RESUMEN
Heat-labile toxin I (LT-I), produced by strains of enterotoxigenic Escherichia coli (ETEC), causes profuse watery diarrhea in humans. Different in vitro and in vivo models have already elucidated the mechanism of action of this toxin; however, their use does not always allow for more specific studies on how the LT-I toxin acts in systemic tracts and intestinal cell lines. In the present work, zebrafish (Danio rerio) and human intestinal cells (Caco-2) were used as models to study the toxin LT-I. Caco-2 cells were used, in the 62nd passage, at different cell concentrations. LT-I was conjugated to FITC to visualize its transport in cells, as well as microinjected into the caudal vein of zebrafish larvae, in order to investigate its effects on survival, systemic traffic, and morphological formation. The internalization of LT-I was visualized in 3 × 104 Caco-2 cells, being associated with the cell membrane and nucleus. The systemic traffic of LT-I in zebrafish larvae showed its presence in the cardiac cavity, yolk, and regions of the intestine, as demonstrated by cardiac edema (100%), the absence of a swimming bladder (100%), and yolk edema (80%), in addition to growth limitation in the larvae, compared to the control group. There was a reduction in heart rate during the assessment of larval survival kinetics, demonstrating the cardiotoxic effect of LT-I. Thus, in this study, we provide essential new depictions of the features of LT-I.
Asunto(s)
Toxinas Bacterianas/toxicidad , Escherichia coli Enterotoxigénica , Enterotoxinas/toxicidad , Proteínas de Escherichia coli/toxicidad , Animales , Toxinas Bacterianas/farmacocinética , Células CACO-2 , Edema/inducido químicamente , Embrión no Mamífero/anomalías , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Enterotoxinas/farmacocinética , Proteínas de Escherichia coli/farmacocinética , Cardiopatías Congénitas/inducido químicamente , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Intestinos/metabolismo , Miocardio/metabolismo , Saco Vitelino/efectos de los fármacos , Pez Cebra/anomalías , Pez Cebra/metabolismoRESUMEN
The cell surface receptor claudin-4 (Cld-4) is upregulated in various tumours and represents an important emerging target for both diagnosis and treatment of solid tumours of epithelial origin. The C-terminal fragment of the Clostridium perfringens enterotoxin cCPE290-319 appears as a suitable ligand for targeting Cld-4. The synthesis of this 30mer peptide was attempted via several approaches, which has revealed sequential SPPS using three pseudoproline dipeptide building blocks to be the most efficient one. Labelling with fluorine-18 was achieved on solid phase using N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) and 4-[18F]fluorobenzoyl chloride as 18F-acylating agents, which was the most advantageous when [18F]SFB was reacted with the resin-bound 30mer containing an N-terminal 6-aminohexanoic spacer. Binding to Cld-4 was demonstrated via surface plasmon resonance using a protein construct containing both extracellular loops of Cld-4. In addition, cell binding experiments were performed for 18F-labelled cCPE290-319 with the Cld-4 expressing tumour cell lines HT-29 and A431 that were complemented by fluorescence microscopy studies using the corresponding fluorescein isothiocyanate-conjugated peptide. The 30mer peptide proved to be sufficiently stable in blood plasma. Studying the in vivo behaviour of 18F-labelled cCPE290-319 in healthy mice and rats by dynamic PET imaging and radiometabolite analyses has revealed that the peptide is subject to substantial liver uptake and rapid metabolic degradation in vivo, which limits its suitability as imaging probe for tumour-associated Cld-4.
Asunto(s)
Claudina-4/antagonistas & inhibidores , Enterotoxinas/síntesis química , Enterotoxinas/farmacocinética , Animales , Claudina-4/química , Claudina-4/metabolismo , Enterotoxinas/química , Enterotoxinas/farmacología , Radioisótopos de Flúor/química , Células HT29 , Humanos , Marcaje Isotópico , Ligandos , Masculino , Ratones , Ratones Desnudos , Imagen Molecular , Imitación Molecular/fisiología , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Tomografía de Emisión de Positrones , Ratas , Ratas Wistar , Técnicas de Síntesis en Fase SólidaRESUMEN
Clostridium difficile TcdB (2366 amino acid residues) is an intracellular bacterial toxin that binds to cells and enters the cytosol where it glucosylates small GTPases. In the current study, we examined a putative cell entry region of TcdB (amino acid residues 1753-1851) for short sequences that function as cell-penetrating peptides (CPPs). To screen for TcdB-derived CPPs, a panel of synthetic peptides was tested for the ability to enhance transferrin (Tf) association with cells. Four candidate CPPs were discovered, and further study on one peptide (PepB2) pinpointed an asparagine residue necessary for CPP activity. PepB2 mediated the cell entry of a wide variety of molecules including dextran, streptavidin, microspheres, and lentivirus particles. Of note, this uptake was dramatically reduced in the presence of the Na+/H+ exchange blocker and micropinocytosis inhibitor amiloride, suggesting that PepB2 invokes macropinocytosis. Moreover, we found that PepB2 had more efficient cell-penetrating activity than several other well-known CPPs (TAT, penetratin, Pep-1, and TP10). Finally, Tf assay-based screening of peptides derived from two other large clostridial toxins, TcdA and TcsL, uncovered two new TcdA-derived CPPs. In conclusion, we have identified six CPPs from large clostridial toxins and have demonstrated the ability of PepB2 to promote cell association and entry of several molecules through a putative fluid-phase macropinocytotic mechanism.
