RESUMEN
AIMS: Antibody formation to therapeutic peptides is common. This analysis characterizes the time-course and cross-reactivity of anti-exenatide antibodies and potential effects on efficacy and safety. METHODS: Data from intent-to-treat patients in 12 controlled (n = 2225,12-52 weeks) and 5 uncontrolled (n = 1538, up to 3 years) exenatide twice-daily (BID) trials and 4 controlled (n = 653,24-30 weeks) exenatide once weekly (QW) trials with 1 uncontrolled period (n = 128,52 weeks) were analysed. RESULTS: Mean titres peaked early (6-22 weeks) and subsequently declined. At 30 weeks, 36.7% of exenatide BID patients were antibody-positive; 31.7% exhibited low titres (≤125) and 5.0% had higher titres (≥625). Antibody incidence declined to 16.9% (1.4% higher titre) at 3 years. Similarly, 56.8% of exenatide QW patients were antibody-positive (45.0% low/11.8% higher titre) at 24-30 weeks, declining to 45.4% positive (9.2% higher titre) at 52 weeks. Treatment-emergent anti-exenatide antibodies from a subset of patients tested did not cross-react with human GLP-1 or glucagon. Other than injection-site reactions, adverse event rates in antibody-positive and antibody-negative patients were similar. Efficacy was robust in both antibody-negative and antibody-positive patients (mean HbA1c change: -1.0 and -0.9%, respectively, exenatide BID; -1.6% and -1.3% exenatide QW). No correlation between change in HbA1c and titre was observed for exenatide BID, although mean reductions were attenuated in the small subset of patients (5%) with higher titres. A significant correlation was observed for exenatide QW with no difference between antibody-negative and low-titre patients, but an attenuated mean reduction in the subset of patients (12%) with higher titres. CONCLUSIONS: Low-titre anti-exenatide antibodies were common with exenatide treatment (32% exenatide BID, 45% exenatide QW patients), but had no apparent effect on efficacy. Higher-titre antibodies were less common (5% exenatide BID, 12% exenatide QW) and within that titre group, increasing antibody titre was associated with reduced average efficacy that was statistically significant for exenatide QW. Other than injection-site reactions, anti-exenatide antibodies did not impact the safety of exenatide.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Glucemia/metabolismo , Diabetes Mellitus Tipo 2/inmunología , Glucagón/farmacología , Hipoglucemiantes/inmunología , Péptidos/inmunología , Ponzoñas/inmunología , Adulto , Anciano , Anticuerpos Antiidiotipos/efectos adversos , Glucemia/efectos de los fármacos , Glucemia/inmunología , Reacciones Cruzadas/efectos de los fármacos , Reacciones Cruzadas/inmunología , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Ensayo de Inmunoadsorción Enzimática , Exenatida , Femenino , Humanos , Hipoglucemiantes/administración & dosificación , Masculino , Persona de Mediana Edad , Péptidos/administración & dosificación , Factores de Tiempo , Resultado del Tratamiento , Ponzoñas/administración & dosificaciónRESUMEN
Diabetes treatment has traditionally focused on correcting insulin deficiency with exogenous insulin and oral agents designed to enhance insulin secretion or insulin sensitivity in peripheral tissues. The more recent view of diabetes as a disease that affects multiple hormones in addition to insulin has led to the development of new therapies more broadly aimed at restoring glucose homeostasis by correcting abnormalities in additional glucoregulatory hormones. Pramlintide, a synthetic analogue of the beta-cell hormone amylin, regulates the appearance of glucose in the circulation following meals through several mechanisms of action: slowing gastric emptying, preventing inappropriate postprandial secretion of glucagon and increasing satiety. Long-term studies have demonstrated that pramlintide improves postprandial glucose fluctuations and A1C while reducing insulin dose and body weight. This combination of benefits associated with pramlintide makes it an attractive new treatment option for patients with diabetes. Clinical Trial Registry Numbers: 137-155 open-label clinical trial: NCT00108004 (Pramlintide long-term, placebo-controlled clinical trials were completed prior to the requirement for NCT registry).
