WO2016090169A1 - Sondes intracellulaires de caspase pour la détection d'apoptose et d'inflammation, et kits contenant de telles sondes - Google Patents

Sondes intracellulaires de caspase pour la détection d'apoptose et d'inflammation, et kits contenant de telles sondes Download PDF

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WO2016090169A1
WO2016090169A1 PCT/US2015/063798 US2015063798W WO2016090169A1 WO 2016090169 A1 WO2016090169 A1 WO 2016090169A1 US 2015063798 W US2015063798 W US 2015063798W WO 2016090169 A1 WO2016090169 A1 WO 2016090169A1
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ome
fmk
bmk
oph
pmk
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David Phelps
Gary Johnson
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Seed Research And Development Llc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids

Definitions

  • the present disclosure relates to cell permeable intracellular probes that bind to active caspases and can be used for detecting apoptosis and/or inflammation.
  • the probes can be used to determine the degree or incidence of caspase-mediated apoptosis, caspase-mediated inflammation, and conditions associated therewith (such as inflammatory response associated with necrosis).
  • the present disclosure additionally relates to kits comprising the present probes and instructions for their use.
  • Apoptosis is a complex mechanism of programmed cell death that is controlled by multiple biochemical events leading to morphological cell changes and eventual cell death.
  • the apoptosis process begins when apoptotic signals cause regulatory proteins to initiate an apoptosis pathway.
  • the primary pathways targeted include mitochondrial functionality, transduced signals via adaptor proteins to the apoptotic mechanism, and drug induced increases in calcium within the cell.
  • Apoptosis culminates in coordinated cell death that requires energy and, unlike cell death occurring by necrosis, does not involve an inflammatory response.
  • Apoptosis is a critical event in numerous processes within the body. For example, embryonic development relies on apoptosis, and tissues that turn over rapidly require tight regulation to avoid serious pathological consequences.
  • Certain medical conditions, such as cancer are characteized by insufficient apoptosis (insufficient cell death) and uncontrolled cell proliferation brought on in part by failure to regulate apoptosis.
  • apoptosis can be induced by various means. For example, chemical induction of apoptosis can be achieved by administering drugs such as chemotherapeutic agents that initiate apoptosis.
  • excess apoptosis can damage organs.
  • Apoptosis can also be indicative of tissue damage, such as damaged heart tissue following ischemia or reperfusion insults.
  • Inflammation is an organism's reaction to harmful stimuli, such as pathogens, damaged cells, or irritants, and an attempt by the organism (e.g., a human body) to initiate a healing process and remove the cause of the inflammation.
  • An inflammatory reaction typically involves an organism's local vascular system, immune system, and cells within the injured tissue. Chronic inflammation is characterized by a shift in cell types at the site of inflammation and simultaneous destruction and healing of tissue.
  • Caspases are a group of highly specific cysteine proteases that cleave aspartic acid peptide bonds within proteins. Caspases collaborate in the proteolytic cascade by activating themselves and each other.
  • Apoptosis-related caspases can be divided into two categories: "initiator” caspases (e.g., caspase-2, caspase-8, caspase-9, and caspase-10), and downstream “effector” caspases (e.g., caspase-3, caspase-6, caspase-7, and caspase-14).
  • Initiator caspases mediate their oligomerization and autoactivation in response to specific upstream signals, and can activate effector caspases by cleaving their inactive pro-forms.
  • Activated effector caspases trigger the apoptotic process by cleaving protein substrates within a cell.
  • Caspase inhibitors can thus regulate the initiation of the apoptotic caspase chain reaction.
  • caspase-1, caspase-4, caspase-5, caspase-11 and caspase-13 are involved in inflammatory pathways.
  • inflammation-related caspase i.e., caspase-1
  • caspase-4 caspase-5
  • caspase-11 and caspase-13 inflammation-related caspase
  • caspase-13 IL-l converting enzyme
  • ICE-I IL-l converting enzyme
  • the detection of active caspases involved in inflammatory pathways indicates an acute or chronic inflammatory response - e.g., inflammation associated with inflammatory diseases such as rheumatoid arthritis or atherosclerosis.
  • caspase probes that bind to active caspases and include a detectable group (e.g., fluorescent label).
  • detectable group e.g., fluorescent label
  • caspase probes that have detectable labels that enable the probes to be stable, safe (e.g., not radioactive), and detectable for a prolonged period of time (i.e., not just immediately after addition and,/or administration).
  • caspase probes that are more cell permeable and have higher caspase binding affinity than the currently available ones.
  • caspase probes that are selective for inflammation-related caspases, such as caspase-1.
  • the present disclosure relates to a caspase probe compound of formula: T-X-NH-CH(R)-CO-CHz-J; wherein T is selected from fluorescent detectable groups (e.g. BODIPY-FL, BODIPY-TMR, BODIPY-TR, 5-FAM, ATT0-655, DY-680, DY-750, DY- 780), compounds containing PET or SPECT suitable radiolabels (e.g. r8F, r23I;, TFA, TFMCBz and non-detectable group Z; X is a caspase recognition sequence comprised of a peptide having 1 to l0 amino acids or synthetic analogs thereof (e.g.
  • X comprises at least one amino acid selected from alanine, aspartic acid, glutamic acid, histidine, isoleucine, leucine, threonine, tryptophan, valine, N-methyltryptophan, tyrosine, glycine and fluorinated synthetic analogs thereof.
  • T-X further comprises one or more labels selected from MRl-active nuclei, radioactive metal ions, non-radioactive isotopes, paramagnetic metal ions, gamma-emitting radioactive halogens, positron-emitting radioactive non-metals, IR-dyes, and long-wavelength fluorescent dyes.
  • the label is leF.
  • J is selected from a halogen atom, a phenol group, abenzoylate group, a BMK group, an FMK group, and a PMK group, in which the groups mentioned herein may be substituted or unsubs i embodiment, the "J" phenol group is a compound of Formula A:
  • Xr, Xz, X3, Xa, and X5 is each individually selected from H, F, Cl, alkyl, aryl, aralkyl, amino, nitro, and carboxy.
  • at least one of Xr, Xz, Xl, &, and X5 is H.
  • alkyl is C1-16 alkyl, for example, Cr-6 alkyl.
  • the "J" beruoylate group is a compound of Formula B:
  • xt, xz, X3, )Q, and X5 is each individually selected from H, F, cl, alkyl, aryl, aralkyl, amino, nitro, or carboxy.
  • at least one of Xr, Xz,X3,)Q, and X5 is H.
  • alkyl is Cr-ro alkyl, for exampl €, Cr-o alkyl.
  • the present disclosure relates to a caspase probe of formula: T-X-NH-CH(R)-CO-CH2-J;
  • T is selected from a fluorescent detectable label, a PET detectable radiolabel, a SPECT detectable radiolabel, an MRI detectable label, or a non-detectable group;
  • X is a peptide having I to 10 amino acids or synthetic analogs thereof
  • R is -CHzCOzCHT or -CH2COzH
  • kits comprising a caspase probe of the invention, at least one buffer, and instructions for using the kit.
  • the kit further comprises at least one control fluorescent dye.
  • the control fluorescent dye is selected from DY 680 carboxylic acid, DY 750 carboxylic acid, DY 780 carboxylic acid, 4TT0-655, 5-FAM, BODIPY-FL, BODIPY-TMR, and BODIpy TR.
