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WO1995020967A1 - Composition de cicatrisation, de croissance des neurones et de vascularisation - Google Patents

Composition de cicatrisation, de croissance des neurones et de vascularisation Download PDF

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Publication number
WO1995020967A1
WO1995020967A1 PCT/US1995/001473 US9501473W WO9520967A1 WO 1995020967 A1 WO1995020967 A1 WO 1995020967A1 US 9501473 W US9501473 W US 9501473W WO 9520967 A1 WO9520967 A1 WO 9520967A1
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group
linoleoyl
oleoyl
bis
pharmaceutical composition
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James A. Bianco
Stuart L. Bursten
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CTI Biopharma Corp
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Cell Therapeutics Inc
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    • 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

  • the present invention provides a compound, pharmaceutical composition and methods for increasing mesenchymal cell migration including smooth muscle cells and endothelial cells and for peripheral nerve regeneration. This will promote tensile strength and rapid vascularization of wounds while accelerating the healing process.
  • the inventive pharmaceutical compositions act as a platelet derived growth factor (PDGF), fibroblast derived growth factor (FGF), epidermal derived growth factor (EGF), vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) agonist, comprising an effective amount of a phosphatidic acid (PA) or bis PA species, or combination thereof.
  • PDGF platelet derived growth factor
  • FGF fibroblast derived growth factor
  • EGF epidermal derived growth factor
  • VEGF vascular endothelial growth factor
  • NGF nerve growth factor
  • the pharmaceutical composition is useful for the treatment of wound healing or surgical healing of skin, vasculature, soft tissue or bone due to physical trauma, surgical procedures, fractures of bone or spinal cord, burns or soft tissue injury.
  • the pharmaceutical composition is also useful for the treatment of tissue regeneration in areas of devascularized tissue or organ trauma, including central nervous system tissue and peripheral nervous tissue, vascularization of the myocardium after an infarction.
  • Angiogenesis is a fundamental process by which new blood vessels are formed. It is essential in reproduction, development and wound repair. Wound repair includes, for example, healing of peptic ulcers, myocardial infarctions and third-degree bums as dependent upon angiogenesis.
  • Wound repair includes, for example, healing of peptic ulcers, myocardial infarctions and third-degree bums as dependent upon angiogenesis.
  • angiogenic agonists only polypeptide molecules have been identified. Such molecules most likely exert their activity outside of the cell. No intracellular-acting angiogenic small molecules have been identified.
  • Angiogenic molecules include, for example, fibroblast growth factors (FGF), platelet derived growth factor (PDGF), transforming growth factor alpha and beta (TGF- ⁇ and TGF- ⁇ ), angiogenin, tumor necrosis factor alpha (TNF- ⁇ ), angiotropin, and vascular endothelial growth factor (VEGF).
  • FGF fibroblast growth factors
  • PDGF platelet derived growth factor
  • TGF- ⁇ and TGF- ⁇ transforming growth factor alpha and beta
  • angiogenin tumor necrosis factor alpha
  • TNF- ⁇ tumor necrosis factor alpha
  • angiotropin vascular endothelial growth factor
  • VEGF vascular endothelial growth factor
  • transfection of cell lines with a cDNA sequence encoding VEGF did not promote transformation, but did facilitate tumor growth in vivo (Ferrara et al., J. Clin. Invest. 91 : 160, 1993). This was likely due to paracrine stimulation of neovasculogenesis.
  • cytokines signal through a common second messenger pathway within the cell, such agonists will have broad therapeutic activity useful for the treatment of wound healing or surgical healing of skin, vasculature, soft tissue or bone due to physical trauma, surgical procedures, fractures of bone or spinal cord, bums or soft tissue injury.
  • the present invention was made by discovering a common signaling mechanism, and discovering a common intracellular signaling intermediate.
  • the present invention provides a compound, pharmaceutical composition and methods for increasing mesenchymal cell migration including smooth muscle cells and endothelial cells and for neuronal cell regeneration and embryogenesis. This will promote tensile strength and rapid vascularization of wounds while accelerating the healing process.
  • the inventive compound and pharmaceutical composition will promote neuronal cell regeneration due to areas of devascularized tissue or traumatic injury (e.g., reinnervation of CNS or peripheral nerves such as the spinal chord) and mimic activity of brain-derived growth factors such as CNTF (ciliary neurotrophic factor).
  • the inventive pharmaceutical compositions act as a platelet derived growth factor (PDGF), CNTF, fibroblast derived growth factor (FGF), epidermal derived growth factor (EGF) and vascular endothelial growth factor (VEGF) agonist, comprising an effective amount of a phosphatidic acid (PA) species, or combination thereof.
