RESUMEN
AF2 is a high affinity murine Ab possessing potent neutralizing activity against human IFN-gamma. In carrying out the modifications to humanize this Ab, we discovered that an initial version displayed affinity for IFN-gamma that was slightly less than that of AF2, but exhibited IFN-gamma-neutralizing activity that was severely diminished. Characterization via site-directed mutagenesis revealed that the majority of this loss in IFN-gamma-neutralizing activity was due to altering the V(H) framework residue at position 11. V(H) position 11 is distal to the binding surface of the Ab; however, it, along with residues 110 and 112, have been identified as forming the socket of a molecular ball-and-socket joint between the V and C domains of the Ig Fab, which influences the elbow angle between these domains. To determine whether disrupting the structure of this joint was the basis for reduced IFN-gamma-neutralizing capacity, we altered residue 148 of C(H1), which with residue 149 comprises the corresponding ball portion of the joint. Changing this single C(H1) domain residue diminished the ability of the Ab to neutralize IFN-gamma to a level similar to that observed with the V(H) alteration. Thus, an intact ball-and-socket joint between the V and C domains in AF2 is required for potent neutralization of IFN-gamma. These results suggest the importance of the elbow angle between Ig V and C domains in Ab activity, and support the hypothesis that this joint can be an important functional element of Ab structure.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Afinidad de Anticuerpos , Sitios de Unión de Anticuerpos , Regiones Constantes de Inmunoglobulina/metabolismo , Región Variable de Inmunoglobulina/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Sustitución de Aminoácidos/inmunología , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Afinidad de Anticuerpos/genética , Sitios de Unión de Anticuerpos/genética , Células COS , Humanos , Regiones Constantes de Inmunoglobulina/química , Regiones Constantes de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/metabolismo , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Inmunosupresores/química , Inmunosupresores/metabolismo , Interferón gamma/antagonistas & inhibidores , Interferón gamma/inmunología , Interferón gamma/metabolismo , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína/genética , Relación Estructura-Actividad , Transfección , Células Tumorales CultivadasRESUMEN
A drug-excipient compatibility screening model was developed by which potential stability problems due to interactions of drug substances with excipients in solid dosage forms can be predicted. The model involved storing drug-excipient blends with 20% added water in closed glass vials at 50 degrees C and analyzing them after 1 and 3 weeks for chemical and physical stability. The total weight of drug-excipient blend in a vial was usually kept at about 200 mg. The amount of drug substance in a blend was determined on the basis of the expected drug-to-excipient ratio in the final formulation. Potential roles of several key factors, such as the chemical nature of the excipient, drug-to-excipient ratio, moisture, microenvironmental pH of the drug-excipient mixture, temperature, and light, on dosage form stability could be identified by using the model. Certain physical changes, such as polymorphic conversion or change from crystalline to amorphous form, that could occur in drug-excipient mixtures were also studied. Selection of dosage form composition by using this model at the outset of a drug development program would lead to reduction of "surprise" problems during long-term stability testing of drug products.
Asunto(s)
Estabilidad de Medicamentos , Excipientes/farmacología , Bloqueadores de los Canales de Calcio/química , Cromatografía Líquida de Alta Presión , Cristalización , Formas de Dosificación , Fosinopril/química , SolubilidadRESUMEN
A monoclonal antibody (AF2) generated against recombinant human interferongamma (IFNgamma) exhibited potent IFNgamma neutralizing activity and prevented human IFNgamma from binding to the cell surface IFNgamma receptor complex. The AF2 antibody also neutralized IFNgamma from higher primates (superfamily Hominoidea) but did not react with IFNgamma from rhesus or other primates in the suborder Anthropoidea IFNgamma from all primates tested, however, could signal via the human IFNgamma receptor complex, as indicated by the ability to upregulate the level of MHC class II molecule expression on the surface of a responsive human cell line. We cloned and sequenced the IFNgamma gene from chimpanzee, gorilla, orangutan, and gibbon, and compared those with the previously reported IFNgamma sequences of human, rhesus, baboon and marmoset. This comparison revealed that, of the primate IFNgammas that were not reactive with AF2, rhesus IFNgamma was most homologous to human IFNgamma, differing at only nine amino acids and containing a one amino acid deletion. Comparing the sequence of human IFNgamma with that of rhesus IFNgamma suggested residues of the human IFNgamma molecule that were involved in the formation of the epitope recognized by the AF2 antibody. Constructing human/rhesus chimeric IFNgamma molecules, combined with site-directed mutagenesis of both human and rhesus IFNgamma revealed that this epitope was dependent upon two non-contiguous amino acids that are juxtaposed in the tertiary structure of IFNgamma. The determinant recognized by AF2 antibody resides in a portion of IFNgamma that is proximal to, but distinct from the surface that interacts with the IFNgamma receptor. Therefore, this neutralizing monoclonal antibody reacts with a conformational determinant that distinguishes primate IFNgammas serologically, but not functionally.
