WO2009055001A2 - Methods of treating aging and methods of screening candidate agents therefor - Google Patents

Abstract

A method of treating aging in a mammalian (e.g., human) subject comprises administering the subject a serotonin 2 (5-HT2) receptor antagonist in an amount effective to treat the aging (e.g., by prolonging or extending lifespan). Assays for identifying active compounds for such methods are also described, along with methods of treating subjects to receive chemotherapy or radiation to combat stress associated therewith.

Description

METHODS OF TREATING AGING AND METHODS OF SCREENING CANDIDATE AGENTS THEREFOR

Michael Petrascheck and Linda B. Buck

Government Support

This invention was made with Government support under a grant from the National Institutes of Health. The US Government has certain rights to this invention.

Field of the Invention

The present invention concerns methods of treating or retarding aging, and methods useful as screens for identifying candidate active agents useful for treating or retarding aging.

Background of the Invention

Numerous treatments that at least partially alleviate the symptoms of aging, or retard the progression of aging, have been reported. US Patent Nos. 7,256,184 and 6,821,997 to Rodriguez describe a method for the treatment and prophylaxis of conditions of aging associated with a decreased presence of cell-specific carbonic anhydrase enzymes in the brain, such as conditions associated with chronic neurodegenerative conditions including dementias such as Alzheimer's disease. The method is carried out by identifying which one or more cell-specific carbonic anhydrase enzymes are present at decreased levels in the blood or brain cells of a subject and then administering a compound that increases the presence of the cell-specific carbonic anydrase enzymes in the blood or brain cells of the subject.

US Patent No. Re39,436 to S. Spindler et al. describes a method of identifying an intervention that mimics the effects of caloric restriction in cells by obtaining a biological sample, exposing the biological sample to an intervention, waiting a specified period of time, assessing changes in gene expression levels, levels of RNA, protein, or protein activity levels related to one or more biomarkers of aging, and identifying the intervention as one that mimics the effects of caloric restriction if one or more changes in the levels also occurs in a reference animal subjected to short term caloric restriction.

US Patent No. 5,602,139 to Rattan describes a method for ameliorating one or more of the adverse effects of aging on mammalian cells in vivo by administering a composition that contains an effective concentration of one or more 6-(substituted amino)purine cytokinins

US Patent No. 4,695,590 to Lippman describes a method for retarding aging in humans by administering to a human a daily dosage of nordihydroguairetic acid, bis(p-hydroxyphenyl)methane, or certain other related compounds. US Patent Application Publication No. 2008/0234310 to Bachurin et al.

(published Sept. 28, 2008) describes dimebon and analogs thereof as geroprotectors which can be used for slowing aging, prolonging lifespan of an individual or cells in an individual, and/or improving quality of life of an individual developing or having a risk of developing age-associated manifestations and/or pathologies. US Patent Application Publication No. 2008/0199865 to Crossman et al

(published August 21 , 2008) describes the treatment aging and aging related conditions with certain IL-I modulators.

US Patent Application Publication No. 2008/0167248 to Witten et al. (published July 10, 2008) with substance P.

Summary of the Invention

A first aspect of the invention is a method of treating aging in a mammalian

(e.g., human) subject in need thereof, comprising administering the subject a serotonin 2 (5-HT₂) receptor antagonist (sometimes referred to as an "active agent" herein) in an amount effective to treat the aging (e.g., by prolonging or extending lifespan).

A second aspect of the invention is, in a method of treating a subject afflicted with cancer with chemotherapy, radiation therapy, or a combination thereof, the improvement comprising concurrently administering said subject a serotonin 2 (5- HT₂) receptor antagonist in an amount effective to treat at least one chemotherapy or radiation therapy side-effect in said subject. A further aspect of the invention is a method of treating a patient in need of radiation therapy or chemotherapy, comprising administering to the patient a protective amount of a serotonin 2 (5-HT₂) receptor antagonist).

In some embodiments, the 5-HT₂ receptor antagonist is SER-4 serotonin receptor antagonist (e.g., in Caenorhabditis elegans (C. elegans)).

In some embodiments, the 5-HT₂ receptor antagonist is a SER-3 octopamine receptor antagonist {e.g., in C. elegans).

In some embodiments, the 5-HT₂ receptor antagonist is selected from the group consisting of Mianserin, Mirtazapine, Methiothepin, Cyproheptadine, 272Nl 8, and pharmaceutically acceptable salts or prodrugs thereof.

A further aspect of the invention is the use of an active agent as described herein for the preparation of a medicament for carrying out a method as described herein.

A further aspect of the invention is a method of screening a candidate agent for activity in treating aging in a mammalian subject, comprising: administering an active agent to a plurality of nematodes {e.g., Caenorhabditis elegans), and then detecting the fraction of the nematodes alive at a plurality of predetermined times after the administering; and then determining the increase in lifespan for the nematode from the detecting at a plurality of predetermined times; with an extension in lifespan of at least 10 percent as compared to corresponding untreated animals indicating the active agent is useful in treating aging in mammalian subjects. In some embodiments, the nematode is grown in liquid medium. In some embodiments, the administering step is carried out on day 1 of adult life. In some embodiments, detecting the fraction of nematodes alive is carried out by detecting body movement of the nematodes. In some embodiments the method is a high-throughput screening method carried out concurrently in multiple wells {e.g., a multiwell plate). In some embodiments, the wells are concurrently seeded with the nematodes as Ll larvae (prior to administration). In some embodiments the wells are seeded with from 5 to 20 nematodes. In some embodiments, the nematodes are administered 5-Fluoro-2'- deoxyuridine in an amount effective to prevent self-fertilization at 42-45 hours after seeding. The present invention is explained in greater detail in the drawings herein and the specification set forth below. The disclosure of all US Patent references cited herein are to be incorporated by reference herein in their entirety.

Brief Description of the Drawings

Figure 1. Increases in C. elegans lifespan by human serotonin receptor antagonists require serotonin and octopamine signaling.

Figure Ia. Structures of one chemical (272Nl 8) and four serotonin receptor antagonists that increased C. elegans lifespan. Figure Ib. Survival curves representing a typical experiment show the fraction of wildtype (N2) or mutant animals alive at different ages when given 50 μM Mianserin (red), 10 μM Methiothepin (blue), or no drug (black). Genes required for lifespan extension by the drugs are highlighted in black. Percent changes in lifespan versus untreated controls are indicated for each drug. Asterisks indicate significant increases (***, P<0.0001, ** P<0.001, no asterisk, P>0.01, sample sizes > 50).

