WO2021224401A1 - Méthodes et compositions de détermination d'une plage de référence d'une plaquette d'exposition du β-galactose - Google Patents

Méthodes et compositions de détermination d'une plage de référence d'une plaquette d'exposition du β-galactose Download PDF

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WO2021224401A1
WO2021224401A1 PCT/EP2021/062020 EP2021062020W WO2021224401A1 WO 2021224401 A1 WO2021224401 A1 WO 2021224401A1 EP 2021062020 W EP2021062020 W EP 2021062020W WO 2021224401 A1 WO2021224401 A1 WO 2021224401A1
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rca
platelet
subject
fsc
ratio
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PCT/EP2021/062020
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Alexandre KAUSKOT
Delphine Borgel
Dominique LASNE
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Assistance Publique-Hôpitaux De Paris (Aphp)
Université Paris-Saclay
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Publication of WO2021224401A1 publication Critical patent/WO2021224401A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/22—Haematology
    • G01N2800/222—Platelet disorders

Definitions

  • the invention is in the field of haematology. More particularly, the invention relates to methods and compositions for determining the quality of platelets.
  • Thrombocytopenia can have various causes, which are usually classified as being 3 central or peripheral.
  • Techniques for exploring central thrombocytopenia include bone marrow assessment, plasma thrombopoietin assays, evaluation of the immature platelet fraction [1]
  • peripheral causes of excessive platelet consumption they can be easily monitored.
  • evidencing an increase in platelet clearance is more challenging because a routine laboratory assay for platelet clearance is not available. Indeed, the in vivo platelet half-life can only be measured in specialized centers after indiumlll labelling, and peripheral destruction can be revealed by a weak platelet transfusion yield.
  • the invention relates to a method for determining a reference range of b-galactose exposure platelet in a biological sample obtained from a subject comprising the steps of: i) determining the concentration of Ricinus communis agglutin (RCA) and the value of Forward SCatter (FSC) in said biological sample; and ii) calculating the ratio between RCA and FSC.
  • RCA concentration of Ricinus communis agglutin
  • FSC Forward SCatter
  • Inventors have developped a standardized flow cytometry assay of b-galactose exposure in platelet-rich plasma by using Ricinus communis agglutinin (RCA) for implementation in a clinical laboratory b-galactose exposure was measured in platelet-rich plasma by using flow cytometry and Ricinus communis agglutinin (RCA).
  • RCA Ricinus communis agglutinin
  • a population of 120 healthy adults was recruited to study variability. They determined an optimal RCA concentration of 12.5 pg/mL. The measure was stable for up to 4 hours (mean fluorescence intensity (MFI)-RCA: 1233 ⁇ 329 at TOh and 1480 ⁇ 410 at T4h).
  • the platelet count did not induce a variation of RCA and the measure of RCA was stable when tested up to 24h after blood collection (MFI-RCA: 1252 ⁇ 434 at day 0 and 1140 ⁇ 29724 hours after blood sampling).
  • results should be expressed as RCA/FSC ratio.
  • This assay may assist in the diagnosis of thrombocytopenic diseases linked to changes in b- galactose exposure.
  • the invention relates to a method for determining a reference range of b-galactose exposure platelet in a biological sample obtained from a subject comprising the steps of: i) determining the concentration of Ricinus communis agglutin (RCA) and the value of Forward SCatter (FSC) in said biological sample; and ii) calculating the ratio between RCA and FSC.
  • a method for determining a reference range of b-galactose exposure platelet in a biological sample obtained from a subject comprising the steps of: i) determining the concentration of Ricinus communis agglutin (RCA) and the value of Forward SCatter (FSC) in said biological sample; and ii) calculating the ratio between RCA and FSC.
  • RCA concentration of Ricinus communis agglutin
  • FSC Forward SCatter
  • the method according to the invention further comprises a step of identifying non-specific populations appearing within the platelet population with a Mitotracker.
  • the invention relates to a method for determining a reference range of b- galactose exposure platelet in a biological sample obtained from a subject comprising the steps of: i) identifying non-specific populations appearing within the platelet population; ii) determining the concentration of Ricinus communis agglutin (RCA) and the value of Forward SCatter (FSC) in said biological sample; and ii) calculating the ratio between RCA and FSC.
  • RCA Ricinus communis agglutin
  • FSC Forward SCatter
  • the term “subject” refers to any mammals, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the subject is a human. In a particular embodiment, the subject is a healthy subject.
  • platelet also called as thrombocytes refers to a component of blood whose function is to react to bleeding from blood vessel injury by clumping, thereby initiating a blood clot. Platelets are made in bone marrow along with white and red blood cells. A normal platelet count is 150,000 to 450,000 platelets per microliter of blood.
  • b-galactose also known as galactose, beta-D-galactose or beta-D-galactopyranose refers to a monosaccharide sugar that is about as sweet as glucose.
  • Galactose molecule linked with a glucose molecule forms a lactose molecule
  • b-galactose is released by an enzyme reaction: B-galactosidase hydrolyzes b-D-galactoside bonds to release D-galactose, and is generally present in microorganisms, animals and plants.
  • b- galactose has the following chemical formula in the art CeHuOe and CAS number: 7296-64-2.
  • platelet b-galactose exposure refers to platelet which have b- galactose on their surface.
  • the term biological sample refers to any sample obtained from a subject, such as a serum sample, a plasma sample, a urine sample, a blood sample, a lymph sample, or a biopsy.
  • the biological sample is a blood sample.
  • the biological sample is plasma sample.
  • the biological sample is venous blood sample.
  • the venous blood is collected in vacutainer® containing 7.5% K3 EDTA.
  • Platelet-rich plasma (PRP) is obtained after two centrifugation steps, i.e. 10 minutes at 38 g and then 10 minutes at 122 g, to enable the recovery of large and giant platelets.
  • non-specific populations appearing within the platelet population refers to impurities and debris present in the biological sample. Said impurities refer to non-specific population. Typically, this step allows to eliminate elements in the blood that may or may not have mitochondria. For example, red blood cells do not have mitochondria. This will be useful to know the background noise before calculation the ratio RCA/FSC.
  • MitoTrackerTM Deep Red FM is a far red-fluorescent dye (abs/em -581/644 nm) that stains mitochondria in live cells and its accumulation is dependent upon membrane potential.
  • MitoTracker ® Red FM is a far red-fluorescent dye (abs/em -581/644 nm) that stains mitochondria in live cells and its accumulation is dependent upon membrane potential.
