WO2003055887A1 - Chromophore conjugue de porphyrine, chlorine ou bacteriochlorine - Google Patents

Chromophore conjugue de porphyrine, chlorine ou bacteriochlorine Download PDF

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Publication number
WO2003055887A1
WO2003055887A1 PCT/GB2002/005867 GB0205867W WO03055887A1 WO 2003055887 A1 WO2003055887 A1 WO 2003055887A1 GB 0205867 W GB0205867 W GB 0205867W WO 03055887 A1 WO03055887 A1 WO 03055887A1
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chromophore
group
protein
poφhyrin
cells
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PCT/GB2002/005867
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English (en)
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Ross William Boyle
John Greenman
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Wellcome Trust Limited
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Publication of WO2003055887A1 publication Critical patent/WO2003055887A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains four or more hetero rings
    • 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 relates to novel po ⁇ hyrin and porphyrin-based chromophores, which may be particularly useful in a range of photodynamic applications, including photochemotherapy and fluorescence analysis and imaging.
  • po ⁇ hyrin and po ⁇ hyrin-based chromophores both as research tools, for example in fluorescence-activated cell sorting (FACS), and as therapeutic agents in photodynamic therapy (PDT) for bringing about the death of undesirable cells in vivo, is wic'ely recognised in the art.
  • FACS fluorescence-activated cell sorting
  • PDT photodynamic therapy
  • Each of these applications is dependent on the ability of the chromophore to be excited by incident light to a singlet excited state, and to decay to a lower energy state with the consequent emission of energy. This energy may be emitted in the form of fluorescent light at a specific wavelength, thereby enabling a cell or biostructure attached to the decaying chromophore to be visualised, and/or sorted by FACS.
  • the energy of excitation may be dissipated by initial conversion of the singlet chromophore into the triplet excited state, followed by the transfer of energy to another triplet such as dioxygen, with the consequent formation of singlet oxygen.
  • Singlet oxygen is a powerful cytotoxic agent, and hence where this latter process occurs in or in the immediate vicinity of a cell, it will usually result in the death of that cell. Accordingly, the chromophore can be exploited both for its fluorescent properties, and for its ability to act as a photosensitiser.
  • non-covalent binding between the po ⁇ hyrin and protein delivery molecules reduce the yield of covalently bound po ⁇ hyrin, but this effect can also hinder the subsequent targeting process.
  • non-covalently bound po ⁇ hyrin can be readily and non-specifically transferred to non-target cell surfaces or proteins, thus destroying the specificity of the targeting process.
  • the problem of non-covalent protein/po ⁇ hyrin binding is alleviated by way of an electroelution purification step carried out on the protein/po ⁇ hyrin mixture after the conjugation step, by which any po ⁇ hyrin non-covalently bound to protein can be separated and discarded.
  • electroelution purification step carried out on the protein/po ⁇ hyrin mixture after the conjugation step, by which any po ⁇ hyrin non-covalently bound to protein can be separated and discarded.
  • this addition purification step is inexpedient and costly, especially when carried out on an industrial scale.
  • po ⁇ hyrin- or po ⁇ hyrin-based chromophores which can be effectively covalently conjugated to protein molecules whilst showing negligible or low levels of non-covalent protein binding, and which when so conjugated display good photodynamic activity, remains a desirable objective.
  • a po ⁇ hyrin, chlorin or bacteriochlorin chromophore which chromophore comprises one conjugating meso substituent which comprises a conjugating group Z for covalently conjugating said chromophore to a protein so as to enable delivery of the chromophore to a selected biological target in vitro or in vivo, and one, two or three hydrophilic meso substituents, wherein the hydrophilicity of the chromophore is such that on carrying out conjugation of said chromophore to said protein by way of incubating said chromophore and said protein for one hour under standard protein conjugation conditions in 10% DMSO/water buffered to pH 9, the percentage of non-covalently bound chromophore out of total protein-bound chromophore is 5% or less.
  • said percentage of non-covalently bound chromophore out of total protein-bound chromophore may be 4% or less; more preferably 3% or less, still more preferably 2% or less, even still more preferably 1% or less; most preferably 0-0.5%.
  • said protein may be a delivery protein with specific affinity for said biological target.
  • said delivery protein may comprise an antibody or a fragment thereof, such as a monoclonal antibody or fragment thereof, a polyclonal antibody or fragment thereof, or a single-chain Fv antibody fragment (ScFv); a ligand or ligand mimetic; or an enzyme or receptor mimetic.
  • said biological target is a cell or a membrane
  • said delivery protein may possess specific affinity for a molecule or structure exposed on the surface of said cell or membrane.
  • said molecule or structure exposed on the surface of said cell or membrane may be a receptor or channel, which receptor or channel is adapted to cause or allow the passage of a molecule bound thereto across said membrane or into said cell.
  • said delivery protein may be adapted to be internalised into said cell upon binding to said cell.
  • said protein may be a bridging polypeptide, which bridging polypeptide is adapted to be bound or linked to a complementary bridging polypeptide, which complementary bridging polypeptide can be bound or linked to said biological target or to a delivery protein with specific affinity for said biological target so as to enable delivery of said protein molecule to said biological target.
  • Said bridging polypeptide may for example comprise avidin streptavidin whilst said complementary bridging polypeptide comprises biotin; or vice versa.
  • said bridging polypeptide may comprise calmodulin whilst said complementary bridging polypeptide comprises calmodulin binding peptide; or vice versa.
  • said protein may be free or substantially free of specific binding sites for po ⁇ hyrin-like molecules, including po ⁇ hyrins, chlorins and bacteriochlorins.
  • the degree of non-covalent protein binding may vary slightly from protein to protein, depending on the structure and properties of each protein. It has however been found that where the degree of non-covalent binding of a po ⁇ hyrin, chlorin or bacteriochlorin chromophore to bovine serum albumin (BSA) under standard protein conjugation conditions, measured in accordance with the invention, is no greater than 20% of total BSA-bound chromophore, the hydrophilicity of the chromophore will always be sufficient for use in accordance with the invention.
  • BSA bovine serum albumin
  • BSA possesses a number of binding sites for po ⁇ hyrin-like molecules, which increases its affinity for po ⁇ hyrin, chlorin and bacteriochlorin chromophores. Accordingly a rate of 20% non-covalent binding between a po ⁇ hyrin, chlorin or bacteriochlorin chromophore and BSA will typically correspond to a significantly lower rate of non-covalent binding between said chromophore and a protein for effecting delivery of said chromophore to a specific biological target.
  • a po ⁇ hyrin, chlorin or bacteriochlorin chromophore which chromophore comprises one conjugating meso substituent which comprises a conjugating group Z for covalently conjugating said chromophore to a protein, and one, two or three hydrophilic meso substituents, wherein the hydrophilicity of the chromophore is sufficient to ensure that on carrying out conjugation of said chromophore to said protein by way of incubating said chromophore and said protein for one hour under standard protein conjugation conditions in 10% DMSO/water buffered to pH 9, the percentage of non-covalently bound chromophore out of total protein-bound chromophore is 20% or less, wherein said protein is bovine serum albumin.
  • said percentage of non- covalently bound chromophore out of total protein-bound chromophore may be 18% or less; more preferably 15% or less; still more preferably 13% or less; yet more preferably 10% or less; even still more preferably 7% or less; even yet more preferably 5% or less; most preferably 3% or less.
  • a chromophore in accordance with the invention is not a 5, 15-di-me.sO-substituted po ⁇ hyrin, chlorin or bacteriochlorin chromophore or a 5,10, 15,20-tetra-mesO-substituted po ⁇ hyrin, chlorin or bacteriochlorin chromophore wherein the 5-meso substituent is a conjugating substituent as hereinbefore defined and the 15-meso substituent and as the case may be the 10- and 20-meso substituents are each the same one of the following groups:
  • a chromophore in accordance with the invention is not a 5,15-di-we-fo-substituted po ⁇ hyrin, chlorin or bacteriochlorin chromophore or a 5,10, 15,20-tetra-mes ⁇ -substituted po ⁇ hyrin, chlorin or bacteriochlorin chromophore wherein the 5-meso substituent is a conjugating substituent as hereinbefore defined and the 15 -me.ro substituent and as the case may be the 10- and 20-meso substituents are each the same one of the following groups:
  • said standard protein conjugation conditions may comprise a mixture of 10% of a stock solution of said chromophore in DMSO with 90% of an aqueous solution of said protein, said mixture being buffered to pH 9 so as to ensure that all lysine residues remain uncharged.
  • said percentage of non-covalently bound chromophore out of total protein-bound chromophore may be measured by separating any unbound chromophore from said protein following said incubation, for example by passing said incubated mix of chromophore and protein down a gel filtration column to obtain said protein and any chromophore bound thereto; loading said protein and any chromophore bound thereto onto a polyacrylamide gel and carrying out SDS-PAGE, so as to separate said non-covalently bound chromophore from said protein; excising from said gel said protein and said non-covalently bound chromophore; and carrying out a spectrophotometric analysis to determine the relative amounts of said non-covalently bound chromophore and of said chromophore covalently bound to said protein, so as to enable calculation of the percentage of non-covalently bound chromophore out of total protein-bound chromophore following said incubation.
  • Said one, two or three hydrophilic meso substituents may comprise any hydrophilic groups which when attached to a po ⁇ hyrin, chlorin or bacteriochlorin chromophore, are capable in combination of increasing the hydrophilicity of said chromophore to a level sufficient to ensure a reduced degree of non-covalent binding of the chromophore to said protein under standard protein conjugation conditions in accordance with the invention.
