WO1992013888A1 - Compositions et procede de cicatrisation des cellules epitheliales - Google Patents

Compositions et procede de cicatrisation des cellules epitheliales Download PDF

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Publication number
WO1992013888A1
WO1992013888A1 PCT/US1992/000558 US9200558W WO9213888A1 WO 1992013888 A1 WO1992013888 A1 WO 1992013888A1 US 9200558 W US9200558 W US 9200558W WO 9213888 A1 WO9213888 A1 WO 9213888A1
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Prior art keywords
epithelial
polypeptide
cells
dexamethasone
growth
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PCT/US1992/000558
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English (en)
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Ted W. Reid
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The Regents Of The University Of California
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Publication of WO1992013888A1 publication Critical patent/WO1992013888A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention generally relates to epithelial cell wound healing, such as for burn and wound problems of the skin and for ocular wounds, such as affecting the cornea, and more particularly to an homogeneous growth factor (designated "ect-GF") characterized by a molecular weight of about 13,000 to about 14,000 daltons and having, among other properties, an ability to protect epithelial cells against the usual growth inhibitory properties of dexamethasone.
  • ect-GF homogeneous growth factor
  • Corneal disease cases are the most painful of all ophthalmic disorders and account for considerable disability. Corneal disease cases account for hospital stays involving a total of 100,000 days and more than 8 million office visits annually for professional eye care. Eye injuries, which also primarily affect the cornea, account for an additional 1.7 million annual visits to physicians.
  • cataract operations there are approximately 3 million cataract operations in the United States each year. This number is increasing since each year a larger proportion of the population is in the age group that would require a cataract operation.
  • One of the major problems with this surgery is the distortion of vision caused by the sutures in the incision required by this surgery. If a drug were available to promote the healing of this wound, then this would be a preferred treatment by the ophthalmologist, since the patient would have clear vision restored more quickly and would have less worry about injury to the eye during the recovery period.
  • An epithelial growth factor could be useful in promoting healing for burn and wound problems of the skin and for ocular wounds. (The outer corneal layer is an epithelial layer.)
  • Growth factors are substances, such as polypeptide hormones, which promote the growth of defined populations of animal cells in vivo or in vitro, but which are not nutrient substances.
  • growth factors presently known are those that can be classified into the insulin family [insulin, insulin-like growth factors (e.g., IGF-I, IGF-II) , mammary stimulating factor (MSF) , and nerve growth factor (NGF) ] ; those classified into the epidermal growth factor family [epidermal growth factor (EGF) and transforming growth factors (TGF ⁇ , TGF ⁇ , TGF ⁇ ) ] ; those classified into the platelet-derived growth factor family [platelet-derived growth factor (PDGF) , osteosarcoma-derived growth factor (ODGF) , and fibroblast growth factor (FGF) ] ; and others [colony stimulating factor (CSF) , T-cell growth factor, tumor angiogenesis factor (TAF) , DNA synthesis promoting factor (DSF) , tumor-derived growth factors, fibroblast- derived growth factor (FDGF) ] .
  • insulin-like growth factors e.g., IGF-I, IGF-II
  • MSF
  • Epidermal growth factors have been found in mammals, can be isolated from human and horse urine, and also from rabbit, rat and mouse sub axillary glands. Adv. Metab. Dis. , Vol. 8, p. 265 (1975).
  • Human epidermal growth factor (hEGF) was isolated from human urine and described as a human-derived factor enhancing both proliferation and keratinization of epidermal tissue by S. Cohen in 1975 [Proc. Natl . Acad. Sci. USA, Vol. 72, p. 1317 (1975)]. This apparently is the same substance as a polypeptide reported as human urogastrone (h-UG) which inhibits gastric acid secretion and was isolated from human urine by H. Gregory et al. [iVature, Vol.
  • the human epidermal growth factor/urogastrone has a molecular weight of about 6000, comprises 53 amino acid residues and has three disulfide bonds in its molecule. [Me aJolis ⁇ . (in Japanese), Vol. 17, 51-58 (1980). This epidermal growth factor is often referred to as "EGF.”
  • Dexamethasone sodium phosphate is used for the treatment of steroid-responsive inflammatory conditions, including corneal injury.
  • U.S. Patent 4,945,089, issued July 31, 1990, inventor Clark describes a method of treating ophthalmic inflammation in a human patient which comprises applying topically to the affected eye a pharmaceutical composition including a glucocorticoid such as dexamethasone.
