RESUMEN
Dengue is a mosquito borne infection, which in recent years has become a major international public health concern. Annually, 100 million dengue virus infections are reported worldwide. The nonstructural protein 1 (NS1) of dengue virus is a useful target for diagnostics of dengue infection since the protein is abundantly circulating in the blood during acute phase of the disease, in both primary and secondary infections. This research paper highlights the development of a panel of Mab and bsMab for dengue NS1 detection. The P148 series of Mabs showed high specificity for recombinant dengue NS1 antigen. These antibodies showed no cross reactivity with recombinant dengue envelope protein and other viral proteins. The hybrid-hybridoma approach to generate the P156.1 and P156.2 bsMabs from the P148 monoclonal antibody method was used during this study. Furthermore, the affinity purification provided good yields of quadromas associated with HRPO in two steps. Direct detection method involved coating of plates with different concentrations of recombinant antigen and detecting with bsMab. Sensitive sandwich assay with Mabs and bsMabs was also done. Detection of nonstructural dengue antigens may be of benefit for early and rapid diagnosis of dengue infection due to their long half-life in the blood.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/aislamiento & purificación , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/aislamiento & purificación , Virus del Dengue/inmunología , Proteínas no Estructurales Virales/sangre , Animales , Anticuerpos Biespecíficos/genética , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Antivirales/genética , Antígenos Virales/sangre , Antígenos Virales/inmunología , Dengue/diagnóstico , Femenino , Ratones Endogámicos BALB C , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas no Estructurales Virales/inmunologíaRESUMEN
Production of antibody fragments in heterologous hosts such as Escherichiacoli provides a unique and cost-effective method to develop engineered vectors for tumor targeting. A single-chain Fragment variable (scFv) of the murine monoclonal antibody MAb-B43.13 targeting CA125 in epithelial ovarian cancer was previously developed, expressed, purified and proposed as a functional targeting entity for biomedical applications. However, the yields from its soluble expression in heterologous systems were very low for any practical use in preclinical translational research; leave alone the defeated objective of convenient and cost-effective production. In the present work, the anti-CA125 scFv gene was re-organized and sub-cloned into pET-22b(+) vector to be in frame with the pelB leader peptide for periplasmic localization and C-terminal hexa-histidine tag to facilitate downstream purification. Six variants of the scFv were constructed to investigate the impact of variable domain orientations, inter-domain peptide linker sequences and codon optimization on the soluble expression of the scFv using Rosetta 2(DE3) as the E. coli host supplemented with tRNAs for rare codons. Expression in shake flask cultures under the control of an inducible T7 promoter and subsequent purification by cobalt based immobilized metal affinity chromatography yielded differential amounts of high purity scFv for all constructs. Here, we report up to 14-fold increase in the soluble expression of the scFv primarily as a result of codon optimization with minor effects from inter-domain peptide linkers and variable domain orientation in the anti-CA125 scFv molecule. All the scFv constructs expressed and purified were found to be immunoreactive for in vitro targeting of CA125 antigen.
Asunto(s)
Antígeno Ca-125/inmunología , Escherichia coli/genética , Proteínas de la Membrana/inmunología , Neoplasias Glandulares y Epiteliales/inmunología , Neoplasias Ováricas/inmunología , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Secuencia de Aminoácidos , Secuencia de Bases , Carcinoma Epitelial de Ovario , Línea Celular Tumoral , Clonación Molecular/métodos , Codón/genética , Humanos , Región Variable de Inmunoglobulina , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Anticuerpos de Cadena Única/química , SolubilidadRESUMEN
Dengue and the more severe dengue hemorrhagic fever have been a very critical public health problem globally. Millions of people especially in the tropical areas get infected with dengue. An efficient diagnostic is very important for early screening of dengue infection. In dengue-infected patients, the nonstructural protein NS1 is present on the surface of infected cells and secreted in plasma. The NS1 antigen is an important target for developing a quick diagnostic largely due to its long presence in the blood. We have developed a simple-to-use immunoswab-based diagnostic procedure employing monoclonal antibodies and the second-generation quadromas. The detection limit for NS1 has been established to be in the subnanogram range. The assay is very sensitive, has a visual end point, and also being extremely inexpensive. With this assay, screening time for a dengue-infected person would be very rapid.
Asunto(s)
Antígenos Virales/sangre , Técnicas de Laboratorio Clínico/métodos , Virus del Dengue/aislamiento & purificación , Pruebas Diagnósticas de Rutina/métodos , Inmunoensayo/métodos , Anticuerpos Monoclonales , Anticuerpos Antivirales , Técnicas de Laboratorio Clínico/economía , Costos y Análisis de Costo , Pruebas Diagnósticas de Rutina/economía , Humanos , Inmunoensayo/economía , Sensibilidad y EspecificidadRESUMEN
PURPOSE: To highlight the expression and purification of the recombinant dengue virus type-1 antigen exploiting the codon optimized full length envelope for increased yield in E. coli. METHODS: A 6x His tag was inserted at the C terminus to facilitate purification. The purified protein was recognized in Western blot by Monoclonal antibody specific for the tag. The in vitro refolded recombinant protein was used to immunize mice for the development of hybridomas and also analyzed for its biological functionality with heparan sulfate binding assay. RESULTS: The polyclonal anti-sera from the immunized mice were found to recognize the envelope protein thereby establishing the immunogenicity of the protein. CONCLUSION: The purified envelope protein could potentially be used towards dengue diagnostics and vaccine development efforts. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.
Asunto(s)
Virus del Dengue/genética , Escherichia coli/genética , Genes env , Proteínas del Envoltorio Viral/genética , Animales , Anticuerpos Antivirales , Clonación Molecular , Humanos , Inmunización , Ratones , Proteínas Recombinantes/metabolismo , Proteínas del Envoltorio Viral/metabolismoRESUMEN
The severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein is known to mediate receptor interaction and immune recognition and thus it is considered as a major target for vaccine design. The spike protein plays an important role in virus entry, virus receptor interactions, and virus tropism. Sensitive diagnosis of SARS is essential for the control of the disease in humans. Recombinant SARS-CoV S1 antigen was produced and purified for the development of monoclonal and bi-specific monoclonal antibodies. The hybridomas secreting anti-S1 antibodies, F26G18 and P136.8D12, were fused respectively with the YP4 hybridoma to generate quadromas. The sandwich ELISA was formed by using F26G18 as a coating antibody and biotinylated F26G18 as a detection antibody with a detection limit of 0.037µg/ml (p<0.02). The same detection limit was found with P136.8D12 as a coating antibody and biotinylated F26G18 as a detection antibody. The sensitivity was improved (detection limit of 0.019µg/ml), however, when using bi-specific monoclonal antibody (F157) as the detection antibody. In conclusion, the method described in this study allows sensitive detection of a recombinant SARS spike protein by sandwich ELISA with bi-specific monoclonal antibody and could be used for the diagnosis of patients suspected with SARS.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Anticuerpos Monoclonales/inmunología , Ensayo de Inmunoadsorción Enzimática , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/inmunología , Síndrome Respiratorio Agudo Grave/diagnóstico , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Proteínas del Envoltorio Viral/análisis , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Antivirales/inmunología , Línea Celular , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Proteínas de la Nucleocápside/análisis , Proteínas de la Nucleocápside/inmunología , Proteínas Recombinantes/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Sensibilidad y Especificidad , Síndrome Respiratorio Agudo Grave/inmunología , Síndrome Respiratorio Agudo Grave/virología , Glicoproteína de la Espiga del Coronavirus , Vacunas Virales/inmunologíaRESUMEN
In this study, we demonstrate a simple strategy for enhanced immune response using a two-component dendritic cell (DC) targeted antigen delivery system. One component consists of a recombinant bifunctional fusion protein (bfFp) used for DC targeting, whereas, the other component is made of biotinylated PLGA nanoparticles that encapsulate the antigen. The fusion protein (bfFp) made of a truncated core-streptavidin fused to anti-DEC-205 single chain antibody (scFv) was mixed with ovalbumin-loaded biotinylated NPs that were formulated using biotin-PEG (2000)-PLGA, and the combination, bfFp functionalized NPs was used for DC targeted antigen delivery. In vitro DC uptake studies revealed a 2-fold higher receptor-mediated uptake of bfFp functionalized NPs when compared to non-targeted NPs. Immunization of the mice with the bfFp functionalized NPs in conjunction with DC maturation stimulus (anti-CD40 mAb) enhanced OVA-specific IgG and IgG subclass responses. Splenocytes of these mice secreted significantly higher levels of Th1 (IFN-γ and IL-2) cytokines upon ex vivo restimulation with OVA. The promising outcomes of the bfFp functionalized DC targeted system support its use as a versatile vaccine delivery system for the design of monovalent or polyvalent vaccines.