Figure Ic. Mean lifespan as a function of increasing Mianserin concentrations. Maximum increase in lifespan of N2 animals (dotted red line) is seen at 50 μM Mianserin. The lifespan of ser-3, ser-4, and tph-1 mutants (solid red lines) is largely unaffected. Strains tested in parallel. Error bars indicate S. E. M. Figure 2. Mianserin and Methiothepin are antagonists of SER-3 octopamine and SER-4 serotonin receptors. HEK293 cells expressing SER-3 or SER-4 were monitored by calcium imaging for responses to potential ligands and drugs. In a-c, time is shown on the abscissa and change in emitted fluorescence (ΔF /F) on the ordinate. Responses are shown for single cells in b and c and for groups of cells in a, d, and e.

Figure 2a. SER-3+ cells showed calcium increases in response to 10 nM octopamine and 10 μM tyramine, but not to serotonin. EC50_octop_ami_ne ⁼ 24 nM ;

EC50,_yramine = 26 μM.

Figure 2b. Mianserin and Methiothepin inhibited the response of SER-3+ cells to 10 nM but not 100 nM octopamine, an effect that was reversible for Mianserin. Figure 2c. SER-4+ cells responded to 0.1 and 0.3 μM serotonin (EC50 -0.1 μM). Mianserin and Methiothepin inhibited these responses, an effect that was reversible for Mianserin.

Figure 2d-3. Bar graphs show the extent to which Mianserin and Methiothepin antagonized responses of SER-3+ and SER-4+ to 10 nM octopamine or 300 nM serotonin. Error bars indicate S. E. M.

Figure 3. Lifespan extension by Mianserin and dietary restriction are related. Mianserin was tested for the ability to extend the lifespan of various aging mutants or animals subjected to dietary restriction (DR). Figure 3a. Left: Pre-exposure to Mianserin reduces serotonin-induced egg laying in strains in which it fails to increase lifespan. Serotonin treated (black bars), pre-exposure to Mianserin + serotonin treated (red bars) (4-6 experiments/condition, ** P<0.001, one-way ANOVA). Right: Pharyngeal pumps/min at different times (days) after receiving Mianserin (red), Methiothepin (blue), or control solvent (black), or in control eat-2 mutants deficient in pumping (light blue).

Figure 3b. Mianserin has different effects when given to animals as both larvae and adults rather than as adults only. Left: Exposure to increasing concentrations of Mianserin starting from the Ll stage leads to progressive larval arrest. Middle: Animals that reach adulthood when exposed to Mianserin as larvae (solid red line, N2/L1) show little increase in lifespan in response to Mianserin as adults compared to animals exposed to the drug only as adults (dotted red line, N2/dl). Mean lifespan in days is shown as a function of increasing Mianserin concentration. Lifespan only determined for animals that reached adulthood. Right: Survival curve for animals exposed to 50 μM Mianserin starting from the larval Ll stage. All controls were assayed in parallel.

Figure 3c. Survival curves representing a typical experiment show the fraction of animals alive at different adult ages (in days) when animals were exposed to Mianserin (red), Methiothepin (blue), or control solvent (black). Percent change and P values are indicated as in Figure 1. (sample sizes > 50). d. Mean lifespan in days for Mianserin-treated and untreated control animals

(cumulative). Mean lifespan and S. E. M. were calculated by averaging the lifespans of identically treated wells (10-15 animals/ well). The number of wells assayed for each condition is indicated underneath each bar.

Detailed Description of Preferred Embodiments The present invention is primarily concerned with the treatment of human subjects, but the invention may also be carried out on animal subjects, particularly mammalian subjects such as dogs, cats, livestock and horses for veterinary purposes. Subjects may be male or female and of any suitable age, the subjects are in some embodiments are adult or geriatric subjects (e.g., human subjects 30, 40, 50, or 60 years old, or more).

"Treat" as used herein refers to any type of treatment that imparts a benefit to an aging patient, particularly delaying or retarding the progression of aging (e.g., by delaying or retarding the onset or progression of one or more age or aging-related condition), or extending or increasing lifespan (e.g., by delaying or retarding the onset or progression of one or more age or aging-related, and typically life-shortening, condition).

"Pharmaceutically acceptable" as used herein means that the compound or composition is suitable for administration to a subject to achieve the treatments described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.

"Concurrently" as used herein means sufficiently close in time to produce a combined effect (that is, concurrently may be simultaneously, or it may be two or more events occurring within a short time period before or after each other).

"Pharmaceutically acceptable prodrugs" as used herein refers to those prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, commensurate with a reasonable risk/benefit ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the invention. The term "prodrug" refers to compounds that are rapidly transformed in vivo to yield the parent compound of the above formulae, for example, by hydrolysis in blood. A thorough discussion is provided in T. Higuchi and V. Stella, Prodrugs as Novel delivery Systems, Vol. 14 of the A. C. S. Symposium Series and in Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated by reference herein. See also US Patent No. 6,680,299. Examples include a prodrug that is metabolized in vivo by a subject to an active drug having an activity of active compounds as described herein, wherein the prodrug is an ester of an alcohol or carboxylic acid group, if such a group is present in the compound; an acetal or ketal of an alcohol group, if such a group is present in the compound; an N-Mannich base or an imine of an amine group, if such a group is present in the compound; or a Schiff base, oxime, acetal, enol ester, oxazolidine, or thiazolidine of a carbonyl group, if such a group is present in the compound, such as described in US Patent No. 6,680,324 and US Patent No. 6,680,322.

1. Active compounds.

Active compounds (also referred to as "active agents" herein) of the present invention are, in general, serotonin-2 (5-HT₂) receptor antagonists. Examples of such compounds include but are not limited to those described in US Patent Application Publication Nos. 2004/0192754 and 2002/0035057. Examples include, but are not limited to, Mianserin, Mirtazapine, Methiothepin, Cyproheptadine, 272Nl 8, and pharmaceutically acceptable salts and prodrugs thereof. Such compounds are again known and described in, for example, US Patent Nos. 7,241,797; 7,038,085; 6,946,141 ; 6,727,242; 6,495,154; and 5,922,341.

Additional examples of serotonin-2 receptor antagonist active agents include, but are not limited to: Amperozide, PAPP (l-[2-(4-Aminophenyl)ethyl]-4-(3- trifluoromethylphenyl)piperazine), dihydroergotaminee, cyclobenzaprin, LY-367,265 (l -(2-(4-(6-fluoro-lH-indol-3-yl)-3,6-dihydro-l(2H)-pyridinyl)ethyl)-5_s6-dihydro IH, 4H-(l,2,5)thiadiazolo(4.3.2-ij)quinoline-2,2-dioxide)), metergoline, keanserin, SB224829, BRLl 5572, 4-Methyl-2,5-dimethoxyamphetamine, Alosetron, Aripiprazole, Azatadine, Cabergolin, Chloroprocaine, Cyclobenzaprine, Dimethyltryptamine, Dolasetron, Frovatriptan, Granisetron, Methotrimeprazine, Methysergide, Nefazodone, Olanzapine, Ondansetron, Paliperidone, Palonosetron, Promazine, Propiomazine, Quetiapine, Risperidone, Sertindole, Thiethylperazine and pharmaceutically acceptable salts and prodrugs thereof.

