Improving the reactivity of hydrazine-bearing MRI probes for: In vivo imaging of lung fibrogenesis

Article abstract of DOI:10.1039/c9sc04821a

Pulmonary fibrosis (PF) is the pathologic accumulation of extracellular matrix components in lung tissue that result in scarring following chronic lung injury. PF is typically diagnosed by high resolution computed tomography (HRCT) and/or invasive biopsy. However, HRCT cannot distinguish old injury from active fibrogenesis. We previously demonstrated that allysine residues on oxidized collagen represent an abundant target during lung fibrogenesis, and that magnetic resonance imaging (MRI) with a small-molecule, gadolinium-containing probe, Gd-Hyd, could specifically detect and stage fibrogenesis in a mouse model. In this work, we present an improved probe, Gd-CHyd, featuring an N,N-dialkyl hydrazine which has an order of magnitude both greater reactivity and affinity for aldehydes. In a paired study in mice with bleomycin induced lung injury we show that the improved reactivity and affinity of Gd-CHyd results in significantly higher lung-to-liver contrast, e.g. 77% higher at 45 min post injection, and slower lung clearance than Gd-Hyd. Gd-CHyd enhanced MRI is >60-fold higher in bleomycin injured mouse lungs compared to uninjured mice. Collectively, our data indicate that enhancing hydrazine reactivity and affinity towards allysine is an effective strategy to significantly improve molecular MRI probes for lung fibrogenesis.

Full text of DOI:10.1039/c9sc04821a

Chemical
Science
View Article Online
View Journal | View Issue
EDGE ARTICLE
Improving the reactivity of hydrazine-bearing MRI
probes for in vivo imaging of lung fibrogenesis†
Cite this: Chem. Sci., 2020, 11, 224
abc
ade
ab
ab
All publication charges for this article Eman A. Akam,
Eric Abston,
Nicholas J. Rotile, Hannah R. Slattery,
have been paid for by the Royal Society
of Chemistry
abc
Michael Lanutice and Peter Caravan *abc
Iris Y. Zhou,
Pulmonary fibrosis (PF) is the pathologic accumulation of extracellular matrix components in lung tissue
that result in scarring following chronic lung injury. PF is typically diagnosed by high resolution
computed tomography (HRCT) and/or invasive biopsy. However, HRCT cannot distinguish old injury
from active fibrogenesis. We previously demonstrated that allysine residues on oxidized collagen
represent an abundant target during lung fibrogenesis, and that magnetic resonance imaging (MRI) with
a
small-molecule, gadolinium-containing probe, Gd-Hyd, could specifically detect and stage
fibrogenesis in a mouse model. In this work, we present an improved probe, Gd-CHyd, featuring an N,N-
dialkyl hydrazine which has an order of magnitude both greater reactivity and affinity for aldehydes. In
a paired study in mice with bleomycin induced lung injury we show that the improved reactivity and
affinity of Gd-CHyd results in significantly higher lung-to-liver contrast, e.g. 77% higher at 45 min post
injection, and slower lung clearance than Gd-Hyd. Gd-CHyd enhanced MRI is >60-fold higher in
bleomycin injured mouse lungs compared to uninjured mice. Collectively, our data indicate that
enhancing hydrazine reactivity and affinity towards allysine is an effective strategy to significantly
improve molecular MRI probes for lung fibrogenesis.
Received 24th September 2019
Accepted 8th November 2019
DOI: 10.1039/c9sc04821a
rsc.li/chemical-science
and pattern of lung scarring but cannot determine current
disease activity or predict disease progression. Progression of
Introduction
7
Tissue brosis is commonly ascribed to wound-healing IPF to its end stage can take several clinical forms from rapid or
1
processes gone astray as a result of chronic injury. Unre- slow decline to stable disease punctuated with periods of rapid
8
solved inammation and oxidative stress lead to tissue decline. Assessment of early disease stages of IPF with HRCT is
remodelling and the accumulation of extracellular matrix further complicated because radiological patterns that deter-
components, scar tissue formation and loss of function in the mine disease are typically those occurring at late stages of the
9
affected organ. In the lungs, this process results in the thick- disease. The limitations of HRCT in IPF diagnosis along with
ening of the interstitium, abolition of alveolar spaces and the unpredictability of disease progression severely limit
2
eventual respiratory failure. Idiopathic pulmonary brosis patient prognostication. A noninvasive method to identify
(
IPF) is the most common and most severe type of idiopathic patients with disease, to identify regions of active disease
interstitial lung disease (ILD) estimated to affect 58.7 per activity, and/or to identify patients likely to progress rapidly
3
1
00 000 individuals. IPF is devastatingly progressive with would also have benet in developing new therapeutics for IPF
4
a median survival of 2.5 to 3.8 years post diagnosis. Recent data because patients could be better stratied for trial enrolment,
suggests that the incidence and mortality of IPF are increasing.
5
10–13
and early measures of treatment response may be observed.
IPF is diagnosed through radiologic patterns on high-
We are interested in developing molecular magnetic reso-
resolution computed tomography (HRCT) scans or using lung nance (MR) probes to asses IPF disease activity by targeting
6
biopsy if HRCT is equivocal. HRCT can characterize the extent biochemical features of active brosis (i.e. brogenesis). MR
imaging is a non-invasive technique that provides three-
dimensional, high resolution anatomical images without
ionizing radiation. Coupled with a targeted MR visible probe,
a
Martinos Center for Biomedical Imaging, Massachusetts General Hospital (MGH),
Boston, USA. E-mail: caravan@nmr.mgh.harvard.edu
b
molecular imaging can be used to specify disease location and
activity, quantify extent of disease and monitor response to
therapy. Molecular MR probes can detect unique biochemical
The Institute for Innovation in Imaging, MGH, Boston, USA
c
Harvard Medical School, Boston, USA
d
Boston University School of Medicine: Pulmonary, Allergy, Sleep & Critical Care
Medicine, Boston, USA
and cellular markers of disease and allow for early assessment
e
14,15
The Division of Thoracic Surgery, MGH, Boston, USA
and diagnosis.
