WO2007145934A1 - Combinaison d'insuline et d'ascorbate pour favoriser la cicatrisation de plaies - Google Patents

Combinaison d'insuline et d'ascorbate pour favoriser la cicatrisation de plaies Download PDF

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Publication number
WO2007145934A1
WO2007145934A1 PCT/US2007/013219 US2007013219W WO2007145934A1 WO 2007145934 A1 WO2007145934 A1 WO 2007145934A1 US 2007013219 W US2007013219 W US 2007013219W WO 2007145934 A1 WO2007145934 A1 WO 2007145934A1
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collagen
ascorbate
insulin
keratocan
accumulation
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PCT/US2007/013219
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English (en)
Inventor
Kurt Musselmann
John R. Hassell
Brad Kane
Bridgette Alexandrou
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University Of South Florida
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins

Definitions

  • the corneal stroma contains keratocytes imbedded in an extracellular matrix consisting primarily of collagen types I and V and of proteoglycans that contain either chondroitin sulfate (CS) or keratan sulfate (KS) chains. Electron microscopic studies show the corneal stroma contains collagen fibrils of small, uniform diameter that are separated by small, uniformly sized spaces. The collagen fibrils in the corneal stroma are heterofibrils of collagen types I and V. Collagen type V is essential for the initiation of fibril formation and the presence of collagen V in the heterofibril has been shown to limit the fibril diameter growth.
  • proteoglycans are in the spaces between the fibrils in vivo (see Muller LJ, Pels E, Schurmans LR, and Vrensen GF.
  • Muller LJ Pels E, Schurmans LR, and Vrensen GF.
  • in vitro assays that measure collagen fibril assembly have shown that these proteoglycans modulate collagen fibril formation.
  • the removal of the GAG side chains did not affect the activity of the proteoglycans, but reduction and alkylation abolished the activity. This indicates that the core protein of these proteoglycans modulates collagen assembly into fibrils.
  • the major proteoglycans of the corneal stroma are decorin, lumican and keratocan. Decorin is a CS proteoglycan, whereas keratocan and lumican are KS proteoglycans. Keratocan (see iu CY, Birk DE, Hassell JR, Kane B, and Kao WW. Keratocan-deficient mice display alterations in corneal structure J Biol Chem.
  • Lumican regulates collagen fibril assembly skin fragility and corneal opacity in the absence of lumican J Cell Biol. 1998; 141 : 1277- 1286; Chakravarti S. Functions of lumican and fibromodulin: lessons from knockout mice Glycoconj J. 2002; 19:287-293 which are incorporated herein by reference) null mice have thinner corneas, and the collagen fibrils in the stromas are larger and less organized than in the stromas of normal mice, confirming the in vitro turbidimetry analysis of collagen fibril assembly:
  • procollagen polypeptides come together to form a left-handed triple helix immediately after synthesis. Stable triple helix formation, however, can only occur if certain lysine and proline residues in the collagen molecule are hydroxylated. This post-translational hydroxylation is performed by either Iysyl or prolyl hydroxylases. These enzymes are found in the lumen of the endoplasmic reticulum and require ascorbic acid as a cofactor. Ascorbate deficiency in cell culture does not affect collagen synthesis but affects fibril formation and the rate of collagen secretion. The u ⁇ hydroxylated collagen molecules denature at a lower temperature and in less stringent environments than properly hydroxylated collagen fibrils.
  • Scorbutic wounds are weaker than non-scorbutic wounds, prone to reopen and contain a mass of irregular, unorganized collagen fibrils.
  • the cornea and anterior segment of the eye contain high levels of ascorbate (see Ringvold A, Anderssen E, and Kjonniksen I. Distribution of ascorbate in the anterior bovine eye Invest Ophthalmol Vis ScL 2000;41 :20-23; which is incorporated herein by reference), where it is thought to function as an antioxidant and protect the cornea from reactive oxygen species that result from UV irradiation (see Brubaker RF, Bourne WM, Bachman LA, and McLaren JW. Ascorbic acid content of human corneal epithelium Invest Ophthalmol Vis ScL 2000;41 :1681-1683; which is incorporated herein by reference).
