Polymer-Tethered Quenched Fluorescent Probes for Enhanced Imaging of Tumor-Associated Proteases.

Fluorescence-based contrast agents enable real-time detection of solid tumors and their neovasculature, making them ideal for use in image-guided surgery. Several agents have entered late-stage clinical trials or secured FDA approval, suggesting they are likely to become the standard of care in cancer surgeries. One of the key parameters to optimize in contrast agents is molecular size, which dictates much of the pharmacokinetic and pharmacodynamic properties of the agent. Here, we describe the development of a class of protease-activated quenched fluorescent probes in which a N-(2-hydroxypropyl)methacrylamide copolymer is used as the primary scaffold. This copolymer core provides a high degree of probe modularity to generate structures that cannot be achieved with small molecules and peptide probes. We used a previously validated cathepsin substrate and evaluated the effects of length and type of linker, as well as the positioning of the fluorophore/quencher pair on the polymer core. We found that the polymeric probes could be optimized to achieve increased overall signal and tumor-to-background ratios compared to the reference small molecule probe. Our results also revealed multiple structure-activity relationship trends that can be used to design and optimize future optical imaging probes. Furthermore, they confirm that a hydrophilic polymer is an ideal scaffold for use in optical imaging contrast probes, allowing a highly modular design that enables efficient optimization to maximize probe accumulation and overall biodistribution properties.

Polymer-tethered quenched fluorescent probes for enhanced imaging of tumor associated proteases.

Fluorescence-based contrast agents enable real-time detection of solid tumors and their neovasculature, making them ideal for use in image-guided surgery. Several agents have entered late-stage clinical trials or secured FDA approval, suggesting they are likely to become standard of care in cancer surgeries. One of the key parameters to optimize in contrast agent is molecular size, which dictates much of the pharmacokinetic and pharmacodynamic properties of the agent. Here, we describe the development of a class of protease-activated quenched fluorescent probes in which a N-(2-hydroxypropyl)methacrylamide copolymer is used as the primary scaffold. This copolymer core provides a high degree of probe modularity to generate structures that cannot be achieved with small molecules and peptide probes. We used a previously validated cathepsin substrate and evaluated the effects of length and type of linker as well as positioning of the fluorophore/quencher pair on the polymer core. We found that the polymeric probes could be optimized to achieve increased over-all signal and tumor-to-background ratios compared to the reference small molecule probe. Our results also revealed multiple structure-activity relationship trends that can be used to design and optimize future optical imaging probes. Furthermore, they confirm that a hydrophilic polymer is an ideal scaffold for use in optical imaging contrast probes, allowing a highly modular design that enables efficient optimization to maximize probe accumulation and overall biodistribution properties.

Iridium-based Polymeric Multifunctional Imaging Tools for Biochemistry.

Herein, we report the development of a macromolecular multifunctional imaging tool for biological investigations, which is comprised of an N-(2-hydroxypropyl)methacrylamide backbone, iridium-based luminescent probe, glutamate carboxypeptidase II (GCPII) targeting ligand, and biotin affinity tag. The iridium luminophore is a tris-cyclometalated complex based on [Ir(ppy)3] with one of its 2-phenylpyridine ligands functionalized to allow conjugation. Synthesized macromolecular probes differed in the structure of the polymer and content of the iridium complex. The applicability of the developed imaging tools has been tested in flow cytometry (FACS) based assay, laser confocal microscopy, and fluorescence lifetime imaging microscopy (FLIM). The FACS analysis has shown that the targeted iBodies containing the iridium luminophore exhibit selective labelling of GCPII expressing cells. This observation was also confirmed in the imaging experiments with laser confocal microscopy. The FLIM experiment has shown that the iBodies with the iridium label exhibit a lifetime greater than 100 ns, which distinguishes them from typically used systems labelled with organic fluorophores exhibiting short fluorescence lifetimes. The results of this investigation indicate that the system exhibits interesting properties, which supports the development of additional biological tools utilizing the key components (iridium complexes, iBody concept), primarily focusing on the longer lifetime of the iridium emitter.

ADR3, a next generation i-body to human RANKL, inhibits osteoclast formation and bone resorption.

