RESUMEN
BACKGROUND: Guillain-Barré syndrome is an acute inflammatory demyelinating polyneuropathy that is characterized histologically by demyelination of peripheral nerves and nerve roots, infiltrates of T lymphocytes, and an inflammatory response that includes macrophage infiltrates. The aim of this study was to evaluate the effects of vasoactive intestinal peptide (VIP) in a rat model of experimental autoimmune neuritis (EAN). METHODS: Forty male Lewis rats were divided into a control group (N=10), an EAN group (N=10), an EAN group treated with 15 nmol of VIP (N=10), and an EAN group treated with 30 nmol of VIP (N=10). The rat model was created by subcutaneous injection of P2 polypeptide (200 µg P257-81) into the base of the tail. Intraperitoneal injection of VIP was given on day 7. Rats were weighed and functionally evaluated using an EAN score (0-10). On day 16, the rats were euthanized. The sciatic nerve was examined histologically and using immunohistochemistry with antibodies against CD8, CD68, and forkhead box p3 (Foxp3). Serum concentrations of IL-17 and interferon-α (IFN-α) were measured by ELISA on day 16 after creating the EAN model. RESULTS: The VIP-treated EAN groups had increased body weight and improved EAN scores compared with the untreated EAN group. CD8-positive and CD68-positive cells were significantly reduced in the EAN group treated with 30 nmol of VIP compared with 15 nmol of VIP. Foxp3-positive cells were significantly decreased in both EAN groups treated with VIP, and serum concentrations of IL-17 and IFN-α were significantly lower compared with the untreated EAN group (P<0.05). CONCLUSION: In a rat model of EAN, treatment with VIP resulted in functional improvement, reduced nerve inflammation, and decreased serum levels of inflammatory cytokines.
Asunto(s)
Modelos Animales de Enfermedad , Síndrome de Guillain-Barré/tratamiento farmacológico , Neuritis Autoinmune Experimental/tratamiento farmacológico , Péptido Intestinal Vasoactivo/uso terapéutico , Vasodilatadores/uso terapéutico , Animales , Relación Dosis-Respuesta a Droga , Síndrome de Guillain-Barré/sangre , Síndrome de Guillain-Barré/patología , Interferón-alfa/sangre , Interleucina-17/sangre , Masculino , Neuritis Autoinmune Experimental/sangre , Neuritis Autoinmune Experimental/patología , Ratas , Ratas Endogámicas Lew , Péptido Intestinal Vasoactivo/síntesis química , Péptido Intestinal Vasoactivo/química , Vasodilatadores/síntesis química , Vasodilatadores/químicaRESUMEN
Vasoactive intestinal peptide receptor 2 (VIPR2, also known as VPAC2) is a class B G-protein coupled receptor (GPCR) and plays important roles in the physiology of central nervous system (CNS) by interaction with natural ligands; vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). Because it has been reported that high-expression and/or overactivation of VIPR2 link to schizophrenic symptoms, VIPR2 antagonists could be good drug candidates for schizophrenia therapeutics. In this study, we discovered several artificial peptides that antagonize both human and rodent VIPR2 with selectivities against receptor subtypes VIPR1 (also known as VPAC1) and pituitary adenylate cyclase-activating polypeptide type-1 receptor (PAC1). Of them, the representative 16-mer cyclic peptide VIpep-3 (Ac-CPPYLPRRLCTLLLRS-OH) exhibited strong binding affinity with KD value of 41â¯nM to extracellular domain of human VIPR2 in SPR analysis and showed potent antagonist activity with IC50 values of 47â¯nM (human), 180â¯nM (mouse), and 44â¯nM (rat) against VIP-VIPR2 signal in cell-based Ca influx assay. This is not only the first report on artificial VIPR2-selective antagonist peptides but also good example of the effective approach to discover novel antagonist against class B GPCR. Our peptides will contribute to study and development of the novel CNS drugs targeting to VIPR2.
