Biomedical subjects

Robert M Straubinger

Publications and source records attributed to Robert M Straubinger.

11 recordsLinked to original sources

PubMed2006

Utility of cleavable isotope-coded affinity-tagged reagents for quantification of low-copy proteins induced by methylprednisolone using liquid chromatography/tandem mass spectrometry.

Gene expression changes underlie important biological and pharmacological responses. Although mRNA expression profiling is routine, quantification of low-abundance proteins, which typically represent key effectors of responses, remains challenging. A novel strategy was developed for sensitive and accurate quantification of low-abundance proteins in highly complex biological matrixes. First, the cysteine specificity of cleavable isotope-coded affinity tags (cICAT) was employed to reduce the complexity of the digested proteome of tissue homogenates and to improve the quantification of low-abundance proteins. Second, cICAT-treated tissue samples were analyzed on a capillary LC coupled to an ion trap MS to screen for the subset of cICAT-peptides, derived from target proteins of interest, that was successfully labeled and retrieved. Third, putatively identified peptides derived from target proteins were synthesized, cICAT-labeled, and used both to optimize multiple reactions monitoring (MRM) analysis and to confirm chromatographic retention time and fragmentation pattern. Finally, batch quantification of target peptides was performed using MRM on a LC/triple-quad MS/MS using (12)C- (control) and (13)C (experimental)-cICAT-labeled tissue mixtures. The utility of this method was demonstrated by elucidating the time-course of tyrosine aminotransferase induction in the liver of rats following treatment with the corticosteroid methylprednisolone (MPL). This approach significantly improved quantitative sensitivity, and the linear range was 10-fold greater than published previously. An additional advantage is that archived samples may be reinterrogated to investigate the regulation of additional targets that become of interest. Stored samples were sucessfully reinterrogated to monitor the induction of ornithine decarboxylase, which is also an MPL-induced protein. To our knowledge, this is the first report of an ICAT-based method that is capable of quantifying low-abundance proteins in highly complex samples, such as tissue homogenates. The approach enables simultaneous quantification of multiple effector proteins induced by biological or pharmacological stimuli, and the processed samples can be interrogated repeatedly as additional targets of interest arise.

Affinity Labels↗

PubMed2005

Effect of repetitive administration of Doxorubicin-containing liposomes on plasma pharmacokinetics and drug biodistribution in a rat brain tumor model.

PURPOSE: The incorporation of doxorubicin in long-circulating sterically stabilized liposomes (SSL-DXR) alters the pharmacokinetics and biodistribution of doxorubicin and therefore has the potential to alter the pharmacologic properties of doxorubicin. Previously, we showed that repetitive administration of SSL-DXR alters tumor vascular permeability. EXPERIMENTAL DESIGN: Here, we investigated the effect of weekly i.v. injections of SSL-DXR on plasma pharmacokinetics and drug biodistribution in the orthotopic 9L rat brain tumor model. RESULTS AND CONCLUSIONS: The pharmacokinetics of free doxorubicin (5.67 mg/kg) did not change with repeat dosing. In contrast, drug concentrations in plasma and brain tumor increased and deposition in liver and spleen decreased after administration of the second of two weekly doses of SSL-DXR. Noncompartmental analysis and descriptive pharmacokinetic models were created to test hypotheses relating to the mechanisms responsible for alterations in SSL-DXR deposition. The analysis suggested that weekly administration of SSL-DXR significantly (P < 0.05) decreased the plasma elimination rate of SSL-DXR (34%) and decreased drug deposition in liver (2-fold) and spleen (3.5-fold). The pharmacokinetic model that best captured the observed 2.5-fold increase in tumor uptake of SSL-DXR mediated by repeat dosing was one that hypothesized that the rates of drug influx/efflux into tumor were increased by the first dose of SSL-DXR. Models that accounted only for residual drug deposited in the tissue or blood by the first weekly injection provided inferior fits to the data. Thus, the effects of repetitive dosing on SSL-DXR deposition in tumor are consistent with a treatment-mediated alteration of tumor vascular permeability.

Animals↗

PubMed2005

Improved sensitivity for quantification of proteins using triply charged cleavable isotope-coded affinity tag peptides.

