Monoclonal Antibody Radiopharmaceuticals : Cationization, Pegylation, Radiometal Chelation, Pharmacokinetics, and Tumor Imaging

Lee, Hwa Jeong,Pardridge, Wiliam M. 梨花女子大學校 藥學硏究所 2003 藥學硏究論文集 Vol.- No.12

The 528 murine monoclonal antibody (MAb) to the human epidermal growth factor receptor (EGFR) was sequentially cationized with hexamethylenediamine and conjugated with diethylenetriamine-pentaacetic acid (DTPA) as a potential antibody radiopharmaceutical for imaging EGFR-expressing cancer. The cationized 528 MAb was characterized with isoelectric focusing and electrophoresis, and an immunoradiometric assay, which showed the affinity of the 528 MAb for the human EGFR was retained following cationization. The native or cationized 528 MAb, labeled with ^(111)In, was injected intravenously in scid mice bearing human U87 flank tumors, which express the EGFR. and tumor imaging was performed with both external detection in live animals and with whole body autoradiography. However, the tumor signal was not increased with the cationized MAb, relative to the native MAb, and this was due to a serum inhibition phenomenon that was confirmed by a pharmacokinctics analysis in control mice. In an attempt to block the serum inhibition, the cationized 528 MAb was pegylated with 2000 Da poly(ethylene glycol), and the cationized/pegylated MAb was conjugated with DTPA and labeled with ^(111)In. However, a pharmacokinetics analysis showed the pegylation did not reverse the serum inhibition of the cationic charge on the MAb. These studies describe methods for reformulating monoclonal antibodies to develop improved radiophamaceuticals, but show that radiolabeling a cationized MAb with DTPA produces a serum neutralization of the initial cationization modification.

Blood-Brain Barrier Disruption Following the Internal Carotid Arterial Perfusion of Alkyl Glycerols

LEE, HWA JEONG,ZHANG, YUN,PARDRIDGE, WILLIAM M. 梨花女子大學校 藥學硏究所 2002 藥學硏究論文集 Vol.- No.11

Non ionic, amphipathic molecules form vesicles and this property correlates with the disruption ofmembranes. In the present studies, high mM concenrations of aliphatic alcohols.1-0-hexyldiglycerol(HDG) and 1-0-heptyltriglycerol (HTG), are shown to cause enhanced drug transport into brain viadisruption of the blood-brain barrier (BBB) in vivo, as determined with an internal carotid arteryperfusion method. The intravenous administration of comparable conceutrations of HDG or HTGcaused no increase in BBB transport of drug. The enhanced transport of drug showed a dependency ormolecular weight as 45 mM HTG increased the transport of sucrose,360Da, but did not increase thetransport of arginine vasopressin (AVP), 1084Da, although AVP transport across the BBB wasincreased by 80mM HDG or HTG. Ouasielastic light scattering measurements provided evidence forthe formation of vesicular structures in aqueous solutions containing high mM concentrations of theHDG or HTG. In summary, these studies demonstrate BBB disruptien following the infernal carotidauerial infusion of high mM concentrations of membrane active alkyl g1ycerols.

Receptor-mediated delivery of an antisense gene to human brain cancer cells

Zhang, Yun,Lee, Hwa Jeong,J. Boado, Ruben,M. Pardridge, William 梨花女子大學校 藥學硏究所 2002 藥學硏究論文集 Vol.- No.11

Background The goal of this work was the development of a gene targetingtechnology that will enable the delivery of therapeutic genes to brain cancercells in vivo following intravenous administration. High-grade brain gliomasoverexpress the epidermal growth factor receptor (EGFR) and EGFR anti-sense gene therapy could reduce the growth of EGFR-dependent gliomas. Methods A human EGFR antisense gene driven by the SV4O promoter ina non-virat plasmid carrying elements that facilitate extra-chromosomalreplication was packaged in the interior of 85 nm pegylated immuno-liposomes (PILs) . The PILs were targeted to U87 human glioma cells withthe 83-14 murine monoclonal antibody (MAb) to the human insulin receptor(HIR). Results Confocal fluorescent microscopy demonstrated that the unconju-gated HIR MAb is rapidly internalized by the glioma cells. Endoytosisfellowed by entry into the nucleus was also demonstrated for the HIR MAbconjugated PILs carrying fluorescein-labeled plasmid DNA. The PILs deliveredenogenous genes to virtually all cells in culture, based on β-galactosidasehistochemistry. The targeting of a luciferasr gene to the U87 cells with thePILs resulted in luciferase levels in excess of 150 pg/mg protein after 72 h ofincubation. The level of luciferase gene expression in the U87 cells acHievedwith the PIL gene targeting system was comparable to that with lipo-fectamine. Targeting the EGFR antisense gene to U87 glioma cells with thePILs resulted in more than 70% reduction in [^3H]thymidine incorporationinto the cells; this was paralleled by a 79% reduction in the level ofimmunoreactive EGFR. Conclusion The present work describes the targeting of an EGFR antisensegene to human brain cancer cells, which results in a 70-80% inhibition incancer cell growth. PILs provide a new approach to gene targeting that iseffective in vivo following intravenous administration without viral vectors.

