WO2012104589A1 - Weight related disorders - Google Patents

Abstract

The present invention concerns the finding that expression of the gene encoding the enzyme, thiosulfate sulphur transferase and the activity or function of the TsT enzyme itself, is elevated in the tissues of lean animals. Consequently, the invention provides compositions, methods and medicaments for treating or preventing weight related disorders as well as methods and assays for diagnosing or detecting a weight related disorder and/or a predisposition/susceptibility thereto.

Description

WEIGHT RELATED DISORDERS

FIELD OF THE INVENTION

The present invention provides compounds for the treatment or prevention of weight related disorders as well as methods for diagnosing or detecting incidences of weight related disorders and/or a susceptibility and/or predisposition thereto.

BACKGROUND OF THE INVENTION

Obesity, with its associated risk of diabetes and heart disease, is a world leading health concern. Nevertheless a significant proportion of the population resists excessive weight gain despite the obesogenic environment of modern society. Recent genome- wide association studies identified key genetic regions and genes that contribute to fat mass. High levels of expression of some of these genes occur in the adipose tissue where they may directly affect adipose distribution and function.

US Patent No 5,601,806 discloses the use of thiotaurine and its analogues as an antioxidant for skin lesions (as occurring in, for example, sunburn). In this patent the authors also describe the use of an alloxan diabetes model in order to determine the effects of thiotaurine administration on alloxan-induced diabetes.. The patent discloses that thiotaurine suppresses the increase in triglycerides and blood sugar in this model of diabetes. US Patent No 5,601,806 does not disclose the mode of action of thiotaurine or any link between the compound, thiotaurine, and the treatment or prevention of weight related disorders.

The present invention aims to provide new and effective treatments for weight related disorders.

SUMMARY OF THE INVENTION

The present invention is based on the finding that expression of the gene encoding the enzyme, thiosulfate sulphur transferase (TsT: the gene being referred to hereinafter as the TsT gene) and the activity or function of the TsT enzyme itself, is elevated in the tissues of lean animals. Consequently, the invention provides compositions, methods and medicaments for treating or preventing weight related disorders as well as methods and assays for diagnosing or detecting a weight related disorder and/or a predisposition/susceptibility thereto.

For simplicity, the remainder of this specification will make reference to TsT only - however, it should be understood that the term "TsT" encompasses (as appropriate) both the enzyme, thiosulfate sulphur transferase and the gene (TsT) encoding the same. As such, a first aspect of this invention provides compounds which modulate TsT activity, function and/or expression for use in treating or preventing weight related disorders.

In a second aspect, the invention provides use of compounds which modulate thiosulfate sulphur transferase TsT activity, function and/or expression for the manufacture of a medicament for treating or preventing weight related disorders.

In a third aspect, the invention relates to a method of treating a weight related disorder, said method comprising the steps of administering to a subject in need thereof, a therapeutically effective amount of a compound which modulates TsT activity, function and/or expression. It should be understood that "a subject in need thereof encompasses subjects suffering or convalescing from a weight related disorder as well as those predisposed or susceptible to, one or more of the weight related disorders described herein.

The phrase "compounds which modulate" should be understood as encompassing compounds which either increase or decrease the expression, function and/Or activity of TsT. In one embodiment, the compounds which modulate TsT activity function and/or expression, comprise compounds which specifically increase, augment or enhance the activity function and/or expression of the TsT enzyme or the gene (TsT) encoding the same.

One of skill will appreciate that increases and/or decreases in TsT activity, function and/or expression may be evaluated or determined by reference to, or comparison with, a control system exhibiting normal or wild-type TsT/TsT function, expression and/or activity.

The term "compounds" may encompass small organic/inorganic compounds, lipids, nucleic acids (RNA and/or DNA), carbohydrates, proteins (including peptides, polypeptides and/or amino acids), antibodies (or fragments (for example Fab or Fab₂ fragments) or antigen binding fragments, thereof) - including monoclonal and/or polyclonal antibodies. In one embodiment, the compounds may comprise agonists or antagonists of TsT activity, function and/or expression.

Where the invention relates to diseases and/or conditions/disorders which result from aberrant or increased TsT expression, the compounds provided by this invention may be designed to specifically inhibit, reduce or antagonise TsT expression, function and/or activity. For example, compounds provided by this invention may comprise antisense, silencing and/or interfering nucleic acids. The skilled person will be familiar with antisense nucleic acids (known as antisense oligonucleotides) which may comprise DNA or RNA and may comprise sequences complementary to mRNA sequences encoding the TsT enzyme or compounds associated with TsT function, activity and/or expression. The skilled person is also familiar with silencing and/or small interfering (si) RNA molecules which may be used to modulate the function, activity and/or expression of the fsf gene or sequences encoding compounds associated with TsT function, activity and/or expression. By analysing wild type TsT gene sequences and with the aid of algorithms such as BIOPREDi and/or siDesign center, one of skill could readily determine or computationally predict oligonucleotide sequences that have an optimal knock-down effect for these genes (see for example: http://www.dharmacon.com/DesignCenter/DesignCenterPage.aspx). Furthermore, the skilled person may generate and test an array or library of different oligonucleotides to determine whether or not they are capable of modulating the expression, function and/or activity of the TsT gene.

In other embodiments, compounds which antagonise TsT function, expression and/or activity may include antibodies which exhibit specificity, selectivity and/or affinity for an epitope of TsT - wherein binding to said epitope results in reduced TsT expression, function and/or activity - perhaps through blocking of the active site of the TsT enzyme.

In other embodiments, the invention relates to compounds which agonise, activate or increase/enhance TsT expression, function and/or activity. Compounds of this type may be referred to as "TsT activators" and may find particular application in the treatment of diseases and/or conditions/disorders which result from reduced TsT expression, function or activity. Without wishing to be bound by theory, the inventors suggest that TsT activator compounds may exert their effects through modulation of sulfur flux. As such, the term "TsT modulator" or "TsT activator" may include compounds which allosterically modulate TsT activity (possibly via modulation of sulfur flux).

One of skill will appreciate that the TsT enzyme itself may be used to treat or prevent conditions which occur as a result of reduced TsT expression, function and/or activity - the TsT enzyme being prepared or purified from samples comprising TsT or produced using recombinant technology and associated affinity purification means. TsT agonists may further include TsT enzyme substrates or analogues thereof, wherein said agonists increase the function, activity and/or expression of the TsT enzyme.

Examples of compounds which increase the function and/or activity of the TsT enzyme and/or TsT gene include compounds comprising thiosulfate, for example sodium thiosulfate (Na2S₂03). One of skill will appreciate that any suitable cation modification to a thiosulfate compound may find utility and the invention encompasses pharmaceutically acceptable salts, hydrates, derivatives and/or variants of thiosulfate. Such variants may include tetrathionate (S₄O ^2") or other polythionates ([S_n(S03)₂]^2" where n is two or more).

Thus in one embodiment, the invention provides thiosulfate, or a variant or derivative thereof as described herein, for use in treating or preventing weight associated disorders.

In a further embodiment, the invention provides use of thiosulfate, or a variant or derivative thereof as described herein, for the manufacture of a medicament for the treatment and/or prevention of weight related disorders.

A yet further embodiment provides a method of treating or preventing a weight related disorder, said method comprising administering a therapeutically ef fective amount of thiosulfate, or a variant or derivative thereof as described herein, to a subject in need thereof.

In other embodiments, compounds of this invention having utility in the treatment of weight related disorders may include those having two directly bonded sulfur atoms thus: Si-S₂, such as the thiosulphate compounds and derivatives mentioned above. Sj may have substituents such as one or more oxygens as in thiosulfate, and/or Si may have organic substituents such as in the compounds of formula I as discussed below. S₂ may be bonded to H, may form a salt with a cation or cations or may be bonded to another atom or group. For example a compound may take the form of a dimer with two Si-S₂ groups bonded together (Si-S2-Sr-S2>)- Such dimeric species might not directly modulate the function and/or activity of TST, however they may be considered to be pro-drug compounds that are metabolised in vivo to form compounds having the S1-S2 feature and which modulate the function and/or activity of TST. Examples of compounds of this invention having utility in the treatment of weight related disorders may include those of the general formula I:

wherein

Y is a hydrocarbyl radical which may be substituted or unsubstituted, saturated or unsaturated, for example alkyl, alkenyl, alkynyl or aryl;

T is a C]-2 hydrocarbyl linking group or may be absent;

M is selected from the group -S, -S(=0) and -S =0)₂;

— is an optional bond; and

Z is sulfur and may form a bond with hydrogen (S-H), or a salt with a cation, for example with a monovalent metal such as lithium (S-Li), sodium (S-Na) or potassium (S-K).

