RESUMEN
BACKGROUND: Nowadays, recombinant therapeutic proteins have been widely produced and consumed. For the safety and effectiveness of the protein production, an auto-inducible expression vector is required to replace inducer interference, which is uneconomic and could be harmful. In this research, an auto-inducible expression plasmid, pCAD2_sod (a pBR322 derivate plasmid), which was under dps (RpoS-dependent gene) promoter control, was modified to provide RpoS at earlier phase. Hence, accumulates more target protein and resulting a new plasmid, pCAD2+_sod. pCAD2_sod had been constructed to automatically induces the expression of recombinant superoxide dismutase (SOD) from Staphylococcus equorum (rMnSODSeq) in the stationary growth phase of Escherichia coli. This work aimed to obtain pCAD2+_sod and determine the expression level of rMnSODSeq on mRNA and protein level. METHOD AND RESULTS: A synthetic rpoS coding region under rpoD promoter control (prpoD_rpoS) was inserted to pCAD2_sod and generated pCAD2+_sod. The rMnSODSeq (24.3 kDa) produced from pCAD2+_sod was ~ 1.5 fold higher at 37 °C and more intense at 43 °C compared to that from pCAD2_sod, likewise shifted to earlier phase (after 1 h of incubation), as shown in the SDS-PAGE. The dismutase activity was also retained after zymography assay. The mRNA level from pCAD2+_sod was determined by qPCR and gave quantification cycle (Cq) values of cDNA lowest among others. It made the relative quantification (RQ) of the mRNA expression towards rho reference gene were high. CONCLUSIONS: The prpoD_rpoS insertion shifts and increases the rMnSODSeq production from stationary to exponential phase. The pCAD2+_sod plasmid is potential for further recombinant protein productions.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/genética , Ingeniería de Proteínas/métodos , Proteínas Bacterianas/genética , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Expresión Génica/genética , Vectores Genéticos/genética , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes/genética , Factor sigma/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
Low physical stability is the limitation of the widespread use of amorphous drugs. The co-amorphous drug system is a new and emerging method for preparing a stable amorphous form. Co-amorphous is a single-phase amorphous multicomponent system consisting of two or more small molecules that are a combination of drugs or drugs and excipients. The co-amorphous system that uses benzoic acid (BA) as an excipient was studied to improve the physical stability, dissolution, and solubility of desloratadine (DES). In this study, the co-amorphous formation of DES and BA (DESâ»BA) was prepared by melt-quenching method and characterized by differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), powder X-ray diffraction (PXRD), and polarized light microscopy (PLM). Dissolution, solubility, and physical stability profiles of DESâ»BA were determined. The DES crystals were converted into DESâ»BA co-amorphous form to reveal the molecular interactions between DES and BA. Solid-state analysis proved that the co-amorphous DESâ»BA system (1:1) is amorphous and homogeneous. The DSC experiment showed that the glass transition temperature (Tg) of tested DESâ»BA co-amorphous had a higher single Tg compared to the amorphous DES. FTIR revealed strong interactions, especially salt formation. The dissolution rate and solubility of co-amorphous DESâ»BA (1:1) obtained were larger than the DES in crystalline form. The PXRD technique was used to assess physical stability for three months at 40 °C with 75% RH. The DESâ»BA co-amorphous system demonstrated better physical stability than a single form of amorphous DES. Co-amorphous DESâ»BA has demonstrated the potential for improving solid-state stability, as the formation of DESâ»BA co-amorphous salt increased solubility and dissolution when compared to pure crystalline DES. This study also demonstrated the possibility for developing a DESâ»BA co-amorphous system toward oral formulations to improve DES solubility and bioavailability.
RESUMEN
This study describes the formation of multicomponent crystal (MCC) of desloratadine (DES). The objective of this study was to discover the new pharmaceutical MCC of DES using several coformers. The MCC synthesis was performed between DES and 26 coformers using an equimolar ratio with a solvent evaporation technique. The selection of the appropriate solvent was carried out using 12 solvents. The preview of the MCC of DES was performed using polarized light microscopy (PLM). The formation of MCC was confirmed using powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). The accelerated stability of MCC at 40 °C and relative humidity of 75% was investigated using PXRD and FTIR. