WO2018172557A1 - Biological methods - Google Patents

Abstract

The present invention relates to methods for determining cholinergic function and the status of associated disorders in patients.

Description

BIOLOGICAL METHODS

The present invention relates to methods for determining cholinergic function and the status of associated disorders in patients.

Dementias - which are an exemplary group of disorders related to cholinergic function - are common and have major clinical and societal consequences, which are expected to increase dramatically during the next three decades. Reliable biomarkers are essential to help patients, caregivers, and health professionals to better plan future care and management.

Cholinergic deficit is known to occur long before any disease symptom arises. For example, Schmitz et al. (2016) Nature Communications 7:13249, provides evidence that early changes in cholinergic neuronal nuclei in basal forebrain predict strongly the atrophic changes that are going to occur in the entorhinal cortex, one the first regions that becomes affected by Alzheimer's disease (AD). Expression of the acetylcholine (ACh) synthesizing enzyme, choline acetyltransferase (ChAT) defines the cholinergic cells/neurons.

Currently, in contrast to the dopaminergic or serotonergic systems, there are no in vivo biomarkers of the health of the cholinergic network.

Thus, there exists a considerable need for a means to determine cholinergic function, which will be useful in determination of the presence and progression of associated disorders.

Against this background, the inventors have discovered that selective ChAT-ligands can be used as in vivo tracers as a biomarker of health/function of cholinergic neuronal network. This will allow for detection of the changes in cholinergic neuronal function earlier than the manifestation of the typical clinical symptoms. The inventors have also specifically identified several FDA-approved drugs with high selectivity and activity for ChAT.

Alzheimer's disease (AD) is the leading cause of dementia. One of the key features of AD is an early selective degeneration of cholinergic neurons/projections in the brain ^(1~4>. Other dementias that share this characteristic are Lewy body disorders (LBD) and Down's syndrome (DS) ⁽⁵· ⁶⁾. LBD include dementia with Lewy bodies and Parkinson's disease dementia). Putatively, pathological events of AD and AD-like disorders are initiated 20-30 years prior to manifestation of clinical symptoms. In the case of the more aggressive autosomal-dominant forms of AD, it is estimated 10-20 years before the onset of clinical symptoms ⁽⁷⁾. Therefore, the discovery that selective ChAT-ligands can be used as in vivo tracers as a biomarker of health/function of cholinergic neuronal network will be useful in relation to monitoring diseases involving cholinergic neurons, such as AD.

In a first aspect of the invention there is provided a method for determining the cholinergic function of one or more cell, comprising the steps of: contacting one or more cell with an agent capable of selectively binding to choline acetyltransferase, and using the agent to measure the amount and/or concentration of choline acetyltransferase in the one or more cell; and - determining the cholinergic function of the one or more cell based on the amount and/or concentration of choline acetyltransferase; wherein the agent is a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein:

Q represents C or N;

each X independently represents -OR^1a, a 5- to 6-membered heteroaryl or a C1-6 alkyl, wherein the latter two groups are optionally substituted with one or more R^2a;

Y represents H or a C1-6 alkyl optionally substituted with one or more R^2b;

each Z independently represents -OR^1b or a O-s alkyl, wherein the latter group is optionally substituted with one or more R2_C;

each of R^1a and R^1b independently represents C1-3 alkyl optionally substituted with one or more fluoro or -OR^3a;

each of R^2a to R^2c independently represents fluoro or -OR^3b;

each R^3a and R^3b independently represents C1-3 alkyl optionally substituted with one or more fluoro;

n represents 0 to 4; and m represents 0 to 4, which compounds (including pharmaceutically acceptable salts) may be referred to herein as the "compounds of the first aspect of the invention".

For the avoidance of doubt, the skilled person will understand that references herein to compounds of particular aspects of the invention (such as the first aspect of the invention, e.g. compounds of formula I) will include references to all embodiments and particular features thereof, which embodiments and particular features may be taken in combination to form further embodiments.

Unless indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

Pharmaceutically acceptable salts include acid addition salts and base addition salts. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound of the invention with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a compound of the invention in the form of a salt with another counter-ion, for example using a suitable ion exchange resin. Particular acid addition salts that may be mentioned include carboxylate salts (e.g. formate, acetate, trifluoroacetate, propionate, isobutyrate, heptanoate, decanoate, cap rate, caprylate, stearate, acrylate, caproate, propiolate, ascorbate, citrate, glucuronate, glutamate, glycolate, a-hydroxybutyrate, lactate, tartrate, phenylacetate, mandelate, phenylpropionate, phenylbutyrate, benzoate, chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate, dinitrobenzoate, o-acetoxybenzoate, salicylate, nicotinate, isonicotinate, cinnamate, oxalate, malonate, succinate, suberate, sebacate, fumarate, malate, maleate, hydroxymaleate, hippurate, phthalate or terephthalate salts), halide salts (e.g. chloride, bromide or iodide salts), sulphonate salts (e.g. benzenesulphonate, methyl-, bromo- or chloro-benzenesulphonate, xylenesulphonate, methanesulphonate, ethanesulphonate, propanesulphonate, hydroxyethanesulphonate, 1 - or 2- naphthalene-sulphonate or 1 ,5-naphthalenedisulphonate salts) or sulphate, pyrosulphate, bisulphate, sulphite, bisulphite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate or nitrate salts, and the like.

Particular base addition salts that may be mentioned include salts formed with alkali metals (such as Na and K salts), alkaline earth metals (such as Mg and Ca salts), organic bases (such as ethanolamine, diethanolamine, triethanolamine, tromethamine and lysine) and inorganic bases (such as ammonia and aluminium hydroxide). More particularly, base addition salts that may be mentioned include Mg, Ca and, most particularly, K and Na salts. Yet more particularly, base addition salts that may be mentioned include Na and Zn salts.

For the avoidance of doubt, compounds of the first aspect of the invention may exist as solids, and thus the scope of the invention includes all amorphous, crystalline and part crystalline forms thereof, and may also exist as oils. Where compounds of the first aspect of the invention exist in crystalline and part crystalline forms, such forms may include solvates, which are included in the scope of the invention. Compounds of the first aspect of the invention may also exist in solution.

Compounds of the first aspect of the invention may contain double bonds and may thus exist as E (entgegen) and Z (zusammen) geometric isomers about each individual double bond. All such isomers and mixtures thereof are included within the scope of the invention.

Compounds of the first aspect of the invention may also exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention.

Compounds of the first aspect of the invention may also contain one or more asymmetric carbon and/or sulphur atoms (e.g. asymmetric sulphur atoms), and may therefore exhibit optical and/or diastereoisomerism. Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers (i.e. enantiomers) may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques. Alternatively the desired optical isomers may be obtained from appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation (i.e. a 'chiral pool' method), by reaction of the appropriate starting material with a 'chiral auxiliary' which can subsequently be removed at a suitable stage, by derivatisation (i.e. a resolution, including a dynamic resolution); for example, with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means such as chromatography, or by reaction with an appropriate chiral reagent or chiral catalyst all under conditions known to the skilled person. All stereoisomers and mixtures thereof are included within the scope of the invention. In particular embodiments, compounds of the first aspect of the invention may possess chiral sulphur atoms, and may therefore be provided as an enantiomericaliy-enriched compound. For example, compounds of the invention may possess chiral sulphur atoms in the S- configuration, and may therefore be provided as an enantiomericaliy-enriched compound (e.g. as for the compound esomeprazole).

Thus, in a particular embodiment, the compound of formula I may be provided as an enantiomericaliy-enriched compound of formula la

(la)

or a pharmaceutically acceptable salt thereof, wherein Q, X, Y, Z, n and m are as defined for compounds of formula I (including all embodiments thereof).

As used herein, references to a compound being enantiomericaliy-enriched may refer to the relevant enantiomer (i.e. the enantiomer as described with reference to the chiral atom) being present in an enantiomeric excess (e.e.) of at least 80%, such as at least 90% (e.g. at least 95%, e.g. at least 98%).

As used herein, references to halo and/or halogen groups will each independently refer to fluoro, chloro, bromo and iodo (for example, fluoro (F) and chloro (CI), such as fluoro).

Unless otherwise specified, Ci_-Z a Iky I groups (where z is the upper limit of the range) defined herein may be straight-chain or, when there is a sufficient number (i.e. a minimum of three) of carbon atoms, be branched-chain, and/or cyclic (so forming a C3-_z-cycloalkyl group). When there is a sufficient number (i.e. a minimum of four) of carbon atoms, such groups may also be part cyclic. Part cyclic alkyl groups that may be mentioned include cyclopropyl methyl and cyclohexylethyl. When there is a sufficient number of carbon atoms, such groups may also be multicyclic (e.g. bicyclic or tricyclic) or spirocyclic. Such alkyl groups may also be saturated or, when there is a sufficient number (i.e. a minimum of two) of carbon atoms, be unsaturated (forming, for example, a C2 alkenyl or a C2 alkynyl group).

For the avoidance of doubt, as used herein, references to heteroatoms will take their normal meaning as understood by one skilled in the art. Particular heteroatoms that may be mentioned include phosphorus, selenium, tellurium, silicon, boron, oxygen, nitrogen and sulphur (e.g. oxygen, nitrogen and sulphur).

For the avoidance of doubt, references to polycyclic (e.g. bicyclic or tricyclic) groups (e.g. when employed in the context of cycloaikyi groups) will refer to ring systems wherein at least two scissions would be required to convert such rings into a straight chain, with the minimum number of such scissions corresponding to the number of rings defined (e.g. the term bicyclic may indicate that a minimum of two scissions would be required to convert the rings into a straight chain). For the avoidance of doubt, the term bicyclic (e.g. when employed in the context of alky! groups) may refer to groups in which the second ring of a two-ring system is formed between two adjacent atoms of the first ring, and may also refer to groups in which two non-adjacent atoms are linked by an alkylene group, which later groups may be referred to as bridged. For the avoidance of doubt, the skilled person will understand that, where Q represents C, that C (i.e. that carbon atom) may, where appropriate, be substituted with an X group or, if not substituted, may be present as a CH group.

The present invention also embraces isotopically-labelled compounds of the first aspect of the invention which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature (or the most abundant one found in nature). All isotopes of any particular atom or element as specified herein are contemplated within the scope of the compounds of the invention. Hence, the compounds of the invention also include deuterated compounds, i.e. in which one or more hydrogen atoms are replaced by the hydrogen isotope deuterium.

In particular, the present invention also embraces isotopically-labelled compounds of the compounds of the first aspect of the invention which contain radioactive isotopes, as described in more detail herein below. For the avoidance of doubt, in cases in which the identity of two or more substituents in a compound of the invention may be the same, the actual identities of the respective substituents are not in any way interdependent. For example, in the situation in which two or more X groups are present, those X groups may be the same or different. Similarly, where two or more X groups are present and each represent halo, the halo groups in question may be the same or different.

The skilled person will appreciate that compounds of the invention that are the subject of this invention include those that are stable. That is, compounds of the invention include those that are sufficiently robust to survive isolation, e.g. from a reaction mixture, to a useful degree of purity.

In particular embodiments (i.e. in relation to compounds of the first aspect of the invention, including compounds of formula I and la), n represents 0 or 1.

In particular embodiments, m represents 1 to 3 (e.g. 2 or 3, such as 3).

In particular embodiments, Y represents H. In more particular embodiments, the compound of formula I (including compounds of formula la) has at least one X substituent in the 5-position of the required benzimidazole ring.

In yet more particular embodiments, the compound of formula I (including compounds of formula la) has at least one Z substituent representing -OR^1b in the 4-position of the required pyridine ring.

Thus, in particular embodiments, the compound of formula I may be a compound of formula II:

such as enantiomerically-enriched compound of formula Ha:

wherein:

X¹ represents H or X;

- t represents 0 to 2;

u represents 0 to 3; and

X, Z and R ^b are as defined for compounds of formula I.

In particular embodiments (i.e. in relation to compounds of the first aspect of the invention, including compounds of formula I, la, II and lla), t represents 0 and/or (e.g. and) X represents -OR^1a or a 5- to 6-membered (e.g. a 5-membered) heteroaryl.

In more particular embodiments:

t represents 0;

X represents -OR^1a or pyrrole (e.g. pyrrol-1-yl); and

R^1a represents Ci alkyl optionally substituted with one or more (e.g. two) fluoro(s) (e.g. -CH₃ or -CHF₂).

In particular embodiments (i.e. in relation to compounds of the first aspect of the invention, including compounds of formula I, la, II and lla), u represents 1 or 2 (e.g. 2) and/or (e.g. and) Z represents -CH3 or -OCH3 (e.g. -CH₃)

In particular embodiments, R^1b represents Ci_₃ alkyl optionally substituted with one or more fluoro or -O e.

In more particular embodiments, R^{1 b} represents -CH3, -CH2CF3 or -(CH2)30CH3.

In yet more particular embodiments, R ^b represents -CH₃. In particular embodiments, Q represents C (e.g. so forming a CH group).

Cholinergic is an abbreviated term which refers to the involvement of acetylcholine-based signalling. The parasympathetic nervous system, which uses acetylcholine (which may be abbreviated as "ACh") almost exclusively to send its messages, is said to be almost entirely cholinergic. Neuromuscular junctions, preganglionic neurons of the sympathetic nervous system, the basal forebrain, and brain stem complexes are also cholinergic. In addition, the receptor for the merocrine sweat glands are also cholinergic, since acetylcholine is released from postganglionic sympathetic neurons. By "cholinergic function" we include the functionality of any system relying on acetylcholine.

The cholinergic machinery consists of ChAT that is localized in the cytoplasm of cholinergic neurons, where it synthesizes ACh. This occurs through transfer of the acetyl-moiety of acetyl-Coenzyme A (A-CoA) to a choline molecule. ChAT contains a catalytic tunnel, within which there is a binding site for choline/ACh and one binding site for A-C0A/-C0A. The synthesized ACh is then transported by vesicular ACh transporter (VAChT) and stored into synaptic vesicles until its release into the synapses to act on its receptors. There are two general types of acetylcholine receptors (AChRs), namely nicotinic AChRs (nAChRs) and muscarinic AChRs (mAChRs). The released ACh is degraded, within the synaptic cleft, to choline and acetic acid by the enzymes, acetylcholinesterase (AChE), and to a lesser extent by butyrylcholinesterase (BChE). The choline is then recycled back into the cytoplasm by uptake by high affinity choline transporter (HChT).

All other cells/neurons that express AChRs and/or choiinesterases (AChE and BuChE) are called cholinoceptive cells/neurons. Thus, these markers, in contrast to ChAT, define the down-stream cholinergic postsynaptic events.

Cholinergic neurons project widely throughout the brain. In addition, autonomic ganglionic neurons in both the PNS and CNS are also cholinergic. Parasympathetic neurons are all cholinergic (eye iris, heart, ciliary muscles, Gl tract, urinary bladder, salivary glands) as well as many sympathetic neurons (sweat glands, etc). In general, all type of muscles one way or the other utilizes ACh. For instance, cholinergic motor neurons also innervate muscle endplates at neuromuscular junction of skeletal muscles. A major part of the enteric nervous system in the gut is also cholinergic, and innervates and controls intestinal motility and function, and may hence be involved in some disorders in the intestinal tracts ⁽¹⁵⁾. Recently, the inventors found age-related changes of ChAT levels in ascending and descending human colon ⁽¹⁶⁾. Furthermore, many other organs/tissues indeed utilize more or less cholinergic signaling, for instance, ChAT is expressed in placenta ^{( 7)}, in seminal fluids and/or spermatozoids) ⁽¹⁸· ¹⁹⁾. The method described in any aspect of the invention may additionally comprise the step of comparing the amount and/or concentration of the choline acetyltransferase to a control value. The control value, for example, may be a predetermined control value, or may represent a value determined in a healthy individual or in an individual known to possess a disorder.

The methods of the present invention may be performed in vitro or in vivo. In some embodiments, the method may be performed on a patient biopsy or sample, or on a cell in culture.

The one or more cell described in any aspect of the invention may include neuronal or non-neuronal cells. The methods may allow determination of any neuronal network and/or cellular clusters anywhere in the body, i.e. in any cell types, as long as they are cholinergic. For instance, lymphocytes, astrocytes and embryonic stem cells express little ChAT under normal conditions but quite high levels of ChAT when activated/stimulated.

In a further aspect of the invention, there is provided the use of an agent, as defined in the first aspect, in determining the cholinergic function of one or more cell, wherein the cholinergic function is determined based on the amount and/or concentration of choline acetyltransferase in the one or more cell.