  • one or more of said hydrophilic meso substituents may comprise a charged substituent, such as a zwitterionic substituent possessing both positively and negatively charged moieties, other than:
  • hydrophilic meso substituents around the core of a po ⁇ hyrin, chlorin or bacteriochlorin chromophore results in enhanced solubility in basic buffer/DMSO or DMF co-solutions which are commonly used in protein bioconjugation. Increased hydrophilicity also produces a marked reduction in the tendency of the chromophore to bind non-covalently to proteins.
  • a decrease in non-covalent binding between the chromophore and the protein will reduce the degree of non-specific transfer of chromophore to cell surfaces, which will substantially increase the accuracy of targeting the chromophore to the cells or tissue of interest.
  • Localisation of a chromophore in or around the mitochondria is especially advantageous for the pu ⁇ oses of photodynamic therapy, as this will enable efficient and targeted disruption of the mitochondria, thereby triggering cellular apoptosis.
  • cell death by apoptosis is to be preferred over cell death by necrosis, as the apoptotic process is naturally succeeded in vivo by the building of healthy tissue, thus minimising formation of scar tissue and organ damage and loss of function.
  • the presence of one, two or three anionic substituents around a chromophore core in accordance with the invention is found to correlate with intracellular localisation of the chromophore principally in or around the lysosomes.
  • said hydrophilic substituent may comprise a quartenised pyridyl (pyridiniumyl) ring. Where said pyridiniumyl ring comprises no charged ring substituents, the hydrophilic substituent will constitute a cationic substituent, owing to the presence of the single positive charge on the quartenised nitrogen ring atom. Where the pyridiniumyl ring comprises a single charged ring substituent which is a negatively charged ring substituent, the hydrophilic substituent will constitute a zwitterionic substituent. Where the pyridiniumyl ring comprises charged substituents which impart to the ring a net positive or a net negative charge, the hydrophilic substituent will constitute a cationic or anionic substituent respectively.
  • Said pyridiniumyl ring may be N-linked to said po ⁇ hyrin, chlorin or bacteriochlorin chromophore core, such that the N-linkage of said pyridiniumyl ring quartenises the nitrogen atom.
  • said pyridiniumyl ring may be linked by a carbon atom in said ring to said po ⁇ hyrin, chlorin or bacteriochlorin chromophore core and may comprise one quartenising ring substituent Q which is N-linked to said pyridiniumyl ring for quartenising said nitrogen atom.
  • Said quartenising ring substituent Q may comprise ethyl, or branched or linear propyl, butyl, pentyl, hexyl, heptyl or octyl, or aryl such as phenyl, or heteroaryl such as pyridyl.
  • said quartenising ring substituent Q may comprise a hydrophilic group W as hereinafter defined.
  • said pyridiniumyl ring may be substituted one or more times by one or more hydrophilic groups W as hereinafter defined.
  • Each hydrophilic group W may be a group selected from R 3 L, or Y ⁇ R , or (R-N-RsJx.
  • R 3 is methyl or ethyl, or branched or linear or cyclised propyl, butyl, pentyl, hexyl, heptyl or octyl, or phenyl, or pyridyl, or pyridiniumyl, or a group R 6 R , where R 6 is methyl or ethyl and R 7 is phenyl or pyridyl or pyridiniumyl; where R 3 does not comprise pyridiniumyl, L represents one or more groups selected from -OH, -COO " (or -COOH) or -SO 3 " (or SO 3 H); where R 3 comprises pyridiniumyl, L represents one or more groups selected from -OH, -COO " (or -COOH) or -SO 3 " (or SO 3 H); where R 3 comprises pyridiniumyl, L represents one or more groups selected from -OH, -COO " (or
  • said R 3 may be methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl or octyl, and said L may include a terminating hydroxy, carboxy or sulfonate group.
  • said hydrophilic group W may be a group - (CH 2 ) a OH, or -(CH 2 ) a COO " (or -(CH 2 ) a COOH), or -(CH 2 ) a SO 3 " (or -(CH 2 ) a SO 3 H) where a is 1, 2, 3, 4, 5, 6, 7 or 8; most preferably 1, 2 or 3.
  • said hydrophilic group W may advantageously be hydroxymethyl, hydroxyethyl, ethylsulfonate or propylsulfonate.
  • said Yi may be O, and said x may be greater than 1.
  • said hydrophilic group W may comprise polyethylene glycol, preferably C 2 . 30 polyethylene glycol.
  • said hydrophilic group W may comprise a glycosyl group.
  • said hydrophilic group W may comprise a glycosyl group which is a sugar such as glucose, mannose, maltose, or a thiosugar such as thiogalactopyranose, thioglucopyranose or thiomannopyranose.
  • said hydrophilic group W may comprise one of the following glycosyl groups, where x is an integer between 1 and 6:
  • said hydrophilic group W may comprise an amino acid.
  • Amino acids are readily available and are well-known and characterised. Amino acids typically have good hydrophilicity owing to their zwitterionic character. Particular amino acids which may be utilised in accordance with the invention include lysine, cysteine, tyrosine, aspartate, glutamate, serine and threonine.
  • Said hydrophilic group W may be further substituted one or more times by hydroxy or oxo, so as further to increase the hydrophilicity of the group.
  • the total number of carbon atoms in said hydrophilic group W may not exceed 30.
  • hydrophilic groups W linked to said pyridiniumyl ring will serve to improve the hydrophilicity of the pyridiniumyl ring.
  • said pyridiniumyl ring may be substituted one or two times by one or two hydrophilic groups W respectively.
  • said hydrophilic substituent may comprise a quartenised amine group -N + Q ⁇ Q 2 Q3 or a quartenised phosphonium group -P + Q ⁇ Q 2 Q3.
  • Qi, Q 2 and Q 3 is selected from methyl, ethyl, branched or linear or cyclised propyl, butyl, pentyl, hexyl, heptyl or octyl, aryl such as phenyl, heteroaryl such as pyridyl, and a hydrophilic group W as hereinbefore defined.
  • said Qi, Q 2 and/or Q 3 may be substituted by a hydrophilic group W as hereinbefore defined, such as by a hydroxy, oxo, sulfonate, or carboxylate group, so as to improve the hydrophilicity of said cationic substituent.
  • a hydrophilic group W as hereinbefore defined, such as by a hydroxy, oxo, sulfonate, or carboxylate group, so as to improve the hydrophilicity of said cationic substituent.
  • At least one, more preferably two, most preferably each of said Qi, Q 2 and Q 3 may comprise an uncharged aryl or heteroaryl moiety, such as a phenyl ring, naphthyl ring, anthracene ring or a pyridyl ring.
  • aryl or heteroaryl moiety such as a phenyl ring, naphthyl ring, anthracene ring or a pyridyl ring.
  • Said uncharged aryl or heteroaryl moiety may be substituted one or more times by one or more substituents which do not interfere with the aromatic character of said moiety, such as (CH 2 ) y R 8 where y is an integer between 0 and 6 and R 8 is hydroxy, halo, sulfonate or carboxylate.
  • said hydrophilic substituent may comprise a phosphate group -P(O)(OR 7 XO " ) or a phosphonate group -OP(O)(OR 7 )(O " ), wherein said R 7 is selected from methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl or octyl.
  • said negatively charged phosphate or phosphonate group may be associated with a counterion such as a sodium or potassium counterion.
  • said phosphate or phosphonate group may comprise a group -P(O)(OR 7 )(OH) or a group -OP(O)(OR 7 )(OH).
  • Said pyridiniumyl ring, quartenised amine group, quartenised phosphonium group, phosphate group or said phosphonate group may be linked to the core of said po ⁇ hyrin, chlorin or bacteriochlorin chromophore by way of a linking group L 2 , which linking group L- 2 may comprise a group -R ⁇ - or -R]R 2 -, where each of Ri and R 2 is independently selected from a single bond, or methyl, or phenyl, or branched or linear ethyl, or branched or linear or cyclised propyl, butyl, pentyl, hexyl, heptyl or octyl, optionally substituted one or more times by one or more hydrophilic substituents W as hereinbefore defined.
  • said linking group L 2 may comprise an ether or thioether chain such as a chain -A]R
  • ether or thioether chain such as a chain -A]R
  • said linking group may be a group R ⁇ R 2 wherein said Ri is phenyl and said R 2 is methyl or ethyl or propyl.
  • said linking group may be a group Ri wherein Ri is a single bond.
  • said hydrophilic substituent may advantageously comprise or consist of a group R 11 R.
  • R ⁇ is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl or octyl
  • R 12 is NH, O, S or CH 2
  • R13 is hydrogen or a hydrophilic group W as hereinbefore defined.
  • Said group RnRnR ⁇ may advantageously be substituted one or more times by hydroxy, so as to improve the hydrophilicity of the group R11R12R.3.
  • said Ru may be ethyl.
  • said R 12 may be O and said R1 3 may be H, and said R1 1 R 12 R 13 may be further substituted one or more times by OH, such that said RnR ⁇ ⁇ constitutes a polyhydroxyalkyl, preferably a dihydroxyalkyl.
  • said R ⁇ 3 may be selected from polyethylene glycol; glycosyl such as glucose, mannose, maltose, thiogalactopyranose, thioglucopyranose or thiomannopyranose; lysine; cysteine; tyrosine; aspartate; glutamate; serine and threonine.
  • said conjugating meso substituent may comprise an aryl moiety R ⁇ 0 which is linked to said conjugating group Z.