  • the term “Ricinus communis agglutin” (RCA) refers to a lectin which binds to b-galactose. This lectin consists of two subunits of 60 kDa which can be dissociated by reducing agents into closely related chains between 27 kDa and 33 kDa. One of the chains appears to be common to the “B” chain of another castor bean lectin, ricin, while the other chain is unique to RCA I.
  • the B chain binds to galactose or N-acetylgalactosamine residues of membrane glycoconjugates.
  • RCA is measured by flow cytometry. In the context of the invention, said RCA is also called as RCAI or RCA120.
  • platelet surface b-galactose exposure was determined using FITC-conjugated RCA (Vector Labs, Burlingame, CA). Briefly, PRP diluted was incubated with RCA for 30 min at room temperature. The reaction was stopped with PBS and then analyzed using flow cytometry. In another embodiment, the samples in which RCA had been incubated with b-lactose were used as negative control.
  • platelet rich plasma PRP
  • PRP platelet rich plasma
  • neuraminidase from Clostridium perfringens (NeuC, Sigma- Aldrich).
  • the stability of platelet b-galactose exposure was studied from 10 controls. On the day of sampling, each blood sample was divided into two aliquots; one was assayed in the two hours, and the other was assayed after 24 hours of storage at room temperature. The stability of RCA binding was evaluated 30 minutes and 1, 2, 3 and 4 hours after the start of the incubation. In each sample, the geometric mean fluorescence intensity (MFI) was determined for a total of 5,000 platelets.
  • MFI geometric mean fluorescence intensity
  • the concentration of RCA is 12.5 pg/mL.
  • the method according to the invention wherein the concentration or quality of RCA can be controlled in lyophilized platelets that are desialylated.
  • platelets are resuspended at a concentration of 150 G/L in PBS and then diluted at 50G/L in the assay. Platelets are then stained with the RCA-FITC at 12.5 pg/mL.
  • Two vials of RCA-FITC from the same lot were then used with a delay of opening of 12 months. Each vial is referred as old (opened 12 months ago) and young vial (recently opened).
  • the signal of RCA- FITC decreased by 2.5 fold times after 12 months of storage. Accordingly, the fixed lyophilized platelets can be used as quality control of RCA.
  • a decrease of ⁇ 10% of the MFI of the RCA- FITC of same RCA vial has to be considered to rule out the vial.
  • FSC Forward Scatter
  • Flow cytometry is a method of single-cell analysis that includes the characterization of a cell's physical properties.
  • the FSC the amount of laser light that passes around the cell, and is proportional to the cell’s diameter
  • the levels of two conventional glycoproteins are measured and the levels of two conventional glycoproteins (GPIba and GPIIb) proportional with the mean platelet volume, at the same time as RCA binding.
  • the RCA/size ratios are stable over the 4 hours of measurement.
  • value refers to the number of cells passed through out of the laser.
  • ratio refers to the relationship between two amounts. Typically, in the context of the invention, ratio refers to the relationship between RCA and FSC as defined above. Typically, the ratio RCA/FSC ratio is determined in a population of healthy subjects. More particularly, the ratio is independent of sex and blood group. In another embodiment, the ratio comprises between 0.237 to 0.941.
  • the ratio of RC/FSC is also called as reference range when it is determined in a population of healthy subjects.
  • the ratio as determined above is the reference range.
  • the ratio or reference range according to the invention comprising between 0.237 to 0.941.
  • the ratio or the reference range calculated in a healthy population according to the invention is: 0.237, 0.238, 0.239, 0.240, 0.241, 0.242, 0.243, 0.244, 0.245, 0.246, 0.247, 0.248, 0.249, 0.250, 0.251, 0.252, 0.253, 0.254, 0.255, 0.256, 0.257, 0.258,
  • the method according to the invention is suitable to measure the number of platelet b-galactose exposure in a biological sample.
  • the invention relates to method according to the invention is suitable to measure the number of platelet b-galactose exposure in a biological sample obtained from a subject comprising the steps of: i) identifying non-specific populations appearing within the platelet population; ii) determining the concentration of Ricinus communis agglutin (RCA) and the value of Forward SCatter (FSC) in said biological sample; and ii) calculating the ratio between RCA and FSC.
  • RCA Ricinus communis agglutin
  • FSC Forward SCatter
  • the method as described above is suitable to measure the quality of platelets.
  • the invention relates to a method for determining the quality of platelets in a biological sample comprising the steps of i) calculating the ratio between RCS and FSC as described above; and ii) comparing said ratio with a reference range.
  • the invention relates to a method for determining the quality of platelets in a biological sample comprising the steps of: i) identifying non-specific populations appearing within the platelet population; ii) determining the concentration of RCA and the value of FSC in said biological sample; iii) calculating the ratio between RCA and FSC; iv) comparing said ratio with a reference range; and, v) concluding that the quality of platelets is stable and/or maintained when the the ratio calculated is in the reference range or concluding that the quality of platelets is not stable and/or maintained when the ratio is outside the reference range.
  • the method according to the invention wherein the concentration or quality of RCA is controlled in lyophilized platelets as described above.
  • the method according to the invention is suitable to measure the quality of platelets during a transfusion.
  • the term “quality” refers to maintain of platelets’ functions in a biological sample.
  • human platelets have a half-life of 8 to 9 days and 150,000 to 450,000 platelets per microliter of blood in the circulation. Platelets’ function is to react to bleeding from blood vessel injury by clumping, thereby initiating a blood clot.
  • the quality of platelets is determined by measuring the ratio between RCA and FSC according to the invention and compared said ratio to reference range as described above.
  • the ratio calculated is in the reference range, it means that the quality of the platelets is stable and/or maintained. More particularly, the platelets’ functions are conserved.
  • the ratio is outside the reference range (inferior to 0.237 or superior to 0.941), it means that the quality of the platelets is not stable and/or maintained. More particularly, the platelets’ functions are not conserved.
  • the term biological sample refers to any sample obtained from a subject, such as a serum sample, a plasma sample, a urine sample, a blood sample, a lymph sample, or a biopsy.
  • the biological sample is a blood sample. More particularly, the biological sample is venous blood sample. In another embodiment, the biological sample is a venous blood sample obtained from a donor. Typically, the biological sample is a storage bag for a transfusion.
  • the quality of platelets can also be measured before a transfusion to a recipient.
  • Platelet transfusion is most frequently used to correct unusually low platelet counts, either to prevent spontaneous bleeding (typically at counts below 10xl0 9 /L) or in anticipation of medical procedures that will necessarily involve some bleeding.
