The invention relates to telmisartan, 2-(4-{[4-methyl-6-(l-methyl-lH-l,3- benzodiazol-2-yl)-2-propyl-lH-l,3-benzodiazol-l-yl] methyl} henyl) benzoic acid, or a pharmaceutically acceptable salt thereof for use in promoting autophagy. The invention also relates to the use of Telmisartan or a pharmaceutically acceptable salt thereof in the treatment of autophagy related disorders, such as various forms of cancer; liver disease, myopathies of various origin; cardiovascular disorders, neurodegenerative disorders. In addition Telmisartan can be used to extend the capabilities of humans or animals of leading a longer life called longevity. The invention further relates to suitable pharmaceutical compositions, which contain telmisartan as a combined preparation for simultaneous, separate or sequential use for the treatment and prevention of these diseases.

Summary

It has been recently discovered that autophagy (a term which is composed of Greek words "auto" - for self - and "phagein" - for eating - and means cellular self- digestion) plays a crucial role in the homeostasis of the cell to maintain its normal function. It has been demonstrated that impaired or attenuated autophagic activity can lead to cancer, liver disease, various myopathies and neurodegenerative disorders. In addition the deterioration of the normal level of autophagy may also be responsible for shortening the life span. In parallel, it has been demonstrated that stimulation of autophagy can lead to longevity.

Although the regulation of autophagy is a complex phenomenon, certain

myotubularin proteins play a key role. It has been shown that two myotubularin proteins, MTMR14 and MTMR6, - have a central role in blocking autophagy by antagonizing the type III phosphatidylinositol 3-kinase VPS34 (vacuolar protein sorting protein 34). This central mechanism of the autophagy pathway is conserved across the various species.

The inventors have demonstrated that telmisartan inhibits MTMR6 in a dose dependent manner and subsequently significantly increases the autophagic activity of the cell. Therefore it is effective in treating autophagy-dependent disorders. The inventors have also demonstrated that by inhibiting MTMR 6, life span is significantly increased in various animal models, and thus telmisartan exerts longevity attributes.

Previously telmisartan— alone or in combination - has been shown to be an effective angiotensin II receptor antagonist (angiotensin receptor blocker, ARB) used in the management of hypertension.

Background

Autophagy is a highly regulated self-degradation process of eukaryotic cells. During autophagy, parts of the cytoplasm are sequestered by a double-membrane structure, thereby forming a vesicle-like structure called an autophagosome. The autophagosome then fuses with a lysosome, and in the resulting structure, called an autolysosome, the sequestered cytoplasm becomes degraded by lysosomal hydrolases (proteases, nucleases, lipases and glycosylases). The end products of autophagic breakdown can serve as building blocks for synthetic processes or provide energy for the cell under starvation conditions. Thus, autophagy plays an essential role in the renewal of cellular components (macromolecule and organelle turnover) and primarily functions as a cell-protecting mechanism. Autophagic degradation is important in cell growth and proliferation, survival of cells, and in defence against intracellular microorganisms. In humans, diverse age-related pathological conditions such as cancer, neurodegenerative diseases (e.g., Alzheimer, Parkinson and Huntington disease), stroke, sarcopenia, immune deficiency and heart attack involve dysregulated autophagy.

The basic biochemical reaction that mediates the formation of the autophagic (isolation) membrane is catalyzed by a conserved kinase, PI3K-III (type III phosphatidylinositol 3-kinase). This enzyme converts phosphatidyl-inositol-3 phosphate into phosphatidyl-inositol-3,5 bisphosphate. Thus, ΡΙ3Κ-ΠΙ is a critical component of the autophagic process. The molecular antagonists of PI3K-III involve certain myotubularin-related (MTMT) phosphatases. These MTMT enzymes can inhibit autophagic degradation. In genetic model systems and cell cultures, inhibition of mtm genes leads to a potent autophagy activation. Loss-of-function mutations in mtm genes can significantly extend lifespan, suppress neuronal cell death, and prevent muscle and other tissues from undergoing atrophy. A myotubularin protein MTMT6, is implicated in the fine tuning of autophagy.

Therefore it is desirable to develop specific MTMT6 inhibitors with the potential to stimulate the autophagic process. The role of autophagy in physiology and pathology

