WO2016157198A1 - Sondes basées sur l'activité photodynamique atténuée et leurs utilisations en imagerie et en thérapie ciblée - Google Patents

Sondes basées sur l'activité photodynamique atténuée et leurs utilisations en imagerie et en thérapie ciblée Download PDF

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WO2016157198A1
WO2016157198A1 PCT/IL2016/050354 IL2016050354W WO2016157198A1 WO 2016157198 A1 WO2016157198 A1 WO 2016157198A1 IL 2016050354 W IL2016050354 W IL 2016050354W WO 2016157198 A1 WO2016157198 A1 WO 2016157198A1
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moiety
compound according
photosensitizer
compound
group
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PCT/IL2016/050354
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WO2016157198A9 (fr
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Galia Blum
Yael BEN-NUN
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Galia Blum
Ben-Nun Yael
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention is generally directed to photodynamic activity probes for use in methods of imaging and targeted therapy.
  • Activity based probes are small molecules that modify a defined set of enzyme targets based on their ability to form specific covalent bonds with key catalytic residues. Since this labeling reaction is mechanism-based and requires enzyme activity, extent of probe modification serves as an indirect readout of activity levels within a given sample. Probes can be designed to target a number of different classes of enzymes through optimization of both reactive functional groups and the scaffolds used to carry the reporter tag.
  • probes A number of classes of such probes have been developed and used to dissect the function of various enzyme families. The most well-established and heavily used probes are those that target proteolytic enzymes. These probes that target serine and cysteine proteases have been applied to studies of protease function in processes such as parasite invasion, prohormone processing, transcriptional regulation, cataract formation, natural killer cell function and cancer progression.
  • the inventors of the present invention designed and developed fluorescent activity-based compounds (probes) for use, e.g., in tissue detection and targeted therapy.
  • the compounds bind to proteases of interest, such as human cathepsin and exert their toxicity when irradiated with light.
  • the compounds of the invention either incorporate a photosensitizer or incorporate a photosensitizer-quencher pair (namely a photosensitizer moiety associated to a fluorescence-quenching moiety).
  • a photosensitizer-quencher pair namely a photosensitizer moiety associated to a fluorescence-quenching moiety.
  • the presence of the fluorescence-quenching moiety renders the compounds of the invention only weakly fluorescent or substantially non-fluorescent due to the quenching of the fluorescence emission by the quencher moiety.
  • the photosensitizer moiety and the quencher are linked by a chemically reactive moiety which contains one or more bonds capable of undergoing cleavage by an enzyme and subsequent conjugation thereto.
  • the cleaving of the one or more bonds causes dissociation of the quencher moiety from the photosensitizer moiety, and subsequent regeneration of fluorescence (dequenching).
  • the compounds of the invention may therefore be regarded as having an "in active" from or state, being the fully intact photosensitizer-quencher pair and an "active" form or state, being generated following dissociation of the quencher moiety.
  • the enzyme-based dissociation of the quencher moiety and subsequent attachment of the enzyme to the photosensitizer moiety typically causes complete restoration of fluorescence which may be detected.
  • the compounds of the invention may be used to detect localities of high enzyme concentrations, typically indicative of a condition associated with increased expression of the enzyme and specifically high expression of proteases, such as cathepsin. As shown herein, such accumulation of the compounds of the invention in such localities results in an amplified signal which when detected provides an indication of a site to be treated.
  • the enzyme-based dissociation of the capping group and subsequent attachment of the enzyme to the photosensitizer moiety may then be used in photodynamic therapy (PDT) as described herein.
  • PDT photodynamic therapy
  • activity based probe refers to small molecules that are engineered to covalently modify enzyme targets in an activity dependent manner.
  • the activity-based probe compounds are driven (targeted) selectively to sites with increased active enzymes expression such as cathepsins (for example cancerous tissue or macrophages in the tumor- microenvironment) and may assist in detection and targeted treatment of such tissue by photodynamic therapy.
  • active enzymes expression such as cathepsins (for example cancerous tissue or macrophages in the tumor- microenvironment) and may assist in detection and targeted treatment of such tissue by photodynamic therapy.
  • the photodynamic therapy in tumors involves the administration of a compound of the invention and subsequent induction of local light delivery.
  • cytotoxic reactive oxygen species ROS
  • ROS cytotoxic reactive oxygen species
  • compounds described herein show a dual function, enabling both detection (i.e. by fluorescence methods) and treatment (i.e. by photodynamic therapy) with minimal side effects.
  • the photosensitizer quenched activity-based probes (PS-qABP) based on cathepsin targeting described herein, have an advantage in vivo as they are specifically targeted to cells showing increased cathepsin expression, e.g., cells in the tumor microenvironment, and can be used for real-time imaging of tumors prior to light activation, in order to better localize the subsequent photodynamic anti-cancer treatment.
  • Such detection of elevated active cathepsins enables precise tumor imaging thereby enabling accuracy in localizing the light treatment that induces targeted cell death, for example of macrophages within tumor microenvironment.
  • the inventors propose that compounds described herein significantly reduce photosensitizers' light toxicity to the skin, since the free unbound probe remains attached to the quencher that absorbs the energy, hence preventing off-target ROS generation similar to the self-quenched probes.
  • the probes described herein have reduced fluorescent background and therefore generate signal very rapidly without the need for clearance of unbound probe.
  • the PS-qABPs bind their targets (i.e. cathepsin) covalently, the detection of the in vivo targets by the probe can be verified and quantified using biochemical analysis.
  • L is a fluorescence-quenching moiety (a quencher), a capping group or an enzyme
  • M is a chemically reactive moiety linking moieties L and V;
  • V is a photosensitizer moiety
  • each "-" designates a covalent bond containing optionally one or multiple intervening atoms that serve as spacers or as selectivity directing moieties.
  • fluorescence emission from said photosensitizer moiety is quenched by said fluorescence-quenching moiety.
  • moiety in the context of the disclosure may refer to an atom, a group of atoms and any functional fragment of a molecule which functions as specifically recited.
  • the moiety may also be in the form of a physical element such as a capsules, spheres, nanoparticles, liposomes etc of at least one material (i.e. of a single atom or multiple atoms) which functions as recited.
  • the photosensitizer moiety is an atom or a group of atoms or otherwise or a physical moiety as defined which functions as a photosensitizer.
  • each moiety may be connected, associated or bonded to another moiety via a bond (such as covalent bond, ionic bond, hydrogen bond, complex and any combination thereof) provided that the function of the compounds is maintained as defined.
  • the moieties L, M and V are covalently bonded to each other.
  • the covalent bonding attaching the moiety L to M and/or M to V contains multiple intervening atoms that serve as spacers.
  • the photosensitizer moiety V is associated to said chemically reactive moiety M via a selectivity determining moiety, D; wherein D may be an amino acid sequence; each amino acid may or may not be a natural amino acid, as further detailed hereinbelow.
  • the compound of Formula (I) is a compound of Formula (II):
  • the "photosensitizer moiety” is a reactive group which is photoactivatable when illuminated with light of an appropriate wavelength.
  • the photosensitizer moiety is selected to exhibit toxicity to the local environment in which it is situated, e.g., a target cell, upon illumination/irradiation (light toxicity), namely the moiety is selected to have light toxicity and is thus photodynamically active.
  • the photodynamic activity is quenched in view of the presence of the fluorescence-quenching moiety.
  • the photosensitizer moiety is selected to have one or more of the following: a strong absorption with high extinction coefficient at longer wavelengths, e.g., between 600 and 900 nm, photochemical reactivity, high quantum yield for singlet oxygen production, no or minimal toxicity when in a compound of the invention at the "in active” state, toxicity in the presence of light, preferential retention by tumor cells and exhibit rapid excretion from the body.
  • a strong absorption with high extinction coefficient at longer wavelengths e.g., between 600 and 900 nm
  • photochemical reactivity e.g., high quantum yield for singlet oxygen production
  • no or minimal toxicity when in a compound of the invention at the "in active” state toxicity in the presence of light, preferential retention by tumor cells and exhibit rapid excretion from the body.
  • compounds of the invention are contemplated in photodynamic "in active” and photodynamic "active” forms, wherein when L is a quencher moiety, potential photodynamic activity is quenched, and
  • L in a compound of Formulae (I) or (II), L is a quencher moiety, as defined. In other embodiments, in a compound of Formulae (I) or (II), L is an enzyme. In other embodiments, in a compound of Formulae (I) or (II), L is a capping group. In some embodiments, the photosensitizer moiety is selected to have absorption at wavelengths between 600 and 900 nm.