Imaging Brain Amyloid of Alzheimer Disease In Vivo in Transgenic Mice With an Aβ Peptide Radiopharmaceutical

Lee, Hwa Jeong,Zhang, Yun,Zhu, Chunni,Duff, Karen,Pardridge, William M. 梨花女子大學校 藥學硏究所 2002 藥學硏究論文集 Vol.- No.11

Aβ^1-40 is a potential peptide radiopharmaceuticalthat coilid be used to image the brain Aβ amyloid of Alaheimerdisease in vivo, should this peptide be made transportablethrough the blood-brain barier in vivo. The blood-brain barriertransport of[^125I]-Aβ^1-40 in a transgenic mouse model wasenabled by conjugation to the rat 8D3 monoclonal antibody tothe mouse transferrin receptor. The Aβ^1-40_8D3 conjugate is abifunctional molecule that binds the blood-brain barrier TfRand unuergoes transport into brain and binds the Aβ amyloidpiaques of AIzheimer disease. App^sw/Psenl double-transgenicand littermate control mice were administered either unconju-gated Aβ^1-40 or the Aβ^1-40_8D3 conjugate intravenously, andbrain scans were obtained 6 hours later. Immunocytochemical analysis showed abundant Aβ immunoreactive plaques in thebrains of the App^sw/Psenl transgenic mice and there was aselective retention of radioactivity in the brains of these mice at6 hours after intravenous administration of the conjugate. Incontrast, there was no selective sequestration either of the con-jugate in control littermate mouse brain or of unconjugatedAβ in transgenic mouse brain. In conclusion, the resultsshow that it is possible to image the Aβ amyloid burden in thebrain in vivo with an amyloid imaging agent, provided themolecule is conjugated to a blood-brain barrier drug-targetingsystem.

Imaging Gene Expression in the Brain In Vivo in a Transgenic Mouse Model of Huntington's Disease with an Antisense Radiopharmaceutical and Drug-Targeting Technology

Lee, Hwa Jeong,Boado, Ruben J.,Brassch, Dwaine A.,Corey, David R.,Pardridge, William M. 梨花女子大學校 藥學硏究所 2002 藥學硏究論文集 Vol.- No.11

Disease-specific genes of unknown function can be imaged invivo with antisense radiopharmaceuticals, providing the trans-cellular transport of these molecules is enabled with drug-tar-geting technology. The current studies describe the productionof 16-mer peptide nucleic acid (PNA) that is antisense aroundthe methionine initiation codon of the huntingtin gene of Hun-tington's disease (HD). Methods: The PNA is biotinylated,which allows for rapid capture by a conjugate of streptavidinand the rat 8D3 monoclonal antibody (mAb) to the mouse trans-ferrin receptor (TfR), and contains a tyrosine residue, whichenables radiolabeling with ^125I. The reformulated PNA antisenseradiopharmaceutical that is conjugated to the 8D3 mAb is des-ignated ^125I-PNA/8D3, This form of the PNA is able to accessendogenous transferrin transport pathways at both the blood-brain barrier and the brain cell membrane and undergoes bothimport from the blood to the brain and export from the brain tothe blood through the TfR. Results: The ability of the PNA tohybridize to the target huntingtin RNA, despite conjugation tothe mAb, was shown both with cell-free translation assays andwith ribonuclease protection assays. The ^123I-PNA/8D3 conju-gate was administered intravenously to either littermate controlmice or to R6/2 transgenic mice, which express the exon 1 ofthe human HD gene. The mice were sacrificed 6 h later forfrozen sectioning of the brain and quantitative autoradiography.The studies showed a 3-fold increase in sequestration of the^125I-PNA/8D3 antisense radiopharmaceutical in the brains of theHD transgenic mice in vivo, consistent with the selective ex-pression of the HD exon-1 messenger RNA in these animals.Conclusion: These results support the hypothesis that geneexpression in vivo can be quantitated with antisense radiophar-maceuticals, providing these molecules are reformulated withdrug-targeting technology. Drug targeting enables access of theantisense agent to endogenous transport pathways, which per-mits passage across the cellular barriers that separate bloodand intracellular compartments of target tissues.

Synthesis of Pegylated Immunonanoparticles

Oliver, Jean-Christophe,Huertas, Ramon,Lee, Hwa Jeong,Calon, Frederic,Pardridge, William M. 梨花女子大學校 藥學硏究所 2002 藥學硏究論文集 Vol.- No.11

Purpose. This work describes the syrthesis of pesylated immu-nonanoparticies by conjugation of an anti-transferrin receptor mono-clonal antibody (MAb) to maleimide-grafted pegylated nanoparticlesprepared from poly(lactic acid) (PLA) and a hi-functional polyeth-yleneglycol (PEG).Methods. Maleimide-PEG_3500-PLA_4000 and methoxy PEG_2600-PLA_4000 copolymers were synthesized by ring opening polymeriza-tion of L-lactide using stannous octoate as catalyst. Pegylated nano-particles were prepared from these copolymers by a multiple emul-sion/solvent evaporation method and thiolated OX26 MAb wasconjugated through the maleimide function located at the distal endof the PEG spacer. The pegylated immunonanoparticles were char-actenzed by quasi-elastic light scattering, gel permeation chromatog-raphy, turbidimetry assays, and transmission electron microscopy.Results. NMR spectroscopy confirmed the synthesis of both copoly-mers and the preservation of the maleimide function. The pegylatedimmunonanoparticles had an auerage diameter of 121 ± 5 nm andappeared spherical by transmission electron microscopy. The numberof OX26 MAb molecules conjugated per individual pegylated nano-particle was 67 ± 4. The MAb conjugated to the surface of the pe-gylated immunonanoparticle was visualized directly by electron mi-croscopy using a conjugate of 10 nm gold and an anti-mouse immu-noglobulin secondary antibody.Conclusion. Pegylated immunonanopartirles can be synthesized withbifunctional PEG derivatives that bridge the nanoparticle and thetargeting MAb. This novel formulation may enable the targeted de-livery of small molecules, protein drugs, and gene medicines.