CGMP-Compliant Automated [18F]FSPG Production for Clinical Testing

Abstract

According to various aspects of the present disclosure, the present disclosure relates to an automated process for the radiosynthesis of [18F]FSPG, a positron emission tracer. The automated optimization process of the present invention enables the centralized and large-scale production of [18F]FSPG for commercial use.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 344,902, filed May 23, 2022, the entire disclosure of which is incorporated herein by reference.

[0002] The present disclosure relates to methods and devices for the automated synthesis of compounds suitable for use as in - vivo contrast agents, and methods of using the same.

Background Art

[0003] Radiolabeled compounds for use as in - vivo contrast agents are currently prepared by automated synthesizers (or “radiosynthesizers”). Such automated synthesizers are commercially available from various suppliers including GE Healthcare; CTI Inc.; Ion Beam Applications S.A. (Chemin du Cyclotron 3, B - 1348 Louvain - La - Neuve, Belgium); Raytest (Germany) and Bioscan (USA). Radiochemistry occurs within a “cassette” or “cartridge” that is removable on the device and designed to be interchangeably attached thereto in such a way that the mechanical movement of the moving parts of the device controls the operation of the cassette. Suitable cassettes can be provided as part of a kit that is assembled on the device in several steps or as a single part that is attached in a single step, thereby reducing the risk of human error. An integrated configuration is generally a disposable single - use cassette that includes all the reagents, eluents, reaction vessels, and devices necessary to carry out the preparation of a given batch of radiopharmaceutical.

[0004] (S)-4-(3 - 18 F - fluoropropyl)-L - glutamic acid( 18 F]FSPG) is a cystine / glutamate antiporter (Xc -A positron emission tomography (PET) tracer that specifically targets 18 and is a biomarker that is frequently overexpressed in cancer and some neuropathies. Pilot studies examining dosimetry and biodistribution in healthy volunteers and tumor detection in patients with non-small cell lung cancer, hepatocellular carcinoma, and brain tumors have shown promising results. In particular, low background uptake was observed in the brain, lung, and intestine, which further

[0005] However, 18F due to the lack of a reliable cGMP-compliant automated process for 18 production, the clinical introduction of this radiotracer has been delayed. Therefore,

Summary of the Invention

[0006] Some aspects of the present disclosure relate to an automated process for the synthesis of (S)-4-(3- 18 Fluoropropyl)-L-glutamic acid ( 18 F-FSPG) in a cassette, the process comprising a. Dissolving an (S)-4-(3- 18 fluoropropyl)-L-glutamic acid precursor in anhydrous acetonitrile and adding the resulting solution to a reaction vessel containing radiolabeled fluoride ( 18 F); b. Heating the reaction vessel at 100°C to 110°C (e.g., for 5 minutes to 15 minutes) to obtain a crude intermediate product; c. Adding a first aliquot of an acid to the reaction vessel, heating at 100°C to 110°C (e.g., for 2 to 10 minutes), subsequently adding an aliquot of a base to the reaction vessel, and heating at 60°C to 80°C (e.g., for 5 to 15 minutes) to remove the protecting group and obtain crude 18 F-FSPG; d. Acidifying the crude 18 F-FSPG; e. loading the acidified crude 18 F-FSPG onto one or more reversed-phase solid-phase extraction cartridges, f. eluting the 18 F-FSPG from one or more reversed-phase solid-phase extraction cartridges, g. purifying the 18 F-FSPG through one or more normal-phase solid-phase extraction cartridges, and including (a)-(g) are each performed within the cassette.

[0007] In some embodiments, 18 the F-FSPG is eluted from one or more reversed-phase solid-phase extraction cartridges using a buffer. In some embodiments, the buffer is phosphate-buffered saline.

[0008] In some embodiments, the acid in step (c) is selected from the group consisting of sulfuric acid, hydrochloric acid, and trifluoroacetic acid. In some embodiments, the acid in step (c) is sulfuric acid or trifluoroacetic acid. In some embodiments, the acid in step (c) is sulfuric acid. In some embodiments, the acid in step (c) is hydrochloric acid. In some embodiments, the acid in step (c) is trifluoroacetic acid.

[0009] In some embodiments, the base in step (c) is sodium hydroxide or potassium hydroxide. In some embodiments, the base in step (c) is sodium hydroxide. In some embodiments, the base in step (c) is potassium hydroxide.

[0010] In some embodiments, the one or more reversed-phase solid-phase extraction cartridges are washed with water to remove by-products prior to step (f).

[0011] In some embodiments, the crude 18 F-FSPG is acidified in step (d) with a second aliquot of acid. In some embodiments, the crude 18 F-FSPG is acidified in step (d) with a second aliquot of sulfuric acid.

[0012] In some embodiments, 18 F-FSPG is purified through one or more normal-phase solid-phase extraction cartridges that are external to the cassette. In some embodiments, 18 the F-FSPG product is further sterilized through a sterile filter to produce the final 18 F-FSPG composition.

[0013] In some embodiments, the final purified 18 F-FSPG has a radiochemical purity of greater than 90%, 95%, 96%, 97%, or 98%.

[0014] In some embodiments, the process produces F-FSPG with an uncorrected radiochemical yield of at least 10%. In some embodiments, the process produces F-FSPG with an uncorrected radiochemical yield of 10% to 40%. 18 In some embodiments, the process produces F-FSPG with an uncorrected radiochemical yield of 13% to 35%. 18 In some embodiments, the process produces F-FSPG with an uncorrected radiochemical yield of 18% to 32%. 18 In some embodiments, the process produces F-FSPG with an uncorrected radiochemical yield of 18% to 32%. 18 In some embodiments, the process produces F-FSPG with an uncorrected radiochemical yield of 18% to 32%.

[0015] In some embodiments, the reaction product is loaded onto a reverse-phase solid-phase extraction at a flow rate of 8 ml / min to 20 ml / min and eluted from the reverse-phase solid-phase extraction cartridge at a flow rate of 4 ml / min to 14 ml / min. In some embodiments, the reaction product is loaded onto a reverse-phase solid-phase extraction at a flow rate of 14 ml / min and eluted from the reverse-phase solid-phase extraction cartridge at a flow rate of 10 ml / min.

[0016] In some embodiments, the reverse-phase solid-phase extraction cartridge includes an MCX Oasis cartridge. In some embodiments, 18 F]FSPG is used as the crude 18Elute from the reverse-phase solid-phase extraction cartridge in the same direction as when loading the FSPG. In some embodiments, the normal-phase solid extraction cartridge includes an alumina Sep-Pak cartridge and / or an ENVI-carb or Hypercarb cartridge. In some embodiments, the normal-phase solid extraction cartridge includes an alumina Sep-Pak cartridge. In some embodiments, the normal-phase solid extraction cartridge includes an ENVI-carb cartridge. In some embodiments, the normal-phase solid extraction cartridge includes a Hypercarb cartridge.

[0017] In some embodiments, the FSPG precursor comprises one of the following structure(s):

Chemical formula

[0018] In some embodiments, 18 F-FSPG has an initial radioactivity of about 10 - 110 GBq.

[0019] In some embodiments, the final 18 F-FSPG composition has a concentration of about 185 - 1850 MBq / ml.

[0020] In some embodiments, the final 18 F-FSPG composition is produced in less than 40 minutes from the start of synthesis (e.g., 5 - 40 minutes, 10 - 40 minutes, 15 - 40 minutes, 5 - 35 minutes, 10 - 30 minutes, 15 - 30 minutes from the start of synthesis).

[0021] In some embodiments, the final 18 F-FSPG composition has a pH of 6.5 - 7.5.

[0022] In some embodiments, the radiolabeled fluoride is a. 18 Irradiate water to 18 generate a solution of 18 F in water, and b. Loading an F solution onto a quaternary ammonium anion exchange cartridge preconditioned with potassium bicarbonate 18 and c. Eluting F from the cartridge into a reaction vessel using an eluent of potassium carbonate / 4,7,13,16,21,24 - hexaoxa - 1,10 - diazabicyclo[8.8.8]hexacosane in a 50% water / acetonitrile solution 18 and d. heating the reaction vessel to remove water, is prepared by a method comprising:

[0023] In some embodiments, 18 the composition comprising F - FSPG is prepared according to any of the processes of the present disclosure.