  • a pharmaceutical composition including a glucocorticoid such as dexamethasone.
  • Effect of Growth Factors with Dexamethasone on Healing of Rabbit Cornea Experimental Eye Research, 40, pp. 47-60 (1985) is described the inhibition of growth by dexamethasone and all the growth factors tested.
  • K ⁇ ssendrup et al. "Influence of Cyclosporin A, Dexamethasone, and Benzalkonium Chloride (BAK) on Corneal Epithelial Wound Healing in the Rabbit and Guinea Pig Eye", Cornea, 4, pp. 177-181 (1985/1986) describe that epithelial wound healing is significantly delayed in guinea pigs under dexamethasone therapy.
  • the authors also found that epithelial wound healing in the contralateral eye of the rabbit was significantly delayed in the presence of dexamethasone.
  • the dexamethasone source used by the authors was commercially available under the trade name "Spersadex” and comprised dexamethasone at 0.1% and benzalkonium chloride at 0.01%.
  • U.S. Patent 4,874,746, issued October 17, 1989, inventors Antoniades et al. discloses a composition with a combination of a platelet-derived growth factor and a transforming growth factor alpha. This composition is said to aid in healing external wounds in a human patient by topical application with the composition promoting the growth of epithelial and connective tissue.
  • U.S. Patent 4,886,786, issued December 12, 1989, inventors Lindstrom et al. discloses an additive concentrate said to be useful in an irrigation solution or a surgical solution for the anterior and posterior chambers of the eye and for corneal preservation media.
  • This additive has, as various essential ingredients, an antioxidant, dextrose or a pyruvate, sodium bicarbonate, and one or more additional components. Inclusion of a growth factor, such as fibroblastic growth factor, is suggested.
  • a growth factor such as fibroblastic growth factor
  • Patent 4,902,782 issued February 20, 1990, inventors Gospodarowicz et al., describe the purification of a mammalian protein they term "basic fibroblast growth factor, or bFGF," which is described as having a molecular weight of about 14,000 daltons and, in its basic form, is said to be mitogenic for corneal and vascular endothelial cells and lens epithelial cells.
  • bFGF basic fibroblast growth factor
  • a purification method for obtaining the basic FGF from brain, pituitary gland, adrenal gland, kidney, or placenta tissue sources by use of Heparin-Sepharose affinity chromatography is described.
  • a growth factor, secreted by a human retino ⁇ blastoma cell line grown in a serum-free media, with an apparent molecular weight of 38,000 daltons has been described by Rubin et al.. Vision Research , Vol. 21, pp. 105-112 (1981) and Tarsio et al., Exp. Cell Res. , Vol. 146, pp. 71-78 (1983).
  • RDGF retinoblastoma cell line
  • a therapeutic composition comprises a biologically active polypeptide having a molecular weight of between about 13,000 and about 14,000 daltons.
  • the biological activity of the polypeptide includes promotion of epithelial cell growth while being resistant to dexamethasone inhibition.
  • the novel polypeptide is sometimes herein designated "ect-GF.”
  • the therapeutic composition preferably includes a pharmaceutically acceptable carrier adapted for topical administration and preferably where the "ect-GF" polypeptide is in an concentration of between about 10 -3 M to about 10-12 M, preferably between about l ⁇ "3 M to about l ⁇ "8 M.
  • Epithelial cell growth promotion by the inventive polypeptide in the presence of dexamethasone is actually potentiated, and has been used to successfully treat human and animal corneal ulcers which had not healed for three months, but which healed when treated in accordance with the invention in less than four days.
  • another aspect of the present invention is a method of treating epithelial cells to promote growth that comprises providing a surgical solution having a therapeutically effective amount of the inventive epithelial growth factor "ect-GF" dissolved therein and contacting cells, such as corneal or skin (epidermal) cells, with said surgical solution for at least about one hour.
  • inventive polypeptide growth factor binds very tightly to its cell surface receptors.
  • Corneal cells for example, may be an actual cornea for a cornea transplant operation and skin cells may be skin transplants or skin grafts.
