U.S. patent application number 11/885254 was filed with the patent office on 2009-05-28 for cytokine modulators using cyclic glycerides of essential polyunsaturated fatty acids.
Invention is credited to Paul Barraclough, Anthony P. Dolan, Laurence S. Harbige, Michael J. Leach.
Application Number | 20090137660 11/885254 |
Document ID | / |
Family ID | 34430537 |
Filed Date | 2009-05-28 |
United States Patent
Application |
20090137660 |
Kind Code |
A1 |
Harbige; Laurence S. ; et
al. |
May 28, 2009 |
Cytokine Modulators Using Cyclic Glycerides of Essential
Polyunsaturated Fatty Acids
Abstract
A method of treating a patient in need of therapy for a cytokine
dysregulation comprising administering to that patient a
therapeutically effective dose of a compound of general formula:
(I) wherein R.sup.1 and R.sup.2 together form a group
--(CH.sub.2).sub.n--CR.sup.4R.sup.5--(CH.sub.2).sub.m-- wherein n
and m are independently selected integers 0, 1 or 2 and R.sup.4 and
R.sup.5 are independently selected from H, C.sub.1-18 alkyl,
C.sub.1-18 alkoxy, C.sub.1-18n hydroxyalkyl, C.sub.2-18 alkenyl and
C.sub.6-18 aryl or aralyalkyl and R.sup.3 is the fatty acyl group
of an essential polyunsaturated fatty acid.
Inventors: |
Harbige; Laurence S.; (Kent,
GB) ; Leach; Michael J.; (Kent, GB) ;
Barraclough; Paul; (Kent, GB) ; Dolan; Anthony
P.; (London, GB) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Family ID: |
34430537 |
Appl. No.: |
11/885254 |
Filed: |
March 2, 2006 |
PCT Filed: |
March 2, 2006 |
PCT NO: |
PCT/GB06/00779 |
371 Date: |
July 16, 2008 |
Current U.S.
Class: |
514/452 ;
514/467; 514/546; 549/372; 549/449 |
Current CPC
Class: |
A61P 37/00 20180101;
C07D 319/06 20130101; A61P 19/02 20180101; A61P 43/00 20180101;
A61P 25/16 20180101; A61P 25/14 20180101; A61P 37/02 20180101; A61P
9/00 20180101; A61P 9/10 20180101; A61P 25/00 20180101; A61P 17/00
20180101; A61P 11/06 20180101; A61P 25/28 20180101; A61P 37/08
20180101; A61K 31/357 20130101; A61P 1/00 20180101; A61P 31/04
20180101; C07D 321/00 20130101 |
Class at
Publication: |
514/452 ;
514/546; 514/467; 549/449; 549/372 |
International
Class: |
A61K 31/357 20060101
A61K031/357; A61K 31/23 20060101 A61K031/23; A61P 25/00 20060101
A61P025/00; A61P 37/00 20060101 A61P037/00; C07D 317/34 20060101
C07D317/34; C07D 319/06 20060101 C07D319/06 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 2, 2005 |
GB |
0504362.5 |
Claims
1. A method of treating a patient in need of cytokine modulation
comprising administering to that patient a therapeutically
effective dose of a glyceride or compound of general formula I
##STR00004## wherein R.sup.1 and R.sup.2 together form a group
--(CH.sub.2).sub.n--CR.sup.4R.sup.5--(CH.sub.2).sub.m-- wherein n
and m are independently selected integers 0, 1 or 2 and R.sup.4 and
R.sup.5 are independently selected from H, C.sub.1-18 alkyl,
C.sub.1-18 alkoxy, C.sub.1-18 hydroxyalkyl, C.sub.2-18 alkenyl and
C.sub.6-18 aryl or aralykyl and R.sup.3 is the a fatty acyl group
of an essential polyunsaturated fatty acid.
2. A method as claimed in claim 1 wherein the polyunsaturated fatty
acyl group is selected from those of n-3, n-6 and n-9 fatty acids,
more preferably n-3 and n-6 fatty acids, still more preferably
being .gamma.-linolenoyl, .gamma.-dihomolinolenoyl and
arachidonoyl.
3. A method as claimed in claim 1 wherein R.sup.1 and R.sup.2 form
a group --CR.sup.4R.sup.5 wherein R.sup.4 and R.sup.5 are
independently selected from H and C.sub.1-6 alkyl.
4. A method as claimed in claim 1 wherein R.sup.1 and R.sup.2 form
a group --CH.sub.2-- or --CH.sub.2CH.sub.2--.
5. A method as claimed in claim 1 wherein the compound of formula
is a 5-essential fatty acyloxy-1,3-dioxans.
6. A method as claimed in claim 1 wherein the compound of claim 1
is an n3-fatty acyloxy-1,3-dioaxan or an n-6 fatty
acyloxy-1,3-dioxans.
7. A method as claimed in claim 1 wherein the compound of formula I
is selected from a 5-.gamma.-linolenoyloxy-1,3-dioxans,
5-dihomo-.gamma.-linolenoyloxy-1,3-dioxans and
5-arachidonoyl-1,3-dioxans.
8. A method as claimed in claim 1 wherein the patient is in need of
therapy for cytokine dysregulation.
9. A method as claimed in claim 1 wherein the patient is in need of
therapy to prevent demyelination in a neurodegenerative
disease.