  • the kit comprises at least one caspase probe selected from (but not limited to):
  • ATTO6 s s -D(OMe)E(OMe)VD(OMe)-BMK;
  • ATTO6S 5-IE(OMe)TD(OMe)-BMK; ATTO6S s -W(NMe)E(OMe)HD(oMe)-BMK; ATT065 5-WAD(OMe)-BMK; 4TT065 5-LE(OMe)HD(OMe)-BMK;
  • ATTO6S 5-LE(OMe)TD(OMe)-OPH; ATTO655-VD(OMe)VAD(OMe)-OPH DY680-VAD(OMe)-FMK; DY680- F6V-AD(OMe)-FMK;
  • BODIPY-FL-VD(OMe)VAD(OMe)-BM BODIPY-FL- FoV-AD(OMe)-BMK BODIPY-FL-D(OMe)E(OMe)VD(OMe)-OPH; BoDIPY-FL-IE(OMe)TD(OMe)- OPH;
  • BODIPY-TR-VAD(OMe)-FMK BOD IPY-TR-WQ. ⁇ Me)E(OMe)HD(OMe)-FMK; BODIPY-TR-wE(OMe)HD(OMe)-FMK;
  • TFA-WE(OMe)HD(OMe)-OPH TFA-YVAD(OMe)-FMK; TFA-YVAD(OMe)- BMK; TFA-WAD(OMe)-PMK; TFA-WAD(OMe)-OPH; TFA- GWE(OMe)HD(OMe)-FMK; TFA-GWE(OMe)HD(oMe)-BMK; TFA-GWE(oMe)HD(OMe)-PMK;
  • TFMCBZ-YVAD(OMe)-FMK TFMCBZ-YVAD(OMe)-BMK
  • TFMCBZ- WAD(OMe)-PM TFMCBZ-YVAD(OMe)-OPH
  • TFMCBZ- GWE(OMe)HD(OMe)-FMK TFMCBZ-GWE(OMe)HD(OMe)-BMK
  • TFMCBZ- GYVAD(OMe)-FMK TFMCBZ-GYVAD(OMe)-BMK
  • TFMCBZ-GYVAD(OMe)-OPH TFMCBZ-GYVAD(OMe)-OPH
  • the kit comprises at least one caspase probe selected from (but not limited to): 5-FAM-VAD(OMe)-FMK; 5-FAM-W(NMe)E(OMe)HD(OMe)-FMK; 780- WQ.,IMe)E(OMe)HD(OMe)-FMK; 780-VAD(OMe)-FMK; BODIPY-FL- D(OMe)E(OMe)VD(OMe)-FMK; BODIPY-FL-IE(OMe)TD(OMe)-FMK; BODIPY-FL- LE(OMe)HD(OMe)-FMK; BODIPY-FL-LE(OMe)TD(OMe)-FMK; BODIPY-FL-FL-VAD(OMe)-FMK; BODIPY-FL-WQ. ⁇ Me)E(OMe)HD(OMe)-FMK; BODIPY-FL-WE(OMe)HD(OMe)-FMK; BODIP
  • the kit comprises a buffer selected from (but not limited to) PBS; tris buffer 20mM, pH 7.4; Hanks balanced salt solutions buffered with 20mM Hepes; and combinations thereof.
  • the buffer is 10X PBS.
  • the present disclosure relates to a caspase probe having the formula: T-X-NH-CH(R)-CO-CH2-J; wherein T is selected from fluorescent detectable groups (e.g. BODIPY-FL, BODIPY-TMR, BODIPY-TR, 5-FAM, 4TT0-655, DY-680, DY-750, DY- 780), a a compound containing a PET or SPECT suitable radiolabel (..g. "F, 123I;, TFA, TFMCBz and non-detectable group Z; X is a caspase recognition sequence comprised of a peptide having I to 10 amino acids or synthetic analogs thereof (e.g.
  • R is -CH2COzCH: or -CHzCOzH; and J is a reactive group that binds to a caspase; provided that J is not FMK when X comprises only natural amino acids.
  • X comprises at least one amino acid selected from (but not limited to) alanine, aspartic acid, glutamic acid, histidine, isoleucine, leucine, threonine, tryptophan, valine, N-methyltryptophan, tyrosine, glycine and fluorinated synthetic analogs thereof.
  • J is selected from a halogen atom, a phenol group, a benzoylate group, a BMK group, an FMK group, and a PMK group, in which the groups mentioned herein may be substituted or unsubstituted.
  • the caspase probe is selected from (but not limited to):
  • ATTO6S s -IE(OMe)TD(OMe)-PMK; ATTO6S 5 -W(NMe)E(OMe)HD(OMe)-PMK; 4.TT065 5-WAD(OMe)-PMK; ATTO655-LE(OMe)HD(OMe)-PMK;
  • DY680-YVAD(OMe)-FMK DY680-LE(OMe)HD(OMe)-FMK; DY680-LE(OMe)TD(OMe)-FMK; DY680-VD(OMe)VAD(OMe)-FMK DY680-VAD(OMe)-PMK; DY680- F6V-AD(OMe)-PMK;
  • TFA-WE(OMe)HD(OMe)-OPH TFA-YVAD(OMe)-FMK
  • TFA-YVAD(OMe)-PMK TFA-YVAD(OMe)-OPH
  • TFA- GWE(oMe)HD(oMe)-FMK TFA- GWE(oMe)HD(oMe)-FMK
  • TFA-GWE(OMe)HD(OMe)-BMK TFA-GWE(OMe)HD(OMe)-PMK;
  • TFMCBZ-YVAD(OMe)-FMK TFMCBZ-YVAD(OMe)-BMK
  • TFMCBZ-YVAD(OMe)-PMK TFMCBZ-YVAD(OMe)-OPH
  • TFMCBZ- GWE(OMe)HD(OMe)-FMK TFMCBZ-GWE(oMe)HD(OMe)-BMK
  • TFMCBZ- GWE(OMe)HD(OMe)-PMK TFMCBZ-GWE(OMe)HD(OMe)-OPH
  • TFMCBZ- GWAD(OMe)-FMK TFMCBZ-GYVAD(OMe)-BMK
  • TFMCBZ-GYVAD(OMe)- PMK TFMCBZ-GYVAD(OMe)-OPH
  • the caspase probe is selected from (but not limited to): BODIPY- FL-D(OMe)E(OMe)VD(OMe)-FMK; BODIPY-FL-IE(OMe)TD(OMe)-FMK BODIPY-FL- LE(OMe)HD(OMe)-FMK; BODIPY-FL-LE(OMe)TD(OMe)-FMK; BODIPY-TMR- D(OMe)E(OMe)VD(OMe)-FMK; BODIPY-TMR-IE(OMe)TD(OMe)-FMK; BODIPY- TMR-LE(OMe)HD(OMe)-FMK; BODIPY-TMR-LE(OMe)TD(OMe)-FMK; BODIPY- TMR-VAD(OMe)-FMK; BODIPY-TMR-W(NMe)E(OMe)HD(OMe)-FMK; BODIPY- TMR-
  • the present disclosure relates to a composition
  • a composition comprising a caspase probe and an excipient.
  • the excipient is selected from sucrose, lactose, cellulose, gelatin, polyvinylpyrrolidone, trehalose, cyclodextrin, hyaluronic acid and polyethylene glycol.
  • the excipient is selected from sucrose, lactose, cellulose, gelatin, pollvinylpyrrolidone, and polyethylene glycol.
  • the present disclosure relates to a compound having the formula: T- X-NH-CH(R)-CO-CH2-S-Cys-Caspase; wherein T is selected from fluorescent detectable groups (e.g. BODIPY-FL, BODIPY-TMR, BODIPY-TR, 5-FAM, ATT0-655, DY-680, DY- 750,DY-780), a PET or SPECT suitable radiolabel (".g. ttF, "'Iy, TFA, TFMCBz and non- detectable group Z;
  • X is a peptide having 1-10 amino acids or synthetic analogs thereof; and
  • R is -CHzCOzCHI or -CHzCOzH.
  • X comprises at least one amino acid selected from (but not limited to) alanine, aspartic acid, glutamic acid, histidine, isoleucine, leucine, threonine, tryptophan, valine, N-methyltryptophan, tyrosine, glycine and fluorinated synthetic analogs thereof.
  • the present disclosure relates to a compound having the formula: T- X-NH-CH(R)-CO-CH2-S-Cys-Caspase; wherein T is selected from detectable groups BODIPY-FL, BODIPY-TMR, and BODIPY TR and non-detectable group Z; X is a peptide having 2-6 amino acids or synthetic analogs thereof; and R is -CH2CO2CH3 or -CHzCOzH.
  • X comprises at least one amino acid selected from alanine, aspartic acid, glutamic acid, histidine, isoleucine, leucine, threonine, tryptophan, valine, N- methyltryptophan, and fl uorinated synthetic analogs thereof.
  • R1 is selected from 780, ABzC, .LECBz, AMCBz, BODIPY-FL, BODIPY-TMR, BODIPY-TR, FAM, N-AE, N-AM, NOTA, ORGn, TFA, TFMCBz, Z, and formula (I)
  • L is a radioisotope
  • n is an integer from 0 to 6
  • the phenyl group of formula (I) is further substituted with one or more substituents selected from H, -OH, -OCH3, halogen, and C1-6 alkyl.
