Targeting VPAC1 Receptors for Imaging Glioblastoma

Molecular Imaging and Biology - Scintigraphic imaging of malignant glioblastoma (MG) continues to be challenging. We hypothesized that VPAC1 cell surface receptors can be targeted for positron...     

Prostate Cancer Theranostics Targeting Gastrin-Releasing Peptide Receptors

Gastrin-releasing peptide receptors (GRPRs), part of the bombesin (BBN) family, are aberrantly overexpressed in many cancers, including those of the breast, prostate, pancreas, and lung, and therefore present an attractive target for cancer diagnosis and therapy. Different bombesin analogs have been radiolabeled and used for imaging diagnosis, staging, evaluation of biochemical recurrence, and assessment of metastatic disease in patients with prostate cancer. Recently, interest has shifted from BBN-like receptor agonists to antagonists, because the latter does not induce adverse effects and demonstrate superior in vivo pharmacokinetics. We review the preclinical and clinical literatures on the use of GRPRs as targets for imaging and therapy of prostate cancer, with a focus on the newer developments and theranostic potential of GRPR peptides.     

Noninvasive PET Imaging of a Ga-68-Radiolabeled RRL-Derived Peptide in Hepatocarcinoma Murine Models

Molecular Imaging and Biology - Tc-99m- and I-131-labeled arginine-arginine-leucine (RRL) peptides have shown the feasibility of tumor imaging in our previous studies. However, there have been no...     

Correlation of the Ga-68-Bombesin Analog Ga-68-BZH3 with Receptors Expression in Gliomas as Measured by Quantitative Dynamic Positron Emission Tomography (dPET) and Gene Arrays

Purpose  

The kinetics of Ga-68-BZH3, a Ga-68-bombesin analog, was compared to molecular biological data obtained from gene arrays in seven patients with a recurrent glioma. The primary aim of this study was the correlation of receptor expression and tracer kinetics.     

Radiolabeling and Preliminary Evaluation of Ga-68 Labeled NODAGA-Ubiquicidin Fragments for Prospective Infection Imaging

Purpose

The present work was aimed at the development of prospective positron emission tomography (PET) agents for infection imaging. Towards this aim, ubiquicidin (UBI) fragments conjugated with the macrocyclic NODAGA chelator were radiolabeled with Ga-68 and evaluated.

Procedures

Conformations of custom synthesized NODAGA-UBI (29–41) and NODAGA-UBI (31–38) conjugates were compared with UBI (29–41) by circular dichroism (CD) spectroscopy. Optimization of labeling of NODAGA conjugates of UBI peptides with Ga-68 was performed and quality control analysis was carried out by chromatography techniques. In vitro uptake of [⁶⁸Ga] NODAGA-UBI (29–41) and [⁶⁸Ga]NODAGA-UBI (31–38) was studied in Staphylococcus aureus cells. In vivo distribution of [⁶⁸Ga]GaCl₃ and [⁶⁸Ga]NODAGA-UBI complexes was performed in normal Swiss mice.

Results

Conformations of NODAGA-UBI (29–41) and NODAGA-UBI (31–38) conjugates were found to be similar to UBI (29–41). NODAGA-UBI conjugates could be consistently labeled with Ga-68 in high radiochemical yields (>95 %) with high radiochemical purity (>95 %). [⁶⁸Ga]NODAGA-UBI (29–41) and [⁶⁸Ga]NODAGA-UBI (31–38) complexes showed retention time of 14 and 14.5 min, respectively, by HPLC radiochromatogram. Specific uptake of [⁶⁸Ga]NODAGA-UBI fragments was observed in S.aureus cells. Greater than 64 % of the injected dose was cleared via the renal route at 1 h post injection, and no significant uptake in vital organs of mice was observed with both the agents.

Conclusion

This is the first report on Ga-68 labeled NODAGA-UBI fragments for infection imaging and the agents hold tremendous prospect in PET imaging.

     

Synthesis and Evaluation of Ga-68-Labeled Rhein for Early Assessment of Treatment-Induced Tumor Necrosis

Molecular Imaging and Biology - This study aimed to synthesize a necrosis-avid agent using rhein as a precursor and labeled with gallium-68 (Ga-68) for positron emission tomography/computed...     

Optimization of a Labeling and Kit Preparation Method for Ga-68 Labeled DOTATATE,Using Cation Exchange Resin Purified Ga-68 Eluates Obtained from a Tin Dioxide <Superscript>68</Superscript>Ge/<Superscript>68</Superscript>Ga Generator

Purpose

The aim of this study was to optimize a radiolabeling method using cationic processed Ga-68 eluates from a SnO₂-based ⁶⁸Ge/⁶⁸Ga generator, followed by the development of DOTA-Tyr³-Thre⁸-octreotide (DOTATATE) kits.

