WO2021199037A1 - Methods of determining activity of activated protein c - Google Patents

Methods of determining activity of activated protein c Download PDF

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Publication number
WO2021199037A1
WO2021199037A1 PCT/IL2021/050355 IL2021050355W WO2021199037A1 WO 2021199037 A1 WO2021199037 A1 WO 2021199037A1 IL 2021050355 W IL2021050355 W IL 2021050355W WO 2021199037 A1 WO2021199037 A1 WO 2021199037A1
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apc
activity
disease
composition
diseases
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PCT/IL2021/050355
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French (fr)
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Joab Chapman
Efrat SHAVIT STEIN
Valeria GOLDERMAN
Nicola MAGGIO
Amir Dori
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Tel Hashomer Medical Research Infrastructure And Services Ltd.
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Publication of WO2021199037A1 publication Critical patent/WO2021199037A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21005—Thrombin (3.4.21.5)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6432—Coagulation factor Xa (3.4.21.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6464—Protein C (3.4.21.69)
    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21006—Coagulation factor Xa (3.4.21.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21069—Protein C activated (3.4.21.69)
    • 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
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/81—Protease inhibitors
    • G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • G01N2333/811—Serine protease (E.C. 3.4.21) inhibitors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96433—Serine endopeptidases (3.4.21)
    • G01N2333/96441—Serine endopeptidases (3.4.21) with definite EC number
    • G01N2333/96461—Protein C (3.4.21.69)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/26—Infectious diseases, e.g. generalised sepsis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention in some embodiments thereof, relates to methods of determining activity of activated protein C.
  • Neural inflammation is a core process in many pathologies, including neurodegenerative diseases [1], neoplasms [2], and nervous system reaction following trauma [3]. Inflammation and coagulation are tightly linked and modify each other in the systemic circulation [4-6]. The role of coagulation system proteins in neural function has been intensively studied and found to affect many processes including synaptic transmission, axonal conduction, learning, memory and behavior [7-9].
  • Activated protein C is a serine protease participating in the coagulation cascade. Following clot formation, thrombin binds thrombomodulin (TM) and activates protein C (PC) to aPC. aPC inhibits FV and FVII which are essential for thrombin generation and thus acts as an anticoagulation factor [10]. Similar to other coagulation factors, aPC has a role in the inflammatory response. aPC inhibits pro-inflammatory cytokines, protects endothelial cells structure and function [11], and maintains normal blood pressure during sepsis [12].
  • aPC requires two receptors for its activation and its cellular effects; the endothelial protein C receptor (EPCR) and protease activator receptor 1 (PARI).
  • EPCR serves as a catalyst of PC activation and mediates aPC activity [10].
  • aPC cleaves PARI, and induce a positive effect in endothelial and Schwann cells [13,14].
  • EPCR and PARI are expressed in both the peripheral nervous system (PNS) [15] and the central nervous system (CNS) [16].
  • the measurement of aPC activity is therefore potentially important for understanding neural function in neuroinflammation.
  • the accepted laboratory method measures aPC activity indirectly, using an in-vitro activation of PC into aPC by exposure to venom of southern copperhead snakes [17]. This method is expensive due to the use of snake’s venom, and only measures aPC activity indirectly. Furthermore, although appropriate for the high plasma levels, this method is not sensitive enough to measure aPC activity levels in neural tissue.
  • Pefafluor PCa Pyr-Pro-Arg-AMC
  • activated Protein C e.g., Biocip Praha, P089-05-25MG
  • aPC activated protein C
  • the conditions in which activity of the thrombin and activity of the Factor Xa (FXa) are inhibited comprise an inhibitor of thrombin and an inhibitor of Factor Xa (FXa).
  • composition of matter comprising a biological sample which putatively comprises aPC, a substrate of the aPC, the substrate comprising a detectable moiety, and an inhibitor of thrombin and an inhibitor of Factor Xa (FXa).
  • the inhibitor of thrombin comprises NAPAP, hirudin and/or TLCK.
  • the inhibitor of Factor Xa comprises apixaban and/or Rivaroxaban.
  • the method further comprises generating a calibration curve for the activity of the aPC.
  • the sample comprises a neural tissue.
  • the sample comprises a neural cell or cell line.
  • the sample comprises a cerebrospinal fluid (CSF).
  • CSF cerebrospinal fluid
  • the sample comprises plasma.
  • the sample comprises a conditioned medium of any one of a neural cell and a neural tissue.
  • the substrate and the detectable moiety compose a molecule of formula I:
  • Z is a protecting group
  • X is an amino acid sequence cleaved by aPC
  • Y is a detectable moiety, wherein Z, X, Y are covalently linked.
  • the detectable moiety comprises any one of a chromogenic, fluorescent, chemiluminescent, or phosphorescent group.
  • the Z is selected from the group consisting of Pyroglutamic acid (Pyr), BOC, (CH3)3COCO-, t-BOC), t-amyloxycarbonyl, adamantyl-oxycarbonyl, and p-methoxybenzyloxycarbonyl, 9-fluorenylmethoxycarbonyl (FMOC), 2-chlorobenzyloxycarbonyl and the like, nitro, tosyl (CH3C6H4S02 — ), benzyloxycarbonyl (CBZ), adamantyloxycarbonyl, 2,2,5,7,8-pentamethylchroman-6-sulfonyl, 2,3,6-trimethyl-4-methoxyphenylsulfonyl, t-butyl benzyl (BZL) or substituted BZL, such as, p- methoxybenzyl, p-nitrobenzyl, p-chlorobenzyl
  • the fluorescent group is selected from the group consisting of AMC and rhodamin-110.
  • the X is Pro-Arg.
  • the molecule comprises Pyr-Pro-Arg-
  • the X is a dipeptide, tripeptide.
  • the X is up to 10 amino acids in length.
  • the method is performed in a multi well plate.
  • the determining comprises photometrically measuring.
  • aPC activated Protein C
  • aPC activated Protein C activity
  • aPC activated Protein C
  • the disease is selected from the group consisting of sepsis, infectious disease, inflammatory disease, neurodegenerative disease and nerve injury.
  • aPC activated Protein C
  • aPC activated protein C
  • FIGs. 1A-D show substrate cleavage and measurement validation:
  • B. Substrate cleavage by FXa: FXa (10 mU) cleaves the APC substrate in a non-APC manner (n 4).
  • NAPAP significantly decreases the non-APC substrate cleavage by FXa (n 4, p ⁇ 0.0005).
  • Apixaban and Apixaban with NAPAP completely block the non-APC substrate cleavage by FXa (n 4, p ⁇ 0.0001).
  • FIGs. 2A-F show APC activity in N9 and C6 cells in normal conditions and LPS model:
  • B. APC activity in the medium and on the cells of N9 and C6 cells: APC activity is significantly higher in the medium compared to APC activity on the cells of N9 and C6 cells (n 8, p ⁇ 0.0001).
  • FIGs. 4A-D show aPC activity in a mice model for systemic inflammation and in human patients:
  • A. Weight decline in EPS injected mice: EPS injected mice demonstrated a significant loss of weight 24 hours following the injection, as a marker for the systemic inflammatory response (n 7, p ⁇ 0.0001).
  • FIG. 5 is a schematic illustration of an embodiment of the present invention.
  • Neural culture secretes various proteases, including activated Protein C (aPC), that maintain homeostasis and respond during inflammatory events.
  • aPC activated Protein C
  • the present inventors developed highly sensitive aPC activity assay, which utilizes florescence labeled substrate and nonspecific proteases inhibitors to measure aPC activity in neural cultures in health and disease.
  • the Figure was created by the software BioRender.
  • FIG. 6 shows the cleavage site composition of aPC.
  • FIGs. 9A-C show the use of aPC activity assay for drug screening:
  • Figure 9A FEAM1-4: novel peptides for modulation of aPC/FVII/EPCR.
  • the present invention in some embodiments thereof, relates to methods of determining activity of activated protein C.
  • aPC activity assays that are available for clinical use are designed mainly for diagnosis of coagulopathies in plasma [25].
  • the present inventors have devised a novel method for aPC activity measurement using the specific amino acid sequence cleaved by aPC in conjugation with a fluorogenic emitting substrate.
  • This novel method is sensitive enough to detect direct aPC activity without the need of prior PC activation.
  • the present inventors measured for the first time, intrinsic aPC activity in neural cell lines, mouse brain and human CSF, and were able to demonstrate significant changes in aPC activity in models of neuroinflammation.
  • the present inventors have established appropriate conditions that enable to distinguish aPC activity from other serine proteases known to cleave the substrate. As is illustrated in Examples 2 and 3 of the Examples section, the present results indicate the use of thrombin and FXa inhibitors in this aPC assay. Indeed, in the presence of these inhibitors, aPC activity levels in human plasma can be measured in a reliable manner. Human plasma contains significantly higher levels of PC compared to aPC [24]. Addition of the potent PC activator, ProtacTM, causes the expected elevation in the measured fluorescence thus supporting the specificity of this method (Figure ID).
  • aPC activity assays that are available for clinical use are designed mainly for diagnosis of coagulopathies in plasma [25].
  • This novel assay indeed enables detection of aPC activity in neural cell lines. Demonstrated herein, for the first time, that N9 microglia cells and C6 glioma cells have intrinsic aPC activity.
  • aPC activity varies between those cell types being higher in N9 microglia cells [27], compared to C6 astrocytic cells.
  • Microglia are immune cells, taking part in inflammatory responses and neuroregenerative changes [28].
  • aPC has been shown to induce neuroregeneration [29] but the possibility that it is produced locally by the neural tissue was not studied before.
  • the present results demonstrating elevated aPC activity in microglia cells may suggest a possible involvement of this pathway in microglial induced neuroregeneration.
  • Further aPC activity was localized to their surrounding medium. aPC activity levels in both cell types are higher in the medium, suggesting the secretion of aPC, as a soluble molecule or as part of an extracellular vesicle. This may be part of a complex interaction process with neighboring cells [24,30].
  • aPC activity was also measured ex-vivo. Measurements of aPC in mice brains show elevated aPC activity levels in the posterior brain sections (see Example 4).
  • aPC activated protein C
  • protein C also known as “autoprothrombin IIA” and “blood coagulation factor XIX”, is a zymogen, the activated form of which plays an important role in regulating anticoagulation, inflammation, and cell death and maintaining the permeability of blood vessel walls in humans and other animals.
  • activated protein C also abbreviated as “PCa” or “aPC” performs the functions of protein C primarily by proteolytically inactivating proteins Lactor V a and Lactor Vill a .
  • aPC is classified as a serine protease since it contains a residue of serine in its active site.
  • protein C is encoded by the PROC gene, which is found on chromosome 2.
  • the present teachings refer to any protein C or aPC of any organism that shares the substrate of human or mouse activated protein C.
  • substrate refers to the amino acid sequence that is cleaved by activated protein C.
  • Activated protein C cleaves natural substrates with arginine in the PI position, whereas the amino acid composition in the P2 and P3 positions are more varied.
  • Ligure 6 shows the substrate structure of aPC.
  • substrates that can be used in accordance with the present teachings include, but are not limited to, Pro-Arg, Glu-Pro-Arg, Val-Leu-Arg, Ile-Pro-Arg, Phe-Pip-Arg.
  • the amino acid sequence of the cleavage site is Pro-Arg.
  • activity refers to serine protease activity.
  • the method is performed without activation of the protein prior to determining the activity.
  • the method is performed with activation of the protein prior to determining the activity.
  • Methods of activating protein C are well known in the art.
  • the determination of protein C can be facilitated by the use of the specific protein C activator, Protac®.
  • the activator is a serine protease isolated and purified from the venom of the southern copperhead snake, Agkistrodon c. contortrix. It rapidly activates both human and bovine protein C, probably via the same mechanism as thrombin, without interfering with other coagulation factors. Since the activation is rapid, it minimizes the efficiency of the protein C inhibitors and thus eliminates the need for isolating protein C in an adsorption step.
  • a biological sample refers to a sample which may comprise protein C preferably in an activated form. This can be a sample derived from a subject. Biological samples include, but are not limited to, body fluids such as whole blood, serum, plasma, cerebrospinal fluid (CSF), urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk as well as white blood cells, tissues, amniotic fluid and chorionic villi. According to a specific embodiment, the sample comprises a tissue/cell culture.
  • body fluids such as whole blood, serum, plasma, cerebrospinal fluid (CSF), urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk as well as white blood cells, tissues, amniotic fluid and chorionic villi.
  • the sample comprises a tissue/cell culture.
  • the sample is freshly tested (e.g., less than 48 or 24 hours following retrieval).
  • the sample is tested following cryopreservation.
  • the sample comprises a neural tissue.
  • neuroneural refers to neurons or glial cells.
  • Glia also called glial cells or neuroglia, are non-neuronal cells in the central nervous system (brain and spinal cord) and the peripheral nervous system that do not produce electrical impulses. They maintain homeostasis, form myelin, and provide support and protection for neurons.
  • glial cells include oligodendrocytes, astrocytes, ependymal cells, and microglia
  • peripheral nervous system glial cells include Schwann cells and satellite cells
  • the sample comprises a cerebrospinal fluid (CSF).
  • CSF cerebrospinal fluid
  • the sample does not comprise plasma.
  • the sample comprises plasma.
  • the sample comprises a conditioned medium of any one of a neural cell and a neural tissue, such a conditioned medium is also referred to a PCa not associated with cells.
  • the sample comprises a neural cell or cell line.
  • neural cell lines are well known in the art, so are listed in the Examples section, e.g., glioma cell lines such as C6 and CNS1, neuronal cells such as PC12 and N2A, macrophages such as J774, RAW264.7, endothelial cell lines such as HUVEC and C9 microglia cells such as N9, Schwann cell lines such as STS26T and ST88-14.
  • a cell refers to a native cell or to a cell line.
  • the cell is genetically modified (GMO).
  • the cell is non-GMO.
  • the protein C activity is associated to a cell (e.g., a neural cell).
  • the protein C activity is in a medium of a cell e.g., in a cell-free sample.
  • the biological sample is contacted with a substrate of aPC under conditions in which the activity of Factor Xa and thrombin are inhibited.
  • Thrombin substrate sequence Gly-Pro-Arg
  • Factor Xa substrate sequence Glu-Gly-Arg
  • aPC share substrate specificity due to similarity in cleavage sites sequences.
  • the background signal of substrate cleavage by non-aPC enzymes is decreased under conditions which decrease the activity of these enzymes such as Factor Xa and Thrombin.
  • Such conditions include the use of specific or non-specific inhibitors of Factor Xa and Thrombin. Measures are taken not to use inhibitors or reaction conditions which inhibit activity of protein C.
  • inhibitors include, but are not limited to, an inhibitor of thrombin which comprises for example, NAPAP, hirudin and/or Na-p-tosyl-F-lysine chloromethyl ketone (TFCK) and an inhibitor of Factor Xa which comprises for example apixaban and/or Rivaroxaban.
  • an inhibitor of thrombin which comprises for example, NAPAP, hirudin and/or Na-p-tosyl-F-lysine chloromethyl ketone (TFCK)
  • TFCK Na-p-tosyl-F-lysine chloromethyl ketone
  • Factor Xa which comprises for example apixaban and/or Rivaroxaban.
  • the substrate comprises a detectable moiety.
  • the detectable moiety comprises a chromogenic group.
