Dejian Huang

A Dual-targeting Anticancer Approach: Soil and Seed Principle

PURPOSE To test the hypothesis that targeting the microenvironment (soil) may effectively kill cancer cells (seeds) through a small-molecular weight sequential dual-targeting theragnostic strategy, or dual-targeting approach. MATERIALS AND METHODS With approval from the institutional animal care and use committee, 24 rats were implanted with 48 liver rhabdomyosarcomas (R1). First, the vascular-disrupting agent combretastatin A4 phosphate (CA4P) was injected at a dose of 10 mg/kg to cause tumor necrosis, which became a secondary target. Then, the necrosis-avid agent hypericin was radiolabeled with iodine 131 to form (131)I-hypericin, which was injected at 300 MBq/kg 24 hours after injection of CA4P. Both molecules have small molecular weight, are naturally or synthetically derivable, are intravenously injectable, and are of unique targetablities. The tumor response in the dual-targeting group was compared with that in vehicle-control and single-targeting (CA4P or (131)I-hypericin) groups with in vivo magnetic resonance imaging and scintigrams and ex vivo gamma counting, autoradiography, and histologic analysis. Tumor volumes, tumor doubling time (TDT), and radiobiodistribution were analyzed with statistical software. P values below .05 were considered to indicate a significant difference. RESULTS Eight days after treatment, the tumor volume of rhabdomyosarcoma in the vehicle-control group was double that in both single-targeting groups (P < .001) and was five times that in the dual-targeting group (P < .0001), without treatment-related animal death. The TDT was significantly longer in the dual-targeting group (P < .0001). Necrosis appeared as hot spots on scintigrams, corresponding to 3.13% of the injected dose of (131)I-hypericin per gram of tissue (interquartile range, 2.92%-3.97%) and a target-to-liver ratio of 20. The dose was estimated to be 100 times the cumulative dose of 50 Gy needed for radiotherapeutic response. Thus, accumulated (131)I-hypericin from CA4P-induced necrosis killed residual cancer cells with ionizing radiation and inhibited tumor regrowth. CONCLUSION This dual-targeting approach may be a simple and workable solution for cancer treatment and deserves further exploitation.

Evaluation of hypericin: effect of aggregation on targeting biodistribution.

Hypericin (Hy) has shown great promise as a necrosis-avid agent in cancer imaging and therapy. Given the highly hydrophobic and π-conjugated planarity characteristics, Hy tends to form aggregates. To investigate the effect of aggregation on targeting biodistribution, nonaggregated formulation (Non-Ag), aggregated formulation with overconcentrated Hy in dimethyl sulfoxide (Ag-DMSO) solution, and aggregated formulation in water solution (Ag-water) were selected by fluorescence measurement. They were labeled with ¹³¹I and evaluated for the necrosis affinity in rat model of reperfused hepatic infarction by gamma counting and autoradiography. The radioactivity ratio of necrotic liver/normal liver was 17.1, 7.9, and 6.4 for Non-Ag, Ag-DMSO, and Ag-water, respectively. The accumulation of two aggregated formulations (Ag-DMSO and Ag-water) in organs of mononuclear phagocyte system (MPS) was 2.62 ± 0.22 and 3.96 ± 0.30 %ID/g in the lung, and 1.44 ± 0.29 and 1.51 ± 0.23 %ID/g in the spleen, respectively. The biodistribution detected by autoradiography showed the same trend as by gamma counting. In conclusion, the Non-Ag showed better targeting biodistribution and less accumulation in MPS organs than aggregated formulations of Hy. The two aggregated formulations showed significantly lower and higher accumulation in targeting organ and MPS organs, respectively.

Necrosis affinity evaluation of 131I-hypericin in a rat model of induced necrosis

Abstract Cancers are often with spontaneous or therapeutic necrosis that could be utilized as a generic target for developing new treatments. The purpose of this study was to investigate the biodistribution and pharmacokinetics of radioiodinated hypericin (Hyp), a naturally occurring compound, after intravenous (i.v.) injection in a rat model of liver and muscle necrosis (n = 42), and evaluate its necrosis affinity. Hyp was labeled with 131I with labeling efficiency >99%. After incubating in solution/rat plasma for 8 days, radiochemical purity of 131I-Hyp remained 98.1 and 97.1%, respectively, indicating good in vitro stability. SPECT-CT images at 24 h after i.v. injection of 131I-Hyp in rats with induced liver and muscle necrosis showed obvious tracer absorption in necrotic tissues. Biodistribution studies revealed that the percentage of the injected dose per gram of tissue (%ID/g) evolved from 1.9 %ID/g at 6 h, through a maximum 3.0 %ID/g at 12 h, to 1.0 %ID/g at 192 h in necrotic liver. Pharmacokinetics studies revealed that the terminal elimination half-life, total body clearance and area under the curve of 131I-Hyp were 32.7 h, 9.2 L/h/kg and 1.6 MBq/L*h, respectively. These results demonstrated that 131I-Hyp features a long blood circulation in animals and persistent retention in necrotic tissues. Therefore, 131I-labeled Hyp could be a broad-spectrum anti-tumor agent with a cost much cheaper relative to the biological agents such as monoclonal antibodies.

