Fluorescently Labeled Cyclodextrin Derivatives as Exogenous Markers for Real-Time Transcutaneous Measurement of Renal Function

Article abstract of DOI:10.1021/acs.bioconjchem.6b00452

Evaluation of renal function is crucial for a number of clinical situations. Here, we reported a novel exogenous fluorescent marker (FITC-HPβCD) to real-time assess renal function by using a transcutaneous fluorescent detection technique. FITC-HPβCD was designed based on the principle of renal clearance of designed drugs. It displays favorable fluorescent properties, high hydrophilicity, low plasma protein binding, and high stability in porcine liver esterase as well as in plasma and nontoxicity. More importantly, FITC-HPβCD can be efficiently and rapidly filtered by glomerulus and completely excreted into urine without proximal tubular reabsorption or secretion in rat models. Additionally, the marker was well-tolerated, with nearly 100% urinary recovery of the given doses, and no metabolism were found. Relying on this novel kidney function marker and transcutaneous devices, we demonstrate a rapid, robust, and convenient approach for real-time assessing renal function without the need of time-consuming blood and urine sample preparation. Our work provides a promising tool for noninvasive real-time monitoring of renal function in vivo.

Full text of DOI:10.1021/acs.bioconjchem.6b00452

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Article
Fluorescently labeled cyclodextrin derivatives as exogenous
markers for real-time transcutaneous measurement of renal function
Jiaguo Huang, Stefanie Weinfurter, Pedro Caetano Pinto, Marc Pretze, Bettina Kränzlin, Johannes Pill,
Rodeghiero Federica, Rossana Perciaccante, Leopoldo Della Ciana, Rosalinde Masereeuw, and Norbert Gretz
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00452 • Publication Date (Web): 09 Sep 2016
Downloaded from http://pubs.acs.org on September 15, 2016
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Fluorescently labeled cyclodextrin derivatives as exogenous markers for
realꢀtime transcutaneous measurement of renal function
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†
,
¶
†
,
¶
‡
#
Jiaguo Huang, Stefanie Weinfurter, Pedro Caetano Pinto, Marc Pretze, Bettina
†
†
▽
▽
Kränzlin, Johannes Pill, Rodeghiero Federica, Rossana Perciaccante, Leopoldo
▽
‡
†*
Della Ciana, Rosalinde Masereeuw, and Norbert Gretz
†
Medical Research Center, Medical Faculty Mannheim, University of Heidelberg,
Mannheim, Germany
‡
Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Utrecht
University, The Netherlands
#
Molecular Imaging and Radiochemistry, Department of Clinical Radiology and
Nuclear Medicine, Medical Faculty Mannheim, University of Heidelberg, Mannheim,
Germany
▽
Cyanagen S.r.l. Bologna, Italy
Email of corresponding author: Norbert.Gretz@medma.uniꢀheidelberg.de
¶
These authors contributed equally to this work.
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Abstract
Evaluation of renal function is crucial for a number of clinical situations. Here, we
reported a novel exogenous fluorescent marker (FITCꢀHPβCD) to realꢀtime assess
renal function by using a transcutaneous fluorescent detection technique.
FITCꢀHPβCD was designed based on the principle of renal clearance of designed
drugs. It displays favorable fluorescent properties, high hydrophilicity, low plasma
protein binding, high stability in porcine liver esterase as well as in plasma and
nonꢀtoxicity. More importantly, FITCꢀHPβCD can be efficiently and rapidly filtered
by glomerulus and completely excreted into urine without proximal tubular
reabsorption or secretion in rat models. Additionally, the marker was well tolerated
and near 100% urinary recovery of the given doses and no metabolism were found.
Relying on this novel kidney function marker and transcutaneous devices, we
demonstrate a rapid, robust and convenient approach for realꢀtime assessing renal
function without the need of timeꢀconsuming blood/urine sample preparation. Our
work provides a promising tool for noninvasive realꢀtime monitoring of renal function
in vivo.
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Keywords: Renal function, Fluorescent markers, Glomerular filtration rate,
Transcutaneous measurement, Cyclodextrin
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Introduction
Renal damage affects the ability of the kidney to remove xenobiotics and
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, 2
metabolic products from blood. Accurate measurement of renal function is
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crucial for detection and treatment of kidney failure. Especially in the case of
impaired kidney function, accurate assessment of renal function is essential for
detecting renal failure in early stage of the disease, evaluating interventions and
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, 4
monitoring changes of function over time. Glomerular filtration rate (GFR) is
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considered as the best indicator for overall renal function. GFR cannot be
measured directly, the most common method of measuring GFR is based on the
concept of clearance, the renal clearance of a substance can be defined as the
volume of plasma from which that substance is completely cleared per unit
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time.
Endogenous markers, such as creatinine and cystatin C, as well as several
99m
exogenous agents including inulin, TcꢀDTPA and iothalamate (Scheme 1 and
Table S2) have been developed to determine GFR in the past several decades.
Plasma creatinine concentration is commonly used to estimate GFR, however,
creatinine is a product of the metabolism of creatine in muscles, therefore this
parameter may be misleading as it is affected by age, gender, muscle mass, diet
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and many other anthropometric variables. Moreover, many studies revealed
that creatinine is not only filtered by the glomerulus, but also secreted by
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, 9, 10
proximal tubule cells, leading to creatinine clearance exceeds GFR.