  • chromogenic group comprises molecules that generate a chromogenic, fluorescent or chemiluminescent signal.
  • the detectable moiety generates a chromogenic signal.
  • the detectable moiety generates a fluorescent signal. According to a specific embodiment, the detectable moiety generates a chemiluminescent signal.
  • chromogenic groups examples include, but are not limited to non-protein organic fluorophores belonging to following major chemical families:
  • Xanthene derivatives fluorescein, rhodamine, Oregon green, eosin, and Texas red; Cyanine derivatives: cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine;
  • Squaraine derivatives and ring-substituted squaraines including Seta and Square dyes; Squaraine Rotaxane derivatives: SeTau dyes;
  • Naphthalene derivatives (dansyl and prodan derivatives);
  • Oxadiazole derivatives pyridyloxazole, nitrobenzoxadiazole and benzoxadiazole;
  • Anthracene derivatives anthraquinones, including DRAQ5, DRAQ7 and CyTRAK Orange;
  • Oxazine derivatives Nile red, Nile blue, cresyl violet, oxazine 170;
  • Arylmethine derivatives auramine, crystal violet, malachite green;
  • Tetrapyrrole derivatives porphin, phthalocyanine, bilirubin;
  • Dipyrromethene derivatives BODIPY, aza-BODIPY;
  • the detectable moity is 7-amino-4-methylcoumarin (AMC) or rhodamine e.g., rhodamine 110.
  • the substrate and the detectable moiety compose a molecule of formula I: Z-X-Y, wherein:
  • Z is a protecting group
  • X is an amino acid sequence cleaved by aPC
  • Y is a detectable moiety, wherein Z, X, Y are linked.
  • Z, X and Y are covalently linked.
  • the protecting moiety is selected from the group consisting of t-butyloxycarbonyl (BOC, (CH3)3COCO-, t-BOC), t-amyloxycarbonyl, adamantyl-oxycarbonyl, and p-methoxybenzyloxycarbonyl, 9-fluorenylmethoxycarbonyl (FMOC), 2-chlorobenzyloxycarbonyl and the like, nitro, tosyl (CH3C6H4S02 — ), benzyloxycarbonyl (CBZ), adamantyloxycarbonyl, 2,2,5,7,8-pentamethylchroman-6-sulfonyl, 2,3,6-trimethyl-4-methoxyphenylsulfonyl, t-butyl benzyl (BZL) or substituted BZL, such as, p- methoxybenzy
  • the detectable moiety may comprise two chromogenic groups e.g., fluorophores, to achieve their quenched ‘off’ state to generate a signal (e.g., by FRET) however in a specific embodiment a single chromogenic group (e.g., fluorophore) is used.
  • FRET fluorophores
  • a single chromogenic group e.g., fluorophore
  • X is Pro-Arg.
  • Other embodiments are described hereinabove.
  • substrate molecules that can be used in accordance with the present teachings include, but are not limited to:
  • the substrate molecule comprises Pyr-Pro-Arg-
  • X is up to 10 amino acids in length. Examples include, but are not limited to, 9, 8, 7, 6, 5, 4, 3, 2 amino acids in length.
  • X is a dipeptide or a tripeptide.
  • determining is effected by photometrically measuring the quantity of colored or fluorescent split product formed as a result of the catalytic hydrolytic action of activated protein C on the substrate which comprises the detectable moiety.
  • a calibration curve is established by means of the dilution series of a control sample with known aPC activity levels.
  • a specific embodiment of a calibration curve is presented in Figure ID, which is based on standardized samples of Protein C containing sera.
  • the detectable moiety is typically measured using optical means, determining the appropriate absorption spectra according to the selected fluorophore (chromogenic group).
  • the release is measured at the selected wavelength, either during the reaction in a photometer cuvette (kinetic method), or discontinuously by stopping the reaction with acetic or citric acid (end-point method).
  • fluorescein and rhodamine-110 exhibit absorption maxima around 490 nm and strong emission around 520 nm.
  • AMC exhibits excitation 360 ⁇ 9nm, emission 465 ⁇ 20nm.
  • the photometric signal is proportional to the enzyme activity in a properly-designed assay.
  • activity is expressed in molar (M).
  • the concertation of the zymogen PC is about 4 pg/ml or 64 nM.
  • the concentration of aPC is 2000-fold lower compared to PC, which is approx. 2.26 ng/ml or 5 pM.
  • the present inventors have found that the sensitivity of the present method is at least as low as 5 pM.
  • the values measured in the cell cultures are in a range of 10-100 times higher than this value. Similarly values measured in CSF are in this level of detection about 5 nM.
  • the present teachings allow determination of aPC levels of as low as 0.1 pM, 1 pM, 5 pM or 1 pM- 1 nM, 1 pM- 5 pM, 1 pM- 5 pM, 0.1 pM -10 pM, 1 pM-100 nM without protein C activation: e.g., 1 pM-1 nM, 1-100 pM, 1-10 pM, 10-100 pM, 5-100 pM, 100-1000 pM, 1-500 pM, 100 pm-1000 nM, 10 pM-500 pM, 1 pM to 10 nM.
  • Reagents described herein for use in assays of aPC activity according to some embodiments of the invention can be included in a kit where at time instructions for use in aPC detection are included.
  • aPC activated protein C
  • Hereditary protein C deficiency is inherited as an autosomal dominant trait. Heterozygotes for protein C deficiency have protein C activity or antigen levels of 30 to 70% of normal, whereas homozygotes (or compound heterozygotes) with a severe defect have levels below 1%. Homozygotes with a mild defect have also been reported with protein C levels of 10- 24%. The normal range of protein C in the adult is 70% to 130% of a normal plasma pool (defined as 100%).
  • the Protein C level is influenced by various diseases and drugs. Acquired protein C deficiency is often associated with disseminated intravascular coagulation (DIC), deep vein thrombosis, severe liver disease, sepsis, vitamin K deficiency, oral anticoagulant therapy and elective surgery.
  • DIC disseminated intravascular coagulation
  • the protein C activity level may in some cases indicate the severity of a disease and can be used as a prognostic parameter.
  • Elevated protein C levels have been reported in diabetic and nephrotic patients, during late pregnancy, and with oral contraceptives and anabolic steroids. Elevated levels have no known clinical significance.
  • inflammatory diseases include, but are not limited to, chronic inflammatory diseases and acute inflammatory diseases.
  • hypersensitivity examples include, but are not limited to, Type I hypersensitivity, Type II hypersensitivity, Type III hypersensitivity, Type IV hypersensitivity, immediate hypersensitivity, antibody mediated hypersensitivity, immune complex mediated hypersensitivity, T lymphocyte mediated hypersensitivity and DTH.
  • Type I or immediate hypersensitivity such as asthma.
  • Type II hypersensitivity include, but are not limited to, rheumatoid diseases, rheumatoid autoimmune diseases, rheumatoid arthritis (Krenn V. el ah, Histol Histopathol 2000 Jul;15 (3):791), spondylitis, ankylosing spondylitis (Jan Voswinkel el ah, Arthritis Res 2001; 3 (3): 189), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Erikson J. et ah, Immunol Res 1998; 17 (l-2):49), sclerosis, systemic sclerosis (Renaudineau Y.
  • myasthenic diseases myasthenic diseases, Lambert-Eaton myasthenic syndrome (Takamori M. Am J Med Sci. 2000 Apr;319 (4):204), paraneoplastic neurological diseases, cerebellar atrophy, paraneoplastic cerebellar atrophy, non-paraneoplastic stiff man syndrome, cerebellar atrophies, progressive cerebellar atrophies, encephalitis, Rasmussen’s encephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles de la Tourette syndrome, polyendocrinopathies, autoimmune polyendocrinopathies (Antoine JC. and Honnorat J.
  • vasculitises necrotizing small vessel vasculitises, microscopic polyangiitis, Churg and Strauss syndrome, glomerulonephritis, pauci-immune focal necrotizing glomerulonephritis, crescentic glomerulonephritis (Noel LH. Ann Med Interne (Paris). 2000 May; 151 (3): 178); antiphospholipid syndrome (Flamholz R. et al, J Clin Apheresis 1999; 14 (4): 171); heart failure, agonist-like b -adrenoceptor antibodies in heart failure (Wallukat G.
  • Type IV or T cell mediated hypersensitivity include, but are not limited to, rheumatoid diseases, rheumatoid arthritis (Tisch R, McDevitt HO. Proc Natl Acad Sci U S A 1994 Jan 18;91 (2):437), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Datta SK., Lupus 1998;7 (9):591), glandular diseases, glandular autoimmune diseases, pancreatic diseases, pancreatic autoimmune diseases, Type 1 diabetes (Castano L. and Eisenbarth GS. Ann. Rev. Immunol. 8:647); thyroid diseases, autoimmune thyroid diseases, Graves’ disease (Sakata S.
  • delayed type hypersensitivity examples include, but are not limited to, contact dermatitis and drug eruption.
  • T lymphocyte mediating hypersensitivity examples include, but are not limited to, helper T lymphocytes and cytotoxic T lymphocytes.
  • helper T lymphocyte-mediated hypersensitivity examples include, but are not limited to, T h l lymphocyte mediated hypersensitivity and T h 2 lymphocyte mediated hypersensitivity.
  • cardiovascular diseases include, but are not limited to, cardiovascular diseases, rheumatoid diseases, glandular diseases, gastrointestinal diseases, cutaneous diseases, hepatic diseases, neurological diseases, muscular diseases, nephric diseases, diseases related to reproduction, connective tissue diseases and systemic diseases.
  • autoimmune cardiovascular diseases include, but are not limited to atherosclerosis (Matsuura E. et al, Lupus. 1998;7 Suppl 2:S135), myocardial infarction (Vaarala O. Lupus. 1998;7 Suppl 2:S132), thrombosis (Tincani A. et al, Lupus 1998;7 Suppl 2:S 107-9), Wegener’s granulomatosis, Takayasu’s arteritis, Kawasaki syndrome (Praprotnik S. et al, Wien Klin Klin Klinschr 2000 Aug 25; 112 (15- 16): 660), anti-factor VIII autoimmune disease (Lacroix- Desmazes S.
  • autoimmune rheumatoid diseases include, but are not limited to rheumatoid arthritis (Krenn V. et al, Histol Histopathol 2000 Jul;15 (3):791; Tisch R, McDevitt HO. Proc Natl Acad Sci units S A 1994 Jan 18;91 (2):437) and ankylosing spondylitis (Jan Voswinkel et al, Arthritis Res 2001; 3 (3): 189).
  • autoimmune glandular diseases include, but are not limited to, pancreatic disease, Type I diabetes, thyroid disease, Graves’ disease, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto’s thyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmune anti-sperm infertility, autoimmune prostatitis and Type I autoimmune polyglandular syndrome diseases include, but are not limited to autoimmune diseases of the pancreas, Type 1 diabetes (Castano L. and Eisenbarth GS. Ann. Rev. Immunol. 8:647; Zimmet P. Diabetes Res Clin Pract 1996 Oct;34 Suppl:S125), autoimmune thyroid diseases, Graves’ disease (Orgiazzi J.
  • autoimmune gastrointestinal diseases include, but are not limited to, chronic inflammatory intestinal diseases (Garcia Herola A. et ah, Gastroenterol Hepatol. 2000 Jan;23 (1): 16), celiac disease (Landau YE. and Shoenfeld Y. Harefuah 2000 Jan 16; 138 (2):122), colitis, ileitis and Crohn’s disease.
  • autoimmune cutaneous diseases include, but are not limited to, autoimmune bullous skin diseases, such as, but are not limited to, pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus.
  • autoimmune hepatic diseases include, but are not limited to, hepatitis, autoimmune chronic active hepatitis (Franco A. et ah, Clin Immunol Immunopathol 1990 Mar;54 (3):382), primary biliary cirrhosis (Jones DE. Clin Sci (Colch) 1996 Nov;91 (5):551; Strassburg CP. et ah, Eur J Gastroenterol Hepatol. 1999 Jun;ll (6):595) and autoimmune hepatitis (Manns MP. J Hepatol 2000 Aug;33 (2):326).
  • autoimmune neurological diseases include, but are not limited to, multiple sclerosis (Cross AH. et ah, J Neuroimmunol 2001 Jan 1 ; 112 (1-2): 1), Alzheimer’s disease (Oron L. et ah, J Neural Transm Suppl. 1997;49:77), myasthenia gravis (Infante AJ. And Kraig E, Int Rev Immunol 1999;18 (l-2):83; Oshima M. et ah, Eur J Immunol 1990 Dec;20 (12):2563), neuropathies, motor neuropathies (Kornberg AJ. J Clin Neurosci.
  • autoimmune muscular diseases include, but are not limited to, myositis, autoimmune myositis and primary Sjogren’s syndrome (Feist E. et al, Int Arch Allergy Immunol 2000 Sep;123 (1):92) and smooth muscle autoimmune disease (Zauli D. et al, Biomed Pharmacother 1999 Jun;53 (5-6):234).
  • autoimmune nephric diseases include, but are not limited to, nephritis and autoimmune interstitial nephritis (Kelly CJ. J Am Soc Nephrol 1990 Aug;l (2): 140).
  • autoimmune diseases related to reproduction include, but are not limited to, repeated fetal loss (Tincani A. et al, Lupus 1998;7 Suppl 2:S 107-9).
  • autoimmune connective tissue diseases include, but are not limited to, ear diseases, autoimmune ear diseases (Yoo TJ. et al, Cell Immunol 1994 Aug;157 (1):249) and autoimmune diseases of the inner ear (Gloddek B. et al, Ann N Y Acad Sci 1997 Dec 29;830:266).
  • autoimmune systemic diseases include, but are not limited to, systemic lupus erythematosus (Erikson J. et al, Immunol Res 1998; 17 (l-2):49) and systemic sclerosis (Renaudineau Y. et al, Clin Diagn Lab Immunol. 1999 Mar;6 (2): 156); Chan OT. et al, Immunol Rev 1999 Jun;169:107).
  • infectious diseases include, but are not limited to, chronic infectious diseases, subacute infectious diseases, acute infectious diseases, viral diseases, bacterial diseases, protozoan diseases, parasitic diseases, fungal diseases, mycoplasma diseases and prion diseases.
  • diseases associated with transplantation of a graft include, but are not limited to, graft rejection, chronic graft rejection, subacute graft rejection, hyperacute graft rejection, acute graft rejection and graft versus host disease.
  • Allergic diseases include, but are not limited to, graft rejection, chronic graft rejection, subacute graft rejection, hyperacute graft rejection, acute graft rejection and graft versus host disease.
  • allergic diseases include, but are not limited to, asthma, hives, urticaria, pollen allergy, dust mite allergy, venom allergy, cosmetics allergy, latex allergy, chemical allergy, drug allergy, insect bite allergy, animal dander allergy, stinging plant allergy, poison ivy allergy and food allergy.
  • cancer examples include but are not limited to carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • cancerous diseases include but are not limited to: Myeloid leukemia such as Chronic myelogenous leukemia. Acute myelogenous leukemia with maturation. Acute promyelocytic leukemia, Acute nonlymphocytic leukemia with increased basophils, Acute monocytic leukemia. Acute myelomonocytic leukemia with eosinophilia; Malignant lymphoma, such as Birkitt's Non-Hodgkin's; Lymphoctyic leukemia, such as Acute lumphoblastic leukemia.
