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1.
J Med Chem ; 63(9): 4496-4505, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32302130

RESUMO

The insertion of single 1,4-disubstituted 1,2,3-triazoles as metabolically stable bioisosteres of trans-amide bonds (triazole scan) was recently applied to the 177Lu-labeled tumor-targeting analog of minigastrin, [Nle15]MG11. The reported novel mono-triazolo-peptidomimetics of [Nle15]MG11 showed either improved resistance against enzymatic degradation or a significantly increased affinity toward the target receptor but never both. To enhance further the tumor-targeting properties of the minigastrin analogs, we studied conjugates with multiple amide-to-triazole substitutions for additive or synergistic effects. Promising candidates were identified by modification of two or three amide bonds, which yielded both improved stability and increased receptor affinity of the peptidomimetics in vitro. Biodistribution studies of radiolabeled multi-triazolo-peptidomimetics in mice bearing receptor-positive tumor xenografts revealed up to 4-fold increased tumor uptake in comparison to the all-amide reference compound [Nle15]MG11. In addition, we report here for the first time a linear peptidomimetic with three triazole insertions in its backbone and maintained biological activity.


Assuntos
Antineoplásicos/farmacologia , Gastrinas/farmacologia , Peptidomiméticos/farmacologia , Compostos Radiofarmacêuticos/farmacologia , Triazóis/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Desenho de Fármacos , Gastrinas/síntese química , Gastrinas/metabolismo , Gastrinas/farmacocinética , Humanos , Lutécio/química , Camundongos , Neoplasias/metabolismo , Peptidomiméticos/síntese química , Peptidomiméticos/metabolismo , Peptidomiméticos/farmacocinética , Ligação Proteica , Radioisótopos/química , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/metabolismo , Compostos Radiofarmacêuticos/farmacocinética , Receptor de Colecistocinina B/metabolismo , Triazóis/síntese química , Triazóis/metabolismo , Triazóis/farmacocinética
2.
J Med Chem ; 63(9): 4484-4495, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32302139

RESUMO

MG11 is a truncated analog of minigastrin, a peptide with high affinity and specificity toward the cholecystokinin-2 receptor (CCK2R), which is overexpressed by different tumors. Thus, radiolabeled MG11 derivatives have great potential for use in cancer diagnosis and therapy. A drawback of MG11 is its fast degradation by proteases, leading to moderate tumor uptake in vivo. We introduced 1,4-disubstituted 1,2,3-triazoles as metabolically stable bioisosteres to replace labile amide bonds of the peptide. The "triazole scan" yielded peptidomimetics with improved resistance to enzymatic degradation and/or enhanced affinity toward the CCK2R. Remarkably, our lead compound achieved a 10-fold increase in receptor affinity, resulting in a 2.6-fold improved tumor uptake in vivo. Modeling of the ligand-CCK2R complex suggests that an additional cation-π interaction of the aromatic triazole moiety with the Arg356 residue of the receptor is accountable for these observations. We show for the first time that the amide-to-triazole substitution strategy offers new opportunities in drug development that go beyond the metabolic stabilization of bioactive peptides.


Assuntos
Antineoplásicos/farmacologia , Gastrinas/farmacologia , Peptidomiméticos/farmacologia , Compostos Radiofarmacêuticos/farmacologia , Triazóis/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Feminino , Gastrinas/síntese química , Gastrinas/metabolismo , Gastrinas/farmacocinética , Humanos , Lutécio/química , Camundongos , Neoplasias/metabolismo , Peptidomiméticos/síntese química , Peptidomiméticos/metabolismo , Peptidomiméticos/farmacocinética , Ligação Proteica , Conformação Proteica , Radioisótopos/química , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/metabolismo , Compostos Radiofarmacêuticos/farmacocinética , Receptor de Colecistocinina B/metabolismo , Triazóis/síntese química , Triazóis/metabolismo , Triazóis/farmacocinética
3.
Dig Dis Sci ; 65(1): 189-203, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31297627

RESUMO

BACKGROUND AND AIMS: Nonalcoholic steatohepatitis (NASH) is a common inflammatory liver condition that may lead to cirrhosis and hepatocellular carcinoma (HCC). Risk factors for NASH include a saturated fat diet, altered lipid metabolism, and genetic and epigenetic factors, including microRNAs. Serum levels of cholecystokinin (CCK) are elevated in mice and humans that consume a high-saturated fat diet. CCK receptors (CCK-Rs) have been reported on fibroblasts which when activated can induce fibrosis; however, their role in hepatic fibrosis remains unknown. We hypothesized that elevated levels of CCK acting on the CCK-Rs play a role in the development of NASH and in NASH-associated HCC. METHODS: We performed a NASH Prevention study and Reversal study in mice fed a saturated fat 75% choline-deficient-ethionine-supplemented (CDE) diet for 12 or 18 weeks. In each study, half of the mice received untreated drinking water, while the other half received water supplemented with the CCK-R antagonist proglumide. CCK-R expression was evaluated in mouse liver and murine HCC cells. RESULTS: CCK receptor antagonist treatment not only prevented NASH but also reversed hepatic inflammation, fibrosis, and steatosis and normalized hepatic transaminases after NASH was established. Thirty-five percent of the mice on the CDE diet developed HCC compared with none in the proglumide-treated group. We found that CCK-BR expression was markedly upregulated in mouse CDE liver and HCC cells compared with normal hepatic parenchymal cells, and this expression was epigenetically regulated by microRNA-148a. CONCLUSION: These results support the novel role of CCK receptors in the pathogenesis of NASH and HCC.