[0024] In some embodiments, 18 the F - FSPG composition, when stored at room - temperature conditions, is not radiolytically degraded for at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours after synthesis.

[0025] In some embodiments, 18 the F - FSPG composition maintains a radiochemical purity of greater than 90%, 95%, 97%, 98%, or 99% after storage at room - temperature conditions for at least 5 hours, at least 6 hours, at least 7 hours, or at least 8 hours. In some embodiments, 18 the F - FSPG composition maintains a radiochemical purity of greater than 90%, 95%, 97%, 98%, or 99% after storage at room temperature for at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0026]

Figure 1

Figure 2

Figure 3

DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention relates to an apparatus and method for automatically synthesizing an [18F] - labeled compound, such as [18F]FSPG, which is suitable for use as an in - vivo contrast agent for positron emission tomography (PET) using a disposable cassette. 18 F], for example, 18 F]FSPG.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, this application, including definitions, will control. Further, unless the context clearly requires otherwise, singular terms shall include pluralities and plural terms shall include singulars. All publications, patents, and other references mentioned in this specification are hereby incorporated by reference in their entirety as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0029] Methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, but the appropriate methods and materials are disclosed below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of this disclosure will be apparent from the detailed description and from the claims.

[0030] I. Definitions To further understand this disclosure, the following terms and definitions are provided.

[0031] Unless the context clearly dictates otherwise, the singular forms “a,” “an,” and “the” include plural referents. The terms “a” (or “an”), “one or more,” and “at least one” are used interchangeably herein. In certain embodiments, the term “a” or “an” means “one.” In other embodiments, the term “a” or “an” includes “two or more” or “plural.”

[0032] The term “about” is used herein to mean approximately, roughly, nearly, or almost. When the term “about” is used in conjunction with a numerical range, this term modifies the range by extending the boundaries to above and below the recited numerical values. In general, the term “about” is used herein with a width of plus or minus (±) 10% of the recited value to modify a numerical value.

[0033] Throughout this disclosure, various aspects of the disclosure are presented in range format. Numerical ranges include the numbers defining the range. When a range of values is recited, it is to be understood that each intervening integer value, and each fractional part thereof, between the recited upper and lower limits of that range, as well as each subrange between such values, is also expressly disclosed. It is also to be understood that the upper and lower limits of any range can independently be included in or excluded from the range, and that each range that includes either, neither, or both of the limits is also encompassed within the disclosure. Accordingly, the ranges recited herein are to be understood to be merely shorthand notations for all of the values falling within the range including the recited endpoints. For example, a range of 1 to 10 is to be understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0034] It should be understood that when values are explicitly enumerated, values that are approximately the same size or amount as the enumerated values are also within the scope of the present disclosure. When a combination is disclosed, each partial combination of the elements of that combination is also specifically disclosed and is included within the scope of the present disclosure. Conversely, when different elements or groups of elements are disclosed individually, their combinations are also disclosed. When any element of the present disclosure is disclosed as having a plurality of alternatives, examples of the disclosure where each alternative is excluded, either alone or in any combination with other alternatives, are also disclosed herein, two or more elements of the disclosure may have such exclusions, and all combinations of elements having such exclusions are disclosed herein.

[0035] As used herein, the term "and / or" should be regarded as a specific disclosure of each of the two specified features or components, regardless of the presence or absence of others. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in statements such as "A, B, and / or C" is intended to include each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0036] It is understood that when an aspect is disclosed herein using the phrase "comprising", similar aspects are also provided with respect to "consisting of" and / or "consisting essentially of" as disclosed separately.

[0037] As used herein, the term "cassette" means a component of a device (e.g., an automated synthesis device) that is designed to fit removably on the device. In some aspects, the cassette can be removed such that the mechanical movement of a movable part of the device (e.g., an automated synthesizer device) controls the operation of the cassette from outside the cassette, i.e., externally. In some aspects, the cassette can be for single use.

[0038] As used with respect to the cassettes of the present disclosure, the term "disposable" means that the cassette is intended to be used once or in a period (e.g., in a single or multiple batches) before disposal.

[0039] As used herein, the term "selectively fluidly connected" means that fluid can be selectively passed to and / or from another mechanism of the cassette, for example, by using a suitable valve. In some embodiments, a suitable valve includes a three-way valve having three ports and means for fluidly blocking a third port while fluidly connecting any two of the three associated ports to each other. In some embodiments, a suitable valve includes a stopcock valve including a rotatable stopcock. In some embodiments, the components of the cassette are selectively fluidly connected along a common path.

[0040] As used herein, the term "common path" refers to a fluid path to which specific components of a system (e.g., an automated synthesizer) and / or specific components of the cassettes of the present disclosure are selectively fluidly connected. In some embodiments, the common path is a linear fluid path. In some embodiments, the common path is made of a rigid pharmaceutical-grade polymer material resistant to radiation. Non-limiting examples of such suitable materials include polypropylene, polyethylene, polysulfone, and ULTEM®. In some embodiments, the common path is made of polypropylene or polyethylene.

[0041] As used herein, the term "automated synthesizer" means an automated module based on the principle of unit operations, for example, as described by Satyamurthy et al (1999 Clin Positr Imag;2(5):233-253), which is hereby incorporated by reference. The term "unit operation" means that a complex process is broken down into a series of more basic or simple operations or reactions, which can be applied to various materials. Such automated synthesizers are preferred for the methods of the present disclosure, particularly when it is desired to prepare radiopharmaceutical compositions. Such automated synthesizers are commercially available from various suppliers, including GE Healthcare; CTI Inc; Ion Beam Applications S.A. (Chemin du Cyclotron 3, B-1348 Louvain-LaNeuve, Belgium); Raytest (Germany) and Bioscan (USA) (Satyamurthy et al, supra). In certain embodiments, the automated synthesizer is designed to be used in a properly configured radioactive work cell or "hot cell" that provides radiation shielding suitable for protecting the operator from potential radiation doses, as well as ventilation for removing chemicals and / or radioactive vapors. Automated synthesizers that use cassettes have the flexibility to produce a variety of different radiopharmaceuticals while minimizing the risk of cross-contamination by allowing for cassette exchange. This approach also has the advantages of simplified setup, thus reducing the risk of operator error, improved compliance with GMP (Good Manufacturing Practice), multi-tracer capabilities, rapid changeover between production runs, automated diagnostic checks of cassettes and reagents prior to the process, and automated barcode verification checks of chemical reagents against the synthesis to be performed, reagent traceability, single-use (thus eliminating the risk of cross-contamination), resistance to tampering and abuse.

[0042] In the context of the present disclosure, a "reaction vessel" is a receptacle of a container or cassette that can deliver the reactants and reagents necessary for PET tracer synthesis and remove the product(s) in the appropriate order. In some embodiments, the reaction vessel has an internal volume suitable for containing the reactants and reagents and is made of a radiation-resistant pharmaceutical-grade material. In some embodiments, the reaction vessel can be heated to catalyze a reaction, increase the reaction rate, or evaporate a solvent internally. In some embodiments, the reaction vessel is heated by a reactor heater (e.g., as a component of an automated synthesizer, such as a GE FASTlab device).

[0043] In the context of the method of the present disclosure, an "aliquot" is a sufficient amount of a particular reagent for use during a step of the method (e.g., during a step of synthesizing a PET tracer).

[0044] As used herein, a "precursor compound" is a non-radioactive derivative of a radiolabeled compound. In some embodiments, the precursor compound is designed such that a chemical reaction with a convenient chemical form of a detectable label occurs site-specifically in a minimal number of steps to obtain the desired radiolabeled compound. In certain embodiments, the precursor compound can have a protecting group for site-specific labeling. In some embodiments, the precursor compound is synthetic and can be conveniently obtained with good chemical purity.

