RU2849465C2 - Compositions of visualising agents for psma - Google Patents

Abstract

FIELD: radiopharmaceutical industry.

SUBSTANCE: group of inventions relates to the field of the radiopharmaceutical industry, namely to an aqueous composition for parenteral administration for radiation therapy and diagnostic imaging, as well as to methods for obtaining said composition. An aqueous composition for parenteral administration for radiation therapy and diagnostic imaging, containing a compound of formula (I) or its salt in the form of a complex with a Cu

,

wherein the composition additionally contains ascorbic acid or its pharmaceutically acceptable salt and gentisic acid or its pharmaceutically acceptable salt. A method for producing the above composition containing a compound of formula (I) in the form of a complex with a Cu radioisotope, wherein the method comprises the following steps: i) adding an amount of the compound of formula (I) to a solution containing an acetate- e buffer and gentisic acid or its pharmaceutically acceptable salt; ii) adding a solution of Cu radioisotope in hydrochloric acid to the compound of formula (I) and the acetate buffer; iii) heating the mixture obtained in step ii) for a period of time and under conditions that ensure the formation of a complex of compound (I) and the Cu radioisotope; iv) adding a sodium ascorbate solution to the mixture of compound (I) and the Cu radioisotope. A method for producing the above composition containing compound (I) in the form of a complex with Cu radioisotope, wherein the method comprises the following steps: i) adding an amount of compound (I) to a solution containing phosphate buffer and gentisic acid or its pharmaceutically acceptable salt; ii) adding a solution of the Cu radioisotope in hydrochloric acid to the compound of formula (I) and the phosphate buffer solution; iii) subjecting the mixture obtained in step ii) to interaction for a period of time and under conditions ensuring the formation of a complex of compound (I) and the Cu radioisotope; iv) adding a sodium ascorbate solution to the mixture of compound (I) and the Cu radioisotope.

EFFECT: obtaining a composition characterised by exceptional stability of the compound of formula (I) with little radiolysis over 48 hours.

18 cl, 1 dwg, 13 ex

Description

Field of technology to which the invention relates

The present invention relates to compositions of radiolabeled compounds that are used in radiation therapy and diagnostic imaging related to prostate-specific membrane antigen (PSMA).

State of the art

Prostate cancer is a leading cause of cancer-related death in men, with its high mortality rate often attributed to difficulties in detection and subsequent treatment. Prostate-associated tumors frequently overexpress prostate-specific membrane antigen (PSMA), an enzyme normally expressed in prostate tissue but frequently upregulated in some types of prostate cancer. This makes PSMA a suitable biomarker or target for imaging, diagnosis, and prognosis. However, because PSMA is also expressed in other tissues, both normal and malignant, effective imaging of prostate cancer remains challenging.

Radiolabeled complexes can be used for imaging and treating cancers such as prostate cancer. However, some complexes containing a radioisotope or radionuclide and a targeting ligand can be unstable and prone to dissociation. If the formed complex is not stable enough, dissociation can occur shortly after its formation, i.e., during the radiolabeling process. Although radiolabeling methods are known, they may not result in complex formation in sufficient yield, or the overall complex solution may not be radiochemically pure. Furthermore, even if a radiolabeled complex can be obtained, purification and isolation procedures that yield the intact complex in good yield are preferred.

Even if a radiolabeled complex can be produced, it may be unstable and susceptible to degradation. This can lead to radioisotope dissociation, reduced radiochemical yield and purity of the composition containing the complex, and limited effectiveness of the composition. If the radioisotope is "lost" and not delivered to the intended tumor site, the quality of imaging and/or treatment is reduced or insufficient.

A radiolabeled complex can also undergo radiolysis, when the activity of the radioisotope causes the ligand to break down and degrade through spontaneous decay of the radioisotope. This results in the release of the radioisotope. Diffusion of the free radioisotope to other sites via the circulatory system can result in the delivery of radioactivity to undesirable sites.

There is a need for stable formulations of radiolabeled complexes suitable for prostate cancer imaging and treatment. There is also a need for efficient methods for preparing such stable formulations.

The essence of the invention

In one aspect, the present invention relates to an aqueous composition for parenteral administration comprising a compound of formula (I) or a salt thereof in the form of a complex with a Cu ion.

formula (I)

wherein the composition further comprises at least one of gentisic acid, ascorbic acid, L-methionine, pyridoxine or a salt thereof.

In a further aspect, the present invention relates to an aqueous composition for parenteral administration comprising a compound of formula (Ia) or a salt thereof in the form of a complex with a Cu ion.

formula (Ia)

wherein the composition further comprises at least one of gentisic acid, ascorbic acid, L-methionine, pyridoxine or a salt thereof.

In another aspect, the present invention relates to an aqueous composition for parenteral administration comprising a compound of formula (I) or a salt thereof in the form of a complex with a Cu ion.

formula (I)

wherein the composition additionally contains a buffer solution.

In a further aspect, the present invention relates to an aqueous composition for parenteral administration comprising a compound of formula (Ia) or a salt thereof in the form of a complex with a Cu ion.

formula (Ia)

wherein the composition additionally contains a buffer solution.

In one embodiment, the aqueous composition comprises gentisic acid or a salt thereof.

In another embodiment, the aqueous composition comprises ascorbic acid or a salt thereof.

In another embodiment, the aqueous composition comprises L-methionine or a salt thereof.

In another aspect, the present invention relates to a method for preparing a composition comprising a compound of formula (I) in the form of a complex with a radioisotope of Cu,

formula (I)

where the method includes the following steps:

i) adding an amount of the compound of formula (I) to the acetate buffer;

ii) adding a solution of a Cu radioisotope in hydrochloric acid to the compound of formula (I) and an acetate buffer; and

iii) heating the mixture obtained in step ii) for a period of time and under conditions ensuring the formation of a complex of the compound of formula (I) and the Cu radioisotope.

In another aspect, the present invention relates to a method for preparing a composition comprising a compound of formula (I) in the form of a complex with a radioisotope of Cu,

formula (I)

where the method includes the following steps:

i) adding an amount of a compound of formula (I) to a phosphate buffer;

ii) adding a solution of a Cu radioisotope in hydrochloric acid to the compound of formula (I) and a phosphate buffer; and

iii) subjecting the mixture obtained in step ii) to reaction for a period of time and under conditions sufficient to form a complex of the compound of formula (I) and the Cu radioisotope.

In one embodiment of the method for preparing a complex as defined herein, the compound of formula (I) has the structure of the compound of formula (Ia)

formula (Ia)

In one embodiment, the Cu radioisotope is ^61Cu .

In one embodiment, the Cu radioisotope is ^64Cu .

In another embodiment, the Cu radioisotope is ^67Cu .

In another embodiment, the pH of the composition is maintained in the range of from about 4 to about 8.

In another aspect, the present invention relates to a method for purifying a compound of formula (I) in the form of a complex with a radioisotope Cu

formula (I)

where the method includes the following steps:

i) loading a solution of the compound of formula (I) in the form of a complex with a Cu radioisotope onto a solid phase extraction cartridge;

ii) eluting the compound of formula (I) as a complex with the Cu radioisotope with an eluent containing water, ethanol and sodium chloride.

In an embodiment of the method for purifying a complex as defined herein, the compound of formula (I) has the structure of the compound of formula (Ia)

formula (Ia)

In one embodiment, a purified compound of formula (I) or a salt thereof in the form of a complex with a radioisotope of Cu purified in accordance with the previously presented aspect is obtained in accordance with another aspect of the present invention.

In one embodiment, a compound of formula (I) or a salt thereof in the form of a complex with a radioisotope of Cu is obtained according to another aspect of the present invention.

Brief description of the figures

Fig. 1: radiochemical purity of a solution of a purified complex of the compound of formula (Ia) and ^64Cu , where the solution contains gentisic acid, ascorbic acid or L-methionine, according to monitoring data for 48 hours.