  • Said aryl moiety R ⁇ 0 may advantageously comprise a phenyl ring, which phenyl ring may preferably be linked by a single bond to the macrocyclic core of said chromophore or may alternatively be linked thereto by a linking group L 2 as hereinbefore defined.
  • said conjugating group Z may be linked to said phenyl ring at the para (4') position thereof.
  • Said conjugating group Z may comprise a group which is capable of bonding covalently to an amine group on a polypeptide molecule; such as an isocyanate, isothiocyanate, or NHS ester group.
  • each of the meso substituents around said po ⁇ hyrin, chlorin or bacteriochlorin should comprise no -NH-, - NH 2 , -NH2 + - or -NH 3 + groups which could become covalently bonded to said conjugating group Z. This will serve to reduce the probability of internal cross-linkage within said chromophore.
  • Said conjugating group Z may alternatively comprise any other protein conjugating group, such as -NH 2 , -NH(C ⁇ - 6 alkyl), maleamide, iodoacetamide, ketone or aldehyde. Methods for achieving the conjugation of such groups to protein molecules are known in the art.
  • said conjugating group Z comprises an isothiocyanato group.
  • Isothiocyanates react readily with lysine residues to produce a stable linkage to proteins, and hence are particularly suitable for bioconjugation of chromophores in accordance with the invention.
  • Said conjugating group Z may be linked directly to said aryl moiety Rio by a single bond.
  • said conjugating group Z may be linked to said aryl moiety Rio by a linking moiety having a relatively high degree of inflexibility and/or steric hindrance.
  • Said linking moiety may, for example, comprise a chain of fused or linked cycloalkyl and/or cycloaryl ring structures having a total molecular weight no greater than lOOOgmol "1 .
  • said linking moiety may comprise an anthracene, acridine, anthranil, napiithyl or naphthalene moiety, or a polyacetylene, phenylacetylene, or polyphenylacetylene moiety.
  • said linking moiety can serve to keep the photoactive core of said chromophore apart from said polypeptide, thereby helping to reduce the degree of fluorescence quenching which may be caused by said polypeptide when said chromophore is caused to fluoresce.
  • Said linking moiety may include a linbking group L 2 as hereinbefore defined.
  • said linking moiety may be substituted one or more times by one or more hydrophilic substituents W as hereinbefore defined, or may include one or more ether or thioether groups -A1R1A2R 2 - as hereinbefore defined. This will help to ensure that the hydrophilicity of the chromophore is not impaired by the presence of said linking moiety.
  • said aryl moiety Rio may be further substituted by one or more hydrophilic substituents, such as hydroxy or oxo, which will serve to improve the hydrophilicity of said chromophore.
  • any of the above-defined groups, substituents or moieties may be further substituted by one or more inert atoms or groupings, such as methyl, ethyl or halo, particularly fluoro, which will not disrupt or substantially affect the properties and functionality of said groups, substituents or moieties.
  • any of the above-defined groups, substituents or moieties may be further substituted one or more times by hydroxy or oxo, so as further to improve hydrophilicity.
  • the total molecular weight of each meso substituent will not exceed lOOOgmol "1 ; more preferably the total molecular weight of each meso substituent will not exceed 700gmol "1 ; still more preferably the total molecular weight of each meso substituent will not exceed SOOgmol "1 ; even still more preferably the total molecular weight of each meso substituent will not exceed 300gmor'.
  • a chromophore in accordance with the invention may be a po ⁇ hyrin chromophore of formula (I) below:
  • Rio is or comprises a conjugating meso substituent as hereinbefore described; at least one of R 2 o, R 30 and R 40 is or comprises a hydrophilic meso substituent as hereinbefore defined; and each of Xi, X 2 , X 3 and X is independently selected from H, OH, halogen, C 1 - 3 alkyl and OC 1 . 3 alkyl, or X] and X 2 and/or X 3 and X 4 together form a bridging moiety selected from O, CH 2 , CH C 1 . 3 alkyl, or C(C ⁇ - 3 alkyl) 2 , such that Xi and X2 and/or X 3 and X with the adjacent C-C bond form an epoxide or cyclopropanyl structure.
  • each or some of X ⁇ - X is H. In particularly preferred embodiments, however, each of Xi - X is OH.
  • said chromophore may be a dihydroxychlorin of formula (II), (III), (IV) or (V) above or a tetrahydroxybacteriochlorin of formula (VI) or (VII) above.
  • the hydrophilicity of dihydroxychlorins and tetrahydroxybacteriochlorins is found to be greater than that of the corresponding po ⁇ hyrins, owing to the presence of extra hydrophilic hydroxy groups around the core of the chromophore.
  • said po ⁇ hyrin, chlorin or bacteriochlorin chromophore will comprise three hydrophilic meso substituents R20. R3o. R 0 and one conjugating meso substituent R ⁇ 0 , such that the chromophore is tetra-meso substituted.
  • Said three hydrophilic meso substituents R 2 o, R30. R ⁇ may be identical one to each other; or may be different one from another.
  • two of said three hydrophilic meso substituents R 20 . R30. R-to may comprise the same group R ⁇ R ⁇ 2 R ⁇ 3 as hereinbefore defined, whilst the remaining one of said three hydrophilic meso substituents may comprise a different hydrophilic substituent in accordance with the invention.
  • hydrophilic meso substituents together with one conjugating meso substituent enables a high degree of hydrophilicity to be imparted to the chromophore.
  • the presence of such substituents can hinder the conversion of po ⁇ hyrins to chlorins and of chlorins to bacteriochlorins within the scope of the invention by way of addition reaction across the exocyclic double bonds around the chromophore core.
  • said R 30 may be a hydrophilic meso substituent as hereinbefore defined, whilst said R 20 and R 0 may each be hydrogen, such that said chromophore is 5,15-di-me-> ⁇ -substituted.
  • each of said R 2 o and said R o is a hydrophilic group R11R12R1 3 as hereinbefore defined, and said R 30 is a group having a total molecular weight less than lOOOgmol "1 , preferably less than 700gmol " ⁇ more preferably less than 500gmol " .
  • said R 3 0 may comprise a phenyl ring, which ring may be substituted one or more times by one or more substituents selected from methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, phenyl, pyridyl, or a hydrophilic group W as hereinbefore defined.
  • Said phenyl ring may be linked directly to the macrocyclic core of said chromophore by way of a single bond, or may be linked thereto by way of a linking group which may comprise methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, phenyl, phenylmethyl, phenylethyl, pyridyl, pyridylmethyl or pyridylethyl.
  • said R 3 0 may comprise a phenyl ring which is linked directly to the macrocyclic core of said chromophore by way of a single bond, and which is para- substituted once by methyl, ethyl, propyl, carboxylate, ethanoate, propanoate, butanoate, sulfonate, methylsulfonate, ethylsulfonate, propylsulfonate, or glycosyl such as glucose, mannose, maltose, thiogalactopyranose, thioglucopyranose or thiomannopyranose.
  • Chromophores in accordance with the invention wherein R 20 , R30. and R-t 0 are identical one to the other may be synthesised in accordance with methods known in the art, for example by acid catalysed condensation of benzaldehydes with pyrrole, or by means of the "MacDonald 2+2" method for synthesising po ⁇ hyrins from dipyrromethanes (Arsenault et al, I. Chem. Soc. 1960, 82:4384-4389 - inco ⁇ orated herein by reference).
  • RX represents a quartenising group selected from CH 2 CH 2 COOH, CH 2 CH 2 OH, CH 2 CH 2 S0 3 H and glycosyl such as glucose, mannose, maltose, thiogalactopyranose, thioglucopyranose or thiomannopyranose:
  • Po ⁇ hyrin, chlorin and bacteriochlorin chromophores in accordance with the present invention wherein at least one of said hydrophilic meso substituents comprises phenylmethylpyridiniumyl may be synthesised in accordance with the generalised reaction scheme set out below as Scheme 3, wherein "R” represents 3,5-(COOH) 2 , 4- CH 2 CH 2 SO 3 H, or 2,6-(CH 2 OH) 2 :
  • Po ⁇ hyrin, chlorin and bacteriochlorin chromophores in accordance with the present invention wherein at least one of said hydrophilic meso substituents comprises alkylphosphonatophenyl or alkylphosphatophenyl may be synthesised in accordance with the generalised reaction scheme set out below as Scheme 4, wherein "R” represents OH, ONa, or 0(C ⁇ - 6 alkyl):
  • a generalised scheme for the production of 5-(4-isothiocyanatophenyl)-10,20- bis(l,2-disubstituted ethyl) po ⁇ hyrin, chlorin and bacteriochlorin is set out in Scheme 6 below, where X represents N, O, S or C; Z represents N or C; R represents polyethylene glycol, glycosyl such as glucose, mannose, maltose, thiogalactopyranose, thioglucopyranose or thiomannopyranose, lysine, cysteine, tyrosine, aspartate, glutamate, serine or threonine; and R 1 represents Me, CH 2 CH 2 COOH, CH 2 CH 2 OH, CH 2 CH 2 S0 3 H or glycosyl such as glucose, mannose, maltose, thiogalactopyranose, thioglucopyranose or thiomannopyranose:
  • a method for synthesising a sugar-substituted meso-a ⁇ y ⁇ po ⁇ hyrin/chlorin/bacteriochlorin comprising the steps of providing a meso-a ⁇ yl po ⁇ hyrin/chlorin/bacteriochlorin in which the meso- aryl substituent is substituted with a leaving group and at least one electron withdrawing group, and reacting this po ⁇ hyrin/chlorin/bacteriochlorin with a nucleophilic sugar such as to displace the leaving group on the meso-ary ⁇ substituent by way of a nucleophilic substitution reaction.