During autophagy, parts of the cytoplasm are sequestered into a double-membrane bound structure called autophagosome, and then delivered into the lysosome lumen for enzymatic degradation. The resulting products of autophagic degradation are later utilized in anabolic processes or as cellular energy. Autophagy is basically responsible for the elimination of damaged or worn-out cellular components (dysfunctional and abnormal macromolecules and organelles). Autophagy also plays a key role in cellular stress response during starvation, in the regulation of cell growth, division and loss, in aging control and in the defense against intracellular pathogens. Defects in autophagy can lead to the development of various geriatric diseases such as cancer, premature aging, various neurodegenerative disorders, tissue atrophy e.g. muscle atrophy (sarcopenia), stroke, heart failure and infections caused by parasitic bacteria or viruses (Levine and Kroemer, 2008; Mizushima et al, 2008). Understanding the mechanisms and regulation of autophagy is therefore of utmost importance for biomedical, social and economic reasons. The most common fatal diseases of mankind normally develop at advanced ages. While the role of pathological functioning of several proteins (such as oncoproteins, tumour suppressors or aggregation-prone proteins) in the development of these diseases has been revealed over the past few decades, understanding the molecular and cytological bases of these processes remains in the forefront of current biological research. Therefore, it is clear that the pathological mechanisms underlying cancer, neurodegeneration and muscle atrophy— all of which are complex, multifactorial processes— are yet to be discovered. Interestingly, these diseases with apparently diverse origin, molecular basis and clinical picture have something else in common apart from the fact that they predominantly develop at advanced ages, and it is that they are all caused by damaged cellular components, including dysfunctional, oxidized, misfolded, cross- linked or aggregated macromolecules. For example, oxidation of DNA may lead to single- or double-stranded breaks, and during the repair of these breaks the nucleotide sequence can change. The resulting mutations can trigger uncontrolled cell division. Protein aggregation can also lead to various neurodegenerative processes. Alzheimer's disease, for instance, is caused by the accumulation of β- amyloid and tau proteins, while Parkinson's disease is accompanied with the aggregation of a-synuclein in dopaminergic neurons. It is the gradual age-related accumulation of molecular damage, which drives the aging process.

Normal metabolic processes result in a continuous generation and accumulation of cellular damage. Various enzymes and the mitochondrial respiratory chain all produce reactive oxygen species (ROS), such as oxygen anions, superoxide and hydroxyl radicals, peroxides, which can oxidize macromolecules. The removal of ROS is essential for the maintenance of cellular homeostasis. Malfunction and deterioration of cellular repair systems are likely to be responsible for aging as well as for the incidence of most age-related diseases. Due to this remarkable molecular convergence, in the future it may be possible to modify (slow down) the rate at which the cells and tissues age and to delay the incidence of numerous age-related degenerative processes. The removal of damaged cellular components primarily occurs through autophagy. During autophagy parts of the cytoplasm are delivered to lysosomes through a regulated process, in which they are degraded by lysosomal hydrolases.

Despite its medical significance, the genetic and molecular basis for the regulation and the mechanism of the autophagy process were only discovered very recently. Autophagic vacuoles are micron-sized and so autophagy in the past could only be examined by electron microscopy. This idiosyncrasy has made it impossible to use efficient genetic methods (genetic screens) to identify autophagy-specific genes. The breakthrough came with the study of autophagy in single-celled yeast. Yeast contains a single autophagic vacuole (an organelle analogous to the lysosome), which can be identified by light microscopy. This finding was followed by a series of genetic screens to identify yeast autophagy-related genes ATG). Identification of metazoan orthologs of yeast autophagy genes has opened the way to the molecular and functional (genetic) analysis of autophagy in higher organisms. During autophagy, cellular components are translocated into the lysosome through a regulated process. Based on the method of translocation, three main types of autophagy can be distinguished: microautophagy, chaperon-mediated autophagy (CMA) and macroautophagy. During microautophagy the lysosomal membrane directly engulfs parts of the cytoplasm (invagination). CMA, which does not occur in plant cells, is responsible for the degradation of proteins containing a specific pentapeptide motif, FERQ. These proteins are marked by molecular chaperones and are transported to the lysosomes though the LAMP-2a (type 2a lysosome-specific membrane protein) receptor. Interestingly, a-synuclein, whose aggregation results in the development of Parkinson's disease contains the KFERQ motif. Qualitatively, macroautophagy is the most significant protein and organelle degradation mechanism. During this process, a double membrane structure called a phagophore is formed inside the cytoplasm, sequestrating cellular components (macromolecules and organelles) from the rest of the cell. When the membrane growth is completed, the resulting structure is called autophagosome. The mature autophagosome then fuses with a lysosome to form an autolysosome, in which the segregated cellular components are degraded into building blocks. As used herein 'autophagy' encompasses microautophagy, chaperone mediated autophagy and macroautophagy. One of the most remarkable features of autophagy is that it is in a tight connection with numerous signal transduction systems, environmental (nutrients, temperature, oxygen) and cellular factors (mitogens, growth factors, ATP levels).