Asunto(s)
Proteínas Bacterianas , Toxinas Bacterianas , Péptidos de Penetración Celular , Clostridioides difficile/química , Enterotoxinas , Amilorida/farmacología , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/farmacocinética , Proteínas Bacterianas/farmacología , Toxinas Bacterianas/química , Toxinas Bacterianas/farmacocinética , Toxinas Bacterianas/farmacología , Células CHO , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacocinética , Péptidos de Penetración Celular/farmacología , Cricetulus , Enterotoxinas/química , Enterotoxinas/farmacocinética , Enterotoxinas/farmacología , Pinocitosis/efectos de los fármacosRESUMEN
Staphylococcal enterotoxin C2 (SEC2) is a classical superantigen (SAg), which can tremendously activate T lymphocytes at very low dosage, thus exerting its powerful antitumor activity. As an intravenous protein drug and a bacterial toxin, SEC2 has some limitations including poor patient compliance and toxic side effects. In this research, we devoted our attention to studying the antitumor activity and toxicity of SEC2 as a potential oral administration protein drug. We proved that His-tagged SEC2 (SEC2-His) could undergo facilitated transcytosis on human colon adenocarcinoma (Caco-2) cells and SEC2-His was detected in the blood of rats after oral administration. Furthermore, oral SEC2-His caused massive cytokine release and immune cell enrichment around tumor tissue, leading to inhibition of tumor growth in vivo. Meanwhile, although SEC2-His was dosed up to 32 mg/kg in mice, no significant toxicity was observed. These data showed that SEC2 can cross the intestinal epithelium in an immunologically integral form, maintaining antitumor activity but with reduced systemic toxicity. Therefore, these results may have implications for developing SEC2 as an oral administration protein drug.
Asunto(s)
Antineoplásicos/farmacología , Enterotoxinas/farmacología , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Transcitosis , Administración Oral , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidad , Células CACO-2 , Relación Dosis-Respuesta a Droga , Enterotoxinas/farmacocinética , Enterotoxinas/toxicidad , Humanos , Intestinos/efectos de los fármacos , Intestinos/ultraestructura , Hígado/efectos de los fármacos , Hígado/ultraestructura , Pruebas de Función Hepática , Masculino , Ratones Endogámicos ICR , Ratas Sprague-Dawley , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Prostate cancer is the second leading cause of non-cutaneous cancer-related death in males, and effective strategies for treatment of metastatic disease are currently limited. The tight junction proteins, claudin 3 and claudin 4, serve as cell-surface receptors for the pore-forming Clostridium perfringens enterotoxin [CPE]. Most prostate cancer cells overexpress claudin 3 and claudin 4, and claudins are aberrantly distributed over the plasma membrane, making these cells particularly sensitive to cytolysis by CPE. Prostate cancer cells secrete PSA locally that is proteolytically active; however, circulating PSA is inactivated via binding to protease inhibitors. To overcome systemic toxicity of CPE, a modified protoxin was constructed with a tethered ligand attached to the C-terminus connected by a flexible linker containing a PSA-specific protease cleavage site. This engineered protoxin selectively and efficiently lyses PSA-producing prostate cancer cells whereas CLDN3 and CLDN4 positive cells that do not express PSA are resistant to cytolysis.
Asunto(s)
Claudina-3/metabolismo , Claudina-4/metabolismo , Clostridium perfringens/metabolismo , Enterotoxinas/farmacología , Calicreínas/biosíntesis , Antígeno Prostático Específico/biosíntesis , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Claudina-3/genética , Claudina-4/genética , Clonación Molecular , Enterotoxinas/genética , Enterotoxinas/farmacocinética , Humanos , Masculino , Datos de Secuencia Molecular , Terapia Molecular Dirigida , Neoplasias de la Próstata/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacocinética , Proteínas Recombinantes de Fusión/farmacología , TransfecciónRESUMEN
Heat-labile enterotoxin subunit B (LTB) is a non-catalytic protein from a pentameric subunit of Escherichia coli. Based on its function of binding specifically to ganglioside GM1 on the surface of cells, a novel nanoparticle (NP) composed of a mixture of bovine serum albumin (BSA) and LTB was designed for targeted delivery of 5-fluorouracil to tumor cells. BSA-LTB NPs were characterized by determination of their particle size, polydispersity, morphology, drug encapsulation efficiency, and drug release behavior in vitro. The internalization of fluorescein isothiocyanate-labeled BSA-LTB NPs into cells was observed using fluorescent imaging. Results showed that BSA-LTB NPs presented a narrow size distribution with an average hydrodynamic diameter of approximately 254±19 nm and a mean zeta potential of approximately -19.95±0.94 mV. In addition, approximately 80.1% of drug was encapsulated in NPs and released in the biphasic pattern. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that BSA-LTB NPs exhibited higher cytotoxic activity than non-targeted NPs (BSA NPs) in SMMC-7721 cells. Fluorescent imaging results proved that, compared with BSA NPs, BSA-LTB NPs could greatly enhance cellular uptake. Hence, the results indicate that BSA-LTB NPs could be a potential nanocarrier to improve targeted delivery of 5-fluorouracil to tumor cells via mediation of LTB.
