Salivary excretion of systemically injected [18F]DCFPyL in prostate cancer patients undergoing PSMA scans.

Introduction: Prostate-specific membrane antigen (PSMA) is present in high amounts in salivary glands, but it is unclear whether labeled binders of PSMA are excreted in the saliva. Methods: Ten patients with prostate cancer underwent whole-body [18F]DCFPyL PET/CT (NCT03181867), and saliva samples were collected between 0-120 minutes post-injection. [18F]DCFPyL salivary excretion was measured over 120 minutes and expressed as %ID/g. Protein-associated binding was estimated by the percentage of [18F]DCFPyL versus parent radiotracer. Results: All PET scans of 10 patients (69 ± 8 years) with histologically confirmed prostate cancer (PSA= 2.4 ± 2.4, and Gleason Grade = 6-9) showed high uptake of [18F]-DCFPyL in salivary glands while 8 patients demonstrated high uptake in the saliva at 45 minutes. The intact [18F]-DCFPyL (98%) was also confirmed in the saliva samples at 120 min with increasing salivary radioactivity between 30-120 min. Conclusion: Systemically injected [18F]DCFPyL shows salivary gland uptake, an increasing amount of which is secreted in saliva over time and is not maximized by 120 minutes post-injection. Although probably insignificant for diagnostic studies, patients undergoing PSMA-targeted therapies should be aware of radioactivity in saliva.

Correction: Tracking the Luminal Exposure and Lymphatic Drainage Pathways of Intravaginal and Intrarectal Inocula Used in Nonhuman Primate Models of HIV Transmission.

[This corrects the article DOI: 10.1371/journal.pone.0092830.].

Preclinical Imaging of Prostate Cancer.

Prostate cancer remains a major cause of mortality and morbidity, affecting millions of men, with a large percentage expected to develop the disease as they reach advanced ages. Treatment and management advances have been dramatic over the past 50 years or so, and one aspect of these improvements is reflected in the multiple advances in diagnostic imaging techniques. Much attention has been focused on molecular imaging techniques that offer high sensitivity and specificity and can now more accurately assess disease status and detect recurrence earlier. During development of molecular imaging probes, single-photon emission computed tomography (SPECT) and positron emission tomography (PET) must be evaluated in preclinical models of the disease. If such agents are to be translated to the clinic, where patients undergoing these imaging modalities are injected with a molecular imaging probe, these agents must first be approved by the FDA and other regulatory agencies prior to their adoption in clinical practice. Scientists have worked assiduously to develop preclinical models of prostate cancer that are relevant to the human disease to enable testing of these probes and related targeted drugs. Challenges in developing reproducible and robust models of human disease in animals are beset with practical issues such as the lack of natural occurrence of prostate cancer in mature male animals, the difficulty of initiating disease in immune-competent animals and the sheer size differences between humans and conveniently smaller animals such as rodents. Thus, compromises in what is ideal and what can be achieved have had to be made. The workhorse of preclinical animal models has been, and remains, the investigation of human xenograft tumor models in athymic immunocompromised mice. Later models have used other immunocompromised models as they have been found and developed, including the use of directly derived patient tumor tissues, completely immunocompromised mice, orthotopic methods for inducing prostate cancer within the mouse prostate itself and metastatic models of advanced disease. These models have been developed in close parallel with advances in imaging agent chemistries, radionuclide developments, computer electronics advances, radiometric dosimetry, biotechnologies, organoid technologies, advances in in vitro diagnostics, and overall deeper understandings of disease initiation, development, immunology, and genetics. The combination of molecular models of prostatic disease with radiometric-based studies in small animals will always remain spatially limited due to the inherent resolution sensitivity limits of PET and SPECT decay processes, fundamentally set at around a 0.5 cm resolution limit. Nevertheless, it is central to researcher's efforts and to successful clinical translation that the best animal models are adopted, accepted, and scientifically verified as part of this truly interdisciplinary approach to addressing this important disease.

Translating a radiolabeled imaging agent to the clinic.