  • PDGF platelet derived growth factor
  • FGF fibroblast derived growth factor
  • EGF epidermal derived growth factor
  • VEGF vascular endothelial growth factor
  • PA phosphatidic acid
  • the present invention provides a method for treating wounds or surgical healing of skin, vasculature, soft tissue or bone due to physical trauma, surgical procedures, fractures of bone or spinal cord, burns or soft tissue injury, comprising administering an effective amount of a non-arachidonoyl phosphatidic acid (PA) selected from the group consisting of Group I PA's, Group II PA's, Group Ila PA's, Group III PA's.
  • PA non-
  • Group IV PA's bis PA's. hemi (bis) PA's, N-acylethanolamines, and combinations thereof.
  • Group I PA's comprise LPAAT-derived (lysophosphatidic acyl transferase) PA's with a >90% Cj-Cig saturated and unsaturated and a >95% linoleate component in the sn-2 position.
  • Group II PA's comprise LPAAT-derived PA's with an alkyl or alkenyl (at least 90% C *
  • Group Ila PA's comprise alkyl myristate or acyl myristate in the sn- 1 position and a C ⁇ g unsaturated (acyl) hydrocarbon in the sn-2 position.
  • Group III PA's comprise an sn-1 alkyl Cj hydrocarbon and any hydrocarbon in the sn-2 position.
  • Group IV PA's comprise an oleoyl (Cjg : ] : ⁇ -9) in the sn-2 position and are specifically derived from PC (phosphatidyl choline) PLD (phospholipase D).
  • Bis PA's comprise bis (diacylglycero) (phosphate) having four acyl chains (R1-R4) according to the following formula I:
  • bis PA's comprise hemi-bis (diacylglycero) (phosphate) having three acyl chains (R' ] -R'3) according to the following formula II:
  • N-acylethanolamines having three acyl chains (R" ⁇ -R"3) according to the following formula HI:
  • R n l R" 2 wherein each of R" ⁇ , R" 2 and R"3 are alkyl or alkenyl chains selected from the group consisting of 18:0, 18:1, 18:2, 18:3 and 20:4.
  • R" ⁇ is 18:0, 18:1 or 18:2
  • R" 2 is 18:2, 18:3 or 20:4
  • R" 3 is 18:2 or 20:4.
  • PA's including, for example, 1-o-octadecanyl 2-oleoyl PA (687), 1- oleoyl 2-linoleoyl PA (697 or 698), 1-o-octadecanyl 2-linoleoyl PA (681), l-o-'en- octadecanyl-9,12-dienyl 2-linoleoyl PA (679), 1-myristoyl 2-oleoyl PA (645), 1-o-myristoyl 2-stearoyl PA (633), 1,2-sn-dilinoleoyl PA (695), 1-stearoyl 2-oleoyl PA (701), 1-o-oleoyl 2- 20:4 PA (707), 1 -o-linoleoyl 2-20:4 PA (705), 1 -o-linoleoyl 2-20:5 PA (703), and combinations thereof comprise the inventive pharmaceutical composition.
  • the present invention further provides a method for promoting tissue regeneration in areas of devascularized tissue and for promoting embryogenesis ex vivo by promoting cellular growth and differentiation, comprising administering an effective amount of a PAs selected from the group consisting of Group I PA's, Group II PA's, Group Ila PA's, Group m PA's, Group IV PA's, bis PA's and combinations thereof.
  • a PAs selected from the group consisting of Group I PA's, Group II PA's, Group Ila PA's, Group m PA's, Group IV PA's, bis PA's and combinations thereof.
  • the inventive method for tissue regeneration in areas of devascularized tissue further comprises healing of neuronal tissue and reineravation of CNS and peripheral nerves due to devascularization or trauma or stroke.
  • the present invention further provides a pharmaceutical composition for local administration to a wound site or a site in or on a patient requiring angiogenic activity, comprising aphosphatidic acid (PA) selected from the group consisting of Group I PA's, Group II PA's, Group Ila PA's, Group El PA's, Group IV PA's, bis PA's and combinations thereof and a pharmaceutically acceptable excipient.
  • aphosphatidic acid selected from the group consisting of Group I PA's, Group II PA's, Group Ila PA's, Group El PA's, Group IV PA's, bis PA's and combinations thereof and a pharmaceutically acceptable excipient.
  • Figure 1 illustrates the stimulation of proliferation of Balb/3T3 cells, a murine fibroblast cell line, by L- ⁇ -Phosphatidic acid.
  • Cellular proliferation was determined by tritiated thymidine incorporation.
  • the PAs were either synthesized or purchased (Avanti Polar-Lipids, Inc. Alabaster AL), dissolved in chloroform, dried under N 2 and stored under argon.
  • the PAs were reconstituted in phosphate buffered saline containing 0.1% fatty acid- free bovine serum albumin by sonication on ice. The concentrations of PAs are shown in the X axis.
  • FIG 2 illustrates the stimulation of proliferation of Balb/3T3 cells by dilauroyl PA (PA2).
