WO2019202149A1

WO2019202149A1 - Sglt2 inhibitors for the treatment of neutropenia

Info

Publication number: WO2019202149A1
Application number: PCT/EP2019/060232
Authority: WO
Inventors: Maria VEIGA DA CUNHA; Emile Van Schaftingen; Guido Bommer
Original assignee: Université Catholique de Louvain
Priority date: 2018-04-19
Filing date: 2019-04-19
Publication date: 2019-10-24

Abstract

The present invention relates to the use of a SGLT2 (sodium glucose cotransporter 2) inhibitor for the treatment of neutropenia, in particular of neutropenia associated with an intracellular accumulation of 1,5-anhydroglucitol-6-phosphate. The present invention further relates to methods for determining whether a subject suffering from neutropenia is eligible for treatment with a SGLT2 inhibitor, for monitoring a neutropenia associated with an intracellular accumulation of 1,5-anhydroglucitol-6-phosphate and for monitoring the effectiveness of a treatment with a SGLT2 inhibitor.

Description

SGLT2 INHIBITORS FOR THE TREATMENT OF NEUTROPENIA

FIELD OF INVENTION

The present invention relates to neutropenia in association with glucose-6-phosphate metabolism, and more particularly to the treatment of neutropenia associated with an intracellular accumulation of l,5-anhydroglucitol-6-phosphate (l,5-AG-6-P) with a SGLT2 (sodium glucose cotransporter 2) inhibitor. The present invention further relates to methods for determining whether a subject suffering from neutropenia is eligible for treatment with a SGLT2 inhibitor, for monitoring a neutropenia associated with an intracellular accumulation of l,5-AG-6-P and for monitoring the effectiveness of a treatment with a SGLT2 inhibitor.

BACKGROUND OF INVENTION

Neutropenia is defined as a reduction in the blood absolute neutrophils count (ANC). Said reduction may be due to the decreased production of white blood cells, the destruction of white blood cells or the marginalization, sequestration and/or migration of white blood cells. Neutrophils are key players of the immune system, notably involved in the defense against bacterial and fungal infections. Depending on its severity, neutropenia may thus increase the risk of bacterial and fungal infection in the affected subject.

Neutropenia is congenital or acquired, the latter including for example drug-induced neutropenia, post-infectious neutropenia, immune neutropenia, neutropenia due to a nutritional deficiency, neutropenia due to hypersplenism or hyperthyroidism, neutropenia due to diseases affecting the bone marrow, and chronic idiopathic neutropenia.

Congenital neutropenia with a monogenic inheritance is rare, and may be X-linked or autosomal, recessive or dominant. So far, mutations in 24 genes have been identified as being responsible for monogenic congenital neutropenia, with or without extra- hematopoietic manifestations (for a recent review, see Donadieu et al., 2017). Among these 24 genes, two encode transmembrane proteins of the endoplasmic reticulum that may play a role in glucose-6-phosphate (G6P) metabolism: the G6PC3 gene, encoding the catalytic unit of the ubiquitous glucose-6-phosphatase complex G6PC3; and the G6PT (or SLC37A4 ) gene, encoding the ubiquitous glucose-6-phosphate transporter (or translocase) G6PT. Neutrophils from patients with either G6PC3 deficiency or G6PT deficiency are characterized by a lower rate of glucose utilization and probably as a consequence of this, decreased respiratory burst, decreased protein glycosylation and increased endoplasmic reticulum stress (Hayee et al., 2011; Gautam el al., 2013; Kiykim et al., 2015). How G6PC3 and G6PT deficiency lead to these perturbations in the neutrophils is still unknown. It has been proposed that a lack of either of these two proteins could prevent the hydrolysis of G6P into glucose in the endoplasmic reticulum of neutrophils, and the subsequent release of said glucose in the cytoplasm, thereby decreasing the amount of cytoplasmic glucose to be converted into G6P available for glycolysis and for the pentose phosphate pathway (Jun et al., 2010). In particular, Jun el al. suggests that G6PT transports G6P in the endoplasmic reticulum, where G6PC3 hydrolyzes it, thus generating glucose. However, one would expect that blocking the transport and/or hydrolysis of G6P would on the contrary increase G6P availability for glycolysis and for the pentose-phosphate pathway. Thus, the pathophysiological mechanism involved in neutropenia linked to a G6PC3 deficiency or to a G6PT deficiency remains to be elucidated. Indeed, elucidating said mechanism may provide an optimal, specific treatment for neutropenia linked to a G6PC3 deficiency or to a G6PT deficiency or for any neutropenia with a similar pathophysiology.

Congenital neutropenia, including neutropenia linked to a G6PC3 deficiency or to a G6PT deficiency, is often treated with the hematopoietic growth factors G-CSF (granulocyte colony stimulating factor). G-CSF promotes granulopoiesis of hematopoietic stem cells to regenerate neutrophils. Treatment with G-CSF has been described to lead to an improvement in neutrophil numbers, a prevention of infections and an improvement of quality of life. However, in some patients G-CSF may fail to control infections even in large doses. Moreover, while tolerability of G-CSF during short-term use has been reported to be good or excellent, long-term use of G-CSF may induce adverse effects, such as thrombocytopenia, splenomegaly, spleen rupture, or osteoporosis (Donadieu et al., 2011). In patients with congenital neutropenia, G-CSF may increase the risk of leukemia (Donadieu et al., 2011).

Therefore, there is still a need for a well-tolerated and cost-effective treatment for congenital neutropenia, and in particular for congenital neutropenia linked to a G6PC3 deficiency or to a G6PT deficiency and for any neutropenia with a similar pathophysiology.

The present invention thus relates to a SGLT2 (sodium glucose cotransporter 2) inhibitor for use in the treatment of neutropenia in a subject in need thereof, said subject having an elevated intracellular level of l,5-anhydroglucitol-6-phosphate (l,5-AG-6P). In particular, the present invention relates to a SGLT2 inhibitor for use in the treatment of neutropenia linked to a G6PC3 deficiency or to a G6PT deficiency. The present invention also relates to methods for determining whether a subject suffering from neutropenia is eligible for treatment with a SGLT2 inhibitor, for monitoring a neutropenia associated with an intracellular accumulation of l,5-AG-6P and for monitoring the effectiveness of a treatment with a SGLT2 inhibitor.

SUMMARY

The present invention relates to a SGLT2 (sodium glucose cotransporter 2) inhibitor for use in the treatment of neutropenia in a subject in need thereof, said subject having an elevated intracellular level of l,5-anhydroglucitol-6-phosphate.

According to one embodiment, said subject suffers from neutropenia linked to a deficiency of the glucose-6-phosphatase encoded by G6PC3 or to a deficiency of the glucose-6-phosphate transporter encoded by G6PT, also known as SLC37A4. In one embodiment, the neutropenia linked to a deficiency of the glucose-6-phosphatase encoded by G6PC3 is a congenital neutropenia selected from the group comprising severe congenital neutropenia type 4 (SCN4) and Dursun syndrome. In one embodiment, the neutropenia linked to a deficiency of the glucose-6-phosphate transporter encoded by G6PT, also known as SLC37A4, is one of the symptoms of the congenital glycogen storage disease type lb. In one embodiment, the neutropenia linked to a deficiency of the glucose-6-phosphatase encoded by G6PC3 or to a deficiency of the glucose-6-phosphate transporter encoded by G6PT, also known as SLC37A4, is drug-induced.

In one embodiment, the SGLT2 inhibitor for use according to the invention is selected from the group comprising empagliflozin, dapagliflozin, canagliflozin, ipragliflozin, ertugliflozin, luseogliflozin, bexagliflozin, tofogliflozin, henagliflozin, sotagliflozin, remogliflozin, sergliflozin and atigliflozin, and any combination thereof, preferably said gliflozin is empagliflozin. In one embodiment, the SGLT2 inhibitor for use according to the invention is selected from the group comprising or consisting of empagliflozin, dapagliflozin, canagliflozin, ipragliflozin, ertugliflozin, and any combination thereof. In one embodiment, the SGLT2 inhibitor for use according to the invention is empagliflozin.

In one embodiment, said SGLT2 inhibitor is to be administrated at a dose ranging from about 0.015 mg per kilo body weight per day (mg/kg/day) to about 4.5 mg/kg/day, preferably at a dose ranging from about 0.035 mg/kg/day to about 1.5 mg/kg/day. In one embodiment, said SGLT2 inhibitor is to be administrated once a day for at least 4 days and subsequently once a week.

The present invention also relates to a method for determining whether a subject suffering from neutropenia is eligible for treatment with a SGLT2 inhibitor, said method comprising measuring the level of l,5-anhydroglucitol-6-phosphate in a biological sample obtained from the subject. In one embodiment, the level of l,5-anhydroglucitol- 6-phosphate in a biological sample obtained from the subject suffering from neutropenia is compared to a reference level. In one embodiment, a subject suffering from neutropenia with a level of l,5-anhydroglucitol-6-phosphate higher than the reference level is determined to be eligible for treatment with a SGLT2 inhibitor.

The present invention also relates to a method for monitoring neutropenia associated with an intracellular accumulation of l,5-anhydroglucitol-6-phosphate in a subject, said method comprising measuring the level of l,5-anhydroglucitol in a biological sample obtained from the subject.

The present invention further relates to a method for monitoring the effectiveness of a SGLT2 inhibitor therapy administered to a subject suffering from neutropenia associated with an intracellular accumulation of l,5-anhydroglucitol-6-phosphate, said method comprising measuring the level of l,5-anhydroglucitol in a biological sample obtained from the subject. In one embodiment, the level of l,5-anhydroglucitol in a biological sample obtained from the subject is compared to a personalized reference level of the subject. In one embodiment, said personalized reference level of the subject is the level of l,5-anhydroglucitol measured in a biological sample obtained from the subject before or at the beginning of the SGLT2 inhibitor therapy.

DEFINITIONS

In the present invention, the following terms have the following meanings:

“1,5-AG” or“AG” refer to l,5-anhydroglucitol (also called l,5-anhydro-D-glucitol or anhydroglucitol), a naturally occurring monosaccharide found in nearly all foods and in the body of mammals. The IUPAC name of l,5-AG is (2R,3S,4R,5S)-2- (hydroxymethyl)oxane-3,4,5-triol and its CAS number is 154-58-5. l,5-AG is structurally similar to D-glucose and its formula is:

“l,5-AG-6-P”,“1,5-AG6P” or“AG6P” refer to l,5-anhydroglucitol-6-phosphate (also called herein anhydroglucitol-6-phosphate), which results from the phosphorylation of l,5-anhydroglucitol. The IUPAC name of l,5-AG is [(2R,3S,4R,5S)-3,4,5-trihydroxyoxan-2-yl]methyl dihydrogen phosphate and its CAS number is 17659-59-5. l,5-AG-6-P is structurally similar to glucose-e- phosphate and its formula is:

“About” preceding a figure encompasses plus or minus 10%, or less, of the value of said figure. It is to be understood that the value to which the term“about” refers is itself also specifically, and preferably, disclosed.

“Absolute neutrophils count (ANC)” refers to the real number of white blood cells (WBC) that are neutrophils. The ANC is not measured directly but is derived by multiplying the WBC count times the percent of neutrophils in the differential WBC count. The percent of neutrophils in the differential WBC count consists of the segmented (fully mature) neutrophils and of the bands (almost mature neutrophils). In a human adult, the normal range for the ANC falls between 1.5 to 8.0 (1500 to 8000/mm³).

“Congenital” in the present invention refers to a disease, in particular neutropenia, or to an enzymatic deficiency, in particular a G6PC3 or G6PT deficiency, caused by a genetic mutation. For example, congenital G6PC3 deficiency refers to a G6PC3 deficiency caused by bi-allelic mutation of the gene G6PC3. Similarly, congenital G6PT deficiency refers to a G6PT deficiency caused by bi-allelic mutation of the gene G6PT (also known as SLC37A4).

“G6PC3 deficiency” refers to a deficiency of the ubiquitous glucose-6-phosphatase encoded by G6PC3, said phosphatase being commonly known as G6PC3, G6Pase-P or G6Pase 3. According to the present invention, a G6PC3 deficiency corresponds to a reduction, an absence or an inhibition of said phosphatase activity. In one embodiment, the G6PC3 deficiency is congenital. In another embodiment the G6PC3 deficiency is drug-induced.

“G6PT deficiency” refers to a deficiency of the glucose-6-phosphate transporter (or translocase) encoded by G6PT, also known as SLC37A4, said transporter being commonly known as G6PT. According to the present invention, a G6PT deficiency corresponds to a reduction, an absence or an inhibition of said transporter activity. In one embodiment, the G6PT deficiency is congenital. In another embodiment the G6PT deficiency is drug-induced. - “Intracellular level of l,5-anhydroglucitol-6-phosphate” may refer herein to the level of l,5-anhydroglucitol-6-phosphate (l,5-AG-6-P) measured in a sample obtained from a subject. Indeed, l,5-AG-6-P is produced within animal cells through the phosphorylation of l,5-AG. Conversely, l,5-AG-6-P is hydrolyzed into l,5-AG within animal cells. l,5-AG-6-P is not secreted from the cells. Thus, according to the present invention, a level of l,5-AG-6-P refers to an intracellular level of l,5-AG-6- P. In particular, a leukocyte level of l,5-AG-6-P refers to a level of l,5-AG-6-P in leukocytes. As neutrophils are the most prevalent circulating leukocytes, the leukocyte level of l,5-AG-6-P is considered in the present invention to reflect the neutrophil level of l,5-AG-6-P, i.e., the level of l,5-AG-6-P in neutrophils.

- “Neutropenia associated with an intracellular accumulation of 1,5- anhydroglucitol-6-phosphate” refers to a neutropenia that is caused by the accumulation of l,5-AG-6-P in the cells, in particular in the neutrophils, of the subject suffering from said neutropenia. In one embodiment, said neutropenia is linked to a G6PC3 deficiency or to a G6PT deficiency. As used herein, the terms“neutropenia associated with an intracellular accumulation of l,5-AG-6-P” encompass neutropenia linked to, caused by, or induced by an intracellular accumulation of l,5-AG-6-P. According to the present invention, one can assess whether a neutropenia is associated with an intracellular accumulation of l,5-AG-6-P by measuring the level or concentration of l,5-AG-6-P in the cells of a subject, in particular in the leukocytes (also called white blood cells) of a subject. Methods for determining the level or concentration of a metabolite in the leukocytes of a subject are well-known to the person skilled in the art. For example, the level or concentration of l,5-AG-6-P in the leukocytes of a subject can be determined by performing LC-MS analysis (liquid chromatography-mass spectrometry) on the huffy coat, i.e., the fraction containing most of the leukocytes and platelets, isolated from a blood sample obtained from the subject. Methods for isolating the buffy coat from a whole blood sample are routinely used in clinical laboratories.

“Neutrophils”, also commonly referred to“granulocytes”, refer to a specific type of leukocytes (or white blood cells) that primarily defend the organism against pathogen infections. Neutrophils constitute up to 70% of the circulating leukocytes. Segmented neutrophils are the most mature neutrophils present in circulating blood and have a lobulated chromatin-dense nucleus. Banded neutrophils (or bands) are slightly less mature than segmented neutrophils and have indented, unsegmented“C” or“S” shaped nuclei. - “Pharmaceutically acceptable excipient” refers to an excipient that does not produce an adverse, allergic or other untoward reaction when administered to a mammal, preferably a human. It includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. 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. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by the regulatory offices such as the FDA or EM A.

