Karen P. Galen

Detection of individual microbubbles of ultrasound contrast agents: imaging of free-floating and targeted bubbles.

Rationale and Objectives:During echo examinations with microbubble contrast, individual “dots” of ultrasound reflection can be visualized. To address the question whether these signals represent individual microbubbles, very dilute suspensions of ultrasound contrast agents or individual microbubbles attached to Petri dishes were prepared and studied by ultrasound imaging. Methods:Microbubble suspensions were diluted in saline and evaluated by a clinical ultrasound imaging system. Microbubble concentration was verified by Coulter counter. Single microbubble preparation on a Petri dish was established by streptavidin–biotin interaction under microscopy control and subjected to ultrasound imaging. Results:Ultrasound of dilute microbubble dispersions demonstrated distinct white foci; concentration of these sites was consistent with signals from individual microbubbles as determined by Coulter. Individual microbubbles immobilized on polystyrene were also visualized by ultrasound. Conclusion:Ultrasound medical systems can resolve backscatter signals from individual microbubbles of ultrasound contrast, both in solution and in the targeted immobilized state, implying picogram sensitivity.

Detection of individual microbubbles of an ultrasound contrast agent: fundamental and pulse inversion imaging.

The use of ultrasound contrast materials in diagnostic imaging has been steadily increasing, with several agents recently approved for clinical application (1). When contrast echo imaging is performed, individual “speckles” of contrast can be often observed in the interrogated tissues. These white foci may represent the images of individual micron-sized bubbles. This implies exceptional detection sensitivity of ultrasound imaging with contrast agents. The capability of echo imaging to detect individual microbubbles is important for the quantification of the amount of bubbles in the tissues, determination of microvascular volume and targeted microbubble imaging. In order to test the ability of ultrasound imaging to detect individual microbubbles, dilute dispersions of microbubbles were prepared and evaluated by ultrasound imaging in vitro.

Hydrophilic pyrazine dyes as exogenous fluorescent tracer agents for real-time point-of-care measurement of glomerular filtration rate.

Various hydrophilic pyrazine-bis(carboxamides) derived from 3,5-diamino-pyrazine-2,5-dicarboxylic acid bearing neutral and anionic groups were prepared and evaluated for use as fluorescent glomerular filtration rate (GFR) tracer agents. Among these, the dianionic d-serine pyrazine derivatives 2d and 2j, and the neutral dihydroxypropyl 2h, exhibited favorable physicochemical and clearance properties. In vitro studies show that 2d, 2h, and 2j have low plasma protein binding, a necessary condition for renal excretion. In vivo animal model results show that these three compounds exhibit a plasma clearance equivalent to iothalamate (a commonly considered gold standard GFR agent). In addition, these compounds have a higher urine recovery compared to iothalamate. Finally, the plasma clearance of 2d, 2h, and 2j remained unchanged upon blockage of the tubular secretion pathway with probenecid, a necessary condition for establishment of clearance via glomerular filtration only. Hence, 2d, 2h, and 2j are promising candidates for translation to the clinic as exogenous fluorescent tracer agents in real-time point-of-care monitoring of GFR.

Exogenous fluorescent tracer agents based on pegylated pyrazine dyes for real-time point-of-care measurement of glomerular filtration rate