10. A method as claimed in claim 1 wherein the modulated cytokines
are one or more of TGF-.beta.1, TNF-.alpha., IL-1, IL4, IL5, IL6,
IL8, IL10, IL13, and .gamma.-IFN.
11. A method as claimed in claim 1 wherein the treatment is for
abnormalities of the immune system.
12. A method as claimed in claim 11 wherein the treatment is for a
condition selected from the group consisting of systemic lupus
erythematosus (SLE), allergy, asthma, crohn's disease and
rheumatoid arthritis, multiple sclerosis, neurodegenerative
diseases sequelae of stroke, head trauma, bleeds and the chronic
abnormalities of Alzheimer's and Parkinson's disease, coronary
heart disease (CHD) abnormalities of pre-mature infants and
sepsis.
13. A compound of formula I ##STR00005## wherein R.sup.1 and
R.sup.2 together form a group
--(CH.sub.2).sub.n--CR.sup.4R.sup.5--(CH.sub.2).sub.m-- wherein n
and m are independently selected integers 0, 1 or 2 and R.sup.4 and
R.sup.5 are independently selected from H, C.sub.1-18 alkyl,
C.sub.1-18 alkoxy, C.sub.1-18 hydroxyalkyl, C.sub.2-18 alkenyl and
C.sub.6-18 aralkyl and R.sup.3 is a fatty acyloxy group of an
essential polyunsaturated fatty acid.
14. A compound as claimed in claim 13 wherein R.sup.3 is a
polyunsaturated fatty acyl groups of an n-3, n-6 and n-9 fatty
acids, more preferably n-3 and n-6 fatty acids, still more
preferably being .gamma.-linolenoyl, .gamma.-dihomolinolenoyl and
arachidonoyl.
15. A compound as claimed in claim 13 wherein R.sup.1 and R.sup.2
form a group --CR.sup.4R.sup.5 where R.sup.4 and R.sup.5 are
independently selected from H and C.sub.1-6 alkyl.
16. A compound as claimed in claim 13, wherein R.sup.1 and R.sup.2
form a group --CH.sub.2-- or --CH.sub.2CH.sub.2--.
17. A compound as claimed in claim 13 being a
5-acyloxy-1,3-dioxan.
18. A compound as claimed in claim 13 being an n-3-fatty acyl or
n-6 fatty acyloxy-1,3-dioxans.
19. A compound as claimed in claim 13 being a
5-.gamma.-linolenyloxy-1,3-dioxan, a
5-dihomo-.gamma.-linolenyloxy-1,3-dioxan or a
5-arachidonyloxy-1,3-dioxan.
20. A compound as claimed in claim 13 for use in therapy.
21. A method for synthesis of a compound of general formula I
comprising reacting a 5-hydroxy-1,3-dioxan with an essential fatty
acid or an essential fatty acid halide.
22. A composition for use in the method of the present invention
comprising a compound of formula I together with a pharmaceutically
or nutraceutically acceptable carrier, coating, capsule, diluent
and/or preservative.
23. A composition as claimed in claim 22 comprising a preservative
which is an antioxidant or inhibitor of transesterification.
24. A composition as claimed in claim 23 comprising 0.05 mg/g or
less of Vitamin E.
25. A pharmaceutical composition for regulating the immune system
comprising a compound of general formula I as defined in claim
1.
26. Use of a compound of formula I as described in claim 1 for the
manufacture of a medicament for the treatment of dysregulation of
cytokines or for the modulation of cytokine disorders.
27. Use as claimed in claim 26 wherein the use is for manufacture
of a medicament for treating neurodegenerative diseases.
28. Use as claimed in claim 27 wherein the medicament is 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.
29. A method as claimed in claim 1 wherein the neurodegenerative
disease involves demyelination.
30. A method as claimed in claim 1 wherein the treatment
specifically arrests underlying neurodegeneration and restores
neuronal function.
31. A method as claimed in claim 1 which normalises neuronal
membrane composition with respect to .gamma.-linolenic acid,
dihomo-.gamma.-linolenic acid and arachidonic acid lipid
content.
32. A method as claimed in claim 1 which restores healthy
TGF-.beta.1/TNF.alpha. ratios as measured from spontaneous release
from peripheral blood mononuclear cell release.
33. A method as claimed in claim 1 wherein the disease is relapsing
remitting multiple sclerosis, primary progressive multiple
sclerosis or chronic progressive multiple sclerosis.
34. A method as claimed in claim 1 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.
35. A method as claimed in claim 1 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.
36. A method as claimed in claim 1 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.
37. A method as claimed in claim 1 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.
38. A method as claimed in claim 1 wherein the amount of compound
administered is between 0.5 and 30 grams, typically 3 to 5 grams,
per day.
39. A method, use or composition as claimed in claim 1 wherein
R.sup.3 is .gamma.-linolenyl or dihomo-.gamma.-linolenyl and the
dose is between 1 and 10 grams/day.
40. A method, use or composition as claimed in claim 39 wherein the
dose is between 2 and 10 grams/day.
Description
[0001] The present invention relates to a method for treating
diseases and disorders in which cytokines are in state of imbalance
or otherwise capable of modulation to provide therapeutic benefit.
Particularly the invention provides a method of treatment of
patients in need of therapy for disorders where the cytokines
TGF-.beta.1, TNF-.alpha., IL-1.beta., IL4, IL5, IL6, IL8, IL10,
IL13, and .gamma.-IFN are dysregulated or capable of modulation to
provide therapeutic benefit.