Molecular Imaging is entering the most fruitful, exciting period in its history with many new agents under development, and several reaching the clinic in recent years. While it is unusual for just one laboratory to take an agent from initial discovery through to full clinical approval the steps along the way are important to understand for all interested participants even if one is not involved in the entire process. Here, we provide an overview of these processes beginning at discovery and preclinical validation of a new molecular imaging agent and using as an exemplar a low molecular weight disease-specific targeted positron emission tomography (PET) agent. Compared to standard drug development requirements, molecular imaging agents may benefit from a regulatory standpoint from their low mass administered doses, they nonetheless still need to go through a series of well-defined steps before they can be considered for Phase 1 human testing. After outlining the discovery and preclinical validation approaches, we will also discuss the nuances of Phase 1, Phase 2 and Phase 3 studies that may culminate in an FDA general use approval. Finally, some post-approval aspects of novel molecular imaging agents are considered.

Near-Infrared Photoimmunotherapy: Photoactivatable Antibody-Drug Conjugates (ADCs).

Cancer treatment has been founded traditionally on the three approaches of surgery, radiation, and chemotherapy with the latter recognized as the obvious systemic treatment approach applicable to disease that has spread. Although significant progress has been made over nearly 100 years of developing systemic treatments, it remains clear that use of the toxic agents involved is a two-edged sword with normal organ toxicities always needing to be balanced with and against administration of relevant therapeutic doses. With the advent of monoclonal antibodies targeted against tumor-associated antigens that could be used as carriers of potently toxic chemotherapy drugs, it was thought that such antibody-drug conjugates (ADCs) could engender the answer to the toxicity/therapeutic equation by shifting the equation more toward beneficial therapeutic efficacy. However, over 40 or so years, antibody-drug conjugates have not significantly affected the toxicity/therapy balance paradigm in most cancer indications, especially in solid tumors. Ideally, a further step may be required in that a non-tumor-targeted antibody-drug conjugate should be essentially nontoxic in its native administered form, with toxic effects unleashed only at the site of targeted tumors. A new approach that employs this principle is the use of an antibody-drug conjugate that is essentially nontoxic to normal tissues by virtue of requiring an extra step of light activation to become potent. We describe the preclinical data and first clinical results gained over the past few years by use of antibody-drug conjugates wherein the drug comprises a near-infrared photoactivatable dye delivered to tumors by a monoclonal antibody and is subsequently activated to a toxic entity solely at sites of tumors.

Ferumoxytol as an intraprostatic MR contrast agent for lymph node mapping of the prostate: a feasibility study in non-human primates.

Background A variety of magnetic resonance (MR) lymphographic agents have been proposed for mapping the lymph nodes draining the prostate. Purpose To investigate the feasibility of using ferumoxytol (an FDA-approved iron oxide agent) for lymph node mapping of the prostate on imaging (MRI) in a non-human primate (NHP) Macaque model. Material and Methods Four NHPs weighing 5-13 kg underwent injection of ferumoxytol after a needle was introduced transrectally under MRI guidance into the prostate using a commercially available intrarectal MRI biopsy guide. Ferumoxytol was administered at dosage in the range of 0.15-0.75 mg Fe/kg in a fixed injection volume of 0.2 mL. T1-weighted MRI was performed at 3 T starting immediately and extending at least 45 min post-injection. Two readers evaluated the images in consensus. The NHPs tolerated the ferumoxytol injections at all doses with no evident side effects. Results It was determined that the lowest dose of 0.15 mg Fe/kg produced the best outcome in terms of lymph node visualization and draining nodes were reliably visualized at this dose and volume. Conclusion Thus, MRI with intraprostatic injection of ferumoxytol may be considered an effective T1 contrast agent for prospective mapping of lymph nodes draining the prostate and, thus, for attempted sentinel lymph node identification in prostate cancer. Large clinical trials to determine safety and efficacy are needed.

A Phase I Dosing Study of Ferumoxytol for MR Lymphography at 3 T in Patients With Prostate Cancer.

