WO1998027975A1

WO1998027975A1 - Poly(adp-ribose) polymerase inhibitors to treat diseases associated with cellular senescence

Info

Publication number: WO1998027975A1
Application number: PCT/US1996/020630
Authority: WO
Inventors: Michael D. West
Original assignee: Geron Corporation
Priority date: 1996-12-20
Filing date: 1996-12-20
Publication date: 1998-07-02
Also published as: AU1743997A

Abstract

Inhibition of the enzyme poly(ADP-ribose) polymerase can delay the onset of senescence and inhibitors of the enzyme can be used to treat diseases caused or exacerbated by cellular senescence.

Description

Title

Poly(ADP-Rιbose) Polymerase Inhibitors to treat Diseases Associated with Cellular Senescence

Background of the Invention Field of the Invention

The present invention relates to the fields of molecular biology, gerontology, and medical pharmacology and diagnostics.

Description of Related Art There is substantial evidence that somatic cells have a finite rep catn e capacity (Hayfhck and Moorhead. 1961. Exp. Cell Res. 25: 585-621 , Hayfhck. 1965. E.xp. Cell Res. 37 614-636. and Hayfhck. 1970, Exp. Geront. 5. 291-303) and that this process is a major etioiogical factor in aging and age-related disease (Goldstein. 1990. Science 249: 1 129- 1 133; Stanuhs-Praeger. 1987, Mech. Ageing Dev 38- 1-48; and Walton. 1982. Mech Ageing Dev 19. 217-244) As cells undergo iephcative senescence in \ itro and in vivo, the cells not only lose the αbi tv to divide in lesponse to growth stimuli, but there are also significant deleterious changes in the pattern of gene expression (West, 1994, Arch. Derm. 130: 87-95) As an individual grows older, senescent cells make up an increasing percentage of the cells present in the tissues of the aging individual. The altered pattern of gene expression exhibited by senescent cells is likely to contribute significantly to age-related pathologies. Reversal of, or a delay in the onset of, senescence should provide an effective therapy for diseases in which rephcative senescence plays a role.

There is growing evidence that the fundamental cause of cellular senescence is the progressive loss or telomeπc repeated DNA in somatic cells that lack the enzyme designated telomerase ( see Harley, 1991, ' elomere loss' Mitotic clock or genetic time bomb " Mut. Res. 25(5:271-282). There is currently no consensus as to the moleculai mechanisms that iecogmze the shortened telomeres in aged cells and cause a cell cvcle arrest at the G [/S inteirace. but this arrest may be caused by a DNA checkpoint arrest in which the senescent cell recognizes the shortened telomere as damaged DNA and causes cell cycle arrest similar to that obsened in normal cells, which arrest their growth in the presence of DNA damage. The mammalian enzyme Poly (ADP-Ribose) Polymerase ( PADPRP) has been implicated in the signaling of DNA damage. PADPRP activity is higher in isolated nuclei of S V40-transformed libroblasts than in those ot untransformed fibroblasts; ieukemic cells show higher enzyme activity than normal leukocytes; and colon cancers show higher enzyme activity than normal colon mucosa (see Miwa et ai. 1977, Arch. Biochem. Biophys. 181: 313-321 ; Burzio et cii. 1975, Proc. Soc. Exp. Biol. Med. 149:933-938: and Hirai et ai, 1983, Cancer Res. ^3:3441-3446. These observations led to the conclusion that the enzyme activity responds to DNA damage and parallels DNA repair Supporting this conclusion is the observation that the leduction ot the activity of the enzyme by certain drugs increases DNA amplification and consequent oncogenesis in cells (see Harris. 1985. Int. J Radiat Biol 48- 675-690) More recent work has tocused on the mechanism by w hich PADPRP modulates DNA lep cation and repair (see Smulson and Sugimura. eds.. "Novel ADP-nbosylations ot regulatoiy enzymes and proteins," Elsevier. N.Y ( 1980)) Such studies have identified PADPRP as an ~ 1 13 kDa piotein that uses NAD as a substrate in the loimation of poly (ADP-nbose ) chains at sites on manv nuclear proteins. The enzyme binds tightly to DNA and requnes DNA stiand breaks for activ ity (see Benjamin and Gill. 1980. J Biol C em. 255- 10502-10508) The PADPRP enzyme system appears to function in response to transient and localized DNA strand breaks in cells that may arise through a variety ot processes including DNA lepan. replication, lecombination. and gene rearrangement (see Alkhatib et ai. 1987, Pi oc Nad. Ac ad. Sci. USA 84 1224- 1228). The LDNA corresponding to the PADPRP gene has also been cloned and sequenced ( see Chernev et ai. 1987. Proc. Natl. Acad. Sci. USA 84 8370), and methods tor detecting a predisposition to cancel arising out of mutations in the PADPRP gene have been reported (see U S Patent No 5.272.057 )

