool: such preferred acids include oleic acid
and palmitic acid.
[0041] Where the sn-1 and sn-3 fatty acid chain (R.sup.1 and
R.sup.3) is unsaturated it may also be that of other essential
fatty acids, such as the n-3 acids such as stearidonic acid,
eicosapentaenoic acid and docosahexanoic acid. Where the fatty acid
is optionally substituted these will preferably be by hydroxy, oxo,
carboxyl, alkyl, alkenyl and alkoxy groups. The hydrocarbyl chain
is preferably one of from 1 to 30 carbon atoms in length, more
preferably from 4 to 28 carbon atoms in length, still more
preferably 4 to 24 carbon atoms in length. Most preferably the
hydrocarbyl chain is that of a fatty acid, more particularly a mono
or polyunsaturated fatty acid.
[0042] Many of the preferred lipids for use in the method of the
invention are known and may be prepared by chemical process known
in the art. For example, many are commercially available, such as
trigamma-linolenin, known as TLG, but herein referred to as GGG,
reflecting the identity of groups R.sup.1R.sup.2R.sup.3 where G
represents .gamma.-linolenic acid residues.
[0043] GGG is commercially available from Nu-Check-Prep Inc. EP
0300844 describes its synthesis using a base-catalysed
trans-esterification of triacetin with methyl gamma linolenate to
give a mixture containing 80% GGG, unreacted methyl
.gamma.-linolenate and 10% mono- and di-glycerides.
[0044] Triarachidin is known and small quantities can be obtained
commercially eg. from Sigma AAA has been synthesised from
arachidonic acid by using immobilised lipase patented for
angiogenisis-enhancing activity U.S. Pat. No. 4,888,324.
[0045] However, whilst the tri and di-.gamma.-linolenic acid,
dihomo-.gamma.-linolenic acid or arachidonic acid di or
triglycerides may be used, the present inventors prefer the use of
the mono-.gamma.-linolenic acid, dihomo-.gamma.-linolenic acid or
arachidonic acid sn-2 ester triglycerides because they administer
less of the immunomodulatory and proinflammatory fatty acids
.gamma.-linolenic acid, dihomo-.gamma.-linolenic acid or
arachidonic acid whilst retaining the enhanced activity that the
sn-2 .gamma.-linolenic acid, dihomo-.gamma.-linolenic acid or
arachidonic acid moiety provides with regard to the desired
membrane normalising and disease modifying effect.
[0046] Novel lipids which are preferred are accessible by processes
and methods set out in the Examples herein. Most preferred lipids
are those where there is just a single .gamma.-linolenic acid,
dihomo-.gamma.-linolenic acid or arachidonic acid moiety esterified
to the glycerol at sn-2, with the flanking sn-1 and sn-3 acids
being unsaturated medium chain or long chain acids.
[0047] Thus a further aspect of the present invention provides
novel lipids disclosed herein including compounds of formula II
##STR00002##
[0048] wherein R.sup.1 and R.sup.3 are the same and are
--C(O)(CH.sub.2).sub.nCH.sub.3 wherein n is selected from 4 to 14,
more preferably 6 to 10 and most preferably 7, 8 or 9 and R.sup.2
is selected from .gamma.-linolenyl, dihomo-.gamma.-linolenyl and
arachidonyl.
[0049] A further aspect of the present invention provides a method
for synthesis of a compound of general formula III
##STR00003##
[0050] wherein R.sup.1 and R.sup.3 are the same and are
--C(O)(CH.sub.2).sub.nCH.sub.3 wherein n is selected from 4 to 14,
more preferably 6 to 10 and most preferably 7, 8 or 9 and R.sup.2
is .gamma.-linolenyl residue, dihomo-.gamma.-linolenyl residue or
arachidonyl residue
comprising reacting 1,3-dihydroxyacetone with a compound of formula
X--C(O)(CH.sub.2).sub.nCH.sub.3 wherein X is selected from Cl, Br
and I, to give the corresponding
1,3-di-(C(O)(CH.sub.2).sub.nCH.sub.3) 2-keto compound reducing the
keto group to the corresponding
1,3-di-(C(O)(CH.sub.2).sub.nCH.sub.3) 2-ol and reacting that with
.gamma.-linolenyl chloride or dihomo-.gamma.-linolenyl chloride or
arachidonyl chloride.
[0051] A still further aspect of the present invention provides a
method for synthesis of a compound of general formula IV
##STR00004##
[0052] wherein R.sup.1 to R.sup.3 are the same and selected from
.gamma.-linolenyl residue, dihomo-.gamma.-linolenyl residue or
arachidonyl residue
[0053] comprising reacting the corresponding .gamma.-linolenyl
chloride, dihomo-.gamma.-linolenyl chloride or arachidonyl chloride
with glycerol.
[0054] Synthesis of some of these compounds is described below and
schemes shown in the figures below.
[0055] For example, a single-step esterification of glycerol using
GLA and a coupling agent, such as DCCI/DMAP
(1.1-Dicylcohexylcarbodiimide/4-dimethylaminopyridine) coupling
reagents may be carried out. This method gives a good yield but
generates impurities that, unless removed, make the final oil
cloudy. This may be circumvented by using a coupling agent such as
EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride)
which gives rise to water-soluble by-products that are easier to
remove. Jpn. Kokai Tokkyo Koho JP 05310638 A2 22 Nov. 1993 Heisei,
6 pp. describes the preparation of tri-.alpha.-linolenin (LnLnLn
where Ln is linoleic acid) using DCCI, and analogous but different
reaction.
[0056] A alternative approach provides a two-step sequence that
utilises reaction of GLA-Cl (prepared from .gamma.-linolenic acid
and oxalyl chloride) and glycerol in dichloromethane/pyridine with
good yields at scale-up to 250 g purified by column chromatography.
Jpn. Kokai Tokkyo Koho JP 04328199 A2 17 Nov. 1992 Heisei, 5 pp.
(Japan) Concentration of .alpha.-linolenic acid triglyceride by
flash chromatography. Ando, Yukiki, Watanebe, Yoichi, Takagi,
Yoshiaki (Nisshin Oil Mills Ltd, Japan) describes a related but
different technique for purification of tri-.alpha.-linolenin
(LnLnLn).
[0057] Comparative example tricaprin (glycerol tridecanate) is a
known compound commercially available from Sigma. It has been
prepared by reaction of methyl decanoate and sodium glyceroxide
with subsequent purification of the crude product by column
chromatography (see E. S. Lutton and A. J. Fehl, Lipids, 5, 90-99
(1970))
[0058] An alternative method involves the acid-catalysed reaction
of glycerol with decanoic acid followed by four crystallisations
(see L. H. Jenson and A. J. Mabis, Acta Cryst., 21, 770
(1966)).
[0059] The applicant further provides an improved process which
allows glycerol to react with more than 3 equivalents of decanoyl
chloride and purified the tricaprin product by
recrystallisation.
[0060] Further aspects of the present invention provide use of
triglyceride oils as described above for the manufacture of a
medicament for the treatment of neurodegenerative diseases as set
out for the method of the invention. Particularly preferred
medicaments are for the arresting and reversing of
neurodegeneration in multiple sclerosis of all types but
particularly relapsing remitting, primary progressive and chronic
progressive and the restoration, in part or completely, of neuronal
integrity function such as measured, eg. By MRI or CAT scan or by
EDSS score. Other TGF-.beta.1 responsive diseases may be treated as
set out previously.
[0061] The lipids for use in the present invention may be
administered by any of the conventional vehicles known in pharmacy.
Most conveniently they are administered as neat oils or in
admixture with foodstuffs, in the form of capsules containing such
oils, or in enterically coated forms. Other forms will occur to
those skilled in the art but Remington Pharmaceutical Sciences
19.sup.th Edition.
[0062] It will be realised by those skilled in the art that other
beneficial agents may be combined with the lipids for use in the
present invention or otherwise form part of a treatment regime with
the lipids. These might be ion channel blockers, eg. sodium channel
blockers, interferons (.alpha., .beta., or .gamma.), T-cell
depleters, steroids or other palliative agents. It will further be
realsied that where the immune and inflammatory responses are being
modulated, such combinations will need to be made carefully, given
the complex nature of these systems. However, given the delayed
response to the present oils, shorter acting agents might be
beneficial in the first months of treatment before the TGF-.beta.1
levels are normalised, as long as the additional treatment does not
impede this normalization process.
[0063] The synthesis of structured lipids for use in the present
invention is described below together with synthesis of comparative
examples. Some of these lipids are novel while others are known but
have not been used for the treatment of the invention.
[0064] The present invention will now be described by way of
Example only by reference to the following non-limiting Tables,
Examples and Figures. Further embodiments falling within the scope
of the invention will occur to those skilled in the art in the
light of these.
TABLES
[0065] Table 1: Shows the compositional % Total fatty acid content
of various triglyceride oils and protective effect in EAE.
[0066] Table 2: Shows the parameters of the three treatment groups
in high sn-2 GLA Borage Oil trial described in PCT/GB04/002089.
[0067] Table 3: Shows the effect of various forms of GLA on EAE
incidence and clinical score in SJL mice: lower score indicating
improved therapeutic effect.
[0068] Table 4: Shows the failure of enriched Blackcurrent oil, a
high GLA, but low sn-2-GLA, plant oil, to match fungal and Borage
oils in EAE.
FIGURES
[0069] FIG. 1: Shows spontaneous peripheral blood mononuclear cell
cytokine production in placebo and high sn-2 .gamma.-linolenic
acid, PCT/GB04/002089 trial oil treated human MS patients at 18
months.
[0070] FIG. 2: Shows the effect of placebo and low dose (5 g/day)
high sn-2 GLA Borage oil on human MS patient EDSS score as compared
to high dose (15 g/day) displayed as a histogram with months
treatment on the x axis.
[0071] FIG. 3: Shows the effect of placebo, low dose and high dose
high sn-2 GLA Borage oil on human MS patient Mean Relapse rate (%)
as histogram with months on x axis.
[0072] FIG. 4: Shows the reaction scheme for synthesis of a single
fatty acid triacylglyceride for use in the method and use of this
invention.
[0073] FIG. 5: Shows the reaction scheme for synthesis of control
compound tricaprin.
[0074] FIG. 6: Shows the reaction scheme for synthesis of CGC, a
mixed fatty acid triacylglyceride of the invention.
[0075] FIG. 7: Shows the reaction scheme for synthesis of
C-DHGLA-C, a mixed fatty acid triacylglyceride of the
invention.
[0076] FIG. 8: Shows the reaction scheme for synthesis of control
compound GCG, 1,3-dicapryl, 2-.gamma.-linolenic acid.
[0077] FIG. 9: Shows the reaction scheme for synthesis of C-AA-C, a
mixed fatty acid triacylglyceride of the invention.
[0078] FIGS. 10 to 19 show the results of EAE studies in SJL and
C57BL mice as set out in the examples below. (DHLA=DHGLA: A=AA)
EXAMPLES
High sn-2 Borage Oil (PCT/GB04/002089) Trial
[0079] Twenty-eight active relapsing-remitting (two relapses in the
preceding 18 months) multiple sclerosis patients (ages ranging from
18 to 65 yrs) were entered into a double-blind placebo controlled
trial to investigate the effects of encapsulated borage oil on
clinical activity and laboratory parameters over 18 months. This
oil was of high sn-2 .gamma.-linolenic (GLA) content (>40% of
sn-2 residues being .gamma.-linolenic acid) with low monene (eg.
erusic acid) content and had no added Vitamin E, a known
immunomodulator.
[0080] Patients were recruited from neurology out-patient clinics
at two inner city hospitals; hospital informed consent was obtained
on first (baseline) visit. Exclusion criteria include any form of
steroid or immunosuppressive drug treatment, pregnancy,
hyperlipidemia, regular use of aspirin or related drugs and vitamin
or fatty acid supplementation within the previous three months.
