The 18‐membered macrocycle H2macropa was investigated for 225Ac chelation in targeted alpha therapy (TAT). Radiolabeling studies showed that macropa, at submicromolar concentration, complexed all ...225Ac (26 kBq) in 5 min at RT. 225Ac(macropa)+ remained intact over 7 to 8 days when challenged with either excess La3+ ions or human serum, and did not accumulate in any organ after 5 h in healthy mice. A bifunctional analogue, macropa‐NCS, was conjugated to trastuzumab as well as to the prostate‐specific membrane antigen‐targeting compound RPS‐070. Both constructs rapidly radiolabeled 225Ac in just minutes at RT, and macropa‐Tmab retained >99 % of its 225Ac in human serum after 7 days. In LNCaP xenograft mice, 225Ac‐macropa‐RPS‐070 was selectively targeted to tumors and did not release free 225Ac over 96 h. These findings establish macropa to be a highly promising ligand for 225Ac chelation that will facilitate the clinical development of 225Ac TAT for the treatment of soft‐tissue metastases.
Actinium in action! A macrocyclic ligand exhibits unprecedented radiolabeling efficiency for the large α‐emitting radionuclide 225Ac3+. This ligand is extremely promising for the implementation of 225Ac in targeted alpha therapy for cancer. RCY=radiochemical yield.
Objective
Heart failure is often preceded by distinct metabolic alterations. However, this shift cannot be sustained indefinitely and typically leads to cardiac dysfunction and arrest. These ...alterations are thought to be caused by mitochondrial dysfunction leading to the accumulation of acyl‐CoAs. To compensate, the injured heart shifts from oxidation of long chain fatty acids (C16) to metabolism of substrates that can replenish the TCA cycle. This is because these fatty acids are restricted to catabolism to acetyl‐CoA, for which the enzymes are saturated. By contrast, odd chain fatty acids are metabolized to propionate. The enzymes specific for propionate are freely available for metabolism to succinate leading to TCA cycle anaplerosis. Propionate conversion to propionyl‐CoA is the first and major step for its sequestration and metabolism. Moreover, the mitochondrial acyl‐CoA synthetase (ACSS) responsible for this conversion is highly expressed in the heart. Hence, we hypothesize that 2‐18F‐fluoropropionate (18F‐FPA) would significantly accumulate in the failing heart and can be imaged by positron emission tomography (PET). We addressed our hypothesis by studying the substrate behavior of FPA for ACSS, and by metabolic profiling of HepG2 cells incubated with FPA. We also successfully imaged heart uptake of 18F‐FPA in healthy rats by PET.
Methods
The ability of acetate, propionate, and FPA to act as ACSS substrates was determined by a colorimetric pyrophosphate assay. Metabolites were determined by LC/MS in lysates of HepG2 cells incubated with basal media, 10 mM propionate or 10 mM FPA. Racemic 18F‐FPA was synthesized from ethyl‐2‐bromopropionate by standard nucleophilic substitution. For PET, Sprague‐Dawley rats were injected with 250‐500 µCi (9.25‐18.5 MBq) of 18F‐FPA, and a 60‐minute dynamic PET scan was immediately acquired.
Results
The kinetic characteristics for the reaction of ACSS acting on acetate (Km = 4.94 mM, Vmax = 0.25 nmols/sec), propionate (Km = 24.20 mM, Vmax = 0.19 nmols/sec), and 2‐fluoropropionoate (V = 0.05 nmols/sec) were determined. We also observed a reduction of free CoA in HepG2 cells incubated in 10 mM propionate (4.4 ± 1.2‐fold) or 10 mM FPA (3.09 ± 0.96‐fold). The kinetics of 18F‐FPA uptake into the healthy heart was assessed by PET and determined to be maximal at 10 minutes post‐injection.
