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`’ WARNING: The information disclosed‘herein may be restricted.
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`lNTERFERENCE-SEARQHED
`--_
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`PATENT APPLICATION SERIAL NO.
`
`US. DEPARTMENT OF COMMERCE
`PATENT AND TRADEMARK OFFICE
`FEE RECORD SHEET
`
`{FIGS/2002 098F001
`
`00000013 10134310
`
`01 FC:301
`
`370.00 0P
`
`PTO-1556
`
`*
`
`'
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`(5/87)
`'U,S, Government Pflntlng Office: 2001 — 481697759173
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`5/95 -
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`69"”63”
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`an: aware;
`
`FISHYt. RICHARDSON P.C.
`
`June 28, 2002
`
`Attorney Docket No; 07917-045003
`
`Box Patent Application
`Commissioner for Patents
`
`Washington, DC 20231
`
`ta , ,
`
`
`
`7.25 Franklin Street
`Boston, Massachusetts
`02110-2804
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`rederick P. Fish
`1855-1930
`WK. Richardson
`1859-1951
`
`Presented for filing is a new continuation patent application of:
`
`Applicant: JAMES G. DOBSON AND MICHAEL F. ETHIER
`
`Title:
`
`TREATMENT OF SKIN WITH ADENOSINE OR ADENOSINE
`
`ANALOG
`
`The prior application is assigned of record to University of Massachusetts,
`a Massachusetts corporation, by virtue of an assignment submitted to the US. Patent
`and Trademark Office and recorded on January 7, 1999 at 9690/0305.
`
`Enclosed are the following papers, including those required to receive a filing date
`under 37 CFR §1.53(b):
`
`6
`BOSTON
`DALLAS
`DELAWARE
`NEW YORK
`SAN DIEGO
`SILICON VALLEY
`TWIN CITIES
`
`WASHINGTON, DC
`
`Specification
`Claims
`Abstract
`Declaration
`
`Drawings
`
`Enclosures:
`
`Pa es
`
`20
`7
`1
`2
`
`2
`
`— Form PTO-1449, 1 page, listing documents cited in the parent
`application(s). Please confirm that these have been considered in this
`
`CERTIFICATE OF MAILING BY EXPRESS MAIL
`
`Express Mail Label N0.
`
`ELS50773342US
`
`I hereby certify under 37 CFR §l.10 that this correspondence is being
`deposited with the United States Postal Service as Express Mail Post
`Office to Addressee with sufficient postage on the date indicated below
`and is addressed to the Commissioner
`for Patents, Washington,
`DC. 20231.
`
`
`
`Date ofw
`
`Signature
`
`Typed or Printed Name of Person Signing Certificate
`
`
`
`6/95
`
`
`
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`
`
`3.. £31 1 £13 @333: 1:31:35 21 39.31 £33: as ESE-5.25 £53.
`
`FISH a: R
`
`:‘IARDSON P.C.
`
`Commissioner for Patents
`June 28, 2002
`Page 2
`
`application by returning a copy of the Form PTO-1449 with the
`examiner’s initials.
`
`— Preliminary amendment, 3 pages.
`— Information Disclosure Statement, 2 pages.
`— Postcard.
`
`This application is a continuation (and claims the benefit of priority under 35 USC
`120) of U.S. application serial no. 09/672,348, filed on September 28, 2000, pending,
`which is a continuation of US. application serial no. 09/179,006, filed on
`October 26, 1998, now abandoned. The disclosure of the prior applications are
`considered part of (and are incorporated by reference in) the disclosure of this
`application.
`
`Basic filing fee
`Total claims in excess of 20 times $9
`Independent claims in excess of 3 times $42
`Fee for multiple dependent claims
`Total filing fee:
`
`’
`
`$370
`$0
`$0
`$0
`. $370
`
`A check for the filing fee is enclosed. Please apply any other required fees or any
`credits to deposit account 06-1050, referencing attorney docket number 07917-
`045003.
`'
`
`If this application is found to be incomplete, or if a telephone conference would
`otherwise be helpful, please call the undersigned at (617) 542-5070.
`
`Kindly acknowledge receipt of this application by returning the enclosed postcard.
`
`Please send all correspondence to:
`
`J. PETER FASSE
`Fish & Richardson RC.