“Subject” refers to a mammal, preferably a human. In one embodiment, the subject is a patient, preferably a human patient, who/which is awaiting the receipt of, or is receiving, medical care or was/is/will be the subject of a medical procedure or is monitored for the development or progression of a disease. In one embodiment, the subject is a human patient who is treated and/or monitored for the development or progression of a neutropenia, preferably a neutropenia associated with an intracellular accumulation of l,5-AG-6-P as defined in the present invention. In one embodiment, the subject is a male. In another embodiment, the subject is a female. In one embodiment, the subject is an adult. In another embodiment, the subject is a child. According to the present invention, the subject has an elevated intracellular level of l,5-AG-6-P. In one embodiment, the subject has an elevated level of l,5-AG-6-P in leukocyte, i.e., an elevated leukocyte level of l,5-AG-6-P. In one embodiment, the subject has an elevated level of l,5-AG-6-P in neutrophils, i.e., an elevated neutrophil level of l,5-AG-6-P. In one embodiment, the subject is suffering from a deficiency of the glucose-6-phosphatase encoded by G6PC3 and/or from a deficiency of the glucose-6-phosphate transporter encoded by G6PT, also known as SLC37A4. Thus, in one embodiment, the subject is suffering from a G6PC3 deficiency and/or from a G6PT deficiency. In a particular embodiment, the subject is suffering from a congenital G6PC3 deficiency or from a congenital G6PT deficiency. In one embodiment, the subject is not suffering from diabetes, i.e., the subject is neither suffering from type 1 diabetes nor from type 2 diabetes. Thus, in one embodiment, the subject is not diabetic. - “Therapeutically effective amount” or“therapeutically effective dose” refer to the amount or concentration of a SGLT2 inhibitor according to the invention that is aimed at, without causing significant negative or adverse side effects to the subject, (1) delaying or preventing neutropenia associated with an intracellular accumulation of l,5-AG-6-P; (2) reducing the severity or incidence of neutropenia associated with an intracellular accumulation of l,5-AG-6-P; (3) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of neutropenia associated with an intracellular accumulation of l,5-AG-6-P; or (4) bringing about ameliorations of the symptoms of neutropenia associated with an intracellular accumulation of l,5-AG-6-P. - “Treating” or“Treatment” refers to therapeutic treatment, to prophylactic (or preventative) measures, or to both, wherein the object is to prevent or slow down (lessen) the development of neutropenia, in particular of neutropenia associated with an intracellular accumulation of l,5-AG-6-P as defined in the present invention. Those in need of treatment include those already suffering from said neutropenia, as well as those susceptible to develop said neutropenia, or those in whom said neutropenia is to be prevented. A subject is successfully“treated” for neutropenia, in particular for neutropenia associated with an intracellular accumulation of l,5-AG-6- P, if, after receiving a therapeutic amount of a SGLT2 inhibitor according to the present invention, the subject shows one or more of the following:

o a decrease of the blood level of 1,5- AG, preferably a decrease of the serum or plasma level of l,5-AG; o a decrease of the intracellular level of l,5-AG-6-P;

o an increase of the absolute neutrophil count;

o a decrease of the susceptibility to bacterial and fungal infections.

DETAILED DESCRIPTION

The present invention relates to a SGLT2 (sodium glucose cotransporter 2) inhibitor for use in the treatment of neutropenia in a subject in need thereof, said subject having an elevated intracellular level of l,5-anhydroglucitol-6-phosphate (l,5-AG-6-P). The present invention thus relates to a SGLT2 inhibitor for use in the treatment of neutropenia associated with an intracellular accumulation of l,5-AG-6-P in a subject in need thereof.

The Applicant surprisingly found that an intracellular accumulation of l,5-AG-6-P occurs in subjects suffering from a deficiency of the glucose-6-phosphatase G6PC3 or of the glucose-6-phosphate transporter G6PT. Without wishing to be bound by any theory, the Applicant suggests that said intracellular accumulation of l,5-AG-6-P is responsible for the neutrophil dysfunction and neutropenia observed in subjects suffering from a G6PC3 deficiency or a G6PT deficiency. According to the present invention, neutropenia associated with an intracellular accumulation of l,5-AG-6-P thus refers to a neutropenia that is caused by the accumulation of l,5-AG-6-P in the cells, in particular in the neutrophils, of the subject suffering from said neutropenia. Subsequently, the Applicant surprisingly showed that a SGLT2 inhibitor, for example empagliflozin, can be used to treat a neutropenia associated with an intracellular accumulation of l,5-AG-6-P, and in particular a neutropenia linked to a G6PC3 deficiency or a G6PT deficiency.

Sodium-glucose co-transporter 2 (SGLT2) inhibitors correspond to a well-known class of compounds initially developed for the treatment of type 2 diabetes. SGLT2 is a low- affinity, high capacity glucose transporter located in the proximal tubule in the kidneys. Sodium-glucose co-transporter 2 (SGLT2) is responsible for 90% of glucose reabsorption. SGLT2 inhibitors block the reabsorption of glucose in the kidney, increase glucose excretion, and thus lower blood glucose levels. Administration of SGLT2 inhibitors to type 2 diabetes patients thus improves the glycemic control in said patients. As used herein, the term“SGLT2 inhibitor” relates to a compound which shows an inhibitory effect on SGLT2, in particular on human SGLT2. The inhibitory effect on SGLT2, preferably hSGLT2, can be determined by methods well-known in the art, for example, such as described in WO 2007/093610 (reference is made to the description from page 23, line 2 to page 24 line 10) or in WO 2010/023594 (reference is made to the description from page 109, line 8 to page 110 line 9).

In the present invention, the term "SGLT2 inhibitor" encompasses any prodrugs, pharmaceutically acceptable salts, hydrates and solvates thereof. The term“SGLT2 inhibitor” also encompasses the crystalline forms of said inhibitor. According to one embodiment of the present invention, SGLT2 inhibitors are gliflozins. Thus, according to one embodiment, the SGLT2 inhibitor for use according to the invention is a gliflozin. In one embodiment, the present invention relates to a gliflozin for use in the treatment of neutropenia in a subject in need thereof, said subject having an elevated intracellular level of l,5-AG-6-P. A gliflozin may for example be represented by the following formula, or a pharmaceutically acceptable salt, hydrate or solvate thereof:

wherein

Y represents O or S;

R¹ represents cyano, halo, hydroxy, Ci-₆-alkyl, Ci-₆-alkoxy or 0-C_3-7-cycloalkyl; R² represents hydrogen, halo, hydroxy, Ci-₆-alkyl or Ci-₆-alkoxy;

n is 1 or 2; Ar represents an aryl or heteroaryl such as for example phenyl, thienyl, or benzothienyl;

wherein the aryl or heteroaryl is optionally substituted by one or more group such as for example cyano, halo, hydroxy, ethinyl, trimethylsilyl, Ci-₆-alkyl, C3-7-cycloalkyl, aryl, heteroaryl, C3-7-heterocyeloalkyl, Ci-₆-alkoxy, OR’,

O-Ci-₆-alkyl-OR’ or 0-C_3-7-cycloalkyl-OR’; wherein R’ is selected from Ci-₆-alkyl, C3-7-cycloalkyl and oxa-C3-7-cycloalkyl;

wherein the alkyl, cycloalkyl, aryl, heteroaryl or alkoxy group is optionally substituted by one or more further substituent selected from cyano, halo and hydroxy.

Examples of gliflozins include, without being limited to, empagliflozin, dapagliflozin, canagliflozin, ipragliflozin, ertugliflozin, luseogliflozin, bexagliflozin, tofogliflozin, henagliflozin, sotagliflozin, remogliflozin, sergliflozin and atigliflozin.

The formulae of the above listed gliflozins are presented in Table 1 below.

In one embodiment, the SGLT2 inhibitor for use according to the invention is selected from the group comprising empagliflozin, dapagliflozin, canagliflozin, ipragliflozin, ertugliflozin, luseogliflozin, bexagliflozin, tofogliflozin, henagliflozin, sotagliflozin, remogliflozin, sergliflozin, atigliflozin, and any combination thereof. In one embodiment, the SGLT2 inhibitor for use according to the invention is selected from the group consisting of empagliflozin, dapagliflozin, canagliflozin, ipragliflozin, ertugliflozin, luseogliflozin, bexagliflozin, tofogliflozin, henagliflozin, sotagliflozin, remogliflozin, sergliflozin, atigliflozin, and any combination thereof.

In one embodiment, the SGLT2 inhibitor for use according to the invention is selected from the group comprising empagliflozin, dapagliflozin, canagliflozin, ipragliflozin, ertugliflozin, luseogliflozin, bexagliflozin, tofogliflozin, henagliflozin, sotagliflozin, remogliflozin, and any combination thereof. In one embodiment, the SGLT2 inhibitor for use according to the invention is selected from the group consisting of empagliflozin, dapagliflozin, canagliflozin, ipragliflozin, ertugliflozin, luseogliflozin, bexagliflozin, tofogliflozin, henagliflozin, sotagliflozin, remogliflozin, and any combination thereof.

In one embodiment, the SGLT2 inhibitor for use according to the invention is selected from the group comprising empagliflozin, dapagliflozin, canagliflozin, ipragliflozin, ertugliflozin, and any combination thereof. In one embodiment, the SGLT2 inhibitor for use according to the invention is selected from the group consisting of empagliflozin, dapagliflozin, canagliflozin, ipragliflozin, ertugliflozin, and any combination thereof.

In one embodiment, the SGLT2 inhibitor for use according to the invention is empagliflozin or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a crystalline form thereof. As used herein, the term“empagliflozin” encompasses prodrugs of empagliflozin.

Thus, in one embodiment, the present invention relates to empagliflozin for use in the treatment of neutropenia in a subject in need thereof, said subject having an elevated intracellular level of l,5-anhydroglucitol-6-phosphate. Empagliflozin (CAS number 864070-44-0) is also known as (2S,3R,4R,5S,6R)-2-[4- chloro-3-({4-[(3S)-oxolan-3-yloxy]phenyl}methyl)phenyl]-6-(hydroxymethyl)oxane- 3,4,5-triol. Empagliflozin is available under the trade name Jardiance®. In one embodiment, the SGLT2 inhibitor for use according to the invention is dapagliflozin or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a crystalline form thereof. As used herein, the term“dapagliflozin” encompasses prodrugs of dapagliflozin. Dapagliflozin (CAS number 461432-26-8) is also known as (2S,3R,4R,5S,6R)-2-(4- Chloro-3-(4-ethoxybenzyl)phenyl)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol. Dapagliflozin is available under the trade names Forxiga®, Farxiga®, and Edistride®.

In one embodiment, the SGLT2 inhibitor for use according to the invention is canagliflozin or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a crystalline form thereof. As used herein, the term“canagliflozin” encompasses prodrugs of canagliflozin.

Canagliflozin (CAS number 842133-18-0) is also known as (2S,3R,4R,5S,6R)-2-(3-{ [5- (4-fluorophenyl)thiophen-2-yl]methyl}-4-methylphenyl)-6-(hydroxymethyl)oxane- 3,4,5-triol. Canagliflozin is available under the trade name Invokana®. In one embodiment, the SGLT2 inhibitor for use according to the invention is ipragliflozin or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a crystalline form thereof. As used herein, the term“ipragliflozin” encompasses prodrugs of ipragliflozin.

Ipragliflozin (CAS number 761423-87-4) is also known as (2S,3R,4R,5S,6R)-2-{3-[(l- benzothiophen-2-yl)methyl]-4-fluorophenyl}-6-(hydroxymethyl)oxane-3,4,5-triol. Ipragliflozin is available under the trade name Steglatro®.

In one embodiment, the SGLT2 inhibitor for use according to the invention is ertugliflozin or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a crystalline form thereof. As used herein, the term“ertugliflozin” encompasses prodrugs of ertugliflozin.

Ertugliflozin (CAS number 1210344-57-2) is also known as (lS,2S,3S,4R,5S)-5-{4- chloro- 3 - [ (4-ethoxyphenyl)methyl] phenyl } - 1 - (hydroxymethyl) -6,8- dioxabicyclo[3.2.l]octane-2,3,4-triol. Ertugliflozin is available under the trade name Suglat®.

In one embodiment, the SGLT2 inhibitor for use according to the invention is luseogliflozin or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a crystalline form thereof. As used herein, the term“luseogliflozin” encompasses prodrugs of luseogliflozin.

Luseogliflozin (CAS number 898537-18-3) is also known as (2S,3R,4R,5S,6R)-2-{5-[(4- ethoxyphenyl)methyl]-2-methoxy-4-methylphenyl}-6-(hydroxymethyl)thiane-3,4,5- triol. In one embodiment, the SGLT2 inhibitor for use according to the invention is bexagliflozin or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a crystalline form thereof. As used herein, the term“bexagliflozin” encompasses prodrugs of bexagliflozin.

Bexagliflozin (CAS number 1118567-05-7) is also known as (2S,3R,4R,5S,6R)-2-(4- chloro- 3 - { [4- (2-cyclopropoxyethoxy)phenyl] methyl } phenyl) - 6- (hydroxymethyl) oxane-

3,4,5-triol.

In one embodiment, the SGLT2 inhibitor for use according to the invention is tofogliflozin or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a crystalline form thereof. As used herein, the term“tofogliflozin” encompasses prodrugs of tofogliflozin.

Tofogliflozin (CAS number 903565-83-3) is also known as (lS,3'R,4'S,5'S,6'R)-6-[(4- ethylphenyl)methyl]-6'-(hydroxymethyl)-3H-spiro[2-benzofuran-l,2'-oxane]-3',4',5'- triol.

In one embodiment, the SGLT2 inhibitor for use according to the invention is henagliflozin or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a crystalline form thereof. As used herein, the term“henagliflozin” encompasses prodrugs of henagliflozin. Henagliflozin (CAS number 1623804-44-3) is also known as (lR,2S,3S,4R,5R)-5-{4- chloro-3-[(4-ethoxy-3-fluorophenyl)methyl]phenyl}-l-(hydroxymethyl)-6,8- dioxabicyclo[3.2.l]octane-2,3,4-triol.

In one embodiment, the SGLT2 inhibitor for use according to the invention is sotagliflozin or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a crystalline form thereof. As used herein, the term“sotagliflozin” encompasses prodrugs of sotagliflozin.

Sotagliflozin (CAS number 1018899-04-1) is also known as (2S,3R,4R,5S,6R)-2-{4- chloro-3-[(4-ethoxyphenyl)methyl]phenyl}-6-(methylsulfanyl)oxane-3,4,5-triol. In one embodiment, the SGLT2 inhibitor for use according to the invention is remogliflozin or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a crystalline form thereof. As used herein, the term“remogliflozin” encompasses prodrugs of remogliflozin, in particular remogliflozin etabonate.

Remogliflozin etabonate (CAS number 442201-24-3) is also known as ethyl [(2R,3S,4S,5R,6S)-3,4,5-trihydroxy-6-{ [5-methyl- l-(propan-2-yl)-4-{ [4-(propan-2- yloxy)phenyl]methyl}-lH-pyrazol-3-yl]oxy}oxan-2-yl]methyl carbonate.

In one embodiment, the SGLT2 inhibitor for use according to the invention is sergliflozin or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a crystalline form thereof. As used herein, the term“sergliflozin” encompasses prodrugs of sergliflozin, in particular sergliflozin etabonate.

Sergliflozin etabonate (CAS number 408504-26-7) is also known as ethyl [(2R,3S,4S,5R,6S)-3,4,5-trihydroxy-6-[2-[(4-methoxyphenyl)methyl]phenoxy]oxan-2- yl] methyl carbonate.

In one embodiment, the SGLT2 inhibitor for use according to the invention is atigliflozin or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a crystalline form thereof. As used herein, the term“atigliflozin” encompasses prodrugs of atigliflozin. Atigliflozin (CAS number 647834-15-9) is also known as (2R,3S,4S,5R,6S)-2- (hydroxymethyl)-6-[2-[(4-methoxyphenyl)methyl]thiophen-3-yl]oxyoxane-3,4,5-triol.

As demonstrated in the Examples hereinafter, the Applicant surprisingly showed that the administration of a SGLT2 inhibitor to a subject, such as, for example empagliflozin, leads to a decrease of the intracellular level of l,5-AG-6-P in said subject. Thus, one object of the present invention is the use of a SGLT2 inhibitor as described hereinabove for decreasing the intracellular level of l,5-AG-6-P in a subject.

Neutropenia is defined as a reduction in the blood absolute neutrophils count (ANC), often leading to an increased susceptibility of the affected subject to bacterial and fungal infections. The severity of neutropenia relates to the relative risk of infection and depends on the neutrophil count. Mild neutropenia is characterized by an absolute neutrophil count ranging from 1000 to 1500/mm³ in a human adult. Moderate neutropenia is characterized by an absolute neutrophil count ranging from 500 to 1000/mm³ in a human adult. Severe neutropenia is characterized by an absolute neutrophil count below 500/mm³ in a human adult or in a human child.

