RESUMEN
Mucopolysaccharidosis Type IIIB (MPS IIIB) is an ultrarare, fatal pediatric disease with no approved therapy. It is caused by mutations in the gene encoding for lysosomal enzyme alpha-N-acetylglucosaminidase (NAGLU). Tralesinidase alfa (TA) is a fusion protein comprised of recombinant NAGLU and a modified human insulin-like growth factor 2 that is being developed as an enzyme replacement therapy for MPS IIIB. Since MPS IIIB is a pediatric disease the safety/toxicity, pharmacokinetics and biodistribution of TA were evaluated in juvenile non-human primates that were administered up to 5 weekly intracerebroventricular (ICV) or single intravenous (IV) infusions of TA. TA administered by ICV slow-, ICV isovolumetric bolus- or IV-infusion was well-tolerated, and no effects were observed on clinical observations, electrocardiographic or ophthalmologic parameters, or respiratory rates. The drug-related changes observed were limited to increased cell infiltrates in the CSF and along the ICV catheter track after ICV administration. These findings were not associated with functional changes and are associated with the use of ICV catheters. The CSF PK profiles were consistent across all conditions tested and TA distributed widely in the CNS after ICV administration. Anti-drug antibodies were observed but did not appear to significantly affect the exposure to TA. Correlations between TA concentrations in plasma and brain regions in direct contact with the cisterna magna suggest glymphatic drainage may be responsible for clearance of TA from the CNS. The data support the administration of TA by isovolumetric bolus ICV infusion to pediatric patients with MPS IIIB.
RESUMEN
BackgroundSanfilippo type B is a mucopolysaccharidosis (MPS) with a major neuronopathic component characterized by heparan sulfate (HS) accumulation due to mutations in the NAGLU gene encoding alfa-N-acetyl-glucosaminidase. Enzyme replacement therapy for neuronopathic MPS requires efficient enzyme delivery throughout the brain in order to normalize HS levels, prevent brain atrophy, and potentially delay cognitive decline.MethodsIn this phase I/II open-label study, patients with MPS type IIIB (n = 22) were treated with tralesinidase alfa administered i.c.v. The patients were monitored for drug exposure; total HS and HS nonreducing end (HS-NRE) levels in both cerebrospinal fluid (CSF) and plasma; anti-drug antibody response; brain, spleen, and liver volumes as measured by MRI; and cognitive development as measured by age-equivalent (AEq) scores.ResultsIn the Part 1 dose escalation (30, 100, and 300 mg) phase, a 300 mg dose of tralesinidase alfa was necessary to achieve normalization of HS and HS-NRE levels in the CSF and plasma. In Part 2, 300 mg tralesinidase alfa sustained HS and HS-NRE normalization in the CSF and stabilized cortical gray matter volume (CGMV) over 48 weeks of treatment. Resolution of hepatomegaly and a reduction in spleen volume were observed in most patients. Significant correlations were also established between the change in cognitive AEq score and plasma drug exposure, plasma HS-NRE levels, and CGMV.ConclusionAdministration of tralesinidase alfa i.c.v. effectively normalized HS and HS-NRE levels as a prerequisite for clinical efficacy. Peripheral drug exposure data suggest a role for the glymphatic system in altering tralesinidase alfa efficacy.Trial registrationClinicaltrials.gov NCT02754076.FUNDINGBioMarin Pharmaceutical Inc. and Allievex Corporation.
