NZ708281B2 - Process for the preparation of complexes of 68ga - Google Patents

Abstract

Disclosed is a process for the preparation of radiolabeled Gallium complexes prepared from 68Ga and chelator molecules (e.g., DOTA, NOTA, PCTA) in an aqueous buffer solution in the presence of compounds capable to sequester metal cations (e.g., phenantroline, crown ethers) to remove competing metal ion impurities, with the proviso that the buffer is not a formic acid/formate buffer solution. ion impurities, with the proviso that the buffer is not a formic acid/formate buffer solution.

Description

Process for the preparation of complexes of 68Ga.

Field of the invention The ion deals with processes for preparing complexes containing isotOpes, in particular complexes useful as radiomarkers containing the isotope 6863.

State of the art Despite the encouraging results of recent clinical studies using abelled radiotracer for PET g in vivo, the short half-life of the isotope (68 minutes) that doesn’t allow a ange distribution together with the need of an equipped “production radiopharmacy” for the labeling process still prohibit their widespread use in nuclear medicine routine.

The labeling with Ga-68 is carried out by complexing the radioactive metal with a suitable chelator in a reaction medium into which are introduced the radioactive dose of 686a driving from the elution of the “Ga generator, the amount of the le to be labeled (referred as chelator—functionalized molecule or precursor in our ation) and a le buffer to assure the optimal pH for the complexation.

The so called 68Ga generator is a resin commercially available and containing Germanium from which the wanted 686a is lly formed by Germanium decay; therefore the elution of the resin, under the appropriate pH conditions, and in the presence of a cheiator—functionalized molecule allows the formation of the wanted complex containing 68Ga; depending on the selected chelator-functionalized molecule , g at 75-90°C can be necessary.

The main limits to the success of the labeling are provided by the fact that the suitable pH must be kept constant and by the competition of the ic impurities with the Get-68 during the complexation process.

In view of the above said, the ch of a suitable buffer capable of assuring a standard pH is obviously a topic subject continuously investigated by those skilled in the 68Ga-labelling and still open.

Such a buffer should be nontoxic, able to buffer in the pH range of 3.550, should not compete with gallium ions and preferentially have a weak metal complexing capacity.

Among the different buffer reported, the ones mainly used up to now are HEPES (sulfonic acid derivative) or acetate buffers; however, they allow working only in a strictly defined range of pH (Publication of Velikyan et al., Bioconjugate Chem., 2008, 19, 569-573) and may no longer retain the required buffer capacity when the eiuate acidity slightly varies.

For example, even a little increase in the eiuate volume coming from the tor cause the pH to turn to values which damage the xation resulting in high amount of free Ga-68. This produces a risk of non-compliance that makes the final purification mandatory. Moreover, about the HEPES buffer no toxicological data are available: the final cation has to be performed also in order to remove, or at least reduce, the HEPES before the administration of the harmaceutical.

Others buffers have been recently proposed () as efficient solution for the Ga-68 complexation, for instance lactate, tartrate and carbonate buffers. These buffers comprise at least two Ga-68 coordination functions ming the prejudice that they could interfere with the labeling. Anyway their use has been successfuily tested with reduced and purified fractions of the generator eluate, without exempting from the pre-labeling treatment of the Ga-68 solution A second important limit is the competition of ic impurities, mainly trivalent and bivalent cations deriving both from the stationary phase and from the Ga-68 decay (Zn). These metals are bound as well as the Ga-68 by the chelator- functionalized molecule reducing the number of molecules actually available for the labeling. This can result in an incomplete complexation of the Ga-68 reducing the final radiochemical purity of the preparation. In the prior art, sometimes the Ga- 68 not xed by the chefator—functionalized molecule during the labeling, is completely tered with the post-labelling addition of an excess of a chelator with ized affinity for the isotope, (eg. the EDTA chelator) in order to avoid the presence of high portion of free metals and to promote their elimination in case of administration of the radiopharmaceutical preparation ( - Example 2). A partial Ga-68 complexation might be differently faced ng from higher amounts of cheiator~functionalized molecule . However, an increase of the amount of chelated precursor produces an undesirable ion of the specific radioactivity (ratio n the radioactive product and the not labeled product) that can worsen the diagnostic results. in fact, due to competition with the labeled molecule for the same receptor, the presence of unlabeled molecule may have a negative effect on the concentration of radioactivity in the target tissue. Hence, a high SRA (Specific Radioactivity) might be critical for providing a sufficient contrast in PET images between the target tissue and its surrounding. In the state of art, the presence of competing metallic ions is usually reduced by pre-purification or fractionation of the eluate before the iabeling (as described by the patent N°WO 2010/092114), but these steps provide a disadvantageous loss of starting activity.