Conclusions
We identified 18F‐FPA, and potentially other odd chain fatty acids, as promising metabolic tracers for imaging anaplerosis in the injured heart. FPA effectively decreases free CoA in HepG2 cells, which indicates the formation of acyl‐CoAs. Moreover, FPA exhibits some substrate activity for ACSS, representing a major conduit by which it is metabolized in the heart. The poor substrate activity of FPA is ideal as it precludes metabolism by the highly active and specific ACSS enzymes in the liver. Albeit we expect FPA to accumulate in the heart due to its high ACSS expression which is induced in energy deficient states that are characteristic of heart failure. In support of this idea, we demonstrated that 18F‐FPA is taken up by the healthy heart. We expect the metabolic alterations resulting from ensuing heart failure are sufficient to increase the uptake of 18F‐FPA in these tissues. Our long‐term aim is to develop 18F‐labeled short chain fatty acids, such as 18F‐FPA, that can be used to diagnose at‐risk patients. The early detection of heart failure by non‐invasive means is critical for the development of interventions to reduce the severity and aid recovery from cardiac disease.
Emerging interest in extending the plasma half-life of small molecule radioligands warrants a consideration of the appropriate radionuclide for PET imaging at longer time points (>8 h). Among ...candidate positron-emitting radionuclides, 66Ga (t1/2 = 9.5 h, β+ = 57%) has suitable nuclear and chemical properties for the labeling and PET imaging of radioligands of this profile. We investigated the value of 66Ga to preclinical screening and the evaluation of albumin-binding PSMA-targeting small molecules. 66Ga was produced by irradiation of a natZn target. 66Ga3+ ions were separated from Zn2+ ions by an optimized UTEVA anion exchange column that retained 99.99987% of Zn2+ ions and allowed 90.2 ± 2.8% recovery of 66Ga3+. Three ligands were radiolabeled in 46.4 ± 20.5%; radiochemical yield and >90% radiochemical purity. Molar activity was 632 ± 380 MBq/µmol. Uptake in the tumor and kidneys at 1, 3, 6, and 24 h p.i. was determined by µPET/CT imaging and more completely predicted the distribution kinetics than uptake of the 68GaGa-labeled ligands did. Although there are multiple challenges to the use of 66Ga for clinical PET imaging, it can be a valuable research tool for ligand screening and preclinical imaging beyond 24 h.
Cancer cells require lipids to fulfill energetic, proliferative, and signaling requirements. Even though these cells can take up exogenous fatty acids, the majority exhibit a dependency on de novo ...fatty acid synthesis. Fatty acid synthase (FASN) is the rate-limiting enzyme in this process. Expression and activity of FASN is elevated in multiple cancers, where it correlates with disease progression and poor prognosis. These observations have sparked interest in developing methods of detecting FASN expression in vivo. One promising approach is the imaging of radiolabeled molecular probes targeting FASN by positron emission tomography (PET). However, although 11Cacetate uptake by prostate cancer cells correlates with FASN expression, no FASN-specific PET probes currently exist. Our aim was to synthesize and evaluate a series of small molecule triazolones based on GSK2194069, an FASN inhibitor with IC50 = 7.7 ± 4.1 nM, for PET imaging of FASN expression. These triazolones were labeled with carbon-11 in good yield and excellent radiochemical purity, and binding to FASN-positive LNCaP cells was significantly higher than FASN-negative PC3 cells. Despite these promising characteristics, however, these molecules exhibited poor in vivo pharmacokinetics and were predominantly retained in lymph nodes and the hepatobiliary system. Future studies will seek to identify structural modifications that improve tumor targeting while maintaining the excretion profile of these first-generation 11C-methyltriazolones.
Background
As
225
Ac-labeled radiopharmaceuticals continue to show promise as targeted alpha therapeutics, there is a growing need to standardize quality control (QC) testing procedures. The ...determination of radiochemical purity (RCP) is an essential QC test. A significant obstacle to RCP testing is the disruption of the secular equilibrium between actinium-225 and its daughter radionuclides during labeling and QC testing. In order to accelerate translation of actinium-225 targeted alpha therapy, we aimed to determine the earliest time point at which the RCP of an
225
Ac-labeled radiopharmaceutical can be accurately quantified.
