Effects of skeleton structure on necrosis targeting and clearance properties of radioiodinated dianthrones

Article abstract of DOI:10.3109/1061186X.2015.1113541

Necrosis avid agents (NAAs) can be used for diagnose of necrosis-related diseases, evaluation of therapeutic responses and targeted therapeutics of tumor. In order to probe into the effects of molecular skeleton structure on necrosis targeting and clearance properties of radioiodinated dianthrones, four dianthrone compounds with the same substituents but different skeletal structures, namely Hypericin (Hyp), protohypericin (ProHyp), emodin dianthrone mesomer (ED-1) and emodin dianthrone raceme (ED-2) were synthesized and radioiodinated. Then radioiodinated dianthrones were evaluated in vitro for their necrosis avidity in A549 lung cancer cells untreated and treated with H₂O₂. Their biodistribution and pharmacokinetic properties were determined in rat models of induced necrosis. In vitro cell assay revealed that destruction of rigid skeleton structure dramatically reduced their necrosis targeting ability. Animal studies demonstrated that destruction of rigid skeleton structure dramatically reduced the necrotic tissue uptake and speed up the clearance from the most normal tissues for the studied compounds. Among these ¹³¹I-dianthrones, ¹³¹I-Hyp exhibited the highest uptake and persistent retention in necrotic tissues. Hepatic infarction could be clearly visualized by SPECT/CT using ¹³¹I-Hyp as an imaging probe. The results suggest that the skeleton structure of Hyp is the lead structure for further structure optimization of this class of NAAs.

Full text of DOI:10.3109/1061186X.2015.1113541

Journal of Drug Targeting
ISSN: 1061-186X (Print) 1029-2330 (Online) Journal homepage: http://www.tandfonline.com/loi/idrt20
Effects of skeleton structure on necrosis targeting
and clearance properties of radioiodinated
dianthrones
Dongjian Zhang, Cuihua Jiang, Shengwei Yang, Meng Gao, Dejian Huang,
Xiaoning Wang, Haibo Shao, Yuanbo Feng, Ziping Sun, Yicheng Ni, Jian Zhang
&
Zhiqi Yin
To cite this article: Dongjian Zhang, Cuihua Jiang, Shengwei Yang, Meng Gao, Dejian Huang,
Xiaoning Wang, Haibo Shao, Yuanbo Feng, Ziping Sun, Yicheng Ni, Jian Zhang & Zhiqi Yin (2015):
Effects of skeleton structure on necrosis targeting and clearance properties of radioiodinated
dianthrones, Journal of Drug Targeting, DOI: 10.3109/1061186X.2015.1113541
To link to this article: http://dx.doi.org/10.3109/1061186X.2015.1113541
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ISSN: 1061-186X (print), 1029-2330 (electronic)
J Drug Target, Early Online: 1–12
2015 Taylor & Francis. DOI: 10.3109/1061186X.2015.1113541
!
ORIGINAL ARTICLE
Effects of skeleton structure on necrosis targeting and clearance
properties of radioiodinated dianthrones
1
,2
1
1
1
1
1
3
Dongjian Zhang , Cuihua Jiang , Shengwei Yang , Meng Gao , Dejian Huang , Xiaoning Wang , Haibo Shao ,
1,5
4
Yuanbo Feng , Ziping Sun , Yicheng Ni , Jian Zhang , and Zhiqi Yin
1,4,5
1
2
1
Laboratory of Translational Medicine, Jiangsu Province Academy of Traditional Chinese Medicine, Nanjing, Jiangsu Province, P.R. China,
2
Department of Natural Medicinal Chemistry & State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, Jiangsu
3
Province, P.R. China, Department of Radiology, the First Affiliated Hospital of China Medical University, Shenyang, Liaoning Province, P.R. China,
4 5
Radiation Medical Institute, Shandong Academy of Medical Sciences, Jinan, Shandong Province, P.R. China, and Theragnostic Laboratory,
Campus Gasthuisberg, KU Leuven, Leuven, Belgium
Abstract
Keywords
Necrosis avid agents (NAAs) can be used for diagnose of necrosis-related diseases, evaluation of Autoradiography, biodistribution, necrosis
therapeutic responses and targeted therapeutics of tumor. In order to probe into the effects of
molecular skeleton structure on necrosis targeting and clearance properties of radioiodinated
dianthrones, four dianthrone compounds with the same substituents but different skeletal
structures, namely Hypericin (Hyp), protohypericin (ProHyp), emodin dianthrone mesomer (ED-
targeting, pharmacokinetics,
radioiodinated dianthrones, SPECT/CT
imaging
1
) and emodin dianthrone raceme (ED-2) were synthesized and radioiodinated. Then
radioiodinated dianthrones were evaluated in vitro for their necrosis avidity in A549 lung
cancer cells untreated and treated with H . Their biodistribution and pharmacokinetic
History
Received 18 July 2015
Revised 12 October 2015
Accepted 25 October 2015
Published online 20 November 2015
2 2
O
properties were determined in rat models of induced necrosis. In vitro cell assay revealed that
destruction of rigid skeleton structure dramatically reduced their necrosis targeting ability.
Animal studies demonstrated that destruction of rigid skeleton structure dramatically reduced
the necrotic tissue uptake and speed up the clearance from the most normal tissues for the
studied compounds. Among these I-dianthrones, I-Hyp exhibited the highest uptake and
persistent retention in necrotic tissues. Hepatic infarction could be clearly visualized by SPECT/
1
31
131
1
31
CT using I-Hyp as an imaging probe. The results suggest that the skeleton structure of Hyp is
the lead structure for further structure optimization of this class of NAAs.
Introduction
(NAAs) are a class of compounds that selectively concentrate
in the necrotic tissue after systemic administration, which can
be used for in vivo imaging of necrotic tissue [3,7] and tumor
necrosis therapy (TNT) [8–12].