Asunto(s)
Antígenos/inmunología , Antígenos/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Portadores de Fármacos/metabolismo , Nanopartículas , Animales , Citocinas/metabolismo , Inmunoglobulina G/sangre , Leucocitos Mononucleares/inmunología , Ratones , Ovalbúmina/inmunología , Ovalbúmina/metabolismo , Unión Proteica , Proteínas Recombinantes/metabolismo , Bazo/inmunologíaRESUMEN
A sensitive double antibody sandwich ELISA (DAS-ELISA) based on chicken anti-gliadin IgY and biotinylated monoclonal antibody (mAb) was developed for the quantification of gliadin in foods. The anti-gliadin IgY and mAb specifically detected gliadin in wheat, barley, and rye by indirect ELISA and Western-blot assay. Using anti-gliadin IgY as capture antibody and biotinylated mAb as detecting antibody, the sensitivity of DAS-ELISA has a linear standard range of 4-40 ng/mL, showing that the limit of detection (LOD) corresponds to 4 ng/mL gliadin in assay buffer, equivalent to 0.8 ppm in foods. The intra-assay expressed as percentage of coefficients of variation (%CV) was 7.25% average of six food samples. The interassay precision was 9.51% in food samples. The combination of anti-gliadin IgY and biotinylated mAb in the DAS-ELISA provides a reliable, sensitive, and inexpensive tool for the detection of gliadin in gluten-free and gluten-containing food products.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Gliadina/análisis , Animales , Anticuerpos Monoclonales/inmunología , Western Blotting , Pollos , Gliadina/inmunología , Inmunoglobulinas/inmunologíaRESUMEN
This work investigates the formulation and in vivo efficacy of dendritic cell (DC) targeted plasmid DNA loaded biotinylated chitosan nanoparticles for nasal immunization against nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) as antigen. The induction of antigen-specific mucosal and systemic immune response at the site of virus entry is a major challenge for vaccine design. Here, we designed a strategy for noninvasive receptor mediated gene delivery to nasal resident DCs. The pDNA loaded biotinylated chitosan nanoparticles were prepared using a complex coacervation process and characterized for size, shape, surface charge, plasmid DNA loading and protection against nuclease digestion. The pDNA loaded biotinylated chitosan nanoparticles were targeted with bifunctional fusion protein (bfFp) vector for achieving DC selective targeting. The bfFp is a recombinant fusion protein consisting of truncated core-streptavidin fused with anti-DEC-205 single chain antibody (scFv). The core-streptavidin arm of fusion protein binds with biotinylated nanoparticles, while anti-DEC-205 scFv imparts targeting specificity to DC DEC-205 receptor. We demonstrate that intranasal administration of bfFp targeted formulations along with anti-CD40 DC maturation stimuli enhanced magnitude of mucosal IgA as well as systemic IgG against N protein. The strategy led to the detection of augmented levels of N protein specific systemic IgG and nasal IgA antibodies. However, following intranasal delivery of naked pDNA no mucosal and systemic immune responses were detected. A parallel comparison of targeted formulations using intramuscular and intranasal routes showed that the intramuscular route is superior for induction of systemic IgG responses compared with the intranasal route. Our results suggest that targeted pDNA delivery through a noninvasive intranasal route can be a strategy for designing low-dose vaccines.
