Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/66—Phosphorus compounds

Definitions

• compositions containing lysophosphatidic acid or analogs and derivatives thereof, which have been found to exhibit anti-apoptotic activity and or to preserve or restore cell, tissue or organ function.
• LPA lysophosphatidic acid or analogs and derivatives thereof
• 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.
• 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.
• LPI lysophosphatidylcholine
• LPC lysophosphatidylcholine
• 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.
• 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.
• apoptosis 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
• HTV hypertension
• 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.
• 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.
• Apoptosis 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. Vvnaxv-ty Molekylyarnaya Biologiya 30:285-295 (1996); VauxandStrasser N S93:2239-2244 (1996); Nunez etal. (1994) Immunol. Today 15:582-588; and Whyte (1996) Trends in Cell Biol. 6:245-148.
• 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 and Differentiation 2:235241; Umansky et al.
• IGF Insulin-like growth factors
• 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.
• 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.
• OPS cold organ preservation solution
• 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.
• the cardiopulmonary bypass is discontinued and the recipient's chest closed.
• Most non-transplant surgical procedures on the heart 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.
• 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.
• the donor organ is cooled rapidly by flushing cold solutions through the organ's vascular system and maintaining the organ at temperatures near 0°C for the purpose of greatly slowing the metabolic rate.
• the flush solution composition is designed to cause the heart to rapidly stop beating as well as to preserve it.
• Wicomb et al. reported the beneficial effects of a solution of PEG 8000 and horseradish peroxidase on rabbit hearts preserved by oxygenated low pressure perfusion for 24 hours. Wicombetal. (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.
• HES hydroxyethyl starch
• 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' [(l-methyl-ethylidene)bis(4, l-phenyleneoxy methylene)]bis-, polymer with (-hydro-(-hydroxypoly(oxy-l,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.
• An improved OPS, CardiosolTM 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 United States Patent No.4,938,961.
• CardiosolTMheart preservation solution contains 5% or 10% by weight PEG 20M (Union Carbide Chemicals and Plastics Co., Inc., Washington, WV), 40 mM sodium, 125 mM potassium, 5 mM magnesium, 25 mM phosphate, 5 mM sulfate, 100 mM lactobionate, 30 mM raffinose, and 3 mM glutathione.
• PEG 20M Union Carbide Chemicals and Plastics Co., Inc., Charleston, WV
• 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 ADDS.
• 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
• the present invention satisfies this need and provides related advantages, as well.
• 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.
• LPA lysophosphatidic acid or analogs and derivatives thereof
• compositions containing a compound of the following formula:
• each W is independently selected from one of the following structures II- V
• 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-carbon bonds are unsaturated, and mixtures thereof. Even more specifically, the composition comprises
• composition contains a compound of the following formula:
• each W is independently SH, OH, OCH 2 CH(NH 2 )C0 2 H, OCHCH 3 CH(NH 2 )C0 2 H, OPO 3 H 2 , OP0 2 HOP0 3 H 2 or Q, wherein when one W is Q, the other W is OH, and wherein Q is one of the following structures:
• Y is O or S
• the composition comprises a compound selected from the group consisting of reverse ester-LPA, reverse thioester-LPA or a salt of either.
• the composition comprises: 3 Oleyl l-thiophosphoryl-2-0- methyl-r ⁇ c-glycerate, or a salt thereof.
• 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.
• 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.
• pharmaceutically effective agents such as, without limitation, drugs, antibiotics, wound healing agents and antioxidants.
• compositions of the present invention 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.
• compositions of the compounds of the present invention 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.
• 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).
• apoptosis ischemia
• traumatic injury or 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 reper
• Treatment can comprise administering to the patient at least one intravenous bolus of an effective amount of the composition.
• methods of the present invention decrease apoptosis-related problems associated with immunosuppressing viruses, chemotherapeutic agents, or radiation and immunosuppressive drugs.
• 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.
• 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.
• "LPA" refers to 18: 1-LPA in all figures.
• the white sections represent adherent cells and the dark sections represent non-adherent cells.
• Fig. 1-8 and 11 the white sections represent adherent cells and the dark sections represent non-adherent cells.
• 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 to serum-free medium to which 18: 1-LPA was added as a 10% (by 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.
• 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.
• Elirex Five Phospholipid Mixture
• PG phosphotidyl glycerol
• PG/PC phosphotidyl glycerol/phosphotidylcholine
• Fig. 4 is a bar graph depicting protection of serum-deprived C3H/10T1/2 cells by 18: 1-LPA and lysophosphotidylserine (LPS).
• Fig.5 is a bar graph depicting protection of C3H/10T1/2 cells from serum-deprivation by soy-derived, 18:1 and 16:0 LPA.
• 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.
• Fig. 7 is abar graph depicting protection of C3H/10T1/2 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,
• 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.
• Fig. 9 depicts an autoradiograph of a non-denaturing polyacrylamide gel of proteins separated following incubation with 3 H-labeled 18: 1-LPA.
• Figs. 10A, 10B, 10C and 10D are graphs showing the elution profiles of 3 H-18: 1-LPA, with and without various proteins, from a Superdex S75 column.
• 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").
• 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
• Fig. 14 is a graph depicting the treatment of rat neonatal cardiomyocytes with PEG plus
• 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).
• 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 (soUd bar) or a dose of the combination of Five PhosphoUpid Mixture and PEG (gray bar, Usted in key as "APM/PEG").
• lysophosphatidic acid is the major active component of phosphoUpid 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.
• LPA lysophosphatidic acid
• 'Treating apoptosis is herein defined as adrninistering 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.
• phosphoUpid structures are weU defined in the Uterature, they can vary with respect to Upid 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.
• LPA has the following general structure:
• 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 ⁇ group, or, in the case of the salt, one or more hydrogens are replaced, for example with Na + .
• the 1 carbon will contain an acyl ester of fatty acids.
• 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.
• LPA includes LPA having any one of a variety of fatty acids esterified at the C 1 position.
• Examples include LPA wherein the fatty acid ester is lauryl, myristyl, palmityl, stearyl, palmitoleyl, oleyl or linoleyl.
• the composition where R is 18 and UB is 1, shall herein be referred to as "18: 1-LPA").
• suitable phospholipids see any chemical catalog of a phospholipid suppUer, for instance, the (1994) Avanti Polar Lipids Inc. catalog, particularly pages 14 and 21.