Radiopharmaceutical evaluation of 131I-protohypericin as a necrosis avid compound

Abstract Hypericin is a necrosis avid agent useful for nuclear imaging and tumor therapy. Protohypericin, with a similar structure to hypericin except poorer planarity, is the precursor of hypericin. In this study, we aimed to investigate the impact of this structural difference on self-assembly, and evaluate the necrosis affinity and metabolism in the rat model of reperfused hepatic infarction. Protohypericin appeared less aggregative in solution compared with hypericin by fluorescence analysis. Biodistribution data of 131I-protohypericin showed the percentage of injected dose per gram of tissues (%ID/g) increased with time and reached to the maximum of 7.03 at 24 h in necrotic liver by gamma counting. The maximum ratio of target/non-target tissues was 11.7-fold in necrotic liver at 72 h. Pharmacokinetic parameters revealed that the half-life of 131I-protohypericin was 14.9 h, enabling a long blood circulation and constant retention in necrotic regions. SPECT-CT, autoradiography, and histological staining showed high uptake of 131I-protohypericin in necrotic tissues. These results suggest that 131I-protohypericin is a promising necrosis avid compound with a weaker aggregation tendency compared with hypericin and it may have a broad application in imaging and oncotherapy.

Trapping effect on a small molecular drug with vascular-disrupting agent CA4P in rodent H22 hepatic tumor model: in vivo magnetic resonance imaging and postmortem inductively coupled plasma atomic emission spectroscopy

Abstract The aim of the present study is to verify the trapping effect of combretastatin A-4-phosphate (CA4P) on small molecular drugs in rodent tumors. Mice with H22 hepatocarcinoma were randomized into groups A and B. Magnetic resonance imaging (MRI) of T1WI, T2WI, and DWI was performed as baseline. Mice in group A were injected with Gd-DTPA and PBS. Mice in group B were injected with Gd-DTPA and CA4P. All mice undergo CE-T1WI at 0 h, 3 h, 6 h, 12 h, and 24 h. Enhancing efficacy of the two groups on CE-T1WI was compared with the signal-to-noise ratio (SNR) calculated. Concentrations of gadolinium measured by ICP-AES in the tumor were compared between groups. On the early CE-T1WI, tumors were equally enhanced in both groups. On the delayed CE-T1WI, the enhancing effect of group A was weaker than that of group B. The SNR and the concentration of gadolinium within the tumor of group A were lower than that of group B at 6 h, 12 h, and 24 h after administration. This study indicates that CA4P could improve the retention of Gd-DTPA in the tumor and MRI allowed dynamically monitoring trapping effects of CA4P on local retention of Gd-DTPA as a small molecular drug.

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

Abstract 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.

Tumor necrosis targeted radiotherapy of non-small cell lung cancer using radioiodinated protohypericin in a mouse model

Lung cancer is the leading cause of cancer-related death. About 80% of lung cancers are non–small cell lung cancers (NSCLC). Radiotherapy is widely used in treatment of NSCLC. However, the outcome of NSCLC remains unsatisfactory. In this study, a vascular disrupting agent (VDA) combretastatin-A4-phosphate (CA4P) was used to provide massive necrosis targets. 131I labeled necrosis-avid agent protohypericin (131I-prohy) was explored for therapy of NSCLC using tumor necrosis targeted radiotherapy (TNTR). Gamma counting, autoradiography, fluorescence microscopy and histopathology were used for biodistribution analysis. Magnetic resonance imaging (MRI) was used to monitor tumor volume, ratios of necrosis and tumor doubling time (DT). The biodistribution data revealed 131I-prohy was delivered efficiently to tumors. Tracer uptake peaked at 24 h in necrotic tumor of 131I-prohy with and without combined CA4P (3.87 ± 0.38 and 2.96 ± 0.34%ID/g). 131I-prohy + CA4P enhanced the uptake of 131I-prohy in necrotic tumor compared to 131I-prohy alone. The TNTR combined with CA4P prolonged survival of tumor bearing mice relative to vehicle control group, CA4P control group and 131I-prohy control group with median survival of 35, 20, 22 and 27 days respectively. In conclusion, TNTR appeared to be effective for the treatment of NSCLC.