Isotope-coded affinity tag (ICAT) methods, in conjunction with capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS), represent a promising approach for accurate protein quantification. However, sensitivity remains a challenge for the quantification of low-copy proteins in complex biological matrices. Here we investigated the electrospray ionization (ESI) and collision-activated dissociation (CAD) behavior of peptides derivatized with the cleavable ICAT (cICAT) reagent. For cICAT-peptides that were either synthesized or obtained by digestion of model proteins, the cICAT moiety showed a tendency toward protonation under positive ESI, producing relatively intense triply charged cICAT-peptide ions ([IP+3H]3+). [IP+3H]3+ exhibited significantly higher CAD reactivity than did the doubly charged cICAT-peptide ([IP+2H]2+), and produced a greater abundance of fragments at lower collision energies. Fragmentation spectra of [IP+3H]3+ showed variable intensities of doubly charged y and b ions, and the amount of sequence information obtained was dependent on the position of the cICAT-labeled cysteine residue in the peptide sequence. However, the absolute abundances of major fragments of [IP+3H]3+ were much higher than for [IP+2H]2+. Although the efficiency of identification of cICAT-peptides was compromised by their charge distribution toward the triply charged state and by the unique CAD behavior of the [IP+3H]3+ ions, it was found that the triply charged ions provided higher sensitivity than [IP+2H]2+ for quantification using multiple reaction monitoring (MRM). ESI and CAD conditions for MRM of [IP+3H]3+ were optimized, and, for all cICAT-peptides studied, MRM using [IP+3H]3+ as precursors showed 2- to 8-fold higher sensitivity than obtained using [IP+2H]2+, without compromising quantitative accuracy. Using this approach, the time course of tyrosine aminotransferase induction by methylprednisolone was monitored in rat livers. A remarkably better signal-to-noise ratio was observed by using [IP+3H]3+ for quantification compared to [IP+2H]2+.

Animals↗

PubMed2005

Preparation and characterization of taxane-containing liposomes.

Drug carriers such as liposomes provide a means to alter the biodisposition of drugs and to achieve concentration-time exposure profiles in tissue or tumor that are not readily accomplished with free drug. These changes in biodisposition can improve treatment efficacy. For hydrophobic drugs, incorporation in liposome carriers can increase drug solubility markedly. The taxanes paclitaxel (taxol) and docetaxel (Taxotere) are members of one of the most important new classes of oncology drugs. However, their poor solubility presents pharmaceutical challenges, and emerging data suggest that specific tissue exposure profiles, such as low drug concentrations for extended times, can enhance beneficial antitumor mechanisms. Incorporation of the taxanes into liposomes eliminates not only the toxic effects of cosolvents required to administer these drugs clinically but also increases drug efficacy in animal tumor models, usually through a reduction in dose-limiting tissue toxicities. Although the taxanes are poorly water soluble, the preparation of physically stabile taxane-liposome formulations requires the balancing of three factors: (1) the drug:lipid ratio, (2) the liposome composition, and (3) the duration of storage in aqueous media. Biophysical evaluation of formulation characteristics, principally using circular dichroism (CD) and differential scanning calorimetry (DSC), can provide the information necessary to develop stable taxane-liposome formulations. These techniques provide information on drug-drug and drug-lipid interactions that underlie the events that lead to taxane formulation instability. Owing to the unusually low solubility of the taxanes, special consideration is necessary to devise methods for resolving drug-containing liposomes from released or precipitated drug to obtain reliable estimates of drug incorporation and retention in liposomes.

Animals↗

PubMed2004

Quantification of Doxorubicin and metabolites in rat plasma and small volume tissue samples by liquid chromatography/electrospray tandem mass spectroscopy.

The anthracycline Doxorubicin (DXR) is used widely for the treatment of human malignancies, and drug delivery technologies are under investigation to enhance antitumor selectivity and effectiveness. A liquid chromatography-tandem mass spectroscopy (LC-MS/MS) method was developed to identify and quantify DXR and key metabolites in small-volume biological samples. The assay was linear over the therapeutically relevant concentration range (0.125-10,000 nM); in brain tissue, the lower limit of quantification was 0.247 nM and the sensitivity was 1.4 pg. The ability to quantify DXR and detect metabolite formation may provide insight into the toxicity and bioavailability of drug incorporated into carriers such as liposomes.

Animals↗

PubMed2003

Pharmacokinetics of paclitaxel-containing liposomes in rats.

