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`U.S. UTILITY PatentApplication —
`
`
`APPLNUM|FILING DATE |cLags |SuSCLASs |GAU _ EXAMINER
`
`voreasia |.
`|
`438
`ChippiwnII4C B—
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`‘CONTINUING DATA-VERIFIED: lox) a be
`THIS APPLICATIONIS A CON OF 09/672,248 09/28/2000 PAT 6,423,327
`WHICH.IS A COM OF09/179,006 10/26/1998 ABN.
`
`be rn pCttificate.
`i
`JUN 22 2019
`:
`}
`of Correction
`
`" FOREIGN APPLICATIONS VERIFIED: on
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`.
`
`CLAIMS ALLOWED
`"Total Claims
`“PrintGlaim for
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`Primary Examiner
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`
`
`7 TERMINAL
`DISCLAMER
`
`
`} WARNING: The information disclosedherein may be restricted.
`
`
`Unauthorized disclosure maybe prohibited by the United States Code Title 35,
`
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`| Sections.122, 181 and 368, Possessionoutside the:U.S,Patesit.& Trademark.
`
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`| Office is restricted:to authorized
`employees and contractors only.
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`INTERNATIONAL
`CLASSIFICATION
`
`INDEX OF CLAIMS
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`PATENT APPLICATION SERIAL NO.
`
`U.S. DEPARTMENT OF COMMERCE
`‘PATENT AND TRADEMARK OFFICE
`FEE RECORD SHEET
`
`G7/O8/2002 GASFAWL
`
`00000013 10184810
`
`OL FCs201
`
`376.00 OP
`
`PTO-1556
`
`(5/87)
`*U.S. Government Printing Office: 2001 — 481-697/59173
`
`
`
`
`
`5/95 -
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`
`
`7 - al-
`!
`FisH.«. RICHARDSON P.c.
`,
`
`ATA 84e40 .oe:
`
`.
`225 Franklin Street
`Boston, Massachusetts
`02110-2804
`Telephone
`=
`6175425070 =
`‘2 Ba
`+ Facsimile
`ae 2c
`617 542-8906 Ss
`aSS
`.
`o
`5S es
`Web Ste
`wwweftcom
`WS" eae
`a
`
`June 28, 2002
`
`oo
`
`Attorney Docket No.: 07917-045003
`
`Box Patent Application
`Commissionerfor Patents
`Washington, DC 20231
`
`Presented forfiling is a new continuation patent application of:
`
`——ES=0
`ose
`i ——
`Pc} =a
`2 = n
`i
`s = ‘i
`Seme:
`=°c
`WK.Richardson
`1859-1951
`
`Applicant: JAMES G. DOBSON AND MICHAELF. ETHIER
`
`e Title:
`BOSTON
`
`TREATMENT OF SKIN WITH ADENOSINE OR ADENOSINE
`ANALOG
`
`DELAWARE
`
`“ewvons
`
`Thepriorapplication is assigned of record to University of Massachusetts,
`a Massachusetts corporation, by virtue of an assignment submitted to the U.S. Patent
`and Trademark Office and recorded on January 7, 1999 at 9690/0305.
`
`SILICON VALLEY
`TWIN CITIES
`WASHINGTON, DC
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`Enclosed are the following papers, including those required to receive a filing date
`under 37 CFR §1,53(b):
`
`Specification
`Claims
`Abstract
`Declaration
`Drawings
`
`Pages
`20
`7
`1
`2
`2
`
`Enclosures:
`— Form PTO-1449,/1 page, listing documents cited in the parent
`application(s). Please confirm that these have been consideredin this
`
`CERTIFICATE OF MAILING BY EXPRESS MAIL
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`Express Mail Label No.___EL950773342US
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`deposited with the United States Postal Service as Express Mail Post
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`and is addressed to the Commissioner
`for Patents, Washington,
`D.C. 20231.
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`
`Date of7,
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`Signature
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`Typed or Printed Name of Person Signing Certificate
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`6/95
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`
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`S433 FSA.ee
`
`Fisu &KR
`
`AARDSON Puc.
`
`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 claimsthe benefit of priority under 35 USC
`120) of U.S. application serial no. 09/672,348,filed on September 28, 2000, pending,
`whichis a continuation of U.S. 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.
`
`Basicfiling fee
`Total claims in excess of 20 times $9
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`A checkforthe filing fee is enclosed. Please apply any other required fees or any
`credits to deposit account 06-1050, referencing attorney docket number 07917-
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`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.
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`Kindly acknowledge receipt of this application by returning the enclosed postcard.