OBJECTIVE: The objective of our study was to determine the optimal dose of ferumoxytol for performing MR lymphography (MRL) at 3 T in patients with prostate cancer. SUBJECTS AND METHODS: This phase I trial enrolled patients undergoing radical prostatectomy (RP) with bilateral pelvic lymph node dissection (PLND). Three groups of five patients each (total of 15 patients) received IV ferumoxytol before RP with bilateral PLND at each of the following doses of iron: 4, 6, and 7.5 mg Fe/kg. Patients underwent abdominopelvic MRI at 3 T before and 24 hours after ferumoxytol injection using T2- and T2*-weighted sequences. Normalized signal intensity (SI) and normalized SD changes from baseline to 24 hours after injection within visible lymph nodes were calculated for each dose level. Linear mixed effects models were used to estimate the effects of dose on the percentage SI change and log-transformed SD change within visible lymph nodes to determine the optimal dose of ferumoxytol for achieving uniform low SI in normal nodes. RESULTS: One patient who was excluded from the study group had a mild allergic reaction requiring treatment after approximately 2.5 mg Fe/kg ferumoxytol injection whereupon the injection was interrupted. The 15 study group patients tolerated ferumoxytol at all dose levels. The mean percentage SI change in 13 patients with no evidence of lymph metastasis was -36.4%, -45.4%, and -65.1% for 4, 6, and 7.5 mg Fe/kg doses, respectively (p = 0.041). CONCLUSION: A dose level of 7.5 mg Fe/kg ferumoxytol was safe and effective in deenhancing benign lymph nodes. This dose therefore can be the starting point for future phase II studies regarding the efficacy of ferumoxytol for MRL.

STAQ: A route toward low power, multicolor nanoscopy.

Nanoscopy has now become a real procedure in fluorescence microscopy of living cells. The STED/RESOLFT family of nanoscopy approaches has the best prospects for delivering high speed imaging, but the history of STED includes a continuing struggle to reduce the deactivation power applied, along with difficulties in achieving simultaneous multicolor images. In this manuscript, we present a concept for a similar real-time nanoscopy, using a new class of bipartite probes that separate the luminescent and quenching functions into two coupled molecules. In particular, the STAQ (Superresolution via Transiently Activated Quencher) example we show herein employs the excited state absorbance (not ground state) of the partner to accept energy from and quench the luminescent dye. The result is that much less deactivation power is needed for superresolved (∼50 nm) imaging. Moreover, the TAQ partner excited by the "donut" beam is shown to quench several different visible dyes via the same mechanism, opening the door to easier multicolor imaging. We demonstrate three dyes sharing the same deactivation and show examples of superresolved multicolor images. We suggest STAQ will facilitate the growth of real-time nanoscopy by reducing confounding photodamage within living cells while expanding the nanoscopist's palette.

Magnetic resonance sentinel lymph node imaging of the prostate with gadofosveset trisodium-albumin: preliminary results in a canine model.

RATIONALE AND OBJECTIVES: To determine if intraprostatic injection of gadofosveset trisodium mixed with human serum albumin (HSA) can identify sentinel lymph nodes (LNs) draining the prostate on magnetic resonance imaging (MRI) in a canine model. MATERIALS AND METHODS: Three male canines weighing between 25.7 and 41.3 kg were anesthetized, placed in a 3-T MRI, and a needle was placed transrectally into one side of the prostate using a commercially available intrarectal needle guide. Gadofosveset trisodium premixed with 10% HSA was then administered at doses ranging from 0.1 to 2.5 mL. T1W MRI was performed immediately after injection, and two readers evaluated images for visualization of LNs draining the prostate. RESULTS: Intraprostatic injection of 0.2 mL gadofosveset trisodium premixed with HSA identified the draining periprostatic LNs in all cases. Delayed images demonstrated upper echelon nodes in the pelvis and the abdomen. Higher volume injections resulted in excessive periprostatic extravasation, whereas lower volume injections resulted in suboptimal visualization of LNs. CONCLUSIONS: We demonstrate that gadofosveset trisodium (premixed with 10% HSA) injected intraprostatically at 0.2 mL visualized LNs draining the prostate. This approach can be readily adapted for clinical applications such as sentinel LN imaging in prostate cancer patients before surgery.

(89)Zr-Oxine Complex PET Cell Imaging in Monitoring Cell-based Therapies.