  • Chronic lymphocytic leukemia Myeloproliferative diseases, such as Solid tumors Benign Meningioma, Mixed tumors of salivary gland, Colonic adenomas; Adenocarcinomas, such as Small cell lung cancer, Kidney, Uterus, Prostate, Bladder, Ovary, Colon, Sarcomas, Liposarcoma, myxoid, Synovial sarcoma, Rhabdomyosarcoma (alveolar), Extraskeletel myxoid chonodrosarcoma, Ewing's tumor; other include Testicular and ovarian dysgerminoma, Retinoblastoma, Wilms' tumor, Neuroblastoma, Malignant melanoma, Mesothelioma, breast, skin, prostate, and ovarian.
  • Adenocarcinomas such as Small cell lung cancer, Kidney, Uterus, Prostate, Bladder, Ovary, Colon, Sarcomas, Lipos
  • the disease is selected from the group consisting of sepsis, infectious disease, inflammatory disease, neurodegenerative disease and nerve injury.
  • the deficiency in aPC is associated with sepsis, hence the disease is sepsis.
  • the disease is a neurodegenerative disease, a neoplasm e.g., head and neck cancer or glioma or a nervous system reaction following trauma, e.g., traumatic brain injury, stroke.
  • a neurodegenerative disease e.g., head and neck cancer or glioma or a nervous system reaction following trauma, e.g., traumatic brain injury, stroke.
  • Additional neural disease states may especially benefit from embodiments of the invention, include, but are not limited to, meningitis, encephalitis due to viral/bacterial/fungal pathogens.
  • Autoimmune nervous system diseases including demyelinating inflammatory diseases of the CNS (e.g., multiple sclerosis) and the PNS (e.g., Guillain-Barre Syndrome), neuronal manifestations of systemic autoimmune diseases such as SLE and sarcoid.
  • diagnosis refers to determining presence or absence of a pathology (e.g., a disease, disorder, condition or syndrome), classifying a pathology or a symptom, determining a severity of the pathology, monitoring pathology progression, forecasting an outcome of a pathology and/or prospects of recovery and screening of a subject for a specific disease.
  • a pathology e.g., a disease, disorder, condition or syndrome
  • screening of the subject for a specific disease is followed by substantiation of the screen results using gold standard methods. For instance in the case of sepsis and other diseases diagnosed by blood tests corroboration is done by immediate white blood cell counts, measuring serum lactate, and obtaining appropriate cultures before starting antibiotics. In other alternative or additional cases e.g., multiple sclerosis imaging, physical tests, and/or molecular markers analysis.
  • a method of diagnosing a disease associated with activated Protein C (aPC) activity in a subject in need thereof comprising determining activity of said aPC in a biological sample of the subject as described herein, said activity being indicative of said disease or disease state.
  • aPC activated Protein C
  • the method further comprising informing the subject of the predicted diagnosis and/or the predicted prognosis of the subject.
  • the phrase “informing the subject” refers to advising the subject that based on the results of the assay (aPC levels) the subject should seek a suitable treatment regimen. For example, if the subject is predicted to respond to heparin, warfarin, and protein C concentrates (in case of deficiency) and is diagnosed or suffers from a pathology requiring it that such a treatment is advisable.
  • the results can be recorded in the subject’s medical file, which may assist in selecting a treatment regimen and/or determining prognosis of the subject.
  • the method further comprising recording the levels of aPC of the subject in the subject’s medical file.
  • the prediction of the diagnosis of a subject based on aPC levels can be used to select the treatment regimen of a subject and thereby treat the subject in need thereof.
  • aPC activated Protein C activity
  • Treatments for protein C deficiency include heparin, warfarin, and protein C concentrates.
  • Acute thrombosis in protein C-deficient individuals should be treated with heparin.
  • Warfarin is used for longer treatment periods to prevent thrombotic recurrences.
  • When initiating the warfarin therapy it must be started at low doses in conjunction with heparin to prevent skin and fat necrosis.
  • Prophylactic anticoagulation is mainly recommended for symptomatic patients in high risk situations (e.g. surgery, pregnancy). Symptom-free relatives of symptomatic patients with protein C deficiency may benefit from prophylactic anticoagulation in similar risk situations, since they run an increased risk of thrombosis compared to nondeficient individuals.
  • the present teachings further provide for a method of identifying an agent suitable for altering an activity of activated Protein C (aPC), the method comprising:
  • agent refers to any chemical entity such as a small molecule, a carbohydrate a biomolecule e.g., peptide, polypeptide, nucleic acid sequence, oligonucleotide, polynucleotide and a physical condition e.g., radiation.
  • control refers to a sample which has not been treated (contacted) with the agent but otherwise incubated under the same assay conditions.
  • change refers to increase or decrease in activity of protein C relative to the control.
  • change is determined in a quantitative manner e.g. at least 5 %, at least 10 %, at least 15 %, at least 20 %, at least 25 %, at least 30 %, at least 35 %, at least 40 %, at least 45 %, at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 2 fold, 3 fold, 5 fold, 10 fold or more as compared to the control.
  • All the methods and assays described herein can be done in a small scale or in a large scale involving the use of multiple well plates e.g., at least 96 wells, robotics, imaging apparat or any means known to facilitate screening in a large scale.
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.
  • the phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • the term “subject” includes mammals, preferably human beings at any age which suffer from the pathology. Preferably, this term encompasses individuals who are at risk to develop the pathology.
  • treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
  • the term “preventing” refers to keeping a disease, disorder or condition from occurring in a subject who may be at risk for the disease, but has not yet been diagnosed as having the disease.
  • C6 rat glioma cells purchased from ATCC, CCL-107) and N9 mouse microglia cells (ATCC) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Bet Haemek, Biological Industries, Israel) supplemented with 10 % fetal bovine serum (Bet Haemek, Biological Industries, Israel), 1% L-Glutamine (Bet Haemek, Biological Industries, Israel) and 0.1% penicillin and streptomycin (Bet Haemek, Biological Industries, Israel). The cells were grown in a 37°C and 5 % CCh-humidified atmosphere.
  • DMEM Dulbecco’s modified Eagle’s medium
  • Bet Haemek fetal bovine serum
  • L-Glutamine Bet Haemek, Biological Industries, Israel
  • penicillin and streptomycin Bet Haemek, Biological Industries, Israel
  • mice were anesthetized using isoflurane. The mouse head was placed on a sponge immobilization board and a 50 gram metal weight was dropped down an 80-cm-high metal tube placed over the head of the mouse on the right anterolateral side. Control mice were anesthetized only. 24 hours following mTBI induction mice were sacrificed and the brain was removed for the aPC activity assay.
  • mice were weighted before LPS induction. Mice were injected intraperitoneal (IP) with LPS, a component of the bacterial wall (. Escherichia co/z 0111:B4, Sigma L4130) 1 mg/kg, diluted in saline. 24 hours following injection, mice were weighted and sacrificed. Brains were removed and prepared as described below.
  • IP intraperitoneal
  • a component of the bacterial wall . Escherichia co/z 0111:B4, Sigma L4130
  • CSL samples were collected from infection chronic and acute inflammatory demyelinating polyneuropathy (CIDP/AIDP) and multiple sclerosis (MS) patients and normal pressure hydrocephalus (NPH) patients by a lumbar puncture, and were kept in -80°C until use. The procedure was approved by the ethical committee of the Chaim Sheba Medical Center (4245-17-SMC), and patients provided written informed consent.
  • aPC activity assay aPC activity was assessed using a fluorogenic substrate synthesized on order by our specifications (Pyr-Pro-Arg-AMC, 20mM, GL Biochem Shanghai Ltd.). The reactions were carried out in black 96- well microplates.
  • the reactions with commercial thrombin, LXa, plasma and brain tissue were performed in Tris buffer (in mM: 150 NaCl, 1 CaC12, 50 Tris-HCl: pH 8.0).
  • Tris buffer in mM: 150 NaCl, 1 CaC12, 50 Tris-HCl: pH 8.0.
  • the cleavage of the substrate was measured at 37°C every 2 minutes over 25 cycles (points at which fluorescence was measured, excitation 360 ⁇ 9nm, emission 465 ⁇ 20nm).
  • the activity was calculated as the linear increase of fluorescence intensity over time infinite 2000 TEC AN).
  • CSL samples (93 pi) were added to the microplate wells.
  • Substrate containing inhibitors (NAPAP-lpM, apixaban— ImM) was added and the fluorescence was measured.
  • NAPAP alpha-naphthylsulphonylglycyl-4- amidinophenylalanine piperidine
  • Apixaban ImM, Selleckchem, S1593
  • mice were sacrificed by lethal phenobarbital injection (CTS Chemical Industries ltd). The organs were rapidly removed and placed on ice. Brain was placed in a steel brain matrix (1 mm, Coronal, Stoelting, IL, USA) on ice. Brains were cut in the sagittal plane to separate right and left hemispheres. Next, brains were cut in the coronal plane into 1 mm slices. Left and right sections of slices #3 to #8 were placed in the wells and the microplate was kept on ice. The microplate was incubated at 37°C for 30 min before the addition of the substrate.. Substrate containing inhibitors (NAPAP, apixaban) was added and the fluorescence was measured.
  • NAPAP arate containing inhibitors
  • the substrate selectivity for APC activity was characterized by evaluation of its cleavage by major confounding proteases.
  • both thrombin and FXa are potentially able to cleave this sequence. Therefore, cleavage of the APC substrate by these proteases was measured.
  • Known concentrations of commercial thrombin (50 mU) and FXa (10 mU) were used, with and without thrombin and FXa specific inhibitors, NAPAP and apixaban, respectively.
  • thrombin and FXa cleaved the substrate.
  • Sensitivity and specificity of the substrate for APC activity measurement was studied in human plasma. As expected, low APC activity was measured in the citrated plasma, due to the anticoagulation effect of citrate. Activation of the coagulation by CaCh caused significant increase in APC activity (1262+74.73, 1+0.26 D in fluorescence intensity respectively, p ⁇ 0.0001, Figure 1C). Further increase was achieved using a specific PC activator Protac (2931+49.48 compared to control, p ⁇ 0.0001, Figure 1C). Next, the substrate was applied on known concentrations of PC, combined with Protac (Figure ID).
  • N9 microglia cell culture presents significantly lower APC activity compared to CaCh activated plasma (14.75+0.96 and 195.1+20.04 aU, respectively, p ⁇ 0.0001, Figure 2A).
  • N9 basal APC activity totally demolished with PMSF (-0.82+0.45 aU, p ⁇ 0.0001, Figure 2A).
  • the next challenge was to examine APC activity in different cell types, and to determine whether APC is secreted into the medium or attached to the cells.
  • Two cells types were used; N9 cells as a model for microglia, and C6 cells as a model for astrocytes [24,25].
  • the serum-free medium was transferred into empty wells 24 hours after initiating the experiment. Fresh medium was added to the remaining cells and activity was measured immediately.
  • Significantly higher APC activity was measured in the medium compared to the cells of both types (30.8+0.9, 8.0+0.8, 9.6+1.2, 1+0.1, N9 and C6, medium versus cells respectively, result are presented relative to APC levels measured in C6 cells, Figure 2B, p ⁇ 0.0001).
  • N9 medium was used in order to determine the kinetic properties of the substrate.
  • APC activity was measured in the cells in response to inflammation induced by LPS (O.lpg/ml) for short (10 minutes) or long duration (24 hours) in the medium and on the cells.
  • short treatment with LPS did not affect APC activity in N9 cells (Figure 2D).
  • C6 cells both short and long treatments with LPS did not change APC activity significantly (Figure 2E).
  • aPC activity was measured ex-vivo.
  • First aPC activity was measured and mapped in control healthy mice brain (Figure 3A and 3B).
  • a specific spatial profile of aPC activity was found in the brain (p ⁇ 0.0001, Figure 3A).
  • a significantly higher aPC activity was found in the posterior slices of the brain compared to the anterior slices (257+12.7, 1163+16.4, p ⁇ 0.0001, Figure 3B).
  • aPC activity was decreased after 24 hours of exposure while, in contrast, elevated aPC activity measured in the brain following systemic LPS administration and mTBI in mice and in acute inflammation in human CSF.
  • the inflammatory response consists of an early phase and a recovery phase. Inflammation resolving molecules are down-regulated in the early phase and up-regulated in the recovery phase [35].
  • aPC has an anti-inflammatory effect, through TF, thrombin inhibition and down-regulation of pro-inflammatory cytokines and chemokines [36].
  • the present inventors measured aPC activity in human CSF samples from patient with various neurological deficits (viral and bacterial infections) compared to NPH (normal pressure hydrocephalus) patients. As can be seen in Figure 7, significantly higher aPC activity was measured in the CSF of the infection patients, AIDP/CIDP patients and MS patients as compared to NPH patients.
  • the present inventors measured aPC activity in the medium of N9 cells following 24 h treatment with four different FX-EPCR-APC-Modulators (FEAMs) ( Figure 9A and Table 1).
  • Table 1 The sequence of FEAM 1-4 and the region in human PC/FVII which the sequence of FEAM was based on.
  • Figure 9B all FEAMs significantly increased aPC activity in the medium of N9 cells. Cells were treated with LPS alone or in combination with FEAMs.
  • Figure 9C in the presence of LPS, significantly lower aPC activity was measured. None of the FEAMs prevented the decrease in aPC activity following LPS treatment.
  • ischemic LTP ischemic LTP

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Abstract

A method of determining activity of activated protein C (aPC) is provided. The method comprising: (a) contacting a biological sample with a substrate of the aPC, the substrate comprising a detectable moiety, and wherein the contacting is under conditions in which activity of thrombin and activity of Factor Xa (FXa) are inhibited; (b) determining an amount of the detectable moiety formed as a result of a catalytic hydrolytic action of the aPC, the amount being indicative of the activity of the aPC.

Description

METHODS OF DETERMINING ACTIVITY OF ACTIVATED PROTEIN C
RELATED APPLICATION:
This application claims the benefit of priority from US Provisional Patent Application No. 63/001,349, filed on March 29, 2020 which is hereby fully incorporated by reference in its entirety.
SEQUENCE LISTING:
The ASCII file, entitled 86381 Sequence Listing.txt, created on 29 March 2021, comprising 4,096 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.
FIELD AND BACKGROUND OF THE INVENTION
The present invention, in some embodiments thereof, relates to methods of determining activity of activated protein C.
Neural inflammation is a core process in many pathologies, including neurodegenerative diseases [1], neoplasms [2], and nervous system reaction following trauma [3]. Inflammation and coagulation are tightly linked and modify each other in the systemic circulation [4-6]. The role of coagulation system proteins in neural function has been intensively studied and found to affect many processes including synaptic transmission, axonal conduction, learning, memory and behavior [7-9].
Activated protein C (aPC) is a serine protease participating in the coagulation cascade. Following clot formation, thrombin binds thrombomodulin (TM) and activates protein C (PC) to aPC. aPC inhibits FV and FVII which are essential for thrombin generation and thus acts as an anticoagulation factor [10]. Similar to other coagulation factors, aPC has a role in the inflammatory response. aPC inhibits pro-inflammatory cytokines, protects endothelial cells structure and function [11], and maintains normal blood pressure during sepsis [12]. aPC requires two receptors for its activation and its cellular effects; the endothelial protein C receptor (EPCR) and protease activator receptor 1 (PARI). EPCR serves as a catalyst of PC activation and mediates aPC activity [10]. When bound to EPCR, aPC cleaves PARI, and induce a positive effect in endothelial and Schwann cells [13,14]. EPCR and PARI are expressed in both the peripheral nervous system (PNS) [15] and the central nervous system (CNS) [16].