In a further aspect of the invention, there is provided an agent, as defined in the first aspect, for use in determining the cholinergic function of one or more cell, wherein the cholinergic function is determined based on the amount and/or concentration of choline acetyltransferase in the one or more cell.

In a further aspect of the invention, there is provided the use of an agent, as defined in the first aspect, for the manufacture of a medicament for determining the cholinergic function of one or more cell, wherein the cholinergic function is determined based on the amount and/or concentration of choline acetyltransferase in the one or more cell.

The one or more cell described in any aspect of the invention may be provided in a sample obtained from an individual. The sample may comprise a biopsy from the relevant tissue/organ types. The sample may comprise blood, lymph fluid, cerebral spinal fluid, saliva, and/or urine.

In some embodiments of the invention, the one or more cell is provided in a cell culture.

There are a large number of cell lines that are cholinergic or that one can transfect with ChAT gene to express it (as experimental models), or that under certain condition may themselves start expressing ChAT (e.g. lymphocytes and astrocytes). ChAT may therefore be detected in any cell types if they express, or when they express, ChAT.

For example, scintillation beads may be used for detection and any samples or cell suspensions may be mixed with a scintillation bead, pre-coated with an antibody (or other reagent) that may selectively bind ChAT. Then adding the labelled ChAT-ligand will emit light when it binds to ChAT that is in close proximity of the scintillation beads (i.e. that been bound to them with the help of antibody or other ChAT-retaining agents). The signal then can be used to quantify levels of ChAT in the samples.

In certain embodiments of any aspect of the invention, the one or more cell is present in an individual, and the step of contacting the agent with the one or more cell comprises administering the agent to the individual. The agent may be administered to the individual by injection, for example directly into the bloodstream. Alternatively, it may be administered by intranasal administration. The skilled person will appreciate that such delivery of therapeutic compounds will deliver them in such a way that permits bypassing of the blood-brain barrier. In certain embodiments of any aspect of the invention, a PET tracer, for example of either 11 C-or 18F-labled compound, wherein the compound is of Formula I as defined above, may be used by IV injection or via nasal delivery.

The administration may comprise one dose of the agent per ChAT-PET assessment. The dosage may be significantly reduced over what is required for a therapeutic effect of the agent. Those skilled in the art of pharmacology and/or medicine will be capable of determining an appropriate dose for such purposes, and will appreciate that the dosages may require optimisation for different types of tracer - as discussed, for example, in Karakatsanis et al. (2015), Am. J . Nuci. Med. Mol. Imaging, 5:527. Dosages may be expressed as a unit of radioactivity per body weight and will typically be in the range of 5MBq per kg to 250MBq/kg, for example: 5MBq per kg; or 10MBq per kg; or 20MBq per kg; or 30MBq per kg; or 40MBq per kg; or 50MBq per kg; or 100MBq per kg; or 150MBq per kg; or 200MBq per kg; or 250MBq per kg.

In certain embodiments of any aspect of the invention, the one or more cell comprises: one or more neuronal cell and/ or one or more non-neuronal cell. The one or more non- neuronal cell may be selected from the group consisting of: lymphocytes; astrocytes; embryonic stem cells. In certain embodiments of any aspect of the invention the one or more cell is a cancer cell. For example, tumours may be detected in this manner, for instance glioblastoma or other cancer forms where a localized ChAT overexpression by cancer cells may help them with proliferation, and/or allowing them to escape immune surveillance by immune suppressing-action of acetylcholine (the phenomena is called the cholinergic-antiinflammatory pathway). Disorders that may cause astrogliosis in the brain may also be detected. In certain embodiments of any aspect of the invention, the one or more neuronal cell is selected from the group consisting of: motor neuron; sensory neuron; interneuron. The one or more neuronal cell may be part of an organ or tissue of the central nervous system. Preferably, the organ or tissue may be selected from the group consisting of: brain; spinal cord; retina; optic nerve; olfactory nerve; olfactory epithelium. Preferably the tissue is selected from the brain, for example the basal forebrain; temporal lobe; hippocampus, olfactory bulb, cerebral cortex and/or amygdala.

In certain embodiments of any aspect of the invention, the one or more neuronal cell is part of an organ or tissue of the peripheral nervous system, such as: part of the somatic nervous system; part of the autonomic nervous system; part of the parasympathetic nervous system; part of the sympathetic nervous system; part of the enteric nervous system.

In certain embodiments of any aspect of the invention, the organ or tissue may be selected from the group consisting of: eye iris; heart; ciliary muscle; upper gastrointestinal tract; lower gastrointestinal tract; colon (ascending and descending); urinary bladder; salivary gland; synovial tissues'⁷⁸⁾; placenta; prostate gland, testes; uterus; tendons'^{79, S0)}; skeletal muscle; skin/keratinocytes'^{87, 82)}; lungs/airways'⁸³'; stem cells'^{84, 85)}, glioblastoma cancer cells'^{86, 87)}; immune cells.

The choline acetyltransferase (ChAT) referred to in any aspect of the invention may be selected from the group consisting of: membrane-bound choline acetyltransferase; soluble choline acetyltransferase; monomeric choline acetyltransferase; dimeric choline acetyltransferase; tetrameric choline acetyltransferase; multimeric choline acetyltransferase. Preferably, the agent referred to in any aspect of the invention may further comprise at least one (e.g. one) detectable moiety. The detectable moiety (or moieties) may be selected from the group consisting of: a fluorescent label; a chemiluminescent label; a paramagnetic label; a radio-isotopic label; or an enzyme label.

In certain embodiments of the aspects of the invention, the paramagnetic isotope is selected from the group consisting of ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr and ⁵⁶Fe.

In particular embodiments, the detectable moiety (or moieties) is a radio-isotopic label. Preferably, the radio-isotopic label comprises (or consists of) a radio-isotope selected from the group consisting of: ³H ¹¹C; ¹⁴C; ¹⁸F; ^99mTc; ¹¹¹ln; ⁶⁷Ga; ⁶⁸Ga; ⁷²As;⁸⁹Zr; ¹²³l; ²⁰¹TI.

In one example, the label comprises a ³H label. Such labels can be used as a reagent in several methods, namely radio-ligand binding, autoradiography, or in scintillation proximity assay (SPA) using certain beads. This can provide a highly sensitive means for determining ChAT containing cells, networks and/or dynamic changes in the expression of ChAT before, during and after various types of stimulation paradigm of the cells in culture. Preferably a ³H label is used together with scintillation proximity assay (SPA) beads or plates to measure the amount of ChAT, for example, in any biological fluids.

In more particular embodiments, the radio-isotopic label comprises (or consists of) a ¹¹C atom. Thus, in particular embodiments, there is provided a compound of formula I (including all embodiments thereof, such as compounds of formula la, II and Ha) comprising at one or more (e.g. one) position therein a ¹¹C.

The skilled person will understand that references to compounds comprising a radioisotope will indicate that the compound is isotopically enriched at the one or more relevant position with an excess of the relevant isotope (i.e. when compared to the natural occurring proportions of the isotopes of the relevant atom), such as at least an 80% excess (e.g. at least a 90% excess, at least a 95% excess, or at least a 99% excess, such as at least a 99.9% excess). For the avoidance of doubt, the skilled person will understand that detectable labels (such as radio-isotopes, e.g. a ¹¹C) may be provided at any suitable point of the compound. For example, where the detectable label is a radio-isotope (such as a ¹¹C), that isotope may be provided at any suitable position (i.e. any position where the corresponding atom is present). Moreover, the skilled person will be aware of numerous ways in which such labelled compounds may be prepared. For example, in the case of radio-labelled compounds (such as those labelled with a ¹¹C), such compounds may be prepared according to the known synthesis of that compound but employing corresponding radio- labelled starting materials in that synthesis, which starting materials may be commercially- available or prepared using techniques known to those skilled in the art.

In certain embodiments, particularly where the detectable label is a ¹¹C, the label may be present of a component of a X group in compounds of formula I or la (i.e. where n represents at least 1 ), or as a component of the essential X¹ group in compounds of formula II and I Is.

In more particular embodiments, an X group in compounds of formula I or la (i.e. where n represents at least 1 ), or the X¹ group in compounds of formula II and I Is, comprises a ¹¹C.

In yet more particular embodiments, an X group in compounds of formula I or la (i.e. where n represents at least 1 ), or the X¹ group in compounds of formula II and Ma, represents -OR^1a wherein represents a C1-3 a Iky I comprising at least one (e.g. one) ¹ C.

In yet more particular embodiments, an X group in compounds of formula I or la (i.e. where n represents at least 1 , such as where n represents 1 ), or the X¹ group in compounds of formula II and Ha, represents -0¹¹CH3. As described herein, the detectable moiety may be detectable by an imaging technique, such as: CT; SPECT; PET; MRI; optical imaging; ultrasound imaging.

In one example, a selective antibody may be used to capture ChAT to the surface of a scintillation bead or a scintillation plate, thereafter using a compound (that has been suitably radio-labelled) as a detecting reagent. This significantly increases the sensitivity and the detection level compared to use of antibodies alone, and makes the assay homogeneous, meaning that one won't need to wash away other undesirable proteins as is the case in an ELISA assay. This in turn would reduce the number of steps in the method.

For CSF and cell medium, the amount/concentration of ChAT measured in any aspect of the invention may be from 1 to 1000 ng/ml. For example, 100 to 500, 10 to 200, 1 to 100, 1 to 10 ng/ml. For plasma and serum, the amount/concentration of ChAT measured in any aspect of the invention may be from 1 to 1000 pg/mL. For example, 100 to 500, 10 to 200, 1 to 100, 1 to 10 pg/mL. For cell and tissue homogenates, the amount/concentration of ChAT measured in any aspect of the invention may be from 0 to 1000 ng/mg total protein. For example, 100 to 500 ng/ml, or 10 to 200 ng/ml, or 1 to 100 ng/ml, or 1 to 10 ng/mg. Exemplary methods for measuring ChAT are discussed in Vijayaraghavan et al. (2013), PLoS One, 8, e65936.

The unit of enzyme activity may be number of mole (of ACh) synthesized per unit of time, per unit of tissue, weight or volume of sample, or unit of total protein in a sample. For instance, in biological fluid such as plasma/serum or CSF, the unit may be given as nmol/min/mL (of sample), and for brain homogenates, may be given as nmol/min/mg total protein. When the aim is to quantify amount (or concentration) of ChAT, the unit may be e.g. ng/mL (e.g. of bodily fluid such as CSF, plasma or serum) or ng/mg total protein (e.g. for brain homogenate). If ChAT is anchored to cell membrane, as it is the case of AChE, on red blood cells, the enzyme activity may be expressed as a unit per number of cells e.g. ng/cell or in term of activity nmol/min/cell. Exemplary methods are discussed in Ellman et al. 1961 , Biochem Pharmacol., 7:88-95. The compound described in any aspect of the invention may be selected from the group consisting of: Esomeprazole; Omeprazole; Lansoprazole; Dexlansoprazole, Pantoprazole; Rabeprazole; Tenatoprazole; llaprazole.

Preferably, the compound is selected from the group consisting of: Omeprazole; Esomeprazole; Tenatoprazole; Rabeprazole; Lansoprazole; Dexlansoprazole; Pantoprazole. More preferably, the compound may be selected from the group consisting of: Omeprazole; Esomeprazole; Tenatoprazole; Rabeprazole; Lansoprazole; Dexlansoprazole. Even more preferably, the compound may be selected from the group consisting of: Omeprazole; Esomeprazole; Tenatoprazole; Rabeprazole.

Most preferably, the compound is selected from the group consisting of: Omeprazole; Esomeprazole.

As described herein, the compound may comprise a detectable label, such as a radio- isotope (e.g. a C¹¹). For example, in particular embodiments, the specific compounds referred to herein (such as Omeprazole and Esomeprazole, e.g. Esomeprazole) may comprise at least one (e.g. one) C¹¹. In particular embodiments, the specific compounds Omeprazole and Esomeprazole (e.g. Esomeprazole) comprise a C¹¹ as a component of one or both (e.g. one) of the essential methoxy groups (i.e. so forming a -OC^{1 1} H3 group), such as at the benzimidazole-bound methoxy group.

In a further aspect of the invention, there is provided a compound of formula I (including all embodiments thereof, such as compounds of formula la, II and Ma), or a pharmaceutically acceptable salt thereof, comprising a detectable label as described herein (including all such embodiments thereof). It will be appreciated that, as such compounds have previously been used in therapy but not as tracers, prior to the present invention it was not been necessary to add a detectable label to those compounds.

Thus, in a particular embodiment there is provided Omeprazole, or Esomeprazole, comprising a C ¹ as a component of one or both (e.g. one) of the essential methoxy groups (i.e. so forming a -OC ¹ H3 group), such as at the benzimidazole-bound methoxy group.

In a further aspect of the invention, there is provided a compound of formula I (including all embodiments thereof, such as compounds of formula la, II and Ma), or a pharmaceutically acceptable salt thereof, comprising a detectable label as described herein (including all such embodiments thereof) for use in medicine (or for use as a pharmaceutical).

Labelled compounds as described herein may be obtained by analogy with conventional synthetic procedures, in accordance with standard techniques, using commercially available starting materials, and using appropriate reagents and reaction conditions. In this respect, the skilled person may refer to inter alia "Comprehensive Organic Synthesis" by B. M. Trost and I. Fleming, Pergamon Press, 1991. Further references that may be employed include "Heterocyclic Chemistry" by J. A. Joule, K. Mills and G. F. Smith, 3^rd edition, published by Chapman & Hall, "Comprehensive Heterocyclic Chemistry II" by A. R. Katritzky, C. W. Rees and E. F. V. Scriven, Pergamon Press, 1996 and "Science of Synthesis", Volumes 9-17 (Hetarenes and Related Ring Systems), Georg Thieme Verlag, 2006. The skilled person may also refer to "Comprehensive Organic Functional Group Transformations" by A. R. Katritzky, O. Meth-Cohn and C. W. Rees, Pergamon Press, 1995 and/or "Comprehensive Organic Transformations" by R. C. Larock, Wiley-VCH, 1999 and/or Suzuki ef al. 2014. PdO-mediated rapid cross-coupling reactions, the rapid C- [11 C]methylations, revolutionarily advancing the syntheses of short-lived PET molecular probes. Chem Rec 14, 516, and/or Erlandsson et a!., 2009. (18)F-labelled metomidate analogues as adrenocortical imaging agents. Nucl Med Biol 36, 435.

Exemplary methods for labelling compounds of the invention are also described in the accompanying Examples.

Labelled compounds as described herein may be isolated from their reaction mixtures and, if necessary, purified using conventional techniques as known to those skilled in the art. In a further aspect of the invention, there is provided a method for determining the presence of a disorder in a patient, comprising the steps of:

- administering to the patient an agent capable of selectively binding to choline acetyltransferase, and using the agent to measure the amount and/or concentration of choline acetyltransferase in one or more cell in the patient;

- determining the cholinergic function based on the amount and/or concentration of choline acetyltransferase; and

- determining the presence of a disorder in the patient on the basis of the cholinergic function of the one or more cell in the patient; wherein the agent is a compound of Formula I, as defined in the first aspect (i.e. including all embodiments thereof, such as compounds of formula la, II and I la, and specific compounds as described herein). As an optional alternative to measuring the amount and/or concentration of choline acetyltransferase in one or more cells, the amount and/or concentration of choline acetyltransferase may be measured in any biological fluid. For example, this may be achieved by using the labelled compound, for example ³H-labeled, together with SPA beads or plates. Examples of such biological fluids include blood, lymph fluid, cerebral spinal fluid, saliva, and/or urine.

In a further aspect of the invention there is provided the use of an agent, as defined in in the first aspect, in determining the presence of a disorder in a patient, the use comprising determining the cholinergic function of one or more cell in the patient based on the amount and/or concentration of choline acetyltransferase in the one or more cell. In a further aspect of the invention there is provided an agent, as defined in the first aspect, for use in determining the presence of a disorder in a patient, the use comprising determining the cholinergic function of one or more cell in the patient based on the amount and/or concentration of choline acetyltransferase in the one or more cell.

In a further aspect of the invention there is provided the use of an agent, as defined in the first aspect, for the manufacture of a medicament for determining the presence of a disorder in a patient, the use comprising determining the cholinergic function of one or more cell in the patient based on the amount and/or concentration of choline acetyltransferase in the one or more cell.

The patient may be determined as having the disorder when the cholinergic function of the one or more cell in the patient is modulated. Optionally, the patient is determined as having the disorder when the cholinergic function of the one or more cell in the patient is reduced. Alternatively, the patient is determined as having the disorder when the cholinergic function of the one or more cell in the patient is increased.