[0045] The term "protecting group" refers to a group that inhibits or suppresses undesired chemical reactions of a molecule, and this group is designed to be sufficiently reactive so that it can be removed (e.g., cleaved) from the functional group of the molecule to produce a product under conditions that do not modify the remainder of the desired molecule. Protecting groups and methods for their removal (i.e., "deprotection") are well known to those skilled in the art and are described in "Protective Groups in Organic Synthesis", Theodora W. Greene and Peter G. M. Wuts, (Fourth Edition, John Wiley & Sons, 2007), which is hereby incorporated by reference. In some embodiments, the deprotecting agent is HCl, NaOH, H 3 PO 4 , trifluoroacetic acid, and H 2 SO 4 selected from. In some embodiments, multiple deprotecting agents are required to remove multiple protecting groups on the precursor compound. In some embodiments, the deprotecting agent is NaOH. In some embodiments, the deprotecting agent is H 2 SO 4 . In some embodiments, the deprotecting agent is trifluoroacetic acid.

[0046] As used herein, the term "reagent" refers to a solvent and / or reactant used in the synthesis of a molecule, compound, or product (e.g., 18 F] labeled PET tracer). In some embodiments, the reagent or reagents are stored in a reagent vial.

[0047] The term "reagent vial" refers to a receptacle or vial containing at least one reagent for use in the methods, apparatuses, or cassettes disclosed herein, e.g., 18 for use in the manufacture of an

[0048] The term "sufficient" means, for example, an appropriate amount of reagent to enable completion of one or more steps or processes that require a reagent.

[0049] The term "solid phase extraction" or "SPE" refers to a sample preparation process that separates compounds in a solution from each other based on their respective affinities for a solid ("solid phase" or "stationary phase") through which the sample passes and a solvent ("mobile phase" or "liquid phase") in which they dissolve. Using the SPE process, the compound of interest is retained either in the solid phase or the mobile phase. The portion that passes through the solid phase can be collected or discarded depending on whether it contains the compound of interest. If the portion retained on the stationary phase contains the compound of interest, it can be removed from the stationary phase for collection in an additional step, and the stationary phase is rinsed with another solution known as an "eluent". In the case of the present disclosure, SPE can be suitably carried out using an "SPE cartridge" (also referred to as an "SPE column"), which is commercially available and readily accessible and is typically in the form of a syringe-type column filled with a solid phase. In some embodiments, the solid phase is based on silica bonded to a specific functional group, such as a hydrocarbon chain of variable length (suitable for reverse phase SPE), a quaternary ammonium or amino group (suitable for anion exchange), and a sulfonic acid or carboxyl group (suitable for cation exchange).

[0050] As used herein, the term "eluting" refers to passing a solution through a solid phase, e.g., via an SPE cartridge, for the purpose of releasing the compound(s) of interest that are or were bound to the solid phase.

[0051] As used herein, the term "capturing" refers to the process by which a particular compound(s) binds to the solid phase of, e.g., an SPE cartridge.

[0052] The term "purifying" or "purification" as used herein refers to obtaining a substantially pure molecule, compound, or product (e.g., 18It can be interpreted to mean a process for obtaining a [[F]]-labeled compound. The term "substantially" refers to a complete or nearly complete degree or extent of an action, characteristic, property, state, structure, article, or result (e.g., suitable for use as a PET tracer). The term "substantially pure" can be interpreted to mean being completely pure, or nearly completely pure, or free of contaminants (e.g., unreacted reagents or by-products) that would interfere with the intended purpose.

[0053] The term "suitable for use as a PET tracer" means that 18 the [[F]]-labeled compound is suitable for intravenous administration to a mammalian subject followed by obtaining one or more useful images of the location and / or distribution of the 18 [[F]]-labeled compound in PET imaging.

[0054] The term "sterilization" refers to a process of removing potential microbial contaminants from a molecule, compound, product, or composition containing it (e.g., 18 the [[F]]FSPG solution).

[0055] II. PET Tracer Certain aspects of the present disclosure relate to components (e.g., cassettes), devices (e.g., automated synthesis devices), and methods of using the same for synthesizing a positron emission tomography (PET) tracer (or radiotracer). In some aspects, the PET tracer is (S)-4-(3- 18 [[F]]-fluoropropyl)-L-glutamic acid ( 18 [[F]]FSPG) radiotracer ( 18 [[F]]FSPG radiotracer). In some aspects, the 18 [[F]]-labeled compound obtained by the methods of the present disclosure is substantially pure. In some aspects, the 18 [[F]]-labeled compound obtained by the methods of the present disclosure is completely pure. In some aspects, the 18 [[F]]-labeled compound obtained by the methods of the present disclosure is nearly pure.

[0056] In some embodiments, the present disclosure provides the production of 18 F]FSPG having at least 90% radiochemical purity. In some embodiments, the present disclosure provides the production of 18 F]FSPG having at least 91% radiochemical purity. In some embodiments, the present disclosure provides the production of 18 F]FSPG having at least 92% radiochemical purity. In some embodiments, the present disclosure provides the production of 18 F]FSPG having at least 93% radiochemical purity. In some embodiments, the present disclosure provides the production of 18 F]FSPG having at least 94% radiochemical purity. In some embodiments, the present disclosure provides the production of 18 F]FSPG having at least 95% radiochemical purity. In some embodiments, the present disclosure provides the production of 18 F]FSPG having at least 96% radiochemical purity. In some embodiments, the present disclosure provides the production of 18 F]FSPG having at least 97% radiochemical purity. In some embodiments, the present disclosure provides the production of 18 F]FSPG having at least 98% radiochemical purity. In some embodiments, the present disclosure provides the production of 18 F]FSPG having at least 90% - 99% radiochemical purity. In some embodiments, the present disclosure provides the production of 18 F]FSPG having at least 91% - 98% radiochemical purity. In some embodiments, the present disclosure provides the production of 18 F]FSPG having at least 92% - 97% radiochemical purity. In some embodiments, the present disclosure provides the production of 18 F]FSPG having at least 93% - 97% radiochemical purity. In some embodiments, the present disclosure provides the production of 18 F]FSPG having at least 93% - 96% radiochemical purity.

[0057] In some embodiments, the present disclosure provides at a radiochemical yield of 10% - 40% (without decay correction)18 F]Provides the production of FSPG. In some embodiments, the present disclosure provides the production of FSPG at a radiochemical yield (without attenuation correction) of 10% to 35% 18 F]Provides the production of FSPG. In some embodiments, the present disclosure provides the production of FSPG at a radiochemical yield (without attenuation correction) of 13% to 35% 18 F]Provides the production of FSPG. In some embodiments, the present disclosure provides the production of FSPG at a radiochemical yield (without attenuation correction) of 18% to 32% 18 F]Provides the production of FSPG. In some embodiments, the present disclosure provides the production of FSPG at a radiochemical yield (without attenuation correction) of 20% to 35% 18 F]Provides the production of FSPG. In some embodiments, the present disclosure provides the production of FSPG at a radiochemical yield (without attenuation correction) of 13% to 35% 18 F]Provides the production of FSPG. In some embodiments, the present disclosure provides the production of FSPG at a radiochemical yield (without attenuation correction) of 25% to 35% 18 F]Provides the production of FSPG. In some embodiments, the present disclosure provides the production of FSPG at a radiochemical yield (without attenuation correction) of at least 10% 18 F]Provides the production of FSPG. In some embodiments, the present disclosure provides the production of FSPG at a radiochemical yield (without attenuation correction) of at least 15% 18 F]Provides the production of FSPG. In some embodiments, the present disclosure provides the production of FSPG at a radiochemical yield (without attenuation correction) of at least 20% 18 F]Provides the production of FSPG. In some embodiments, the present disclosure provides the production of FSPG at a radiochemical yield (without attenuation correction) of at least 25% 18 F]Provides the production of FSPG. In some embodiments, the present disclosure provides the production of FSPG at a radiochemical yield (without attenuation correction) of at least 30% 18 F]Provides the production of FSPG. In some embodiments, the present disclosure provides the production of FSPG at a radiochemical yield (without attenuation correction) of at least 35% 18 F]Provides the production of FSPG.