The measurement of aPC activity is therefore potentially important for understanding neural function in neuroinflammation. The accepted laboratory method measures aPC activity indirectly, using an in-vitro activation of PC into aPC by exposure to venom of southern copperhead snakes [17]. This method is expensive due to the use of snake’s venom, and only measures aPC activity indirectly. Furthermore, although appropriate for the high plasma levels, this method is not sensitive enough to measure aPC activity levels in neural tissue.
Additional Background Art:
Pefafluor PCa, Pyr-Pro-Arg-AMC, is a commercially available substrate for activated Protein C (e.g., Biocip Praha, P089-05-25MG).
U.S. Patent No. 4,849,403 teaching determination of plasma levels of aPC in the absence of background inhibitors.
SUMMARY OF THE INVENTION
According to an aspect of some embodiments of the present invention there is provided a method of determining activity of activated protein C (aPC), the method comprising:
(a) contacting a biological sample with a substrate of the aPC, the substrate comprising a detectable moiety, and wherein the contacting is under conditions in which activity of thrombin and activity of Factor Xa (FXa) are inhibited;
(b) determining an amount of the detectable moiety formed as a result of a catalytic hydrolytic action of the aPC, the amount being indicative of the activity of the aPC.
According to some embodiments of the invention, the conditions in which activity of the thrombin and activity of the Factor Xa (FXa) are inhibited comprise an inhibitor of thrombin and an inhibitor of Factor Xa (FXa).
According to an aspect of some embodiments of the present invention there is provided a composition of matter comprising a biological sample which putatively comprises aPC, a substrate of the aPC, the substrate comprising a detectable moiety, and an inhibitor of thrombin and an inhibitor of Factor Xa (FXa).
According to some embodiments of the invention, the inhibitor of thrombin comprises NAPAP, hirudin and/or TLCK.
According to some embodiments of the invention, the inhibitor of Factor Xa comprises apixaban and/or Rivaroxaban.
According to some embodiments of the invention, the method further comprises generating a calibration curve for the activity of the aPC.
According to some embodiments of the invention, the sample comprises a neural tissue.
According to some embodiments of the invention, the sample comprises a neural cell or cell line. According to some embodiments of the invention, the sample comprises a cerebrospinal fluid (CSF).
According to some embodiments of the invention, the sample comprises plasma.
According to some embodiments of the invention, the sample comprises a conditioned medium of any one of a neural cell and a neural tissue.
According to some embodiments of the invention, the substrate and the detectable moiety compose a molecule of formula I:
Z-X-Y, wherein:
Z is a protecting group;
X is an amino acid sequence cleaved by aPC;
Y is a detectable moiety, wherein Z, X, Y are covalently linked.
According to some embodiments of the invention, the detectable moiety comprises any one of a chromogenic, fluorescent, chemiluminescent, or phosphorescent group.
According to some embodiments of the invention, the Z is selected from the group consisting of Pyroglutamic acid (Pyr), BOC, (CH3)3COCO-, t-BOC), t-amyloxycarbonyl, adamantyl-oxycarbonyl, and p-methoxybenzyloxycarbonyl, 9-fluorenylmethoxycarbonyl (FMOC), 2-chlorobenzyloxycarbonyl and the like, nitro, tosyl (CH3C6H4S02 — ), benzyloxycarbonyl (CBZ), adamantyloxycarbonyl, 2,2,5,7,8-pentamethylchroman-6-sulfonyl, 2,3,6-trimethyl-4-methoxyphenylsulfonyl, t-butyl benzyl (BZL) or substituted BZL, such as, p- methoxybenzyl, p-nitrobenzyl, p-chlorobenzyl, o-chlorobenzyl, and 2,6-dichlorobenzyl.
According to some embodiments of the invention, the fluorescent group is selected from the group consisting of AMC and rhodamin-110.
According to some embodiments of the invention, the X is Pro-Arg.
According to some embodiments of the invention, the molecule comprises Pyr-Pro-Arg-
AMC.
According to some embodiments of the invention, the X is a dipeptide, tripeptide.
According to some embodiments of the invention, the X is up to 10 amino acids in length.
According to some embodiments of the invention, the method is performed in a multi well plate.
According to some embodiments of the invention, the determining comprises photometrically measuring. According to an aspect of some embodiments of the present invention there is provided a method of diagnosing a disease associated with activated Protein C (aPC) activity in a subject in need thereof, the method comprising determining activity of the aPC in a biological sample of the subject as described herein, the activity being indicative of the disease or disease state.
According to an aspect of some embodiments of the present invention there is provided a method of treating a disease associated with activated Protein C activity (aPC) in a subject in need thereof, the method comprising:
(a) determining activity of the aPC in a biological sample of the subject as described herein, the activity being indicative of the disease or disease state;
(b) treating the subject according to indication or state of the disease.
According to an aspect of some embodiments of the present invention there is provided a method of monitoring treatment of a disease associated with activated Protein C (aPC) activity in a subject in need thereof, the method comprising:
(a) determining activity of the aPC in a biological sample as described herein of a subject having been treated for the disease, the activity being indicative of a disease state;
(b) treating the subject according to the state of the disease.
According to some embodiments of the invention, the disease is selected from the group consisting of sepsis, infectious disease, inflammatory disease, neurodegenerative disease and nerve injury.
According to an aspect of some embodiments of the present invention there is provided a method of identifying an agent suitable for altering an activity of activated Protein C (aPC), the method comprising:
(a) contacting a biological sample comprising aPC with an agent;
(b) determining an activity of the aPC following or concomitant with the contacting as described herein, wherein a change in the activity compared to a control sample not contacted with the agent is indicative that the agent is suitable for altering an activity of activated protein C (aPC).
According to an aspect of some embodiments of the present invention there is provided a kit for measuring the activity of activated protein C (aPC), the kit comprising:
(a) a substrate for aPC, the substrate being a molecule of formula I;
(b) a thrombin inhibitor;
(c) a Factor Xa inhibitor;
(d) optionally reagents for a calibration curve;
(e) at least one buffer; and (f) instructions for measuring the activity of activated protein C (aPC).
Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.
In the drawings:
FIGs. 1A-D show substrate cleavage and measurement validation: A. Substrate cleavage by thrombin: Thrombin (50 mU) cleaves the APC substrate in a non-APC manner (n=3). Apixaban significantly decreases the non-APC substrate cleavage by thrombin (n=4, p<0.0001). NAPAP and NAPAP with Apixaban completely block the non-APC substrate cleavage by thrombin (n=4, p<0.0001). B. Substrate cleavage by FXa: FXa (10 mU) cleaves the APC substrate in a non-APC manner (n=4). NAPAP significantly decreases the non-APC substrate cleavage by FXa (n=4, p <0.0005). Apixaban and Apixaban with NAPAP completely block the non-APC substrate cleavage by FXa (n=4, p<0.0001). C. APC activity in human plasma: Human citrated plasma presents low APC activity (n=2), that significantly increases after activation in the presence of CaCh (n=4, p<0.0001) and further increases in the presence of Protac (n=4, p<0.0001). D. Standard curve: APC activity plotted versus known concentration of PC activated by Protac, show positive linear fit (0-1.38 %PC: slope=13.25, R2=0.95; 1.38-138 %PC: slope=26.78, R2=0.92).
FIGs. 2A-F show APC activity in N9 and C6 cells in normal conditions and LPS model: A. APC activity in activated plasma compared to N9 cells: APC activity in activated plasma (n=5) is significantly higher compared to APC activity in N9 cell culture (n=5, p<0.0001). APC activity in N9 culture is completely inhibited following PMSF treatment (n=5, p<0.0001). B. APC activity in the medium and on the cells of N9 and C6 cells: APC activity is significantly higher in the medium compared to APC activity on the cells of N9 and C6 cells (n=8, p<0.0001). APC activity in the medium of N9 cells is significantly higher compared to APC activity in the medium of C6 cells (n=8, p<0.0001). APC activity on the cells of N9 cells is significantly higher compared to APC activity on the cells of C6 cells (n=8, p=0.0003). C. Michaelis-Menten saturation curve of the substrate: The measured activity shoved a good fit to Michaelis-Menten saturation curve with Km=65.26- 117.3 and Vmax=413.8-534.2 (R2=0.972). D. APC activity in N9 cells in LPS model: APC activity in the medium and on the cells of N9 did not changed significantly after 10 minutes treatment of LPS (0.1 pg/ml). 24 hours treatment of LPS significantly decreased APC activity in the medium and on the cells of N9 (n=24, p<0.0001 and p=0.0006 respectively). E. APC activity in C6 cells in LPS model: APC activity did not change significantly after short or long LPS treatment in the medium and on the cells of C6 (n=16). F. APC protein levels in N9 cells in LPS model: APC/actin levels are significantly higher in the medium compared to cells (n=2, p=0.0259) and significantly decrease in the medium following LPS treatment (n=2, p=0.0052).
FIGs. 3A-D show aPC activity in mouse brain: A. aPC activity across healthy mouse brain: aPC activity varies across the brain (n=17-20, p<0.0001). The highest activity was measured in brain slice #6, and it's significantly higher compared to brain slice #5 (n=19, p<0.0001). B. aPC activity in anterior versus posterior healthy brain slices: Posterior brain slices (#6-8) presents significantly higher aPC activity compares to anterior brain slices (#3-5) (n=55- 58, p<0.0001). C. aPC activity in the brain 24 hours after mTBI: aPC activity in brain slices following mTBI was significantly higher in the right and the left hemispheres, compared to control (n=58, 56, and 27 respectively, p<0.0001and 0.0053 respectively). D. Average aPC activity 24 hours after mTBI: The average aPC activity across the brain was significantly higher after mTBI compared to control (n=5 and n=10 respectively, p<0.0001).
FIGs. 4A-D show aPC activity in a mice model for systemic inflammation and in human patients: A. Weight decline in EPS injected mice: EPS injected mice demonstrated a significant loss of weight 24 hours following the injection, as a marker for the systemic inflammatory response (n=7, p<0.0001). B. aPC activity in anterior versus posterior brain slices of EPS injected mice: Posterior brain slices of systemically injected mice showed a trend towards elevated aPC activity compared to posterior brain slices of control mice (n=35 and 58 respectively, p=0.06). C. aPC activity in brain slices of LPS injected mice: aPC activity measurements conducted in slices number 7# and 8# of the brain showed significantly elevated activity compared to control (n=9-20 , p=0.04and p=0.017 respectively). D. Elevation of aPC activity in human CSF of patients suffering from systemic inflammation: CSF samples taken from patients with systemic inflammation showed significantly elevated aPC activity compare to healthy controls (n=5 and 4 respectively, p=0.01).
FIG. 5 is a schematic illustration of an embodiment of the present invention. Neural culture secretes various proteases, including activated Protein C (aPC), that maintain homeostasis and respond during inflammatory events. The present inventors developed highly sensitive aPC activity assay, which utilizes florescence labeled substrate and nonspecific proteases inhibitors to measure aPC activity in neural cultures in health and disease. The Figure was created by the software BioRender.
FIG. 6 shows the cleavage site composition of aPC.
FIG. 7 shows aPC activity in human CSF. Increased aPC activity was found in CSF of patients with infection, GBS/CIDP and MS compared to NPH control. (N=14,7,7 and 15 respectively, * p<0.05, ** p <0.01)
FIG. 8 shows aPC activity in various tissues of ICR mice. Detectable aPC activity was found in the skin, hippocampus, cornea and sciatic nerve of healthy ICR mice. (N=9,10,6 and 6 respectively).
FIGs. 9A-C show the use of aPC activity assay for drug screening: Figure 9A. FEAM1-4: novel peptides for modulation of aPC/FVII/EPCR. Figure 9B. aPC activity in N9 cells in the presence of FEAM1-4 (SEQ ID NOs. 1-4) . All FEAMS significantly increased aPC activity (N=24, **p<0.01, ****p<0.0002). Figure 9C. aPC activity in N9 cells following LPS and FEAM1-4 treatment. aPC activity was significantly decreased following LPS treatment. The LPS induced decreased aPC activity was not significantly prevented by FEAM 1-4 treatment (N=l 18-23, ****p<0.0001).
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
The present invention, in some embodiments thereof, relates to methods of determining activity of activated protein C.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. aPC activity assays that are available for clinical use are designed mainly for diagnosis of coagulopathies in plasma [25]. The contemporary studies on the positive effect of aPC on neurons in the cellular and synaptic levels [16,26] require a more sensitive assay. Whilst conceiving embodiments of the invention, the present inventors have devised a novel method for aPC activity measurement using the specific amino acid sequence cleaved by aPC in conjugation with a fluorogenic emitting substrate. This novel method is sensitive enough to detect direct aPC activity without the need of prior PC activation. Using this method, the present inventors measured for the first time, intrinsic aPC activity in neural cell lines, mouse brain and human CSF, and were able to demonstrate significant changes in aPC activity in models of neuroinflammation.
Specifically the present inventors have established appropriate conditions that enable to distinguish aPC activity from other serine proteases known to cleave the substrate. As is illustrated in Examples 2 and 3 of the Examples section, the present results indicate the use of thrombin and FXa inhibitors in this aPC assay. Indeed, in the presence of these inhibitors, aPC activity levels in human plasma can be measured in a reliable manner. Human plasma contains significantly higher levels of PC compared to aPC [24]. Addition of the potent PC activator, Protac™, causes the expected elevation in the measured fluorescence thus supporting the specificity of this method (Figure ID).
As mentioned, aPC activity assays that are available for clinical use are designed mainly for diagnosis of coagulopathies in plasma [25]. The contemporary studies on the positive effect of aPC on neurons in the cellular and synaptic levels [16,26] require a more sensitive assay. This novel assay indeed enables detection of aPC activity in neural cell lines. Demonstrated herein, for the first time, that N9 microglia cells and C6 glioma cells have intrinsic aPC activity. Interestingly, aPC activity varies between those cell types being higher in N9 microglia cells [27], compared to C6 astrocytic cells. Microglia are immune cells, taking part in inflammatory responses and neuroregenerative changes [28]. aPC has been shown to induce neuroregeneration [29] but the possibility that it is produced locally by the neural tissue was not studied before. The present results demonstrating elevated aPC activity in microglia cells may suggest a possible involvement of this pathway in microglial induced neuroregeneration. Further aPC activity was localized to their surrounding medium. aPC activity levels in both cell types are higher in the medium, suggesting the secretion of aPC, as a soluble molecule or as part of an extracellular vesicle. This may be part of a complex interaction process with neighboring cells [24,30]. aPC activity was also measured ex-vivo. Measurements of aPC in mice brains show elevated aPC activity levels in the posterior brain sections (see Example 4). This may be due to high concentration of microglia in these sections [31]. An expected elevation of aPC activity was measured following mTBI in mice. This probably represents the inflammatory response initiated by the trauma [32] and the involvement of microglia in this pathophysiology. Systemic injection of LPS creates a general inflammatory response, including brain involvement. Elevated brain thrombin activity, together with PC and EPCR upregulation, following LPS injection has been previously described [34]. aPC activity following LPS injection was tested (see Example 5). In contrast to the results in the mTBI model, elevation of aPC activity in the LPS model is prominent mostly in the posterior brain. High concentration of microglia cells in the posterior parts of the brain and the present findings, suggest that LPS leads to elevated aPC activity levels through local microglia activation.