Assuntos
Carcinoma Hepatocelular/prevenção & controle , Antagonistas de Hormônios/farmacologia , Neoplasias Hepáticas/prevenção & controle , Fígado/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Proglumida/farmacologia , Receptor de Colecistocinina B/antagonistas & inibidores , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Deficiência de Colina/complicações , Modelos Animais de Doenças , Epigênese Genética , Etionina , Feminino , Regulação Neoplásica da Expressão Gênica , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Receptor de Colecistocinina B/genética , Receptor de Colecistocinina B/metabolismo , Transdução de Sinais
4.
PLoS One ; 14(8): e0221456, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31430329

RESUMO

Gastrin is a peptide hormone, which in combination with other factors such as TGFα, EGF or GLP-1, is capable of increasing beta cell mass and lowering blood glucose levels in adult diabetic mice. In humans, administration of a bolus of gastrin alone induces insulin secretion suggesting that gastrin may target islet cells. However, whether gastrin alone is sufficient to exert an effect on isolated human islets has been controversial and the mechanism remained poorly understood. Therefore, in this study we started to examine the effects of gastrin alone on cultured adult human islets. Treatment of isolated human islets with gastrin I for 48 h resulted in increased expression of insulin, glucagon and somatostatin transcripts. These increases were significantly correlated with the levels of donor hemoglobin A1c (HbA1c) but not BMI or age. In addition, gastrin treatment resulted in increased expression of PDX1, NKX6.1, NKX2.2, MNX1 and HHEX in islets from donors with HbA1c greater than 42 mmol/mol. The addition of YM022, an antagonist of the gastrin receptor cholecystokinin B receptor (CCKBR), together with gastrin eliminated these effects, verifying that the effects of gastrin are mediated through CCKBR.CCKBR is expressed in somatostatin-expressing delta cells in islets from all donors. However, in the islets from donors with higher HbA1c (greater than 42 mmol/mol [6.0%]), cells triple-positive for CCKBR, somatostatin and insulin were detected, suggesting a de-differentiation or trans-differentiation of endocrine cells. Our results demonstrate a direct effect of gastrin on human islets from prediabetic or diabetic individuals that is mediated through CCKBR+ cells. Further, our data imply that gastrin may be a potential treatment for diabetic patients.


Assuntos
Gastrinas/farmacologia , Hemoglobina A Glicada/metabolismo , Ilhotas Pancreáticas/metabolismo , Doadores de Tecidos , Adolescente , Adulto , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Receptor de Colecistocinina B/metabolismo , Somatostatina/metabolismo , Transcrição Genética/efeitos dos fármacos
5.
Nanoscale ; 11(29): 13714-13719, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31314031

RESUMO

The selective formation of nanomedicines around cancer cells is very important for cancer therapy because it increases the inhibitory capacity and decreases the systemic toxicity. However, successful examples are rare. Taking advantage of the overexpression of both the enzyme alkaline phosphatase (ALP) and the cell membrane receptor (CCK2R), we demonstrated in this study the selective formation of supramolecular nanofibers and hydrogels in the pericellular space of two cancer cell lines (HeLa and HepG2 cells). Both cell lines showed high expression levels of extracellular ALP and membrane-bound CCK2R. ALP efficiently converted Comp. 1 to a self-assembling molecule (Comp. 2). Comp. 2 interacted with CCK2R, thereby facilitating the self-assembly and formation of hydrogels around the cancer cells. The selective pericellular hydrogelations efficiently inhibited cancer cells. Pericellular hydrogelation around cancer cells is a promising strategy to control the formation of nanomedicines spatiotemporally in cellular microenvironments for cancer therapy and diagnostics.