  • Ascorbic acid has been used to study collagen synthesis in culture, but it is easily oxidized in solution and its metabolic by-products are cytotoxic in extended cell culture.
  • a stable, non-toxic phosphate derivative of ascorbic acid (2-phospho-L-ascorbic acid) was developed and has been shown to stimulate collagen accumulation by skin fibroblasts in culture and also to enhance the secretion of type I and type III collagen peptides by rabbit keratocytes cultured in medium containing fetal bovine serum. Serum, however, contains mitogens and morphogens that cause keratocytes in culture to proliferate, acquire a fibroblastic morphology and cease keratocan expression.
  • a chemically defined medium containing insulin has been previously shown to stimulate keratocyte proliferation while maintaining their dendritic morphology as well as keratocan expression (see Musselmann K, Alexandrou B 3 Kane B, and Hassell JR. Maintenance of the keratocyte phenotype during cell proliferation stimulated by insulin J Biol Chem. 2005; 280:32634-32639; which is incorporated herein by reference).
  • keratocytes were cultured in this defined medium containing 2-phospho-L-ascorbic acid to determine its effects on the synthesis and accumulation of collagen and KS proteoglycans.
  • Collagenase isolated keratocytes were cultured with or without insulin with or without ascorbate.
  • Collagen and glycosaminoglycan synthesis were determined by collagenase digestion of incorporated 3 H-glycine and by chondroitinase ABC or endo- ⁇ -galactosidase digestion of incorporated 35 SCv KSPGs were detected by Western blot.
  • Collagen stability was determined by pepsin digestion.
  • Ethyl- 3,4-dihydroxybenzoate (EDB) was used to inhibit collagen hydroxylation.
  • Insulin stimulated the synthesis of collagen but did not affect the accumulation of iumican and keratocan.
  • Insulin plus ascorbate stimulated the synthesis of collagen and increased the accumulation of these proteoglycans.
  • the accumulation of PGDS 1 a KSPG that does not interact with collagen was not affected by ascorbate. Only the collagen made in the presence of ascorbate was pepsin resistant. EDB overrode the effects of ascorbate on pepsin resistance and proteoglycan accumulation.
  • the invention includes a method of stimulating collagen synthesis comprising contacting a cell with a therapeutically effective amount of a compound comprising insulin and ascorbate.
  • the compound further comprises 2-phospho-L-ascorbic acid.
  • the invention includes a method of stimulating wound healing, comprising contacting the wound with a therapeutically effective amount of a compound comprising insulin and ascorbate.
  • a compound comprising insulin and ascorbate.
  • the compound further comprises 2-phospho-L-ascorbic acid.
  • the invention includes a method of inducing the secretion of collagen from a cell comprising contacting the cell with an effective amount of a compound comprising insulin and ascorbate.
  • the compound further comprises 2-phospho-L-ascorbic acid.
  • the invention includes a method of inducing the secretion of a proteoglycan from a cell comprising contacting the cell with an effective amount of a compound comprising insulin and ascorbate.
  • the compound further comprises 2-phospho-L-ascorbic acid and the proteoglycan is a keratin sulfate proteoglycan selected from the group consisting of iumican and keratocan.
  • the invention includes a method of stimulating the synthesis of pepsin resistant collagen comprising contacting a cell with an effective am ount of a compound comprising ascorbate.
  • the compound further comprises insulin and/or 2-phospho-L-ascorbic acid.
  • Radioactivity incorporated into collagen present in the media (black bar) and cell layer (open bar) was determined using a collagenase specific for fibrillar regions of collagen.
  • Adding insulin to the culture medium significantly increased collagen in both the cell layer and the medium.
  • Insulin plus ascorbate show a significant increase collagen in secreted into the medium and a significant decrease in collagen in the cell layer compared to insulin alone.
  • n 3 Figure 3.
  • the medium of cells cultured in insulin (I), insulin plus ascorbate (IA) or insulin, ascorbate and EDB (0.4mM) was collected, concentrated and digested with pepsin in 0.5M acetic acid.
  • Figure 4 Accumulation of keratan sulfate proteoglycans in the medium.
