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EP1053005A2 - Activation de la proliferation des keratinocytes - Google Patents

Activation de la proliferation des keratinocytes

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Publication number
EP1053005A2
EP1053005A2 EP99905876A EP99905876A EP1053005A2 EP 1053005 A2 EP1053005 A2 EP 1053005A2 EP 99905876 A EP99905876 A EP 99905876A EP 99905876 A EP99905876 A EP 99905876A EP 1053005 A2 EP1053005 A2 EP 1053005A2
Authority
EP
European Patent Office
Prior art keywords
seq
group
active agent
tyr
ala
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP99905876A
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German (de)
English (en)
Inventor
Kathleen E. Rodgers
Gere Dizerega
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University of Southern California USC
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University of Southern California USC
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Application filed by University of Southern California USC filed Critical University of Southern California USC
Publication of EP1053005A2 publication Critical patent/EP1053005A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0629—Keratinocytes; Whole skin
    • 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
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1808—Epidermal growth factor [EGF] urogastrone
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0629—Keratinocytes; Whole skin
    • C12N5/063—Kereatinocyte stem cells; Keratinocyte progenitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/32—Angiotensins [AT], angiotensinogen

Definitions

  • This present invention relates to methods to accelerate the proliferation of epithelial stem cells and keratinocytes, to promote more rapid and efficient cellular response to stratified epithelial injury, and to decrease scarring and loss of function at the injury site.
  • the epidermis is composed of a continually renewing stratified layer of epithelial cells, called keratinocytes.
  • the basal layer of the epidermis which is in direct contact with the basal lamina and dermal connective tissue, contains epithelial stem cells that divide and give rise to keratinocytes, which produce keratin as they differentiate and are "pushed" to the surface of the epidermis.
  • keratinocytes As the keratinocytes approach the surface of the epidermis, the cells die and the keratin contributes to the cornified skin surface, which is substantially impermeable to water and acts to prevent bacterial infection.
  • the dermis is a well vascularized tissue that provides support for the epidermis.
  • the dermis contains fibroblasts, which produce various elements of the connective tissue, including extracellular matrix proteins such as collagens, fibronectin and elastin that contribute to the strength and flexibility of the skin.
  • fibroblasts which produce various elements of the connective tissue, including extracellular matrix proteins such as collagens, fibronectin and elastin that contribute to the strength and flexibility of the skin.
  • Blood vessels present in the dermis transport nutrients to the epithelial cells in the epidermis and carry away waste products of cell metabolism.
  • the basement membrane serves, in part, to attach the epidermis to the dermis.
  • the skin also contains various accessory organs such as hair follicles and sweat glands.
  • An injury to the skin due, for example, to a laceration, a puncture or a burn results in a wound that can extend into or through the skin. If the wound is fairly small and localized, normal healing processes can close the wound and restore normal function to the tissue. (Id.) In some cases, however, an injury results in a deep wound or a wound that affects a large area. Such wounds can require clinical intervention for healing to occur. For example, a burn that covers a significant portion of the body requires, at a minimum, extensive cleaning of the injured tissue and application of a dressing to prevent infection of the tissue, and may be accompanied by scar formation, possibly producing disfigurement and loss of function at the injured region. (Id.) In addition, skin grafting using undamaged skin from the patient or a skin substitute often
  • Wound closure is achieved by the combined action of keratinocyte migration into the wound site and contraction of specialized fibroblasts in the tissue underneath the wound site, which pulls the edges of the wound closer together.
  • Inefficient remodeling of the wound bed leaves a scar, and in severe cases can lead to loss of tissue function at the wound site.
  • the ability to survive injury to the skin over a significant surface area is directly dependent upon the efficiency with which an epithelial lining conferring appropriate barrier function is restored over the wound site. (Id.)
  • the present invention provides methods that promote ESC and keratinocyte proliferation by contacting the cells with angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues thereof, angiotensin II (All) analogues, All fragments or analogues thereof or All AT type 2 receptor agonists, either alone or in combination with other growth factors and cytokines.