Asunto(s)
Descubrimiento de Drogas , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , Receptores de Tipo II del Péptido Intestinal Vasoactivo/antagonistas & inhibidores , Péptido Intestinal Vasoactivo/farmacología , Animales , Técnicas Biosensibles , Células CHO , Cricetulus , Humanos , Ligandos , Ratones , Biblioteca de Péptidos , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/síntesis química , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/química , Ratas , Receptores de Tipo II del Péptido Intestinal Vasoactivo/metabolismo , Proteínas Recombinantes/metabolismo , Resonancia por Plasmón de Superficie , Péptido Intestinal Vasoactivo/síntesis química , Péptido Intestinal Vasoactivo/químicaRESUMEN
Neuroblastoma (NB) is a pediatric cancer. New therapies for high-risk NB aim to induce cell differentiation and to inhibit MYCN and ALK signaling in NB. The vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP) are 2 related neuropeptides sharing common receptors. The level of VIP increases with NB differentiation. Here, the effects of VIP and PACAP analogs developed for therapeutic use were studied in MYCN-amplified NB SK-N-DZ and IMR-32 cells and in Kelly cells that in addition present the F1174L ALK mutation. As previously reported by our group in IMR-32 cells, VIP induced neuritogenesis in SK-N-DZ and Kelly cells and reduced MYCN expression in Kelly but not in SK-N-DZ cells. VIP decreased AKT activity in the ALK-mutated Kelly cells. These effects were PKA-dependent. IMR-32, SK-NDZ and Kelly cells expressed the genes encoding the 3 subtypes of VIP and PACAP receptors, VPAC1, VPAC2 and PAC1. In parallel to its effect on MYCN expression, VIP inhibited invasion in IMR-32 and Kelly cells. Among the 3 PACAP analogs tested, [Hyp(2)]PACAP-27 showed higher efficiency than VIP in Kelly cells. These results indicate that VIP and PACAP analogs act on molecular and cellular processes that could reduce aggressiveness of high-risk NB.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neuronas/efectos de los fármacos , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , Péptido Intestinal Vasoactivo/farmacología , Quinasa de Linfoma Anaplásico , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Mutación , Proteína Proto-Oncogénica N-Myc/genética , Proteína Proto-Oncogénica N-Myc/metabolismo , Neuronas/metabolismo , Neuronas/patología , Especificidad de Órganos , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/síntesis química , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/genética , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/metabolismo , Receptores de Tipo II del Péptido Intestinal Vasoactivo/genética , Receptores de Tipo II del Péptido Intestinal Vasoactivo/metabolismo , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/genética , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/metabolismo , Transducción de Señal , Relación Estructura-Actividad , Péptido Intestinal Vasoactivo/síntesis químicaRESUMEN
A new component for the solid phase peptide synthesis of lipopeptide, 2-[(4R,5R)-5-({[(9H-fluoren-9-yl)methoxy]carbonylaminomethyl}-2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]acetic acid (2), was designed and synthesized from (-)-2,3-O-isopropylidene-D-threitol (3) in 4 steps. The key step was the selective alkylation of 3 with benzyl bromoacetate in the presence of Cs2CO3. Vasoactive intestinal peptide (VIP)-lipopeptide (1) incorporating this linker was synthesized by solid phase peptide synthesis.
Asunto(s)
Dioxolanos/síntesis química , Portadores de Fármacos/química , Liposomas/química , Péptido Intestinal Vasoactivo/síntesis química , Acetatos/química , Alquilación , Carbonatos/química , Cesio/química , Dioxolanos/química , Técnicas de Síntesis en Fase Sólida , Péptido Intestinal Vasoactivo/químicaRESUMEN
A model octapeptide segment derived from vasoactive intestinal peptide (VIP) was utilised to investigate the effect of several conventional cyclisation methods on the α-helical conformation in short peptide fragments. Three of the classical macrocyclisation techniques (i.e. lactamisation, ring-closing metathesis and Huisgen cycloaddition) were applied, and the conformations of the resulting cyclic peptides, as well as their linear precursors, were compared by CD analysis. The visibly higher folding propensity of the triazole-tethered peptide after azide-alkyne CuAAC macrocyclisation illustrates that the secondary structure of a short peptide fragment can differ significantly depending on the chemical strategy used to covalently cross-link side chain residues in a 'helical' fragment.