Recent results suggest that autophagy acts as a downstream effector process in the regulation of cell growth, proliferation and death. On the other hand, depending on the actual cellular milieu, autophagy is one of the most important means of cell survival. For example, the effect of genetic pathways regulating cell division (such as the Ras, insulin/IGF-1, TGF-β, JNK, G-protein mediated and TOR signal transduction systems) are mediated by the autophagic process. Signal transduction pathways regulating aging (e.g. insulin/IGF-1, TGF -beta, JNK, TOR and Ras/ERK signalling) also converge on the autophagy gene cascade. In addition, biomedically highly important proteins such as p53, FoxO, E2F (a component of the retinoblastoma complex), FoxA, Sirtl (a sirtuin) regulate the activity of certain autophagy genes directly (i.e. they function as transcription factors of autophagy genes). Therefore, it is evident that autophagy plays a role in the processes of aging, cell division and death. Autophagy genes are vital in Drosophila and in C. ekgans under both normal and starvation-stress induced conditions. In C. ekgans, reduced levels of insulin/IGF-1 (insulin-like growth factor 1), TOR signal transduction pathways, mitochondrial respiration or caloric restriction each increase lifespan. The increased lifespan of these animals is autophagy-dependent: inactivation of autophagy genes suppresses the extension of lifespan (Toth et al, 2008). Furthermore, it has been demonstrated in insects that the activity (expression) of autophagy genes gradually decreases as the animal ages (as part of the normal aging process) and that overexpression of the autophagy protein Atg8 in the nervous system increases lifespan by 50% (Simonsen et al, 2008). Autophagy genes hence form an "anti- aging" pathway, onto which the effects of the signal transduction systems regulating longevity converge. Autophagy is, therefore, a central regulatory mechanism of animal aging.

The mechanisms of autophagy

Based on their function, the -ATG genes can be classified into four groups: 1, genes mediating induction (nucleation); 2, genes that mediate isolation membrane growth; 3, members of the Atg8 conjugation system; and 4, genes involved in recyclization. Induction of autophagy is regulated by an Atgl kinase complex. This complex contains other proteins, including Atgl 3 and Atgl 7. Under normal conditions, Atgl 3 is phosphorylated by the kinase target of rapamycin (TOR); in this state the complex is not able to initiate autophagy. Under starvation, however, TOR becomes inactivated, resulting in the dephosphorylated state of Atgl 3. Under these circumstances the Atgl complex promotes autophagosome formation.

After induction, another kinase complex, whose central component is VPS34 (vacuolar protein sorting-associated protein), a type III phosphatidylinositol-3 kinase, mediates the synthesis of the growing isolation membrane (phagophore). In addition to VPS34, this complex also includes Atg6, Atgl4 and Atgl5 proteins, and participates in the synthesis of other, non-autophagosomal membranes.

The growing isolation membrane should be identified as an autophagosomal membrane. This can be achieved by covalent binding (conjugation) of Atg8, a ubiquitin-like protein, to the membrane. Initially, Atg8 is a cytosolic, soluble protein (Atg8-I). Upon induction, the last amino acid (a glycine) of Atg8 becomes cleaved off from the protein, leaving a free carboxyl terminus that can bind to a membrane component, phosphatidyl-ethanolamine (PE). The PE-bound form of Atg8 is insoluble (Atg8-II). It binds to the forming autophagosomal membranes. In the conjugation process of Atg8, numerous Atg proteins participate, including Atg3, 4, 5, 7, 12 and 16.

After autophagosome formation, its outer membrane fuses with a lysosome, generating thereby a structure called autolysosome, where the cargo (sequestered cytoplasmic materials) is degraded by acidic hydrolases. After autolysosome formation, several components of the autophagic structure can be regained through recyclization.

Myotubularin phosphatases

The catalyst of the initial biochemical process during autophagy is a lipid kinase, ΡΙ3Κ-ΠΙ, which phosphorylates phosphatidylinositol 3-phosphate (PtdIns3P) to phosphatidylinositol 3,5-bisphosphate (PtdInsi,5F), which is essential for membrane formation. Thus, PI3K-III activity stimulates the formation of autophagosomes. The chemical process catalyzed by PI3K-III is an equilibrated biochemical reaction:

myotubularin-related (MTMT) phosphatases dephosphorylate Ptdlnsi to Ptdlns.

Ptdlns Pt€lIns3P . . . i i i . i ^ Autophagy kinase (PI3K-III), VPS34 (vacuolar sorting protein). PI3K converts Ptdlns to MTMT myotubularins act as antagonists of the type III phosphatidyl-inositol-3

PtdIns3P, while MTMT proteins reverse this reaction. In this way, MTMTs function as negative regulators of autophagy.

MTMT activity, therefore, results in the suppression of autophagy. This suggests that inhibition of MTMT activity can lead, in theory, to stimulation of autophagy. Indeed, it has been demonstrated that in C. ekgans the suppression of certain mtm genes activates autophagy, to salvage the larval mortality of PI3K-III- mutant animals (Xue et a/., 2003).