• R can be an unsubstituted or substituted, saturated or unsaturated, straight or branched chain alkyl having from about 10 to about 24 carbon atoms.
• 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.
• 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.
• W is OH;
• Z is OH, SH, NH 2 , halogen, OPOjH H,
• 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 2 ) ⁇ n O) p (CH 2 ) I -W 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 0(CH 2 ) q CH 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.
• 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.
• ethers and thioethers at the Cl position having the structure: o
• X is O, S, or CH 2 ;
• W is OH
• Z is OH, CH 2 OH, SH, NH 2 , halogen, OPOsHj, H or SOjH
• 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 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 0(CH 2 ) q CH 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.
• 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.
• glycero LPA analogs having the structure:
• W is OH
• Z is OH, SH, NH 2 , halogen, OPO ⁇ , H or SO ⁇
• 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 ⁇ p OJ ⁇ CH ⁇ p V 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 0( ⁇ ⁇ 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.
• R is between aboutlO 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.
• LPA analogs containing an amide bond and having the structure:
• 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 2 ) m O) p (CH 2 ) I -W 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 0(CH 2 ) q CH 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.
• 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.
• each W is independently selected from one of the following structures (XI, XH, Xffl, XTV): X Z
• W is not ethanolamine, glycerol, or choline.
• LPA 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. CycUc derivatives can also be synthesized by methods known in the art.
• salts of the phosphoUpids 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(2hydroxyethyl)ammonium, and fromethamine (tris(hydroxymemyl)aminomethane). Particularly preferred are sodium and ammomum salts.
• 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.
• phospholipids of the present invention can be isolated from a particular species of plant, plant extract or organ within a plant.
• U.S. Patent No. 3,365,440 describes extraction oflipids from soybeans.
• U.S. Patent 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.
• 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 phosphoUpids from plant sources.
• PhosphoUpids can also be derived from animal sources.
• the animal is a mammal.
• the phosphoUpids are derived from Uver cells.
• Such phosphoUpids 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.
• PhosphoUpids in general, and LPAs in particular, can also be derived from blood.
• the phosphoUpids 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 l-0-[3H]Palmitoyl-L-glycerin-3-phosphate (L-3-Lysophosphatidsaure), ChemJBer., 347 (1966) 94-101. The synthesis of various cyclic phosphate LPAs is described in
• 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 Upids, phospholipids or phosphate. Various suitable methods are outlined in Kates (1972).
• the phosphoUpids must be of sufficient purity such that, when mixed at a total concentration of about 10 mg/rnL, the mixture can be sonicated as described below to provide a relatively translucent solution.
• the phospholipids are at least 90% pure, more preferably, they are at least 95% pure and, most preferably, they are at least 99% pure.
• the references cited herein are incorporated by reference in their entirety.
• 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), Upid membrane structure forming compounds, polypeptides, modified polypeptides and polymers.
• 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.
• 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.
• the phosphoUpids PA; PI; lysophosphatidic acid; LPI; and LPC are present in the composition in a range of ratios from 0-20:5-20:2-16:0-4:0-8, respectively.
• these phosphoUpids are in a ratio of approximately 2-15:8-15:6-10:2-4:2-8, respectively.
• these phospholipids are in a ratio of approximately 10:10:8:2:4, respectively.
• 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-l,2-ethanediyl)), is known by numerous designations including macrogel; PEG; Carbowax; Jeffox; Nycoline; Pluracol E; Poly-G; Polyglycol E; and
• PEG refers to the Uquid and solid polymers of the general formula H(OCH 2 -CH 2 ) folkOH, where n is greater than or equal to 4. In general, each PEG is foUowed by a number which corresponds to its average MW. PEG syntheses are described for instance in Hibbert (1939) J. Am. Chem. Soc. 61:1905-1910. For review, see also, PoweU, III in Handbook of Water-Soluble Gums &Resins, R.L. Davidson ed. (McGraw-HiU, 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.
• PEGs are clear, viscous Uquids or white soUds that dissolve in water to form transparent solutions. They are soluble in many 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 Usted herein are given in number averages rather than weight averages.
• 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' [(l-methyl-ethylidene)bis(4,l-phenyleneoxy methylene)]bis-, polymer with (-hydro-(-hydroxypoly(oxy-l,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, ClariantHoechst 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.
• PEG has been used in a number of pharmacologic applications.
• Conjugation to PEG also increases the solubility and biological half-Ufe of cytokines.
• Conjugates of PEG and glucocerebroside have been formulated for treating Gaucher's disease.
• PEG has also been conjugated to such enzymes as adenosine deaminase, amidase bovine and asparaginase, 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.
• compositions comprising therapeutically effective amounts of LPA and PEG.
• the PEG to LPA weight ratio is such that the LPA therapeutic activity is potentiated by the PEG.
• the PEG to LPA weight ratio is such that the LPA therapeutic activity is potentiated by the PEG.
• LPA weight ratio of the composition is preferably 1-100,000: 1 and most preferably 10-10,000: 1.
• PEG can be in the molecular weight range from about 6,000 to about 500,000.
• 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.
• PEG20L is meant a substantiaUy 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.
• 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 deUvery.
• 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.
• molecular weight refers to the average molecular weight of a number of molecules in any given sample, as commonly used in the art.
• 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. PEG20L is suppUed 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 availabiUty of oxygen.
• GPC gel permeation chromatography
• PEG PEG is present in an effective concentration and is essentially free of impurities.
• impurities is meant the products produced when PEG is oxidized.
• smaU molecular fragments are formed such as formate, methyl formate, formaldehyde, acetaldehyde, etc., all of which are defined here as impurities.
• 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.
• dialysis which removes not only small molecular impurities such as formaldehyde, but also removes hydroperoxides, as evidenced by spectrophotometry.
• conductivity is measured. When dialysis is effective, conductivity drops markedly to 10-20 microsiemens.
• Removal of impurities by dialysis can be performed through a CeUulose Acetate HoUow Fiber Dialyzer (Baxter Model CA 110) but the same procedure can also be performed by
• a 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.
• PEG is present in a therapeutically effective amount.
• an effective amount is the amount required to improve the ability of an OPS to preserve organs.
• the preferred composition is where phosphoUpids 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%.
• 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.