Furthermore, plasma clearance of exogenous agents including iothalamate and
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9m
TcꢀDTPA, have been used to measure GFR. Those methods suffer from
complexity of ex-vivo handling of blood/urine samples or disposal of
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radioactivity. GFR can be determined by injecting inulin, since inulin is neither
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reabsorbed nor being secreted by the proximal tubule after glomerular filtration,
so its rate of excretion is directly proportional to the rate of filtration of water
and solutes across the glomerular filter. Therefore, inulin is considered as the
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gold standard’ agent. Nevertheless, it is difficult to handle due to poor water
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solubility and limited availability. Also, determinations of the plasma/urinary
clearance of exogenous renal markers are cumbersome, invasive and very
timeꢀconsuming, due to the requirement of multiple blood/urine sampling and
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tedious samples analysis by HPLC.
Many attempts had been made to overcome those limitations. Recently, fluorescent
GFR markers have gained much attention. However, very few fluorescent compounds
have been reported as true GFR markers so far. Only two kinds of fluorescent GFR
markers have been reported from animal experiments. First, Dorshow et al. made
great efforts by focusing on the development of fluorescent GFR markers based on
two general approaches, one of the approaches involved enhancing the fluorescence
of known or existing renal agents, which are intrinsically poor emitters, such as
lanthanide metal complexes. However, the results suggested that these complexes are
excreted not only via glomerular filtration, but also through tubular secretion, leading
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to a bias in GFR measurement, the other approach was based on transforming highly
fluorescent dyes into hydrophilic and anionic substances to force them to be excreted
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by kidneys. Although hydrophilic pyrazineꢀdicaboxylic acid derivatives were
developed, the sophisticated synthesis routes made them difficult to produce and
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expensive.
Secondly, earlier, inulin has been engineered in its fluorescein
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isothiocyanate conjugated variant for a singleꢀbolus injection method in conscious
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animals,
its elimination kinetics is typically obtained by measuring the fluorescent
values in plasma over a specific period time after the bolus injection. Despite the
highly reproducible results provided that no longer required urine sampling, the
repeated blood sampling makes the method be stressful and still in an invasive manner,
moreover, the necessity to heat and dialyze the FITCꢀinulin solution for removing
17, 18
residual unbound FITC makes the procedure be cumbersome.
In keeping with
these observations, it is urgent to overcome the poor solubility of FITCꢀinulin, as well
as plasma sample collection and analysis. In our previous studies, we, on one hand,
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successfully developed FITCꢀsinistrin instead of FITCꢀinulin,
due to its better
water solubility and no need to dialyze before injection; On the other hand, we
developed a noninvasive transcutaneous measurement for the elimination kinetics of
fluorescent FITCꢀsinistrin marker based on a miniaturized electronic device attached
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to the skin.
The major advantage of this approach is their independence from
blood/urine sampling and laboratory assays. Therefore, allowing renal function
assessment in realꢀtime and making the evaluation of rapid changes in renal function
possible, for example, in acute renal failure. Importantly, more precise results of the
plasma clearance can be obtained via transcutaneous realꢀtime measurement, which
relies on a high number of dataꢀpoints rather than a limited number of dataꢀpoints
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from blood/urine sampling. Nevertheless, both inulin and sinistrin suffered from their
inherent limitations such as high cost, limited availability, sophisticated extraction and
purification from plants roots. Therefore, there is an unmet challenge to develop novel
fluorescent GFR markers for noninvasive realꢀtime assessing renal function.
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(a) Exogenous renal function markers
(b) Endogenous renal function markers
OH
OH
CH2OH
O
O
ONa
I
O
NH
I
I
I
OH
O
OH
H
H
N
OH
N
OH
H3C
N
H
CH3
HO
N
OH
O
I
O
OH
I
O
O
CH2OH
O
OH
Iothalamate
Iohexol
Cystatin C
CH2
OH
n
O
COO-
COO-
COO-
CH2OH
O
N
N
OH
CH2OH
COO-
N
CH3
99mTc5+
51Cr3+
OH
-OOC
COO-
COO-
N
N
COO-
NH
N
N
Inulin
n = ~35
COO-
O
H
51Cr-EDTA
Creatinine
99mTc-DTPA
(c) Exogenous markers in this work
R1O
n = 6, 7, or 8
O
CH3
R1 =
R2 =
H
H
or
or Fluorophore
OR1
O
OR2
OR1
n
or Fluorophore
S
NH
S
NH
COOH
O
O
HO
O
O
HO
O
HO
O
O
Fluorescein-HPCDs
FITC-HPCDs
XITC-HPCDs
2
9
Scheme 1. Structures of (a) exogenous, (b) endogenous renal function markers and
(
c) novel exogenous markers FluoresceinꢀHPCDs, FITCꢀHPCDs and XITCꢀHPCDs.
Here, we report 2ꢀhydroxylpropylꢀcyclodextrins (HPCDs) based fluorescent markers
(Scheme 1c) for transcutaneous assessment of kidney function. FITCꢀHPCDs markers
are judiciously designed by combining the basic principle of renally cleared drugs and
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the knowledge of a cyclodextrinꢀbased drug delivery system. These markers can be
easily synthesized, have favorable fluorescent properties, high hydrophilicity, low
plasma protein binding (PPB), high stability in porcine liver esterase as well as in
plasma and nonꢀtoxicity. More significantly, FITCꢀHPβCD can be excreted efficiently
and rapidly through kidneys into urine without reabsorption and secretion in kidney
tubule. This is in agreement with a previous study that 99% administered cyclodextrin
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can be excreted to urine within 12 h. High urinary recovery further demonstrates
that it is completely renally cleared without metabolism in vivo. Due to these
favorable properties, FITCꢀHPβCD has a high potential to be an exogenous marker
for noninvasive realꢀtime transcutaneous assessment of renal function.