Detailed description of the invention

Compositions of complexes of the compound of formula (I)

The present invention relates to stable compositions of a specific radioisotope-ligand complex. The inventors of the present invention have discovered that the compositions of the complex disclosed herein minimize the dissociation of the radioisotope from the ligand and/or minimize the radiolysis of the ligand caused by the radioisotope.

The radioisotope-ligand complex compositions described herein are stable in solution and under physiological conditions for a period of time. The stability of the composition is related to the stability of the complex. The radioisotope can dissociate from the complex, resulting in a decrease in the radioactivity delivered to the site to which the ligand binds. Because the radioisotope undergoes spontaneous decay or releases energy, this energy, when released, can lead to degradation of the ligand, a process known as radiolysis. The radiostability of the complex can be measured by considering the radiochemical purity of the composition. Radiochemical purity is defined as the amount of radioisotope complexed with the sarcophagin ligand, expressed as a percentage of the total amount of radioisotope present in the composition. The radioisotope can be present in the composition as a complex with the sarcophagin ligand, as a free radioisotope, or as part of a radiolysis product.

Ligands containing a urea-based motif have previously been found to bind to the catalytic site of prostate-specific membrane antigen (PSMA), which is commonly expressed in prostate tissue and is upregulated in some types of prostate cancer. An example of a ligand containing such a motif is Sar-bisPSMA, which is a macrocyclic ligand 1,8-diamino-3,6,10,13,16,19-hexaazabicyclo[6.6.6.]icosane (also known as sarcophagin or "Sar"), where each terminal amino group is attached to a linker group and a urea-based motif.

Sar-bisPSMA is represented by formula (I)

formula (I)

The compound of formula (I) can be prepared via a series of coupling reactions between a sarcophagin ligand, a linker, and a urea-based motif. Procedures for preparing the compound of formula (I) can be found in WO 2018/223180.

A compound of formula (I) may have a structure of formula (Ia) as shown below, where the stereochemistry of the compound is defined

formula (Ia)

Unless otherwise stated, any reference below to a compound of formula (I) shall include a reference to a compound of formula (Ia).

The present invention relates to the use of compounds of formula (I) and (Ia) in compositions. The compounds of formula (I) and (Ia) can be used in the form of a pharmaceutically acceptable salt. The compounds of formula (I) and (Ia) contain two urea-based motifs that are independently capable of binding to the catalytic site of PSMA. The inventors believe that the increased binding affinity of the compounds of formula (I) at the desired site is due to the presence of the second urea motif. Without wishing to be bound by theory, the inventors believe that the additional binding affinity of the compound of formula (I), which is more effective than using twice the amount of a similar compound having only one urea motif, is due to the presence of the second urea motif. Accordingly, the compositions described herein containing a compound of formula (I) or (Ia) show better efficacy than compositions of a similar compound containing a urea motif.

The term "pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the above compounds and include pharmaceutically acceptable acid addition salts and base addition salts. Suitable pharmaceutically acceptable acid addition salts of the compounds of formula (I) and (Ia) can be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, sulfuric and phosphoric acids. Suitable organic acids can be selected from the groups of organic acids such as aliphatic, cycloaliphatic, aromatic, heterocyclic carboxylic and sulfonic acids, examples of which are formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, fumaric, maleic, alkylsulfonic and arylsulfonic acids. Additional information on pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Co., Easton, PA 1995. In the case of agents that are solids, those skilled in the art will appreciate that the compounds, agents, and salts of the invention may exist in various crystalline or polymorphic forms, all of which are within the scope of the present invention and the defined formula.

In a preferred embodiment, the compound of formula (I) is present as an acetate salt.

The compositions of the present invention comprise a compound of formula (I) or a salt thereof and a radioisotope. The radioisotope, which may also be referred to as a radionuclide, may be a metal or a metal ion. It has been found that the compound of formula (I) of the present invention is particularly effective in complexing with copper ions, in particular, Cu ²⁺ ions. One skilled in the art understands that a complex of a compound of formula (I) can be obtained by contacting a compound of formula (I) with the desired radioisotope, wherein the radioisotope is a Cu ²⁺ ion.

In one embodiment, the ligand forms a complex with a Cu ion. The copper ion may be radioactive and, therefore, a radionuclide or radioisotope of copper. In one embodiment, the ligand forms a complex with ⁶⁰ Cu. In another embodiment, the ligand forms a complex with ⁶¹ Cu. In another embodiment, the ligand forms a complex with ⁶⁴ Cu. In yet another embodiment, the ligand forms a complex with ⁶⁷ Cu. In a preferred embodiment, the ligand forms a complex with ⁶⁴ Cu. In yet another preferred embodiment, the ligand forms a complex with ⁶⁷ Cu.

A complex of a compound of formula (I) with a Cu radioisotope may be unstable and susceptible to radiolysis when in solution. The present inventors have discovered that the solubilized complex can be stabilized by adding one or more stabilizing agents to a composition containing the complex. Such stabilizing agents include gentisic acid, ascorbic acid, L-methionine, pyridoxine, and their salts.

The compositions of the present invention may contain at least one of gentisic acid, ascorbic acid, L-methionine, and pyridoxine, or salts thereof. The inventors have found that the addition of gentisic acid, ascorbic acid, L-methionine, and/or pyridoxine to the compositions of the present invention helps prevent or minimize radiolysis of the complex of the compound of formula (I), thereby increasing the radiostability of the complex and its composition.

Gentisic acid is also known as 2,5-dihydroxybenzoic acid, 5-hydroxysalicylic acid, or hydroquinone carboxylic acid. Salts of gentisic acid may include the sodium salt and the sodium salt hydrate. Any reference to gentisic acid may include its salts, where appropriate. Other isomers of dihydroxybenzoic acid are also considered. Examples of other isomers include 2,4-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid and their salts.

In one embodiment, gentisic acid is present in the composition in an amount of from about 0.02% to about 0.1% (w/v). In one embodiment, gentisic acid or a salt thereof is present in the composition in an amount of about 0.02% (w/v). In another embodiment, gentisic acid or a salt thereof is present in the composition in an amount of about 0.025% (w/v). In another embodiment, gentisic acid or a salt thereof is present in the composition in an amount of about 0.03% (w/v). In another embodiment, gentisic acid or a salt thereof is present in the composition in an amount of about 0.035% (w/v). In another embodiment, gentisic acid or a salt thereof is present in the composition in an amount of about 0.04% (w/v). In another embodiment, gentisic acid or a salt thereof is present in the composition in an amount of about 0.045% (w/v). In another embodiment, gentisic acid or a salt thereof is present in the composition in an amount of about 0.05% (w/v). In another embodiment, gentisic acid or a salt thereof is present in the composition in an amount of about 0.055% (w/v). In another embodiment, gentisic acid or a salt thereof is present in the composition in an amount of about 0.6% (w/v). In another embodiment, gentisic acid or a salt thereof is present in the composition in an amount of about 0.065% (w/v). In another embodiment, gentisic acid or a salt thereof is present in the composition in an amount of about 0.07% (w/v). In another embodiment, gentisic acid or a salt thereof is present in the composition in an amount of about 0.075% (w/v). In another embodiment, gentisic acid or a salt thereof is present in the composition in an amount of about 0.08% (w/v). In another embodiment, gentisic acid or a salt thereof is present in the composition in an amount of about 0.085% (w/v). In another embodiment, gentisic acid or a salt thereof is present in the composition in an amount of about 0.09% (w/v). In another embodiment, gentisic acid or a salt thereof is present in the composition in an amount of about 0.095% (w/v). In another embodiment, gentisic acid or a salt thereof is present in the formulation in an amount of about 0.1% (w/v). In still other embodiments, the present invention also provides for gentisic acid or a salt thereof to be present in the ranges between the above amounts. In a preferred embodiment, gentisic acid or a salt thereof is present in the composition in an amount of no more than 0.056% (w/v).

L-methionine is an amino acid containing a thiol ester in its side chain, also known as Met or L-Met. Salts of L-methionine include the sodium salt. Any reference to L-methionine may include its salts, where appropriate.

In one embodiment, L-methionine or a salt thereof is present in the composition in an amount of from about 1 mg/mL to about 4 mg/mL. In one embodiment, L-methionine or a salt thereof is present in the composition in an amount of about 1.0 mg/mL. In another embodiment, L-methionine or a salt thereof is present in the composition in an amount of about 1.5 mg/mL. In another embodiment, L-methionine or a salt thereof is present in the composition in an amount of about 2.0 mg/mL. In another embodiment, L-methionine or a salt thereof is present in the composition in an amount of about 2.5 mg/mL. In yet another embodiment, L-methionine or a salt thereof is present in the composition in an amount of about 3.0 mg/mL. In yet another embodiment, L-methionine or a salt thereof is present in the composition in an amount of about 3.5 mg/mL. In another embodiment, L-methionine or a salt thereof is present in the composition in an amount of about 4.0 mg/mL. In other embodiments, the present invention also provides for a content of L-methionine or a salt thereof in ranges between the above amounts. In a preferred embodiment, L-methionine is present in the composition in an amount of about 3 mg/ml.

Ascorbic acid is also known as 2,3-didehydro-L-threohexano-1,4-lactone or vitamin C. Salts of ascorbic acid include sodium ascorbate, potassium ascorbate, calcium ascorbate, and magnesium ascorbate. Any reference to ascorbic acid may include a reference to its salts, where appropriate.

In one embodiment, ascorbic acid or a salt thereof is present in the composition in an amount of from about 5 mg/mL to about 50 mg/mL. In one embodiment, ascorbic acid or a salt thereof is present in the composition in an amount of about 5 mg/mL. In another embodiment, ascorbic acid or a salt thereof is present in the composition in an amount of about 6 mg/mL. In another embodiment, ascorbic acid or a salt thereof is present in the composition in an amount of about 7 mg/mL. In another embodiment, ascorbic acid or a salt thereof is present in the composition in an amount of about 8 mg/mL. In another embodiment, ascorbic acid or a salt thereof is present in the composition in an amount of about 9 mg/mL. In another embodiment, ascorbic acid or a salt thereof is present in the composition in an amount of about 10 mg/mL. In another embodiment, ascorbic acid or a salt thereof is present in the composition in an amount of about 11 mg/mL. In another embodiment, ascorbic acid or a salt thereof is present in the composition in an amount of about 12 mg/ml. In another embodiment, ascorbic acid or a salt thereof is present in the composition in an amount of about 13 mg/ml. In another embodiment, ascorbic acid or a salt thereof is present in the composition in an amount of about 14 mg/ml. In another embodiment, ascorbic acid or a salt thereof is present in the composition in an amount of about 15 mg/ml. In another embodiment, ascorbic acid or a salt thereof is present in the composition in an amount of about 20 mg/ml. In another embodiment, ascorbic acid or a salt thereof is present in the composition in an amount of about 25 mg/ml. In another embodiment, ascorbic acid or a salt thereof is present in the composition in an amount of about 30 mg/ml. In another embodiment, ascorbic acid or a salt thereof is present in the composition in an amount of about 35 mg/ml. In another embodiment, ascorbic acid or a salt thereof is present in the composition in an amount of about 40 mg/ml. In another embodiment, ascorbic acid or a salt thereof is present in the composition in an amount of about 45 mg/mL. In yet another embodiment, ascorbic acid or a salt thereof is present in the composition in an amount of about 50 mg/mL. In other embodiments, the present invention also provides for ascorbic acid or a salt thereof in ranges between the above amounts. In a preferred embodiment, ascorbic acid is present in the composition in an amount of about 10 mg/mL.

Pyridoxine is also known as 4,5-bis(hydroxymethyl)-2-methylpyridin-3-ol or vitamin B6. Pyridoxine salts may include the hydrochloride salt.

In one embodiment, pyridoxine or a salt thereof is present in the composition in an amount of from about 5 mg/mL to about 15 mg/mL. In one embodiment, pyridoxine or a salt thereof is present in the composition in an amount of about 5 mg/mL. In another embodiment, pyridoxine or a salt thereof is present in the composition in an amount of about 6 mg/mL. In another embodiment, pyridoxine or a salt thereof is present in the composition in an amount of about 7 mg/mL. In another embodiment, pyridoxine or a salt thereof is present in the composition in an amount of about 8 mg/mL. In another embodiment, pyridoxine or a salt thereof is present in the composition in an amount of about 9 mg/mL. In another embodiment, pyridoxine or a salt thereof is present in the composition in an amount of about 10 mg/mL. In another embodiment, pyridoxine or a salt thereof is present in the composition in an amount of about 11 mg/mL. In another embodiment, pyridoxine or a salt thereof is present in the composition in an amount of about 12 mg/mL. In another embodiment, pyridoxine or a salt thereof is present in the composition in an amount of about 13 mg/mL. In another embodiment, pyridoxine or a salt thereof is present in the composition in an amount of about 14 mg/mL. In yet another embodiment, pyridoxine or a salt thereof is present in the composition in an amount of about 15 mg/mL. In a preferred embodiment, pyridoxine is present in the composition in an amount of about 10 mg/mL.

The compositions of the present invention may contain ethanol as a component. The ethanol used in the composition may be anhydrous ethanol. Alternatively, the ethanol used in the composition may not be subjected to drying processes and may be hydrated. The ethanol is preferably pharmaceutical grade ethanol. Ethanol present in the composition may further help prevent radiolysis of the radiolabeled complex of the compound of Formula (I).

In one embodiment, ethanol is present in the composition in an amount of from about 7% to about 13% (v/v). In one embodiment, ethanol is present in the composition in an amount of about 7% (v/v). In another embodiment, ethanol is present in the composition in an amount of about 8% (v/v). In another embodiment, ethanol is present in the composition in an amount of about 9% (v/v). In another embodiment, ethanol is present in the composition in an amount of about 10% (v/v). In another embodiment, ethanol is present in the composition in an amount of about 11% (v/v). In another embodiment, ethanol is present in the composition in an amount of about 12% (v/v). In another embodiment, ethanol is present in the composition in an amount of about 13% (v/v). In a preferred embodiment, ethanol is present in the composition in an amount of about 10% (v/v). In yet other embodiments, the present invention also contemplates ethanol contents in ranges between the above amounts.

The compositions of the present invention may also contain sodium chloride as a component. Sodium chloride in the composition of the present invention may be provided in the form of a saline solution. A saline solution is defined as an aqueous solution of sodium chloride. For example, a physiological saline solution is defined as an aqueous solution of sodium chloride with a concentration of 0.9% (w/v). In one embodiment of the present invention, the sodium chloride in the composition is provided by a saline solution.

In one embodiment, sodium chloride is present in the composition in an amount of from about 0.6% to about 1.2% (w/v). In one embodiment, sodium chloride is present in an amount of about 0.6% (w/v). In another embodiment, sodium chloride is present in an amount of about 0.7% (w/v). In another embodiment, sodium chloride is present in an amount of about 0.8% (w/v). In another embodiment, sodium chloride is present in an amount of about 0.9% (w/v). In another embodiment, sodium chloride is present in an amount of about 1.0% (w/v). In another embodiment, sodium chloride is present in an amount of about 1.1% (w/v). In another embodiment, sodium chloride is present in an amount of about 1.2% (w/v). In a preferred embodiment, sodium chloride is present in the composition in an amount of about 0.9% (w/v). In still other embodiments, the present invention also provides for sodium chloride content in ranges between the above amounts.

