**3. Development of fluorescent tracer agents**

252 Chronic Kidney Disease

partially cleared by tubular secretion along with glomerular filtration, and, as Diskin17 recently remarked, "Creatinine clearance is not and has never been synonymous with GFR, and all of the regression analysis will not make it so because the serum creatinine depends upon many factors other than filtration." More recently, endogenous cystatin-C has been suggested as an improvement over creatinine,15,20 but this marker also suffers from the same limitations as

In the past several decades, exogenous tracers such as inulin (**2**), iothalamate (**3**), iohexol (**4**), 99mTc-DTPA (diethylenetriaminepentaacetate) (**5**), and 51Cr-EDTA (ethylenediaminetetraacetate) (**6**) (Fig.1), have been developed to determine GFR, but all of them require either radiometric, HPLC (high performance liquid chromatography), or X-ray fluorescence methods for detection and quantification.22-29 Unfortunately, all of these markers suffer from various undesirable properties including the use of radioactivity, ionizing radiation, and the laborious ex-vivo handling of blood and urine samples, and the use of HPLC method that render them unsuitable for continuous monitoring of renal function in the clinical setting. Furthermore, inulin as well as other polysaccharides are polydisperse polymers, and availability of these substances in a reliable, uniform batches is a serious limiting factor for their use as GFR markers. Currently, iothalamate and iohexol are the accepted standard for the assessment of GFR. However, iothalmate requires the collection of blood samples and requires HPLC method, which is not well suited for continuous monitoring. Continuous monitoring of GFR has been accomplished via radiometric12 and magnetic resonance imaging30 techniques, but these are not suitable at the bedside. Hence, the availability of an exogenous marker for the measurement of GFR under specific yet changing circumstances would represent a substantial improvement over any currently available or widely practiced method. Moreover, a method that depends solely on the renal elimination of an exogenous chemical entity would provide an absolute and continuous pharmacokinetic measurement requiring less

creatinine, and thus it remains questionable whether it is really an improvement.

subjective interpretation based upon age, muscle mass, blood pressure, etc.

Fig. 1. Structures of Currently Known Exogenous GFR Markers.

Accordingly, there has been some effort on developing exogenous GFR tracer agents that absorb and emit in the visible or near infrared (NIR) region, which includes small molecules as well as macromolecular bioconjugates such FITC (fluorescien isothicyanate)-inulin and FITC- and Texas Red-dextrans.31-37 The key requirements for an ideal fluorescent tracer agent are: (a) must be excited at and emit in the visible region ( ≥ ~425 nm); (b) must be highly hydrophilic; (c) must be either neutral or anionic; (c) must have very low or no plasma protein binding; (d) must not be metabolized in vivo, and (e) must clear exclusively via glomerular filtration as demonstrated by equality of plasma clearance with and without a tubular secretion inhibitor such as probenecid.38 The selection of the lead clinical candidate(s) may be based on secondary considerations such as the ease of synthesis, lack of toxicity, and stability. The secondary screening criteria should further take into account the tissue optics properties and the degree of extracellular distribution of the fluorescent tracers. Volume of distribution is an important parameter in the assessment of hydration state of the patient, whereas the absorption/emission properties provide essential information for the design of the probe.

This chapter focuses on the most recent development on luminescent tracers for GFR measurement. There are basically two principal pathways for the design of fluorescent tracers for GFR determination. The first method involves enhancing the fluorescence of known renal agents that are intrinsically poor emitters such as lanthanide metal complexes; and the second involves transforming highly fluorescent dyes (which are intrinsically lipophilic) into hydrophilic, anionic species to force them to clear via the kidneys.32 In the first approach, several europium-DTPA complexes endowed with various molecular 'antenna' to induce ligand-to-metal fluorescence resonance energy transfer (FRET) were prepared and tested.32 Some of metal complexes (e.g. compound **7** exhibited high (c.a. 2000 fold) enhancement of europium fluorescence and underwent clearance exclusively through the kidneys, but whether they cleared exclusively via glomerular filtration remains uncertain. Moreover, the excitation maxima of these complexes remained in the violet or UV-A region.

Fig. 2. Eu-DTPA-Quinoline Complex.

Exogenous Fluorescent Agents for the Determination of Glomerular Filtration Rate 255

Absorption Maxima, (max, nm) 435 437 488 499 NA Emission Maxima, (max, nm) 557 558 597 604 NA Plasma Protein Binding (%) 0 0 0 3 10 Plasma Clearance Half-Life (min) 29 25 20 19 32 Injected Dose Recovered in Urine at 6 Hrs (%) 90 71 88 97 80 Clearance – No probenecid (mL/min) 2.5 NA 3.0 3.1 2.5 Clearance – Probenecid, 70 mg/kg (mL/min) 2.6 NA 2.4 3.3 2.2

Table 2. Physicochemical and Pharmacokinetic Properties of Pyrazine Tracers.

high percent of injected dose recovered in urine given in Table 2.

**4. Real-time monitoring of renal clearance** 

An in vivo fluorescence image of the renal clearance of compound **13** is shown in Fig. 5. The panel contains images of three mice. The mouse in the middle was administered 300 L of a 2 mM solution in phosphate-buffered saline (PBS) of compound **13**. The other mice served as controls where the mice received only PBS. Compound **13** distributed throughout the body and then concentrated in one spot in the abdomen. Surgery after the 60 minute time point verified that this highly fluorescent spot in the abdomen was the bladder. Thus, this observation of fluorescence only appearing at the bladder is a visual demonstration of the

In vivo noninvasive real-time monitoring of renal clearance, with eventual translation to commercial development, has been demonstrated in the rodent model. A schematic of an apparatus is shown in Fig. 6. A 445 nm solid state laser was directed into one leg of a silica

**12 13 14 15 Iothalamate** 

Fig. 4. Hydrophilic Pyrazine Derivatives.