  • Platelets are either isolated from collected units of whole blood and pooled to make a therapeutic dose, or collected by platelet apheresis: blood is taken from the donor, passed through a device which removes the platelets, and the remainder is returned to the donor in a closed loop.
  • the term “quality” refers to maintain of platelets’ functions during the storage period for a transfusion.
  • transfusion refers to an event where blood is removed from one individual (donor), animal, or human, and transfused to another in need (recipient).
  • the term “donor” refers to a human or animal, donating blood.
  • the donor is a healthy human.
  • the donor is a healthy animal.
  • the term “recipient” refers to a corresponding human or animal receiving blood.
  • the recipient is a subject suffering from thrombocytopenia (elicited by chemotherapy and/or other diseases).
  • the recipient is an elderly person.
  • the recipient is pregnant.
  • the recipient is a human susceptible to have a surgery.
  • the recipient is a human susceptible to have an organ transplantation.
  • the invention in a third aspect, relates to a method for determining whether platelets in a biological sample are desialylated or hyposialylated comprising the steps of: i) calculating the ratio between RCA and FSC as described above; and ii) concluding that platelets are desialylated or hyposialylated when the ratio is outside the reference range according to the invention.
  • the invention relates to the invention relates to a method for determining whether platelets in a biological sample obtained from a subject are desialylated or hyposialylated comprising the steps of: i) identifying non-specific populations appearing within the platelet population; ii) determining the concentration of RCA and the value of FSC in said biological sample; ii) calculating the ratio between RCA and FSC; iii) comparing said ratio with a reference range; and iv) concluding that platelets are desialylated or hyposialylated when the ratio is outside the reference range according to the invention.
  • the terms “desialylated”, “hyposialyted” desialylation or “hyposialation” refer to any reaction that deplete a sialyl acid from a molecule.
  • Sialic acids are terminal sugar components of glycoproteins and glycolipids and are incorporated during the sialylation process. Sialic acids are monosaccharides with a shared 9-carbon backbone, and they are typically found at the outermost end of the glycan chains in all cell types. Sialic acids play pivotal roles in many physiologically and pathologically important processes. They play numerous roles in several aspects of immunity, and they might serve as “self’ markers in different physiological and pathophysiological functions. Typically, the hyposialylation/desialylation on platelets is characterized by abnormally high b-galactose exposure that accelerates platelet clearance and can lead to many diseases such as thrombocytopenia.
  • outside the reference range refers to when the ratio is inferior to 0.237 or superior to 0.941.
  • the ratio calculated is in the reference range, it means that that platelets are not desialylated or hyposialylated.
  • the ratio is outside the reference range (inferior to 0.237 or superior to 0.941), it means that that platelets are desialylated or hyposialylated.
  • the invention a method for determining whether platelets in a biological sample are hypersialylated comprising the steps of: i) calculating the ratio between RCA and FSC as described above; and ii) concluding that platelets are hypersialylated when the ratio is inferior to the reference range according to the invention.
  • the invention relates to the invention relates to a method for determining whether platelets in a biological sample obtained from a subject are hypersialylated comprising the steps of: i) identifying non-specific populations appearing within the platelet population; ii) determining the concentration of RCA and the value of FSC in said biological sample; ii) calculating the ratio between RCA and FSC; iii) comparing said ratio with a reference range; and iv) concluding that platelets are hypersialylated when the ratio is inferior to the reference range according to the invention.
  • hypothalalylation refers to an increase of the immature platelet fraction or young platelets.
  • the ratio calculated is inferior to the reference range (e.g. inferior to 0.237), it means that subject the platelets are hypersialylated.
  • the method according to the invention is suitable to diagnosis a platelet desialylation related disease.
  • the invention relates to a method for diagnosing a platelet desialylation related disease in a subject comprising the steps of: i) calculating the ratio between RCA and FSC as described above; and ii) concluding that the subject is suffering or is susceptible to suffer from a platelet desialylation related disease when the ratio is outside the reference range according to the invention.
  • the invention relates to the invention relates to a method for diagnosing a platelet desialylation related disease in a subject comprising the steps of: i) identifying non-specific populations appearing within the platelet population; ii) determining the concentration of RCA and the value of FSC in said biological sample; iii) calculating the ratio between RCA and FSC; iv) comparing said ratio with a reference range; and v) concluding that the subject is suffering or is susceptible to suffer from a platelet desialylation related disease when the ratio is outside the reference range according to the invention.
  • the method according to the invention wherein the concentration or quality of RCA is controlled in lyophilized platelets as described above.
  • diagnosis refers to classifying a disease or a symptom, determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease and/or prospects of recovery.
  • the term biological sample refers to any sample obtained from a subject, such as a serum sample, a plasma sample, a urine sample, a blood sample, a lymph sample, or a biopsy.
  • the biological sample is a blood sample.
  • the biological sample is plasma sample.
  • the biological sample is plasma sample with platelets called Platelet-Rich Plasma (PRP). More particularly, the biological sample is venous blood sample.
  • PRP Platelet-Rich Plasma
  • the biological sample is obtained from thrombocytopenic suspected samples.
  • the non-specific populations appearing within the platelet population is determined by Mitotracker. This will be useful to know the background noise before calculation the ratio RCA/FSC in thrombocytopenic suspected subjects.
  • platelet desialylation related disease refers to diseases elicited by platelet desialylation.
  • the ratio calculated is in the reference range (between 0.237 and 0.941), it means that subject does not suffer from a platelet desialylation related disease.
  • the ratio is outside the reference range (inferior to 0.237 or superior to 0.941), it means that the subject suffers from a platelet desialylation related disease.
  • the platelet desialylation related disease is selected from the group consisting of but not limited to: thrombocytopenia, sepsis, immune thrombocytopenia (ITP), gestational thrombocytopenia, congenital disorders of glycosylation, VWF-type 2B disease, bacterial infections, viral infection (e.g. influenza virus A infection, dengue), parasitic infection (e.g. malaria), liver diseases (e.g. NAFLD, NASH), allogenic hematopoietic stem cell transplantation, disorders related to Trans Aortic Valve Implantation (TAVI) or Mechanical Circulatory Support Implantation (MCSI).
  • TAVI Trans Aortic Valve Implantation
  • MCSI Mechanical Circulatory Support Implantation
  • the platelet desialylation related disease is thrombocytopenia.