  • the photosensitizer moiety is selected to match the absorption wavelength of the fluorescence-quenching moiety.
  • L is a quencher moiety selected from moieties listed in Scheme 1 below.
  • SCHEME 1 Non- limiting examples of quencher moieties in accordance with embodiments of the invention.
  • the quencher moieties are connected to the carbonyl group, as shown in Formulae (IV) or (V) via the linker X, at the quencher position designated in the Scheme structures with an asterisk (*).
  • the L is a quencher moiety known as DyLight quencher.
  • V is a photosensitizer moiety selected from such moieties having emission at 600 nm and higher.
  • L is a quencher moiety selected from moieties listed in Scheme 1 herein and V is a photosensitizer moiety selected from such moieties having emission at 600 nm and higher.
  • L is a capping group
  • L is a capping group and V is a photosensitizer moiety selected from such moieties having emission at 600 nm and higher.
  • fluorescence-quenching moiety or “quencher” refers to a non-fluorescent moiety capable of absorbing energy emitted by the photosensitizer moiety upon illumination; thus quenching emission of fluorescence by said photosensitizer moiety.
  • capping group refers to a atom or group of atoms which is an end group (terminal). In some embodiment the end group is reactive. In some embodiment, the end group is non-reactive. In case where the capping group is functional it may be selected among any of the active moieties disclosed herein. In some embodiments, he capping group may be a protecting group.
  • the capping group is selected from H, an aryl group (e.g. optionally substituted phenyl), an alkyl group, an amino acid (AA) group or a non fluorescence-quenching moiety. In some embodiments, the capping group does not include a fluorescence-quenching moiety.
  • the capping group may be dimethyl benzoyl.
  • a chemically reactive moiety M refers to an atom or a group of atoms that is capable of undergoing bond cleavage by an enzyme and subsequent attachment (chemical bonding) thereto.
  • the reactive moiety is an electrophile or nucleophile that can form a covalent bonding upon exposure to a nucleophilic or electrophilic group on the enzyme, respectively.
  • the chemically reactive moiety acting as a linker moiety linking the quencher and the photosensitizer moiety, is of a length suitable for placing the quencher and photosensitizer moieties in sufficient proximity for quenching to occur.
  • M is a group containing an electrophilic atom (or group) susceptible for nucleophilic attack and formation of a non-labile (non- hydrolizable) covalent bond
  • M is an acyloxymethyl ketone moiety.
  • M is an electrophilic atom (or group) which is susceptible for nucleophilic attack and which results in expulsion of a leaving group and formation of a new non-labile (non-hydrolizable) covalent bond.
  • M may be one or more phosphonate groups.
  • the chemically active moiety M containing, e.g., the acyloxymethyl ketone functional group
  • D is a selectivity determining moiety, which in some embodiments is a peptide moiety selected amongst mono-, di-, tri- or tetra-peptide scaffold moieties. These peptide moieties are typically designed to bind specifically to the enzyme, e.g., protease.
  • the peptide moiety D acts as a linker associating the photosensitizer moiety V to the acyloxymethyl ketone functional group, or as a side chain on which the photosensitizer moiety is pendent.
  • selectivity determining moiety refers to a part of the compounds of the invention which is specifically recognized by the enzyme, e.g., protease. In some embodiments, the selectivity determining moiety is recognized by cathepsin. As described herein, D may be a single amino acid or a peptide. Further, as described herein, D may be lysine.
  • the compound of the Formula (II) is a compound of the general Formula (III):
  • each V, D and L are as defined herein and p is an integer.
  • p is between 0 and 5. In some embodiments, p is 0 (i.e. absent). In some other embodiments, p is 1.
  • Cleavage by the enzyme e.g., protease
  • loss of a carboxylate group separates the part of the molecule containing the photosensitizer moiety V from the other part of the molecule containing the quencher moiety L.
  • L is a quencher moiety
  • such cleavage generates a signal associated with the active form (a compound of Formulae (I) or (II), wherein L is an enzyme and p is 0).
  • the quencher moiety or the capping group L may optionally be connected to the carbonyl via a carbon-containing linker group X of between 0 and 10 carbon atoms; such group may be selected amongst alkyl, alkenyl, alkynyl, aryl, alkylaryls, heteroaryl and others.
  • the carbon-containing linker X may comprise one or more natural or non-natural amino acids.
  • X is PEG.
  • the selectivity-determining moiety D is an amino acid sequence, AA, as defined, to which the photosensitizer moiety V is connected.
  • the point of connection to V may be any of the nitrogen atoms of the AA sequence or any alpha-carbon of the AA sequence.
  • AA is selected from a single amino acid, a dipeptide, a tripeptide, a tetrapeptide or any peptide having between 1 and 10 amino acids.
  • amino acid refers to amino acid residues, connected by peptide bonds. A peptide sequence is generally reported from the N-terminal end containing free amino group to the C-terminal end containing free carboxyl group.
  • amino acid as used herein refer to naturally occurring and non- natural synthetic amino acids, as well as amino acid analogs and amino acid mime tics that function in a manner similar to the naturally occurring amino acids.
  • the amino acid moiety is derived from an amino acid of the general formula H 2 NCHRCOOH, wherein R is an organic substituent.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same fundamental chemical structure as a naturally occurring amino acid, i.e., an alpha carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • the amino acid is selected amongst natural amino acids, non natural amino acids and any combination thereof. In some embodiments, the amino acid is at least one natural amino acid. In some other embodiments, the amino acid is at least one of alanine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine, tyrosine, arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine.
  • AA contains at least hydrophobic amino acid or groups. In further embodiments, AA comprises at least one of lysine and phenylalanine. In some other embodiments, the amino acid is lysine. In some embodiments, the amino acid is phenylalanine.
  • the compound of the invention is a compound of Formula (IV):
  • V, AA, X and L are as defined herein and wherein p is an integer between 0 and 5 as described herein.
  • the compound is of Formula (V):
  • R is a carbon-containing group or an extension group of a natural or non- natural amino acid, said R being bonded to said photosensitizer moiety V; in some embodiments, R is the amine extension group (at the alpha carbon) of the amino acid lysine;
  • n is an integer defining the number of amino acids in the peptide sequence, n may be between 1 and 10; and
  • V, AA, X and L are as defined hereinabove.
  • n 1, 2, or 3.
  • AA is selected amongst natural and non natural amino acids, e.g., lysine, phenylalanine, and combinations thereof.
  • AA is capped at the end of the peptide chain with any capping group known in the art.
  • the capping group is carboxybenzyl (CBZ) group forming a CBZ-AA peptide moiety; the amino acid may or may not be natural amino acid.
  • the AA is one or more hydrophobic amino acids.
  • the amino acids are phenylalanine and/or lysine.
  • AA is CBZ-AA containing at least one amino acid.
  • the at least one amino acid is lysine phenylalanine.
  • the at least one amino acid is lysine.
  • the at least one amino acid is phenylalanine.
  • the photosensitizer moiety V is bonded to lysine or to phenylalanine.
  • the photosensitizer moiety V may be selected from macrocyclyl moieties. In some embodiments, the photosensitizer moiety V comprises a macrocyclyl moiety.
  • macrocyclyl denotes a cyclic moiety or a moiety having a cyclic structure comprising carbon atoms and one or more heteroatoms selected from N, O and S.
  • the macrocyclyl may be selected from macrocyclyl moieties having betweenl8n electrons and 22 ⁇ electrons.
  • the macrocyclyl moiety may be selected from coordination moieties wherein at least one of the heteroatoms in the macrocyclyl is coordinated (associated, bonded to) to at least one metal atom.
  • the photosensitizer moiety, V is selected from porphyrin, hematoporphyrin, chlorin, bacteriochlorin, isobacteriochlorin or any derivative thereof.
  • the photosensitizer moiety may be any derivative of the above macrocyclyl having the same number of ⁇ electrons or having additional exo-macrocyclyl or endo- macrocyclyl electron and substitutes.
  • the photosensitizer moiety is selected from porphyrin, hematoporphyrin or any derivative thereof (e.g., a macrocyclyl containing 22 ⁇ electrons).
  • the photosensitizer moiety is selected from porphyrin or any derivative thereof.
  • the photosensitizer moiety is chlorin or any derivative thereof (e.g., a macrocyclyl containing 20 ⁇ electrons). In some further embodiments, the photosensitizer moiety is bacteriochlorin, isobacteriochlorin or any derivative thereof (e.g., a macrocyclyl containing 18 ⁇ electrons).
  • the photosensitizer moiety is a group having a general Formula (la) or Formula (lb).