[0002] Particular diseases that are treatable by the present method
are disorders such as abnormalities of the immune system, for
example systemic lupus erythematosus (SLE), allergy, asthma,
crohn's disease and rheumatoid arthritis, but particularly multiple
sclerosis, and also neurodegenerative diseases such as sequelae of
stroke, head trauma, bleeds and the chronic abnormalities of
Alzheimer's and Parkinson's disease. Further disorders that can be
pretreated both prophylcatically and therapeutically are coronary
heart disease (CHD) abnormalities of pre-mature infants and
sepsis.
[0003] The inventor's copending patent application WO2004/100943
and WO2005/018632, incorporated herein by reference, relate to the
use of synthetic, plant and fungal oils for the treatment of
neurodegenerative diseases, particularly multiple sclerosis,
stroke, head trauma, Alzheimer's and Parkinson's disease.
WO2004/100943 relates to oils characterised by having at 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. WO2005/018632 relates
to structured lipids having an sn-2 fatty acid residue selected
from .gamma.-linolenic acid (GLA), dihomo-.gamma.-linolenic acid
(DHGLA) and arachidonic acid (AA).
[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] 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.
[0006] 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.
[0007] 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 (EPO: 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.
[0008] 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
WO2004/100943 study.
[0009] 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.
[0010] In the WO2004/100943 study the present inventors Harbige and
Leach, with coinventor Sharief, 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.
[0011] The WO2004/100943 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.
[0012] 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.
[0013] Thus 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.
[0014] The present inventors now set out, in view of the results
obtained with 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 monoglyceride, particularly an sn-2
monoglyceride, or a metabolic precursors thereof, that gives it
efficacy in treating cytokine dysregulation. Noting that the
triglycerides themselves are of nearly three times the weight, and
thus dose, of monoglyceride counterparts, they have determined that
it is possible to administer essential fatty acids of the n-3, n-6
and n-9 type, particularly the n-6 type, as metabolic precursors or
analogues of sn-2 monoglycerides and still obtain beneficial
cytokine changes. Particularly these compounds are believed to be
more stable than the equally less bulky monoglycerides.
[0015] It has been reported that 2-arachidonyl glycerol has
activity in decreasing TNF-.alpha. and reactive oxygen species
induced disease (see WO 01/97793), however, the corresponding
.gamma.-linolenoyl and palmitoyl compounds are said to be inactive.
An sn-2-arachidonyl structured lipid has further been reported to
be active in `brain hypofunction` such as in mild cognitive
impairment (see EP 1419768).
[0016] The dose advantages of use of monoglycerides over
triglycerides may be offset in part by possible increased
instability of certain forms as compared with the sn-1, sn-3
saturated acyl group sn-2 EFA triglyceride exemplified in
PCT/GB2004/003524. Such instability may be due eg. to
transesterification and oxidation. This issue may be addressed by
producing the monoglyceride in a more stable form, eg a solid or
semi solid rather than a liquid oil.
[0017] The present inventors have now further provided compounds,
known and novel, which may be metabolic precursors of the
monoglyceride as claimed below that are either metabolised to give
that form in vivo, or are directly active, but which resist
transesterification and other degradations in storage.
[0018] Preliminary studies by the inventors have shown that the
compounds selected for the use of the present invention are more
resistant to degredation by at least some mammalian lipases than
the previously used triglyceride based Borage, Evening Primrose and
structured lipids and more resistant to rearrangement than
monoglycerides.
[0019] These compounds are particularly cyclic glycerols bearing
essential fatty acids, particularly n-3, n-6 and n-9, but
particularly n-6 fatty acids, in a position equivalent to the sn-2
position on the glycerol. The inventors have determined that these
compounds are capable of regulating cytokines in a favourable
manner to patient health based upon their previous clinical studies
with Borage oil and the structured lipid in vivo and in vitro
work.
[0020] Cyclic glycerols are known as a class eg see. Chem Abstracts
no 65:8747b and have been used as intermediates in the synthesis of
monoacylglycerides eg see Chemical & Pharmaceutical Bulletin
(2000), 48(7) 903-907. Recently US2005/0020679 has proposed that
all members of this class bearing a polyunsaturated fatty acyl
group, particularly an arachidonyl group, have been proposed as
having utility as anandamide transport inhibitors having
pharmacological use in treatment of pain, peripheral pain,
glaucoma, epilepsy, nausea, AIDS wasting, cancer,
neurodegeneration, Multiple Sclerosis, Parkinson's Disease,
Huntington's Chorea and Alzheimer's disease, enhancement of
appetite, reduction of fertility, Tourettes and other motor
function disorders, for neuroprotection, peripheral vasodilation
and suppress memory.
[0021] This document and its predecessor WO99/64389 (directed to
analgesia) provide no teaching of the particular compounds of the
present invention and significantly teaches that a therapeutically
effective amount of cyclic glycerol is 10 mg/day to 1000 mg/day.
The present inventors studies on relative effect in animals and on
cells has led them to determine that such a dose will be
ineffective in the cytokine regulating indication now disclosed.
The effective dose in man is expected to be above 1000 mg/day,
particularly between 1 g to 10 g per day. More preferably being 2
to 6 g/day, still more preferably 2 to 4 g/day.
[0022] Without the cytokine regulation such dosages provide,
treatment of diseases such as demyelinating diseases Multiple
Sclerosis and Alzheimer's, Parkinson's, Huntingdon's Chorea and
trauma and stroke with a view to halting ongoing immune cell and
cytokine destruction of tissues cannot be achieved. Mere
enhancement of natural anandamide levels by blocking reuptake
cannot provide relief where natural anandamide is not present in
effective amount, eg. in T-cells and around CNS lesions.
[0023] In a first aspect the present invention provides a method of
treating a patient in need modulation of cytokines, particularly
TGF-.beta.1, TNF-.alpha., IL-1.beta., IL4, IL5, IL6, IL8, IL10,
IL13 and/or .gamma.-IFN, comprising administering to that patient a
therapeutically effective dose of a compound of general formula
I
##STR00001## [0024] wherein R.sup.1 and R.sup.2 together form a
group
[0024] --(CH.sub.2).sub.n--CR.sup.4R.sup.5--(CH.sub.2).sub.m--
[0025] wherein n and m are independently selected integers 0, 1 or
2
[0026] and R.sup.4 and R.sup.5 are independently selected from H,
C.sub.1-18 alkyl, C.sub.1-18 alkoxy, C.sub.1-18 hydroxyalkyl,
C.sub.2-18 alkenyl and C.sub.6-18 aryl or aralykyl
[0027] and R.sup.3 is the a fatty acyl group of an essential
polyunsaturated fatty acid.
[0028] Preferred polyunsaturated fatty acyl groups are those of
n-3, n-6 and n-9 fatty acids, more preferably n-3 and n-6 fatty
acids, still more preferably being .gamma.-linolenoyl,
.gamma.-dihomolinolenoyl and arachidonoyl.
[0029] Preferably R.sup.1 and R.sup.2 are selected from H or a
group --CR.sup.4R.sup.5 where R.sup.4 and R.sup.5 are independently
selected from H and C.sub.1-6 alkyl. Most advantageously R.sup.1
and R.sup.2 form a group --CH.sub.2-- or --CH.sub.2CH.sub.2--.
[0030] Particularly preferred are compounds that are 5-essential
fatty acyloxy-1,3-dioxans, more particularly n-3-fatty acyloxy or
n-6 fatty acyloxy-1,3-dioxans, most preferably
5-.gamma.-linolenoyloxy, 5-dihomo-.gamma.-linolenoyloxy and
5-arachidonoyloxy-1,3-dioxans. Most preferable are the
non-inflammatory 5-.gamma.-linolenoyloxy and
5-dihomo-.gamma.-linolenoyloxy-1,3-dioxans.
[0031] Particularly treated are dysregulated cytokine abnormalities
of the immune system, for example diseases such as systemic lupus
erythematosus (SLE), allergy, asthma, crohn's disease and
rheumatoid arthritis, but particularly multiple sclerosis, and also
neurodegenerative diseases such as sequelae of stroke, head trauma,
bleeds and the chronic abnormalities of Alzheimer's and Parkinson's
disease. Further disorders that can be pretreated both
prophylcatically and therapeutically are coronary heart disease
(CHD) abnormalities of premature infants and sepsis.
[0032] Particularly advantageously treated neurodegenerative
diseases are those involving demyelination. The present method is
specifically addressed at arresting underlying neurodegeneration
and restoring neuronal function. Particularly the method preferably
normalises neuronal membrane composition, and restores healthy PBMC
stimulated or spontaneously 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.
[0033] Preferably the compound of the present invention is
administered for a duration and at a dose sufficient to maintain or
elevate TGF-.beta.1 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.
[0034] The present invention further provides a method of treating
a patient in need of remyeleination in a demyelinating disease
comprising administering to that patient a therapeutically
effective amount of a compound of formula I wherein R.sup.3 is
selected from .gamma.-linolenoyl, .gamma.-dihomolinolenoyl and
arachidonoyl; most preferably .gamma.-linolenoyl and
.gamma.-dihomolinolenoyl.
[0035] For all the methods of the invention, the amount of compound
administered daily will be between 0.5 and 30 grams, orally dosed,
still more preferably between 0.75 and 20 grams and most preferably
between 1 and 18 grams, typically 2 to 5 grams. This dose may be
given as one single dose or in two or more doses together totally
this amount per day.
[0036] Where the sn-2 moiety is that of a .gamma.-linolenic acid
residue, the dose may be toward the higher end of these ranges, but
is preferably between 1 and 10 grams/day, more preferably 2 to 6
grams/day. Where the sn-2 moiety is that of a
dihomo-.gamma.-linolenic acid residue, the dose may be less, whilst
where the sn-2 moiety is that of an arachidonic acid residue,
efficacy is higher, but dosing should be more cautious, due to
possibilities of unwanted side effects at higher levels and the
pro-inflammatory nature of this PUFA.
A second aspect of the present invention provides novel compound of
formula I
##STR00002##
[0037] wherein R.sup.1 and R.sup.2 together form a group
(CH.sub.2).sub.n--CR.sup.4R.sup.5--(CH.sub.2).sub.m--
[0038] wherein n and m are independently selected integers 0, 1 or
2
[0039] and R.sup.4 and R.sup.5 are independently selected from H,
C.sub.1-18 alkyl, C.sub.1-18 alkoxy, C.sub.1-18 hydroxyalkyl,
C.sub.2-18 alkenyl and C.sub.6-18 aralkyl
[0040] and R.sup.3 is a fatty acyl group of an essential
polyunsaturated fatty acid.