For example, the patient may be determined as having the disorder when the cholinergic function of the one or more cell in the patient is increased or reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%, preferably, increased or reduced by 50, 60, 70, 80, 90 or 100%.

Additionally or alternatively, the patient is determined as having the disorder when the amount and/or concentration of choline acetyltransferase measured is increased or reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%, preferably, increased or reduced by 50, 60, 70, 80, 90 or 100% .

The cholinergic function of the one or more cell in the patient may be determined by measuring the number of cells in the patient which have a specified amount and/or concentration of choline acetyltransferase.

Optionally, the one or more cell may be provided in a sample obtained from the patient. The sample may be a biopsy from the relevant tissue/organ types. The sample may also be blood, lymph fluid, cerebral spinal fluid, saliva, and/or urine. In certain embodiments, the one or more cell is provided in a cell culture.

In certain embodiments of the aspects of the invention, the disorder is a neurodegenerative disorder. The one or more cell may be a neuronal cell as defined above. By "neurodegenerative disorder", we include any disorder associated with the progressive loss of structure or function of neurons, which may also be characterised by neuron death. The neurodegenerative disorder may be selected from the list consisting of: Alzheimer's disease; Lewy's bodies disorder's dementia (such as dementia with Lewy bodies and Parkinson's disease dementia); fronto-temporal dementia; vascular dementia; traumatic brain injury; brain cancers; degenerative nerve diseases; encephalitis; epilepsy; genetic brain disorders; head and brain malformations; hydrocephalus; stroke; Parkinson's disease; multiple sclerosis (MS); amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease); HIV dementia; Huntington's disease; Sjogren's syndrome; prion diseases (such as Creutzfeld-Jacob disease (CJD)); Down's syndrome; myasthenia gravis. The neurodegenerative disorder is optionally Alzheimer's disease (AD). Alternatively, the neurodegenerative disorder is not Alzheimer's disease (AD). The AD may be the "familia form or the "sporadic" form of the disease. Sporadic AD may be divided into "early onset" and "late onset" sporadic AD, which may be classified according to the age of the individual in which clinical symptoms develop and/or based on the presence or absence of the APOE4 allele (since APOE4 carriers typically develop clinical symptoms around 5-10 years earlier than non-carriers, and are typically classified as having "early-onset" disease). The AD may be selected from the group consisting of: asymptomatic preclinical AD (i.e. subject has the initial stages of disease: this may include subjects with amyloid in the brain but showing no clinically assessable cognitive impairment); prodromal AD (early symptoms emerge); AD (full manifestation of clinical disease, dementia stage); subjects with mild cognitive impairment; individuals with subjective cognitive impairment (the subjects themselves perceive reduced cognitive ability but cognitive tests show no impairment); mixed AD (usually with vascular changes/dementia);

In one embodiment of any aspect of the invention, the neurodegenerative disorder is early- stage or "prodromal" Alzheimer's disease. Prodromal AD is defined, for example, in Welsh-Bohmer, 2008, Neuropsychol Rev 18, 70. A major characterization feature of prodromal AD is the presence of clinical symptoms, accompanied by positive amyloid-PET scan, low CSF amyloid-beta and/or high CSF tau; in more problematic cases, it may additionally be characterised by brain atrophy (determined, for example, by MRI), together with clinical cognitive symptoms. These examinations are done in subjects who seek or are sent to a geriatric clinic. In one embodiment of the invention, the disorder may be defined as one in which the subject shows cholinergic degeneration without having yet reached the manifestation of any clinical symptoms. In embodiment of the invention, the disorder is an inflammatory disorder. In such embodiments, the one or more cell may be any inflammatory cell. The inflammatory disorder may be selected from the group consisting of: rheumatoid arthritis (RA); multiple sclerosis (MS); tendonitis; atopic dermatitis; general inflammation in the brain; brain trauma; spinal injury. Preferably the inflammatory disorder is selected from the group consisting of: rheumatoid arthritis (RA); multiple sclerosis (MS); tendonitis; atopic dermatitis.

In another embodiment of the invention, the disorder is cancer. In such embodiments, the one or more cell may be any cancerous cell, for example, as defined above. Those skilled in the art will be aware that changes in cholinergic activity may occur in cancer, as discussed for example in: Chernyavsky et al. (2015) BMC Cancer 15, 152; Jonsson et al. (2007) Inflamm. Bowel Dis., 13: 1347-1356; Xie et al., (2009), Am. J. Physiol. Gastrointes. Liver Physiol., 296, G755-763. The cancer may comprise solid phase tumours/malignancies, locally advanced tumours, human soft tissue sarcomas, metastatic cancer, including lymphatic metastases, blood cell malignancies including multiple myeloma, acute and chronic leukemias, and lymphomas, head and neck cancers including mouth cancer, larynx cancer and thyroid cancer, lung cancers including small cell carcinoma and non-small cell cancers, breast cancers including small cell carcinoma and ductal carcinoma, gastrointestinal cancers including esophageal cancer, stomach cancer, colon cancer, colorectal cancer and polyps associated with colorectal neoplasia, pancreatic cancers, liver cancer, urologic cancers including bladder cancer and prostate cancer, malignancies of the female genital tract including ovarian carcinoma, uterine (including endometrial) cancers, and solid tumour in the ovarian follicle, kidney cancers including renal cell carcinoma, brain cancers including intrinsic brain tumours, neuroblastoma, astrocytic brain tumours, gliomas, metastatic tumour cell invasion in the central nervous system, bone cancers including osteomas, skin cancers including malignant melanoma, tumour progression of human skin keratinocytes, squamous cell carcinoma, basal cell carcinoma, hemangiopericytoma and Kaposi's sarcoma. The cancer may be selected from pancreatic cancer, ovarian cancer, breast cancer, prostate cancer, liver cancer, chondrosarcoma, lung cancer, head and neck cancer, colon cancer, sarcoma, leukaemia, myeloma, lymphoma, kidney cancer, thyroid cancer and brain cancers such as glioblastoma.

The cancer may preferably be selected from the group consisting of: prostate cancer; colon cancer; glioblastoma; breast cancer; lung cancer; bladder cancer. Preferably the cancer is selected from the group consisting of: prostate cancer; colon cancer; glioblastoma. More preferably, the cancer is prostate cancer.

In certain embodiments, an additional step of selecting one or more appropriate treatment for the patient or individual is performed.

Optionally, an additional step of identifying and/or selecting one or more treatments which is not appropriate for the patient or individual may be performed. For example, when the amount and/or concentration of ChAT is reduced by 50, 60, 70, 80, 90 or 100%, preferably 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79 or 80%, cholinergic function may be inadequate to make its stimulation by a particular treatment meaningful. In that situation that particular treatment might then not be performed and an alternative treatment may be performed instead. Such treatments may be selected by an appropriate clinician. An additional step of administering the selected treatment to the patient or individual may be performed.

Where the disorder is Alzheimer's Disease or "mixed" Alzheimer's Disease, the treatment may comprise a cholinesterase inhibitor (ChEI), optionally in combination with an NMDA antagonist - for example, a ChEI (such as donepezil, galantamine and/or rivastigmine) administered alone (at MCI, mild, and moderate AD) or in combination with memantine (an NMDA antagonist).

Commonly, Alzheimer's Disease and/or Vascular Dementia (VaD) and/or Lewy body dementias (LBD) will coexist - such conditions are termed "mixed AD", and may be detected by clinical evaluation, patient history of stroke/brain trauma, and/or MRI.

In clinical trials, Vascular Dementia has traditionally been diagnosed by the Hachinski Score and its modified versions, or the criteria of the National Institute of Neurological Disorders and Stroke - Association Internationale pour la Recherche et I'Enseignement en Neurosciences (NINDS-AIREN). This is a guideline similar to the NINCDS-ADRDA criteria for AD. Where the disorder is a Lewy body disorder (which includes dementia with Lewy bodies (DLB), and Parkinson's disease with dementia (PDD)), the treatment may comprise a cholinesterase inhibitor (ChEI), preferably rivastigmine or donepezil.

Where the disorder is Down's syndrome dementia, the treatment may comprise a cholinesterase inhibitor (ChEI)

Where the disorder is amyotrophic lateral sclerosis (ALS), the treatment may comprise Riluzole (Rilutek).

Where the disorder is multiple sclerosis (MS), the treatment may comprise one or more treatment selected from the list consisting of: interferon beta- 1a (Avonex, Rebif); peginterferon beta- 1a (Plegridy); teriflunomide (Aubagio); natalizumab (Tysabri); fingolimod (Gilenya); cytostatics; mitoxantrone (Novantrone); dimethyl fumarate (Tecfidera).

Where the disorder is rheumatoid arthritis (RA), the treatment may comprise one or more treatment selected from the list consisting of: non-steroidal anti-inflammatory drugs (NSAIDs); methotrexate; TNF-a blocker/inhibitors, such as Abatacept (Orencia), Adalimumab (Humira), and/or Etanercept (Enbrel).

New therapeutic interventions aiming to activate/revive the cholinergic neuronal system, such as deep brain stimulation of nucleus basalis of Meynert or NGF ^(50"53) may provide additional or alternative treatments to those mentioned above.

Preferably the ChEI is selected from the group consisting of: donepezil; galantamine; and rivastigmine. In a further aspect of the invention there is provided a method for determining the risk of an individual developing a disorder, comprising the steps of:

- administering to the individual an agent capable of selectively binding to choline acetyltransferase, and using the agent to measure the amount and/or concentration of choline acetyltransferase in one or more cell in the individual;

- determining the cholinergic function based on the amount and/or concentration of choline acetyltransferase; and - determining the risk of the individual developing the disorder on the basis of the cholinergic function of the one or more cell in the individual;

wherein the agent is a compound of Formula I, as defined in the first aspect. As an alternative to measuring the amount and/or concentration of choline acetyltransferase in one or more cells, the amount and/or concentration of choline acetyltransferase may be measured in any biological fluid, e.g. a biological fluid as defined above. For example, this may be achieved by using the labelled compound, for example ³H-labeled, together with SPA beads or plates.

In a further aspect of the invention, there is provided the use of an agent, as defined in the first aspect, in determining the risk of an individual developing a disorder, the use comprising determining the cholinergic function of one or more cell in the patient based on the amount and/or concentration of choline acetyltransferase in the one or more cell.

In a further aspect of the invention, there is provided an agent, as defined in the first aspect, for use in determining the risk of an individual developing a disorder, the use comprising determining the cholinergic function of one or more cell in the patient based on the amount and/or concentration of choline acetyltransferase in the one or more cell.

In a further aspect of the invention, there is provided a use of an agent, as defined in the first aspect, for the manufacture of a medicament for determining the risk of an individual developing a disorder, the use comprising determining the cholinergic function of one or more cell in the patient based on the amount and/or concentration of choline acetyltransferase in the one or more cell.

The disorder may be any disorder as defined herein.

The level of risk may preferably be correlated with the cholinergic function of the one or more cell. For example, the correlation may be a negative correlation. Alternatively, the correlation may be a positive correlation.

The cholinergic function of the one or more cell in the individual may be determined by measuring the number of cells in the patient which have a specified amount and/or concentration of choline acetyltransferase. The particular amount and/or concentration of choline acetyltransferase will vary depending on the precise method used, for example if it is an in vivo PET assessment or determining ChAT amount in CSF/plasma/or any other biological fluids; or autopsy tissue, post-mortem tissue, cell culture, organ culture.

For cells, organ, or autopsy tissue the number of ChAT-positive cells can be counted, and thereby changes noted in the expected number of ChAT-positive cells. If homogenates are used then the output will in e.g. ng/mg tot protein. If used as radio-labelled the methods will be more sensitive (e.g. more than what is expected by means of antibody).

For example, the patient may be determined as being at risk of developing the disorder when the cholinergic function of the one or more cell in the patient is increased or reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%, preferably, increased or reduced by 50, 60, 70, 80, 90 or 100%.

Additionally or alternatively, the patient may be determined as being at risk of developing the disorder when the amount and/or concentration of choline acetyltransferase measured is increased or reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%, preferably, increased or reduced by 50, 60, 70, 80, 90 or 100%.

In a further aspect of the invention there is provided a method for determining the progression of a disorder in a patient, comprising the steps of:

- administering to the patient an agent capable of selectively binding to choline acetyltransferase, and using the agent to measure the amount and/or concentration of choline acetyltransferase in one or more cell in the patient over time;

- determining the change in cholinergic function based on the amount and/or concentration of choline acetyltransferase over time; and

- determining the progression of the disorder in the patient on the basis of the change in cholinergic function of the patient over time;

wherein the agent is a compound of Formula I, as defined in the first aspect.

In a further aspect of the invention there is provided a use of an agent, as defined in the first aspect, in determining the progression of a disorder in a patient, the use comprising determining the cholinergic function of one or more cell in the patient based on the amount and/or concentration of choline acetyltransferase in the one or more cell. In a further aspect of the invention there is provided an agent, as defined the first aspect, for use in determining the progression of a disorder in a patient, the use comprising determining the cholinergic function of one or more cell in the patient based on the amount and/or concentration of choline acetyltransferase in the one or more cell.

In a further aspect of the invention there is provided a use of an agent, as defined in the first aspect, for the manufacture of a medicament for determining the progression of a disorder in a patient, the use comprising determining the cholinergic function of one or more cell in the patient based on the amount and/or concentration of choline acetyltransferase in the one or more cell.

The disorder in the patient may be determined as having regressed if there is a modulation in cholinergic function in the patient over time. Alternatively, the disorder in the patient may be determined as having progressed if there is a modulation in cholinergic function in the patient over time.

The disorder may be any disorder as defined herein. The modulation may correspond to a reduction in cholinergic function. Alternatively, the modulation may correspond to an increase in cholinergic function.

For example, the disorder may be determined as having regressed when the cholinergic function of the one or more cell in the patient is increased or reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%, preferably, increased or reduced by 50, 60, 70, 80, 90 or 100%.

Additionally or alternatively, the disorder may be determined as having regressed when the amount and/or concentration of choline acetyltransferase measured is increased or reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%, preferably, increased or reduced by 50, 60, 70, 80, 90 or 100%.

For example, the disorder may be determined as having progressed when the cholinergic function of the one or more cell in the patient is increased or reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%, preferably, increased or reduced by 50, 60, 70, 80, 90 or 100%.

Additionally or alternatively, the disorder may be determined as having progressed when the amount and/or concentration of choline acetyltransferase measured is increased or reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%, preferably, increased or reduced by 50, 60, 70, 80, 90 or 100%.

The disorder in the patient may be determined as being stable if there is no change in cholinergic function in the patient over time.

The change in cholinergic function may be determined by measuring the number of cells in the patient which have a specified amount and/or concentration of choline acetyltransferase.

The agent may be administered to the patient at regular intervals over time and the amount and/or concentration of choline acetyltransferase in one or more cell is measured in the patient at the same regular intervals over time. In a further aspect of the invention there is provided a method for determining the response to therapy of a disorder in a patient, comprising the steps of: providing a patient with a disorder that is undergoing therapy for the disorder;

- administering to the patient an agent capable of selectively binding to choline acetyltransferase, and using the agent to measure the amount and/or concentration of choline acetyltransferase in one or more cell from the patient over time;

- determining the change in cholinergic function of the one or more cell from the patient based on the amount and/or concentration of choline acetyltransferase over time;

wherein the agent is administered to the patient before and/or during and/or after therapy;

and wherein the response to therapy of the disorder in the patient is determined on the basis of the change in cholinergic function of the one or more cell from the patient over time during and/or after therapy;

and wherein the agent is a compound of Formula I, as defined in the first aspect.

In a further aspect of the invention there is provided a use of an agent, as defined in the first aspect, in determining the response to therapy of a disorder in a patient, the use comprising determining the cholinergic function of one or more cell in the patient based on the amount and/or concentration of choline acetyltransferase in the one or more cell. In a further aspect of the invention there is provided an agent, as defined in the first aspect, for use in determining the response to therapy of a disorder in a patient, the use comprising determining the cholinergic function of one or more cell in the patient based on the amount and/or concentration of choline acetyltransferase in the one or more cell.

In a further aspect of the invention there is provided a use of an agent, as defined in the first aspect, for the manufacture of a medicament for determining the response to therapy of a disorder in a patient, the use comprising determining the cholinergic function of one or more cell in the patient based on the amount and/or concentration of choline acetyltransferase in the one or more cell.

