Objectives: Recently, a new type of MRI contrast mechanism that relies on direct chemical exchange of protons with bulk water has been developed and is referred to as chemical exchange saturation ...transfer (CEST) MRI. The specificity of CEST agents is improved over relaxation based agents because of the dependence of CEST on labeling frequency. Furthermore, diamagnetic organic compounds, which are termed diaCEST agents, can be employed to generate contrast instead of metals, avoiding the well-known toxicity of the latter. Salicylic acid (SA)and its analogues are a promising class of highly sensitive, nontoxic diaCEST contrast agents. The goal of this project is to enhance the sensitivity of such agents for receptor-targeted imaging employing an established polymeric platform to increase the concentration of salicylic acid moieties within a single molecular entity. We report here the exchange properties of the agents in solution and in an experimental murine model to demonstrate the feasibility of receptor-targeted CEST MRI of prostate cancer. Methods: Four polymeric diaCEST agents (1 - 4) were synthesized. Agents 1 and 3 were the control agents with polymer and SA (~1: 40) and SA and 1,4,7,10-tetra-azacylcododecane-N,N',N',N‴-tetraacetic acid (DOTA)-monoamide chelating moieties in ratio ~1: 40: 4 (polymer:SA: DOTA) respectively. Agents 2 and 4 were grafted with SA and a known high-affinity ligand targeting prostate-specific membrane antigen (PSMA) in molar ratios ~1: 40: 4, (polymer:SA:urea) and ~1: 40: 4:4 (polymer:SA:urea:DOTA) respectively to provide optimal magnetic resonance (MR) imaging sensitivity and target specificity. The presence of salicylic acid moieties on the polymer backbone was confirmed by 1H-NMR spectroscopy. PSMA binding affinities of agents were evaluated by a competitive protein binding assay. In vivo CEST-MR images were acquired on a Bruker Biospec 11.7 T MR scanner. Male SCID/NOD mice bearing both PSMA+ PC3 PIP and PSMA- PC3 flu xenografts on opposite upper flanks were injected with 125 mg/kg of contrast agent 1 or 2 in PBS MR imaging. Compounds 3 and 4 were radiolabeled with 111In for SPECT imaging and tissue biodistribution (BioD) studies. Results: Binding affinities of the targeted contrast agents 2 and 4 were significantly higher (~ 100 fold) compared to untargeted agents 1 or 3. All agents demonstrated significant CEST contrast at 9.2 ppm as anticipated, along with additional peaks at 3.5 ppm and 2.2 ppm. A linear relationship between the contrast and the concentration of the agent was observed at low concentrations. In vivo CEST imaging showed significantly higher contrast enhancement at 90 min post-injection for the targeted contrast agent 2 in PSMA+ PIP tumor compared to PSMA- flu tumor. Minimal contrast enhancement was demonstrated in both PSMA+ PC3 PIP and PSMA- PC3 flu tumors for untargeted polymeric contrast agent 1 during a similar time frame as for 2. CEST MRI data were further validated by SPECT imaging and tissue BioD results of 111In-3 and 111In-4, demonstrating specific and high uptake in PSMA+ tumors for 111In-4. Conclusion: These results define that SA-based PSMA-targeting polymeric contrast agents can permit selective CEST MRI of PSMA-positive tumors in vivo.
