Activatable two-component photosensitizers

Patent metadata

Field	Value
Publication number	US-10434177-B2
Application number	US-201515527061-A
Country	US
Kind code	B2
Filing date	Nov 17, 2015
Priority date	Nov 17, 2014
Publication date	Oct 8, 2019
Grant date	Oct 8, 2019

Abstract

Provided herein is a two-component photosensitizer, which demonstrated robust and selective killing effects for transfected HEK cells and affibody targeted A431 cancer cells when exposed to near infrared light excitation. Free MG2I is a pure and stable fluorogen; it is easy to synthesize and modify, and has no toxicity to cells. Unlike conventional photosensitizers, the dye and FAP itself has no photosensitizing effect until they are bound. Also unlike other activation methods, the activation step is achieved by adding the fluorogen, not the presence of the targeted molecule, requiring an ‘active’ activation instead of a ‘passive’ activation. This method offers the ability to locally switch-on and selective generation of singlet oxygen at the target site and can be used for a wide variety of molecular targets.

First claim

We claim:

1. A method of targeting and killing cells, comprising:
a. contacting cells with a targeting activator composition comprising a targeting moiety that selectively binds a target compound of the cell, and an activator moiety that selectively binds a heavy atom-modified malachite green derivative having an excitation wavelength so that the heavy atom-modified malachite green derivative produces singlet oxygen when bound by the targeting activator and exposed to light at the excitation wavelength;
b. contacting cells with the heavy atom-modified malachite green derivative; and
c. exposing the cells to light at an excitation wavelength of the targeting activator-bound heavy atom-modified malachite green derivative,

wherein the heavy atom-modified malachite green derivative has the structure:

where X and X′ are, independently, Br, I, As, Se, Ga, Ge, or Sb; R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are, independently H or F, R11, R12, R13 and R14 are, independently, methyl, H, aziridine or azetidine, wherein when R11, R12, R13, and/or R14 are aziridine or azetidine, R11 and R12 form a single ring and/or R13 and R14 form a single ring, and where R is selected from —H, —OH, —COO − , —SO 3 − ,-PO 4 − , —NO 2 , —NH 2 ,-N(CH 3 )(R15),-OR16, alkyl, ether, polyether, PEG 1-30 , —(C 1 -C 4 alkyl)-R17, heterocyles containing N, S or O atoms, substituted acetylenic groups, cyano, and carbohydrate groups, wherein R15 and R16 are: straight- or branched-chain alkyl; straight or branched-chain C 1-6 alkyl; straight-chain or branched poly(C 1 -C 4 alkyl amide); straight-chain or branched poly(C 1 -C 4 alkyl amide) having from 2 to 6 amide moieties; poly(C 1 -C 4 alkylene glycol); poly(C 1 -C 4 alkylene glycol) having from 2 to 30 or from 2-10 C 1 -C 4 alkylene glycol moieties; straight-chain or branched poly(C 1 -C 4 alkyl amide):poly(C 1 -C 4 alkylene glycol) diblock copolymer; straight-chain or branched poly(C 1 -C 4 alkyl amide):poly(C 1 -C 4 alkylene glycol) diblock copolymer having from 2 to 6 amide moieties and from 2 to 10 C 1 -C 4 alkylene glycol moieties; sulfonyl- or bis-sulfonyl-terminated straight-chain or branched poly(C 1 -C 4 alkyl amide); bis-taurine-terminated branched poly(C 1 -C 4 alkyl amide); ethyl butyrate; C 1-6 alkyl C 1-6 alkanoate; —(CH 2 ) n —C(O)—O—(CH 2 ) m —CH 3 , where n=1-4 and m=0-3, and wherein R17 is selected from H, —OH,-COO − , —SO 3 − , —PO 4 − , —NO 2 , or —NH 2 , or a pharmaceutically acceptable salt thereof,
wherein the activator moiety is a fusion protein of an scFv activator moiety and an affibody targeting moiety, and the targeting moiety is selective for an epidermal growth factor receptor.

2. The method of claim 1 , in which the scFv is an L5-MG scFv peptide.

3. The method of claim 1 , in which the epidermal growth factor receptor is HER1 (human epidermal growth factor receptor 1) or HER2 (human epidermal growth factor receptor 2).

4. The method of claim 1 , in which the targeting activator comprises a sequence selected from SEQ ID NOS: 1-4, 10-15, 17 and 18.

5. The method of claim 1 , in which R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 of the heavy atom-modified malachite green derivative are H.

