Evaluation of necrosis avidity of radioiodinated 5-hydroxytryptophan and its potential applications in myocardial infarction imaging

Lin M,Lingqio Ci,Qiomi Jin,Jiji Ling,Dongjin Zhng,Wi Liu,Yichng Ni,Zhiqi Yin,Jin Zhng,*,K Pn,**

a Department of Natural Medicinal Chemistry&Department of TCMs Pharmaceuticals,School of Traditional Chinese Pharmacy,China Pharmaceutical University,Nanjing 10009,China

b Af fl iated Hospital of Integrated Traditional Chinese and Western Medicine,Nanjing University of Chinese Medicine,Nanjing 10028,China

c Laboratories of Translational Medicine,Jiangsu Province Academy of Traditional Chinese Medicine,Nanjing 210028,China

d College of Pharmacy,Nanjing University of Chinese Medicine,Nanjing 210023,China

e Department of Nuclear Medicine,The First Af fi liated Hospital of Nanjing Medical University,Nanjing 210029,China

[38_TD DIFF]f Theragnostic Laboratory,Campus Gasthuisberg,KU Leuven,3000 Leuven,Belgium

Key words:Radioiodinated 5-hydroxytryptophan Necrosis avidity DNA Groove binding Necrotic myocardium imaging

ABSTRACT This study aimed to seek necrosis avid agents with high safety from DNA binding agents.The interaction of 5-hydroxytryptophan(5-HTP)with DNA[41_TD DIFF]wasinvestigated by a series of spectroscopic studies.Then,5-HTPwaslabeled with iodine-131([131I]5-HTP)and the necrosisavidity of[131I]5-HTPwasevaluated by in vitro cell binding assays,in vivo biodistribution experiments and blocking experiment.Finally,the potential of[131I]5-HTP to image necrotic myocardium was explored in rat models with myocardial infarction by SPECT/CTimaging.Results showed that 5-HTPbound to DNA in groove binding mode and the binding constant was 1.69[42_TD DIFF]?104 L/mol.[131I]5-HTP showed specific af fi nity to necrotic A549 cells,which might be related to cell nucleus.Biodistribution and autoradiography results showed preferential accumulation of[131I]5-HTP in necrotic muscle(necrotic/viable muscle ratio:6.95?0.68 at 3 h postinjection(p.i.)),which could be blocked by 5-HTPwith 66.7%decline,indicating that[131I]5-HTPmight share the same necrotic targetswith 5-HTP.On SPECT/CTimages,a hotspot wasclearly observed at 3h p.i.in the necrotic myocardium while not in the control myocardium.In conclusion,[131I]5-HTP is a novel necrosis avid agent and can rapidly visualize necrotic myocardium at 3h p.i.The necrosis avidity mechanism of[131I]5-HTPmay be attributed to its interactions with exposed DNA in the necrotic tissues.?2018 Chinese Chemical Society and Institute of Materia Medica,Chinese Academy of Medical Sciences.

Necrosis is described as the passive death of cellsthat subjected to illness or serious injury[1].As a form of cell death,necrosis is w idespread in various diseases,such as cancer,myocardial infarction(MI),neurodegenerative diseases,and stroke,etc.[2].Necrosis avid agents(NAAs)can specifically accumulate in the necrotic area of tissues and show promising practical prospect in many fields,such as the diagnosis and therapy of necrosis-related diseases,as well as the evaluation of therapeutic ef fi cacy[3,4].

Previous studies have revealed that hypericin (Hyp),a naphthodianthrone compound,can specifically accumulate in necrotic tissues[5].[123I]Hyp could visualize myocardial infarcts with small animal SPECT(single-photon emission computed tomography)imaging[6].However,the self-aggregate property and high blood pool activity of Hyp have limited its clinical applications[6–8].Recently,we found that monomeric anthraquinones(AQs)also exhibited peculiar af fi nity to necrotic tissues by splitting the chemical structure of Hyp[8].Among them,rhein emerged as the most promising compound for imaging necrotic tissues due to its optimal pharmacokinetics properties and had been labeled with iodine-131 and Tc-99m to visualize the necrotic myocardium[8,9].However,the hepatotoxicity and poor solubility of rhein limit to some extent the clinical applications of rheinbased NAAs[10].Therefore,there is still a pressing need to seek NAAs with high safety and suitability for clinical applications.