Inhibitois ot PADPRP have also been developed, pπmaiily toi the purpose ot enzymatic studies ( see Banasik et ai. 1992 J Biol Client. 267 1569- 1575) and toi use m cancel and antiviral theiapies ( see PCT patent publication No 91/18591 ) PADPRP inhibitois have been reported to be effective in radiosensitizing hypoxic tumor cells (see U S. Patent Nos. 5,032.617. 5.215.738: and 5.041 ,653) These compounds can also be used to prevent tumoi cells from recov ering from potentially lethal damage ot DNA alter iadiation therapy, presumably by their abi t} to prevent DNA repair.

One weak inhibitor ot PADPRP known as kinetin (Althaus, F.R., and Richter, C. 1987. ^■'ADP-πbosylation of Proteins^'* (Springer- Verlag); see p. 26) has also been reported to delay the onset ot aging characteristics in human tibroblasts (see Rattan and Claik. 1994. Biochem. Biophys. Res. Comm. 201(2). 665-672). However, the researchers speculated that kinetin acted through receptor-mediated action on the components ot protein synthetic machinery, improving the efficiency ot various maintenance and lepair pathways such as fidelity ot protein synthesis, scavenging tiee ladicals. and removing abnormal and damaged maciomolecules Moreover, the researchers stated that the anti-aging effects of kinetin were not accompanied b> an increase in cell culture hfespan in terms ot maximum prohferative capacity m vitro. Consequently, there lemains a need for compounds that can delay the onset of senescence and extend the maximum prohferative capacity of cells in xivo and in vitro The present invention meets this and other needs.

Summary of the Invention In a fust aspect, the present invention provides a method to extend the hfespan and prohferative capacity of cells, which method comprises administering a therapeutically effective amount of a PADPRP inhibitor to said cells under conditions such that PADPRP activity is inhibited. The method is especially useful in treating disease or disease conditions induced or exacei bated by cellular senescence. In particular the method of the invention is useful in treating skin aging. Alzheimer s disease, atherosclerosis, osteoarthπtis. osteoporosis, age-related macular degeneration. Duchenne muscular dystrophy or other degenerative diseases ot skeletal muscle involving rep cative senescence, and immune senescence, including diseases, such as AIDS, that result in immune senescence In a second aspect, the present invention provides a method to alter gene expression of senescent cells, which method comprises administering a therapeutically effective amount of a PADPRP inhibitor to said cells under conditions such that PADPRP activity is inhibited. Senescent gene expression can be altered by increasing expression of young cell specific genes and/or decreasing expression of senescent cell specific genes This method is paiticulaily useful in treating diseases and conditions associated with cellular senescence and aging.

In a third aspect, the present invention provides compounds and formulations useful in the above methods. Preferred compounds for use in the present method include 3-hydroxybenzamιde, 3-acetamιdobenzamιde. 3-methoxybenzamιde. 3-methylbenzamιde. 3-fluorobenzamιde. 2- methoxybenzamide. 3-chlorobenzamιde. benzamide. 4-dmιno- 1.8-naphthahmιde. 2H- benz[c]ιsoquιnohn- l -one [6(5H)-phenanthπdιnone], 2-nιtiO-6( 5H)-phenanthπdιnone. 1.5- dihydroxyisoquinoline. 2H-benz[de]ιsoquιnohne- 1.3-dιone, ( 1.8-naphthalιmιde). 2-methyl- 4(3H)-quιnazohnone. l -hydroxyisoquinohne ( lsocarbostyπi). 2,4( l H.3H)-quιnazolιnedιone (benzoyleneurea), chlorthenoxazin. 4-hydiOxyquιnazohne, l (2H)-phthalazιnone, 2- phenylchromone (flavone), 3-amιnophthalhydrazιde (luminol). N-formyl luminol. arachidonic acid, oleic acid, hnoleic acid, and nicotinamide.

These and other aspects of the invention are described in more detail below, beginning with a brief description of the accompanying drawings.

Brief Description of the Drawings

Figure 1 shows the results of treating human tibroblast cells (W 138 cells) //z i itro with varying concentrations of 3-amιnobenzamιde ( 3-AB) as measured in the maximum achievable cumulative population doublings. The X-axis shows the number of days in culture, while the Y- axis shows the cumulative population doublings observed for each of the treated cell cultures. A significant increase in the maximum achievable cumulative population doublings was observed with cells treated with 1 miM 3-AB.