`225 Franklin Street
`
`, Boston, Massachusetts 02110-2804
`
`Respectfully submitted,
`
`éf’eter Fasse, Esq.
`
`eg. No. 32,983
`Enclosures
`
`JPF/lng/kxh
`20457832.doc
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`7/95 .
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`#7
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`113331-12 {Li-35% i 343 m 1323 23:32: £531 5% 31231 $32
`(
`
`TREATMENT OF SKIN WITH ADENOSINE OR ADENOSINE ANALOG
`Abstract of the Disclosure
`Methods for enhancing the condition of non-diseased
`
`skin by application of compositions containing adenosine or
`an adenosine analog are disclosed. Also disclosed are
`
`methods for increasing DNA synthesis or protein synthesis in
`dermal cells, and methods for increasing dermal cell size,
`by application of compositions containing adenosine.
`
`227728.311
`
`I
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`8/95
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`#
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`!
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`31:1? flflegfl 3M3 Ii 1144 95;?” “2:335
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`Attorney‘s Docket N\
`
`‘917-045003 / UMMC 97-32
`
`APPLICATION
`
`FOR
`
`UNITED STATES LETTERS PATENT
`
`TITLE:
`
`TREATMENT OF SKIN WITH ADENOSINE OR
`ADENOSINE ANALOG
`
`APPLICANT:
`
`JAMES G. DOBSON AND MICHAEL F. ETHIER
`
`CERTIFICATE OF MAILING BY WRESS MAIL
`
`Express Mail Label No.
`
`EL95077§342US
`
`I hereby certify under 37 CFR §l.10 that this correspondence is being
`deposited with the United States Postal Service as Express Mail Post
`Office to Addressee with sufficient postage on the date indicated below
`and is addressed to the Commissioner
`for Patents, Washington,
`DC 20231.
`at;
`
`Signature
`
`6 V0
`
`Typed or Printed Name of
`son Signing Certificate
`
`’L
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`
`
`9/95
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`
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`
`PATENT
`ATTORNEY DOCKET NO: 07917/045002
`
`TREATMENT OF SKIN WITH ADENOSINE OR ADENOSINE ANALOG
`
`C1\
`S onsored Research
`tatement as to Federall
`
`
`Work on this invention was supported by funds from
`
`the United States government
`
`(Public Health Service Grants
`
`10
`
`HL-22828 and AG-ll491).
`rights in this invention.
`
`The government therefore has certain
`
`.Field of the Invention
`
`This invention relates to dermatology and cell
`biology.
`
`15
`
`20
`
`25
`
`30
`
`Background of the Invention
`Skin includes a surface layer, known as the
`
`epidermis, and a deeper connective tissue layer, known as the
`dermis.
`The epidermis undergoes continuous turnover as the‘
`
`outermost cells are exfoliated and replaced by cells that
`
`arise from inner dermal layers.
`
`The dermis is composed of a
`
`including fibroblasts.
`variety of cell types,
`skin thickness begins to decline in humans after the age
`of 20 as the dermis becomes thinner and the number of skin
`fibroblasts declines. As skin ages, or is exposed to UV
`
`light and other environmental insults, changes in the
`
`underlying dermis can lead to the functional and
`
`morphological changes associated with damaged skin.
`Decreases in the abundance and function of products of the
`
`fibroblasts, which include collagen and proteoglycans, are
`
`believed to play major roles in wrinkled and damaged skin.
`
`
`
`$
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`Summary of the Invention
`
`We have discovered that adenosine stimulates DNA
`
`increases protein synthesis, and increases cell
`synthesis,
`size in cultures of human skin fibroblasts. Based on this
`
`the invention provides methods and compositions
`discovery,
`for enhancing the condition of skin.
`
`the invention provides a method for
`In general,
`enhancing the condition of non-diseased skin of a mammal,
`e.g., a human.
`The method includes topically applying a
`therapeutically effective amount of a composition including
`adenosine or an adenosine analog to non-diseased skin of the
`mammal.
`
`The invention also provides a method for promoting
`
`healing of broken, non-diseased skin in a mammal by
`
`topically administering a composition including a
`
`therapeutically effective amount of adenosine or an
`adenosine analog to the mammal.