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Mucopolisacaridosis III , Humanos , Mucopolisacaridosis III/tratamiento farmacológico , Mucopolisacaridosis III/genética , Heparitina Sulfato , Encéfalo , Hígado , BazoRESUMEN
OBJECTIVE: To characterize the longitudinal natural history of disease progression in pediatric subjects affected with mucopolysaccharidosis (MPS) IIIB. STUDY DESIGN: Sixty-five children with a confirmed diagnosis of MPS IIIB were enrolled into 1 of 2 natural history studies and followed for up to 4 years. Cognitive and adaptive behavior functions were analyzed in all subjects, and volumetric magnetic resonance imaging analysis of liver, spleen, and brain, as well as levels of heparan sulfate (HS) and heparan sulfate nonreducing ends (HS-NRE), were measured in a subset of subjects. RESULTS: The majority of subjects with MPS IIIB achieved an apex on both cognition and adaptive behavior age equivalent scales between age 3 and 6 years. Development quotients for both cognition and adaptive behavior follow a linear trajectory by which subjects reach a nadir with a score <25 for an age equivalent of 24 months by age 8 years on average and by 13.5 years at the latest. All tested subjects (n = 22) had HS and HS-NRE levels above the normal range in cerebrospinal fluid and plasma, along with signs of hepatomegaly. Subjects lost an average of 26 mL of brain volume (-2.7%) over 48 weeks, owing entirely to a loss of cortical gray matter (32 mL; -6.5%). CONCLUSIONS: MPS IIIB exists along a continuum based on cognitive decline and cortical gray matter atrophy. Although a few individuals with MPS IIIB have an attenuated phenotype, the majority follow predicted trajectories for both cognition and adaptive behavior. TRIAL REGISTRATION: ClinicalTrials.gov identifiers NCT02493998, NCT03227042, and NCT02754076.
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Mucopolisacaridosis III , Atrofia/patología , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Sustancia Gris , Heparitina Sulfato , Humanos , Imagen por Resonancia Magnética , Mucopolisacaridosis III/diagnósticoRESUMEN
Mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo syndrome B; OMIM #252920) is a lethal, pediatric, neuropathic, autosomal recessive, and lysosomal storage disease with no approved therapy. Patients are deficient in the activity of N-acetyl-alpha-glucosaminidase (NAGLU; EC 3.2.150), necessary for normal lysosomal degradation of the glycosaminoglycan heparan sulfate (HS). Tralesinidase alfa (TA), a fusion protein comprised of recombinant human NAGLU and a modified human insulin-like growth factor 2, is in development as an enzyme replacement therapy that is administered via intracerebroventricular (ICV) infusion, thus circumventing the blood brain barrier. Previous studies have confirmed ICV infusion results in widespread distribution of TA throughout the brains of mice and nonhuman primates. We assessed the long-term tolerability, pharmacology, and clinical efficacy of TA in a canine model of MPS IIIB over a 20-month study. Long-term administration of TA was well tolerated as compared with administration of vehicle. TA was widely distributed across brain regions, which was confirmed in a follow-up 8-week pharmacokinetic/pharmacodynamic study. MPS IIIB dogs treated for up to 20 months had near-normal levels of HS and nonreducing ends of HS in cerebrospinal fluid and central nervous system (CNS) tissues. TA-treated MPS IIIB dogs performed better on cognitive tests and had improved CNS pathology and decreased cerebellar volume loss relative to vehicle-treated MPS IIIB dogs. These findings demonstrate the ability of TA to prevent or limit the biochemical, pathologic, and cognitive manifestations of canine MPS IIIB disease, thus providing support of its potential long-term tolerability and efficacy in MPS IIIB subjects. SIGNIFICANCE STATEMENT: This work illustrates the efficacy and tolerability of tralesinidase alfa as a potential therapeutic for patients with mucopolysaccharidosis type IIIB (MPS IIIB) by documenting that administration to the central nervous system of MPS IIIB dogs prevents the accumulation of disease-associated glycosaminoglycans in lysosomes, hepatomegaly, cerebellar atrophy, and cognitive decline.
