WO2017004686A1 - Utilisation de plaquettes, procédé de détection et/ou de suivi, kit de détection et/ou de suivi - Google Patents

Utilisation de plaquettes, procédé de détection et/ou de suivi, kit de détection et/ou de suivi Download PDF

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WO2017004686A1
WO2017004686A1 PCT/BR2016/050119 BR2016050119W WO2017004686A1 WO 2017004686 A1 WO2017004686 A1 WO 2017004686A1 BR 2016050119 W BR2016050119 W BR 2016050119W WO 2017004686 A1 WO2017004686 A1 WO 2017004686A1
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platelets
platelet
chemical
monolayers
monolayer
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Portuguese (pt)
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Marcos Emílio FRIZZO
José Cláudio FONSECA MOREIRA
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Universidade Federal do Rio Grande do Sul UFRGS
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Universidade Federal do Rio Grande do Sul UFRGS
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells

Definitions

  • the present invention describes the use of platelets in an in vitro chemical and / or biological substance evaluation process, an in vitro chemical and / or biological substance detection and / or tracking process, and a detection and / or in vitro screening of chemical and / or biological substance.
  • the platelets are arranged in a monolayer.
  • the present invention is in the fields of Pharmacy, Medicine and Chemistry.
  • Human platelets have several functional similarities with neural cells (neurons and astrocytes) and have therefore been used as peripheral indicators of central nervous system (CNS) changes associated with different pathologies such as Alzheimer's (Ferrarese C, Begni B, Canevari C, Zoia C, Piolti R, Frigo M, Appollon I, Frattola L. 2000. Glutamate uptake is decreased in platelets from Alzheimer's disease patients. Ann Neurol 47: 641 -643.), Parkinson's (Ferrarese C, Zoia C, Pecora N, Piolti R, Frigo M, Bianchi G, Room G, Begni B, Riva R, Frattola L.
  • CNS central nervous system
  • US 6,087,118 (Aronson et al.) Discloses a method for diagnosing Alzheimer's disease using human blood platelets for diagnosing Alzheimer's disease.
  • the invention describes a method for immobilizing human platelets in a filter where pharmacological tests are performed to verify the behavior of calcium-dependent potassium channels.
  • US 6,087,118 to Aronson et al. does not disclose or suggest detection and / or screening chemical and / or biological substances with the step of contacting said substance with monolayer platelets which are not activated.
  • the present invention aims at solving the constant problems in the state of the art from the use of platelets in chemical and / or biological evaluation or screening processes / methods and wherein said platelets are in monolayer. and are not enabled.
  • Another object of the present invention is a chemical and / or biological in vitro detection and / or screening method / method comprising the step of contacting at least one chemical and / or biological substance with a monolayer platelet culture in vitro.
  • Another object of the present invention is a chemical and / or biological in vitro detection and / or tracking kit comprising:
  • inventive concept common to all claimed protection contexts refers to the use of platelets in chemical and / or biological evaluation or screening processes / methods.
  • Figure 1 shows an image of the human platelet monolayer in vitro. Platelets were seeded in 96 well plates at a density of 6 40.10 per well and maintained at room temperature for 4 h or for five days at 4 ° C.
  • Figure 3 shows the amount of protein in human platelet monolayers. Platelets were seeded in 96-well plates and kept at room temperature for 24 h, incubation medium was removed and the amount of protein in the monolayers was determined by the Bradford method. Samples from each donor containing the same cell density were evaluated immediately after obtaining the PC, without being plated (time 0 h). The amount of protein obtained in these monolayers was considered to be 100% and compared to the 24 h monolayer data from the respective donors.
  • Figure 4 shows the determination of the effect of repeated washes on the amount of human platelet monolayer protein.
  • Platelets were seeded in 96 well plates at a density of 6 20:10 and kept at room temperature for 4 h.
  • the incubation medium 150 ⁇ _
  • This washing procedure was performed three times and after the monolayers were lysed to determine the amount of protein by the Bradford method.
  • Samples from each donor containing the same cell density were evaluated immediately after 4 h without being washed. The amount of protein obtained in these unwashed monolayers was considered to be 100% and compared to the washed donor monolayer data of the respective donors.
