RESUMEN
The multiple sclerosis (MS) treatment landscape in the United States has changed dramatically over the past decade. While many disease-modifying therapies (DMTs) have been approved by the US Food and Drug Administration (FDA) for the treatment of relapsing forms of MS, DMT costs continue to rise. The availability of generics and biosimilars in the MS-treatment landscape is unlikely to have a major impact on clinical benefit. However, their availability will provide alternative treatment options and potentially lower costs through competition, thus increasing the affordability of and access to these drugs. In April 2015, the first generic version of the complex drug glatiramer acetate (Glatopa® 20 mg/mL) injection was approved in the United States as a fully substitutable generic for all approved indications of the 20 mg/mL branded glatiramer acetate (Copaxone®) dosage form. Despite glatiramer acetate's complex nature-being a chemically synthesized (ie, nonbiologic) mixture of peptides-the approval occurred without conducting any clinical trials. Rather, extensive structural and functional characterization was performed to demonstrate therapeutic equivalence to the innovator drug. The approval of Glatopa signifies an important milestone in the US MS-treatment landscape, with the hope that the introduction of generic DMTs and eventually biosimilar DMTs will lead to future improvements in the affordability and access of these much-needed treatments for MS.
Asunto(s)
Adyuvantes Inmunológicos/uso terapéutico , Aprobación de Drogas/métodos , Desarrollo de Medicamentos/métodos , Medicamentos Genéricos/uso terapéutico , Acetato de Glatiramer/uso terapéutico , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Adyuvantes Inmunológicos/síntesis química , Biosimilares Farmacéuticos/síntesis química , Biosimilares Farmacéuticos/uso terapéutico , Aprobación de Drogas/legislación & jurisprudencia , Desarrollo de Medicamentos/legislación & jurisprudencia , Acetato de Glatiramer/síntesis química , Humanos , Inmunosupresores/síntesis química , Inmunosupresores/uso terapéutico , Esclerosis Múltiple Recurrente-Remitente/inmunología , Péptidos/síntesis química , Péptidos/uso terapéutico , Estados UnidosRESUMEN
Intravenous immunoglobulin (IVIg) is a complex mixture drug comprising diverse immunoglobulins and non-IgG proteins purified from the plasma of thousands of healthy donors. Approved IVIg products on the market differ regarding source of plasma, isolation process, and formulation. These products are used widely, and often interchangeably, for the treatment of immunodeficiency and autoimmune and inflammatory diseases, but their mechanisms of action in different indications are not well understood. A primary limitation to understanding the therapeutic relevance of specific components within IVIg has been the limited resolution of analytics historically implemented to characterize its complex mixture. In this study, high-resolution analytics were applied to better understand the composition of IVIg and product variations. We characterized three approved IVIg products: Gammagard®, Privigen®, and Octagam®. Differences in the distribution of molecular weight species, IgG sequence variants, isoforms, glycoforms, and the repertoire of previously reported antibody specificities were identified. We also compared the effect of aging on these products to identify changes in size distribution and posttranslational modifications. This type of characterization may provide insights into the specific factors and components of IVIg that may influence its activity and ultimately lead to optimization of IVIg products for use in autoimmune diseases.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Inmunoglobulina G/uso terapéutico , Inmunoglobulinas Intravenosas/uso terapéutico , Síndromes de Inmunodeficiencia/tratamiento farmacológico , Envejecimiento , Humanos , Fragmentos de Inmunoglobulinas/química , Inmunoglobulina G/química , Espectrometría de MasasRESUMEN
Autoantibody immune complex (IC) activation of Fcγ receptors (FcγRs) is a common pathogenic hallmark of multiple autoimmune diseases. Given that the IC structural features that elicit FcγR activation are poorly understood and the FcγR system is highly complex, few therapeutics can directly block these processes without inadvertently activating the FcγR system. To address these issues, the structure activity relationships of an engineered panel of multivalent Fc constructs were evaluated using sensitive FcγR binding and signaling cellular assays. These studies identified an Fc valency with avid binding to FcγRs but without activation of immune cell effector functions. These observations directed the design of a potent trivalent immunoglobulin G-Fc molecule that broadly inhibited IC-driven processes in a variety of immune cells expressing FcγRs. The Fc trimer, Fc3Y, was highly efficacious in three different animal models of autoimmune diseases. This recombinant molecule may represent an effective therapeutic candidate for FcγR-mediated autoimmune diseases.
