Copper-64 Labeled PEGylated Exosomes for In Vivo Positron Emission Tomography and Enhanced Tumor Retention.

Exosomes have attracted tremendous attention due to their important role in physiology, pathology, and oncology, as well as promising potential in biomedical applications. Although great efforts have been dedicated to investigating their biological properties and applications as natural cancer drug-delivery systems, the systemic biodistribution of exosomes remains underexplored. In addition, exosome-based drug delivery is inevitably hindered by the robust liver clearance, leading to suboptimal tumor retention and therapeutic efficiency. In this study, we report one of the first examples using in vivo positron emission tomography (PET) for noninvasive monitoring of copper-64 (64Cu)-radiolabeled polyethylene glycol (PEG)-modified exosomes, achieving excellent imaging quality and quantitative measurement of blood residence and tumor retention. PEGylation not only endowed exosomes with a superior pharmacokinetic profile and great accumulation in the tumor versus traditionally reported native exosomes but also reduced premature hepatic sequestration and clearance of exosomes, findings that promise enhanced therapeutic delivery efficacy and safety in future studies. More importantly, this study provides important guidelines about surface engineering, radiochemistry, and molecular imaging in obtaining accurate and quantitative biodistribution information on exosomes, which may benefit future exploration in the realm of exosomes.

Metformin Reduces Renal Uptake of Radiotracers and Protects Kidneys from Radiation-Induced Damage.

Metformin is the most widely prescribed drug for type 2 diabetes. Chemically, metformin is a hydrophilic base that functions as an organic cation, suggesting that it may have the capacity to inhibit the tubular reabsorption of peptide radiotracers. The purpose of this study was to investigate whether metformin could reduce renal uptake of peptidyl radiotracers and serve as a radioprotective agent for peptide receptor radionuclide therapy (PRRT). METHODS: We used two radiolabeled peptides: a 68Ga-labeled cyclic (TNYL-RAW) peptide (68Ga-NOTA-c(TNYL-RAW) (NOTA: 1,4,7 triazacyclononane-1,4,7-trisacetic acid) targeting EphB4 receptors and an 111In- or 64Cu-labeled octreotide (111In/64Cu-DOTA-octreotide) (DOTA: 1,4,7,10 triazacyclododecane-1,4,7,10-tetraacetic acid) targeting somatostatin receptors. Each radiotracer was injected intravenously into normal Swiss mice or tumor-bearing nude mice in the presence or absence of metformin administered intravenously or orally. Micropositron emission tomography or microsingle-photon emission computed tomography images were acquired at different times after radiotracer injection, and biodistribution studies were performed at the end of the imaging session. To assess the radioprotective effect of metformin on the kidneys, normal Swiss mice received two doses of 111In-DOTA-octreotidein the presence or absence of metformin, and renal function was analyzed via blood chemistry and histology. RESULTS: Intravenous injection of metformin with 68Ga-NOTA-c(TNYL-RAW) or 111In-DOTA-octreotide reduced the renal uptake of the radiotracer by 60% and 35%, respectively, compared to uptake without metformin. These reductions were accompanied by greater uptake in the tumors for both radiolabeled peptides. Moreover, the renal uptake of 111In-DOTA-octreotide was significantly reduced when metformin was administered via oral gavage. Significantly more radioactivity was recovered in the urine collected over a period of 24 h after intravenous injection of 64Cu-DOTA-octreotide in mice that received oral metformin than in mice that received vehicle. Finally, coadministration of 111In-DOTA-octreotide with metformin mitigated radio-nephrotoxicity. CONCLUSION: Metformin inhibits kidney uptake of peptidyl radiotracers, protecting the kidney from nephrotoxicity. Further studies are needed to elucidate the mechanisms of these finding and to optimize mitigation of radiation-induced damage to kidney in PRRT.

Integrin αvß3-Targeted [64Cu]CuS Nanoparticles for PET/CT Imaging and Photothermal Ablation Therapy.