Figure 2 shows the results ot treating W 138 cells with varying concentrations of 1, 5- dihydroxyisoquinoline ( 1 , 5-DHI: also known as 1, 5-isoquιnolιnediol). The X-axis shows the number of days in culture, and the Y-axis shows the maximum achievable cumulative population doublings. At 100 μM (DMSO concentration 0.1 %), the cells displayed an extension of their prohferative lifespan. Description of the Preferred Embodiments

The present invention piovides a method to extend the hfespan and pro teiative capacity ot cells, which method comprises administering a therapeutically effectiv e amount ot a PADPRP inhibitor to said cells under conditions such that PADPRP activity is inhibited The method is especially useful in treating disease oi disease conditions induced oi exacerbated bv celiulai senescence In particular, the method of the invention is useful in tieating skin aging. Alzheimer s disease, atherosclerosis, osteoaithπtis. osteoporosis, muscular dystrophy, age-related macular degeneration, and immune senescence, including diseases, such as AIDS, that iesult in immune senescence

Compounds for use in the piesent method include any compound that specifically inhibits PADPRP. such as those disclosed in Banasik et al . 1992. 7 Biol Chen 267 1569- 1575. and U S Patent Nos 5.032.617 5 215.738. and 5.041 653 Pieteiied compounds ot the inv ention are shown in Table 1. below

Table

PADPRP ihibitoi s

Compound IC50 ( μ )

Benzamide analo^gues

3-Hydroxybenzamιde 9 1

3-Acetamidobenzamide 12

3-Methoxybenzamιde 17

3-Methylbenzamιde 19

3-Fluorobenzamιde 20

2-Methoxybenzamιde 20

3-Chlorobenzamιde n

Benzamide ^">2

3-Amιnobenzamιde 33

5-Acetamιdosaiιcylamιde 45 m-Phthalamide (isophthalamide) 50

3-Bromobenzamιde 55

2-Hydιoxybenzamιde (sahcylamide) 82

Fattv Acids

Arachidonic acid 44

L oleic acid 48

Oleic acid 82 Other Compounds.

4-amιno- 1.8-naphthalιmιde 0 18 2H-Benz[c]ιsoquιnolιn- 1 -one

[6(5H)-phenanthπdιnone] 0.30

2-Nιtro-6(5H)-phenanthπdιnone 0.35

1.5-Dιhydroxyιsoquιnohne 0.39 2H-Benz[de]ιsoquιnolιne- 1 ,3-dιone

( 1 ,8-naphthahmιde) 1 4

2-Methyl-4(3H)-quιnazohnone 5.6

Table 1

PADPRP Inhibitors (cont )

1 -Hydroxy isoquinoline ( isocaibostv iil ) 7 0

2,4( lH.3H)-Quιnazolιnedιone

(benzoyleneurea) 8.1

Chlorthenoxazin 8.5

4-Hydroxyquιnazohne 9 5 l(2H)-Phthalazιnone 12

2-Phenylchromone (flavone) 22

3-Amιnophthalhydrazιde (luminol) 23

2.3-Dιhydro- 1 ,4-phthalazιnedιone

(Phthalhydrazide ) 30 5-Iodouπdιne 43

2-Mercapto-4(3H -quιnazohnone 44

2-Methyl- 1.4-benzopyrone

(2-methylchromone) 45

5-Iodouracιl 71 3-Nitrophthalhydrazιde 72

4-Hydroxy-2-methylquιnohne 74

4-Hydroxyquιnolιne 80

Nicotinamide 210

Preferred compounds of the invention include 3-amιnobenzamιde and 1 , 5-dιhydιoxyιsoquιnolιne. To demonstrate the etfectiveness of the present method for extending the prohferative capacity and hfespan of cells, human tibroblast cells lines (either W 138 at Population Doubling (PDL) 23 or BJ cells at PDL 71 ) were thawed and plated on T75 flasks and allowed to grow in normal medium ( DMEM/M 199 plus 10% bovine calf sei urn) for about a week, at which time the cells weie confluent, and the cultuies weie theiefoie readv to be subdivided Λt the time of subdi ision, the media was aspirated, and the cells were nnsed with phosphate outfeied saline (PBS) and then trypsinized The cells weie counted w ith a Coulter counter and plated at a density ot 10^ cells per cm^ in 6-well tissue cultuie plates in DMEM/199 medium supplemented with 10% bovine calf serum and varving amounts (0. 10 μM. 100 U.M, and 1 niM, hom a 100X stock solution in DMEM/M 199 medium) ot a PADPRP inhibitoi ( 3-amιnobenzamιde purchased fiom

Sigma) This process was repeated every ~7 days until the cells appealed to stop div iding

