Changes over the years in radiopharmaceutical design.

Of the many uses of radiopharmaceuticals, developing radiotracers that contribute significantly to diagnosis and therapy of patients has been a major focus. This requires a broad spectrum of expertise including that of the attending physician who lends insight to an unmet clinical need neither addressed by other imaging techniques nor by analysis of tissue, blood, and urine for diagnostics and addressed by pharmaceuticals for therapeutic applications. The design criteria have depended on radiochemistry, on matching the radiopharmaceutical with the imaging devices, and basing the design on current pharmaceuticals. The chelates of technetium-99m were based on radiochemistry rather than clinical need yet are still used today in >70% of the clinical studies. Targeted radiotracers in neurologic and psychiatric disorders, inflammation, cardiovascular disease, and oncology have all been studied with the goal of determining the change in the density of a target protein as a function of disease or treatment or, especially in oncology, detection of the total extent of disease. In the latter approach, PET in university settings leads the way; however, the use of SPECT/CT has increased the specificity of SPECT imaging to complement the cost-effective generator and instant kits already available. Remarkable advances have been achieved in radionuclide therapy using theragnostic agents, with the exclusive domain of oncology. For this application the design of radionuclide therapy follows that used for diagnostics. The increased impact of the discipline depends on the opportunity to continue the search for the most appropriate radiopharmaceutical for each individual patient.

[68Ga]Ga-P16-093 as a PSMA-Targeted PET Radiopharmaceutical for Detection of Cancer: Initial Evaluation and Comparison with [68Ga]Ga-PSMA-11 in Prostate Cancer Patients Presenting with Biochemical Recurrence.

PURPOSE: This study was undertaken to evaluate radiation dosimetry for the prostate-specific membrane antigen targeted [68Ga]Ga-P16-093 radiopharmaceutical, and to initially assess agent performance in positron emission tomography (PET) detection of the site of disease in prostate cancer patients presenting with biochemical recurrence. PROCEDURES: Under IND 133,222 and an IRB-approved research protocol, we evaluated the biodistribution and pharmacokinetics of [68Ga]Ga-P16-093 with serial PET imaging following intravenous administration to ten prostate cancer patients with biochemical recurrence. The recruited subjects were all patients in whom a recent [68Ga]Ga-PSMA-11 PET/X-ray computed tomography (CT) exam had been independently performed under IND 131,806 to assist in decision-making with regard to their clinical care. Voided urine was collected from each subject at ~ 60 min and ~ 140 min post-[68Ga]Ga-P16-093 injection and assayed for Ga-68 content. Following image segmentation to extract tissue time-activity curves and corresponding cumulated activity values, radiation dosimetry estimates were calculated using IDAC Dose 2.1. The prior [68Ga]Ga-PSMA-11 PET/CT exam (whole-body PET imaging at 60 min post-injection, performed with contrast-enhanced diagnostic CT) served as a reference scan for comparison to the [68Ga]Ga-P16-093 findings. RESULTS: [68Ga]Ga-P16-093 PET images at 60 min post-injection provided diagnostic information that appeared equivalent to the subject's prior [68Ga]Ga-PSMA-11 scan. With both radiopharmaceuticals, sites of tumor recurrence were found in eight of the ten patients, identifying 16 lesions. The site of recurrence was not detected with either agent for the other two subjects. Bladder activity was consistently lower with [68Ga]Ga-P16-093 than [68Ga]Ga-PSMA-11. The kidneys, spleen, salivary glands, and liver receive the highest radiation exposure from [68Ga]Ga-P16-093, with estimated doses of 1.7 × 10-1, 6.7 × 10-2, 6.5 × 10-2, and 5.6 × 10-2 mGy/MBq, respectively. The corresponding effective dose from [68Ga]Ga-P16-093 is 2.3 × 10-2 mSv/MBq. CONCLUSIONS: [68Ga]Ga-P16-093 provided diagnostic information that appeared equivalent to [68Ga]Ga-PSMA-11 in this limited series of ten prostate cancer patients presenting with biochemical recurrence, with the kidneys found to be the critical organ. Diminished tracer appearance in the urine represents a potential advantage of [68Ga]Ga-P16-093 over [68Ga]Ga-PSMA-11 for detection of lesions in the pelvis.

Chemistry and biology of radiotracers that target changes in sympathetic and parasympathetic nervous systems in heart disease.