`Also included in the invention is a method for
`
`10
`
`15
`
`increasing DNA synthesis in a dermal cell of non-diseased
`
`2,0
`
`skin of a mammal.
`
`The method includes topically
`
`administering a therapeutically effective amount of
`
`adenosine or an adenosine analog to a region of nonediseased
`
`skin of the mammal containing dermal cell.
`
`The adenosine is
`
`added so that it does not cause proliferation of the dermal
`cell.
`
`25
`
`The invention also features a method of increasing
`
`protein synthesis in a dermal cell of non—diseased skin of a
`mammal.
`The method includes topically administering a
`
`30
`
`composition including a therapeutically effective amount of
`adenosine or an adenosine analog to a region of skin of the
`
`mammal containing the dermal cell.
`
`The adenosine or
`
`adenosine analog does not cause proliferation of the dermal
`cell.
`
`1W95»
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`Also provided in the invention is a method of
`increasing cell size in a dermal cell in non—diseased skin
`
`of a mammal, e.g., a human.
`
`The method includes topically
`
`administering a composition including a therapeutically
`effective amount of adenosine to a region of skin of the
`
`mammal containing the dermal cell, wherein addition of the
`adenosine does not cause proliferation of the dermal cell,
`wherein addition bf the adenosine does not cause
`
`proliferation of the dermal cell.
`
`The invention also includes a method for enhancing
`’
`skin condition in a mammal, e.g., a human.
`The method
`
`includes providing fibroblasts from the mammal ex vivo,
`culturing the fibroblasts in the presence of adenosine, and
`
`reintroducing the
`
`fibroblasts into the mammal.
`
`The therapeutically effective amount of adenosine
`used in the above-described methods is preferably 10” M to
`
`10‘7 M, more preferably 10‘4 M to 10‘6 M, and most preferably
`about 10‘A M.
`
`‘
`
`The composition used in the above-described methods
`
`can include a second agent
`
`in addition to adenosine.
`
`‘The
`
`second agent can be, e.g. an agent that promotes binding of
`adenosine or an adenosine analog to an adenosine receptor,
`
`an angiogenic factor such as vascular endothelial cell
`
`10
`
`15
`
`2O
`
`25
`
`growth factor (VEGF), basic fibroblast growth factor (BFGF),
`an agent that itself enhances skin condition, such as
`
`tretoinin or another known conditioning agent such as an
`
`emollient, a humectant, or an occlusive agent.
`
`In preferred embodiments of the invention,
`
`the
`
`adenosine or an adenosine analog does not promote skin cell
`
`30
`
`proliferation.
`
`The invention also provides a composition including
`
`about 10'3 M to about 10‘7 M adenosine and a therapeutically
`effective amount of an angiogenesis factor.
`In some
`
`-
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`embodiments,
`M.
`
`the composition of the adenosine is about 10“
`
`As used herein, "enhancement of skin condition".
`
`means a noticeable decrease in the amount of wrinkling,
`
`roughness, dryness, laxity, sallowness, or pigmentary
`mottling in skin.
`
`As used herein, a "therapeutically effective amount"
`
`of adenosine or an adenosine analog means an amount that
`
`enhances skin condition when applied to skin.‘
`As used herein, "non-diseased skin" means skin free
`
`10
`
`15
`
`20
`
`of any proliferatiVe disorder observable by visual
`inspection.
`
`The present
`
`invention advantageously allows for
`
`enhancement of skin condition. This results in skin that
`
`shows a less wrinkled,
`
`rough, or dry complexion.
`
`For
`
`example,
`
`the invention provides for enhancing the condition
`
`of skin damaged due to exposure to the sun or skin whose
`
`condition has deteriorated due to normal aging.
`Unless otherwise defined, all technical and
`
`scientific terms used herein have the same meaning as\
`commonly understood by one of ordinary skill in the art to
`
`which this invention belongs. Although methods and
`
`materials similar or equivalent to those described herein
`
`can be used in the practice or testing of the present
`invention, suitable methods and materials are described
`
`25
`
`below. All publications, patent applications, patents, and
`
`other references mentioned herein are incorporated by
`
`reference in their entirety.
