Examination of Charge Modifications of an Endolysosomal Trapping Inhibitor in an Antagonistic NTSR1-Targeted Construct for Colon Cancer.

Many low-molecular weight targeted radiotherapeutics (TRTs) are capable of rapidly achieving exceptional tumor to non-target ratios shortly after administration. However, the low tumor residence time of many TRTs limits therapeutic dose delivery and has become the Achilles heel to their clinical translation. To combat the tumor efflux of these otherwise promising agents, we have previously presented a strategy of equipping low-molecular weight TRTs with irreversible cysteine cathepsin inhibitors (e.g., E-64 analogues). These inhibitors are capable of forming irreversible adducts with cysteine proteases within the endolysosomal compartments of cells. Using these endolysosomal trapping agents (ETs), the receptor-targeted constructs are able to increase tumor retention and, thus, deliverable therapeutic doses. In this study, we examine this approach in the development of agents targeting the neurotensin receptor subtype 1 (NTSR1), a receptor overexpressed in numerous cancers. Using an antagonistic NTSR1-targeting vector, we explore the impact of charge modification of the ETs on the in vitro and in vivo biological performance of the constructs using HT-29 colon cancer models. Four ETs (based on the epoxysuccinyl peptide E-64) with various charge states were synthesized and incorporated into the structures of the NTSR1-targeted antagonist. These four 177Lu-labeled, ET-enhanced, NTSR1-targeted agents (177Lu-NA-ET1-4), along with the structurally analogous 177Lu-3BP-227, currently in clinical trials, underwent a battery of in vitro assays using HT-29 xenograft colon cancer cells to examine their NTSR1 binding, internalization and efflux, inhibition, and adduct formation properties. The biodistribution profile of these constructs was studied in an HT-29 mouse model. Charge modification of the terminal carboxylic acid and arginine of the ETs had deleterious effects on inhibition kinetics and in vitro adduct formation. Contrastingly, deletion of the arginine resulted in a modest increase in inhibition kinetics. Incorporation of ETs into the NTSR1-targeted agents was well-tolerated with minimal impact on the in vivo NTSR1 targeting but resulted in increased renal uptake. This study demonstrates that the ETs can be successfully incorporated into antagonistic NTSR1-targeted constructs without compromising their adduct formation capabilities. Based on these results, further exploration of the endolysosomal trapping approach is warranted in NTSR1- and other receptor-targeted antagonistic constructs.

Access to Highly Strained Tricyclic Ketals Derived from Coumarins.

Synthesis of highly strained fused substituted dihydrobenzopyran cyclopropyl lactones derived from coumarin carboxylates are reported. The substrate scope tolerates a variety of 6- and 8-substituents on the coumarin ring. Substitution at the 5- or 7-position is resistant to tricyclic lactone formation except with 7-methyl substitution. Benzamide-containing coumarins afford the tricyclic ketal. A plausible mechanism is proposed for the formation of the fused lactone: intramolecular rearrangement of trans cyclopropyl methyl ketones with phenolic acetate via the formation of a hemiacetal.

Tricyclic Imidazolidin-4-ones by Witkop Oxidation of Tetrahydro-ß-carbolines.

1-Substituted and 1,1-disubstituted tetrahydro-ß-carbolines undergo sodium periodate oxidative ring expansion in the presence of formaldehyde and other aldehydes to form 5,6-dihydro-7H-1,4-methanobenzo[e][1,4]diazonine-2,7(3H)-diones in 30-81% yield. In most cases, the reaction to form this new 6/8/5-tricyclic ring system proceeds with high diastereoselectivity. These benzannulated medium-ring keto imidazolidin-4-ones expand the menu of tetrahydro-ß-carboline oxidation products.

In Vitro Evaluation and Biodistribution Studies of HPMA Copolymers Targeting the Gastrin Releasing Peptide Receptor in Prostate Cancer.