Fibrosis is accompanied by upregulation of
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c9sc04821a
16
lysyl oxidase (Lox) and Lox-like (LoxL) enzymes which catalyze
1
224 | Chem. Sci., 2020, 11, 224–231
This journal is © The Royal Society of Chemistry 2020

View Article Online
Edge Article
Chemical Science
oxidation of lysine 3-amino groups to allysine aldehydes thereby
allowing for the crosslinking of collagen and formation of
17
scar. The electrophilic aldehydes of allysine residues provide
an abundant target for reaction with nucleophiles and thus
a mechanism by which MRI probes can be targeted to brosing
18,19
tissue.
Hydrazines selectively react with aldehydes and ketones to
form hydrazones under physiological conditions with water
being the sole by-product. Hydrazines present excellent reactive
handles for bioorthogonal reactions linking targets and probes
20
to biomolecules of interest. Recently a turn-on chemical
exchange saturation transfer (CEST) MR probe that detects
21
small bioactive aldehydes in vitro was reported, as well as
some aldehyde targeting positron emission tomography
22,23
probes.
We have demonstrated that the hydrazine-equipped
aldehyde-reactive small molecule gadolinium chelate Gd-Hyd
can bind aldehydes in vivo and robustly stage and quantify
18
hepatic and pulmonary brogenesis.
Gd-Hyd is hydrophilic, anionic, and exhibits very low non-
specic protein binding which results in a short, 5 minute
18
blood half-life in vivo in mice. This short residency time results
in efficient whole body elimination and low background signal,
Fig. 1 Conjugation of the probes to 2-formyl pyridine, allysine-rich
protein (BSA-Ald) and allysine-rich tissue (porcine aorta). (a) Structures
however it does limit the time that the complex is exposed to of probes evaluated. (b) Relaxivity values at 60 MHz (PBS, pH 7.40, 37
ꢀ
target tissue. Increasing the rate of reaction with aldehydes
should lead to an increase of the on-target accumulation of the
probe. Extensive work by Kool and co-workers on the reactivity
of hydrazines reveals that electron-poor acyl hydrazines such as
that featured in Gd-Hyd react with aldehydes at a slower rate
C) for Gd-Hyd and Gd-CHyd in the presence or absence of BSA and
aldehyde-rich BSA-Ald. (c) Concentration-dependent binding of the
probes to 2-formyl pyridine as determined by LC-ICP-MS (d) binding
of probes to allysine-rich porcine aorta as determined by ICP-MS
following tissue digestion.
20,24,25
than electron-rich alkyl hydrazines.
As such, we sought to
improve the reactivity of the prototype probe Gd-Hyd by
substituting the reactive hydrazine moiety from a hydrazide to
alkyl hydrazine. In this work, we present the aldehyde-reactive
gadolinium chelate Gd-CHyd, featuring a secondary alkyl
hydrazine. We compare the in vitro reactivity of Gd-CHyd to that
of Gd-Hyd and assess the consequences of the differing reac-
tivities in vivo in a bleomycin (BM) lung-injury model of lung
using catalytic amounts of palladium on carbon resulted in the

nal product Gd-CHyd (5).
We compared the rates of reactivity of Gd-CHyd and Gd-Hyd
with a small-molecule model aldehyde under physiologically
relevant conditions. We chose 2-formyl pyridine in order to

brosis.
Results and discussion
Synthesis and reactivity of Gd-CHyd with aldehydes
The allysine-reactive probe Gd-CHyd features a Gd-DOTA scaf-
fold conjugated to a hydrazine arm that reacts reversibly with
aldehydes (Fig. 1a). Unlike the prototype probe Gd-Hyd
18
(
Fig. 1a), Gd-CHyd features an electron-rich piperazino-
hydrazine which we hypothesized would undergo condensa-
tion reactions at a faster rate than the acyl hydrazide of Gd-Hyd
(Fig. 1a). As depicted in Scheme 1, we rst synthesized the
carboxybenzyl (CBZ)-protected piperazino-hydrazine arm 1 via
an alkylation/cyclization reaction of bis(2-chloroethyl)amine
hydrochloride with benzyl carbazate. We then prepared the
activated ester of t-butyl protected DOTAGA using N-hydrox-
0
ysuccinamide (NHS) and N,N -dicyclohexylcarbodiimide (DCC)
and coupled the active ester with 1 to give compound 2.
Removal of the t-butyl groups using triuoroacetic acid resulted
in the CBZ-protected scaffold 3 which we used to chelate
Scheme
1 Synthetic route of the aldehyde-reactive Gd-CHyd.
gadolinium resulting in 4. Finally, a hydrogenolysis reaction Charges on Gd-complexes are omitted for clarity.
This journal is © The Royal Society of Chemistry 2020
Chem. Sci., 2020, 11, 224–231 | 225

View Article Online
Chemical Science
Edge Article
follow the condensation reaction spectrophotometrically as concentration-dependent conjugation of probe in allysine-rich
24
previously reported. We found that Gd-CHyd reacts with 2- tissue at equilibrium. We found that the amount of probe
formyl pyridine an order of magnitude faster than Gd-Hyd bound to porcine aorta aer 72 h incubation was higher for Gd-
(Table 1) under pseudo-rst order conditions at physiological CHyd than for Gd-Hyd but neither binding isotherm reached
pH and temperature. We conrmed the relative rates of reac- saturation (Fig. 1d).
tivity of the two probes using a chromatographic method with
Collectively, these data demonstrate that Gd-CHyd reacts
ICP-MS detection under slightly different conditions and found with small molecule aldehydes in solution, aldehyde-containing
that the relative reactions rates remain consistent (ESI, Fig S5 proteins in solution, and aldehyde-rich tissue. Gd-CHyd
and Table S1†).
undergoes hydrazone condensation reactions at an order of
The extent of formation of the condensation product, i.e. the magnitude faster rate than Gd-Hyd, and Gd-CHyd results in an
equilibrium dissociation constant K , is another factor that order of magnitude more thermodynamically stable hydrazone
d
should impact the accumulation of the probe in brosing product than Gd-Hyd.
tissue. We evaluated the dissociation constant for the reaction
of Gd-Hyd and Gd-CHyd with 2-formyl pyridine at various Bleomycin lung injury mouse model of lung brogenesis
concentrations. We quantied the amount of Gd-containing
species using a chromatographic method with ICP-MS detec-
We evaluated the effects of the superior reactivity of Gd-CHyd in
vivo in the bleomycin-induced lung-injury model (BM) in mice.
tion and calculated the corresponding dissociation constants
We previously demonstrated that Gd-Hyd can quantify and
(
Fig. 1c). We found that the dissociation constants for these
stage pulmonary brogenesis in the lungs of BM mice, and that
26
reactions fall within literature-reported ranges, with K for Gd-
d
Gd-Hyd enhanced MRI can be used to monitor disease
CHyd being 9-fold smaller than that for Gd-Hyd (Table 1).
