METHOD FOR EXTRACTION AND PURIFICATION OF ⁶⁸ Ga

The present application claims the priority benefit of United States provisional application number 62/714,190, filed August 3, 2018, the entire contents of which are incorporated herein by reference.

BACKGROUND

I. Field of the Invention

The present invention relates generally to the fields of chemistry and biological imaging. More particularly, it concerns methods for preparing purified ⁶⁸ Ga, systems for preparing purified

⁶⁸ Ga, compositions comprising purified ⁶⁸ Ga, and methods of use thereof.

II. Description of Related Art

The noticeable increase in PET imaging with ⁶⁸ Ga-labeled tracers over recent years has been marginally supported by the increased accessibility of ⁶⁸ Ge/ ⁶⁸ Ga generators (Prata, 2012). Gallium-68 conveniently possesses two desirable properties, a short half-life (t½: 68 min) and a high branching ratio for positron emission (b+%: 89%). To date, ⁶⁸ Gahas been widely applied into various pre-clinical and clinical imaging studies. For instance, ⁶⁸ Ga-labeled somatostatin analogs have already demonstrated their value in imaging patients diagnosed with neuroendocrine tumors (Ambrosini and Fanti, 2014; Sharnim et al., 2015; Virgolini et al., 2016). Recent reports indicate ⁶⁸ Ga-PSMA is superior to ¹¹ C/ ¹⁸ F-Choline in detecting more prostate cancer lesions, especially at lower prostate-specific antigen (PSA) levels in biochemically recurrent patients (Lindenberg et al., 2016; Schwenck et al., 2017). Given these results, the clinical promise of ⁶⁸ Ga-labeled radiopharmaceutical s clearly warrants the need to increase the availability of ⁶⁸ Ga for PET imaging applications.

Although current ⁶⁸ Ge/ ⁶⁸ Ga generators can nominally deliver up to 1.85 GBq (50 mCi) of ⁶⁸ Ga per elution, nonetheless ⁶⁸ Ga activity decreases over time due to the decay of the parent nuclide ⁶⁸ Ge (ti/2: 271 d). Furthermore, elution efficiencies around 80% of present activity are typical. In addition, the potential breakthrough of ⁶⁸ Ge with eluted ⁶⁸ Ga remains a concern (Belosi et al., 2013). Therefore, cyclotron production of ⁶⁸ Ga not only can provide an alternative way to meet the increasing demand for ⁶⁸ Ga, but also eliminates the possibility of ⁶⁸ Ge breakthrough.

Jensen and Clark reported the first attempt to produce ⁶⁸ Ga using a cyclotron with a liquid target filled with a ⁶⁸ ZnCh solution (Jensen and Clark, 2011). Since then, Engle et al. have used a solid natZn target to produce ^66/68 Ga (Engle et al., 2012). The thermal diffusion method (Tolmachev and Lundqvist, 1996) to recover ^66/68 Ga from natural Zn foils presents advantages in processing time when thin (< 0.1 mm) foils are considered (Siikanen, 2013). Pandey et al. tried to optimize ⁶⁸ Ga production tlirough liquid targetry (Pandey et al., 2014). However, due to lengthy ⁶⁸ Ga separation steps reported by Engle et al. along with the relatively low production yields of ⁶⁸ Ga observed using liquid targetry, available ⁶⁸ Ga at end of processing is not significantly higher than generator produced isotope . Recently, medical cyclotron production of ⁶⁸ Ga from a ⁶⁸ Zn solid target has been shown to produce Curie quantities of ⁶⁸ Ga with low levels of ⁶⁸ Ge breakthrough.

Despite the improvement in methods to produce ⁶⁸ Ga, present methods for the purification of ⁶⁸ Ga produced by liquid or solid targetry require lengthy processing times due to the need for multiple purification steps. Due to the short half-life of ⁶⁸ Ga, there is a need for single-step purification protocols that can expediently deliver higher quantities of purified ⁶⁸ Ga for medical imaging applications.

SUMMARY OF THE INVENTION

The present disclosure provides methods for the purification of radionuclide solutions, including ⁶⁸ Ga solutions, and imaging agents prepared by these methods.

In some embodiments, the present disclosure provides methods of preparing a purified, carrier-free ⁶⁸ Ga solution comprising:

a) obtaining a ⁶⁸ Ga solution to be purified comprising ⁶⁸ Ga;

b) contacting the ⁶⁸ Ga solution with an anion-exchange resin; and

c) eluting the ⁶⁸ Ga from the anion-exchange resin to obtain the purified, carrier-free 6 ⁸ Ga solution.

In some aspects, the method comprises contacting the ⁶⁸ Ga solution to be purified with a single anion-exchange resin. In some aspects, the method does not comprise contacting the ⁶⁸ Ga solution to be purified or the purified, carrier-free ⁶⁸ Ga solution with another ion-exchange resin. In some aspects, the ⁶⁸ Ga in the ⁶⁸ Ga solution to be purified is prepared by synthesizing the ⁶⁸ Ga in the ⁶⁸ Ga solution. In further aspects, a cyclotron is used to synthesize the ⁶⁸ Ga. In further aspects, the cyclotron comprises a metal sample in the form of a liquid target. In still further aspects, the metal sample comprises ⁶⁸ Zn ²⁺ . In other aspects, the cyclotron comprises a metal sample in the form of a solid target. In further aspects, the metal sample comprises ⁶⁸ Zn. In some aspects, the ⁶⁸ Ga solution to be purified comprises an acid. In further aspects, the acid has a concentration of greater than about 2 N. In still further aspects, the acid has a concentration of from about 3 N to about 10 N, such as about 6 N. In some aspects, the acid is HCl or is derived from the dissociation of HCl. In some aspects, the ⁶⁸ Ga solution to be purified is an aqueous solution.

In some aspects, the anion-exchange resin comprises a phosphonate or a substituted phosphonate. In some aspects the anion-exchange resin comprises a phosphonate, such as dipentyl pentylphosphonate. In some aspects, the methods further comprise washing the anion-exchange resin with a washing solution. In some aspects, the washing step occurs before the eluting step. In some aspects, the washing solution comprises an acid. In some aspects, the acid has a concentration of greater than about I N. In further aspects, the acid has a concentration of from about 2 N to about 8 N, such as about 4 N. In some aspects, the acid is HCl or is derived from the dissociation of HCl. In some aspects, the washing solution is an aqueous solution.

In some aspects, eluting comprises contacting the anion-exchange resin with an eluting solution. In some aspects, the eluting solution comprises as acid. In some aspects, the acid has a concentration of from about 0.005 N to about 2 N. In further aspects, the acid has a concentration from about 0.01 N to about 1 N, such as about 0.05 N. In some aspects, the acid is HCl or is derived from the dissociation of HCl. In some aspects, the eluting solution is an aqueous solution.