The active compounds disclosed herein can, as noted above, be prepared in the form of their pharmaceutically acceptable salts. Pharmaceutically acceptable salts are salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects. Examples of such salts are (a) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; (b) salts formed from elemental anions such as chlorine, bromine, and iodine, and (c) salts derived from bases, such as ammonium salts, alkali metal salts such as those of sodium and potassium, alkaline earth metal salts such as those of calcium and magnesium, and salts with organic bases such as dicyclohexylamine and N-methyl-D-glucamine.

2. Pharmaceutical formulations.

The active compounds described above may be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (9^th Ed. 1995). In the manufacture of a pharmaceutical formulation according to the invention, the active compound (including the physiologically acceptable salts thereof) is typically admixed with, inter alia, an acceptable carrier. The carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the patient. The carrier may be a solid or a liquid, or both, and is preferably formulated with the compound as a unit-dose formulation, for example, a tablet, which may contain from 0.01 or 0.5% to 95% or 99% by weight of the active compound. One or more active compounds may be incorporated in the formulations of the invention, which may be prepared by any of the well known techniques of pharmacy comprising admixing the components, optionally including one or more accessory ingredients.

The formulations of the invention include those suitable for oral, rectal, topical, buccal (e.g., sub-lingual), vaginal, parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous), topical {i.e., both skin and mucosal surfaces, including airway surfaces) and transdermal administration, although the most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular active compound which is being used. Formulations suitable for oral administration may be presented in discrete units, such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. Such formulations may be prepared by any suitable method of pharmacy which includes the step of bringing into association the active compound and a suitable carrier (which may contain one or more accessory ingredients as noted above). In general, the formulations of the invention are prepared by uniformly and intimately admixing the active compound with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture. For example, a tablet may be prepared by compressing or molding a powder or granules containing the active compound, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, the compound in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface active/dispersing agent(s). Molded tablets may be made by molding, in a suitable machine, the powdered compound moistened with an inert liquid binder.

Formulations suitable for buccal (sub-lingual) administration include lozenges comprising the active compound in a flavoured base, usually sucrose and acacia or tragacanth; and pastilles comprising the compound in an inert base such as gelatin and glycerin or sucrose and acacia.

Formulations of the present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injection solutions of the active compound(s), which preparations are preferably isotonic with the blood of the intended recipient. These preparations may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions may include suspending agents and thickening agents. The formulations may be presented in unit\dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water- for-injection immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. For example, in one aspect of the present invention, there is provided an injectable, stable, sterile composition comprising an activ e compound(s), or a salt thereof, in a unit dosage form in a sealed container. The compound or salt is provided in the form of a lyophilizate which is capable of being reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection thereof into a subject. The unit dosage form typically comprises from about 10 mg to about 10 grams of the compound or salt. When the compound or salt is substantially water- insoluble, a sufficient amount of emulsifying agent which is physiologically acceptable may be employed in sufficient quantity to emulsify the compound or salt in an aqueous carrier. One such useful emulsifying agent is phosphatidyl choline. Formulations suitable for rectal administration are preferably presented as unit dose suppositories. These may be prepared by admixing the active compound with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.

Formulations suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which may be used include petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.

Formulations suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Formulations suitable for transdermal administration may also be delivered by iontophoresis (see, for example, Pharmaceutical Research 3 (6):318 (1986)) and typically take the form of an optionally buffered aqueous solution of the active compound. Suitable formulations comprise citrate or bis\tris buffer (pH 6) or ethanol/water and contain from 0.1 to 0.2M active ingredient.

Further, the present invention provides liposomal formulations of the compounds disclosed herein and salts thereof. The technology for forming liposomal suspensions is well known in the art. When the compound or salt thereof is an aqueous-soluble salt, using conventional liposome technology, the same may be incorporated into lipid vesicles. In such an instance, due to the water solubility of the compound or salt, the compound or salt will be substantially entrained within the hydrophilic center or core of the liposomes. The lipid layer employed may be of any conventional composition and may either contain cholesterol or may be cholesterol- free. When the compound or salt of interest is water-insoluble, again employing conventional liposome formation technology, the salt may be substantially entrained within the hydrophobic lipid bilayer which forms the structure of the liposome. In either instance, the liposomes which are produced may be reduced in size, as through the use of standard sonication and homogenization techniques.

Of course, the liposomal formulations containing the compounds disclosed herein or salts thereof, may be lyophilized to produce a lyophilizate which may be reconstituted with a pharmaceutically acceptable carrier, such as water, to regenerate a liposomal suspension. Other pharmaceutical compositions may be prepared from the water-insoluble compounds disclosed herein, or salts thereof, such as aqueous base emulsions. In such an instance, the composition will contain a sufficient amount of pharmaceutically acceptable emulsifying agent to emulsify the desired amount of the compound or salt thereof. Particularly useful emulsifying agents include phosphatidyl cholines, and lecithin.

In addition to active compound(s), the pharmaceutical compositions may contain other additives, such as pH-adjusting additives. In particular, useful pH- adjusting agents include acids, such as hydrochloric acid, bases or buffers, such as sodium lactate, sodium acetate, sodium phosphate, sodium citrate, sodium borate, or sodium gluconate. Further, the compositions may contain microbial preservatives. Useful microbial preservatives include methylparaben, propylparaben, and benzyl alcohol. The microbial preservative is typically employed when the formulation is placed in a vial designed for multidose use. Of course, as indicated, the pharmaceutical compositions of the present invention may be lyophilized using techniques well known in the art. 3. Dosage and routes of administration.

As noted above, the present invention provides pharmaceutical formulations comprising the active compounds (including the pharmaceutically acceptable salts thereof), in pharmaceutically acceptable carriers for oral, rectal, topical, buccal, parenteral, intramuscular, intradermal, intravenous, or transdermal administration, etc.

The therapeutically effective dosage of any specific compound, the use of which is in the scope of present invention, will vary somewhat from compound to compound, and patient to patient, and will depend upon the condition of the patient and the route of delivery. In general, the active agent is in some embodiments given in a dose of from 0.1 or 1 mg/Kg up to 50 or 100 mg/Kg subject body weight, or given in a dose of from 0.1 or 1 mg up to 50 or 100 mg. For example, in some embodiments, Mianserin may be given in a dose of 1 or 2 mg/Kg up to 25 or 50 mg/Kg; Mirtazapin may be given in a dose of from 5 or 10 mg up to 50 or 100 mg; Methiothepin may be given in a dose of from 0.1 or 1 mg/Kg up to 10 or 20 mg/Kg; and Cyproheptadine may be given in a dose of from 1 or 2 mg/ up to 50 or 80 mg. Dosages may be once or twice daily for as long as indicated, e.g., once or twice daily each day for a time of at least one month, at least six months, or a year or more.