  • Thrombocytopenia is characterized by abnormally low levels of platelets in the blood. Thrombocytopenia might occur as a result of a bone marrow disorder such as leukemia or an immune system problem. Or it can be a side effect of taking certain medications. Thrombocytopenia signs and symptoms may include: easy or excessive bruising (purpura), superficial bleeding into the skin that appears as a rash of pinpoint-sized reddish-purple spots (petechiae), usually on the lower legs, prolonged bleeding from cuts, bleeding from gums or nose, blood in urine or stools, unusually heavy menstrual flows, fatigue or enlarged spleen. Factors that can decrease platelet production include: leukemia and other cancers, some types of anemia, viral infections, such as hepatitis C or HIV, chemotherapy drugs and radiation therapy or heavy alcohol consumption.
  • the thrombocytopenia is elicited by chemotherapy agents used for treating a cancer.
  • chemotherapeutic agent refers to chemical compounds that are effective in inhibiting tumor growth.
  • chemotherapeutic agents include multkinase inhibitors such as sorafenib and sunitinib, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolo melamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its ad
  • calicheamicin especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Inti. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolin
  • paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisp latin and carhop latin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,
  • the platelet desialylation related disease is idiopathic or Immune thrombocytopenia (ITP). It is also known as immune (idiopathic) thrombocytopenic purpura (ITP), refers to a clinical disorder in which thrombocytopenia manifests as a bleeding tendency, purpura, or petechiae.
  • ITP Immune thrombocytopenia
  • Desialylation-related thrombocytopenia might also be present in other acquired disorders related to VWF-platelet binding during common clinical procedures, such as the implantation of transaortic valves and mechanical circulatory support.
  • the platelet desialylation related disease is a disorder related to Trans Aortic Valve Implantation (TAVI).
  • TAVI Trans Aortic Valve Implantation
  • TAVR transcatheter aortic valve replacement
  • the aortic valve usually opens when blood is pumped from heart to the rest of body.
  • Aortic stenosis is a condition where the aortic valve cannot open and close properly. This condition puts extra strain on heart and can result in breathlessness, swollen ankles, chest pain, dizziness, and sometimes, blackouts.
  • the platelet desialylation related disease is a disorder related to Mechanical Circulatory Support Implantation (MCSI).
  • MSCI refers to devices that assist the ventricle and are attached to it in use. These are called Ventricular Assist Devices (VADs), and are designed to drive a flow of blood that is in parallel with flow within the native heart, between the ventricle and the aorta. In other words, they are designed as left (or right) ventricular assist devices (LVADs or RVADs), pumping devices that directly unload the respective ventricle.
  • VADs Ventricular Assist Devices
  • LVADs or RVADs left (or right) ventricular assist devices
  • the platelet desialylation related disease is allogeneic hematopoietic cell transplantation (HSCT) related disease.
  • HSCT refers to the transplantation of hematopoietic stem cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood. It may be autologous (the patient's own stem cells are used), allogeneic (the stem cells come from a donor) or syngeneic (from an identical twin). In the context of the invention, the hematopoietic stem cells are from a donor (allogenic).
  • the platelet desialylation related disease is liver diseases.
  • liver disease refers to any disturbance of liver function that causes illness.
  • the liver is responsible for many critical functions from protein production and blood clotting to cholesterol, glucose (sugar), and iron metabolism. When it becomes diseased or injured, the loss of those functions can cause significant damage to the body.
  • Liver disease is also referred to as hepatic disease.
  • the liver disease is selected from the group consisting of: Nonalcoholic fatty liver disease (NAFLD), Non-alcoholic steatohepatitis (NASH), or fibrotic NASH.
  • the platelet desialylation related disease is VWF-type 2B.
  • VWF-type 2B refers to a subtype of type 2 VWD characterized by a bleeding disorder associated with an increase in the affinity of the Willebrand factor (von Willebrand factor; VWF) for platelets. This anomaly results in spontaneous binding of high molecular weight VWF multimers to platelets leading to rapid clearance of both the platelets (increasing the risk of thrombocytopenia) and the high molecular weight VWF multimers from the plasma.
  • Willebrand factor von Willebrand factor
  • the platelet desialylation related disease is sepsis.
  • sepsis has its general meaning in the art and is used to identify the continuum of the clinical response to infection. Patients with sepsis present evidences of infection and clinical manifestations of inflammation. Sepsis is defined as SIRS secondary to documented or suspected infection.
  • the platelet desialylation related disease is viral infection.
  • the platelet desialylation related disease is an influenza virus A infection.
  • influenza virus type A infection refers to any infection caused by an influenza virus type A without consideration of serotype based on hemagglutinine (HI to H 15) and neuraminidase (N1 to N9) expression.
  • influenza virus type A include but are not limited to H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, and H10N7.
  • the platelet desialylation related disease is dengue virus infection.
  • dengue virus as is general meaning in the art and denotes an RNA virus of the family Flaviviridae; genus Flavivirus. Other members of the same genus include yellow fever virus, West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, tick-home encephalitis virus, Kyasanur forest disease virus, and Omsk hemorrhagic fever virus. Most are transmitted by arthropods (mosquitoes or ticks), and are therefore also referred to as arboviruses (arthropod-borne viruses).
  • the dengue virus may be of any serotype, i.e. serotype 1, 2, 3 or 4.
  • the platelet desialylation related disease is parasitic infection.
  • the platelet desialylation related disease is malaria.
  • malaria refers to a parasitic disease caused by a protozoon of the genus Plasmodium.
  • the five subspecies active in humans are P. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesiwithP. falciparumbeing responsible for most malaria-related death.
  • the term "subject” refers to any mammals, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the subject is a human.
  • the subject is a human who is susceptible to have a platelet desialylation related disease.
  • the subject is a human who is susceptible to have thrombocytopenia.
  • the subject is a human who is susceptible to have sepsis.
  • the subject is a human who is susceptible to have immune thrombocytopenia (ITP).
  • ITTP immune thrombocytopenia
  • the subject is a human who is susceptible to have allogenic hematopoietic stem cell transplantation.
  • the subject is a human who is susceptible to have a sepsis.
  • the subject is a human who is susceptible to have an influenza virus A infection.
  • the subject is a human who is susceptible to have a dengue virus infection.
  • the subject is a human who is susceptible to have malaria.
  • the subject is a human who is susceptible to have VWF type 2B.
  • the subject is a human who is susceptible to have liver disease such as NAFLD or NASH.
  • the subject is a human who is susceptible to have disorder related to Trans Aortic Valve Implantation (TAVI) or Mechanical Circulatory Support Implantation (MCSI).
  • TAVI Trans Aortic Valve Implantation
  • MCSI Mechanical Circulatory Support Implantation
  • the method according to the invention is suitable to diagnosis a platelet hypersialylation related disease.