In some aspects, the purified, carrier-free ⁶⁸ Ga solution has an activity of from about 37 MBq to about 370 GBq. In further aspects, the purified, carrier-free ⁶⁸ Ga solution has an activity of from about 370 MBq to about 370 GBq. In still further aspects, the purified, carrier-free ⁶⁸ Ga solution has an activity of from about 3.7 GBq to about 370 GBq. In some aspects, the purified, carrier-free ⁶⁸ Ga solution comprises less than about 1 mg/GBq of zinc. In further aspects, the purified, carrier-free ⁶⁸ Ga solution comprises less than about 0.1 mg/GBq of zinc. In still further aspects, the purified, carrier-free ⁶⁸ Ga solution comprises less than about 0.01 mg/GBq of zinc. In some aspects, the purified, carrier-free ⁶⁸ Ga solution comprises less than about 10 mg/GBq of iron. In further aspects, the purified, carrier-free ⁶⁸ Ga solution comprises less than about 1 mg/GBq of iron. In still further aspects, the purified, carrier-free ⁶⁸ Ga solution comprises less than about 0.5 mg/GBq of iron. In some aspects, the purified, carrier-free ⁶⁸ Ga solution comprises less than about 1 mg/GBq of germanium. In further aspects, the purified, carrier-free ⁶⁸ Ga solution comprises less than about 0.01 mg/GBq of germanium. In still further aspects, the purified, carrier-free ⁶⁸ Ga solution comprises less than about 0.0001 mg/GBq of germanium.

In some aspects, the purified, carrier-free ⁶⁸ Ga solution has a specific activity of greater than about 25 GBq/mg. In further aspects, the purified, carrier-free ⁶⁸ Ga solution has a specific activity of from about 25 GBq/mg to about 100,000 GBq/mg. In further aspects, the purified, carrier-free ⁶⁸ Ga solution has a specific activity of from about 100 GBq/mg to about 100,000 GBq/mg. In further aspects, the purified, carrier-free ⁶⁸ Ga solution has a specific activity of from about 1,000 GBq/mg to about 100,000 GBq/mg. In further aspects, the purified, carrier-free ⁶⁸ Ga solution has a specific activity of from about 10,000 GBq/mg to about 80,000 GBq/mg. In further aspects, the purified, carrier-free ⁶⁸ Ga solution has a specific activity of from about 25,000 GBq/mg to about 50,000 GBq/mg. In still further aspects, the purified, carrier-free ⁶⁸ Ga solution has a specific activity of from about 31,000 GBq/mg to about 36,000 GBq/mg. In some embodiments, the present disclosure provides purified, carrier-free ⁶⁸ Ga solutions prepared according to methods disclosed herein.

In some embodiments, the present disclosure provides systems for preparing a purified, carrier-free ⁶⁸ Ga solution comprising:

a) a solid target comprising ⁶⁸ Zn;

b) a cyclotron, wherein the solid target is irradiated using the cyclotron; and c) an anion-exchange resin. In some aspects, the system comprises a single anion-exchange resin. In some aspects, the system does not comprise another ion-exchange resin . In some aspects, the anion-exchange resin comprises a phosphonate or a substituted phosphonate. In some aspects, the anion-exchange resin comprises a phosphonate, such as dipentyl pentylphosphonate . In some aspects, the purified, carrier-free ⁶⁸ Ga solution has a specific activity of from about 25 GBq/mg to about 100,000 GBq/mg. In further aspects, the purified, carrier-free ⁶⁸ Ga solution has a specific activity of from about 100 GBq/mg to about 100,000 GBq/mg. In further aspects, the purified, carrier-free ⁶⁸ Ga solution has a specific activity of from about 1,000 GBq/mg to about 100,000 GBq/mg. In further aspects, the purified, carrier-free ⁶⁸ Ga solution has a specific activity of from about 10,000 GBq/mg to about 85,000 GBq/mg. In further aspects, the purified, carrier-free ⁶⁸ Ga solution has a specific activity of from about 25,000 GBq/mg to about 50,000 GBq/mg. In still further aspects, the purified, carrier-free ⁶⁸ Ga solution has a specific activity of from about 31,000 GBq/mg to about 36,000 GBq/mg.

In some embodiments, the present disclosure provides compositions comprising the purified, carrier-free ⁶⁸ Ga solutions disclosed herein. In some aspects, the compositions further comprise a carrier molecule, wherein the carrier molecule is radiolabeled with the ⁶⁸ Ga of the purified, carrier-free ⁶⁸ Ga solution. In further aspects, the carrier molecule is an antibody or a fragment thereof. In other aspects, the carrier molecule is a peptide or a fragment thereof. In other aspects, the carrier molecule is prostate-specific membrane antigen (PSMA), 1,4,7- triazacyclononane-N,N',N"-triacetic acid (NOTA),

1 ,4,7, 10-tetraazacyclododecane- 1 ,4,7, 10-tetraacetic acid (DOTA), diethylene triamine pentaacetic acid (DTP A), desferrioxamine, DOTA-Tyr(3)-octreotide (DOTATOC), DOTA-Tyr(3)-Tyr(8)- octreotide (DOTATATE), DOTA- 1 -naphtyl-alanine (DOTANOC), DOT A-benzothienyl-alanine (DOTA-BOC), DOTA-bombesin, DOTA-arginine-glycine-aspartic acid-bombesin (DOTA-RGD-bombesin), NOTA-RGD,

3,6,9,l5-tetraazabicyclo[9.3.l]pentadeca-l(l5)-,l l,l3-triene-3,6,9-triacetic acid-RGD (PCTA-RGD), DOTA-albumin, DOTA-human epidermal growth factor, 1,4,7 -triazacyclononane- l-[methyl(2-carboxyethyl)phosphinic

acid]-4,7-bis[methyl(2-hydroxymethyl)phosphinic acid-integrin alpha(IIb)beta(3)-specific cyclic hexapeptide (NOPO-RGDfK), l,4,7-triazacyclononane-l,4-bis(acetic acid)-7-(2-glutaric acid) (NODAGA), NOPO-NaI(3)-octreotide conjugate (NOPO-NOC),

1,4,7 -triazacyclononane- 1 ,4,7-tris [(2-carboxyethyl)methylenephosphinic acid] (TRAP(RGD)3), or citrate. In further aspects, the carrier molecule is PSMA, DOTATATE, DOTATOC, DOTANOC, or citrate. In some aspects, the carrier molecule is PSMA. In other aspects, the carrier molecule is DOTATATE. In still other aspects, the carrier molecule is citrate. In some aspects, the carrier molecule targets a human tissue. In some aspects, the human tissue is selected from the following: thyroid, brain, gastrointestinal, pancreas, spleen, kidney, neuroendocrine tumors, renal cell carcinoma, lung cancer, breast cancer, prostate cancer, and malignant lymphoma.

In some embodiments, the present disclosure provides methods of imaging comprising administering a composition disclosed herein to a patient. In some aspects, the patient is a mammal. In further aspects, the mammal is a human. In some aspects, the composition is administered more than once. In other aspects, the composition is administered once. In some aspects, the composition is formulated for administration intraarterially, intraarticularly, intracranially, intrapericardially, intraperitoneally, intratumorally, intravenously, intravesicularlly , parenterally, via injection, via local delivery, or via localized perfusion. In some aspects, the composition is formulated for administration intravenously. In some aspects, the composition is formulated as a unit dose. In some aspects, the methods further comprise imaging a tumor. In some aspects, the tumor is a neuroendocrine tumor. In some aspects, the tumor expresses elevated levels of PSMA. In further aspects, the tumor is a pancreatic tumor, a prostate tumor, or a lung tumor. In some aspects, the tumor is a pancreatic tumor. In other aspects, the tumor is a prostate tumor. In still other aspects, the tumor is a lung tumor.

In other embodiments, the present disclosure provides methods of preparing a purified, carrier-free ⁶⁸ Ga solution having a specific activity of greater than about 25 GBq/mg comprising: a) obtaining a ⁶⁸ Ga solution to be purified comprising ⁶⁸ Ga;

b) contacting the ⁶⁸ Ga solution with an ion-exchange resin; and

c) eluting the ⁶⁸ Ga from the ion-exchange resin to obtain the purified, carrier-free ⁶⁸ Ga solution.