We then examined the reaction of Gd-Hyd with an aldehyde-
bearing protein, oxidized bovine serum albumin (BSA-Ald) and
evaluated the effect of protein-conjugation on relaxivity (ability
to effect MR signal change) in solution. BSA is a soluble, lysine
rich protein that can serve as a useful model. We oxidized BSA
using reported procedures and quantied the aldehyde load,
conrming the presence of one aldehyde motif per protein
18
progression and response to treatment of PF. We sought to
directly compare Gd-CHyd enhanced lung MRI to that gener-
ated by Gd-Hyd. The BM model is known for its heterogeneity
both in location of brosis and extent of disease. We therefore
designed a pair-wise study to mitigate the heterogeneity of the
disease in this model. We imaged mice 14 days aer initial
bleomycin injury rst with Gd-Hyd and then 24 h later we per-
formed the same imaging protocol in the same mouse with Gd-
CHyd (Fig. 2a). Aer the second imaging session, the mice were
euthanized and the lungs and other tissues harvested for
analysis. We chose to image with Gd-CHyd second in the two-
probe sequence in order to determine the biodistributions of
this new probe while adhering to the 3R principles in the ethical
27,28
(ESI†).
We then determined the relaxivity of Gd-Hyd or Gd-
CHyd aer 72 h incubation with BSA, BSA-Ald or in the
ꢀ
absence of protein. In PBS at 1.41 T and 37 C, Gd-Hyd and Gd-
CHyd exhibit similar relaxivities of 3.9 ꢁ 0.2 and 4.3 ꢁ 0.3
ꢂ1 ꢂ1
mM
s
respectively, decreasing slightly at 4.7 T to 3.4 ꢁ 0.1
ꢂ1 ꢂ1
and 4.1 ꢁ 0.1 mM
s
respectively. At 1.41 T in the presence of
unmodied BSA protein, the relaxivity increases slightly due to
non-specic protein interaction. Incubation of Gd-Hyd or Gd-
CHyd with BSA-Ald resulted in a 30% increase of relaxivity
due to covalent interaction with protein aldehydes (Fig. 1b). We
conrmed binding of probes to BSA-Ald by passing the sample
though an ultracentrifugation 5000 Da lter and quantifying
the unbound gadolinium probe in the ltrate by ICP-MS (ESI,
Fig. S7†).
The aorta is a tissue rich in allysine because it is constantly
28,29
being remodelled due to high shear stress.
We evaluated the
binding of Gd-Hyd and Gd-CHyd to porcine aorta to assess the
Table 1 Kinetic and thermodynamic parameters measured for the
condensation reactions of hydrazine-bearing gadolinium probes. The Fig. 2 Study time line and imaging sequences (a) Bleomycin admin-
pseudo-first order rate constant (kobs) for the reaction Gd-CHyd with istration to initiate lung injury at day 0 followed by 14 days of fibrosis
2
-formyl pyridine at pH 7.40 in PBS is 11 times larger than that development. On day 14, the animals are imaged dynamically with Gd-
measured for Gd-Hyd. The K
for this reaction indicates that the Gd- Hyd. After 24 hours, the same animals are dynamically imaged with
CHyd hydrazone is 9 times more stable than the analogous Gd-Hyd Gd-CHyd, and then euthanized and the organs collected for analyses.
d
hydrazone
(b) Anatomical 2D-RARE and T1-weighted (T1W) 3D-FLASH images are
acquired before probe injection and are used along with the 3D-FLASH
images post injection to define regions of interest (ROIs). The T1W 3D-
UTE images are used to quantify the signal in the lung ROIs before and
after probe injection. The same imaging protocol was followed for
a cohort with bleomycin-na ¨ı ve mice that were imaged with Gd-CHyd
only.
ꢂ1
k
obs, (min
)
Relative kobs
K
d
(mM)
Relative K
d
Gd-hyd
Gd-CHyd 0.0314 ꢁ 0.0002
0.0028 ꢁ 0.0006
1
11
415 ꢁ 63
1
45 ꢁ 16 0.11
226 | Chem. Sci., 2020, 11, 224–231
This journal is © The Royal Society of Chemistry 2020

View Article Online
Edge Article
Chemical Science
30
use of animals: replacement, reduction, and renement. We (Fig. 3). Conrming the presence of brosing tissue in
veried complete clearance of Gd-Hyd from bleomycin-injured bleomycin-injured lungs.
lungs 24 hours post injection by harvesting the tissue and
In the imaging protocol, we rst acquired 2D Rapid Acqui-
determining the gadolinium content by ICP-MS. We found that sition with Relaxation Enhancement (RARE) images, T1-
the lung gadolinium concentration 24 hours post Gd-Hyd weighted 3D Fast Low Angle Shot (FLASH) images, and 3D T1-
injection was below the detection limit of our instrument. We weighted ultrashort time to echo (UTE) images. Mice were
ꢂ1
also imaged BM mice (n ¼ 2) with Gd-Hyd on two consecutive then injected with probe (0.1 mmol kg ) as a bolus via an
days and found that there was no signicant change in the MR indwelling tail vein line. Then the FLASH sequence was
signal obtained on the different days. This conrms that disease repeated 10 times for a total of 15 minutes to follow clearance of
progression within 24 hours is negligible.
the probe and to measure the blood half-life. The UTE sequence
We veried brosis in BM animals by histology and by was repeated at 15, 30, and 45 minutes aer probe injection.
quantifying the biochemical markers hydroxyproline and ally- Aer the last UTE acquisition, the mouse was removed from the
sine in the lungs of na ¨ı ve and BM mice. Aer imaging, the right scanner. The UTE sequence overcomes the inherently low MR
lung was xed in formalin and stained with hematoxylin and signal in the lung due to the very short T2* of the lung protons.
eosin (H&E) and sirius red/fast green (S/F) (Fig. 3a). H&E However, the UTE images do not provide strong contrast
staining show increased tissue density and cellularity in the between the lung parenchyma, large vessels in the lung, or
bleomycin injured lungs. S/F staining shows increased regions airways. Therefore, we used the RARE (black blood, black lung)
of tissue stained red, indicative of brosis. We quantied the and the rst post probe FLASH (bright blood) images to dene
area of the slides that stained positive with S/F as the collagen regions of interest (ROIs) in the lung that excluded vessels and
proportional area (CPA, Fig. 3b), and observed a 10-fold and airways. A total of 8 lung ROIs were dened on sagittal image
signicantly higher CPA in the bleomycin injured lungs. The slices spanning both lungs. As a reference tissue, we also
le lungs of the animals were homogenized and the amounts of dened ROIs in the liver in each slice. We averaged the lung (or
hydroxyproline and allysine were determined using previously liver) signal from the 8 slices to get signal intensity (SI) at a given
18,28
reported assays.