Necrosis represents a principal feature of numerous pathol-
ogies or disorders such as ischemic cardiac disease, athero-
sclerosis, stroke, sepsis, pancreatitis and malignant tumors
A common strategy for effective molecular imaging and
targeted therapeutics is to employ a suitably designed
molecular probe that is composed of a targeting group
conjugated to a reporter group or therapeutic group. A
number of molecular probes with high avidity for necrotic
tissue have been explored for diagnosis and treatment of
[
1,2]. It is often classified as an uncontrolled, accidental
process of cell or tissue death that results from chemical or
physical noxious insults and ischemic or inflammatory injury
[3]. Differing from apoptosis, necrosis usually involves
groups of cells and is frequently followed by granulation
development and fibrosis replacement with distortion of the
local tissue’s architecture [4–6]. Thus the presence of necrotic
tissue may provide an anchorage for the diagnosis, prognosis
or treatment of aforementioned diseases. Necrosis avid agents
9
9m
necrosis-related pathologies such as
Tc-pyrophosphate
In-antimyosin monoclonal
I-hypericin monocarboxylic acid
9
9m
111
[
13],
antibody [17,18],
Tc-glucarate [14–16],
1
23
1
23
99m
(
1
I-HMA) [19],
23 131
Tc(CO) -bis-DTPA-pamoate [20],
3
123 131
I/ I-hypericin( I/ I-Hyp) [21,22], Hoechst-IR [1]
Address for correspondence: Dr. Jian Zhang, Laboratory of
Translational Medicine, Jiangsu Province Academy of Traditional
Chinese Medicine, No. 100, Shizi Street, Hongshan Road, Nanjing
64
and Cu-bis-DOTA-Hyp [23], etc. Among them, the most
1
23
studied is Hyp.
tissue and may serve as a potential necrosis-avid diagnostic
I-Hyp allowed visualization of necrotic
2
10028, Jiangsu Province, P.R. China. Tel: +86 25 52362107. E-mail:
131
zjwonderful@hotmail.com; Dr. Zhiqi Yin, Department of Natural
Medicinal Chemistry & State Key Laboratory of Natural Medicines,
China Pharmaceutical University, No. 24, Tongjiaxiang, Gulou District,
Nanjing 210009, Jiangsu Province, P.R. China. Tel: +86 25 86185371.
E-mail: chyzq2005@126.com
agent for assessment of tissue viability [24–27]. I-Hyp has
been explored for diagnostic and therapeutic utilities in
experimental malignancies [8,10,11]. The inherent fluores-
cence properties of Hyp have also been exploited for

2
D. Zhang et al.
J Drug Target, Early Online: 1–12
concentration was 370 MBq/mL and the radionuclidic purity
was greater than 99%. Thin layer chromatography (TLC) was
performed on Merck aluminum-precoated silica gel 60 F254
plates or Silica gel 60 RP-18 F254 plates with spots visualized
by UV light (254 nm or 365 nm) and/or by development
with magnesium acetate, or aluminum chloride. Column
chromatography was carried out with Merck silica gel 60
1
normal phase (45–75 mm) or Sephadex LH20. H NMR
was recorded on a Bruker 300 or 500 MHz spectrometer and
1
3
C NMR spectra were recorded at 75 MHz. Chemical shifts
are reported as parts per million (ꢁ) relative to tetramethylsi-
lane (TMS, 0.00 ppm), which was used as an internal
standard. MS spectra were measured by HP1100 mass
spectrometer utilizing electrospray ionization (ESI) (Agilent,
Santa Clara, CA).
Figure 1. The chemical structures of sennidin A (SA) and sennoside B
SB), glc ¼ ꢂ-D-glucopyranosyl.
(
microscopic necrosis imaging [28,29]. However, Hyp is a
lipophilic compound that sparingly dissolves and forms
aggregates in aqueous solution [30]. It makes systemic
administration problematic and, in turn, reduces its biodis-
tribution to necrotic tissue and shows unwanted biodistribu-
tion in normal tissue [31,32].
Synthesis of emodin anthrone
Emodin anthrone was synthesized following a literature
procedure [34] with slight modification. A solution of tin
(II) chloride hydrate (29.1 g, 129 mmol) in conc. hydrochloric
acid (149 mL) was added to a suspension of emodin (3.49 g,
In recent years, we have committed to discover new
necrosis avid probes for noninvasive imaging diagnosis of
necrosis-related pathologies and dual-targeting theragnostic
strategy of tumors. In addition to Hyp, we found that median
dianthrone compounds sennidin A (SA) [12] and sennoside B
1
2.9 mmol) in acetic acid (270 mL) to give the red-colored
ꢀ
solution. After the solution was stirred at 120 C for 24 h, it
was poured into 1000 mL of ice-water and the precipitate was
ꢀ
collected by filtration and dried in vacuo at 60 C to give
1 (3.2 g, 96.9%). The resulting emodin anthrone was directly
used in the next reaction without further purification.
(SB) [33] (Figure 1) also had necrosis targetability. Compared
with Hyp, SA and SB both have better water solubility and
giant ꢀ-conjugation damaged parent nucleus structure. The
31
1
131
fewer uptakes of
1
I-SA and
I-Hyp suggested that SA and SB may be prone to
I-SB in the lung compared
Synthesis of ProHyp
31
with
aggregation to a lesser degree than Hyp.
ProHyp (1.25 g, 63.2%) was synthesized following a literature
protocol [35] and purified by preparative reversed phase-high
performance liquid chromatography (RP-HPLC) (see
Although dianthrone compounds, Hyp, HMA, SA and SB
all reveal necrosis targeting property, the necrosis targeting
ability of them is different. This is mainly due to the
difference of their parent nucleus structures and their
peripheral substituents. To seek for new necrosis targeting
compounds and to provide the basis for further structural
optimization, we decided to first explore the effects of
skeleton structure on necrosis targeting and clearance proper-
ties of radioiodinated dianthrones. Therefore, we synthesized
four dianthrone compounds with the same substituents but
different skeletal structures, namely Hyp, protohypericin
1
Supplementary data). Data for ProHyp: H NMR (300 MHz,
DMSO-d ) ꢁ 14.38 (s, 2H), 12.87 (s, 2H), 7.20 (s, 2H), 6.74
6
(s, 2H), 6.33 (s, 2H), 2.05 (s, 6H), assignments referencing to
the literature [36,37]; C NMR (75 MHz, DMSO-d ) ꢁ (15
pairs of signals are overlapping) 184.4, 173.8, 168.3, 160.0,
1
3
6
142.5, 136.2, 129.7, 127.7, 125.8, 119.6, 118.7, 116.1, 113.3,
104.2, 21.3, assignments referencing to the literature [38]. MS
ꢁ
(ESI) m/z: 505.2 (M–H) .