Assuntos
Fosfatase Alcalina/metabolismo , Hidrogéis/química , Receptor de Colecistocinina B/metabolismo , Fosfatase Alcalina/química , Fosfatase Alcalina/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Microscopia Confocal , Peptídeos/química , Peptídeos/farmacologia , Receptor de Colecistocinina B/química , Receptor de Colecistocinina B/genética
6.
Cell Commun Signal ; 17(1): 68, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31215470

RESUMO

BACKGROUND: Cholecystokinin (CCK) is implicated in the regulation of nociceptive sensitivity of primary afferent neurons. Nevertheless, the underlying cellular and molecular mechanisms remain unknown. METHODS: Using patch clamp recording, western blot analysis, immunofluorescent labelling, enzyme-linked immunosorbent assays, adenovirus-mediated shRNA knockdown and animal behaviour tests, we studied the effects of CCK-8 on the sensory neuronal excitability and peripheral pain sensitivity mediated by A-type K+ channels. RESULTS: CCK-8 reversibly and concentration-dependently decreased A-type K+ channel (IA) in small-sized dorsal root ganglion (DRG) neurons through the activation of CCK type B receptor (CCK-BR), while the sustained delayed rectifier K+ current was unaffected. The intracellular subunit of CCK-BR coimmunoprecipitated with Gαo. Blocking G-protein signaling with pertussis toxin or by the intracellular application of anti-Gß antibody reversed the inhibitory effects of CCK-8. Antagonism of phosphatidylinositol 3-kinase (PI3K) but not of its common downstream target Akts abolished the CCK-BR-mediated IA response. CCK-8 application significantly activated JNK mitogen-activated protein kinase. Antagonism of either JNK or c-Src prevented the CCK-BR-mediated IA decrease, whereas c-Src inhibition attenuated the CCK-8-induced p-JNK activation. Application of CCK-8 enhanced the action potential firing rate of DRG neurons and elicited mechanical and thermal pain hypersensitivity in mice. These effects were mediated by CCK-BR and were occluded by IA blockade. CONCLUSION: Our findings indicate that CCK-8 attenuated IA through CCK-BR that is coupled to the Gßγ-dependent PI3K and c-Src-mediated JNK pathways, thereby enhancing the sensory neuronal excitability in DRG neurons and peripheral pain sensitivity in mice.


Assuntos
Proteína Tirosina Quinase CSK/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinase/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Receptor de Colecistocinina B/metabolismo , Células Receptoras Sensoriais/citologia , Células Receptoras Sensoriais/efeitos dos fármacos , Animais , Gânglios Espinais/citologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Nociceptividade/efeitos dos fármacos , Dor/patologia , Dor/fisiopatologia , Sincalida/farmacologia
7.
Int J Mol Sci ; 20(10)2019 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-31108898

RESUMO

BACKGROUND: Studies on the regulation of gastric and pancreatic secretion began more than 100 years ago. Secretin was the first hormone postulated to exist, initiating the field of endocrinology. Gastrin produced in the antral mucosa was the second postulated hormone, and together with histamine and acetylcholine, represent the three major gastric acid secretagogues known since 1920. For a long time, the mast cell was the only recognized histamine-producing cell in the oxyntic mucosa and, in the mid-1980s, the ECL cell was recognized as the cell producing histamine, taking part in the regulation of gastric acid secretion. METHODS: This review is based upon literature research and personal knowledge. RESULTS: The ECL cell carries the gastrin receptor, and gastrin regulates its function (histamine release) as well as proliferation. Long-term hypergastrinemia results in gastric neoplasia of variable malignancies, implying that gastric hypoacidity resulting in increased gastrin release will induce gastric neoplasia, including gastric cancer. CONCLUSIONS: The trophic effect of gastrin on the ECL cell has implications to the treatment with inhibitors of acid secretion.


Assuntos
Celulas Tipo Enterocromafim/metabolismo , Mucosa Gástrica/metabolismo , Gastrinas/metabolismo , Acetilcolina/metabolismo , Animais , Celulas Tipo Enterocromafim/patologia , Mucosa Gástrica/patologia , Histamina/metabolismo , Humanos , Receptor de Colecistocinina B/metabolismo
8.
Br J Pharmacol ; 176(15): 2678-2690, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31012948

RESUMO

BACKGROUND AND PURPOSE: A cholecystokinin (CCK) system has been identified in white adipose tissue (WAT). Nevertheless, the endocrine actions of CCK on WAT remain unknown. Our goal was to investigate the role of CCK in regulating the production of adiponectin, an adipokine expressed in WAT, which is pivotal in preserving energy homeostasis. EXPERIMENTAL APPROACH: The effect of the bioactive CCK fragment CCK-8 on adiponectin production was studied both in vivo and in vitro. CCK-8 effects were characterized in rats treated with selective CCK1 and CCK2 receptor antagonists as well as in pre-adipocytes carrying the selective silencing of either CCK1 or CCK2 receptors. The influence of insulin on CCK-8 responses was also analysed. KEY RESULTS: In WAT, CCK-8 increased plasma adiponectin levels and the expression of the adiponectin gene (Adipoq). In pre-adipocytes, CCK-8 up-regulated adiponectin production. CCK-8 effects were abolished by L-365,260, a selective CCK2 receptor antagonist. CCK2 receptor knockdown also abolished the effects of CCK-8 in pre-adipocytes. Moreover, in vitro CCK-8 effects were blocked by triciribine, a specific inhibitor of protein kinase B (Akt) and by the PPARγ antagonist T0070907. Silencing the expression of the insulin receptor inhibited CCK-8-induced Adipoq expression in pre-adipocytes. Furthermore, insulin potentiated the effect of CCK-8. CONCLUSION AND IMPLICATIONS: CCK-8 stimulates adiponectin production in WAT by acting on CCK2 receptors, through a mechanism involving both Akt and PPARγ. Moreover, CCK-8 actions are only observed in the presence of insulin. Our results could have translational value in the design of new insulin-sensitizing therapies.


