IT202000032603A1 - COLD KITS BASED ON PSMA LIGANDS FOR THE PREPARATION OF RADIO-PHARMACEUTICALS. - Google Patents

Description

DESCRIPTION of the invention entitled:

?Cold kits based on PSMA ligands for the production of radiopharmaceuticals?

Summary of the invention

The present invention relates to a new formulation, based on Prostate-Specific Membrane Antigen (PSMA) ligands, to be used as radiopharmaceutical precursors (cold kits). The formulation of the invention? designed to allow the preparation of the radiopharmaceutical instantaneously and in a single step by direct addition of radioactive isotopes, to be used for both diagnostic and therapeutic purposes.

Technical context

In recent years, as a direct consequence of the increase in the incidence of prostate cancer, the global demand for diagnostic, therapeutic and theranostic radiopharmaceuticals with which to intervene has also intensified.

In particular, one of the major challenges in prostate cancer ? represented by the possibility to early identify the biochemical marker that characterizes it, to date identified in an increase in PSA (Prostate Specific Antigen) levels. In the serum for? the PSA ? present in small quantities, and increases, sometimes very slightly, only in pathological cases, such as in the case of an enlarged prostate (benign prostatic hypertrophy) or, as mentioned, in the case of prostate cancer. The current gold standard used for PET/CT imaging of prostate cancer is today represented by the drug [<18>F] Choline which for? shows a reduced sensitivity? and specificity, especially in cases where PSA levels are very low.

The protein PSMA (Prostate-Specific Membrane Antigen) ? a zinc membrane protein overexpressed in prostate cancer cells. For this reason it could represent a very interesting molecule both for obtaining good PET images of prostate cancer and for its treatment. Recent studies have in fact shown how the radiopharmaceutical [<68>Ga]PSMA (Prostate-Specific Membrane Antigen) can represent a promising alternative in early diagnosis and, for this reason, some methods have been developed which allow the radiolabelling of said specific antigen with radioisotopes, both for diagnostic and therapeutic purposes.

One of the disadvantages related to the use of the isotope [<68>Ga] ? its short half-life (t1/2=68 minutes) which implies the need? to set up the radiolabelled compound in a very short time, preferably instantaneously.

WO2016030103 discloses a kit for the radiolabelling of various possible ligands based on Gallium-68 which comprises, in addition to the agent to be radiolabelled (functionalized with a chelating agent of radioactive Gallium), also an acetate salt or buffer and a co-chelating agent in capable of inactivating any other metals present in the solution eluted by the Gallium-68 generator. The radiolabelling reaction takes place at room temperature, pH between 3 and 5 and following an incubation time of about 10 minutes with a yield of 85% which varies according to the type of ligand and chelator considered.

EP3064224 discloses a method for radiolabelling a PSMA ligand with radioactive isotopes for the diagnosis and/or therapy of prostate cancer. In particular, the method uses a sodium-based buffering agent and provides for the possible further correction of the pH by adding NaOH up to a value of 4-8. The incubation of the reaction ? maintained for 5-10 minutes. Does the need still exist today? to have available a kit for radioisotope labeling of PSMA which is easy to use, for example it provides for the direct addition of the labeled isotope in any sterile acid solution it contains, which can be applied to various radionuclides, which does not need to adjust the pH n? of any incubation time and allows to obtain yields of complexation and radiochemical purity higher than 95%.

Purposes of the invention

? object of the present invention to provide a new formulation, based on Prostate-Specific Membrane Antigen (PSMA) ligands, to be used as radiopharmaceutical precursors (cold kits), designed to allow the preparation of the radiopharmaceutical in a single step by direct addition of radioactive isotopes .

? another object of the present invention is to provide a formulation, based on PSMA ligands, which can be used for the preparation of radiopharmaceuticals to be used for diagnostic and/or therapeutic purposes following the direct addition of the radioisotope pi? appropriate, without necessity? of pH adjustments of the reaction solution.

? another object of the present invention is to make available a production process of the formulation based on PSMA ligands (cold kit) object of the present invention. Another purpose of the present invention ? provide a method for preparing a radiopharmaceutical based on PSMA ligands in a single step, by direct addition of the appropriate radioactive isotope, without the need for of pH adjustments of the reaction solution and with yields and radiochemical purity higher than 95%.

These and other purposes are achieved by the object of the present invention which provides a new composition based on PSMA ligands to be used as a kit for radiopharmaceutical preparations.

