RESUMEN
Mongersen is a 21-mer antisense oligonucleotide designed to downregulate Mothers against decapentaplegic homolog 7 (SMAD7) expression to treat Crohn's disease. Mongersen was manufactured in numerous batches at different scales during several years of clinical development, which all appeared identical, using common physicochemical analytical techniques, while only phosphorous-31 nuclear magnetic resonance (31P-NMR) in solution showed marked differences. Close-up analysis of 27 mongersen batches revealed marked differences in SMAD7 downregulation in a cell-based assay. Principal component analysis of 31P-NMR profiles showed strong correlation with SMAD7 downregulation and, therefore, with pharmacological efficacy in vitro. Mongersen contains 20 phosphorothioate (PS) linkages, whose chirality (Rp/Sp) was not controlled during manufacturing. A different diastereomeric composition throughout batches would lead to superimposable analytical data, but to distinct 31P-NMR profiles, as indeed we found. We tentatively suggest that this may be the origin of different biological activity. As similar manifolds are expected for other PS-based oligonucleotides, the protocol described here provides a general method to identify PS chirality issues and a chemometric tool to score each preparation for this elusive feature.
Asunto(s)
Enfermedad de Crohn , Oligonucleótidos Antisentido , Enfermedad de Crohn/tratamiento farmacológico , Enfermedad de Crohn/metabolismo , Regulación hacia Abajo , Humanos , Oligonucleótidos , Oligonucleótidos Antisentido/farmacología , Oligonucleótidos Fosforotioatos/químicaRESUMEN
PURPOSE: To compare the changes in postoperative peripapillary retinal nerve fiber layer (p-RNFL) thickness after vitrectomy for epiretinal membrane in eyes with preexisting posterior vitreous detachment (PVD) and eyes with surgically induced PVD. METHODS: This study included consecutive patients who underwent 25-gauge vitrectomy for epiretinal membrane. Eyes were divided, according to intraoperative PVD status, into a preexisting PVD group and surgically induced PVD group. Best-corrected visual acuity, p-RNFL thickness, and central retinal thickness were performed before and at 1, 3, and 6 months after surgery. RESULTS: One hundred and twenty eyes of 120 patients were enrolled: 64 eyes in the preexisting PVD group and 56 eyes in the surgically induced PVD group. In the preexisting PVD group at 6 months, the mean global p-RNFL thickness did not change, whereas it was reduced in the temporal sector (P = 0.034). In the surgically induced PVD group at 6 months, significant decreases were observed in global p-RNFL thickness (P = 0.027), temporal (P = 0.021), temporal inferior (P = 0.030), and nasal inferior sectors (P = 0.010). At 6 months, the two groups differed significantly in temporal (P < 0.001) and temporal inferior sectors (P = 0.004). The preoperative mean best-corrected visual acuity improved significantly at 6 months in both groups. CONCLUSION: Postoperative p-RNFL thickness after vitrectomy for epiretinal membrane tended to decrease in the temporal sector in all eyes and in the temporal inferior and nasal inferior sectors in eyes with surgically induced PVD.
Asunto(s)
Membrana Epirretinal/cirugía , Fibras Nerviosas/patología , Células Ganglionares de la Retina/patología , Tomografía de Coherencia Óptica/métodos , Vitrectomía/instrumentación , Desprendimiento del Vítreo/cirugía , Anciano , Membrana Epirretinal/complicaciones , Membrana Epirretinal/diagnóstico , Diseño de Equipo , Femenino , Estudios de Seguimiento , Humanos , Masculino , Disco Óptico , Periodo Posoperatorio , Estudios Retrospectivos , Factores de Tiempo , Desprendimiento del Vítreo/complicaciones , Desprendimiento del Vítreo/diagnósticoRESUMEN
PURPOSE: The aim of this study was to compare the efficiency and safety of ultrahigh-speed cut rate 25-gauge system and standard cut rate 25-gauge vitrectomy system. METHODS: In this single-center, prospective randomized study, all consecutive eyes that underwent 25-gauge vitrectomy at the Eye Clinic of the University of Ancona from September 2014 to November 2014 were randomized to receive 25-gauge vitrectomy with 7,500 cuts per minute (cpm) probes (7,500 Group) or 25-gauge vitrectomy with 5,000 cpm probes (Standard Group). Exclusion criteria were previously vitrectomized eye, trauma cases, retinal detachment with proliferative vitreoretinopathy, and endophthalmitis. Main outcome measure was core vitrectomy duration. Secondary outcome was the incidence of iatrogenic retinal breaks and other complications related to surgery. RESULTS: Overall, 62 eyes were enrolled; 31 eyes received 25-gauge 7,500 cpm vitrectomy and 31 eyes received 25-gauge 5,000 cpm vitrectomy. The duration of core vitrectomy was significantly lower in the 7,500 Group (P = 0.030, t-test for independent samples). Mean ± standard deviation core vitrectomy time was 161.32 ± 39.10 seconds in the 7,500 Group and 184.10 ± 41.69 seconds in the Standard Group. The observed difference in mean core vitrectomy duration between subjects treated with 7,500 cpm probes and those in the Standard Group was equal -22 seconds (95% confidence interval: -43.3 to -2.2). There was no difference in the incidence of iatrogenic breaks between the 2 groups, and there were no other complications over a 3-month follow-up period. CONCLUSION: The 25-gauge 7,500 cpm vitrectomy is an effective and safe surgical procedure, and it can significantly reduce core vitrectomy time in eyes undergoing vitreoretinal surgery.
Asunto(s)
Vitrectomía/métodos , Cirugía Vitreorretiniana , Anciano , Femenino , Humanos , Implantación de Lentes Intraoculares , Masculino , Microcirugia/métodos , Tempo Operativo , Facoemulsificación , Estudios Prospectivos , Resultado del Tratamiento , Vitrectomía/instrumentaciónRESUMEN
PURPOSE: To compare the clinical outcomes and the rate of complications of 27-gauge transconjunctival nonvitrectomizing vitreous surgery (NVS) and of 25-gauge transconjunctival sutureless vitrectomy surgery for idiopathic epiretinal membrane removal. METHODS: In this prospective randomized study, 83 phakic eyes of 83 consecutive patients with an idiopathic epiretinal membrane were randomized to receive 27-gauge NVS (NVS-group) or 25-gauge vitrectomy (Standard-group). Main outcome measures were best-corrected visual acuity, central retinal thickness, nuclear density units' changes, and rate of complications. RESULTS: Thirty-nine eyes of the Standard-group and 40 of the NVS-group were considered in final analysis. Mean best-corrected visual acuity improved significantly in both groups, with a significant better result at 12 months in NVS-group (P = 0.039; t-test). Central retinal thickness decreased significantly in both groups (P < 0.001, Tukey test), without significant difference between the two groups at any time point. At 12 months, nuclear density increased significantly in the Standard-group (analysis of variance, P < 0.001), and it did not change in the NVS-group (analysis of variance, P = 0.537). Epiretinal membrane recurred in 5.1% of eyes in the Standard-group and in 7.5% of eyes in the NVS-group (Fisher's exact test, P = 1.000). CONCLUSION: The 27-gauge NVS is an effective surgical procedure in eyes with epiretinal membrane and it induces less progression of nuclear sclerosis than 25-gauge vitrectomy.
Asunto(s)
Membrana Epirretinal/cirugía , Vitrectomía , Cuerpo Vítreo/cirugía , Anciano , Conjuntiva/cirugía , Membrana Epirretinal/diagnóstico , Membrana Epirretinal/fisiopatología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias , Estudios Prospectivos , Recurrencia , Técnicas de Sutura , Agudeza Visual/fisiologíaRESUMEN
PURPOSE: To investigate, with spectral-domain optical coherence tomography, if the preoperative status of the retinal pigment epithelium (RPE) affects the postoperative foveal morphology and visual outcomes in eyes with surgically closed macular holes (MHs). METHODS: In 52 eyes with surgically closed MHs, preoperative RPE morphology was evaluated and graded based on the measurement of the largest hyperreflective protrusions above the RPE line. Foveal microstructural features and best-corrected visual acuity (BCVA) were evaluated 12 months after surgery. RESULTS: At 12 months, a significant correlation was found between postoperative degree of integrity of the photoreceptors with preoperative RPE morphology, and base diameter of the hole (p = 0.003 and p = 0.028, respectively); mean BCVA at 12 months in eyes with diffuse RPE alteration was significantly lower than in eyes with small or no RPE alteration (p < 0.05). CONCLUSIONS: Preoperative RPE integrity may be indicative of good photoreceptor restoration and visual recovery in patients with surgically closed MHs.