Assuntos
Tecido Adiposo Branco/metabolismo , Colecistocinina/metabolismo , PPAR gama/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Colecistocinina B/agonistas , Adipócitos/metabolismo , Adiponectina/sangue , Adiponectina/genética , Animais , Benzamidas/farmacologia , Masculino , PPAR gama/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Piridinas/farmacologia , Ratos Sprague-Dawley , Receptor de Colecistocinina B/metabolismo , Ribonucleosídeos/farmacologia
9.
Proc Natl Acad Sci U S A ; 116(13): 6397-6406, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30850520

RESUMO

Memory is stored in neural networks via changes in synaptic strength mediated in part by NMDA receptor (NMDAR)-dependent long-term potentiation (LTP). Here we show that a cholecystokinin (CCK)-B receptor (CCKBR) antagonist blocks high-frequency stimulation-induced neocortical LTP, whereas local infusion of CCK induces LTP. CCK-/- mice lacked neocortical LTP and showed deficits in a cue-cue associative learning paradigm; and administration of CCK rescued associative learning deficits. High-frequency stimulation-induced neocortical LTP was completely blocked by either the NMDAR antagonist or the CCKBR antagonist, while application of either NMDA or CCK induced LTP after low-frequency stimulation. In the presence of CCK, LTP was still induced even after blockade of NMDARs. Local application of NMDA induced the release of CCK in the neocortex. These findings suggest that NMDARs control the release of CCK, which enables neocortical LTP and the formation of cue-cue associative memory.


Assuntos
Colecistocinina/metabolismo , Potenciação de Longa Duração/fisiologia , Memória/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Córtex Auditivo/metabolismo , Comportamento Animal , Colecistocinina/genética , Estimulação Elétrica , Córtex Entorrinal/metabolismo , Feminino , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , N-Metilaspartato/metabolismo , Neocórtex/metabolismo , Neurônios/metabolismo , Ratos Sprague-Dawley , Receptor de Colecistocinina B/efeitos dos fármacos , Receptor de Colecistocinina B/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Sinapses/metabolismo
10.
Sci Rep ; 9(1): 3978, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30850691

RESUMO

Animals that communicate using sound are found throughout the animal kingdom. Interestingly, in contrast to human vocal learning, most animals can produce species-specific patterns of vocalization without learning them from their parents. This phenomenon is called innate vocalization. The underlying molecular basis of both vocal learning in humans and innate vocalization in animals remains unknown. The crowing of a rooster is also innately controlled, and the upstream center is thought to be localized in the nucleus intercollicularis (ICo) of the midbrain. Here, we show that the cholecystokinin B receptor (CCKBR) is a regulatory gene involved in inducing crowing in roosters. Crowing is known to be a testosterone (T)-dependent behavior, and it follows that roosters crow but not hens. Similarly, T-administration induces chicks to crow. By using RNA-sequencing to compare gene expression in the ICo between the two comparison groups that either crow or do not crow, we found that CCKBR expression was upregulated in T-containing groups. The expression of CCKBR and its ligand, cholecystokinin (CCK), a neurotransmitter, was observed in the ICo. We also showed that crowing was induced by intracerebroventricular administration of an agonist specific for CCKBR. Our findings therefore suggest that the CCK system induces innate vocalization in roosters.


Assuntos
Galinhas/metabolismo , Galinhas/fisiologia , Colecistocinina/metabolismo , Corvos/metabolismo , Corvos/fisiologia , Animais , Comportamento Animal/fisiologia , Expressão Gênica/fisiologia , Masculino , Neurotransmissores/metabolismo , Receptor de Colecistocinina B/metabolismo , Som , Testosterona/metabolismo , Regulação para Cima/fisiologia , Vocalização Animal/fisiologia
11.
Am J Physiol Regul Integr Comp Physiol ; 316(5): R628-R639, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30892908

RESUMO

We investigated expression of cholecystokinin (CCK) in humans and mice, and the bitter taste receptor TAS2R14 in the human placenta. Because CCK and gastrin activate the CCKBR receptor, we also explored placental gastrin expression. Finally, we investigated calcium signaling by CCK and TAS2R14. By RT-PCR, we found CCK/Cck and GAST/Gast mRNA expression in both normal human and mouse placentas, as well as in human trophoblast cell lines (TCL). Although both Cckar and -br mRNA were expressed in the mouse placenta, only CCKBR mRNA was detected in the human placenta and TCL. mRNA expression for TAS2R14 was also observed in the human placenta and TCL. Using immunohistochemistry, CCK protein was localized to the syncytiotrophoblast (ST) and extravillous trophoblast (EVT) in the human term placenta, and to trophoblast glycogen cells in mouse and human placentas. Gastrin and TAS2R14 proteins were also observed in ST and EVT of the human placenta. Both sulfated and nonsulfated CCK elicited a comparable rise in intracellular calcium in TCL, consistent with CCKBR expression. Three TAS2R14 agonists, flufenamic acid, chlorhexidine, and diphenhydramine, also evoked rises in intracellular calcium in TCL. These results establish CCK, gastrin, and their receptor(s) in both human and mouse placentas, and TAS2R14 in the human placenta. Both CCK and TAS2R14 agonists increased intracellular calcium in human TCL. Although the roles of these ligands and receptors, and their potential cross talk in normal and pathological placentas, are currently unknown, this study opens new avenues for placental research.


