KEYWORDS: Kidney, Injuries, Plasma, Biomedical optics, Point-of-care devices, Skin, Fiber optics, Systems modeling, In vivo imaging, Analog electronics
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.
Accurate measurement of glomerular filtration rate (GFR) at the bedside is highly desirable in order to assess renal
function in real-time, which is currently an unmet clinical need. In our pursuit to develop exogenous fluorescent tracers
as GFR markers, various hydrophilic derivatives of 3,6-diaminopyrazine-2,5-dicarboxylic acid with varying molecular
weights and absorption/emission characteristics were synthesized. These include polyhydroxyalkyl based small
molecules and poly(ethylene glycol) (PEG) substituted moderate molecular weight compounds, which were further sub-grouped
into analogs having blue excitation with green emission, and relatively longer wavelength analogs having green
excitation with orange emission. Lead compounds were identified in each of the four classes on the basis of structure-
activity relationship studies, which included in vitro plasma protein binding, in vivo urine recovery of administered dose,
and in vivo optical monitoring. The in vivo optical monitoring experiments with lead candidates have been correlated
with plasma pharmacokinetic (PK) data for measurement of clearance and hence GFR. Renal clearance of these
compounds, occurring exclusively via glomerular filtration, was established by probenecid blocking experiments. The
renal clearance property of all these advanced candidates was superior to that of the iothalamate, which is currently an
accepted standard for the measurement of GFR.
Folate receptors are over expressed in many types of cancers, including, ovarian, breast, and cervical. In our continuing
efforts toward the development of targeted Type 1 phototherapeutic agents, an azide-based Type 1 photosensitizer and a
pyrzine-based fluorophore that absorb and emits in the visible region, and a dual diagnostic-therapeutic probe consisting
of the fluorophore and the photosensitizer were prepared and independently conjugated to two folate receptor specific
vectors: γ-carboxyl-modified folic acid and anti-human FOLR1 (folate receptor-1) antibody In vitro receptor binding
study showed that all the conjugates had high (ca 1-7 nM) affinity to the folate receptor. Confocal microscopy images
indicated that the pyrazine conjugates were selectively taken up by the folate receptor expressing ovarian cancer KB
cells.
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-), and oxaza (-N-O-) functional groups that
produce reactive intermediates such as radicals and nitrenes upon photoexcitation with visible light were prepared and
tested for cell viability using U397 leukemia cell line. 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.
Photodynamic therapy involving Type 2 photosenstizers has been conspicuously successful in the treatment of various lesions. Type 1 process, in contrast has not received much attention despite its considerable potential. We have prepared several classes of molecules containing fragile bonds that will produce reactive intermediates such as radicals and nitrenes upon photoexcitation with UV-A and visible light. In a primary screen, many of these compounds had a significant concentration and light dose dependent effect on the cell viability on U937. The chemistry of these
compounds differentiated their ability to induced cytotoxic effects. To further establish these findings, we tested one compound representing each class of chemistry in three different tumor cell lines. Cells were incubated with three different concentrations (100μM, 25μM and 6.25μM) and exposed to UV light dose of 4J/cm2 and 8J/cm2. The compounds showed varying effects on viability in different cell lines. Finally we also investigated the ability to induce apoptosis as the mechanism of cell death in HCT116 cell lines using the experimental conditions mentioned. The type 1 photosensitizers induced apoptosis as early as 4 hours after exposure in HCT116 cells and the rate of apoptosis increased with time with majority of cells in late apoptotic or necrotic stage.
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.
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.
Lori Chinen, Karen Galen, K. Kuan, Mary Dyszlewski, Hiroaki Ozaki, Hiroaki Sawai, Raghootama Pandurangi, Frederick Jacobs, Richard Dorshow, Raghavan Rajagopalan
Real-time, non-invasive assessment of glomerular filtration rate (GFR) is essential not only for monitoring critically
ill patients at the bedside, but also for staging and monitoring patients with chronic kidney disease. In our pursuit to
develop exogenous luminescent probes for dynamic optical monitoring of GFR, we have prepared and evaluated Eu3+
complexes of several diethylenetriamine pentaacetate (DTPA)-monoamide ligands bearing molecular "antennae" to
enhance metal fluorescence via the intramolecular ligand-metal fluorescence resonance energy transfer (FRET) process.
The results show that Eu-DTPA-monoamide complex 13a, which contains a quinoxanlinyl antenna, exhibits large (c.a.
2700-fold) Eu3+ fluorescence enhancement over Eu-DTPA (4c). Indeed, complex 13a exhibits the highest fluorescent
enhancement observed thus far in the DTPA-type metal complexes. The renal clearance profile of the corresponding
radioactive 111In complex 13c is similar to that of 111In-DTPA, albeit 13c clears slower than 111In-DTPA. The biodistribution
data indicates that 13c, and, by inference, 13a clear via a complex mechanism that includes glomerular filtration.
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.
Biocompatible organic dyes, in general, exhibit poor photostability in aqueous solution. This is especially true at the high concentrations needed for bolus administration in a clinical application. Several years ago, we showed that by using carefully chosen macromolecular additives, the stability of aqueous dye solutions may be enhanced significantly. We now report the observation of an unexpected long-term stability of a near infrared dye-peptide conjugate. The Mallinckrodt developed dye-peptide conjugate known as Cytate is an indocyanine type dye attached to the peptide octreotate. We have previously shown that this compound targets somatostatin receptor rich tumors and provides unambiguous contrast for optical imaging. A freshly prepared aqueous solution of Cytate was mixed with blood (from a rat) in a small glass bottle. An optical image was taken soon after, along with several control samples. The Cytate-blood bottle fluoresced quite nicely. The sample bottles were then left in a lab for three plus years, at room temperature with ordinary exposure to lab lights. The sample bottles were imaged after the three year period, and the Cytate-blood bottle fluoresces still.
