Synthesis and Evaluation of 131I-Skyrin as a Necrosis Avid Agent for Potential Targeted Radionuclide Therapy of Solid Tumors

Article abstract of DOI:10.1021/acs.molpharmaceut.5b00630

An innovative anticancer approach targeted to necrotic tissues, which serves as a noncancerous and generic anchor, may present a breakthrough. Necrosis avid agents with a flat conjugate aromatic structure selectively accumulate in necrotic tissues, but they easily form aggregates that undesirably distribute to normal tissues. In this study, skyrin, a dianthraquinone compound with smaller and distorted -cores and thus decreased aggregates as compared with hypericin (Hyp), was designed to target necrosis for tumor therapy. Aggregation studies of skyrin by UV/vis spectroscopy showed a smaller self-association constant with skyrin than with Hyp. Skyrin was labeled by iodine-131 with a radiochemical purity of 98% and exhibited good stability in rat serum for 72 h. In vitro cell uptake studies showed significant difference in the uptake of ¹³¹I-skyrin by necrotic cells compared to normal cells (P < 0.05). Compared in rats with liver and muscle necrosis, radiobiodistribution, whole-body autoradiography, and SPECT/CT studies revealed higher accumulation of ¹³¹I-skyrin in necrotic liver and muscle (p < 0.05), but lower uptake in normal organs, relative to that of ¹³¹I-Hyp. In mice bearing H22 tumor xenografts treated with combretastatin A4 disodium phosphate, the highest uptake of ¹³¹I-skyrin was found in necrotic tumor. In conclusion, ¹³¹I-skyrin appears a promising agent with reduced accumulation in nontarget organs for targeted radionuclide therapy of solid tumors.

Full text of DOI:10.1021/acs.molpharmaceut.5b00630

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Article
Synthesis and Evaluation of 131I-skyrin as a Necrosis Avid Agent
for Potential Targeted Radionuclide Therapy of Solid Tumors
Cong Wang, Qiaomei Jin, Shengwei Yang, Dongjian Zhang, Qin Wang,
Jindian Li, Shaoli Song, Ziping Sun, Yicheng Ni, Jian Zhang, and Zhiqi Yin
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00630 • Publication Date (Web): 08 Dec 2015
Downloaded from http://pubs.acs.org on December 15, 2015
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Synthesis and Evaluation of 131Iꢀskyrin as a Necrosis
Avid Agent for Potential Targeted Radionuclide Therapy
of Solid Tumors
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Cong Wang,†,‡ Qiaomei Jin,‡ Shengwei Yang‡, Dongjian Zhang,‡ Qin Wang,‡,§
Jindian Li,†,‡ Shaoli Song,∥ Ziping Sun,⊥ Yicheng Ni,# Jian Zhang,*,‡ Zhiqi Yin,* †
†Department of Natural Medicinal Chemistry & State Key Laboratory of Natural
Medicines, China Pharmaceutical University, Nanjing 210009, Jiangsu Province,
P.R.China
‡Laboratories of Translational Medicine, Jiangsu Province Academy of Traditional
Chinese Medicine, Nanjing 210028, Jiangsu Province, P.R.China
§College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, Jiangsu,
China
∥Department of Nuclear Medicine, the Affiliated Renji Hospital of Shanghai Jiaotong
University
⊥Radiation Medical Institute, Shandong Academy of Medical Sciences, Jinan 250062,
Shandong Province, P.R.China
#Theragnostic Laboratory, Campus Gasthuisberg, KU Leuven, 3000 Leuven, Belgium
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ABSTRACT
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An innovative anticancer approach targeted to necrotic tissues, which serves as a
noncancerous and generic anchor, may present a breakthrough. Necrosis avid agents
with a flat conjugate aromatic structure selectively accumulate in necrotic tissues, but
they easily forms aggregates that undesirably distribute to normal tissues. In this study,
skyrin, a dianthraquinone compound with smaller and distorted πꢀcores and thus deꢀ
creased aggregates as compared with Hypericin (Hyp), was designed to target necroꢀ
sis for tumor therapy. Aggregation studies of skyrin by UV/Vis spectroscopy showed
a smaller selfꢀassociation constant with skyrin than with Hyp. Skyrin was labeled by
Iodineꢀ131 with a radiochemical purity of 95% and exhibited good stability in rat seꢀ
rum for 72 h. In vitro cell uptake studies showed significant difference in the uptake
of 131Iꢀskyrin by necrotic cells than by normal cells (P < 0.05). Compared in rats with
liver and muscle necrosis, radiobiodistribution, wholeꢀbody autoradiography and
SPECT/CT studies revealed higher accumulation of 131Iꢀskyrin in necrotic liver and
muscle (p < 0.05), but lower uptake in normal organs, relative to that of 131IꢀHyp. In
mice bearing H22 tumor xenografts treated with combretastatin A4 disodium phosꢀ
phate, the highest uptake of 131Iꢀskyrin was found in necrotic tumor. In conclusion,
131Iꢀskyrin appears a promising agent with reduced accumulation in nontarget organs
for targeted radionuclide therapy of solid tumors.