Copper sulfide (CuS) nanoparticles have been considered one of the most clinical relevant nanosystems because of their straightforward chemistry, small particle size, low toxicity, and intrinsic theranostic characteristics. In our previous studies, radioactive [64Cu]CuS nanoparticles were successfully developed to be used as efficient radiotracers for positron emission tomography and for photothermal ablation therapy of cancer cells using near-infrared laser irradiation. However, the major challenge of CuS nanoparticles as a theranostic platform is the lack of a means for effective targeted delivery to the tumor site. To overcome this challenge, we designed and synthesized angiogenesis-targeting [64Cu]CuS nanoparticles, which are coupled with cyclic RGDfK peptide [c(RGDfK)] through polyethylene glycol (PEG) linkers using click chemistry. In assessing their tumor-targeting efficacy, we found that the tumor uptakes of [64Cu]CuS-PEG-c(RGDfK) nanoparticles at 24 h after intravenous injection were significantly greater (8.6% ± 1.4% injected dose/gram of tissue) than those of nontargeted [64Cu]CuS-PEG nanoparticles (4.3% ± 1.2% injected dose/gram of tissue, p < 0.05). Irradiation of tumors in mice administered [64Cu]CuS-PEG-c(RGDfK) nanoparticles induced 98.7% necrotic areas. In contrast, irradiation of tumors in mice administered nontargeted CuS-PEG nanoparticles induced 59% necrotic areas ( p < 0.05). The angiogenesis-targeting [64Cu]CuS nanoparticles may serve as a promising platform for image-guided ablation therapy with high efficacy and minimal side effects in future clinical translation of this novel class of multifunctional nanomaterials.

Design, Synthesis, and Biological Evaluation of 68Ga-DOTA-PA1 for Lung Cancer: A Novel PET Tracer for Multiple Somatostatin Receptor Imaging.

Most of the radiolabeled somatostatin analogues (SSAs) are specific for subtype somatostatin receptor 2 (SSTR2). Lack of ligands targeting other subtypes of SSTRs, especially SSTR1, SSTR3, and SSTR5, limited their applications in tumors of low SSTR2 expression, including lung tumor. In this study, we aimed to design and synthesize a positron emission tomography (PET) radiotracer targeting multi-subtypes of SSTRs for PET imaging. PA1 peptide and its conjugate with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator or fluorescein isothiocyanate (FITC) at the N-terminal of the lysine position were synthesized. 68Ga was chelated to DOTA-PA1 to obtain 68Ga-DOTA-PA1 radiotracer. The stability, lipophilicity, binding affinity, and binding specificity of 68Ga-DOTA-PA1 and FITC-PA1 were evaluated by various in vitro experiments. Micro-PET imaging of 68Ga-DOTA-PA1 was performed in nude mice bearing A549 lung adenocarcinoma, as compared with 68Ga-DOTA-(Tyr3)-octreotate (68Ga-DOTA-TATE). Histological analysis of SSTR expression in A549 tumor tissues and human tumor tissues was conducted using immunofluorescence staining and immunohistochemical assay. 68Ga-DOTA-PA1 had high radiochemical yield and radiochemical purity of over 95% and 99%, respectively. The radiotracer was stable in vitro in different buffers over a 2 h incubation period. Cell uptake of 68Ga-DOTA-PA1 was 1.31-, 1.33-, and 1.90-fold that of 68Ga-DOTA-TATE, which has high binding affinity only for SSTR2, after 2 h incubation in H520, PG, and A549 lung cancer cell lines, respectively. Micro-PET images of 68Ga-DOTA-PA1 showed that the PET imaging signal correlated with the total expression of SSTRs, instead of SSTR2 only, which was measured by Western blotting and immunofluorescence analysis in mice bearing A549 tumors. In summary, a novel PET radiotracer, 68Ga-DOTA-PA1, targeting multi-subtypes of SSTRs, was successfully synthesized and was confirmed to be useful for PET imaging. It may have potential as a noninvasive PET radiotracer for imaging SSTR-positive tumors.

Hepatocellular Carcinoma: Intra-arterial Delivery of Doxorubicin-loaded Hollow Gold Nanospheres for Photothermal Ablation-Chemoembolization Therapy in Rats.