Linally, the method was utilized for measuring aPC activity in human samples. It is suggested that changes in brain aPC activity, following neuroinflammation, would be measurable in the CSL. As expected, aPC activity was significantly higher in CSL taken from patients with viral meningoencephalitis, compared to NPH controls (see Ligure 4D). These results demonstrate that this novel method can be used for diagnosis and drug efficacy evaluation in human subjects.
Thus, according to an aspect of the invention there is provided a method of determining activity of activated protein C (aPC), the method comprising:
(a) contacting a biological sample which putatively comprises PCa with a substrate of the aPC, the substrate comprising a detectable moiety, and wherein the contacting is under conditions in which activity of thrombin and activity of Lactor Xa (LXa) are inhibited;
(b) determining an amount of the detectable moiety formed as a result of a catalytic hydrolytic action of the aPC, the amount being indicative of the activity of the aPC.
As used herein “protein C” also known as “autoprothrombin IIA” and “blood coagulation factor XIX”, is a zymogen, the activated form of which plays an important role in regulating anticoagulation, inflammation, and cell death and maintaining the permeability of blood vessel walls in humans and other animals.
As used herein “activated protein C” also abbreviated as “PCa” or “aPC” performs the functions of protein C primarily by proteolytically inactivating proteins Lactor Va and Lactor Villa. aPC is classified as a serine protease since it contains a residue of serine in its active site. In humans, protein C is encoded by the PROC gene, which is found on chromosome 2. The present teachings refer to any protein C or aPC of any organism that shares the substrate of human or mouse activated protein C.
As used herein “substrate” refers to the amino acid sequence that is cleaved by activated protein C. Activated protein C cleaves natural substrates with arginine in the PI position, whereas the amino acid composition in the P2 and P3 positions are more varied. Ligure 6 shows the substrate structure of aPC. Examples of substrates that can be used in accordance with the present teachings include, but are not limited to, Pro-Arg, Glu-Pro-Arg, Val-Leu-Arg, Ile-Pro-Arg, Phe-Pip-Arg. According to a specific embodiment the amino acid sequence of the cleavage site is Pro-Arg. As used herein “activity” refers to serine protease activity.
According to a specific embodiment, the method is performed without activation of the protein prior to determining the activity.
According to another specific embodiment, the method is performed with activation of the protein prior to determining the activity. Methods of activating protein C are well known in the art. For instance, the determination of protein C can be facilitated by the use of the specific protein C activator, Protac®. The activator is a serine protease isolated and purified from the venom of the southern copperhead snake, Agkistrodon c. contortrix. It rapidly activates both human and bovine protein C, probably via the same mechanism as thrombin, without interfering with other coagulation factors. Since the activation is rapid, it minimizes the efficiency of the protein C inhibitors and thus eliminates the need for isolating protein C in an adsorption step.
As used herein “a biological sample” refers to a sample which may comprise protein C preferably in an activated form. This can be a sample derived from a subject. Biological samples include, but are not limited to, body fluids such as whole blood, serum, plasma, cerebrospinal fluid (CSF), urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk as well as white blood cells, tissues, amniotic fluid and chorionic villi. According to a specific embodiment, the sample comprises a tissue/cell culture.
According to a specific embodiment, the sample is freshly tested (e.g., less than 48 or 24 hours following retrieval).
According to a specific embodiment, the sample is tested following cryopreservation.
According to a specific embodiment, the sample comprises a neural tissue.
As used herein “neural” refers to neurons or glial cells. Glia, also called glial cells or neuroglia, are non-neuronal cells in the central nervous system (brain and spinal cord) and the peripheral nervous system that do not produce electrical impulses. They maintain homeostasis, form myelin, and provide support and protection for neurons. In the central nervous system, glial cells include oligodendrocytes, astrocytes, ependymal cells, and microglia, and in the peripheral nervous system glial cells include Schwann cells and satellite cells
According to a specific embodiment, the sample comprises a cerebrospinal fluid (CSF).
According to a specific embodiment, the sample does not comprise plasma.
According to a specific embodiment, the sample comprises plasma. According to a specific embodiment, the sample comprises a conditioned medium of any one of a neural cell and a neural tissue, such a conditioned medium is also referred to a PCa not associated with cells.
According to a specific embodiment, the sample comprises a neural cell or cell line. Examples of neural cell lines are well known in the art, so are listed in the Examples section, e.g., glioma cell lines such as C6 and CNS1, neuronal cells such as PC12 and N2A, macrophages such as J774, RAW264.7, endothelial cell lines such as HUVEC and C9 microglia cells such as N9, Schwann cell lines such as STS26T and ST88-14.
As used herein “a cell” refers to a native cell or to a cell line.
According to a specific embodiment, the cell is genetically modified (GMO).
According to a specific embodiment, the cell is non-GMO.
According to a specific embodiment, the protein C activity is associated to a cell (e.g., a neural cell).
According to another specific embodiment, the protein C activity is in a medium of a cell e.g., in a cell-free sample.
As mentioned, the biological sample is contacted with a substrate of aPC under conditions in which the activity of Factor Xa and thrombin are inhibited. This is because Thrombin (substrate sequence Gly-Pro-Arg), Factor Xa (substrate sequence Glu-Gly-Arg) and aPC share substrate specificity due to similarity in cleavage sites sequences.
To enhance the specificity of the assay, the background signal of substrate cleavage by non-aPC enzymes is decreased under conditions which decrease the activity of these enzymes such as Factor Xa and Thrombin.
Such conditions include the use of specific or non-specific inhibitors of Factor Xa and Thrombin. Measures are taken not to use inhibitors or reaction conditions which inhibit activity of protein C.
Examples of such inhibitors, include, but are not limited to, an inhibitor of thrombin which comprises for example, NAPAP, hirudin and/or Na-p-tosyl-F-lysine chloromethyl ketone (TFCK) and an inhibitor of Factor Xa which comprises for example apixaban and/or Rivaroxaban.
According to a specific embodiment, the substrate comprises a detectable moiety.
According to a specific embodiment, the detectable moiety comprises a chromogenic group.
As used herein “chromogenic group” comprises molecules that generate a chromogenic, fluorescent or chemiluminescent signal. Thus according to a specific embodiment, the detectable moiety generates a chromogenic signal.
According to a specific embodiment, the detectable moiety generates a fluorescent signal. According to a specific embodiment, the detectable moiety generates a chemiluminescent signal.
Examples of chromogenic groups that can be used in accordance with the present teachings include, but are not limited to non-protein organic fluorophores belonging to following major chemical families:
Xanthene derivatives: fluorescein, rhodamine, Oregon green, eosin, and Texas red; Cyanine derivatives: cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine;
Squaraine derivatives and ring-substituted squaraines, including Seta and Square dyes; Squaraine Rotaxane derivatives: SeTau dyes;
Naphthalene derivatives (dansyl and prodan derivatives);
Coumarin derivatives;
Oxadiazole derivatives: pyridyloxazole, nitrobenzoxadiazole and benzoxadiazole; Anthracene derivatives: anthraquinones, including DRAQ5, DRAQ7 and CyTRAK Orange;
Pyrene derivatives: cascade blue;
Oxazine derivatives: Nile red, Nile blue, cresyl violet, oxazine 170;
Acridine derivatives: proflavin, acridine orange, acridine yellow;
Arylmethine derivatives: auramine, crystal violet, malachite green;
Tetrapyrrole derivatives: porphin, phthalocyanine, bilirubin;
Dipyrromethene derivatives: BODIPY, aza-BODIPY;
Resofurin;
Fluorescein;
Umbelliferone.
Each possibility represents a separate embodiment of the present invention.
Drake et al. Curr Org Synth. 2011 August; 8(4): 498-520. Teach various possibilities for the generation of such probes and the content of which is hereby incorporated in its entirety.
According to a specific embodiment the detectable moity is 7-amino-4-methylcoumarin (AMC) or rhodamine e.g., rhodamine 110.
According to a specific embodiment, the substrate and the detectable moiety compose a molecule of formula I: Z-X-Y, wherein:
Z is a protecting group;
X is an amino acid sequence cleaved by aPC;
Y is a detectable moiety, wherein Z, X, Y are linked.
According to a specific embodiment, Z, X and Y are covalently linked.
Various protecting groups can be used and one of ordinary skill in the art would know which to select. In some embodiments the protecting moiety is selected from the group consisting of t-butyloxycarbonyl (BOC, (CH3)3COCO-, t-BOC), t-amyloxycarbonyl, adamantyl-oxycarbonyl, and p-methoxybenzyloxycarbonyl, 9-fluorenylmethoxycarbonyl (FMOC), 2-chlorobenzyloxycarbonyl and the like, nitro, tosyl (CH3C6H4S02 — ), benzyloxycarbonyl (CBZ), adamantyloxycarbonyl, 2,2,5,7,8-pentamethylchroman-6-sulfonyl, 2,3,6-trimethyl-4-methoxyphenylsulfonyl, t-butyl benzyl (BZL) or substituted BZL, such as, p- methoxybenzyl, p-nitrobenzyl, p-chlorobenzyl, o-chlorobenzyl, and 2,6-dichlorobenzyl. Each possibility represents a separate embodiment of the present invention.
It will be appreciated that the detectable moiety may comprise two chromogenic groups e.g., fluorophores, to achieve their quenched ‘off’ state to generate a signal (e.g., by FRET) however in a specific embodiment a single chromogenic group (e.g., fluorophore) is used. Conceptually this is simpler than the dual-fluorophore systems with the action of the target enzymes converting the non-fluorescent probe into a fluorescent reporter.
Methods of engineering such molecules are well known in the art and some molecules are commercially available from various vendors including Diapharma, Sigma-Aldrich, Biocip Praha and more.
According to a specific embodiment X is Pro-Arg. Other embodiments are described hereinabove.
Examples of substrate molecules that can be used in accordance with the present teachings include, but are not limited to:
• Chromogenix S-2366™ pyroGlu-Pro-Arg-pNA;
• Chromogenix S-2266™ H-D-Val-Leu-Arg-pNA;
• Chromogenix S-2288™ H-D-Ile-Pro-Arg-pNA;
• Chromogenix S-2238™ H-D-Phe-Pip-Arg-pNA; or
. Pyr-CHG-Arg-AMC-AcOH (Interchim). According to a specific embodiment, the substrate molecule comprises Pyr-Pro-Arg-
AMC.
According to a specific embodiment, X is up to 10 amino acids in length. Examples include, but are not limited to, 9, 8, 7, 6, 5, 4, 3, 2 amino acids in length.
According to a specific embodiment, X is a dipeptide or a tripeptide.
Regardless of the specific configuration whenever a chromogenic group is used, determining is effected by photometrically measuring the quantity of colored or fluorescent split product formed as a result of the catalytic hydrolytic action of activated protein C on the substrate which comprises the detectable moiety.
According to a specific embodiment, a calibration curve is established by means of the dilution series of a control sample with known aPC activity levels. A specific embodiment of a calibration curve is presented in Figure ID, which is based on standardized samples of Protein C containing sera.
As mentioned the detectable moiety is typically measured using optical means, determining the appropriate absorption spectra according to the selected fluorophore (chromogenic group).
According to a specific embodiment, the release is measured at the selected wavelength, either during the reaction in a photometer cuvette (kinetic method), or discontinuously by stopping the reaction with acetic or citric acid (end-point method).
For instance, in aqueous solutions both fluorescein and rhodamine-110 exhibit absorption maxima around 490 nm and strong emission around 520 nm. AMC exhibits excitation 360±9nm, emission 465±20nm.
The photometric signal is proportional to the enzyme activity in a properly-designed assay.
According to a specific embodiment, activity is expressed in molar (M).
In human plasma, the concertation of the zymogen PC is about 4 pg/ml or 64 nM. According to Gruber and Griffin (Blood, 1992, 79: 2340-2348), the concentration of aPC is 2000-fold lower compared to PC, which is approx. 2.26 ng/ml or 5 pM. The present inventors have found that the sensitivity of the present method is at least as low as 5 pM. The values measured in the cell cultures are in a range of 10-100 times higher than this value. Similarly values measured in CSF are in this level of detection about 5 nM.
According to a specific embodiment, the present teachings allow determination of aPC levels of as low as 0.1 pM, 1 pM, 5 pM or 1 pM- 1 nM, 1 pM- 5 pM, 1 pM- 5 pM, 0.1 pM -10 pM, 1 pM-100 nM without protein C activation: e.g., 1 pM-1 nM, 1-100 pM, 1-10 pM, 10-100 pM, 5-100 pM, 100-1000 pM, 1-500 pM, 100 pm-1000 nM, 10 pM-500 pM, 1 pM to 10 nM. Reagents described herein for use in assays of aPC activity according to some embodiments of the invention can be included in a kit where at time instructions for use in aPC detection are included.
Thus according to an aspect there is provided a kit for measuring the activity of activated protein C (aPC), the kit comprising:
(a) a substrate for aPC as described herein e.g., the substrate of formula I;
(b) a thrombin inhibitor;
(c) a Factor Xa inhibitor;
(d) optionally reagents for a calibration curve;
(e) at least one buffer; and
(f) as mentioned, instructions for measuring the activity of activated protein C (aPC). Protein C activity is associated with various physiological/ pathological conditions and therefore is imperative for the provision of diagnosis, prognosis and the like.
Protein C deficiency:
Hereditary protein C deficiency is inherited as an autosomal dominant trait. Heterozygotes for protein C deficiency have protein C activity or antigen levels of 30 to 70% of normal, whereas homozygotes (or compound heterozygotes) with a severe defect have levels below 1%. Homozygotes with a mild defect have also been reported with protein C levels of 10- 24%. The normal range of protein C in the adult is 70% to 130% of a normal plasma pool (defined as 100%).
Acquired protein C deficiency
The Protein C level is influenced by various diseases and drugs. Acquired protein C deficiency is often associated with disseminated intravascular coagulation (DIC), deep vein thrombosis, severe liver disease, sepsis, vitamin K deficiency, oral anticoagulant therapy and elective surgery. The protein C activity level may in some cases indicate the severity of a disease and can be used as a prognostic parameter.
Conditions and drugs associated with an elevated or decreased protein C level
Decreased levels
DIC
Deep vein thrombosis Pulmonary embolus Severe liver disease Post-operative patients Infection Malignancy L-asparaginase therapy Adult respiratory distress syndrome Hemolytic uremic syndrome Thrombotic thrombocytopenic purpura Oral anticoagulants Vitamin K-deficiency Neonatal period Elevated PC levels Diabetes
Nephrotic syndrome Late pregnancy Oral contraceptives Anabolic steroids Elevated protein C levels
Elevated protein C levels have been reported in diabetic and nephrotic patients, during late pregnancy, and with oral contraceptives and anabolic steroids. Elevated levels have no known clinical significance.
As used herein “inflammatory diseases” include, but are not limited to, chronic inflammatory diseases and acute inflammatory diseases.
Inflammatory diseases associated with hypersensitivity.
