EP1294726A2 - Verbesserungen in bezug auf chromophore - Google Patents

Verbesserungen in bezug auf chromophore

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Publication number
EP1294726A2
EP1294726A2 EP01949625A EP01949625A EP1294726A2 EP 1294726 A2 EP1294726 A2 EP 1294726A2 EP 01949625 A EP01949625 A EP 01949625A EP 01949625 A EP01949625 A EP 01949625A EP 1294726 A2 EP1294726 A2 EP 1294726A2
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EP
European Patent Office
Prior art keywords
chromophore
alkyl
chromophores
porphyrin
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP01949625A
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English (en)
French (fr)
Inventor
Ross William Boyle
Oliver James Clarke
Jonathan Mark Sutton
John Greenman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wellcome Trust Ltd
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Catalyst Biomedica Ltd
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Publication date
Priority claimed from GB0113784A external-priority patent/GB0113784D0/en
Application filed by Catalyst Biomedica Ltd filed Critical Catalyst Biomedica Ltd
Publication of EP1294726A2 publication Critical patent/EP1294726A2/de
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0036—Porphyrins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
    • B01D15/424—Elution mode
    • B01D15/426—Specific type of solvent
    • 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
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K9/00—Tenebrescent materials, i.e. materials for which the range of wavelengths for energy absorption is changed as a result of excitation by some form of energy
    • C09K9/02—Organic tenebrescent materials

Definitions

  • the present invention relates to novel porphyrin and porphyrin-based chromophores and sets of porphyrin and porphyrin-based chromophores, which may be particularly useful in a range of photodynamic applications, including photochemotherapy and fluorescence analysis and imaging.
  • porphyrin and porphyrin-based chromophores both as research tools, for example in fluorescence-activated cell sorting (FACS), and as therapeutic agents in photodynamic therapy (PDT) f ⁇ f bringing about the death of targeted cells in vivo, is widely recognised in the art.
  • FACS fluorescence-activated cell sorting
  • PDT photodynamic therapy
  • 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.
  • Photofrin® a photosensitising agent comprising a mixture of porphyrin structures derived from hematoporphyrin-IX by treatment with acids which is commercially used in the treatment of carcinomas and sarcomas, is, for example, conventionally administered systemically to patients without any targeting vehicle or means. This is evidently undesirable, as incorrect localisation of the photosensitiser will not only decrease the efficiency of the photochemotherapy, but may also result in the death of healthy cells.
  • R 2 is a hydrophilic aryl moiety
  • R 3 is H or a hydrophilic aryl or hydrophilic non-aromatic moiety
  • each of Xi, X 2 , X 3 and X 4 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 together form a bridging moiety selected from O, CH 2 , CH Ci alkyl, or C(C ⁇ -3 alkyl) 2 , such that Xi and X 2 and/or X 3 and X with the adjacent C-C bond form an epoxide or cyclopropanyl structure; wherein each of said Rj, R
  • hydrophilic substituents around the core of a chromophore in accordance with the invention 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 nonspecific transfer of chromophore to cell surfaces, which will substantially increase the accuracy of targeting the chromophore to the cells or tissue of interest.
  • a method for separating a mixture which comprises one or more hydrophilic chromophores each having a hydrophilic or amphiphilic moiety, and a plurality of less hydrophilic substances and/or molecules, comprising the step of introducing said mixture to a hydrophobic eluting solvent, and passing said mixture and said eluting solvent over a hydrophilic or polar solid phase, such that said one or more chromophores are arrested on said solid phase whilst said substances and/or molecules are eluted or substantially eluted from said solid phase by said eluting solvent.
  • Said method may, for example, comprise chromatography on a Sephadex® (dextran) column, or reverse-phase HPLC.
  • said mixture of less hydrophilic substances and/or molecules may comprise a mixture of cells and/or membranes.
  • said one or more hydrophilic chromophores include one or more chromophores in accordance with the present invention.
  • each or some of Xi- X4 is H. In particularly preferred embodiments, however, each of Xi - X 4 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 porphyrins, owing to the presence of extra hydrophilic hydroxy groups around the core of the chromophore.
  • said aryl moiety Rj may 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 C1.6 branched or linear alkyl chain.
  • 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 porphyrin, chlorin or bacteriochlorin should comprise no -NH-, - NH 2 , -NH 2 + - 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 Ri by a single bond.
  • said conjugating group Z may be linked to said aryl moiety Ri 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, naphthyl 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 hydrophilic or amphiphilic moiety of the kind described above, such as a C 2 -C 30 polyethylene glycol moiety. This will help to ensure that the hydrophilicity of the chromophore is not impaired by the presence of said linking moiety.
  • said aryl moiety Ri may be further substituted by one or more hydrophilic substituents, such as hydroxy, which will serve to improve the hydrophilicity of said chromophore.
  • Said hydrophilic aryl moiety R 2 may comprise a phenyl ring, which phenyl ring may be substituted one or more times, preferably at least two times, by one or more hydrophilic substituents which serve to increase the hydrophilicity of said aryl moiety R 2 .
  • Said 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 C e branched or linear alkyl chain.
  • said hydrophilic aryl moiety R 2 may comprise a heteroaryl ring, such as a pyridyl or quaternised pyridyl (pyridiniumyl) ring, which heteroaryl ring may be substituted one or more times, preferably at least two times, by one or more hydrophilic substituents which serve to increase the hydrophilicity of said aryl moiety R .
  • Said heteroaryl ring may preferably be linked by a single bond to the macrocyclic core of said chromophore or may alternatively be linked thereto by a C ⁇ -6 branched or linear alkyl chain.
  • Said one or more hydrophilic substituents may advantageously be selected from hydroxy; alkoxy such as methoxy or ethoxy; C 2 - 5 polyethylene glycol; quatenised pyridyl (pyridiniumyl) such as N-methylpyridiniumyl; mono-, di- or poly-saccharide; Ci. ealkyl sulfonate; a phosphonium group R 4 P(R 5 )(R 6 )(R7), wherein R 4 is a single bond or Ci.
  • each of R 5 , Rg and R 7 is independently selected from hydrogen, an aryl ring such as a phenyl ring, a heteroaryl ring such as a pyridyl ring, and a C 1 - 6 alkyl chain, which aryl ring, heteroaryl ring or C ⁇ .s alkyl chain is unsubstituted or is substituted one or more times by hydroxy, C t .
  • each of said R 5 , Rg and R 7 may be the same, and may advantageously be unsubstituted phenyl.
  • said R 8 may be methyl.
  • said R 9 and said Rio may be the same, and/or may be methyl or ethyl.
  • said hydrophilic aryl moiety R 2 is selected from m,m-(dihydroxy)phenyl
  • EtO OEt m- or p-(C ⁇ . 6 alkylphosphonato-di-alkoxy)phenyl such as p-methylphosphonato- di-ethoxy)phenyl
  • meta- or para- sugar-substituted phenyl such as pentose-, hexose- or disaccharide-substituted phenyl
  • said hydrophilic aryl moiety R 2 comprises a quaternised pyridyl (pyridiniumyl) group such as a p-N-(C ⁇ - 6 alkyl)pyridiniumyl group or m-N-(C ⁇ . ⁇ alkyl)pyridiniumyl group.
  • Quaternised pyridyl (pyridiniumyl) groups are highly hydrophilic and display advantageous properties when incorporated into chromophores in accordance with the invention.
  • Particularly preferred groups in this regard are m- or p-N-((C ⁇ - 6 )alkyl)pyridiniumyl, such as m-N-methylpyridiniumyl or p-N-methylpyridiniumyl
  • said quaternised pyridiniumyl group may comprise a zwitterionic group, such as p-N-(C ⁇ -6alkylsulfonate)pyridiniumyl or m- N-(Ci. 6 alkylsulfonate)pyridiniumyl; in particular, p-N-(propylsulfonate)pyridiniumyl
  • the or each quaternised pyridiniumyl group R 2 may be associated with a halide counterion, such as an iodide counterion .or, in most preferred embodiments, a chloride counterion.
  • a halide counterion such as an iodide counterion .or, in most preferred embodiments, a chloride counterion.
  • R 3 is H, such that said chromophore constitutes a 5, 15-diaryl-porphyrin, -chlorin or -bacteriochlorin.
  • said R 3 is a hydrophilic aryl or non-aromatic moiety.
  • said R 3 may comprise a hydrophilic aryl moiety as defined above in relation to R .
  • Said hydrophilic aryl moiety R 3 may be the same as said hydrophilic aryl moiety R 2 , such that the chromophore possesses the same substituents at the 10, 15 and 20 positions thereof; or may be different from said hydrophilic aryl moiety R 2 .