Additionally compounds of the formula ^ TrrrrM Z _may _aj_{so ex}j_st j_n a dimeric form ¥— ^" z M—S— S— ΜτττΤ— Y (or (γ— "ErrrM— ) · It will be understood that in the dimeric forms mentioned above the groups Y, T and M may be the same or different for each occurrence and the optional bond— may be present or absent for each occurrence.

While such compounds might not directly modulate the function and/or activity of TST, they may be considered to be pro-drug compounds that are metabolised in vivo to form compounds having the formula ^ TrrrrM Z _m^ which modulate the function and or activity of TST.

By alkyl is meant herein a saturated hydrocarbyl radical, which may be straight-chain, cyclic or branched. A carbon-carbon double bond provides an alkenyl group; the presence of a carbon-carbon triple bond provides an alkynyl group. As with the alkyl groups, alkenyl and alkynyl groups may be straight-chain, cyclic or branched. Typically alkyl, alkenyl and alkynyl groups Y will comprise from 1 to 25 carbon atoms, more usually 1 to 10 carbon atoms, more usually still 1 to 6 carbon atoms or even 1 to 4 carbon atoms. It being of course understood that the lower limit in alkenyl and alkynyl groups is 2 carbon atoms and in cycloalkyl groups, 3 carbon atoms. Typically aryl groups Y will comprise a ring or rings (that may be fused) of from 3 to 10 carbons that may include heteroatoms in the ring such as Ο,Ν or S. Groups Y may be substituted in more than one position. Where Y is alkyl, alkenyl or alkynyl, substituents may be (independently for each occurrence) selected from the group consisting of -C0₂H, -CN, -CON¾, -CONHR^x, -CONR^x ₂, halogen (-F, CI , Br or I; in particular_-F), -CF_3f -OH, -OR^x, -OCFj , NH₂, -NHR^X, -NR^X ₂, - NHCOR , and -NR^xCOR ; wherein R^x is hydrocarbyl, such as alkyl as defined above, for example. In particular R may be C1 alkyl.

Where Y is aryl it may be substituted with one or more substituents (independently for each occurrence) selected from the group consisting of -R^x - C¾OH, -CO2H, halogen ( -F, -CI, -Br, or -I ), CF₃, -OH, -OR , -OCF3, -N¾, -NHR^X, -NR 2, -NO2, NHCOR^x, NR COR^x, and -CN; wherein R is hydrocarbyl, such as alkyl as defined above, for example. In particular R^x may be CM alkyl. Where Y is aryl it may be phenyl or substituted phenyl. Where Y is heteroaryl it may be a five or six membered ring including one or more heteroatoms each independently selected from N, S and O. Examples of heteroaryl Y include substituted or unsubstituted pyridyl or pyrimidyl.

Accordingly, the invention may extend to uses, medicaments and methods comprising one or more of the compounds provided in Tables 1-12 below. The invention may also extend to uses, medicaments and methods employing a salt or a dimer (as discussed above) of these compounds. The invention may even extend to uses, medicaments and methods employing two different examples of these compounds coupled together by an S-S linkage i.e. a dimer with different substituents to either side of the S-S linkage.

Table 1: Y=alkyl, M=S

Table 2: Y=alkyl, M=S(=0)

Table 4: Y=substituted alkyl, M=S

Table 5: Y-substituted alkyl, M-S(=0)

Table 6: Y=substituted alkyl, M=S(=0)₂

Table 7: Y=aryl, M=S, T = Cj ₀r 2-alkyl

Table 8: Y=aryl, M=S(=0), T = C, _{or 2}-alkyl

Table 9: Y=aryl, M=S(=0)₂, T = Ci _or 2-alkyl

Table 10: Y=heteroaryl, M=S

Table 11: Y=heteroaryl, M=S(=0)

Table 12: Y=heteroaryl, M=S(=0)₂

It should be understood that insofar as the invention extends to variants or derivatives of thiosulfate or any of the other compounds described herein, those variants or derivatives should possess the ability to modulate TsT function, activity and/or expression.

Compounds provided by this invention may be formulated as compositions to be administered for the treatment and/or prevention of weight related disorders. Such compositions may take the form of (sterile) pharmaceutical compositions comprising, for example, the compounds of the fourth aspect of this invention together with a pharmaceutically acceptable diluent, excipient or carrier.

Thus a fourth aspect of this invention provides pharmaceutical compositions comprising one or more compounds selected from those described herein in association with, a pharmaceutically acceptable excipient, carrier or diluent.

In one embodiment, the pharmaceutical compositions do not comprise the compounds disclosed in Table 1-12.

Pharmaceutical formulations include those suitable for oral, topical (including dermal, buccal and sublingual), rectal or parenteral (including subcutaneous, intradermal, intramuscular and intravenous), transdermal, nasal and pulmonary (for example by inhalation) administration. The formulation may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy. Methods typically include the step of bringing into association an active compound with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation. Pharmaceutical formulations suitable for oral administration wherein the carrier is a solid are most preferably presented as unit dose formulations such as boluses, capsules or tablets each containing a predetermined amount of active compound. A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine an active compound in a free-flowing form such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, lubricating agent, surface-active agent or dispersing agent. Moulded tablets may be made by moulding an active compound with an inert liquid diluent. Tablets may be optionally coated and, if uncoated, may optionally be scored. Capsules may be prepared by filling an active compound, either alone or in admixture with one or more accessory ingredients, into the capsule shells and then sealing them in the usual manner. Cachets are analogous to capsules wherein an active compound together with any accessory ingredient(s) is sealed in a rice paper envelope. An active compound may also be formulated as dispersible granules, which may for example be suspended in water before administration, or sprinkled on food. The granules may be packaged, e.g., in a sachet. Formulations suitable for oral administration wherein the carrier is a liquid may be presented as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water liquid emulsion.

Formulations for oral administration include controlled release dosage forms, e.g., tablets wherein an active compound is formulated in an appropriate release-controlling matrix, or is coated with a suitable release-controlling film. Such formulations may be particularly convenient for prophylactic use.

Pharmaceutical formulations suitable for rectal administration wherein the carrier is a solid are most preferably presented as unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories may be conveniently formed by admixture of an active compound with the softened or melted carrier(s) followed by chilling and shaping in moulds.

Pharmaceutical formulations suitable for parenteral administration include sterile solutions or suspensions of an active compound in aqueous or oleaginous vehicles.

Injectable preparations may be adapted for bolus injection or continuous infusion. Such preparations are conveniently presented in unit dose or multi-dose containers, which are sealed after introduction of the formulation until required for use. Alternatively, an active compound may be in powder form that is constituted with a suitable vehicle, such as sterile, pyrogen-free water, before use.

An active compound may also be formulated as long-acting depot preparations, which may be administered by intramuscular injection or by implantation, e.g., subcutaneously or intramuscularly. Depot preparations may include, for example, suitable polymeric or hydrophobic materials, or ion-exchange resins. Such long-acting formulations are particularly convenient for prophylactic use.

Formulations suitable for pulmonary administration via the buccal cavity are presented such that particles containing an active compound and desirably having a diameter in the range of 0.5 to 7 microns are delivered in the bronchial tree of the recipient.

As one possibility such formulations are in the form of finely comminuted powders which may conveniently be presented either in a pierceable capsule, suitably of, for example, gelatin, for use in an inhalation device, or alternatively as a self- propelling formulation comprising an active compound, a suitable liquid or gaseous propellant and optionally other ingredients such as a surfactant and/or a solid diluent. Suitable liquid propellants include propane and the chlorofluorocarbons, and suitable gaseous propellants include carbon dioxide, Self-propelling formulations may also be employed wherein an active compound is dispensed in the form of droplets of solution or suspension.