In some aspects, the method comprises a single ion-exchange resin. In some aspects, the method does not comprise another ion-exchange resin.

In still other embodiments, the present disclosure provides methods of preparing a purified, carrier-free ⁶⁸ Ga solution having a specific activity of greater than about 25 GBq/mg comprising: a) obtaining a ⁶⁸ Ga solution to be purified comprising ⁶⁸ Ga;

b) contacting the ⁶⁸ Ga solution with an anion-exchange resin; and

c) eluting the ⁶⁸ Ga from the anion-exchange resin to obtain the purified, carrier-free 6 ⁸ Ga solution. In some aspects, the method comprises a single anion-exchange resin. In some aspects, the method does not comprise another ion-exchange resin.

In still other embodiments, the present disclosure provides systems for preparing a purified, carrier-free ⁶⁸ Ga solution having a specific activity of greater than about 25 GBq/mg comprising: a) a solid target comprising ⁶⁸ Zn;

b) a cyclotron, wherein the solid target is irradiated using the cyclotron; and c) an ion-exchange resin.

In some aspects, the system comprises a single ion-exchange resin. In some aspects, the system does not comprise another ion-exchange resin.

In still other embodiments, the present disclosure provides systems for preparing a purified, carrier-free ⁶⁸ Ga solution having a specific activity of greater than about 25 GBq/mg comprising: a) a solid target comprising ⁶⁸ Zn;

b) a cyclotron, wherein the solid target is irradiated using the cyclotron; and c) a single anion-exchange resin.

In some aspects, the system comprises a single anion-exchange resin. In some aspects, the system does not comprise another ion-exchange resin.

Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description lt should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIGS. 1A-1C show HPLC traces of DOTA-Oct labeling tests with cyclotron-produced ⁶⁸ Ga (conditions: pH 4.5, 95 °C for 10 minutes). FIG. 1A shows labeling test with 100 mg of DOTA-Oct and 100 mCi of ⁶⁸ Ga (radiochemical yield = 98.4%, radiochemical purity = 94.2%). FIG. 1B shows labeling test with 50 mg of DOTA-Oct and 100 mCi of ⁶⁸ Ga (radiochemical yield = 99.2%, radiochemical purity = 96.7%). FIG. 1C shows labeling test with 25 mg of DOTA-Oct and 100 mCi of ⁶⁸ Ga (radiochemical yield = 96.5%, radiochemical purity = 88.1%).

FIGS.2A-2C show HPLC traces of PSMA-l 1 labeling tests with cyclotron-produced ⁶⁸ Ga (conditions: pH 4, 95 °C for 10 minutes). FIG. 2A shows labeling test with 100 mg of PSMA-l 1 and 100 mCi of ⁶⁸ Ga (radiochemical yield = 100%, radiochemical purity = >99%). FIG. 2B shows labeling test with 50 mg of PSMA-l 1 and 100 mCi of ⁶⁸ Ga (radiochemical yield = 100%, radiochemical purity = >99%). FIG. 2C shows labeling test with 25 mg of PSMA-l 1 and 100 mCi of ⁶⁸ Ga (radiochemical yield = 100%, radiochemical purity = >99%).

FIGS.3A-3C show HPLC traces of PSMA-l 1 labeling tests with cyclotron-produced ⁶⁸ Ga (conditions: pH 4, 95 °C for 10 minutes). FIG. 3A shows labeling test with 10 mg of PSMA-11 and 100 mCi of ⁶⁸ Ga (radiochemical yield = 100%, radiochemical purity = >99%). FIG. 3B shows labeling test with 5 mg of PSMA-l 1 and 100 mCi of ⁶⁸ Ga (radiochemical yield = 100%, radiochemical purity = >99%). FIG. 3C shows labeling test with 1 mg of PSMA-l 1 and 100 mCi of ⁶⁸ Ga (radiochemical yield = 74%, radiochemical purity = 38.1%).

FIGS. 4A & 4B show HPLC traces of large-scale labeling tests with DOTA-Oct with cyclotron-produced ⁶⁸ Ga (conditions: 1 Ci ⁶⁸ Ga, 500 mg DOTA-Oct, pH 4.5, 95 °C for 10 minutes). FIG. 4A shows radiochemical yield = 87.4% and radiochemical purity = 62.9% at 0 h; FIG. 4B shows radiochemical purity = 17% at 3 h.

FIGS. 5A-5C show HPLC traces of large-scale labeling tests with PSMA-1 1 with cyclotron-produced ⁶⁸ Ga (conditions: 0.5 Ci ⁶⁸ Ga, 125 mg PSMA-l 1, pH 4, 95 °C for 10 minutes). FIG. 5A shows labeling test at 0 h: radiochemical yield = 100%, radiochemical purity = >99%; FIG. 5B shows labeling test at 1 h: radiochemical purity = -66.6%; FIG. 5C shows labeling test at 3 h: radiochemical purity = -47.9%. FIGS. 6A-6C show HPLC traces of DOTA-Oct labeling tests with cyclotron-produced ⁶⁸ Ga (conditions: 0.5 Ci ⁶⁸ Ga, 250 mg DOTA-Oct, 200 mg L-ascorbic acid, pH 4.5, 95 °C for 10 minutes) after 0 h (radiochemical purity = >99%, FIG. 6A), 1 h (radiochemical purity = >99%, FIG. 6B), and 4 h (radiochemical purity = >99%, FIG. 6C).

FIGS. 7A-7C show HPLC traces of PSMA-l 1 labeling tests with cyclotron-produced ⁶⁸ Ga (conditions: 0.5 Ci ⁶⁸ Ga, 125 mg PSMA-l 1, 200 mg L-ascorbic acid, pH 4, 95 °C for 10 minutes) after 0 h (radiochemical purity = >99%, FIG. 7A), 1 h (radiochemical purity = >99%, FIG. 7B), and 4 h (radiochemical purity = >99%, FIG. 7C).

FIGS. 8A & 8B show HPLC traces of PSMA-l 1 labeling tests with generator-produced ⁶⁸ Ga. FIG. 8A shows the HPLC trace of a standard of ⁶⁸ Ga-PSMA-l 1. FIG. 8B shows the HPLC trace of PSMA-l 1 with generator-produced ⁶⁸ Ga (conditions: 0.5 18 mCi ⁶⁸ Ga, 50 mg PSMA-l 1, pH 4, 95 °C for 10 minutes) and shows radiochemical yield = 100% and radiochemical purity = >99%.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

I. Introduction

Disclosed herein are methods and systems for the rapid purification of ⁶⁸ Ga solutions, compositions comprising ⁶⁸ Ga solutions prepared using said methods and systems, as well as methods of using said compositions to image biological tissues, such as tumors. The methods of purification comprise contacting a solution of ⁶⁸ Ga to be purified with a single anion-exchange resin.

Gallium-68 is a positron-emitting radioisotope that can be produced from a ⁶⁸ Ge/ ⁶⁸ Ga generator or from proton beam bombardment of ⁶⁸ Zn in a cyclotron. ⁶⁸ Ga-labeled peptides are a valuable class of radiopharmaceuticals exhibiting fast target localization and blood clearance. ⁶⁸ Ga-DOTATOC, ⁶⁸ Ga-DOTATATE, ⁶⁸ Ga-DOTANOC, are the most prominent radiopharmaceuticals currently in use for imaging and differentiating lesions of various somatostatin receptor subtypes, overexpressed in many neuroendocrine tumors. The number of clinical trials with ⁶⁸ Ga has increased dramatically, including within the United States.