Pyrazines (Fig. 3) are one the very few classes of photostable small molecules having highly desirable properties for various biomedical and non-medical optical applications.39-41 Pyrazine derivatives **8** containing electron donating groups (EDG) in the 2,5 positions and electron withdrawing groups (EWG) in the 3,6 positions such as compounds **9-11** are shown to absorb and emit in the visible region with a large Stokes shift on the order of ~ 100 nm and with fluorescence quantum yields of about 0.4.39,40 For example, conversion of the carboxyl group in **8** to the secondary amide derivatives **9** produces a bathochromic (red) shift of about 40 nm, and alkylation of the amino group in **9** produces further red shift of about 40 nm. Thus, the pyrazine nucleus offers considerable opportunity to 'tune' the electronic properties by even simple modifications. Furthermore, the relative small size of pyrazine renders it an ideal scaffold to introduce hydrophilic substituents to bring about renal clearance.

Fig. 3. Pyrazine Derivatives.

Based on the structure and properties of known GFR tracer agents, and on the primary and secondary considerations stated earlier, the set of GFR tracer agents can be divided finto our categories as outlined in Table 1. The upper and lower quadrants address the tissue optics differences, and the left and right quadrants address volume of distribution (Vd) differences. (Vd) is important not only in affecting clearance rates, but also in the assessment of hydration state of a patient. Tissue optics parameters are important in instrument design in that the longer the wavelength of light, the deeper the penetration into the tissue. Recently, low and high molecular weight hydrophilic pyrazine derivatives **12-15** (Fig. 4) bearing neutral and anionic side chains such as alcohols, carboxylic acids, and polyethylene glycol (PEG) units were reported.41 The structures of the candidates from each of the four quadrants above are shown in Fig. 2. Unlike inulin, dextran, and other polymers, compounds **13** and **15** are monodisperse. The photophysical and biological properties of these compounds are given in Table 2. Both plasma protein binding and urinary clearance properties are superior to iothalamate, which is a currently used 'gold standard' for clinical GFR measurement. Furthermore, all four compounds displayed insignificant biodegradation.


Table 1. Design of Exogenous Fluorescent GFR Tracers.

Fig. 4. Hydrophilic Pyrazine Derivatives.

Pyrazines (Fig. 3) are one the very few classes of photostable small molecules having highly desirable properties for various biomedical and non-medical optical applications.39-41 Pyrazine derivatives **8** containing electron donating groups (EDG) in the 2,5 positions and electron withdrawing groups (EWG) in the 3,6 positions such as compounds **9-11** are shown to absorb and emit in the visible region with a large Stokes shift on the order of ~ 100 nm and with fluorescence quantum yields of about 0.4.39,40 For example, conversion of the carboxyl group in **8** to the secondary amide derivatives **9** produces a bathochromic (red) shift of about 40 nm, and alkylation of the amino group in **9** produces further red shift of about 40 nm. Thus, the pyrazine nucleus offers considerable opportunity to 'tune' the electronic properties by even simple modifications. Furthermore, the relative small size of pyrazine renders it an ideal scaffold to introduce hydrophilic substituents to bring about

Based on the structure and properties of known GFR tracer agents, and on the primary and secondary considerations stated earlier, the set of GFR tracer agents can be divided finto our categories as outlined in Table 1. The upper and lower quadrants address the tissue optics differences, and the left and right quadrants address volume of distribution (Vd) differences. (Vd) is important not only in affecting clearance rates, but also in the assessment of hydration state of a patient. Tissue optics parameters are important in instrument design in that the longer the wavelength of light, the deeper the penetration into the tissue. Recently, low and high molecular weight hydrophilic pyrazine derivatives **12-15** (Fig. 4) bearing neutral and anionic side chains such as alcohols, carboxylic acids, and polyethylene glycol (PEG) units were reported.41 The structures of the candidates from each of the four quadrants above are shown in Fig. 2. Unlike inulin, dextran, and other polymers, compounds **13** and **15** are monodisperse. The photophysical and biological properties of these compounds are given in Table 2. Both plasma protein binding and urinary clearance properties are superior to iothalamate, which is a currently used 'gold standard' for clinical GFR measurement. Furthermore, all four compounds displayed insignificant

> *Short Wavelength Low Molecular Weight*

> *Long Wavelength Low Molecular Weight*

Table 1. Design of Exogenous Fluorescent GFR Tracers.

**Volume of Distribution** 

*Short Wavelength High Molecular Weight* 

*Long Wavelength High Molecular Weight* 

renal clearance.

biodegradation.

**Tissue Optics** 

Fig. 3. Pyrazine Derivatives.


Table 2. Physicochemical and Pharmacokinetic Properties of Pyrazine Tracers.

An in vivo fluorescence image of the renal clearance of compound **13** is shown in Fig. 5. The panel contains images of three mice. The mouse in the middle was administered 300 L of a 2 mM solution in phosphate-buffered saline (PBS) of compound **13**. The other mice served as controls where the mice received only PBS. Compound **13** distributed throughout the body and then concentrated in one spot in the abdomen. Surgery after the 60 minute time point verified that this highly fluorescent spot in the abdomen was the bladder. Thus, this observation of fluorescence only appearing at the bladder is a visual demonstration of the high percent of injected dose recovered in urine given in Table 2.