Examples of hypersensitivity include, but are not limited to, Type I hypersensitivity, Type II hypersensitivity, Type III hypersensitivity, Type IV hypersensitivity, immediate hypersensitivity, antibody mediated hypersensitivity, immune complex mediated hypersensitivity, T lymphocyte mediated hypersensitivity and DTH.
Type I or immediate hypersensitivity, such as asthma.
Type II hypersensitivity include, but are not limited to, rheumatoid diseases, rheumatoid autoimmune diseases, rheumatoid arthritis (Krenn V. el ah, Histol Histopathol 2000 Jul;15 (3):791), spondylitis, ankylosing spondylitis (Jan Voswinkel el ah, Arthritis Res 2001; 3 (3): 189), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Erikson J. et ah, Immunol Res 1998; 17 (l-2):49), sclerosis, systemic sclerosis (Renaudineau Y. et ah, Clin Diagn Lab Immunol. 1999 Mar;6 (2): 156); Chan OT. et ah, Immunol Rev 1999 Jun;169:107), glandular diseases, glandular autoimmune diseases, pancreatic autoimmune diseases, diabetes, Type I diabetes (Zimmet P. Diabetes Res Clin Pract 1996 Oct;34 Suppl:S125), thyroid diseases, autoimmune thyroid diseases, Graves’ disease (Orgiazzi J. Endocrinol Metab Clin North Am 2000 Jun;29 (2):339), thyroiditis, spontaneous autoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol 2000 Dec 15; 165 (12):7262), Hashimoto’s thyroiditis (Toyoda N. et al, Nippon Rinsho 1999 Aug;57 (8): 1810), myxedema, idiopathic myxedema (Mitsuma T. Nippon Rinsho. 1999 Aug;57 (8): 1759); autoimmune reproductive diseases, ovarian diseases, ovarian autoimmunity (Garza KM. et al, J Reprod Immunol 1998 Feb;37 (2):87), autoimmune anti-sperm infertility (Diekman AB. et al, Am J Reprod Immunol. 2000 Mar;43 (3): 134), repeated fetal loss (Tincani A. et al, Lupus 1998;7 Suppl 2:S 107-9), neurodegenerative diseases, neurological diseases, neurological autoimmune diseases, multiple sclerosis (Cross AH. et al, J Neuroimmunol 2001 Jan 1 ; 112 (1-2): 1), Alzheimer’s disease (Oron L. et al, J Neural Transm Suppl. 1997;49:77), myasthenia gravis (Infante AJ. And Kraig E, Int Rev Immunol 1999;18 (l-2):83), motor neuropathies (Kornberg AJ. J Clin Neurosci. 2000 May;7 (3): 191), Guillain-Barre syndrome, neuropathies and autoimmune neuropathies (Kusunoki S. Am J Med Sci. 2000 Apr;319 (4):234), myasthenic diseases, Lambert-Eaton myasthenic syndrome (Takamori M. Am J Med Sci. 2000 Apr;319 (4):204), paraneoplastic neurological diseases, cerebellar atrophy, paraneoplastic cerebellar atrophy, non-paraneoplastic stiff man syndrome, cerebellar atrophies, progressive cerebellar atrophies, encephalitis, Rasmussen’s encephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles de la Tourette syndrome, polyendocrinopathies, autoimmune polyendocrinopathies (Antoine JC. and Honnorat J. Rev Neurol (Paris) 2000 Jan;156 (1):23); neuropathies, dysimmune neuropathies (Nobile- Orazio E. et al, Electroencephalogr Clin Neurophysiol Suppl 1999;50:419); neuromyotonia, acquired neuromyotonia, arthrogryposis multiplex congenita (Vincent A. et al, Ann N Y Acad Sci. 1998 May 13;841:482), cardiovascular diseases, cardiovascular autoimmune diseases, atherosclerosis (Matsuura E. et al, Lupus. 1998;7 Suppl 2:S135), myocardial infarction (Vaarala O. Lupus. 1998;7 Suppl 2:S132), thrombosis (Tincani A. et al, Lupus 1998;7 Suppl 2:S 107-9), granulomatosis, Wegener’s granulomatosis, arteritis, Takayasu’s arteritis and Kawasaki syndrome (Praprotnik S. et al, Wien Klin Wochenschr 2000 Aug 25 ; 112 (15-16):660); anti factor VIII autoimmune disease (Lacroix-Desmazes S. et al, Semin Thromb Hemost.2000;26 (2): 157); vasculitises, necrotizing small vessel vasculitises, microscopic polyangiitis, Churg and Strauss syndrome, glomerulonephritis, pauci-immune focal necrotizing glomerulonephritis, crescentic glomerulonephritis (Noel LH. Ann Med Interne (Paris). 2000 May; 151 (3): 178); antiphospholipid syndrome (Flamholz R. et al, J Clin Apheresis 1999; 14 (4): 171); heart failure, agonist-like b -adrenoceptor antibodies in heart failure (Wallukat G. et al, Am J Cardiol. 1999 Jun 17;83 (12A):75H), thrombocytopenic purpura (Moccia F. Ann Ital Med Int. 1999 Apr- Jun;14 (2): 114); hemolytic anemia, autoimmune hemolytic anemia (Efremov DG. el al, Leuk Lymphoma 1998 Jan;28 (3-4):285), gastrointestinal diseases, autoimmune diseases of the gastrointestinal tract, intestinal diseases, chronic inflammatory intestinal disease (Garcia Herola A. et al, Gastroenterol Hepatol. 2000 Jan;23 (1): 16), celiac disease (Landau YE. and Shoenfeld Y. Harefuah 2000 Jan 16; 138 (2): 122), autoimmune diseases of the musculature, myositis, autoimmune myositis, Sjogren’s syndrome (Feist E. et al, Int Arch Allergy Immunol 2000 Sep;123 (1):92); smooth muscle autoimmune disease (Zauli D. et al, Biomed Pharmacother 1999 Jun;53 (5-6):234), hepatic diseases, hepatic autoimmune diseases, autoimmune hepatitis (Manns MP. J Hepatol 2000 Aug;33 (2):326) and primary biliary cirrhosis (Strassburg CP. et al, Eur J Gastroenterol Hepatol. 1999 Jun;ll (6):595).
Type IV or T cell mediated hypersensitivity, include, but are not limited to, rheumatoid diseases, rheumatoid arthritis (Tisch R, McDevitt HO. Proc Natl Acad Sci U S A 1994 Jan 18;91 (2):437), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Datta SK., Lupus 1998;7 (9):591), glandular diseases, glandular autoimmune diseases, pancreatic diseases, pancreatic autoimmune diseases, Type 1 diabetes (Castano L. and Eisenbarth GS. Ann. Rev. Immunol. 8:647); thyroid diseases, autoimmune thyroid diseases, Graves’ disease (Sakata S. et al, Mol Cell Endocrinol 1993 Mar;92 (1):77); ovarian diseases (Garza KM. et al, J Reprod Immunol 1998 Feb;37 (2):87), prostatitis, autoimmune prostatitis (Alexander RB. et al, Urology 1997 Dec;50 (6):893), polyglandular syndrome, autoimmune polyglandular syndrome, Type I autoimmune polyglandular syndrome (Hara T. et al, Blood. 1991 Mar 1;77 (5): 1127), neurological diseases, autoimmune neurological diseases, multiple sclerosis, neuritis, optic neuritis (Soderstrom M. et al, J Neurol Neurosurg Psychiatry 1994 May;57 (5):544), myasthenia gravis (Oshima M. et al, Eur J Immunol 1990 Dec;20 (12):2563), stiff-man syndrome (Hiemstra HS. et al, Proc Natl Acad Sci U S A 2001 Mar 27;98 (7):3988), cardiovascular diseases, cardiac autoimmunity in Chagas’ disease (Cunha-Neto E. et al, J Clin Invest 1996 Oct 15;98 (8): 1709), autoimmune thrombocytopenic purpura (Semple JW. et al, Blood 1996 May 15;87 (10):4245), anti-helper T lymphocyte autoimmunity (Caporossi AP. et al, Viral Immunol 1998; 11 (1):9), hemolytic anemia (Sallah S. et al, Ann Hematol 1997 Mar;74 (3): 139), hepatic diseases, hepatic autoimmune diseases, hepatitis, chronic active hepatitis (Franco A. et al, Clin Immunol Immunopathol 1990 Mar;54 (3):382), biliary cirrhosis, primary biliary cirrhosis (Jones DE. Clin Sci (Colch) 1996 Nov;91 (5):551), nephric diseases, nephric autoimmune diseases, nephritis, interstitial nephritis (Kelly CJ. J Am Soc Nephrol 1990 Aug;l (2): 140), connective tissue diseases, ear diseases, autoimmune connective tissue diseases, autoimmune ear disease (Yoo TJ. et al, Cell Immunol 1994 Aug;157 (1):249), disease of the inner ear (Gloddek B. et al, Ann N Y Acad Sci 1997 Dec 29;830:266), skin diseases, cutaneous diseases, dermal diseases, bullous skin diseases, pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus.
Examples of delayed type hypersensitivity include, but are not limited to, contact dermatitis and drug eruption.
Examples of types of T lymphocyte mediating hypersensitivity include, but are not limited to, helper T lymphocytes and cytotoxic T lymphocytes.
Examples of helper T lymphocyte-mediated hypersensitivity include, but are not limited to, Thl lymphocyte mediated hypersensitivity and Th2 lymphocyte mediated hypersensitivity.
Autoimmune diseases
Include, but are not limited to, cardiovascular diseases, rheumatoid diseases, glandular diseases, gastrointestinal diseases, cutaneous diseases, hepatic diseases, neurological diseases, muscular diseases, nephric diseases, diseases related to reproduction, connective tissue diseases and systemic diseases.
Examples of autoimmune cardiovascular diseases include, but are not limited to atherosclerosis (Matsuura E. et al, Lupus. 1998;7 Suppl 2:S135), myocardial infarction (Vaarala O. Lupus. 1998;7 Suppl 2:S132), thrombosis (Tincani A. et al, Lupus 1998;7 Suppl 2:S 107-9), Wegener’s granulomatosis, Takayasu’s arteritis, Kawasaki syndrome (Praprotnik S. et al, Wien Klin Wochenschr 2000 Aug 25; 112 (15- 16): 660), anti-factor VIII autoimmune disease (Lacroix- Desmazes S. et al, Semin Thromb Hemost.2000;26 (2): 157), necrotizing small vessel vasculitis, microscopic polyangiitis, Churg and Strauss syndrome, pauci-immune focal necrotizing and crescentic glomerulonephritis (Noel LH. Ann Med Interne (Paris). 2000 May; 151 (3): 178), antiphospholipid syndrome (Flamholz R. et al, J Clin Apheresis 1999; 14 (4): 171), antibody- induced heart failure (Wallukat G. et al, Am J Cardiol. 1999 Jun 17;83 (12A):75H), thrombocytopenic purpura (Moccia F. Ann Ital Med Int. 1999 Apr-Jun;14 (2): 114; Semple JW. et al, Blood 1996 May 15;87 (10):4245), autoimmune hemolytic anemia (Efremov DG. et al, Leuk Lymphoma 1998 Jan;28 (3-4):285; Sallah S. et al, Ann Hematol 1997 Mar;74 (3): 139), cardiac autoimmunity in Chagas’ disease (Cunha-Neto E. et al, J Clin Invest 1996 Oct 15;98 (8): 1709) and anti-helper T lymphocyte autoimmunity (Caporossi AP. et al, Viral Immunol 1998; 11 (1):9).
Examples of autoimmune rheumatoid diseases include, but are not limited to rheumatoid arthritis (Krenn V. et al, Histol Histopathol 2000 Jul;15 (3):791; Tisch R, McDevitt HO. Proc Natl Acad Sci units S A 1994 Jan 18;91 (2):437) and ankylosing spondylitis (Jan Voswinkel et al, Arthritis Res 2001; 3 (3): 189). Examples of autoimmune glandular diseases include, but are not limited to, pancreatic disease, Type I diabetes, thyroid disease, Graves’ disease, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto’s thyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmune anti-sperm infertility, autoimmune prostatitis and Type I autoimmune polyglandular syndrome diseases include, but are not limited to autoimmune diseases of the pancreas, Type 1 diabetes (Castano L. and Eisenbarth GS. Ann. Rev. Immunol. 8:647; Zimmet P. Diabetes Res Clin Pract 1996 Oct;34 Suppl:S125), autoimmune thyroid diseases, Graves’ disease (Orgiazzi J. Endocrinol Metab Clin North Am 2000 Jun;29 (2):339; Sakata S. el ah, Mol Cell Endocrinol 1993 Mar;92 (1):77), spontaneous autoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol 2000 Dec 15; 165 (12):7262), Hashimoto’s thyroiditis (Toyoda N. et ah, Nippon Rinsho 1999 Aug;57 (8): 1810), idiopathic myxedema (Mitsuma T. Nippon Rinsho. 1999 Aug;57 (8):1759), ovarian autoimmunity (Garza KM. et ah, J Reprod Immunol 1998 Feb;37 (2):87), autoimmune anti sperm infertility (Diekman AB. et ah, Am J Reprod Immunol. 2000 Mar;43 (3): 134), autoimmune prostatitis (Alexander RB. et ah, Urology 1997 Dec;50 (6):893) and Type I autoimmune polyglandular syndrome (Hara T. et ah, Blood. 1991 Mar 1;77 (5): 1127).
Examples of autoimmune gastrointestinal diseases include, but are not limited to, chronic inflammatory intestinal diseases (Garcia Herola A. et ah, Gastroenterol Hepatol. 2000 Jan;23 (1): 16), celiac disease (Landau YE. and Shoenfeld Y. Harefuah 2000 Jan 16; 138 (2):122), colitis, ileitis and Crohn’s disease.
Examples of autoimmune cutaneous diseases include, but are not limited to, autoimmune bullous skin diseases, such as, but are not limited to, pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus.
Examples of autoimmune hepatic diseases include, but are not limited to, hepatitis, autoimmune chronic active hepatitis (Franco A. et ah, Clin Immunol Immunopathol 1990 Mar;54 (3):382), primary biliary cirrhosis (Jones DE. Clin Sci (Colch) 1996 Nov;91 (5):551; Strassburg CP. et ah, Eur J Gastroenterol Hepatol. 1999 Jun;ll (6):595) and autoimmune hepatitis (Manns MP. J Hepatol 2000 Aug;33 (2):326).