[0058] In some embodiments, the present disclosure is not subject to radiolysis after 1 hour when stored at room temperature 18 F]Provides the production of FSPG. In some embodiments, the present disclosure is not subject to radiolysis after 2 hours when stored at room temperature18 F]Provides the generation of FSPG. In some embodiments, the present disclosure does not undergo radiolysis after 3 hours when stored at room temperature 18 F]Provides the generation of FSPG. In some embodiments, the present disclosure does not undergo radiolysis after 4 hours when stored at room temperature 18 F]Provides the generation of FSPG. In some embodiments, the present disclosure does not undergo radiolysis after 5 hours when stored at room temperature 18 F]Provides the generation of FSPG.

[0059] In some embodiments, the present disclosure has a radiochemical purity of at least 90% after being stored at room temperature for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours 18 F]Provides the generation of FSPG. In some embodiments, the present disclosure has a radiochemical purity of at least 91% after being stored at room temperature for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours 18 F]Provides the generation of FSPG. In some embodiments, the present disclosure has a radiochemical purity of at least 92% after being stored at room temperature for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours 18 F]Provides the generation of FSPG. In some embodiments, the present disclosure has a radiochemical purity of at least 93% after being stored at room temperature for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours 18 F]Provides the generation of FSPG. In some embodiments, the present disclosure has a radiochemical purity of at least 94% after being stored at room temperature for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours 18 F]Provides the generation of FSPG. In some embodiments, the present disclosure has a radiochemical purity of at least 95% after being stored at room temperature for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours 18 F]Provides the generation of FSPG. In some embodiments, the present disclosure has a radiochemical purity of at least 96% after being stored at room temperature for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours 18Provide the production of [¹⁸F]FSPG. In some embodiments, the present disclosure has a radiochemical purity of at least 97% after storage at room temperature for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours 18 Provide the production of [¹⁸F]FSPG. In some embodiments, the present disclosure has a radiochemical purity of at least 98% after storage at room temperature for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours 18 Provide the production of [¹⁸F]FSPG. In some embodiments, the present disclosure has a radiochemical purity of at least 95% after storage at room temperature for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours 18 Provide the production of [¹⁸F]FSPG. In some embodiments, the present disclosure has a radiochemical purity of at least 95% after storage at room temperature for 8 hours 18 Provide the production of [¹⁸F]FSPG.

[0060] In some embodiments, the final 18 [¹⁸F]FSPG composition obtained by the method of the present disclosure is provided as [¹⁸F]FSPG in phosphate buffered saline. 18 In some embodiments, the final 18 [¹⁸F]FSPG composition obtained by the method of the present disclosure has a pH of about 6 - 8. In some embodiments, the final 18 [¹⁸F]FSPG composition obtained by the method of the present disclosure has a pH of about 6.5 - 7.5.

[0061] In some embodiments, the final 18 [¹⁸F]FSPG composition obtained by the method of the present disclosure has a concentration of 185 - 2035 MBq / ml (5 - 55 mCi / ml). In some embodiments, the final 18 [¹⁸F]FSPG composition obtained by the method of the present disclosure has a concentration of 185 - 1850 MBq / ml (5 - 50 mCi / ml). In some embodiments, the final 18 [¹⁸F]FSPG 18The [¹⁸F]FSPG has a concentration of 260 - 1925 MBq / ml (7 - 52 mCi / ml). In some embodiments, the final 18 [¹⁸F]FSPG 18 [¹⁸F]FSPG has a concentration of 260 - 1850 MBq / ml (7 - 50 mCi / ml).

[0062] III. Cassette In some embodiments, the cassette of the present disclosure is a disposable single - part type cassette pre - loaded with a reagent including a linear array of valves, and each valve is connected to a port to which a container or vial can be attached (e.g., by either needle puncture of an inverted septum - sealed vial or an airtight coupling joint). In one embodiment, each valve is a three - way valve. In some embodiments, the cassette has 25 identical three - way valves in a linear array, an example of which is shown in FIG. 1. In one embodiment, each valve is a valve - type valve including a rotatable stopper. In certain embodiments, each valve has a male - female joint that interfaces with a corresponding movable arm of an automated synthesizer. Thus, the external rotation of the arm controls the opening and closing of the valve when the cassette is attached to the automated synthesizer. Additional movable parts of the automated synthesizer can be clipped to a syringe plunger tip and thus can be designed to raise or lower the syringe barrel. The cassette is multi - purpose and, in some embodiments, has several positions where reagents can be attached, and some positions are suitable for the attachment of reagent syringe vials or chromatography columns. In some embodiments, the cassette includes a reaction vessel, and generally, three or more ports of the cassette are connected thereto, configured to enable the transfer of reagents or solvents from various ports on the cassette and to enable the transfer of reaction products from the reaction vessel to various ports on the cassette.

[0063] In some embodiments, the reagent vial is made of a rigid pharmaceutical-grade polymer that is resistant to radiation. In some embodiments, suitable reagents contained in the reagent vial can include ethanol, acetonitrile, a deprotecting agent, a buffer, or any combination thereof. In some embodiments, the reagent includes a deprotecting agent. In some embodiments, the reagent includes a buffer. In some embodiments, the buffer is based on a weak acid selected, for example, from citrate, phosphate, acetate, and ascorbate. In some embodiments, the buffer is phosphate buffered saline (PBS). In some embodiments, each reagent is contained in a separate reagent vial. For example, in some embodiments, the single-use cassette of the present disclosure includes a reagent vial containing acetonitrile, another reagent vial containing NaOH, one or more reagent vials containing H 2 SO 4 and another reagent vial containing PBS.

[0064] In certain embodiments, the cassette is designed to be suitable for use in the manufacture of radiopharmaceuticals and is therefore made of a pharmaceutical-grade material that is resistant to radiolysis. In one embodiment, the single-use cassette is suitable for use with a GE Healthcare FASTlab™ automated synthesizer. In some embodiments, the various elements of the cassette are selectively fluidly connected.

[0065] FIG. 1 shows a cassette suitable for synthesizing 18 F]FSPG in an automated synthesizer (e.g., the FASTlab™ of GE Healthcare).

[0066] In some embodiments of the present disclosure, position 1 on the cassette includes a tube connected to a vial or container. In certain embodiments of the present disclosure, the tube at position 1 is a silicone tube. In certain embodiments of the present disclosure, the tube at position 1 is a silicone tube that is 12 - 16 cm (e.g., about 14 cm).

[0067] In some aspects of the present disclosure, position 2 on the cassette includes a vial or container containing an eluent. In some aspects of the present disclosure, the vial or container at position 2 contains an eluent of K 2 CO 3 / Kryptofix™ 222 in about 50% water / acetonitrile.

[0068] In some aspects of the present disclosure, positions 3, 11, and 24 on the cassette each include a syringe. In some aspects of the present disclosure, the movable part of the automated synthesizer is clipped to a syringe plunger tip and is thus designed to raise or lower the syringe barrel.

[0069] In some aspects of the present disclosure, position 4 on the cassette includes an SPE cartridge. In some aspects, the SPE cartridge is an anion exchange SPE cartridge. In some aspects, the anion exchange SPE cartridge is a quaternary ammonium anion exchange (QMA) SPE cartridge. In some aspects of the present disclosure, the anion exchange SPE cartridge is preconditioned with an eluent. In some aspects, the anion exchange SPE cartridge is preconditioned with acetonitrile. In some aspects, the anion exchange SPE cartridge is a QMA SPE cartridge preconditioned with acetonitrile. In some aspects, the SPE cartridge at position 4 is connected to a tube at position 5 of the cassette. In certain aspects of the present disclosure, the tube at position 5 is a silicone tube. In certain aspects of the present disclosure, the tube at position 5 is a 12 - 16 cm (e.g., about 14 cm) silicone tube.

[0070] In some aspects of the present disclosure, position 6 on the cassette 18 includes means for transferring [F] fluoride to the cassette. In some aspects, the conical reservoir 18 The means for transferring [F] fluoride to the cassette is a conical reservoir.