  • PA2 dilauroyl PA
  • Cellular proliferation was determined by tritiated thymidine incorporation.
  • the PAs were either synthesized or purchased (Avanti Polar-Lipids, Inc. Alabaster AL), dissolved in chloroform, dried under N 2 and stored under argon.
  • the PAs were reconstituted in phosphate buffered saline containing 0.1% fatty acid-free bovine serum albumin by sonication on ice. The concentrations of PAs are shown in the X axis.
  • Figure 3 illustrates the stimulation of proliferation of Balb/3T3 cells by Dioleoyl PA.
  • the PAs were either synthesized or purchased (Avanti Polar-Lipids, Inc. Alabaster AL), dissolved in chloroform, dried under N 2 and stored under argon.
  • the PAs were reconstituted in phosphate buffered saline containing 0.1% fatty acid-free bovine serum albumin by sonication on ice. The concentrations of PAs are shown in the X axis.
  • Figure 4 illustrates the stimulation of proliferation of Balb/3T3 cells by 1-alkyl-oleoyl 2-oleoyl PA.
  • Cellular proliferation was determined by tritiated thymidine incorporation.
  • the PAs were either synthesized or purchased (Avanti Polar-Lipids, Inc. Alabaster AL), dissolved in chloroform, dried under N 2 and stored under argon.
  • the PAs were reconstituted in phosphate buffered saline containing 0.1 % fatty acid-free bovine serum albumin by sonication on ice. The concentrations of PAs are shown in the X axis.
  • Figures 5-7 illustrate a mass spectrograph of a designated lipid fraction isolated from Balb 3T3 fibroblasts stimulated with PDGF (25 ng/ml).
  • Figure 5 shows a mass spec of a PA HPLC peak 5 seconds after stimulation with PDGF including the 1-o-alkyl C18 PA derivatives including 679 (l-o-'en-octadeca-9,12-dienyl 2 linoleoyl PA), 681 (l-o- octadeca-9,12-dienyl 2-linoleoyl PA), 683 (l-o-octadeca-9-enyl 2-linoleoyl PA), and related PA derivatives with C Q sn-2 components such as 703 (l-o-'en-octadeca-9,12-dienyl 2- arachidonoyl PA), and 707 (l-o-octadeca-9-enyl 2-arachidonoyl PA).
  • Figure 6 shows that synthesis of Group E PA species (especially 679 and 681) was maintained after 15 seconds of stimulation with PDGF.
  • Group E PA species include, for example, 1-o-octadecanyl 2- oleoyl PA (687), 1-oleoyl 2-linoleoyl PA (697 or 698), 1-o-octa 9,12-decadiennyl 2-linoleoyl PA (681), l-o-'en-octadecanyl-9,12-dienyl 2-linoyl PA (679), 1-myristoyl 2-oleoyl PA (645), and 1-o-myristoyl 2-stearoyl PA (633) PA species.
  • Figure 7 further shows the 15 second stimulation maintaining the Group E PA species and, in addition, 673 (1-palmitoyl, 2- oleoyl PA) and 671 (1-palmitoyl, 2-linoleoyl PA) PA species.
  • Figure 8 is a four-part figure showing mass spec plots from thin layer chromatographed lipids from neutrophils. Briefly, neutrophils were stimulated with GM- CSF in order to stimulate PC-directed phospholipase D (PLD), in the presence of excess diacylglycerol (DG) in order to generate bis(PA). Neutrophils were stimulated in the presence or absence of CT-3501. a PA signaling antagonist. The incubation with GM-CSF was stopped by addition of ice cold MeOH. the lipids were extracted and separated on thin layer chromatography (TLC). Panel A illustrates the bis (PA) that was made (due to ⁇ H-DG to follow the appropriate TLC fraction).
  • PLD PC-directed phospholipase D
  • DG diacylglycerol
  • the receptors are phosphorylated at various sites in their intracellular domain by intrinsic tyrosine kinase activity of the receptor. This leads to the creation of additional binding sites for intracellular proteins.
  • these include phospholipase C- ⁇ -1 (PLC- ⁇ -1), the ras GTPase activating protein (GAP), phosphatidylinositol 3 kinase (PDkinase), pp ⁇ Oc-src, p62c-yes, p50-fyn, Nek, and CRB2 as well as a 120 kd and a 64 kd species.
  • PLC- ⁇ -1 is a specific phosphodiesterase that produces diacylgycerol (DAG) and inositol triphosphate, two second messengers that activate a serine/threonine specific protein kinase protein kinase C (PKC) and increase intracellular calcium levels.
  • DAG diacylgycerol
  • PLC protein kinase C
  • PDkinase is a lipid kinase that phosphorylates the D3 position of phosphatidylinositol phosphatidylinositol-4-phosphate, or PI 4,5,P2.