  • said R 3 may comprise a hydrophilic alkyl moiety, such as a C 1-6 alkyl chain which is substituted one or more times by one or more hydrophilic substituents such as hydroxy or C 2- ⁇ s polyethylene glycol
  • said R 3 comprises polyhydroxy(C ⁇ -6 alkyl), such as 1,2-dihydroxy ethyl
  • Chromophores in accordance with the invention wherein R 2 is the same as R 3 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 porphyrins from dipyrromethanes (Arsenault et al, J. Chem. Soc. 1960, 82 4384-4389 - incorporated herein by reference)
  • Scheme 1 A generalised scheme for the synthesis of .5-isothiocyanatophenyl-15- methylphosphoniumphenyl porphyrins, chlorins and bacteriochlorins in accordance with the present invention is set out as Scheme 2 below, wherein R represents hydrogen, C e alkyl, a heterocyclic group or an aromatic group
  • Porphyrin, chlorin and bacteriochlorin chromophores in accordance with the present invention wherein said R 2 and optionally said R 3 comprises pyridiniumylphenyl may be synthesised in accordance with the generalised reaction scheme set out below as Scheme 3, wherein "R” represents hydrogen or one or more hydrophilic substituents as defined above in relation to formulas (I) to (VII) :
  • Porphyrin, chlorin and bacteriochlorin chromophores in accordance with the present invention wherein said R 2 and optionally said R 3 comprise alkylphosphonatophenyl or alkylphosphonophenyl may be synthesised in accordance with the generalised reaction scheme set out below as Scheme 4, wherein "R” represents OH, ONa, or O(C i- 6 alkyl):
  • R i3 is vinyl or aryl, such as a hydrophilic aryl moiety as hereinbefore defined in relation to R 3 ; and reacting said chromophore with said coupling reagent in the presence of a base selected from potassium phosphate, sodium phosphate, caesium carbonate and barium hydroxide, and a Pdo catalyst; such that said R ⁇ 3 replaces said leaving group Q at the 10- and 20- meso positions of said chromophore.
  • a base selected from potassium phosphate, sodium phosphate, caesium carbonate and barium hydroxide, and a Pdo catalyst
  • Suzuki-coupling proceeds rapidly and successfully at the 10- and 20- meso-positions of the starting porphyrin, chlorin or bacteriochlorin chromophore.
  • This method thereby enables convenient synthesis of tetra- meso-substituted porphyrins, chlorins or bacteriochlorins by Suzuki-coupling.
  • Said leaving group Q may be chloride, bromide, iodide or triflate (trifluoromethanesulfonate).
  • said leaving group Q may be bromide.
  • Methods for the meso-bromination of di-meso-substituted porphrins, chlorins or bacteriochlorins are known in the art.
  • said 5, 15-di-meso-substituted porphyrin, chlorin or bacteriochlorin chromophore may be halogenated at the 10- and 20- meso-positions thereof by way of reaction with halosuccinimide, such as bromosuccinimide.
  • Said coupling reagent may comprise a boronic ester or a boronic acid.
  • each of said Rn and R i2 is H, such that said coupling reagent is a boronic acid.
  • said 5,15-di-meso-substituted porphyrin, chlorin or bacteriochlorin chromophore is a chromophore in accordance with the invention, or a protected form thereof.
  • said 5,15-di-meso-substituted porphyrin, chlorin or bacteriochlorin chromophore may be selected from a porphyrin chromophore of formula (VIII) below:
  • R 5 is a group R 2 as defined above in relation to formulas (I) to (VII) or a protected form thereof or a group convertible thereto; and each of Xi, X , X 3 and X* is independently selected from H, OH, halogen, C 1 - 3 alkyl and OC ⁇ -3 alkyl, or Xi and X 2 and/or X 3 and X4 together form a bridging moiety selected from O, CH 2 , CH C1.3 alkyl, or C(C ⁇ . 3 alkyl) 2 , such that Xi and X 2 and/or X 3 and X with the adjacent C-C bond form an epoxide or cyclopropanyl structure.
  • R ⁇ 3 is a hydrophilic aryl substituent as defined above in relation to R 3
  • said 5, 10, 15,20-tetra-meso-substituted porphyrin, chlorin or bacteriochlorin chromophore may also constitute a chromophore in accordance with the present invention.