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KEYWORDS
necrosis, targeted radionuclide therapy, skyrin, 131I, SPECT/CT, cancer
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INTRODUCTION
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Targeted therapies revitalized the field of cancer therapeutics with the promise of
improved cure rates accompanied by reduced toxicity.1 However, the unpredictable
mutations, histological diversity and developed drug resistance of the growing tumor
still impose challenges to target therapies.2ꢀ4 Rather than directly targeting viable
cancer cells, an innovative anticancer approach targeting to necrotic tissues may preꢀ
sent a breakthrough.5ꢀ7 Necrotic tissues, which locate always close to the viable cancer
cells, comprise 30−80% of a fastꢀgrowing solid tumor8ꢀ10 and the proportion can be
even larger after certain anticancer therapies,6, 11 can function as a generic anchor for
targeting solid malignancies. In addition, necrotic tissues are noncancerous and more
stable stromal targets that may confront less drug resistance.7
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Necrosis avid agents (NAAs), a class of compounds that selectively accumulate
in the necrotic tissues, have been explored over the past decade for estimating progꢀ
nosis, assessing therapeutic responses to drug treatment and formulating therapeutic
strategies.12ꢀ15 To date, the majority of NAAs are developed for molecular imaging of
16ꢀ18
necrotic tissues, such as hoechstꢀIR, zincꢀdipicolylamine, 99mTcꢀglucarate etc.
only a handful are therapeutic or theragnostic agents.7, 19
Currently, Hypericin (Hyp, Figure 1B), a naphthodianthrone compound, is the
most studied NAA and found to selectively accumulate in necrotic tissues with high
necroticꢀtoꢀviable tissue ratios (up to 80).20, 21 131IꢀHyp has been proved as a promisꢀ
ing theragnostic radiopharmaceutical with a high sustaining accumulation in tumor
necrotic areas for cancer therapy.7, 15, 19 However, Hyp easily forms aggregates in
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aqueous environment,22 which may strongly weaken the targeting biodistribution and
lead to the undesired uptake in normal organs.23, 24
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The aggregation of Hyp in aqueous solvent is mainly attributed to the strongly πꢀπ
interactions and hydrophobic effect of the nearly planar πꢀconjugate aromatic core
(Figure 1D).25, 26 Distorting πꢀconjugated system or reducing the size of πꢀcores will
decrease the πꢀπ stacking energy, making the formation of aggregates more difficult.27,
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28
Our groups recently synthesized protohypericin that has the same anthraquinone
base structure but a smaller and slightly twisted aromatic core.29 It presents a minor
aggregation behavior and comparable necrosis affinity compared with Hyp.29 Howevꢀ
er, protohypericin still tends to form aggregates and easily converts into Hyp under
light.29
In order to further inhibit the aggregate formation, we designed skyrin (Figure 1A)
that is a diphenyl compound sharing the same anthraquinone fragment but different
molecular skeleton compared with Hyp. Due to the twisted πꢀcores, the dihedral angle
of skyrin is 81.25°30(22.96° for Hyp). Hence, we speculate that skyrin may share simꢀ
ilar necrosis targetability, but present a dramatically reduced tendency in forming agꢀ
gregates with better tissue distribution.
To validate this hypothesis, we synthesized and studied the hydrophilicity and agꢀ
gregation of skyrin, preliminarily evaluated the necrosis avidity of 131Iꢀskyrin in rats
with reperfused liver infarction (RLI) and ethanolꢀinduced muscular necrosis (MN),
and compared with Hyp (or 131IꢀHyp). Furthermore, the therapeutic potential of
131Iꢀskyrin was assessed on a H22 murine hepatoma mouse model.
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EXPERIMENTAL SECTION
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General. All reagents and solvents used were purchased from Sinopharm Chemical
Reagent Co., Ltd (Shanghai, China) or Nanjing Chemical Reagent Co., Ltd (Nanjing,
China) with the exception of Emodin (purity > 98%), which was purchased from
Chengdu Huaxia Chemical Reagent Co., Ltd (Chengdu, China), and Hyp (purity >
98%), which was purchased from Chengdu Biopurify Phytochemicals Ltd (Chengdu,
China). Iodogen (1, 3, 4, 6ꢀtetrachloroꢀ3α, 6αꢀdiphenylglycouril) was obtained from
Pierce Biotechnology (ZI Camp Jouven, France). Sodium iodide (Na131I) was supꢀ
plied by HTA Co., Ltd (Beijing, China) with a radionuclidic purity over 99% and the
specific activity was 370 MBq/mL. Gadoliniumꢀbased contrast agent Magnevist was
obtained from Bayer Schering Pharma AG (Berlin, Germany). The human hepatoma
cell line HepG2 and murine hepatoma cell line H22 were obtained from American
Type Culture Collection (Manassas, VA). Kunming mice (25−28 g) and Sprague
Dawley (SD) rats (250−300 g) were furnished by Experimental Animal Center,
Jiangsu Academy of Traditional Chinese Medicine (Nanjing, China). Animal proceꢀ
dures utilizing rats and mice were approved by the Institutional Animal Care and Use
Committee.
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All reaction mixtures were monitored using thin layer chromatography (TLC) on
silica gel 60 F254 plates (Merck, Germany), and visualized by ultraviolet (UV) irradiaꢀ
tion at 254 nm or staining with aluminum chloride or magnesium acetate. The syntheꢀ
sized compounds were purified by silica gel chromatography and Sephadex LH20.
SepꢀPak plus C18 cartridges (Waters, USA) were preconditioned with ethanol (2 mL)
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and water (2 mL) before use. H and 13C NMR spectra were carried out on a Bruker
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300 MHz NMR (Switzerland) at 303K using DMSOꢀd6 as an internal standard.