Age or aging-related conditions that may be treated by the methods of the invention include, but are not limited to, one or more of those described in US Patent Application Publication No. 2008/0199865 to Crossman et al (published August 21, 2008): impaired skin and/or connective tissue function, cardiovascular disease, age- related cancer, abnormal immune system function and impaired neurological function. In some cases aging-related conditions of the invention are conditions that result, at least in part, from the accumulation of calcified products and amyloid, oxidative damage or increased production of reactive oxygen species. In preferred embodiments, aging-related conditions are in part the result of cellular senescence, including increased apoptosis, decreased ability to undergo cell division, mutations in cellular repair systems, and changes in cell behavior caused by the buildup of undesired by-products, such as byproducts of oxidative damage or glycation. In particular embodiments, age-related conditions of the invention include, but are not limited to: osteoporosis, osteoarthritis, decreased chondrocyte proteoglycan synthesis, decreased wound healing, wrinkled skin, rheumatoid arthritis, amyloidosis, Alzheimer's disease, type 2 diabetes mellitus, reduced T cell proliferation, increased IL-I production, decreased responsiveness to IL-I, decreased resistance to infection, impaired long-term potentiation in hippocampal neurons, decreased synaptic plasticity, memory loss, hearing loss, changes in the eye, including but not limited to retinal degeneration, depression, insomnia, impaired learning, endometrial cancer, prostate cancer, ovarian cancer, breast cancer, coronary artery disease, cerebrovascular disease (such as, but not limited to, stroke), peripheral artery disease, atherosclerosis, congestive heart failure and hypertension..

In some embodiments, compounds of the invention are administered in an amount effective to treat one or more of the conditions described in US Patent Application Publication No. 2008/0167248 to Witten et al. (published July 10, 2008): that is, to improve sleep patterns, reduce residual muscle pain following exercise, ameliorate short-term memory loss, improve a human's visual accommodation, increasing a human's muscle strength, reducing pain due to arthritis.

In some embodiments, the invention may be carried out to achieve one or more of the treatments or goals described in US Patent Application Publication No. 2008/0234310 to Bachurin et al. (published Sept. 28, 2008), including but not limited to: prolonging the lifespan of an individual,. prolonging the lifespan of cells in an individual, such as cells that respond to calcium influx, including cardiac cells, neurons, glial cells and the like (The cells may be normal cells. The cells may be uninjured cells.); slowing aging in an individual (for example by delaying the onset and/or slowing the progression of an aging-associated or age-related manifestation and/or pathology or condition, including, but not limited to, disturbance in skin-hair integument (such as hair-loss, baldness or alopecia), vision disturbance (such as development of cataracts), and weight loss (including weight loss due to the death of muscular and/or fatty cells)); improving the quality of life of an individual (such as an individual developing or at risk of developing these aging-associated or age-related manifestations and/or pathologies (where the aging-associated pathologies or conditions may or may not be life-threatening).

In some embodiments, the active agent is administered to a subject afflicted with cancer concurrently with chemotherapy and/or radiation therapy for that cancer, with cancer (e.g., either during or sufficiently close in time before and/or after the chemotherapy or radiation therapy), in an amount effective to reduce or inhibit at least one side-effect from the chemotherapy and/or radiation therapy. Examples of such side-effects include, but are not limited to, those set forth in Table A below (each to be taken individually or as any combination thereof).

Table A: Chemotherapy/radiation therapy side-effects.

I

Abdominal Pain Impotence

Acid Indigestion Incoordination

Acid Reflux Infection

Allergic Reactions Injection Site Reactions

Alopecia Injury

Anaphylasix Insomnia

Anemia Iron Deficiency Anemia

Anxiety Itching

Appetite (Lack Of)

Arthralgias J

Asthenia Joint Pain

Ataxia

Azotemia K

Kidney Problems τ>

D

Balance & Mobility Changes L

Bilirubin Blood Level Leukopenia

Bone Pain Libido (Loss Of)

Bladder Problems Liver Dysfunction

Bleeding Problems Liver Problems

Blood Clots Loss of Libido

Blood Pressure Changes Low Blood Counts

Blood Test Abnormalities Low Blood Pressure (Hypotension)

Breathing Problems Low Platelet Count

Bronchitis Low Red Blood Cell Count

Bruising Low White Blood Cell Count

Lung Problems t_

Cardiotoxicity M

Cardiovascular Events Memory Loss

Cataracts Menopause

Central Neurotoxicity Metallic Taste

Chemo Brain Mouth Sores

Chest Pain Mucositis

Chills Muscle Pain

Cognitive Problems Myalgias

Cold Symptoms Myelosuppression

Confusion Myocarditis

Conjunctivitis (Pink Eye)

Constipation N

Cough Nail Changes Table A: Chemotherapy/radiation therapy side-effects.

Cramping Nausea

Cystitis Nephrotoxicity

Nervousness

D Neutropenia

Deep Vein Thrombosis (DVT) Neutropenic Fever

Dehydration Nosebleeds

Depression Numbness

Diarrhea

Dizziness O

Drug Reactions Ototoxicity

Dry Eye Syndrome

Dry Mouth P

Dry Skin Pain

Palmar-Plantar Erythrodysesthesia

Dyspepsia (PPE)

Dyspnea Pancytopenia

Pericarditis

E Peripheral Neuropathy

Early Satiety Pharyngitis

Edema Photophobia

Electrocardiogram (ECG/EKG)

Photosensitivity Changes

Electrolyte Imbalance Pneumonia

Esophagitis Pneumonitis

Eye Problems Post-nasal Drip

Proteinuria

F Pulmonary Embolus (PE)

Fatigue Pulmonary Fibrosis

Feeling Faint Pulmonary Toxicity

Fertility

Fever R

Flatulence Radiation Recall

Flu-like Syndrome Rash

Flushing Rapid Heart Beat

Rectal Bleeding

G Restlessness

Gas Rhinitis

Gastric Reflux Ringing Ears

Gastroesophageal Reflux Disease

Runny Nose (GERD)

Genital Pain

Granulocytopenia S

Gynecomastia Sadness

Glaucoma Seizures

Sexuality

H Shortness of Breath

Hair Loss Sinusitis Table A: Chemotherapy/radiation therapy side-effects.