Such self-propelling formulations are analogous to those known in the art and may be prepared by established procedures. Suitably they are presented in a container provided with either a manually-operable or automatically functioning valve having the desired spray characteristics; advantageously the valve is of a metered type delivering a fixed volume, for example, 25 to 100 microlitres, upon each operation thereof.

As a further possibility an active compound may be in the form of a solution or suspension for use in an atomizer or nebuliser whereby an accelerated airstream or ultrasonic agitation is employed to produce a fine droplet mist for inhalation.

Formulations suitable for nasal administration include preparations generally similar to those described above for pulmonary administration. When dispensed such formulations should desirably have a particle diameter in the range 10 to 200 microns to enable retention in the nasal cavity; this may be achieved by, as appropriate, use of a powder of a suitable particle size or choice of an appropriate valve. Other suitable formulations include coarse powders having a particle diameter in the range 20 to 500 microns, for administration by rapid inhalation through the nasal passage from a container held close up to the nose, and nasal drops comprising 0.2 to 5% w/v of an active compound in aqueous or oily solution or suspension.

It should be understood that in addition to the aforementioned carrier ingredients the pharmaceutical formulations described above may include, an appropriate one or more additional carrier ingredients such as diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like, and substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient.

Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline. Additionally, pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as oiive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.

Formulations suitable for topical formulation may be provided for example as gels, creams or ointments.

Liquid or powder formulations may also be provided which can be sprayed or sprinkled directly onto the site to be treated, e.g. a wound or ulcer. Alternatively, a carrier such as a bandage, gauze, mesh or the like can be impregnated, sprayed or sprinkled with the formulation and then applied to the site to be treated.

Therapeutic formulations for veterinary use may conveniently be in either powder or liquid concentrate form. In accordance with standard veterinary formulation practice, conventional water-soluble excipients, such as lactose or sucrose, may be incorporated in the powders to improve their physical properties. Thus particularly suitable powders of this invention comprise 50 to 100% w/w and preferably 60 to 80% w/w of the active ingredient(s) and 0 to 50% w/w and preferably 20 to 40% w/w of conventional veterinary excipients. These powders may either be added to animal feedstuffs, for example by way of an intermediate premix, or diluted in animal drinking water.

Liquid concentrates of this invention suitably contain the compound or a derivative or salt thereof and may optionally include an acceptable water-mi scible solvent for veterinary use, for example polyethylene glycol, propylene glycol, glycerol, glycerol formal or such a solvent mixed with up to 30% v/v of ethanol. The liquid concentrates may be administered to the drinking water of animals.

in general, a suitable dose of the one or more compounds of the invention may be in the range of about 1 to about 5000 /kg body weight of the subject per day, e.g., 1 , 5, 10, 25, 50, 100, 250, 1000, 2500 or 5000 per day. Where the compound(s) is a salt, solvate, prodrug or the like, the amount administered may be calculated on the basis the parent compound and so the actual weight to be used may be increased proportionately.

Transdermal administration may be achieved with the use of impregnated coverings dressings, bandages or the like or via the use of some form of transdermal delivery device.

Examples of transdermal delivery devices may include, for example, a patch, dressing, bandage or plaster adapted to release a compound or substance through the skin of a patient. A person of skill in the art would be familiar with the materials and techniques which may be used to transdermally deliver a compound or substance and exemplary transdermal delivery devices are provided by GB2185187, US3249109, US3598122, US4144317, US4262003 and US4307717.

By way of example, any of the compounds provided by this invention may be combined with some form of matrix or substrate, such as a non-aqueous polymeric carrier, to render it suitable for use in a bandage, dressing, covering or transdermal delivery system. The compound matrix or substrate mixture may be further strengthened by the use of a woven or knit, non-woven, relatively open mesh fabric, to produce a patch, bandage, plaster or the like which may be reversibly attached to a particular region of a patient's body, in this way, while in contact with a patient's skin, the transdermal delivery device releases the compound or substance through the skin.

A weight related disorder may include any disease, condition or syndrome directly and/or indirectly associated with the weight of a subject. For example, the compositions, medicaments and methods described herein may find application in the treatment and/or prevention of obesity and/or diabetes, especially type 2 diabetes - a condition known to be associated with obesity. Other diseases and/or conditions which might be embraced by the term "weight related disorders" include, for example, obesity and being overweight, dyslipidaemia, insulin resistant syndromes, Metabolic Syndrome, steatohepatitis/fatty liver and non-alcoholic fatty liver disease. Further conditions embraced by the term weight elated disorders may include, for example, hyperglycaemia, glucose intolerance and impaired glucose tolerance, insulin resistance, hyperlipidaemia, hypertriglyceridaemia, hypercholesterolaemia, low HDL levels, high LDL levels and abdominal obesity. Additionally, the term "weight related disorders" may encompass cardiovascular complications resulting as a consequence of conditions such as obesity, diabetes and/or hyperglycaemia; disorders of this type may include endothelial dysfunction, atherosclerosis, hypertension, cardiovascular disease and certain cancer.

In view of the above, one embodiment of the invention provides compounds which modulate TsT function, activity and/or expression for use in treating or preventing obesity and/or type 2 diabetes.

While the primary focus of this invention is the treatment and/or prevention of disorders affecting humans, the invention may also extend to the treatment of the same or similar conditions occurring in other species, in particular mammals such as canine, rodent and/or feline species.

It should be understood that any of the compounds, compositions, medicaments and/or methods of treatment described herein, may be combined with one or more other compounds, compositions, medicaments and/or methods of treatment for treating weight related disorders and/or other diseases and or conditions. Said other compounds, compositions, medicaments and/or methods may be administered or practiced concurrently with the compounds, compositions, medicaments and/or methods of present invention or separately and at different times. In a fifth aspect, the present invention provides a method of diagnosing a weight related disorder, disease, condition or syndrome or a susceptibility and/or predisposition thereto, said method comprising the steps of:

providing a sample from a subject to be tested; and

detecting a level of the TsT enzyme of TsT expression in said sample, wherein a decreased level of TsT activity, expression and/or function detected in the sample, might indicate that the subject is suffering from, or predisposed/susceptible to, a weight related disorder.

A "sample" for use in the methods described herein may comprise a sample of a biological fluid such as blood, including whole blood or a component or fraction thereof such as, for example, serum or plasma. Other samples of biological fluid may comprise saliva, sweat and/or semen.

In other embodiments "samples" such as tissue biopsies and/or scrapings may be used. In particular, adipose, muscle, kidney, gut or liver tissue biopsies and/or scrapings may be used. Advantageously such biopsies may comprise cells, for example adipose or hepatocyte cells. In addition, a sample may comprise a tissue or gland secretion and washing protocols may be used to obtain samples of fluid secreted into or onto various tissues. One of skill in this field will appreciate that the samples described above may yield or comprise quantities of TsT protein or TsT nucleic acid (i.e. DNA or RNA).

As stated, subjects diagnosed as suffering from a weight related disorder or having a susceptibility or predisposition thereto, may yield samples which exhibit modulated and/or aberrant TsT expression, function or activity. The term "aberrant" or "modulated" expression, function and/or activity should be understood to encompass levels expression, function or activity that are either increased and/or decreased relative to the expression, function and/or activity of TsT genes/proteins detected or identified in samples derived from healthy subjects or from subjects of known status (either overweight, suffering from a weight related disorder and/or prone to leanness). As such, all of the diagnostic methods described herein may further comprise the optional step of comparing the results with those obtained from reference or control samples, wherein aberrant or modulated TsT function, expression and/or activity in a sample tested, manifests as a level of TsT expression, function and/or activity which is different from (i.e. higher or lower than) the level of TsT expression, function and/or activity identified in the reference or control sample.

One of skill in the art will be familiar with the techniques that may be used to identify levels of the TsT enzyme (or fragments or portions thereof) and/or the TsT gene (and fragments or portions thereof) in samples such as those listed above.