It was reported that exposed DNA(E-DNA)is one of the most classical molecular targets for NAAs ow ning to its abundance and specificity in necrotic areas[11].Recently,a seriesof NAAs targeted to E-DNA have been reported,such as Hoechst-IR[11],Hoechst-Gemcitabine[12],and Gd-TO[13].Besides,our previous studies also found that the necrosis avidity of Hyp derivatives and AQs might be related to the intercalations with E-DNA[9,14,15].Hence,it might be practicable to develop potential NAAs from DNA binding agents.

With this in mind,5-hydroxytryptophan(5-HTP),a kind of natural amino acids belonging to indole compounds,drew our attentions.Firstly,indole derivatives can interact with DNA.Both ellipticine and bis-indole alkaloid fascaplysin are reported as DNA intercalators[16].Secondly,bis-indole derivative,ECIV-7,has been investigated to showextraordinary necrosisavidity[17].Therefore,we speculated that 5-HTPmight also feature necrosis avidity as a DNA binding agent.More importantly,5-HTP is a kind of endogenous substances and has been used as over-the-counter drug or supplements with high safety[18].

UV-vis absorption spectroscopy is one of the most effective techniques to investigate the interactions of small molecules with DNA.Generally,the absorbance and position of the peak show changes w hen small molecules interact with DNA.Hypochromic effect and red shift are associated with the intercalative interactions.For groove binding mode,hypochromic effect can be observed but the position of the absorption band changes hardly.However,hyperchromic effect can be caused w hen electrostatic attractions take place[19].With the increasing concentration of calf thymus DNA(CTDNA),the absorption spectra of 5-HTP exhibited an apparent hyperchromism and blue shift(Fig.1a).However,the CTDNAband at 260[46_TD DIFF]nm obviously interfered with the 5-HTPabsorption band at 275nm.Therefore,there might be errors in the calculation of binding constant and binding mode via UV–vis absorption spectroscopy,and further experiments were required to understand the interactions between 5-HTPand DNA.

Fluorescence experiments were performed to clarify the interaction and binding constant of 5-HTP with CT DNA.The fluorescence intensity of small molecule reduces w hen interacts with DNA.Such a reduction is called fluorescence quenching.Stern-Volmer equation is used to assess the fluorescence quenching ef fi ciency:F0/F=1+KSV[Q](1)w here F0and F are the fluorescence intensities in the absence and presence of the quencher respectively.KSVis the quenching constant and[Q]is the concentration of the quencher[19].Besides,the fluorescence quenching could be classi fied as dynamic quenching and static quenching[20].Dynamic quenching depends upon molecule diffusion.Higher temperatures w ill increase the diffusion coef ficient and result in a larger KSVvalues,but lower the KSVvalues for static quenching due to the decrease of complexes stability[20].Equation Kq=KSV/t0(2)was used to investigate the quenching process of small molecules and DNA,w here Kqis the bimolecular quenching rate constant and t0is the fluorescence lifetime of the biomolecule in the absence of quencher(t0[49_TD DIFF]=10?8s)[19].

The fluorescence intensity of 5-HTP decreased gradually with the addition of CT DNA at 298[50_TD DIFF]K in Fig.1b,suggesting that DNA interacted with 5-HTPand quenched the intrinsic fluorescence of 5-HTP.The KSVvalues calculated from the Stern-Volmer plots(Fig.1c)showed a tiny change from 7.42[51_TD DIFF]?103to 7.45?103L/mol at different temperatures(290,298 and 310 K)and were listed in Table S1(Supporting information).The Kqvalues were found to be in the range of 7.42?1011-7.45?1011L mol?1s-1at different temperatures(290,298 and 310[54_TD DIFF]K),higher than the limiting diffusion rate constant of biomolecules(2.0?1010).These results indicated that the fluorescence quenching process was initiated by static quenching.

Equation(3)can describe the relationship between fluorescence intensity and concentration of the quencher in the process of static quenching[20].Log (F0-F)/F]=log[57_TD DIFF]K+n log[Q](3)w here K is the binding constant,n is the number of binding sites and Q is the concentration of the quencher.From the plot of log(F0-F)/Fversus log[CTDNA]in Fig.1d,the K value of 5-HTPand DNAwasestimated to be 1.69[58_TD DIFF]?104L/mol at 298 K.