Asunto(s)
Toxinas Bacterianas/farmacocinética , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/metabolismo , Enterotoxinas/farmacocinética , Proteínas de Escherichia coli/farmacocinética , Fluorouracilo/administración & dosificación , Nanocápsulas/química , Nanocompuestos/administración & dosificación , Albúmina Sérica Bovina/química , Antimetabolitos Antineoplásicos/administración & dosificación , Antimetabolitos Antineoplásicos/química , Apoptosis/efectos de los fármacos , Toxinas Bacterianas/administración & dosificación , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Difusión , Enterotoxinas/administración & dosificación , Proteínas de Escherichia coli/administración & dosificación , Fluorouracilo/química , Calor , Humanos , Nanocápsulas/administración & dosificación , Nanocápsulas/ultraestructura , Nanocompuestos/química , Nanocompuestos/ultraestructura , Tamaño de la Partícula , Resultado del TratamientoRESUMEN
The ability of Clostridium perfringens type C to cause human enteritis necroticans (EN) is attributed to beta toxin (CPB). However, many EN strains also express C. perfringens enterotoxin (CPE), suggesting that CPE could be another contributor to EN. Supporting this possibility, lysate supernatants from modified Duncan-Strong sporulation (MDS) medium cultures of three CPE-positive type C EN strains caused enteropathogenic effects in rabbit small intestinal loops, which is significant since CPE is produced only during sporulation and since C. perfringens can sporulate in the intestines. Consequently, CPE and CPB contributions to the enteropathogenic effects of MDS lysate supernatants of CPE-positive type C EN strain CN3758 were evaluated using isogenic cpb and cpe null mutants. While supernatants of wild-type CN3758 MDS lysates induced significant hemorrhagic lesions and luminal fluid accumulation, MDS lysate supernatants of the cpb and cpe mutants caused neither significant damage nor fluid accumulation. This attenuation was attributable to inactivating these toxin genes since complementing the cpe mutant or reversing the cpb mutation restored the enteropathogenic effects of MDS lysate supernatants. Confirming that both CPB and CPE are needed for the enteropathogenic effects of CN3758 MDS lysate supernatants, purified CPB and CPE at the same concentrations found in CN3758 MDS lysates also acted together synergistically in rabbit small intestinal loops; however, only higher doses of either purified toxin independently caused enteropathogenic effects. These findings provide the first evidence for potential synergistic toxin interactions during C. perfringens intestinal infections and support a possible role for CPE, as well as CPB, in some EN cases.
Asunto(s)
Toxinas Bacterianas/farmacocinética , Toxinas Bacterianas/toxicidad , Enterotoxinas/farmacocinética , Enterotoxinas/toxicidad , Intestino Delgado/efectos de los fármacos , Animales , Anticuerpos Antibacterianos/inmunología , Femenino , Regulación Bacteriana de la Expresión Génica , Intestino Delgado/microbiología , Intestino Delgado/patología , Masculino , Mutación , ConejosRESUMEN
BACKGROUND: Emerging evidence has suggested that the capability to sustain tumor formation, growth, and chemotherapy resistance in ovarian as well as other human malignancies exclusively resides in a small proportion of tumor cells termed cancer stem cells. During the characterization of CD44(+) ovarian cancer stem cells, we found a high expression of the genes encoding for claudin-4. Because this tight junction protein is the natural high-affinity receptor for Clostridium perfringens enterotoxin (CPE), we have extensively investigated the sensitivity of ovarian cancer stem cells to CPE treatment in vitro and in vivo. METHODS: Real-time polymerase chain reaction and flow cytometry were used to evaluate claudin-3/-4 expression in ovarian cancer stem cells. Small interfering RNA knockdown experiments and MTS assays were used to evaluate CPE-induced cytotoxicity against ovarian cancer stem cell lines in vitro. C.B-17/SCID mice harboring ovarian cancer stem cell xenografts were used to evaluate CPE therapeutic activity in vivo. RESULTS: CD44(+) ovarian cancer stem cells expressed claudin-4 gene at significantly higher levels than matched autologous CD44(-) ovarian cancer cells, and regardless of their higher resistance to chemotherapeutic agents died within 1 hour after exposure to 1.0 µg/mL of CPE in vitro. Conversely, small-interfering RNA-mediated knockdown of claudin-3/-4 expression in CD44(+) cancer stem cells significantly protected cancer stem cells from CPE-induced cytotoxicity. Importantly, multiple intraperitoneal administrations of sublethal doses of CPE in mice harboring xenografts of chemotherapy-resistant CD44(+) ovarian cancer stem cells had a significant inhibitory effect on tumor progression leading to the cure and/or long-term survival of all treated animals (ie, 100% reduction in tumor burden in 50% of treated mice; P < .0001). CONCLUSIONS: CPE may represent an unconventional, potentially highly effective strategy to eradicate chemotherapy-resistant cancer stem cells.
