NZ622071B2 - 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 a buffer formic acid/formate aqueous solution in the presence of compounds capable to sequester metal cations.

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 6363.

Despite the encouraging results of recent clinical studies using GBGa-labelled radiotracer for PET imaging in vivo, the short half-life of the isotOpe (68 minutes) that doesn’t allow a long-range distribution together with the need of an ed “production radiopharmacy" for the labeling process still prohibit their read useinnudearmedcmeroufine 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 “Ga driving from the elution of the 686a generator, the amount of the molecule to be labeled (referred as chelator-functionalized le or precursor in our application) and a suitable buffer to assure the optimal pH for the complexation.

The so called 68Gua generator is a resin cially available and containing Germanium from which the wanted “Ga is naturally formed by Germanium decay; therefore the elution of the resin, under the appropriate pH conditions, and in the presence of a chelator-functionalized le allows the formation of the wanted complex containing 68Ga; ing on the selected chelator-functionalized molecule at 75-90°C can be necessary.

The main limits to the s of the labeling are provided by the fact that the le pH must be kept constant and by the competition of the ic impurities with the Ga—68 during the complexation process. in view of the above said, the research 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 3.5-5.0, should not compete with gallium ions and preferentially have a weak metal complexing capacity.

Among the ent 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, 3) and may no longer retain the required buffer capacity when the eluate acidity slightly .

For example, even a little increase in the eluate volume coming from the generator cause the pH to turn to values which damage the complexation 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 purification has to be performed also in order to remove, or at least reduce, the HEPES before the administration of the radiopharmaceutical.

Others buffers have been recently proposed () as efficient solution for the Ga-68 xation, for instance lactate, tartrate and carbonate buffers. These buffers comprise at least two Ga—68 coordination functions overcoming the prejudice that they could interfere with the labeling. Anyway their use has been successfully tested with reduced and purified fractions of the generator eluate, t exempting from the pre-labeling treatment of the Ga-68 solution A second important limit is the competition of metallic 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 ng the number of molecules actually available for the labeling. This can result in an incomplete complexation of the Ga—68 reducing the final hemical purity of the preparation. In the prior art, sometimes the Ga- 68 not complexed by the chelator-functionalized le during the labeling, is completely sequestered with the post-labelling addition of an excess of a chelator with ized affinity for the e, (e.g. 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 ( — 3O Example 2). A partial Ga-68 xation might be ently faced starting from higher amounts of chelator-functionalized molecule of the . However, an increase amount of cheiated precursor produces an undesirable reduction of the specific radioactivity (ratio between the radioactive product and the not d product) that can worsen the stic results. In fact, due to competition with the d molecule for the same receptor, the presence of unlabeled molecule may have a negative effect on the concentration of radioactivity in the target . Hence, a high SRA (Specific Radioactivity) might be critical for providing a ient contrast in PET images between the target tissue and its surrounding. in the state of art, the presence of ing metallic ions is usually reduced by pre-purification or fractionation of the eluate before the labeling (as described by the patent N‘WO 2010/092114), but these steps provide a disadvantageous loss of starting activity.

Moreover, if pre-labeiling steps as well as the final purification cannot be avoided, the Ga-68 labeling will be always based, in some extent, on the automation, by using a synthesis module, making the kit strategy ible. Beside the technical expertise needed, this e unfavorable prolonged time for the labeling. Due to short ife of the radionuclide (t1l2=68 s) and the limited activity provided by the generator, any improvement aimed to obtain a very rapid, direct and high- yielding complexation is highly desirable.

From all the above said it is clear the need of a process allowing the preparation of “Ga complexes overcoming the above said problems.

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

Detailed description of the invention The present invention allows to overcome the above said problem through a process wherein the Ga-68 is ively complexed by a cheiator-functicnalized molecule in an aqueous buffer formic acid/formate.

The above said buffer formic acid/formats not only allows to establish the right pH but also to tolerate the eluate volume/acidity variation. in fact, its buffering capacity is centered at a pH value suitable for the Ga-68 complexation and it has no metal complexing capacity, so it doesn't provide interference with the labeling. Moreover, this buffer should be ible with the pharmaceutical application e the formic acid is classified as class 3 (solvents with low toxic potential) residual solvent in the copoeia for which a limit of 5 mg/ml (5000 ppm) is ed. ly as e sodium tormate is preferred but also any other metallic salt of the formic acid can be used.