Results and discussion
Design and synthesis
An ideal exogenous fluorescent marker for transcutaneous assessment of GFR should
14
meet certain requirements such as 1) the excitation and emission wavelength in the
visible region or near infrared range; 2) highly hydrophilicity and neutral or anionic
charge; 3) very low or no PPB; 4) nonꢀtoxicity and no metabolism in vivo; 5) no
reabsorption and no secretion in kidney tubules, filtrated only by the glomerulus; and
6
) easily to produce with low costs. It is necessary to take all of these characteristics
into account for a rational GFR marker design. To meet these criteria, a series of
fluorescent markers were designed comprising the two key functional components:
2ꢀhydroxylpropylꢀCDs (HPCDs) and fluorophores. Firstly, introducing HPCDs is
attempted to increase water solubility, decrease PPB and accelerate excretion for
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fluorescent markers. Among various native cyclodextrins (CDs) and CDs derivatives,
2
ꢀhydroxylpropylꢀαCD, ꢀβCD and ꢀγCD (abbreviated as HPαCD, HPβCD and
HPγCD in below) not only have better water solubility than their native CDs (Table
S3), but are also more stable to resist the hydrolysis by αꢀamylases of either porcine
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3
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or human origin. γCD can be hydrolyzed relatively fast and extensively (80% in 24
3
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h) by αꢀamylases. However, the degradation rates of αCD, βCD, HPαCD, HPβCD
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are limited and slow (only 2% in 24 h) by porcine pancreatic αꢀamylases. The merits
32ꢀ34
of no toxicity resulted in a FDA approval ten years ago.
Additionally, their narrow
32ꢀ35
molecular weight distribution, low costs, sufficient availability
make them be an
ideal backbone of GFR agents. Secondly, fluorescent dyes belonging to the xanthene
family with appealing properties such as physiological stability and safety profile,
were employed for labeling HPCDs. Furthermore, we expanded our panel of xanthene
fluorophores based on decarboxylated FITC, referred as XITC (Scheme 2a), to study
the different conjugation bonds and sites between fluorophore and HPCDs
systematically. It is noticeable that anionic and neutral substances are preferentially
14
cleared through the renal system, therefore, rhodamine based dyes were not chosen
as fluorophores for labeling due to the positive charge of the aniline nitrogens or
diethylaniline group. The three resulting markers possess different covalent
conjugation bonds and net molecular charge, which are likely to show different
pharmacokinetics and pharmacodynamics.
The general synthetic routes of FITCꢀHPCDs, FluoresceinꢀHPCDs and XITCꢀHPCDs
are illustrated in Scheme 2. The synthesis of compound XITC was carried out
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6ꢀ38
following an efficient synthetic procedure
depicted in Scheme 2a. Bisphenolic
precursor 2 can be easily accomplished under high temperature in special pressurized
flasks. Based on a nucleophilic addition of Grignard derivative to a TBDMSꢀprotected
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, 6ꢀdihydroxyꢀxanthenone and a subsequent dehydration with aqueous hydrochloric
36, 39
acid,
the corresponding amine product 4 was obtained. Treatment of this
compound with an excess of thiophosgene under basic conditions yielded the
isothiocyanate product XITC. The synthesis of three different markers is easily done
40ꢀ42
in a oneꢀstep reaction (Scheme 2b).
All intermediates and markers were
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13
characterized by HꢀNMR, CꢀNMR and LRꢀMS.
a
H
N
O
O
HO
OH
Si
Cl
H2O, 200 °C
,
N
Si
Si
HO
O
2
OH
DMF, rt
O
O
3
O
OH
O
1
OH
TMS
N
NH2
NCS
TMS
S
MgBr
Cl Cl
CH2Cl2, rt
HCl, THF, rt
HO
O
4
OH
HO
O
O
XITC
6-hydroxy-9-(4-isothiocyanatophenyl)
-3H-xanthen-3-one
b
R3
1) NaH/DM, 45 °C for 1 h,
and rt for 18 h
OR1
O
OR4
R2
O
R1O
R4O
OR1
O
or 2) EDC, DMAP, DMF, rt
OR4
O
n
HO
O
O
n
R1= H or 2-hydroxylpropyl
HP CD, n = 6, DS = 0.6
HP CD, n = 7, DS = 1.0
HP CD, n = 8, DS = 0.6
DS: degree of substitution
FITC: R2 = COOH; R3 = NCS
Fluorescein: R2 = COOH; R3 = H
XITC: R2 = H; R3 = NCS
n = 6, 7 or 8
R4= H or 2-hydroxylpropyl or
S
NH
S
NH
R4=
COOH
O
O
O
OH
O
O
OH
O
O
OH
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Scheme 2. (a) Synthesis of decarboxylated FITC, XITC. (b) Synthesis of
FluoresceinꢀHPCDs, FITCꢀHPCDs and XITCꢀHPCDs.
Physicochemical characteristics and optical properties
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The spectroscopic and physicochemical properties are summarized in Table 1. All
conjugated compounds showed absorption peaks in the wavelength range between
490 nm to 500 nm either in PBS or Sprague Dawley (SD) rat plasma (Table 1, Figure
1a and S2), by irradiation at 480 nm, all the markers displayed emission peaks around
520 to 530 nm, which is consistent with the spectra of their free dye. Therefore, the
introduction of HPCDs on the fluorophore moiety did not cause shift on the spectra.
Notably, their spectrum is effectively matching with the configuration of the
transcutaneous device, which is composed of two lightꢀemitting diodes with
excitation wavelength at 480 nm and a photodiode for emission wavelength detection
at 520 nm. Despite XITC is having a simple structure, it is a novel compound in the
xanthene family. Compared to FITC, the removal of carboxylic acid group on the
meso aryl ring for eliminating the negative charge did not result in change in
absorbance and emission spectra. However, the extinction coefficient of XITC is
43
44
clearly lower than that of FITC and fluorescein (Table 1). This result is consistent
with previous studies showing, that the removal of a carboxylic acid group on the
meso aryl ring allows the benzene moiety a free rotation, leading to a reduction of the
39
extinction coefficient as well as a fluorescent quantum yield.