  • Said Pd 0 catalyst may, for example, comprise Pd(PPh 3 ) , PdCl 2 (PPh 3 ) 2 , or Pd(OAc) 2 .
  • said Pdo catalyst may comprise Pd(PPh 3 ) .
  • Said coupling reaction is performed in a solvent, which may be selected from toluene or dry THF. It is found that the coupling reaction proceeds swiftly in dry THF, and so dry THF is preferred as solvent.
  • said 5,10,15,20-tetra-meso-substituted porphyrin, chlorin or bacteriochlorin chromophore may be subjected following said coupling reaction to an osmylation reaction utilising OsO , such as to convert said 10- and 20- vinyl substituents to hydroxyalkyl.
  • Said osmylation reaction may be carried out under conditions identical to those suitable for converting a porphyrin to a di-beta- hydroxy-chlorin and then to a tetra-beta-hydroxy-bacteriochlorin.
  • this step may be performed in accordance with the invention on 5, 10(v ⁇ nyl), 15,20(v ⁇ nyl)-meso- substituted porphyrin, chlorin or bacteriochlorin chromophores which are obtained otherwise than in accordance with the method of the invention, such as by way of Pd- catalysed Stille coupling performed on said 5, 15-di-meso-substituted chromophore in accordance with the method described in DiMagno et al, J Org Chem 1993 58, 5983- 5993, (incorporated herein by reference) wherein vinyl t ⁇ butyl tin is used as a coupling reagent
  • said chromophore may be respectively converted to a chlorin or bacteriochlorin chromophore or to a bacteriochlorin chromophore in accordance with methods known to the man skilled in the art
  • said porphyrin or chlorin chromophore may be osmylated by way of reaction with OsO , such as to produce a di- beta-hydroxy-chlo ⁇ n or a tetra-beta-hydroxy-bacte ⁇ ochlo ⁇ n
  • a 5, 15- diphenylporphyrin, 5,15-diphenylchlorin or 5, 15-diphenylbacteriochlorin chromophore wherein each of the ortho-, meta-, and/or para- positions of each of the 5- and 15- phenyl groups is substituted by a substituent P 1 -P 5 and Q1-Q5 respectively which is independently H or an inert substituent which in combination with the other substituents P1-P5 and Qi-Qs does not substantially impair the fluorescent properties of the chromophore; and the chromophore further comprises a conjugating group Z which is capable of conjugating the chromophore to a polypeptide molecule for delivering said chromophore to a specific biological target in vitro or in vivo.
  • Such chromophores 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 targetted 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.
  • said fluorochrome is selected from the following compounds:
  • each of Xi, X 2 , X 3 and X4 are as defined above in relation to the first aspect of the invention.
  • said chromophore may be further substituted at one or more of the 2, 3, 7, 8, 12, 13, 17 or 18 positions thereof by a C ⁇ - 3 alkyl substituent.
  • Z has been omitted for clarity.
  • said Z substituent may be attached to any of the 1-4, 6-14, or 16-20 positions of each chromophore, or may be one of the substituents Pi-P 5 or Q1-Q5, or may be attached to one of the 5- or 15- phenyl groups through one of said substituents Pi-P 5 or Qi-Q 5 .
  • each of P 1 -P5 is the same or substantially the same as the corresponding one of Q 1 -Q 5 , such that said two primary phenyl rings are symmetrically substituted.
  • one or more of P1-P5 is not the same as the corresponding one of Q 1 -Q5, such that said two primary phenyl rings are not symmetrically substituted.
  • all of P1-P5 and/or all of Q1-Q5 may comprise H, such that one or both of said two primary phenyl rings is or are unsubstituted.
  • said substituents P 1 -P5 and Q 1 -Q 5 collectively provide a degree of steric hindrance around the core of said chromophore which is sufficient to reduce the rate of spontaneous oxidation of said chromophore, such that said chromophore is substantially inert in air, but which does not to a substantial extent inhibit selective addition or substitution at the 2, 3, 7, 8, 12, 13, 17 or 18 positions around the core of said chromophore.
  • each of Pi, P 5 , Qi and Q 5 may be H.
  • the total cumulative molecular weight of said substituents P 1 -P 5 does not exceed lOOOgmol "1
  • the total cumulative molecular weight of said substituents 1-Q5 does not exceed lOOOgmol "1 .