PURPOSE: The development of diagnostic and therapeutic agents utilizing small peptides (e.g., bombesin (BBN)) to target the overexpression of the gastrin-releasing peptide receptor (GRPR) in cancers has been widely investigated. Herein, we examine the capabilities of BBN-modified HPMA copolymers to target the GRPR. METHODS: Four positive, four negative, and two zwitterionic BBN HPMA copolymer conjugates of varying peptide content and charge were synthesized. In vitro and in vivo studies were conducted in a GRPR-overexpressing prostate cancer cell line (PC-3) and a normal CF-1 mouse model, respectively. RESULTS: Cellular uptake of the conjugates were found to be charge and BBN density dependent. The positively-charged conjugates illustrated a direct relationship between the extent of cellular internalization, ranging from 0.7 to 20%, and BBN-incorporation density. The negative and zwitterionic conjugates showed low PC-3 uptake values. Blocking studies confirmed the GRPR-targeting effect of the positively-charged constructs. In vivo studies of the positively-charged copolymers resulted in rapid blood clearance by the mononuclear phagocyte system (MPS)-associated tissues (e.g., liver and spleen). CONCLUSION: Positively-charged BBN-HPMA copolymer conjugates demonstrated good GRPR-targeting and internalization in vitro. However, the impact of peptide density and charge on in vivo MPS recognition are parameters that must be optimized in future agent development.

Investigation into the Biological Impact of Block Size on Cathepsin S-Degradable HPMA Copolymers.

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers have been studied as an efficient carrier for drug delivery and tumor imaging. However, as with many macromolecular platforms, the substantial accumulation of HPMA copolymer by the mononuclear phagocyte system (MPS)-associated tissues, such as the blood, liver, and spleen, has inhibited its clinical translation. Our laboratory is pursuing approaches to improve the diagnostic and radiotherapeutic effectiveness of HPMA copolymers by reducing the nontarget accumulation. Specifically, we have been investigating the use of a cathepsin S (Cat S)-cleavable peptidic linkers to degrade multiblock HPMA copolymers to increase MPS-associated tissue clearance. In this study, we further our investigation into this area by exploring the impact of copolymer block size on the biological performance of Cat S-degradable HPMA copolymers. Using a variety of in vitro and in vivo techniques, including dual labeling of the copolymer and peptide components, we investigated the constructs using HPAC pancreatic ductal adenocarcinoma models. The smaller copolymer block size (S-CMP) demonstrated significantly faster Cat S cleavage kinetics relative to the larger system (L-CMP). Confocal microscopy demonstrated that both constructs could be much more efficiently internalized by human monocyte-differentiated macrophage (hMDM) compared to HPAC cells. In the biodistribution studies, the multiblock copolymers with a smaller block size exhibited faster clearance and lower nontarget retention while still achieving good tumor targeting and retention. Based on the radioisotopic ratios, fragmentation and clearance of the copolymer constructs were higher in the liver compared to the spleen and tumor. Overall, these results indicate that block size plays an important role in the biological performance of Cat S-degradable polymeric constructs.

Development of hypoxia enhanced 111In-labeled Bombesin conjugates: design, synthesis, and in vitro evaluation in PC-3 human prostate cancer.