Assuntos
Colecistocinina/metabolismo , Gastrinas/metabolismo , Receptor de Colecistocinina B/metabolismo , Receptores da Colecistocinina/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , Trofoblastos/metabolismo , Animais , Sinalização do Cálcio , Linhagem Celular , Colecistocinina/genética , Colecistocinina/farmacologia , Feminino , Gastrinas/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor de Colecistocinina B/genética , Receptores da Colecistocinina/agonistas , Receptores da Colecistocinina/genética , Receptores Acoplados a Proteínas-G/agonistas , Receptores Acoplados a Proteínas-G/genética , Transdução de Sinais/efeitos dos fármacos , Trofoblastos/efeitos dos fármacos
12.
Bioconjug Chem ; 30(3): 657-666, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30608664

RESUMO

Derivatized minigastrin analogues make up a promising class of candidates for targeting cholecystokinin receptor subtype 2 (CCK2R), which is overexpressed on cancer cells of various neuroendocrine tumors. The pentaglutamic acid sequence of minigastrin influences its biological properties. In particular, it plays a crucial role in the kidney reuptake mechanism. However, the importance of the binding affinity and interaction of this region with the receptor on a molecular level remains unclear. To elucidate its structure-activity relationship with CCK2R, we replaced this sequence with various linkers differing in their amount of anionic charge, structural characteristics, and flexibility. Specifically, a flexible aliphatic linker, a linker with only three d-Glu residues, and a structured linker with four adjacent ß3-glutamic acid residues were evaluated and compared to the lead compound PP-F11N (DOTA-[d-Glu1-6,Nle11]gastrin-13). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the minigastrin derivatives, which allowed radiolabeling with Lutetium-177. The levels of In vitro internalization into MZ-CRC1 cells and in vivo tumor uptake as well as human blood plasma stability increased in the following order: aliphatic linker < three d-Glu < (ß3-Glu)4 < (d-Glu)6. The in vitro and in vivo behavior was therefore significantly improved with anionic charges. Computational modeling of a CCK2 receptor-ligand complex revealed ionic interactions between cationic residues (Arg and His) of the receptor and anionic residues of the ligand in the linker.


Assuntos
Gastrinas/química , Gastrinas/farmacologia , Receptor de Colecistocinina B/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Estabilidade de Medicamentos , Gastrinas/farmacocinética , Humanos , Camundongos , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade , Distribuição Tecidual
13.
J Nucl Med ; 60(7): 1010-1016, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30530828

RESUMO

Molecular imaging and targeted radiotherapy with radiolabeled cholecystokinin-2 receptor (CCK2R) targeting peptide probes holds high promise to improve the clinical management of patients with metastatic medullary thyroid carcinoma and other CCK2R-expressing malignancies. Low stability and suboptimal targeting of currently available radiolabeled peptide analogs has prompted us to seek new stabilization strategies. In this study, we present a new minigastrin analog with site-specific C-terminal modifications showing a highly optimized targeting profile. Methods: DOTA-D-Glu-Ala-Tyr-Gly-Trp-(N-Me)Nle-Asp-1-Nal-NH2 (DOTA-MGS5) radiolabeled with 111In, 68Ga, and 177Lu was evaluated in extensive in vitro stability studies. For 177Lu-DOTA-MGS5, additional metabolic studies were performed on BALB/c mice. Receptor affinity and cell uptake were studied in A431 human epidermoid carcinoma cells transfected with human CCK2R (A431-CCK2R), as well as the same cell line transfected with the empty vector (A431-mock). A431-CCK2R/A431-mock xenografted athymic BALB/c nude mice were used for biodistribution studies and small-animal SPECT/CT. Results: DOTA-MGS5 radiolabeled with 111In and 177Lu showed a highly increased stability against enzymatic degradation in different media up to 24 h of incubation. Similar results were observed for 68Ga-DOTA-MGS5 incubated up to 4 h. In the blood of mice injected with 177Lu-DOTA-MGS5, at least 70% intact radiopeptide was detected up to 1 h after injection. The unlabeled peptide and the complexes with the natural isotopes showed retained receptor affinity, and the radiopeptides showed unexpectedly high cell uptake in A431-CCK2R cells (>60% at 4 h). Regardless of the radiometal used for labeling, impressively high uptake in A431-CCK2R xenografts was found (∼20% injected activity/g 1-4 h after injection), whereas the uptake in A431-mock xenografts was negligible. Low background activity and favorable tumor-to-kidney ratios (4-6) allowed for high image contrast in small-animal SPECT/CT. Conclusion: The excellent targeting properties of DOTA-MGS5 support future clinical studies evaluating the diagnostic and therapeutic potential in patients with progressive or metastatic medullary thyroid carcinoma, as well as other advanced-stage CCK2R-expressing malignancies.


