WO2012045451A1

WO2012045451A1 - Novel therapeutic treatment of progranulin-dependent diseases

Info

Publication number: WO2012045451A1
Application number: PCT/EP2011/004976
Authority: WO
Inventors: Anja Capell; Christian Haass; Katrin Fellerer
Original assignee: Ludwig-Maximilians-Universitaet Muenchen
Priority date: 2010-10-05
Filing date: 2011-10-05
Publication date: 2012-04-12

Abstract

The present invention relates an alkalizing agent for use in the treatment, acceleration, or amelioration of a disease and/or the improvement of the outcome of a disease, wherein said disease is a disease which benefits from an enhanced and/or restored progranulin level. Said alkalizing drug finds particularly use in the treatment of wound healing, inflammatory diseases, chronic pain and/or neurodegenerative diseases. It is also envisaged that said disease is caused by haploinsufficiency of the progranulin gene (GRN). The present invention also relates to a pharmaceutical composition, package or kit for use in the treatment, acceleration, amelioration of a disease (or symptoms associated thereto) and/or the improvement of the outcome of a disease, wherein said disease is a disease which benefits from an enhanced and/or restored progranulin level, and wherein said kit or package comprises an alkalizing drug and optionally a package insert or instructions. In a further embodiment, the present invention relates to a method of screening for a pharmaceutically active drug for use in the treatment, acceleration, amelioration of a disease (or symptoms associated thereto) and/or the improvement of the outcome of a disease, wherein said disease is a disease which benefits from an enhanced and/or restored progranulin level, said method comprising the following steps: (a) contacting a test-compound with a test cell, and (b) evaluating an alteration in the intracellular pH of said test-cell, wherein an increase in the intracellular pH indicates that said test compound is a pharmaceutically active drug for use in the treatment, acceleration, amelioration of a disease (or symptoms associated thereto) and/or the improvement of the outcome of a disease, wherein said disease is a disease which benefits from an enhanced and/or restored progranulin level.

Description

Novel therapeutic treatment of Progranulin-dependent diseases

The present invention relates an alkalizing agent for use in the treatment, acceleration, or amelioration of a disease and/or the improvement of the outcome of a disease, wherein said disease is a disease which benefits from an enhanced and/or restored progranulin level. Said alkalizing drug finds particularly use in the treatment of wound healing, inflammatory diseases, chronic pain and/or neurodegenerative diseases. It is also envisaged that said disease is caused by haploinsufficiency of the progranulin gene (GRN). The present invention also relates to a pharmaceutical composition, package or kit for use in the treatment, acceleration, amelioration of a disease (or symptoms associated thereto) and/or the improvement of the outcome of a disease, wherein said disease is a disease which benefits from an enhanced and/or restored progranulin level, and wherein said kit or package comprises an alkalizing drug and optionally a package insert or instructions. In a further embodiment, the present invention relates to a method of screening for a pharmaceutically active drug for use in the treatment, acceleration, amelioration of a disease (or symptoms associated thereto) and/or the improvement of the outcome of a disease, wherein said disease is a disease which benefits from an enhanced and/or restored progranulin level, said method comprising the following steps: (a) contacting a test-compound with a test cell, and (b) evaluating an alteration in the intracellular pH of said test-cell, wherein an increase in the intracellular pH indicates that said test compound is a pharmaceutically active drug for use in the treatment, acceleration, amelioration of a disease (or symptoms associated thereto) and/or the improvement of the outcome of a disease, wherein said disease is a disease which benefits from an enhanced and/or restored progranulin level.

Progranulin (GRN), also known as proepithelin, acrogranin, or prostate cancer cell- derived growth factor, is a secreted protein with important functions in several processes, including immune response, wound healing and embryonic development (Tolkatchev et al., 2008). Local administration of progranulin potently inhibits inflammation, such as neutrophilic inflammation, in vivo, which demonstrates that this protein represents a crucial inflammation-suppressing mediator (Kessenbrock et al., J. Clin. Invest. 118:2438-2447 (2008); Yin et al., J. Exp. Med. Vol. 207 No. 1 117-128). The role of Progranulin as a mediator of the wound response (wound related growth factor) was demonstrated in He et al., Nature Medicine, Vol 9(2), 225 (2003)). It was also reported that progranulin play a pivotal role in chronic pain and in particular in neuropathic pain (WO2009/010045).

GRN is expressed by epithelial cells, macrophages, and neurons but also in strongly proliferating tissues such as adenoid tissue, spleen, skin epithelium, gastrointestinal mucous membranes, and haematopoietic cells. It is a 68.5 kDa pre cursor protein, consisting of 593-amino acid which are composed of seven granulin-like domains. Progranulin appears in strongly glycosylated form (in vivo) and therefore has a size of approximately 90 kDa. The protein undergoes proteolysis to generate seven mutually homologous 6-kD peptides, called granulins or epithelins. These smaller cleavage products are named granulin A, granulin B, granulin C, etc. Epithelins 1 and 2 are synonymous with granulins A and B, respectively. Until now no specific receptors, which would obtain the effect of Progranulin or the granulins are known.

Frontotemporal lobar degeneration (FTLD) is the second most abundant form of dementia in people under the age of 60 years after Alzheimer's disease (AD) (Graff- Radford and Woodruff, 2007). While about 40% of FTLD patients are pathologically characterized by tau positive inclusions, the remaining patients present with tau and alpha-synuclein-negative, ubiquitin-positive nuclear or cytoplasmic inclusions (frontotemporal lobar degeneration with ubiquitin-positive inclusions; FTLD-U) (Mackenzie and Rademakers, 2007, Cruts and Van Broeckhoven, 2008). Deposited proteins observed in FTLD-U brains include the TAR-DNA binding protein 43 (TDP-43; FTLD-TDP) (Neumann et al., 2006) and the fused in sarcoma protein (FUS; FTLD-FUS) (Kwiatkowski et al., 2009). Genetic linkage studies and/or mutation screening identified approximately 70 mutations in the progranulin (GRN) gene in patients with familial FTLD- TDP (http://www.molgen.ua.ac.be/FTDMutations/) (Gijselinck et al., 2008). Most of the mutations identified are loss of function mutations, which lead to a severe reduction of GRN levels in tissues and biological fluids of patients (Baker et al., 2006, Cruts et al., 2006, Cruts and Van Broeckhoven, 2008, Sleegers et al., 2009). Additionally, missense mutations (e.g. (Schymick et al., 2007, Van Der Zee et al., 2007), (Brouwers et al., 2008)) lead to cytoplasmic missorting and degradation of GRN (Mukherjee et al., 2008, Shankaran et al., 2008) or to reduced secretion probably due to misfolding (Shankaran et al., 2008). Thus all FTLD-TDP associated GRN mutations investigated so far result in a deficiency of GRN. Reduced GRN levels in biological fluids such as cerebrospinal fluid and plasma or serum are not only sensitive biomarkers but also predict GRN mutations and a significantly enhanced risk for FTLD-TDP (Ghidoni et al., 2008, Finch et al., 2009, Sleegers et al., 2009). Together with the observation that GRN has neurotrophic properties (Van Damme et al., 2008) these findings strongly indicate that GRN haploinsufficiency is causally related to neurodegeneration.

Having regard to the above it is evident that Progranulin plays a pivotal role in many medical conditions (including inflammation, wound healing and neurodegenerative diseases) where it appears to act as a key mediator. GRN as such was therefore already suggested as a medicament for the treatment of some of these diseases. The use of recombinantly produced progranulin as a therapeutic effective ingredient of a pharmaceutical composition is, however, unwanted as it cannot be excluded that the addition of an exogenous protein to a human or animal subject provokes undesirable adverse reactions such as allergic or immunological side effects. Thus, the technical problem underlying the present invention was to provide means and methods which can improve the outcome (chances of recovery) of wound healing, inflammatory diseases and/or neurodegenerative diseases by way of enhancing the production and/or secretion of progranulin. The present inventors addressed this need and surprisingly discovered compounds that are capable of stimulating GRN production and/or secretion. These compounds can thus be used to restore or enhance the GRN level in a subject. Most to our surprise, the present inventors were able to show that it is possible to enhance the GRN secretion and/or to restore the physiological GRN level when administering alkalizing agents to relevant cells and/or tissues. In view of that it is proposed to employ drugable alkalizing agents for stimulating the progranulin production and/or secretion in a subject. These drugable alkalizing compounds enhance the production and/or secretion of progranulin and, thereby, improve the outcome (chances of recovery) of diseases which benefit from an enhanced and/or restored progranulin level. "Diseases which benefit from an enhanced and/or restored progranulin level" include at least wound healing, inflammatory diseases neurodegenerative diseases, and/or chronic pain such as neuropathic pain. A particularly preferred disease which can be treated or ameliorated in accordance with the present invention is a disease caused by haploinsufficiency of the progranulin gene (GRN).

The term "diseases which benefit from an enhanced and/or restored progranulin level" as used herein is exchangeable with "disease associated with a decreased progranulin level".

The term "subject" when used herein includes animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In preferred embodiments, the subject is a human. The compositions, compounds, uses and methods of the present invention are thus applicable to both human therapy and veterinary applications.

Disclosed herein are also methods of determining whether a subject has or is at risk for a disease which benefits from a restored progranulin level. Specifically provided is a method of determining whether a subject has or is at risk for a disease which benefits from a restored progranulin level, comprising determining the progranulin level (protein level) in a biological sample obtained from said subject, wherein a decrease in expression or activity of progranulin as compared to a control indicates the subject has or is at risk for a disease associated with a decreased progranulin level. The mentioned biological sample is preferably a sample comprising or consisting of a biological fluid. Said biological fluid is preferably cerebrospinal fluid, or a blood based fluid such as plasma or serum (obtained from the patient).

It is likewise envisaged that the alkalizing agent of the present invention increases the progranulin protein (GRN) level in the extracellular space. "Extracellular space" means "outside the cell". This space is usually taken to be outside the plasma membranes, and occupied by fluid.

The above methods may also be carried out before and after the administration of an alkalizing drug (for example chloroquine), in order to verify that the alkalizing drug enhances or restores the biological level of progranulin in the above mentioned biological samples, thereby indicating that the alkalizing drug is therapeutically effective (i.e. enhances or restores the progranulin level). The term "restores" means that at least the "normal" progranulin level is achieved once the alkalizing drug(s) of the present invention has/have been administered to the subject (which subject had a decreased progranulin protein level prior to said administration). The term "decrease" means a negative deviation from the "normal" progranulin level (protein) in the biological samples, i.e. a negative deviation from the standard value or "control" which is characteristic for (a) healthy subject(s). Said control value is sometimes also denoted as "physiological" or "biological" value, and can be determined in one healthy subject or in a cohort of healthy subjects.

