Structure-Activity Relationship of (18)F-Labeled Phosphoramidate Peptidomimetic Prostate-Specific Membrane Antigen (PSMA)-Targeted Inhibitor Analogues for PET Imaging of Prostate Cancer.

A series of phosphoramidate-based prostate specific membrane antigen (PSMA) inhibitors of increasing lipophilicity were synthesized (4, 5, and 6), and their fluorine-18 analogs were evaluated for use as positron emission tomography (PET) imaging agents for prostate cancer. To gain insight into their modes of binding, they were also cocrystallized with the extracellular domain of PSMA. All analogs exhibited irreversible binding to PSMA with IC50 values ranging from 0.4 to 1.3 nM. In vitro assays showed binding and rapid internalization (80-95%, 2 h) of the radiolabeled ligands in PSMA(+) cells. In vivo distribution demonstrated significant uptake in CWR22Rv1 (PSMA(+)) tumor, with tumor to blood ratios of 25.6:1, 63.6:1, and 69.6:1 for [(18)F]4, [(18)F]5, and [(18)F]6, respectively, at 2 h postinjection. Installation of aminohexanoic acid (AH) linkers in the phosphoramidate scaffold improved their PSMA binding and inhibition and was critical for achieving suitable in vivo imaging properties, positioning [(18)F]5 and [(18)F]6 as favorable candidates for future prostate cancer imaging clinical trials.

A high-affinity [(18)F]-labeled phosphoramidate peptidomimetic PSMA-targeted inhibitor for PET imaging of prostate cancer.

INTRODUCTION: In this study, a structurally modified phosphoramidate scaffold, with improved prostate-specific membrane antigen (PSMA) avidity, stability and in vivo characteristics, as a PET imaging agent for prostate cancer (PCa), was prepared and evaluated. METHODS: p-Fluorobenzoyl-aminohexanoate and 2-(3-hydroxypropyl)glycine were introduced into the PSMA-targeting scaffold yielding phosphoramidate 5. X-ray crystallography was performed on the PSMA/5 complex. [(18)F]5 was synthesized, and cell uptake and internalization studies were conducted in PSMA(+) LNCaP and CWR22Rv1 cells and PSMA(-) PC-3 cells. In vivo PET imaging and biodistribution studies were performed at 1 and 4 h post injection in mice bearing CWR22Rv1 tumor, with or without blocking agent. RESULTS: The crystallographic data showed interaction of the p-fluorobenzoyl group with an arene-binding cleft on the PSMA surface. In vitro studies revealed elevated uptake of [(18)F]5 in PSMA(+) cells (2.2% in CWR22Rv1 and 12.1% in LNCaP) compared to PSMA(-) cells (0.08%) at 4 h. In vivo tumor uptake of 2.33% ID/g and tumor-to-blood ratio of 265:1 was observed at 4 h. CONCLUSIONS: We have successfully synthesized, radiolabeled and evaluated a new PSMA-targeted PET agent. The crystal structure of the PSMA/5 complex highlighted the interactions within the arene-binding cleft contributing to the overall complex stability. The high target uptake and rapid non-target clearance exhibited by [(18)F]5 in PSMA(+) xenografts substantiates its potential use for PET imaging of PCa. ADVANCES IN KNOWLEDGE: The only FDA-approved imaging agent for PCa, Prostascint®, targets PSMA but suffers from inherent shortcomings. The data acquired in this manuscript confirmed that our new generation of [(18)F]-labeled PSMA inhibitor exhibited promising in vivo performance as a PET imaging agent for PCa and is well-positioned for subsequent clinical trials. Implications for Patient Care Our preliminary data demonstrate that this tracer possesses the required imaging characteristics to be sensitive and specific for PCa imaging in patients at all stages of the disease.

Framework selection can influence pharmacokinetics of a humanized therapeutic antibody through differences in molecule charge.