Asunto(s)
Dicroismo Circular , Péptido Intestinal Vasoactivo/química , Ciclización , Estabilidad Proteica , Estructura Secundaria de Proteína , Péptido Intestinal Vasoactivo/síntesis químicaRESUMEN
The effects of vasoactive intestinal peptide-camptothecin (VIP-CPT) conjugates were investigated on breast cancer cells and cells transfected with VIP receptors (R). (Ala(2,8,9,19,24.25.27), Nle(17), Lys(28))VIP, (A-NL-K)VIP, was synthesized and Lys(28) was coupled to a linker, N-methyl-amino-ethyl-glycine, L2, which formed a carbamate bond with CPT. The resulting (A-NL-K)VIP-L2-CPT was cytotoxic for MCF7 breast cancer cells, which have VPAC(1)-R, with IC(50) values of 380 and 90 nM using the MTT and clonogenic assays, respectively. (A-NL-K)VIP, (A-NL-K)VIP-L2 and (A-NL-K)VIP-L2-CPT inhibited specific binding of (125)I-VIP to 3T3 cells transfected with VPAC(1)-R with IC(50) values of 1.9, 56 and 126 nM, respectively. In contrast, (A-NL-K)VIP, (A-NL-K)VIP-L2 and (A-NL-K)VIP-L2-CPT inhibited specific binding of (125)I-Ro25-1553 to 3T3 cells transfected with VPAC(2)-R with IC(50) values of 3.9, 3162 and 2690 nM, respectively. (A-NL-K)VIP, (A-NL-K)VIP-L2 and (A-NL-K)VIP-L2-CPT caused increased cAMP after addition to MCF7 cells. (125)I-(A-NL-K)VIP-L2-CPT was internalized by MCF7 cells at 37 degrees C but not 4 degrees C. These results indicate that (A-NL-K)VIP-L2-CPT is a VPAC(1)-R agonist which is cytotoxic for breast cancer cells.
Asunto(s)
Camptotecina/farmacología , Proliferación Celular/efectos de los fármacos , Péptido Intestinal Vasoactivo/farmacología , Células 3T3 , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Camptotecina/síntesis química , Camptotecina/química , Línea Celular , Línea Celular Tumoral , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Péptido Intestinal Vasoactivo/síntesis química , Péptido Intestinal Vasoactivo/químicaRESUMEN
BACKGROUND: Radiolabelled vasoactive intestinal peptide (VIP) and its analogues have shown their potential as imaging agents for diagnosing tumours expressing VIP receptor. However, the fast proteolytic degradation in vivo has limited their clinical use. AIM: To prepare the 18F-labelled (R8,15,21, L17)-VIP analogue in a convenient way and to evaluate its potential as an imaging agent for VIP receptor-positive tumours. METHODS: Radiolabelled (R8,15,21, L17)-VIP was obtained by conjugation with N-succinimidyl 4-([18F]fluoromethyl) benzoate and purified by HPLC. Radiochemical purity and specific radioactivity were measured by analytical HPLC. In-vitro stability of the product was carried out in HSA solution and analysed by HPLC. Biodistribution study was carried out in mice bearing C26 colorectal tumours. RESULTS: 18F-(R8,15,21, L17)-VIP was obtained in greater than 99% radiochemical purity within 60 min in decay-for-corrected radiochemical yields of 21.8+/-4.7% (n=5) and a specific activity of 17.76 GBq x mumol(-1) at the end of synthesis (EOS). Results of in-vitro studies demonstrated a high stability in human serum albumin (HSA) solution. Biodistribution data showed a rapid blood clearance and specific binding towards receptor-positive tumours. CONCLUSION: 18F-(R8,15,21, L17)-VIP was prepared by a convenient method. Preliminary biodistribution results showed its potential for imaging tumours over-expressing VIP receptors and encouraged further investigation.
Asunto(s)
Neoplasias Colorrectales/diagnóstico por imagen , Radioisótopos de Yodo , Péptido Intestinal Vasoactivo/síntesis química , Animales , Humanos , Ratones , Ratones Endogámicos BALB C , Cintigrafía , Receptores de Péptido Intestinal Vasoactivo , Péptido Intestinal Vasoactivo/análogos & derivadosRESUMEN
Vasoactive intestinal peptide (VIP) receptors are expressed abundantly on many types of tumors and, hence, radiolabeled VIP analogues are being explored for tumor imaging and therapy. Here, we report synthesis of three VIP analogues and their radiolabeling with (99m)Tc via a novel tricarbonyl synthon. The radiolabeled product could be prepared in high yields (>95%) and stability. In vitro studies showed significant uptake of (99m)Tc(CO)((3))-VP05 in human colon carcinoma cells. Biodistribution studies in animal tumor model showed 0.4-1%ID/g tumor uptake.