  • the present disclosure relates to a method of determining in vivo the incidence of apoptosis in a living organism, comprising the steps of: (i) administering in vivo a probe to a living organism; (ii) subjecting the living organism to a means for in vivo detection of caspase-probe complexes; and (iii) determining the incidence of apoptosis as indicated by the presence or absence of caspase-probe complexes.
  • the in vivo detection is performed on at least one portion of the living organism selected from eye, breast, heart, brain and central nervous system, kidney, lung, liver, skin, pancreas, skeletal system, connective tissue, stomach, upper gastrointestinal tract, lower gastrointestinal tract, circulatory system, lymphatic system, sexual organ, prostate, embryologic tissue, muscular system, and gallbladder.
  • the method fuither comprises the steps of: (a) before administering the probe in step (i), administering in vivo a candidate therapy intended to induce apoptosis in the living organism; and (b) after step (iii), determining whether the candidate therapy induces or inhibits apoptosis as indicated by the detected presence or absence of caspase-probe complexes, respectively.
  • the candidate therapy is a chemotherapy, immunotherapy or radiation therapy.
  • the present disclosure relates to a method of determining in vivo the incidence of inflammation in a living organism, comprising the steps of: (i) administering in vivo a probe to a living organism; (ii) subjecting the living organism to ameans for invivo detection of caspase-1-probe complexes; and (iii) determining the incidence of inflammation as indicated by the presence or absence of caspase-l-probe complexes.
  • the in vivo detection is performed on at least one portion of the living organism selected from eye, breast, heart, brain and central nervous system, kidney, lung, liver, skin, pancreas, skeletal system, connective tissue, stomach, upper gastrointestinal tract, lower gastrointestinal tract, circulatory system, lymphatic system, sexual organ, prostate, embryologic tissue, muscular system, and gallbladder.
  • the method further comprises the steps of: (a) before administering the probe in step (i), administering in vivo a candidate therapy intended to induce inflammation in the living organism; and (b) after step (iii), determining whether the candidate therapy induces or inhibits inflammation as indicated by the detected presence or absence of caspase-1-probe complexes, respectively.
  • the present disclosure relates to a method of determining ex vivo the incidence of apoptosis in a biological sample extracted from a living organism, comprising the steps of: (i) administering in vivo a probe to a portion of a living organism; (ii) extracting a biological sample from the portion of the living organism; (iii) subjecting the biological sample to a means for ex vivo detection of caspase-probe complexes; and (iv) determining the incidence of apoptosis as indicated by the presence or absence of caspase-probe complexes.
  • the biological sample is a biopsy.
  • the present disclosure relates to a method of determining ex vivo the incidence of inflammation in a biological sample extracted from a living organism, comprising the steps of: (i) administering in vivo a probe to a portion of a living organism; (ii) extracting a biological sample from the portion of the living organism; (iii) subjecting the biological sample to a means for ex vivo detection of caspase-l-probe complexes; and (iv) determining the incidence of apoptosis as indicated by the presence or absence of caspase-l- probe complexes.
  • the present disclosure relates to a method of determining in vitro the incidence of apoptosis in a biological sample, comprising the steps of: (i) adding a probe to a biological sample; (ii) incubating the sample with the probe under conditions sufficient to form caspase-probe complexes; (iii) subjecting the sample of step (ii) to a means for detecting caspase-probe complexes; and (iv) determining the incidence of apoptosis as indicated by the presence or absence of caspase-probe complexes.
  • the method further comprises the step of inducing apoptosis in the biological sample before adding the probe in step (i).
  • the biological sample is selected from a blood sample, tissue sample, cell suspension, cellular extract, and tissue homogenate.
  • the present disclosure relates to a method of determining in vitro the incidence of inflammation in a biological sample, comprising the steps of: (i) adding a probe to a biological sample; (ii) incubating the sample with the probe under conditions sufficient to form caspase-l-probe complexes; (iii) subjecting the sample of step (ii) to a means for detecting caspase-l-probe complexes; and (iv) determining the incidence of inflammation as indicated by the presence or absence of caspase-l-probe complexes.
  • the method further comprises the step of inducing inflammation in the biological sample before adding the probe in step (i).
  • the biological sample is selected from a blood sample, tissue sample, cell suspension, cellular extract, and tissue homogenate.
  • Figure lA-F shows nea.r infrared in vivo imaging of retinal apoptosis in rats (Example l).
  • Figure 2A-B shows the results of the HPLC ( Figure 2A) and Mass Spectral ( Figure 28) analyses for the 780-VAD(OMe)-PMK compound (see; Example 2).
  • Figure 3A-B shows the results of the HPLC ( Figure 3.4.) and Mass Spectral ( Figure 3B) analyses for the 780-VAD(OMe)-OPH compound (see, Example 3).
  • the present disclosure relates to intracellular caspase probes that covalently bind to active caspases, and methods of using such probes to detect caspase-mediated apoptosis, caspase-mediated inflammation, and conditions associated therewith.
  • the present disclosure further relates to kits comprising the present probes and instructions for their use.
  • biological sample refers to any type of material of biological origin, including but not limited to a blood sample, tissue sample, cell suspension, cellular extract, or tissue homogenate.
  • ex vivo refers to processes or procedures performed on a biological sample (e.g., tissue, cells, blood, etc.) extracted from a living, multicellular organism following the in vlvo administration of a caspase probe to the living organism.
  • ex vivo detection includes obtaining a biopsy (e.g., tumor cells) extracted from a living subject (e.g., a mammal, such as a human) subsequent to in vivo administration of a caspase probe to that subject, and subjecting the biopsy to a means for detecting caspase- probe complexes (e.g., IR imaging, MRI, PET, SPECT, etc.), which would be indicative of apoptosis occurring in the subject.
  • a biological sample e.g., tissue, cells, blood, etc.
  • ex vivo detection includes obtaining a biopsy (e.g., tumor cells) extracted from a living subject (e.g., a mammal, such as a human) subsequent to in vivo
  • in vitro refers to processes or procedures performed on a biological sample outside a living organism.
  • in vitro administration includes administering (i.e., delivering, applying, etc.) a caspase probe to a biological sample that is outside a living organism; and in vitro detection includes subjecting a biological sample (e.g., cells in a test tube or cultured dish) to a means for detecting caspase-probe complexes (e.g., flow cytometry), which could reflect whether a candidate therapy present in the sample induces or inhibits apoptosis.
  • a biological sample e.g., cells in a test tube or cultured dish
  • a means for detecting caspase-probe complexes e.g., flow cytometry
  • in vivo refers to processes or procedures performed inside a living, multicellular organism.
  • in vivo administration includes administering a caspase probe to a living subject (e.g., a mammal, such as a human); and in vivo detection includes subjecting a living subject to a means for detecting caspase-probe complexes (e.g., magnetic resonance imaging (MRI), etc.), which would be indicative of apoptosis occurring5 in the subject.
  • MRI magnetic resonance imaging
  • incidence refers to the occurrence rate, frequency of an event, or the quantifiable degree to which an event occurs.
  • incubating when used with respect to incubating a sample with a caspase probe, refers to exposure conditions (e.g., time, temperature, pH, etc.)0 sufficient for the formation of caspase-probe complexes.
  • label refers to a detectable moiety comprising one or more atoms.
  • N-terminal group refers to a moiety attached to the N- terminal position of the recognition sequence of the caspase probes.
  • the N-terminal group5 can be a detectable or non-detectable group.
  • the term "reactive group” refers to, in one embodiment, a portion of the caspase probe that covalently binds to the active catalytic site of a caspase.
  • a reactive group is capable of interacting with or reacting with the catalytic site of a caspase.
  • a reactive group is a leaving group that can be displaced to0 form a covalent bond with a caspase.
  • the reactive group can be FMK, PMK, or OPH.
  • the reactive group can be a halogen atom, a substituted or unsubstituted phenol group, a substituted or unsubstituted benzoylate group, a substituted or unsubstituted BMK group, a substituted or unsubstituted FMK group, and a substituted or unsubstituted PMK group.