MTMR proteins form a conserved family of phosphatases. The human genome encodes 13 MTMR proteins (Robinson and Dixon, 2006). These paralogs differ in their structure and only certain types are suitable for efficiently dephosphorylating Ptdlnsi . The lack of certain MTMR proteins during ontogeny might lead to the development of mendelian inherited diseases (e.g. myopathy, neuropathy or Charcot- Marie-Tooth syndrome). Out of the 13 human MTMR proteins only MTMR1 {myotubular myopathy), MTMR2 (type 4B 1 Charcot-Marie-Tooth syndrome) and MTMR5/13 (infertility in mice) have so far been linked to pathological processes. The lack of MTMR proteins in adulthood (that is, after ontogeny) has not yet been linked to known human disease. Thus, the specific-suppression of MTMR proteins does not result in degenerative disorders. Description of the Invention

In one aspect the present invention relates to the use of telmisartan or a pharmaceutically acceptable salt thereof in promoting autophagy. In one embodiment the invention relates to a method of promoting autophagy, comprising administering to the subject a therapeutically effective amount of telmisartan or a pharmaceutically acceptable salt thereof. Autophagy is the major catabolic process of eukaryotic cells that degrades and recycles damaged macromolecules and organelles.

„Promoting autophagy" as used herein means increasing the rate of autophagy within a cell or organism as compared to the rate of autophagy in the absence of treatment. Preferably, the invention relates to telmisartan or a pharmaceutically acceptable salt thereof for use in a method of treating an autophagy related disorder. As used herein an "autophagy related disorder" is selected from cancer, stroke, sarcopenia, infection, immune system deficiencies, liver disease, neurodegenerative diseases and cardiac disorders.

As used herein, a "neurodegenerative disease" is selected from, but not limited to Adrenoleukodystrophy (ALD), Alexander's disease, Alper's disease, Alzheimer's disease, Amyotrophic lateral sclerosis (Lou Gehrig's Disease), Ataxia telangiectasia, Batten disease (also known as Spielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiform encephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease, Frontotemporal lobar degeneration, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabbe's disease, Lewy body dementia, Neuroborreliosis, Machado-Joseph disease (Spinocerebellar ataxia type 3), MELAS - Mitochondrial Encephalopathy, Lactic Acidosis and Stroke, Multiple System Atrophy, Multiple sclerosis, Niemann Pick disease, Parkinson's disease, Pelizaeus-Merzbacher Disease, Pick's disease, Primary lateral sclerosis, Prion diseases, Progressive Supranuclear Palsy, Refsum's disease, Sandhoff disease, Schilder's disease, Spinocerebellar ataxia (multiple types with varying characteristics), Spinal muscular atrophy, Steele-Richardson-Olszewski disease, Tabes dorsalis, Tay-Sachs Disease, and Toxic encephalopathy.

The cancer can be selected from, but no limited to bone cancer, colon cancer, multiple myeloma, gastric cancer, colorectal cancer, prostate cancer, cervical cancer, lung cancer, pancreatic cancer, medulloblastoma, liver cancer, parathyroid cancer, endometrial cancer or breast cancer. Liver disease includes, but is not limited to hepatitis, including viral hepatitis and autoimmune hepatitis; hepatitis steatosis; and cirrhosis.

Cardiac disorders include but are not limited to stroke, cardiac atrophy, heart attacks (myocardial infections), cardiomyopathy and transient ischemic attacks (TIAs). Preferably the cardiac disorder is a stroke or heart attack. In a further aspect the present invnetion relates to telmisartan or a pharmaceutically acceptable salt thereof for use in a method of promoting longevity or for alleviating or preventing premature ageing.

As used herein "promoting longevity" means increasing the expected life span of a patient. For humans the expected life span is 77-90 years in developed countries and 32— 80 years in developing countries. The use of telmisartan may increases the expected life span by 1-5%.

Promoting longevity also includes increasing the length of time a person can lead an active lifestyle without suffering from conditions associated with old age such as dementia, painful or reduced movement of limbs for example due to arthritis, or decreased cardiovascular function. The 'active' phase of a subject's life can be increased by 1-10 years, preferably 3-8 years, or 4-6 years.

"Premature ageing" as used herein refers to appearance of the signs of aging earlier than expected i.e. before old age. This includes early onset of conditions associated with old age such as degeneration of eyesight, dementia, impaired movement, cardiac conditions, as well as diseases such as Cockayne's syndrome. Premature ageing in the skin can be associated with the appearance of wrinkles, sun or liver spots, and thinning of the skin, at an earlier age than expected.

Telmisaratan or the pharmaceutically acceptable salt thereof for use as described in the present invention may form part of a pharmaceutical composition which may be presented in unit dose forms containing a predetermined amount of each active ingredient per dose. Such a unit may be adapted to provide 5-100mg/day of the compound, preferably either 5-15mg/day, 10-30mg/day, 25-50mg/day 40-80mg/day or 60-1 OOmg/ day. For compounds of formula I, doses in the range 100-lOOOmg/ day are provided, preferably either 100-400mg/day, 300-600mg/day or 500-1000mg/day. Such doses can be provided in a single dose or as a number of discrete doses. The ultimate dose will of course depend on the condition being treated, the route of administration and the age, weight and condition of the patient and will be at the doctor's discretion.

The compositions comprising telmisartan for use in the invention may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier (s) or excipient(s). Pharmaceutical formulations adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.

Pharmaceutical formulations adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research, 3(6), 318 (1986).

Pharmaceutical formulations adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.

For applications to the eye or other external tissues, for example the mouth and skin, the formulations are preferably applied as a topical ointment or cream. When formulated in an ointment, the active ingredient may be employed with either a paraffmic or a water-miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in-oil base.

Pharmaceutical formulations adapted for topical administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent. Pharmaceutical formulations adapted for topical administration in the mouth include lozenges, pastilles and mouth washes.

Pharmaceutical formulations adapted for rectal administration may be presented as suppositories or enemas. Pharmaceutical formulations adapted for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.

Pharmaceutical formulations adapted for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurised aerosols, nebulizers or insufflators. Pharmaceutical formulations adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

Pharmaceutical formulations adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which 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 water for injections, immediately prior to use.

Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

Preferred unit dosage formulations are those containing a daily dose or sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient.

It should be understood that in addition to the ingredients particularly mentioned above, the formulations may also include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.

Telmisartan for use in the present invention may be administered in combination with one or more other active ingredients known to treat the disease of interest. Telmisartan or a pharmaceutically acceptable salt thereof can be adapted for the simultaneous, separate or sequential use with one or more other active ingredients for the treatment and prevention of these diseases

The invention will now be described with reference to the following non-limiting examples which refer to the Figures described below.

Description of Figures

Figure la shows the basal activity of autophagy in HeLa cells used as a negative control (0.1% DMSO). Figure lb shows the effect of 100m Bafilomycin Al on HeLa cells which inhibits the fusion process between autophagasomes and lysosomes.

Figure lc shows the results of using 200 nM rapamycin which is a known autophagy inducer, as a positive control.

Figure Id shows the results of treating HeLa cells with 1.0 μΜ Telmisartan. Figure le shows the results of treating the HeLa cells with 10 μΜ Telmisartan.

Figure 2 compares to change in the cytoplasmic GFP/ GFP ratio induced by Telmisartan.

Figure 3a shows the results for the control non-treated, feeding wandering (90 hours) L3 larva (fat body). Figure 3b shows the results following treatment with 1.0 uM Telmisartan for feeding wandering (90 hours) L3 larva (fat body).

Figure 3c shows the results of treatment with 10 uM Telmisartan in starved, wandering (90 hours) L3 larva (fat body).

Figure 4 shows the effect of Telmisartan (20 uM) treatment on lifespan in

Drosophilia. Figure 5 compares the effect of Telmisartan, at a dose of 10 uM or 20 uM on life span in C. Elegans.

Figure 6a shows the pancreas of a mouse from the control group treated with 100 uM DMSO. Figures 6b and 6c show autophagasomes and autolysosomes (indicated by arrows) present in the pancreas of mice treated with 50 uMol Telmisartan.

Figures 6d and 6e show authophagasomes and autolysosomes (indicated by arrows) in the liver of mice treated with 50 uMol Telmisarten.

Examples

Example 1) The effects of telmisartan on human cell line (HeLa) with autophagy reporting proteins

Methods:

The GFP-RFP-LC3 HeLa cell line (Settembre et al, Science, 2011) was cultured in DMEM (Dulbecco's Modified Eagle's Medium, Sigma, D7777) containing 4500 mg/1 glucose, 10% heat inactivated FCS (Merck), 40 μg/ml gentamycin

(Hungaropharma) and 600 μg/ml G418 (Sigma, G8168).

3xl0 ⁴ cells were plated onto 13 mm diameter poly-D-lysine coated coverslips in 24- well plates (Greiner) with 24 hours before the treatment. Cells were exposed to 1 or 10 μΜ telmisartan for 6 hours. As controls, 0.1 and 1% DMSO, 200 nM rapamycin (autophagy inductor) and 100 nM bafilomycin Al (an autophagy inhibitor) were used.

For fluorescence microscopy, cells were fixed in 4% paraformaldehyde (Taab) and mounted in mowiol 4.88 (Polysciences) supplemented with bis-benzimide (Sigma) for nuclei staining. 5 epifluorescent pictures were taken in each condition by a BX51 microscope (Olympus) fitted with a FluoViewII camera and the AnalysisPro software (Olympus), using a 60X/1.4 oil Plan objective and the appropriate filter sets (DAPI: BP330-385/DM400/BA420; GFP: BP460-500/DM505/BP510-560; RFP: BP480-550/DM570/BA590).