  • Fig. 1 is a bar graph where the vertical axis measures the amount of tritium incorporated into DNA, and thus is a measure of DNA synthesis, or cell growth, and where each of the paired bar graphs represents use of a different growth factor with or without the presence of dexamethasone;
  • Fig. 2 graphically illustrates the dose response data for a prior art growth factor (at differing concentrations) with and without the presence of dexamethasone;
  • Fig. 3 graphically illustrates use of the "ect-GF" inventive growth factor at differing concentrations with and without the presence of dexamethasone;
  • Fig. 4 graphically compares dose response for the highly purified and homogenous "ect-GF" inventive growth factor by comparison to impure growth factor where the horizontal axis is logmetric (concentrations) ;
  • Fig. 5 illustrates bar graphs of data from receptor binding studies by comparison with insulin. Detailed Description of the Preferred Embodiments.
  • this invention provides a novel polypeptide that may be isolated and purified from a human retinoblastoma cell line, preferably grown in serum-free culture.
  • This human retinoblastoma cell line has been designated "Y-79", which is available from the ATCC as HTB-18.
  • Non-homogenous or only partially puri ied growth factor or growth factors from this cell line have been reported in the Rubin et al. and Tarsio et al. articles, supra, of 1981 and 1983, and by Schweigerer et al., supra , 1987 article; however, the subject polypeptide is homogeneous, highly purified, and has a number of unique properties which will be more fully described hereinafter.
  • the homogeneous, highly purified inventive polypeptide has been designated "ect- GF", and may be isolated and purified as follows.
  • Retinoblastoma cells of the Y-79 cell line available from the ATCC are grown in serum free media (RPMI 1640) in a low oxygen atmosphere. This forces the cells to produce their own growth factor.
  • the cells are fed fresh media every three days and the old media is frozen.
  • This conditioned media is then concentrated on a Pelicon concentrator using a 10,000 MW cutoff filter. The media is concentrated approximately 200 fold.
  • the concentrated material is passed through a Sephacryl S- 200 column at 0.1 M NaCl. Since at this ionic strength the growth factor aggregates, it moves through the column with an apparent molecular weight of approximately 40,000, and both smaller material and larger material can be thrown away.
  • the semi-purified factor is then passed through the same column at 0.4 M NaCl.
  • the inventive factor "ect- GF” does not aggregate and moves through the column with a molecular weight of about 13,000.
  • the "ect-GF” factor is then bound to a reactive Green column (reactive Green Agarose - Sigma Chemical Cat #R4004) .
  • the inventive factor elutes from the column by a salt gradient at 1.5M NaCl. These procedures result in material that yields one band on an SDS polyacrylamide gel, at approximately 13,000 to 14,000 MW.
  • the factor is assayed by the DNA synthesis assay outlined below, which determines its ability to synthesize DNA in lens epithelial cells.
  • Nakano mouse lens epithelial cells were obtained from Paul Russell (National Eye Institute, NIH, Bethesda, MD) . The particular cells used in these experiments were a clone obtained from these cells (NK- 11) which were found to grow in a reproducible manner.
  • the rabbit cornea epithelial cells were obtained from the American Type Culture Collection (ATTC CCL-60) .
  • the NK-11 cells are grown in RPMI 1640 supplemented with 10% Calf Serum.
  • the SIRC cells were grown in (“MEM”) supplemented with 5% new born serum and 5% fetal calf serum.
  • the NK-11 cells were plated in 96 well plates at a density of 10,000 per well in RPMI 1640 plus 10% calf serum. The cells were fed three days later with fresh RPMI 1640 containing 10% calf serum. After two days the cells were fed with RPMI 1640 (no serum) and allowed to reach quiescence. After two to three days the cells were then used for the assay.
  • the SIRC cells were plated in 96 well plates at a density of 10,000 per well in MEM plus 5% new born serum and 5% calf serum. After three days the cells were fed with MEM plus 5% new born serum and 5% calf serum. After three to four days the cells were then fed with MEM and allowed to become quiescent. After three days the cells were ready to use in the assay.
  • the cells were refed fresh media (RPMI 1640 or MEM depending on the cell type) and then the growth factor was added. After sixteen hours 0.1 ⁇ c of tritiated- thymidine was added to each well. After a total of forty hours the cells were washed twice with 0.15 M NaCl and then fixed with 0.3 M HCL. The cells were allowed to set for twenty minutes at 4° and then washed twice with 0.3M HC1 and then with 95% ethanol 5% water. The cells were then solubilized in 0.5 M NaOH and counted in a scintillation counter. This radioactivity was then used as a measure of DNA synthesis.
