177Lu‐labeled monomeric,dimeric and multimeric RGD peptides for the therapy of tumors expressing α(ν)β(3) integrins

The conjugation of peptides to gold nanoparticles (AuNPs) produces biocompatible and stable multimeric systems with target‐specific molecular recognition. Peptides based on the cyclic Arg‐Gly‐Asp (RGD) sequence have been reported as high‐affinity agents for the α(ν)β(3) integrin. The aim of this research was to prepare a multimeric system of ¹⁷⁷Lu‐labeled gold nanoparticles conjugated to c(RGDfK)C (cyclo(Arg‐Gly‐Asp‐Phe‐Lys)Cys) and to compare the radiation‐absorbed dose with that of ¹⁷⁷Lu‐labeled monomeric and dimeric RGD peptides to α(ν)β(3) integrin‐positive U87MG tumors in mice. DOTA‐GGC (1,4,7,10‐tetraazacyclododecane‐N‐N′,N″,N?‐tetraacetic acid‐Gly‐Gly‐Cys) and c(RGDfK)C peptides were synthesized and conjugated to AuNPs by a spontaneous reaction of the thiol groups. Transmission electron microscopy, ultraviolet–visible, X‐ray photoelectron spectroscopy, Raman and far‐infrared spectroscopy techniques demonstrated that AuNPs were functionalized with the peptides. For the ¹⁷⁷Lu‐AuNP‐c(RGDfK)C to be obtained, the ¹⁷⁷Lu‐DOTA‐GGC radiopeptide was first prepared and added to a solution of AuNPs followed by c(RGDfK)C (25 µl, 5 µ m ) at 18 °C for 15 min. ¹⁷⁷Lu‐DOTA‐GGC, ¹⁷⁷Lu‐DOTA‐cRGDfK and ¹⁷⁷Lu‐DOTA‐E‐c(RGDfK)₂ were prepared by adding ¹⁷⁷LuCl₃ (370 MBq) to 5 µl (1 mg/ml) of the DOTA derivative diluted with 50 µl of 1 m acetate buffer pH 5. The mixture was incubated at 90 °C in a block heater for 30 min. Radiochemical purity was determined by ultrafiltration and HPLC analyses. Biokinetic studies were accomplished in athymic mice with U87MG‐induced tumors. The radiochemical purity for all ¹⁷⁷Lu‐RGD derivatives was 96 ± 2%. ¹⁷⁷Lu‐absorbed doses per injected activity delivered to U87MG tumors were 0.357 ± 0.052 Gy/MBq (multimer), 0.252 ± 0.027 Gy/MBq (dimer) and 0.102 ± 0.018 Gy/MBq (monomer). ¹⁷⁷Lu‐labeled dimeric and multimeric RGD peptides demonstrated properties suitable for targeted radionuclide therapy of tumors expressing α(ν)β(3) integrins. Copyright © 2012 John Wiley & Sons, Ltd.     

Evaluation of a 188Re‐radiolabeled Arg‐Gly‐Asp peptide for tumor overexpressed αvβ3 receptors

To develop a peptide‐based radiopharmaceutical for the therapy of α_vβ₃ receptors overexpressed tumors, we have prepared a novel Arg‐Gly‐Asp (RGD) peptide (HCRGDCF(D)CRGDC, P12) radiolabeled with ¹⁸⁸Re. With His acid at the end of the peptide containing RGD, the label efficiency was more than 95% within 30 min. The peptide binds to human glioblastoma U87MG cells with high affinity [IC₅₀ = 86.3 nm]. The stability of ¹⁸⁸Re‐P12 in vitro was also investigated. More than 80% of radioactivity was kept in the peptide after 4 h incubation in phosphate buffer solution (pH = 7.4) or calf serum under physiological conditions. Biodistribution of this radiocompound was carried out in mice bearing S180 tumor. Fast clearance of ¹⁸⁸Re‐peptide from blood and specific uptakes by tumors realized higher tumor‐to‐blood ratio (1.80) 4 h post‐injection. Obvious difference was observed between the blocking and unblocking experiments in whole body autoradiography imaging. These results have demonstrated the potential of ¹⁸⁸Re‐labeling RGD as a radiotherapeutic agent.     