Asunto(s)
Neoplasias del Colon/diagnóstico por imagen , Compuestos de Organotecnecio/síntesis química , Péptido Intestinal Vasoactivo/análogos & derivados , Secuencia de Aminoácidos , Animales , Humanos , Marcaje Isotópico , Ratones , Compuestos de Organotecnecio/farmacocinética , Cintigrafía , Péptido Intestinal Vasoactivo/síntesis química , Péptido Intestinal Vasoactivo/farmacocinéticaRESUMEN
In an effort to develop a peptide-based radiopharmaceutical for the detection of tumors overexpressed vasoactive intestinal peptide receptors with positron emission tomography, we have prepared a novel [R(8,15,21), L17]-VIP peptide for 18F-labeling. This peptide inhibited 125I-VIP binding to rats lung membranes with high affinity [half-maximal inhibitory concentrations (IC50) of 0.12 nm]. Additionally, [R(8,15,21), L17]-VIP showed higher stability than native vasoactive intestinal peptide in vivo of mice. With N-succinimidyl 4-[18F] fluorobenzoate as labeling prosthetic group, [18F]FB-[R(8,15,21), L17]-VIP was obtained in >99% radiochemical purity within 100 min in decay-for-corrected radiochemical yield of 33.6 +/- 3% (n = 5) and a specific radioactivity 255 GBq/micromol at the end of synthesis. Stability of [18F]FB-[R(8,15,21), L17]-VIP in vitro and in vivo were investigated. Biodistribution of this trace was carried out in mice with induced C26 colorectal tumor. Fast clearance of [18F]FB-[R(8,15,21), L17]-VIP from non-target tissues and specific uptakes by tumors realized higher tumor-to-muscle ratio (3.55) and tumor-to-blood ratio (2.37) 60 min postinjection. Clear difference was observed between the blocking and unblocking experiments in biodistribution and whole body radioautography. [18F]FB-[R(8,15,21), L17]-VIP has demonstrated its potential for diagnosing tumors overexpressed vasoactive intestinal peptide receptors both in vitro and in vivo.
Asunto(s)
Neoplasias/diagnóstico por imagen , Tomografía de Emisión de Positrones , Radiofármacos , Receptores de Péptido Intestinal Vasoactivo/análisis , Péptido Intestinal Vasoactivo/análogos & derivados , Secuencia de Aminoácidos , Animales , Membrana Celular/química , Neoplasias Colorrectales/química , Radioisótopos de Yodo/análisis , Radioisótopos de Yodo/química , Pulmón/química , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/química , Radiofármacos/síntesis química , Radiofármacos/química , Ratas , Ratas Endogámicas , Péptido Intestinal Vasoactivo/síntesis química , Péptido Intestinal Vasoactivo/químicaRESUMEN
Vasoactive intestinal peptide (VIP) exerts many biological functions through interaction with the VPAC1 receptor, a class II G protein-coupled receptor. Photoaffinity labeling studies associated with receptor mapping and three-dimensional molecular modeling demonstrated that the central part of VIP (6-24) interacts with the N-terminal ectodomain of VPAC1 receptor. However, the domain of the VPAC1 receptor interacting with the C-terminus of VIP is still unknown. A photoaffinity probe, Bpa28-VIP, was synthetized by substitution of amidated Asn28 of VIP by amidated photoreactive para-benzoyl-L-Phe (Bpa). Bpa28-VIP was shown to be a hVPAC1 receptor agonist in CHO cells expressing the recombinant VPAC1 receptor. After obtaining a covalent 125I-[Bpa28-VIP]/hVPAC1 complex, it was cleaved by CNBr, PNGase F, and endopeptidase Glu-C and the cleavage products were analyzed by electrophoresis. The data demonstrated that 125I-[Bpa28-VIP] was covalently bonded to the 121-133 fragment within the N-terminal ectodomain of the receptor. This fragment is adjacent to those covalently attached to the central part (6-24) of VIP.
Asunto(s)
Fragmentos de Péptidos/farmacología , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/metabolismo , Péptido Intestinal Vasoactivo/farmacología , Adenilil Ciclasas/metabolismo , Animales , Células CHO , Cricetinae , Humanos , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Unión Proteica , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo/genética , Péptido Intestinal Vasoactivo/síntesis química , Péptido Intestinal Vasoactivo/químicaRESUMEN
The mechanism by which vasoactive intestinal peptide (VIP)-ellipticine (E) conjugates are cytotoxic for human lung cancer cells was investigated. VIP-alanyl-leucyl-alanyl-leucyl-alanine (ALALA)-E and VIP-leucyl-alanyl-leucyl-alanine (LALA)-E inhibited (125)I-VIP binding to NCI-H1299 cells with an IC50 values of 0.5 and 0.1 microM, respectively. VIP-ALALA-E and VIP-LALA-E caused elevation of cAMP in NCI-H1299 cells with ED50 values of 0.7 and 0.1 microM. Radiolabeled VIP-LALA-E was internalized at 37 degrees C and delivered the cytotoxic E into NCI-H1299 cells. VIP-LALA-E inhibited the growth of NCI-H1299 cells in vitro. Three days after the addition of VIP-LALA-E to NCI-H1299 cells, cell viability decreased based on trypan blue exclusion and reduced 3H-thymidine uptake. These results suggest that VIP-E conjugates are internalized in lung cancer cells as a result of VPAC1 receptor-mediated endocytosis.