RFP intensity shows both soluble LC3 molecules and activated LC3 along the whole autophagy process (late stages included). While GFP intensity shows soluble LC3 molecules and LC3 only in the early stages of autophagy, as GFP fluorescence is bleached by the acidic pH of the lysosome in late autophagosomes.

RFP: red fluorescence protein - autolysosome (mature autophagic compartment)

GFP: green fluorescence protein

Atg8: Autophagy-related protein 8— the most frequently used marker for autophagy Yellow: RFP and GFP (merged) - autophagosome (the primary autophagic structure)

Results

In control (DMSO), Figure la some background expression shows basal activity of autophagy in human HeLa cells. Yellow dots correspond to growing

autophagosomes (before fusing with lysosomes); in these compartments both RFP and GFP are active, resulting in yellow coloring. Red dots indicate autolysosomes (which result from the fusion of autophagosomes with lysosomes), whose lumen is acidic that leads to the degradation of GFP. Thus, autolysosomes are red.

As shown in Figure lb Bafilomycin Al inhibits the fusion process between autophagosomes and lysosomes. Thus, the autophagic process is blocked at an early stage upon treatment with this compound. All autophagic structures are represented by autophagosomes, thereby causing the appearence of yellow dots only (merged from red and green colors). As autophagy cannot proceed further yellow foci accumulate in large quantities. Figure lc shows the Positive control: Rapamycin is known as a potent autophagy inducer. It actually inhibits the kinase target of rapamycin (TOR), which serves as an important upstream negative regulator of the autophagic process. Rapamycin treatment causes a huge accumulation of autolysosomes (red dots), but the presence of several autophagosomes (yellow dots) is also evident. As can be seen from Figure Id 1.0 μΜ Telmisartan increases the number of red foci (autolysosomes), compared to negative control (0.1% DMSO, see Figure la).

Telmisartan induces the accumulation the autolysosomes - red dots - while autophagosome activation (yellow dots) is relatively unchanged. A larger dose (10.0 μΜ) Telmisartan (Figure le) significantly increased the appearance of autolysosomes - red dots, as a clear demonstration of autophagy induction.

The numeric anaysis of the samples using AnalysisPro software demonstrated markedly significant autophagy induction (p < 0,0001). The change of the cytoplasmic RFP/ GFP ratio induced by the administration of various doses of Telmisartan was calculated (Figure 2).

The following table shows the raw data of the experiments:

0.1% 200nM ΙμΜ 10μΜ

DMSO Ra amycin Telmisartan Telmisartan

1.020743134 1.053843055 1.043953577 1.336837452

1.083489708 1.220474495 1.095677964 1.212362215

1.038112472 1.184322703 1.165061348 1.272548704

1.019265026 1.039528378 1.109163728 1.144783847

0.964024718 1.002585819 1.112501615 1.36138068

0.953534275 1.0347821 1.403654161 1.586179428

1.082269354 1.052116961 1.261276856 1.115804619

1.030581156 0.980272025 1.141803239 1.632860068

0.960742339 1.231350883 1.273252844 1.236525932

0.957775968 1.050641332 1.289536047 1.350729468

1.042305231 1.076109108 1.201269598 1.328025411

1.081853056 1.217253448 1.131773139 1.277387834

1.057233997 1.059122551 1.224275613 1.200992539

1.078244773 1.046841663 1.112858422 1.233420744

1.268164555 1.075916102 1.2137908 1.270549486

1.138522249 1.005572025 1.235003964 1.064206935

1.03991095 1.070771278 1.305988136 1.151627596

1.069634088 1.061056029 1.183307544 1.133810373

1.153024766 1.064430171 1.077213013 1.146814636

1.139580166 1.002726574 1.168034493 1.081940469

1.061175269 1.019385699 1.073916145 1.087648006

1.19099555 1.058369893 1.31868289 1.118248894

1.02116226 1.005394646 1.169144536 1.098864993

1.042760696 1.029544849 1.247748376 1.05703016

1.116447569 1.104231747 1.176216802 1.060267851

1.172340914 1.050405714 1.308469328 1.115438442 1.216178028 1.097273481 1.271571253 1.101544928 1.082331484 1.089585906 1.112071904 1.071567531 1.098005392 1.095017997 1.193202801 1.089110593 1.184586118 1.072098438 1.031426535 1.119362604 1.070855625 1.165200726 1.128578688 1.248609517 0.976312987 1.289395879 1.268572955 1.156753253 0.956258901 1.036066984 1.066123768 1.041388139 0.957549531 1.213349858 1.083200219 1.095387241 0.972938021 1.215703337 1.046878091 1.199677215