RGD‐based PET tracers for imaging receptor integrin αvβ3 expression

Positron emission tomography (PET) imaging of receptor integrin α_vβ₃ expression may play a key role in the early detection of cancer and cardiovascular diseases, monitoring disease progression, evaluating therapeutic response, and aiding anti‐angiogenic drugs discovery and development. The last decade has seen the development of new PET tracers for in vivo imaging of integrin α_vβ₃ expression along with advances in PET chemistry. In this review, we will focus on the radiochemistry development of PET tracers based on arginine–glycine–aspartic acid (RGD) peptide, present an overview of general strategies for preparing RGD‐based PET tracers, and review the recent advances in preparations of ¹⁸F‐labeled, ⁶⁴Cu‐labeled, and ⁶⁸Ga‐labeled RGD tracers, RGD‐based PET multivalent probes, and RGD‐based PET multimodality probes for imaging receptor integrin α_vβ₃ expression.     

Integrin α5β1: a new purification strategy based on immobilized peptides

Abstract: Novel efficient and robust affinity chromatography material: There are several strategies known for the purification of integrins by affinity chromatography, but the disadvantages of common strategies like insufficient selectivity or compelling conditions for the elution still require alternatives. A new strategy, based on the immobilized C‐terminally modified peptide Ac‐Gly‐Ala‐c‐(Cys^SS‐Arg‐Arg‐Glu‐Thr‐Ala‐Trp‐Ala‐Cys^SS)‐Gly‐Ala‐O(CH₂CH₂O)₂CH₂CH₂‐NH₂ allows for the affinity purification of the integrin α₅β₁. While RGD peptides have been proven in the past to be inappropriate for selective purification of integrins by affinity chromatography, the new peptide can be efficiently used for selective enrichment of the integrin α₅β₁. It is a specific ligand of the target protein, but does not contain an RGD sequence. The application of well‐characterized affinity chromatography material with a site‐specifically immobilized peptide allows to obtain integrin α₅β₁ in a single chromatography step without contamination by other integrins. This process combines the advantages of a selective and monospecific protein‐ligand recognition with mild elution conditions and a low sensitivity of the immobilized ligand with respect to column regeneration.     

Cerenkov luminescence imaging of αvβ6 integrin expressing tumors using 90Y‐labeled peptides

Cerenkov luminescence imaging (CLI) is an emerging preclinical molecular imaging modality that tracks the radiation emitted in the visible spectrum by fast moving charged decay products of radionuclides. The aim of this study was in vitro and in vivo evaluation of the two radiotracers, ⁹⁰Y‐DOTA‐PEG₂₈‐A20FMDV2 (⁹⁰Y‐1) and ⁹⁰Y‐DOTA‐Ahx‐A20FMDV2 (⁹⁰Y‐2) (>99% radiochemical purity, 3.7 GBq/µmol specific activity) for noninvasive assessment of tumors expressing the integrin α_vβ₆ and their future use in tumor targeted radiotherapy. Cell binding and internalization in α_vβ₆‐positive cells was ⁹⁰Y‐1: 10.1 ± 0.8%, 50.3 ± 2.1%; ⁹⁰Y‐2: 22.4 ± 1.7%, 44.7 ± 1.5% with <5% binding to α_vβ₆‐negative control cells. Biodistribution studies showed maximum α_vβ₆‐positive tumor uptake of the radiotracers at 1‐h post injection (p.i.) (⁹⁰Y‐1: 0.64 ± 0.15% ID/g; ⁹⁰Y‐2: 0.34 ± 0.11% ID/g) with high renal uptake (>25% ID/g at 24 h). Because of the lower tumor uptake and high radioactivity accumulation in kidneys (that could not be reduced by pre‐administration of either lysine or furosemide), the luminescence signal from the α_vβ₆‐positive tumor was not clearly detectable in CLI images. The studies suggest that CLI is useful for indicating major organ uptake for both radiotracers; however, it reaches its limitation when there is low signal‐to‐noise ratio.     

