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.     

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.     

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 and radiolabelling of Re(CO)3‐β‐elemene derivatives as potential therapeutic radiopharmaceuticals

β‐Elemene, (1S, 2S, 4R)‐(?)‐(1‐methy‐1‐vinyl‐2,4‐diisopropenyl cyclohexane) is an anticancer agent from the Traditional Chinese Herb Medicinal. Three novel Re(CO)₃‐β‐elemene derivatives including their radioactive conjugates containing N,N,N tridentate ligands and tricarbonyl rhenium (complex 12, 13, 14) were synthesized. Their structures were characterized by infrared (IR), ¹H‐NMR and HRMS. Good radioactive yield (above 90%) and radioactive chemical purity with Re‐188 (above 95%) were obtained for all of the three derivatives (complex 15, 16, 17). The antiproliferative activity of non‐radioactive β‐elemene‐Re(CO)₃ derivatives on Lewis lung cancer cells and HeLa cell lines were evaluated by WST‐1 methods. The result shows substantial decrease in IC₅₀ values compared with the parent compound β‐elemene. The synthesis and radiosynthesis of β‐elemene tricarbonyl rhenium conjugates provide the possibility to find a new kind of potential radiopharmaceuticals on β‐elemene. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

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.     

Design,synthesis and biological evaluation of peptide‐NSAID conjugates for targeted cancer therapy

Linear arginine‐glycine‐aspartic acid (RGD) and asparagine‐glycine‐arginine (NGR) peptide‐nonsteroidal anti‐inflammatory drug conjugates were synthesized to evaluate their anticancer effect. Two well‐known targeting peptide sequences, RGD and NGR, were conjugated with naproxen and ibuprofen. It is expected that the RGD peptide selectively binds to α_v‐integrin receptors, which are highly expressed in cancer cells, and that the NGR peptide selectively targets aminopeptidase N (APN/CD13, EC 3.4.11.2), which is overexpressed in blood vessels of tumors. To investigate the impact of possible steric hindrance due to the attachment of the drug to the peptide, a linear six‐carbon linker (hexanoic acid) was also used as a spacer. Cytotoxic effects of the synthesized compounds were evaluated against several cancer cell lines, including MCF‐7, A2780 (α_vβ₃ positive), OVCAR3 (high α_vβ₃), HT‐1‐80, and SKOV‐3 cells (CD13 positive). The NGR conjugate forms of both ibuprofen and naproxen showed better activity against the SKOV‐3 tumor cell line. The improved binding of these conjugates to their receptors was confirmed by docking studies.     

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.     

Conformational investigation of α, β-dehydropeptides

The crystal structure of Ac-Pro-ΔVal-NHCH₃ was examined to determine the influence of the α,β-dehydrovaline residue on the nature of peptide conformation. The peptide crystallizes from methanol-diethyl ether solution at 4° in needle-shaped form in orthorhombic space group P2₁2₁2₁ with a= 11.384(2) Å, b = 13.277(2) Å, c = 9.942(1) Å. V = 1502.7(4) ų Z = 4, D_m= 1.17 g cm^?3 and D_c=1.18 g cm^?3 The structure was solved by direct methods using SHELXS-86 and refined to an R value of 0.057 for 1922 observed reflections. The peptide is found to adopt a β-bend between the type I and the type III conformation with φ₁=?68.3(4)°, ψ₁=? 20.1(4)°, φ₂=?73.5(4)°= and Ψ₂=?14.1(4)°=. An intramolecular hydrogen bond between the carbonyl oxygen of ith residue and the NH of (i+ 3)th residue stabilizes the β-bend. An additional intermolecular N.,.O hydrogen bond joins molecules into infinite chains. In the literature described crystal structures of peptides having a single α,β-dehydroamino acid residue in the (i+ 2) position and forming a β-bend reveal a type II conformation.     

Synthesis of 68Ga‐labeled NOTA‐RGD‐GE11 heterodimeric peptide for dual integrin and epidermal growth factor receptor‐targeted tumor imaging

Radiolabeled Arg‐Gly‐Asp (RGD) peptide analogs have been extensively studied for α_vβ₃ integrin‐targeted angiogenesis imaging. According to recently presented evidence, the dodecapeptide GE11 has high affinity to the epidermal growth factor receptor (EGFR), which is overexpressed in many types of cancer. Dual‐receptor molecular imaging probes with two different heterodimeric peptides exhibit improved cancer targeting efficacy. In the present study, the design and synthesis of a new RGD‐GE11 peptide heterodimer for dual α_vβ₃ integrin/EGFR‐targeted cancer imaging are described. The RGD‐GE11 heterodimer was linked with 6‐aminohexanoic acid (6‐Ahx) and cysteine and conjugated with 1,4,7‐triazacyclononane‐N,N′,N″‐triacetic acid (NOTA) to form NOTA‐RGD‐cys‐6‐Ahx‐GE11. The monomeric peptides, NOTA‐cys‐6‐Ahx‐GE11 and c(RGDyK), were formed by a peptide synthesizer. The peptide heterodimer NOTA‐RGD‐GE11 was obtained by NOTA‐cys‐6‐Ahx‐GE11 and maleimidopropyl‐c(RGDyK) conjugation with a thioether linkage. The NOTA peptide conjugate was labeled with freshly eluted ⁶⁸Ga and purified using reversed‐phase high‐performance liquid chromatography. The ⁶⁸Ga‐NOTA‐RGD‐cys‐6‐Ahx‐GE11 was successfully prepared, in this study, with a radiochemical yield of 85% and a radiochemical purity of >98%. These results warrant further investigation of this heterodimeric peptide's binding affinity to the receptors.     