  • each dashed line " " represents a single bond or double bond, such that the peripheral bonds (represented by the dashed lines) may also be bonds bridging two ring system or bonds fusing two ring systems and wherein, each carbon atom in the representative Formula (la) may be substituted with one or more substituents as exemplified with regard to substituents R1-R15 in Formula (2), Formula (3) or Formula (4).
  • Formula (la) is a general representative of certain macrocyclyl moieties, any alterative to the macrocyclyl moieties of Formula (la) may have an oxygen atom or a sulfur atom instead of any of the nitrogen atoms depicted in formal (la).
  • the nitrogen atoms (or alternatively oxygen or sulfur atoms) positioned as shown in Formal (la) may be coordinated to an atom, Me, as shown in Formula (lb), wherein Me may be selected from metal or hydrogen.
  • Me is selected from Cd, Co, Cr, Cu, Fe, Mg, Mn, Ni, Os, Pd, Pt, Ru, Sn, and Zn.
  • each dashed line " " represents a single bond or double bond as described for Formula (la) and wherein each curved line connecting a nitrogen atom to "Me” may be present or absent.
  • Each carbon atom in Formula (lb) may be substituted with any of Rl to R15 as described herein below in Formula (2), Formula (3) and Formula (4).
  • each one of Formula (2) to (15) may or may not have a coordinating central atom Me, as described above.
  • the photosensitizer moiety is selected from a compound having a general Formulae (lc), (Id) or (le).
  • Ri5 is as described below for Formulae (2) to (4).
  • the photosensitizer moiety is selected from a group of the general Formulae (2), (3) and (4).
  • each dashed line "— " represents a single bond or a double bond
  • n may be an integer between 0 to 8;
  • Ri6 may be selected independently from H, Ci-Ci 2 alkyl, C 2 -Ci 2 alkenyl, C 2 -C 12 alkynyl, d-C 12 alkoxy, COOH, halogen, and S0 3 .
  • each one of Rj to R J S may be further substituted with R 17 ;
  • R 17 may be selected independently from H, Ci-Ci 2 alkyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, -C C 12 alkoxy, COOH, S0 3 , OH, halogen, N0 2 , and NH 2 .
  • each one of Rj to R J S represents "one or two substitutions on the macrocyclyl moiety". Specifically, it should be noted that one moiety may be substituted by a single substituent (for example Rj) and the other moiety may be substituted by more than one substituent (for example Rj), as defined above.
  • the photosensitizer moiety of the Formula (lc) is a photosensitizer moiety of the general Formula (2).
  • the photosensitizer moiety of the Formula (Id) is a photosensitizer moiety of the general Formula (3).
  • the photosensitizer moiety of the Formula (le) is a photosensitizer moiety of the general Formula (4).
  • the photosensitizer moiety is selected from a compound having general Formula (3) or Formula (5) to Formula (9).
  • each " " ", Ri to R15 are as described herein for Formula (2), Formula (3) or Formula (4).
  • Rj may be independently selected from H or (CH2)n- COOR 16 , as defined above. In some other embodiments, Rj may be (CH2)n-COOH. In some further embodiments, n is 2. In some further embodiments, Rj in Formula (1) to Formula (9) may be -(CH2)-(CH2)-COOH. In some other embodiments, Rj in Formula (1) to Formula (9) may be H. In some embodiments, at least one Rj in Formula (1) to Formula (9) may be may be -(CH 2 )-(CH 2 )-COOH. In some further embodiments, the hoto sensitizer moiety of Formula has a structure provided by Formula ( 10) or Formula (11).
  • Rj6 may be selected from C3 ⁇ 4 or O-CH 3 .
  • each one of Rio , R 11 and R 1 2 may be H.
  • each one of R2, R3, R4, R5, R6, and R7 may be independently of each other selected from Ci-Ci2alkyl, C2-Ci2alkenyl.
  • Rs is O.
  • the photosensitizer moiety is a moiety of pyropheophorbide-a. In some other embodiments, the photosensitizer moiety is a moiety of pheophorbide-a.
  • the photosensitizer moiety has a structure provided by Formula (12):
  • each one of R2, R3, R4, R7, R9, Rio, R 11 , R 1 2, Ri 3 , Ri4 and R J S may be independently of each other selected from H, C 1 -C 1 2 alkyl, C2-C 1 2 alkenyl, C 2 -Ci2alkynyl.
  • each one of R9, Rjo, Rn, R 1 2, R13, R14 may be independently of each other selected from H.
  • one of R2, R3, R4, R7 and R J S may be independently of each other selected from C 1 -C 1 2 alkyl, C2-C 1 2 alkenyl.
  • each one of R2, R4, R7 and R J S may be independently of each other selected from CH 3 .
  • the photosensitizer moiety is a moiety of Visudyne.
  • the photosensitizer moiety has a structure provided by Formula (13), Formula (14) or Formula (15).
  • Ri 6 may be selected from C 1 -C 1 2 alkyl, Ci-Ci 2 alkoxy, COOH, halogen, SO 3 .
  • R 16 may be selected from Cj-C 12 alkyl, Cj-C 12 alkoxy.
  • n may be 0, 1, 2.
  • each one of Rjo, Rn and R 12 may be independently of each other selected from H.
  • each one of R 2 , R3, R5, R6, and R7 may be independently of each other selected from H, C 1 -C 1 2 alkyl, C 2 -Ci 2 alkenyl, C 2 -Ci 2 alkynyl. In some embodiments, each one of R 2 , R3, R5, R6, and R 7 , may be independently of each other selected from H, C 1 -C 1 2 alkyl, C 2 -Ci 2 alkenyl. In some embodiments, each one of R 2 , R3, R5, and R7, may be independently of each other selected from CH 3 . In some further embodiments, each one of R 2 , and R5, may be independently of each other selected from H.
  • R6 may be independently of each other selected from C 2 alkyl, optionally substituted. In some embodiments, R6 may be selected from C 2 I3 ⁇ 4. In some embodiments, R6 may be -CH(OH)-CH 3 . In some embodiments, R6 may be H.
  • R 4 may be independently of each other selected from C2H5.
  • n 0.
  • n 2.
  • Rj6 is H.
  • Rj6 is S03, OH, halogen, NO2, NH2.
  • Rj6 may be CH 3 .
  • Rg may be COOH. In some other embodiments, Rg may be -CH2-COOH.
  • R9 may be H. In some other embodiments, R may be -CH2-COOH.
  • the photosensitizer moiety is a moiety of Chlorin-e6. In some embodiments, the photosensitizer moiety is a moiety of Porfimer Sodium. In the structural Formulae given herein and throughout the present specification, the following terms have the indicated meaning:
  • alky refers to a linear, branched saturated hydrocarbon having from 1 to 20 carbon atoms.
  • C1-C12 alkyr or "Ci-Cn alkylene” refers to a linear (straight), branched saturated hydrocarbon having from 1 to 12 carbon atoms, in some embodiments, contain from 2 to 8 carbons, in yet some embodiments from 2 to 5 carbons, in yet some further embodiments, from 1 to 3 carbon atoms.
  • alkyl refers to an alkyl end chain and alkylene refers to a middle chain alkyl, namely divalent, branched or straight hydrocarbon group.
  • Representative C1-Q2 alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, sec-butyl, iso-butyl, tert-butyl, cyclobutyl, pentyl, iso-pentyl, neo-pentyl, tert- pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, cycloheptyl, octyl, sec-octyl (1- methylheptyl), and cyclooctyl.
  • alkeny refers to a linear (straight), branched unsaturated hydrocarbon having from 2 to 20 carbon atoms and at least one carbon- carbon double bond.
  • C2-C12 alkenyl or C2-C12 alkenylene refers to a linear, branched unsaturated hydrocarbon having from 2 to 12 carbon atoms and at least one carbon-carbon double bond, in some embodiments from 3 to 8 carbons, in yet some further embodiments, from 3 to 5 carbon atoms and at least one double bond.
  • alkenyl refers to an alkyl end chain and alkenylene refers to a middle chain alkyl, namely divalent, branched or straight hydrocarbon.
  • Examples of such groups include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, isopropenyl, 1,3-butadienyl, 1-butenyl, 2-butenyl, 1-pentenyl, 2-pentenyl, 1-hexenyl, 2- hexenyl and the like.
  • alkynyl refers to a linear, branched unsaturated hydrocarbon having from 2 to 20 carbon atoms and at least one carbon-carbon triple bond.
  • C2-C12 alkynyl or “C2-C12 alkynylene” as used herein refers to a linear, branched unsaturated hydrocarbon having from 2 to 12 carbon atoms in certain embodiments, from 3 to 8 carbons, and at least one triple bond (at least one carbon- carbon triple bond). It should be noted that alkynyl refers to an alkyl end chain and alkynylene refers to a middle chain alkyl.
