1990 — 1994 |
Straubinger, Robert M |
R29Activity Code Description: Undocumented code - click on the grant title for more information. |
Cell Biology/Therapy of Mycobacterial Infection in Aids @ State University of New York At Buffalo
Opportunistic infection (OI) by a variety of organisms represents the most frequent life-threatening manifestation of AIDS. Lacking a cure for the underlying viral infection, effective management of OI is required to sustain a high quality of life for those with AIDS. Infections such as M. avium-intracellular complex (MAI/MAC) are increasingly prevalent in AIDS, and may exacerbate the depression of host resistance to infection. Tissues rich in cells of the reticuloendothelial system (RES) typically contain high concentrations of MAI. At the cellular level, macrophages are replete with undigested intracellular bacteria. MAI is resistant to anti-tuberculosis drugs, and often to a wide range of antibiotics. New pharmaceuticals, optimized formulations, and a detailed understanding of the biology of intracellular parasites are required to attain the short- and long-term goals of control and cure. This research will contribute to the therapy of AIDS-related OI in two ways: (1) explore the cell biology of MAI, to develop new points of therapeutic attack on MAI specifically, and mycobacterial infections generally; (2) improve the effectiveness of therapy, using liposomes, a promising drug carrier system, to target the macrophage reservoir of MAI/MAC. (1) The mechanism by which intracellular parasites evade intracellular degradation is unknown, but important clinically. Intracellular vesicle traffic, phagosome-lysosome fusion, and lysosomal pH may be affected. Novel fluorescence assays, detecting phagocytosis of MAI, vesicular pH, and lysosomal enzyme content have been devised to examine the process of infection. Video fluorescence microscopy and image analysis will show MAI effects on specific compartment of living cells; previous assays often suffer the shortcoming of reporting an average effect on multiple cell compartments, and may hinder study of the true lesion in specific compartments. Understanding the effect of MAI on cellular antibacterial functions may reveal new points for pharmacologic intervention, and allow screening for biological response modifiers that reverse the inhibition of macrophage degradative capacity. (2) Liposomes are undergoing clinical trials as drug carriers for improved therapy of parasitic, fungal, and neoplastic diseases; antibiotics and a variety of biological response modifiers encapsulated in liposomes may be useful for treating OI of P. carinii, M. avium and T. gondii. Strong rationale for liposomes includes efficient natural targeting to the RES site of infection, reduction of adverse effects via site-specific delivery, prolonged lifetime of labile bio-active agents, and improved formulation of active but highly lipophilic drugs. Recent therapeutic experiments show efficacy of antibiotic-containing liposomes against MAI infections in liver and spleen. However, nothing is known of the efficiency with which MAI-infected cells bind and endocytose liposomes. Since endocytosis is required for optimal drug delivery, uptake and intracellular processing of liposomes by infected cells will be examined. Improving therapy of MAI in the lung, using aerosolized or lung-targeted liposomes, is a major objective for later years of the project, since high lung levels of liposomes are achieved by inhalation or intravenous injection.
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1 |
1991 — 1993 |
Straubinger, Robert M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Pharmocology and Efficacy of Novel Taxol Formulations @ State University of New York At Buffalo
We seek to improve the efficacy of a promising anticancer agent, taxol, by reformulation in a carrier-based system. Ongoing clinical trials show activity of taxol in the treatment of refractory human ovarian cancer,melanoma, and others. Dose-limiting side effects of taxol include myelosuppression, non-hematologic effects such as peripheral neuropathy, and alopecia; life-threatening epithelial necrosis in the gastrointestinal (GI) tract also has been noted. In addition to toxicity of the Cremophor vehicle, taxol is given by prolonged infusion; this limits not only comfort and convenience to the patient, but, more importantly, limits the opportunities for widening the taxol therapeutic index through alterations in route and schedule of administration that could optimize taxol pharmacokinetics. Several modern drug carrier systems, including cyclodextrins, polyethylene glycols, and lipid vesicles (liposomes), presently are under evaluation to reduce dose-limiting side effects of anticancer agents. Among these carriers, liposomes represent a mature, versatile technology for improved solubilization of lipophilic drugs such as adriamycin (ADR), Amphotericin B, and cyclosporine. ADR:liposome formulations have been examined in Phase I human trials, and show a reduction in ADR toxic side effects, particularly myelosuppression, epithelial necrosis in the GI tract, and alopecia. Human pharmacokinetic studies also reveal a reduction in biotransformation of liposome-encapsulated ADR to a metabolite believed to be inactive. Thus the reduced toxicity observed in clinical trials suggest the feasibility of further dose escalation for AFR reformulated in a liposome carrier system, as well as a prolonged circulation time for active drug. We propose to develop carrier formulations of taxol, with the primary aim of eliminating the toxic effects attributed to the Cremophor vehicle, and the secondary aim of modulating the toxicity, and perhaps the pharmacology, of taxol itself. First priority is given to optimizing formulations of taxol:liposomes already identified in preliminary studies; second priority is given to developing new-generation carrier formulations such as taxol:cyclodextrins, which may be more amenable to large-scale pharmaceutical production, and which may possess additional unique pharmacological properties. The development scheme first will evaluate cytostatic and cytotoxic potency of taxol formulations in vitro, on human ovarian cancer cell lines, and a murine melanoma and colon carcinoma. Sexond, we will optimize antitumor activity of the taxol:carrier formulations in murine tumor systems, and subsequently evaluate efficacy against a human ovarian tumor in athymic nude mice. Third, we will examine whether carrier formulations alter the pharmacokinetics and tissue distribution of active taxol.