In one embodiment, the neutropenia to be treated according to the invention is a mild neutropenia. In another embodiment, the neutropenia to be treated according to the invention is a moderate neutropenia. In another embodiment, the neutropenia to be treated according to the invention is a severe neutropenia. As shown by the Applicant, neutropenia may be observed in a subject with an elevated intracellular level of l,5-anhydroglucitol-6-phosphate (l,5-AG-6-P). Thus, another object of the invention is a SGLT2 inhibitor as described hereinabove for use in the treatment of neutropenia in a subject in need thereof, said subject having an elevated intracellular level of l,5-AG-6-P. In one embodiment, the subject has an elevated level of l,5-AG-6-P in leukocyte, i.e., an elevated leukocyte level of l,5-AG-6-P. In one embodiment, the subject has an elevated level of l,5-AG-6-P in neutrophils, i.e., an elevated neutrophil level of l,5-AG-6-P.

Another object of the invention is a SGLT2 inhibitor as described hereinabove for use in the treatment of neutropenia in a subject in need thereof, wherein said subject is selected for treatment if the intracellular level of l,5-AG-6-P is elevated in a biological sample obtained from said subject.

Methods for determining the intracellular level of l,5-AG-6-P, in particular the level of l,5-AG-6-P in leukocytes reflecting the level of l,5-AG-6-P in neutrophils, are well- known in the art. For example, the level of l,5-AG-6-P in the leukocytes of a subject can be determined by performing LC-MS analysis (liquid chromatography-mass spectrometry) on the huffy coat, i.e., the fraction containing most of the leukocytes and platelets, isolated from a blood sample obtained from the subject. Methods for determining the concentration or level of a metabolite using LC-MS analysis are well- known to the person skilled in the art (see for example Coulier L et al, 2006). Alternatively, the level of l,5-AG-6-P in cells may also be determined by performing GC-MS analysis (gas chromatography-mass spectrometry) as described in Mizuno et al., 1995.

According to one embodiment, the elevated intracellular level of l,5-AG-6-P is reflected by an elevated serum or plasma level of l,5-AG-6-P. Without wishing to be bound by any theory, the Applicant suggests that upon death, neutrophils containing elevated levels of l,5-AG-6-P may burst and release l,5-AG-6-P in the blood. Thus, in one embodiment, the level of l,5-AG-6-P is determined on a serum or plasma sample obtained from the subject. According to one embodiment, to determine whether a subject has an elevated intracellular level of l,5-AG-6-P, the intracellular level of l,5-AG-6-P of the subject is compared to a predetermined intracellular level of l,5-AG-6-P.

As used herein, the term“predetermined intracellular level of l,5-AG-6-P” broadly encompasses any suitable reference levels which may be used as a basis for comparison with respect to the intracellular level of l,5-AG-6-P assessed in a subject to determine whether said subject has an elevated intracellular level of l,5-AG-6-P.

As used herein, a reference level can be relative to a number or value derived from population studies including for example, but without being limited to, such subjects having similar age range, or subjects in the same or similar ethnic group, or subjects having family histories of neutropenia. In one embodiment, the reference level is constructed using algorithms and other methods of statistical and structural classification.

In one embodiment, the reference level is derived from the measure of the intracellular level of 1 ,5- AG-6-P in one or more subjects who are substantially healthy. As used herein, a“substantially healthy subj ect” has not been previously diagnosed or identified as having or suffering from neutropenia, from a G6PC3 deficiency, from a G6PT deficiency and/or from any deficiency susceptible to affect the metabolism of 1,5- AG and/or l,5-AG-6-P.

In another embodiment, the reference level is derived from the measure of the intracellular level of l,5-AG-6-P in one or more subjects who are diagnosed or identified as having or suffering from neutropenia, from a G6PC3 deficiency, from a G6PT deficiency and/or from any deficiency susceptible to affect the metabolism of 1,5 -AG and/or l,5-AG-6-P.

According to one embodiment, in a subject, preferably a human subject, an elevated intracellular level of l,5-AG-6-P is an intracellular level of l,5-AG-6-P at least about 10- fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 75-fold, lOO-fold, 200-fold, 250- fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 750-fold, 800-fold, 900-fold, or 1000-fold higher than the reference level, preferably the reference level is derived from the measure of the intracellular level of l,5-AG-6-P in one or more subjects who are substantially healthy. In another embodiment, in a subject, preferably a human subject, an elevated intracellular level of l,5-AG-6-P is an intracellular level of l,5-AG-6-P at least about 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 75% higher than the reference level, preferably the reference level is derived from the measure of the intracellular level of l,5-AG-6-P in one or more subjects who are substantially healthy. In another embodiment, in a subject, preferably a human subject, an elevated intracellular level of l,5-AG-6-P is an intracellular level of l,5-AG-6-P greater than about 0.1 mM, 0.15 mM, 0.2 mM, or 0.25 mM of l,5-AG-6-P. In another embodiment, in a subject, preferably a human subject, an elevated intracellular level of l,5-AG-6-P is an intracellular level of l,5-AG-6-P at least about 10-fold higher than a reference level derived from the measure of the intracellular level of l,5-AG-6-P in one or more subjects who are substantially healthy and greater than about 0.1 mM of 1.5-AG-6-P.

As shown by the Applicant, in a subject, neutropenia may be associated with an intracellular accumulation of l,5-anhydroglucitol-6-phosphate (l,5-AG-6-P), in particular in neutrophils. Thus, another object of the invention is a SGLT2 inhibitor as described hereinabove for use in the treatment of neutropenia associated with an intracellular accumulation of l,5-AG-6-P in a subject in need thereof.

Methods for determining the intracellular level of l,5-AG-6-P, in particular the level of l,5-AG-6-P in leukocytes reflecting the level of l,5-AG-6-P in neutrophils are described hereinabove.

According to one embodiment, in a subject, preferably a human subject, an intracellular accumulation of l,5-AG-6-P corresponds to an intracellular level of l,5-AG-6-P that is at least about 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 75-fold, lOO-fold, 200-fold, 250-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 750-fold, 800-fold, 900-fold, or 1000-fold higher than the reference level as described hereinabove.

In another embodiment, in a subject, preferably a human subject, an intracellular accumulation of l,5-AG-6-P corresponds to an intracellular level of l,5-AG-6-P that is at least about 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 75% higher than the reference level as described hereinabove.

In one embodiment, in a subject, preferably a human subject, an intracellular accumulation of l,5-AG-6-P corresponds to an intracellular level of l,5-AG-6-P that is greater than about 0.1 mM, 0.15 mM, 0.2 mM, or 0.25 mM of l,5-AG-6-P.

In one embodiment, in a subject, preferably a human subject, an intracellular accumulation of l,5-AG-6-P corresponds to an intracellular level of l,5-AG-6-P that is at least about 10-fold higher than a reference level derived from the measure of the intracellular level of l,5-AG-6-P in one or more subjects who are substantially healthy and greater than about 0.1 mM of l,5-AG-6-P.

In one embodiment, a subject with an elevated intracellular level of l,5-AG-6-P is a subject suffering from neutropenia linked to a deficiency of the glucose-6-phosphatase G6PC3 and/or linked to a deficiency of the glucose-6-phosphate transporter G6PCT. Thus, in one embodiment, neutropenia associated with an intracellular accumulation of l,5-AG-6-P is linked to a G6PC3 deficiency and/or to a G6PT deficiency.

In one embodiment, the SGLT2 inhibitor as described hereinabove is for use in the treatment of neutropenia linked to a G6PC3 deficiency and/or to a G6PT deficiency. In one embodiment, the present invention relates to a SGLT2 inhibitor as described hereinabove for use in the treatment of neutropenia linked to a G6PC3 deficiency and/or to a G6PT deficiency. In a particular embodiment, the present invention relates to empagliflozin for use in the treatment of neutropenia linked to a G6PC3 deficiency and/or to a G6PT deficiency. In one embodiment, the SGLT2 inhibitor as described hereinabove is for use in the treatment of neutropenia linked to a G6PC3 deficiency or to a G6PT deficiency wherein said neutropenia is congenital. In one embodiment, said congenital neutropenia is selected from the group comprising or consisting of severe congenital neutropenia type 4 (SCN4) Dursun syndrome and glycogen storage disease type lb. In one embodiment, a subject with an elevated intracellular level of l,5-AG-6-P is a subject suffering from neutropenia linked to a G6PC3 deficiency, in particular to a congenital G6PC3 deficiency. Thus, in one embodiment, neutropenia associated with an intracellular accumulation of l,5-AG-6-P is linked to a G6PC3 deficiency, in particular to a congenital G6PC3 deficiency. The G6PC3 gene encodes the catalytic unit of the ubiquitous glucose-6-phosphatase complex, G6Pase-P or G6Pase 3 or G6PC3. The presence of bi-allelic mutations in G6PC3 (also referred to as deficiency in G6PC3 or G6PC3 deficiency) is responsible for a multi-system disorder autosomal recessive disorder also called severe congenital neutropenia type 4 (SCN4), or Dursun syndrome. Congenital G6PC3 deficiency (reference OMIM® 612541) is characterized by severe congenital neutropenia, recurrent bacterial infections, intermittent thrombocytopenia in many patients, a prominent superficial venous pattern and a high incidence of congenital cardiac defect and uro genital anomalies (Banka and Newman, 2013; Chou et al., 2014). In one embodiment, the SGLT2 inhibitor as described hereinabove is for use in the treatment of neutropenia linked to a G6PC3 deficiency, wherein said neutropenia is a congenital neutropenia selected from the group comprising or consisting of severe congenital neutropenia type 4 (SCN4) and Dursun syndrome.

In one embodiment, a subject with an elevated intracellular level of l,5-AG-6-P is a subject suffering from neutropenia linked to a G6PT deficiency, in particular to a congenital G6PT deficiency. Thus, in one embodiment, neutropenia associated with an intracellular accumulation of l,5-AG-6-P is linked to a G6PT deficiency, in particular to a congenital G6PT deficiency.

The G6PT (or SLC37A4 ) gene encodes the ubiquitous glucose-6-phosphate transporter, also called glucose-6-phosphate translocase or G6PT. The presence of bi-allelic mutations in G6PT is responsible for a deficiency in G6PT (or G6PT deficiency) causing a multi-system autosomal recessive disorder also referred to as glycogen storage disease type lb. Congenital G6PT deficiency (reference OMIM® 602671) is characterized by an impaired glucose homeostasis, manifesting as an accumulation of glycogen in the liver and kidneys, hypoglycemia and lactic acidosis, and neutropenia. Thus, neutropenia is one of the symptoms of congenital glycogen storage disease type lb.

In one embodiment, the SGLT2 inhibitor as described hereinabove is for use in the treatment of neutropenia linked to a G6PT deficiency, wherein said neutropenia is glycogen storage disease type lb. In one embodiment, the SGLT2 inhibitor as described hereinabove is for use in the treatment of neutropenia linked to a G6PT deficiency, wherein said neutropenia is one of the symptoms of congenital glycogen storage disease type lb. In one embodiment, a subject with an elevated intracellular level of l,5-AG-6-P is a subject suffering from drug-induced neutropenia. Thus, in one embodiment, neutropenia associated with an intracellular accumulation of l,5-AG-6-P is drug-induced.

In one embodiment, the SGLT2 inhibitor as described hereinabove is for use in the treatment of neutropenia as described hereinabove, said neutropenia being drug-induced.

In one embodiment, the SGLT2 inhibitor as described hereinabove is for use in the treatment of neutropenia linked to a G6PC3 deficiency and/or to a G6PT deficiency as described hereinabove, said neutropenia being drug-induced. In other words, in one embodiment, the neutropenia to be treated with a SGLT2 inhibitor according to the invention is caused by a drug-induced inhibition of the glucose-6-phosphatase G6PC3 or of the glucose-6-phosphate transporter G6PT.

Drugs that may induce neutropenia in a subject include both chemotherapeutic agents and non-chemotherapeutics agents.

Examples of chemotherapeutic agents that may induce neutropenia in a subject include, without being limited to, alkylating agents, anthracyclines, antimetabolites, camptothecins, epipodophyllotoxins, hydroxyurea, mitomycin C, taxanes, and vinblastine.

Examples of non-chemotherapeutic agents that may induce neutropenia in a subject include, without being limited to, carbimazole, chlorpromazine, clozapine, dapsone, hydroxychloroquine, flecainide, indomethacin, infliximab, lamotrigine, methimazole, oxacillin, penicillin G, phenytoin, procainamide, propylthiouracil, quinidine/quinine, rituximab, sulfasalazine, ticlodipine, trimethoprim-sulfamethoxazole (cotrimoxazole), and vancomycin.

Another object of the present invention is a method for treating neutropenia in a subject in need thereof, said method comprising administering a SGLT2 inhibitor as described hereinabove to the subject, said subject having an elevated intracellular level of 1,5- anhydroglucitol-6-phosphate. The present invention also relates to a method for treating neutropenia associated with an intracellular accumulation of l,5-AG-6-P as described hereinabove, in a subject in need thereof, said method comprising administering a SGLT2 inhibitor as described hereinabove to the subject. In one embodiment, the present invention relates to a method for treating neutropenia linked to a G6PC3 deficiency or to a G6PT deficiency in a subject in need thereof, said method comprising administering a SGLT2 inhibitor as described hereinabove to the subject. In a particular embodiment, the present invention relates to a method for treating neutropenia linked to a G6PC3 deficiency or to a G6PT deficiency in a subject in need thereof, said method comprising administering empagliflozin to the subject.

Another object of the invention is a method for decreasing the intracellular level of 1,5- AG-6-P in a subject in need thereof, said method comprising administering a SGLT2 inhibitor as described hereinabove to the subject.

Another object of the present invention is the use of a SGLT2 inhibitor as described hereinabove for the manufacture of a medicament for the treatment of neutropenia in a subject in need thereof, said subject having an elevated intracellular level of 1,5- anhydroglucitol-6-phosphate as described hereinabove. The present invention also relates to the use of a SGLT2 inhibitor as described hereinabove for the manufacture of a medicament for the treatment of neutropenia associated with an intracellular accumulation of l,5-AG-6-P as described hereinabove. In one embodiment, the present invention relates to the use of a SGLT2 inhibitor as described hereinabove for the manufacture of a medicament for the treatment of neutropenia linked to a G6PC3 deficiency or to a G6PT deficiency. In a particular embodiment, the present invention relates to the use of empagliflozin for the manufacture of a medicament for the treatment of neutropenia linked to a G6PC3 deficiency or to a G6PT deficiency.

Another object of the present invention is a pharmaceutical composition comprising a SGLT2 inhibitor as described hereinabove, and at least one pharmaceutically acceptable excipient, for use in the treatment of neutropenia as described hereinabove. In one embodiment, the present invention relates to a pharmaceutical composition comprising empagliflozin, and at least one pharmaceutically acceptable excipient, for use in the treatment of neutropenia as described hereinabove. Pharmaceutically acceptable excipients that may be used in the pharmaceutical composition of the invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances (for example sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene- polyoxypropylene- block polymers, polyethylene glycol and wool fat.

Another object of the invention is a medicament comprising a SGLT2 inhibitor or a pharmaceutical composition as described hereinabove, for use in the treatment of neutropenia as described hereinabove. In one embodiment, the present invention relates to a medicament comprising empagliflozin for use in the treatment of neutropenia as described hereinabove.

According to one embodiment, the SGLT2 inhibitor, pharmaceutical composition or medicament according to the invention will be formulated for administration to the subject. The SGLT2 inhibitor, pharmaceutical composition or medicament according to the invention may be administered orally, parenterally, topically, by inhalation spray, rectally, nasally, buccally, vaginally or via an implanted reservoir.

In one embodiment, the SGLT2 inhibitor, pharmaceutical composition or medicament according to the invention is in a form adapted for oral administration. Thus, in one embodiment, the SGLT2 inhibitor, pharmaceutical composition or medicament according to the invention is to be administered orally to the subject. Examples of forms adapted for oral administration include, without being limited to, liquid, paste or solid compositions, and more particularly tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like.

In one embodiment, the SGLT2 inhibitor, pharmaceutical composition or medicament according to the invention is in a form adapted for rectal administration. Thus, in one embodiment, the SGLT2 inhibitor, pharmaceutical composition or medicament according to the invention is to be administered rectally.