PURPOSE: To develop a clinically translatable method of cell labeling with zirconium 89 ((89)Zr) and oxine to track cells with positron emission tomography (PET) in mouse models of cell-based therapy. MATERIALS AND METHODS: This study was approved by the institutional animal care committee. (89)Zr-oxine complex was synthesized in an aqueous solution. Cell labeling conditions were optimized by using EL4 mouse lymphoma cells, and labeling efficiency was examined by using dendritic cells (DCs) (n = 4), naïve (n = 3) and activated (n = 3) cytotoxic T cells (CTLs), and natural killer (NK) (n = 4), bone marrow (n = 4), and EL4 (n = 4) cells. The effect of (89)Zr labeling on cell survival, proliferation, and function were evaluated by using DCs (n = 3) and CTLs (n = 3). Labeled DCs (444-555 kBq/[5 × 10(6)] cells, n = 5) and CTLs (185 kBq/[5 × 10(6)] cells, n = 3) transferred to mice were tracked with microPET/CT. In a melanoma immunotherapy model, tumor targeting and cytotoxic function of labeled CTLs were evaluated with imaging (248.5 kBq/[7.7 × 10(6)] cells, n = 4) and by measuring the tumor size (n = 6). Two-way analysis of variance was used to compare labeling conditions, the Wilcoxon test was used to assess cell survival and proliferation, and Holm-Sidak multiple tests were used to assess tumor growth and perform biodistribution analyses. RESULTS: (89)Zr-oxine complex was synthesized at a mean yield of 97.3% ± 2.8 (standard deviation). It readily labeled cells at room temperature or 4°C in phosphate-buffered saline (labeling efficiency range, 13.0%-43.9%) and was stably retained (83.5% ± 1.8 retention on day 5 in DCs). Labeling did not affect the viability of DCs and CTLs when compared with nonlabeled control mice (P > .05), nor did it affect functionality. (89)Zr-oxine complex enabled extended cell tracking for 7 days. Labeled tumor-specific CTLs accumulated in the tumor (4.6% on day 7) and induced tumor regression (P < .05 on day 7). CONCLUSION: We have developed a (89)Zr-oxine complex cell tracking technique for use with PET that is applicable to a broad range of cell types and could be a valuable tool with which to evaluate various cell-based therapies.

Preparation and long-term biodistribution studies of a PAMAM dendrimer G5-Gd-BnDOTA conjugate for lymphatic imaging.

AIMS: To demonstrate the use of gadolinium (Gd)-labeled dendrimers as lymphatic imaging agents and establish the long-term biodistribution (90-day) of this type of agent in mice. MATERIALS & METHODS: A G5-Gd-BnDOTA dendrimer was prepared and injected into mice and monkeys for MR lymphangiography, and long-term biodistribution of the conjugate was studied. RESULTS: Administration of G5-Gd-BnDOTA in mice demonstrated a rapid uptake in the deep lymphatic system while injection in monkeys showed enhanced internal iliac nodes, indicating its general utility for lymphatic tracking. Biodistribution studies to 90 days showed that gadolinium conjugate is slowly being eliminated from the liver and other organs. CONCLUSION: The use of G5-Gd-BnDOTA holds great promise for lymphatic imaging, but its slow clearance from the body might hamper its eventual clinical translation.

Synthesis of fluorine-18 radio-labeled serum albumins for PET blood pool imaging.

We sought to develop a practical, reproducible and clinically translatable method of radiolabeling serum albumins with fluorine-18 for use as a PET blood pool imaging agent in animals and man. Fluorine-18 radiolabeled fluoronicotinic acid-2,3,5,6-tetrafluorophenyl ester, [(18)F]F-Py-TFP was prepared first by the reaction of its quaternary ammonium triflate precursor with [(18)F]tetrabutylammonium fluoride ([(18)F]TBAF) according to a previously published method for peptides, with minor modifications. The incubation of [(18)F]F-Py-TFP with rat serum albumin (RSA) in phosphate buffer (pH9) for 15 min at 37-40 °C produced fluorine-18-radiolabeled RSA and the product was purified using a mini-PD MiniTrap G-25 column. The overall radiochemical yield of the reaction was 18-35% (n=30, uncorrected) in a 90-min synthesis. This procedure, repeated with human serum albumin (HSA), yielded similar results. Fluorine-18-radiolabeled RSA demonstrated prolonged blood retention (biological half-life of 4.8 hours) in healthy awake rats. The distribution of major organ radioactivity remained relatively unchanged during the 4 hour observation periods either by direct tissue counting or by dynamic PET whole-body imaging except for a gradual accumulation of labeled metabolic products in the bladder. This manual method for synthesizing radiolabeled serum albumins uses fluorine-18, a widely available PET radionuclide, and natural protein available in both pure and recombinant forms which could be scaled up for widespread clinical applications. These preclinical biodistribution and PET imaging results indicate that [(18)F]RSA is an effective blood pool imaging agent in rats and might, as [(18)F]HSA, prove similarly useful as a clinical imaging agent.