Hydroxyproline is a quantitative marker of time point. We computed the lung-to-liver contrast-to-noise
total collagen in brosing tissue commonly used to gauge the ratio as (SIlung ꢂ SIliver)/SDair, where SDair is the standard devi-
severity of brosis. Allysine is the aldehyde target for Gd-Hyd ation in an ROI drawn outside of the animal. We then calculated
and Gd-CHyd and is formed from the oxidation of collagen the change in CNR (DCNR) at each time point post probe
29
lysine through lysyl oxidase enzymes. We found that the lungs injection compared to the image before probe injection.
of BM mice had signicantly higher levels of collagen, hydrox- We found that the signal generated by Gd-CHyd was signif-
yproline and allysine compared to the lungs of na ¨ı ve mice icantly higher than that generated by Gd-Hyd at all three time
points post injection of contrast agent (Fig. 4a). Notably, the
percent difference in DCNR between Gd-Hyd and Gd-CHyd
increases with time, i.e. 34% aer 15 minutes, 53% aer 30
minutes and 77% aer 45 minutes indicating slower washout
from the brotic lung with Gd-CHyd compared to Gd-Hyd. Since
the same animal was imaged with both probes, we could also
compare the pairwise effect (Fig. 4b). We found that the lung
Fig. 4 Quantification of the change in lung-to-liver CNR of BM mice
Fig. 3 Ex vivo assessment of lung fibrosis. (a) Hematoxylin and eosin after injection of Gd-Hyd or Gd-CHyd 14 and 15 days after bleomycin
(
H&E)- and sirius red/fast green (S/F)-stained sections of lung tissues injury, respectively. Mice were imaged prior to and 15, 30 and 45 min
from mice that were challenged with bleomycin intratracheally 2 post probe injection. (a) Gd-CHyd lung enhancement is always greater
weeks prior (BM, n ¼ 8) or na ¨ı ve mice (n ¼ 4). (b) Lung injury as than Gd-Hyd enhancement at all three time points post contrast (n ¼
assessed by collagen proportional area (CPA), (c) lung hydroxyproline 8). (b) Pair-wise analysis of the DCNR in the lungs of BM animals
content and (d) allysine content. *p < 0.05, **p < 0.01, ***p < 0.001, t- observed arising from Gd-Hyd or Gd-CHyd injection. **p <0.05, ***p
test. Scale bar ¼ 1 mm.
<0.001, paired t-test.
This journal is © The Royal Society of Chemistry 2020
Chem. Sci., 2020, 11, 224–231 | 227

View Article Online
Chemical Science
Edge Article
signal from Gd-CHyd enhanced MRI was higher than that from post injection of Gd-CHyd (ESI, Fig S10†). In a previous report,
Gd-Hyd enhanced MRI for all eight animals. Gd-Hyd showed only a 3.7-fold enhancement in DCNR in the
There was very little signal enhancement in other tissues. lungs of BM animals compared to na ¨ı ve animals at a similar
18
Gd-CHyd undergoes rapid clearance from the blood through the time point (12 min post injection).
kidneys with a half-life of 5.6 ꢁ 1.3 min, which was not statis- We quantied the gadolinium content in the le lungs of
tically signicantly different than the 4.7 ꢁ 1.7 min blood half- mice 75 min post Gd-CHyd administration. We found that the
18
life measured for Gd-Hyd. Apart from the initial transient lung gadolinium content of BM mice was 10 times higher than
increase in signal immediately post injection due to blood that measured in na ¨ı ve animals, in line with the observed MR
distribution, there was no increase in signal in the liver signal changes (Fig. 5b)
consistent with exclusive renal elimination (ESI, Fig. S9†).
Because Gd-Hyd and Gd-CHyd have similar structures, blood
half-lives, in vivo clearance routes (renal), and are both stable in
Conclusions
blood plasma (ESI, Fig S6†), the lung imaging results collec- Gd-CHyd is an improved allysine-targeted small molecule MRI
tively indicate that Gd-CHyd has greater uptake and persists molecular probe suitable for imaging lung brogenesis. Gd-CHyd
longer in brosing lung tissue compared to Gd-Hyd. We also is equipped with a secondary alkyl hydrazine which resulted in an
compared the lung MR signal and DCNR using Gd-CHyd 11-fold increase in aldehyde binding rate constant and an order
enhanced MRI of na ¨ı ve (control) mice. We found that there of magnitude higher affinity for aldehydes compared to our
was a remarkable difference in the signal in the lungs of BM earlier prototype the acyl-hydrazine Gd-Hyd which employed an
animals compared to the lungs of na ¨ı ve animals 30 minutes acyl hydrazide for targeting. The relaxivities of Gd-Hyd and Gd-
aer injection of Gd-CHyd (Fig. 5a)
CHyd were similar and increased by 10% when conjugated to
In na ¨ı ve mice, there is essentially no lung enhancement with the aldehyde groups of proteins. Both Gd-CHyd and Gd-Hyd 60-
Gd-CHyd as expected. MR images unambiguously reect the fold higher signal in bleomycin-injured lungs, exhibit rapid blood
notable contrast enhancement in the lungs of BM animals clearance through the kidneys with very low signal enhancement
injected with Gd-CHyd compared to the same animal injected in the liver, heart, muscle, or normal lung tissue. The greater
with Gd-Hyd (Fig. 5c) and the same differences are visually aldehyde affinity and reactivity of Gd-CHyd translates in signi-
observable in the case of BM vs. na ¨ı ve animals imaged with Gd- cantly higher lung-to-liver DCNR values than Gd-Hyd at 15 min
CHyd. Notably, we observed a 12-fold enhancement in DCNR in post injection (30% higher) and at 45 min post injection (77%
the lungs of BM animals compared to na ¨ı ve animals 15 min higher). We previously reported MR probes and methods to
31
quantify the burden of brosis in the lung, inammation in the
32
33,34
lung, as well as vascular permeability in the context of IPF.