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 ).     

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,biology, NMR and conformation studies of the topographically constrained δ‐opioid selective peptide analogs of [β‐iPrPhe3]deltorphin I

Abstract: Replacement of Phe³ in the endogenous δ‐opioid selective peptide deltorphin I with four optically pure stereoisomers of the topographically constrained, highly hydrophobic novel amino acid β‐isopropylphenylalanine (β‐iPrPhe) produced four pharmacologically different deltorphin I peptidomimetics. Radiolabeled ligand‐binding assays and in vitro biological evaluation indicate that the stereoconfiguration of the iPrPhe residue plays a crucial role in determining the binding affinity, bioactivity and selectivity of [β‐iPrPhe³]deltorphin I analogs: a (2S,3R) configuration of the iPrPhe³ residue in [β‐iPrPhe³]deltorphin I provided the most desirable biological properties with binding affinity (IC₅₀ = 2 n m ), bioassay potency (IC₅₀ = 1.23 n m in MVD assay) and exceptional selectivity for the δ‐opioid receptor over the µ‐opioid receptor (30 000). Further conformational studies based on two‐dimensional NMR and computer‐assisted molecular modeling suggested a model for the possible bioactive conformation in which the Tyr¹ and (2S,3R)‐β‐iPrPhe³ residues adopt trans side‐chain conformations, and the linear peptide backbone favors a distorted β‐turn conformation.     

Protective effects of β‐sheet breaker α/β‐hybrid peptide against amyloid β‐induced neuronal apoptosis in vitro

Alzheimer's disease is most common neurodegenerative disorder and is characterized by increased production of soluble amyloid‐β oligomers, the main toxic species predominantly formed from aggregation of monomeric amyloid‐β (Aβ). Increased production of Aβ invokes a cascade of oxidative damages to neurons and eventually leads to neuronal death. This study was aimed to investigate the neuroprotective effects of a β‐sheet breaker α/β‐hybrid peptide (BSBHp) and the underlying mechanisms against Aβ₄₀‐induced neurotoxicity in human neuroblastoma SH‐SY5Y cells. Cells were pretreated with the peptide Aβ₄₀ to induce neurotoxicity. Assays for cell viability, cell membrane damage, cellular apoptosis, generation of reactive oxygen species (ROS), intracellular free Ca²⁺, and key apoptotic protein levels were performed in vitro. Our results showed that pretreatment with BSBHp significantly attenuates Aβ₄₀‐induced toxicity by retaining cell viability, suppressing generation of ROS, Ca²⁺ levels, and effectively protects neuronal apoptosis by suppressing pro‐apoptotic protein Bax and up‐regulating antiapoptotic protein Bcl‐2. These results suggest that α/β‐hybrid peptide has neuroprotective effects against Aβ₄₀‐induced oxidative stress, which might be a potential therapeutic agent for treating or preventing neurodegenerative diseases.     

Therapeutic 188Re‐lanreotide: determination of radiopharmacokinetic parameters in rats

Objectives The radiopharmacokinetic parameters of the therapeutic radiopharmaceutical ¹⁸⁸Re‐lanreotide were compared in rats implanted with hepatocarcinoma tumours (n= 18) and healthy rats (n= 18). Methods Rats were injected with approximately 1.8 MBq ¹⁸⁸Re‐lanreotide (0.1 ml) via the tail vein and blood samples were obtained. The activity per gram of tissue (%IA/g) was calculated and the radiopharmacokinetic parameters determined. Data were fitted using a two‐compartment model. Key findings Significant differences were found between healthy and hepatoma rats for beta elimination half‐life (22.56 vs 48.14 h); transference constants K₁₀ (k_e) (6.44 vs 3.05 h^‐1) and K₁₂ (2.76 vs 7.09 h^‐1); volume of distribution (2.06 vs 5.45 ml); mean residence time (66.58 vs 95.50 h) and apparent volume of distribution at steady state (131.30 vs 810.37 ml). The tumour/organ ratios after 24 h were 11.20 for tumour/muscle, 8.00 for tumour/liver and 7.72 for tumour/bone. The scintigraphic images obtained therefore had high resolution. Conclusions ¹⁸⁸Re‐lanreotide had a prolonged beta elimination half‐life and increased volume of distribution in rats with hepatocellular carcinoma. This may be beneficial in the diagnosis and therapy of metastatic lesions in patients with cancer.     