Asunto(s)
Clostridium perfringens/química , Enterotoxinas/farmacología , Receptores de Hialuranos/biosíntesis , Neoplasias Glandulares y Epiteliales/tratamiento farmacológico , Neoplasias Glandulares y Epiteliales/patología , Células Madre Neoplásicas/efectos de los fármacos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Adulto , Anciano , Animales , Carcinoma Epitelial de Ovario , Línea Celular Tumoral , Chlorocebus aethiops , Claudina-3 , Claudinas/genética , Claudinas/metabolismo , Clostridium perfringens/metabolismo , Enterotoxinas/biosíntesis , Enterotoxinas/farmacocinética , Femenino , Citometría de Flujo , Humanos , Inyecciones Intraperitoneales , Ratones , Ratones SCID , Persona de Mediana Edad , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Glandulares y Epiteliales/metabolismo , Células Madre Neoplásicas/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Células Vero , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Treatment of tumors with macromolecular toxins directed to cytoplasmic targets requires selective endocytosis followed by release of intact toxin from the endosomal/lysosomal compartment. The latter step remains a particular challenge. Claudins 3 and 4 are tight junction proteins that are over-expressed in many types of tumors. This study utilized the C-terminal 30 amino acid fragment of C. perfringens enterotoxin (CPE), which binds to claudins 3 and 4, to deliver a toxin in the form of recombinant gelonin (rGel) to the cytoplasm of the human ovarian carcinoma cell line 2008. RESULTS: CPE was fused to rGel at its N-terminal end via a flexible G4S linker. This CPE-G4S-rGel molecule was internalized into vesicles from which location it produced little cytotoxicity. To enhance release from the endosomal/lysosomal compartment a poly-arginine sequence (R9) was introduced between the CPE and the rGel. CPE-R9-rGel was 10-fold more cytotoxic but selectivity for claudin-expressing cells was lost. The addition of a poly-glutamic acid sequence (E9) through a G4S linker to R9-rGel (E9-G4S-R9-rGel) largely neutralized the non-selective cell membrane penetrating activity of the R9 motif. However, introduction of CPE to the E9-G4S-R9-rGel fusion protein (CPE-E9-G4S-R9-rGel) further reduced its cytotoxic effect. Treatment with the endosomolytic reagent chloroquine increased the cytotoxicity of CPE-E9-G4S-R9-rGel. Several types of linkers susceptible to cleavage by furin and endosomal cathepsin B were tested for their ability to enhance R9-rGel release but none of these modifications further enhanced the cytotoxicity of CPE-E9-G4S-R9-rGel. CONCLUSION: We conclude that while a claudin-3 and -4 ligand serves to deliver rGel into 2008 cells the delivered molecules were entrapped in intracellular vesicles. Incorporation of R9 non-specifically increased rGel cytotoxicity and this effect could be masked by inclusion of an E9 sequence. However, the putative protease cleavable sequences tested were inadequate for release of R9-rGel from CPE-E9-G4S-R9-rGel.
