Case 1:17-cv-00868-CFC-SRF Document 35-1 Filed 03/22/19 Page 1 of 9 PageID #: 1267
`Case 1:17-cv-00868—CFC-SRF Document 35-1 Filed 03/22/19 Page 1 of 9 PageID #: 1267
`
`EXHIBIT A
`EXHIBIT A
`
`

`

`Case 1:17-cv-00868-CFC-SRF Document 35-1 Filed 03/22/19 Page 2 of 9 PageID #: 1268
`
`(12) United States Patent
`D0bs0n, Jr. et al.
`
`US006423327B1
`US 6,423,327 B1
`Jul. 23, 2002
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`(54) TREATMENT OF SKIN WITH ADENOSINE
`OR ADENOSINE ANALOG
`
`(75) Inventors: James G. Dobson, J r., Auburn;
`Michael F. Ethier, Grafton, both of
`MA (US)
`
`(73) Assignee: University of Massachusetts, Boston,
`MA (US)
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21) Appl. No.: 09/672,348
`(22) Filed:
`Sep. 28, 2000
`
`Related US. Application Data
`
`(63) Continuation of application No. 09/179,006, ?led on Oct.
`26, 1998, now abandoned.
`
`(51) Int. Cl.7 ................................................ .. A61K 7/00
`(52) US. Cl. ..................... .. 424/401; 424/447; 424/448;
`424/449; 514/46
`(58) Field of Search ............................... .. 424/407, 447,
`424/448, 449; 514/46
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`5/1978 Voorhees .................. .. 424/180
`4,088,756 A
`6/1984 Stramentionoli et al.
`424/180
`4,454,122 A
`3/1995 Paunescu et al. ...... .. 424/195.1
`5,399,349 A
`5,460,959 A 10/1995 Mulligan et al. .
`435/1723
`5,618,544 A * 4/1997 Brown ..................... .. 424/401
`5,770,582 A
`6/1998 Von Borstel et al. .
`514/45
`5,785,978 A * 7/1998 Porter et al. ....... ..
`424/401
`
`. . . .. 514/75
`5,821,237 A 10/1998 Bissett et al. . . . . .
`514/46
`5,932,558 A
`8/1999 Crostein et al. ..
`5,998,423 A 12/1999 Manneth et al. .......... .. 514/260
`
`FOREIGN PATENT DOCUMENTS
`
`DE
`
`19545107
`
`* 6/1997
`
`OTHER PUBLICATIONS
`
`HartZshtark et al. The use of indentometry to study the effect
`of agents known to increase skin cAMP content. EXperentia.
`41(3), 378—379, 1985*
`
`Adair et al., “Vascular development in chick embryos: a
`possible role for adenosine” American Physiological Soci
`ety; 0363—6135/89 1989.
`Ahmed et al., “Presence of Both A1 and A2 Adenosine
`Receptors in Human Cells and Their Interaction,” Bio
`chemical and Biophysical Research Communications,
`208:871—878, 1995.
`Ethier et al., “Adenosine Stimulation of DNA Synthesis in
`Human Endothelial Cells,” The American Physiological
`Society, 272:H1470—H1479, 1997.
`Grove et al., “Optical pro?lometry: An objective method for
`quanti?cation of facial Wrinkles,” Journal of the American
`Academy of Dermatology, 21:631—637, 1989.
`Gruber et al., “Increased Adenosine Concentration in Blood
`From Ischemic Myocardium by AICA Riboside,” Circula
`tion, 80:1400—1411, 1989.
`Kollias—Baker et al., Journal Pharmacology and Experimen
`tal Therapeutics, 281: 761—768, 1997.
`NeWby et al., Critical Evaluation of the Role of Ecto—and
`Cytosolic 5’ Nucleotidase in Adenosine Formation Topics
`and Perspectives in Adenosine Research, 155—168, 1987.
`Olsen et al, “Tretinoin emollient cream: a neW therapy for
`photodamaged skin,” Journal of the American Academy of
`Dermatology, 26:215—224, 1992.
`Olsen et al., “Tretinoin emollient cream for photodamaged
`skin: Results of 48—Week, multicenter, double—bir studies,”
`Journal of the American Academy of Dermatology,
`37:217—226, 1997.
`
`* cited by examiner
`
`Primary Examiner—Thurman K. Page
`Assistant Examiner—Lakshmi Channavajjala
`(74) Attorney, Agent, or Firm—Fish & Richardson PC.
`(57)
`ABSTRACT
`
`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.
`
`10 Claims, 2 Drawing Sheets
`
`HUMAN SKIN FlBROBLASTS
`
`HUMAN LUNG FIBROBLASTS
`
`150
`
`[3H] THYMIDINE 125
`INCORPORATION
`(% OF CONI'ROL) 100
`
`75
`
`50
`
`ADENOSINE
`
`( Ill-4M)
`
`'
`
`+
`
`150
`
`[3H] THYMIDINE 125
`INCORPORATION
`(% OF CONTROL) 100
`
`75
`
`50
`ADENOSINE
`(10»4M)
`
`‘
`
`1'
`
`