Synthesis of Hyp
(
ProHyp), emodin dianthrone mesomer (ED-1) and emodin
dianthrone raceme (ED-2) (Figure 2). After radioiodination,
necrosis targeting and pharmacokinetic properties of the four
Hyp (1.19 g, 60.4%) was synthesized following a literature
1
protocol [35]. Data for Hyp: H NMR (300 MHz, DMSO-d
)
ꢁ 14.65 (s, 2H), 14.01 (s, 2H), 7.28 (s, 2H), 6.41 (s, 2H), 2.63
6
1
31
I-dianthrones were examined in rat models with reperfused
partial liver infarction (RPLI) and absolute ethanol induced
muscular necrosis. Thereafter, we further evaluated the ability
1
3
(s, 6H); C NMR (75 MHz, DMSO-d ) ꢁ (15 pairs of signals
6
are overlapping) 183.1, 174.3, 168.0, 161.1, 143.2, 126.7,
125.9, 121.1, 120.5, 119.2, 118.5, 108.3, 105.3, 101.8, 23.5,
assignments referencing to the literature [38]. MS (ESI) m/z:
1
31
of I-Hyp to image necrosis in vivo in rats with RPLI.
ꢁ
Materials and methods
503.2 (M–H) .
Chemistry
Synthesis of ED-1 and ED-2
All reagents and solvents used, unless stated otherwise, were
purchased from Sinopharm Chemical Reagent Co., Ltd or
Nanjing Chemical Reagent Co., Ltd (Nanjing, China) and
were used without further purification. The starting material
emodin was purchased (Chengdu Huaxia Chemical Reagent
A solution of iron (III) chloride hydrate (1.95 g, 7.02 mmol) in
ethanol (180 mL) was added drop wise to a solution of emodin
anthrone (1.58 g, 5.85 mmol) in ethanol (375 mL) over
30 min. After heating for 4 h under reflux, the solution was
poured into an aqueous solution of hydrochloric acid (5%,
750 mL). It was cooled to room temperature and the product
1
31
Co., Ltd, Chengdu, China). Sodium iodide (Na I) was
supplied by HTA Co., Ltd (Beijing, China). The radioactivity

DOI: 10.3109/1061186X.2015.1113541
Effects of skeleton structure on necrosis
3
Figure 2. The chemical structures of the target compounds.
was extracted with ether. After drying over sodium sulfate and
removal of the solvent, the residue was purified by column
chromatography (chloroform:methanol ¼ 50:1) to give
emodin dianthrone 1.73 g, 58%) as a diastereomeric mixture.
The diastereomeric mixture was further separated by pre-
serum proteins. The resulting mixture was centrifuged at
12 000 rpm for 10 min to collect the supernatant. The
supernatant was filtered through 0.22 -mm pore-size filters
(Membrana, Germany) and then injected into RP-HPLC for
analysis using the method described above.
parative RP-HPLC (see Supplementary data) to yield ED-1
1
and ED-2. Data for ED-1; H NMR (500 MHz, DMSO-d ) ꢁ
Induction of cell necrosis
6
1
1.86 (s, 2H), 11.77 (s, 2H), 10.71 (s, 2H), 6.66 (s, 2H), 6.20
s, 2H), 6.17 (d, J ¼ 1.5 Hz, 2H), 6.02 (s, 2H), 4.45 (s, 2H),
.22 (s, 6H), assignments referencing to the literature [36];
An in vitro model of necrosis was set up whereby A549 cells
were treated with the H O and analysed for FITC-annexin V
(
2
2
2
and propidium iodide (PI) binding by flow cytometry. The
human lung cancer A549 cell line was obtained from
American Type Culture Collection (Manassas, VA). The
A549 cells were seeded into a six-well cell culture plate at a
1
3
C
NMR (75 MHz,DMSO-d6)
ꢁ
167.14, 138.32,
1
1
1
37.16 137.01, 136.94, 136.79, 131.64, 130.97, 129.36,
29.22, 128.82, 128.39, 127.98, 127.69, 127.12, 123.32,
22.61, 115.81, 114.25, 110.16, 108.56, 102.68, 102.27,
5
ꢀ
density of 2.5 ꢂ 10 cells/well and incubated at 37 C for 24 h.
ꢁ
5
2.56, 50.07. MS (ESI) m/z: 509.1 (M–H) . Data for ED-2:
H NMR (500 MHz, DMSO-d ) ꢁ 11.92 (s, 2H), 11.68 (s,
After being washed once with phosphate-buffered saline
ꢀ
1
6
(PBS), cells were incubated at 37 C for 1 h in the presence of
increasing concentrations of H O (0, 20, 40, 60, 80, 100,
2
H), 10.79 (s, 2H), 6.61 (s, 2H), 6.22 (s, 2H), 6.22 (s, 2H),
.00 (s, 2H), 4.43 (s, 2H), 2.17 (s, 6H), assignments
2
2
6
referencing to the literature [36];
120 mM) in 1 mL of RPMI-1640 medium. After incubation,
1
3
C NMR (75 MHz,
DMSO-d ) ꢁ 167.14, 138.32, 137.16, 137.01, 136.94,
the reaction medium was aspirated and the cells were rinsed
once with 1 mL of cold pH 7.4 PBS to remove the remaining
H O . Necrosis and apoptosis were both assessed by using the
6
1
1
1
5
36.79, 131.64, 130.97, 129.36, 129.22, 128.82, 128.39,
27.98, 127.69, 127.12, 123.32, 122.61, 115.81, 114.25,
10.16, 108.56, 102.68, 102.27, 52.56, 50.07. MS (ESI) m/z:
2
2
annexin V/PI kit according to the manufacturer’s instructions
5
(
Roche, France). Briefly, cells (5 ꢂ 10 ) were washed twice
ꢁ
09.0 (M–H) .
with PBS and suspended in annexin V and PI staining solution
for 15 min at room temperature. Cells were analysed imme-
diately on a flow cytometer (FACS Calibur; BD Biosciences,
Franklin Lakes, NJ).