Our data indicate that improving the aldehyde reactivity and
affinity of hydrazine-bearing gadolinium contrast agents trans-
lates to enhanced and prolonged accumulation in brosing lung
tissue and improved specicity. We are currently expanding our
library of allysine-reactive probes to determine the relationship of
modifying the rate of reactivity on in vivo efficacy.
Experimental methods
General methods and instrumentation
Detailed descriptions, purication methods, characterizations,
assay procedures, and imaging sequences can be found in the
ESI.†
NMR spectra were recorded on a JEOL ECZ 500R 11.7 T NMR
1
system equipped with a 5 mm broadband probe ( H: 499.81
1
3
MHz, C: 125.68 MHz). HPLC-MS purity analysis was carried
out on an Agilent 1260 system coupled to Agilent 6130 LC-MS
system. Quantication of gadolinium was carried out using an
Agilent 8800-QQQ ICP-MS system. HPLC-ICP-MS was carried
out on an Agilent 1260 HPLC system coupled to an Agilent 8800-
QQQ ICP-MS system. Longitudinal (T1) relaxation measure-
Fig. 5 Selectivity of Gd-CHyd for actively fibrosing lung vs. healthy
lung. (a) Quantification of the MR signal in the lungs of bleomycin-
injured (BM, n ¼ 8) mice or na ¨ı ve mice (na ¨ı ve, n ¼ 4) 30 min after Gd-
CHyd administration revealing 63-fold higher signal in bleomycin-
injured lungs. (b) Quantification of the gadolinium content in the left
lungs of BM or na ¨ı ve animals 75 minutes post injection of Gd-CHyd ments were recorded using a Bruker mq60 Minispec at 1.41 T
showing 10-fold higher amounts of gadolinium in the lungs of BM
mice. (c) Coronal RARE MR images overlaid with false color image of
lung enhancement generated by subtraction of the pre-injection UTE
image from the post injection UTE image. Enhancement is highest in
the lungs of BM-treated mouse injected with Gd-CHyd. *p < 0.05,
ꢀ
and 37 C. UV-Vis spectra were recorded on SpectraMax® plus
instrument. High resolution mass spectra were acquired with
a high-resolution time-of-ight mass spectrometer (AccuTOF-
DART, JEOL). Mice were imaged on a 4.7 Tesla MRI scanner
(Bruker, Billerica MA) using a custom-built volume coil.
***p <0.001, unpaired t-test.
228 | Chem. Sci., 2020, 11, 224–231
This journal is © The Royal Society of Chemistry 2020

View Article Online
Edge Article
Chemical Science
1
3
Gd-Hyd and 2-(R)-2-(4,7,10-tris-tert-butylcarboxymethyl- 2H), 2.79 (m, 4H), 2.62 (m, 1H), 1.94 (bs, 2H). C NMR (126
,4,7,10-tetraazacyclododec-1-yl)-pentanedioic acid-1- MHz, D O) d 179.50, 176.17, 176.13, 176.06, z160.10, 139.11,
tertbutylester ( butyl-DOTAGA) were synthesized as previously 131.79, 131.46, 130.73, 70.09, 59.31, 57.84, 57.46, 54.05, 53.66,
1
2
t
1
8,35
described.
All other reagents were obtained from commer- 47.95, 47.82, 44.11, 32.89, 28.14. LC-MS (method 2, ESI†): t ¼
R
ꢂ
+
cial sources and used without additional purication.
3.55 min, HRMS: m/z: [M ꢂ H] calcd for [C31
46 7
H N O11] ,
692.3255 found 692.3254.
Synthesis of 4. To a solution of 3 (30 mg, 0.04 mmol) dis-
solved in water (1 mL) was added gadolinium chloride (20.9 mg,
Synthetic procedures
Synthesis of 1. Bis(2-chloroethyl)amine hydrochloride (1 g, 0.06 mmol) and the pH was adjusted to 6.8 with 1 M NaOH. The
.6 mmol) was combined with benzyl carbazate (1.0 g, 5.9 mixture was stirred overnight then puried by HPLC using
5
mmol) and potassium carbonate (2.4 g, 16.8 mmol) and sus- method 5. The product was isolated as a white powder aer
pended in ethanol (20 mL). The suspension brought to reux freeze-drying (20.8 mg, 54%). LC-MS (method 2, ESI†): t
¼
+
R
+
7
under argon for 18 h. Following removal of the resulting 8.01 min, m/z: HRMS [M] Calcd for [C31H44GdN O11] ,
precipitate by ltration, the reaction mixture was concentrated 848.2347; found 848.2356. Purity (>97%) was assessed by LC-
under reduced pressure and then puried via Combiash ICP-MS detecting gadolinium species as shown in Fig S1 (ESI†).
method 4 with C and D (ESI†) as solvents to give 1 as an off-
Synthesis of Gd-CHyd (5). Compound 4 (40 mg) was dis-
O): d 7.28 (m, solved in anhydrous methanol (3 mL) and activated palladium
H), 5.14 (s, 2H), 3.36 (t, J ¼ 5.0 Hz, 4H), 3.06 (t, J ¼ 5.0 Hz, 4H). on carbon (10%, 1 mg) was added to the solution. The
3
1
white solid (408 mg, 31% yield). H NMR (D
2
5
1
C NMR (126 MHz, D
O) d 160.07, 138.9, 131.75, 131.47 130.74, suspension was placed under vacuum then connected to
2
7
0.21, 54.52, 46.07. LC-MS (method 1, ESI†): t
R
¼ 2.68 min, a balloon containing hydrogen gas. The reaction mixture was
+
+
HRMS m/z: [M + H] calcd for [C12
H
18
N
3
O
2
] , 236.1399; found stirred for 18 h and progress was monitored by LC-MS. The
reaction mixture was passed through a Celite pad and the
236.1397.