[0081] Only patients meeting all the following criteria were
included in the trial: (a) able to provide informed consent prior
to treatment, with the full understanding that consent may be
withdrawn at any time without prejudice; (b) male or female
out-patients aged 18 to 60 years inclusive; (c) have confirmed
diagnosis of clinically definite relapsing MS; (d) have had at
least three documented clinical relapses in the past two years; (e)
have a baseline Expanded Disability Scoring Scale (EDSS) score of
0.0-5.5 inclusive, provided they have well documented
exacerbations; and (f) healthy, apart from the MS-related symptoms,
as confirmed by the medical history, physical examination and
clinical chemistry, urine and haematological tests.
[0082] Patients were randomly allocated by the Pharmacy Department
to one of three groups each containing 12 patients: [0083] One
clinical group (n=12) to receive placebo (5 g of Polyethylene
Glycol 400) [0084] Second clinical group (n=12) to receive low-dose
(5 g) refined Borage officinalis [0085] Third clinical group (n=12)
to receive high-dose (15 g) refined Borage officinalis
[0086] Supplementation was in the form of one gram oil capsules
daily (5/day for low dose, 15/day high dose) for 18 months
duration. Borage officinalis oil and omega-6 polyunsaturated fatty
acids are food ingredients that are generally recognised as safe
for human consumption (GRAS). There are no classification or
labelling requirements under EC regulations. Clinical assessment
included: Extended Disability Scale Scores (EDSS) and clinical
relapse record. Venous blood (50 mls) was obtained for laboratory
studies on the 1.sup.st, 3.sup.rd, 6.sup.th, 12.sup.th, 15.sup.th,
and 18.sup.th month of supplementation.
[0087] The following biochemical and immunological parameters were
investigated on each visit for comparison with pre-treatment data
and between group data: [0088] Stimulated and unstimulated ex vivo
peripheral blood mononuclear cell cytokine production: changes in
TGF-.beta.1, IFN-.gamma., TNF-.alpha., IL-1.beta., IL-6 and
IFN-.beta., which are implicated in the pathogenesis of MS.
Cytokine and related gene expression. [0089] Soluble adhesion
molecules in serum particularly ICAM-1 and VCAM-1 [0090] Peripheral
blood mononuclear cell membrane fatty acids and plasma phospholipid
fatty acid composition. Results are shown in Tables 1 and 2 and
FIGS. 1 to 5.
[0091] The primary outcome parameter was the number of clinical
relapses between baseline (Month 0) and the end of treatment (Month
18). Secondary outcome parameters included: the time to first
clinical relapse; severity of relapses, as assessed by EDSS score
and the use of steroid treatment; and changes in EDSS at Month 3,
6, 9, 12, and 18 compared to baseline and defined as at least 1.0
point increase in the EDSS that is sustained for 3 months or at
least 1.5 point increase on the EDSS from the baseline EDSS that is
sustained for 3 months.
[0092] Eleven patients were in the placebo group, seven patients
had been taking low-dose Borage oil, and ten patients had been
taking high-dose Borage oil. The study drug was well-tolerated, and
there were no serious adverse events during the 18-month trial.
Isolation and Culture of PBMC
[0093] Heparinised whole blood was diluted with an equal volume of
Hanks' balanced salt solution (Sigma, UK) and the resulting diluted
blood layered onto Lymphoprep (Nycomed, Oslo, Norway). Following
density centrifugation at 800 g for 30 minutes the PBMC were
removed from the interface and diluted in Hanks' solution. The
cells were then washed twice by centrifugation for 10 minutes at
250 g. The resulting final pellet was then resuspended in culture
medium consisting of RPMI-1640 medium (Sigma, UK) supplemented with
2 mM L-glutamine, 100 U penicillin and 100 .mu.g streptomycin
(Sigma, UK) and 10% autologous plasma. 2.times.10.sup.6 per ml
PBMC, >95% viable as judged by trypan blue exclusion, were added
to tissue culture tubes (Bibby Sterilin Ltd, Stone, UK) and
incubated for 24 h at 37.degree. C. with 5% CO.sub.2. The
concentration of antigen, cell density and time of culture were all
determined in previous kinetic experiments to determine maximum
cytokine production (data not shown). Routine cytospin preparations
were also prepared for subsequent differential counts. Following
incubation the cells were removed from culture by centrifugation at
250 g for 10 minutes, the resulting supernatants were then removed,
aliquoted and stored at -70.degree. C.
Preparation of Plasma Samples
[0094] 10 ml of heparinised blood was spun at 250 g for 10 minutes.
The resulting plasma layer was then removed, aliquoted and stored
at -70.degree. C.
Detection of Pro-Inflammatory Cytokines
[0095] TNF-.alpha., IL-1.beta. and IFN-.gamma. in cell culture
supernatants and plasma were detected using commercially available
paired antibodies enabling cytokine detection in an ELISA format
(R&D systems Ltd, Abingdon, UK). The sensitivities for the
TNF-.alpha. and TFN-.gamma. ELISAs were 15.6-1000 pg/ml and 3.9-250
pg/ml for IL-1.beta..
Detection of Biologically Active TGF-.beta.1
[0096] Biologically active TGF-.beta.1 in cell culture supernatants
and plasma were detected using the commercially available E.sub.max
ELISA system with a sensitivity of 15.6-1000 pg/ml (Promega,
Southampton, UK).
Statistical Analysis
[0097] Differences in cytokine production were compared using
Student's t-test and Mann-Whitney U-test and were considered
significant when p values were less than 0.05.
Results
[0098] Two patients had developed diarrhoea, both of whom were
later confirmed to have been taking high-dose Borage oil. The
diarrhoea was mild in one patient, but was moderately severe in the
second patient, who later discontinued the study drug. The code was
not broken and the diarrhoea had stopped after the discontinuation
of the drug, but reappeared upon re-challenge. Therefore, this
patient was withdrawn from the trial. The remaining patients who
were treated with high-dose Borage oil showed excellent clinical
improvement on all primary and secondary outcome criteria. For
example, their mean EDSS score after 6 months of treatment had
improved from baseline EDSS (FIG. 1). More importantly, the mean
number of clinical relapses had significantly reduced after 6
months of treatment when compared to the number of relapses in the
placebo group (FIG. 2). In contrast, patients who had been
receiving low-dose Borage oil did not show any clinical improvement
when compared to the placebo group. In addition to its beneficial
effect on MS disease activity, high dose Borage oil provided some
symptomatic relief of muscle spasticity (stiffness) and painful
sensory symptoms, and also improved cognitive functions.
[0099] As can be seen for the figures below, relapse rate after 9,
12 and 18 months was down to zero in the high dose group. The
increase seen at 15 months was due to the patient dropping out of
this group.
[0100] The following are three brief case histories to illustrate
the therapeutic benefits of high dose high sn-2 GLA Borage oil. The
first two are from the trial while the third is a post trial
patient for whom MRI studies were obtained.
Patient 1 (Treatment):
[0101] The first patient was a 48 year old woman who had had a
clinically active, relapsing remitting MS for 9 years. She had
originally worked as a full-time administrator at the local Health
Authority, but she was unable to perform her duties because of her
severe MS. Therefore, she later worked as a part-time secretary,
but still had difficulties in mobilization because of muscles
stiffness and sensory disturbances. She was also experiencing
severe clinical relapses at an average of one relapse every nine
months. Most of these relapses had resulted in hospital admissions
for steroid therapy. In view of her active MS, she was recruited
into the Borage oil trial. There were no adverse events relating to
the study, and after taking the medication for four months, she
experienced good improvement in her walking and sensory
symptoms.
[0102] About nine months after therapy, she was well enough to
start full-time employment. In addition, she remained relapse-free
for the 18-month duration of the clinical trial. Following the
conclusion of the trial, the treatment code revealed that she was
taking high-dose Borage oil.
Patient 2 (Control):
[0103] The second case was a 46-year old woman who also had a
clinically active relapsing remitting MS for 8 years. She had
originally worked as a shop assistant, but became unemployed after
MS was diagnosed.
[0104] Her symptoms included difficulty with mobilisation and
painful sensory symptoms in both legs. She had experienced three
clinical relapses in the two years preceding the clinical trial,
and had been admitted to hospital twice for steroid therapy.
Consequently, she was recruited into the Borage oil trial, but her
walking continued to deteriorate. Six months into the trial, she
need to use a walking stick and also received treatment with
Baclofen to reduce low limb spasticity. Approximately ten months
after starting the Borage oil trial, she was admitted to hospital
because of severe clinical relapse, which was treated with
steroids. She later developed bladder disturbances and began to use
a wheelchair for long journeys. The treatment code was broken after
the conclusion of the 18-month trial, and she was found to have
been taking placebo. Since then, she started using a walking frame
for journeys exceeding 50 yards.
Patient 3: Treatment (Additional to Trial)
[0105] The third case was a 26 year-old man who was diagnosed with
definite MS in April 2001. His symptoms had started in 1999 when he
complained of diffuse, intractable pain affecting various parts of
his body, particularly the left side of the chest and abdomen. This
was followed by intermittent numbness in the hands and feet,
associated with fluctuating weakness. There were also distressing
bladder symptoms in the form of urinary frequency and urgency. The
diagnosis of MS in 2001 was based on his relapsing remitting
symptoms, and was confirmed by positive cerebrospinal fluid
analysis and magnetic resonance imaging (MRI) of the brain, which
showed multiple white matter abnormalities in both cerebral
hemispheres. Symptoms did not respond to various pharmaceutical
therapies.
[0106] In April 2003, oral supplementation with the present high
dose Borage oil was commenced. The patient reported dramatic
improvement in his symptoms within three months of starting this
oral supplementation. His painful sensory symptoms disappeared
completely. He reported no numbness or weakness since May 2003, and
noticed significant improvement in his bladder control. The oral
supplementation caused no adverse events. A repeat brain MRI was
undertaken to verify the reported improvement in Mr N's symptoms.
The repeat MRI showed a reduction in the size and distribution of
the white matter abnormalities.
EXAMPLES
Structured sn-2 Lipids
[0107] In all the examples below higher purity is obtained by use
of higher purity starting material .gamma.-linolenic,
dihomo-.gamma.-linolenic or arachidonic acid, such as is available
eg from Sigma Aldrich. GLA 95 indicates 95% pure .gamma.-linolenic
acid.
Synthesis Example 1
Synthesis of Trigammalinolenin
1) Acid Chloride Method
[0108] 2.0 g (7.2 mmol, 3.1 equiv) GLA95 (95% pure
.gamma.-linolenic acid) was dissolved in 10 ml DCM. 1.01 g (0.71
ml, 8.0 mmol, 3.4 equiv) oxalyl chloride in 5 ml DCM added dropwise
over 2-3 min under nitrogen. Stirred at RT overnight. Reaction
mixture concentrated in vacuo to remove DCM and excess oxalyl
chloride. This acid chloride was then added dropwise over 2-3 min
to a stirred mixture of 215 mg (2.3 mmol, 1 equiv) of glycerol,
0.5-8 ml (3.1 equiv) pyridine and 10 ml DCM under nitrogen. The
mixture was stirred at RT overnight. The pyridine hydrochloride
formed was then filtered off and washed with DCM. The solution was
washed 1.times.4 ml water, 0.1N HCl, 5% sodium bicarbonate and 5%
NaCl. Dried over magnesium sulphate, filtered and concentrated in
vacuo to a yellow oil. This oil was purified on a silica column
using 10% ether in hexane as eluting solvent. A clear colourless
oil was obtained, a sample of which was trans-esterified and
subsequently analysed by GC. The product contained 96.3% GLA
2) DCCI Method
[0109] 2.19 g GLA95 (3.15 equiv), 230 mg (1 equiv) glycerol, 153 mg
DMAP (0.5 equiv) were stirred in 10 ml DCM under nitrogen. 1.85 g
DCCI (3.6 equiv) in 5 ml DCM was added. The reaction mixture was
stirred at RT fader nitrogen overnight. The DCU formed was filtered
and washed with DCM. DCM washed 1.times.5 mls N HCl, water, 5%
sodium bicarbonate and water. Dried over magnesium sulphate,
filtered and concentrated in vacuo to an oil. This oil was then
purified on a silica column using 10% ether in hexane as eluting
solvent. 1.47 g (67%) of a slightly cloudy oil was obtained. A
sample of this product was trans-esterified and subjected to GC
analysis. The product contained 95.8% GLA.