Results
Six ligands were conjugated to macrocyclic metal chelators and labeled with actinium-225 under conditions designed to generate diverse incorporation yields. RCP was determined by radio thin layer chromatography (radioTLC) followed by exposure of the TLC plate on a phosphor screen either 0.5, 2, 3.5, 5, 6.5, or 26 h after the plate was developed. The dataset was used to create models for predicting the true RCP for any pre-equilibrium measurement taken at an early time point. The 585 TLC measurements span RCP values of 1.8–99.5%. The statistical model created from these data predicted an independent data set with high accuracy. Predictions made at 0.5 h are more uncertain than predictions made at later time points. This is primarily due to the decay of bismuth-213. A measurement of RCP > 90% at 2 h predicts a true RCP > 97% and guarantees that RCP will exceed 90% after secular equilibrium is reached. These findings were independently validated using NaI(Tl) scintillation counting and high resolution gamma spectroscopy on a smaller set of samples with 10% ≤ RCP ≤ 100%.
Conclusions
RCP of
225
Ac-labeled radiopharmaceuticals can be quantified with acceptable accuracy at least 2 h after radioTLC using various methods of quantifying particle emissions. This time point best balances the need to accurately quantify RCP with the need to safely release the batch as quickly as possible.
A good-manufacturing-practices (GMP) (68)Ge/(68)Ga generator that uses modified dodecyl-3,4,5-trihydroxybenzoate hydrophobically bound to a octadecyl silica resin (C-18) as an adsorbent has been ...developed that allows for dilute HCl (0.05N) to efficiently elute metal-impurity-free (68)Ga(3+) ready for peptide labeling. We characterized the performance of this generator system over a year in conjunction with the production of (68)Ga-labeled DOTATOC and Glu-NH-CO-NH-Lys(Ahx)-HBED-CC (PSMA-HBED-CC) intended for clinical studies and established protocols for batch release.
A 2,040-MBq self-shielded (68)Ge/(68)Ga generator provided metal-free (68)GaCl3 ready for peptide labeling in the fluidic labeling module after elution with 4 mL of 0.05N HCl. The compact system was readily housed in a laminar flow cabinet allowing an ISO class-5 environment. (68)Ga labeling of peptides using GMP kits was performed in 15-20 min, and the total production time was 45-50 min. Batch release quality control specifications were established to meet investigational new drug submission and institutional review board approval standards.
Over a period of 12 mo, (68)Ga elution yields from the generator averaged 80% (range, 72.0%-95.1%), and (68)Ge breakthrough was less than 0.006%, initially decreasing with time to 0.001% (expressed as percentage of (68)Ge activity present in the generator at the time of elution), a unique characteristic of this generator. The radiochemical purity of both (68)Ga-DOTATOC and (68)Ga-PSMA-HBED-CC determined by high-performance liquid chromatography analysis was greater than 98%, with a minimum specific activity of 12.6 and 42 GBq/μmol, respectively. The radionuclidic ((68)Ge) impurity was 0.00001% or less (under the detection limit). Final sterile, pyrogen-free formulation was provided in physiologic saline with 5%-7% ethanol.
The GMP-certified (68)Ge/(68)Ga generator system was studied for a year. The generator system is contained within the fluidic labeling module, and it is compact, self-shielded, and easy to operate using simple manual techniques. The system provides radiolabeled peptides with high (>98%) radiochemical purity and greater than 80% radiochemical yield. The (68)Ge levels in the final drug products were under the detection limits at all times. (68)Ga-DOTATOC and (68)Ga-PSMA-HBED-CC investigational radiopharmaceuticals are currently being studied clinically under investigational new drug (IND) applications submitted to the U.S. Food and Drug Administration.
Determining chemokine receptor CXCR4 expression is significant in multiple diseases due to its role in promoting inflammation, cell migration and tumorigenesis.