  • the reactive group can be a halogen atom (e.g. F or Cl) or a (C1-Ca) alkyl group that is substituted with one or more halogen atoms.
  • the reactive group can be a portion of the caspase probe that covalently binds to the active catalytic site of a caspase.
  • the term “recognition sequence” refers to a portion of the caspase probes comprising a sequence of I to l0 natural amino acids or synthetic analogs thereof, which is selective for one or more caspases. In one embodiment, the term “recognition sequence” refers to a portion of the caspase probes comprising a sequence of 1 to 6 natural amino acids or synthetic analogs thereof, which is selective for one or more caspases.
  • substituted means the group is substituted withone or more (e.g., 1, 2,3,4 or 5) substituents independently selected from halo, cyano, nitro, carboxy, (Cr-Ca) alkyl, (Cr-C+) haloalkyl, (Cr-C+) alkoxy, (Cr-C+) haloalkoxy, (Cr-C+) alkanoyl, (Cr-C+) alkoxycarbonyl, (C1-Ca) alkanoyloxy, amino, (Cr-Cq) alkylamino, and ((C1-Ca)alkyl)2 amino.
  • substituents independently selected from halo, cyano, nitro, carboxy, (Cr-Ca) alkyl, (Cr-C+) haloalkyl, (Cr-C+) alkoxy, (Cr-C+) haloalkoxy, (Cr-C+) alkanoyl, (C
  • amino acid As used herein, the term “synthetic,” with respect to an amino acid, means that the amino acid is not naturally occurring.
  • BODIPY-FL boron-dipyrromethene fluorescent dye (structure shown below)
  • BODIPY-TMR boron-dipyrromethene tetramethylrhodamine dye (structure shown below)
  • BODIPY-TR boron-dipyrromethene dye (structure shown below)
  • BODIPY-FL BODIPY-TMR BODIPY-TR 780 DylightrM 780 Infrared Dye (infrared specialty dye having a benzopyrillium core and 1-3 sulfonates)
  • ATTO 655 far red excitation dye (manufactured by ATTO-TEC GmbH, catalog # AD 655)
  • DY 680 far red excitation dye (structure shown below)
  • DY 750 near-infrared excitation dye (structure shown below)
  • DY 780 near-infrared excitation dye (structure shown below)
  • LAD L-leucyl-L-alanyl-L-aspartic acid
  • VAD L-valyl-L-alanyl-L-aspartic acid
  • VD L-v alyl-L-aspartic acid
  • AMBzC 4-aminomethylbenzyloxycarbonyl
  • F6V-AD L-hexafluorovalyl-L-alanyl-L-aspartic acid
  • TETA p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid
  • the N-terminal group may serve to protect the recognition sequence during synthesis of a caspase probe. Additionally, the N-terminal group may include a label. In at least one embodiment, an N-terminal protecting group may be present during synthesis in an intermediate precursor of a caspase probe, but may be removed and replaced with an N- terminal labeled moiety - i.e., the N-terminal group of a caspase probe intermediate may be different from the N-terminal group of a final caspase probe.
  • Each recognition sequence comprises one or more amino acids and is able to bind to one or more caspases.
  • "capable of binding” i.e. binding affinity for a certain caspase
  • a recognition sequence may allow the caspase probe to target structurally similar caspases with the same or different affinities and kinetics.
  • the recognition sequence VAD valine-alanine-aspartic acid
  • VAD valine-alanine-aspartic acid
  • caspase probes containing the VAD recognition sequence will covalently bind to all or most active caspases.
  • the caspase probe is designed to react with multiple caspases to allow detection of all or most caspases.
  • the caspase probe is designed to react selectively0 with caspase-l, thus allowing for detection of inflammation in particular.
  • caspase probes containing the recognition sequence WEHD, YVAD, GWEHD or GWAD will bind covalently to caspase-1 over other apoptotic caspases.
  • the caspase probe is designed to react with two caspases that have similar substrate selectivity.
  • caspase probes containing the5 recognition sequence DEVD will recognize and allow detection of both caspase-3 and caspase-7.
  • the caspase probe is selected from probes listed in Table 1.
  • Table I identifies recognition sequences of caspase probes disclosed herein and their corresponding ability to selectively bind to all or most active caspases (i.e., poly-caspase or0 pan-caspase), inflammation-related active caspases (e.g., caspase-l), or apoptosis-related active caspases (e.g., caspase-317 or caspase-8).
  • the caspase probe is fluorinated.
  • One way to fluorinate a caspase probe and render it detectable by leF imaging is to incorporate a synthetic fluorinated amino acid into the recognition sequence. Due to the unique properties of fluorine, biopolymers with high fluorine content simultaneously exhibit both hydrophobic and lipophobic characteristics. Fluorinated analogs of hydrophobic amino acids (e.g., trifluoro- methionine, hexafluoro-valine, hexafluoro-leucine and pentafluoro-phenylalanine) are sterically similar to the corresponding nonfluorinated amino acids and can be used to substitute their hydrogen counterparts.
  • a non-exhaustive list of suitable synthetic amino acid analogs, some of which are fluorinated amino acids, is provided in Table 2, and are either commercially available or can be synthesized using known procedures.
  • a multi-step binding mechanism results in the formation of a covalent bond between the reactive group and the -SH moiety of the cysteine residue in the active catalytic site of the caspase.
  • the caspase probe binds irreversibly and covalently to the caspase.
  • the caspase probes comprise a reactive group that enables the probe to covalently bind to an active caspase.
  • the reactive group includes a moiety that leaves as the reactive group reacts with the active caspase.
  • the reactive group can be tailored to bind to specific types of caspases, such as caspases associated with apoptosis, caspases associated with inflammation, or caspases associated with both.
  • caspase probes of the present disclosure bind to active caspases, which are expressed in active form during apoptosis and/or inflammation, the disclosed caspase probes have good signal-to-noise ratio.
  • the caspase probes comprise detectable labels that may be incorporated into the N- terminal group.
  • the label allows a probe to be detected by one or more detection techniques-e.g., MRI (".g., "F MRI), PET (e.g. l8F or r23I imaging), SpECT, IR, luminescence (e.g., bioluminescence or chemiluminescence), optical imaging including confocal microscopy, flow cytometry, fluorescence, and others.
  • detection techniques e.g., MRI (".g., "F MRI), PET (e.g. l8F or r23I imaging), SpECT, IR, luminescence (e.g., bioluminescence or chemiluminescence), optical imaging including confocal microscopy, flow cytometry, fluorescence, and others.
  • Suitable labels include, but are not limited to, MRl-active nuclei, fluorescent dyes (including long-wavelength far red and near infrared fluorescent dyes), PET suitable radiolabels, SPECT suitable radiolabels.
  • the label is a dye, such as a chromophore or a fluorescent compound, that interacts with light in the electromagnetic spectrum with wavelengths from ultraviolet light to near infrared.
  • Suitable organic chromophoric and fluorophoric labels include, but are not limited to, moieties having an extensive delocalized electron system -0 €.9., cyanines, merocyanines, indocyanines, phthalocyanines, naphthalocyanines, triphenylmethines, porphyrins, pyrilium dyes, thiapyrilium dyes, squarylium dyes, croconium dyes, azulenium dyes, indoanilines, benzophenoxaziniurn dyes, benzothiaphenothiazinium dyes, anthraquinones, napthoquinones, indathrenes, phthaloylacridones, trisphenoquinones, azo dyes, intramolecular and
  • the label is fluorescent.
  • Suitable fluorescent labels include, but are not limited to, fluorescent proteins, such as green fluorescent protein (GFP) and modifications of GFP that have different absorption/emission properties, phycobiliproteins, complexes of certain rare earth metals (e.g., europium, samarium, terbium, or dysprosium),0 and fluorescent nanocrystals (quantum dots).
  • Suitable fluorescent dyes include, but are not limited to, fluorescein (e.g.5-FAM), sulforhodamine l0l (Texas Red), rhodamine B, rhodamine 6G, rhodamine 19, indocyanine green, Cy2,Cy3, Cy3.5, Cy5, Cy5.5,Cy7, Marina Blue, Pacific Blue, Oregon Green 88, Oregon Green 514, tetrarrrethylrhodamine, BODIPY dyes (e.g., BODIPY-FL), Alexa Fluor dyes such as
  • the fluorescent dye has an absorption maximum in the visible (400-800 nm) or near infrared (800-2500 nm) region.