The compositions of the present invention have a pH of from about 4 to about 8. One skilled in the art understands that the pH of the composition is an intrinsic characteristic of the composition associated with the combination of a compound of Formula (I) or a complex thereof, and the remaining excipients of the composition. Alternatively, the pH of the composition can be adjusted to a desired value by adding one or more buffering agents. Examples of a suitable buffer solution include an acetate buffer, which may contain a mixture of sodium acetate and acetic acid. In some embodiments, the compositions of the present invention contain an acetate buffer. Another suitable buffer solution includes a phosphate buffer, which may contain a mixture of various phosphate salts or hydrates thereof. Examples of suitable phosphate salts include sodium dihydrogen phosphate (NaH ₂ PO ₄ ), sodium hydrogen phosphate (Na ₂ HPO ₄ ), potassium dihydrogen phosphate (KH ₂ PO ₄ ), and potassium hydrogen phosphate (K ₂ HPO ₄ ). In one embodiment, the phosphate buffer contains sodium phosphate salts. In another embodiment, the phosphate buffer comprises potassium phosphate salts. In yet another embodiment, the phosphate buffer comprises a mixture of sodium and potassium phosphate salts.

For the purposes of the present invention, the term "buffer" refers to a component that maintains a constant pH level in the medium to which it is added. Gentisic acid, ascorbic acid, L-methionine, and pyridoxine, their salts, or aqueous solutions are not considered buffers in the context of this description.

In one embodiment, the pH of the composition is from about 4 to about 8. In one embodiment, the pH of the composition is about 4. In another embodiment, the pH of the composition is about 4.5. In another embodiment, the pH of the composition is about 5.0. In one embodiment, the pH of the composition is about 5.5. In another embodiment, the pH of the composition is about 5.6. In another embodiment, the pH of the composition is about 5.7. In another embodiment, the pH of the composition is about 5.8. In another embodiment, the pH of the composition is about 5.9. In another embodiment, the pH of the composition is about 6.0. In another embodiment, the pH of the composition is about 6.1. In another embodiment, the pH of the composition is about 6.2. In another embodiment, the pH of the composition is about 6.3. In another embodiment, the pH of the composition is about 6.4. In another embodiment, the pH of the composition is about 6.5. In another embodiment, the pH of the composition is about 7.0. In another embodiment, the pH of the composition is about 7.5. In another embodiment, the pH of the composition is about 8.0. In a preferred embodiment, the pH of the composition is about 6.0. In yet another preferred embodiment, the pH of the composition is about 5.0.

The present inventors have discovered that when a compound of formula (I) is formulated as an aqueous solution, the compound is relatively unstable and susceptible to oxidation and degradation. One approach to overcoming the observed instability may be to add one or more components that are antioxidants and/or stabilizing agents to the composition. However, the inclusion of additional components in the composition creates potential reactivity issues between the compound of formula (I) and these added components. For example, the addition of an antioxidant may actually lead to an interaction with the compound of formula (I), thereby potentially altering the structure and function of the compound, which is undesirable.

However, the present inventors have now discovered that the addition of certain stabilizing agents may in some cases be sufficient to produce a composition containing a compound of formula (I). As disclosed herein, compositions of compounds of formula (I) are provided that contain a stabilizing agent such as gentisic acid, ascorbic acid, L-methionine, or pyridoxine, since they do not interact with the compound of formula (I) and can provide the necessary stability.

However, it has now been discovered that a composition containing a buffer and a compound of formula (I) provides the necessary stability of the compound. Thus, the present inventors have discovered that the presence of a buffer, in addition to providing a composition with a suitable pH for parenteral administration, provides a composition in which the compound of formula (I) has the necessary stability.

The present inventors have surprisingly discovered that, although the addition of a stabilizing agent such as gentisic acid, ascorbic acid and other agents described herein can provide the required stability, the stability of the composition can also be achieved by using only one buffer, i.e., in the absence of a stabilizing agent.

Methods for preparing complexes of the compound of formula (I)

The present invention also relates to methods for preparing a radiolabeled complex of compounds of formula (I) and compositions thereof. As discussed above, compounds of formula (I) can form a complex with a radioisotope, such as a Cu ion. Therefore, the present invention relates to a method for preparing a composition comprising a compound of formula (I) in the form of a complex with a Cu radioisotope.

formula (I),

where the method includes the following steps:

i) adding an amount of the compound of formula (I) to the acetate buffer;

ii) adding a solution of a Cu radioisotope in hydrochloric acid to the compound of formula (I) and an acetate buffer; and

iii) heating the mixture obtained in step ii) for a period of time and under conditions ensuring the formation of a complex of the compound of formula (I) and the Cu radioisotope.

The present invention also relates to a method for preparing a composition comprising a compound of formula (I) in the form of a complex with a radioisotope of Cu

formula (I),

where the method includes the following steps:

i) adding an amount of a compound of formula (I) to a phosphate buffer solution;

ii) adding a solution of a Cu radioisotope in hydrochloric acid to the compound of formula (I) and a phosphate buffer solution; and

iii) subjecting the mixture obtained in step ii) to reaction for a period of time and under conditions sufficient to form a complex of the compound of formula (I) and the Cu radioisotope.

In some embodiments, the compound of formula (I) has the structure of the compound of formula (Ia)

formula (Ia).

In one embodiment, the method further comprises the step of adding a sodium ascorbate solution to the mixture of the compound of formula (I) and the Cu radioisotope after completion of the reaction between the compound of formula (I) and the Cu radioisotope.

The compound of formula (I) can be provided as a component of a stock solution. Prior to preparing the stock solution, the compound of formula (I) can be subjected to drying steps, such as lyophilization. The compound of formula (I) can be dissolved in a mixture of ethanol and water to form a stock solution of the compound of formula (I). In one embodiment, the compound of formula (I) is dissolved in a mixture of ethanol and water, wherein ethanol and water are in a ratio of about 1:1. In one embodiment, the compound of formula (I) is provided as a stock solution with a concentration of about 1 nmol/μL.

The compound of formula (I) may be present in an amount of from about 1 nmol to about 10 nmol. In one embodiment, the compound of formula (I) is present in an amount of about 1 nmol. In another embodiment, the compound of formula (I) is present in an amount of about 2 nmol. In another embodiment, the compound of formula (I) is present in an amount of about 3 nmol. In another embodiment, the compound of formula (I) is present in an amount of about 4 nmol. In another embodiment, the compound of formula (I) is present in an amount of about 5 nmol. In another embodiment, the compound of formula (I) is present in an amount of about 6 nmol. In another embodiment, the compound of formula (I) is present in an amount of about 7 nmol. In another embodiment, the compound of formula (I) is present in an amount of about 8 nmol. In another embodiment, the compound of formula (I) is present in an amount of about 9 nmol. In another embodiment, the compound of formula (I) is present in an amount of about 10 nmol. One skilled in the art understands that the required volume of the stock solution of the compound of formula (I) depends on the initial concentration of the stock solution. One skilled in the art also understands that larger amounts of the compound of formula (I) can be used, and then the amounts of other reagents, buffers, and solvents can be adjusted accordingly.

In one embodiment, the buffer solution can be an acetate buffer. The acetate buffer used in the method can be prepared from sodium acetate and acetic acid. The acetate buffer maintains the pH in a range suitable for complexation of the compound of formula (I) with the Cu radioisotope. The pH of the buffer solution can be about 5.0. The acetate buffer can have a concentration of about 1.0 M. The acetate buffer can also contain ethanol. In one embodiment, the acetate buffer contains ethanol in an amount of from about 10% to about 30%. In one embodiment, the acetate buffer contains ethanol in an amount of about 10%. In one embodiment, the acetate buffer contains ethanol in an amount of about 20%. In one embodiment, the acetate buffer contains ethanol in an amount of about 30%. In a preferred embodiment, the acetate buffer contains ethanol in an amount of about 20%.