[0041] Preferred polyunsaturated fatty acyl groups are those of
n-3, n-6 and n-9 fatty acids, more preferably n-3 and n-6 fatty
acids, still more preferably being .gamma.-linolenoyl,
.gamma.-dihomolinolenoyl and arachidonyl.
[0042] Preferably R.sup.1 and R.sup.2 form a group
--CR.sup.4R.sup.5 where R.sup.4 and R.sup.1 are independently
selected from H and C.sub.1-6 alkyl. Most advantageously R.sup.1
and R.sup.2 form a group --CH.sub.2-- or --CH.sub.2CH.sub.2--.
[0043] Thus compounds of formula IIa are preferred
##STR00003##
[0044] wherein R.sup.1 and R.sup.2 are independently selected from
hydrogen and C.sub.1-6 alkyl.
[0045] Particularly preferred are compounds that are
5-acyloxy-1,3-dioxans, more particularly n-3-fatty acyl or n-6
fatty acyloxy-1,3-dioxans, most preferably 5-.gamma.-linolenoyloxy,
5-dihomo-.gamma.-linolenoyloxy and
5-arachidonoyloxy-1,3-dioxans.
[0046] A third aspect of the present invention provides a method
for synthesis of a compound of general formula II comprising
reaction a 5-hydroxy-1,3-dioxan with an essential fatty acid or an
essential fatty acid halide.
[0047] A fourth aspect of the present invention provides
compositions for use in the method of the present invention
comprising the compounds of formula I together with a
pharmaceutically or nutraceutically acceptable carrier, coating,
capsule, diluent and/or preservative. The compounds 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
[0048] By preservative is meant an antioxidant or inhibitor of
transesterification. It is particularly preferred that the
composition does not include Vitamin E, or includes only levels of
Vitamin E that are 0.05 mg/g or less, eg 0.005 to 0.05 mg/g.
[0049] A fifth aspect of the present invention provides a
pharmaceutical composition for regulating the immune system,
particularly by modulating cytokines TGF-.beta.1, TNF-.alpha., IL4,
IL5, IL6, IL8, IL10, IL13, and/or .gamma.-IFN comprising a compound
of general formula I as defined for the method of treatment of the
invention.
[0050] A sixth aspect of the present invention provides use of the
compounds of formula I, formula II and formula IIa as described
above for the manufacture of a medicament for the treatment of
cytokine dysregulation and 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. Particularly treated is demyelination.
[0051] It will be realised by those skilled in the art that other
beneficial agents may be combined with the compounds for use in the
present invention or otherwise form part of a treatment regime.
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 realised
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 potential
for delayed response to the present compounds, shorter acting
agents might be beneficial in the first months of treatment before
the cytokine levels are normalised, as long as the additional
treatment does not impede this normalization process.
[0052] The synthesis of compounds and compositions for use in the
present invention is described below together with synthesis of
comparative examples. Whilst the Examples exemplify compounds where
R.sup.3 is .gamma.-linolenoyl, the dihomo-.gamma.-linolenoyl and
arachidonoyl and n-3 and n-9 analogues of these are readily
synthesised through analogous starting materials.
[0053] 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
FIGURES
[0054] FIG. 1: Shows spontaneous peripheral blood mononuclear cell
cytokine production in placebo and high sn-2 .gamma.-linolenic
acid, WO2004/100943 trial oil treated human MS patients at 18
months. The left hand column in each case is placebo: right
treated.
[0055] 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.
[0056] 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.
[0057] FIG. 4: Shows synthesis of 2-GLA glycerol formal (aka:
5-.gamma.-linolenoyloxy)-1,3-dioxan and
5-(1,3-dioxanyl)methyl-.gamma.-linolenate)
EXAMPLES
Background
[0058] High sn-2 Borage Oil (WO2004/100943) Trial.
Isolation and Culture of PBMC
[0059] 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
[0060] 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
[0061] 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 IFN-.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
[0062] 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
[0063] 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
See FIG. 1
Experimental Procedure
[0064] 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 14.89s 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.
Synthesis of Compounds for Use in the Present Invention.
Example 1
1a) Preparation of 1,3-O-benzylidene glycerol 2-octa-6Z, 9Z,
12Z-trienoate. (Intermediate and Known Compound for Use in the
Invention where R4 is H and R5 is Benzyl)
[0065] Oxaloyl chloride (7.8 ml, 11.3 g, 0.089 mol, 0.95
equivalents) was added over 2-3 minutes to a stirred solution of
.gamma.-linolenic acid (GLA95, 16.7 g, 0.060 mol, 0.64
equivalents-Scotia) in dichloromethane (100 ml) under N.sub.2. The
mixture was stirred overnight at room temperature and then
concentrated in vacuo to give a tan oil. This crude
.gamma.-linolenoyl chloride was added over ca 10 minutes to a
stirred solution of 1,3-O-benzylidene glycerol (13.0 g, 0.094 mol,
1 equivalent), dry pyridine (30 ml, 29.3 g, 0.37 mol, 4
equivalents) and dichloromethane (DCM, 120 ml) at 5.degree. C. and
the mixture then stirred at room temperature for 2 hours. The
reaction mixture was filtered and then the filtrate washed with
DCM. The combined filtrate and washings were then washed with water
(2.times.20 ml) and the DCM extract dried over Mg SO4 and
concentrated in vacuo to give a crude product as a tan oil (purity
>90% by HPLC). The oil was purified by column chromatography on
silica gel (300 g). Elution with DCM gave the product as a yellow
oil (19.2 g (73%), 96.3% purity by HPLC).