  • Suitable MRl-active nuclei have a non-zero nuclear spin.
  • an MRI label contait s 'eF and is detectable by NMR or MRI reF imaging.
  • Examples of a label detectable by t'F imaging include, but are not limited to, a fluorinated Z group,TFA (trifluoroacetyl) and TFMCBz (4-trifluoromethylcarbobenzyloxy). Because the resonance frequency of leF is close to that of 1H, measurements can be performed using a MRI apparatus suitable for lH.
  • MRI labels may be present in one or more parts of the caspase probe. Additionally, more than one label may be included in one particular part of the caspase probe - e.g., the N-terminal group may be dual labeled, or the N-terminal group may be dual labeled and the recognition sequence may be labeled as well.
  • a caspase probe may be labeled with one or more trifluoromethyl groups such that signals from each fluorine atom are nearly identical, which produce an additive0 effect and more intense signal. That is, the signals from each fluorine will be additive rather than produce different chemical shifts, thereby broadening the signal.
  • Other 'eF-label"d caspase probes produce additive MRI signals from discrete sites of fluorination on the probes by design, not simply by attaching fluorine anywhere on the molecule.
  • leF in the caspase5 probes can stabilize the probe, protect against photobleaching (when the leF is attached to a fluorescent dye, such as Oregon Green), enhance cell permeability, and/or enhance probe- caspase binding.
  • a fluorescent dye such as Oregon Green
  • fluorine is a small and dense atom, only about 10% bigger in diameter than hydrogen; and fluorine-containing molecules are hydrophobic.
  • Fluorinated amino acids tend to bind to proteins more strongly than non-fluorinated amino acids, likely0 due to the neutral, electron dense, and hydrogen-similar size of fluorine atoms.
  • suitable fluorescent labels include, but are not limited to BODIPY-FL, BODIPY-TMR, BODIPY-TR, 780, and FAM.
  • Suitable detection techniques include techniques used to detect "F (".g., leF Mzu; and5 fluorescence, such as luminescence, optical imaging including confocal microscopy, and flow cytometry.
  • the detection means is leF imaging, which is particularly advantageous because it need not be performed immediately after administration of the caspase probe since an leF-containing probe will remain bound to active caspases in the0 apoptotic cell for the life of the cell (i.e., until the cell becomes compromised and eliminated through phagocytosis).
  • Detection can be performed using equipment designed for in vitro use, such as optical imaging, flow cytometry, and luminescence detector; and detection can also be performed using equipment designed for in vivo detection, such as MRL
  • detection means will depend, in part, on the type of label to be detected.
  • Table 3 provides examples of detection means suitable for various labels used in caspase probes disclosed herein. Additional detection means other than those listed in Table 3 may be suitable as well.
  • the caspase probes of the present disclosure have the following general structure:
  • T is a detectable N-terminal group selected from (for example) BODIPY-FL,
  • X is a peptide having l-10 amino acids or synthetic analogs thereof
  • R is -CHzCOzCHT or -CHzCOzH
  • J is a reactive group that binds to a caspase.
  • T comprises the N-terminal group
  • X-NH-CH(R)-CO-CH2 comprises the recognition sequence
  • J comprises the reactive group
  • T is a detectable N-terminal group selected from BODIPY-FL, BODIPY-TMR, BODIPY-TR, 5-FAM, 780, TFA, or a non-detectable group Z;
  • X is a peptide having 2-6 amino acids or synthetic analogs thereof
  • R is -CHzCOzCH: or -CHzCOzH
  • J is a reactive group that binds to a caspase.
  • T is selected from (for example) BODIPY-FL, BODIPY- TMR, BODIPY-TR, s-FAM, 4TT0-655, DY-680, DY-750, DY-780, ,'F, ",I, TFA ANd TFMCBz.
  • T is a non-detectable Z group.
  • T is selected from BODIPY-FL, BODIPY-TMR, BODIPY-TR, 5-FAM, 780, and TFA.
  • T is a non-detectable Z group.
  • X is selected from one or more naturally occurring amino acids, one or more synthetic amino acids and combinations thereof.
  • X is one or more amino acids selected from alanine, aspartic acid, glutamic acid, histidine, isoleucine, leucine, threonine, tryptophan, valine, N-methyltryptophan, tyrosine, glycine and fluorinated synthetic analogs thereof.
  • J is capable of reacting and/or binding with at least one caspase selected from caspase-l, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, and caspase-I0, caspase-l 1, caspase-l2, caspase-l3, and caspase-14.
  • J is selected from BMK, FMK, and PMK.
  • the "J" phenol group is a compound of Formula A: Formula A
  • X1 Xz, X3, )Q, and X5 is each individually selected from H, F, Cl, alkyl, aryl, aralkyl, amino, nitro, and carboxy.
  • at least one of X1, X2,X3, &, and Xs is H.
  • alkyl is C1-16 alkyl, for example, Cr-o alkyl.
  • the "J" benzoylate group is a compound of Formula B:
  • Xr, X2, X3, X4, and X5 is each individually selected from H, F, Cl, alkyl, aryl, aralkyl, amino, nitro, or carboxy.
  • at least one of X1, Xz,Xt, )Q, and X5 is H.
  • alkyl is Cr-ro alkyl, for example, Ct6 alkyl.
  • the caspase probe has a molecular weight of about 300- 1500 Daltons. In at least one embodiment, the caspase probe has a molecular weight of about 400-1200 Daltons.
  • the caspase probes are cell permeant (i.e., exhibit good cell membrane permeability) and can selectively target caspases of interest inside the cells of a living organism or a biological sample.
  • the caspase probes do not undergo facile metabolism, and possess a long half-life (e.g., more than 6 hours) or remain detectable throughout the life of the permeated cell.
  • each caspase probe forms a metabolite having the formula T-X-NH-CH(R)-Co-CH2-S-Cys-Enzyme, wherein T, x, and R are as defined above.
  • the caspase probes can be synthesized using liquid phase peptide synthesis (Bodanszky, PnrNcrI-ES oF PEeTIDE S ⁇ .NrHesrs (1993) or solid phase peptide synthesis (Menifield, J Amer Chem Soc 85(14):2149-54 (1963); Amblard et al., Mol. Biotechnol.,33(3):239-54 (2006)).
  • a suitable liquid phase peptide synthetic pathway is shown below, and generally involves building oligo peptides from the N-terminus of an amino acid by providing a first amino acid (1); protecting or blocking the end terminus of the amino acid using a protecting or blocking group (e.g., BOC or FMOC); coupling the first amino acid with a C-terminal ester of a second amino acid (2) by reacting the amino acids in the presence of a coupling agent (e.g., dicyclohexyl carbodiimide (DCC), diisopropyl carbodiimide (DIC), usually in the presence of N-hydroxysuccinimide or l- hydroxybenzotriazole) to form a dipeptide (3); and deprotecting the dipeptide without removing any other protecting groups to yield the free dipeptide acid (4).
  • a coupling agent e.g., dicyclohexyl carbodiimide (DCC), diisopropyl carbodiimide (DIC), usually in the presence
  • Dipeptide (4) can be coupled to the suitably derivatized L-aspartic acid B-methyl ester to yield a desired caspase inhibitor or it can be coupled with a suitably protected amino acid to yield a fully protected tripeptide (5). If desired, by sequential deprotection of the C-terminus of 5 and analogous coupling to another suitably protected amino acid, a fully protected tetrapeptide may be constructed. Synthesized caspase probes disclosed herein may be in the form of a waxy material or lyophilized powder.
  • the caspase probe is prepared by finishing the peptide chain with an aspartic acid portion and adding a fluoromethyl ketone (FMK), benzoyloxymethyl ketone (BMK) or phenoxymethyl ketone (PMK) to the end of the peptide chain as a leaving group that is part of the reactive group and is positioned at the C-terminus of the recognition sequence of the caspase probe.
  • FMK fluoromethyl ketone
  • BMK benzoyloxymethyl ketone
  • PMK phenoxymethyl ketone
  • the synthetic strategy also applies to fluorescent dyes (such as BODIPY-FL, BODIPY-TMR and BODIPY-TR) and protecting groups (such as a Z group). They may be attached to the fully protected peptides by known peptide coupling techniques, deprotected as required and coupled to the suitably derivatized L-aspartic acid B-methyl ester to comprise the final, desired cell-permeable caspase inhibitor.