Asunto(s)
Complejo Antígeno-Anticuerpo/inmunología , Enfermedades Autoinmunes/terapia , Enfermedades del Complejo Inmune/terapia , Fragmentos Fc de Inmunoglobulinas/inmunología , Receptores de IgG/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Artritis/inmunología , Artritis/terapia , Artritis Experimental/inmunología , Artritis Experimental/terapia , Autoanticuerpos/inmunología , Enfermedades Autoinmunes/inmunología , Línea Celular , Epidermólisis Ampollosa Adquirida/inmunología , Epidermólisis Ampollosa Adquirida/terapia , Humanos , Enfermedades del Complejo Inmune/inmunología , Inmunoglobulina G/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/citología , Fagocitos , Activación Plaquetaria , Púrpura Trombocitopénica Idiopática/inmunología , Púrpura Trombocitopénica Idiopática/terapia , Transducción de SeñalRESUMEN
Despite the beneficial therapeutic effects of intravenous immunoglobulin (IVIg) in inflammatory diseases, consistent therapeutic efficacy and potency remain major limitations for patients and physicians using IVIg. These limitations have stimulated a desire to generate therapeutic alternatives that could leverage the broad mechanisms of action of IVIg while improving therapeutic consistency and potency. The identification of the important anti-inflammatory role of fragment crystallizable domain (Fc) sialylation has presented an opportunity to develop more potent Ig therapies. However, translating this concept to potent anti-inflammatory therapeutics has been hampered by the difficulty of generating suitable sialylated products for clinical use. Therefore, we set out to develop the first, to our knowledge, robust and scalable process for generating a well-qualified sialylated IVIg drug candidate with maximum Fc sialylation devoid of unwanted alterations to the IVIg mixture. Here, we describe a controlled enzymatic, scalable process to produce a tetra-Fc-sialylated (s4-IVIg) IVIg drug candidate and its qualification across a wide panel of analytic assays, including physicochemical, pharmacokinetic, biodistribution, and in vivo animal models of inflammation. Our in vivo characterization of this drug candidate revealed consistent, enhanced anti-inflammatory activity up to 10-fold higher than IVIg across different animal models. To our knowledge, this candidate represents the first s4-IVIg suitable for clinical use; it is also a valuable therapeutic alternative with more consistent and potent anti-inflammatory activity.
Asunto(s)
Antiinflamatorios/uso terapéutico , Diseño de Fármacos , Inmunoglobulinas Intravenosas/uso terapéutico , Ácido N-Acetilneuramínico/metabolismo , Receptores Fc/metabolismo , Animales , Antiinflamatorios/farmacocinética , Antiinflamatorios/farmacología , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/patología , Vesícula/complicaciones , Vesícula/tratamiento farmacológico , Vesícula/patología , Modelos Animales de Enfermedad , Epidermólisis Ampollosa Adquirida/complicaciones , Epidermólisis Ampollosa Adquirida/tratamiento farmacológico , Epidermólisis Ampollosa Adquirida/patología , Glicosilación/efectos de los fármacos , Células HEK293 , Humanos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Inmunoglobulinas Intravenosas/farmacocinética , Inmunoglobulinas Intravenosas/farmacología , Ratones , Púrpura Trombocitopénica Idiopática/tratamiento farmacológico , Púrpura Trombocitopénica Idiopática/patología , Distribución Tisular/efectos de los fármacos , Resultado del TratamientoRESUMEN
Relaxation dispersion spectroscopy is one of the most widely used techniques for the analysis of protein dynamics. To obtain a detailed understanding of the protein function from the view point of dynamics, it is essential to fit relaxation dispersion data accurately. The grid search method is commonly used for relaxation dispersion curve fits, but it does not always find the global minimum that provides the best-fit parameter set. Also, the fitting quality does not always improve with increase of the grid size although the computational time becomes longer. This is because relaxation dispersion curve fitting suffers from a local minimum problem, which is a general problem in non-linear least squares curve fitting. Therefore, in order to fit relaxation dispersion data rapidly and accurately, we developed a new fitting program called GLOVE that minimizes global and local parameters alternately, and incorporates a Monte-Carlo minimization method that enables fitting parameters to pass through local minima with low computational cost. GLOVE also implements a random search method, which sets up initial parameter values randomly within user-defined ranges. We demonstrate here that the combined use of the three methods can find the global minimum more rapidly and more accurately than grid search alone.