Moreover, if pre-iabeliing steps as well as the final purification cannot be d, the Ga-68 labeiing will be aiways based, in some extent, on the automation, by using a synthesis module, making the kit strategy unfeasible. Beside the technical expertise needed, this require unfavorable prolonged time for the labeling. Due to short half-life of the radionuclide (t1,2=68 minutes) and the limited activity provided by the generator, any improvement aimed to obtain a very rapid, direct and high- yielding compiexation is highly desirabie.

From all the above said it is clearthe need of a process ng the preparation of 68Ga compiexes ming the above said problems.

Summary of the ion A process for the preparation of complexes containing 68Ga wherein a buffer formic acid/formate, ly in the presence of compounds capable to sequester metal cations, is used in the compiexion reaction, is described.

Detailed ption of the invention The present invention allows to overcome the above said m through a s wherein the Ga-68 is effectively complexed by a chelator—functionalized molecule in an s buffer formic acid/formats.

The above said buffer formic acid/formats not only allows to establish the right pH but also to tolerate the eluate /acidity variation.

In fact, its buffering ty is centered at a pH value suitable for the Ga-68 complexation and it has no metal complexing capacity, so it doesn’t provide erence with the labeling. Moreover, this buffer shouid be compatible with the pharmaceutical application because the formic acid is classified as class 3 (solvents with low toxic potential) residual solvent in the Pharmacopoeia for which a limit of 5 mg/ml (5000 ppm) is ed.

Normally as e sodium formate is preferred but aiso any other ic salt of the formic acid can be used.

The ratio formic ormats is normally comprised between 1 and 3.5.

Moreover, in order to face the problem of the presence of metallic impurities, instead of increasing the amount of chelator-functionalized molecuie (providing a reduction of the SRA) or pre-treating the generator eluate with time- and radioactivity—consuming purification steps, as it is the normal praxis in the art, it was found that sequestering agent can be used in the process in order to neutralize the ering species leaving the Ga-68 more free to react with the chelator functionalized molecule.

These sequestering agents, if present. act as t chelator—functionaiized molecule that temporarily or permanently subtract the competing metals to the on with the cheiated-functionalized molecules. it is worth noticing that the function of the sequestering agents in the present ion is opposite to the function of the sequestering agents used in the prior art, as described above.

In fact, according to the known procedures, at the end of the labeling a sequestering agent with particuiar affinity for the gallium can be added in order to chelate the not reacted portion of the isotope, while, according to the present invention, a sequestering agent able to minimize the competition of metallic impurities is added at the beginning of the reaction. sly the sequestering agents used in the present invention should bind preferentialiy the competing metals rather than Ga-68 ion in order to avoid the interference with the main labeling reaction or the formation of e labeled species.

Moreover, according to a particular embodiment, the invention refers also to processes for complexing radioisotopes, and in particular 586a, wherein buffered solutions are used in combination with sequestering agents as above and hereinafter described.

According to the invention with or—functionalized molecules it is intended any molecule with targeting ability functionalized with a chelate able to complex radioactive isotopes such as Ga-68. red chelates for the complexation of Ga-68 according to the invention can be chosen among: DOTA and its derivatives, NOTA and its derivatives, PCTA and its tives.

Use may also be made, in general, of any cheiate able to form a sufficiently stable cage around Ga“ in particular any aliphatic, macrocyclic or linear amine, or macrocycie amine with tertiary .

As molecule with targeting ability it is intended a molecule able to target a biological process of diagnostic or therapeutic st, advantageously an amino acid, a peptide, advantageously sing 4 to 15, or 4 to 10 amino acids, a polypeptide, a protein, a vitamin, a monosaccharide or polysaccharide, an antibody, a nucleic acid or an aptamer.