Novel pyrazine carboxamides bearing hydrophilic poly(ethylene glycol) (PEG) moieties were designed, synthesized, and evaluated for use as fluorescent glomerular filtration rate (GFR) tracer agents. Among these, compounds 4d and 5c that contain about 48 ethylene oxide units in the PEG chain exhibited the most favorable physicochemical and renal clearance properties. In vitro studies show that these two compounds have low plasma protein binding, a necessary condition for renal excretion. In vivo animal model results show that 4d and 5c have a higher urine recovery of the injected dose than iothalamate (a commonly considered gold standard GFR agent). Pharmacokinetic studies show that these two compounds exhibit a plasma clearance equivalent to iothalamate, but with a faster (i.e. lower) terminal half-life than iothalamate (possibly from restricted distribution into the extracellular space due to large molecular size and hydrodynamic volume). Furthermore, the plasma clearance of 4d and 5c remained unchanged upon blockage of the tubular secretion pathway with probenecid, a necessary condition for establishment of clearance via glomerular filtration exclusively. Finally, noninvasive real-time monitoring of this class of compounds was demonstrated by pharmacokinetic clearance of 5c by optical measurements in rat model, which correlates strongly with plasma concentration of the tracer. Hence, 4d and 5c are promising candidates for translation to the clinic as exogenous fluorescent tracer agents in real-time point-of-care monitoring of GFR.

Albumin-binding MR blood pool agents as MRI contrast agents in an intracranial mouse glioma model

Intravenous MRI contrast agents are commonly used to improve the detection of intracranial tumors and other central nervous system (CNS) lesions for diagnosis and treatment planning. Two small‐molecule, albumin‐binding blood pool contrast agents (MP‐2269 and MS‐325) of potential clinical significance were evaluated at 1.5 Tesla in a mouse glioma model and compared with an extracellular contrast agent (OptiMARK). Tumor image contrast was significantly enhanced and long‐lived following administration of 30 μmole/kg of the blood pool agents: specifically, contrast enhancement peaked slowly at 25–30 min following administration, remained constant for >3 hr, and returned to baseline within 20 hr. Comparable but “transient” enhancement was achieved using 100 μmole/kg OptiMARK: specifically, contrast enhancement peaked rapidly at 2–5 min following administration and then declined over 40 min. The blood pool contrast agents demonstrated an approximately threefold increased dose‐effectiveness and a lengthened window of tumor contrast enhancement in comparison to commonly available extracellular contrast agents. This demonstrates the potential of alternative contrast‐enhanced (CE) MRI examination protocols for tumor detection. Magn Reson Med 49:586–590, 2003.

Cardiovascular effects caused by rapid administration of gadoversetamide injection in anesthetized dogs.

Wible JH, Galen KP, Wojdyla J. Cardiovascular effects caused by rapid administration of gadoversetamide injection in anesthetized dogs. Invest Radiol 2001;36:292–298. rationale and objectives. This study assessed the cardiovascular effects of gadoversetamide and other gadolinium chelates administered at high rates of injection. methods.Anesthetized beagles were instrumented to record the electrocardiogram and to measure arterial blood pressure. In part 1, each animal was injected with gadoversetamide at rates of 1.0, 3.0, and 10 mL/s. In part 2, each animal was injected with gadoversetamide, gadopentetate dimeglumine, gadodiamide, and gadoteridol at a dose of 0.6 mmol/kg delivered at a rate of 3.0 mL/s. results.Intravenous administration of gadoversetamide caused transient decreases in both heart rate and blood pressure. The rate of injection did not affect the magnitude of the heart rate or blood pressure changes. Administration of gadoversetamide, gadopentetate dimeglumine, and gadodiamide elicited equivalent changes in cardiovascular function. Injection of gadoteridol caused a similar degree of hypotension, but the changes lasted longer. conclusions.Rapid administration of gadoversetamide caused no potentiation in cardiovascular changes. Our data support the initiation of a clinical trial to demonstrate the safety of rapidly administering gadoversetamide with the use of a power injector.