Asunto(s)
Amiloide/uso terapéutico , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/uso terapéutico , Amiloide/metabolismo , Glucemia/metabolismo , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 2/sangre , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos , Periodo Posprandial , Resultado del TratamientoRESUMEN
Transport of proteins through the ALP (alkaline phosphatase) pathway to the vacuole requires the function of the AP-3 adaptor complex and Vps41p. However, unlike other adaptor protein-dependent pathways, the ALP pathway has not been shown to require additional accessory proteins or coat proteins, such as membrane recruitment factors or clathrin. Two independent genetic approaches have been used to identify new mutants that affect transport through the ALP pathway. These screens yielded new mutants in both VPS41 and the four AP-3 subunit genes. Two new VPS41 alleles exhibited phenotypes distinct from null mutants of VPS41, which are defective in vacuolar morphology and protein transport through both the ALP and CPY sorting pathways. The new alleles displayed severe ALP sorting defects, normal vacuolar morphology, and defects in ALP vesicle formation at the Golgi complex. Sequencing analysis of these VPS41 alleles revealed mutations encoding amino acid changes in two distinct domains of Vps41p: a conserved N-terminal domain and a C-terminal clathrin heavy-chain repeat (CHCR) domain. We demonstrate that the N-terminus of Vps41p is required for binding to AP-3, whereas the C-terminal CHCR domain directs homo-oligomerization of Vps41p. These data indicate that a homo-oligomeric form of Vps41p is required for the formation of ALP containing vesicles at the Golgi complex via interactions with AP-3.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Portadoras/farmacología , Proteínas de la Membrana/metabolismo , Proteínas de Ensamble de Clatrina Monoméricas , Proteínas Nucleares , Proteínas de Unión al ARN/farmacología , Proteínas de Saccharomyces cerevisiae , Proteínas de Transporte Vesicular , Proteínas Adaptadoras del Transporte Vesicular , Alelos , Secuencia de Aminoácidos , Sitios de Unión , Proteínas Portadoras/genética , Clatrina/genética , Clatrina/farmacología , Cadenas Pesadas de Clatrina , Dimerización , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/farmacología , Proteínas de la Membrana/genética , Proteínas de la Membrana/farmacología , Datos de Secuencia Molecular , Mutación , Fenotipo , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/química , Alineación de Secuencia , Vesículas Transportadoras/efectos de los fármacosAsunto(s)
Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Transporte de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Bioquímica/métodos , Escherichia coli/metabolismo , Vectores Genéticos , Peróxido de Hidrógeno/metabolismo , Plásmidos/metabolismo , Levaduras/metabolismo , beta-FructofuranosidasaRESUMEN
Aminopeptidase I (API) is imported into the yeast vacuole/lysosome by a constitutive non-classical vesicular transport mechanism, the cytoplasm to vacuole targeting (Cvt) pathway. Newly synthesized precursor API is sequestered in double-membrane cytoplasmic Cvt vesicles. The Cvt vesicles fuse with the vacuole, releasing single-membrane Cvt bodies containing proAPI into the vacuolar lumen, and maturation of API occurs when the Cvt body is degraded, releasing mature API. Under starvation conditions, API is transported to the vacuole by macroautophagy, an inducible, non-selective mechanism that shares many similarities with the Cvt pathway. Here we show that Tlg2p, a member of the syntaxin family of t-SNARE proteins, and Vps45p, a Sec1p homologue, are required in the constitutive Cvt pathway, but not in inducible macroautophagy. Fractionation and protease protection experiments indicate that Tlg2p is required prior to or at the step of API segregation into the Cvt vesicle. Thus, the early Vps45-Tlg2p-dependent step of the Cvt pathway appears to be mechanistically distinct from the comparable stage in macroautophagy. Vps45p associates with both the Tlg2p and Pep12p t-SNAREs, but API maturation is not blocked in a pep12(ts) mutant, indicating that Vps45p independently regulates the function of multiple t-SNARES at distinct trafficking steps.