Chemical shifts were reported in parts per million (δ, ppm) values. Elecꢀ
trosprayꢀionization mass spectrometry (ESIꢀMS) was carried out on HP1100 mass
spectrometer (Agilent, Santa Clara, CA, USA). Reversedꢀphase high performance liqꢀ
uid chromatography (RPꢀHPLC) was performed on a Waters Alliance 2695 Separaꢀ
tions module equipped with Waters 2998 UV/Visible detector (Waters Cooperation,
Milford, MA, USA) and Berthold HERM LB500 radiometric detector (Millipore,
Billerica, MA, USA). The RPꢀHPLC column was GRACE AlltimaTM C18 analytical
column (250 mm × 4.6 mm, 5 µm). The radiochemical purity was assayed by analytiꢀ
cal HPLC using methanol and water containing 0.04% ammonium acetate (v/v =
70:30 for 131Iꢀskyrin and 88:12 for 131IꢀHyp) in an isocratic mode at a flow rate of 1
mL/min under the column temperature of 30 °C. Magnetic resonance imaging (MRI)
was performed using a 1.5T wholeꢀbody MR magnet (Echo speed; GE Co., NY).
Synthetic Approach.
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Synthesis of Skyrin. The synthesis of emodinbianthrone was performed using
emodin as the starting material as reported in the literature31 (Scheme 1). Then emodꢀ
inbianthrone (100 mg) was dissolved in 0.2 M potassium hydroxide solution (100
mL). The solution was heated at 100 °C, while a strong stream of oxygen was passed
through it for 10 min. Then the solution was poured into excess 2 M hydrochloric acid,
the red precipitate was so obtained and subjected to silica gel column chromatography
(chloroform: methanol = 55:1) to give compound 4. Yield: 53 mg, 52%. The final
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product was characterized by H, 13C NMR and mass spectrometry. H NMR (300
MHz, DMSOꢀd6) δ (ppm): 2.32 (s, 6H, CH3), 6.72 (s, 2H, Hꢀ7,7’), 7.12 (s, 2H,
Hꢀ4,4’), 7.27 (s, 2H, Hꢀ2,2’), 11.14 (brs, 2H, OH), 12.01, 12.77 (s, 4H, OH); 13C
NMR (75 MHz, DMSOꢀd6) δ (ppm): 21.49 (CH3), 107.23 (CH), 109.01 (C), 113.10
(C), 120.42 (CH), 123.45 (CH), 123.51 (C), 131.29 (C), 133.23 (C), 148.17 (C),
161.05 (C), 164.35 (C), 181.99 (CO), 189.63 (CO); HRꢀESIꢀMS: calculated for
[C30H18O10]: 538.0900, found [M−H]ꢀ: 537.0824.
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Synthesis of 127I-Skyrin. Iodine (25 mg) was added to a stirred suspension of
skyrin (107 mg) in a mixed solvent of tetrahydrofuran (THF, 15 mL) and H2O (15 mL)
at 0°C. Then NaHCO3 (25 mg) was stepwise added and the mixture was stirred for 30
min. CH3COOCH3 (300 mL) was used to extract the organic phase. Purification of the
reaction mixture was carried out using silica gel column chromatography (chloroform:
methanol = 100:1) and Sephadex LH20 column chromatography. Compound 5 was
obtained as a red powder (13 mg, 10%). 1H NMR (300 MHz, DMSO) δ (ppm): 2.29
(s, 3H, CH3), 2.33 (s, 3H, CH3), 6.69 (s, 1H, Hꢀ7’), 7.04, 7.13 (s, 2H, Hꢀ4,4’), 7.19,
7.27 (s, 2H, Hꢀ2,2’), 12.07, 12.79 (s, 2H, OH); HRꢀESIꢀMS: calculated for
[C30H17O10I]: 663.9866, found [M−H]ꢀ: 662.9801.
Aggregation Studies by UV/Vis Spectroscopy. The UV/Vis spectras of skyrin and
Hyp were carried out using a Cary 5000 spectrophotometer (Varian, Australia).22
Measurements were conducted in the function of concentration ranging from 1.3 ×
10ꢀ6 M to 2.7 × 10ꢀ5 M at 20 °C, in water solution containing 50% DMSO. The measꢀ
urements were carried out at wavelength ranging from 300 to 800 nm. The apparent
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molar absorption coefficients at suitable wavelengths were fitted by nonlinear
leastꢀsquare regression analysis to the isodesmic (equal K) model according to equaꢀ
tion (1)
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ꢇꢈ ꢊꢋꢌꢍ ꢏꢈ ꢊꢋꢌ
ꢎ
ꢐ
ꢉ
ꢉ
ꢀ
ꢁ
ꢂ
ꢀ ꢁ
ꢀ ꢁꢅ
ꢀ ꢁ
ꢑ + εꢄ λ C (1)
A λ, C = εꢃ λ − εꢄ λ ꢆ
ꢇꢈ
ꢉ
ꢊ
Where A(λ,C) ꢀ absorbance at wavelength λ of the measured sample at the concenꢀ
tration of C in solution; εM(λ) and εA(λ) ꢀ the extinction coefficients for the monomerꢀ
ic and the aggregated compounds, respectively; Ka ꢀ aggregation constants.
The degree (or fraction) of aggregated molecules αagg can be obtained according to
the following equation (2), where cT is the concentration of the compound.