  • angiotensinogen angiotensin I
  • AI analogues AI fragments and analogues thereof
  • angiotensin II (All) analogues All fragments or analogues thereof or All AT type 2 receptor agonists
  • an improved cell culture medium for the proliferation of ESC and keratinocytes, wherein the improvement comprises addition to the cell culture medium of an effective amount of angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, All analogues, All fragments or analogues thereof or All AT 2 type 2 receptor agonists.
  • kits for the propagation of ESC and keratinocytes comprising an effective amount of angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, All analogues, All fragments or analogues thereof or All AT 2 type 2 receptor agonists, and instructions for using the amount effective of active agent as a cell culture medium supplement.
  • the kit further comprises a cell culture media.
  • the kit further comprises a sterile container for cell culturing.
  • Figure 1 is a graph demonstrating the effect of All fragments and analogues on
  • Figure 2 is a graph demonstrating the effect of All fragments, analogues, and fragment analogues on keratinocyte proliferation on Day 7 post-epithelial burn.
  • epithelium As defined herein, the term “epithelial stem cell (ESC)” refers to cells that are long-lived, relatively undifferentiated, have a great potential for cell division, and are ultimately responsible for the homeostasis of epithelium.
  • Cells of this type include, but are not limited to, those described in U.S. Patent No. 5,556,783; U.S. Patent No. 5,423,778; Rochat et al., Cell 76:1063 (1994); Jones et al. Cell 73:713 (1993); Jones et al., Cell 80:83 (1995); all references hereby incorporated in their entirety.
  • proliferation encompasses both cell self renewal and cellular proliferation with accompanying differentiation.
  • active agents refers to the group of compounds comprising angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues thereof, All analogues, All fragments or analogues thereof and All AT 2 type 2 receptor agonists.
  • angiotensin The biological formation of angiotensin is initiated by the action of renin on the plasma substrate angiotensinogen (Circulation Research 60:786-790 (1987); Clouston et al, Genomics 2:240-248 (1988); Kageyama et al, Biochemistry 23:3603-3609; Ohkubo et al., Proc. Natl. Acad. Sci. 80:2196-2200 (1983); all references hereby incorporated in their entirety).
  • the substance so formed is a decapeptide called angiotensin I (AI) which is converted to All by the converting enzyme angiotensinase which removes the C-terminal His-Leu residues from AI, Asp- Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu [SEQ ID NO:37]. All is a known pressor agent and is commercially available.
  • AI angiotensin I
  • AII(l-7) activity on a particular cell type cannot be predicted based solely on the effect of All on the same cell type.
  • angiotensinogen, AI, AI analogues, and/or AI fragments and analogues thereof, All analogues, All fragments or analogues thereof, and or All AT 2 type 2 receptor agonists accelerate the proliferation
  • a peptide agonist selective for the AT2 receptor (All has 100 times higher affinity for AT2 than ATI) is p-aminophenylalanine6-AII ["(p-NH 2 -Phe)6-AII)"], Asp- Arg-Val-Tyr-Ile-Xaa-Pro-Phe [SEQ ID NO.36] wherein Xaa is p-NH 2 -Phe (Speth and Kim, BBRC 169:997-1006 (1990).
  • This peptide gave binding characteristics comparable to AT2 antagonists in the experimental models tested (Catalioto, et al., Ewr. J. Pharmacol. 256:93-97 (1994); Bryson, et al, Eur. J. Pharmacol. 225:119-127
  • AII(l-7) (All residues 1-7) or other fragments of All to evaluate their activity.
  • AII(l-7) elicits some, but not the full range of effects elicited by AIL Pfeilschifter, et al, Eur. J. Pharmacol. 225:57-62 (1992); Jaiswal, et al., Hypertension 19(Supp. II):II-49-II-55 (1992); Edwards and Stack, J. Pharmacol. Exper. Ther. 266:506-510 (1993); Jaiswal, et al., J. Pharmacol. Exper. Ther.