  • Examples of such groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2- pentynyl, 1-hexynyl, 2-hexynyl and the like.
  • alkoxy refers to an alkyl group bonded to an oxygen atom, radical -O-C 1 -C 12 alkyl, wherein C 1 -Q 2 alkyl is as defined above.
  • C 1 -C 12 alkoxy as used herein refers to a C 1 -Q 2 alkyl group linked to an oxygen, wherein C 1 -Q 2 alkyl is as defined above.
  • the alkyl group may include one to twelve carbon atoms, at times between one to eight carbon atoms, at times one to five carbon atoms and at times one to three carbon atoms.
  • Representative examples are methoxy, ethoxy, n-propoxy, isopropoxy, butoxy, sec-butoxy, tert-butoxy, pentoxy, isopentoxy, hexoxy, isohexoxy and the like.
  • the alkoxy is ethoxy.
  • aryl as used herein is intended to include carbocyclic aromatic ring systems such as phenyl, biphenylyl, naphthyl, anthracenyl, phenanthrenyl, fluorenyl, indenyl, pentalenyl, azulenyl and the like.
  • Aryl is also intended to include the partially hydrogenated derivatives of the carbocyclic systems enumerated above. Non-limiting examples of such partially hydrogenated derivatives are 1,2,3,4-tetrahydronaphthyl, 1,4- dihydronaphthyl and the like.
  • heteroaryl as used herein is intended to include heterocyclic aromatic ring systems containing one or more heteroatoms selected from nitrogen, oxygen and sulfur such as furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, isoxazolyl, isothiazolyl, 1,2,3-triazolyl, 1 ,2,4-triazolyl, pyranyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, 1,2,3-oxadiazolyl, 1,2,4- oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,3-thiadiazolyl, 1 ,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-
  • Heteroaryl is also intended to include the partially hydrogenated derivatives of the heterocyclic systems enumerated above.
  • partially hydrogenated derivatives are 2,3-dihydrobenzofuranyl, pyrrolinyl, pyrazolinyl, indanyl, indolinyl, oxazolidinyl, oxazolinyl, oxazepinyl and the like.
  • aralkyl refers refers to an alkyl-aryl, wherein aryl is as defined above. In other words, one of the hydrogen atoms of the alkyl is replaced by an aryl group.
  • halogen refers to F, CI, Br or I.
  • carbon number refers to the carbon backbone and carbon branching, but does not include carbon atoms of the substituents, such as alkoxy substitutions, OH and the like.
  • the photosensitizer moiety may comprise macrocyclyl which may be considered as a chelating agent, optionally forming a complex with a metal, as shown schematically in Formula (lb).
  • chelation relates to a particular way, by which ions and molecules bind metal ions.
  • complex refers to a chemical compound composed of a ligand having multiple binding sites (multidentate ligand) and a metal ion.
  • the ligand being a chelating agent is in association with at least one metal ion.
  • association refers to the chemical or physical force which holds two entities together (i.e. the ligand and the metal ion). Such force may be any type of chemical or physical bonding interaction known to a person skilled in the art. Non-limiting examples of such association interactions are ionic bonding, covalent bonding, coordination bonding, complexation, hydrogen bonding, van der Waals bonding, hydrophobicity-hydrophilicity interactions, etc.
  • the association may be via covalent bonding. In other specific embodiments, the association may be via coordinative bonding. As used herein the term coordinative bonding or coordinate bond refers to a type of covalent bond in which two shared electrons originate from the same atom (known as dative bond). In the context of the present disclosure, the association between the ligand and the metal may comprise association via multiple atoms in the ligand (i.e. nitrogen atoms) to several sites on the metal.
  • a metal When referring to a metal it is to be understood as including any one or more of the elements commonly known as, transition metals, post transition metal/s, lanthanide/s, actinide/s and metalloids. In the context of the present disclosure, the metal may be referred as a "central metal" .
  • Non limiting examples include Cd, Co, Cr, Cu, Fe, Mg, Mn, Ni, Os, Pd, Pt, Ru, Sn or Zn.
  • the photosensitizer moiety is selected from isohematoporphyrin; tetrasulfonated meso-tetraphenyl porphyrin (H 2 TPPS 4 ); 5,10,15,20-tetrakis (4-sulphonato-phenyl)-21,23-dichalcogenapo hyrin; isomers of tetra(hydroxyphenyl) -porphyrin; benzoporphyrin derivatives and others.
  • the photosensitizer moiety is selected from pheophorbide- a; visudyne; chlorin-e6; and pyropheophorbide-a.
  • the photosensitizer moiety is selected from such listed in Scheme 2 below.
  • SCHEME 2 Non-limiting examples of photosensetizer moieties in accordance with certain embodiments of the invention.
  • the photosensitizer moieties are connected via the bond marked with a wavy line.
  • compounds designated herein as YBN1, YBN2, YBN3, YBN4, YBN5, YBN6, YBN7, YBN8, YBN26 are provided. The structure of each of the compounds exemplified herein is given in Figs. 3F-3N
  • composition of the invention may be formulated in a pharmaceutical composition. More specifically, the composition of the invention comprises as an active ingredient at least one of the compound of the invention as described above, or any combinations thereof, and at least one pharmaceutically acceptable carrier/s, diluent/s, excipient/s.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated.
  • the pharmaceutical composition of the invention is suitable for systemic administration.
  • the pharmaceutical composition of the invention can be administered and dosed by the methods of the invention, in accordance with good medical practice. More specifically, the compositions used in the methods and kits of the invention, described herein after, may be adapted for administration by systemic, parenteral, intraperitoneal, transdermal, oral (including buccal or sublingual), rectal, topical (including buccal or sublingual), vaginal, intranasal and any other appropriate routes.
  • Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s).
  • systemic administration means the administration of a compound, drug or other material other than directly into the central blood system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • Systemic administration includes parenteral injection by intravenous bolus injection, by intravenous infusion, by sub-cutaneous, intramuscular, intraperitoneal injections or by suppositories, by patches, or by any other clinically accepted method, including tablets, pills, lozenges, pastilles, capsules, drinkable preparations, ointment, cream, paste, encapsulated gel, patches, boluses, or sprayable aerosol or vapors containing these complexes and combinations thereof, when applied in an acceptable carrier.
  • any pulmonary delivery as by oral inhalation such as by using liquid nebulizers, aerosol-based metered dose inhalers (MDI's), or dry powder dispersion devices.
  • MDI's aerosol-based metered dose inhalers
  • the pharmaceutical composition is adapted for topical administration.
  • topical administration it is meant that the pharmaceutical composition and the carrier may be adapted to any mode of topical administration including: epicutaneous, oral, bronchoalveolar lavage, ophtalmic administration, enema, nasal administration, administration to the ear, administration by inhalation.
  • the compounds of the present invention which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.
  • compositions of the invention may further comprise least one of diluent/s, carrier/s and excipient/s. It should be appreciated that in certain embodiments, the compositions of the invention may comprise several different compounds as described herein.
  • the compounds of the invention selectively bind to cathepsin and the binding is inhibited by a cathepsin inhibitor (Example 3 and Example 7).
  • a cathepsin inhibitor Example 3 and Example 7
  • an in vivo signal was detected with several compounds, from tumors after injection of the compounds of the invention resulting in a significant tumor-specific fluorescent signal.
  • the compounds of the invention triggered massive apoptosis in tumor tissue. Light treatment that induces targeted cell death of macrophages within the tumor microenvironment. Based on these results, the inventors have suggested that the compounds described herein are targeted to the tissue that expresses high levels of active cysteine cathepsin proteases.
  • compositions comprising an effective amount of the compound of the invention may be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered to a patient already affected by increased cathepsin in an amount sufficient to cure or at least partially arrest the condition and its complications.
  • the present disclosure provides, in another of its aspects, use of the compounds described herein in a method of diagnosis (detection) and/or treatment of a pathological condition characterized by increased enzyme expression and specifically increased cathepsin expression in a subject.
  • another aspect of the present invention concerns a method of treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a pathological condition or a disorder in a subject in need thereof.
  • the method comprises the step of administering to the treated subject a therapeutically effective amount of at least one compound, or any pharmaceutical compositions, carriers, matrix or vehicles comprising the same.
  • these disorders and/or diseases may be characterized as a pathology associated with increased cathepsin expression.