Means and methods to test/evaluate/determine the progranulin protein level in a biological sample are also well-known. Test systems which allow the reliable determination of the progranulin protein level in a biological sample are even commercially available (for example ELISA test systems) and furthermore described in sufficient detail in the appended examples. In fact, the present inventors analyzed in a pre-study the plasma progranulin level over four weeks in nine healthy volunteers. Plasma samples were taken twice a week and progranulin was measured by ELISA in triplicates. Aim of the study was to evaluate the steadiness of the progranulin plasma concentration within one patient, because high differences among patients have been reported before. However, from Figure 14 it is apparent that, though differences in the progranulin level may exist between patients, the progranulin level in each patient does not at all significantly vary, i.e., it is stable. Accordingly, progranulin plasma levels are quite stable over time within one healthy volunteer although high variations may occur between different volunteers. However, it is commonly known in the art to use the median value as reference or control value. Accordingly, in the uses and methods of the present invention preferably the median value calculated on the basis of preferably a plurality of progranulin levels determined from healthy volunteers, i.e., subjects not having a disease caused by haploinsufficiency of progranulin (GRN) is applied as a reference or control value.

Accordingly, a reference (or control) value may be determined as the median or as specific percentile (i.e., 0.90 or 0.95) of the measured levels in a population of healthy individuals not suffering from a disease caused by haploinsufficiency of the progranulin gene (GRN). Evaluating the levels in further individuals or patients, e.g. in cohort studies, can help to refine the known levels or ratios. In the simplest case, the reference value is the same as the level measured in the control sample or the average of the levels measured in a multitude of control samples. However, the reference value may also be calculated from more than one control sample. E.g., the reference value may be the arithmetic average of the level in control samples representing the control status (e.g. healthy). Preferably, the reference value relates to a range of values that can be found in a plurality of comparable control samples (control samples representing the same or similar disease status), e.g. the average one or more times the standard deviation.

Similarly, the reference value may also be calculated by other statistical parameters or methods, for example as a defined percentile of the level found in a plurality of control samples, e.g. a 90 %, 95 %, 97.5%, or 99 % percentile. The choice of a particular reference value may be determined according to the desired sensitivity, specificity or statistical significance (in general, the higher the sensitivity, the lower the specificity and vice versa). Calculation may be carried out according to statistical methods known and deemed appropriate by the person skilled in the art.

Another method to test whether a subject has or is at risk for a disease which benefits from a restored progranulin level is to evaluate the mRNA expression level of progranulin (with well-known methods such as mRNA-profiling, RT-PCR, quantitative RT-PCR, Northern blot, fluorescence in situ hybridization-techniques etc). Provided that the mRNA expression profile of a relevant sample obtained from a patient is decreased (negative deviation from the normal progranulin level (mRNA) in a cell or tissue sample when compared to a control sample) then it is likely that the respective disease benefits from a restored progranulin level. Alternatively or additionally it is possible to evaluate whether the subject is characterized by a haploinsufficiency for the progranulin gene. Haploinsufficiency occurs when a diploid organism has one single functional copy of a gene only (the other copy being inactivated by mutation) and the single functional copy of the gene does not produce enough of a gene product (progranulin) to bring about a wild-type condition, leading to an abnormal or diseased state. It is well-known that GRN mutations frequently result in haploinsufficiency (Baker, ., et ah, Nature 442:916-919 (2006); Cruts, M., et ah, Nature 442:920-924 (2006)). It is for example well-established that GRN mutations are present in nearly 50% of familial FTD cases, making GRN mutation a major genetic contributor to FTD (Cruts, M. & Van Broeckhoven, C, Trends Genet. 24:186-194 (2008); Le Ber, I., et ah, Brain 129:3051- 3065 (2006)). The loss-of- function heterozygous character of GRN mutations implies that in healthy subjects, GRN expression plays a dose-dependent, critical role in protecting healthy subjects. Loss-of- function mutations are the result of gene product having less or no function. When the allele has a complete loss of function (null allele) it is often called an amorphic mutation. Phenotypes associated with such mutations are most often recessive. Exceptions are when the organism is haploid, or when the reduced dosage of a normal gene product is not enough for a normal phenotype (this is then called haploinsufficiency). Put differently, A haploinsufficient gene is described as needing both alleles to be functional in order to express the wild type. A mutation as such is not haploinsufficient, but dominant loss of function mutations are the result of mutations in haploinsufficient genes.

An example for a disease which benefits from a restored progranulin level is frontotemporal lobar degeneration with tau-negative, ubiquitin-positive inclusions (FTDL- U).

The skilled person is also well-aware which medical conditions are covered by the term "disease which benefits from an enhanced progranulin level". The term "enhanced" thereby denotes a situation where the "normal" progranulin level is increased. The term "increase" means a positive deviation from the "normal" progranulin level (protein) in the biological samples, i.e. a positive deviation from the standard value or "control" which is characteristic for (a) healthy subject(s).

As mentioned hereinbefore, the administration of progranulin potently inhibits inflammation (i.e. progranulin is anti-inflammatory), (Kessenbrock et al., J. Clin. Invest. 118:2438-2447 (2008); Yin et al., J. Exp. Med. Vol. 207 No. 1 117-128), mediates the wound response (i.e. progranulin is a wound related growth factor) (He et al., Nature Medicine, Vol 9(2), 225 (2003), and alleviates pain (for example chronic pain (WO2009/010045)). It turned out that progranulin mRNA was unregulated in these medical conditions (see for example WO2009/010045 or He et al., Nature Medicine, Vol 9(2), 225 (2003)), indicating the decisive influence of progranulin in these medical conditions.

A "disease or medical condition which benefits from an enhanced progranulin level" is thus typically a disease which is anyway characterized by an increased expression level of progranulin (either on protein and/or mRNA level) during a certain stage in the course of the disease. The mentioned stage is typically the point of time when the progranulin protein exerts a wanted effect in the course of an unwanted medical condition (progranulin switches off, for example, the inflammatory phenotype of leukocytes - see Kessenbrock et al., J. Clin. Invest. 1 18:2438-2447 (2008)).

An example for a disease which benefits from an enhanced progranulin level is for example wound healing, inflammatory diseases, chronic pain and/or neurodegenerative diseases. It will be understood that some diseases might not just benefit from a restored but also from an enhanced progranulin level.

In view of the above, it is evident that the present specification contains a teaching in the form of testable criteria allowing the skilled person to recognize which medical conditions fall within the functional definition of a disease which benefits from an enhanced and/or restored progranulin level and accordingly within the scope of the present invention.

It is envisaged that the step of obtaining the biological sample is as such not necessarily included in the scope of the present invention.

The present invention is based on the unexpected discovery of certain compounds which are capable of stimulating GRN production and/or secretion in biological samples. Most to our surprise, it came as a surprise for the inventors to show that it is possible to enhance the GRN secretion and/or to enhance/restore the physiological GRN level when administering alkalizing agents to the respective cells and/or tissues. In view of that it is proposed to employ drugable alkalizing agents for stimulating the progranulin production and/or secretion in a subject.

The term "drugable" or drugable alkalizing agents or alkalizing drug(s) includes all kinds of alkalizing agents which (a) can be formulated into a pharmaceutical formulation and (b) which are usable as a medicament in human or veterinary medicine to treat, accelerate, ameliorate and/or improve the outcome of the diseases (or symptoms associated therewith) which benefit from an enhanced and/or restored progranulin level. "Usable" means that the drugable compounds disclosed herein exert a therapeutic effect which effect can at least be characterized by the capability of the drugable alkalizing agent to restore or increase the progranulin level (protein) in said subject, which increase can be determined for example in a biological sample obtained from the subject before and after the administration of the respective drug. Preferably, said use is approved by a regulatory authority such as the EMEA or the FDA.

Alternatively it is also possible to test the capability of the drugable alkalizing agent to increase the progranulin level (protein) in an in vitro system based, for example, on lymphoblasts derived from healthy control subjects. Said capability of the alkalizing drugs disclosed herein can thus be tested or verified by established models, for example the test assays described in the appended examples and herein. For example, in order to test whether progranulin can be increased to physiological levels in a disease stage, lymphoblasts derived from four healthy controls and six patients with confirmed familial FTLD-TDP associated GRN loss-of-function mutations (Brouwers et al., 2007, Gijselinck et al., 2008) were treated with BafA1 and chloroquine (CQ). CQ is of special interest as it is frequently used for malaria prophylaxis and treatment, in auto-immune disorders and in sensitizing cancer therapy (Solomon and Lee, 2009). As expected, absolute levels of GRN were reduced in conditioned media obtained from cells with GRN loss-of-function mutations in comparison to cells derived from healthy controls (Fig. 9A & B). Levels of GRN were significantly increased upon stimulation with BafA1 to levels at least similar to untreated cells of healthy control subjects (Fig. 9A). CQ treatment led to a less pronounced but still significant increase of GRN secretion (Fig. 9B). Moreover, the two additional FDA approved drugs (Bepridil and Amiodarone) which have a known alkalizing potential were able to increase the progranulin level (protein) in this experimental setting (Fig. 9C). Thus, our data demonstrate that GRN deficiency can be rescued with alkalizing drugs in lymphoblasts derived from patients with familial FTLD- TDP and in brain slices of GRN*^1' mice, which both recapitulate disease associated GRN deficiency.

The ability of the compounds of the present invention to act as enhancers of the progranulin expression/secretion makes them useful pharmacological agents for disorders that involve progranulin in humans and animals, but particularly in humans.

Thus, in a first aspect, the present invention relates to an alkalizing agent for use in the treatment, acceleration, amelioration of a disease (or symptoms associated thereto) and/or the improvement of the outcome of a disease, wherein said disease is a disease which benefits from an enhanced and/or restored progranulin level. It will be understood that the alkalizing drugs disclosed herein enhance and/or restore the physiological progranulin level in a subject and thereby treat, accelerate, and/or ameliorate the mentioned diseases (which diseases are caused by progranulin (GRN) -deficiency).

In a preferred embodiment, said diseases which benefit from an enhanced and/or restored progranulin level include at least wound healing, inflammatory diseases neurodegenerative diseases, and/or chronic pain such as neuropathic pain. Methods to determine a disease which benefits from a restored progranulin level have been disclosed herein before.