• Lipid Membrane Structures Lipid membrane structures (LMSs), including Uposomes, micelles, multilamellar vesicles and cellular membrane isolates, have been used as vehicles for delivering therapeutic agents.
• LMSs Lipid membrane structures
• LMSs are lamellar lipid particles wherein polar head groups of a polar Upid are arranged to face an aqueous phase of an interface to form membrane structures.
• a "Uposome” or “Upid vesicle” is a small vesicle bounded by at least one and possibly more than one bilayer Upid membrane. Liposomes are completely closed Upid bilayer membranes which contain entrapped aqueous volume. Liposomes are vesicles which can be unilameUar (single membrane) or multilamellar (onion-like structures characterized by multiple membrane bUayers, each separated from the next by an aqueous layer).
• the bilayer is composed of two Upid monolayers having a hydrophobic "tail” region and a hydrophilic "head” region.
• liposomes In the membrane bilayer, the hydrophobic (nonpolar) "tails” of the Upid 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. These Upid 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 lipiddetergent complexes.
• a Uposome can also optionally comprise additional components associated with the outer surface, such as a tissue targeting component.
• a "Upid 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, and other amphipathic molecules, providing the general structure of the membrane is a sheet of two hydrophilic surfaces sandwiching a hydrophobic core.
• Suitable lipids include, but are not limited to, those discussed in Lasic (1993)
• the lipid bilayer making up the Uposome can comprise phospholipids, glycolipids, steroids, and their equivalents .
• the lipid bilayer can also be made of amphipathic proteins and lipidsoluble chemicals.
• a composition is chosen that allows the membrane to be formed with reproducible quaUties, such as diameter, and is stable in the presence of elements expected to occur where the Uposome is to be used, such as physiological buffers and circulating molecules.
• the Uposome is resilient to effects of manipulation by storage, freezing, and mixing with pharmaceutical excipients.
• Lipids suitable for incorporation into lipid membrane structures include, but are not Umited to, natural, semi-synthetic or synthetic mono- or di-glycerophospholipids including phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidylinositols, phosphatidic acids, phosphatidylserines, glycero- and cardioUpins. Sphingolipids such as sphingomyelin and cerebrosides can also be incorporated.
• lipids can have alternative stereochemistry with, e.g., the phospho group at the sn-l or sn-2 positions.
• the hydrophobic chains can be attached to the glycerol backbone by acyl, ether, alkyl or other linkages.
• Derivatives of these Upids are also suitable for incorporation into Uposomes.
• 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.
• cholesterol and other amphipathic steroids, bolaamphiphiles 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.
• cholesterol and other amphipathic steroids, bolaamphiphiles include, but are not limited to, haloalkyl derivatives, including those
• Liposomes can be composed of a single Upid or mixtures of two or more different Upids.
• the Upid bilayer of the Uposome is formed primarily 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 sphingomyeUn (SM).
• the LMS can further comprise additional Upids such as phosphatidylinositol (PI), phosphatidylserine (PS), or cardioUpin (diphosphatidylglycerol).
• PI phosphatidylinositol
• PS phosphatidylserine
• cardioUpin diphosphatidylglycerol
• SM can be replaced with a greater proportion of PC, PE, or a combination thereof.
• PS can optionally be replaced with phosphatidylglycerol (PG).
• PG phosphatidylglycerol
• 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 phosphoUpid 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 phosphoUpid can be used. However, phospholipids containing particular fatty acid moieties may be preferred for certain applications.
• 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 Upid is between about 0.5 and about 1.2 moles of cholesterol per mole of Upid. 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 crossUnked phospholipids.
• Liposomes for use in vivo are those with an enhanced ability to evade the reticuloendotheUal system, thereby giving them a longer period in which to reach the target cell.
• Effective Upid 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 monosialogangUoside GMl, glucuronide, or PEG.
• 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.
• Liposomes can also be provided with molecules at the surface that target them to the ceU of interest.
• Such smaU molecules can be attached by incorporating into the Upid bilayer a functionaUzed phosphoUpid (US . Patents 5,052,421 and 5,540,935) or a functionalized cholesterol
• Polypeptides can be attached covalently to the Upid bilayer (EP Patent 0036277), to a glycophosphohpid (U.S. Patent 5,374,548), to a carboxylated phosphoUpid (U.S. Patent 4,762,915), to a derivatized sterol (U.S. Patent 5,000,960), or to a peptide anchor (U.S. Patent 5,109,113).
• 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-Jensenetal. (1994) J. Membrane Biol. 140: 215-23; EP Patent 0047480; and U.S. Patent 5,252,348.
• Liposomes have been prepared with mammalian-derived peptides such as cytokines (U.S. Patent 5,258,499), transferrin (Stavridis et al.), antibody (Laukkanen et al. (1994) Biochem.
• mammalian-derived peptides such as cytokines (U.S. Patent 5,258,499), transferrin (Stavridis et al.), antibody (Laukkanen et al. (1994) Biochem.
• 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. Patent 5,252,348), reovirus M cell attachment protein (Rubas etal. (1990) ⁇ ⁇ /cro ⁇ «c ⁇ pj «/ ⁇ 'o j 7:385-395), influenza virus surface protein (WO 92/19267; EP 0047480; and Nussbaum etal.
• RSV respiratory syncytial virus
• the present invention also encompasses compositions comprising micelles and methods of using such compositions.
• MiceUes in aqueous solution both non-ionic, cationic and anionic, have been described in the literature in numerous pubUcations. Mittal (1977) Micellization, Solubilization andMicroemulsions 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) Ace.
• Micelles is a term appUed 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.
• a process for preparing liposomes containing LPA is as foUows.
• An aqueous dispersion of Uposomes is prepared from membrane components, such as phosphoUpids (e.g. phosphatidylcholine, phosphatidylglycerol, sphingomyelin and phosphatidylethanolamine) and glycoUpids according to known methods as disclosed. Ann. Rev. Biophys. Bioeng. 9:467 (1980).
• the Uposomes can further contain sterols (e.g., cholesterol and cholestanol), dialkylphosphates, diacylphosphatidic acids, stearylamine, (-tocopherol, etc., in the liposomal membrane.
• 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°C, followed by cooling to produce liposomes containing LPA in the liposomal membrane.
• 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.
• the Upid vesicles can be prepared by any suitable technique known in the art. Methods include, but are not Umited 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 unilameUar 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.