Hand-Foot Syndrome Skin Reactions

Headache Sleep Problems

Hearing Loss Sore Mouth

Hearing Problems Stomach Sour

Heart Failure Stomach Upset

Heart Palpitations Stomatitis

Heart Problems Swelling

Heart Rhythm Changes

Heartburn

Hematoma Taste Changes

Hemorrhagic Cystitis Thrombocytopenia

Hepatotoxicity Thyroid Hormone Levels

High Blood Pressure (Hypertension) Tingling

High Liver Enzymes Tinnitus

Hyperamylasemia (High Amylase) Trouble Sleeping

Hypercalcemia (High Calcium)

Hyperchloremia (High Chloride) U

Hyperglycemia (High Blood Sugar) Urinary Tract Infection

Hyperkalemia (High Potassium)

Hyperlipasemia (High Lipase) V

Hypermagnesemia (High Magnesium) Vaginal Bleeding

Hypernatremia (High Sodium) Vaginal Dryness

Hyperphosphatemia (High Phosphous) Vaginal Infection

Hyperpigmentation Vertigo Hypersensitivity Skin Reactions Vomiting Hypertriglyceridemia (High Triglycerides)

Hyperuricemia (High Uric Acid) W Hypoalbuminemia (Low Albumin) Water Retention Hypocalcemia (Low Calcium) Watery Eyes Hypochloremia (Low Chloride) Weakness Hypoglycemia (Low Blood Sugar) Weight Changes Hypokalemia (Low Potassium) Weight Gain Hypomagnesemia (Low Magnesium) Weight Loss Hyponatremia (Low Sodium) Hypophosphatemia (Low Phosphous) X

Xerostomia

Compiled by Chemocare (2005)(a program of the Scott Hamilton CARES initiative and the Cleveland Clinic Cancer Center).

Chemotherapy and radiation therapy (and the side effects thereof as set forth in Table A) are known. Examples include, but are not limited to, those methods and procedures described in US Patents Nos. 4,436,741 ; 4,631 ,289; 5,017,371 ; 5,354,782 The present invention is explained in greater detail in the following non-limiting Examples.

EXAMPLE 1 An antidepressant that extends lifespan in adult C. elegans

The nematode Caenorhabditis elegans is a model organism with a lifespan of ~3 weeks. Its lifespan can be increased by dietary restriction, an effect seen in many organisms, as well as by alterations in a number of different genes, some with analogous effects in fruitflies and/or mice²'³. These observations suggest that mechanisms that underlie aging and lifespan may be common to many organisms, including mammals. One long-term aim of aging research is to find drugs that could slow aging and delay the onset of age-associated disease. Several chemicals have been found to increase lifespan in invertebrates, including one identified by testing C. elegans with 19 compounds and another that also increases lifespan in yeast and fish^{4" 8}. However, no large-scale screens have been conducted for longevity enhancing drugs. The identification of such drugs and their endogenous targets in invertebrates could provide additional insights into aging mechanisms and ultimately point to drugs suitable for testing in mammals.

Based on these considerations, we conducted a high throughput screen for chemicals that increase lifespan when given to adult C. elegans. Using animals grown in liquid medium in 384-well plates, we tested 88,000 diverse chemicals for effects on lifespan. Starting at day 1 of adult life, animals in each well were continuously exposed to a single chemical at 30-90 μM. Based on the fraction of live animals per well relative to controls, 1083 chemicals were retested on larger populations. Of these, 1 15 compounds statistically increased lifespan, with 13 increasing lifespan by 30-60%, 18 by 20-29%, 27 by 10-19%, and 57 by 3-9%. The number of chemicals that actually entered the animal is unknown as is the number that increased lifespan by acting on the same endogenous target(s).

One compound that increased lifespan by 20%, "272Nl 8" (3-(3-nitrophenyl)- 1 1 -phenyl-2,3,4,5, 10, 1 1 -hexahydro- 1 H-dibenzo[b,e] [ 1 ,4]diazepin- 1 -one dihydrochloride) is structurally related to certain antidepressant drugs (Fig. Ia). In humans, these antidepressants affect intercellular signaling by serotonin, a neurotransmitter found in many animals, including C. elegans^9'u. This finding raised the possibility that serotonin signaling might affect C. elegans longevity.

To investigate this possibility, we tested C. elegans with 20 different compounds known to affect serotonin signaling pathways '' . Four of the compounds increased lifespan by 20-33%: Mianserin, Mirtazapine, Methiothepin, and Cyproheptadine (Figure 1). In humans, all four compounds are antagonists of serotonin 2 (5-HT₂) receptors and, to a lesser and variable extent, of certain adrenergic and histamine receptors⁹'¹². Mianserin and Mirtazapine are used to treat depression and Cyproheptadine to treat migraine and allergies whereas Methiothepin is not used clinically¹²'¹³. The effect of Mianserin on lifespan was dose dependent with a 31±3% maximal increase in lifespan obtained with 50 μM drug (Fig. Ic). Not all antidepressants increased lifespan: the serotonin specific reuptake inhibitors (SSRIs) Fluoxetine, Sertraline, and Paroxetine all decreased lifespan and the reuptake inhibitors Imipramine and Desipramine had no discernible effect. To determine whether human serotonin receptor antagonists that increase C. elegans longevity do so by altering serotonin signaling, we tested Mianserin on animals with mutations in different serotonin signaling components. In these and most subsequent studies, we also tested Mirtazapine, Methiothepin, or Cyproheptadine and observed effects similar to those seen with Mianserin. Mianserin failed to increase lifespan in tph- 1 (mg280) mutants that lack tryptophan hydroxylase, a key enzyme in serotonin synthesis¹⁴ (Fig.lb,c, Table 1). And, in animals lacking the serotonin reuptake transporter, MOD-5, maximum lifespan extension by Mianserin was 14% compared to 31±3% in wildtype animals¹⁵(Fig. Ib, c, Table 1). Thus, full lifespan extension by Mianserin requires both the production of serotonin and its normal reuptake at synapses.

We next asked whether lifespan extension by Mianserin requires a specific type of serotonin receptor. We tested the drug on animals mutant for each member of the serotonin/octopamine/tyramine receptor family of G protein-coupled receptors (GPCRs) (SER-I, SER-2, SER-3, SER-4, and SER-7) or for MOD-I, the serotonin- gated chloride channel¹⁶'¹⁷. Mianserin binds both SER-2 and MOD-I, and genetic studies suggest it may bind SER-4. The ser-l(ok345) mutant was recently reported to be long lived¹⁰'¹⁶'¹⁸. Table 1. Effects of 50 M Mianserin on the lifespan of wild type, DR and mutant animals.

* Drug added to N2 animals on day 1 of adulthood. * Drug added to N2 animals starting from the Ll stage. ⁺ Drug added to N2 animals on day 5 of adulthood. ^δ N2 animals subjected to dietary restriction. % change calculated relative to untreated controls in same experiment. Values of mean lifespan and % change in lifespan were averaged over all experiments.

Mianserin caused increases in lifespan similar to, or greater than, those seen in wildtype animals in ser-l(ok345), ser-2(pkl357), ser-7(tml325), and mod-l(okl03) mutants. In contrast, the drug failed to extend the lifespan of two different ser-3 mutants (Fig. 1, Table 1) and it produced only a slight (7±2%) lifespan increase in a ser-4(ok512) mutant. Moreover, when a wild type ser-4 transgene was expressed in the ser-4(ok512) mutant, Mianserin caused a 16% lifespan increase versus a 4% increase in the ser-4(ok512) mutant alone. These results were not due to a decrease in Mianserin uptake by ser-3 and ser-4 mutants, since, as with wildtype animals, preincubation of the mutants with Mianserin decreased serotonin-induced egg laying ¹⁰(Fig. 3a). SER-4 is activated by serotonin¹⁶ and genetic studies suggest that SER-3 is activated by octopamine, which is proposed to be the invertebrate equivalent of vertebrate noradrenalin¹⁷'¹⁹ . Our results thus indicate that lifespan extension by Mianserin requires both a serotonin receptor, SER-4, and a likely octopamine receptor, SER-3^{16J 7}.