Asunto(s)
Quitosano/química , Células Dendríticas/metabolismo , Nanopartículas/química , Proteínas de la Nucleocápside/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Vacunas de ADN/inmunología , Vacunas Virales/inmunología , Animales , Femenino , Inmunidad Humoral/inmunología , Ratones , Ratones Endogámicos BALB CRESUMEN
The re-emergence of tuberculosis (TB) as a global public health threat highlights the necessity of rapid, simple and inexpensive point-of-care detection of the disease. Early diagnosis of TB is vital not only for preventing the spread of the disease but also for timely initiation of treatment. The later in turn will reduce the possible emergence of multi-drug resistant strains of Mycobacterium tuberculosis. Lipoarabinomannan (LAM) is an important non-protein antigen of the bacterial cell wall, which is found to be present in different body fluids of infected patients including blood, urine and sputum. We have developed a bispecific monoclonal antibody with predetermined specificities towards the LAM antigen and a reporter molecule horseradish peroxidase (HRPO). The developed antibody was subsequently used to design a simple low cost immunoswab based assay to detect LAM antigen. The limit of detection for spiked synthetic LAM was found to be 5.0 ng/ml (bovine urine), 0.5 ng/ml (rabbit serum) and 0.005 ng/ml (saline) and that for bacterial LAM from M. tuberculosis H37Rv was found to be 0.5 ng/ml (rabbit serum). The assay was evaluated with 21 stored clinical serum samples (14 were positive and 7 were negative in terms of anti-LAM titer). In addition, all 14 positive samples were culture positive. The assay showed 100% specificity and 64% sensitivity (95% confidence interval). In addition to good specificity, the end point could be read visually within two hours of sample collection. The reported assay might be used as a rapid tool for detecting TB in resource constrained laboratory settings.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Técnicas de Laboratorio Clínico/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Recursos en Salud , Tuberculosis/diagnóstico , Tuberculosis/inmunología , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Antígenos Bacterianos/inmunología , Biotina/metabolismo , Western Blotting , Bovinos , Línea Celular , Electroforesis en Gel de Poliacrilamida , Humanos , Límite de Detección , Lipopolisacáridos/inmunología , Mycobacterium tuberculosis/inmunología , Conejos , Ratas , Sensibilidad y Especificidad , Albúmina Sérica Bovina/metabolismo , Coloración y Etiquetado , Tuberculosis/microbiologíaRESUMEN
Gene therapy concept has been being overcome massive challenges from 1972 in ethical, socio-economical and developmental issues. In this review, we have attempted to go through almost all the arenas and described in a methodical way that reflects not only the initial ethical and scientific thoughts but also adorned a solid depiction of gene therapy related physico-chemical barriers, approaches and strategies till to date.
Asunto(s)
Técnicas de Transferencia de Gen , Terapia Genética , Adenoviridae/genética , Técnicas de Transferencia de Gen/tendencias , Terapia Genética/tendencias , Vectores Genéticos , Humanos , Nanopartículas/química , Plásmidos/metabolismo , Polímeros/química , Retroviridae/genética , Simplexvirus/genéticaRESUMEN
This review highlights the advantages and current status of plant-derived vaccine development with special reference to the dengue virus. There are numerous problems involved in dengue vaccine development, and there is no vaccine against all four dengue serotypes. Dengue vaccine development using traditional approaches has not been satisfactory in terms of inducing neutralizing antibodies. Recently, these issues were addressed by showing a very good response to inducing neutralizing antibodies by plant-derived dengue vaccine antigens. This indicates the feasibility of using plant-derived vaccine antigens as a low-cost method to combat dengue and other infectious diseases. The application of new methods and strategies such as dendritic cell targeting in cancer therapy, severe acute respiratory syndrome, tuberculosis, human immune deficiency virus, and malaria might play an important role. These new methods are more efficient than traditional protocols. It is expected that in the near future, plant-derived vaccine antigens or antibodies will play an important role in the control of human infectious diseases.
Asunto(s)
Antígenos de Plantas/inmunología , Antígenos Virales/inmunología , Vacunas contra el Dengue/inmunología , Virus del Dengue/inmunología , Dengue/prevención & control , Plantas Modificadas Genéticamente/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Antígenos de Plantas/biosíntesis , Antígenos de Plantas/genética , Antígenos Virales/biosíntesis , Antígenos Virales/genética , Dengue/inmunología , Dengue/virología , Vacunas contra el Dengue/biosíntesis , Vacunas contra el Dengue/genética , Virus del Dengue/genética , Humanos , Inmunización , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismoRESUMEN
Tuberculosis (TB) is a major threat in global public health. The emergence of HIV and also multi drug resistant (MDR) and extremely drug resistant (XDR)-TB poses a vital challenge to the control of the disease. For the last 40 years, no new anti-TB drug has been discovered. This literature review provides a brief discussion of existing drugs and emerging drug targets, and also of the advantages of incorporating modern drug delivery systems and immune modulators in order to improve the existing treatment regimen in terms of better efficacy, reduced drug administration frequency, shortened period of treatment and reduced drug related toxicity. The investigation for new a drug target is essential in carrying on the fight against MDR and XDR-TB. However, owing to the enormous cost and time involved in new drug development, improvement of the existing treatment regimen is seen to be a valid alternative.
Asunto(s)
Antituberculosos/uso terapéutico , Tuberculosis/tratamiento farmacológico , Animales , Antituberculosos/administración & dosificación , Humanos , Factores Inmunológicos/administración & dosificación , Factores Inmunológicos/uso terapéutico , NanocápsulasRESUMEN
Five monoclonal antibodies (MAbs) against recombinant nucleocapsid protein (NP) of severe acute respiratory syndrome (SARS)-causing coronavirus (CoV) were developed by hybridoma technology. Epitope mapping by Western blotting showed that these anti-SARS-CoV NP MAbs bind to distinct domains of NP. These anti-SARS-CoV NP MAbs, with their high specificity, are potentially ideal candidates for developing early and sensitive diagnostic assays for SARS-CoV.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Proteínas de la Nucleocápside/antagonistas & inhibidores , Proteínas de la Nucleocápside/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Western Blotting , Proteínas de la Nucleocápside de Coronavirus , Mapeo Epitopo , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
A new class of anti-inflammatory (AI) cupferron prodrugs was synthesized wherein a diazen-1-ium-1,2-diolato ammonium salt, and its O(2)-methyl and O(2)-acetoxyethyl derivatives, nitric oxide (NO) donor moieties were attached directly to an aryl carbon on a celecoxib template. The percentage of NO released from the O(2)-methyl and O(2)-acetoxyethyl compounds was higher (18.0-37.8% of the theoretical maximal release of one molecule of NO/molecule of the parent compound) upon incubation in the presence of rat serum, relative to incubation with phosphate buffer saline (PBS) at pH 7.4 (3.8-11.6% range). All compounds exhibited weak inhibition of the COX-1 isozyme (IC(50)=5.8-17.0 microM range) in conjunction with weak or modest inhibition of the COX-2 isozyme (IC(50)=1.6-14.4 microM range). The most potent AI agent 5-[4-(O(2)-ammonium diazen-1-ium-1,2-diolato)phenyl]-1-(4-sulfamoylphenyl)-3-trifluoromethyl-1H-pyrazole exhibited a potency that was about fourfold and twofold greater than that observed for the respective reference drugs aspirin and ibuprofen. These studies indicate that use of a cupferron template constitutes a plausible drug design approach targeted toward the development of AI drugs that do not cause gastric irritation, or elevate blood pressure and induce platelet aggregation that have been associated with the use of some selective COX-2 inhibitors.