  • Figure 7 shows the transport of glutamate by suspended and monolayer platelets.
  • Panel A represents glutamate uptake obtained in tubes immediately after obtaining cells and in plates after three days.
  • Figure 8 shows the transport of glutamate in monolayers.
  • Figure 10 shows the viability of platelets after the incubation period, assessed by determining the reducing activity with the MTT technique.
  • Figure 11 shows the viability of platelets after the incubation period assessed by determining MTT reduction.
  • BDNF brain derived neurotrophic factor
  • the chemical and / or biological substance is a pharmacologically active substance.
  • the pharmacologically active substance is of action in the central and / or peripheral nervous system.
  • the pharmacologically active substance is glutamatergic, GABAergic, serotonergic and / or dopaminergic.
  • the pharmacologically active substance is a neuropharmaceutical selected from the group consisting of: riluzole, sertraline, fluoxetine, ceftriaxone, carbamazepine, topiramate and lamotrigine.
  • Another object of the present invention is a chemical and / or biological in vitro detection and / or screening method / method comprising the step of contacting at least one chemical and / or biological substance with a monolayer platelet culture in vitro.
  • the chemical and / or biological substance is a pharmacologically active substance.
  • the pharmacologically active substance is of action in the central and / or peripheral nervous system.
  • the pharmacologically active substance is glutamatergic, GABAergic, serotonergic and / or dopaminergic.
  • the pharmacologically active substance is a neuropharmaceutical selected from the group consisting of: riluzole, sertraline, fluoxetine, ceftriaxone, carbamazepine, topiramate and lamotrigine.
  • Another object of the present invention is a chemical and / or biological substance in vitro detection and / or tracking kit comprising:
  • the chemical and / or biological substance is a pharmacologically active substance.
  • the pharmacologically active substance is of action in the central and / or peripheral nervous system.
  • the pharmacologically active substance is glutamatergic, GABAergic, serotonergic and / or dopaminergic.
  • the pharmacologically active substance is a neuropharmaceutical selected from the group consisting of: riluzole, sertraline, fluoxetine, ceftriaxone, carbamazepine, topiramate and lamotrigine.
  • Chemical and / or biological substance refers to any chemicals, for example, that are of interest for toxicological evaluation and also refers to drugs such as, for example. , neuropharmaceuticals comprising riluzole, sertraline, fluoxetine, ceftriaxone, carbamazepine, topiramate and lamotrigine, as well as other central nervous system (CNS) or peripheral (SNP) drugs, such as glutamatergic drugs, GABAergic, serotoninergic and / or dopaminergic action, among other types of action.
  • Biological substances include proteins, peptides, antibodies, hormones, biological synthesis intermediates, as well as biological nanocomposites and biological nanostructures.
  • the term "In vitro chemical and / or biological substance evaluation process” refers to an in vitro (ie ex vivo, out-of-body) evaluation process / method for evaluation of chemicals such as, but not limited to, chemicals of interest for toxicological and / or pharmacological evaluation.
  • Chemicals include drugs such as riluzole, sertraline, fluoxetine, ceftriaxone, carbamazepine, topiramate and lamotrigine, as well as other central nervous system (CNS) or peripheral (SNP) drugs glutamatergic, GABAergic, serotonergic and / or dopaminergic drugs, among others.
  • Chemical and / or biological in vitro screening methods / methods as well as chemical and / or biological in vitro detection methods / methods are also encompassed by said term.
  • In vitro chemical and / or biological substance detection and / or tracking process refers to a process / method for identifying potential pharmacological applications of the chemical and / or biological, including bioactive or non-endogenous substances and synthetic drugs.
  • Platelets being arranged in a monolayer
  • Platelets being arranged in a monolayer refers to the fact that platelets are grown ex vivo and / or preserved ex vivo in culture medium suitable for survival. platelets, said platelets being in a single layer on the cultivation surface (ie being in a monolayer) even without being activated. It is also to be understood that, in the context of the present patent application, platelets may be of human or other mammalian origin. It should also be understood that the surface on which the monolayer is formed may be any surface suitable for such purpose, for example plastic surfaces.
  • neuropharmaceutical refers to any type of chemical and / or biological substance having action on the central nervous system (CNS) and / or peripheral nervous system (PNS).