Scale-Up
[0110] 20 g (0.072 mol, 3.1 equiv) of GLA95 (gamma linolenic acid,
95%) was dissolved in 100 ml DCM. 13.7 g (9.3 ml, 0.11 mol, 4.78
equiv) oxalyl chloride was added over 3-4 min under nitrogen. The
reaction mixture was stirred under nitrogen overnight. It was then
concentrated in vacuo to remove DCM and excess oxalyl chloride.
This oil was then added dropwise over ca 5 min to a stirred mixture
of 2.14 g (0.023 mol, 1 equiv) of glycerol, 100 ml DCM and 5.8 ml
(5.68 g, 0.072 mol, 3.1 equiv) of pyridine under nitrogen. 85 mg
(0.7 mmol, 0.03 equiv) of DMAP (4-dimethylaminopyridine) catalyst
was added. The mixture was stirred at RT overnight. Pyridine
hydrochloride was filtered off and washed with DCM. The DCM
solution was washed 1.times.25 ml: water, 10% sodium bicarbonate,
0.1N HCl, 5% NaCl. (Emulsions formed during this process,
especially at first). The DCM was dried over magnesium sulphate,
filtered and concentrated in vacuo to a brown oil (.about.21
g).
[0111] The oil was purified on a silica column using 5% ether in
hexane at first and then 10%. 15.6 g (77% yield) of a clear oil was
obtained. By tlc this material contained a small amount of free
GLA. (This material was repurified at a later date)
Large Scale-Up
[0112] The above reaction was repeated on 10 times scale. Thus, 200
g of GLA95, 1 L DCM, 137 g of oxalyl chloride, and 21.4 g of
glycerol were used. On the addition of the acid chloride the
reaction mixture was cooled in a cold water bath and the
temperature kept below 35.degree. C. 250 g of a brown oil were
produced. This was initially purified on a 500 gram silica column.
The oil was dissolved in 200 ml hexane and applied to the column.
The column was eluted at first with hexane, then 5% ether in hexane
and then 10%. Fractions were collected and analysed by tlc
eventually yielding two batches of oils. The first A (66 g)
contained a small amount of front running impurity and a little GLA
(slower running than TGL), the second fraction B (99 g) was clear
of front running impurity and contained a little GLA.
[0113] The large scale reaction was repeated using 169 g of GLA and
gave two fractions as above. This time there was 85 g of `A`
fraction and 54 g of `B` fraction. Both batches of `A` were
combined and re-purified on a 500 g silica column. The `B`
fractions were treated in a similar manner (15 g of material from
the small-scale reaction were also added to this batch).
[0114] Some fractions from the above were again re-purified to
eventually give 259 grams of oil. The oil was pumped down on a
rotary evaporator under high vacuum to constant weight--256 g. This
represents an overall yield of 65%.
Analysis of Product.
GC
[0115] A small sample was trans-esterified and subjected to GC
analysis:
[0116] The GLA content was 97.1%. The main impurity was linoleic
acid--1.91%.
[0117] Note: The original GLA95 that was used for the synthesis
contained 96.2% GLA and 2.42% linoleic acid.
HPLC
[0118] An HPLC method was developed using a reversed phase column
(Hypersil C18 4.6.times.100 mm), eluting with 80/20
acetonitrile/THF. Detection was by UV at 210 nm. This showed the
product to be a mixture of three components. The main peak (93.6%)
was the required product. A slower running impurity (representing
5.0% of the product) was probably a GGLI triglyceride (Ll=linoleic
acid). A second impurity was slightly faster running and
represented 1.4% of the product.
[0119] Note: Absorption at 210 nm varies considerably between
triglycerides of differing fatty acid content. For example
trigammalinolenin has a UV absorbtion 5-6 times greater than that
of trilinolenin
Summary
[0120] 254 g of glycerol tri-6,9,12-linolenate (gamma linolenic
acid triglyceride, trigammalinolenin, GGG) was prepared from 96.2%
GLA by a two-step acid chloride route. It is a clear, pale yellow
oil and was stored under nitrogen in the freezer. The GLA content
was 97.1% and no C20:1, C22:1, or C24:1 acids were detected). The
HPLC purity was 93.6%.
[0121] Synthesis of higher purity GGG would is readily achievable
using GLA 98 (98% .gamma.-linolenic acid: Scotia) or higher
starting material.
Comparative Lipid 1: synthesis Tricaprin (Glycerol
Tridecanoate)
Small Scale
[0122] Glycerol (3.0 g, 0.0325 mol, 1 eq) pyridine (8.1 ml, 0.10
mol, 3.1 eq) and dichloromethane (100 ml) were stirred at room
temperature under nitrogen. Decanoyl chloride (21 ml, 19.25 g, 0.10
mol, 3.1 equiv) was then added dropwise over 5 min, with external
cooling in a water bath to keep the temperature at 30-35.degree. C.
When the addition was complete 4-dimethylaminopyridine (DMAP (0.12
g, 1 mmol, 0.03 eq) was added and the mixture stirred under
nitrogen at room temperature overnight. The precipitated pyridine
hydrochloride was removed by filtration and washed with
dichloromethane. The combined washing and filtrate was then washed
with aqueous solutions (20 ml) of 5% sodium chloride, 5% sodium
bicarbonate, 0.1N hydrochloric acid, and 5% sodium chloride. The
dichloromethane layer was then dried over MgSO.sub.4 and the
solvent removed in vacuo. The residual oil crystallised on
standing. This material was recrystallised from isopropanol (40 ml)
to give 15.6 g (86% yield) of a waxy white solid.
Analysis
[0123] GC--99.8% pure
HPLC
[0124] (C18 4.6.times.100 mm, ACN/THF 85/15 1 ml/min, .lamda.210
nm)--94.9% pure
Large Scale
[0125] The above was repeated on 15 times the scale. Glycerol (45.0
g, 0.49 mol, 1 eq), pyridine (121.5 ml, 1.50 mol, 3.1 eq) and
dichloromethane (1.5 L) were stirred at room temperature under
nitrogen. Decanoyl chloride (315 ml, 288.8 g, 1.50 mol, 3.1 equiv)
was then added dropwise over 15 min, with external cooling in a
water bath to keep the temperature at 30-35.degree. C. When the
addition was complete 4-dimethylaminopyridine (DMAP (1.8 g, 15
mmol, 0.03 eq) was added and the mixture stirred under nitrogen at
room temperature overnight. The precipitated pyridine hydrochloride
was removed by filtration and washed with dichloromethane. The
combined washing and filtrate was then washed with aqueous
solutions (300 ml) of 5% sodium chloride, 5% sodium bicarbonate,
0.1N hydrochloric acid, and 5% sodium chloride. The dichloromethane
layer was then dried over MgSO.sub.4 and the solvent removed in
vacuo. The residual oil crystallised on standing. This material was
recrystallised from isopropanol (400 ml) to give 228 g (86% yield)
of a waxy white solid.
Analysis
[0126] GC--99.8% pure
[0127] HPLC
[0128] (C18 4.6.times.100 mm, ACN/THF 85/15 1 ml/min, .lamda.210
nm)--94.9% pure
[0129] A further batch was made and combined with the small-scale
batch above and recrystallised from isopropanol to give 44 g of
product. The above batches were combined (268 g) and
reanalysed:
[0130] GC
[0131] 99.9% pure
[0132] HPLC
[0133] 97.9%
Summary
[0134] 263 g of glycerol tridecanoate (tricaprin, CCC) was been
prepared from decanoyl chloride (98%) by a one-step process (scheme
given below). It is a white, low-melting solid and was stored under
nitrogen in the freezer. The C content was 99.9% of fatty acid
content and the HPLC purity was 97.9%.
Synthesis Example 2
1,3-Dicaprin 2-gammalinolenoate (Glycerol 1,3-didecanoate
2-octadecatri(6-Z,9-Z,12-Z)enoate or CGC)
[0135] This triglyceride is novel. Unlike CGC, its isomer CLnC
(Ln=.alpha.-linolenic acid), has been identified (see K. Long et al
Biotechnol. Lett., 20, 369-372 (1998).sub.7 and H. Mu, P. Kalo et
al, Eur. J. Lipid Sci. Technol., 102, 202-211 (2000). as a
component of coconut oil. In addition, CLxC (Lx=a linolenic acid of
unspecified double bond position) has been described (see J. Gresti
et al. J. Dairy Sci., 76, 1850-1869 (1993)),
[0136] The two intermediates used in the synthesis of CGC are known
(see L. El Kihel et al Arzneim-Forsch./Drug Res., 46, 1040-1044
(1996) and U.S. Pat. No. 4,178,299. The last step described below
is novel and the first two stages are also inventive since they are
more suitable for large scale production than those previously
reported.
[0137] CGC was prepared by reaction of 1,3-Dicaprin with
GLA-chloride in dichloromethane-pyridine. 1,3-Dicaprin was prepared
by sodium borohydride reduction of 1,3-didecanoyloxypropan-2-one,
which was in turn prepared by reaction of decanoyl chloride with
1,3-dihydroxyacetone. The intermediate 1,3-dicaprin must be handled
with care since it can undergo acyl migration on exposure to acids,
bases and heat. An older method of making 1,3-dicaprin has been
described (see A. P. J. Mank et al Chem. Physics Lipids, 16,
107-114 (1976).
[0138] A versatile, flexible synthesis of 1,3-diglycerides and
triglycerides. by catalysed addition of decanoic acid to a glycidol
ester (from epichlorohydrin) is less attractive because of more
severe reaction conditions and acyl migration problems. The final
product, CGC, was purified by careful column chromatography on
silica which removed by-products.
Small Scale
[0139] 1,3-didecanoyloxypropan-2-one
[0140] Decanoyl chloride (40.0 ml, 36.8 g, 0.19 mol, 1.98 equiv)
was added dropwise over 10-15 min to a stirred suspension of
1,3-dihydroxyacetone dimer (8.68 g, 0.048 mol, 1.0 equiv), pyridine
(15.6 ml, 0.19 mol), 4-dimethylaminopyridine (0.18 g, 0.0014 mol,
0.03 equiv) and dichloromethane (DCM, 150 ml) at room temperature
under nitrogen. The temperature of the reaction mixture was kept
below 30.degree. C. by cooling in a cold water bath. The reaction
mixture was stirred at RT under nitrogen overnight. The pyridine
hydrochloride formed was removed by filtration and washed with DCM.
The combined filtrate and washings were then washed with 1.times.25
ml portions of 5% NaCl, 5% NaHCO.sub.3, 0.1N HCl, 5% NaCl. The
solution was then dried over MgSO.sub.4 and concentrated in vacuo
to a yellowish semi-solid. This was then crystallised from methanol
(150 ml) to give a white solid. The yield was 28.2 g (73%).