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1 |
1996 — 1999 |
Straubinger, Robert M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Pharmacology and Efficacy of Novel Taxol Formulations @ State University of New York At Buffalo
Taxol, the first of an important new class of chemotherapeutic compounds, is highly-promising for a variety of malignancies, including refractory ovarian and breast cancer. However, efficacy is limited by delayed and acute toxicity. Myelosuppression, peripheral and sensory neuropathy, gastrointestinal epithelial necrosis, and alopecia are examples of delayed toxicity. Acute toxicity arises from the low solubility of taxol in water, which necessitates administration in Cremophor EL (polyethoxylated castor oil), an excipient that causes life-threatening hypersensitivity reactions (HSR). Severe pain is an additional dose-limiting toxicity hindering the use of this otherwise-promising agent in regional therapy (eg. i.p. for ovarian cancer). Prophylaxis against HSR involves slow drug infusion and premedication with antihistamines and glucocorticoids. Conceivably, pharmacokinetics and therapeutics of taxol may be complicated by interactions of the premedication regimen. We have developed taxol-liposome (T-lip) formulations that eliminate acute toxicity, reduce chronic toxicity, and in some cases enhance antitumor activity of taxol. The formulations are prepared in the lyophilized state, providing a scaleable, pharmaceutically-rational dosage form. Mice tolerate bolus i.v. doses of equal to or more than 5O mg/kg (or equal to or greater than 2OO mg/kg in split doses over 4 h), whereas conventional taxol is lethal at equal to or greater than 35 mg/kg. Delayed toxicity likewise is reduced, with a 2-7-fold increase in Maximum Tolerated Dose (MTD) for T-lip. Cumulative doses of 360 mg/kg T-lip were tolerated, whereas 180 mg/kg free taxol was lethal. Antitumor potency of T-lip formulations was tested against subcutaneous (s.c.) taxol-resistant murine Colon-26 (C-26), human ovarian tumors (A121a), and primary human lung tumors, as well as i.p. P388 leukemia. Potency of T-lip equals or exceeds that of free drug; against C-26, no dose of free taxol had a discernible effect, up to a lethal cumulative dose. In contrast, T-lip showed a significant reduction in tumor growth at doses equal to or greater than the MTD of free taxol. We have also developed formulations of other taxanes, with similar retention of antitumor effect. We have two main objectives: (l) investigate the pharmacology and pharmaceutics of existing T-lip formulations to determine the mechanism by which beneficial effects have been obtained; (2) develop new formulations, either optimized in general or for specific applications. Essential to achieving (1) is a comprehensive investigation of taxol biopharmaceutics. Efforts relating to T-lip pharmacology will investigate mechanisms responsible for the observed increase in MTD and efficacy. Pharmacokinetic approaches will be used to investigate the mechanisms of drug transport to tumor and tissues, and the stability of the drug:liposome complex in vivo. Physiologic pharmacodynamic models for T- lip action are the eventual objective of these studies. Objective (2), development of new formulations, builds on extensive ongoing biophysical studies, and includes aims to increase taxol content of T-lip, extend their circulating half-life, increase the deposition of drug in tumors, and investigate alternate technologies for production of T-lip with specific properties.
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1 |
1998 — 2000 |
Straubinger, Robert M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Taxol--Microtubule Physical Interactions @ State University of New York At Buffalo
The taxanes are an important new class of compounds showing great promise for cancer therapy. Taxol (paclitaxel), the prototype, is a complex diterpenoid natural product that has significant activity in refractory ovarian and breast cancer. Taxotere (docetaxel), a semisynthetic analogue, is in advanced clinical trial, and a large number of new active taxanes are in various stages of development. The subcellular target of taxane action is microtubules (MT), but the mechanism of action is unique, involving stabilization, rather than disassembly of microtubules. Mane details of taxane interaction with microtubules are unknown. Taxol binds to assembled MT, but not to tubulin heterodimer. Binding studies show maximal effects on MT at approximately 1:1 (mole:mole) taxol:tubulin heterodimer. Photoaffinity studies identified two peptide domains on beta tubulin as possible taxol contact sites: the N-terminal 33 amino acids, and residues 217-231. Most studies show photolabeling of beta tubulin alone; some show alpha tubulin labeling also. The molecular structure of beta tubulin and details of alpha/beta tubulin interaction are unknown. Thus the feasibility of a single taxane molecule contacting both peptides simultaneously is unclear. Nor is it known whether a single taxane molecule could interact simultaneously with both alpha and beta-tubulin. Recent solution conformation studies show that taxol in hydrophobic and aqueous environments adopts a "stacked" conformation. Hydrophobic forces and a mesh of net-like intermolecular H-bonds (involving both the taxane ring system and the C13 side chain) stabilize the structure. The stack conformation is amenable to infinite propagation by the addition of taxane monomer at the stack ends, and therefore could bridge considerable distances. Taxotere has a similar ability to form stacks, but inactive taxanes (Baccatin III) do not. We will investigate the conformation of taxol in the presence of MT, and test the hypothesis that taxane binding to microtubules involves taxane:taxane interaction. Fluorescent taxanes will be synthesized to probe microtubule interaction at low concentration. CD (Circular Dichroism), fluorescence, and NMR spectroscopy are the primary tools to be used to probe taxane conformation in the presence of MT, with the ultimate aim of providing molecular details of taxane-microtubule interaction. Preliminary CD results show the unique spectral signature of hydrophobically-localized taxol in the presence of MT. Elucidation of taxane: MT interaction details would have considerable relevance to understanding MT structure and function, and for the rational design of new taxanes.