  • CNS central nervous system
  • PNS peripheral nervous system
  • the term includes, but is not limited to, glutamatergic, GABAergic, serotonergic and / or dopaminergic drugs.
  • pharmacologically active substance refers to any chemical and / or biological substance that has potential pharmacological action / effect in humans or any other living being.
  • pharmacologically active substance includes neuropharmaceuticals such as riluzole, sertraline, fluoxetine, ceftriaxone, carbamazepine, topiramate and lamotrigine as well as other central nervous system (CNS) drugs such as for example, glutamatergic, GABAergic, serotonergic and / or dopaminergic drugs, among others.
  • CNS central nervous system
  • in vitro chemical and / or biological substance detection kit refers to a kit for the purpose of detection and / or tracking in in vitro detection processes / methods.
  • chemical and / or biological substance such as, for example, neuropharmaceuticals comprising riluzole, sertraline, fluoxetine, ceftriaxone, carbamazepine, topiramate and lamotrigine, as well as other glutamatergic drugs, as well as endogenous substances such as hormones, peptides , proteins, etc.
  • the advantages provided by the present invention are numerous. Among the advantages are: applications in toxicological evaluations, application as a substitute model for the use of animals in pharmacological tests, application in screening of drugs or chemical agents, pharmacological tests of equivalence or second use (new therapeutic applications for drugs already reprofiling) and other technical advantages that will be readily recognized by one of ordinary skill in the art based on the knowledge disclosed herein in this patent application.
  • Platelet parameters are determined using a hematology analyzer.
  • PRP platelet rich plasma
  • PC platelet concentrate
  • Platelet parameters are determined on the PC using a hematology analyzer.
  • a known density of human platelets is transferred in sucrose buffer to multiwell plate wells.
  • Examples 1-4 relate to the conditions of platelet monolayers and demonstrate fundamental characteristics for understanding the invention, such as cell parameters, adhesiveness and resistance to incubation medium changes.
  • Examples 5-8 demonstrate that platelet monolayers maintain an important energy-dependent biological function (glutamate uptake), which has been widely used in the scientific literature as a correspondence of a CNS condition.
  • Example 6 even compares the transport of glutamate obtained with the method described in the scientific literature (suspended cells) with that obtained with the present invention (adhered cells).
  • Example 8 demonstrates an as yet undescribed biological effect of a commercial drug on glutamate uptake.
  • Examples 9 and 10 demonstrate the viability of platelet monolayers at different times and conditions, including with pharmacological treatment.
  • Examples 14 and 15 determine BDNF secretion of platelet monolayers at different times and conditions, including with pharmacological treatment.
  • Example 15 demonstrates a novel biological effect of a commercial drug on BDNF release discovered using the platelet monolayer model.
  • sucrose buffer and Tris citrate are used, but the use of alternative solutions is not ruled out.
  • Sucrose Buffer (mM composition): Sucrose (320); NaCl (137); KCI (2.7); KH 2 PO 4 (1.5); NaH 2 PO 4 (8.1).
  • Tris-citrate Buffer NaCl (11.8); KCI (4.5); MgSO 4 (1.1); KH 2 PO 4 (1,1); Tris-HCl (25); Na-citrate (11); D-glucose (10,2).
  • Results obtained from 8 ml blood samples are described in the present invention, but the volume may be smaller (4 ml) since the model uses a known cell density.
  • the mean platelet volume (MPV), platelet distribution range (PDW), platelet count (PCT), and platelet number by volume (PLT) indices are obtained with a hematology analyzer, eg Horiba (Micros ES60) and are essential for adjusting platelet density, assessing whether platelets are activated and whether any subpopulation has been selected during sample processing.
  • the VPM, PDW, PCT and PLT indices are determined in whole blood and again after cell processing in platelet concentrate (PC), thus, possible variations in these parameters can be evaluated (Table 1).
  • VPM, PDW, PCT and PLT indices of the sample are determined in whole blood using the hematology analyzer.
  • the tubes are centrifuged (1400 rpm for 5 min at 4 Q C) to obtain platelet - rich plasma (PRP).
  • minitube are centrifuged (8400 rpm for 10 min at 4 Q C). The supernatant from both tubes is discarded.
