MONO-, DI- OR POLYSACCHARIDE USED AS METAL INHIBITOR IN THE PREPARATION OF 68GA-CHELATE-FUNCTIONALIZED TARGETING AGENT

Abstract

The present invention relates the use of metal inhibitor in radiolabelling reactions using radioactive metals.

Claims

1-39. (canceled)

40. A method for radiolabelling a chelate-functionalized targeting agent for use in in-vivo PET imaging with a metal radionuclide, comprising the steps of: a) providing a chelate-functionalized targeting agent, able to chelate the metal radionuclide in a radiolabelling reaction, wherein the chelate functional group of the chelate-functionalized targeting agent is selected from the group consisting of DOTA, NOTA, NODAGA, HBED, DFO, EDTA, 6SS, B6SS, PLED, TAME, YM103 and H.sub.2dedpa; b) adding a metal inhibitor not chemically linked to said chelate-functionalized targeting agent, to the radiolabelling reaction obtained in a), said metal inhibitor being a co-chelating agent selected from the group consisting of: beta-cyclodextrin, a disaccharide and a monosaccharide, capable of inactivating metals other than metal radionuclides without interfering with the chelation between the metal radionuclide and the chelate-functionalized targeting agent, under the conditions of the radiolabelling reaction, thereby obtaining a mixture of the chelate-functionalized targeting agent of a) and the metal inhibitor of b) which are not chemically linked to each other when present in said mixture; and c) adding a metal radionuclide that is an isotope of a metal selected from the group consisting of copper, gallium, lutetium, indium, scandium, and zirconium to the mixture of a) and b) in an acetate buffer allowing to maintain the pH in the range 3-7; wherein the targeting agent of said chelate-functionalized targeting agent is selected from the group consisting of a peptide, a peptide comprising 2 to 20 amino acids, a polypeptide, an antibody, a nanobody, a diabody, an antibody fragment, a nucleic acid, an aptamer, an antisense oligonucleotide, and an organic molecule.

41. The method according to claim 40, wherein said metal inhibitor is present in micromolar amounts.

42. The method according to claim 40, wherein the radiolabelling reaction is carried out at a temperature of below 50° C.

43. The method according to claim 40, wherein the radiolabelling reaction is performed at a pH between 3 and 7.

44. The method according to claim 40, wherein the metal radionuclide is selected from the group consisting of copper-64, gallium-68, gallium-67, gallium-66, lutetium-177, indium-114, indium-111, scandium-47, scandium-44, scandium-43, and zirconium-89.

45. The method according to claim 40, wherein the monosaccharide is selected from the group consisting of: Glucose, Fructose, and D-Mannose.

46. A kit for radiolabelling of a chelate-functionalized targeting agent for use in in-vivo PET imaging comprising: a chelate-functionalized targeting agent, able to chelate a metal radionuclide in a radiolabelling reaction, wherein the chelate-functional group of the chelate-functionalized targeting agent is selected from the group consisting of DOTA, NOTA, NODAGA, HBED, DFO, EDTA, 6SS, B6SS, PLED, TAME, YM103 and H.sub.2dedpa; a metal inhibitor, which is not chemically linked to said chelate-functionalized targeting agent and which is a co-chelating agent selected from the group consisting of beta-cyclodextrin, a disaccharide and a monosaccharide, capable of inactivating metals other than metal radionuclides without interfering with the chelation between the metal radionuclide and the chelate-functionalized targeting agent, under the conditions of the radiolabelling reaction; a metal radionuclide that is an isotope of a metal selected from the group consisting of copper, gallium, lutetium, indium, scandium, and zirconium; and an acetate buffer, allowing to maintain the pH in the range 3-7, wherein the targeting agent of said chelate-functionalized targeting agent is selected from the group consisting of a peptide, a peptide comprising 2 to 20 amino acids, a polypeptide, an antibody, a nanobody, a diabody, an antibody fragment, a nucleic acid, an aptamer, an antisense oligonucleotide, and an organic molecule.

47. The kit according to claim 46, wherein said metal inhibitor is present in micromolar amounts.

48. The kit according to claim 46, wherein the metal radionuclide is selected from the group consisting of copper-64, gallium-68, gallium-67, gallium-66, lutetium-177, indium-114, indium-111, scandium-47, scandium-44, scandium-43, and zirconium-89.