Brief description of the Figures

Figure 1 shows the image of the TLC plate on the ? the radiopharmaceutical preparation of Example 2.1 was run according to the conditions described in Example 2.2.1. Figure 2 shows the chromatogram deriving from the HPLC analysis of the radiopharmaceutical preparation of Example 2.1, carried out according to the conditions described in Example 2.2.2.

Description of the invention

Object of the present invention ? a formulation based on Prostate-Specific Membrane Antigen (PSMA) ligands, to be used as a kit for radiopharmaceutical preparations. This formulation, also called ?cold kit?, ? contained in a single vial containing the PSMA molecule, functionalized with a chelating group of the radiopharmaceutical, in a quantity in the range between 10 and 30 ?g, together with suitable pharmaceutical excipients.

In the present invention, the PSMA molecule is present in a form characterized by the presence of a functionalisation which allows it to bind to the radionuclide; in the text can then be for brevity? indicated as ?PSMA functionalized?. The expert of the branch? fully aware of the different functionalized forms of this membrane protein currently used and will select? the "P? appropriate depending on the type of radiopharmaceutical you intend to use.

In a preferred embodiment, the present invention involves the use of PSMA-11 which has a chelating group HBED-CC capable of coordinating the radioactive ions of interest, in particular Ga-68, Lu-177 and Ac-225, even at room temperature.

The formulation of the invention is, as mentioned, contained in a single vial/vial and is presented in the form of a white, sterile and apyrogenic lyophile.

Said vial/vial also represents the container in which the complexation and radiolabelling reaction takes place, without the need to of addition of other reagents n? need no incubation or warming time.

In particular, in addition to the functionalized PSMA molecule, the formulation contains buffering excipients which, following the addition of concentrated hydrochloric acid in which ? normally conveyed the radioisotope, are able to maintain the pH of the solution that is formed in a range between 3 and 6, plus? preferably between 5 and 6, considered optimal for the radiolabelling reaction.

In a preferred embodiment of the invention, inside the vial/vial there are also excipients able to protect the functionalized PSMA molecule from degradation, increasing its stability. in the formulation itself and reducing the formation of peroxides and free radicals, as well as? agents capable of assisting/coordinating chelation. In a preferred aspect said further excipients are multifunctional excipients capable of simultaneously carrying out several? of one of the actions listed above.

In this text the words ?radiomarkers?, ?radioisotopes? and ?radionuclides? are considered synonyms.

The labeling of the functionalized PSMA molecule can be carried out with different types of radioisotopes, particularly preferred are Ga-68, Lu-177 and Ac-225. Said radioisotopes are added directly to the reaction vial/vial containing the functionalized PSMA molecule and the other excipients. For example, Ga-68 is added in the form of sterile acid eluate from the radionuclide generator. For ?eluated? here it is intended to indicate the strongly acid solution produced by the machinery which generates the radioisotopes and in which said radioisotopes are conveyed.

Said sterile acid eluate can? have an HCl concentration of, for example, 0.05 N, 0.1 N or 0.6 N, and be introduced directly into the reaction vial in different volumes, for example from 1 mL to 5 mL.

As previously mentioned, in the present invention, the buffering systems which form part of the formulation contained within the reaction vial/vial allow the stabilization of the pH within values in the desired range, i.e. between 3 and 6, preferably between 5 and 6, also following the introduction of the sterile acid eluate deriving from the radioisotope generator.

According to a preferred aspect of the present invention, said buffering systems are preferably characterized by at least one, preferably more? of one of the following aspects:

- they are suitable for use in injectable pharmaceutical preparations, they are not toxic and can be administered intravenously to humans;

- have a capacity? buffer capable of reaching and maintaining a pH between 3 and 6 within the reaction vial/vial, even after the addition of different volumes (in the range between 1 mL and 5 mL) of a concentrated solution of HCl, for example a 0.05 N, 0.1 N or 0.6 N solution, in the presence of functionalized PSMA and a radioisotope, preferably selected from Ga-68, Lu-177 and Ac-225;

- are they able to tolerate and compensate for small variations in terms of volume and/or degree of acidity? of the acid solution containing radionuclides (i.e. the eluate coming from the generator), determined, for example, by elutions and additions to the reaction vial/vial performed manually by the operator and not automatically by a machine;

- are able to determine high yields, higher than 95%, in the complexation reaction which takes place instantaneously and without need? of heating. In the present invention with the term ?buffer system? is meant to indicate the excipients that allow to bring and maintain the pH of the solution in the range of values more? suitable for the complexation reaction between the functionalized PSMA molecule and the radionuclide, both when present as a single acid/base pair and in the case said pair is accompanied by other excipients which support the activity? buffering, the chelation reaction and / or activity? stabilizer for the formulation itself.

