Synthesis and comparative in vivo evaluation of 99mTc(CO)3‐labeled PEGylated and non‐PEGylated cRGDfK peptide monomers

This work aimed at studying the effect of insertion of medium PEG (PEG₇) on the pharmacokinetic behavior of cRGDfK peptide in comparison with the non‐PEGylated analogue. The cRGDfK peptide has thus been derivatized at ε‐amino group of lysine by conjugation with N₃–PEG₇–COOH/N₃–CH₂–COOH to prepare a PEGylated and a non‐PEGylated analogue of cRGDfK. A tridentate chelator was then incorporated by click chemistry conjugation of the two peptide azides for radiolabeling with [^99mTc(CO)₃(H₂O)₃]⁺ precursor. Comparative in vivo evaluation of the two ^99mTc(CO)₃‐labeled radiotracers, ^99mTc(CO)₃–Pra–Tz–CH₂–cRGDfK 5 and ^99mTc(CO)₃–Pra–Tz–PEG₇–cRGDfK 6 , was carried out in C57BL/6 mice bearing α_vβ₃‐positive melanoma tumors to determine their potential toward targeting integrin α_vβ₃ receptors. The radiotracers exhibited excellent stability in saline as well as in serum. Maximum tumor uptake for the two radiotracers was observed at 30 min p.i. ( 5 : 3.0 ± 0.7% ID/g; 6 : 4.1 ± 0.5% ID/g). The two neutral ^99mTc(CO)₃ radiotracers prepared exhibited receptor‐mediated uptake in melanoma tumor. The increase in the tumor uptake on introduction of PEG₇ unit was accompanied by slower clearance from other organs which resulted in decreased target‐to‐background ratios. The in vivo kinetics of ^99mTc(CO)₃‐labeled radiotracer, ^99mTc(CO)₃–Pra–Tz–CH₂–cRGDfK 5 with only methylene unit as the spacer, was found to be more favorable due to higher tumor/blood, tumor/liver, tumor/kidney, and tumor/lung ratios.     

An improved kit formulation for one‐pot synthesis of [99mTc]Tc‐HYNIC‐E[c(RGDfK)]2 for routine clinical use in cancer imaging

Radiolabeled Arg‐Gly‐Asp (RGD) peptide derivatives have immense potential for non‐invasive monitoring of malignancies overexpressing integrin α_vβ₃ receptors. Easy availability of suitable radiotracers would augment the utility of this class of molecular imaging agents. Towards this, the present article describes the development of an improved lyophilized kit for the routine clinical formulation of [^99mTc]Tc complex of HYNIC‐conjugated dimeric cyclic RGD peptide derivative E‐[c(RGDfK)]₂ (E = glutamic acid, f = phenyl alanine, K = lysine) without using Sn²⁺ and systematic evaluation of its efficacy. Five batches of the kits were prepared, and [^99mTc]Tc‐HYNIC‐E[c(RGDfK)]₂ radiotracer was synthesized with high radiochemical purity (98.6 ± 0.5%) and specific activity (124.8 GBq/μmol) using the kits. Biodistribution studies in C57BL/6 mice bearing melanoma tumor exhibited significant accumulation of the radiotracer in tumor (5.32 ± 0.56 %ID/g at 60 min p.i.), and this uptake was also found to be receptor‐specific by blocking studies. Preliminary human clinical investigations carried out in 10 breast cancer patients revealed high radiotracer uptake in the tumor along with good tumor‐to‐background contrast. The developed kit formulation showed an exceptionally high shelf‐life of at least 18 months. These results demonstrated promising attributes of the developed kit formulation and warrant more extensive clinical investigations.     

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.     

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.     

Synthesis of [18F]3‐[1‐(3‐fluoropropyl)‐(S)‐pyrrolidin‐2‐ylmethoxy]pyridine ([18F]NicFP): a potential α4β2 nicotinic acetylcholine receptor radioligand for PET

Nicotinic acetylcholine receptors are widely distributed throughout the human brain and are believed to play a role in several neurological and psychiatric disorders. In order to identify an effective PET radioligand for in vivo assessment of the α4β2 subtype of nicotinic receptor, we synthesized [¹⁸F]3‐[1‐(3‐fluoropropyl)‐(S)‐pyrrolidin‐2‐ylmethoxy]pyridine (NicFP). The in vitro K_D of NicFP was determined to be 1.1 nM, and the log P value obtained by HPLC analysis of the unlabelled standard was found to be 2.2. The radiosynthesis of [¹⁸F]NicFP was carried out by a nucleophilic substitution reaction of anhydrous [¹⁸F]fluoride and the corresponding mesylate precursor. After purification by HPLC, the radiochemical yield was determined to be 11.3±2.1% and the specific activity was 0.47±0.18 Ci/μmol (EOS, n = 3). The time of synthesis and purification was 99±2 min. The final product was prepared as a sterile saline solution suitable for in vivo use. Copyright © 2003 John Wiley & Sons, Ltd.     