49. The kit according to claim 46, wherein the monosaccharide is selected from the group consisting of Glucose, Fructose, and D-Mannose.

50. The kit according to claim 46, wherein said chelate-functionalized targeting agent and metal inhibitor are lyophilised.

51. An in vivo method for imaging a subject comprising the steps of: 1) producing a radiolabelled chelate-functionalized targeting agent using the method of claim 40; 2) administering said radiolabelled chelate-functionalized targeting agent to the subject; and 3) detecting said radiolabelled chelate-functionalized targeting agent in the subject using positron emission tomography (PET).

Description

DETAILED DESCRIPTION

[0071] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0072] The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms also encompass “consisting of” and “consisting essentially of”.

[0073] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0074] The term “about” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of and from the specified value, in particular variations of +1-10% or less, preferably +/−5% or less, more preferably +/−1% or less, and still more preferably +/−0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” refers is itself also specifically, and preferably, disclosed.

[0075] Whereas the term “one or more”, such as one or more members of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any ≥3, θ4, θ5, ≥6 or ≥7 etc. of said members, and up to all said members.

[0076] All documents cited in the present specification are hereby incorporated by reference in their entirety.

[0077] Unless otherwise specified, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions may be included to better appreciate the teaching of the present invention.

[0078] In the following passages, different aspects or embodiments of the invention are defined in more detail. Every aspect or embodiment so defined may be combined with each of the other aspects or embodiments unless stated otherwise. In particular, any feature indicated as being preferred or advantageous in one embodiment may be combined with any other embodiment or embodiments indicated as being preferred or advantageous.

[0079] The present invention overcomes one or more of the problems identified and observed in the state of the art and allows the direct radiolabeling of a chelate-functionalized targeting agents with radioactive metal.

[0080] The present invention is related to the use of a metal inhibitor for improving radiolabelling yields and reliability of radioactive metal-based radiotracer synthesis, the radiolabelling being performed with: [0081] A chelate-functionalized targeting agent, able to chelate the radioactive metal in the radiolabeling conditions; [0082] A metal inhibitor, which is a co-chelating agent, capable of inactivating metals other than radioactive metal without interfering with the chelation between the radioactive metal and the said chelate-functionalized targeting agent, under the conditions of the labelling reaction. In other words, said metal inhibitor is selected for its ability to chelate contaminating metals interfering and competing with the chelation of the radioactive metal while being mostly unable to chelate the radioactive metal in the said conditions of the labelling reaction as opposed to the chelate-functionalized targeting agent; [0083] A radioactive metal; and [0084] Optionally a buffer.

[0085] Furthermore, the present inventors have found that a metal inhibitor can be used in the radiolabeling method for neutralizing, at least partially, interfering species and allowing the radioactive metal to react with the chelate-functionalized targeting agent. These metal inhibitors may temporarily or permanently remove metals that compete with radioactive metal for the reaction with the chelate-functionalized targeting agent. Said metal inhibitor is thus unable to chelate the radioactive metal in the said conditions of the labelling reaction, but chelates other metals interfering with the chelation of radioactive metal by the chelate-functionalized targeting agent. The presence of metal inhibitors during the radiolabeling reaction provides an advantageous alternative to current approaches for managing the presence of metallic impurities such as increasing the amount of chelate-functionalized targeting agent or the pre-treatment of the eluate of the generator, these additional purification steps consume time (and radioactivity).

[0086] The aspects as described herein advantageously allow to obtain an appropriate chelation yield, particularly above 90%, and therefore a sufficient radiochemical purity without any preliminary or further final purification.

[0087] The presence of a chelate-functionalized targeting agent, optionally a buffer and a metal inhibitor in the labelling medium advantageously allows to directly transfer the radioactive metal to the targeting agent and to perform the radiolabeling reaction without the need for any prior or subsequent operation or purification.

[0088] In addition, all kit components as described herein can be lyophilized altogether or frozen which ensures a longer shelf life.

[0089] Thus, the main advantages of the invention as disclosed herein that differentiate from the state of the art are: [0090] The possibility of radiolabeling without the need for an automated synthesizer; [0091] The possibility of a radiolabelling without the need for heating; [0092] The presence of a metal inhibitor which advantageously allows to use less chelate-functionalized targeting agent and allowing the implementation of more affordable radiopharmaceutical synthesis; and [0093] The presence of a metal inhibitor which advantageously allows improving the radiolabelling 1.5 yields.