αꢒꢓꢓ = 1 − ꢇꢔꢕ ꢋꢌꢍ ꢏꢔꢕ ꢋꢌ (2)
ꢎ
ꢖ
ꢖ
ꢐ
ꢐ
ꢇꢈ ꢕ
ꢖ
131I Radiolabeling. In order to prolong the time of evaluation and investigate the
potential for therapy, we choose Iodineꢀ131 that has a longer halfꢀlife than Iodineꢀ123
(8 days vs 13 h) and emits γꢀrays and βꢀparticles for imaging diagnosis and tumor inꢀ
ternal radiotherapy. The Iodogen coating method was employed to radioiodinate
skyrin/Hyp to form 131Iꢀskyrin/131IꢀHyp. Briefly, the waterꢀinsoluble oxidant “Iodogen”
was dissolved in dichloromethane and coated on the walls of the Eppendorf tube. Raꢀ
dioiodination was conducted by adding 14.8 MBq of Na131I and 1 × 10ꢀ7 mol of skyrꢀ
in/Hyp dissolved in 300 ꢁl dimethylsulfoxide (DMSO). Afterwards, the mixture was
incubated at 30 °C for 20 min, and the reaction was terminated by transference of reꢀ
action solution to another clean tube. The product was passed through a SepꢀPak C18
cartridge, washed with 1 mL of water and eluted with 1 mL of methanol. The methaꢀ
nol eluent was collected and assayed by analytical HPLC using the conditions as deꢀ
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scribed above with 127Iꢀskyrin as a reference.
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Stability Studies in Rat Serum. For in vitro stability study, 0.1 mL of the solution
of 131Iꢀskyrin or 131IꢀHyp (7.4 MBq) was added to 0.9 mL of rat serum and incubated
at 37 °C for 72 h. Plasma proteins were precipitated by adding 300 ꢁL of ethanol and
removed by centrifugation (12000 rpm, 8 min). The radiochemical stability was asꢀ
sayed by HPLC using the isocratic analysis method.
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OctanolꢀWater Partition Coefficient. Log P was determined at physiological conꢀ
dition by the “shakeꢀflask” method.32 131Iꢀskyrin or 131IꢀHyp (10 ꢁL, 1.85 MBq) was
added to a solution containing octanol (500 ꢁL) and phosphateꢀbuffered saline (PBS,
500 ꢁL, pH 7.4). The resulting solution was vigorously vortexed at room temperature
for 3−5 min and then centrifuged at 14000 rpm for 5 min. The counts in 10 ꢁL aliꢀ
quots of both octanol and saline phases were measured on a γ counter (PerkinElmer
1480; Waltham, MA). Two independent experiments were performed in triplicate.
The octanol/buffer partition coefficient P was calculated as P = (cpm in the organic
phase − background cpm)/(cpm in the aqueous phase − background cpm). The partiꢀ
tion coefficient value was expressed as log P.
In Vitro Cell Binding Assay. The HepG2 cells were seeded into a 6ꢀwell cell culꢀ
ture plate at a density of 5 × 105 cells/well and incubated at 37 °C in a humidified CO2
incubator (5% CO2) in RPMI 1640 supplemented with 10% fetal bovine serum (FBS)
and 1% (v/v) penicillin/streptomycin overnight. After being washed with PBS conꢀ
taining 10% FBS, cells were incubated for 1 h under intense hyperthermia at 57 °C to
induce necrosis33 and at 37 °C as a control. Then the cells were transferred into 4 mL
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Eppendorf tubes and coꢀincubated at 37 °C for 15 min with 37 KBq of 131Iꢀskyrin or
131IꢀHyp in 1 mL of RPMIꢀ1640 medium. After incubation, the mixture was transꢀ
ferred into 1.5 mL tubes and centrifuged at 12000 rpm for 10 min. Then 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 the radioactivity was measꢀ
ured using a gamma counter. The radioactivity values were calculated and expressed
as the percentage uptake per 108 cells. Experiments were performed twice with tripliꢀ
cate wells.
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Animal Models of Necrosis
Rat Model of Reperfused Liver Infarction and Muscular Necrosis (RLI&MN).
RLI&MN was conducted as previously described.34 Simply, male SD rats were
anaesthetized with intraꢀperitoneal injection of sodium pentobarbital at a dose of 40
mg/kg. Under laparotomy, the hilum of the right liver lobes was ligated with a suture
to generate ischemia for 3 h. Then released hepatic inflow obstruction and massaged
the liver lobe for reperfusion, and closed the abdominal cavity with twoꢀlayered suꢀ
tures. The coexisting normal liver lobes were used as control for intraꢀindividual
comparison.
As a sclerotic agent, ethanol is used for tumor ablation in human patients and inꢀ
ducing tissue necrosis for optical imaging studies.17 Each RLI model rat was intraꢀ
muscularly injected with 0.2 mL ethanol in the left leg for creating chemically inꢀ
duced muscle necrosis, and animals were allowed to recover for 6−8 h after the proꢀ
cedure.
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In Vivo Tumor Models. Induction of necrosis in an H22 hepatocarcinoma cancer
model was achieved as described previously with minor modification.7 Briefly, mice
were injected subcutaneously in the unilateral flank region with 1 × 107 H22 cells.
Tumor volume was determined by caliper measurements with the formula: tumor
volume = (minimum diameter)2 × (maximum diameter) × 1/2. When the tumors
reached approximately 6 to 8 mm in diameter (2−3 weeks), mice were injected via the
tail vein with Combretastatin A4 disodium phosphate (CA4P) at a dose of 10 mg/kg.