[0141] 1,3-Dicaprin
[0142] The above ketone (28.2 g, 0.071 mol) was dissolved in
tetrahydrofuran (THF, 200 ml). Water (10 ml) was then added, the
solution cooled to 5.degree. C., and sodium borohydride (5.38 g,
0.14 mol) added portionwise below 10.degree. C. The reaction
mixture was stirred at RT for 1 h and then concentrated in vacuo to
remove THF. The residue was partitioned between ethyl acetate and
5% sodium chloride solution. The aqueous phase was re-extracted
with ethyl acetate and the combined extracts dried over MgSO.sub.4
and concentrated in vacuo to a waxy solid. This was crystallised
twice from hexane to give 11.2 g (40%) of a white solid. (99%+pure
by HPLC)
[0143] 1,3-Dicaprin 2-gammalinolenoate (CGC)
[0144] Gamma-linolenic acid (GLA95, 8.34 g, 0.03 mol) was dissolved
in dichloromethane (DCM, 60 ml). The resulting solution was stirred
at RT under nitrogen and oxalyl chloride (3.9 ml, 5.67 g, 0.044
mol) added dropwise over 5 mins. The mixture was stirred at RT
overnight and then concentrated in vacuo to remove DCM and excess
oxalyl chloride. The residual oily acid chloride (GLA-Cl) was then
added dropwise over 15 min (ice/water cooling) to a stirred
solution of 1,3-dicaprin (11.2 g, 0.028 mol), DCM (50 ml), pyridine
(2.42 ml, 2.37 g, 0.03 mol) and 4-dimethylaminopyridine (0.10 g,
0.0008 mol, 0.03 equiv) at 10-15.degree. C. The temperature was
maintained by ice-water cooling. The reaction mixture was stirred
at RT under nitrogen overnight. Pyridine hydrochloride was removed
by filtration and washed with DCM. The combined washing and
filtrate was washed with 1.times.20 ml portions of 5% NaCl, 5%
NaHCO.sub.3, 0.1N HCl, 5% NaCl. The solution was then dried over
MgSO.sub.4 and the solvent removed in vacuo. The residual brown oil
was purified by column chromatography on silica. Elution with
hexane and then with 5%-ether/hexane gave 10.3 g (56%) of a
colourless oil. The structure was confirmed by .sup.13C NMR and
GLC. Purity determined by HPLC.
Large Scale
[0145] 1,3-didecanoyloxypropan-2-one
[0146] Decanoyl chloride (272 ml, 250 g, 1.3 mol, 2 equiv) was
added dropwise over 10-15 min to a stirred suspension of
1,3-dihydroxyacetone dimer (59.1 g, 0.65 mol, 1.0 equiv), pyridine
(106 ml, 103.7 g 1.3 mol), 4-dimethylaminopyridine (2.38 g, 0.02
mol, 0.03 equiv) and dichloromethane (DCM, 750 ml) at room
temperature under nitrogen. The temperature of the reaction mixture
was kept below 30.degree. C. by cooling in a cold water bath. The
reaction mixture was stirred at RT under nitrogen overnight. The
pyridine hydrochloride formed was removed by filtration and washed
with DCM. The combined filtrate and washings were then washed with
1.times.150 ml portions of 5% NaCl, 5% NaHCO.sub.3, 0.1N HCl, 5%
NaCl. The solution was then dried over MgSO.sub.4 and concentrated
in vacuo to a yellowish semi-solid. This was then crystallised from
methanol (500 ml) to give a white solid. The yield was 158 g
(60%).
[0147] 1,3-Dicaprin
[0148] The above ketone (158 g, 0.40 mol) was dissolved in
tetrahydrofuran (THF, 2.25 L). Water (50 ml) was then added, the
solution cooled to 5.degree. C., and sodium borohydride (5.66 g,
1.5 eq) added portionwise below 10.degree. C. The reaction mixture
was monitored by HPLC (C18, eluted with ACN at 1 ml/min .lamda.210
nm) (Note: only about 4.5 g of the borohydride was in fact added,
as all SM had reacted). The reaction mixture was stirred at RT for
1 h and then concentrated in vacuo to remove THF. The residue was
partitioned between ethyl acetate and 5% sodium chloride solution.
The aqueous phase was re-extracted with ethyl acetate and the
combined extracts dried over MgSO.sub.4 and concentrated in vacuo
to a waxy solid. This was crystallised twice from hexane to give 96
g (60%) of a white solid. (98% pure by HPLC)
[0149] 1,3-Dicaprin 2-gammalinolenoate (CGC)
[0150] Gamma-linolenic acid (GLA95, 120.2 g, 0.43 mol) was
dissolved in dichloromethane (DCM, 750 ml). The resulting solution
was stirred at RT under nitrogen and oxalyl chloride (55.7 ml, 82.3
g, 0.65 mol, 1.5 eq) added dropwise at 15-20.degree. C. over 15
mins. The mixture was stirred at RT overnight and then concentrated
in vacuo to remove DCM and excess oxalyl chloride. The residual
oily acid chloride (GLA-Cl) was then added dropwise over 30-40 min
at 10-15.degree. C. (ice/water cooling) to a stirred solution of
1,3-dicaprin (164.7 g, 0.41 mol), DCM (650 ml), pyridine (33.3 ml,
32.5 g, 0.41 mol) and 4-dimethylaminopyridine (1.50 g, 0.012 mol,
0.03 equiv) at 10-15.degree. C. The reaction mixture was stirred at
RT under nitrogen overnight. Pyridine hydrochloride was removed by
filtration and washed with DCM. The combined washing and filtrate
was washed with 1.times.150 ml portions of 5% NaCl, 5% NaHCO.sub.3,
0.1N HCl, 5% NaCl. The solution was then dried over MgSO.sub.4 and
the solvent removed in vacuo to a brown oil (275 g).
[0151] The scale of the above three reactions was the largest on
which each was carried out. The borohydride reduction produced, in
addition to 1,3-dicaprin, a by-product in variable yield. The
presence of this by-product greatly affected the yield of the
isolated pure 1,3-dicaprin; the by-product could only be removed by
two crystallisations of the crude product. Since the final product,
CGC, is purified by column chromatography, it is imperative that
the 1,3-dicaprin used for the final step is as pure as
possible!
[0152] From the above reactions about 440 g of crude CGC was
produced as a brown oil. This was purified on a series of silica
columns using hexane followed by 2-3% ether/hexane. The
purification required 7 or 8 columns, using 3-4 kilos of silica,
25-30 litres of solvent (recycling solvent kept this figure low--in
practice over 100 litres were used)
[0153] The resulting product, a clear almost colourless oil, (264
grams) was 96.4% pure by HPLC (C18 4.6.times.100 mm, eluted with
85/15 ACN/THF at 1 ml/min. UV detection .lamda.210 nm). GC
indicated a ratio of 66.1/33.9 C/G. NMR analysis indicated the
product to have the correct CGC structure and be of at least 95%
purity: .delta..sub.c (500 MHz, CDCl.sub.3) 172.65 (2-GLA
carbonyl), 173.25 (1,3-capric carbonyl). Ratio of signals 2.04:1.
No signal at 173.0 indicating absence of 1.3-GLA. Trace signal at
172.79 could be oleic acid impurity in GLA or 2-capric acid.
Summary
[0154] 264 g of glycerol 1,3-didecanoate-2-gammalinolenoate
(1,3-dicaprin-2-GLA, CGC) has been prepared from decanoyl chloride
(98%) by a three-step process (scheme given below). It is an almost
colourless oil (slight yellow tinge) and was stored under nitrogen
in the freezer. The HPLC purity was 96.4%.
Synthesis Example 3
1,3-Didecanoate-2-dihomo-.gamma.-linolenoate (Glycerol
1,3-didecanoate2-eicosa-(8Z,11Z,14Z)-trienoate or C(DHLA)C
[0155] This triglyceride appears to be novel--no reference to it
has been found.
[0156] DHLA (3.93 g, 12.8 mmol, 1 eq) was dissolved in
dichloromethane (DCM, 20 ml) and stirred at room temperature under
a nitrogen atmosphere. Oxalyl chloride (1.69 ml, 2.46 g, 19.4 mmol,
1.5 eq) was added dropwise over 1-2 min, and left stirring at room
temperature overnight. The resulting solution was concentrated in
vacuo to remove DCM and excess oxalyl chloride. The residual oily
acid chloride (DHLA-Cl) was then added dropwise over 5 min at
25.degree. C. to a stirred mixture of 1,3-dicaprin (4.91 g, 12.2
mmol, 0.95 eq), pyridine (0.98 ml, 0.96 g 12.1 mmol, 0.95 eq) and
4-dimethylaminopyridine (DMAP, 8 mg, 0.07 mmol, 0.03 eq). The
reaction temperature rose to 32.degree. C. during the addition. The
reaction was stirred at 30-35.degree. C. and monitored by HPLC. The
reaction was stopped after 1.5 h. The precipitated pyridine
hydrochloride was filtered off and washed with DCM. The combined
filtrate and washings were then washed with 1.times.10 ml portions
of 5% NaCl, 5% NaHCO.sub.3, 0.1N HCl, 5% NaCl. The solution was
then dried over MgSO.sub.4 and concentrated in vacuo to give the
crude product as a yellow-orange oil (8.9 g, 86% purity by HPLC).
This oil was chromatographed on silica gel (250 g). Elution with
hexane and diethyl ether-hexane (2-6%) gave a purified product as a
pale yellow oil. Treatment of a hexane solution with decolourising
charcoal and removal of the solvent in vacuo gave C(DHLA)C as a
clear colourless oil (6.48 g, 98.9% purity by HPLC).
Synthesis Example 4
Triarachidin (Glycerol trieicosotetra5-Z,8-Z,11-Z,14Z-eneoate) or
AAA
[0157] Arachidonic acid (50.9 g, 0.17 mol, 3 eq) was dissolved in
dichloromethane (DCM, 175 ml) and stirred at room temperature under
a nitrogen atmosphere. Oxalyl Chloride (21.9 ml, 31.9 g, 0.25 mol,
4.4 eq) was then added to the stirred solution over 5 min and the
temperature increased by 4.degree. C. The resulting yellow-green
mixture was stirred at RT overnight and then concentrated in vacuo
to remove DCM and excess oxalyl chloride. The residual oily acid
chloride (A-Cl) was then added dropwise over 15 min to a pre-warmed
(25.degree. C.) stirred mixture of glycerol (5.11 g, 0.055 mol, 1
eq), pyridine (13.5 ml, 13.2 g, 0.17 mol, 3 eq) and 4-dimethylamino
pyridine (DMAP, 0.20 g, 0.002 mol, 0.03 eq). The temperature of the
reaction mixture rose to 42.degree. C. during the addition and a
gentle reflux was observed. The mixture was stirred at
30-40.degree. C. and monitored by HPLC. After 2 h, no further
product formation was observed. The precipitated pyridine
hydrochloride was filtered off and washed with DCM. The combined
filtrate and washings were then washed with 1.times.50 ml portions
of 5% NaCl, 5% NaHCO.sub.3, 0.1N HCl, 5% NaCl. The solution was
then dried over MgSO.sub.4 and concentrated in vacuo to give the
crude product as a yellow-orange oil (57 g). This oil was purified
by column chromatography on silica gel (ca. 600 g). Elution with
hexane and diethyl ether (2-4%)-hexane gave 22.8 g of the product
as an oil. A second batch (17.8 g) was produced from 39.8 g of
arachidonic acid, The two batches were combined and residual
solvents removed under vacuo to give 40.5 g (43%) of a mobile pale
yellow oil. HPLC purity 84.8% GLC analysis 94.3% AA (arachidonic
acid).
Comparative Lipid 2
1,3-Di(octadeca-6Z,9Z,12Z-enoyloxy)propan-2-one
[0158] (1,3-Di(.gamma.-linolenoyloxy)propan-2one, GonG) Stage 1
intermediate for GCG
[0159] Gamma-linolenic acid (GLA95, 197 g, 0.71 mol, 2.2 equiv) was
dissolved in dichloromethane (DCM, 600 ml) contained in a 2 L 3
necked flask. The resulting solution was stirred at RT under
nitrogen. Oxalyl chloride (93 ml, 136 g, 1.07 mol, 3.3 eq) was
added dropwise at 15-20.degree. C. over 15 min. The brown mixture
was stirred at RT overnight and then concentrated in vacuo to
remove DCM and excess oxalyl chloride. The residual oily acid
chloride (GLA-Cl) was then added dropwise over 20 min at 25.degree.