The disorder may be any disorder as defined above. The patient may be determined as having responded to therapy if there is a modulation in cholinergic function in the patient over time. Alternatively, the patient may be determined as having not responded to therapy if there is a modulation in cholinergic function in the patient over time. The modulation may correspond to a reduction in cholinergic function. Alternatively, the modulation corresponds to an increase in cholinergic function. For example, patient may be determined as having responded to therapy when the cholinergic function of the one or more cell in the patient is increased or reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%, preferably, increased or reduced by 50, 60, 70, 80, 90 or 100%. Additionally or alternatively, patient may be determined as having responded to therapy when the amount and/or concentration of choline acetyltransferase measured is increased or reduced by 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%, preferably, increased or reduced by 50, 60, 70, 80, 90 or 100%. The change in cholinergic function may be determined by measuring the number of cells in the patient which have a specified amount and/or concentration of choline acetyltransferase.

In any of the aspects described above, the one or more cell may be provided in a sample obtained from the patient or individual. In a further aspect of the invention there is provided a kit comprising an agent as defined in the first aspect and a detectable moiety. The detectable moiety may be as defined above. The following examples embody various aspects of the invention. It will be appreciated that the specific antibodies and/or antigens used in the examples serve to illustrate the principles of the invention and are not intended to limit its scope.

The present invention and Examples are described with reference to the following figures.

Figure 1. Estimated remaining ChAT activity in the AD brain as percentage of the control brain. This figure is modified and adapted from Procter et a/., 1988. Topographical distribution of neurochemical changes in Alzheimer's disease. J Neurol Sci 84, 125 <² >.

Figure 2. Three-dimensional docking poses of the compounds Esomeprazole (A), R_Omperazole (B), SJIaprazole (C) and RJIaprazole (D). The residues involved in important interactions are represented as stick model. The 2D ligand-interaction diagram illustrates the major interactions between the ligand and the active sites amino acid residues of ChAT.

Figure 3. Three-dimensional docking poses of the compounds SJLansoprazole (A), (^.Lansoprazole (B), S_Pantoprazole (C) and R_Pantoprazole (D). The residues involved in important interactions are represented as stick model. The 2D ligand- interaction diagram illustrates the major interactions between the ligand and the active sites amino acid residues of ChAT.

Figure 4. Three-dimensional docking poses of the compounds S_Rabeprazole (A), R_Rabeprazole (B), S_Tenatoprazole (C) and R_Tenatoprazole (D). The residues involved in important interactions are represented as stick model. The 2D ligand- interaction diagram illustrates the major interactions between the ligand and the active sites amino acid residues of ChAT.

Figure 5. IC50 analysis of the leads, CH-24, CH-26 and CH-26S. Comparison of the IC₅o values indicates that CH-26 (Omeprazole) and CH-26S (Esomeprazole) are 15 and 30 fold more potent than CH-24, respectively. The enzyme assay was run kinetically in the presence of a constant concentration of rhChAT protein, 150μΜ choline, and 10 μΜ acetyl- Coenzyme A at room temperature at pH 7.5.

Figure 6. Enzyme kinetic analysis with and without the Lead CH-26S. A. Omeprazole (also termed "CH-26" herein). B. Results of non-linear regression analysis for changes in the recombinant human ChAT activity at three different concentration of CH-26S and at various concentration of the enzyme substrate, choline. This analysis estimated a binding affinity, Ki, of 141 nM (ranging between 125-159 nM). C. Lineweaver-Burk plot illustrating that CH-26S behaves as a mixed competitive ligand. The enzyme assay was run kinetically in the presence of a constant concentration of rhChAT protein (75ng/well) and 10 μΜ acetyl-Coenzyme A at room temperature at pH 7.5.

Figure 7. Enzyme kinetic analysis with and without the Lead CH-F17. A. Molecular structure of CH-F17. B. Inhibition-dose response analysis at varying concentrations of the compound and choline substrate. C. Results of non-linear regression analysis for changes in the recombinant human ChAT activity. This analysis estimated a Ki of 17.5 nM (ranging between 13-23nM). D. Lineweaver-Burk plot showing that CH-F17 behaves as a noncompetitive ligand of ChAT. All of the enzyme assays were run kinetically in the presence of a constant final concentration of rhChAT protein (75ng/well) and 10 μΜ acetyl- Coenzyme A at room temperature at pH 7.5.

Figure 8. Enzyme kinetic analysis with and without the Lead CH-F18. A. Molecular structure of CH-F18. B. Inhibition-dose response analysis at varying concentrations of the compound and choline substrate. C. Results of non-linear regression analysis for changes in the recombinant human ChAT activity. This analysis estimated a Ki of 12.8 nM (ranging between 10-16nM). D. Lineweaver-Burk plot showing that CH-F18 behaves as a mixed- competitive ligand of ChAT. All of the enzyme assays were run kinetically in the presence of a constant final concentration of rhChAT protein (75ng/well) and 10 μΜ acetyl- Coenzyme A at room temperature at pH 7.5.

Figure 9. Enzyme-Inhibition analysis. A. Changes in the BuChE activity of human plasma with a total dilution of 400X. All compounds had a final concentration of 100μΜ. B. Corresponding changes in the partially purified human blood AChE activity. All compounds had a final concentration of 500μΜ. C. Dose-inhibition analyses of a-NETA for ChAT, AChE and BuChE. Figure 10. Association of PPI use and incident dementia. This figure is adapted from Gomm et al., 2016. Association of Proton Pump Inhibitors With Risk of Dementia: A Pharmacoepidemiological Claims Data Analysis. JAMA Neurol 73, 410. Figure 11. Potential clinical relevance of ChAT as a multifaceted biomarker in dementia as depicted in three independent studies. I: Changes in CSF ChAT activity and in cognition in AD patients participating in a 12-months pilot study of NGF-releasing cells (EC-NGF) implant therapy ⁽⁵¹⁾. II: Correlation between cognition (MMSE test) and plasma ChAT activity in AD patients after 12 months of treatment with a cholinesterase inhibitor. Ill: Differential levels of ChAT in the plasma of patients with Alzheimer's disease (AD), mild cognitive impairment (MCI) and subjective cognitive impairment (SCI). IV: Differential levels of ChAT in CSF of patients with the neuroinflammatory disease, multiple sclerosis (MS) compared to an aged-matched group of patients with other neurological disorders (OND) <¹ >

Figure 12. Changes in three hallmarks of Alzheimer's disease. A. Illustrates the immunostaining of ChAT as marker of the relative changes in cholinergic neurons of nucleus basalis of Meynert (NBM) in the brain from subjects with no cognitive decline (NCD), compared to the brain from subjects with very mild to moderate (MCD) and severe to very severe cognitive decline (SCD). B. Illustrates relative levels of AT8-antibody immunostaining of tau deposits in the NBM of post-mortem brain. C. This closely resembles the accumulation of amyloid-beta (Αβ) deposits in the brain. In contrast to relative changes in ChAT, both tau and Αβ deposits show a clear disconnection to the stage of the disease (both clinically and pathologically. The figure is modified from different figures from Zhu et al., 2016⁽²²⁾. The blue curves are drawn for illustrative purpose. The groups are defined based on the Reisberg scale⁽⁸⁸⁾: No cognitive decline group (NCD), R1 ; Mild cognitive decline group (MCD, very mild to moderately severe CD) R2-5; Severe cognitive decline group (SCD, who had severe/very severe CD) R6-7. Figure 13. Comparison of concentrations of omeprazole in blood versus the brain.

The figure is adopted from Cheng F. C. et al., 2002. The following calculations are made based on the graph in this reference. Based on a Mw of 345.42 g/mol for omeprazole, the values are converted here to molar concentrations. The concentration of omeprazole are given as molality unit (pg/mL or mg/L) in the graph. As is marked by the arrows, the highest brain concentration (at t min) is -0.3 pg/rnl, which (after dividing by Mw) corresponds to a brain concentration of -870 nM. At tsomm the brain concentration is reduced to - 0.1 pg/ml, which corresponds to -300 nM. ^{1 1}The Lower limit is calculated at tso m since a PET assessment is expected to have in average a duration of 45-50min. Cbrain =0.1 to 0.3 (mg/L), then C/Mw= 0.1χ10 ³/345.42=290ηΜ. Similarly C/Mw= 0.3x10^"3/345.42 = 869nM. Thus Cbrain range is ~300-870nM. Figure 14. Protein level of ChAT in paired synovial fluids and plasma samples of patients with rheumatoid arthritis. ChAT protein was measured by the ELISA assay described in the methods in the accompanying Examples. Plasma samples were diluted 50-times before use but the synovial samples were undiluted. Protein concentration is calculated from a known set pf standard proteins of recombinant human ChAT.

Figure 15. MRI and PET summation images of ¹¹C-CH-26 in non-human primate. The assessments were run at KI-PET center on Siemens HRRT.

Figure 16. Horizontal view of NHP-PET using [1 1 C]CH-26 tracer PET assessment in the female cynomolgus primate. The PET data is summation images for 2 hours.

Figure 17. Coronal view of NHP-PET using [1 1 C]CH-26 tracer PET assessment in the female cynomolgus primate. The PET data is summation images for 2 hours. Figure 18. Sagittal view of NHP-PET using [1 1 C]CH-26 tracer PET assessment in the female cynomolgus primate. The PET data is summation images for 2 hours.

Figure 19. Projection of the cholinergic cranial nerves of the parasympathetic nervous system throughout the body. Illustration is from Anatomy & Physiology, Connexions Web site (http://cnx.org/content/col1 1496/1.6/), Jun 19, 2013.

Figure 20. Transaxial, coronal and sagittal PET images of ¹C-CH-26 binding and distribution in the brain of a non-human primate. The images were prepared by summation of the frames 20-34 for each PET scan, and subtraction of PET2 from PET1. Data are calculated as differences in SUV (standard uptake values), representing a measure of displacement of the hot-compound (¹¹C-CH-26) by the pretreatment with the cold- compound (un-labeled CH-26). The PET assessments were run at KI-PET center on Siemens HRRT. Figure 21. Comparison of the net binding of the ¹¹C-CH-26 PET tracer in the brain of a non-human primate (sagittal PET images). PET binding is highlighted by the dashed line in each image. EXAMPLES

Example 1 - Validity of the ChAT enzyme as a prominent target for PET Dementias are common and have major clinical and societal consequences, which are expected to increase dramatically during the next three decades. Reliable biomarkers are essential to help patients, caregivers, and health professionals to better plan future care and management. Alzheimer's disease (AD) is the leading cause of dementia. One of the key features of AD is an early selective degeneration of cholinergic neurons/projections in the brain ^(1_4). Other dementias that share this characteristic are Lewy body disorders (LBD) and Down's syndrome (DS) ⁽⁵· ⁶⁾. LBD include dementia with Lewy bodies and Parkinson's disease dementia).

Putatively, pathological events of AD and AD-like disorders are initiated 20-30 years prior to manifestation of clinical symptoms. In the case of the more aggressive autosomal- dominant forms of AD, it is estimated 10-20 years before the onset of clinical symptoms

(7)_

Deposits of Αβ peptides and tau's neurofibrillary tangle (NFT) seem to be present (and have reached ceiling or plateau levels) already in the brain of patients with mild cognitive impairment (MCI, considered a p re-stage of AD, and which will convert to AD within five years). This has led to the putative view that the brain compensates for these abnormalities until there are no resources left due to the progressive neuronal degeneration/brain atrophy.

However, there are no associations between the Αβ (and tau) deposits in the brain and severity of clinical symptoms of dementia. Because of this, some are now arguing that soluble Αβ and/or tau oligomers rather than their deposits are most noxious for brain function. As yet, there are no methods for determining the levels of soluble Αβ and/or tau oligomers in the brain (or even in the cerebrospinal fluids, CSF). In addition, the brain dysfunction is expected to manifest itself at neuronal levels much earlier than actual occurrence of neuronal death. Indeed, choline acetyltransferase (ChAT) level, as the most specific marker of cholinergic neuronal system, is reduced in the brain of subject suspected for early onset of the disease ⁽⁸⁾. Thus, the cholinergic system is the most vulnerable neuronal network, showing early and selective degeneration in AD-like diseases (Tables 1 and 2, and Fig. 1) <²-⁵· ⁹- ²>.

Cholinergic definition, machinery and distribution

Expression of the acetylcholine (ACh) synthesizing enzyme, choline acetyltransferase (ChAT) defines the cholinergic cells/neurons. In the CNS, four "ChAT-containing" neuronal nuclei (Ch1-Ch4) exist in the basal forebrain ⁽¹³⁾. Ch1 and Ch2 innervate the hippocampal complex, Ch3 the olfactory bulb and Ch4 the rest of cerebral cortex and amygdala ⁽¹³⁾. Ch4-neurons are located in the nucleus basalis of Meynert (NBM). In addition there is now enough evidence to extend the cholinergic definition to diverse ChAT-expressing non- excitable cells, such as immune cells and astrocytes. The latter was recently documented

(14)_ The cholinergic machinery consists of ChAT that is localized in the cytoplasm of cholinergic neurons, where it synthesizes ACh. This occurs through transfer of the acetyl-moiety of acetyl-Coenzyme A (A-CoA) to a choline molecule. ChAT contains a catalytic tunnel, within which there is a binding site for choline/ACh and one binding site for A-C0A/-C0A. The synthesized ACh is then transported by vesicular ACh transporter (VAChT) and stored into synaptic vesicles until its release into the synapses to act on its receptors. There are two general types of acetylcholine receptors (AChRs), namely nicotinic AChRs (nAChRs) and muscarinic AChRs (mAChRs). The released ACh is degraded, within the synaptic cleft, to choline and acetic acid by the enzymes, acetylcholinesterase (AChE), and to a lesser extent by butyrylcholinesterase (BChE). The choline is then recycled back into the cytoplasm by uptake by high affinity choline transporter (HChT).

All other cells/neurons that express AChRs and/or cholinesterases (AChE and BuChE) are called cholinoceptive cells/neurons. Thus, these markers, in contrast to ChAT, define the down-stream cholinergic postsynaptic events.

Cholinergic neurons project widely throughout the brain. In addition, autonomic ganglionic neurons in both the PNS and CNS are also cholinergic. Parasympathetic neurons are all cholinergic (eye iris, heart, ciliary muscles, Gl tract, urinary bladder, salivary glands) as well as many sympathetic neurons (sweat glands, etc). In general, all type of muscles one way or the other utilizes ACh. For instance, cholinergic motor neurons also innervate muscle endplates at neuromuscular junction of skeletal muscles. A major part of the enteric nervous system in the gut is also cholinergic, and innervates and controls intestinal motility and function, and may hence be involved in some disorders in the intestinal tracts ⁽¹⁵⁾. Recently, the inventors found age-related changes of ChAT levels in ascending and descending human colon ⁽¹⁶⁾. Furthermore, many other organs/tissues indeed utilize more or less cholinergic signaling, for instance, ChAT is expressed in placenta ⁽¹⁷⁾, in seminal fluids and/or spermatozoids) ^{(18, 19)}.

Longitudinal changes in Cholinergic signaling in dementia

Reduction in the ChAT levels by enzymatic or immune-histochemical assays of the cortical and subcortical AD brain regions is well-documented ⁽⁴· ²⁰-²²⁾. The reduction in ChAT level is estimated between 50-60% in the majority of brain regions, but 75-98% in brain regions that become affected early and severely in dementia ^{(4, 10)}, such as the regions of medial temporal lobe ⁽²³⁾ involved in memory consolidation. Similar reduction is observed in LBD and DS ^{(5 6)}. Consistently, cholinergic cell densities in the dementia brain seem to also be reduced by about 50% ⁽¹²⁾.

Although the reason why the cholinergic network shows such sensitivity to the AD pathology remains elusive, recent reports provide evidence that closely links cholinergic signaling in general and, in particular its deficit, to the key player in AD, namely the amyloid-β (Αβ) peptides and their pathological aggregates ⁽²⁴· ²⁵⁾. Intra-neuronal Αβ seems to preferentially be present in the basal forebrain cholinergic neurons regardless of age or disease ⁽²⁴⁾, indicating a non-toxic function for Αβ peptides in cholinergic cells. Αβ seems to act as regulator of cholinergic signaling by allosteric modulation of the activity of cholinesterases ^{(H 26}· ²⁷⁾ through formation of hybrid complexes, termed ΒΑβΑΰε ⁽²⁸⁾, which are formed by physical interactions of BuChE and/or AChE with Αβ peptides and ApoE proteins ⁽²⁶⁾.