Osteoporosis is a chronic skeletal condition characterized by low bone mass and deteriorated microarchitecture of bone tissue and puts tens of millions of people at high risk of fractures. New therapeutic agents like i-bodies, a class of next-generation single-domain antibodies, are needed to overcome some limitations of conventional treatments. An i-body is a human immunoglobulin scaffold with two long binding loops that mimic the shape and position of those found in shark antibodies, the variable new antigen receptors of sharks. Its small size (∼12 kDa) and long binding loops provide access to drug targets, which are considered undruggable by traditional monoclonal antibodies. Here, we have successfully identified a human receptor activator of nuclear factor-κB ligand (RANKL) i-body, ADR3, which demonstrates a high binding affinity to human RANKL (hRANKL) with no adverse effect on the survival or proliferation of bone marrow-derived macrophages. Differential scanning fluorimetry suggested that ADR3 is stable and able to tolerate a wide range of physical environments (including both temperature and pH). In addition, in vitro studies showed a dose-dependent inhibitory effect of ADR3 on osteoclast differentiation, podosome belt formation, and bone resorption activity. Further investigation on the mechanism of action of ADR3 revealed that it can inhibit hRANKL-mediated signaling pathways, supporting the in vitro functional observations. These clues collectively indicate that hRANKL antagonist ADR3 attenuates osteoclast differentiation and bone resorption, with the potential to serve as a novel therapeutic to protect against bone loss.

Half-life extension and non-human primate pharmacokinetic safety studies of i-body AD-114 targeting human CXCR4.

Single domain antibodies that combine antigen specificity with high tissue penetration are an attractive alternative to conventional antibodies. However, rapid clearance from the bloodstream owing to their small size can be a limitation of therapeutic single domain antibodies. Here, we describe and evaluate the conjugation of a single domain i-body, AD-114, which targets CXCR4, to a panel of half-life extension technologies including a human serum albumin-binding peptide, linear and branched PEG, and PASylation (PA600). The conjugates were assessed in murine, rat and cynomolgus monkey pharmacokinetic studies and showed that the branched PEG was most effective at extending circulating half-life in mice; however, manufacturing limitations of PEGylated test material precluded scale-up and assessment in larger animals. PA600, by comparison, was amenable to scale-up and afforded considerable half-life improvements in mice, rats and cynomolgus monkeys. In mice, the circulating half-life of AD-114 was extended from 0.18 h to 7.77 h following conjugation to PA600, and in cynomolgus monkeys, the circulating half-life of AD-114-PA600 was 24.27 h. AD-114-PA600 was well tolerated in cynomolgus monkeys at dose rates up to 100 mg/kg with no mortalities or drug-related clinical signs.

Sputum induction method for studying total IgE levels in atopics asthamtic patients / 천식및알레르기

BACKGROUND: IgE antibodies have been considered to play an important role in the pathogenesis of atapic asthma. However, there have been only few studies on the role of IgE in airway secretion in the pathogenesis of bronchial asthma. This might be partly due to difficulty in sampling of airway seceretion from asthmatic patients. Recently, sputum induction method by inhalation of nebulized hypertonic saline was developed, and proved to be valid and useful method for obtaining airway secretion from asthmatic patients for studying airway inflammation. OBJECTIVE AND METHOD: To evaluate the usefulness of sputum induction method for studying IgE antibodies in airway secretion from atopic asthmatic patients, total IgE levels in induced sputum from 54 atopic asthmatics were measured by enzyme-linked immunosorbent assay(ELISA) and tried to find an association with sputum eosinophilia. RESULT: Total IgE levels in induced sputum were significantly higher in atopic asthmatic patients(1.27+82.066 IU/ml) than in controls(0.203+0.291 IUgmP)(p5% of 200 counted leukocytes). There was a significant correlation of total IgE levels between induced sputum and serum in atopic asthmatic patients(r=0.60, p<0.05). Total IgE levels in induced sputum(1.278+ 2.066) were significantly higher than saliva sample(0.504 + 1.111 IU/ml) from atopic asthmatic patients(p<0.05). CONCLUSION: These results suggest that total IgE levels are increased in the induced sputum of atopic asthmat,ic patients and sputum induction method is a useful tool for studying IgE antibodies in airway secretion from asthmatic patients.

The Effect of Systemic PUVA on the Proliferation of Melanocytes and the Titer of Anti - Pigment Cell Autoantibodies in Vitiligo Patients / 대한피부과학회지