Asunto(s)
Anticuerpos Monoclonales , Interferón gamma/inmunología , Primates/inmunología , Receptores de Interferón/inmunología , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/genética , Mapeo Epitopo , Humanos , Interferón gamma/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Pruebas de Neutralización , Primates/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Receptor de Interferón gammaRESUMEN
The two polymorphic modifications of fosinopril sodium have been characterized as to their differences in melting behaviour, powder X-ray diffraction patterns, Fourier transform infrared spectra (FTIR), and solid-state 31P- and 13C-NMR spectra. The polymorphs were found to be enantiotropically related based upon melting point, heat of fusion, and solution mediated transformation data. Analysis of the solid-state FTIR and 13C-NMR data indicated that the environment of the acetal side chain of fosinopril sodium differed in two polymorphs, and that there might be cis-trans isomerization about the C6-N peptide bond. These conformational differences are postulated as the origin of the observed polymorphism.
Asunto(s)
Fosinopril/análisis , Espectroscopía de Resonancia Magnética , Espectroscopía Infrarroja por Transformada de Fourier , Cristalización , Estereoisomerismo , Difracción de Rayos XRESUMEN
Fosinopril sodium (I), a new angiotensin converting enzyme inhibitor, is a diester prodrug of the active moiety II. We report here a novel transformation of fosinopril into beta-ketoamide, III, and a phosphonic acid, IV, mediated through metal ion participation. The interaction of fosinopril with magnesium ions was studied in a solution model system in which methanol was used as the solvent and magnesium acetate as the source of metal ions. Kinetic analysis indicated the degradation to be a bimolecular process, with the rate being first order in both metal ion and fosinopril concentration. The degradation products II, III, and IV effectively retarded the magnesium ion mediated reaction of fosinopril. Based on the results of 31P-NMR, 1H-NMR, Mn(II)-EPR spectroscopy experiments and mass spectrometry, a mechanism is postulated for this transformation. A key reactive intermediate has been characterized that supports the proposed mechanism. The results can account for the observed degradation profile of the fosinopril sodium in a prototype tablet formulation.
Asunto(s)
Fosinopril/química , Magnesio/química , Espectroscopía de Resonancia por Spin del Electrón , Hidrólisis , Cinética , Espectrometría de Masas , Metanol/química , Modelos Moleculares , Isótopos de Fósforo , Solventes , ComprimidosRESUMEN
The mechanism of possible incompatibility between commercially available metronidazole parenteral solutions and the injectable aztreonam leading to the development of pink color in their intravenous admixtures was studied. It was demonstrated that nitrite ions may be produced in metronidazole solutions at the time of preparation or during storage by the effects of temperature and light. Under acidic pH conditions of admixtures the aminothiazole moiety of aztreonam was diazotized by the nitrite ion contributed by metronidazole solutions. The diazotized molecule, in turn, reacted with another aztreonam molecule by diazo-coupling. The resultant pink-colored product was isolated by chromatography and its structure was determined by mass spectral and NMR analyses. Other beta-lactam antibiotics containing the 2-aminothiazole moiety also react in acidic media in a similar manner.
Asunto(s)
Aztreonam/química , Metronidazol/química , Color , Interacciones Farmacológicas , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Nitritos/análisisRESUMEN
Physicochemical factors involved in the development of a topical solution of a novel corticosteroid, tipredane (1), are described. A cosolvent system consisting of polyethylene glycol 400 (PEG 400), propylene glycol, and water was used to dissolve the concentration (0.1% w/w) of 1 required for the formulation. The solvent mixture was also nonirritating to the skin. Buffering agent, antioxidant, and metal-chelating agent were required to stabilize the drug. Solubilities of hydrophilic and lipophilic excipients were ensured by careful adjustment of their concentrations, as well as that of PEG 400. Two formulations, one containing potassium citrate and the other tromethamine as the buffering agents, were identified. Upon storage, sodium metabisulfite, an antioxidant used in the formulation, oxidized to form K2SO4 in the formulation containing potassium citrate. Potassium citrate decreased the solubility and resulted in the precipitation of K2SO4 by exerting a common ion effect. Lowering of the concentrations of potassium citrate, sodium metabisulfite, and PEG 400 ensured the solubility of K2SO4 formed. There was no such precipitation of K2SO4 in the formulation buffered with tromethamine, thus indicating that tromethamine is a good buffering agent in cosolvent systems.