Examples of forms adapted for rectal administration include, without being limited to, suppository, micro enemas, enemas, gel, rectal foam, cream, ointment, and the like. In one embodiment, the SGLT2 inhibitor, pharmaceutical composition or medicament according to the invention is in a form adapted for topical administration. Thus, in one embodiment, the SGLT2 inhibitor, pharmaceutical composition or medicament according to the invention is to be administered topically to the subject.

Examples of forms adapted for topical administration include, without being limited to, liquid, paste or solid compositions, and more particularly aqueous solutions, drops, dispersions, sprays, microcapsules, micro- or nanoparticles, polymeric patch, or controlled-release patch, and the like.

In one embodiment, the SGLT2 inhibitor, pharmaceutical composition or medicament according to the invention is in a form adapted for parenteral administration. Thus, in one embodiment, the SGLT2 inhibitor, pharmaceutical composition or medicament according to the invention is to be administered parenterally.

In one embodiment, the SGLT2 inhibitor, pharmaceutical composition or medicament according to the invention is in a form adapted for injection, such as, for example, for intravenous, subcutaneous, intramuscular, intradermal, transdermal injection or infusion. Thus, in one embodiment, the SGLT2 inhibitor, pharmaceutical composition or medicament according to the invention is to be administered by injection to the subject, such as, for example, by intravenous, subcutaneous, intramuscular, intradermal, transdermal injection or infusion.

Sterile injectable forms of the SGLT2 inhibitor, pharmaceutical composition or medicament according to the invention may be a solution or an aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non- toxic pharmaceutically acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation. In one embodiment, the pharmaceutical composition or the medicament as described hereinabove for use according to the invention comprises a therapeutically effective amount of a SGLT2 inhibitor as described hereinabove. In a particular embodiment, the pharmaceutical composition or the medicament as described hereinabove for use according to the invention comprises a therapeutically effective amount of empagliflozin. In one embodiment, the SGLT2 inhibitor as described hereinabove for use according to the invention is to be administered at a daily dose ranging from about 1 mg to about 300 mg, preferably at a daily dose ranging from about 2.5 mg to about 100 mg. In another embodiment, the SGLT2 inhibitor as described hereinabove for use according to the invention is to be administered at a dose ranging from about 0.015 mg per kilo body weight per day (mg/kg/day) to about 4.5 mg/kg/day, preferably at a dose ranging from about 0.035 mg/kg/day to about 1.5 mg/kg/day.

In one embodiment, the SGLT2 inhibitor as described hereinabove for use according to the invention is to be administered at a daily dose of about 2.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, or 300 mg. In another embodiment, the SGLT2 inhibitor as described hereinabove for use according to the invention is to be administered at a dose of about 0.035, 0.07, 0.15, 0.2, 0.28, 0.35, 0.42, 0.55, 0.7, 0.85, 1, 1.07, 1.15, 1.28, 1.45, 1.78, 2.15, 2.5, 2.85, 3.20, 3.57, 3.92, or 4.5 mg/kg/day. It will be understood that the total daily usage of the SGLT2 inhibitor according to the invention will be decided by the attending physician within the scope of sound medical judgment. The specific dose for any particular patient will depend upon a variety of factors such as the severity of the neutropenia to be treated; the specific SGLT2 inhibitor employed, the age, body weight, general health, sex and diet of the patient; and like factors well-known in the medical arts.

In one embodiment, the SGLT2 inhibitor for use according to the invention is empagliflozin and is to be administered at a daily dose ranging from about 2.5 mg to about 25 mg, preferably at a daily dose ranging from about 5 mg to about 15 mg, more preferably at a daily dose of about 10 mg. In one embodiment, the SGLT2 inhibitor for use according to the invention is empagliflozin and is to be administered at a dose ranging from about 0.035 mg/kg/day to about 0.35 mg/kg/day, preferably at a dose ranging from about 0.07 mg/kg/day to about 0.2 mg/kg/day, more preferably at a dose of about 0.15 mg/kg/day.

In another embodiment, the SGLT2 inhibitor for use according to the invention is empagliflozin and is to be administered at a daily dose of about 1 mg, 2 mg, 3 mg, 4mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, or 25 mg, preferably at a daily dose of about 10 mg.

In one embodiment, the SGLT2 inhibitor for use according to the invention is empagliflozin and is to be administered at a daily dose of less than about 15 mg, preferably less than about 12 mg, more preferably less than about 10 mg. In one embodiment, the SGLT2 inhibitor for use according to the invention is empagliflozin and is to be administered at a dose of less than about 0.2 mg/kg/day, preferably less than about 0.17 mg/kg/day, more preferably less than about 0.15 mg/kg/day. In one embodiment, the SGLT2 inhibitor, pharmaceutical composition or medicament according to the invention is to be administered to the subject in need thereof at least once a day, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days. For example, the SGLT2 inhibitor, pharmaceutical composition or medicament according to the invention may be administered once a day, twice a day, or three times a day, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days.

In another embodiment, the SGLT2 inhibitor, pharmaceutical composition or medicament according to the invention is to be administered to the subject in need thereof at least once a week. For example, the SGLT2 inhibitor, pharmaceutical composition or medicament according to the invention may be administered once a week, twice a week, three times a week, four times a week or up to seven times a week.

In another embodiment, the SGLT2 inhibitor, pharmaceutical composition or medicament according to the invention is to be administered to the subject in need thereof once a month, two times a month, every two months, every two or three months, two times a year or once a year.

In one embodiment, the SGLT2 inhibitor, pharmaceutical composition or medicament according to the invention is to be administered to the subject in need thereof at least once a day, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days, preferably for at least 4 days, and subsequently once a week. The present invention also relates to a method for determining whether a subject suffering from neutropenia is eligible for treatment with a SGLT2 inhibitor as described hereinabove, said method comprising measuring the level of l,5-anhydroglucitol-6- phosphate in a biological sample obtained from the subject.

Another object of the invention is a method for determining whether a subject suffering from neutropenia is susceptible to be a responder to a treatment with a SGLT2 inhibitor as described hereinabove, said method comprising measuring the level of l,5-AG-6-P in a biological sample obtained from the subject. Another object of the present invention is a method for determining whether a subject is a responder to a treatment with a SGLT2 inhibitor as described hereinabove, said method comprising measuring the level of l,5-AG-6-P in a biological sample obtained from the subject. Another object of the present invention is a method for determining the probability of response of a subject suffering from neutropenia to a treatment with a SGLT2 inhibitor as described hereinabove, said method comprising measuring the level of l,5-AG-6-P in a biological sample obtained from the subject.

Another object of the present invention is a method for assessing responsiveness to a treatment with a SGLT2 inhibitor as described hereinabove in a subject suffering from neutropenia, said method comprising measuring the level of l,5-AG-6-P in a biological sample obtained from the subject.

Another object of the present invention is a method for predicting whether a subject suffering from neutropenia will respond to treatment with a SGLT2 inhibitor as described hereinabove, said method comprising measuring the level of l,5-AG-6-P in a biological sample obtained from the subject.

According to the present invention, a subject suffering from neutropenia is a responder or responds to a treatment with a SGLT2 inhibitor as described hereinabove if the administration of said SGLT2 inhibitor to the subject results in a reduction or alleviation of at least one adverse effect or symptom of neutropenia in said subject.

In one embodiment, a subject suffering from neutropenia is a responder or responds to a treatment with a SGLT2 inhibitor as described hereinabove if the administration of said SGLT2 inhibitor to the subject results in a decrease of the blood level of l,5-AG of the subject, preferably a decrease of the serum or plasma level of 1,5 -AG of the subject. In another embodiment, a subject suffering from neutropenia is a responder or responds to a treatment with a SGLT2 inhibitor as described hereinabove if the administration of said SGLT2 inhibitor to the subject results in a decrease of the intracellular level of l,5-AG- 6-P of the subject. In another embodiment, a subject suffering from neutropenia is a responder or responds to a treatment with a SGLT2 inhibitor as described hereinabove if the administration of said SGLT2 inhibitor to the subject results in an increase of the absolute neutrophil count of said subject. In another embodiment, a subject suffering from neutropenia is a responder or responds to a treatment with a SGLT2 inhibitor as described hereinabove if the administration of said SGLT2 inhibitor to the subject results in a decrease of the susceptibility of said subject to bacterial and fungal infections.

Similarly, according to the present invention, a response to a SGLT2 inhibitor treatment as described hereinabove is defined as a reduction or alleviation of at least one adverse effect or symptom of neutropenia in said subject after administration of said SGLT2 inhibitor. In one embodiment, a response to a SGLT2 inhibitor treatment as described hereinabove is defined as a decrease of the blood level of l,5-AG of the subject, preferably a decrease of the serum or plasma level of 1,5- AG of the subject, a decrease of the intracellular level of l,5-AG-6-P of the subject, an increase of the absolute neutrophil count of said subject, and/or a decrease of the susceptibility of said subject to bacterial and fungal infections. Methods for determining the level of l,5-AG-6-P, i.e., the intracellular level of l,5-AG- 6-P, are described hereinabove. Methods for determining the blood level of l,5-AG are described hereinafter. Methods for assessing the absolute neutrophil count of a subject are routinely used in clinical laboratories.

According to the present invention, the methods of the invention as described hereinabove are carried out for a subject who is suffering from neutropenia. In one embodiment, the subject is suffering from congenital neutropenia. In another embodiment, the subject is suffering from drug-induced neutropenia. In one embodiment, the subject is suffering from mild, moderate or severe neutropenia.

In one embodiment, the methods of the invention as described hereinabove do not comprise obtaining a biological sample from a subject. In one embodiment, the biological sample of the subject is a sample previously obtained from the subject. Said biological sample may be conserved in adequate conditions before being used in the method of the invention. In one embodiment, the biological sample obtained from the subject is a body fluid sample. Examples of body fluids include, without being limited to, blood, plasma, serum, lymph, urine, cerebrospinal fluid or sweat.

In a preferred embodiment, the biological sample obtained from the subject is a blood sample. In one embodiment, the biological sample obtained from the subject is a whole blood sample. In one embodiment, the whole blood sample obtained from the subject is processed to obtain the buffy coat, i.e., the blood fraction containing most of the leukocytes and platelets. Methods for isolating the buffy coat from a whole blood sample are routinely used in clinical laboratories. Thus, in one embodiment, the level of l,5-AG- 6-P measured in a biological sample obtained from the subject is the leukocyte level of l,5-AG-6-P measured in a blood sample obtained from the subject.

According to one embodiment, the elevated intracellular level of l,5-AG-6-P is reflected by an elevated serum or plasma level of l,5-AG-6-P. Without wishing to be bound by any theory, the Applicant suggests that upon death, neutrophils containing elevated levels of l,5-AG-6-P may burst and release l,5-AG-6-P in the blood. In one embodiment, the whole blood sample obtained from the subject is processed to obtain a plasma sample or a serum sample. Methods for isolating the plasma sample or the serum sample from a whole blood sample are routinely used in clinical laboratories. Thus, in one embodiment, the level of l,5-AG-6-P measured in a biological sample obtained from the subject is the serum or plasma level of l,5-AG-6-P measured in a blood sample obtained from the subject.

As mentioned hereinabove, methods for determining the level of l,5-AG-6-P, i.e., the intracellular level of l,5-AG-6-P, in a biological sample are well-known in the art. For example, the level of l,5-AG-6-P in the leukocytes of a subject can be determined by performing LC-MS analysis (liquid chromatography-mass spectrometry) on the buffy coat, i.e., the fraction containing most of the leukocytes and platelets, isolated from a blood sample obtained from the subject. In one embodiment, the methods of the invention as described hereinabove comprise comparing the level of l,5-AG-6-P in a biological sample obtained from the subject to a reference level.

As mentioned hereinabove, a reference level can be relative to a number or value derived from population studies including for example, but without being limited to, such subjects having similar age range, or subjects in the same or similar ethnic group, or subjects having family histories of neutropenia. In one embodiment, the reference level is constructed using algorithms and other methods of statistical and structural classification.

In one embodiment, the reference level is derived from the measure of the intracellular level of l,5-AG-6-P in one or more subjects who are substantially healthy.

According to one embodiment, a subject suffering from neutropenia with a level of 1,5- AG-6-P higher than the reference level is determined to be eligible for treatment with a SGLT2 inhibitor as described hereinabove, is determined to be susceptible to be a responder to a treatment with a SGLT2 inhibitor as described hereinabove, is determined to be responder to a treatment with a SGLT2 inhibitor as described hereinabove, is determined to have significant probability of response to a treatment with a SGLT2 inhibitor, is determined to have a good responsiveness to a treatment with a SGLT2 inhibitor as described hereinabove, or is determined to respond to a treatment with a SGLT2 inhibitor as described hereinabove. According to one embodiment, a level of l,5-AG-6-P higher than the reference level is a level of l,5-AG-6-P at least about 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50- fold, 75-fold, lOO-fold, 200-fold, 250-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700- fold, 750-fold, 800-fold, 900-fold, or 1000-fold higher than the reference level, preferably the reference level is derived from the measure of the intracellular level of l,5-AG-6-P in one or more subjects who are substantially healthy.

In another embodiment, a level of l,5-AG-6-P higher than the reference level is a level of l,5-AG-6-P at least about 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 75% higher than the reference level, preferably the reference level is derived from the measure of the level of l,5-AG-6-P in one or more subjects who are substantially healthy.

In another embodiment, a level of l,5-AG-6-P higher than the reference level is a level of l,5-AG-6-P greater than about 0.1 mM, 0.15 mM, 0.2 mM, or 0.25 mM of l,5-AG- 6-P. In another embodiment, a level of l,5-AG-6-P higher than the reference level is a level of l,5-AG-6-P at least about 10-fold higher than a reference level derived from the measure of the level of l,5-AG-6-P in one or more subjects who are substantially healthy and greater than about 0.1 mM of l,5-AG-6-P.

The present invention thus relates to a method for identifying whether a subject is eligible for treatment with a SGLT2 inhibitor as described hereinabove, said method comprising the steps of:

a) measuring the leukocyte level of l,5-AG-6-P in a blood sample obtained from the subject;

b) comparing the leukocyte level of 1 ,5- AG-6-P in the blood sample obtained from the subject to a reference leukocyte level of l,5-AG-6-P, preferably to a reference level derived from the measure of the leukocyte level of l,5-AG-6-P in one or more subjects who are substantially healthy; and

c) identifying the subject as eligible for treatment with a SGLT2 inhibitor as described hereinabove if the leukocyte level of l,5-AG-6-P in the blood sample obtained from the subject is higher than the reference leukocyte level of 1,5- AG-6-P, preferably if the leukocyte level of l,5-AG-6-P in the blood sample obtained from the subject is at least about 10-fold higher than the reference leukocyte level of l,5-AG-6-P. The present invention also relates to a method for monitoring neutropenia associated with an intracellular accumulation of l,5-anhydroglucitol-6-phosphate in a subject, said method comprising measuring the level of l,5-anhydroglucitol in a biological sample obtained from the subject. As mentioned hereinabove, the intracellular 1 ,5- AG-6-P results from the phosphorylation of 1,5- AG in animal cells. Without wishing to be bound by any theory, the Applicant suggests that measuring the level of 1,5- AG in a biological sample obtained from a subject suffering from neutropenia associated with an intracellular accumulation of l,5-AG-6-P allows the monitoring of said neutropenia in the subject. According to the present invention, the method of the invention as described hereinabove is carried in a subject who is suffering from neutropenia associated with an intracellular accumulation of l,5-AG-6-P, i.e., in a subject having an elevated intracellular level of l,5-AG-6-P. In one embodiment, said neutropenia is linked to a G6PC3 deficiency or to a G6PT deficiency. In one embodiment, said neutropenia is congenital. In another embodiment, said neutropenia is drug-induced.

In one embodiment, the method of the invention as described hereinabove does not comprise obtaining a biological sample from a subject. In other words, in one embodiment, the method of the invention as described hereinabove does not comprise taking or collecting a biological sample from the subject, such as, for example, a blood sample. In one embodiment, the biological sample of the subject is a sample previously obtained from the subject. Said biological sample may be conserved in adequate conditions before being used in the method of the invention.

In one embodiment, the biological sample obtained from the subject is a body fluid sample. Examples of body fluids include, without being limited to, blood, plasma, serum, lymph, urine, cerebrospinal fluid or sweat.