  • fluorescent dyes such as BODIPY-FL, BODIPY-TMR and BODIPY-TR
  • protecting groups such as a Z group
  • the caspase probes may be synthesized by a method that includes the steps of forming the recognition sequence and then (in any sequence or combination) attaching an N-terminal protecting group, attaching a reactive group, and incorporating a label. Renresentative Eram of Casoase Probes
  • caspase probes of the present disclosure containing FMK reactive groups include, but are not limited to, the compounds listed below.
  • aspartic acid can exist in every configuration, includinEL,D, or D,L.
  • BODIPY-TMR-VAD(OMe)-FMK BODlPY-TMR-L-valyl-L-alanyl-L-aspartic acid methyl ester fluoromethyl ketone
  • BODIPY-TR-VAD(OMe)-FMK BoDlPY-TR-L-valyl-L-alanyl-L-aspartic acid methyl ester fluoromethyl ketone
  • BODIPY-TMR-WE(OMe)HD(OMe)-FMK BoDlPY-TMR-L-tryptophanyl-L-glutamic acid methyl ester-L-histidine-L-as artic acid methyl ester fluoromethyl ketone
  • BODIPY-TMR-W(I ⁇ Me)E(OMe)HD(OMe)-FMK BODIPY-TMR-N-methyl -L- tryptophanyl-L-glutamic acid methyl ester-L-histidine-L-aspartic acid methyl ester fluoromethyl ketone
  • BODIPY-TR-wQ'.IMe)E(OMe)HD(oMe)-FMK BoDlPY-TR-N-methyl-L-tryptophanyl-L- glutamic acid methyl ester-L-histidine-L-aspartic acid methyl ester fluoromethyl ketone
  • BODIPY-TR-WE(OMe)HD(OMe)-FMK BoDlPY-TR-L-tryptophanyl-L-glutamic acid methyl ester-L-histidine-L-as artic i meth l ester fluorometh l ketone
  • BODIPY-TMR-D(OMe)E(OMe)VD(OMe)-FMK BODIPY-TMR-L-aspartic acid methyl ester-L-glutamic acid methyl ester-L-valyl-L-aspartic acid methyl ester fluoromethyl ketone
  • Z-IE(OMe)TD(OMe)-FMK Carbobenzyloxy-L-isoleucyl-L-glutamic acid methyl ester-L- threonine-L-aspartic acid methyl ester fl uoromethyl ketone
  • BODIPY-FL-IE(OMe)TD(OMe)-FMK BoDlPY-Fl-L-isoleucyl-L-glutamic acid methyl ester-L-threonine-L-aspartic acid methyl ester fluoromethyl ketone
  • BODIPY-TMR-IE(OMe)TD(OMe)-FMK BODIPY-TMR-L-isoleucyl-L-glutamic acid methyl ester-L-threonine-L-aspartic acid methyl ester fl uoromethyl ketone zcH:
  • BODIPY-TR-IE(OMe)TD(OMe)-FMK BoDlPY-TR-L-isoleucyl-L-glutamic acid methyl ester-L-threonine-L-aspartic acid methyl ester fl uoromethyl ketone
  • BODIPY-FL-LE(OMe)TD(OMe)-FMK BODIPY-FL-L-leucyl-L-glutamic acid methyl ester-L-threonine-L-aspartic acid methyl ester fluoromethyl ketone
  • BODIPY-TMR-LE(OMe)TD(OMe)-FMK BODIPY-TMR-L-leucyl-L-glutamic acid methyl ester-L-threonine-L-aspartic acid methyl ester fluoromethyl ketone
  • BODIPY-TR-LE(OMe)HD(OMe)-FMK BODIPY-TR-L-leucyl-L-glutamic acid methyl ester-L-histi
  • Z-VAD(OMe)-FMK Carbobenzyloxy-L-valyl-L-alanyl-L-aspartic acid methyl ester fluoromethyl ketone
  • 5-FAM-VAD(OMe)-FMK 5-FAM-L-valyl-L-alanyl-L-aspartic acid methyl ester fluoromethyl ketone
  • TFA-VAD(OMe)-FMK Trifl uoroacetyl-L-valyl-L-alanyl-L-aspartic acid methyl ester fluoromethyl ketone
  • BODIPY FL-w(NMe)E(oMe)HD(oMe)-FMK BoDlPY-Fl-N-methyl-L-tryptophanyl-L- glutamic acid meth l ester-L-histidine-L-aspartic acid meth l ester fluoromethyl ketone
  • 5-FAM-W(NMe)E(OMe)HD(OMe)-FMK 5-FAM-N-methyl-L-tryptophanyl-L-glutamic acid methyl ester-L-histidine-L-as artic acid meth l ester fluorometh l ketone
  • caspase probe is selected from the compounds listed below: 780-VAD(OMe)-FMK
  • the caspase probes are used for in vivo detection of caspase-mediated apoptosis to assess diseases/conditions associated with apoptosis. Excessive apoptosis is associated with a wide range of disorders, and the importance of caspases in the progression of these disorders has been demonstrated.
  • the caspase probes may be used to assess caspase-mediated apoptosis associated with a wide range of diseases and conditions, including but not limited to: eye diseases (e.g., glaucoma, diabetic retinopathy, macular degeneration), neurodegenerative diseases (e.g., neuropathy, Alzheimer's disease, multiple sclerosis, Huntington's disease), cancers (bladder cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, leukemia, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, thyroid cancer), cardiac diseases (e.g. cardiomyopathy), cardiac conditions (myocardial infarction), drug related toxicity and trauma.
  • eye diseases e.g., glaucoma, diabetic retinopathy, macular degeneration
  • neurodegenerative diseases e.g., neuropathy, Alzheimer's disease, multiple sclerosis, Huntington's disease
  • cancers bladder cancer, breast cancer,
  • the caspase probes are used for in vivo detection of caspase-mediated inflammation to assess diseases/conditions associated with inflammation. Inflammation is associated with a wide range of chronic and acute disorders.
  • the caspase probes may be used to assess caspase-mediated inflammation associated with a0 wide range of diseases and conditions, including but not limited to: ulcerative colitis, endotoxic shock, rheumatoid arthritis, juvenile arthritis, osteoarthritis, psoriasis, Crohn's disease, inflammatory bowel disease, multiple sclerosis, insulin dependent diabetes mellitus, gout, psoriatic arthritis, reactive arthritis, viral or post-viral arthritis, and ankylosing spondylarthritis.
  • diseases and conditions including but not limited to: ulcerative colitis, endotoxic shock, rheumatoid arthritis, juvenile arthritis, osteoarthritis, psoriasis, Crohn's disease, inflammatory bowel disease, multiple sclerosis, insulin dependent diabetes mellitus, gout, psoriatic arthritis, reactive arthritis, viral or post-viral arthritis, and ankylosing spondylarthritis.
  • the caspase probes are used for in vitro or in vivo detection of caspase-mediated apoptosis to assess the health of a transplant organ or transplanted tissue (pre- and post-transplant), a diseased organ or diseased tissue, or a traumatized organ or traumatized tissue since apoptosis can be indicative of impairment.
  • the caspase probes are used for in viyo monitoring of apoptosis during medical procedures.
  • Medical devices designed to detect fluorescence can be used in combination with the caspase probes to detect and monitor apoptosis during medical procedures.
  • cardiomyocyte apoptosis is associated with the pathogenesis of5 heart failure as well as ischemic and inflammatory myocardial conditions. Thus, it is critical to prevent or attenuate apoptosis in patients undergoing cardiac surgery.
  • apoptosis is one of the central aspects of the pathology.
  • cells resistant to apoptosis are responsible for the development of many types of malignancies.
  • tumor cells resistant to chemotherapy, and thus apoptosis constitute a subcellular population thought to play a crucial role in tumor recurrence, as well as in metastatic progression and colonization of distant organs.
  • signaling mechanisms that govern apoptotic resistance during these processes remain unclear.
  • caspase probes disclosed in this invention provide a valuable in vivo and in vitro research tool for studying apoptotic cascade under diverse conditions.