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular , Proteínas/química , Programas Informáticos , Algoritmos , Método de Montecarlo , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: There is an urgent need to determine whether oversulfated chondroitin sulfate (OSCS), a compound contaminating heparin supplies worldwide, is the cause of the severe anaphylactoid reactions that have occurred after intravenous heparin administration in the United States and Germany. METHODS: Heparin procured from the Food and Drug Administration, consisting of suspect lots of heparin associated with the clinical events as well as control lots of heparin, were screened in a blinded fashion both for the presence of OSCS and for any biologic activity that could potentially link the contaminant to the observed clinical adverse events. In vitro assays for the activation of the contact system and the complement cascade were performed. In addition, the ability of OSCS to recapitulate key clinical manifestations in vivo was tested in swine. RESULTS: The OSCS found in contaminated lots of unfractionated heparin, as well as a synthetically generated OSCS reference standard, directly activated the kinin-kallikrein pathway in human plasma, which can lead to the generation of bradykinin, a potent vasoactive mediator. In addition, OSCS induced generation of C3a and C5a, potent anaphylatoxins derived from complement proteins. Activation of these two pathways was unexpectedly linked and dependent on fluid-phase activation of factor XII. Screening of plasma samples from various species indicated that swine and humans are sensitive to the effects of OSCS in a similar manner. OSCS-containing heparin and synthetically derived OSCS induced hypotension associated with kallikrein activation when administered by intravenous infusion in swine. CONCLUSIONS: Our results provide a scientific rationale for a potential biologic link between the presence of OSCS in suspect lots of heparin and the observed clinical adverse events. An assay to assess the amidolytic activity of kallikrein can supplement analytic tests to protect the heparin supply chain by screening for OSCS and other highly sulfated polysaccharide contaminants of heparin that can activate the contact system.
Asunto(s)
Anafilaxia/inducido químicamente , Sulfatos de Condroitina/análisis , Sulfatos de Condroitina/farmacología , Activación de Complemento/efectos de los fármacos , Contaminación de Medicamentos , Heparina/química , Calicreínas/efectos de los fármacos , Animales , China , Sulfatos de Condroitina/efectos adversos , Complemento C3a/biosíntesis , Complemento C3a/efectos de los fármacos , Complemento C5a/biosíntesis , Complemento C5a/efectos de los fármacos , Industria Farmacéutica , Femenino , Alemania , Heparina/efectos adversos , Humanos , Hipotensión/inducido químicamente , Calicreínas/metabolismo , Persona de Mediana Edad , Sus scrofa , Estados Unidos , United States Food and Drug AdministrationRESUMEN
Recently, certain lots of heparin have been associated with an acute, rapid onset of serious side effects indicative of an allergic-type reaction. To identify potential causes for this sudden rise in side effects, we examined lots of heparin that correlated with adverse events using orthogonal high-resolution analytical techniques. Through detailed structural analysis, the contaminant was found to contain a disaccharide repeat unit of glucuronic acid linked beta1-->3 to a beta-N-acetylgalactosamine. The disaccharide unit has an unusual sulfation pattern and is sulfated at the 2-O and 3-O positions of the glucuronic acid as well as at the 4-O and 6-O positions of the galactosamine. Given the nature of this contaminant, traditional screening tests cannot differentiate between affected and unaffected lots. Our analysis suggests effective screening methods that can be used to determine whether or not heparin lots contain the contaminant reported here.
Asunto(s)
Sulfatos de Condroitina/análisis , Sulfatos de Condroitina/química , Contaminación de Medicamentos/prevención & control , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Heparina/análisis , Heparina/química , Evaluación Preclínica de Medicamentos , HumanosRESUMEN
Dynamic processes are implicit in the catalytic function of all enzymes. To obtain insights into the relationship between the dynamics and thermodynamics of protein fluctuations and catalysis, we have measured millisecond time scale motions in the enzyme dihydrofolate reductase using NMR relaxation methods. Studies of a ternary complex formed from the substrate analog folate and oxidized NADP+ cofactor revealed conformational exchange between a ground state, in which the active site loops adopt a closed conformation, and a weakly populated (4.2% at 30 degrees C) excited state with the loops in the occluded conformation. Fluctuations between these states, which involve motions of the nicotinamide ring of the cofactor into and out of the active site, occur on a time scale that is directly relevant to the structural transitions involved in progression through the catalytic cycle.