  • Medium was collected on day 4, digested with endo- ⁇ -galactosidase, separated by SDS-PAGE, transferred to nitrocellulose and probed with antibodies to keratocan, lumican or PGDS.
  • the Western blots using antibodies against keratocan, lumican and PGDS were scanned to determine their pixel density and the net pixel density divided by the micrograms of DNA in each culture. Bars represent the mean of 3 determinations plus S.D.
  • Keratocan accumulation in medium containing ascorbate increased 4- fold (p ⁇ 0.005) compared to control.
  • Medium containing insulin and ascorbate contained 9-fold more keratocan and lumican (p ⁇ 0.005) than control medium.
  • the addition of ascorbate did not have an effect on PGDS levels with or without insulin.
  • n 3
  • FIG. 5 Accumulation of keratan sulfate proteoglycans in culture medium of cells treated with ethyl- 3,4-dihydroxybenzoate (EDB). Keratocytes were cultured in medium containing either 0.08mM or 0.4mM EDB in insulin containing growth medium with or without ascorbate. Samples were processed as described in Figure 6. A dose dependent significant decrease (p ⁇ 0.01) at 0.08mM EDB was detected in both lumican and keratocan accumulation between cells cultured in insulin + ascorbate and insulin + ascorbate + EDB. At 0.4mM, lumican and keratocan levels fell below the levels of the insulin alone control.
  • EDB ethyl- 3,4-dihydroxybenzoate
  • FIG. 7 Chromatography Of 35 SO 4 radiolabeled proteoglycans on Superose 6. Culture medium from keratocytes incubated with 35 SO 4 for 72-hours was harvested and equal amounts of incorporated radioactivity was fractionated on Superose 6 to determine incorporation into intact proteoglycans. Legend: insulin: filled squares, solid line; insulin plus ascorbate: open squares, dashed line. Inlay shows percent CS/KS of fractions 15 to 23 from media of insulin plus ascorbate treated cells. Inlay legend: CS, solid line; KS, dashed line. Most of the incorporated radioactivity elutes at the position of the intact proteoglycans. Ascorbate preferentially stimulates the incorporation Of 35 SO 4 into KSPG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
  • DMEM/F12 Dulbecco's modified Eagle's medium and Ham's F-12 Medium, 1:1;
  • HRP horse-radish peroxidase
  • SDS sodium dodecyl sulfate
  • PRK photorefractive keratectomy
  • ER endoplasmic reticulum
  • UV ultraviolet light
  • ECL electrochemiluminescence
  • Ascorbate is required for the hydroxylation of collagen that is present in the corneal stroma.
  • Ascorbate was added to a defined medium containing insulin and its effects on the synthesis of collagen and KSPGs by keratocytes was determined.
  • Collagenase isolated keratocytes were cultured with or without insulin with or without ascorbate.
  • Collagen and glycosam ⁇ noglycan synthesis were determined by collagenase digestion of incorporated 3 H-glycine and by chondroitinase ABC or end ⁇ - ⁇ -galactosidase digestion of incorporated 35 SO ⁇ .
  • KSPGs were detected by Western blot.
  • Collagen stability was determined by pepsin digestion.
  • Ethyl- 3,4-dihydroxybenzoate (EDB) was used to inhibit collagen hydroxylation.
  • Insulin stimulated the synthesis of collagen but did not affect the accumulation of lumican and keratocan. Insulin plus ascorbate, however, stimulated the synthesis of collagen and increased the accumulation of these proteoglycans.
  • ascorbate to culture medium containing insulin not only stimulated collagen synthesis, but also increased keratocan and lumican accumulation 9-fold and KS synthesis 1 1 -fold.
  • the increase in lumican and keratocan accumulation correlates with the increased synthesis of hydroxylated collagen.
  • Culture in insulin alone stimulated collagen synthesis, but did not affect lumican and keratocan accumulation.
  • insulin stimulated collagen synthesis in the absence of ascorbate the collagen did not form stable triple helices, as shown by a greater proportion of the total collagen made accumulating with the cell layer, or pericellularly, and by the susceptibility of the collagen secreated into the medium to degradation by pepsin.
