Multivalent targeting fluorescent tracer in the near infrared range for optical imaging

Abstract

A fluorescent tracer for targeting tumors, comprises: at least one first fluorophore fluorescing in a range of wavelengths of between 700 and 1000 nm, a targeting assembly comprising at least two identical targeting molecules, and a cyclic oligopeptide: configured so as to define a mean plane defining a first upper face and a second lower face, comprising at least one first lysine amino acid residue on the second lower face, the targeting molecules being fixed to the first upper face of the mean plane, the fluorophore being fixed to the second lower face of the mean plane via a spacer arm connecting a carbon of the sequence of the at least three double bonds and the lysine amino acid residue of the oligopeptide.

Claims

1. A fluorescent tracer for targeting tumors, comprising: a fluorophore chosen from the following fluorophores: S0121, S0306, S0456, S2180, IR775 chloride, IR780 iodide, IR786, IR806, IR820; an assembly for targeting .sub.v3 integrin consisting of 4 targeting molecules being cyclic pentapeptides of sequence [RGDfK]; and a cyclic decapeptide comprising the following sequence of amino acid residues: [-G.sub.a-P.sub.b-K.sub.c-A.sub.d-K.sub.e-G.sub.f-P.sub.g-K.sub.h-K.sub.i-K.sub.j-], the cyclic decapeptide being configured to define a mean plane defining a first upper face consisting of the four lysine residues K.sub.c, K.sub.e, K.sub.h and K.sub.j, and a second lower face consisting of the residues G.sub.a, P.sub.b, A.sub.d, G.sub.f, P.sub.g and K.sub.i, each of the targeting molecules being fixed to a different lysine amino acid residue of the four lysine residues K.sub.c, K.sub.e, K.sub.h and K.sub.j via oxime bonds, the fluorophore being fixed to the second lower face of the mean plane via a spacer arm being 5-(4-hydroxyphenyl)pentanoic acid, the spacer arm being fixed to the fluorophore via an ether bond resulting from the reaction between the phenol function of the spacer arm and the chlorine element of the fluorophore and the spacer arm being fixed to the lysine residue K.sub.i via an amide bond.

2. A process for synthesizing a fluorescent tracer for targeting tumors of claim 1 comprising: (i) preparation of a modified fluorophore in which the spacer arm is coupled to the fluorophore, wherein fluorophore is selected from the group consisting of S0121, S0306, S0456, S2180, IR775 chloride, IR780 iodide, IR786, IR806 and IR820; (ii) preparation of a regioselectively addressable functionalized (RAFT) template, wherein RAFT is a cyclic decapeptide represented by sequence of amino acid residues: G.sub.a-P.sub.b-K.sub.c-A.sub.d-K.sub.e-G.sub.f-P.sub.g-K.sub.h-K.sub.i-K.sub.j; (iii) coupling of RAFT template to the modified fluorophore to form a fluorescent template; (iv) grafting an oxime bond precursor onto the RAFT template on the lysine residues to form a modified fluorescent template; (v) synthesis of targeting molecules; and (vi) coupling between the targeting molecules and the modified fluorescent template.

Description

LIST OF FIGURES

(1) The invention will be better understood and other advantages will become apparent on reading the following description, which is given by way of non-limiting example, and by virtue of the appended figures in which:

(2) FIG. 1, already described, represents a RAFT template,

(3) FIG. 2, already described, illustrates a targeting molecule comprising a cyclic pentapeptide [-RGDfK-] specific for the .sub.v.sub.3 integrin,

(4) FIG. 3, already described, represents a fluorescent tracer according to the known art,

(5) FIG. 4 illustrates a representative of the family of fluorescent tracers according to the invention,

(6) FIGS. 5a, 5b, and 5c represent the general formulae of fluorophores used in a tracer according to the invention,

(7) FIGS. 6a, 6b, 6c, 6d, 6e, 6f, 6g, and 6h represent examples of chemical formulae of commercial fluorophores which are potentially of use according to the invention,

