Selection of small pretargeting peptides with affinity for radiochelates and separation and purification of radiolanthanides

"May 2014."Dissertation Supervisor: Dr. Silvia S. Jurisson.Dissertation Supervisor: Dr. Cathy S. Cutler.Includes vita.The first objective of this dissertation was to develop metallated chelates with Michael acceptor groups for selecting small peptides from a random phage display library (RPL). The selection is for cysteine bearing peptides, so that the sulfhydryl on cysteine reacts with the reactive electrophilic group conjugated to the 1, 4, 7, 10- tetraazacyclododecane N, N', N'', N''', N'''' -tertraacetic acid (DOTA) chelate. Upon selection of peptides with affinity for the radiochelates, the antibody-conjugated peptides are used in pretargeting tumors. Three radionuclides (?�??�?Ga, ??????In, and ???�??�?Lu) were selected for labeling DOTA due to their nuclear properties applicable for radiopharmaceuticals, as well as their ability to form unique coordination geometries with the DOTA chelate. The latter variance is expected to contribute to selecting a peptide that binds specifically to each of the three metallated complexes. The newly synthesized chelate 2,2',2'',2'''-(2-(4-(4-amino-4-oxobut-2-enamido)benzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10- tetrayl)tetraacetic acid (AmMABD) was radiolabeled with ?�??�?Ga and ???�??�?Lu and the complexes were evaluated for their in vitro and in vivo stabilities and compared to previously reported radiolabeled complexes without electrophilic functional groups. The result from these studies showed the radiolabled (?�??�?Ga and ???�??�?Lu) AmMABD complexes were in vitro and in vivo stable. The encouraging biodistribution profile and fast blood clearance of the radiocongugates suggest that they are potential candidates for radiodiagnostic and radiotherapeutic agents. The second objective of this dissertation focuses on separation and purification of radiolanthanides to achieve high specific activity radioisotopes applicable in nuclear medicine. The reactor produced radiolanthanides, such as ???�??�?Lu, ???�???Tb and ???�??�?Ho, have nuclear properties useful for diagnosIncludes bibliographical references

Reduction of rhenium (V) oxo Schiff Base Complexes with triphenyl phosphine ligands

Abstract only availableOne approach to the treatment of cancer is to direct beta-emitting radionuclide to the cancer site where the radiation destroys the cancer cells. This can be achieved by coordinating the radioisotope in a very stable environment and linking it to a specific biological targeting molecule, which interacts specifically with particular cancer cells. It is necessary to have extremely stable in vivo radionuclide complexes so that limited amounts of radiation are released to other parts of the body before the radionuclide can reach the cancer cells. Isotopes of radioactive Rhenium are characteristic of such a nuclide. Our emphasis was to obtain a Rhenium (III) metal ligand complex since the lower oxidation state is more kinetically inert relative to Rhenium (V). The method employed was to first produce the ReV-ligand complex, [ReVOCl(Sal2phen)], by reacting a 1:2 molar ratio of TBA[ReVIIOCl4] to Sal2phen. Next, [ReVOCl(Sal2phen)] was reacted with three equivalents of triphenylphosphine to determine whether a mono-substituted ReV complex or a di-substituted ReIII complex was formed. After purifying the product by solvent extraction, the coordinated complex was reacted with ammonium hexaflurophoshate, NH4PF6, to induce crystallization of the target compound, [ReIII(PPh3)2(Sal2phen)][PF6]. Preliminary 1H NMR, and FT-IT spectra suggest formation of trans-[ReIII(PPh3)2(sal2phen)]PF6. The Re=O stretch at 951.36 cm-1 observed for [ReOCl(sal2phen)] in the IR spectrum is missing from our product, implying the Re (III) product has been formed.Stevens' Chemistry Progra

Labeling and purification studies on cancer targeting DOTA-TATE labeled with radiolanthanides

Abstract only availableMolecular imaging and targeted radiotherapy are emerging fields for cancer treatment. DOTA-Tyr (3)-Thr(9)-octreotate (DOTA-TATE) is used for peptide receptor-mediated radionuclide therapy (PRMRT) in neuroendocrine tumours. These biomolecules can be radiolabeled with an appropriated radioisotope to produce radiopharmaceuticals for diagnostic and therapeutic applications. The DOTA-TATE molecule is comprised of a bifunctional chelate (DOTA) that is capable of stably binding a radiolanthanide as well as being covalently attached to a targeting biomolecule (e.g.,octreotate). Among the radiolanthanides, Ho-166, Tb-161 and Lu-177, that were used to label the peptide, Lu-177 was used to obtain optimum conditions. Direct neutron capture on Lu-176 produces Lu-177. The indirect production of Lu-177 proceeds by neutron capture on Yb-176 producing Yb-177, which beta decays to Lu-177. Chromatographic separation yields high specific activity Lu-177 that minimizes the presence of cold Lu-176. Lanthanides have similar chemical properties that allow further studies to apply similar conditions as those developed for Lu-177. In addition, longevity of half-life of Lu-177 enables longer periods of dose delivery to targeted tumors. This research focused on identifying appropriate buffer solutions and volumes that could neutralize the acidic radioisotope to appropriate pH levels to label the peptide in high yield. The sample was purified from the unlabeled peptide by using HPLC separations methods and adding stabilizing agents (ascorbic and gentisic acid) to prevent radiolysis of the radiolabeled peptide. The results for the labeled peptide with various radioisotopes shows that 0.4 M NH4OAc, 0.4 M NaOAc, and 0.01 M HEPES buffer solution in 500 µL yields 99% labeling at pH ranging from 6.0 to 7.5. The labeled ligand at equimolar ratio with the metal yields 3 mCi/µg of the ligand, whereas as high specific activity sample can label up to 6.68 mCi/µg of the ligand. Carrier free Lu-177-DOTA-TATE was labeled using 0.01 M HEPES buffer at pH 6.0 and remains stable after using ascorbic acid; gentisic acid shows interference on HPLC which may cause some purification problems. (Ho- holmium, Tb- terbium Lu- lutetium)U.S. Dept. of Energy Innovations in Nuclear Infrastructure and Education Summer MURR Undergraduate Research Scholarshi

