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WEST CHESTER, PA, United States

Patent
Molecular Targeting Technologies, Inc. | Date: 2013-11-21

The invention provides novel multi-modality probes for pathologic cell tracking which allow labeling of dying cells with new probes and tracking them via non-invasive imaging techniques to diagnose ocular diseases, determine disease progression and evaluate effectiveness of treatment. The molecular probes of the invention can be topically, locally, or systemically administered for diagnosing and monitoring improvement or progression of any ocular diseases.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 222.58K | Year: 2014

Not Available


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 219.94K | Year: 2013

DESCRIPTION: Tracing neuronal connections with lipophilic carbocyanine dyes has revolutionized neuroanatomical tract tracing and is an essential feature to understand development of brain connections in both control and mutant mice in particular since multiple colors of dyes can be used. More recently, lipophilic dyes have also gained ground in labeling blood vessels by directly staining the endothelial cell membranes upon contact. A persistent problem that blocks even wider use of these extremely successful dyes is their limited combination with other procedures such as immunostaining or detailed histology that requires dehydration for embedding, since the dye molecules are not permanently bound to the membranes and can either leak out or be easily washedaway by lipophilic solvents or detergents. Attempts to overcome these problems have thus far been at best partially successful. Therefore, to broaden even further the use of carbocyanine lipophilic dyes we propose to develop fixable carbocyanine dyes thatcan be bonded to lysine groups in membrane bound proteins thereby retaining the dyes in the membranes even after the lipid bilayers has been removed with detergents or organic solvents. We plan to optimize the use of these dyes by developing a protocol that allows combination of multiple distinct fluorophores to maximize the information gained from a given model organism. Specifically, our Specific Aims are: (1) Synthesize four spectrally distinct fixable lipophilic dyes, three for nerve tract tracing andone or blood vessel labeling, that are compatible with standard fixation techniques used in tissue processing and immunocytochemistry protocols. Dyes synthesized will feature an aromatic N-hydroxysuccinimide ester group to provide covalent anchoring to membrane proteins. (2) Evaluate fixable dyes in standardized test systems in fixed tissue using processing techniques needed for high resolution histology at the light microscope level. In this aim we will characteriz how long these dyes are retained in tissue after treatment with organic solvents and detergents and how washing out progresses over time. We will also characterize the conditions under which these dyes can be best combined with each other and with immunochemistry and/or staining for dying cells.(3) Create test products to market each dye alone or in combination to the research community. We will develop test products to be sent to 5-10 collaborators in the neuroscience community along with the published protocol developed in SA2. Combined, thesethree aims will provide novel reagents useful for studies related to breaches in the blood-brain barrier such as tumors, trauma and neurodegenerative diseases as well as normal development. PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: Studying the normal and diseased brain with fluorescence imaging techniques requires probes with improved performance. We will generate high performance fluorescent dyes for membrane labeling of nerve tracts and blood vessels compatible with long-term tissuepreservation techniques and other known fluorescent cell markers. The possible multicolor labeling will provide detailed knowledge of the developing brain and disease models such as brain tumors, fetal alcohol syndrome, neurodegenerative diseases, traumaor other disruptions of the blood-brain barrier.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 193.51K | Year: 2009

DESCRIPTION (provided by applicant): Optical imaging is an advanced technique to image biotechnology products like gels, sensors, and micro arrays. It is also used increasingly to monitor specific molecular pathways in animals. This latter technique uses fluorescent dyes that absorb and emit near-infrared (NIR) radiation (700-900 nm) a spectral window where hemoglobin and water absorb minimally and therefore allow photons to penetrate several centimeters through tissue. Cyanine based NIR dyes (Cy7) are the popular ones among the very few commercially available fluorescent dyes showing NIR photophysical features. Unfortunately they are highly susceptible to degradation under biological conditions, and also known to undergo photochemical isomerization leading to nonfluorescent products. This proposal builds on our recent discovery that squaraine rotaxanes (SRs) are among the world's brightest and most stable fluorescent dyes. Current generation of SR dyes show absorption and emission profiles close to the Cy5 dyes. Herein we are proposing the synthesis of new generation NIR SR dyes which can be a superior substitute for Cy7 dyes. Our plan is to develop NIR absorbing bright and stable squaraine rotaxane dyes which can readily undergo conjugation with various biomolecules. They are likely to become superior replacements for the popular cyanine (Cy7) dyes and become extremely useful probes for both in vitro and in vivo imaging applications. The overall workplan for Phase I is to prepare novel bright and stable NIR-SR dyes and compare the properties of various bioconjugates that have been labeled with NIR-SR or Cy7. The two Specific Aims of the present proposal are: 1) SA1 to be performed at MTTI - Synthesize novel bright and stable NIR SR NHS esters and conjugate to 3 different types of biomolecules, a zinc coordinated dipicolylamine ligand (Zn-DPA), an antibody and a protein for comparison with Cy7 analogues. The first goal is to produce bright and stable NIR SR dyes and identifying the best two based on the chemical stability and brightness. This will be followed by conjugation of the squaraine rotaxane probes as well as Cy7 as a control probe to Zn-DPA, IgG protein and streptavidin . 2) SA2 to be perfomed at UND - In vitro evaluation of bioconjugates. In vitro molecular recognition performance of the NIR-SR and Cy7 fluorescent IgG and streptavidin bioconjugates developed at MTTI will be characterized using standard gel electrophoresis and streptavidin/biotin protocols. NIR-SR- and Cy7-Zn-DPA probes will be investigated for their interaction with S aureus and E coli bacteria membranes and their photostability using fluorescence microscopy. The goal is to demonstrate the advantage of using molecular probes labeled with an NIR-SR dye in 3 different systems compared with Cy7 dye. PUBLIC HEALTH RELEVANCE: The fluorescent probe developed by this research will be used in biotechnology as a stain for in vitro diagnostic devices, and in medical science as an in vivo probe to image disease in living animals. This optical imaging technology will help researchers discover new therapies and eventually it will help physicians choose the most appropriate therapy for a specific patient. This latter application is an example of how optical imaging will contribute to the evolving concept of personalized medicine.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 224.44K | Year: 2012