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Mucopolisacaridosis III , Animales , Encéfalo/metabolismo , Niño , Modelos Animales de Enfermedad , Perros , Terapia de Reemplazo Enzimático , Glicosaminoglicanos/metabolismo , Heparitina Sulfato/líquido cefalorraquídeo , Heparitina Sulfato/uso terapéutico , Humanos , Mucopolisacaridosis III/tratamiento farmacológico , Mucopolisacaridosis III/patologíaRESUMEN
BACKGROUND AND PURPOSE: Triptans are 5-HT1B/1D receptor agonists (that also display 5-HT1F receptor affinity) with antimigraine action, contraindicated in patients with coronary artery disease due to their vasoconstrictor properties. Conversely, lasmiditan was developed as an antimigraine 5-HT1F receptor agonist. To assess the selectivity and cardiovascular effects of lasmiditan, we investigated the binding, functional activity, and in vitro/in vivo vascular effects of lasmiditan and compared it to sumatriptan. EXPERIMENTAL APPROACH: Binding and second messenger activity assays of lasmiditan and other serotoninergic agonists were performed for human 5-HT1A , 5-HT1B , 5-HT1D , 5-ht1E , 5-HT1F , 5-HT2A , 5-HT2B , and 5-HT7 receptors, and the results were correlated with their potency to constrict isolated human coronary arteries (HCAs). Furthermore, concentration-response curves to lasmiditan and sumatriptan were performed in proximal and distal HCA, internal mammary, and middle meningeal arteries. Finally, anaesthetized female beagle dogs received i.v. infusions of lasmiditan or sumatriptan in escalating cumulative doses, and carotid and coronary artery diameters were measured. KEY RESULTS: Lasmiditan showed high selectivity for 5-HT1F receptors. Moreover, the functional potency of the analysed compounds to inhibit cAMP increase through 5-HT1B receptor activation positively correlated with their potency to contract HCA. In isolated human arteries, sumatriptan, but not lasmiditan, induced contractions. Likewise, in vivo, sumatriptan decreased coronary and carotid artery diameters at clinically relevant doses, while lasmiditan was devoid of vasoconstrictor activity at all doses tested. CONCLUSIONS AND IMPLICATIONS: Lasmiditan is a selective 5-HT1F receptor agonist devoid of vasoconstrictor activity. This may represent a cardiovascular safety advantage when compared to the triptans.
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Benzamidas/farmacología , Vasos Coronarios/efectos de los fármacos , Arterias Meníngeas/efectos de los fármacos , Contracción Muscular/efectos de los fármacos , Piperidinas/farmacología , Piridinas/farmacología , Receptores de Serotonina/metabolismo , Agonistas de Receptores de Serotonina/farmacología , Vasoconstricción/efectos de los fármacos , Animales , Unión Competitiva , Células CHO , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Vasos Coronarios/metabolismo , Vasos Coronarios/fisiopatología , Cricetulus , Perros , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Técnicas In Vitro , Masculino , Arterias Meníngeas/metabolismo , Arterias Meníngeas/fisiopatología , Unión Proteica , Ensayo de Unión Radioligante , Sumatriptán/farmacología , Receptor de Serotonina 5-HT1FRESUMEN
OBJECTIVE: Topical interleukin (IL)-1 receptor (R)1 blockade is therapeutically active in reducing signs and symptoms of dry eye disease. Herein, we describe in vitro and in vivo nonclinical Investigational New Drug (IND)-enabling studies of EBI-005, a novel protein chimera of IL-1ß and IL-1 receptor antagonist (IL-1Ra or anakinra) that potently binds IL-1R1 and blocks signaling. These studies provide an assessment of receptor affinity, drug bioavailability, immunogenic response, safety, and tolerability in mice and rabbits. METHODS: In vitro and in silico along with Good Laboratory Practices (GLP) and non-GLP in vivo studies in mice and rabbits assessed the topical ocular and systemic immunogenicity and toxicology of EBI-005. Animals were treated with EBI-005 once daily subcutaneously or four times daily by topical ocular administration for up to 6 weeks (with 2-week recovery phase). RESULTS: EBI-005 has 500 times higher affinity than anakinra to IL-1R1. Predictive immunogenicity testing suggested that EBI-005 is not more immunogenic. Systemic bioavailability of EBI-005 is low (1.4% in mice and 0.2% in rabbits) after topical ocular administration. EBI-005 penetrated into the anterior ocular tissues within 15 min of topical ocular administration. However, it is low or undetectable after 4 hr and does not form a depot after repeated topical ocular administration. EBI-005 was safe and well tolerated, and exposure to drug was maintained despite an antidrug antibody response after systemic administration, based on IND-enabling toxicology and safety pharmacology studies. CONCLUSIONS: Ocular doses of EBI-005 at 50 mg/mL in mice and rabbits totaling 0.15 mg/eye in mice and 1.5 mg/eye in rabbits, administered 4 times daily, did not produce adverse effects, and demonstrated excellent bioavailability in target tissues with low systemic exposure. In addition, immunogenic response to the drug did not cause adverse effects or diminish the drug's activity in most cases. The results support drug administration of the highest anticipated human clinical study dose of a 20 mg/mL solution (40 µL 3 times daily in each eye).