  • PRP is resuspended in 0.5 mL sucrose buffer (0.32 M, pH 7.4). Is added another 0.5 ml of the same buffer (4 C Q). Identical procedure is performed in the other mini-tube.
  • minitubes are centrifuged (8400 rpm for 5 min at 4 Q C). The supernatant from the two mini-tubes is discarded.
  • VPM, PDW, PCT and PLT are determined on the PC using hematology analyzer.
  • Example 2 Representative Image of Platelet Monolayer
  • VPM, PDW, PCT and PLT indices of the sample are determined in whole blood using the hematology analyzer.
  • minitube are centrifuged (8400 rpm for 10 min at 4 Q C). The supernatant from both tubes is discarded.
  • PRP is resuspended in 0.5 mL sucrose buffer (0.32 M, pH 7.4). Is added another 0.5 ml of the same buffer (4 C Q). Identical procedure is performed in the other mini-tube.
  • minitubes are centrifuged (8400 rpm for 5 min at 4 Q C). The supernatant from the two mini-tubes is discarded.
  • VPM, PDW, PCT and PLT are determined on the PC using hematology analyzer.
  • a density 40.10 6 platelets is transferred to a well of a 96 well plate in a volume of 50 ⁇ sucrose buffer (320 mM, pH 7.4) at 4 Q C.
  • VPM, PDW, PCT, and PLT indices of the sample are determined in whole blood using the blood analyzer.
  • the tubes are centrifuged (1400 rpm for 5 min at 4 Q C) to obtain the PRP.
  • minitube are centrifuged (8400 rpm for 10 min at 4 Q C). The supernatant from both tubes is discarded.
  • PRP is resuspended in 0.5 mL sucrose buffer (0.32 M, pH 7.4). Is added another 0.5 ml of the same buffer (4 C Q). Identical procedure is performed in the other mini-tube.
  • minitubes are centrifuged (8400 rpm for 5 min at 4 Q C). The supernatant from the two mini-tubes is discarded.
  • the volumes of the 2 minitubes are pooled originating the PC.
  • Platelet parameters VPM, PDW, PCT and PLT are determined on the PC using hematology analyzer. 1 1. Densities of 20, 40 and 60.10 6 platelets are transferred to wells of a 96 well plate in amounts of 50 ⁇ _ sucrose buffer (320 mM, pH 7.4) at 4 Q C.
  • total protein quantification can be performed on monolayers maintained for times greater than 04 (four) hours, for example, example 24 (twenty four) hours ( Figure 3).
  • VPM, PDW, PCT and PLT indices of the sample are determined in whole blood using the hematology analyzer.
  • PRP is resuspended in 0.5 mL sucrose buffer (0.32 M, pH 7.4). Is added another 0.5 ml of the same buffer (4 C Q). Identical procedure is performed in the other mini-tube.
  • minitubes are centrifuged (8400 rpm for 5 min at 4 Q C). The supernatant from the two mini-tubes is discarded.
  • VPM, PDW, PCT and PLT are determined on the PC using hematology analyzer.
  • Tris-citrate buffer pH 6.5
  • Example 5 Uptake of qlutamate in platelet monolayers
  • an energy dependent cellular transport system is used. The results show that platelet monolayers are biologically functional ( Figures 5 and 6).
  • VPM, PDW, PCT and PLT indices of the sample are determined in whole blood using the hematology analyzer.
  • minitube are centrifuged (8400 rpm for 10 min at 4 Q C). The supernatant from both tubes is discarded.
  • PRP is resuspended in 0.5 mL sucrose buffer (0.32 M, pH 7.4). Is added another 0.5 ml of the same buffer (4 C Q). Identical procedure is performed in the other mini-tube.
  • minitubes are centrifuged (8400 rpm for 5 min at 4 Q C). The supernatant from the two mini-tubes is discarded.
  • VPM, PDW, PCT and PLT are determined on the PC using hematology analyzer.
  • Example 6 Uptake of Glutamate by Suspended and Monolayer Platelets
  • glutamate (10 ⁇ ) transport capacity of suspended (so-called classical) and monolayer platelets (the present invention) was compared.
  • the protocol performed on tubes was described by Mangano & Schwarcz, 1981.