Immuno-PET imaging of tumor endothelial marker 8 (TEM8).

Tumor endothelial marker 8 (TEM8) is a cell surface receptor that is highly expressed in a variety of human tumors and promotes tumor angiogenesis and cell growth. Antibodies targeting TEM8 block tumor angiogenesis in a manner distinct from the VEGF receptor pathway. Development of a TEM8 imaging agent could aid in patient selection for specific antiangiogenic therapies and for response monitoring. In these studies, L2, a therapeutic anti-TEM8 monoclonal IgG antibody (L2mAb), was labeled with (89)Zr and evaluated in vitro and in vivo in TEM8 expressing cells and mouse xenografts (NCI-H460, DLD-1) as a potential TEM8 immuno-PET imaging agent. (89)Zr-df-L2mAb was synthesized using a desferioxamine-L2mAb conjugate (df-L2mAb); (125)I-L2mAb was labeled directly. In vitro binding studies were performed using human derived cell lines with high, moderate, and low/undetectable TEM8 expression. (89)Zr-df-L2mAb in vitro autoradiography studies and CD31 IHC staining were performed with cryosections from human tumor xenografts (NCI-H460, DLD-1, MKN-45, U87-MG, T-47D, and A-431). Confirmatory TEM8 Western blots were performed with the same tumor types and cells. (89)Zr-df-L2mAb biodistribution and PET imaging studies were performed in NCI-H460 and DLD-1 xenografts in nude mice. (125)I-L2mAb and (89)Zr-df-L2mAb exhibited specific and high affinity binding to TEM8 that was consistent with TEM8 expression levels. In NCI-H460 and DLD-1 mouse xenografts nontarget tissue uptake of (89)Zr-df-L2mAb was similar; the liver and spleen exhibited the highest uptake at all time points. (89)Zr-L2mAb was highly retained in NCI-H460 tumors with <10% losses from day 1 to day 3 with the highest tumor to muscle ratios (T:M) occurring at day 3. DLD-1 tumors exhibited similar pharmacokinetics, but tumor uptake and T:M ratios were reduced ∼2-fold in comparison to NCI-H460 at all time points. NCI-H460 and DLD-1 tumors were easily visualized in PET imaging studies despite low in vitro TEM8 expression in DLD-1 cells indicating that in vivo expression might be higher in DLD-1 tumors. From in vitro autoradiography studies (89)Zr-df-L2mAb specific binding was found in 6 tumor types (U87-MG, NCI-H460, T-47D MKN-45, A-431, and DLD-1) which highly correlated to vessel density (CD31 IHC). Westerns blots confirmed the presence of TEM8 in the 6 tumor types but found undetectable TEM8 levels in DLD-1 and MKN-45 cells. This data would indicate that TEM8 is associated with the tumor vasculature rather than the tumor tissue, thus explaining the increased TEM8 expression in DLD-1 tumors compared to DLD-1 cell cultures. (89)Zr-df-L2mAb specifically targeted TEM8 in vitro and in vivo although the in vitro expression was not necessarily predictive of in vivo expression which seemed to be associated with the tumor vasculature. In mouse models, (89)Zr-df-L2mAb tumor uptakes and T:M ratios were sufficient for visualization during PET imaging. These results would suggest that a TEM8 targeted PET imaging agent, such as (89)Zr-df-L2mAb, may have potential clinical, diagnostic, and prognostic applications by providing a quantitative measure of tumor angiogenesis and patient selection for future TEM8 directed therapies.

Simultaneous ECG-gated PET imaging of multiple mice.