  • the inventive factor "ect-GF” that may be obtained in homogenous, highly purified form from the above procedures is labile in 0.2 M sodium chloride solution at 60°C in 10 minutes, but is stable for 30 minutes at 60 ⁇ C in 0.01 M sodium chloride solution.
  • the inventive factor is also stabilized in 2 M sucrose or 20% glycerol.
  • the inventive factor disaggregates at pH 2.6 but loses only 50% of its activity in one hour at room temperature at pH 2.6.
  • the inventive "ect-GF” factor is inactivated by treatment with 0.1% trypsin or chymotrypsin for one hour.
  • the inventive factor may be stored in 0.15 M sodium chloride solution at -20 ⁇ C, for example stored for one year or more, as it has been shown to be stable in such a salt solution for over two years.
  • ect-GF differs from the growth factor described by Schweigerer et al. , supra , designated by them as “bFGF,” not just by molecular weight (the inventive factor being between about 13,000 to about 14,000 daltons and the bFGF being at 16,000 daltons), but also in various important properties.
  • Fig. 1 graphically represents cell growth stimulation (or inhibition) in the presence of the inventive factor "ect-GF” with or without the presence of dexamethasone (at 10 " M) and of the prior art bFGF factor also by itself and in the presence of dexamethasone (10 _6 M) .
  • the inventive factor "ect-GF” stimulated cell growth in the absence of dexamethasone much better than did the bFGF.
  • the inventive factor was actually potentiated so that cell growth was even higher than without the presence of dexamethasone.
  • the bFGF was considerably inhibited by the presence of dexamethasone. Further details of this experiment are found in Example 1.
  • Fig. 2 illustrates dose response data for the prior art bFGF with and without the presence of dexamethasone (l ⁇ '6 M) and shows that over an entire dose range up to 100 pg/ml of bFGF there is damping, or inhibition, due to the presence of the dexamethasone except at the very end of the dose response range.
  • inventive growth factor "ect-GF” is yet further different in properties from the "bFGF” described by Schweigerer et al. , supra , as the inventive factor does not stimulate either bovine aortic or corneal endothelial cell growth over a range of concentrations that are quite stimulatory for epithelial cells, yet the "bFGF” is quite stimulatory for both types of endothelial cell growth.
  • Fig. 4 illustrates a comparison of the inventive factor "ect-GF” with the relatively high molecular weight (38,000 daltons) growth factor designated "RDGF” described by Rubin et al., supra, in the 1981 and 1983 articles.
  • the inventive growth factor is up to 6,000 times more potent in growth promoting activity and further provides a growth promoting activity response over a wide range of relatively dilute concentrations.
  • the inventive factor "ect-GF” protects epithelial cells completely against growth inhibition by dexamethasone, and in fact appears to work better when dexamethasone is present, which means that steroids (such as dexamethasone) can be used to block inflammation while the inventive growth factor promotes healing of the wound.
  • a model was developed to test the tensile strength of a stromal wound in the cornea.
  • This model required a six millimeter incision in a rabbit cornea be made (which is about 90% of the way through the cornea) .
  • the incision is performed by using a pachy eter to determine the thickness of the cornea, followed by the use of a radial keratotomy knife to make the incision to the desired depth.
  • These eyes were then treated twice a day with a control, the inventive or prior art growth factors for one week, and then allowed to heal further for two weeks. At the end of the three weeks, the corneas were removed and a second incision was made to the back of the eye 20% of the way through the original wound.
  • the inventive growth factor "ect-GF” binds to its receptor with no appreciable loss in activity for over three days, and thus the use of the inventive factor as a topical treatment to a wound will have an excellent chance of being effective.
  • a surgical solution having a therapeutically effective amount of the inventive epithelial growth factor dissolved therein, and contacts cells, such as corneal cells, with the surgical solution, then the contacting preferably is for at least about one hour but needs not be appreciably longer than one hour (unless desired or convenient) due to the excellent binding properties of the inventive growth factor with its receptor.
  • Example 5 further illustrates receptor binding, and is the protocol from which the Fig. 5 data was taken.