  • One or more of said substituents P 1 -P5 and Q 1 -Q 5 may comprise -OH, -CN, -NO , halogen, -T or -OT, where T is a C 1 -C 15 alkyl, cycloalkyl or aryl group or a hydroxylated, halogenated, sulphated or aminated derivative thereof or a carboxylic acid, ester, ether, poly ether, amide, aldehyde or ketone derivative thereof.
  • substituents P1-P5 and Q1-Q5 may additionally or alternatively comprise a C3-C ⁇ cycloalkyl and/or aryl ring structures, or between two and six, preferably two - three, fused or linked C 3 -C ⁇ 2 cycloalkyl and/or aryl ring structures, each of which ring structures may optionally comprise one or more N, O or S atoms.
  • substituents P1-P5 and QrQ 5 may comprise a quatenised amine or pyridyl group, such as an N-methyl pyridyl (pyridiniumyl) group.
  • one of P1-P5 and Q 1 -Q 5 is a conjugating substituent which comprises said conjugating group Z.
  • said conjugating substituent is P 3 or Q 3 , such that said conjugating group Z is provided on the para- position of one of the two primary phenyl rings.
  • said conjugating group is as defined above in relation to the first aspect of the invention.
  • one or more of said substituents P1-P5 and Q1-Q5, not being said conjugating substituent may consist of a member independently selected from the group consisting of A1 Z1 A ⁇ ; wherein Z ⁇ is Z2, 2A5 or Z2A5A6; ⁇ and A5 are independently selected from -(CA 2 A 3 ) n - , -C(Y)(CA 2 A 3 ) n -, -C(Y)Y'(CA 2 A3) n - -
  • Z 3 - Z3 is selected from Z4, Z5 and Zg, wherein Z3 is unsubstituted or substituted one or more times by OH, halo, CN, NO 2 , Ai A 10 , A 6 A 8 , NAI QA! C(Y)A 7 , C(Y)Y'A 7 ,
  • NA 10 C(NCN)SA 9 NA ⁇ oC(NCN)NA 10 A ⁇
  • NA 10 S(O) 2 A 93 S(O) r A 9 NA 10 C(NCN)SA 9 , NA ⁇ oC(NCN)NA 10 A ⁇ , NA 10 S(O) 2 A 93 S(O) r A 9 ,
  • said chromophore may comprise a chromophore having a structure set out as (x), (y) or (z) below:
  • R and R' may be any of the following combinations:
  • said chromophore may comprise a porphyrin chromophore having the structure set out below:
  • a set of fluorochromic markers for multicolour fluorochromic analysis comprising at least two chromophores selected from the group consisting of a porphyrin chromophore, a chlorin chromophore and a bacteriochlorin chromophore, each of which chromophores comprises the same porphyrin skeleton, each of which chromophores comprises one or more substituents on said porphyrin skeleton, one of which substituents is a conjugating substituent L comprising a conjugating group Z, wherein Z is a conjugating group capable of conjugating each of said chromophores to a polypeptide molecule for delivering each chromophore to one of a plurality of different specific biological targets.
  • each of the other of said substituents on the skeleton is independently H or an inert substituent R which together with said conjugating substituent L and all of the other core substituents does not substantially impair the fluorescent properties of each chromophore.
  • each of the chromophores in a set in accordance with the present invention on excitation, will 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 bioconjugation to a biological target 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 set of chromophores may in particular comprise two or more of a porphyrin chromophore in accordance with any aspect of the present invention, the corresponding chlorin chromophore, and the corresponding bacteriochlorin chromophore.
  • corresponding herein is meant having the same meso-substituents around the macrocyclic core of the molecule).
  • said conjugating group Z may be conjugated to a binding 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 binding protein which is adapted to bind specifically to said biological target. Accordingly, said chromophore may be covalently linked to said binding 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 binding 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 binding protein with specificity for said specific biological target.
  • Said binding 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.
  • said antibody may be a phage antibody, that is an antibody expressed on the surface of a bacteriophage.
  • said binding 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 binding 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 binding 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 binding 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 internalisation receptor is capable of binding said binding 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 group such as a quatenised amine or pyridyl (pyridiniumyl) group, or a phosphonium group, so as to promote the intracellular accumulation of said chromophore around the mitochondria of the cell, owing to the net negative charge on the mitochondrial membrane. This will result in the rapid and efficient killing of the cell, on production of singlet oxygen by decay of the chromophore.