Synthesis of a non‐peptidic PET tracer designed for α5β1 integrin receptor

Arginine–glycine–aspartic acid (RGD)‐containing peptides have been traditionally used as PET probes to noninvasively image angiogenesis, but recently, small selective molecules for α₅β₁ integrin receptor have been developed with promising results. Sixty‐one antagonists were screened, and tert‐butyl (S)‐3‐(2‐((3R,5S)‐1‐(3‐(1‐(2‐fluoroethyl)‐1H‐1,2,3‐triazol‐4‐yl)propanoyl)‐5‐((pyridin‐2‐ylamino)methyl)pyrrolidin‐3‐yloxy)acetamido)‐2‐(2,4,6‐trimethylbenzamido)propanoate (FPMt) was selected for the development of a PET tracer to image the expression of α₅β₁ integrin receptors. An alkynyl precursor (PMt) was initially synthesized in six steps, and its radiolabeling was performed according to the azide–alkyne copper(II)‐catalyzed Huisgen's cycloaddition by using 1‐azido‐2‐[¹⁸F]fluoroethane ([¹⁸F]12). Different reaction conditions between PMt and [¹⁸F]12 were investigated, but all of them afforded [¹⁸F]FPMt in 15 min with similar radiochemical yields (80–83%, decay corrected). Overall, the final radiopharmaceutical ([¹⁸F]FPMt) was obtained after a synthesis time of 60–70 min in 42–44% decay‐corrected radiochemical yield. Copyright © 2014 John Wiley & Sons, Ltd.     

Methadone is a Non‐Competitive Antagonist at the α4β2 and α3* Nicotinic Acetylcholine Receptors and an Agonist at the α7 Nicotinic Acetylcholine Receptor

Nicotine–methadone interactions have been studied in human beings and in various experimental settings regarding addiction, reward and pain. Most methadone maintenance treatment patients are smokers, and methadone administration has been shown to increase cigarette smoking. Previous in vitro studies have shown that methadone is a non‐competitive antagonist at rat α3β4 nicotinic acetylcholine receptors (nAChR) and an agonist at human α7 nAChRs. In this study, we used cell lines expressing human α4β2, α7 and α3* nAChRs to compare the interactions of methadone at the various human nAChRs under the same experimental conditions. A [³H]epibatidine displacement assay was used to determine whether methadone binds to the nicotinic receptors, and ⁸⁶Rb⁺ efflux and changes in intracellular calcium [Ca²⁺]_i were used to assess changes in the functional activity of the receptors. Methadone displaced [³H]epibatidine from nicotinic agonist‐binding sites in SH‐EP1‐hα7 and SH‐SY5Y cells, but not in SH‐EP1‐hα4β2 cells. The K_i values for methadone were 6.3 μM in SH‐EP1‐hα7 cells and 19.4 μM and 1008 μM in SH‐SY5Y cells. Methadone increased [Ca²⁺]_i in all cell lines in a concentration‐dependent manner, and in SH‐EP1‐hα7 cells, the effect was more pronounced than the effect of nicotine treatment. In SH‐EP1‐hα4β2 cells, the effect of methadone was negligible compared to that of nicotine. Methadone pre‐treatment abolished the nicotine‐induced response in [Ca²⁺]_i in all cell lines expressing nAChRs. In SH‐EP1‐hα4β2 and SH‐SY5Y cells, methadone had no effect on the ⁸⁶Rb⁺ efflux, but it antagonized the nicotine‐induced ⁸⁶Rb⁺ ion efflux in a non‐competitive manner. These results suggest that methadone is an agonist at human α7 nAChRs and a non‐competitive antagonist at human α4β2 and α3* nAChRs. This study adds further support to the previous findings that opioids interact with nAChRs, which may underlie their frequent co‐administration in human beings and might be of interest to the field of drug discovery.     