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Figure 1. (a) Absorption and emission spectra of FITCꢀHPβCD in rat plasma. (b) The
size of FITCꢀHPβCD was analyzed by DLS.
Investigations of matrixꢀassisted laser desorption/ionization (MALDI) mass
spectrometry provided further evidence for fluorophore functionalized HPCDs. In
Figure 5b the mass of FITCꢀHPβCD features five peaks on the right part of the
spectrum at m/z = 1720.235, 1778.272, 1836.320, 1894.366 and 1952.403 Da,
corresponding to the calculated mass of native HPβCD plus FITC, respectively. A
distribution corresponding to the mass of unmodified native HPβCD is also observed
at m/z = 1331.368, 1389.395, 1447.423, 1505.459, 1563.495 Da on the left part of the
1
1
spectrum. 2D ( Hꢀ H NOESY) spectra of FITCꢀHPβCD and FluoresceinꢀHPβCD
were investigated as shown in Figure S23 and S24, the crossꢀpeak in the NOESY
spectrum of both FITCꢀHPβCD and FluoresceinꢀHPβCD clearly demonstrates that the
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HPCD’s interior protons gave the NOE correlations with the aromatic protons of
xanthene moiety in either FITC or Fluorescein. The protons of δ 6.7ꢀ7.1 ppm were
assigned to the xanthene protons and exhibit cross peaks with the HPCD protons (δ
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3
ꢀ4 ppm), suggesting a part fluorophore moiety is encapsulated into the cavity of
HPβCD. Distribution coefficient calculations using the JChem plugin of ChemAxon
indicated that all the markers had excellent hydrophilicity with the Log D value at pH
7
.4 below ꢀ5 and varied with different dyes conjugation and HPCDs (Table 1). The net
charge of the compounds spans a range from 0 to ꢀ1, depending on the absence or
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functionalization of carboxylic acids. The degree of labeling (DOL) is defined as the
average number of dye molecules coupled to HPβCD, the DOL of all the markers
were determined as shown in Table 1 and the low DOL (< 0.13) is aim to preserve the
properties of HPCDs. Although the DOL of XITCꢀHPCDs is higher than that of
FITCꢀHPCDs and FluoresceinꢀHPCDs, their absorbance and emission intensity is
lower than FITCꢀHPCDs and FluoresceinꢀHPCDs in the same concentration. This is
ascribed to the low extinction coefficient and fluorescent quantum yield of XITC,
suggesting a higher dose of XITCꢀHPCDs might be needed in animal experiments.
Aggregation analysis
It has been postulated that macromolecules over 70 kDa or big nanoparticles cannot
46
pass through the glomerular filter into urine under normal conditions. In addition,
the possible presence of βCD aggregates in a critical aggregation concentration 3 mM
47ꢀ50
βCD was determined by Dynamic light scattering (DLS) in previous studies.
Therefore, DLS measurements of FITCꢀsinistrin, FITCꢀHPCDs
and
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FluoresceinꢀHPCDs at 3 mM concentrations in aqueous solutions were performed to
further investigate their aggregation phenomenon (Figure 1b, S5 and Table S8). The
hydrodynamic diameter of FITCꢀsinistrin is roughly 21.9 nm, which is likely due to a
molecular weight range of 2000 Da to 6000 Da. Mean diameters in size distribution
by volume ranging from 0.6 nm to 3 nm for FITCꢀHPCDs and FluoresceinꢀHPCDs
were observed. Based on these results and the actual value of the outer diameter (1.46
nm, 1.54 nm and 1.75 nm) and the height (0.79 nm) of αCD, βCD and γCD,
respectively (Table S3), we conclude that each of the markers is in its monomeric unit
or containing 2 or 3 units at most.
0
1
2
3
4
5
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7
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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1
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9
0
1
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9
0
Table 1 Photophysical properties of free dyes and their HPCD based markers.
λabs (nm)
λem (nm)
Stokes shift
(nm)
ε
Net charge
(pH = 7.0)
MW
[Da]
Log D
a
Entry
DOL
ꢀ1
ꢀ1
PBS/Plasma
PBS/Plasma
(M cm )
(pH=7.4)
c
FITC
494/496
490/494
490/494
490/494
494/498
498/500
498/500
498/500
494/500
498/500
498/500
520/523
522/525
522/525
522/525
520/525
534/537
534/537
534/537
530/531
530/531
530/531
530/531
26
32
32
32
26
36
36
36
36
32
32
70000
ꢀ1
ꢀ1
ꢀ1
ꢀ1
ꢀ1
0
389.38
ꢀ0.70
ꢀ9.30
ꢀ10.70
ꢀ10.95
ꢀ1.30
ꢀ5.53
ꢀ6.93
ꢀ8.33
3.06
NA
b
FITCꢀ HPαCD
FITCꢀ HPβCD
FITCꢀ HPγCD
Fluorescein
ND
1400 ꢀ 1800
1700 ꢀ 2100
2000 ꢀ 2400
332.31
0.081
0.080
0.082
NA
ND
ND
68000
ND
Fluorescein ꢀ HPαCD
Fluorescein ꢀ HPβCD
Fluorescein ꢀ HPγCD
XITC
1400 ꢀ 1750
1700 ꢀ 2050
2000 ꢀ 2350
345.04
0.055
0.043
0.049
NA
ND
0
ND
0
16800
ND
0
XITCꢀ HPαCD
XITCꢀ HPβCD
0
1400 ꢀ 1760
1700 ꢀ 2060
2000 ꢀ 2360
ꢀ5.55
ꢀ6.95
ꢀ7.20
0.101
0.123
0.114
ND
0
XITCꢀ HPγCD
500/500
30
ND
0
a
b
c
DOL: degree of labeling. ND: not determined. NA: not applicable.