Example 2
2-GLA glycerol formal (aka: 5-.gamma.-linolenoyloxy)-1,3-dioxan and
5-(1,3-dioxanyl)-methyl-.gamma.-linolenate)
[0066] For the purposes of this exemplification, the synthesis of
this ester was carried out by acylation of the commercially
available glycerol formal mixture using .gamma.-linolenoyl
chloride. By this method a mixture of two products is formed and
these were separable by column chromatography. The undesired
by-product is eluted off the column first; further elution gives
the desired acetal-ester. It is a yellow oil at room temperature
and appears to have stability properties similar to GLA, stable in
air at room temperature for short periods (days) but is best stored
long term in a cool place under nitrogen.
Experimental
[0067] Oxalyl chloride (2.6 ml, 3.78 g, 30 mmol, 1.5 equiv) was
added to a solution of .gamma.-linolenic acid (GLA, 5.56 g, 20
mmol. 1.0 equiv) in dichloromethane (DCM, 40 ml). The resulting
solution was stirred under N.sub.2 at room temperature overnight
and then concentrated in vacuo. The residual oily
.gamma.-linolenoyl chloride was added dropwise over 10 min to a
stirred solution of glycerol formal (2.50 g, 24 mmol, 1.2 equiv) in
DCM (40 ml) containing pyridine (10 ml, 9.78 g, 0.12 mol, 6 equiv)
at 5.degree. C. The reaction mixture was stirred at room
temperature for 2 h, the precipitated pyridine hydrochloride
filtered off, and the filtrate washed with water (2.times.). After
drying over MgSO.sub.4 the solvent was removed in vacuo to give a
light tan oil (6.5 g). This material was chromatographed on silica
(60 g). Elution with hexane-ether (94:6) gave 5.2 g of an oil
consisting of two components (TLC, HPLC). These were separated on a
second silica column (60 g). Elution with hexane-ether (98:2 then
95:5) gave 4-(.gamma.-linolenoyloxymethyl)-1,3-dioxolane as a
yellow oil (1.2 g, 98% by HPLC). .delta..sub.H (500 MHz,
CDCl.sub.3) 0.89 (3H, t, J=7.0 Hz, CH.sub.3), 1.24-1.45 (8H,
complex m, 4.times.CH.sub.2), 1.65 (2H, p, J=7.5 Hz,
CH.sub.2--C--CO), 2.08 (4H, m, 2.times.CH.sub.2C.dbd.C), 2.35 (2H,
t, J=7.5 Hz, CH.sub.2CO), 2.80 (4H, t, J=6.0 Hz,
2.times.C.dbd.CCH.sub.2C.dbd.C), 3.67 (1H, m, OCH.sub.AH.sub.B),
3.97 (1H, m, OCH.sub.AH.sub.B), 4.14 (2H, m, OCH.sub.AH.sub.B),
4.26 (1H, p, J=3.5 Hz, CHO), 4.89 and 5.02 (2H, 2.times.s,
OCH.sub.2O), 5.36 (6H, m, 3.times.CH.dbd.CH). .delta..sub.C (126.8
MHz, CDCl.sub.3) 14.09 (CH.sub.3), 22.60, 24.51, 25.65, 26.85,
27.23, 29.16, 29.34, 31.53, 33.97, 63.93 (CH.sub.2O), 66.72
(CH.sub.2O), 73.31 (CHO), 95.44 (OCO), [127.60, 128.04, 128.32,
128.41, 129.50, 130.41, olefinic C], 173.26 (carbonyl).
[0068] Further elution gave 5-(.gamma.-linolenoyloxy)-1,3-dioxan as
a yellow oil (1.6 g, 97.8%) by HPLC). .delta..sub.H (500 MHz,
CDCl.sub.3) 0.89 (3H, t, J=7.0 Hz, CH.sub.3), 1.24-1.46 (8H,
complex m, 4.times.CH.sub.2), 1.67 (2H, p, J=7.5 Hz,
CH.sub.2--C--CO), 2.05 (4H, m, 2.times.CH.sub.2C.dbd.C), 2.40 (2H,
t, J=7.5 Hz, CH.sub.2CO), 2.81 (4H, t, J=6.0 Hz,
2.times.C.dbd.CCH.sub.2C.dbd.C), 3.91 (2H, m, OCH.sub.2), 3.99 (2H,
m, OCH.sub.2), 4.73 (1H, p, J=3.5 Hz, CHO), 4.80 (1H, d, J=6.0 Hz,
OCH.sub.AH.sub.BO), 4.93 (1H, d, J=6.0 Hz, OCH.sub.AH.sub.BO), 5.37
(6H, m, 3.times.CH.dbd.CH). .delta..sub.C (126.8 MHz, CDCl.sub.3)
14.08 (CH.sub.3), 22.59, 24.53, 25.65, 26.86, 27.22, 29.06, 29.34,
31.52, 34.12, 65.54 (CHO), 68.55 (CH.sub.2O), 93.66 (OCO), [127.60,
128.05, 128.32, 128.42, 129.51, 130.42, olefinic C], 173.12
(carbonyl). Some fractions containing both compounds were obtained
during the chromatography and these could be recycled if necessary
to give more material. The reaction scheme for this synthesis is
shown in the figures below.