Among the molecules with targeting ability useful for the invention, we can mention (as example and not as limiting list): - Molecules targeting VEGF receptors - Bombesin analogs or molecules ing GRP receptors - Molecules targeting somatostatin ors - RGD peptides or av[33 and owBS ing molecules - Annexin V or molecules targeting apoptotic processes - Molecules targeting estrogen receptors - Molecules targeting atheroma plaque - The targeting molecules recalled in Topics in Current Chemistry, voi.222, 260-274, Fundamentals of Receptor-based Diagnostic Metailopharmaceuticals, The sequestering agents, if present, are preferably chosen in the group consisting of: - glycine and other cheiating aminoacids (for example methionine, cystein, etc...) - crown ethers and nitrogen crown ethers - eterocyciic organic compound e.g. 1,10—phenantroline, 2,2'—Bipyridine - caiixarenes - polydentate cheiator e.g. proteins, polysaccharides, and polynucleic acids - l cheiating agents e.g. catechins, tannin, porphyrin - in general linear or macrocyciic chelating agents (for example podands or kryptands) Normally micromolar or, more advantageously nanomoiar amounts of sequestering agent are used preferably less than 100 nanomolar, for example in a range of 20 and 25 nanomolar.

It is important to note that the sequestering agents as above explained can be advantageously utilized also in complexing reaction wherein other buffers are used.

Therefore it is another embodiment of the present invention a process sing herein complexing on of radioactive es, in particular 686a, wherein sequestering agents as above defined are added to the reaction buffer.

Preferably the complexing reaction is carried out in a pH range between 3 and 4.5, more preferably between 3.2 and 4.2, most preferably between 3.4 and 4.0.

The complexes obtained according to the process bed above are also an embodiment of the present invention; they can contain formic acid/formate below mg/ml and the sequestering agent (if used) below 100 nmols.

As said a commercial generator sting of a column of resin bearing ium) is eluted with an eluent containing an acid (normaliy HCL) ly into a vial containing buffer e and a base.

A chelator—functionaiized molecule (normally in the ce of a metals sequestering agent, as for example phenanthroline) is added into the vial and the reaction vial is heated for a short time; the product solution is collected and checked by reversed phase HPLC and ITLC (MeOH/ammonium e 1M 1/1).

The addition order can also be inverted.

For example the commercial generator can be eluted with an eluent containing an acid (normally HCI) directly in a vial containing a chelator—functionalised molecule rably in the presence of a metal sequestering agent, as for example a phenanthroiine).

The formate buffer and the base are added in the vial and the on mixture is heated for a short time.

The acid eluate is normally tuted by an aqueous solution of a strong acid as for exampie HCI, while the base is an aqueous solution of a strong base as for example NaOH.

On the whole, the use of formate buffer guarantees a suitable pH even if variations in the eluate acidity occur and, in this way reduces, the amount of not complexed Ga-68 due to a too iow or a too high pH resulting in high content of free 686a3+ or “(Be ides respectively. Moreover the on of a sequestering agent allows to bring down the amount of chelator-functionalized molecule needed to obtain a complete Ga—68 complexation.

These two aspects enabled the applicant to e a suitable degree of complexation, advantageously at least 92%, 95% and 97%, and consequently a sufficient purity (at least 92%, 95% and 97%) without any kind of pre- or final purification. Since the results obtained confirm the feasibility of a direct Ga—68 labeling that t require manipulation or purification, the formulation can be applied to the production of a ic kit.

Therefore, according to a particular embodiment the ion reiates also to a kit comprising: - a siliconized vial containing the chelator—functionalized molecule and the selected sequestering agent; a siliconized viai or a syringe containing a suitable ultra-pure formic acid/ sodium formate mixture.

Moreover the ion relates also to a single vial containing the chelator— functionaiized molecule, the selected sequestering agent and a suitable uitra-pure formic acid/sodium formate mixture.

Example 1 “GaDOTApeptide labelling with 3 ml HCI 0.6M eluate A 30 mCi cial generator (from IDB) having a SnOz stationary phase was eluted with 3 ml eluate of ultrapure HCI 0.6 M directly into a vial containing 200 ul of ultrapure buffer formate 1.5 M and ultrapure 400 ul of NaOH 4.5 M. Then 30 of DOTA-peptide and 4.5 ug of 1,10-phenantroline are added and the reaction vial is heated at 95°C for 7 minutes. The product was checked by reversed phase HPLC and ITLC ammonium acetate 1M. 1/1) and the radiochemical purity resulted 98% in both tests.

Example 2 GBGaDOTApeptide labelling with 3.2 ml HCI 0.6M eluate A 30 mCi commercial generator (from IDB) having a SnOz stationary phase was eluted with 3.2 ml eluate of ultrapure HCI 0.6 M directly into a vial containing 200 ul of ultrapure buffer formate 1.5 M and ultrapure 400 ul of NaOH 4.5 M. Then 30 ug of DOTA-peptide and 4.5 ug of 1,10-phenantroline are added and the reaction vial is heated at 95°C for 7 s. The product was checked by reversed phase HPLC and lTLC (MeOH/ammonium acetate 1M. 1/1) and the radiochemical purity resulted 97% in both tests.