As demand for ⁶⁸ Ga increases for use in radiopharmaceuticals, die need for improved methods for the synthesis and subsequent processing of ⁶⁸ Ga has grown significantly. Current methods for processing ⁶⁸ Ga require multiple purification steps (i.e. passing the crude ⁶⁸ Ga solution through more than one ion-exchange resin) and/or additional concentration and reconstitution steps in order to achieve a purity level and concentration sufficient to radiolabel carrier molecules for administration to patients. Such additional processing steps take time and leads to a krner specific activity of the purified ⁶⁸ Ga as a result of the short half-life of ⁶⁸ Ga (~68 minutes). As such, there is a need for methods that can produce purified ⁶⁸ Ga quickly while removing unwanted impurities such as zinc, iron, and/or germanium.

This invention is based, in part, on the discovery of a fully defined process of preparing purified ⁶⁸ Ga using a single anion-exchange resin which rapidly and directly provides the purified ⁶⁸ Ga in a solution sufficient such that no further processing is required prior to use in radiolabeling. As shown in the Examples, there is provided a method to prepare a purified, carrier-free solution of ⁶⁸ Ga with very high specific activity.

II. Definitions

The use of the word“a” or“an,” when used in conjunction with the term“comprising” in the claims and/or the specification may mean“one,” but it is also consistent with the meaning of “one or more,”“at least one,” and“one or more than one.” Throughout this application, the term“about” is used to indicate that a value includes the inherent variation of error for the device employed to determine the value, the method being employed to determine the value, or the variation that exists among the study subjects or patients.

The terms“comprise,”“have” and“include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as“comprises,”“comprising,”“has,”“having,” “includes” and“including,” are also open-ended. For example, any method that“comprises,” “has” or“includes” one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps.

When used in the context of a chemical group:“hydrogen” means -H;“hydroxy” means -OH;“oxo” means =O;“carbonyl” means -C(=O)-;“carboxy” means -C(=O)0H (also written as -COOH or -CO ₂ H);“halo” means independently -F, -Cl, -Br or -1;“amino” means -NH2; “hydroxyamino” means -NHOH;“nitro” means -NO ₂ ; imino means =NH;“cyano” means -CN; “isocyanyl” means -N=C=O;“azido” means -N ₃ ; in a monovalent context“phosphate” means -OP(O)(OH) ₂ or a deprotonated form thereof; in a divalent context “phosphate” means -OP(O)(OH)O- or a deprotonated form thereof;“mercapto” means -SH; and“thio” means =S;

“sulfbnyl” means -S(O)2 ^~ ; and“sulfinyl” means -S(O)-.

Unless specified otherwise, any chemical group or compound class listed in a claim set has a carbon atom limit of less than or equal to twelve.

In the context of chemical formulas, the symbol“-” means a single bond, “=” means a double bond, and“º” means triple bond. The symbol“ ----” represents an optional bond, which if present is either single or double. The symbol represents a single bond or a double bond.

Thus, the formula covers, for example, And it is

understood that no one such ring atom forms part of more than one double bond. Furthermore, it is noted that the covalent bond symbol when connecting one or two stereogenic atoms, does not indicate any preferred stereochemistry. Instead, it covers all stereoisomers as well as mixtures thereof. The symbol when drawn perpendicularly across a bond (e.g. , for methyl)

indicates a point of attachment of the group. It is noted that the point of attachment is typically only identified in this manner for larger groups in order to assist the reader in unambiguously identifying a point of attachment. The symbol means a single bond where the group attached to the thick end of the wedge is“out of the page.” The symbol means a single bond where the group attached to the thick end of the wedge is“into the page”. The symbol means a single bond where the geometry around a double bond (e.g., either E or Z) is undefined. Both options, as well as combinations thereof are therefore intended. Any undefined valency on an atom of a structure shown in this application implicitly represents a hydrogen atom bonded to that atom. A bold dot on a carbon atom indicates that the hydrogen attached to that carbon is oriented out of the plane of the paper.

When a variable is depicted as a“floating group” on a ring system, for example, the group “R” in the formula:

then the variable may replace any hydrogen atom attached to any of the ring atoms, including a depicted, implied, or expressly defined hydrogen, so long as a stable structure is formed. When a variable is depicted as a“floating group” on a fused ring system, as for example the group“R” in the formula:

then the variable may replace any hydrogen attached to any of the ring atoms of either of the fused rings unless specified otherwise. Replaceable hydrogens include depicted hydrogens (e.g., the hydrogen attached to the nitrogen in the formula above), implied hydrogens (e.g., a hydrogen of the formula above that is not shown but understood to be present), expressly defined hydrogens, and optional hydrogens whose presence depends on the identity of a ring atom (e.g., a hydrogen attached to group X, when X equals -CH-), so long as a stable structure is formed. In the example depicted, Rmay reside on either the 5-membered or the 6-membered ring of the fused ring system. In the formula above, the subscript letter“y” immediately following the R enclosed in parentheses, represents a numeric variable. Unless specified otherwise, this variable can be 0, 1, 2, or any integer greater than 2, only limited by the maximum number of replaceable hydrogen atoms of the ring or ring system.

The term“saturated” when used to modify a compound or chemical group means the compound or chemical group has no carbon-carbon double and no carbon-carbon triple bonds, except as noted below. When the term is used to modify an atom, it means that the atom is not part of any double or triple bond In the case of substituted versions of saturated groups, one or more carbon oxygen double bond or a carbon nitrogen double bond may be present. And when such a bond is present, then carbon-carbon double bonds that may occur as part of keto-enol tautomerism or imine/enamine tautomerism are not precluded. When the term“saturated” is used to modify a solution of a substance, it means that no more of that substance can dissolve in that solution. The term“aliphatic” signifies that the compound or chemical group so modified is an acyclic or cyclic, but non-aromatic compound or group. In aliphatic compounds/groups, the carbon atoms can be joined together in straight chains, branched chains, or non-aromatic rings (alicyclic). Aliphatic compounds/groups can be saturated, that is joined by single carbon-carbon bonds (alkanes/alkyl), or unsaturated, with one or more carbon-carbon double bonds (alkenes/alkenyl) or with one or more carbon-carbon triple bonds (alkynes/alkynyl).

The term“aromatic” signifies that the compound or chemical group so modified has a planar unsaturated ring of atoms with 4 n +2 electrons in a fully conjugated cyclic p system.