  • said meso-ary ⁇ substituent may be a phenyl ring or a pyridyl ring.
  • Said po ⁇ hyrin, chlorin or bacteriochlorin may comprise one, two, three or four me-jo-aryl substituents, some or each of which may be substituted with a leaving group and at least one electron withdrawing group in accordance with the invention.
  • said nucleophilic sugar acts as a nucleophile in displacing said leaving group from said meso- ⁇ ry] substituent.
  • Said nucleophilic sugar may be a deprotonated sugar such as deprotonated glucose, mannose, or maltose, having an anomeric hydroxyl group, which anomeric hydroxyl group is deprotonated.
  • said nucleophilic sugar may be a thiosugar, such as thiogalactopyranose, thioglucopyranose or thiomannopyranose.
  • Electron withdrawing groups suitable for use in the method of the invention are known in the art. Examples include fluoro, nitro and cyano groups.
  • said aryl substituent may be substituted by more than one electron withdrawing group, such as by 2, 3, 4, or 5 fluoro, nitro and/or cyano groups. This will improve the rate of the nucleophilic substitution reaction.
  • said aryl substituent may be substituted by a plurality of fluoros, such as by 3 or 5 fluoros. The presence of a plurality of fluoros around said meso-aryl substituent will have a minimal effect on the properties and functions of said po ⁇ hyrin, chlorin or bacteriochlorin.
  • Leaving groups suitable for use in the method of the invention are also known in the art. Examples include fluoro, mesylate, tosylate and triflate groups. In preferred embodiments, said aryl substituent may be para-substituted by one leaving group, such as by fluoro.
  • said me-> ⁇ -aryl substituent may be ortho- and/or meta-substituted by a plurality of fluoros by way of electron withdrawing groups, and may be para-substituted by fluoro by way of a leaving group.
  • said meso-aryl substituent may be pentafluoro-substituted.
  • Said n ⁇ cleophilic sugar may comprise protecting groups, such as acyl protecting groups; and said method may further include the step of deprotecting said sugar, such as to remove said protecting groups.
  • the deprotection of said sugar will reduce the solubility of said sugar-substituted po ⁇ hyrin, chlorin or bacteriochlorin in tetrahydrofuran and in dichloromethane, which are solvents used in a number of important reactions including in the formation of NCS groups.
  • said method may further include the step of dissolving said sugar- substituted po ⁇ hyrin, chlorin or bacteriochlorin in tetrahydrofuran or dichloromethane, and forming an NCS group on a meso-aryl substituent of said sugar-substituted po ⁇ hyrin, chlorin or bacteriochlorin, in order to obtain a sugar-substituted NCS-linked meso-aryl po ⁇ hryin, chlorin or bacteriochlorin in accordance with the invention.
  • the chromophores of the invention are novel, and are each capable on excitation of emitting fluorescent light at different and substantially non-overlapping wavelengths.
  • conjugating group Z enables a chromophore in accordance with the invention to be specifically targeted to a specific biological target, thus facilitating control over the localisation of the chromophore in vitro or in vivo.
  • Chromophores in accordance with the invention may therefore be usefully employed in fluorescence analysis and imaging applications (including FACS), or in PDT.
  • a set of fluorochromic markers for multicolour fluorochromic analysis comprising at least two chromophores selected from the group consisting of a po ⁇ hyrin chromophore, a chlorin chromophore and a bacteriochlorin chromophore, each of which chromophores is a chromophore in accordance with the invention and each of which chromophores comprises the same 5, 10, 15 and 20 meso substituents.
  • each of the chromophores in a set in accordance with the present invention will on excitation emit fluorescent light at a different discrete wavelength.
  • all of the chromophores within the set can be excited by a single laser, producing separate emission bands which can be substantially individually resolved.
  • all of the chromophores provided in said set share substantially the same molecular structure, and will accordingly share substantially the same biochemical and physicochemical properties, including substantially the same degree of efficiency of protein conjugation and target delivery under given conditions.
  • a set of chromophores in accordance with the present invention may be usefully employed in fluorescence analysis and sorting applications, including FACS, for the convenient sorting and analysis of several types of cells or other biological targets.
  • the components of such a set may, for example, be introduced to a mixture comprising one or more of said different specific biological targets, under conditions which will allow the delivery of each chromophore to its respective specific biological target; and said mixture may be exposed to light so as to cause said chromophores to fluoresce.
  • a multicolour analysis may then be carried out for identifying the different emission bands produced by each chromophore, thereby permitting counting and visualisation of the location of each of the different biological targets.
  • said conjugating group Z may be conjugated to a delivery protein which is adapted to bind specifically to said biological target.
  • said conjugating group Z may be conjugated to a bridging polypeptide which is adapted to bind to a complementary bridging polypeptide so as to couple said chromophore to said complementary bridging polypeptide.
  • said bridging polypeptide may be bound to said complementary bridging polypeptide, and said complementary bridging polypeptide may comprise or be coupled to or fused with a delivery protein which is adapted to bind specifically to said biological target. Accordingly, said chromophore may be covalently linked to said delivery protein by means of said bridging polypeptide and said complementary bridging polypeptide.
  • a kit comprising a chromophore in accordance with the present invention or a set of chromophores in accordance with the present invention, wherein said chromophore or each chromophore is conjugated to a bridging polypeptide that is adapted to bind to a complementary bridging polypeptide so as to couple the chromophore to said complementary bridging polypeptide; and a construct or plurality of constructs each of which comprises said complementary bridging polypeptide fused or coupled to a delivery protein which is adapted to bind specifically to said biological target; the arrangement being such that said chromophore or each chromophore in the kit is adapted to bind to a different construct in the kit with specificity for said specific biological target, so as to link said or each chromophore to a delivery protein with specificity for said specific biological target.
  • Said delivery protein may, for example, be an antibody such as a monoclonal or polyclonal antibody or a fragment thereof with specificity for a target specific molecule on the surface of said biological target.
  • Panels of antibodies which recognise colorectal cell lines are, for example, commercially available.
  • panels of antibodies against selected cell lines, such as lung cancer cell lines include COR L23, L51, L105 and L283, can be generated using the bacteriophage display library technique, as described in Hoogenboom et al, (2000) Natural and designer binding sites made by phage display technology (Immunology Today) 21(8): 371-378. This technology enables isolation of antibody fragments, such as Mab or scFv fragments, with particular binding activity.
  • the fragments are individually displayed on the surface of phage and can be subsequently characterised and produced in quantity.
  • This technique has been used to raise antibody fragments against human colorectal cell lines (Topping et al, (2000) Isolation of human colorectal tumour reactive antibodies using phage display technology. Int. J. Oncol. 16(1): 187-195).
  • the murine scDv antibody MFE-23 which reacts with high specificity with the carcinoembyronic antigen (CEA/CD66e) (Begent et al, 1996 Nat. Med. 2: 979-984) is suitable for use in connection with the present invention.
  • said antibody may be a phage antibody, that is an antibody expressed on the surface of a bacteriophage.
  • said delivery protein may be a protein which is adapted to bind to one or more cell surface molecules or receptors, such as a serum albumin protein.
  • said delivery protein may comprise a low density lipoprotein, such as a fatty acid chain, which is adapted for insertion into a cell membrane. When conjugated to a chromophore, such a lipoprotein can serve to anchor the chromophore to a cell membrane.
  • Said bridging polypeptide may comprise calmodulin, and said complementary bridging polypeptide may comprise calmodulin binding peptide; or vice versa.
  • said bridging polypeptide may comprise avidin or streptavidin, and said complementary bridging polypeptide may comprise biotin; or vice versa.
  • said or each chromophore in a kit in accordance with the present invention may be conjugated to avidin, and said or each construct may comprise a biotinylated monoclonal antibody with specificity for a target specific molecule on the surface of said biological target.
  • said avidin-linked chromophore when allowed to bind said biotinylated antibody, said chromophore will become firmly linked to said antibody.
  • said or each biotinylated monoclonal antibody in the kit may be selected and/or readily substituted, so as to enable said or each chromophore to be delivered to any desired biological target. Methods for the preparation of monoclonal antibodies and for the biotinylation thereof are well known in the art.
  • a method for attaching a chromophore in accordance with the invention or a set of chromophores in accordance with the invention to said specific biological target or targets comprising the steps of providing a kit in accordance with the present invention, and introducing the components of said kit into the vicinity of said specific biological target or targets, under conditions suitable for enabling the binding of said or each delivery protein to said specific biological target or targets.
  • the components of said kit may be allowed to associate with one another prior to introduction to said target or targets, so as to enable the bridging polypeptide conjugated to said or each chromophore to bind to a complementary bridging polypeptide provided on one of said constructs in the kit. This will ensure that said or each chromophore in the kit is linked to a delivery protein prior to introduction of said chromophore to said target or targets.
  • the components of said kit may be introduced sequentially to said target or targets.
  • said specific biological target may be a cell or a membrane.
  • Said specific biological target may be in vivo or in vitro (ex vivo).
  • Said biological target may, for example, be a cancer cell, a tumour cell, a cell infected with HIV or with any other microbe or virus, a cell responsible for detrimental activity in auto-immune disease, a foreign or diseased cell, or any other such cell.
  • said biological target is a cell in vitro
  • said target specific molecule comprises a molecule exposed on the surface of said cell, such as a polypeptide, carbohydrate, fatty acid, lipoprotein, phospholipid or other biological molecule.