  • the protocol used to obtain and treat the monolayers was described in Example 5.
  • platelets obtained from the same donor were used, and the same cell density was used in both methods. The results obtained with both protocols are presented in table 2 below and figure 7.
  • VPM, PDW, PCT and PLT indices of the sample are determined in whole blood using the hematology analyzer.
  • minitube are centrifuged (8400 rpm for 10 min at 4 Q C). The supernatant from both tubes is discarded.
  • PRP is resuspended in 0.5 mL sucrose buffer (0.32 M, pH 7.4). Is added another 0.5 ml of the same buffer (4 C Q). Identical procedure is performed in the other mini-tube.
  • minitube are centrifuged (8400 rpm for 5 min at 4 Q C). The supernatant from the two mini-tubes is discarded. 8) The resulting pellet (obtained PRP) of each minitubo resuspended in 1/5 initial volume of PRP obtained (centrifugation described under item 3) sucrose in buffer (320 mM, pH 7.4) at 4 Q C.
  • VPM, PDW, PCT and PLT are determined on the PC using hematology analyzer.
  • Tris-citrate buffer pH 6.5
  • the glycogen of human platelet monolayers was quantified at different times ( Figure 14). Platelets are obtained according to example 1 and the glycogen content assay comprises the following steps: (1) cell lysis and glycogen extraction, (2) glycogen precipitation and washing, (3) glycogen to glucose conversion free, (4) neutralization, and (5) free glucose detection.
  • Step 1 To lyse the platelets, 0.25 mL KOH (30%) is added and the suspension is homogenized with repeated passes (15x) by 1 mL plastic tip. The volume is transferred to a conical tube to which an additional 0.25 mL KOH (30%) is added and the mixture is again homogenized (15x). For glycogen extraction the tube is boiled for 1 h and 50 ⁇ saturated Na 2 SO 4 (35%) is added.
  • Step 2 The obtained glycogen is precipitated with 1 mL of anhydrous ethanol and the tube is shaken rapidly on the vortex shaker. Then the tube is centrifuged 750 x g for 10 min, the supernatant is carefully discarded and the precipitate resuspended in 0.5 mL of distilled water. The precipitation and washing steps were repeated as described above and glycogen was finally resuspended in 0.3 mL of distilled water for determination.
  • Step 3 The obtained total glycogen was converted to free glucose by acid hydrolysis.
  • a standard curve is established from a stock solution (1 mg.mL "1 ) of glycogen type III (Sigma), and different amounts of glycogen, 50 (S1), 100 (S2), 150 (S3) and 200 pg (S4) are used as a reference.Glycogen obtained from the sample and that used on the standard curve are hydrolyzed with boiling HCl (4 N) for 1 h.In this process 0.3 mL of HCl is added to S1, S2, S3, S4 and the sample.
  • Step 4 After the mixture has cooled, 0.3 ml Na 2 CO 3 (2 M) is slowly added at each point of the standard curve and also in the sample to neutralize the solution. This process produces large bubble formation and must be performed carefully.
  • Step 5 After neutralization, free glucose obtained from standard glycogen hydrolysis (S1-S4) and sample is determined using commercial glucose-oxicity kit. For this, a 40 ⁇ _ aliquot of the sample (or standard curve) is incubated with the reagent enzyme for 5 min at 37 ° C. The absorbance is read at 505 nm.
  • Platelets are obtained according to example 1. Platelet parameters VPM, PDW, PCT and PLT are determined on the PC using hematology analyzer. A certain density platelets is transferred to a well of a 96 well plate in a volume of 50 ⁇ _ sucrose buffer (320 mM, pH 7.4) at 4 Q C. is added a volume of 100 ⁇ _ Tris buffer citrate (pH 6.5) at 4 Q C. After 4 h at room temperature The medium is replaced by lysis buffer for cytosolic ATP extraction ( Figure 15). Total ATP quantification utilizes Bioluminescent Commercial Kit (Example: ATP Determination Invitrogen Kit, A22066) or other commercial detection technique.
  • Lysis Buffer PBS (pH 7.8); sodium azide (2 mM); Triton-X 100 (1%). Commercial product may be used.