Asunto(s)
Aminopeptidasas/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas de Transporte Vesicular , Autofagia , Lisosomas/metabolismo , Microscopía Electrónica , Proteínas Munc18 , Mutación , Precursores de Proteínas/metabolismo , Proteínas Qa-SNARE , Proteínas SNARE , Saccharomyces cerevisiae , Sirolimus/farmacología , Vacuolas/metabolismoRESUMEN
Transport of a subset of membrane proteins to the yeast vacuole requires the function of the AP-3 adaptor protein complex. To define the molecular requirements of vesicular transport in this pathway, we used a biochemical approach to analyse the formation and content of the AP-3 transport intermediate. A vam3tsf (vacuolar t-SNARE) mutant blocks vesicle docking and fusion with the vacuole and causes the accumulation of 50-130-nanometre membrane vesicles, which we isolated and showed by biochemical analysis and immunocytochemistry to contain both AP-3 adaptors and alkaline phosphatase (ALP) pathway cargoes. Inactivation of AP-3 or the protein Vps41 blocks formation of this vesicular intermediate. Vps41 binds to the AP-3 delta-adaptin subunit, suggesting that they function together in the formation of ALP pathway transport intermediates at the late Golgi.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Ensamble de Clatrina Monoméricas , Proteínas Nucleares , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas de Transporte Vesicular , Subunidades alfa de Complejo de Proteína Adaptadora , Proteínas Adaptadoras del Transporte Vesicular , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Sitios de Unión , Transporte Biológico , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Compartimento Celular , Membrana Celular/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Proteínas de la Membrana/genética , Mutagénesis , Proteínas Qa-SNARE , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/fisiología , Vacuolas/metabolismoRESUMEN
The transport of newly synthesized proteins through the vacuolar protein sorting pathway in the budding yeast Saccharomyces cerevisiae requires two distinct target SNAP receptor (t-SNARE) proteins, Pep12p and Vam3p. Pep12p is localized to the pre-vacuolar endosome and its activity is required for transport of proteins from the Golgi to the vacuole through a well defined route, the carboxypeptidase Y (CPY) pathway. Vam3p is localized to the vacuole where it mediates delivery of cargoes from both the CPY and the recently described alkaline phosphatase (ALP) pathways. Surprisingly, despite their organelle-specific functions in sorting of vacuolar proteins, overexpression of VAM3 can suppress the protein sorting defects of pep12Delta cells. Based on this observation, we developed a genetic screen to identify domains in Vam3p (e.g., localization and/or specific protein-protein interaction domains) that allow it to efficiently substitute for Pep12p. Using this screen, we identified mutations in a 7-amino acid sequence in Vam3p that lead to missorting of Vam3p from the ALP pathway into the CPY pathway where it can substitute for Pep12p at the pre-vacuolar endosome. This region contains an acidic di-leucine sequence that is closely related to sorting signals required for AP-3 adaptor-dependent transport in both yeast and mammalian systems. Furthermore, disruption of AP-3 function also results in the ability of wild-type Vam3p to compensate for pep12 mutants, suggesting that AP-3 mediates the sorting of Vam3p via the di-leucine signal. Together, these data provide the first identification of an adaptor protein-specific sorting signal in a t-SNARE protein, and suggest that AP-3-dependent sorting of Vam3p acts to restrict its interaction with compartment-specific accessory proteins, thereby regulating its function. Regulated transport of cargoes such as Vam3p through the AP-3-dependent pathway may play an important role in maintaining the unique composition, function, and morphology of the vacuole.