  • the present invention provides a method for treating, preventing, reducing, attenuating, inhibiting and eliminating a pathology associated with increased cathepsin expression by administering to said a subject in need thereof a therapeutically effective amount of at least one compound, optionally with at least one metal, and optionally with at least one of carriers, matrix or vehicles comprising the same.
  • pathology associated with increased cathepsin expression refers to any condition in which cathepsin is over-expressed as indicated herein. Moreover, this term also encompasses conditions in which cathepsin plays a role. Such roles can be directly related to the pathological condition or can be indirectly related to the condition.
  • the pathologies associated with increased cathepsin may be a proliferative disorder. In some other embodiments, the pathologies associated with increased cathepsin may be an inflammatory disorder.
  • cathepsins are a family of enzymes including cathepsins B cathepsin C, cathepsins F, cathepsins H, cathepsins K, cathepsins LI, cathepsins L2, cathepsins O, cathepsins S, cathepsins W, cathepsins Z (X).
  • These proteins regulate several biological processes including, inflammation, antigen presentation, epidermal homeostasis, angiogenesis, extracellular matrix (ECM) turnover and more.
  • Cathepsins are thought to be involved in several processes of cancer progression, including angiogenesis initiation, tumor growth and invasiveness. It was suggested that the compounds described herein may bind to the family of cathepsins.
  • proliferative disorder is a disorder displaying hyper proliferation. This term means cell division and growth that is not part of normal cellular turnover, metabolism, growth, or propagation of the whole organism. Unwanted proliferation of cells is seen in tumors and other pathological proliferation of cells, does not serve normal function, and for the most part will continue unbridled at a growth rate exceeding that of cells of a normal tissue in the absence of outside intervention.
  • hypo proliferative disease A pathological state that ensues because of the unwanted proliferation of cells is referred herein as a "hyper proliferative disease” or “hyper proliferative disorder.”
  • proliferative disorder cancer
  • tumor tumor-associated fibroblasts
  • malignancy all relate equivalently to a hyperplasia of a tissue or organ.
  • compositions and methods of the present invention may be used in the treatment of non-solid and solid tumors.
  • Non-limiting examples include at least one of blastoma, carcinoma, lymphoma, leukemia, sarcoma, mesothelioma, glioma, germinoma, choriocarcinoma, melanoma, glioblastoma, lymphoid malignancies, squamous cell cancer (e.g.
  • lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer.
  • lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer,
  • inflammatory disorder is a disorder encompassing any immune response.
  • the inflammatory disorder may be an infectious or a non- infectious disorder.
  • Non-infectious inflammatory disorders denote any disorder which the activation of macrophages or activated macrophages play a role such as auto-immune disorders and inflammatory disorders which are not infection related, i.e. nonpathogenic, caused by other than an infectious agent (e.g. auto-antigen, hypersensitivity, wound).
  • inflammatory diseases of the gastrointestinal tract such as Crohn's disease, inflammatory bowel disease, gastritis, colitis, ulcerative colitis, colon irritable, gastric ulcer and duodenal ulcer, inflammatory diseases of the skin such as psoriasis, inflammatory diseases of the respiratory system such as asthma, allergic rhinitis or chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, sarcoidosis, inflammatory diseases of the musculoskeletal system such as rheumatoid arthritis, osteomyelitis, osteoporosis, or neuritis, systemic sclerosis, inflammatory diseases of the kidneys such as glomerulonephritis, renal ischemia, or renal inflammation; inflammatory diseases of the nervous system such as multiple sclerosis, Alzheimer's disease and HIV- 1 -associated dementia; autoimmune diseases such as diabetes, type 1 and 2 diabetes mellitus and graft versus host reaction; infectious disease such as nephritis, seps
  • the pathologies associated with increased cathepsin may be cancer. In some embodiments, the pathologies associated with increased cathepsin may be a pathology associated with angiogenesis, degradation of vascular basement membrane, and activation of angiogenic growth factors, macrophage-targeted treatment.
  • the pathologies associated with increased cathepsin may be a skin disorder. In some further embodiments, the pathologies associated with increased cathepsin may be a cardiovascular disease. In some further embodiments, the pathologies associated with increased cathepsin may be atherosclerosis, osteoarthritis, arthritis, Alzheimer, psoriasis. In some further embodiments, the pathologies associated with increased cathepsin may be parasite infection.
  • the present invention is appropriate for a subject suffering from a disorder such as inflammatory, infectious, proliferative, neuro-degenerative, ischemic, metabolic, spinal cord injury, trauma, autoimmune disorders and acute or chronic wound or injury.
  • a disorder such as inflammatory, infectious, proliferative, neuro-degenerative, ischemic, metabolic, spinal cord injury, trauma, autoimmune disorders and acute or chronic wound or injury.
  • a compound of the invention may be applied topically, injected locally or administered by injection into the bloodstream of a subject to be treated.
  • the compound in its "inactive" form is absorbed by cells of the subject's body and becomes attached (i.e. covalently bound) to protease enzymes (forming the "active" form).
  • protease enzymes forming the "active” form.
  • the enzymes are more abundant in cancer cells, the absorption and association of the compound of the invention to cancer cells is more efficient and is maintained over longer periods of time as compared to normal cells.
  • the subject's body is exposed to non-thermal light (e.g., laser) of a suitable wavelength, adapted to the absorption spectrum of the photosensetizer moiety to be excited.
  • non-thermal light e.g., laser
  • the intensity and period of exposure of said light may be adapted to permit identification of said localities without substantially inducing treatment.
  • the tumors may be selectively exposed to light at the same or different and appropriate intensity and for a period of time permitting light absorption by the tumors, production of oxygen active forms and subsequent destruction of the tumor.
  • the method permits one or more of direct killing of cells, for example cell within cancerous tissue, shrinking the size of tumors, damaging cells within tumor, preventing cancer cells from proliferating.
  • the light source used in the photodynamic method of the invention may be any light source having the appropriate intensity and wavelength. Irradiation may be achieved from the surface of the skin or by means permitting delivery of light to areas inside the body of the subject (by using e.g., fiber optic cable).
  • the compounds of the present disclosure may be administrated to a subject in need thereof in an effective amount.
  • the "effective amount" for purposes herein may be determined by such considerations as known in the art. The amount must be effective to achieve the desired therapeutic effect, depending, inter alia, on the type and severity of the disease to be treated and the treatment regime.
  • the effective amount depends on a variety of factors including for example the distribution profile within the body, a variety of pharmacological parameters such as half life in the body, on undesired side effects, if any, on factors such as age and gender, and others.
  • the compounds of the present disclosure may be used for diagnosis and may be administered at a diagnostically effective amount.
  • diagnostically effective amount refers to an amount of the compound, radiopharmaceutical or diagnostic composition, as defined herein, which allows for efficient molecular imaging depending on the type of the imaging technique (e.g. fluoresce, PET, SPECT etc) used, the acquisition parameters of the specific imaging technique used, the area of the body scanned, the physical condition of the subject, the purpose of the test or any other factors which are apparent to the person skilled in art.
  • the effective amount may be the same as the diagnostically effective amount indicating the amount of the compounds that may be sufficient for diagnosis and treatment.
  • the methods of the invention may be used for the treatment of endoscopically accessible tumors such as lung, bladder, gastrointestinal and gynaecological neoplasms, and also in dermatology for the treatment of non-melanoma skin cancers (basal cell carcinoma) and precancerous diseases (e.g., actinic keratosis).
  • the cancer to be treated is selected from breast cancer, prostate cancer and head and neck cancer.
  • treatment or prevention refers to the complete range of therapeutically positive effects of administrating to a subject including inhibition, reduction of, alleviation of, and relief from, pathologies associated with increased cathepsin. More specifically, treatment or prevention includes the prevention or postponement of development of the disease, prevention or postponement of development of symptoms and/or a reduction in the severity of such symptoms that will or are expected to develop. These further include ameliorating existing symptoms, preventing- additional symptoms and ameliorating or preventing the underlying metabolic causes of symptoms.
  • the terms “inhibition”, “moderation”, “reduction” or “attenuation” as referred to herein, relate to the retardation, restraining or reduction of a process by any one of about 1% to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%.
  • any method of treatment according to the invention may comprise a step of irradiation with a light at an appropriate wavelength, as described herein.
  • the light is actinic radiation.
  • the wavelength of the irradiated light is selected to match the absorption wavelength of the fluorescence-quenching moiety.
  • the compounds of the invention may be used to diagnose tissue of subject that may benefit from the activity based probe of the invention.
  • the compound of the invention may be formulated for a variety of administrations as known in the art.
  • the formulation for use in medicine e.g., oncology, may be diagnostic or therapeutic (diagnostic composition/formulation, or pharmaceutical composition/formulation).