6. The method of claim 1 , in which X and X′ of the heavy atom-modified malachite green derivative are I.

7. The method of claim 6 , wherein R of the heavy atom-modified malachite green derivative is-OR16, and R16 is:
ethylbutyrate; or
sulfonyl- or bis-sulfonyl-terminated straight-chain or branched poly(C 1 -C 4 alkyl amide) having from 2 to 6 amide moieties.

8. The method of claim 1 , in which R11, R12, R13 and R14 of the heavy atom-modified malachite green derivative are, independently, methyl or H, or R11, R12, R13 and R14 are methyl.

9. The method of claim 1 , in which R of the heavy atom-modified malachite green derivative is-OR16, and R16 is:
ethylbutyrate; or
sulfonyl- or bis-sulfonyl-terminated straight-chain or branched poly(C 1 -C 4 alkyl amide) having from 2 to 6 amide moieties.

10. The method of claim 9 , in which R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 of the heavy atom-modified malachite green derivative are H and R11, R12, R13 and R14 are methyl.

11. The method of claim 10 , in which X and X′ of the heavy atom-modified malachite green derivative are I.

12. The method of claim 1 , wherein the heavy atom-modified malachite green derivative has the structure:

13. The method of claim 12 , wherein X and X′ are I.

14. The method of claim 1 , wherein the heavy atom-modified malachite green derivative has the structure:

15. A method of targeting and killing cells in a patient, comprising
a. administering to the patient an effective amount of a targeting activator composition comprising a targeting moiety that selectively binds to targeted cells, and an activator moiety that selectively binds a heavy atom-modified malachite green derivative having an excitation wavelength so that the heavy atom-modified malachite green derivative produces singlet oxygen when bound by the targeting activator and exposed to light at the excitation wavelength;
b. administering to the patient an effective amount of the heavy atom-modified malachite green derivative; and
c. exposing the cells to light at an excitation wavelength of the targeting activator-bound heavy atom-modified malachite green derivative, thereby killing the cells,

wherein the heavy atom-modified malachite green derivative has the structure:

where X and X′ are, independently, Br, I, As, Se, Ga, Ge, or Sb; R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are, independently H or F, R11, R12, R13 and R14 are, independently, methyl, H, aziridine or azetidine, wherein when R11, R12, R13, and/or R14 are aziridine or azetidine, R11 and R12 form a single ring and/or R13 and R14 form a single ring, and where R is selected from-H, —OH,-COO − ,-SO 3 − ,-PO 4 − , —NO 2 , —NH 2 ,-N(CH 3 )(R15),-OR16, alkyl, ether, polyether, PEG 1-30 , —(C 1 -C 4 alkyl)-R17, heterocyles containing N, S or O atoms, substituted acetylenic groups, cyano, and carbohydrate groups, wherein R15 and R16 are: straight- or branched-chain alkyl; straight or branched-chain C 1-6 alkyl; straight-chain or branched poly(C 1 -C 4 alkyl amide); straight-chain or branched poly(C 1 -C 4 alkyl amide) having from 2 to 6 amide moieties; poly(C 1 -C 4 alkylene glycol); poly(C 1 -C 4 alkylene glycol) having from 2 to 30 or from 2-10 C 1 -C 4 alkylene glycol moieties; straight-chain or branched poly(C 1 -C 4 alkyl amide):poly(C 1 -C 4 alkylene glycol) diblock copolymer; straight-chain or branched poly(C 1 -C 4 alkyl amide):poly(C 1 -C 4 alkylene glycol) diblock copolymer having from 2 to 6 amide moieties and from 2 to 10 C 1 -C 4 alkylene glycol moieties; sulfonyl- or bis-sulfonyl-terminated straight-chain or branched poly(C 1 -C 4 alkyl amide); bis-taurine-terminated branched poly(C 1 -C 4 alkyl amide); ethyl butyrate; C 1-6 alkyl C 1-6 alkanoate; —(CH 2 ) n —C(O)—O—(CH 2 ) m —CH 3 , where n=1-4 and m=0-3, and wherein R17 is selected from H,-OH, —COO − , —SO 3 − , —PO 4 − , —NO 2 , or —NH 2 , or a pharmaceutically acceptable salt thereof, wherein the activator moiety is a fusion protein of an scFv a

Assignees

• Univ Carnegie Mellon

Inventors

• Bruchez Marcel P
• He Jianjun
• Wang Yi

Classifications

• A61P35/00

  Antineoplastic agents · CPC title

• C07C233/40

  having the carbon atom of the carboxamide group bound to an acyclic carbon atom of a carbon skeleton containing six-membered aromatic rings · CPC title

• C07C233/36

  having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton · CPC title

• C09B11/22

  containing OH groups bound to an aryl nucleus {and their ethers and esters} · CPC title

• C07K2317/622

  Single chain antibody (scFv) · CPC title