Synthesis of 2. DOTAGA (100 mg, 0.14 mmol) was combined ltrate was subjected to rotary evaporation. The resulting solid
with N-hydroxysuccinimide (NHS, 18.1 mg, 0.15 mmol) and was puried by HPLC using method 5. Purity and reactivity were
0
N,N -dicyclohexylcarbodiimide (DCC, 29.4 mg, 0.14 mmol) and conrmed using LC-ICP-MS in the presence or absence of
purged with argon. Anhydrous dichloromethane (2 mL) was butyraldehyde (method 6, R of 5 : 14.1 min; R of butyraldehyde
t t
added and reaction mixture was stirred for 10 h. The insoluble condensation product: 15.1 min. The gadolinium content was
urea by-product was removed by ltration and the ltrate was assessed by ICP-MS to determine the yield of the reaction (40.0
concentrated under reduced pressure resulting in a foamy off- mmol, 87% isolated yield (30.1 mg 87%.). LC-MS (method 3,
+
white solid. Diisopropyl ethylamine (DIPEA, 27 mL, 0.16 ESI†):
t
R
¼
8.01 min, HRMS m/z: calcd for [M]
mmol) in dichloromethane (anhydrous, 1.5 mL) and 1 (26.9 mg, [C23 37GdN
H
7
O
9
]+, 713.1899; found 713.1919. Purity (>98%) was
0
(
.1 mmol) were added to a portion of this resulting solid assessed by LC-ICP-MS with gadolinium detection as shown in
82.9 mg, 0.1 mmol) and the mixture was stirred for 1 h under Fig. S1 (ESI†).
argon. Reaction completion was monitored by LC-MS (method
, ESI†). The solvent was removed under reduced pressure and
the resulting residue was puried via CombiFlash (method 4
with A and B as solvents, ESI†) to give 2 as a yellow semi-solid First-order rate constants for reaction of Gd-Hyd and Gd-CHyd
1
In vitro reaction rates
1
(91 mg, 95% yield). H NMR (500 MHz, CDCl ) d 7.43–7.27 (m, with 2-formylpyridine were determined by UV-vis spectroscopy
3
24
5
2
H), 6.45 (s, 1H), 5.13 (s, 2H), 3.76–3.55 (m, 8H), 3.42–3.31 (m, as previously described. The absorbance at 300 nm arising
H), 3.11 (m, 8H), 3.00–2.82 (m, 11H), 2.45 (m, 2H), 2.12–1.98 from the hydrazone condensation product was monitored in
1
3
(
m, 2H), 1.96–1.80 (m, 2H), 1.54–1.36 (m, 36H). C NMR (126 30–60 second intervals for up to 3 h. Reactions were conducted
MHz, CDCl ) d 171.72, 170.42, 170.03, 169.36, 166.33, 136.16, in phosphate-buffered saline (pH 7.40) under pseudo-rst order
28.55, 128.48, 128.19, 128.15, 82.00, 81.73, 81.63, 66.85, 63.15, conditions with 2-formylpyridine at 1000 mM and gadolinium-
6.30, 55.60, 55.28, 52.97, 52.36, 50.58, 44.69, 41.10, 29.41, containing probes at 166 mM. Pseudo-rst-order reaction
8.28, 28.21, 28.13, 28.11, 24.46. LC-MS (method 1, ESI†): t
kinetics were extracted from linear least-squares ts as previ-
3
1
5
2
6
9
R
¼
+
+
24
.18 min, HRMS: m/z: [M + H] calcd for [C H N O ] , ously described and reported as the mean of triplicate set of
18.5916; found 918.5916.
4
7
80 7 11
experiments ꢁ the standard deviation. Rates of reactions of
Synthesis of 3. Compound 2 (91 mg, 0.1 mmol) was dissolved allysine-targeted probes with 2-formyl pyridine were also
in dichloromethane (1 mL) along with triuoroacetic acid (0.7 determined using a HPLC with ICP-MS detection (ESI, Fig S5
mL) and stirred for 48 h. Reaction progress was followed by LC- and Table S1†).
MS. The solution was concentrated by rotary evaporation and
the product was precipitated by addition of diethyl ether (10
mL). The precipitate was isolated and washed repeatedly with
Determination of dissociation constants with LC-ICP-MS
diethyl ether then dried under vacuum. This material was used Dissociation constants were determined by incubating 2-for-
1
without further purication (56 mg, 81% yield). H NMR (500 mylpyridine (PBS, pH 7.40, 500 mM) with varying concentrations
ꢀ
MHz, D
2
O) d 7.42 (m, 5H), 5.14 (bs, 2H) 3.95–3.90 (m, 1H), 3.84, of Gd-Hyd or Gd-CHyd (10–300 mM) for 72 h at 37 C and using
(m, 4H), 3.75–3.53 (m, 7H), 3.36 (m, 9H), 3.07 (m, 5H), 2.88 (m, LC-ICP-MS to determine ratios of Gd-containing species. The
This journal is © The Royal Society of Chemistry 2020
Chem. Sci., 2020, 11, 224–231 | 229

View Article Online
Chemical Science
Edge Article
½
Aꢃ½Bꢃ
where [A]
regions of interest (ROI). The FLASH image immediately post
injection was also used to identify large vessels. ROIs of the lungs
dissociation constant (K
d
) is calculated as Kd ¼
½
ABꢃ
is the concentration of unreacted 2-formylpyridine, [B] is the excluding large vessels and airways were drawn on eight sagittal
concentration of unreacted probe (Gd-Hyd or Gd-CHyd) and slices. Additional ROIs were placed over the forearm muscle, the
[
AB] is the concentration of the resulting condensation product. liver, and an ROI was placed in the eld of view without any tissue
to measure the variation in background (air) signal. These ROIs
were then copied onto the UTE images to quantify signal intensity
SI). The analysis was repeated for the pre-probe and post-probe
UTE data sets. Image visualization and quantication was per-
formed using the Horos soware package.
To quantify signal enhancement in the lung, contrast to
noise ratio (CNR) was calculated as:
Binding to porcine aorta and BSA-Ald
(
Binding of Gd-CHyd and Gd-Hyd to protein and tissue aldehydes
18
was carried out as previously described, with minor modica-
tions as outlined in the ESI.† Solution of Gd-Hyd or Gd-CHyd
ranging from 0.05 to 1 mM were prepared in PBS (pH 7.40)
and 500 mL aliquots were added to 25 mg portions of puried
aorta. The aorta segments were then incubated at 37 C for 72
hours. The aorta pieces were then washed twice with PBS (pH
ꢀ
CNR ¼ (SIlung ꢂ SIliver)/SDair
7
.40), placed in concentrated nitric acid overnight for digestion.
