GLCNACYLATION IN HORSES

FIELD OF THE INVENTION:

The invention is in the field of horses, more particularly the invention relates to method and composition for determining the O-GlcNAcylation blood level in horses.

BACKGROUND OF THE INVENTION:

Digestive diseases (colic and diarrhea) are among the most important diseases in horses, both from a medical and economic point of view (Archer et Proudman 2006). They can be responsible for considerable economic and sporting losses for the equine sector due to their frequency and possible complications (Taylor 2015). While in many cases they resolve with medical treatment in the field, for some horses they can become the leading cause of death, especially when associated with a serious complication such as sepsis. It is defined as organ dysfunction caused by a deregulated host response to infection (Singer et al., 2016). As for human, cardiovascular shock secondary to sepsis generates organic dysfunctions that can lead to acute cardiac decompensation and endothelial dysfunction (disseminated intravascular coagulation, coagulopathies, etc.), which cause excess mortality. A few studies have looked at sepsis in horses with digestive diseases and have demonstrated its negative impact on survival prognosis (Roy et al., 2017 ; Borde et al., 2014 ; McConachie, Giguere, et Barton 2016). Indeed, more than 30% of horses with colic or diarrhea develop sepsis, and among them, about 40% die (Taylor 2015). Despite recent advances in the diagnosis and treatment of digestive disorders in horses, there are still many unknowns in sepsis. This has led to numerous scientific studies to understand its pathophysiology and to develop new diagnostic, prognostic and therapeutic strategies.

SUMMARY OF THE INVENTION:

The invention relates to a method for diagnosing whether a horse is at risk of or is susceptible to have a risk of sepsis comprising following steps: i) quantifying the expression level of O-GlcNAcylation in a biological sample obtained from the horse; ii) comparing the expression level quantified at step i) with its predetermined reference value; and iii) concluding that the horse is at risk of or is susceptible to have a risk of sepsis when the expression level of O-GlcNAcylation quantified at step i) is lower than its predetermined reference value.

In particular, the invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Inventors have used three cohorts of horses: a cohort consisting of healthy horses (n=20), a cohort consisting of horses hospitalized for colic without signs of sepsis until discharge (n=17) and horses hospitalized for colic with signs of sepsis (n=20). Blood sample and clinical data were collected at admission to the equine emergency service of Oniris and during the 3 first days of hospitalization. O-GlcNAc levels in blood were evaluated by western blot. Data were analyzed with ImageLab and Prism software.

Their analysis confirms the presence of O-GlcNAc in blood of horses. At admission O- GlcNAc levels are 2 times higher in colic induced sepsis group than in control or colic groups (control 0.65±0.42; colic 0.55±0.13; sepsis 1.09±0.35, p<0.05 Mann-Whitney test). O-GlcNAc levels decrease by 80% over time in septic horses (p<0.05 One way-ANOVA test).

O-GlcNAc levels in septic horses tended to decrease between admission and the first day (DI) after admission. This decrease became significant from the second day (D2) of hospitalization (Control D2: 80.52 ± 52.04; Sepsis D2: 27.86 ± 28.40, p < 0.05; Control D3: 84.91 ± 51.30; Sepsis D3: 27.86 ± 30.33, p < 0.01 Two way ANOVA test with repeated measure).

Accordingly, inventors have demonstrated that the O-GlcNAc levels in septic horses tended to decrease between admission and the first day (DI) after admission. This decrease became significant from the second day (D2) of hospitalization.

Accordingly, in a first aspect, the invention relates to a method for diagnosing whether a horse is at risk of or is susceptible to have a risk of sepsis comprising following steps: i) quantifying the expression level of O-GlcNAcylation in a biological sample obtained from the horse; ii) comparing the expression level quantified at step i) with its predetermined reference value; and iii) concluding that the horse is at risk of or is susceptible to have a risk of sepsis when the expression level of O-GlcNAcylation quantified at step i) is lower than its predetermined reference value. In a particular embodiment, the invention relates to a method for diagnosing whether a horse is at risk of or is susceptible to have a risk of sepsis comprising following steps: i) quantifying the expression level of O-GlcNAcylation in a biological sample obtained from the horse at the admission and/or at the first day (DI) after admission; ii) quantifying the expression level of O-GlcNAcylation in a biological sample obtained from the horse at 2h, 4h, 6h, 8h, lOh, 12h, 14h, 16h, 18h, 20h, 22h, 24h, 26h, 28h, 30h, 32h, 34h, 36h, 38h, 40h, 42h, 44h, 44h, 46h or 48h after admission; iii) comparing the expression level quantified at step i) with the expression level quantified at step ii) and iv) concluding that the horse is at risk of or is susceptible to have a risk of sepsis when the expression level of O-GlcNAcylation is lower at step ii) than the expression level quantified at step i) or concluding that the horse is not at risk of or is not susceptible to have a risk of sepsis when the expression level of O-GlcNAcylation quantified at step ii) is higher than the expression level quantified at step i).

In a particular embodiment, the method according to the invention, wherein the step ii) is performed at second day (D2) after admission.

As used herein term “diagnosing” refers to classifying a disease or a symptom, determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease and/or prospects of recovery. The present invention relates to a method for diagnosing sepsis in a horse.

As used herein, the term “sepsis” means morbid condition induced by a toxin, the introduction or accumulation of which is caused by infection or trauma, and includes the early stage of sepsis, severe sepsis and the acute phase of septic shock. Sepsis is one of the complication of colic. The term “colic” encompasses all forms of gastrointestinal conditions which cause pain as well as other causes of abdominal pain not involving the gastrointestinal tract. The most common forms of colic are gastrointestinal in nature and are most often related to colonic disturbance.

The term “early stage sepsis” refers to the stage of the disease with the onset of clinical symptoms of severe infectious disease that typically include chills, profuse sweat, irregularly remittent fever, prostration and the like. The term “severe sepsis” refers to the stage of the disease with the clinical symptoms of early stage sepsis in addition to persistent fever, lymphopenia, disseminated intravascular coagulation, respiratory distress syndrome, multiple organ failure and hypotension leading to shock.

The term “acute phase of septic shock” refers to peripheral circulatory collapse, resulting in hemodynamic, metabolic and visceral disorders that almost invariably lead to death.

As used herein, the term “risk” refers to the probability that an event will occur over a specific time period, as in the conversion to sepsis, and can mean a subject's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation postmeasurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l- p) where p is the probability of event and (1- p) is the probability of no event) to no conversion. Alternative continuous measures, which may be assessed in the context of the present invention, include time to sepsis conversion risk reduction ratios.

"Risk evaluation," or "evaluation of risk" in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another, i.e., from a normal condition to a sepsis condition or to one at risk of developing a sepsis. Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of sepsis, such as cellular population determination in peripheral tissues, in serum or other fluid, either in absolute or relative terms in reference to a previously measured population. The methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion to sepsis, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk for a sepsis. In the categorical scenario, the invention can be used to discriminate between normal and other subject cohorts at higher risk for sepsis. In other embodiments, the present invention may be used so as to help to discriminate those having sepsis from normal. As used herein, the term “O-GlcNAcylation” also known as O-N-acetyl glucosaminylation refers to a reversible posttranslational modification (PTM) that involves the addition of the monosaccharide b-D-Nacetylglucosamine to serine and threonine residues of proteins. Protein O-GlcNAcylation is catalyzed by O-GlcNAc transferase (OGT), and the O- GlcNAc group on proteins can be removed with the catalysis of P-N-acetylglucosaminidase (O- GlcNAcase, OGA). O-GlcNAcylation is regulated dynamically by these two enzymes and by the intracellular concentration of UDP-GlcNAc, a product of glucose metabolism through the hexosamine biosynthetic pathway (HBP).