111In‐ and 203Pb‐labeled cyclic arginine‐glycine‐aspartic acid peptide conjugate as an αvβ3 integrin‐binding radiotracer

Methodology for site‐specific modification and chelate conjugation of a cyclic arginine‐glycine‐aspartic acid (cRGD) peptide for the preparation of a radiotracer molecular imaging agent suitable for detecting α_vβ₃ integrin is described. The method involves functionalizing the peptide with an aldehyde moiety and conjugation to a 1,4,7,10‐tetraazacyclododecane‐N,N′,N″,N?‐tetraacetic acid derivative that possesses an aldehyde reactive aminooxy group. The binding assay of the ¹¹¹In‐labeled peptide conjugate with α_vβ₃ integrin showed 60% bound when four equivalents of the integrin was added, a reasonable binding affinity for a monovalent modified RGD peptide.     

Noninvasive imaging of c(RGD)2‐9R as a potential delivery carrier for transfection of siRNA in malignant tumors

The purpose of our study was to develop and evaluate a novel integrin α_vβ₃‐specific delivery carrier for transfection of siRNA in malignant tumors. We adopted arginine‐glycine‐aspartate (RGD) motif as a tissue target for specific recognition of integrin α_νβ₃. A chimaeric peptide was synthesized by adding nonamer arginine residues (9‐arginine [9R]) at the carboxy terminus of cyclic‐RGD dimer, designated as c(RGD)₂‐9R, to enable small interfering RNA (siRNA) binding. To test the applicability of the delivery carrier in vivo, c(RGD)₂‐9R was labeled with radionuclide of technetium‐99m. Biodistribution and γ‐camera imaging studies were performed in HepG2 xenograft‐bearing nude mice. As results, an optimal 10:1 molar ratio of ^99mTc‐c(RGD)₂‐9R to siRNA was indicated by the electrophoresis on agarose gels. ^99mTc‐c(RGD)₂‐9R/siRNA remained stable under a set of conditions in vitro. For in vivo study, tumor radioactivity uptake of ^99mTc‐c(RGD)₂‐9R/siRNA in nude mice bearing HepG2 xenografts was significantly higher than that of control probe (P  < .05). The xenografts were clearly visualized at 4 hours till 6 hours noninvasively after intravenous injection of ^99mTc‐c(RGD)₂‐9R/siRNA, while the xenografts were not visualized at any time after injection of control probe. It was concluded that c(RGD)₂‐9R could be an effective siRNA delivery carrier. Technetium‐99m radiolabeled‐delivery carrier represents a potential imaging strategy for RNAi‐based therapy.     

A simple and efficient synthesis of [3α‐3H]5α‐androst‐16‐en‐3β‐ol

An efficient one step synthesis of [3α‐³H]5α‐androst‐16‐en‐3β‐ol by NaBT₄ reduction of a ketone precursor is described. The specific activity of the product was 21.6 Ci/mmol with a radiochemical purity >99%. Synthesis of the precursor, 5α‐androst‐16‐en‐3‐one, from commercially available 5α‐androst‐16‐en‐3α‐ol is also presented. Copyright © 2006 John Wiley & Sons, Ltd.     

Design and evaluation of benzophenone‐containing conformationally constrained ligands as tools for photoaffinity scanning of the integrin αVβ3–ligand bimolecular interaction

Abstract: Integrins are cell‐surface adhesion molecules involved in mediating cell–extracellular matrix interactions. High‐resolution structural data are not available for these heterodimeric receptors. In order to generate tools for photoaffinity scanning of the RGD‐binding site of human integrin α_Vβ₃, new conformationally constrained ligands were designed. The ligands were based on five different cyclic peptidic or peptidomimetic scaffolds with high affinity for α_Vβ₃. A single photoreactive group (a benzophenone moiety) was introduced at different positions relative to the RGD triad. In addition, an ¹²⁵I or a biotin group was introduced as a reporting tag. Twenty‐four cyclic ligands were prepared and their binding affinity for α_Vβ₃ was determined. In most cases, the modifications resulted in a 5‐ to 500‐fold decrease in affinity relative to the unmodified scaffold. Analogs representing three of the five families were screened for their cross‐linking efficiency. Ligands with submicromolar affinities cross‐linked efficiently and specifically to the integrin receptor, whereas ligands with weaker affinities gave specific cross‐linking, but with lower efficiency. Almost all of the screened ligands cross‐linked predominantly to the β₃ subunit.     