Radiolabeling and in vitro stability
1
31
The compounds were radiolabelled with
I via a direct
oxidation reaction using iodogen as an oxidation agent.
Briefly, iodogen (1, 3, 4, 6-tetrachloro-3a, 6a-diphenylgly-
couril; Pierce Biotechnology, ZI Camp Jouven, France) is first
dissolved in dichloromethane and is coated on the wall of the
Eppendorf tube according to the required dose (40–60 mg). To
an iodogen-coated tube were added 400 mL of 0.5 mg/mL of
In vitro necrotic binding assay
1
31
131
131
131
The binding of I-Hyp, I-ProHyp, I-ED-1 and I-ED-
2 for the cell were determined in A549 cells. The cell binding
1
31
assay was replicated in triplicate for each I-compound.
Control group cells and 100 mM H O treated cells were
respectively transferred into 4 mL tubes and coincubated at
2
2
1
31
each compound in DMSO and 100 mL of fresh Na I solution
37 MBq). The method of controlling variables (temperature,
ꢀ
131
131
(
37 C for 15 min with 0.037 MBq of
1
I-Hyp,
I-ED-1 or I-ED-2 in 1 mL of RPMI-1640 medium. After
I-ProHyp,
31
131
reaction time and the amount of iodogen) was used to
optimize the radiolabeling yields. Radiochemical yield of
ꢀ
incubation at 37 C for 15 min, the mixture was transferred
into 1.5 mL tubes and centrifuged at 12 000 rpm for 10 min.
The tubes were frozen in liquid nitrogen, the bottom tips
containing the cell pellet were cut off, and the cell pellets and
supernatants were collected and counted for radioactivity in a
1
31
each
lysis methods are seen in the Supplementary data).
I-dianthrone was determined using RP-HPLC (ana-
1
In vitro stabilities of I-compounds were determined by
31
ꢀ
incubation in rat serum at 37 C for 72 h and monitored for
degradation by RP-HPLC. Briefly, 100 mL of each reaction
2
WIZARD 2470 automated gamma counter (PerkinElmer,
solution was added into 900 mL of rat serum and incubated at
37 C for 72 h. After the incubation, 1 mL of a mixture of
ethanol and acetonitrile (1:1, v/v) was added to precipitate the
Maltham, MA). The data were expressed as activity ratios of
the cell pellets to the cell pellets and supernatants ([cpm of
cell pellets/cpm of cell pellets and supernatants] ꢂ 100%).
ꢀ

4
D. Zhang et al.
J Drug Target, Early Online: 1–12
31 131
1
Animal models of necrosis
animals. After IV administration of
31
I-Hyp,
I-ED-1 or I-ED-2, the urine and feces were collected for
I-ProHyp,
1
131
Adult male SD rats weighing 250–300 g were provided by
Experimental Animal Center, Jiangsu Academy of Traditional
Chinese Medicine (Nanjing, China). All animal studies were
approved by the Institutional Animal Care and Use
Committee.
gamma counting during the whole experiment and then the
rats were sacrificed at 24 h and 72 h post injection (p.i.) (n ¼ 4
per time point). Organs of interest were isolated, weighed and
2
counted in a WIZARD 2470 automated gamma counter
(PerkinElmer, Waltham, MA). The stomach and intestines
Reperfused partial liver infarction: Adult male SD rats were
anaesthetized with intraperitoneal injection of pentobarbital
were emptied of food contents and the bladder was emptied of
urine prior to radioactivity measurements. The necrotic liver
and muscle tissues that were labeled by Evans blue were
carefully sampled for gamma counting. Corrections were
made for background radiation and physical decay during
counting. Radioactive uptake in the organs was expressed as
percentage of the injected dose per gram of tissues (%ID/g)
while radioactive content in excretion was expressed as
percentage of the injected dose (%ID).
(Nembutal Veterinary; Sanofi Sante Animale, Paris, France) at
a dose of 40mg/kg. Under laparotomy, reperfused hepatic
infarction was induced by temporarily clamping the hilum of
the right liver lobes for 3 h. After reperfusion by declamping
hepatic inflow and by giving massage to the liver lobe, the
abdominal cavity was closed with two-layer sutures and the
animals were allowed to recover overnight. Moreover, sham
operated rats, which were subjected to the same surgery except
for not clamping the hilum of the right liver lobes, were also
established to be used for control in SPECT/CT imaging study.
Ethanol induced muscle necrosis: Each rat of reperfused
hepatic infarction was then intramuscularly injected with
After gamma counting, representative tissues of necrotic
liver, normal liver and necrotic muscle were cryosectioned
into 30 mm thick slices with a cryotome (Shandon Cryotome
FSE; Thermo Fisher Scientific Co., Waltham, MA).
Autoradiography were performed by exposing the sections
to a high performance phosphor screen (PerkinElmer,
Waltham, MA) for 4–48 h. Digital autoradiographic images
were obtained by reading out the phosphor screen using a
Cyclone Plus Phosphor Imager (PerkinElmer, Waltham, MA).
0
.2 mL absolute alcohol in the left hind limb to establish the
model of chemically induced muscle necrosis. Thus, each rat
model had both liver necrosis and muscle necrosis and was
allowed to recover overnight after the procedure.
Regions of interest (ROI) from the images were analyzed with
TM
In vivo MRI examination
OptiQuant
Meriden, CT). Ratios of radioactivity concentration (DLU/
software (version 5.0; Canberra-Packard,
Magnetic resonance imaging (MRI) was performed to exam-
ine whether the model of liver necrosis and muscle necrosis
were successfully established. Under anesthesia with 2%
isoflurane in a mixture of 20% oxygen and 80% room air, the
rat models placed in a wrist coil were scanned using a 1.5 T
whole body MRI scanner (Echo speed; GE Co., New York,
NY). MRI with T1-, T2- and DWI sequences was performed
to acquire T1-weighted imaging (T1WI), T2-weighted
imaging (T2WI) and diffusion-weighted imaging (DWI).