In a preferred embodiment, the biological sample obtained from the subject is a blood sample. In one embodiment, the biological sample obtained from the subject is a whole blood sample. In a preferred embodiment, the biological sample obtained is a plasma sample or a serum sample. In one embodiment, the whole blood sample obtained from the subject is processed to obtain a plasma sample or a serum sample. Methods for isolating the plasma sample or the serum sample from a whole blood sample are routinely used in clinical laboratories.

Methods for determining the level of 1,5- AG in a biological sample, preferably in a blood sample, more preferably in a plasma or serum sample, are well-known in the art. For example, the level of 1,5- AG of a subject can be determined by performing LC-MS analysis (liquid chromatography-mass spectrometry) on a plasma sample or a serum sample obtained from the subject. Alternatively, the level of 1,5 -AG in a serum or plasma sample of a subject can be determined by using the l,5-anhydroglucitaol GLYCOMARK® assay (Diazyme Laboratories). Briefly, the l,5-anhydroglucitaol GLYCOMARK® assay is an enzymatic assay relying on the use of two enzymes: a pyranose oxidase (PROD) to oxidize the second position hydroxyl group of l,5-AG, and a peroxidase (POD) to detect the generated hydrogen peroxide by colorimetry.

According to one embodiment, the method of the invention as described hereinabove comprises repeating the measure of the level of 1,5- AG in a biological sample obtained from the subject.

In one embodiment, the measure of the level of 1,5- AG in a biological sample obtained from the subject is repeated at least once, at least twice, at least three times or more. In another embodiment, the measure of the level of l,5-AG in a biological sample obtained from the subject is repeated at regular intervals. In another embodiment, the measure of the level of 1,5- AG in a biological sample obtained from the subject is repeated every week, every two weeks, every three weeks or every four weeks. In another embodiment, the measure of the level of 1,5- AG in a biological sample obtained from the subject is repeated every month, every two months, every three months, every six months, every nine months or every twelve months. In another embodiment, the measure of the level of 1,5- AG in a biological sample obtained from the subject is repeated every year.

According to one embodiment, the method of the invention as described hereinabove comprises comparing the level of 1,5- AG in a biological sample obtained from the subject to a reference level. As mentioned hereinabove, a reference level can be relative to a number or value derived from population studies including for example, without being limited to, such subjects having similar age range, or subjects in the same or similar ethnic group, or subjects having family histories of neutropenia. In one embodiment, the reference level is constructed using algorithms and other methods of statistical and structural classification.

In one embodiment, the reference level is derived from the measure of the blood level of 1,5- AG in one or more subjects who are substantially healthy.

In another embodiment, the reference level is derived from the derived from the measure of the blood level of 1,5- AG in one or more subjects who are diagnosed or identified as having or suffering from neutropenia, from a G6PC3 deficiency, from a G6PT deficiency and/or from any deficiency susceptible to affect the metabolism of l,5-AG and/or 1,5- AG-6-P.

In one embodiment, the reference level is a personalized reference level. In one embodiment, said personalized reference level is a level of 1,5 -AG previously determined for the subject. In one embodiment, said personalized reference level is the first level of 1,5- AG determined for the subject.

The present invention thus relates to a method for monitoring neutropenia associated with an intracellular accumulation of l,5-AG-6-P in a subject, said method comprising the steps of:

a) measuring the level of l,5-anhydroglucitol in a plasma or serum sample obtained from the subject; and

b) comparing the level of l,5-AG in a plasma or serum sample obtained from the subject to a reference level of 1,5- AG, preferably said reference level of 1,5- AG is the first plasma or serum level of 1,5- AG determined for the subject. The present invention also relates to a method for monitoring the effectiveness of a SGLT2 (sodium glucose cotransporter 2) inhibitor therapy administered to a subject suffering from neutropenia associated with an intracellular accumulation of 1,5- anhydroglucitol-6-phosphate, said method comprising measuring the level of 1,5- anhydroglucitol in a biological sample obtained from the subject. As mentioned hereinabove, the intracellular l,5-AG-6-P results from the phosphorylation of 1,5- AG in animal cells. Without wishing to be bound by any theory, the Applicant suggests that measuring the level of 1,5- AG in a biological sample obtained from a subject suffering from neutropenia associated with an intracellular accumulation of l,5-AG-6-P allows the monitoring of the effectiveness of a SGLT2 inhibitor therapy administered to said subject.

According to the present invention, the method of the invention as described hereinabove is carried in a subject who is suffering from neutropenia associated with an intracellular accumulation of l,5-AG-6-P, i.e., in a subject having an elevated intracellular level of l,5-AG-6-P. In one embodiment, said neutropenia is linked to a G6PC3 deficiency or to a G6PT deficiency. In one embodiment, said neutropenia is congenital. In another embodiment, said neutropenia is drug-induced.

In one embodiment, the method of the invention as described hereinabove does not comprise obtaining a biological sample from a subject. In other words, in one embodiment, the method of the invention as described hereinabove does not comprise taking or collecting a biological sample from the subject, such as, for example, a blood sample. In one embodiment, the biological sample of the subject is a sample previously obtained from the subject. Said biological sample may be conserved in adequate conditions before being used in the method of the invention. In one embodiment, the biological sample obtained from the subject is a body fluid sample. Examples of body fluids include, without being limited to, blood, plasma, serum, lymph, urine, cerebrospinal fluid or sweat.

In one embodiment, the biological sample obtained from the subject is a urine sample. In a preferred embodiment, the biological sample obtained from the subject is a blood sample. In one embodiment, the biological sample obtained from the subject is a whole blood sample. In a preferred embodiment, the biological sample obtained is a plasma sample or a serum sample. In one embodiment, the whole blood sample obtained from the subject is processed to obtain a plasma sample or a serum sample. Methods for isolating the plasma sample or the serum sample from a whole blood sample are routinely used in clinical laboratories.

As mentioned hereinabove, methods for determining the level of l,5-AG in a biological sample, preferably in a blood sample, more preferably in a plasma or serum sample, are well-known in the art.

In one embodiment, the measure of the level of 1,5- AG in a biological sample obtained from the subject is repeated at least once, at least twice, at least three times or more. In a preferred embodiment, the level of 1,5- AG in a biological sample obtained from the subject is measured before or at the beginning of SGLT2 inhibitor therapy and at least once, at least twice, at least three times or more after the beginning of SGLT2 inhibitor therapy. In another embodiment, the level of 1,5- AG in a biological sample obtained from the subject is measured before or at the beginning of SGLT2 inhibitor therapy and is measured at regular intervals after the beginning of SGLT2 inhibitor therapy. In one embodiment, the measure of the level of 1,5- AG in a biological sample obtained from the subject is repeated every week, every two weeks, every three weeks or every four weeks after the beginning of SGLT2 inhibitor therapy. In another embodiment, the measure of the level of 1,5- AG in a biological sample obtained from the subject is repeated every month, every two months, every three months, every six months, every nine months or every twelve months after the beginning of SGLT2 inhibitor therapy. In another embodiment, the measure of the level of 1,5- AG in a biological sample obtained from the subject is repeated every year after the beginning of SGLT2 inhibitor therapy. According to one embodiment, the method of the invention as described hereinabove comprises comparing the level of 1,5- AG in a biological sample obtained from the subject to a reference level.

As mentioned hereinabove, a reference level can be relative to a number or value derived from population studies including for example, without being limited to, such subjects having similar age range, or subjects in the same or similar ethnic group, or subjects having family histories of neutropenia. In one embodiment, the reference level is constructed using algorithms and other methods of statistical and structural classification.

In one embodiment, the reference level is a personalized reference level. In one embodiment, said personalized reference level is a level of 1,5 -AG previously determined for the subject. In a preferred embodiment, said personalized reference level is the level of 1,5- AG measured in a biological sample obtained from the subject before or at the beginning of the SGLT2 inhibitor therapy.

According to one embodiment, a decrease of the level of 1,5- AG measured in a biological sample obtained from the subject after the beginning of the SGLT2 inhibitor therapy when compared to the level of l,5-AG measured in a biological sample obtained from the subject before or at the beginning of the SGLT2 inhibitor therapy indicates that said SGLT2 inhibitor therapy is effective. In one embodiment, a decrease of at least about 30%, 40%, 50%, 60%, 70%, 75% or 80%, preferably of at least about 50%, of the level of 1,5- AG measured in a biological sample obtained from the subject after the beginning of the SGLT2 inhibitor therapy when compared to the level of 1,5- AG measured in a biological sample obtained from the subject before or at the beginning of the SGLT2 inhibitor therapy indicates that said SGLT2 inhibitor therapy is effective.

The present invention thus relates to a method for monitoring the effectiveness of a SGLT2 inhibitor therapy administered to a subject suffering from neutropenia associated with an intracellular accumulation of l,5-AG-6-P as described hereinabove, said method comprising the steps of: a) measuring the level of l,5-anhydroglucitol in a plasma or serum obtained from the subject; and

b) comparing the level of l,5-AG in a plasma or serum sample obtained from the subject to a personalized reference level, preferably said personalized reference level is the level of 1,5 -AG measured in a plasma or serum sample obtained from the subject before or at the beginning of the SGLT2 inhibitor therapy.

The present invention relates to a SGLT2 inhibitor for use in the treatment of neutropenia associated with an intracellular accumulation of l,5-AG-6-P as described hereinabove.

Indeed, the Applicant demonstrated that a SGLT2 inhibitor can be used for the treatment of neutropenia linked to a G6PC3 deficiency or to a G6PT deficiency, in particular congenital neutropenia linked to a G6PC3 deficiency or to a G6PT deficiency.

Congenital neutropenia requires a long-term treatment, notably to minimize the risk of bacterial or fungal infection in the affected subjects. Currently, congenital neutropenia, including neutropenia linked to a G6PC3 deficiency or to a G6PT deficiency, is often treated with the hematopoietic growth factors G-CSF administered parenterally. The G- CSF administered to the subjects is a recombinant protein and its manufacture and parenteral administration are associated with a significant cost. Moreover, long-term administration of G-CSF can induce adverse effects, such as thrombocytopenia, splenomegaly, spleen rupture, or osteoporosis. Long-term administration of G-CSF is also suspected to increase the risk of leukemia.

By contrast, SGLT2 inhibitors are small synthetic compounds and their manufacture is associated with a lower cost. SGLT2 inhibitors such as empagliflozin, dapagliflozin, or canagliflozin are currently approved for the treatment of type 2 diabetes. The most frequently reported adverse effects are urogenital infections which tend to be mild to moderate and easily manageable with standard treatment.

Thus, the use of a SGLT2 inhibitor according to the present invention represents a cost- effective, well-adapted treatment for neutropenia associated with an intracellular accumulation of l,5-AG-6P, particularly neutropenia linked to a G6PC3 deficiency or to a G6PT deficiency. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A is a western blot showing wild type (WT) and mutated (H176A or H167A) human G6PC1 and G6PC3 expressed in HEK293T cells. A membrane fraction was isolated by centrifugation and analyzed (10 pg/well) by western blotting with anti-6xHis antibody. Figure 1B-C is a group of graphs showing phosphatase activity assays, carried out with the membrane preparations comprising G6PC1 (B) or G6PC3 (C) and with the indicated substrates at 100 pM. Controls were run with the mutated proteins (H176A G6PC1 and H167A G6PC3). G6P: glucose-6-phosphate, AG6P: l,5-anhydroglucitol-6- phosphate, Man6P: mannose-6-phosphate, R5P: ribose-5-phosphate, Rol5P: ribitol-5- phosphate, PPi: inorganic pyrophosphate.

Figure 2 is a group of graphs showing the glucose-6-phosphatase (G6Pase) and ribose- 5-phosphatase (R5Pase) activities of microsomes obtained from different rat tissues (A) and the effect of the G6PT inhibitor S3483 on the activity of the G6PC3 phosphatase in rat skeletal muscle microsomes (B). (A) Phosphatase activities in the microsomes derived from rat skeletal muscle, heart, spleen and liver were determined using 10 pM radiolabeled ribose-5-phosphate or glucose-6-phosphate as substrate. (B) The G6PC3 activity in skeletal muscle microsomes was measured with 100 pM of the indicated substrates (G6P: glucose-6-phosphate, l,5-AG6P: l,5-anhydroglucitol-6-phosphate, R5P: ribose-5-phosphate, Rol5P: ribitol- 5 -phosphate and PPi: inorganic pyrophosphate) in the presence or in the absence of the glucose-6-phosphate translocase (G6PT) inhibitor S3483 (100 pM), with or without 7.5 mM of the detergent octylglucoside (OG).

Figure 3 is a graph showing the outcome of the accumulation of l,5-anhydroglucitol-6- phosphate on glucose consumption, on depletion of glycolysis and pentose -phosphate pathway intermediates and on cell survival in G6PC3- or G6PT-deficient Hapl-cells in the presence of l,5-anhydroglucitol or its precursor l,5-anhydrofructose. (A and C) Intracellular concentration of l,5-anhydroglucitol-6-phosphate (AG6P) in HAP1 cells either wild-type (WT), G6PT-deficient (G6PT-A4 KO) or G6PC3-deficient (G6PC3-D7 KO and G6PC3-A6 KO), after incubation with the indicated concentration of 1,5- anhydroglucitol (AG) or l,5-anhydrofructose. (B and D) Glucose consumption in the indicated HAP1 cells after incubation with the indicated concentration of 1,5- AG or 1,5- AF. (E-J) Intracellular level of the indicated metabolites in the indicated HAP1 cells after incubation with the indicated concentration of l,5-AF. G6P: glucose-6-phosphate, F6P: fructose-6-phosphate, 6PG: 6-phosphogluconate, R5P: ribose-5-phosphate, and aKG: alpha-ketoglutarate. (K) Survival of the indicated HAP1 cells after incubation with the indicated concentration of l,5-AG or l,5-AF.

Figure 4 is a group of graphs showing the effect of l,5-anhydroglucitol (1,5- AG or AG) or its precursor l,5-anhydrofructose, on mouse wild-type neutrophil progenitors (WT) and on mouse neutrophil progenitors deficient in G6PC3 (G6PC3 KO). (A) The level of

1.5-anhydroglucitol-6-phosphate (l,5-AG6P) in the neutrophil progenitors was assessed after incubation with and without a physiological concentration of 1,5- AG. The results shown correspond to two experiments with two biological replicas in each of them; statistical significance estimated using the Holm-Sidak method, with alpha = 0.05; *** p = 0.0006. (B) The rate of glucose-6-phosphate production from glucose by the neutrophil progenitors was assessed after adding or not 0.2 mM of l,5-AG to the growth medium for the indicated time. The results shown are expressed as mean ± SEM for 3 different experiments; *** p < 0.001 for the comparison between G6PC3 KO and G6PC3 KO +

AG. (C) The survival of neutrophil progenitors was assessed after culturing the cells for 72h in growth medium containing the indicated concentration of l,5-anhydroglucitol or of l,5-anhydrofructose, a precursor of l,5-anhydroglucitol. Figure 5 is a group of graphs showing the kinase activity of recombinant human hexokinases HK1, HK2 and HK3 and of recombinant human ADP-GK. The kinase activities were assessed using 10 mM radiolabeled glucose (A) and 10 pM radiolabeled

1.5-anhydroglucitol (B) as substrates. ADP-GK is much better at phosphorylating 1,5- anhydroglucitol than HK1, HK2 and HK3. The results shown are expressed as means ± SD for 3 determinations.