"Wound healing" or wound repair, is an intricate process in which the skin or another organ repairs itself after injury.Said term includes chronic and acute wounds. A "chronic wound" refers a wound that does not heal, see, e.g., Lazarus et al, Definitions and guidelines for assessment of wounds and evaluation of healing, Arch. Dermatol. 130:489-93 (1994). Chronic wounds include, but are not limited to, e.g., arterial ulcers, diabetic ulcers, pressure ulcers, venous ulcers, etc. An acute wound can develop into a chronic wound. Acute wounds include, but are not limited to, wounds caused by, e.g., thermal injury, trauma, surgery, excision of extensive skin cancer, deep fungal and bacterial infections, vasculitis, scleroderma, pemphigus, toxic epidermal necrolysis, etc. See, e.g., Buford, Wound Healing and Pressure Sores, HealingWell.com, published on: October 24, 2001. A "normal wound" refers a wound that undergoes normal wound healing repair. The term "inflammatory disease", e.g, an acute or chronic inflammatory disorder, includes in accordance with the present invention Crohn's disease, reactive arthritis, including Lyme disease, insulin-dependent diabetes, organ- specific auto immunity, Hashimoto's thyroiditis and Grave's disease, contact dermatitis, psoriasis, organ transplant rejection, graft rejection, graft versus host disease, sarcoidosis, atopic conditions, gastrointestinal allergies, including food allergies, pancreatitis, eosinophilia, conjunctivitis, glomerular nephritis, multiple vasculitides, myasthenia gravis, asthrria, chronic obstructive pulmonary disease, myocardial infarction, stroke, transplant rejection, reperfusion injury, autoimmune disease (e.g, Ankylosing spondylitis, systemic lupus erythematosus (SLE), or the like) inflammatory bowel disease, psoriasis, arthritis (including, e.g., rheumatoid arthritis), allergic rhinitis, berillium disease, bronchiectasis, bronchitis, bronchopneumonia, cystic fibrosis, diphtheria, dyspnea, emphysema, allergic bronchopulmonary aspergillosis, pneumonia, acute pulmonary edema, pertussis, pharyngitis, atelectasis, Wegener's granulomatosis, Legionnaires disease, pleurisy, rheumatic fever, sinusitis or the like, but is not limited thereto.

-Neutrophilic inflammation'' is preferred. Said term includes and denotes inflammation mediated, at least in part, by the function and/or activity of neutrophilic cells, including, but not limited to, the release of mediators by neutrophilic cells, the death of neutrophilic cells and the activity of cells and processes activated or inhibited by the function of neutrophilic cells. Optionally, the neutrophilic inflammation is chronic neutrophilic inflammation. Diseases associated with neutrophilic inflammation include, but are not limited to asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), pulmonary transplantation rejection, chronic bronchitis, emphysema, bronchiectasis, bronchiolitis obliterans syndrome (BOS), interstitial pneumonia, pulmonary fibrosis, bacterial infection and viral infection.

The term "chronic pain" can be divided into "nociceptive pain" (caused by activation of nociceptors), and "neuropathic pain" (caused by damage to or malfunction of the nervous system), neuropathic pain being preferred.

The term "neurodegenerative diseases" are a group of disorders characterized by changes in neuronal function, leading in the majority of cases to loss of neuron function and cell death. Neurodegenerative disorders (diseases) include, but are not limited to, Alzheimer's diseases, Pick's disease, diffuse Lewy Body disease, progressive supranuclear palsy (Steel-Richardson syndrome), multisystem degeneration (Shy- Drager syndrome), motor neuron diseases including amyotrophic lateral sclerosis, degenerative ataxias, cortical basal degeneration, ALS-Parkinson's-Dementia complex of Guam, subacute sclerosing panencephalitis, Huntington's disease, Parkinson's disease, synucleinopathies, primary progressive aphasia, striatonigral degeneration, Machado -Joseph disease/spinocerebellar ataxia type 3, or olivopontocerebellar.

Consistent with the neutralizing effect of BafA1 on acidic cellular compartments which is shown in more detail in the appended examples, monitoring of the intracellular pH after treatment with BafAl and in untreated cells proved that BafAl rapidly and reversibly affects the cellular pH. While in control cells lysosomes and other acidic vesicles were readily stained with LysoSensor, no labeling was observed immediately after treatment (Fig. 4). However, upon withdrawal of BafAl the intracellular pH rapidly normalized as proven by the robust vesicular staining with lysosensor after 4, 8, and 24 h (Fig. 4). Thus BafAl prevents vesicular acidification in our cellular system and this effect is fully reversible. Consistent with the alkalizing activity of BafAl , other alkalizing reagents such as NH4CI and chloroquine (CQ) independently elevated endogenous GRN levels in cell lysates and in conditioned media of neuronal and non-neuronal cell lines (Fig. 5A & Fig. 10). BafAl reaches its maximal effect on GRN at 25 - 50 nM (Fig. 5B) without affecting general protein secretion (Fig. 5B). CQ increased GRN levels dose-dependently up to 50 μΜ (Fig. 5C). Even at high doses of CQ no general effect on total protein secretion was observed (Fig. 5C). It could thus be shown that alkalizing agents positively influence the expression of progranulin and thereby the amount of progranulin in the extracellular space.

The term "alkalizing agent" which is preferably an alkalizing drug (i.e. a drugable alkalizing agent), means in the context of the present invention that the respective agent is able to modify the pH gradient in a test cell leading to an increase of the pH in said test cell. The mentioned "test cell" is for example a neuronal primary culture (such as a cortical slice culture - see also the appended examples) or a peripheral cell line such as HEK293 although the present invention is not limited thereto. The skilled person is well aware that several cells/cell lines might be employed in order to test the alkalizing capabilities of a potential alkalizing agent, i.e. an agent which is capable of increasing the intracellular pH.

Said increase of the intracellular pH which is achieved by the alkalizing agents of the present invention (including the pharmaceutical compositions of the present invention) is preferably between about 0,4 to 0.6 (including the limits) although it is not excluded (but not preferred) that the increase of the pH exceeds a value of 0,6. It is preferred that the "alkalinizing agent" of the present invention when contacted in vitro or in vivo with a cell causes the intracellular pH to be maintained above pH 6.8, but still within acceptable physiological limits (preferably below pH 7.6) following administration of an alkalinizing agent. It is preferred that said increase in the intracellular pH occurs in the endolysosomal pathway or endolysosomal system. Means and methods to evaluate the pH in a cell or in a cell compartment are well-known to the skilled person and also exemplified herein.

The "endolysosomal system" comprises a series of membrane-bound intracellular compartments, within which extracellular material flow vectorially, proceeding through a series of vesicle-like organelles, the main ones being the early endosome, the endosome carrier vesicle, the late endosome and the lysosome. The different components of the endolysosomal system are competent for specific proteolytic activities, and the whole process is highly dependent from the calcium concentration and the pH inside the vesicles (Pillai and Panchagnula, Cur Opin Chem Biol, 5: 447- 451 ,2001.; Sachse M et al., Histochem Cell Biol, 117: 91-104,2002. It is envisaged that the alkalizing agents of the present invention either directly or indirectly increase the pH of the respective cells (leading to an increase of progranulin protein level in a biological fluid). "Directly" means that the compound (alkalizing agent) as such is able to alkalize the pH itself (due to its chemical capabilities for example due to its pKa value (preferably >8.0) and log P values preferably >2.4). "Indirectly" means that the compound is able to inhibit/block for example proton pumps which are responsible for the acidification of the cell (for example the V-ATPase) or chloride ion channel exchanger proteins which also trigger the intracellular pH of a cell (see for example WO/1996/034604). WO/1996/034604 is a document which provides several alkalizing agents all of which are incorporated herein by way of reference.

An alkalinizing agent can be an organic base which can permeate the cell or be delivered to the cytosol to maintain the intracellular pH above pH 6.8. Another biochemical characteristic and an advantage of an organic base useful as an alkalinizing agent of the invention is that the agent is uncharged at physiologic pH (approximately pH 7.4) which allows it to permeate the cell during in vivo and in vitro administration. Alkalinizing agents of the invention specifically include organic bases such as imidazole, or ethanolamine. Furthermore, chloroquine (including hydroxychloroquine and chloroquine phosphate) (see Figure 13 for the potential of hydroxychloroquine to increase progranulin), chloroquine diphosphate and fluoroquinolones (such as but not limited to, moxifloxacin, ciprofloxacin, ofloxacin, levofloxacin, lomefloxacin, norfloxacin, enoxacin, gatifloxacin, and sparfloxacin) polyether ionophore antibiotics such as monensin, macrolide antibiotic derived from Streptomyces griseus, preferably pleco- macrolides such as bafilomycin A1 and tamoxifen have been shown to prevent acidification of vesicles and are therefore specifically included within the scope of the present invention. Means and methods to screen for such agents are well known - WO/2003/014386 discloses for example means and methods to screen for macrolide antibiotics.

Further compounds which are envisaged in the context of the present invention are bepridil, amiodarone, amlodipine, astemizole, benztropine, camylofin, chlorprothixene, clomiphene, cloperastine, cyclobenzaprine, cyproheptadine, doxepine, drofenine, fendiline, fluoxetine, maprotiline, norfluoxetine, nortriptyline, paroxetine, pimethixene, promazine, promethazine, protriptyline, sertraline, suloctidil, terfenadine, and/or triflupromazine.

The pH of intracellular compartments and extracellular space is carefully controlled by the proton pump V-ATPase, a multiprotein complex, which is ubiquitously expressed in all cell types and is localized in cellular organelles such as Golgi, lysosomes, endosomes and at the plasmamenbrane (summarized by (Forgac, 2007)). Vacuolar-type H+-ATPase (V-ATPase) is a highly conserved evolutionarily ancient enzyme with remarkably diverse functions in eukaryotic organisms. V-ATPases acidifiy a wide array of intracellular organelles and pump protons across the plasma membranes of numerous cell types. These proton pumps couple the energy of ATP hydrolysis to proton transport across intracellular and plasma membranes of eukaryotic cells.