Objectives: Prostate-specific membrane antigen (PSMA) is an important target for radiopharmaceutical therapy (RPT) for the treatment of mCRPC. The goal of this project is to develop optimized agents ...employing the low and high linear energy transfer (LET) radiation of β- and a-particles, respectively, for treating mCRPC. Based on our extensive structure-activity relationship studies using urea-based inhibitors of PSMA, we selected a low molecular weight ligand conjugated with 1,4,7,10-tetra-azacylcododecane-N,N',N',N‴-tetraacetic acid (DOTA)-monoamide, ligand 1. We synthesized radiometal-labeled agents, including β-emitting 177Lu-1 and a-emitting 213Bi-1 and 225Ac-1 for RPT. We also synthesized an a-emitting Pb-212-1,4,7,10-tetraaza-1,4,7,10-tetra-(2-carbamoylmethyl)-cyclododecane (TCMC) analog, 212Pb-2. We evaluated those radiotherapeutics in solution, in vitro and in vivo in experimental models of prostate cancer. Methods: PSMA binding affinities of 1, 2 and the corresponding non-radioactive metal analogs were evaluated by a competitive protein binding assay. Cellular uptake, internalization and clonogenic survival studies were undertaken using PSMA+ PC3 PIP and PSMA- PC3 flu cells at 2-24 h post-incubation. Ligand 1 was selected based on head-to-head comparison studies between177Lu-1, 177Lu-PSMA I&T and 177Lu-PSMA-617 employing our standard mouse model bearing PSMA+ PC3 PIP and PSMA- PC3 flu tumors in opposite upper flanks. Tissue biodistribution studies were performed for all radiometal analogs of 1 using the same flank-tumor model. A Pb-203 analog of 2 was prepared for biodistribution and SPECT-CT imaging studies. Antitumor efficacy all agents were tested in vivo in the aforementioned flank-tumor model. Therapeutic efficacy of 177Lu-1 and 212Pb-2 was investigated head-to-head in mice bearing micrometastases from a PC3-ML cell line engineered to express both firefly luciferase and PSMA. Sub-organ distribution of 213Bi-1 and 225Ac-1 was evaluated using an a-camera. Results: All radiotherapeutics and 203Pb-2 displayed PSMA-specific cellular uptake and internalization. Decreased clonogenic survival in vitro was observed in PSMA+ PC3 PIP cells for 177Lu-1, 213Bi-1 and 225Ac-1. PSMA+ PC3 PIP tumor/kidney ratios at 2h post-injection for 177Lu-1,213Bi-1, 225Ac-1 and 203Pb-2 were 6.4 ± 2.1, 1.8 ± 1.0, 8.2 ± 0.8 and 0.4 ± 0.1 respectively. All radiotherapeutics demonstrated significant tumor growth delay in vivo in PSMA+ PC3 PIP flank tumors. Direct comparison of 212Pb-2 and 177Lu-1 in the PSMA+ micrometastatic PC3-ML model demonstrated improved survival for the group treated with 212Pb-2 (P < 0.0001). a-Camera images of 213Bi-1 showed rapid clearance from renal cortex and increased PSMA+ PC3 PIP tumor uptake from 10-30 min post-injection. By contrast, a-Camera images of 225Ac-1 demonstrated high renal cortical uptake at 2 h, which completely cleared by 24 h while maintaining high PSMA+ PC3 PIP tumor uptake. Conclusion: PSMA-targeted low- and high-LET RPT enabled improved survival in mice bearing primary tumor xenografts, while high-LET RPT is promising in treating micrometastases after systemic administration.
Antimicrobial peptides have received increasing attention as a new pharmaceutical substance, because of their broad spectrum of antimicrobial activities and the rapid development of ...multidrug-resistant pathogenic microorganisms. The main obstacle to the wide application of antimicrobial peptides has been the lack of a cost-effective, mass-production method. A novel mass-production method for an antimicrobial peptide of 21 amino acids, buforin II, which was isolated from the stomach of the amphibian Bufo bufo gargarizans, has been developed. This method is based on the neutralization of the positive charges of buforin II by fusing to an acidic peptide to avoid the lethal effect of the expressed antimicrobial peptide on the host cells. The fusion peptide was expressed in Escherichia coli as tandem repeats to increase the product yield. Multimers of the acidic peptide-buforin II fusion peptide were expressed at high levels without causing damage to the cells. The presence of cysteine residues in the acidic peptide was critical for the high level expression of the fusion peptide multimers. Multimers of this fusion peptide were expressed as inclusion bodies, and about 107 mg of pure buforin II was obtained from 1 L of E. coli culture by cleaving the multimers with CNBr. Recombinant buforin II had an antimicrobial activity identical to that of natural buforin II. These results may lead to a general, cost-effective solution to the mass production of antimicrobial peptides and other basic peptides which are lethal to the host strain.
Abstract
Carbonic anhydrase IX (CAIX) is a cell surface enzyme that is over-expressed in approximately 95% of clear cell renal cell carcinomas (ccRCCs), the most common renal cancer. CAIX is also a ...surrogate marker of hypoxic tumors for various human cancers; however, a clinically viable molecular imaging agent targeting CAIX is not available. We have synthesized and performed in vitro and in vivo evaluation of a dual-motif, low-molecular-weight inhibitor of CAIX, 64Cu XYIMSR-06, for imaging CAIX expression on ccRCC tumors using positron emission tomography (PET). 64Cu XYIMSR-06 was synthesized in two steps from reported dual-motif precursor 1. Upon radiolabeling, 64Cu XYIMSR-06 was evaluated in immunocompromised mice bearing CAIX-expressing SK-RC-52 tumors for in vivo PET imaging and biodistribution. 64Cu XYIMSR-06 was generated in radiochemical yields of 51.0 ± 4.5% (n = 5) and specific radioactivity of 6.0 GBq/μmol (170Ci/mmol ± 70, n = 5). Tumor could be visualized on PET images by 1 h post-injection with high tumor-to-background levels achieved within 24 h. Biodistribution studies of 64Cu XYIMSR-06 demonstrated a maximum tumor uptake of 19.3 ± 4.51% injected dose per gram of radioactivity at 4 h. Tumor-to- blood, muscle and kidney ratios were 129.6 ± 18.8, 84.3 ± 21.0 and 2.1 ± 0.26, respectively, at 8 h post-injection. At 24 h, a tumor-to-kidney ratio of 7.1±2.5 was achieved. These findings represent increased tumor-to-background ratios at the indicated times relative to previously reported CAIX-targeted imaging agents. 64Cu XYIMSR-06 is a promising candidate for PET imaging of CAIX expressing tumors, especially ccRCC.