INTRODUCTION: We describe and illustrate a method for creating ECG-gated PET images of the heart for each of several mice imaged at the same time. The method is intended to increase "throughput" in PET research studies of cardiac dynamics or to obtain information derived from such studies, e.g. tracer concentration in end-diastolic left ventricular blood. METHODS: An imaging bed with provisions for warming, anesthetic delivery, etc., was fabricated by 3D printing to allow simultaneous PET imaging of two side-by-side mice. After electrode attachment, tracer injection and placement of the animals in the scanner field of view, ECG signals from each animal were continuously analyzed and independent trigger markers generated whenever an R-wave was detected in each signal. PET image data were acquired in "list" mode and these trigger markers were inserted into this list along with the image data. Since each mouse is in a different spatial location in the FOV, sorting of these data using trigger markers first from one animal and then the other yields two independent and correctly formed ECG-gated image sequences that reflect the dynamical properties of the heart during an "average" cardiac cycle. RESULTS: The described method yields two independent ECG-gated image sequences that exhibit the expected properties in each animal, e.g. variation of the ventricular cavity volumes from maximum to minimum and back during the cardiac cycle in the processed animal with little or no variation in these volumes during the cardiac cycle in the unprocessed animal. CONCLUSION: ECG-gated image sequences for each of several animals can be created from a single list mode data collection using the described method. In principle, this method can be extended to more than two mice (or other animals) and to other forms of physiological gating, e.g. respiratory gating, when several subjects are imaged at the same time.

Inhibition of glutathione peroxidase mediates the collateral sensitivity of multidrug-resistant cells to tiopronin.

Multidrug resistance (MDR) is a major obstacle to the successful chemotherapy of cancer. MDR is often the result of overexpression of ATP-binding cassette transporters following chemotherapy. A common ATP-binding cassette transporter that is overexpressed in MDR cancer cells is P-glycoprotein, which actively effluxes drugs against a concentration gradient, producing an MDR phenotype. Collateral sensitivity (CS), a phenomenon of drug hypersensitivity, is defined as the ability of certain compounds to selectively target MDR cells, but not the drug-sensitive parent cells from which they were derived. The drug tiopronin has been previously shown to elicit CS. However, unlike other CS agents, the mechanism of action was not dependent on the expression of P-glycoprotein in MDR cells. We have determined that the CS activity of tiopronin is mediated by the generation of reactive oxygen species (ROS) and that CS can be reversed by a variety of ROS-scavenging compounds. Specifically, selective toxicity of tiopronin toward MDR cells is achieved by inhibition of glutathione peroxidase (GPx), and the mode of inhibition of GPx1 by tiopronin is shown in this report. Why MDR cells are particularly sensitive to ROS is discussed, as is the difficulty in exploiting this hypersensitivity to tiopronin in the clinic.

Tracking the luminal exposure and lymphatic drainage pathways of intravaginal and intrarectal inocula used in nonhuman primate models of HIV transmission.

Over 80% of sexual HIV-1 transmissions originate from a single viral variant, but the underlying basis for this transmission bottleneck remains to be elucidated. Nonhuman primate models of mucosal virus transmission allow opportunities to gain insight into the basis of this mucosal bottleneck. We used simulated inocula consisting of either non-infectious vital dye or contrast dye with non-invasive magnetic resonance imaging (MRI) to visualize mucosal exposure and passive lymphatic drainage patterns following vaginal and rectal exposures in Indian origin rhesus macaques. Results revealed a limited overall distance of dye coverage from the anal verge following 1 ml (n = 8) intrarectally administered, which greatly increased with a 3 ml (n = 8) volume. Intravaginal dye exposure using 2 ml revealed complete coverage of the mucosa of the vagina and ectocervix, however dye was not detectable in the endocervix, uterus, fallopian tubes or ovaries in nuliparous sexually mature rhesus macaques (n = 9). In addition, following submucosal and intranodal injections of vital dye or MRI contrast dye in the rectum (n = 9), or distal and proximal vagina (n = 4), the lymphatic drainage pathways were identified as first the internal then common iliac chain followed by para-aortic lymph nodes. Drainage from the distal descending colon (n = 8) was via the para-colonic lymph nodes followed by the inferior mesenteric and para-aortic lymph nodes. Analysis after vaginal challenge with infectious SIVmac239 followed by euthanasia at day 3 revealed a pattern of viral dissemination consistent with the imaging results. These results provide insights into potential patterns of viral dissemination that can help guide efforts to better elucidate the earliest events of virus transmission and potential intervention strategies.

Magnetic resonance lymphography of the thoracic duct after interstitial injection of gadofosveset trisodium: a pilot dosing study in a porcine model.

