Improved in vivo Targeting Capability and Pharmacokinetics of 99mTc

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Improved in vivo Targeting Capability and Pharmacokinetics of 99mTclabeled isoDGR by Dimerization and Albumin-Binding for Glioma imaging Hannan Gao, Chuangwei Luo, Guangjie Yang, Shuaifan Du, Xiaoda Li, Huiyun Zhao, Jiyun Shi, and Fan Wang Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/ acs.bioconjchem.9b00323 • Publication Date (Web): 07 May 2019 Downloaded from http://pubs.acs.org on May 8, 2019

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Bioconjugate Chemistry

Improved in vivo Targeting Capability and Pharmacokinetics of 99mTc-labeled

isoDGR by Dimerization and Albumin-Binding for Glioma imaging

Hannan Gao†, Chuangwei Luo†, Guangjie Yang†, Shuaifan Du†, Xiaoda Li‡, Huiyun Zhao‡, Jiyun Shi§*, Fan Wang†, §* †Medical

Isotopes Research Center and Department of Radiation Medicine, State Key

Laboratory of Natural and Biomimetic Drugs, School of Basic Medical Sciences, Peking University, Beijing 100191, China ‡Medical §Key

and Healthy Analytical Center, Peking University, Beijing 100191, China

Laboratory of Protein and Peptide Pharmaceuticals, CAS Center for Excellence

in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China

Running title: Improved 99mTc-labeled isoDGR dimer probe for glioma detection ____________________________________________________________________ *Correspondence should be addressed to: 1) Dr. Fan Wang, Medical Isotopes Research Center and Department of Radiation Medicine, Peking University, 38 Xueyuan Road, Beijing 100191, China, E-mail: [email protected]; and 2) Dr. Jiyun Shi, Key Laboratory of Protein and Peptide Pharmaceuticals, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China, E-mail: [email protected].

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Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT Previously, we have successfully developed the c(phg-isoDGRk) peptide as a novel integrin α5β1-targeted SPECT imaging probe imaging. However, the fast clearance of

99mTc-HisoDGR

99mTc-HisoDGR

for Glioma

in blood reduced its tumor

accumulation and retention, which would be the obstacles for further application in clinical. Dimerization and albumin-binding strategies have been proved as effective approaches to improve tumor targeting capability and blood circulation time of radiotracers. In this study, the novel PEGylated dimeric isoDGR peptides (termed 3PisoDGR2) with an albumin binder (termed AB-3PisoDGR2) were designed, and the 99mTc-3PisoDGR

corresponding radiotracers

2

and

99mTc-AB-3PisoDGR

2

were

fabricated and assessed for tumor-targeting and in vivo pharmacokinetics properties in subcutaneous and orthotopic tumor models. The dimerization of isoDGR peptide provided higher binding affinity to tumor cells and longer blood circulation time than the original monomeric isoDGR peptide, resulting in twice increased tumor uptake (99mTc-3PisoDGR2 2.51 ± 0.17 %ID/g vs. 99mTc-PisoDGR 1.17 ± 0.21 %ID/g, P10000 nmol), providing the

approaches for the integrin α5β1-targeted diagnosis and treatment of glioma.12 Our data demonstrated that the highest tumor uptake was achieved at 0.5 h p.i., with fast blood clearance via a renal pathway, as evidenced by high amounts of activity in the bladder. We therefore hypothesize that there would be benefit in further increasing tumor uptake of the tracer, with the aim of enhancing tumor to non-target organ contrast and thus the sensitivity of the probe as a diagnostic imaging agent. Over the past decades, the PEGylation and multimerization concepts have been extensively utilized in peptides-based radiotracers.13-15 PEGylation can help to increase the hydrophilicity of the hydrophobic tracers, which improve the tracer excretion kinetics from noncancerous organs, such as liver and lungs.16,17 PEG group may also help the tracers to prolong the circulation time, which result in better tumor accumulation. Multimerization brings enhanced receptor binding affinity, which lead to better tumor uptake and longer tumor retention.13,14,18 Especially, the dimerization bridged by two PEG4 linkers resulted in tremendously enhanced tumor targeting and