  • PDGF intracellular lipid species
  • PDGF induces activation of the serine/threonine kinase MAP kinase, via MAP kinase kinase, which may be activated by activation of ras/raf pathway.
  • MAP kinase acts to activate the nuclear transcription factors c-jun, c-fos and possibly c-myc.
  • PDGF also up regulates increased transcription of these transcription factors.
  • PA phosphatidic acid
  • PA can be produced by either a membrane associated lysophosphatidic acyl transferase
  • PAAT phospholipase D catalyzed hydrolysis of phosphatidyl choline or phosphatidyl- ethanolamine, or via DAG kinase conversion of diacyl glycerol (DAG) to PA.
  • DAG diacyl glycerol
  • PA is a potent intracellular signaling lipid and can be converted to DAG by phosphatidyl phosphohydrolase (PAPH).
  • PA's including L-a PA (derived from natural sources), 1,2 dilauroyl-sn-glycero-3-phosphate, 1,2 dioleoyl-sn-glycero-3- phosphate, l-stearolyl-2-aracidonyl-sn-glycero-3-phosphate and l-alkyl-oleoyl-2-oleoyl-PA, are all mitogenic in Balb/3T3 cells, a fibroblast cell line that is predictive of would healing, including vascularization and improved tensile strength.
  • L-a PA derived from natural sources
  • 1,2 dioleoyl-sn-glycero-3-phosphate 1,2 dioleoyl-sn-glycero-3- phosphate
  • l-stearolyl-2-aracidonyl-sn-glycero-3-phosphate l-alkyl-oleoyl-2-oleoyl-PA
  • the present invention provides a compound, pharmaceutical composition and methods for increasing mesenchymal cell migration including smooth muscle cells and endothelial cells. This will promote tensile strength and rapid vascularization of wounds while accelerating the healing process.
  • the inventive pharmaceutical compositions act as a platelet derived growth factor (PDGF), fibroblast derived growth factor (FGF), epidermal derived growth factor (EGF) and vascular endothelial growth factor (VEGF) agonist, comprising an effective amount of a phosphatidic acid (PA) species, or combination thereof.
  • PDGF platelet derived growth factor
  • FGF fibroblast derived growth factor
  • EGF epidermal derived growth factor
  • VEGF vascular endothelial growth factor
  • the present invention provides a method for treating wounds or surgical healing of skin, vasculature, soft tissue or bone due to physical trauma, surgical procedures, fractures of bone or spinal cord, burns or soft tissue injury, comprising, comprising administering an effective amount of a phosphatidic acid (PA) selected from the group consisting of Group I PA's, Group II PA's, Group Ea PA's, Group IE PA's, Group IV PA's, bis PA's, hemi (bis) PA's, N-acylethanolamines, and combinations thereof.
  • PA phosphatidic acid
  • Group I PA's comprise LPAAT- derived (lysophosphatidic acyl transferase) PA's with a >90% Cj-Cig saturated and unsaturated and a >95% linoleate component in the sn-2 position.
  • Group E PA's comprise LPAAT-derived PA's with an alkyl or alkenyl (at least 90% Ci g) in the sn-1 position and >80% linoleate in the sn-2 position.
  • Group Ea PA's comprise alkyl myristate or acyl myristate in the sn-1 position and a Cjg unsaturated (acyl) hydrocarbon in the sn-2 position.
  • Group IE PA's comprise an sn-1 alkyl Cjg hydrocarbon and any hydrocarbon in the sn-2 position.
  • Group IV PA's comprise an oleoyl (C ⁇ -. ⁇ : ⁇ £ 9) in the sn-2 position and are specifically derived from PC (phosphatidyl choline) PLD (phospholipase D).
  • Bis PA's comprise bis (diacylglycero) (phosphate) having four acyl chains (R1-R4) according to the following formula I:
  • bis PA's comprise hemi-bis (diacylglycero) (phosphate) having three acyl chains
  • N-acylethanolamines having three acyl chains (R" ⁇ -R"3) according to the following formula IE:
  • R" -. , R" 2 and R"3 are alkyl or alkenyl chains selected from the group consisting of 18:0, 18:1, 18:2, 18:3 and 20:4.
  • R" j is 18:0, 18:1 or 18:2;
  • R" 2 is 18:2, 18:3 or 20:4;
  • R" 3 is 18:2 or 20:4.
  • PA's including, for example, 1 -o-octadecanyl 2-oleoyl PA (687), 1- oleoyl 2-linoleoyl PA (697 or 698), 1 -o-octadecanyl 2-linoleoyl PA (681), l-o-'en- octadecanyl-9,12-dienyl 2-linoleoyl PA (679), l-myristoyl 2-oleoyl PA (645), 1-o-myristoyl 2-stearoyl PA (633), 1,2-sn-dilinoleoyl PA (695), 1-stearoyl 2-oleoyl PA (701), 1-o-oleoyl 2- 20:4 PA (707), 1 -o-linoleoyl 2-20:4 PA (705), 1 -o-linoleoyl 2-20:5 PA (703), and combinations thereof comprise the inventive pharmaceutical composition.
