Determination of plasma levels of the active thiol form of the direct-acting PrC-210 ROS-scavenger using a fluorescence-based assay

Article abstract of DOI:10.1016/j.ab.2021.114100

PrC-210 is a direct-acting ROS-scavenger. It's active when administered orally, IV, or topically; it has none of the nausea/emesis nor hypotension side effects that have precluded human amifostine use. PrC-210 confers 100% survival to mice and rats that received an otherwise 100% lethal radiation dose and 36% reduction of ischemia-reperfusion-induced mouse myocardial infarct damage, and thus is a viable candidate to prevent human ROS-induced ischemia-reperfusion and ionizing radiation toxicities. We report the first assay for the pharmacologically active PrC-210 thiol in blood. PrC-210 has no double-bonds nor light absorption, so derivatizing the thiol with a UV-absorbing fluorochrome enables quantification. This assay: i) is done on the benchtop; it's read with a fluorescence plate reader, ii) provides linear product formation through 60 min, iii) quantifies μM to low mM rodent blood levels of PrC-210 that confer complete radioprotection, iv) accurately reflects PrC-210 thiol formation of mixed disulfides with other thiols in blood, and v) shows excellent between-day assay outcome with very low standard deviation and coefficient of variation. A fluorescence assay quantifying formation of a PrC-210 thiol-bimane adduct enables measurement of blood PrC-210 thiol. A blood assay will help in the development of PrC-210 for use in the human clinical setting.

Full text of DOI:10.1016/j.ab.2021.114100

Analytical Biochemistry 616 (2021) 114100
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Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio
Determination of plasma levels of the active thiol form of the direct-acting
PrC-210 ROS-scavenger using a fluorescence-based assay
Emma Dreischmeier, William E. Fahl*
Wisconsin Institutes of Medical Research, University of Wisconsin-Madison, Madison, WI, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
PrC-210 is a direct-acting ROS-scavenger. It’s active when administered orally, IV, or topically; it has none of the
nausea/emesis nor hypotension side effects that have precluded human amifostine use. PrC-210 confers 100%
survival to mice and rats that received an otherwise 100% lethal radiation dose and 36% reduction of ischemia-
reperfusion-induced mouse myocardial infarct damage, and thus is a viable candidate to prevent human ROS-
induced ischemia-reperfusion and ionizing radiation toxicities. We report the first assay for the pharmacologi-
cally active PrC-210 thiol in blood. PrC-210 has no double-bonds nor light absorption, so derivatizing the thiol
with a UV-absorbing fluorochrome enables quantification. This assay: i) is done on the benchtop; it’s read with a
Ischemia-reperfusion injury suppression
Radioprotector
Aminothiol
fluorescence plate reader, ii) provides linear product formation through 60 min, iii) quantifies μM to low mM
rodent blood levels of PrC-210 that confer complete radioprotection, iv) accurately reflects PrC-210 thiol for-
mation of mixed disulfides with other thiols in blood, and v) shows excellent between-day assay outcome with
very low standard deviation and coefficient of variation. A fluorescence assay quantifying formation of a PrC-210
thiol-bimane adduct enables measurement of blood PrC-210 thiol. A blood assay will help in the development of
PrC-210 for use in the human clinical setting.
1. Introduction
added to naked DNA 30 s before the ⋅OH insult [5]. For these reasons of
efficacy and low toxicity in preclinical animal models, PrC-210 is a
In many clinical settings, including ischemia-reperfusion organ
injury [1] and exposure to ionizing radiation [2], the primary toxic
event in affected cells is induced by a bolus of reactive oxygen species
(ROS), such as hydroxyl radical (⋅OH). A low toxicity, efficient
ROS-scavenger that could be easily and safely administered would have
broad application in suppressing or preventing these ROS toxicities.
PrC-210 is a new, direct-acting, aminothiol ROS-scavenger [3–5]
that has neither the nausea/emesis nor hypotension side effects [6] that
have precluded use of previous aminiothiols like amifostine in humans
[7]. In animal models, PrC-210 is orally, IV and topically bioactive; a
single systemic dose conferred, 30 min later, 100% survival to mice and
rats that received an otherwise 100% lethal radiation dose [3,4].