Following the discovery of the sympathetic and parasympathetic nervous system, numerous adrenoceptor drugs were radiolabeled and potent radioligands were prepared in order to image the ß-adrenergic and the muscarinic systems. But the greatest effort has been in preparing noradrenaline analogs, such as norepinephrine, (11)C-metahydroxyephedrine, and (123)I-metaiodobenzylguanidine that measure cardiac sympathetic nerve varicosities. Given the technical and clinical challenges in designing and validating targeted adrenoceptor-binding radiotracers, namely the heavily weighted flow dependence and relatively low target-to-background ratio, both requiring complicated mathematic analysis, and the inability of targeted adrenoceptor radioligands to have an impact on clinical care of heart disease, the emphasis has been on radioligands monitoring the norepinephrine pathway. The chemistry and biology of such radiotracers, and the clinical and prognostic impact of these innervation imaging studies in patients with heart disease, are examined.

Rational development of radiopharmaceuticals for HIV-1.

The global battle against HIV-1 would benefit from a sensitive and specific radiopharmaceutical to localize HIV-infected cells. Ideally, this probe would be able to identify latently infected host cells containing replication competent HIV sequences. Clinical and research applications would include assessment of reservoirs, informing clinical management by facilitating assessment of burden of infection in different compartments, monitoring disease progression and monitoring response to therapy. A "rational" development approach could facilitate efficient identification of an appropriate targeted radiopharmaceutical. Rational development starts with understanding characteristics of the disease that can be effectively targeted and then engineering radiopharmaceuticals to hone in on an appropriate target, which in the case of HIV-1 (HIV) might be an HIV-specific product on or in the host cell, a differentially expressed gene product, an integrated DNA sequence specific enzymatic activity, part of the inflammatory response, or a combination of these. This is different from the current approach that starts with a radiopharmaceutical for a target associated with a disease, mostly from autopsy studies, without a strong rationale for the potential to impact patient care. At present, no targeted therapies are available for HIV latency, although a number of approaches are under study. Here we discuss requirements for a radiopharmaceutical useful in strategies targeting persistently infected cells. The radiopharmaceutical for HIV should be developed based on HIV biology, studied in an animal model and then in humans, and ultimately used in clinical and research settings.

Mood stabilizer treatment increases serotonin type 1A receptor binding in bipolar depression.

Abnormal serotonin type 1A (5-HT1A) receptor function and binding have been implicated in the pathophysiology of mood disorders. Preclinical studies have consistently shown that stress decreases the gene expression of 5-HT1A receptors in experimental animals, and that the associated increase in hormone secretion plays a crucial role in mediating this effect. Chronic administration of the mood stabilizers lithium and divalproex (valproate semisodium) reduces glucocorticoid signaling and function in the hippocampus. Lithium has further been shown to enhance 5-HT1A receptor function. To assess whether these effects translate to human subject with bipolar disorder (BD), positron emission tomography (PET) and [18F]trans-4-fluoro-N-(2-[4-(2-methoxyphenyl) piperazino]-ethyl)-N-(2-pyridyl) cyclohexanecarboxamide ([(18)F]FCWAY) were used to acquire PET images of 5-HT1A receptor binding in 10 subjects with BD, before and after treatment with lithium or divalproex. Mean 5-HT1A binding potential (BPP) significantly increased following mood stabilizer treatment, most prominently in the mesiotemporal cortex (hippocampus plus amygdala). When mood state was also controlled for, treatment was associated with increases in BPP in widespread cortical areas. These preliminary findings are consistent with the hypothesis that these mood stabilizers enhance 5-HT1A receptor expression in BD, which may underscore an important component of these agents' mechanism of action.

99mTc-labeled small-molecule inhibitors of prostate-specific membrane antigen for molecular imaging of prostate cancer.