The ratio formic acid/formate is normally comprised between 1 and 3.5.

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

These sequestering agents, if present, act as support chelator-functionalized molecule that temporarily or permanently subtract the competing metals to the reaction with the chelated-functionaiized les.

It is worth noticing that the function of the sequestering agents in the t invention is opposite to the function of the tering 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 particular 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.

Obviously the sequestering agents used in the present invention should bind preferentially the competing metals rather than Ga-68 ion in order to avoid the interference with the main ng on or the formation of by-side labeled species.

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

According to the invention with chelator-functionalized molecules it is intended any molecule with ing ability functionalized with a chelate able to complex radioactive isotopes such as Ga—68.

Preferred chelates for the xation of Ga-68 according to the invention can be chosen among: DOTA and its derivatives, NOTA and its tives, PCTA and its derivatives.

Use may also be made, in general, of any chelate able to form a sufficiently stable cage around Ga3*' in particular any aliphatic, macrocyclic or linear amine, or ycle amine with tertiary amines.

As le with targeting ability it is intended a molecule able to target a biological process of diagnostic or therapeutic interest, advantageously an amino acid, a peptide, advantageously comprising 4 to 15, or 4 to to 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 ion, we can mention (as example and not as limiting list): - Molecules targeting VEGF ors - Bombesin analogs or molecules targeting GRP receptors - Molecules targeting somatostatin receptors - RGD es or de3 and avBS targeting molecules - Annexin V or molecules targeting apoptctic processes — Molecules targeting estrogen receptors - les targeting atheroma plaque ~ The targeting molecules recalled in Topics in t Chemistry, vo|.222, 260-274, Fundamentals of Receptor-based Diagnostic Metallopharmaceuticals, The sequestering agents, if present, are preferably chosen in the group consisting — glycine and other chelating aminoacids (for example methionine, cystein, etc...) - crown ethers and nitrogen crown ethers - eterocyclic organic compound e.g. 1,10-phenantroline, 2,2‘-Bipyridine — calixarenes - polydentate chelator e.g. proteins, polysaccharides, and polynucleic acids - natural chelating agents e.g. catechins, tannin, porphyrin - in general linear or macrocyclic ing agents (for e podands or nds) Normally micromolar or, more advantageously nanomolar amounts of sequestering agent are used preferably less than 100 nanomolar, for e 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 on wherein other buffers are used.

Therefore it is another embodiment of the present invention a process comprising herein complexing reaction of radioactive isotopes, in particular £53Ga, 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 described above are also an embodiment of the present invention; they can contain formic acid/formate below mg/mt and the sequestering agent (if used) below 100 nmols.

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

A chelator-functionalized molecule (normally in the presence 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 ted and checked by reversed phase HPLC and lTLC (MeOH/ammonium e 1M 1/1).

The addition order can also be inverted. 3O For example the cial generator can be eluted with an eluent containing an acid (normally HCI) directly in a vial containing a or—functionalised molecule (preferably in the presence of a metal sequestering agent, as for e a throline).

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

The acid eluate is normaiiy constituted by an aqueous solution of a strong acid as for example HCi, white the base is an aqueous solution of a strong base as for example NaOH.

On the whoie, the use of formate buffer guarantees a le pH even if variations in the eiuate acidity occur and, in this way reduces, the amount of not complexed Ga-68 due to a too low or a too high pH resulting in high content of free 68Get“ or 68Ga hydroxides respectively. Moreover the addition of a sequestering agent allows to bring down the amount of chelator-functionalized moiecule needed to obtain a complete Ga—68 complexation.

These two aspects enabled the applicant to achieve 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 ility of a direct Ga-68 labeling that doesn’t require manipulation or purification, the formulation can be applied to the production of a specific kit.

Therefore, according to a particuiar ment the ion relates also to a kit comprising: - a nized viai containing the or—functionalized molecule and the selected sequestering agent; a nized vial or a syringe containing a suitable ultra-pure formic acid/ sodium formate mixture.