The invention demonstrates that specific inhibitors of the V-ATPase like the well known plecomacrolides BafA1 and concanamycin A as well as the novel inhibitors archazolid B and apicularen A (summarized by (Huss and Wieczorek, 2009)) increase intracellular and thus consequently extracellular GRN levels at nanomolar concentrations. These compounds including the compounds disclosed in the relevant documents cited herein are also envisaged in the context of the present invention. Off target effects are unlikely since inhibitors of different chemical classes consistently increase GRN expression. Moreover apicularen A and plecomacrolides are known to bind to distinct sites of the V- ATPase (Huss et al., 2005). It can be ruled out that inhibitors of V-ATPase affect putative receptor mediated uptake of GRN, since GRN accumulates upon treatment with BafA1 not only in conditioned media but also in cell lysates. Moreover, even when cellular transport through the secretory pathway is blocked beyond the Golgi, GRN still accumulates in cell lysates even in the absence of any secretion. Finally, it can be excluded that transcription of GRN is upregulated upon inhibition of V-ATPase. The present inventors' findings therefore suggest a translational upregulation of GRN caused by intracellular pH changes induced by V-ATPase inhibitors or other alkalizing drugs. Indeed increased GRN transcription as observed upon aberrant lysosomal conditions (Sardiello et al., 2009) could be ruled out. It is demonstrated that the GRN increase evoked by the alkalizing drug CQ or by BafA1 can compensate reduced levels of GRN in lymphoblasts of GRN-associated FTLD-TDP patients and in organotypic slice cultures derived from a mouse model for GRN deficiency. Therefore specific V-ATPase inhibitors, such as those currently discussed in cancer therapy (Fais et al., 2007), or alkalizing drugs may be developed or further improved to increase GRN levels in FTLD-TDP patients with GRN-mutation to physiologically normal levels. Such drugs may be tolerated without major adverse side effects, as shown for CQ a frequently used malaria drug (Solomon and Lee, 2009). To ensure correct dosing of such drugs, established ELISA assays are available for convenient monitoring of GRN levels in plasma and cerbral spinal fluid (Ghidoni et al., 2008, Finch et al., 2009, Sleegers et al., 2009). The above findings and observations are backed up by knock-down experiments conducted by the present inventors. In particular, siRNA was used for the knock down of the cytoplasmic v-ATPase subunit c (ATP6V0C). siRNA was transfected twice (at day 1 +3) and GRN expression was analyzed at day 5 by ELISA. V-ATPase subunit c knock down was verified by qRT-PCR. It turned out that knock down of the ATPaseVoC subunit results in progranulin increase (see Figure 12). ATPases are described in "Handbook of ATPases" Ed. Futai, Wada and Kaplan, Wiley-VCH Verlag GmbH & Co. KGaA, 2004 (see in particular Figure 15.1 ). Accordingly, as an alternative to an alkalizing agent, siRNA or antisense RNA directed against ATPaseVoC, in particular against v-ATPase subunit c (ATP6V0C) (see "Handbook of ATPases", cited above) may be used in the uses and methods of the present invention. The alkalizing agent of the present invention includes all kinds of V-ATPase inhibitors which are meanwhile very well-known to the skilled person and also commercially available. Such inhibitors are exemplarily disclosed in WO/2008/124072, WO/2007/048848, WO/1994/004161 , WO/2007/035734 or WO/2008/058897 to name some, all of which are included herein by way of reference thereto. Several V-ATPase inhibitors have been synthesised, cf. Curr.Pharm. Design _n8,2033-2048, 2002. For V- ATPase inhibitor it is meant in a preferred embodiment a compound which, when assayed according to J Nadler, G. et al- Bioorg. Med. Chem. Letters 8, 3621-3626, 1998, causes at least 50% inhibition of the bafilomycin-sensitive ATPase activity at concentrations of 5 μΜ. V-ATPase inhibitors may be of natural, semi-synthetic or entirely synthetic origin. Examples of V-ATPase inhibitors of natural origin are the macrolides bafilomycins, concanamycins, depsipeptide mycotoxins derived from the fungi Metharisium anisopliae, destruxin, lobatamides, saiicylihaiamides, oximidines; examples of semi-synthetic derivatives are the sulphonamide derivatives of bafilomycins, 7,21- O- disubstituted bafilomycins, 2-methoxy-2,4-pentadienoic esters of bafilomycins, etc.; examples of entirely synthetic V-ATPase inhibitors are N-ethylmaleimide, 7- chloro-4- nitrobenzo-2-oxa-1 ,3-diazole, and derivatives thereof, the compounds WY 47766, SB242784, aminoquinoline derivatives like the compound FR167356. A number of V- ATPase inhibitors have been described for use in preventing bone loss and inhibiting bone resorption, thereby being useful in the treatment of osteoporosis and other conditions related to osteoclast hyperactivity (Acta Physiol.Scand., 163(suppl.), 195, 1998.; J.Clin. Invest, 106, 309, 2000). Other V- ATPase inhibitors are disclosed in US2002099080, WO9801443, WO0100587, WO0102388, PCT/EP2005/051908, and PCT/EP2005/051910 all of which are incorporated herein by way of reference. The invention is however not limited to the above V-ATPase inhibitors which are just exemplary of some well-known V-ATPase inhibitors.

An alkalinizing agent can also be a cellular ion channel blocker, an interleukin-1-beta converting enzyme (ICE) inhibitor, an ionophore or other agent which alters the intracellular/extracellular ion concentrations such as agents which activate proton ATPase, cellular proton pumps or other alkalinizing biochemical pathways, or such as agents which inhibit acidifying biochemical pathways. Alteration of ion balance by the alkalinizing agent results in a change in the intracellular pH to a physiological pH above 6.8. Indirect alkalinizing agents therefore further include blockers of chloride ion channel exchanger proteins such as mefenamic acid, flufenamic acid, mefamic acid, diphenylamine carboxylate, and other phenylanthranilic acids; 4,4 diisothiocyanostilbene-2,2 ' disulfonic acid (DIDS) , 4,4 ' -dibenzamidostilbene, 2,2 ' - disulfonic acid, 4,4 ' -dinitrostilbene-2, 2 ' -disulfonic acid, 4-amino-4'- nitrostilbene-2, 2 ' - disulfonic acid, 4-acetamido-4 ' - isothiocyanatestilbene-2,2 ' -disulfonic acid, and other derivatives of stilbene disulfonic acids; and probenecid.

The alkalizing agent of the present invention may be administered together with a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" means a vehicle for delivering an alkalinizing agent to a target cell, in which the vehicle is compatible with cell viability. Pharmaceutically acceptable carriers suitable for use in the administration of alkalinizing agents of the invention are well known to those skilled in the art. Selection of the pharmaceutically acceptable carrier will depend upon a variety of factors including the alkalinizing agent to be administered, the route of administration, and the condition to be treated. Pharmaceutically acceptable carriers suitable for use with the alkalinizing agents of the invention include, but are not limited to, 0.01-0.1 and preferably 0.05 M succinate buffer or 0.8% saline. Additionally, such phamriaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Further, pharmaceutically acceptable carriers may include detergents, phospholipids, fatty acids, or other lipid carriers. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Pharmaceutically acceptable carriers for use with the alkalinizing agents of the invention also include lipid carriers. Lipid carriers can be in the form of sterile solutions or gels, or can be detergents or detergent- containing biological surfactants. Examples of nonionic detergents include polysorbate 80 (also known as TWEEN 80 or polyoxyethylenesorbitan monooleate). Examples of ionic detergents include, but are not limited to, alykltrimethylammonium bromide.

As already mentioned herein before, frontotemporal lobar degeneration (FTLD) is the second most abundant form of dementia in people under the age of 60 years after Alzheimer's disease (AD) (Graff-Radford and Woodruff, 2007). Numerous loss-of- function mutations in the progranulin (GRN) gene cause frontotemporal lobar degeneration (FTLD) with ubiquitin and TAR-DNA binding protein 43 positive inclusions by reduced production and secretion of GRN. Consistent with the observation that GRN has neurotrophic properties pharmacological stimulation of GRN production is a hopeful approach to rescue GRN haploinsufficiency and prevent disease progression. The inventors therefore searched for compounds capable to selectively increase GRN levels. Herein it is demonstrated that four independent and highly selective inhibitors of vacuolar ATPase (Bafilomycin A1 , concanamycin A, archazolid B and apicularen A) significantly elevate intracellular and secreted GRN. Furthermore, alkalizing reagents including chloroquine, a frequently used anti-malaria drug, similarly stimulate GRN production. Elevation of GRN levels occurs via a translational mechanism independent of lysosomal degradation, autophagocytosis or endocytosis. Importantly, Bafilomycin A1 as well as chloroquine rescue GRN deficiency in organotypic cortical slice cultures of a mouse model for GRN deficiency and in primary cells derived from human patients with GRN loss-of-function mutations. Thus alkalizing reagents, specifically those already used in humans for other applications, and vacuolar ATPase inhibitors may be therapeutically employed to prevent GRN dependent neurodegeneration.

In view of the above, the present invention also relates to an alkalizing agent as defined herein for use in the treatment, acceleration, or amelioration of a disease and/or the improvement of the outcome of a disease, wherein said disease is a disease which benefits from an enhanced and/or restored progranulin level and wherein said disease is caused by haploinsufficiency of the progranulin gene (GRN).

Haploinsufficiency occurs when a diploid organism has one single functional copy of a gene only (the other copy being inactivated by mutation) and the single functional copy of the gene does not produce enough of a gene product (progranulin) to bring about a wild-type condition, leading to an abnormal or diseased state.

It is well-known that GRN mutations frequently result in haploinsufficiency (Baker, M., et ah, Nature 442:916-919 (2006); Cruts, M., et ah, Nature 442:920-924 (2006)). Genetic linkage studies and/or mutation screening identified approximately 70 mutations in the progranulin (GRN) gene in patients with familial FTLD-TDP (http://www.molgen.ua.ac.be/FTDMutations/) (Gijselinck et al., 2008). Most of the mutations identified are loss of function mutations, which lead to a severe reduction of GRN levels in tissues and biological fluids of patients (Baker et al., 2006, Cruts et al., 2006, Cruts and Van Broeckhoven, 2008, Sleegers et al., 2009). Additionally, missense mutations (e.g. (Schymick et al., 2007, Van Der Zee et al., 2007), (Brouwers et al., 2008)) lead to cytoplasmic missorting and degradation of GRN (Mukherjee et al., 2008, Shankaran et al., 2008) or to reduced secretion probably due to misfolding (Shankaran et al., 2008). Thus all FTLD-TDP associated GRN mutations investigated so far result in a deficiency of GRN.