The gastrin-releasing peptide receptor (BB2r) has shown great promise for tumor targeting due to the increase of the receptor expression in a variety of human cancers including prostate, breast, small-cell lung, and pancreatic cancer. From clinical investigations, prostate cancer has been shown to be among the most hypoxic of the cancers investigated. Many solid tumors contain regions of hypoxia due to poor organization and efficiency of the vasculature. However, hypoxia is typically not present in normal tissue. Nitroimidazoles, a thoroughly investigated class of hypoxia selective drugs, have been shown to be highly retained in hypoxic tissues. The purpose of this study is to determine if the incorporation of hypoxia trapping moieties into the structural paradigm of BB2r-targeted peptides will increase the retention time of the agents in prostate cancer tumors. The present work involves the design, syntheses, purification, and in vitro investigation of hypoxia enhanced (111)In-BB2r-targeted radioconjugates. A total of four BB2r-targeted conjugates (1-4) were synthesized and coupled with increasing numbers of 2-nitroimidazoles, a hypoxia trapping moiety. Conjugates were radiolabeled with (111)In and purified by HPLC prior to in vitro studies. Receptor saturation assays under both normoxic and hypoxic conditions showed that the BB2r receptor expression on the PC-3 human prostate cancer cell line was not significantly affected by oxygen levels. Competitive binding assays revealed that incorporation of 2-nitroimidazoles had a detrimental effect to BB2r binding when adequate spacer groups, between the hypoxia trapping agent and the pharmacophore, were not employed. All of the 2-nitroimidazole containing BB2r-targeted agents exhibited significantly higher longitudinal retention in PC-3 cells under hypoxic conditions compared to the analogous normoxic studies. Protein association analysis revealed a 3-fold increase in binding of a 2-nitroimidazole containing BB2r-targeted agent under hypoxic relative to normoxic conditions. The positive nature of these results indicate that further exploration into the potential of hypoxia selective trapping agents for BB2r-targeted agents, as well as other targeted compounds, is warranted.

Examination of the impact molecular charge has on NTSR1-targeted agents incorporated with cysteine protease inhibitors.

Our laboratory has previously reported a strategy of employing cysteine cathepsin (CC) inhibitors as adduct forming, trapping agents to extend the tumor residence time of neurotensin receptor subtype 1 (NTSR1)-targeted radiopharmaceuticals. As a follow-up, we herein report a small library of CC trapping agent (CCTA)-incorporated, NTSR1-targeted conjugates with structural modifications that reduce the number of charged functional groups for both the CCTA and the peptide targeting sequence. These modifications were pursued to reduce the renal uptake and increase the translational potential of the CCTA-incorporated, NTSR1-targeted agents as radiotherapeutics. The biological performance of these constructs was examined using a battery of in vitro and in vivo studies employing the NTSR1-positive HT-29 human colon cancer cell line as our model. In vitro studies confirmed the ability of these constructs to target the NTSR1 and efficiently form intracellular adducts with cysteine proteases. Biodistribution studies using an HT-29 xenograft mouse model revealed that truncation (removal of Lys6-Pro7) of the NTSR1-targeted peptide (177Lu-NE2a) had the greatest (3.7-fold) effect at lowering renal recognition/uptake relative to our previously reported construct. Other charge-reducing modifications to the CCTA resulted in unexpected increases in renal uptake. All of the constructs demonstrated similar levels of in vivo NTSR1-positive tumor targeting with the highest tumor residualization resulting from the construct containing the zwitterionic CCTA (177Lu-NE2a). In vivo adduct formation of the conjugates was confirmed using autoradiographic SDS-PAGE analysis.

In Vitro and In Vivo Evaluation of DTPA-HPMA Copolymers as Potential Decorporating Agents for Prophylactic Therapy of Actinide Contamination.

The release of actinides into the environment represents a significant potential public health concern. Chelation therapy utilizing diethylenetriamine pentaacetate (DTPA) is a U.S. Food and Drug Administration (FDA)-approved therapy capable of mitigating the deposition of some absorbed actinides in the body. However, the pharmacokinetic profile of DTPA is not ideal for prophylactic applications. In this study, we examine the incorporation of DTPA into a HPMA copolymer (P-DTPA) to investigate if the enhanced blood circulation time can offer superior prophylactic protection and of improving in vivo radiometal decorporation. Utilizing lutetium-177 (177Lu) as an actinide model, the performance of P-DTPA and DTPA (control) were evaluated using selectivity studies in the presence of competing biological metals, chelation and stability assays in human serum and cytotoxicity studies using human umbilical vein endothelial cells (HUVEC). The in vivo decorporation efficiency of P-DTPA relative to DTPA and untreated controls was also evaluated over two weeks in CF-1 mice. In the experimental groups, the mice were prophylactically treated with P-DTPA or DTPA (30 µmol/kg) 6 or 24 h prior to 177LuCl3 administration. The in vitro results reveal that P-DTPA gives efficient complexation yields relative to DTPA with a tolerable cytotoxicity profile and good serum stability. The in vivo decorporation studies demonstrated enhanced total excretion of the 177Lu using P-DTPA compared to DTPA in both the 6 and 24 h prophylactic treatment study arms. This enhanced decorporation effect is certainly attributable to the expected prolonged biological half-life of DTPA when grafted to the HPMA polymer.

