RESUMO
Although therapeutically efficacious, ipilimumab can exhibit dose-limiting toxicity that prevents maximal efficacious clinical outcomes and can lead to discontinuation of treatment. We hypothesized that an acidic pH-selective ipilimumab (pH Ipi), which preferentially and reversibly targets the acidic tumor microenvironment over the neutral periphery, may have a more favorable therapeutic index. While ipilimumab has pH-independent CTLA-4 affinity, pH Ipi variants have been engineered to have up to 50-fold enhanced affinity to CTLA-4 at pH 6.0 compared to pH 7.4. In hCTLA-4 knock-in mice, these variants have maintained anti-tumor activity and reduced peripheral activation, a surrogate marker for toxicity. pH-sensitive therapeutic antibodies may be a differentiating paradigm and a novel modality for enhanced tumor targeting and improved safety profiles.
Assuntos
Neoplasias , Microambiente Tumoral , Animais , Concentração de Íons de Hidrogênio , Ipilimumab/uso terapêutico , Camundongos , Índice TerapêuticoRESUMO
Protein glycosylation can impact the efficacy, safety, and pharmacokinetics of therapeutic proteins. Achieving uniform and consistent protein glycosylation is an important requirement for product quality control at all stages of therapeutic protein drug discovery and development. The development of a new microfluidic CE device compatible with MS offers a fast and sensitive orthogonal mode of high-resolution separation with MS characterization. Here, we describe a fast and robust chip-based CE-MS method for intact glycosylation fingerprinting of a therapeutic fusion protein with complex sialylated N and O-linked glycoforms. The method effectively separates multiple sialylated glycoforms and offers a rapid detection of changes in glycosylation profile in 6 min.
Assuntos
Eletroforese Capilar/instrumentação , Dispositivos Lab-On-A-Chip , Espectrometria de Massas/instrumentação , Polissacarídeos/análise , Proteínas Recombinantes de Fusão , Glicosilação , Mapeamento de Peptídeos/instrumentação , Mapeamento de Peptídeos/métodos , Polissacarídeos/química , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
Lyophilization was used to prepare dry, glassy solid vaccine formulations of recombinant ricin toxin A-chain containing suspensions of colloidal aluminum hydroxide adjuvant. Four lyophilized formulations were prepared by using combinations of rapid or slow cooling during lyophilization and one of two buffers, histidine or ammonium acetate. Trehalose was used as the stabilizing excipient. Aggregation of the colloidal aluminum hydroxide suspension was reduced in formulations processed with a rapid cooling rate. Aluminum hydroxide particle size distributions, glass transition temperatures, water contents, and immunogenicities of lyophilized vaccines were independent of incubation time at 40 °C for up to 15 weeks. Mice immunized with reconstituted ricin toxin subunit A (RTA) vaccines produced RTA-specific antibodies and toxin-neutralizing antibodies (TNAs) regardless of the length of high temperature vaccine storage or the degree of aluminum adjuvant aggregation that occurred during lyophilization. In murine studies, lyophilized formulations of vaccines conferred protection against exposure to lethal doses of ricin, even after the lyophilized formulations had been stored at 40 °C for 4 weeks. A corresponding liquid formulation of vaccine stored at 40 °C elicited RTA-specific antibody titers but failed to confer immunity during a ricin challenge.
Assuntos
Estabilidade de Medicamentos , Proteínas Recombinantes/química , Ricina/química , Vacinas de Subunidades Antigênicas/química , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Adjuvantes Farmacêuticos/química , Adjuvantes Farmacêuticos/farmacologia , Hidróxido de Alumínio/química , Animais , Anticorpos Neutralizantes/imunologia , Formação de Anticorpos/imunologia , Soluções Tampão , Química Farmacêutica/métodos , Armazenamento de Medicamentos , Excipientes/química , Feminino , Liofilização/métodos , Temperatura Alta , Camundongos , Tamanho da Partícula , Proteínas Recombinantes/imunologia , Ricina/imunologia , Temperatura de Transição , Trealose/química , Vacinas de Subunidades Antigênicas/imunologia , Água/químicaRESUMO
An all-PDMS on-line microdialysis-microchip electrophoresis with on-chip derivatization and electrophoretic separation for near real-time monitoring of primary amine-containing analytes is described. Each part of the chip was optimized separately, and the effect of each of the components on temporal resolution, lag time, and separation efficiency of the device was determined. Aspartate and glutamate were employed as test analytes. Derivatization was accomplished with naphthalene-2,3,-dicarboxyaldehyde/cyanide (NDA/CN(-)), and the separation was performed using a 15-cm serpentine channel. The analytes were detected using LIF detection.