C. to a stirred mixture of 1,3-dihydroxyacetone dimer (28.99 g,
0.32 mol, 1.0 equiv), pyridine (52 ml, 50.9 g 0.64 mol, 2.0 equiv),
4-dimethylaminopyridine (2.36 g, 0.02 mol, 0.06 equiv) and
dichloromethane (DCM, 600 ml) at room temperature under nitrogen.
The temperature of the reaction mixture was allowed to rise to
40.degree. C. and the mixture was stirred for a further 2 h under
nitrogen (monitored by HPLC). The pyridine hydrochloride that
formed was removed by filtration and washed with DCM. The combined
filtrate and washings were then washed with 1.times.150 ml portions
of 5% NaCl, 5% NaHCO.sub.3, 0.1N HCl, 5% NaCl. The solution was
then dried over MgSO.sub.4 and concentrated in vacuo to give ca.
200 g of a yellow oil. This material was partially purified by
column chromatography on silica (600 g). Elution with hexane and
then ether-hexane mixtures (2-15%) gave 42 g of a pale yellow oil.
This oil was chromatographed again on silica (600 g) and eluted
with hexane and then 1-10% ether-hexane to give the product (95.9%
purity) as a pale yellow oil. The yield was 42 g (17%).
1,3-Di(octadeca-6Z,9Z,12Z-enoyloxy)propan-2-ol
[0160] (1,3-Di(.gamma.-linolenoyloxy)propan-2-ol or
1,3-Di-gamma-linolenin GolG) Stage 2 intermediate for GCG
[0161] 13-Di(.gamma.-linolenoyloxy)propan-2-one (GonG, 25.5 g, 0.04
mol, 1 eq) was dissolved in tetrahydrofuran (THF, 375 ml) and water
(12.7 ml). The solution was vigorously stirred at -20.degree. C.,
care was taken to keep the reaction temperature below -15.degree.
C. Sodium borohydride (790 mg, 0.02 mol, 1.25-eq) was added
portionwise to the stirred solution over 3 mins. The reaction
mixture was stirred for a further 10 mins at -20.degree. C. and
hexane (380 ml) then added. The still cold mixture was then washed
with water (2.times.200 ml), dried over MgSO.sub.4 and concentrated
in vacuo to give the title compound as a brown oil (27.8 g) (82.6%
purity by HPLC, less than 1% migrated material). Another batch was
prepared and combined with the first to give 50 g of crude product.
This material was purified by column chromatography on silica gel
(400 g). Elution with hexane and diethyl ether-hexane mixture
(5-20%) gave 36.1 g of the product as a pale oil (91.5%
purity).
[0162] (N.B. Care should be taken not to leave the compound on the
silica overnight as it appears to undergo a migration reaction,
giving GGoI)
1,3-Di-.gamma.-linolenin 2-decanoate (Glycerol
1,3-dioctadeca-(6Z,9Z,12Z)-trienoate 2-decanoate or GCG)
[0163] Decanoyl chloride (13.5 ml, 12.4 g, 0.065 mol, 1.1 eq) was
added to a stirred solution of 1,3-di-.gamma.-linolenin (36.1 g,
0.059 mol, 1 eq), dry pyridine (5.7 ml, 5.6 g, 0.07 mol, 1.1 eq),
4-dimethylaminopyridine (0.2 g, 0.002 mol, 0.03 eq) and
dichloromethane (DCM, 150 ml) over ca. 10 mins. The temperature was
maintained at 17.degree. C.-23.degree. C. during addition. The
reaction was then stirred at 30-35.degree. C. and monitored by
HPLC. A further 1-2 ml of decanoyl chloride was added after 1 h,
1.5 h and 2 h. Further addition appeared to increase the conversion
to product as determined by HPLC. After 3 h the reaction mixture
was filtered and the filtrate washed with DCM. The combined
filtrate and washings were then washed with 1.times.50 ml portions
of 5% NaCl, 5% NaHCO.sub.3, 0.1N HCl, 5% NaCl. The DCM extract was
then dried over MgSO.sub.4 and concentrated in vacuo to give the
crude product as a pale yellow oil; (purity 90% by HPLC). The oil
was purified by column chromatography on silica gel (600 g).
Elution with hexane and diethyl ether-hexane (1.5-2.5 then 3.5%)
gave the product (GCG.) as a clear oil; (35.5 g 96.1% purity by
HPLC). Another 7.5 g of pure lipid was obtained by further
chromatography on some of the fractions containing only a small
amount of impurity.
Synthesis Example 5
1,3-Dicaprin 2-arachidonate (Glycerol 1,3-didecanoate
2-eicosatetra-(5-Z,8-Z,11-Z,14-Z)enoate or CAC)
[0164] This triglyceride is known. CAC has been identified as a
constituent of lymph lipids following administration of safflower
oil to rats. WO 03 013,497 describing an arachidonic acid
containing triglyceride (produced by culturing Mortierella alpina)
useful for diseases caused by brain hypofunction, but specifically
for cognition enhancement. The two intermediates used in the
synthesis of CAC are known.
[0165] The synthesis of CAC from 1,3-dicaprin, and the purification
of this are all novel.
[0166] Here CAC was prepared by reaction of 1,3-Dicaprin with
arachidonyl chloride in dichloromethane-pyridine. 1,3-Dicaprin was
prepared by sodium borohydride reduction of
1,3-didecanoyloxypropan-2-one, which was in turn prepared by
reaction of decanoyl chloride with 1,3-dihydroxyacetone. The
intermediate 1,3-dicaprin must be handled with care since it can
undergo acyl migration on exposure to acids, bases and heat. An
older method.sup.6 of making 1,3-dicaprin, by catalysed addition of
decanoic acid to a glycidol ester (from epichlorohydrin) was deemed
less attractive because of more severe reaction conditions and acyl
migration problems. The final product, CAC, was purified by careful
column chromatography on silica which removed by-products.
1,3-Dicaprin 2-arachidonate (CAC)
[0167] Arachidonic acid (AA96, 8.34 g, 0.03 mol) was dissolved in
dichloromethane (DCM, 60 ml). The resulting solution was stirred at
RT under nitrogen and oxalyl chloride (3.9 ml, 5.67 g, 0.044 mol)
added dropwise over 5 mins. The mixture was stirred at RT overnight
and then concentrated in vacuo to remove DCM and excess oxalyl
chloride. The residual oily acid chloride (GLA-Cl) was then added
dropwise over 15 min (ice/water cooling) to a stirred solution of
1,3-dicaprin (11.2 g, 0.028 mol), DCM (50 ml), pyridine (2.42 ml,
2.37 g, 0.03 mol) and 4-dimethylaminopyridine (0.10 g, 0.0008 mol,
0.03 equiv) at 10-15.degree. C. The temperature was maintained by
ice-water cooling. The reaction mixture was stirred at RT under
nitrogen overnight. Pyridine hydrochloride was removed by
filtration and washed with DCM. The combined washing and filtrate
was washed with 1.times.20 ml portions of 5% NaCl, 5% NaHCO.sub.3,
0.1N HCl, 5% NaCl. The solution was then dried over MgSO.sub.4 and
the solvent removed in vacuo. The residual brown oil was purified
by column chromatography on silica. Elution with hexane and then
with 5% ether/hexane gave 10.3 g (56%) of a colourless oil. The
structure was confirmed by .sup.13C NMR and GLC. Purity determined
by HPLC.
Large Scale
[0168] 1,3-didecanoyloxypropan-2-one
[0169] Decanoyl chloride (272 ml, 250 g, 1.3 mol, 2 equiv) was
added dropwise over 10-15 min to a stirred suspension of
1,3-dihydroxyacetone dimer (59.1 g, 0.65 mol, 1.0 equiv), pyridine
(106 ml, 103.7 g 1.3 mol), 4-dimethylaminopyridine (2.38 g, 0.02
mol, 0.03 equiv) and dichloromethane (DCM, 750 ml) at room
temperature under nitrogen. The temperature of the reaction mixture
was kept below 30.degree. C. by cooling in a cold water bath. The
reaction mixture was stirred at RT under nitrogen overnight. The
pyridine hydrochloride formed was removed by filtration and washed
with DCM. The combined filtrate and washings were then washed with
1.times.150 ml portions of 5% NaCl, 5% NaHCO.sub.3, 0.1N HCl, 5%
NaCl. The solution was then dried over MgSO.sub.4 and concentrated
in vacuo to a yellowish semi-solid. This was then crystallised from
methanol (500 ml) to give a white solid. The yield was 158 g
(60%).
1,3-Dicaprin
[0170] The above ketone (158 g, 0.40 mol) was dissolved in
tetrahydrofuran (THF, 2.25 L). Water (50 ml) was then added, the
solution cooled to 5.degree. C., and sodium borohydride (5.66 g,
1.5 eq) added portionwise below 10.degree. C. The reaction mixture
was monitored by HPLC(C18, eluted with ACN at 1 ml/min .lamda.210
nm) (Note: only about 4.5 g of the borohydride was in fact added,
as all SM had reacted). The reaction mixture was stirred at RT for
1 h and then concentrated in vacuo to remove THF. The residue was
partitioned between ethyl acetate and 5% sodium chloride solution.
The aqueous phase was re-extracted with ethyl acetate and the
combined extracts dried over MgSO.sub.4 and concentrated in vacuo
to a waxy solid. This was crystallised twice from hexane to give 96
g (60%) of a white solid. (98% pure by HPLC)
1,3-Dicaprin 2-arachidonate (CAC)
[0171] Arachidonic acid (AA96, 78.8 g, 0.26 mol) was dissolved in
dichloromethane (DCM, 425 ml). The resulting solution was stirred
at RT under nitrogen and oxalyl chloride (33.9 ml, 49.4 g, 0.39
mol, 1.5 eq) added dropwise at 15-20.degree. C. over 15 mins. The
mixture was stirred at RT overnight and then concentrated in vacuo
to remove DCM and excess oxalyl chloride. The residual oily acid
chloride (GLA-Cl) was then added dropwise over 30-40 min at
10-15.degree. C. (ice/water cooling) to a stirred solution of
1,3-dicaprin (94.2 g, 0.24 mol), DCM (450 ml), pyridine (19.1 ml,
18.6 g, 0.24 mol) and 4-dimethylaminopyridine (1.72 1.50 g, 0.014
mol, 0.06 equiv) at 10-15.degree. C. The reaction mixture was
stirred at RT under nitrogen overnight. Pyridine hydrochloride was
removed by filtration and washed with DCM. The combined washing and
filtrate was washed with 1.times.150 ml portions of 5% NaCl, 5%
NaHCO.sub.3, 0.1N HCl, 5% NaCl. The solution was then dried over
MgSO.sub.4 and the solvent removed in vacuo to a brown oil (171
g).
[0172] The scale of the above three reactions was the largest on
which each was carried out. The borohydride reduction produced, in
addition to 1,3-dicaprin, a by-product in variable yield. The
presence of this by-product greatly affected the yield of the
isolated pure 1,3-dicaprin; the by-product could only be removed by
two crystallisation of the crude product. Since the final product,
CAC, is purified by column chromatography, it is imperative that
the 1,3-dicaprin used for the final step is as pure as
possible!
[0173] 412 g of crude CAC was produced as a brown oil from the
above reactions. This material was purified on a series of silica
columns using hexane followed by 1-3% ether/hexane. The
purification required 7 or 8 columns, using 3-4 kilos of silica,
and 100 litres of solvent.
[0174] The resulting product, a clear very pale yellow oil, (295
grams) was 95.8% pure by HPLC(C18 4.6.times.100 mm, eluted with
85/15 ACN/THF at 1 ml/min. UV detection .lamda.210 nm). GC
indicated a ratio of 66.3/32.1 C/A (1.6% impurity carried through
from the 5% impurity in A).
Summary
[0175] 295 g of glycerol 1,3-didecanoate-2-arachidonate
(1,3-dicaprin-2-AA, CAC) has been prepared from decanoyl chloride
(98%) and Arachidonic acid (95%) by a three-step process (scheme
given below). It is a very pale yellow oil and was stored under
nitrogen in the freezer. The HPLC purity is 95.8%.