`
`In case of conflict,
`
`the
`
`30
`
`including definitions, will control.
`present specification,
`In addition,
`the materials, methods, and examples are
`
`illustrative only and not
`
`intended to be limiting.
`
`13/95
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`
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`Other features and advantages of this invention will
`
`be apparent from the following description of the preferred
`embodiments thereof, and from the claims.
`
`Brief Description of the Drawings
`
`Figs. 1A and 1B are histograms showing the effect of
`
`adenosine on [3H1thymidine incorporation in cultures of
`
`normal human skin (Fig. 1A) and lung fibroblasts (Fig. 18).
`After incubation in serum—free medium for 24 hours, cells
`
`were exposed to 10* M adenosine for 18 hours. Medium was
`
`10
`
`replaced with serum—free medium without adenosine, and
`
`PH1thymidine was added. Results are expressed as percent
`
`PHIthymidine incorporation compared to control cultures
`without adenosine and are means 1 SEM for 4-5 experiments.
`
`15
`
`"*" denotes value was significantly different from control
`value without adenosine.
`
`Figs. 2A and 28 are histograms showing concentration
`
`responses of adenosine—stimulated protein synthesis in human
`
`skin fibroblasts from a young (Fig. 2A) and aged (Fig. 2B)
`
`donor. Cells were grown to 75% confluence. Medium was then
`
`replaced with serum-free medium with or without adenosine.
`
`[3H1phenylalanine incorporation was
`After 48 hours,
`determined as described. Results are expressed as
`
`%[’H]phenylalanine incorporation compared to control
`cultures without adenosine and are means iSEM for 6—25
`
`experiments. "*"_denotes value was significantly different
`from control value without adenosine.
`
`Detailed Description
`
`The invention is suitable for treating skin of a
`
`mammal, e.g.,
`
`a human, for which promotion of fibroblast-
`
`associated dermal functions is desired.
`
`For example,
`
`-5-
`
`20
`
`25
`
`3O
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`promotion of fibroblast-associated functions is desirable in
`
`enhancing the condition of aged skin, which is associated
`with a decrease in dermal cell function and is characterized
`
`The method can
`by increased dryness or roughness, or both.
`also be used on subjects having otherwise damaged skin,
`
`e.g., wrinkled skin and skin with a non~proliferative
`
`disorder:' The method can may further be used
`
`10
`
`prophylactically on a subject to minimize deterioration of
`skin condition associated with aging or environmental
`factors, such as photodamage.
`Adenosine and suitable adenosine analogs are
`
`suitable for use in enhancing skin condition. Adenosine
`
`analogs iuch as adenosine agOnists, adenosine receptor
`
`agonists, and compounds that increase intracellular or
`extracellular adenosine levels are suitable for use in the
`
`15
`
`invention.
`
`20
`
`25
`
`3O
`
`Agonists of adenosine include 2’-deoxyadenosine;
`
`2',3’-isopropoylidene adenosine;
`
`toyocamycin;
`
`l—
`
`methyladenosine; N—6-methyladenosine; adenosine N-oxide; 6-
`
`methylmercaptopurine riboside; 6—chloropurine riboside, S’-
`
`adenosine monophosphate, 5'—adenosine diphosphate, or 5'—
`
`adenosine triphosphate. Adenosine receptor agonists include
`
`phenylisopropyl-adenosine ("PIA"),
`
`l-Methylisoguanosine,
`
`ENBA (S(—), NG—Cyclohexyladenosine (CHA), N5—
`
`Cyclopentyladenosine (CPA), 2—Chloro-Ny
`
`cyclopentyladenosine, 2—chloroadenosine, and adenosine amine
`
`congener
`
`(ADAC), all of which are agonists for the adenosine
`
`A1 receptor. Other receptor agonists include 2-p—(2-
`carboxy-ethyl) phenethyl—amino—S’-N-ethylcarboxamido—
`
`adenosine (CGS—21680), N-ethylcarboxamido-adenosine (NECA)
`and napthyl—substituted aralkoxyadenosine (SKA—082), S’(N-
`Cyclopropyl)-carboxamidoadenosine, DPMA (PD 129,944),
`Metrifudil, which are agonists for the adenosine A2
`
`-6-
`
`
`
`“W95 ,
`
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`receptor. Other adenosine receptor agonists include those
`which preferentially bind the A1 receptor relative to the A2
`
`receptor, such as 2¥Chloroadenosine, N‘—Phenyladenosine, and
`Ns—Phenylethyladenosine; and those which preferentially bind
`
`the A2 receptor relative to the A.1 receptor, such as 2-
`
`Phenylaminoadenosine and MECA.‘
`Also suitable for use are compounds that increase
`
`intracellular adenosine concentration by inhibiting the
`cellular uptake of adenosine or the breakdown of adenosine.