Overall, compelling old and new observations impose the view that cholinergic deficit may be a cause rather than a consequence of the AD-type pathological events. For instance, exposure to drugs with strong anti-cholinergic activity increases the risk of developing dementia ^(29"31), indicating that the effect is not just symptomatic. Thereby a decline (be it age-related and/or pathologically related) in the central cholinergic signaling may at least be a vulnerability determinant for the neurodegenerative dementia disorders. Thus, a bio-functional marker of the health of central cholinergic network could identify susceptible subjects. Altered Cholinergic signaling in other disorders

ChAT expression and activity, through the cholinergic anti-inflammatory pathway (CAP), has been linked to an involvement in acute and chronic inflammation ^{( 18}' ^{9, 32"34)}. This may represent another link, that is an observed low-systemic (most likely chronic) neuroinflammation in AD which might be propagated through malfunctioning of cholinergic anti-inflammatory pathway (CAP), in particular of the cross-talk interfaces between immune system and the cholinergic Ch4-neurons of nucleus basalis in the brain ⁽³⁵⁾. Strictly speaking, ChAT expression defines all type of cholinergic cells. Reports show that lymphocytes and astrocytes under stimulatory conditions start expressing and releasing ChAT ⁽¹⁴⁾. In other words, they become cholinergic cells on demand, and use ACh as a signaling molecule. Thus, the term "cholinergic signaling" rather than cholinergic neurotransmission is used here to cover these aspects of cholinergic action that may not merely be a function of cholinergic neurons.

The compelling evidence that suggests acetylcholine is a key anti-inflammatory molecule has led to major attempts in developing effective cholinergic agonists as treatment for various inflammatory disorders ^{(34, 36~38)}. Stimulation of lymphocytes with lipopolysaccharides (LPS) or anti-CD3 antibody causes a dramatic changes in the expression and release of ChAT by lymphocytes ⁽¹⁴⁾.

ChAT is reduced in several other diseases with dementia as one of their final features. For instance, ChAT is reduced in the brain of an HIV animal model ⁽³⁹⁾, and in amyotrophic lateral sclerosis (ALS), a motor neuron disease with significant loss of ChAT immunoreactivity ⁽⁴⁰⁾.

Considering that cholinergic signaling is involved in regulation of inflammatory processes (again both in the CNS, the PNS and peripheral tissues), it can be expected to also play a crucial role in other inflammatory disorders, such as rheumatoid arthritis (RA) and multiple scleroses (MS). In patients with MS, there is a significant increase in ChAT levels in CSF compared to controls, which also correlates strongly with several factors of the complement immune system ^{(1 )}. However, in such cases an increase in the expression of ChAT or an increase in the number of ChAT-positive non-neuronal cells, such as astroglial cells ^{(H 41 )} would be expected. ChAT and ACh-receptors are also expressed by lymphocytes ⁽⁴²⁾. Direct evidence suggests acute expression of ChAT upon stimulation of lymphocytes ^{(14, 43)}. Treatment with AChE inhibitors also seems to alter the release of oncostatin-M and other cytokine from lymphocytes ⁽⁴⁴⁾.

There is also evidence that cholinergic signaling is involved in the etiology of some types of cancer, in particular colon and prostate cancer. Indeed, ChAT overexpression is identified in several cancer types, and evidence indicates that ACh may act as an autocrine stimulator of cell proliferation ^{( 5)}. High amount of choline is accumulated in the prostate and/or lymph-nodes of subjects having prostate cancer ^(46"48). The putative reason is that choline is needed by cancer cells for their growth as it is required for synthesis of several fatty acids of the lipid bilayer in the cell membrane <⁴⁶-⁴⁸⁾. However, several studies show the presence of high ChAT activity in seminal fluids and/or spermatozoids ⁽¹⁸· ¹⁹⁾. Thus the factor that triggers accumulation of choline in cancerous prostate tissue may also reflect a malfunction in ChAT levels, and/or acetylcholine synthesis and signaling rather than mere cell proliferation (of cancer cells). Otherwise, accumulation of choline should be common among all cancerous cells and not only prostate cancer.

Thus, ChAT is also expected to be a useful biomarker target for detecting peripheral inflammation, neuroinflammation and cancerous tissues. Cholinergic enhancing therapies

Currently, three out of four drugs that are available for AD-like dementia are cholinesterase inhibitors (ChEls) ⁽⁴⁹⁾. ChEls (e.g. donepezil, galantamine or rivastigmine) are used alone for treatment of patients with MCI, mild, and moderate AD (or mixed AD) or in combination with memantine (an NMDA antagonist) for treatment of patients at severe stages of AD. Rivastigmine and donepezil are also used in patients with Lewy body disorders (both dementia with Lewy bodies, DLB, and Parkinson's disease with dementia, PDD). ChEls has been used also in patients with Down's syndrome dementia. Vascular dementia has little or no cholinergic deficit and ChEls do not work in such patients. However, as mentioned, in mixed AD (which usually has vascular abnormalities) ChEls are used.

Other cholinergic enhancing therapeutic strategies that have shown positive results in AD patients are deep brain stimulation (DBS) of the nucleus basalis of Meynert ⁽⁵⁰⁾, and nerve growth factor (NGF) therapy ^{(51 > 52)}. All other non-cholinergic therapeutic strategies that have been tested have failed to show clinical benefit, including anti-Αβ vaccination/antibody that aimed to clear Αβ deposits.

Altogether, the well-established facts of early and selective cholinergic neuronal dysfunction in AD-like dementias (AD, LBD and DS), as well as the role of cholinergic signaling in memory and cognition, suggest that any new intervention (symptomatic or disease-modifying) needs to show a regression of the observed deficit in the cholinergic signaling or a slowing of the degeneration of central cholinergic neurons. Responders/non-responders

Not all patients respond well to cholinergic enhancing therapies. There are therefore responders and non-responders. For instance, patients diagnosed with vascular dementia (VD) do not respond to ChEls, most likely, because these drugs are designed to strengthen cholinergic signaling which is not greatly affected in VD. Similarly, there is evidence that patients with severe cholinergic neurodegeneration may not respond well to ChEls because too few cholinergic neurons remain in the brain so that the stimulation by therapeutic interventions is incapable to produce a meaningful clinical response. Similarly, such patients would not be able to respond to other new more experimental therapeutic interventions aiming to activate/revive the cholinergic neuronal system, such as deep brain stimulation of nucleus basalis of Meynert ^{(50, 53)} or NGF therapy ^{(51, 52)}.

This issue is also of importance for new drugs claiming to have a disease-modifying effect as it is unlikely that a disease-modifying drug will produce meaningful biological or clinical changes in a brain that had acquired severe and irreversible neuronal degeneration. Such therapeutic approaches should be most effective in subjects at risk, but before extensive irreversible damage/neuronal death has occurred in their brain. Simply, the neuronal functions in the brain are likely to have more plasticity at earlier stages than later on when extensive degeneration has accu m u I ated/acq u i red in the brain. In this way a clinical study can be enriched by including subjects that may respond most/best to such treatments. This would increase the probability of a successful clinical outcome if the drug is as effective as it had been intended.

It may on the other hand reduce the cost of giving drug to patients that will not respond. Perhaps, most importantly it may reduce unnecessary risk/suffering of the patients from adverse events associated with these drugs. Therefore, there exists a considerable need for a means to determine cholinergic function which will also be useful in determining the presence and progression of associated disorders. Currently, in contrast to the dopaminergic or serotonergic systems, there are no in vivo biomarkers of the health of the cholinergic network.

It is well established that cholinergic neuronal networks are closely associated with different cognitive domains of the brain function, in particular memory, attention, arousal and its deficit correlate best with severity of dementia. Indeed, changes in the cholinergic neuronal function as deduced by severe alteration of ChAT levels in the brain show good agreement with clinical feature of dementia ⁽⁴· ⁸· ¹°· ²⁰- ²¹· ⁵⁴· ⁵⁵⁾. The changes in ChAT also occur early in the course of the disease ^{(8, 22)}.

The reduction of ChAT activity is more severe than other neuronal markers, and topographical changes in the cholinergic projections are well-established (Tables 1 and 2, and Fig. 1) ⁽²⁰· ^{21 )}. For instance, AD brain biopsy investigation show no evidence of loss of other neurotransmitters, such as aspartate, glutamate and gamma-aminobutyric acid, even five years after emergence of symptoms ⁽⁵⁵- ⁵⁶⁾.

Table 1. ChAT activity (nmoi/100mg protein/min) in cortex of postmortem brain.

Data are from Procter et al., 1988. Topographical distribution of neurochemical changes in Alzheimer's disease. J Neurol Sci 84, 125.

Brain regions

Temporal lobe PT FT IT ST AT

Ctrl 7.5 ±3.3 (16) 5.3 ± 2.0 (16) 5.6 ± 1.9 (16) 11.3 ± 3.6 (18) 8.4 ± 2.8 (15)

AD 3.3 ± 2.2 (16)^** 1.8 ± 0.9 (16)^** 2.1 ± 1.0 (14)^** 3.8 ± 2.4 (14)*^* 2.8 ± 1.3 (15)^**

Reduction 56% 66% 63% 66% 67%

Frontal lobe PF OF SF

Ctrl 22.2 ± 12.0 (15) 9.1 ± 3.3 (16) 7.8 ± 2.5 (17)

AD 9.1 ± 5.2 (15)^** 4.6 ± 2.8 (16)^** 3.9 ± 1.6 (15)^**

Reduction 59% 55% 50%

Parietal lobe IP SP

Ctrl 5.3 ± 2.0 (16) 5.9 ± 2.3 (16)

AD 2.9 ± 1.2 (16)^** 3.1 ± 1.7 (15)*^*

Reduction 45% 47%

Occipital lobe LO

Ctrl 6.0 ± 2.5 (16)

AD 2.7 ± 1.6 (15)^**

Reduction 55%

Cingulate cortex PC AC

Ctrl 6.6 ± 2.8 (17) 7.7 ± 2.8 (17)

AD 3.4 ± 1.6 (16)^* 4.5 ± 2.3 (16)^**

Reduction 48% 42%

Results are means ± SD (number of subjects in parentheses). Controls (Ctrl) and AD samples were from the parahippocampal gyms (PT, in the region of Brodmann area, BA 28), fusiform gyms (FT), inferior temporal gyms (IT, BA 20), middle and superior temporal gyri (ST, BA 21/22), temporal pole (AT, BA 38), paraterminal gyms (PF, BA 25), orbital gyms (OF, BA 11 ), superior frontal gyms (SF, BA 9), superior parietal lobule (SP, BA 7), inferior parietal lobule (IP, BA 40), lateral occipital gyms (LO, BA 18), posterior cingulate gyms (PC, BA 23) and anterior cingulate gyms (AC, BA 24). ^*P < 0.05 ^**P < 0.005

Table 2. ChAT activity (μιηοΙ/hr/g protein) in the cerebral cortex of control and AD brains

Brain regions Control SDAT % of control

Frontal Cortex

4 0.79±0.02 (6.1) 0.53+0.05(3.4) 55 **

6 0.80±0.03 (6.4) 0.5410.05 (3.7) 55 **

8 0.79±0.02 (5.9) 0.46+0.05 (3.1) 47 **

9 0.81±0.02 (6.4) 0.50+0.05 (3.5) 49 **

Brodmann area 10 0.82±0.02 (6.6) 0.58+0.05 (4.2) 57 **

11 0.85±0.02 (7.2) 0.57+0.05 (4.4) 52 **

24 1.04±0.02 (11.5) 0.77+0.04 (6.3) 54 **

25 1.24±0.03 (16.8) 1.03+0.06 (8.9) 62 *

32 0.91±0.02 (8.6) 0.66+0.04 (5.5) 56 **

Temporal Cortex

20 0.81±0.03 (6.8) 0.42+0.04 (2.5) 41 **

21 0.8410.03 (7.0) 0.4310.05 (2.4) 39 **

Brodmann area 22 0.89±0.04 (8.2) 0.48+0.05 (2.8) 39 **

28 1.01±0.04 (11.9) 0.6310.05 (4.4) 42 **

38 0.93±0.03 (8.1) 0.53+0.05 (2.9) 40 *#

Parietal Cortex

Brodmann area 7 0.70±0.03 (5.1) 0.40+0.05 (2.8) 51 **

Occipital Cortex

Brodmann area 17 0.5010.03 (3.1) 0.17+0.06 (1.4) 47 *«

Anterior hippocampus 1.2810.04 (17.9) 0.94+0.06 (8.4) 46 **

Posterior hippocampus 1.2410.03 (16.6) 0.8610.07 (8.1) 42 **

Subiculum 1.0910.03 (12.1) 0.75+0.03 (5.4) 46 **

Table 2 is adapted from Rossor et ai, 1982 ⁽²⁰⁾. A post-mortem study of the cholinergic and GABA systems in senile dementia. Brain 105, 313. Data are means ± SEM of the logarithmically transformed data with medians of the untransformed data in parenthesis. The value for the SDAT mean expressed as a percentage of control was calculated from the antilog of the difference between the means of the transformed data. Two-tailed student's t-test. *P < 0.01 , **P < 0.001. The numbers of observations were 22-26.

In addition, usage of drugs with anticholinergic activity is associated with not merely cognitive decline but also an increased risk of incident dementia ^{(29, 30)}. These facts together with emerging reports strongly suggest that the cholinergic deficit may play a causative role than being one of the consequences of the AD-type neuropathological events ^{(24, 26, 57)}. In conclusion, ChAT as a target of a PET tracer to map the health of and/or changes in the core cholinergic neuronal projections in the central nervous system is highly justified.

There are ongoing attempts to target vesicular ACh-transporter (VAChT) for developing PET-tracer of cholinergic synapses ^{(¾ 59)}. The ligands are derivatives of vesamicol (a VAChT inhibitor). However, as yet no tracer with good clinical potential has been developed. The major problem with VAChT as PET target seems to be its limited distribution (to the synaptic interfaces) and density in the brain, allowing good signal mainly in striatum. Indeed, preliminary analyses at our lab on brain homogenates indicate that VAChT levels at a given total protein concentration are much less than ChAT levels. This might be due to possibility that not all cholinergic cells express VAChT, in particular non- neuronal cholinergic cells, such as astrocytes which evidently express and release ChAT at demand ⁽¹⁴· ⁴¹⁾. A few PET-tracers targeted at AChRs or ChEs exist. However, in contrast to ChAT, expression of AChRs and/or ChEs defines the cholinoceptive cells, and thereby these tracers do not necessarily provide insight on the core physiological and/or the initial pathological events affecting cholinergic cells in the brain, but rather the later downstream events.

Altogether, ChAT is a suitable target for developing a PET tracer as an in vivo biomarker of the health of the cholinergic system. Methods

In silica analyses

Docking was performed using the crystal structure of ChAT (PDB ID: 2FY3) ⁽⁶⁰⁾ to get insight into the binding mode of identified drug substances in the active site of ChAT and performed with the Surflex-Dock module interfaced in SYBYL-X 2.1.1 which is a fully automatic flexible molecular docking algorithm with a combination of empirical scoring function and a surface based molecular similarity based search engine ^{<61 )}. Prior to docking, the 3D structure of receptor was prepared which included addition of hydrogens, repairing side chain, treating termini, fixing atom type, setting protonation state, fixing bond order, adding charge, and fixing side chain amide. Finally the prepared structure is minimized in order to remove the strain produced during the earlier protein preparation steps. The 'protomol' which is the defined binding pocket of receptor was generated using the co-crystallized ligand in the active site of ChAT. The chemical structure of different substances were sketched and converted into 3D conformation covering both R and S stereoisomers of the drug substances. Finally, the prepared dataset of compounds were docked into the active site of ChAT using Surflex-Dock GeomX (SFXC) module and the compounds were ranked using Total_Score (-!ogKd). Enzyme-inhibition analyses

Purification of recombinant human ChAT

Recombinant human ChAT was produced in E Coli according to standard procedures using pProEXHTa-ChAT plasmid ⁽⁶²⁾. Briefly, DYT media (16 g/l Tryptone, 10 g/l yeast extract, 5 g/l NaCI, 100 pg/ml ampicillin, 34 g/ml chloramphenicol) was inoculated with a preculture of E. Coli BL21 Rosetta2 transformed with pProEXHTa-ChAT (a generous gift from Brian Shilton ⁽⁶²⁾). The bacteria were grown at 37°C and 200 rpm until the optical density at 600 nm reached 0.5. After which 0.5 mM IPTG was added and Hise-ChAT was expressed for circa 16 h at 18 °C. The bacteria was harvested and stored at -80°C. His6- ChAT was purified with "Ni-NTA fast start Kit" (Qiagen) following the manufacturer's instructions. The elution buffer was exchanged to storage buffer (10 mMTris pH 7.4, 500 mMNaCI, 10% (v/v) glycerol) using Amicon Ultra concentrators (Merck Millipore) with a molecular cutoff of 30 kDa. The protein preparation was aliquoted, frozen at dry ice and stored at -80°C. The absence of contaminating proteins was determined using sodium dodecyl sulfate PAGE and Coomassie staining. The total protein concentration was measured with BioRad DC protein Assay (BioRad). Experimental enzymatic activity assays

Cholinesterase assays

A modified version of Ellman^'s colorimetric assay was used for the enzymatic activity of BuChE and AChE, as described previously ^{(63, 64)}. Briefly, a pooled human plasma sample was prepared, aliquoted in small Eppendorf tubes (SOMlJtube) and kept frozen at -80C until assay. For BuChE activity, an aliquot was diluted 1 :400 in Na/K-phosphate buffer (50mM, pH 7.4). 50plJwell of the 1 :400 diluted solution of the pooled human plasma sample was applied to the wells of a 96-well plate. For AChE activity, the 1 :400 diluted pooled human plasma samples had been supplied with 50ng/ml of purified AChE protein (Sigma). To the blank wells (negative controls) only 50μΙ_/ννβΙΙ of the buffer was added. These were preincubated with δΟμΙ-Λ/νβΙΙε of different concentrations of the hits for 30 minutes at room temperature. In the positive control wells (without hits but containing plasma as the enzyme source) just 50 l_/well of buffer was added. Then, 100μ1_ of a cocktail (Na/K phosphate buffer, containing 5,5' -dithiobis(2-nitrobenzoic acid) (DTNB, final concentration 0.4 mM) and butyrylthiocholine iodide (Sigma, final concentration 5 mM) or acetylthiocholine iodide (Sigma, final concentration 0.5 mM) were added and the changes in absorbance was monitored at one minutes intervals at 412 nm wavelength. For the AChE activity, the cocktail also contained the selective BuChE inhibitor ethopropazine (Sigma, final concentration 0.1 mM).