MRI was performed to evaluate whether the necrosis model was successful or not.
See the supplemental material for the detailed procedures.
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Tracer Administration. The thyroid of model rats and mice was blocked with
Lugol’s solution (1.2 g/L) in their drinking water 3 days before and till the end of exꢀ
periment. In order to facilitate the administration, the reaction solution was diluted
with polyethylene glycol 400 (PEG400) and propylene glycol (v/v =1:1) to formulate
preparations immediately before injection. Each rats and mice was administrated inꢀ
travenously with 131Iꢀskyrin or 131IꢀHyp formulation (14.8 MBq/Kg) for in vivo animal
experiments.
Biodistribution Studies. For biodistribution studies, 24 model rats of RLI&MN (n
= 4/group) were intravenously administered with 131Iꢀskyrin or 131IꢀHyp under anaesꢀ
thesia and sacrificed at 12 h, 24 h and 72 h p.i., respectively. Organs of interest were
removed, weighed, and counted for radioactivity using a γ counter. The deꢀ
cayꢀcorrected activity per mass of tissue was calculated. Uptake of the tracers was
calculated as the percentage of the injected dose per gram of tissue (% ID/g) with the
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values expressed as mean ± standard deviation (SD) and percentage of injected dose (%
ID) with the values expressed as mean ± SD.
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Ex vivo Autoradiography and Histochemical Staining. For ex vivo autoradiogꢀ
raphy studies, representative tissues of necrotic liver, normal liver and necrotic muscle
at each time point were removed and washed thoroughly with 0.9% saline (4 °C) to
remove blood pool activity. Sections of 10 and 30 ꢁm were cut using a cryostat miꢀ
crotome (Shandon Cryotome FSE, Thermo Fisher Scientific Co., MA) at −20 °C and
were thawꢀmounted on glass slides. Autoradiographs of these slides were obtained by
exposing the sections for 6−32 h to a high performance phosphor screen (Cyclone;
CanberraꢀPackard, Ontario, Canada). After the exposure, the screen was read using a
Phosphor Imager scanner and analyzed using Optiquant software (CycloneTM, Canꢀ
berraꢀPackard, CT). Relative tracer concentration was estimated by regions of interest
(ROI) analysis for the necrotic and viable tissues on all autoradiographs. The slides
were stained with haematoxylinꢀeosin (H&E) using a conventional procedure and
digitally photographed to confirm the presence or absence of necrosis. Photomicroꢀ
graphs were obtained from an optical microscope (Axioskop; Zeiss, Oberkochen,
Germany) with magnification at × 200.
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SPECT/CT Imaging Studies. The tomographic acquisitions were done on a comꢀ
bined SPECT/CT scanner (Precedence 6; Philips, The Netherlands) which hybrids a
standꢀalone dualꢀhead γꢀcamera equiped with a pinhole highꢀenergy collimator with a
6ꢀslice spiral CT camera installed within the same gantry. Model rats (n = 3/group)
were injected via the tail vein with approximately 3.7 MBq (100 ꢁCi) of 131Iꢀskyrin or
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131IꢀHyp. At 12 h, 24 h and 72 h p.i., rats were anesthetized with sodium pentobarbital
at a dose of 40 mg/kg and placed in the prone position near the center of the field of
view (FOV) of the scanner. The following protocol was used for SPECT acquisition:
128 × 128 matrix, 180◦ SPECT with 6◦ steps, 25 s/frame and a variable radius of rotaꢀ
tion adapting to the closest possible distance from the phantom surface. Prior to each
CT imaging, 0.1 mL Iobitridol was injected intravenously. For CT image acquisition,
the scan parameters were 240 mA, 120 kV, a rotation time of 0.75 s, and 1mm slice
thickness and a 0.5 × 1.5 mm collimation. Both SPECT and CT data were reconꢀ
structed and displayed as transaxial and coronal slices using an Astonish bone appliꢀ
cation package (Philips, The Netherlands). The SPECT and CT fusion images were
obtained using the Syntegra software (Philips, The Netherlands).
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Whole-body Autoradiography. For wholeꢀbody autoradiography studies, 6 model
rats of RLI were intravenously injected with 3.7 MBq (100 ꢁCi) of 131Iꢀskyrin or
131IꢀHyp. At 72 h p.i., rats were euthanized with overdose of sodium pentobarbital,
embedded in 4% carboxymethylcellulose and stored at −20 °C until sectioning. Indiꢀ
vidual rat legs were removed, and coronal sections of 150 ꢁm thickness were obtained
using a cryostat macrotome (Leica SM2500, Wetzlar, Germany) at −20 °C. Sections
were lyophilized and mounted on clear tape and exposed at −20 °C for 0.5−2 h to
phosphorꢀimaging plates. Autoradiographic images were obtained and analyzed as
described above.
Experiment on Mice Bearing H22 Tumors. The mice received 131Iꢀskyrin (15 ꢁCi)
24 h after CA4P treatment. Twenty mice (n = 10/group) were sacrificed at 24 h and 72
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h p.i. for biodistribution studies, wholeꢀbody autoradiography, ex vivo autoradiogꢀ
raphy and histochemical staining as described above.
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Statistical analysis. Numerical results were reported as the mean ± SD. Means were
compared using Student’s t test and groups with a P value less than 0.05 were considꢀ
ered significant differences.
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Chemistry. Skyrin and 127Iꢀskyrin were synthesized as shown in Scheme 1. Comꢀ
pound 2 was synthesized using tin (II) chloride hydrate in high yield. Coupling of
emodin anthrone was realized using iron (III) chloride hydrate as the catalyst and
skyrin was obtained when emodin bianthrone dissolved in alkali solution in the presꢀ
ence of sufficient quantities of oxygen. In all cases, intermediate and final products
were purified on silica gel or Sephadex LH20 column chromatography and the > 95%
purity was verified by RPꢀHPLC.
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Aggregation in WaterꢀDMSO. For a comparison of the aggregation behaviour of
skyrin and Hyp, waterꢀDMSO (1:1) proved to be an ideal solvent. Hyp is insoluble in
water that is the main components of blood, DMSO appears an ideal solvent to introꢀ
duce it into the aqueous medium and was chosen to dissolve Hyp. The aggregation
constants of skyrin and Hyp determined according to the isodesmic model were (3.17
± 0.52) × 105 M−1 and (1.13 ± 0.34) × 106 M−1, respectively. Figure 2 presents the
concentrationꢀdependent transitions from monomeric to aggregated molecules and the
calculated regression lines according to the isodesmic model. All these indicated a
downward tendency in forming aggregates of skyrin compared that of Hyp.
Radiochemistry. 131I labeling was performed in a straightforward method with a
preparation time of 20 min for 131Iꢀskyrin/131IꢀHyp at room temperature. After purifiꢀ
cation, the specific activity of 131I tracers were typically about 5ꢀ10 Ci/mmol. Radioꢀ
chemical purity greater than 98% (labeling yield more than 90%) could be obtained as
measured by analytic RPꢀHPLC for both tracers. The structure of 131Iꢀskyrin was deꢀ
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termined by using 127Iꢀskyrin as reference compound (Figure 3).
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Partition Coefficient and in Vitro Stability. The partition coefficient, log P (pH
7.4), of 131Iꢀskyrin and 131IꢀHyp were measured as 1.88 ± 0.08 (n = 3) and 2.76 ± 0.03
(n = 3), respectively. For in vitro serum stability analysis, both tracers were intact for
72 h at 37 °C in rat serum (Figure 3), suggesting that they had very good stability in
vitro.
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In Vitro Binding to Necrotic Cells. Cell binding of 131Iꢀskyrin and 131IꢀHyp were
performed using HepG2 cells and the results are shown in Table 1. Both tracers were
found to have a significantly higher uptake with hyperthermia treated cells compared
to nonꢀtreated cells (P < 0.05). The ratios of radioactivity in the pellet for the treated
cells compared to nonꢀtreated cells were 3.52 ± 0.70 for 131Iꢀskyrin and 3.79 ± 0.61
for 131IꢀHyp.
Biodistribution. The biological distribution results of 131Iꢀskyrin and 131IꢀHyp in
rat models with RLI&MN were showed in Table 2 and Table S1. 131Iꢀskyrin had high
uptake in necrotic tissues (3.19 ± 0.64% ID/g for necrotic liver and 1.52 ± 0.48% ID/g
for necrotic muscle at 12 h p.i., respectively) and good retention (3.85 ± 0.27% ID/g
for necrotic liver and 1.21 ± 0.36% ID/g for necrotic muscle at 72 h p.i., respectively).
Necrotic to viable liver and muscle ratios increased from 6.06 ± 1.27 and 4.92 ± 1.18
at 12 h to 20.87 ± 3.98 and 10.59 ± 0.84 at 72 h. Except for the blood, the clearance of
131Iꢀskyrin from the nontarget tissues was fast and the accumulations were lower than
0.8% ID/g at 12 h p.i. in most of the examined normal organs.
When compared with 131Iꢀskyrin, 131IꢀHyp had comparable necrotic liver uptake at
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earlier time points but lower at later time points. 131IꢀHyp also showed lower uptake in
necrotic muscle but higher lung and spleen accumulation (P < 0.05). For all of the
other tissues, these two radiotracers showed comparable radioactivity uptake except
slower blood clearance with 131Iꢀskyrin (P < 0.05).
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Autoradiography and Histochemical Staining. Representative autoradiographic
images (upper row), photographs (lower row) and photomicrographs (bottom row) of
the corresponding H&E stained slices of necrotic muscle, necrotic liver and normal
liver slices comparing two tracers at 24 h p.i. are shown in Figure 4. On H&E stained
slices, purple areas correspond to viable tissue while pink areas correspond to necrotic
tissue. The radioactivity signal from both 131Iꢀskyrin and 131IꢀHyp colocalized with
necrotic tissues detected with H&E assay. Analysis of the autoradiogram images
(comparison of the DLU/mm2 for necrotic and normal tissue) showed that both
131Iꢀskyrin and 131IꢀHyp were able to accurately delineate necrotic tissue with necrotic
to viable tissue activity ratios up to 9−25/6−31 for the liver and 8−15/5−6 for muscle,
respectively.
SPECT/CT Imaging. Representative coronal and transaxial SPECT/CT images of
model rats of RLI&MN (n = 3/group) at 72 h after administration of 131Iꢀskyrin or
131IꢀHyp are shown in Figure 5. The enhanced CT displayed the necrotic liver/muscle
with lower signal than normal due to the contrast agents (Iobitridol) inaccessible into
necrotic areas. SPECTꢀCT showed that the necrotic liver and muscle could be clearly
visualized after intravenous injection of 131Iꢀskyrin and demonstrated high contrast
with low contralateral background. Most of the nontargeted organs demonstrated low
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signal, indicating relatively low accumulation of the tracer. When compared with
131Iꢀskyrin, similar findings were observed with 131IꢀHyp except that there was no deꢀ
tectable uptake of 131IꢀHyp in necrotic muscle with high radioactivity accumulation in
intestines.
