U.S. patent application number 11/834505 was filed with the patent office on 2008-03-27 for compositions containing lysophosphatidic acids which inhibit apoptosis and uses thereof.
This patent application is currently assigned to SKY HIGH, LLC. Invention is credited to Anthony D. Baxter, Edward A. Boyd, John G. Goddard, Donald H. Picker, Steven Price, Samuil R. Umansky, Jac C. Wijkmans.
Application Number | 20080076736 11/834505 |
Document ID | / |
Family ID | 34992618 |
Filed Date | 2008-03-27 |
United States Patent
Application |
20080076736 |
Kind Code |
A1 |
Goddard; John G. ; et
al. |
March 27, 2008 |
COMPOSITIONS CONTAINING LYSOPHOSPHATIDIC ACIDS WHICH INHIBIT
APOPTOSIS AND USES THEREOF
Abstract
The invention provides anti-apoptotic compositions containing
lysophosphatidic acids and methods for making and using the
compositions. Such compositions may also contain LPA potentiating
agents, including proteins, lipid membrane structures and polymers
such as polyethylene glycols. The compositions can additionally
contain other pharmaceutically effective agents such as drugs,
antibiotics, wound healing agents and antioxidants.
Inventors: |
Goddard; John G.; (San
Francisco, CA) ; Picker; Donald H.; (Warren, NJ)
; Umansky; Samuil R.; (Richmond, CA) ; Price;
Steven; (Aylesbury, GB) ; Wijkmans; Jac C.;
(Abingdon, GB) ; Boyd; Edward A.; (Didcot, GB)
; Baxter; Anthony D.; (Abingdon, GB) |
Correspondence
Address: |
SHERIDAN ROSS PC
1560 BROADWAY
SUITE 1200
DENVER
CO
80202
US
|
Assignee: |
SKY HIGH, LLC
1560 Sherman Avenue Suite 900
Evanston
IL
60201
|
Family ID: |
34992618 |
Appl. No.: |
11/834505 |
Filed: |
August 6, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11236429 |
Sep 26, 2005 |
7259273 |
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11834505 |
Aug 6, 2007 |
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09646599 |
Jan 10, 2001 |
6949528 |
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PCT/US99/05943 |
Mar 17, 1999 |
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11236429 |
Sep 26, 2005 |
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60078375 |
Mar 18, 1998 |
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Current U.S.
Class: |
514/75 ; 435/375;
435/383; 568/15 |
Current CPC
Class: |
A61P 43/00 20180101;
C07F 9/091 20130101 |
Class at
Publication: |
514/075 ;
435/375; 435/383; 568/015 |
International
Class: |
A61K 31/66 20060101
A61K031/66; A61P 43/00 20060101 A61P043/00; C07F 9/02 20060101
C07F009/02; C12N 5/06 20060101 C12N005/06 |
Claims
1-57. (canceled)
58. A compound of the following formula: ##STR16## or a salt
thereof, wherein, each W is independently OH,
OCH.sub.2CH(NH.sub.2)CO.sub.2H, OCHCH.sub.3CH(NH.sub.2)CO.sub.2H,
OPO.sub.3H.sub.2, or OP(O)(OH)--O--PO.sub.3H.sub.2, and Q is the
following structure: ##STR17## wherein, Y is O or S; R is
substituted or unsubstituted, saturated or unsaturated, straight or
branched-chain alkyl, or ((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.pT
where q is an integer from 1 to 900 and where each p is
independently an integer from 2 to 10 and T is OH, or
O(CH.sub.2).sub.bCH.sub.3 where b is an integer from 0 to 10; V is
independently OH, SH, H, NH.sub.2, halogen, OPO.sub.3H.sub.2, or
OSO.sub.3H; n is an integer from 0 to 10; m is an integer from 0 to
10; Z is OH, SH, NH.sub.2, OPO.sub.3H.sub.2, halogen, HN,
O(CH.sub.2).sub.dCH.sub.3 where d=0 to 2, or SO.sub.3H; and L is
independently O or S.
59. A composition comprising a compound of claim 58, and at least
one pharmaceutically acceptable excipient.
60. A method of making a composition comprising a compound of claim
58 and a potentiating component, comprising: a) forming a lipid
dispersion comprising a compound of claim 58; b) combining the
lipid dispersion with at least one potentiating component to form a
composition.
61. The method according to claim 60, wherein the lipid dispersion
is formed by: a) dissolving a compound of claim 58 and any other
lipids in organic solvent; b) removing the solvent from any lipid
compounds dissolved therein to form dried lipid; and, c) dispersing
the dried lipid into aqueous media by the steps of: i) forming an
even lipid dispersion; and ii) forming an even dispersion of lipid
membrane structures.
62. The method according to claim 60, further comprising the step
of c) sterilizing the composition.
63. A method of inhibiting apoptosis of fibroblasts comprising
contacting fibroblasts with a compound of claim 58.
64. A method of culturing cells comprising contacting cells in
culture with a compound of claim 58.
65. A method of inhibiting apoptosis of cells present within an
organ that has been removed from a donor comprising contacting said
organ with a compound according to claim 58.
66. A method of inhibiting apoptosis of cells present within an
organ of a donor comprising administering to said donor at least
one intravenous bolus of an effective amount of a compound
according to claim 58.
67. A compound of claim 58, wherein R is substituted or
unsubstituted, saturated or unsaturated, straight or branched-chain
alkyl having from about 5 to 30 carbons.
68. A compound of claim 58, wherein R is substituted or
unsubstituted, saturated or unsaturated, straight or branched-chain
alkyl having about 10 carbons.
Description
TECHNICAL FIELD
[0001] This invention relates, to therapeutically effective
compositions of matter. More specifically, it encompasses
compositions containing lysophosphatidic acid or analogs and
derivatives thereof (collectively "LPA") which have been found to
exhibit anti-apoptotic activity and/or to preserve or restore cell,
tissue or organ function. This invention specifically relates to
therapeutically effective reverse ester, reverse thioester, and
bis-LPA analogs encompassed by the term LPA. Additionally, this
invention relates to methods of use of these therapeutically
effective compositions.
BACKGROUND OF THE INVENTION
[0002] Phospholipids.
[0003] Phospholipids are a class of amphipathic
phosphorus-containing lipids which are essential constituents of
biological membranes. Various phospholipid preparations have been
used for cooking, drug delivery (liposomes), slow release delivery
systems, carrier media for hydrophobic drugs, gene transfer and
replacement therapy, sunscreens, emulsions, anti-foaming agents,
replacement of damaged or absent pulmonary surfactants, detergents
and membrane stabilization. Phosphatidic acid (PA),
phosphatidylinositol (PI), lysophosphatidic acid,
lysophosphatidylinositol (LPI), and lysophosphatidylcholine (LPC)
are found in a variety of plant and animal products.
Lysophosphatidic acid analogs have been reported to have a variety
of physiological activities including mitogenesis (i.e. prevention
of hyperproliferative diseases), vasodilation, growth factor, wound
healing and to be an anti-wrinkle agent. In addition, previous
studies have shown that lysophosphatidic acid, when bound to serum
albumin, can activate membrane currents in Xenopus oocytes and
induce neurite retraction in PC12 pheochromocytoma cells.
[0004] Apoptosis.
[0005] Cell death can occur by necrosis and apoptosis. is generally
results from catastrophic irreversible cell damage. It is
characterized by early swelling of the cell and its cytoplasmic
organelles, with subsequent rupture of the cellular membrane.
[0006] Apoptosis is a normal physiologic process that leads to
individual cell death. This process of programmed cell death is
involved in a variety of normal and pathogenic biological events
and can be induced by a number of unrelated stimuli. Changes in the
biological regulation of apoptosis also occur during aging and are
responsible for many of the conditions and diseases related to
aging. Apoptotic cell death appears to play a significant role in
the tissue damage that occurs in association with, for example,
ischemia, organ transplantation, and various gastrointestinal
disorders.
[0007] Studies of apoptosis suggest that a common metabolic pathway
leading to apoptosis can be initiated by a wide variety of signals,
including hormones, serum growth factor deprivation,
chemotherapeutic agents, ionizing radiation, and infection by human
immunodeficiency virus (HIV). Apoptosis can also be induced by
mild, non-catastrophic cell injury and can be concomitant with
adjacent necrosis. Agents that affect the biological control of
apoptosis thus have therapeutic utility in numerous clinical
indications.
[0008] Apoptotic cell death is characterized by morphologic changes
such as cellular shrinkage, chromatin condensation and margination,
cytoplasmic blebbing, and increased membrane permeability. Specific
internucleosomal DNA fragmentation is a hallmark for many, but not
all, instances of apoptosis.
[0009] Several genes and gene families involved in signal
transduction and modulation of apoptosis have been described.
Apoptosis, however, is an active cellular response to a physiologic
or external signal and can be modulated by interfering with the
apoptotic pathway. Conversely, by definition, necrosis can be
prevented only by decreasing cell injury. Prevention of apoptosis
by upregulation of bcl-2 and bcl-x expression, or by inhibitors of
ICE-like proteases are typical examples of modulation of cell
death. Umansky Molekylyarnaya Biologiya 30:285-295 (1996); Vaux and
Strasser PNAS 93:2239-2244 (1996); Nunez et al. (1994) Immunol.
Today 15:582-588; and Whyte (1996) Trends in Cell Biol.
6:245-148.
[0010] Ischemia and Reperfusion.
[0011] Ischemia is the result of decreased blood flow to a
particular area or organ of the body. Ischemia is responsible for
several important types of physiologic damage such as brain damage,
spinal cord trauma and myocardial ischemia. Acute myocardial
ischemia leads to the death of individual heart cells which can
result in organ dysfunction. Although early reperfusion (i.e.,
restoration of blood flow) decrease heart damage caused by
ischemia, cell death by apoptosis can occur upon reperfusion. In
this instance, the cells that die are those that remained viable at
the end of ischemia. Support for the role of apoptosis in heart
injury induced by ischemia and subsequent reperfusion has been
provided by numerous laboratories. Gottlieb et al. (1994) J. Clin.
Invest. 94:16211628; Umansky et al. (1995) Cell Death an
Differentiation 2:235241; Umansky et al. (1996) Basic and Applied
Myology 6:227235; and Itoh et al. (1995) Am. J. Pathol.
146:13251331. Severe cell damage during prolonged ischemia appears
to result in necrotic death of myocardial cells. However, if the
ischemia is relatively limited in extent and duration, the
apoptotic pathway is initiated. Restoration of blood flow
(reperfusion) allows apoptosis to proceed. Insulin-like growth
factors (IGF) and calpain inhibitors, which are capable of
preventing apoptosis in different systems, also inhibited apoptosis
of cardiomyocytes following ischemia and reperfusion both in vivo
and in vitro. Umansky et al. (1995); and Buerke et al. (1995) Proc.
Natl. Acad. Sci. USA 92:80318035.
[0012] Organ Preservation.
[0013] Transplantation of vital organs such as the heart, liver,
kidney, pancreas, and lung has become increasingly successful and
sophisticated in recent years. Because mammalian organs
progressively lose their ability to function during storage, even
at freezing temperatures, transplant operations need to be
performed soon after obtaining a donor organ to minimize the period
of time that the organ is without blood flow. This need diminishes
the availability of organs for transplantation.
[0014] In clinical practice, the two major situations in which
cardiac preservation is required are heart transplantation and
cardioplegia for open heart surgery. In heart transplantation, the
donor heart is flushed with approximately 1 liter of cold organ
preservation solution (OPS) to arrest the heart. Cooling is
supplemented by surrounding the heart with iced saline. The
chilled, arrested heart is then surgically excised, immersed in
cold OPS, packed in ice and rushed to the recipient center.
[0015] After placing the recipient on cardiopulmonary bypass, the
diseased heart is excised. The preserved donor heart is then
removed from the OPS, trimmed appropriately and transplanted into
the recipient. Blood is allowed to flow to the transplanted heart.
The transplanted heart will then either resume beating
spontaneously or will require chemical and/or electrical treatment
to restore normal rhythm. When the heart is ready to take over the
circulation, the cardiopulmonary bypass is discontinued and the
recipient's chest closed.
[0016] Most non-transplant surgical procedures on the heart, such
as coronary artery bypass grafting, require that the heart be
arrested for a period ranging from 1 to 4 hours. During this time,
the heart is kept cool by external cooling as well as by
periodically reflushing an OPS through the coronary arteries. The
OPS composition is designed to rapidly arrest the heart and to keep
it in good condition during the period of standstill so that it
will resume normal function when the procedure is finished.
[0017] In cardioplegic procedures, the heart is exposed in the
chest and, at a minimum, the aortic root is isolated. A vascular
clamp is applied across the aorta and approximately 1 liter of cold
OPS is flushed into the aortic root through a needle. Venting is
provided through the left ventricle, pulmonary artery or the right
atrium and the effluent, which can contain high levels of
potassium, is suctioned out of the chest. This, together with
external cooling, produces rapid cessation of contractions. During
the period of arrest the patient's circulation is maintained
artificially using cardiopulmonary bypass.
[0018] After completion of the surgical procedure, blood flow is
restored to the coronary circulation and heartbeat returns either
spontaneously or after chemical and electric treatment. The ease
with which stable function is restored depends to a large extent on
the effectiveness of the OPS to preserve the heart. Once the heart
is beating satisfactorily, cardiopulmonary bypass is discontinued
and the chest closed.
[0019] It is generally understood at "living" organs, including the
heart, continue the process of metabolism after removal from the
donor so that cell constituents are continuously metabolized to
waste products. If the storage technique is inadequate, the
accumulation of these metabolic waste products, depletion of cell
nutrients and consequent derangement of cell composition lead to
progressive loss of function and ultimately to cell death, with
loss of adequate function after transplantation into the recipients
Several procedures have been explored to successfully enable organ
preservation ex vivo for useful time periods. In one method, the
donor organ is cooled rapidly by flushing cold solutions through
the organ's vascular system and maintaining the organ at
temperatures near 0.degree. C. for the purpose of greatly slowing
the metabolic rate. In the case of the mammalian heart, the flush
solution composition is designed to cause the heart to rapidly stop
beating as well as to preserve it.
[0020] In 1988, University of Wisconsin (UW) solution was
introduced. Belzer et al. (1988) Transplantation 45:673-676. This
solution capable of preserving the pancreas and kidney for 72
hours, and the liver for 30 hours, subsequently became the standard
organ preservation solution (OPS) for transplant surgery and the
benchmark against which other OPS compositions were measured.
However, the heart is more recalcitrant to long-term storage than
other organs, and UW solution is unreliable for storage of hearts
for as short a period as 24 hours. Wicomb et al. (1989)
Transplantation 47:733-734.
[0021] Improvements in the design of OPS compositions, as reviewed
in Collins et al. (1992) Kidney International 42:S-197-S-202 and
others described in the art, have proceeded along several paths,
including: (1) modification and simplification of UW solution; (2)
investigation of organ-specific requirements; (3) addition of
pharmacologic agents, particularly calcium antagonists for control
of acidosis; (5) the use of a terminal rinse solution; and (6) the
use of solutions containing PEG.
[0022] Wicomb et al. reported the beneficial effects of a solution
of PEG 8000 and horseradish peroxidase on rabbit hearts preserved
by oxygen low pressure perfusion for 24 hours. Wicomb et al. (1989)
Transplantation Proceedings 21:1366-1368. The substitution of
PEG20M as the colloid for hydroxyethyl starch (HES) of the UW
solution also yielded excellent cardiac function. PEG20M consists
of two or more molecules of PEG 6000-8000 joined by a bisphenol
epoxide linker (CAS # 37225-26-6; CAS name Oxirane,
2,2'[(1-methyl-ethylidene)bis(4,1-phenyleneoxy methylene)]bis-,
polymer with (-hydro-(-hydroxypoly(oxy-1,2-ethanediyl). The
substitution of PEG20M for HES also allowed baboon heart storage up
to 48 hours and increased cardiac output (CO) under conditions of
microperfusion. Wicomb et al. (1986) J. Surg. Res. 40:276; and
Wicomb et al. (1989) Transplantation 48:6-9. "Microperfusion" is a
hypoxic, very-low-flow perfusion with flowrates such as 3 ml/g
heart wt/24 hour, which is 1/500 of that typical of conventional
continuous perfusion. Wicomb et al. (1989) Transplantation
48:6-9.
[0023] An improved OPS, Cardiosol.TM. heart preservation solution,
contained the substitution of PEG20M for HES and eliminated five
components of UW solution penicillin, dexamethasone, insulin,
allopurinol, and adenosine). Wicomb et al. (1990) Transplantation
49:261-264; and U.S. Pat. No. 4,938,961. Cardiosol.TM. heart
preservation solution contains 5% or 10% by weight PEG 20M (Union
Carbide Chemicals and Plastics Co., Inc., Charleston, W. Va.), 40
mM sodium, 125 mM potassium, 5 mM magnesium, 25 mM phosphate, 5 mM
site, 100 mM lactobionate, 30 mM raffinose, and 3 mM glutathione.
Collins et al., The Lancet 338:890-891 (1991); and Wicomb et al.
(1994) J. Heart Lung Transplantation 13:891-894. This solution was
found to be superior to UW solution both for 4-hour hypothermic and
24-hour microperfusion storage. Collins et al. (1992).
[0024] Gastrointestinal Disorders.
[0025] A variety of food supplements containing, in part, partially
processed plant extracts have been used to ameliorate the
gastrointestinal disorders that often accompany chemotherapy,
radiation and AIDS. The supplements generally contain
carbohydrates, fat and plant protein hydrolysates. See, e.g., Tomei
and Cope et al. in Apoptosis: The Molecular Basis of Cell Death
(1991) Cold Spring Harbor Laboratory Press. PCT Publication No. WO
95/15173, U.S. Pat. Nos. 5,620,885, 5,567,425, 5,635,186 and
5,624,672 describe plant-derived extracts that produce an
anti-apoptotic effect. It has now been found that these extracts
contain the following phospholipids: LPA, LPC, LPI, PA and PI in
the ratios of approximately 2:1:2:20:20, by weight in addition to
various optional protein and carbohydrate constituents.
[0026] A need exists for improved solutions and methods for
preserving or restoring cell, tissue, or organ function, and/or
preventing apoptosis for a variety of therapeutic uses,
particularly organ preservation. The present invention satisfies
this need and provides related advantages, as well.
SUMMARY OF THE INVENTION
[0027] This invention relates to therapeutically effective
compositions of matter. More specifically, it encompasses
compositions containing lysophosphatidic acid or analogs and
derivatives thereof, (collectively "LPA") which have been found to
exhibit anti-apoptotic activity or to preserve or restore cell,
tissue or organ function. Additionally, this invention relates to
methods of use of these therapeutically effective compositions.
[0028] The present invention encompasses compositions, containing a
compound of the following formula: ##STR1## [0029] wherein M is O
or S, each W is independently selected from one of the following
structures II-V ##STR2## [0030] wherein Y is O or S; R is
unsubstituted or substituted, saturated or unsaturated, straight or
branched-chain alkyl having from preferably 5-7, more preferably
8-10 and most preferably, about 10 to preferably 24-30, more
preferably 24-28 and most preferably about 24 carbon atoms, or
((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.pT where q is an integer
from 1 to about 900 and where each p is independently an integer
from 2 to about 10 and T is OH or O(CH.sub.2).sub.bCH.sub.3 where b
is an integer from 0 to about 10; each V is independently OH, SH,
H, NH.sub.2, halogen, OPO.sub.3H.sub.2, or OSO.sub.3H; n is an
integer from 0 to about 10; m is an integer from 0 to about 10; Z
is OH, SH, NH.sub.2, halogen, OPO.sub.3H.sub.2, H,
O(CH.sub.2).sub.bCH.sub.3 where b=0 to about 2, or SO.sub.3H; L is
independently O, S, CH.sub.2; and X is independently O or S; or a
salt thereof.
[0031] In specific embodiments, R is an alkyl having between about
10 and about 24 carbon atoms, wherein between 0 and 11, inclusive,
of the carbon-carbon bonds are unsaturated. More specifically, R is
an alkyl having 18 carbon atoms, wherein 1 or 2 of the carbon on
bonds are unsaturated and mixtures thereof. Even more specifically,
the composition comprises Bis(3-O-oleyl-2-O-methyl-rac-glyceryl)
thiophosphate, or a salt thereof.
[0032] In another embodiment of the present invention, a
composition contains a compound of the following formula: ##STR3##
[0033] wherein each W is independently SH, OH,
OCH.sub.2CH(NH.sub.2)CO.sub.2H, OCHCH.sub.3CH(NH.sub.2)CO.sub.2H,
OPO.sub.3H.sub.2, OPO.sub.2HOPO.sub.3H.sub.2 or Q, wherein when one
W is Q, the other W is OH, and wherein Q is one of the following
structures: ##STR4## [0034] wherein Y is O or S; R is unsubstituted
or substituted, saturated or unsaturated, straight or
branched-chain allyl having from preferably 5-7, more preferably
8-10 and most preferably, about 10 to preferably 24-30, more
preferably 24-28 and most preferably about 24 carbon atoms, or
((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.pT where q is an integer
from 1 to about 900 and where each p is independently an integer
from 2 to about 10 and T is OH or O(CH.sub.2).sub.pCH.sub.3 where p
is an integer from 0 to about 10; each V is independently OH, SH,
H, NH.sub.2, halogen OPO.sub.3H.sub.2, or OSO.sub.3H; n is an
integer from 0 to about 10; m is an integer from 0 to about 10; Z
is OH, SR, NH.sub.2, halogen, OPO.sub.3H.sub.2, H,
O(CH.sub.2).sub.pCH.sub.3 where p=0 to about 2, or SO.sub.3H; L is
independently O, S, or CH.sub.2; X is independently O or S; and M
is P or S, where when M is S, one W is (.dbd.O) and the other W is
SH, OH, OCH.sub.2CH(NH.sub.2)CO.sub.2H,
OCHCH.sub.3CH(NH.sub.2)CO.sup.2H, OPO.sub.3H.sub.2, or
OPO.sub.2HOPO.sub.3H.sub.2; or a salt thereof.
[0035] In another embodiment, the composition comprises a compound
selected from the group consisting of reverse ester-LPA, reverse
thioester-LPA or a salt of either.
[0036] In a specific embodiment, the composition comprises: 3 Oleyl
1-thiophosphoryl-2-O-methyl-rac-glycerate, or a salt thereof.
[0037] The present invention also provides methods of treating
apoptosis, preserving or restoring function in a cell, tissue or
organ comprising administering in vivo or in vitro a
therapeutically effective amount of a pharmaceutically acceptable
composition of the present invention. The present composition can
also contain a potentiating component including, for example, a
polyethylene glycol, a protein, or a lipid membrane structure.
[0038] In specific embodiments of the present invention, the
compositions of the present invention also comprise
pharmaceutically acceptable excipients, which can be, without
limitation, selected from the group encompassing topical
pharmaceutically acceptable carrier, cosmetic carrier, sterile
solutions, sterile isotonic solutions, ingestable liquids,
pharmaceutically acceptable aerosols and solutions for
organ/tissue/cell preservation and/or transplantation.
[0039] In specific embodiments of the present invention, the
compositions of the present invention also comprise
pharmaceutically effective agents, such as, without limitation,
drugs, antibiotics, wound healing agents and antioxidants.
[0040] Also encompassed by the present invention are methods of
making compositions of the present invention, and the compositions
obtained therefrom, comprising the steps of: forming a lipid
dispersion comprising LPA; providing at least one of said
components; and combining the products of steps a) and b), and more
specifically, wherein the lipid dispersion is formed by the steps
of: a) dissolving LPA and any other lipids in organic solvent; b)
removing the solvent to form dried lipid; and c) dispersing the
dried lipid into aqueous media by the steps of: i) forming an even
lipid dispersion; and ii) forming an even dispersion of lipid
membrane structures.
[0041] Additionally encompassed are methods of treating apoptosis
or preserving or restoring function, in a cell, tissue or organ,
comprising administering a therapeutically effective amount of a
pharmaceutically acceptable composition of the compounds of the
present invention. Such composition can further comprise a
potentiating component.
[0042] The compounds of the present invention can be administered
to a patient suffering from a condition related to apoptosis,
ischemia, traumatic injury or reperfusion damage (wherein the
reperfusion damage can be, without limitation, associated with
coronary artery obstruction, stroke, cerebral infarction,
spinal/head trauma and concomitant severe paralysis, frostbite,
coronary angioplasty, blood vessel attachment, limb attachment,
organ attachment, or kidney reperfusion), and gastrointestinal
perturbation (wherein the gastrointestinal perturbation can be
caused by, without limitation, a stimulus such as viruses,
chemotherapeutic agents, radiation, infectious diseases such as
human immunodeficiency virus, inflammatory bowel disease, and
diarrhea-causing organisms). Treatment can comprise administering
to the patient at least one intravenous bolus of an effective
amount of the composition.
[0043] In another embodiment, methods of the present invention
decrease apoptosis-related problems associated with
immunosuppressing viruses, chemotherapeutic agents, or radiation
and immunosuppressive drugs.
[0044] Also encompassed by the present invention are methods of
culturing cells comprising treating cells with an amount of at
least one of the compositions of the present invention effective to
prevent apoptosis and/or preserve the cells, including preserving
an organ.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] FIG. 1 is a bar graph depicting the effect of various
concentrations of 18:1-LPA analog on protection of C3H/10T1/2 cells
from serum deprivation. LPA was prepared with or without a
potentiating component (bovine serum albumin or phosphatidyl
glycerol vesicles). The open section of the bars represents
adherent cells and the solid section of the bars represents
non-adherent cells. Where not specified as a particular analog,
"LPA" refers to 18:1-LPA in all figures. In FIGS. 1-8 and 11, the
white sections represent adherent cells and the dark sections
represent non-adherent cells.
[0046] FIG. 2 is a bar graph depicting the percentage of adherent
cells (open sections) and non-adherent cells (solid sections) in
the C3H/10t1/2 assay after 24 hours exposure serum-free medium to
which 18:1-LPA was added as a 10% (y weight) mixture in various
phospholipid membrane structures. For all treatments, 18:1-LPA was
delivered at 0.25, 0.75, 2.25 or 6.75 (g/mL. The following list
provides the compositions represented by each column in FIG. 2:
[0047] 1 BME [0048] 2 Five Phospholipid Mixture (0.25 (g/ml LPA)
[0049] 3 Five Phospholipid Mixture (0.75 (g/ml LPA) [0050] 4 Five
Phospholipid Mixture (2.25 (g/ml LPA) [0051] 5 Five Phospholipid
Mixture (6.75 (g/ml LPA) [0052] 6 10% LPA in PG (0.25 (g/ml LPA)
[0053] 7 10% LPA in PG (0.75 g/ml LPA) [0054] 8 10% LPA in PG (2.25
(g/ml LPA) [0055] 9 10% LPA in PG (6.75 (g/ml LPA) [0056] 10 PG
only 67.5 (g/mL [0057] 11 10% LPA in PG/5% PE-PEG (0.25 (g/ml LPA)
[0058] 12 10% LPA in PG/5% PE-PEG (0.75 (g/ml LPA) [0059] 13 10%
LPA in PG/5% PE-PEG (2.25 (g/ml LPA) [0060] 14 10% LPA in PG/5%
PE-PEG (6.75 (g/ml LPA) [0061] 15 PG/5% PE-PEG only 67.5 (g/ml
[0062] 16 PG/5% PE-PEG only 22.5 (g/ml [0063] 17 10% LPA in PG/10%
PE-PEG (0.25 (g/ml LPA) [0064] 18 10% LPA in PG/10% PE-PEG (0.75
(g/ml LPA) [0065] 19 10% LPA in PG/ 10% PE-PEG (2.25 (g/ml LPA)
[0066] 20% LPA in PG/ 10% PE-PEG (6.75 (g/ml LPA) [0067] 21 PG/10%
PE-PEG only 67.5 (g/ml [0068] 22 PG/10% PE-PEG only 22.5 (g/ml
[0069] 23 10% LPA in PC (0.25 (g/ml LPA) [0070] 24 10% LPA in PC
(0.75 (g/ml LPA) [0071] 25% LPA in PC (2.25 (g/ml LPA) [0072] 26
10% LPA in PC (6.75 (g/ml LPA) [0073] 27 PC only 67.5 (g/ml [0074]
28 PC only 22.5 (g/ml [0075] 29 10% LPA in PC/TAP (0.25 (g/ml LPA)
[0076] 30 10% LPA in PC/TAP (0.75 (g/ml LPA) [0077] 31 10% LPA in
PC/TAP (2.25 (g/ml LPA) [0078] 32 10% LPA in PC/TAP (6.75 (g/ml
LPA) [0079] 33 PC/TAP only 67.5 (g/ml [0080] 34 PC/TAP only 22.5
(g/ml [0081] 35 10% LPA in PS (0.25 (g/ml LPA) [0082] 36 10% LPA in
PS (0.75 (g/ml LPA) [0083] 37 10% LPA in PS (2.25 (g/ml LPA) [0084]
38 10% LPA in PS (6.75 (g/ml LPA) [0085] 39 PS only 67.5 (g/ml
[0086] 40 BME only
[0087] FIG. 3 is a bar graph depicting protection of serum-deprived
C3H/10T1/2 cells by Five Phospholipid Mixture (referred to as
"Elirex" in the figure) and 18:1-LPA formulations in phosphotidyl
glycerol (PG) and phosphotidyl glycerol/phosphotidylcholine (PG/PC)
membrane structures.
[0088] FIG. 4 is a bar graph depicting protection of serum-deprived
C3HT1/2 cells by 18:1-LPA and lysophosphotidylserine (LPS).
[0089] FIG. 5 is a bar graph depicting protection of C3HT1/2 cells
from serum-deprivation by soy-derived, 18:1 and 16:0 LPA.
[0090] FIG. 6 is a bar graph depicting C3H/10T1/2 cell protection
from serum-deprivation by 18:1 LPA and 18:0 LPA alone or in PG
membrane structures.
[0091] FIG. 7 is a bar graph depicting protection of C3HT1/2 cells
from serum-deprivation by lysophosphatidic acid incorporated at
different concentrations into Phosphatidic acid
(PA)/phosphatidylinositol (PI) (1:1) membrane structures. Controls
include concentrations of 0.3, 1, 3, and 10 (g/ml of the Five
Phospholipid Mixture (referred to in the figure as "Elirex").
[0092] FIG. 8 is a bar graph depicting protection of serum-deprived
C3H/10T1/2 cells by 18:1-LPA in Five Phospholipid Mixture (labeled
"Elirex") and PC or chemically modified PC membrane structures.
[0093] FIG. 9 depicts an autoradiograph of a non-denaturing
polyacrylamide gel of proteins separated following incubation with
.sup.3H-labeled 18:1-LPA.
[0094] FIGS. 10A, 10B, 10C and 10D are graphs showing the elution
profiles of .sup.3H-18:1-LPA, with and without various proteins,
from a Superdex S75 column.
[0095] FIG. 11 is a bar graph depicting the effect of polyethylene
glycol (PEG) molecular weight on protection of C3H/10T1/2 cells
from serum deprivation at 1 mM in the presence and absence of 1.5
.mu.M 18:1-LPA. PEG of molecular weights 200, 1,000, 3,000, 6,000,
8,000, 20,000 and 35,000 were used.
[0096] FIG. 12 is a bar graph depicting the effect of the actual
infarct size compared to the area at risk in the pig heart model
treated with control (solid bar), PEG 20L (striped bar) and a dose
of Five Phospholipid Mixture and PEG (gray bar--labeled
"PEG+Elirex").
[0097] FIG. 13 is a graph depicting the prevention of cardiomyocyte
death induced by serum/glucose deprivation by PEG, Five
Phospholipid Mixture (referred to in the figure as "Elirex") and a
mixture of PEG and Five Phospholipid Mixture. The squares represent
no PEG, the circles represent 0.3% PEG and the triangles represent
2% PEG.
[0098] FIG. 14 is a graph depicting the treatment of rat neonatal
cardiomyocytes with PEG plus Five Phospholipid Mixture (closed
circles) and PEG plus 18:1-LPA (open circles) following ischemia to
evaluate the effect of treatment on cardiomyocyte death induced by
ischemia and reperfusion. The amount of LPA corresponds to its
quantity in the indicated amount of Five Phospholipid Mixture.
[0099] FIG. 15 is a bar graph depicting the actual infarct size
compared to the area at risk in the dog heart model treated with
placebo (solid bar), a mixture of superoxide dismutase (SOD) and
catalase (striped bar) and a dose of the combination of Five
Phospholipid Mixture (12.5 (g/kg) and PEG (25 mg/kg) (white
bar).
[0100] FIG. 16 is a bar graph depicting the actual infarct size
compared to the area at risk in the rat heart model treated with
placebo (solid bar) or a dose of the combination of Five
Phospholipid Mixture and PEG (gray bar, listed in key as
"APM/PEG").
DETAILED DESCRIPTION OF THE INVENTION
[0101] It has now been found that lysophosphatidic acid is the
major active component of phospholipid mixtures extracted from
plants that have anti-apoptotic activity. This invention
encompasses methods of use of therapeutically effective
compositions containing lysophosphatidic acid, its analogs and
derivatives (collectively "LPA"), which have been found to exhibit
anti-apoptotic activity and to preserve or restore cell, tissue and
organ function.