  • the compound or a composition comprising same may be used in a method of detection of tumor cells or tumor regions by tumor- targeting photodynamic imaging and/or in a method of treatment of said tumors by tumor-targeting photodynamic.
  • the present disclosure also provides a kit for photodynamic therapy, the kit comprising a compound of the invention in a form suitable for direct administration to a subject, and instructions for use.
  • the activity based probe described herein or a composition comprising it may be administrated by any known method in the art. These include, but are not limited to, injection (e.g., using a subcutaneous, intramuscular, intravenous, or intradermal injection), intranasal administration and oral administration.
  • injection e.g., using a subcutaneous, intramuscular, intravenous, or intradermal injection
  • intranasal administration e.g., intranasal administration
  • oral administration e.g., oral administration.
  • the present invention relates to the treatment of subjects, or patients, in need thereof.
  • patient or “subject in need' it is meant any organism who may be infected by the above-mentioned pathogens, and to whom the preventive and prophylactic kit/s, system/s and methods herein described is desired, including humans, domestic and non- domestic mammals such as canine and feline subjects, bovine, simian, equine and murine subjects, rodents, domestic birds, aquaculture, fish and exotic aquarium fish. It should be appreciated that the treated subject may be also any reptile or zoo animal. More specifically, the kit/s and method/s of the invention are intended for preventing pathologic condition in mammals.
  • mamalian subject any mammal for which the proposed therapy is desired, including human, equine, canine, and feline subjects, most specifically humans. It should be noted that specifically in cases of non- human subjects, the method of the invention may be performed using administration via injection, drinking water, feed, spraying, oral lavage and directly into the digestive tract of subjects in need thereof.
  • any component of a composition of the invention should be read to encompass one, two, three, four, five, six, seven, eight, nine or ten different occurrences of said component in a composition of the invention.
  • Figs. 1A to ID present synthesis of the compouds (probes) according with the invention
  • Fig. lA-Fig.lC show detailed sysnethis of PS-ABP and PS-qABP probes
  • Fig. ID shows general structure of the probes (quenched and non-quenched).
  • Figs. 2A and 2B presents PS moieties used in the generation of probes according to the invention
  • Fig. 2A shows photosensitizer moiety
  • Fig. 2B shows quenching moiety.
  • Figs. 3A-3N present LCMS characterization of intermediates and compounds including compounds designated herein as YBN1-YBN8 and YBN26 such that each one of Figs. 3A-3N shows a chemical structure and characterization by HPLC and MS (indicating the molecular weight of the major peak).
  • Figs. 4A and 4B show representative quenching efficiency plots YBN6 (Fig. 4A) and YBN4 (Fig. 4B), fluorescent units were plotted versus probe concentration and the ratio between the slopes was determined as quenching efficiency.
  • Fig. 5 shows labeling of recombinant cathepsin B, cathepsin L and cathepsin S by PS-ABP (designed herein as YBN1, YBN3, YBN5 and YBN7) and PS-qABP probes (designed herein as YBN2, YBN4, YBN6 and YBN8).
  • PS-ABP designed herein as YBN1, YBN3, YBN5 and YBN7
  • PS-qABP probes designed herein as YBN2, YBN4, YBN6 and YBN8.
  • Figs. 6Aand 6B presents probe cell permeability
  • Fig. 6A shows detection of cathepsin inhibition in intact cells by labeling of residual protease activities in NIH-3T3 cells treated for 4 hours with probes
  • Fig. 6B is a bar graph showing average of proteases inhibition by YBN1-8 at 4 to 8 hour time points.
  • Figs. 7A to 7C present direct labeling of endogenous cathepsin in intact RAW 264.7 cells (Fig. 7A) and 4T1 cells (Fig. 7B), Fig. 7C shows identification of cathepsin B, cathepsin L and cathepsin S expression in cells.
  • Figs. 8A to 8D are fluorescent images in live cells
  • Figs. 8A and 8C are images of NIH-3T3 cells before washing
  • Figs. 8B and 8D are images of NIH-3T3 cells after a 16 hour wash with growth medium
  • Figs. 8A and 8B show results obtained with YBN5 and YBN6
  • Figs. 8C and 8D show results obtained with YBN7 and YBN8, LysoTracker being lysosomal marker showing lysosomal compartments.
  • Fig. 9 is a bar graph showing evaluation of non-quenched (YBN5), and quenched (YBN6), PS-ABPs for cell killing after light treatment.
  • Figs. 10A and 10B are optical (fluorescence) images (non-invasive imaging) of the compounds described herein
  • Fig. 10A presents images from Balb/C mice bearing subcutaneous 4T1 cell tumors injected with compounds designated herein as YBN7 and YBN8 or the corresponding free photosensitizer
  • Fig. 10B are images from nude mice bearing subcutaneous MDA-MB -231 cell tumors injected with compounds designated herein as YBN5 and YBN6 or the corresponding free photosensitizer. The tumors are marked by arrows.
  • Fig. 11 presents schematic representation of PS-ABP and PS-qABP probes including compounds designated herein as YBN5, YBN6 and YBN26.
  • Figs. 12A and 12B show representative quenching efficiency for YBN6 and YBN26
  • Fig. 12A is a plot of fluorescent units versus probe concentration and the ratio between the slopes was determined as quenching efficiency
  • Fig, 12B shows a fluorescence image of a dilution series of YBN5, YBN6 and YBN26.
  • Fig. 13 shows labeling of recombinant cathepsin B, cathepsin L and cathepsin S by YBN26.
  • Figs. 14Aand 14B presents probe cell permeability in 4T1 cells
  • Fig. 14A shows a dose response of YBN5, YBN6 and YBN26
  • Fig. 14B is a bar graph showing quantification the data of Fig. 14A
  • Fig. 15 show non-invasive imaging of YBN26 in a 4T1 tumor model in Balb/C mice (tumors are marked by arrows).
  • Figs. 16Aand 16B are ex vivo images showing accumulation of YBN26 in different organs.
  • Figs. 17A to 17C are fluorescent images showing induction of apoptosis by YBN26 in a 4T1 tumor model in mice treated with only a single dose of YBN26 followed by two light treatments, DAPI specifically shows cell's nucleus, cleaved caspase-3 being an apoptosis marker and F4/80 a macrophage marker, Fig. 17A are images of cells treated with YBN26 and stained as detailed in this figure with light and without (dark), Fig. 17B are images of cells treated with DMSO and stained as detailed in this figure with light and without (dark), Fig. 17C are images of cells treated with visudyne and stained as detailed in this figure with light and without (dark). DETAILED DESCRIPTION OF EMBODIMENTS
  • the compounds of the invention namely the photodynamic quenched activity based probes (herein referred to in short as PS-qABP) target cathepsin B, L and S.
  • PS-qABP photodynamic quenched activity based probes
  • the compounds of the invention have been prepared by both solid and solution phase methodologies. The evaluation of the selectivity, potency and photosentization properties of the compounds of the invention using in vitro and culture model systems is further demonstrated.
  • the compounds are capable of inducing cell death by activation of the photodynamic tag, e.g., in cancer mouse models, skin maladies (psoriasis, melanoma, AK, acne), atherosclerosis or arthritis.
  • the compounds developed herein contain an acyloxymethyl ketone electrophile that enables removal of the quencher upon enzyme binding.
  • the selection of PS moieties for attachment was based on two criteria: 1) high wavelength fluorescence in the near infrared range to enable non-invasive imaging, and 2) presence of an acid moiety enabling chemical binding to the core probe structure.
  • synthesis of the probes was carried out using solid-phase peptide synthesis (SPPS) combined with solution synthesis.
  • SPPS solid-phase peptide synthesis
  • the precursor compound GB111- NH2 [1], compound 3 was used for generating the non-quenched PS-ABP, its synthesis was modified from [1] as described in Fig. 1A.
  • the commercial photosensitizer acid moiety was activated to an ester to enable attachment to the lysine ⁇ free amine of compound 3 as shown in Fig. 1C.
  • Fig. IB Compound 6, is a free amine of GB137 described in [2], which contains a quencher moiety and as noted above was used as a precursor for the quenched PS-ABPs.
  • the activated PS moiety was coupled in the same manner.
  • General structure of the probes is presented in Fig. ID. Initially a library of eight probes was generated out of which four are quenched and four are non-quenched. The activation of the photosensitizers to succinimide ester and a select number of such moieties are depicted in Fig. 2 A.
  • Fig. 2B shows representative quenchers.