  • the present invention further provides a pharmaceutical composition for local administration to a wound site or a site in or on a patient requiring angiogenic activity, comprising a phosphatidic acid (PA) selected from the group consisting of Group I PA's, Group II PA's, Group Ea PA's, Group IE PA's, Group IV PA's, bis PA's, hemi (bis) PA's, N- acylethanolamines, and combinations thereof and a pharmaceutically acceptable excipient.
  • PA phosphatidic acid
  • Activation of phospholipase D does not always produce PA but may also produce either bis (diacylglycerol) phosphate (BisfPAj), bis (monoacylglycerol) phosphate (lyso(bis) PA), or closely-related species derived from phosphatidylglycerol. This was observed for a variety of glycerol forms containing free hydroxyl moieties (Tettenbron et al., Biochem. Biophys. Res. Comm. 155:249, 1988; and Guillemain et al., Amer. J. Physiol. 266:C692, 1994).
  • inventive pharmaceutical compositions are signaling agonists that work by acting as intracellular messengers to up-regulate cellular activity and have a utility as therapeutic agents.
  • the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient (i.e., PA, bis PA, hemi (bis PA, or acylethanolamine) with which it is to be combined, the route of administration and other well-known variables.
  • a PA, bis PA, hemi (bis) PA, or acylethanolamine compound or a pharmaceutically acceptable salt or hydrate or solvate thereof is administered to a patient in an amount sufficient to increase mesenchymal cell migration.
  • the route of administration of the compound or composition is not critical but is usually local, topical, sustained release, impregnated in a suture, sponge or wound covering, oral or parenteral.
  • parenteral includes intravenous, intramuscular, subcutaneous, intranasal, intrarectal, transdermal, opthalmic, intravaginal or intraperitoneal administration.
  • the subcutaneous and intramuscular forms of parenteral administration are generally preferred.
  • the daily parenteral dosage regimen will preferably be from about 0.01 mg/kg to about 25 mg/kg of total body weight, most preferably from about 0.1 mg/kg to about 4 mg/kg.
  • each parenteral dosage unit will contain the active ingredient in an amount of from about 0.1 mg to about 400 mg.
  • the pharmaceutical composition is formulated for sustained release and interdispursed into an injectable matrix, such as collagen or alginate (or other complex polysaccharide) and injected or locally applied (e.g., during a surgical procedure) to a site in need of angiogenic wound healing.
  • an injectable matrix such as collagen or alginate (or other complex polysaccharide)
  • the pharmaceutical composition may be administered in the form of a skin patch, a surgical sponge or in a polymeric matrix for local organ application to an organ in need of angiogenic wound healing.
  • the pharmaceutical composition is impregnated in surgical sutures to augment surgical wound healing.
  • compositions are generally active when given orally and can be formulated as liquids, for example, syrups, suspensions or emulsions, tablets, capsules and lozenges.
  • a liquid formulation will generally consist of a suspension or solution of the compound or pharmaceutically acceptable salt in a suitable liquid carrier(s), for example, ethanol, glycerine, non-aqueous solvent, for example polyethylene glycol, oils, or water with a suspending agent, preservative, flavoring or coloring agent.
  • a composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations. Examples of such carriers include magnesium stearate, starch, lactose, sucrose and cellulose.
  • a composition in the form of a capsule can be prepared using routine encapsulation procedures.
  • pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatin capsule.
  • a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), for example, aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule.
  • the daily oral dosage regimen will preferably be from about 0.01 mg/kg to about 40 mg/kg of total body weight.
  • each oral dosage unit will contain the active ingredient in an amount from about 0.1 mg to about 1000 mg.
  • the optimal quantity and spacing of individual dosages of a compound or a pharmaceutically acceptable salt or hydrate or solvate thereof will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular patient being treated, and that such optimums can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment (i.e., the number of doses of a compound or a pharmaceutically acceptable salt or hydrate or solvate thereof given per day and duration of therapy) can be ascertained by those skilled in the art using conventional course of treatment determination tests. Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following examples are, therefore, to be construed as merely illustrative and not a limitation of the scope of the present invention in any way.
  • Example 1 This example illustrates that various species of PA and lyso-phosphatidic acid induced Balb/3T3 cells to proliferate ( Figures 1-4). The various species of PA are indicated on each figure.
  • Phosphatidic Acid (PA's) derivatives were synthesized or purchased from Avanti Polar-Lipids, Inc., Alabaster, AL. PA's were dissolved in chloroform, dried under N 2 and stored under Argon. PA's were reconstituted in phosphate buffered saline containing 0.1% fatty-acid free bovine serum albumin by sonication on ice.