  • the method for diagnosing a platelet hypersialylation related disease in a subject comprising the steps of: i) calculating the ratio between RCA and FSC as described above; and ii) concluding that the subject is suffering or is susceptible to suffer from a platelet hypersialylation related disease when the ratio is inferior to the reference range according to the invention.
  • the invention relates to the invention relates to a method for diagnosing a platelet hypersialylation related disease in a subject comprising the steps of: i) identifying non-specific populations appearing within the platelet population; ii) determining the concentration of RCA and the value of FSC in said biological sample; iii) calculating the ratio between RCA and FSC; iv) comparing said ratio with a reference range; and v) concluding that the subject is suffering or is susceptible to suffer from a platelet hypersialylation related disease when the ratio is inferior the reference range according to the invention.
  • the ratio is inferior to 0.237.
  • the platelet hypersialylation-related diseases could be observed in case of compensation mechanisms from the bone marrow to recover in thrombocytopenic patients a normal platelet count.
  • This boost of the bone marrow stimulates the production of young platelets also called immature platelet fraction that are hypersialylated compared to old platelets.
  • the invention relates to a method for predicting whether a subject suffering from a platelet desialylation related disease will achieve a response with a sialidase inhibitor comprising the steps: i) calculating the ratio between RCA and FSC as described above; and ii) concluding that the subject will achieve a response with a sialidase inhibitor when the ratio is in the reference range according to the invention or concluding that the subject will not achieve a response with a sialidase inhibitor when the ratio is outside the reference range according to the invention.
  • the invention relates to the invention relates to a method for predicting whether a subject suffering from a platelet desialylation related disease will achieve a response with a sialidase inhibitor comprising the steps of: i) identifying non-specific populations appearing within the platelet population; ii) determining the concentration of RCA and the value of FSC in said biological sample; iii) calculating the ratio between RCA and FSC; iv) comparing said ratio with a reference range; and v) concluding that the subject will achieve a response (responder) with a sialidase inhibitor when the ratio is in the reference range according to the invention or concluding that the subject will not achieve a response with a sialidase inhibitor when the ratio is outside the reference range according to the invention.
  • the method according to the invention wherein the concentration or quality of RCA is controlled in lyophilized platelets as described above.
  • the term "predicting" means that the subject to be analyzed by the method of the invention is allocated either into the group of subjects who will achieve a response, or into a group of subjects who will not achieve as response for the treatment with a sialidase inhibitor.
  • the terms “will achieve a response” or “responder” in the context of the invention refer to a subject that will achieve a response, i.e. a subject where the platelet desialylation related disease is eradicated, reduced or improved.
  • the responders have an objective response and therefore the term does not encompass subjects having a stabilized platelet desialylation related disease such that the disease is not progressing after the treatment with the sialidase inhibitor.
  • a non- responder or refractory patient includes subjects for whom the platelet desialylation related disease does not show reduction or improvement after the treatment with the sialidase inhibitor.
  • the term“non-responder” also includes subjects having a stabilized platelet desialylation related disease.
  • the characterization of the subject as a responder or non-responder can be performed by reference to a standard or a training set.
  • the standard may be the profile of a subject who is known to be a responder or non-responder or alternatively may be a numerical value.
  • Such predetermined standards may be provided in any suitable form, such as a printed list or diagram, computer software program, or other media.
  • the ratio calculated is in the reference range (between 0.237 and 0.41), it means that subject will achieve a response with a sialidase inhibitor.
  • the ratio is outside the reference range (inferior to 0.237 or superior to 0.941), it means that the subject will not achieve a response with a sialidase inhibitor.
  • the term "subject” refers to any mammals, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the subject is a human.
  • the subject is a human who is susceptible to have a platelet desialylation related disease.
  • the subject is a human who is susceptible to have thrombocytopenia.
  • the subject is a human who is susceptible to have sepsis.
  • the subject is a human who is susceptible to have immune thrombocytopenia (ITP).
  • ITTP immune thrombocytopenia
  • the subject is a human who is susceptible to have allogenic hematopoietic stem cell transplantation.
  • the subject is a human who is susceptible to have a sepsis.
  • the subject is a human who is susceptible to have an influenza virus A infection.
  • the subject is a human who is susceptible to have a dengue virus infection.
  • the subject is a human who is susceptible to have malaria.
  • the subject is a human who is susceptible to have VWF type 2B.
  • the subject is a human who is susceptible to have liver disease such as NAFLD or NASH.
  • the subject is a human who is susceptible to have disorder related to Trans Aortic Valve Implantation (TAVI) or Mechanical Circulatory Support Implantation (MCSI).
  • TAVI Trans Aortic Valve Implantation
  • MCSI Mechanical Circulatory Support Implantation
  • sialidase also called as neuraminidase refers to enzymes which are glycoside hydrolase enzymes that cleave the glycosidic linkages of neuraminic acids.
  • Neuraminidase enzymes are a large family, found in a range of organisms. Human sialidase homologues have been described in the human genome (NEU1, NEU2, NEU3, NEU4).
  • sialidase inhibitor refers to a natural or synthetic compound that has a biological effect to inhibit or significantly reduce the activity or expression of the transcripts and/or proteins.
  • sialidase inhibitor refers to a natural or synthetic compound that has a biological effect to inhibit or significantly reduce the activity or expression of sialidase transcripts and/or proteins.
  • the inhibitor of sialidase expression has a biological effect on one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.
  • the inhibitor of sialidase activity has a biological effect on the binding or activity of sialidase. Inhibition can be partial or total, resulting in a reduction or modulation in the activity of the enzyme as detected.
  • the sialidase inhibitor activity is a peptide, petptidomimetic, small organic molecule, antibody or aptamers.
  • peptidomimetic refers to a small protein-like chain designed to mimic a peptide.
  • the inhibitor of sialidase is an aptamer.
  • Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
  • the sialidase inhibitor activity is an antibody.
  • antibody is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
  • the term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical” scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"
  • Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments.
  • Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et ak, 2006; Holliger & Hudson, 2005; Le Gall et ak, 2004; Reff & Heard, 2001 ; Reiter et ak , 1996; and Y oung et ak , 1995 further describe and enable the production of effective antibody fragments.
  • the antibody is a “chimeric” antibody as described in U.S. Pat. No. 4,816,567.
  • the antibody is a humanized antibody, such as described U.S. Pat. Nos. 6,982,321 and 7,087,409.
  • the antibody is a human antibody.
  • a “human antibody” such as described in US 6,075,181 and 6,150,584.
  • the antibody is a single domain antibody such as described in EP 0368 684, WO 06/030220 and WO 06/003388.