DESCRIPTION (provided by applicant): Although a rapid and accurate diagnosis is crucial to the management of patients suspected of bacterial infection, the currently available radiopharmaceuticals are not capable of distinguishing between sterile inflammation and bacterial infections. Our goal is to develop an infection-specific PET/SPECT radiopharmaceutical for eventual use in clinical practice. In Phase I, we will evaluate two independent approaches. In a covalent approach, we will conjugate a radionuclide chelator for PET/SPECT imaging with a Zn-DPA targeting moiety that is known to selectively target the negatively charged bacterial envelope, to provide a novel small molecule nuclear imaging agent. In an alternative, non-covalent radiolabeling approach,we will use streptavidin (SA) as a linker between the biotinylated Zn-DPA targeting motif and a biotinylated chelator to form an imaging agent which may have improved bacterial lesion accumulation over the covalent approach due to: (i) its slower pharmacokinetics because of increased size, and (ii) its potential to bind up to three DPA groups for affinity enhancement. Our Specific Aims include: 1) Synthesize and characterize DOTA- DPA-(1 Zn) for the covalent conjugation approach, DOTA/SA/DPA-(1 Zn) for thenon-covalent approach and radiolabel the DOTA containing agents with the PET isotope 68Ga, as well as the SPECT isotope 111In. 2) Serum stability assays and in vitro evaluation of the covalent [68Ga /111In-DOTA-DPA-(1 Zn)] and non- covalent [68Ga/111In-DOTA/SA/DPA-(1 Zn)] agents to S. pyogenes. Specific binding to bacteria will be evaluated by measuring binding to bacteria with increasing concentrations of unlabeled DOTA-DPA-Zn. Thereafter, labeled bacteria will be evaluated in 37o C serum environments to determine the stability of both radionuclide within the chelate and the stability of both agents to the bacteria. 3) Evaluate the covalent and non-covalent approaches in infection and inflammation mouse models for evidence of specific accumulations. SKH1 hairless mice will be injected in the thigh with live S. pyogenes to provide the bacterial infection model or lipopolysaccharide to provide the inflammation model. We will evaluate the agents radiolabeled with 68Ga as well as 111In in the mouse models usingsmall animal PET and SPECT/CT cameras respectively. In all cases, the location and extent of infection will be monitored by co-injecting PSVue(R) 794 (a fluorescent bacteria targeting probe) and imaging on a small animal optical camera. Agents will be evaluated for their pharmacokinetics, their accumulation in the target, their target thigh/contralateral normal thigh accumulation, evidence of specific infection imaging and sensitivity of detection. At sacrifice, full biodistributions of each radiolabel willbe done to supplement the imaging results. Key benchmarks for Phase I will be to obtain using either 68Ga/ 111In-DOTA- DPA-Zn or 68Ga/111In-DOTA/SA/DPA-Zn an infected thigh/normal thigh ratio of greater than 5 within a 10 h (68Ga) or 24 h (111In) period,obtain a statistically higher accumulation in the infected thighs compared to the inflammation thighs, and obtain an estimate of the lower limits of detection in the infection model. PUBLIC HEALTH RELEVANCE: Bacterial infection is one of the major causes of morbidity and mortality not only in developing countries but globally. Early diagnosis of infection and an ability to distinguish between bacterial infection and sterile inflammation is critical to the effective management of these patients. However, despite the efforts of many international imaging groups, there is currently no validated bacterial imaging agent that can distinguish infection from sterile inflammation. Obviously, the development of such an agent would greatly advance our ability todetect, localize, and quantify infections, to prescribe the appropriate treatment and to follow the patient throughout the treatment. In this project we propose to evaluate two novel approaches aimed at developing a new radiopharmaceutical which would allow noninvasive imaging of bacterial infections with the sensitivity that nuclear imaging approaches promise and would also allow infectious and inflammatory abscesses to be distinguished.

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