UNLABELLED: Prostate-specific membrane antigen (PSMA) is highly expressed in prostate cancer, and small-molecule radiopharmaceuticals targeting PSMA rapidly detect the location and extent of disease. Here we evaluated preclinically 4 novel (99m)Tc-labeled small-molecule inhibitors of PSMA with the potential for clinical translation for molecular imaging of prostate cancer in humans. METHODS: Four PSMA inhibitors derived from the glutamate-urea-glutamate or glutamate-urea-lysine pharmacophores conjugated to CIM or TIM chelators were radiolabeled with (99m)Tc and evaluated in vitro and in vivo. RESULTS: High-affinity, saturable binding to PSMA on LNCaP cells was observed with Kd values of 0.64 ± 0.46 nM for (99m)Tc-MIP-1427, 1.07 ± 0.89 nM for (99m)Tc-MIP-1404, 1.75 ± 0.32 nM for (99m)Tc-MIP-1428, and 4.35 ± 0.35 nM for (99m)Tc-MIP-1405. (99m)Tc-labeled PSMA inhibitors did not bind human prostate cancer PC3 cells, which lack PSMA, demonstrating specificity, and binding was abolished with 2-(phosphonomethyl)pentanedioic acid (PMPA), a structurally unrelated PSMA inhibitor. (99m)Tc-labeled PSMA inhibitors were shown to internalize at 37 °C. Uptake in LNCaP xenografts ranged from 9.3% to 12.4% injected dose per gram at 1 h after injection and from 7.2% to 11.0% at 4 h, with tumor-to-blood ratios ranging from 29:1 to 550:1 and tumor-to-skeletal muscle ratios ranging from 31:1 to 157:1 at 4 h. (99m)Tc-MIP-1404 exhibited the best combination of high tumor uptake and rapid clearance from kidney and nontarget tissues. (99m)Tc-MIP-1404 specifically bound to PSMA in vivo as demonstrated by the absence of uptake in PC3 xenografts and by competition with PMPA. SPECT/CT imaging corroborated the tissue distribution results, demonstrating uptake only in PSMA-expressing kidney and tumor tissue and clearance through the urinary bladder. CONCLUSION: These (99m)Tc-labeled radiopharmaceuticals targeting PSMA may provide a SPECT molecular imaging option to assist in the initial diagnosis of prostate cancer and the management of patient care by monitoring disease progression.

Progress using Tc-99m radiopharmaceuticals for measuring high capacity sites and low density sites.

Technetium-99m (Tc-99m) has long been a mainstay in clinical nuclear medicine, primarily monitoring biological processes in the heart, kidney, liver, and brain. More recently, Tc-99m chelates have been used as the reporter in targeted nuclear medicine probes that monitor changes in specific protein expression products. The strengths remain the inexpensive source of Tc-99m from the Mo-99/Tc-99m generator, its rich chemistry, high-yield kit formulation, and its widespread availability. Hardware and software advances, such as OSEM reconstructions with scatter and attenuation corrections, have led to quantitation of the injected radioactivity in terms of kBq/cm.

Reduced post-synaptic serotonin type 1A receptor binding in bipolar depression.

Multiple lines of evidence suggest that serotonin type 1A (5-HT(1A)) receptor dysfunction is involved in the pathophysiology of mood disorders, and that alterations in 5-HT(1A) receptor function play a role in the mechanisms of antidepressant and mood stabilizer treatment. The literature is in disagreement, however, as to whether 5-HT(1A) receptor binding abnormalities exist in bipolar disorder (BD). We acquired PET images of 5-HT(1A) receptor binding in 26 unmedicated BD subjects and 37 healthy controls using [¹8F]FCWAY, a highly selective 5-HT(1A) receptor radio-ligand. The mean 5-HT(1A) receptor binding potential (BP(P)) was significantly lower in BD subjects compared to controls in cortical regions where 5-HT(1A) receptors are expressed post-synaptically, most prominently in the mesiotemporal cortex. Post-hoc assessments involving other receptor specific binding parameters suggested that this difference particularly affected the females with BD. The mean BPP did not differ between groups in the raphe nucleus, however, where 5-HT(1A) receptors are predominantly expressed pre-synaptically. Across subjects the BPP in the mesiotemporal cortex was inversely correlated with trough plasma cortisol levels, consistent with preclinical literature indicating that hippocampal 5-HT(1A) receptor expression is inhibited by glucocorticoid receptor stimulation. These findings suggest that 5-HT(1A) receptor binding is abnormally reduced in BD, and this abnormality may particularly involve the postsynaptic 5-HT(1A) receptor system of individuals with a tendency toward cortisol hypersecretion.

Synthesis and SAR of 99mTc/Re-labeled small molecule prostate specific membrane antigen inhibitors with novel polar chelates.

Prostate specific membrane antigen (PSMA) is recognized as an attractive molecular target for the development of radiopharmaceuticals to image and potentially treat metastatic prostate cancer. A series of novel (99m)Tc/Re-tricarbonyl radiolabeled PSMA inhibitors were therefore synthesized by the attachment of glutamate-urea-lysine (Glu-urea-Lys) and glutamate-urea-glutamate (Glu-urea-Glu) pharmacophore to single amino acid chelate (SAAC) where the SAAC ligand was either bis(pyridin-2-ylmethyl)amino (DPA), bis((1-methyl-1H-imidazol-2-yl)methyl)amino (NMI), bis((1-(carboxymethyl)-1H-imidazol-2-yl)methyl)amino (CIM) or bis((1-(2-(bis(carboxymethyl)amino)-2-oxoethyl)-1H-imidazol-2-yl)methyl)amino (TIM). The in vitro binding affinity of the rhenium complexes was evaluated using PSMA-expressing human prostate cancer LNCaP cells. IC(50) values ranged from 3.8 ± 2 to >2000 nM. A linker between the SAAC chelate and pharmacophore was required for high affinity binding. However, extending the length of the linker did not substantially improve binding. PSMA binding was also influenced by the nature of the SAAC chelate. One of the most potent compounds, 23b (IC(50)=4.8 ± 2.7 nM), was radiolabeled with technetium tricarbonyl ({(99m)Tc(CO)(3)}(+)) to afford the {(99m)Tc(CO)(3)}(+) complex in excellent yield and high purity. This effort has led to the identification of a diverse series of promising high affinity {(99m)Tc(CO)(3)}(+) radiolabeled PSMA inhibitors.