  • said target specific molecule is specifically expressed by, or is over-expressed by, said cell.
  • Said target specific molecule may, for example, be a T cell marker such as CD4 or CD8.
  • a chromophore in accordance with the present invention or a chromophore forming part of a set of chromophores in accordance with the present invention is attached to said cell, and said cell is illuminated so as to cause fluorescence of said chromophore, the fluorescence of the chromophore will enable said cell to be visualised and counted and/or sorted by FACS.
  • a method for fluorescence-activated sorting of target cells from a mixture of cells comprising the step of attaching to said target cells a chromophore in accordance with the invention or a set of chromophores in accordance with the invention, illuminating said mixture of cells so as to cause fluorescence of one or more of said chromophores attached to said target cells, imparting a charge to the fluorescing cells, and passing said mixture of cells through a polarised environment so as to cause or allow said charged cells to be separated from said mixture.
  • a method for the visualisation and/or counting of a plurality of target cells comprising the steps of providing a chromophore set in accordance with the present invention, which chromophore set comprises two or three chromophores each of which is adapted to be delivered to a different one of said cell types; attaching said chromophores in the set to said target cells in accordance with the method of the present invention; illuminating said target cells so as to cause the emission of fluorescence by said chromophores; detecting the fluorescent emission bands produced by each of said chromophores; and optionally measuring for each of said bands the area under an emission/wavelength curve, so as to obtain a measure of the number of fluorescent cells of each respective cell type.
  • said target cell is a cell in vivo, such as a cancer cell, tumour cell, or an infected, foreign or diseased cell
  • said target specific molecule is a target cell specific molecule which is specifically expressed by, or is over-expressed by, or is attached to, and is exposed on, the surface of said target cell; such as a target cell specific membrane protein. Accordingly, when a chromophore in accordance with the invention is delivered to said target specific molecule, said chromophore will be caused to be attached to said cell.
  • said chromophore attached to said cell may be caused to be excited, and this may result in the production of singlet oxygen in the immediate vicinity of said cell, hence bringing about the death of the cell.
  • said target cell specific molecule comprises an internalisation receptor on the surface of said cell, which intemahsation receptor is capable of binding said delivery protein and thereby mediating the internalisation of said chromophore within said cell. Accordingly, subsequent illumination of said cell with light at a wavelength suitable for causing excitation of said chromophore may result in the production of singlet oxygen within said cell, hence bringing about the death of said cell.
  • the present invention therefore comprehends a method for causing the death of a target cell, comprising the step of attaching a chromophore in accordance with the present invention to said cell and illuminating said cell so as to cause the production of singlet oxygen in the vicinity of said cell, thereby causing the death of the cell.
  • said chromophore is attached to an internalisation receptor on the surface of said cell, which internalisation receptor is capable of mediating the internalisation of said chromophore within said cell, and said cell is thereafter illuminated such as to cause the production of singlet oxygen within said cell, thereby causing the death of the cell.
  • said chromophore comprises a cationic or a zwitterionic group such as a quartenised amine or pyridyl (pyridiniumyl) group, or a quartenised phosphonium group, in particular a phosphonium group wherein the phosphorous atom is directly linked to three aryl groups such as three phenyl rings.
  • a cationic or a zwitterionic group such as a quartenised amine or pyridyl (pyridiniumyl) group, or a quartenised phosphonium group, in particular a phosphonium group wherein the phosphorous atom is directly linked to three aryl groups such as three phenyl rings.
  • a method for treating a disease or disorder which is characterised by the presence in the body of diseased or undesired cells, such as tumours, cancers, in particular lung cancer or colorectal cancer, viral infections such as HIV infection, or autoimmune disorders such as rheumatoid arthritis, or a disease or disorder which can be effectively treated by photodynamic therapy such as age related macular degeneration (AMD), comprising the step of administering to a patient in need thereof an effective amount of a chromophore in accordance with the invention, which chromophore is adapted to be targeted to a target cell specific molecule on the surface of said diseased or undesired cells for attachment thereto, such that the chromophore is caused to be attached to said cells, and illuminating said cells with light so as to cause the production of singlet oxygen in the vicinity of said cells, thereby killing said cells.
  • said target cell specific molecule comprises an internalisation receptor, and said chrom
  • Said chromophore may be administered topically or systemically to said patient.
  • said chromophore may be administered by injection.
  • a pharmaceutical composition for administration to a patient for the treatment of a disease or disorder which is characterised by the presence in the body of diseased or undesired cells, such as tumours, cancers, in particular lung cancer or colorectal cancer, viral infections such as HIV infection, or autoimmune disorders such as rheumatoid arthritis, or a disease or disorder which can be effectively treated by photodynamic therapy such as age related macular degeneration (AMD), which composition comprises a chromophore in accordance with the present invention that is adapted to be delivered to said diseased or undesired cells, and a suitable carrier.
  • a disease or disorder which is characterised by the presence in the body of diseased or undesired cells, such as tumours, cancers, in particular lung cancer or colorectal cancer, viral infections such as HIV infection, or autoimmune disorders such as rheumatoid arthritis, or a disease or disorder which can be effectively treated by photodynamic therapy such as age related macular degeneration (AMD), which composition comprises a chrom
  • Yet another aspect of the invention envisages a chromophore in accordance with the invention for use in the production of a medicament, for use in the treatment of patients suffering from a disease or disorder which is characterised by the presence in the body of diseased or undesirable cells, such as tumours, cancers, in particular lung cancer or colorectal cancer, viral infections including HIV infection, and autoimmune disorders including rheumatoid arthritis or a disease or disorder which can be effectively treated by photodynamic therapy such as age related macular degeneration (AMD); said chromophore being adapted for delivery to said diseased or undesired cells.
  • a disease or disorder which is characterised by the presence in the body of diseased or undesirable cells, such as tumours, cancers, in particular lung cancer or colorectal cancer, viral infections including HIV infection, and autoimmune disorders including rheumatoid arthritis or a disease or disorder which can be effectively treated by photodynamic therapy such as age related macular degeneration (AMD); said chromophore being adapted for delivery
  • the DPP was synthesised according to the method of Dolphin et al. (1998 5- Phenyldipyrromethane and 5, 15-Diphenylpo ⁇ hyrin Org. Synth. 76, 287-293)
  • the resulting mixture of three po ⁇ hyrins was chromatographed, eluting initially with DCM to allow removal of 5,15-(4-pyridyl)-DPP, and then continuing with ethyl acetate/DCM (1:4) to elute the required product as pu ⁇ le crystals.
  • the crude pu ⁇ le solid was chromatographed, eluting with DCM, and gave 5-(4- Aminophenyl)-15-(4-methoxyphenyl)po ⁇ hyrin, as a pu ⁇ le crystalline solid.
  • the amino group was protected as follows: To a stirred solution of 5-(4-Aminophenyl)-15-(4- methoxyphenyl)po ⁇ hyrin, (28 mg, 55 ⁇ mol) in anhydrous 1,4-dioxane (2.5 mL) was added solid sodium hydrogen carbonate (6 eq., 28 mg, 0.33 mmol).
  • the required po ⁇ hyrin was obtained by chromatography, eluting with DCM.
  • the desired po ⁇ hyrin was obtained as pu ⁇ le crystals.
  • the resulting (Fmoc) protected po ⁇ hyrin was then used as a stock for the preparation of 5-(4-isothiocyanato)- 15-(4-pyridiniumyl) po ⁇ hyrins, 5-(4-isothiocyanato)-15-(4-pyridiniumyl) dihydroxychlorins and 5-(4-isothiocyanato)-15-(4-pyridiniumyl) tetrahydroxybacteriochlorins as follows: 5-(4-isothiocyanato)-15-(4-pyridiniumyl) po ⁇ hyrin (35 mg, 48.0 ⁇ mol) was converted into the required mixture of chlorin or bacteriochlorin stereoisomers by minor modification of the procedure of Sutton et al.
  • the reaction was stirred under argon for 3 hours at room temperature, monitored by TLC (silica) in a water/saturated aqueous potassium nitrate/acetonitrile (1: 1:8) solvent system. Upon reaction completion excess DMF was evaporated in vacuo (0.1 torr) at 30-40 °C to yield the above compound as a lustrous pu ⁇ le solid.
  • the pyridiniumyl-sugars were deprotected in the following way: After redissolution in a mixture of dichloromethane and methanol (4:1). Sodium methanolate in dry methanol(1.5 equivalents per OAc group) was added and the mixture stirred for 1 hour.
  • the (4- carbomethoxvphenyl) group was converted to a (4-(l-bromomethyl)phenyl) group using the following standard procedure: the po ⁇ hyrin (0.2 mmol) was dissolved in dry THF (25 ml) at 0°C and stirred under argon for 10 minutes. Lithium aluminium hydride (0.7 mmol) was added and the stirring continued for 24 hours. The reaction was monitored by TLC and, when the reaction was complete ethyl acetate (2 ml) was added and the mixture washed with aqueous HC1 (0.2 M, 20 ml), saturated sodium bicarbonate solution (30 ml) and finally, brine (20 ml).
  • the organic layer was dried (MgS0 ) and evaporated to dryness to yield the corresponding (4-(l-hydroxymethyl)phenyl) substituted po ⁇ hyrin.
  • the stock po ⁇ hyrin was then used to generate the corresponding dihydroxychlorin or tetrahydroxybacteriochlorin by treatment with Os0 following the procedure of Sutton et al.(2000 Functionalised diphenylchlorins and bacteriochlorins - their synthesis and bioconjugation for targeted photodynamic therapy and tumour cell imaging I. Porphyrin and Phthalocyanines 4, 655-658).