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`Please send all correspondenceto:
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`LOAG4E 10.Pride
`¢
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`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.B11
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`404184840 .feaeson
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`Attorney’s Docket Ns
`
`4917-045003 / UMMC 97-32
`
`APPLICATION
`
`FOR
`
`UNITED STATES LETTERS PATENT
`
`TITLE:
`
`TREATMENTOF SKIN WITH ADENOSINE OR
`ADENOSINE ANALOG
`
`APPLICANT:
`
`JAMES G. DOBSON AND MICHAELF. ETHIER
`
`CERTIFICATE OF MAILING BY EXPRESS MAIL
`
`Express Mail Label No.
`EL950773342U8
`L hereby certify under 37 CFR §1.10 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,
`D.C. 20231.
`
`
`
`Typed or Printed Nameof
`
`Iferson Signing Certificate
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`
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`9/95
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`7
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`LOLS1 .oeafaoe
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`PATENT
`ATTORNEY DOCKET NO: 07917/045002
`
`TREATMENT OF SKIN WITH ADENOSINE OR ADENOSINE ANALOG
`
`C\
`tatement as to Federally Sponsored Research
`
`
`Work on this invention was supported by funds from
`the United States government
`(Public Health Service Grants
`HL-22828 and AG-11491).
`The government therefore has certain
`rights in this invention.
`
`, Field of the Invention
`This invention relates to dermatology and cell
`
`biology.
`
`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
`variety of cell types,
`including fibroblasts.
`‘
`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.
`
`10
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`Ai 2S oO.ea
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`¢
`
`Summary of the Invention
`
`We have discovered that adenosine stimulates DNA
`synthesis,
`increases protein synthesis, and increases cell
`size in cultures of human skin fibroblasts. Based on this
`discovery,
`the invention provides methods and compositions
`for enhancing the condition of skin.
`In general,
`the invention provides a method for
`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
`
`
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`10
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`15
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`increasing DNA synthesis in a dermal cell of non-diseased
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`20
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`25
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`skin of a mammal.
`The method includes topically
`administering a therapeutically effective amount of
`adenosine or an adenosine analog to a region of non-diseased
`skin of the mammal containing dermal cell.
`The adenosine is
`added so that it does not cause proliferation of the dermal
`cell.
`
`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
`
`composition including a therapeutically effective amount of
`adenosine or an adenosine analog to a region of skin of the
`
`30
`
`mammal containing the dermal cell.
`The adenosine or
`adenosine analog does not cause proliferation of the dermal
`cell.
`
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`aN
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`A G AL ee LEE a Ee]
`¢
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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 of 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
`
`fibroblasts into the mammal.
`reintroducing the
`The therapeutically effective amount of adenosine
`used in the above-described methods is preferably 10° M to
`10°77 M, more preferably 10* M to 10° M, and most preferably
`about 10% 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
`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.
`the
`In preferred embodiments of the invention,
`adenosine or an adenosine analog does not promote skin cell
`proliferation.
`The invention also provides a composition including
`about 10°? M to about 10°’ M adenosine and a therapeutically
`effective amount of an angiogenesis factor.
`In some
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`AG LEYS 2 eros
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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
`of any proliferative disorder observable by visual
`inspection.
`invention advantageously allows for
`The present
`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
`
`10
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`15
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`20
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`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
`
`the
`In case of conflict,
`reference in their entirety.
`present specification,
`including definitions, will control.
`In addition,
`the materials, methods, and examples are
`
`30
`
`illustrative only and not
`
`intended to be limiting.
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`ps
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`10 Leeo.ooaoe
`¢
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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 [H]thymidine incorporation in cultures of
`normal human skin (Fig. 1A) and lung fibroblasts (Fig. 1B).
`After incubation in serum-free medium for 24 hours, cells
`
`were exposed to 10% M adenosine for 18 hours. Medium was
`
`replaced with serum-free medium without adenosine, and
`CH] thymidine was added. Results are expressed as percent
`CH] thymidine incotporation compared to control cultures
`without adenosine and are means + SEM for 4-5 experiments.
`"*" denotes value was significantly different from control
`value without adenosine.
`Figs. 2A and 2B 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.
`After 48 hours,
`[?H]phenylalanine incorporation was
`determined as described. Results are expressed as
`
`% CH] phenylalanine incorporation compared to control
`cultures without adenosine and are means +SEM for 6-25
`
`"*" denotes value was significantly different
`experiments.
`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,
`
`- § -
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`am
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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
`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 such as adenosine agonists, adenosine receptor
`agonists, and compounds that increase intracellular or
`extracellular adenosine levels are suitable for use in the
`
`invention.