The results are show n in Figure 1 As can be seen om the Figure, untreated (Control) cells reached senescence and stopped dividing atter about 40 days in cultuie \λ hile no effect was observ ed using 10 uM 3-AB. cells tieated with 100 μM 3-AB did appear to hav e a longei hfespan than control cells, and cells tieated with 1 miM 3-AB showed a diamatic increase in hfespan and prohferative capacity The cells tieated with 1 niM 3- AB weie still dividing atter 60 davs in culture, a lemarkable etfect as compaied with control cells In a second example ot the method. the results ot which aie show n in Figuie 2. the same procedure w as conducted using the PADPRP inhibitoi 1 5-dιhvdroxvιsoquιnohne. and the lesults again show that the tieated cells had increased prohfeiative capacity

The invention also provides a method to alter gene expression ot senescent cells, which method comprises administeimg a therapeutically ettective amount ol a PADPRP inhibitor to said cells under conditions such that PADPRP activity is inhibited PADPRP inhibitois can be used to normalize the expression ot those genes tor which expression is altered in senescence and the altered expression ot which contnbutes to age-related pathologies and so can be used to treat age i elated diseases and conditions

Senescent gene expression can be alteied by increasing the expression ot v oung cell specific genes and/or decieasing expression ot senescent cell specific genes These cell specific genes aie collectively leteied to as 'senescence lelated genes^" A "senescence-ielated gene ^' refeis to a gene that is expressed at a ditfeient lev el in a senescent cell than in a non-senescent cell ot the same cell type

Northern analysis ot the mRNA populations ot cell populations, l e . compound treated senescent cells and nontreated senescent cells, can be employed to examine the etfects ot a PADPRP inhibitor on panels ot genes that show altered expression or abundance in senescence

Various techniques known to those ot skill in the art can be used to identify senescence related genes and/or gene products, such as Enhanced Differential Display (EDD) described in U S

Patent No 5.580,726, issued Dec 3, 1996, and/or other techniques described in PCT Pub No

96/13610. published May 9, 1996, both ot which aie incorporated herein by leterence Alternatively, the effects ot a PADPRP inmbitor on gene expression in senescent cells can be monitored by lmmunohistochemistiy and these assays can be used to select optimal compounds among a tamily ot PADPRT inhibitois

Thus, the methods ot the inv ention can be used to increase the hfespan ot cells in vin o and alter gene expression ot senescent cells While this aspect ot the invention is important and of value for a wide variety ot applications in cell culture methodology, a perhaps more important application of the present method involves the treatment ot human and other disease. Because cell senescence is implicated in a wide variety of diseases, and may be proven to have a role in essentially all diseases affecting the aged, the present invention offers remarkable promise in the treatment of disease.

In general, a suitable effective dose of a compound of the invention will be in the range ot 0 001 to 100 milligram (mg) per kilogram ( kg) of body weight of recipient per day. preferably in the range of 0.1 to 10 mg per kg of body weight per day. The desired dosage is preferably presented in one. two, three, four, or more subdoses administered at appropriate intervals throughout the day. These subdoses can be administered as unit dosage forms, for example, containing 5 to 10,000 mg. preferably 10 to 1000 mg of active ingredient per unit dosage form. The composition used in these therapies can be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspensions, hposomes. and injectable and infusible solutions. The preferred form depends on the intended mode ot administration and therapeutic application. The compositions also preferably include conv entional pharmaceutically acceptable carriers and adjuv ants, as is well known to those of skill in the art. See. e <.,'., Remington 's Pharmaceutical Sciences. Mack Publishing Co.: Easton. PA. 17th Ed. ( 1985) Generallv . administration will be by oral or parenteral (including subcutaneous, intramuscular, intravenous, and intradermal ) routes. The therapeutic methods and agents of this invention can ot course be used concomitantly or in combination with other methods and agents for treating a particular disease or disease condition.

While it is possible to administer the active ingredient of this invention alone, it is preferable to present it as part of a pharmaceutical formulation. The formulations of the present invention comprise at PADPRP inhibitor of this invention in a therapeutically or pharmaceutically effective dose together with one or more pharmaceutically oi therapeutically acceptable carriers and optionally other therapeutic ingredients. Various considei ations are described, . u . in Gilman et al. (eds. ) ( 1990) Goodman and Gilman 77/ e Pharmacological Bases of Therapeutics. 8th Ed.. Pergamon Press: and Remington's supra, each of which is incorporated herein bv reference. Methods for administration are discussed therein, e.g., for oral, intravenous, lntrapeπtoneal, or intramuscular administration, and others.

The pharmaceutical compositions will be administered by topical, parenteral. or oral administration for prophylactic and/or therapeutic treatment. The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable for oral administration include powder, tablets, pills, and capsules.