Examples of autoimmune neurological diseases include, but are not limited to, multiple sclerosis (Cross AH. et ah, J Neuroimmunol 2001 Jan 1 ; 112 (1-2): 1), Alzheimer’s disease (Oron L. et ah, J Neural Transm Suppl. 1997;49:77), myasthenia gravis (Infante AJ. And Kraig E, Int Rev Immunol 1999;18 (l-2):83; Oshima M. et ah, Eur J Immunol 1990 Dec;20 (12):2563), neuropathies, motor neuropathies (Kornberg AJ. J Clin Neurosci. 2000 May;7 (3): 191); Guillain- Barre syndrome and autoimmune neuropathies (Kusunoki S. Am J Med Sci. 2000 Apr;319 (4):234), myasthenia, Lambert-Eaton myasthenic syndrome (Takamori M. Am J Med Sci. 2000 Apr;319 (4):204); paraneoplastic neurological diseases, cerebellar atrophy, paraneoplastic cerebellar atrophy and stiff-man syndrome (Hiemstra HS. et al, Proc Natl Acad Sci units S A 2001 Mar 27;98 (7):3988); non-paraneoplastic stiff man syndrome, progressive cerebellar atrophies, encephalitis, Rasmussen’s encephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles de la Tourette syndrome and autoimmune polyendocrinopathies (Antoine JC. and Honnorat J. Rev Neurol (Paris) 2000 Jan;156 (1):23); dysimmune neuropathies (Nobile- Orazio E. et al, Electroencephalogr Clin Neurophysiol Suppl 1999;50:419); acquired neuromyotonia, arthrogryposis multiplex congenita (Vincent A. et al, Ann N Y Acad Sci. 1998 May 13;841:482), neuritis, optic neuritis (Soderstrom M. et al, J Neurol Neurosurg Psychiatry 1994 May;57 (5):544) and neurodegenerative diseases.
Examples of autoimmune muscular diseases include, but are not limited to, myositis, autoimmune myositis and primary Sjogren’s syndrome (Feist E. et al, Int Arch Allergy Immunol 2000 Sep;123 (1):92) and smooth muscle autoimmune disease (Zauli D. et al, Biomed Pharmacother 1999 Jun;53 (5-6):234).
Examples of autoimmune nephric diseases include, but are not limited to, nephritis and autoimmune interstitial nephritis (Kelly CJ. J Am Soc Nephrol 1990 Aug;l (2): 140).
Examples of autoimmune diseases related to reproduction include, but are not limited to, repeated fetal loss (Tincani A. et al, Lupus 1998;7 Suppl 2:S 107-9).
Examples of autoimmune connective tissue diseases include, but are not limited to, ear diseases, autoimmune ear diseases (Yoo TJ. et al, Cell Immunol 1994 Aug;157 (1):249) and autoimmune diseases of the inner ear (Gloddek B. et al, Ann N Y Acad Sci 1997 Dec 29;830:266).
Examples of autoimmune systemic diseases include, but are not limited to, systemic lupus erythematosus (Erikson J. et al, Immunol Res 1998; 17 (l-2):49) and systemic sclerosis (Renaudineau Y. et al, Clin Diagn Lab Immunol. 1999 Mar;6 (2): 156); Chan OT. et al, Immunol Rev 1999 Jun;169:107).
Infectious diseases
Examples of infectious diseases include, but are not limited to, chronic infectious diseases, subacute infectious diseases, acute infectious diseases, viral diseases, bacterial diseases, protozoan diseases, parasitic diseases, fungal diseases, mycoplasma diseases and prion diseases.
Graft rejection diseases
Examples of diseases associated with transplantation of a graft include, but are not limited to, graft rejection, chronic graft rejection, subacute graft rejection, hyperacute graft rejection, acute graft rejection and graft versus host disease. Allergic diseases
Examples of allergic diseases include, but are not limited to, asthma, hives, urticaria, pollen allergy, dust mite allergy, venom allergy, cosmetics allergy, latex allergy, chemical allergy, drug allergy, insect bite allergy, animal dander allergy, stinging plant allergy, poison ivy allergy and food allergy.
Cancerous diseases
Examples of cancer include but are not limited to carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Particular examples of cancerous diseases but are not limited to: Myeloid leukemia such as Chronic myelogenous leukemia. Acute myelogenous leukemia with maturation. Acute promyelocytic leukemia, Acute nonlymphocytic leukemia with increased basophils, Acute monocytic leukemia. Acute myelomonocytic leukemia with eosinophilia; Malignant lymphoma, such as Birkitt's Non-Hodgkin's; Lymphoctyic leukemia, such as Acute lumphoblastic leukemia. Chronic lymphocytic leukemia; Myeloproliferative diseases, such as Solid tumors Benign Meningioma, Mixed tumors of salivary gland, Colonic adenomas; Adenocarcinomas, such as Small cell lung cancer, Kidney, Uterus, Prostate, Bladder, Ovary, Colon, Sarcomas, Liposarcoma, myxoid, Synovial sarcoma, Rhabdomyosarcoma (alveolar), Extraskeletel myxoid chonodrosarcoma, Ewing's tumor; other include Testicular and ovarian dysgerminoma, Retinoblastoma, Wilms' tumor, Neuroblastoma, Malignant melanoma, Mesothelioma, breast, skin, prostate, and ovarian.
According to a specific embodiment, the disease is selected from the group consisting of sepsis, infectious disease, inflammatory disease, neurodegenerative disease and nerve injury.
According to a specific embodiment, the deficiency in aPC is associated with sepsis, hence the disease is sepsis.
According to a specific embodiment, the disease is a neurodegenerative disease, a neoplasm e.g., head and neck cancer or glioma or a nervous system reaction following trauma, e.g., traumatic brain injury, stroke.
Additional neural disease states may especially benefit from embodiments of the invention, include, but are not limited to, meningitis, encephalitis due to viral/bacterial/fungal pathogens. Autoimmune nervous system diseases including demyelinating inflammatory diseases of the CNS (e.g., multiple sclerosis) and the PNS (e.g., Guillain-Barre Syndrome), neuronal manifestations of systemic autoimmune diseases such as SLE and sarcoid.
As used herein the term “diagnosing” refers to determining presence or absence of a pathology (e.g., a disease, disorder, condition or syndrome), classifying a pathology or a symptom, determining a severity of the pathology, monitoring pathology progression, forecasting an outcome of a pathology and/or prospects of recovery and screening of a subject for a specific disease.
According to some embodiments of the invention, screening of the subject for a specific disease is followed by substantiation of the screen results using gold standard methods. For instance in the case of sepsis and other diseases diagnosed by blood tests corroboration is done by immediate white blood cell counts, measuring serum lactate, and obtaining appropriate cultures before starting antibiotics. In other alternative or additional cases e.g., multiple sclerosis imaging, physical tests, and/or molecular markers analysis.
Thus, according to an aspect of the invention there is provided a method of diagnosing a disease associated with activated Protein C (aPC) activity in a subject in need thereof, the method comprising determining activity of said aPC in a biological sample of the subject as described herein, said activity being indicative of said disease or disease state.
For example, in meningitis much elevated levels of aPC are evident in the CSF as compared to a control healthy sample.
According to some embodiments of the invention, the method further comprising informing the subject of the predicted diagnosis and/or the predicted prognosis of the subject.
As used herein the phrase “informing the subject” refers to advising the subject that based on the results of the assay (aPC levels) the subject should seek a suitable treatment regimen. For example, if the subject is predicted to respond to heparin, warfarin, and protein C concentrates (in case of deficiency) and is diagnosed or suffers from a pathology requiring it that such a treatment is advisable.
Once the diagnosis is determined, the results can be recorded in the subject’s medical file, which may assist in selecting a treatment regimen and/or determining prognosis of the subject.
According to some embodiments of the invention, the method further comprising recording the levels of aPC of the subject in the subject’s medical file.
As mentioned, the prediction of the diagnosis of a subject based on aPC levels can be used to select the treatment regimen of a subject and thereby treat the subject in need thereof.
Thus, according to an aspect of the invention there is provided a method of treating a disease associated with activated Protein C activity (aPC) in a subject in need thereof, the method comprising:
(a) determining activity of said aPC in a biological sample of the subject as described herein, said activity being indicative of said disease or disease state;
(b) treating said subject according to indication or state of said disease. According to an additional or alternative aspect there is provided a method of monitoring treatment of a disease associated with activated Protein C (aPC) activity in a subject in need thereof, the method comprising:
(a) determining activity of said aPC in a biological sample as described herein of a subject having been treated for said disease, said activity being indicative of a disease state;
(b) treating said subject according to said state of said disease.
According to specific embodiments provided below are some exemplary treatment options.
Treatments for protein C deficiency include heparin, warfarin, and protein C concentrates. Acute thrombosis in protein C-deficient individuals should be treated with heparin. Warfarin is used for longer treatment periods to prevent thrombotic recurrences. When initiating the warfarin therapy it must be started at low doses in conjunction with heparin to prevent skin and fat necrosis.
Prophylactic anticoagulation is mainly recommended for symptomatic patients in high risk situations (e.g. surgery, pregnancy). Symptom-free relatives of symptomatic patients with protein C deficiency may benefit from prophylactic anticoagulation in similar risk situations, since they run an increased risk of thrombosis compared to nondeficient individuals.
Replacement of protein C can be carried out with fresh frozen plasma or pure protein C concentrates produced either from plasma or by recombinant techniques. There is currently major interest in the use of APC preparations in the management of acute DIC. Studies of APC in animal models show that it is a powerful antithrombotic agent, without the risk of bleeding as a side effect.
The present teachings further provide for a method of identifying an agent suitable for altering an activity of activated Protein C (aPC), the method comprising:
(a) contacting a biological sample comprising aPC with an agent;
(b) determining an activity of said aPC following or concomitant with said contacting as described herein, wherein a change in said activity compared to a control sample not contacted with said agent is indicative that said agent is suitable for altering an activity of activated protein C (aPC).
As used herein “agent” refers to any chemical entity such as a small molecule, a carbohydrate a biomolecule e.g., peptide, polypeptide, nucleic acid sequence, oligonucleotide, polynucleotide and a physical condition e.g., radiation.
As used herein “control” refers to a sample which has not been treated (contacted) with the agent but otherwise incubated under the same assay conditions. As used herein “change” refers to increase or decrease in activity of protein C relative to the control.
As used herein “change” is determined in a quantitative manner e.g. at least 5 %, at least 10 %, at least 15 %, at least 20 %, at least 25 %, at least 30 %, at least 35 %, at least 40 %, at least 45 %, at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 2 fold, 3 fold, 5 fold, 10 fold or more as compared to the control.
All the methods and assays described herein can be done in a small scale or in a large scale involving the use of multiple well plates e.g., at least 96 wells, robotics, imaging apparat or any means known to facilitate screening in a large scale.
As used herein the term “about” refers to ± 10 %.
The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".
The term “consisting of’ means “including and limited to”.
The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.
Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
As used herein, the term “subject” includes mammals, preferably human beings at any age which suffer from the pathology. Preferably, this term encompasses individuals who are at risk to develop the pathology.
As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
As used herein, the term “preventing” refers to keeping a disease, disorder or condition from occurring in a subject who may be at risk for the disease, but has not yet been diagnosed as having the disease.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
EXAMPLES
Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et ah, (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et ah, "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et ah, "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); “Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. L, ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1- 317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.
EXAMPLE 1 Methods
Cell culture
C6 rat glioma cells (purchased from ATCC, CCL-107) and N9 mouse microglia cells (ATCC) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Bet Haemek, Biological Industries, Israel) supplemented with 10 % fetal bovine serum (Bet Haemek, Biological Industries, Israel), 1% L-Glutamine (Bet Haemek, Biological Industries, Israel) and 0.1% penicillin and streptomycin (Bet Haemek, Biological Industries, Israel). The cells were grown in a 37°C and 5 % CCh-humidified atmosphere.
Animals
Adult ICR mice (Envigo, Jerusalem, Israel) were housed in standard conditions and fed standard diet and water available ad libitum. Ambient temperature was set to 22°C to 23 °C with day/night light control. The protocols of this study were approved by the Sheba Medical Center Committee on the Use and Care of Animals (1084-17, 1000/15) according to the ARRIVE Guidelines. mTBI induction
Mice were anesthetized using isoflurane. The mouse head was placed on a sponge immobilization board and a 50 gram metal weight was dropped down an 80-cm-high metal tube placed over the head of the mouse on the right anterolateral side. Control mice were anesthetized only. 24 hours following mTBI induction mice were sacrificed and the brain was removed for the aPC activity assay.
Mice lipopoly saccharide (LPS) treatment
Mice were weighted before LPS induction. Mice were injected intraperitoneal (IP) with LPS, a component of the bacterial wall (. Escherichia co/z 0111:B4, Sigma L4130) 1 mg/kg, diluted in saline. 24 hours following injection, mice were weighted and sacrificed. Brains were removed and prepared as described below.
Human patients
CSL samples were collected from infection chronic and acute inflammatory demyelinating polyneuropathy (CIDP/AIDP) and multiple sclerosis (MS) patients and normal pressure hydrocephalus (NPH) patients by a lumbar puncture, and were kept in -80°C until use. The procedure was approved by the ethical committee of the Chaim Sheba Medical Center (4245-17-SMC), and patients provided written informed consent. aPC activity assay aPC activity was assessed using a fluorogenic substrate synthesized on order by our specifications (Pyr-Pro-Arg-AMC, 20mM, GL Biochem Shanghai Ltd.). The reactions were carried out in black 96- well microplates. The reactions with commercial thrombin, LXa, plasma and brain tissue were performed in Tris buffer (in mM: 150 NaCl, 1 CaC12, 50 Tris-HCl: pH 8.0). The cleavage of the substrate was measured at 37°C every 2 minutes over 25 cycles (points at which fluorescence was measured, excitation 360±9nm, emission 465±20nm). The activity was calculated as the linear increase of fluorescence intensity over time infinite 2000 TEC AN).
CSL samples (93 pi) were added to the microplate wells. Substrate containing inhibitors (NAPAP-lpM, apixaban— ImM) was added and the fluorescence was measured.
Experimental design:
Thrombin/FXa/Plasma
Pooled human citrated plasma from healthy donors was purchased from Instrumentation Laboratory (normal control Assayed, 0020003110) and reconstituted using standard laboratory methods. Bovine thrombin (50mU, Sigma, T4648), bovine LXa (ImU, Hematologic Technologies, BCXA-1060) or human plasma were added to the wells (final volume of IOOmI). In the relevant wells, the samples were incubated with Protac (Technoclone, 5346212) for 5 min in order to activate PC. Substrate with the inhibitors alpha-naphthylsulphonylglycyl-4- amidinophenylalanine piperidine (NAPAP, ImM, Santa Cruz, sc-208083) and Apixaban (ImM, Selleckchem, S1593) was added and the fluorescence was measured.
Cells
Cells were seeded (1X105 cells/ml, 200 pl/well) and allowed to attach for 24 hours. The medium was then replaced by serum free Dulbecco’s Modified Eagle Media (DMEM, 100 pi). FEAM(10pM)/LPS (0.1 pg/ml, 0111:B4, Sigma, L4130) were added for 24 hours incubation period. The medium was transferred to empty wells, substrate with inhibitors (NAPAP, apixaban) was added immediately and the fluorescence was measured. The results are presented as the linear slope of the fluorescent intensity/time.
Mice tissues
Mice were sacrificed by lethal phenobarbital injection (CTS Chemical Industries ltd). The organs were rapidly removed and placed on ice. Brain was placed in a steel brain matrix (1 mm, Coronal, Stoelting, IL, USA) on ice. Brains were cut in the sagittal plane to separate right and left hemispheres. Next, brains were cut in the coronal plane into 1 mm slices. Left and right sections of slices #3 to #8 were placed in the wells and the microplate was kept on ice. The microplate was incubated at 37°C for 30 min before the addition of the substrate.. Substrate containing inhibitors (NAPAP, apixaban) was added and the fluorescence was measured.