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improved in vivo pharmacokinetics.15,19,20 Besides, albumin-binding has also been considered as a simple but powerful strategy to enhance the tumor uptake of small molecules via prolonging its blood circulation time.21,22 Recently, the albumin binders (such as truncated Evans blue (EB) and 4-(p-iodophenyl)butyric acid) have been appended to ligands of prostate-specific membrane antigen (PSMA), folic acid and TATE motifs, achieving improved diagnostic imaging and long-acting radiotherapy in tumor-bearing animal models and cancer patients.23,24 In this study, the PEG4 linker-bridged dimerization strategy was firstly introduced to the original monomeric c(phg-isoDGRk) peptide to improve the targeting capability and in vivo pharmacokinetics, creating a novel PEGylated dimeric isoDGR peptide (termed 3PisoDGR2). Moreover, an albumin binder (AB) of 4-(p-iodophenyl)butyric acid was further inserted into the PEGylated isoDGR dimer (termed AB-3PisoDGR2), which was expected to further prolong the blood circulation time and increase tumor delivery.

99mTc

is a very cost-effective nuclide for

single-photon emission computed tomography (SPECT), which can be easily obtained from a

99Mo/99mTc

generator and quickly labeled to peptides via a kit formulation,

enabling availability for clinical applications. Therefore, the

99mTc-labeled

3PisoDGR2 and AB-3PisoDGR2 were fabricated and assessed for tumor detecting and in vivo pharmacokinetics in subcutaneous and orthotopic glioma xenograft mice models. Compared to monomeric achieved

improved

99mTc-AB-3PisoDGR

2

tumor

99mTc-PisoDGR

uptake

and

tracer, the

tumor

99mTc-3PisoDGR

retention,

while

2

the

obtained additional tumor uptake enhancement.

RESULTS Chemistry. The products were prepared following the synthesis routes (Supplemental Fig. S1~3). The detailed procedures and results were elaborated in the Supporting Information. Briefly, 2PisoDGR2 was made from two motifs of PEGylated isoDGR peptide PisoDGR by conjugating to one L-glutamate (E). For albumin-binding purpose, albumin binder (AB) was added into dimeric peptide 2PisoDGR2 via D-lysine (k) to obtain AB-2PisoDGR2. Radiolabeling precursors including


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HYNIC-PisoDGR, HYNIC-3PisoDGR2, HYNIC-AB-3PisoDGR2 (Fig. 1) were prepared by conjugating PEGylated bifunctional chelator HYNIC-PEG4-NHS to different isoDGR peptides (isoDGR, 2PisoDGR2 and AB-2PisoDGR2), respectively. All final products were obtained after HPLC purification with more than 95% purity and confirmed with correct molecular weight via mass spectrum (Supporting Information S4, S5 and S6).

Figure 1. Chemical structures of the HYNIC-PEG4-c(phg-isoDGRk) (termed as HYNIC-PisoDGR), HYNIC-PEG4-E[PEG4-c(phg-isoDGRk)]2 (termed as HYNIC-3PisoDGR2) and HYNIC-PEG4-k(AB)-E[PEG4-c(phg-isoDGRk)]2 (termed as HYNIC-AB-3PisoDGR2) and their components (Albumin Binder; PEG4; HYNIC).

Radiochemistry. The bifunctional chelator conjugated peptides (HYNIC-PisoDGR, HYNIC-3PisoDGR2, HYNIC-AB-3PisoDGR2) were radiolabeled in 50 mM pH5.0 succinate buffer solution with Na99mTcO4 using TPPTS and Tricine as coligands to obtain

99mTc-radiolabed

trinary

[99mTc-(HYNIC-peptide)(Tricine)(TPPTS)] 99mTc-3PisoDGR

radio-HPLC

2

and

99mTc-AB-3PisoDGR

chromatograms

of

(termed 2,

complex as

respectively)

99mTc-PisoDGR,


of

99mTc-PisoDGR,

(Fig.

2A).

99mTc-3PisoDGR

2

The and


99mTc-AB-3PisoDGR

2

were determined at the same RP-HPLC condition (Fig. 2B~D).