In another embodiment _, the buffer solution may be a phosphate buffer. The phosphate buffer may comprise a mixture of various phosphate salts or hydrates thereof. Examples of suitable phosphate salts include sodium dihydrogen phosphate ( _NaH2PO4 ), sodium hydrogen phosphate ( _{Na2HPO4 )} , potassium dihydrogen phosphate ( _{KH2PO4 )} , and potassium hydrogen phosphate ( _K2HPO4 ). In one embodiment, the phosphate buffer _comprises sodium phosphate salts. In another embodiment, the phosphate buffer comprises potassium phosphate salts. In yet another embodiment, the phosphate buffer comprises a mixture of sodium and potassium phosphate salts. The phosphate buffer may also contain a salt and/or water. In one embodiment, the phosphate buffer comprises a mixture of sodium hydrogen phosphate salts and a salt.

An aliquot containing an amount of the compound of formula (I) is removed from a stock solution of the compound of formula (I) in a mixture of ethanol and water and mixed with an amount of acetate buffer. In one embodiment, the compound of formula (I) is added to the acetate buffer, wherein the acetate buffer contains approximately 20% ethanol. In one embodiment, the compound of formula (I) is added to the acetate buffer at room temperature.

As discussed above, the compound of formula (I) forms complexes with Cu ions. In one embodiment, the Cu ion is a Cu radioisotope. In one embodiment, the Cu radioisotope is ⁶⁰ Cu. In another embodiment, the Cu radioisotope is ⁶¹ Cu. In another embodiment, the Cu radioisotope is ⁶⁴ Cu. In yet another embodiment, the Cu radioisotope is ⁶⁷ Cu. The Cu radioisotope is in the form of a Cu salt. In one embodiment, the Cu salt is in the form of a Cu ²⁺ chloride salt. In one embodiment, the Cu salt is in the form of a [ ⁶⁴ Cu]CuCl ₂ salt. The Cu radioisotope is in the form of a hydrochloric acid solution. In one embodiment, the Cu radioisotope is ^64Cu and is provided as a hydrochloric acid solution, wherein the hydrochloric acid has a concentration of about 0.02 M. In one embodiment, the Cu radioisotope is provided as a solution of [ ^64Cu ] _CuCl2 in a hydrochloric acid solution, wherein the hydrochloric acid has a concentration of about 0.02 M. One skilled in the art understands that the Cu salt can be provided in a hydrochloric acid solution of other concentrations. In another embodiment, the Cu radioisotope is provided as a Cu2 ⁺ acetate salt. In one embodiment, the Cu salt is present as a [ ^64Cu ]Cu(OAc) ₂ salt.

A solution of a copper salt provided as a hydrochloric acid solution will have a certain initial radioactivity. The initial activity of the solution may vary depending on the specific batch of radioisotope. One skilled in the art understands that the final activity of a compound of formula (I) complexed with a copper ion will depend on the activity of the copper salt used to form the complex of the compound of formula (I), and that this, in turn, will depend on the activity of the copper salt solution in hydrochloric acid. The total radiochemical yield of the complex of the compound of formula (I) and the copper salt can be determined relative to the amount of radioactivity initially present in the copper salt solution. An aliquot of the copper radioisotope in hydrochloric acid solution is added to the compound of formula (I) in acetate buffer. One skilled in the art understands that radiochemical purity can be determined by radio-HPLC or a similar method.

In one embodiment, the ^64Cu radioisotope solution has a radioactivity of about 100 to about 5000 MBq. In one embodiment, the ^64Cu radioisotope solution has a radioactivity of about 100 MBq. In another embodiment, the ^64Cu radioisotope solution has a radioactivity of about 250 MBq. In another embodiment, the ^64Cu radioisotope solution has a radioactivity of about 500 MBq. In another embodiment, the ^64Cu radioisotope solution has a radioactivity of about 750 MBq. In another embodiment, the ^64Cu radioisotope solution has a radioactivity of about 1000 MBq. In another embodiment, the ^64Cu radioisotope solution has a radioactivity of about 1500 MBq. In another embodiment, the ^64Cu radioisotope solution has a radioactivity of about 2000 MBq. In another embodiment, the solution of the radioactive isotope ^64Cu has a radioactivity of about 2500 MBq. In another embodiment, the solution of the radioactive isotope ^64Cu has a radioactivity of about 3000 MBq. In another embodiment, the solution of the radioactive isotope ^64Cu has a radioactivity of about 4000 MBq. In another embodiment, the solution of the radioactive isotope ^64Cu has a radioactivity of about 5000 MBq.

In one embodiment, the ^61Cu radioisotope solution has a radioactivity of about 100 to about 5000 MBq. In one embodiment, the ^61Cu radioisotope solution has a radioactivity of about 100 MBq. In another embodiment, the ^61Cu radioisotope solution has a radioactivity of about 250 MBq. In another embodiment, the ^61Cu radioisotope solution has a radioactivity of about 500 MBq. In another embodiment, the ^61Cu radioisotope solution has a radioactivity of about 750 MBq. In another embodiment, the ^61Cu radioisotope solution has a radioactivity of about 1000 MBq. In another embodiment, the ^61Cu radioisotope solution has a radioactivity of about 1500 MBq. In another embodiment, the ^61Cu radioisotope solution has a radioactivity of about 2000 MBq. In another embodiment, the solution of the radioactive isotope ^61Cu has a radioactivity of about 2500 MBq. In another embodiment, the solution of the radioactive isotope ^61Cu has a radioactivity of about 3000 MBq. In another embodiment, the solution of the radioactive isotope ^61Cu has a radioactivity of about 4000 MBq. In another embodiment, the solution of the radioactive isotope ^61Cu has a radioactivity of about 5000 MBq.

In one embodiment, the ^67Cu radioisotope solution has a radioactivity of about 100 to about 3000 MBq. In one embodiment, the ^67Cu radioisotope solution has a radioactivity of about 100 MBq. In another embodiment, the ^67Cu radioisotope solution has a radioactivity of about 250 MBq. In another embodiment, the ^67Cu radioisotope solution has a radioactivity of about 500 MBq. In another embodiment, the ^67Cu radioisotope solution has a radioactivity of about 750 MBq. In another embodiment, the ^67Cu radioisotope solution has a radioactivity of about 1000 MBq. In another embodiment, the ^67Cu radioisotope solution has a radioactivity of about 1500 MBq. In another embodiment, the ^67Cu radioisotope solution has a radioactivity of about 2000 MBq. In another embodiment, the solution of the radioactive isotope 67Cu has a radioactivity of about 2500 MBq. In another embodiment, the solution of the radioactive isotope ^67Cu has a radioactivity of about 3000 MBq. In another embodiment, the solution of the radioactive isotope ^67Cu has a radioactivity of about 4000 MBq. In another embodiment, the solution of ^{the radioactive isotope 67Cu has} a radioactivity of about 5000 MBq.

The Cu radioisotope can be provided in the form of a solution in hydrochloric acid. In one embodiment, the Cu radioisotope is provided in a hydrochloric acid solution having a concentration of from about 0.01 M to about 0.05 M. In one embodiment, the concentration of the hydrochloric acid solution is about 0.01 M. In another embodiment, the concentration of the hydrochloric acid solution is about 0.02 M. In another embodiment, the concentration of the hydrochloric acid solution is about 0.03 M. In another embodiment, the concentration of the hydrochloric acid solution is about 0.04 M. In another embodiment, the concentration of the hydrochloric acid solution is about 0.04 M. In another embodiment, the concentration of the hydrochloric acid solution is about 0.05 M. In another embodiment, the concentration of the hydrochloric acid solution is about 0.02 to 0.05 M.