`One pathway of adenosine metabolism is the conversion of
`adenosine to inosihe by adenosine deaminase. An example of
`an adenosine deaminase inhibitor is erythro—9—(2—hydroxy-3—
`
`nonyl) adenine {:EE§::;> Adenosine kinase inhibitors can
`
`e kinase converts adenosine to
`
`also be used. Adeno '
`
`adenosine monophosphate by adenosine kinase. An example of
`an adenosine kinase inhibitor is iodotubercidin. Other
`
`suitable compounds include those that inhibit the
`
`dipyridamole-sensitive nucleoside transporter, which exports
`adenosine from the cytoplasm, and agents that promote the
`
`activity of a 5'-nucleotidase, e.g.,
`
`the ATPeactivated 5’-
`
`Compounds that
`nucleotidase, which forms adenosine.
`increase tissue adenosine and ATP levels include acadesine
`', which is described in Gruber et a1.,
`Circulation 80:1400-1411 (1989).
`Adenosine can be also be administered with a second
`
`10
`
`15
`
`20
`
`25
`
`The second compound can enhance the action of
`compound.
`adenosine or the adenosine analog, e.g., by enhancing
`
`binding of adenosine or an adenosine analog to an adenosine
`
`3O
`
`receptor. An example of such a compound is PD 81,728, which
`is described in Kollias-Baker et al. J. Pharmacol. Exp.
`
`Ther. 281:761—68. Alternatively,
`
`the second agent can
`
`itself act to enhance skin condition.
`
`Examples of these
`
`types of agents include tretinoin,
`
`a recognized skin
`
`-7-
`
`16/95
`
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`
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`o
`
`conditioning agent
`(see, e.g., Olsen et al., J. Amer. Acad.
`Dermatol. 37:217—26, 1997), an angiogenic factor such as
`vascular endothelial cell growth factor (VEGF) or basic
`
`fibroblast growth factor (BFGF), or a conditioning agent.
`: The second compound can also be a conditioning agent
`such as an emollient, humectant, or occlusive agent.
`
`Numerous examples of particular conditioning agents are
`
`provided‘in the CTFA Cosmetic Ingredient Handbook (Cosmetic
`Toiletries and Fragrances Association, Washington, D.D.,
`
`1988). Emollients help to maintain the soft, smooth, and
`pliable appearance of skin and function by remaining on the
`skin surface or in the stratum corneum to act as lubricants,
`
`to reduce flaking, and to improve the skin's appearance.
`
`Examples of emollients include acetyl trioctyl citrate,
`
`cetyl alcohol, butyl myristate, cetyl alcohol, and mineral
`oil.
`
`Humectants act to increase the water content of the
`
`top layers of the skin. Humectants include, e.g., acetamide
`MEA,
`fructose, and xylitol. Occlusive agents inhibit the
`
`thereby increasing the water
`evaporation of water from skin,
`contend of the skin. Acetylated castor oil, mineral oil,
`
`and lauryl stearate are examples of occlusive agents.
`
`A subject can be treated by applying adenosine or an
`
`adenosine analog in a pharmaceutical composition in an
`
`effective amount and for a period of time sufficient to
`
`improve the condition of the skin.
`The pharmaceutical c0mposition may be formulated
`
`using conventional methods to prepare pharmaceutically
`
`10
`
`15
`
`20
`
`25
`
`30
`
`Such compositions preferably include
`useful compositions.
`at least one pharmaceutically acceptable carrier, such as
`
`those described in Remington's Pharmaceutical Sciences (E.w.
`
`the compositions preferably include a
`In addition,
`Martin).