Figure 6 is a group of graphs showing the levels of l,5-anhydroglucitol (AG) and 1,5- anhydroglucitol-6-phosphate (AG6P) in the indicated mouse tissues (brain, liver, kidney, white blood cells (WBC), heart, lung, spleen and pancreas). The levels of AG and AG6P were determined in the tissues of G6PC3 knockout mice (black) and in the tissues of control heterozygous mice (white). (A) Level of l,5-anhydroglucitol (AG) in the tissues of untreated mice. (B) Level of l,5-anhydroglucitol (AG) in the tissues of mice treated with l,5-anhydroglucitol (AG). (C) Level of l,5-anhydroglucitol-6-phosphate (AG6P) in the tissues of untreated mice. (D) Level of l,5-anhydroglucitol-6-phosphate (AG6P) in the tissues of mice treated with l,5-anhydroglucitol (AG). Figure 7 is a group of graphs showing the effect of a treatment of mice, either heterozygous control mice (heteroz) or G6PC3-deficient mice (KO), with 1,5- anhydroglucitol (1,5- AG or AG) or with empagliflozin (empa) on the l,5-anhydroglucitol (l,5-AG or AG) level in serum (A) or in plasma (B), on the neutrophil counts (C) and on the leukocyte l,5-anhydroglucitol-6-phosphate (l,5-AG6P) level (D). The experiment was repeated with a larger cohort of G6PC3-deficient mice (G6PC3 KO), and the effect of empagliflozin (empa) or 0.9% NaCl (control) was assessed on the plasma 1,5- anhydroglucitol (l,5-AG) level (E), on the neutrophil count (F), on the segmented neutrophil count (G) on the leukocyte l,5-anhydroglucitol-6-phosphate (l,5-AG6P) level (D), on the blood l,5-anhydroglucitol (I) and on Mac-l/Gr-l labeled granulocytes showed by flow cytometry of white blood cells analysis (J). A, B, C, D: white circle = untreated heterozygous control mice; black circle = untreated G6PC3-deficient mice; white square = heterozygous control mice treated with empagliflozin; black square = G6PC3-deficient mice treated with empagliflozin; white triangle = heterozygous control mice treated with l,5-AG; black triangle = G6PC3-deficient mice treated with l,5-AG. E, F, G, H: black circle = G6PC3-deficient mice treated with 0.9 % NaCl (control); black square = G6PC3-deficient mice treated with empagliflozin. Six mice per group. The results shown are expressed as mean values ± SD; individual values are also shown in panels B-D. A-H: Data are means and error bars are ± SD. 3 p < 0.01; $ p < 0.0001. TIC: Total Ion Current. I, J: open circle = G6PC3-deficient mice treated with 0.9 % NaCl (control); black circle = G6PC3-deficient mice treated with empagliflozin. Six mice per group.

Figure 8 is a graph showing l,5-anhydroglucitol-6-phosphate accumulation in neutrophils from patients deficient in G6PT or G6PC3. Serum l,5-anhydroglucitol (1,5 AG) in two GSDIb (filled symbols) and one G6PC3-deficient (open symbols) patients and seven healthy controls (CT) was determined by LC-MS (each symbol represents a different control; for some individuals, blood samples were taken on two different occasions to estimate variability and the two values are shown).

Figure 9 is a scheme illustrating the mechanisms leading to the accumulation of 1,5- anhydroglucitol-6-phosphate (l,5-AG6P) in the neutrophils of subjects with a G6PC3 deficiency or a G6PT deficiency. l,5-anhydroglucitol (l,5-AG) is transported into the neutrophils where it is converted into l,5-AG6P by a side activity of at least ADP-GK. A deficiency of the G6PT transporter prevents the transport of l,5-AG6P into the endoplasmic reticulum (ER). A deficiency of the G6PC3 phosphatase prevents the hydrolysis of l,5-AG6P and its conversion into l,5-AG. Thus, G6PT deficiency and G6PC3 deficiency both result in the accumulation of l,5-AG6P in the cytoplasm of the neutrophils. Said accumulation of l,5-AG6P inhibits the phosphorylation of the glucose into glucose-6-phosphate (G6P) by hexokinases (HK1, HK3). Subsequently, there is less G6P available for glycolysis and for the pentose phosphate pathway (Pentose-P- Pathway), and thus the production of NAPDH and ATP is decreased. Said decrease in the production of NAPDH and ATP can lead to neutrophil dysfunction and stress notably through endoplasmic reticulum stress, glycosylation defects reduced respiratory burst and increased apoptosis.

EXAMPLES

The present invention is further illustrated by the following examples. Example 1:

Materials and Methods Material

Production of HAP1 cell lines deficient in G6PT and G6PC3

The CRISPR/Cas9 constructs generated to inactivate G6PC3 and G6PT were prepared starting from two different primer pairs, as indicated below.

For G6PC3-target 1 (to remove the catalytic histidine Hisl67):

CRISP-hG6PC3-Tl-sl: CACCGgegacaagccaaccgceaaa (SEQ ID NO: 1); CRISP-hG6PC3-T 1 -as 1 : AAAQttggcggttggcttgtcgcC (SEQ ID NO: 2);

CRISP-hG6PC3-Tl-s2: CACCGgacatttcccccaccaggtgc (SEQ ID NO: 3); and

CRISP-hG6PC3-Tl-as2: AAACgcacctggtgagggaaatgtcC (SEQ ID NO: 4).

For G6PC3 -target 2 (to target the ATG region):

CRISP-hG6PC3-T2-s 1 : CACCGgcgctacagaaecagctagce (SEQ ID NO: 5);

CRISP-hG6PC3-T2-as 1 : AAACggctagetggttctgtagegcC (SEQ ID NO: 6);

CRISP-hG6PC3-T2-s2: CACCGgcecagcgtggactceatgg (SEQ ID NO: 7); and

CRISP-hG6PC3-T2-as2: AAACecatggagtccacgetgggcC (SEQ ID NO: 8).

For G6PT (target region = exon 4):

CRISP-hG6PT-sl: CACCGgetgaccagatgagtgctcge (SEQ ID NO: 9);

CRISP-hG6PT-asl: AAACgegagcactcatctggteagcC (SEQ ID NO: 10);

CRISP-hG6PT-s2: CACCGgataagctgecgactggctge (SEQ ID NO: 11); and

CRISP-hG6PT-as2: AAACgeagccagtcggcagettatcC (SEQ ID NO: 12).

Annealed primer pairs were ligated into the vector pSpCas9n(BB)-2A-Puro (PX462) V2.0 (a gift from F. Zhang, Massachusetts Institute of Technology; Addgene plasmid no.

62987) (Ran FA, et ah, 2013. Nat Protoc 8: 2281-2308) digested by Bbsl. Constructs were validated by sequencing (Beckman Coulter Genomics).

HAP1 cells (Horizon Discovery Austria) were cultured in IMDM (Iscove’s Modified Dulbecco’s Medium) containing 10% FBS, 2 mM L-glutamine and penicillin/streptomycin (Life Technologies). Cells were transfected with the CRISP constructs essentially as previously described (Zheng et al., 2014, Biotechniques 57: 115- 124). Genomic DNA from puromycin resistant clones was used to amplify by PCR the regions encompassing the targeted sites and the PCR products were sequenced to assess the presence of the gene modification in each clone.

The following clones, with the indicated mutations, were used:

G6PTA4: change of reading frame after Gly50 (exon 4; sequence context 50GFIT); G6PC3A6: 32 bp deletion encompassing the initiator ATG;

G6PC3D7: 48 bp deletion after amino acid Leu 155 leading to a premature stop codon (in the following context: CTFLL). Animals

G6PC3 knockout mice: G6PC3 KO mice were produced by injection of plasmids allowing the expression of guide RNAs and Cas9. The oligonucleotides used for the construction of the guide RNAs were:

CRISP-mG6PC3H 167-s 1 : CACCGgccaggaatcaccctcaccc (SEQ ID NO: 13);

CRISP-mG6PC3H 167-as 1 AAACgggtgagggtgattcctggcC (SEQ ID NO: 14);

CRISP-mG6PC3Hl67-s2 CACCGgcatttccctcaccaagtgtt (SEQ ID NO: 15); and

CRISP-mG6PC3Hl67-as2 AAACaacacttggtgagggaaatgcC (SEQ ID NO: 16).

Mice carrying mutations were genotyped by PCR analysis of the mutated region. Two nul alleles were used for founding the colonies. A 90 bp deletion (strain G6PC3-904) removed the catalytic histidine (H167), while the other mutation (strain G6PC3-912) caused an 8 bp deletion after the Pro 120 codon, leading to a change in reading frame and a premature stop codon. Experiments were performed mostly with the G6PC3-904 strain either in the homozygous form or in the heterozygous form as a control (G6PC3 heterozygous mice do not display a neutropenia phenotype).

Methods

Molecular cloning and expression of recombinant proteins

The coding sequences of human G6PC1 and human G6PC3 were PCR-amplified from human liver cDNA and inserted in the pEF6/Myc-His A or pEF6/His B, enabling the expression of the proteins with a C-terminal His tag and a N-terminal His tag, respectively. Plasmids allowing the expression of untagged proteins were derived from the pEF6/Myc-His A plasmids. Additionally, plasmids carrying human G6PC1 and G6PC3 with a mutation of the highly conserved catalytic histidine (H176A in G6PC1; H167A in G6PC3) were prepared as negative controls. All plasmids were checked by sequencing. The different plasmids were transfected in HEK293T cells using jetPEI® (Polyplus-transfection® SA). Cells were collected after 48 h and lysed. A membrane fraction containing the recombinant proteins was obtained by centrifugation at 15000 x g for 15 min. The resulting pellet was washed with buffer containing 25 mM Hepes, pH 7.2, 0.5 mM PMSF, 2 pg/ml leupeptin and 2 pg/ml antipain, recentrifuged as above and the resulting pellet resuspended in the same buffer and used as an enzyme source. The different tagged protein preparations were analyzed by western blotting using an anti-His tag antibody (N-terminal 6xHis-tag: Anti-His antibody 27-4710-01, Amersham; C- terminal 6xHis-tag, PentaHis antibody 34660, Qiagen) to compare the level of expression of the recombinant proteins. In vitro assay of G6PC1 and G6PC3 activities

Assays were carried out by incubating said enzyme preparations (typically 25 pg/ml with 100 pM (unless otherwise indicated) of the different substrates to be tested. Controls with the corresponding H176A or H 167 A mutants were run in parallel. The incubation was performed for 10 min at 30°C in 50 mM cacodylate, pH 5.8, 2 mM EDTA, 0.25 mg/ml BSA, 2.5 mM octylglucoside, in a final volume of 50 mΐ. The reaction was stopped by addition of HC1 and the inorganic phosphate (Pi) released was determined with a Malachite green assay (Itaya and Ui, 1966). The amount of enzyme and incubation times used in the assays were such that < 30 % of the substrate was consumed at the end of the incubation. Preparation of microsomes and assay of enzymatic activities

Tissue microsomes of liver, spleen, heart and skeletal muscle from overnight starved 270 g rats were prepared by differential centrifugation essentially as previously described (de Duve et al., 1955, Biochem. J. 60: 604-617). Briefly, tissues were homogenized with 3 volumes of 25 mM Hepes 7.1, 25 mM KC1, 250 mM sucrose, 2.5 pg/ml each Leupeptin and Antipain (skeletal muscle and heart were first minced on ice). The homogenates were centrifuged for 20 min at 1400 rpm and the resulting supernatants were centrifuged at 60 min at 40000 rpm at l0°C. The resulting pellets were resuspended in the initial volume of homogenizing buffer with the help of Dounce homogenizer, and recentrifuged at 40000 rpm. The pellets were resuspended with a Dounce homogenizer in 0.2 ml of the homogenizing buffer per g of initial tissue.

To determine the substrate specificity of the phosphatase(s) endogenously present in the tissue microsomes, the preparations were incubated with 100 mM ribose-5-phosphate or 100 mM glucose-6-phosphate for 30 min at 30°C as described above in 50 mM cacodylate, pH 5.8, 2 mM EDTA, 7.5 mM octylglucoside, and 250 mM sucrose in a final volume of 50 pl. Preparation of HAP1 cell extracts for GC-MS analysis

0.7 x 10⁶ HAP1 cells were seeded in 6-well plates and grown for 24h in DMEM medium (lg glucose) containing 10% FBS, 2 mM L-glutamine, penicillin/streptomycin and either no, or 0.1 or 1 mM l,5-anhydroglucitol or 0.05 or 0.5 mM l,5-anhydrofructose. 5 hours before extraction, the media was removed (and kept for measuring glucose consumption) and replaced by equivalent fresh media. For metabolite extraction, the medium was removed and cells were immediately washed with ice-cold NaCl (0.9%), followed by addition of 500 pL dry-ice-cold methanol and 500 pL cold water per well. The cells were then scraped. Extracts were mixed with 1 ml chloroform and shaken vigorously for 30 min at 4°C. After separating the organic and aqueous phases by centrifugation, the latter was taken up and frozen at -80°C until used. The extracts were then dried by speed-vac, re-dissolved in 15 pl of pyridine containing methoxamine (40 mg/ml) and incubated for 90 min at 30°C, under agitation. Finally, samples were derivatized with 30 pl of TMSFA at 37°C for 30 min and GC-MS analysis was performed as described in (Collard F, et al. 2016, Nat Chem Biol 12: 601-607). Metabolites were identified based on their retention time and characteristic ions. Selected ion monitoring (SIM) chromatograms for appropriate m/z were integrated using Masshunter software (Agilent) and areas were normalized to total ion current (Peracchi et al, 2017, Proc Natl Acad Sci U S A. 114(16):E3233-E324). Hapl cells viability assays

Viability of Hapl cells was assessed after 72 h culture in 96-well plates (2500 cells/ well) in DMEM medium (containing 5.5 mM glucose) under described conditions.

The CellTiter-Glo® Luminescent Cell Viability assay (100 pl) was used to assess cell survival following the manufacturer’s instructions. Experiments with neutrophil progenitors

Mouse wild type and G6PC3-deficient (G6PC3 ^/_) neutrophil progenitors were obtained from Georg Hacker (Freiburg, Germany). In this model, progenitor lines differentiate into neutrophils when Hoxb8 is turned off (Gautam et al., 2013). Cells were seeded in 96 well plates (2500 cells /well) in 100 pl OptiMEM-Glutamax containing 6% FCS, 30 pM beta- mercaptoethanol, 10 ng/ml SCF and 1 pM beta estradiol (which allows expression of Hoxb8) as well as the indicated concentrations of l,5-anhydroglucitol or 1,5- anhydrofructose. After 72h, cell survival was assessed using the CellTiter-Glo® Luminescent Cell Viability assay following the manufacturer’s instructions. Viability was similarly assessed in HAP1 cells (2500 cells/ well) using DMEM medium (containing 5.5 mM glucose).

Expression and analysis of hexokinases and ADP-GK

Prokaryotic vectors were constructed for expressing human hexokinases 1, 2 and 3 and human ADP-dependent glucokinase (ADP-GK), as proteins fused to a N-terminal his tag. The peptide which allows binding of hexokinase 1 and hexokinase 2 to the mitochondrial membrane was not included in the expressed sequence. Hexokinase 1 and hexokinase 2 were thus expressed as proteins missing the first 20 and 28 amino acids, respectively, i.e., A20-hHKl and A28-hHK2. Similarly, the signal propeptide of ADP-GK (first 50 amino acids) was omitted from the expressed protein (A50-hADPGK). The proteins were expressed in Escherichia coli and purified by affinity chromatography on a His-trap column.

Radiolabeled anhydroglucitol was produced by reduction of 1, 5 -anhydro fructose with tritiated sodium borohydride. The resulting radiolabeled polyol was converted by phosphorylation with ADPGK to l,5-anhydroglucitol-6-phosphate, purified by anion exchange chromatography to remove the unreacted radiolabeled l,5-anhydromannitol and dephosphorylated with alkaline phosphatase. The final radiolabeled product was used in radiochemical assays of the kinase activity of hexokinases and ADP-GK.

Mouse assays

Mice (~ 28 g for males; 21 g for females; 3-month old) were gavaged with 10 pg Empagliflozin/g body weight on 8 different days over a 12-day period. The drug was each time administered in 100 mΐ as a crushed suspension in 0.9 % NaCl. 1,5- Anhydroglucitol was also administered by gavage (100 mΐ of a 50 mM NaCl solution; 5 doses over a 6-day period). Control mice were either untreated or were administered 100 mΐ of 0.9 % NaCl. Blood was taken from the mice tail on several days to determine the serum concentration of l,5-anhydroglucitol by LC/MS. The experiments were terminated by deep anesthesia of the mice ( 10 pl/g body weight of a mixture containing 10 mg/ml Ketamine and 1 mg/ml Xylazine), removal of blood from the vena cava in EDTA tubes (for counting of the blood cell formula and determination of the neutrophil count by cell flow cytometry using MAC1 and GR1 antibodies. The blood sample was centrifuged to isolate the buffy coat (white blood cells, used for LC-MS analysis) and the plasma (used for 1,5- anhydroglucitol analysis by LC/MS).