  • a preferred class of AT2 agonists for use in accordance with the present invention comprises All, All analogues or active fragments thereof having p-NH-Phe in a position corresponding to a position 6 of AIL
  • nonpeptidic agents e.g., peptidomimetics
  • the active All analogues, fragments of All and analogues thereof of particular interest in accordance with the present invention comprise a sequence consisting of at least three contiguous amino acids of groups R -R in the sequence of general formula I R 1 -R 2 -R 3 -R 4 -R 5 -R 6 -R 7" R 8 in which R 1 and R 2 together form a group of formula
  • R A is suitably selected from Asp, Glu, Asn, Acpc (1-aminocyclopentane carboxylic acid), Ala, Me 2 Gly, Pro, Bet, Glu(NH 2 ), Gly, Asp(NH 2 ) and Sue,
  • R B is suitably selected from Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys,
  • R 3 is selected from the group consisting of Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc and Tyr, while Lys has also been found effective at this residue;
  • R 4 is selected from the group consisting of Tyr, Tyr(PO 3 ) , Thr, Ser, homoSer, azaTyr, and Ala;
  • R 5 is selected from the group consisting of He, Ala, Leu, norLeu, Val and Gly;
  • R 6 is His, Arg or 6-NH 2 -Phe;
  • R 7 is Pro or Ala;
  • R 8 is selected from the group consisting of Phe, Phe(Br), He and Tyr, excluding sequences including R 4 as a terminal Tyr group, and wherein the sequence is not AIL
  • compositions falling within the category of AT2 agonists useful in the practice of the invention include the AH analogues set forth above subject to the restriction that R 6 is p-NH 2 -Phe.
  • R A and R B are Asp- Arg, Asp-Lys, Glu- Arg and Glu-Lys.
  • Particularly preferred embodiments of this class include the following: AIII or AII(2-8), Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:2]; AII(3-8),
  • scope of the invention is a peptide having the sequence Asp-Arg-Pro-Tyr-Ile-His-Pro- Phe [SEQ ID NO:31].
  • AII(6-8), His-Pro-Phe [SEQ ID NO:14] and AII(4-8), Tyr-Ile- His-Pro-Phe [SEQ ID NO: 15] were also tested and found not to be effective.
  • Rl-Arg-R2-Tyr-R3-His-Pro-R4 wherein Rl is selected from the group consisting of H and Asp;
  • R2 is selected from the group consisting of Val and Pro
  • R3 is selected from the group consisting of Ala, He, Leu, norLeu, and Val;
  • R4 is selected from the group consisting of He, Phe, and H; and and wherein the active agent is not AIL
  • R 2 is selected from the group consisting of H, Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys;
  • R 3 is selected from the group consisting of Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc and Tyr;
  • R 4 is selected from the group consisting of Tyr, Tyr(PO 3 ) 2 , Thr, Ser, homoSer, azaTyr, and Ala;
  • R 5 is selected from the group consisting of He, Ala, Leu, norLeu, Val and Gly;
  • R 6 is His, Arg or 6-NH 2 -Phe
  • R is Pro or Ala; and R 8 is selected from the group consisting of Phe, Phe(Br), He and Tyr.
  • a particularly preferred subclass of the compounds of general formula II has the formula
  • R , R and R are as previously defined.
  • Particularly preferred is angiotensin III of the formula Arg-Val-Tyr-lle-His-Pro-Phe [SEQ ID NO:2].
  • Other preferred compounds include peptides having the structures Arg-Val-Tyr-Gly-His-Pro- Phe [SEQ ID NO: 17] and Arg-Val-Tyr-Ala-His-Pro-Phe [SEQ ID NO: 18].
  • the fragment AII(4-8) was ineffective in repeated tests; this is believed to be due to the exposed tyrosine on the N-terminus.
  • AH and its analogues adopt either a gamma or a beta turn (Regoli, et al., Pharmacological Reviews 26:69 (1974).
  • neutral side chains in position R 3 , R 5 and R 7 may be involved in maintaining the appropriate distance between active groups in positions R 4 , R 6 and R 8 primarily responsible for binding to receptors and/or intrinsic activity.
  • Hydrophobic side chains in positions R 3 , R 5 and R 8 may also play an important role in the whole conformation of the peptide and/or contribute to the formation of a hypothetical hydrophobic pocket.