Moreover the invention relates also to a single vial containing the chelator- functionalized moiecule, the selected sequestering agent and a suitable pure formic acid/sodium formate mixture.

Example 1 3O asGaDOTApeptide labelling with 3 ml HCI 0.6M eluate A 30 mCi commercial generator (from IDB) having a SnOe stationary phase was eluted with 3 mi eluate of ultrapure HCI 0.6 M directly into a vial containing 200 ul of ultrapure buffer formate 1.5 M and ure 400 U! of NaOH 4.5 M. Then 30 ug of DOTA-peptide and 4.5 ug of 1,10-phenantroiine 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 iM. t/1) and the radiochemical purity resulted 98% in both tests.

Example 2 “GaDOTApeptide iabelling with 3.2 ml HCI 0.6M eluate A 30 mCi commercial generator (from IDB) having a Sn02 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 e 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 on 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 resulted 97% in both tests.

Example 3: 68GaDOTApeptide tabelling 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 200 ul of ultrapure buffer formate 1.5 M and uitrapure 400 ul of NaOH 4.5 M. Then 30 ug of DOTA-peptide and 15 ug of wn-4 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 radiochemical purity resulted respectively 98% and 96%.

Exampte 4: 68GaDOTApeptide labelling with 3 ml HCI 0.6 M eluate A 30 mCi commercial generator (from IDB) having a Sn02 stationary phase was eluted with 3 mi eluate of ultrapure HCI 0.6 M directly into a vial ning 30 ug of eptide and 15 ug of 12-crown-4 . Then 200 ui of ultrapure buffer formate 1.5 M and uitrapure 400 ul of NaOH 4.5 M 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 hemica! purity resulted respectively 98% and 96%.

Claims (14)

Claims

1. Process for the preparation of complexes of 68Ga wherein the complexing reaction between a chelator—functionalized molecule and 686a is carried out in a buffer formic acid/ e aqueous solution possibly in the presence of a compound capable of sequestering metal s, wherein said compound able to sequester metal cations, if used, is added at the beginning of the complexing reaction.

2. A process according to Claim 1 wherein said chelator—functionaiized molecule is chosen in the group consisting of: DOTA and its derivatives, NOTA and its derivatives, PCTA and its derivatives. white said formate is sodium formate.

3. A s according to Claims 1 and 2 wherein the ratio formic acid/formate in the labeling mixture is comprised between 1 and 3.5.

4. A process ing to Claim 1 wherein said the sequestering agent is chosen in the group consisting of: glycine and other chelating aminoacids, crown ethers and nitrogen crown ethers, heterocyclic organic nd, renes, poiydentate chelator, natural chelating agents eg. catechins, tannin, porphyrinin, linear or macrocyciic chelating .

5. A process according to Claim 1 n the xing reaction is carried out in a pH range between 3 and 4.5.

6. A process according to Claim 5 wherein the reaction pH is comprised between 3.2 and 4.2.

7. A process according to Claim 5 wherein the reaction pH is n 3.4 and 4.0.

8. Process ing 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 containing buffer formate and a base; - a chelator-functionalized molecule is added into the vial and the reaction vial is heated for a short time; - the product is collected.

9. Process according to any one of Claims 1 to 7 wherein: - a commercial generator of 636a is eluted with an eluate containing an acid directly into a vial containing a chelator—functionalized molecule; 2282809vl — buffer e and a base are added into the vial and the reaction vial is heated for a short time; - the product is collected.

10. Process according to Claim 8 or 9 wherein the acid eluate is an aqueous solution of HCl, white the base is an aqueous solution of NaOH.

11. A reaction kit comprising: - a vial containing the chelator—functionaiized molecule and a compound capable of sequestering metal cations; - a vial or a syringe containing a suitable ultra-pure formic acid/ sodium formate mixture.

12. A vial containing a or—functionalized molecule, a selected compound capable of sequestering metal cations and a suitable ultra-pure formic acid/ sodium e mixture.

13. A reaction kit according to Claim 11 and a vial according to claim 12 wherein said vials are siliconized vials.

14. xes of 68Ga, obtained by the process, according to any one of Claims 1 to 10, wherein the complexes contain iess than 10 mg/ml formate/formic acid and, if used, less than 100 nmols of tering agent. 2282809v}