Where SI ¼ signal intensity, SD ¼ standard deviation. An
average of all image slices was calculated for the pre-injection
images (CNRpre) and for the post-injection images (CNRpost).
The lung enhancement for each mouse is expressed as DCNR,
the difference between the pre-injection CNR and the post-
injection CNR
The gadolinium content was then assessed by ICP-MS.
Animal model
All experiments and procedures were performed in accordance
with the National Institutes of Health's “Guide for the Care and
Use of Laboratory Animals” and were approved by the Massa-
chusetts General Hospital Institutional Animal Care and Use
Committee. C57Bl/6 adult male mice at 8 weeks of age (Jackson
Laboratories, Barr Harbor, ME) received a single intratracheal
dose of bleomycin, 1 units per kg body weight (50 mL total volume)
DCNR ¼ CNRpost ꢂ CNRpre
MR lung image representation
36
A difference image of pre and post contrast in the lungs was
generated in Horos soware by subtracting the UTE pre probe
injection image from the UTE post probe injection image. A
mask was applied to the lung based on the anatomical scan. The
resulting difference image was then false colored, and super-
imposed on the corresponding anatomical (2D-RARE) image.
(
Fresenius Kabi, Lake Zurich, Il) as previously described.
MR imaging with Gd-Hyd was carried out 14 days aer
bleomycin injury (N ¼ 8) and the same mice were imaged again
4 hours later with Gd-CHyd. An additional cohort of bleomy-
2
cin-na ¨ı ve mice (N ¼ 4) were imaged with Gd-CHyd. All animals
ꢂ1
were dosed via tail vein injection at 100 nmol g body weight
from 30 mM solutions of gadolinium probes as determined by
ICP-MS. All animals were sacriced aer the imaging for
biochemical analysis and histological evaluations
Ex vivo tissue analyses
Following imaging, animals were euthanized and the le lung was
collected and homogenized, and then analyzed for allysine,
hydroxyproline and gadolinium content. The right lung was har-
vested, xed in neutral 10% buffered formalin, embedded in
paraffin, and sectioned into 5 mm-thick slices for hematoxylin and
eosin (H&E) and sirius sed staining. Sirius red stained slides were
scanned and images were digitally acquired and were analyzed by
thresholding to determine the collagen proportional area (CPA),
MR images acquisition
For all animals, images were acquired prior to, during, and post
injection of probe. Animals were anesthetized with 1–2% iso-
urane and air/oxygen mixture to maintain constant respiration
rate at 60 breaths/minute monitored by a small animal physio-
logical monitoring system (SA Instruments Inc., Stony Brook
NY). Animals were placed in the scanner the following images
were acquired: axial and coronal 2-dimensional (2D) Rapid
Acquisition with Relaxation Enhancement (RARE) images, T1w
%
area stained collagen positive, using ImageJ (National Institute
18
of Health, Bethesda MD) as previously described.
3
D Fast Low Angle Shot (FLASH) images, and T1w 3D Ultra Short Conflicts of interest
Time to Echo (UTE) images. During and immediately following
ꢂ1
There are no conicts to declare.
0
.1 mmol kg probe injection, a series of 10 T1w FLASH images
were acquired to monitor probe injection and clearance. UTE
images were then acquired again at 15, 30 and 45 min post
injection. Sequence parameter details are outlined in the ESI.†
Acknowledgements
Research support from the National Heart Lung and Blood
Institute (T32HL007208 to E. A. A.; R01HL116315,
R01HL131907 to P. C.), the National Institute for Diabetes and
MR lung image analysis
We rst visualized the vasculature of the lungs on the anatomical Digestive, and Kidney Diseases (R01DK121789, U01DK104302
coronal images (T1-weighted RARE sequence) to identify lung to P. C.) and the NIH Office of the Director (S10OD010650,
parenchyma and exclude vessels and airways and to dene the S10OD025234 to P. C.) is gratefully acknowledged.
230 | Chem. Sci., 2020, 11, 224–231
This journal is © The Royal Society of Chemistry 2020

View Article Online
Edge Article
Chemical Science
1
9 P. A. Waghorn, C. M. Jones, N. J. Rotile, S. K. Koerner,
D. S. Ferreira, H. H. Chen, C. K. Probst, A. M. Tager and
P. Caravan, Angew. Chem., Int. Ed., 2017, 56, 9825–9828.
References
1
2
3
M. L. Urban, L. Manenti and A. Vaglio, N. Engl. J. Med., 2015,
73, 95–96.
C. Kuhn, J. Boldt, T. E. King, E. Crouch, T. Vartio and
3
20 D. K. K ¨o lmel and E. T. Kool, Chem. Rev., 2017, 117, 10358–
10376.
J. A. McDonald, Am. Rev. Respir. Dis., 1989, 140, 1693–1703. 21 T. Dang, M. Suchy, Y. J. Truong, W. Oakden, W. W. Lam,
D. B. Esposito, S. Lanes, M. Donneyong, C. N. Holick,
J. A. Lasky, D. Lederer, S. D. Nathan, S. O'Quinn, J. Parker
C. Lazurko, G. Facey, G. J. Stanisz and A. J. Shuhendler,
Chem.–Eur. J., 2018, 24, 9148–9156.
and T. N. Tran, Am. J. Respir. Crit. Care Med., 2015, 192, 22 J. Wahsner, P. D ´e sog `e re, E. Abston, K. A. Graham-O'Regan,
1
200–1207.
J. Wang, N. J. Rotile, M. D. Schirmer, D. D. Santos Ferreira,
J. Sui, B. C. Fuchs, M. Lanuti and P. Caravan, J. Am. Chem.
Soc., 2019, 141, 5593–5596.
4
G. Raghu, H. R. Collard, J. J. Egan, F. J. Martinez, J. Behr,
K. K. Brown, T. V. Colby, J. F. Cordier, K. R. Flaherty,
J. A. Lasky, D. A. Lynch, J. H. Ryu, J. J. Swigris, A. U. Wells, 23 A. Kirby, M. Such ´y , A. Brouwer and A. Shuhendler, Chem.
J. Ancochea, D. Bouros, C. Carvalho, U. Costabel, M. Ebina,
Commun., 2019, 55, 5371–5374.