  • R 2 Appropriate side chains on the amino acid in position R 2 may contribute to affinity of the compounds for target receptors and/or play an important role in the conformation of the peptide. For this reason, Arg and Lys are particularly preferred as R 2 .
  • R 3 may be involved in the formation of linear or nonlinear hydrogen bonds with R 5 (in the gamma turn model) or R (in the beta turn model). R 3 would also participate in the first turn in a beta antiparallel structure (which has also been proposed as a possible structure). In contrast to other positions in general formula I, it appears that beta and gamma branching are equally effective in this position. Moreover, a single hydrogen bond may be sufficient to maintain a relatively stable conformation. Accordingly, R 3 may suitably be selected from Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc and Tyr.
  • R 4 is preferably selected from Tyr, Thr, Tyr (PO 3 ) 2 , homoSer, Ser and azaTyr.
  • Tyr is particularly preferred as it may form a hydrogen bond with the receptor site capable of accepting a hydrogen from the phenolic hydroxyl (Regoli, et al. (1974), supra). It has also been found that R 4 can be Ala.
  • an amino acid with a ⁇ aliphatic or alicyclic chain is particularly preferred
  • Gly is suitable in position R ⁇ ⁇ it is preferred that the amino acid in this position be selected from He, Ala, Leu, norLeu, Gly and Val.
  • R 6 is His, Arg or 6-NH 2 -Phe.
  • the unique properties of the imidazole ring of histidine e.g., ionization at physiological pH, ability to act as proton donor or acceptor, aromatic character) are believed to contribute to its particular utility as R 6 .
  • conformational models suggest that His may participate in hydrogen bond formation (in the beta model) or in the second turn of the antiparallel structure by influencing the orientation of R 7 .
  • R 7 should be Pro in order to provide the most desirable orientation of R 8 .
  • R 8 both a hydrophobic ring and an anionic carboxyl terminal appear to be particularly useful in binding of the analogues of interest to receptors; therefore, Tyr and especially Phe are preferred for purposes of the present invention.
  • Analogues of particular interest include the following: TABLE 2
  • polypeptides of the instant invention may be synthesized by any conventional method, including, but not limited to, those set forth in J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford, 111. (1984) and J. Meienhofer, Hormonal Proteins and Peptides, Vol. 2, Academic Press, New York, (1973) for solid phase synthesis and E. Schroder and K. Lubke, The Peptides, Vol. 1, Academic Press, New York, (1965) for solution synthesis.
  • the disclosures of the foregoing treatises are incorporated by reference herein.
  • these methods involve the sequential addition of protected amino acids to a growing peptide chain (U.S. Patent No. 5,693,616, herein incorporated by reference in its entirety). Normally, either the amino or carboxyl group of the first amino acid and any reactive side chain group are protected. This protected amino acid is then either attached to an inert solid support, or utilized in solution, and the next amino acid in the sequence, also suitably protected, is added under conditions amenable to formation of the amide linkage. After all the desired amino acids have been linked in the proper sequence, protecting groups and any solid support are removed to afford the crude polypeptide. The polypeptide is desalted and purified, preferably chromatographically, to yield the final product.
  • peptides are synthesized according to standard solid-phase methodologies, such as may be performed on an Applied Biosystems Model 430A peptide synthesizer (Applied Biosystems, Foster City, Calif), according to manufacturer's instructions. Other methods of synthesizing peptides or peptidomimetics, either by solid phase methodologies or in liquid phase, are well
  • a method of increasing in vitro and ex vivo ESC and keratinocyte proliferation by exposure to angiotensinogen, AI, AI analogues, and/or AI fragments and analogues thereof, AH analogues, All fragments and analogues thereof, and/or All AT type 2 receptor agonists ("active agents") is disclosed, either alone or in combination with other growth factors and cytokine.
  • growth factors and cytokines include, but are not limited to lymphokines, interleukins - 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, keratinocyte growth factor, tumor necrosis factor, epidermal growth factor ("EGF"), HB-EGF, fibroblast growth factors 1, 2 and 4, and transforming growth factor alpha.
  • EGF epidermal growth factor
  • HB-EGF HB-EGF
  • fibroblast growth factors 1, 2 and 4 transforming growth factor alpha.