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.     

Labedipinedilol‐A: A vanilloid‐based α/β‐adrenoceptor blocker with calcium entry blocking and long‐acting antihypertensive properties

The combination of β‐adrenoceptor blockade and vasodilator action have proved highly useful in antihypertensive therapy. Studies of the mechanisms of action of labedipinedilol‐A that combine these effects within a single molecule are described in this report. Intravenous labedipinedilol‐A (0.1–1.0 mg/kg) produced dose‐dependent hypotensive and bradycardia responses for above 1.0 h, significantly different from nifedipine (0.5 mg/kg, i.v.)‐induced hypotensive and reflex tachycardia activities in pentobarbital‐anesthetized Wistar rats. Pretreatment with labedipinedilol‐A also inhibited phenylephrine (20 μg/kg, i.v.)‐induced hypertensive and (‐)isoprenaline (0.5 μg/kg, i.v.)‐induced tachycardia effects. Oral administration of labedipinedilol‐A (5–50 mg/kg) in spontaneously hypertensive rats (SHR) reduced the blood pressure and heart rate for 24 h but did not increase heart rate. Labedipinedilol‐A (10^–7–10^–5 M) competitively antagonized (‐)isoprenaline (10^–10–10^–4M)‐induced positive chronotropic and inotropic effects of the isolated rat atria and tracheal relaxation responses of the isolated guinea pig tissues. Labedipinedilol‐A also prevented the rate‐increasing effects of increased extracellular Ca²⁺ (3.0–9.0 mM) in a concentration‐dependent manner. In the isolated rat aorta, labedipinedilol‐A competitively antagonized CaCl₂ and norepinephrine‐induced contractions with pKCa^–1 and pA₂ values of 8.46 ± 0.05 and 8.28 ± 0.03 and had a potent effect of inhibiting high K⁺‐induced vasocontraction. Furthermore, labedipinedilol‐A, in an equal antagonist activity, inhibited norepinephrine‐induced phasic and tonic contraction. In the cultured blood vessel smooth muscle cell (A7r5 cell line), KCl, norepinephrine, and Bay K 8644‐induced intracellular calcium changes were decreased after application of labedipinedilol‐A (10^–9–10^–6 M). The binding characteristics of labedipinedilol‐A were evaluated in [³H]CGP‐12177 binding to ventricle and lung and [³H]nitrendipine and [³H]prazosin binding to brain membranes in rats. The ‐logIC₅₀ values of labedipinedilol‐A for β₁‐, β₂‐, and α₁‐adrenoceptor and calcium channel, were 8.17 × 10^–7 M, 8.20 × 10^–7 M, 2.20 × 10^–8 M, and 2.46 × 10^–8 M, respectively. Labedipinedilol‐A‐induced sustained depressor effect was mainly attributed to its calcium entry and α‐adrenoceptor blocking activities in the blood vessel. Sustained bradycardia effect resulted from β‐adrenoceptor and calcium entry blocking, which deleted the sympathetic activation‐associated reflex tachycardia in the heart. Drug Dev. Res. 49:94–108, 2000. © 2000 Wiley‐Liss, Inc.     

Role of azaamino acid residue in β‐turn formation and stability in designed peptide

Abstract: The structural perturbation induced by C^αH→N^α exchange in azaamino acid‐containing peptides was predicted by ab initio calculation of the 6‐31G* and 3‐21G* levels. The global energy‐minimum conformations for model compounds, For‐azaXaa‐NH₂ (Xaa = Gly, Ala, Leu) appeared to be the β‐turn motif with a dihedral angle of φ = ± 90°, ψ = 0°. This suggests that incorporation of the azaXaa residue into the i + 2 position of designed peptides could stabilize the β‐turn structure. The model azaLeu‐containing peptide, Boc‐Phe‐azaLeu‐Ala‐OMe, which is predicted to adopt a β‐turn conformation was designed and synthesized in order to experimentally elucidate the role of the azaamino acid residue. Its structural preference in organic solvents was investigated using ¹H NMR, molecular modelling and IR spectroscopy. The temperature coefficients of amide protons, the characteristic NOE patterns, the restrained molecular dynamics simulation and IR spectroscopy defined the dihedral angles [ (φ_i+1, ψ_i+1) (φ_i+2, ψ_i+2)] of the Phe‐azaLeu fragment in the model peptide, Boc‐Phe‐azaLeu‐Ala‐OMe, as [(?59^°, 127^°) (107^°, ?4^°)]. This solution conformation supports a βII‐turn structural preference in azaLeu‐containing peptides as predicted by the quantum chemical calculation. Therefore, intercalation of the azaamino acid residue into the i + 2 position in synthetic peptides is expected to provide a stable β‐turn formation, and this could be utilized in the design of new peptidomimetics adopting a β‐turn scaffold.     

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.