In one embodiment, molecular detection techniques such as PCR, or PCR based protocols, may be used. Such methods may be particularly useful when the user wishes to detect levels of TsT expression, function and/or activity. In other embodiments, immunological detection techniques may be used including, for example ELISA and the like. Immunological detection techniques are most useful where the user wishes to probe samples for levels of TsT expression, function and/or activity.

PCR based techniques may be used to detect levels of TsT gene expression or gene quantity in a sample. Useful techniques may include, for example, polymerase chain reaction (PCR) using genomic DNA as template or reverse transcriptase (RT)- PCR (see below) based techniques in combination with real-time PCR (otherwise known as quantitative PCR). In the present case, real time-PCR may used to determine the level of expression of the TsT genes. Typically, and in order to quantify the level of expression of a particular nucleic acid sequence, RT-PCR may be used to reverse transcribe the relevant mRNA to complementary DNA (cDNA). Preferably, the reverse transcriptase protocol may use primers designed to specifically amplify an mRNA sequence of interest (in this case TsT gene derived mRNA). Thereafter, PCR may be used to amplify the cDNA generated by reverse transcription. Typically, the cDNA is amplified using primers designed to specifically hybridise with a certain sequence and the nucleotides used for PCR may be labelled with fluorescent or radiolabelled compounds.

One of skill in the art will be familiar with the technique of using labelled nucleotides to allow quantification of the amount of DNA produced during a PCR. Briefly, and by way of example, the amount of labelled amplified nucleic acid may be determined by monitoring the amount of incorporated labelled nucleotide during the cycling of the PCR.

Further information regarding the PCR based techniques described herein may be found in, for example, PCR Primer: A Laboratory Manual, Second Edition Edited by Carl W. Dieffenbach & Gabriela S. Dveksler: Cold Spring Harbour Laboratory Press and Molecular Cloning: A Laboratory Manual by Joseph Sambrook & David Russell: Cold Spring Harbour Laboratory Press.

Other techniques that may be used to determine a level TsT gene expression in a sample include, for example, Northern and/or Southern Blot techniques. A Northern blot may be used to determine the amount of a particular mRNA present in a sample and as such, could be used to determine an amount or level of TsT gene expression. Briefly and in one embodiment, mRNA may be extracted from, for example, a cell using techniques known to the skilled artisan, and subjected to electrophoresis. A nucleic acid probe, designed to hybridise (i.e. complementary to) an mRNA sequence of interest - in this case mRNA encoding all or part of the TsT gene, may then be used to detect and quantify the amount of a particular mRNA present in a sample.

Additionally, or alternatively, a level of TsT gene expression may be identified by way of microarray analysis. Such a method would involve the use of a DNA or RNA microarray comprising nucleic acid derived the TsT gene - perhaps a representation of the human genome. To identify a level of TsT gene expression, one of skill in the art may contact a sample, optionally processed to extract nucleic acid present therein, with a microarray comprising TsT nucleic acid at one or more defined loci. Additionally, or alternatively, nucleic acid preferably mRNA may be extracted from the sample and subjected it to an amplification protocol such as, RT-PCR to generate cDNA.

The amplified TsT cDNA may be subjected to a further amplification step, optionally in the presence of labelled nucleotides (as described above). Thereafter, the optionally labelled amplified cDNA may be contacted with the microarray under conditions which permit binding with the DNA of the microarray. In this way, it may be possible to identify a level of TsT gem expression.

In addition, other techniques such as deep sequencing and/or pyrosequencing may be used to detect TsT sequences in any of the samples described above. Further information on these techniques may be found in "Applications of next-generation sequencing technologies in functional genomics", Olena Morozovaa and Marco A. Marra, Genomics Volume 92, Issue 5, November 2008, Pages 255-264 and "Pyrosequencing sheds light on DNA sequencing", Ronaghi, Genome Research, Vol. 11, 2001, pages 3-11.

In other embodiments, it may be possible to subject a sample to a protocol which utilises an enzyme based detection process. For example, the sample might be contacted with a system comprising a known TsT substrate, where presence of functional or active TsT in the sample causes metabolism of the substrate. In such cases, detection of the reaction metabolites - perhaps by, for example ELISA, might serve to indicate a level of TsT present in the sample.

In addition to the molecular detection methods described above, one of skill will also appreciate that immunological detection techniques such as, for example, may be used to identify aberrant levels of TsT function, expression and/or activity in samples. In other embodiments, immunological detection techniques such as enzyme- linked immunosorbent assays/spot (ELISA/ELISPOT), dot blot and/or Western blot techniques may also be used.

Immunological detection techniques, may require the use of a substrate to which an antibody and/or antigen may be bound, conjugated or otherwise immobilised. The substrates provided by this invention may comprise a TsT enzyme or TsT protein (for example a portion or fragment of the whole TsT enzyme) bound, conjugated and/or immobilised thereto. In other embodiments, the substrate may comprise an agent, for example an antibody, capable of binding a TsT protein - substrates of this type may be used in capture type immunological detection methods.

Suitable substrates may comprise, for example, glass, nitrocellulose, paper, agarose and/or plastic. A substrate which comprises, for example, a plastic material, may take the form of a microtitre plate.

It should be understood that references to agents capable of binding a TsT protein, may include antibodies and in particular polyclonal and/or monoclonal antibodies. Techniques used to generate antibodies are well known in the art and may involve the use of TsT proteins in animal immunisation protocols or as a basis for the generation of hybridomas. Further information on the preparation and use of polyclonal and/or monoclonal antibodies may be obtained from Using Antibodies: A Laboratory Manual by Harlow & Lane (CSHLP: 1 99) and Antibodies: A Laboratory Manual by Harlow & Lane (CSHLP: 1988) - both of which are incorporated herein by reference.

Immunological detection techniques such as, ELISA, may be classed as

"indirect capture" assays or "direct" assays - both forms of ELISA are useful here. An indirect/capture ELISA may exploit the use of a substrate coated with an agent capable of binding a TsT protein whereas a direct ELISA may utilise substrates with a TsT protein bound, conjugated or immobilised thereto. An ELISA may involve contacting a sample with a substrate (such as a substrate described above) under conditions which permit binding between proteins (for example TsT proteins) present in the sample and the substrate and/or substances bound or immobilised to the substrate.

One familiar with these techniques will appreciate that immunological detection techniques such as ELISA, may utilise blocking steps to reduce or prevent non-specific binding.

An ELISA may comprise the further step of contacting the substrate with a secondary antibody having specificity or affinity for TsT protein bound thereto (either directly or via an antibody which is itself immobilised, bound or conjugated to the substrate and which has affinity for the TsT protein). Secondary antibodies for use in this invention may be rodent or ruminant antibodies (polyclonal or monoclonal) specific to particular forms of antibody present within the sample being tested. Furthermore, secondary antibodies may be conjugated to moieties which permit them to be detected - such moieties being referred to hereinafter as detectable moieties. By way of example, a secondary antibody may be conjugated to an enzyme capable of being detected via a colourmetric/chemiluminescent reaction. Such conjugated enzymes may include but are not limited to Horse radish Peroxidase (H P) and alkaline phosphatise (AlkP). Additionally, or alternatively, the secondary antibodies may be conjugated to a fluorescent molecule such as, for example, a fluorophore, such as FITC, rhodamine or Texas Red. Other types of detectable moiety include radiolabelled moieties.

One of skill will appreciate that the amount of secondary antibody detected as bound to the substrate (via other moieties which are themselves bound directly or indirectly to the substrate) may be representative of the amount of TsT protein present in the sample being tested.

Alternatively, in order to identify a level of TsT protein in a sample, a substrate (optionally comprising an agent capable of binding a TsT protein) may be contacted with a sample to be tested. Any TsT protein bound to the substrate (perhaps via an agent capable of binding a TsT protein) may be detected with the use of a further agent capable of binding a TsT protein - referred to hereinafter as a primary antibody. The primary binding agent may be an antibody, optionally conjugated to a detectable moiety as described above. One of skill will appreciate that many variations of the ELISA protocols described above may be used in order to detect a level of TsT protein present in a sample and further information regarding ELISA procedures and protocols relating to the other immunological techniques described herein may be found in Using Antibodies: A Laboratory Manual by Harlow & Lane (CSHLP: 1 99) and Antibodies: A Laboratory Manual by Harlow & Lane (CSHLP: 1988).