Choline-acetyltransferase assay

ChAT activity was measured depending on the objective at hand with a new colorimetric assay as described previously⁰⁴⁾, or with a new fluorometric assay using recombinant ChAT protein. Briefly, for the colorimetric ChAT assay 20 LJwells of 1.5 Mg/ml of the recombinant ChAT was incubated with 20μΙ_Λ/νβΙΙ of different concentrations of the ligands for 30 minutes at room temperature in dilution buffer (10 mM Tris-HCI, pH 7.4, 150 mM NaCI, 1.0 mM EDTA, 0.05 % (v/v) Triton X-100). Then 60 μΙ of a cocktail-A [dilution buffer containing choline chloride (Sigma, final concentration 250 μΜ), eserine (E8625, Sigma- Aldrich, final concentration 60 μΜ), acetyl coenzyme-A (A2181 , Sigma-Aldrich, final concentration 50 μΜ), phosphotransacetylase (P2783, Sigma-Aldrich, final concentration 1.02 U/ml), lithium potassium acetyl-phosphate (#01409, Sigma-Aldrich, final concentration 12 mM)] was added to the samples. The final concentration of ChAT in the wells was O^g/mL. In separate wells, 100 μΙ of a serial two-fold dilution of choline chloride (500-ΟμΜ) was applied in triplicates, which were used as standards for determining choline concentration in the wells after reaction with ChAT. The plate was incubated for 20 minutes at 37 °C. Then 50 μΙ of a cocktail-B [phosphate buffered saline, containing 0.93 U/ml choline oxidase (C5896, Sigma-Aldrich), 1/5000 U streptavidin-horseradish peroxidase, 6.3 mM phenol, and 3 mM 4-aminoantipyrine (A4382, Sigma-Aldrich)] was added to each sample including the standards. Absorbance was then monitored using a microplate s pectrop h oto m ete r reader (Infinite M1000, Tecan) at 500 nm wavelength. ChAT activity (nmol/min/mg of recombinant protein) was calculated according to the following formula: ChAT activity [ChBL-Chs] I t l m, where CIIBL is the measured number of mole of choline in control wells lacking the inhibitor and ChAT, Chs is the measured number of mole of in the samples, the t is the incubation time and m is the mass of ChAT protein added per sample. Inhibition was given as compared to a ChAT sample incubated with only buffer.

The fluorometric ChAT assay was employed when real-time kinetic of the enzyme was assessed. Human recombinant ChAT was produced, purified, aliquoted in small Eppendorf tubes (50 LJtube, containing 10% glycerol) and kept at - 80°C. For the assay, an aliquot were used to prepare enough enzyme solution at the concentration noted above. 50pLJwell of this enzyme solution was applied to the wells of a 96-well Nunc black plate. To the blank wells (negative controls) only 50pUwell of the buffer was added. Then 50 μΙ_ of a series of choline concentrations were added to their corresponding wells. These were preincubated with 50pL/wells of different concentrations of the ligands for 30 minutes at room temperature. Meanwhile, a cocktail solution was freshly prepared by mixing certain amount of a 10mM stock solution of acetyl-coenzyme A (in double distilled water), to get a final concentration of 10μΜ when applied to the wells. This cocktail also contained CPM [(7-Diethylamino-3-(4'-Maleimidylphenyl)-4-Methylcoumarin) from a DMSO stock solution] to get a final concentration of 10μΜ when applied to the wells. Then 50 μ-Jwell of this cocktail was added to all the wells. Immediately, the plate was placed in a Tecan Infinite M1000 spectrophotometer, and the change in fluorescent was kinetically monitored at 1 min intervals for about 30 minutes at the excitation and emission wavelengths of 390nm and 479nm, respectively. The excitation/emission bandwidths, the Flash frequency, the Gain, and the Z-Position height were 20nm, 400Hz, 30 and 20000μηη, respectively.

Results

In silico docking analyses

In silico molecular docking analysis on crystal structure of ChAT (PDB ID: 2FY3) ⁽⁶⁰⁾ revealed high scores for proton pump inhibitors. All of the drug substances were docked into the active site of ChAT, and the docking score (Total-Score), which represent -logKd are given in Table 3. Table 3. In silico molecular docking scores for the representative identified ligands of ChAT.

Compound Name Total_Score (-log Cd) Crash - Polar

SJIaprazole 7.55 -1.714 2.478

R_omeprazole 9.457 -4.158 3.373

R _pantoprazole 8.457 -1.253 3.297

S_Tena toprazole 8.782 -1.236 1.586

R_Lansoprazole 7.803 -0.766 0.547

Esomeprazole 9.374 -2.706 1.653

S_Rabeprazole 9.013 -3.44 1.912

R_Tenatoprazole 8.433 -2.257 1.262

RJIaprazole 8.774 -2.376 2.378

S_Pantoprazole 9.151 -2.409 2.248

R_rabeprazole 9.902 -2.659 3.855

The compounds with -logKd value larger than 6 are deemed to be the most active. In order to probe the mechanism of interaction of the ChAT ligands, all the compounds from the dataset were docked into the active site of the enzyme using Surflex-Dock GeomX (SFXC) module of SYBYL-X2.1.1 suite ⁽⁶⁵⁾. The 3D docked pose and 2D ligand interaction diagram of the compound are shown in Figures 2-4. Most of the compounds showed docking scores greater than eight, and an interaction with the important amino acid residue, HIS324 which is the catalytic amino acid responsible for transfer of acetyl group from acetyl-CoA to choline. In addition, the TYR₈₅, ASN95, SER540, VAL555, SER₅₃8, and GLY₅₆i residues seemed to form a pocket to accommodate the bulkier groups.

ICso and K, for the ChAT ligands, CH-24, CH-26 and CH-26S

IC50 analyses indicate that omeprazole (CH-26) with an IC50 of ~1 OOnM for rhChAT protein has -15 folds higher affinity than lansoprazole, which exhibit and IC50 of 1.5μΜ (Fig. 5a). Omeprazole is a racemate (a mixture of two enantiomers). The S-enantiomer, esomeprazole (CH-26S) exhibited the IC50 values of ~50nM (Fig. 5b), which is half of that for omeprazole, indicating that this enantiomer possesses the full activity of this compound. Further enzyme-kinetic analyses indicated that CH-26 behaves as reversible mixed- competitive ChAT ligands with a binding affinity (K) of 140nM with regards to choline concentration (Fig. 6b-c). This means that CH-26/26S by definition may bind to both the free and choline-bounded enzyme. This in turn means that the total binding potential (ChAT levels) in the brain is most likely fully accessible to CH-26 even at the expected physiological concentration of choline. Being a reversible ChAT ligand has another important implication as a PET-tracer, namely it suggests that radiolabeled-CH-26 is unlikely to be retained in the brain of subjects for a prolonged time-period compared to an irreversible ligand.

Similar analyses with varying concentrations of Acetyl-CoA did not indicate any competing interaction with CH-26 for the binding site of this cofactor on ChAT (data not shown). This is important with regard to the potential selectivity of CH-26 towards ChAT relative to numerous other enzymes that utilize Acetyl-CoA as a cofactor. In contrast, there are only two known enzymes that utilize choline as a substrate, namely choline-oxidase (ChOX) and choline-kinase. Since ChOX was one of the axillary enzymes that was used in the ChAT assay (as described in the Methods section), we found no inhibition of ChOX by the Leads. This means that the leads should have negligible (if any at all) affinity for ChOX, and thereby presence of this enzyme should not pose any problem for tracing ChAT in the brain.

Ki for the ChAT ligand, CH-F17

The chemical structure of rabeprazole (CH-F17) is shown in Fig. 7 A. Enzyme-ligand kinetic assessments were done by non-linear regression analyses at various concentrations of the ligand and choline (Fig. 7B and C). These analyses suggest that CH-F17 has a high affinity for ChAT as may be deduced by a K, of 17.5nM. A Lineweaver- Burk plot suggests (Fig. 7C) that this highly potent lead behaves like a reversible noncompetitive ChAT ligand, with equal affinity for both free and choline-bound enzyme. This property is expected to grant this lead maximum ChAT binding potential that could be available in the brain.

Ki for the ChAT ligand, CH-F18

The chemical structure of tenatoprazole (CH-F18) is shown in Fig. 8A. Non-linear enzyme- ligand kinetic analyses were done at various concentrations of the ligand and choline (Fig. 8B and C). These analyses suggest that CH-F18 has a high affinity for ChAT as may be deduced by a K_t of 12.8nM. A Lineweaver-Burk plot suggests (Fig. 8C) that this highly potent lead like CH-26 behaves as reversible mixed-competitive ligand of ChAT, with high affinity for both free and choline-bound enzyme. This property is therefore expected to grant this lead the maximum ChAT binding potential that could be available in the brain Selectivity of the Leads

Relative affinity of PPIs for ChAT vs ChEs

There are two known enzymes that breakdown ACh, namely AChE and BuChE. These two enzymes are highly abundant as soluble or membrane-anchored in blood circulation, at the neuromuscular junctions, and in the CNS. Thereby it is important that the ChAT- ligands possess low affinity for these two enzymes.

Enzyme-inhibition analyses indicated that all the Leads affect negligibly the activities of human AChE and BuChE at 100-500 μΜ concentration (Fig. 9). Considering that the IC50 of these Leads for ChAT is between 80-17nM, the Leads exhibit at least 12500 to 30000 folds selectivity for ChAT compared to AChE or BuChE. As a comparison, a-NETA (the only known ChAT inhibitor that commercially is available) shows an IC50 of 88nM for ChAT, 34 μΜ for AChE and 34μΜ for BuChE, indicating merely 386 folds selectivity toward ChAT. Relative affinity of PPIs for ChA T vs A TPases

The leads had been developed as selective inhibitors of the proton pump, H⁺-K⁺-ATPase, which are mainly present in the parietal cells that produce the acidic environment in the stomach. Although immunohistological analyses indicate that this enzyme is unique to gastric mucosa ⁽⁶⁶⁾, there are several other ATPases that are present in the CNS, namely Na⁺-K⁺-ATPase and bicarbonate-activated ATPase (HCCvr -ATPase). An study with omeprazole suggests that this drug at concentrations of 2 m , 200 and 2 μΜ reduces the Na⁺-K⁺-ATPase activity by about 50%, 10% and 0%, respectively^. A comparison between IC50 of omeprazole for ChAT (~100nM) and for Na⁺-K⁺-ATPase (~2mM) suggests that omeprazole has about 20 thousand folds higher affinity for ChAT than for Na⁺-K⁺-ATPases. Similarly, omeprazole did not affect the HCO3- -ATPase at a concentration of 200μΜ ⁽⁶⁷⁾, suggesting an at least similar fold level of selectivity for ChAT versus HCC -ATPase.

Relative affinity of PPIs for ChAT vs OCTs

Proton pump inhibitors may also act as inhibitors of organic cation transporters (OCTs) ⁽⁶⁸⁾. These transporters show some affinity for choline and/or Ach ⁽⁶⁹⁾. High affinity cation transporter for choline (HChT) is present in the CNS. Although these sodium- and chloride- dependent transporter proteins are the main organic cation transporters involved in the choline uptake/re uptake by the cholinergic neurons, other OCTs with low to intermediate affinities for choline may also be present in the CNS ⁽⁶⁷· ^70>. An in vitro examination of changes in choline uptake in choroid plexus tissues indicated that omeprazole at 20μΜ concentration did not reduced choline uptake. At 2 mM concentration, omeprazole however reduced choline uptake in plexus tissues by -70%. Nonetheless, these findings suggest that omeprazole has (similar to its relative affinity for Na⁺-K⁺-ATPases) about 20 thousand folds less affinity for these OCTs than for ChAT. In conclusion, comparisons of the relative affinities of PPIs indicate that the main high affinity target of PPIs in the CNS is ChAT.

Evidence for the potential of leads and ChAT as a functional biomarker

In light of our findings of high affinities of the so called PPIs for ChAT and the nature of experimental procedures and the high μΜ to mM omeprazole concentrations used in Bader et al study ^{70 it became evident that a direct inhibition of ChAT rather than ACh-transport by OCTs is most likely the underlying cause for the observed reduction in the propionate- induced ACh release. Indeed, in the same study bromoacetylcholine (a known potent inhibitor of ChAT) was used at 50μΜ concentration, which like omeprazole strongly reduced propionate-induced ACh release ⁽⁷⁰⁾. The second line of evidence comes from a recent epidemiological report conducted on a large German population of -74,000 subjects showing that prolonged usage of PPI significantly increased the risk of incident dementia (Fig. 10)^{(71 )}. The estimated hazard ratio (HR) was 1.58 for pantoprazole, 1.51 for omeprazole, and 2.12 for the S-enantiomer of omeprazole, esomeprazole ⁽⁷¹⁾. Once more, in the light of our finding that these compounds, in particular esomeprazole, are highly potent ChAT inhibitors it is highly plausible that the underlying mechanism for the observed increased risk of incident dementia is mediated through inhibition of ChAT and thereby dysfunctional cholinergic system. Indeed, this is consistent with the well-established observations that drugs with known anti-cholinergic activity increase the risk of developing dementia ^{(29_31 )}.

Consequently, these compelling old and new observations impose the view that cholinergic deficit is the cause rather than consequence of the AD-type pathological events. Thereby, a decline (be it age-related and/or pathologically related) in the central cholinergic signaling may at least be a vulnerability determinant for the neurodegenerative dementia disorders. Thus, a ChAT-PET tracer should potentially be able to map changes in the cholinergic system. Other more direct evidence for ChAT as a potentially functional biomarker comes from several pilot studies in patients. In one pilot investigation, we measured ChAT levels in CSF of AD patients participating in a unique clinical trial of nerve growth factor (NGF)- releasing cells' implants ⁽⁷²⁾. The results indicated that patients who showed >25% increases in CSF ChAT after 12 months of the NGF treatment, were the responders. The changes in ChAT also correlated with changes in cognition (Fig. 11 I) and several in vivo measures by PET and MRI ^{(51 )}. Most noteworthy, although the number of patients were admittedly, too small, the assessment of CSF ChAT activity provided a coherent pattern suggestive of the treatment effect ^{(5 )}.

In another pilot study, we found strong correlation between plasma ChAT level and the patients' cognitive test's result after 12-mouths of cholinergic-enhancing therapy by ChEls (Fig. 11 II). In the third pilot study, we found highly differential levels of plasma ChAT level and clinical diagnosis of the patients (Fig. 11 III).