Synthesis Example 7
1,3-Dioleoin 2-gammalinolenoate (Glycerol 1,3-dioctadeca-9Z-enoate
2-octadecatri(6-Z,9-Z,12-Z)enoate or OGO)
[0176] This triglyceride is known: a carbon-14 labelled version has
been prepared by normal chemical synthesis and the normal
unlabelled form by biochemical synthesis using lipases. OGO is not
a major component of borage oil but its isomer OOG is (9%). The two
intermediates used in the synthesis of CGC are known. The last step
is novel.
[0177] The use of, the synthesis of from 1,3-dioleoin, and the
purification of CGC are all believed novel. In general
triglycerides CXC are preferred over OXO on patent and cost of
goods grounds.
[0178] OGO was here prepared by reaction of 1,3-Doleoin with
GLA-chloride in dichloromethane-pyridine. 1,3-Diolein was prepared
by sodium borohydride reduction of 1,3-dioleoylpropan-2-one, which
was in turn prepared by reaction of oleoyl chloride with
1,3-dihydroxyacetone. The intermediate 1,3-dioleolin must be
handled with care since it can undergo acyl migration on exposure
to acids, bases and heat. Older methods.sup.7,8 of making
1,3-dioleoin, via mono-tritylglycerols or glycidyl esters was
deemed less attractive because of more steps and acyl migration
problems. The final product, OGO, was purified by careful column
chromatography on silica which removed by-products.
Small Scale
[0179] 1,3-dioleoylpropan-2-one
[0180] 155.1 g Oleic acid (155.1 g, 0.55 mol, 1.0 equiv, Croda 094
RV05192) was dissolved in dichloromethane (DCM, 500 ml). The
solution was stirred at room temperature (RT) under nitrogen and
104.4 g (1.5 eq 71 mls) oxalyl chloride (104.4 g, 71.8 ml, 0.82
mol, 1.5 equiv) was added dropwise at 15-20.degree. C. over about
20 mins. The reaction mixture was stirred overnight at RT. The
excess oxalyl chloride and DCM were removed in vacuo and the
residual oily acid chloride was added dropwise over 15-20 min to a
stirred suspension of 1,3-dihydroxyacetone dimer (22.5 g, 0.24 mol
of monomer), pyridine (40.4 ml), 4-dimethylaminopyridine (1.83 g)
and dichloromethane (DCM, 500 ml) at room temperature under
nitrogen. The temperature of the reaction mixture was kept below
20.degree. C. by cooling in an ice/water bath. The reaction mixture
was stirred at RT under nitrogen overnight. The pyridine
hydrochloride formed was removed by filtration and washed with DCM.
The combined filtrate and washings were then washed with
1.times.150 ml portions of 5% NaCl, 5% NaHCO.sub.3, 0.1N HCl, 5%
NaCl. The solution was then dried over MgSO.sub.4 and concentrated
in vacuo to an orange/brown semi-solid. This was triturated in
methanol and stored in the `fridge overnight. The solid deposited
(90% pure by HPLC) was then crystallised from diisopropyl ether
(DIPE) and methanol to give 51.3 g of an off white solid which was
95% pure by HPLC. Further crystallisation from DIPE/methanol
yielded 41 g (27%) of a 98% pure product.
1,3-Diolein
[0181] The above ketone (32.8 g, 0.053 mol) was dissolved in
tetrahydrofuran (THF, 250 ml). Water (10 ml) was then added, the
solution cooled to 5.degree. C., and sodium borohydride added
portionwise below 10.degree. C. The reaction was followed by HPLC
(C18, ACN/THF 90/10 at 2 mls/min, .lamda.210 nm) and after all the
starting ketone had reacted the addition of the borohydride was
stopped (830 mg, 0.022 mol added). The mixture was then
concentrated in vacuo to remove THF. The residue was partitioned
between ethyl acetate and water. The aqueous phase was re-extracted
with ethyl acetate and the combined extracts dried over MgSO.sub.4
and concentrated in vacuo to an oil (.about.33 g) which solidified
on cooling. The product (68% pure by HPLC) was crystallised from
100 ml hexane at -20.degree. C. (in the freezer) overnight This
product (92% pure 21.1 g) was recrystallised from hexane (50 ml) to
give 18.28 g (56% yield) of a product 97.5% pure by HPLC.
1,3-Diolein 2-gammalinolenoate (O-G-O)
[0182] .gamma.-Linolenic acid (GLA95, 41.2 g, 0.15 mol, 1.1 equiv)
was dissolved in dichloromethane (DCM, 250 ml). The resulting
solution was stirred at RT under nitrogen and oxalyl chloride (19.1
ml, 28.2 g, 0.22 mol, 1.65 equiv) added dropwise over 5 mins. The
mixture was stirred at RT overnight and then concentrated in vacuo
to remove DCM and excess oxalyl chloride. The residual oily acid
chloride (GLA-Cl) was then added dropwise over 15 min (ice/water
cooling) to a stirred solution of 1,3-diolein (83.5 g, 0.13 mol),
DCM (250 ml), pyridine (10.9 ml, 10.6 g, 0.14 mol) and
4-dimethylaminopyridine (0.49 g, 0.004 mol, 0.15 equiv) at
10-15.degree. C. The temperature was maintained by ice-water
cooling. The reaction mixture was stirred at RT under nitrogen
overnight. Pyridine hydrochloride was removed by filtration and
washed with DCM. The combined washing and filtrate was washed with
1.times.80 ml portions of 5% NaCl, 5% NaHCO.sub.3, 0.1N HCl, 5%
NaCl. The solution was then dried over MgSO.sub.4 and the solvent
removed in vacuo. The residual brown oil was purified by column
chromatography on silica. Elution with hexane and then with 5%
ether/hexane gave 63.6 g (54%) of a colourless oil. Purity
determined by HPLC.
Summary
[0183] 64 g of glycerol 1,3-oleoate-2-gammalinolenoate
(1,3-dioleate-2-GLA, OGO) was prepared from oleoyl chloride (98%)
by a three-step process (scheme given below). It was an almost
colourless oil (slight yellow tinge) and is being stored under
nitrogen in the freezer. The HPLC purity was 89.4%.
.sup.13C NMR Data for Structured Lipids
[0184] GGG .delta..sub.C (125.7 MHz, CDCl.sub.3) 172.69 (1C, C-2
carbonyl), 173.09 (2C, C-1, C-3 carbonyls) CGC .delta..sub.C (125.7
MHz, CDCl.sub.3) 172.76 (1C, C-2 carbonyl), 173.17 (2C, C-1, C-3
carbonyls) CAC .delta..sub.C (125.7 MHz, CDCl.sub.3) 172.65 (1C,
C-2 carbonyl), 173.28 (2C, C-1, C-3 carbonyls) C(DHLA)C
.delta..sub.C (125.7 MHz, CDCl.sub.3) 172.83 (1C, C-2 carbonyl),
173.30 (2C, C-1, C-3 carbonyls) GCG .delta..sub.C (125.7 MHz,
CDCl.sub.3) 172.91 (1C, C-2 carbonyl), 173.11 (2C, C-1, C-3
carbonyls) OGO .delta..sub.C (125.7 MHz, CDCl.sub.3) 172.69 (1C,
C-2 carbonyl), 173.25 (2C, C-1, C-3 carbonyls) AAA .delta..sub.C
(125.7 MHz, CDCl.sub.3) 172.66 (1C, C-2 carbonyl), 173.04 (2C, C-1,
C-3 carbonyls) CCC .delta..sub.C (125.7 MHz, CDCl.sub.3) 172.81
(1C, C-2 carbonyl), 173.21 (2C, C-1, C-3-carbonyls)
Experimental Procedure
[0185] The proton-decoupled .sup.13C NMR spectra with suppressed
NOE were collected at 21.degree. C. in a 5-mm broadband probe on a
Joel 500 MHz spectrometer operating at 125.728 MHz. Waltz
decoupling was the chosen mode of decoupling and was gated on only
during the 14.89 s acquisition time. The relaxation delay was set
at 30 secs and the pulse angle was 90.degree.. The spectral window
used was ca.35 ppm (from 173.5 to 172.6 ppm) with a 170 ppm offset.
The spectra were internally referenced to CDCl.sub.3 at 77.0 ppm.
Typically, the approximate number of scans collected for adequate
signal-to-noise ranged from 300 to 1200 scans depending on the
concentration and purity of the sample. The total acquisition time
for the experiments ranged between 2-8 h e.g 1272 scans; data
points 65,536. Concentrated solutions up to 20% w/v were employed
when possible to reduce the acquisition time The chemical shifts
quoted vary with the concentration of the solution.
Biological Studies.
Chronic Relapsing Experimental Autoimmune Encephalomyelitis (CREAE)
Studies.
Induction and Clinical Assessment of EAE
[0186] CREAE was induced in C57B1/6 and SJL mice. Animals were
injected subcutaneously with 100 .mu.g of the neuroantigen peptide
MOG 35-55 (amino acid sequence MEVGWYRSPFSRVVHLYRNGK Genemed
Synthesis, Inc) or 1 mg of mouse spinal cord homogenate (SCH), in
phosphate buffered saline (PBS), emulsified by sonication for 10
min at room temperature, in incomplete Freund's adjuvant (DIFCO,
Detroit, USA) supplemented with 480 .mu.g of mycobacteria
tuberculosis and 60 .mu.g of Mycobacteria butyricium (DIFCO,
Detroit, USA) on days 0 and 7 as described previously
(Morris-Downes, MM., et al 2002). In addition to optimise the
disease mice also received 200 ng (intraperitoneally) of Bordetella
pertussis toxin dissolved in PBS administered 1 hr and 24 hrs after
immunization with the MOG neuroantigen and for SCH days 0, 1, 7 and
8.
[0187] Animals were weighed from day 5 onwards and examined daily
for clinical neurological signs by two experienced investigators
and graded according to a previously validated grading scheme
(Morris-Downes, M M. et al 2002 and others): 0=normal; 1=limp tail
and feet; 2=impaired righting reflex; 3=partial hind limb
paralysis; 4=complete hindlimb paralysis; 5=moribund; 6=death.
Animals exhibiting clinical signs of a lesser severity grade than
typically observed were scored as 0.5 less than the indicated
grade.
Reference
[0188] Morris-Downes, M M., et al (2002). Pathological and
regulatory effects of anti-myelin antibodies in experimental
allergic encephalomyelitis in mice. J. Neuroimmunol.
125.114-124.
[0189] The mean group EAE score was compared for each test group
compared to a respective control group by non-parametric
statistical analysis (Mann Whitney U Test).
[0190] All MOG-CREAE studies comprised a treatment control group
(C--C--C or saline as selected from the above study). Each
structured lipid was tested at 3 dose levels, all treatments being
orally administered for 2 weeks from day 7 after inoculation. All
treatment groups will contained 10 animals. On completion of
studies (day 21), brain and spinal cord were be removed and half of
the samples were processed for signs of CNS perivascular
mononuclear leucocyte-infiltrated sites and demyelination.