In a preferred embodiment, it is envisaged that said disease is caused by haploinsufficiency of the progranulin gene is frontotemporal lobar degeneration with tau- negative, ubiquitin-positive inclusions (FTDL-U). Said disease is sometimes also denoted as frontotemporal lobar degeneration with ubiquitin-positive, tau- and alpha- synuclein negative inclusions.

In an even more preferred embodiment, said FTDL-U is further characterized by TAR DNA binding protein 43 positive inclusions (FTDL-TDP).

The present invention also relates to a pharmaceutical composition, package or kit for use in the treatment, acceleration, amelioration of a disease (or symptoms associated thereto) and/or the improvement of the outcome of a disease, wherein said disease is a disease which benefits from an enhanced and/or restored progranulin level, and wherein said kit or package comprises an alkalizing drug and optionally a package insert and/or instructions.

The term "package insert and/or instructions' is used to refer to instructions customarily included in commercial packages of pharmaceutical products, that contain information about the methods, usage, storage, handling, and/or warnings concerning the use of such products. It is also envisaged that the pharmaceutical package or kit of the present invention, further comprises means to administer the alkalizing agent to a patient and/or buffers, vials, teflon bags or infusion bags which are normally used for the administration of therapeutic agents. "Means" thereby includes one or more article(s) selected from the group consisting of a syringe, a hypodermic needle, a cannula, a catheter, an infusion bag for intravenous administration, intravenous vehicles, vials, buffers, stabilizers, written instructions which aid the skilled person in the preparation of the respective doses and infusions of the invention etc. In another embodiment, the present invention relates to the use of an alkalizing drug for the preparation of a pharmaceutical composition/package/kit for use in the treatment, acceleration, amelioration of a disease (or symptoms associated thereto) and/or the improvement of the outcome of a disease, wherein said disease is a disease which benefits from an enhanced and/or restored progranulin level.

In another embodiment, the present invention relates to a method of treatment or prophylaxis of a disease (or symptoms associated thereto) and/or the improvement of the outcome of a disease, wherein said disease is a disease which benefits from an enhanced and/or restored progranulin level, which method is characterized by the administration of a therapeutically effective amount of an alkalizing drug to said patient.

In a further embodiment, the present invention relates to a method of screening for a pharmaceutically active drug for use in the treatment, acceleration, amelioration of a disease (or symptoms associated thereto) and/or the improvement of the outcome of a disease, wherein said disease is a disease which benefits from an enhanced and/or restored progranulin level, said method comprising the following steps:

(a) contacting a test-compound with a test cell, and

(b) evaluating an alteration in the intracellular pH of said test-cell,

wherein an increase in the intracellular pH indicates that said test compound is a pharmaceutically active drug for use in the treatment, acceleration, amelioration of a disease (or symptoms associated thereto) and/or the improvement of the outcome of a disease, wherein said disease is a disease which benefits from an enhanced and/or restored progranulin level.

Possible test cells which might be employed in the context of this method have been disclosed herein elsewhere (for example HEK 293 cells). It is preferred that the alteration in the intracellular pH of said test cell is evaluated in the endolysosomal system of said cell.

* * * * * *

It must be noted that as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein. Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. At least one includes for example, one, two, three, four, or five or even more.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention. Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

This disclosure may best be understood in conjunction with the accompanying drawings, incorporated herein by references. Furthermore, a better understanding of the present invention and of its many advantages will be had from the following examples, given by way of illustration and are not intended as limiting.

The figures show: Figure 1

BafA1 but none of the tested protease inhibitor increases endogenous intracellular and secreted GRN levels. A, B, HeLa and SH-SY5Y cells were treated for 16 h with DMSO, BafA1 (50 nM), phenanthroline (10 mM), EDTA (15 mM), MG132 (10 μΜ), epoxomicin (1 pM), pepstatin A (1 μΜ), E64 10μΜ), antipain (5 μΜ), ALLN (5μΜ), mix 1 (E64, leupeptin, antipain), mix 2 (mix 1 plus pepstatin A and ALLN). Cell lysates (A) and conditioned media (B) were analyzed for GRN by immunoblotting. Equal loading of cell lysates was confirmed by probing for D-actin (A). C, HeLa cells treated with 25 nM BafA1 or DMSO were stained with a monoclonal anti-GRN antibody (red) and co-stained with antibodies against calnexin (green, ER), lamp-1 (green, lysosomes), GM130 (green, cis-Golgi) and TMEM59 (green, Golgi). The BafA1 mediated increase of GRN does not affect intracellular GRN localization. Increased excitations are shown to visualize the low levels of GRN under control conditions. Scale bar = 10 pm.

Figure 2

The BafA1 mediated GRN increase is independent of lysosomal degradation and autophagy. A, BafA1 treatment was carried out at 20°C to block forward transport through the secretory pathway or under control conditions (37°C). Media and cell lysates were subjected to GRN immunoblotting followed by quantification. GRN levels were normalized to untreated control cells. Note that at 20°C no secretion is observed, therefore no GRN is detected in conditioned media. B, MEF from ATG5 knockout and wild type (wt) mice (Mizushima et al., 2001) were treated with and without 25 nM of BafA1 (20 h). The complete loss of autophagosome formation was verified by LC3II immunobloting. Note that inhibition of autophagy did not interfere with the BafA1 mediated GRN increase. C, HeLa cells transfected with GFP-LC3 (green) fusion construct were immunostained for GRN in the absence and presence of BafA1. No co- localization of GRN with autophagosomes was observed.

Figure 3

BafA1 causes a posttranscriptional increase of GRN expression and secretion. A, Quantification of GRN mRNA in BafA1 (25 nM; 16 h) treated and untreated HeLa and N2a cells by qRT-PCR. GRN mRNA levels were normalized to GAPDH mRNA and are presented as the ratio to the untreated control. Parallel experiments were carried out in the presence of the transcription inhibitor actinomycin D (ActD; 1 μΜ). B, Northern blot of BafA1 (25nM; 16h) treated and non-treated HeLa cells probed for GRN and re-probed for GAPDH. Quantification of triplicates was performed with a Phosphorlmager. C, Conditioned media of the samples used for RNA extraction in (A) were analyzed for protein levels of GRN by immunoblotting and quantified. For (A), (B) and (C) normalized values are shown as means (n=3) ± s.d. of independent experiments (^*** p<0.001 ; ^**** p<0.0001, ***** p<0.00001 ; statistical analysis: Student^'s two-tailed, unpaired t-test). Figure 4

Inhibition of lysosomal acidification by BafA1 is reversible. HeLa cells pretreated with BafA1 (30 nM) and non-treated control cells (16h) were incubated with 100 nM LysoSensor for 30 min, at indicated time points after BafA1 treatment. Figure 5

Alkalizing reagents increase intracellular and secreted GRN levels. A, HeLa cells were treated with BafA1 (50 nM), CQ (50 μΜ) and 25 mM NH CI. Conditioned media and cell lysates were analyzed for GRN expression by immunoblotting. GRN increase was quantified in conditioned media and cell lysates of HeLa ceils. Data are expressed as fold increase of untreated control cells and shown as means (n=3) ±s.d. (^* p<0.05; ^** p<0.01 ; ^*** p<0.001; statistical analysis: one-way ANOVA post-hoc Dunnetf s test). For dose response curves HeLa cells were treated for 16 h with BafA1 (B) or CQ (C) at indicated concentrations. Secreted GRN was analyzed by immunoblot and quantified (B and C, left panel). Potential effects of BafA1 or CQ treatment on total protein secretion were quantified by metabolic labeling. Secreted ^S-methionine labeled proteins were measured by TCA precipitation and scintillation counting. Data were normalized to untreated cells (means (n=3) ± s.d.) (B and C, right panel).

Figure 6

V-ATPase inhibitors increase intracellular and secreted GRN levels. HeLa cells were treated with the highly specific V-ATPase inhibitors concanamycin A (ConA; 50 nM), archazolid B (ArcB; 50 nM) and apicularen A (ApiA; 100 nM). Conditioned media and cell lysates were analyzed for GRN expression by immunoblotting. GRN increase was quantified in conditioned media and cell lysates. Data are expressed as fold increase of untreated control cells and shown as means (n=3) ±s.d. (^** p<0.01 ; ^*** p<0.001 ; statistical analysis: one-way ANOVA post-hoc Dunnetfs test).

Figure 7

A, HeLa cells were treated with BafA1 (30 nM) for 16 h and conditioned media and lysates were investigated for GRN (time point Oh). A parallel set of culture dishes were then kept without BafA1 for additional 72 h. Conditioned media were collected during the last 16h of this time period. Control cells were treated with DMSO for the same time points (control). B, Vesicle acidification was monitored by LysoTracker staining (30 min). Impairment of vesicular acidification is observed immediately after BafA1 treatment but not 72 h after treatment. Scale = 20 pm.

Figure 8

Rescue of reduced GRN levels in organotypic cortical slice cultures of heterozygous GRN knockout mice. Cortical slice cultures (n=6 for each mouse), GRN^*'^* (n=1 ) or GRN^*'^~ (n=8), were prepared. After 72h, media were collected (A.day 0-3) and exchanged with drug containing media. Two cortical slice cultures of each mouse were treated with BafA1 (25 nM), CQ (10μΜ) or DMSO for 48 h (B, day 3-5). After 48 h media were replaced and secreted proteins were collected for 96 h in the absence of drugs (C, day 5-9). GRN levels were measured in triplicates using a sandwich ELISA specific for mouse GRN (means ±s.d., n=3).

Figure 9

BafA1 and CQ treatment rescues GRN haploinsufficiency primary human lymphoblasts. Lymphoblasts of loss-of-function mutation carriers (DR-lines) and control lymphoblasts from non-mutation carriers (CR-lines) were cultured at equal cell density. Triplicates of each DR-cell line were treated with CQ (10 μΜ) (A), BafA1 (25 nM) (B) or DMSO for 16 h. GRN levels of conditioned media were determined by ELISA. For CR cell lines GRN levels of untreated cells are shown. Data represent means ± s.d.. Note that all cell lines derived from GRN mutation carriers displayed a significant increase of GRN upon treatment with both drugs. Fig. 9C depicts results which were obtained with Bepridil (BEP) and Amiodarone (AMI) Figure 10

Consistent with the alkalizing activity of BafA1 , other alkalizing reagents such as NH₄CI and chloroquine (CQ) independently elevated endogenous GRN levels in cell lysates and in conditioned media of neuronal and non-neuronal cell lines.