Experimental evaluation of radioiodinated sennoside B as a necrosis-avid tracer agent.

Abstract Necrosis-avid agents are a class of compounds that selectively accumulate in the necrotic tissues after systemic administration, which can be used for in vivo necrosis imaging and targeted therapies. In order to search for a necrosis-avid tracer agent with improved drugability, we labelled iodine-131 on sennoside B (SB) as a naturally occurring median dianthrone compound. The necrosis targetability and clearance properties of 131I-SB were evaluated in model rats with liver and muscle necrosis. On SPECT/CT images, a “hot spot” in the infarcted liver lobe and necrotic muscle was persistently observed at 24 h and 72 h post-injection (p.i.). Gamma counting of the tissues of interest revealed a radioactivity ratio of necrotic to viable liver at 4.6 and 3.4 and of necrotic to viable muscle at 7.0 and 8.8 at 24 h and 72 h p.i., respectively. The good match of autoradiographs and fluoromicroscopic images with corresponding histochemical staining suggested preferential uptake of 131I-SB in necrotic tissue. Pharmacokinetic study revealed that 131I-SB has an elimination half-life of 8.6 h. This study indicates that 131I-SB shows not only prominent necrosis avidity but also favourable pharmacokinetics, which may serve as a potential necrosis-avid diagnostic agent for assessment of tissue viability.

Biodistribution and anti-tumor efficacy of intratumorally injected necrosis-avid theranostic agent radioiodinated hypericin in rodent tumor models

Abstract Hypericin has an excellent necrosis-specific targeting capacity; thus, we explored small-molecular tumor necrosis therapy (SMTNT) for inhibiting tumor growth in rodent tumor models. H22 and S180 tumor-bearing Kunming (KM) mice were intratumorally injected with 131I-monoiodohypericin (131I-MIH) to investigate the biodistribution of 131I-MIH as a function of time. Single-photon emission computed tomography (SPECT), autoradiography, fluorescence microscopy and hematoxylin and eosin (H&E) staining were performed to determine the intra-tumoral distribution of 131I-MIH. A therapeutic evaluation study was also performed in the tumor-bearing KM mice using saline and a positive drug as controls. Gamma counting, SPECT images, autoradiography and fluorescence microscopy and H&E staining results revealed intense retention of 131I-MIH in the necrotic tumor over 168 h and good in vivo stability of the agent. Therapy with a single dose of intra-tumoral administration of 131I-MIH caused significant tumor growth delay. A histopathological analysis of the tumors and normal organs further validated the therapeutic efficacy and limited systemic toxicity of 131I-MIH. The prolonged tumor retention and effective therapy indicated that 131I-MIH may be a promising intratumorally injected SMTNT agent.

Combretastatin-A4 phosphate improves the distribution and antitumor efficacy of albumin-bound paclitaxel in W256 breast carcinoma model

Nanomedicine holds great promise for fighting against malignant tumors. However, tumor elevated interstitial fluid pressure (IFP) seriously hinders convective transvascular and interstitial transport of nanomedicines and thus damages its antitumor efficacy. In this study, combretastatin-A4 phosphate (CA4P) was utilized to reduce tumor IFP, and thereby to improve the intratumoral distribution and antitumor efficacy of nanoparticle albumin-bound paclitaxel (nab-paclitaxel). IFP was measured using the wick-in-needle method in tumors growing subcutaneously pretreatment and posttreatment with a single intravenous injection of CA4P. The tracing method of iodine 131 isotope was used for biodistribution analysis of nab-paclitaxel. Liquid chromatography coupled with tandem mass spectrometry was used to detect the intratumoral concentration of paclitaxel. Magnetic resonance imaging was applied to monitor tumor volume and ratios of necrosis. The tumor IFP continued to decline gradually over time following CA4P treatment, reaching approximately 31% of the pretreatment value by 1 h posttreatment. Biodistribution data indicated that both 131I-nab-paclitaxel and paclitaxel exhibited higher tumor uptake in CA4P + 131I-nab-paclitaxel group compared with I131-nab-paclitaxel group. Nab-paclitaxel combined with CA4Pshowed significant tumor growth inhibition and higher tumor necrosis ratio relative to PBS, CA4P and nab-paclitaxel group, respectively. In conclusion, CA4P improved the intratumoral distribution and antitumor efficacy of nab-paclitaxel in W256 tumor-bearing rats.