  • Figs. 3A to 3N Characterization of all compounds synthesized (structure, UV trace, mass spectrometry analysis of the major peak and maximal fluorescence absorption) including the exemplary probes of the invention, designated herein as YBN1-YBN8 and YBN26 are shown in Figs. 3A to 3N.
  • the exemplary probes of the invention, designated herein as YBN1-YBN8 and YBN26 were purified to over 95% purity as shown in Figs. 3F-3N.
  • Each one of Figs. 3A to 3N shows a chemical structure of a compound (either an intermediate or a compound of the invention), HPLC data and MS data (molecular weight of the major peak).
  • the inventors further evaluated the fluorescent properties and quenching efficiency of the probes.
  • Each pair of probes, PS-ABP and the corresponding PS-qABP were evaluated for quenching efficiency in physiological pH 7.5, and lysosomal pH 5.5 (where the cathepsins reside in the cell).
  • the fluorescence was compared at increasing concentrations of the quenched probes relative to their non-quenched PS-ABPs counterpart at physiological or lysosomal pHs.
  • YBN4 and YBN6 Representative quenching efficiency plots for YBN4 and YBN6 are presented in Figs. 4A-4B.
  • Visudyne containing probes, YBN5 (non-quenched) and YBN6 (quenched) were excited at 430 nm and emission was measured at 690 nm.
  • YBN6 was found to have more than 360-fold lower fluorescence in PBS (pH 7.5), when compared to YBN5 and more than 160-fold quenching in acetate buffer (Figure 4A). Similar pattern was observed for YBN3 and YBN4 (Fig. 4B). All of the PS- qABPs were excited at the wavelengths of their maximum absorption peak and were found significantly quenched (14 to 363-fold) in comparison to the corresponding non- quenched PS-ABPs as shown in Table 1.
  • Example 3 Binding of PS-ABPs to recombinant Cathepsin B, L and S.
  • the inventors evaluated the ability of the various PS-qABPs and PS-ABPs to bind to the target cathepsins by incubating them with recombinant human cathepsins B, L, or S.
  • the free probes were separated from the enzyme -probe complex using SDS- PAGE.
  • the detection of the probe-enzyme complex was done by a fluorescence scan of the gel.
  • a control was performed in the presence of a potent active-site cathepsin inhibitor GB111-NH2.
  • the inventors set out to determine the probes' cell permeability, specificity and their ability to target the endogenous cathepsins.
  • the labeling of endogenous cathepsin targets in intact NIH 3T3 cells was tested. This experiment determines the probes cell permeability and ability to reach and inhibit the cathepsin activity in the lysosomes.
  • a competition study was performed, in which cells were first treated with each one of the probes for four hours followed by labeling of the residual cathepsin activity with GB 123 (the inventors' published Cy5 labeled probe that is easily detected) [2].
  • Cell permeable probes inhibit the cellular cathepsin activity resulting in minimal residual activity as seen in YBN 1, 3, 5 and 7, while treatment with probes that have limited permeability lead to the detection of pronounced residual activity as seen in YBN 2, 4, 6 and 8, (Fig. 6A).
  • the inventers investigated cathepsin labeling in cells that have increased cellular uptake such as Raw 264.7 macrophages (Fig. 7A) and in cells that possess elevated cathepsin activity such as 4T1 breast cancer cells (Fig. 7B).
  • YBN5 and YBN6 managed to enhance cell death by light illumination and enabled cell killing to the same extent as the free photosensitizer, as shown in Fig. 9.
  • Figs. 10A and 10B show representative non-invasive images of Balb/C mice bearing subcutaneous 4T1 cell tumors and nude mice bearing subcutaneous MDA-MB-231 cell tumors, respectively, that were injected with YBN5-8 or the corresponding free photosensitizers.
  • the various YBN probes can clearly be detected because of accumulation at the tumor site post injection.
  • Example 7 An oxidized QC-1 quencher - in vitro data
  • the QSY-21 quencher was replaced with the commercial QC-1 quencher that the inventors found to be cell permeable.
  • the chlorine atom in QC-1 quencher was oxidized to an OH to enable better absorbance of the energy omitted by Visudyne photosensitizer.
  • the chlorine of QC-1 was naturally oxidized to OH after overnight shaking in DMSO.
  • the inventors then used oxidized QC-1 to generate a new quenched probe YBN26 containing the PS Visudyne using similar methods described above, YBN26 structure seen in Fig. 11.
  • the quenching efficiency of YBN26 was measured in comparison to YBN5 and YBN6 and is shown in Fig. 12A as described above. A dramatic reduction in fluorescence was detected in the quenched probe YBN26 by 11 fold. Although the reduction in fluorescence was lower in YBN26 than in YBN6, the inventors found it sufficient for in vivo applications. A fluorescent image of a dilution series of YBN5, YBN6 and YBN26 scanned by IVIS-Kinetic can be seen in Fig 12B, significant quenching is visible in both YBN6 and YBN26.
  • YBN26 was then tested for its ability to label cellular cathepsins in intact 4T1 cells. Modifying the quencher from QSY21 to oxidized QC-1 should enable better cell permeability of the quenched probe. Indeed, YBN26 shows high cell permeability and efficient labeling of the intact cathepsins after incubation of the probe in the growth media of the growing cells. High selectivity of the probe to the cathepsins is detected by the fluorescent bands between 20 and 37 kDa.
  • Fig. 14A Quantification of the 22kDa and 28 kDa fluorescent bands form Fig 14A show high fluorescent generated by labeling with YBN26, Fig 14B.
  • the inventors monitored the pharmacokinetics and non-invasive capabilities of the probe in Balb/C mice injected with 4T1 tumor cells (each mouse bearing two tumors on its back) relative toYBN6 and the free phosphsensitizer Visudyne.
  • a clear signal from the tumors could be detected already after one -hour post YBN26 injection, the fluorescence of the free i.e. un-bound probe (namely the probe which was not bound to an enzyme) distributed throughout the body could not de detected because of its quenched fluorescent properties.
  • the cancerous tissue rapidly bound the probe resulting in a significant tumor- specific fluorescent signal.
  • One of the two tumors was treated with light at indicated times points post probe injection, marked with a thunder cartoon, while the other was kept in the dark, Fig 15.
  • tumor sections were stained for cleaved caspase-3, an apoptosis marker, and F4/80, a macrophage marker.
  • Significant signal for cleaved caspase-3 was detected in tumors treated with only a single dose of YBN26 followed by two light treatments, indicating that YBN26 triggered massive apoptosis in tumor tissue after light treatment.
  • the apoptotic cell death was mostly colocalized with the tumor macrophages known to exhibit high cathepsin activity (Fig. 17). Tumors that were kept in dark showed no apoptosis similar to the vehicle control.
  • positive control light treatment of tumors treated with the free photosensitizer Visudyne two hours post injection resulted in tumor apoptosis.
  • Fmoc-Lys(Boc)-BMK, 1 As described in [1], detailed below. Fmoc- Lys(Boc)-OH (1 eq., 3gr, 6.4mmol) was dissolved in anhydrous Tetrahydrofuran (THF) at 0°C in an inert atmosphere under Argon, N-methylmorpholine (NMM; 1.25 eq., 880 ⁇ 1, 8.0mmol) and isobutyl chloroformate (IBCF; 1.15eq., 857 ⁇ 1, 7.36mmol) were sequentially added. The solution was stirred at 0°C for 10 minutes and additional two hours at -10°C.
  • THF Tetrahydrofuran
  • Fmoc-Lys(Boc)-AOMK 2: based on the synthesis of carboxybenzyl phenylalanine lysine (Boc) AOMK described in [1] with modification, detailed below.
  • Fmoc-Lys(Boc)-BMK (1 eq., 495mg, 0.9mmol) and 2,6-dimethylbenzoic acid (5 eq, 690mg, 4.5mmol) were dissolved in dry dimethylformamide (DMF) under argon. KF (10 eq., 534mg, 9.2mmol) was added and reaction was kept at room temperature for 2 hours while stirring.
  • the peptide was elongated by addition of a solution of N -benzyloxycarbonyl-phenyalanine (3 eq., 226mg), Hydroxybenzotriazole (HOBT; 3 eq., 101.3mg) and diisopropylcarbodiimide (DIC; 3 eq., 116mg) in DMF for 2 hours.
  • the resin was washed with DCM and DMF.
  • the final peptide product was cleaved from resin by addition of 5% TFA/DCM (v/v) for 7 to 15 minutes. The cleaved solution was collected and solvent was removed by co- evaporation with toluene.