Synthesis and evaluation of 90Y‐DOTA‐Colchicine conjugate in murine fibrosarcoma model

Colchicine is a cytotoxic bioactive alkaloid that exhibits its action by microtubular binding. With an aim to develop a tumor targeted radio‐therapeutic agent, colchicine has been functionalized to trimethylcolchicinic acid and conjugated to the isothiocyanato derivative of DOTA (1,4,7,10‐tetraaza cyclododecane tetracetic acid). DOTA coupled colchicine was radiolabeled with ⁹⁰Y, one of the most commonly used therapeutic radioisotope. Complexation of 200 µg of the conjugate with ⁹⁰Y was carried out at pH 4.5 with an incubation time of 45 min at 70°C. Complexation yield of ⁹⁰Y‐DOTA‐NCS‐colchicine was confirmed to be >98% using C‐18 reverse phase HPLC system. ⁹⁰Y‐colchicine complex could be differentiated from ⁹⁰Y‐p‐NCS‐benzyl‐DOTA on the basis of difference in their retention times 8 and 4 min, respectively in a standardized HPLC system. Biodistribution studies in Swiss mice fibrosarcoma tumor model showed an uptake of ～0.8% ID/g tumor at 3 h.p.i. that was retained till 24 h.p.i. ⁹⁰Y‐DOTA‐NCS‐colchicine complex showed excellent pharmacokinetics with major portion of the radioactivity being excreted out within 3 h.p.i. and no accumulation of radioactivity in vital organs. Copyright © 2006 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.     

Analogues of arginine vasopressin and its agonist and antagonist modified in the N‐terminal part of the molecule with l‐β‐homophenylalanine

Abstract: In continuation of our efforts to elucidate the role of positions 2 and 3 in arginine vasopressin (AVP) and its analogues, we designed and synthesized peptides modified in these positions with l ‐β‐homophenylalanine (β‐Hph). Two of them had just this single modification, the next two peptides are analogues of the V₂ agonist, namely [3‐mercaptopropionic acid (Mpa)¹]AVP (dAVP). The last two compounds were designed by substitution of positions 2 or 3 of a potent V_1a antagonist, [1‐mercaptocyclohexaneacetic acid (Cpa)¹]AVP, with β‐Hph. All the peptides were tested for their pressor and antidiuretic and uterotonic in vitro activities in the rat. All the activities tested have been found to be significantly decreased. Three analogues, i.e. [Mpa¹,β‐Hph²]AVP, [Cpa¹,β‐Hph²]AVP, [Cpa¹,β‐Hph³]AVP, turned out to be uterotonic antagonists with pA₂ = 6.3 ± 0.2, 6.3 ± 0.1, 6.0 ± 0.3 respectively. The last one exhibited antipressor properties also (pA₂ = 6.4 ± 0.1).     

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 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.     

Synthesis of an 18F‐labelled high affinity β1‐adrenoceptor PET radioligand based on ICI 89,406

To date, some non‐selective β‐adrenoceptor (β‐AR) positron emission tomography (PET) radioligands are in clinical use, but no PET radioligand for the selective imaging of cardiac β₁‐ARs is clinically available. Therefore, the aim of this study was to develop a potential high‐affinity PET radioligand for the β₁‐subtype of ARs. Here, the synthesis and in vitro evaluation of (S)‐ and (R)‐N‐[2‐[3‐(2‐cyano‐phenoxy)‐2‐hydroxy‐propylamino]‐ethyl]‐N′‐[4‐(2‐fluoro‐ethoxy)‐phenyl]‐urea ( 8a–b ), derivatives of the well‐characterized β₁‐AR selective antagonist, ICI 89,406, are described. The (S)‐isomer 8a shows both higher β₁‐AR selectivity and β₁‐AR affinity than the (R)‐enantiomer 8b (selectivity: 40 800 vs 1580; affinity: K_I1=0.049 nM vs K_I1=0.297 nM). Therefore, the ¹⁸F‐labelled analogue 8e of compound 8a was synthesized. While the direct nucleophilic ¹⁸F‐fluorination of the tosylate precursor 8d produced 8e in low radiochemical yields (?2.9% decay‐corrected) and specific activities (?3.5 GBq/µmol at the end of synthesis (EOS), n=9) the alternative two‐step synthesis of 8e from ethylene glycol di‐p‐tosylate 9 , [¹⁸F]fluoride ion and phenol precursor 8f gave satisfying results (16.4±3.2% radiochemical yield (decay‐corrected), 99.7±0.5% radiochemical purity, 40±8 GBq/µmol specific activity at the EOS within 174±3 min from the end of bombardment (EOB) (n=5)). Copyright © 2006 John Wiley & Sons, Ltd.     

