Abstract 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 by177 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.
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.
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.
Abstract 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), β-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 using177 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 of177 Lu-N1 demonstrated excellent accumulation (3.1 ± 0.4%ID/g) in the NTR1-positive tumors at 4 h 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 that177 Lu-N1 could be used for imaging and radiotherapy of NTR1-positive tumors.
Hematological deficiencies increase with aging, including anemias, reduced responses to hematopoietic stress and myelodysplasias. This investigation tested the hypothesis that increased bone marrow ...(BM) fat content in humans with age was associated with decreased numbers of side population (SP) hematopoietic stem cells, and this decrease correlated with changes in cytokine levels. BM was obtained from the femoral head and trochanteric region of the femur removed at surgery for total hip replacement (N = 100 subjects). In addition, BM from cadavers (N = 36), with no evidence of hip disease, was evaluated for fat content. Whole trabecular marrow samples were ground in a sterile mortar and pestle, and cellularity and lipid content determined. Marrow cells were stained with Hoechst dye and SP profiles were acquired. Plasma levels of insulin‐like growth factor (IGF)‐1, stromal‐derived factor (SDF)‐1 and interleukin (IL)‐6 were measured using ELISA. Fat content in the BM of human subjects and cadavers increased with age. The numbers of SP stem cells in BM as well as plasma IGF‐1 and SDF‐1 levels decreased in correlation with increased BM fat. IL‐6 had no relationship to changes in marrow fat. These data suggest that increased BM fat may be associated with a decreased number of SP stem cells and IGF‐1 and SDF‐1 levels with aging. These data further raise a more general question as to the role of adipose cells in the regulation of tissue stem cells.
N-(2-hydroxypropyl)-methacrylamide (HPMA) copolymers have shown promise for application in the detection and staging of cancer. However, non-target accumulation, particularly in the liver and spleen, ...hinders the detection of resident or nearby metastatic lesions thereby decreasing diagnostic effectiveness. Our laboratory has pursued the development of cathepsin S susceptible linkers (CSLs) to reduce the non-target accumulation of diagnostic/radiotherapeutic HPMA copolymers. In this study, we ascertain if the length of the linking group impacts the cleavage and clearance kinetics, relative to each other and a non-cleavable control, due to a reduction in steric inhibition. Three different CSLs with linking groups of various lengths (0, 6 and 13 atoms) were conjugated to HPMA copolymers. In vitro cleavage studies revealed that the longest linking group (13 atoms) led to more rapid cleavage when challenged with cathepsin S. The CSL incorporated HPMA copolymers demonstrated significantly higher levels of excretion and a significant decrease in long-term hepatic and splenic retention relative to the non-cleavable control. Contrary to in vitro observations, the length of the linking group did not substantially impact the non-target in vivo clearance. In the case of HPAC tumor retention, the CSL with the null (0 atom) linker demonstrated significantly higher levels of retention relative to the other CSLs. Given these results, we find that the length of the linking group of the CSLs did not substantially impact non-target clearance, but did influence tumor retention. Overall, these results demonstrate that the CSLs can substantially improve the non-target clearance of HPMA copolymers thereby enhancing clinical potential.
Objective To determine the effects of exercise and/or training on hematologic indices, circulating side population (SP) cells, and cytokines. Specifically hemoglobin (Hgb), hematocrit (Hct), white ...blood cell (WBC) and platelet (Plt) numbers, SP cells and plasma vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) levels were analyzed before and following exercise to maximal fatigue. Materials and Methods Thirty-seven nonsmoking subjects, aged 19 to 35 years, free of cardiopulmonary disease were enrolled and characterized as “trained” or “untrained.” Standard hematologic indices were measured. Blood cells were stained with Hoechst 33342 vital dye and analyzed using flow cytometry for enumeration of SP cells. The levels of IL-6 and VEGF were determined by enzyme-linked immunosorbent assay. Results Trained individuals had higher oxygen utilization and significantly longer exercise times than untrained individuals. Following exercise, significant increases were observed in Hgb, Hct, Plt, SP cell numbers, and IL-6 levels. These changes occurred in both trained and untrained individuals of both genders. No significant change in WBC numbers or VEGF levels was observed. Although circulating SP cell numbers were significantly increased, the “quality” of SP cells, defined by the ratio of lower SP to upper SP cells, was unchanged. Increases in SP cells did not correlate with cytokine levels. Conclusion Exercise increased Hgb, Hct, and Plt numbers, circulating SP cell numbers and IL-6 levels in young, healthy individuals of both genders and all fitness levels. These changes in hematologic, hematopoietic, and cytokine parameters, suggest that exercise can have a physiologic impact by potentially mobilizing stem cells and thereby enhancing tissue repair mechanisms.