As used herein, the term “level of O-GlcNAcylation” refers to the expression or level of O-GlcNAcylation in the biological sample. Typically, the said expression level may be expressed as any arbitrary unit that reflects the amount of the protein of interest that has been detected in the biological sample, such as intensity of a radioactive or of a fluorescence signal emitted by a labeled antibody specifically bound to the protein of interest. Alternatively, the value obtained at the end of step a) may consist of a concentration of protein(s) of interest that could be measured by various protein detection methods well known in the art, such as ELISA, SELDI-TOF, FACS or Western blotting. Typically, blood proteins were extracted according to the protocol used by lin and collaborators. Western blots were performed on the protein extracts using an antibody directed against the O-GlcNAc (Ac-RL2-HRP ref : 201995 Abeam). Briefly proteins were quantified using a BCA protein assay kit. 25 mg of each sample was separated on an SDS-PAGE gel and transferred to a nitrocellulose membrane. The membranes were blocked with 3% Bovine serum albumin (BSA) in TBS IX -Tween 0.5X (TBS-T) and then incubated with Anti-O-Linked N-acetylglucosamine antibody (clone RL2, 1 :20 000) overnight at 4°C. Analyses were performed using Image Lab software (Bio-Rad, California, United States). A ratio to the stain-free intensity was calculated.

In a particular embodiment, the horse is at risk of or is susceptible to have a risk of sepsis when the level of O-GlcNAcylation decreases at two days after admission at hospital.

As used herein, the term “biological sample” refers to any sample obtained from a horse, such as a serum sample, a plasma sample, a urine sample, a blood sample, a lymph sample, or a tissue sample. In a particular embodiment, the biological sample is a blood sample obtained from the horse. As used herein, the term “predetermined reference value” refers to a threshold value or a cut-off value. In the context of the invention, the predetermined reference value refers to the expression level of O-GlcNAcylation measured at step i). Typically, the predetermined reference value corresponds to the level of O-GlcNAc level at the admission.

In a particular embodiment, the expression level of O-GlcNAcylation measured at step i) relates to the quantification of O-GlcNAc level at admission and/or the first day (DI) after admission.

In a particular embodiment, the expression level of O-GlcNAcylation measured at step ii) relates to the quantification of O-GlcNAc level at second day (D2) of hospitalization.

Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognised by a person of ordinary skilled in the art. For example, retrospective measurement of the O-GlcNAcylation level in properly banked historical subject samples may be used in establishing the reference value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. The full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic or prognostic tests. ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1- specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER. SAS, CREATE-ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), Stata/Se version 12.0 software (StataCorp LP, College Station, TX, USA), etc.

In some embodiments, the reference value is the level of O-GlcNAcylation in a healthy subject (i.e that has not been diagnosed for a sepsis). In some embodiments, the reference value is the level of O-GlcNAcylation detected in previous samples obtained from the subject. In some embodiment, the reference value is the upper limit of values determined in healthy subjects (i.e that has not been diagnosed for a sepsis).

In a second aspect, the invention relates to a method for increasing O-GlcNAcylation levels in a horse having or is susceptible to have a sepsis comprising administering said horse with a therapeutically effective amount of a compound that increases O-GlcNAcylation level.

In other words, the invention relates to a method for treating sepsis in a horse comprising administering said horse with a therapeutically effective amount of a compound that increases O-GlcNAcylation level.

In a particular embodiment, the method according to the invention wherein the horse is diagnosed as having a risk of sepsis according to the method described as above.

In a particular embodiment, the method according to the invention wherein the compound that increases O-GlcNAcylation level is administered when the horse has a low expression level of O-GlcNAcylation during the second time.

As used herein, the term “increasing” refers to an increase in O-GlcNAcylation levels. In the context of the invention (sepsis in horse), an increase in O-GlcNAcylation levels allows to reduce production of lactates, creatininemia and blood urea nitrogen (BUN) and arterial bicarbonates.

As used herein, the term “compound that increases O-GlcNAcylation levels” refers to a natural or synthetic compound that has a biological effect to increase the levels of - GlcNAcylation.

In a particular embodiment, the compound that increases O-GlcNAcylation levels is a peptide, peptidomimetic or a small organic molecule.