(8) FIGS. 7a, 7b, 7c and 7d represent spacer arms used in a tracer according to the invention,

(9) FIG. 8 represents a RAFT template enabling the grafting of two fluorophores to the lower face Fi,

(10) FIG. 9 represents the coupling reactions between a spacer arm and a fluorophore according to the invention,

(11) FIGS. 10a and 10b represent the reactions for synthesizing the RAFT template, and for coupling between the fluorophore provided with the spacer arm and the RAFT template, according to the invention,

(12) FIG. 11 represents the reactions for synthesizing the targeting molecule according to the invention,

(13) FIG. 12 represents the coupling reaction between the fluorescent RAFT template and the targeting molecules via an amide bond, according to the invention;

(14) FIG. 13a illustrates the tissue distribution of a tracer Tf according to the known art and FIG. 13b illustrates the tissue distribution of a tracer Tf according to an embodiment of the invention.

DETAILED DESCRIPTION

(15) FIG. 4 is a semi-expanded formula of a representative of the fluorescent tracer Tf family according to the invention.

(16) In the case in point, the fluorescent tracer Tf comprises a RAFT template 1 comprising, as in the document by Boturyn, a sequence of ten amino acid residues [K-K-K-P-G-K-A-K-P-G-] in cyclic form. The sequences of the amino acid residues [-Glycine-Proline] form bends 2 so as to define a mean plane Pm, on either side of which a fluorophore 4 and a targeting assembly 3 may be fixed. Generally, the targeting molecules 3 may be molecules for targeting tumors. These molecules may be peptides, and more particularly peptides able to target at least one integrin, preferentially .sub.v.sub.3.

(17) In the case in point, the targeting assembly 3 comprises cyclic pentapeptide molecules 3a comprising a specific sequence of RGD type. The cyclic pentapeptides 3a are connected to the RAFT template 1 at the lysine residues K.sub.c, K.sub.e, K.sub.h, and K.sub.i of the upper face F.sub.s of the RAFT template 1. In the case in point, they are connected via an oxime bond, enabling good stability in vivo and in vitro. Nonetheless, it is also possible to connect them via an amide bond. Alternatively, it is possible to connect at least one targeting molecule 3a via a thioether bond, with the proviso that a lysine residue is replaced by a cysteine residue, in which case it is possible to graft one type of targeting molecules onto the lysine residues and another type of molecule onto the cysteine residue, thereby enabling the regioselective grafting of targeting molecules.

(18) The fluorophore 4 is fixed to the template 1 via a spacer arm 8. In the case in point, the fluorophore 4 is IR775 (registered trademark) or 2-[(E)-2-{(3E)-2-chloro-3-[(2Z)-2-(1,3,3-trimethyl-1,3-dihydro-2H-indol-2-ylidene)ethylidene]cyclohex-1-en-1-yl}ethenyl]-1,3,3-trimethyl-3H-indolium chloride, depending on the nomenclature.

(19) The spacer arm 8 comprises a sequence of five carbons, a phenolate function at one end 8.sub.84 of the carbon-based chain connecting the spacer arm 8 to the fluorophore 4 and a carboxylic function 8.sub.81 connecting the spacer arm 8 to the RAFT template 1.

(20) More specifically, FIGS. 5a, 5b and 5c represent general formulae of fluorophores 4 potentially of use according to the invention.

(21) The fluorophore 4 according to the invention must have properties of absorption in the near-infrared wavelength range of between 750 nm and 1000 nm and an emission peak having a maximum of between 770 nm and 870 nm. The fluorophore 4 also has a linear or non-linear carbon-based chain comprising a sequence of conjugated covalent double bonds, the electrons of the double bonds being able to delocalize. Moreover, the fluorophore 4 has, on one of the carbons of the carbon-based chain, a halogen group X which may react with a group.