Assuntos
Compostos Heterocíclicos com 1 Anel/química , Imagem Molecular/métodos , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Radioterapia/métodos , Receptor de Colecistocinina B/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Feminino , Camundongos , Oligopeptídeos/farmacocinética , Oligopeptídeos/uso terapêutico , Distribuição Tecidual
14.
Pain ; 160(2): 345-357, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30281531

RESUMO

Pain is associated with negative emotions such as anxiety, but the underlying neurocircuitry and modulators of the association of pain and anxiety remain unclear. The neuropeptide cholecystokinin (CCK) has both pronociceptive and anxiogenic properties, so we explored the role of CCK in anxiety and nociception in the central amygdala (CeA), a key area in control of emotions and descending pain pathways. Local infusion of CCK into the CeA of control rats increased anxiety, as measured in the light-dark box test, but had no effect on mechanical sensitivity. By contrast, intra-CeA CCK infusion 4 days after Complete Freund's Adjuvant (CFA) injection into the hindpaw resulted in analgesia, but also in loss of its anxiogenic capacity. Inflammatory conditions induced changes in the CeA CCK signaling system with an increase of CCK immunoreactivity and a decrease in CCK1, but not CCK2, receptor mRNA. In CFA rats, patch-clamp experiments revealed that CCK infusion increased CeA neuron excitability. It also partially blocked the discharge of wide dynamic range neurons in the dorsal spinal cord. These effects of CCK on CeA and spinal neurons in CFA rats were mimicked by the specific CCK2 receptor agonist, gastrin. This analgesic effect was likely mediated by identified CeA neurons projecting to the periaqueductal gray matter that express CCK receptors. Together, our data demonstrate that intra-CeA CCK infusion activated a descending CCK2 receptor-dependent pathway that inhibited spinal neuron discharge. Thus, persistent pain induces a functional switch to a newly identified analgesic capacity of CCK in the amygdala, indicating central emotion-related circuit controls pain transmission in spinal cord.


Assuntos
Tonsila do Cerebelo/metabolismo , Colecistocinina/metabolismo , Dor/patologia , Receptor de Colecistocinina B/metabolismo , Transdução de Sinais/fisiologia , Tonsila do Cerebelo/patologia , Animais , Adaptação à Escuridão/efeitos dos fármacos , Modelos Animais de Doenças , Comportamento Exploratório/efeitos dos fármacos , Adjuvante de Freund/toxicidade , Gastrinas/uso terapêutico , Glutamato Descarboxilase/metabolismo , Inflamação/induzido quimicamente , Inflamação/complicações , Masculino , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Nociceptividade/efeitos dos fármacos , Dor/etiologia , Limiar da Dor/efeitos dos fármacos , Substância Cinzenta Periaquedutal/efeitos dos fármacos , Substância Cinzenta Periaquedutal/fisiologia , Ratos , Ratos Sprague-Dawley , Receptor de Colecistocinina B/agonistas , Receptor de Colecistocinina B/antagonistas & inibidores , Receptor de Colecistocinina B/genética , Transdução de Sinais/efeitos dos fármacos , Sincalida/uso terapêutico , Tetragastrina/análogos & derivados , Tetragastrina/uso terapêutico
15.
Brain Res ; 1708: 200-206, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30571983

RESUMO

Recently, we reported that non-sulfated cholecystokinin-8 (NS CCK-8) reduces food intake by cholecystokinin-B receptors (CCK-BR). To examine a possible site of action for this peptide, and based on the fact that both NS CCK-8 and CCK-BR are found centrally and peripherally, in the current study we hypothesized that NS CCK-8 increases Fos-like immunoreactivity (Fos-LI, a neuronal activation marker) in the dorsal vagal complex (DVC) of the hindbrain and the myenteric and submucosal plexuses of the small intestine. We found that intraperitoneal NS CCK-8 (0.5 nmol/kg) increases Fos-LI in the DVC, the myenteric and the submucosal plexuses of the duodenum and the myenteric plexus of the jejunum. The findings suggest, but does not prove, a potential role for the DVC and the enteric neurons in the feeding responses evoked by NS CCK-8.