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1 |
1999 |
Straubinger, Robert M |
S10Activity Code Description: To make available to institutions with a high concentration of NIH extramural research awards, research instruments which will be used on a shared basis. |
Circular Dichroism Spectropolarimeter @ State University of New York At Buffalo
In ten NIH-supported core projects conducted by the seven co- investigators of this proposal, Circular Dichroism (CD) spectropolarimetry provides a unique window into dynamic aspects of protein conformation, conformational transitions accompanying peptide- membrane interaction and insertion, nucleic acid-protein interaction, and investigations of the active conformation of drug molecules bound to their protein or nucleic acid targets. We seek to acquire a state-of- the-art CD spectropolarimeter to support and enhance these research programs. This instrument would replace an obsolete reconditioned instrument having only analog (chart paper) output and no programming or analysis capabilities, and which represents the sole CD resource for NIH-supported investigators in 7 departments and 4 divisions of this University who have extensive, demonstrable need, as well as experience with CD. The transition to modern instrumentation, which would provide digital data acquisition and computer-controlled experiment management, will not only enhance productivity by reducing the need for constant operator intervention, but also will make possible experimental approaches that are not feasible with the existing instrument. Repetitive scanning and digital averaging of spectra, accurate and programmable temperature control, stopped flow for kinetic studies, and an automated titrator for denaturation/refolding studies represent capabilities that will enhance greatly the existing NIH-supported projects.
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1 |
2000 |
Straubinger, Robert M |
S10Activity Code Description: To make available to institutions with a high concentration of NIH extramural research awards, research instruments which will be used on a shared basis. |
Liquid Chromatography/Mass Spectroscopy System @ State University of New York At Buffalo
In ten NIH-supported core projects conducted by the nine co- investigators of this proposal, the enhanced analytical sensitivity, high- throughput, and precise molecular mass determination of Liquid Chromatography/Tandem Mass Spectroscopy (LC/MS-MS) would provide essential capabilities to enhance these projects, given the common need for rapid and precise quantitation from biological samples of pharmacologica1 agents, their in vivo metabolites, or fragments of proteins and peptides. We seek to acquire state-of-the-art LC/MS-MS instrumentation to support and enhance these research programs, as well as open new research avenues in basic molecular biochemistry. This instrument would represent the only research-grade LC/MS-MS available in a wide geographical area. It will be integrated into a central core facility, which would provide these services to a concentrated population of researchers engaged in basic and applied biomedical research who have extensive, demonstrable need for LC/MS-MS capabilities. The availability of this modem instrumentation, which would provide high- throughput analytical capabilities and computer-controlled data acquisition/management, would not only enhance productivity by eliminating more tedious, labor intensive, and less sensitive Liquid Chromatography (LC) -based assays, but would also make possible experimental approaches that are not feasible with any existing instrument. Furthermore, acquisition of this instrument would have a direct impact on human well-being, given the role of some of these investigators in ongoing clinical trials of the NIH, in the area of pharmacotherapy for MDS and drug abuse. Therefore, these existing and potential research capabilities will enhance greatly a substantial number of existing, pending, and planned NIH-supported projects of broad scope and impact.
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1 |
2001 |
Straubinger, Robert M |
S10Activity Code Description: To make available to institutions with a high concentration of NIH extramural research awards, research instruments which will be used on a shared basis. |
Chemical, Biological and Drug Discovery Computing System @ State University of New York At Buffalo
For 135 users in 45 faculty laboratories engaged in more than 50 NIH- supported projects, computation plays a central role in advancing knowledge in the chemical, biological, and pharmaceutical fields. Computational chemistry, investigations of molecular interactions, and molecular visualization are major applications. There exists a pressing need for a distributed system of state-of-the-art computational hardware to support the needs of these projects. This need has been heightened by several factors. First, multiple redundant software licenses for computational packages have been coalesced, providing additional software capabilities but increasing the number of users and straining workstation capacity. Second, the elimination of redundant software from multiple servers has placed greater load on a single temporary server. Third, upgrading of the software has made obsolete some lower-powered workstations in centralized computational locations. We seek to address this need through the purchase of (1) a high-performance multi-processor server with adequate memory and disk storage resources to support the needs of the user group, and (2) adequately-equipped workstations and a graphics output device which will be located in an existing central site. System support will be provided by an experienced, centralized staff of 13, and financial operations will be managed through a structure that has provided for shared computational facilities for more than 12 years. The proposed hardware acquisition, coupled with user-supported software, would provide greater processing power, enhanced visualization capabilities, and a platform for development of parallel processing approaches that can be transitioned to the University's supercomputers. Its availability would not only enhance productivity, but also make a wide range of advanced software available to a significant number of NIH-supported users.
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1 |
2002 |
Straubinger, Robert M |
S10Activity Code Description: To make available to institutions with a high concentration of NIH extramural research awards, research instruments which will be used on a shared basis. |
Stopped Flow Spectrofluorometer @ State University of New York At Buffalo
In ten NIH-supported core projects conducted by the 9 co-investigators of this proposal, fluorescence spectroscopy provides a unique window into dynamic aspects of protein conformational transitions accompanying peptide-membrane interaction and insertion, nucleic acid- protein interaction, and investigations of the active conformation of drug molecules bound to their protein or nucleic acid targets. We seek to acquire a state-of-the-art fluoresc4ence spectrometer to support and enhance these research programs. This instrument would replace an aging fluorescence spectrometer that is now prone to breakdown. Also, it is controlled by an obsolete, non-Y2K-compliant computer for which replacement parts are unavailable. The existing instrument has been operated as part of a shared facility for approximately 6 years; in that time it has served more than 20 investigators from in 8 departments and 4 divisions of this University, and over time has provided support to 20 or more NIH-supported projects. The instrument to be purchased will be equipped to enable fluorescence anisotropy measurements, and will have a stopped-flow accessory to support those projects requiring rapid kinetics measurements. Diode array detectors and dual monochromators will enable rapid acquisition of entire spectra at short time scales. Absorbance measurements will be possible, enabling the monitoring of self-assembly reactions or aggregation. This instrument will support a principal group of 7 NIH-supported investigators and two additional core investigators and two additional core investigators. Among them there is both have extensive, demonstrable need, as well as experience with fluorescence and stopped-flow techniques. The transition to modern, reliable instrumentation with computer-controlled data acquisition will not only make possible experimental approaches that are not feasible with the existing instrument, but also enhance productivity by reducing down time for repairs and the fairly constant drain of time devoted to scavenging functional parts for the obsolete computer system. Over all, extensive computer-controlled data acquisition, rapid data acquisition, and stopped-flow fluorescence represent capabilities that will enhance greatly the existing NIH0-supported projects.