Plasma protein binding
Of particular significance for the suitability of fluorescent markers as GRF markers is
the knowledge of their interaction with plasma proteins, because binding to proteins
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influences the resulting pharmacokinetics, including bioꢀdistribution and excretion.
PPB studies were carried out for all of the markers using the assay described
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before.
Each of the markers was incubated with rat plasma at 37 °C for 1 h. The
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incubated markers were separated from nonabsorbed protein by equilibrium dialysis
of PBS against dyeꢀprotein stock solution using a twoꢀchamber dialysis setꢀup. After
2
4 h the concentrations of markers in both PBS and plasma were determined. As
shown in Table 2 and Table S9ꢀS17, all the markers exhibited very low PPB (< 10%),
comparable to or even lower (i.e., better) than some of the ‘gold standard’ renal
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99m
markers such as iothalamate, CrꢀEDTA and
TcꢀDTPA (Table S2). Furthermore,
the free fluorophores adsorb to a larger amount of protein than HPCDs based markers,
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for example, fluorescein exhibits 93.1% PPB, thereby reflecting a greater tendency
for proteins to bind to hydrophobic compound. In fact, most oral drugs have a
lipophilic physicochemical property, which is associated with higher PPB and hepatic
clearance; on the contrary, renally cleared drugs are hydrophilic and usually have a
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low PPB. In this study, the extremely low PPB of HPCD based markers was
attributed to the introduction of hydrophilic HPCDs on those hydrophobic
fluorophores structures, which increases their hydrophilicity and minimizes
nonꢀspecific interactions with serum proteins.
Stability studies in porcine liver esterase and plasma
To determine the stability of conjugation bonds between HPCDs and fluorophore, we
coꢀincubated HPβCD based markers with porcine liver esterase (PLE) for 24 h, and
then analyzed their stability with HPLC. No degradation was observed for all of
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HPβCD based markers (Figure S6), as indicated by no appearance of free fluorophore
peak in HPLC chromatographs when compared to three control measurements,
including PLE, free fluorophore and the corresponding marker, respectively. The ester
bond of FluoresceinꢀHPβCD in PLE is stable, which may be attributed to the effect of
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steric hindrance. To gain further insight into the stability of these markers in plasma,
we coꢀincubated all the HPβCD based markers with plasma. Similarly, no degradation
was found after 24 h of incubation with rat plasma (Figure S7). The results revealed
that HPβCD based markers are stable in both PLE condition and plasma in vitro
.
Cell viability evaluated by MTT assay
The cytotoxicity of FITCꢀHPβCD and FluoresceinꢀHPCDs was evaluated by using
3ꢀ(4, 5ꢀdimethylꢀ2ꢀthiazoly)ꢀ2, 5ꢀdiphenyltetrazolium bromide (MTT) assay, in
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conditionally immortalized human renal proximal tubule cell line (ciPTEC). The
results (Figure 2 and S8) show that FITCꢀHPβCD and FluoresceinꢀHPCDs did not
affect the viability of both of the two cell types tested (organic anion transporters 1 or
3 (OAT1 or OAT3) transfected), as compared to the control group with untreated cells,
suggesting these markers have no significant cytotoxic effect in representative human
renal cell lines. These nonꢀtoxicity results are also in agreement with previous reports
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using βCD derivatives on other cell lines.
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Figure 2. (a) Cell viability of ciPTECꢀOAT1 and (b) ciPTECꢀOAT3 cells incubated
with FITCꢀHPβCD and FluoresceinꢀHPβCD in MTT assays.
Table 2 PPB, urinary recovery and halfꢀlife with or without probenecidꢀtreatment
b
c
PPB
%]
Urinary Recovery
[%]
T
1/2 without Probenecid
[min]
T
1/2 with Probenecid
Changes in
Substance
[
[min]
halfꢀlife [%]
d
FITCꢀ HPαCD
FITCꢀ HPβCD
7.1
2.3
2.8
0
99.1 ± 2.6 (3)
103.4± 4.1 (3)
100 ± 6.7 (3)
57.7 ± 2.1 (3)
52.2 ± 8.5 (3)
44.3 ± 3.1 (3)
38.0 ± 4.7 (6)
32.2 ± 5.8 (6)
23.6 ± 6.6 (6)
18.1 ± 2.7 (9)
13.3 ± 6.6 (6)
9.6 ± 3.7 (7)
9.4 ± 2.5 (7)
ND
ꢀ15.3
ꢀ2.1
24.1 ± 3.2 (6)
20.2 ± 3.2 (6)
7.8 ± 4.3 (6)
7.3 ± 2.8 (7)
6.3 ± 1.3 (7)
ND
FITCꢀ HPγCD
ꢀ10.9
+70.5
+31.5
+49.2
ND
Fluorescein ꢀ HPαCD
Fluorescein ꢀ HPβCD
Fluorescein ꢀ HPγCD
XITCꢀ HPαCD
0
2.4
9.5
7.7
9.6
a
ND
XITCꢀ HPβCD
103.2 ± 5.4 (3)
ND
31.3 ± 7.0 (3)
ND
32.2 ± 4.8 (3)
ND
+2.9
ND
XITCꢀ HPγCD
a
b
c
ND: not determined.
T
1/2: clearance halfꢀlife. Changes in halfꢀlife: [T1/2 Probenecidꢀ T1/2 without Probenecid]/[T1/2
d
Probenecid]. (n): number of rats.