  • Experimental conditions for the isolation, purification, ex vivo growth and in vivo mobilization of ESC and keratinocytes have been reported (U.S. Patent No. 5,423,778; U.S. Patent No. 5,561,107; U.S. Patent No. 5,686,307; Rheinwald et al., in: The Formation of Keratinizing Colonies from Cells. Cell G. 331-343, 1975; all references hereby incorporated in their entirety).
  • ESC and keratinocytes are obtained by trypsinization of skin samples from patients undergoing plastic surgery, as previously described (Svendsen et al., Pharmacology and Toxicology 80:49-56 (1997)).
  • ESC are isolated from the upper hair follicle, as described in U.S. Patent No. 5,556,783, herein incorporated by reference in its entirety. Isolated ESC and keratinocytes are then cultured under appropriate growth conditions, in the presence of the active agents of the invention.
  • Cell proliferation is assessed at various time points during culture using methods well known in the art, including, but not limited to, measuring the rate of DNA synthesis according to the method of Nakamura and coworkers (Nakamura et al., J. Biochem. (Tokyo) 94:1029 (1982); Nakamura et al., Biochem. Biophys. Res. Comm. 122:1450 (1984)) Trypan blue dye exclusion/hemocytometer counting (Omori et al., Hepatology 26:720 (1997)), or flow cytometry (Drakes, 1997).
  • ESC and keratinocytes are suspended in standard culture medium, including but not limited to, keratinocyte-basal growth medium (Clonetics Corp., San Diego, CA) and incubated in the presence of, preferably, between about 0.1 ng/ml and about 10 mg/ml of the active agents.
  • the cells are expanded for a period of between 8 and 21 days, and cellular proliferation is monitored by measuring the rate of DNA synthesis (Nakamura et al., 1982; Nakamura et al., 1984).
  • ESC and keratinocyte proliferation is measured by 3 [H]thymidine incorporation as described in Yang et al., J. of Investig. Dermatol. 107:367-372 (1996).
  • ESC and keratinocyte cell proliferation is assessed periodically by immunohistochemistry using an antibody directed against a protein known to be present in higher concentrations in proliferating cells than in non-proliferating cells, such as proliferating cell nuclear antigen (PCNA or cyclin; Zymed Laboratories) (Rodgers et al., 1997).
  • PCNA proliferating cell nuclear antigen
  • cyclin Zymed Laboratories
  • Differentiation of keratinocytes to squames is detected by measuring the expression of typical differentiated keratinocyte markers including, but not limited to, transglutaminase type I (TG-I) (Svendsen et al., 1997) and keratin (Martin et al., 1997)
  • Methods of detecting these markers include, but are not limited to, Northern blot analyses or reverse transcriptase-polymerase chain reaction (RT-PCR) with marker- specific DNA primers (Song et al., Biochem. Biophys. Res. Commun. 235:10-14 (1997); Takahashi et al., J. Biol. Chem. 270:18581-92 (1995)), and antibody detection
  • ESC and keratinocytes that have been cultured in the presence of active agents, are used for ex vivo ESC- and keratinocyte- directed gene therapy, as described in Morgan et al., Science 237:1476-1479 (1987), and Tenmer et al., FASEB J. 4:3245-3250 (1990).
  • the cells Prior to ex vivo ESC- or keratinocyte-directed gene therapy, the cells are rinsed to remove all traces of culture fluid, resuspended in an appropriate medium and then pelleted and rinsed several times. After the final rinse, the cells are resuspended at between 0.7 x 10 6 and 50 x 10 6 cells per ml in an appropriate medium and re-introduced into a subject, as described below.
  • the active agents are used to increase in vivo ESC and keratinocyte proliferation, which can be measured by taking biopsy samples and using the methods described above.
  • biopsy samples are taken between about 1 day to about 2 weeks after treatment.
  • the active agents may be administered by any suitable route, including orally, parentally, by inhalation spray, rectally, transdermally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles.
  • parenteral as used herein includes, subcutaneous, intravenous, intramuscular, intrasternal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques
  • the active agents may be made up in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions), and may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents,
  • the active agents can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more other compounds.