To directly examine the effects of potential ligands and Mianserin on SER-3 and SER-4 receptors, we used calcium imaging²⁰. HEK293 cells were transfected with an expression vector²¹ encoding SER-3, or vectors encoding SER-4 and Gαl5, a G protein that couples to many GPCRs and induces increases in intracellular calcium²². Cells expressing SER-3 responded to octopamine at concentrations as low as 10 nM and to tyramine at a thousand-fold higher concentration (10 μM), but showed no response to serotonin (Fig. 2a). Cells expressing SER-4 responded to serotonin at concentrations as low as 100 nM (Fig. 2c). Neither SER-3+ nor SER-4+ cells responded to Mianserin, excluding its possible role as an agonist. We next asked whether Mianserin acts as a SER-3 or SER-4 antagonist.

Receptor-expressing cells were pre-incubated with Mianserin (or Methiothepin), and then exposed to octopamine (SER-3) or serotonin (SER-4) in the presence of the drug. Mianserin and Methiothepin both inhibited the response of SER-3+ cells to octopamine and the response to SER-4+ cells to serotonin (Fig. 2). The inhibitory effect of Mianserin was reversible within 5 minutes of removal, but inhibition by Methiothepin lasted longer (Fig. 2b, c). These results indicate that Mianserin, an antidepressant that inhibits serotonin signaling in humans, extends C. elegans lifespan by blocking signaling through SER-4 serotonin receptors and SER-3 octopamine receptors (Fig. 2d, e). Interestingly, 80-90% inhibition of the SER-3 response to octopamine required a ten-fold higher concentration of Mianserin than a comparable inhibition of the SER- 4 response to serotonin (10 uM versus 1 uM) (Fig. 2d, e). The difference was even greater for Methiothepin. Thus both Mianserin and Methiothepin are more potent antagonists of SER-4 than SER-3. This suggests that exposure of animals to certain concentrations of these drugs would fully inhibit SER-4, but only partially inhibit SER-3 whereas very high concentrations might completely block both receptors. Interestingly, Mianserin produced a large increase in lifespan at 50 μM, but had little or no effect when used at 250 μM (Fig. Ic, 3b). Together, these findings suggest that the lifespan-increasing effects of Mianserin may result from a greater inhibition of SER-4 than SER-3, and that some activity of SER-3 may be necessary to achieve lifespan extension by Mianserin. While 50 μM Mianserin increased lifespan by 31±3% when given only during adult life, it increased it by only 10±5% when the drug was given during both larval and adult life (Fig. 3b, Table 1). No further increase in lifespan was obtained by varying the drug's concentration, but increasing its concentration caused developmental arrest (non-dauer) at the L2 larval stage (Fig. 3b). In addition, although ser-3 and ser-4 are required for lifespan increases in response to Mianserin given to adults, lifespan increases in mutants not given the drug were only 6±3% for ser-3 (ok2007) and 19±2% for ser-4 (ok512) mutants, with the latter showing an 11% lower lifespan increase when it contained a wildtype ser-4 transgene (see above). Thus, neither removing ser-3 or ser-4 nor blocking SER-3 and SER-4 receptors with Mianserin during both larval and adult life recapitulates the lifespan-increasing effects of Mianserin given only during adulthood. One possible explanation for these findings is that SER-3 and/or SER-4 loss of function in larvae induces compensatory mechanisms that alter the animal's physiology and, thereby, Mianserin's effects on adults. Age-associated differences in loss of function phenotypes have previously been seen in mice²³.

Does Mianserin increase longevity via processes previously linked to aging in C. elegans? To explore this question, we tested Mianserin on animals that have altered lifespans as a result of dietary restriction (DR) or because of mutations in genes encoding DAF-2 or DAF- 16, two components of the IGF-I /insulin signaling pathway²'³, CLK-I , a mitochondrial protein ²⁴, or EAT-2, an ion channel subunit required for proper pumping of food into the pharynx²⁵.

Our results indicate that lifespan extension by Mianserin involves aging mechanisms associated with dietary restriction. The combination of Mianserin and DR increased lifespan by only 4±2% more than DR alone (Fig. 3c, d, Table 1). In addition, consistent with reported links between DR aging mechanisms and increased lifespan in eat-2(adl 116) and clk-l(e2519) mutants²⁵, Mianserin increased the lifespans of these two mutants by only 2±5% and 3±4%, respectively (Fig. 3c, d, Table I)). This was not due to a lack of Mianserin uptake, as evidenced by the ability of Mianserin to reduce serotonin-induced egg laying in both mutants (Fig. 3a, left).

Although serotonin is involved in pharyngeal pumping^{1 1}'²⁶, two findings indicate that Mianserin does not reduce food intake. First, while eat-2(adlll6) mutants defective in pharyngeal pumping showed an 80-90% decrease in pharyngeal pumping²⁵, pumping rates were the same in Mianserin-treated and untreated animals (Fig. 3a). Second, while DR can increase lifespan even when initiated as late as day 10 of adulthood ⁷, exposure to Mianserin beginning on day 5 had no effect on lifespan (Fig. 3c, d and Tablel). These results indicate that, although Mianserin extends lifespan through mechanisms associated with DR, it does not do so by decreasing food intake.

In daf-2(el370) and daf-16(mu86) mutants, Mianserin caused 1 1±4% and 14±1% increases in lifespan, respectively, raising the possibility that the two genes might also influence Mianserin-induced lifespan extension to some extent (Fig. 3c, d). However, these results do not compare to the dramatic results obtained when Mianserin was tested on DR animals or on eat-2(adlll6) and clk-l(e2519) mutants. Interestingly, these findings contrast with an absolute requirement for daf-16 for the increased reproductive period ('reproductive longevity') seen in tph~l(mg280) mutants deficient in serotonin synthesis¹⁴. In our studies, tph-l(mg280) mutants showed only a 4±5% increase in lifespan compared to the 31±3% lifespan increase seen in response to Mianserin, further distinguishing the effects of serotonin signaling on reproductive longevity versus lifespan¹⁴.