Tissues were collected as rapidly as possible, freeze-clamped in liquid nitrogen and maintained at ~ - 70°C until further processing for LC/MS analysis.

G6PC3-deficient mice (n=8) were treated with 1,5 -anhydroglucitol (l,5AG) (5 x 100 mΐ of 50 mM) during seven days. Then they were split in two groups (n = 4) that received either Empagliflozin (10 mg/kg in 100 mΐ 0.9% NaCl; five times over eight days) to lower plasma l,5AG or vehicle (100 mΐ 0.9% NaCl). On the day of sacrifice white blood cell (WBC) counts were obtained from a Sysmex XN1000 hematology analyzer and blood differentials come from manual reclassification on a Sysmex Di60 digital microscope. Blood 1,5 AG was monitored by LC-MS analysis of serum from 15 mΐ tail blood taken on the indicated days or from plasma of blood collected after euthanasia on day 15. Flow cytometry of white blood cells analysis showing Mac-l/Gr-l labeled granulocytes was performed either on 25 mΐ of tail blood (days 0, 7 and 13) or on 50 mΐ of EDTA-blood collected after euthanasia.

Flow cytometry assays

Mouse blood granulocytes were analyzed by flow cytometry as previously described (16). In brief, 100 mΐ of whole blood collected in EDTA-tubes was diluted in 2 ml PBS (without Ca2+ and Mg2+), separated in two tubes (1 ml/tube) and centrifuged at 20°C (5 min at 400 g). The supernatant was immediately removed and the recovered pellet was stained with 50 mΐ of PBS + 2% FBS containing or not 0.25 pg of FITC -labeled Ly-6G (Gr-l) monoclonal antibody (clone RB6-8C5, eBioscience™, Invitrogen) and 0.12 pg of PE- labeled CDl lb (Mac-l) monoclonal antibody (clone Ml/70, eBioscience™, Invitrogen). Samples were incubated for 15 min in the dark. Red blood cells were lysed by adding 0.6 ml of red blood cells lysis buffer consisting of 155 mM NH4C1, 10 mM KHC03, 1 mM EDTA, adjusted to pH 7.2 (freshly made from a filter- sterilized lO-fold concentrated solution). After incubation for 20 min, data acquisition was on a flow cytometer (FACSVERSE, BD Biosciences) and data analysis using the FlowJo software package (FlowJo LLC, Oregon, USA).

Isolation of human leukocytes and quantification of l,5-anhydroglucitol-6-phosphate in human leukocytes

PMN leukocytes (consisting mostly of neutrophil granulocytes) present in 2.5 ml of freshly collected blood were separated from PBMC (peripheral blood mononuclear cells) by centrifugation on 2.5 ml Polymorphprep™ (AXIS-SHIELD, Oslo, Norway) following the manufacturer's instructions. Control cells behaved as expected, and a perfect separation of PMN from red blood cells was achieved. PMN cells and contaminating red blood cells were resuspended in red blood cell lysis buffer (155 mM NH4C1, 10 mM KHC03, 1 mM EDTA, adjusted to pH 7.2) to purify PMN cells. After red blood cell lysis and centrifugation (10 min at 400 g), the purified PMN cells and the PBMC were washed by centrifugation in 0.9% NaCl (10 min at 400 g). To extract intracellular metabolites, the pellets were carefully resuspended in 1 ml (50 % methanol/H20) followed by addition of 1 ml chloroform and the metabolites were extracted by shaking the tubes for 40 min at 20000 rpm at 4°C in a horizontal shaker followed by centrifugation at 4°C (10 min at 16000 g). Metabolites present in the aqueous phase were recovered and kept at -80°C until analysis by LC-MS as described for HAP1 cells above.

To estimate absolute concentrations, each sample was typically spiked with 0.05 to 0.2 mM deuterated l,5-anhydroglucitol-6-phosphate (2-[D]-l,5AG6P) as internal standard. These variable concentrations were necessary to account for the vastly different l,5-anhydroglucitol-6-phosphate concentrations between samples and the limited isotopic purity (98.5 %) of our internal standard. Absolute concentrations in the cellular lysates were determined by comparing the integrated extracted ion chromatograms corresponding to l,5-anhydroglucitol (m/z = 163.0612) with the ones of the standard 2- [D]-l,5-anhydroglucitol (m/z = 164.0674), taking into account the natural isotopomer distribution of l,5-anhydroglucitol and the isotopic purity of the synthesized 2-[D]-l,5- anhydroglucitol. Since we knew the number of PMN cells or PBMC that were used to prepare the extracts, based on published intracellular volumes for PMN (17) and PBMC (18) we calculated the intracellular concentration of l,5-anhydroglucitol-6-phosphate in

PMN cells and PBMC. Of note, particularly for patients with lower neutrophil counts, we cannot exclude a small contamination of the PBMC fraction with the PMN cell fraction, which likely explains the variability observed in the ratio of l,5-anhydroglucitol-6- phosphate found in PMN cells versus PBMC (4.3 to 13 - see results), for the three analysed patients. LCMS analysis of 1.5AG6P in mouse white blood cells extracts

To prepare mouse white blood cells extracts, a volume of - 0.6 ml of whole blood collected in EDTA tubes was centrifuged, the plasma recovered and a volume of - 2 ml of red blood cell lysis buffer was added (155 mM NH4C1, 10 mM KHC03, 1 mM EDTA, pH 7.2) to approximately 0.6 ml of whole blood. After red blood cell lysis (- 15 min), the tubes were centrifuged (5 min at 600 g) and the pellet containing the white blood cells washed with 2 ml 0.9% NaCl and centrifuged as above. After removing the supernatant, the pellet was resuspended in 1 ml (50% methanol/H20) followed by addition of 1 ml chloroform and the metabolites were extracted by shaking the tubes for 40 min at 20000 rpm at 4°C in a horizontal shaker followed by centrifugation at 4°C (10 min at 16000 g). The metabolites present in the aqueous phase were recovered and kept at -80°C until analysis by the same ion pairing LC-MS method as used for the analysis of HAP1 cells (see above). Extracted-ion chromatograms of the [M-H]- forms were integrated using MassHunter software (Agilent, Santa Clara, CA, USA), and areas under the curve were normalized to total ion current. Data is shown as means ± SD across the indicated number of animals.

Quantification of l,5-anhydroglucitol in mouse and human plasma by LC-MS

1,5 AG in mouse and human plasma was quantified by LC-MS. Practically, a volume of 4 mΐ of plasma was added to 191 mΐ of (20% H2O/80% of a solution containing 90% methanol and 10% chloroform) and spiked it with 5 mΐ of 30 mM 2-[D]-l,5AG (0.75 mM final concentration), used as an internal standard. After centrifugation at 4°C (5 min at 16000 g) the supernatant was kept at -80°C until used for LC-MS analysis. LC-MS analysis was performed with a rapid version of the ion-pairing approach described above using a 5 mM ODS(2) InertClone column (100 x 4.6 mm, Phenomenex) and the same buffers as described above. The mobile phase profile consisted of the following steps and linear gradients: 0 - 4 min at 0 % B; 4 - 10 min from 0 to 100 %; 10 - 15 min at 100 % B; 15 - 16 min from 100 to 0 %; 16 - 20 min at 0 % B. Flow rate was 0.5 ml/min between 0 and 4 min, and 1 ml/min for the remaining time. Mass spectrometric analysis was performed as described above, but absolute concentrations were determined by comparing the integrated extracted ion chromatograms corresponding to 1,5 AG (m/z = 163.0612) with the ones of the standard 2-[D]-l,5AG (m/z = 164.0674), taking into account the natural isotopomer distribution of 1,5 AG and the isotopic purity of the synthesized 2- [D]- 1,5 AG.

Results

Specificity of human recombinant G6PC1 and G6PC3 G6PC3 and G6PC1 are integral membrane proteins of the endoplasmic reticulum. As such, they are extremely difficult if not impossible to purify in a stable, active form (Van Schaftingen and Gerin, 2002, Biochem. J. 362: 513-532). Thus, human G6PC3 and G6PC1 were expressed in HEK293T cells and their kinetic properties were assessed in partially purified membrane fractions, in the presence of small amounts of detergents added to avoid any limitation in the access of substrates to the catalytic site. The proteins were tagged with a C-terminal His tag in order to perform quantification by western blotting (Figure 1A) and thereby compare the relative specific activity of G6PC1 and G6PC3. As controls, inactivated forms of human G6PC3 and G6PC1 were expressed, wherein the catalytic histidine (H176 in G6PC1; H167 in G6PC3), which transiently accepts a phosphoryl group during the catalytic cycle, was substituted by an alanine.

The phosphatase activities of G6PC1 and G6PC3 on a series of phosphate ester substrates were assessed through the quantification of inorganic phosphate (Pi) released after incubation of the partially purified membrane fractions with 0.1 mM of the indicated phosphate ester substrates (see Figure 1B-C). As shown in Figure IB, G6PC1 acted best on glucose-6-P, mannose-6-P and inorganic pyrophosphate (PPi) but did not hydrolyze, or almost not, the other phosphate esters tested. By contrast, as shown in Figure 1C, G6PC3 had a much wider substrate specificity but did not hydrolyze glucose-6-phosphate very efficiently. Indeed, inorganic pyrophosphate (PPi), ribose-5-phosphate, ribitol-5- phosphate and l,5-anhydroglucitol-6-phosphate were hydrolyzed 4 to 8 times faster than glucose-6-phosphate. Other phosphate esters like ribulose-5-P, xyhilose-5-P, mannose-6- P and dihydroxyacetone-P were also hydrolyzed by G6PC3 but at a slower rate. Controls run in parallel with the inactive forms of G6PC3 or G6PC1 indicated that the phosphatase activities observed with all the substrates tested could be specifically attributed to G6PC1 or G6PC3 (data not shown).

Table 2 below illustrates the kinetic properties of G6PC1 and G6PC3 determined through incubation with different concentrations of glucose-6-P, ribose-5-P, l,5-anhydroglucitol- 6-P and inorganic pyrophosphate (PPi). The enzymatic activities were assayed by measuring the release of inorganic phosphate (Pi) (Itaya and Ui, 1966, Clin Chim Acta 14: 361-366). For G6PC1, glucose-6-phosphate (glucose-6-P) and inorganic pyrophosphate (PPi) were equally good substrates, while ribose-5-phosphate (ribose-5- P) and l,5-anhydroglucitol-6-phosphate (l,5-AG-6-P) were hydrolyzed 10 to 15 times more slowly. For G6PC3, the best substrate in terms of V_max was inorganic pyrophosphate (PPi), followed by l,5-anhydroglucitol-6-phosphate, ribose-5-phosphate and glucose-6- phosphate. Remarkably, the K_m for glucose-6-phosphate was much higher than for the three other substrates, making it a much poorer substrate of G6PC3. Overall, when using the relative V_max/K_m value as a criterion, glucose-6-phosphate was twelve times poorer as a substrate for G6PC3 than for G6PC1, while ribose-5-phosphate and 1,5- anhydroglucitol-6-phosphate were seven times better for G6PC3 than for G6PC1. Table 2: Characterization of the enzymatic activities of human G6PC1 and G6PC3

⁽¹⁾ The“Vmax” values correspond to the“apparent Vmax” because the recombinant proteins were not purified. Endogenous activities of G6PC1 and G6PC3

To confirm that the specificity described hereinabove also applied to G6PC1 and G6PC3 when expressed in tissues, microsomes were prepared from rat skeletal muscle, heart, spleen (which express G6PC3 but no G6PC1) and from rat liver (which expresses G6PC1 but almost no G6PC3) and their capacity of hydrolyzing radiolabeled glucose-6- phosphate and ribose-5-phosphate were determined (see Figure 2A). When tested in the presence of a detergent (octylglucoside) to prevent any limitation in the entry of substrate, microsomes from muscle, heart and spleen, expressing G6PC3 only, hydrolyzed ribose- 5-phosphate between 2.5 and 4-fold faster than glucose-6-phosphate. By contrast, in the same conditions, liver microsomes, expressing mostly G6PC1, acted about 20 times faster on glucose-6-phosphate than on ribose-5-phosphate. The ratio of phosphatase activity on ribose-5-phosphate/phosphatase activity on glucose-6-phosphate was 4.02 for skeletal muscle microsomes, 2.6 for heart microsomes and 3 for spleen microsomes, similar to the ratio of 4.6 observed for human G6PC3. By contrast, said ratio was only 0.05 for liver microsomes, similar to the ratio of 0.02 for human G6PC1. Taken all together, these results thus confirm the specificity of G6PC1 for glucose-6-P and the lack of preference of G6PC3 for glucose-6-P.

Involvement of the glucose-6-phosphate translocase in the hydrolysis of 1,5- anhydroglucitol-6-phosphate

The transporter G6PT is inhibited by S3483, a pharmacological compound belonging to the family of chlorogenic acid (Arion el al., 1998). As the effect of S3483 is best demonstrated in tissue microsomes, rat skeletal muscle microsomes were used. As shown in Figure 2B, the phosphatase activity of skeletal muscle microsomes, i.e., G6PC3 activity, on glucose-6-phosphate and l,5-anhydroglucitol-6-phosphate was significantly inhibited by S3483. By contrast, the phosphatase activity of skeletal muscle microsomes, i.e., G6PC3 activity, on ribose-5-phosphate, ribitol- 5 -phosphate and inorganic pyrophosphate (PPi) was not inhibited by S3483. This indicates that G6PT transports glucose-6-phosphate and its close structural analogue l,5-anhydroglucitol-6-phosphate, but does not transport ribose-5-phosphate, ribitol- 5 -phosphate and inorganic pyrophosphate (PPi). This conclusion is also consistent with the effect of detergent addition, which strongly increased the hydrolysis of ribose-5-phosphate but barely affected that of l,5-anhydroglucitol-6-phosphate and had a lesser effect on the hydrolysis of glucose-6-phosphate. Indeed, entry of ribose-5-phosphate in the microsomes is rate limiting in the absence of detergents, but this is not (or less) the case for entry of glucose- 6-phosphate and l,5-anhydroglucitol-6-phosphate, owing to the presence of G6PT which can actively transport them in the microsomes. Taken together, these findings suggest that

1.5-anhydroglucitol-6-phosphate, a known inhibitor of low K_m hexokinases, might be the common metabolite that accumulates in both G6PC3 and G6PT deficiencies and accounts for the neutropenia linked to these deficiencies. Accumulation of 1 ,5 -anhydroglucitol-6-phosphate in G6PC3 and G6PT deficient cells

To verify that G6PC3 or G6PT deficiency causes accumulation of anhydroglucitol-6-P, HAP1 cells deficient in either of these two proteins were prepared: one cell line G6PT- deficient (G6PT-A4 KO) and two G6PC3-deficient cell lines (G6PC3-D7 KO and G6PC3-A6 KO). Sequencing of the DNA confirmed the presence of mutations incompatible with a functional protein.

As shown in Figure 3A, incubation of the HAP1 cells with l,5-anhydroglucitol during 24 hours led to the progressive accumulation of l,5-anhydroglucitol-6-phosphate in cells that were deficient in G6PT or G6PC3. By contrast, l,5-anhydroglucitol-6-phosphate remained almost undetectable in the non-deficient HAP1 cells (WT cells). These findings confirmed the involvement of G6PC3 and G6PT in the hydrolysis of 1 ,5-anhydroglucitol- 6-phosphate and further showed that l,5-anhydroglucitol is phosphorylated to 1,5- anhydroglucitol-6-phosphate in intact cells.

Remarkably, as shown in Figure 3C, an approximately 10-fold higher accumulation of

1.5-AG-6-P was observed when cells were grown in the presence of l,5-anhydrofructose, a sugar derivative that is readily converted to 1 ,5-anhydroglucitol by 1 ,5-anhydrofructose reductase (Sakuma et al., 1998), a widely distributed enzyme expressed in HAP1 cells. LC-MS analysis did not reveal the presence of l,5-anhydrofructose-phosphate in the HAP1 cells, indicating that the formation of l,5-AG-6-P from l,5-anhydrofructose only occurs via the conversion of l,5-anhydrofructose to the intermediate l,5-anhydroglucitol. The about 10-fold higher levels of l,5-AG-6-P observed after incubation with 1,5- anhydrofructose are likely explained by a more efficient entry of l,5-anhydrofructose.