  • the active agents and other compounds can be formulated as separate compositions that are given at the same time or different times, or the active agents and other compounds can be given as a single composition.
  • the active agents are ordinarily combined with one or more adjuvants appropriate for the indicated route of administration.
  • the compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration.
  • the compounds of this invention may be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers.
  • Other adjuvants and modes of administration are well known in the pharmaceutical art.
  • the carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well
  • the dosage regimen for increasing in vivo proliferation of ESC and keratinocytes with the active agents is based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the individual, the severity of the condition, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely by a physician using standard methods. Dosage levels of the order of between 0.1 ng/kg and 10 mg/kg of the active agents per body weight are useful for all methods of use disclosed herein.
  • the active agents are administered transdermally or topically.
  • a suitable transdermal or topical dose of active ingredient of the active agents is preferably between about 0.1 ng/kg and about 10 mg/kg administered twice daily.
  • the active ingredient may comprise from 0.001% to 10%) w/w, e.g., from 1%> to 2% by weight of the formulation, although it may comprise as much as 10% w/w, but preferably not more than 5% w/w, and more preferably from 0.1% to 1% of the formulation.
  • a formulation suitable for topical administration includes liquid or semi-liquid preparations suitable for penetration through the skin (e.g., liniments, lotions, ointments, creams, or pastes) and drops suitable for administration to the eye, ear, or nose.
  • liquid or semi-liquid preparations suitable for penetration through the skin e.g., liniments, lotions, ointments, creams, or pastes
  • drops suitable for administration to the eye, ear, or nose e.g., liniments, lotions, ointments, creams, or pastes
  • Transdermal means including, but not limited to, transdermal patches may be utilized to deliver the active agents to the treatment site.
  • Transdermal formulations may be prepared by incorporating the active agent in a thixotropic or gelatinous carrier including, but not limited to, a cellulose medium, e.g., methyl cellulose or hydroxyethyl cellulose, with the resulting formulation then being packed in a transdermal device adapted to be secured in dermal contact with the skin of a wearer.
  • an improved cell culture medium for the proliferation of ESC and keratinocytes, wherein the improvement comprises addition to the cell culture medium of an effective amount of the active agents as described above.
  • Any cell culture media that can support the growth of ESC and keratinocytes can be used with the present invention.
  • Such cell culture media include, but are not limited to Keratinocyte Basal Growth Media, Basal Media Eagle, Dulbecco's Modified Eagle Medium, Iscove's Modified Dulbecco's Medium, McCoy's Medium, Minimum Essential Medium, F-10 Nutrient Mixtures, Opti-MEM® Reduced- Serum Medium, and RPMI Medium, or combinations thereof.
  • the improved cell culture medium can be supplied in either a concentrated (ie:
  • the cell culture may be either chemically defined, or may contain a serum supplement.
  • Culture media and serum supplements are commercially available from many sources, such as GIBCO BRL (Gaithersburg, MD) and Sigma (St. Louis, MO)
  • kits for the propagation of ESC and keratinocytes wherein the kits comprise an effective amount of the active agents of the invention, and instructions for using the amount effective of active agent as a cell culture medium supplement.
  • the kit further comprises cell culture growth media.
  • Any cell culture media that can support the growth of ESC and keratinocytes can be used with the present invention. Examples of such cell culture media are described above.
  • the improved cell culture medium can be supplied in either a concentrated (ie: 10X) or non-concentrated form, and may be supplied as a liquid, a powder, or a lyophilizate.
  • the cell culture may be either chemically defined, or may contain a serum
  • the kit further comprises a sterile container.
  • the sterile container can comprise either a sealed container, such as a cell culture flask, a roller bottle, or a centrifuge tube, or a non-sealed container, such as a cell culture plate or microtiter plate (Nunc; Naperville, IL).
  • a sealed container such as a cell culture flask, a roller bottle, or a centrifuge tube
  • a non-sealed container such as a cell culture plate or microtiter plate (Nunc; Naperville, IL).
  • the kit further comprises an antibiotic supplement for inclusion in the reconstituted cell growth medium.