In particular, the present invention uses as a basic buffering system the boric acid/sodium borate pair, obtained in situ in aqueous solution by using sodium tetraborate in the anhydrous or hydrated form, preferably in the decahydrated form. Other inorganic or organic salts of the tetraborate can be used. Alternatively, the pair boric acid / sodium borate can? be obtained in situ also starting from any other suitable analogous precursor. The boric acid compound ? been already? also used in formulations for the intravenous administration of drugs to humans.

Sodium tetraborate decahydrate can be used in the present invention in a range of quantities? between 80 and 100 mg, preferably between 85 and 88.5 mg. The quantities? of boric acid/borate currently authorized for human use by the EMA are still able to determine the activity? buffer within the formulation of the invention.

To said acid/base pair capable of buffering the solution of the reaction environment, other excipients can be added, as previously indicated, which will implement, on the one hand, the capacity? buffering of the pair acid/base, and on the other hand to improve the capacity? of coordination between the functionalized PSMA molecule and the radiomarkers, assisting what? chelation and increasing the yield and radiochemical purity of the final compound.

In one of its configurations the formulation of the invention comprises, for example, in addition to the boric acid/sodium borate pair, the addition of at least one sugar or a mixture of sugars, the sugar preferably ? a monosaccharide or disaccharide. Non-limiting examples of suitable sugars include glucose, sucrose, fructose, trehalose, mannitol and sorbitol. In some preferred embodiments, the sugar is a reduced sugar, pi? preferably mannitol or sorbitol. Preferably, the mannitol or sorbitol has the D configuration, although the L configuration can also be used.

In another of its configurations, the formulation of the invention comprises, for example, in addition to the boric acid/sodium borate pair, the addition of at least one compound having at least two hydroxyl groups separated by at least two atoms connected to each other in a chain or ring, said chain or ring containing carbon atoms and, optionally, a heteroatom or heteroatoms such as N, S, or O. Said compounds having at least two hydroxyl groups can be, for example but not limited to, pinandiol, pinacol, ethylene glycol, diethylene glycol, catechol, 1,2-cyclohexanediol, 1,3-propanediol, 2,3-butanediol, 1,2-butanediol, glycerol and diethanolamine. For purposes of the present invention, the compound having two hydroxyl groups ? preferably a pharmaceutically acceptable compound and ? preferably soluble or miscible with water.

In a preferred aspect of the invention said compound having the two hydroxyl groups ? a sugar, as described above, preferably a monosaccharide or a disaccharide, plus? preferably a reduced sugar, and even more? preferably mannitol or sorbitol. In some particularly preferred embodiments, said compound ? the mannitol, pi? preferably D-mannitol.

Said compound having two hydroxyl groups, or a mixture of more? compounds, preferably two compounds, having two hydroxyl groups, and the sodium tetraborate decahydrate can be introduced into the formulation in a mixture in molar ratio with the sodium tetraborate decahydrate in a range between about 0.5:1 and 100 :1, more preferably between 0.5:1 and 50:1. In the preferred embodiments, the compound having the two hydroxyl groups and the sodium tetraborate decahydrate are present in a ratio of between 1:1 and 10:1, preferably between 1:1 and 5:1, even more preferably between 1:1 and 4:1.

The introduction, in the formulation of the invention, of at least one compound having at least two hydroxyl groups can be desired and/or advantageous since? their concomitant presence with boric acid leads to the formation of esters of the latter. The boric acid ester ? an acid pi? stronger than the corresponding non-esterified acid (characterized by a lower pKa acid/base equilibrium constant) and therefore a better capacity is obtained? buffering the solution, it makes cio? more it is easy to reach the desired pH values for the solution contained in the reaction vial once the sterile acid eluate deriving from the radioisotope generator has been added.

If desired, the sodium tetraborate decahydrate of the formulation of the invention can be added, alone or together with the sugar or compound having two hydroxyl groups, pyruvic acid as an acid species or in its salified form, preferably sodium pyruvate, in an amount? ranging from about about 1 mg to 50 mg, preferably from 10 mg to 50 mg. The pyruvic acid? a ?-ketoacid naturally present in living organisms, including man, and involved in various cellular processes, whose safety of intravenous use is? proven in the literature. In solution with the other components of the invention, tender? to come to create the couple pyruvic acid/pyruvate, which will contribute? to the activity buffering agent of the buffer system of which it forms part, bringing and maintaining the pH of the solution in the range of desired values between 3 and 6, preferably between 5 and 6.