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1 |
2004 — 2008 |
Straubinger, Robert M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Drug Carrier of Blood/Tumor/Brain Permeability @ State University of New York At Buffalo
DESCRIPTION (provided by applicant): Drug carriers can alter pharmacology and confer novel mechanisms of action upon agents having properties that compliment the characteristics of the carrier. Doxorubicin encapsulated within long-circulating liposomes by means of a "remote loading" method (L-DXR) represents the first in a new class of anticancer agents, and was recently approved by the FDA. However, its full spectrum of action and mechanisms remain to be defined. Previously we demonstrated substantial extension of lifespan for rats bearing an orthotopically-implanted drug-resistant brain tumor when administered L-DXR, but not free DXR. We have recently observed that repetitive doses of L-DXR (but not equivalent doses of free DXR) increase tumor vascular permeability and mediate vascular barrier breakdown. Normal vasculature appears to be unaffected. This effect, unprecedented in the literature, has important clinical and basic implications. The objectives of this proposal are (a) to understand the mechanistic basis by which this drug carrier system confers upon DXR the property of compromising vascular permeability, and (b) to determine the functional consequences of this effect. In particular, we will (c) explore the potential to enhance therapy through rational application of tumor vascular barrier breakdown, not only to optimize tumor deposition and therapeutic effect of L-DXR itself, but also to promote the therapeutic effects of other agents. Selective tumor vascular barrier breakdown could improve the penetration and effects of gene-carrier systems, and could increase the sensitivity of tumors to drugs having complimentary mechanisms of action, such as antiangiogenic agents. Vascular permeability changes resulting from repetitive L-DXR treatment will be investigated using a series of permeability probes and immunohistological approaches. Free DXR will be used as a control. Functional Magnetic Resonance (fMR) imaging will enable dynamic observation of tumor perfusion and vascular permeability changes. Effects of the vascular permeability compromise on the activity of potentially complementary agents will be investigated using TNP-470 and paclitaxel; the former is a well-characterized antiangiogenic agent, while the latter is an active chemotherapeutic agent having some antiangiogenic properties. The effect of vascular permeability changes on delivery by other macromolecular carriers will be probed using viral vectors carrying model genes.
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1 |
2005 |
Straubinger, Robert M |
S10Activity Code Description: To make available to institutions with a high concentration of NIH extramural research awards, research instruments which will be used on a shared basis. |
Lc/Quadrupole Ion Trap Mass Spectroscopy System: Pharamaceutical Sciences @ State University of New York At Buffalo |
1 |
2005 |
Straubinger, Robert M |
S10Activity Code Description: To make available to institutions with a high concentration of NIH extramural research awards, research instruments which will be used on a shared basis. |
Lc/Quadrupole Ion Trap Mass Spectroscopy System: Proteomics @ State University of New York At Buffalo |
1 |
2005 |
Straubinger, Robert M |
S10Activity Code Description: To make available to institutions with a high concentration of NIH extramural research awards, research instruments which will be used on a shared basis. |
Lc/Quadrupole Ion Trap Mass Spectroscopy System: Aids @ State University of New York At Buffalo |
1 |
2005 |
Straubinger, Robert M |
S10Activity Code Description: To make available to institutions with a high concentration of NIH extramural research awards, research instruments which will be used on a shared basis. |
Lc/Quadrupole Ion Trap Mass Spectroscopy System @ State University of New York At Buffalo
DESCRIPTION (provided by applicant): For the selection of nine core NIH-supported projects described in this proposal, the liquid chromatography/quadrupole ion trap (LC-ion trap MS-n) mass spectroscopy system proposed would provide major enhancement. For the 14 co-investigators, key features of the proposed instrument include high combined sensitivity/scan speed in full-scan mode, multi-stage fragmentation (Ms-n), and data-dependent acquisition in automated scan mode. The projects have in common the need for identification of pharmacological agents and their in vivo metabolites, as well as their effect on cellular responses at the protein level. A major use of the instrument will be the identification and characterization of low copy number proteins and peptides in biological matrices, using methods we are developing. The specific instrument we propose is the Thermo Finnigan LCQ Deca XP ion trap MS. It would be unique in the extended geographic region of Western New York, and would be the only instrument of its kind available on a shared basis for investigators who require the direct access to the instrument and the extended use time necessary to develop new analytical approaches for protein and peptide analysis. It will be integrated into the existing Pharmaceutical Sciences mass spectrometry facility, consisting of two triple quadrupole LC/MS-MS instruments. This facility provides instrument access to a diverse population of researchers engaged in basic, applied, and clinical biomedical research, as well as analytical mass spectrometry. The facility is highly utilized, providing investigators with 4,000 hours of data acquisition on the existing LC/triple quad instruments in the last calendar year. Mass spectrometric analysis is a mainstay of many NIH-supported projects, but this region and university lacks the complementary capabilities that the Deca XP ion trap instrument will provide. Proteomics and protein chemical analysis projects are hindered by the lack of an instrument well suited for identification and characterization of proteins and peptides. Furthermore, no facility exists to provide the extended access necessary to support the development of new methods for quantitation of low-copy cellular protein transcripts or therapeutically important protein drugs. The proposed instrumentation would also enable more facile identification and characterization of small-molecule drugs and their metabolites. Given the involvement of our investigators in studies of high clinical relevance, the proposed instrument would have direct, major impact on the development of new therapies for life-threatening diseases.
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1 |
2007 |
Straubinger, Robert M |
S10Activity Code Description: To make available to institutions with a high concentration of NIH extramural research awards, research instruments which will be used on a shared basis. |
High Sensitivity Liquid Chromatography Tandem Mass Spectrometry System @ State University of New York At Buffalo
[unreadable] DESCRIPTION (provided by applicant): For 8 core NIH-supported laboratories and 11 investigators described in this proposal, liquid chromatography/triple quadrupole mass spectrometry (LC/MS-MS) plays an important role in the quantification of drugs metabolites, and peptides. Sensitivity and selectivity often presents a problem because the analytes are either intrinsically low in abundance, or sample size is very limited, and the analytes generally are present in highly complex biological matrices. The major use of proposed instrument will be the quantification of conventional small-molecule analytes in both basic and clinical NIH-supported projects. A second use will be the quantification of therapeutic and functional proteins in complex biological matrices such as tissues, using methods we are developing. Two instruments were considered for this purpose: the Thermo Finnigan Quantum Ultra AM MS/MS, with the accurate mass measurement option, and the Applied Biosystems API5000 MS/MS. Both instruments represent a significant advance in the state-of-the-art, but present distinct advantages and compromises. Our evaluation of these instruments and the decision as to which instrument best serves the needs of our users is described. The proposed instrument will be integrated into the existing Pharmaceutical Sciences mass spectrometry facility, which is the most comprehensive and heavily utilized academic research facility in the extended geographic region of Western New York. It currently houses three triple quadrupole (LC-MS/MS) instruments, an ion trap (LC/MSn) system, and a newly-acquired linear trap/quadrupole system with multidimensional LC capabilities (MDLC/LTQ). This facility provides instrument access to a diverse population of researchers engaged in basic, applied, and clinical biomedical research. The facility is highly utilized, and has provided investigators with an average of at least 1600 hours of useful data acquisition per instrument over the last 3 calendar years. However, the facility lacks an instrument possessing the sensitivity and selectivity that the proposed instrument would bring, and limitations in the sensitivity and selectivity of the existing MS equipment hinders the described projects in key ways, most often when sample volumes are limited, e.g. in pediatric clinical trials or in small rodent disease models. Furthermore, quantification of marker peptides for target proteins of interest is a growing activity in our facility, and often the proteins of greatest interest are those of low abundance. No other regional facility exists to provide the extended access necessary to support the development of new methods for quantification of low-copy cellular protein transcripts or therapeutically important protein drugs. Given the involvement of our investigators in studies of both basic and clinical sciences, the proposed instrument would have direct, major impact on the development of new therapies for life-threatening diseases, as well as on our basic understanding of biochemical and physiological processes. Relevance The ability to quantify drugs, their metabolites, and/or protein biomarkers in cells and tissues, with high sensitivity and selectivity, is an essential and common need of many research projects that aim to improve the therapy or diagnosis of diseases, or to understand the basic mechanisms biological processes. The proposed instrument will bring to this geographic area the state-of-the art instrumentation necessary, thereby advancing many projects that seek to improve the therapy of serious diseases such as AIDS and cancer. [unreadable] [unreadable] [unreadable]
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1 |
2009 — 2010 |
Qu, Jun Straubinger, Robert M Thompson, Alexis Cassandra (co-PI) [⬀] |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Peripheral Biomarkers of Cocaine Dependence and Relapse @ State University of New York At Buffalo
DESCRIPTION (provided by applicant): The goal of this application is to identify candidate biomarkers which, in combination, may constitute a lingering biosignature that is informative of cocaine abuse history over a protracted period of time. The neurochemical and behavioral manifestations of chronic cocaine abuse are known to endure for a considerable duration, and given the role of neuropeptides and other secreted proteins in compulsive behaviors, drug abuse, and addiction, we hypothesize that the proteome of biophases that can be obtained, by minimally-invasive means, may contain families of biomarkers that reflect the well-established neurochemical alterations and constitute a peripherally-accessable cocaine abuse biosignature. Our approach is to apply recent inter-related analytical advances in sample processing, nano-scale liquid chromatography (nano-LC), and mass spectrometry (MS) that enhance the suitability of nano-LC/MS approaches for the analysis of highly complex proteomic samples such as tissues or bodily fluids. These advances provide: (i) highly quantitative recovery of tissue or sample proteins, (ii) highly reproducible, selective, and sensitive expression profiling of proteins, (iii) quantification of significantly greater numbers of proteins, and (iv) highly sensitive, multiplexed quantification of specific proteins of interest and (v) their PTM status. In the context of a well-controlled and validated rat model of cocaine abuse and withdrawal, samples will be obtained from naive- and drug-withdrawn animals both before and during a relapse of cocaine administration. Proteomes of select neural centers and blood will be contrasted to assemble lists of abuse-selective biomarker candidates, and their importance will be ranked by the commonality among replicates, the magnitude of change, and bioinformatic criteria relating them plausibly as drug-abuse responsive. In the second phase, the top-prioritized candidates will be confirmed and absolute quantification will be performed;matched plasma samples will be interrogated for these candidate biomarkers to identify promising constituents of a biosignature. Finally, using a rat model of addiction involving self- administration, we will attempt to refine biosignature candidates in terms of use/abuse vs. dependence/addiction, to determinate the exclusivity of these markers for drug addiction. By employing these linked analytical technologies and a well-validated animal model, we aim to investigate, identify, and validate potential biosignatures of cocaine abuse and relapse, based upon differential comparison of proteins in peripheral blood samples, which could aid in diagnosis, evaluation of therapy, and monitoring of recovery. PUBLIC HEALTH RELEVANCE: Chronic use of drugs of abuse represents a scourge to users and society. The ability to obtain information about use patterns beyond the period of time in which drug or metabolites are detectable in blood would have broad applications in diagnosis, estimation of prognosis, personalization of treatment, evaluation of drug effects, and detection of relapse. We propose to combine novel analytical advances in liquid chromatography, mass spectrometry (LC/MS), and sample preparation, along with a series of validated animal models, to interrogate samples that are obtainable by minimally-invasive means, such as blood, to achieve the elusive goal of identifying families of persistent biomarkers, comprising a biosignature, of chronic cocaine abuse and addiction.
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1 |
2009 |
Straubinger, Robert M |
S10Activity Code Description: To make available to institutions with a high concentration of NIH extramural research awards, research instruments which will be used on a shared basis. |
High Performance Computational System to Support Lcms/Proteomics Analysis @ State University of New York At Buffalo
DESCRIPTION (provided by applicant): The University at Buffalo (UB) and regional partners Roswell Park Cancer Inst. (RPCI) and Hauptman-Wood- ward Research Inst. (HWI) are joined in a major continuing investment in infrastructure to support proteomic research directed toward basic, applied, and clinical biomedical applications. A distributed regional network of instrumentation provides access to state-of-the-art liquid chromatography/mass spectrometry (LC/MS) instrumentation. Key instruments generating proteomics data for our consortium of investigators include a Waters LC-QTOF Premier (RPCI), a Thermo LTQ-XL linear ion trap equipped with Electron Transfer Dissociation and multidimensional nano-flow LC (UB Pharmaceutical Sciences Instrumentation Core), and a Thermo LTQ Orbitrap with ion chromatography fractionation and nano-flow LC in UB's new NY State Center of Excellence in Bioinformatics &Life Sciences (CBLS), located physically contiguous to the RPCI campus. Additional LC/MS instruments support the ongoing proteomics research and provide enhanced quantitative capabilities. These include four LC/triple-quadrupole MS (2 API3000 and two Thermo Quantum Ultra EMR) in the UB Pharmaceutical Sciences Core, an API3000 and Thermo Quantum Ultra at RPCI. For this multi-institutional consortium of NIH-supported laboratories, computational analysis of the large proteomics data sets acquired from basic and clinical research samples presents a severe bottleneck. Because of the nature of the research, analyses of experimental data using SEQUEST can require hours to days of computation on standard high-end workstations, depending on the matrix complexity and peptide modifications expected, the use of reagents such as ICAT or iTRAQ to quantify relative expression, and the proteome under study. To alleviate this bottleneck, we propose to acquire a dedicated high-performance computing (HPC) cluster specifically designed to accelerate LC/MS proteomic data analysis. Cluster-enabled software is in hand or committed for acquisition, and includes SEQUEST, X!Tandem, Mascot, and Scaffold. The proposed grid- enabled computing system will be housed in the UB Center for Computational Research (CCR), an established HPC research center located in close proximity to the LC/MS proteomics facilities. Adequate mass storage and backup facilities will be integrated into the proposed HPC cluster. Workstations placed strategically at the sites of data acquisition will provide decentralized access to the HPC system for investigators. Benchmarking of the proteomics software reveals that a considerable acceleration of MS data analysis can be achieved by acquisition of the proposed system, which exploits advances in quad-core processor technology and leverages very substantial existing CCR infrastructure. Given the emphasis of our investigators in both basic and clinical sciences, the proposed equipment will have direct, major impact on the development of new therapies for life-threatening diseases, as well as on our understanding of basic biochemical and physiological processes. PUBLIC HEALTH RELEVANCE: The analysis of protein expression and post-translational modifications (PTMs) on a proteome-wide scale represents an important emerging technique that will assist in our understanding of fundamental biochemical processes, networks of cellular responses, and the impact of disease processes upon them. This information can also contribute to the development of new, mechanistically-targeted drugs. Current and pending NIH-supported research projects are hampered by bottlenecks in the computational analysis of the large data sets and/or complex PTMs that are encountered in proteomics research. The proposed grid-enable high-performance computing cluster and distributed processing network will effectively address this computational bottleneck for our regional proteomics research consortium, and thereby directly advance the numerous NIH- supported projects aimed at improving the therapy of serious diseases such as AIDS, cardiovascular disease, and cancer.
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1 |
2010 |
Straubinger, Robert M |
S10Activity Code Description: To make available to institutions with a high concentration of NIH extramural research awards, research instruments which will be used on a shared basis. |
Circular Dichroism Spectropolarimeter With Fluorescence Acquisition and Temperatu @ State University of New York At Buffalo
DESCRIPTION (provided by applicant): In the NIH-supported core projects conducted by the eight co-investigators of this proposal, Circular Dichroism (CD) spectropolarimetry provides a unique window into dynamic aspects of protein conformation and unfolding, conformational transitions accompanying peptide-membrane interaction and insertion, the development of less immunogenic, more effective therapeutic protein formulations, and the discovery of alternative formulations for poorly-soluble anticancer drugs. We seek to acquire a state-of-the-art CD spectropolarimeter to support and enhance these research programs. This instrument will provide convenient access to a well-supported but under-served group of investigators on one of three university campuses that are spread across this regional geographic area. Specific accessories on the instrument will make possible unique experimental approaches, such as programmed repetitive, simultaneous analysis of multiple samples, programmed thermal ramping studies to probe macromolecule conformation, acquisition of fluorescence emission data interleaved with CD measurements, and inclusion of an automated titrator for denaturation/refolding studies. These capabilities will enhance greatly the existing NIH-supported projects. The instrument will be operated by the Pharmaceutical Sciences Instrumentation Facility, which has operated successfully since 1995, when it brought the first shared-access CD instrument to the Western New York region. The facility has provided open access to users from the University at Buffalo (UB), our regional partners Roswell Park Cancer Inst. (RPCI) and Hauptman-Woodward Research Inst. (HWI), and occasional local college users. The current active user base is approximately 24 lab groups, the majority of which are NIH-funded. The facility also provides professional staff to train and assist new users, or perform collaborative work with less frequent users. Faculty associated with this facility and proposal also includes CD-related instruction in graduate-level courses, to provide graduate- and postdoctoral trainees with a sound basis of the theory and the potential applications of CD in their research projects.