Asunto(s)
Enterotoxinas/administración & dosificación , Proteínas de la Membrana/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Inactivadoras de Ribosomas Tipo 1/administración & dosificación , Línea Celular Tumoral , Claudina-3 , Claudina-4 , Enterotoxinas/química , Enterotoxinas/genética , Enterotoxinas/farmacocinética , Femenino , Humanos , Proteínas de la Membrana/biosíntesis , Terapia Molecular Dirigida/métodos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacocinética , Proteínas Inactivadoras de Ribosomas Tipo 1/química , Proteínas Inactivadoras de Ribosomas Tipo 1/genética , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacocinéticaRESUMEN
IMPORTANCE OF THE FIELD: New agents that specifically engage the immune system are being tested in a variety of malignancies. This review provides an overview of naptumomab, an immunotoxin, with encouraging clinical activity in Phase I trials. AREAS COVERED IN THIS REVIEW: This review examines the preclinical and the published clinical data with regards to naptumomab. WHAT THE READER WILL GAIN: This review provides the reader with an understanding of the mechanism of action, immunology, pharmacokinetics and clinical activity of this agent. TAKE HOME MESSAGE: Naptumomab has a unique mechanism of action and appears to be an active agent in the treatment of refractory solid tumors such as renal cell carcinoma.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Enterotoxinas/uso terapéutico , Inmunoconjugados/uso terapéutico , Neoplasias/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales/farmacología , Antineoplásicos/efectos adversos , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Ensayos Clínicos Fase I como Asunto , Diseño de Fármacos , Enterotoxinas/efectos adversos , Enterotoxinas/química , Enterotoxinas/farmacocinética , Enterotoxinas/farmacología , Humanos , Inmunoconjugados/efectos adversos , Inmunoconjugados/química , Inmunoconjugados/farmacocinética , Inmunoconjugados/farmacologíaRESUMEN
PURPOSE: To conduct a phase II trial in chemoresistant hairy cell leukemia (HCL) with BL22, a recombinant anti-CD22 immunotoxin which showed phase I activity in HCL. PATIENTS AND METHODS: Eligible patients had relapsed/refractory HCL and needed treatment based on blood counts. Patients were stratified into three groups: response to cladribine less than 1 year, those with a response lasting 1 to 4 years, or no response and uncontrolled infection. Patients received BL22 40 microg/kg every other day for three doses on cycle 1. Those achieving hematologic remission (HR), defined as neutrophils > or = 1,500/mm(3), hemoglobin > or = 11 g/dL, and platelets > or = 100,000/mm(3), were observed. Patients without HR were re-treated at 30 microg/kg every other day for three doses every 4 weeks beginning at least 8 weeks after cycle 1. RESULTS: Thirty-six patients were enrolled including 26, nine, and one in groups 1 to 3. The response after one cycle (CR, 25%; PR, 25%) improved when 56% were re-treated (CR, 47%; PR, 25%). CR rate was similar in groups 1 and 2 (P = .7). Twenty-two with baseline spleen height lower than 200 mm had higher CR (64% v 21%; P = .019) and OR rates (95% v 36%; P = .0002) compared to 14 with spleens either absent or higher than 200 mm. The only serious toxicity was reversible grade 3 hemolytic uremic syndrome, not requiring plasmapheresis, in two patients (6%). High neutralizing antibodies were observed in four patients (11%) and prevented re-treatment. CONCLUSION: BL22 activity in HCL is confirmed. Best responses to BL22 after cladribine failure are achieved before the patients develop massive splenomegaly or undergo splenectomy.
Asunto(s)
Anticuerpos/uso terapéutico , Enterotoxinas/uso terapéutico , Leucemia de Células Pilosas/tratamiento farmacológico , Adulto , Anciano , Anticuerpos/administración & dosificación , Anticuerpos/toxicidad , Antineoplásicos/uso terapéutico , Cladribina/uso terapéutico , Resistencia a Medicamentos , Enterotoxinas/administración & dosificación , Enterotoxinas/farmacocinética , Enterotoxinas/toxicidad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Bazo , Resultado del TratamientoRESUMEN
Recent studies have shown that cholesterol plays a significant role in the ability of Toxin A from Clostridium difficile to enter eukaryotic cells. The translocation process is one of three major steps during intoxication that could be targeted for intervention against the severe antibiotic-associated diarrhea caused by C. difficile.
Asunto(s)
Clostridioides difficile/metabolismo , Enterocolitis Seudomembranosa/epidemiología , Infecciones Oportunistas/microbiología , Proteínas Bacterianas/química , Proteínas Bacterianas/farmacocinética , Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/química , Toxinas Bacterianas/farmacocinética , Toxinas Bacterianas/toxicidad , Colesterol/deficiencia , Clostridioides difficile/patogenicidad , Infección Hospitalaria/epidemiología , Infección Hospitalaria/prevención & control , Diarrea/microbiología , Enterotoxinas/química , Enterotoxinas/farmacocinética , Enterotoxinas/toxicidad , Humanos , Lípidos de la Membrana/fisiologíaRESUMEN
BACKGROUND: Radiolabeled analogs of the E. coli heat-stable enterotoxin (ST(h)) are currently under study as imaging and therapeutic agents for colorectal cancer. The aim of these studies is to compare in vitro and in vivo characteristics of two novel ST(h) analogs with appended DOTA chelating moieties. MATERIALS AND METHODS: ST(h) analogs were synthesized with pendant N-terminal DOTA moieties and radiolabeled with indium-111. In vitro cell binding was studied using cultured T-84 human colorectal cancer cells, and in vivo biodistribution studies were carried out using T-84 human colorectal tumor xenografts in SCID mice. RESULTS: Competitive radioligand binding assays employing T-84 human colon cancer cells demonstrated similar IC50 values for the F19-ST(h)(2-19) and F9-ST(h)(6-19) analogs. Addition of DOTA to the N-terminus of these peptides elicited distinctly different effects on binding affinities in vitro, effects that were largely unchanged by metallation with nonradioactive (nat)In. In vivo pharmacokinetic studies in SCID mice bearing T-84 human colon cancer-derived tumor xenografts demonstrated tumor uptake of 0.74 +/- 0.1% ID/g at 4 h post-injection (p.i.) for the 111In-DOTA-F19-ST(h)(2-19) analog, and significantly reduced tumor localization (0.27 + 0.08 % ID/g) for the 111In-DOTA-F9-ST(h)(6-19) analog. CONCLUSION: These results demonstrate that placement of a DOTA moiety immediately adjacent to Cys 6 in ST(h) significantly inhibits receptor binding in vitro and in vivo, highlighting the need for intervening spacer residues between the pharmacophore and the DOTA chelating moiety in effective ST(h)-based radiopharmaceutical constructs.