Example 3
Production of Monoglyceride Enriched Compositions from Synthetic
CGC Structured-Lipid. Model Compound for Metabolite of Compounds of
the Invention
[0069] 1. 2-GLA MG (.gamma.-linolenic acid monoglyceride) from CGC
(Glycerol 1,3-didecanoate-2-.gamma.-linolenoate)
[0070] Lipase acrylic resin from Candida antarctica (Sigma,
Novozyme, 0.1 g) was added to a solution of CGC (0.25 g) in ethanol
(0.75 ml). The mixture was stirred at 3540.degree. C. and monitored
by HPLC. After 3 h the resin was removed by filtration and washed
with ethanol. The filtrate and washings were concentrated in vacuo.
Analysis of the residual oil by HPLC indicated the formation of two
major products. These were separated by chromatography on
silica-boric acid. Elution with dichloromethane (DCM) gave an oil
(fraction A, 160 mg). Further elution with DCM-MeOH (9:1) gave an
oil (fraction B, 80 mg). HPLC comparison with authentic materials
indicated that B was the required product i.e. 2-GLA MG (8%
rearranged 1-isomer also present). The main (>90%) component of
fraction A was found (by HPLC comparison and NMR) to be ethyl
decanoate. The minor component was found (by HPLC and NMR) to be
ethyl .gamma.-linolenoate. These esters are expected to be formed
under the reaction conditions from the corresponding acids (C and
GLA) and ethanol.
Biological Studies.
Example 4
[0071] Solublization of Sn-2 monoglyceride was performed using
ethyl alcohol or DMSO for in vitro work on human peripheral blood
mononuclear cells (PBMCs). A tendancy to precipitate at acid pH may
have been the cause of some animals regurgitating solid material
after gavage suggesting that enterically coated formulation may be
preferred. SJL mice were fed sn-2 GLA of Example 1 at three doses
(50, 125 and 250 .mu.l) for seven days by gavage. Mice receiving
higher doses were prone to regurgitation. After seven days animals
were killed and the brain, liver and spleen were removed the liver
and brain frozen at -70.degree. C. and mononuclear cells were
isolated from spleens by sieving and density centrifugation on
Lymphoprep (Sigma Chemical Co) and cultured at 37.degree. C. in 5%
CO.sub.2 atmosphere in 511 culture tubes at a cell density of
1.times.10.sup.6 cells/ml in RPMI 1640 medium in 5% foetal calf
serum (FCS). Cells were cultured with and without 1 .mu.g/ml or 25
.mu.g/ml concanavalin A (Con A) for approximately 20 hours and the
supernatants removed and stored at -70.degree. C. until required.
Mouse TGF-.beta.1 was measured in supernatants using a commercially
available ELISA (Promega, Madison Wis.).
TABLE-US-00001 TABLE 1 Stimulated (Con A) and unstimulated
TGF-.beta.1 production pg/ml from spleen PBMCs in response to
feeding of sn-2-.gamma.-linolenoyl-glycerol monoglyceride of
Example 3 Con A (.mu.g/ml Monoglyceride 0 1 25 50 .mu.g 377 409 480
Control 209 228 393 Change % 80 79 42
[0072] This shows that the metabolite of the .gamma.-linolenoyl
compound of formula of Example 2 is active in raising cytokine
TGF-.beta.1 in the same manner as Borage oil of the trial. This is
consistent with modulation of all the cytokines previously shown
modulated in this matter.
Example 5
In Vivo Activity of Monoglyceride Formal (Compound of Example
2)
[0073] C57/BL mice were dosed orally with 2 GLA MG formal at two
doses of 50 and 150 ul for eight days. The compound was supplied in
8 separate tubes to be stored at 4.degree. C. prior to dosing but
warmed to 25.degree. C. before use. After eight days the animals
were killed and the brain, liver and spleen were removed. Liver and
brain were frozen at -70.degree. C. and mononuclear cells were
isolated from spleens by sieving and density centrifugation on
Lymphoprep (Sigma Chemical Co) and cultured at 37.degree. C. in 5%
CO.sub.2 atmosphere in 5 ml culture tubes at a cell density of
2.times.10.sup.6 cells/ml in RPMI 1640 medium in 5% fetal calf
serum (FCS). Cells were cultured with and without 1 .mu.g/ml or 25
.mu.g/ml concanavalin A (Con A) for approximately 20 hrs and the
supernatants removed and stored at -70.degree. C. until required.
Mouse TGF-.beta.1 was measured in supernatants using a commercially
available ELISA system (Pomega, Madison, Wis.).
Results:
[0074] Concanavalin A did not significantly stimulate TGF-.beta.1
production in this study. Data were therefore combined for the
purposes of statistical analysis (n=9/group). Data were analysed
using Graphpad Instat.
TABLE-US-00002 TABLE 2 Spleen mononuclear cell TGF.beta. production
(pg/ml) Controls 77.9 .+-. 4.1 GLA 2-MG-F50 ul 90.9 .+-. 5.2 P =
0.0702 GLA 2-MG-F 150 ul 158.6 .+-. 7.5 P < 0.0001 .uparw.103%
[Data analysed using unpaired t-test; data are mean .+-. SEM]
REFERENCES
[0075] Amor S, Groome N, Linington C, Morris M M, Dornmair K,
Gardinier 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. [0076] 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. [0077] 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.