  • the sialidase inhibitor activity is a monoclonal antibody.
  • Monoclonal antibodies can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique, the human B-cell hybridoma technique and the EBV-hybridoma technique.
  • the inhibitor is an intrabody having specificity for sialidase.
  • the term "intrabody” generally refer to an intracellular antibody or antibody fragment.
  • Antibodies in particular single chain variable antibody fragments (scFv), can be modified for intracellular localization. Such modification may entail for example, the fusion to a stable intracellular protein, such as, e.g., maltose binding protein, or the addition of intracellular trafficking/localization peptide sequences, such as, e.g., the endoplasmic reticulum retention.
  • the intrabody is a single domain antibody.
  • the antibody according to the invention is a single domain antibody.
  • single domain antibody sdAb or "VHH” refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb.
  • the inhibitor of sialidase activity is a small organic molecule.
  • small organic molecule refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals.
  • small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.
  • the sialidase inhibitor includes, but is not limited to one or more of the following: fetuin, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA) or a pharmaceutically acceptable salt thereof; Oseltamivir (ethyl (3R,4R,5S)-5-amino-4-acetamido- 3-(pentan-3-yloxy)-cyclohex-l-ene-l-carboxylate); Zanamivir ((2R,3R,4S)-4-guanidino-3- (prop-l-en-2-ylamino)-2-((lR,2R)-l,2,3-trihydroxypropyl)-3,4-dihydro-2H-pyran-6- carboxylic acid); Laninamivir ((4S,5R,6R)-5-acetamido-4-carbamimidamido-6-[(lR,2R)-3- hydroxy-2
  • DANA 2,
  • the sialidase inhibitor is the sodium salt of 2,3-dehydro-2- deoxy-N-acetylneuraminic acid or a combination thereof.
  • Sialidase inhibitors used with the present invention include those known in the art or those later developed.
  • the sialidase inhibitor is Oseltamivir or a variant thereof also known as ethyl (3R,4R,5S)-5-amino-4-acetamido-3-(pentan-3-yloxy)-cyclohex-l-ene-l- carboxylate (Tamiflu, Genentech, Cambridge, Mass.)
  • Oseltamivir has the following Cas number in the art: 196618-13-0.
  • the sialidase inhibitor is Zanamivir or a variant thereof also known as ((2R,3R,4S)-4-guanidino-3-(prop-l-en-2-ylamino)-2-((lR,2R)-l,2,3- trihydroxypropyl)-3,4-dihydro-2H-pyran-6-carboxylic acid) (Relenza; Glaxo Smith Kline, Research Triangle Park, N.C.). Zanamivir has the following Cas number in the art: 139110-80- 8
  • the sialidase inhibitor is Peramivir or a variant thereof ((lS,2S,3S,4R)-3-[(lS)-l-acetamido-2-ethyl-butyl]-4-(diaminomethylideneamino)-2- hydroxy-cyclopentane- 1 -carboxylic acid) (BioCry st, Birmingham, Ala.).
  • Peramivir has the following Cas number in the art: 229614-55-5.
  • the sialidase inhibitor is Laninamivir or a variant thereof also known as (2R,3R,4S)-3-acetamido-4-(diaminomethylideneamino)-2-[(lR,2R)-2,3- dihydroxy-l-methoxypropyl]-3,4-dihydro-2H-pyran-6-carboxylic acid (marketed under the name Inavir by Daiichi Sankyo). It is currently in Phase III clinical trials Laninamivir has the following Cas number in the art: 203120-17-6.
  • Small molecules as described above may also be derivatized, for example, bearing modifications other than insertion, deletion, or substitution of amino acid residues, thus resulting in a variation of the original product (a variant).
  • modifications can be covalent in nature, and include for example, chemical bonding with lipids, other organic moieties, inorganic moieties, and polymers.
  • the method according to the invention wherein the sialidase inhibitor gene expression is siRNA, shRNA, miRNA, antisense oligonucleotide, or a ribozyme.
  • the sialidase inhibitor expression is an antisense oligonucleotide.
  • Anti-sense oligonucleotides including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of sialidase mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of sialidase proteins, and thus activity, in a cell.
  • antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding sialidase can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous, or subcutaneous injection.
  • the sialidase inhibitor expression is a shRNA.
  • shRNA is generally expressed using a vector introduced into cells, wherein the vector utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited.
  • the shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it is bound.
  • RISC RNA-induced silencing complex
  • the sialidase inhibitor expression is a small inhibitory RNAs (siRNAs).
  • Sialidase expression can be reduced by contacting the subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that ialidase 1 expression is specifically inhibited (i.e. RNA interference or RNAi).
  • dsRNA small double stranded RNA
  • RNAi RNA interference
  • Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT.
  • the siRNA is ALN-PCS02 developed by Alnylam (phase 1 ongoing).
  • the sialidase inhibitor expression is a ribozyme.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of sialidase mRNA sequences are thereby useful within the scope of the present invention.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.
  • the sialidase inhibitor expression is an endonuclease.
  • the term “endonuclease” refers to enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as Deoxyribonuclease I, cut DNA relatively nonspecifically (without regard to sequence), while many, typically called restriction endonucleases or restriction enzymes, and cleave only at very specific nucleotide sequences.
  • endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the errorprone nonhomologous end-joining (NHEJ) and the high-fidelity homology-directed repair (HDR).
  • NHEJ errorprone nonhomologous end-joining
  • HDR high-fidelity homology-directed repair
  • the endonuclease is CRISPR- cas.
  • CRISPR-cas has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences.
  • the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes.
  • the CRISPR/Cas9 system has been described in US 8697359 B1 and US 2014/0068797.
  • the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).
  • the invention relates to a method for treating a subject suffering from a platelet desialylation related disease, comprising the steps of: i) calculating the ratio between RCA and FSC as described above and ii) administering said subject a sialidase inhibitor when the platelets are considered as desialylated according to the invention.
  • the invention relates to the invention relates to a method for treating a subject suffering from a platelet desialylation related disease, comprising the steps of: i) identifying non-specific populations appearing within the platelet population; ii) determining the concentration of RCA and the value of FSC in said biological sample; iii) calculating the ratio between RCA and FSC; iv) comparing said ratio with a range; and v) administering said subject a sialidase inhibitor when the platelets are considered as desialylated according to the invention.
  • the method according to the invention wherein the concentration or quality of RCA is controlled in lyophilized platelets as described above.
  • treating refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).
  • the term "subject” refers to any mammals, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the subject is a human.
  • the subject is a human who is susceptible to have a platelet desialylation related disease.