In summary, we found that C. elegans lifespan is increased by giving the adult animal Mianserin, a drug used as an antidepressant in humans. This effect requires the presence of serotonin as well as two neurotransmitter receptors, the SER-4 serotonin receptor and the SER-3 octopamine receptor. Similar to its antagonistic action on human serotonin receptors, Mianserin inhibits both SER-4 and SER-3. Serotonin and octopamine are thought to serve as physiological antagonists that signal the presence of food (serotonin) versus starvation (octopamine) in C. elegans ^{l l >17}'²⁶. It may be that these two neurotransmitters exist in a dynamic equilibrium that is tipped in the direction of a starvation response by Mianserin, possibly because of the greater inhibitory effect of Mianserin on SER-4 than SER-3. In this way, Mianserin could potentially create a 'perceived' state of starvation that, despite adequate food intake, would activate mechanisms of lifespan extension downstream of dietary restriction. Interestingly, Mianserin has been used to treat anorexia as well as depression in humans, suggesting a possible evolutionary link between appetite and lifespan in C. elegans and humans²⁸.

METHODS SUMMARY

Lifespan assay. Lifespans were assessed in liquid medium⁵'²⁹ at 20 ⁰C in 384 or 96 well plates. Age-synchronized C. elegans were distributed (seeded) in wells as Ll larvae (10-15_% or 6-12₃₈₄ animals/well) together with E. coli OP50. To prevent self-fertilization 5-Fluoro-2'-deoxyuridine (Sigma) was added 42-45 hours after seeding (0.12 mM final). Unless otherwise specified, drugs were added 68 hours after seeding, which corresponded to day 1 of adult life and the lifespan assay. The fraction of animals alive was scored on the basis of body movement.

Statistical analysis. Comparisons and P value calculations were made between treated and untreated animals of the same strain using the log-rank test (Mantel-Haenszel).

Calcium imaging. HEK293 cells were transfected with expression plasmids encoding SER-4 or Ga 15 and SER-3. Potential ligands were applied for 4 sec, with 2 min separating different applications. Fluorescence emission was determined every 4 seconds. To test the effects of Mianserin and Methiothepin, cells were first exposed to a ligand and responses recorded. Drugs were then applied for 5 min, after which responses to the ligands were tested again. Inhibition of responses by Mianserin or Methiothepin was calculated by the following equation: % inhibition = 100 x ((response in the presence of inhibitor)/ (uninhibited response)). Additional Methods. Further details and additional methods, including strains, cloning strategies, dietary restriction, egg-laying, are presented in Methods.

METHODS

Strains and genetics. All strains were maintained at 20 °C as described in "The genetics of Caenorhabditis elegans". C. elegans strains used were: Bristol strain (N2), CB4876 clk-l(e2519) III, GR1321 tph-l(mg280) II, CF1038 daf-

16(mu86) I,CB1370 daf-2(el370)l\\, DAl 1 16 eat-2(adll 16) II, AQ866 ser-4(ok512)

III, ser-4(ok512)\yzEx2Q5[seτ-4(+); Rol-6], OHO313 ser-2(pkl357) X, DA1814 ser- I(ok345) X, MT9668 mod-l(okl03) V, MT9772 mod-5(n3314) I, CB1370 daf- 2(el370) III, DA2100 ser-7(tml325) X, RB1631 ser-3(ok2007) I and VNI l ser- 3(adl 774);tzls3 [ere: :gfp, Hn- 15(+)].

Lifespan assay. Lifespans were assessed in liquid medium⁵'² (S-complete, 50 μg/ml carbenicillin, 0.1 ug/ml fungizone, 60 μl₃g₄ or 150 μl₉₆ total volume) at 20 °C in either 96 or 384 well plates (Falcon, Nunc). Synchronized worms were seeded as Ll larvae (10-15g₆ or 6-12₃₈₄ animals/well) together with freshly prepared E. coli OP50 (6mg/ml₉₆ or 9 mg/ml₃g₄ wet weight). To avoid evaporation plates were sealed with tape (Nunc). To prevent self-fertilization 5-Fluoro-2'-deoxyuridine (Sigma) was added 42-45 hours after seeding (0.12 mM final). Drugs were added 68 hours after seeding (day 1 of adult life) unless otherwise specified. Day 1 of the lifespan assay started 68 hours after seeding the animals into plates.

The fraction of animals alive was scored based on movement. Prior to counting, each plate was put onto a plate rotator for 1-2 minutes. Strong light (visual or UV) effectively stimulated movement even in old animals. Using this assay, daf- 16, eat-2, and clk-1 aging mutants showed alterations in lifespan similar those reported using standard conditions (agar plates) ' ' .

Dietary restriction. Animals were grown in liquid medium in the presence of food until day 3 of adulthood, when food was reduced 25-fold by serial dilution²⁷. Statistical analysis. STATA8 software was used for analysis. Comparisons and P value calculations were made between treated and untreated animals of the same strain using the log-rank test (Mantel-Haenszel). We observed the death of 98.6% of the animals (excluding screen). Animals that were still alive at the end of an experiment (1.4%) were analyzed as alive up to this point with unknown time of death (censoring). Wells containing more than 19 animals were excluded from analysis.

Pharyngeal pumping. Animals were grown in liquid medium at 20 °C (+/- drug). After the animals were transferred to bacteria-coated agar plates (+/- drug) and then left for 30 min, the grinder movements within a 10 second interval were counted. Animals were assayed on 3 consecutive days following drug treatment. Numbers of animals tested on the three days were: controls, n=67/67/58, Mianserin, n=57/68/57, Methiothepin, n=26/27/24.

Egg-laying assay. The egg-laying assay was performed as described in Dempsey et al.¹⁰ but using 50 μM Mianserin instead of 20 μM. Numbers of animals tested (+/- Mianserin): N2 (64/54), clk-1 (26/26), eat-2 (38/29), mod-5 (40/40), ser-3 (39/29), ser-4 (48/38), tph- J (47 /40).

Expression vectors. The ser-3 or ser-4 coding region was amplified by PCR from cDNA prepared from C. elegans RNA, and then cloned into the pcDNA3.1(-) expression vector (Invitrogen) to give the SER-3 (pMP513#6) or SER-4 (pMP509#6) expression vector. In pMP509#6, sequence encoding the first 20 amino acids of bovine rhodopsin was added to the 5' end of the ser-4 coding region to enhance cell surface expression²¹.