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Wholeꢀbody Autoradiography. Wholeꢀbody autoradiography proved as a preꢀ
ferred method for conducting tissue biodistribution studies. The autoradiographic reꢀ
sults are compared in Figure 6. For both probes, the necrotic liver was the organ with
the highest uptake, reflecting the extraordinary avidity in necrotic tissues. Relatively
low uptake in the lung, gut cavities and high uptake in the heart (blood) was observed
with 131Iꢀskyrin, which was in good agreement with the results of ex vivo biodistribuꢀ
tion by γ counting.
Experiment on Mice Bearing H22 Tumors. Treatment of mice bearing tumors
with CA4P induces substantial necrosis.6 Spontaneous necrosis existed in H22 tumor,
tumor necrosis ratio as measured from contrast enhanced T1ꢀweighted (CEꢀT1W)
images at baseline was about 17% (Figure S7 in the Supporting Information) in each
group. The necrosis ratio increased to 52% 24h after the CA4P treatment. 131Iꢀskyrin
showed highest uptake in the necrotic tumors at both time points and the actual value
of necrotic tumor should be higher due to the fact that viable cells were intermixed
with necrotic cells (Table S2 in the Supporting Information). Wholeꢀbody and intraꢀ
tumoral distribution of 131Iꢀskyrin at 72 h in mice bearing H22 tumors were shown in
both autoradiographic and H&E images (Figure 7). The highest uptake in tumor was
clearly visualized after chemotherapy (Figure 7A and 7B). There was also relatively
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high accumulation in the liver, but the tumor to liver was 3.49 ± 0.52 as determined
by autoradiography. For slices of tumors, there was little signal in the viable tumors
and the necrotic to viable ratio was up to 11.52 ± 1.25 (Figure 7C−F), which further
confirmed the excellent necrosis affinity.
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DISCUSSION
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In the present study, we synthesized a tracer and evaluated its targetability to neꢀ
crotic tissues, especially tumor necrosis for potential tumor therapy. Skyrin presented
a better hydrophilicity and downward tendency in forming aggregates compared with
Hyp, leading to its lower uptake in the monocyte macrophage system (MPS). Simulꢀ
taneously encouraging result is that 131Iꢀskyrin showed prominent necrotic targetabilꢀ
ity with high sustaining necrotic to viable activity ratios.
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Skyrin appeared less aggregative in aqueous solution by UV/Vis analysis with a
selfꢀassociation constant that is less than one third of the corresponding value of Hyp.
The aggregation of molecules might be affected by many factors such as solvent, the
size and electronic nature of the πꢀconjugated system and the peripheral substituents
attached to the πꢀconjugated core.35ꢀ37 Dispersion forces, an important contributor to
the overall energy of πꢀπ interactions, are favored by increased surface areas. Accordꢀ
ingly, it is reasonable that skyrin with a decreased core size had a lower propensity to
form aggregates. Furthermore, due to the twisted core, the πꢀπ contact area may also
dramatically reduce, which can be attributed to an increase of the πꢀπ distance or a
longitudinal displacement between aggregates. In addition, the hydrophobic effect of
131Iꢀskyrin, an important contribution to the Gibbs free aggregation energy, is also deꢀ
creased with a smaller octanolꢀwater partition coefficient. Hyp is poorly soluble and
forms aggregates in aqueous environment. The coarse aggregates are intercepted by
pulmonary capillary network and small ones are cleaned and retained in the liver and
spleen. Liu et al. revealed that the nonꢀaggregated formulation of Hyp showed higher
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accumulation in target organ and lower accumulation in MPS compared with two agꢀ
gregated formulations (over concentrated Hyp in DMSO and in water solution).24 The
lessened aggregation led to a significantly less radioactive uptake of 131Iꢀskyrin in the
MPS than that of 131IꢀHyp, especially in the lung and spleen (0.40 ± 0.03% ID/g and
0.26 ± 0.07% ID/g vs 1.97 ± 0.20% ID/g and 1.05 ± 0.15% ID/g at 12 h p.i.). These
were also clearly observed on the wholeꢀbody autoradiographic image, while 131IꢀHyp
showed higher signal in the lung.
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The necrosis targetability of 131Iꢀskyrin was preliminarily investigated and comꢀ
pared with 131IꢀHyp in model rats with RLI&MN to acquire reliable evidences. Acute
and localized tissue damage in rats was achieved by injecting ethanol into the rear leg
muscle and performing ischemiaꢀreperfusion operation on the right liver lobe. Altꢀ
hough this in vivo tissue necrosis model has limited relevance with clinic, it serves as
a highly reproducible and reliable experimental system for quantitative comparison of
radiolabeled probes.17, 38 Both tracers showed high necrotic to viable tissue activity
ratios (up to 20−24 for liver and 5−11 for muscle) and sustaining uptake in necrotic
liver (3.85 ± 0.27% ID/g for 131Iꢀskyrin and 3.26 ± 0.23% ID/g for 131IꢀHyp, at 72 h).
