Thermodynamics And Biodistribution Evaluation Of The 1,3-Diamino-2-hydroxypropane-N, N, N', N'-Tetraacetic Acid With Lanthanide Metals

Objective:Lanthanide radioisotopes ¹⁷⁷Lu,¹⁶⁶Ho,¹⁶¹Tb and ¹⁴⁹Tb are important medical nuclides,1,3-diamino-2-hydroxypropane-N,N,N',N'-tetraacetic acid?DHPTA?is a polyaminocarboxylic acid ligand.Studies have shown that DHPTA has a strong binding ability with lanthanides.We will explore the thermodynamic stability of DHPTA with lanthanides(Tb³⁺,Ho³⁺,Lu³⁺),and preliminary analysis whether DHPTA has the potential to be a lanthanide radioisotope chelator.At present,the coordination structure of Ln³⁺(Tb³⁺,Ho³⁺,Lu³⁺)/DHPTA complex is controversial.We aim to investigate the coordination structure of DHPTA with lanthanides(Tb³⁺,Ho³⁺and Lu³⁺)by spectrophotometry,luminescence spectroscopy,and nuclear magnetic resonance spectroscopy?NMR?to provide basic chemical theory for follow-up research.At last we will explore the the biological distribution characteristics of DHPTA/Lu³⁺complex.Methods:The protonation constants of DHPTA and the equilibrium constants of lanthanides(Tb³⁺,Ho³⁺and Lu³⁺)with DHPTA was mesured by potentiometric experiments.Solutions containing metals,DHPTA ligand,NaOH?or HCl?,and 0.5 mol·L^–11 NaCl were titrated by standard NaOH solution/HCl solution with known concentration.About 50 points were collected for each titration.Usually,three to five titrations were conducted by changing the amount of metals,DHPTA ligand,NaOH?or HCl?,and 0.5mol·L^–11 NaCl to minimize systematic errors and to check the repeatability of the measurements.The complex formation constants(log₁₀?)were calculated by the Hyperquad program.Absorption spectra of Ho³⁺were collected on a UV–VIS–NIR spectrophotometer.Using the equilibrium constants obtained by the Hyperquad2008 program,we prepared Tb(H_–1L)^2–and Ho(H_–1L)^2–solutions.Ho(H_-1L)^2-was titrated by appropriate amounts of Tb(H_-1L)^2-solution,and a series spectrums of Ho³⁺were recorded in each titration.The emission spectra and lifetime of Tb³⁺/DHPTA solution changed with the addition of NaOH solution were acquired on a fluorometer.The fluorescence lifetime of Tb(H_-1L)^2-in H₂O and D₂O were acquired to calculate the number of coordinated water in Tb(H_-1L)^2-complexes.The luminescence spectra of Tb(H_–1L)^2–changed with the addition of Ho(H_–1L)^2–solution were acquired on a fluorometer.Lu³⁺/DHPTA ratios and pH,were carefully selected through simulation by the Hyss2009 program so that a desired complex speciation was dominated in the individual samples.Dominant speciations were L^4–?100%?and Lu(H_–1L)^2–?100%?in the solution?A?and?B?,respectively.¹H–NMR,¹³C–NMR,2D ¹H–¹H COSY and ¹³C–¹H HSQC data were collected on a 400 MHz Bruker Advance spectrometer.The assignment of peaks in the ¹H–NMR and ¹³C–NMR spectra were accomplished with the help of 2D ¹H–¹H COSY and ¹³C–¹H HSQC data.¹⁷⁷Lu(H_–1L)^2–solution was prepared according to the thermodynamic experimental data.Then we measured the radiolabeling rate.Twelve healthy Kunming mice were randomly divided into 4 groups.20?Ci?0.1mL?of¹⁷⁷Lu(H_–1L)^2–was injected through the tail vein.Mice were sacrificed at 1h,1d,2d,and 7d.The weight and corresponding radioactivity of blood,heart,liver,spleen,lung,kidney,small intestine,femur,muscle,skull were measured at each time point.Then calculated the percentage activity of injection dose per gram of tissue?%ID/g?,which expressed as mean±standard deviation??.Results:Potentiometric titration indicated Tb³⁺,Ho³⁺,Lu³⁺have similar coordination structures with DHPTA.The dominant species at pH 4¹0 is Ln(H_–1L)^2–?metal:H⁺:L=1:-1:1?.Because the alkoxide of DHPTA hydroxyl group directly binds to lanthanide atom,The equilibrium constant of the Lu(H_–1L)^2–was calculated to be 25.01 at 25^oC,which is equivalent to the Lu³⁺/DOTA complexes(log₁₀?