  • the (4-carbomethoxyphenyl) groups on these po ⁇ hyrins were then converted to (4-(l-bromomethyl)phenyl) groups using the following standard procedure: the po ⁇ hyrin (0.2 mmol) was dissolved in dry THF (25 ml) at 0°C and stirred under argon for 10 minutes. Lithium aluminium hydride (0.7 mmol) was added and the stirring continued for 24 hours. The reaction was monitored by TLC and, when the reaction was complete ethyl acetate (2 ml) was added and the mixture washed with aqueous HC1 (0.2 M, 20 ml), saturated sodium bicarbonate solution (30 ml) and finally, brine (20 ml).
  • the product was then metallated by refluxing in a chloroform/methanol (9: 1) solution of zinc acetate dihydrate (80 mmol). The metallation was followed by visible spectroscopy and, upon completion, was passed through a short column of neutral alumina to remove uncoordinated zinc.
  • the zinc 5,15-dibromo-10, 20-diarylpo ⁇ hyrin (0,6 mmol) was dissolved in dry THF to which had been added tetrakis(triphenylphosphine)-palladium(0) (0.6 mmol) and vinyltributyltin (1.4 mmol).
  • Zinc 5-(Fmoc aminophenyl)-15- aryl-10,20-diethenyl po ⁇ hyrin was demetallated by dissolution in a solution of trifluoroacetic acid in dichloromethane (1% v/v) to give 5-(Fmoc aminophenyl)-15-aryl- 10,20-diethenyl po ⁇ hyrin after extracting with water and evaporation of solvent from the organic layer in vacuo. Finally the 10 and 20 ethenyl groups were hydroxylated by osmium tetroxide as described (Sutton J, Fernandez N, Boyle RW (2000) J.
  • the 15-aryl group can be substituted either before or post hydro cylations depending upon the nature of the substituent to be introduced and example of this is given for a 15-(4-pyridyl) group which can be quarternised post hydroxylations as follows: Stocks of 5-(4-isothiocyanatophenyl)-10,20-(l,2 dihydroxyethyl)-15-(4-pyridyl) po ⁇ hyrin, chlorin or bacteriochlorin, respectively, were split into batches and reacted independently with methyl iodide, 2-bromoethanoic acid, 2- bromoethanol, 2-bromosulphonic acid, 2,3,4,6-tetra-O-acetyl- ⁇ -glucopyranosyl bromide, 2,3,4,6-tetra-O-acetyl- ⁇ -mannopyranosyl bromide, 2,3,4,6-tetra-O-acetyl- ⁇ - galactopyranosyl
  • the reaction was stirred under argon for 3 hours at room temperature, monitored by TLC (silica) in a water/saturated aqueous potassium nitrate/acetonitrile (1:1:8) solvent system. Upon reaction completion excess DMF was evaporated in vacuo (0.1 torr) at 30-40 °C to yield the above compound as a lustrous pu ⁇ le solid.
  • the pyridiniumyl-sugars were deprotected in the following way: After redissolution in a mixture of dichloromethane and methanol (4:1). Sodium methanolate in dry methanol (1.5 equivalents per OAc group) was added and the mixture stirred for 1 hour. The fully deprotected po ⁇ hyrin, chlorin or bacteriochlorin was recovered by precipitation with hexane.
  • methylphosphono-di-ethoxy groups were then deprotected to either methylphosphono-mono-ethoxy sodium groups by sonication in aqueous sodium hydroxide for 1 hour followed by reversed phase medium pressure chromatography (C ⁇ 8 ; gradient elution 0.1% aqueous TFA to methanol), 4 or to the fully deprotected methylphosphonic acids by treatment with bromotrimethylsilane (2 equivalents per methylphosphono-di-ethoxy group) for 2 hours followed by reversed phase chromatographic purification chromatography (C ⁇ 8 ; gradient elution 0.1% aqueous TFA to methanol). 5 Fmoc deprotection (see above) followed by conversion of the unmasked 4-(aminophenyl) group to it's isothiocyanato analogue was performed using standard procedures. 3
  • the residue was purified by reversed phase medium pressure chromatography ( 8 ; gradient elution 0.1% aqueous TFA to methanol) to yield the N-Boc protected 4- aminophenyl compounds.
  • Deprotection of the aminophenyl group(s) and conversion to the isothiocyanato analogue(s) were conducted as follows: The Boc protecting group was removed by dissolution of the po ⁇ hyrin in chloroform or acetonitrile, depending upon solubility, and addition of trimethylsilyl iodide (5.0 equivalents), after 30 minutes the reaction was quenched with methanol (10 ml). Removal of solvent by evaporation, followed by purification by flash column chromatography (silica; gradient: dichloromethane to methanol) gave the corresponding 4-(aminophenyl) po ⁇ hyrin in quantitative yield.
  • the product was then metallated by refluxing in a chloroform/methanol (9:1) solution of zinc acetate dihydrate (80 mmol). The metallation was followed by visible spectroscopy and, upon completion, was passed through a short column of neutral alumina to remove uncoordinated zinc.
  • the zinc 5,15-dibromo-10, 20-diarylpo ⁇ hyrin (0,6 mmol) was dissolved in dry THF to which had been added tetrakis(triphenylphosphine)-palladium(0) (0.6 mmol) and vinyltributyltin (1.4 mmol).
  • Zinc 5-(Fmoc aminophenyl)- 15- aryl- 10,20-diethenyl po ⁇ hyrin was demetallated by dissolution in a solution of t ⁇ fluoroacetic acid in dichloromethane (1% v/v) to give 5-(Fmoc aminophenyl)- 15-aryl- 10,20-d ⁇ ethenyl po ⁇ hy ⁇ n after extracting with water and evaporation of solvent from the organic layer in vacuo
  • MCPBA meta-perchlorobenzoic acid
  • the reaction was stirred under argon for 3 hours at room temperature, monitored by TLC (normal phase silica) in a water/saturated aqueous potassium nitrate/acetonitrile (1 : 1:8) solvent system. Upon reaction completion excess DMF was evaporated in vacuo (0.1 ton) at 30-40 °C to yield the above compound as a lustrous pu ⁇ le solid.
  • the pyridiniumyl-sugars were deprotected in the following way: After redissolution in a mixture of dichloromethane and methanol (4: 1). Sodium methanolate in dry methanol (1.5 equivalents per OAc group) was added and the mixture stirred for 1 hour. The fully deprotected po ⁇ hyrin, chlorin or bacteriochlorin was recovered by precipitation with hexane.
  • the solid was re- dissolved in a mixture of DCM/triethylamine (9: 1) (200 mL) and stirred for 10 min at room temperature. The solution was then washed with water (3 x 200 mL), saturated brine (200 mL) and the organic layer separated and dried (Na2S0 ), then concentrated in vacuo. The crude pu ⁇ le solid was chromatographed, eluting with DCM, and gave 5-(4- Aminophenyl)-15-(4-methoxyphenyl)po ⁇ hyrin, as a pu ⁇ le crystalline solid.
  • the (4- carbomethoxyphenyl) group was converted to a (4-(l-bromomethyl)phenyl) group using the following standard procedure: the po ⁇ hyrin (0.2 mmol) was dissolved in dry THF (25 ml) at 0°C and stirred under argon for 10 minutes. Lithium aluminium hydride (0.7 mmol) was added and the stirring continued for 24 hours. The reaction was monitored by TLC and, when the reaction was complete ethyl acetate (2 ml) was added and the mixture washed with aqueous HCl (0.2 M, 20 ml), saturated sodium bicarbonate solution (30 ml) and finally, brine (20 ml).
  • the organic layer was dried (MgS0 4 ) and evaporated to dryness to yield the corresponding (4-(l-hydroxymethyl)phenyl) substituted po ⁇ hyrin.
  • the stock po ⁇ hyrin was then used to generate the corresponding dihydroxychlorin or tetrahydroxybacteriochlorin by treatment with OsO following the procedure of Sutton et ⁇ /.(2000 Functionalised diphenylchlorins and bacteriochlorins - their synthesis and bioconjugation for targeted photodynamic therapy and tumour cell imaging I. Porphyrin and Phthalocyanines 4, 655-658).
  • the resulting sugar substituted po ⁇ hyrins were deprotected in the following way: After redissolution in a mixture of dichloromethane and methanol (4: 1). Sodium methanolate in dry methanol(1.5 equivalents per OAc group) was added and the mixture stirred for 1 hour. The fully deprotected po ⁇ hyrin, chlorin or bacteriochlorin was recovered by precipitation with hexane. Finally the Boc protecting group was removed by dissolution of the po ⁇ hyrin in chloroform or acetonitrile, depending upon solubility, and addition of trimethylsilyl iodide (5.0 equivalents), after 30 minutes the reaction was quenched with methanol (10 ml). Removal of solvent by evaporation, followed by purification by flash column chromatography (C ⁇ 8 silica; gradient: dichloromethane to methanol) gave the corresponding 4-(aminophenyl) po ⁇ hyrins.
  • Pentafluorobenzaldehyde was condensed with 4-nitrobenzaldehyde and pyrrole using Lindsey conditions or Alder Longo procedure and the crude reaction mixture purified by column chromatography to give 5-(4-nitrophenyl)-10,15,20-tri(pentafluorophenyl)- po ⁇ hyrin.