  • the subject is a human who is susceptible to have thrombocytopenia.
  • the subject is a human who is susceptible to have sepsis.
  • the subject is a human who is susceptible to have immune thrombocytopenia (ITP).
  • ITTP immune thrombocytopenia
  • the subject is a human who is susceptible to have allogenic hematopoietic stem cell transplantation.
  • the subject is a human who is susceptible to have a sepsis.
  • the subject is a human who is susceptible to have an influenza virus A infection.
  • the subject is a human who is susceptible to have disorder related to Trans Aortic Valve Implantation (TAVI) or Mechanical Circulatory Support Implantation (MCSI).
  • TAVI Trans Aortic Valve Implantation
  • MCSI Mechanical Circulatory Support Implantation
  • the subject is a human who is susceptible to have a dengue virus infection.
  • the subject is a human who is susceptible to have malaria.
  • the subject is a human who is susceptible to have VWF type 2B.
  • the subject is a human who is susceptible to have liver disease such as NAFLD or NASH.
  • sialidase inhibitor refers to a natural or synthetic compound that has a biological effect to inhibit or significantly reduce the activity or expression of the transcripts and/or proteins.
  • sialidase inhibitor refers to a natural or synthetic compound that has a biological effect to inhibit or significantly reduce the activity or expression of sialidase transcripts and/or proteins.
  • sialidase inhibitors are described above.
  • the ratio calculated is in the reference range (between 0.237 and 0.941)
  • the ratio is outside the reference range (inferior to 0.237 or superior to 0.941), it means that the subject will not achieve a response with a sialidase inhibitor and thus the method according to the invention is not suitable to treat said subject with a sialidase inhibitor.
  • inhibitors of sialidase as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising inhibitors of sialidase and pharmaceutically acceptable excipients.
  • the terms “pharmaceutically” or “pharmaceutically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to anon-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • saline solutions monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts
  • dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists.
  • Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine,
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions
  • the preferred methods of preparation are vacuum drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or inj ected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • the invention a kit for performing the method according to the invention, wherein said kit comprises i) means for identifying non-specific populations appearing within the platelet population (for example with a Mitotracker), ii) means for determining RCA and FSC in a biological sample obtained from a subject; and iii) instructions notice with a reference range determined in a healthy population as described above.
  • the kit according to the invention allows to measure the concentration or quality of RCA in lyophilized platelets.
  • the means for identifying non-specific populations appearing within the platelet population is a Mitotracker, such as MitoTrackerTM Deep Red FM.
  • the means for determining RCA in a biological sample is FITC- conjugated RCA.
  • FIGURES are a diagrammatic representation of FIGURES.
  • Figure 1 RCA labeling, concentrations and specificity
  • A) A flow cytometry analysis of RCA binding on PRP containing various concentrations of RCA (1.56, 3.12, 6.25, and 12.5 pg/mL) (n 3 experiments).
  • C) A histogram of the flow cytometric analysis of RCA binding in PRP incubated (or not) with 200 mM b-lactose (n 4 experiments). The mean ⁇ SD values were compared using Student’s t-test.
  • a flow cytometry analysis of (A) RCA binding, (B) FSC parameter, (C) anti-GPIba binding and (D) anti-GPIIb binding in PRP at the indicated time points. Analyses performed at 30, 60, 120, 180 and 240 min after the incubation had been stopped with PBS (n 3 experiments), and the RCA/size parameter ratios were calculated: (E) RCA/FSC, (F) RCA/GPIba and (G) RCA/GPIIb. The mean ⁇ SD values were compared using a one-way ANOVA and Dunnett’s post test.
  • Figure 5 Values of RCA, FSC and RCA/FSC in healthy subjects
  • RCA, FSC, and RCA/FSC values for males and females were compared using a Mann-Whitney test.
  • RCA, FSC, and RCA/FSC values for blood groups were compared using a one-way ANOVA and Dunnett’s post test.
  • FIG. 6 Platelets staining with mitotracker.
  • A Platelets labeling with mitotracker at different concentrations (25, 100, 250, 500 mM), the signal was measured in the FL4-H channel of the flow cytometer. The dashed line indicated the signal of red blood cells (RBC) in the presence of mitotracker, as RBC are anucleated, this signal corresponded to the background.
  • B Mitotracker (100 nM) was used in thrombocytopenic patients (without disease classification). The % of events (i), and the RCA (ii) were measured in the platelet gate with and without mitotracker.
  • Figure 7 Variation of RCA staining on lyophilized platelets. Platelets were resuspended at a concentration of 150 G/L in PBS and then diluted at 50G/L in the assay. Platelets were then stained with the RCA-FITC at 12.5 pg/niL. Two vials of RCA-FITC from the same lot were then used with a delay of opening of 12 months. Each vial was referred as old (opened 12 months ago) and young vial (recently opened).
  • Platelet-rich plasma was obtained after two centrifugation steps, i.e. 10 minutes at 38 g and then 10 minutes at 122 g, to enable the recovery of large and giant platelets.
  • Platelet surface b-galactose exposure was determined using FITC-conjugated RCA (Vector Labs, Burlingame, CA). Briefly, PRP diluted 1/20 with PBS (95 pL) was incubated with RCA for 30 min at room temperature in a final volume of 100 pL. The reaction was stopped with PBS (400 pL) and then analyzed using flow cytometry. In some experiments, samples in which RCA had been incubated with b-lactose (200 mM) (Sigma-Aldrich, Saint- Quentin Fallavier, France) were used as negative controls [25, 28] As a positive control of platelet desialylation (i.e.
  • PRP was incubated with 0.5 U/mL neuraminidase from Clostridium perfringens (NeuC, Sigma-Aldrich) for 20 minutes at 37°C.
  • neuraminidase from Clostridium perfringens (NeuC, Sigma-Aldrich) for 20 minutes at 37°C.
  • the stability of platelet b-galactose exposure was studied from 10 controls. On the day of sampling, each blood sample was divided into two aliquots; one was assayed in the two hours, and the other was assayed after 24 hours of storage at room temperature. The stability of RCA binding was evaluated 30 minutes and 1, 2, 3 and 4 hours after the start of the incubation.
  • GPIba and GPIIb expression was measured with mouse anti-GPIba (clone SZ2) or anti-GPIIb (clone P2) antibodies, respectively, or a negative isotype control (Biocytex, Marseille, France), and detected with a FITC-labeled secondary antibody. After 30 minutes of incubation at room temperature, samples were diluted with PBS and analyzed using flow cytometry.