`

`Case 1:17-cv-00868-CFC-SRF Document 35-1 Filed 03/22/19 Page 3 of 9 PageID #: 1269
`
`U.S. Patent
`
`Jul. 23, 2002
`
`Sheet 1 0f 2
`
`US 6,423,327 B1
`
`HUMAN SKIN FIBROBLASTS
`
`150
`
`[3H] THYMIDINE 125 '
`INCORPORATION
`(% OF CONTROL) 100_
`
`75
`
`ADENOSINE
`(10-4M)
`
`150
`
`[3H] THYMIDINE 125 '
`INCORPORATION
`(% 0F CONTROL) 100_
`
`75
`
`50
`ADENOSINE
`(IO-4M)
`
`FIG. 1A
`
`HUMAN LUNG FIBROBLASTS
`
`

`

`Case 1:17-cv-00868-CFC-SRF Document 35-1 Filed 03/22/19 Page 4 of 9 PageID #: 1270
`
`U.S. Patent
`
`Jul. 23, 2002
`
`Sheet 2 0f 2
`
`US 6,423,327 B1
`
`YOUNG DONOR
`
`[3H] PHENYLALANINE
`INCORPORATION
`(% OF CONTROL)
`
`120
`
`110*
`
`100)
`
`90-"
`
`80
`
`0
`
`-5
`-6
`[ADENOSINE] (M)
`
`-4
`
`FIG. 2A
`
`AGED DONOR
`
`120 -
`
`[3H1PHENYLALANINE 110'
`INCORPORATION
`(% OF CONTROL)
`
`.1
`
`.
`
`.
`
`-
`
`I
`
`-5
`-6
`[ADENOSINE] (M)
`
`-4
`
`FIG. 2B
`
`

`

`Case 1:17-cv-00868-CFC-SRF Document 35-1 Filed 03/22/19 Page 5 of 9 PageID #: 1271
`
`US 6,423,327 B1
`
`1
`TREATMENT OF SKIN WITH ADENOSINE
`OR ADENOSINE ANALOG
`
`This application is a continuation of application Ser. No.
`09/179,006, ?led Oct. 26, 1998, noW abandoned.
`
`STATEMENT 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.
`
`10
`
`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 ?broblasts.
`Skin thickness begins to decline in humans after the age
`of 20 as the dermis becomes thinner and the number of skin
`?broblasts 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 ?broblasts, Which include
`collagen and proteoglycans, are believed to play major roles
`in Wrinkled and damaged skin.
`
`SUMMARY OF THE INVENTION
`
`15
`
`20
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`25
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`
`We have discovered that adenosine stimulates DNA
`synthesis, increases protein synthesis, and increases cell siZe
`in cultures of human skin ?broblasts. 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 therapeu
`tically effective amount of a composition including adenos
`ine 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 therapeu
`tically effective amount of adenosine or an adenosine analog
`to the mammal.
`Also included in the invention is a method for increasing
`DNA synthesis in a dermal cell of non-diseased skin of a
`mammal. The method includes topically administering a
`therapeutically effective amount of adenosine or an adenos
`ine 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 mammal
`containing the dermal cell. The adenosine or adenosine
`analog does not cause proliferation of the dermal cell.
`
`50
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`55
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`65
`
`2
`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 ?broblasts from the mammal ex vivo, culturing
`the ?broblasts in the presence of adenosine, and reintroduc
`ing the ?broblasts into the mammal.
`The therapeutically effective amount of adenosine used in
`the above-described methods is preferably 10'3 M to 10'7
`M, more preferably 10'3 M to 10'6 M, and most preferably
`about 10'4 M.
`The composition used in the above-described methods
`can include a second agent in addition to adenosine. The
`second agent can be, eg 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 ?broblast 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 prolif
`eration.
`The invention also provides a composition including
`about 10'3 M to about 10'7 M adenosine and a therapeuti
`cally effective amount of an angiogenesis factor. In some
`embodiments, the composition of the adenosine is about
`10-4 M.
`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.
`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 de?ned, all technical and scienti?c
`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 beloW. All publications, patent
`applications, patents, and other references mentioned herein
`are incorporated by reference in their entirety. In case of
`con?ict, the present speci?cation, including de?nitions, Will
`control. In addition, the materials, methods, and examples
`are illustrative only and not intended to be limiting.
`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 [3H]thymidine incorporation in cultures of
`
`