  • 5-(4-nitrophenyl)dipyrromethane and 5-(pentafluorophenyl)dipyrromethane were condensed to give 5-(4-nitrophenyl)-15-( pentafluorophenyl)-po ⁇ hyrin.
  • the crude reaction mixture was purified by flash column chromatography. Reduction of the nitro group of these po ⁇ hyrins was performed by dissolution in THF and addition of 10% palladium on carbon. Stirring of the mixture under H 2 for 5 hours followed by filtration through Celite and purification by flash column chromatography gave the corresponding amino po ⁇ hyrins, which were N-protected by reaction with Fmoc chloride (2 equivalents) in anhydrous 1,4-dioxane in the presence of sodium bicarbonate (6 equivalents) under argon. The reaction was monitored by TLC and, upon completion, diluted with dichloromethane and washed with water then brine before drying the organic layer (MgS0 ).
  • the core po ⁇ hyrin was either deprotected (see below) or converted to the corresponding di hydroxychlorin or tetrahydroxybacteriochlorin by treatment with Os0 4 followed by reduction of the intermediate osmate ester with H 2 S, as described using the standard procedure described for other 5,15-diphenylpo ⁇ hyrins.
  • N and O protecting groups were removed by dissolution of the po ⁇ hyrin in dichloromethane/mo ⁇ holine (1/1) and stirring for 1 hour. Removal the solvent by evaporation in vacuo was followed by redissolution of the residue in a mixture of dichloromethane and methanol (4/1).
  • a stock solution of a PITC in accordance with the invention in DMSO was prepared to a molarity of 0.027, this solution was desiccated and stored at 0°C until required.
  • a solution of antibody was extensively dialysed against sterilised PBS to remove any trace of azide. The dialysed antibody solution was then adjusted to a concentration of 10 mg/mL via centrifugal concentration and separated into 250 ⁇ L aliquots.
  • a I M solution of sodium bicarbonate was prepared and adjusted to pH 9.0 with 2 M sodium hydroxide.
  • a 250 ⁇ L aliquot of antibody was added 30 ⁇ L of 1 M sodium bicarbonate.
  • a predetermined volume of PITC stock solution was then added to give a desired molar ratio (MR) of po ⁇ hyrin to antibody.
  • MR molar ratio
  • an MR of 20 was achieved via the addition of 10 ⁇ L of stock solution to 250 ⁇ L of antibody at 10 mg/mL.
  • all aliquots of stock solution were diluted to 25 ⁇ L with further portions of DMSO.
  • the bioconjugation reaction was agitated gently for 1 hour at 25°C. After 1 hour the crude bioconjugation reaction mixture was loaded directly onto the top of a prepacked PD10 size exclusion column pre-equilibrated with sterile PBS (25 mL). The column was eluted with sterile PBS. Antibody-po ⁇ hyrin conjugate was eluted in the first coloured band/fraction. The antibody-po ⁇ hyrin conjugate concentration following dilution during chromatography was determined as 1.25 mg/mL. The degree of labelling (DOL) of po ⁇ hyrin to antibody was calculated via standard spectroscopic methods using known constants of molar abso ⁇ tivity for both po ⁇ hyrin and protein.
  • DOL degree of labelling
  • Antibody-po ⁇ hyrin conjugates were stored, without further concentration, in PBS + azide at 0°C unless otherwise stated.
  • Cells are grown to confluence or appropriate density then washed 2 times with PBS (phosphate buffered saline) to eliminate all trace of FBS (fcetal bovine serum). Cell density is adjusted to 1.5x106 cells/ml in medium without FBS and these are then incubated for 1 hour in the dark (37 degrees C, 5% C02 ) with a range of photosensitiser/conjugate concentrations. Post incubation, cells are washed further with medium (without FBS )to eliminate unbound photosensitiser, then resuspended and seeded in 96 wells plates (lxlO 5 cells/well) in quadruplate.
  • PBS phosphate buffered saline
  • Plates are then either irradiated (3.6J/cm2 of filtered red light >600nm ) or left in the dark as "dark toxicity controls" for the same period of time (-14 minutes).Five microliters (5%/well) of FBS is added after the irradiation/dark period and the plates are returned to the incubator overnight. Twenty to 24 hours after treatment, 10 ⁇ l of MTT solution (Sigma Thiazolyl blue, 4.8x10 " ⁇ in PBS)is added per well and the plates are returned to the incubator until color develops (between 1 and 4 hours). A solution of acid-alcohol (lOO ⁇ l/well of 0.04N HCL in isopropanol) is the added and mixed thoroughly to dissolve the dark blue crystals. Plates are then read at 570nm in a microplate reader and the % cell survival calculated against controls.
  • MTT solution Sigma Thiazolyl blue, 4.8x10 " ⁇ in PBS
  • the two fluorochromic probes were generated from separate reactions of 2,3- dihydroxy-5-(4-methylphosphoniumtriethyl)-15-(4-isothiocyanatophenyl)chlorin (higher R f regioisomer) and 2,3, 12,13-tetrahydroxy-5-(4-isothionatophenyl)-15-(4- methylphosphoniumtriethyl) bacteriochlorin (lower R f cis stereoisomer) with avidin under the standard bioconjugation protocols given earlier.
  • Biorad Protean 2 equipment was used in accordance with manufacturer's instructions
  • Samples (total volume 15-20 ⁇ L containing 1-10 ⁇ g sample protein) were loaded onto a gel.
  • Target antibodies and antigens All antibodies are purchased from Serotec Ltd, Kidlington , Oxford, 0X5 1JE
  • CD 104 ( ⁇ -4-INTEGRIN)
  • ⁇ -4-Integrin is a 205kDa glycoprotein member of the integrin family that associates with ⁇ 6 integrin to form the ⁇ 6 ⁇ 4 complex.
  • Integrins are cell surface adhesion molecules that interact with the extracellular matrix and affect cellular mo ⁇ hology and function. The polarized distribution of integrins is often lost in carcinomas and there have been several reports that ⁇ 6 ⁇ 4 integrin is upregulated in skin, colorectal and bladder carcinomas. It has also been reported that an increase in ⁇ 6 ⁇ 4-integrin leads to a more invasive, and hence aggressive, phenotype in colorectal carcinoma.
  • EpCAM Epithelial cell adhesion molecule
  • 17-1A is the most widely used antibody and has been as adjuvant treatment following surgery to kill residual tumour cells.
  • Phage scFv antibodies have been generated against EpCAM to give better tumour penetration and retention; these antibodies have also been conjugated to give a T-cell/EpCAM bispecific antibody.
  • the GA733 and 323/A3 antibodies have also undergone trials but as they are of higher affinity than 17-1 A they cause more damage to normal cells.
  • the 17-1A antibody has been conjugated to photodynamic molecules and used in PDT by us and others.
  • MUC-18 (also called Mel-CAM) is a 100-1 lOkDa cell adhesion molecule which is a member of the immunoglobulin gene superfamily. MUC-18 is not expressed in most normal tissues, except in basal cells of bronchial epithelium and endothelial cells. It has been found up-regulated on the surface of numerous tumour types including malignant melanoma and is thought to promote tumour progression, implantation and metastases. Conversely, in some breast cancers low levels of MUC-18 expression were associated with good prognosis. MUC-18 antibodies may have applications in tumour targeting due to the protein core being exposed after loss of the carbohydrate moieties from the molecule during malignancy. A MUC-18 antibody, ABX-MA1, has been shown to inhibit melanoma tumour growth and metastases.
  • the CD4 molecule is a single chain transmembrane glycoprotein of 59kDa expressed principally on helper/inducer T cells and monocytes.
  • the CD4 molecule plays a role in the T cell receptor binding of Class II MHC complexes that are found on cells involved in immune regulation, e.g. dendritic cells, B cells and macrophages.
  • the CD8 molecule is composed of two glycoprotein chains and has a molecular weight of 32kDa. It is expressed on cytotoxic T cells and NK cells. The CD8 molecule plays a role in the T cell receptor binding of Class I MHC complexes that are found on a large number of human cells.
  • HEPP-NCS was prepared as described in Example 1 above.
  • Antibody 17.1 A was selected for the bioconjugation procedure.
  • 17.1 A is an antibody which reacts specifically with a receptor that is over-expressed on colorectal cancer cells, in particular Colo 320 cells (ECACC, deposit no. 87061205).
  • ECACC Colo 320 cells
  • any antibody which reacts against any antigen that is over-expressed on a suitable cell line may be utilised in accordance with the invention.
  • Examples of such antibodies include Ber-EP4 and MOK-31, each of which is commercially available from DAKO Ltd, Ely, Cambridgeshire, and each of which is reactive against an antigen that is over-expressed on epithelial cells.
  • the monoclonal antibody preparation was either buffer-exchanged from a phosphate to an acetate buffer using a Centricon centrifuge or was subjected to dialysis so as to exchange the phosphate buffer for an acetate buffer.
  • HEPP-NCS was conjugated with 17.1 A monoclonal antibody in accordance with the method described in Methodology Description 1, to obtain a range of conjugation dilutions having respective MRs of 2.5, 5, 10 and 20..
  • OX-34 has been found to lack specificity for any antigens expressed on the surface of Colo 320 cells. Accordingly, as expected these control experiments show no photocytotoxicity following irradiation.
  • Protocols for performing and assessing photodynamic therapy in vivo, utilising the conjugates of the invention are variously described in R Boyle et al, Br. I. Cancer (1992) 65:813-817; R Boyle et al, Br. I. Cancer (1993) 67:1177-1181; R Boyle et al, Br. I. Cancer (1996) 73:49-53; and Lapointe et al, I. Nuclear Medicine, Vol. 40, No. 5 (May 1999) 876-882; the contents of each of which are inco ⁇ orated herein by reference.