The term“alkyl” when used without the“substituted” modifier refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, a linear or branched acyclic structure, and no atoms other than carbon and hydrogen. The groups -CH ₃ (Me), -CH ₂ CH ₃ (Et), -CH ₂ CH ₂ CH ₃ (n-Pr or propyl), -CH(CH ₃ ) ₂ (i-Pr, ⁱ Pr or isopropyl), -CH ₂ CH ₂ CH ₂ CH ₃ (n-Bu), -CH(CH ₃ )CH ₂ CH ₃ (sec-butyl), -CH ₂ CH(CH ₃ ) ₂ (isobutyl), -C(CH ₃ ) ₃ (tort-butyl, t-butyl, t-Bu or ^t Bu), and -CH ₂ C(CH ₃ ) ₃ (neo-pentyl) are non-limiting examples of alkyl groups. The term “alkanediyl” when used without the“substituted” modifier refers to a divalent saturated aliphatic group, with one or two saturated carbon atom(s) as the point(s) of attachment, a linear or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups -CH ₂ - (methylene), -CH ₂ CH ₂ -, -CH ₂ C(CH ₃ )2CH ₂ -, and -CH ₂ CH ₂ CH ₂ - are non-limiting examples of alkanediyl groups. The term“alkylidene” when used without the “substituted” modifier refers to the divalent group =CRR' in which R and R' are independently hydrogen or alkyl. Non-limiting examples of alkylidene groups include: =CH ₂ , =CH(CH ₂ CH ₃ ), and =C(CH ₃ ) ₂ . An“alkane” refers to the class of compounds having the formula H-R, wherein R is alkyl as this term is defined above. When any of these tenns is used with the“substituted” modifier, one or more hydrogen atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH ₂ , -NO ₂ , -CO ₂ H, -CO ₂ CH ₃ , -CN, -SH, -OCH ₃ , -OCH ₂ CH ₃ , -C(O)CH ₃ , -NHCH ₃ ,

-NHCH ₂ CH ₃ , -N(CH ₃ )2, -C(O)NH ₂ , -C(O)NHCH ₃ , -C(O)N(CH ₃ ) ₂ , -OC(O)CH ₃ ,

-NHC(O)CH ₃ , -S(O)20H, or -S(O) ₂ NH ₂ . The following groups are non-limiting examples of substituted alkyl groups: -CH ₂ OH, -CH ₂ Cl, -CF ₃ , -CH ₂ CN, -CH ₂ C(O)OH, -CH ₂ C(O)OCH ₃ , -CH ₂ C(O)NH ₂ , -CH ₂ C(O)CH ₃ , -CH ₂ OCH ₃ , -CH ₂ OC(O)CH ₃ , -CH ₂ NH ₂ , -CH ₂ N(CH ₃ )2, and -CH ₂ CH ₂ Cl.

The term“aryl” when used without the“substituted” modifier refers to a monovalent unsaturated aromatic group with an aromatic carbon atom as the point of attachment, said carbon atom forming part of a one or more aromatic ring structures, each with six ring atoms that are all carbon, and wherein the group consists of no atoms other than carbon and hydrogen. If more than one ring is present, the rings may be fused or unfused. Unfused rings are connected with a covalent bond. As used herein, the term aryl does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl, -C ₆ H ₄ CH ₂ CH ₃ (ethylphenyl), naphthyl, and a monovalent group derived from biphenyl (e.g., 4-phenylphenyl). The temi“arenediyl” when used without the“substituted” modifier refers to a divalent aromatic group with two aromatic carbon atoms as points of attachment, said carbon atoms forming part of one or more six-membered aromatic ring structures, each with six ring atoms that are all carbon, and wherein the divalent group consists of no atoms other than carbon and hydrogen. As used herein, the term arenediyl does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. If more than one ring is present, the rings may be fused or unfused. Unfused rings are connected with a covalent bond. Non-limiting examples of arenediyl groups include:

The term“aralkyl” when used without the“substituted” modifier refers to the monovalent group -alkanediyl-aryl, in which the terms alkanediyl and aryl are each used in a manner consistent with the definitions provided above. Non-limiting examples are: phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl. When the term aralkyl is used with the“substituted” modifier one or more hydrogen atom from the alkanediyl and/or the aryl group has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH ₂ , -NO ₂ , -CO ₂ H, -CO ₂ CH ₃ , -CN, -SH, -OCH ₃ , -OCH ₂ CH ₃ , -C(O)CH ₃ , -NHCH ₃ , -NHCH ₂ CH ₃ , -N(CH ₃ ) ₂ , -C(O)NH ₂ , -C(O)NHCH ₃ , -C(O)N(CH ₃ ) ₂ , -OC(O)CH ₃ , -NHC(O)CH ₃ , -S(O) ₂ OH, or -S(O) ₂ NH ₂ . Non-limiting examples of substituted aralkyls are: (3-chlorophenyl)-methyl, and 2-chloro-2-phenyl-eth- 1 -yl .

The term“phosphonate” when used without the “substituted” modifier refers to a compound with the formula R-P(O)(0R')(0R"), in which R, R', and R" can be the same or different alkyl, and, or aralkyl groups, as those terms are defined above, or R' and R" can be taken together to represent an alkanediyl. Non-limiting examples of phosphonate include: Et-P(O)(0Me)(0Bn) and C5H1 I-P(O)(0C5HI 1)2. When any of these terms is used with the “substituted” modifier, one or more hydrogen atom has been independently replaced by -OH, -F,

-Cl, -Br, -1, -NH ₂ , -NO ₂ , -CO ₂ H, -CO ₂ CH ₃ , -CN, -SH, -OCH ₃ , -OCH ₂ CH ₃ , -C(O)CH ₃ , -NHCH ₃ , -NHCH ₂ CH ₃ , -N(CH ₃ )2, -C(O)NH ₂ , -C(O)NHCH ₃ , -C(O)N(CH ₃ ) ₂ , -OC(O)CH ₃ , -NHC(O)CH ₃ , -S(O) ₂ 0H, or -S(O) ₂ NH ₂ .

In the present specification reference is made to specific isotopes of elements, e.g. gallium- 68. Isotopes are designated either with a superscripted mass number prefix or a hyphenated, non- superscripted suffix, e.g. gallium-68 or ⁶⁸ Ga. Isotopes as disclosed herein should not necessarily be taken to refer to the neutral elements but may refer to ions of these isotopes or to complexes thereof. Thus, for example“ ⁶⁸ Ga” should be taken to refer to a ⁶⁸ Ga species, e.g. ⁶⁸ Ga ³⁺ or a ⁶⁸ Ga complex, for example a citrate complex. Such complexes may themselves be neutral or may be electrically charged.

An“excipien t” is a pharmaceutically acceptable substance formulated along with the active ingredient(s) of a medication, pharmaceutical composition, formulation, or drug delivery system. Excipients may be used, for example, to stabilize the composition, to bulk up the composition (thus often referred to as "bulking agents,”“fillers,” or“diluents” when used for this purpose), or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, or enhancing solubility. Excipients include pharmaceutically acceptable versions of antiadherents, binders, coatings, colors, disintegrants, flavors, glidants, lubricants, preservatives, sorbents, sweeteners, and vehicles. The main excipient that serves as a medium for conveying the active ingredient is usually called the vehicle. Excipients may also be used in the manufacturing process, for example, to aid in the handling of the active substance, such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation or aggregation over the expected shelf life. The suitability of an excipient will typically vary depending on the route of administration, the dosage form, the active ingredient, as well as other factors.

As used herein, the term“patient” or“subject” refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof In certain embodiments, the patient or subject is a primate. Non-limiting examples of human patients are adults, juveniles, infants and fetuses.

An“active ingredient” (AI) or active pharmaceutical ingredient (API) (also referred to as an active compoimd, active substance, active agent, pharmaceutical agent, radiological agent, imaging agent, biologically active molecule, or a therapeutic compound) is the ingredient in a pharmaceutical drug that is biologically or radiologically active.

As generally used herein“pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.