  • the caspase probes can be used for research purposes in order to gain a better understanding of the molecular mechanisms responsible for the resistance to apoptosis during carcinogenesis.
  • the caspase probes allow detection and inhibition of specific caspases, they can be used to delineate the degree to which individual caspases contribute to certain pathological states. For instance, identifying the type of caspase critical for the occurrence of apoptotic resistance during chemotherapy could lead to the development of new therapies targeting that specific caspase.
  • many neurodegenerative disorders are associated with excessive apoptosis and neuronal death. For example, neuronal death underlines various diseases including Alzheimer's, Parkinson's, and Huntington's disease.
  • the caspase probes can also be used in drug development.
  • the process of drug development, from start to commercialization is very long and involves numerous steps including identifying in vitro lead drug candidates from a million of compounds, pre-clinical development using in vivo animal models, and finally, clinical trials in humans.
  • High-throughput in vitro screening is a widely used method during the initial stages of drug development, and allows for the simultaneous evaluation of millions of compounds under a given condition. It involves the screening of the candidate therapy compound library against the specific drug target directly or in a more complex assay system.
  • high throughput screen can involve screening a candidate therapy compound library in a cell-based assay, where the activity of the candidate compound is intended to affect the incidence of apoptosis or inflammation.
  • This tlpe of screen can be carried out using cells cultured in multi-well plates with automated operation. Since the ultimate outcome desired by the candidate compound is the effect on apoptosis, the caspase probes can be used in cell based screens to quantifu the incidence of apoptosis in response to tested drug candidates.
  • a library of candidate therapy compounds0 can be added individually to particular cancer cells cultured in a multi-well plate.
  • the caspase probe is applied to the cells before the therapy compound in order to generate a baseline of apoptosis.
  • the caspase probe can be applied to cancer cells containing a candidate therapy compound, and incubated with cells to allow the formation of caspase-probe5 complexes. Subjecting the cells to a means for detecting caspase-probe complexes would determine whether a candidate therapy induces apoptosis as indicated by the presence of caspase-probe complexes.
  • the caspase probes can be used to detect the incidence of apoptosis during the pre-clinical and clinical development.
  • candidate therapy0 compounds are administered in vivo to animals used to model a specific disease. Therefore, under the circumstances where a desired outcome of the candidate therapy compound is the induction of apoptosis, the caspase probes can be administered to animals following the treatment with the candidate therapy compound. Subjecting the animals to a means for detecting caspase-probe complexes would determine whether a candidate therapy induces5 apoptosis as indicated by the presence of caspase-probe complexes.
  • the caspase probes can be used in clinical trials, where patients receiving candidate therapy compound can be administered the caspase probes in order to detect the incidence of apoptosis caused by the candidate therapy.
  • the caspase probes can also be used for evaluating and/or predicting the efficacy ofa0 particular therapeutic treatment (e.g., chemotherapy or an anti-inflammatory), as well as for determining drug-induced impairment or toxicity (e.g., evaluating side effects of a drug treatment by assessing collateral damage to non-target organs).
  • a therapeutic treatment could be assessed by, after administration of the therapy, administering in vivo an effective amount of a caspase probe to a subject, and then detecting the degree of apoptosis, such that detection of apoptosis at or above a predetermined level indicates efficacy of the therapeutic treatment.
  • the subject's naturally occurring level of apoptosis could be assessed both before and after therapeutic treatment in order to determine how much apoptosis increases due to administration of the therapy.
  • the efficacy of an anti-inflammatory could be assessed by administering the anti-inflammatory to a subject, administering in vivo an effective amount of a caspase probe to the subject, and then detecting the degree of inflammation, such that detection of inflammation at or below a predetermined level indicates efficacy of the anti-inflammatory treatment.
  • the subject's naturally occurring level of inflammation could be assessed before the anti- inflammatory treatment as well in order to determine how much inflammation decreases due to administration of the therapy.
  • organs or targets that may be assessed using the caspase probes include, but are not limited to: eye, breast, heart, brain and central nervous system (CNS), kidneys, lungs, liver, skin, pancreas, skeletal system, connective tissue (e.g., joints), stomach, upper gastrointestinal tract, lower gastrointestinal tract, circulatory system (e.g., blood), lymphatic system, sexual organs (male and female), prostate, embryologic tissue, muscular system, and gallbladder.
  • the caspase probes may also be used for whole-body imaging to assess apoptosis and/or inflammation throughout a living organism.
  • the caspase probe is delivered in vitro to a biological sample (e.g., a blood sample, tissue sample, cell suspension, cellular extract, or tissue homogenate) by direct application of the probe to the sample.
  • a biological sample e.g., a blood sample, tissue sample, cell suspension, cellular extract, or tissue homogenate
  • the caspase probes exhibit good cell permeability, there is no need to use additional reagents to facilitate the entry of the probes into cells.
  • the caspase probe is reconstituted in DMSO, further diluted in phosphate buffered saline (PBS) or cell culture media and applied directly to cells in a cell culture dish.
  • PBS phosphate buffered saline
  • caspase-probe complexes After an incubation period under the conditions sufficient for the formation of caspase-probe complexes (l hour at 37oC), cells are washed with PBS containing lYo of bovine serum albumin (BSA). To determine whether cells have undergone apoptosis, the incidence of apoptosis can be evaluated by subjecting the biological sample to a means for detecting caspase-probe complexes.
  • BSA bovine serum albumin
  • the caspase probe is administered in vivo to a subject (e.g., animal or human) orally, intravenously, intraperitoneally, intramuscularly, subcutaneously, topically, and/or by direct application to a target organ.
  • the caspase probe is diluted in 10X injection buffer.
  • the injection buffer is PBS.
  • the caspase probes are administered in an effective amount, which is an amount that is sufficient to provide meaningful results with respect to the intended purpose - €.g., diagnostic, drug development, etc.
  • the caspase probe is present in a composition, wherein the composition comprises at least a caspase probe and an excipient.
  • the excipient is a pharmaceutically acceptable excipient. Suitable excipients include, but are not limited to, adhesives, binders, bulking agents, carriers, colors, diluents, disintegrating agents, fillers, glidants, granulating agents, lubricating agents, polymers, preservatives, wetting agents, and combinations thereof.
  • one or more excipients is selected from sucrose, lactose, cellulose, gelatin, polyvinylpyrrolidone, trehalose, cyclodextrin, hyaluronic acid and polyethylene glycol. In one embodiment, one or more excipients is selected from sucrose, lactose, cellulose, gelatin, polyvinylpyrrolidone, and polyethylene glycol. Kits
  • kits of the present disclosure are used for detecting and quantifuing the incidence of apoptosis and/or inflammation in a biological sample.
  • the kit may comprise one or more caspase probes, one or more buffers (e.g., buffers for delivery of caspase probes to samples), and optionally one or more non-specific fluorescence probes that can be used as a control.
  • Suitable non-specific fluorescence control probes include, but are not limited to DY 680 carboxylic acid, DY 750 carboxylic acid, DY 780 carboxylic acid, s-FAM, 4TT0-655, BODIPY-FL, BODIPY-TIvIR, and BODIPY-TR.
  • the kit may further include packaging materials with instructions for using the components of the kit, such as how to combine caspase probes with buffers, how to use the caspase probes provided in the kit, storage conditions, etc. Components of the kit may be provided in separate containers (e.g., vials) or combined.
  • the buffer is an injection buffer (e.g., in vivo) or a wash buffer (e.g., in vitro).
  • the buffer may be 10X PBS, which is suitable for use as either an injection buffer or wash buffer.
  • the buffer is provided in an amount of 10-50 mL, or in another embodiment, in an amount of 15-30 mL.
  • the probe is provided in an amount of 25-500 Fg, or in another embodiment, in an amount of 30-350 pg.
  • the caspase probe is reconstituted in DMSO, and then optionally diluted in sterile lX PBS.
  • a lOX PBS buffer can be diluted to lX PBS using water - e.g., distilled water, water-for-injection, etc.
  • the buffer is supplied at a 2X concentration, and is diluted to lX using an equal amount of water.
  • Suitable buffers include, but are not limited to, tris (tris-hydroxymethylamino- methane) buffer (20mM, pH 7. ⁇ and Hanks balanced salt solutions (available from Life Technologies) buffered with 20mM Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).