In animal models, liposomal formulations of paclitaxel possess lower toxicity and equal antitumor efficacy compared with the clinical formulation, Taxol. The goal of this study was to determine the formulation dependence of paclitaxel pharmacokinetics in rats, in order to test the hypothesis that altered biodistribution of paclitaxel modifies the exposure of critical normal tissues. Paclitaxel was administered intravenously in either multilamellar (MLV) liposomes composed of phosphatidylglycerol/phosphatidylcholine (L-pac) or in the Cremophor EL/ethanol vehicle used for the Taxol formulation (Cre-pac). The dose was 40 mg/kg, and the infusion time was 8 to 9 minutes. Animals were killed at various times, and pharmacokinetic parameters were determined from the blood and tissue distribution of paclitaxel. The area under the concentration vs time curve (AUC) for blood was similar for the 2 formulations (L-pac: 38.1 +/- 3.32 microg-h/mL; Cre-pac: 34.5 +/- 0.994 microg-h/mL), however, the AUC for various tissues was formulation-dependent. For bone marrow, skin, kidney, brain, adipose, and muscle tissue, the AUC was statistically higher for Cre-pac. For spleen, a tissue of the reticuloendothelial system that is important in the clearance of liposomes, the AUC was statistically higher for L-pac. Apparent tissue partition coefficients (K(p)) also were calculated. For bone marrow, a tissue in which paclitaxel exerts significant toxicity, K(p) was 5-fold greater for paclitaxel in Cre-pac. The data are consistent with paclitaxel release from circulating liposomes, but with efflux delayed sufficiently to retain drug to a greater extent in the central (blood) compartment and reduce penetration into peripheral tissues. These effects may contribute to the reduced toxicity of liposomal formulations of paclitaxel.

Animals↗

PubMed2002

Antivasculature effects of doxorubicin-containing liposomes in an intracranial rat brain tumor model.

Increased neovascularization and vascular hyperpermeability are integral processes in tumors, and various therapeutic strategies seek to reverse the angiogenic phenotype. Long-circulating liposomes extravasate in tumors such as the rat 9L gliosarcoma and accumulate in perivascular areas. Under such conditions, liposome-encapsulated doxorubicin (DOX) provides approximately 30% increase in life span, but free DOX is no more beneficial than a saline control. However, the relationship between drug deposition and therapeutic effect is understood poorly. In the present work, magnetic resonance (MR) and functional MR (fMR) imaging were used for noninvasive, serial evaluation of intracranial 9L tumor responses to repetitive doses of free DOX or DOX in sterically stabilized long-circulating liposomes (SSL-DOX). After multiple doses of SSL-DOX, MR imaging revealed the induction of intratumor hemorrhage in 63-75% of rats (n = 8). No hemorrhage was observed by MR imaging after a single dose of SSL-DOX, in normal brain regions in animals treated with free DOX (n = 3) or in saline controls (n = 9). Histological sections from rats sacrificed immediately after MR imaging verified the putative hemorrhagic regions and revealed necrotic and apoptotic tumor cells surrounding the area of the hemorrhage. fMR maps were obtained by comparing paired images acquired during air and Carbogen (7% CO2 and 93% oxygen) breathing. These blood oxygenation level-dependent fMR maps showed enhanced image intensity after both single and multiple doses of SSL-DOX, which suggested increased and progressive vascular permeabilization. The results suggest that the breakdown of tumor vasculature induced by SSL-DOX may arise from the perivascular accumulation of liposomes in tumor and cytotoxic effects on tumor vascular endothelium.

Angiogenesis Inhibitors↗

PubMed2002

Noncovalent dimerization of paclitaxel in solution: evidence from electrospray ionization mass spectrometry.

Paclitaxel, a unique antimitotic chemotherapy agent that inhibits cell division by binding to microtubules and prevents them from "depolymerizing," has received widespread interest because of its efficacy in fighting certain types of cancer, including breast and ovarian cancer. Paclitaxel undergoes aggregation at millimolar concentrations in both aqueous media and solvents of low polarity (mimicking hydrophobic environments). Its aggregation may have impact on its aqueous stability and its ability to stabilize microtubules. Here, we investigated the dimerization phenomenon of paclitaxel by electrospray ionization mass spectrometry (ESI-MS). Paclitaxel dimers were stable in solutions of acetonitrile/aqueous ammonium acetate (80/20) and aqueous sodium acetate/acetonitrile (92/8 or 95/5) at various pH values. Additional experiments using solution-phase hydrogen/deuterium exchange were employed to ascertain whether or not the observed dimers were formed in solution or as an artifact of the ESI process by ion-molecule reaction. The evidence supports formation of the dimer in solution, and the approach used can be extended to investigation of other types of drug-drug interactions.