Moreover, Dotarem (Gd-DOTA, Guerbet, France) was intra-
venously injected at a dose of 0.2 mmol/kg to obtain contrast
enhanced-T1WI (CE-T1WI).
2
mm ) between ROIs on necrotic and viable areas were
calculated. Afterwards, the sections were stained with hema-
toxylin and eosin (H&E) using the conventional procedure
and were digitally photographed for macroscopic inspection.
Pharmacokinetics study
Another six rat models with RPLI and muscle necrosis was
used for pharmacokinetics study. After IV administration of
1
31
131
131
131
I-Hyp,
blood samples were collected via a tail incision at 5 min,
0 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 24 h and 48 h
I-ProHyp,
I-ED-1 or
I-ED-2, 10 mL of
1
p.i. Blood samples were measured for radioactive counts in a
2
WIZARD 2470 automated gamma counter (PerkinElmer,
Drug delivery protocol
In order to facilitate intravenous (IV) administration, the
solution after radioactive labeling reaction was diluted with
polyethylene glycol 400 (PEG400) and propylene glycol (1:1,
v/v) to formulate preparation immediately before injection.
On day 1 after reperfusion of the partial liver infarction and
Waltham, MA) and corrections were made for its background
radiation and physical decay during counting. Radioactivity
concentrations in blood samples were calculated and
expressed as megabecquerel per liter (MBq/L). The pharma-
cokinetic parameters including area under the curve
1
31
131
131
induction of muscle necrosis, I-Hyp, I-ProHyp, I-ED-
1
(
AUC(0–t), AUC
), elimination half-life (t1/2z), peak time
Tmax), clearance (CL ), peak concentration (Cmax) and
(0–1)
131
or I-ED-2 preparation was administered via the tail vein
(
apparent volume of distribution (V ) were calculated by
z
1
14.8 MBq/kg according to I dose and 0.2 mg/kg according
31
(
z
to compound dose) under anaesthesia for biodistribution and
pharmacokinetic studies. For biodistribution study, each rat
model was intravenously injected with 0.1 mL of 1% Evans
blue solution for intravital staining of necrosis at 8 h before
being euthanized.
Drug and Statistics for windows 2.0 software.
SPECT/CT imaging
1
31
I-Hyp was evaluated for its ability to image necrosis in vivo
in rats with RPLI or sham surgery. Approximately, 14.8 MBq/kg
1
31
of I-Hyp was injected into rats with RPLI or sham surgery
via the tail vein. SPECT/CT imaging was performed using
a variable-angle dual-detector SPECT with 6-slice CT
(Symbia T6; Siemens Medical Systems Inc., Chicago, IL).
Rats were anaesthetized by intraperitoneal injection of
Biodistribution, autoradiography and histochemical
staining
1
31
For each I-compound, rat models with RPLI and muscle
necrosis were randomly allocated into two groups of four

DOI: 10.3109/1061186X.2015.1113541
Effects of skeleton structure on necrosis
5
sodium pentobarbital at a dose of 30 mg/kg and then secured
to the head holder of the patient bed in supine position.
SPECT/CT images were acquired at 24 h and 72 h after
H O treated A549 cells were performed. All four tracers
2
2
were found to have a significantly higher uptake for 100 mM
H O treated cells compared to non-treated cells (Figure 5).
2
2
1
31
intravenous injection of
I-Hyp using the following acqui-
The ratio of percentage of radioactivity in the pellet for the
treated cells compared with non-treated cells were 8.10 for
sition parameters: static image matrix size 128 ꢂ 128, acqui-
1
31
131
131
sition count limit 50000, SPECT tomographic image matrix
6
I-Hyp, 5.27 for
1
I-ProHyp, 3.08 for
I-ED-1 and 2.22
I-ED-2. These results demonstrated that the skeleton
31
4 ꢂ 64, and continuous acquisition 15 s/frame ꢂ 24 frames.
for
structure had a profound influence on necrosis targeting
ability for the four radiotracers.
Statistical analysis
Numerical data were expressed as mean ± standard deviation
(SD), unless otherwise stated. Statistical significance in all
experiments was determined by unpaired two-tailed Student’s
In vivo MRI examination
All rats survived the anesthesia and surgical procedures.
Model rats with RPLI and absolute ethanol induced muscular
necrosis were successfully established as proven by MRI.
The necrotic liver lobe showed homogeneously isointense
on T1WI but hyperintense relative to the adjacent normal
liver on T2WI, DWI and contrast enhanced T1WI. The
necrotic area in the left leg showed iso- or slightly
hyperintense on T1WI but hyperintense on T2WI, DWI and
contrast enhanced T1WI compared with the healthy right leg
t test, p50.05 was considered significant.
Results
Synthesis
All the target compounds were successfully synthesized
starting with the coupling of two molecules of emodin
anthrone. The desired intermediate emodin anthrone was
prepared from commercially available emodin in high yields.
Emodin dianthrone was synthesized as a diastereomeric
mixture in the presence of iron (III) chloride hydrate in
anhydrous ethanol. The diastereomers are inseparable in silica
gel TLC plate and slightly separable in reversed phase TLC
plate. They were successfully separated into ED-1 and ED-2
by preparative RP-HPLC.
(
Figure 6).
Biodistribution and excretion
1
The biodistribution properties of I-Hyp, I-ProHyp, I-
31
131
131
1
31
ED-1 and I-ED-2 are illustrated in Figure 7 with the results
expressed as mean %ID/g ± SD. The excretion results are
depicted in Figure 8, where the radioactivity of urine and
feces are expressed as mean %ID ± SD. Among these four
Radiolabeling and in vitro stability
1
31
131
I-dianthrones,
and necrotic muscle uptake both at 24 h and 72 h p.i. Despite
I-Hyp showed the highest necrotic liver
The radiolabeling method [39] used for radioiodination of the
target compounds was simple and effective in this work. All
131
higher radioactivity uptake in most normal tissues, I-Hyp
1
31
target compounds were readily radiolabeled with
greater than 95% radiolabeling yields. The retention times of
I with
had lowest radioactivity retention in normal liver, which
results in the highest necrotic to normal liver ratio. Although
131
it had higher radioactivity uptake in normal muscle, I-Hyp
1
31
131
131
131
I-Hyp, I-ProHyp, I-ED-1 and I-ED-2 were 14.40,
3.52, 12.03 and 12.66 min, respectively. Stabilities of all four
1
still exhibited the highest necrotic/normal muscle ratio. The
higher radioactivity uptake and slow clearance in the lung
131
demonstrated that I-Hyp may form coarse aggregates in the
1
31
ꢀ
I-dianthrones were favorable in rat serum at 37 C for 72 h
Figure 3).