  • the buffer is a pharmaceutically acceptable buffer.
  • a suitable injection buffer may be prepared according to the following recipe in endotoxin free DI Hzo:87.66 g/L of NaCl (1.5 M),60.53 glL of Na2HPo4.12H2o+4.84 g/L of NaH2PO+.2HzO (0.2 M phosphate), pH 6.9.
  • caspase probes for caspase-l The relative specificity of caspase probes for caspase-l is tested.
  • the structure of YEHD is similar to the caspase-4 binding peptide YVAD, and FEHD is similar to the caspase-9 binding peptide LEHD. Therefore, the ability of the caspase probes to affect the binding of BODIPY-FL-YVAD-FMK, BODIPY-FL-LEHD-FMK, and BODIPY-FL-VAD- FMK in JURKAT T cells induced to undergo apoptosis as described below is determined. Importantly, JURKAT T cells do not express caspase-I, so binding of the caspase probes would indicate binding to one or more apoptosis-related caspases.
  • the JURKAT T cells are stimulated with staurosporine in the presence or absence of the test caspase-l caspase probe. After 4 hours of incubation, the cells are washed and stained with the BODIPY-FL-conjugated caspase-4, caspase-9, or poly caspase reagent. The cells will then be assessed by flow cytometry or fluorimetry. In the absence of any binding by the caspase-l caspase probe, the strong level of staining should remain unchanged in cells induced in the presence of the test caspase probes.
  • test caspase-l caspase probes are capable of binding active caspases in apoptotic cells, preventing binding of the BODIPY-FL-labeled reagent.
  • caspase probes One of the clinically relevant uses for the caspase probes is the determination of the efficacy of cancer chemotherapy - e.g., in animal models of disease. Accordingly, the function of the caspase probes is tested in vivo using a simple murine model of human multiple myeloma.
  • the MOPC-I5 plasmacytoma is a recognized tumor model with growth characteristics and responses to chemotherapy that are well documented.
  • Balb/c mice bearing large (>20 mm) syngeneic MOPC-315 tumors subcutaneously are treated intraperitoneally with 100 mglkg cyclophosphamide that is curative for 99oh of the treated mice within 10 days.
  • 24 hrs post chemotherapy mice are injected with 300 nanomoles of labeled poly- caspase (also called pan-caspase) probe per kg of wet weight (roughly 10 pg per 25g mouse, depending on the dye). Two hours later, the mice are sacrificed and their tumors excised.
  • poly- caspase also called pan-caspase
  • the tumors are minced and subjected to the appropriate readout protocol to determine the binding of the caspase probe to apoptotic cells within the tumors.
  • Fluorophore-labeled caspase probes are detected by assessing the tumor cell suspensions using flow cytometry or fluorimetry.
  • Caspase probes utilizing the leF label are detected in tumor cell suspensions using micro MRI. It is expected that the label in chemo-induced apoptotic tumor cells will be readily detected.
  • caspase-l caspase probes Another clinically relevant use for the caspase-l caspase probes is the determination of the extent of collagen-induced arthritis.
  • a model system in mice or rats is readily available. The model is widely used to address questions of disease pathogenesis and to validate therapeutic targets. Accordingly, rodents are injected with heterologous type II collagen plus an adjuvant. The presence of pro-inflammatory cytokines including IL-1 beta is greatly enhanced in the affected host indicating the activation of caspase-l in the response.
  • the caspase probes are injected into model animals at the site of joint inflammation. It is expected that the binding of the
  • test caspase probe in inflammatory monocytes present within the inflammatory response will be readily detected using small animal imaging machines to detect either fluorophores or leF (MRr).
  • MRr leF
  • kits to analyze caspase activity in response to chemotherapy
  • breast cancer is a highly heterogeneous disease, in which various subtypes of tumors differ significantly among each other.
  • the heterogeneous nature of tumor subtypes is reflected in the substantial variation among different subtypes with respect to response to therapy and treatment.
  • understanding the underlying molecular disparities between5 different tumor types will provide critical insight when developing novel drug targets and therapies.
  • triple-negative breast cancer encompasses a subtype of tumors that lack the expression of estrogen receptor (ER), progesterone receptor (PR), or HER-2 genes.
  • Triple-negative breast cancer is one of the most aggressive breast carcinomas, and does not respond to endocrine therapy or other available targeted agents. Since therapy resistance can0 be attributed to acquisition of anti-apoptotic mechanisms by cancer cells, it would be advantageous to delineate differences in apoptosis between different subtypes of breast cancer.
  • the overall degree of apoptosis is evaluated in breast cancer cells.
  • a kit containing poly-caspase probe BODIPY-TMR-VAD(OMe)-FMK is used to determine the incidence of apoptosis among breast cancer cells that exhibit different molecular signatures. Initially, BODIPY-TMR-VAD(OMe)-FMK probe is resuspended in DMSO (under sterile conditions) until the probe is dissolved. Further dilution of the caspase probe is carried out according to the instructions provided in the kit, which may comprise all or part of the 5 following instructions:
  • kits with control fluorescent dye to confirm caspase-selective activity
  • This example uses a control fluorescent dye provided in the kit to confirm that BODIPY-TMR-VAD(OMe)-FMK detects caspase activity and does not emit non-selective fluorescence, which can lead to false-positive signals.
  • MCF-7 cells which are predicted to undergo apoptosis, are treated with cyclophosphamide for 24 hours. The next day, BODIPY- TMR-VAD(OMe)-FMK is added to one plate of MCF-7 cells, and BODIPY-TMR (control fluorescent dye) is added to another plate of MCF-7 cells.
  • test refers to a volume of 300 pL (0.3 mL), which is sufficient for one in vitro test.
  • poly-caspase kit components include, but are not limited to
  • caspase-l/caspase-3/7 kit components include, but are not limited to:
  • retinal apoptosis was induced by exposing rats to blue light for 8 hours. Control animals did not receive any blue light exposure. Following blue light exposure, photoreceptors and Retinal Pigment Epithelium (RPE) cells began a process of inflammation, apoptosis and phagocytosis lasting 2 to 5 days, resulting in regions of significant retinal thinning similar to Geographic Atrophy. 780-VAD(OMe)-FMK tracer was injected into control and test animals 2 days post blue light exposure at 100 nMlkg. Animals were fundus imaged using a Heidelberg Spectralis confocal scanning laser ophthalmoscope, 5 to 10 minutes post injection, with the tracer visualized using ICG angiography mode.
  • RPE Retinal Pigment Epithelium
  • OCT Optical Coherence Tomography

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Abstract

La présente invention concerne des sondes intracellulaires à perméabilité cellulaire, qui se lient à des caspases actives et peuvent être utilisées pour détecter une apoptose et une inflammation. En particulier, les sondes peuvent être utilisées pour déterminer le degré ou l'incidence d'apoptose induite par caspase, d'inflammation induite par caspase, et d'états associés à ces derniers. La présente invention concerne également des kits comprenant les présentes sondes et des instructions pour leur utilisation.
PCT/US2015/063798 2014-12-03 2015-12-03 Sondes intracellulaires de caspase pour la détection d'apoptose et d'inflammation, et kits contenant de telles sondes WO2016090169A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017053864A1 (fr) * 2015-09-23 2017-03-30 Intracellular Technologies, Llc Inhibiteurs de protéases à cystéine
WO2018234557A1 (fr) * 2017-06-23 2018-12-27 Nanotemper Technologies Gmbh Procédés de mesure d'interactions inter- et/ou intra-moléculaires
EP3845902A1 (fr) * 2017-06-23 2021-07-07 NanoTemper Technologies GmbH Procédés de mesure d'interactions inter- et/ou intra-moléculaires
US11994521B2 (en) 2017-06-23 2024-05-28 Nanotemper Technologies Gmbh Methods for measuring inter- and/or intra-molecular interactions
CN111773394A (zh) * 2019-04-04 2020-10-16 复旦大学 一种β-半乳糖苷酶荧光探针纳米微球及其制备方法和用途
WO2024066540A1 (fr) * 2022-09-30 2024-04-04 江苏省原子医学研究所 Sonde moléculaire pet de reconnaissance ciblée par une protéase d'acide aspartique et utilisation

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