[0102] "Treating apoptosis" is herein defined as administering to a
cell, tissue, organ or organism exhibiting apoptosis, or at risk of
apoptosis, a treatment to effect beneficial or desired clinical
results, including, but not limited to preventing or diminishing
apoptosis.
[0103] I. Compounds of the Present Invention.
[0104] Although phospholipid structures are well defined in the
literature, they can vary with respect to lipid chain length and
saturation. "Lysophosphatidic acids" or "LPAs" as used herein shall
encompass the following structures and descriptions and will also
include related structures known in the art provided they are
effective in producing therapeutic response.
[0105] A. Structures of Compounds of the Present Invention.
[0106] LPA has the following general structure: ##STR5##
[0107] An LPA is an acid in which only one of the hydroxyl groups
of the glycerol is esterified to a fatty acid. LPA is a
phosphatidic acid in which the 2 carbon is not esterified and the 3
carbon is bound to the O--PO.sub.3H.sub.2 group, or, in the case of
the salt, one or more hydrogens are replaced, for example with
Na.sup.+. The 1 carbon will contain an acyl ester of fatty
acids.
[0108] The term "UB" is used in reference to the various structures
herein to describe the number of unsaturated carbon atoms in R. For
example, if R is 18 and UB is 1, R contains 18 carbon atoms, with 1
unsaturated bond. Some LPA analogs are also referred to herein as
R:UB-LPA (i.e. 18:1-LPA, wherein R is 18 carbon atoms with 1
unsaturated bond).
[0109] As used herein, LPA includes LPA having any one of a variety
of fatty acids esterified at the C1 position, Examples include LPA
wherein the fatty acid ester is lauryl, myristyl, palmityl,
stearyl, palmitoleyl, oleyl or linoleyl. (In structure I, above,
the composition where R is 18 and UB is 1, shall herein be referred
to as "18:1-LPA"). For a representative example of suitable
phospholipids see any chemical catalog of a phospholipid supplier,
for instance, the (1994) Avanti Polar Lipids Inc. catalog,
particularly pages 14 and 21.
[0110] R can be an unsubstituted or substituted saturated or
unsaturated straight or branched chain alkyl having from about 10
to about 24 carbon atoms. For all of the structures referenced
herein, R can have between 0 and (n-2)/2 unsaturated bonds, wherein
n is the number of carbon atoms in R. Substitutions include, but
are not limited to, halogen, hydroxy, phenyl, amino or
acylamino.
[0111] As used herein, "LPA" also encompasses LPA analogs. Given
the examples provided herein, it can be determined readily if an
LPA analog exerts sufficient anti-apoptotic activity to be suitable
for the uses contemplated herein. A wide variety of LPA analogs are
known in the art and many of these can be purchased from commercial
sources such as Avanti Polar Lipids Inc. (Alabaster, Ala.), or they
can be synthesized by methods known in the art.
[0112] LPA analogs include, but are not limited to, the following
structure:
[0113] or a cyclic phosphate derivative thereof having the
structure:
[0114] or pharmaceutically acceptable salts thereof, wherein each X
is independently O or S; M is P or S, where when M is S, one W is
(.dbd.O); each W is independently SH, OH,
OCH.sub.2CH(NH.sub.2)CO.sub.2H, OCHCH.sub.3CH(NH.sub.2)CO.sub.2H,
OPO.sub.3H.sub.2, or OPO.sub.2HOPO.sub.3H.sub.2, where if one W
##STR6## is OPO.sub.3H.sub.2 or OPO.sub.2HOPO.sub.3H.sub.2, the
remaining W is OH; Z is OH SH, NH.sub.2, halogen OPO.sub.3H.sub.2,
H, ##STR7## O(CH.sub.2).sub.bCH.sub.3 where b=0 to about 2, or
SO.sub.3H; R is unsubstituted or substituted, saturated or
unsaturated, straight or branched-chain alkyl having from
preferably 5-7, more preferably 8-10 and most preferably, about 10
to preferably 24-30, more preferably 24-28 and most preferably
about 24 carbon atoms, or
((CH.sub.2).sub.mO).sub.p(CH.sub.2).sub.mW where p is an integer
from 1 to about 900 and where each m is independently an integer
from 2 to about 10 and W is OH or O(CH.sub.2).sub.qCH.sub.3 where q
is an integer from 0 to about 10; Y is O or S; and n is an integer
from 0 to about 10. Preferably, R is between about 10 and 24, UB is
0-11, and mixtures thereof. More preferably, R is between about 14
and 20, UB is 0-6, and mixtures thereof. Even more preferably, R is
between about 16 and 18, UB is 0-3, and mixtures thereof. Most
preferably, R is 18, UB is 1 or 2, and mixtures thereof.
[0115] Methods of preparation of substitutions at the phosphate
group of LPA have been described, and are included herein. Tokumura
et al. (1981) J. Pharm. Exp. Therap. 219:219-224.
[0116] Also included in LPA analogs are ethers and thioethers at
the C1 position having the structure: ##STR8##
[0117] or a cyclic phosphate derivative thereof having the
structure: ##STR9##
[0118] or pharmaceutically acceptable salts thereof, wherein X is
O, S, or CH.sub.2; M is P or S, where when M is S, one W is
(.dbd.O); each W is independently SH, OH,
OCH.sub.2CH(NH.sub.2)CO.sub.2H, OCHCH.sub.3CH(NH.sub.2)CO.sub.2H,
OPO.sub.3H.sub.2, or OPO.sub.2HOPO.sub.3H.sub.2, where if one W is
OPO.sub.3H.sub.2 or OPO.sub.2HOPO.sub.3H.sub.2, the remaining W is
OH; Z is OH, CH.sub.2OH, SH, NH.sub.2, halogen, OPO.sub.3H.sub.2, H
or SO.sub.3H; R is unsubstituted or substituted, saturated or
unsaturated, straight or branched-chain allyl having from
preferably 5-7, more preferably 8-10 and most preferably, about 10
to preferably 24-30, more preferably 24-28 and most preferably
about 24 carbon atoms, or
((CH.sub.2).sub.mO).sub.p(CH.sub.2).sub.mW where p is an integer
from 1 to about 900 and where each m is independently an integer
from 2 to about 10 and W is OH, or O(CH.sub.2).sub.qCH.sub.3 where
q is an integer from 0 to about 10; Y is O or S; and n is an
integer from 0 to about 10. Simon et al. (1982) Biochem. Biophys.
Res. Comm. 108:1743-1750.
[0119] Preferably, R is between about 10 and 24, UB is 0-11, and
mixtures thereof. More preferably, R is between about 14 and 20, UB
is 06, and mixtures thereof. Even more preferably, R is between
about 16 and 18, UB is 0-3, and mixtures thereof. Most preferably,
R is 18, UB is 1 or 2, and mixtures thereof.
[0120] Also included are glycero LPA analogs having the structure:
##STR10##
[0121] or a cyclic phosphate derivative thereof having the
structure: ##STR11##
[0122] or pharmaceutically acceptable salts thereof, wherein each V
is independently OH, SH, H, NH.sub.2, halogen, OPO.sub.3H.sub.2, or
OSO.sub.3H; each X is independently O or S; M is P or S, where when
M is S, one W is (.dbd.O); each W is independently SH, OH,
OCH.sub.2CH(NH.sub.2)CO.sub.2H, OCHCH.sub.3CH(NH.sub.2)CO.sub.2H,
OPO.sub.3H.sub.2, or OPO.sub.2HOPO.sub.3H.sub.2, where if one W is
OPO.sub.3H.sub.2 or OPO.sub.2HOPO.sub.3H.sub.2, the remaining W is
OH; Z is OH, SH, NH.sub.2, halogen, OPO.sub.3H.sub.2, H or
SO.sub.3H; R is unsubstituted or substituted, saturated or
unsaturated, straight or branched-chain alkyl having from
preferably 5-7, more preferably 8-10 and most preferably, about 10
to preferably 24-30, more preferably 24-28 and most preferably
about 24 carbon atoms, or
((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.pV where q is an integer
from 1 to about 900 and where each p is independently an integer
from 2 to about 10 and V is OH, or O(CH.sub.2).sub.bCH.sub.3 where
b is an integer from 0 to about 10. Y is O or S; n is an integer
from 0 to about 10; and m is an integer from 0 to about 10.
Preferably, R is between about 10 and 24, UB is 0-11, and mixtures
thereof. More preferably, R is between about 14 and 20, UB is 06,
and mixtures thereof. Even more preferably, R is between about 16
and 18, UB is 0-3, and mixtures thereof. Most preferably, R is 18,
UB is 1 or 2, and mixtures thereof.
[0123] Also included are LPA analogs containing a amide bond and
having the structure: ##STR12##
[0124] or a cyclic phosphate derivative thereof having the
structure: ##STR13##
[0125] or the reverse amide [structures XVI and XVII, having
R--NH--C(--O)(CH.sub.2).sub.n-- in place of
R--C(.dbd.O)--NH--(CH.sub.2).sub.n--](NH--CH.sub.2).sub.n-- or
pharmaceutically acceptable salts thereof, wherein Z is OH, SH,
NH.sub.2, halogen, OPO.sub.3H.sub.2, H or SO.sub.3H; M is P or S,
where when M is S, one W is (.dbd.O); each W is independently SH,
OH, OCH.sub.2CH(NH.sub.2)CO.sub.2H,
OCHCH.sub.3CH(NH.sub.2)CO.sub.2H, OPO.sub.3H.sub.2, or
OPO.sub.2HOPO.sub.3H.sub.2, where if one W is OPO.sub.3H.sub.2 or
OPO.sub.2HOPO.sub.3H.sub.2, the remaining W is OH; R is an amino
acid side chain or a branched amino acid side chain, or an
alkylated amino acid side chain, unsubstituted or substituted,
saturated or unsaturated, straight or branched-chain alkyl having
from preferably 5-7, more preferably 8-10 and most preferably,
about 10 to preferably 24-30, more preferably 24-28 and most
preferably about 24 carbon atoms, or
((CH.sub.2).sub.mO).sub.p(CH.sub.2).sub.mW where p is an integer
from 1 to about 900 and where each m is independently an integer
from 2 to about 10 and W is OH, or O(CH.sub.2).sub.qCH.sub.3 where
q is an integer from 0 to about 10; Y is O or S; and n is an
integer from 0 to about 10. Preferably, R is between about 10 and
24, UB is 0-11, and mixtures thereof. More preferably, R is between
about 14 and 20, UB is 0-6, and mixtures thereof. Even more
preferably, R is between about 16 and 18, UB is 0-3, and mixtures
thereof. Most preferably, R is 18, UB is 1 or 2, and mixtures
thereof.
[0126] Also included are "bis" LPA compounds, having the following
structure (I), ##STR14## [0127] wherein M is O or S, each W is
independently selected from one of the following structures (XI,
XII, XIII, XIV): ##STR15## [0128] wherein Y is O or S; R is
unsubstituted or substituted, saturated or unsaturated, straight or
branched-chain alkyl having from preferably 5-7, more preferably
8-10 and most preferably, about 10 to preferably 24-30, more
preferably 24-28 and most preferably about 23 carbon atoms, or
((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.pT where q is an integer
from 1 to about 900 and where each p is independently an integer
from 2 to about 10 and T is OH, or O(CH.sub.2).sub.bCH.sub.3 where
b is an integer from 0 to about 10; each V is independently OH, SH,
H, NH.sub.2, halogen, OPO.sub.3H.sub.2, or OSO.sub.3H; n is an
integer from 0 to about 10; m is an integer from 0 to about 10; Z
is OH, SH, NH.sub.2, halogen, OPO.sub.3H.sub.2, H,
O(CH.sub.2).sub.bCH.sub.3 where b=0 to about 2, or SO.sub.3H; L is
independently O, S, CH.sub.2 and X is independently O or S; or a
salt thereof.
[0129] Also encompassed by the term LPA are the compounds herein
referred to as "reverse ester-LPA" or "reverse thioester-LPA",
wherein the R--C--(.dbd.O)--O--CH.sub.2-- group is replaced by
R--CH.sub.2--O--C(.dbd.O)-- in Structure X, and wherein the
R--C(.dbd.X)--X--(CH.sub.2).sub.n-- group is replaced by
R--(CH.sub.2).sub.n--X--C(.dbd.X)-- in Structure X, XI, XIV, XV,
II, and IV.
[0130] In all analogs containing W, it is preferred that W is not
ethanolamine, glycerol, or choline.
[0131] In the above structures, where one W is SH, some molecules
will exist as resonance structures, alternating between the
(.dbd.O) and (.dbd.S) structures.
[0132] Naturally occurring derivatives are also encompassed in the
term "LPA." Such derivatives include, but are not limited to,
PHYPLA or cLPA, Murakami-Murofushi et al. (1992) J. Biol. Chem.
267:21512-21517. Cyclic derivatives can also be synthesized by
methods known in the art.
[0133] Pharmaceutically acceptable salts of the phospholipids
encompassed by the present invention, include, but are not limited
to, the free acid form, alkali metal salts, such as sodium and
potassium; alkaline earth metal salts, such as calcium and
magnesium; non-toxic heavy metal salts; ammonium salts;
trialkylammonium salts, such as trimethyl-ammonium and
triethylammonium; and alkoxyammonium salts, such as
triethanolammonium, tri(2-hydroxyethyl)ammonium, and
tromethamine(tris(hydroxymethyl)aminomethane). Particularly
preferred are sodium and ammonium salts.
[0134] B. Obtaining Compounds of the Present Invention.
[0135] The phospholipids can be obtained from any source including,
but not limited to, commercial, isolated from a variety of
different plants (including plant organs) and animals or created
synthetically. Preferably the plants are in the soybean family, but
the phospholipids can be isolated from other plants including, but
not limited to, those in the leguminosae (beans and peas etc.). The
phospholipids can also be isolated from partially purified plant
extracts including, but not limited to, soy molasses, lecithin
(fluid, deoiled or other forms), partially purified protein
concentrates, partially purified protein hydrolysates, defatted soy
flakes, refined soy oils, soy grits, soy flours and other soy
fractions from which lipids can be extracted. It is within the
skill of one in the art, utilizing the methods described herein, to
determine whether the phospholipids of the present invention can be
isolated from a particular species of plant, plant extract or organ
within a plant. In addition, U.S. Pat. No. 3,365,440 describes
extraction of lipids from soybeans. U.S. Pat. Nos. 5,567,425;
5,602,885; 5,624,675; 5,635,186; 5,635,187 have further general
descriptions of a variety of techniques useful for the present
invention.
[0136] The phospholipids can be obtained from plant sources by any
method known in the art provided it results in purification of at
least one of the phospholipids of the invention. A variety of
methods are known in the art for purifying and analyzing
phospholipids from plant sources. For review, see Bligh and Dyer
(1959) Can. J. Biochem. Physiol 37:911-917; Patton et al. (1982) J.
Lipid Res. 23:190-196; Jungalwala (1985) Recent Developments in
Techniques for Phospholipid Analysis, in Phospholipids in Nervous
Tissues (ed. Eichberg) John Wiley and Sons, pp. 1-44; Hamilton et
al. (1992) in the series, A Practical Approach (Rickwood et al.
eds.) IRL Press at Oxford University Press; and Kates (1986)
Techniques of Lipidology: Isolation, Analysis and Identification in
Laboratory Techniques in Biochemistry and Molecular Biology (Burdon
et al. ads.) Elsevier.
[0137] Phospholipids can also be derived from animal sources.
Preferably, the animal is a mammal. Even more preferably, the
phospholipids are derived from liver cells. Such phospholipids are
commercially available or can be purified from animal tissue by
methods known in the art, for instance from animal and egg lecithin
or from the compositions described in WO 95/15173. Phospholipids in
general, and LPAs in particular, can also be derived from
blood.
[0138] The phospholipids of the invention can also be synthesized
by methods known in the art. Suitable semi-synthetic phospholipids
and their synthesis are described in Kates, Techniques of
Lipidology (1972). A synthesis of lysophosphatidic acid is
described in W. Stoffel and G. D. Wolf, Chemische Synthese von
1-O-[3H]Palmitoyl-L-glycerin-3-phosphate (L-3-Lysophosphatidsaure),
Chem. Ber., 347 (1966) 94-101. The synthesis of various cyclic
phosphate LPAs is described in A. J. Slotboom, et al., Synthesis of
Lysophosphoglycerides, Chem. Phys. Lipids 1 (1967) 317-336; PCT
Publication No. WO 92/21323; and U.S. Pat. No. 5,565,439. The
synthesis of a phosphonate analog of
1-O-hexadecyl-2-O-methyl-glycero-phosphate is described in Z. Li,
et al., Phosphonate isosteres of phospholipids, Tetrahedron Lett.,
34 (1993) 3539-3542.
[0139] Procedures for synthesis of functionalized glycerol ether
derivatives which can be used in the synthesis of compounds
suitable for use in the present invention are described in K.
Agarwal, et al. Synthesis of carbamyl and ether analogs of
phosphatidylcholines, Chem. Phys. Lipids, 39 (1984) 169-177, and H.
Eibl and P. Woolley, A general synthetic method for
enantiomerically pure ester and ether lysophospholipids, Chem.
Phys. Lipids. 47 (1988) 63-68.
[0140] The preparation of 1-O-benzyl-2-deoxy-2-bromo glycerol, a
starting material for the synthesis of 2-bromo LPA Compound 37, is
described in W. L. F. Armarego, B. A. Milloy and W. Pendergast, A
highly stereospecific synthesis of (R)- and (S)-[2-2H]glycine, J.
C. S. Perkin I, (1976) 2229-2237.
[0141] The synthesis of 2-deoxy-2-bromo-phosphatidylcholine is
described in C. J. Lacey and L. M. Loew, Phospholipid synthesis
based on new sequential phosphate and carboxylate ester bond
formation steps, J. Org. Chem., 48 (1983) 5214-5221.
[0142] The synthesis of bisphosphatidic acid and its conversion to
bis-lysophosphatidic acid using phospholipase A2 from pig pancreas
is described in Q. Quan Dang, et al., Synthesis and identification
of bis(diacylglycero)phosphoric acid and
bis(monoacylglycero)phosphoric acid, Lipids, 17 (1982) 798-802, and
Q. Quan Dang and L. Douste-Blazy, Synthesis and stereochemical
study of some biologically relevant phosphoglycerides: dicatoxylic
phosphatidyl cholines and bis(diacylglycero)phosphoric acids,
Phosphorus and Sulfur, 18 (1983) 377-380.
[0143] A method for the preparation of lysophosphatidic acid or
lysophosphatidates by reacting glycidyl esters with anhydrous
phosphoric acid is described in U.S. Pat. No. 3,423,440.
[0144] A synthesis of lysothiophosphatidic acid is described in N.
V. Heeb and K. P. Nambiar, Synthesis of (R)-lysothiophosphatidic
acid and (R)-thiophosphatidic acid, Tetrahedron Lett., 34 (1993)
6193-6196
[0145] The preparation of LPA amide analogs and 2-deoxy LPA plus
various derivatives is described in D. W. Hopper, et al., Facile
synthesis of lysophospholipids containing unsaturated fatty acid
chains, Tetrahedron Lett., 37 (1996) 7871-7874; and K. R. Lynch, et
al., Structure/activity relationships in lysophosphatidic acid: the
2-hydroxyl moiety, Mol. Pharmacol., 52 (1997) 75-81.
[0146] The following papers describe synthetic routes which can be
used for the synthesis of additional LPA analogs: M. Fuji, et al.,
A stereoselective and highly practical synthesis of cytosolic
phospholipase A2 substrate, 2-S
arachidonoyl-1-O-hexadecyl-sn-tioglycero-3-O-phosphocholine, J.
Org. Chem., 62 (1997) 6804-6809; (Strategy for the preparation of
2-thioglycero phosphocholines and guidance for the synthesis of
2-deoxy-2-thiol LPA); and A. Markowska, et al., Etheranaloge der
Thio-und Dithiophospholipide mit C--S--P-Binding, Liebigs Ann.
Chem., (1993) 1327-1329 (synthesis of
1-O-hexadecyl-2-O-methyl-3-thioglycero-3-phosphocholine and
1-S-hexadecyl-2-O-methyl-1,3-dithioglycero-3-phosphocholine,
guidance for the preparation of LPA analogs containing sulfur
linked phosphates).
[0147] Various degrees of purity of the phospholipids can be used.
Purity can be assayed by any method known in the art such as two
dimensional TLC or HPLC and assayed for total lipids, phospholipids
or phosphate. Various suitable methods are outlined in Kates
(1972). Preferably, the phospholipids must be of sufficient purity
such that when mixed at a total concentration of about 10 mg/mL,
the mixture can be sonicated as described below to provide a
relatively translucent solution. Preferably, the phospholipids are
at least 90% pure, more preferably, they are at least 95% pure and,
most preferably, they are at least 99% pure.
[0148] The references cited herein are incorporated by reference in
their entirety.
[0149] C. Potentiating Components.
[0150] One factor that can influence the therapeutic activity of
the compositions of the present invention is the presence of a
potentiating component. Under certain conditions LPA alone has
displayed mitogenic activity and a short half life in vivo and
under certain storage conditions. The present invention provides
novel compositions in which one or more potentiating components are
combined with LPA that have been found to potentiate the
therapeutic activity of LPA. A "potentiating component" is defined
as a molecule which potentiates the therapeutic activity of LPA.
Potentiating components include, but are not limited to, proteins,
other phospholipids, polyethylene glycols (PEG), lipid membrane
structure forming compounds, polypeptides, modified polypeptides
and polymers.
[0151] In some cases, the presence of calcium has been found to
inhibit the anti-apoptotic properties of LPA. However, certain
potentiating components have proven to counteract the inhibition by
calcium. Therefore, in one embodiment LPA is presented in
combination with a potentiating component such as BSA and/or PEG.
Alternative potentiating components appropriate to protect the
activity of LPA can be identified by performing a screen as
described in Example 2, in the presence and absence of the
potentiating component to be tested.
[0152] An appropriate potentiating component for use in the present
invention can easily be selected by combining the composition to be
evaluated with a therapeutic amount of LPA in a therapeutically
acceptable solution and evaluating the combination for its
mitogenic activity, storage stability, in vivo half life, and for
anti-apoptotic activity by any method known in the art, including
those described herein. If the mixture has acceptable stability, is
not therapeutically unacceptable due to mitogenic activity, and
displays anti-apoptotic activity, or preserves or restores cell,
tissue or organ function, the potentiating component is appropriate
for use in the present invention.
[0153] D. Other Phospholipids.
[0154] In one embodiment of the present invention, the
phospholipids PA; PI; lysophosphatidic acid; LPI; and LPC are
present in the composition in a range of ratios from
0-20:5-20:2-16:0-4:08, respectively. Preferably, these
phospholipids are in a ratio of approximately
2-15:8-15:6-10:2-4:2-8, respectively. Most preferably, these
phospholipids are in a ratio of approximately 10:10:8:2:4,
respectively.
[0155] 2. Polyethylene Glycol.
[0156] In one embodiment of the present invention, LPA is combined
with PEG prior to therapeutic use. PEGs constitute a diverse group
of molecules. Only those that potentiate the therapeutic efficacy
of LPA are encompassed herein. It is within the skill of one in the
art to determine whether a particular PEG is suitable for use in
the claimed compositions. Such a determination can be made, for
instance, by the methods described herein. Polyethylene glycol
("PEG"), ((-Hydro-(-hydroxypoly(oxy-1,2-ethanediyl)), is known by
numerous designations including macrogel; PEG; Carbowax; Jeffox;
Nycoline; Pluracol E; Poly-G; Polyglycol E; and Solbase. PEG refers
to the liquid and solid polymers of the general formula
H(OCH.sub.2--CH.sub.2).sub.nOH, where n is greater than or equal to
4. In general, each PEG is followed by a number which corresponds
to its average MW. PEG syntheses are described for instance in a
Hibbert (1939) J. Am. Chem. Soc. 61:1905-1910. For review, see
also, Powell, III in Handbook of Water-Soluble Gums & Resins,
R. L. Davidson ed. (McGraw-Hill, New York, 1980) pp. 18/1-18/31.
PEGs have found use as water-soluble lubricants for rubber molds,
textile fibers, and metal-forming operations, in food and food
packaging, in hair preparations and in cosmetics in general and as
ointment and suppository bases in pharmaceutical compositions.
[0157] Typically, PEGs are clear, viscous liquids or white solids
that dissolve in water to form transparent solutions. They are
soluble in many or organic solvents and readily soluble in aromatic
hydrocarbons. They are only slightly soluble in aliphatic
hydrocarbons. Typically, they do not hydrolyze on storage. PEGs
have low toxicity. The molecular weights of PEG compositions listed
herein are given in number averages rather than weight
averages.
[0158] PEG20M consists of two or more molecules of PEG having
approximate molecular weights of 6000-10,000 joined by a bisphenol
epoxide linker (CAS # 37225-26-6; CAS name Oxirane,
2,2'[(1-methyl-ethylidene)bis(4,1-phenyleneoxy methylene)]bis-,
polymer with (-hydro-(-hydroxypoly(oxy-1,2-ethanediyl). PEG20L is a
substantially linear PEG having an average molecular weight of
about 20,000 Daltons (available from several commercial sources
including, but not limited to, Clariant/Hoechst Celanese, Fluka and
Nippon Oils and Fats). The molecular weights of PEG compositions
listed herein are given in number averages rather than weight
averages. Various other molecular weights of linear PEG are also
available from several commercial sources.
[0159] More recently, PEG has been used in a number of
pharmacologic applications. The conjugation of PEG to foreign
proteins, such as cytokines and antibodies, reduces the immune
response triggered when the proteins are administered into test
mammals. U.S. Pat. Nos. 5,447,722; 4,902,502; 5,089,261; 5,595,732;
5,559,213; and 4,732,863. Conjugation to PEG also increases the
solubility and biological half-life of cytokines. WO 8700056 and
U.S. Pat. No. 5,089,261. Conjugates of PEG and glucocerebroside
have been formulated for treating Gaucher's disease. WO 9413311.
PEG has also been conjugated to such enzymes as adenosine
deaminase, amidase bovine and asparagase, for therapeutic use. See
Delgado et al. (1992) Crit. Rev. Ther. Drug. Carrier Syst.
9:249-304; and Burnham (1994) Am. J. Hosp. Pharm. 51:210-218, for
review.
[0160] The invention further encompasses compositions comprising
therapeutically effective amounts of LPA and PEG. In compositions
comprising LPA and PEG, the PEG to LPA weight ratio is such that
the LPA therapeutic activity is potentiated by the PEG. Typically,
the PEG to LPA weight ratio of the composition is preferably
1-100,000:1 and most preferably 10-10,000:1.
[0161] PEG can be in the molecular weight range from about 6,000 to
about 500,000. Preferably, the PEG has an average molecular weight
of about 8,000 to about 40,000. More preferably, the PEG has an
average molecular weight of from about 20,000 to about 40,000. Most
preferably, the PEG has an average molecular weight of 20,000. Even
more preferably, the PEG is 20L PEG. By "PEG20L" is meant a
substantially linear PEG having an average molecular weight of
about 1,000 to 100,000 Daltons, preferably about 8,000 to 35,000
Daltons, and most preferably about 20,000 Daltons. By "linear PEG"
or "linear polymer" is meant that each PEG molecule comprises a
single polymeric subunit without molecular linkages such as those
found in PEG20M. PEG of 35,000 molecular weight is also preferred.
PEG of higher molecular weight may have clearance problems when
administered in vivo. Thus, PEG of molecular weight greater than
35,000 is preferably used in compositions for topical delivery.
[0162] With reference to PEG20L, "linear PEG" or "linear polymer"
means that each PEG molecule comprises a single subunit without
molecular linkages. By "having an average molecular weight of about
20,000 Daltons" is meant that individual linear polymers can vary
in length, but the average molecular weight is about 20,000
Daltons. Those skilled in the art will appreciate that synthetic
polymers such as PEG cannot be prepared practically to have exact
molecular weights, and that the term "molecular weight" as used
herein refers to the average molecular weight of a number of
molecules in any given sample, as commonly used in the art. For
example, a sample of PEG 2,000 might contain a statistical mixture
of polymer molecules ranging in weight from, for example, 1,500 to
2,500 Daltons with one molecule differing slightly from the next
over a range. Specification of a range of molecular weight
indicates that the average molecular weight can be any value
between the limits specified, and can include molecules outside
those limits. The molecular weight distribution of a PEG can be
determined by gel permeation chromatography (GPC), a technique
known in the art, using, for example, a combination of columns to
achieve resolution from 1,000,000 to 200 molecular weight. PEG
standards from 100,000 to 1,400 molecular weight can be used for
calibration.
[0163] PEG20L is supplied as a white flake, and is readily soluble
in water. The oxidation rate of PEG is dependent on storage
conditions including: (1) temperature; (2) exposure to light; and
(3) the availability of oxygen.
[0164] Selection of the appropriate PEG depends on the intended use
and can be readily determined by those skilled in the art.
[0165] By "anoxically" is meant the reduction of ambient oxygen, a
condition which can be maintained by purging with argon or nitrogen
gas, and then packaging in a gas-impervious container. The absence
of oxygen need not be total. Preferably, it is below about 10% of
the total gas present in the sample. More preferably, it is below
about 1% of total gas present in the sample.
[0166] Preferably, PEG is present in an effective concentration and
is essentially free of impurities. By "impurities" is meant the
products produced when PEG is oxidized. In addition, small
molecular fragments are formed such as formate, methyl formate,
formaldehyde, acetaldehyde, etc., all of which are defined here as
impurities. These impurities can be removed by any method known in
the art, including, but not limited to, dialysis, which removes not
only small molecular impurities such as formaldehyde, but also
removes hydroperoxides, as evidenced by spectrophotometry. In order
to determine the effectiveness of dialysis, conductivity is
measured. When dialysis is effective, conductivity drops markedly
to 10-20 microsiemens.
[0167] Removal of impurities by dialysis can be performed through a
Cellulose Acetate Hollow Fiber Dialyzer (Baxter Model CA 110) but
the same procedure can also be performed by Ultrafiltration using a
Regenerated Cellulose Ultrafiltration Membrane or a
Polyethersulphone Ultrafiltration Membrane or using other dialysis
methods known in the art. The membrane should have a molecular
weight cut off of approximately 20,000 Daltons to allow removal of
impurities, metals and other contaminants.
[0168] By "therapeutically effective amount" is meant an amount
sufficient to effect beneficial or desired clinical results. A
therapeutically effective amount can be administered in one or more
administrations.
[0169] Preferably, PEG is present in a therapeutically effective
amount. In the case of OPS, an effective amount is the amount
required to improve the ability of an OPS to preserve organs.
[0170] In the case of compositions comprising LPA and PEG, where
the compositions include other phospholipids, the preferred
composition is where phospholipids are combined in a ratio of about
10:10:8:2:4 by weight. A ratio of "about" means that the ratios of
the phospholipids can range approximately up to 15% but preferably
not more than 5%. More preferably, the ratios are within
.+-.0.5%.
[0171] Just as PEG has been used as a potentiating component for
therapeutic agents, so the capacity of other macromolecules and
macromolecular structures to serve as potentiating components has
been explored. Included among these are various lipid membrane
structures and proteins.
[0172] 3. Lipid Membrane Structures.
[0173] Lipid membrane structures (LMSs), including liposomes,
micelles, multilamellar vesicles and cellular membrane isolates,
have been used as vehicles for delivering therapeutic agents. U.S.
Pat. Nos. 5,045,530; 5,141,751; 5,100,662; 5,292,499; 5,213,804;
5,449,513; 5,190,822; 5,540,925; and 5,395,619. LMSs are lamellar
lipid particles wherein polar head groups of a polar lipid are
arranged to face an aqueous phase of an interface to form membrane
structures.
[0174] As used herein, a "liposome" or "lipid vesicle" is a small
vesicle bounded by at least one and possibly more than one bilayer
lipid membrane. Liposomes are completely closed lipid bilayer
membranes which contain entrapped aqueous volume. Liposomes are
vesicles which can be unilamellar (single membrane) or
multilamellar (onion-like structures characterized by multiple
membrane bilayers, each separated from the next by an aqueous
layer). The bilayer is composed of two lipid monolayers having a
hydrophobic "tail" region and a hydrophilic "head" region. In the
membrane bilayer, the hydrophobic (nonpolar) "tails" of the lipid
monolayers orient toward the center of the bilayer, whereas the
hydrophilic (polar) "heads" orient toward the aqueous phase. The
basic structure of liposomes can be made by a variety of techniques
known in the art.
[0175] These lipid vesicles are made artificially from
phospholipids, glycolipids, lipids, steroids such as cholesterol,
bolaamphiles related molecules, or a combination thereof by any
technique known in the art, including, but not limited to
sonication, extrusion, or removal of detergent from lipid detergent
complexes. A liposome can also optionally comprise additional
components associated with the outer surface, such as a tissue
targeting component. It is understood that a "lipid membrane" or
"lipid bilayer" need not consist exclusively of lipids, but can
additionally contain any percentage of other components, including,
but not limited to, cholesterol and other steroids, proteins of any
length ad other amphipathic molecules, providing the general
structure of the membrane is a sheet of two hydrophilic surfaces
sandwiching a hydrophobic core. For a general discussion of
membrane structure, see The Encyclopedia of Molecular Biology by J.