In one embodiment, the methods for detecting TsT proteins and/or TsT genes or levels of expression, function and/or activity thereof, may take the form of an immunochromatographic test - otherwise known as a "dip-stick" or "pen" tests, where a substrate, or portion thereof, is contacted with a sample to be tested. Thereafter, the test sample flows through and/or along a substrate (perhaps guided by microfluidic channels) under capillary action and is brought into contact with an agent or agents which enables detection of any TsT/TsT gene/proteins or fragments thereof in the sample. Such tests can offer rapid result and exemplary devices may include those known as lateral flow devices. The results of a dip-stick or lateral flow test may be revealed in a "test line" where, for example, a change in appearance of the test line may indicate a positive result (i.e. presence of a TsT nucleic acid or TsT protein).

Agents capable of effecting detection of TsT genes or TsT proteins in a sample may include particles such as, for example latex or gold particles optionally coated with compounds capable of binding the target analyte (namely TsT of a TsT gene/nucleic acid) in a sample. Other forms of particle such as, for example, fluorescent and/or magnetic particles may also be used.

Other useful immunological techniques may include, for example, Western blot or dot blot. A Western blot may involve subjecting a sample to electrophoresis so as to separate or resolve the components, for example the proteinaceous components, of the sample. In other embodiments, electrophoresis techniques may be used to separate proteins purified from recombinant (perhaps microbial) systems. The resolved components/proteins may then be transferred to a substrate, such as nitrocellulose.

In order to identify any TsT proteins in a sample, the substrate (for example nitrocellulose substrate) to which the resolved components and/or proteins have been transferred, may be contacted with an agent capable of binding TsT proteins under conditions which permit binding between any TsT protein in the sample (or transferred to the substrate) and the agents capable of binding the TsT protein. Advantageously, the agents capable of binding the TsT protein may be conjugated to a detectable moiety.

Other immunological techniques which may be used to identify a level of TsT protein in a sample include, for example, immunohistochemistry wherein binding agents, such as labelled antibodies capable of binding TsT, are contacted with a sample such as those described above, under conditions which permit binding between any TsT protein present in the sample and the binding agent. Typically, prior to contacting the sample with the binding agent, the sample is treated with, for example a detergent such as Triton XI 00. Such a technique may be referred to as "direct" immunohistochemical staining.

In all cases, the level of TsT protein identified in a sample may be compared with a level identified in a control or reference sample.

In a sixth aspect, the present invention provides a transgenic animal genetically modified to aberrantly express the TsT gene. Such animals may be modified to exhibit markedly reduced (i.e. no) TsT gene expression or, in other cases, substantially increased TsT expression. One of skill will appreciate that transgenic animals provided by this invention are particularly useful in the study of weight related disorders.

Such animals or indeed cells derived therefrom may be used as the basis of methods for testing agents for potential use in the treatment of a weight related disorder. By contacting a transgenic animal, or cell derived therefrom with a test agent and thereafter monitoring the level of TsT (gene/protein) expression, function and/or activity, it may be possible to screen libraries of test agents for those for use in the treatment or prevention of weight related disorders.

In a seventh aspect, the present invention provides a kits comprising reagents and compositions suitable for diagnosing, detecting or evaluating levels of TsT in subjects. Kits according to this invention may be used to identify and/or detect aberrant or modulated levels of TsT protein/gene expression, function or activity in samples. Depending on whether or not the kits are intended to be used to identify or analyse TsT gene function, expression and/or activity and/or TsT protein function, expression and/or activity in samples, the kits may comprise substrates having TsT proteins or agents capable of binding TsT proteins, bound thereto. In addition, the kits may comprise agents capable of binding TsT proteins - particularly where the kit is to be used to identify levels TsT protein in samples. In other embodiments, the kit may comprise polyclonal antibodies or monoclonal antibodies which exhibit specificity and/or selectivity for one or more TsT proteins. Antibodies for inclusion in the kits provided by this invention may be conjugated to detectable moieties. Kits for use in detecting the expression of the TsT gene may comprise one or more oligonucleotides/primers for detecting/amplifying/probing samples (particularly samples comprising nucleic acid for TsT protein) for TsT encoding sequences. The kits may also comprise other reagents to facilitate, for example, sequencing, PCR and/or RFLP analysis. All kits described herein may further comprise instructions for use.

DETAILED DESCRIPTION

The present invention will now be described in detail with reference to the following Figures which show.

Figure 1 : TsT is elevated in adipose from lean mice and reduced in adipose tissue from obese mice, a) Fold expression of Tst mRNA levels in a microarray comparison of Lean (L)- versus Fat (F)- line tissues (SC, subcutaneous, EPI, epididymal, MES, mesenteric adipose tissues; Liv, liver; Muse, muscle; Kid, kidney), b) Tst mRNA levels in SC adipose tissue of F, L and congenic mice with Chrl5 Fob3b (U12) and Fob3a (W) L-line chromosomal segments introgressed onto the F genetic background (n=6, ***=P<0.001 L versus F, **=P<0.01 U12 c) Effects of chronic 18 week high fat feeding (HF) on fat mass, corrected for body weight, in L and F mice, d) Effects of HF on Tst mRNA levels (microarray) in SC adipose tissue (n=4, ***=P<0.001 effect of diet,†††=P<0.001 effect of line).e) The effects of HF on Tst protein levels in SC fat of L and F mice. Upper panel, western blot showing 33kDa Tst band and upper non-responsive but Tst-related band.

Figure 2: The effects of adipose-specific Tst overexpression on weight gain and diabetes in mice, a) Quantification of mRNA (upper panel) and protein levels (lower panel) in adiponectin promoter-driven Tst transgenic mice (Ad-Tst). Longitudinal weight gain, b) in B6N (solid lines and bars) and Ad-Tst mice (broken lines and diagonal hatched bars), (n=5, **=P<0.01) with total 8 week weight gain, c) total 8 week feeding efficiency (weight gain per gram eaten) in HF fed B6N and Ad-Tst mice (n=5, **=P<0.01) and Glucose tolerance (GTT, d), insulin excursion of GTT e), 15 minute post-GTT NEFA suppression (f) and final plasma high molecular weight (HMW) adiponectin levels (g) in HF fed B6N and Ad-Tst mice. Figure 3: TsT levels in adipose tissue of obese rodents and humans, a) Tst mRNA levels in control diet (black bar) or 18 week high fat-fed (HF) C57BL/6J (6J) mice (white bar), b) Tst mRNA levels in 10 week old C57BI/6J (black bar) or genetically obese leptin deficient (Lepob: C51&L16l'^epob/oh) mice (grey bar), c) Tst protein levels in SC adipose tissue of 6J, HF fed 6J (6JHF) and in Lep^o mice. Note the more pronounced reduction in the lower 33kDa (Tst) band and a slight reduction in the upper Tst-related band with obesity, d) TST mRNA levels in human SC and visceral (Vise) adipose tissue from lean (black bars) or obese (horizontal hatched bars) patients.

Figure 4: Knock-down of adipocyte Tst accelerates fat accumulation and impairs mature fat cell function whereas Tst activation promotes adiponectin release, a)

Expression pattern of Tst mRNA (lower panel, n=6) and protein (upper panel, n=3) throughout differentiation of 3T3-L1 preadipocytes to mature adipocytes. Note: Increased mRNA levels are highly significant from day 4, the specific 33kDa Tst protein band is highly expressed by day 10. b) Effect of Tst knockdown using siRNA transfection in differentiating (day3-8) 3T3-L1 cells on fat accumulation at day 8 of differentiation. SCR, scrambled siRNA (control) and si42, si23, si43 are three different siRNAs directed against Tst. (n=6, *=P<0.05, **=PP<0.01). -c) Effects of Lentiviral shRNAmir-mediated Tst knockdown on mitochondrial ROS production in H 0₂-exposed mature (day 10) 3T3-L1 adipocytes. (n=6, *=P<0.05, knockdown efficiency was -50%). d) TST mRNA is expression throughout differentiation of clonal human adipocytes (SGBS cells), e) Effects of the Tst activator thiosulfate (on adiponectin release from mature human SGBS adipocytes. (n=6, ***=P<0.001) Figure 5: The Tst activator thiosulfate (sodium thiosulfate, STS) ameliorates weight gain and diabetes in chronically high fat fed (HF) mice. The effects of 3mg/ml STS (administered in drinking water) on (a) longitudinal weight gain (b) total 8 week feeding efficiency and (c) final glucose tolerance in HF-fed 6N (solid lines, open symbols, open bars) HF-fed 6N mice with STS (grey square or circle symbols, grey bars) and Ad-Tst transgenic mice on STS (closed triangle or circle symbols and hatched bars) n=5, **=P<0.01 , ***=P<0.001.