  • antibiotic supplements include, but are not limited to actimonycin D, Fungizone®, kanamycin, neomycin, nystatin, penicillin, streptomycin, or combinations thereof
  • the present invention by providing methods for enhanced proliferation of ESC and keratinocytes, will be clinically useful in accelerating ESC and keratinocyte growth
  • the method of the present invention also increases the potential utility of ESC and keratinocytes as vehicles for gene therapy, by more efficiently providing a large number of such cells for transfection, and also by providing a more efficient means to rapidly expand transfected ESC and keratinocytes.
  • Example I Effect of All and AH analogues and analogue fragments on keratinocyte proliferation in vitro as measured by cell number
  • KGM Keratinocyte Basal Medium containing human recombinant epidermal growth factor, human recombinant insulin, hydrocortisone, epinephrine, prostaglandin
  • the cells were trypsinized from the culture flask and re-seeded into 24 well plates at 100 cells per well in KGM. After allowing adherence of the cells for 24 hours, the cells were placed in
  • Ala4AIII (Arg-Val-Tyr-Ala-His-Pro-Phe) (SEQ ID NO: 18) was added to the wells. As shown in Table 1, exposure of keratinocytes to the various peptides increased the number of keratinocytes per well over KBM alone.
  • Example 2 Effect of All and All analogues and analogue fragments on keratinocyte proliferation in vitro as measured by thymidine incorporation
  • keratinocytes Normal human keratinocytes were purchased from Clonetics (San Diego, CA) and cultured in Keratinocyte Growth Medium (KGM) as described in Example 1 , until confluence at 37°C in 5% CO . The cells were harvested at confluence with 0.02%o trypsin/0.05% ETA. Thereafter, the cells were seeded into 24 well plates at a density of
  • Each burn was treated with 10% low viscosity carboxymethylcellulose (CMC sodium salt, Sigma Chemical CO., St. Louis, MO) in 0.05 M phosphate buffer, pH 7.2, and sterilized by autoclaving, with and without either 1 mg/ml AII(l-7) (SEQ ID NO:4), Ala4-AIII (SEQ ID NO: 18), Pro3-AII (SEQ ID NO:31), or Ile8-AII (Asp-Arg- Val-Tyr-Ile-His-Pro-Ile) (SEQ ID NO:38).
  • Each burn was subsequently dressed with a Hilltop Chamber (Hilltop Research, Madison, WI) and covered with Tegaderm (Western Medical Supply, Arcadia, CA).
  • the bandages were checked and changed daily for the first 5 days and every other day until necropsy on day 7 after injury.
  • the guinea pigs were given 20 ⁇ g/kg bupronex (buprenophine hydrochloride) intramuscularly for pain on the day of injury and the first 3 days after injury.
  • the guinea pigs were euthanized, the burned areas were excised en bloc and the tissues were placed in 10% buffered formaldehyde solution overnight.
  • the tissues were embedded in paraffin and 5 ⁇ m sections were prepared.
  • the sections were processed for immunohistochemical analysis with a primary antibody to cyclin (MIB-1) followed by recognition of the primary antibody with a DAKO kit (see following text).
  • the paraffin embedded sections were baked in an oven overnight at 60°C. De- paraffmization was performed by four 5 minute incubations in "fresh" xylene followed by two 5 minute incubations with 100% ethanol, two 5 minute incubations with 95 % ethanol, and one 5 minute incubation with water.
  • PBS saline solution
  • Immunohistochemical staining was performed with the avidin-biotin-peroxidase conjugate method with some modifications. The slides were placed in a PBS bath for 5 minutes and then laid in humidified incubation chambers. Blocking for nonspecific antibody binding was performed by incubating the sections in 5% horse serum in PBS.
  • the solution was decanted and replaced with the solution of primary antibody MIB-1, used at a dilution of 1 :100 in PBS, and incubated for 60 minutes at room temperature.
  • the slides were washed with PBS for 5 minutes and then incubated with a biotinylated horse-anti-mouse antibody. After a 5 minute wash in PBS, the avidin-biotin complex was applied to the slides at a dilution of 1:100 in PBS at room temperature and was incubated for 1 hour.