  • Lectin or antibody binding was determined on a FACS CANTO-IITM flow cytometer, and the resulting data were analyzed using Diva 6 software (both from BD Biosciences, San Jose, CA). The flow cytometer were routinely calibrated with SPHEROTM Rainbow Calibration Particles (BD Biosciences, Le Pont de Claix, France). In each sample, the geometric mean fluorescence intensity (MFI) was determined for a total of 5,000 platelets. Statistical analysis
  • Quantitative data were graphed as dot plots (showing the mean ⁇ standard deviation (SD)) or as box-and-whisker plots (with the lower and upper hinge defining the interquartile range, the line inside the box denoting the median, and the whiskers corresponding to the maximum and the minimum). All statistical analyses were performed with Prism 6 for Mac software (version 6; GraphPad, Inc., San Diego, CA). If only two groups were compared, Student’s t-test (for data with a Gaussian distribution, according to a D’Agostino-Person test) or Mann-Whitney test (for data with a non-Gaussian distribution) was used.
  • Student’s t-test for data with a Gaussian distribution, according to a D’Agostino-Person test
  • Mann-Whitney test for data with a non-Gaussian distribution
  • RCA data > 25 pg/mL were not usable.
  • the flow cytometry histogram for FITC showed a large peak at 25 pg/mL in the last decade of the FL1-H filter (data not shwon) and for 50 pg/mL RCA, a non specific platelet population (P2, corresponding to platelet agglutination) was found outside the platelet gate (i.e. outside the forward scatter (FSC)/SSC window) (data not shwon).
  • Standardization of the RCA-based assay influence of the platelet count, RCA binding stability, platelet size, and sample storage conditions
  • Platelet count was assessed the effect of the platelet count by comparison of MFI- RCA from 3 healthy subjects before adjustment of the PRP’s platelet count (525 ⁇ 52 xlO 9 L ') and after adjustment (100, 50, 20 and 10 xlO 9 L 1 ). There were no significant differences in the MFI-RCA between the various dilutions; the MFI-RCA was 1109 ⁇ 233 with a platelet count of 10 xlO 9 L 1 and 874 ⁇ 208 in the unadjusted PRP ( Figure 2). These data demonstrate that a platelet count as low as 10 xlO 9 L 1 does not induce a variation with a RCA concentration of 12.5 pg/mL.
  • the possible presence of large and/or giant platelets means that the MFI for RCA must be normalized against platelet size [9, 25]
  • FSC the amount of laser light that passes around the cell, and is proportional to the cell’s diameter
  • GPIIb the levels of two conventional glycoproteins
  • the FSC parameter and the two glycoprotein levels were stable up for to 240 min after the incubation had been stopped ( Figure 3B-D).
  • the RCA/size ratios (RCA/FSC, RCA/GPIba and RCA/GPIIb were stable over the 4 hours of measurement ( Figure 3E-G).
  • the MFI for GPIIb was 11548 ⁇ 3172 on DO and 5963 ⁇ 2155 on Dl (p O.OOl), whereas the MFI for GPIba was 7033 ⁇ 740 on DO and 8040 ⁇ 1006 on Dl (p ⁇ 0.05).
  • the FSC parameter was the same on DO and Dl ( Figure 4D); hence, the RCA/FSC ratio was also the same on DO and Dl ( Figure 4E).
  • the median [95%CI] MFI-RCA was 1232 [652-2874] ( Figure 5A, Table 3). As expected in a population of healthy subjects, the MFI-RCA was not correlated with the platelet count or the FSC variable (data not shown). Interestingly, there were no differences between men and women or between ABO blood groups ( Figure 5B-C). The median [95%CI] FSC was 2310 [1282-3894] ( Figure 5D, Table 3). Again, there were no differences with regard to sex or ABO blood group ( Figure 5E-F). We next evaluated the variability of the RCA/FSC ratio. After rejecting outliers (7 outliers have been identified), we determined reference values for 120 healthy subjects. We found that the RCA/FSC values were not normally distributed.
  • Table 3 Statistic values of RCA, FSC and RCA/FSC ratio in the healthy population.
  • Example 2 Platelets staining with mitotracker
  • Mitochondrion in platelets were stained with the mitotracker (MitoTrackerTM Deep Red FM, Thermo Fischer Scientific, numiro de catalogue: M22426 .
  • mitotracker Mitsubishi Chemical Company, Japan LaishawTM Deep Red FM, Thermo Fischer Scientific, numiro de catalogue: M22426 .
  • PRP diluted 1/20 with PBS 95 pL was incubated with Mitotracker for 30 min at room temperature in a final volume of 100 pL. The reaction was stopped with PBS (400 pL) and then analyzed using flow cytometry (Accuri BD).
  • results The % of events measured without mitotracker was 90 ⁇ 15%, when mitotracker was added the number of events was 52 ⁇ 16%. This data suggested that in thrombocytopenic PRP, a non-specific population (without mitochondrion) was present.
  • the RCA values (MFI) were 21279 ⁇ 8626 without mitotracker and 9178 ⁇ 3546 in the presence of mitotracker (ii). These data demonstrated that using mitotracker allow to remove non-specific population that are RCA positive. The RCA values were therefore refined.

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Abstract

La présente invention se rapporte à un procédé de détermination d'une plage de référence de plaquettes d'exposition du β-galactose dans un échantillon biologique obtenu chez un sujet, consistant : i) à déterminer la concentration de ricinus communis agglutine (RCA) et la valeur de diffusion aux petits angles (FSC) dans ledit échantillon biologique ; et ii) à calculer le rapport entre RCA et FSC. Les inventeurs ont déterminé une concentration de RCA optimale de 12,5 µg/mL pour le dosage. Il est à noter que la mesure était stable pendant jusqu'à 4 heures (intensité de fluorescence moyenne (MFI)-RCA : 1233±329 à T0h et 1480±410 à T4h). Le comptage de plaquettes n'a pas induit une variation de RCA et la mesure de RCA était stable lors du test jusqu'à 24 h après la collecte de sang, ce qui démontre la robustesse du dosage (MFI-RCA : 17 1252±434 au jour 0 et 1140±297 24 heures après la prise de sang). Afin de tenir compte de la taille des plaquettes, les résultats doivent être exprimés sous la forme d'un rapport RCA/FSC. Le dosage a été ensuite utilisé pour étudier la variabilité de l'exposition du β-galactose des plaquettes dans une population de 120 adultes sains, il a été découvert que le rapport est indépendant du sexe et du groupe sanguin et une plage normale a pu être définie, ce qui constitue une étape importante avant une potentielle utilisation à grande échelle du dosage.
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