(
physiological environment and were subsequently trapped by
131
Induction of cell necrosis
pulmonary capillary network.
1
I-ProHyp exhibited similar
31
Necrosis was induced by H O with concentrations of 20, 40,
2
necrosis uptake pattern to I-Hyp. Although it showed much
lower radioactivity uptake and rapid clearance in the lung,
2
6
0, 80, 100, 120 mM for 1 h. As shown in Figure 4(A), the
1
31
percentage of necrotic cells as evaluated by flow cytometry
increased with the concentration of H O from 20 mM to
I-ProHyp also exhibited much lower radioactivity uptake in
1
necrotic liver and muscle in comparison with I-Hyp. I-
31
131
2
2
1
31
1
00 mM while slightly declined with 120 mM. Untreated
ED-1 and
pattern in both normal and necrotic tissues. Among these
I-ED-2 exhibited similar radioactivity uptake
A549 cells (control cells) showed 4.71% of necrotic cells,
whereas cells treated with increasing H O concentrations for
1
31
131
131
studied
I-dianthrones,
I-ED-1 and
I-ED-2 presented
2
2
1
a maximum of 78.13% of necrotic cells with 100 mM H O
h showed gradually increasing necrotic cells from 20.84% to
the lowest radioactivity uptake in necrotic tissues and had the
lowest necrotic/normal tissue ratio.
2
2
1
31
(Figure 4). The percentage of apoptotic cells reached a
maximum of 13.60% with 20 mM H O but there was no
As described in Figure 8,
through the hepatobiliary pathway resulting in increasing
I-Hyp was mainly cleared
2
2
1
feces radioactivity with time. More than 61.0%ID of I-Hyp
31
proportional change with H O concentration. It is worth
2
2
1
31
noting that there is no significant difference on the percentage
of apoptotic cells between controls and treated with 100 mM
H O cells (6.22% versus 6.64%, p40.5).
was excreted via feces and only 11.1%ID of
I-Hyp were
I-ProHyp was also mainly
cleared via the hepatobiliary pathway and to a lesser extent
1
31
excreted via urine at 72 h p.i.
2
2
1
31
via the renal pathway, resulting in 31.3%ID of I-ProHyp in
1
feces and 26.4%ID of I-ProHyp in urine within 72 h. Unlike
31
In vitro necrotic binding assay
131
131
131
131
I-Hyp and
I-ProHyp, excretion of
I-ED-1 and
I-
In order to determine the effect of structure on necrosis
1
ED-2 was mainly via the renal pathway and to a lesser extent
via the hepatobiliary pathway.
31
targeting of
I-dianthrones, in vitro binding studies with

6
D. Zhang et al.
J Drug Target, Early Online: 1–12
1
Figure 3. Radioactive HPLC profiles of I-Hyp (A), I-ProHyp (B), I-ED-1 (C) and I-ED-2 (D) in rat serum after incubation at 37 C for 72 h.
31
131
131
131
ꢀ
1
The arrows denote the peaks corresponding to intact I-Hyp (14.40 min), I-ProHyp (13.52 min), I-ED-1 (12.03 min) and I-ED-2 (12.66 min).
31
131
131
131

DOI: 10.3109/1061186X.2015.1113541
Effects of skeleton structure on necrosis
7
Figure 5. Histogram representation of in vitro binding experiment. The
1
31
131
percentage of radioactivity in the pellet is shown for
1
I-Hyp,
O
2 2
I-
ProHyp, I-ED and I-ED in non-treated (control) and H -treated
31
131
A549 cells. For all four tracers, the percentage of radioactivity in the
pellet is significantly increased for the treated cells in comparison to
control cells.
1
31
was significantly higher than that for I-ProHyp (p50.01)
and the same is true for the AUC(0–t) (p50.01) and AUC
(0–1)
I-ED-2 was similar to
1
31
Figure 4. (A) In vitro determination of the percentage of viable,
apoptotic and necrotic cells by flow cytometry after the treatment of
A549 cells with increasing concentrations of H O . The results are
2 2
(
that of I-ED-1, as the t1/2z, CL and V with no statistically
p50.01). The blood clearance of
131
z
z
significant difference (p40.05). Although the C
value for
expressed as the percentage relative to the mean of fluorescence,
mean ± SD, n ¼ 3. (B) Cytogramms obtained with untreated A549 cells
max
131
1
31
I-ED-2 was significantly higher than that for
p50.01), both AUC(0–t) (p40.05) and AUC
I-ED-1
(p40.05)
2 2
and those treated with 100 mM H O for 1 h. Apoptotic cells (annexin-V-
(
were similar to each other.
(0–1)
positive, PI-negative) were on the lower right gate, necrotic cells
annexin-V-positive or -negative, PI-positive) were on the upper right
(
and left gate and viable cells (annexin-V-negative, PI-negative) were on
the lower left gate.
SPECT/CT imaging
1
31
As
normal liver uptake than the other three
4 h and 72 h p.i., we further evaluated its ability for imaging
I-Hyp showed higher necrotic liver uptake and lower
1
31
I-dianthrones at
Postmortem autoradiography and histochemical
staining
2
necrosis in vivo in rats with RPLI or sham surgery.