[0071] In some aspects of the present disclosure, positions 7, 8, and 25 on the cassette include tubes connected to three ports on the reaction vessel. In some aspects, the tube at position 7 is connected to the port on the left side of the reaction vessel, the tube at position 8 is connected to the central port of the reaction vessel, and the tube at position 25 is connected to the port on the right side of the reaction vessel. In certain aspects of the present disclosure, the tubes at positions 7, 8, and / or 25 are silicone tubes. In certain aspects of the present disclosure, the tubes at positions 7 and / or 8 are silicone tubes that are 12 - 16 cm (e.g., about 14 cm). In certain aspects of the present disclosure, the tube at position 25 is a silicone tube that is 40 - 44 cm (e.g., about 42 cm).

[0072] In some aspects of the present disclosure, positions 9 and 10 on the cassette include tubes connected to a vial or container. In certain aspects of the present disclosure, the tubes at positions 9 and / or 10 are silicone tubes. In certain aspects of the present disclosure, the tubes at positions 9 and / or 10 are silicone tubes that are 14 cm. In some aspects, the vial and / or container contains a reagent. In some aspects, the vial or container connected to position 9 via the tube contains 2 - 6 ml of an acid (e.g., 4 ml of 1 M sulfuric acid). In some aspects, the vial or container connected to position 10 via the tube contains 15 - 30 ml (e.g., about 20 ml) of phosphate buffered saline.

[0073] In some aspects of the present disclosure, position 12 on the cassette includes a vial or container containing a reagent. In some aspects of the present disclosure, the vial or container at position 12 contains a PET tracer precursor dissolved in a reagent. In some aspects, the vial or container contains an FSPG precursor in acetonitrile.

[0074] In some aspects of the present disclosure, position 14 on the cassette includes a vial or container containing a reagent. In some aspects of the present disclosure, the vial or container at position 14 contains a reagent selected from the group consisting of sulfuric acid, hydrochloric acid, and trifluoroacetic acid. In some aspects of the present disclosure, the vial or container at position 14 contains trifluoroacetic acid. In some aspects of the present disclosure, the vial or container at position 14 contains hydrochloric acid. In some aspects of the present disclosure, the vial or container at position 14 contains sulfuric acid (e.g., 1M sulfuric acid). In some aspects, the vial or container contains about 1.5 - 2.0 ml of an acid (e.g., 1M sulfuric acid). In some aspects, the vial or container contains 1.7 ml of 1M sulfuric acid. In some aspects, the vial is a 13 mm vial containing 1.7 ml of 1M sulfuric acid.

[0075] In some aspects of the present disclosure, position 15 on the cassette includes means for connecting the cassette to a reagent and / or eluent container. In some aspects, the means for connecting the cassette to the container is a spike. In some aspects, the container is a bag containing sterile water for injection. In some aspects, the water bag is connected by the spike at position 15.

[0076] In some aspects of the present disclosure, position 16 on the cassette includes a vial or container containing a reagent. In some aspects of the present disclosure, the vial or container at position 16 contains potassium hydroxide. In some aspects of the present disclosure, the vial or container at position 16 contains sodium hydroxide (e.g., 4N sodium hydroxide). In some aspects, the vial or container contains 1.5 - 2.0 ml (e.g., about 1.7 ml) of 4N sodium hydroxide. In some aspects, the vial is a 13 mm vial containing 1.7 ml of 4N sodium hydroxide.

[0077] In some aspects of the present disclosure, positions 18 and 20 on the cassette include SPE cartridges. In some aspects, the SPE cartridge is a reversed-phase SPE cartridge. Reversed-phase SPE uses a nonpolar modified solid phase and a polar mobile phase. In the case of reversed-phase SPE, compounds are retained by hydrophobic interactions and are eluted using a nonpolar elution solvent to disrupt the forces binding the compounds to the solid phase. Non-limiting examples of reversed-phase SPE cartridges include C18, tC18, CS, CN, Dial, HLB, Porapak, RDX, and NH 2 include SPE cartridges. In some aspects, the SPE cartridge has a mixed-mode cation exchange and reversed-phase adsorbent. In some aspects, the reversed-phase / cation exchange SPE cartridge is an Oasis® MCX cartridge. In some aspects of the present disclosure, the reversed-phase SPE cartridge is a tC18 or HLB SPE cartridge. In some aspects, the reversed-phase SPE cartridge is an HLB SPE cartridge. In another aspect of the present invention, the reversed-phase SPE cartridge is a tC18 cartridge. In some aspects of the present invention, the tC18 cartridge is an environmental tC18 cartridge that may also be referred to as a long tC18 cartridge or a tC18 plus cartridge. In some aspects of the present disclosure, the reversed-phase SPE cartridge is preconditioned with an eluent. In some aspects, the reversed-phase or reversed-phase / cation exchange SPE cartridge is preconditioned with a phosphate buffered saline or a sodium bicarbonate solution. In some aspects, the reversed-phase / cation exchange SPE cartridge is an Oasis® MCX SPE cartridge preconditioned with a sodium bicarbonate solution.

[0078] In some aspects, the SPE cartridge at position 18 is connected to the tube at position 17 of the cassette. In a particular aspect of the present disclosure, the tube at position 17 is a silicone tube. In a particular aspect of the present disclosure, the tube at position 17 is a silicone tube that is 12 to 16 cm (e.g., about 14 cm).

[0079] In some embodiments, the SPE cartridge at position 20 is connected to the tube at position 19 of the cassette. In certain embodiments of the present disclosure, the tube at position 19 is a silicone tube. In certain embodiments of the present disclosure, the tube at position 19 is a silicone tube that is 12 - 16 cm (e.g., about 14 cm).

[0080] In some embodiments of the present disclosure, position 22 on the cassette contains an SPE cartridge. In some embodiments, the SPE cartridge is a normal-phase SPE cartridge. Normal-phase SPE uses a polar modified stationary phase and a nonpolar mobile phase. In the case of normal-phase SPE, compounds are retained by hydrophilic interactions and eluted using an eluent that is more polar than the original mobile phase to disrupt the binding mechanism. Non-limiting examples of normal-phase SPE cartridges include alumina, diol, and silica SPE cartridges. In some embodiments of the present disclosure, the normal-phase SPE cartridge is an alumina SPE cartridge. In some embodiments of the present disclosure, the normal-phase SPE cartridge is a Sep-Pak® Alumina N Plus Long Cartridge. In some embodiments, the normal-phase SPE cartridge is preconditioned with an eluent. In some embodiments, the normal-phase SPE cartridge is preconditioned with phosphate-buffered saline. In some embodiments, the normal-phase SPE cartridge is an alumina SPE cartridge preconditioned with phosphate-buffered saline.

[0081] In some aspects of the present disclosure, position 23 on the cassette includes a tube connected to the SPE cartridge. In certain aspects of the present disclosure, the tube at position 23 is a silicone tube. In certain aspects of the present disclosure, the tube at position 23 is a silicone tube that is 12 to 16 cm (e.g., about 14 cm). In some aspects, the SPE cartridge is a reverse-phase SPE cartridge. In some aspects, the SPE cartridge contains graphitized non-porous carbon. In some aspects, the cartridge is an ENVI-carb or Hypercarb cartridge. In some aspects, the SPE cartridge is a Supelco® Superclean™ ENVI-Carb™ or Fisher Scientific HyperSep™ Hypercarb™ cartridge.

[0082] Methods for sterilizing compositions intended for parenteral administration are well known to those skilled in the art. In some aspects, the final 18 F]FSPG solution can be filtered through a sterile syringe filter (e.g., having a pore size of 0.22 μm) and a mixed cellulose ester membrane before being collected in the final product vial. In some aspects, the final product vial can be subjected to high heat, such as in an autoclave.

[0083] IV. Automatic synthesizer Certain aspects of the present disclosure relate to a cassette for use in an automatic synthesizer for 18 F]FSPG production. In some aspects, the automatic synthesizer is a reliable cGMP-compliant device.

[0084] Non-limiting examples of commercially available automatic synthesizers include GE Healthcare; CTI Inc.; Ion Beam Applications S.A. (Chemin du Cyclotron 3, B-1348 Louvain-La-Neuve, Belgium); Raytest (Germany) and Bioscan (USA).