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Conjuntivitis Alérgica/tratamiento farmacológico , Síndromes de Ojo Seco/tratamiento farmacológico , Soluciones Oftálmicas/uso terapéutico , Proteínas/uso terapéutico , Receptores de Interleucina-1/antagonistas & inhibidores , Administración Tópica , Animales , Modelos Animales de Enfermedad , Inmunoglobulina G/análisis , Inmunoglobulina M/análisis , Proteína Antagonista del Receptor de Interleucina 1/metabolismo , Masculino , Proteínas/inmunología , ConejosRESUMEN
OBJECTIVES: Many allergic conjunctivitis (AC) patients are inadequately treated with conventional therapies or require steroids. EBI-005 was developed to address the late phase allergic response. This study's objectives were to evaluate two adapted clinical models for this indication and to assess safety and biological activity of EBI-005 in AC. METHODS: In this randomized, double-masked, vehicle-controlled study, 159 subjects with moderate-to-severe AC were randomized to topical EBI-005 (5 mg/mL) or vehicle control given 3 times per day and repeatedly challenged with allergen using an adaptation of 2 clinical models of AC. Subjects were assigned to repetitive aerosolized challenge in an allergy chamber (Environmental Exposure Chamber, EEC), or repetitive challenges with a direct conjunctival allergen challenge (Conjunctival Allergen Provocation Test, CAPT). RESULTS: In the EEC, the prespecified primary endpoint of ocular itching was not met. In the CAPT, EBI-005-treated subjects showed clinically meaningful, statistically significant improvements in ocular itching compared with vehicle control at the final 2 efficacy time points, visit 6 (P=0.033) and visit 7 (P=0.046). EBI-005-treated subjects showed statistically significant improvement compared with vehicle control for ocular tearing (P=0.027 and P=0.044) and nasal symptoms (P=0.004 and P=0.011) at visit 6 and visit 7. EBI-005 was well tolerated. CONCLUSIONS: These results support use of an adapted, multiple-challenge, direct conjunctival allergen model to assess efficacy of EBI-005 in late phase AC. In the CAPT, EBI-005 showed statistically significant improvements in clinically meaningful symptoms (ocular itching, tearing, and nasal symptoms) at multiple time points for moderate-to-severe AC subjects.
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Antialérgicos/uso terapéutico , Conjuntivitis Alérgica/tratamiento farmacológico , Inhibidores Enzimáticos/uso terapéutico , Proteínas/uso terapéutico , Receptores de Interleucina-1/antagonistas & inhibidores , Adolescente , Adulto , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Soluciones Oftálmicas/uso terapéuticoRESUMEN
The long circulating half-life of serum albumin, the most abundant protein in mammalian plasma, derives from pH-dependent endosomal salvage from degradation, mediated by the neonatal Fc receptor (FcRn). Using yeast display, we identified human serum albumin (HSA) variants with increased affinity for human FcRn at endosomal pH, enabling us to solve the crystal structure of a variant HSA/FcRn complex. We find an extensive, primarily hydrophobic interface stabilized by hydrogen-bonding networks involving protonated histidines internal to each protein. The interface features two key FcRn tryptophan side chains inserting into deep hydrophobic pockets on HSA that overlap albumin ligand binding sites. We find that fatty acids (FAs) compete with FcRn, revealing a clash between ligand binding and recycling, and that our high-affinity HSA variants have significantly increased circulating half-lives in mice and monkeys. These observations open the way for the creation of biotherapeutics with significantly improved pharmacokinetics.
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Antígenos de Histocompatibilidad Clase I/química , Receptores Fc/química , Albúmina Sérica/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Unión Competitiva , Femenino , Humanos , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Fragmentos Fc de Inmunoglobulinas/química , Inmunoglobulina G/química , Cinética , Ligandos , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Imitación Molecular , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de Proteína , Ratas , Homología de Secuencia , Albúmina Sérica/genética , Microglobulina beta-2/químicaRESUMEN
Through a parallel approach of tracking product quality through fermentation and purification development, a robust process was designed to reduce the levels of product-related species. Three biochemically similar product-related species were identified as byproducts of host-cell enzymatic activity. To modulate intracellular proteolytic activity, key fermentation parameters (temperature, pH, trace metals, EDTA levels, and carbon source) were evaluated through bioreactor optimization, while balancing negative effects on growth, productivity, and oxygen demand. The purification process was based on three non-affinity steps and resolved product-related species by exploiting small charge differences. Using statistical design of experiments for elution conditions, a high-resolution cation exchange capture column was optimized for resolution and recovery. Further reduction of product-related species was achieved by evaluating a matrix of conditions for a ceramic hydroxyapatite column. The optimized fermentation process was transferred from the 2-L laboratory scale to the 100-L pilot scale and the purification process was scaled accordingly to process the fermentation harvest. The laboratory- and pilot-scale processes resulted in similar process recoveries of 60 and 65%, respectively, and in a product that was of equal quality and purity to that of small-scale development preparations. The parallel approach for up- and downstream development was paramount in achieving a robust and scalable clinical process.
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Proteínas Bacterianas/aislamiento & purificación , Biotecnología/métodos , Técnicas de Cultivo de Célula/métodos , Receptores de Interleucina-1/antagonistas & inhibidores , Proteínas Recombinantes/aislamiento & purificación , Proteínas Bacterianas/química , Reactores Biológicos , Cromatografía por Intercambio Iónico , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentación , Proyectos Piloto , Proteínas Recombinantes/análisis , TemperaturaRESUMEN
IL-1 is a key inflammatory and immune mediator in many diseases, including dry-eye disease, and its inhibition is clinically efficacious in rheumatoid arthritis and cryopyrin-associated periodic syndromes. To treat ocular surface disease with a topical biotherapeutic, the uniqueness of the site necessitates consideration of the agent's size, target location, binding kinetics, and thermal stability. Here we chimerized two IL-1 receptor ligands, IL-1ß and IL-1Ra, to create an optimized receptor antagonist, EBI-005, for topical ocular administration. EBI-005 binds its target, IL-1R1, 85-fold more tightly than IL-1Ra, and this increase translates to an â¼100-fold increase in potency in vivo. EBI-005 preserves the affinity bias of IL-1Ra for IL-1R1 over the decoy receptor (IL-1R2), and, surprisingly, is also more thermally stable than either parental molecule. This rationally designed antagonist represents a unique approach to therapeutic design that can potentially be exploited for other ß-trefoil family proteins in the IL-1 and FGF families.