[0078] 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. [0079]
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. [0080]
Brosnan C F and Raine C S. Mechanisms of immune injury in multiple
sclerosis. Brain Pathol. 1996:6, 243-257. [0081] 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. [0082] Cannella B, Raine C S.
The adhesion molecule and cytokine profile of multiple sclerosis
lesions. Ann Neurol 1995; 37:424-35. [0083] 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. [0084]
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. [0085]
Ewing C, Bernard C C. Insights into the aetiology and pathogenesis
of multiple sclerosis. Immunol Cell Biol 1998; 76:47-54. [0086]
Fazakerly J K. Molecular biology of multiple sclerosis. Wiley and
Sons Ltd. 1997, 255-273. [0087] 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. [0088] Genain C P., Cannella B., hauser S L
and Raine C S. Identification of autoantibodies associated with
myelin damage in multiple sclerosis. Nature Med 1999:5, 170-175.
[0089] 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. [0090] Harbige, L S, 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. [0091] Harbige L S.
Nutrition and immunity with emphasis on infection and autoimmune
disease. (1996) Nutr Health, 10(4):285-312. [0092] 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. [0093]
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. [0094] 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. [0095] 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. [0096] 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. [0097] 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-inflammatory
and anti-inflammatory cytokines during relapse and remission.
Autoimmunity, 2003 36(3):133-141. [0098] 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. [0099] 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. [0100] 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.
[0101] 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. [0102] 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. [0103] Khalil N.
TGF-beta: from latent to active. Microbes Infect 1999; 1:1255-63.
[0104] 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. [0105] Kuroda Y, Shimamoto
Y. Human tumor necrosis factor-alpha augments experimental allergic
encephalomyelitis in rats. J Neuroimmunol 1991; 34:159-64. [0106]
Lu C Z, Jensen M A, Arnason B G. Interferon gamma and
interleukin-4-secreting cells in multiple sclerosis. J Neuroimmunol
1993; 46:123-8. [0107] 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.
[0108] Marino G, Hartung H-P. Immunopathogenesis of multiple
sclerosis: the role of T cells. Curr Opin Neurol 1999; 12:309-21.
[0109] 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. [0110] McDonald W
I, Compston A, Edan G, Goodkin D, Harting 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. [0111] 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. [0112] 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. [0113] Merrill J E,
Zimmerman R P. Natural and induced cytotoxicity of oligodendrocytes
by microglia is inhibitable by TGF beta. Glia 1991; 4:327-31.
[0114] Miyazono K, Hellman U, Wernstedt 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. [0115] 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. [0116] Navikas V, Link H.
Review: cytokines and the pathogenesis of multiple sclerosis. J
Neurosci Res 1996; 45:322-33. [0117] Noseworthy J H. Progress in
determining the causes and treatment of multiple sclerosis. Nature
1999:399(6738 Suppl), A40-47. [0118] 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. [0119]
Perkin G D, Wolinsky J S. Fast facts-Multiple Sclerosis, 1st Edn.
Oxford, UK: Health Press, 2000. [0120] 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. [0121] 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. [0122] Prehn J 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. [0123] 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. [0124]
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. [0125] 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. [0126] 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.
[0127] 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. [0128] 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. [0129]
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. [0130] 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. [0131]
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. [0132] Selmaj K, Raine C S,
Farooq M et al. Cytokine cytotoxicity against oligodendrocytes.
Apoptosis induced by lymphotoxin. J Immunol 1991; 147:1522-9.
[0133] 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.
[0134] 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 J Immunol 2001;
31:907-17. [0135] 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.
[0136] 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.
[0137] 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. [0138] Japanese Patent 6172263 (1994) Y. Kosugi
et al, Agency of Industrial Science & Technology High-purity
arachidonic acid triglyceride and its production. [0139] U.S. Pat.
No. 4,888,324 (1989) N. Catsimpoolas et al, Angio-Medical
Corporation Method for enhancing angiogenesis with lipid containing
molecules. [0140] Y. Kosugi and N. Azuma, J. Amer. Oil Chem. Soc.,
71, 1397-1403 (1994). Synthesis of Triacylglycerol from
polyunsaturated fatty acid by immobilized ipase. [0141] J. W.
Hageman et al, J. Amer, Oil Chem. Soc., 49, 118-xxx (1972)
Preparation of Glycerin and their Uses. [0142] E. S. Lutton and A.
J. Fehl, Lipids, 5, 90-99 (1970). The polymorphism of odd and even
saturated single acid triglycerides, C.sub.8-C.sub.22. [0143] D.
Horrobin, A. McMordie, M. S. Manku (Scotia Holdings PLC UK) Eur.
Pat. Appl EP 609078 3 Aug. 1994. Phospholipids containing two
different unsaturated fatty acids for use in therapy, nutrition,
and cosmetics. [0144] 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 [0145] 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. [0146] 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. [0147] 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 [0148] P. H.
Bentley and W. McCrae, J. Org. Chem. 35, 2082-2083 (1970) An
Efficient Synthesis of Symmetrical 1,3-Diglycerides. [0149] 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. [0150] 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. [0151] L.
Hartman, Chem. Rev., 58, 845-867 (1958) and references therein.
Advances in the Synthesis of Glycerides of Fatty Acids
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