Structure activity relationship (SAR) study identifies a quinoxaline urea analog that modulates IKKß phosphorylation for pancreatic cancer therapy.

Genetic models validated Inhibitor of nuclear factor (NF) kappa B kinase beta (IKKß) as a therapeutic target for KRAS mutation associated pancreatic cancer. Phosphorylation of the activation loop serine residues (S177, S181) in IKKß is a key event that drives tumor necrosis factor (TNF) α induced NF-κB mediated gene expression. Here we conducted structure activity relationship (SAR) study to improve potency and oral bioavailability of a quinoxaline analog 13-197 that was previously reported as a NFκB inhibitor for pancreatic cancer therapy. The SAR led to the identification of a novel quinoxaline urea analog 84 that reduced the levels of p-IKKß in dose- and time-dependent studies. When compared to 13-197, analog 84 was ∼2.5-fold more potent in TNFα-induced NFκB inhibition and ∼4-fold more potent in inhibiting pancreatic cancer cell growth. Analog 84 exhibited ∼4.3-fold greater exposure (AUC0-∞) resulting in ∼5.7-fold increase in oral bioavailability (%F) when compared to 13-197. Importantly, oral administration of 84 by itself and in combination of gemcitabine reduced p-IKKß levels and inhibited pancreatic tumor growth in a xenograft model.

Coordination chemistry of mercury(ii) halide complexes: a combined experimental, theoretical and (ICSD & CSD) database study on the relationship between inorganic and organic units.

From the viewpoint of inorganic crystal engineering (ICE), the coordination sphere of the metal centre can be affected by two main parts of inorganic and organic units in complexes. Database study can play a significant role in the explanation of the relationship between various parameters related to these parts. For the first time, we have investigated this relationship through the concomitant studies of the inorganic crystal structure database (ICSD) and the cambridge structural database (CSD) for mercury(ii) halide compounds. The results of CSD analysis are divided into two categories of metal halide complexes (MHC or mercury halide compounds with ligands) and metal halide only (MHO or mercury halide compounds without ligands). MHC (970, 460, and 521 metal centres as HgCl2, HgBr2, and HgI2, respectively) and MHO (419, 141, and 201 metal centres as HgCl2, HgBr2, and HgI2, respectively) were structurally investigated. The coordination number, polymerization mode, coordination geometry of the metal centre, type of donor atom in ligands, and the chelation mode of the ligand for all MHC and MHO compounds were extracted as effective factors in inorganic and organic units. To rationalize the effect of ICE in the design of the coordination sphere, eleven new mercury halide complexes, including the ester ligands of L1, naphthalene-5-yl nicotinate (complexes 1-3), L2, naphthalene-6-yl nicotinate (complexes 4-6), L3, naphthalene-5-yl pyrazine-2-carboxylate (complexes 7-9), and L4, naphthalene-6-yl pyrazine-2-carboxylate (complexes 10-11) were synthesized and fully characterized. The various parameters of substitution, C-H to nitrogen replacement, counteranion, and symmetry effects were investigated for all of the complexes. The results show that there is a meaningful relationship between inorganic and organic units. According to the findings of the CSD and ICSD analyses, most of the complexes obeyed the same relationship. Despite the predominant role of the inorganic unit in determining the coordination geometry, the organic unit can also change the coordination sphere of complexes with one major effect or the cooperativity of minor effects.