Figure 6: The Tst substrate activator thiosulfate is anti-diabetic.

Thiosulfate was administered (3mg/ml in drinking water for 6 weeks) to leptin receptor deficient mice (Lepr^{db db}) that had established obesity (BW>=40g, note age- matched normal mice are ~23g) and diabetes (fasting blood glucose >15mmole/L, normal range 6mmole/L). Thiosulfate ameliorated A. polydipsia (excessive fluid intake) and B. polyuria (excessive urine production) without affecting C. food/calorie intake or D. final body weight or E. final organ weight, F. Plasma glucose levels were significantly reduced in thiosulfate-treated mice and they showed a greater response (insulin sensitisation) to exogenously administered insulin (ITT: 2mU/g body weight, HumulinS, Novo Nordisk, i.p.). G. Thiosulfate reduced the levels of glycosylated haemoglobin (HbAlc by HPLC, clinical biochemistry, NRIE, Edinburgh), a marker of chronic hyperglycaemia and diabetes. Key: grey bars, control normal mice with blood glucose ~6mM, white bars, control (water-treated) Lepr mice, black bars, thiosulfate-treated mice. **, ***=P<0.01, PO.001 respectively by students t-test or RM-Anova.†=P<0.05 difference between final ITT glucose levels in treated versus control Lepr.†††=P<0.001 significant difference in HbAlc between a normal mouse and an untreated Lepr mouse.

Methods and Materials

F and L mice

All animal experiments were performed according to local ethical guidelines and within the Scientific Procedure Act (1986) of the UK Government Home Office. The long term selection and further development of the F and L lines and details of the genetic basis of the line divergence and the inbreeding period are described elsewhere [Horvat et ah, 2000, Stylianou et al., 2005]. Mice derived from the inbred lines were used in this study. Extensive characterisation of our lines in previous studies determined that body weight gain is highly correlated with fat mass accretion in fat mice. This was substantiated in several genetic mapping experiments [Horvat et al., 2000, Stylianou et al., 2005] demonstrating that the fat% trait and body weight trait co-localise to the same QTL regions with significant LOD scores. C57BL/6J mice, 05Ί&ΊΙ6η&ρ^οί,Ιο>) and C57BL/6N (the genetic strain of the Ad-TsT transgenic mice) were purchased from Charles River (UK). Animals were fed on pelleted rat and mouse breeder and grower diet (Special Diets Services, SDS, UK Ltd., Witham, Essex, UK) or with defined low (11% calories as fat with sucrose; D 12329) and high fat (58% calories as fat with sucrose; D12331) diets (Research Diets, New Brunswick, New Jersey). Feeding efficiency (weight gain for food eaten) was routinely monitored throughout our studies.

Adipose-specific TsT overexpressing mice The mouse 0.9kb TsT cDNA was excised from the MCS of EX-Mm05875-Lv08 lentiviral expression vector (Genecopoeia, Open Biosystems) using EcoRl and Nhel. The overhang was filled and the insert was blunt-end cloned into the EcoRV site of a pBS plasmid containing a 5.4kbp region of the upstream adiponectin promoter (Adipro, ref) including a 400bp artificial intron fragment from the untranslated adiponectin exon 2 sequence and before a 1.3 kb rabbit β-globin 3 '-untranslated region was (gift of PE Scherer, USW Texas). The correctly aligned Adipro-5'TsT3'- 3'UTR was identifed using restriction digestion and this clone was maxi-prepped and purified. The Adipro-TsT was excised from the plasmid backbone using Pvul and SacII digestion to give a ~8kbp transgene fragment that was purified by agarose gel electrophoresis and then microinjected into the pronuclei of fertilized oocytes from C57BL/6N mice by standard techniques (Turku Center for Disease Modelling, Department of Physiology, Institute of Biomedicine, University of Turku, Finland). Offspring were screened by PCR on genomic DNA extracted from tail tips using Adipro-TsT specific primers that amplified a unique transgenic band. 4 male and 1 female founders were identified and transferred to the Biomedical facility, University of Edinburgh. 3 lines exhibited Germline transmission of which 2 gave a modest overexpression of adipose TsT mRNA (Supplemental Fig). Line 3. which showed a 2- fold increase in TsT mRNA and protein, was used for the HF feeding and thiosulfate administration in the present study.

Tissue, medium and plasma measurements

At the end of the experiment, mice were killed within 1 minute of disturbing the home cage by cervical dislocation, to avoid stress-induced changes in metabolic parameters. Blood was collected in EDTA coated tubes (Sarstedt, Numbrecht, Germany). Plasma insulin was measured by ELISA (Crystalchem, Downers Grove, IL, USA), glucose by (Infinity reagent, Sigma, Dorset, Uk) and free fatty acids (Wako Diagnostics, Neuss, Germany) levels (FFA) were measured with a colorimetric method. Adiponectin (high molecular weight) was measure by ELISA (Alpco, Salem, US). Liver (L), muscle (quadriceps, M), kidney ( ), and epididymal (E), subcutaneous (S), and mesenteric (M) adipose tissues were collected, weighed and stored at -80°C.

Glucose tolerance tests

Mice were fasted for 6 hours at 8am then injected with 2mg/g D-glucose (25% stock solution in saline) intraperitoneally. Blood was sampled into EDTA-coated tubes at 0, 2, 5, 10, 15, 30, 60, and 120min intervals after glucose bolus. Glucose levels were determined immediately with a OneTouch glucose meter (Lifescan, High Wicombe, UK) and NEFA and insulin levels were determined as above.

Microarray analysis

Tissue NA for microarray was prepared using Qiagen RNeasy kits (Quiagen, Crawley, UK) and hybridized to Affymetrix Mouse Genome 430 2.0 GeneChips (n=4 per group, Affymetrix, High Wycombe, UK). RNAs were processed through standard Affymetrix protocols and imported into BioConductor for background subtraction and normalization with the Robust Multichip Average (RMA) algorithm. Differential expression was determined using the Bioconductor Limma tool and the Benjamini and Hochberg FDR method. Annotation data for the genes were obtained from NetAffx. A web accessible front-end query tool generated normalised expression, fold-changes and p-values. Clustering and pathways analyses were carried out using WebGestalt (http:^ioinfo.vanderbilt.edu/webgestalt, Vanderbilt University, USA), David and Genego.

Snap-shot microarray. Our first experiment was designed to look across a panel of tissues of the F and L mice including 3 white adipose tissue depots, muscle, liver and kidney for broad and large qualitative fold-changes in gene expression. Tissues were pooled from 3 chow fed mice of each line. RNA was hybridised to Affymetrix Genechip 2.0 arrays as described above. We confirmed the previously described differences in gene expression (Morton et al., 2005) as validatory transcriptome 'landmarks' for the qualitative microarray data. The snap-shot approach allowed us to 1. Assess which genes were grossly different between the Fat and Lean lines in all tissues tested. 2. Provide information on which genes were divergently expressed selectively across all white adipose depots. 3. Apply stricter criteria for genes that were specifically altered in the 3 white fat depots but not in other metabolic tissues to increase the likelihood of identifying adipose-specific causal lean genes. Note the original selection criteria of the F and L mice was on divergent fat pad mass and that the obesity is not the result of increased food intake (Simoncic et al., 2008) . Moreover, this was useful since the mixed genetic background of the base population may have carried 'bystander' genes that are differentially expressed between the lines in both adipose and non-adipose tissues, but that are not related to the divergent obesity and metabolic phenotype. Our second microarray experiment was designed to look at the adipose tissue depot which showed the greatest divergence in mass between the lines in response to chronic high fat feeding (Morton et al., 2005). We took subcutaneous fat from control diet and high fat fed F and L mice after 18 weeks on the diets (n=4) which allowed us to 1.Validate changes in subcutaneous fat from experiment 1 and 2. Perform quantitative analyses on the changes in gene expression between the lines and 3. Identify line-specific effects of the diet.