  • 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, viral infections such as HIV infection, or autoimmune disorders such as rheumatoid arthritis or multiple sclerosis 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
  • said chromophore is adapted to be internalised within said cells on delivery to said internalisation receptor, such as to enable the production of singlet oxygen within said
  • Said chromophore may be administered topically or systemically to said patient.
  • said chromophore may be administered by injection.
  • 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, viral infections including HIV infection, and autoimmune disorders including rheumatoid arthritis or multiple sclerosis; said chromophore being adapted for delivery to said diseased or undesired cells
  • Porphyrin 1 (500 mg, 0.587 mmol) was dissolved in 18% HCl (100 mL) and the solution heated for 2 hours under reflux. Upon cooling the reaction mixture was evaporated in vacuo to yield a crude green solid. The solid was redissolved in a 9:1 mixture of dichloromethane/triethylamine (200 mL) and stirred for 10 min at room temperature. The solution was then washed with water (3 x 200 mL) and brine (200 mL), the organic layer separated and dried (Na 2 SO 4 ). Excess solvent was evaporated in vacuo and the crude purple solid purified by flash chromatography (silica, eluent: CH 2 Cl 2 EtOAc, 4:1).
  • Porphyrin 6 (300 mg, 0.45 mmol) was dissolved in 18% HCl (100 mL) and the solution heated for 2 hours under reflux. Upon cooling the reaction mixture was evaporated in vacuo to yield a crude green solid. The solid was redissolved in a 9: 1 mixture of dichloromethane/triethylamine (200 mL) and stirred for 10 min at room temperature. The solution was then washed with water (3 x 200 mL) and brine (200 mL), the organic layer separated and dried (Na 2 SO 4 ). Excess solvent was evaporated in vacuo and the purple crude solid purified by flash chromatography (silica, eluent: CHCl 3 /MeOH, 20: 1).
  • the DDP was synthesised according to the method of Dolphin et al (1998 5- Phenyldipyrromethane and 5, 15-Diphenylporphyrin Org. Synth. 76, 287-293 incorporated herein by reference)
  • Porphyrin 12 (35 mg, 48.0 ⁇ mol) was converted into the required mixture of bacteriochlorin stereoisomers by minor modification of the procedure of Sutton et al. (2000 Functionalised diphenylchlorins and bacteriochlorins - their synthesis and bioconjugation for targeted photodynamic therapy and tumour cell imaging J. Porphyrin and Phthalocyanines 4, 655-658 - incorporated herein by reference) (reaction carried out using 1,4-dioxane (5 mL) to allow dissolution of 12). The crude reaction mixture was chromatographed, eluting initially with 1% MeOH in DCM to remove chlorin byproducts.
  • the dipyrromethane was synthesised using the general procedure detailed above using the same molar quantity of starting aldehyde
  • the dipyrromethane was synthesised using the general procedure detailed above using the same molar quantity of starting aldehyde
  • Example 4 To a solution of Example 4 (30 mg, 0.027 mmol) in anhydrous methanol (30 mL) was added Amberlite ® IRA 400 (lg) and the mixture stirred for 1 hour at room temperature. Amberlite ® IRA 400 resin was filtered under vacuum and the porphyrin filtrate recovered, dried (Na 2 SO ) and evaporated in vacuo to yield the above compound as a water soluble purple solid (22 mg, 96.4%). Porphyrins of Examples 4 and 6 were distinguished only by their respective solubility in water.
  • Example 8 17,18-Dihydroxy-5-(4-isothiocyanatophenyl)-15-(4-methoxyphenyl)chlorin,
  • the higher R f regioisomeric chlorin 10 (17.5 mg, 23.2 ⁇ mol) was converted into the corresponding isothiocyanate according to the following method.
  • Porphyrin 8 (100 mg, 0 18 mmol) was converted, in a single reaction, to a mixture of chlorin diols/bacte ⁇ ochlo ⁇ n tetrols following the procedure of Sutton et al. After 38 h the reaction was stopped The crude reaction mixture was then chromatographed, eluting with 2% MeOH in DCM to give first, some un-reacted starting material then the higher Ri chlorin isomer of Example 10 as a brown-purple crystalline solid (5 mg, 5%) The lower R t isomer of Example 11 was obtained by further elution with 3 5% MeOH in DCM and gave also a brown-purple crystalline solid (7 0 mg, 7%) Further elution with 5% MeOH in DCM afforded the required tr ⁇ ws/ z.y-bacteriochlorin tetrols of Examples 12 and 13 respectively as pink/green solids (5 0 mg, 5%) and (7 0 mg, 7%) respectively High R
  • 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-diarylporphyrin (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 porphyrin 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 porphyrin 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.