Kendrew (1994). Suitable lipids include, but are not limited to,
those discussed in Lasic (1993) "Liposomes: from Physics to
Applications" Elsevier, Amsterdam. The lipid bilayer making up the
liposome can comprise phospholipids, glycolipids, steroids, and
their equivalents. The lipid bilayer can also be made of
amphipathic proteins and lipid soluble chemicals. Preferably, a
composition is chosen that allows the membrane to be formed with
reproducible qualities, such as diameter, and is stable in the
presence of elements expected to occur where the liposome is to be
used, such as physiological buffers and circulating molecules.
Preferably, the liposome is resilient to effects of manipulation by
storage, freezing, and mixing with pharmaceutical excipients.
[0176] Lipids suitable for incorporation into lipid membrane
structures include, but are not limited to, natural, semi-synthetic
or synthetic mono- or di-glycerophospholipids including
phosphatidylcholines, phosphatidylethanolamines,
phosphatidylglycerols, phosphatidylinositols, phosphatidic acids,
phosphatidylserines, glycero- and cardiolipins. Sphingolipids such
as sphingomyelin and cerebrosides can also be incorporated. While
natural phospholipids occur with the phospho moiety at the sn-3
position and hydrophobic chains at the sn-1 and sn-2 positions,
synthetic lipids can have alternative stereochemistry with, e.g.,
the phospho group at the sn-1 or sn-2 positions. Furthermore, the
hydrophobic chains can be attached to the glycerol backbone by
acyl, ether, alkyl or other linkages. Derivatives of these lipids
are also suitable for incorporation into liposomes. Derivatives
suitable for use include, but are not limited to, haloalkyl
derivatives, including those in which all or some of the hydrogen
atoms of the alkyl chains are substituted with, for example,
fluorine. In addition, cholesterol and other amphipathic steroids,
bolaamphiphiles (lipids with polar moieties at either end of the
molecule which form monolayer membranes) and
polyglycerolmonoalkylthers can also be incorporated. Liposomes can
be composed of a single lipid or mixtures of two or more different
lipids.
[0177] In one preferred embodiment, the lipid bilayer of the
liposome is formed primary from phospholipids. More preferably, the
phospholipid composition is a complex mixture, comprising a
combination of phosphatidylcholine (PC), phosphatidic acid (PA),
phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and
sphingomyelin (SM). The LMS can further comprise additional lipids
such as phosphatidylinositol (PI), phosphatidylserine (PS), or
cardiolipin (diphosphatidylglycerol). If desired, SM can be
replaced with a greater proportion of PC, PE, or a combination
thereof. PS can optionally be replaced with phosphatidylglycerol
(PG). Preferably, at least PC and PE are included; more preferably,
at least three of the group PC, PS, PE, and SM are included. The
composition is chosen so as to confer upon the LMS stability during
both storage and administration. Each phospholipid described above
can vary in its structure depending on the fatty acid moieties that
are esterified to the glycerol moiety of the phospholipid.
Generally, most commercially available forms of a particular
phospholipid can be used. However, phospholipids containing
particular fatty acid moieties may be preferred for certain
applications.
[0178] Preferably, the LMS also includes cholesterol or a related
steroid to improve the rigidity of the membrane. Any amount of
cholesterol can be used. A preferred ratio of total cholesterol to
lipid is between about 0.5 and about 1.2 moles of cholesterol per
mole of lipid. More preferred is a molar ratio of about 0.8 to
about 1.2:1; even more preferred is a molar ratio of about 0.9 to
about 1.1:1; still more preferred is a molar ratio of about
1.0:1.0. Other molecules that can be used to increase the rigidity
of the membrane include crosslinked phospholipids.
[0179] Other preferred liposomes for use in vivo are those with an
enhanced ability to evade the reticuloendothelial system, thereby
giving them a longer period in which to reach the target cell.
Effective lipid compositions in this regard are those with a large
proportion of SM and cholesterol, or SM and PI. Liposomes with
prolonged circulation time also include those that comprise the
monosialoganglioside GM1, glucuronide, or PEG. For example,
cholesterol can be added at the ratios indicated above to a lipid
mixture consisting of any combination of SM, PI, glucuronide, PEG,
and other suitable components.
[0180] Methods of making LMSs are well known in the art. A number
of publications describe a variety of methods for preparing
liposomes of different structure and lipid composition. Gregoriadis
(1988) Liposomes as Drug Carriers Wiley, New York; Gregoriadis
(1993) Liposome Technology 2nd Ed. Vol. 1: Liposome Preparation and
Related Techniques CRC Press, Boca Raton; Watwe et al. (1995) Curr.
Sci. 68:715-724; Vemuri et al. (1995) Pharm. Acta Helvetiae
70:95-111; Elorza et al. (1993) J. Microencapsulation 10:237-248;
and U.S. Pat. Nos. 4,737,323 and 5,008,050.
[0181] Liposomes can also be provided with molecules at the surface
that target them to the cell of interest. Such small molecules can
be attached by incorporating into the lipid bilayer a
functionalized phospholipid (U.S. Pat. Nos. 5,052,421 and
5,540,935) or a functionalized cholesterol (U.S. Pat. No.
4,544,545). Polypeptides can be attached covalently to the lipid
bilayer (EP Patent 0036277), to a glycophospholipid (U.S. Pat. No.
5,374,548), to a carboxylated phospholipid (U.S. Pat. No.
4,762,915), to a derivatized sterol (U.S. Pat. No. 5,000,960), or
to a peptide anchor (U.S. Pat. No. 5,109,113). Alternatively, if
the polypeptide comprises a hydrophobic domain, it can be
incorporated directly into the lipid bilayer, either by forming the
liposome in its presence, or by performing the liposome and
inserting the polypeptide subsequently using a suitable detergent.
Tranum-Jensen et al. (1994) J. Membrane Biol. 140: 215-23; EP
Patent 0047480; and U.S. Pat. No. 5,252,348.
[0182] Liposomes have been prepared with mammalian-derived peptides
such as cytokines (U.S. Pat. No. 5,258,499), transferrin (Stavridis
et al.), antibody (Laukkanen et al. (1994) Biochem.
33:11664-11670), asialofetuin and other galactose-terminated side
chains (Ishihara (1990) Pharm. Res. 7:542-546; and Ghosh et al.
(1991) In: Liver Diseases, Targeted Diagnosis and Therapy Using
Specific Receptors and Ligands Wu et al., Ed. Marcel Dekker, New
York), a fusogenic protein from rat brain microsomal membranes
(Rakowska et al. (1994) J. Membrane Biol. 142:35-42), and
surfactant protein A (Walther et al. (1993) Am. J. Physiol.
265:L330-339). Liposomes have been prepared with artificial
peptides, such as a 14-residue amphipathic sequence which is a
fusogenic GALA-type peptide. Puyal et al. (1994) Biochim. Biophys.
Acta 1195:259-266. Liposomes have also been prepared with viral
components: for example, the F and G glycoprotein of respiratory
syncytial virus (RSV) (U.S. Pat. No. 5,252,348), reovirus M cell
attachment protein (Rubas et al. (1990) J. Microencapsulation
7:385-395), influenza virus surface protein (WO 92/19267; EP
0047480; and Nussbaum et al. (19897) J. Virol. 61:2245-2252), viral
membrane fusion proteins, particularly hemagglutinin (WO 95/32706),
and the influenza hemagglutinin D loop and K loop peptides (Friede
et al. (1994) Vaccine 12:791-797). Rapid uptake of liposomes in
vivo by cells of the reticuloendothelial system has restricted
their therapeutic utility. This problem has been overcome by
incorporation of lipids derivatized with various synthetic
polymers, for example, polyethylene glycol (PEG), polylactic acid,
polyglycolic acid, or combinations thereof. Woodle and Lasic (1992)
Biochim. Biophys. Acta 1113:171-199; Zalipsky et al. (1994) FEBS
Letters 353:71-74; and U.S. Pat. No. 5,395,619.
[0183] The present invention also encompasses compositions
comprising micelles and methods of using such compositions.
Micelles in aqueous solution, both non-ionic, cationic and anionic,
have been described in the literature in numerous publications.
Mittal (1977) Micellization, Solubilization and Microemulsions
Plenum Press, New York; Mittal (1979) Solution Chemistry of
Surfactants Plenum Press, New York; Menger (1977) In Biorganic
Chemistry III. Macro-and Multicomponent Systems Van Tanelen, Ed.
Academic Press, New York; and Menger (1979) Acc. Chem. Res.
12:111-117, "Micelles" is a term applied to aggregates which form
from tenside molecules in aqueous solutions above a specific
temperature or a characteristic concentration. This concentration
is called the critical micellization concentration, or cmc. When
the cmc is exceeded, the monomer concentration remains practically
constant and the excess tenside molecules form micelles. They can
occur in various shapes (spheres, rods, discs) depending on the
chemical constitution of the tenside and on the temperature,
concentration or ionic strength of the solution. The micelles have
characteristic aggregation numbers with usually only a small
distribution spread. Reaching the cmc is manifest by abrupt changes
in the surface tension, the osmotic pressure, the electrical
conductivity and the viscosity. Micelles are thermodynamically
stable association colloids of surfactant substances in which the
hydrophobic radicals of the monomers lie in the interior of the
aggregates and are held together by hydrophobic interaction; the
hydrophilic groups face the water and by solvation provide the
solubility of the colloid.
[0184] A process for preparing liposomes containing LPA is as
follows. An aqueous dispersion of liposomes is prepared from
membrane components, such as phospholipids (e.g.
phosphatidylcholine, phosphatidylglycerol, sphingomyelin and
phosphatidylethanolamine) and glycolipids according to known
methods as disclosed. Ann. Rev. Biophys. Bioeng. 9:467 (1980). The
liposomes can further contain sterols (e.g., cholesterol and
cholestanol), dialkylphosphates, diacylphosphatidic acids,
stearylamine, (-tocopherol, etc., in the liposomal membrane.
[0185] An aqueous solution of LPA is added to the liposomal
dispersion at a concentration sufficient to produce a
therapeutically effective final product. The mixture is allowed to
stand for a given period of time, preferably under warming at a
temperature more than the phase transition temperature of the
membrane or above 40.degree. C., followed by cooling to produce
liposomes containing LPA in the liposomal membrane.
[0186] Alternatively, the desired liposomes can also be prepared by
previously mixing the above-described membrane components and LPA
and treating the mixture in accordance with known methods for
preparing liposomes.
[0187] The lipid vesicles can be prepared by any suitable technique
known in the art. Methods include, but are not limited to,
microencapsulation, microfluidization, LLC method, ethanol
injection, freon injection, the "bubble" method, detergent
dialysis, hydration, sonication, and reverse-phase evaporation
(reviewed in Watwe et al.). For example, ultrasonication and
dialysis methods generally produce small unilamellar vesicles,
while extrusion and reverse-phase evaporation generally produce
larger sized vesicles. Techniques can be combined in order to
provide vesicles with the most desirable attributes. One
particularly useful method is microfluidization.
[0188] The invention encompasses LMSs containing tissue or cellular
targeting components. Such targeting components are components of a
LMS that enhance its accumulation at certain tissue or cellular
sites in preference to other tissue or cellular sites when
administered to an intact animal, organ, or cell culture. A
targeting component is generally accessible from outside the
liposome and is, therefore, generally either bound to the outer
surface or inserted into the outer lipid bilayer. A targeting
component can be, for example, a peptide, a region of a larger
peptide, an antibody or antigen binding fragment thereof, a nucleic
acid, a carbohydrate, a region of a complex carbohydrate, a special
lipid, or a small molecule such as a drug, hormone, or hapten,
attached to any of the aforementioned molecules.
[0189] Attached to targeting components, the LMSs can be targeted
to any cell type that is undergoing an inappropriate level of
apoptosis. Such cells include, but are not limited to,
cardiomyocytes, endothelial cells, neuronal cells, hepatocytes,
glomerulocytes, lung cells, mucosal cells, skin cells, heart cells
and cancer cells.
[0190] LMSs can be targeted to such cell types in various ways. For
example, a LMS can be modified to contain an antibody, or a
fragment of an antibody, specific for a cell surface molecule, or
marker, found solely or primarily on a given cell type. Antibodies
with specificity toward cell type-specific cell surface markers are
known in the art.
[0191] The compositions of the present invention can additionally
comprise surfactants. Surfactants can be cationic, anionic,
amphiphilic, or nonionic. A preferred class of surfactants are
nonionic surfactants; particularly preferred are those that are
water soluble. Nonionic, water soluble surfactants include
polyoxyethylene derivatives of fatty alcohols, fatty acid ester of
fatty alcohols and glyceryl esters, wherein the polyoxyethylene
group is coupled via an ether linkage to an alcohol group. Examples
include, but are not limited to, polyoxyethylene sorbitan fatty
acid esters, polyoxyethylene castor oil derivatives,
polyoxyethylene hardened castor oil derivatives, fatty acid sodium
salts, sodium cholates, polyexyethylene fatty acid ester and
polyoxyethylene alkyl ethers.
[0192] 4. Proteins.
[0193] Proteins other than serum albumin have been characterized or
implicated as having fatty acid/lipid binding capabilities. These
include the fatty acid binding proteins (FABPs), the lipid binding
proteins, long-chain fatty acyl-CoA (LCFA-CoA) binding proteins,
phospholipid transfer proteins, and Ca.sup.2+/lipid binding
proteins. Lipid-binding proteins are a family of fatty acid and
retinoid transport proteins. Some are intracellular, for example,
while others are secreted from the cell. Intracelluar lipid binding
proteins include cellular retinoic acid binding proteins, CRABP I
and CRABP II, which belong to a family of small cytosolic lipid
binding proteins and appear to play a role in regulating transport
and metabolism of retinoic acid in the developing embryo and
throughout adult life. Banaszak et al. (1994) Adv. Prot. Chem.
45:89-151; and Donovan et al. (1995) J. Steroid Biochem. Mol. Bol.
53:459-465. The lipid-binding protein family includes many FABP.
The FABP are relatively small proteins (13-15 kDa) capable of
binding long-chain fatty acids (LCFA) and their coenzyme A and
L-carnitine esters. They are believed to have major functions in
the metabolism of LCFA for energy production or storage, and are
abundantly present in tissues such as the intestine, liver and
heart, which are actively involved in the uptake or utilization of
LCFA. Bass (1993) Mol. Cell. Biochem. 123:191-202; and Glatz and
van der Vusse (1989) Mol. Cell. Biochem. 88:37-44. A liver form of
FABP has been shown to stimulate export of LPA from mitochondria.
Vancura and Haldar (1992) J. Biol. Chem. 267:14353-14359.
[0194] LCFA-CoA play an important physiological role in
intermediary metabolism of fatty acids, but recent data indicate
that they also can be potent regulators of cell functions. LCFA-CoA
typically exist within a cell bound to membrane lipids and/or
proteins. Proteins which bind LCFA-CoA include LCFA CoA binding
protein, FABP and sterol carrier protein-2. Gossett et al. (1996)
Lipids 31:895-918.
[0195] A protein that enhances a therapeutic effect of LPA can be a
naturally-occurring or a synthetic protein, a protein fragment, or
can contain lipid or sugar moieties. Preferably, the protein is a
lipid-binding or carrier protein. The protein can contain other
modifications including, but not limited to glycosylation,
phosphorylation, myristylation, sulfation and hydroxylation. The
protein can be a hybrid protein, part of which confers the property
of enhancing a therapeutic effect of LPA, and another part of which
confers some other desirable property such as targeting to a
particular cell typo, enhanced in vivo stability, and the like.
Preferred proteins include, but are not limited to, albumin, soy
and plant proteins, cytochrome C, low density lipoprotein, acyl
carrier protein and alphafeto-protein.
[0196] Suitable polypeptides include any known in the art. Modified
polypeptides contain any of a variety of modifications, including,
but not limited to glycosylation, phosphorylation, myristylation,
sulfation and hydroxylation. As used herein, a suitable polypeptide
is one that will protect LPA to preserve its activity. Examples of
binding proteins include, but are not limited to, fatty acid
binding proteins, albumins such as bovine serum albumin (BSA) and
pea albumin.
[0197] Proteins active in enhancing anti-apoptotic activity of LPA
can bind to LPA. "Binding" as used herein means that the LPA and
the protein form non-covalent complexes with one another. Binding
of a protein to LPA can be determined by a variety of methods known
in the art, including a non-denaturing gel electrophoresis binding
assay as described in Example 3 or a size shift assay as described
in Example 3.
[0198] The protein/LPA compositions optionally exhibit a reduction
in the mitogenic activity of LPA. Mitogenic activity can be
measured by any method known in the art, for instance, measurement
of uptake of .sup.3H-thymidine by cells treated with LPA with or
without the addition of protein.
[0199] LPA/protein compositions of the present invention can be
prepared in a variety of ways. LPA and a protein can be suspended
in any biocompatible buffer, for example, bicarbonate buffered
saline, at a ratio of about 0.00001% to 10%, more preferably about
0.001% to 1%. The mixtures can then be sonicated for about 5
minutes or until the mixture is clear or can be sterile
filtered.
[0200] The protein concentration of the LPA/protein compositions
can be from about 0.001 to about 50 mg/mL, more preferably from
about 0.01 to about 10 mg/mL, even more preferably from about 0.05
to about 1.0 mg/mL.
[0201] The protein component of the LPA/protein compositions of the
present invention are substantially pure, i.e., the protein is
substantially free of lipids, other proteins, or any other material
that might affect the activity of the LPA/protein complex.
Preferably, the protein is at least about 75% pure, more preferably
at least about 85% pure and still more preferably at least about
95% pure.
[0202] 5. Other Optional Components.
[0203] Suitable polymers can be any known in the art of
pharmaceuticals and include, but are not limited to,
naturally-occurring polymers such as dextrans, hydroxyethyl starch,
and polysaccharides; and synthetic polymers.
[0204] Examples of naturally occurring polymers include proteins,
glycopeptides, polysaccharides, dextran and lipids. The additional
polymer can be a synthetic polymer. Examples of synthetic polymers
which are suitable for use in the present invention include, but
are not limited to, polyalkyl glycols (PAG) such as PEG,
polyoxyethylated polyols (POP) such as polyoxyethylated glycerol
(POG), polytrimethylene glycol (PTG) polypropylene glycol (PPG),
polyhydroxyethyl methacrylate, polyvinyl alcohol (PVA), polyacrylic
acid, polyethyloxazoline, polyacrylamide, polyvinylpyrrolidone
(PVP), polyamino acids, polyurethane and polyphosphazene. The
synthetic polymers can also be linear or branched, substituted or
unsubstituted, homopolymeric, copolymers, or block copolymers of
two or more different synthetic monomers.
[0205] The synthetic polymers can have the following generic
structure. R.sub.1--(X--R.sub.2).sub.a--R.sub.3 (XVIII)
[0206] where R.sub.1 and R.sub.3 are the same or different and are
H, H.sub.3C, OH, R.sub.2 or a reactive group (as described below);
where R.sub.2 is a linear or branched substituted or unsubstituted
alkyl group; where X is O (in which case the synthetic polymer can
be a polyoxyalkylene) or X is NH(C.dbd.O) (in which case the
synthetic polymer can be a polyamine), or X is absent (in which
case the synthetic polymer can be a polyalkylene); and a is an
integer between 1 and about 1,000.
[0207] Biodegradable polymers can also be included in the
compositions. These include, but are not limited to poly(lactide),
poly(glycolide) poly(vinyl alcohol), crosslinked collagen. The
polymers can also include polyglycolic acid, polyethylene
terephthalate, polybutyl lactose, polycaprolactone, D-polylactic
acid, L-polylactic acid and poly-L-lysine and polymeric mixtures
thereof.
[0208] Suitable polymers also include polysaccharides. Suitable
polysaccharides include, but are not limited to, trehalose,
glucose, maltose, lactose, maltulose, isomaltulose, lactulose,
mono-reducing glycosides of polyhydroxy compounds selected from
sugar alcohols, other straight chain polyalcohols, raffinose,
stachyose, melezitose, dextran, sucrose and sugar alcohols thereof,
maltitol, lactitol, iso-maltulose, palatinit,
2-D-glucopyranosyl-1-(-6-mannitol and their individual sugar
alcohols.
[0209] The compositions can further include pharmaceutically
acceptable excipients. Pharmaceutically acceptable excipients
include, but are not limited to, topical pharmaceutically
acceptable carrier, cosmetic carrier, sterile solutions, sterile
isotonic solutions, ingestable liquids, pharmaceutically acceptable
aerosols and other constituents used in solutions for
organ/cell/cell preservation and/or transplantation.
[0210] The compositions can further include additional
pharmaceutically effective agents. Suitable classes of
pharmaceutically effective agents include, but are not limited to,
drugs, antibiotics, wound heating agents and antioxidants.
[0211] Suitable drugs include, but are not limited to, those listed
in Table 1 and from the following classes: antipyretic and
anti-inflammatory drugs, analgesics, antiarthritics,
antispasmodics, antidepressants, antipsychotics, tranquilizers,
antianxiety drugs, narcotic antagonists, antiparkinsonism agents,
cholinergic antagonists, chemotherapeutic agents,
immuno-suppressive agents, antiviral agents, parasiticides,
appetite suppressants, antiemetics, antihistamines, antimigraine
agents, coronary vasodilators, cerebral vasodilators, peripheral
vasodilators, hormonal agents, contraceptives, antithrombotic
agents, diuretics, antihypertensive agents, cardiovascular drugs,
opioids, and vitamins are all included in the compositions
described herein. TABLE-US-00001 TABLE 1 Cardiac glycosides
Digitalis digitoxin lanatoside C digoxin Anticancer Azathioprine
Bleomycin Byclophosphamide Adriamycin Daunoribicin Vincristine
Antibiotic Penicillin Tetracycline Erythromycin Cephalothin
Imipenem Cefofaxime Carbenicillin Vancomycin Gentamycin Tobramycin
Piperacillin Moxalactam Amoxicillin Ampicillin Cefazolin Cefadroxil
Cefoxitin other aminoglycosides other cephalosporins Antiasthma
Metaproterenol Aminophylline Theophylline Terbutaline Tegretol
Ephedrine Isoproterenol Adrenaline Norepinephrine Antihypertensives
Apresoline Etanolol Immunotherapies Interferon interleukin-2
monoclonal antibodies gammaglobulin Steroids Prednisone
Triamcinolone Hydrocortisone Dexamethasone Betamethosone
Prednisolone Hormones Antidiuretic Corticosteroids Testosterone
Estrogen Thyroid Growth ACTH Progesterone Gonadotropin
Mineralocorticoid Antihistamines Pyribenzamine Chlorpheniramine
Diphenhydramine Antiparasitic Praziquantel Metronidazole
Pentamidine Sedatives & Analgesic Morphine Dilaudid Codeine
codeine-like synthetics Demerol Oxymorphone Phenobarbital
Barbiturates Antidiabetic Diabenese Insulin Antifungal amphotericin
B myconazole muramyl dipeptide clotrimazole Antiarrhythimic
propanolol etanolol verapamil captopril isosorbide Antiviral
acyclovir and derivatives Winthrop-51711 ribavirin
rimantadine/amantadine azidothymidine & derivatives adenine
arabinoside amidine-type protease inhibitors Vaccines influenza
respiratory syncytial virus Hemophilus influenza vaccine
Antihypotension dopamine dextroamphetamine Tranquilizers
chlorpromazine benzodiazepine butyrophenomes hydroxyzines
meprobamate phenothiazines reserpine thioxanthines Other Receptor
agonists and antagonists
[0212] Suitable antibiotics include, but are not limited to,
ampicillin tetracycline, chloramphenicol, erythromycin,
amphotericin B and penicillin. Suitable wound healing agents
include, but are not limited to, transforming growth factors,
platelet-derived growth factors, epidermal growth factors and
fibroblast growth factors. Suitable antioxidants include, but are
not limited to, naturally-occurring antioxidants such as
tocopherols, (e.g., (-tocopherol, vitamin E), ascorbic acid
(Vitamin C), (-carotene (vitamin A), dihydrolipoamide and
flavenoids; and synthetic antioxidants such as butylated
hydroxytoluene, butylated hydroxyanisole, Trolox.RTM., propyl
gallate, other phenolic antioxidants and phenothiazines; and
chelators such as desferoxamide, HBED and CP130.
[0213] II. Methods of Formulating Compositions for Use in the
Present Invention.
[0214] The invention further includes methods of formulating
compositions for use in the present invention, some of which appear
below and some of which appear throughout the text of the
specification.
[0215] The compositions of the present invention can be in either a
liquid or solid form. In the liquid form, the LPA can be
concentrated for dilution prior to use. Preferably, the components
are in a concentration suitable for immediate use. In the case of a
solid, addition of a predetermined amount of an aqueous solution
will result in the appropriate concentration of the components. The
solid can also be in powder form, for use in therapies including,
but not limited to inhalation therapies. In the case where the
composition is a solution, the LPA is preferably present in an
amount of from about 0.00001% to about 10% (weight/volume). More
preferably, the LPA is present in an amount of from about 0.0001%
to about 1% (weight/volume). Most preferably, the LPA is present in
an amount of from about 0.005% to about 1% (weight/volume).
[0216] Where the composition is a solid, preferably the LPA is
present in an amount of from about 0.00001% to 50% (weight/weight).
More preferably, the LPA is present in an amount of from about
0.001% to 1% (weight/weight).
[0217] LPA can be suspended in any buffered solution that is
preferably free of divalent cations having a pH range of 2-10, more
preferably, about 4-8 and most preferably about 6-8. Suitable
buffers include, but are not limited to, D-PBS (phosphate buffered
saline, free of calcium and magnesium salts; Gibco BRL) or 50 mM
ammonium bicarbonate containing isotonic sodium chloride. When the
compositions are to be used therapeutically, the buffered solution
is preferably physiologically acceptable. A wide range of pH values
are effective. Preferably the pH is between about 5.5 to about 8.
However, any pH at which the composition is at least minimally
therapeutically effective is suitable for use. The mixture has been
found to be most active at pH 8. Preferably, the phospholipids are
suspended in 50 mM ammonium bicarbonate/0.154 M sodium chloride,
250 (M EDTA with a pH of 7.7-8.0.
[0218] Preferably, if there is a mixture of phospholipids/lipids,
the mixture is dispersed in order to achieve ma activity. Any
method of dispersion that forms particles of about 5-450 microns is
acceptable and about 30-100 microns is preferred. These methods
include, but are not limited to, microfluidization, extrusion and
sonication, provided that the method does not denature or otherwise
chemically modify the phospholipids in such a manner as to render
them toxic or of substantially diminished therapeutic activity.
Typically, when small batches are prepared, the mixture is
sonicated until optical clarity is attained although sonication can
be continued beyond this point provided the mixture is not
overheated. The preferred sonication parameters are those provided
in the examples herein. As used herein, "optical clarity" indicates
that the mixture changes from opaque to translucent. This change is
readily monitored visually; no further measurements are necessary.
However, "translucent" can be defined as when the mixture has an
O.D. 600 of less than about 0.2 AU. Concentrations of up to
approximately 50 mg/mL phospholipids can be prepared. Preferably,
10 mg/mL solutions are used. Typically, sonication, if used, is in
5 minute alternating cycles, with 5 minutes of sonication followed
by 5 minutes of equilibration. However, this can be varied,
depending on the volume of mixture being sonicated and the heat
generated by sonication.
[0219] The total length of sonication depends on the concentration
and volume of the mixture being sonicated and the power output of
the sonicator. Sonication should proceed until the mixture has
become translucent. Typically, mixtures are sonicated for 3 to 90
minutes. Preferably, sonication proceeds by several periods of 5
minutes each, 6 to 12 total periods, with 1 to 5 minutes between
each period to allow equilibration and dissipation of heat. The
temperature of the water bath should not exceed about 60.degree. C.
Preferably, the temperature of the water bath is not allowed to
exceed 37.degree. C. Preferably, the sonicated mixture is passed
through a sterile filter before use. Preferably, the sterile filter
has a 0.2 micron cut off.
[0220] The compositions can be sterilized at any point.
Sterilization can be by any method known in the art, and
encompasses, but is not limited to, heat sterilization, steam
sterilization, ultrafiltration, sterile filtration and ultraviolet
light sterilization. Sterilization is essential for most of the
methods of treatment, although for certain applications, it may not
be necessary, or the level of sterilization required can be
reduced. The compositions can also be prepared and dried to form a
solid. The solid is suitable for use as a powder or pill, or in
solution upon reconstitution. Any method of drying is suitable for
use herein, including, but not limited to, freeze-drying, air
drying, spray drying and fluidized bed evaporation, vacuum drying
and rotary evaporation
[0221] Preferably, the compositions, both liquid and solid, are
stored under anoxic conditions. Any method of such storage known in
the art is suitable for use herein, including, but not limited to,
storage under an inert gas such as argon.
[0222] III. Prevention of Apoptosis and Preservation or Restoration
of Cell, Tissue and Organ Function.
[0223] The anti-apoptotic activity of the compositions of the
present invention can be measured in many anti-apoptosis assays
known in the art. These include, but are not limited to, the serum
deprivation of the C3H/10T1/2 cell assay described in detail in
Example 2. Furthermore, in vivo apoptosis inhibition can be
measured by any method known in the art. Methods for determining
therapeutic efficacy in treating an ischemic event are known in the
art and described herein. Methods for determining efficacy in organ
storage and transplantation are known in the art and described
herein.
[0224] The therapeutic activity of the compositions described
herein can be measured or determined by any method known in the
art. For instance, there are a variety of wound healing assays
described in the art and cited herein.
[0225] The invention further comprises any of the above-described
compositions in combination with a pharmaceutically acceptable
vehicle. The level of purity of the components necessary for the
composition can be determined empirically and is within the skill
of one in the art, The compositions are suitable for use in a
variety of disorders, as described below, and in both human and
veterinary applications.
[0226] In general, the compositions are pharmaceutically acceptable
due to their low toxicity in the therapeutic dosage range,
stability and ability to be incorporated into a wide variety of
vehicles for numerous routes of administration. The compositions
can be administered alone or in combination with other
pharmaceutically effective agents including, but not limited to,
antibiotics, wound healing agents, antioxidants and other
therapeutic agents as previously described.
[0227] The compositions can contain at least a therapeutically
effective amount of at least one of the above-described
compositions and at least one physiologically acceptable carrier. A
physiologically acceptable carrier is one that does not cause an
adverse physical reaction upon administration and one in which the
compositions are sufficiently soluble to deliver a therapeutically
effective amount of the compound. The therapeutically effective
amount of the compositions depends in part upon the manner of
introduction and the indication to be treated and other criteria
evident to one of ordinary skill in the art. Typically, a
therapeutically effective amount is one sufficient to ameliorate or
cure the condition being treated as evidenced by diminishment of
the symptoms compared to a control. Typically, a therapeutically
effective amount is from about 0.0001% or 1 .mu.g/mL by weight of
the phospholipid mixture although a wide range of effective amounts
can be used for different indications and can be determined
empirically. The route(s) of administration useful in a particular
indication are discussed below and are well known to one of skill
in the art.
[0228] IV. Biological Materials Suitable for Treatment and Routes
of Administration to These Materials.
[0229] Suitable cell types for treatment and/or preservation
include, but are not limited to, eukaryotic and prokaryotic cells,
such as bacterial cells, plant cells, yeast cells, fungi cells,
insect cells, mammalian cells, and human cells in particular.
Mammalian cell types encompass cardiomyocytes, endothelial cells,
neuronal cells, hepatocytes, renal cells, lung cells, mucosal
cells, pancreatic cells, gastrointestinal cells, corneal cells and
skin cells. These cell types, and the tissues and organs they form,
are suitable for treatment and/or preservation by the methods of
the present invention. These cell types can be treated either in
vivo or in vitro using methods of the present invention.
[0230] Routes of administration include, but are not limited to,
topical, transdermal, parenteral, gastrointestinal, transbronchial,
transalveolar, and in vitro treatment of cells, tissues or organs
followed by in vitro administration of treated cells, tissues or
organs. Internal routes of administration encompass any method of
in vivo administration other than solely by topical application to
the skin. Surface administration is accomplished via application of
a cream, gel, rinse, etc. containing a therapeutically effective
amount of the compositions. Transdermal administration is
accomplished by application of a cream, rinse, gel, etc. capable of
allowing the active components to penetrate the skin and enter the
blood stream. Parenteral routes of administration include, but are
not limited to, direct injection such as intravenous,
intramuscular, intraperitoneal or subcutaneous injection.
Gastrointestinal routes of administration include, but are not
limited to, ingestion and rectal. Transbronchial and transalveolar
routes of administration include, but are not limited to,
inhalation, either via the mouth or intranasally (for example, of a
mist or a dry powder) and direct injection into an airway, such as
through a tracheotomy. While the compositions can be topically
administered alone, it may be desirable to administer them in a
mixture with a topical physiologically or cosmetically acceptable
carrier. "Topical pharmaceutically acceptable carrier" as used
herein is any substantially non-toxic carrier conventionally
useable for topical administration of pharmaceutical agents in
which the compositions will remain stable and bioavailable when
applied directly to skin or mucosal surfaces. For example, the
compositions can be dissolved in a liquid, dispersed or emulsified
in a medium in a conventional manner to form a liquid preparation
or mixed with a semi-solid (gel) or solid carrier to form a paste,
powder, ointment, cream, lotion or the like.