  • tumours may be induced or transplanted into animals such as mice, and the animal may then be injected with a quantity of photosensitiser in accordance with the invention conjugated to an antibody with specificity for an antigen which is specifically expressed or over-expressed on the surface of the tumour cells. Thereafter, the animal may be subjected to irradiation, and the effects on the tumour assessed, qualitatively or metrically, with reference to tumour metabolism (as described in Lapointe et al, I. Nuclear Medicine, Vol. 40, No. 5 (May 1999) 876- 882). As described in R Boyle et al, Br. I. Cancer (1996) 73:49-53, the distribution of the photosensitiser in vivo may also be measured, by biodistribution and/or vascular stasis assays.
  • Fluorescence images of cells were obtained with a Bio-Rad Rad ⁇ ance2000 confocal laser scanning microscope (Bio-Rad Microscience, Camb ⁇ dge, MA) on an inverted Olympus 1X70 microscope using a 60x (NA 1.4) oil immersion objective lens.
  • the illumination source was the 514 nm line from a 25 mW argon ion laser.
  • Po ⁇ hy ⁇ ns were visualised with a 514 nm band-pass excitation filter, a 510 nm dichroic mirror, and a 570 nm long-pass emission filter.
  • Each field of cells was sectioned 3-d ⁇ mens ⁇ onally by recording images from a se ⁇ es of focal planes. Movement from one focal plane to another was achieved by a stepper motor attached to the fine focus control of the microscope, the step sizes (in the range 0.5 ⁇ m to 1 25 ⁇ m) being chosen with regard to the aperture size being used, so that there would be some overlap between adjacent sections. Enough vertical sections were taken so that the tops and bottoms of all the cells in each field would be recorded. Each image collected was the average of four scans at the confocal microscope's normal scan rate.
  • FIG. 14 shows the UV-visible spectrum of HEPP-NCS identifying its p ⁇ ncipal abso ⁇ tion bands. Unfortunately, no laser line was available in order to excite HEPP-NCS at its Soret band ⁇ max .
  • Figure 15 demonstrates the relative intensities of fluorescence emission for HEPP-NCS when excited at 422 nm (optimal), and at the four wavelengths of the argon ion laser, 457, 476, 488, and 514 nm.
  • FIG. 16 A Z-series fluorescence image of HeLa cells incubated with HEPP-NCS-BSA is shown in Figure 16 (this Figure should be viewed from top left to bottom right). Consecutive sections were scanned with a 2 ⁇ M step between each focal plane resolved by the microscope, thus enabling three dimensional visualisation of the localisation of the conjugate within the cell. Clearly the conjugate HEPP-NCS-BSA had entered the cell, no studies of the nature of cellular uptake were conducted, however it is most likely that uptake had taken place via endocytosis. It can be seen that the conjugate has not entered the nucleus and appears to be largely distributed throughout the cytoplasm.

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Abstract

La présente invention concerne un chromophore de porphyrine, chlorine ou bactériochlorine qui comprend un substituant méso de conjugaison comprenant un groupe de conjugaison Z destiné à conjuguer par covalence ledit chromophore avec une protéine de façon à permettre la délivrance du chromophore sur une cible biologique choisie in vitro ou in vivo, et un, deux ou trois substituants méso hydrophiles, l'hydrophilité du chromophore étant telle que : lorsque l'on effectue la conjugaison dudit chromophore avec ladite protéine en incubant le chromophore et la protéine pendant une heure dans des conditions normales de conjugaison de protéines dans 10 % DMSO/eau tamponnés à un pH de 9, le pourcentage de chromophore non lié par covalence est inférieur ou égal à 5 % du total du chromophore lié à la protéine ; ou bien lorsque ladite protéine est une albumine de sérum bovin, lors de la conjugaison dudit chromophore avec ladite protéine que l'on effectue en incubant pendant une heure dans des conditions normales de conjugaison de protéines dans 10 % DMSO/eau tamponnés à un pH de 9, le pourcentage de chromophore non lié par covalence est inférieur ou égal à 20 % du total du chromophore lié à la protéine. L'invention concerne également des procédés de synthèse des chromophores précités et des procédés d'utilisation desdits chromophores dans la thérapie et l'analyse.
PCT/GB2002/005867 2001-12-21 2002-12-20 Chromophore conjugue de porphyrine, chlorine ou bacteriochlorine WO2003055887A1 (fr)

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WO2005120573A2 (fr) * 2004-06-07 2005-12-22 Yeda Research And Development Co. Ltd. Derives de bacteriochlorophylles cationiques et utilisation de ceux-ci
WO2006107598A2 (fr) * 2005-04-01 2006-10-12 Allergan, Inc. Agents et methodes visant a ameliorer la therapie photodynamique
WO2010033678A3 (fr) * 2008-09-18 2010-07-01 Ceramoptec Industries, Inc. Nouveau procédé et application de porphyrines méso-substituées de façon non symétrique et de chlorines pour thérapie photodynamique
EP2438847A3 (fr) * 2004-06-18 2012-11-07 David R. Elmaleh Agent de contraste et son utilisation dans un dispositif d'imagerie intravasculaire
CZ306495B6 (cs) * 2014-09-16 2017-02-15 Vysoká škola chemicko- technologická v Praze 5,15-Bis-pentafluorfenylporfyrin substituovaný v polohách 10 a 20 perbenzylovanými molekulami sacharosy jako substrát matricí asistované supramolekulární amplifikace chirality
CN109651383B (zh) * 2019-01-25 2021-11-16 华东理工大学 用于光敏剂的化合物及其应用
CN114206388A (zh) * 2019-05-24 2022-03-18 株式会社糖锁工学研究所 新的人工蛋白质催化剂

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WO2005120573A3 (fr) * 2004-06-07 2006-01-26 Yeda Res & Dev Derives de bacteriochlorophylles cationiques et utilisation de ceux-ci
WO2005120573A2 (fr) * 2004-06-07 2005-12-22 Yeda Research And Development Co. Ltd. Derives de bacteriochlorophylles cationiques et utilisation de ceux-ci
AU2005251556B2 (en) * 2004-06-07 2011-04-14 Yeda Research And Development Co. Ltd. Cationic bacteriochlorophyll derivatives and uses thereof
EP2322173A1 (fr) * 2004-06-07 2011-05-18 Yeda Research And Development Co., Ltd. Dérivés de bactériochlorophylles cationiques et utilisations associées
NO339304B1 (no) * 2004-06-07 2016-11-21 Yeda Res And Development Company Ltd Kationiske bakterieklorofyllderivater, farmasøytiske sammensetninger inneholdende slike, og anvendelse derav.
CN1980661B (zh) * 2004-06-07 2011-09-28 耶达研究及发展有限公司 阳离子细菌叶绿素衍生物及其用途
US8207154B2 (en) 2004-06-07 2012-06-26 Yeda Research And Development Co., Ltd. Catatonic bacteriochlorophyll derivatives
EP2438847A3 (fr) * 2004-06-18 2012-11-07 David R. Elmaleh Agent de contraste et son utilisation dans un dispositif d'imagerie intravasculaire
WO2006107598A2 (fr) * 2005-04-01 2006-10-12 Allergan, Inc. Agents et methodes visant a ameliorer la therapie photodynamique
WO2006107598A3 (fr) * 2005-04-01 2007-07-12 Allergan Inc Agents et methodes visant a ameliorer la therapie photodynamique
WO2010033678A3 (fr) * 2008-09-18 2010-07-01 Ceramoptec Industries, Inc. Nouveau procédé et application de porphyrines méso-substituées de façon non symétrique et de chlorines pour thérapie photodynamique
RU2574062C2 (ru) * 2008-09-18 2016-02-10 Биолитек Унтернеменсбетайлигунгс 11 Аг НОВЫЙ СПОСОБ И ПРИМЕНЕНИЕ НЕСИММЕТРИЧНО мезо -ЗАМЕЩЕННЫХ ПОРФИРИНОВ И ХЛОРИНОВ ДЛЯ ФДТ
RU2574062C9 (ru) * 2008-09-18 2016-10-27 Биолитек Унтернеменсбетайлигунгс Ii Аг НОВЫЙ СПОСОБ И ПРИМЕНЕНИЕ НЕСИММЕТРИЧНО мезо-ЗАМЕЩЕННЫХ ПОРФИРИНОВ И ХЛОРИНОВ ДЛЯ ФДТ
CN102159569A (zh) * 2008-09-18 2011-08-17 塞拉莫普泰克工业公司 用于pdt的不对称内消旋取代的卟啉和二氢卟酚的新方法及其应用
EP3459955A1 (fr) * 2008-09-18 2019-03-27 biolitec Unternehmensbeteiligungs II AG Nouveau procédé et application de porphyrines méso-substituées de façon non symétrique et de chlorines pour thérapie photodynamique
CZ306495B6 (cs) * 2014-09-16 2017-02-15 Vysoká škola chemicko- technologická v Praze 5,15-Bis-pentafluorfenylporfyrin substituovaný v polohách 10 a 20 perbenzylovanými molekulami sacharosy jako substrát matricí asistované supramolekulární amplifikace chirality
CN109651383B (zh) * 2019-01-25 2021-11-16 华东理工大学 用于光敏剂的化合物及其应用
CN114206388A (zh) * 2019-05-24 2022-03-18 株式会社糖锁工学研究所 新的人工蛋白质催化剂

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