Topical administration typically involves the delivery of a PADPRP inhibitor tor percutaneous passage of the drug into the systemic circulation of the patient. The skin sites include anatomic regions for transdermallv administering the drug as represented by the forearm, abdomen, chest, back, buttock, mastoidal area and the like The PADPRP inhibitor is administered to the skin by placing on the skin a topical formulation comprising the PADPRP inhibitor or a transdermal drug delivery device that administers the diug. and which bandage is designed, shaped, sized, and adapted for easy placement and comfortable retention on the skin. A variety of transdermal drug delivery devices can be employed w ith the compounds described herein. For example, a simple adhesive patch comprising a backing material and an acrylate adhesive can be prepared The drug and any penetration enhancer can be formulated into the adhesive casting solution The adhesive casting solution can be cast directlv onto the backing material or can be applied to the skin to torm an adherent coating. See. e.g.. U S. Patent Nos. 4.310.509. 4.560.555. and 4.542.012. In other embodiments, the PADPRP inhibitor ill be dehv eied using a liquid reservoir system ding delivery device These systems typically comprise a backing material, a membrane, an acrylate based adhesive, and a ielea.se liner. The membrane is sealed to the backing to form a reservoir. The drug and any vehicles, enhancers, stabilizers, gelling agents, and the like are then incorporated into the reservoir See. e g . U S. Patent Nos. 4.597.961. 4.485.097. 4.608.249. 4.505.89 1. 3.843.480. 3.948.254. 3.948.262. 3.053.255. and 3.993.073

Matrix patches comprising a backing, a drug/penetration enhancer matrix, a membrane, and an adhesive can also be employed to dehvei PADPRP inhibitors transdermally The matrix material typically will comprise a poiyurethane toam The drug, any enhancers, v ehicles, stabilizers, and the like aie combined with the toam precursors. The foam is allowed to cure to produce a tacky, elastomenc matrix which can be directly affixed to the backing material. See, e.g., U.S. Patent Nos. 4.542,013, 4.460.562, 4,466.953, 4,482.534, and 4,533,540.

Also included within the invention are preparations for topical application to the skin comprising a PADPRP inhibitor, typically in concentrations in the range of from about 0 001 % to 10%. together with a non-toxic, pharmaceutically acceptable topical carrier. These topical preparations can be prepared by combining an active ingredient according to this invention with conventional pharmaceutical diluents and carriers commonly used in topical dry. liquid, and cream formulations. Ointment and creams may. tor example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Such bases may include water and/or an oil such as liquid paraffin or a vegetable oil such as peanut oil or castor oil. Thickening agents which may be used according to the nature of the base include soft paraffin, aluminum stearate, cetostearyl alcohol, propylene glycol, polyethylene glycols. woolfat. hydrogenated lanolin, beeswax, and the like.

Lotions may be formulated with an aqueous or oily base and will, in general, also include one or more of the following: stabilizing agents, emulsifying agents, dispersing agents, suspending agents, thickening agents, coloring agents, perfumes, and the like. A preferred composition of the invention is a lotion containing a PADPRP inhibitor, such as arachidonic acid, noleic acid, oleic acid, and/or nicotinamide. that is applied topically to treat skin aging. A more potent version ot the lotion would include a PADPRP inhibitor such as 1. 5-DHI. Powders may be formed with the aid ot any suitable powder base, e g.. talc, lactose, starch, and the like Drops may be formulated with an aqueous base oi non-aqueous base also comprising one or more dispersing agents, suspending agents, solubihzing agents, and the like.

The topical pharmaceutical compositions according to this invention mav also include one or more preservatives or bacteπostatic agents, e.g., methyl hydroxybenzoate. piopyl hydroxybenzoate. chlorocresol. benzalkonium chlorides, and the like. The topical pharmaceutical compositions also can contain other active ingredients such as antimicrobial agents, particularly antibiotics, anesthetics, analgesics, and antipruntic agents.

The compounds of the present invention can also be delivered through mucosal membranes. Transmucosal ( i.e.. subhngual. buccal. and vaginal) drug delivery provides for an efficient entry of active substances to systemic circulation and reduces immediate metabolism by the liver and intestinal wall flora. Tiansmucosal drug dosage forms (e.g.. tablet, suppository, ointment, pessary, membrane, and powder) aie typically held in contact with the mucosal membrane and disintegrate and/or dissolve rapidly to allow immediate systemic absorption.