All the radiochemical yields (RCY) were measured to be above 97.0% at the retention time of 13.1 min, 17.2 min and 23.0 min, respectively. The LogP values of 99mTc-PisoDGR, 99mTc-3PisoDGR

2

and 99mTc-AB-3PisoDGR2 were tested in an equal

volume mixture of n-octanol and 25 mM phosphate buffer (pH = 7.4) and calculated to be -3.68 ± 0.05, -3.71 ± 0.04 and -3.40 ± 0.03, respectively, suggesting all tracers remained well hydrophilicity. The in vitro stabilities of all the three tracers were determined in saline and 1 mg/mL cysteine at room temperature (25 °C). All three tracers remained more than 99% intact in saline and cysteine solution for at least 8 hours. The in vivo metabolism stability was tested in urine collected at 1 h p.i. from the mice administered the radiotracers intravenously. For all three tracers, no evidential radioactive metabolite was found in radio-HPLC chromatography, indicating excellent in vivo metabolism stability (Fig. 2).

Figure 2. (A) Chelating structure of [99mTc(HYNIC-Peptide)(tricine)(TPPTS)]. (B-D) Typical 99mTc-HYNIC-PisoDGR radio-HPLC chromatograms of (99mTc-PisoDGR), 99mTc-HYNIC-3PisoDGR 99mTc-HYNIC-AB-3PisoDGR (99mTc-3PisoDGR2) and 2 2 99m ( Tc-AB-3PisoDGR2) in saline, cysteine and the urine sample obtained from tracer injected mice, respectively.


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Cell Binding Assay. Previously, the integrin binding property of isoDGR was determined via

99mTc-labeled

isoDGR incubating with integrin α5β1-positive U87MG

glioma cells.11 Here, the displacement curves of isoDGR and 2PisoDGR2 against the binding of 125I-c(phg-isoDGRy) on U87MG cells were shown and compared (Fig. 3A). The IC50 values were measured to be 93.36 ± 5.50 nM for isoDGR and 13.63 ± 0.56 nM for 2PisoDGR2.

Figure 3. (A) The displacement curves of isoDGR and 2PisoDGR2 against the binding of 125I-c(phg-isoDGRy) on U87MG cells. That leads 99mTc-3PisoDGR to bind integrin α β in a 2 5 1 univalent or a bivalent manner. (B) The blood clearance curves of 99mTc-PisoDGR, 99mTc-3PisoDGR and 99mTc-AB-3PisoDGR . The evidently prolonged pharmacokinetics in blood 2 2 99m for Tc-AB-3PisoDGR2 was due to the albumin binder.

Blood Clearance. The blood clearance studies were evaluated in normal KM mice. The

blood

clearance

99mTc-AB-3PisoDGR

2

curves

of

99mTc-PisoDGR,

99mTc-3PisoDGR

2

and

were shown and compared (Fig. 3B). The results were given as

percentage injected dose per gram (%ID/g), fitting with nonlinear regression of two phase decay.

99mTc-PisoDGR

min; T1/2β = 13.79 min),

showed particularly fast blood half-life (T1/2α = 1.42

99mTc-3PisoDGR

1.56 min; T1/2β = 24.77 min), and

2

showed prolonged blood half-life (T1/2α =

99mTc-AB-3PisoDGR

2

showed further prolonged

blood half-life (T1/2α = 3.75 min; T1/2β = 97.38 min). Biodistribution. 99mTc-3PisoDGR

The 2

and

in

vivo

targeting

99mTc-AB-3PisoDGR

model at 0.5, 1 and 2 h p.i. Compared to

2

capabilities

of

99mTc-PisoDGR,

were evaluated in U87MG xenograft

99mTc-PisoDGR,


the

99mTc-3PisoDGR

2


Page 8 of 27

showed significantly increased tumor uptake (2.51 ± 0.34 vs. 1.20 ± 0.37 %ID/g, p 95%), the pharmacokinetics characteristics of

99mTc-3PisoDGR

2

and

99mTc-AB-3PisoDGR

2

were assessed in

comparison with monomer tracer 99mTc-PisoDGR by blood clearance experiments. As the results,