A solution containing a mixture of a Cu radioisotope, a compound of formula (I), and an acetate buffer is then stirred for a period of time and at a certain temperature to ensure complexation of the compound of formula (I) with the Cu radioisotope. The solution can be mixed using a suitable device. For example, given the small volumes used, an Eppendorf tube may be a suitable vessel, so that an Eppendorf Thermomixer can be used both for mixing and, if desired, for heating the solution. In one embodiment, the solution is stirred at room temperature. In one embodiment, the solution is stirred at a temperature of about 40°C. The inventors of the present invention have found that complexation of the radioisotope is complete within about 5 minutes when the solution is stirred at a temperature of about 40°C. Although a lower temperature of about 21°C, i.e., room temperature, can be used for mixing, the complexation reaction may not be complete after 5 minutes, but rather complete in about 15 minutes. The present inventors also found that temperatures above about 40°C, such as 60°C, lead to some degradation of the compound of formula (I), thereby reducing the yield of the complex. In one embodiment, the solution is stirred at about 40°C for about 5 minutes. In one embodiment, the solution is stirred at about 40°C for about 10 minutes. In another embodiment, the solution is stirred at about 40°C for about 15 minutes. In another embodiment, the solution is stirred at about 21°C for about 10 minutes. In yet another embodiment, the solution is stirred at about 21°C for about 15 minutes.

In some embodiments, the compound of formula (I) is added to a phosphate buffer solution to which a solution containing a Cu radioisotope in hydrochloric acid is added. The mixture containing the compound of formula (I), the phosphate buffer, and the Cu radioisotope is reacted for a period of time and under conditions that allow the formation of a complex of the compound of formula (I) and the Cu radioisotope. In one embodiment, the solution is stirred at room temperature. In one embodiment, the solution is stirred at room temperature for about 10 minutes. In another embodiment, the solution is stirred at room temperature for about 15 minutes. In another embodiment, the solution is stirred at room temperature for about 20 minutes. In another embodiment, the solution is stirred at room temperature for about 25 minutes.

After the complex of the compound of formula (I) and the Cu radioisotope is formed, the solution is diluted with a sodium ascorbate solution. When sodium ascorbate is added to the mixture, a reducing agent is introduced, which in turn provides a radiostabilizing effect for the complex of the compound of formula (I) and the Cu radioisotope. In turn, this increases the stability of the composition as a whole and allows for an extended shelf life of the composition containing the complex. The sodium ascorbate solution can have a concentration of from about 25 mg/mL to about 75 mg/mL. In one embodiment, the sodium ascorbate solution can have a concentration of about 25 mg/mL. In another embodiment, the sodium ascorbate solution can have a concentration of about 50 mg/mL. In yet another embodiment, the sodium ascorbate solution can have a concentration of about 75 mg/mL. The sodium ascorbate solution of a certain concentration is added in an amount such as to ensure sufficient dilution of any remaining Cu radioisotope. One skilled in the art understands that the volume of solution added will depend on the amount of uncomplexed Cu radioisotope and the concentration of the sodium ascorbate solution.

The present inventors have discovered that the method for preparing a compound of formula (I) as a complex with a Cu radioisotope, as disclosed herein, provides efficient radiolabeling of the compound and allows for a high radiochemical yield. The present inventors have discovered that the complexation of the compound of formula (I) with the Cu radioisotope occurs more rapidly when an acetate buffer containing some ethanol is used. According to an embodiment of the present invention, the method comprises adding a compound of formula (I) to an acetate buffer containing ethanol.

In one embodiment, the method comprises the following steps:

i) adding an amount of a compound of formula (I) to an acetate buffer containing ethanol;

ii) adding a solution of [ ⁶⁴ Cu]CuCl ₂ in hydrochloric acid to the compound of formula (I) and an acetate buffer containing ethanol; and

iii) heating the mixture obtained in step ii) for about 5 min at 40°C.

In another embodiment, the method includes the following steps:

i) adding an amount of a compound of formula (I) to a phosphate buffer containing ethanol and sodium gentisate;

ii) adding a solution of [ ^64Cu ] _CuCl2 in hydrochloric acid to the compound of formula (I) and a phosphate buffer containing sodium gentisate; and

iii) subjecting the mixture obtained in step ii) to reaction for a period of time and under conditions sufficient to form a complex of the compound of formula (I) and the Cu radioisotope.

A method for purifying a complex of a compound of formula (I)

After completing the process for preparing a compound of formula (I) as a complex with a Cu radioisotope, the complex must be purified and isolated. Those skilled in the art understand that loss of substance may occur during purification and isolation, which reduces the overall chemical and radiochemical yield. These losses may be due to mechanical processing steps, loss of substance in syringes and other devices used in the purification process, or retention of the substance in the reaction vessel. The purification process typically includes a filtration step using a solid-phase medium; retention of the substance on the solid phase often leads to a decrease in yield. The purification process often relies on washing the solid phase with various solvents to elute the complex; however, the use of large amounts of solvent leads to dilution of the complex solutions, which is undesirable. Degradation of the complex can also occur during purification, which can lead to a decrease in the yield of the complex, as well as the loss of the free radioisotope.

The present inventors have found that the purification of a complex of a compound of formula (I) and a Cu radioisotope is advantageously achieved, and the complex is isolated in high chemical and radiochemical yield.

According to another aspect, the present invention relates to a method for purifying a compound of formula (I) in the form of a complex with a radioisotope Cu

formula (I),

where the method includes the following steps:

i) loading a solution of the compound of formula (I) in the form of a complex with a Cu radioisotope onto a solid phase extraction cartridge;

ii) eluting the compound of formula (I) as a complex with the Cu radioisotope with an eluent containing water, ethanol and sodium chloride.

In one embodiment of the method, the compound of formula (I) has the structure of the compound of formula (Ia)

formula (Ia),

In one embodiment, a solution of a compound of formula (I) in the form of a complex with a Cu radioisotope is obtained according to another embodiment of the present invention. After completion of the reaction for obtaining a complex of the compound of formula (I) and the Cu radioisotope, the resulting solution is subjected to a purification method.

A solution containing a complex of a compound of formula (I) with a Cu radioisotope is loaded onto a solid-phase extraction cartridge. The solid-phase extraction cartridge contains a stationary phase that retains the complex and other components present in the solution. For the purposes of this invention, the term "stationary phase" refers to the resinous material contained in the solid-phase extraction cartridge, which allows for the separation of compounds based on their polarity.

The solid-phase extraction method described herein may utilize a reversed-phase stationary phase. As used herein, the term "reversed-phase" with respect to a stationary phase refers to a stationary phase that is hydrophobic in nature, such that the stationary phase has an affinity for hydrophobic or uncharged molecules. Examples of a reversed-phase stationary phase may include Waters Sep-Pak cartridges, such as C8, C18, light C18, light CN, light tC2, or HLB cartridges. Prior to loading the solution containing the complex of the compound of formula (I), the cartridge is washed with ethanol, air-dried, and equilibrated with water. In one embodiment, the solid-phase extraction cartridge is a Waters C18 cartridge. In another embodiment, the solid-phase extraction cartridge is a Waters tC2 cartridge. In yet another embodiment, the solid-phase extraction cartridge is a Waters CN cartridge. In another embodiment, the solid phase extraction cartridge is a Waters HLB cartridge.

The purified solution containing the complex of the compound of formula (I) with a Cu radioisotope can then be used to prepare a composition containing the complex. For example, one or more pharmaceutically acceptable diluents, adjuvants, and/or excipients can be added to the solution containing the complex of the compound of formula (I) with a Cu radioisotope. The diluents, adjuvants, and excipients must be "acceptable" in terms of compatibility with the other ingredients of the composition and not toxic to the recipient. Pharmaceutical carriers for the preparation of pharmaceutical compositions are well known in the art and are described in monographs such as Remington's Pharmaceutical Sciences, 20th Edition, Williams & Wilkins, Pennsylvania, USA. The carrier will depend on the route of administration, and, again, one skilled in the art can readily determine the most suitable composition for each specific case.