New optical probes for the continuous monitoring of renal function

The ability to continuously monitor renal function via the glomerular filtration rate (GFR) in the clinic is currently an unmet medical need. To address this need we have developed a new series of hydrophilic fluorescent probes designed to clear via glomerular filtration for use as real time optical monitoring agents at the bedside. The ideal molecule should be freely filtered via the glomerular filtration barrier and be neither reabsorbed nor secreted by the renal tubule. In addition, we have hypothesized that a low volume of distribution into the interstitial space could also be advantageous. Our primary molecular design strategy employs a very small pyrazine-based fluorophore as the core unit. Modular chemistry for functionalizing these systems for optimal pharmacokinetics (PK) and photophysical properties have been developed. Structure-activity relationship (SAR) and pharmacokinetic (PK) studies involving hydrophilic pyrazine analogues incorporating polyethylene glycol (PEG), carbohydrate, amino acid and peptide functionality have been a focus of this work. Secondary design strategies for minimizing distribution into the interstitium while maintaining glomerular filtration include enhancing molecular volume through PEG substitution. In vivo optical monitoring experiments with advanced candidates have been correlated with plasma PK for measurement of clearance and hence GFR.

Novel type 1 photosensitizers: viability of leukemia cells exposed to reactive intermediates generated in situ by in vitro photofragmentation

Photodynamic therapy of tumors involving Type 2 photosenstizers has been conspicuously successful, but the Type 1 process, in contrast, has not received much attention despite its considerable potential. Accordingly, several classes of molecules containing fragile bonds such as azido (-N=N=N), azo (-N=N-), sulfenato (-S-O-) and oxaza (-N-O-) functional groups that produce reactive intermediates such as radicals and nitrenes upon photoexcitation were prepared and tested for cell viability using U397 leukemia cell line. The azido photosensitizer was conjugated to leukemia cell binding peptide, SFFWRLS, for targeted cell viability study. The cells were incubated with the photosensitizer at various concentrations, and were illuminated for 5, 10, and 20 minutes. The results show that all the photosensitizers caused cell death compared to the controls when exposed to both the photosensitizers and light. Most importantly, selective cell death was observed with the azido peptide conjugate 6, which clearly demonstrates that these Type 1 sensitizers are useful for phototherapeutic applications.

Real-time point-of-care measurement of impaired renal function in a rat acute injury model employing exogenous fluorescent tracer agents

Renal function assessment is needed for the detection of acute kidney injury and chronic kidney disease. Glomerular filtration rate (GFR) is now widely accepted as the best indicator of renal function, and current clinical guidelines advocate its use in the staging of kidney disease. The optimum measure of GFR is by the use of exogenous tracer agents. However current clinically employed agents lack sensitivity or are cumbersome to use. An exogenous GFR fluorescent tracer agent, whose elimination rate could be monitored noninvasively through skin would provide a substantial improvement over currently available methods. We developed a series of novel aminopyrazine analogs for use as exogenous fluorescent GFR tracer agents that emit light in the visible region for monitoring GFR noninvasively over skin. In rats, these compounds are eliminated by the kidney with urine recovery greater than 90% of injected dose, are not broken down or metabolized in vivo, are not secreted by the renal tubules, and have clearance values similar to a GFR reference compound, iothalamate. In addition, biological half-life of these compounds measured in rats by noninvasive optical methods correlated with plasma derived methods. In this study, we show that this noninvasive methodology with our novel fluorescent tracer agents can detect impaired renal function. A 5/6th nephrectomy rat model is employed.

N-alkylated aminopyrazines for use as hydrophilic optical agents

Rapid assessment of glomerular filtration rate (GFR), which measures the amount of plasma filtered through the kidney within a given time, would greatly facilitate monitoring of renal function for patients at the bedside in the clinic. In our pursuit to develop exogenous fluorescent tracers for real-time monitoring of renal function by optical methods, N-alkylated aminopyrazine dyes and their hydrophilic conjugates based on poly (ethylene glycol) (PEG) were synthesized via reductive amination as the key step. Photophysical properties indicated a bathochromic shift on the order of 50 nm in both absorption and emission compared to naked aminopyrazines which could be very useful in enhancing both tissue penetration as well as easier detection methods. Structure-activity relationship (SAR) and pharmacokinetic (PK) studies, and the correlation of in vivo optical data with plasma PK for measurement of clearance (and hence GFR) are focus of the current investigation.