Synthesis of [3α‐3H] 17α‐Hydroxy pregnenolone and [3α‐3H] Pregnenolone

For the first time, [3α‐³H] 17α‐hydroxy pregnenolone (1) was synthesized through a multiple step sequence. The presence of [3β‐³H] isomer in RP‐HPLC purified product was identified by tritium NMR. The [3β‐³H] isomer was then separated from [3α‐³H] 17α‐hydroxy pregnenolone with chiralPAK AD‐H column. [3α‐³H] pregnenolone (2) was synthesized from commercial available 5‐pregnen‐3,20‐dione in one step with an improved procedure.     

18F labeled RGD‐A7R peptide for dual integrin and VEGF‐targeted tumor imaging in mice bearing U87MG tumors

The aim of this study is to develop a novel Arg‐Gly‐Asp acid (RGD) and Ala‐Thr‐Trp‐Leu‐Pro‐Pro‐Arg (ATWLPPR A7R) peptide‐containing ligand for ¹⁸F labeling as αvβ3 and vascular endothelial growth factor receptor‐targeted imaging agent. ¹⁸F‐RGD‐A7R was prepared by conjugation with ¹⁸F‐SFB. The final product was purified by high‐performance liquid chromatography and tested in vitro and in vivo. Cell‐binding assays of RGD‐A7R, RGD and RGD‐A7R, A7R were tested in U87MG cells (¹²⁵I‐RGDyK and ¹²⁵I‐A7RY as radioligand, respectively). Preliminary biodistribution of the ¹⁸F‐RGD‐A7R was also evaluated. The RGD‐A7R had good integrin binding affinity (50% inhibitory concentration (IC₅₀) = 21.67 and 23.68 nM, slightly lower than unmodified RGD (40.02 nM) and A7R (50.18 nM)). The radiotracer had receptor‐mediated activity accumulation in U87MG tumor (1.90 ± 0.34 percentage of injected dose per gram (%ID/g) at 0.5 h postinjection), which is known to be integrin positive. After blocking with RGD‐A7R, the tumor uptake was reduced to 0.47 ± 0.06 %ID/g at 0.5 h postinjection. ¹⁸F‐RGD‐A7R exhibited dual receptor targeting properties both in vitro and in vivo. The favorable characterizations of ¹⁸F‐RGD‐A7RY, such as convenient synthesis, high specific activity, and high tumor uptake, warrant its further investigation for clinical cancer imaging.     

Synthesis,crystal structure and molecular conformation of Nα‐Boc‐l‐leucyl‐(Z)‐β‐(3‐pyridyl)‐α,β‐ dehydroalanyl‐l‐leucine methyl ester*

Abstract: A protected tridehydropeptide containing (Z)‐β‐(3‐pyridyl)‐α,β‐dehydroalanine (Δ^Z3Pal) residue, Boc‐Leu‐Δ^Z3Pal‐Leu‐OMe ( 1 ), was synthesized via Erlenmeyer azlactone method. X‐ray crystallographic analysis revealed that the peptide 1 adopts an extended conformation, which is similar to that of a Δ^ZPhe analog, Boc‐Leu‐Δ^ZPhe‐Leu‐OMe ( 2 ).     

99mTc‐HYNIC‐(Ser)3‐J18 peptide: A radiotracer for non‐small‐cell lung cancer targeting

Radiolabeled peptide could be a useful tool for the diagnosis of non‐small‐cell lung cancer (NSCLC). In this study, HYNIC‐(Ser)₃‐J18 peptide was labeled with ^99mTc using EDDA/tricine as coligands. The in vitro and in vivo studies of this radiolabeled peptide were performed for cellular‐specific binding and tumor targeting in A‐549 cells and tumor‐bearing mice, respectively. The high radiochemical purity was obtained and this radiolabeled peptide exhibited high stability in buffer and serum. The radiolabeled peptide showed high affinity for the A‐549 cells with a dissociation constant value (K_D) of 4.4 ± 0.8 nm . The tumor–muscles ratios were 2.7 and 4.4 at 1 and 2 hr after injection of ^99mTc‐(EDDA/tricine)‐HYNIC‐(Ser)₃‐J18 in tumor‐bearing mice. The tumor uptake was decreased after preinjection with non‐labeled peptide for this radiolabeled peptide in blocking experiment. The results of this study showed the ^99mTc‐(EDDA/tricine)‐(Ser)₃‐HYNIC‐J18 peptide might be a promising radiolabeled peptide for NSCLC targeting.     