Asunto(s)
Antineoplásicos/síntesis química , Elipticinas/síntesis química , Péptido Intestinal Vasoactivo/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , AMP Cíclico/biosíntesis , Diseño de Fármacos , Elipticinas/química , Elipticinas/farmacocinética , Elipticinas/farmacología , Endocitosis , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Receptores de Péptido Intestinal Vasoactivo/agonistas , Receptores de Péptido Intestinal Vasoactivo/metabolismo , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo , Timidina/metabolismo , Péptido Intestinal Vasoactivo/química , Péptido Intestinal Vasoactivo/farmacocinética , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
C-terminally truncated human VPAC(1) receptors were constructed and stably transfected in Chinese hamster ovary (CHO) cells. Selected clones expressing comparable receptor densities were studied for ligand's binding properties, basal and stimulated adenylate cyclase activity. The wild-type (1-457) receptor served as reference. The binding properties of all the constructions were preserved. As judged by the intrinsic activity of the partial agonist Q(3)-VIP, the shortest receptors have a moderate impairment of the coupling efficacy to G(alpha s) protein. Cells expressing the VPAC(1) (1-436) and (1-441) truncated receptors had a two- to three-fold higher basal adenylate cyclase activity than those expressing the wild-type or the VPAC(1) (1-444), (1-433), (1-429), (1-421) and (1-398) receptor. The stimulatory effect of VIP and other agonist was preserved. This suggested that VPAC(1) (1-436) and (1-441) receptors had a constitutive activity. The selective VPAC(1) receptor antagonist Ac His(1) [D-Phe(2), K(15), R(16), L(27)] VIP (3-7)/GRF (8-27) reduced by 60% the basal activity with an EC(50) value of 3 nM comparable to its IC(50) value for binding. This agonist behaved thus like an inverse agonist on the constitutively active VPAC(1) receptors generated by C-terminal truncation and expressed in CHO cells.
Asunto(s)
Receptores de Péptido Intestinal Vasoactivo/química , Péptido Intestinal Vasoactivo/farmacología , Adenilil Ciclasas/metabolismo , Secuencia de Aminoácidos , Animales , Unión Competitiva , Células CHO , Células Clonales , Cricetinae , Cricetulus , Humanos , Concentración 50 Inhibidora , Radioisótopos de Yodo , Ligandos , Datos de Secuencia Molecular , Péptidos Cíclicos/farmacología , Conformación Proteica , Receptores de Péptido Intestinal Vasoactivo/metabolismo , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transfección , Péptido Intestinal Vasoactivo/síntesis química , Péptido Intestinal Vasoactivo/química , Péptido Intestinal Vasoactivo/aislamiento & purificaciónRESUMEN
We synthesized a VIP analog that combines mutations that decrease the affinity for the VPAC1 receptor but maintain a high affinity for the VPAC2 receptor with an amino-terminal hexanoylation that increases the affinity for the VPAC2 receptor with a limited decrease in the affinity of the VPAC1 receptor. The resulting Hexanoyl[A19,K(27,28)]VIP had the expected properties of a high affinity for the VPAC2 receptor and a low affinity for the VPAC1 receptor and also a low affinity for the PAC1 and secretin receptors. With a 1000-fold preference for the VPAC2 receptor and a IC50 value of binding of 1 nM, this compound is the most potent and the most selective agonist presently described.
