Researches On The Anthraquinones-based Necrosis Avid MRI Contrast Agent

Early and accurate assessment of therapeutic response to anticancer therapy plays an important role in determining the treatment strategy and keeping patients from suffering unnecessary side effects and curative costs.The imaging of tissue necrosis provides a new way to solve this problem.Anthraquinones have been discovered as a class of necrosis avid agents（NAAs）.Among of them,rhein showed prominent targetability and better pharmacokinetic properties,which can serve as a good carrier for imaging necrotic tissue.In this study,we synthesize three Magnetic resonance imaging（MRI）contrast agents with three different length linkers based rhein and evaluate their necrosis targetability.We also preliminarily evaluate the treatment efficacy of combretastatin A-4 phosphate（CA4P）on rats bearing W256 breast carcinoma by using a novel necrosis-avid contrast agent.Ligands L₁、L₂ and L₃ were synthesized by two steps of acylation and deprotection reaction.The paramagnetic contrast agents GdL₁、GdL₂ and GdL₃ were obtained by coordination of Gd3+with the synthesized ligand respectively.Relaxivities,in vitro stabilities and water-solubility of those contrast agents were studied.The results showed that the relaxivities of the three contrast agents were 7.28、7.35 and 8.03 respectively,which were all higher than Gd-DOTA of 4.28.The contrast agents were incubated with the plasma for 24 h and maintained high integrity.The LogP of those agents were-2.15 ± 0.01、-1.32 ± 0.01 and-0.71 ± 0.01,respectively.It can be deduced that the hydrophobicity improves with the increase in the number of methylene and GdL₁ has the best water solubility.In virtue of a large number of DNA were exposure after cell necrosis and anthraquinones were well-known intercalators of DNA,we speculated that anthraquinone-based necrosis-avid compounds may bind to exposed DNA for targeting necrotic tissues.In this study,the binding ability of GdL₁ and DNA was investigated by spectroscopic and relaxation rate measurements.Binding mode and bonding strength of anthraquinones were determined by measuring the EB-DNA quenching constants of rhein,Ln and GdLn（n = 1,2,3）.Quenching constants of those seven compounds were 1.01,1.36,1.32,1.40,2.28,2.19 and 2.30 × 104 moL-1,respectively.It suggested that the seven compounds can bind to DNA in intercalative mode.Moreover,Gd3+ has positive influence on the intercalation between anthraquinones and DNA.From the results above,GdL₁ was chosen for the following research.The interaction of GdL₁ with DNA was simultaneously determined by relaxometry with the addition of DNA（from 0 to 0.3 1mM）,R1 increased from 1.43 ± 0.02 to 1.78 ± 0.02（mM.sec）-1.These results indicated that GdL₁ could bind to DNA.In this study,the necrosis affinity of GdL₁ was evaluated on A549 that heated in 57℃ for 1 h and rat models of liver infarction and muscular necrosis.Firstly,hyperthermia-treated cells and non-treated cells were incubated at 37℃ for 30 min with the GdL₁,and then detected by MRI.The results showed that hyperthermia-treated cells performed hyperintense but non-treated cell performed relatively hypointense.The result indicates that GdL₁ can image necrosis specifically.Further,in vivo MRI was performed on rat models of liver infarction and muscular necrosis before administration of GdL₁ and during 0 h to 12 h after administration of GdL₁（0.1 mmol/kg）respectively,and Gd-DOTA was used as control.L₁ was then labeled by 64Cu with a radiochemical purity of 90%after purification.Biodistribution were performed with 64CuL₁ at 3,12,and 24 h p.i.The results from MRI on model rats showed that necrotic liver was significantly enhanced from 0.5 to 12 h postinjection of GdL₁.The contrast ratios of necrotic liver/normal liver were 2.09 ± 0.22 and 1.95 ± 0.15 at 3 h and 12 h postinjection of GdLj（0.1 mmol/kg）respectively,demonstrating a significant difference compared with before administration of GdL₁（1.29 ± 0.07;P<0.05）.The similiar results were obtained from necrotic muscles.The biodistribution results showed that necrotic to viable liver and muscle ratios were 6.85 ± 0.51 and 6.08 ±0.64 at 3 h postinjection.These findings together suggested that GdL₁ possessed the necrosis targeting and imaging capability.The last purpose in this study is to assess the CA4P therapy response on Walker 256 rats bearing W256 breast carcinoma with GdL₁.MR imaging studies were performed at 24 h after CA4P administration.After MRI scaning,tumor tissues were cut into 10 μm frozen section and then were stained with H&E.The results from MRI showed that the signal in necrotic center of tumor was obviously increased at 3 h after administration of GdL₁.The contrast ratios of necrotic center/periphery was 1.63±0.11,demonstrating a significant difference compared with before administration of GdL₁（0.98±0.04;P<0.05）.These findings revealed that GdL₁ possessed the potential for early assessment of treatment response to CA4P by MRI imaging of necrotic tissue.In conclusion,GdL₁ has good necrosis targetability and the potential of early assessment on CA4P therapeutic effect,serving as a promising necrosis-avid contrast agent.