In a particular embodiment, the compound that increases O-GlcNAcylation levels is a small organic molecule. The term “small organic molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da. In a particular embodiment, the compound that increases O-GlcNAcylation levels is 1,2- dideoxy-2 ^z -propyl- a -d-glucopyranoso-[2, 1-D]- A 2-thiazoline (NButGT) and its derivatives. The CAS number of NButGT is 863918-55-2.

In a particular embodiment, the compound that increases O-GlcNAcylation levels is (3aR,5R,6S,7R,7aR)-2-(ethylamino)-3a,6,7,7a-tetrahydro-5-(hy droxymethyl)-5H-Pyrano[3,2- d]thiazole-6,7-diol) (ThiametG) and its derivatives. The CAS number of ThiametG is 1009816- 48-1.

In a particular embodiment, the compound that increases O-GlcNAcylation levels is a compound that increases the Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc).

As used herein, the term “UDP-GlcNAc” refers to a nucleotide sugar and a coenzyme in metabolism. It is synthesized de novo from glucose by the hexosamine synthesis pathway (HXBP), but can also be produced via salvage pathways.

In a particular embodiment, the compound that increases O-GlcNAcylation levels is UDP-GlcNAc.

In a particular embodiment, compound that increases O-GlcNAcylation levels is a ccompound that increases the hexosamine biosynthesis pathway flux that bypasses glutamine/fructose-6-phosphate amidotransferase 2.

In a particular embodiment, the compound that increases O-GlcNAcylation levels is glutamine or glucosamine.

In a particular embodiment, the compound that increases O-GlcNAcylation levels is an inhibitor of O-GlcNAcase (OGA).

As used herein, the term “O-GlcNAcase” is an enzyme with systematic name (protein)- 3-O-(N-acetyl-D-glucosaminyl)-L-serine/threonine N-acetylglucosaminyl hydrolase. OGA is encoded by the OGA gene. This enzyme catalyzes the removal of the O-GlcNAc post- translational modification. OGA catalyzes O-GlcNAc hydrolysis via an oxazoline reaction intermediate.

Inhibitor of OGA is well known in the art. Tests for determining the capacity of a compound to be OGA inhibitor are well known to the person skilled in the art. In a preferred embodiment, the inhibitor specifically binds to OGA in a sufficient manner to inhibit the biological activity of OGA. Binding to OGA and inhibition of the biological activity OGA may be determined by any competing assays well known in the art. For example, the assay may consist in determining the ability of the agent to be tested as OGA inhibitor to bind to OGA. The binding ability is reflected by the Kd measurement. . The term "KD", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e. Kd/Ka) and is expressed as a molar concentration (M). KD values for binding biomolecules can be determined using methods well established in the art.

In a particular embodiment, the inhibitor of OGA is selected from the group consisting of but not limited to: MK-8719, LY3372689 or ASN-120,290 and their derivatives.

In a particular embodiment, the inhibitor of OGA is MK-8719 as described in Seinick et al 2019 (DOI: 10.1021/acs.jmedchem.9b01090; J. Med. Chem. 2019, 62, 10062-10097).

In a particular embodiment, the inhibitor of OGA is LY3372689 as described in WO2018/140299 and in Shcherbinin et al 2020 (Alzheimer's & Dementia: The Journal of the Alzheimer's Association , Dec2020 Supplement SI 1, Vol. 16 Issue 11, pl-2, 2p. Publisher: John Wiley & Sons, Inc).

In a particular embodiment, the inhibitor of OGA is ASN-120,290 (previously ASN- 561) as described in WO2017/144639.

In a particular embodiment, the inhibitor of OGA is described in W02006/092049, WO 2008/025170, W02010/012106, WO2010/012106, W02010/012107, WO2010/037207, WO201 1/140640, W02012/064680, WO2012/083435, WO2013/166654, W02014/100934, WO2014/032184, WO2014/032185, WO2014/032187, WO2014/067003, WO2017106254, and WO2022/108377..

As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., a compound that increases O-GlcNAcylation levels) into the subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

A “therapeutically effective amount” is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount" to a subject is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder. It will be understood that the total daily usage of the compounds of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound 15 employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

The compound that increases O-GlcNAcylation levels as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.