Asunto(s)
Receptores de la Hormona Gastrointestinal/agonistas , Receptores de Péptido Intestinal Vasoactivo/agonistas , Péptido Intestinal Vasoactivo/química , Animales , Sitios de Unión , Humanos , Unión Proteica , Receptores Acoplados a Proteínas G , Receptores de Tipo II del Péptido Intestinal Vasoactivo , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo , Péptido Intestinal Vasoactivo/análisis , Péptido Intestinal Vasoactivo/síntesis químicaRESUMEN
Currently, there is no effective pharmacotherapy against exaggerated mobilisation of neutrophils in human airway diseases such as chronic obstructive pulmonary disease and asthma. We evaluated the effect of two synthetic vasoactive intestinal peptide (VIP)-like analogues on cytokine-induced neutrophil recruitment in airways in vivo. Recombinant interleukin (IL)-1 beta was administered intratracheally (i.t.) to intubated, spontaneously breathing Sprague-Dawley rats. The rats were pretreated either with a VIP synthetic peptide analogue, a pituitary adenylate cyclase-activating peptide (PACAP)-1-27 synthetic analogue, the beta(2)-adrenoceptor agonist salbutamol or vehicle, systemically or locally. Differential cell counts were performed on bronchoalveolar lavage fluid (BALf) cytospins. Effects on mean arterial blood pressure (MAP) were monitored in separate experiments. Systemic administration of the VIP analogue, the PACAP analogue and salbutamol attenuated the cytokine-induced increase in BALf neutrophil number. Local administration of the VIP analogue and salbutamol, but not the PACAP analogue, also decreased the neutrophil number in BALf. Local administration of the VIP analogue and salbutamol caused a transient decrease in MAP. Systemic or local administration of a synthetic VIP peptide analogue inhibits cytokine-induced neutrophil recruitment in airways in vivo. This action is exerted without severe, sustained cardiovascular side effects, and deserves to be further evaluated in obstructive pulmonary diseases in human.
Asunto(s)
Pulmón/efectos de los fármacos , Infiltración Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Péptido Intestinal Vasoactivo/farmacología , Albuterol/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Citocinas/antagonistas & inhibidores , Citocinas/farmacología , Humanos , Pulmón/citología , Masculino , Neuropéptidos/farmacología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Ratas , Ratas Sprague-Dawley , Péptido Intestinal Vasoactivo/síntesis químicaRESUMEN
Targeted delivery of radionuclides and therapeutic agents to specific biomarkers of breast cancer has important implications for the diagnosis and therapy of breast cancer. Vasoactive intestinal peptide receptors (VIP-R) are approximately five times more expressed in human breast cancer, compared to normal breast tissue. We have used VIP, a 28 amino acid mammalian neuropeptide, as a breast cancer targeting moiety for targeted imaging of breast cancer. VIP was covalently attached to the surface of sterically stabilized liposomes (SSL) that encapsulated a radionuclide, Tc99m-HMPAO. Rats with n-methyl nitrosourea (MNU)-induced in situ breast cancers were used to test this targeted liposomal imaging agent. Specifically, the pharmacokinetics and biodistribution of Tc99m-HMPAO encapsulating SSL with and without VIP were determined together with their ability to image breast cancer. The presence of VIP did not alter the size and Tc99m-HMPAO encapsulation ability of SSL. It also did not alter the pharmacokinetic profile of SSL. Long-circulating liposomes with and without VIP on their surface accumulated at significantly higher quantities in breast cancer when compared to normal breast, indicating passive targeting of these constructs to cancer tissues. Importantly, in breast cancer, Tc99m-HMPAO encapsulating SSL with VIP showed significantly more accumulation than SSL without VIP. The tumor to non-tumor ratio was also significantly higher for Tc99m-HMPAO encapsulating VIP-SSL than Tc99m-HMPAO encapsulating SSL without VIP, suggesting active targeting of VIP-SSL to breast cancer. Collectively, these data showed that Tc99m-HMPAO encapsulating VIP-SSL can be successfully used for the targeted imaging of breast cancer.
Asunto(s)
Neoplasias Mamarias Experimentales/diagnóstico por imagen , Radiofármacos/síntesis química , Péptido Intestinal Vasoactivo/síntesis química , Animales , Carcinógenos , Portadores de Fármacos , Composición de Medicamentos , Femenino , Procesamiento de Imagen Asistido por Computador , Liposomas , Neoplasias Mamarias Experimentales/inducido químicamente , Metilnitrosourea , Neoplasias/diagnóstico por imagen , Neoplasias/metabolismo , Tamaño de la Partícula , Cintigrafía , Ratas , Exametazima de Tecnecio Tc 99m , Distribución TisularRESUMEN
Submandibular secretory and vascular responses to stimulation of the parasympathetic innervation and the output of vasoactive intestinal peptide (VIP) were investigated in anaesthetized sheep in the presence and absence of atropine (>/=0.5 mg/kg). In the absence of atropine, parasympathetic stimulation caused an increase in the flow of saliva and a decrease in submandibular vascular resistance; the latter response persisted after the administration of atropine and was then significantly reduced at the lowest but not at the higher frequencies tested. The output of VIP from the gland was frequency dependent over the range of 10-20 Hz (continuously) and significantly increased after atropine (P < 0.02). Furthermore, the fall in vascular resistance was linearly related to log VIP output after total muscarinic blockade. Intracarotid infusions of synthetic VIP produced dose-dependent falls in submandibular vascular resistance, together with a corresponding increase in submandibular blood flow. It is concluded that the atropine-resistant vasodilatation that occurs in this gland during parasympathetic stimulation is likely to be due largely, if not entirely, to the release of VIP.