Competitive displacement assays,iodide quenching studies,comparison interactions of 5-HTPwith double-stranded DNA and single-stranded DNA,circular dichroism studies and viscosity measurements were performed to further elucidate the binding mode of 5-HTPon CTDNA.All the results(Supporting information)indicated that 5-HTPbound to CTDNAin the groove binding mode.

The radiochemical purity of[131I]5-HTP measured by radio-HPLC was 99.53%(Fig.S3a in Supporting infromation).92.47%of the intact tracer remained in the rat serum at 12[59_TD DIFF]h(Fig.S3b in Supporting infromation),indicating good stability of[131I]5-HTP in vitro.

As show n in Fig.2a,necrotic A549 cells(with cell nucleus)exhibited 2.43-fold increase in the uptake of[131I]5-HTPcompared with viable A549 cells.It revealed that[131I]5-HTP features necrosis avidity in vitro.However,the uptake of[131I]5-HTP in necrotic red blood cells(without cell nucleus)showed no signi fi cant difference with that in viable red blood cells(P>0.05),but was obviously lower than that in necrotic A549 cells(P<0.01).These results indicated that the high accumulation of[131I]5-HTP in necrotic A549 cells might be related to cell nucleus.

The biologic distribution results in mice model of muscular necrosis are show n in Table 1.At each time point,the uptake of[131I]5-HTPin necrotic muscle was significantly higher than that in normal muscle(P<0.01).The radioactivity ratios of necrotic and viable muscles increased from 3.59[60_TD DIFF]?0.47 at 1 h to 6.95?0.68 at 3 h.Lower uptake of[131I]5-HTP was detected in normal organs/tissues such as viable muscle,heart and brain,except for kidney,bladder,and stomach at each time point.Besides,the biodistribution data displayed an obvious clearance of radioactivity from normal organs within 3h except for the kidneys and bladder,indicating that[131I]5-HTP might prefer to metabolize in a renal metabolistic w ay.

Fig.2b represents TTCstaining and autoradiography outcomes.Higher tracer signals in the autoradiographs primarily appeared in necrotic regions of muscle,which were consistent with the data from biodistribution studies.These results also suggested that[131I]5-HTP could specifically localize in necrotic tissue.

Fig.1.(a)UV–vis absorption spectra of 5-HTPwith CTDNA.(b)Fluorescence emission spectra of 5-HTPwith CTDNA at 298K.(c)Stern-Volmer plot for interactions of 5-HTP with CTDNAat 290,298,and 310K.(d)Stern-Volmer plot of log[(F0-F)/F]versus log[13_TD DIFF]CTDNA]at 298 K to calculate the binding constants.Binding constant was obtained from the Y-intercept.

Fig.2.(a)Uptake of[131I]5-HTP in A549 cells and red blood cells.Data were displayed as average percentage uptake per 105 cells plus or minus standard deviation(%uptake/105 cells?SD).*P<0.01 compared with necrotic A549 cells.?P>0.05 compared with necrotic red blood cells.(b)TTC images and corresponding autoradiographs(aut)of partially necrotic muscle sections from muscular necrosis model mice at 1 h and 3 h after administration with[131I]5-HTP.(c)Uptake of[131I]5-HTPin unblocked and 20 mg/kg 5-HTPblocked(5-HTP-blocked)necrotic and viable muscle.Data are presented as average percentage of injected dose per gram(%ID/g)?SDat 3 h after coinjection.Necrotic M=necrotic muscle;Viable M=viable muscle.*P<0.01 compared with unblocked[66_TD DIFF]Necrotic M group.?P>0.05 compared with unblocked[67_TD DIFF]Viable M group.(d)TTC images and corresponding aut of partially necrotic muscle sections from unblocked and 5-HTP-blocked groups.

Fig.3.(a)SPECT/CTimages of control rats and rat models with MIat 3h p.i.of[131I]5-HTP.(b)Biodistribution data after SPECT/CTimaging.Data are%ID/g,expressed as mean?SD(n=5).Viable M=viable myocardium;Necrotic M[23_TD DIFF]=necrotic myocardium.*P<0.01 compared with necrotic myocardium.**P<0.05 compared with necrotic myocardium.(c)Aut,corresponding H&Estaining and micrograph(Micro)of 10[24_TD DIFF]m m heart slices from rat models.N[25_TD DIFF]=necrotic area,V=viable area.Scale bar=200 m m.