• the invention encompasses LMSs containing tissue or cellular targeting components.
• tissue or cellular targeting components are components of a LMS that enhance its accumulation at certain tissue or cellular sites in preference to other tissue or ceUular sites when administered to an intact animal, organ, or ceU 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.
• 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 ceUs and cancer cells.
• LMSs can be targeted to such cell types in various ways.
• 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.
• compositions of the present invention can additionally comprise surfactants.
• Surfactants can be cationic, anionic, amphiphiUc, 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 Umited to, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene castor oil derivatives, polyoxyethylene hardened castor oU derivatives, fatty acid sodium salts, sodium cholates, polyexyethylene fatty acid ester and polyoxyethylene alkyl ethers.
• Proteins other than serum albumin have been characterized or impUcated as having fatty acid/Upid binding capabiUties. These include the fatty acid binding proteins (FABPs), the Upid binding proteins, long-chain fatty acyl-CoA (LCFA-CoA) binding proteins, phosphoUpid transfer proteins, and Ca + /lipid binding proteins.
• FABPs fatty acid binding proteins
• LCFA-CoA long-chain fatty acyl-CoA binding proteins
• phosphoUpid transfer proteins and Ca + /lipid binding proteins.
• Lipid-binding proteins are a family of fatty acid and retinoid transport proteins. Some are intraceUular, for example, while others are secreted from the cell. IntraceUular lipid binding proteins include cellular retinoic acid binding proteins, CRABP
• the lipid-binding protein family includes many FABP.
• the FABP are relatively smaU proteins (13-15 kDa) capable of binding long-chain fatty acids (LCFA) and their coenzyme A and L-camitine esters .
• LCFA-CoA play an important physiological role in intermediary metaboUsm of fatty acids, but recent data indicate that they also can be potent regulators of ceU 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.
• 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.
• the protein is a lipid-binding or carrier protein.
• the protein can contain other modifications including, but not Umited 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 type, enhanced in vivo stability, and the like.
• Preferred proteins include, but are not Umited to, albumin, soy and plant proteins, cytochrome C, low density Upoprotein, acyl carrier protein and alphafeto-protein.
• Suitable polypeptides include any known in the art. Modified polypeptides contain any of a variety of modifications, including, but not Umited to glycosylation, phosphorylation, myristylation, sulfationandhydroxylation. 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.
• BSA bovine serum albumin
• 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.
• 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 3 H-thymidine by cells treated with LPA with or without the addition of protein.
• LP Aprotein 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.
• the protein concentration of the LP A/protein compositions can be from about 0.001 to about 50 mg/mL, more preferably from about 0.01 to about lO mg/mL, even more preferably from about 0.05 to about l.Ojmg/mL.
• the protein component of the LP A/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 LP A/protein complex.
• the protein is at least about 75% pure, more preferably at least about 85% pure and still more preferably at least about 95% pure. 5.
• Other Optional Components are also be from about 0.001 to about 50 mg/mL, more preferably from about 0.01 to about lO mg/mL, even more preferably from about 0.05 to about l.Ojmg/mL.
• the protein component of the LP A/protein compositions of the present invention are substantially pure, i.e., the protein is substantially free of
• Suitable polymers can be any known in the art of pharmaceuticals and include, but are not Umited to, naturaUy-occurring polymers such as dextrans, hydroxyethyl starch, and polysaccharides; and synthetic polymers. 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 Umited 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), polyacryhc acid, polyethyloxazoUne, polyacrylamide, polyvinylpyrroUdone (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.
• the synthetic polymers can have the following generic structure:
• R, and R 3 are the same or different and are H, H 3 C, OH, R 2 or a reactive group (as described below); where R 2 is a linear or branched substituted or unsubstituted alkyl group; where
• Biodegradable polymers can also be included in the compositions. These include, but are not Umited to poly(lactide), poly(glycoUde) 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.
• Suitable polymers also include polysaccharides.
• Suitable polysaccharides include, but are not limited to, trehalose, glucose, maltose, lactose, maltulose, iso-maltulose, 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.
• the compositions can further include pharmaceutically acceptable excipients.
• compositions 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/tissue/cell preservation and/or transplantation.
• the compositions can further include additional pharmaceutically effective agents.
• Suitable classes of pharmaceuticaUy effective agents include, but are not Umited to, drugs, antibiotics, wound healing agents and antioxidants.
• Suitable drugs include, but are not Umited to, those listed in Table 1 and from the foUowing classes: antipyretic and anti-inflammatory drugs, analgesics, antiarthritics, antispasmodics, antidepressants, antipsychotics, tranquiUzers, 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 vasodUators, peripheral vasodilators, hormonal agents, contraceptives, antithrombotic agents, diuretics, antihypertensive agents, cardiovascular drugs, opioids, and vitamins are all included in the compositions described herein.
• Suitable antibiotics include, but are not limited to, ampicillin, tetracycline, chloramphenicol, erythromycin, amphotericin B and penicillin.
• Suitable wound healing agents include, but are not Umited to, transforming growth factors, platelet-derived growth factors, epidermal growth factors and fibroblast growth factors .
• Suitable antioxidants include, but are not limited to, naturaUy-occurring antioxidants such as tocopherols, (e.g., (-tocopherol, vitamin E), ascorbic acid (Vitamin_C), (-carotene (vitamin A), dihydroUpoamide and flavenoids; and synthetic antioxidants such as butylated hydroxytoluene, butylated hydroxyanisole, Trolox®, propyl gallate, other phenoUc antioxidants and phenothiazines; and chelators such as desferoxamide, HBED and
• compositions for Use in the Present Invention 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.
• compositions of the present invention can be in either a Uquid or soUd form.
• the LPA can be concentrated for dilution prior to use.
• the components are in a concentration suitable for immediate use.
• a soUd addition of a predetermined amount of an aqueous solution will result in the appropriate concentration of the components.
• the soUd can also be in powder form, for use in therapies including, but not Umited to inhalation therapies.
• 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).
• 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).
• 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 Umited to, D-PBS (phosphate buffered saline, free of calcium and magnesium salts; Gibco BRL) or 50 mM ammomum bicarbonate containing isotonic sodium chloride.
• the buffered solution is preferably physiologically acceptable.
• a wide range ofpH values are effective.
• the pH is between about 5.5 to about 8.