Conformational properties of hybrid peptides containing α‐ and ω‐amino acids*

Abstract: This review briefly surveys the conformational properties of guest ω‐amino acid residues when incorporated into host α‐peptide sequences. The results presented focus primarily on the use of β‐ and γ‐residues in αω sequences. The insertion of additional methylene groups into peptide backbones enhances the range of accessible conformations, introducing additional torsional variables. A nomenclature system, which permits ready comparisons between α‐peptides and hybrid sequences, is defined. Crystal structure determination of hybrid peptides, which adopt helical and β‐hairpin conformations permits the characterization of backbone conformational parameters for β‐ and γ‐residues inserted into regular α‐polypeptide structures. Substituted β‐ and γ‐residues are more limited in the range of accessible conformation than their unsubstituted counterparts. The achiral β,β‐disubstituted γ‐amino acid, gabapentin, is an example of a stereochemically constrained residue in which the torsion angles about the C^β–C^γ (θ₁) and C^α–C^β (θ₂) bonds are restricted to the gauche conformation. Hybrid sequences permit the design of novel hydrogen bonded rings in peptide structures.     

Synthesis,Biological Activity,and NMR‐Based Structural Studies of Deltorphin I Analogs Modified in Message Domain with a New α,α‐Disubstituted Glycines

This article describes new deltorphin I analogs in which phenylalanine residues were replaced by the corresponding (R) or (S)‐α‐benzyl‐β‐azidoalanine, α‐benzyl‐β‐(1‐pyrrolidinyl)alanine, α‐benzyl‐β‐(1‐piperidinyl)alanine, and α‐benzyl‐β‐(4‐morpholinyl)‐alanine residues. The potency and selectivity of the new analogs were evaluated by a competitive receptor binding assay in the rat brain using [³H]DAMGO (a μ ligand) and [³H]DELT (a δ ligand). The affinity of analogs containing (R) or (S)‐α‐benzyl‐β‐azidoalanine in position 3 to δ‐receptors strongly depended on the chirality of the α,α‐disubstituted residue. The conformational behavior of peptides modified with (R) or (S)‐α‐benzyl‐β‐(1‐piperidinyl)Ala, which displays the opposite selectivity, was analyzed by ¹H and ¹³C NMR. The μ‐selective Tyr‐d ‐Ala‐(R)‐α‐benzyl‐β‐(1‐piperidinyl)Ala‐Asp‐Val‐Val‐Gly‐NH₂ lacks the helical conformation observed in the δ‐selective Tyr‐d ‐Ala‐(S)‐α‐benzyl‐β‐(1‐piperidinyl)Ala‐Asp‐Val‐Val‐Gly‐NH₂. Our results support the proposal that differences between δ‐ and μ‐selective opioid peptides are attributable to the presence or absence of a spatial overlap between the N‐terminal message domain and the C‐terminal address domain.     

Agonist function of the recombinant α4β3δ GABAA receptor is dependent on the human and rat variants of the α4‐subunit