99mTc-3PisoDGR

2

half-life (T1/2β) compared to

showed almost 2-fold prolonged elimination phase 99mTc-PisoDGR,

and

99mTc-AB-3PisoDGR

2

showed

further prolonged blood half-life with more than 2-fold distribution phase half-life 99mTc-3PisoDGR

2

99mTc-AB-3PisoDGR

2

(T1/2α) and 4-fold elimination phase half-life (T1/2β) compared to (Fig 3B). By conjugating the reversible albumin binder (AB), could be deemed as a slow-release

99mTc-3PisoDGR

2

analog, which possessed the

binding property to integrin α5β1. The ultrafiltration experiment of blood samples from mice injected with tracer suggested that

99mTc-AB-3PisoDGR

2

bound to serum

protein other than dissociating in the blood stream (Fig. S8A). Since the tumor uptake is highly related to the tracer’s targeting capability, the results of biodistribution studies revealed that the targeting capability to U87MG tumor was ranged by

99mTc-AB-3PisoDGR

2

99mTc-3PisoDGR

>

2

>

99mTc-PisoDGR.

Both PEG-bridged-dimerization and albumin-binding strategies contributed to the tumor uptake enhancement, while the dimerization strategy corresponding tracer 99mTc-3PisoDGR 99mTc-PisoDGR

2

caused

2-fold

increase

compared

to

monomer

tracer

(p 98% purity. MALDI-TOF-MS found m/z =1140.41 for [M+H]+ consistent with 1140.47 for [C50H70N13O16S]+ (Supporting Information, Figure S1 and S4). b) Synthesis of HYNIC-3PisoDGR2. Synthesis of Boc-E[PEG4-c(phg-isoDGRk)]2. PEG4-c(phg-isoDGRk) (16.6 mg, 19.8 μmol) and Boc-Glu(NHS)-NHS (4.3 mg, 10 μmol) were dissolved in 1.0 mL DMF. After addition of 20 μL DIEA, the mixture was stirred at room temperature overnight. The product was isolated by HPLC (The gradient of solvent B started with 10 % to 10% at 10 min to 30% at 15 min to 80% at 25 min to 10% at 30 min). The fraction at 17.2 min was collected. Lyophilization of the collection afforded the product as a white powder. The yield was 9.4 mg (~50%) with >98% purity. MALDI-TOF-MS found m/z = 1885.13 for [M+H]+ consistent with 1884.96 for [C84H133N21O28]+ (Supporting Information, Figure S2 and S5). Preparation of E[PEG4-c(phg-isoDGRk)]2. Boc-E[PEG4-c(phg-isoDGRk)]2 (9.4 mg, 5.0 μmol) was dissolved and reacted in 1.0 mL TFA. The mixture was stirred at room temperature for 5 min. Solvent was removed by a stream of nitrogen. After that, 4.0 mL water was added to dissolve the residue. Lyophilization of the solvent afforded white powder. The product was tested by HPLC (The gradient of solvent B started with 5% to 5% at 5 min to 30% at 25 min to 5% at 30 min). The retention time was 17.3 min. The yield was 8.9 mg (~99%) with >98% purity. MALDI-TOF-MS found m/z = 1785.12 for [M+H]+ consistent with 1784.91 for [C79H126N21O26]+ (Supporting Information, Figure S2 and S5). Synthesis of HYNIC-3PisoDGR2. E[PEG4-c(phg-isoDGRk)]2 (2.0 mg, 1.1 μmol)