Development of Improved Tumor-Residualizing, GRPR-Targeted Agents: Preclinical Comparison of an Endolysosomal Trapping Approach in Agonistic and Antagonistic Constructs.

Receptor-targeted radiopharmaceuticals based on low-molecular-weight carriers offer many clinically advantageous attributes relative to macromolecules but have generally been hampered by their rapid clearance from tumors, thus diminishing tumor-to-nontarget tissue ratios. Herein, we present a strategy using irreversible inhibitors (E-64 derivative) of cysteine cathepsins (CCs) as trapping agents to increase the tumor retention of receptor-targeted agents. Methods: We incorporated these CC-trapping agents into agonistic and antagonistic pharmacophores targeting the gastrin-releasing peptide receptor (GRPR). The synthesized radioconjugates with either an incorporated CC inhibitor or a matching control were examined using in vitro and in vivo models of the GRPR-positive, PC-3 human prostate cancer cell line. Results: From the in vitro studies, multiple techniques confirmed that the CC-trapping, GRPR-targeted constructs were able to increase cellular retention by forming intracellular macromolecule adducts. In PC-3 tumor-bearing xenograft mice, the CC-trapping, GRPR-targeted agonistic and antagonistic constructs led to an approximately 2-fold increase in tumor retention with a corresponding improvement in most tumor-to-nontarget tissue ratios over 72 h. Conclusion: CC endolysosomal trapping provides a pathway to increase the efficacy and clinical potential of low-molecular-weight, receptor-targeted agents.

Enhanced tumor retention of NTSR1-targeted agents by employing a hydrophilic cysteine cathepsin inhibitor.

We explored the approach of using an analog of E-64, a well-known and hydrophilic cysteine cathepsin (CC) inhibitor, as a potent cysteine cathepsin-trapping agent (CCTA) to improve the tumor retention of low-molecular-weight, receptor-targeted radiopharmaceuticals. The synthesized hydrophilic CCTA-incorporated, NTSR1-targeted agents demonstrated a substantial increase in cellular retention upon uptake into the NTRS1-positive HT-29 human colon cancer cell line. Similarly, biodistribution studies using HT-29 xenograft mice revealed a significant and substantial increase in tumor retention for the CCTA-incorporated, NTSR1-targeted agent. The intracellular trapping mechanism of the CCTA-incorporated agents by macromolecular adduct formation was confirmed using multiple in vitro and in vivo techniques. Furthermore, utilization of the more hydrophilic CCTA greatly increased the hydrophilicity of the resulting NTSR1-targeted constructs leading to substantial decreases in most non-target tissues in contrast to our previously reported dipeptidyl acyloxymethyl ketone (AOMK) constructs. This work further confirms that the CCTA trapping approach can make significant improvements in the clinical potential of NTSR1-and other receptor-targeted radiopharmaceuticals.

Comparative Study of Subcutaneous and Orthotopic Mouse Models of Prostate Cancer: Vascular Perfusion, Vasculature Density, Hypoxic Burden and BB2r-Targeting Efficacy.

The gastrin-releasing peptide receptor (BB2r) is overexpressed in a variety of cancers including prostate cancer. As a consequence, the development of BB2r-targeted diagnostic/therapeutic radiopharmaceuticals has been widely explored. Both subcutaneous and orthotopic mouse models have been extensively used in BB2r-targeted agent development, but side-by-side studies examining how biological parameters (tumor perfusion efficacy, hypoxic burden and microvasculature density) impact BB2r-targeted agent delivery has not been reported. Herein, we examine these biological parameters using subcutaneous and orthotopic PC-3 xenografts. Using a dual isotope biodistribution study, tumor perfusion was accessed using [99mTc]NaTcO4 and BB2r-targeted uptake evaluated by utilization of a novel 177Lu-labeled conjugate ([177Lu]Lu-DOTA-SP714). Immunofluorescence, immunohistochemistry and autoradiography were utilized to examine the tumor vascular density, hypoxic burden and microdistribution of the BB2r-targeted agent. Our studies demonstrated that compared to the subcutaneous model the PC-3 orthotopic tumors had significantly higher levels of perfusion that led to higher BB2r-targeted uptake and lower levels of hypoxia burden. It is anticipated that our results will allow researchers to better understand the biological variables affecting drug delivery and assist them in more clearly interpreting their results in this common prostate cancer mouse model.