Procedures

Diluted generator eluates were adsorbed on a SCX resin and desorbed with acidified 5 M NaCl solution. Optimized labeling conditions were determined by variation of pH, using 35 μg DOTATATE and sodium acetate buffer. DOTATATE kits were developed based on optimized radiolabeling conditions, were labeled, and evaluated.

Results

Optimized labeling conditions resulted in a radiolabeling efficiency of around 99 % and radiochemical yield of almost 85 %. Different kit preparation methods did not significantly influence the radiolabeling results. Kits were found to be stable over 3 months.

Conclusion

A labeling method using SCX-processed Ga-68 eluates was optimized. DOTATATE kits specifically for these SCX-processed Ga-68 eluates were successfully formulated. A post-labeling Sep-Pak C18 purification should be optional.

     

Synthesis and Application of a Long-Circulating Radiolabeled Peptide for Targeting of Osteosarcoma

Molecular Imaging and Biology - The small peptide TMTP1 (NVVRQ) has been proved to target a series of highly metastatic tumor cells. The aim of this study was to develop a new agent based on TMTP1...     

Elucidation of the vasoactive intestinal peptide pharmacophore for VPAC(2) receptors in human and rat and comparison to the pharmacophore for VPAC(1) receptors

Vasoactive intestinal peptide (VIP) functions as a neurotransmitter involved in a number of physiological and pathological conditions. The actions of VIP are mediated through VPAC(1) and VPAC(2). In contrast to VPAC(1), which has been extensively studied, little is known about the pharmacology of VPAC(2). In this study we investigated the VIP pharmacophore for VPAC(2) by using alanine and D-amino acid scanning. We found significant species differences, and the human VPAC(2) (hVPAC(2)) expressed in Chinese hamster ovary (CHO) cells, which have been used in previous studies, differed significantly from the native hVPAC(2) in Sup T(1) cells and hVPAC(2) expressed in PANC1 cells. There was a close agreement between binding affinities and potencies for VPAC(2) activation. The amino acids whose backbone or side chain orientations were most important for high affinity potency are Asp(3), Phe(6), Thr(7), Tyr(10), Arg(12), Tyr(22), and Leu(23), whereas the side chains of Ser(2), Asp(8), Asn(9), Gln(16), Val(19), Lys(20), Lys(21), Asn(24), and Ser(25) are not essential. Comparison of the VIP pharmacophore between hVPAC(1) and hVPAC(2) demonstrated that the side chains of Thr(7), Tyr(10), Thr(11), and Tyr(22) were much more critical for high affinity for the hVPAC(2) than the hVPAC(1). In contrast, the orientation of the side chain of Asn(24) was more important for high affinity for the hVPAC(1). This study shows that in assessing the pharmacophore of VIP analogs for the VPAC(2), important species differences need to be considered as well as the expression system used. These results of our study should be useful for designing VPAC subtype-selective analogs, simplified analogs, and possibly metabolically stable analogs.     

Pulmonary and Systemic Pharmacokinetics of Inhaled and Intravenous Colistin Methanesulfonate in Cystic Fibrosis Patients: Targeting Advantage of Inhalational Administration

The purpose of this study was to define the pulmonary and systemic pharmacokinetics of colistin methanesulfonate (CMS) and formed colistin following intravenous (i.v.) and inhaled administration in cystic fibrosis (CF) patients. Six CF subjects were administered nebulized CMS doses of 2 and 4 million IU and an i.v. CMS infusion of 150 mg of colistin base activity. Blood plasma, sputum, and urine samples were collected for 12 to 24 h postdose. To assess the tolerability of the drug, lung function tests, blood serum creatinine concentrations, and adverse effect reports were recorded. All doses were well tolerated in the subjects. The pharmacokinetic parameters for CMS following i.v. delivery were consistent with previously reported values. Sputum concentrations of formed colistin were maintained at <1.0 mg/liter for 12 h postdose. Nebulization of CMS resulted in relatively high sputum concentrations of CMS and formed colistin compared to those resulting from i.v. administration. The systemic availability of CMS was low following nebulization of 2 and 4 million IU (7.93% ± 4.26% and 5.37% ± 1.36%, respectively), and the plasma colistin concentrations were below the limit of quantification. Less than 2 to 3% of the nebulized CMS dose was recovered in the urine samples in 24 h. The therapeutic availability and drug targeting index for CMS and colistin following inhalation compared to i.v. delivery were significantly greater than 1. Inhalation of CMS is an effective means of targeting CMS and formed colistin for delivery to the lungs, as high lung exposure and minimal systemic exposure were achieved in CF subjects.     