Page 18 of 27

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and HYNIC-PEG4-NHS (1.0 mg, 1.5 μmol) were dissolved in 200 μL DMF. After addition of 10 μL DIEA, the mixture was stirred at room temperature overnight. The product was isolated by HPLC (The gradient of solvent B started with 5% for 5 min to 30% at 25 min to 5% at 30 min). The fraction at 24.3 min was collected. Lyophilization of the collection afforded the product as a white powder. The yield was 1.6 mg (~ 62%) with >98% purity. MALDI-TOF-MS found m/z =2335.27 for [M+H]+ consistent with 2335.08 for [C103H155N25O35S]+ (Supporting Information, Figure S2 and S5). c) Synthesis of HYNIC-AB-3PisoDGR2. Preparation of AB-NHS. 4-(p-Iodophenyl)butyric acid (AB) (20 mg, 68.7 μmol), EDC.HCL (17.1 mg, 89.3 μmol) and NHS (10.3 mg, 89.3 μmol) were dissolved in 0.5 mL DMF. The mixture was stirred at room temperature overnight. The product was isolated by HPLC (The gradient of solvent B started with 60% to 60% at 5 min to 80% at 15 min to 60% at 20 min). The fractions at 8.9 min were collected. Lyophilization of the fractions afforded white powder. The yield was 20.1 mg (~75%) with >98% purity. Synthesis of Fmoc-k(AB)-COOH. AB-NHS (20.1 mg, 51.9 μmol) and Fmoc-k(NH2)-COOH·HCl (20.9 mg, 51.9 μmol) were dissolved in 0.5 mL DMF. After addition of 20 μL DIEA, the reaction mixture was stirred at room temperature overnight. The product was isolated by HPLC (The gradient of solvent B started with 60% to 60% at 5 min to 80% at 15 min to 60% at 20 min). The fraction at 11.7 min was collected. Lyophilization of the collection afforded the product as a white powder. The yield was14.4 mg (~43%) with >99% HPLC purity. ESI-TOF-MS found m/z = 641.15 for [M+H]+ consistent with 641.14 for [C31H34IN2O5]+ (Supporting Information, Figure S3 and S6). Preparation of Fmoc-k(AB)-NHS. Fmoc-k(AB)-COOH (14.4 mg, 22.5 μmol), EDC.HCl (5.5 mg, 28.8 μmol) and NHS (3.4 mg, 29.5μmol) were dissolved in 0.5 mL DMF. The reaction mixture was stirred at room temperature overnight. The product was isolated by HPLC (The gradient of solvent B started with 60% to 60% at 5 min to 80% at 15 min to 60% at 20 min). The fraction at 13.6 min was collected. Lyophilization of the collection afforded the product as a white powder. The yield was 8.1 mg (~48.8%) with >99% purity. ESI-TOF-MS found m/z = 738.16 for [M+H]+ consistent with 738.16 for [C35H37IN3O7]+ (Supporting Information, Figure S3 and S6).



Synthesis

of

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Fmoc-k(AB)-E[PEG4-c(phg-isoDGRk)]2.

E[PEG4-c(phg-isoDGRfk)]2 (4.28 mg, 2.4 μmol) and Fmoc-k(AB)-NHS (3.1 mg, 4.2 μmol) were dissolved in 0.2 mL DMF. After addition 50 μL of NaHCO3 (0.05 mol/L), the reaction mixture was stirred at room temperature overnight. Any precipitate was dissolved by adding 5 μL TFA and the product was isolated by HPLC (The gradient of solvent B started with 15% to 15% at 5 min to 70% at 20 min to 70% at 25 min to 15% at 30 min). The fraction at 18.3 min was collected. Lyophilization afforded the product as a white powder. The yield was 2.42 mg (41.8%) with >99% purity. MALDI-TOF-MS found m/z = 2406.42 for [M+H]+ consistent with 2407.04 for [C110H157IN23O30]+ (Supporting Information, Figure S3 and S6). Preparation

of

NH2-k(AB)-E[PEG4-c(phg-isoDGRk)]2.