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2012 — 2013 |
Ma, Wen Wee Straubinger, Robert M |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Pancreas Cancer Combination Therapy Based On Stromal Modulators and Nano-Carriers @ State University of New York At Buffalo
DESCRIPTION (provided by applicant): Pancreatic cancer is the 4th leading cause of cancer deaths and the mortality rate matches the incidence. The majority of patients have advanced disease at diagnosis and the 2-year survival is a dismal 6%. A number of factors contribute to the challenges in the treatment. One is the disease's genetic heterogeneity such that one size fit all approach to treatment development has yielded limited progress and personalized treatment based on individual patient's cancer biology is needed. The other significant challenge is the poor blood supply and perfusion to pancreatic tumor that inhibits the delivery of effective chemotherapeutic drugs. Tumor stroma is increasingly recognized as a collaborator with malignant cells, establishing the microenvironment that promotes metastasis, invasion, and treatment resistance. Stroma is thus a novel target in the treatment of pancreatic cancer. Here we propose to investigate a novel sequential pharmacologic approach to exploit this potential vulnerability: pretreatment with an anti-stromal agent that breaches the tumor-vascular barrier thereby increasing tumor perfusion and permeability, and then followed by administration of an FDA-approved nanoparticulate carrier. The latter can deposit significant concentrations of drug, thereby establishing a persistent slow-release depot in the tumor enabling continuous cell kill. Previously we have demonstrated in a brain tumor model that this intra-tumor depot leads to progressive compromise of tumor vascular integrity. Our preliminary data establish that inhibitors of Sonic Hedgehog (sHH) signaling mediate an increase in tumor vascular permeability in low-passage, patient-derived pancreatic cancers in SCID mice, which have very low intrinsic vascularization. The intra-tumoral deposition of a sterically-stabilized nano-liposomal formulation containing doxorubicin (SSL-DXR) is dramatically increased in animals pre-treated with an sHH inhibitor, leading to an initial shutdown in vascular permeability/perfusion and subsequent tumor regression. Here we propose to investigate the mechanisms by which this sequential therapy mediates the observed effects, optimize the effectiveness of treatment, and survey a broader range of patent-derived pancreatic cancers to ascertain the general effectiveness of this approach. We will also examine the effects of treatment in a genetically engineered model of pancreatic cancer that faithfully recapitulates the stromal amplification and poor vascular permeability/perfusion that are hallmarks of the human disease. Our objective is to lay the groundwork that would provide strong rationale for clinical evaluation of sequential pharmacologic-based treatment of pancreatic cancer by combining SHH inhibitors in a specific sequence with a first-in-class, FDA- approved, nanoparticulate drug delivery agent.
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2015 — 2016 |
Barron, Niall Ma, Wen Wee Scott, Chris Straubinger, Robert M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Tumor Priming Sequences Combined With Novel Nanoparticle Drug Carriers For Enhanced Therapeutic Efficacy in Pancreatic Cancer: a Tripartite Usa/Northern Ireland/Republic of Ireland Consortium @ State University of New York At Buffalo
? DESCRIPTION (provided by applicant): Pancreatic cancer (PaCA) has significant incidence, and is usually advanced at diagnosis. Regardless of treatment, 94% die within 2 yrs. Treatment challenges include tumor genetic heterogeneity, so that one size fit all treatment approaches are a dismal failure. Personalizing treatment based on patients' cancer biology is needed. Another challenge is that poor tumor blood perfusion limits delivery of effective chemotherapeutic drugs. Tumor stromal cells collaborate with malignant cells to establish a microenvironment that promotes metastasis, invasion, and treatment resistance, and therefore constitutes an additional target in PaCA treatment. We hypothesize that a specific, sequential, pharmacology-based combination approach will exploit this potential vulnerability: first, a `tumor priming' step will be employed to compromise vascular barrier properties. When vascular compromise is achieved, a persistent, intra-tumor drug depot will be established to extend pharmacological exposure of the tumor cells, even if the barrier is re-established. Nanoparticulate drug carriers containing high drug cargo loads will be employed to achieve this objective. Employing low-passage, patient-derived (PDX) tumor models, Aim 1 will investigate the tumor priming characteristics of two distinct types of agents: a stroma-modifying drug and a clinically-approved chemotherapeutic that primes by inducing tumor decompression. The key objectives are to understand the temporal interrelationships of (i) the primary pharmacological effects that lead to (ii) the tumor priming effect and (iii) enhanced nanoparticle deposition. Of particular interest is (iv) the role of discharging tumor interstitial pressure in the temporal onst (and waning) of the priming effect. A novel, quantitative pharmacological model will be developed to interrelate all these constituent effects into a predictive framework so that mathematical simulation can be used to develop testable hypotheses as to the appropriate priming and therapeutic sequence. In Aim 2 we will investigate distinct drug-containing nanoparticles for their efficacy; high drug-content sterically-stabilized liposomes (SSL) and PEGylated polymeric nanoparticles will be investigated for their therapeutic and mechanistic effects in a tumor- priming regimen. These formulations enable us to test the hypothesis that the aggregate properties of drug and carrier, rather than those of drug alone, determine the tumor pharmacological effects observed. In Aim 3 we will test the hypothesis that under tumor priming conditions, tumor clearance (efflux) of therapeutic nanoparticles can be reduced by employing a tumor- or stroma-targeting ligand, resulting in significant increases in efficacy. Cetuximab- (anti EGFR) bearing nanoparticles will provide proof-of-concept, owing to the predominance of EGFR expression in PaCA. Novel targeting ligands (such as anti-Death Receptor 5) will be explored also. These studies will provide a comprehensive understanding of the `tumor priming' phenomenon to enable their rational design, as well as a strong rationale for therapeutic combinations and agents that could rapidly transition to clinical evaluation in PaCA, a cancer of nearly uniform lethality.