The fourth pilot study illustrates further clinical potential of ChAT in another neurodegenerative inflammatory disease, namely multiple sclerosis (Fig. 11 IV), supporting the well-established role of cholinergic signaling in inflammatory processes*¹⁴*. Evidence for brain permeability

PPIs have been developed to inhibit H+/Na+ ATPase, which appears to be restricted to the parietal cells in the stomach. Thereby few studies are available for these drugs on the extent of brain permeability. Figure 10 is taken from Gomm et al., which observed an association between patients treated with PPIs and the risk of dementia. In our view, the study results provide a strong indication that PPIs do pass the blood/brain barrier, and have an influence on brain activity - although that is not recognized by the authors of that study. Figure 13 relates to a study in rats, in which the brain/blood concentration ratio indicates 15% brain permeability for omeprazole ⁽⁷³⁾. In human it seems to be about the same ratio, -10% ⁽⁶⁷⁾. The peak plasma concentrations of omeprazole in human are between 0.7-4.6 μΜ, following a single 20 mg oral dosage ⁽⁷⁴⁾. Thus, the concentration range of omeprazole in the brain is expected to be around 0.07-0.46 μΜ in human. This is also in line with the aforementioned report of increased risk of incident dementia associated with usage of omeprazole, pantoprazole and in particular esomeprazole ^{(71 )}. Example 2 - Changes in three hallmarks of Alzheimer's disease

A recent post-mortem study reveals (Fig. 12 A) ⁽²²⁾, in agreement with numerous previous reports ^{(75, 76} that cholinergic degeneration occurs in line with the clinical and/or pathological stages of the disease. A comparison between Fig.12A and 12B reveals that while changes in ChAT show robust disease-stage dependent alterations, the tau deposit displays a sudden increase that remains unchanged regardless of the progression of the disease. This resembles the well-established lack of association between the degrees of deposition of Αβ peptides (Fig. 12C), and/or the absence of longitudinal changes in the Αβ deposits in the brain with the clinical manifestation of disease in untreated and treated patients.

It is worth noting that a recent report shows that cholinergic failure triggers tauopathy in both animal models and human brains (Kolisnyk et a/., 2016) ⁽⁵⁷⁾, providing strong evidence that cholinergic dysfunction might be the underlying cause for hyperphosphorylation of tau and thereby the formation neurofibrillary tangles of tau in the AD brain.

Finally, in agreement with this paper, and in confirmation of the idea that cholinergic dysfunction is one of the earliest signs of AD development, another recent paper in a large cohort of older adults showed that the volume of basal forebrain (where cholinergic circuitry projects to the cerebral cortex and hippocampus) could predict degeneration of entorhinal cortex (the first site where tauopathy is putatively considered to be initiated), and hence which was until now thought to be the first area to be affected in the disease. In other words, this latter study provides the first in vivo evidence that basal forebrain pathology precedes and predicts both entorhinal pathology and memory impairment, thus challenging the hypothesis that AD has a cortical origin (Schmitz et al. (2016) Nature Commun., 7: 13249).

Thus these observations suggest that cholinergic neuronal degeneration is expected to precede the manifestation of intracellular deposits of tau, i.e. NFT.

Furthermore, evolution of tau deposits indicate that tau must first form intracellular aggregates to later on build up the NFT to finally manifest themselves as "ghost tangles" as marker of neuronal death in the brain ^{7T>. In contrast, any alteration in ChAT levels is expected to immediately and directly cause dysfunctional cholinergic neuronal activity as this enzyme is responsible for the synthesis of acetylcholine. Thus, a ChAT tracer is expected to detect changes in ChAT levels and thereby altered cholinergic function in the brain prior to manifestation of enough number of NFT and/or "ghost tangles" to allow detectable signal intensities in the brain.

Example 3 - Presence of ChAT in Rheumatoid Arthritis

Preliminary data demonstrates that ChAT protein is present and readily measurable in the synovial fluid and in the plasma of patients with Rheumatoid Arthritis (Figure 14). Experiments were performed on paired synovial fluid and plasma samples taken from two RA patients, using the methods described in the preceding Examples. Our data show that ChAT protein is present in the synovial fluid and in the plasma of patients with RA. Based on the data in the preceding examples (in which CSF and plasma samples from MS and AD patients were compared with non-diseased controls), the indication is that ChAT levels are increased in the synovial fluid of RA patients.

Example 4 - Preparation of labelled compounds

As described herein, compounds having a detectable label (such as compounds labelled with a radio-isotope) may be prepared using techniques known to those skilled in the art.

For example, C ¹ labelled Omeprazole (also referred to herein as CH-26) may be prepared in accordance with the following reaction (wherein the * label denotes the position of the

C ¹).

5-0-desmethyl-CH-26 ( *C)-CH-26

Such reactions as described above may be performed using techniques and materials as known to those skilled in the art, and as described in references as mentioned herein, such as "Comprehensive Organic Synthesis" by B. M. Trost and I. Fleming, Pergamon Press, 1991. Starting materials may be commercially available or prepared from commercially available materials in accordance with techniques known to those skilled in art. Example 5 - Radio-labelling of exemplary compounds Radiosynthesis and Purification:

The radioactive starting material, [¹¹C]CH₄, was produced in a cyclotron by irradiating the target chamber containing H₂ (10%) in N₂ with a 16.4 MeV proton beam at 35 bar using the ¹⁴N(p, a) ¹C nuclear reaction. [¹¹C]CH₄ was released into a recirculation system and converted to [¹¹C]CH₃I through a free radical reaction. [¹¹C]Methyliodide ([¹ C]CH₃I) was transferred into a solution of precursor (desmethyl omeprazole, 0.8 -1.2 mg) in DMF (500 μΙ_) and NaH (2-4 mg) by a helium stream (shown in schematic below). The reaction mixture was kept at 70 °C for 4 minutes and diluted with water (3 mL) before injecting in to the HPLC.

The crude reaction mixture was subjected to purification using reversed phase semi- preparative HPLC equipped with C-18 ACE semi preparative HPLC column (10 pm, 250x7.8 mm). Aceton itri le/Am mon i umf ormate (0,1 M): 30/70 was use as mobile phase. The effluent was monitored for radioactivity and UV absorbance at 254 nm. The [¹¹C]- omeprazole fraction was collected in sterile water (50 mL) and the solvents in the mobile phase are removed by SPE (tC-18 plus). Subsequently [¹¹C]-omeprazole was eluted by EtOH (1.0 mL) and formulated in sterile water (9 mL).

Precursor

Desmethyl omeprazole [¹¹C]omeprazole

Schematic illustration of carbon 11 [¹ C]-labelling of the lead compound, Omeprazole (also termed "CH-26" herein). Quality control

The radiochemical purity, identity and stability of [¹¹C]-omeprazole was determined by analytical HPLC system which included a C-18 ACE analytical HPLC column (C18, 3.9 0 x 250 mm, 10 pm particle size), Merck-Hitachi L-7100 Pump, L-7400 UV detector and GM- tube for radioactivity detection (VWR International). The mobile phase CH3CN/AMF (0,1 M) with gradient 20%-80% in 8 min and flow rate of 2 mL/min was used to elute the product. The effluent was monitored with an UV absorbance detector (λ = 214 nm) coupled to a radioactive detector (b-flow, Beckman, Fullerton, CA). The retention time (tf¾) of [¹¹C]- omeprazole was 4,5-5,5 min. The identity of [¹¹C]-omeprazole was confirmed by co- injection with the authentic non-radioactive omeprazole.

Molar activity (MA) determination:

The MA of the final product was measured by analytical HPLC using mobile phase mobile phase CH₃CN/AMF (0,1 M) (35/65) at flow rate of 2 mL/min. SA was calibrated for UV absorbance (Λ = 254 nm) response per mass of ligand and calculated as the radioactivity of the radioligand (GBq) divided by the amount of the associated carrier substance (pmol). Each sample was analysed three times and compared to a reference standard also analysed three times.

Example 6 - PET study in non-human primate

Subject: One female cynomolgus monkey (body weight 6150g) was included in this study (see Table 4 below). That non-human primate (NHP) was housed in the Astrid Fagraeus Laboratory (AFL) of the Swedish Institute for Infectious Disease Control, Solna, Sweden. The study was approved by the Animal Ethics Committee of the Swedish Animal Welfare Agency and was performed according to "Guidelines for planning, conducting and documenting experimental research" (Dnr 4820/06-600) of Karolinska Institutet.

PET measurements:

NHP was scanned twice; one baseline scan and one pretreatment scan on the same day. NHP was administrated intravenously 20 minutes before PET scanning with bolus infusion (1.0 mg/kg for 20 minutes). Anaesthesia was induced by intramuscular injection of ketamine hydrochloride (approximately 10 mg/kg) at AFL, and maintained by administration of a mixture of sevoflurane, oxygen and medical air with endotracheal intubation at Karolinska Institutet PET centre. The head was immobilized with a fixation device. Body temperature was maintained by a Bair Hugger model 505 (Arizant Healthcare, MN, USA) and monitored by an esophageal thermometer. ECG, heart rate, blood pressure, respiratory rate and oxygen saturation were continuously monitored throughout the experiments. Fluid balance was maintained by a continuous infusion of saline. PET measurements were conducted using the High Resolution Research Tomograph (HRRT) (Siemens Molecular Imaging, TN, USA). A transmission scan of 6 min using a single ¹³⁷Cs source was performed before the emission scan. List mode data were acquired continuously for 123 min immediately after intravenous injection of the [¹¹C]- compound. The injected radioactivities were 161 and 167 MBq at baseline and pretreatment scans, respectively. Images were reconstructed with a series of 34 frames (20 sec *9, 1 min ^χ3, 3 min *5, and 6 min *17).

Data analysis:

The regions of interest (ROIs) were delineated manually on the MRI images of NHP for caudate, putamen, thalamus, cerebellum, occipital cortex, frontal cortex, temporal cortex, parietal cortex, amyglada, and hippocampus. The summed PET images of whole scanning were co-registered to the MRI image of the individual NHP. After applying the co-registration parameters to the dynamic PET data, the time-activity curves (TAG) of brain regions were generated for each PET measurement. The analyses of imaging data were performed using PMOD (version 3.6; PMOD Technologies, Zurich, Switzerland).

Table 4. Summary of the NHP-PET measurement.

Body Injected Specific Injected

Cynomolgus

Sex PET date weight radioactivity radioactivity mass Condition

NHP

(9) (MBq) (GBq/ mol) 9)

2017.09.06 161 273.3 0.20 Baseline

Pretreatment

0609088 F 6150 with 1.0

2017.09.06 167 73.4 0.79

mg/kg of omeprazole Result:

Labelling of CH-26: TOQmeprazole: The product yield for [¹¹C]Omeprazole was -2000 MBq from 20 min with 35 μΑ beam current. Radiochemical purity was >98%. [¹¹C]Omeprazole was found to be stable in the formulated solution up to 90 min after the end of synthesis. The total synthesis time including purification was 40 min. Molar activity was 173 ±141 GBq/ mol. It should be noted that the labelling protocol described above and illustrated as the above schematic, can be readily used to in principle label the compounds with any isotopes [such as tritium (³H), ⁸F, etc.] by replacing [¹¹C]methyliodide with for instance [³H]methyliodide or [¹⁸F]fluoromethyl iodide (92). It also should be noted that this same protocol can be used label CH-26S, using the S- form of desmethyl -omeprazole precursor. This in turn can be prepared by passing the race mate of the CH-26 precursor through a chiral HPLC column (commercially available e.g. from Chiral Technologies, Exton, PA, USA) to separate the (S)-enantiomer from the racemic mixture using conditions reported in the literature for unlabelled material (93). Nonetheless, the protocol was used to prepare [ C]Omeprazole with specific radioactivity required for the NHP-PET assessment, as shown in Table 4, above.

Preliminary NHP-PET assessment:

Upon the successful ¹¹C-labeling of the lead, CH-26, we run the NHP-PET assessment of ¹¹C-CH-26 first at baseline condition, and then following pretreatment with the cold-CH-26. The preliminary results are summarized in Figure 15. The images are calculated in Standard Uptake Values (SUV), according to conventional procedures (88).

The data indicated that: - CH-26 passess Blood-Brain Barrier ("BBB") by exhibiting a brain-permeability of approximately 1.5 SUV at peak;

- CH26 exhibit regional brain binding and distribution pattern that might be expected of a ChAT-PET tracer; - A comparison between the PET1 and PET2 images in Figure. 15, it is appreciated that there was a decrease in the uptake of ¹¹C-CH-26 in PET2, which reflect the blocking effect of the cold-CH-26. This will be confirmed with pretreatment with higher doses of cold-CH-26.

- The uptake observed outside the brain is also expected of a ChAT-PET tracer (Figure 18). It is well-estabilished that all cranial nerves, which consitutes the parasympathetic fibers, are cholinergic (Figure 19). Thus the uptake ouside the CNS might reflect the binding of ChAT to the end-terminal projections of the cholinergic cranial nerves (CN 0, and CN III, VII and/or IX) (94, 95). This will be corroborated in vitro by a combination of immunohistochemistry and auto-radiography. - A preliminary quantitative image analysis by comparison between the ¹¹C-CH-

26 uptake in the PET1 and PET2 images is shown in Figure 20, providing good preliminary evidence for the potential and feasibitly of the Lead ¹¹C-CH-26 as a ChAT-PET tracer. - Noteworhty, the specific ¹¹C-CH-26 uptake and displacement by the cold- compound (as depicted in Figure. 20) was also observed outside the brain. However, this is as noted before expected due to the known cholinergic innervations of facial, tung and oculomotor muscles, as well as the olfactory epthelium in nasal cavities by projections originated from cholinergic motorneurons and/or cranial nerves 0, I (olfactory nerve), III, VII and/or IX, etc

(see also Figure 19) (94, 95). This can be investigated further and confirmed by, for instance, auto-radiographic analysis.

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Claims

Claims

1. A method a method for determining the cholinergic function of one or more cell, comprising the steps of: contacting one or more cell with an agent capable of selectively binding to choline acetyltransferase, and using the agent to measure the amount and/or concentration of choline acetyltransferase in the one or more cell; and

determining the cholinergic function of the one or more cell based on the amount and/or concentration of choline acetyltransferase; wherein the agent is a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein:

Q represents C or N;

each X independently represents -OR^1a, a 5- to 6-membered heteroaryl or a Ci-6 alkyl, wherein the latter two groups are optionally substituted with one or more R^2a;

Y represents H or a C1-6 alkyl optionally substituted with one or more R^2b; each Z independently represents -OR^1b or a C1-6 alkyl, wherein the latter group is optionally substituted with one or more R2_C;

each of R^1a and R^1b independently represents C1-3 alkyl optionally substituted with one or more fluoro or -OR^3a;

each of R^2a to R^2c independently represents fluoro or -OR^3b; each R^3a and R^3b independently represents C1-3 alkyl optionally substituted with one or more fluoro;

n represents 0 to 4; and

m represents 0 to 4, Use of an agent, as defined in Claim 1 , in determining the cholinergic function of one or more cell, wherein the cholinergic function is determined based on the amount and/or concentration of choline acetyltransferase in the one or more cell.

An agent, as defined in Claim 1 , for use in determining the cholinergic function of one or more cell, wherein the cholinergic function is determined based on the amount and/or concentration of choline acetyltransferase in the one or more cell.

Use of an agent, as defined in Claim 1 , for the manufacture of a medicament for determining the cholinergic function of one or more cell, wherein the cholinergic function is determined based on the amount and/or concentration of choline acetyltransferase in the one or more cell.

The method, use or compound for use according to any one of Claims 1 to 4 wherein the one or more cell is provided in a sample obtained from an individual.

The method, use or compound for use according to any one of Claims 1 to 4 wherein the one or more cell is present in an individual, and the step of contacting the agent with the one or more cell comprises administering the agent to the individual.

The method, use or compound for use according to any preceding claim wherein the one or more cell comprises one or more neuronal cell; or one or more non- neuronal cell.

The method, use or compound for use according to Claim 7 wherein the one or more non-neuronal cell is selected from the group consisting of: lymphocytes; astrocytes; embryonic stem cells.

The method, use or compound for use according to any of Claims 1-7 wherein the one or more neuronal cell is selected from the group consisting of: motor neuron; sensory neuron; interneuron.

The method, use or compound for use according to any of Claims 1-7 or 9 wherein the one or more neuronal cell is part of an organ or tissue of the central nervous system.

11. The method, use or compound for use according to Claim 10 wherein the organ or tissue is selected from the group consisting of: brain; spinal cord; retina; optic nerve; olfactory nerve; olfactory epithelium.

12. The method, use or compound for use according to any of Claims 1-7 or 9 wherein the one or more neuronal cell is part of an organ or tissue of the peripheral nervous system, such as: part of the somatic nervous system; part of the autonomic nervous system; part of the parasympathetic nervous system; part of the sympathetic nervous system; part of the enteric nervous system.