Evaluation of the pharmacokinetic effects of various linking group using the 111In-DOTA-X-BBN(7-14)NH2 structural paradigm in a prostate cancer model.

The high incidence of BB2 receptor (BB2r) expression in various cancers has prompted investigators to pursue the development of BB2r-targeted agents for diagnostic imaging, chemotherapy, and radiotherapy. Development of BB2r-targeted agents, based on the bombesin (BBN) peptide, has largely involved the use of the bifunctional chelate approach in which the linking group serves several key roles including pharmacokinetic modification. Understanding the in vivo properties of the various pharmacokinetic modifying linking groups is crucial for developing BB2r-targeted agents with improved targeting and clearance characteristics. The goal of this study was to systematically evaluate the pharmacokinetic profile of aliphatic hydrocarbon, aromatic, and poly(ethylene glycol) (ether) functional groups in order to obtain a better understanding of the in vivo properties of these pharmacokinetic modifiers. Specifically, we synthesized six radioconjugates with the structure 111In-DOTA- X-BBN(7-14)NH2, where X = 8-aminooctanoic acid (8-AOC), 5-amino-3-oxapentyl-succinamic acid (5-ADS), 8-amino-3,6-dioxaoctyl-succinamic acid (8-AOS), p-aminobenzoic acid (AMBA), Gly-AMBA, and Gly- p-aminomethylbenzoic acid (Gly-AM2BA). All of the (nat)In-conjugates demonstrated nanomolar binding affinities to the BB2r. In CF-1 mice, the BB2r uptake in the pancreas of radioconjugates containing aromatic linking groups was found to be significantly higher at 1 h postinjection than the radioconjugates with ether linker moieties. For PC-3 tumor-bearing SCID mice, the tumor uptake was found to be 6.66 +/- 2.00, 6.21 +/- 1.57, 6.36 +/- 1.60, 4.46 +/- 0.81, and 7.76 +/- 1.19 %ID/g for the 8-AOC, 8-ADS, AMBA, Gly-AMBA, and Gly-AM2BA radioconjugates, respectively, at 15 min postinjection. By 24 h postinjection, the radioconjugates containing aromatic groups exhibited the highest percentage tumor retention with 11.4%, 19.8%, 26.6%, 25.8%, and 25.5% relative to the 15 min values remaining in the tumor tissue for the 8-AOC, 8-ADS, AMBA, Gly-AMBA, and Gly-AM2BA radioconjugates, respectively. Fused Micro-SPECT/CT imaging studies performed at 24 h postinjection revealed substantial accumulation of radioactivity in the tumor tissue for all radioconjugates. In both biodistribution and Micro-SPECT/CT imaging studies, the radioconjugates containing aromatic linking groups typically exhibited significantly higher G.I. tract retention than the hydrocarbon or ether linking moieties. In conclusion, our studies indicate that radioconjugates incorporating aromatic linking groups, of the type investigated, generally demonstrated enhanced retention in BB2r expressing tissues in comparison to either the hydrocarbon or ether linking moieties. Furthermore, this investigation clearly demonstrates the significance of the linking group upon not only the in vivo clearance of the radiopharmaceutical, but also on the in vivo uptake and retention of the BB2r-targeted agent in tumor tissue. Future designs of BB2r-targeted agents should include a careful consideration of the effect linking group functionality has upon tumor targeting and retention.