  • PA's Phosphatidic Acid
  • PA's including L-a PA (derived from natural sources), 1,2 dilauroyl-sn-glycero-3-phosphate, 1,2 dioleoyl-sn-glycero-3-phosphate, l-stearoyl-2- arachidonyl-sn-glycero-3-phosphate and l-alkyl-oleoyl-2-oleoyl-PA, were mitogenic in Balb/3T3 cells.
  • L-a PA derived from natural sources
  • 1,2 dioleoyl-sn-glycero-3-phosphate 1,2 dioleoyl-sn-glycero-3-phosphate
  • l-stearoyl-2- arachidonyl-sn-glycero-3-phosphate and l-alkyl-oleoyl-2-oleoyl-PA
  • Figures 5-7 illustrate a mass spectrograph of a designated lipid fraction isolated from Balb 3T3 fibroblasts stimulated with PDGF (25 ng/ml).
  • Figure 5 shows a mass spec of a PA HPLC peak 5 seconds after stimulation with PDGF including the 1-o-alkyl C18 PA derivatives including 679 (l-o-'en-octadeca-9,12-dienyl 2 linoleoyl PA), 681 (l-o- octadeca-9,12-dienyl 2-linoleoyl PA), 683 (l-o-octadeca-9-enyl 2-linoleoyl PA), and related PA derivatives with C20 sn-2 components such as 703 (l-o-'en-octadeca-9,12-dienyl 2- arachidonoyl PA), and 707 (l-o-octadeca-9-enyl 2-arachidonoyl PA).
  • FIG. 6 shows that synthesis of Type IB PA species (especially 679 and 681) was maintained after 15 seconds of stimulation with PDGF.
  • Type IB PA species include, for example, 1 -o-octadecanyl 2- oleoyl PA (687), 1-oleoyl 2-linoleoyl PA (697 or 698), l-o-octa9,12-decadienyl 2-linoleoyl PA (681), l-o-'en-octadecanyl-9,12-dienyl 2-linoleoyl PA (679), 1-myristoyl 2-oleoyl PA (645), and 1-o-myristoyl 2-stearoyl PA (633) PA species.
  • Figure 7 further shows the 15 second stimulation maintaining the Type IB PA species and, in addition, 673 (1-palmitoyl, 2- oleoyl PA) and 671 (1-palmitoyl, 2-linoleoyl PA)
  • Example 2 This example illustrates a synthesis procedure for l-o-alkyl-2-acyl PA (C:18:l, C: 18: 1) in four steps. Initially, 1-o-oleyl glycerol (1 g) was reacted with 1.2 ols of trityl chloride. The resulting l-o-alkyl-3 trityl glycerol was purified by column chromatography (yield 1.81 g). Trityl protected compound was reacted with oleic anhydride (1.25 mole) and the product, l-0-alkyl-2-acyl-3-trityl, was purified by column chromatography (yield 1.637 g).
  • the product was detritylated and again purified on a silica column (yield 500 mg) and reacted with phosphorous oxychloride to obtain the required phosphatidic acid (yield 200 mg).
  • the PA was partially purified on silica column (yield 100 mg) and gave two spots on TLC.
  • the final purification was achieved by HPLC (22 mg).
  • the final characterization of the compound was achieved by FAB/MS.
  • Example 3 This example illustrates a different synthesis of PA using the procedure described in example 2 except a different procedure for acylation. Instead of using commercially available oleic anhydride, oleic acid and 1,1 carbonyldiimidazol was used for acylation.
  • Example 4 This example illustrates a synthesis of PA ( 18:2,18;2) l-o-(9-12) octadecyl-2- linoleoyl-PA in 6 steps.
  • Commercially available 2,3 isopropyhdene glycerol (Sigma ) was reacted with linoleoyl methane sulfonate in the presence of sodium hydride to obtain l-o-(9- 12) octadecyl-2,3 isopropyhdene glycerol.
  • the resulting compound was deacetonated in the presence of 10% HC1 to obtain l-o-(9-12) octadecyl glycerol.
  • the remaining steps are same as described in example 2.
  • Example 5 This example illustrates an experiment with lipid analysis of stimulated neutrophils stimulated with GM-CSF. Briefly, neutrophils were stimulated with GM-CSF in order to stimulate PC-directed phospholipase D (PLD), in the presence of excess diacylglycerol (DG) in order to generate bis (PA). Bis (PA) was formed from PA and DG conjugation by PLD using DG as an alcohol to transphosphatidylate PA. Neutrophils were stimulated in the presence or absence of CT-3501, a PA signaling antagonist. The incubation with GM-CSF was stopped by addition of ice cold MeOH, the lipids were extracted and separated on thin layer chromatography (TLC).
  • TLC thin layer chromatography
  • Panel A illustrates the bis (PA) that was made (due to -1H-DG to follow the appropriate TLC fraction).