First-in-man evaluation of 2 high-affinity PSMA-avid small molecules for imaging prostate cancer.

UNLABELLED: This phase 1 study was performed to determine the pharmacokinetics and ability to visualize prostate cancer in bone, soft-tissue, and the prostate gland using (123)I-MIP-1072 and (123)I-MIP-1095, novel radiolabeled small molecules targeting prostate-specific membrane antigen. METHODS: Seven patients with a documented history of prostate cancer by histopathology or radiologic evidence of metastatic disease were intravenously administered 370 MBq (10 mCi) of (123)I-MIP-1072 and (123)I-MIP-1095 2 wk apart in a crossover trial design. (123)I-MIP-1072 was also studied in 6 healthy volunteers. Whole-body planar and SPECT/CT imaging was performed and pharmacokinetics studied over 2-3 d. Target-to-background ratios were calculated. Absorbed radiation doses were estimated using OLINDA/EXM. RESULTS: (123)I-MIP-1072 and (123)I-MIP-1095 visualized lesions in soft tissue, bone, and the prostate gland within 0.5-1 h after injection, with retention beyond 48 h. Target-to-background ratios from planar images averaged 2:1 at 1 h, 3:1 at 4-24 h, and greater than 10:1 at 4 and 24 h for SPECT/CT. Both agents cleared the blood in a biphasic manner; clearance of (123)I-MIP-1072 was approximately 5 times faster. (123)I-MIP-1072 was excreted in the urine, with 54% and 74% present by 24 and 72 h, respectively. In contrast, only 7% and 20% of (123)I-MIP-1095 had been renally excreted by 24 and 72 h, respectively. Estimated absorbed radiation doses were 0.054 versus 0.110 mGy/MBq for the kidneys and 0.024 versus 0.058 mGy/MBq for the liver, for (123)I-MIP-1072 and (123)I-MIP-1095, respectively. CONCLUSION: (123)I-MIP-1072 and (123)I-MIP-1095 detect lesions in soft tissue, bone, and the prostate gland at as early as 1-4 h. These novel radiolabeled small molecules have excellent pharmacokinetic and pharmacodynamic profiles and warrant further development as diagnostic and potentially when labeled with (131)I therapeutic radiopharmaceuticals.

Synthesis and SAR of novel Re/99mTc-labeled benzenesulfonamide carbonic anhydrase IX inhibitors for molecular imaging of tumor hypoxia.

Carbonic anhydrase IX (CA-IX) is upregulated in cancer in response to the hypoxic tumor microenvironment, making it an attractive molecular target for the detection of hypoxic solid tumors. A series of small molecule benzenesulfonamide based CA-IX inhibitors containing novel tridentate chelates complexed with the M(CO)(3) core (M = Re or (99m)Tc) were designed and synthesized. The in vitro binding affinity of the benzenesulfonamide rhenium complexes yielded IC(50) values ranging from 3 to 116 nM in hypoxic CA-IX expressing HeLa cells. One of the most potent compounds, 3d (IC(50) = 9 nM), was radiolabeled with technetium tricarbonyl ({(99m)Tc(CO)(3)}(+)) to afford the {(99m)Tc(CO)(3)}(+) complex in excellent yield and high purity. (99m)Tc(CO)(3)-3d bound specifically to CA-IX expressing hypoxic HeLa cells. This effort led to the identification of a diverse series of promising high affinity {(99m)Tc(CO)(3)}(+) radiolabeled CA-IX inhibitors with the potential to significantly impact diagnosis, staging, and treatment selection of hypoxic solid tumors.

The synthesis and in vivo pharmacokinetics of fluorinated arachidonic acid: implications for imaging neuroinflammation.