GaPentixafor is a promising ligand ...for imaging CXCR4 expression in multiple tumor types, but its utility is limited by the physical properties of
Ga. We screened a library of >200 fluorine-containing structural derivatives of AMD-3465 to identify promising candidates for in vivo imaging of CXCR4 expression by positron emission tomography (PET). Compounds containing fluoroethyltriazoles consistently achieved higher docking scores. Six of these higher scoring compounds were radiolabeled by click chemistry and evaluated in PC3-CXCR4 cells and BALB/c mice bearing bilateral PC3-WT and PC3-CXCR4 xenograft tumors. The apparent CXCR4 affinity of the ligands was relatively low, but tumor uptake was CXCR4-specific. The tumor uptake of
FRPS-534 (7.2 ± 0.3 %ID/g) and
FRPS-547 (3.1 ± 0.5 %ID/g) at 1 h p.i. was highest, leading to high tumor-to-blood, tumor-to-muscle, and tumor-to-lung ratios. Total cell-associated activity better predicted in vivo tumor uptake than did the docking score or apparent CXCR4 affinity. By this metric, and on the basis of their high yielding radiosynthesis, high tumor uptake, and good contrast to background,
FRPS-547, and especially
FRPS-534, are promising
F-labeled candidates for imaging CXCR4 expression.
Background
Here we report on the comprehensive quality control of a 4.04 GBq (109 mCi) generator supplied by itG (Munich, Germany), and used for routine production of
68
GaGa-PSMA-11 for clinical ...imaging. The performance of the 4.04 GBq itG
68
Ge/
68
Ga generator was studied for a year and parameters including elution yield, elution profile, radioactive and stable contaminants were collected. The production yields of a series of 175
68
GaGa-PSMA-11 clinical batches are also reported herein.
Results
This first-of-its-kind GMP grade
68
Ge/
68
Ga generator from itG with a nominal activity of 4.04 GBq (109 mCi) showed a stable
68
Ga elution profile with elution efficiency averaging 58.3 ± 3.7%.
68
Ge contaminant in the eluent slightly increased over time but remained 100x lower than those reported for comparable 1.85 GBq (50 mCi) itG generators. Metal impurities were found in concentrations lower than 100 ng/ml (ppb) throughout the study.
68
GaGa-PSMA-11 was obtained in 89 ± 4% radiochemical yields and > 99% radiochemical and chemical purities.
Conclusion
4.04 GBq (109 mCi) itG
68
Ge/
68
Ga generator is suitable for routinely produced
68
Ga tracers used in the clinic. Up to 30% higher amount of final drug product was obtained as compared to the 1.85 GBq (50 mCi) itG generator, and as a result larger number of studies could be performed, while reducing the synthetic burden.
Purpose
Current clinical imaging of PSMA-positive prostate cancer by positron emission tomography (PET) mainly features
68
Ga-labeled tracers, notably
68
GaGa-PSMA-HBED-CC. The longer half-life of ...fluorine-18 offers significant advantages over Ga-68, clinically and logistically. We aimed to develop high-affinity PSMA inhibitors labeled with fluorine-18 as alternative tracers for prostate cancer.
Methods
Six triazolylphenyl ureas and their alkyne precursors were synthesized from the Glu-urea-Lys PSMA binding moiety. PSMA affinity was determined in a competitive binding assay using LNCaP cells. The
18
Ftriazoles were isolated following a Cu(I)-catalyzed click reaction between the alkynes and
18
Ffluoroethylazide. The
18
F-labeled compounds were evaluated in nude mice bearing LNCaP tumors and compared to
68
GaGa-PSMA-HBED-CC and
18
FDCFPyL. Biodistribution studies of the two tracers with the highest imaged-derived tumor uptake and highest PSMA affinity were undertaken at 1 h, 2 h and 4 h post-injection (p.i.), and co-administration of PMPA was used to determine whether uptake was PSMA-specific.