• 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.
• the phosphoUpids are suspended in 50 mM ammomum bicarbonate / 0.154 M sodium chloride, 250_(M EDTA with a pH of 7.7-8.0.
• the mixture is dispersed in order to achieve maximal 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 Umited to, microfluidization, extrusion and sonication, provided that the method does not denature or otherwise chemicaUy modify the phosphoUpids in such a manner as to render them toxic or of substantially diminished therapeutic activity.
• 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.
• optical clarity indicates that the mixture changes from opaque to translucent. This change is readily monitored visually; no further measurements are necessary.
• 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 phosphoUpids can be prepared. Preferably, 10 mg/mL solutions are used.
• sonication if used, is in 5 minute alternating cycles, with 5 minutes of sonication foUowed by 5 minutes of equihbration. However, this can be varied, depending on the volume of mixture being sonicated and the heat generated by sonication.
• 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 equihbration and dissipation of heat.
• the temperature of the water bath should not exceed about 60°C. Preferably, the temperature of the water bath is not allowed to exceed 37°C.
• the sonicated mixture is passed through a sterile filter before use.
• the sterile filter has a 0.2 micron cut off.
• 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 soUd 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
• compositions are stored under anoxic conditions.
• Any method of such storage known in the art is suitable for use herein, including, but not Umited to, storage under an inert gas such as argon.
• 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 Umited to, the serum deprivation of the C3H/10T1/2 ceU assay described in detaU 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.
• 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.
• 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 skiU 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 appUcations.
• compositions are pharmaceutically acceptable due to their low toxicity in the therapeutic dosage range, stability and ability to be inco ⁇ orated 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.
• 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.
• a therapeutically effective amount is from about 0.0001% or 1 ⁇ g/mL by weight ofthe phospholipid mixture although a wide range of effective amounts can be used for different indications and can be determined empiricaUy.
• the route(s) of administration useful in a particular indication are discussed below and are weU known to one of skiU in the art.
• 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 ceUs, mammaUan cells, and human cells in particular.
• Mammalian cell types encompass cardiomyocytes, endotheUal cells, neuronal cells, hepatocytes, renal ceUs, 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.
• Routes of administration include, but are not limited to, topical, transdermal, parenteral, gastrointestinal, transbronchial, transalveolar, and in vitro treatment of cells, tissues or organs foUowed by in vitro administration of treated ceUs, 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 Umited 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 Umited 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 topicaUy administered alone, it may be desirable to administer them in a mixture with a topical physiologically or cosmetically acceptable carrier.
• Topical pharmaceutically acceptable carrier is any substantiaUy non-toxic carrier conventionally useable for topical administration of pharmaceutical agents in which the compositions wiU remain stable and bioavailable when appUed directly to skin or mucosal surfaces .
• the compositions can be dissolved in a liquid, dispersed or emulsified in a medium in a conventional manner to form a Uquid preparation or mixed with a semi-solid (gel) or soUd carrier to form a paste, powder, ointment, cream, lotion or the like.
• Suitable topical pharmaceutically acceptable carriers include water, petroleum jeUy (Vaseline), petrolatum, mineral oil, vegetable oil, animal oil, organic and inorganic waxes, such as microcrystaUine, paraffin and ozocerite wax, natural polymers, such as xanthanes, gelatin, ceUulose, 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 substantiaUy miscible in water.
• Such water miscible topical pbarmaceuticaUy 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.
• 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.
• 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.
• 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, fatty or waxy environment include thermoplastic or flexible thermoset resin or elastomer including thermoplastic resins such as polyvinyl halides, polyvinyl esters, polyvinyUdene halides and halogenated polyolefins, elastomers such as brasiUensis, polydienes, and halogenated natural and synthetic rubbers, and flexible thermoset resins such as polyurethanes, epoxy resins and the like.
• the sustained or delayed release carrier is a liposome, microsponge, microsphere or gel.
• 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.
• 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, propeUants, antimicrobial agents, sunscreens, enzymes and the like.
• 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, reticuUn, 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
• topical carrier can also be a topical cosmeticaUy acceptable carrier.
• topical cosmeticaUy acceptable carrier as used herein is meant any substantiaUy non-toxic carrier conventionaUy Useable for topical administration of cosmetics in which the compositions will remain stable and bioavailable when appUed directly to the skin surface.
• Suitable cosmeticaUy acceptable carriers are known to those of skill in the art and include, but are not limited to, cosmeticaUy acceptable Uquids, creams, oUs, lotions, ointments, gels, or soUds, such as conventional cosmetic night creams, foundation creams, suntan lotions, sunscreens, hand lotions, make-up and make-up bases, masks and the like.
• cosmeticaUy acceptable carriers and pharmaceuticaUy acceptable carriers are similar, if not often identical, in nature so that most of the earlier discussion on pharmaceuticaUy acceptable carriers also appUes to cosmeticaUy acceptable carriers.
• compositions can contain other ingredients conventional in cosmetics including perfumes, estrogen, Vitamins A, 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.
• 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 phosphoUpids 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.
• 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.
• 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.
• the topical composition is administered by applying a coating or layer to the skin or mucosal area desired to be treated.
• a coating or layer is applied to the skin or mucosal area desired to be treated.
• 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.
• compositions suitable for transdermal administration including, but not Umited to, pharmaceuticaUy 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 appUed directly to the skin or inco ⁇ orated into a protective carrier such as a transdermal device (so-called "patch").
• a transdermal device so-called "patch”
• suitable creams, ointments etc. can be found, for instance, in the Physician's Desk Reference.
• suitable transdermal devices are described, for instance, in U.S. Patent No. 4,818,540 (
• the present invention includes compositions suitable for parenteral administration including, but not Umited to, pharmaceuticaUy acceptable sterile isotonic solutions. Such solutions include, but are not Umited to, saline and phosphate buffered saline for intravenous, intramuscular, intraperitoneal or subcutaneous injection.
• compositions suitable for gastrointestinal administration including, but not limited to, pharmaceuticaUy acceptable powders, pills or Uquids for ingestion and suppositories for rectal administration.
• the present invention includes compositions suitable for transbronchial and transalveolar administration including, but not Umited 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.
• the present invention encompasses methods of delivery including, but not Umited to, delivery by catheter inserted into a vessel. Where deUvery 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.
• the present invention further encompasses devices suitable for transbronchial and transalveolar administration of the compositions.