`pharmaceutically acceptable buffer, preferably phosphate
`
`_
`
`3 -
`
`17/95
`
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`
`4
`
`5
`
`6
`
`7
`
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`
`0
`
`33213131113331? as £11333: F3 $3333
`
`buffered saline,
`
`together with a pharmaceutically acceptable
`
`compound for adjusting isotonic pressure, such as,
`
`for
`
`example, sodium chloride, mannitol, or sorbitol.
`Adenosine or an adenosine agonist can also be
`
`provided in carriers and adjuvants such as ion exchangers,
`alumina, aluminum stearate,
`lecithin,
`serum proteins, such
`
`as human serum albumin, buffer substances, such as
`
`phosphates, glycine, sorbic acid, potassium sorbate, partial
`
`glyceride mixtures of saturated vegetable fatty acids,.
`
`water, salts or electrolytes, such as protamine sulfate,
`disodium hydrogen phosphate, potassium hydrogen phosphate,
`sodium chloride, zinc salts, colloidal silica, magnesium
`
`trisilicate, polyvinyl pyrrolidone, cellulose—based
`
`substances and polyethylene glycol. Adjuvants for topical
`
`or gel base forms of adenosine or adenosine analogs may, for
`
`example, be selected from the group consisting of sodium
`
`carboxymethylcellulose, polyacrylates, polyoxythylene-
`
`polyoxypropylene-block polymers, polyethylene glycol and
`wood wax alcohols.
`For all administrations, conventional
`
`depot forms may be used.
`The adenosine or adenosine analog-containing
`
`compositions may be in any pharmaceutically acceptable
`
`dosage form.
`
`”They are preferably applied by topical routes
`
`to exert local therapeutic effects.
`
`For topical
`
`the penetration of the adenosine into skin
`application,
`tissue may be enhanced by a variety of methods known to
`
`thOSe of ordinary skill in the art.
`
`For example, adenosine
`
`may be applied directly and mechanically rubbed into the
`skin.- Alternatively, adenosine or adenosine analogs may be
`
`incorporated into a transdermal patch that is applied to the
`skin. Preferably,
`the penetration resulting from these
`
`methods is enhanced with a chemical
`
`transdermal delivery
`
`agent such as dimethyl sulfoxide (DMSO) or the nonionic
`
`-9-
`
`10
`
`15
`
`20
`
`25
`
`30
`
`18/95
`
`3
`
`8
`
`5
`
`6
`
`7
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`
`
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`
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`
`surfactant, n—decylmethyl sulfoxide (NDMS), as described in
`
`Choi et al., Pharmaceutical Res., 7(11):1099, 1990.
`
`Other modes of administration include, e.g., oral,
`subdermal,
`intradermal, or intravenous. When oral
`
`administration is used, it is critical that the adenosine or
`
`adenosine analog be delivered to that it is not degraded
`
`3
`prior to exiting the digestive system.
`The most effective mode of administration and dosage
`
`regimen of adenosine or the adenosine analog will depend
`
`the subject's
`upon the skin condition, previous therapy,
`the judgment of
`health status,
`response to the adenosine,
`the treating physician and the mode in which the adenosine
`
`For example, dosages for a therapeutically
`is applied;
`effective amount for topical application would be in the
`
`range of 100 mg to 10 mg per treated surface area per day.
`
`The adenosine may be administered to the patient at one time
`or over a series of treatments. when adenosine or the
`
`adenosine analog is administered in conjunction with a
`
`second agent,
`
`they can be administered either concurrently
`
`or sequentially, and can be administered in the same mode or
`
`a different mode, e.g.,
`
`topical or oral.
`
`Adenosine or an adenosine analog enhances skin
`condition when there is a noticeable decrease in noticeable
`
`decrease in the amount of wrinkling,
`
`roughness, dryness,
`
`laxity, sallowness, or pigmentary mottling of the treated
`skin. Methods of measuring improvements in skin condition
`
`are well known in the art
`
`(see, e.g., Olsen et al., J. Amer.
`
`Acad. Dermatol. 262215-24, 1992), and can include subjective
`
`evaluations by the patient or a second party, e.g., a
`
`treating physician. Objective methods can include skin
`
`topography measurements, such as those described in Grove et
`al., J. Amer. Acad. Dermatol. 21:631-37 (1989).