PrC-210 administration to mice during 45 min coronary artery
ligation-induced myocardial infarcts resulted in a 36% reduction in
ischemia-reperfusion-induced cardiac muscle death 24 h later. In ex vivo
human tissue studies, PrC-210 significantly suppressed diagnostic
x-ray-induced DNA double-strand breaks (γ-H2AX foci), and also pro-
vided 100% suppression of ROS-induced naked DNA damage when
potential candidate for development as a human pre-exposure and
co-exposure ROS-scavenger.
In both preclinical animal development as well as any human clinical
trial path, measuring levels of the active PrC-210 thiol drug form in
blood, plasma, tissue homogenates and urine is a requirement. Initial
studies using [14C]-labeled PrC-210 in both pigs and rats [6] indicated
straightforward blood pharmacokinetics and elimination of the small
molecule (MW: 148) in urine and feces. Because the PrC-210 molecule
has i) no double-bonds, and hence no UV absorbance, and ii) has a free
thiol group, bimane derivitization of the thiol to produce a covalent
adduct that has both UV absorbance and fluorescence is a logical
strategy for measuring levels of the active PrC-210 thiol in plasma and
other biological specimens.
In this report we identify reaction conditions in which a stable PrC-
210 thiol-bimane adduct is formed, and this reaction enables an efficient
and highly sensitive assay for the PrC-210 thiol in a 96 well format to
enable measurement of the PrC-210 thiol in biological specimens.
* Corresponding author.
E-mail address: fahl@oncology.wisc.edu (W.E. Fahl).
https://doi.org/10.1016/j.ab.2021.114100
Received 8 October 2020; Received in revised form 31 December 2020; Accepted 2 January 2021
Available online 5 January 2021
0003-2697/© 2021 Elsevier Inc. All rights reserved.

E. Dreischmeier and W.E. Fahl
Analytical Biochemistry 616 (2021) 114100
Fig. 1. Chemical structures and characterization of the PrC-210-monochlorobimane covalent conjugate using mass spectrometry. (A) Chemical structures of the
pharmacologically-active PrC-210 thiol and the monochlorobimane fluorochrome. (B) Mass spectrograms (left to right) of the PrC-210 thiol form, mono-
chlorobimane, and PrC-210-monochlorobimane conjugate.
2. Materials and methods
2.2. PrC-210 thiol fluorescence assay
2.1. Materials
To measure PrC-210 thiol in aliquots of a liquid, such as plasma, a
PrC-210 standard curve was first constructed. Aliquots of a 6.66 mM
PrC-210 stock were diluted with water to create standards ranging from
Synthesis of the PrC-210 aminothiol (3-(methylamino)-2-(methyl-
aminomethyl)propane-1-thiol) is described separately [8,9]. Crystals
are stored under a nitrogen atmosphere at ꢀ 20 ◦C, and even with routine
thawing, use, nitrogen flush of the vial, and re-storage at ꢀ 20 ◦C,
crystalline PrC-210 is stable for >4 yr by mass spectrometry analysis.
Purity of the PrC-210 thiol and disulfide forms was greater than 99% as
assayed by mass spectrometry and nmr. Mass spectrometry was per-
formed at the Mass Spectrometry facility in the University of Wisconsin
Biotechnology Center. Monochlorobimane (#69899), cysteine
(#C5360), glutathione (#G4251) and all other chemical reagents were
obtained from Sigma Aldrich (St. Louis, MO). Sprague-Dawley (SD) rats
(female, 35–40 g b.w.) that were used as blood donors were from Envigo
(Madison, WI). ICR mice (female 20–25 g b.w.) used for plasma PrC-210
concentration and radiation protection experiments were from Envigo
(Madison, WI). Rats and mice were maintained on 12 h light/dark cycle
and provided ad lib water and food. All animal procedures were con-
ducted according to a protocol (#M05744) approved by the University
of Wisconsin Institutional Animal Care and Use Committee.