[0094] Metal inhibitors used in the present invention are selected for their ability to block the competing metals in the radiolabelling reaction without inhibiting the radioactive metal ions in their chelation reaction with the chelate-functionalized targeting agent. Indeed, these metal inhibitors should not interfere negatively on the main radiolabeling reaction or lead to the formation of secondary radiolabeled species. In other words metal inhibitors should have a limited or no capacity to complex radioactive metal in the conditions used for the radiolabelling reaction. Limited means at least 100 times less then the chelating agent used for the radiolabelling of the chelate-functionalized targeting agent.

[0095] It is interesting to note that the function of metal inhibitors in the present invention is the opposite of the function of the sequestering agents used in the prior art. Indeed, according to known methods, at the end of the labelling reaction, a sequestering agent having a particular affinity for e.g. the radioactive gallium may be added to chelate the unreacted portion of the isotope, whereas, according to the present invention an agent capable of reducing the competition of metallic impurities other than the radioactive metal is added at the beginning of the reaction.

[0096] As used herein, an “inhibitor of metal” refers to any molecule capable of interacting with, or competing metals, or the chelating moiety of the chelate-functionalized targeting agent or with radioactive metal directly, to inhibit wholly or partially the chelation the chelate-functionalized targeting agent said competing metals and/or promote the chelating of radioactive metal by said targeting agent.

[0097] Metal inhibitors are preferably selected from the group of sugars. Sugars used as agents metal inhibitors in the kit of the invention can be monosaccharides or derivatives of monosaccharides such as tetracetose, pentacetose, hexacetose, tetrose, pentose, hexose, D-mannose, D-fructose, and derivatives; and/or disaccharides and their derivatives such as maltose and its derivatives; and/or polysaccharides and their derivatives such as dextrins, cyclodextrins, sulfated sugars, cellulose and derivatives thereof.

[0098] Preferably, the metal inhibitor is present in the kit as described herein in micromolar amounts, preferably in nanomolar quantities, preferably in an amount of less than 500 nanomolar, still more preferably in an amount less than 100 nanomoles.

[0099] The metal inhibitory agent may also be chemically bound to the chelate-functionalized targeting agent. This chemical bond can or cannot be a labile bond under the conditions of radiolabeling with the chelate-functionalized targeting agent like manner as in the conditions of radiolabeling the metal inhibitor is formed and released in situ.

[0100] As used herein, a “chelate-functionalized targeting agent” refers to a targeting agent capable of being labelled with a radioisotope such as for example radioactive metal, by means of an chelation agent to which this targeting agent is bound.

[0101] Preferred chelation agents for functionalizing a targeting agent to be radiolabeled with radioactive metals are those which form stable complexes at least for a time sufficient for diagnostic investigations using radiolabelled targeting agents. Suitable chelating agents include aliphatic amines, linear or macrocyclic such as macrocyclic amines with tertiary amines. While these examples of suitable chelating agents are not limited, they preferably include the DOTA and its derivatives, such as, DOTAGA, TRITA, DO3A-Nprop, BisDO3A and TrisDO3A; DTPA and its derivatives such as tetra-tBu-DTPA, p-SCN-Bz-DTPA, MX-DTPA and CHX-DTPA; NOTA and its derivatives, such as TACN, TACN-TM, DTAC, H3NOKA, NODASA, NODAGA, NOTP, NOTPME, PrP9, TRAP, Trappist Pr, NOPO, TETA; chelates open chain such as HBED, DFO, EDTA, 6SS, B6SS, PLED, TAME, YM103; NTP (PRHP) 3; the H2dedpa and its derivatives such as H2dedpa-1, 2-H2dedpa, H2dp-bb-NCS, and H2dp-N-NCS; (4,6-MeO2sal) 2-BAPEN; and citrate and derivatives thereof.

[0102] The chelate-functionalized targeting agent can be a peptide, for example, a peptide comprising 2 to 20 amino acids, a polypeptide, a protein, a vitamin, a saccharide, for example a monosaccharide or a polysaccharide, an antibody, nucleic acid, an aptamer, an antisense oligonucleotide, or an organic molecule.