Accordingly, in a third aspect, the invention relates to a pharmaceutical composition comprising a compound that increases O-GlcNAcylation levels.

In particular embodiment, the invention relates to the pharmaceutical composition comprising a compound that increases O-GlcNAcylation levels for use in the treatment of sepsis in horse.

In particular embodiment, the invention relates to a pharmaceutical composition comprising the compound that increases O-GlcNAcylation levels as described above and excipients.

"Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

In a particular embodiment, the invention relates to a pharmaceutical composition comprising NButGT in the context of sepsis in horse.

In a particular embodiment, the invention relates to a pharmaceutical composition comprising ThiametG in the context of sepsis in horse. In a particular embodiment, the invention relates to a pharmaceutical composition comprising glutamine or glucosamine in the context of sepsis in horse.

In a particular embodiment, the invention relates to a pharmaceutical composition comprising MK-8719, LY3372689 or ASN-120,290 and their derivatives

A further object of the present invention relates to a method of screening a compound that increases O-GlcNAcylation levels comprising i) providing a test compound and ii) determining the ability of said test compound to increase O-GlcNAcylation levels.

Any biological assay well known in the art could be suitable for determining the ability of the test compound to increase O-GlcNAcylation levels. In some embodiments, the assay first comprises determining the ability of the test compound to bind to a protein O-GlcNAcylation. In some embodiments, a population of cells is then contacted and activated so as to determine the ability of the test compound to increase O-GlcNAcylation. In particular, the effect triggered by the test compound is determined relative to that of a population of immune cells incubated in parallel in the absence of the test compound or in the presence of a control agent either of which is analogous to a negative control condition. The term "control substance", "control agent", or "control compound" as used herein refers a molecule that is inert or has no activity relating to an ability to modulate a biological activity or expression. It is to be understood that test compounds capable of increasing O-GlcNAcylation levels, as determined using in vitro methods described herein, are likely to exhibit similar modulatory capacity in applications in vivo. Typically, the test compound is small organic molecules. For example, the test compound according to the invention may be selected from a library of compounds previously synthesised, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesised de novo. In some embodiments, the test compound may be selected form small organic molecules.

In a fourth aspect, the invention relates to a kit suitable diagnosis whether a horse is at risk of or is susceptible to have a risk of sepsis comprising a reagent that specifically reacts with O-GlcNAcylation and instructions to perform the diagnosing method of sepsis according to the method as described above.

In another aspect, the invention relates to a method of screening a compound that increases O-GlcNAcylation levels comprising i) providing a test compound and ii) determining the ability of said test compound to increase O-GlcNAcylation levels. Any biological assay well known in the art could be suitable for determining the ability of the test compound to increase the O-GlcNAcylation levels. In some embodiments, the assay first comprises determining the ability of the test compound to bind to O-GlcNAcylation level. In some embodiments, a population of cells is then contacted and activated so as to determine the ability of the test compound to increase the O-GlcNAcylation levels. In particular, the effect triggered by the test compound is determined relative to that of a population of immune cells incubated in parallel in the absence of the test compound or in the presence of a control agent either of which is analogous to a negative control condition. The term "control substance", "control agent", or "control compound" as used herein refers a molecule that is inert or has no activity relating to an ability to modulate a biological activity or expression. It is to be understood that test compounds capable of increasing the O-GlcNAcylation levels, as determined using in vitro methods described herein, are likely to exhibit similar modulatory capacity in applications in vivo. Typically, the test compound is selected from the group consisting of peptides, petptidomimetics, small organic molecules, aptamers or nucleic acids. For example, the test compound according to the invention may be selected from a library of compounds previously synthesised, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesised de novo. In some embodiments, the test compound may be selected form small organic molecules. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: Variation in blood O-GlcNAc levels of horses admitted with sepsis. Evaluation by western blot of O-GlcNAcylation levels of blood proteins throughout treatment (Controladmission, J1-8H, J2-8H and J3-8H, n = 20, Colic admission, J1-8H, J2-8H n = 17 and J3-8H n = 14, Sepsis admission n = 10, Jl-8H n=10, J2-8H n = 9 and J3-8H n= 8) Quantification of O-GlcNAcylationlevels is normalized to stain free. Results expressed as a mean ± SEM. Colic J2-8h vs Sepsis J2-8h, *p<0.05; Control J2 vs Sepsis J2, *p< 0.05 and Control J3-8h vs Sepsis J3-8h, ** p<0.01 Tow wayAnova test (Mixted effect model) with repeated mesure. Figure 2: Variation in blood O-GlcNAc levels of horses admitted with sepsis. Evaluation by western blot of O-GlcNAcylation levels of blood proteins throughout treatment at admission. Control n=9, colique n=7, sepsis n=4, sepsis mort n=5. Quatification of O- GlcNAcylation levels is normalizedto stain free. Results expressed as a mean ± SEM. NS, Test one way Anova, post hoc test Bonferonni.