Figure 11

The three V-ATPase inhibitors Concanamycin A, archazolid B and apicularen A increase intracellular and extracellular levels of GRN similar to BafA1 in neuronal and non- neuronal cells.

Figure 12

siRNA-mediated ATPaseVoC subunit result in progranulin increase.

siRNA for the knock down of the cytoplasmic v-ATPase subunit c (ATP6V0C) were transfected twice (day 1 +3). GRN expression was analyzed at day 5 by ELISA. V- ATPase subunit c knock down was verified by qRT-PCR.

Figure 13

FDA approved drug Hydroxychloroquine result in progranulin increase in HEKT cells. Since Hydroxychloroquine is considered a disease-modifying anti-rheumatic drug (D ARD), which can decrease the pain and swelling of arthritis, it was tested whether Hydroxchloroquine is also sufficient to increase Progranulin levels. For dose response curve HEK293T cells were treated for 16 h with Hydroxychloroquine (HCQ) at indicated concentrations. Secreted GRN was analyzed by immunoblot and quantified. Data are expressed as fold increase of untreated control cells and shown as means (n=3) ±s.d. of independent experiments

Figure 14

Progranulin plasma levels are quite stable over time within one healthy volunteer although high variations occur between volunteers.

In a small pre-study we analyzed the plasma progranulin level over four weeks in nine healthy volunteers. Plasma samples were taken twice a week and progranulin was measured by ELISA in triplicates. Aim of the study was to evaluate the steadiness of the progranulin plasma concentration within one patient, because high differences among patients have been reported before and were confirmed in our study. However, since the progranulin plasma concentration is quite stable over time, a drug facilitated progranulin increase might easily be detected. Progranulin concentration shown as means (n=3) ± s.d.

The invention can also be characterized by the following items:

1. An alkalizing agent for use in the treatment, acceleration, or amelioration of a disease and/or the improvement of the outcome of a disease, wherein said disease is a disease which benefits from an enhanced and/or restored progranulin level.

2. The alkalizing drug of item 1 , wherein said disease is wound healing, inflammatory diseases, chronic pain and/or neurodegenerative diseases.

3. The alkalizing drug of item 1 wherein said disease is caused by haploinsufficiency of the progranulin gene (GRN).

4. The alkalizing drug of item 3 wherein said disease which is caused by haploinsufficiency of the progranulin gene, is frontotemporal lobar degeneration with tau-negative, ubiquitin-positive inclusions (FTDL-U).

5. The alkalizing drug of item 4, wherein said FTDL-U is further characterized by TAR DNA binding protein 43 positive inclusions (FTDL-TDP).

6. A pharmaceutical composition, package or kit for use in the treatment, acceleration, amelioration of a disease (or symptoms associated thereto) and/or the improvement of the outcome of a disease, wherein said disease is a disease which benefits from an enhanced and/or restored progranulin level, and wherein said kit or package comprises an alkalizing drug and optionally a package insert or instructions.

. Use of an alkalizing agent in the manufacture of a pharmaceutical composition, package or kit for use in the treatment, acceleration, amelioration of a disease (or symptoms associated thereto) and/or the improvement of the outcome of a disease, wherein said disease is a disease which benefits from an enhanced and/or restored progranulin level.

. A method of treatment or prophylaxis of a disease (or symptoms associated thereto) and/or the improvement of the outcome of a disease, wherein said disease is a disease which benefits from an enhanced and/or restored progranulin level, which method is characterized by the administration of a therapeutically effective amount of an alkalizing drug to said patient. Any one of the preceding items, wherein said patient is a human subject.

Any one of the preceding items, wherein said alkalizing agent increases the progranulin protein (GRN) level in biological fluids of said patient. Any one of the preceding items, wherein said alkalizing agent increases the progranulin protein (GRN) level in the extracellular space.

The alkalizing agent of item 10, wherein said biological fluid is cerebrospinal fluid, plasma or serum (obtained from the patient).

Any one of the preceding items, wherein said alkalizing agent is capable of increasing the intracellular pH of neuronal primary cultures or HEK293.

Any one of the preceding items, wherein said alkalizing agent increases the intracellular pH of the cells defined in item 13 by about 0,4 to 0,6 (preferably in the endolysosomal pathway).

Any one of the preceding items, wherein said alkalizing agent is selected from the group consisting of chloroquine, bepridil, amiodarone, chloroquine diphosphate.amlodipine, astemizole, benztropine, camylofin, chlorprothixene, clomiphene, cloperastine, cyclobenzaprine, cyproheptadine, doxepine, drofenine, fendiline, fluoxetine, maprotiline, norfluoxetine, nortriptyline, paroxetine, pimethixene, promazine, promethazine, protriptyline, sertraline, suloctidil, terfenadine, and/or triflupromazine.

Any one of items 1 to 15, wherein said alkalizing agent is an inhibitor of the vacuolar ATPase.

A method of screening for a pharmaceutically active drug for use in the treatment, acceleration, amelioration of a disease (or symptoms associated thereto) and/or the improvement of the outcome of a disease, wherein said disease is a disease which benefits from an enhanced and/or restored progranulin level, said method comprising the following steps:

(a) contacting a test-compound with a test cell, and

(b) evaluating an alteration in the intracellular pH of said test-cell,

wherein an increase in the intracellular pH indicates that said test compound is a pharmaceutically active drug for use in the treatment, acceleration, amelioration of a disease (or symptoms associated thereto) and/or the improvement of the outcome of a disease, wherein said disease is a disease which benefits from an enhanced and/or restored progranulin level. 18. The method of item 17, wherein said test cell is the test cell defined in item 13.

19. The method of item 17 or 18, wherein said increase in the intracellular pH is evaluated in the endolysosomal pathway of said cell. Examples:

The following examples illustrate the invention. These examples should not be construed as to limit the scope of this invention. The examples are included for purposes of illustration and the present invention is limited only by the claims. Materials and Methods

Cell culture

Human cervical carcinoma (HeLa) cells, human embryonic kidney (HEK 293T) cells and mouse embryonic fibroblasts (MEF) from ATG5 knockout and wild type (wt) mice (Mizushima et al., 2001 ) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) with Glutamax I (Invitrogen). Lymphoblasts, immortalized by Ebstein Barr Virus transformation of lymphocytes collected from whole blood on lithium heparin according to standard procedures (Brouwers et al., 2007, Gijselinck et al., 2008), were cultured in RPMI 1640 medium (Invitrogen) with glutamine (Invitrogen). Mouse neuroblastoma cells (N2a) were cultured in Modified Eagle's Medium (MEM) with glutamine. Human neuroblastoma (SH-SY5Y) cells were cultured in DMEM/F12 with glutamine (Cambrex) supplemented with non-essential amino acids (Invitrogen). All media were supplemented with 10% (vol/vol) fetal calf serum (Invitrogen) and penicillin/streptomycin (PAA).

Organotypic slice culture of mouse neocortex

Organotypic slice cultures of mouse neocortex were prepared according to the protocol of Del Turco and Deller (Del Turco and Deller, 2007) with minor modifications. Mouse pups (GRN*^ ^~*^~) at postnatal day 3-5 (P3-P5) were decapitated, brains gently removed, neocortex dissected and cut on a chopper (Mcllwain Tissue Chopper, Mickle Laboratory Engineering) into sections of 400 μπι. Four sections were transferred to one membrane insert (Millicell, 30 mm, 0.4 μιτι pore size, Millipore) and cultured in a six-well plate. Cultures were kept in a humidified incubator (95% air, 5% C0₂, 35°C) and allowed to adjust to culture conditions for 3 days. Thereafter media were exchanged for media supplemented with compounds and incubated for 48 h. Supernatants were collected, immediately frozen and stored at -80°C. Slice culture medium: 50% MEM, 25% heat inactivated normal horse serum, 25% Basal Medium Eagle (BME), 25 mM HEPES, 2 mM Glutamax I, 0.65% Glucose, 0.1 mg/ml Streptomycin and 100 U/ml Penicillin, 0.15% sodium bicarbonate, pH 7.3. Inhibitors and reagents

The following inhibitors were used: Bafilomycin A1 (BafA1 ), pepstatin A, antipain, ALLN (all Merck, Calbiochem), MG132, epoxomicin (Biomol), concanamycin A, bepridil, amiodarone (all Sigma), archazolid B and apicularen A were dissolved in DMSO. Leupeptin (Merck, Calbiochem), chloroquine (CQ) NH₄CI, EDTA, and phenanthroline (Sigma) were dissolved in H₂0. E64 (Biomol) was dissolved in 50% ethanol, and actinomycin D and cycloheximide (Sigma) were dissolved in methanol. Concentrations and length of treatment are indicated in the figure legends.

Antibodies

The following antibodies were used for immunoblotting: Rabbit polyclonal antibody to human GRN (Invitrogen, 1 :700); sheep polyclonal antibody to mouse GRN (R&D Systems, 1 :1 ,000); mouse monoclonal antibody to beta-actin, (Sigma, 1 :2,000); rabbit polyclonal antibody to ATG5 (Cell Signaling, 1 :2,000) and a mouse monoclonal antibody to LC3 (Nanotools, 1 :1 ,200). Secondary antibodies were HRP-conjugated goat anti- mouse, goat anti-rabbit IgG (Promega, 1 :10,000) or anti-sheep IgG (Santa Cruz, 1 :5,000). For immunocytochemistry mouse monoclonal antibody to GRN (R&D Systems, 1 :500), rabbit polyclonal antibody to calnexin (1 :500), Alexa-488 and Alexa- 647 conjugated monoclonal antibodies to GM-130 (BD Pharmingen, 1 :10) and LAMP-1 (Santa Cruz, 1 :50) were used. The rabbit antibody to TMEM59 (1 :300) was a generous gift from Dr. Lichtenthaler and described elsewhere (Ullrich et al., 2010).