`

`Case 1:17-cv-00868-CFC-SRF Document 35-1 Filed 03/22/19 Page 6 of 9 PageID #: 1272
`
`US 6,423,327 B1
`
`3
`normal human skin (FIG. 1A) and lung ?broblasts (FIG.
`1B). After incubation in serum-free medium for 24 hours,
`cells Were exposed to 10'4 M adenosine for 18 hours.
`Medium Was replaced With serum-free medium Without
`adenosine, and [3H]thymidine Was added. Results are
`expressed as percent [3H]thymidine incorporation compared
`to control cultures Without adenosine and are IIIGZIIISISEM
`for 4—5 experiments. “*” denotes value Was signi?cantly
`different from control value Without adenosine.
`FIGS. 2A and 2B are histograms shoWing concentration
`responses of adenosine-stimulated protein synthesis in
`human skin ?broblasts from a young (FIG. 2A) and aged
`(FIG. 2B) donor. Cells Were groWn to 75% con?uence.
`Medium Was then replaced With serum-free medium With or
`Without adenosine. After 48 hours, [3H]phenylalanine incor
`poration Was determined as described. Results are expressed
`as % [3H]phenylalanine incorporation compared to control
`cultures Without adenosine and are IIIGZIHSISEM for 6—25
`experiments. “*” denotes value Was signi?cantly different
`from control value Without adenosine.
`
`10
`
`15
`
`DETAILED DESCRIPTION
`
`The invention is suitable for treating skin of a mammal,
`e.g., a human, for Which promotion of ?broblast-associated
`dermal functions is desired. For example, promotion of
`?broblast-associated functions is desirable in enhancing the
`condition of aged skin, Which is associated With a decrease
`in dermal cell function and is characteriZed by increased
`dryness or roughness, or both. The method can 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 com
`pounds 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 recep
`tor agonists include phenylisopropyl-adenosine (“PIA”),
`1-Methylisoguanosine,
`ENBA
`(S(-),
`N?-Cyclohexyladenosine (CHA), N6-Cyclopentyladenosine
`(CPA), 2-Chloro-N6-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-5‘-N-ethylcarboxamido-adenosine (CGS-21680),
`N-ethylcarboxamido-adenosine (NECA) and napthyl
`substituted aralkoxyadenosine (SHA-082), 5‘
`(N-Cyclopropyl)-carboxamidoadenosine, DPMA (PD 129,
`944), Metrifudil, Which are agonists for the adenosine A2
`receptor. Other adenosine receptor agonists include those
`Which preferentially bind the A1 receptor relative to the A2
`receptor, such as 2-Chloroadenosine, N6-Phenyladenosine,
`and N6-Phenylethyladenosine; and those Which preferen
`tially bind the A2 receptor relative to the A1 receptor, such
`as 2-Phenylaminoadenosine and MECA.
`Also suitable for use are compounds that increase intra
`cellular adenosine concentration by inhibiting the cellular
`uptake of adenosine or the breakdoWn of adenosine. One
`
`25
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`
`4
`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 (“EHN ”). Adenosine kinase
`inhibitors can also be used. Adenosine 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 activity of a 5‘-nucleotidase, e.g., the
`ATP-activated 5‘-nucleotidase, Which forms adenosine.
`Compounds that increase tissue adenosine and ATP levels
`include acadesine (AICA-riboside), Which is described in
`Gruber et al., Circulation 80:1400—1411 (1989).
`Adenosine can be also be administered With a second
`compound. The second compound can enhance the action of
`adenosine or the adenosine analog, e.g., by enhancing bind
`ing 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 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 ?broblast
`groWth factor (BFGF), or a conditioning agent.
`The second compound can also be a conditioning agent
`such as an emollient, humectant, or occlusive agent. Numer
`ous examples of particular conditioning agents are provided
`in the CTFA Cosmetic Ingredient Handbook (Cosmetic
`Toiletries and Fragrances Association, Washington, DD,
`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 ?aking, 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 suf?cient to
`improve the condition of the skin.
`The pharmaceutical composition may be formulated
`using conventional methods to prepare pharmaceutically
`useful compositions. Such compositions preferably include
`at least one pharmaceutically acceptable carrier, such as
`
`those described in Remington’s Pharmaceutical Sciences W. Martin). In addition, the compositions preferably include
`a pharmaceutically acceptable buffer, preferably phosphate
`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
`
`