`In skin
`
`topography measurements, silicone_rubber replicas are made
`
`-
`
`lO _
`
`10
`
`15
`
`20
`
`25
`
`3O
`
`19/95
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`(
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`
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`
`Jfliflflflfiifl sflfiflfififlfi
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`
`of a small area of skin, e.g., a 1 cm diameter circular
`
`area.
`
`The Silicone rubber replicas capture fine lines and
`
`wrinkles on the skin, These specimens are then analyZed
`using computerized digital image processing to provide an
`objective measurement of the skin's topography.
`Skin
`
`topography measurements generated following digital~image
`
`processing can be measured using the values RB and R2 as
`
`described in Olsen et al., J. Amer. Acad. Dermatol. 37:217-
`26, 1997, where Ra represents the area of deviation of skin
`
`surface features above and below an average central line,
`
`and R.z represents the difference between the maximum and
`
`minimum heights in five equal segments_of the skin surface
`profile.
`A statistically significant decline (e.g., P <
`0.05)
`in Ra and R1 values in skin treated with adenosine or
`an adenosine analog compared to untreated skin indicates an
`enhancement of skin condition.
`
`Fibroblasts treated with adenosine or adenosine
`
`analogs can also be incorporated into a matrix and implanted
`in the body, e.g., as part of a skin graft.
`In addition,
`
`fibroblasts can be genetically engineered ex vivo to
`increase the amount of intracellular adenosine levels and
`
`then re-introduced into a human patient.
`
`(See, for example,
`
`Anderson et al. U.s. Patent No. 5,399,349; and Mulligan &
`Wilson, U.S. Patent No. 5,460,959, each of which is
`
`incorporated by reference herein in its entirety).
`
`c_el_l_Cu_l_tLu-”s
`
`Experimental Information
`
`Human skin fibroblasts and human lung fibroblasts
`
`were supplied by the N.I.A. Aging Culture Repository Center
`
`(Camden, NJ).
`
`For skin fibroblasts, primary cultures had
`
`been initiated from explants obtained from a 3 mm punch
`
`biopsy of the mesial aspect of the upper left arm.
`
`Human
`
`-11-
`
`10
`
`15
`
`20
`
`25
`
`3O
`
`20/95
`
`9
`
`:
`
`
`
`
`
`
`
`
`
`
`
`4"»
`
`giiflfiiififliffiilifi Eiiiaéflfigiiifi
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`
`lung fibroblasts (IMR-90) were established from a 16-week
`normal female fetus. All cells displayed a normal diploid
`karyotype.and all cells tested negative for bacteria,
`fungi
`and mycoplasma contamination.
`I
`Cells were grown in Eagle’s minimal essential medium
`(MEM) supplemented with 10% fetal bovine serum (FBS), 100
`U/ml penicillin and 100 mg/ml streptomycin in a 37°C,
`5%
`co2/95% air environment. After reaching confluence, cells
`were subcultivated with 0.25% trypsin in MEM with no added
`Ca2+ or Mg“.
`‘
`
`I
`
`Incorporation of ‘3H Thymidine
`As an index of DNA synthesis incorporation of
`
`PH1thymidine was measured as described in Ethier et al.,
`Am. J. Physiol. 272:H1470-79 (1997). Confluent monolayers
`of human skin fibroblasts in MEM plus 10% PBS were seeded
`
`into 15 mm diameter culture wells (24-well plates) at a
`
`density of l x 10‘ cells/cwfi. Cells were grown at 37°C
`under standard culture conditions (5% cog-95% air) until
`they were approximately 75% confluent. Medium was then
`removed and the cells were made “serum—free" by incubation
`in MEM with no FBS for 24 hours. Adenosine or vehicle (MEM)
`was added for an additional 18 hours. This medium was then
`replaced with fresh MEM, and the cells were pulsed with
`lmCi/ml
`[3H]
`thymidine (6.7 Ci/mmol). After a 2 hour
`incubation period,
`the medium was discarded and the cells
`
`were rinsed twice with cold (4°C) Hank’s balanced salt
`solution (HESS) and incubated for 5 minutes with 0.5 ml cold
`
`10% (w/v)
`
`trichloroacetic acid (TCA).