Immunocvtochemistry

HeLa cells were grown on poly-lysine coated cover slips, fixed for 20 min with 4% paraformaldehyde (PFA) and 4% sucrose in PBS, permeabilized for 10 min with 0.2 % Triton X-100, 50 mM NH₄CI in PBS and subsequently blocked for 1 h in PBS with 5% BSA. Cells were then double-stained with the indicated antibodies for 2 h. After washing repeatedly with PBS, cells were incubated with Alexa-488 and Alexa-555 (Invitrogen) coupled secondary anti-mouse, anti-rat or anti-rabbit antibodies for 1 h. Subsequently cells were washed with PBS and the cover slips were mounted on glass slides using Mowiol (Hoechst) supplemented with 0.5% DABCO (Sigma). Images were obtained on a Zeiss confocal laser scanning microscope (LSM 510 META) using an oil immersion IOOx/1.4 objective and the LSM software v3.5 (Carl Zeiss Microimaging). For LC3 staining the GFP-LC3 cDNA construct (Schmid et al., 2007) was transfected into HeLa cells grown on cover slips, using Lipofectamine 2000 (Invitrogen) according to the manufacturer^'s instructions. 24 h post transfection, cells were subject to BafA1 treatment (30 nM) for 16 h. Immunocytochemistry was carried out as described above. LysoSensor DND-189 and LysoTracker DND-99 (Molecular Probes, Invitrogen) dyes were used for labelling acidic cell organelles. Therefore cells were incubated with the indicated dye for 30 min according to the manufacturer^'s instructions. Cells were imaged directly after incubation with the indicated dye, using an oil immersion 40x/1.4 objective or a 10x objective.

Metabolic labeling and TCA precipitation on filter

To analyze total protein secretion, HeLa cells were incubated for 16 h with 5 MBq/ml 35S-methionine/cysteine (Hartmann Analytic) in methionine-, cysteine- and serum free medium, in the presence of DMSO, BafA1 or CQ at the indicated concentrations. 10 μΙ conditioned media were pipetted on Whatman filter paper and proteins were precipitated by boiling the filter in 5% TCA for 10 min followed by extensive washing in acetone. Quantification was performed in a scintillation counter (Beckman).

Preparation of conditioned media, cell Ivsates and immunoblotting

Conditioned media were collected, immediately cooled down and centrifuged at 15,000 g for 15 min at 4°C. Supernatants were either directly or after TCA-precipitation subjected to standard 10% SDS-PAGE. For cell lysates, cells were washed twice with PBS, scraped off and pelleted at 1 ,000 g, 5 min. Cell pellets were lysed for 15 min in ice cold STEN lysis-buffer (150 mM NaCI, 50 mM Tris-HCI pH 7.6, 2 mM EDTA, 1% NP-40), freshly supplemented with protease inhibitor cocktail (Sigma) and clarified by centrifugation at 4°C for 30 min at 15,000 g. Equal amounts of protein were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. For detection, the indicated antibodies were used. Bound antibodies were visualized by horseradish peroxidase-conjugated secondary antibody using enhanced chemiluminescence technique (Amersham Bioscience). Quantifying mRNA with real time RT-PCR

For qRT-PCR total RNA was prepared using the RNeasy Kit (Qiagen). RNA preparations were treated with DNase (DNase I, RNase-free, Qiagen) and 1 pg of total RNA was used for reverse transcription with oligo dT primer and M-MLV RT (Ambion) according to the manufacturer's protocols. qRT-PCRs were carried out on a 7500 Fast Real-Time PCR System (Applied Biosystems) with TaqMan technology, using primer sets from Applied Biosystems: for mouse GRN Mm00433848_m1 (exon boundary 4-5); for mouse GAPDH (4352339E); for human GRN Hs00173570_m1 (exon boundary 1-2); for human GAPDH (4326317E). Each sample was analyzed in triplicate and levels of GRN cDNA were normalized to GAPDH cDNA according to the AACt method using the equation ₂-(CtGRN-CtGAPDH)treatment -(CtGRN-CtGAPDH)control

Northern blotting

For Northern blot analysis, quality of total RNA was controlled using the Agilent 2100 Bioanalyser (data not shown). 3 pg of total RNA were separated on a formaldehyde- containing agarose gel. Transfer onto a HyBond N membrane (Amersham Biosciences) and hybridization were carried out as described (Lammich et al., 2004). Templates of GRN and GAPDH for generating the radioactive probes were amplified by PCR using following the primer pairs: For GRN 5'- GAGCTCGGATCCGTCGACCCACGCGTCCGCAAGGTAC-3' (SEQ ID No:1 ) and 5- AAACTGCAGTGGAAGCCCCGTGGGCAGCAG-3" (SEQ ID No:2); for GAPDH 5'- GGAAGCTTGTCATCAATGG-3' (SEQ ID No:3) and S'-CAGGGATGATGTTCTGGAG-S' (SEQ ID No:4). [32P]-dCTP (Hartmann Bioanalytics)-labeled probes were generated using the Random Primers DNA Labeling System (Invitrogen). Labeled RNA was detected by exposure of the blot to Super RX film (Fuji) and quantified by Phosphorlmager (Molecular Dynamics).

ELISA for human and mouse GRN

Secreted GRN in conditioned media was quantified by a sandwich immunoassay using the Meso Scale Discovery Sector Imager 2400. Streptavidin-coated 96-well Multi-Array plates were blocked in blocking buffer (0.5% bovine serum albumin, 0.05% Tween-20 in PBS, pH 7.4) over night. For detection of human GRN, plates were incubated for 1 h at room temperature with a biotinylated goat anti-human GRN capture antibody (R&D Systems) diluted 1 :100 in blocking buffer. Plates were washed four times with washing buffer (0.05% Tween-20 in PBS) before addition of the samples or the standards (GenWay Biotech) and the first detection antibody (mouse monoclonal anti-human GRN antibody (R&D Systems, 1 :2,000 diluted in blocking buffer). Plates were incubated at room temperature for 2 h followed by three washing steps. For detection, a SULFO- TAG- labeled secondary anti-mouse antibody (Meso Scale Discovery, 1 :1 ,000) was added and plates were incubated for 1 h in the dark. After three washes Meso Scale Discovery Read buffer was added, and the light emission at 620 nm after electrochemical stimulation was measured using the Meso Scale Discovery Sector Imager 2400 reader. For detection of mouse GRN, mouse specific anti-GRN antibodies and the appropriate secondary detection antibody were used: Biotinylated sheep anti- mouse GRN antibody (R&D Systems, 1 :200); rat anti-mouse GRN antibody (R&D Systems, 1 :1 ,000); a SULFO-TAG- labeled secondary anti-rat antibody diluted 1 :500 (SULFO-TAG was coupled to anti-rat IgG (Sigma) using Meso Scale Discovery SULFO- TAG-NHS-ester according to the manufacture's protocol), respectively.

Animal husbandry

AW experiments were performed in compliance with the guidelines of the German Council on Animal Care. Results

Numerous loss-of-function mutations in the progranulin (GRN) gene cause frontotemporal lobar degeneration with ubiquitin and TAR-DNA binding protein 43 positive inclusions by reduced production and secretion of GRN. Consistent with the observation that GRN has neurotrophic properties pharmacological stimulation of GRN production is a hopeful approach to rescue GRN haploinsufficiency and prevent disease progression. The inventors therefore searched for compounds capable to selectively increase GRN levels. Herein it is demonstrated that four independent and highly selective inhibitors of vacuolar ATPase (Bafilomycin A1 , concanamycin A, archazolid B and apicularen A) significantly elevate intracellular and secreted GRN. Furthermore, alkalizing reagents including chloroquine, a frequently used anti-malaria drug, similarly stimulate GRN production. Elevation of GRN levels occurs via a translational mechanism independent of lysosomal degradation, autophagocytosis or endocytosis. Importantly, Bafilomycin A1 as well as chloroquine rescue GRN deficiency in organotypic cortical slice cultures of a mouse model for GRN deficiency and in primary cells derived from human patients with GRN loss-of-function mutations. Thus alkalizing reagents, specifically those already used in humans for other applications, and vacuolar ATPase inhibitors may be therapeutically employed to prevent GRN dependent neurodegeneration.

Bafilomvcin A1 increases intracellular and secreted GRN

Strong evidence supports the finding that GRN haploinsufficiency is the cause of neurodegeneration observed in all patients with familial GRN associated missense and nonesense mutations. Increasing GRN levels by influencing its turnover or production is consequently a hopeful therapeutic approach. The inventors specifically screened for compounds capable to inhibit proteolytic degradation of GRN since GRN may be metabolized during its passage through the secretory pathway or upon receptor mediated uptake. Cells were treated with a variety of protease inhibitors and cell lysates as well as conditioned media were analyzed for an increase in GRN levels. In two different cell lines, HeLa cells and neuronal SH-SY5Y cells, none of the tested compounds, including inhibitors of lysosomal proteases such as E64, pepsatin A, and leupeptin, had a significant effect on the amount of intracellular (Fig. 1A) or secreted GRN (Fig. 1 B) except bafilomycin A1 (BafA1). In addition inhibition of the ubiquitin proteasome system with MG132 or epoxomycine also failed to increase GRN levels in cell lysates or conditioned media (Fig. 1A & B). Moreover, blocking neutrophil elastase, an enzyme involved in processing of progranulin into granulin peptides (Bateman and Bennett, 2009) had no effect on GRN levels as well (data not shown). Thus BafA1 was the only compound found to significantly elevate intracellular and secreted GRN levels. Bafilomvcin A1 increases GRN independent of lysosomal and autophagosomal degradation

BafA1 selectively inhibits the vacuolar ATPase (V-ATPase), which among other cellular consequences leads to impaired lysosomal degradation (Bowman et al., 2004). Since inhibitors of lysosomal serine-, cysteine- and aspartyl proteases had surprisingly no effect on GRN levels, it was investigated the subcellular localization of GRN in presence and absence of BafA1 to confirm that GRN does not accumulate in lysosomes. This indeed revealed no co-localization of GRN with the lysosomal marker LAMP-1 after BafA1 treatment (Fig. 1C). However, upon BafA1 treatment i a significant increase of intracellular GRN was observed in a vesicular compartment co-stained with Golgi marker antibodies to GM130 and TME 59 respectively and to some extend with the ER marker antibody to calnexin (Fig. 1 C). These findings suggest that intracellular GRN levels are already increased early within the secretory pathway. To further proof that BafA1 increases GRN levels independently of lysosomal degradation, protein transport beyond the Golgi complex was blocked by incubating cells at 20oC (Griffiths et al., 1989). In control cells kept at 37oC, BafA1 treatment leads to the expected intra- and extracellular increase of GRN (Fig. 2A). In cells incubated at 20oC no GRN secretion was observed (Fig. 2A, upper panel) as expected. However, GRN still accumulated intracellularly upon BafA1 treatment (Fig. 2A, lower panel) confirming that this effect is independent of lysosmal degradation. Moreover, since GRN is retained within the Golgi under these conditions, this also excludes the possibility that the increased levels of GRN in conditioned media of cells treated with BafA1 are due to the inhibition of putative receptor mediated endocytosis of GRN.