  • bis (PA) was suppressed and hemi (lyso) bis PA was produced.
  • panel C the species of PA produced by GM-CSF stimulation of neutrophils are seen. Such species are suppressed by CT-3501 (panel D).

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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un composé, une composition pharmaceutique ainsi que des procédés permettant d'augmenter la migration des cellules mésenchymateuses y compris les cellules des muscles lisses ainsi que les cellules endothéliales, et de régénérer les nerfs périphériques, afin de favoriser la résistance à la traction et la vascularisation rapide des lésions tout en accélérant le processus de cicatrisation. Les compositions pharmaceutiques de l'invention agissent en tant que facteur mitogénique plaquettaire, facteur de croissance dérivé du fibroblaste, facteur de croissance épidermique, facteur de croissance endothéliale vasculaire, agoniste du facteur de croissance des nerfs. Lesdites compositions pharmaceutiques comprennent une quantité efficace d'une espèce d'acide phosphatidique ou d'acide bis phosphatidique, ou bien une association de ceux-ci. Elles conviennent au traitement de la cicatrisation de lésions ou bien à la cicatrisation chirurgicale de la peau, du système vasculaire, des tissus mous ou des os résultant de trauma physique, aux interventions chirurgicales, aux fractures des os ou de la moelle épinière, des brûlures ou des lésions des tissus mous. La composition pharmaceutique se prête également au traitement de la régénération tissulaire aux niveaux de zones de tissus ou d'organes dévascularisés, y compris le tissu du système nerveux central et le tissu nerveux périphérique.
PCT/US1995/001473 1994-02-04 1995-02-03 Composition de cicatrisation, de croissance des neurones et de vascularisation Ceased WO1995020967A1 (fr)

Priority Applications (1)

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AU17434/95A AU1743495A (en) 1994-02-04 1995-02-03 Composition for wound healing, neuron growth and vascularization

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US19184394A 1994-02-04 1994-02-04
US08/191,843 1994-02-04
US31018794A 1994-09-21 1994-09-21
US08/310,187 1994-09-21

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WO1997041874A1 (fr) * 1996-05-08 1997-11-13 Modus Biological Membranes Ltd. Compositions contenant de l'acide phosphatidique
WO1999003479A1 (fr) * 1997-07-16 1999-01-28 Modus Biological Membranes Ltd. Composition immuno-modulatrice a base de lipides
EP1069895A4 (fr) * 1998-03-18 2002-07-31 Lxr Biotechnology Inc Compositions contenant des acides lysophosphatidiques qui inhibent l'apoptose et leurs utilisations
EP1232740A3 (fr) * 2001-02-16 2003-07-02 Kyowa Hakko Kogyo Co., Ltd. Agent pour la croissance des cheveux
EP1214928A4 (fr) * 1999-08-18 2004-03-24 Kyowa Hakko Kogyo Kk Stimulants pour la pousse de cheveux
WO2005044176A3 (fr) * 2003-11-10 2005-09-09 Lipogen Ltd Compositions contenant un acide phosphatidique et leurs procedes d'utilisation et de production, et articles de fabrication contenant lesdites compositions
US7192616B2 (en) * 2000-04-28 2007-03-20 L'oreal Plant extract of the Olea europaea species as NO-synthase inhibitor and uses
US7473678B2 (en) 2004-10-14 2009-01-06 Biomimetic Therapeutics, Inc. Platelet-derived growth factor compositions and methods of use thereof
EP2428211A4 (fr) * 2009-05-07 2013-04-03 Moon & J Inc Composition pharmaceutique pour prévenir ou traiter une lésion neuronale ou des maladies neurologiques
US8870954B2 (en) 2008-09-09 2014-10-28 Biomimetic Therapeutics, Llc Platelet-derived growth factor compositions and methods for the treatment of tendon and ligament injuries
US9161967B2 (en) 2006-06-30 2015-10-20 Biomimetic Therapeutics, Llc Compositions and methods for treating the vertebral column
US9642891B2 (en) 2006-06-30 2017-05-09 Biomimetic Therapeutics, Llc Compositions and methods for treating rotator cuff injuries
US10258566B2 (en) 2004-10-14 2019-04-16 Biomimetic Therapeutics, Llc Compositions and methods for treating bone
US10835543B2 (en) * 2013-05-31 2020-11-17 Sports Nutrition Research, Ltd. Method of increasing muscle mass and strength and compositions therefor
US10869843B2 (en) 2010-11-23 2020-12-22 Chemi Nutra Method for increasing muscle mass and strength