[0085] In some embodiments, the automated synthesis apparatus comprises a cassette as disclosed herein. In some embodiments, 18 For F]FSPG production, the radiochemistry is performed within a removable cassette or cartridge designed to fit the apparatus. In some embodiments, the cassette fits into the apparatus in such a way that the mechanical movement of the moving parts of the apparatus controls the operation of the cassette. In some embodiments, the automated synthesis apparatus has means for connecting to a cyclotron to receive a desired isotope. In some embodiments, the automated synthesis apparatus has an external arm that can rotate and thus controls the opening and closing of valves when an appropriate cassette for the automated synthesis apparatus is attached. Additional moving parts of the automated synthesis apparatus are clipped to the syringe plunger tip and can thus be designed to raise or lower the syringe barrel.

[0086] V. Synthesis Method Certain aspects of the present disclosure relate to 18 a cGMP-compliant process to enable the large-scale production required for the clinical and commercial use of F]FSPG radiotracers. The synthesis of F]FSPG in the cassettes (e.g., as shown by the cassette of FIG. 1) disclosed herein 18 is 18 performed by nucleophilic fluorination using 18 F]fluoride generated by the reaction 18 O(p,n)

[0087] In some embodiments, 18 F]fluoride suitable for use in the synthesis of PET tracers 18 is obtained from the nuclear reaction 18 O(p,n) 18 F as an aqueous solution (see, for example, Hess, E et al 2001, Radiochim. Acta 89, 357-362). In some embodiments, a cyclotron is used to 18 generate 18To increase the reactivity of the fluoride and / or to reduce or minimize hydroxylation by-products resulting from the presence of water, prior to the reaction 18 water can be removed from the fluoride. In some embodiments, the fluorination reaction is carried out using an anhydrous reaction solvent (see Aigbirhio et al 1995 J Fluor Chem;70:279-87). In some embodiments, additional steps can be used to improve the reactivity of the 18 fluoride for the radiofluorination reaction, including adding a cationic counterion prior to water removal. This cationic counterion can be dissolved in an aqueous organic solution, which solution 18 can be used as an eluent to elute the 18 fluoride from an anion exchange column in which the fluoride is bound or trapped. In some embodiments, the aqueous organic solution is an aqueous solution of acetonitrile, methanol, and / or water. In some embodiments, the aqueous organic solution is an aqueous acetonitrile solution. In some embodiments, the aqueous organic solution is an aqueous solution of acetonitrile and water. In some embodiments, the counterion can have sufficient solubility in the anhydrous reaction solvent to maintain the solubility of the 18 fluoride. Thus, commonly used counterions include large but soft metal ions such as rubidium or cesium, potassium complexed with a cryptand such as Kryptofix™ 222, or tetraalkylammonium salts, with potassium complexed with a cryptand such as Kryptofix™ 222 or tetraalkylammonium salts being preferred. The term Kryptofix™ 222 (or K222) as used herein refers to a commercially available preparation of the compound 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane. In some embodiments, the potassium is in the form of potassium carbonate. In some embodiments, 18 the eluent used to elute the 18 fluoride from an anion exchange SPE cartridge in which the fluoride is trapped is K in 50% water / acetonitrile2 CO 3 is a solution of / Kryptofix(trademark) 222.

[0088] In some embodiments, the 18 F] fluoride thus generated enters the cassette at position 6 and is loaded onto the SPE cartridge. In some embodiments, the SPE cartridge is a quaternary methylammonium anion exchange (QMA) solid phase extraction (SPE) cartridge. In some embodiments, 18 the F] fluoride is retained by an ion exchange reaction within the QMA cartridge, 18 and the O - water is flowed through the common path of the cassette and recovered at position 1. In some embodiments, the 18 F] fluoride retained by the QMA cartridge is then eluted into the reaction vessel with an eluent (e.g., Kryptofix(trademark) 222 and potassium carbonate in acetonitrile at position 2).

[0089] In some embodiments, the reaction vessel is heated to evaporate water. In some embodiments, the reaction vessel is heated to about 120 °C for 5 - 10 minutes. In some embodiments, additional acetonitrile is added and the reaction vessel is heated again to complete the removal of water. In some embodiments, 18 the initial radioactivity of the F] fluoride is 0.25 - 3.5 Ci (10 - 130 GBq). In some embodiments, 18 the initial radioactivity of the F] fluoride is 1.0 - 3.3 Ci (37 - 122 GBq).

[0090] In some embodiments, the FSPG precursor dissolved in anhydrous acetonitrile (from position 12) is 18Add to a reaction vessel containing fluoride. In some embodiments, 6 mg to 12 mg of the FSPG precursor is dissolved in acetonitrile before adding to the reaction vessel. In some embodiments, 6 to 12 mg of the FSPG precursor is dissolved in 1.3 ml of acetonitrile. In some embodiments, 6 mg of the FSPG precursor is dissolved in 1.3 ml of anhydrous acetonitrile. In some embodiments, the FSPG precursor is di-tert-butyl (2S,4S)-2-(3-((naphthalene-2-ylsulfonyl)oxy)propyl)-4-(tritylamino)pentaenedioate or di-tert-butyl (2S,4S)-2-(3-((naphthalene-2-ylsulfonyl)oxy)propyl)-4-(boc-amino)pentaenedioate. In some embodiments, the FSPG precursor comprises one of the following chemical structure(s): [Chemical formula] Processes for preparing the FSPG precursor are well known in the art (see, e.g., Shih et al, PLoS One. 2020;15(12):e0243831, which is incorporated herein by reference).

[0091] In some embodiments, the FSPG precursor has a chemical purity of at least 90%. In some embodiments, the FSPG precursor has a chemical purity of at least 95%. In some embodiments, the FSPG precursor has a chemical purity of at least 97%. In some embodiments, the FSPG precursor has a chemical purity of at least 98%. In some embodiments, the FSPG precursor has a chemical purity of at least 99%. In some embodiments, 6 mg of di-tert-butyl (2S,4S)-2-(3-((naphthalene-2-ylsulfonyl)oxy)propyl)-4-(tritylamino)pentaenedioate or di-tert-butyl (2S,4S)-2-(3-((naphthalene-2-ylsulfonyl)oxy)propyl)-4-(boc-amino)pentaenedioate is dissolved in 1.0 to 1.5 ml (e.g., about 1.3 ml) of anhydrous acetonitrile and then added to the reaction vessel.

[0092] In some embodiments, the reaction vessel is heated at a temperature in the range of 100°C to 120°C for 5 to 15 minutes to 18 generate the F-labeled FSPG precursor. In some embodiments, the reaction vessel is heated at a temperature of 105°C to 115°C for 5 to 15 minutes. In some embodiments, the reaction vessel is heated at a temperature of 105°C to 110°C for 5 to 15 minutes. In some embodiments, the reaction vessel is heated at a temperature in the range of 100 to 120°C for 5 to 10 minutes. In some embodiments, the reaction vessel is heated at a temperature in the range of 100 to 120°C for about 5 minutes. In some embodiments, the reaction vessel is heated at a temperature of 105°C for about 5 minutes.

[0093] In some embodiments, next, an acid is added to the reaction vessel (e.g., from position 14), and the mixture is reheated to 105°C to 115°C for 2 to 10 minutes. In some embodiments, the acid is selected from the group consisting of sulfuric acid, hydrochloric acid, and trifluoroacetic acid. In some embodiments, the acid is sulfuric acid. In some embodiments, the acid is hydrochloric acid. In some embodiments, the acid is trifluoroacetic acid. In some embodiments, the reaction vessel is heated at a temperature of 105°C to 110°C for 3 to 7 minutes. In some embodiments, the reaction vessel is heated at a temperature in the range of 100 to 120°C for 3 to 10 minutes. In some embodiments, the reaction vessel is heated at a temperature in the range of 100 to 120°C for 4 minutes. In some embodiments, the reaction vessel is heated at a temperature of 105°C for about 4 minutes.