For delivery to the buccal or subhngual membranes, typically an oral formulation, such as a lozenge, tablet, oi capsule w ill be used. The method of manutactuie ot these rormulations is known in the art. including but not limited to. the addition ot the pharmacological agent to a pre- manufactured tablet: cold compression ot an inert lillei, a binder, and either a pharmacological agent or a substance containing the agent ( as described in U.S. Patent No 4,806.356); and encapsulation. Another oral formulation is one that can be applied with an adhesive, such as the cellulose derivative, hydroxvpropyl cellulose, to the oral mucosa. for example, as described in

U.S. Pat. No. 4,940.587. This buccal adhesive formulation, when applied to the buccal mucosa, allows tor controlled release ot the pharmacological agent into the mouth and thiough the buccal mucosa.

Parenteral administration is generally characterized by injection, either subcutaneously, intramuscularly or intravenously Thus, this invention provides compositions tor intravenous administration which comprise a solution ot the PADPRP inhibitoi dissolved oi suspended in an acceptable carrier. Injectables can be piepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable foi solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are. for example, water, buffered water, saline, dextrose, glycerol, ethanol or the like. These compositions will sometimes be sterilized by conventional, well known, sterilization techniques, or can be sterile filtered. The resulting solutions can be packaged for use as is. or lyophilized. the lyophilized preparation being combined with a sterile solution prior to administration. In addition, if desired, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents. pH buffering agents and the like, such as tor example, sodium acetate, sorbitan monolaurate. tπethanolamine oleate. etc.

Another approach for parenteral administration employs the implantation of a slow-release or sustained-release system, such that a constant level of dosage is maintained. See, e.g.. U.S. Patent No 3.710.795. which is incorporated herein by reference. Liquid pharmaceutically administeiable compositions can. toi example, be prepared bv dissolving, dispersing, etc.. an active compound as defined above and optional pharmaceutical adjuvants in a excipient, such as. foi example, water, saline, aqueous dextiose. glyceroi, ethanol. olive oil and other hpophilic solvents, and the like, to thereby toim a solution oi suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting oi emulsifying agents. pH buffeting agents and the like, for example, sodium acetate, soibitan monolaurate. tπethanolamine sodium acetate, tπethanolamine oleate. etc. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this ait: for example, see Remington 's, supra The composition or formulation to be administered will, in any event, contain an effective amount of the active compound.

For solid compositions, conventional nontoxic solid earners can be used, w hich include, for example, pharmaceutical grades ot mannitol. lactose, starch, magnesium stearate. sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium caibonate. and the like. For oial administration, a pharmaceuticailv acceptable nontoxic composition is toimed bv incoiporating anv ot the normally employed excipients. such as those carπeis previously listed, and generally 0 1 - 95% ot active ingredient, preferably about 20%

The compositions containing the compounds can be administeied for prophylactic and/or therapeutic treatments. In therapeutic applications, compositions are administered to a patient already suffering from a disease, as described above, in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. An amount adequate to accomplish this is defined as a "therapeutically effective amount or dose." Amounts effective tor this use will depend on the severity of the disease and the weight and general state of the patient Once improvement of the patient s conditions has occurred, a maintenance dose is administered if necessary Subsequently, the dosage oi the fiequency ot administration, oi both, can be reduced, as a function of the symptoms, to a level at which the improved condition is retained. When the symptoms have been alleviated to the desued level, treatment can cease. Patients can, however, require intermittent treatment on a long-term basis upon any recurrence of the disease symptoms. In prophylactic applications, compositions containing the compounds of the invention aie administered to a patient susceptible to or otheiwise at risk of a particular disease Such an amount is defined to be a "prophylactically effective amount or dose " In this use, the precise amounts again depend on the patient s state of health and weight.

With regard to the prophylactic applications of the present invention, the present invention provides useful methods to delay the progression of AIDS While not wishing to be bound by theory, the present inventor believes that immune cell senescence may induce HIV expression, which is known to be increased upon DNA damage (see. e g , Valerie et cil . 1988. Nature 333:7 - 81. and Stein et ai. 1989. Mol. Cell Biol 9 5169-5181 ) One can theielore administer a PADPRP inhibitor according to the method ot the in ention to those infected w uh HIV to delay the onset ot AIDS

One can also employ a novel screening method of the invention to find more effective inhibitors of PADPRP and to find any compound that delays the onset ot senescence and/or delays expression of HIV in HIV-intected cells. In this method, one fust constructs a lecombinant v ector that comprises the HIV long teiminal repeat (LTR) piomotei positioned to di n e expression ot a reporter gene product (a reportei gene is a gene that produces a gene product that can be leadily assayed, i.e., the beta-galactosidase gene, the alkaline phosphatase gene, etc ) This vector is used to transform suitable cell lines, such as eukaiyotic tibroblast oi lymphocyte cell lines, w hich when grown to senescence should show induction ot expiession ot the reporter gene In the screen, one assays whether test compounds delay, inhibit, oi prevent the induction ot expiession of the leportei gene caused by senescence Compounds that could delay senescence ould delay, inhibit, or prevent the induction of expiession ot the reportei gene and would be useful for purposes of the piesent invention not only geneiallv to prevent cell senescence but also specifically to treat AIDS.