[0231] Suitable topical pharmaceutically acceptable carriers
include water, petroleum jelly (Vaseline), petrolatum, mineral oil,
vegetable oil, animal oil, organic and inorganic waxes, such as
microcrystalline, paraffin and ozocerite wax, natural polymers,
such as xanthanes, gelatin cellulose, collagen, starch, or gum
arabic, synthetic polymers, such as discussed below, alcohols,
polyols, and the like. The carrier can be a water miscible carrier
composition that is substantially miscible in water. Such water
miscible topical pharmaceutically acceptable carrier composition
can include those made with one or more appropriate ingredients set
forth above but can also include sustained or delayed release
carriers, including water containing water dispersible or water
soluble compositions, such as liposomes, microsponges, microspheres
or microcapsules, aqueous base ointments, water-in-oil or
oil-in-water emulsions, gels or the like.
[0232] In one embodiment of the invention, the topical
pharmaceutically acceptable carrier comprises a sustained release
or delayed release carrier. The carrier is any material capable of
sustained or delayed release of the compositions to provide a more
efficient administration resulting in one or more of less frequent
and/or decreased dosage of the compositions, ease of handling, and
extended or delayed effects on dermatologic conditions. The carrier
is capable of releasing the compositions when exposed to any oily,
fatty, waxy, or moist environment on the area being treated or by
diffusing or by release dependent on the degree of loading of the
compositions to the carrier in order to obtain release thereof.
Non-limiting examples of such carriers include liposomes,
microsponges, microspheres, or microcapsules of natural and
synthetic polymers and the like.
[0233] Examples of suitable carriers for sustained or delayed
release in a moist environment include gelatin, gum arabic,
xanthane polymers; by degree of loading include lignin polymers and
the like; by oily, fifty or waxy environment include thermoplastic
or flexible thermoset resin or elastomer including thermoplastic
resins such as polyvinyl halides, polyvinyl esters, polyvinylidene
halides and halogenated polyolefins, elastomers such as
brasiliensis, polydienes, and halogenated natural and synthetic
rubbers, and flexible thermoset resins such as polyurethanes, epoxy
resins and the like. Preferably, the sustained or delayed release
carrier is a liposome, microsponge, microsphere or gel.
[0234] The compositions used in the method of treating dermatologic
conditions of the invention are applied directly to the areas to be
treated. While not required, it is desirable that the topical
composition maintain the active components at the desired location
for about 24 to 48 hours, or a length of time sufficient to exert
therapeutic efficacy.
[0235] If desired, one or more additional ingredients
conventionally found in topical pharmaceutical or cosmetic
compositions can be included with the carrier, such as a
moisturizers, humectants, odor modifiers, buffers, pigments,
preservatives, Vitamins such as A, C and E, emulsifiers, dispersing
agents, wetting agents, odor-modifying agents, gelling agents,
stabilizers, propellants, antimicrobial agents, sunscreens, enzymes
and the like. Those of skill in the art of topical pharmaceutical
formulations can readily select the appropriate specific additional
ingredients and amounts thereof. Suitable non-limiting examples of
additional ingredients include superoxide dismutase, stearyl
alcohol, isopropyl myristate, sorbitan monooleate, polyoxyethylene
stearate, propylene glycol, water, alkali or alkaline earth lauryl
sulfate, methylparaben, octyl dimethyl-p-amino benzoic acid
(Padimate O), uric acid, reticulin, polymucosaccharides,
hydroxyethyl starch (such as, DuPont Pentafraction), hyaluronic
acids, aloe vera, lecithin, polyoxyethylene sorbitan monooleate,
Vitamin A or C, tocopherol (Vitamin E), alpha-hydroxy of alpha-keto
acids such as pyruvic, lactic or glycolic acids, or any of the
topical ingredients disclosed in U.S. Pat. No. 4,340,586,
4,695,590, 4,959,353 or 5,130,298 and 5,140,043. Because
dermatologic conditions to be treated may be visible, the topical
carrier can also be a topical cosmetically acceptable carrier. By
"topical cosmetically acceptable carrier" as used herein is meant
any substantially non-toxic carrier conventionally useable for
topical administration of cosmetics in which the compositions will
remain stable and bioavailable when applied directly to the skin
surface. Suitable cosmetically acceptable carriers are known to
those of skill in the art and include, but are not limited to,
cosmetically acceptable liquids, creams, oils, lotions, ointments,
gels, or solids, such as conventional cosmetic night creams,
foundation creams, suntan lotions, sunscreens, hand lotions,
make-up and make-up bases, masks and the like. Thus, to a
substantial extent, topical cosmetically acceptable carriers and
pharmaceutically acceptable carriers are similar, if not often
identical, in nature so that most of the earlier discussion on
pharmaceutically acceptable carriers also applies to cosmetically
acceptable carriers. The compositions can contain other ingredients
conventional in cosmetics including perfumes, estrogen, V, C, or E,
alpha-hydroxy or alpha-keto acids such as pyruvic, lactic or
glycolic acids, lanolin, vaseline, aloe vera, methyl or propyl
paraben, pigments and the like.
[0236] The effective amount of the compositions used to treat
dermatologic conditions or diseases can vary depending on such
factors as condition of the skin, age of the skin, the particular
ratio of phospholipids or degree of the purity of phospholipids
employed, the type of formulation and carrier ingredients used,
frequency of administration, overall health of the individual being
treated and the like. The precise amount for any particular patient
use can be determined by those of skill in the dermatologic art
taking into consideration these factors and the present disclosure.
Preferably the composition is administered in at least two doses
and no more than about six doses per day, or less when a sustained
or delayed release form is used.
[0237] The compositions for topical, oral and parenteral
administration usually contain from about 0.001% to about 10% by
weight of the LPA compared to the total weight of the composition,
preferably from about 0.01% to about 2% by weight of the mixture to
the pharmaceutical composition, and especially from about 0.1% to
about 1.5% by weight of the mixture to the pharmaceutical
composition.
[0238] The topical composition is administered by applying a
coating or layer to the skin or mucosal area desired to be treated.
As a practical matter of convenience, the applied material is
rubbed into the area. Applications need not be rubbed into the skin
and the layer or coating can be left on the skin overnight.
[0239] The present invention provides compositions suitable for
transdermal administration including, but not limited to,
pharmaceutically acceptable lotions, suspensions, oils, creams,
ointments, rinses, gels and liposomal carriers suspended in a
suitable vehicle in which a therapeutically effective amount of the
compositions has been admixed. Such compositions are applied
directly to the skin or incorporated into a protective carrier such
as a transdermal device (so-called "patch"). Examples of suitable
creams, ointments etc. can be found, for instance, in the
Physician's Desk Reference. Examples of suitable transdermal
devices are described, for instance, in U.S. Pat. No. 4,818,540
(Chien et al.).
[0240] The present invention includes compositions suitable for
parenteral administration including, but not limited to,
pharmaceutically acceptable sterile isotonic solutions. Such
solutions include, but are not limited to, saline and phosphate
buffered saline for intravenous, intramuscular, intraperitoneal or
subcutaneous injection.
[0241] The present invention includes compositions suitable for
gastrointestinal administration including, but not limited to,
pharmaceutically acceptable powders, pills or liquids for ingestion
and suppositories for rectal administration.
[0242] The present invention includes compositions suitable for
transbronchial and transalveolar administration including, but not
limited to, various types of pharmaceutically acceptable aerosols
for inhalation, both liquid and powder forms. An example of a drug
administered in the form of an aerosol is pentamidine which is
administered to AIDS patients by inhalation to prevent pneumonia
caused by Pneumocystis carnii.
[0243] In some cases it may be desirable to perform internal
delivery of LPA containing compositions in a localized area of the
body, organ or tissue. The present invention encompasses methods of
delivery including, but not limited to, delivery by catheter
inserted into a vessel. Where delivery of the LPA containing
compositions is desired to prevent or minimize damage resulting
from cardiac ischemia the present invention encompasses
intracoronary delivery by guide catheter.
[0244] The present invention further encompasses devices suitable
for transbronchial and transalveolar administration of the
compositions. Such devices include, but are not limited to,
atomizers and vaporizers. The present invention also includes
devices for electrical or direct injection. Electrical injection,
or iontophoresis, is the process of using a small electrical
current to drive charged elements, compounds and drugs through the
skin to deliver the therapeutic compound to the local tissues or to
the whole body without breaking the skin.
[0245] The present invention encompasses solutions suitable for
flushing, perfusion, and storage of organs and tissues prior to or
during transplantation. Such solutions are described in Chien et
al. (1993) "Hibernation Induction Trigger for Organ Preservation"
in Medical Intelligence Unit, R. G. Landes Co. Austin, Tex. The
compositions described herein can be used, for instance, to replace
and improve on much more impure soy preparations currently in
use.
[0246] By "organ preservation solution" (OPS) is meant an aqueous
solution specifically designed to preserve organs. Preferably the
organ is the heart. Preferably, the solutions are used in organ
transplantation, but are also useful for use in cardioplegia during
open heart surgery. The OPS can also be used to flush the organ to
be transplanted either prior to or after harvesting, or both.
Preferably, this solution contains between about 0.00001% to about
10%, preferably about 0.001% to about 1%, more preferably about
0.005% to about 0.1%. Preferably, this solution additionally
contains between about 0.1% and 20% by weight PEG. More preferably,
this solution contains between about 2% and 15% by weight PEG and
most preferably, this solution contains about 8% by weight PEG.
Preferably, the PEG is PEG20L.
[0247] Preferably, this solution contains between about 2% and 15%
by weight PEG and can also contain effective amounts of: (a) a
buffer such as NaOH, preferably about 30-40 mM (or sufficient to
result in pH of 7.2 to 7.9); (b) an impermeant anion such as
Lactobionic acid, preferably about 100 mM; (c) a component
providing phosphate such as KH.sub.2PO.sub.4, preferably about 25
mM; (d) a component providing potassium such as KOH, preferably
about 100 mM; and (e) a component controlling cell swelling such as
Raffinose, preferably about 30 mM.
[0248] Optionally, the OPS also contains effective amounts of any
component known in the art of organ preservation. These include,
but are not limited to glutathione, parahydroxyanisole (PHA),
desferoxamine, and nitroglycerin.
[0249] The above-mentioned compositions are meant to describe, but
not limit, the compositions suitable for use in the invention. The
methods of producing the various compositions are within the
ability of one skilled in the art and are not described in detail
here. The methods of producing suitable devices for injection,
topical application, atomizers and vaporizers are known in the art
and will not be described in detail.
[0250] The invention further provides methods of treatment
comprising administering an amount of the compositions effective to
inhibit apoptosis or to preserve or restore cell, tissue or organ
function. These methods entail administration of a therapeutically
effective amount of the above-described compositions.
[0251] Various indications which can be treated, include, but are
not limited to, those related to apoptosis, ischemia, traumatic
injury and reperfusion damage. Those conditions related to
apoptosis include, but are not limited to, dermatological effects
of aging, the effects of reperfusion after an ischemic event,
immunosuppression, gastrointestinal perturbations, cardiovascular
disorders, rejection of tissue transplantation, wound healing and
Alzheimer's disease. The treatment can also diminish the
apoptosis-related problems associated with immunosuppressing
viruses, chemotherapeutic agents, or radiation and
immunosuppressive drugs.
[0252] The compositions are also suitable for use in organ
transplantation during all phases of transplantation. The
compositions can be used to prepare the organ by administering an
amount of the compositions to the donor effective to stabilize or
preserve the organ. The organ can be perfused and/or preserved in
OPS containing the compositions. The organ recipient can then be
administered an amount of the compositions effective to enhance
organ stability and function. The compositions are also
particularly suitable for use in treating cardioplegia whether
related to transplantation or other surgical intervention.
[0253] Apoptosis related problems are caused by a variety of
stimuli which include, but are not limited to, viruses including,
but not limited to, HIV, chemotherapeutic agents, and radiation.
These stimuli trigger apoptosis in a variety of disorders,
including, but not limited to, those of the digestive tract tissues
and associated gastrointestinal perturbations.
[0254] Gastrointestinal perturbations include, but are not limited
to, damage to the lining of the gut, severe chronic ulcers,
colitis, radiation induced damage, chemotherapy induced damage, and
the perturbation of the gastrointestinal tract caused by parasites,
and diarrhea from any other cause. Various viral and bacterial
infections are known to result in gastrointestinal perturbations;
the compositions are also suitable for use in treatment of the side
effects associated with these infections. The compositions are
particularly suited for use in ameliorating the gastrointestinal
disturbances associated with chemotherapy. Thus, the compositions
are suitable for use not only in preventing the diarrhea associated
with chemotherapy but also the nausea. The compositions are
particularly suited to treatment of various gastrointestinal
conditions in animals, including, but not limited to livestock and
domesticated animals. Such conditions, particularly diarrhea,
account for the loss of or many calves and puppies to dehydration
and malnutrition. Treatment of gastrointestinal conditions is
preferably by gastrointestinal administration. In the case of
cattle and domesticated animals, an effective amount of the
compositions can be conveniently mixed in with the feed. In humans,
administration can be by any method known in the art of
gastrointestinal administration. Preferably, administration is
oral.
[0255] In addition, the compositions can be administered to
immunodeficient patients, particularly HIV-positive patients, to
prevent or at least mitigate apoptotic death of T cells associated
with the condition, which results in the exacerbation of
immunodeficiencies as seen in patients with AIDS. Preferably,
administration to such patients is parenterally, but can also be
transdermally or gastrointestinally.
[0256] The compositions can also be administered to treat apoptosis
associated with reperfusion damage involved in a variety of
conditions, including, but not limited to, coronary artery
obstruction; cerebral infarction; spinal/head trauma and
concomitant severe paralysis; reperfusion damage due to other
insults such as frostbite; coronary angioplasty, blood vessel
attachment, limb attachment, organ attachment and kidney
reperfusion.
[0257] Myocardial and cerebral infarctions (stroke) are caused
generally by a sudden insufficiency of arterial or venous blood
supply due to emboli, thrombi, or pressure that produces a
macroscopic area of necrosis; the heart, brain, spleen, kidney,
intestine, lung and testes are likely to be affected. Cell death
occurs in tissue surrounding the infarct upon reperfusion of blood
to the area; thus, the compositions are effective if administered
at the onset of the infarct, during reperfusion, or shortly
thereafter. The invention includes methods of treating reperfusion
damage by administering a therapeutically effective amount of the
compositions to a patient in need of such therapy.
[0258] The invention further encompasses a method of reducing the
damage associated with myocardial and cerebral infarctions for
patients with a high risk of heart attack and stroke by
administering a therapeutically effective amount of the
compositions to a patient in need of such therapy. Preferably,
treatment of such damage is by parenteral administration of the
compositions of the invention. Any other suitable method can be
used, however, for instance, direct cardiac injection in the case
of myocardial infarct. Devices for such injection are known in the
art, for instance the Aboject cardiac syringe.
[0259] The invention further provides methods of limiting and
preventing apoptosis in cells, or otherwise preserving cells,
during the culture or maintenance of mammalian organs, tissues, and
cells, by the addition of an effective amount of the compositions
to any media or solutions used in the art of culturing or
maintaining mammalian organs, tissues, and cells.
[0260] The invention further encompasses media and solutions known
in the art of culturing and maintaining mammalian organs, tissues
and cells, which comprise an amount of the compositions effective
to preserve or restore cell, tissue or organ function, or limit or
prevent apoptosis of the cells in culture.
[0261] These aspects of the invention encompass mammalian cell
culture media comprising an effective amount of at least one
composition and the use of such media to preserve or restore cell,
tissue or organ function, or to limit or prevent apoptosis in
mammalian cell culture. An effective amount is one which decreases
the rate of apoptosis and/or preserves the cells, tissue or organ.
The compositions have been found to limit or prevent apoptosis
under circumstances in which cells are subjected to mild traumas
which would normally stimulate apoptosis. Such traumas can include,
but are not limited to, low level irradiation, thawing of frozen
cell stocks, rapid changes in the temperature, pH, osmolarity, or
ion concentration of culture media, prolonged exposure to
non-optimal temperature, pH, osmolarity, or ion concentration of
the culture media, exposure to cytotoxins, disassociation of cells
from an intact tissue in the preparation of primary cell cultures,
serum deprivation (or growth in serum-free media). Thus, the
invention encompasses compositions comprising tissue culture medium
and an effective amount of the compositions. Serum-free media to
which the compositions can be added as anti-apoptotic media
supplements include, but are not limited to, AIM V.RTM. Media,
Neuman and Tytell's Serumless Media, Trowell's T8 Media, Waymouth's
MB 752/1 and 705/1 Media, and Williams' Media E. In addition to
serumfree media, suitable mammalian cell culture media to which the
compositions can be added as anti-apoptotic media supplements
include, but are not limited to, Basal Media Eagles, Fischer's
Media, McCoy's Media, Media 199, RPMI Media 1630 and 1640, Media
based on F-10 & F-12 Nutrient Mixtures, Leibovitz's L-15 Media,
Glasgow Minimum Essential Media, and Dulbecco's Modified Eagle
Media. Mammalian cell culture media to which the compositions can
be added further comprise any media supplement known in the art,
including, but not limited to, sugars, Vitamins, hormones,
metalloproteins, antibiotics, antimycotics, growth factors,
lipoproteins and sera.
[0262] The invention further encompasses solutions for maintaining
mammalian organs prior to transplantation, which comprise an
effective amount of the compositions, and the use of such solutions
to preserve or restore organ function or to limit or prevent
apoptosis in such mammalian organs during their surgical removal
and handling prior to transplantation. The solutions can be used to
flush, perfuse and/or store the organs. In all cases,
concentrations of the compositions required to limit or prevent
damage to the organs can be determined empirically by one skilled
in the art by methods such as those found in the example provided
below, as well as other methods known in the art.
[0263] It has now been found that the compositions can be topically
applied to the skin to treat a variety of dermatologic conditions.
These conditions include, but are not limited to, hair loss and
wrinkling due to age and/or photo damage. The present invention
thus encompasses methods of treating dermatological conditions.
Furthermore, hair loss can be caused by apoptosis of the cells of
the hair follicles. Therefore, the compositions are suitable for
use in topical treatment of the skin to prevent continued hair
loss. Stenn et al. (1994) J. Invest. Dermatol. 103:107-111. As
discussed above, these conditions are preferably trod by topical
application of a composition comprising an effective amount of the
compositions. An effective amount of the compositions is one which
ameliorates or diminishes the symptoms of the dermatologic
conditions. Preferably, the treatment results in resolution of the
dermatologic condition or restoration of normal skin function;
however, any amelioration or lessening of symptoms is encompassed
by the invention.
[0264] The following examples are provided to illustrate but not
limit the invention. Note that references to specific LPAs using
compound numbers in this document refer to the compound numbers
assigned in Example 1, below.
EXAMPLE 1
Structure and Synthesis of Various Analogs of Lysophosphatidic
Acid
[0265] The following example sets forth the synthetic methodology
and analytical data used in the construction and characterization
of several lysophosphatidic acid analogs and derivatives. In
addition experimental procedures and analytical data has been
provided for intermediates used in the construction of these
LPAs.
[0266] Contained at the end of the experimental procedures and
analytical data are reaction schemes which show the synthetic
routes used in compound construction. The following nomenclature
and abbreviations are used in the naming of the compounds: [0267]
Bn benzyl [0268] BSA bis(trimethylsilyl)acetamide [0269] t-BuOOH
tert.-butylhydroperoxide [0270] CNE cyanoethyl [0271] DMAP
N,N-dimethylaminopyridine [0272] DMF N,N-dimethylformamide [0273]
Ile L-isoleucine [0274] Me methyl [0275] MeI methyl iodide [0276]
MeOH methanol [0277] sat. saturated [0278] TBAF tetra-butylammonium
fluoride [0279] TBS tert.-butyldimethylsilyl [0280] TBTU
O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate
[0281] TEA triethylamine [0282] THF tetrahydrofuran [0283] TLC thin
layer chromatography [0284] TMSBr trimethylsilyl bromide [0285] Tr
trityl [0286] Ts tosyl [0287] Val L-valine [0288] Z
benzyloxycarbonyl
[0289] 1-O-Decyl-rac-glycerol (Compound 1) To a sting mixture of
NaH (0.36 g, 15.1 mmol) in dry DMF (15 ml) under N.sub.2 was added
solketal (0.94 ml, 7.6 mmol) dropwise over a 30 min period. After
30 min. NaI (0.30 g, 2.0 mmol) was added followed by the addition
of 1-chlorodecane (1.34 g, 7,6 mmol) and stirring was continued at
50.degree. C. for 16 hours. The reaction mixture was diluted with
diethyl ether (50 mL) and washed with H.sub.2O (2.times.50 mL),
dried (MgSO.sub.4) and concentrated to give a crude oil (1.48 g).
The crude oil (1.06 g, 3.9 mmol) was treated with 1/1 2M HCl/THF
(14 mL, v/v) for 2 hours, and the resulting solution was
concentrated and redissolved in ethyl acetate (80 ml), washed with
H.sub.2O (2.times.20 mL), dried (MgSO.sub.4) and concentrated to
give a yellow oil. The oil was subjected to silica-gel column
chromatography [eluent: ethyl acetate/hexane, 50/50, v/v] to give
the title diol (Compound 1) (58 mg, 65%) as a colorless oil.
[0290] .sup.1H NMR (360 MHz; CDCl3): (0.87 (3H, t, J 7 Hz, Me),
1.26 (14H, br s, 7.times.CH.sub.2), 1.53-1.59 (2-H, m, (--H.sub.2),
3.41-3.55 (4H, m, (--H.sub.2 and 1-H.sub.2 or 1-H.sub.2), 3.64 (1H,
dd, J 11.5 and 5 Hz, 1-H or 3-H), 3.72 (1H, dd, J 11.5 and 4 Hz,
1-H or 3-H) and 3.83-3.88 (1H, m, 2-H; ESI-MS (m/z, +ve): 231
(MH.sup.+, 100%).
[0291] 1-O-Tetradecyl-rac-glycerol (Compound 2) To a mixture of
solketal (5.0 mL, 40 mmol), 1-chlorotetraecane (10.9 mL, 40 mmol)
and a catalytic amount of NaI in DMF (200 mL) was added NaH (3.1 g,
80 mmol) and the reaction was stirred for 16 hours at 50.degree. C.
After removal of the solvent in vacuo, the residue was redissolved
in ethyl a (150 mL), washed with H.sub.2O (3.times.50 mL), dried
(MgSO.sub.4) and evaporated to dryness. The obtained oil was tread
with 1/1 2M HCl/THF (150 mL, v/v) for 16 hours. The resulting
mixture was concentrated and redissolved in ethyl acetate (150 mL),
washed with H.sub.2O (3.times.50 mL), dried (MgSO.sub.4) and
evaporated to dryness. The crude product was purified by silica-gel
column chromatography [eluent: CH.sub.2Cl.sub.2(ethyl
acetate/CH.sub.2Cl.sub.2, 50/50, v/v] to yield diol Compound 2 (8.4
g, 73%) as a white solid.
[0292] .sup.1H NMR (360 MHz; CDCl): (0.87 (3H, t, J 7 Hz, Me), 1.25
(22H, br s, 11.times.CH.sub.2), 1.57 (2H, quintet, J 7 Hz,
(--H.sub.2), 2.38 and 2.77 (each 1H, br s, 2.times.OH), 3.46 (2H,
dt, J 6.5 and 2 Hz, 1-H.sub.2 or (--H.sub.2), 3.49-3.55 (2H, m,
(--H.sub.2 or 1-H.sub.2), 3.64 (1H, dd, J 11.5 and 5 Hz, 3-H), 3.71
(1H, dd, J 11.5 and 4 Hz 3-H) and 3.85-3.86 (1H, m, 2-H).
[0293] Dimethyl-3-O-tetradecyl-rac-glycerol-1-phosphate (Compound
3) To a solution of diol Compound 2 (1.0 g, 3.5 mmol) and
N-methylimidazole (0.45 mL, 5.6 mmol) in dry CH.sub.2Cl.sub.2 (25
mL) was added dimethyl chlorophosphate (0.42 ml, 3.9 mmol). After
stirring for 3 days, the mixture was concentrated and purified by
silica-gel column chromatography [eluent: hexane/ethyl acetate,
60/40, v/v] to yield phosphate Compound 3 (0.10 g, 7%) as an
oil.
[0294] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.87 (3H, t, J 7 Hz,
Me), 1.25 (22H, br s, 11.times.CH.sub.2), 1.55-1.58 (2H, m,
(--H.sub.2), 3.43-3.53 (4H, m, 3-H.sub.2 and (--H.sub.2), 3.80 and
3.83 (each 3H, s, 2.times.OMe), 4.00-4.02 (1H, m, 2-H) and
4.10-4.20 (2H, m, 1-H.sub.2); ESI-MS (m/z, +ve): 397 (MH.sup.+,
100%) and 419 MH.sup.+, 66).
[0295] 3-O-Tetradecyl-rac-glycerol-phosphate (Compound 4) To a
solution of diol 2 (1.0 g, 3.5 mmol) and dibenzyl
N,N-diisopropylphosphoramidite (1.16 mL, 3.5 mmol) in dry
CH.sub.3CN (30 mL) was added 1H-tetrazole (0.12 g, 1.7 mmol). After
stirring for 2 hours, t-BuOOH (2 mL) was added and after another 30
min the mixture was concentrated. The residue was purified by
silica-gel column chromatography [eluent: hexane/ethyl acetate,
66/33, v/v) to give dibenzyl 3-O-tetradecyl-rac-glycero-1-phosphate
(0.27 g, 14%) as an oil. Subsequent hydrogenolysis in MeOH (30 mL)
in the presence of 10% Pd/C (0.5 g), followed by filtration over
Celite and evaporation to dryness, afforded the title phosphate
(Compound 4) (0.18 g, 99%) as a glass.
[0296] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.87 (3H, t, J 6.5 Hz,
Me), 1.25 (22H, br s, 11.times.CH.sub.2), 1.55 (2H, br s,
(--H.sub.2), 3.45 (4H, br s, (--H.sub.2 and 3-H.sub.2 and 4.05-4.14
(3H, m, 2-H and 1-H.sub.2); .sup.31P NMR (146 MHz; CDCl.sub.3):
(1.11; ESI-MS (m/z, +ve): 367 (M-H.sup.+, 100%).
[0297] 1-O-Decyl-glycidol (Compound 6) To a stirring solution of
glycidyl tosylate (2.0 g, 8.8 mmol) and decyl alcohol (1.34 mL, 7.0
mmol) in dry CH.sub.2Cl.sub.2 (40 mL) under N2 was added a
BF3.OEt.sub.2 solution (.about.2.2 mL, .about.10% in CH2Cl2). After
48 hours, TLC analysis showed the reaction to be complete and the
solvent was removed in vacuo to give
1-O-decyl-rac-glycero-3-p-toluenesulfonate (Compound 5) as an
oil.
[0298] Crude Compound 5 was taken up in 50% aqueous MeOH (30 mL),
NaOH (1.4 g, 35.0 mmol) was added and the mixture was left to stir
for 16 hours. TLC analysis showed the reaction to be complete and
the mixture was concentrated to remove MeOH. The remaining aqueous
phase was extracted with diethyl ether (2.times.40 ml) and the
combined organic phases were washed with H.sub.2O (30 mL), sat.
NaHCO.sub.3 (30 mL), dried MgSO.sub.4) and concentrated. The
residue was subjected to silica-gel column chromatography [eluent:
ethyl acetate/hexane, 91/9, v/v] to give epoxide Compound 6 (0.93
g, 62%) as a colourless oil.
[0299] .sub.1H NMR (360 MHz; CDCl.sub.3): (0.87 (3H, t, J 7 Hz,
Me), 1.26 (14H, br s, 7.times.CH.sub.2), 1.58 (2H, m, (--H.sub.2),
2.61 (1H, dd, J 5 and 2.5 Hz, 3-H), 2.80 (1H, t, J 4.5 Hz, 3-H),
3.13-3.17 (1H, m, 2-H), 3.38 (1H, dd, J 11.5 and 6 Hz, 1-H),
3.42-3.54 (2H, m, (--H.sub.2) and 3.70 (1H, dd, J 11.5 and 3 Hz,
1-H).
[0300] 1-O-Tetradecyl-glycidol (Compound 8) To a solution of
glycidyl tosylate (2.0 g, 8.8 mmol) and 1-tetradecanol (1.5 g, 7.0
mmol) in dry CH.sub.2Cl.sub.2 (40 mL) under N.sub.2 was added a
BF.sub.3.OEt.sub.2 solution (.about.2.2 mL, .about.10% in
CH.sub.2Cl.sub.2). After 48 hours, TLC analysis showed the reaction
to be complete and the solvent was removed in vacuo to give
1-O-tetradecyl-rac-glycero-3-p-toluenesulfonate (Compound 7) as an
oil.
[0301] Crude Compound 7 was taken up in 50% aq. MeOH (30 mL), NaOH
(1.4 g, 35.0 mmol) was added and the mixture was left to stir for
16 hours. TLC analysis showed the reaction to be complete and the
mixture was concentrated to remove MeOH. The remaining aqueous
phase was extracted with diethyl ether (2.times.40 mL) and the
combined organic phases were washed with H.sub.2O (30 mL), sat.
NaHCO.sub.3 (30 mL), dried MgSO.sub.4) and evaporated to dryness.
The residue was subjected to silica-gel column chromatography
[eluent: ethyl acetate/hexane, 91/9, v/v] to give epoxide Compound
8 (1.57 g, 83%) as a colourless oil.
[0302] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.88 (3H, t, J 7 Hz,
Me), 1.25 (22H, m, 11.times.CH.sub.2), 1.57 (2H, m, (--H.sub.2),
2.61 (1H, dd, J 5.5 and 3 Hz, 3-H), 2.80 (1H, t, J 4.5 Hz, 3-H,
3.13-3.17 (1H, m 2-H), 3.38 (1H, dd, J 11.5 and 6 Hz, 1-H),
3.42-3.54 (1H, m, (--H.sub.2) and 3.70 (1H, dd, J 11.5 and 6 Hz,
1-H).
[0303] 1-O-Oleyl-glycidol (Compound 10) To a solution of glycidyl
tosylate (2.0 g, 8.8 mmol) and oleyl alcohol (1.88 mL, 7.0 mmol) in
dry CH.sub.2Cl.sub.2 (40 mL) under N.sub.2 was added a
BF3.OEt.sub.2 solution (.about.2.2 mL, .about.10% in
CH.sub.2Cl.sub.2). After 48 hours, TLC analysis showed the reaction
to be complete and the solvent was removed in vacuo to give
1-O-oleyl-rac-gycero-3-p-toluenesulfonate (Compound 9) as an
oil.
[0304] Crude Compound 9 was taken up in 50% aq. MeOH (30 mL) to
which NaOH (1.4 g, 35.0 mmol) was added. After 16 hours, TLC
analysis showed the reaction to be complete and the mixture was
concentrated to remove MeOH. The remaining aqueous phase was
extracted with diethyl ether (2.times.40 mL) and the combined
organic phases were washed with H.sub.2O (30 mL), sat. NAHCO.sub.3
(30 mL), dried (MgSO.sub.4) and concentrated. The residual oil was
purified by silica-gel column chromatography [eluent: ethyl
acetate/hexane 91/9, v/v] to furnish epoxide Compound 10 (1.65 g,
72%) as a colourless oil.
[0305] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.88 (3H, t, J 6.5 Hz,
Me), 1.28 (22H, apparent br d, separation 9 Hz,
--(CH.sub.2).sub.5-- and --(CH.sub.2).sub.6--), 1.58 (2H, br s,
(--H.sub.2), 2.01 (4H, apparent br d, separation 4.5 Hz,
--CH.sub.2CH.dbd.CHCH.sub.2--), 2.61 (1H, br s, 3-H), 2.80 (1H, br
t, J 4 Hz, 3-H), 3.15 (1H, br s, 2-H), 3.38 (1H, dd, J 11.5 and 6
Hz, 1-H), 3.45-3.54 (2H, m, (--H.sub.2), 3.69-3.72 (1H, m, 1-H) and
5.34 (2H, br s, --CH.sub.2CH.dbd.CHCH.sub.2--).
[0306] 3-O-Decyl-rac-glycero-1-phosphate (Compound 11) A mixture of
98% phosphoric acid (0.18 g, 1.9 mol) and decyl glycidol (Compound
6) (0.4 g, 1.9 mmol) in dry CH.sub.2Cl.sub.2 was refluxed for 2
hours until TLC analysis showed the reaction to be complete. Then,
the reaction mixture was concentrated to afford phosphate Compound
11 (0.56 g, 78%) as a colourless oil.