BACKGROUND: PUVA has been used effectively in the treat,ment of vitiligo, but the mechanism by which PUVA stimulat.es melanocyte proliferation in vitiligo is not known. Several mechanisms have been suggested to be involved in the process of repigmentation of vitiligo. First, UV light, with or without psoralen, directly stimulates the proliferation of melanocytes. Secondly, PUVA may act. on epidermal keratinocytes or dermal components to stimulate t,hem to release certain melanocyte growth st,inulation factors that enhance the proliferation of melanocytes in depigmented lesions. Thirdly, PUVA irnmunologically leads to the impairment of epidermal Langerhans cell function and alteration of circulating T and B cell function, which results in the suppression of the stimuli is for rnelanocyte destruction during the therapy. OBJECTIVE: To test, th hypothesis that PUVA induced repigmentation in vitiligo results from the stimulation of growth factors that induce melanocyte proliferation, and that PUVA may suppress the immune reacticin to melanocytes, especially in autoantibody synt,hesis, we examined the effects of sera on the growth of epidermal melanocytes and control cells, and t,he incidence of antibodies to melanocyte and melanoma cells(SK-Mel 2~3) in the sera of patients with vitiligo. We also had normal control individuals and studied the changes of the antibody titer in the sera of patients with vitiligo. METHODS: The rate of H thymidine uptake was estimat,ed in cultured melanocytes and fibroblasts t,reated by patients sera before and after PUVA treatment. SDS-PAGE and immunoblotting analysis were used to idcntify anti pigment cell autoantibodies and were compared to the titers of autoantibodies after PUVA. RESULTS: 1. Melanocyte and fibrablast proliferation was increased by PUVA treated sera. Their proliferation was in proportion to the duration of the PUVA treatment. Melanocytes proliferated more than fibroblasts. 2. Significant differences between vitiligo patients and normal controls were found in the inci dence of anti-pigment cell antibodies. The antibodies were predominantly directed to melanocyte antigens of 110 kD, 65 kD, 45 kD and melanoma cell antigens of 110 kD, 103 kD, 88kD, 70 kD, 56 kD, 41 kD. 3. The titer of anti piment cell antibodies showed a tendency to decrease after PUVA treat- ment in most patients regardless of clinical improvement. Conclusion ; PUVA treated sera induced proliferation of melanocytes and fibroblasts and the production of aut,oantibodies was suppressed against pigment cell antigens through irnmunosuppression, which might help in the repigmentation of vitiligo.

The Effect of Systemic PUVA on the Proliferation of Melanocytes and the Titer of Anti - Pigment Cell Autoantibodies in Vitiligo Patients / 대한피부과학회지

BACKGROUND: PUVA has been used effectively in the treat,ment of vitiligo, but the mechanism by which PUVA stimulat.es melanocyte proliferation in vitiligo is not known. Several mechanisms have been suggested to be involved in the process of repigmentation of vitiligo. First, UV light, with or without psoralen, directly stimulates the proliferation of melanocytes. Secondly, PUVA may act. on epidermal keratinocytes or dermal components to stimulate t,hem to release certain melanocyte growth st,inulation factors that enhance the proliferation of melanocytes in depigmented lesions. Thirdly, PUVA irnmunologically leads to the impairment of epidermal Langerhans cell function and alteration of circulating T and B cell function, which results in the suppression of the stimuli is for rnelanocyte destruction during the therapy. OBJECTIVE: To test, th hypothesis that PUVA induced repigmentation in vitiligo results from the stimulation of growth factors that induce melanocyte proliferation, and that PUVA may suppress the immune reacticin to melanocytes, especially in autoantibody synt,hesis, we examined the effects of sera on the growth of epidermal melanocytes and control cells, and t,he incidence of antibodies to melanocyte and melanoma cells(SK-Mel 2~3) in the sera of patients with vitiligo. We also had normal control individuals and studied the changes of the antibody titer in the sera of patients with vitiligo. METHODS: The rate of H thymidine uptake was estimat,ed in cultured melanocytes and fibroblasts t,reated by patients sera before and after PUVA treatment. SDS-PAGE and immunoblotting analysis were used to idcntify anti pigment cell autoantibodies and were compared to the titers of autoantibodies after PUVA. RESULTS: 1. Melanocyte and fibrablast proliferation was increased by PUVA treated sera. Their proliferation was in proportion to the duration of the PUVA treatment. Melanocytes proliferated more than fibroblasts. 2. Significant differences between vitiligo patients and normal controls were found in the inci dence of anti-pigment cell antibodies. The antibodies were predominantly directed to melanocyte antigens of 110 kD, 65 kD, 45 kD and melanoma cell antigens of 110 kD, 103 kD, 88kD, 70 kD, 56 kD, 41 kD. 3. The titer of anti piment cell antibodies showed a tendency to decrease after PUVA treat- ment in most patients regardless of clinical improvement. Conclusion ; PUVA treated sera induced proliferation of melanocytes and fibroblasts and the production of aut,oantibodies was suppressed against pigment cell antigens through irnmunosuppression, which might help in the repigmentation of vitiligo.