Design, synthesis, and biological evaluation of an antagonist-bombesin analogue as targeting vector.

The gastrin releasing peptide receptor (GRP-R) is overexpressed on a number of tumors and cancer cell lines including pancreas, prostate, breast, gastrointestinal, and small cell lung cancer (SCLC). Radiolabeled bombesin (BBN) analogues have exhibited high binding affinity and specificity to the GRP-R. A bombesin analogue with an antagonist targeting vector at the C-terminus, DOTA-aminohexanoyl-[D-Phe(6), Leu-NHCH 2CH 2CH3(13), des Met(14)] BBN[6-14] (1, "Bomproamide"), has been synthesized and displays high binding affinity (IC50 = 1.36 +/- 0.09 nM) against (125)I-Tyr (4)-BBN in in vitro competitive assays using PC-3 cells. Maximum internalization of (111)In-1 reached 14% in PC-3 cells after 45 min of incubation. Rapid (0.25 h PI) and high (12.21 +/- 3.2%ID/g) pancreatic uptake of (111)In-1 was observed in healthy CF-1 mice, and 90% of the activity was blocked by coinjection of 100 mug of BBN. Rapid (0.25 h PI) and high uptake (6.90 +/- 1.06%ID/g) was observed in PC-3 prostate cancer xenografts in SCID mice, as well as visualized clearly in a SPECT/CT study. These results support the use of a bombesin construct with an antagonist C-terminal vector as a candidate of choice for specific in vivo imaging of tumors overexpressing GRP-receptors.

Increasing time on target: utilization of inhibitors of cysteine cathepsins to enhance the tumor retention of receptor-targeted agents.

We report a strategy of utilizing irreversible cysteine cathepsin inhibitor as trapping agent to increase the tumor residence time of receptor-targeted agents. The targeted constructs incorporating these cysteine cathepsin trapping agents were able to form high molecular weight adducts with intracellular cysteine cathepsins, thus achieving superior retention in tumor tissues.

Formation of 2-Imino Benzo[e]-1,3-oxazin-4-ones from Reactions of Salicylic Acids and Anilines with HATU: Mechanistic and Synthetic Studies.

We describe a new 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU)-mediated coupling reaction to produce 2-imino benzo[e]-1,3-oxazin-4-ones from salicylic acids and anilines. Mechanistic studies support a reaction pathway in which HATU mediates carbon transfer to the initially formed salicylanilides to form in succession reactive tetramethylisouronium and N-acyl(dimethyl)isouronium intermediates, which then undergo imine-iminium exchange to generate the desired oxazinones.

Bioimaging predictors of rilpivirine biodistribution and antiretroviral activities.

Antiretroviral therapy (ART) has changed the outcome of human immunodeficiency virus type one (HIV-1) infection from certain death to a life free of disease co-morbidities. However, infected people must remain on life-long daily ART. ART reduces but fails to eliminate the viral reservoir. In order to improve upon current treatment regimens, our laboratory created long acting slow effective release (LASER) ART nanoformulated prodrugs from native medicines. LASER ART enables antiretroviral drugs (ARVs) to better reach target sites of HIV-1 infection while, at the same time, improve ART's half-life and potency. However, novel ARV design has been slowed by prolonged pharmacokinetic testing requirements. To such ends, tri-modal theranostic nanoparticles were created with single-photon emission computed tomography (SPECT/CT), magnetic resonance imaging (MRI) and fluorescence capabilities to predict LASER ART biodistribution. The created theranostic ARV probes were then employed to monitor drug tissue distribution and potency. Intrinsically 111Indium (111In) radiolabeled, europium doped cobalt-ferrite particles and rilpivirine were encased in a polycaprolactone core surrounded by a lipid shell (111InEuCF-RPV). Particle cell and tissue distribution, and antiretroviral activities were sustained in macrophage tissue depots. 111InEuCF-PCL/RPV particles injected into mice demonstrated co-registration of MRI and SPECT/CT tissue signals with RPV and cobalt. Cell and animal particle biodistribution paralleled ARV activities. We posit that particle selection can predict RPV distribution and potency facilitated by multifunctional theranostic nanoparticles.