Asunto(s)
Fóvea Central/ultraestructura , Perforaciones de la Retina/patología , Epitelio Pigmentado de la Retina/patología , Anciano , Femenino , Estudios de Seguimiento , Humanos , Masculino , Periodo Preoperatorio , Perforaciones de la Retina/cirugía , Estudios Retrospectivos , Tomografía de Coherencia Óptica/métodos , Agudeza Visual , VitrectomíaRESUMEN
PURPOSE: To evaluate the incidence rates of iatrogenic retinal breaks in eyes that underwent 25-gauge vitrectomy under air compared with 25-gauge standard vitrectomy for idiopathic macular holes or idiopathic epiretinal membranes. METHODS: In this retrospective, comparative interventional study, 435 eyes were enrolled. In all patients after core vitrectomy and epiretinal/inner limiting membrane peeling, complete vitrectomy of the base was performed, respectively under air (air group) or under fluid infusion (standard group). RESULTS: The number of eyes with iatrogenic retinal breaks was significantly lower in the air group than in standard group (4/197 and 16/238, 2% and 7%, respectively; P = 0.035). A postoperative retinal detachment developed in 2 eyes (1%) in the standard group, and in no eyes of the air group (0%). Factors related to the occurrence of retinal breaks were surgically induced posterior vitreous detachment (P = 0.006), standard vitrectomy (P = 0.023), and surgery for macular hole (P = 0.030). CONCLUSION: The 25-gauge vitrectomy under air is associated with a lower incidence rate of retinal breaks compared with the standard 25-gauge vitrectomy.
Asunto(s)
Aire , Membrana Epirretinal/cirugía , Complicaciones Intraoperatorias , Complicaciones Posoperatorias , Perforaciones de la Retina/epidemiología , Vitrectomía/métodos , Anciano , Membrana Basal/cirugía , Femenino , Humanos , Enfermedad Iatrogénica/epidemiología , Incidencia , Masculino , Perforaciones de la Retina/cirugía , Estudios RetrospectivosRESUMEN
We report an atypical presentation of Antiphospholipid syndrome (APS) with concomitant subhyaloid hemorrhage, engorged and tortuous retinal veins, intraretinal hemorrhages, and cotton wool spots in a 38-year-old female. Medical treatment was preferred to any invasive treatment. The subhyaloid hemorrhage resolved spontaneously and the patient recovered a visual acuity of 20/20 in her right eye 3 months after the initial episode. A prompt diagnosis of this condition is fundamental to consider a systemic treatment to avoid any further thrombosis.
RESUMEN
In the gut of patients with Crohn's disease (CD), high Smad7 blocks the immune-suppressive activity of transforming growth factor (TGF)-ß1, thereby contributing to amplify inflammatory signals. In vivo in mice, knockdown of Smad7 with a Smad7 antisense oligonucleotide (GED0301) attenuates experimental colitis. Here, we provide results of a phase 1 clinical, open-label, dose-escalation study of GED0301 in patients with active, steroid-dependent/resistant CD, aimed at assessing the safety and tolerability of the drug. Patients were allocated to three treatment groups receiving oral GED0301 once daily for 7 days at doses of 40, 80, or 160 mg. A total of 15 patients were enrolled. No serious adverse event was registered. GED0301 was well tolerated and no patient dropped out during the study. Twenty-five adverse events were documented in 11 patients, the majority of whom were judged to be of mild intensity and unrelated to treatment. GED0301 treatment reduced the percentage of inflammatory cytokine-expressing CCR9-positive T cells in the blood. The study shows for the first time that GED0301 is safe and well tolerated in patients with active CD.