The foregoing text describes vanous aspects ot the invention and how the invention can be practiced. The description ot the invention is not intended to piovide an exhaustive descnption ot the many different embodiments of the invention All publications and patent applications cited above aie hereby incorpoiated heiein by reteience as if each individual publication or patent application were specifically and individually indicated to be incorporated bv leterence.

Examples

The following examples demonstrate how PADPRP inhibitois can be used to alter senescence-related gene expression and also provide methods tor identifying compounds that alter senescence-related gene expiession. The examples should not be construed as limiting the invention, as the examples meiely piovide specific alternative methods useful in understanding and practicing the invention.

Example 1 Measuring Altered Gene Expression in (mRNA) Senescent Cells

Human fibroblast BJ cells, at Population Doubling (PDL) 94. were plated in regular growth medium and then changed to low serum medium to reflect more physiological conditions as described by Linskens. et ai, 1995, Nucleic Acids Res. 23, No. 16:3244-3251 ( 1995). The medium was DMEM/199 supplemented with 0 5% bovine calf serum. The cells were treated daily tor 13 days with the PADPRP inhibitoi 1 ,5-dιhydιoxyιsoquιnohne ( 100 μM) The control cells were treated with and without DMSO (dimethylsuifoxide), the solvent used in the drag treated cells to administer the PADPRP inhibitoi. In addition, old and young control cells w hich were not treated with DMSO weie tested for comparison. RNA was prepared from the tieated and control cells according to the techniques descnbed in PCT Publication No 96/13610. supra, and Northern blotting was conducted. Probes specific for senescence-related genes wei e analyzed, and treated and control cells were compaied. The results are shown in Table 1. in which gene expression is normalized in each row. with one control (of the three ) showing the lowest level of gene expression arbitrarily set at 1 to provide basis for comparing expression (in this test) levels of a specific gene in one cell with the levels of that same gene in another cell. Three genes that are particularly relevant to age-related changes in skin are collagen la l . collagenase. and elastin (West. 1994. Arch. Derm. 130:87-95 ) As can be seen from Table 1 , elastin expression of the treated cells was significantly increased in the 1.5- dihydroxyisoqumohne treated senescent cells relative to the control cells. Elastin expression is significantly higher in young cells ( 16 0 in young BJ cells. PDL 38. without DMSO) compared to senescent cells ( 1 4 in senescent BJ cells. PDL 93. without DMSO). Thus, treatment with 1.5- dihydroxyisoquinolme caused elastin expression levels in senescent cells to change to levels similar to those tound in much younger cells. Similarly, a beneficial effect is seen in collagenase and collagen la l expression with 1.5- dihydroxyisoqumohne an effect that is improved using the DMSO v ehicle

Table 1. Changes in Gene Expiession upon Tieatment ot Senescent Cells with 1.5- Dihydroxyisoquinoline .

Young Old Old Old

BJ 38 BJ 93 BJ 94 BJ 94

Probe No DMSO No DMSO DMSO 1 ,5-dιhydroxy

0.5% Serum Control Control Control ^a isoquinohne

Collagen lal 3.5 1 0 1 7 2 0

ALDH 1 3.2 1 .0 1 0 1 6

LAMΓNIN A 3.4 1 .4 1 .0 1 .2

ELASTIN 16 1 4 1 .0 6.0

EPC- 1 1 .0 1 .7 1 .2 0.7

SOKL 2.0 1.0 1.3 1.3

COLLAGENASE 1 .2 19.2 1 .0 0.4

PAI-1 1.0 3.3 5.5 6.6

UPA 1.0 10.0 3.3 3.5

PAI- 1 1 1.0 4. 1 1.7 3.0 a. normalized values Example 2

Measuring Altered Gene Expression ( Protein) in Senescent Cells

Approximately. 10 BJ cells at PDL 95-100 were plated and grown in 15 cm dishes. The growth medium was DMEM/199 supplemented with 10% bovine calf serum. The cells were treated daily for 24 hours with the PADPRP inhibitor ( 100 μg / 1 mL of medium) 1.5- dihydroxyisoquinoline. The cells were washed with phosphate buffered solution ( PBS), then permeablized with 4% paraformaldehyde for 5 minutes, then washed with PBS. and treated with 100% cold methanol for 10 minutes. The methanol was removed, and the cells were washed with PBS. and then treated with 10% serum to block nonspecific antibody binding. About 1 mL of the appropriate commercially available antibody solutions ( 1 :500 dilution. Vector) was added to the cells and the mixture incubated for 1 hour. The cells were rinsed and washed three times with PBS. A secondary antibody, goat anti-mouse IgG ( 1 mL), with a biotin tag was added along with 1 mL of a solution containing streptavidin conjugated to alkaline phosphatase and 1 mL of NBT reagent ( Vector). The cells were then washed and changes in gene expression were noted colorimetπcally.