Assuntos
Comportamento Alimentar/fisiologia , Rombencéfalo/metabolismo , Sincalida/metabolismo , Animais , Encéfalo/metabolismo , Colecistocinina , Sistema Nervoso Entérico/metabolismo , Intestino Delgado , Masculino , Plexo Mientérico/metabolismo , Neurônios/metabolismo , Fragmentos de Peptídeos , Proteínas Proto-Oncogênicas c-fos/imunologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor de Colecistocinina B/metabolismo , Sincalida/fisiologia , Plexo Submucoso/metabolismo , Nervo Vago/metabolismo
16.
J Nucl Med ; 60(3): 393-399, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30002107

RESUMO

Patients with metastatic medullary thyroid cancer (MTC) have limited systemic treatment options. The use of radiolabeled gastrin analogs targeting the cholecystokinin-2 receptor (CCK2R) is an attractive approach. However, their therapeutic efficacy is presumably decreased by their enzymatic degradation in vivo. We aimed to investigate whether the chemically stabilized analog 177Lu-DOTA-PP-F11N (177Lu-DOTA-(dGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2) performs better than reference analogs with varying in vivo stability, namely 177Lu-DOTA-MG11 (177Lu-DOTA-dGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2) and 177Lu-DOTA-PP-F11 (177Lu-DOTA-(dGlu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2), and whether the use of protease inhibitors further improves CCKR2 targeting. First human data on 177Lu-DOTA-PP-F11N are also reported. Methods: In vitro stability of all analogs was assessed against a panel of extra- and intracellular endoproteases, whereas their in vitro evaluation was performed using the human MTC MZ-CRC-1 and the transfected A431-CCK2R(+) cell lines. Biodistribution without and with the protease inhibitors phosphoramidon and thiorphan was assessed 4 h after injection in MZ-CRC-1 and A431-CCK2R(+) dual xenografts. Autoradiography of 177Lu-DOTA-PP-F11N (without and with phosphoramidon) and NanoSPECT/CT were performed. SPECT/CT images of 177Lu-DOTA-PP-F11N in a metastatic MTC patient were also acquired. Results: natLu-DOTA-PP-F11N is less of a substrate for neprilysins than the other analogs, whereas intracellular cysteine proteases, such as cathepsin-L, might be involved in the degradation of gastrin analogs. The uptake of all radiotracers was higher in MZ-CRC-1 tumors than in A431-CCK2R(+), apparently because of the higher number of binding sites on MZ-CRC-1 cells. 177Lu-DOTA-PP-F11N had the same biodistribution as 177Lu-DOTA-PP-F11; however, uptake in the MZ-CRC-1 tumors was almost double (20.7 ± 1.71 vs. 11.2 ± 2.94 %IA [percentage injected activity]/g, P = 0.0002). Coadministration of phosphoramidon or thiorphan increases 177Lu-DOTA-MG11 uptake significantly in the CCK2R(+) tumors and stomach. Less profound was the effect on 177Lu-DOTA-PP-F11, whereas no influence or even reduction was observed for 177Lu-DOTA-PP-F11N (20.7 ± 1.71 vs. 15.6 ± 3.80 [with phosphoramidon] %IA/g, P < 0.05 in MZ-CRC-1 tumors). The first clinical data show high 177Lu-DOTA-PP-F11N accumulation in tumors, stomach, kidneys, and colon. Conclusion: The performance of 177Lu-DOTA-PP-F11N without protease inhibitors is as good as the performance of 177Lu-DOTA-MG11 in the presence of inhibitors. The human application of single compounds without unessential additives is preferable. Preliminary clinical data spotlight the stomach as a potential dose-limiting organ besides the kidneys.


Assuntos
Gastrinas/química , Gastrinas/metabolismo , Compostos Heterocíclicos com 1 Anel/química , Lutécio , Inibidores de Proteases/farmacologia , Radioisótopos , Receptor de Colecistocinina B/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular Tumoral , Estabilidade de Medicamentos , Feminino , Gastrinas/farmacocinética , Humanos , Camundongos , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único , Distribuição Tecidual/efeitos dos fármacos
18.
J Med Chem ; 61(22): 10173-10184, 2018 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-30395477

RESUMO

In the search for an alternative strategy to the radioactivity measurement conventionally performed to probe receptor-ligand interactions in pharmacological assays, we demonstrated that selenium labeling of the studied ligand combined with elemental mass spectrometry was as efficient and robust as the reference method but devoid of its environmental and health hazards. The proof-of-concept was illustrated on two GPCR receptors, vasopressin (V1A) and cholecystokinin B (CCK-B), involving peptides as endogenous ligands. We proposed several methodologies to produce selenium-labeled ligands according to peptide sequences along with binding affinity constraints. A selection of selenopeptides that kept high affinities toward the targeted receptor were engaged in saturation and competitive binding experiments with subsequent sensitive RP-LC-ICP-MS measurements. Experimental values of affinity constant ( Ki) were perfectly correlated to literature data, illustrating the general great potency of replacing radioactive iodine by selenium for ligand labeling to further undergo unaffected pharmacology experiments efficiently monitored by elemental mass spectrometry.