13. The method, use or compound for use according to Claim 12 wherein the organ or tissue is selected from the group consisting of: eye iris; heart; ciliary muscle; upper gastrointestinal tract; lower gastrointestinal tract; colon (ascending and descending); urinary bladder; salivary gland; synovial tissues*⁷⁸'; placenta; prostate gland, testes; uterus; tendons⁽⁷⁹' ⁸⁰⁾; skeletal muscle; skin/keratinocytes'⁸'- ⁸²⁾; lungs/airways⁽⁸³⁾; stem cells⁽⁸⁴' ⁸⁵⁾; glioblastoma cancer cells^{(8R 87)}; immune cells.

14. The method, use or compound for use according to any preceding claim wherein the choline acetyltransferase is selected from the group consisting of: membrane- bound choline acetyltransferase; soluble choline acetyltransferase; monomeric choline acetyltransferase; dimeric choline acetyltransferase; tetrameric choline acetyltransferase; multimeric choline acetyltransferase.

15. The method, use or compound for use according to any preceding claim wherein the agent further comprises a detectable moiety.

16. The method, use or compound for use according to Claim 15 wherein the detectable moiety is selected from the group consisting of: a fluorescent label; a chemiluminescent label; a paramagnetic label; a radio-isotopic label; or an enzyme label.

17. The method, use or compound for use according to Claim 16 wherein the radioisotopic label comprises a radio-isotope selected from the group consisting of: ³H

11Q. 14_C; 18_F; 99m_Tc; 111 ,_{n ;} 67_{Qa ;} eSQg. 72_As;89_Zr; 123] . 201J_L

18. The method, use or compound for use according to Claim 16 wherein the paramagnetic isotope is selected from the group consisting of ^{1 7}Gd, ⁵⁵Mn, ¹⁶²Dy, 5²Cr and ⁵⁶Fe.

19. The method, use or compound for use according to any of Claims 15-18 wherein the detectable moiety is detectable by an imaging technique, such as: CT; SPECT; PET; MRI; optical imaging; ultrasound imaging.

20. The method, use or compound for use according to any preceding claim wherein the amount and/or concentration of choline acetyltransferase is: (i) for CSF and cell medium, from 1 to 1000 ng/ml; (ii) for plasma and serum, from 1 to 1000 Mg/mL; (ii) for cell and tissue homogenates, from 0 to 1000 ng/mg total protein.

21 . The method, use or compound for use according to any preceding claim wherein the compound is selected from the group consisting of: Esomeprazole; Omeprazole; Lansoprazole; Dexlansoprazole; Pantoprazole; Rabeprazole; Tenatoprazole; llaprazol.

22. A method for determining the presence of a disorder in a patient, comprising the steps of:

- administering to the patient an agent capable of selectively binding to choline acetyltransferase, and using the agent to measure the amount and/or concentration of choline acetyltransferase in one or more cell in the patient;

- determining the cholinergic function based on the amount and/or concentration of choline acetyltransferase; and

determining the presence of a disorder in the patient on the basis of the cholinergic function of the one or more cell in the patient;

wherein the agent is a compound of Formula I, as defined in Claim 1 .

23. Use of an agent, as defined in Claim 1 , in determining the presence of a disorder in a patient, the use comprising determining the cholinergic function of one or more cell in the patient based on the amount and/or concentration of choline acetyltransferase in the one or more cell.

24. An agent, as defined in Claim 1 , for use in determining the presence of a disorder in a patient, the use comprising determining the cholinergic function of one or more cell in the patient based on the amount and/or concentration of choline acetyltransferase in the one or more cell.

25. Use of an agent, as defined in Claim 1 , for the manufacture of a medicament for determining the presence of a disorder in a patient, the use comprising determining the cholinergic function of one or more cell in the patient based on the amount and/or concentration of choline acetyltransferase in the one or more cell.

26. The method, use or compound for use according to any one of Claims 22 to 25 wherein the patient is determined as having the disorder when the cholinergic function of the one or more cell in the patient is modulated.

27. The method, use or compound for use of Claim 26 wherein the patient is determined as having the disorder when the cholinergic function of the one or more cell in the patient is reduced.

28. The method, use or compound for use of Claim 26 wherein the patient is determined as having the disorder when the cholinergic function of the one or more cell in the patient is increased.

29. The method, use or compound for use according to any one of Claims 22 to 28 wherein the cholinergic function of the one or more cell in the patient is determined by measuring the number of cells in the patient which have a specified amount and/or concentration of choline acetyltransferase.

30. The method, use or compound for use according to any one of Claims 22 to 29 wherein the one or more cell is provided in a sample obtained from the patient.

31. The method, use or compound for use according to any one of Claims 22 to 30 wherein the disorder is a neurodegenerative disorder.

32. The method, use or compound for use according to Claim 31 wherein the neurodegenerative disorder is selected from the list consisting of: Alzheimer's disease; Lewy's bodies disorder's dementia (such as fronto-temporal dementia and dementia with Lewy bodies and Parkinson's disease dementia); fronto-temporal dementia; vascular dementia; traumatic brain injury; brain cancers; degenerative nerve diseases; encephalitis; epilepsy; genetic brain disorders; head and brain malformations; hydrocephalus; stroke; Parkinson's disease; multiple sclerosis (MS); amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease); HIV dementia; Huntington's disease; Sjogren's syndrome; prion diseases (such as Creutzfeld-Jacob disease (CJD)); Down's syndrome; myasthenia gravis.

33. The method, use or compound for use according to Claim 32 wherein the neurodegenerative disorder is Alzheimer's disease.

34. The method, use or compound for use according to Claim 32 wherein the neurodegenerative disorder is early-stage or prodromal Alzheimer's disease.

35. The method, use or compound for use according to any one of Claims 22 to 34 wherein the one or more cell is as defined in any one of Claims 7 or 9 to 11. 36. The method, use or compound for use according to any one of Claims 22 to 30 wherein the disorder is an inflammatory disorder.

37. The method, use or compound for use according to Claim 36 wherein the inflammatory disorder is selected from the list consisting of: rheumatoid arthritis (RA); multiple sclerosis (MS); tendonitis; atopic dermatitis; topic dermatitis; general inflammation in the brain; brain trauma; spinal injury.

38. The method, use or compound for use according to any one of Claims 36 or 37 wherein the one or more cell is as defined in any one of Claims 7 or 8.

39. The method, use or compound for use according to any one of Claims 22 to 30 wherein the disorder is cancer.

40. The method, use or compound for use according to Claim 39 wherein the cancer is prostate cancer; colon cancer; glioblastoma.

41. The method, use or compound for use according to any one of Claims 39 or 40 wherein the one or more cell is a cancer cell. 42. The method, use or compound for use according to any one of Claims 22 to 41 wherein the choline acetyltransferase is as defined in claim 14.

43. The method, use or compound for use according to any one of Claims 22 to 42 wherein the agent further comprises a detectable moiety.

44. The method, use or compound for use according to Claim 43 wherein the detectable moiety is as defined in any one of claims 16-19.

45. The method, use or compound for use according to any one of Claims 22 to 44 wherein the compound is as defined in claim 21.

46. The method, use or compound for use according to any one of Claims 22 to 45 comprising the additional step of selecting one or more appropriate treatment for the patient or individual.

47. The method, use or compound for use according to any one of Claims 22 to 46 comprising the additional step of selecting one or more treatments which is not appropriate for the patient or individual.

48. The method, use or compound for use according to Claim 47 comprising the additional step of selecting an alternative appropriate treatment for the patient or individual.

49. The method, use or compound for use according to Claim 46 or 48, further comprising the step of administering the selected treatment to the patient or individual.

50. The method, use or compound for use according to any one of Claims 46 to 49 wherein:

(i) the disorder is Alzheimer's Disease or mixed Alzheimer's Disease and the treatment comprises a cholinesterase inhibitor (ChEI), optionally in combination with an NMDA antagonist; and/or

(i) the disorder is a Lewy body disorder, including dementia with Lewy bodies (DLB) and Parkinson's disease with dementia (PDD) and the treatment comprises a cholinesterase inhibitor (ChEI), preferably rivastigmine or donepezil; and/or

(ii) the disorder is Down's syndrome dementia and the treatment comprises a cholinesterase inhibitor (ChEI); and/or (iii) the disorder is amyotrophic lateral sclerosis (ALS) and the treatment comprises Riluzole (Rilutek); and/or

(iv) the disorder is multiple sclerosis (MS) and the treatment comprises one or more selected from the list consisting of: interferon beta-1a (Avonex, Rebif); peginterferon beta- 1a (Plegridy); teriflunomide (Aubagio); natalizumab (Tysabri); fingo!imod (Gilenya); cytostatics; mitoxantrone (Novantrone); dimethyl fumarate (Tecfidera); and/or

(v) the disorder is rheumatoid arthritis (RA) and the treatment comprises one or more selected from the list consisting of: non-steroidal anti-inflammatory drugs (NSAIDs); methotrexate; TNF-a blocker/inhibitors, such as Abatacept (Orencia), Adalimumab (Humira), and/or Etanercept (Enbrel).

51. The method, use or compound for use according to Claim 50 wherein the ChEI is selected from the group consisting of: donepezil; galantamine; and rivastigmine.

52. A method for determining the risk of an individual developing a disorder, comprising the steps of:

- administering to the individual an agent capable of selectively binding to choline ace tyltransf erase, and using the agent to measure the amount and/or concentration of choline acetyltransferase in one or more cell in the individual;

- determining the cholinergic function based on the amount and/or concentration of choline acetyltransferase; and

- determining the risk of the individual developing the disorder on the basis of the cholinergic function of the one or more cell in the individual;

wherein the agent is a compound of Formula I, as defined in Claim 1.

53. Use of an agent, as defined in Claim 1 , in determining the risk of an individual developing a disorder, the use comprising determining the cholinergic function of one or more cell in the patient based on the amount and/or concentration of choline acetyltransferase in the one or more cell.

54. An agent, as defined in Claim 1 , for use in determining the risk of an individual developing a disorder, the use comprising determining the cholinergic function of one or more cell in the patient based on the amount and/or concentration of choline acetyltransferase in the one or more cell.

55. Use of an agent, as defined in Claim 1 , for the manufacture of a medicament for determining the risk of an individual developing a disorder, the use comprising determining the cholinergic function of one or more cell in the patient based on the amount and/or concentration of choline acetyltransferase in the one or more cell.

56. The method, use or compound for use according to any one of Claims 52 to 55 wherein the level of risk is correlated with the cholinergic function of the one or more cell.

57. The method, use or compound for use according to Claim 56 wherein the correlation is a negative correlation.

58. The method, use or compound for use according to Claim 56 wherein the correlation is a positive correlation.

59. The method, use or compound for use according to any one of Claims 52 to 58 wherein the cholinergic function of the one or more cell in the individual is determined by measuring the number of cells in the patient which have a specified amount and/or concentration of choline acetyltransferase.

60. A method for determining the progression of a disorder in a patient, comprising the steps of:

- administering to the patient an agent capable of selectively binding to choline acetyltransferase, and using the agent to measure the amount and/or concentration of choline acetyltransferase in one or more cell in the patient over time;

- determining the change in cholinergic function based on the amount and/or concentration of choline acetyltransferase over time; and

- determining the progression of the disorder in the patient on the basis of the change in cholinergic function of the patient over time;

wherein the agent is a compound of Formula I, as defined in Claim 1.

61. Use of an agent, as defined in Claim 1 , in determining the progression of a disorder in a patient, the use comprising determining the cholinergic function of one or more cell in the patient based on the amount and/or concentration of choline acetyltransferase in the one or more cell.

62. An agent, as defined in Claim 1 , for use in determining the progression of a disorder in a patient, the use comprising determining the cholinergic function of one or more cell in the patient based on the amount and/or concentration of choline acetyltransferase in the one or more cell.

63. Use of an agent, as defined in Claim 1 , for the manufacture of a medicament for determining the progression of a disorder in a patient, the use comprising determining the cholinergic function of one or more cell in the patient based on the amount and/or concentration of choline acetyltransferase in the one or more cell.

64. The method, use or compound for use according to any one of Claims 60 to 63 wherein the disorder in the patient is determined as having regressed if there is a modulation in cholinergic function in the patient over time.

65. The method, use or compound for use according to any one of Claims 60 to 63 wherein the disorder in the patient is determined as having progressed if there is a modulation in cholinergic function in the patient over time.

66. The method, use or compound for use according to Claim 64 or 65 wherein the modulation corresponds to a reduction in cholinergic function.

67. The method, use or compound for use according to Claim 64 or 65 wherein the modulation corresponds to an increase in cholinergic function.

68. The method, use or compound for use according to any one of Claims 60 to 63 wherein the disorder in the patient is determined as being stable if there is no change in cholinergic function in the patient over time.

69. The method, use or compound for use according to any one of Claims 60 to 68 wherein the change in cholinergic function is determined by measuring the number of cells in the patient which have a specified amount and/or concentration of choline acetyltransferase.

70. The method, use or compound for use according to any one of Claims 60 to 69 wherein the agent is administered to the patient at regular intervals over time and the amount and/or concentration of choline acetyltransferase in one or more cell is measured in the patient at the same regular intervals over time.

71. A method for determining the response to therapy of a disorder in a patient, comprising the steps of:

- providing a patient with a disorder that is undergoing therapy for the disorder;

- administering to the patient an agent capable of selectively binding to choline acetyltransferase, and using the agent to measure the amount and/or concentration of choline acetyltransferase in one or more cell from the patient over time;

- determining the change in cholinergic function of the one or more cell from the patient based on the amount and/or concentration of choline acetyltransferase over time;

wherein the agent is administered to the patient before and/or during and/or after therapy;

and wherein the response to therapy of the disorder in the patient is determined on the basis of the change in cholinergic function of the one or more cell from the patient over time during and/or after therapy;

and wherein the agent is a compound of Formula I, as defined in Claim 1.

72. Use of an agent, as defined in Claim 1 , in determining the response to therapy of a disorder in a patient, the use comprising determining the cholinergic function of one or more cell in the patient based on the amount and/or concentration of choline acetyltransferase in the one or more cell.

73. An agent, as defined in Claim 1 , for use in determining the response to therapy of a disorder in a patient, the use comprising determining the cholinergic function of one or more cell in the patient based on the amount and/or concentration of choline acetyltransferase in the one or more cell.

74. Use of an agent, as defined in Claim 1 , for the manufacture of a medicament for determining the response to therapy of a disorder in a patient, the use comprising determining the cholinergic function of one or more cell in the patient based on the amount and/or concentration of choline acetyltransferase in the one or more cell.

75. The method, use or compound for use according to any one of Claims 71 to 74 wherein the patient is determined as having responded to therapy if there is a modulation in cholinergic function in the patient over time.

76. The method, use or compound for use according to any one of Claims 71 to 74 wherein the patient is determined as having not responded to therapy if there is a modulation in cholinergic function in the patient over time.

77. The method, use or compound for use according to Claim 75 or 76 wherein the modulation corresponds to a reduction in cholinergic function.

78. The method, use or compound for use according to Claim 75 or 76 wherein the modulation corresponds to an increase in cholinergic function.

79. The method, use or compound for use according to any one of Claims 71 to 78 wherein the change in cholinergic function is determined by measuring the number of cells in the patient which have a specified amount and/or concentration of choline acetyltransferase.

80. The method, use or compound for use according to any one of Claims 52 to 79 wherein the one or more cell is provided in a sample obtained from the patient or individual.

81 . The method, use or compound for use according to any one of Claims 52 to 80 wherein the disorder and/or the one or more cell is as defined in any one of Claims 31 to 41.

82. The method, use or compound for use according to any one of Claims 52 to 81 wherein the choline acetyltransferase is as defined in Claim 15.

83. The method, use or compound for use according to any one of Claims 52 to 82 wherein the agent further comprises a detectable moiety.

84. The method, use or compound for use according to Claim 83 wherein the detectable moiety is as defined in any one of Claims 16-19. 85. The method, use or compound for use according to any one of Claims 52 to 84 wherein the compound is as defined in Claim 21.

86. The method, use or compound for use according to any one of Claims 52 to 85 comprising one or more additional steps as defined in any one of Claims 46 to 51.

87. A kit comprising an agent as defined in Claim 1 and a detectable moiety.

88. The kit according to claim 87 wherein the detectable moiety is as defined in any one of claims 16 to 19.

89. A method, use, agent for use or kit substantially as described herein with reference to the accompanying description, drawings, examples, and/or claims.