(22) FIG. 5a presents a fluorophore 4 comprising a linear carbon chain 4a having conjugated double bonds, the electrons of the double bonds being able to delocalize over the whole of the carbon-based chain 4a. The ends of the carbon-based chain 4a have tertiary amine and quaternary ammonium groups.

(23) FIG. 5b also presents a fluorophore 4 potentially of use for producing a fluorescent tracer according to the invention. FIG. 5b differs from FIG. 5a in that the quaternary ammonium end has an aromatic group. FIG. 5c has, at each end of the carbon-based chain 4a, an aromatic group.

(24) The aromatic group, such as pyrrole or indole, optionally comprises an aliphatic group having a functional group which is able to modify the physicochemical characteristics of the fluorescent tracer Tf. For example, the addition of a sulfonate group may increase hydrophilicity and thereby increase the solubility of the fluorescent tracer Tf in an aqueous solvent.

(25) A number of examples of commercial fluorophores which are potentially of use are represented in FIG. 6: IR786 (registered trademark), or 2-(2-[2-Chloro-3-([1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylidene]ethylidene)-1-cyclohexen-1-yl]ethenyl)-1,3,3-trimethylindolium perchlorate (FIG. 6a), IR806 (registered trademark) or internal salt (sodium salt) of 2-[2-[2-Chloro-3-[[1,3-dihydro-1,1-dimethyl-3-(4-sulfobutyl)-2H-benzo[e]indol-2-ylidene]-ethylidene]-1-cyclohexen-1-yl]-ethenyl]-1,1-dimethyl-3-(4-sulfobutyl)-1H-benzo[e]indolium hydroxide (FIG. 6b), IR820 (registered trademark) or internal salt (sodium salt) of 2-[2-[2-Chloro-3-[[1,3-dihydro-1,1-dimethyl-3-(4-sulfobutyl)-2H-benzo[e]indol-2-ylidene]-ethylidene]-1-cyclohexen-1-yl]-ethenyl]-1,1-dimethyl-3-(4-sulfobutyl)-1H-benzo[e]indolium hydroxide (FIG. 6c), S0456 (registered trademark) or internal salt (trisodium salt) of 3,3-Dimethyl-2-[2-[2-chloro-3-[2-[1,3-dihydro-3,3-dimethyl-5-sulfo-1-(4-sulfobutyl)-2H-indol-2-ylidene]-ethylidene]-1-cyclohexen-1-yl]-ethenyl]-5-sulfo-1-(4-sulfobutyl)-3H-indolium hydroxide (FIG. 6d), S2180 (registered trademark) or internal salt (trisodium salt) of 2-[2-(2-Chloro-3-[2-[1,1-dimethyl-7-sulfo-3-(4-sulfobutyl)-1,3-dihydro-benzo[e]indol-2-ylidene]-ethylidene]-cyclohex-1-enyl)-vinyl]-1,1-dimethyl-7-sulfo-3-(4-sulfobutyl)-1H-benzo[e]indolium hydroxide (FIG. 6e), S0306 (registered trademark) or internal salt (sodium salt) of 2-[2-[2-Chloro-3-[[1,3-dihydro-1,1-dimethyl-3-(4-sulfobutyl)-2H-benzo[e]indol-2-ylidene]-ethylidene]-1-cyclohexen-1-yl]-ethenyl]-1,1-dimethyl-3-(4-sulfobutyl)-1H-benzo[e]indolium hydroxide (FIG. 6f), S0121 (registered trademark) or internal salt (sodium salt) of 2-[2-[2-Chloro-3-[2-[1,3-dihydro-3,3-dimethyl-1-(4-sulfobutyl)-2H-indol-2-ylidene]-ethylidene]-1-cyclohexen-1-yl]-ethenyl]-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium hydroxide (FIG. 6g), IR780 iodide (registered trademark) or 2-[2-[2-Chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2H-indol-2-ylidene)ethylidene]-1-cyclohexen-1-yl]ethenyl]-3,3-dimethyl-1-propylindolium iodide (FIG. 6h).