  • inventive growth factor "ect-GF” Additional advantageous properties include chemotactic ability toward cells even at quite low concentrations, which ability is important for cellular migration into a wound as a first step in wound healing. Further, the inventive growth factor is synergistic with other growth factors and causes a persistence of action. This means that the inventive growth factor can interact with other growth factors that are both naturally present or which can be added with it. However, as earlier noted the inventive growth factor does not stimulate endothelial cell growth, and thus has no angiogenic properties. This is particularly desirable for the healing of cornea wounds.
  • inventive growth factor "ect-GF” as a material secreted by the Y-79 human retinoblastoma cell line
  • cloning techniques can be used to synthesize the inventive polypeptide, such as by isolating the gene for the inventive growth factor and then cloning it into a suitable vector for expression in large quantities.
  • a plasmid library is available from the National Eye Institute, and is potentially preferable because the gene would then be obtained in a plasmid and would shorten the time involved to clone the gene into a suitable factor.
  • Preferred embodiments of the invention are therapeutic compositions in which the inventive polypeptide includes a pharmaceutically acceptable carrier in which the inventive polypeptide is present in a therapeutically effective amount.
  • Typical such carriers are aqueous based and include commercially available balanced salt solution bases known and used for irrigation solutions or surgical solutions.
  • Therapeutically effective amounts of the polypeptide have concentrations of at least about 10 " M, preferably are between about l ⁇ "3 M to about l ⁇ " T ⁇ .
  • Additional additives, known to the art, such as antioxidants, hormones (e.g., insulin), energy sources (e.g., pyruvate) and the like may be included in preparing compositions of the invention.
  • EXAMPLE 1 The protocol from which the Fig. 1 data was taken was is follows. The standard DNA synthesis assay earlier described was used. The following growth factors were added at time zero to lens epithelial cells.
  • Fibroblast growth factor (bFGF) was added at a concentration of 50 pg/ml, both with and without l ⁇ "6 M dexamethasone.
  • Epidermal growth factor (EGF) was added at a concentration of 10 ng/ml, both with and without 10 * M dexamethasone.
  • Fetal calf serum was added at a concentration of 10% both with and without l ⁇ "6 M dexamethasone.
  • EXAMPLE 4 Again the experiment was carried out in the same manner as that seen for Example 1 and Fig. 1. The only difference is that different concentrations of "ect-GF” were used for the assay.
  • the purified "ect-GF” was obtained by the purification procedure already described. As can be seen the dose response curve for the purified "ect-GF” is shifted over 3 orders of magnitude to the left from that seen for impure material. In this case the concentrations are given in milligrams of protein per ml.
  • Fig. 5 data can be explained as follows; 1. This data shows that the effect of the addition of "ect-GF” lasts even if it is removed from the cells after as short a time as 1 hour, since the synthesis of DNA does not occur until about 16 hours later.
  • Insulin on the other hand shows a loss of its activity even if it is allowed to remain on the cells for 7 hours before it is removed (for the purpose of Fig. 6 it was only removed after 1 hour) .
  • the assay was allowed to run for 40 hours.
  • the protocol for this assay is the same as that outlined earlier, except in some cases the cells were washed with fresh media one hour after the addition of the growth factors in question.
  • the "ect-GF” was added at a concentration that was the same as that found in the supernatant solution over the Y79 cells.
  • the insulin concentration was 2 ⁇ g/ml.

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Abstract

Un polypeptide bioactif d'une masse moléculaire comprise entre environ 13 000 et environ 14 000 daltons stimule la croissance des cellules épithéliales en présence de dexaméthasone, et il est utile dans le traitement des cellules cornéennes. Ledit polypeptide hautement purifié, homogène, peut être isolé et purifié à partir d'une lignée cellulaire humaine de rétinoblastome appelée 'Y-79', disponible auprès de l'ATCC sous la référence HTB-18.
PCT/US1992/000558 1991-02-01 1992-01-23 Compositions et procede de cicatrisation des cellules epitheliales WO1992013888A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4886786A (en) * 1985-08-01 1989-12-12 Lindstrom Richard L Additive for irrigation solution or surgical solution
US4902782A (en) * 1986-12-10 1990-02-20 The Salk Institute For Biological Studies Isolation of fibroblast growth factor

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4886786A (en) * 1985-08-01 1989-12-12 Lindstrom Richard L Additive for irrigation solution or surgical solution
US4902782A (en) * 1986-12-10 1990-02-20 The Salk Institute For Biological Studies Isolation of fibroblast growth factor

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