Although 131Iꢀskyrin had similar accumulation in necrotic liver at early time points, it
had higher uptake at 72 h and in necrotic muscle than 131IꢀHyp. Encouraged by these
results, 131Iꢀskyrin was then evaluated in xenograft model. Biodistribution studies,
autoradiography and corresponding H&E demonstrated that 131Iꢀskyrin may accumuꢀ
late highly in necrotic tumors and be a promising agent for cancer therapy.
The similar higher uptake with hyperthermia treated cells compared to nonꢀtreated
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cells indicated that both tracers had favourable necrosis targetability. Being a proꢀ
nounced hydrophobic compound, 131IꢀHyp tended more to nonspecifically bind to
cellular lipophilic membrane structures. 39, 40 Once cell become necrotic, the binding
of 131IꢀHyp to the intracellular membrane structures added up. For these reasons, the
absolute values of accumulation in both treated and nonꢀtreated cells for 131IꢀHyp
seem to be higher.
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Currently, the majority of targeted probes are designed to bind to the receptors
overexpressed on the membrane of viable tumor cells. However, probes that targeted
to the necrosis tissues are rare but have following advantages. Necrosis is widely preꢀ
sent in numerous disorders or pathologies, including cardiac disease and cancer. Furꢀ
thermore, probes can be designed with extraordinarily high avidity and specificity due
to the failure of necrotic cells to preserve the integrity of cell membrane and exchange
of macromolecules (such as exposed DNA, heat shock proteins, phosphatidylserine
etc.) between the intracellular and extracellular environment which are absent in viaꢀ
ble cells.41 In addition, being more stable and generic targets with less heterogeneity
and drug resistance, better disease diagnosis and therapeutic response can be acquired
by attaching γ rayꢀemitting or βꢀ particleꢀemitting radioisotopes to the NAAs.
As a promising anticancer strategy has been applied in clinical treatment, tumor
necrosis therapy (TNT) uses a genetically engineered, 131I chimeric mAb, which tarꢀ
gets naturally occurring nuclear antigen histone present only on dying or dead cells.6
However, potential immunogenicity and poor stability owing to its property as a large
protein have limited its clinical application.42 Being a small molecule compound,
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131Iꢀskyrin would show little immunogenicity as well as good stability.
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Rational tumor combination therapy can improve treatment efficacy and enable the
reduction of side effects.43 Due to the insufficient blood supply, tumor necrosis is
ubiquitous in the solid tumor as a result of hypoxia and lack of prompt clearance.
However, the degree and location of necrosis is closely related to the type of the tuꢀ
mor and it is recommended to combine with other types of treatments. If radiofreꢀ
quency ablation or chemotherapy drugs are first used to cause massive tumor necrosis,
then NAAs carrying radioisotopes can be systemically delivered and specifically acꢀ
cumulate at intratumoral necrosis, irradiate the adjacent remnant tumor cells with βꢀ
particle to prevent malignant cell repopulation. Being a promising molecular agent for
clinical translation and future development, we plan to combine 131Iꢀskyrin with
chemotherapy drugs for treating various solid tumors in future experiments. In addiꢀ
tion, the exact mechanism of NAAs binding to necrotic tissues should be further eluꢀ
cidated and more efficient way of synthesis needs to be exploited.
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CONCLUSIONS
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In this study, 131Iꢀskyrin exhibited comparable necrosis targeting properties with
131IꢀHyp in vitro and in vivo. However, skyrin presented a smaller selfꢀassociation
constant compared with Hyp and the uptake of 131Iꢀskyrin in normal organs, especialꢀ
ly lungs and spleen, was significantly reduced. In tumor models, 131Iꢀskyrin showed
the highest uptake in chemotherapyꢀinduced necrotic tumors. All these results indicate
that 131Iꢀskyrin is a promising agent for targeted radionuclide therapy of solid tumors.
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ASSOCIATED CONTENT
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Supporting Information
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1H, 13C NMR, high resolution mass spectra of skyrin and 127Iꢀskyrin. Biodistribution
of 131Iꢀskyrin in rats of RLI&MN and CA4P treated liver cancer model. Magnetic
resonance images of mice bearing H22 tumors before and 24 h after CA4P treatment.
This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
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Corresponding Author
*(J.Z.) Phone: +86ꢀ25ꢀ52362107. Fax: +86ꢀ25ꢀ85637817. Eꢀmail: zjwonderꢀ
ful@hotmail.com.
*(Z.Y.)
Phone:
+86ꢀ25ꢀ86185371.
Fax:
+86ꢀ25ꢀ85301528.
Eꢀmail:
chyzq2005@126.com.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was supported, in part, by the National Natural Science Foundation of
China (No.81473120), the Ninth Batch of “Six Talent Peaks” Project of Jiangsu
Province (No.2012ꢀYYꢀ008), and A Project Funded by the Priority Academic Proꢀ
gram Development of Jiangsu Higher Education Institutions. We thank Mr Yinyan
Zhu, Department of Nuclear Medicine, the Affiliated Renji Hospital of Shanghai
Jiaotong University, for his wonderful work in SPECT/CT scanning.
ABBREVIATIONS
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NAAs, necrosis avid agents; Hyp, Hypericin; SPECT/CT, single photon emission
computed tomography/computed tomography; RPꢀHPLC, reverse phaseꢀhigh perforꢀ
mance liquid chromatography; RLI, reperfused liver infarction; MN, muscular necroꢀ
sis; KI, potassium iodide; H&E, haematoxylinꢀeosin; p.i., postinjection; CA4P, comꢀ
bretastatin A4 disodium phosphate.
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