0
μ
M to 666
either plasma or standards, in a 150
90–120 l of potassium phosphate buffer (0.25 M, pH 7.0) was delivered
to wells of black 96 well plates, ii) 15 l of a 5 mM monochlorobimane
stock solution (in methanol:water, 90:10) was added to each of the 96
wells to create a 500 M final concentration of monochlorobimane, iii)
15 l of PrC-210 standards or 10–40 l aliquots of plasma were added to
μM. For the fluorescence assay of known volume aliquots of
μl total volume incubation, i)
μ
μ
μ
μ
μ
wells via multipipettor, and iv) foil-covered plates were then vigorously
rotated at room temperature for 60 min. The pH of the complete reaction
mixture, whether it contained PrC-210 standards or plasma, was
7.0–7.1. Fluorescence in the 96 wells (390 excitation, 478 emission) was
then determined using a CLARIOStar plate reader.
2.3. Irradiation and PrC-210 treatment
Mice were irradiated in a J.L. Shepherd 137Cs irradiator. For mouse
radiation survival assays, unanesthetized mice were placed in small
2

E. Dreischmeier and W.E. Fahl
Analytical Biochemistry 616 (2021) 114100
irradiation. There were 10–12 mice in each PrC-210 treatment group.
2.4. Statistical analysis
Graphpad Prizm software, was used for plotting data and for analysis
of statistical differences between treatment groups.
3. Results
3.1. A stable, fluorescent PrC-210 thiol-bimane adduct
In an initial experiment, we found that mixing monochlorobimane,
dissolved in an alcohol:water solution (methanol:water, 90:10) with an
equimolar solution of PrC-210 in water at room temperature resulted in
the formation of a stable product of the appropriate mass and charge
(Fig. 1) to indicate formation of the PrC-210-bimane adduct. Formation
of this adduct was expected in the context of published reports of other
thiol-bimane adducts [10–14]. PrC-210, when dissolved in aqueous
buffer shows the single m/z mass at 149 indicating the pure PrC-210
thiol form. At pH 7.2, PrC-210 thiol conversion to disulfide form (m/z
peaks at 295,148) occurs with a half-life of 3.5 h; in water at pH ≤ 6, the
PrC-210 thiol is stable for more than a week. The m/z peak at 339 in-
dicates formation of the PrC-210-monochlorobimane conjugate, pre-
sumably through a thiol bridge, as previously described for other
bimane-thiol adducts [10,11].
Fig. 2. Conjugation reaction pH optimum. Aliquots of 0.25 M potassium
phosphate buffer, at the specified pH’s, were combined with 333 μM PrC-210
(final concentration) in water and 500 μM monochlorobimane (final concen-
tration) in methanol:water (90:10) and incubated in the dark at room tem-
perature for 1 h. Fluorescence of the PrC-210-monochlorobimane adduct was
monitored on a plate reader with excitation at 390 nm and emission at 470 nm
(see Methods).
3.2. pH-Dependence of the conjugation reaction
To maintain a stable pH within the reaction mixture, to enable both
stable reaction rate and stable fluorescence excitation and emission,
whether it contained PrC-210 standards in water or various plasma pH-
modifiers, 0.25 M potassium phosphate was used. Titration of the
KH2PO4 stock solution created a family of buffer solutions from pH 4.3
plastic containers centered in the irradiator and received 9 Gy of whole-
body radiation at a dose rate of 2.1 Gy/min. Radiation dose was cali-
brated using thermoluminescent dosimeters. Mice received either
intraperitoneal water or intraperitoneal PrC-210 dissolved in water (pH
~6.0) 30 min prior to irradiation. Mice were observed for 30 days post-
to 7.7 (Fig. 2). Addition of 333
μM PrC-210 and 500 μM mono-
chlorobimane to these buffer aliquots showed that peak adduct
Fig. 3. PrC-210 adduct formation as a function of both incubation time and PrC-210 concentration. (A) A standard curve containing 7–666 μM PrC-210 thiol and
500 μM monochlorobimane in pH 7.0 0.25 M phosphate buffer was shaken at room temperature for the indicated times and adduct fluorescence was monitored. (B)
Parallel reaction incubations, with PrC-210 added at the specified concentrations to buffer, either in its thiol form or in its disulfide form, were mixed and fluo-
rescence was determined after 1 h. For illustration purposes, standard deviation bars are shown for three data points on the 60 min curve in panel A.