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Citocinas/antagonistas & inhibidores , Diseño de Fármacos , Administración Tópica , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Citocinas/química , Estabilidad de Medicamentos , Femenino , Humanos , Proteína Antagonista del Receptor de Interleucina 1/antagonistas & inhibidores , Proteína Antagonista del Receptor de Interleucina 1/química , Proteína Antagonista del Receptor de Interleucina 1/genética , Interleucina-1beta/antagonistas & inhibidores , Interleucina-1beta/química , Interleucina-1beta/genética , Cinética , Ligandos , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Datos de Secuencia Molecular , Soluciones Oftálmicas , Conformación Proteica , Receptores Tipo I de Interleucina-1/antagonistas & inhibidores , Receptores Tipo I de Interleucina-1/química , Receptores Tipo I de Interleucina-1/genética , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Homología de Secuencia de Aminoácido , Electricidad EstáticaRESUMEN
The random amino acid copolymer poly(Y,E,A,K)(n) (Copaxone®) is widely used in multiple sclerosis treatment and a second generation copolymer poly(Y,F,A,K)(n) with enhanced efficacy in experimental autoimmune encephalomyelitis in mice has been described. A major mechanism through which copolymers function to ameliorate disease is the generation of immunosuppressive IL-10-secreting regulatory T cells entering the CNS. In addition, the antigen presenting cell to which these copolymers bind through MHC Class II proteins may have an important role. Here, both CCL22 (a Th2 cell chemoattractant) in large amounts and CXCL13 in much smaller amounts are shown to be secreted after administration of YFAK to mice and to a smaller extent by YEAK parallel to their serum concentrations. Moreover, bone marrow-derived macrophages secrete CCL22 in vitro in response to YFAK and to higher concentrations of YEAK. Strikingly, these chemokines are also secreted into serum of MHC Class II -/- mice, indicating that an innate immune receptor on these cells also has an important role. Thus, both the innate and the adaptive immune systems are involved in the mechanism of EAE amelioration by YFAK. The enhanced ability of YFAK to stimulate the innate immune system may account for its enhanced efficacy in EAE treatment.
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Aminoácidos/farmacología , Quimiocinas/metabolismo , Encefalomielitis Autoinmune Experimental/inmunología , Inmunidad Innata/inmunología , Macrófagos/inmunología , Péptidos/farmacología , Secuencia de Aminoácidos , Aminoácidos/administración & dosificación , Aminoácidos/sangre , Aminoácidos/inmunología , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Línea Celular , Quimiocina CCL22/metabolismo , Quimiocina CXCL13/metabolismo , Quimiocinas/sangre , Encefalomielitis Autoinmune Experimental/patología , Acetato de Glatiramer , Antígenos de Histocompatibilidad Clase II/inmunología , Inmunidad Innata/efectos de los fármacos , Interleucina-3/metabolismo , Cinética , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos/efectos de los fármacos , Péptidos/administración & dosificación , Péptidos/sangre , Péptidos/inmunología , Bazo/citología , Bazo/efectos de los fármacos , Bazo/inmunologíaRESUMEN
PI-2301 is an amino acid copolymer acting as an immunomodulator for the treatment of autoimmune diseases. The present study evaluated the safety, pharmacokinetics (PK), and pharmacodynamics of PI-2301 in a single ascending dose, first-in-human study involving healthy, male adult volunteers. A total of 56 subjects were given a subcutaneous injection of PI-2301 ranging from 0.035 to 60 mg. The only consistent side effect was transient injection site reactions. We describe, for the first time, a pharmacokinetic assay to monitor amino acid copolymer concentration in human serum. PI-2301 was detected in the serum of subjects in the 10-, 30-, and 60-mg cohorts. Maximum serum concentration was achieved between 10 and 30 minutes postdosing with some compound detected 4 hours after dosing. PI-2301's lasting immunological properties were evident by an ex vivo recall assay showing T-cell proliferation and IL-13 production in subjects dosed with 1, 3, or 10 mg of PI-2301, up to 6 months after dosing. A transient increase in chemokine CXCL9 and CXCL10 plasma levels was seen in subjects dosed with 30 or 60 mg of PI-2301. These results are highly consistent with our preclinical findings and suggest that PI-2301 could facilitate the expansion of a favorable immune posture in patients with autoimmune disorders.