Asunto(s)
Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Proteínas de Ensamble de Clatrina Monoméricas , Proteínas del Tejido Nervioso/química , Fosfoproteínas/química , Proteínas de Saccharomyces cerevisiae , Proteínas de Transporte Vesicular , Proteínas Adaptadoras del Transporte Vesicular , Fosfatasa Alcalina/fisiología , Transporte Biológico/fisiología , Carboxipeptidasas/fisiología , Catepsina A , Endosomas/fisiología , Proteínas Fúngicas/metabolismo , Leucina/química , Mutación/genética , Proteínas Qa-SNARE , Proteínas SNARE , Saccharomyces cerevisiae , Proteína 3 de Membrana Asociada a VesículasRESUMEN
A genetic screen to isolate gene products required for vacuolar morphogenesis in the yeast Saccharomyces cerevisiae identified VAM7, a gene which encodes a protein containing a predicted coiled-coil domain homologous to the coiled-coil domain of the neuronal t-SNARE, SNAP-25 (Y. Wada and Y. Anraku, J. Biol. Chem. 267:18671-18675, 1992; T. Weimbs, S. H. Low, S. J. Chapin, K. E. Mostov, P. Bucher, and K. Hofmann, Proc. Natl. Acad. Sci. USA 94:3046-3051, 1997). Analysis of a temperature-sensitive-for-function (tsf) allele of VAM7 (vam7(tsf)) demonstrated that the VAM7 gene product directly functions in vacuolar protein transport. vam7(tsf) mutant cells incubated at the nonpermissive temperature displayed rapid defects in the delivery of multiple proteins that traffic to the vacuole via distinct biosynthetic pathways. Examination of vam7(tsf) cells at the nonpermissive temperature by electron microscopy revealed the accumulation of aberrant membranous compartments that may represent unfused transport intermediates. A fraction of Vam7p was localized to vacuolar membranes. Furthermore, VAM7 displayed genetic interactions with the vacuolar syntaxin homolog, VAM3. Consistent with the genetic results, Vam7p physically associated in a complex containing Vam3p, and this interaction was enhanced by inactivation of the yeast NSF (N-ethyl maleimide-sensitive factor) homolog, Sec18p. In addition to the coiled-coil domain, Vam7p also contains a putative NADPH oxidase p40(phox) (PX) domain. Changes in two conserved amino acids within this domain resulted in synthetic phenotypes when combined with the vam3(tsf) mutation, suggesting that the PX domain is required for Vam7p function. This study provides evidence for the functional and physical interaction between Vam7p and Vam3p at the vacuolar membrane, where they function as part of a t-SNARE complex required for the docking and/or fusion of multiple transport intermediates destined for the vacuole.
Asunto(s)
Proteínas Fúngicas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , Vacuolas/fisiología , Proteínas de Transporte Vesicular , Secuencia de Aminoácidos , Proteínas Portadoras/metabolismo , Fraccionamiento Celular , Genotipo , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestructura , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Microscopía Electrónica , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas Sensibles a N-Etilmaleimida , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Mutación Puntual , Proteínas Qa-SNARE , Proteínas Qc-SNARE , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructura , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteína 25 Asociada a Sinaptosomas , Temperatura , Vacuolas/ultraestructuraRESUMEN
Protein transport in eukaryotic cells requires the selective docking and fusion of transport intermediates with the appropriate target membrane. t-SNARE molecules that are associated with distinct intracellular compartments may serve as receptors for transport vesicle docking and membrane fusion through interactions with specific v-SNARE molecules on vesicle membranes, providing the inherent specificity of these reactions. VAM3 encodes a 283-amino acid protein that shares homology with the syntaxin family of t-SNARE molecules. Polyclonal antiserum raised against Vam3p recognized a 35-kD protein that was associated with vacuolar membranes by subcellular fractionation. Null mutants of vam3 exhibited defects in the maturation of multiple vacuolar proteins and contained numerous aberrant membrane-enclosed compartments. To study the primary function of Vam3p, a temperature-sensitive allele of vam3 was generated (vam3(tsf)). Upon shifting the vam3(tsf) mutant cells to nonpermissive temperature, an immediate block in protein transport through two distinct biosynthetic routes to the vacuole was observed: transport via both the carboxypeptidase Y pathway and the alkaline phosphatase pathway was inhibited. In addition, vam3(tsf) cells also exhibited defects in autophagy. Both the delivery of aminopeptidase I and the docking/ fusion of autophagosomes with the vacuole were defective at high temperature. Upon temperature shift, vam3(tsf) cells accumulated novel membrane compartments, including multivesicular bodies, which may represent blocked transport intermediates. Genetic interactions between VAM3 and a SEC1 family member, VPS33, suggest the two proteins may act together to direct the docking and/or fusion of multiple transport intermediates with the vacuole. Thus, Vam3p appears to function as a multispecificity receptor in heterotypic membrane docking and fusion reactions with the vacuole. Surprisingly, we also found that overexpression of the endosomal t-SNARE, Pep12p, suppressed vam3Delta mutant phenotypes and, likewise, overexpression of Vam3p suppressed the pep12Delta mutant phenotypes. This result indicated that SNAREs alone do not define the specificity of vesicle docking reactions.