(26) These fluorophores have a much lower cost than the fluorophores used for synthesizing fluorescent tracers Tf according to the prior art.

(27) The difference in cost may be explained on the one hand by the fact that these fluorophores are structurally simpler than the fluorophores used formerly, of IRDye 800 type, and one the other hand by the fact that it is not necessary to activate them.

(28) Moreover, FIGS. 7a, 7b, 7c and 7d present semi-expanded formulae of spacer arms 8 which are potentially of use.

(29) Generally, the spacer arm 8 according to the invention comprises a carbon-based chain 8a, a first end group 8.sub.84 connecting the fluorophore 4 to the spacer arm 8 and a second end group 8.sub.81 connecting the spacer arm 8 to the RAFT template 1.

(30) Although the prior art presents a spacer arm consisting of a tyrosine, it has been demonstrated that it is not possible to graft a fluorophore 4 onto a RAFT template 1 according to the invention via a spacer arm consisting of a tyrosine residue, especially because of steric hindrance or spacing problems.

(31) Preferentially, the number of carbons of the carbon-based chain 8a is greater than or equal to 4, thereby facilitating the grafting of the fluorophore 4 and thereby limiting the problems associated with steric hindrance.

(32) The first end group 8.sub.84 may be an amide group (corresponding to an amine group before a reaction between the spacer arm 8 and the fluorophore 4), an ether group (corresponding to an alcohol group before a reaction between the spacer arm 8 and the fluorophore 4) or a thioether group (corresponding to a thiol group before a reaction between the spacer arm 8 and the fluorophore 4). The fixing of the spacer arm 8 via the first end group 8.sub.84 substantially modifies the emission maximum. Indeed, an amine first end group reduces the wavelength of the maximum fluorescence peak by 100 to 150 nm. Thus, this type of first end group will not be favored. An ether first end group (FIGS. 7a and 7c, corresponding to an alcohol group before a reaction between the spacer arm 8 and the fluorophore 4) reduces the wavelength of the emission peak maximum by 10 to 15 nm. A thioether first end group (FIGS. 7b and 7d, corresponding to a thiol group before a reaction between the spacer arm 8 and the fluorophore 4) increases the wavelength of the emission peak maximum by 10 to 15 nm. In addition, the use of a first end group of phenol type (FIG. 7a and FIG. 7c) increases delocalization of the electrons of the carbon-based chain 4a of the fluorophore, which increases the quantum yield. Generally, an end group 8.sub.84 of the spacer arm is able to increase delocalization of the electrons of the carbon-based chain 4a of the fluorophore 4, and thereby to increase the quantum yield.

(33) The second end group 8.sub.81 may be a carboxylic acid (FIG. 7a and FIG. 7b) or succinimide ester (FIG. 7c and FIG. 7d).

(34) The spacer arm 8 is fixed, via the second end group 8.sub.81, to a lysine residue on what is referred to as the lower face F.sub.i of the RAFT template 1.

(35) According to a variant of the invention represented in FIG. 8, the alanine residue located on what is referred to as the lower face F.sub.i of the RAFT template 1 may be substituted by a lysine residue, in which case a second fluorophore may additionally be fixed so as to enable FRET fluorescence, for example.

(36) According to another aspect of the invention, a process is proposed for producing the tracer Tf comprising a step of coupling the fluorophore 4 with the RAFT template 1 so as to form a fluorescent RAFT template prior to the step of coupling the targeting molecules 3.

(37) This production process reduces starting material losses, and more particularly losses of the targeting molecules, and makes it possible to avoid the step of activating the fluorophore before the coupling step.