• Such devices include, but are not Umited 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.
• 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 InteUigence Unit,
• compositions described herein can be used, for instance, to replace and improve on much more impure soy preparations currently in use.
• organ preservation solution an aqueous solution specifically designed to preserve organs.
• the organ is the heart.
• 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.
• this solution contains between about 0.00001% to about 10%, preferably about
• 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.
• the PEG is PEG20L.
• 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 2 P0 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.
• a buffer such as NaOH, preferably about 30-40 mM, (or sufficient to result in pH of 7.2 to 7.9)
• an impermeant anion such as Lactobionic acid, preferably about 100 mM
• a component providing phosphate such as KH 2 P0 4 , preferably about 25 mM
• a component providing potassium such as KOH, preferably
• the OPS also contains effective amounts of any component known in the art of organ preservation. These include, but are not Umited to glutathione, parahydroxyanisole (PHA), desferoxamine, and nitroglycerin.
• PHA parahydroxyanisole
• desferoxamine desferoxamine
• nitroglycerin any component known in the art of organ preservation.
• 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.
• suitable devices for injection, topical application, atomizers and vaporizers are known in the art and will not be described in detail.
• 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 therapeuticaUy effective amount of the above-described compositions.
• Various indications which can be treated include, but are not Umited to, those related to apoptosis, ischemia, traumatic injury and reperfusion damage.
• Those conditions related to apoptosis include, but are not Umited 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.
• compositions are also suitable for use in organ transplantation during aU 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 stabiUty and function.
• the compositions are also particularly suitable for use in treating cardioplegia whether related to transplantation or other surgical intervention.
• Apoptosis related problems are caused by a variety of stimuli which include, but are not Umited to, viruses including, but not Umited to, HTV, chemotherapeutic agents, and radiation. These stimuli trigger apoptosis in a variety of disorders, including, but not Umited to, those of the digestive tract tissues and associated gastrointestinal perturbations.
• Gastrointestinal perturbations include, but are not Umited 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.
• 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
• gastrointestinal conditions are preferably by gastrointestinal administration.
• an effective amount of the compositions can be conveniently mixed in with the feed.
• administration can be by any method known in the art of gastrointestinal administration.
• administration is oral.
• compositions can be administered to immunodeficient patients, particularly HIV-positive patients, to prevent or at least mitigate apoptotic death of T ceUs associated with the condition, which results in the exacerbation of immunodeficiencies as seen in patients with AIDS.
• administration to such patients is parenterally, but can also be transdermaUy or gastrointestinaUy.
• compositions can also be administered to treat apoptosis associated with reperfusion damage involved in a variety of conditions, including, but not Umited 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.
• Myocardial and cerebral infarctions 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.
• CeU 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.
• 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.
• 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.
• the invention further provides methods of limiting and preventing apoptosis in cells, or otherwise preserving cells, during the culture or maintenance of mammaUan organs, tissues, and ceUs, by the addition of an effective amount of the compositions to any media or solutions used in the art of culturing or maintaining mammaUan organs, tissues, and cells.
• the invention further encompasses media and solutions known in the art of culturing and maintaining mammaUan 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 ceUs in culture.
• mammaUan 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 Umited 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).
• 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® Media, Neuman and Tytell's Serumless Media, TroweU's T8 Media,
• suitable mammalian cell culture media to which the compositions can be added as anti-apoptotic media supplements include, but are not Umited to, Basal Media Eagle's, Fischer's Media, McCoy's Media, Media 199,RPMI Media 1630and 1640, Media based on F-10&F-12 Nutrient Mixtures, Leibovitz's L-15 Media, Glasgow Minimum Essential Media, and Dulbecco's Modified Eagle
• MammaUan cell culture media to which the compositions can be added further comprise any media supplement known in the art, including, but not Umited to, sugars, Vitamins, hormones, metaUoproteins, antibiotics, antimycotics, growth factors, lipoproteins and sera.
• the invention further encompasses solutions for maintaining mammaUan 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 mammaUan organs during their surgical removal and handling prior to transplantation.
• the solutions can be used to flush, perfuse and/or store the organs.
• concentrations of the compositions required to limit or prevent damage to the organs can be determined empirically by one skiUed in the art by methods such as those found in the example provided below, as well as other methods known in the art.
• compositions can be topically appUed 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.
• hair loss can be caused by apoptosis of the ceUs 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.Jnvest. Dermatol. 103:107-111. As discussed above, these conditions are preferably treated by topical application of a composition comprising an effective amount of the compositions.
• An effective amount of the compositions is one which ameliorates or dirninishes the symptoms of the dermatologic conditions.
• 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.
• EXAMPLE 1 Structure and Synthesis of Various Analogs of Lysophosphatidic Acid
• the following example sets forth the synthetic methodology and analytical data used in the construction and characterization of several lysophosphatidic acid analogs and derivatives.
• experimental procedures and analytical data has been provided for intermediates used in the construction of these LPAs.
• reaction schemes which show the synthetic routes used in compound construction.
• the following nomenclature and abbreviations are used in the naming of the compounds:
• the obtained oil was treated 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 2 O (3 x 50 mL), dried (MgS0 4 ) and evaporated to dryness.
• the crude product was purified by siUca-gel column chromatography [eluent: CH 2 Cl 2 (ethyl acetate/CH 2 Cl 2 , 50/50, v/v] to yield diol Compound 2 (8.4 g, 73%) as a white soUd.
• the residual oU 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.
• 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, Mel (10 mL, 0.17 mol) was added and, after another 3 hours, the mixture was quenched with H 2 0 (150 mL), extracted into diethyl ether (500 mL), dried (MgS0 4 ) and concentrated.
• ESI-MS (m/z, +ve): 581 (MSa + , 100%); ESI-MS (m/z, -ve): 504 ( ⁇ f-NCCtf 2 CH 2 , 100%).
• Example 2 Anti-apoptotic Activity Assay 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. Patent 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 C3B/10T1/2 cells (clone 8) were obtained from ATCC (RockviUe, MD) and were maintained in exponential growth phase in which the cell cycle is randomly distributed and no cells are arrested in Go, and in quiescence.
• AU 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.
• SDR serum deprived released cells
• the remaining adherent cells were exposed to a buffered, typicaUy pB_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 cUaminetetraacetic 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.