Asunto(s)
Toxinas Bacterianas/farmacocinética , Neoplasias del Colon/tratamiento farmacológico , Enterotoxinas/farmacocinética , Proteínas de Escherichia coli/farmacocinética , Calor , Animales , Toxinas Bacterianas/uso terapéutico , Unión Competitiva , Neoplasias del Colon/diagnóstico por imagen , Neoplasias del Colon/metabolismo , Enterotoxinas/química , Enterotoxinas/uso terapéutico , Proteínas de Escherichia coli/uso terapéutico , Femenino , Compuestos Heterocíclicos con 1 Anillo , Humanos , Radioisótopos de Indio/farmacocinética , Radioisótopos de Indio/uso terapéutico , Ligandos , Ratones , Ratones Endogámicos ICR , Ratones SCID , Unión Proteica , Desnaturalización Proteica , Cintigrafía , Radiofármacos/química , Radiofármacos/farmacocinética , Radiofármacos/uso terapéutico , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
OBJECTIVES: The current study examined the ability of a new adsorbent, CTR, to remove enterotoxins, toxic shock syndrome toxin-1 (TSST-1), and cytokines from blood and/or serum in vitro and the effects of the extracorporeal treatment with CTR column on mortality rate and inflammatory responses to endotoxic shock in vivo. DESIGN: Laboratory investigation. SETTING: University and company experimental laboratory. MATERIALS: CTR is composed of porous cellulose beads to which a hydrophobic organic compound with a hexadecyl alkyl chain has been covalently bound to the surface as a ligand. Human/bovine serum and human blood samples in vitro and Male Wistar rats were used. INTERVENTIONS: CTR's ability to adsorb bacterial toxins and cytokines related to sepsis in serum and/or blood was examined with an in vitro batch adsorption protocol. In vivo, male Wistar rats were anesthetized and assigned to one of three groups (n=14 per group): Escherichia coli endotoxin (15 mg/kg intravenously) alone (endotoxemic), apheresis with control column without CTR for 120 mins (control column), or extracorporeal treatment with CTR column for 120 mins (CTR treatment). MEASUREMENTS AND MAIN RESULTS: With use of the CTR adsorbent, the adsorption rates were 50% to 90% for enterotoxins, TSST-1, and cytokines such as TNF-alpha and interleukin (IL)-6 in the batch tests. In vivo, the mortality rates at 8 hrs after endotoxin injection were 92%, 92%, and 14% for the endotoxemic, control column, and CTR treatment groups, respectively. Hypotension and elevated plasma cytokine concentrations and the infiltration of neutrophils of the lungs were less conspicuous in the CTR treatment group than in the other two groups. CONCLUSIONS: CTR, a novel adsorbent, effectively adsorbed small- to middle-sized proteins, such as cytokines, enterotoxins, and TSST-1 in vitro. Direct hemoperfusion apheresis with CTR column reduced mortality and had inhibitory effects on the inflammatory responses during endotoxemia in vivo. These findings suggest that extracorporeal blood purification with CTR column may be available to use for patients with sepsis and/or endotoxemia.
Asunto(s)
Toxinas Bacterianas/farmacocinética , Citocinas/farmacocinética , Enterotoxinas/farmacocinética , Hemoperfusión/métodos , Choque Séptico/terapia , Adsorción , Análisis de Varianza , Animales , Celulosa/análogos & derivados , Celulosa/uso terapéutico , Humanos , Técnicas In Vitro , Pulmón/patología , Masculino , Distribución Aleatoria , Ratas , Ratas Wistar , Sepsis/terapia , Superantígenos , Análisis de SupervivenciaRESUMEN
Conventional influenza vaccines currently in use are administered parenterally and generally confer good protection against systemic disease through the induction of high titers of serum virus-neutralizing antibodies. Parenteral vaccines are suboptimal in that they fail to induce a local mucosal response that may prevent the early stages of virus infection. Thus, the intranasal administration of a vaccine may provide a viable alternative to the parenteral route. Indeed, intranasal administration of vaccine antigens when formulated with an appropriate mucosal adjuvant (e.g., bacterial toxins), results in a vigorous local and systemic immune response. This review discusses the nonclinical safety evaluation of Escherichia coli heat-labile toxin as a mucosal adjuvant for an intranasally administered influenza vaccine.