Purpose To determine if combretastatin A-4 phosphate disodium (CA4P) can enhance the tumor uptake of doxorubicin (Dox)-loaded, polyethylene glycol (PEG)-coated hollow gold nanospheres (HAuNS) mixed with ethiodized oil for improved photothermal ablation (PTA)-chemoembolization therapy (CET) of hepatocellular carcinoma (HCC) in rats. Materials and Methods Animal experiments were approved by the institutional animal care and use committee and performed from February 2014 to April 2015. Male Sprague-Dawley rats (n = 45; age, 12 weeks) were inoculated with N1S1 HCC cells in the liver, and 8 days later, were randomly divided into two groups of 10 rats. Group 1 rats received intrahepatic arterial injection of PEG-HAuNS and ethiodized oil alone; group 2 received pretreatment with CA4P and injection of PEG-HAuNS and ethiodized oil 5 minutes later. The gold content of tumor and liver tissue at 1 hour or 24 hours after injection was quantified by using neutron activation analysis (n = 5 per time point). Five rats received pretreatment CA4P, PEG-copper 64-HAuNS, and ethiodized oil and underwent micro-positron emission tomography (PET)/computed tomography (CT). In a separate study, three groups of six rats with HCC were injected with saline solution (control group); CA4P, Dox-loaded PEG-coated HAuNS (Dox@PEG-HAuNS), and ethiodized oil (CET group); or CA4P, Dox@PEG-HAuNS, ethiodized oil, and near-infrared irradiation (PTA-CET group). Temperature was recorded during laser irradiation. Findings were verified at postmortem histopathologic and/or autoradiographic examination. Wilcoxon rank-sum test and Pearson correlation analyses were performed. Results PEG-HAuNS uptake in CA4P-pretreated HCC tumors was significantly higher than that in non-CA4P-pretreated tumors at both 1 hour (P < .03) and 24 hours (P < .01). Mean ± standard deviation of tumor-to-liver PEG-HAuNS uptake ratios at 1 hour and 24 hours, respectively, were 5.63 ± 3.09 and 1.68 ± 0.77 in the CA4P-treated group and 1.29 ± 2.40 and 0.14 ± 0.11 in the non-CA4P-treated group. Micro-PET/CT allowed clear delineation of tumors, enabling quantitative imaging analysis. Laser irradiation increased temperature to 60°C and 43°C in the tumor and adjacent liver, respectively. Mean HCC tumor volumes 10 days after therapy were 1.68 cm3 ± 1.01, 3.96 cm3 ± 1.75, and 6.13 cm3 ± 2.27 in the PTA-CET, CET, and control groups, respectively, with significant differences between the PTA-CET group and other groups (P < .05). Conclusion CA4P pretreatment caused a higher concentration of Dox@PEG-HAuNS to be trapped inside the tumor, thereby enhancing the efficacy of anti-HCC treatment with PTA-CET in rats. © RSNA, 2016 Online supplemental material is available for this article.

Development of a targeted gene vector platform based on simian adenovirus serotype 24.

Efforts to develop adenovirus vectors suitable for genetic interventions in humans have identified three major limitations of the most frequently used vector prototype, human adenovirus serotype 5 (Ad5). These limitations--widespread preexisting anti-Ad5 immunity in humans, the high rate of transduction of normal nontarget tissues, and the lack of target-specific gene delivery--justify the exploration of other Ad serotypes as vector prototypes. In this paper, we describe the development of an alternative vector platform using simian Ad serotype 24 (sAd24). We found that sAd24 virions formed unstable complexes with blood coagulation factor X and, because of that, transduced the liver and other organs at low levels when administered intravenously. The overall pattern of biodistribution of sAd24 particles was similar, however, to that of Ad5, and the intravenously injected sAd24 was cleared by Kupffer cells, leading to their depletion. We modified the virus's fiber protein to design a Her2-specific derivative of sAd24 capable of infecting target human tumor cells in vitro. In the presence of neutralizing anti-Ad5 antibodies, Her2-mediated infection with targeted sAd24 compared favorably to that with the Ad5-derived vector. When used to target Her2-expressing tumors in animals, this fiber-modified vector achieved a higher level of gene transfer to metastasis-containing murine lungs than to tumor-free lungs. In aggregate, these studies provide important insights into sAd24 biology, identify its advantages and limitations as a vector prototype, and are thus essential for further development of an sAd24-based gene delivery platform.

Iodine scanning of a phenazine inhibitor of vacuolar sorting.

Affinity reagents are often used to address the target identification problem in chemical genetics. The design of such reagents so that the linker does not occlude interactions with protein targets is an ongoing challenge. This work describes a systematic approach to synthesize derivatives of a bioactive that should avoid interference with binding to targets and be readily converted to affinity reagents.