(38) The process comprises: a first step, illustrated in FIG. 9, for preparing a modified fluorophore 4 in which the spacer arm 8 is coupled to the fluorophore 4, a second step, represented in FIG. 10, in which the RAFT template 1 is synthesized, a third step, represented in FIG. 10, in which the RAFT template 1 is coupled to the modified fluorophore 4 so as to form a fluorescent template 10, a fourth step, illustrated in FIG. 10, in which an oxime bond precursor 11 is grafted onto the RAFT template 1 on the lysine residues of what is referred to as the upper face F.sub.s so as to form a modified fluorescent template 10, a fifth step, represented in FIG. 11, in which the targeting molecules 3a are synthesized, and a sixth step, illustrated in FIG. 12, for coupling between the targeting molecules 3a and the modified fluorescent template 10.

(39) More specifically, FIG. 9 presents the steps for preparing the modified fluorophore 4, described especially in the document by Hyun, H. et al., c-GMP-compatible preparative scale of near-infrared fluorophores. Contrast Media Mol. Imaging, 2012, 7: 516. The spacer arm 8, 5-(4-hydroxyphenyl)pentanoic acid, comprises a first phenol end group 8.sub.81. The phenol is converted to phenolate, which is more reactive than phenol, in a solution of sodium hydroxide in methanol, to form a modified spacer arm 8. The spacer arm 8 obtained is then mixed with the fluorophore 4, in this case S0456 (or internal salt (trisodium salt) of 3,3-Dimethyl-2-[2-[2-chloro-3-[2-[1,3-dihydro-3,3-dimethyl-5-sulfo-1-(4-sulfobutyl)-2H-indol-2-ylidene]-ethylidene]-1-cyclohexen-1-yl]-ethenyl]-5-sulfo-1-(4-sulfobutyl)-3H-indolium hydroxide) in DMSO (acronym for DiMethyl SulfOxide) to obtain the modified fluorophore 4 consisting of the fluorophore 4 onto which the spacer arm 8 is grafted.

(40) FIG. 10a presents the second, third and fourth sub-steps of the process. Firstly, a linear decapeptide comprising the sequence of amino acid residues [K(Boc)-K(Alloc)-K(Boc)-P-G-K(Boc)-A-K(Boc)-P-G-] is synthesized on resin, the Boc and Alloc groups being protecting groups so as to subsequently enable regioselective functionalization of the fluorescent template. After detachment from the resin, the decapeptide is cyclized and the lysine residue protected by the (Alloc) group is deprotected in the presence of palladium)(Pd.sup.0) and phenylsilane so as to form a RAFT template 1 having a mean plane Pm. The modified fluorophore 4 is then grafted onto the deprotected lysine residue. The Boc protecting groups on the lysine residues of what is referred to as the upper face F.sub.s are cleaved in acid medium then a protected oxyamine precursor 11 is grafted onto the lysine residues via an amide bond, to form the modified fluorescent RAFT template 10. It should be noted that the third step and the fourth step may be switched around, as illustrated in FIG. 10b. In the case in point, firstly the Boc protecting groups on the lysine residues of what is referred to as the upper face F.sub.s are cleaved in acid medium then protected oxyamine precursors 11 are grafted onto the lysine residues via an amide bond. The lysine residue located on the lower face F.sub.i protected by the (Alloc) group is deprotected so as to enable the grafting of the modified fluorophore 4 to form a modified fluorescent template 10.

(41) Moreover, it is interesting to note that the sub-step of activating the fluorophore 4, which is essential in the process according to the prior art, is not necessary in the process according to the invention. In the invention, the step of activating the fluorophore 4 carried out in the prior art is in part bypassed by a step of preparing a modified fluorophore 4 in which an arm 8 reacts with a halogen group X present on one of the carbons of the carbon-based chain. In this way, the spacer arm 8 connects a carbon of the sequence of the at least three double bonds of the fluorophore 4. This arrangement enables a reduction in the cost of synthesizing the tracer, with equivalent and/or better performance.

(42) FIG. 13a illustrates the tissue distribution of a tracer Tf according to the known art, described in FIG. 3, at different times post-injection.