Transcutaneous measurement of renal function in rat models
Encouraged by those excellent properties in vitro, we proceeded to investigate
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whether these HPCDs based markers can be excreted by kidneys in rats by using
noninvasive transcutaneous measurements. The principles and methods of the
transcutaneous technique are described in experimental section and shown in Figure
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aꢀc. Fluorescence elimination curves and kinetic parameters of all the markers can be
found in Figure 3, Figure S9 and Table 2. Taken FITCꢀHPβCD as an example (Figure
d), the clearance curves of FITCꢀHPβCD returned to background within 2 h after
3
intravenous injection, suggesting it was excreted completely in this period and has a
clearance halfꢀlife of 24.1 ± 3.2 min (Table 2). Furthermore, to determine whether
FITCꢀHPβCD can be reabsorbed or secreted in addition to glomerular filtration in
kidneys, probenecid, an inhibitor of organic anion transporter (OAT) proteins in
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kidney tubules,
was administered to block a tubular reabsorption and secretion
pathway, then fluorescence elimination curves were also measured transcutaneously
for the same rats. The fluorescent signals descended to baseline and it has a similar
clearance halfꢀlife (23.6 ± 6.6 min) when compared to without probenecid treatment
(Figure 3e and Table 2). The clearance halfꢀlife of FITCꢀHPCDs either in the absence
or presence of probeneicd follow the order FITCꢀHPαCD > FITCꢀHPβCD >
FITCꢀHPγCD, supporting that the elimination goes faster with increased size of
HPCDs.
Similar phenomena
were observed
for FluoresceinꢀHPCDs
(FluoresceinꢀHPαCD > FluoresceinꢀHPβCD > FluoresceinꢀHPγCD). The clearance
halfꢀlife of the tested markers also varied with the fluorophore scaffold used, for
example, XITCꢀHPβCD > FITCꢀHPβCD > FluoresceinꢀHPβCD. Either with or
without probenecid treatment, FluoresceinꢀHPCDs were excreted extremely fast
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(
Figure 3f and 3g) and have a shorter clearance halfꢀlife than FITCꢀHPCDs or
XITCꢀHPβCD. In FITCꢀHPCDs, FITCꢀHPαCD and FITCꢀHPγCD have a slight
tubular reabsorption, because shorter clearance halfꢀlifes were observed in the
presence of probenecid (Figure 4a). In contrast, FluoresceinꢀHPCDs showed a
significant higher halfꢀlife with probenecid treatment, which indicates tubular
secretion in kidneys. Surprisingly, a negligible difference in halfꢀlife between the
absence and presence of probenecid treatment for FITCꢀHPβCD and XITCꢀHPβCD
was observed (Figure 4a), suggesting that FITCꢀHPβCD and XITCꢀHPβCD exhibit
no tubular reabsorption or secretion, and are cleared by glomerular filtration solely.
BlandꢀAltman plots for transcutaneous measurement in the presence and absence
probenecid treatment are depicted in Figure S10.
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Figure 3. (a) Miniaturized transcutaneous devices and a battery used for the
transcutaneous measurement of kidney function. (b) Fluorescent markers diffused
from the vascular space to the interstitium after intravenously administration and its
excretion was determined using a miniaturized electronic transcutaneous device. (c) A
conscious SD rat is under transcutaneous measurement with an attached device.
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Elimination curves of FITCꢀHPβCD (d and e, n=6), FluoresceinꢀHPβCD (f and g,
n=7) and XITCꢀHPβCD (h and i, n=3) by transcutaneous measurements in SD rats
models in the absence and presence of probenecid treatment.
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Urinary recovery and metabolism studies
A mandatory prerequisite for an ideal renal function marker is to recover completely
in urine and have no metabolism in vivo, therefore we conducted recoveries of the
injected doses using in vivo experiments with metabolic cages. Each marker was
injected intravenously into rats. As shown in Figure 4, Figure S11 and Table S18, a
high urinary recovery of FITCꢀHPαCD, FITCꢀHPβCD and FITCꢀHPγCD with 99.1 ±
2
.6%, 103.4 ± 4.1% and 100 ± 6.7%, respectively, were observed. Considering low
extinction coefficient of XITC and a higher dose of XITCꢀHPCDs might be needed in
rats, XITCꢀHPβCD was chosen only for this trial. A similar tendency of urinary
recovery (103.2 ± 5.4%, Figure 4d and Table S20) was also determined in the case of
XITCꢀHPβCD. Indeed, urinary recovery of the given dose was almost completed at 6
to 9 h postꢀinjection for FITCꢀHPβCD and FITCꢀHPγCD, which is in agreement with
their short clearance halfꢀlife. These high urinary excretion rates indicated all the
injected FITCꢀHPCDs and XITCꢀHPβCD are excreted into urine. However, only 40
to 60% of the injected doses were recovered for FluoresceinꢀHPCDs (Figure 4c,
Figure S12 and Table S19). These results suggested that FluoresceinꢀHPCDs are not
completely excreted through the kidneys, but also via other routes including
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metabolism by enzymes in vivo. The excretion profiles of FluoresceinꢀHPCDs are
consistent with their extremely short clearance halfꢀlife. Although
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FluoresceinꢀHPCDs are stable under coꢀincubation with PLE in vitro, the aromatic
carboxylic acid ester on FluoresceinꢀHPCDs is prone to hydrolysis in a complex
environment in vivo.
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Figure 4. (a) Clearance halfꢀlife for each marker in the presence and absence of
probenecid in rat models. (b) Urinary recovery for each marker in SD rats (n=3) after
2
4 h. Urinary recoveryꢀtime curves of FITCꢀHPβCD (c), FluorescentinꢀHPβCD (d)
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and XITCꢀHPβCD (e) in SD rats (n=3) over a period of 24 h.