[0307] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.87 (3H, t, J 6.5 Hz,
Me), 1.25 (14H, br s, 7.times.CH.sub.2), 1.56 (2H, br s,
(--H.sub.2), 3.44-3.53 (4H, m, (--H.sub.2 and 3-H.sub.2) and
3.61-4.13 (3H, m, 2-H and 1-H.sub.2); .sup.31P NMR (146 MHz;
CDCl.sub.3): (1.41; ESI-MS (m/z, -ve): 311 (M-H.sup.+, 100%).
[0308] 3-O-Oleyl-rac-glycero-1-phosphate (Compound 12) A mixture of
98% phosphoric acid (0.18 g, 1.9 mol) and oleyl glycidol (Compound
10) (0.4 g, 1.2 mmol) in dry CH.sub.2Cl.sub.2 was refluxed for 16
hours. TLC analysis showed the reaction to be complete and the
mixture was concentrated to give the title phosphate (Compound 12)
(0.501 g, 96%) as a colourless oil.
[0309] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.87 (3H, t J 6.5 Hz,
Me), 1.27 (22H, apparent br d, separation 6 Hz,
--(CH.sub.2).sub.5-- and --(CH.sub.2).sub.6--), 1.55 (2H, br s,
(--H.sub.2), 1.98-2.03 (4H, m, --CH.sub.2CHCH.sub.2--), 3.44-3.51
(4H, m, 3-H2 and (--H2), 3.53-4.12 (3H, m, 2-H and 1-H.sub.2),
5.30-5.38 (2H, m, CH.sub.2.dbd.CHCH.sub.2--) and 6.43 (2H, br s,
2.times.OH); .sup.31P NMR (146 MHz; CDCl.sub.3): (1.68; ESI-MS
(m/z, -ve): 421 (M-H, 100%).
[0310] Dimethyl 3-oleyloxypropyl-1-phosphate (Compound 15) To a
suspension of NaH (0.40 g, 13.1 mmol) and anhydrous NaI (1.98 g,
13.4 mmol) in dry DMF (30 mL) under N.sub.2 was added dropwise
1,3-propanediol (0.95 mL, 13.1 mmol) over a 30 min period, Next,
oleyl tosylate (13) (5.6 g, 13.1 mmol) was added and stirring was
continued for 16 hours at 50.degree. C. The reaction mixture was
quenched with water (50 mL) and extracted with diethyl ether
(2.times.100 mL). The combined organic phases were dried
(MgSO.sub.4) and concentrated to give crude alcohol Compound
14.
[0311] To a solution of Compound 14 (0.51 g, 1.6 mmol) in dry
CH.sub.2Cl.sub.2 (10 mL) was added N-methylimidazole (0.28 mL, 3.5
mmol) followed by the addition of dimethyl chlorophosphate (0.2 mL,
1.9 mmol). After 16 hours, the reaction was quenched with sat.
KHSO.sub.4 (20 mL) and extracted with ethyl acetate (3.times.50
mL). The organic phases were washed with sat. NaCl (40 mL), dried
(MgSO.sub.4) and concentrated to give protected phosphate Compound
15 as a yellow oil.
[0312] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.88 (3H, t J 7 Hz, Me),
1.27 (22H, apparent br d, separation 12H, --(CH.sub.2).sub.5-- and
--(CH.sub.2).sub.6--), 1.55-1.57 (2H, m, b-H2), 1.95-2.01 (6H, m,
--CH.sub.2CH.dbd.CHCH.sub.2-- and 2-H.sub.2), 3.41-3.43 (2H, t, J 6
Hz, --H.sub.2), 3.57 (2H, t, J 6.5 Hz, 3-H.sub.2), 3.77 and 3.80
(each 3H, s, 2.times.OMe), 4.20 (2H, m, 1-H.sub.2) and 5.33-5.38
(2H, m, CH.sub.2CH--CHCH.sub.2--); ESI-MS (m/z, +ve): 435
(MH.sup.+, 100%).
[0313] 3-O-Oleyloxypropyl-1-phosphate (Compound 16) To a solution
of dimethyl phosphate Compound 15 (0.26 g, 0.59 mmol) in dry
CH.sub.2Cl.sub.2 (8 mL) was added BSA (0.30 mL, 1.2 mmol) followed
by the addition of TMSBr (0.24 mL, 1.8 mmol). After 15 min, TLC
analysis showed complete consumption of the starting material and
the reaction was quenched with 1/1 MeOH/H.sub.2O (2 mL, v/v) for 15
min, followed by the addition of sat. KHSO.sub.4 (5 mL). The
reaction mixture was subsequently extracted with ethyl acetate
(2.times.30 mL), the organic extracts were combined, dried
(MgSO.sub.4) and concentrated to give the title phosphate (Compound
16) as an oil.
[0314] .sup.1H NMR (360 MHz, CDCl.sub.3): (0.88 (3H, t, J 7 Hz,
Me), 1.27 (22H, apparent br d, separation 6 Hz,
--(CH.sub.2).sub.5-- and --(CH.sub.2).sub.6--), 1.56 (2H, br s,
(--H.sub.2), 1.95-2.01 (6H, m, 2-H.sub.2 and
--CH.sub.2CH.dbd.CHCH.sub.2--), 3.43 (2H, t, J 6 Hz, (--H.sub.2),
3.57 (2H, br s, 3-H.sub.2), 4.12 (2H, br s, 1-H.sub.2), 5.33-5.38
(2H, m, --CH.sub.2CH.dbd.CHCH.sub.2--) and 8.06 (2H, br s,
2.times.OH); .sup.31P NMR (146 MHz; CDCl.sub.3): (1.95; ESI-MS
(m/z, -ve): 405 (M-H.sup.+, 100%).
[0315] 3-Hydroxypropyl decyl ether (Compound 17) To a mixture of
NaH (2.6 g, 70 mmol) and anhydrous NaI (9.9 g, 70 mmol) in dry DMF
(80 mL) under N.sub.2 was added dropwise a solution of
1,3-propanediol (4.75 mL, 70 mmol) in DMF (20 mL). The mixture was
stirred until hydrogen evolution had ceased, decyl chloride (1.34
g, 7.6 mmol) was added and stirring was continued at 50.degree. C.
for 18 hours. Then, the reaction mixture was poured into H.sub.2O
(300 mL) and extracted with diethyl ether (3.times.250 mL). The
organic extracts were combined, washed with sat. NaCl (100 mL),
dried (MgSO.sub.4) and evaporated to dryness. The residue was
subjected to silica-gel column chromatography [eluent: ethyl
acetate/hexane, 30/70, v/v] to furnish decyl ether Compound 17 as a
colourless oil, .sup.1H NMR (360 MHz; CDCl.sub.3): (0.87 (3H, t, J
7 Hz, Me), 1.25 (14H, br s, 7.times.CH.sub.2), 1.55 (2H, quintet, J
7 Hz (--H.sub.2) 1.82 (2H, quintet, J 5.5 Hz, 2-H.sub.2), 2.62 (1H,
br s, OH), 3.41 (2H, t, J 7.5 Hz, (--H.sub.2), 3.60 (2H, t, J 5.5
Hz, 1H.sub.2), and 3.77 (2H, t, J 5.5 Hz, 3-H.sub.2); ESI-MS (m/z,
+ve): 217 (MH.sup.+, 100%).
[0316] 3-Decyloxypropyl-1-phosphate (Compound 19) To a solution of
alcohol Compound 17 (0.5 g, 2.3 mmol) in dry CH.sub.2Cl.sub.2 (20
mL) was added TEA (0.48 ml, 3.5 mmol) followed by the addition of
trimethyl phosphite (0.41 mL, 3.5 mmol). After 90, the reaction was
cooled to -40.degree. C., pyridinium tribromide (0.89 g, 2.8 mmol)
was added and the reaction was allowed to warm to 20.degree. C.
overnight. The mixture was subsequently quenched with sat.
KHSO.sub.4 (30 mL) and extracted with ethyl acetate (2.times.50
mL). The organic extracts were combined, washed with sat. NaCl (30
mL), dried (MgSO.sub.4) and concentrated to give phosphate triester
Compound 18.
[0317] .sup.1H NMR (360 MHz; CDCl.sub.3): 0.87 (3H, t, J 6.5 Hz,
Me), 1.25 (14H, br s, 7.times.CH.sub.2), 1.50-1.56 (2H, m,
(--H.sub.2), 1.93 (2H, quintet, J 6 Hz 2-H.sub.2), 3.39 (2H, t, J
6.5 Hz, (--H.sub.2 or 3-H.sub.2), 3.49 (2H, t, J 6 Hz, (--H.sub.2
or 3-H.sub.2), 3.74 and 3.77 (each 3H, s, 2.times.OMe) and 4.14 (2
quartet, J 6.5 Hz, 1-H.sub.2).
[0318] To a stirring solution of phosphate triester Compound 18
(0.25 g, 0.72 mmol) in dry CH.sub.2Cl.sub.2 (5 mL) was added BSA
(0.25 mL, 1.0 mmol) followed by the addition of TMSBr (0.3 mL, 2.3
mmol). After 15 min, TLC analysis showed complete consumption of
the starting material and the reaction was quenched with 1/1
MeOH/H.sub.2O (2 mL, v/v) for 15 min, followed by the addition of
sat. KHSO.sub.4 (5 mL). The reaction mixture was subsequently
extracted with ethyl acetate (2.times.30 mL), and the combined
organic extracts were dried (MgSO.sub.4) and concentrated to give
the title phosphate (Compound 19) as an oil.
[0319] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.88 (3H, t, J 7 Hz,
Me), 1.27 (14H, br s, 7.times.CH.sub.2), 1.55-1.58 (2H m,
(--CH.sub.2), 1.95 (2H, br s, 2-H.sub.2), 3.42-3.48 (4H m,
(--H.sub.2 and 3-H.sub.2), 4.10 (2H, br s, 1-H.sub.2) and 6.35 (2H,
br s, 2.times.OH); ESI-MS (m/z, -ve): 295 (M-H.sup.+, 100%).
[0320] Dimethyl 1-O-oleyl-2-O-methyl-rac-glycero-1-phosphate
(Compound 23) To a solution of tosyl chloride (19.1 g, 0.10 mol) in
dry CH.sub.2Cl.sub.2 (200 mL) at 0.degree. C. was added TEA (15.3
mL, 0.10 mol) and oleyl alcohol (37.2 mL, 0.10 mol). The reaction
mixture was allowed to warm to 20.degree. C. and was subsequently
stirred for 3 days. Next, the solvent was removed in vacuo and the
resulting residue was redissolved in ethyl acetate (400 mL), washed
with sat. NaHCO.sub.3 (40 mL), dried (MgSO.sub.4), concentrated and
subjected to silica-gel column chromatography [eluent:
hexane/CH.sub.2Cl.sub.2, 66:33, v/v] to give oleyl tosylate
(Compound 13) as an oil.
[0321] To a mixture of NaH (4.8 g, 120 mmol) and NaI (0.2 g in dry
DW (20 mL) was added solketal (5.0 mmol) 40 mmol) dropwise over a
30 min period. Once the effervescence had stopped, oleyl tosylate
(Compound 13) (16.9 g, 40 mmol) was added and the reaction mixture
was left for 3 days at 50.degree. C. Then, H.sub.2O (10 mL) was
added and the mixture was extracted with diethyl ether (2.times.100
mL), the combined organic phases were dried (MgSO.sub.4) and
evaporated to dryness. The residue was taken up in THF (150 mL), 2M
HCl was added until the solution turned turbid and the reaction was
left to stir for 16 hours. The mixture was concentrated and
subjected to silica-gel column chromatography [eluent: ethyl
acetate/hexane, 50/50, v/v] to give 1-O-oleyl-rac-glycerol
(Compound 20) as an oil.
[0322] Diol Compound 20 (2.0 g, 5.97 mmol) was added dropwise to a
solution of trityl chloride (1.66 g, 6.0 mmol) in anhydrous
pyridine (10 mL). The reaction was left to stir for 16 hours after
which the solvent was removed by co-evaporation with toluene
(2.times.5 mL). Then, part of the residue (1.0 g, 1.7 mmol) was
added dropwise to a suspension of NaH (0.14 g, 3.4 mmol) in THF (50
mL), followed by the addition of MeI (0.22 mL, 3.4 mmol). After 16
hours, TLC analysis showed that the reaction had gone to completion
and H.sub.2O (5 mL) was added followed by concentration of the
reaction mixture. The residue was partitioned between H.sub.2O (20
mL) and diethyl ether (100 .mu.mL), the organic extract was washed
with sat. NaHCO.sub.3 (10 ml), dried (MgSO.sub.4) and concentrated
to give 1-oleoyl-2-methyl-3-O-trityl glycerol (Compound 21) as an
oil. Crude Compound 21 (1.16 g) was dissolved in CH.sub.2Cl.sub.2
(10 mL) after which trifluoroacetic acid (2 mL) was added. The
reaction mixture was stirred for 5 min and excess TFA was quenched
by adding solid NaHCO.sub.3 until effervescence ceased. The mixture
was diluted with H.sub.2O (10 mL), extracted with CH.sub.2Cl.sub.2
(2.times.50 mL) and the combined organic phases were dried
MgSO.sub.4), concentrated and subjected to silica-gel column
chromatography [eluent: hexane/ethyl acetate, 80/20, v/v] to give
glycerol derivative Compound 22 (0.28 g, 42%) as an oil.
[0323] .sup.1H NMR (360 M CDCl.sub.3): (0.88 (3H, t J 7 Hz, Me),
1.27 (22H, apparent br d, separation 9.5 Hz, --(CH.sub.2).sub.5--
and --(CH.sub.2).sub.6--), 1.53-1.60 (2H, m, (--H.sub.2), 1.98-2.01
(4H, m, --CH.sub.2CH.dbd.CHCH.sub.2--), 3.37-3.46 (3H, m, 2-H and
(--H.sub.2), 3.47 (3H, s, OMe), 3.52 (1H, dd, J 10 and 5 Hz, 1-H),
3.56 (1H, dd, J 10 and 5 Hz, 1-H), 3.65 (1H, dd, J 11.5 and 5.5 Hz,
3-H, 3.76 (1H, dd, J 11.5 and 4 Hz, 3-H and 5.30-5.39 (2H m,
--CH.sub.2CH.dbd.CHCH.sub.2--).
[0324] To a solution of compound Compound 22 (0.26 g, 0.76 mmol) in
dry CH.sub.2Cl.sub.2 (8 mL) was added N-methylimidazole (67 (1,
0.84 mmol) followed by the addition of dimethyl chlorophosphate
(0.12 mL, 1.22 mmol). After 1 hours, TLC analysis showed the
reaction to be complete, sat. KHSO.sub.4 (20 mL) was added and the
mixture was extracted with ethyl acetate (3.times.50 mL). The
organic phases were combined and washed with sat. NaCl (40 mL),
dried (MgSO.sub.4), concentrated and subjected to silica-gel column
chromatography [eluent: ethyl acetate/hexanes, 50/50, v/v] to give
the title dimethyl phosphate (Compound 23) as an oil.
[0325] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.88 (3H, t J 7 Hz, Me),
1.27 (22H, apparent br d, separation 12 Hz, --(CH.sub.2).sub.5--
and --(CH.sub.2).sub.6--), 1.54-1.59 (2H, m, (--H.sub.2), 1.98-2.01
(4H, m, --CH.sub.2CH.dbd.CHCH.sub.2--), 3.41-3.56 (5H, m, 2-H,
3-H.sub.2 and (--H.sub.2), 3.47 (3H, s, OMe), 3.77 (3H, d, J 2 Hz,
OMe), 3.80 (3H, d, J 2 Hz, OMe), 4.08 (1H, ddd, J 11, 7.0 and 5.5
Hz, 1-H), 4.19 (1H, ddd, J 11, 7.0 and 4 Hz, 1-H) and 5.30-5.39
(2H, m, --CH.sub.2CH.dbd.CHCH.sub.2--); .sup.31P NMR (146 MHz;
CDCl.sub.3): (2.60; ESI-MS (m/z, +ve): 465 (MH.sup.+, 100%).
[0326] 3-O-Oleyl-2-O-methyl-rac-glycerol-phosphate (Compound 24) To
a stirring solution of protected phosphate Compound 23 (85 mg, 0.18
mmol) in dry CH.sub.2Cl.sub.2 (5 mL) was added BSA (140 (L, 0.55
mmol) and TMSBr (50 (L, 0.05 mmol). After 30 min, TLC analysis
showed the reaction to be complete, 1/1 MeOH/H.sub.2O (1 mL, v/v)
was added and left for 30 min. Then, sat. KHSO.sub.4 (5 mL) was
added and the mixture was extracted with ethyl (2.times.30 mL), the
combined organic extracts were dried (MgSO.sub.4), evaporated to
dryness and subjected to Sephadex LH-20 column chromatography
[eluent: MeOH/CH.sub.2Cl.sub.2, 50/50, v/v] to give phosphate
Compound 24 as an oil.
[0327] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.87 (3H, t J 7 Hz, Me),
1.25 (22H, apparent br d, separation 4 Hz, --(CH.sub.2).sub.5-- and
--(CH.sub.2).sub.6--), 1.55-1.56 (2H, m (--H.sub.2), 1.98-2.03 (4H,
m, --CH.sub.2CH.dbd.CHCH.sub.2--), 3.40-3.58 (5H, m, 2-H, 3-H.sub.2
and (--H.sub.2), 3.46 (3H, s, OMe), 3.99-4.05 (2H, m, 1-H.sub.2),
5.30-5.37 (2H, --CH.sub.2CH.dbd.CHCH.sub.2--) and 5.75 (2H, br s,
2.times.OH); .sup.31P NMR (146 MHz; CDCl.sub.3): (1.68; ESI-MS
(m/z, -ve): 435 (M-H.sup.+, 100%).
[0328] Dimethyl 3-oleyl-rac-glycero-1-thiophosphate (Compound 25)
To a solution of 1-O-Oleyl-glycerol (Compound 20) (1.5 g, 4.4 mmol)
and dimethyl chlorothiophosphate (0.80 mL, 6.57 mmol), dried by
azeotropic removal of H.sub.2O with CH.sub.3CN (2.times.15 mL), in
CH.sub.2Cl.sub.2 (20 mL) under N.sub.2 was added N-methylimidazole
(0.70 mL, 8.8 mmol). After 3 days, the mixture was concentrated and
subjected to silica-gel column chromatography [eluent: hexane:ethyl
acetate, 90/10, v/v] to give protected thiophosphate Compound 25
(0.17 g, 25%) as an oil.
[0329] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.87 (3H, t, J 6 Hz,
Me), 1.27 (22H, apparent br d, separation 12 Hz;
--(CH.sub.2).sub.5-- and --(CH.sub.2).sub.6--), 1.54-1.58 (2H, m,
(--H.sub.2), 1.98-2.04 (4H, m, --CH.sub.2CH.dbd.CHCH.sub.2--),
3.44-3.53 (2.times.H, m, 3-H.sub.2), 3.75 and 3.79 (each 3H, s,
2.times.OMe), 3.97-4.03 (1H, m, 2-H), 4.04-4.18 (2H, m 1-H.sub.2)
and 5.30-5.35 (2H, m, --CH.sub.2CH--CHCH.sub.2--); ESI-MS (m/z,
+ve): 467 (MH.sup.+, 52%), 489 (MNa.sup.+, 100) and 505 (MK.sup.+,
20%).
[0330] 3-Hydroxypropyl oleate (Compound 28) To a solution of
1,3-propanediol (3.61 mL, 50.0 mmol) in pyridine (100 mL) was added
chlorotriphenylmethane (14 g, 50.0 mmol) and the mixture was
stirred at 70.degree. C. for 16 hours. The solvent was removed by
co-evaporation with dry toluene (2.times.50 mL), and the resulting
residue was redissolved in diethyl ether (150 mL), washed with
H.sub.2O (2.times.50 mL), dried (MgSO.sub.4), concentrated and
purified by silica-gel column chromatography [eluent: hexane/ethyl
acetate, 90/10, v/v] to give monotritylated diol Compound 26.
[0331] .sup.1H NMR (360 MHz; CDCl.sub.3): (1.82 (2H, quintet, J 6
Hz, 2-H.sub.2), 3.23 (2H, t, J 6 Hz, 1-H.sub.2 or 3-H.sub.2), 3.72
(2H, t, J 5.5 Hz, 1-H.sub.2 or 3-H.sub.2), 7.18-7.28 (10H, m,
2.times.Ph) and 7.34-7.40 (5H, m, Ph).
[0332] To a solution of Compound 26 (3.2 g, 10 mmol) and DMAP (50
mg) in pyridine (10 mL) was added oleoyl chloride (3.3 g, 10 mmol).
After 4 hours, pyridine was removed by co-evaporation with dry
toluene (2.times.50 ml) and the obtained residue was partitioned
between diethyl ether (150 mL) and H.sub.2O (50 mL). The diethyl
ether layer was subsequently washed with sat. NaCl (50 mL), dried
(MgSO.sub.4) and evaporated to dryness. The residue was purified by
silica-gel column chromatography [eluent: hexane/diethyl ether,
90/10, v/v] to give trityl ester 27.
[0333] Treatment of a solution of Compound 27 (3.72 g, 6.39 mmol)
in dry CH.sub.2Cl.sub.2 (100 mL) with trifluoroacetic acid (5 mL)
resulted in a bright yellow solution. Subsequently, the mixture was
quenched by the addition of H.sub.2O (10 mL) and solid NaHCO.sub.3
(5 g). The product was extracted into CH.sub.2Cl.sub.2 (2.times.100
mL), the organic phases were combined, dried (MgSO.sub.4) and
concentrated. Purification of the residue by silica-gel column
chromatography [eluent: ethyl acetate/hexanes, 30/70, v/v]
furnished the title alcohol (Compound 28) (2.1 g, 95%) as an
oil.
[0334] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.87 (3H, t, J 7 Hz,
Me), 1.28 (20H, apparent br d, separation 13.5 Hz,
--(CH.sub.2).sub.4-- and CH.sub.2).sub.6--), 1.60-1.63 (2H, m, in
(--H.sub.2) 1.86 (2H, quintet, J 6 Hz 2-H.sub.2), 1.98-2.01 (4H, m,
--CH.sub.2CH.dbd.CHCH.sub.2--), 2.31 (2H, m, t, J 7.5 Hz,
--H.sub.2), 3.68 (2H, t, J 6 Hz, 3-H.sub.2, 4.24 (2H, J 6 Hz,
1-H.sub.12, and 5.29-5.39 (2H, m,
--CH.sub.2CH.dbd.CHCH.sub.2--).
[0335] Dimethyl 1-(3-propyl oleoate)phosphate (Compound 29) To a
solution of alcohol Compound 28 (0.52 g, 1.5 mmol) and
N-methylimidazole (0.27 mL, 3.3 mmol) in dry CH.sub.2Cl.sub.2 (5
mL) was added dimethyl chlorophosphate (0.2 mL, 1.8 mmol). After 1
hours, TLC analysis showed the reason to be complete, sat.
KHSO.sub.4 (20 mL) was added and the mixture was extracted with
ethyl acetate (3.times.50 mL), the combined organic phases were
washed with sat. NaCl (40 mL), dried (MSgSO.sub.4) and
concentrated. The residue was subjected to silica-gel column
chromatography [eluent: ethyl acetate/hexanes, 50/50, v/v] to give
protected phosphate Compound 29 as an oil.
[0336] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.87 (3H, t, J 7 Hz,
Me), 1.28 (20H, apparent br d, separation 13 Hz,
--(CH.sub.2).sub.5-- and --CH.sub.2).sub.6--), 1.59-1.64 (2H, m,
(--H.sub.2), 1.98-2.04 (6H, m, 2-H.sub.2 and
--CH.sub.2CH.dbd.CHCH.sub.2--), 2.30 (2H, t, J 7.5 Hz, (--H.sub.2),
3.75 and 3.78 (each 3H, s, 2.times.OMe), 4.11-4.20 (4H, m,
1-H.sub.2 and 3-H.sub.2), 5.29-5.39 (2H, m,
--CH.sub.2CH.dbd.CHCH.sub.2--); .sup.31P NMR (146 MHz; CDCl.sub.3):
(2.54; ESI-MS (m/z, +ve): 449 MH.sup.+, 100%) and 466 (M+18.sup.+,
89).
[0337] 1-(3-propyl oleoate)phosphate (Compound 30) To a stirring
solution of dimethyl phosphate 29 (0.31 g, 0.7 mmol) in dry
CH.sub.2Cl.sub.2 (4 mL) was added BSA (0.52 mL, 2.1 mmol) followed
by the addition of TMSBr (0.19 mL, 1.4 mmol). After 30 ruin, TLC
analysis showed complete consumption of the starting material, the
reaction was quenched with 1/1 MeOH/H.sub.2O (1 mL, 1/1) for 30
min, followed by the addition of sat. KHSO.sub.4 (5 mL). The
mixture was extracted with ethyl acetate (2.times.30 mL), the
organic extracts were combined, dried (MgSO.sub.4) and evaporated
to dryness. Purification of the residue by Sephadex LH-20 column
chromatography [eluent: MeOH/CH.sub.2Cl.sub.2, 50/50, v/v] gave the
title phosphate (Compound 30) as an oil.
[0338] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.87 (3H, t, J 7 Hz,
Me), 1.28 (20H, apparent br d, separation 11 Hz,
--(CH.sub.2).sub.4-- and --(CH.sub.2).sub.6--), 1.57-1.61 (2H, m,
(--H.sub.2), 2.00 (6H, apparent br d, separation 3.5 Hz,
--CH.sub.2CH.dbd.CHCH.sub.2-- and 2-H.sub.2), 2.30 (2H, t, J 7.5
Hz, 4 (--H.sub.2), 4.11 (2H, quartet, J 6 Hz, 3-H.sub.2), 4.19 (2H,
t, J 6.5 Hz, 1-H.sub.2), 5.29-5.38 (2H, m,
CH.sub.2CH--CHCH.sub.2--) and 9.24 (2H, br s, 2.times.OH); .sup.31P
NMR (146 MHz; CDCl.sub.3): (1.80; ESI-MS (m/z, -ve): 419
(M-H.sup.+, 100%).
[0339] Dimethyl 3-oleoyl-2-deoxy-2-bromo-rac-glycero-1-phosphate
(Compound 36) To a stirring solution of
1-O-benzyl-2-deoxy-2-bromo-rac-glycerol (0.5 g, 2.0 mmol), dimethyl
chorophosphate (270 (L, 2.5 mmol) in dry CH.sub.2Cl.sub.2 (20 mL)
under N.sub.2 was added N-methylimidazole (180 (L, 2.2 mmol). After
3 days, the mixture was concentrated and purified by silica-gel
column chromatography [eluent: ethyl acetate/hexane, 50/50, v/v] to
yield dimethyl 3-O-benzyl-2-deoxy-2-bromo-rac-glycero-1-phosphate
(Compound 34) (0.43 g, 59%) as an oil.
[0340] .sup.1H NMR (360 MHz; CDCl.sub.3): (3.73-3.81 (2H, m,
3-H.sub.2), 3.75 (3H, apparent d, separation 13.5 Hz, OMe), 3.78
(3H, apparent d, separation 14 Hz, OMe), 4.21 (1H, quintet, J 5.5
Hz, 2-H), 4.28-4.40 (2H, m, 1-H.sub.2), 4.57 (2H, br s,
--OCH.sub.2Ph) and 7.26-7.37 (5H, m, -Ph); .sup.31P NMR (146 MHz;
CDCl.sub.3): (1.88; ESI-MS (m/z, +ve): 353 and 355 (MH.sup.+,
100%).
[0341] A mixture of bromo Compound 34 (0.43 g, 1.2 mmol) and 10%
palladium on activated carbon (0.43 g) in ethyl acetate (50 mL) was
evacuated using an aspirator pump and filled with hydrogen. After 2
hours, TLC analysis showed the reaction to be complete and the
mixture was filtered through Celite. The filtrate was evaporated to
dryness to furnish dimethyl 2-deoxy-2-bromo-rac-glycero-1-phosphate
(Compound 35) (0.28 g, 88%) as a colourless oil.
[0342] .sup.1H NMR (360 MHz; CDCl.sub.3): (2.35 (1H, br s, OH),
3.80 (3H, d, J 7 Hz, OMe), 3.83 (3H, d, J 6.5 Hz, OMe), 3.89 (1H,
dd, J 12.5 and 6 Hz, 3-H), 3.94 (1H, dd, J 12.5 and 5 Hz, 3-H),
4.13-4.19 (1H, m, 2-H), 4.31 (1H, ddd, J 11.5, 9.5 and 6 Hz, 1-H)
and 4.46 (1H, ddd, J 11.5, 9 and 4.5 Hz, 1-H).
[0343] To a solution of alcohol Compound 35 (0.28 g, 1.1 mmol) in
dry CH.sub.2Cl.sub.2 (5 mL) under N.sub.2 was added oleoyl chloride
(0.39 mL, 1.2 mmol) followed by the addition of pyridine (0.09 mL,
1.2 mmol). After 1 hour, the mixture was concentrated and subjected
to silica-gel column chromatography [eluent: ethyl acetate (20-50%)
in hexane, v/v] to give phosphate Compound 36 (0.48 g, 85%) as an
oil. .sup.1H NMR (360 MHz; CDCl.sub.3): (0.86 (3H, t, J 7 Hz, Me),
1.25 (20H, apparent doublet, separation 14 Hz, --(CH.sub.2).sub.4--
and <(CH.sub.2).sub.6--), 1.58-1.62 (2H, m, (--H.sub.2),
1.97-1.98 (4H, m, --CH.sub.2CH.dbd.CHCH.sub.2--), 2.33 (2H, t, J
7.5 Hz (--H.sub.2), 3.76 (3H, d, J 1.5 Hz, OMe), 3.79 (3H, d, J 2
Hz, OMe), 4.20-4.31 (3H, m, 2-H and 3-H.sub.2), 4.36 (1H, dd, J 12
and 5 Hz, 1-H), 4.40 (1H, dd, J 12 and 5.5 Hz, 1-H) and 5.26-5.36
(2H, m, --CH.sub.2CH.dbd.CHCH.sub.2--); .sup.31P NMR (146 MHz;
CDCl.sub.3): (1.82; ESI-MS (m/z, +ve): 527 and 529 (MH.sup.+, 29%),
549 and 551 (MH.sup.+, 49) and 565 and 567 (MH.sup.+, 100).
[0344] 3-O-Oleoyl-2-deoxy-2-bromo-rac-glycero-1-phosphate (Compound
37) To a solution of bromo alcohol Compound 36 (0.20 g, 0.38 mmol)
in dry CH.sub.2Cl.sub.2 (20 mL) under N.sub.2 was added BSA (0.21
ml, 0.83 mmol) followed by the addition of TMSBr (0.11 mL, 0.83
mmol). The reaction was carefully monitored by TLC and after 85 min
all the start material had been consumed. Then, 1/1 MeOH/H.sub.2O
(3 mL, v/v) was added and the mixture was stirred for a subsequent
15 min after which sat. KHSO.sub.4 (5 mL) was added. The mixture
was subsequently extracted with ethyl acetate (3.times.10 mL), the
organic extracts were combined, dried (MgSO.sub.4), concentrated
and subjected to Sephadex LH-20 column chromatography [eluent:
MeOH/CH.sub.2Cl.sub.2, 50/50, v/v] to give the title phosphate
(Compound 37) (0.19 g, 99%) as a yellow oil.
[0345] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.87 (3H, t, J 7 Hz,
Me), 1.28 (20H, apparent doublet, separation 13.5 Hz,
--CH.sub.2).sub.4-- and --(CH.sub.2).sub.6--), 1.61-1.62 (2H, m,
(--H.sub.2), 1.99-2.01 (4H, m, --CH.sub.2CH.dbd.CHCH.sub.2--), 2.36
(2H, t, J 7 Hz, (--HA, 4.19-4.28 (3H, m, 2-H and 3-H.sub.2), 4.37
(1H, dd, J 12 and 5 Hz, 1-H), 4.47 (1H, dd, J 12 and 5 Hz, 1-H),
5.29-5.38 (2H, m, --CH.sub.2CH.dbd.CHCH.sub.2--) And 9.05 (2H, S,
2.times.OH); .sup.31P NMR (146 MHz; CDCl.sub.3): (1.08; ESI-MS
(m/z, -ve): 499 and 501 (M-H.sup.+, 100%).
[0346] 1-O-Oleoyl-2-O-methyl-rac-glycerol (Compound 44) To a
mixture of 1-O-benzyl-glycerol (27 mL, 0.16 mol) and
t-butyldimethylsilyl chloride (25 g, 0.17 mol) in dry
CH.sub.2Cl.sub.2 (250 mL) under N.sub.2 was added DMAP (0.8 g, 6.6
mmol) followed by the addition of TEA (23 mL, 0.17 mol). After 3
hours, the mixture was washed with H.sub.2O (2.times.100 mL), dried
(MgSO.sub.4) and concentrated to give
1-O-benzyl-3-O-t-butyldimethylsilyl-rac-glycerol (40) (48.8 g,
100%) as a yellow oil. .sup.1H NMR (360 MHz; CDCl.sub.3): (0.06
(6H, S, Me.sub.2Si), 0.89 (9H, S, Bu.sup.tSi), 3.48-3.56 (2H, m,
1-H.sub.2 or 3-H.sub.2), 3.64 (1H, dd, J 10 and 5.5 Hz, 1-H or 3-H,
3.68 (1H, dd, J 10 and 5 Hz, 1-H or 3-H), 3.86 (1H, quintet, J 5.5
Hz, 2-H), 4.56 (2H, s, --OCH.sub.2Ph) and 7.27-7.35 (5H, m,
-Ph).