(43) In the case in point, the fluorescence was measured at times post-injection of 4 h, 24 h, 48 h then seven days and the organs or tissues studied were: the heart, the lungs, the muscles, the kidney, the skin, the brain, the adrenal glands, the bladder, the spleen, the stomach, the intestines, the ovaries and the uterus, the pancreas, fat, the liver and a subcutaneous murine mammary tumor (Ts/Apc).

(44) The diagram shows that the fluorescence intensity is greatest at a time, post-injection of the tracer Tf according to the known art, of 4 h, and decreases after 24 h. After seven days, the fluorescence intensity in the organs and tissues is virtually zero.

(45) In addition, the diagram shows that the tracer Tf according to the known art is particularly well-suited for targeting a tumor which is overexpressing the .sub.v.sub.3 integrin. This diagram also shows a significant accumulation of the tracer Tf according to the known art in the kidneys from 4 hours post-injection, demonstrating rapid renal elimination of the product.

(46) FIG. 13b illustrates the tissue distribution of a tracer Tf according to an embodiment of the invention. FIG. 13b illustrates in particular the fluorescence intensity of a tracer Tf according to an embodiment of the invention, for example illustrated in FIG. 4, in the same organs and tissues as those studied in FIG. 13a.

(47) The tissue distribution of the tracer Tf according to the invention is similar to the distribution observed for the tracer Tf according to the known art.

(48) Moreover, the affinity of the tracer Tf according to the invention is similar to the affinity of a tracer Tf according to the known art, such as Cy5-RAFT-(c[RGDfK]).sub.4. This is explained by the fact that the targeting molecules are identical and represented in identical amounts in the tracer Tf according to the known art and the tracer Tf according to the invention.

(49) On the other hand, the tracer Tf according to the invention has a much greater affinity than a monomeric tracer having the targeting molecule represented in a single example.

(50) FIGS. 13a and 13b illustrate similar, equivalent and/or superior performance of a tracer Tf according to an embodiment of the invention compared to a trace Tf according to the known art.

(51) FIG. 11 presents the steps for synthesizing the targeting molecule 3a. Firstly, the linear pentapeptide comprising the sequence of amino acid residues RGD specific for integrins is synthesized on a resin. In the case in point, the pentapeptide comprises the sequence [-D(tBu)-f-K(Alloc)-R(Pbf) G-]. After detaching the peptide from the resin, said peptide is cyclized then the lysine residue protected by the Alloc group is subsequently deprotected in the presence of palladium)(Pd.sup.0) and phenylsilane. A protected serine residue is the grafted onto the lysine residue before total deprotection of the pentapeptide in acid medium. The alcohol function of the serine is then oxidized with sodium periodate in water, to obtain an aldehyde group.

(52) FIG. 12 illustrates the step of coupling between the modified fluorescent template 10 and the cyclic pentapeptides 3a.

(53) In the claimed process, the commercial fluorophore 4 is involved very early on in the process for synthesizing the fluorescent tracer Tf compared to the process of the prior art.

(54) The requirements for purity and quality relating to starting materials incorporated into formulations intended for human administration involved early on in the synthesis process are less stringent than for starting materials involved at the end of the process, as is the case in the process of the prior art. In the case in point, the fluorophore 4 may therefore be of lesser quality and purity than those required during the synthesis according to the process of the prior art, which contributes significantly to lowering the purchase cost of the fluorophore 4.

(55) Moreover, unlike the process described in the prior art, the step of activating the fluorophore prior to the step of coupling between the modified fluorophore 10 and the RAFT template 1 is not necessary, which makes it possible to further reduce the costs of producing the fluorescent tracer Tf according to the invention.

(56) Thus, the synthesis of 15 g of fluorescent tracer Tf requires 30 g of RAFT, 105 g of RGD and 13 g of fluorophore.

(57) The novel family of fluorescent tracers proposed in the present invention makes it possible to significantly reduce costs, on the one hand, by enabling the use of less expensive materials, but also by improving the production process, thereby making it possible to do away with reaction steps.