Results
F-18-labeled triazolylphenyl ureas were prepared with a decay-corrected RCY of 20–40 %, >98 % radiochemical and chemical purity, and specific activity of up to 391 GBq/μmol. PSMA binding (IC
50
) ranged from 3–36 nM. The position of the triazole influenced tumor uptake (3 > 4 > 2), and direct conjugation of the triazole with the phenylurea moiety was preferred to insertion of a spacer group. Image-derived tumor uptake ranged from 6–14 %ID/g at 2 h p.i., the time of maximum tumor uptake; uptake of
68
GaGa-PSMA-HBED-CC and
18
FDCFPyL was 5–6 %ID/g at 1–3 h p.i., the time of maximum tumor uptake. Biodistribution studies of the two most promising compounds gave maximum tumor uptakes of 10.9 ± 1.0 % and 14.3 ± 2.5 %ID/g, respectively, as compared to 6.27 ± 1.44 %ID/g for
68
GaGa-PSMA-HBED-CC.
Conclusions
Six
18
Ftriazolylphenyl ureas were prepared in good radiochemical yield. Compounds showed PSMA-specific uptake in LNCaP tumors as high as 14 % ID/g, more than a 2-fold increase over
68
GaGa-PSMA-HBED-CC. The facile and high-yielding radiosynthesis of these
18
F-labeled triazoles as well as their promising in vitro and in vivo characteristics make them worthy of clinical development for PET imaging of prostate cancer.
Purpose
Treatment of late-stage prostate cancer by targeted radiotherapeutics such as
131
I-MIP-1095 and
177
Lu-PSMA-617 has shown encouraging early results. Lu-177 is preferred to I-131 in clinical ...settings, but targeted radioligand therapy (RLT) with
177
Lu-PSMA-617 has not reached its full potential due to insufficient dose delivery to the tumor. We recently developed a dual-targeting radioiodinated ligand, RPS-027, that targets PSMA and uses albumin binding to enable good tumor uptake and significantly reduced kidney uptake in a preclinical model. Further development of this ligand is limited by the inability to independently modify PSMA and albumin binding and the requirement of I-131 for therapeutic application. We therefore sought to devise a new class of trifunctional ligands for RLT with (1) a high-affinity PSMA-binding domain, (2) an albumin-binding group (ABG), and (3) a chelator for radiometals such as
68
Ga
3+
,
177
Lu
3+
and
225
Ac
3+
.
Methods
Ligands incorporating a triazolylphenylurea-containing PSMA-targeting group, an
N
ε
-(2-(4-iodophenyl)acetyl)lysine ABG and the bifunctional chelator
p
-SCN-Bn-DOTA linked by a PEG-containing polymer containing 0,3,4,6,8 or 12 repeats were prepared. PSMA affinity was determined in LNCaP cells and uptake and tissue distribution was studied in mice bearing LNCaP tumor xenografts and compared to
177
Lu-PSMA-617. Imaging studies were performed up to 24 h post-injection (p.i.) using
66
Ga
3+
and biodistribution studies at 4 h, 24 h and 96 h p.i. with
177
Lu
3+
.
Results
PSMA affinity was high (IC
50
= 1–10 nM) and inversely proportional to the linker length. Tumor uptake correlated with binding affinity and was significantly greater than for
177
Lu-PSMA-617 over 96 h. The highest uptake was achieved with
177
Lu-RPS-063 (30.0 ± 6.9 %ID/g; 4 h p.i.). Kidney uptake was generally high, with the exception of the lowest affinity ligand
177
Lu-RPS-067. Each of the compounds showed slower blood clearance than
177
Lu-PSMA-617, with clearance proportional to linker length.
Conclusions
The high tumor uptake achieved with these trifunctional ligands predicts larger (up to 4×) doses delivered to the tumor than can be achieved with
177
Lu-PSMA-617. Although PSMA-mediated kidney uptake was also observed, the exceptional area under the curve (AUC) in the tumor warrants further investigation of these novel ligands as candidates for RLT.
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