Bioinformatics analysis of microarray data: 1. Experiment 1 looked at qualitative fold changes in gene expression between the lines in the 3 WAT depots (subcutaneous (S), epididymal (E) and mesenteric (M), liver (L), muscle (M) and kidney ( ). We set the fold-difference threshold for changes of interest to >±1.5 (negative numbers, denotes genes that are pregulated in Lean, positive numbers '+' denotes upregulated in Fat lines, respectively). We set the fold change within B, L, M or K to be within <±1.5. This search narrowed our targets to white adipose tissue-specific changes in gene expression, with the caveat that many gene pathways maybe linked between or operate differently/reciprocally from white fat and non- adipose tissues. We excluded all genes whose absolute signal was below the threshold of 100 (genes unlikely to be meaningfully expressed in the adipose), with the caveat that it is possible some genes may be effectively switched on or off in adipose tissue of one or other line. Quantitative microarray analysis of subcutaneous fat in experiment 2 looked at fold changes in gene expression in a single fat depot, but allowed us to further assess the effects of chronic high fat feeding between and within the lines. We set the fold difference threshold to ±1.5 as before. Both experiment 1 and experiment 2 benefitted from the previously identified major QTL information (Horvat et al., 2000), which we introduced as a Filter in our bioinformatics analysis to provide more information on which genes were more likely to represent causal genes (within QTL boundaries) and those which were more likely secondary (outwith the major QTLs).

Clonal adipocyte cell experiments

The 3T3-L1 preadipocyte cell line (Green and Meuth 1974) was cultured in Dulbecco's modified Eagle medium (DMEM) (Cambrex, Venders, Belgium) supplemented with 10% new bora calf serum (NCS), 2mM L-glutamine, penicillin (50U ml) and streptomycin (50 ug/ml) (Invitrogen, Paisley, UK) at 37°C in humidified atmosphere with 5% C02. Confluent 3T3-L1 cells were differentiated by replacing NCS with 10% FBS and supplementing with 0.5mM isobutylmethylxanthine (IBMX), 0.25 μΜ dexamethasone (Dex), ^g ml insulin and ΙΟΟηΜ Rosiglitazone. Adipocyte TsT knock down in differentiating preadipocytes was performed using siRNA-mediated transfection with the DeliverX Plus kit (Panomics, Freemont, CA, USA) with lOnM each of 3 different inventoried TsT siRNAs (Applied Biosystems: s75542, s202323, and s75543, of which the latter was the most effective in our system). Gapdh (ID 430849) and a non-specific scrambled siRNA (ID 4390843) were used as controls. Knockdown was initiated at day 3 post differentiation and cells remained exposed to the reagent for 72 hours before continuing to day 8 for oil red O lipid accumulation analysis. For improved efficiency of TsT knockdown in mature fat cells we used the Expression Arrest GIPZ lentiviral shRNAmir-mediated plasmid based system (Open Biosystems, Thermofisher, Epsom, UK). Lentiviral particles were produced using a shTsTmir or a control scrambled shRNAmir by transfection of HEK293T cells with packaging vectors according to instructions (Open Biosystems). For all subsequent treatments (insulin, p3-agonist, ROS, STS) fully differentiated 3T3-L1 cells were cultured over night in phenol red free differentiation medium with charcoal stripped FBS. Mitochondrial ROS studies were performed by exposing the 3T3-L1 cells, that had been lentiviral-transfected for 2 days (shTsTmir or shScramblemir), to medium containing 1 % or 10% H₂0₂ in the medium for 6 hours followed, after careful rinsing with normal medium, hy exposure to 37°C to medium containing Mitotracker Red-CM-H₂XROS (M7513, final concentration 250nM) for 30 minutes according to instructions (Molecular Probes, Invitrogen, Paisley, UK). The medium was replaced and the excitation(579nm)- emission(599nm) was quantified using an Ml 000 fluorescence reader and analyzed using Magellan software (Tecan Uk Ltd, Reading, UK). NEFA release was measured using the Wako NEFA kit (Wako Diagnostics, VA, US) and adiponectin secretion was measured at 1 :75 dilution with an EL1SA (Quantikine, R&D Systems, UK). 3T3- Ll cell lipid accumulation was measured at day 8 of differentiation following siRNA mediated knockdown from day 3 to day 6. Cells were fixed in 10% formalin overnight followed by successive rinses in water, 100% ethanol and then Oil redO in isopropanol 6:4 water for 25 minutes, after further rinses with water and 100% ethanol before extraction of the stained lipid with 4% IGEPAL (Sigma Aldrich, MO, USA). The lipid content was quantified by absorbance at 550nm in a full spectrum plate reader (Molecular Dynamics). Human Simpson-Golabi-Behmel syndrome (SGBS) human clonal preadipocytes were maintained at 37°C, 5% C0₂ in DMEM/F12 supplemented with 10% (vol/vol) FBS, penicillin/streptomycin (50 U/ml and 50 pg ml, respectively), 33μΜ biotin and 17μ pantothenic acid. To differentiate SGBS cells, maintenance medium was removed from confluent cells and replaced with differentiation medium (DMEM/F12 supplemented with 10% heat -inactivated FBS, penicillin/streptomycin (50 U/ml and 50 pg/ml, respectively), 33μΜ biotin, 17μΜ pantothenic acid, 0.5mM IBMX, 0.25μ DEX, 20nM insulin, 2μΜ rosiglitazone, O.Olmg/ml transferrin, 0.1 μΜ Cortisol and 200pM triiodothyronine (T3). Cells were maintained in this for 4 days with the medium changed every 48 hours, after which the differentiation medium (as above) was used but without rosiglitazone. From day 7 to day 21, cells were maintained in day 4 differentiation medium but without IBMX and DEX and the medium were changed every 48 hours. After 21 days, cells were fully differentiated and subsequently incubated in medium made with stripped FBS overnight before treatments the next day. Adiponectin release in response to a 6 hour thiosulfate exposure was measured using and ELISA kit (Alpco).

Gene expression

RNA was extracted using 800μ1 TRIzol reagent (Invitrogen, Paisley, UK) per 50mg tissue. Cell RNA was extracted using 1ml trizol (Invtrogen) per well of a 12 well plate. Total RNA was isolated and putified using an RNA binding matrix method (RNaid+ kit, BIO 101, Anachem, UK) and RNA was eluted in diethylpyrocarbonate- pretreated (Promega, Southampton, UK) water containing lOOmM dithiothreitol. RNA quality and quantitiy was determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Wilmington, Deleware, USA). cDNA was synthesised from 2 g RNA using Reverse Transcription system (Promega, Southampton, UK) with oligo(dT) primer, according to the manufacturer's instructions. Realtime PCR was performed on cDNA made from tissue or cell RNA as described above using a Roche Lightcycler 480 mastermix (Roche Diagnostic Ltd, West Sussex, UK) and primer/probe sets from Applied Biosystems (Foster City, CA) or TaqMan® Gene expression Assays (Applera, Cheshire, UK). Probes used in this study were mouse TsT: Mm00726109_mL Gapdh (internal control): Mm99999915_gl and TBP (internal control): Mm0000446973_ml. Human TsT was measured using Hs00361812_ml in SGBS cells and in human adipose.

Statistical analyses Gene expression differences in validation realtime was analysed using 2-way ANOVA for line and diet effects followed by post-hoc Holm-Sidak multiple comparison tests using Sigmastat version 3.5 (Systat Software). Effects of treatments on biological parameters such as fatty acids, tissue glycogen or palmitate uptake were assessed using 1-way ANOVA. P-values below 0.05 were accepted as statistically significant.