This study enumerated CD45hi /CD34+ and CD45hi /CD133+ human hematopoietic stem cells (HSCs) and progenitor granulocyte-macrophage colony forming cells (GM-CFCs) in blood and trochanteric and femoral ...bone marrow in 233 individuals. Stem cell frequencies were determined with multiparameter flow cytometry and using an internal control to determine the intrinsic variance of the assays. Progenitor cell frequency was determined using a standard colony assay technique. The frequency of outliers from undetermined methodological causes was highest for blood, but less than 5% for all values. The frequency of CD45hi /CD133+ cells correlated highly with the frequency of CD45hi /CD34+ cells in trochanteric and femoral bone marrow. The frequency of these HSC populations in trochanteric and femoral bone marrow rose significantly with age. In contrast, there was no significant trend of either of these cell populations with age in the blood. Trochanteric marrow progenitor GM-CFCs showed no significant trends with age, but femoral marrow GM-CFCs trended downward with age, potentially because of the reported conversion of red marrow at this site to fat with age. Hematopoietic stem and progenitor cells exhibited changes in frequencies with age that differed between blood and bone marrow. We previously reported that side population (SP) multipotential HSC, which includes the precursors of CD45hi /CD133+ and CD45hi /CD34+ , decline with age. Potentially the increases in stem cell frequencies in the intermediate compartment between SP and GM progenitor cells observed in this study represent a compensatory increase for the loss of more potent members of the HSC hierarchy.
The purpose of this study was to assess the inflammatory nature of obesity and its effect on blood and bone marrow endothelial cell populations. Obese patients (BMI ≥30) had significantly higher ...concentrations of the inflammatory marker C‐reactive protein (CRP) (P = 0.03) and lower concentrations of the anti‐inflammatory cytokine interleukin‐10 (IL‐10) (P = 0.05). This cytokine profile is consistent with obesity being an inflammatory condition and is further supported by the significant correlation between total white blood cell count and BMI (r = 0.15; P = 0.035). High BMI was associated with significantly lower numbers of early endothelial cells (CD45−/CD34+) in the bone marrow (r = −0.20; P = 0.0068). There was also a significant inverse correlation between BMI and a more mature endothelial cell phenotype (CD45−/31+) in the blood (r = −0.17; P = 0.02). In addition, there was a significant correlation between BMI‐ and endothelial‐related cells of hematopoietic origin (CD133+/VEGFR‐2+) in the bone marrow (r = −0.26; P = 0.0007). Patients with higher plasma IL‐10 and insulin‐like growth factor (IGF) concentrations had higher numbers of endothelial phenotypes in the bone marrow suggesting a protective effect of these anti‐inflammatory cytokines. In conclusion, this work confirms the inflammatory nature of obesity and is the first to report that obesity is associated with reduced endothelial cell numbers in the bone marrow of humans. These effects of obesity may be a potential mechanism for impaired tissue repair in obese patients.
Receptor-targeted agents, such as gastrin-releasing peptide receptor (BB2r)-targeted peptides, have been investigated extensively in preclinical and clinical studies. In an attempt to increase the ...effectiveness of diagnostic or radiotherapeutic agents, we have begun to explore the incorporation of the hypoxia-selective prodrug 2-nitroimidazole into receptor-targeted peptides. Hypoxia is a well-known characteristic of many solid tumors, including breast, prostate, and pancreatic cancers. The aim of this approach is to use the hypoxia-trapping capability of 2-nitroimidazoles to increase the retention of the agent in hypoxic, BB2r-positive tumors. We have demonstrated that incorporation of one or more 2-nitroimidazoles into the BB2r-targeted peptide significantly increases the in vitro retention of the agent in hypoxic prostate cancer cells. The study described herein represents our first investigation of the in vivo properties of these hypoxia-enhanced BB2r-targeted agents in a PC-3 xenograft mouse model.
Four (111)In-labeled BB2r-targeted conjugates--(111) IN-1, (111) IN-2, (111) IN-3, and (111) IN-4, composed of 2-nitroimidazole moieties of 0, 1, 2, and 3, respectively--were synthesized, labeled, and purified. The BB2r binding affinities, externalization, and protein-association properties of these radioconjugates were assessed using the BB2r-positive PC-3 human prostate cancer cell line under hypoxic and normoxic environments. The in vivo biodistribution and micro-SPECT/CT imaging of the (111) IN-1, (111) IN-2, and (111) IN-4 radioconjugates were investigated in PC-3 tumor-bearing severely combined immunodeficient mice.
All conjugates and (nat)In-conjugates demonstrated nanomolar binding affinities. (111) IN-1, (111) IN-2, (111) IN-3, and (111) IN-4 demonstrated 41.4%, 60.7%, 69.1%, and 69.4% retention, correspondingly, of internalized radioactivity under hypoxic conditions relative to 34.8%, 35.3%, 33.2%, and 29.7% retention, respectively, under normoxic conditions. Protein-association studies showed significantly higher levels of association under hypoxic conditions for 2-nitroimidazole-containing BB2r-targeted radioconjugates than for controls. On the basis of the initial 1-h uptake in the PC-3 tumors, (111) IN-1, (111) IN-2, and (111) IN-4 demonstrated tumor retentions of 1.5%, 6.7%, and 21.0%, respectively, by 72 h after injection. Micro-SPECT/CT imaging studies of (111) IN-1, (111) IN-2, and (111) IN-4 radioconjugates resulted in clear delineation of the tumors.
On the basis of the in vitro and in vivo studies, the BB2r-targeted agents that incorporated 2-nitroimidazole moieties demonstrated improved retention. 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.
PDF