Besides inhibition of lysosomal degradation blocking of vesicle acidification also leads to impaired autophagy. To investigate if GRN undergoes autophagic degradation it was investigated GRN metabolism in the presence and absence of BafA1 in mouse embryonic fibroblasts derived from an ATG-5 knockout mice (Mizushima et al., 2001 ).

Prevention of autophagy by eliminating the critical gene product of the autophagy-related gene (Atg) 5 still allowed the BafA1 induced increase in intracellular and extracellular levels of GRN (Fig. 2B). This is confirmed by double immunohistochemistry demonstrating that GRN does not co-localize with GFP-LC3, a marker protein of autophagosomes, before or after BafA1 treatment (Fig. 2C).

Bafilomycin A1 increases GRN levels independent of transcription

Recently, it has been demonstrated that under aberrant lysosomal storage conditions GRN mRNA among many others is transcriptionally upregulated (Sardiello et al., 2009). The inventors therefore investigated whether transcriptional mechanisms increase GRN levels upon treatment with BafA1 . However, GRN mRNA levels were only moderately increased upon treatment with BafA1 (Fig. 3A, B). In contrast, GRN protein still increased several-fold, upon BafA1 treatment even when transcription was blocked by actinomycin D (Fig. 3A & C). Moreover, the generation of alternatively spliced mRNAs could also be excluded, as only one mRNAs species of identical length before and after treatment with BafA1 (Fig. 3B) was detected. Thus, besides a moderate and probably cell line-dependent transcriptional upregulation, posttranscriptional mechanisms are likely causing the significant and increase in GRN expression and secretion upon BafAI treatment.

Alkalizing reagents increase GRN

Consistent with the neutralizing effect of BafAI on acidic cellular compartments, monitoring of the intracellular pH after treatment with BafAI and in untreated cells proved that BafAI rapidly and reversibly affects the cellular pH. While in control cells lysosomes and other acidic vesicles were readily stained with LysoSensor, no labeling was observed immediately after treatment (Fig. 4). However, upon withdrawal of BafAI the intracellular pH rapidly normalized as proven by the robust vesicular staining with lysosensor after 4, 8, and 24 h (Fig. 4). Thus BafAI prevents vesicular acidification in our cellular system and this effect is fully reversible. Consistent with the alkalizing activity of BafAI , other alkalizing reagents such as NH4CI and chloroquine (CQ) independently elevated endogenous GRN levels in cell lysates and in conditioned media of neuronal and non-neuronal cell lines (Fig. 5A & Fig. 10). BafAI reaches its maximal effect on GRN at 25 - 50 nM (Fig. 5B) without affecting general protein secretion (Fig. 5B). CQ increased GRN levels dose-dependently up to 50 μΜ (Fig. 5C). Even at high doses of CQ no general effect on total protein secretion was observed (Fig. 5C). V-ATPase is the cellular target of BafAI

V-ATPase was confirmed as a cellular target of BafAI that mediates the effect on increased intracellular and extracellular GRN levels by treatment of cells with three independent and highly selective V-ATPase inhibitors, namely concanamycin A, archazolid B and apicularen A (Huss et al., 2005). All three V-ATPase inhibitors increased intracellular and extracellular levels of GRN similar to BafAI in neuronal and non-neuronal cells (Fig. 6 & Fig. 11 ).

Inhibition of V-ATPase leads not only to a robust intracellular and extracellular increase of GRN but also has sustainable effects. Even 72 h after the initial treatment with BafAI significantly increased levels of GRN compared to controls were observed in cell lysates or conditioned media respectively (Fig. 7A). Importantly, 72 h after BafAI treatment was terminated LysoTracker staining revealed robust labeling of those cells, which were originally treated with BafAI , demonstrating that BafAI was washed out and that lysosomal pH recovered (Fig. 7B). Thus the long lasting post treatment increase of GRN secretion is independent the current lysosomal intracellular pH and lysosomal degradation, consistent with the data presented above.

Targeting of V-ATPase or increasing intracellular pH rescues GRN deficiency in an animal model and in primary cells from human patients

Next it was investigated if reduced GRN levels due to disease causing GRN haploinsufficiency could be elevated to physiological levels by alkalizing reagents. The only currently available animal model for GRN deficiency is the heterozygous GRN knockout mouse (Kayasuga et al., 2007, Yin et al., 2010). The inventors used organotypic cortical slice cultures to monitor GRN levels in the presence and absence of BafA1. As expected, an approximately 60-70% reduction of GRN was observed in conditioned media from organotypic cortical slice cultures derived from GRN+/- mice (Fig. 8A) similar to human patients with GRN mutations (Ghidoni et al., 2008, Finch et al., 2009, Sleegers et al., 2009). Upon BafA1 treatment of organotypic cortical slice cultures derived from GRN+/- mice, an increase in GRN levels was achieved (Fig. 8B), whereas a smaller but still significant stimulation was observed with CQ (Fig. 8B). Again, the effects were long lasting and an even more pronounced increase was monitored four days after BafA1 treatment (Fig. 8C).

To test whether GRN can be increased to physiological levels in a disease stage, lymphoblasts derived from four healthy controls and six patients with confirmed familial FTLD-TDP associated GRN loss-of-function mutations (Brouwers et al., 2007, Gijselinck et al., 2008) were treated with BafA1 and CQ. CQ is of special interest as it is frequently used for malaria prophylaxis and treatment, in auto-immune disorders and in sensitizing cancer therapy (Solomon and Lee, 2009). As expected, absolute levels of GRN were reduced in conditioned media obtained from cells with GRN loss-of-function mutations in comparison to cells derived from healthy controls (Fig. 9A & B). Levels of GRN were significantly increased upon stimulation with BafA1 to levels at least similar to untreated cells of healthy control subjects (Fig. 9A). CQ treatment led to a less pronounced but still significant increase of GRN secretion (Fig. 9B). Moreover, additional drugs with a known alkalizing potential like FDA approved Amiodarone, and Bepridl increase GRN levels (Fig. 9C).

Thus, our data demonstrate that GRN deficiency can be rescued with FAD approved drugs in lymphoblasts derived from patients with familial FTLD-TDP and in brain slices of GRN+/- mice, which both recapitulate disease associated GRN deficiency. It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.

The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background of the Invention, detailed Description, and Examples is hereby incorporated herein by reference.

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Claims

1. An alkalizing agent for use in the treatment or amelioration of a disease and/or the improvement of the outcome of a disease, wherein said disease is caused by haploinsufficiency of the progranulin gene (GRN).

2. The alkalizing drug of claim 1 wherein said disease which is caused by haploinsufficiency of the progranulin gene, is frontotemporal lobar degeneration with tau-negative, ubiquitin-positive inclusions (FTDL-U).

3. The alkalizing drug of claim 2, wherein said FTDL-U is further characterized by TAR DNA binding protein 43 positive inclusions (FTDL-TDP).

4. A pharmaceutical composition, package or kit for use in the treatment, acceleration, amelioration of a disease (or symptoms associated thereto) and/or the improvement of the outcome of a disease, wherein said disease is caused by haploinsufficiency of the progranulin gene (GRN), and wherein said kit or package comprises an alkalizing drug and optionally a package insert or instructions.

5. Use of an alkalizing agent in the manufacture of a pharmaceutical composition, package or kit for use in the treatment, acceleration, amelioration of a disease (or symptoms associated thereto) and/or the improvement of the outcome of a disease, wherein said disease is caused by haploinsufficiency of the progranulin gene (GRN).

6. The alkalizing drug, pharmaceutical composition, package, kit or use of any one of the preceding claims, wherein said patient is a human subject.

7. The alkalizing drug, pharmaceutical composition, package, kit or use of any one of the preceding claims, wherein said alkalizing agent increases the progranulin protein (GRN) level in biological fluids of said patient.

8. The alkalizing drug, pharmaceutical composition, package, kit or use of any one of the preceding claims, wherein said alkalizing agent increases the progranulin protein (GRN) level in the extracellular space.

9. The alkalizing drug, pharmaceutical composition, package, kit or use of claim 7, wherein said biological fluid is cerebrospinal fluid, plasma or serum (obtained from the patient).

10. The alkalizing drug, pharmaceutical composition, package, kit or use of any one of the preceding claims, wherein said alkalizing agent is capable of increasing the intracellular pH of neuronal primary cultures or HEK293.

11. The alkalizing drug, pharmaceutical composition, package, kit or use of any one of the preceding claims, wherein said alkalizing agent increases the intracellular pH of the cells defined in claim 13 by about 0,4 to 0,6 (preferably in the endolysosomal pathway).

12. The alkalizing drug, pharmaceutical composition, package, kit or use of any one of the preceding claims, wherein said alkalizing agent is selected from the group consisting of chloroquine, bepridil, amiodarone, chloroquine diphosphate.amlodipine, astemizole, benztropine, camylofin, chlorprothixene, clomiphene, cloperastine, cyclobenzaprine, cyproheptadine, doxepine, drofenine, fendiline, fluoxetine, maprotiline, norfluoxetine, nortriptyline, paroxetine, pimethixene, promazine, promethazine, protriptyline, sertraline, suloctidil, terfenadine, and/or triflupromazine.

13. The alkalizing drug, pharmaceutical composition, package, kit or use of any one of the preceding claims, wherein said alkalizing agent is an inhibitor of the vacuolar ATPase.

14. A method of screening for a pharmaceutically active drug for use in the treatment, acceleration, amelioration of a disease (or symptoms associated thereto) and/or the improvement of the outcome of a disease, wherein said disease is caused by haploinsufficiency of the progranulin gene (GRN), said method comprising the following steps:

(a) contacting a test-compound with a test cell, and

(b) evaluating an alteration in the intracellular pH of said test-cell,

wherein an increase in the intracellular pH indicates that said test compound is a pharmaceutically active drug for use in the treatment, acceleration, amelioration of a disease (or symptoms associated thereto) and/or the improvement of the outcome of a disease, wherein said disease is caused by haploinsufficiency of the progranulin gene (GRN).

15. The method of claim 14, wherein said test cell is the test cell defined in claim 10.

16. The method of claim 14 or 15, wherein said increase in the intracellular pH is evaluated in the endolysosomal pathway of said cell.

17. The method of any one of claims 14 to 16, wherein said disease which is caused by haploinsufficiency of the progranulin gene, is frontotemporal lobar degeneration with tau-negative, ubiquitin-positive inclusions (FTDL-U).