In one embodiment, four senescence-specific genes (collagen I. collagen III. collagenase. and interferon gamma) in senescent cells treated with 1,5-dihydroxyisoquinoline were monitored, and the results showed a decrease in interferon gamma expression with no observable change in the expression levels of the other three genes, demonstrating that PADPRP inhibitors can alter senescence-specific gene expression.

The reagents employed in the examples are commercially available or can be prepared using commercially available instrumentation, methods, or reagents known in the art. The foregoing examples illustrate how PADPRP inhibitors can alter senescent gene expression and provide methods to determine additional compound that alter senescent gene expression. The examples are not intended to provide an exhaustive description of the many different embodiments of the invention. Thus, although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, those of ordinary skill in the art will realize readily that many changes and modification can be made thereto without departing from the spirit or scope of the appended claims.

Claims

We Claim:

1 A method to extend the hfespan and proliferate e capacity of cells, w hich method comprises administering a therapeutically effective amount ot a PADPRP inhibitor to said cells under conditions such that PADPRP activity is inhibited.

2. The method of Claim 1 , wherein said cells are in in vitro culture.

3. The method ot Claim 1. wherein said cells are m

o. and said method is used to treat a disease or disease conditions induced or exacerbated by cellular senescence.

4. The method ot Claim 3. wherein said disease is a disease selected from the group consisting of skin aging, Alzheimer s disease, atheiosclerosis. osteoarthntis. osteoporosis. muscular dystrophy, age-related macular degeneration, immune senescence, and AIDS

5. The method of Claim 1. wherein said inhibitor is selected from the group of inhibitors consisting of 3-hv droxybenzamιde. 3-acetamιdobenzamιde. 3-methoxybenzamιde. 3- methylbenzamide. 3-fluorobenzamιde. 2-methoxybenzamιde. 3-ehlorobenzamιde. benzamide. 4- arnιno- 1.8-naphthalιmιde. 2H-bcnz[c]ιsoquιnolιn- l -one [6( 5H )-phenanthπdιnone]. 2-nιtro-6( 5H)- phenanthπdinone. l .S-dihydroxyisoquinoline, 2H-benz[dc]ιsoquιnolιne- 1.3-dιone. ( 1.8- naphthahmide), 2-methyl-4( 3H)-quιnazohnone, 1 -hydroxyιsoquιnohne ( lsocarbostynl), 2,4( 1 H,3H)-quιnazohnedιone (benzoyleneurea). chlorthenoxazin. 4-hydroxyquιnazolme, 1 (2H)- phthalazinone. 2-phenylchromone ( fiavone), and 3-amιnophthalhydrazιde (luminol )

6. The method of Claim 1 , wherein said inhibitor is 3-hydroxybenzamιde.

7. The method of Claim 1. wherein said inhibitor is 3-amιnobenzamιde.

8. A pharmaceutical formulation comprising a PADPRP inhibitor in a therapeutically or pharmaceutically effectiv e dose together with one or more pharmaceutically oi therapeutically acceptable carriers.

9 The pharmaceutical formulation oi Claim 8. herein said inhibitoi is selected from the group of inhibitors consisting ot 3-hydroxybenzamιde. 3-acetamιdobenzamιde. 3- methoxybenzamtde. 3-methylbenzamιde. 3-fluorobenzamιde. 2-methoxybenzamιde. 3- chlorobenzamide. benzamide. 4-amιno- 1.8-naphthahmιde. 2H-benz[c]ιsoquιnolιn- l -one [6(5H)- phenanthπdinone], 2-nιtro-6(5H)-phenanthridinone. 1.5-dιhydroxyιsoquιnolιne. 2H- benz[de]isoquιnolιne- 1.3-dione. ( 1.8-naphthahmιde ). 2-methyI-4(3H)-quιnazohnone. 1- hydroxyisoquinolme (lsocarbostynl), 2,4( l H,3H)-quιnazolιnedιone ( benzoyleneurea). chlorthenoxazin. 4-hydroxyquιnazohne. l (2H)-phthaiazιnone. 2-phenylchromone ( fiavone), nicotinamide. and 3-amιnophthalhydrazιde (luminol).

10. A method to alter gene expression of senescent cells, which method comprises administering a therapeutically effective amount of a PADPRP inhibitor to said cells under conditions such that PADPRP activity is inhibited.