Site specific delivery of drugs and contrast agents to tumors protects normal tissues from the cytotoxic effect of drugs, and enhances the contrast between normal and diseased tissues. In optical medicine, biocompatible dyes can be used as phototherapeutics or as contrast agents. Previous studies have shown that the use of covalent or non-covalent dye conjugates of carriers such as antibiodies, liposomes, and polysaccharides improves the delivery of such molecules to tumors. However, large biomolecules can elicit adverse immunogenic reactions and also result in long blood clearance times, delaying visualization of target tissues. A viable alternative to this strategy is to use small bioactive molecule-dye conjugates. These molecules have several advantages over large biomolecules, including ease of synthesis of a variety of high purity compounds for combinatorial screening of new targets, enhanced diffusivity to solid tumors, and the ability to affect the pharmacokinetics of the conjugates by minor structural changes. Thus, we conjugated a near infrared absorbing dye to several bioactive peptides that specifically target overexpressed tumor receptors in established rat tumor lines. High tumor uptake of the conjugates was obtained without loss of either the peptide receptor affinity or the dye fluorescence. These findings demonstrate the efficacy of a small peptide-dye conjugate strategy for in vivo tumor imaging. Site-specific delivery of photodynamic therapy agents may also benefit from this approach.
Five peptides binding to somatostatin and bombesin receptors were conjugated to 4-azido-2,3,4,6-tetrafluorophenylbenzoic acid, a Type 1 photosensitizer, at the N-terminal position. The receptor affinities were determined by competition binding assay using two different pancreatic tumor cell lines, CA20948 and AR42-J, that expresses somatostatin-2 (SST-2) and bombesin receptors receptively. All compounds exhibited high receptor specificity, i.e., the IC50 values ranged between 1.0 to 64.0 nM. These conjugates may be useful for targeted Type 1 phototherapy via the generation of nitrenes at the cell surfaces expressing these receptors.
Site-specific delivery of drugs and contrast agents to tumors protects normal tissues from the cytotoxic effect of drugs, and enhances the contrast between normal and diseased tissues. In optical medicine, biocompatible dyes can be used as photo therapeutics or as contrast agents. Previous studies have shown that the use of covalent or non-covalent dye conjugates of carries such as antibodies, liposomes, and polysaccharides improves the delivery of such molecules to tumors. However, large biomolecules can elicit adverse immunogenic reactions and also result in prolonged blood circulation times, delaying visualization of target tissues. A viable alternative to this strategy is to use small bioactive molecule-dye conjugates. These molecules have several advantages over large biomolecules, including ease of synthesis of a variety of high purity compounds for combinatorial screening of new targets, enhanced diffusivity to solid tumors, and the ability to affect the pharmocokinetics of the conjugates by minor structural changes. Thus, we conjugated a near IR light absorbing dye to bioactive peptides that specifically target over expressed tumor receptors in established rat tumor lines. High tumor uptake of the conjugates was obtained without loss of either the peptide receptor affinity or the dye fluorescence. These findings demonstrate the efficacy of a small peptide-dye conjugate strategy for in vivo tumor imaging. Site-specific delivery of photodynamic therapy agents may also benefit form this approach.
Fluorescein dye covalently attached to anionic and cationic carriers of molecular weight 0.5 to 30 kDa were prepared and evaluated as potential fluorescent tracer agents for monitoring real-time dynamic renal function. These fluorescein bioconjugates were generally prepared by the reaction of fluorescein isothiocyanate with the desired polypeptides. The mode of clearance from the blood, and the clearance rate, is substantially altered in these conjugated compounds with respect to the dye alone. For example, the blood clearance of fluorescein is known to be through both the hepatic and renal systems, while several of the newly synthesized anionic fluorescein bioconjugates exhibit renal clearance only. Increasing the number of anionic residues in such compounds yields an increase in the renal clearance rate until a lower limit (saturation value) is reached. Thus, an optimum fluorescent renal tracer agent with respect to clearance rate and chain length can be identified. More lipophilic anionic conjugates clear at slower rates, and are not exclusively renal. In contrast, fluorescein bioconjugates composed of cationic carriers do not clear from the blood within the time frame of measurement.
Several dyes are currently used for various biomedical applications due to their biocompatibility and high molar absorptivity. Localization of dyes in tumors may be mediated by several factors such as leaky vasculature and high metabolic activity in proliferating cells. However, these mechanisms of action make it difficult to differentiate inflammation from benign or malignant tumors. In order to enhance their tumor specificity, dyes have been conjugated to biomolecules that target unique factors in various diseased state. However, such large biomolecules can elicit adverse immunogenic reactions in humans, and are often preferentially taken up by the liver. Furthermore, for solid tumors which may rely on diffusion of the biomarkers from the vascular, penetration of large dye conjugates is not favorable. To overcome these problems, we designed and synthesized novel dye-peptide conjugates that are receptor specific. The efficacy of these new fluorescent contrast agents was tested in vivo in well-characterized rat tumor lines. The resulting optical images demonstrate that successful specific tumor targeting was achieved.
An invasive in vivo fluorescence detection scheme was employed to continuously monitor exogenous dye clearance from the vasculature. This invasive physiological monitoring technique was successfully demonstrated in a rabbit model. A commercially available catheter with embedded fiber optics was employed to transmit the excitation light and detect the emitted fluorescence. The clearance of indocyanine green, known to be exclusively cleared from the blood stream by the liver, was determined invasively. The clearance curves determined by this novel invasive method replicated the clearance curves in the same animals employing the non- invasive method established previously. Thus, the feasibility of a new invasive method for physiological function assessment was established.
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