Pyruvic acid also has properties antioxidants thanks to its ability to neutralize the peroxides; could these molecules form during the preparation and storage of the lyophilisate as well as? in the radiolabelling phase. So this compound? capable of acting not only as an adjuvant in maintaining the pH within the pre-established range, but also as a stabilizer of the formulation, preventing the degradation of the functionalized PSMA molecule by the peroxides.

Alternatively, pyruvic acid can? be replaced by another analogue ?-ketoacid, or its salt, which is able to perform the same action.

A further aspect of the present invention provides for the presence of sodium hydroxide which allows the buffer pair to respond in a more positive way. efficient to the possible variations/differences of acidity? and/or volume of acid solution containing the radionuclide, maintaining the pH of the radiolabelling solution within the desired range. Said sodium hydroxide pu? be present in quantity? in a range between 0 mg and 50 mg, preferably between 1 mg and 30 mg, more? preferably between about 1 and 15 mg.

In a preferred form of the invention the formulation comprises sodium tetraborate decahydrate, D-mannitol, sodium pyruvate and sodium hydroxide in relative ratios expressed as a weight/weight percentage of about 28%, 52%, 16%, 4 % respectively, preferably 27.7%, 52.3%, 15.8%, 4.2% respectively.

The formulation of the invention, contained, as mentioned, within a single vial/phial, is prepared by subjecting to a freeze-drying process between 2 and 5 mL of a solution containing the functionalized PSMA molecule in a quantity between 10 and 30 ?g, sodium tetraborate decahydrate in a quantity? between 80 and 100 mg and possibly other desired or necessary excipients. This freeze-drying process can take place, for example, in a LIO5Pascal lyophilizer, for a time of 48 hours, at a temperature of -50°C and a pressure of 0.06 mbar.

The present invention discloses the formulation for a ?cold kit? for the radiolabelling of PSMA ligands containing a specific buffering system capable of maintaining the pH of the solution in which the radiolabelling takes place in a range between 3 and 6 following the direct addition of the acid eluate deriving from the radioisotope generator.

Said buffering system included in the formulation of the invention ? constituted by an appropriate acid/base pair, in particular by the boric acid/borate pair, and possibly by other pharmaceutical excipients which contribute to improve the ability? buffering the pair of acid/base as well as? to assist the chelation reaction and stabilize/protect the functionalized PSMA molecule from any degradation caused, for example, by the possible presence of peroxides.

The formulation of the invention does s? that the radiolabelling reaction can take place instantly, without the need? of heating, with a yield between 95 and 99% and a radiochemical purity between 95 and 99%.

The present invention also describes a process for the radiolabelling of the PSMA molecule with Ga-68, Lu-177 or Ac-225, which provides for the use of the ?cold kit? for the diagnosis and treatment of prostate cancer.

The invention will come now exemplified, in a non-limiting way, by the experimental section that follows.

Experimental section

Example 1

Preparation of a lyophilized formulation containing the functionalized PSMA molecule, sodium tetraborate decahydrate, D-mannitol, sodium pyruvate and sodium hydroxide 2 mL of a solution containing 30 µg of the PSMA molecule are transferred into a single vial/vial functionalized, 88 mg of sodium tetraborate decahydrate, 166 mg of D-mannitol, 50 mg of sodium pyruvate and 325 ?L of a 1 M NaOH solution. This solution is stored in a refrigerating cell at -20°C until completely frozen. The vial/vial? was subsequently placed in a lyophilizer at a temperature between about -40?C and -50?C and a pressure of 0.06 mbar, for about 42 hours, until the ice completely disappeared. In a second phase of the freeze-drying cycle, is the temperature of the vial/vial ? gradually increased from about -40?C to about 25?C, over a period of about 2 hours. The secondary drying process of the sample contained in the vial/vial ? was carried out for about 4 hours at a temperature of 25?C. The champion later ? was sealed with a rubber stopper under a stream of nitrogen and removed from the freeze dryer.