[0103] Chelate-functionalized targeting agent as described herein preferably have a capacity of biological targeting. Non-limiting examples of suitable targeting agents include molecules that target VEGF receptors, analogs of bombesin or GRP receptor targeting molecules, molecules targeting somatostatin receptors, RGD peptides or molecules targeting αvβ3 and αvβ5 annexin V or molecules targeting the apoptotic process, molecules targeting estrogen receptors, biomolecules targeting the plaque . . . . More generally, a list targeting molecules, organic or not, functionalized by a chelating can be found in the journal of Velikyan et al., Theranostic 2014, Vol. 4, Issue 1 “Prospective of 68Ga-Radiopharmaceutical Development.”

[0104] The term “radioactive metal” as used herein for radioactive labelling of the functionalised targeting agent(s) encompasses all radioactive metal ions suitable for use in medical imaging or radionuclides therapy. The radioactive metals are typically radioisotopes or radionuclides such as: copper-64, gallium-68, gallium-67, gallium-66, lutecium-177, yttrium-86, yttrium-90, indium-114, indium-111, scandium-47, scandium-44, scandium-43, zirconium-89, bismuth-213, bismuth-212, actinium-225, lead-212, rhenium-188, rhenium-186, rubidium-82 and the like. Many of these radionuclides are issued from nuclear reactor sub-products, cyclotron or from their specific radionuclide generator. In one embodiment, the radioactive metal is not gallium-68.

[0105] The term “radioactive metal” as used herein for radioactive labelling of the functionalised targeting agent(s) also encompasses metal linked to a radioactive species such as for example Fluorine-18-based metallic fluorides.

[0106] After addition of the radioactive metal solution to the metal inhibitor and the chelate-functionalized targeting agent, optionally containing a buffer, the solution obtained is left to the radiolabeling reaction for a short period of time, in particular between about 2 minutes and about 60 minutes, preferably from about 2 minutes to about 30 minutes, for example about 15 minutes.

[0107] The invention also discloses a radiolabeled targeting agent with radioactive metal, obtained by a method as described herein.

EXAMPLES

Example 1: Ga-68 Generator E & Z/NODAGA Peptide without Metal Inhibitor

[0108] Labelling a Peptide with a 68Ga Eluate of 5 ML of 0.1 M HCl

[0109] A solution of 1850 MBGa-68 of 6 (Eckert & Ziegler) is eluted with 5 mL of 0.1M HCl (Ultrapure grade) directly into a flask containing 150 mg of sodium acetate (Ultrapure grade) lyophilized, 240 μl of HCl 3M (Ultrapure grade), 760 μl of Milli-0 and 50 μg lyophilized NODAGA-NOC. The flask was left for 10 min at room temperature. The product is obtained with a radiochemical purity of 64% according to TLC analysis of the reaction medium.

Similarly to What was Done in Example 1 Different Combinations were Tested and are Summarized in the Table Below:

TABLE-US-00001 Chelating agent use in the chelate- Radiolabelling yield vs Radioactive functionalized Metal Labelling radiolabelling yield # metal targeting agent Inhibitor conditions without inhibitor 1 Cu-64 DOTA 25 μg Glucose 10 minutes, 65° C. 82% vs 51% 2 Ga-58 NODAGA 25 μg Fructose 10 min, R.T. 97% vs 61% 3 Ga68 NOTA 25 μg Beta- 10 minutes, RT 83% vs 51% cyclodextrin 4 Sc-47 DOTA 85 μg Beta- 30 minutes, 60° C. 85% vs 64% cyclodextrin S Zr-89 DFO Beta- 30 minutes, RT 91% vs 77% cyclodextrin 6 Lu-177 DOTA 100 μg Fructose 30 minutes, 65° C. 94% vs 77% 7 In-111 DOTA 100 μg Fructose 30 minutes, 65° C. 9 Lu-177 NODAGA 85 μg Beta- 30 minutes, 40° C. 95% vs 55% cyclodextrin 9 Lu-177 DOTA 100 μg Beta- 30 minutes, 40° C. 87% vs 51% cyclodextrin 10 Ga-58 NODAGA 25 μg D-Mannose 10 minutes, RT 97% vs 76% 11 Ga-68 DOTA 100 μg D-Mannose 30 minutes, RT 80% vs 15% 13 Ga-68 NODAGA 25 μg Fucoidan 10 minutes, RT 93% vs 76%

[0110] Said targeting agent and metal inhibitor were present in a buffer consisting of phosphate, nitrate. HEPES, acetate. TRIS, ascorbate, or citrate, or a mixture thereof.