We found that Tst mRNA and protein was elevated in adipose tissue of a genetically lean mouse strain where there is good evidence it is causal for a degree of their leanness. Conversely, Tst was low in adipose tissue from a number of obese mouse strains and in human fat from obese patients. We hypothesised that high Tst levels were beneficial for adipose tissue function and promoted leanness, which we showed in a transgenic animal overexpressing Tst under and adipose-specific promoter. We showed that knocking down Tst in adipocytes in vitro caused fat cells to accumulate more fat and secrete less of the insulin-sensitizing hormone adiponectin, suggesting that higher levels of the enzyme were important in normal healthy fat function and lipid storage. We then stimulated Tst using a known simple activator, thiosulfate, and showed the beneficial effect of stimulation on adiponectin release in cultured fat cells in vitro as well as prevention of excessive weight gain and correction of diabetes in mice in vivo. Taken together these data show that increased Tst levels and or activity promote healthy leanness. Tst activators represent a novel medicament for the treatment of weight related disorders. Since thiosulfate is nontoxic and has been used clinically to effectively treat a number of unrelated disorders from cyanide detoxification (Baskin et al., 1999) to prevention of serious tissue calcium deposition (including coronary artery disease) in humans (Hayden and Goldsmith 2010, Adirekkiat et al., 2010) and rats (Pasch et al., 2008) and heart failure in mice (Sen et al., 2008), we suggest in the first instance that this compound could be re-positioned for the treatment of weight-related disorders such as obesity and type 2 diabetes. Remarkably, although never directly linked because of the belief that thiosulfate works directly chelating calcium or indirectly through metabolism of H₂S, direct beneficial effects of Tst activation by thiosulfate may be a (previously unrecognised) beneficial modality in the aforementioned disease processes, as well as those we propose for weight-related disorders. Novel potent selective Tst activators could be developed to increase the efficacy of Tst therapeutics. Recombinant Human TST Assay

For the measurement of TST enzyme activity in vitro, recombinant full length human TST was used. Assays were carried out in 96-well microplates in total volume of ΙΟΟμΙ. Assays contained the following components:

Sodium phosphate (30mM, pH 7.5)

Human TST (400nM)

Substrate (0-50mM)

Microplates were incubated at room temperature for 5 minutes and KCN (SOmM) added to each well. Following a further 5 minute incubation, 67μ1 of solution was removed and transferred to a new mcroplate. 7μ1 formaldehyde and 30μ1 Fe(N0 )₃ were then added. The microplate was centrifuged for 5 minutes at 4000rpm and the contents transferred to a new microplate. Absorbance at 460nM was measured in a tunable microplate reader.

The calculation of K_m and kc_at parameters for each substrate was performed by plotting A460 vs substrate concentration and fitting data to the Michaelis-Menten equation using GraphPad Prism® software.

Results

The following compounds were tested using the recombinant human TST assay described above.

The results shown the Table 13 above, demonstrate that compounds 001 through 003 are metabolised by human TST with greater efficiency than the prototype substrate sodium thiosulphate. REFS:

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Claims

Claims

1. Compounds which modulate TsT activity, function and/or expression for use in treating or preventing weight related disorders.

2. A method of treating a weight related disorder, said method comprising the steps of administering to a subject in need thereof, a therapeutically effective amount of a compound which modulates TsT activity, function and/or expression.

3. The compound for use of claim 1 , or method of claim 2, wherein the compound either increases or decreases the expression, function andfor activity of TsT.

4. The compound for use or method of any one of claims 1-3, wherein the compound increases., augments or enhances the activity function aud or expression of the TsT enzyme or the gene (TsT) encoding the same.

5. The compound for use or method of claims 1 -4, wherein the compound is selected from the group consisting of (i) small organic/inorganic compounds; (ii) lipids; (iii) nucleic acids; (iv)carbohydrates; (v) proteins peptides, polypeptides and/or amino acids; and (vi) antibodies or antigen binding fragments, thereof.

6. The compound for use or method of claim 5, wherein the nucleic acid comprises an antisense, silencing and/or interfering nucleic acid.

7. The compound for use or method of claim 5, wherein the antibody or antigen binding fragment thereof exhibits specificity, selectivity and/or affinity for an epitope ofTsT.

8. The compound for use or method of any one of claims 1 -5, wherein the compound comprises, consists or substantially consists of the TsT enzyme.

9. The compound for use or method of any one of claims 1-5, wherein the compound comprises thiosulfate and/or a pharmaceutically acceptable salt, hydrate, derivative and/or variant thereof.

10. The compound for use or method of any one of claims 1 -5 or 9, wherein the compound comprises sodium thiosulfate (Na₂S₂C>3).

11. The compound for use or method of any one of claims 1 -5 or 9, wherein the compound has general formula I:

Y rr-rrM Z

( I ) wherein

Y is a hydrocarbyl radical which may be substituted or unsubstituted, saturated or unsaturated, for example alkyl, alkenyl, alkynyl or aryl;

T is a Cj-2 hydrocarbyl linking group or may be absent;

M is selected from the group -S, -S(=0) and -S(=0)₂;

-— is an optional bond; and Z is sulfur and may form a bond with hydrogen (S-H), or a salt with a cation, for example with a monovalent metal such as lithium (S-Li), sodium (S-Na) or potassium (S-K).

12. compound for use or method of any one of claims 1-5 or 9, wherein the compound has general formula II

Y T...M— S— S— MrrrT— Y

(Π)

Wherein Y, T,— and M are defined as per claim 11 and S is sulphur.

13. Compounds which modulate TsT activity, function and/or expression for use in treating or preventing weight related disorders, wherein the compound is selected from the group consisting of:

(i) thiosulfate;

(ii) sodium thiosulfate (Na2S₂0₃);

(iii) one or more of the compounds presented in Tables 1-12;

(iv) a pharmaceutically acceptable salt, hydrate, derivative and/or variant of any of (i)-(iii).

14. A pharmaceutical composition comprising one or more of thiosulfate, sodium thiosulfate (Na2S₂C>3) and/or a pharmaceutically acceptable salt, hydrate, derivative and/or variant of either, in association with, a pharmaceutically acceptable excipient, carrier or diluent.

15. The compound for use or method of any one of claims 1-13, wherein the weight related disorder is one or more selected from the group consisting of: (i) obesity; (ϋ) diabetes;

(iii) type 2 diabetes;

(iv) dyslipidaemia;

(v) insulin resistant syndromes;

(vi) Metabolic Syndrome;

(vti) steatohepatitis/fatty liver and non-alcoholic fatty liver disease;

(viii) hyperglycaemia;

(ix) glucose intolerance and impaired glucose tolerance;

(x) insulin resistance;

(xi) hyperlipidaemia;

(xii) hypertriglyceridaemia;

(xiii) hypercholesterolaemia;

(xiv) low HDL levels;

(XV) high LDL levels;

(xvi) abdominal obesity;

(xvii) atherosclerosis;

(xviii) hypertension;

(xix) cardiovascular disease; and

(XX) cancer.

16. The compound for use or method of any one of claims 1-13, wherein the weight related disorder is one or more selected from the group consisting of:

(i) Obesity; and

(ii) Type 2 diabetes

17. A method of diagnosing a weight related disorder, disease, condition or syndrome or a susceptibility and/or predisposition thereto, said method comprising the steps of:

(i) providing a sample from a subject to be tested; and

(ii) detecting a level of the TsT enzyme of TsT expression in said sample, wherein a decreased level of TsT activity, expression and/or function detected in the sample, might indicate that the subject is suffering from, or predisposed/susceptible to, a weight related disorder.

18. A transgenic animal genetically modified to aberrantly express the TsT gene.

19. Use of the transgenic animal of claim 18 in the study of weight related disorders.

20. A kit comprising one or more components selected from the group consisting of:

(i) substrates having TsT proteins or agents capable of binding TsT proteins, bound thereto;

(ii) agents capable of binding TsT proteins;

antibodies which exhibit specificity and/or selectivity for one or more TsT proteins;

(iii) one or more oligonucleotides/primers for detecting/amplifying/probing samples;

(iv) reagents to facilitate, for example, sequencing, PCR and/or RFLP analysis; and

(v) instructions for use.