Peptidyl Aldehyde NK-1.8k Suppresses Enterovirus 71 and Enterovirus 68 Infection by Targeting Protease 3C

Enterovirus (EV) is one of the major causative agents of hand, foot, and mouth disease in the Pacific-Asia region. In particular, EV71 causes severe central nervous system infections, and the fatality rates from EV71 infection are high. Moreover, an outbreak of respiratory illnesses caused by an emerging EV, EV68, recently occurred among over 1,000 young children in the United States and was also associated with neurological infections. Although enterovirus has emerged as a considerable global public health threat, no antiviral drug for clinical use is available. In the present work, we screened our compound library for agents targeting viral protease and identified a peptidyl aldehyde, NK-1.8k, that inhibits the proliferation of different EV71 strains and one EV68 strain and that had a 50% effective concentration of 90 nM. Low cytotoxicity (50% cytotoxic concentration, >200 μM) indicated a high selective index of over 2,000. We further characterized a single amino acid substitution inside protease 3C (3C^pro), N69S, which conferred EV71 resistance to NK-1.8k, possibly by increasing the flexibility of the substrate binding pocket of 3C^pro. The combination of NK-1.8k and an EV71 RNA-dependent RNA polymerase inhibitor or entry inhibitor exhibited a strong synergistic anti-EV71 effect. Our findings suggest that NK-1.8k could potentially be developed for anti-EV therapy.     

Imaging Characteristics and First Experience of [<Superscript>68</Superscript>Ga]THP-PSMA,a Novel Probe for Rapid Kit-Based Ga-68 Labeling and PET Imaging: Comparative Analysis with [<Superscript>68</Superscript>Ga]PSMA I&amp;T

Purpose

[⁶⁸Ga]Trishydroxypyridinone (THP)–prostate-specific membrane antigen (PSMA) is a novel tracer that can be labeled in one step by cold reconstitution of a kit with unprocessed generator eluate, targeting PSMA via the lysine-urea-glutamate (KuE) motif. The aim of this study was to evaluate the human imaging characteristics of [⁶⁸Ga]THP-PSMA.

Procedures

[⁶⁸Ga]THP-PSMA positron emission tomography (PET)/x-ray computed tomography (CT) was performed in 25 patients with biochemical recurrence after radical prostatectomy for prostate cancer. Urinary and biliary excretion and tumor lesion uptake were quantified using standardized uptake values (SUVs). Imaging characteristics were assessed in terms of non-target organ uptake, background activity, target-to-background ratios (TBRs) of tumor lesions, and frequency of bladder halo artifacts. Findings were compared to a matched cohort of 25 patients undergoing PET/CT with the established agent [⁶⁸Ga]PSMA I&T.

Results

Physiologic uptake of [⁶⁸Ga]THP-PSMA was significantly lower in salivary glands (P?<?0.0001), liver (P?<?0.0001), spleen (P?<?0.0001), and kidneys (P?<?0.0001) than with [⁶⁸Ga]PSMA I&T. While biliary tracer excretion of [⁶⁸Ga]THP-PSMA was negligible, urinary tracer excretion of [⁶⁸Ga]THP-PSMA was fast, and significantly higher than for [⁶⁸Ga]PSMA I&T, contributing to a higher frequency of bladder artifacts. Malignant lesion uptake of [⁶⁸Ga]THP-PSMA assessed as either SUV or TBR was significantly lower than with [⁶⁸Ga]PSMA I&T.

Conclusion

[⁶⁸Ga]THP-PSMA yields suitable in vivo uptake characteristics. The simplified synthesis method for [⁶⁸Ga]THP-PSMA may facilitate wider application and higher patient throughput with PSMA imaging. However, direct intraindividual comparison studies are needed to assess the relative performance of [⁶⁸Ga]THP-PSMA vs other PSMA ligands in terms of clinical detection rate and image quality.

     

68Ga-Triacetylfusarinine C and 68Ga-Ferrioxamine E for Aspergillus Infection Imaging: Uptake Specificity in Various Microorganisms

Purpose

⁶⁸Ga-triacetylfusarinine C (⁶⁸Ga-TAFC) and ⁶⁸Ga-ferrioxamine E (⁶⁸Ga-FOXE) showed excellent targeting properties in Aspergillus fumigatus rat infection model. Here, we report on the comparison of specificity towards different microorganisms and human lung cancer cells (H1299).