Conformational investigation of α,β-dehydropeptides. IX. N-Acetyl-(E)-α,β-dehydrobutyrine N′-methylamide: stereoelectronic properties from infrared and theoretical studies*

Abstract: : The Fourier transform infrared spectra of Ac-(E)-ΔAbu-NHMe were analyzed to determine the predominant solution conformation (s) of this (E)-α,β-dehydropeptide-related compound and the electron density perturbation in its amide groups. The measurements were performed in dichloromethane and acetonitrile in the region of mode v_s (N–H), amide I, amide II and v_s (C^α= C^β). The equilibrium geometrical parameters, calculated by a method based on the density functional theory with the B3LYP functional and the 6–31G* basis set, were used to support spectroscopic interpretation and gain some deeper insight into the molecule. The experimental and theoretical data were compared with those of three previously described molecules: isomeric Ac-(Z)-ΔAbu-NHMe, Ac-ΔAla-NHMe, which is deprived of any β-substituent, and saturated species Ac-Abu-NHMe. The titled compound assumes two conformational states in equilibrium in the DCM solution. One conformer is extended almost fully and like Ac-ΔAla-NHMe is C₅ hydrogen-bonded. The other adopts a warped C₅ structure similar to that of Ac-(Z)-ΔAbu-NHMe. The C₅ hydrogen bond, unlike the H-bond in Ac-ΔAla-NHMe, is disrupted by acetonitrile. The resonance within the N-terminal amide groups in either of the (E)-ΔAbu conformers is not as well developed as the resonance in Ac-Abu-NHMe. However, these N-terminal groups, compared with the other unsaturated compounds, constitute better resonance systems in each conformationally related couple: the C₅ hydrogen-bonded Ac-(E)-ΔAbu-NHMe/Ac-ΔAla-NHMe and the warped C₅ Ac-(E)-ΔAbu-NHMe/Ac-(Z)-ΔAbu-NHMe. The resonance within the C-terminal groups of the latter couple apparently is similar, but less developed than the resonance in Ac-Abu-NHMe. The electron distribution within the C-terminal group of the hydrogen-bonded C₅ (E)-ΔAbu conformer apparently is determined mainly by the electron influx from the C^α= C^β double bond.     

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.     

Expression and regulation of the bile acid transporter,OSTα‐OSTβ in rat and human intestine and liver

The regulation of the OSTα and OSTβ expression was studied in the rat jejunum, ileum, colon and liver and in human ileum and liver by ligands for the farnesoid X receptor (FXR), pregnane X receptor (PXR), vitamin D receptor (VDR) and glucocorticoid receptor (GR) using precision cut tissue slices. The gradient of protein and mRNA expression in segments of the intestine for rOSTα and rOSTβ paralleled that of rASBT. OSTα and OSTβ mRNA expression, quantified by qRT‐PCR, in rat jejunum, ileum, colon and liver, and in human ileum and liver was positively regulated by FXR and GR ligands. In contrast, the VDR ligand, 1,25(OH)₂D₃ decreased the expression of rOSTα‐rOSTβ in rat intestine, but had no effect on human ileum, and rat and human liver slices. Lithocholic acid (LCA) decreased the expression of rOSTα and rOSTβ in rat ileum but induced OSTα‐OSTβ expression in rat liver slices, and human ileum and liver slices. The PXR ligand, pregnenolone‐16α carbonitrile (PCN) had no effect. This study suggest that, apart from FXR ligands, the OSTα and OSTβ genes are also regulated by VDR and GR ligands and not by PXR ligands. This study show that VDR ligands exerted different effects on OSTα‐OSTβ in the rat and human intestine and liver compared with other nuclear receptors, FXR, PXR, and GR, pointing to species‐ and organ‐specific differences in the regulation of OSTα‐OSTβ genes. Copyright © 2009 John Wiley & Sons, Ltd.     