Asunto(s)
Sistema Nervioso Parasimpático/fisiología , Glándula Submandibular/fisiología , Animales , Atropina/farmacología , Fenómenos Fisiológicos Cardiovasculares , Arterias Carótidas , Nervio de la Cuerda del Tímpano/fisiología , Relación Dosis-Respuesta a Droga , Resistencia a Medicamentos , Estimulación Eléctrica/métodos , Inyecciones Intraarteriales , Antagonistas Muscarínicos/farmacología , Flujo Sanguíneo Regional/efectos de los fármacos , Saliva/efectos de los fármacos , Saliva/metabolismo , Ovinos , Glándula Submandibular/irrigación sanguínea , Glándula Submandibular/metabolismo , Resistencia Vascular , Péptido Intestinal Vasoactivo/administración & dosificación , Péptido Intestinal Vasoactivo/síntesis química , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
Vasoactive intestinal peptide (VIP) and its two G protein-coupled receptors, VPAC1 and VPAC2, are quantitatively prominent and functionally critical in the immune system. Transgenic (T) mice constitutively expressing VPAC2 selectively in CD4 T cells, at levels higher than those found after maximal induction in CD4 T cells of wild-type (N) mice, have elevated blood concentrations of IgE, IgG1, and eosinophils; enhanced immediate-type hypersensitivity; and reduced delayed-type hypersensitivity. In contrast, VPAC2-null (K) mice manifest decreased immediate-type hypersensitivity and enhanced delayed-type hypersensitivity. The phenotypes are attributable to opposite skewing of the Th2/Th1 cytokine ratio, but no studies were conducted on the roles of T cell-derived VIP and altered expansion of the Th subsets. Dependence of the Th phenotype of T mice, but not of N or K mice, on T cell-derived VIP now is proven by showing that eliminating VIP from TCR-stimulated T cell cultures with VIPase IgG normalizes the elevated number of IL-4-secreting CD4 T cells, decreases the secretion of IL-4 and IL-10, and increases the secretion of IFN-gamma. Flexible responsiveness of CD4 T cells from N and K mice, but not T mice, to exogenous VIP in vitro and in vivo is shown by increased numbers of IL-4-secreting CD4 T cells, greater secretion of IL-4 and IL-10, and lesser secretion of IFN-gamma after TCR stimulation with VIP. The level of VIP recognized by CD4 T cells thus is a major determinant of the relative contributions of Th subsets to the immune effector phenotype.
Asunto(s)
Regulación de la Expresión Génica/inmunología , Inmunofenotipificación , Receptores de Péptido Intestinal Vasoactivo/biosíntesis , Receptores de Péptido Intestinal Vasoactivo/genética , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Péptido Intestinal Vasoactivo/farmacología , Animales , Anticuerpos Catalíticos/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , Citocinas/biosíntesis , Citocinas/metabolismo , Humanos , Hipersensibilidad Tardía/genética , Hipersensibilidad Inmediata/genética , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Biosíntesis de Péptidos/genética , Biosíntesis de Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/fisiología , Receptores de Péptido Intestinal Vasoactivo/deficiencia , Receptores de Tipo II del Péptido Intestinal Vasoactivo , Péptido Intestinal Vasoactivo/síntesis química , Péptido Intestinal Vasoactivo/inmunología , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
OBJECTIVE: To develop a tumor imaging agent for vasoactive intestinal peptide (VPAC) receptor and evaluate its biological activity and pharmacokinetics of radiolabeled peptide. METHODS: VIP(28) was modified at the carboxyl terminal by the addition of His-tag which was the chelating site of (99m)Tc(I) and the general purification tag for immobilized metal ion affinity chromatography. Biological activity of the modified VIP(28) analogue MY34 was examined in vitro by radiological cell-binding assay, rabbit internal anal sphincter (IAS) smooth muscle relaxing assay and immunocytochemical stain. The pharmacokinetics of this labeled peptide was examined in C57 mice. RESULTS: MY34 could relax the IAS smooth muscle and bind VPAC receptors on tumor cell membranes. (99m)Tc- MY34, with a yield of about 90%, was stable enough for practical use. Both MY34 and VIP(28) could inhibit the binding between the labeled peptide and VPAC receptor. The pharmacokinetics of [(99m)Tc(H(2)O)(3)(CO)(3)]-MY34 was studied in mice conformed well with the two-compartment model (Wi = 1/C(2)), with a t(1)/(2alpha) of 16.35 min and a t(1)/(2beta) of 1013.56 min. CONCLUSION: MY34 possesses physiological activities and specific receptor binding characteristics similar to those of natural VIP(28).
Asunto(s)
Receptores de Péptido Intestinal Vasoactivo/análisis , Células 3T3 , Animales , Unión Competitiva , Marcaje Isotópico , Ratones , Ratones Endogámicos C57BL , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Compuestos de Organotecnecio , Péptidos/síntesis química , Péptidos/metabolismo , Péptidos/farmacología , Conejos , Cintigrafía , Neoplasias Gástricas , Células Tumorales Cultivadas , Péptido Intestinal Vasoactivo/síntesis química , Péptido Intestinal Vasoactivo/metabolismo , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
Vasoactive intestinal peptide (VIP) functions as a neurotransmitter involved in a number of physiological and pathological conditions. The actions of VIP are mediated through VPAC(1) and VPAC(2). In contrast to VPAC(1), which has been extensively studied, little is known about the pharmacology of VPAC(2). In this study we investigated the VIP pharmacophore for VPAC(2) by using alanine and D-amino acid scanning. We found significant species differences, and the human VPAC(2) (hVPAC(2)) expressed in Chinese hamster ovary (CHO) cells, which have been used in previous studies, differed significantly from the native hVPAC(2) in Sup T(1) cells and hVPAC(2) expressed in PANC1 cells. There was a close agreement between binding affinities and potencies for VPAC(2) activation. The amino acids whose backbone or side chain orientations were most important for high affinity potency are Asp(3), Phe(6), Thr(7), Tyr(10), Arg(12), Tyr(22), and Leu(23), whereas the side chains of Ser(2), Asp(8), Asn(9), Gln(16), Val(19), Lys(20), Lys(21), Asn(24), and Ser(25) are not essential. Comparison of the VIP pharmacophore between hVPAC(1) and hVPAC(2) demonstrated that the side chains of Thr(7), Tyr(10), Thr(11), and Tyr(22) were much more critical for high affinity for the hVPAC(2) than the hVPAC(1). In contrast, the orientation of the side chain of Asn(24) was more important for high affinity for the hVPAC(1). This study shows that in assessing the pharmacophore of VIP analogs for the VPAC(2), important species differences need to be considered as well as the expression system used. These results of our study should be useful for designing VPAC subtype-selective analogs, simplified analogs, and possibly metabolically stable analogs.
Asunto(s)
Receptores de Péptido Intestinal Vasoactivo/efectos de los fármacos , Péptido Intestinal Vasoactivo/análogos & derivados , Péptido Intestinal Vasoactivo/farmacología , Adenilil Ciclasas/biosíntesis , Adenilil Ciclasas/genética , Alanina/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Aminoácidos/química , Animales , Células CHO , Cricetinae , Humanos , Radioisótopos de Yodo , Datos de Secuencia Molecular , Péptidos/química , Péptidos/farmacología , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/farmacología , Ratas , Receptores de Péptido Intestinal Vasoactivo/química , Receptores de Tipo II del Péptido Intestinal Vasoactivo , Receptores de Tipo I del Polipéptido Intestinal Vasoactivo , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Transfección , Células Tumorales Cultivadas , Péptido Intestinal Vasoactivo/agonistas , Péptido Intestinal Vasoactivo/síntesis químicaRESUMEN
Since numerous tumor cells overexpress the vasoactive intestinal peptide (VIP) receptor subtype 1 (VPAC(1)), VIP-dye conjugates would be useful as contrast agents for in vivo imaging. However, proteolytic degradation of VIP in vivo limits their diagnostic use and highlights the need for structurally optimized VIP derivatives with improved pharmacokinetics. Here, we applied parallel nano-synthesis of cleavable peptides on cellulose membranes to perform a complete VIP substitutional analysis. The resulting 504 different VIP-dye analogs were tested for cell binding by flow cytometry. They provided a detailed analysis of amino acid positions essential for binding to VPAC(1) overexpressing cells. A generalized VIP-dye binding motif derived from the substitutional analysis results served as a reference point for further optimization. An [Arg8]-VIP-dye analog showed increased stability towards proteolytic degradation, good tumor-to-tissue contrast in mice and a longer half-life in vivo.