A “pharmaceutically acceptable carrier,” “drug carrier,” or simply “carrier” is a pharmaceutically acceptable substance formulated along with the active ingredient that is involved in carrying, delivering and/or transporting a chemical agent. Carriers may be used to improve the delivery and the effectiveness of drugs, including for example, controlled-release technology to modulate drug bioavailability, decrease drug metabolism, and/or reduce drug toxicity. Some carriers may increase the effectiveness of drug delivery to the specific target sites. Some carriers may target specific tissues such as thyroid, brain, gastrointestinal, pancreas, spleen, kidney, neuroendocrine tumors, renal cell carcinoma, lung cancer, breast cancer, prostate cancer, and malignant lymphoma. Non-limiting examples of carriers include: peptides, small molecules, antibodies, or antibody-drug conjugates, or fragments thereof. Further non-limiting examples of carrier molecules include prostate-specific membrane antigen (PSMA), 1,4,7- triazacyclononane-N,N',N"-triacetic acid (NOTA), 1,4,7, lO-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid (DOTA), diethylene triamine pentaacetic acid (DTP A), desferrioxamine, DOTA-Tyr(3)-octreotide (DOTATOC), DOTA-Tyr(3)-Tyr(8)- octreotide (DOTATATE), DOTA- 1 -naphtyl-alanine (DOTANOC), DOT A-benzothienyl-al anine (DOTA-BOC), DOTA-bombesin, DOTA-arginine-glycine-aspartic acid-bombesin (DOTA-RGD-bombesin), NOTA-RGD, 3,6,9,l5-tetraazabicyclo[9.3.l]pentadeca-l(l5)-,l l,l3-triene-3,6,9-triacetic acid-RGD

(PCTA-RGD), DOTA-albumin, DOTA-human epidermal growth factor, 1,4,7 -triazacyclononane- l-[methyl(2-carboxyethyl)phosphinic

acid]-4,7-bis[methyl(2-hydroxymethyl)phosphinic acid-integrin alpha(IIb)beta(3)-specific cyclic hexapeptide (NOPO-RGDfK), l,4,7-triazacyclononane-l,4-bis(acetic acid)-7-(2-glutaric acid) (NODAGA), NOPO-NaI(3)-octreotide conjugate (NOPO-NOC),

1,4,7 -triazacyclononane- 1 ,4,7-tris [(2-carboxyethyl)methylenephosphinic acid] (TRAP(RGD)3), or citrate.

A“pharmaceutical drug” (also referred to as a pharmaceutical, pharmaceutical preparation, pharmaceutical composition, pharmaceutical formulation, pharmaceutical product, medicinal product, medicine, medication, medicament, or simply a drug, agent, or preparation) is a composition used to diagnose, image, cure, treat, or prevent disease, which comprises an active pharmaceutical ingredient (API) (defined above) and optionally contains one or more inactive ingredients, which are also referred to as excipients (defined above).

“Prevention” or“preventing” includes: (1) inhibiting the onset of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease, and/or (2) slowing the onset of the pathology or symptomatology of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease.

“Treatment” or“treating” includes (1) inhibiting a disease in a subject or patient experiencing or displaying the pathology or symptomatology of the disease (e.g., arresting further development of the pathology and/or symptomatology), (2) ameliorating a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease (e.g., reversing the pathology and/or symptomatology), and/or (3) effecting any measurable decrease in a disease or symptom thereof in a subj ect or patient that is experiencing or displaying the pathology or symptomatology of the disease.

The term“unit dose” refers to a formulation of the compound or composition such that the formulation is prepared in a manner sufficient to provide a single therapeutically effective dose of the active ingredient to a patient in a single administration. Such unit dose formulations that may be used include but are not limited to a single tablet, capsule, or other oral formulations, or a single vial with a syringeable liquid or other injectable formulations.

The above definitions supersede any conflicting definition in any reference that is incorporated by reference herein. The fact that certain terms are defined, however, should not be considered as indicative that any term that is undefined is indefinite. Rather, all terms used are believed to describe the invention in terms such that one of ordinary skill can appreciate the scope and practice the present invention.

Ill. Production of Gallium-68 to be Purified

In the first step of the method provided herein for preparing a purified, carrier-free ⁶⁸ Ga solution, a ⁶⁸ Ga solution comprising ⁶⁸ Gato be purified is obtained. The ⁶⁸ Ga solution comprising ⁶⁸ Ga to be purified may be prepared through, or a byproduct of, the synthesis of ⁶⁸ Ga. The ⁶⁸ Ga to be purified may be synthesized using a cyclotron. A cyclotron is understood to be an apparatus in which charged atomic and subatomic particles are accelerated by an alternating electric field while following an outward spiral or circular path in a magnetic field. Without wishing to be bound by any theory, the ⁶⁸ Ga to be purified is synthesized via irradiation of a metal sample comprising ⁶⁸ Zn, which upon irradiation is converted into ⁶⁸ Ga. The metal sample may be in the fonn of a liquid target or in the form of a solid target, e.g. a solution of ZnNO ₂ or solid ⁶⁸ Zn. When a liquid target is employed, the ⁶⁸ Zn is dissolved and the liquid target may further comprise an acidic solution. When a solid target is employed, the solid target may be suspended in an acidic solution or an acidic solution may be added upon or after irradiation. The acidic solution may comprise HCl or an acid derived from the dissociation of HCl. Such acids are understood to be the protonated form of a solvent, e.g. H ₃ O ⁺ in the case of water as a solvent or CH ₃ OH ₂ ⁺ in the case of methanol as a solvent. The acid may have a concentration of about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 N. The ⁶⁸ Ga solution comprising ⁶⁸ Ga to be purified may be an aqueous solution. IV. Purification of Gallium-68

In another step, the ⁶⁸ Ga solution comprising ⁶⁸ Ga to be purified is contacted with an anion- exchange resin. The anion-exchange resin may be treated with an acidic solution prior to contacting the resin with the ⁶⁸ Ga solution comprising ⁶⁸ Ga to be purified. The anion-exchange resin should have a higher affinity for ⁶⁸ Ga than for zinc, iron, germanium or other impurities so that the impurities are eluted from the anion-exchange resin while the majority, preferably substantially all, of the ⁶⁸ Ga is retained on the anion-exchange resin under an initial set of conditions. The anion-exchange resin should have an affinity for ⁶⁸ Ga that can be modulated so as to allow for the ⁶⁸ Ga to be eluted from the resin under a second set of conditions. The anion- exchange resin may comprise an amine group or phosphonate, e.g. phosphonate, such as the UTEVA ^® resin. The resin should be resistant to radiation. The methods and systems disclosed herein employ a single anion-exchange resin. Whereas other methods employ a single cation- exchange resin or a combination of two different ion-exchange resins, the methods disclosed herein comprise a single anion-exchange resin. Use of a single anion-exchange resin reduces the amoimt of time required to purify ⁶⁸ Ga and provides ⁶⁸ Ga with a higher specific activity. Commonly, the anion-exchange resins are in particulate form, so that a resin column contains a packed bed of the particulate resin. The resin may be porous, e.g. microporous, mesoporous, nanoporous etc. A typical resin column may contain from about 0.01 g to about 2 g of resin, e.g. about 0.01, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 1, 1.5, or 2 g.

After contacting the anion-exchange resin with the ⁶⁸ Ga solution comprising ⁶⁸ Ga to be purified, the resin may be optionally washed with a washing solution. The washing solution may comprise an acid, including but not limited to HCl, or an acid derived from the dissociation of the acid, e.g. H ₃ O ⁺ . The acid of the washing solution may have a concentration of greater than 1 N, such as from about 2 N to 8 N, e.g. 2 N, 3 N, 4 N, 5 N, 6 N, 7 N, or 8 N. The washing solution may be an aqueous solution.

Following the optional washing step, ⁶⁸ Ga is eluted from the anion-exchange resin by contacting the anion-exchange resin with an eluting solution. Without wishing to be bound by theory, the eluting solution modulates the affinity of the anion-exchange resin for ⁶⁸ Ga. The eluting solution may comprise an acid, including but not limited to HCl, or an acid derived from the dissociation of the acid, e.g. H ₃ O ⁺ . The acid of the eluting solution may have a concentration of from about 0.005 N to about 2 N, e.g. 0.005 N, 0.01 N, 0.02 N, 0.03 N, 0.04 N, 0.05 N, 0.06 N, 0.07 N, 0.08 N, 0.09 N, 0.1 M, 0.5 N, 1 N, or 2 N. The eluting solution may be an aqueous solution.

The resulting purified, carrier-free ⁶⁸ Ga solution has a specific activity of greater than about 25 GBq/mg. The specific activity may be from about 25 GBq/mg to about 100,000 GBq/mg, e.g. 25, 100, 500, 1000, 10,000, 15,000, 20,000, 25,000, 30,000, 31,000, 32,000, 33,000, 33,100,

33,200, 33,300, 33,400, 33,500, 33,600, 33,700, 33,800, 33,900, 34,000, 35,000, 36,000, 37,000, 38,000, 39,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or 100,000 GBq/mg. Specific activity values present in this disclosure are correlated to the End of Bombardment (EOB) unless explicitly noted otherwise.

The present disclosure also provides systems for preparing a purified, carrier-free ⁶⁸ Ga solution. These systems comprise a solid target comprising ⁶⁸ Zn, a cyclotron, wherein the solid target is irradiated using the cyclotron, and a single anion-exchange resin. The system may be portable or may be fixed in a specific location.

V. Compositions of Gallium-68 for Imaging

The present disclosure also provides compositions comprising the purified, carrier-free solution of ⁶⁸ Ga. Compositions may further comprise a carrier molecule. The compositions disclosed herein may be useful for biological imaging, e.g. PET imaging. Compositions may be prepared by the addition of the purified, carrier-free solution of ⁶⁸ Ga to a carrier molecule or a solution comprising a carrier molecule. Further processing of the composition may be performed prior to administration to a patient. Such processing may include purification, concentration, or dilution. Compositions of the present disclosure may also comprise pharmaceutically acceptable excipients.

The compositions disclosed herein may be formulated for administration intraarterially, intraarticularly, intracranially, intrapericardially, intraperitoneally, intratumorally, intravenously, intravesicularlly, parenterally, via injection, via local delivery, or via localized perfusion. The compositions may be administered once or more than once. The compositions may be used to image tissue, such as a tumor, e.g. a prostate, pancreatic, lung, or neuroendocrine tumor. The compositions may be used to image a tumor that exhibits elevated levels of PSMA.

VI. Examples

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result wi thout departing from the spirit and scope of the invention.

Example 1 : Synthesis, Purification, and Analysis of ⁶⁸ Ga

All reagents and solvents were purchased from commercial vendors and used directly unless otherwise noted. Enriched ⁶⁸ Zn metal powder (99+% purity) was purchased from ISOFLEX USA (San Francisco, CA). Milli-Q water (18 MW cm) was obtained from a Millipore Milli-Q Integral 5 water purification system (Billerica, MA). The ITG ⁶⁸ Ge/ ⁶⁸ Ga Generator (GMP) was obtained from RadioMedix (Houston, TX). UTEVA ^® resin (50-100 mm) was purchased from Eichrom (Lisle, IL). Hydrochloric acid (33% min., 99.999999% metal basis) was obtained from Alfa Aesar (Pittsburgh, PA) and was diluted to 10 N, 4 N, and 0.05 N with Milli-Q water. 1 ,4,7, 10-Tetraazacyclododecane- 1 ,4,7, 10-tetraacetic acid (DOTA) was purchased from Sigma-Aldrich (St. Louis, MO). Instant thin-layer chromatography paper (iTLC-SG) was acquired from Fisher Scientific (Hampton, NH). The labeling efficiency of ⁶⁸ Ga-DOTA was determined using radio-TLC with the mobile phase composed of 10% ammonium acetate and methanol in one-to-one ratio. The radio-TLC plates were scanned using a Bioscan AR-2000 radio-TLC scanner (Washington, DC).

The Cyclotron Radiochemistry Facility (CRF) is equipped with a 16.5 MeV proton energy cyclotron (PETrace, GE Healthcare, Waukesha, WI). Prior to irradiation, a 7 mm diameter of enriched ⁶⁸ Zn (60-120 mg) was electrodeposited on a platinum disc using a solution comprised of ⁶⁸ ZnCl ₂ and 0.05 N HCl (concentration of ⁶⁸ Zn: 25-30 mg/mL) and a current density of 25 mA/cm ² . The target was then transferred and mounted in the PETtrace cyclotron through the Comecer EDS/PTS module (Castel Bolognese RA, Italy). The target was irradiated at an incident proton-beam energy of 14.5 MeV with beam currents of 15-40 pA. After irradiation, the target was pneumatically retrieved. Chemical separation of ⁶⁸ Ga was performed and labeling procedures were conducted using the Comecer EDS and GE Healthcare TRACERlab FX modules.

Gallium-68 was separated from ⁶⁸ Zn using a column containing 100 mg of UTEVA ^® resin, in which the resin was pre-conditioned with 10 mL of 6 N HCl. Upon retrieving the irradiated ⁶⁸ Zn target from the cyclotron into the Comecer EDS module, the target was completely dissolved with 10 mL of 6 N HCl imder 10 min. The crude ⁶⁸ Ga/ ⁶⁸ Zn solution was passed through the column containing the UTEVA ^® resin. After rinsing the transfer lines in the EDS module with 5 mL of 6 N HCl, the HCl rinse was also passed through the column followed by 40 mL of 4 N HCl to remove residual ⁶⁸ Zn and any additional metal contaminants. The [ ⁶⁸ Ga]GaCl ₃ was desorbed from the resin with 2 mL of 0.05 N HCl and readily available to be used in a labeling reaction without any further processing.

The effective specific activity (GBq/mmol) of [ ⁶⁸ Ga]GaCl ₃ was determined through a series of labeling reactions with a chelating solution. Since DOTA is one of the chelators that is often conjugated to a biomolecule for labeling with ⁶⁸ Ga, multiple dilutions of DOTA solution (2.48 x 10 ^-5 - 2.48 x 10 ² mM) in 0.5 M acetate buffer (pH 4.5) were used for the study. A volume of 5 mL (~74 MBq) of [ ⁶⁸ Ga]GaCl ₃ was first diluted with 1 mL of 0.05 M HCl as a stock solution. The study was performed by adding 50 mL of [ ⁶⁸ Ga]GaCl ₃ stock solution into 200 mL of DOTA solution. The reactions were allowed to precede at 95 °C for 20 min. After incubation was completed, the effective specific activity was determined by measuring labeling efficiency using radio-TLC analysis (mobile phase: 10% ammonium acetate/methanol, 1: 1 (v/v)).

Samples containing activities of 0.5-1.0 mCi (18.5-37 KBq) of [ ⁶⁸ Ga]GaCl ₃ in 2 mL microcentrifuge tubes were diluted with Milli-Q water to bring the final volume to 1 mL. The radionuclidic purity was then determined by gamma-ray spectroscopy using a cryogenically cooled germanium (HPGe) detector (ORTEC, Oak Ridge, TN). Samples were counted for 1 h starting immediately after End of Synthesis (EOS) and the measurement was repeated after 24 h. The radionuclidic purity of ⁶⁸ Ga was based on the analysis of the regions of interest (ROI) at EOS and of decayed samples.

ICP-MS analysis of the ⁶⁸ Ga samples, after decay, were conducted at the University of Missouri Research Reactor (MURR). All samples were analyzed on a PerkinElmer NexION 300X ICP-MS operated in kinetic energy discrimination (KED) mode with two different helium cell gas flows at 2.5 and 4.5 mL/min. The amount of gallium, germanium, iron, and zinc in each sample were detennined.

Example 2: Evaluation of Cyclotron Irradiated ⁶⁸ Zn Targets and ⁶⁸ Ga Separation and ICP-MS Analysis

Due to the unique characteristics and availability of ⁶⁸ Ge/ ⁶⁸ Ga generators, several efforts have been made to translate ⁶⁸ Ga-labeled compounds from preclinical evaluations into routine clinical practice (Amor-Coarasa et al., 2016; Velikyan, 2015; Vis et al., 2015). In order to satisfy the increasing demand for ⁶⁸ Ga in preclinical and clinical applications, an approach to reliably produce ⁶⁸ Ga via ⁶⁸ Zn(p,n) ⁶⁸ Ga reaction in significantly large quantities was investigated. The methods described herein have been used successfully for the cyclotron production and chemical separation of ⁶⁸ Ga. See Table 1 for a comparison of ⁶⁸ Ga production at various facilities. Table 1. Comparison of ⁶⁸ Ga Production at Various Facilities

Due to the relatively low melting point of Zn metal (419.5 °C), initial tests with natZn targets were conducted for short time intervals (5 min) at an incident proton-beam energy of 14.5 MeV while increasing beam currents from 15 to 40 mA. No significant heat effects on the electrodeposited natZn were observed throughout the tests indicating efficient heat removal from the target by the EDS/PTS module cooling system. To ensure production reliability during long irradiations targets were tested with beam currents up to 40 mA. No visible heat effects or loss of yield was observed. All subsequent measurements reported here were conducted at a beam current of 30 mA. To optimize target thickness for maximum yield, targets with a diameter of 7 mm and a mass of ⁶⁸ Zn between 60 and 120 mg were bombarded for 20-90 min. The ⁶⁸ Ga activity at End of Bombardment (EOB) correlated well with irradiation time and amount of ⁶⁸ Zn metal present. Targets containing (l04.l±2.7) mg ⁶⁸ Zn (n = 3) irradiated for 1 h at a beam current of 30 mA resulted in 60.9±l.8 GBq (1645±51 mCi) of ⁶⁸ Ga at EOB. The production yield was found to be 2.72±0.08 GBq/mA h (73.5±2.3 mCi/mA h). Sadeghi et al. reported production of ⁶⁸ Ga using a 6° grazing incidence target geometry with a proton-beam energy of 15.0 MeV and a beam current of 150 mA (Sadeghi et al., 2009). They report a production yield for a target mass of 434 mg of 5.03 GBq/mA h (136 mCi/mA h) at EOB for a short 15 min irradiation. Their result is not far from the calculated value of 5.81 GBq/mA h (157 mCi/mA h) presented by Szelecsenyi et al. derived from cross section measurements (Szelecsenyi et al., 2012). These results compare well with these reported values taking into account the lower incident proton-energy employed herein to minimize 6 ⁷ Ga impurities and a reduced target density of (4.8±0.4) g/cm ³ based on target thickness measurements.

During initial development the time required to dissolve the irradiated ⁶⁸ Zn target was investigated. The target was dissolved using 10 mL of 10 N HCl. The completion of the dissolution process was monitored using the flow meter integrated into the system. At the beginning of the dissolution process hydrogen gas was released partially interrupting the overall flow rate of circulating HCl solution. Once the target was completely dissolved and no more hydrogen gas was being released, the flow rate was re-stabilized. Based on these observations (and posterior verification) we found the total dissolution time required was less than 5 min. High zinc and HCl acid contents in the final ⁶⁸ Ga solution are two potential challenges for cyclotron- produced ⁶⁸ Ga. Although previous reports indicate that most ⁶⁸ Zn can be removed by using the AG ^® 50W cation resin alone (Engle et al., 2012; Pandey et al., 2014), lower specific activity is obtained as it takes a relatively long time (i.e., usually more than an hour) to evaporate the ⁶⁸ Ga solution and reconstitute it with a low concentration of HCl for radiolabeling use. McAlister and Horwitz have established a two-column system which utilizes AG ^® 50W cation resin and UTEVA ^® anion resin to minimize ⁶⁸ Ge breakthrough from a ⁶⁸ Ge/ ⁶⁸ Ga generator (McAlister and Philip Horwitz, 2009). However, the two-column system again results in lower specific activity due to processing times. By adapting this concept for processing ⁶⁸ Ga from an irradiated ⁶⁸ Zn target, over 75 % of ⁶⁸ Ga produced activity (decay corrected) can be recovered while the cyclotron- produced ⁶⁸ Ga solution results in lower (p<0.01) zinc content (0.004±0.002 mg/GBq) when compared to that of generator-produced ⁶⁸ Ga (0.37±0.09 mg/GBq) after ICP-MS analysis. In addition, low iron content was observed in both cyclotron and generator-produced ⁶⁸ Ga. The germanium was below detection limits for cyclotron-produced ⁶⁸ Ga (See Table 2). It was found that the UTEVA ^® anion-exchange resin could be used to generate ⁶⁸ Ga solutions with high purity and very high specific activity without the need for additional processing. All of these data suggest that the method presented herein not only minimizes zinc and other metal impurities, but also allows the reconstitution of the final ⁶⁸ Ga solution in a low concentration of HCl as well. The whole separation process only takes less than 10 min, which is especially important for ⁶⁸ Ga production.

Table 2. Comparison of Generator- vs. Cyclotron-Produced ⁶⁸ Ga

The effective specific activity of cyclotron and generator-produced ⁶⁸ Ga was detennined by DOTA titration using radio-TLC. While the ⁶⁸ Ga-DOTA radio-peak migrates to the solvent front, the unreacted [ ⁶⁸ Ga]GaCl ₃ remains at the origin. In all experiments (n = 3 for each study), the effective specific activity of cyclotron and generator-produced [ ⁶⁸ Ga]GaCl ₃ was fomid to be 6.7±0.8 and 7.1±1.6 GBq/pmol, respectively. No statistically significant difference (p = 0.775) was found in the effective specific activity between cyclotron and generator-produced ⁶⁸ Ga. To confirm the radionuclidic purity of the ⁶⁸ Ga solution produced via the ⁶⁸ Zn(p,n) ⁶⁸ Ga reaction, gamma-ray spectroscopy was performed. A small fraction (< 0.2%) of ⁶⁷ Ga was observed when the target was irradiated up to 90 min with 14.5 MeV protons.

* * * * * * * * * * * * * * * * All of the compounds, systems, compositions, and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the disclosure may have focused on several embodiments or may have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications may be applied to the compounds, compositions, and methods without departing from the spirit, scope, and concept of the invention. All variations and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

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