Procedures

The in vitro uptake of ⁶⁸Ga-TAFC and ⁶⁸Ga-FOXE was studied in various fungal, bacterial and yeast cultures as well as in H1299 cells. The in vivo imaging was studied in fungal and bacterial rat infection and inflammation models.

Results

⁶⁸Ga-TAFC and ⁶⁸Ga-FOXE showed rapid uptake in A. fumigatus cultures, significantly lower in other fungal species and almost no uptake in other microorganisms and H1299 cells, except for ⁶⁸Ga-FOXE in Staphylococcus aureus. ⁶⁸Ga-TAFC and ⁶⁸Ga-FOXE revealed rapid uptake in the lungs of A. fumigatus-infected rats, low accumulation in sterile inflammation and no uptake in bacterial abscess.

Conclusions

We have shown that ⁶⁸Ga-FOXE and ⁶⁸Ga-TAFC have high uptake in A. fumigatus both in vitro and in vivo. ⁶⁸Ga-TAFC showed higher specificity, while ⁶⁸Ga-FOXE showed higher sensitivity.     

Evaluation of WLBU2 Peptide and 3-O-Octyl-sn-Glycerol Lipid as Active Ingredients for a Topical Microbicide Formulation Targeting Chlamydia trachomatis

Topical microbicides for prevention of sexually transmitted diseases (STDs) would be especially useful for women who are not able to persuade their partner(s) to take precautions. Many topical microbicides are in various stages of development, based on a variety of active ingredients. We investigated the in vitro activity of an engineered antimicrobial peptide (WLBU2) and a lipid (3-O-octyl-sn-glycerol [3-OG]) which could potentially be used as active ingredients in such a product. Using commercially available cytotoxicity reagents [Alamar Blue, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and lactate dehydrogenase (LDH)], we first determined the toxicity of WLBU2 and 3-OG to the host cells in our assay procedure and excluded toxic concentrations from further testing. To determine activity against Chlamydia trachomatis, we used an assay previously developed by our laboratory in which chlamydial elementary bodies (EBs) were exposed to microbicides prior to contact with epithelial cells: the minimum (microbi)cidal concentration (MCC) assay. To further simulate conditions of transmission, we carried out the same assay in the presence of a simulated vaginal fluid, a simulated seminal fluid, human serum albumin, and a range of pH values which might be found in the human vagina at the time of exposure. Last, we tested WLBU2 and 3-OG in combination to determine if adding them together resulted in synergistic activity. We found that WLBU2 and 3-OG both have excellent activity in vitro against C. trachomatis and significantly more activity when added together. The simulated fluids reduced activity, but the synergy seen is good evidence that they would be effective when combined in a microbicide formulation.Because vaccines are not available for most sexually transmitted diseases (STDs), alternative prevention methods are needed. Topical microbicides are STD prevention products currently under development that would be applied before sex, similar to a spermicide. Initial microbicide candidates included commercially available spermicides that contained nonoxynol-9 (non-9) as an active ingredient, but it has become clear that non-9 can actually increase a woman''s risk of contracting HIV infection due to the lesions it causes when used frequently (10, 43). Thus, surfactants (like non-9) are no longer leading candidates for formulated products. Other compounds, such as lipids and peptides, have received recent attention as possible topical microbicide candidates (26, 49, 50). In this study, we focused on the antichlamydial activity of the antimicrobial peptide WLBU2 and lipid 3-O-octyl-sn-glycerol (3-OG). WLBU2 is an engineered cationic amphipathic 24-residue peptide that contains only arginine, valine, and tryptophan, whose sequence (RRWVRRVRRWVRRVVRVVRRWVRR) is optimized for formation of an amphipathic helix conformation (9). 3-OG is a synthetic lipid that is modeled after antibacterial lipids found in human breast milk (18, 23). Both WLBU2 and 3-OG have already been found to have excellent activity against many Gram-negative and Gram-positive bacteria (9, 30, 31). WLBU2 has been found to be highly active against herpesvirus and HIV (22; unpublished data). 3-OG has also been found to be active against HIV (21, 34). In our previous published and unpublished research, we found that lipids similar to 3-OG and peptides similar to WLBU2 have good activity against Chlamydia trachomatis specifically (1, 26). A combination microbicide with dual modes of action has the potential to be more potent and less likely to induce resistant strains than one containing a single active ingredient. Synergistic activity between lipids and peptides has already been demonstrated against the herpesvirus using a lipid that is similar to 3-OG and peptides that are similar to WLBU2 (19, 22). Thus, in this study we not only evaluated the activities of these two compounds individually but also examined their combined effect in vitro.C. trachomatis is the most commonly reported bacterial STD in the United States, and the incidence continues to grow each year. In 2007, more than 1,100,000 new Chlamydia infections were reported to the CDC (5). Antibiotics can be used to cure Chlamydia infections, but many are asymptomatic and go unnoticed. If left untreated, these infections can lead to many serious complications, especially in women (5). C. trachomatis is an obligate intracellular parasite with a unique biphasic developmental cycle. The infectious form, the elementary body (EB), is fairly resistant to changes in environmental conditions. EBs are found in genital secretions and would be exposed to microbicides during transmission. Antibiotics, meant to cure existing infections, must be able to enter the host cell and target the metabolically active and vulnerable reticulate bodies (RBs). Topical microbicides are designed to prevent infection, and thus, we utilized our minimum (microbi)cidal concentration (MCC) assay, previously developed to mimic the order of events in the human vagina during exposure to C. trachomatis, to examine topical microbicide activity directly on extracellular EBs before infection occurs. In this study, we have also modified our MCC assay to simulate the presence of vaginal fluids, seminal fluids, and blood, body fluids that a topical microbicide might come into contact with during a transmission event.We have found that WLBU2 peptide and 3-OG lipid show excellent potential as active ingredients in a topical microbicide formulation. Both are very active against multiple serovars of C. trachomatis in vitro and are somewhat resistant to simulated body fluids and changes in pH. Additionally, when tested in combination, they act synergistically to inhibit C. trachomatis more than these compounds do individually. This synergistic activity makes them especially desirable for use in a topical microbicide product because it reduces the amount of each compound needed to provide protection, decreasing costs, potential toxicity to the user, and potential for inducing resistant strains of C. trachomatis.     