3

E. Dreischmeier and W.E. Fahl
Analytical Biochemistry 616 (2021) 114100
formation was achieved in the pH 7.1–7.6 range (Fig. 2).
3.3. A 96 well fluorescence assay
A 96 well assay format was designed to enable routine analysis of
small (microliter) aliquots of liquids, such as plasma, serum, tissue
culture media, etc. Both the monochlorobimane reactant and the PrC-
210 thiol are sufficiently stable to enable routine reagent preparation
and dilutions of standards to the 96 well format. Unknown samples, in
which PrC-210 thiol may be less stable because of basic pH or other
reasons, can be immediately thawed and microliter aliquots added
directly to 96 wells pre-charged with monochlorobimane, so that the
thiol-bimane reaction can immediately proceed upon admixture in the
well. With standardized mixing of PrC-210 thiol standards (0–666 μM
final concentration) and monochlorobimane, both linear PrC-210 con-
centration-dependent, and linear time-dependent, formation of the
fluorescent thiol-bimane product were observed (Fig. 3A, and inset).
Addition of the pure PrC-210 disulfide, instead of the PrC-210 thiol, to
the other reactants produced no detectable fluorescent product
(Fig. 3B).
3.4. PrC-210 in blood workup
Three approaches were compared to determine the best way to
quickly process rat blood that had been spiked with PrC-210. These
included: i) spike blood with PrC-210, immediately microfuge the blood
(13,000 rpm, 45 s) and immediately transfer and freeze the supernate
plasma using dry ice, i(a)) same as i), except allow the blood to sit at
room temperature for 30 min before microfuge, ii) spike blood with PrC-
210, wait 30 min at room temperature, add 4 ◦C ethanol (to achieve 55%
ethanol final), microfuge and freeze the supernate, iii) spike blood with
PrC-210, wait 30 min at room temperature, add 0.5 M perchloric acid to
achieve 0.25 M final concentration, microfuge and neutralize the
supernate with added NaOH, and freeze the supernate. The supernates
were then thawed and assayed in pH 7.0 reactions, and Fig. 4 shows the
adduct formation associated with each procedure. Simple collection of
blood into EDTA- or heparin-tubes, spiking with PrC-210, and then rapid
microfuge spin and freezing the supernate gives the most consistent
outcome. The downward displacement of the curve with a 30 min wait
before microfuging is consistent with the previously determined PrC-
210 half-life of 3.5 h at pH 7.25. Precipitation steps with either
ethanol or perchloric acid were not productive.
Fig. 4. Determining an optimized processing strategy for PrC-210-spiked into
3.5. PrC-210 fate in plasma
rat blood. 100 μl aliquots of rat blood were spiked with the indicated concen-
trations of PrC-210. Samples were then processed as indicated in the figure. In
two sets, either 55% ethanol (final concentration) or 0.25 M perchloric acid
(final concentration) were added to precipitate blood constituents. For all
samples, centrifugation at 14,000 rpm in a microfuge for 5 min resulted in clear
supernates. A predetermined amount of 5 N NaOH was added to perchloric acid
supernates to yield a final pH of 7.0. Aliquots of all final supernates were then
added to standard incubations containing pH 7.0 potassium phosphate buffer
and monochlorobimane, and adduct fluorescence after 1 h was determined.
To validate this adduct-formation assay, PrC-210 was spiked into rat
plasma to achieve three known plasma concentrations, 0.5, 1.0 and 3.0
mM, the tubes sat at room temperature for 15 min, and aliquots of the
spiked plasma were then frozen, thawed, and assayed in the 96 well
assay described here. At 0.5, 1.0 and 3.0 mM spiking, the assayed values
were 0%, 10.5% or 14.5% lower than the specified spike concentration
(Fig. 5B).
To explore at least two explanations for “the loss” of PrC-210 thiol
spiked into plasma, equimolar concentrations of PrC-210 thiol, cysteine-
thiol or glutathione-thiol were incubated together in water, and aliquots
of both the pre- and post-reaction mixtures were analyzed by mass
spectrometry (Fig. 5C). Ion masses consistent with formation of both
PrC-210-cysteine and PrC-210-glutathione mixed disulfides were
observed.