UNLABELLED: Arachidonic acid (AA) is found in high concentrations in brain phospholipids and is released as a second messenger during neurotransmission and much more so during neuroinflammation and excitotoxicity. Upregulated brain AA metabolism associated with neuroinflammation has been imaged in rodents using [1-(14)C]AA and with PET in Alzheimer disease patients using [1-(11)C]AA. Radiotracer brain AA uptake is independent of cerebral blood flow, making it an ideal tracer despite altered brain functional activity. However, the 20.4-min radioactive half-life of (11)C-AA and challenges of routinely synthesizing (11)C fatty acids limit their translational utility as PET biomarkers. METHODS: As a first step to develop a clinically useful (18)F-fluoroarachidonic acid ((18)F-FAA) with a long radioactive half-life of 109.8 min, we report here a high-yield stereoselective synthetic method of nonradioactive 20-(19)F-FAA. We tested its in vivo pharmacokinetics by infusing purified nonradioactive (19)F-FAA intravenously for 5 min at 2 doses in unanesthetized mice and measured its plasma and brain distribution using gas chromatography-mass spectrometry. RESULTS: Incorporation coefficients of injected (19)F-FAA into brain phospholipids (ratio of brain (19)F-FAA concentration to plasma input function) were 3- to 29-fold higher for choline glycerophospholipid and phosphatidylinositol than for ethanolamine glycerophospholipid and phosphatidylserine at each of the 2 tested doses. The selectivities and values of incorporation coefficients were comparable to those reported after [1-(14)C]AA (the natural arachidonate) infusion in mice. CONCLUSION: These results suggest that it would be worthwhile to translate our stereoselective synthetic method for (19)F-FAA to synthesize positron-emitting (18)F-FAA for human brain AA metabolism in neuroinflammatory disorders such as Alzheimer disease.

Essential parameters to consider for the characterization of optical imaging probes.

The Molecular Imaging and Contrast Agents Database (MICAD) was launched in 2005 to promote the development and application of imaging and contrast agents (probes) to advance the field of molecular imaging. As of March 2012, there are approximately 1170 agents available in MICAD. Based on the modality used for imaging, the largest category of probes described in MICAD are those used for PET (41.6%), followed by agents used for single-photon emission computed tomography (30.3%), optical imaging (12.0%), MRI (9.3%), multimodality imaging (3.4%), ultrasound (2.4%) and x-ray/computed tomography (1.0%). This article is intended to be a guideline for new investigators and students who wish to characterize an optical imaging probe that will be used to perform in vivo molecular imaging studies. It is necessary, however, to ensure that these agents meet certain quality control parameters before they are used in various in vitro and in vivo applications.

A receptor-targeted fluorescent radiopharmaceutical for multireporter sentinel lymph node imaging.

PURPOSE: To determine the imaging and receptor-binding properties of a multireporter probe designed for sentinel lymph node (SLN) mapping via nuclear and fluorescence detection. MATERIALS AND METHODS: The animal experiments were approved by the institutional animal care and use committee. A multireporter probe was synthesized by covalently attaching cyanine 7 (Cy7), a near-infrared cyanine dye, to tilmanocept, a radiopharmaceutical that binds to a receptor specific to recticuloendothelial cells. In vitro binding assays of technetium 99m (99mTc)-labeled Cy7 tilmanocept were conducted at 4°C by using receptor-bearing macrophages. Optical SLN imaging after foot pad administration was performed by using two molar doses of Cy7 tilmanocept. Six mice were injected with 0.11 nmol of 99mTc-labeled Cy7 tilmanocept (low-dose group); an additional six mice were injected with 31 nmol of 99mTc-labeled Cy7 tilmanocept (high-dose group) to saturate the receptor sites within the SLN. After 2.5 hours of imaging, the mice were euthanized, and the sentinel and distal lymph nodes were excised and assayed for radioactivity for calculation of SLN percentage of injected dose and extraction. Four mice were used as controls for autofluorescence. Standard optical imaging software was used to plot integrated fluorescence intensity against time for calculation of the SLN uptake rate constant and scaled peak intensity. Significance was calculated by using the Student t test. RESULTS: In vitro binding assays showed subnanomolar affinity (mean dissociation constant, 0.25 nmol/L±0.10 [standard deviation]). Fluorescence imaging showed a detection sensitivity of 1.6×10(3) counts·sec(-1)·µW(-1) per picomole of Cy7. All four imaging metrics (percentage of injected dose, SLN extraction, SLN uptake rate constant, and expected peak fluorescence intensity) exhibited higher values (P=.005 to P=.042) in the low-dose group than in the high-dose group; this finding was consistent with receptor-mediated image formation. CONCLUSION: The multireporter probe 99mTc-labeled Cy7 tilmanocept exhibits in vitro and in vivo receptor-binding properties for successful receptor-targeted SLN mapping with nuclear and optical imaging.