  • the peptide was elongated by addition of a solution of N-benzyloxycarbonyl-phenyalanine (3 eq., 336mg), HOBT (3 eq., 151.7mg) and DIC (3 eq., 175 ⁇ 1) in DMF for overnight shaken. The resin was washed with DCM and DMF. The final, fully-protected peptide product was cleaved from resin by addition of 2% TFA/DCM (v/v) for 5 minutes. The cleavage solution was collected and solvent was removed by co-evaporation with toluene. The crude peptide was further dried in lyophilization to yield a white solid, over 95% purity by LC-MS, and was used without further purification.
  • N-benzyloxycarbonyl-phenyalanine 3 eq., 336mg
  • HOBT eq., 151.7mg
  • DIC eq., 175 ⁇ 1
  • the resin was washed
  • the resin was washed with DMF and DCM, Cbz-Phe-Fmoc-Lys(Boc)-BMK (3 eq., 152mg) and potassium fluoride (10 eq., 49mg) in dry DMF mixture were added to the resin for 2 hours under Argon.
  • the crude product ZFK(Boc) 2,6 dimethyltherephthalic amide 6-aminohexane was cleaved from resin by addition of 2% TFA/DCM. The cleavage solution was collected and solvent was removed by co- evaporation with toluene. The product was further dried by lyophilization to yield a white solid that was over 95% pure by LC-MS and was used without further purification. 17.73mg, 0.022mmol, 26% yield, M/z+1 816.
  • PS-NHS succinimide ester
  • DCC ⁇ , ⁇ '- Dicyclohexylcarbodiimide
  • TSTU 0-(N-Succmimidyl)- l,l,3,3-tetramethyluronium tetrafluoroborate
  • Quenching efficiency Increasing concentrations of YBN1-8, 26 in acetate buffer (50 mM acetate, 5 mM MgCl 2 , 2 mM DTT, pH 5.5) containing 1% DMSO were prepared in a 96-well plate. Fluorescence was imaged and quantified using an IVIS-Kinetics system equipped with an excitation filter of 430 nm and an emission filter of 690 nm for all compounds, or using SpectraMax M5 plate reader with a suitable excitation/emission wavelength for each probe. Arbitrary fluorescent units were plotted versus probe concentrations, and the ratio between the slope of the non-quenched probe and the corresponding quenched probe was determined as the quenching efficiency. Similarly, quenching efficiency of the probes was evaluated in PBS.
  • Recombinant cathepsin labeling Recombinant human Cathepsin L (0.6 ⁇ g), Cathepsin B (0.7 ⁇ g) or Cathepsin S (0.7 ⁇ g) were pretreated with 1 ⁇ inhibitor GBI I I-NH 2 or vehicle for 30 min (indicated samples) in reaction buffer (50 mM acetate, 2 mM DTT and 5 mM MgCi 2 , pH 5.5) at room temperature. Indicated concentrations of YBN1-8, 26 were added to samples for 90 minutes at 37°C.
  • sample buffer x4 50% glycerol, 0.2 M Tris/HCl pH 6.8, 20% beta-mercaptoethanol, 12% SDS and 0.4 mg/ml bromophenol blue. Samples were then boiled, separated on a 12.5% SDS gel and scanned for fluorescence by the Typhoon scanner FLA 9500 at excitation/emission wavelengths of 635/670 nm for YBN1-8, and Odyssey scanner at excitation/emission wavelengths of 680/700 nm for YBN26.
  • sample buffer x4 50% glycerol, 0.2 M Tris/HCl pH 6.8, 20% beta-mercaptoethanol, 12% SDS and 0.4 mg/ml bromophenol blue.
  • NIH-3T3 mouse fibroblast cells Raw 264.7 mouse macrophage cells or 4T1 murine mammary gland epithelial cells were cultured in DMEM (Dulbecco's modified eagle's medium) supplemented with 10% fetal bovine serum (FBS), 1% penicillin and 1% streptomycin. All cells were cultured in a humidified atmosphere of 95% air and 5% C0 2 at 37°C.
  • DMEM Dulbecco's modified eagle's medium
  • FBS fetal bovine serum
  • streptomycin All cells were cultured in a humidified atmosphere of 95% air and 5% C0 2 at 37°C.
  • NIH-3T3 cells (3xl0 5 cells/well) were seeded in a six-well plate one day before treatment. Cells were treated with vehicle or 20 ⁇ probes that were pre-dissolved in 10% DMSO, 90% ethanol in culture medium. After 4, 6 or 8 hours of probe incubation residual cathepsin activity was labeled with GB123 [2] (5 ⁇ ) (final DMSO concentration was maintained at 0.2% and 0.9% ethanol). Cells were washed with PBS and lysed by addition of sample buffer. Lysates were boiled for 5 minutes, centrifuged, and separated by 12.5% SDS-PAGE. Residual labeled proteases in cells were visualized by scanning the gel with an Odyssey Scanner at excitation/emission wavelengths of 680/700 nm.
  • YBN5, YBN6 and YBN26 probes in intact cell by direct labeling assay Similar to the described above with minor changes, 4T1 cells (lxlO 5 cells/well) were seeded in a twelve -well plate one day before treatment. Cells were pre-treated with inhibitor GB111-NH 2 (5 ⁇ ) or vehicle (0.05% DMSO) for 1 hr and 45 minutes followed by 5 hours incubation with the probes (10 or 20 ⁇ , pre-dissolved in 90% ethanol / 10% DMSO). Final DMSO and ethanol concentrations were maintained throughout at 0.1% and 0.9%, respectively. Cells were then washed with PBS and lysed by addition of sample buffer. Lysates were boiled for 5 minutes, centrifuged separated by 12.5% SDS-PAGE. Labeled proteases were visualized by scanning the gel with an Odyssey Scanner at excitation/emission wavelengths of 680/700 nm.
  • NIH-3T3 cells were seeded in an 8 well cover-slip chamber containing growth medium one day before treatment. Cells were either pretreated with inhibitor GB I I I-NH2 (5 ⁇ ) or with vehicle DMSO (0.1%) for 1 hr. Cells were labeled by addition of probes (10 ⁇ , 0.1% DMSO, 0.9% ethanol) to growth media (1%FBS) for 7 hours. Medium was then replaced with growth medium without phenol red. Fluorescent images were taken at 100X magnification using an Olympus inverted fluorescent microscope 1X51 equipped with a Cy5 filter. Cells were either imaged directly or washed with growth medium for 16 hrs and imaged in the presence of LysoTracker.
  • YBN5 and YBN6 for cell killing: Tumor cells (PyMT, 6,500 cells/well) were cultured in a 96 well plate one day prior to treatment. Triplicate samples of cells were incubated with 20 ⁇ YBN5 or YBN6 or free photosensitizer, Visudyne, pre- dissolved in DMSO/ethanol as described above, in growth medium without phenol red (1% FBS) for 4 hours. Cells were illuminated from the bottom with a 15 mW light for 11 minutes, or kept in the dark. Cell survival was determined a day after illumination by methylene blue assay, relative to DMSO dark control. Probes demonstrated efficient cell killing after light treatment.
  • In vivo imaging Tumor cells were grown to subconfluency, followed by detachment with trypsin, spinning down and resuspending in 0.5% BSA in sterile PBS and 25% matrigel.
  • Fig 10A and 10B Balb/C or Balb/C nude mice were injected subcutaneously under isoflurane anesthesia with 1X10 6 4T1 or MDA-MB-231 cells per spot, in a total volume of 20 ⁇ , respectively.
  • Balb/C mice were injected with two tumor spots subcutaneously under isoflurane anesthesia, 7.6X10 5 4T1 cells per spot in a total volume of 20 ⁇ .
  • Tumors were typically established 7-13 days after cells injections, the fur was removed from mice (Balb/C) and compounds were injected intravenously via the tail vein as follows: YBN probes, Visudyne and chlorin e6, (75 nmol) dissolved in 50% DMSO in PBS in a total volume of 100 ⁇ . Mice anesthetized with isoflurane were then imaged at indicated time points after probe injection using the IVIS kinetic imaging system (Perkin Elmer) equipped with a 640nm/Cy5 excitation/emission filter.
  • IVIS kinetic imaging system Perkin Elmer
  • F4/80 Molecular Probes
  • Caspase-3 Cell Signaling
  • Cas-Block Invitrogen

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Abstract

La présente invention concerne des composés comprenant un photosensibilisateur, des compositions les comprenant ainsi que des utilisations et des procédés associés pour la détection et le traitement par thérapie photodynamique.
PCT/IL2016/050354 2015-03-31 2016-03-31 Sondes basées sur l'activité photodynamique atténuée et leurs utilisations en imagerie et en thérapie ciblée WO2016157198A1 (fr)

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