Example 3: ssGaDOTApeptide labelling with 3 ml HCI 0.6 M eluate A 30 mCi commercial generator (from lDB) having a SnOz stationary phase was eluted with 3 ml eluate of ultrapure HCI 0.6 M ly into a vial containing 200 ul of ure buffer formate 1.5 M and ultrapure 400 ill of NaOH 4.5 M. Then 30 ug of DOTA—peptide and 15 ug of 12-crown—4 are added and the reaction vial is heated at 95°C for 7 minutes. The product was checked by reversed phase HPLC and ITLC (MeOH/ammonium acetate 1M. 1/1) and the radiochemical purity ed respectively 98% and 96%.

Example 4: SBGaDOTApeptide labelling with 3 ml HCI 0.6 M eluate A 30 mCi commercial generator (from IDB) having a SnOz stationary phase was eluted with 3 ml eluate of ultrapure HCI 0.6 M directly into a vial containing 30 of eptide and 15 ug of 12-crown—4 . Then 200 ill of ultrapure buffer formate 1.5 M and uttrapure 400 u! of NaOH 4.5 M are added and the reaction vial is heated at 95°C for 7 minutes. The product was checked by ed phase HPLC and ITLC (MeOH/ammonium acetate 1M. 1/1) and the radiochemicai purity resuited respectively 98% and 96%.

Claims (14)

Claims

1. A process for the preparation of complexes of 686a wherein a complexing reaction between a chelator—functionalized molecule and 686a is carried out in a buffered aqueous solution having a pH range of 3.5-5.0, wherein said complexing reaction is carried out in the presence of a compound capable of sequestering metal cations added at the beginning of the complexing reaction and with the proviso that said buffered aqueous solution is not a formic acid/formate buffer.

2. The process according to Claim 1 wherein the buffers are selected from the group consisting of: sulfonic acid derivatives, acetate buffers, lactates, tartrates 10 and carbonate s.

3. The process ing to any one of Claim 1 or 2 wherein said chelator— functionalized molecule is selected from the group ting of: DOTA and its tives, NOTA and its derivatives, PCTA and its derivatives.

4. The process according to any one of Claim 1 or 2 n said the sequestering 15 agent is selected from the group consisting of: glycine and other chelating amino acids, crown ethers and nitrogen crown ethers, heterocyclic organic compounds, calixarenes, ntate chelators, catechins, tannins, porphyrinins, linear or macrocyciic chelating agents.

5. The process according to Claim 1 wherein the complexing reaction is carried 20 out in a pH range between 3 and 4.5.

6. The s according to Claim 5 wherein the reaction pH range is between 3.2 and 4.2.

7. The process according to Claim 6 wherein the reaction pH range is between 3.4 and 4.0. 25

8. The process according to any one of Claims 1 to 7 wherein: - a commercial generator of 68Ga is eluted with an eluate containing an acid directly into a vial ning the buffer and a base; — a sequestering agent is added; - a chelator~functiona|ized moiecule is added into the vial and the reaction vial is 30 heated for a short time; - the product is collected.

9. The process according to any one of Claims 1 to 7 wherein: - a ciai generator of 6"Ga is eluted with an eluate containing an acid directly into a vial containing a chetator—functionalized molecuie ; - a sequestering agent is added - a buffer and a base are added into the vial and the reaction vial is heated for a short time; - the product is collected.

10. The process according to Claim 8 or 9 wherein the acid eluate is an aqueous 10 solution of HCi, while the base is an aqueous solution of NaOH.

11. A on kit comprising: - a vial containing the cheiator—functionalized molecule and a compound capable of sequestering metal cations; - a vial or a syringe containing a suitable ultra—pure buffer solution. 15

12. A vial containing a chelator—functionalized molecule, a selected nd capable of sequestering metai cations and a suitable ultra—pure buffer solution

13. The reaction kit according to Claim 11 and the viai according to claim 12 n said vials are siliconized vials.

14. xes of 686a, obtained by the process, according to any one of Claims 1 20 to 10, wherein the complexes contain less than 10 mg/ml buffer solution and, less than 100 nmois of sequestering agent.