[0347] Alcohol Compound 40 (48.8 g, 0.16 mmol) was added to a
mixture of NaH (4.0 g, 0.17 mol) in dry THF (500 mL). After 5 min,
MeI (10 mL, 0.17 mol) was added and, after another 3 hours, the
mixture was quenched with H.sub.2O (150 mL), extracted into diethyl
ether (500 mL), dried (MgSO.sub.4) and concentrated. Purification
of the residue by silica-gel column chromatography [eluent:
hexane/ethyl acetate, 90/10, v/v] yielded
1-O-benzyl-3-O-t-butyldimethylsilyl-2-O-methyl-rac-glycerol
(Compound 41) (37 g, 74%) as a yellow oil.
[0348] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.05 and 0.05 (each 3H,
s, Me.sub.2Si), 0.88 (9H, s, Bu.sup.tSi), 3.42 (1H apparent
quintet, separation 4.5 Hz, 2-H), 3.47 (3H, s, OMe), 3.51 (1H, dd,
J 10 and 5.5 Hz, 1-H or 3-H), 3.61 (1H dd, J 10 and 4 Hz, 1-Hz, 1-H
or 3-H), 3.68 (2H, d, J 5.5 Hz, 1-H or 3-H), 4.54 (2H apparent d,
separation 12 Hz, --OCHHAr), 4.58 (2H apparent d, separation 12 Hz,
--OCHHAr), 7.28 (I H, m, p-H) and 7.34 (4H, apparent d, separation
4.5 Hz, o-H.sub.2 and m-H.sub.2); ESI-MS (m/z, +ve): 333
(MNa.sup.+, 100%).
[0349] A mixture of the protected glycerol derivative Compound 41
(5.0 g, 16 mmol) and 10% palladium on activated carbon (wet Degussa
type E101 NE/W) (0.43 g) in MeOH (50 mL) was evacuated using an
aspirator pump and filled with hydrogen. After 2 hours, TLC
analysis showed the reaction to be complete, the mixture was
filtered through Celite and the filtrate was evaporated to dryness
to give 1-O-t-butyldimethylsilyl-2-O-methyl-rac-glycerol (Compound
42) (99%) as a colourless oil.
[0350] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.07 (6H, s,
Me.sub.2Si), 0.89 (9H, s, Bu.sup.tSi), 3.30-3.36 (I H, m, 2-H),
3.46 (3H s, OMe), 3.63 (1H dd, J 11.5 and 5.5 Hz, 1- or 3-H), 3.65
(1H dd, J 10.5 and 6.5 Hz, 1- or 3-H), 3.74 (1H, dd, J 10.5 and 5
Hz, 1- or 3-H and 3.75 (1H, dd, J 11.5 and 4H, 1- or 3-H).
[0351] To a solution of alcohol Compound 42 (1.1 g, 5.0 mmol) in
CH.sub.2Cl.sub.2 (10 mL) was added oleoyl chloride (1.8 mL, 5.5
mmol) followed, after 5 min, by the addition of pyridine (0.44 mL,
5.5 mmol). After TLC analysis indicated that the reaction had gone
to completion, the mixture was partitioned between ethyl acetate
(100 mL) and H.sub.2O (30 mL), the organic extract was dried
MgSO.sub.4) and concentrate to give
1-O-t-butyldimethylsilyl-2-O-methyl-3-O-oleoyl-rac-glycerol
(Compound 43) (2.4 g, 99%) as a dark brown oil.
[0352] .sup.1H NMR (360; CDCl.sub.3): (0.06 (6H, s, Me.sub.2Si),
0.88 (3H t, J 7 Hz, Me), 0.89 (9H, s, Bu.sup.tSi), 1.28 (20H,
apparent br d, separation 11.5 Hz, --(CH.sub.2).sub.4-- and
--(CH.sub.2).sub.6--), 1.60-1.64 (2H, m, (--H.sub.2), 1.98-2.03
(4H, m, --CH.sub.2CH--CHCH.sub.2--), 2.33 (2H, t, J 7.5 Hz,
(--H.sub.2), 3.41-3.46 (1H, br t, 2-H), 3.45 (3H, s, OMe), 3.64
(1H, dd, J 10.5 and 6 Hz, 3-H), 3.68 (1H, dd, J 10.5 and 5.5 Hz,
3-H), 4.09 (1H dd, J 11.5 and 5.5 Hz, 1-H), 4.27 (1H, dd, J 11.5
and 4 Hz, 1-H) and 5.32-5.36 (2H, m,
CH.sub.2CH.dbd.CHCH.sub.2--).
[0353] 1M TBAF (6 mL, 6.0 mmol) was added to a solution of compound
Compound 43 (2.4 g, 5.0 mmol) in THF (50 mL). After 30 min, TLC
analysis showed that all the starting material had been consumed.
Then the reaction mixture was partitioned between diethyl ether
(100 mL) and sat. NaCl solution (30 mL). The organic extract was
dried (MgSO.sub.4), concentrated and subjected to silica-gel column
chromatography [eluent: hexane/ethyl acetate, 75/25, v/v] to give
glycerol derivative Compound 44 (1.52 g, 79%) as an oil
[0354] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.88 (3H, J 7 Hz, Me),
1.28 (20H, apparent br d, separation 13 Hz, --(CH.sub.2).sub.4--
and --(CH.sub.2).sub.6--), 1.60-1.64 (2H, m, (--H.sub.2), 1.98-2.03
(4H, m, --CH.sub.2CH.dbd.CHCH.sub.2--), 2.33 (2H, t, J 7.5
(--H.sub.2), 3.45-3.51 (1H, br t, 2-H), 3.47 (3H, s, OMe), 3.61
(1H, dd, J 11.5 and 6 Hz, 3-H), 3.69 (1H, dd, J 11.5 and 4.5 Hz
3-H), 4.20 (2H, apparent d, separation 5 Hz, 1-H.sub.2) and
5.29-5.39 (2H, m, --CH.sub.2CH.dbd.CHCH.sub.2--).
[0355] Dimethyl 3-O-oleoyl-2-O-methyl-rac-glycerol-phosphate
(Compound 45) To a solution of alcohol Compound 44 (0.79 g, 2.2
mmol) in dry CH.sub.2Cl.sub.2 (8 mL) was added N-methylimidazole
(0.2 mL, 2.4 mmol) followed by the addition of dimethyl
chlorophosphate (0.26 mL, 2.4 mmol). After stirring for 1 hour, the
mixture was quenched with sat. KHSO.sub.4 (20 mL) and extracted
into ethyl acetate (3.times.50 mL). The combined organic phases
were subsequently washed with sat. NaCl (40 mL), dried (MgSO.sub.4,
concentrated and subjected to silica-gel column chromatography
[eluent: ethyl acetate/hexanes, 50/50, v/v] to give protected
phosphate Compound 45 as an oil.
[0356] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.87 (3H, t, J 7 Hz,
Me), 1.28 (20H, R apparent br d, separation 12.5 Hz,
--CH.sub.2).sub.4-- and --(CH.sub.2).sub.6--), 1.60-1.64 (2H, m,
(--H.sub.2), 1.98-2.01 (4H, m, --CH.sub.2CH.dbd.CHCH.sub.2--), 2.33
(2H, t, J 7.5H, (--H.sub.2), 3.47 (3H, s, OMe), 3.61 (1H, quintet,
J 5 Hz, 2-H), 3.76 (3H, d, J 2 Hz, OMe), 3.80 (3H, d, J 2 Hz, OMe),
4.05-4.19 (3H, m, 1-H.sub.2 or 3-H.sub.2 and 3-H or 1-H), 4.25 (1H,
dd, J 12 and 4.5 Hz, 1-H or 3-H) and 5.29-5.39 (2H, m,
--CH.sub.2CH.dbd.CHCH.sub.2--); .sup.31P NMR (146 MHz; CDCl.sub.3):
(2.52; ESI-MS (m/z, +ve): 479 (MH.sup.+, 100%) and 496 (M+18.sup.+,
83).
[0357] 3-O-Oleoyl-2-O-methyl-rac-glycero-1-phosphate (Compound 46)
To a solution of dimethyl phosphate Compound 45 (0.10 g, 0.22 mmol)
in dry CH.sub.2Cl.sub.2 (5 mL) was added BSA (0.16 ml, 0.65 mmol)
followed by the addition of TMSBr (60 (L, 1.4.times.mmol). After 30
min, the reaction was treated with 1/1 MeOH/H.sub.2O 1 mL, v/v) for
30 min after which sat. KHSO.sub.4 (5 mL) was added. The mixture
was subsequently extracted with ethyl acetate (2.times.30 mL, the
combined organic extracts were dried (MgSO.sub.4), concentrated and
subjected to Sephadex LH-20 column chromatography [eluent:
MeOH/CH.sub.2Cl.sub.2, 50/50, v/v] to give the title phosphate
(Compound 46) as an oil.
[0358] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.86-0.87 (3H, m, Me),
1.28 (20H, apparent br d, separation 12 Hz, --(CH.sub.2).sub.4--
and --(CH.sub.2).sub.6--), 1.61 (2H, br s, (--H.sub.2), 2.00 (4H,
apparent br d, separation 5 Hz, --CH.sub.2CH.dbd.CHCH.sub.2--),
2.34 (2H, t, J 7.5 Hz, (--H.sub.2), 3.48 (3H, s, OMe), 3.66 (1H, br
s, 2-H), 4.15 (3H, m, 1-H.sub.2 or 3-H.sub.2 and 3-H or 1-H), 4.27
(1H, m, 1-H or 3-H), 5.30-5.34 (2H, m,
--CH.sub.2CH.dbd.CHCH.sub.2--) and 8.41 (1H, br s, 2.times.OH);
.sup.31P NMR (146 MHz; CDCl.sub.3): (1.82; ESI-MS (m/z, -ve): 449
(M-H.sup.+, 100%) and 463 (Mna.sup.+, 37).
[0359] Bis-(2-cyanoethyl)
3-O-oleoyl-2-O-methyl-rac-glycero-1-thiophosphate (Compound 47)
2-Cyanoethyl N,N,N,N'-tetraisopropylphosphorodiamidite (0.53 mL,
1.7 mmol) was added under N.sub.2 to a solution of
3-hydroxypropionitrile (94 (1, 1.4 mmol) and 1H-tetrazole (0.12 g,
1.7 mmol) in dry CH.sub.2Cl.sub.2 (10 mL). After stirring for 1
hour, a subsequent portion of 1H-tetrazole (0.19 g, 2.8 mmol) was
added followed by the addition of alcohol Compound 44 (0.51 g, 1.4
mmol). After an additional 30 min, elemental sulfur (1 g) and 1/1
CS.sub.2/pyridine (1 mL, v/v) was added. After 2 hours, the
reaction mixture was filtered through a short silica-gel plug,
concentrated and subjected to silica-gel column chromatography
[eluent: ethyl acetate (20-100%) in hexane, v/v] to yield protected
thiophosphate Compound 47 (0.29 g, 37%) as a pale yellow oil.
[0360] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.88 (3H, t, J 7 Hz,
Me), 1.28 (20H, apparent br d, separation 13.5 Hz,
--(CH.sub.2).sub.4-- and --(CH.sub.2).sub.6--), 1.60-1.64 (2H, m,
(--H.sub.2), 1.98-2.01 (4H, m, --CH.sub.2CH.dbd.CHCH.sub.2--), 2.34
(2H, t, J 7.5 Hz, (--H.sub.2), 2.78 (4H, t, J 6 Hz,
2.times.NCCH.sub.2CH.sub.2--), 3.47 (3H, s, OMe), 4.11-4.48 (8H, m,
1-H.sub.2, 3-H.sub.2, and 2.times.NCCH.sub.2CH.sub.2--) and
5.29-5.39 (2H, m, --CH.sub.2CH.dbd.CHCH.sub.2--); .sup.31P NMR (146
MHz; CDCl.sub.3): (69.08; ESI-MS (m/z, +ve): 595 (MNa.sup.+,
100%).
[0361] 3-O-Oleoyl-2-O-methyl-rac-glycero-1-thiophosphate (Compound
48) To a solution of Compound 47 (100 mg, 0.18 mmol) in CH.sub.3CN
(1.5 mL) under N.sub.2 was added TEA (1.5 mL) followed by the
addition of BSA (0.11 mL, 0.44 mmol). After 24 hours, the reaction
mixture was concentrated and subjected to Sephadex LH-20 column
chromatography [eluent: MeOH/CH.sub.2Cl.sub.2, 50/50, v/v] to give
thiophosphate Compound 48 (75 mg, 92%) as a pale yellow oil.
[0362] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.87 (3H t, J 7 Hz, Me),
1.28 (20H apparent br d, separation 12.5 Hz, --(CH.sub.2).sub.6--),
1.60-1.61 (2H, m, (--H.sub.2), 2.00-2.01 (4H, m,
--CH.sub.2CH.dbd.CHCH.sub.2--), 2.35 (2H, t, J 7.5 Hz, (--H.sub.2),
3.51 (3H, s, OMe), 3.69 (1H, br t, 2-H), 4.13-4.31 (4H, m,
1-H.sub.2 and 3-H.sub.2), 5.18 (2H, br s, 2.times.OH) and 5.29-5.39
(2H, m, --CH.sub.2CH--CHCH.sub.2--); .sup.31P NMR (146 MHz;
CDCl.sub.3): (56.72 ESI-MS (m/z, -ve): 465 (M-H.sup.+, 100%);
ESI-MS (m/z, +ve): 467 (MH.sup.+, 100%).
[0363] 3-Hydroxypropyl oleoyl amide (Compound 51) To a solution of
3-amino-propan-1-ol (3.1 mL, 40 mmol) in dry THF (150 mL) was added
oleoyl chloride (4.4 mL, 13 mmol). After 16 hours, the reaction
mixture was diluted with ethyl acetate (100 mL) and washed with
sat. NH.sub.4Cl (2.times.100 mL). The organic extract was dried
(MgSO.sub.4), concentration and subjected to silica-gel column
chromatography [eluent: ethyl acetate (25-100%) in hexane, v/v] to
give amide Compound 51 (4.4 g, 95%) as a white solid.
[0364] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.88 (3H, t, J 7 Hz,
Me), 1.27 (20H, apparent br d, separation 12.5 Hz,
--(CH.sub.2).sub.4-- and --(CH.sub.2).sub.6--), 1.60-1.69 (4H, m,
2-H.sub.2 and (--H.sub.2), 1.99-2.00 (4H, m,
--CH.sub.2CH.dbd.CHCH.sub.2--), 2.19 (2H, t, J 7.5 Hz, (--H.sub.2),
3.41 (2H, quartet, J 6 Hz, 1-H.sub.2), 3.61 (2H, t, 5.5 Hz,
3-H.sub.2), 5.29-5.38 (2H, m, --CH.sub.2CH.dbd.CHCH.sub.2--) and
5.90 (1H, br s, N--H); ESI-MS (m/z, +ve): 340 (MH.sup.+, 100%).
[0365] Dibenzyl 3-amino-3-N-oleoyl-propyl phosphate (Compound 52)
To a mixture of Compound 51 (0.34 g, 1.0 mmol) and 1H-tetrazole
(0.14 g, 2.0 mmol) in dry CH.sub.2Cl.sub.2 (5 mL) under N.sub.2 was
added dibenzyl N,N-diisopropylphosphoramidite (1.1 mL, 3.0 mmol).
After 45 min t-BuOOH (2 mL) was added and the mixture was stirred
for a subsequent 35 min after which the mixture was concentrated
and subjected to silica-gel column chromatography [eluent: ethyl
acetate in hexane, 50/50, v/v] to give dibenzyl phosphate Compound
38 (0.27 g, 44%) as an oil.
[0366] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.87 (3H, t, J 7 Hz,
Me), 1.28 (20H, br s, --(CH.sub.2).sub.4-- and
--(CH.sub.2).sub.6--), 1.57 (2H, br s, (--H.sub.2) 1.77-1.82 (2H,
m, 2-H.sub.2), 1.98-2.00 (4H, m, --CH.sub.2CH--CHCH.sub.2--), 2.11
(2H, t, J 8 HZ, (--H.sub.2), 3.29 (2H, quartet, J 6 Hz, 1-H.sub.2),
4.10-4.15 (2H, t, J 5.5 Hz, 3-H.sub.2), 4.99-5.09 (4H, m,
--OCH.sub.2Ph), 5.28-5.38 (2H, m, H.sub.2CH.dbd.CHCH.sub.2--), 6.25
(1H, br s, N--H) and 7.35 (10H, br s, 2.times.-Ph); .sup.31P NMR
(146 MHz; CDCl.sub.3): (0.78 ESI-MS (m/z, +ve): 600 (MH.sup.+,
100%).
[0367] Dimethyl 3-amino-3N-oleoyl-propyl phosphate (Compound 53) To
a mixture of amide Compound 51 (0.30 g, 0.9 mmol) and dimethyl
chlorophosphate (0.14 mL, 1.3 mmol) in dry CH.sub.2Cl.sub.2 (5 mL)
under N.sub.2 was added N-methylimidazole (0.14 mL, 1.8 mmol).
After 40 hours, TLC analysis showed the reaction to be complete and
the mixture was partitioned between ethyl acetate (20 mL) and sat.
NaCl (4 mL). The organic phase was dried (MgSO.sub.4), concentrated
and subjected to silica-gel column chromatography [eluent: 5% MeOH
in CH.sub.2Cl.sub.2, v/v] to give dimethyl phosphate Compound 53
(0.35 g, 87%) as a pale yellow oil.
[0368] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.87 (3H, t, J 7 Hz,
Me), 1.28 (20H, apparent br d, separation 11.5 Hz,
--(CH.sub.2).sub.4-- and --(CH.sub.2--).sub.6--), 1.61-1.78 (2H, m,
(--H.sub.2, 1.88 (2H, quintet, J 6 Hz, 2-H.sub.2, 1.99-2.01 (4H, m,
--CH.sub.2CH.dbd.CHCH.sub.2--).sub.6--), 2.16 (2H, t, J 8 Hz,
(--H.sub.2), 3.38 (2H, quartet, J 6 Hz, 3-H.sub.2, 3.76 and 3.79
(each 3H, s, 2.times.OMe), 4.10-4.15 (2H, t, J 5.5 Hz, 1-H.sub.2),
5.33-5.35 (2H, m, --CH.sub.2CH CHCH.sub.2--) and 6.25 (1H, br s,
N--H); .sup.31P NMR (146 MHz; CDCl.sub.3): (3.19; ESI-MS (m/z,
+ve): 340 (Mna.sup.+, 32%) and (M+83, 100).
[0369] 1-O-Palmitoyl-rac-glycidol (Compound 57) To a solution of
glycidol (0.45 mL, 6.7 mmol) in dry CH.sub.2Cl.sub.2 (50 ml) under
N.sub.2 at -78.degree. C. was added palmitoyl chloride (2.0 mL, 7.3
mmol). After 5 min, pyridine (1.4 mL, 16.8 mmol) was added and the
reaction mixture was allowed to stir for 1 hour at -78.degree. C.
before being allowed to warm to room temperature over a 1.5 hours
period. Then, the mixture was diluted with CH.sub.2Cl.sub.2 (100
mL), washed with sat. KHSO.sub.4 (10 mL), sat. NaHCO.sub.3 (10 mL)
and sat. NaCl (20 mL), dried (MgSO.sub.4) and concentrated to give
an oil. The residue was subjected to silica-gel column
chromatography [eluent: ethyl acetate/hexane, 10/90, v/v] to give
epoxide Compound 57 (1.3 g, 63%) as a colourless oil.
[0370] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.88 (3H, t, J 7 Hz,
Me), 1.25 (24H, br s, 12.times.CH.sub.2), 1.63 (2H, quintet, J 7.5
Hz, (--H.sub.2), 2.35 (2H, t, J 7.5 Hz, (--H.sub.2), 2.65 (1H, dd,
J 5 and 2.5 Hz, 3-H), 2.85 (1H, t, J 4.5 Hz, 3-H), 3.19-3.23 (1H,
m, 2-H), 3.91 (1H, dd, J 12.5 and 6.5 Hz, 1-H) and 4.42 (1H, dd, J
12.5 and 3 Hz, 1-H); ESI-MS (m/z, +ve): 335 MNa.sup.+, 100%).
[0371] 1-O-Palmitoleoyl-rac-glycidol (Compound 58) To a solution of
glycidol (0.04 mL, 0.8 mmol) in dry CH.sub.2Cl.sub.2 (10 ml) under
N.sub.2 at -78.degree. C. was added palmitoleoyl chloride (0.20 g,
0.7 mmol). After 5 min, pyridine (0.15 mL, 1.8 mmol) was added and
the reaction mixture was allowed to stir for 1 hour at -78.degree.
C. before being allowed to warm to room temperature over a 1.5
hours period. Next, the reaction mixture was diluted with
CH.sub.2Cl.sub.2 (50 mL), washed with sat. KHSO.sub.4 (5 mL), sat.
NaHCO.sub.3 (5 ml) and sat. NaCl (10 mL), dried MgSO.sub.4) and
concentrated to give an oil. The residue was purified by silica-gel
column chromatography [eluent: ethyl acetate/hexane, 10/90, v/v] to
furnish epoxide Compound 58 as a colourless oil.
[0372] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.88 (3H, t, J 6.5 Hz,
Me), 1.30 (16H, br s, --(CH.sub.2).sub.4-- and
--(CH.sub.2).sub.4--), 1.61-1.65 (2H, m, (--H.sub.2), 2.00-2.01
(4H, m, --CH.sub.2CH.dbd.CHCH.sub.2--), 2.35 (2H, t, J 7.5 Hz,
(--H.sub.2), 2.65 (1H, dd, J 4.5 and 2.5 Hz, 3-H), 2.85 (1H, t, J
4.5 Hz, 3-H), 3.20-3.22 (1H, m, 2-H), 3.91 (1H dd, J 12.5 and 6.5
Hz, 1-H), 4.41 (1H, dd, J 12.5 and 2.5 Hr, 1-H) and 5.29-5.39 (2H,
m, --CH.sub.2CH.dbd.CHCH.sub.2--); ESI-MS (m/z, +ve): 311
(MH.sup.+, 30%), 333 (MNa.sup.+, 100%).
[0373] Dimethyl 2-amino-2-N-oleoyl ethyl phosphate (Compound 59) To
a mixture of amide Compound 51 (1.0 g, 3.1 mmol) and dimethyl
chlorophosphate (0.50 mL, 4.6 mmol) in dry CH.sub.2Cl.sub.2 (100
mL) under N.sub.2 was added N-methylimidazole (0.73 mL, 9.2 mmol).
After 40 hours, TLC analysis showed the reaction to be complete and
the mixture was washed with sat, NaHCO.sub.3 (3.times.20 mL), dried
(MgSO.sub.4) and evaporated to dryness to give dimethyl phosphate
Compound 59 (1.2 g, 89%) as a pale yellow oil.
[0374] .sup.1H NMR (360; CDCl.sub.3): (0.75 (3H, t, J 6.5 Hz, Me),
1.16 (20H, apparent br d, separation 11.5 Hz, --(CH.sub.2).sub.4--
and --(CH.sub.2).sub.6--), 1.50 (2H, br s, (--H.sub.2), 1.88 (4H,
apparent br d, separation 5.5 Hz, --CH.sub.2CH.dbd.CHCH.sub.2--),
2.08 (2 Hz, t, J 7.5 Hz, (--H.sub.2), 3.34-3.38 (2H, m, 2-H.sub.2),
3.64 and 3.67 (each 3H, s, 2.times.OMe), 3.97-4.02 (2H, m,
1-H.sub.2), 5.16-5.26 (2H, m, --CH.sub.2CH.dbd.CHCH.sub.2--) and
6.79 (1H, br s, N--H); .sup.31P NMR (146 MHz; CDCl.sub.3): (2.49;
ESI-MS (m/z, +ve): 434 MH.sup.+, 100%).
[0375] Di-tert-butyl 2-amino-2-N-oleoyl-propyl phosphate (Compound
60) To a mixture of Compound 51 (1.0 g, 2.9 mmol) and 1H-tetrazole
(0.41 g, 5.9 mmol) in dry CH.sub.2Cl.sub.2 (10 mL) under N.sub.2
was added di-tert.-butyl N,N-diisopropylphosphoramidite (1.2 g, 4.4
mmol). After 1.5 hours, t-BuOOH (2 mL) was added and the mixture
was stirred for a another 30 min. Next, the mixture was
concentrated and subjected to silica-gel column chromatography
[eluent: ethyl acetate in hexane, 25/75, v/v] to give
di-tert.-butyl phosphate Compound 60 (1.3 g, 81%) as an oil.
[0376] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.87 (3H, t, J 7 Hz,
Me), 1.27 (20, apparent br d, separation 10 Hz,
--(CH.sub.2).sub.4-- and --(CH.sub.2).sub.6--), 1.49 (18H, s,
2.times.Bu.sup.tO), 1.59-1.66 (2H, m, (--H.sub.2), 1.83 (2H,
quintet, J 6 Hz, 3-H.sub.2), 1.97-2.00 (4H, m,
CH.sub.2CH.dbd.CHCH.sub.2--), 2.16 (2H, t, J 7.5H, (--H.sub.2),
3.39 (2H, quintet, J 6 Hz, 3-H), 4.03 (2H, dt, J 7.5 and 6 Hz;
1-H.sub.2), 5.32-5.35 (2H, m, --CH.sub.2CH.dbd.CHCH.sub.2--) and
6.47 (1H, m, N--H); .sup.31P NMR (146 MHz; CDCl.sub.3): (-7.47;
ESI-MS (m/z, +ve): 420 (M-2Bu.sup.t+3H.sup.+, 78%), 476
(M-Bu.sup.t+2H.sup.+, 30), 532 (MH.sup.+, 100).
[0377] 2-Amino-2-N-oleoyl-propyl phosphate (Compound 61) A solution
of the protected phosphate Compound 60 (250 mg, 0.47 mmol) was
treated with 1/4 TFA/CH.sub.2Cl.sub.2 (15 mL, v/v) for 3 hours.
Next, the reaction mixture was concentrated and the residue was
purified by Sephadex LH-20 column chromatography [eluent:
MeOH/CH.sub.2Cl.sub.2, 50/50, v/v] to give phosphate Compound 61
(130 mg, 67%) as a white solid.
[0378] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.81 (3H, t, J 6.7 Hz,
Me), 1.20 (20H apparent br d, --(CH.sub.2).sub.4-- and
--(CH.sub.2).sub.6--), 1.51 (2H, br s, (--H.sub.2), 1.79 (2H, br s,
2-H.sub.2), 1.9 (4H, m, CH.sub.2CH.dbd.CHCH.sub.2--), 2.16 (2H, br
s, (--H.sub.2), 3.3 (2H, br s, 2-H), 3.9 (2H, m, 1-H, 5.25 (2H, m,
--CH.sub.2CH.dbd.CHCH.sub.2--) and 7.2 (1H, br s, N--H); .sup.31P
NMR (146 Mhz; CDCl.sub.3): (1.10; ESI-MS (m/z, -ve): 418-M-H.sup.+,
100%); ESI-MS (m/z, +ve): 420 MH.sup.+, 100%).
[0379] 1-O-Oleyl-2-O-methyl glycerol (Compound 64) 20-Methyl
glycerol (1.54 g, 14.5 mmol) was added to a suspension of NaH (0.38
g, 15.9 mmol) in dry DMF (20 mL) under N.sub.2. After 2 min, oleyl
bromide (2.4 g, 7.2 mmol) was added and stirring was continued for
0.5 hour. Then, the reaction mixture was quenched with water (20
mL) and extracted with diethyl ether (150 mL). The combined organic
phases were dried (MgSO.sub.4) and concentrated to give an oil
which was subjected to silica-gel column chromatography [eluent:
EtOAc/Hexane, 50/50, v/v] to give alcohol Compound 64 (1.3 g,
50%).
[0380] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.88-0.89 (3H, m, Me),
1.29 (22H, br s, --(CH.sub.2).sub.5-- and --(CH.sub.2).sub.6--),
1.59 (2H, br s, (--H, 2.01 (4H, br s,
--CH.sub.2CH.dbd.CHCH.sub.2--), 3.44-3.76 (7H, m, 3-H.sub.2,
1-H.sub.2, 2-H and (--H.sub.2), 3.47 (3H, s, OMe), and 5.34 (2H, m,
--CH.sub.2CH.dbd.CHCH.sub.2--); ESI-MS (m/z, +ve): 357 (MH.sup.+,
100%), 379 (MNa.sup.+, 53).
[0381] Bis-(2-cyanoethyl)
3-oleyl-2-O-methyl-rac-glycero-1-thiophosphate (Compound 65) To a
solution of 3-hydroxypropionitrile (0.16 mL, 2.4 mmol) and
1H-tetrazole (0.18 g, 2.5 mmol) in dry CH.sub.2Cl.sub.2 (8 mL)
under N.sub.2 was added 2-cyanoethyl
N,N,N'N'-tetraisopropylphosphorodiamidite (0.71 mL, 2.2 mmol).
After stirring for 1 hour, a second portion of 1H-tetrazole (0.20
g, 2.8 mmol) was added followed by the addition of alcohol Compound
64 (0.50 g, 1.4 mmol). After an additional 30 min, elemental sulfur
(0.5 g) and 1/1 CS.sub.2/pyridine (0.5 mL, v/v) were added. After
14 hours, the reaction mixture was filtered through a plug of
silica-gel, concentrated and purified by silica-gel column
chromatography [eluent: EtOAc/hexane, 25/75 v/v] to yield protected
thiophosphate Compound 65 (0.33 g, 42%) as a pale yellow oil.
[0382] .sup.1H NMR (360 CDCl.sub.3): (0.88 (3H, t, J 7 Hz, Me),
1.28 (22H, m, --(CH.sub.2).sub.4-- and --(CH.sub.2).sub.6--),
1.54-1.58 (2H, m, (--H.sup.2), 1.98-2.03 (4H, m,
--CH.sub.2CH.dbd.CHCH.sub.2--), 2.78 (4H, t, J 6 Hz,
2.times.NCCH.sub.2CH.sub.2--), 3.44 (2H, t, J 7 Hz, (--H.sub.2),
3.46 (3H, s, OMe), 3.49-3.51 (2H, m, 3-H.sub.2), 3.53-3.58 (1H,
2-H), 4.15 (1H, dd, J 15 and 5 Hz, 1-H), 4.23-4.36 (5H, m,
2.times.NCCH.sub.2CH.sub.2-- and 1-H) and 5.30-5.39 (2H, m,
--CH.sub.2CH.dbd.CHCH.sub.2--); .sup.31P NMR (146 MHz; CDCl.sub.3):
(69.04; ESI-MS (m/z, +ve): 581 (MNa.sup.+, 100%); ESI-MS (m/z,
-ve): 504 (M-NCCH.sub.2CH.sub.2, 100%).
[0383] 3-O-Oleyl-2-O-methyl-rac-glycerol-thiophosphate (Compound
66) To a solution of compound Compound 65 (50 mg, 87 (mol) in
CH.sub.3CN (0.5 mL) under N.sub.2 at 30.degree. C. was added TEA
(0.5 mL) followed by the addition of BSA (54 (L, 0.22 mmol). After
24 hours, the reaction mixture was concentrated and subjected to
Sephadex LH-20 column chromatography [eluent:
MeOH/CH.sub.2Cl.sub.2Cl.sub.2, 50/50, v/v] to give a mixture of
thiophosphate Compound 66 and mono-deprotected Compound 65 (80 mg)
in a 3/2 ratio.
[0384] .sup.31P NMR (146 MHz; CDCl.sub.3): (59.24, 56.74 (ratio
3/2, respectively); ESI-MS (m/z, -ve): 451 (M-H.sup.+, 80%) and 504
(M+NCCH.sub.2CH.sub.2CH.sub.2-2H.sup.+, 100).
[0385] Bis-(2-cyanoethyl) 1-(3-propyl oleoate) thiophosphate
(Compound 67) To a solution of 3-hydroxypropionitrile (0.16 mL, 2.4
mmol) and 1H-tetrazole (0.19 g, 2.6 mmol) in dry CH.sub.2Cl.sub.2
(7 mL) under N.sub.2 was added 2-cyanoethyl
N,N,N'N'-tetraisopropylphosphorodiamidite (0.65 mL, 2.1 mmol).
After stirring for 1 hour, a second portion of 1H-tetrazole (0.21
g, 2.9 mmol) was added followed by the addition of alcohol Compound
28 (0.50 g, 1.5 mmol). After an additional 30 min, elemental sulfur
(0.5 g) and 1/1 CS.sub.2/pyridine (1 ml, v/v) were added. After 14
hours, the reaction mixture was filtered through a plug of
silica-gel, concentrated and purified by silica-gel column
chromatography [eluent: EtOAc/hexane, 25/75 v/v] to yield protected
thiophosphate Compound 67 (0.27 g, 34%) as a pale yellow oil.