Pharmacokinetic (PK) testing of a humanized (κI, VH3 framework) and affinity matured anti-hepatitis C virus E2-glycoprotein (HCV-E2) antibody (hu5B3.κ1VH3.v3) in rats revealed unexpected fast clearance (34.9 mL/day/kg). This antibody binds to the rat recycling receptor FcRn as expected for a human IgG1 antibody and does not display non-specific binding to baculovirus particles in an assay that is correlated with fast clearance in cynomolgus monkey. The antigen is not expressed in rat so target-dependent clearance does not contribute to PK. Removal of the affinity maturation changes (hu5B3.κ1VH3.v1) did not restore normal clearance. The antibody was re-humanized on a κ4, VH1 framework and the non-affinity matured version (hu5B3.κ4VH1.v1) was shown to have normal clearance (8.5 mL/day/kg). Since the change in framework results in a lower pI, primarily due to more negative charge on the κ4 template, the effect of additional charge variation on antibody PK was tested by incorporating substitutions obtained through phage display affinity maturation of hu5B3.κ1VH3.v1. A variant having a pI of 8.61 gave very fast clearance (140 mL/day/kg) whereas a molecule with pI of 6.10 gave slow clearance (5.8 mL/kg/day). Both antibodies exhibited comparable binding to rat FcRn, but biodistribution experiments showed that the high pI variant was catabolized in liver and spleen. These results suggest antibody charge can have an effect on PK through alterations in antibody catabolism independent of FcRn-mediated recycling. Furthermore, introduction of affinity maturation changes into the lower pI framework yielded a candidate with PK and virus neutralization properties suitable for clinical development.

Comparative physiology of mice and rats: radiometric measurement of vascular parameters in rodent tissues.

A solid understanding of physiology is beneficial in optimizing drug delivery and in the development of clinically predictive models of drug disposition kinetics. Although an abundance of data exists in the literature, it is often confounded by the use of various experimental methods and a lack of consensus in values from different sources. To help address this deficiency, we sought to directly compare three important vascular parameters at the tissue level using the same experimental approach in both mice and rats. Interstitial volume, vascular volume, and blood flow were radiometrically measured in selected harvested tissues of both species by extracellular marker infusion, red blood cell labeling, and rubidium chloride bolus distribution, respectively. The latter two parameters were further compared by whole-body autoradiographic imaging. An overall good interspecies agreement was observed for interstitial volume and blood flow on a weight-normalized basis in most tissues. In contrast, the measured vascular volumes of most rat tissues were higher than for mouse. Mice and rats, the two most commonly utilized rodent species in translational drug development, should not be considered as interchangeable in terms of vascular volume per gram of tissue. This will be particularly critical in biodistribution studies of drugs, as the amount of drug in the residual blood of tissues is often not negligible, especially for biologic drugs (e.g., antibodies) having long circulation half-lives. Physiologically based models of drug pharmacokinetics and/or pharmacodynamics also rely on accurate knowledge of biological parameters in tissues. For tissue parameters with poor interspecies agreement, the significance and possible drivers are discussed.

Iron accumulation during cellular senescence.

Iron accumulates as a function of age and is associated with the pathology of numerous age-related diseases. These changes may be caused by altered iron homeostasis at the cellular level, yet this is poorly understood. Therefore, changes in iron content in primary human fibroblasts were studied in culture models of cellular senescence. Total iron content increased exponentially during cellular senescence, reaching approximately 10-fold higher levels than young cells. Increasing intracellular iron levels through iron-citrate supplementation or decreasing intracellular iron levels using iron-selective chelators had little effect on cellular life span and markers of cellular senescence when used at subtoxic doses. However, accelerating cellular senescence with low-dose H(2)O(2) also accelerated senescence-associated iron accumulation. Delaying cellular senescence with N-tert-butyl-hydroxylamine (NtBHA) attenuated senescence-associated iron accumulation. Furthermore, H(2)O(2) or NtBHA had no effect on iron intracellular levels in immortalized fibroblasts. Thus, iron accumulation is not a cause, but a consequence of normal cellular senescence in vitro. Senescence-associated iron accumulation may contribute to the increased oxidative stress and cellular dysfunction seen in senescent cells.