1.01571793 1.111613066 1.316930455 1.373249409 1.356126382 1.141254105 1.282967782 1.196097936 1.056546361 1.126123463 1.211788815 1.340223691 1.068593783 1.215793941 1.14231823 1.355139699 1.125489413 1.094002014 1.182360971 1.37556736 1.044048511 1.157696296 1.147934292 1.168225596 1.051738929 1.072318788 1.106559953 1.062201342 1.119389494 1.072948284 1.13241011 1.077690608 1.086323738 1.015502107 0.977701465 1.146106978 1.140114275 1.121866571 1.006228251 1.057033791 1.124842349 1.060962198 1.083915667 1.081587194 1.0962467 1.160457773 1.050482734 1.223395132 1.081941956 1.097412838 1.093008856 1.086416974 1.063589688 1.026499147 1.059287248 1.003017448 0.998317137 1.214474348 1.074212554 1.093996903 1.084251609 1.131006969 1.012593935 1.216322612 1.021184576 1.095307527 1.237350565 1.181056338 1.168067238 1.209660543 1.114418706 1.079557621 1.195563312 1.084754882 1.072036225 1.189663962 1.037115136 1.128182264 1.199242884 1.12271783 1.074900115 1.073025352 1.131183126 1.103325556 1.132710863 1.221666947 1.204116537 1.147903906 1.035097142 1.387528725 1.170647046 1.085910389 1.116968369 1.2691039 1.239038843 1.321138625

1.13573382 1.15673084 1.314228378 1.338298671 1.059322483 1.045877522 1.403001329 1.139734401 1.105843487 1.107836838 1.004132346 1.105932643

0.99009803 1.069701424 1.373630525 1.35100637 0.942471093 1.07399123 1.086500664 1.266653004 1.070688687 1.173859924 1.25928339 1.20880551

1.07321344 1.127576548 0.999949003 1.326822756 1.088191382 1.107620261 1.006603014 1.306664742 1.094600965 1.108837501 1.255578898 1.17089986 1.112891119 1.161046698 1.285136053 1.313993694 1.053821066 1.007786257 1.011433241 1.312185453 1.110656421 0.96315944 1.155888431 1.186658892 1.130897111 1.05001406 1.09072431 1.329425885 1.125923726 1.111438911 1.013554569 1.357062383 1.354063687 1.103801687 1.179676672 1.292710676

1.233881756 1 .104682192 1.015920325 1.223152117

1.103490041 1 .106593015 1.144960258 1.222073482

1.159138334 1 .047659717 1.027918154 1.290556962

1.035965897 1 .106026951 0.981899469 1.141692486

1.036497309 1 .132426139 1.034048636 1.223087937

1.060470353 1 .061198422 1.093855181 1.108591836

1.024100946 1 .176459336 1.1025635 1.160215559

1.027873884 1 .209551772 1.074368496 1.103150775

0.981316807 1 .072385369 1.146890396 1.174213791

1.056869581 1 .163559188 1.134018666 1.202589849

1.086107044 1 .186326027 1.178485938 1.11560651

1.099597349 1 .304445262 1.191473255 1.259659859

1.193186713 1 .249828149 1.189675426 1.347747475

1.314452019 1 .065906309 1.289678989 1.228897978

1.170872619 1 .072981636 1.179171779 1.169280613

1.05354306 1 .213158544 1.104992571 1.127684439

1.153581229 1 .114658201 1.215715974 1.143418921

Example 2 The effects of Telmisartan in Drosophila melanogaster transgenic for an mCherry::Atg8 reporter

Methods: Flies with the genotype of hsFlppAct<CD2<Gal4, UAS-nlsGFP,r4::mCherry::Atg8a were used. Microscopic images were taken from the fat body of wandering larvae at the L3 stage. Note that in well-fed larvae at the same stage, no autophagic activity is visible. 2 hours before the test, 90-94 hours larvae were transferred onto yeast suspension (0.5 g yeast extract 2.5 ml water, homogenization and boiling). 1 g in tant medium (Carolina, Formula 4-24 Instant Drosophila medium) and 4 ml H ₂ 0 were added to the suspension, Then, 1 mM active sustances dissolved in DMSO were added, supplemented to 6.5 ml of final volume (final concentration is 10-100 mM). Larvae were cultured in this medium for 2 hours. Preparation of fat bodies was performed in PBS solution. For covering, 8:2 ratio of glycerine:PBS was used (supplemented with lOmM Hoechst solution).

Imaging was taken with a Zeiss Axiolmager Zl fluorescence microscope, supplemented with Apotome semiconfocal setup, 400x magnification, the same exposition time. Examining T544-1567 (inhibitor), hsFlp _; pAct<CD2<Gal4, UAS-n/sGFP, t4::mCherry::Atg8a flies were crossed with animals of yw; (EP)EDTP ^EY22967 genotype (the autophagy antagonist EDTP/Jumpy is clonally overexpressed in fat body).