• typicaUy 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 ceUs as described in U.S. Patent Nos.
• 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 ceUs released on serum free treatment.
• Example 3 Preparation of Five PhosphoUpid Mixture
• PA, PI, LPI, LPC available, for example, from Avanti® Polar Lipids, Inc.
• PA, PI, LPI, LPC available, for example, from Avanti® Polar Lipids, Inc.
• PA, PI, LPI, LPC available, for example, from Avanti® Polar Lipids, Inc.
• PA, PI, LPI, LPC available, for example, from Avanti® Polar Lipids, Inc.
• 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.
• the phosphoUpid mixtures are suspended in a buffer and the mixture is placed in a disposable borosilicate glass, preferably 1-2 mL in a 16 x 100 mm tube or 0.5-2 mL in a 13 x 100 mm tube, or up to 1 mL in a 12 x 75 mm tube.
• the combination of phosphoUpids is then sonicated.
• a small bath sonicator is used, such as a that sold by Laboratory SuppUes, HicksviUe, NY.
• the temperature of the water bath is between about 21 and 50°C, preferably between about 21 °C and about 40°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 phosphoUpid mixture in the glass tube(s).
• a probe sonicator can be used (Fisher Scientific Sonic Dismembrator model 550), as long as care is taken to prevent overheating of the mixture.
• the mixture was sonicated for between 3 and 90 minutes, with alternating 5 minute intervals of sonication foUowed 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.
• sonication is for 5-10 minutes.
• compositions were stored for one week at 4°C, room temperature, and 65 °C .
• results show loss of activity after storage at 65°C, while the compositions stored at 4°C or at room temperature do not have a significant loss of activity.
• the concentration of LPA was varied as was the chain length to determine the effects of these parameters on activity.
• LPA Anti-apoptotic activity of LPA
• Log phase cells were seeded in 60 mm Perri dishes at 175-350 ceUs per cm2 and maintained in Basal Medium Eagle (BME) supplemented with 10% Beat Inactivated Fetal Bovine Serum (HIFBS). 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.
• BME Basal Medium Eagle
• HIFBS Beat Inactivated Fetal Bovine Serum
• SDR serum deprived released
• ADH adherent
• Adherent cells were remove by treatment with 5 mL of Hanks Balanced Salt Solution (HBSS) without ions containing 0.05%trypsin and 0.53 mMEDTAand were counted with the lower threshold setting at 11.01 (m. AU samples were tested in tripUcate and serum-deprived controls (BME only) were assayed at both the beginning and end of each experiment.
• HBSS Hanks Balanced Salt Solution
• the dose shown is the lowest concentration at which cytotoxic effects were observed.
• LPA LPA was incorporated into Upid membrane structures of various Upid compositions . Except in the Five PhosphoUpid 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
• Positively charged particles containing 18: 1-LPA combined with the neutral phosphoUpid phosphatidyl choline (PC) and 20 mol% of the positively charged lipid l,2-dioleoyl-3-trimethylammonium-propane (TAP) were efficacious in protecting 70 to 80 % of ceUs, compared with PC/TAP alone, offered no protection over control.
• 18:1 -LPA into the negatively charged phosphatidyl glycerol (PG) gave even better protection, with up to 90% of ceUs protected as compared to alone which offered no protection over control.
• inclusion in the 18:1-LPA/PG particles of 5 to 10 mol% of the neutral phosphoUpid phosphatidylethanolamine (PE) with a long polyethylene glycol (2000 MW) covalently linked to the polar head group resulted in reduced efficacy of 18 : 1 -LPA.
• PE neutral phosphoUpid phosphatidylethanolamine
• LPA was then tested in two different weight ratios with PG, using 18: 1-LPA in concentrations representing LPA equivalents to Five PhosphoUpid Mixture at 1, 3, or 10 (g/ml. As shown in Fig. 3, LPA equivalent to 10 (g/ml Five PhosphoUpid Mixture incorporated into particles at 10% by weight with PG protected approximately 65% of ceUs from apoptosis. This degree of protection was roughly the same as that afforded by Five Phospholipid Mixture alone.
• LPS lysophosphatidyl serine
• Soy derived LPA (soy-LPA) (Avanti® Polar Lipids, Inc., Alabaster, AL) was stored in an organic solution and was dried immediately prior to assay using a Speed Vac (Savant Instruments, HicksvUle, NY). Tested proteins were resuspended at lOjmg/mL in a bicarbonate buffered saline EDTA solution (50_mM NH,BC0 3 , 104 mM NaCl, 250_(M EDTA) prior to being added to dried soy-LPA at a 1 : 10_soy-LPA to protein (weightweight) ratio.
• the mixture was then sonicated for 5 minutes using a high power 80 Watt sonication bath (Laboratory SuppUes Co., Hicksville, NY). Following sonication, concentrated LP A/protein mixtures were left standing for 15 minutes at room temperature prior to dilution in cell culture assay media.
• SDR serum deprived release
• ADH adherent
• 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 11.0 l_(m. AU samples were tested in tripUcate and serum-deprived controls (BME only) were assayed at both the beginning and end of each experiment.
• soy-LPA When soy-LPA was added together with protein, the degree of protection from apoptosis was, for certain proteins, more than additive.
• FAD fatty acid depleted bovine serum albumin
• BSA bovine serum albumin
• soy-LPA alone protected 8.0% of cells, wh ⁇ e 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 Upoprotein (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 ceUs.
• Binding of LPA to protein was examined using an electrophoresis radiobinding detection assay and a size shift analysis assay.
• Electrophoresis radiobinding assay Proteins were analyzed on native polyacrylamide gels foUowing incubation with 3 B-labeled 18: 1-LPA. 3 B-labeled lysophosphatidic acid (1 -oleyl) (DuPont NEN Products, Boston, MA) was added to tested proteins at 1 nM labeled 18:l-LPA/45_(g protein. Mixtures were incubated in a bicarbonate buffered saline/EDTA solution at 37°C for 60 minutes. Incubated samples were mixed with native gel sample buffer (312_mM Tris pB 6.8, 50% glycerol, 0.05% bromophenol blue) and loaded entirely onto an 8% discontinuous native polyacrylamide gel.