Table 1 Biodistribution data of[131I]5-HTPin muscular necrosis models.

Compared with the unblocked group,about 66.7%of the radioactivity in the necrotic muscle was blocked by unlabeled 5-HTP(Fig.2c).However,the uptakes of[131I]5-HTP in the viable muscles had no signi fi cant differences between the unblocked and 5-HTP-blocked groups(P>0.05).Results were consistent with the images of autoradiography(Fig.2d).These results indicated that[131I]5-HTPmay share the same specific targets with 5-HTP in vivo.Considering that the higher accumulation of[131I]5-HTPin necrotic A549 cells might relate to cell nucleus and that 5-HTPbinds to DNA in groove binding mode,we speculate that the necrosis avidity mechanism of[131I]5-HTPmight be attributable to its interactions with E-DNA in necrotic cells.

Fig.3a shows the representative SPECT/CT images of control and MI model rats at 3h post-injection (p.i.).Hotspot was visualized in the heart of rats with MI,while no obvious uptake was observed in the control rats,indicating that[131I]5-HTPcould specifically accumulate in necrotic myocardium at 3h p.i.

The biodistribution data after SPECT/CTimaging are illustrated in Fig.3b.The radioactivity ratio of necrotic and viable myocardium was 4.87.Besides,the uptake of[131I]5-HTPin blood and lungs was less than that in necrotic myocardium significantly(P<0.05).These results suggested that[131I]5-HTP may be a promising imaging probe of the necrotic myocardium.Prominent activity was detected in the kidney and liver,consistent with the SPECT/CT imaging.H&Estaining and autoradiography of heart slices from rat models with MI are displayed in Fig.3c.Higher tracer signals in autoradiographs primarily matched well with the pink parts in H&E staining.The result indicated that[131I]5-HTP could speci fically localize in necrotic myocardium and evaluate myocardial viability.

In the present study,[131I]5-HTP(3[61_TD DIFF]h p.i.)enabled the rapid imaging of necrotic myocardium compared with[123I]Hyp(9[62_TD DIFF]h p.i.)[6]and[131I]Rhein(6[63_TD DIFF]h p.i.)[8].Meanwhile,[131I]5-HTPimproved the necrotic myocardium-to-blood ratio(from 0.96 for[131I]Rhein to 1.99 for[131I]5-HTP),which greatly influences the image quality.It may be attributed to the better water solubility of[131I]5-HTP,which w ould result to the rapid distribution in necrotic myocardium and fast clearance from the circulation.Therefore,[131I]5-HTP may be more bene fi cial and potential for the early imaging and diagnosis of necrosis related diseases with high quality of image.

As a kind of endogenous substance,5-HTP show s high safety and has been used as dietary supplements or neurotransmitters in clinic for over 30 years[18].Moreover,the small dose of[131I]5-HTP(about 0.2[64_TD DIFF]10F]mg/kg of 5-HTP)for SPECT/CT imaging in this study is much less than the administration dosage of 5-HTP in clinic(?3.3 mg10F]kg?1day?1)[18].All the above facts indicate that[131I]5-HTPis a promising necrosis avid agent with high safety.Especially,the predominant necrosis avidity and necrotic myocardium imaging property of[131I]5-HTP were evaluated for the fi rst time in the present paper.These results supplement the literature data on extending the applications of[131I]5-HTP in necrosis related pathologies.

Additionally,most of the NAAs are dark in color due to their big conjugate planar structures[3].The unchangeable natures have largely limited their further commercial development.By contrast,colorless 5-HTPcan be easily accepted by patients compared with the dark NAAs.

In conclusions,[131I]5-HTP is a promising necrosis avid agent with the advantages of high safety,good solubility and colorlessness.Furthermore,this study provides an effective w ay to develop novel NAAs from DNA binding agents.

Acknow ledgments

This work was partially supported by the National Natural Science Foundation of China(Nos.81473120,81501536,81473120).We appreciate Mr.Changwen Fu for his kindly work in SPECT/CT scanning.

Appendix A.Supplem entary data

Supplementary material related to this article can be found,in the online version, at doi:https://doi.org/10.1016/j.cclet.2018.06.005.