Example 2

Radiopharmaceutical preparation <68>Ga-PSMA-11

2.1 - PREPARATION OF THE RADIOPHARMACEUTICAL PREPARATION <68>Ga-PSMA-11

Inside the single vial/vial containing the lyophilized formulation deriving from Example 1, 5 mL of <68>GaCl3 eluate coming from the <68>Ge/<68>Ga generator eluted with a 0.1N HCl solution are transferred .

The solution is mixed by inverting the vial pi? times until complete solubilization of the lyophile.

Are immediately taken from the radiopharmaceutical preparation set up the quantities? necessary to verify the radiochemical purity by analytical methods in TLC and HPLC.

2.2 - QUALITY CONTROLS ABOUT THE RADIOPHARMACEUTICAL PREPARATION <68>Ga-PSMA-11

2.2.1 TLC method:

? Stationary phase: iTLC SG chromatography paper (10cm)

? Mobile phase: Ammonium acetate 77g/L in water / Methanol (50:50 volume/volume) ? Sample to be tested: 1.5 ?L

? Retention factors:

or [<68>Ga]gallium in colloidal form: 0.0 ? 0.1

or [<68>Ga]PSMA-11: 0.8 ? 1.0.

How can you? find from the numerical results in Table 1, which shows the data obtained using the TLC plate shown in Figure 1, the Gallium-68 present in the radiopharmaceutical preparation appears to be almost exclusively in the form linked to the functionalized PSMA molecule (retention times larger, evident spot at the top of the plate) compared to a much lower percentage of Gallium-68 in colloidal form (much smaller spot at the base of the plate).

Table 1: Numerical results from TLC analysis of the radiopharmaceutical preparation

<68>Ga-PSMA-11

2.2.2 HPLC method

? Stationary phase: Deactivated C18 silica gel column

? Mobile phase: [A] Trifluoracetic acid / Water 0.1:99.9 (volume/volume); [B] Trifluoracetic acid / Acetonitrile 0.1:99.9 (volume/volume)

? Sample to be tested: 5 ?L

? Retention times:

o [<68>Ga]PSMA-11: between 6.5 and 7.5 minutes

o Relative retention time Free ion [<68>Ga]Gallium (III): 0.3 minutes How can this be done? find from the numerical data reported in Table 2, deriving from the analysis of the chromatogram reported in Figure 2, in the analyzed sample there is no presence of free Gallium-68 ions but only of the form linked to the functionalized PSMA molecule, in the two respective stereoisomeric forms .

Table 2: Numerical results from HPLC analysis of the radiopharmaceutical preparation

<68>Ga-PSMA-11

Claims (10)

Claims 1. Pharmaceutical formulation based on functionalized PSMA for the preparation of radiopharmaceutical precursors (cold kits) comprising a buffering system, said buffering system comprising the boric acid/borate pair. 2. Pharmaceutical formulation according to claim 1 characterized in that said buffering system ? generated from anhydrous or hydrated sodium tetraborate, preferably decahydrate. 3. Pharmaceutical formulation according to claim 1 or 2 characterized in that said buffering system further comprises at least one sugar or at least one compound containing at least two hydroxyl groups separated by two atoms connected to each other in a chain or ring, or a mixture thereof. 4. Pharmaceutical formulation according to any one of the preceding claims characterized in that said buffering system comprises a ?-ketoacid, preferably pyruvic acid or a salt thereof. 5. Pharmaceutical formulation according to any one of the preceding claims characterized in that said buffering system comprises sodium hydroxide. 6. Pharmaceutical formulation according to any one of the preceding claims characterized in that said buffering system comprises sodium tetraborate decahydrate, D-mannitol, sodium pyruvate and sodium hydroxide in a relative w/w% ratio equal to about 28%, 52%, 16%, 4% respectively, preferably 27.7%, 52.3%, 15.8%, 4.2% respectively. 7. Pharmaceutical formulation according to any one of the preceding claims characterized in that said functionalized PSMA ? a PSMA-11 comprising a chelating group HBED-CC. 8. Use of the pharmaceutical formulation according to any one of the preceding claims for obtaining a radiolabeled form of the PSMA molecule functionalized with a radionuclide, said radionuclide preferably selected from Ga-68, Lu-177, Ac-225. 9. Method for the preparation of a radiolabelled form of the functionalized PSMA molecule which comprises the direct addition of a sterile acid solution containing a radionuclide to the pharmaceutical formulation according to any one of claims 1-7, said radionuclide preferably selected from Ga-68, Lu -177, Ac-225. The radiolabelled form of the PSMA molecule functionalized according to the method of claim 9 for its use in the diagnosis or therapy of prostate cancer.