Reduction of rheniumV oxo Schiff base complexes with triethylphosphine

Abstract only availablePioneering techniques for therapeutic treatment of cancers involve targeting cancer sites with strong beta-emitting radionuclides, thereby destroying the cancer cells. This is achieved by coordinating the radioisotope to a very chemically stable environment and linking it to a specific biologically active targeting molecule, which interacts with particular cancer cells. Radioactive isotopes of rhenium possess characteristics of such a nuclide. The focus of our research is to investigate two possible pathways for the reaction of [ReOX(Schiff base)] with phosphine ligands, one a mono-substituted ReV complex and one a di-substituted ReIII complex. The preferred ReIII complex is lower in oxidation state and more kinetically inert or stable relative to ReV. For practical applications it is necessary to have an extremely stable in vivo radionuclide complex which can be conjugated to a suitable biological targeting agent. The rigid sal2phen ligand, where Sal2phen is a tetradentate Schiff base ligand, was investigated to determine if the ReIII could be synthesized from the ReV starting complex [ReVOCl(Sal2phen)]. [ReVOCl(Sal2phen)] was reacted with triethylphosphine (PEt3) in attempts to yield the ReIII complex trans-[ReIII(PEt3)2(Sal2phen)][X]. Previous work indicated that the strongly reducing and strongly nucleophilic PEt3 might yield the ReV product from [ReVOCl(Sal2phen)]. The synthesized coordinated complex was reacted with an quaternary ammonium salt, ammonium hexaflurophosphate (NH4PF6), to induce crystallization of target compound [ReIII(PEt3)2(Sal2phen)][PF6]. Preliminary 1H-NMR, 31P-NMR, and infrared spectroscopy spectra indicate the formation of cis-[ReVO(PPh3)(Sal2phen)][X]. FTIR shows the presence of the Rhenium oxo group; 31P-NMR and 1H-NMR indicate the presence of ReV and a 1:1 PEt3 : Sal2phen complex. Single crystal x-ray diffraction, mass spectroscopy, and elemental analysis are additional methods of characterization.NSF-REU/NIH Program in Radiochemistr

Separation of radiolanthanides by high performance liquid chromatography [abstract]

Abstract only availableStudies were carried out to develop a method for the separation of radiolanthanides using high performance liquid chromatography (HPLC). Radiolanthanides are essential for radiotherapy and/or imaging of cancer and their metastases. Specifically, a method was developed to separate lutetium-177 (177Lu) from samarium-153 (153Sm) using a cation-exchange column on the HPLC. 177Lu and 153Sm are obtained through neutron activation of 176Lu and 152Sm respectively at the University of Missouri-Columbia Research Reactor. A good separation of Lu-177/Sm-153 allowed us to decide on the selection and conditions of solvents to be used for other lanthanides. Additionally, studies were also carried out for the separation of promethium-149 (149Pm) from cold neodymium-148 (147Nd used as a radioactive trace). As Pm and Nd are located next to each other on the periodic table their very similar chemical properties pose a challenge in performing a complete separation. For example, varying the concentration and pH of solvents, ?-hydroxy-?- methylbutyric acid (?-H-? -MBA) and ?-hydroxy isobutyric acid (?-HIBA), can effectively produce good chromatographic separation of the lanthanides. High purity germanium counting was used to analyze the fractions of the isolated 149Pm from 147Nd. Solvent pH was determined to be the dominant factor in obtaining a good separation of lanthanides by HPLC

Reducution of rhenium(V)-oxo schiff base complexesith triphenyl phosphine ligands [abstract]

Abstract only availableFaculty Mentor: Dr. Silvia Jurisson, ChemistryThe purpose of this research is to produce radiopharmaceutical drugs for the possible use in therapy and diagnosis of cancer. A radiopharmaceutical drug is composed of a radioactive element contained within chelating agent and linked to a biological targeting molecule. This can be achieved by complexing the radioisotope and conjugating the complex to a biologically active targeting molecule, such as a peptide antibody or antibody fragment. The biological targeting molecule directs radiation to specific peptides, antibodies and antibodies' fragments. The amount of dosage is limited to non-targeted tissue that occurs when free radionuclide is released from a decomposing molecule. Higher kinetic stability will maximize the localization of the radioisotope to the cancer sites and minimize the radiation dose to non-target tissues. This research was a continuation to seek the best pathway that would easily enable us to synthesize a kinetically inert metal complex [Re(Sal2Phen)] attached to a ligand. The rigid Rhenium(V)-oxo Schiff base complex is found in the following form: [ReO(Sal2Phen)Cl]. Initially salicylic aldehyde reacted with bis-2-phenlinediamine to get sal2phen. This tetradentate Schiff base ligand was reacted with TBA [ReOCl4] yielding [ReOCl(Sal2Phen)]. The reduction of Rhenium(V)-oxo core to Rhenium is obtained by reacting [ReO(Sal2Phen)Cl] with a triphenyl phosphine (PPh3) in dichloromethane and ethanol. Products obtained from this reaction were 1[Re(Sal2Phen)(PPh3)2], 2ReCl(Sal2Phen)PPh3, and 3[ReO(Sal2Phen)PPh3].Analysis of the major product were performed through crystals through mass spectrometry, hydrogen and phosphorus NMR, infrared spectra and x-ray crystallography