Asunto(s)
Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolato Deshidrogenasa/metabolismo , Fenómenos Biofísicos , Biofisica , Catálisis , Dominio Catalítico , Escherichia coli/enzimología , Ácido Fólico/metabolismo , Cinética , Modelos Biológicos , Modelos Moleculares , NADP/metabolismo , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , TermodinámicaRESUMEN
One of the principal promises of solid-state NMR (SSNMR) magic angle spinning (MAS) experiments has been the possibility of determining the structures of molecules in states that are not accessible via X-ray or solution NMR experiments-e.g., membrane or amyloid proteins. However, the low sensitivity of SSNMR often restricts structural studies to small-model compounds and precludes many higher-dimensional solid-state MAS experiments on such systems. To address the sensitivity problem, we have developed experiments that utilize dynamic nuclear polarization (DNP) to enhance sensitivity. In this communication, we report the successful application of MAS DNP to samples of cryoprotected soluble and membrane proteins. In particular, we have observed DNP signal enhancements of up to 50 in 15N MAS spectra of bacteriorhodopsin (bR) and alpha-lytic protease (alpha-LP). The spectra were recorded at approximately 90 K where MAS is experimentally straightforward, and the results suggest that the described protocol will be widely applicable.
Asunto(s)
Bacteriorodopsinas/química , Resonancia Magnética Nuclear Biomolecular/métodos , Serina Endopeptidasas/química , SolubilidadRESUMEN
The role of proline residues in the photocycle of bacteriorhodopsin (bR) is addressed using solid-state NMR. (13)C and (15)N chemical shifts from X-Pro peptide bonds in bR are assigned from REDOR difference spectra of pairwise labeled samples, and correlations of chemical shifts with structure are explored in a series of X-Pro model compounds. Results for the three membrane-embedded X-Pro bonds of bR indicate only slight changes in the transition from the resting state of the protein to either the early or late M state of the protonmotive photocycle. These results suggest that the buried prolines serve a principally structural role in bR.
Asunto(s)
Bacteriorodopsinas/química , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Oligopéptidos/química , Fotoquímica , Prolina/química , EstereoisomerismoRESUMEN
Active transport requires the alternation of substrate uptake and release with a switch in the access of the substrate binding site to the two sides of the membrane. Both the transfer and switch aspects of the photocycle have been subjects of magnetic resonance studies in bacteriorhodopsin. The results for ion transfer indicate that the Schiff base of the chromophore is hydrogen bonded before, during, and after its deprotonation. This suggests that the initial complex counterion of the Schiff base decomposes in such a way that the Schiff base carries its immediate hydrogen-bonding partner with it as it rotates during the first half of the photocycle. If so, bacteriorhodopsin acts as an inward-directed hydroxide pump rather than as an outward-directed proton pump. The studies of the access switch explore both protein-based and chromophore-based mechanisms. Combined with evidence from functional studies of mutants and other forms of spectroscopy, the results suggest that maintaining access to the extracellular side of the protein after photoisomerization involves twisting of the chromophore and that the decisive switch in access to the cytoplasmic side results from relaxation of the chromophore when the constraints on the Schiff base are released by decomposition of the complex counterion.
Asunto(s)
Bacteriorodopsinas/química , Imagen por Resonancia Magnética/métodos , Animales , Aniones , Anisotropía , Transporte Biológico Activo , Hidróxidos , Iones , Modelos Químicos , Protones , Retina/metabolismoRESUMEN
In recent years, structural information about bacteriorhodopsin has grown substantially with the publication of several crystal structures. However, precise measurements of the chromophore conformation in the various photocycle states are still lacking. This information is critical because twists about the chromophore backbone chain can influence the Schiff base nitrogen position, orientation, and proton affinity. Here, we focus on the C14-C15 bond, using solid-state nuclear magnetic resonance spectroscopy to measure the H-C14-C15-H dihedral angle. In the resting state (bR(568)), we obtain an angle of 164 +/- 4 degrees, indicating a 16 degrees distortion from a planar all-trans chromophore. The dihedral angle is found to decrease to 147 +/- 10 degrees in the early M intermediate (M(o)) and to 150 +/- 4 degrees in the late M intermediate (M(n)). These results demonstrate changes in the chromophore conformation undetected by recent X-ray diffraction studies.