3.6. PrC-210 fate in whole blood
When 1.0 mM PrC-210 was spiked into rat plasma the assay standard
curve (PrC-210 dissolved in water) extrapolated a concentration of 0.89
mM in the plasma sample (Figs. 5A and 6, center bar). When 1.0 mM
PrC-210 was spiked into rat whole blood, the blood was immediately
4

E. Dreischmeier and W.E. Fahl
Analytical Biochemistry 616 (2021) 114100
Fig. 5. Fate of PrC-210 thiol when spiked into rat plasma. (A) 95
μ
l aliquots of rat plasma were spiked with 5
μ
l aliquots of water containing PrC-210 thiol to achieve
the final indicated concentrations. Samples sat at room temperature for 3 min to simulate blood recovery and micro-centrifugation from a rodent or human, and were
then frozen on dry ice. Aliquots of the thawed plasma (30 μl) were then assayed versus a PrC-210 standard curve (0–666 μM PrC-210 in buffer), and after a 1 h
incubation at room temperature, adduct fluorescence was determined, and the PrC-210 thiol concentrations in plasma, extrapolated from the standard curve were
plotted. Shaded boxes highlight the PrC-210 “shortfall” that was observed in the 1.0 mM and 3.0 mM samples. (B) The observed concentration shortfalls in the
assayed plasma samples, plotted against the spiked plasma PrC-210 concentration, are shown. (C) In separate, water incubations, PrC-210 thiol was incubated with
either cysteine-thiol or glutathione-thiol for 24 h, and reaction samples were then analyzed by mass spectrometry.
5

E. Dreischmeier and W.E. Fahl
Analytical Biochemistry 616 (2021) 114100
Table 1
Validation parameters of PrC-210 assay in rat plasma.
Spiked Sample Conc (mM)
Measured concentration (mM)
Within Day (n = 5) Between Days (n = 5)
0.50
Mean
SD
0.502
0.015
Mean
SD
0.498
0.016
microfuged, and the plasma supernate was then assayed, the assay
determined a concentration of 1.36 mM in the plasma (Fig. 6, left bar). If
the PrC-210 could not access a portion of the whole blood volume in the
time before microfuging and pelleting the blood cells, then its concen-
tration in the available liquid volume would be incrementally higher. If
the 0.89 mM PrC-210 concentration found in 0.1 ml of plasma is divided
by 0.6, to account for a standard 40% hematocrit in rat blood, the
calculated PrC-210 concentration is then 1.49 mM (Fig. 6, right bar).
The 0.12 mM difference in PrC-210 (“*” in Fig. 6) between measured
blood plasma and hematocrit-corrected blood plasma could result from
passage of the PrC-210 molecule into blood cells in the few minutes
before microfuging.
3.7. PrC-210 assay validation
The spiked addition of 0.5 mM PrC-210 to rat plasma (Fig. 5A)
yielded samples that measured 0.502 mM ± 0.015 SD (n = 5) by assay
when the samples were freeze/thawed on the day of the spiking, and
0.498±0.016 SD when the samples were thawed on the day after the
spiking. This is a standard deviation for the assay of 3.0% (Table 1).
3.8. PrC-210-conferred radioprotection
Fig. 7A shows that a single intraperitoneal dose of PrC-210 (252 μg/
gm b.w.) administered between 7.5 and 60 min before whole-body ra-
diation conferred highly significant radioprotection (P values of
<0.0001 to 0.001) versus the sham-treated 0 min controls. In Fig. 7B
experiment, we treated mice with the same intraperitoneal dose of PrC-
210 as used in Fig. 7A radioprotection experiment, and then collected
blood, prepared plasma, and measured the PrC-210 concentrations to
provide both a picture of the pharmacokinetics of PrC-210 absorption
from the peritoneal cavity into blood and its clearance; these blood
concentrations and the associated pharmacokinetic parameters that
determined them conferred 100% radioprotection. The peak PrC-210
plasma level of 2.3 mM was achieved rapidly, just 3 min following IP
injection.