Acetates↗

PubMed2002

Propofol, a general anesthetic, promotes the formation of fluid phase domains in model membranes.

The molecular site of anesthetic action remains an area of intense research interest. It is not clear whether general anesthetics act through direct binding to proteins or by perturbing the membrane properties of excitable tissues. Several studies indicate that anesthetics affect the properties of either membrane lipids or proteins. However, gaps remain in our understanding of the molecular mechanism of anesthetic action. Recent developments in membrane biology have led to the concept of small-scale domain structures in lipid and lipid--protein coupled systems. The role of such domain structures in anesthetic action has not been studied in detail. In the present study, we investigated the effect of anesthetics on lipid domain structures in model membranes using the fluorescent spectral properties of Laurdan (6-dodecanoyl-2-dimethylamino naphthalene). Propofol, a general anesthetic, promoted the formation of fluid domains in model membranes of dipalmitoyl phosphatidyl choline (DPPC) or mixtures of lipids of varying acyl chains (DPPC:DMPC dimyristoyl phosphatidyl choline 1:1). The estimated size of these domains is 20--50 A. Based on these studies, we speculate that the mechanism of anesthetic action may involve effects on protein--lipid coupled systems through alterations in small-scale lipid domain structures.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2002

Pharmacodynamics and pharmacogenomics of diverse receptor-mediated effects of methylprednisolone in rats using microarray analysis.

Corticosteroids such as methylprednisolone (MPL) produce many of their anti-inflammatory, immunosuppressive, and exaggerated physiological effects by receptor and gene-mediated mechanisms. The temporal pattern of change in four genes in rat tissues was measured by quantitative Northern hybridization and rtPCR after a single dose of MPL. Two profiles were observed: two genes with enhanced expression showed a slow onset and moderate rate of decline within a 24 hr time frame while two genes with reduced expression exhibited a rapid onset and prolonged suppression over a > or = 72 hr time span. These patterns are consistent with and rationalized by pharmacodynamic expectations based on earlier models. cDNA microarrays used to assess the expression levels of 5200 genes at one optimal time-point showed marked variation in baseline values. Of these, 20 genes showed statistically significant enhanced expression with increases ranging from 130 to 1690%, 31 genes exhibited reduced expression ranging from 31 to 72% of control. Many genes could be categorized as affecting acute phase/immune response, energy metabolism, microsomal metabolism, and hepatic function. These studies provide the first simultaneous assessment of the diversity in pharmacogenomic effects of corticosteroids. They also provide some insight into the advantages and limitations of microarray measurements in regard to the pharmacodynamics of drugs having complex, multi-faceted, and integrated mechanisms of action.

Animals↗

PubMedDate not supplied

Antivascular and antitumor activities of liposome-associated drugs.

Particulate drug carriers offer unique opportunities to improve tumor therapy through several different mechanisms. Liposomes may (1) assist in formulation of poorly-soluble therapeutic agents, (2) provide a slow-release vehicle to achieve pharmacokinetic profiles that maximize the therapeutic index, or (3) behave as long-circulating nano-particulates that can extravasate in the hyperpermeable regions of tumor vasculature. For paclitaxel, liposomes provide an aid to formulation. In the intracranial rat 9L brain tumor model, paclitaxel liposomes reduced dose-limiting toxicity and mediated a 40% increase in median survival. Free drug did not extend survival. Doxorubicin entrapped within sterically-stabilized liposomes (SSL-DXR) represents a long-circulating formulation that can extravasate within tumors and enhance drug deposition. Repetitive dosing with SSL-DXR mediated a 30% extension in median lifespan of animals bearing advanced 9L tumors. Fluorescence microscopic imaging revealed non-uniform, sporadic deposition of liposomes within the tumor. Magnetic resonance imaging showed that repetitive dosing with SSL-DXR, but not free drug, resulted in vascular collapse and microhemorrhage within tumors. Exploiting this antivascular effect may provide a new means to enhance tumor therapy, and suggests the utility of combination therapy with agents such as paclitaxel that have antiangiogenic effects on tumors.

Angiogenesis Inhibitors↗