Metabolism studies
To evaluate whether these markers can be metabolized in vivo, urine samples were
collected and investigated by HPLC. The HPLC results (Figure S14) indicate that
metabolites were not formed. In order to better understand these results, we carried
out an additional experiment by MALDIꢀTOF to confirm these markers in urine
samples. The obtained MALDIꢀTOF data demonstrated that the mass distribution of
FITCꢀHPβCD recovered from urine sample is the same as that of before injection
(Figure 5c and S14). Therefore, we concluded that four substances, including
FITCꢀHPαCD, FITCꢀHPβCD, FITCꢀHPγCD and XITCꢀHPβCD, have high urinary
recoveries of nearly 100% of the injected doses and do not undergo metabolism in
vivo. However, FITCꢀHPαCD and FITCꢀHPγCD are reabsorbed slightly in kidney
tubule and XITC suffers from undesirable optical properties, thus a higher dose of
XITCꢀHPCDs might be required for in vivo studies. It should be emphasized that
neither premature deaths nor adverse clinical signs in behaviors, by measurement of
body weight and food consumption of rats were observed during the whole
experiments. Taken together, relying on the attractive features of FITCꢀHPβCD in
vivo including completely and rapidly excreted through kidneys into urine,
exclusively filtered via glomerulus, no reabsorption and secretion in kidney proximal
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tubule, it is considered as a promising novel exogenous fluorescent GFR marker. In
contrast, FITCꢀHPαCD and FITCꢀHPγCD have a potential to determine the capacity
of reabsorption function in kidney tubules.
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Figure 5. MALDI spectrum of (a) HPβCD, (b) FITCꢀHPβCD and (c) FITCꢀHPβCD
recovered from urine samples in SD rats.
Conclusions
To summarize, by considering the basic principle of renally cleared drug design and
the knowledge of CDꢀbased drug delivery system, we developed a type of novel
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fluorescent markers based on fluorophore labeled HPCDs. Through a rational
screening approach, FITCꢀHPβCD is considered as the most promising exogenous
marker for transcutaneous GFR measurements. This marker can be easily synthesized
and exhibits desirable fluorescence properties. In in vitro experiments, low plasma
protein binding, good stability in plasma and esterase, as well as no cytotoxicity were
found. In rat models, a high urinary recovery of the given doses and no metabolism in
vivo were observed. Noninvasive realꢀtime monitoring of bolus clearance was
determined in combination with a miniaturized electronic device for the
transcutaneous fluorescence intensity detection in conscious rats. The results show no
significant differences in clearance halfꢀlife of the markers in the absence and
presence of probenecid treatment, so neither reabsorption nor secretion happened in
kideny proximal tubule, demonstrated it is filtrated by glomerulus only. To the best of
our knowledge, this is the first use of cyclodextrin derivatives as a backbone for
developing a novel GFR marker. Our platform of novel fluorescent markers combined
with transcutaneous fluorescent detection technique enables a rapid, robust and
convenient monitoring of renal function without the need of timeꢀconsuming
blood/urine sample preparation. Therefore in future this method allows the assessment
of renal function in realꢀtime and the evaluation of rapid changes in renal function, for
example, in acute renal failure. Formal preclinical development studies are in progress.
Finally, we noted that the development of near infrared transcutaneous devices and
markers for deeper penetration depth will be an improvement for assessing kidney
function.
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Experimental Section
Materials
Reagents including fluorescent dyes (European Pharmacopoeia grade), anhydrous
solvents (Purity ≥ 99.9%), and deuterated solvents (99.96 atom % D, contains 0.03 %
0
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(
v/v) TMS) were purchased from Sigma Aldrich or Carl Roth. All other solvents were
used as supplied (HPLC grade), without prior purification. Silica gel (Silicycle,
30ꢀ400 mesh) was used for column chromatography. 2ꢀhydroxylpropylꢀαCD, ꢀβCD
2
and ꢀγCD have the degree of substitution (DS) 0.6, 1.0, and 0.6, respectively, and
average molecular weight 1180, 1540, and 1580 Da, respectively. The catalog number
of 2ꢀhydroxylpropylꢀαCD, ꢀβCD and ꢀγCD from Sigma Aldrich is 390690ꢀ25G,
389145ꢀ25G and 390704ꢀ25G, respectively. NMR spectra were recorded on a Bruker
3
00 MHz NMR instrument. Chemical shifts are reported in ppm relative to residual
protic solvent resonances. Mestre Nova LITE v5.2.5ꢀ4119 software (Mestre lab
Research S.L.) was used to analyze the NMR spectra. Matrixꢀassisted laser
desorption/ionization timeꢀofꢀflight (MALDIꢀTOF) analyses were collected on a
Bruker microflex instrument. UVꢀvis and fluorescence spectra were acquired using a
microplate reader (Tecan Infinite M200) and an Eppendorf biospectrometer kinetic
device using quartz cuvettes (1 cm path length). HPLC analysis and separations were
carried out on a Thermo scientific ultimate 3000 liquid chromatography using
Ascentis® C18 columns. The pH of samples solution was tested by Mettler Toledo
FiveEasy™ FE20pH bench meter. Dynamic light scattering studies were conducted
using a Malvern Zetasizer Nano S90 equipment. IUPAC names of all compounds are
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provided and were determined using CS ChemBioDraw Ultra 12.0. The
transcutaneous devices are available from Mannheim Pharma & Diagnostics,
Mannheim, Germany.
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Synthesis
Synthesis of compound 2. A suspension of 2,2’,4,4’ꢀtetrahydroxybenzophenone
2
(compound 1, 2.46 g, 10 mmol) in 16 mL H O was stirred and heated to 200 °C for 48
h. This reaction was accomplished in special pressure flask. The mixture was cooled
to 60 °C, then it was poured into 60 °C hot water and kept stirring 20 min. The yellow
residue was filtered and extensively washed by 60 °C hot water until no start material
was left over (monitored by TLC, EtOAc/nHex, 1/1). 3, 6ꢀdihydroxyxanthenꢀ9ꢀone
(compound 2, 1.74 g) was obtained in a 76.3% yield. TLC (silica gel, EtOAc/nHex,
1
2
/1) R
f
= 0.28, H NMR (300 MHz, DMSOꢀd
6
) δ (ppm): 10.84 (s, 2H), 7.97 (d, J = 8.7
) δ (ppm): 173.87, 163.32,
57.43, 127.72, 113.96, 113.61, 102.05. LRMS (m/z): calcd: 228.05, found: 228.13.