Statistics
Statistical analyses and graphs were conducted using GraphPad Prism (version 7.00 for Windows, GraphPad Software, La Jolla California USA, www(dot)graphpad(dot)com). Paired t- tests or one-way ANOVA followed by a post hoc test were applied on normally distributed data sets. Mann Whitney test was applied on non-normal distributed data sets. Results are expressed as mean +SEM, p values <0.05 were considered significant.
EXAMPLE 2 Assay validation
The substrate selectivity for APC activity was characterized by evaluation of its cleavage by major confounding proteases. In addition to APC, both thrombin and FXa are potentially able to cleave this sequence. Therefore, cleavage of the APC substrate by these proteases was measured. Known concentrations of commercial thrombin (50 mU) and FXa (10 mU) were used, with and without thrombin and FXa specific inhibitors, NAPAP and apixaban, respectively. As can be seen in Figure 1A and IB, both thrombin and FXa cleaved the substrate. The cleavage of the substrate by thrombin was totally blocked by NAPAP and partially blocked by apixaban (7500+393, -140.6+50.4, 1967+217 aU, respectively, p<0.0001, Figure 1A). Similarly, FXa activity was fully blocked by apixaban and partially blocked by NAPAP (25.3+3.9, -9.2+1.6 and 0.83+4 aU, respectively, p<0.0005, Figure IB). The results indicate that both thrombin and FXa cleave the substrate to a certain degree, and therefore it was concluded that in order to selectively measure the APC activity, it is necessary to add NAPAP and apixaban to the assay.
Sensitivity and specificity of the substrate for APC activity measurement was studied in human plasma. As expected, low APC activity was measured in the citrated plasma, due to the anticoagulation effect of citrate. Activation of the coagulation by CaCh caused significant increase in APC activity (1262+74.73, 1+0.26 D in fluorescence intensity respectively, p<0.0001, Figure 1C). Further increase was achieved using a specific PC activator Protac (2931+49.48 compared to control, p<0.0001, Figure 1C). Next, the substrate was applied on known concentrations of PC, combined with Protac (Figure ID). Using the substrate, it was possible to detect positive APC activity, which was demonstrated by a positive slope, in samples of 138% PC (compared to normal plasma) down to 0.26% PC. The results demonstrate that the assay is sensitive to values of around 0.1% plasma PC which corresponds to a calculated concentration of approximately 5pM. Values above this can be calculated from this calibration curve.
EXAMPLE 3 Cell cultures
APC activity in human plasma after activation is relatively high, and measurable even by the current routine laboratory methods. Based on our previous experience, the coagulation proteases activity in the neural tissues and cells is much lower, compared to plasma, and requires a more sensitive method for detection [20-23]. In order to validate the use of the new assay in neural tissue, it was applied to N9 microglia cell culture. As expected, N9 cell culture presents significantly lower APC activity compared to CaCh activated plasma (14.75+0.96 and 195.1+20.04 aU, respectively, p<0.0001, Figure 2A). In addition, N9 basal APC activity totally demolished with PMSF (-0.82+0.45 aU, p<0.0001, Figure 2A).
The next challenge was to examine APC activity in different cell types, and to determine whether APC is secreted into the medium or attached to the cells. Two cells types were used; N9 cells as a model for microglia, and C6 cells as a model for astrocytes [24,25]. In order to distinguish between medium and cells derived activity, the serum-free medium was transferred into empty wells 24 hours after initiating the experiment. Fresh medium was added to the remaining cells and activity was measured immediately. Significantly higher APC activity was measured in the medium compared to the cells of both types (30.8+0.9, 8.0+0.8, 9.6+1.2, 1+0.1, N9 and C6, medium versus cells respectively, result are presented relative to APC levels measured in C6 cells, Figure 2B, p<0.0001). Interestingly, APC activity in the medium and on the cells of N9 is significantly higher compared to APC activity in the medium and on the cells of C6 (p<0.0001 and p=0.0003).
Next N9 medium was used in order to determine the kinetic properties of the substrate. The substrate was applied, at a rising concentrations, on N9 medium and calculated from the curve the Km and the Vmax of the substrate (R2=0.972, Km=65.26-117.3 and Vmax=413.8-534.2, Figure 2C).
APC activity was measured in the cells in response to inflammation induced by LPS (O.lpg/ml) for short (10 minutes) or long duration (24 hours) in the medium and on the cells. Long duration LPS treatment significantly decreased APC activity in N9 medium and cells compared to control (0.43+0.02 versus 1+0.03 aU in medium, 0.26+0.01 versus 0.36+0.01 aU in cells, p<0.0001 and p=0.0006 respectively). In contrast, short treatment with LPS did not affect APC activity in N9 cells (Figure 2D). In C6 cells, both short and long treatments with LPS did not change APC activity significantly (Figure 2E).
In order to verify the findings regarding APC activity in N9 cells, Western blot analysis was done on N9 cells and medium samples following LPS treatment. As can be seen in Figure 2F, APC protein can be detected both on the cells and in the medium. Similar protein amounts were loaded for each sample but due to the high levels of albumin in the medium the apparent amount of APC seems much lower in these samples. However both APC and actin levels are lower in the medium compared to the cells, and therefore when APC levels are corrected to actin levels, the results demonstrate significantly higher APC/actin levels in the medium compared to the cells (1.89+0.29 and 1+0.0195 aU, respectively, Figure 2E, p=0.0259). This suggests that, in the medium, most APC is present on actin containing structures such as vesicles. Following LPS treatment the APS/actin levels are significantly lower in the medium, but not on the cells, compared to controls (0.4955+0.057 and 1.89+0.29 aU, respectively, Figure 2E, p=0.0052). EXAMPLE 4
Mice, healthy control and mTBI
After establishing the method in-vitro , aPC activity was measured ex-vivo. First aPC activity was measured and mapped in control healthy mice brain (Figure 3A and 3B). A specific spatial profile of aPC activity was found in the brain (p<0.0001, Figure 3A). A significantly higher aPC activity was found in the posterior slices of the brain compared to the anterior slices (257+12.7, 1163+16.4, p<0.0001, Figure 3B).
Changes in aPC activity in the brain, in response to neuroinflammation associated with trauma, were evaluated. mTBI was induced in mice, followed by aPC activity measurements in the brain 24 hours after the injury. mTBI mice had a significantly higher aPC activity in the right hemisphere of the brain compare to control (410+48.3, 233+18.2 respectively, p<0.0001, Figure 3C). A significant elevation in aPC activity was measured in the left hemisphere as well (315.6+27.9, 193.9+13.6 respectively, p=0.0053, Figure 3C). In addition, analysis of the whole brain showed significantly higher aPC activity in the brains of mTBI mice compared to control (358.9+19.2, 203.8+17.9, p<0.0001, Figure 3D).
EXAMPLE 5
The effect of inflammation, LPS in Mice and human inflammatory CSF :
Systemic inflammation was modeled using LPS. As expected, LPS injected mice lost weight compared to healthy controls 24 hours following injection, as the result of general inflammation (31.8+0.9gr, 34+0.7gr, p<0.0001 Figure 4A). aPC activity was measured in the brains of LPS injected mice. A trend towards elevation was measured in the posterior brain of LPS injected mice (309.1+23.4, 257.2+16.4, p=0.06 Figure 4B). Measurements conducted specifically in slices 7 and 8 in the posterior brain showed a significant aPC activity elevation (337.4+45.5, 229.2+22.7 and 387.5+41.8, 254.6+32.2, p=0.04, p=0.017, respectively, Figure 4C).
Finally, aPC activity measurements were conducted in human CSF. Samples taken from viral meningoencephalitis patients showed significantly elevated aPC activity compared to NPH controls (50.3+12.4, 4.17+1.1, p=0.01, Figure 4D).
It is important to consider the opposite responses of aPC activity that were seen in-vitro and ex- vivo in the different inflammation models in this study. In the N9 cell line LPS model in vitro, aPC activity was decreased after 24 hours of exposure while, in contrast, elevated aPC activity measured in the brain following systemic LPS administration and mTBI in mice and in acute inflammation in human CSF. The inflammatory response consists of an early phase and a recovery phase. Inflammation resolving molecules are down-regulated in the early phase and up-regulated in the recovery phase [35]. aPC has an anti-inflammatory effect, through TF, thrombin inhibition and down-regulation of pro-inflammatory cytokines and chemokines [36]. Accordingly, one may assume that aPC will act as an inflammation resolving molecule and therefore its activity will vary over the inflammatory response. Hence the present results represent two different points in time of the inflammatory response. aPC activity in CSF was further assessed as shown in Figure 7.
The present inventors measured aPC activity in human CSF samples from patient with various neurological deficits (viral and bacterial infections) compared to NPH (normal pressure hydrocephalus) patients. As can be seen in Figure 7, significantly higher aPC activity was measured in the CSF of the infection patients, AIDP/CIDP patients and MS patients as compared to NPH patients.
EXAMPLE 6
Activity of aPC in various mouse tissues
As can be seen in Figure 8, detectable levels of aPC actvity were measured in various mouse tissues - skin, hippocampus, cornea and sciatic nerve. The highest aPC activity was measured in the skin and the hippocampus. The lowest aPC activity was measured in the cornea. The results were not corrected to protein levels.
EXAMPLE 7
Use of the aPC activity assay of some embodiments of the invention in drug screening
The present inventors measured aPC activity in the medium of N9 cells following 24 h treatment with four different FX-EPCR-APC-Modulators (FEAMs) (Figure 9A and Table 1).
Table 1: The sequence of FEAM 1-4 and the region in human PC/FVII which the sequence of FEAM was based on.
Figure imgf000034_0001
As can be seen in Figure 9B, all FEAMs significantly increased aPC activity in the medium of N9 cells. Cells were treated with LPS alone or in combination with FEAMs. As can be seen in Figure 9C, in the presence of LPS, significantly lower aPC activity was measured. None of the FEAMs prevented the decrease in aPC activity following LPS treatment. Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is/are hereby incorporated herein by reference in its/their entirety.
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Claims

WHAT IS CLAIMED IS:
1. A method of determining activity of activated protein C (aPC), the method comprising:
(a) contacting a biological sample with a substrate of said aPC, said substrate comprising a detectable moiety, and wherein said contacting is under conditions in which activity of thrombin and activity of Factor Xa (FXa) are inhibited;
(b) determining an amount of said detectable moiety formed as a result of a catalytic hydrolytic action of said aPC, said amount being indicative of said activity of said aPC.
2. The method of claim 1, wherein said conditions in which activity of said thrombin and activity of said Factor Xa (FXa) are inhibited comprise an inhibitor of thrombin and an inhibitor of Factor Xa (FXa).
3. A composition of matter comprising a biological sample which putatively comprises aPC, a substrate of said aPC, said substrate comprising a detectable moiety, and an inhibitor of thrombin and an inhibitor of Factor Xa (FXa).
4. The method or composition of any one of claims 2-3, wherein said inhibitor of thrombin comprises alpha-naphthylsulphonylglycyl-4-amidinophenylalanine piperidine (NAPAP), hirudin and/or Na-p-tosyl-F-lysine chloromethyl ketone (TFCK).
5. The method or composition of any one of claims 2-3, wherein said inhibitor of Factor Xa comprises apixaban and/or Rivaroxaban.
6. The method of any one of claims 1-2 and 4-5, further comprising generating a calibration curve for said activity of said aPC.
7. The method or composition of any one of claims 1-6, wherein said sample comprises a neural tissue.
8. The method or composition of any one of claims 1-6, wherein said sample comprises a neural cell or cell line.
9. The method or composition of any one of claims 1-6, wherein said sample comprises a cerebrospinal fluid (CSF).
10. The method or composition of any one of claims 1-6, wherein said sample comprises plasma.
11. The method or composition of any one of claims 1-6, wherein said sample comprises a conditioned medium of any one of a neural cell and a neural tissue.
12. The method or composition of any one of claims 1-11, wherein said substrate and said detectable moiety compose a molecule of formula I:
Z-X-Y, wherein:
Z is a protecting group;
X is an amino acid sequence cleaved by aPC;
Y is a detectable moiety, wherein Z, X, Y are covalently linked.
13. The method or composition of any one of claims 1-12, wherein said detectable moiety comprises any one of a chromogenic, fluorescent, chemiluminescent, or phosphorescent group.
14. The method or composition of claim 12, wherein said Z is selected from the group consisting of Pyroglutamic acid (Pyr), BOC, (CH3)3COCO-, t-BOC), t-amyloxycarbonyl, adamantyl-oxycarbonyl, and p-methoxybenzyloxycarbonyl, 9-fluorenylmethoxycarbonyl (FMOC), 2-chlorobenzyloxycarbonyl and the like, nitro, tosyl (CH3C6H4S02 — ), benzyloxycarbonyl (CBZ), adamantyloxycarbonyl, 2,2,5,7,8-pentamethylchroman-6-sulfonyl, 2,3,6-trimethyl-4-methoxyphenylsulfonyl, t-butyl benzyl (BZL) or substituted BZL, such as, p- methoxybenzyl, p-nitrobenzyl, p-chlorobenzyl, o-chlorobenzyl, and 2,6-dichlorobenzyl.
15. The method or composition of claim 13, wherein said fluorescent group is selected from the group consisting of AMC and rhodamin-110.
16. The method or composition of any one of claims 12-15, wherein said X is Pro-
Arg.
17. The method or composition of any one of claims 1-16, wherein said molecule comprises Pyr-Pro-Arg-AMC.
18. The method or composition of any one of claims 12-15, wherein said X is a dipeptide, tripeptide.
19. The method or composition of any one of claims 12-15, wherein said X is up to 10 amino acids in length.
20. The method or composition of any one of claims 1-19, performed in a multi-well plate.
21. The method or composition of any one of claims 13-20, wherein said determining comprises photometrically measuring.
22. A method of diagnosing a disease associated with activated Protein C (aPC) activity in a subject in need thereof, the method comprising determining activity of said aPC in a biological sample of the subject according to any one of claims 1-21, said activity being indicative of said disease or disease state.
23. A method of treating a disease associated with activated Protein C activity (aPC) in a subject in need thereof, the method comprising:
(a) determining activity of said aPC in a biological sample of the subject according to any one of claims 1-21, said activity being indicative of said disease or disease state;
(b) treating said subject according to indication or state of said disease.
24. A method of monitoring treatment of a disease associated with activated Protein C (aPC) activity in a subject in need thereof, the method comprising:
(a) determining activity of said aPC in a biological sample according to any one of claims 1-21 of a subject having been treated for said disease, said activity being indicative of a disease state; (b) treating said subject according to said state of said disease.
25. The method of any one of claims 22-24, wherein said disease is selected from the group consisting of sepsis, infectious disease, inflammatory disease, neurodegenerative disease and nerve injury.
26. A method of identifying an agent suitable for altering an activity of activated Protein C (aPC), the method comprising:
(a) contacting a biological sample comprising aPC with an agent;
(b) determining an activity of said aPC following or concomitant with said contacting according to any one of claims 1-21, wherein a change in said activity compared to a control sample not contacted with said agent is indicative that said agent is suitable for altering an activity of activated protein C (aPC).
27. A kit for measuring the activity of activated protein C (aPC), the kit comprising:
(a) a substrate for aPC, the substrate being a molecule of formula I;
(b) a thrombin inhibitor;
(c) a Factor Xa inhibitor;
(d) optionally reagents for a calibration curve;
(e) at least one buffer; and
(f) instructions for measuring the activity of activated protein C (aPC).
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