UNLABELLED: BACKGROUND-RATIONALE: To investigate whether interstitial injection of gadofosveset trisodium (Ablavar®, Lantheus Medical, North Billerica, MA) would be suitable for thoracic duct (TD) imaging in a pig model. METHODS AND RESULTS: Gadofosveset trisodium alone or premixed with 10% human serum albumin (HSA) was administered intradermally in the extremities of pigs at varying doses to visualize the TD by MRI. Two blinded readers evaluated MRIs for TD visibility. The inter-observer variability for all MR imaging sessions was assessed using the Spearman rank correlation test. MR lymphography using gadofosveset trisodium premixed with HSA yielded superior visualization of the TD compared to gadofosveset trisodium alone, with a high inter-observer agreement (correlation coefficient of 0.88 (p=0.00000115)). CONCLUSIONS: We demonstrate that gadofosveset trisodium (premixed with 10%HSA) can be injected intradermally in order to perform MR lymphography of the thoracic duct. Since this agent is already FDA approved for MR imaging, the off-label use of it for imaging of the thoracic duct in humans is feasible, and the approach may prove to be beneficial for patients with TD abnormalities.

Endosomolytic anionic polymer for the cytoplasmic delivery of siRNAs in localized in vivo applications.

The use of small interfering RNAs (siRNAs) to down-regulate the expression of disease-associated proteins carries significant promise for the treatment of a variety of clinical disorders. One of the main barriers to the widespread clinical use of siRNAs, however, is their entrapment and degradation within the endolysosomal pathway of target cells. Here we report the trafficking and function of PP75, a non-toxic, biodegradable, lipid membrane disruptive anionic polymer composed of phenylalanine derivatized poly(L-lysine iso-phthalamide). PP75 is readily endocytosed by cells, safely permeabilizes endolysosomes in a pH dependent manner and facilitates the transfer of co-endocytosed materials directly into the cytoplasm. The covalent attachment of siRNAs to PP75 using disulfide linkages generates conjugates that effectively traffic siRNAs to the cytoplasm of target cells both in vitro and in vivo. In a subcutaneous malignant glioma tumor model, a locally delivered PP75-stathmin siRNA conjugate decreases stathmin expression in tumor cells and, in combination with the nitrosourea chemotherapy carmustine, is highly effective at inhibiting tumor growth. PP75 may be clinically useful for the local delivery of siRNAs, in particular for the treatment of solid tumors.

Synthesis of ApoSense compound [18F]2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-(fluoromethyl)butanoic acid ([18F]NST732) by nucleophilic ring opening of an aziridine precursor.

INTRODUCTION: The small molecule 2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-(fluoromethyl)butanoic acid (NST732) is a member of the ApoSense family of compounds, capable of selective targeting, binding and accumulation within cells undergoing apoptotic cell death. It has application in molecular imaging and blood clotting particularly for monitoring antiapoptotic drug treatments. We are investigating a fluorine-18-radiolabeled analog of this compound for positron emission tomography studies. METHODS: We prepared the tosylate precursor methyl 2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-(tosyloxymethyl)butanoate (4) to synthesize fluorine-18-labeled NST732. Fluorination reaction of the tosylate precursor in 1:1 acetonitrile:dimethylsulfoxide with tetrabutyl ammonium fluoride proceeds through an aziridine intermediate (4A) to afford two regioisomers: 2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-fluorobutanoate (5) and methyl 2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-(fluoromethyl)butanoate (6). Acid hydrolysis of the fluoromethylbutanoate (6) isomer produced NST732. As the fluorination reaction of the tosylate precursor proceeds through an aziridine intermediate (4A) and the fluorination conceivably could be done directly on the aziridine, we have separately prepared an aziridine precursor (4A). Fluorine-18 labeling of the aziridine precursor (4A) was performed with [(18)F]tetrabutyl ammonium fluoride to afford the same two regioisomers (5 and 6). The [18F]2-((5-dimethylamino)naphthalene-1-sulfonamido)methyl)-2-fluorobutanoic acid (NST732) was then obtained by the hydrolysis of corresponding [18F]-labeled ester (6) with 6 N hydrochloric acid. RESULTS: Two regioisomers obtained from the fluorination reaction of aziridine were easily separated by high-performance liquid chromatography. The total radiochemical yield was 15%±3% (uncorrected, n=18) from the aziridine precursor in a 70-min synthesis time with a radiochemical purity>99%. CONCLUSION: Fluorine-18-labeled ApoSense compound [18F]NST732 is prepared in moderate yield by direct fluorination of an aziridine precursor.