Asunto(s)
Enfermedad de Crohn/terapia , Oligonucleótidos Antisentido/uso terapéutico , Proteína smad7/metabolismo , Adulto , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Oligonucleótidos Antisentido/administración & dosificación , Farmacocinética , Proteína smad7/genética , Adulto JovenRESUMEN
To describe a case of macular pucker treated by non-vitrectomizing vitreoretinal surgery using a 25-gauge high-flow infusion cannula with a 27-gauge illumination source (Photon II; both from Synergetics USA Inc., O'Fallon, MO). A 42-year-old man complaining of visual reduction and metamorphopsia was referred to the Vitreoretinal Surgery Unit, where he underwent full ophthalmic examination. Best corrected visual acuity (BCVA) at baseline was 0.5 in the left eye (LE) and 1.0 in the right eye. He was diagnosed with epiretinal membrane (ERM) in the LE. The surgical procedure envisaged two-port sclerotomy, one for the infusion line of the 27-gauge Photon II light source, the other for the 25-gauge trocar in the superotemporal quadrant. The ERM was removed with a 25-gauge disposable forceps. The infusion line was placed for safety in the event of additional intraocular pressure being needed, but was not required. The 27-gauge light source afforded good visualization of the posterior pole and the ERM. At 12 months, the patient's BCVA had improved to 1.0, and his cataract has not progressed. A 25-gauge infusion line used with the photon illumination may further improve non-vitrectomizing surgery, minimizing risk and optimizing outcome. Further trials are required to validate these preliminary observations.
Asunto(s)
Mácula Lútea/cirugía , Enfermedades de la Retina/cirugía , Vitrectomía/métodos , Adulto , Humanos , Mácula Lútea/patología , Masculino , Retina/patología , Instrumentos Quirúrgicos , Agudeza VisualRESUMEN
UNLABELLED: The aim of this study was to target the angiogenesis-associated extracellular matrix protein ED-B fibronectin for molecular imaging of solid tumors. Recombinant and chemically modified derivatives of the single-chain antibody fragment (scFv) L19, capable of being labeled with 99mTc, were synthesized and radiolabeled. The resulting compounds 99mTc-AP39, 99mTc-L19-His, and 99mTc-L19-Hi20 were assessed for their imaging properties in vivo. METHODS: L19 was genetically modified by inserting either the (Gly)3-Cys-Ala (AP39) or a (His)6 tag (L19-His) sequence at the C-terminal end. Chemical modifications were performed by conjugating the bifunctional chelator Hi20 (L19-Hi20) at epsilon-Lys-NH2 residues of the molecule to allow for a direct chelator-based labeling with 99mTc. Tumor-targeting, pharmacokinetic, and scintigraphic imaging properties of the radiolabeled scFvs were evaluated in nude mice bearing murine F9 teratocarcinoma. RESULTS: 99mTc labeling of the L19 derivatives yielded radiochemically pure proteins maintaining high immunoreactivity to ED-B fibronectin, as measured by affinity chromatography. Size-exclusion high-performance liquid chromatographic analysis of labeled L19 derivatives demonstrated either dimeric species (L19-His) or a mixture of predominantly associative dimeric and monomeric species (AP39, L19-Hi20). 99mTc-AP39 showed the most favorable biodistribution and imaging properties with high and fast tumor uptake (8.3 percentage injected dose per gram at 3 h after injection), rapid blood clearance and renal excretion, leading to high signal-to-noise ratios (tumor-to-blood ratio of 6.4 at 3 h after injection), and excellent planar scintigraphy in vivo. CONCLUSION: ED-B fibronectin can be efficiently targeted by 99mTc-AP39 and scintigraphically visualized in tumor-bearing mice, providing a potentially useful clinical tool for imaging of angiogenesis-associated ED-B fibronectin-expressing human tumors.