Studies were as Follows: Study 2: Spinal cord homogenate(SCH) EAE
in SJL mice. EAE Induction: 1 mg SCH day 0+day 7 sc. 200 ng
Pertussis toxin day 0, 1, 7 & 8 ip. 10 mice/group. Mice were
treated from day 7 to 21 with CCC or CGC. Study 3: SCH EAE in SJL
mice: Treatment was from PSD 7 to 21, both days inclusive. Study 4:
MOG EAE in C57BL mice: Treatment was from PSD 7 to 21, both days
inclusive. Study 5: SCH EAE in SJL mice: Treatment was from PSD 5
to 18, both days inclusive. Study 6: MOG EAE in C57BL mice:
Treatment was from Days 5 to 21 inclusive except C-DHLA-C group
where treatment was from days 5 to 15 inclusive. Animals were
culled on PSD 25. [Five animals from an untreated group, 3 animals
from control CCC treatment group, 5 animals from GG G 150 ul
treatment group and 2 animals from GGG 350 ul treatment group were
sampled for histological analysis on PSD 20]. Study 7: SCH EAE in
SJL mice Treatment was from Days 6 to 20 inclusive. Study 2--Spinal
cord homogenate (SCH) in SJL mice:--tested [0191] CGC (50/150/350
ul); CCC (350 ul). [0192] GGG. (50/350 ul) [0193] [Severe disease
observed] Study 3--SCH/SJL mice:--tested [0194] CCC (50/150/350 ul)
[0195] CGC (25/50/150/350 ul) [0196] GGG (50/150/350 ul) [0197]
OGO. (25/50/150/350 ul) [0198] [Severe disease observed] Study
4--MOG/C57BL mice:--tested [0199] CCC (50/150/350 ul) [0200] CGC
(25/50/150/350 ul) [0201] GGG (50/150/350 ul) [0202] OGO.
(25/50/150/350 ul) Study 6--MOG/C57BL mice:--tested [0203] CCC (150
ul) [0204] C-DHLA-C (50 ul) [0205] CAC (50/350 ul) [0206] AAA
(50/150 ul) [0207] GCG (50 ul) [0208] CGC (50 ul) [0209] GGG.
(150/350 ul)
[Pathology: CCC; GGG]
[0210] Histological examination of the submitted samples of brain
and spinal cord showed lesions typical of experimental allergic
encephalomyelitis.
[0211] Localised and diffuse lesions were characterised by gliosis,
myelin vacuolation, axonal degeneration and perivascular cuffing
with lymphocytes, macrophages and neutrophils.
[0212] Spinal cord lesions were mostly located in subpial white
matter and brain lesions mostly occurred in the cerebellar white
matter. Lesions were more severe in the spinal cords than in the
brains and whereas all animals with brain lesions had lesions in
the spinal cord, not all animals with cord lesions had lesions in
the brain.
[0213] Variation in the severity of changes between individual mice
is summarised using a semi-quantitative five point grading
system.
[0214] Untreated mice had histological scores of 3-4 which
correlated with EAE scores of 1.5-3. One mouse showed little
pathological change with a zero score. In the GGG treated mice, the
majority showed no abnormalities. Two mice from this group had
histological scores of 2 and 3 respectively which correlated with
EAE severity. scores of 1 and 1.5
[0215] The results of the four studies are shown in FIGS. 11 to 20
below
[0216] These show that the compounds G-G-G, A-A-A, C-G-C,
C-DHGLA-C, and C-A-C are all capable of reducing severity of CREAE
whereas compounds G-C-G and C--C--C failed to treat the condition.
Compound O-G-O is believed to work if the dose is adjusted.
[0217] As cautioned in the description, the arachidnoic acid
compounds are effective, but lead to death of some animals.
Surviving animals had much reduced disease. It is believed that the
dose of these compounds may be reduced still further to provide
survival with satisfactory treatment.
[0218] Some of the studies show a bell shaped response curve for
compounds C-G-C and G-G-G, suggesting that very high doses are not
optimal, as set out above. Such dosing can be conveniently
determined by those skilled art, eg. By dose escalatio and
monitoring TGF-.beta.1/TNF-.alpha. spontaneously release ratio
changes from PBMCs.
[0219] Given the PCT/GB04/002089 high sn-2 .gamma.-linolenic acid
results, the lack of efficacy of low sn-2 black-current oil and
G-C-G in CREAE and the low dose efficacy of C-G-C and C-DHGLA-C in
FIG. 20, it can be seen that sn-2-.gamma.-linolenic acid,
dihomo-.gamma.-linolenic acid and arachidonic acid lipids provide a
novel treatment for MS that far exceeds any current therapy outcome
in that lesions are repaired and difficult symptoms are resolved:
decreasing EDSS over a period of years being so far unachieved in
other treatments.
REFERENCES
[0220] Amor S, Groome N, Linington C, Morris M M, Dornmair K,
Gardiner M V, Matthieu J M, Baker D. Identification of epitopes of
myelin oligodendrocyte glycoprotein for the induction of
experimental allergic encephalomyelitis in SJL and Biozzi AB/H
mice. J Immunol. 1994 Nov. 15; 153(10):4349-56. [0221] Beck J,
Rondot P, Catinot L et al. Increased production of interferon gamma
and tumor necrosis factor precedes clinical manifestation in
multiple sclerosis: do cytokines trigger off exacerbations? Acta
Neurol Scand 1988; 78:318-23. [0222] Bertolotto A, Capobianco M,
Malucchi S et al. Transforming growth factor beta1 (TGFbeta1) mRNA
level correlates with magnetic resonance imaging disease activity
in multiple sclerosis patients. Neurosci Lett 1999; 263:21-4.
[0223] Bertolotto A, Malucchi S, Capobianco M et al. Quantitative
PCR reveals increased levels of tumor necrosis factor-alpha mRNA in
peripheral blood mononuclear cells of multiple sclerosis patients
during relapses. J Interferon Cytokine Res 1999; 19:575-81. [0224]
Brosnan C F, Selmaj K and Raine C S. Hypothesis: a role for tumor
necrosis factor in immune-mediated demyelination and its relevance
to multiple sclerosis. J Neuroimmunol 1988:18, 87-94. [0225]
Brosnan C F and Raine C S. Mechanisms of immune injury in multiple
sclerosis. Brain Pathol. 1996:6, 243-257. [0226] Burns J,
Bartholomew B, Lobo S. Isolation of myelin basic protein-specific T
cells predominantly from the memory T-cell compartment in multiple
sclerosis. Ann Neurol 1999; 45:33-9. [0227] Cannella B, Raine C S.
The adhesion molecule and cytokine profile of multiple sclerosis
lesions. Ann Neurol 1995; 37:424-35. [0228] Chou Y K, Bourdette D
N, Offner H et al. Frequency of T cells specific for myelin basic
protein and myelin proteolipid protein in blood and cerebrospinal
fluid in multiple sclerosis. J Neuroimmunol 1992; 38:105-14. [0229]
De Stefano N., Narayanan S., Francis G S., Arnaoutelis R.,
Tartaglia M C., Antel J P., matthews P M and Arnold D L. Evidence
of axonal damage in the early stages of multiple sclerosis and its
relevance to disability. Arch Neurol. 2001: 58(1), 65-70. [0230]
Ewing C, Bernard C C. Insights into the aetiology and pathogenesis
of multiple sclerosis. Immunol Cell Biol 1998; 76:47-54. [0231]
Fazakerly J K. Molecular biology of multiple sclerosis. Wiley and
Sons Ltd. 1997, 255-273. [0232] Fredrikson S, Soderstrom M, Hillert
J et al. Multiple sclerosis: occurrence of myelin basic protein
peptide-reactive T cells in healthy family members. Acta Neurol
Scand 1994; 89:184-9. [0233] Genain C P., Cannella B., hauser SL
and Raine C S. Identification of autoantibodies associated with
myelin damage in multiple sclerosis. Nature Med 1999:5, 170-175.
[0234] Gross C E, Bednar M M, Howard D B and Spom M B (1993)
Transforming growth factor beta I reduces infarct size after
experimental cerebral ischemia in a rabbit model. Stroke 24,
558-562. [0235] Harbige, LS, Crawford M A, Jones J, Preece A W and
Forti A. Dietary intervention studies on the phosphoglyceride fatty
acids and electrophoreitic mobility of erythrocytes in multiple
sclerosis. Prog. Lipid Res 1986:25, 243-248. [0236] Harbige L S.
Nutrition and immunity with emphasis on infection and autoimmune
disease. (1996) Nutr Health, 10(4):285-312. [0237] Harbige L S
(1998) Dietary n-6 and n-3 fatty acids in immunity and autoimmune
disease. Proceedings of the Nutrition Society 57, 555-562. [0238]
Harbige L S, Yeatman N, Amor S & Crawford M A (1995) Prevention
of experimental autoimmune encephalomyelitis in Lewis rats by a
novel source of .gamma.-linolenic acid. British Journal of
Nutrition 74, 701-715. [0239] Harbige L S., Layward L.,
Morris-Downes M M., Dumonde D C and Amor S. The protective effects
of omega-6 fatty acids in experimental autoimmune encephalomyelitis
(EAE) in relation to transforming growth factor-beta 1 (TGF-beta1)
up-regulation and increased prostaglandin E2 (PGE2) production.
Clin Exp Immunol 2000:122, 445-452. [0240] Henrich Noack P, Prehn J
H, and Kriegistein J. (1996) TGF-beta I protects hippocampal
neurons against degeneration caused by transient global ischaemia.
Dose-response relationship and potential neuroprotective
mechanisms. Stroke, 27, 1609-1614. [0241] Hirsch R L, Panitch H S,
Johnson K P. Lymphocytes from multiple sclerosis patients produce
elevated levels of gamma interferon in vitro. J Clin Immunol 1985;
5:386-9. [0242] Hollifield R D, Harbige L S, PhM-Dinh D, Sharief M.
Evidence for cytokine Dysregulation in Multiple Sclerosis:
Peripheral Blood Mononuclear cell production of pro-inflammmatory
and anti-inflammatory cytokines during relapse and remission.
Autoimmunity,2003 36(3):133-141. [0243] Imamura K, Suzumura A,
Hayashi F et al. Cytokine production by peripheral blood
monocytes/macrophages in multiple sclerosis patients. Acta Neurol
Scand 1993; 87:281-5. [0244] Issazadeh S, Lorentzen J C, Mustafa M
I et al. Cytokines in relapsing experimental autoimmune
encephalomyelitis in DA rats: persistent mRNA expression of
proinflammatory cytokines and absent expression of interleukin-10
and transforming growth factor-beta. J Neuroimmunol 1996;
69:103-15. [0245] Johns L D, Sriram S. Experimental allergic
encephalomyelitis: neutralizing antibody to TGF beta 1 enhances the
clinical severity of the disease. J Neuroimmunol 1993; 47:1-7.
[0246] Kerlero de Rosbo N, Hoffman M, Mendel I et al. Predominance
of the autoimmune response to myelin oligodendrocyte glycoprotein
(MOG) in multiple sclerosis: reactivity to the extracellular domain
of MOG is directed against three main regions. Eur J Immunol 1997;
27:3059-69. [0247] Kerlero de Rosbo N, Milo R, Lees M B et al.
Reactivity to myelin antigens in multiple sclerosis. Peripheral
blood lymphocytes respond predominantly to myelin oligodendrocyte
glycoprotein. J Clin Invest 1993; 92:2602-8. [0248] Khalil N.
TGF-beta: from latent to active. Microbes Infect 1999; 1:1255-63.
[0249] Krupinski J, Kumar P, Kumar S, and Kaluza J. (1996)
Increased expression of TGF-beta I in brain tissue after ischemic
stroke in humans. Stroke, 27, 852-857. [0250] Kuroda Y, Shimamoto
Y. Human tumor necrosis factor-alpha augments experimental allergic
encephalomyelitis in rats. J Neuroimmunol 1991; 34:159-64. [0251]
Lu C Z, Jensen M A, Amason B G. Interferon gamma- and
interleukin-4-secreting cells in multiple sclerosis. J Neuroimmunol
1993; 46:123-8. [0252] Maimone D, Reder A T, Gregory S. T cell
lymphokine-induced secretion of cytokines by monocytes from
patients with multiple sclerosis. Cell Immunol 1993; 146:96-106.
[0253] Martino G, Hartung H-P. Immunopathogenesis of multiple
sclerosis: the role of T cells. Curr Opin Neurol 1999; 12:309-21.