[0094] In some embodiments, next, a base is added to the reaction vessel (e.g., from position 16), and it is heated to 60°C to 80°C for 5 to 15 minutes to 18Generate the F-labeled FSPG precursor. In some embodiments, the base is sodium hydroxide. In some embodiments, the base is potassium hydroxide. In some embodiments, the reaction vessel is heated at a temperature of 65°C to 75°C for 5 to 15 minutes. In some embodiments, the reaction vessel is heated at a temperature in the range of 60 to 80°C for 5 to 10 minutes. In some embodiments, the reaction vessel is heated at a temperature in the range of 65 to 75°C for 5 to 10 minutes. In some embodiments, the reaction vessel is heated at a temperature of 70°C for about 5 minutes.

[0095] In some embodiments, next, raise the pH of the reaction mixture, i.e., acidify it. In some embodiments, raise the pH of the reaction mixture with sulfuric acid, hydrochloric acid, or trifluoroacetic acid. In some embodiments, acidify the reaction mixture with sulfuric acid (e.g., 1 M sulfuric acid). In some embodiments, acidify the reaction mixture with hydrochloric acid. In some embodiments, acidify the reaction mixture with trifluoroacetic acid.

[0096] In some embodiments, the crude 18 F-labeled FSPG is captured and separated from by-products on two consecutive anion exchange SPE cartridges. In some embodiments, the crude 18 F-labeled FSPG is loaded onto an anion exchange SPE cartridge at a flow rate of 8 ml / min to 20 ml / min. In some embodiments, the loading flow rate is 10 ml / min to 18 ml / min. In some embodiments, the loading flow rate is 12 ml / min to 16 ml / min. In some embodiments, the loading flow rate is about 14 ml / min. In some embodiments, the cartridge is first washed with water, for example, to remove by-products. In some embodiments, 18 F-labeled FSPG is eluted from the cartridge with a phosphate buffered saline eluent (e.g., from position 10). In some embodiments, 18 F-labeled FSPG is eluted from the cartridge at a flow rate of 4 ml / min to 16 ml / min. In some embodiments, the elution flow rate is 6 ml / min to 14 ml / min. In some embodiments, the elution flow rate is 8 ml / min to 12 ml / min. In some embodiments, the elution flow rate is about 10 ml / min.

[0097] Potential residue 18 F]The fluoride removal can be carried out on an alumina SPE column (e.g., at position 22 via the tube at position 21). The product can be further purified with a normal phase SPE cartridge (e.g., via the tube at position 23). In some embodiments, the product can be sterilized by passing it through an optional sterile syringe filter (e.g., having a pore size of 0.22 μm). In some embodiments, the finally purified and sterilized product is collected in a receptacle (e.g., a vial). In some embodiments, the position of the cassette (e.g., position 13) can be empty. In some embodiments, a cap is placed on the valve at the empty position.

[0098] In some embodiments, as disclosed herein 18 F]The automated process for FSPG generation takes 10 - 90 minutes. In some embodiments, as disclosed herein 18 F]The automated process for FSPG generation takes 20 - 90 minutes. In some embodiments, as disclosed herein 18 F]The automated process for FSPG generation takes 30 - 90 minutes. In some embodiments, as disclosed herein 18 F]The automated process for FSPG generation takes 10 - 80 minutes. In some embodiments, as disclosed herein 18 F]The automated process for FSPG generation takes 20 - 80 minutes. In some embodiments, as disclosed herein 18 F]The automated process for FSPG generation takes 30 - 80 minutes. In some embodiments, as disclosed herein 18 F]The automated process for FSPG generation takes 10 - 70 minutes. In some embodiments, as disclosed herein 18 F]The automated process for FSPG generation takes 20 - 70 minutes. In some embodiments, as disclosed herein 18 F]The automated process for FSPG generation takes 30 - 70 minutes. In some embodiments, as disclosed herein 18 F]The automated process for FSPG generation takes 10 - 60 minutes. In some embodiments, as disclosed herein18 F]The automated process for FSPG generation takes 20 to 60 minutes. In some embodiments, as disclosed herein 18 F]The automated process for FSPG generation takes 30 to 60 minutes. In some embodiments, as disclosed herein 18 F]The automated process for FSPG generation takes 10 to 50 minutes. In some embodiments, as disclosed herein 18 F]The automated process for FSPG generation takes 20 to 50 minutes. In some embodiments, as disclosed herein 18 F]The automated process for FSPG generation takes 30 to 50 minutes.

[0099] In some embodiments, as disclosed herein 18 F]The automated process for FSPG generation takes less than 90 minutes. In some embodiments, as disclosed herein 18 F]The automated process for FSPG generation takes less than 80 minutes. In some embodiments, as disclosed herein 18 F]The automated process for FSPG generation takes less than 70 minutes. In some embodiments, as disclosed herein 18 F]The automated process for FSPG generation takes less than 60 minutes. In some embodiments, as disclosed herein 18 F]The automated process for FSPG generation takes less than 50 minutes. In some embodiments, as disclosed herein 18 F]The automated process for FSPG generation takes less than 40 minutes.

Example

[0100] Example 1. Automated Process for Radiolabeling of FSPG The following table 18 details the cassette positions for the automated radiosynthesis of F-FSPG. The cassette positions correspond to the labeled positions of the cassette shown in Figure 1.

Table 1-1

Table 1-2

[0101] Figure 2 shows a flow diagram of an automated manufacturing process for radiolabeling FSPG. Using the cassette configuration shown in Figure 1, the following method was used to 18 generate F]FSPG (the numbers in this method are reference numbers in Figure 1 unless otherwise described as "position", and are one of positions 1 to 25 from left to right on the cassette of Figure 1): (i) By high-energy proton beam extracted from PET Trace (GE Healthcare) 18 O]-H 2 impact on O to 18 obtain F]fluoride and transfer it to the cassette via the conical reservoir at position 6. (ii) 18 Capture F]fluoride at a position on the QMA column, separate it from the concentrated water, and collect this in an external vial via position 5-4-1. (iv) Withdraw an eluent containing potassium carbonate and Kryptofix™ 222 in 50% water / acetonitrile from the vial at position 2 into the syringe at position 3, and 18 release F]fluoride through the QMA cartridge and send it to the reaction vessel. (v) Catalyze the evaporation of water in the reaction vessel by heating the reaction vessel at 120 °C for 5 minutes to complete the removal of water. (vi) Add 6 - 12 mg of the FSPG precursor in 1.3 ml of anhydrous acetonitrile from the vial at position 12 to the reaction vessel. The labeling reaction is carried out by heating the reaction vessel at 105 °C for 5 minutes. (vii) Add 2.2 ml of a first aliquot of 1 M sulfuric acid from the vial at position 14 to the reaction vessel, and heat the reaction vessel at 105 °C for 4 minutes to remove the protecting group from the radiolabeling precursor. (viii) Add 1.7 ml of an aliquot of 4 M sodium hydroxide solution from the vial at position 16 to the reaction vessel, and heat the reaction vessel at 70 °C for 5 minutes. (ix) The reaction mixture is added to the 10 mL vial at position 9 containing 4 mL of 1 M sulfuric acid to acidify the solution. (x) The resulting crude 18 F]FSPG is loaded at a flow rate of 14 mL / min and captured on two Oasis® MCX cartridges, via the tube at position 17 to position 18 and via the tube at position 19 to position 20. The cartridges are first washed with water to remove by-products. 18 F]FSPG is eluted from the cartridges using PBS eluent (from position 10) at a rate of 10 mL / min and passed in the same direction as the crude 18 F]FSPG loaded onto the cartridges. (xi) Next, 18 F]FSPG solution is passed through an alumina SPE column at position 22 via a tube at position 21 to remove residual 18 F]fluoride. (x) The final product is passed through an external ENVI-carb cartridge connected to the cassette by a tube at position 23. The final product is then passed through a sterile syringe filter with a pore size of 0.22 μm and collected in a sterile final product vial.

[0102] This cassette configuration has a concentrated water recirculation path on the cassette (possibility of manifold contamination by concentrated water) and engages at four positions on the cassette (i.e., position 6 for the radioactivity inlet, position 1 with the connection for the concentrated water vial, position 4 for the QMA cartridge, and position 5 for the tube of the QMA cartridge).