  • Porphyrins and Phthalocyanines 4, 655) due to the rapidity of the reaction between the ethenyl groups and osmium tetroxide it was possible to selectively hydroxylate these groups by control of reaction time and stoichiometry.
  • reaction vessel was flushed with N 2 and sealed with a lightly greased glass stopper, then covered in aluminium foil to exclude the light and left to stir for 72 h at room temperature. After this period the reaction vessels glass stopper was replaced with a plastic stopper and a continuous stream of hydrogen sulfide gas was bubbled through the reaction mixture for 5 min., (a gas outlet needle was attached and allowed excess hydrogen sulfide gas to escape into a series of Dreshel bottles filled with mineral oil and a bleach solution respectively). After this time the reaction mixture was filtered through Celite® and then concentrated in vacuo. Any excess pyridine was removed under high vacuum.
  • Example 24 Unsymmetrical Porphyrin/ Chlorin Diol/ Bacteriochlorin Tetrol Fluorochrome Sets for Bioconjugation 5-(4-Acetomidophenyl)-15-(4-methoxyphenyl)porphyrin
  • the 1,4-dioxane was removed in-vacuo and the residue partitioned between water (25 ml) and DCM (2 x 25 ml).
  • the combined organic extracts were washed with saturated brine (25 ml) then dried (anhyd. Na 2 SO 4 ), filtered and concentrated in vacuo.
  • the required porphyrin was obtained by chromatography on silica-gel ( 100 ml), (dry loaded on to 10 ml flash silica-gel from DCM and a little methanol for solubility) eluting with DCM.
  • a stock solution of hexahydroxy PITC 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 v a centrifugal concentration and separated into 250 ⁇ L aliquots
  • MR molar ratio
  • Antibody-porphyrm conjugates were stored, without further concentration, in PBS + azide at 0 ⁇ C unless otherwise stated
  • a stock solution of N-methylpy ⁇ dinium chloride PITC in DMSO was prepared to a mola ⁇ ty 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
  • Antibody-porphyrin conjugates were stored, without further concentration, in PBS + azide at 0 ⁇ C unless otherwise stated
  • the two fluorochromic probes were generated from separate reactions of 2,3- d ⁇ hydroxy-5-(4-methoxyphenyl)- 15-(4- ⁇ soth ⁇ ocyanatophenyl)chlo ⁇ n (higher R f regioisomer) and 2,3, 12, 13-tetrahydroxy-5-(4- ⁇ soth ⁇ onatophenyl)-15-(4-methoxyphenyl) bacteriochlorin (lower Rf cis stereoisomer) with avidin under the standard bioconjugation protocols given earlier
  • An initial flow experiment has been undertaken utilising these separate avidin conjugates with RAJI cells and biotin monoclonal antibodies (HLA-DRl, L243), (laser excitation 488 nm, collecting emissions at ⁇ 640 nm (FL2) > 670 nm (FL3))
  • 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.
  • Antibody 17.1A 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 deposit no. 87061205
  • 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.
  • Each of OH6 and PYR was separately 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..
  • the acetate-buffered antibody preparation and range of conjugation dilutions obtained therefrom were subjected to SDS-PAGE in accordance with the method described in Methodology Description 6. The results are shown in Figures 1-3 respectively.
  • Figure 1 shows a gel loaded with buffer-exchanged 17.1 A antibody (lane 1), and buffer-exchanged antibody/OH6 conjugations at MRs 2.5 (lanes 2, 3), 5 (lanes 4, 5), 10 (lanes 6, 7) and 20 (lanes 8, 9) and molecular weight markers (lane 10).
  • Figure 2 shows a gel loaded with dialysed 17 1 A antibody (lane 1), and dialysed antibody/OH6 conjugations at MRs 2.5 (lanes 2, 3), 5 (lanes 4, 5), 10 (lanes 6, 7) and 20 (lanes 8, 9) and molecular weight markers (lane 10).
  • Figure 3 shows a gel loaded with buffer-exchanged 17 1 A antibody (lane 1), and buffer-exchanged antibody/PYR conjugations at MRs 2.5 (lanes 2, 6), 5 (lanes 3, 7), 10 (lanes 4, 8) and 20 (lanes 5, 9) and molecular weight markers (lane 10).