Fig. 6. Fate of PrC-210 thiol when spiked into rat blood or plasma. 95 μl ali-
quots of rat whole blood or rat plasma were spiked with 5 μl aliquots of water
containing PrC-210 to achieve a final concentration of “1 mM” After 3 min at
room temperature, blood was micro-centrifuged for 30 s, and both the blood
plasma supernate from the spin, and the separate plasma sample were frozen on
dry ice, then thawed, and then assayed for PrC-210 content versus a PrC-210
standard curve prepared in pH 7.0 phosphate buffer.
4. Discussion
We report here the first assay for the pharmacologically active form,
PrC-210 thiol, in blood and plasma. The assay is also well-suited for
assays of the molecule in tissue culture media and other biological fluids
to establish half-life and effective drug concentrations. The small PrC-
210 molecule, with no double-bonds, has no light absorption, so deriv-
atization of the free thiol with a UV-absorbing fluorochrome provides
many options to quantify PrC-210 without the substantial cost, or the
accessibility issues during an extended pandemic, of accessing
technically-staffed, liquid chromatography-mass spectrometry in-
struments. The assay described here: i) is conveniently done on the
benchtop and read with a standard fluorescence plate reader, ii) pro-
vides linear product formation over 1 h, iii) detects and quantifies the
μ
M to low mM blood levels of PrC-210 thiol found in rodents in which
PrC-210 confers complete radioprotection, iv) accurately reflects PrC-
210 thiol formation of mixed disulfides with other thiols present in
blood, and v) shows excellent between-day storage and assay outcome
with very low standard deviation and coefficient of variation.
Derivitization of a thiol group with monochlorobimane to form a
6

E. Dreischmeier and W.E. Fahl
Analytical Biochemistry 616 (2021) 114100
Fig. 7. Mouse plasma PrC-210 thiol levels and their radioprotective impact upon mouse survival following a single intraperitoneal dose of PrC-210. (A) 30-Day
survival in groups of mice that were given a single intraperitoneal injection of PrC-210 (252 g/gm b.w.) at the indicated times prior to a single-dose of whole-
μ
body radiation (9 Gy). (B) Individual levels of PrC-210 thiol in plasma samples of individual mice (at indicated times post intraperitoneal injection) that
received a single intraperitoneal injection of PrC-210 at “0 Min.” Mice received a single dose of 252 μg/gm bw, which equals the 0.5 X Maximum Tolerated PrC-210
Dose [4]. Different symbols represent different experiments.
fluorescent, UV-absorbing adduct of a thiol molecule has been widely
used before to quantify plasma and cell levels of the thiols of interest
[10–14]. The pH optimum of 7.4 for formation of the
PrC-210-monochlorobimane adduct is similar to that of the previous
examples as well. There was no detectable reaction between mono-
chlorobimane and the PrC-210 disulfide, so the derivatization reaction
for use in a variety of human clinical settings.
Author contributions
Both authors contributed equally to this manuscript.
provides
a
simple means to measure levels solely of the
pharmacologically-active thiol form of the drug molecule. As described
with previous derivatization assays of thiols [14,16], i) reaction of the
thiol of interest with physiologic thiols in blood, principally cysteine and
glutathione, and ii) reaction of the derivatizing agent with these same
thiols to create possible fluorescent background, are always-present
aspects of assays such as the one presented here for PrC-210 thiol.
PrC-210 is the prototype of a new family of direct-acting, small
molecule aminothiol ROS scavengers [3,4], which can be administered
orally or IV, and it has none of the nausea/emesis nor hypotension side
effects [6] in animal models that have prevented significant use of
earlier aminothiols like amifostine in humans. Because PrC-210 is orally
bioactive, where it confers: i) 100% survival to mice and rats that
received an otherwise 100% lethal radiation dose, and ii) 36% reduction
in ischemia-reperfusion-induced mouse cardiac tissue damage [15], it is
a viable candidate for use in humans to suppress or prevent ROS-induced
toxicities from both ischemia-reperfusion [1] and ionizing radiation [2].
Having an efficient assay to enable blood and tissue levels of the active
PrC-210 thiol form will support and hasten clinical development of this
new drug molecule.
Declaration of competing interest
The authors declare no conflicts of interest.
Acknowledgements
Supported by US National Cancer Institute grant#R03CA176799 to
William Fahl.
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