Calcium imaging. 4 x 10⁵ HEK293 cells were seeded into individual wells of 6-well plates containing coverslips coated with poly D-lysine, and then transfected with pMP509#6 (SER-4, 200 ng/well) and a Ga 15 expression plasmid²² (150 ng/well), or with pMP513#6 (SER-3, 200ng/well) using lipofectamine (Invitrogen, cat# 1514-015 according to the manufacturer's directions). After 24 hours, cells were loaded with the calcium indicator, calcium 3 (Molecular Devices) in HBSS/20 mM HEPES (Gibco) for 1 h prior to imaging. Calcium imaging was done on coverslips in a perfusion chamber mounted on an inverted microscope (Olympus 1x70) using a 1 Ox/0.3 NA objective (Olympus UplanFI) to maximize the number of imaged neurons. During imaging, cells were continuously perfused with HBSS and intermittently exposed to HBSS containing ligands and/or drugs. Ligands were applied for 4 sec, with 2 min separating different applications. Fluorescence emission was determined every 4 sec using a CCD camera (Hamatsu C4742-95-10NR) and a standard filter set (high Q filter set (R.P.I.): 470/40 excitation filter; 495/LP nm dichroic mirror; 525/50 nm emissions filter). To test the drugs Mianserin and Methiothepin, cells were first exposed to a ligand and responses recorded. Drugs were added to the perfusion buffer and continuously applied for 5 min, after which responses to the ligands were tested again. The perfusion buffer was changed back to pure HBSS for a 5 min washout, after which ligands were applied again. Image analysis was done using Metafluor (Molecular Devices) software. Fluorescent signals were normalized using the following equation: ΔF/F = (F_t-Fo)/F_o. Inhibition of responses by Mianserin or Methiothepin was calculated by the following equation: % inhibition = 100 x ((response in the presence of inhibitor)/ (uninhibited response)). REFERENCES

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Satoshi Suo kindly provided us with the VN 1 1 strain (ser-

3(adl 774);tzte3[cτe::gfp, lin-15(+)]) and Ji Ying Sze for the ser-

¥foλ:Ji2_>);yzΕx205[ser-4(+); Rol-6] strain. All other strains were provided by the Caenorhabditis Genetics Center.

The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

What is Claimed is:

1. A method of treating aging in a mammalian subject in need thereof, comprising administering said subject a serotonin 2 (5-HT₂) receptor antagonist in an amount effective to treat said aging.

2. The method of claim 1, wherein said subject is a human subject.

3. The method of claim 1-2, wherein said 5-HT₂ receptor antagonist is SER-4 serotonin receptor antagonist (e.g., in C. elegans).

4. The method of claim 1-3, wherein said 5-HT₂ receptor antagonist is a SER- 3 octopamine receptor antagonist fe..g., in C. elegans).

5. The method of claim 1-2, wherein said 5-HT₂ receptor antagonist is selected from the group consisting of Mianserin, Mirtazapine, Methiothepin, Cyproheptadine, 272Nl 8, Amperozide, PAPP (l-[2-(4-Aminophenyl)ethyl]-4-(3- trifluoromethylphenyl)piperazine), dihydroergotaminee, cyclobenzaprin, LY-367,265 ( 1 -(2-(4-(6-fluoro- 1 H-indol-3-yl)-3,6-dihydro- 1 (2H)-pyridinyl)ethyl)-5,6-dihydro 1 H, 4H-(1 ,2.5)thiadiazolo(4.3.2-ij)quinoline-2,2-dioxide)), metergoline, keanserin, SB224829, BRLl 5572, 4-Methyl-2,5-dimethoxyamphetamine, Alosetron, Aripiprazole, Azatadine, Cabergolin, Chloroprocaine, Cyclobenzaprine, Dimethyltryptamine, Dolasetron, Frovatriptan, Granisetron, Methotrimeprazine, Methysergide, Nefazodone, Olanzapine, Ondansetron, Paliperidone, Palonosetron, Promazine, Propiomazine, Quetiapine, Risperidone, Sertindole, Thiethylperazine, and pharmaceutically acceptable salts or prodrugs thereof.

6. The use of a compound as described in claims 1-5 above for the preparation of a medicament for carrying out a method as described in claims 1-5 above.

7. A method of screening a candidate agent for activity in treating aging in a mammalian subject, comprising: administering an active agent to a plurality of nematodes, and then detecting the fraction of said nematodes alive at a plurality of predetermined times after said administering; and then determining the increase in lifespan for said nematode from said detecting at a plurality of predetermined times; with an extension in lifespan of at least 10 percent as compared to corresponding untreated animals indicating said active agent is useful in treating aging in mammalian subjects.

8. The method of claim 7, wherein said nematode is Caenorhabditis elegans.

9. The method of claim 7, wherein said nematode is grown in liquid medium.

10. The method of claim 7, wherein said administering step is carried out on day 1 of adult life.

1 1. The method of claim 7, wherein said detecting the fraction of nematodes alive is carried out by detecting body movement of said nematodes.

12. The method of claim 7, wherein said method is a high-throughput screening method carried out concurrently in multiple wells.

13. The method of claim 12, wherein said wells are concurrently seeded with said nematodes as Ll larvae.

14. The method of claim 13, wherein said wells are seeded with from 5 to 20 nematodes.

15. The method of claim 14, wherein said nematodes are administered 5- Fluoro-2'-deoxyuridine in an amount effective to prevent self-fertilization at 42-45 hours after seeding.

16. In a method of treating a subject afflicted with cancer with chemotherapy, radiation therapy, or a combination thereof, the improvement comprising: concurrently administering said subject a serotonin 2 (5-HT₂) receptor antagonist in an amount effective to treat at least one chemotherapy or radiation therapy side-effect in said subject.

17. A method of treating a patient in need of radiation therapy or chemotherapy, comprising administering to said patient a protective amount of a serotonin 2 (5-HT₂) receptor antagonist.

18. The method of claim 16-17, wherein said side-effect comprises alopecia.

19. The method of claim 16-18, wherein said side effect comprises mucositis.

20. The method of claim 16-19, wherein said side effect comprises fatigue.

21. The method of claim 16-20, wherein said side effect comprises anemia.

22. The method of claim 16-21, wherein said side effect comprises low white blood cell counts.

23. The method of claim 16-22, wherein said 5-HT₂ receptor antagonist is selected from the group consisting of Mianserin, Mirtazapine, Methiothepin, Cyproheptadine, 272Nl 8, Amperozide, PAPP (l-[2-(4-Aminophenyl)ethyl]-4-(3- trifluoromethylphenyl)piperazine), dihydroergotaminee, cyclobenzaprin, LY-367,265 (l-(2-(4-(6-fluoro-l H-indol-3-yl)-3,6-dihydro-l(2H)-pyridinyl)ethyl)-5,6-dihydro I H, 4H-( 1.2,5)thiadiazolo(4.3.2-ij)quinoline-2,2-dioxide)), metergoline, keanserin, SB224829, BRL 15572, 4-Methyl-2,5-dimethoxyamphetamine, Alosetron, Aripiprazole, Azatadine, Cabergolin, Chloroprocaine, Cyclobenzaprine, Dimethyltryptamine, Dolasetron, Frovatriptan, Granisetron, Methotrimeprazine, Methysergide, Nefazodone, Olanzapine, Ondansetron, Paliperidone, Palonosetron, Promazine, Propiomazine, Quetiapine, Risperidone, Sertindole, Thiethylperazine, and pharmaceutically acceptable salts or prodrugs thereof.

24. The use of a compound as described in claims 16-23 above for the preparation of a medicament for carrying out a method as described in claims 16-23 above.