Reference in this specification to any prior publication (or information derived therefrom) or to any prior art does not constitute and should not be construed as an endorsement or admission, or any form of suggestion, that such prior publication (or information derived therefrom) or prior art forms part of the general knowledge in the technical field to which the present invention pertains.

In the present description and claims, unless the context otherwise requires, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of the stated integer, step, or group of integers or steps, but not the exclusion of any other integer, steps, or group of integers or steps.

Examples

General experimental details

Sodium acetate-based buffers for radiolabeling were prepared using sodium acetate (TraceSELECT, Fluka, lot number BCBM4793V), acetic acid (TraceSELECT, Fluka, lot number BCBM5177V), and MilliQ water in acid-washed glass bottles and stored in acid-washed plastic bottles. All buffers were stored at 2°C-4°C until used.

Phosphate buffers for radiolabeling were prepared using dibasic (anhydrous) sodium phosphate, monobasic sodium phosphate, and TraceSELECT water. All buffers were stored at room temperature until used.

Copper-64 ( ^64Cu ) was obtained from SAHMRI, SA, Australia as [ ^64Cu ] _CuCl2 in 0.02 M HCl, batch no. 19-0075-902R, with an initial activity of 2.39 GBq @ 08:39 in a volume of 450 µl.

Radiolabeled products were purified by SPE using Waters Sep-Pak light C8 cartridges (lot number: 002836047A). The cartridges were conditioned by rinsing with EtOH (10 mL), then air (3 × 10 mL), then equilibrated with MilliQ _H2O (10 mL) and air (3 × 10 mL).

HPLC grade MeCN (Honeywell, lot number S1RA1H) and HPLC grade trifluoroacetic acid (TFA, ReagentPlus, 99%, Sigma Aldrich, lot number SHBG2783V), (+)-sodium L-ascorbate (Sigma Aldrich, >99%, lot number BCBV4424), L-methionine (Sigma Aldrich, >99.5%, lot number BCBS2107V), and sodium gentisic acid hydrate (Sigma Aldrich, >99%, lot number MKCC2280) were used as received. All HPLC mobile phases were prepared before use, filtered (using a 0.45 μm aqueous or organic filter), and degassed using a combination of ultrasonic degassing and vacuum for 10 min. All EtOH used was 100% ethyl alcohol (molecular biology grade). All syringes used were B Braun Injekt-F.

The elution buffer was prepared as a mixture of EtOH: _H2O , 1:1+0.9% NaCl.

All reaction vessels were acid-washed before use. Plastic microcentrifuge tubes were filled with 4 M HCl and left overnight. The 4 M HCl was removed, the vials were thoroughly rinsed with MilliQ _H2O and dried in an oven at 50°C. After drying, the vials were hermetically sealed to prevent further contamination. Glassware was acid-washed by soaking in 4 M _HNO3 for at least 12 h. After pouring the 4 M _HNO3 into a suitable waste container, the glassware was thoroughly rinsed with MilliQ _H2O and dried in an oven at 50°C. After drying, the glassware was hermetically sealed to prevent further contamination.

A stock solution of Sar-bis(PSMA), i.e., the compound of formula (Ia), was prepared in a mixture of EtOH:H ₂ O (1:1) to obtain a solution of the compound of formula (Ia) with a concentration of 1 nmol/μl.

Radiolabeling of a compound of formula (I) with a radioisotope Cu

Example 1

Labeling buffer (acetate, 50 µl, 1 M, pH 5.0) was added to a 500 µl acid-washed microcentrifuge tube, followed by 10 µl of a stock solution of the compound of Formula I. [ ⁶⁴ Cu]CuCl ₂ in 0.02 M HCl (25 µl, 116 MBq) was added to the buffer solution. The microcentrifuge tube was sealed and the radioactivity present in the reaction mixture was measured using a dose calibrator. The tube was transferred to an Eppendorf Thermomixer C and heated at 40°C for 20 min. After 20 min, the reaction mixture was removed from the thermomixer, and a sample (5 µL) was removed from the reaction mixture, diluted with 1:1 EtOH: _H2O (5 µL), and injected into radio-HPLC systems (QC1, 5 µL). The reaction mixture was maintained at room temperature while a final analysis was performed to determine whether the radiochemical yield was > 95% at 7 min.

Example 2

Labeling buffer (acetate, 50 µl, 1 M, pH 5.0) was added to a 500 µl acid-washed microcentrifuge tube, followed by 5 µl of a stock solution of the compound of Formula I. [ ^64Cu ] _CuCl2 in 0.02 M HCl (25 µl, 109 MBq) was added to the buffer solution. The microcentrifuge tube was sealed and the radioactivity present in the reaction mixture was measured using a dose calibrator. The tube was transferred to an Eppendorf Thermomixer C and heated at 40°C for 20 min. After 20 min, the reaction mixture was removed from the thermomixer, and a sample (5 µL) was removed from the reaction mixture, diluted with 1:1 EtOH: _H2O (5 µL), and injected into radio-HPLC systems (QC1, 5 µL). The reaction mixture was maintained at room temperature while a final analysis was performed to determine whether the radiochemical yield was > 95% at 7 min.

Example 3

Labeling buffer (acetate, 600 µl, 1 M, pH 5.0) was added to an acid-washed 1500 µl microcentrifuge tube, followed by 20 µl of a stock solution of the compound of Formula I. [ ^64Cu ] _CuCl2 in 0.02 M HCl (300 µl, 1136 MBq) was added to the buffer solution. The microcentrifuge tube was sealed and the radioactivity present in the reaction mixture was measured using a dose calibrator. The tube was transferred to an Eppendorf Thermomixer C and heated at 40°C for 20 min. After 20 min, the reaction mixture was removed from the thermomixer, and a sample (5 µL) was removed from the reaction mixture, diluted with 1:1 EtOH: _H2O (5 µL), and injected into radio-HPLC systems (QC1, 5 µL). The reaction mixture was maintained at room temperature while a final analysis was performed to determine whether the radiochemical yield was > 95% at 7 min.

Example 4

To a 500 µl acid-washed microcentrifuge tube was added labeling buffer (20% EtOH in acetate buffer, 100 µl, 1 M, pH 5.0), followed by 10 µl of the stock solution of compound of Formula I. To the buffer solution was added [ ⁶⁴ Cu]CuCl ₂ in 0.02 M HCl (50 µl, 183 MBq). The microcentrifuge tube was sealed and the radioactivity present in the reaction mixture was measured using a dose calibrator. The tube was transferred to an Eppendorf Thermomixer C and heated at 40°C for 20 min. After 20 min, the reaction mixture was removed from the thermomixer, and a sample (5 µL) was removed from the reaction mixture, diluted with 1:1 EtOH: _H2O (5 µL), and injected into radio-HPLC systems (QC1, 5 µL). The reaction mixture was maintained at room temperature while a final analysis was performed to determine whether the radiochemical yield was > 95% at 7 min.

Example 5

To an acid-washed 500 µl microcentrifuge tube was added labeling buffer (acetate, 100 µl, 1 M, pH 5.0), followed by 5 µl of a stock solution of the compound of Formula I. To the buffer solution was added [ ⁶⁴ Cu]CuCl ₂ in 0.02 M HCl (50 µl, 174 MBq). The microcentrifuge tube was sealed and the radioactivity present in the reaction mixture was measured using a dose calibrator. The tube was transferred to an Eppendorf Thermomixer C and heated at 21°C for 20 min. After 5 and 15 min, aliquots (5 µl) were removed for analysis to determine the completion of the reaction. These samples were diluted 1:1 with EtOH: _H2O (5 µL) and injected into radio-HPLC systems (QC1, 5 µL). The reaction mixture was maintained at room temperature while final analysis was performed to determine whether the radiochemical yield was >95% at 7 min.