D. M. Hansell, T. Johkoh, D. S. Kim, T. E. King, Y. Kondoh, 24 E. Kool, P. Crisalli and K. Chan, Org. Lett., 2014, 16, 1454–
J. Myers, N. L. M u¨ ller, A. G. Nicholson, L. Richeldi,
1457.
M. Selman, R. F. Dudden, B. S. Griss, S. L. Protzko and 25 E. Kool, D. Park and P. Crisalli, J. Am. Chem. Soc., 2013, 135,
H. J. Sch u¨ nemann, Am. J. Respir. Crit. Care Med., 2011, 183,
88–824.
J. Hutchinson, A. Fogarty, R. Hubbard and T. McKeever, Eur.
Respir. J., 2015, 46, 795–806.
D. A. Lynch, N. Sverzellati, W. D. Travis, K. K. Brown,
T. V. Colby, J. R. Galvin, J. G. Goldin, D. M. Hansell,
17663–17666.
7
26 A. Dirksen and P. E. Dawson, Bioconjugate Chem., 2008, 19,
2543–2548.
27 R. L. Levine, D. Garland, C. N. Oliver, A. Amici, I. Climent,
A. G. Lenz, B. W. Ahn, S. Shaltiel and E. R. Stadtman,
Methods Enzymol., 1990, 186, 464–478.
5
6
Y. Inoue, T. Johkoh, A. G. Nicholson, S. L. Knight, S. Raoof, 28 P. A. Waghorn, B. L. Oliveira, C. M. Jones, A. M. Tager and
L. Richeldi, C. J. Ryerson, J. H. Ryu and A. U. Wells, Lancet
Respir. Med., 2018, 6, 138–153.
P. Caravan, J. Chromatogr. B: Anal. Technol. Biomed. Life
Sci., 2017, 1064, 7–13.
7
A. Levin, M. Tonelli, J. Bonventre, J. Coresh, J. A. Donner, 29 S. R. Pinnell and G. R. Martin, Proc. Natl. Acad. Sci. U. S. A.,
A. B. Fogo, C. S. Fox, R. T. Gansevoort, H. J. L. Heerspink,
1968, 61, 708–716.
M. Jardine, B. Kasiske, A. K ¨o ttgen, M. Kretzler, A. S. Levey, 30 W. M. S. Russell, R. L. Burch and C. W. Hume, The principles
V. A. Luyckx, R. Mehta, O. Moe, G. Obrador, N. Pannu,
C. R. Parikh, V. Perkovic, C. Pollock, P. Stenvinkel,
K. R. Tuttle, D. C. Wheeler and K. U. Eckardt, Lancet, 2017,
of humane experimental technique, Altweb (Online service),
Johns Hopkins Bloomberg School of Public Health., Johns
Hopkins Center for Alternatives to Animal Testing and
Universities Federation for Animal Welfare, 2005.
390, 1888–1917.
8
9
B. Ley, H. R. Collard and T. E. King, Am. J. Respir. Crit. Care 31 P. Caravan, Y. Yang, R. Zachariah, A. Schmitt, M. Mino-
Med., 2011, 183, 431–440.
V. Cottin and L. Richeldi, Eur. Respir. Rev., 2014, 23, 106–110.
0 S. L. F. Walsh, Curr. Opin. Pulm. Med., 2018, 24, 445–452.
Kenudson, H. H. Chen, D. E. Sosnovik, G. Dai, B. C. Fuchs
and M. Lanuti, Am. J. Respir. Cell Mol. Biol., 2013, 49,
1120–1126.
1
1
1 L. Richeldi, H. R. Collard and M. G. Jones, Lancet, 2017, 389, 32 A. Rodr ´ı guez-Rodr ´ı guez, S. Shuvaev, N. Rotile, C. M. Jones,
1
941–1952.
C. K. Probst, D. Dos Santos Ferreira, K. Graham-O Regan,
E. Boros, R. S. Knipe, J. W. Griffith, A. M. Tager,
A. Bogdanov and P. Caravan, ACS Sens., 2019, 4(9), 2412–
2419.
1
1
1
1
2 S. B. Montesi, P. D ´e sog `e re, B. C. Fuchs and P. Caravan, J.
Clin. Invest., 2019, 129, 24–33.
3 P. D ´e sog `e re, S. B. Montesi and P. Caravan, Chem.–Eur. J.,
2
019, 25, 1128–1141.
4 R. Weissleder and U. Mahmood, Radiology, 2001, 219, 316–
33.
33 S. B. Montesi, R. Rao, L. L. Liang, H. E. Goulart, A. Sharma,
S. R. Digumarthy, B. S. Shea, R. T. Seethamraju, P. Caravan
and A. M. Tager, Eur. Respir. J., 2018, 51, 1800171.
3
5 J. Wahsner, E. M. Gale, A. Rodr ´ı guez-Rodr ´ı guez and 34 B. S. Shea, C. K. Probst, P. L. Brazee, N. J. Rotile, F. Blasi,
P. Caravan, Chem. Rev., 2019, 119, 957–1057.
6 J. M. M ¨a ki, Histol. Histopathol., 2009, 24, 651–660.
7 M. L. Tanzer, Science, 1973, 180, 561–566.
P. H. Weinreb, K. E. Black, D. E. Sosnovik, E. M. Van Cott,
S. M. Violette, P. Caravan and A. M. Tager, JCI Insight,
2017, 2, e86608.
1
1
1
8 H. H. Chen, P. A. Waghorn, L. Wei, L. F. Tapias, 35 S. Levy, V. Jacques, K. Zhou, S. Kalogeropoulos,
D. T. Sch u€ hle, N. J. Rotile, C. M. Jones, R. J. Looby,
G. Zhao, J. M. Elliott, C. K. Probst, M. Mino-Kenudson,
G. Y. Lauwers, A. M. Tager, K. K. Tanabe, M. Lanuti,
B. C. Fuchs and P. Caravan, JCI Insight, 2017, 2, e91506.
K. Schumacher, J. Amedio, J. Scherer, S. Witowski,
R. Lombardy and K. Koppetsch, Org. Process Res. Dev.,
2009, 13, 535–542.
36 P. Desogere, L. Tapias, T. Rietz, N. Rotile, F. Blasi, H. Day,
J. Elliott, B. Fuchs, M. Lanuti and P. Caravan, J. Nucl. Med.,
2017, 58, 1991–1996.
This journal is © The Royal Society of Chemistry 2020
Chem. Sci., 2020, 11, 224–231 | 231