Asunto(s)
Antiinflamatorios/síntesis química , Hidrazinas/química , Donantes de Óxido Nítrico/química , Óxido Nítrico/metabolismo , Profármacos/síntesis química , Pirazoles/química , Sulfonamidas/química , Animales , Antiinflamatorios/química , Antiinflamatorios/farmacología , Celecoxib , Ciclooxigenasa 1/química , Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/química , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa/síntesis química , Inhibidores de la Ciclooxigenasa/química , Inhibidores de la Ciclooxigenasa/farmacología , Diseño de Fármacos , Humanos , Nitrosaminas/síntesis química , Nitrosaminas/química , Profármacos/química , Profármacos/farmacología , Pirazoles/síntesis química , Pirazoles/farmacología , Ratas , Sulfonamidas/síntesis química , Sulfonamidas/farmacologíaRESUMEN
A new group of acetic acid (7a-c, R(1) = H), and propionic acid (7d-f, R(1) = Me), regioisomers wherein a N-difluoromethyl-1,2-dihydropyrid-2-one moiety is attached via its C-3, C-4, and C-5 position was synthesized. This group of compounds exhibited a more potent inhibition, and hence selectivity, for the cyclooxygenase-2 (COX-2) relative to the COX-1 isozyme. Attachment of the N-difluoromethyl-1,2-dihydropyrid-2-one ring system to an acetic acid, or propionic acid, moiety confers potent 5-LOX inhibitory activity, that is, absent in traditional arylacetic acid NSAIDs. 2-(1-Difluoromethyl-2-oxo-1,2-dihydropyridin-5-yl)acetic acid (7c) exhibited the best combination of dual COX-2 and 5-LOX inhibitory activities. Molecular modeling (docking) studies showed that the highly electronegative CHF(2) substituent present in 7c, that showed a modest selectivity for the COX-2 isozyme, is oriented within the secondary pocket (Val523) present in COX-2 similar to the sulfonamide (SO(2)NH(2)) COX-2 pharmacophore present in celecoxib, and that the N-difluoromethyl-1,2-dihydropyrid-2-one pharmacophore is oriented close to the region containing the LOX enzyme catalytic iron (His361, His366, and His545). Accordingly, the N-difluoromethyl-1,2-dihyrdopyrid-2-one moiety possesses properties suitable for the design of dual COX-2/5-LOX inhibitory drugs.
Asunto(s)
Araquidonato 5-Lipooxigenasa/metabolismo , Inhibidores de la Ciclooxigenasa/química , Inhibidores de la Ciclooxigenasa/farmacología , Inhibidores de la Lipooxigenasa/química , Inhibidores de la Lipooxigenasa/farmacología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Acetatos/síntesis química , Acetatos/química , Acetatos/farmacología , Animales , Araquidonato 5-Lipooxigenasa/química , Inhibidores de la Ciclooxigenasa/síntesis química , Humanos , Isomerismo , Inhibidores de la Lipooxigenasa/síntesis química , Modelos Moleculares , Prostaglandina-Endoperóxido Sintasas/química , Piridonas/síntesis química , Piridonas/química , Piridonas/farmacologíaRESUMEN
A new group of hybrid nitric oxide (NO) releasing anti-inflammatory (AI) coxib prodrugs (NO-coxibs) wherein the para-tolyl moiety present in celecoxib was replaced by a N-(4-nitrooxybutyl)piperidyl 15a-b, or N-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyridyl 17a-b, NO-donor moiety was synthesized. All compounds released a low amount of NO upon incubation with phosphate buffered saline (PBS) at pH 7.4 (2.4-5.8% range). In comparison, the percentage NO released was higher (3.1-8.4% range) when these nitrate prodrugs were incubated in the presence of L-cysteine. In vitro COX-1/COX-2 isozyme inhibition studies showed this group of compounds are moderately more potent, and hence selective, inhibitors of the COX-2 relative to the COX-1 enzyme. AI structure-activity relationship data acquired showed that compounds having a MeSO2 COX-2 pharmacophore exhibited superior AI activity compared to analogs having a H2NSO2 substituent. Compounds having a MeSO2 COX-2 pharmacophore in conjunction with a N-(4-nitrooxybutyl)piperidyl (ED50=132.4 mg/kg po), or a N-(4-nitrooxybutyl)-1,2,3,6-tetrahydropyridyl (ED50=118.4 mg/kg po), moiety exhibited an AI potency profile that is similar to aspirin (ED50=128.7 mg/kg po) but lower than ibuprofen (ED50=67.4 mg/kg po).