=25.4),higher than the Lu³⁺/EDTA complexes?19.8?and Lu³⁺/DTPA complexes?22.44?.Moreover,the reaction conditions of Lu(H_–1L)^2–complex are more relaxed than DOTA,indicating that DHPTA has the potential to be a lanthanide metal chelator in the field of radioimmunity.Since the results of potentiometric titration only represent the ratio of Ln³⁺?H⁺and ligands,the actual quantities cannot be determined.We have conducted spectral titration,fluorescence titration and nuclear magnetic experiment to explored the coordination structure of Ln(H_–1L)^2–.The normalized intensity of molar Ho³⁺increases as the solution of Tb(H_–1L)^2–were added into the solution of Ho(H_–1L)^2–.The fluorescence intensity of the Tb(H_–1L)^2–solution is effectively quenched by the addition of Ho(H_–1L)^2–.Both the spectral titration experiment and luminescence quenching experiment confirmed the dimeric or multimeric complexes indirectly.According to the fluorescence lifetime of Tb(H_–1L)^2–in H₂O and D₂O,respectively,we calculated the number of waters bound to the inner coordination sphere of Tb³⁺in Tb(H_–1L)^2–was 0.4.Measurements based on the luminescence lifetime suggest that the major fraction of the hydration number of Tb³⁺in H₂O to be 9.Assuming Tb(H_–1L)^2–exists as a monomer,two additional water molecules are needed to meet the coordination requirements of Tb³⁺,which is far from the experimental results.We assume that Tb(H_–1L)^2–could be present as a dimeric or polymeric complex,deprotonated hydroxyl or carboxylic acid oxygen forms O-bridge and coordinates with Tb³⁺to occupy water coordination sites.So,the number,=0.4,observed by experiment should be reasonable.NMR analysis shows that there is only one ¹³C peaks for carboxlate in^-solution L⁴.However,several new peaks were observed for this group in solution Lu(H_-1L)^2-,which indicating there are multiple different coordination environments of carboxylic acids.The Lu³⁺/DHPTA would take very complicated polymer structure and bridged by carboxylato group such as Nd³⁺/DHPTA tetranuclear.The structures consists of four Nd³⁺,four DHPTA ligands,and four coordinated water molecules.Under the condition that the activity of ¹⁷⁷Lu³⁺was 0.2 mCi and the amount of DHPTA was 200 ug,adjusting pH to 9.0 and allowing reactions at room temperature for 5 min.100%radiolabeling rate was obtained.Biodistribution experiments shows that ¹⁷⁷Lu(H_–1L)^2–is basically cleared from the blood in about 24 hours.¹⁷⁷Lu(H_–1L)^2–is mainly cleared through kidneys and liver.And the uptake of bone is pretty high,which may be related to the osteophilicity of ¹⁷⁷Lu.Conclusion:Lanthanides(Tb³⁺,Ho³⁺,Lu³⁺)form three complexes with DHPTA,they are LnL^–,Ln(H_–1L)^2–,and Ln?OH?(H_–1L)^3–.It is worth noting that the dominant speciation in the pH 4¹0 range is Ln(H_–1L)^2-.The formation constant of the Ln(H_–1L)^2–was quite high because the alkoxide of DHPTA hydroxyl group directly binds to lanthanide atom.In terms of thermodynamics and binding dynamics,Lu(H_–1L)^2–complex is higher than Lu³⁺/EDTA complex and Lu³⁺/DTPA complex,and to be equivalent to the stability constant of Lu³⁺/DOTA complex.Moreover,It suggests the binding reaction are much easier than Lu³⁺/DOTA.So DHPTA has the potential to be a lanthanide radioisotope chelator in radioimmunity.Through spectral titration,fluorescence titration,and NMR experiment we proved that Lu(H_–1L)^2–exists as a dimer or multimer bridged by oxygen atom from deprotonated hydroxyl and carboxylic acid.Biodistribution experiments showed that Lu(H_–1L)^2–clears from blood through the kidneys and liver.The uptake of bones are quite high,which may be related to the osteopathic nature of ¹⁷⁷Lu.