1. It is known that the α₄‐subunit is likely to occur in the brain predominantly in α₄β₃δ receptors at extrasynaptic sites. Recent studies have revealed that the α₁‐, α₄‐, γ₂‐ and δ‐subunits may colocalize extrasynaptically in dentate granule cells of the hippocampus. In the present study, we characterized a series of recombinant GABA_A receptors containing human (H) and rat (R) α₁/α₄‐, β₂/β₃‐ and γ_2S/δ‐subunits in Xenopus oocytes using the two‐electrode voltage‐clamp technique. 2. Both Hα₁β₃δ and Hα₄β₃γ_2S receptors were sensitive to activation by GABA and pentobarbital. Contrary to earlier findings that the α₄β₃δ combination was more sensitive to agonist action than the α₄β₃γ_2S receptor, we observed extremely small GABA‐ and pentobarbital‐activated currents at the wild‐type Hα₄β₃δ receptor. However, GABA and pentobarbital activated the wild‐type Rα₄β₃δ receptor with high potency (EC₅₀ = 0.5 ± 0.7 and 294 ± 5 μmol/L, respectively). 3. Substituting the Hα₄ subunit with Rα₄ conferred a significant increase in activation on the GABA and pentobarbital site in terms of reduced EC₅₀ and increased I_max. When the Hα₄ subunit was combined with the Rβ₃ and Rδ subunit in a heteropentameric form, the amplitude of GABA‐ and pentobarbital‐activated currents increased significantly compared with the wild‐type Hα₄β₃δ receptor. 4. Thus, the results indicate that the Rα₄β₃δ, Hα₁β₃δ and Hα₄β₃γ_2S combinations may contribute to functions of extrasynaptic GABA_A receptors. The presence of the Rα₄ subunit at recombinant GABA_A receptors containing the δ‐subunit is a strong determinant of agonist action. The recombinant Hα₄β₃δ receptor is a less sensitive subunit composition in terms of agonist activation.     

Preparation of clinical‐scale 177Lu‐Rituximab: Optimization of protocols for conjugation,radiolabeling, and freeze‐dried kit formulation

Rituximab is a monoclonal chimeric antibody, which has been approved by the US Food and Drug Administration for immunotherapy of non–Hodgkin lymphoma. Bexxar and Zevalin are the two other approved radiolabeled antibodies for radioimmunotherapy of non–Hodgkin lymphoma; however, they are of murine origin that reduces their treatment efficacy. So as to circumvent this, efforts have been made to radiolabel Rituximab with various therapeutic radioisotopes. In the present study, an effort has been made to optimize the conjugation (bifunctional chelating agent and antibody) and radiolabeling procedures for the preparation of clinical‐scale ¹⁷⁷Lu‐labeled Rituximab. An attempt was also made to prepare the freeze‐dried Rituximab kit for the easy and convenient clinical translation of the agent. Clinical‐scale ¹⁷⁷Lu‐Rituximab (40 mCi, 1.48 GBq) was prepared with >95% radiochemical purity using the kit. Biological evaluation of ¹⁷⁷Lu‐Rituximab was performed by in vitro cell binding studies in Raji cell lines, which showed satisfactory binding at 4°C and 37°C. Pharmacokinetic behavior of the agent, evaluated by biodistribution studies in normal Swiss mice, revealed high blood and liver uptake at the initial time points, although it exhibited slow and gradual clearance with time. The study indicates that clinical‐scale ¹⁷⁷Lu‐Rituximab could be conveniently formulated using the methodology described in the present article.     

Synthesis of [18F]‐labeled 2′‐deoxy‐2′‐fluoro‐5‐methyl‐1‐β‐D‐arabinofuranosyluracil ([18F]‐FMAU)

Synthesis of 2′‐deoxy‐2′‐[¹⁸F]fluoro‐5‐methyl‐1‐β‐D‐arabinofuranosyluracil ([¹⁸F]‐FMAU) is reported. 2‐Deoxy‐2‐[¹⁸F]fluoro‐1,3,5‐tri‐O‐benzoyl‐α‐D‐arabinofuranose 2 was prepared by the reaction of the respective triflate 1 with tetrabutylammonium[¹⁸F]fluoride. The fluorosugar 2 was converted to its 1‐bromo‐derivative 3 and coupled with protected thymine 4 . The crude product mixture ( 5a and 5b ) was hydrolyzed in base and purified by HPLC to obtain the radiolabeled FMAU 6a . The radiochemical yield of 6a was 20–30% decay corrected (d.c.) in four steps with an average of 25% in four runs. Radiochemical purity was >99% and average specific activity was 2300 mCi/μmol at the end of synthesis (EOS). The synthesis time was 3.5–4.0 h from the end of bombardment (EOB). Copyright © 2002 John Wiley & Sons, Ltd.