  • each section of the biopsy specimen was separated into either (a) areas on the burn edge or (b) the actual burn areas.
  • the area of the burn was excised and serially cut into three to five sections of 4-5 mm in thickness.
  • the entire area of the bum and edge of the bum was embedded and examined histologically.
  • the cells that stained with the MIB-1 antibody were a distinct brown color.
  • To count the MIB-1 stained cells each section on the slide was separated into individual 100X fields. Each field was then determined to be either a section on the edge of the bum or a part of the bum area itself. An edge was indicated by a positive stain showing brown epithelial cells along the edge of the section.
  • the brown cells in the basal layer of the epidermis (proliferating keratinocytes) located at the 100X field adjacent to the bum site were
  • the depth of the bums was determined to be deep partial-thickness to full- thickness by (1) analysis of vessel patency with intraaortic injection of India Ink, and (2) the appearance of the cells in the hair follicles by microscopic analysis of hematoxylin-eosin-stained sections. This analysis revealed that most of the preexisting blood vessels and cells were destroyed in the bum site, although the injuries did not extend through the panniculus camosus.

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Abstract

L'invention concerne des procédés, des milieux de culture cellulaire améliorés et des ensembles de matériel permettant d'activer la prolifération et la différentiation des cellules souche épithéliales et des kératinocytes par une croissance effectuée en présence d'antgiotensinogènes, d'AI, d'analogues d'AI et/ou de fragments d'AI et d'analogues de ces derniers, d'analogues d'AII, de fragments ou d'analogues d'AII, d'agonistes de récepteurs d'AII AT2 de type 2, utilisés seuls ou combinés à d'autres facteurs de croissance et cytokines.
EP99905876A 1998-02-09 1999-02-08 Activation de la proliferation des keratinocytes Withdrawn EP1053005A2 (fr)

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US7410598P 1998-02-09 1998-02-09
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AU755943B2 (en) 1998-08-13 2003-01-02 University Of Southern California Methods to increase blood flow to ischemic tissue
US6730775B1 (en) 1999-03-23 2004-05-04 University Of Southern California Methods for limiting scar and adhesion formation
US6747008B1 (en) 2000-06-19 2004-06-08 University Of Southern California Methods for treating and preventing alopecia
DK1395566T3 (da) 2001-05-31 2008-01-07 Vicore Pharma Ab Tricycliske forbindelser, der er nyttige som angiotensin Il-agonister
JP4623967B2 (ja) 2002-01-14 2011-02-02 ザ・ボード・オブ・トラスティーズ・オブ・ザ・ユニバーシティ・オブ・イリノイ 幹細胞の移動および増殖を促進するための修飾されたピリミジン化合物の使用
US7998737B2 (en) 2006-09-12 2011-08-16 Deutsches Krebsforschungszentrum Cell culture of keratinocytes under non-differentiating conditions
AT508569A1 (de) * 2009-07-23 2011-02-15 Affiris Ag Pharmaceutical compound
EP2455388A1 (fr) 2010-11-23 2012-05-23 LanthioPep B.V. Nouveaux agonistes du récepteur 2 (AT2) de type angiotensine et leurs utilisations
EA026440B1 (ru) 2011-02-02 2017-04-28 Университи Оф Сатерн Калифорния Способ лечения диабетической язвы стопы
WO2016107879A2 (fr) * 2014-12-30 2016-07-07 Vicore Pharma Ab Nouvelle utilisation d'agonistes du récepteur de l'angiotensine ii
US20220257698A1 (en) 2019-08-02 2022-08-18 Lanthiopep B.V. Angiotensin type 2 (at2) receptor agonists for use in the treatment of cancer

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US5955430A (en) * 1993-09-24 1999-09-21 University Of Southern California Use of angiotensin II fragments and analogs thereof in tissue repair
US5693332C1 (en) * 1995-08-11 2001-01-09 Univ California Human keratinocytes supported on a hydrophilic membrane and methods of using same to effect wound closure

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WO1999040107A3 (fr) 1999-09-30

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