Representative SPECT/CT images at 24 h and 72 h p.i. of
The representative autoradiographs and corresponding H&E
staining images of the liver (Figure 9A) and muscle slices
1
31
I-Hyp are presented in Figure 11. On the SPECT/CT
1
Figure 9B) at 24 h and 72 h p.i. for each I-dianthrone are
31
(
images, the uptake of radioactivity in the infarcted liver lobe
was apparently much higher than that in the non-infarcted
liver lobe and that in the liver of the sham-operated rat. Right
shown in Figure 9. Hepatic infarct and muscle necrosis
was identified by H&E staining. Autoradiography showed
that radioactivity uptake in necrotic liver was much higher
infarcted liver lobes could be clearly visualized by SPECT/CT
131
1
31
than that in normal liver for each I-dianthrone, particularly
1
using
specific high uptake of
I-Hyp as an imaging probe, which suggested that
131
31
131
for
I-Hyp. Moreover, necrosis targetability of
I-
I-Hyp in the infarcted liver was
1
31
dianthrones was further verified in muscle tissue as higher
radioactivity uptake was seen in the necrotic areas than
that in the viable areas. By a semiquantitative analysis,
ratios of radioactivity in necrotic over normal tissues obtained
from the representative autoradiographs are presented in
Table 1. At 24 h and 72 h p.i., the necrotic to normal
mediated by necrosis and I-Hyp can detect necrosis in the
liver after a partial liver infarction. The SPECT/CT images of
rats with RPLI showed high contrast of infarcted liver lobes to
normal liver lobes, which was consistent with the biodistribu-
tion results. Moreover, higher radioactivity uptake was seen
especially in the intestines, indicating the major excretion
pathways of the compound.
1
31
liver uptake ratios for I-Hyp were 20.9 and 46.4, respect-
ively; for necrotic to normal muscle, these values were 9.0
and 13.8.
Discussion
In our previous reports [33,40,41], we have perceived the
important effect of skeleton structure of dianthrones on their
necrosis targeting. In order to ascertain in more detail the
relationship between molecular skeleton structures of dia-
nthrones and their necrosis targeting and clearance properties,
Pharmacokinetics
The mean plasma radioactive concentration–time profiles of
1
31
I-dianthrones from 0 to 48 h in rat models with RPLI and
muscle necrosis are shown in Figure 10. The major
pharmacokinetic parameters are presented in Table 2.
1
31
131
131
we synthesized and evaluated I-Hyp, I-ProHyp, I-ED-
131
1 and I-ED-2 in in vitro cell of induced necrosis and in
in vivo model rats of induced necrosis.
1
31
Although there was no significant difference in t
1
for I-
/2z
1
31
131
Hyp and I-ProHyp (p40.05), the Cmax value for I-Hyp

8
D. Zhang et al.
J Drug Target, Early Online: 1–12
Figure 6. In vivo MRI transverse section images of rat model with RPLI and muscle necrosis. The upper row was T1WI, CE-T1WI, T2WI and DWI of
the liver with infarcted right lobe (arrow) and the bottom row was that of the leg with muscle necrosis (arrow). Infarcted liver lobe and necrotic muscle
appeared hyperintense on CE-T1WI, T2WI and DWI.
1
31
Figure 7. Biodistribution of I-dianthrones in rat models with RPLI and muscle necrosis at 24 h (A) and 72 h (B) p.i. (n ¼ 4 per time point).

DOI: 10.3109/1061186X.2015.1113541
Effects of skeleton structure on necrosis
9
1
31
Figure 8. Excretion of I-dianthrones by the feces (A) and urine (B) in rat models of induced necrosis within 72 h p.i. (n ꢃ 4 per time point).
Figure 9. Representative autoradiographs
(
corresponding H&E images (lower row for
131
upper row for each I-dianthrones) and
1
31
each I-dianthrones) of liver slices (A) and
muscle slices (B) from the rat models at 24 h
and 72 h p.i., respectively. The left column
represents the normal liver and the right
column represents the necrotic liver at each
time point in A. Each muscle section contains
both normal muscle and necrotic muscle in B.
N ¼ necrotic area, V ¼ viable area.
The present study showed that the disruption of molecular
planarity has a dramatic impact on the necrosis targeting of
Table 1. Ratios of radioactivity uptake between ROIs on necrotic and
viable areas from rat models with RPLI and muscle necrosis at 24 h and
7
131
131
2 h after IV injection of I-dianthrones*.
the
molecular planarity, the necrosis targeting also continues to
I-dianthrones. With the increasing distortion of
1
31
131
reduce. Among the four I-dianthrones, I-Hyp displayed
the highest percentage of radioactivity in the pellet for the
Necrotic liver/
normal liver
Necrotic muscle/
normal muscle
1
treated cells followed by I-ProHyp, whereas I-ED-1 and
31
131
Compounds
24 h
72 h
24 h
72 h
1
31
I-ED-2 exhibited significantly lower percentage of radio-
1
1
1
1
31
31
31
31
I-Hyp
20.9
4.3
2.8
2.4
46.4
7.9
4.1
3.2
9.0
6.3
5.0
4.5
13.8
9.1
7.5
5.4
activity in the pellet for the treated cells. In vivo biodistribu-
tion study displayed a similar necrotic tissue uptake pattern.
Up to now, the mechanism of necrosis avidity behind Hyp and
other NAAs has not yet been fully elucidated, although
several thoughts or hypotheses were raised to explain the
phenomenon, such as cholesterol or phospholipid binding in
lipid membranes, or a compound-specific mechanism behind
I-ProHyp
I-ED-1
I-ED-2
*
Regions of interest (ROI) from the autoradiographic images was analyzed
with Optiquant software. Radioactivity uptake on ROIs on necrotic and
viable areas was expressed as DLU/mm . Ratios were calculated by
2
2
dividing DLU/mm of ROI on necrotic areas by that on viable areas.

1
0
D. Zhang et al.
J Drug Target, Early Online: 1–12
Figure 10. The mean plasma radioactive
concentration–time profiles following a
1
31
single IV administration of I-dianthrones
in rat models with RPLI and muscle necrosis.
gD r ao t ua p a) r. e expressed as mean ± SD (n ¼ 6, each
1
31
Table 2. Major pharmacokinetic parameters of I-dianthrones obtained by non-compartmental modeling after IV injection in rat models with RPLI
and muscle necrosis within 48h (n ¼ 6).