EXAMPLE:

Material & Methods

Horse Patient

Clinical routine data and blood samples were collected at the equine emergency service of Oniris. Horses admitted for digestive syndrome (also called colic) or diarrhea were included in the study. If these horses had a SIRS score greater than or equal to 4 (Table I) and a Multiple Organ Dysfunction Score (MODS) (Table II) greater than or equal to 6 then horses were considered to be in sepsis.

Table I : Scoring of systemic inflammatory response syndrome (SIRS)

Using these criteria, three cohorts were designed: a cohort consisting of healthy horses (n=20), a cohort consisting of horses hospitalized for colic without signs of sepsis until discharge (n=17) and horses hospitalized for colic with signs of sepsis (n=20). Blood samples and clinical data of healthy horses were collected at the Daniel Brother Agricultural High

5 School (Bouaye) and at the Feuriaye stables (Carquefou).

Table II: sepsis criteria adapted from the sepsis score used in human medicine (Singer et al.,

2016) and the multi-organ dysfunction score described for horses in colic (McConachie, Giguere, et Barton 2016). To be included in one of the 2 sepsis groups in the study, horses 0 will need to have at least 2 criteria of exaggerated systemic inflammatory response syndrome (SIRS) and an organ dysfunction score >2.

Blood sampling and analyses

4-mL volume of arterial blood was collected on admission of the horse (TD) and then each morning for 3 days after admission (data not shown). Blood samples were placed in a 4 5 mL heparin tube. NFS and blood gases were performed on admission. Aliquots of 1.5 mL of whole blood were made and stored at -80°C.

Protein extraction and western blot analyses

Blood proteins were extracted according to the protocol used by lin and collaborators. Western blots were performed on the protein extracts using an antibody directed against the O- GlcNAc (201995 Abeam). Briefly proteins were quantified using a BCA protein assay kit. 25 mg of each sample was separated on an SDS-PAGE gel and transferred to a nitrocellulose membrane. The membranes were blocked with 3% Bovine serum albumin (BSA) in TBS IX - Tween 0.5X (TBS-T) and then incubated with Anti-O-Linked N-acetylglucosamine antibody (clone RL2, 1 :20 000) overnight at 4°C. Analyses were performed using Image Lab software (Bio-Rad, California, United States). A ratio to the stain-free intensity was calculated.

Statistical analyses

Analyses of Western blots were expressed in relation to the average of the protein quantification (stain free) and then reduced to the average of the control samples. Data of O- GlcNAc level at admission and following levels of O-GlcNAc over time were analyzed with Mann Whitney test and with Kruskal-Wallis test with repeated measures, respectively.

Results

Demographic and clinic results: The demographic and clinical data of the horses included in the study are described in

Table III below.

Table III: Summary table of demographic and clinical data of horses included in the study. N represents the number of patients in each group. SD represents standard deviation. The rows labeled N-miss show the number of missing data. A 1-way Anova test was used. Study of O-GlcNAc levels

Western blot analysis confirmed the presence of O-GlcNAc in the blood of the horses and showed that, on admission of the horses to the veterinary emergency department.

In addition, O-GlcNAc levels decreased by 80% over time in septic horses (p<0.05 Kruskall Wallis test with repeated measures) (Figure 2).