[0254] McCarron R M, Wang L, Racke M K et al. Cytokine-regulated
adhesion between encephalitogenic T lymphocytes and cerebrovascular
endothelial cells. J Neuroimmunol 1993; 43:23-30. [0255] McDonald W
I, Compston A, Edan G, Goodkin D, Hartung H P, Lublin F D,
McFarland H F, Paty D W, Polman C H, Reingold S C,
Sandberg-Wollheim M, Sibley W, Thompson A, van den Noort S,
Weinshenker B Y, Wolinsky J S. [0256] Recommended diagnostic
criteria for multiple sclerosis: guidelines from the International
Panel on the diagnosis of multiple sclerosis. Ann Neurol. 2001
July; 50(1):121-7. [0257] Merrill J E, Strom S R, Ellison G W et
al. In vitro study of mediators of inflammation in multiple
sclerosis. J Clin Immunol 1989; 9:84-96. [0258] Merrill J E,
Zimmerman R P. Natural and induced cytotoxicity of oligodendrocytes
by microglia is inhabitable by TGF beta. Glia 1991; 4:327-31.
[0259] Miyazono K., Hellman U, Wemstedt C et al. Latent high
molecular weight complex of transforming growth factor beta 1.
Purification from human platelets and structural characterization.
J Biol Chem 1988; 263:6407-15. [0260] Mokhtarian F, Shi Y,
Shirazian D et al. Defective production of anti-inflammatory
cytokine, TGF-beta by T cell lines of patients with active multiple
sclerosis. J Immunol 1994; 152:6003-10. [0261] Navikas V, Link H.
Review: cytokines and the pathogenesis of multiple sclerosis. J
Neurosci Res 1996; 45:322-33. [0262] Noseworthy J H. Progress in
determining the causes and treatment of multiple sclerosis. Nature
1999:399(6738 Suppl), A40-47. [0263] Ota K, Matsui M, Milford E L
et al. T-cell recognition of an immunodominant myelin basic protein
epitope in multiple sclerosis. Nature 1990; 346:183-7. [0264]
Perkin G D, Wolinsky J S. Fast facts-Multiple Sclerosis, 1st Edn.
Oxford, UK: Health Press, 2000. [0265] Philippe J, Debruyne J,
Leroux-Roels G et al. In vitro TNF-alpha, IL-2 and IFN-gamma
production as markers of relapses in multiple sclerosis. Clin
Neurol Neurosurg 1996; 98:286-90. [0266] Phylactos A C,
Ghebremeskel K, Costeloe K, Leaf A A, Harbige L S, Crawford M A.
(1994) Polyunsaturated fatty acids and antioxidants in early
development. Possible prevention of oxygen-induced disorders. Eur J
Clin Nutr. 48Suppl 2:S17-23. [0267] Prehn T H, Peruche B, Unsicker
K and Kriegistein J. (1993) Isoform-specific effects of
transforming growth factor-beta on degeneration of primary neuronal
cultures induced by cytotoxic hypoxia or glutamate. J. Neurochem.
60, 1665-1672. [0268] Rack M K, Sriram S, Calrimi J, Cannella B,
Raine C S & McFarim D E (1993) Long-term treatment of chronic
relapsing experimental allergic encephalomyelitis by transforming
growth factor-p2. Journal of Neuroimmunology, 46, 175-183. [0269]
Racke M K, Cannella B, Albert P et al. Evidence of endogenous
regulatory function of transforming growth factor-beta 1 in
experimental allergic encephalomyelitis. Int Immunol 1992;
4:615-20. [0270] Rieckmann P, Albrecht M, Kitze B et al. Cytokine
mRNA levels in mononuclear blood cells from patients with multiple
sclerosis. Neurology 1994; 44:1523-6. [0271] Rieckmann P, Albrecht
M, Kitze B et al. Tumor necrosis factor-alpha messenger RNA
expression in patients with relapsing-remitting multiple sclerosis
is associated with disease activity. Ann Neurol 1995; 37:82-8.
[0272] Ruddle N H, Bergman C M, McGrath K M et al. An antibody to
lymphotoxin and tumor necrosis factor prevents transfer of
experimental allergic encephalomyelitis. J Exp Med 1990;
172:1193-200. [0273] Santambrogio L, Hochwald G M, Saxena B, Leu C
H, Martz J E, Carlino J A, Ruddle N H, Palladino M A, Gold L I
& Thorbecke G J (1993) Studies on the mechanisms by which
Transforming Growth Factor-p protects against allergic
encephalomyelitis. Journal of Immunology 151, 1116-1127. [0274]
Schiefer H B, Hancock D S, Loew F M. Long-term effects of partially
hydrogenated herring oil on the rat myocardium. Drug Nutr Interact.
1982; 1(2):89-102. [0275] Schluesener H J, Lider O. Transforming
growth factors beta 1 and beta 2: cytokines with identical
immunosuppressive effects and a potential role in the regulation of
autoimmune T cell function. J Neuroimmunol 1989; 24:249-58. [0276]
Selmaj K, Raine C S, Cannella B et al. Identification of
lymphotoxin and tumor necrosis factor in multiple sclerosis
lesions. J Clin Invest 1991; 87:949-54. [0277] Selmaj K, Raine C S,
Farooq M et al. Cytokine cytotoxicity against oligodendrocytes.
Apoptosis induced by lymphotoxin. J Immunol 1991; 147:1522-9.
[0278] Sharief M K, Thompson E J. In vivo relationship of tumor
necrosis factor-alpha to blood-brain barrier damage in patients
with active multiple sclerosis. J Neuroimmunol 1992; 38:27-33.
[0279] Tejada-Simon M V, Hong J, Rivera V M et al. Reactivity
pattern and cytokine profile of T cells primed by myelin peptides
in multiple sclerosis and healthy individuals. Eur Immunol 2001;
31:907-17. [0280] Vartanian T, Li Y, Zhao M et al.
Interferon-gamma-induced oligodendrocyte cell death: implications
for the pathogenesis of multiple sclerosis. Mol Med 1995; 1:732-43.
[0281] Vivien D, Bemaudin M, Buisson A, Divoux D, MacKenzie ET and
Nouvelot A. (1998) Evidence of type I and type II transforming
growth factor-beta receptors in central nervous tissues: changes
induced by focal cerebral ischemia. J. Neurochem. 70, 2296-2304.
[0282] Zhang J, Markovic-Plese S, Lacet B et al. Increased
frequency of interleukin 2-responsive T cells specific for myelin
basic protein and proteolipid protein in peripheral blood and
cerebrospinal fluid of patients with multiple sclerosis. J Exp Med
1994; 179:973-84. [0283] Japanese Patent 6172263 (1994) Y. Kosugi
et al, Agency of Industrial Science & Technology High-purity
arachidonic acid triglyceride and its production. [0284] U.S. Pat.
No. 4,888,324 (1989) N. Catsimpoolas et al, Angio-Medical
Corporation Method for enhancing angiogenesis with lipid containing
molecules. [0285] Y. Kosugi and N. Azuma, J Amer. Oil Chem. Soc.,
71, 1397-1403 (1994). Synthesis of Triacylglycerol from
polyunsaturated fatty acid by immobilized lipase. [0286] J. W.
Hageman et al, J. Amer. Oil Chem. Soc., 49, 118-xxx (1972)
Preparation of Glycerin and their Uses. [0287] E. S. Lutton and A.
J. Fehl, Lipids, 5, 90-99 (1970). The polymorphism of odd and even
saturated single acid triglycerides, C8-C22. [0288] D. Horrobin, A.
McMordie, M. S. Manku (Scotia Holdings PLC UK) [0289] Eur. Pat.
Appl EP 609078 3 Aug. 1994. Phospholipids containing two different
unsaturated fatty acids for use in therapy, nutrition, and
cosmetics. [0290] Y.-S. Huang, X. Lin, P. R. Redden and D. F.
Horrobin, J. Am. Oil Chem. Soc., 72, 625-631, (1995). In vitro
Hydrolysis of Natural and Synthetic .gamma.-Linolenic
Acid-Containing Triacylglycerols by Pancreatic Lipase [0291] K.
Osada, K. Takahashi, M. Hatano and M. Hosokawa, Nippon Suisan
Gakkaishi., 57, 119-125 (1991). Chem. Abstr., 115:278299 Molecular
Species of Enzymically-synthesized Polyunsaturated Fatty acid-rich
Triglycerides. [0292] J.-W. Liu, S. DeMichele, M. Bergana, E.
Bobik, Jr., C. Hastilow, Lu-Te Chuang, P. Mukerji and J.-S. Huang.,
J. Am. Oil Chem. Soc., 78, 489-493. (2001) Characterization of Oil
Exhibiting High .gamma.-Linolenic Acid from a Genetically
transformed Canola Strain. [0293] D. R. Kodali, D. Atkinson, T. G.
Redgrave and D. Small, J. Lipid Res., 28, 403-413 (1987). Structure
and polymorphism of 18-Carbon Fatty Acid Triacylglycerols: Effect
of Unsaturation and Substitution in the 2-Position [0294] P. H.
Bentley and W. McCrae, J. Org. Chem. 35, 2082-2083 (1970) An
Efficient Synthesis of Symmetrical 1,3-Diglycerides. [0295] M.
Berger, K. Laumen and M. P. Schneider, J. Am. Oil. Chem. Soc., 69,
955-959, (1992). Enzymatic Esterification of Glycerol 1.
Lipase-Catalyzed Synthesis of Regioisomerically Pure
1,3-sn-Diacylglycerols. [0296] A. P. J. Mank, J. P. Ward and D. A.
van Dorp, Chem. Physics Lipids, 16, 107-114 (1976). A versatile,
flexible synthesis of 1,3-diglycerides and triglycerides. [0297] L.
Hartman, Chem. Rev., 58, 845-867 (1958) and references therein.
Advances in the Synthesis of Glycerides of Fatty Acids
TABLE-US-00001 [0297] TABLE 1 Compositional (% Total FAs)
Characteristics of Various Oils and their Protective Effects in EAE
INCIDENCE Treatment 18:2n-6 18:3n-6 18:2n-6/18:3n-6 18:1n-9 OF EAE
FGO 17 20 0.6 35 0/10 BOO 37 24 1.5 15 3/10 EPO 71 9.4 7.5 9 7/10
SAF 66 -- -- 17 9/10 Controls -- -- -- -- 9/10 FGO, Fungal Oil;
BOO, Borage Oil; EPO, Evening Primrose Oil, SAF, Safflower Oil.
TABLE-US-00002 TABLE 2 Treatment Groups- PCT/GB04/002089 Borage
oil-MS trial Mean Relapse Rate Mean (in past Base Female Male two
years) EDSS Number Group Placebo 7 4 2.6 3.9 11 Low Dose 5 2 2.9
3.5 7 High Dose 8 2 3.4 2.8 10 Total 20 8 2.9 3.4 28
TABLE-US-00003 TABLE 3 Molecular Species Comparison of
Triacylglycerol-GLA (TG-GLA), Ethyl-Ester-GLA (EE-GLA) and
PCT/GB04/002089 Borago Officinalis Oil-GLA (BOR-GLA) in MOG-induced
CREAE in SJL Mice Treatment No. with EAE Mean Clinical Score
Control 10/11 3.3 .+-. 1.3.sup. EE-GLA.sup.a 5/6 3.0 .+-. 0.8.sup.
TG-GLA.sup.a 3/6 1.0 .+-. 1.3.sup.c BOR-GLA.sup.b 3/6 1.0 .+-.
1.2.sup.c .sup.aAnimals given 100 .mu.l of test lipid; .sup.b250
.mu.l BOR-GLA given. Significance of difference compared with
controls, .sup.cp < 0.05
TABLE-US-00004 TABLE 4 Effect of enriched black-currant seed oil
(73% GLA) on the incidence of EAE % Incidence of EAE (Days after
immunisation) 13 17 21 Controls (n = 10) 60 90 10 Blackcurrant (n =
10) 10 80 70 Note: Blackcurrant oil delays the incidence but does
not provide full protection. Animals were fed 7 days after
sensitization (immunisation).
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