  • EDB ethyl-3,4-dihydroxybenzoate
  • the ascorbic acid mediated stimulation of keratan sulfate proteoglycan accumulated by keratocytes was limited to keratocan and lumican. Both keratocan and lumican have core proteins containing leucine rich repeats (LRR). Homology modeling of proteins with LRR to ribonuclease inhibitor show that these proteins to fold to a solenoid tertiary structure, that interacts with the collagen fibril and regulates fibril formation and diameter.
  • PGDS is made as a keratan sulfate proteoglycan by keratocytes in culture, but it does not contain LRR and does not interact with collagen.
  • PGDS accumulation was not affected by insulin, ascorbate or the combination of both.
  • the invention shows that while insulin stimulated collagen synthesis, most of this increase was in the collagen associated with the cell layer and that when ascorbic acid was included with insulin, most of the increase was in the collagen secreted into the medium, where the proteoglycans are also secreted.
  • Proteoglycans such as lumican and keratocan that interact with collagen may depend on the formation of a stable collagen helix for their own stability.
  • the core protein of these proteoglycans interact with specific regions of the collagen fibril in a manner similar to the interaction of collagen with decorin.
  • ascorbate besides stabilizing the collagen triple helix, also increases the accumulation of lumican and keratocan proteoglycans in the medium.
  • This data suggests that the prophylactic action of ascorbate in alkali wound treatment, and in the prevention of late onset corneal haze after PRK by the use of topical ascorbate, may be due to the effect of ascorbate on increasing the stability of the triple helix for collagen I and V which then acts to increase the stability and therefore the accumulation of the keratan sulfate proteoglycans lumican and keratocan.
  • the increased accumulation of keratan sulfate proteoglycans may accelerate the restoration of stromal transparency by regulating the assembly of the stably folded collagen triple helices into fibrils of the correct diameter.
  • Insulin which has been shown to stimulate collagen synthesis and which has been shown to improve wound healing, may further enhance wound healing if used in combination with ascorbate, particularly in humans, since they lack the ability to synthesize ascorbate.
  • the DNA content of the keratocyte cultures was measured over a 10-day culture period to determine if ascorbate stimulated cell accumulation.
  • the hydroxylation of proline in the procollagen molecule is a post-translational modification required for the formation of a stable triple helix in the endoplasmic reticulum.
  • Prolyl hydroxylases are the enzymes required for the hydroxylation of the 4-position of proline.
  • Ethyl-3,4-dihydroxy-benzoate (EDB) is a selective inhibitor of prolyl hydroxylase. EDB is not toxic to the cells and has been previously shown to inhibit collagen deposition in a dose-dependent manner. Since most of the collagen made by keratocytes cultured in ascorbate plus insulin was in the media ( Figure 2), the collagen in the media was analyzed for stability by resistance to pepsin digestion.
  • Keratocytes were cultured in insulin or insulin plus ascorbate-containing medium with or without 0.4mM EDB.
  • the medium of the cells was collected, adjusted to 0.5M acetic acid, digested with pepsin and analyzed by SDS-PAGE ( Figure 3).
  • the medium of keratocytes cultured in insulin plus ascorbate contained pepsin-resistant collagen types I and V fibrils, demonstrated by the prominent bands for ocl(I) and ⁇ 2(I), as well as a clear band for ⁇ l(V) between the 64 and 19IkDa markers ( Figure 3, lane IA).
  • the accumulation of lumican and keratocan in the culture medium was determined by Western blot.
  • the medium was digested with endo- ⁇ -galactosidase to remove the KS side chain before SDS-PAGE to facilitate transfer in Western blot.
  • Antibodies to the core protein of the proteoglycan detect the core protein as a sharp band in a Western blot, and the pixel density for each band was determined (Figure 4). Treating cells with ascorbate alone increased keratocan and lumican levels in the medium 4-fold (p ⁇ 0.005), but insulin alone had no effect on the levels of these proteoglycans. A 9-fold increase in both lumican and keratocan was observed (p ⁇ 0.005 for both), when cells were cultured with insulin plus ascorbate.