Targeted photothermal ablation of murine melanomas with melanocyte-stimulating hormone analog-conjugated hollow gold nanospheres.

PURPOSE: To develop melanoma-targeted hollow gold nanospheres (HAuNS) and evaluate their potential utility for selective photothermal ablation in melanoma. EXPERIMENTAL DESIGN: A new class of photothermal coupling agents based on HAuNS was synthesized. HAuNS were stabilized with polyethylene glycol (PEG) coating and attached with alpha-melanocyte-stimulating hormone (MSH) analog, [Nle4,D-Phe7]alpha-MSH (NDP-MSH), which is a potent agonist of melanocortin type-1 receptor overexpressed in melanoma. The intracellular uptake of the NDP-MSH-conjugated PEGylated HAuNS (NDP-MSH-PEG-HAuNS) and the distribution of beta-arrestin were examined in murine B16/F10 melanoma cells. The biodistribution of NDP-MSH-PEG-HAuNS was assessed at 4 hours post i.v. injection in tumor-bearing nude mice. Photothermal ablation effect of the nanoparticles was evaluated both histologically using excised tissue and functionally by [18F]fluorodeoxyglucose positron emission tomography. RESULTS: NDP-MSH-PEG-HAuNS consist only of a thin gold wall with hollow interior (outer diameter, 43.5 +/- 2.3 nm; shell thickness, 3-4 nm), which displays strong and tunable resonance absorption in near-IR region (peak, 808 nm). The nanoparticles were specifically taken up by melanoma cells, which initiated the recruitment of beta-arrestins, the adapters to link the activated G-protein-coupled receptors to clathrin, indicating the involvement of receptor-mediated endocytosis. This resulted in enhanced extravasation of NDP-MSH-PEG-HAuNS from tumor blood vessels and their dispersion into tumor matrix compared with nonspecific PEGylated HAuNS. Successful selective photothermal ablation of B16/F10 melanoma with targeted HAuNS was confirmed by histologic and [18F]fluorodeoxyglucose positron emission tomography evaluation at 24 hours post near IR-region laser irradiation at a low-dose energy of 30 J/cm2. CONCLUSION: NDP-MSH-PEG-HAuNS have the potentials to mediate targeted photothermal ablation of melanoma.

Evaluation of Pan-SSTRs Targeted Radioligand [64Cu]NOTA-PA1 Using Micro-PET Imaging in Xenografted Mice.

64Cu-labeled new pan-somatostatin receptors (pan-SSTRs) probe PA1 was synthesized, characterized, and evaluated by in vitro and in vivo experiments. [64Cu]NOTA-PA1 was obtained with high specific activity, high radiochemical purity, and good stability. Cell uptake of [64Cu]NOTA-PA1 was higher than that of [64Cu]DOTA-TATE in MCF-7, A549, BGC823, and HT-29 cell lines. [64Cu]NOTA-PA1 showed high binding affinity for SSTRs expressed in A549 cells. The in vivo biodistribution and micropositron emission tomography (micro-PET) imaging studies of [64Cu]NOTA-PA1 revealed good detection ability in MCF-7 and A549 xenografted nude mice. The radiosynthesis, quality control, and preliminary biological evaluation of [64Cu]NOTA-PA1 have broaden the application of radiolabeled octreotide for SSTRs imaging, which could act as a potential multisubtypes targeted radiotracer for imaging SSTRs-positive tumors.

Concurrent Injection of Unlabeled Antibodies Allows Positron Emission Tomography Imaging of Programmed Cell Death Ligand 1 Expression in an Orthotopic Pancreatic Tumor Model.