Assuntos
Espectrometria de Massas , Selênio/química , Animais , Células CHO , Cricetulus , Marcação por Isótopo , Ligantes , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Receptor de Colecistocinina B/metabolismo , Vasopressinas/metabolismo
19.
PLoS One ; 13(7): e0201224, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30059514

RESUMO

Positron emission tomography (PET) with radiolabelled peptide-based tracers has attracted great interest in oncology over the past decades. The success of imaging is closely related to sufficient uptake of the radiotracer in malignant tissue and for this sufficient biological half-life, particularly in the bloodstream, is mandatory. Fast enzymatic degradation during circulation leading to insufficient imaging abilities of peptide-based radioligands remains a major issue. The design of multimeric constructs, bearing multiple targeting moieties, has been widely applied to improve target interaction. This concept may also be applied to prolong the biological half-life of peptide-based radiopharmaceuticals as enzymatic degradation can result in formation of metabolites still capable to interact with the target binding site. In this study we aimed to identify such metabolites and therefore we utilized the siderophore-based bifunctional chelator fusarinine C (FSC) for the design of novel mono- and multimeric constructs, bearing minigastrin (MG) analogues as targeting moieties to address cholecystokinin-2 receptors (CCK2R) which are overexpressed in a variety of cancerous diseases and are well known for fast enzymatic degradation, particularly for truncated des-(Glu)5-MG members, such as MG11. FSC-based imaging probes were radiolabelled with gallium-68 and characterized in vitro (logD, protein binding, affinity and cell-uptake studies, stability and metabolite studies, as well as generation of corresponding metabolites by artificial enzymatic degradation) and in vivo (biodistribution in A431-CCK2R/A431-mock tumour xenografted BALB/c nude mice and stability in blood of living BALB/c mice and analysis of corresponding organ homogenates and urine to identify degradation products). In summary, multimerization was accompanied by partial improvement towards targeting abilities. Identified metabolites formed by artificial enzymatic cleavage of trimeric FSC-MG conjugates in vitro contained intact binding sequences for the receptor. Furthermore, the 68Ga-labelled trimers exhibiting increasing uptake of radioligand in tumour tissue over time and improved in vivo stability in blood samples of living animals of the trimers compared to corresponding mono- and dimers, strongly supporting our hypothesis.


Assuntos
Gastrinas , Compostos Radiofarmacêuticos , Receptor de Colecistocinina B/metabolismo , Animais , Linhagem Celular Tumoral , Quelantes/química , Feminino , Compostos Férricos/química , Radioisótopos de Gálio , Gastrinas/química , Humanos , Ácidos Hidroxâmicos/química , Rim/metabolismo , Fígado/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Imagem Molecular , Transplante de Neoplasias , Estudo de Prova de Conceito , Multimerização Proteica , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/química , Ratos Sprague-Dawley
20.
J Am Heart Assoc ; 7(14)2018 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-30005556

RESUMO

BACKGROUND: Ischemia/reperfusion injury (IRI) is one of the most predominant complications of ischemic heart disease. Gastrin has emerged as a regulator of cardiovascular function, playing a key protective role in hypoxia. Serum gastrin levels are increased in patients with myocardial infarction, but the pathophysiogical significance of this finding is unknown. The purpose of this study was to determine whether and how gastrin protects cardiac myocytes from IRI. METHODS AND RESULTS: Adult male Sprague-Dawley rats were used in the experiments. The hearts in living rats or isolated Langendorff-perfused rat hearts were subjected to ischemia followed by reperfusion to induce myocardial IRI. Gastrin, alone or with an antagonist, was administered before the induction of myocardial IRI. We found that gastrin improved myocardial function and reduced the expression of myocardial injury markers, infarct size, and cardiomyocyte apoptosis induced by IRI. Gastrin increased the phosphorylation levels of ERK1/2 (extracellular signal-regulated kinase 1/2), AKT (protein kinase B), and STAT3 (signal transducer and activator of transcription 3), indicating its ability to activate the RISK (reperfusion injury salvage kinase) and SAFE (survivor activating factor enhancement) pathways. The presence of inhibitors of ERK1/2, AKT, or STAT3 abrogated the gastrin-mediated protection. The protective effect of gastrin was via CCK2R (cholecystokinin 2 receptor) because the CCK2R blocker CI988 prevented the gastrin-mediated protection of the heart with IRI. Moreover, we found a negative correlation between serum levels of cardiac troponin I and gastrin in patients with unstable angina pectoris undergoing percutaneous coronary intervention, suggesting a protective effect of gastrin in human cardiomyocytes. CONCLUSIONS: These results indicate that gastrin can reduce myocardial IRI by activation of the RISK and SAFE pathways.


Assuntos
Gastrinas/farmacologia , Coração/efeitos dos fármacos , Hormônios/farmacologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Idoso , Angina Instável/sangue , Angina Instável/cirurgia , Animais , Apoptose/efeitos dos fármacos , Feminino , Gastrinas/sangue , Humanos , Preparação de Coração Isolado , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Intervenção Coronária Percutânea , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Receptor de Colecistocinina B/antagonistas & inibidores , Receptor de Colecistocinina B/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Troponina I/sangue
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