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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Renal iron accumulation occurs in lupus nephritis and iron chelation delays the onset of albuminuria.

Proteins involved in iron homeostasis have been identified as biomarkers for lupus nephritis, a serious complication of systemic lupus erythematosus (SLE). We tested the hypothesis that renal iron accumulation occurs and contributes to renal injury in SLE. Renal non-heme iron levels were increased in the (New Zealand Black x New Zealand White) F1 (NZB/W) mouse model of lupus nephritis compared with healthy New Zealand White (NZW) mice in an age- and strain-dependent manner. Biodistribution studies revealed increased transferrin-bound iron accumulation in the kidneys of albuminuric NZB/W mice, but no difference in the accumulation of non-transferrin bound iron or ferritin. Transferrin excretion was significantly increased in albuminuric NZB/W mice, indicating enhanced tubular exposure and potential for enhanced tubular uptake following filtration. Expression of transferrin receptor and 24p3R were reduced in tubules from NZB/W compared to NZW mice, while ferroportin expression was unchanged and ferritin expression increased, consistent with increased iron accumulation and compensatory downregulation of uptake pathways. Treatment of NZB/W mice with the iron chelator deferiprone significantly delayed the onset of albuminuria and reduced blood urea nitrogen concentrations. Together, these findings suggest that pathological changes in renal iron homeostasis occurs in lupus nephritis, contributing to the development of kidney injury.

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.

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.

Cathepsin S-cleavable, multi-block HPMA copolymers for improved SPECT/CT imaging of pancreatic cancer.

This work continues our efforts to improve the diagnostic and radiotherapeutic effectiveness of nanomedicine platforms by developing approaches to reduce the non-target accumulation of these agents. Herein, we developed multi-block HPMA copolymers with backbones that are susceptible to cleavage by cathepsin S, a protease that is abundantly expressed in tissues of the mononuclear phagocyte system (MPS). Specifically, a bis-thiol terminated HPMA telechelic copolymer containing 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. Three maleimide modified linkers with different sequences, including cathepsin S degradable oligopeptide, scramble oligopeptide and oligo ethylene glycol, were subsequently synthesized and used for the extension of the HPMA copolymers by thiol-maleimide click chemistry. All multi-block HPMA copolymers could be labeled by (177)Lu with high labeling efficiency and exhibited high serum stability. In vitro cleavage studies demonstrated highly selective and efficient cathepsin S mediated cleavage of the cathepsin S-susceptible multi-block HPMA copolymer. A modified multi-block HPMA copolymer series capable of Förster Resonance Energy Transfer (FRET) was utilized to investigate the rate of cleavage of the multi-block HPMA copolymers in monocyte-derived macrophages. Confocal imaging and flow cytometry studies revealed substantially higher rates of cleavage for the multi-block HPMA copolymers containing the cathepsin S-susceptible linker. The efficacy of the cathepsin S-cleavable multi-block HPMA copolymer was further examined using an in vivo model of pancreatic ductal adenocarcinoma. Based on the biodistribution and SPECT/CT studies, the copolymer extended with the cathepsin S susceptible linker exhibited significantly faster clearance and lower non-target retention without compromising tumor targeting. Overall, these results indicate that exploitation of the cathepsin S activity in MPS tissues can be utilized to substantially lower non-target accumulation, suggesting this is a promising approach for the development of diagnostic and radiotherapeutic nanomedicine platforms.

Liver-targeted antiviral peptide nanocomplexes as potential anti-HCV therapeutics.

Great success in HCV therapy was achieved by the development of direct-acting antivirals (DAA). However, the unsolved issues such as high cost and genotype dependency drive us to pursue additional therapeutic agents to be used instead or in combination with DAA. The cationic peptide p41 is one of such candidates displaying submicromolar anti-HCV potency. By electrostatic coupling of p41 with anionic poly(amino acid)-based block copolymers, antiviral peptide nanocomplexes (APN) platform was developed to improve peptide stability and to reduce cytotoxicity associated with positive charge. Herein, we developed a facile method to prepare galactosylated Gal-APN and tested their feasibility as liver-specific delivery system. In vitro, Gal-APN displayed specific internalization in hepatoma cell lines. Even though liver-targeted and non-targeted APN displayed comparable antiviral activity, Gal-APN offered prominent advantages to prevent HCV association with lipid droplets and suppress intracellular expression of HCV proteins. Moreover, in vivo preferential liver accumulation of Gal-APN was revealed in the biodistribution study. Altogether, this work illustrates the potential of Gal-APN as a novel liver-targeted therapy against HCV.

Evaluation of DOTA-chelated neurotensin analogs with spacer-enhanced biological performance for neurotensin-receptor-1-positive tumor targeting.

INTRODUCTION: Neurotensin receptor 1 (NTR1) is overexpressed in many cancer types. Neurotensin (NT), a 13 amino acid peptide, is the native ligand for NTR1 and exhibits high (nM) affinity to the receptor. Many laboratories have been investigating the development of diagnostic and therapeutic radiopharmaceuticals for NTR1-positive cancers based on the NT peptide. To improve the biological performance for targeting NTR1, we proposed NT analogs with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelation system and different lengths of spacers. METHODS: We synthesized four NTR1-targeted conjugates with spacer lengths from 0 to 9 atoms (null (N0), ß-Ala-OH (N1), 5-Ava-OH (N2), and 8-Aoc-OH (N3)) between the DOTA and the pharmacophore. In vitro competitive binding, internalization and efflux studies were performed on all four NT analogs. Based on these findings, metabolism studies were carried out on our best performing conjugate, (177)Lu-N1. Lastly, in vivo biodistribution and SPECT/CT imaging studies were performed using (177)Lu-N1 in an HT-29 xenograft mouse model. RESULTS: As shown in the competitive binding assays, the NT analogs with different spacers (N1, N2 and N3) exhibited lower IC50 values than the NT analog without a spacer (N0). Furthermore, N1 revealed higher retention in HT-29 cells with more rapid internalization and slower efflux than the other NT analogs. In vivo biodistribution and SPECT/CT imaging studies of (177)Lu-N1 demonstrated excellent accumulation (3.1 ± 0.4%ID/g) in the NTR1-positive tumors at 4h post-administration. CONCLUSIONS: The DOTA chelation system demonstrated some modest steric inhibition of the pharmacophore. However, the insertion of a 4-atom hydrocarbon spacer group restored optimal binding affinity of the analog. The in vivo assays indicated that (177)Lu-N1 could be used for imaging and radiotherapy of NTR1-positive tumors.