Purpose: Among the treatment options for pancreatic ductal adenocarcinoma (PDAC) are antibodies against the programmed cell death receptor 1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway. Positron emission tomography (PET) has been successfully used to assess PD-1/PD-L1 signaling in subcutaneous tumor models, but orthotopic tumor models are increasingly being recognized as a better option to accurately recapitulate human disease. However, when PET radiotracers have high uptake in the liver and spleen, it can obscure signals from the adjacent pancreas, making visualization of the response in orthotopic pancreatic tumors technically challenging. In this study, we first investigated the impact of radioisotope chelators on the biodistribution of 64Cu-labeled anti-PD-1 and anti-PD-L1 antibodies and compared the distribution profiles of anti-PD-1 and anti-PD-L1 antibodies. We then tested the hypothesis that co-injection of unlabeled antibodies reduces uptake of 64Cu-labeled anti-PD-L1 antibodies in the spleen and thereby permits accurate delineation of orthotopic pancreatic tumors in mice. Procedures: We established subcutaneous and orthotopic mouse models of PDAC using KRAS* murine pancreatic cancer cells with a doxycycline-inducible mutation of KRASG12D. We then (1) compared the biodistribution of 64Cu-labeled anti-PD-1 with 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA) and 2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) used as the chelators in the orthotopic model; (2) compared the biodistribution of [64Cu]Cu-NOTA-anti-PD-1 and [64Cu]Cu-NOTA-anti-PD-L1 in the orthotopic model; and (3) imaged subcutaneous and orthotopic KRAS* tumors with [64Cu]Cu-NOTA-anti-PD-L1 with and without co-injection of unlabeled anti-PD-L1 as the blocking agent. Results: [64Cu]Cu-NOTA-anti-PD-L1 was a promising imaging probe. By co-injection of an excess of unlabeled anti-PD-L1, background signals of [64Cu]Cu-NOTA-anti-PD-L1 from the spleen were significantly reduced, leading to a clear delineation of orthotopic pancreatic tumors. Conclusions: Co-injection with unlabeled anti-PD-L1 is a useful method for PET imaging of PD-L1 expression in orthotopic pancreatic cancer models.

Targeting Forward and Reverse EphB4/EFNB2 Signaling by a Peptide with Dual Functions.

The tyrosine kinase receptor EphB4 is frequently overexpressed in ovarian and other solid tumors and is involved in interactions between tumor cells and the tumor microenvironment, contributing to metastasis. Trans-interaction between EphB4 and its membrane-bound ligand ephrin B2 (EFNB2) mediates bi-directional signaling: forward EFNB2-to-EphB4 signaling suppresses tumor cell proliferation, while reverse EphB4-to-EFNB2 signaling stimulates the invasive and angiogenic properties of endothelial cells. Currently, no small molecule-based, dual-function, EphB4-binding peptides are available. Here, we report our discovery of a bi-directional ephrin agonist peptide, BIDEN-AP which, when selectively internalized via receptor-mediated endocytosis, suppressed invasion and epithelial-mesenchymal transition of ovarian cancer cells. BIDEN-AP also inhibited endothelial migration and tube formation. In vivo, BIDEN-AP and its nanoconjugate CCPM-BIDEN-AP significantly reduced growth of orthotopic ovarian tumors, with CCPM-BIDEN-AP displaying greater antitumor potency than BIDEN-AP. Both BIDEN-AP and CCPM-BIDEN-AP compromised angiogenesis by downregulating epithelial-mesenchymal transition and angiogenic pathways. Thus, we report a novel EphB4-based therapeutic approach against ovarian cancer.

Cisplatin-loaded hollow gold nanoparticles for laser-triggered release.

BACKGROUND: Hollow gold nanoparticles (HGNPs) exposed to near-infrared (NIR) light yield photothermal effects that can trigger a variety of biological effects for potential biomedical applications. However, the mechanism of laser-triggered drug release has not been studied before. METHODS: A tripeptide Ac-Glu-Glu-Cys-NH2 (Ac-EEC) was directly linked to the surface of HGNPs. The EEC-HGNPs conjugate was then complexed with cisplatin Pt(II) to give Ac-EEC(Pt)-HGNPs. Folic acid was introduced to the gold surface of Ac-EEC-HGNPs through a thioctic acid-terminated polyethylene glycol linker (F-PEG-TA) followed by complexation with Pt(II) to give F-Ac-EEC(Pt)-HGNPs. Laser treatment was instituted with a 15-ns pulsed laser at a repetition rate of 10 Hz. The released Pt(II) was quantified by inductively coupled plasma mass spectroscopy, and the nature of the released Pt-containing species was characterized by liquid chromatography-mass spectroscopy. The cytotoxicity was studied using the MTT assay. RESULTS: Pt(II) was released from Ac-EEC(Pt)-HGNPs via two modes: (1) sustained release through an inverse ligand exchange reaction with chloride ions and (2) rapid release through cleavage of the Au-S bond between the tripeptide linker and Au surface upon NIR laser irradiation. The folate (F) conjugate of the nanoconstruct, F-Ac-EEC(Pt)-HGNPs, in combination with laser treatment showed a significantly greater effect on cell mortality against folate-overexpressing human epidermoid carcinoma KB cells than F-Ac-ECC(Pt)-HGNPs alone after 24 h of incubation. CONCLUSIONS: These results demonstrate that the photothermal property of HGNPs can be used for dual-modality photothermal therapy and NIR laser-triggered platinum-based chemotherapy.