`
`Agonists of adenosine include 2’ -deoxyadenosine;
`
`2',3’-isopropoylidene adenosine;
`
`toyocamycin; 1-
`
`methyladenosine; N-6-methyladenosine; adenosine N-oxide; 6-
`methylmercaptopurine riboside; 6-chloropurine riboside, 5’-
`adenosine monophosphate, 5’-adenosine diphosphate, or 5‘-
`
`adenosine triphosphate. Adenosine receptor agonists include
`phenylisopropyl-adenosine ("PIA"), 1-Methylisoguanosine,
`ENBA (S(-), N®-Cyclohexyladenosine (CHA), N&-
`Cyclopentyladenosine (CPA), 2-Chloro-N,-
`cyclopentyladenosine, 2-chloroadenosine, and adenosine amine
`congener
`(ADAC), all of which are agonists for the adenosine
`A, receptor. Other receptor agonists include 2-p-(2-
`carboxy-ethyl) phenethyl-amino-5’ -N-ethylcarboxamido-
`adenosine (CGS-21680), N-ethylcarboxamido-adenosine (NECA)
`and napthyl-substituted aralkoxyadenosine (SHA-082), 5? (N-
`Cyclopropyl) -carboxamideoadenosine, DPMA (PD 129,944),
`Metrifudil, which are agonists for the adenosine A,
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`receptor. Other adenosine receptor agonists include those
`which preferentially bind the A, receptor relative to the A,
`receptor, such as 2-Chloroadenosine, N®-Phenyladenosine, and
`N°~Phenylethyladenosine; and those which preferentially bind
`
`aeS OD eeae
`
`the A, receptor relative to the A, 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 inosine by adenosine deaminase. An example of
`an adenosine deaminase inhibitor is erythro-9- (2-hydroxy-3-
`
`nonyl) adenine a, Adenosine kinase inhibitors can
`
`also be used. AdenoSifie kinase converts adenosine to
`
`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
`
`the ATP-activated 5’-
`activity of a 5’-nucleotidase, e.g.,
`Compounds that
`nucleotidase, which forms adenosine.
`increase tissue adenosine and ATP levels include acadesine
`“RICA-riboside)/, which is described in Gruber et al.,
`Circulation 80:1400-1411 (1989).
`Adenosine can be also be administered with a second
`
`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
`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
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`c .
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`O
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`OLE. ie Seo.
`
`
`(see, e.g., Olsen et al., J. Amer. Acad.
`conditioning agent
`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
`evaporation of water from skin,
`thereby increasing the water
`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 composition may be formulated
`
`using conventional methods to prepare pharmaceutically
`useful compositions.
`Such compositions preferaply include
`at least one pharmaceutically acceptable carrier, such as
`
`those described in Remington’s Pharmaceutical Sciences (E.W.
`Martin).
`In addition,
`the compositions preferably include a
`pharmaceutically acceptable buffer, preferably phosphate
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`AOSis Us.at SS
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`together with a pharmaceutically acceptable
`buffered saline,
`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
`application,
`the penetration of the adenosine into skin
`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
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`©
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`AAO AGG a. eea
`
`e@
`
`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
`prior to exiting the digestive system.
`:
`The most effective mode of administration and dosage
`regimen of adenosine or the adenosine analog will depend
`upon the skin condition, previous therapy,
`the subject's
`health status,
`response to the adenosine,
`the judgment of
`the treating physician and the mode in which the adenosine
`is applied.
`For example, dosages for a therapeutically
`effective amount for topical application would be in the
`
`range of 100 ng 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
`
`roughness, dryness,
`decrease in the amount of wrinkling,
`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. 26:215-24, 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
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`(
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`LG LSS) hese
`
`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 R, and R, as
`described in Olsen et al., J. Amer. Acad. Dermatol. 37:217-
`26, 1997, where R, represents the area of deviation of skin
`surface features above and below an average central line,
`and R, 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 R, and R, values in skin treated with adenosine or
`an adenosine analog compared to untreated skin indicatesan
`enhancement of skin condition.
`
`Fibroblasts treated with adenosine or adenosine
`analogs can also be incorporated into a matrix and implanted
`in the body,
`€.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).
`
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`Experimental Information
`
`CellCulture
`
`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
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`e
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`AD LB4S430 .062cn ae
`
`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.
`.
`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%
`co,/95% air environment. After reaching confluence, cells
`were subcultivated with 0.25% trypsin in MEM with no added
`Ca2+ or Mg?*.
