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.
The aim of this work was to establish measurement conditions under which endogenous skin fluorescence ("auto-fluorescence")
is relatively invariant, so that changes in exogenous agents can be accurately determined. Fluorescence
emission was measured on the volar forearm of 36 subjects, chosen to be equally representative of all 6 Fitzpatrick skin
types. All subjects were exposed to approximately 40 minutes of optical excitation at 450 and 500 nm with 4 irradiances
between 0.3 and 9 mW/cm2. Both non-optically-induced (e.g. tissue settling and fluctuation) and optically-induced
variations were observed in the measured fluorescence and mechanisms explaining these effects are proposed. The
optically-induced auto-fluorescence decay was independent of skin type when excited at 450 nm, but significantly
dependent on skin type when excited at 500 nm. Further, the extent of decay over time was linearly related to irradiance
at 500 nm, but at 450 nm was non-linear, with the extent of decay rolling off between 2 and 9 mW/cm2. In order to
maintain the auto-fluorescence signal within 95% of its original value over a 30 minute period, the excitation at 450 nm
would need to be limited to 1.5 mW/cm2, while excitation at 500 nm should be limited to 5 mW/cm2.
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.
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.
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.
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