Cells with overactivated EDTP/Jumpy are in green due to the presence of the GFP transgene. Blue colour (DAP staining) indicates the nuclei (DNA). Red dots

(mCherry::Atg8) indicate autophagy structures (autophagosomes + autolysosomes). The fat body was prepared form control (non-treated) and treated flies - this tissue consists of large, polyploid cells, in which stress-induced autophagy can relatively easily be visualized. Results:

Figure 3a shows the results for the central Control (non-treated, feeding wandering) (90 hours) L3 larva (fat body). The blue colour (DAP staining) indicates the nucleic DNA). Red dots (mCherry::Atg8) indicate autophagy structures (autophagosomes and autolysosomes). The image was taken on negative control - non treated - larvae. Only a very few red foci are visible, representing a basal level of autophagy in a well- fed larva.

Figure 3b shows the results for larva treated with 1.0 μΜ telmisartan, feeding wandering (90 hours) L3 larva (body fat). The blue colour (DAP staining) indicates the nuclei (DNA). Red dots (mCherry::Atg8) indicate autophagy structures

(autophagosomes + autolysosomes). The image was taken on a larva treated with 1.0 μΜ telmisartan. The number of red dots have significantly increased number of red dots are visible, representing an increased autophagic activity in response to the Telmisartan treatment

Figure 3c shows the results for larva treated with 10 uM telmisartan, starved, wandering (90 hours) L3 larva (fat body). In Figure 3c the blue colour (DAP staining) indicates the nuclei (DNA). Red dots (mCherry::Atg8) indicate autophagy structures (autophagosomes + autolysosomes). The image was taken on a larva treated with 10.0 μΜ telmisartan. An increased showing a markedly intensified autophagic activity in response to the Telmisartan treatment Example 3 The effects of Telmisartan in Drosophila on life-span essays.

Methods:

For lifespan assays, flies with the w (wild-type) genotype were used. 10 and 20 uM telmisartan were added to yeast suspensions, from which 100 ul were taken and dried on the surface of normal solid media. Each test contained 100 male and 100 female flies (animals were cultured in glass tubes - 20-20 males and females/ tube). Animals were transferred to new tubes with freshly prepared agents in every 2-days, and dead animals were counted by 2 days. Flies were maintained at 25 °C. Active substances were dissolved in DMSO (controls also contained DMSO). Statistics were performed by SPSS software, Kaplan-Meyer curves were generated for survival data.

Results

As shown in Figure 4 Telmisartan extends lifespan in Drosophila, and this longevity effect is particularly evident at advanced ages. The differences are statistically highly significant (Mantel-Cox longrank test, p < 0,0001)

Example 4 The effects of Telmisartan in C Elemns on life-span essays.

Telmisartan dose dependently extends lifespan in C elegans (Figure 5). The differences are statistically highly significant (Mantel-Cox longrank test, p < 0,0001)

Example 5 The effects of Telmisartan on autophagic activity in mice Methods:

50 μΜοΙ Telmisartan was dissolved in 10% DMSO and administered

intraperitoneally. After 2.5 hours the animals were sacrificed and their organs removed. The organs were transfered to tissue cultivating solution.

The organs were prepared for electronmicroscopic studies by standard preparation techniques. The ultrathin slices were prepared using Reichert-Jung Ultracut-E type ultramicrotome. The slides were investigated by JEM Jeol 1011 electron microscope. Results:

Figure 6a shows the results obtained for the Control: 100 μΐ DMSO. No autophagic structures are visible (pancreas)

The administration of 50 μΜοΙ Telmisartan shows pronounced autophagic activity represented by autophagosomes and autolysosomes (pancreas) (Figures 6b and 6c)

Autophagosomes appear in the liver as well (Figures 6d and 6e)

References:

1. Levine B, Kroemer G (2008). Autophagy in the pathogenesis of disease. Cell 132, 27-42

2. Mizushima N, Levine B, Cuervo AM, Klionsky DJ (2008). Autophagy fights disease through cellular self-digestion. Nature. 451,1069-1075.

3. Toth, M.L. et al. (2008). Longevity pathways converge on autophagy genes to regulate life span in Caenorhabditis elegans. Autophagy 4, 330-338. 4. Simonsen, A. et al. (2008). Promoting basal levels of autophagy in the nervous system enhances longevity and oxidative stress in adult Drosophila. Autophagy 4, 176— 184.

5. Xue Y et al. (2003). Genetic Analysis of the Myotubularin Family of Phosphatases in Caenorhabditis elegans. ]. Biol Chem. 278, 34380-34386, 6. Robinson FL, Dixon JE (2006). Myotubularin phosphatases: policing 3- phosphoinositides. Trends Cell Biol. 16, 403-408.