Example 6

To a solution of Sar-bis(PSMA) (50 μg, 24.8 nmol) in 0.1 M Na/Na phosphate buffer (5 ml) containing sodium gentisate (5 mg, 0.03 mmol) was added [ ⁶⁴ Cu]CuCl ₂ in 0.02 M-0.05 M HCl (NMT 500 μl, NMT 5000 MBq) at room temperature. The resulting mixture was reacted at room temperature for 25 min. Upon completion, the reaction mixture was quenched with 50 mg/ml sodium ascorbate solution (15 ml). The resulting mixture was then transferred through a vented 0.22 μm filter into a sterile vial to afford the compound of formula (I) as a complex with the radioisotope ⁶⁴ Cu.

Example 7

To a solution of Sar-bis(PSMA) (50 μg, 24.8 nmol) in 0.1 M Na/Na phosphate buffer (4.5 mL) containing sodium gentisate (5 mg, 0.03 mmol) and ethanol (neat, 0.5 mL) was added [ ⁶⁴ Cu]CuCl ₂ in 0.02 M-0.05 M HCl (NMT 500 μL, NMT 5000 MBq) at room temperature. The resulting mixture was reacted at room temperature for 25 min. Upon completion, the reaction mixture was quenched with 50 mg/mL sodium ascorbate solution (15 mL). The resulting mixture was then transferred through a vented 0.22 μm filter into a sterile vial to afford the compound of formula (I) as a complex with the radioisotope ⁶⁴ Cu.

Cleaning procedures

Example 8

The solution obtained in Example 1 was purified using a C8 SPE cartridge, eluting the product with 0.5 mL of 1:1 EtOH: _H2O +0.9% NaCl. 62% of the product was eluted by SPE, and the product was shown to have a high radiochemical purity of 94.3% with no "free copper." 4% was lost in the dilution/loading syringe, 12% remained in the reaction vessel, and 9% was lost during SPE. <1% was lost during the loading and washing steps of SPE.

Example 9

The solution obtained in Example 2 was purified using a C8 SPE cartridge, eluting the product with 0.5 mL of 1:1 EtOH: _H2O +0.9% NaCl. 73% of the product was eluted by SPE, and the product was shown to have a high radiochemical purity of 94.3% with 0.2% "free copper." 7% was lost in the dilution/loading syringe, 1% remained in the reaction vessel, and 14% was lost during SPE. <1% was lost during the loading and washing steps of SPE.

Example 10

The solution obtained in Example 3 was purified using a C8 SPE cartridge, eluting the product with 0.5 mL of a 1:1 mixture of EtOH: _H2O + 0.9% NaCl. 64% of the product was eluted by SPE, and the product was shown to have a high radiochemical purity of 96.6% with 0.1% "free copper." 4% was lost in the dilution/loading syringe, leaving 1.5% in the reaction vessel.

Example 11

The solution obtained in Example 4 was purified using a C8 SPE cartridge, eluting the product with 0.5 mL of 1:1 EtOH: _H2O +0.9% NaCl. 71% of the product was eluted by SPE, and the product was shown to have a high radiochemical purity of 96.3% with zero free copper. 6% was lost in the dilution/loading syringe, 3% remained in the reaction vessel, and 15% was lost during SPE. <1% was lost during the loading and washing steps of SPE.

Example 12

The solution obtained in Example 5 was purified using a C8 SPE cartridge, eluting the product with 0.5 mL of 1:1 EtOH: _H2O +0.9% NaCl. 59% of the product was eluted by SPE, and the product was shown to have a high radiochemical purity of 96.9% with 0.1% "free copper." 11% was lost in the dilution/loading syringe, 7% remained in the reaction vessel, and 20% was lost during SPE. <1% was lost during the loading and washing steps of SPE.

Preparation of compositions

Example 13

Aliquots of the purified solution from Example 10 were taken and diluted with a mixture of ethanol and saline to obtain solutions with a final ethanol concentration of approximately 10% in saline. One of gentisic acid (0.63 mg/mL), ascorbic acid (10 mg/mL), or L-methionine (3 mg/mL) was added, and the radiochemical purity of each sample was monitored for 48 hours.

Claims (39)

1. An aqueous composition for parenteral administration for radiation therapy and diagnostic imaging, containing a compound of formula (I) or a salt thereof in the form of a complex with a Cu ion , wherein the composition further comprises ascorbic acid or a pharmaceutically acceptable salt thereof and gentisic acid or a pharmaceutically acceptable salt thereof. 2. An aqueous composition according to claim 1, wherein the composition further comprises a buffer solution. 3. An aqueous composition according to claim 1, wherein the compound of formula (I) has a structure of formula (Ia) . 4. An aqueous composition according to any one of claims 1-3, wherein the composition further comprises L-methionine or a salt thereof. 5. An aqueous composition according to any one of claims 1 to 4, wherein the Cu ion is a radioisotope of Cu. 6. The aqueous composition of claim 5, wherein the Cu radioisotope is ^64Cu . 7. The aqueous composition of claim 5, wherein the Cu radioisotope is ^67Cu . 8. An aqueous composition according to any one of claims 1 to 7, wherein the pH of the composition is from about 4 to about 8. 9. A method for producing a composition containing a compound of formula (I) in the form of a complex with a Cu radioisotope according to claim 1 , where the method includes the following steps: i) adding an amount of a compound of formula (I) to a solution containing an acetate buffer and gentisic acid or a pharmaceutically acceptable salt thereof; ii) adding a solution of the radioisotope Cu in hydrochloric acid to the compound of formula (I) and an acetate buffer; iii) heating the mixture obtained in step ii) for a period of time and under conditions allowing the formation of a complex of the compound of formula (I) and the Cu radioisotope; and iv) adding a solution of sodium ascorbate to the mixture of the compound of formula (I) and the Cu radioisotope. 10. A method for producing a composition containing a compound of formula (I) in the form of a complex with a Cu radioisotope according to claim 1 , where the method includes the following steps: i) adding an amount of a compound of formula (I) to a solution containing a phosphate buffer and gentisic acid or a pharmaceutically acceptable salt thereof; ii) adding a solution of a Cu radioisotope in hydrochloric acid to the compound of formula (I) and a phosphate buffer solution; iii) subjecting the mixture obtained in step ii) to reaction for a period of time and under conditions allowing the formation of a complex of the compound of formula (I) and the Cu radioisotope; and iv) adding a solution of sodium ascorbate to the mixture of the compound of formula (I) and the Cu radioisotope. 11. The method according to claim 9 or 10, wherein the compound of formula (I) has a structure of formula (Ia) . 12. The method according to any one of claims 9-11, wherein the Cu radioisotope is ^64Cu . 13. The method according to any one of claims 9 to 11, wherein the Cu radioisotope is ^61Cu . 14. The method according to any one of claims 9-11, wherein the Cu radioisotope is ^67Cu . 15. The method according to any one of paragraphs .9-11, wherein the Cu radioisotope is [ ⁶⁴ Cu]CuCl ₂ . 16. The method of any one of claims 9-15, wherein the pH of the composition is maintained in the range of from about 4 to about 8. 17. The method according to any one of claims 9 to 16, wherein the method further comprises the step of purifying the compound of formula (I) in the form of a complex with a radioisotope of Cu by: i) loading a solution of a compound of formula (I) complexed with a Cu radioisotope onto a solid phase extraction cartridge; and ii) eluting the compound of formula (I) as a complex with the Cu radioisotope with an eluent containing water, ethanol and sodium chloride. 18. The method according to claim 17, wherein the compound of formula (I) has a structure of formula (Ia) .