131
Major pharmacokinetic parameter value for I-dianthrones
1
31
I-Hyp
131
I-ProHyp
131
I-ED-1
131
I-ED-2
Parameters
Unit
AUC(0-t)
MBq/L*h
MBq/L*h
h
1178.41 ± 62.92
1268.36 ± 48.63
14.73 ± 2.77
0.15 ± 0.04
0.01 ± 0
200.57 ± 18.38
0.25 ± 0.05
864.80 ± 129.92
955.14 ± 151.52
15.05 ± 1.28
0.13 ± 0.05
0.02 ± 0
114.86 ± 11.84
0.34 ± 0.05
509.71 ± 71.50
546.83 ± 79.04
12.71 ± 2.70
0.14 ± 0.04
0.03 ± 0
68.73 ± 12.81
0.50 ± 0.12
540.82 ± 104.35
557.92 ± 108.16
10.05 ± 1.25
0.08 ± 0
0.03 ± 0.01
96.56 ± 10.84
0.40 ± 0.09
AUC(0-1)
t
1/2z
T
max
CL
h
z
L/h/kg
MBq/L
L/kg
C
max
V
z
The values are expressed as mean ± SD.
131
Figure 11. I-Hyp can image tissue necrosis
in vivo after a partial liver infarction. (A) and
(C) Representative SPECT/CT images of rat
with sham surgery at 24 h and 72 h post
1
31
injection of I-Hyp, respectively. (B) and
D) Representative SPECT/CT images of rat
with RPLI at 24 h and 72 h post injection of
(
1
31
I-Hyp, respectively.
the necrosis targeting of different NAAs [22]. Although we
have demonstrated that the Hyp compounds are indeed
more effective than the ED compounds both in vitro and
in vivo, we don’t know the exact cause yet. As we know, Hyp
has a nearly planar rigid skeleton. The skeleton structure of
ProHyp is less planar than that of Hyp as a result of the
absence of a single bond. Emodin dianthrones can be free to
rotate around a single bond connecting two molecules of
emodin anthrone and the overall rigidity was completely
broken. Therefore, by analyzing structure–necrosis targeting
relationship of the four dianthrone compounds, we can at least
draw the conclusion that rigid skeleton structure may play an

DOI: 10.3109/1061186X.2015.1113541
Effects of skeleton structure on necrosis 11
important role in necrosis targeting and molecular morph-
ology of a rigid nearly planar configuration may be more
beneficial to its binding to the target site.
the next step, we attempt to study whether DNA binding is
responsible for high Hyp targeting to necrosis region.
The difference in pharmacokinetic behavior suggested that
1
Conclusions
31
skeleton structures of I-dianthrones have also a profound
influence on their pharmacokinetic properties. Fonge et al.
The present study demonstrated that the destruction of rigid
skeleton structure dramatically reduced the necrosis targeting
1
31
[
42] confirmed that
I-Hyp bind mainly to low-density
131
131
ability of
higher necrosis tissue uptake and lower normal liver uptake
I-bianthrones.
I-Hyp exhibited sustaining
lipoprotein (LDL) and to a lesser extent to high-density
lipoprotein (HDL), which was similar to plasma protein
binding profile of Hyp. Considering the similarity in structure
131
than those of the other three
infarction could be clearly visualized by SPECT/CT using
I-dianthrones. Hepatic
1
31
131
that both I-Hyp and I-ProHyp maintain a nearly planar
1
131
I-Hyp as an imaging probe. The preliminary SAR study
31
structure, we have reason to believe that
have a similar plasma lipoprotein binding profile to I-Hyp.
I-ProHyp could
1
indicated that the skeleton structure of Hyp was the most
attractive for further developing NAAs. Future work will
focus on reducing the self-aggregation degree of Hyp and
understanding the mechanism that yields necrosis targeting in
molecular level.
31
This may explain why they showed similar t1 (p40.05)
/2z
1
31
despite the fact that lipophility of
(
I-ProHyp
1
I-Hyp
31
log P ¼ 2.40 ± 0.18) [42] is less than that of
(
lipophilicity but also other parameters have a significant
log P ¼ 3.08 ± 0.03) [43]. This indicates that not only
1
31
Acknowledgements
impact on the pharmacokinetic properties of the
1
I-
I-Hyp was
31
bianthrones. Furthermore, the Cmax value for
1
We thank Mr Changwen Fu and Mr Yu Fu, Department of
Nuclear Medicine, the First Affiliated Hospital of Nanjing
Medical University, for their wonderful work in SPECT/CT
scanning.
31
nearly two times higher than that for I-ProHyp, which may
ensure sufficient hypericin that could be delivered to the
1
31
131
target tissues. Although I-ED-2 displayed
similar plasma radioactive concentration–time profiles, the
I-ED-1 and
1
31
131
higher Cmax (p50.01) for
I-ED-2 relative to I-ED-1
suggested stereoisomerism have an influence on the pharma-
Declaration of interest
The authors report no declaration of interest. This work was
financially supported by the National Natural Science
Foundation of China (81473120 and 21171092), the fourth
phase of 333 projects in Jiangsu Province (BRA2012211), the
Project Program of State Key Laboratory of Natural
Medicines, China Pharmaceutical University (ZJ11175) and
the Ninth Batch of ‘‘Six Talent Peaks’’ Project of Jiangsu
Province (2012-YY-008).
cokinetic parameters. Moreover, both the AUC(0–t) (p40.05)
and AUC(
(p40.05) values were not significantly
0–1)
different, which suggested they may have similar bioavail-
ability after IV injection.
The results of present study showed that the skeleton
structure of Hyp was the most attractive for the development
of promising NAAs both for necrosis imaging and targeted
radionuclide therapy. Further SAR study will involve the
effect of various substituent groups on necrosis targeting of
Hyp. Owing to the hydrophobic effect of its nearly planar
highly conjugated structure and the cofacial ꢀ–ꢀ stacking
between the molecular skeletons, Hyp is prone to form
aggregates in an aqueous environment [30,44]. After intra-
venous administration, parts of the Hyp might form the
aggregates and even coarse pellets in the blood circulation.
The formed coarse aggregates are intercepted by mononuclear
phagocyte system, especially by pulmonary capillary net-
work, which not only reduces its biodistribution to necrotic
tissues but also shows unwanted biodistribution in normal
tissues [31,32]. Although distortion and even destruction of
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