US11235030B2 (en) 2010-02-22 2022-02-01 Biomimetic Therapeutics, Llc Platelet-derived growth factor compositions and methods for the treatment of tendinopathies
CN114668778A (zh) * 2022-04-19 2022-06-28 西南民族大学 一种促进创面愈合的微针

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Cited By (30)

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US6051564A (en) * 1996-05-08 2000-04-18 Modus Biological Membranes Ltd. Phosphatidic acid-comprising compositions
WO1997041874A1 (fr) * 1996-05-08 1997-11-13 Modus Biological Membranes Ltd. Compositions contenant de l'acide phosphatidique
WO1999003479A1 (fr) * 1997-07-16 1999-01-28 Modus Biological Membranes Ltd. Composition immuno-modulatrice a base de lipides
US6288047B1 (en) 1997-07-16 2001-09-11 Modus Biological Membranes Ltd. Lipid-based immune modulator composition
EA002364B1 (ru) * 1997-07-16 2002-04-25 Модус Байолоджикал Мембрейнс Лтд. Применение иммуномодулирующей композиции на основе липидов
EP1069895A4 (fr) * 1998-03-18 2002-07-31 Lxr Biotechnology Inc Compositions contenant des acides lysophosphatidiques qui inhibent l'apoptose et leurs utilisations
EP1214928A4 (fr) * 1999-08-18 2004-03-24 Kyowa Hakko Kogyo Kk Stimulants pour la pousse de cheveux
US7015209B2 (en) 1999-08-18 2006-03-21 Kyowa Hakko Kogyo Co., Ltd. Hair-growing agent
US7192616B2 (en) * 2000-04-28 2007-03-20 L'oreal Plant extract of the Olea europaea species as NO-synthase inhibitor and uses
EP1232740A3 (fr) * 2001-02-16 2003-07-02 Kyowa Hakko Kogyo Co., Ltd. Agent pour la croissance des cheveux
WO2005044176A3 (fr) * 2003-11-10 2005-09-09 Lipogen Ltd Compositions contenant un acide phosphatidique et leurs procedes d'utilisation et de production, et articles de fabrication contenant lesdites compositions
US7473678B2 (en) 2004-10-14 2009-01-06 Biomimetic Therapeutics, Inc. Platelet-derived growth factor compositions and methods of use thereof
US11571497B2 (en) 2004-10-14 2023-02-07 Biomimetic Therapeutics, Llc Platelet-derived growth factor compositions and methods of use thereof
US11364325B2 (en) 2004-10-14 2022-06-21 Biomimetic Therapeutics, Llc Platelet-derived growth factor compositions and methods of use thereof
US10258566B2 (en) 2004-10-14 2019-04-16 Biomimetic Therapeutics, Llc Compositions and methods for treating bone
US11318230B2 (en) 2004-10-14 2022-05-03 Biomimetic Therapeutics, Llc Platelet-derived growth factor compositions and methods of use thereof
US9545377B2 (en) 2004-10-14 2017-01-17 Biomimetic Therapeutics, Llc Platelet-derived growth factor compositions and methods of use thereof
US9161967B2 (en) 2006-06-30 2015-10-20 Biomimetic Therapeutics, Llc Compositions and methods for treating the vertebral column
US9642891B2 (en) 2006-06-30 2017-05-09 Biomimetic Therapeutics, Llc Compositions and methods for treating rotator cuff injuries
US10456450B2 (en) 2006-06-30 2019-10-29 Biomimetic Therapeutics, Llc Compositions and methods for treating rotator cuff injuries
US11058801B2 (en) 2006-06-30 2021-07-13 Biomimetic Therapeutics, Llc Compositions and methods for treating the vertebral column
US11135341B2 (en) 2008-09-09 2021-10-05 Biomimetic Therapeutics, Llc Platelet-derived growth factor composition and methods for the treatment of tendon and ligament injuries
US8870954B2 (en) 2008-09-09 2014-10-28 Biomimetic Therapeutics, Llc Platelet-derived growth factor compositions and methods for the treatment of tendon and ligament injuries
US9168282B2 (en) 2009-05-07 2015-10-27 Dongkook Pharmaceutical Co., Ltd. Method for treating neuronal damage and neurological diseases
EP2428211A4 (fr) * 2009-05-07 2013-04-03 Moon & J Inc Composition pharmaceutique pour prévenir ou traiter une lésion neuronale ou des maladies neurologiques
US11235030B2 (en) 2010-02-22 2022-02-01 Biomimetic Therapeutics, Llc Platelet-derived growth factor compositions and methods for the treatment of tendinopathies
US10869843B2 (en) 2010-11-23 2020-12-22 Chemi Nutra Method for increasing muscle mass and strength
US10835543B2 (en) * 2013-05-31 2020-11-17 Sports Nutrition Research, Ltd. Method of increasing muscle mass and strength and compositions therefor
CN114668778A (zh) * 2022-04-19 2022-06-28 西南民族大学 一种促进创面愈合的微针
CN114668778B (zh) * 2022-04-19 2023-08-29 西南民族大学 一种促进创面愈合的微针

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