Interestingly, the presence of 0.5 mM l,5-anhydrofructose in the culture medium caused in G6PC3- and G6PT-defient HAP1 cells, though not in control cells, a marked decrease in glucose utilization (Figure 3B and D) and a striking decrease in the intracellular metabolites resulting from the metabolism of glucose (Fig. 3E: glucose-6-phosphate, Fig. 3F: fructose-6-phosphate, Fig. 3G: 6-phosphogluconate and Fig. 3H: ribose-5- phosphate) while the concentration of Krebs cycle intermediates was not significantly affected (Fig. 31: a-ketoglutarate and Fig. 3J: succinate). This suggested that cells were not dying, but instead could use other compounds that were present in the medium (such as pyruvate or glutamine) to feed the Krebs cycle. However, as shown in Figure 3K, cell viability was decreased when G6PC3- or G6PT-deficient cells were cultured for 72h in the same medium in presence of 0,5 and 1 mM l,5-anhydrofructose. No such effect was observed with l,5-anhydroglucitol (up to 2 mM), presumably because the 1,5- anhydroglucitol entry is slower than that of l,5-anhydro fructose, thus causing a much lower accumulation of l,5-anhydroglucitol-6-phosphate that did not reach toxicity levels for these cells.

Accumulation of 1,5 -anhydroglucitol-6-phosphate in neutrophil precursors derived from G6PC3 -deficient mice Studies similar to the ones described hereinabove were performed with neutrophil precursors derived from G6PC3 -deficient mice (Gautam et al., 2013). Thus, as shown in Figure 4, incubation of neutrophil precursors derived from G6PC3-deficient mice with l,5-anhydroglucitol induced (A) the accumulation of l,5-anhydroglucitol-6-phosphate, (B) a decreased rate of glucose metabolism as assessed with the detritiation of 2-³H- glucose, and (C) cell death, in G6PC3-deficient cells but not in control cells. Notably, l,5-anhydroglucitol was about as potent as l,5-anhydrofructose to induce cell death in neutrophil precursors and the concentrations at which it acted were in the physiological range for mice. This difference when compared to HAP1 cells (see Figure 3B) is presumably due to the fact that 1,5 -anhydroglucitol is much better transported in neutrophils than in HAP1 cells. A possible mechanism for the toxic effect of l,5-anhydroglucitol-6-phosphate observed in G6PC3- or G6PT-deficient cells might be that the accumulation of 1,5- anhydroglucitol-6-phosphate inhibits glucose phosphorylation in these cells. Thus, the rate of glucose detritiation was determined in neutrophil precursors derived from G6PC3- deficient mice that were incubated with l,5-anhydroglucitol. As shown in Figure 4B, incubation with l,5-anhydroglucitol caused a progressive decrease in the rate of glucose phosphorylation. These findings support a negative effect of the accumulation of 1,5- anhydroglucitol-6-phosphate in G6PC3- or G6PT-deficient cells on glucose phosphorylation. Action of hexokinases on 1 ,5-anhydroglucitol and inhibition by 1 ,5-anhydroglucitol-6- phosphate

Human hexokinases 1, 2 and 3 and human ADP-GK were expressed in Escherichia coli and purified by affinity chromatography on a His-trap column. The peptide which allows binding of hexokinase 1 and hexokinase 2 to the mitochondrial membrane was not included in the expressed sequence, which were therefore expressed as proteins missing the first 20 and 28 amino acids, respectively, i.e., A20-hHKl and A28-hHK2. Similarly, the signal propeptide of ADP-GK (first 50 amino acids) was omitted from the expressed protein (A50-hADPGK). To assess their kinase activity, purified A20-hHKl, A28-hHK2, hHK3 and A50-hADPGK were incubated 10 min at 30°C either with radiolabeled D-[U- ¹⁴C]-glucose (and 10 mM of D-glucose) or with radiolabeled 2-[³H]-l,5-anhydroglucitol (and 10 pM l,5-anhydroglucitol) in the following assay mixture (100 pl final volume): 25 mM Hepes, pH 7.2, 25 mM KC1 , 5 mM MgCl₂, 1 mM DTT, 0.1 mM di-adenosine- penta-phosphate, 0.5 mM ATP or ADP, 20 mM NaF, 0.5 mg/ml BSA, 30 000 cpm of the radioactive substrate. The glucose kinase activities of A20-hHKl, A28-hHK2, hHK3 and A50-hADPGK were also assessed in a spectrophotometric assay that coupled the oxidation of the glucose-6-phosphate produced during ATP (or ADP) phosphorylation of glucose by HK1-3 or ADP-GK to NADPH production in a G6PDH (glucose-6-phosphate dehydrogenase) coupled assay. Practically, assays were done at 30°C, in 1 ml final volume, containing 25 mM Hepes, pH 7.2, 25 mM KC1, 5 mM MgCl₂, 1 mM DTT, 0.5 mM ATP or ADP, 0.5 mg/ml BSA, 0.01 - 1 mM glucose, 0.3 mM NADP and 1 mΐ of G6PDH (glucose-6-phosphate dehydrogenase) from L. mesentewides.

As shown in Figure 5A, A20-hHKl, A20-hHKl, hHK3 and A50-hADPGK were all able to phosphorylate glucose, with A20-hHKl and hHK3 having the strongest kinase activity on glucose. However, only A50-hADPGK displayed a side-activity on 1,5- anhydroglucitol, as shown in Figure 5B and in Table 3 below. Thus, the ratio of kinase activity on glucose/kinase activity on l,5-anhydroglucitol was 14 000 for A20-hHKl, 9 000 for A28-hHKl and 17 000 for hHK3, while said ratio was only 14 for D50- hADPGK. These findings thus strongly suggest that ADP-GK is responsible for the formation of l,5-anhydroglucitol-6-phosphate.

To verify whether l,5-anhydroglucitol-6-phosphate can inhibit glucose phosphorylation, purified A20-hHKl, A28-hHK2, hHK3 and A50-hADPGK were incubated with 10 mM radiolabeled glucose in the presence of 150 mM l,5-anhydroglucitol-6-phosphate. As shown in Table 3 below, phosphorylation of glucose by the low K_m hexokinases-hHKl, hHK2, and hHK3 was strongly inhibited by l,5-anhydroglucitol-6-phosphate. By contrast, l,5-anhydroglucitol-6-phosphate did not inhibit the phosphorylation of glucose by hADP-GK.

Table 3: Characterization of the enzymatic activities of human hexokinases HK1, HK2 and HK3 and of human ADP-GK

⁽¹⁾ measured using spectrophotometric assay

⁽²⁾ measured using radiochemical assay

⁽³⁾ substrate used to assess the enzymatic activity of the indicated kinase

⁽⁴⁾ noncompetitive inhibition versus glucose

¾ no inhibition of hADPGK detected in the presence of 1 mM l,5AG6P

Treatments modulating the 1,5-anhydroglucitol concentration in mouse blood affect neutropenia and the l,5-anhydroglucitol-6-phosphate concentration in neutrophils

The levels of l,5-anhydroglucitol and l,5-anhydroglucitol-6-phosphate were assessed in G6PC3-deficient mice (G6PC3 ^/_ mice) and in control mice (heterozygous G6PC3^+/ mice) (Figure 6). The level of l,5-anhydroglucitol in neutrophils (WBC) and in tissues was not significantly different between G6PC3-deficient mice and control mice (Figure 6A). The level of l,5-anhydroglucitol was increased both in G6PC3-deficient mice and control mice upon administration of l,5-anhydroglucitol to the mice (Figure 6B). As shown in Figure 6C, the level of l,5-anhydroglucitol-6-phosphate in neutrophils (WBC) and in tissues was about 200-fold higher in G6PC3-deficient mice than in control mice. Furthermore, as shown in Figure 6D, administration of 1,5- anhydroglucitol induced a higher increase of the level of l,5-anhydroglucitol-6-phosphate in neutrophils (WBC) and in tissues in G6PC3-deficient mice than in control mice.

Next, G6PC3-deficient mice (G6PC3 ^/_ mice) and control mice (heterozygous G6PC3^+/ mice) were administered l,5-anhydroglucitol or empagliflozin, a SGLT2 inhibitor. As a control, G6PC3-deficient mice and control mice were either untreated or administered NaCl, as indicated in Figure 7. The serum level (see Figure 7A) and plasma level (see Figure 7B) of l,5-anhydroglucitol were increased by the administration of 1,5- anhydroglucitol, both in G6PC3 -deficient and control mice. By contrast, the serum and plasma levels of l,5-anhydroglucitol level were reduced by the administration of empagliflozin, both in G6PC3 -deficient and control mice (see Figures 7A, 7B, and 7E). The leukocyte l,5-anhydroglucitol-6-phosphate level was significantly increased in untreated G6PC3-deficient mice as compared to control mice (see Figure 7D). The leukocyte l,5-anhydroglucitol-6-phosphate level was further increased in G6PC3- deficient mice, but not in control mice, following the administration of 1,5- anhydroglucitol (see Figure 7D), and reduced in G6PC3 -deficient mice following the administration of empagliflozin (see Figures 7D and 7H). Most interestingly, as shown in Figure 7C, the neutrophil count, which was about 2-fold lower in G6PC3-deficient mice than in control mice, was further decreased in G6PC3-deficient mice to barely detectable levels following administration of l,5-anhydroglucitol. Conversely, as shown in Figures 7C, 7F and 7G, the neutrophil count of G6PC3-deficient mice was increased following administration of empagliflozin.

The reversibility of the granulocyte maturation arrest was also tested by treating G6PC3- deficient mice that were first treated with l,5-anhydroglucitol with either empagliflozin (+ EMPA), to rapidly lower the levels of l,5-anhydroglucitol, or with saline as a control (+ saline). After day 7, when administration of l,5-anhydroglucitol was stopped, blood l,5-anhydroglucitol either declined slowly in mice that were administered saline (Figure 71, open circles) or declined sharply in those administered Empagliflozin (Figure 71, black circles). The rapid decrease in blood l,5-anhydroglucitol, as a result of empagliflozin administration (see above), massively increased the number of circulating granulocytes (Figure 7J and Table 4) and reversed the maturation arrest in the bone marrow (data not shown). These effects were not observed after the administration of saline.

Table 4: Peripheral blood cell counts of G6PC3 -deficient mice force-fed with 1,5- anhydroglucitol followed by either saline or Empagliflozin

Immature granulocytes include promyelocytes, myelocytes and metamyelocytes. Data equals mean ± SD. These experiments demonstrated unequivocally that the modulation of 1,5- anhydroglucitol concentrations with empagliflozin is of therapeutic value in G6PC3- deficient mice.

Accumulation of 1,5 -anhydroglucitol-6-phosphate in neutrophils from patients deficient in G6PT or G6PC3

To assess whether comparable pathogenic mechanisms are at play in neutrophils of human patients, blood samples from one patient with a mutation in G6PC3 (PT3) and from two GSDIb patients with mutations in G6PT (PT1 and PT2) were analyzed. All three patients were neutropenic. PT2 was liver-transplanted ~8 years ago and is currently taking G-CSF. Polymorphonuclear cells (PMNs; mostly neutrophils) were separated from peripheral blood mononuclear cells (PBMCs; mostly lymphocytes), and the concentration of l,5-anhydroglucitol-6-phospohate (l,5AG6P) was quantified. Leukocytes from all three patients had a concentration of l,5AG6P that was more than 500-fold higher than that measured in controls (Table 5), while the average concentration of 1,5- anhydroglucitol (1,5 AG) in plasma was not significantly different between patients and healthy controls (Figure 8). Furthermore, the concentration of l,5AG6P was always higher in PMN cells compared with PBMCs in the same patient (4.3- to l3-fold higher depending on the sample) (Table 5).

Table 5: Concentration of l,5-anhydroglucitol-6-phosphate (l,5AG6P) in granulocytes (PMNs) and lymphocytes (PBMCs) obtained from patients and healthy controls

Taken together, these results support the idea that G6PT and G6PC3 are involved in the breakdown of l,5AG6P in human neutrophils and that, in their absence, l,5AG6P reaches concentrations that are likely to inhibit hexokinases.

Conclusion Taken all together, the results presented above support that an intracellular accumulation of l,5-AG-6-P is responsible for the neutrophil dysfunction and neutropenia observed in subjects suffering from a G6PC3 or G6PT deficiency (see Figure 9). Moreover, the results presented above demonstrate that the administration of a SGLT2 inhibitor, such as empagliflozin, results in a decrease in the leukocyte level of l,5-AG-6-P and in an increase of the neutrophil count.

Claims

1. A SGLT2 (sodium glucose cotransporter 2) inhibitor for use in the treatment of neutropenia in a subject in need thereof, said subject having an elevated intracellular level of l,5-anhydroglucitol-6-phosphate.

2. The SGLT2 inhibitor for use according to claim 1, wherein said subject suffers from neutropenia linked to a deficiency of the glucose-6-phosphatase encoded by G6PC3 or to a deficiency of the glucose-6-phosphate transporter encoded by G6PT, also known as SLC37A4.

3. The SGLT2 inhibitor for use according to claim 2, wherein said neutropenia linked to a deficiency of the glucose-6-phosphatase encoded by G6PC3 is a congenital neutropenia selected from the group comprising severe congenital neutropenia type 4 (SCN4) and Dursun syndrome.

4. The SGLT2 inhibitor for use according to claim 2, wherein said neutropenia linked to a deficiency of the glucose-6-phosphate transporter encoded by G6PT, also known as SLC37A4, is one of the symptoms of the congenital glycogen storage disease type lb.

5. The SGLT2 inhibitor for use according to claim 2, wherein said neutropenia linked to a deficiency of the glucose-6-phosphatase encoded by G6PC3 or to a deficiency of the glucose-6-phosphate transporter encoded by G6PT, also known as SLC37A4, is drug-induced.

6. The SGLT2 inhibitor for use according to any one of claims 1 to 5, wherein said SGLT2 inhibitor is selected from the group comprising empagliflozin, dapagliflozin, canagliflozin, ipragliflozin, ertugliflozin, luseogliflozin, bexagliflozin, tofogliflozin, henagliflozin, sotagliflozin, remogliflozin, sergliflozin and atigliflozin, and any combination thereof, preferably said gliflozin is empagliflozin.

7. The SGLT2 inhibitor for use according to any one of claims 1 to 6, wherein said SGLT2 inhibitor is selected from the group comprising or consisting of empagliflozin, dapagliflozin, canagliflozin, ipragliflozin and ertugliflozin, and any combination thereof.

8. The SGLT2 inhibitor for use according to any one of claims 1 to 7, wherein said

SGLT2 inhibitor is to be administrated at a dose ranging from about 0.015 mg per kilo body weight per day (mg/kg/day) to about 4.5 mg/kg/day, preferably at a dose ranging from about 0.035 mg/kg/day to about 1.5 mg/kg/day.

9. A method for determining whether a subject suffering from neutropenia is eligible for treatment with a SGLT2 (sodium glucose cotransporter 2) inhibitor, said method comprising measuring the level of l,5-anhydroglucitol-6-phosphate in a biological sample obtained from the subject.

10. The method of claim 9, wherein the level of l,5-anhydroglucitol-6-phosphate in a biological sample obtained from the subject suffering from neutropenia is compared to a reference level.

11. The method of claim 9, wherein a subject suffering from neutropenia with a level of l,5-anhydroglucitol-6-phosphate higher than the reference level is determined to be eligible for treatment with a SGLT2 inhibitor.

12. A method for monitoring neutropenia associated with an intracellular accumulation of l,5-anhydroglucitol-6-phosphate in a subject, said method comprising measuring the level of l,5-anhydroglucitol in a biological sample obtained from the subject.

13. A method for monitoring the effectiveness of a SGLT2 (sodium glucose cotransporter 2) inhibitor therapy administered to a subject suffering from neutropenia associated with an intracellular accumulation of l,5-anhydroglucitol- 6-phosphate, said method comprising measuring the level of l,5-anhydroglucitol in a biological sample obtained from the subject.

14. The method according to claim 13, wherein the level of l,5-anhydroglucitol in a biological sample obtained from the subject is compared to a personalized reference level of the subject.

15. The method according to claim 14, wherein the personalized reference level of the subject is the level of 1 ,5-anhydroglucitol measured in a biological sample obtained from the subject before or at the beginning of the SGLT2 inhibitor therapy.