  • Prostaglandin D synthase is also synthesized as a KSPG by keratocytes in vitro. Consequently, the inventors performed Western blots for PGDS as well and found that neither ascorbate nor insulin affected PGDS levels. Since only keratocan and lumican have been shown to interact with collagen fibrils, these results suggest that the increased accumulation of KSPGs in the medium is limited to those with core proteins that interact with collagen.
  • the results of this experiment also confirm a correlation between hydroxylation and increased levels of lumican and keratocan production.
  • Keratocyte cultures were radiolabeled with 35 SO 4 , and the amount of incorporated radiolabel in the CS and KS secreted into the media was determined by digestion with chondroitinase ABC and endo- ⁇ - galactosidase to determine if ascorbate would also increase the incorporation Of 35 SO 4 into KS ( Figure
  • fractions 19 to 23 There was, however, relatively greater incorporation in fractions 19 to 23 for keratocytes cultured in insulin plus ascorbate.
  • Fractions 15-23 were digested with chondroitinase ABC or endo- ⁇ -galactosidase to determine the glycosaminoglycan chain composition.
  • the inlay shows that over 80% of the incorporated counts in fractions 15-18 were released by chondroitinase ABC digestion, whereas over 60% of the incorporated counts in fractions 19-23 were released by endo- ⁇ -galactosidase digestion.
  • Example Chemicals - Chemicals were purchased from Sigma (St. Louis, MO) unless otherwise indicated. Isotopes were obtained from PerkinElmer (Boston, MA). Invitrogen (Carlsbad, CA) gels, reagents and equipment were used to separate proteins and for transfer onto nitrocellulose.
  • DNA quantitation - Cell layers were harvested on days 1, 4, 7 and 10 to measure DNA content using Cyquant (Invitrogen, Carlsbad, CA). Briefly, the cell layers were rinsed with PBS, frozen, thawed and solubilized in a lysis buffer supplemented with a DNA binding dye. The DNA content was determined at 480/535nm by measuring 4 wells in triplicate and comparing the values to a calf thymus DNA standard. The DNA content of parallel cultures was measured for experiments that utilized the cell layer.
  • Cell proliferation Cultures were radiolabeled with 20 ⁇ Ci 3 H-thymidine/ml of medium for 72 hours beginning on days 1 and 4. The labeled medium was removed upon harvesting, and the cell layers washed with cold PBS. The cell layers were processed as described under DNA quantitation. Incorporation into DNA was determined as previously described.
  • Collagen synthesis The incorporation of 3 H-glycine into collagen was determined as previously described. In summary, cultures were incubated for 72 hours in medium containing 25 ⁇ Ci 3 H- glycine/ml beginning on day I . Media was adjusted to 4M guanidine HCl and the cell layers extracted in 4M guanidine HCl. Unincorporated isotope in media and cell layers was removed by chromatography on PDlO columns equilibrated and eluted with 4M guanidine HCl (GE Healthcare, Piscataway, NJ).
  • Collagen helix stability- Medium from cells was adjusted to 0.5 M acetic acid and concentrated 8-fold using Amicon Ultra spin-concentrators MWCO 10,000(Millipore Corp., Bedford, MA). 50ul of a 4mg/ml pepsin solution (in 0.5M acetic acid) per 3 ml was added and the samples rocked overnight at 4'C. Each sample received a second 50 ⁇ l aliquot from the pepsin stock, and digestion was allowed to continue for an additional six hours at 4°C. The samples were titrated with IN NaOH to pH 8.0 to inactivate the enzyme, dialyzed against water overnight, lyophilized, reconstituted in IX SDS running buffer and separated on 10% bis-tris gels under reducing conditions. The gels were stained with Safestain following the manufacturers protocol.
  • Proteoglycan synthesis - Cultures were labeled with 50 ⁇ Ci/ml Of 35 SO 4 for 72 hours on day 1. The medium was collected, frozen, lyophilized and reconstituted in 4M guanidine HCI. Unincorporated isotope was removed using PDlO columns. Fractions containing incorporated 35 S ⁇ 4 were combined and concentrated using Amicon Ultra spin-concentrators. Incorporation into CS and KS was determined by digestion with chondroitinase ABC or endo- ⁇ -galactosidase (Seikagaku, Associates of Cape Cod, E. Falmouth, MA) as previously described.