Citation Format: IL MINN, Soo Min Koo, Hye Soo Lee, Mary Brummet, Steven P. Rowe, Michael A. Gorin, Polina Sysa-Shah, William D. Lewis, Hye-Hyun Ahn, Yuchuan Wang, Sangeeta Ray, Ronnie C. Mease, Sridhar Nimmagadda, Mohamad E. Allaf, Martin G. Pomper, Xing Yang. A dual-motif CAIX inhibitor, 64CuXYIMSR-06, for PET imaging of clear cell renal cell carcinoma. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4206.
Three antimicrobial peptides, which had strong antimicrobial activity against a broad spectrum of microorganisms, were isolated from the stomach of the bullfrog,
Rana catesbeiana. Two of the ...antimicrobial peptides were found to be derived from the N-terminal sequences of pepsinogen A and C prosequences. The amino acid sequences of the new antimicrobial peptides, named bullfrog pepsinogen A-derived antimicrobial peptide (bPaAP) and bullfrog pepsinogen C-derived antimicrobial peptide (bPcAP), were Gly-Val-Val-Lys-Val-Ser-Arg-Leu-Lys-Gly-Glu-Ser-Leu-Arg-Ala-Arg-Leu (MW 1865.5) and Ile-Ile-Lys-Val-Pro-Leu-Lys-Lys-Phe-Lys-Ser-Met-Arg-Glu-Val-Met-Arg-Asp-His-Gly-Ile-Lys-Ala-Pro-Val-Val-Asp-Pro-Ala-Thr-Lys-Tyr (MW 3691.6), respectively. The bPaAP and bPcAP adopted 35% and 42% amphipathic α-helical structure in 50% trifluoroethanol, respectively, and were non-hemolytic up to a concentration of 200 μg/ml. Synthesized pepsinogen C prosequences of monkey and human, which had similar structural characteristics as bPaAP and bPcAP, also showed antimicrobial activity at concentrations of 10–200 μg/ml. The third peptide was buforin I, previously found in the stomach of the Asian toad,
Bufo bufo gargarizans. These findings strongly suggest that peptides derived from the prosequences of pepsinogens, along with buforin I, may contribute to the antimicrobial function of the gastrointestinal mucosa of vertebrates, including human.
Inspired by the Catholic intellectual tradition, these essays are the fruit of a series of seminars sponsored by the Center for Catholic Studies and the Saint Paul Seminary School of Divinity at the ...University of St. Thomas in Saint Paul, Minnesota. With a special focus on the works of John Paul II (especially Veritatis Splendor and Fides et Ratio), the authors bring to light a host of considerations that set the work of his pontificate within the illuminating light of the living intellectual tradition.
Although the incidence of brain metastasis in gastric cancer is relatively low, its prevalence may increase with improved therapy and longer survival tumors. The molecular mechanisms underlying brain ...metastases are not well understood. To gain insight into the mechanism of brain metastasis, we studied differences in microRNA (miRNA) expression levels in 8 cases of matched primary gastric adenocarcinoma and brain metastatic adenocarcinoma using the Illumina microRNA microarray chip. We identified 6 upregulated and 2 downregulated miRNAs in all 8 cases simultaneously. Interestingly, 2 out of 8 miRNAs (hsa-miR-141-3p and hsa-miR-200b-3p) belonged to the miR-200 family. Online microRNA database searching revealed that ZEB2 is the top-ranked target gene for hsa-miR141-3p and hsa-miR-200b-3p, prompting us to focus ZEB2 expression in brain metastatic adenocarcinoma. We confirmed that ZEB2 expression was markedly downregulated in some brain metastatic samples. In addition, decreased ZEB2 expression was noted by western blot analysis of 2 metastatic gastric adenocarcinoma cell types that were derived by in vivo selection following intracardiac injection of gastric cancer cell lines. In conclusion, we demonstrate that expression of miRNA-200 family members and ZEB2 are associated with brain metastases of gastric adenocarcinoma, not only in matched patient samples, but also in metastatic cell lines that were derived by in vivo selection.
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