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Adyuvantes Inmunológicos/administración & dosificación , Toxinas Bacterianas/administración & dosificación , Enterotoxinas/administración & dosificación , Proteínas de Escherichia coli , Vacunas contra la Influenza/administración & dosificación , Adyuvantes Inmunológicos/farmacocinética , Adyuvantes Inmunológicos/toxicidad , Administración Intranasal , Animales , Toxinas Bacterianas/farmacocinética , Toxinas Bacterianas/toxicidad , Parálisis de Bell/etiología , Enterotoxinas/farmacocinética , Enterotoxinas/toxicidad , Hurones , Humanos , Inmunidad Mucosa , Vacunas contra la Influenza/farmacocinética , Vacunas contra la Influenza/toxicidad , Ratones , Bulbo Olfatorio/metabolismo , Papio , Conejos , Ratas , Seguridad , Porcinos , Porcinos Enanos , Virosis/etiologíaRESUMEN
New human Escherichia coli heat-stable peptide (ST(h)) analogues containing a DOTA chelating group were synthesized by sequential and selective formation of disulfides bonds in the peptide. This synthetic approach utilizes three orthogonal thiol-protecting groups, Trt, Acm, and t-Bu, to form three disulfide bonds by successive reactions using 2-PDS, iodine, and silyl chloride-sulfoxide systems. The DOTA-ST(h) conjugates exhibiting high guanylin/guanylate cyclase-C (GC-C) receptor binding affinities were obtained with >98% purity. In vitro competitive binding assays, employing T-84 human colon cancer cells, demonstrated the IC(50) values of <2 nM for GC-C receptor binding suggesting that the new synthetic ST(h) analogues are biologically active. In vitro stability studies of the (111)In-DOTA-Phe(19)-ST(h) conjugate incubated in human serum at 37 degrees C under 5% CO(2) atmosphere revealed that this conjugate is extremely stable with no observable decomposition at 24 h postincubation. HPLC analysis of mouse urine at 1 h pi of the (111)In-DOTA-Phe(19)-ST(h) conjugate showed only about 15% decomposition suggesting that the (111)In-DOTA-Phe(19)-ST(h) conjugate is highly stable, even under in vivo conditions. In vivo pharmacokinetic studies of the (111)In-DOTA-Phe(19)-ST(h) conjugate in T-84 human colon cancer derived xenografts in SCID mice conducted at 1 h pi showed an initial tumor uptake of 2.04 +/- 0.30% ID/g at 1 h pi with efficient clearance from the blood pool (0.23 +/- 0.14% ID/g, 1 h pi) by excretion mainly through the renal/urinary pathway (95.8 +/- 0.2% ID, 1 h pi). High tumor/blood, tumor/muscle, and tumor/liver ratios of approximately 9:1, 68:1, and 26:1, respectively, were achieved at 1 h pi The specific in vitro and in vivo uptake of the radioactivity by human colonic cancer cells highlights the potential of radiometalated-DOTA-ST(h) conjugates as diagnostic/therapeutic radiopharmaceuticals.
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Toxinas Bacterianas/síntesis química , Toxinas Bacterianas/farmacocinética , Neoplasias del Colon/tratamiento farmacológico , Disulfuros/metabolismo , Enterotoxinas/síntesis química , Enterotoxinas/farmacocinética , Escherichia coli/química , Compuestos Heterocíclicos con 1 Anillo/química , Radiofármacos/síntesis química , Radiofármacos/farmacocinética , Animales , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/uso terapéutico , Unión Competitiva , Cromatografía Líquida de Alta Presión , Neoplasias del Colon/metabolismo , Enterotoxinas/metabolismo , Enterotoxinas/uso terapéutico , Proteínas de Escherichia coli , Femenino , Humanos , Radioisótopos de Indio/química , Concentración 50 Inhibidora , Ratones , Ratones SCID , Estructura Molecular , Trasplante de Neoplasias , Radiofármacos/metabolismo , Radiofármacos/uso terapéutico , Células Tumorales Cultivadas , Proteína Tumoral Controlada Traslacionalmente 1RESUMEN
UNLABELLED: Guanylyl cyclase C (GC-C) is a transmembrane receptor expressed by human intestinal cells and primary and metastatic colorectal adenocarcinomas but not by extraintestinal tissues or tumors. The Escherichia coli heat-stable enterotoxin analog, STa (5--18), is a 14--amino acid peptide that selectively binds to the extracellular domain of GC-C with subnanomolar affinity. This study examined the utility of a radiolabeled conjugate of STa (5--18) to selectively target and image extraintestinal human colon cancer xenografts in vivo in nude mice. METHODS: The STa conjugate, ethoxyethyl-mercaptoacetamidoadipoylglycylglycine-STa (5--18) (NC100586), was synthesized and labeled with (99m)Tc to produce (99m)Tc-NC100586. This compound was intravenously administered to nude mice bearing human colon cancer xenografts, and specific targeting was evaluated by biodistribution and gamma camera imaging. RESULTS: In CD-1 nude mice, biodistribution and scintigraphic imaging analyses showed selective uptake of (99m)Tc-NC100586 into human colon cancer xenografts that express GC-C but not into normal tissues that do not express GC-C. Similarly, (99m)Tc-NC100586 injected intravenously into CD-1 nude mice with human colon cancer hepatic metastases selectively accumulated in those metastases, and about 5-mm foci of tumor cells were visualized after ex vivo imaging of excised livers. Accumulation of (99m)Tc-NC100586 in human colon cancer xenografts reflected binding to GC-C because (99m)Tc-NC100588, an inactive analog that does not bind to GC-C, did not selectively accumulate in cancer xenografts compared with normal tissues. Also, coadministration of excess unlabeled STa (5--18) prevented accumulation of (99m)Tc-NC100586 in human colon cancer xenografts. Furthermore, (99m)Tc-NC100586 did not selectively accumulate in Lewis lung tumor xenografts, which do not express GC-C. CONCLUSION: This study showed that intravenously administered STa (5--18) selectively recognizes and binds to GC-C expressed by human colon cancer cells in vivo. Also shown was the ability to exploit this selective interaction to target imaging agents to extraintestinal human colon tumors in nude mice. These results suggest the utility of STa and GC-C for the development of novel targeted imaging and therapeutic agents with high specificity for metastatic colorectal tumors in humans.