  • compositions and/or of the subject invention can be formulated according to known methods for preparing pharmaceutically useful compositions.
  • pharmaceutically acceptable carrier means any of the standard pharmaceutically acceptable carriers.
  • the pharmaceutically acceptable carrier can include diluents, adjuvants, and vehicles, as well as implant carriers, and inert, non-toxic solid or liquid fillers, diluents, or encapsulating material that does not react with the active ingredients of the invention. Examples include, but are not limited to, phosphate buffered saline, physiological saline, water, and emulsions, such as oil/water emulsions.
  • the carrier can be a solvent or dispersing medium containing, for example, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • ethanol for example, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • polyol for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like
  • suitable mixtures thereof for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like
  • Formulations suitable for parenteral administration include, for example, aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteri ostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the condition of the sterile liquid carrier, for example, water for injections, prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powder, granules, tablets, etc. It should be understood that in addition to the ingredients particularly mentioned above, the formulations of the subject invention can include other agents conventional in the art having regard to the type of formulation in question.
  • the pharmaceutical composition can be adapted for various forms of administration. Administration can be continuous or at distinct intervals as can be determined by a person skilled in the art.
  • the administration of the compound comprising insulin, ascorbate, or any combination thereof is administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight, and other factors known to medical practitioners.
  • a therapeutically effective amount of the compound comprising insulin, ascorbate, or any combination thereof is that amount necessary to provide a therapeutically effective result in vivo.
  • the amount of the compound comprising insulin, ascorbate, or any combination thereof must be effective to achieve a response, including but not limited to increased collagen synthesis within the cell layer, increased collagen synthesis pericellularly, increased resistance of the synthesized collagen to degradation by pepsin, increased synthesis and/or secretion of lumican and/or keratocan or other indicators as are selected as appropriate measures by those skilled in the art.
  • a suitable single dose size is a dose that is capable of preventing or alleviating (reducing or eliminating) a symptom or condition in a patient when administered one or more times over a suitable time period.
  • One of skill in the art can readily determine appropriate single dose sizes for systemic administration based on the size of a mammal and the route of administration.

Abstract

La présente invention concerne un procédé permettant de stimuler la synthèse du collagène et l'accumulation de protéoglycane (lumicane et kératocane). Les kératocytes isolés par la collagénase ont été cultivés avec ou sans insuline et avec ou sans ascorbate. L'insuline stimule la synthèse de collagène mais ne modifie pas l'accumulation de lumicane et de kératocane. L'insuline additionnée d'ascorbate stimule cependant la synthèse de collagène et favorise l'accumulation de ces protéoglycanes. L'accumulation de PGDS, un KSPG qui n'interagit pas avec le collagène, n'est pas modifiée par l'ascorbate. Seul le collagène fabriqué en présence d'ascorbate s'est révélé résistant à la pepsine. L'EDB a annulé les effets de l'ascorbate sur la résistance à la pepsine et l'accumulation de protéoglycane.
PCT/US2007/013219 2006-06-05 2007-06-05 Combinaison d'insuline et d'ascorbate pour favoriser la cicatrisation de plaies WO2007145934A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6359007B1 (en) * 1999-04-07 2002-03-19 Chronorx, Llc Clinical uses for L-arginine ascorbate and various metalloarginate complexes
US20030181386A1 (en) * 2000-08-10 2003-09-25 Teruo Nishida Skin wound healing promoters
US20040248871A1 (en) * 2001-08-03 2004-12-09 Jean Farjanel Use of lysyl oxidase inhibitors for cell culture and tissue engineering

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6359007B1 (en) * 1999-04-07 2002-03-19 Chronorx, Llc Clinical uses for L-arginine ascorbate and various metalloarginate complexes
US20030181386A1 (en) * 2000-08-10 2003-09-25 Teruo Nishida Skin wound healing promoters
US20040248871A1 (en) * 2001-08-03 2004-12-09 Jean Farjanel Use of lysyl oxidase inhibitors for cell culture and tissue engineering

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