The

Fmoc-k(AB)-E[PEG4-c(phg- isoDGRk)]2 (2.42 mg, 1.0 μmol) was dissolved in 100 μL DMF. After adding 25 μL piperidine (20%), the reaction mixture was stirred at room temperature for 10 min. The precipitate was then dissolved by adding 50 μL TFA and the product was isolated by HPLC (The gradient of solvent B started with 15% to 15% at 5 min to 70% at 20 min to 70% at 25 min to 15% at 30 min). The fraction of 14.6 min was collected. Lyophilization of the collection afforded the product as a white powder. The yield was 1.91 mg (87%) with >99% purity. MALDI-TOF-MS found m/z = 2185.08 for [M+H]+ consistent with 2184.98 for [C95H147IN23O28]+ (Supporting Information, Figure S3 and S6). Synthesis of HYNIC-AB-3PisoDGR2. HYNIC-PEG4-NHS (1.0 mg, 1.5 μmol) and NH2-k(AB)-E[PEG4-c(phg-isoDGRk)]2 (1.91 mg, 0.87 μmol) were dissolved in 100 μL DMF. After addition 100 μL NaHCO3 (0.05 M), the reaction mixture was stirred at room temperature overnight. The product was isolated by HPLC (The gradient of solvent B started with 15% to 15% at 5 min to 50% at 25 min to 15% at 30 min). The fraction at 21.4 min was collected. Lyophilization of the collection afforded the product as a white powder. The yield was 1.0 mg (42%) with >99% purity. MALDI-TOF-MS found m/z = 2735.12 for [M+H]+ consistent with 2735.15 for [C119H177IN27O37S]+ (Supporting Information, Figure S3 and S6). Cell Culture and Animal Model. The human glioma U87MG cell line was purchased from ATCC (ATCC® HTB-14™) and cultured in DMEM with 10% FBS. Female BALB/c nude mice (4 weeks of age) and KM mice (6 weeks of age) were


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purchased from the Department of Animal Experiment, Peking University Health Science Center. All animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Peking University. The tumor xenografted models were established by implanting U87MG cells (2.5 × 106) subcutaneously into the right upper flanks of nude mice. When tumors had reached 200-300 mm3 (2 ~ 3 weeks after inoculation), the mice were able to be used for biodistribution and SPECT/CT imaging studies. Integrin Binding Affinity. Cell binding assays were using U87MG cells and the binding buffer (20 mM Tris, 150 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, 0.1% BSA, pH = 7.4). The 125I-c(phg-isoDGRy) was prepared in high specific activity (∼600 Ci/mmol) according to the literature.37 Cells were seeded in 96-well multiscreen filter plates (1×105 cells/well) and incubated with 9 KBq of 125I-c(phg-isoDGRy)

in various concentrations of competitive inhibitors (2PisoDGR2

and isoDGR) for 2 h at 4 °C. After several washes, the filters were collected and measured in a γ-counter (Wizard 2470, Perkin Elmer Inc.). The IC50 values were determined by fitting displacement binding inhibition values using non-linear regression via Graph Pad Prism 6.0™. Experiments were carried out twice with triplicate samples. Radiochemistry. HYNIC-PisoDGR (10 μg, 0.88 nmol), HYNIC-3PisoDGR2 (20 μg, 0.85 nmol), and HYNIC-AB-3PsioDGR2 (25 μg, 0.91 nmol) were added in 2.0 mg tricine, 3.0 mg Tris(3-sulfonatophenyl) phosphine sodium salt (TPPTS) and 400 μL succinate buffer (250 mM, pH = 4.5), respectively. The mixed formula was then lyophilized in a 10 mL vial. Approximately 740 MBq of Na99mTcO4 solution (500 ~ 1000 μL) were added into the ready to label formulated kits. The vials were heated at 99 °C for 20 min. The radiochemical purity (RCP) of the tracers were determined by radio-HPLC, elaborating in the supporting information. The in vitro placement and reduction stability of them was measured in phosphate saline buffer and cysteine solution (1 mg/mL). The sample was subjected to radio-HPLC for RCP determination at 0, 2 and 6 h post placement. Partition Coefficient (Log P). The radiotracers were prepared and purified by



Page 22 of 27

Sep-Pak-C18 cartridges to thoroughly remove free Na99mTcO4. The elution was then removed under nitrogen stream, the concentrate was dissolved in an equal volume (3 mL:3 mL) mixture of n-octanol and 25 mM phosphate buffer (pH = 7.4). After stirring vigorously for 2 h at room temperature, the mixture was centrifuged at 10,000 rpm for 10 min. Samples (in triplets) in both octanol and aqueous layers were pipetted out and measured in γ-counter. Experiments were carried out twice with triplicate samples. Metabolic stability. The metabolic stability was evaluated in normal KM mice. Mice were administered with 37 MBq of 99mTc-AB-3PisoDGR

2

99mTc-PisoDGR,

99mTc-3PisoDGR

2

or

through tail vein. The urine samples were collected at 1 h p.i.