Asunto(s)
Donantes de Óxido Nítrico , Óxido Nítrico/química , Piperidinas/síntesis química , Profármacos , Pirazoles/química , Sulfonamidas/química , Animales , Antiinflamatorios/síntesis química , Antiinflamatorios/química , Antiinflamatorios/farmacología , Aspirina/farmacología , Celecoxib , Cristalografía por Rayos X , Inhibidores de la Ciclooxigenasa 2/química , Humanos , Concentración de Iones de Hidrógeno , Ibuprofeno/farmacología , Concentración 50 Inhibidora , Estructura Molecular , Donantes de Óxido Nítrico/síntesis química , Donantes de Óxido Nítrico/química , Donantes de Óxido Nítrico/farmacología , Piperidinas/química , Piperidinas/farmacología , Profármacos/síntesis química , Profármacos/química , Profármacos/farmacología , Ratas , Ovinos , Relación Estructura-ActividadRESUMEN
A novel class of phenylacetic acid regioisomers possessing a N-difluoromethyl-1,2-dihydropyrid-2-one pharmacophore attached to its C-2, C-3 or C-4 position was designed for evaluation as anti-inflammatory (AI) agents. A number of compounds exhibited a combination of potent in vitro cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) inhibitory activities. 2-(1-Difluoromethyl-2-oxo-1,2-dihydropyridin-4-yl)phenylacetic acid (9a) exerted the most potent AI activity among this group of compounds. Molecular modeling studies showed that the N-difluoromethyl-1,2-dihydropyridin-2-one moiety present in 9a inserts into the secondary pocket present in COX-2 to confer COX-2 selectivity, and that the N-difluoromethyl-1,2-dihydropyrid-2-one group (9a) binds close to the region of the 15-LOX enzyme containing catalytic iron (His361, His366). Accordingly, the N-difluoromethyl-1,2-dihyrdopyrid-2-one moiety possesses properties that make it an attractive pharmacophore suitable for the design of dual COX-2/5-LOX inhibitory AI drugs.
Asunto(s)
Antiinflamatorios no Esteroideos/química , Inhibidores de la Ciclooxigenasa/química , Inhibidores de la Lipooxigenasa , Inhibidores de la Lipooxigenasa/química , Fenilacetatos/química , Animales , Antiinflamatorios no Esteroideos/farmacología , Araquidonato 5-Lipooxigenasa/metabolismo , Sitios de Unión/fisiología , Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa/farmacología , Evaluación Preclínica de Medicamentos/métodos , Humanos , Inhibidores de la Lipooxigenasa/farmacología , Fenilacetatos/farmacología , Estructura Secundaria de Proteína , Ovinos , EstereoisomerismoRESUMEN
A novel class of salicylic acid and N-acetyl-2-carboxybenzenesulfonamide regioisomers possessing a N-difluoromethyl-1,2-dihydropyrid-2-one pharmacophore attached to its C-4 or C-5 position was designed for evaluation as anti-inflammatory (AI) agents. Replacement of the 2,4-difluorophenyl ring in diflunisal by the N-difluoromethyl-1,2-dihydropyrid-2-one moiety provided compounds showing dual selective cyclooxygenase-2 (COX-2)/5-lipoxygenase (5-LOX) inhibitory activities. AI structure-activity studies showed that the C-4 (14a) and C-5 (14b) salicylate regioisomers were 1.4- and 1.6-fold more potent than aspirin, and the C-5 N-acetyl-2-carboxybenzenesulfonamide regioisomer (22b) was 1.3- and 2.8-fold more potent than ibuprofen and aspirin, respectively. In vivo ulcer index (UI) studies showed that the 4- and 5-(N-difluoromethyl-1,2-dihydropyrid-2-one-4-yl)salicylic acids (14a and 14b) were completely non-ulcerogenic since no gastric lesions were present (UI=0) relative to aspirin (UI=57) at an equivalent mumol/kg oral dose. The N-difluoromethyl-1,2-dihydropyridin-2-one moiety provides a novel 5-LOX pharmacophore for the design of cyclic hydroxamic mimetics for exploitation in the development of dual COX-2/5-LOX inhibitory AI drugs.