`

`Case 1:17-cv-00868-CFC-SRF Document 35-1 Filed 03/22/19 Page 7 of 9 PageID #: 1273
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`US 6,423,327 B1
`
`5
`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 composi
`tions may be in any pharmaceutically acceptable dosage
`form. They are preferably applied by topical routes to exert
`local therapeutic effects. For topical application, the pen
`etration 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 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 admin
`istration 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 condi
`tion 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. 26:215—24, 1992), and can include subjec
`tive 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
`of a small area of skin, e.g., a 1 cm diameter circular area.
`The silicone rubber replicas capture ?ne lines and Wrinkles
`on the skin. These specimens are then analyZed using
`computeriZed digital image processing to provide an objec
`tive measurement of the skin’s topography. Skin topography
`measurements generated folloWing digital-image processing
`can be measured using the values Ru and R, 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 R2
`represents the difference betWeen the maximum and mini
`mum heights in ?ve equal segments of the skin surface
`
`10
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`6
`pro?le. Astatistically signi?cant decline (e.g., P<0.05) in Ru
`and R2 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, ?broblasts 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. Pat.
`No. 5,399,349; and Mulligan & Wilson, U.S. Pat. No.
`5,460,959, each of Which is incorporated by reference herein
`in its entirety).
`
`Experimental Information
`
`Cell Culture
`Human skin ?broblasts and human lung ?broblasts Were
`supplied by the N.I.A. Aging Culture Repository Center
`(Camden, NJ For skin ?broblasts, primary cultures had
`been initiated from explants obtained from a 3 mm punch
`biopsy of the mesial aspect of the upper left arm. Human
`lung ?broblasts (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% CO2/95% air environment. After reaching con?uence,
`cells Were subcultivated With 0.25% trypsin in MEM With no
`added Ca2+ or Mg2+.
`Incorporation of [3H]Thymidine
`As an index of DNA synthesis incorporation of [3H]
`thymidine Was measured as described in Ethier et al., Am. J.
`Physiol. 272:H1470—79 (1997). Con?uent monolayers of
`human skin ?broblasts in MEM plus 10% PBS were seeded
`into 16 mm diameter culture Wells (24-Well plates) at a
`density of 1><104 cells/cm2. Cells Were groWn at 37° C. under
`standard culture conditions (5% CO2—95% air) until they
`Were approximately 75% con?uent. Medium Was then
`removed and the cells Were made “serum-free” by incuba
`tion 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.) Hanks
`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 radioac
`tivity of this 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 percent of their respective mean control value.
`Incorporation of [3H]phenylalanine
`Incorporation of [3H]phenylalanine Was measured as an
`index of protein synthesis. Human skin ?broblasts Were
`seeded into 24-Well culture plates in MEM containing 10%
`FBS. When cells had groWn to approximately 75% con?u
`ence the culture medium Was replaced With serum-free
`MEM With or Without adenosine. After 48 hours, 2 pCi/ml
`
`