[0386] .sup.1H NMR (360 M; CDCl.sub.3): (0.88 (3H, t, J 6.5 Hz,
Me), 1.28 (20H, apparent br d, separation 13.5 Hz,
--(CH.sub.2).sub.4-- and --(CH.sub.2).sub.6--), 1.61 (2H, br s,
(--H.sub.2), 2.02-2.05 (6H, m, 2-H.sub.2 and
--CH.sub.2CH.dbd.CHCH.sub.2--), 2.31 (2H, t, J 7.5 Hz, (--H.sup.2),
2.78 (4H, t, J 6 Hz, 2.times.NCCH.sub.2CH.sub.2--), 4.17-4.24 (4H,
m, 1-H.sub.2 and 3-H.sub.2 or 2.times.NCCH.sub.2CH.sub.2--),
4.17-4.24 (4H, m, 1-H.sub.2 and 3-H.sub.2 or
2.times.NCCH.sub.2CH.sub.2--), and 5.34 (2H, br s,
--CH.sub.2CH.dbd.CHCH.sub.2--); .sup.31P NMR (146 MHz; CDCl.sub.3):
(68.19; ESI-MS (m/z, +ve): 543 MH.sup.+, 77%) and 565 (MN.sup.+,
100).
[0387] 1-(3-propyl oleoate) thiophosphate (Compound 68) To a
solution of Compound 67 (220 ma, 0.41 mmol) in CH.sub.3CN (2 mL)
under N.sub.2 was added TEA (2 mL) followed by the addition of BSA
(0.25 mL, 1.01 mmol). After 66 hours, the reaction mixture was
concentrated and subjected to Sephadex LH-20 column chromatography
[eluent: MeOH/CH.sub.2Cl.sub.2, 50/50, v/v] to give thiophosphate
Compound 68 (144 mg, 69%) as a pale yellow oil.
[0388] .sup.1H NMR (360 M; CDCl.sub.3): (0.85 (3H, t, J 7 Hz, Me),
125 (20H, apparent br d, separation 11.5 Hz, --(CH.sub.2).sub.4--
and --(CH.sub.2).sub.6--), 1.58 (2H, br s, (--H.sub.2), 1.97-2.02
(6H, m, 2-H.sub.2 and --CH.sub.2CH.dbd.CHCH.sub.2--), 2.30 (2H, t,
J 7.5 Hz, (--H.sub.2), 4.11-4.17 (2H, m, 1-H.sub.2 or 3-H.sub.2),
4.21 (2H, t, J 6 Hz, 1-H.sub.2 or 3-H.sub.2), 5.31 (2H, br s,
--CH.sub.2CH.dbd.CHCH.sub.2--) and 7.26 (2H, br s, 2.times.OH);
.sup.31P NMR (146 MHz; CDCl.sub.3): (48.60 ESI-MS (m/z, -ve): 465
M-H.sup.+, 100%); ESI-MS (m/z, +ve): 323 (100%) and 437 (MH.sup.+,
95).
[0389] Di-tert.-butyl
3-O-oleyl-2-O-methyl-rac-glycero-1-thiophosphate (Compound 69) To a
mixture of Compound 64 (0.3 g, 0.84 mmol) and 1H-tetrazole (0.12 g,
1.7 mmol) in dry CH.sub.2Cl.sub.2 (5 mL) under N.sub.2 was added
di-tert.-butyl N,N-diisopropylphosphoraidite (0.34 mL, 1.1 mmol).
After 2 hours, elemental sulfur (0.5 g) and 1/1 CS.sub.2/pyridine
(1.0 mL, v/v) were added and the mixture was stirred for a another
2 hours after. Then, the mixture was filtered through a plug of
silica-gel, concentrated and subjected 10 silica-gel column
chromatography [eluent: ethyl acetate in hexane, 1/15, v/v] to give
di-tert.-butyl thiophosphate Compound 69 (0.48 g, quant.) as an
oil.
[0390] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.87 (3H, t, J 7 Hz,
Me), 1.26-1.30 (22H, m, --(CH.sub.2).sub.4-- and
--(CH.sub.2).sub.6), 1.53 (20H, m, 2.times.Bu.sup.tO and
(--H.sub.2), 1.97-2.01 (4H, m, --CH.sub.2CH.dbd.CHCH.sub.2--), 3.43
(2H, t, J 7 Hz, (--H.sub.2), 3.46 (3H, s, OMe), 3.48-3.57 (3H, m,
3-H.sub.2 and 2-H), 4.01-4.14 (2H, m, 1-H.sub.2) and 5.34 (2H,
apparent t, separation 5 Hz, --CH.sub.2CH.dbd.CHCH.sub.2--);
.sup.31P NMR (146 MHz; CDCl.sub.3): (52.39; ESI-MS (m/z, -ve): 507
(M-Bu.sup.t, 100%); ESI-MS (m/z, +ve): 589 (MNa.sup.+, 100%).
[0391] Dimethyl erucyl phosphate (Compound 70) To a solution of
erucyl alcohol (0.50 g, 1.5 mmol) in dry CH.sub.2Cl.sub.2 (10 mL)
was added N-methylimidazole (0.14 mL, 1.7 mmol) followed by the
addition of dimethyl chlorophosphate (0.20 mL, 1.9 mmol). After 20
hours, the reaction mixture was concentrated and subjected to
silica-gel column chromatography [eluent: hexane/ethyl acetate,
66/33, v/v] to give dimethyl phosphate Compound 70 (0.59 g, 89%) as
an oil.
[0392] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.88 (3H, t, J 7 Hz,
Me), 1.26 (30H, br s, --(CH.sub.2).sub.9-- and
--(CH.sub.2).sub.6--), 1.68 (2H, quintet, J 7 Hz (--H.sub.2), 2.01
(4H, apparent br s, separation 5 Hz,
--CH.sub.2CH.dbd.CHCH.sub.2--), 3.75 and 3.78 (each 3H, s,
2.times.OMe), 4.04 (2H, quartet, J 7 Hz, (--H.sub.2), and 5.30-5.39
(2H, m, --CH.sub.2CH.dbd.CHCH.sub.2); .sup.31P NMR (146 Mhz;
CDCl.sub.3): (2.65; ESI-MS (m/z, +ve): 433 (MH.sup.+, 100).
[0393] Erucyl phosphate (Compound 71) To a solution of protected
phosphate Compound 70 (150 mg, 0.35 mmol) in dry CH.sub.2Cl.sub.2
(6 mL) was added BSA (0.19 mL, 0.76 mmol) followed by the addition
of TMSBr (0.10 mL, 0.76 mmol). After 30 min, TLC analysis showed
complete consumption of the starting material and the reaction was
quenched with 1/1 MeOH/H.sub.2O (2 mL, v/v) for 15 min, followed by
the addition of sat. KHSO.sub.4 (5 mL). Subsequently, the reaction
mixture was extracted with ethyl (2.times.30), the organic
extracted were combined, dried (MgSO.sub.4) and concentrated to
give an oil which was subjected to Sephadex LH-20 column
chromatography [eluent. MeOH/CH.sub.2Cl.sub.2, 50/50, v/v] to give
phosphate Compound 71 (90 mg, 64%) as an oil.
[0394] .sup.1H NMR (360 M CDCl.sub.3): (0.88 (3H, t, J 7 Hz, Me),
1.26 (30H, br s, --(CH.sub.2).sub.9-- and --(CH.sub.2).sub.6--),
1.66 (2H, quintet, J 6.5 Hz (--H.sub.2), 1.98-2.03 (4H, m,
--CH.sub.2CH.dbd.CHCH.sub.2--), 3.94-4.00 (2H, m, (--H.sub.2),
5.30-5.39 (2H, m, --CH.sub.2CH.dbd.CHCH.sub.2--) and 7.32 (2H, br
s, 2.times.OH); .sup.31P NMR (146 Mhz; CDCl.sub.3): (2.59; ESI-MS
(m/z, -ve): 403 (M-H.sup.+, 100).
[0395] Oleyl 2-O-methyl-rac-glycerate (Compound 76) To a solution
of oxalyl chloride (87 (L, 1.0 mmol) in CH.sub.2Cl.sub.2 (7 mL) at
-78.degree. C. was added DMSO (71 (L, 1.0 mmol). After 20 min
alcohol Compound 42 (0.20 g, 0.91 mmol) was added followed after
another 20, by the addition of triethylamine (0.63 mL, 4.6 mmol).
The reaction was warmed to room temperature over a 10 min period,
filtered through a plug of silica-gel, which was washed with ethyl
a ethyl (50 mL) and concentrated to give
3-O-t-butyldimethylsilyl-2-O-methyl-rac-glyceraldehyde Compound 73
(0.20 g, 100%) as a colourless oil.
[0396] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.07 (6H, s,
Me.sub.2Si), 0.88 (9H, s, Bu.sup.tSi), 3.51 (3H, s, OMe), 3.67-3.68
(1H, m, 2-H), 3.92 (2H, br d, J 6.5 Hz, 3-H.sub.2), 9.72 (1H, s,
CHO).
[0397] To a vigorously stirring mixture of CH.sub.2Cl.sub.2 (20
mL), water (20 mL) and aldehyde Compound 73 (0.93 g, 4.3 mmol) at
0.degree. C. was added sulfamic acid (0.63 g, 6.8 mmol) and
2-methyl-2-butene (0.60 g, 8.5 mmol). After 5 min, sodium chlorite
(0.77 g, 8.5 mmol) was added and the reaction was monitored by TLC
until no starting material remained. The reaction mixture was
extracted with CH.sub.2Cl.sub.2 (.times.20 mL), and the combined
organic layers were washed with sat. NaCl (2.times.20 mL), dried
MgSO.sub.4) and concentrated to give a colourless oil. The oil was
redissolved in CH.sub.2Cl.sub.2 (50 mL) after which sat.
NaHCO.sub.3 (10 mL) was added. The aqueous phase was isolated,
CH.sub.2Cl.sub.2 (50 mL) was added and the mixture was acidified by
adding 10% aqueous acetic acid (v/v). The organic phase was dried
(MgSO.sub.4) and concentrated to give
3-O-t-butyldimethylsilyl-2-O-methyl-rac-glyceric acid Compound 74
(0.39 g, 38%) as a colourless oil.
[0398] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.07 and 0.07 (each 3H,
s, Me.sub.2Si), 0.88 (9H, s, Bu.sup.tSi), 3.52 (3.times.H, s, OMe)
and 3.87-3.99 (3H, m, 2-H and 3-H.sub.2).
[0399] To a mixture of oleyl alcohol (0.52 mL, 1.6 mmol), DIC (0.27
mL, 1.7 mmol) and glyceric acid Compound 74 (0.39, 1.6 mmol) in DMF
(10 mL) was added DMAP (.about.20 mg). After 16 hours, the solvent
was removed in vacuo and the residue was partitioned between EtOAc
(50 mL) and water (10 mL). The organic phase was dried (MgSO.sub.4)
and concentrated to give an oil which was purified by silica-gel
column chromatography [eluent: EtOAc/hexane, 10/90, v/v] to give
oleyl 3-O-t-butyldimethylsilyl-2-O-methyl-rac-glycerate Compound 75
(0.33 g, 41%) as a colourless oil.
[0400] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.05 (6H, s,
Me.sub.2Si), 0.86-0.89 (12H, m, Bu.sup.tSi and Me), 1.28 (22H,
apparent br d, separation 10.5 Hz, --(CH.sub.2).sub.5-- and
--(CH.sub.2).sub.6--), 1.61-1.67 (2H, m, (--H.sub.2), 1.98-2.04
(4H, m, --CH.sub.2CH.dbd.CHCH.sub.2--), 3.44 (3H, s, OMe),
3.83-3.90 (3H m, 1-H and 2-H.sub.2), 4.15 (2H, t, J 7H,
(--H.sub.2), and 5.33-5.39 (2H, m,
--CH.sub.2CH.dbd.CHCH.sub.2--).
[0401] 1M TBAF (0.7 mL, 0.7 mmol) was added to a solution of
Compound 75 in THF (8 mL). After 40 min, TLC analysis showed that
all the starting material had been consumed. Then, the reaction
mixture was partitioned between diethyl ether (100 mL) and sat.
NaCl solution (15 mL). The organic extract vas dried (MgSO.sub.4)
and concentrated to give a yellow oil which was subjected to
silica-gel column chromatography [eluent: hexane/ethyl acetate,
80/20, v/v] to give the title ester (Compound 76) (0.18 g,
71%).
[0402] .sup.1H NMR (360 M; CDCl.sub.3): (0.87 (3H, t, J 7 Hz, Me),
1.28 (22H, apparent br d, separation 11 Hz, --(CH.sub.2).sub.5--
and --(CH.sub.2).sub.6--, 1.65 (2H, quintet, J 7 Hz, (--H.sub.2),
2.01 (4H quintet, J 6.5 Hz, --CH.sub.2CH.dbd.CHCH.sub.2--), 2.24
(1H, m, OH), 3.49 (3H, s, OMe), 3.75-3.81 (1H, m, 2-H.sub.2), 3.89
(2H, quintet J 3.5 Hz, 3-H.sub.2 or (--H.sub.2), 4.11-4.22 (2H, m,
3-H.sub.2 or (--H.sub.2) and 5.29-5.40 (2H, m,
CH.sub.2CH.dbd.CHCH.sub.2--); ESI-MS (m/z, +ve): 393 (MNa.sup.+,
100), 763 (2MNa.sup.+, 20).
[0403] Oleyl
1-(bis-(2-cyanoethyl)thiophosphoryl)-2-O-methyl-rac-glycerate
(Compound 77) To a solution of 3-hydroxypropionitrile (55 (L, 0.80
mmol) and 1H-tetrazole (60 mg, 0.85 mmol) in dry CH.sub.2Cl.sub.2
(4 mL) under N.sub.2 was added 2-cyanoethyl
N,N,N',N'-tetraisopropylphosphorodiamidite (0.24 mL, 0.76 mmol).
After stirring for 1 hour, a second portion of 1H-tetrazole (66 mg,
0.94 mmol) was added followed by the addition of ester Compound 76
(175 mg, 0.47 mmol). After 1 hour, elemental sulfur (0.25 g) and
1/1 CS.sub.2/pyridine (0.5 mL, v/v) were added. After an addition 2
hours, the reaction mixture was filtered rough a plug of
silica-gel, concentrated and purified by silica-gel column
chromatography [eluent: EtOAc/hexane, 25/75 v/v] to yield protected
thiophosphate 77 (78 mg, 29%) as a pale yellow oil and recovered
ester Compound 76 (73 mg, 42%).
[0404] .sup.1H NMR (360 MHz, CDCl.sub.3): (0.86 (3H, t, J 7 Hz,
Me), 1.27 (22H, apparent br d, separation 12.5 Hz,
--(CH.sub.2).sub.4-- and --(CH.sub.2).sub.6--), 1.65 (2H, quintet,
(--H.sub.2), 1.98-2.02 (4H, m, --CH.sub.2CH.dbd.CHCH.sub.2--), 2.76
and 2.78 (each 2H, t, J 6.5 Hz, 2.times.NCCH.sub.2CH.sub.2--), 3.4
(3H, s, OMe), 3.99 (1H, t, J 4.2 Hz, 2-H), 4.17 (2H, J 7 Hz,
(--H.sub.2), 4.22-4.47 (6H, m, 3-H.sub.2 and
2.times.NCCH.sub.2CH.sub.2--) and 5.28-5.37 (2H, m,
--CH.sub.2CH.dbd.CHCH.sub.2--); .sup.31P NMR (146 MHz; CDCl.sub.3):
(69.04; ESI-MS (m/z, +ve): 573 (M-H.sup.+, 25%), 595 (MNa.sup.+,
100).
[0405] Oleyl 1-thiophosphoryl-2-O-methyl-rac-glycerate (Compound
78). To a solution of Compound 77 (100 mg, 0.18 mmol) in CH.sub.3CN
(0.5 mL) under N.sub.2 at 30.degree. C. added TEA (0.5 mL) followed
by the addition of BSA (0.11 mL, 0.45 mmol). After 24 hours, the
reaction mixture was concentrated and subjected to Sephadex LH-20
column chromatography [eluent: MeOH/CH.sub.2Cl.sub.2, 50/50, v/v]
to give thiophosphate Compound 78 (40 mg, quant.) as a pale yellow
oil.
[0406] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.87 (3H, t, J 7 Hz,
Me), 1.24-1.35 (22H, --(CH.sub.2).sub.4-- and
--(CH.sub.2).sub.6--), 1.65 (2H, quintet, (--H.sub.2), 1.98-2.03
(4H, --CH.sub.2CH.dbd.CHCH.sub.2--), 3.47 (3H, OMe), 4.09 (1H, dd,
J 5 and 3.5 Hz, 2-H), 4.09-4.38 (4H, m, 1-H.sub.2 and (--H.sub.2),
5.32-5.38 (2H, m, --CH.sub.2CH.dbd.CHCH.sub.2--), 5.51 (2H, br s,
2.times.OH); .sup.31P NMR (146 MHz; CDCl.sub.3): (55.28; ESI-MS
(m/z, -ve): 465 .mu.M-H.sup.+, 100%).
[0407] 3-O-Oleoyl-2-O-methyl-rac-glycero-1-sulfate (Compound 81) To
a solution of alcohol Compound 44 (0.30 g, 0.8 mmol) in DMF (10 mL)
was added triethylamine sulfur trioxide complex (0.74 g, 4.1 mmol)
and the mixture was stirred for 5.5 h at 40.degree. C. Next,
NaHCO.sub.3 (0.3 g, 3.6 mmol) was added and, after stirring for
another 30 min, the mixture was filtered through a glass sinter.
The filtrate was concentrated to give a light brown oil which was
subjected to Sephadex LH-20 column chromatography [eluent:
MeOH/CH.sub.2Cl.sub.2, 50/50, v/v] to furnish, after extensive
drying in vacuo, sulfate Compound 81 (0.52 g) as its triethylamine
salt.
[0408] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.87 (3H, t, J 7 Hz,
Me), 1.27 (20H apparent br d, separation J 11 Hz,
--(CH.sub.2).sub.4-- and --(CH.sub.2).sub.6--), 1.46 (15H, t,
(CH.sub.3CH.sub.2).sub.3N, J 7 Hz), 1.59 (2H, m, (--H.sub.2),
1.97-2.02 (4H, m, --CH.sub.2CH.dbd.CHCH.sub.2--), 2.31 (2H, t, J
7.6 Hz, (--H.sub.2), 3.21-3.13 (10H, m, (CH.sub.3CH.sub.2).sub.3N),
3.45 (3H, s, OMe), 3.69-3.74 (1H, m, 2H), 4.06-4.29 (4H, m,
1-H.sub.2 and 3-H.sub.2) and 5.28-5.37 (2H, m,
--CH.sub.2CH.dbd.CHCH.sub.2--); .sup.31P NMR (146 MHz, CDCl.sub.3):
(63.69 (m); ESI-MS (m/z, +ve): 656 (MH.sup.+2TEA, 100%); ESI-MS
(m/z, -ve): 449 (M-H.sup.+, 100%).
[0409] Bis(oleyl-3-O-methyl-rac-glyceryl) thiophosphate (Compound
80) To a solution of 1H-tetrazole (0.31 g, 4.0 mmol) and 2
cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0.53 mL, 1.7
mmol) in dry CH.sub.2Cl.sub.2 (5 mL) under N.sub.2 was added
alcohol Compound 64 (0.38 g, 1.1 mmol). After stirring for 30 ml
elemental sulfur (0.5 g) and 1/1 CS.sub.2/pyridine (0.5 mL, v/v)
were added. After 2 h, the reaction mixture was filtered through a
plug of silica-gel, concentrated and purified by silica-gel column
chromatography [eluent: EtOAc/hexane, 25/75 v/v] to yield
2-cyanoethyl bis(3-oleyl-O-methyl-rac-glyceryl)thiophosphate
(Compound 79) (0.15 g, 34%) as a pale yellow oil.
[0410] To a solution of compound 79 (0.15 g, 0.18 mmol) in
CH.sub.3CN (1.3 mL) under N.sub.2 was added TEA (1.5 mL) followed
by the addition of BSA (0.17 mL, 0.68 mmol). After 60 h, the
reaction mixture was concentrated and subjected to Sephadex LH-20
column chromatography [eluent: MeOH/CH.sub.2Cl.sub.2, 50/50, v/v]
to give thiophosphate Compound 80 (0.12 g, 81%).
[0411] .sup.1H NMR (360 MHz; CDCl.sub.3): (0.87 (6H, t, J 7 Hz,
2.times.Me), 1.26 (44H apparent br d, separation J 6 Hz,
2.times.--(CH.sub.2).sub.4-- and 2.times.--(CH.sub.2).sub.6--),
1.55 (4H, m, 2.times.(--H.sub.2), 1.97-2.00 (8H, m,
2.times.--CH.sub.2CH.dbd.CHCH.sub.2--), 3.43 (4H, t, J 7 Hz,
2.times.(--H.sub.2), 3.47 (6H, 2.times.s, 2.times.OMe), 3.49-3.59
(6H, m, 2.times.2-H and 2.times.3-H.sub.2), 3.99-4.20 (4H,
2.times.1-H.sub.2) 5.33 (4H, m,
2.times.CH.sub.2CH.dbd.CHCH.sub.2--) and 5.68 (2H, br s,
2.times.OH); .sup.31P NMR (146 MHz; CDCl.sub.3): (63.69 (m/z);
ESI-MS (M/z, +ve): 792 (MH.sup.+, 100%); ESI-MS (m/z, -ve): 790
(M-H.sup.+, 100%).
EXAMPLE 2
Anti-Apoptotic Activity Assay
[0412] In order to determine the apoptotic activity of the claimed
invention, the following method of analysis was used. The cell
assay is described in detail in U.S. Pat. Nos. 5,637,486,
5,620,888, and 5,681,703, and Tomei et al. (1993) Proc Natl. Acad.
Sci. 90:853-857. Briefly, mouse fibroblast C3HT1/2 cells (clone 8)
were obtained from ATCC (Rockville, Md.) and were maintained in
exponential growth phase in which the cell cycle is randomly
distributed and no cells are arrested in G.sub.0, and in
quiescence. Exponential growth phase was assured by seeding at 2000
cells per 1 ml (5 ml for a 60 mm culture plate) five days prior to
the beginning of the experiment. Assays were performed on cells
only up to passage 15. At T=0, cultures were transferred to
serum-free medium, as an apoptosis stimulus, and seed extracts were
added. Controls included 10.sup.-7 and 5.times.10.sup.-8 M
12-O-tetradecanoyl phorbol-13-acetate (TPA) to ensure the
responsiveness of the cell culture. The samples were added to serum
free medium and sterile filtered prior to addition to the cultures.
Assays were performed in triplicate or quadruplicate. Analyses of
the cell responses were made between 18 and 28 hours of serum
deprivation. Two assays were performed on each cell culture plate
consisting of differential cell counts.
[0413] 1. All non-adherent or loosely adherent cells were removed
from the culture dish and counted by appropriate techniques,
typically counting by electronic particle counting instrument.
These are the apoptotic cells, the serum deprived released cells
(SDR), released by the action of cultivation in serum-free medium.
Approximately 95% of these released cells are apoptotic as shown by
both ultrastructure analysis and DNA fragmentation analysis.
[0414] 2. The remaining adherent cells (ADH) were exposed to a
buffered, typically pH 7.3 balanced salt solution such as Hanks
Balanced Salt Solution without calcium and magnesium salts
containing 0.05% trypsin and 0.53 mM ethylene diaminetetraacetric
acid (EDTA). Each culture was incubated at either room temperature
or 37 (C on a rocking platform to ensure uniform distribution of
the trypsin reagent over the culture surface. After a standardized
period of time, typically 10 minutes, the released cells were
removed from each culture dish and measured by the same means as
described above, typically electronic particle counting. This ADH
cell count is comprised of both trypsin resistant and trypsin
sensitive cells as described in U.S. Pat. Nos. 5,637,486,
5,620,888, and 5,681,703.
[0415] Anti-apoptotic activity is expressed in the following
examples as the calculated concentration of material ((g/ml of
media) required to save 50% of the cells released on serum free
treatment.
EXAMPLE 3
Preparation of Five Phospholipid Mixture
[0416] Commercially available purified soy phospholipids containing
lysophosphatidic acid and the following other phospholipids: PA,
PI, LPI, LPC (available, for example, from Avanti.RTM. Polar
Lipids, Inc.) were suspended in 50 mM ammonium bicarbonate pH 8.0
containing 154 mM NaCl or buffered aqueous solutions free of
divalent cations having a pH range of 5 to 8. Total concentrations
of phospholipids of greater than 10 mg/mL can be used provided that
clarity is obtainable upon sonication. Total concentrations of up
to about 50 mg/mL have been utilized.
[0417] Typically, the phospholipid mixtures are suspended in a
buffer and the mixture is placed in a disposable borosilicate
glass, preferably 1-2 mL in a 16.times.100 mm tube or 0.5-2 mL in a
13.times.100 mm tube, or up to 1 mL in a 12.times.75 mm tube. The
combination of phospholipids is then sonicated. Preferably, a small
bath sonicator is used, such as a that sold by Laboratory Supplies,
Hicksville, N.Y. The temperature of the water bath is between about
21 and 50.degree. C., preferably between about 21.degree. C. and
about 40.degree. C. The optimal temperature depends on the
phospholipids used and can be determined empirically. The water
level is adjusted so that it is approximately the same height as
the phospholipid mixture in the glass tube(s). Alternatively, a
probe sonicator can be used Fisher Scientific Sonic Dismembrator
model 550), as long as care is taken to prevent overheating of the
mixture.
[0418] The mixture was sonicated for between 3 and 90 minutes, with
alternating 5 minute intervals of sonication followed by 5 minutes
of thermal equilibration, in a 1-2 ml volume until the mixture
became translucent and passed readily through a filter attached to
a 5 ml syringe with a pore size of 0.22 (m. Preferably, sonication
is for 5-10 minutes.
[0419] The stability of the compositions at various temperature was
determined. The compositions were stored for one week at 4.degree.
C., room temperature, and 65.degree. C. The results show loss of
activity after storage at 65.degree. C., while the compositions
stored at 4.degree. C. or at room temperature do not have a
significant loss of activity.
[0420] Optimization of each constituent phospholipid was determined
by mixing the purified phospholipids in various ratios, varying one
phospholipid at a time. Each mixture was analyzed for
anti-apoptotic activity as described in Example 2. When the
apparent optimized ratio was obtained, the ratio of the most active
ingredient was varied to find the absolute optimize activity. Table
2 shows the final ratios tasted (10:10:8:2:4 is the "Five
Phospholipid Mixture" referred to herein). TABLE-US-00002 TABLE 2
PA: PI: LPA: LPI:LPC 10: 10: 2: 2:1 10: 10: 2: 2:2 10; 10: 2: 2:4
10: 10: 4: 2:4 10: 10: 4: 2:1 10: 10: 4: 2:2 10: 10: 8: 2:4
[0421] The concentration of LPA was varied as was the chain length
to determine the effects of these parameters on activity.
EXAMPLE 4
Anti-Apoptotic Activity of LPA
[0422] The ability of 18:1-LPA and other LPAs to protect
serum-stared cells from apoptotic death was measured using the
C3H/10T/1/2 cell assay, performed as described in Example 2. The
effect of various compounds on the ability of lysophosphatidic acid
to protected serum-starved cells from apoptotic death was also
measured. Log phase cells were seeded in 60 mm Petri dishes at
175-350 cells per cm2 and maintained in Basal Medium Eagle (BME)
supplemented with 10% Heat Inactivated Fetal Bovine Scrum (MFBS).
On day 3 the cells were given fresh media. Treatment began on day 5
when the media containing serum was removed and replaced with the
LPA mixture to be tested. After 24 hours of treatment, day 6, serum
deprived released (SDR) cells (the apoptotic cell population) and
adherent (ADH) cells were separated and counted using an electronic
cell counter (Coulter Corporation, Hialea, Fla.). SDR cells were
counted with the lower threshold setting at 6.3 (m and were defined
as the apoptotic cells dying in response to cultivation in serum
free media. Approximately 95% of the SDR cells were confirmed to be
apoptotic as previously shown by size, ultrastructure and DNA
fragmentation analysis. Adherent cells were remove by treatment
with 5 mL of Hanks Balanced Salt Solution (HBSS) without ions
containing 0.05% trypsin and 0.53 mM EDTA and were counted with the
lower threshold setting at 11.01 (m. All samples were tested in
triplicate and serum-deprived controls (BME only) were assayed at
both the beginning and end of each experiment.
[0423] To test the efficacy of each LPA to inhibit apoptosis or
preserve function of serum-deprived cells, 2.5 (Mol of dried LPA
was dissolved (via approximately 5 minute sonication) in 1 mL of
citrate-saline (10 mM Na Citrate, 110 mM NaCl, pH 6.5) to give a
2.5 mM stock solution and presented to the cells at four
concentrations: 1, 3, 10 and 30 (M LPA. The results are shown in
FIG. 1 (lysophosphatidic acid) and Table 3. The effects of LPA
alone or in combination with various proteins or liposomes are
expressed as percent cells saved. TABLE-US-00003 TABLE 3
Concentration for Maximum effect Compound # Max Effect (.mu.M)
relative to 18:1 LPA 78 1 160% 68 1 150% 48 1 130% 66 1 130% 80 3
100% 18:1 LPA 10 100% 18:2 LPA 10 100% 24 10 100% 12 10 100% 16 10
100% 30 30 100% 16:0 LPA 10 80% 14:0 LPA 30 80% 71 100 76% 46 30
75% 37 30 75% 61 30 70% 19 30 50% 10:0 LPA 30 30% 24:1 LPA 30 30%
23 30 17% 33 ND 70 ND 11 ND 39 ND 38 ND 6:0 LPA ND 15 ND 29 ND 25
ND 45 ND 36 ND 4 10 Toxic 59 30 Toxic 8 30 Toxic 3 30 Toxic 53 100
Toxic ND: no protection detected.
[0424] For those compounds exhibiting toxicity, the dose shown is
the lowest concentration at which cytotoxic effects were
observed.
[0425] In medium alone, approximately 80% of serum-deprived 10T1/2
cells were non adherent following 24 hour serum deprivation.
However, 18:1-LPA at various concentrations (1, 3, 10, 30 (M)
showed anti-apoptotic activity, protecting 35% to approximately 53%
of the cells. 18:1-LPA combined with polyethylene glycol or BSA
also protects serum-deprived 10T/1/2 cells from apoptosis.
[0426] When 18:1-LPA (at the same various concentrations) was
combined with BME (containing calcium) and filtered prior to
presentation to serum-deprived 10T1/2 cells, the anti-apoptotic
activity of 18:1-LPA appeared to be inhibited. The addition of BSA
apparently preserved the anti-apoptotic activity of 18:1-LPA
despite the presence of calcium, as seen when 18:1-LPA (at the same
various concentrations) was combined with BSA in BME (containing
calcium) and presented to serum-deprived 10T1/2 cells.
[0427] To test the efficacy of LPA, presented in a lipid membrane
structure, to inhibit apoptosis in serum-deprived cells, LPA was
incorporated into lipid membrane structures of various lipid
compositions. Except in the Five Phospholipid Mixture treatments,
LPA was presented to the cells as 18:1-LPA, and was tested in all
preparations at four concentrations: 0.25, 0.75, 2.25 and 6.75
(g/ml. The results are shown in FIG. 2. In medium alone,
approximately 50% of the cells died of serum deprivation-induced
apoptosis. Phosphatidylserine (PS) alone was slightly toxic. When
18:1-LPA at 0.75 (g/ml was added together with PS, approximately
75% of cells were protected. Positively charged particles
containing 18:1-LPA combined with the neutral phospholipid
phosphatidyl choline (PC) and 20 mol % of the positively charged
lipid 1,2-dioleoyl-3-trimethylammonium-propane (TAP) were
efficacious in protecting 70 to 80% of cells, compared with PC/TAP
alone, offered no protection over control. Incorporation of
18:1-LPA into the negatively charged phosphatidyl glycerol (PG)
gave even better protection, with up to 90% of cells protected as
compared to alone which offered no protection over control.
However, inclusion in the 18:1-LPA/PG particles of 5 to 10 mol % of
the neutral phospholipid phosphatidylethanolamine (PE) with a long
polyethylene glycol (2000 MW) covalently linked to the polar head
group resulted in reduced efficacy of 18:1-LPA. Taken together,
these data suggest that LPA, presented in particles as a 10% by
weight mixture with either PC or PG, afforded a degree of
protection similar to that achieved with Five Phospholipid Mixture
against serum deprivation-induced apoptosis.
[0428] LPA was then tested in two different weight ratios with PG,
using 18:1-LPA in concentrations representing LPA equivalents to
Five Phospholipid Mixture at 1, 3, or 10 (g/ml. As shown in FIG. 3,
LPA equivalent to 10 (g/ml Five Phospholipid Mixture incorporated
into particles at 10% by weight with PG protected approximately 65%
of cells from apoptosis. This degree of protection was roughly the
same as that afforded by Five Phospholipid Mixture alone.