Asunto(s)
Fibronectinas/inmunología , Región Variable de Inmunoglobulina , Neoplasias Experimentales/irrigación sanguínea , Neovascularización Patológica/diagnóstico por imagen , Radiofármacos , Proteínas Recombinantes , Animales , Línea Celular Tumoral , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Cintigrafía , Radiofármacos/farmacocinética , Proteínas Recombinantes/farmacocinética , Tecnecio/farmacocinética , Distribución Tisular , Trasplante HeterólogoRESUMEN
PURPOSE: The expression of extra domain B (ED-B) fibronectin is always associated with angiogenic processes and can be exclusively observed in tissues undergoing growth and/or extensive remodeling. Due to this selective expression, ED-B fibronectin is an interesting target for radioimmunotherapy of malignant diseases. The aim of this study was to identify the most appropriate ED-B-targeting radioimmunoconjugate for the therapy of solid tumors. EXPERIMENTAL DESIGN: Three ED-B fibronectin-binding human antibody formats of L19 were investigated: dimeric single-chain Fv (approximately 50 kDa), "small immunoprotein" (SIP, approximately 80 kDa), and immunoglobulin G1 (IgG1, approximately 150 kDa). These L19 derivatives were either labeled with I-125 or with In-111 (using MX-diethylenetriaminepentaacetic acid, MX-DTPA). Pharmacokinetics and tumor accumulation of the radiolabeled immunoconjugates were investigated in F9 (murine teratocarcinoma) tumor-bearing mice. Subsequently, dosimetry for the corresponding therapeutic isotopes I-13-1 and Y-90 was done. After testing the myelotoxicity of I-131-L19-SIP and I-131-L19-IgG1 in non-tumor-bearing mice, the therapeutic efficacy of these iodinated antibody formats was finally investigated in F9 tumor-bearing mice. RESULTS: The most favorable therapeutic index was found for I-131-L19-SIP followed by I-131-L19-IgG1. The therapeutic index of all In-111-labeled derivatives was significantly inferior. Considering the bone marrow as the dose-limiting organ, it was calculated that activities of 74 MBq I-131-L19-SIP and 25 MBq I-131-L19-IgG1 could be injected per mouse without causing severe myelotoxicity. The best therapeutic efficacy was observed using I-131-L19-SIP, resulting in significant tumor growth delay and prolonged survival after a single injection. CONCLUSION: Compared with other L19-based radioimmunoconjugates, I-131-L19-SIP is characterized by superior antitumor efficacy and toxicity profile in the F9 teratocarcinoma animal model. These results indicate that ED-B fibronectin-targeted radioimmunotherapy using I-131-L19-SIP has potential to be applied to treatment of solid cancers.
Asunto(s)
Fibronectinas/química , Inmunoconjugados/farmacología , Neoplasias/terapia , Radioinmunoterapia/métodos , Animales , Médula Ósea/metabolismo , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Dimerización , Electroforesis en Gel de Poliacrilamida , Fibronectinas/metabolismo , Humanos , Inmunoconjugados/química , Fragmentos de Inmunoglobulinas/química , Inmunoglobulina G/química , Lisina/química , Ratones , Ácido Pentético/análogos & derivados , Ácido Pentético/química , Estructura Terciaria de Proteína , Radiometría , Factores de TiempoRESUMEN
Advances in proteomic research allow the identification of several hundred protein components in complex biological specimens. Structural information is typically lost during proteomic investigations. For this reason, the rapid isolation of monoclonal antibodies specific to proteins of interest would allow the study of structurally intact biological specimens, thus providing complementary proteomic information. Here, we describe the design, construction, characterization, and use of a large synthetic human antibody phage display library (ETH-2-Gold) containing three billion individual antibody clones. A large repertoire of antibodies with similar biochemical properties was produced by appending short variable complementarity-determining region 3 (CDR3) onto three antibody germline segments (DP47, DPK22, and DPL16), which are frequently found in human antibodies. The ETH-2-Gold library exhibits efficient display of antibody fragments on filamentous phage, as assessed by immunoblot. Furthermore, the library is highly functional, since >90% of clones express soluble antibodies in bacteria and since good quality monoclonal antibodies have been isolated against 16 different antigens. The usefulness of the library as a tool for generating monoclonal antibodies for biomedical applications was tested using the C-domain of tenascin-C (a marker of angiogenesis) as antigen and showing that specific antibodies to this target were able to stain vascular structures in tumor sections.