Synthesis of compound 3. To a solution of 3, 6ꢀdihydroxyxanthenꢀ9ꢀone (compound 2,
.70 g, 7.45 mmol) and tertꢀbutylchlorodimethyl silane (TBDMSꢀCl, 6.90 g, 45.78
13
Hz, 2H), 6.84 (m, 4H). C NMR (75 MHz, DMSOꢀd
6
1
1
mmol) in DMF (30 mL), imidazole (5.20 g, 76.30 mmol) was added. The mixture was
stirred at room temperature 12 h. The reaction was diluted by toluene (75 mL) and
then extensively washed and extracted by water. The product was recrystallized from
ethanol to recieve the diꢀprotected product (compound 3, 3.12 g) with a yield of 91%.
1
TLC (silica gel, EtOAc/nHex, 2/1) R
f
= 0.9, H NMR (300 MHz, DMSOꢀd
6
) δ (ppm):
7
.94 (d, J = 9.1 Hz, 2H), 6.80 (m, 4H), 1.23 (s, 18H), 0.84 (s, 12H). LRMS (m/z):
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calcd: 456.22, found: 456.91. The characterization data is comparable with the
3
8
previous reports.
Synthesis of compound 4. To a solution of xanthone diTBDMS ether (compound 3,
.10 g, 6.78 mmol) in anhydrous THF was added phenylmagnesium bromide
derivative (25 mL, 0.50 mol/mL in THF). This mixture was stirred under gas N
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protection for 4 h. The reaction was quenched by adding 10 mL of 2 N HCl (aq) and
stirred for 30 min, then 5 mL NaOH (3 mol/mL) was added dropwise to rise the pH of
the mixture to 7.5 at ice bath. The resulting yellow precipitate was filtrated, washed
with a small volume of distilled THF and dried in vacuum, to yield a product
(compound 4, 1.81 g, yield 87.6%). Further purification by silica gel column
chromatography was done if required. LRMS (m/z): calcd: 303.09, found: 303.25.
Synthesis of compound XITC. Thiophosgene (6 mL, 78 mmol) was dissolved in
anhydrous CH
2 2
Cl (10 mL).This mixture was chilled to 0 °C in an ice bath. Product
amine (compound 4, 1.80 g) was dissolved in anhydrous CH Cl and added slowly in
2
2
sequence. This mixture was allowed to warm to room temperature over 3 h. The
mixture was concentrated under reduced vacuum and extensively washed by acetone
to obtain 9ꢀ(4ꢀisothiocyanatophenyl)ꢀ9Hꢀxantheneꢀ3, 6ꢀdiol (compound XITC). TLC
1
(silica gel, MeOH/ EtOAc, 1/9) R
f
= 0.5, H NMR (300 MHz, DMSOꢀd
6
) δ (ppm):
8.13 (d, J =9, 1H), 7.71(d, J =9, 1H), 7.63 (s, 2H), 7.48 (m, 4H), 7.02 (d, J =9, 2H).
LRMS (m/z): calcd: 345.05.22, found: 345.57.
General procedures for the synthesis of FITCꢀHPαCD, FITCꢀHPβCD and
FITCꢀHPγCD. To a solution of (2ꢀhydroxypropyl)ꢀαꢀcyclodextrin (HPαCD, 800 mg)
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in DMF (8 mL), NaH (400 mg, 10 mmol, 60% susp.) was added slowly and stirred at
room temperature. The reaction was stirred at 45 °C for 30 min. Afterwards, FITC
(
100 mg, 0.26 mmol) was added and the mixture was stirred at 45 °C for another 30
min and then kept at room temperature in the dark for 18 h. The red solution was
cooled in an ice bath, saturated NH Cl solution was added then to adjust pH near 10.
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4
Then using 5 % acetic acid was added dropwise to obtain a pH of 7.5. The solvent
was concentrated under reduced pressure in a rotary evaporator (bath temperature
4
5 °C). The crude product was washed by acetone two times (300 mL in each time)
and filtered; the orange remains were purified by gel silica chromatographic column
with gradient eluent (EtOAc, MeOH/EtOAc (1/2) and MeOH/H O (8/2)). The weight
2
amount of silica gel per gram of crude products is 210 g. The eluent was concentrated
under vacuum. To transfer this into a more handable form, the material is dissolved in
100 mL H
2
O at room tempertaure, filtered, and freezeꢀdried to yield a yellow solid
FITCꢀHPαCD. According to the same procedure of FITCꢀHPαCD, FITCꢀHPβCD and
1
13
FITCꢀHPγCD were obtained. HꢀNMR; CꢀNMR and Mass data are available in
supporting information.
General procedures for the synthesis of FluoresceinꢀHPαCD, FluoresceinꢀHPβCD and
FluoresceinꢀHPγCD. To a solution of (2ꢀhydroxypropyl)ꢀαꢀcyclodextrin (HPαCD, 2.0
g) and fluorescein (300 mg, 0.90 mmol) in DMF (10 mL), EDC (300 mg, 1.56 mmol)
and DMAP (27 mg, 0.22 mmol) was added. The reaction was stirred without light at
room temperature for 18 h. The resulting solution was concentrated under reduced
pressure. The residue was purified by a gel silica chromatographic column with
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