Targeting BAFF: immunomodulation for autoimmune diseases and lymphomas

In an effort to develop more effective treatments for inflammatory diseases, immunologists have targeted numerous molecular pathways, but with limited success. Notable exceptions are anti-TNF agents, which have proved efficacious in a proportion of rheumatoid arthritis (RA) patients. Another TNF family member, termed BAFF ("B cell-activating factor belonging to the TNF family"), plays a central role in autoimmune diseases, as well as in B cell maturation, survival, and T cell activation. Agents that block BAFF have proven to be highly effective in the treatment of certain autoimmune conditions in mice. In addition, phase II data in human clinical trials for RA appear very promising. BAFF is also a survival factor for certain B cell lymphomas. Despite the relatively recent identification of BAFF, this molecule has provided considerable new insight into B cell homeostasis and immune function, and represents an important new molecular target for treatment of autoimmune diseases and lymphomas.     

Evaluating ethnography: considerations for analysis

The informed evaluation of ethnographic reports is essential to the practitioner who is working towards research-based practice. It is also part of the process of developing and refining nursing knowledge. While there are features common to the critical examination of all research, an understanding of ethnographic design and, in particular, of issues of validity and reliability, is a prerequisite for evaluation of ethnographic research reports.     

Discovery of a Linear Peptide for Improving Tumor Targeting of Gene Products and Treatment of Distal Tumors by IL-12 Gene Therapy

Like many effective therapeutics, interleukin-12 (IL-12) therapy often causes side effects. Tumor targeted delivery may improve the efficacy and decrease the toxicity of systemic IL-12 treatments. In this study, a novel targeting approach was investigated. A secreted alkaline phosphatase (SEAP) reporter gene-based screening process was used to identify a mini-peptide which can be produced in vivo to target gene products to tumors. The coding region for the best peptide was inserted into an IL-12 gene to determine the antitumor efficacy. Affinity chromatography, mass spectrometry analysis, and binding studies were used to identify a receptor for this peptide. We discovered that the linear peptide VNTANST increased the tumor accumulation of the reporter gene products in five independent tumor models including one human xenogeneic model. The product from VNTANST-IL-12 fusion gene therapy increased accumulation of IL-12 in the tumor environment, and in three tumor models, VNTANST-IL-12 gene therapy inhibited distal tumor growth. In a spontaneous lung metastasis model, inhibition of metastatic tumor growth was improved compared to wild-type IL-12 gene therapy, and in a squamous cell carcinoma model, toxic liver lesions were reduced. The receptor for VNTANST was identified as vimentin. These results show the promise of using VNTANST to improve IL-12 treatments.