• native gel sample buffer 312_mM Tris pB 6.8, 50% glycerol, 0.05% bromophenol blue
• 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 3 HANCE, DuPontNEN Products, Boston, MA), and dried onto 3M chromatography paper (Whatman, Clifton, NJ) and exposed to autoradiography film (X-Omat, Kodak, Rochester, NY) for 72 hours at -80°C.
• autoradiography enhancer EN 3 HANCE, DuPontNEN Products, Boston, MA
• Size shift assay Size analysis of LPA both with and without protein was performed using a Superdex® S75 column (Pharmacia Biotech, Uppsala, Sweden). Soy LPA was combined in a chloroform solution with 0.5 Ci 3 B-labeled 18:l-LPA(DuPontNENProducts,Boston,MA),dried under vacuum and resuspended by sonication in a buffered solution (50 mM ammomum bicarbonate, 154 mM sodium chloride, pB 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 scintiUant solution using a Uquid scintillation counter (Beckman
• soy-LPA alone eluted at 22-26 minutes after sample injection, corresponding to a size of approximately 1.3 kD.
• radioactivity eluted primarily in the 10 to 12-minute fractions, indicating that most of the LPA was bound to BSA.
• a LP A/FAD soy protein mixture appUed to the column resulted in an elution profile with two peaks, one corresponding to the position of unbound LPA, and the other shifted.
• 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.
• the foUowing experiment was performed.
• TPA 12-0-tetradecanoyl phorbol- 13 -acetate
• AU non-adherent or loosely adherent cells were removed from the culture dish and counted by appropriate techniques, typicaUy 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.
• SDR serum deprived released cells
• the remaining adherent cells were exposed to a buffered, typicaUy 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 maminetetraacetic 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.
• typicaUy 10_minutes the released cells were removed from each culture dish and measured by the same means as described above, typicaUy 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.
• 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.
• 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 NB ⁇ CO ⁇ 104 mM
• the septa Prior to use, the septa had been subjected to 2 hours of high vacuum then sealed in an aluminum bag filled with Argon in order to displace as much dissolved oxygen as possible.
• the Five PhosphoUpid Mixture/PEG vials were labeled then individually packaged into aluminum bags filled with Argon and stored at 4°.
• phospholipid mixtures were prepared by combining the desired phosphoUpids in solution with organic solvent then drying down the mixture with gentle heat (45 °C) and high vacuum foUowed by sonication into aqueous suspension at 5 to 20 mg/mL.
• 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 cardiomvocvtes 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, lxMEM vitamins (Gibco), 5% fetal bovine serum and 50 U/ml peniciUin-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 3.5xl0 5 viable cells per ml. The cells were aUowed to adhere for 16-24 hours in a 37°C/5% C0 2 humidified incubator.
• 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 2 -ceramide was accomplished by the addition of the agent prepared in serum-free RPMI.
• 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 2 /5% C0 2 for 8 hours at 37°C.
• 10% fetal bovine serum, 2 g/L of glucose were added and the cultures were returned to a normal oxygen gas overlay (37°C/5% CO 2 ) in a humidified incubator for 16 hours.
• 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 because of their rapid lysis foUowing release from adhesion substrate.
• Adherent cardiomyocytes were coUected from culture dishes using 0.25% Trypsin/0.05% EDTA and counted on Coulter Counter ZM and Coulter Channelyzer 256.
• 15(M ceramide was added to cardiomyctes 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 PhosphoUd Mixture plus 0.4% PEG, and (e) 5 (M LPA presented as Five PhosphoUd Mixture plus 2% PEG.
• LPA has anti-apototic activity as shown in the cardiomyocyte assay, and that the combination of LPA with other phosphoUpids retains that activity. Additionally, the presence of PEG improves the activity of LPA.
• 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 ofthe 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 ofthe ischemic period, and continuing for 75 minutes in total (approximately 67 (IJmin., approximately 3.3 (g Five PhosphoUpid Mixture/min.). The total dose was 250 (g Five PhosphoUpid Mixture and 500 mg PEG20L.
• the atria and right ventricle are removed from the left ventricle. After removal ofthe apex, the portion ofthe 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 infarcted 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 ofthe circumflex, viable LAD and infarcted LAD territories. Calculation of infarct size is based upon the assumption that the area of infarction in the sectioned plane is representative of the mass of infarcted tissue in that plane.
• PEG 100_mg/ml as a bolus plus infusion (at 4_n ⁇ 7hr) starting 15 min before reperfusion and going on for 60 min into the reperfusion period.
• Placebogroup dogs receive placebo foUowing the same modalities described for the study drug group.
• SOD-Catalase group dogs receive SOD plus catalase following the same modaUties described by Simpson et_al. (1987) Fed. Proc. 46:7, 2413-21.
• Heart function was monitored by EKG and cardiac output was determined by contrast imaging. The animal was euthenized 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 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.
• LAD left anterior descending coronary artery
• the chest was opened through the left fourth intercostal space. Then, the pericardium was incised and the heart was exposed. A large anterolateral branch ofthe circumflex artery, or the circumflex artery itself was identified and encircled with a 4-0 silk suture. The ends ofthe 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 ofthe procedure.
• 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 ofthe Five PhosphoUpid Mixture, described in Example 3, above, 8 mg/ml PEG 20L, in 10 mM citrate 110 mMNaCl.
• the control solution contained 10 mM citrate and 110 mM NaCl.
• 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.
• 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.
• the artery was occluded for 30 minutes. FoUowing 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.
• the coronary artery is reoccluded.
• Unisperse blue (Ciby-Geigy, Hawthorne, NY) 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) foUowed by 12 mEq of potassium chloride given into the left atrium.
• Prospective exclusion criteria included an ischemic risk zone of less than 10% ofthe 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).
• 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 triphenyltetrazoUum chloride pre-heated to 37 °C for 10 minutes and rephotographed for analysis of area of necrosis. AU sections were later fixed in formalin. These photographic slides were projected and areas of risk (AR) and areas of necrosis (AN) were traced by planimetry. The planimetered areas of each sUce were multiplied by the weight ofthe sUce and then summed. Because infarct size is measured from photographic sUdes, the entire left ventricle was used for the analysis.
• AR area of risk
• AN necrosis
• RMBF Regional myocardial blood flow
• Radioactive microspheres were injected via the atrial catheter.
• the catheter was then reprimed with approximately 0.2 ml ofthe drug treatment, and the catheter was reconnected to the pump and infusion continued.

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