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Factores Inmunológicos/farmacocinética , Oligopéptidos/farmacocinética , Polímeros/farmacocinética , Proteínas/farmacocinética , Adolescente , Adulto , Anciano , Anticuerpos/sangre , Biomarcadores/sangre , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CXCL10/sangre , Quimiocina CXCL9/sangre , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Francia , Humanos , Factores Inmunológicos/administración & dosificación , Factores Inmunológicos/efectos adversos , Factores Inmunológicos/sangre , Factores Inmunológicos/inmunología , Inyecciones Subcutáneas , Interferón gamma/metabolismo , Interleucina-13/metabolismo , Activación de Linfocitos/efectos de los fármacos , Masculino , Persona de Mediana Edad , Oligopéptidos/administración & dosificación , Oligopéptidos/efectos adversos , Oligopéptidos/sangre , Oligopéptidos/inmunología , Polímeros/administración & dosificación , Polímeros/efectos adversos , Proteínas/administración & dosificación , Proteínas/efectos adversos , Proteínas/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Adulto JovenRESUMEN
Injury causes tissue breakdown, which releases large quantities of intracellular contents into the extracellular space. Some of these materials are well-established activators of the immune system and include heat shock proteins (HSPs), uric acid, nucleotides, High Mobility Group Box-1 protein (HMGB-1), and DNA. Here, we show that in vivo delivery of HSPs into BALB/cJ mice with full-thickness wounds accelerates the rate of wound closure by 60% as compared with control-treated mice. The onset is rapid and the effect is sustained, dose dependent, and protein specific. Adoptive transfer of RAW264 macrophages pretreated with HSP70 into naïve recipients with a wound transfers the HSP-mediated effect on the rate of wound closure. Further, we demonstrate that part of the mechanism by which HSP70 accelerates wound closure is through the stimulation of macrophage-mediated phagocytosis of wound debris. Disabling the HSP70-mediated enhancement of phagocytosis abrogates the HSP-mediated acceleration of the healing process. These findings create two opportunities: one, therapeutic, wherein HSP70 could be used in the clinical management of wounds; and two, pathophysiologic, to decode signals by which the host defenses recognize and respond to injury.
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Proteínas HSP70 de Choque Térmico/farmacología , Macrófagos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , Animales , Células Cultivadas , Quimiocina CCL2/metabolismo , Femenino , Proteínas HSP90 de Choque Térmico/farmacología , Interleucina-6/metabolismo , Glicoproteínas de Membrana/farmacología , Ratones , Ratones Endogámicos BALB C , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia ArribaRESUMEN
Immunization with high doses of heat shock protein, gp 6 given in vivo, has been shown to mediate the activation of CD4+ T cell, which in turn suppresses an ongoing CD8+ immune response. Here we demonstrate that high doses of gp 6 (HDgp 6) (100 microg given subcutaneously on days 0 and 7 following skin graft transplantation) improve survival of skin grafts with both minor and major histocompatibility. First, skin from male C57BL/6 donors was grafted onto female C57BL/6 recipients that were subsequently treated with either HDgp 6 or buffer and graft survival was monitored. At 64 days post-transplantation, graft recipients treated with HDgp 6 showed 56% graft survival compared to 10% in those treated with buffer. Further, HDgp 6 down regulates a secondary immune response as well. Skin graft obtained from a male C57BL/6 donor was transplanted to a female C57BL/6 recipient that had previously rejected a male skin graft. Recipients that were treated with HDgp 96 showed a 75% graft survival of the secondary graft at 64 days post-transplantation, whereas recipients treated with buffer rejected their secondary grafts within 37 days. Finally, the effect of HDgp 96 treatment on survival of graft with major histoincompatibility was tested. Skin grafts from female BALB/c donors were transplanted to female C57BL/6 recipients previously treated with either HDgp 96 or buffer. Although all the grafts were ultimately rejected, mice treated with HDgp96 showed a delay in the rejection as compared with buffer controls. Our findings indicate that HDgp 96 improves survival of grafts with both minor and major antigenic disparity and could lead to developing novel therapies in transplant medicine.