Asunto(s)
Aspirina/análogos & derivados , Inhibidores de la Ciclooxigenasa 2/química , Inhibidores de la Lipooxigenasa/química , Piridonas/química , Salicilatos/química , Sulfonamidas/química , Aspirina/síntesis química , Aspirina/química , Aspirina/farmacología , Inhibidores de la Ciclooxigenasa 2/síntesis química , Inhibidores de la Ciclooxigenasa 2/farmacología , Humanos , Isomerismo , Inhibidores de la Lipooxigenasa/síntesis química , Inhibidores de la Lipooxigenasa/farmacología , Piridonas/síntesis química , Piridonas/farmacología , Salicilatos/síntesis química , Salicilatos/farmacología , Sulfonamidas/síntesis química , Sulfonamidas/farmacologíaRESUMEN
A new class of hybrid nitric oxide-releasing anti-inflammatory (AI) ester prodrugs (NONO-coxibs) wherein an O(2)-acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,2-diolate (13a-b), or O(2)-acetoxymethyl-1-(2-methylpyrrolidin-1-yl)diazen-1-ium-1,2-diolate (16a-b), NO-donor moiety was covalently coupled to the COOH group of 5-(4-carboxymethylphenyl)-1-(4-methane(amino)sulfonylphenyl)-3-trifluoromethyl-1H-pyrazole (11a-b) was synthesized. The percentage of NO released from these diazen-1-ium-1,2-diolates was significantly higher (59.6-74.6% of the theoretical maximal release of 2 molecules of NO/molecule of the parent hybrid ester prodrug) upon incubation in the presence of rat serum, relative to incubation with phosphate buffer (PBS) at pH 7.4 (5.0-7.2% range). These incubation studies suggest that both NO and the AI compound would be released from the parent NONO-coxib upon in vivo cleavage by non-specific serum esterases. All compounds were weak inhibitors of the COX-1 isozyme (IC(50)=8.1-65.2microM range) and modest inhibitors of the COX-2 isozyme (IC(50)=0.9-4.6microM range). The most potent parent aminosulfonyl compound 11b exhibited AI activity that was about sixfold greater than that for aspirin and threefold greater than that for ibuprofen. The ester prodrugs 13b, 16b exhibited similar AI activity to that exhibited by the more potent parent acid 11b when the same oral mumol/kg dose was administered. These studies indicate hybrid ester AI/NO donor prodrugs of this type (NONO-coxibs) constitute a plausible drug design concept targeted toward the development of selective COX-2 inhibitory AI drugs that are devoid of adverse cardiovascular effects.
Asunto(s)
Antiinflamatorios/química , Antiinflamatorios/farmacología , Donantes de Óxido Nítrico/química , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico/metabolismo , Profármacos/química , Profármacos/farmacología , Animales , Antiinflamatorios/síntesis química , Antiinflamatorios/uso terapéutico , Compuestos Azo/química , Inhibidores de la Ciclooxigenasa/síntesis química , Inhibidores de la Ciclooxigenasa/química , Inhibidores de la Ciclooxigenasa/farmacología , Inhibidores de la Ciclooxigenasa/uso terapéutico , Edema/inducido químicamente , Edema/tratamiento farmacológico , Pie/patología , Humanos , Donantes de Óxido Nítrico/síntesis química , Donantes de Óxido Nítrico/uso terapéutico , Profármacos/síntesis química , Profármacos/uso terapéutico , Prostaglandina-Endoperóxido Sintasas/metabolismo , Ratas , OvinosRESUMEN
A new group of hybrid nitric oxide (NO) releasing anti-inflammatory (AI) ester prodrugs (NONO-NSAIDs) wherein a 1,3-dinitrooxy-2-propyl (12a-c), or O(2)-acetoxymethyl-1-[2-(methyl)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (14a-c), NO-donor moiety is directly attached to the carboxylic acid group of aspirin, indomethacin or ibuprofen were synthesized. NO release from the dinitrooxypropyl, or diazen-1-ium-1,2-diolate, ester prodrugs was increased substantially upon incubation in the presence of l-cysteine (12a-c) or rat serum (14a-c). The ester prodrugs (12a-c, 14a-c), which did not inhibit the COX-1 isozyme, exhibited modest inhibitory activity against the COX-2 isozyme. The NONO-NSAIDs 12a-c and 14a-c exhibited in vivo AI activity that was similar to that exhibited by the parent drug aspirin, indomethacin or ibuprofen when the same oral dose (micromol/kg) was administered. These similarities in oral potency profiles suggest these NONO-NSAIDs act as classical prodrugs that require metabolic activation by esterase-mediated hydrolysis. Hybrid NO-donor/anti-inflammatory prodrugs of this type (NONO-NSAIDs) offer a potential drug design concept targeted toward the development of anti-inflammatory drugs with reduced adverse gastrointestinal effects.