`
`'
`
`;
`Incorporation of DH] Thymidine
`As an index of DNA synthesis incorporation of
`GH] thymidine was measured as described in Ethier et al.,
`am. d. Physiol. 272:H1470-79 (1997). Confluent monolayers
`of human skin fibroblasts in MEM plus.10% FBS were seeded
`into 16 mm diameter culture wells (24-well plates) at a
`density of 1 x 10‘ cells/cm’. Cells were grown at 37°C
`under standard culture conditions (5% CO,-95% air) until
`they were approximately 75% confluent. Medium was then
`removed and the cells were made “serum-free” by incubation
`in MEM withno 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
`imCi/ml
`(H]
`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 (HBSS) 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 ml of a solution of 0.2M NaOH and 0.2%
`sodium decyl sulfate (SDS).
`
`The radioactivity of this
`;
`&
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`(
`)
`
`

`

`
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`¢
`
`AO LEE .oeasoe
`
`fraction was determined by standard liquid scintillation
`spectrometric techniques.
`_
`thymidine was expressed as
`Incorporation of
`(*H]
`counts per minute (cpm) of 7H 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 percent of their respective mean control value.
`
`10
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`
`7H] phenylalanine
`Incorporation of
`Incorporation of
`[(H] phenylalanine 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
`({H] phenylalanine 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.
`[7H] phenylalanine was expressed as
`Incorporation of
`cpm of *H 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.
`
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`
`Determination of Cell Size
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`Human fibroblasts in MEM 10% FBS were seeded into 25
`cm? culture flasks at a density of 1x10‘ cells/cm’. 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 1x10‘. cells per experiment.
`
`Experimental Materials
`trypsin, and
`MEM, FBS, penicillin, streptomycin,
`HBSS were obtained from GIBCO (Grand Island, NY),
`[H]
`thymidine (6.7 Ci/mmol) and phenylalanine, L-ring-2,3,4,5,6-
`3H]
`(92 Ci/mmol) were obtained from Dupont NEN (Boston, MA).
`
`Adenosine was from Boehringer Mannheim, SDS was from
`National Diagnostics,
`(Highland Park, NJ) and TCA and
`ethanol were obtained from Fisher Scientific
`
`(Pittsburgh, PA).
`
`Data Analysis
`
`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. pp. 150-153, 1984.
`In addition,
`the
`Wilcoxon test was employed to verify differences between
`
`30
`
`values expressed as a percentage. Differences were
`considered statistically different when P < 0.05.
`
`- 14 -
`
`
`
`<
`
`
`
`
`
`
`
`

`

`
`
`SABE 10 .oeaseaue
`
`DNA Synthesis
`
`Exposure to 10°*M adenosine increased (H]thymidine
`incorporation by 43 49% in five studies on cultures of human
`fibroblasts (AG607720B) made quiescent by serum removal.
`These results are summarized in Fig. 1A.
`In contrast,
`adenosine (10°M) had no effect on [H]thymidine
`incorporation in cultures of human lung fibroblasts (IMR-90)
`(Fig. 1B). Concentrations of adenosine ranging from 10-7 M
`to 107M also failed to stimulate (?H] thymidine incorporation
`in IMR-90 lung fibroblasts (data not shown).
`The effect of adenosine on DNA synthesis was
`additionally determined on skin fibroblast cultures from six
`different human donors. Adenosine (10 “M) stimulated DNA
`synthesis in all three cultures derived from young human
`donors (Table 1). Values shown are means +SEM, where n is
`number of experiments. Exposure to adenosine and
`determination of
`[3H]
`thymidine incorporation were as
`described above.
`The asterisk denotes a value significantly
`
`different from the corresponding control
`
`(100%).
`
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`>
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`

`
`
`fn
`
`
`
`reeset
`
`LOLS418 .opPeaae
`
`Table 1. Effect of adenosine on [*H] thymidine incorporation
`into cultured human skin fibroblasts derived from young
`donors
`.
`
`Cell
`Strain
`
`Adenosine
`(107% M)
`
`(H] thymidine
`incorporation
`-
` (% of
`control)
`
`
`
`AGO7720B
`
`scorsoea_ |
`
`AGo9605 —
`peak stimulation of
`
`[H]thymidine incorporation
`
`10
`
`15
`
`20
`
`(93+420%, n=6) was achieved in human skin fibroblast cultures
`
`derived from a 28 year old female (AGO7306A).
`Adenosine (10M) stimulated DNA synthesis in 2 of 3
`cultures derived from aged human donors (Table 2).
`As in
`Table 1, values are means +SEM, and n is the number of
`
`:
`
`The asterisk denotes a measurement
`experiments performed.
`significantly different from the corresponding control
`{100%}. Adenosine exposure increased [?H] thymidine
`incorp

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