  • Platelets are obtained according to example 1. Platelet parameters VPM, PDW, PCT and PLT are determined on the PC using a hematology analyzer. A density platelet 20:10 6 is transferred to a well of a 96 well plate in a volume of 50 ⁇ _ sucrose buffer (320 mM, pH 7.4) at 4 Q C. is added a volume of 100 ⁇ _ Tris buffer -citrate (pH 6.5) at 4 Q C. The drug was diluted in Tris-citrate (pH 6.5). After 24 h at room temperature the medium is replaced by lysis buffer for cytosolic ATP extraction (Figure 16). Total ATP quantification utilizes Bioluminescent Commercial Kit (eg ATP Determination Invitrogen Kit, A22066) or other commercial detection technique.
  • Bioluminescent Commercial Kit eg ATP Determination Invitrogen Kit, A22066
  • Lysis Buffer PBS (pH 7.8); sodium azide (2 mM); Triton-X 100 (1%). Commercial product may be used.
  • Platelets are obtained according to example 1. Platelet parameters VPM, PDW, PCT and PLT are determined on the PC using hematology analyzer. A density platelet 20:10 6 is transferred to a well of a 96 well plate in a volume of 50 ⁇ _ sucrose buffer (320 mM, pH 7.4) at 4 Q C. is added a volume of 100 ⁇ _ Tris buffer -citrate (pH 6.5) at 4 Q C. After 4 h (or later time) at room temperature through an aliquot is removed for determination of BDNF released (figure 17). Note: In case of drug addition it should be diluted in Tris-citrate (pH 6.5).
  • BDNF levels in the supernatant were measured with the ChemiKine TM Brain Derived Neurotrophic Factor Sandwich ELISA kit (Millipore, USA) following the manufacturer's instructions, however, another commercial kit may also be used. All measurements were performed in triplicate in 96-well plates and a standard curve was calculated for each experiment. In the determination of BDNF, supernatant samples were diluted 1: 16 in PBS (pH 7.4). The optical density of each well was measured using a microplate reader (EZ Read 400, Biochrom, UK) at 450 nm and data were analyzed with Galapagos software (Biochrom, UK).
  • Platelets are obtained according to example 1. Platelet parameters VPM, PDW, PCT and PLT are determined on the PC using hematology analyzer. A density platelet 20:10 6 is transferred to a well of a 96 well plate in a volume of 50 ⁇ _ sucrose buffer (320 mM, pH 7.4) at 4 Q C. is added a volume of 100 ⁇ _ Tris buffer -citrate (pH 6.5) at 4 Q C.
  • BDNF Brain Derived Neurotrophic Factor Sandwich ELISA kit
  • All measurements were performed in triplicate in 96-well plates and a standard curve was calculated for each experiment.
  • supernatant samples were diluted 1: 16 in PBS (pH 7.4).
  • the optical density of each well was measured using a Microplate reader (EZ Read 400, Biochrom, UK) at 450 nm and data were analyzed with Galapagos software (Biochrom, UK).
  • the exemplary embodiments of the invention described above prove the use of platelets in an in vitro chemical and / or biological substance evaluation process and it is therefore entirely feasible to apply the present invention to any bioactive drug / molecule. (not limited to neuropharmaceuticals only) and also for any efficacy comparison and drug equivalence testing (eg comparison between generic drug and a reference drug).

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Abstract

La présente invention concerne l'utilisation de plaquettes dans un procédé d'évaluation in vitro d'une substance chimique et/ou biologique, un procédé de détection et/ou de suivi in vitro d'une substance chimique et/ou biologique, et un kit de détection et/ou de suivi in vitro d'une substance chimique et/ou biologique. Plus particulièrement, les plaquettes sont disposées dans une monocouche. La présente invention trouve une application dans les domaines de la pharmacie, de la médecine et de la chimie.
PCT/BR2016/050119 2015-07-08 2016-06-01 Utilisation de plaquettes, procédé de détection et/ou de suivi, kit de détection et/ou de suivi Ceased WO2017004686A1 (fr)

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BRBR102015016451-3 2015-07-08
BR102015016451A BR102015016451A2 (pt) 2015-07-08 2015-07-08 uso de plaquetas, processo de detecção e/ou de rastreamento, kit de detecção e/ou de rastreamento

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