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2017 — 2019 |
Barron, Niall Ma, Wen Wee Scott, Chris Straubinger, Robert M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Tumor Priming Sequences Combined With Novel Oparticle Drug Carriers For Enhanced Therapeutic Efficacy in Pancreatic Cancer: a Tripartite Usa/Northern Ireland/Republic of Ireland Consortium @ State University of New York At Buffalo
? DESCRIPTION (provided by applicant): Pancreatic cancer (PaCA) has significant incidence, and is usually advanced at diagnosis. Regardless of treatment, 94% die within 2 yrs. Treatment challenges include tumor genetic heterogeneity, so that one size fit all treatment approaches are a dismal failure. Personalizing treatment based on patients' cancer biology is needed. Another challenge is that poor tumor blood perfusion limits delivery of effective chemotherapeutic drugs. Tumor stromal cells collaborate with malignant cells to establish a microenvironment that promotes metastasis, invasion, and treatment resistance, and therefore constitutes an additional target in PaCA treatment. We hypothesize that a specific, sequential, pharmacology-based combination approach will exploit this potential vulnerability: first, a `tumor priming' step will be employed to compromise vascular barrier properties. When vascular compromise is achieved, a persistent, intra-tumor drug depot will be established to extend pharmacological exposure of the tumor cells, even if the barrier is re-established. Nanoparticulate drug carriers containing high drug cargo loads will be employed to achieve this objective. Employing low-passage, patient-derived (PDX) tumor models, Aim 1 will investigate the tumor priming characteristics of two distinct types of agents: a stroma-modifying drug and a clinically-approved chemotherapeutic that primes by inducing tumor decompression. The key objectives are to understand the temporal interrelationships of (i) the primary pharmacological effects that lead to (ii) the tumor priming effect and (iii) enhanced nanoparticle deposition. Of particular interest is (iv) the role of discharging tumor interstitial pressure in the temporal onst (and waning) of the priming effect. A novel, quantitative pharmacological model will be developed to interrelate all these constituent effects into a predictive framework so that mathematical simulation can be used to develop testable hypotheses as to the appropriate priming and therapeutic sequence. In Aim 2 we will investigate distinct drug-containing nanoparticles for their efficacy; high drug-content sterically-stabilized liposomes (SSL) and PEGylated polymeric nanoparticles will be investigated for their therapeutic and mechanistic effects in a tumor- priming regimen. These formulations enable us to test the hypothesis that the aggregate properties of drug and carrier, rather than those of drug alone, determine the tumor pharmacological effects observed. In Aim 3 we will test the hypothesis that under tumor priming conditions, tumor clearance (efflux) of therapeutic nanoparticles can be reduced by employing a tumor- or stroma-targeting ligand, resulting in significant increases in efficacy. Cetuximab- (anti EGFR) bearing nanoparticles will provide proof-of-concept, owing to the predominance of EGFR expression in PaCA. Novel targeting ligands (such as anti-Death Receptor 5) will be explored also. These studies will provide a comprehensive understanding of the `tumor priming' phenomenon to enable their rational design, as well as a strong rationale for therapeutic combinations and agents that could rapidly transition to clinical evaluation in PaCA, a cancer of nearly uniform lethality.
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2019 — 2020 |
Mager, Donald E (co-PI) [⬀] Qu, Jun Straubinger, Robert M |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Large-Scale Proteome-Wide Analysis With High Accuracy/Precision to Guide Pancreatic Cancer Therapy Development @ State University of New York At Buffalo
Quantitative profiling of the expressed proteome of cancer/cancer-associated cells has potentially transforma- tive applications in cancer research, treatment development, and individualization of therapy, potentially provid- ing (i) a molecular-level understanding of cancer subtypes and how they respond to standard-of-care (SoC) therapy or new agents, (ii) insights into tumor cell/stromal interactions that govern responses to therapy, and (iii) how signaling and drug response networks are `wired' in both genetically-mutated cancer cells and in non- cancer stromal cells. Given the prevalent discordance between genetic/transcriptomic analysis and expressed tissue proteomes, we hypothesize that large scale, quantitative proteomic analysis that interrogates the expressed proteome can provide an in-depth, high-resolution understanding of tumor and associated cells respond to chemotherapy drugs. We hypothesize that detailed understanding of SoC chemotherapy responses will also assist in selection or repurposing of new agents into treatment regimens to enhance response by influencing specific cellular response networks. We will employ a novel workflow, IonStar, that incorporates analytical, technological and informatics advances to enable robust simultaneous quantification of a majority of the expressed tumor proteome at high sensitivity, accuracy, and reproducibility, in large sample cohorts, in order to capture concentration- and time-dependent responses of patient-derived xenograft (PDX) models of pancreatic adenocarcinoma (PDAC) to SoC regimens. IonStar is a robust, well-validated sample processing, analysis, and informatics workflow that currently quantifies 5-7,000 proteins in 80-100 biological sample batches, with >95% of proteins free of missing data, at high accuracy and reproducibility, and a <5% false- positive biomarker discovery rate, without prior sample fractionation. IonStar permits discrimination of tumor (human cell) and stromal (mouse) responses. We will obtain rich data sets that capture responses of multiple PDAC PDX isolates to SoC drugs gemcitabine and paclitaxel (Abraxane) at high temporal resolution, providing a foundation for understanding tumor and stromal responses to these agents, and how PDAC PDX isolates differ in their responses. The phosphoproteome and transcriptome will also be interrogated at specific times to provide overlapping data. Novel mathematical, systems-level pharmacodynamic models integrated with traditional network informatics approaches will accelerate knowledge extraction by imposing quantitative pharmacodynamic response mechanisms upon individual and clustered protein responses. We hypothesize this approach will address the challenge of proteomic analysis that key signaling and response network nodes may not always be represented within datasets, confounding conventional network analysis. With well- validated, sensitive, accurate, and reproducible high-throughput workflows coupled with novel systems pharmacological analysis, we will use the insights garnered to create testable predictions of chemotherapy combinations that are more efficacious in PDAC, or in particular PDAC patient isolates.
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