`
`The wells were then
`
`rinsed with 8% TCA and the TCA-insoluble material was
`
`solubilized with 0.5 m1 of a solution of 0.2M NaOH and 0.2%
`
`sodium decyl sulfate (SDS).
`
`The radioactivity of this
`
`10
`
`15
`
`2O
`
`25
`
`30
`
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`
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`
`fraction was determined by standard liquid scintillation
`
`spectrometric techniques.
`Incorporation of
`
`’
`[3H]
`
`thymidine was expressed as
`
`counts per minute (cpm) of 3H per culture. Data in each
`
`experiment was derived from 4 identically treated wells.
`Since the cpm/well exhibited variation between experiments,
`
`data representing combined experiments are expressed herein
`as a pertent of their respective mean control value.
`
`10
`
`15
`
`20
`
`25
`
`3H phenylalanine
`Incorporation of
`Incorporation of
`[JHIphenylalanine was measured as
`an index of protein synthesis; Human Skin fibroblasts were
`
`seeded into 24—well culture plates in MEM containing 10%
`
`FBS. When cells had grown to approximately 75% confluence
`
`the culture medium was replaced with serum-free MEM with or
`without adenosine. After 48 hours, 2uCi/ml
`
`PHJphenylalanine was added to the cultures. Unlabeled
`
`phenylalanine (0.36 mM) was also added to equalize
`concentrations of intracellular and extracellular
`
`phenylalanine. After 8 hours, medium was removed and the
`cells were washed twice with cold (4°C) HBSS and incubated
`
`for 20 minutes in cold 10% (w/v) TCA. Cells were then
`
`incubated 5 minutes in 95% ethanol
`
`(4°C) and the TCA-
`
`insoluble material was solubilized with a solution of 0.2M
`
`NaOH and 0.2% SDS.
`
`The radioactivity of this fraction was
`
`determined by standard liquid scintillation spectrometric
`
`techniques.
`
`Incorporation of
`
`[3H] phenylalanine was expressed as
`
`3O
`
`cpm of 3H per culture well and data in each experiment were
`derived from six identically treated wells.
`Since the
`
`cpm/well exhibited variation between experiments, data
`
`representing combined experiments are expressed as a percent-
`of their respective mean control value.
`
`:13—
`
`
`
`
`
`
`
`
`
`22/95
`
`
`
`
`
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`
`Determination of Cell Size
`
`Human fibroblaSts in MEM 10% PBS were seeded into 25
`
`cm2 culture flasks at a density of lxlO‘| cells/CUP. When the
`cells had groWn to approximately 80% confluence the culture
`medium was removed and the cells were incubated in serum—
`
`free MEM for 24 hours. Adenosine or vehicle (MEM) was added
`
`for 18 hours and cells were then washed twice with cold
`
`(4°C) HBSS.> Cells were removed with 0.25% trypsin in
`calcium-and magnesium—free MEM and diluted in cold (4°C)
`HBSS for measurement of relative cell size with a
`fluorescence-activated cell sorter (FACS; Becton Dickinson
`
`Vantage). Cell size was determined by forward light scatter
`
`on a minimum of 1x104.cells per experiment.
`
`Experimental Materials
`
`23/95
`
`
`
`10
`
`15
`
`2O
`
`trypsin, and
`MEM, FBS, penicillin, streptomycin,
`HESS were obtained from GIBCO (Grand Island, NY),
`PH]
`thymidine (6.7 Ci/mmol) and phenylalanine, L-ring-2,3,4,5,6e
`3H]
`(92 Ci/mmol) were obtained from Dupont NEN (Boston, MA).
`
`Adenosine was from Boehringer Mannheim, SDS was from
`
`(Highland Park, NJ) and TCA and
`National Diagnostics,
`ethanol were obtained from Fisher Scientific
`
`(Pittsburgh, PA).
`
`Data Anal sis
`
`Analysis of variance (ANOVA) was used to determine
`statistical differences between means.
`The Dunett’s test
`
`25
`
`was applied for multiple comparisons as described in Zar,
`
`J H., Biostatistical Analysis. Englewood Cliffs, N.J.,
`
`Prentice Hall,
`
`Inc
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