Assuntos
Aminas/análise , Aminoácidos/análise , Eletroforese em Microchip/instrumentação , Eletroforese em Microchip/métodos , Ácido Aspártico/análise , Ácido Glutâmico/análise , Microdiálise/instrumentação , Microdiálise/métodos , Naftalenos/análise , Naftalenos/químicaRESUMO
The blood-brain barrier (BBB) is an important interface between the peripheral and central nervous systems. It protects the brain against the infiltration of harmful substances and regulates the permeation of beneficial endogenous substances from the blood into the extracellular fluid of the brain. It can also present a major obstacle in the development of drugs that are targeted for the central nervous system. Several methods have been developed to investigate the transport and metabolism of drugs, peptides, and endogenous compounds at the BBB. In vivo methods include intravenous injection, brain perfusion, positron emission tomography, and microdialysis sampling. Researchers have also developed in vitro cell-culture models that can be employed to investigate transport and metabolism at the BBB without the complication of systemic involvement. All these methods require sensitive and selective analytical methods to monitor the transport and metabolism of the compounds of interest at the BBB.
Assuntos
Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Animais , Transporte Biológico , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Cromatografia Líquida/métodos , Eletroforese Capilar/métodos , Desenho de Equipamento , Humanos , Injeções Intravenosas/métodos , Espectrometria de Massas/métodos , Microdiálise/instrumentação , Microdiálise/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Perfusão/métodos , Tomografia por Emissão de Pósitrons/métodosRESUMO
A PDMS-based microfluidic system for online coupling of microdialysis sampling to microchip electrophoresis with fluorescence detection for in vivo analysis of amino acid neurotransmitters using naphthalene-2,3-dicarboxaldehyde and sodium cyanide as the derivatization reagents is described. Fabricating chips from PDMS rather than glass was found to be simpler and more reproducible, especially for chips with complex designs. The microchip incorporated a 20-cm serpentine channel in which sample plugs were introduced using a "simple" injection scheme; this made fluid handling and injection on-chip easier for the online system compared with gated or valve-based injection. The microchip was evaluated offline for the analysis of amino acid standards and rat brain microdialysis samples. Next, precolumn derivatization was incorporated into the chip and in vivo online microdialysis-microchip electrophoresis studies were performed. The system was employed for the continuous monitoring of amino acid neurotransmitters in the extracellular fluid of the brain of an anesthetized rat. Fluorescein was dosed intravenously and monitored simultaneously online as a marker of in vivo blood-brain barrier permeability. The microdialysis-microchip electrophoresis system described here will be employed in the future for simultaneous monitoring of changes in blood-brain barrier permeability and levels of amino acid neurotransmitters in the rat stroke model.
Assuntos
Aminoácidos/análise , Dimetilpolisiloxanos/química , Eletroforese em Microchip/instrumentação , Eletroforese em Microchip/métodos , Microdiálise/métodos , Neurotransmissores/análise , Nylons/química , Animais , Química Encefálica , Fluoresceína/química , Concentração de Íons de Hidrogênio , Naftalenos/química , Ratos , Ratos Sprague-Dawley , Cianeto de Sódio/química , Espectrometria de Fluorescência/métodosRESUMO
Microdialysis (MD) is a sampling technique that can be employed to monitor biological events both in vivo and in vitro. When it is coupled to an analytical system, microdialysis can provide near real-time information on the time-dependent concentration changes of analytes in the extracellular space or other aqueous environments. Online systems for the analysis of microdialysis samples enable fast, selective and sensitive analysis while preserving the temporal information. Analytical methods employed for online analysis include liquid chromatography (LC), capillary (CE) and microchip electrophoresis and flow-through biosensor devices. This review article provides an overview of microdialysis sampling and online analysis systems with emphasis on in vivo analysis. Factors that affect the frequency of analysis and, hence, the temporal resolution of these systems are also discussed.
Assuntos
Microdiálise/tendências , Animais , Técnicas Biossensoriais , Cromatografia Líquida de Alta Pressão , Eletroforese Capilar , Procedimentos Analíticos em Microchip , Microdiálise/instrumentação , Microdiálise/métodos , Sistemas On-Line/instrumentação , RatosRESUMO
The integration of rapid on-chip sample derivatization employing naphthalene 2,3-dicarboxaldehyde and 2-mercaptoethanol (NDA/2ME) with an easily assembled microdialysis/microchip electrophoresis device was carried out. The microchip device consisted of a glass layer with etched microfluidic channels that was sealed with a layer of poly(dimethylsiloxane) (PDMS) via plasma oxidation. This simple sealing procedure alleviated the need for glass thermal bonding and allowed the device to be re-sealed in the event of blockages within the channels. The device was used for analysis of a mixture of amino acids and peptides derivatized on-chip with NDA/2ME for laser-induced fluorescence (LIF) detection. A 0.6 mM NDA/1.2 mM 2ME mixture was simply added into the buffer reservoir for dynamic on-column derivatization of sample mixtures introduced at a flow rate of 1.0 microl/min. Using this scheme, sample injection plugs were derivatized and separated simultaneously. Injections of ca. 12 fmol of 5 mM amino acid and peptide samples were conducted using the system. Finally, a three-component mixture of Arg, Gly-Pro, and Asp was sampled from a vial using microdialysis, derivatized, separated and detected with the system. The ultimate goal of this effort is the creation of a micro-total analysis system for high-temporal resolution monitoring of primary amines in biological systems.