[0103] The starting radioactivity, final radioactivity, and residual radioactivity were measured by a calibrated ionization chamber CAPINTEC (CRC25-PET). To determine the yield, the following yield calculation was performed. Delta Tf = elapsed time (min) after the start of synthesis Af = final radioactivity (mCi) cAf = final activity corrected with respect to the start of synthesis (min) = Af * Exp(ln(2) * (Delta Tf / 110)) (where 110 = 18Half-life of F (minutes) cAi = Corrected starting radioactivity (mCi) with respect to the start of synthesis (mCi) Delta Ts = Duration of synthesis Corrected yield (CY) = (cAf / cAi) × 100 Uncorrected yield (NCY) = CY × Exp(ln(2) × (-Delta Ts / 110))

[0104] According to the disclosed method, 18 an [F]FSPG composition was prepared. The [F]FSPG composition prepared according to the disclosed method 18 had the following characteristics. Starting radioactivity = 1.0 - 3.3 Ci Final concentration = 7.0 - 52.0 mCi / ml pH = 6.0 - 8.0 Radiochemical yield = 18% - 32%, without decay correction

[0105] 18 The radiochemical purity of the [F]FSPG composition was tested at the end of synthesis and up to a maximum of 5 hours after storage at room temperature. Reverse-phase HPLC was performed using an Agilent 1260 HPLC system equipped with a 1200 Series Diode Array Detector and a Raytest GABI Star NaI(Tl) scintillation detector. The following process parameters were used: Column: Phenomenex Luna C-18(2) 250 mm × 4.6 mm (5 μm) Mobile phase A: 40 μM aqueous sodium phosphate solution Mobile phase B: Acetonitrile / methanol / water (45:45:10) (% w / v) Flow rate: 1.5 mL / min Gradient: Initial: 100% mobile phase A, 0% mobile phase B 2 minutes: 96% mobile phase A, 4% mobile phase B 5 minutes: 88% mobile phase A, 12% mobile phase B 15 minutes: 88% mobile phase A, 12% mobile phase B 20 minutes: 40% mobile phase A, 60% mobile phase B ​22 minutes: 0% mobile phase A, 100% mobile phase B 24 minutes: 0% mobile phase A, 100% mobile phase B 25 minutes: 100% mobile phase A, 0% mobile phase B 30 minutes: 100% mobile phase A, 0% mobile phase B Total runtime: 35 minutes UV detector: 340 nm

[0106] 18 F]FSPG solution (80 μl) was mixed with o-phthalaldehyde reagent solution (20 μl). Before HPLC analysis, the mixture was saturated for 1 minute.

[0107] At the end of synthesis, 18 F]The radiochemical purity of the FSPG composition was >95%. When stored at room temperature, 18 F]the FSPG composition maintained a radiochemical purity of >95% over 5 hours (Figure 3).​

Claims

1. (S)-4-(3- １８ F-Fluoropropyl)-L-Glutamic Acid ([ １８ An automated process for the synthesis of F)FSPG), a. (S)-4-(3- １８ The F-fluoropropyl)-L-glutamic acid precursor is dissolved in anhydrous acetonitrile, and the resulting solution is radiolabeled with fluoride ( １８ To add to the reaction vessel containing F), b. The reaction vessel is heated at 100°C to 110°C for 5 to 15 minutes to obtain a crude intermediate product. c. Add a first aliquot of the acid to the reaction vessel and heat at 100°C to 110°C for 2 to 10 minutes, then add an aliquot of the base to the reaction vessel and heat at 60°C to 80°C for 5 to 15 minutes to remove the protecting group, and crude [ １８ F) Obtaining FSPG, d. The aforementioned rough [ １８ F) Acidifying FSPG, e. Acidified crude [ １８ F) Loading FSPG and f. From one or more reversed-phase solid-phase extraction cartridges, the [ １８ F) Eluting FSPG, g. Purifying the １８ F]FSPG through one or more carbon phase / reversed-phase solid-phase extraction cartridges, and Includes, The process, wherein each of (a) to (g) is performed within the cassette.

2. The above [ １８ The process according to claim 1, wherein FSPG is eluted from one or more reversed-phase solid-phase extraction cartridges using a buffer solution.

3. The process according to claim 2, wherein the buffer solution is phosphate-buffered saline.

4. The process according to any one of claims 1 to 3, wherein the first aliquot of the acid in step (c) is selected from the group consisting of sulfuric acid, hydrochloric acid, and trifluoroacetic acid.

5. The process according to any one of claims 1 to 3, wherein the base in step (c) is sodium hydroxide or potassium hydroxide.

6. (a) One or more reversed-phase solid-phase extraction cartridges are washed with water before step (f) to remove by-products. (b) The rough [ １８ F) FSPG is acidified in step (d) with a second aliquot of sulfuric acid, (c) The process is performed by passing the [ １８ F) Further comprising purifying FSPG, and / or (d) The process is final [ １８ F] Pass the FSPG composition through a sterile filter to produce the [ １８ F) Further comprising sterilizing the FSPG product, The process according to any one of claims 1 to 3.

7. The final purified [ １８ F) The process according to any one of claims 1 to 3, wherein the FSPG has a radiochemical purity of more than 90%, more than 95%, more than 96%, more than 97%, or more than 98%.

8. The process yields at least 10% uncorrected radiochemical yield, 13% to 35% uncorrected radiochemical yield, or 18% to 32% uncorrected radiochemical yield [ １８ F) A process according to any one of claims 1 to 3 for generating an FSPG.

9. The process according to any one of claims 1 to 3, wherein the reaction product is loaded into the reversed-phase solid-phase extractor at a flow rate of 8 ml / min to 20 ml / min and eluted from the reversed-phase solid-phase extractor cartridge at a flow rate of 4 ml / min to 14 ml / min.

10. (a) The one or more solid-phase extraction cartridges in step (e) contain a reverse-phase / cation exchange adsorbent, (b) The above [ １８ F]FSPG is the rough [ １８ F) Elutes from the reversed-phase solid-phase extraction cartridge in the same direction as when FSPG was loaded. (c) The one or more reversed-phase solid extraction cartridges in step (f) contain aluminum oxide, (d) The one or more carbon phase / reverse phase solid extraction cartridges in step (g) include a carbon adsorbent, (e) The FSPG precursor has one of the following structures: 【Chemistry 1】 (f) Said [ １８ F)FSPG has an initial radioactivity of approximately 10 to 125 GBq, (g) The last [ １８ F) The FSPG composition has a concentration of about 185 to 1850 MBq / ml, (h) The last [ １８ F) The FSPG composition is produced in less than 40 minutes from the start of synthesis, and / or (i) The last [ １８ F) The FSPG composition has a pH of 6.5 to 7.

5. The process according to any one of claims 1 to 3.

11. The aforementioned radiolabeled fluoride is a. １８ Irradiate with water, １８ O in water １８ To produce solution F, b. The quaternary ammonium anion exchange cartridge preconditioned with potassium bicarbonate is used １８ Loading solution F, c. Using a 50% water / acetonitrile solution of potassium carbonate / 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane as an eluent, the cartridge is transferred to the reaction vessel. １８ To elute F, d. Heating the reaction vessel to remove water, The process according to any one of claims 1 to 3, prepared by a method comprising:

12. The process according to claim 11, wherein the reaction vessel is heated to 120°C for about 5 minutes in step (d).

13. Prepared by the process described in any one of claims 1 to 3, １８ F) A composition containing FSPG.

14. The composition is (a) When stored at room temperature, at least 1 hour after synthesis, (b) If stored at room temperature, at least 2 hours after synthesis, (c) When stored at room temperature, at least 3 hours after synthesis, (d) If stored at room temperature, at least 4 hours after synthesis, (e) If stored at room temperature, at least 5 hours after synthesis, or (f) When stored at room temperature, at least 6 hours, 7 hours, or 8 hours after synthesis, The composition according to claim 13, which is not subjected to radiolysis.

15. The composition is (a) Radiochemical purity of more than 90% for at least 5 hours after storage at room temperature, (b) Radiochemical purity of more than 95% for at least 5 hours after storage at room temperature, or (c) Radiochemical purity of more than 95% for at least 6 hours, 7 hours, or 8 hours after storage at room temperature. The composition according to claim 13, which maintains the following:

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