and centrifuged at 10,000 rpm for 10 min. The supernatant was analyzed by radio-HPLC. The HPLC chromatograms were compared with the initial results at 0 h post placement to find whether any new radio-metabolite forms. Blood Clearance. Female KM mice were randomly divided into three groups (n = 5). Mice of each group were intravenously injected with 0.74 MBq of the radiotracers, respectively. The Blood samples were harvested through canthus vein at 1 to 600 min p.i., weighed and counted via a γ-counter. The results were given as percentage per injected dose per gram (%ID/g) and analyzed by nonlinear regression of two phase decay by Graph Pad Prism 6.0™. Biodistribution. The mice bearing U87MG xenograft models were randomly divided into 11 groups (n = 4). The one optional group of mice were administrated with 0.74 MBq of 99mTc-PisoDGR and sacrificed at 0.5 h p.i. The three optional groups of mice were administered with 0.74 MBq of 99mTc-3PisoDGR2 and sacrificed at 0.5, 1 and 2 h p.i. The five optional groups of mice were administrated with 1.11 MBq of 99mTc-AB-3PisoDGR

2

and sacrificed at 0.5, 2, 4, 8 and 12 h p.i. The two optional

group of mice were administrated with 0.74 MBq of 99mTc-AB-3PisoDGR

2

99mTc-3PisoDGR

2

and

by co-injecting with 500 μg c(phg-isoDGRk) as the blocking

agent, and sacrificed at 0.5 h p.i., respectively. Blood, organs and tissues were harvested, weighed, and measured via a γ-counter. The tissue and organ uptake were calculated as %ID/g. The T/NT ratios were calculated as the %ID/g of tumor divided


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the those of normal tissues. SPECT/CT Imaging. SPECT/CT imaging was using small-animal SPECT/CT imaging system (Mediso Inc.). Each mouse bearing U87MG tumors was injected with ~37 MBq of any radiotracer. The mice were imaged at 0.5 h p.i. by injecting isoDGR tracers. The mice were imaged at 0.5, 1 and 2 h p.i. by injecting

99mTc-3PisoDGR

2.

And mice were imaged at 0.5, 1, 2, 4, 8 and 12 h p.i. by injecting 99mTc-AB-isoDGR2. The binding specificity of

99mTc-3PisoDGR

2

and

99mTc-AB-3PisoDGR

2

was

demonstrated by the blocking experiments co-injecting with excess c(phg-isoDGRk) peptide (~500 μg), which were imaged at 0.5 h p.i. The raw SPECT data was reconstructed in a whole-body region (99mTc-3PisoDGR2 and

99mTc-AB-3PisoDGR

2)

or a sectional region excluded the bladder (99mTc-PisoDGR). The SPECT and CT images were then fused by the Nucline v2.01 (Mediso Inc.). The maximum intensity projection (MIP) was given for whole-body imaging by posterior view. Orthotropic Glioma. The orthotropic brain tumor model was established by intracranial injection of ∼105 U87MG tumor cells into the frontal white matter of nude mice (4 weeks of age). Mice with brain carcinoma in situ were able to be used for SPECT imaging at 10∼14 days post-inoculation. The SPECT/CT imaging of 99mTc-3PisoDGR

2

and 99mTc-AB-3PisoDGR2 was performed at 1 h p.i.

SUPPORTING INFORMATION The Supporting Information is available free of charge on the ACS Publications website. Method of albumin binding and blood-cerebrospinal fluid (BCSF) barrier assay, Figures S1-S8 and Table S1 are provided in the Supporting Information. ACKNOWLEDGMENTS This research was supported by National Natural Science Foundation of China (NSFC) projects (81630045, 81571727, 81420108019, 81621063, 81427802), the National Key R&D Program of China (2017YFA0205600), and Strategic Priority Research Program of the Chinese Academy of Sciences (XDA12020110), the Youth Innovation



Promotion Association of Chinese Academy of Sciences (YIPACAS) project (2016090), and the Beijing Natural Science Foundation (BJNSF) project (7142086).


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Improved in vivo Targeting Capability and Pharmacokinetics of 99mTc

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