New tools for the control of peptide conformation: the helicogenic Cα‐methyl,Cα‐cyclohexylglycine*

Abstract: The novel C^α‐tetrasubstituted α‐amino acid C^α‐methyl, C^α‐cyclohexylglycine was prepared by hydrogenation of its C^α‐methyl, C^α‐phenylglycine precursor. Terminally protected homodi‐, homotri‐, and homotetrapeptides from C^α‐methyl, C^α‐cyclohexylglycine and co‐oligopeptides to the pentamer level in combination with Gly or α‐aminoisobutyric acid residues were prepared by solution methods and fully characterized. The results of a conformational analysis, performed by use of Fourier transform infrared (FT‐IR) spectrophotomet absorption, ¹H NMR, and X‐ray diffraction techniques, support the contention that this C^α‐methylated, C^β‐trisubstituted aliphatic α‐amino acid is an effective β‐turn and 3₁₀‐helix inducer in tri‐ and longer peptides as its C^α‐methyl valine parent compound, but partially divergent from the corresponding aromatic C^α‐methyl, C^α‐diphenylmethylglycine residue, known to promote folded and fully extended structures to a significant extent in these oligomers.     

Preparation and preliminary biological evaluation of 177Lu‐labeled GluDTPA‐cyclo(RGDfK) for integrin ανβ3 receptor‐positive tumor targeting

Integrin α_νβ₃ is a receptor and is highly expressed on activated and proliferating endothelial cells during the growth and metastasis of solid tumors but not on resting endothelial cells and normal organs. Because RGD peptide binds to integrin α_νβ₃ receptor, a variety of radiolabeled RGD peptides have been evaluated for non‐invasive imaging of integrin α_νβ₃‐positive tumors. In an attempt to develop RGD‐based radiopharmaceuticals, a novel GluDTPA‐cyclo arginine‐glycine‐aspartic acid‐d ‐phenylalanine‐lysine (GluDTPA‐cycloRGDfK) was simply synthesized and radiolabeled with ¹⁷⁷Lu. Also, tumor targeting and retention of the radiolabeled complex were evaluated in U87MG glioma‐bearing mice. The ¹⁷⁷Lu‐labeled GluDTPA‐cyclo(RGDfK) was formulated with a high radiolabeling yield (>98%) under mild condition, and the radiochemical purity was sustained in both saline and serum for over 4 days at 37°C. The radiolabeled compounds were rapidly cleared from the blood pool and non‐target tissue. Tumor‐to‐blood ratio was 12.09 at 2 h post injection and increased to 134.67 at 24 h, while tumor to liver ratio was 2.01 at 24 h similar to that of 2 h. Though it is inappropriate for targeted therapy due to its low uptake in tumor (~ 1 %ID/g), the acceptable results on radiochemistry and biodistribution propose to take a further assessment for non‐invasive imaging and detection of integrin α_νβ₃‐positive tumors by applying diagnostic radionuclides. Copyright © 2011 John Wiley & Sons, Ltd.     

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 ).     

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 of (R)‐ and (S)‐[O‐methyl‐11C]N‐[2‐[3‐(2‐cyano‐phenoxy)‐2‐hydroxy‐propylamino]‐ethyl]‐N′‐(4‐methoxy‐phenyl)‐urea as candidate high affinity β1‐adrenoceptor PET radioligands

Molecular imaging and quantification of myocardial β₁‐adrenoceptor (AR) rather than total β‐AR density is of great clinical interest since cardiac biopsy studies suggest that myocardial β₁‐AR density is reduced in patients with chronic heart failure whereas cardiac β₂‐AR density may vary. Positron emission tomography (PET), with appropriate radioligands, offers the possibility to assess β‐AR density non‐invasively in humans. However, no PET radioligand for the selective imaging of cardiac β₁‐ARs is clinically available. Here some derivatives of the well characterized β₁‐AR selective antagonist, ICI 89,406, namely the enantiomers of N‐[2‐[3‐(2‐cyano‐phenoxy)‐2‐hydroxy‐propylamino]‐ethyl]‐N′‐(4‐hydroxy‐phenyl)‐urea ( 5a and 5b ) were synthesized and evaluated in vitro. The (R)‐isomer 5a was more β₁‐selective but has lower affinity than its (S)‐enantiomer 5b (β₁‐AR selectivity: 6100 vs 1240; β₁‐affinity: K₁ = 0.288 nM vs K₁ = 0.067 nM). Etherification of the analogous desmethyl precursors, 5e and 5f , respectively, with [¹¹C]iodomethane gave ¹¹C‐labelled versions of 5a and 5b , namely 5g and 5h , in 44 ± 5% radiochemical yield (decay‐corrected) and 97.4 ± 1.3% radiochemical purity with specific radioactivities of 26.4 ± 9.4 GBq/µmol within 41.2 ± 3.4 min from the end of bombardment (n = 14). 5g and 5h are now being evaluated as candidate radioligands for myocardial β₁‐ARs. Copyright © 2005 John Wiley & Sons, Ltd.     

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.