  • Figure 4 shows results derived utilising FITC-labelled 17.1 A and Colo 320 cells (3 repeats) and indicates that binding of the antibody to the cells has occurred (ie the Colo 320 cells express the antigen specific to 17 1A).
  • Figure 5 shows results derived utilising OH6/17.1A conjugate and Colo 320 cells with a FITC-labelled anti-17.1A antibody for detection (3 repeats) and indicates that the OH6/17.1A conjugate has bound to the cells.
  • Figure 6 shows results derived utilising PYR/17.1A conjugate and Colo 320 cells with a FITC-labelled anti-17.1 A antibody for detection (3 repeats) and indicates that the PYR/17.1A conjugate has bound to the cells.
  • Figure 7 shows results derived utilising FITC-labelled OX-34 which is an antibody of the same class (IgG2a) as 17.1 A but with a different antigen specificity (3 repeats). The results indicate that OX-34 has not bound to the Colo 320 cells and hence that there are no binding sites for OX-34 on Colo 320 cells.
  • FIGS 8 and 9 show the results of control experiments performed using OH6/OX-34 and PYR/OX-34 conjugates respectively. As described in Example 16 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.
  • Figures 10 and 1 1 show the results of further control experiments performed using "capped” OH6 and PYR respectively.
  • the "capping" procedure involved reacting the NCS group on each chromophore with propylamine, so as to block serum protein conjugation.
  • Figure 10 shows no cytotoxicity in the dark, indicating that OH6 is nontoxic to Colo 320 cells. On irradiation, however, some photocytotoxicity is observed, indicating that an amount of the capped OH6 has been transferred to the surface of the Colo 320 cells.
  • Figure 1 1 meanwhile shows some cytotoxicity in the dark, suggesting that PYR is to some extent cytotoxic to Colo 320 cells, and increased photocytotoxicity on irradiation, which again indicates that an amount of the capped PYR has been transferred to the surface of the Colo 320 cells.
  • Figures 12 and 13 show results obtained using OH6/17.1A and PYR/17.1A conjugates respectively, at various conjugation dilutions (2.5, 5, 10, 20 for OH6/17.1A; 10 and 20 for PYR/17.1 A).
  • the results indicate a significant increase in cytotoxicity on irradiation, indicating that the binding of the bioconjugates to the cell surface confers photosensitivity upon the cells. Hence, these species are suitable candidates for PDT.
  • Protocols for performing and assessing photodynamic therapy in vivo, utilising the conjugates of the invention are variously described in R Boyle et al, Br. J. Cancer (1992) 65:813-817; R Boyle et al, Br. J. Cancer (1993) 67: 1177-1181; RBoyle et al, Br. J. Cancer (1996) 73:49-53; and Lapointe et al, J. Nuclear Medicine, Vol. 40, No. 5 (May 1999) 876-882; the contents of each of which are incorporated 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, J. Nuclear Medicine, Vol. 40, No. 5 (May 1999) 876- 882). As described in R Boyle et al, Br. J. Cancer (1996) 73:49-53, the distribution of the photosensitiser in vivo may also be measured, by biodistribution and/or vascular stasis assays.
  • a preliminary examination of the intracellular localisation of a conjugate of 10, 15,20-tris(3,5-dihydroxyphenyl)5-isothiocyanatophenylporphyrin (OH6-NCS) with BSA was carried out using confocal laser scanning microscopy.
  • the readily available epithelial human carcinoma cell line HeLa was selected for incubation with the conjugate. All incubations were performed ' , in triplicate with sub-confluent cultures of HeLa cells, including a series of control solutions of unlabelled BSA, 10,15,20-tris(3,5- dihydroxyphenyl)5-aminophenylporphyrin porphyrin (OH6-NH2, amino precursor of OH6-NCS), and PBS on its own. Cells were seeded onto coverslips in 35 mm dishes.
  • Fluorescence images of cells were obtained with a Bio-Rad Radiance2000 confocal laser scanning microscope (Bio-Rad Microscience, Cambridge, 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.
  • Porphyrins 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-dimensionally by recording images from a series 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 OH6-NCS identifying its principal absorption bands. Unfortunately, no laser line was available in order to excite OH6-NCS at its Soret band ⁇ max .
  • Figure 15 demonstrates the relative intensities of fluorescence emission for OH6-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 OH6-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 OH6-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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