`

`Case 1:17-cv-00868-CFC-SRF Document 35-1 Filed 03/22/19 Page 8 of 9 PageID #: 1274
`
`US 6,423,327 B1
`
`10
`
`15
`
`20
`
`25
`
`30
`
`7
`[3H]phenylalanine Was added to the cultures. Unlabeled
`phenylalanine (0.36 mM) Was also added to equalize con
`centrations 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
`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 repre
`senting combined experiments are expressed as a percent of
`their respective mean control value.
`Determination of Cell SiZe
`Human ?broblasts in MEM 10% FBS Were seeded into 25
`cm2 culture ?asks at a density of 1><104 cells/cm2. When the
`cells had groWn to approximately 80% con?uence the cul
`ture 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 ?uorescence-activated cell sorter (FACS; Becton Dickin
`son Vantage). Cell siZe Was determined by forWard light
`scatter on a minimum of 1><104 cells per experiment.
`Experimental Materials
`MEM, FBS, penicillin, streptomycin, trypsin, and HBSS
`Were obtained from GIBCO (Grand Island, NY), [3H]
`thymidine (6.7 Ci/mmol) and phenylalanine, L-ring-2,3,4,
`5,6-3H ] (92 Ci/mmol) Were obtained from Dupont NEN
`35
`(Boston, Mass.). Adenosine Was from Boehringer
`Mannheim, SDS Was from National Diagnostics, (Highland
`Park, N] and TCA and ethanol Were obtained from Fisher
`Scienti?c (Pittsburgh, PA).
`Data Analysis
`Analysis of variance (ANOVA) Was used to determine
`statistical differences betWeen means. The Dunett’s test 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 values
`expressed as a percentage. Differences Were considered
`statistically different When P<0.05.
`DNA Synthesis
`Exposure to 10—4M adenosine increased [3H]thymidine
`incorporation by 43:9% in ?ve studies on cultures of human
`?broblasts (AG607720B) made quiescent by serum
`removal. These results are summariZed in FIG. 1A. In
`contrast, adenosine (10_4M) had no effect on [3H] thymidine
`incorporation in cultures of human lung ?broblasts (IMR
`90) (FIG. 1B). Concentrations of adenosine ranging from
`10-7 M to 10'3 M also failed to stimulate [3H]thymidine
`incorporation in IMR-90 lung ?broblasts (data not shoWn).
`The effect of adenosine on DNA synthesis Was addition
`ally determined on skin ?broblast cultures from six different
`human donors. Adenosine (10_4M) stimulated DNA synthe
`sis in all three cultures derived from young human donors
`(Table 1). Values shoWn are IIICZIHSISEM, Where n is number
`of experiments. Exposure to adenosine and determination of
`[3H] thymidine incorporation Were as described above. The
`asterisk denotes a value signi?cantly different from the
`corresponding control (100%).
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`8
`
`TABLE 1
`
`Effect of adenosine on [3H] thymidine incorporation
`into cultured human skin ?broblasts derived from young
`donors
`
`Cell
`
`Strain
`
`AG07720B
`
`AG07306A
`
`AG09605
`
`Adenosine
`
`Donor
`
`[3H] thymidine
`incorporation
`(% of
`
`(104M)
`
`Age
`
`Sex
`
`control)
`
`—
`+
`—
`+
`—
`+
`
`24
`
`28
`
`30
`
`F
`
`F
`
`M
`
`100
`124 r 7*
`100
`193 r 20*
`100
`133 r 15*
`
`n
`
`24
`24
`6
`6
`12
`12
`
`Peak stimulation of [3H] thymidine incorporation
`(93:20%, n=6) Was achieved in human skin ?broblast
`cultures derived from a 28 year old female (AG07306A).
`Adenosine (10_4M) stimulated DNA synthesis in 2 of 3
`cultures derived from aged human donors (Table 2). As in
`Table 1, values are meanszSEM, and n is the number of
`experiments performed. The asterisk denotes a measurement
`signi?cantly different from the corresponding control
`(100%). Adenosine exposure increased [3H] thymidine
`incorporation by 53:31% and 54:22% in human skin ?bro
`blast cultures derived from a 70 year-old male and a 84
`year-old male, respectively. Adenosine had no effect on
`cultures derived from a 67-year old female.
`
`TABLE 2
`
`Effect of adenosine on [3H] thymidine incorporation
`into cultured human skin ?broblasts derived from aged
`donors
`
`Cell
`
`Adenosine
`
`Donor
`
`[3H] thymidine
`incorporation
`(% of
`
`Strain
`
`(104M)
`
`Age
`
`Sex
`
`control)
`
`AG11728
`
`AG12949
`
`AG11730
`
`—
`+
`—
`+
`—
`+
`
`F
`
`67
`
`70
`
`84
`
`100
`91 r 6
`100
`150 + 31*
`100
`154 r 22*
`
`n
`
`6
`6
`11
`11
`10
`10
`
`Protein Synthesis
`The effect of adenosine on protein synthesis Was deter
`mined by measuring [3H]phenylalanine incorporation into
`cultures of human ?broblasts from a young and aged donor.
`Cultures made quiescent by serum removal Were exposed to
`adenosine (10_6M to 10_4M) for 48 hours and then pulsed
`With phenylalanine. In skin ?broblast cultures derived from
`a 28-year old female (AG073060A) and an 84-year old male
`(AG11730), adenosine(10_4M) increased protein synthesis
`by 13:4% (n=25) and 13:6% (n=17), respectively (FIG. 2).
`Cell SiZe
`The effect of adenosine on cell siZe Was determined on
`human skin ?broblasts from young and aged donors by
`measuring forWa