Asunto(s)
Anticuerpos Monoclonales , Biblioteca de Péptidos , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/genética , Especificidad de Anticuerpos , Bacterias/metabolismo , Proteínas de la Cápside , Clonación Molecular , Proteínas de Unión al ADN/genética , Ensayo de Inmunoadsorción Enzimática , Glioblastoma/irrigación sanguínea , Humanos , Región Variable de Inmunoglobulina/genética , Inovirus/metabolismo , Neovascularización Patológica/metabolismo , Estructura Terciaria de Proteína , Tenascina/análisis , Tenascina/inmunología , Proteínas Virales de Fusión/genéticaRESUMEN
The dissociation rate constant of bimolecular complexes between macromolecules (k(off)) is often measured in solution by competition experiments and is generally expected to follow first-order kinetics.When measuring k(off) constants by competition for three complexes of high-affinity recombinant antibody fragments with the cognate antigen and for one calmodulin/peptide complex, a surprising dependence between apparent dissociation rate and concentration of competitor (antigen or calmodulin-binding peptide) was observed. Our results may be characteristic for macromolecules consisting of two domains (such as single-chain Fv fragments) and may reflect a transient opening of the two domains which are involved in the binding reaction, and which are connected by a polypeptide linker.
Asunto(s)
Complejo Antígeno-Anticuerpo/inmunología , Anticuerpos/inmunología , Antígenos/inmunología , Unión Competitiva , Calmodulina/metabolismo , Relación Dosis-Respuesta Inmunológica , Ensayo de Inmunoadsorción Enzimática , Fibronectinas/inmunología , Cinética , Ligandos , Sustancias Macromoleculares , Modelos Inmunológicos , Muramidasa/inmunología , Péptidos/metabolismo , Unión ProteicaRESUMEN
PURPOSE: ED-B fibronectin is expressed only during angiogenic processes and in tissues undergoing growth and/or extensive remodeling. We demonstrated previously the possibility to target and selectively deliver therapeutic substances to tumor vasculature in experimental animal models using a human recombinant antibody fragment, L19, specific for the ED-B domain of fibronectin. Here we evaluate the possibility of targeting primary tumors and metastatic lesions in cancer patients through immunoscintigraphy using (123)I-labeled dimeric L19 [L19(scFv)(2)]. EXPERIMENTAL DESIGN: Twenty patients (34-79 years of age) with lung, colorectal, or brain cancer, whose tumors had been confirmed by imaging techniques and/or histologically, were admitted to the immunoscintigraphic investigation. RESULTS: The dimeric L19 antibody selectively localized in tumor lesions in aggressive types of lung cancer and colorectal cancer. Because ED-B fibronectin is expressed only during angiogenic processes and in tissues undergoing growth and/or extensive remodeling, L19(scFv)(2) is able to distinguish between quiescent and actively growing lesions. No side effects were observed. CONCLUSIONS: The ability of L19(scFv)(2) to target tumors in patients provides the foundations for new therapeutic applications, in which the L19 antibody is engineered to selectively deliver bioactive molecules to primary tumors as well as to metastases.
Asunto(s)
Fibronectinas/análisis , Fibronectinas/química , Neoplasias/irrigación sanguínea , Neovascularización Patológica/diagnóstico por imagen , Adulto , Anciano , Biomarcadores/análisis , Neoplasias Encefálicas/irrigación sanguínea , Neoplasias Encefálicas/diagnóstico por imagen , Neoplasias Encefálicas/patología , Neoplasias Colorrectales/irrigación sanguínea , Neoplasias Colorrectales/diagnóstico por imagen , Neoplasias Colorrectales/patología , Dimerización , Humanos , Inmunohistoquímica , Radioisótopos de Yodo , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/patología , Persona de Mediana Edad , Neoplasias/diagnóstico por imagen , Neoplasias/patología , RadioinmunodetecciónRESUMEN
Angiogenesis, the sprouting of new blood vessels from preexisting ones, is a characteristic feature of many aggressive solid tumors. Advances in the identification of markers of angiogenesis and new methodologies for the production of human recombinant antibodies are making it possible to deliver bioactive molecules to the tumor neovasculature in a selective fashion. This review illustrates how recombinant antibody derivatives may open new avenues for the imaging and therapy of angiogenesis-related diseases, such as cancer, blinding ocular disorders and rheumatoid arthritis.