Increasing the weight percent of 18:1-LPA to 23.5 resulted in a
reduction of the proportion of cells saved to about 50%.
Incorporation of 18:1-LPA into particles containing 1:1 ratio PG:PC
with 23.5% by weight 18:1-LPA gave particles that protected about
57% of cells. In this assay, in medium alone, only about 30% of
cells survived serum-deprivation, and when PC or PG/PC mixtures
alone were added to the culture medium, only about 25% of cells
survived.
[0429] Various other natural lysophospholipids were tested for
ability to protect cells from serum deprivation-induced cell death.
Of the phospholipids tested only lysophosphatidyl serine (LPS)
showed any significant activity in the C3HT1/2 assay. On a molar
basis, LPS was approximately 50% as active as 18:1-LPA (FIG. 4).
Palmitoyl-LPA (16:0 LPA) at 15 (M protected 63% of cells, which was
similar to the protection afforded by 15 (M 18:1-LPA. However,
differences were, seen at lower concentrations of the two LPAs: 5
(M 18:1-LPA protected about 62% of cells, while 5 (M palmitoyl-LPA
protected only about 42% of cells, compared with a medium alone
control of 30% protection (FIG. 5). Similarly, stearyl-LPA at 15 (M
and at 5 (M protected 65 and 63% of cells, respectively, while 18:1
LPA at 15 (M and 5 (M protected 80% and 75%, respectively (FIG. 6).
Stearyl-LPA and 16:1-LPA as 10% dispersions in PG gave similar
degrees of protection.
[0430] The efficacy of various mixtures of oleoyl-lysophosphatidic
acid and phosphatidic acid (PA):phosphatidyl inositol (PI) was also
tested. oleoyl-lysophosphatidic acid was mixed at 10, 20 and 30% by
weight with PA:PI at a 1:1 by weight) ratio and tested in the
C3HT1/2 serum deprivation assay. The results (FIG. 7) indicate that
oleoyl-lysophosphatidic acid presented to C3HT1/2 cells at the time
of serum deprivation as a 30% (by weight) mixture with 1:1 PA:PI in
a final concentration equivalent to Five Phospholipid Mixture at 10
(g/ml afforded nearly the same degree of protection (about 82% of
cells protected) as Five Phospholipid Mixture at 10 (g/ml (about
87% of cells protected), compared to a medium alone control value
of 38%,
[0431] 18:1-LPA was presented to C3HT101/2 cells at the time of
serum deprivation as a 10% (by weight) mixture with PC, PC
containing 5 mol % phosphatidyl ethanolamine-N-polyethylene glycol
(PE-PEG), or PC containing 20 mol %
1,2-di-oleoyl-sn-glycero-3-ethylphosphocholine (EtPC). The results,
in FIG. 8, show that preparations containing LPA incorporated into
PC, PC/PE-PEG, or PC/EtPC were all effective in protecting cells
from serum deprivation-induced cell death, wt 18:1-LPA/PC/EtPC
liposomes providing as much protection (approximately 85% of cells
protected) as Five Phospholipid Mixture.
[0432] The analogs and derivatives of lysophosphatidic acid that
were test (Table 3) generally showed activity similar to
lysophosphatidic acid. One genus of LPAs, the phosphothionate
analogs, (exemplified by the following compositions:
3-O-Oleoyl-2-O-methyl-rac-glycero-1-thiophosphate, Oleyl
1-thiophosphoryl-2-O-methyl-rac-glycerate, and
3-O-Oleyl-2-O-methyl-rac-glycero-1-thiophosphate), showed activity
significantly higher than lysophosphatidic acid.
EXAMPLE 5
Preparation of Protein/LPA Mixtures
[0433] Soy derived LPA (soy-LPA) (Avanti.RTM. Polar Lipids, Inc.,
Alabaster, Ala.) was stored in an organic solution and was dried
immediately prior to assay using a Speed Vac (Savant Instruments,
Hicksville, N.Y.). Tested proteins were resuspended at 10 mg/mL in
a bicarbonate buffered saline/EDTA solution (50 mM
NH.sub.4HCO.sub.3, 104 mM NaCl, 250 (M EDTA) prior to being added
to dried soy-LPA at a 1:10 soy-LPA to protein (weight:weight)
ratio. The mixture was then sonicated for 5 minutes using a high
power 80 Watt sonication bath (Laboratory Supplies Co., Hicksville,
N.Y.). Following sonication, concentrated LPA/protein mixtures were
left standing for 15 minutes at room temperature prior to dilution
in cell culture assay media.
EXAMPLE 6
Effects of Protein on Anti-Apoptotic Activity of LPA
[0434] The effect of various proteins on the ability of LPA to
protect serum-starved cells from apoptotic death was measured using
the C3H/10T1/2 cell assay, performed as described in Example 2,
above. Log phase cells were seeded in 60 mm Petri dishes at 175-350
cells per cm.sup.2 and maintained in Basal Medium Eagle (MEB)
supplemented with 10% fetal calf serum (Hyclone, Logan, Utah). At
time zero, serum containing media was replaced with sterile
filtered BME containing the LPA/protein mixture to be tested. After
24 hours of treatment, serum deprived release (SDR) cells (the
apoptotic cell population) and adherent (ADH) cells were separated
and counted using an electronic cell counter (Coulter Corporation,
Hialea, Fla.). SDR cells were counted with the lower threshold
setting at 6.3 (m and were defined as the apoptotic cells dying in
response to cultivation in serum-free-media. Approximately 95% of
the SDR cells are confirmed to be apoptotic as previously shown by
size, ultrastructure and DNA fragmentation analysis. Adherent cells
were removed by treatment with a Hanks' balanced salt solution
containing 0.05% trypsin and 0.53 mM EDTA and were counted with the
lower threshold setting at 11.01 (m, AU samples were tested in
triplicate and serum-deprived controls (BME only) were assayed at
both the beginning and end of each experiment.
[0435] The results are shown in Table 4. The effects of various
proteins alone or in combination with LPA are expressed as percent
cells saved from apoptosis. When C3HT1/2 cells were treated with 10
(g/mL soy LPA without protein, 8.0% of the cells were saved. The
degree of protection from apoptosis afforded by protein alone,
added to the medium at a concentration of 0.1% (wt/vol.), varied
from 0.9% to 27.7% of cells saved from apoptosis. An unusually high
percentage of cells were protected from serum-starvation-induced
apoptosis by casein. This value may be artificially high, as casein
preparations have been known to contain growth factors, including
insulin-like growth factors (IGF) IGF1 and IGF2. IGF2 is a mitogen
that can mask the serum deprivation-induced apoptotic response in
these cells.
[0436] When soy-LPA was added together with protein, the degree of
protection from apoptosis was, for certain proteins, more than
additive. Thus, way acid depleted (FAD)) bovine serum albumin (BSA)
alone protected 0.9% of cells from serum-induced apoptosis, soy-LPA
alone protected 8.0% of cells, while FAD BSA plus soy-LPA saved
58.7% of the cells. Similar effects were seen for mixtures of
soy-LPA with FAD soy protein, cytochrome c and low density
lipoprotein (LDL) and, to a lesser degree, acyl carrier protein,
casein and myoglobin. Soybean trypsin inhibitor, ovalbumin, retinol
binding protein and myelin basic protein did not enhance the
anti-apoptotic activity of LPA, while bromolain was toxic to the
cells. TABLE-US-00004 TABLE 4 Detectable Protein Protein +10
Increase Acrylamide 3H-LPA Size Alone (g/mL with Gel .sup.3H LPA
Shift Analysis Protein Source (0.01%) Soy LPA LPA Binding (+/-)
(+/-) Albumin (Fatty Bovine 0.9 58.7 +57.8 + + Acid Depleted) serum
Soy Protein Soybean 5.6 51.6 +46.0 did not + (Fatty Acid resolve on
Depleted) native gel Cytochrome C Bovine 16.2 54.6 +38.4 did not -
heart resolve on native gel Low Density Human 25.9 63.4 +37.5 - Not
tested Lipoprotein plasma (LDL) Acyl Carrier E. coli 8.1 36.2 +28.1
not tested Not tested Protein Casein Bovine 63.4 84.3 +20.9 - Not
tested milk Myoglobin Horse 9.3 27.8 +18.5 - Not tested heart
Trypsin Soybean 4.9 12.3 +7.4 - - Inhibitor Ovalbumin Chicken 7.9
15.1 +7.2 + + Egg Retinol Binding Human 27.7 33.3 +5.6 - Not tested
Protein (RBP).sup.3 urine Myelin Basic Rabbit 16.8 17.0 +0.2 - Not
tested Protein.sup.3 brain Bromolain Pineapple -100 -100 0 - Not
tested stem .alpha.-fetoprotein Fetal not not not + Not tested
bovine tested tested tested serum
EXAMPLE 7
Binding of LPA to Protein
[0437] Binding of LPA to protein was examined using an
elecetrophoresis radiobinding detection assay and a size shift
analysis assay.
[0438] Electrophoresis radiobinding assay. Proteins were analyzed
on native polyacrylamide gels following incubation with
.sup.3H-labeled 18:1-LPA. .sup.3H-labeled lysophosphatidic acid
(1-oleyl) (DuPont NEN Products, Boston, Mass.) was added to tested
proteins at 1 nM labeled 18:1-LPA/45 (g protein. Mixtures were
incubated in a bicarbonate buffered saline/EDTA solution at
37.degree. C. for 60 minutes. Incubated samples were mixed with
native gel sample buffer (312 mM Tris pH 6.8, 50% glycerol, 0.05%
bromophenol blue) and loaded entirely onto an 8% discontinuous
native polyacrylamide gel. Following electrophoresis, proteins were
fixed into the gels using an aqueous solution consisting of glacial
acetic acid, 10% (vol/vol) and methanol 30% (vol./vol.) solution.
Fixed gels were then incubated for 60 minutes in autoradiography
enhancer EN.sup.3HANCE, DuPont NEN Products, Boston, Mass.), and
dried onto 3M chromatography paper (Whatman, Clifton, N.J.) and
exposed to autoradiography film (X-Omat, Kodak Rochester, N.Y.) for
72 hours at -80.degree. C.
[0439] The results, shown in FIG. 9, demonstrate that BSA and
(-fetoprotein bind to 18:1-LPA, whereas bromolain and acyl carrier
protein did not yield detectable bands. In other experiments,
ovalbumin was shown to produce a detectable band (see table 4).
Other experiments indicate that FAD soy protein can bind LPA.
[0440] Size shift assay. Size analysis of LPA both with and without
protein was performed using a Superdex.RTM. S75 column Pharmacia
Biotech, Uppsala, Sweden). Soy LPA was combined in a chloroform
solution with 0.5 Ci .sup.3H-labeled 18:1-LPA (DuPont NEN Products,
Boston, Mass.), dried under vacuum and resuspended by sonication in
a buffered solution (50 mM ammonium bicarbonate, 154 mM sodium
chloride, pH 8.0) in the presence or absence of test protein. The
ammonium bicarbonate/sodium chloride solution was used as the
mobile phase at a flow rate of 1 ml/min. One-minute fractions were
collected for 30 minutes following sample injection. Fractions were
counted in scintillant solution using a liquid scintillation
counter (Beckman Instruments, Irvine, Calif.). Calibration was
performed using a standard solution containing purified proteins
(BioRad, Hercules, Calif.).
[0441] As shown in FIGS. 10A, 10B, 10C and 10D, soy-LPA alone
eluted at 22-26 minutes after sample injection, corresponding to a
size of approximately 1.3 kD). When a LPA/BSA mixture was applied
to the column, radioactivity eluted primarily in the 10 to
12-minute fractions, indicating that most of the LPA was bound to
BSA. A LPA/FAD soy protein mixture applied to the column resulted
in an elution profile with two peaks, one corresponding to the
position of unbound LPA, and the other shied. A cytochrome c/LPA
mixture gave an elution profile similar to that of soy-LPA alone,
indicating that LPA does not bind to this protein.
EXAMPLE 8
Anti-Apoptotic Activity of Five Phospholipid Mixture in an Organ
Reservation Solution
[0442] In order to determine the apoptotic activity of the claimed
invention, the following experiment was performed. The cell assay
is described in Example 2. Exponential growth phase was assured by
seeding at 2000 cells per 1 ml (5 ml for a 60 mm culture plate)
five days prior to the beginning of the experiment. At T=0,
cultures were transferred to serum-free medium, as an apoptosis
stimulus, and seed extracts were added. Controls included 10.sup.-7
and 5.times.10.sup.-8 M 12-O-tetradecanoyl phorbol-13-acetate TPA)
to ensure the responsiveness of the cell culture. The samples were
added to serum free medium and sterile filtered prior to addition
to the cultures. Assays were performed in triplicate or
quadruplicate. Analyses of the cell responses were made between 18
and 28 hours of serum deprivation with Five Phospholipid Mixture
alone in Basal Medium Eagle's (BME) culture medium or supplemented
with 5% or 25% of the Cardiosol.TM. organ preservation solution
described in U.S. Pat. No. 4,938,961. Two assays were performed on
each cell culture plate consisting of differential cell counts.
[0443] 1. All non-adherent or loosely adherent cells were removed
from the culture dish and counted by appropriate techniques,
typically counting by electronic particle counting instrument.
These were the apoptotic cells, the serum deprived released cells
(SDR), released by the action of cultivation in serum-free medium.
Approximately 95% of these released cells were apoptotic as shown
by both ultrastructure analysis and DNA fragmentation analysis.
[0444] 2. The remaining adherent cells (ADH) were exposed to a
buffered, typically pH 7.3, balanced salt solution such as Hanks
Balanced Salt Solution without calcium and magnesium salts
containing 0.05% trypsin and 0.53 -mM ethylene diaminetetraacetric
acid (EDTA). Each culture was incubated at either room temperature
or 37.degree. C. on a rocking platform to ensure uniform
distribution of the trypsin reagent over the culture surface. After
a standardized period of time, typically 10 minutes, the released
cells were removed from each culture dish and measured by the same
means as described above, typically electronic particle counting.
This ADH cell count was comprised of both trypsin resistant and
trypsin sensitive cells as described in PCT Publication No. WO
94/25621.
[0445] Anti-apoptotic activity is expressed as the calculated
concentration of material ((g/ml of media) required to save 50% of
the cells released on serum free treatment.
EXAMPLE 9
Preparation and use of Five Phospholipid Mixture in PEG20L
[0446] Twenty milligrams of Five Phospholipid Mixture stored in
CHCl.sub.3 (20 mg/mL) under Argon was dried in a glass tube with
vacuum concentrator (Savant) with gentle heat. This was sonicated
for 5 minutes under an Argon blanket in 1 mL of buffer (50 mM
NH.sub.4HCO.sub.3, 104 mM NaCl, 250 (M EDTA) 5 mL of which had been
bubbled for 5 minutes with Argon.
[0447] 250 (L (5 mg) of the Five Phospholipid Mixture sonicate was
added under a stream of Argon to 100 mL of 10% PEG20L which had
been prepared in water, ultrafiltered then sterilized by filtration
(0.22 (M) and stored anaerobically (under Argon in Ar-filled bags).
The resulting 50 (g/mL Five Phospholipid Mixture in 10% PEG20L was
packaged under a stream of Argon into 10 mL autoclaved amber glass
vials in 2 mL and 10 mL amounts and sealed with autoclaved butyl
rubber septa/aluminum rings. Prior to use, the septa had been
subjected to 2 hours of high vacuum then scaled in an aluminum bag
filled with Argon in order to displace as much dissolved oxygen as
possible. The Five Phospholipid Mixture/PEG vials were labeled then
individually packaged into aluminum bags filled with Argon and
stored at 4.degree..
EXAMPLE 10
Activity of LPA and PEG in In Vitro C3H/10T1/2 Assays
[0448] All the phospholipid mixtures were prepared by combining the
desired phospholipids in solution with organic solvent then drying
down the mixture with gentle heat (45.degree. C.) and high vacuum
followed by sonication into aqueous suspension at 5 to 20 mg/mL. In
some cases, the phospholipids were then mixed with serum-free
culture medium containing PEG of the indicated molecular weight,
concentration and type of PEG noted in the figures and prepared as
described above. The results obtained are depicted in FIG. 11 and
Table 3.
EXAMPLE 11
In Vitro Cardiomyocyte Assays Isolation of Rat Neonatal
Cardiomyocytes
[0449] Cardiomyocytes were prepared from hearts of day-old Sprague
Dawley rats by trypsinization and mechanical disaggregation
(Simpson, (1985)) Circ. Res. 56: 884-894. The cells were
resuspended in MEM, 1.times.MEM vitamins (Gibco), 5% fetal bovine
serum and 50 U/ml penicillin-G and pre-plated for 30 minutes to
reduce contamination of non-myocytes. The non-adherent cardiac
myocytes were separated and seeded in 2 ml in 35 mm dishes at a
density of 3.5.times.10.sup.5 viable cells per ml. The cells were
allowed to adhere for 16-24 hours in a 37.degree. C./5% CO.sub.2
humidified incubator.
Cardiomyocyte Treatment
[0450] For serum deprivation, the medium was replaced with fresh
serum-free RPMI, whereas serum/glucose deprivation was performed
using glucose-free RPMI. The induction of cell death by adriamycin
or C.sub.2-Ceramide was accomplished by the addition of the agent
prepared in serum-free RPMI. As a model of ischemia, cultures in
serum and glucose free RPMI were placed in an airtight chamber and
the latter was continuously perfused with oxygen-free gas overlay
of 95% N.sub.25% CO.sub.2 for 8 hours at 37.degree. C. To model
reperfusion of the ischemic cells, 10% fetal bovine sewn, 2 g/L of
glucose were added and the cultures were returned to a normal
oxygen gas overlay (37.degree. C./5% CO.sub.2) in a humidified
incubator for 16 hours.
[0451] To investigate effects of LPA, LPA plus other phospholipids,
PEG or their mixtures on cell death, various combinations of these
components were added to cells in serum free medium at the
beginning of cytotoxic treatment.
Measurement of Cell Death
[0452] Because cardiomyocytes are terminally differentiated
non-dividing cells, viability was determined by measurement of the
decrease in the relative number of adherent cells. The measurement
of non-adherent cells was found to be less reproducible cause of
their rapid lysis following release from adhesion substrate.
Adherent cardiomyoctes were collected from culture dishes using
0.25% Trypsin/0.05% EDTA and counted on Coulter Counter ZM and
Coulter Channelyzer 256.
[0453] The results were as follows:
[0454] 1. Five Phospholipid Mixture prevents cardiomyocyte death
induced by serum/glucose deprivation or by ischemia/reperfusion
(FIG. 14).
[0455] 2. 0.3-2% PEG with molecular weight 20,000 kDa enhances the
protective effect of Five Phospholipid Mixture against
serum/glucose deprivation induced cell death (FIG. 13).
[0456] 3. Mixtures of PEG with Five Phospholipid Mixture and
18:1-LPA equally protect cardiomyocyte death induced by ischemia
and reperfusion (FIG. 14). TABLE-US-00005 TABLE 5 Concentration
ranges Maximum effect relative for cardiomyocyte to 18:1 LPA in
Compound # protection (.mu.M) cardiomyocytes 30 .sup. 1-10 150% 78
.sup. .01-3 130% 68 .sup. .003-10 100% 48 .sup. .03-10 100% 18:1
LPA .sup. 0.3-10 100% 12 .sup. 1-10 100% 11 .sup. 1-10 100% 19
.sup. 1-10 100% 16 .sup. 1-3 100% 66 .sup. .03-1 60% 45 30 30% 46
ND 24 ND 10:0 LPA ND 23 ND 15 ND 29 ND 25 ND 36 ND 3 ND 4 Toxic
(3-30 .mu.M) ND: No protection detected.
[0457] 4. Various LPAs prevent cardiomyocyte death induced by
serum/glucose deprivation (results appear in Table 5).
[0458] 5. Five Phospholipid Mixture educes cardiomyocyte death
induced by ceramide and the addition of PEG to the Five
Phospholipid Mixture enhances this effect.
[0459] In these experiments, 10 (M ceramide was added to cell
cultures of cardiomyoctes alone or in the presence of: (a) 0.3%
PEG, (b) 2% PEG, (c) 5 (M LPA presented as Five Phospholid Mixture,
(d) 5 (M LPA presented as Five Phospholid Mixture plus 0.4% PEG,
and (e) 5 (M LPA presented as Five Phospholid Mixture plus 2% PEG.
Controls also included only serum and glucose without any active
ingredients. In other experiments, 15 (M ceramide was added to
cardiomyoctes alone or in the presence of: (a) 0.3% PEG, (b) 2%
PEG, (c) 5 (M LPA presented as Five Phospholipid Mixture, (d) 5 (M
LPA presented as Five Phospholid Mixture plus 0.4% PEG, and (e) 5
(M LPA presented as Five Phospholid Mixture plus 2% PEG.
[0460] It is clear from these results that LPA has anti-apoptotic
activity as shown in the cardiomyocyte assay, and that the
combination of LPA with other phospholipids retains that activity.
Additionally, the presence of PEG improves the activity of LPA.
EXAMPLE 12
Regional Ischemic Dog Heart Model
[0461] The experimental model used was a regional ischemic dog
heart (mongrel male hound, approximately 20 kg) with the left
anterior descending coronary artery clamped distal to the first
diagonal branch rendering a portion of the left ventricle ischemic.
The clamp was maintained for 90 minutes then released gradually
over a 5 minute period. A narrow canula was placed into the left
main coronary artery through which Five Phospholipid Mixture (5 mL
of 50 (g/mL in 10% PEG) was infused starting 75 minutes after the
start of the ischemic period, and continuing for 75 minutes in
total (approximately 67 (L/min., approximately 3.3.times.g Five
Phospholipid Mixture/min.). The total dose was 250 (g Five
Phospholipid Mixture and 500 mg PEG20L.
Determination of Infarct Size
[0462] After staining and fixation, the atria and right ventricle
are removed from the left ventricle. After removal of the apex, the
portion of the left ventricle distal to the mitral apparatus is cut
into 5 transverse sections of equal thickness. Evans Blue dye
injected into the circumflex vessel stains the circumflex territory
blue. TTC stains viable LAD territory red, while the infracted
myocardium will remain unstained (white). Sections are weighed and
photographed for documentation and future verification of results.
Computer assisted planimetry is used to measure the areas of the
circumflex, viable LAD and infracted LAD territories. Calculation
of infarct size based upon the assumption that the area of
infarction in the sectioned plane is representative of the mass of
infracted tissue in that plane.
Study Protocols
[0463] 1. Study-drug group; dogs are treated with Five Phospholipid
Mixture 50 (g/ml and PEG 100 mg/ml as a bolus plus infusion (at 4
ml/hr) starting 15 min before reperfusion and going on for 60 min
into the reperfusion period.
[0464] 2. Placebo group: dogs receive placebo following the same
modalities described for the study drug group.
[0465] 3. SOD-Catalase group: dogs receive SOD plus catalase
following the same modalities described by Simpson et al. (1987)
Fed. Prod. 46:7, 2413-21.
[0466] The rationale for using a third group of dogs in the present
protocol resides in the fact that the SOD+Catalase group represents
a positive control. In fact it has been shown that the combination
of oxygen radical scavenger is very efficacious in limiting
myocardial reperfusion injury in the canine mode.
[0467] The results obtained in the dog heart experiments are
depicted in FIG. 15.
EXAMPLE 13
Evaluation of Infarct Measurement and EKG Alterations in a Regional
Ischemic Pig Heart Model
[0468] This example evaluates the anti-apoptotic activity of PEG20L
in a regional ischemic pig heart model. Adult Yucatan miniature
pigs (approx. 30 kg each) were treated with general anesthesia
followed by balloon catheterization to create an occlusion of the
first obtuse marginal branch of the circumflex coronary artery.
After 105 minutes of occlusion treatment with control, infusion of
PEG20L (100 mg/mL), or PEG20L and Five Phospholipid Mixture (100
mg/mL PEG+50 (g/mL Five Phospholipid Mixture) solution was started
through the guide catheter into the aortic root in close
approximation to the occlusion. The solutions were introduced
initially by a bolus equal to 25% of the hourly drug dose, followed
by continuous infusion through the guide catheter for 135 minutes.
The occlusion was removed after 120 minutes (15 minutes after the
start of drug delivery).
[0469] Heart function was monitored by EKG and cardiac output was
determined by contrast imaging. The animal was euthanized 1-3 days
after the occlusion. The heart was removed for histology and
examined. Infarct size was determined by a method of triphenyl
tetrazolium chloride (TTC) staining for the area at risk and actual
infarct. The area not at risk was stained with a blue pigment or
Evan's Blue dye.
[0470] Pigs were treated with one of the following test regimens
(displayed as total dose): [0471] a) 250 mg/kg PEG20L only (10%
ultrafiltered in water) (n=2); [0472] b) 125 .mu.g/kg Five
Phospholipid Mixture+250 mg/kg PEG20L (n=3).
[0473] Four additional pigs were occluded but not treated with any
solution, for use as controls.
[0474] Of the four controls the mean infarct size (as % risk zone)
was 34.5% (7.2%. The controls also showed a marked unresolved S-T
segment shift during the ischemic period which reflects severe
myocardial ischemia and infarction.
[0475] In contrast to the controls, two effects were noted in the
treatment groups. First, in some animals, within 2 minutes of the
start of drug infusion there was a normalization of the EKG signal
disturbances in the occluded hearts. During the ischemic period the
normal EKG signal shifted to one in which there was an elevation in
the S-T segment indicating an impairment of myocyte repolarization.
This elevation disappeared in several pigs treated with PEG20L and
Five Phospholipid Mixture. Some pigs that were treated were not
included in the infarct measurement data due to technical
difficulties in determining the infarct size. However, some of
these animals did exhibit a reduction in S-T segment elevation.
[0476] The second effect noted was a decrease in infarct size in
the treated animals when compared to the control animals (FIG. 12).
In three Five Phospholipid Mixture treated pigs with measurable
infarcts, the infarct sizes were reduced to only 12.3% (5.1%, in
contrast to 34.5% in the controls.
[0477] It is clear from this example that LPA and LPA in
combination with PEG are effective therapeutics for the example of
cardiac ischemia, as both protect tissue from infarct damage and
reduce the level of S-T segment shift measured by
electrocardiogram.
EXAMPLE 14
Evaluation of Infarct Measurement in a Regional Ischemic Rat Heart
Model
[0478] The left anterior descending coronary artery (LAD) of each
Sprague dawley female rat (225-250 grams) was occluded for 20
minutes followed by 2 hours of blood reperfusion. The heart was
then removed and the non-ischemic area not at risk for developing
an infarction was delineated by perfusing Evan's Blue through all
but the LAD of the coronary vasculature. The infarct and area at
risk for infarction was identified by perfusing the LAD with
triphenyltetrazolium chloride which is metabolized to a red dye in
viable tissues while remaining uncolored within the necrotic zone.
The heart was then fixed and sliced in order to permit planimetry
measurements of both the area at risk for developing the infarction
and the actual infarct size itself. The results are expressed as
infarct size as a proportion of the area at risk.
[0479] At start of reperfusion a mixture of Five Phospholipid
Mixture (1 mg/kg) (preparation described in Example 3) and PEG (8
mg/kg) was injected over an approximately two minute period into
the left ventricle through a 35 gauge needle. Control rats received
a similar volume injection of placebo containing 10 mM citrate/110
mM NaCl, pH 6.5.
[0480] The results, in FIG. 16, show that treatment with LPA, in a
phospholipid mixture, combined with PEG resulted in a 42% reduction
in infarct size relative to placebo-treated rats (p=0.005, n=4 in
both groups, two tailed T-test). The areas at risk in the two
groups were similar (p=0.42).
EXAMPLE 15
Evaluation of Infarct Measurement in a Regional Ischemic Rabbit
Heart Model
[0481] Male New Zealand white rabbits were initially anesthetized
using a mixture of ketamine (400 mg per rabbit) and xylazine (20 mg
per rabbit) administered intramuscularly in two doses,
approximately 10 minutes apart. Throughout the study, a level of
deep anesthesia is maintained using sodium pentobarbital given
intrapeitonally at a dose of approximately 50 mg/hour. All rabbits
were intubated and mechanically ventilated using room air
supplemented with oxygen. Fluid filled catheters were placed into
the jugular vein to administer fluids. A catheter was also placed
into the left carotid artery to measure heart rate and blood
pressure and to obtain reference blood samples during regional
myocardial blood flow measurements. The chest was opened through
the left fourth intercostal space. Then, the pericardium was
incised and the heart was exposed. A large anterolateral branch of
the circumflex artery, or the circumflex artery itself was
identified and encircled with a 4-0 silk suture. The ends of the
suture were threaded through a piece of flanged tubing, forming a
snare, which was later used to occlude the artery. A catheter was
then placed into the left atrial appendage to inject the control or
test solution, the radioactive microspheres and blue pigment at the
conclusion of the procedure.
[0482] After the surgical preparation, the rabbits were stabilized
for ten minutes. At this time, the rabbits were randomized into
either the treatment or control group. The treatment solution
contained 1 mg/ml of the Five Phospholipid Mixture, described in
Example 3, above, 8 mg/ml PEG 20L, in 10 mM citrate 110 mM NaCl.
The control solution contained 10 mM citrate and 110 mM NaCl. Five
minutes before occlusion, a bolus dose of 1 mg/kg body weight of
either the Five Phospholipid Mixture/PEG solution or the control
solution was administered into the left atrium. Immediately
following the bolus dose, infusion was started at the rate of 1
mg/kg for one hour into the left atrium. Baseline hemodynamics and
core body temperature were recorded. Next, the artery was occluded
for 30 minutes. Following this, the hearts were reperfused for
three hours. Heart rate and arterial blood pressure were recorded
at 20 minutes of occlusion and at time points during reperfusion at
a 25 mm/second paper speed. Regional myocardial blood flow
measurements were performed on two occasions; during coronary
occlusion (20 minutes) to confirm no blood flow in the ischemic
zone, and during early reperfusion (30 minutes) to confirm reflow
in the same zone. At the end of three hours of reperfusion, the
coronary artery is reoccluded. Next, 4 ml of 50% Unisperse blue
(Ciby-Geigy, Hawthorne, N.Y.) were infused through the left atrial
catheter and allowed to circulate throughout the vascular system.
The rabbit was then euthanized by an overdose intravenous injection
of xylazine (300 mg) followed by 12 mEq of potassium chloride given
into the left atrium. Prospective exclusion criteria included an
ischemic risk zone of less than 10% of the left ventricular weight,
a regional blood flow of more than 0.2 ml/minute/g in the risk zone
during coronary artery occlusion (lack of ischemia), or a regional
blood flow of less than 0.4 ml/min/g in the risk zone at 30 minutes
of reperfusion (failure to reperfuse).
[0483] Infarct size was evaluated as follows. The right ventricle
was trimmed off and the left ventricle was sliced transversely into
seven or eight sections, approximately two millimeters in
thickness. These slices were photographed to identify the ischemic
risk regions (uncolored by the blue pigment) and the non-ischemic
regions (colored by the blue pigment). The slices were then
incubated in a 1% solution of triphenyltetrazolium chloride
preheated to 37.degree. C. for 10 minutes and rephotographed for
analysis of area of necrosis. All sections were later fixed in
formalin. These photographic slides were projected and areas of
risk (AR) and areas of necrosis (AN) were tad by planimetry. The
planimetered areas of each slice were multiplied by the weight of
the slice and then summed. Because infarct size is measured from
photographic slides, the entire left ventricle was used for the
analysis.
[0484] Regional myocardial blood flow (RMBF) was measured as
follows. Just before measuring RMBF, during occlusion, the atrial
catheter was disconnected from the treatment infusion pump.
Radioactive microspheres were injected via the atrial catheter. The
catheter was then reprimed with approximately 0.2 ml of the drug
treatment, and the catheter was reconnected to the pump and
infusion continued.
[0485] Regional myocardial blood flow was measured with 11 (m
radioactive microspheres labeled with .sup.141Ce, .sup.96Nb or
.sup.103Ru (New England Nuclear, North Billerica, Mass.), using
approximately 500,000 per injection. These microspheres were
injected into the left atrial catheter. At the same time, a
reference blood sample was obtained from the carotid artery at 2.06
ml/minute. The blood removed during RMBF measurement was about 5
ml. These volume changes do not cause changes in systemic arterial
pressure. At the end of the protocol, after the photographic slides
had been taken and the heart weight, myocardial samples were cut
from the center of the non-ischemic and the ischemic regions,
weighed and counted with the reference blood samples in a well
gamma counter. Blood flows at each interval, for ischemic and
non-ischemic tissues, were then computed and expressed in
ml/minute/g.
[0486] The results were as follows. With a risk zone of
approximately 25% of the left ventricle, treatment with the control
solution resulted in infarct size of approximately 40% of the risk
zone on average, as compared to an average of only approximately
25% of the risk zone in the subjects treated with the LPA
containing PEG and other phospholipids.
[0487] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be apparent to those skilled in the art
that certain changes and modifications can be practiced. Therefore,
the descriptions and examples should not be construed as limiting
the scope of the invention, which is delineated by the appended
claims.
* * * * *