Native and engineered tropism of vectors derived from a rare species D adenovirus serotype 43.

Unique molecular properties of species D adenoviruses (Ads)-the most diverse yet underexplored group of Ads-have been used to develop improved gene vectors. The low seroprevalence in humans of adenovirus serotype 43 (Ad43), an otherwise unstudied species D Ad, identified this rare serotype as an attractive new human gene therapy vector platform. Thus, in this study we wished to assess biological properties of Ad43 essential to its vectorization. We found that (1) Ad43 virions do not bind blood coagulation factor X and cause low random transduction upon vascular delivery; (2) they clear host tissues more quickly than do traditionally used Ad5 vectors; (3) Ad43 uses CD46 as primary receptor; (4) Ad43 can use integrins as alternative primary receptors. As the first step toward vectorization of Ad43, we demonstrated that the primary receptor specificity of the Ad43 fiber can be altered to achieve infection via Her2, an established oncotarget. Whereas this modification required use of the Ad5 fiber shaft, the presence of this domain in chimeric virions did not make them susceptible for neutralization by anti-Ad5 antibodies.

Erythropoietin Stimulates Tumor Growth via EphB4.

While recombinant human erythropoietin (rhEpo) has been widely used to treat anemia in cancer patients, concerns about its adverse effects on patient survival have emerged. A lack of correlation between expression of the canonical EpoR and rhEpo's effects on cancer cells prompted us to consider the existence of an alternative Epo receptor. Here, we identified EphB4 as an Epo receptor that triggers downstream signaling via STAT3 and promotes rhEpo-induced tumor growth and progression. In human ovarian and breast cancer samples, expression of EphB4 rather than the canonical EpoR correlated with decreased disease-specific survival in rhEpo-treated patients. These results identify EphB4 as a critical mediator of erythropoietin-induced tumor progression and further provide clinically significant dimension to the biology of erythropoietin.

Stereoselective synthesis of novel dipeptide beta-turn mimetics targeting melanocortin peptide receptors.

[reaction: see text] The novel dipeptide beta-turn mimetic, 4,8-disubstituted azabicyclo[4.3.0]nonane amino acid ester (15), has been synthesized to serve as a peptide mimetic of the dipeptide Phe-Arg, which contains two important pharmacophore elements in melanotropin peptides. Introduction of side-chain functionality at C-8 was achieved by using beta-functionalized pyroglutamate (8) as a synthetic precursor. The side chain at C-4 was introduced by bromination of dehydroamino acid intermediate (10) followed by Suzuki cross-coupling.

Stereoselective bromination-suzuki cross-coupling of dehydroamino acids to form novel reverse-turn peptidomimetics: substituted unsaturated and saturated indolizidinone amino acids.

A general and efficient methodology has been developed to prepare the C4-substituted dipeptide reverse-turn mimetics unsaturated (9a, 10a) and saturated (11a) azabicyclo[4.3.0] alkane amino acid derivatives. The side chain was introduced by bromination of dehydroamino acid intermediates followed by Suzuki coupling. Hydrogenation of the bicyclic dehydroamino acid 9a afforded saturated bicyclic lactam 11a. This approach can be further explored for the synthesis of a variety of such beta-turn mimetics with aryl and alkyl side chain functionalities. [reaction: see text]

Stereoselective synthesis of individual isomers of Leu-enkephalin analogues containing substituted beta-turn bicyclic dipeptide mimetics.

Novel constrained beta-turn dipeptide mimetics, 8-phenyl thiaindolizidinone amino acids 3, have been synthesized stereoselectively and incorporated into Leu-enkephalin peptides as a replacement of dipeptide Gly3-Phe4 to afford four individual isomers of Leu-enkephalin analogues 6.