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Antígenos de Neoplasias/inmunología , Supervivencia de Injerto/inmunología , Proteínas de Choque Térmico/farmacología , Trasplante de Piel/inmunología , Animales , Antígenos de Neoplasias/farmacología , Relación Dosis-Respuesta Inmunológica , Femenino , Rechazo de Injerto/inmunología , Rechazo de Injerto/prevención & control , Supervivencia de Injerto/efectos de los fármacos , Proteínas de Choque Térmico/inmunología , Inmunización , Masculino , Ratones , Ratones Endogámicos BALB C , Trasplante HomólogoRESUMEN
The extracellular presence of endotoxin-free heat shock protein 70 (HSP70) enhances the rate and capacity of macrophage-mediated phagocytosis at 6 times the basal rate. It is protein-specific, dose- and time-dependent and involves the internalization of inert microspheres, Gram-positive and -negative bacteria and fungi. Structurally, exogenous HSP70 binds the macrophage plasma membrane, specifically on its lipid raft-microdomain. Disruption of lipid rafts, HSP70-LR interaction, or denaturing HSP70 abrogates the HSP-mediated increase in phagocytosis. Further, HSP70-mediated phagocytosis directly enhances the processing and presentation of internalized antigens via the endocytic MHC class-II pathway to CD4+ T lymphocytes. Modulating the HSP70-LR interaction presents an opportunity to intervene at the level of host-pathogen interface: a therapeutic tool for emerging infections, especially when conventional treatment with antibiotics is ineffective (antibiotic resistance) or unavailable (rapidly spreading, endemic). These results identify a new role for HSP70, a highly conserved molecule in stimulating phagocytosis: a primordial macrophage function, thereby influencing both innate and adaptive immune responses.
Asunto(s)
Proteínas HSP70 de Choque Térmico/farmacología , Antígenos de Histocompatibilidad Clase II/genética , Microdominios de Membrana/fisiología , Fagocitos/fisiología , Fagocitosis/fisiología , Animales , Humanos , Complejo Mayor de Histocompatibilidad , Microdominios de Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fagocitos/efectos de los fármacos , Fagocitos/inmunología , Fagocitosis/efectos de los fármacos , Receptores de Antígenos de Linfocitos T alfa-beta/genéticaRESUMEN
The ability of mature T lymphocytes to develop effector capacity after encounter with cognate Ag is generally dependent upon inflammatory signals associated with infection that induce dendritic cell activation/maturation. These inflammatory signals can derive directly from pathogens or can be expressed by host cells in response to infection. Heat shock proteins (HSPs) are a class of host-derived inflammatory mediators that perform the dual function of both chaperoning MHC class I-restricted epitopes into the cross-presentation pathway of DCs and inducing the activation/maturation of these DCs to allow priming of cognate CD8(+) T cell effector responses. Although the ability of HSPs to elicit effector CD8 cell responses has been well established, their potential to prime CD4 cell effector responses has been relatively unexplored. In the current study we compared the ability of the endoplasmic reticulum-resident HSP gp96 to prime CD4 vs CD8 cells using TCR transgenic adoptive transfer systems and soluble gp96-peptide complexes. As expected, gp96 facilitated the cross-presentation of a class I-restricted peptide and priming of effector function in cognate CD8 cells. Interestingly, gp96 also facilitated the in vivo presentation of a class II-restricted peptide; however, the resulting CD4 cell response did not involve the development of effector function. Taken together, these data suggest that gp96 is an inflammatory mediator that selectively primes CD8 cell effector function.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos de Neoplasias/fisiología , Proteínas de Choque Térmico/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Chaperonas Moleculares/fisiología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Anergia Clonal , Citotoxicidad Inmunológica , Epítopos de Linfocito T/metabolismo , Mediadores de Inflamación/metabolismo , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Immunization with heat-shock protein (HSP) gp96 elicits protective immunity to the cancer or virus-infected cells from which it is derived. Low doses of gp96 generate immunity, while doses 10 times the immunizing dose do not. We show here that injection of high doses of gp96 generates CD4(+) T cells that down-regulate a variety of ongoing immune responses. Immunization with high doses of gp96 prevents myelin basic protein- or proteolipid protein-induced autoimmune encephalomyelitis in SJL mice and the onset of diabetes in non-obese diabetic mice. The suppression of immune response can be adoptively transferred with CD4(+) cells and does not partition with the CD25 phenotype. The immunomodulatory properties of gp96 (and possibly other HSP) may be used for antigen-specific activation or suppression of cellular immune responses. The latter may form the basis for novel immunotherapies for autoimmune diseases.