In vivo evaluation and small-animal PET/CT of a prostate cancer mouse model using 64Cu bombesin analogs: side-by-side comparison of the CB-TE2A and DOTA chelation systems.

UNLABELLED: The BB2 receptor subtype, of the bombesin family of receptors, has been shown to be highly overexpressed in a variety of human tumors, including prostate cancer. Bombesin (BBN), a 14-amino acid peptide, has been shown to target the BB2 receptor with high affinity. 64Cu (half-life = 12.7 h, beta+: 18%, E(beta+ max) = 653 keV; beta-: 37%, E(beta- max) = 578 keV) is a radioisotope that has clinical potential for application in both diagnostic imaging and radionuclide therapy. Recently, new chelation systems such as 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A) have been reported to significantly stabilize the 64Cu radiometal in vivo. The increased stability of the 64Cu-CB-TE2A chelate complex has been shown to significantly reduce nontarget retention compared with tetraazamacrocycles such as 1,4,7,10-tetraazacyclodoadecane-N,N',N'',N'''-tetraacetic acid (DOTA). The aim of this study was to determine whether the CB-TE2A chelation system could significantly improve the in vivo stability of 64Cu bombesin analogs. The study directly compares 64Cu bombesin analogs using the CB-TE2A and DOTA chelation systems in a prostate cancer xenograft SCID (severely compromised immunodeficient) mouse model. METHODS: The CB-TE2A-8-AOC-BBN(7-14)NH2 and DOTA-8-AOC-BBN(7-14)NH2 conjugates were synthesized and radiolabeled with 64Cu. The receptor-binding affinity and internalization profile of each metallated conjugate was evaluated using PC-3 cells. Pharmacokinetic and small-animal PET/CT studies were performed using female SCID mice bearing PC-3 xenografts. RESULTS: In vivo BB2 receptor targeting was confirmed by tumor uptake values of 6.95 +/- 2.27 and 4.95 +/- 0.91 %ID/g (percentage injected dose per gram) at the 15-min time point for the 64Cu-CB-TE2A and 64Cu-DOTA radioconjugates, respectively. At the 24-h time point, liver uptake was substantially reduced for the 64Cu-CB-TE2A radioconjugate (0.21 +/- 0.06 %ID/g) compared with the 64Cu-DOTA radioconjugate (7.80 +/- 1.51 %ID/g). The 64Cu-CB-TE2A-8-AOC-BBN(7-14)NH2 radioconjugate demonstrated significant clearance, 98.60 +/- 0.28 %ID, from the mouse at 24 h after injection. In contrast, only 67.84 +/- 5.43 %ID of the 64Cu activity was excreted using the 64Cu-DOTA-8-AOC-BBN(7-14)NH2 radioconjugate because of nontarget retention. CONCLUSION: The pharmacokinetic and small-animal PET/CT studies demonstrate significantly improved nontarget tissue clearance for the 64Cu-CB-TE2A8-AOC-BBN(7-14)NH2. This is attributed to the improved in vivo stability of the 64Cu-CB-TE2A chelate complex as compared with the 64Cu-DOTA chelate complex.

Structure-Activity Relationship Studies with Tetrahydroquinoline Analogs as EPAC Inhibitors.

EPAC proteins are therapeutic targets for the potential treatment of cardiac hypertrophy and cancer metastasis. Several laboratories use a tetrahydroquinoline analog, CE3F4, to dissect the role of EPAC1 in various disease states. Here, we report SAR studies with tetrahydroquinoline analogs that explore various functional groups. The most potent EPAC inhibitor 12a exists as a mixture of inseparable E (major) and Z (minor) rotamers. The rotation about the N-formyl group indeed impacts the activity against EPAC.