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Neoplasias del Colon/diagnóstico por imagen , Enterotoxinas , Guanilato Ciclasa/metabolismo , Compuestos de Organotecnecio , Radiofármacos , Receptores de Péptidos/metabolismo , Animales , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Enterotoxinas/farmacocinética , Humanos , Neoplasias Hepáticas/diagnóstico por imagen , Neoplasias Hepáticas/secundario , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Compuestos de Organotecnecio/farmacocinética , Cintigrafía , Radiofármacos/farmacocinética , Receptores de Enterotoxina , Receptores Acoplados a la Guanilato-Ciclasa , Distribución TisularRESUMEN
OBJECTIVE: To make a study on the preparation and tissue distribution of staphylococcal enterotoxin A (SEA) liposomes and to provide scientific basis for the therapy of liver cancer by using SEA liposomes. METHODS: SEA liposomes were prepared by reverse-phase evaporation; the diameter and entrapment efficiency (EC) of SEA liposomes were determined. 125I labeled SEA solution and 125I-SEA liposomes were administrated intravenously to mice, respectively. The radioactivity of the organs was determined by gamma-counter. RESULTS: The mean diameter and EC of SEA liposomes were 505 +/- 34 nm and 44.1% +/- 4.8%, respectively. SEA liposomes were found mainly distributed in the liver and spleen. SEA liposomes had a higher blood clearance, compared with SEA solution; SEA solution had high-radio-activity in plasma and kidney; there was statistical significance between the two groups (P < 0.05). CONCLUSION: The preparation method of SEA liposomes is simple and repeatable. SEA liposomes possess liver-targeting properties and may provide a new application foreground for the treatment of liver cancer.
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Enterotoxinas/administración & dosificación , Hígado/metabolismo , Animales , Portadores de Fármacos , Sistemas de Liberación de Medicamentos/métodos , Enterotoxinas/farmacocinética , Femenino , Liposomas , Masculino , Ratones , Distribución Aleatoria , Distribución TisularRESUMEN
Nasalflu Berna is a trivalent influenza virus vaccine for active immunization against influenza by the nasal route. It consists of influenza virosomes which are formulated from inactivated influenza strains and heat-labile toxin from aseptic Escherichia coli bacteria strain, as an adjuvant (HLT). The results of preclinical studies in ferrets, baboons, minipigs, mice and rabbits are presented here, and issues concerning route of administration, mechanism of action (preventing the disease and halting further spread of the disease), and the specific safety issues of the adjuvant itself (possible neurological activity of HLT) are examined. No clinical signs were detected in the animals, and hematological values were in the normal range. In particular, there was no evidence of any systemic adverse reaction, including sensitization to the test substances, and no evidence of possible neurological activity of the HLT. Further clinical studies in humans conducted over five influenza seasons using this virosome-formulated intranasal vaccine, elicited high levels of influenza-specific hemagglutination inhibition IgG antibody titers to the strains incorporated in the administered vaccine. In addition, IgA antibodies were also elicited in the nasal mucosa, and in the saliva. In addition to the systemic IgG antibody titers, the nasal mucosal IgA antibody response may provide additional local protection by the inhibition of viral replication and further spread in the respiratory tract. Nasalflu was well tolerated by most of the vaccinated subjects, both in terms of nasal symptoms and possible vaccination-mediated systemic symptoms. Both local and systemic symptoms were primarily mild, with only an occasional subject reporting moderate intensity. Out of four serious adverse events seen during the clinical development, only one was thought to be remotely related to the test vaccine.Nasalflu, developed by the Swiss Serum and Vaccine Institute, is a novel, highly immunogenic and safe influenza subunit vaccine which is easily administered as a nasal spray. This new route of administration is likely to increase compliance to vaccination, and could become an important tool to promote vaccination in population groups which show high resistance to vaccination.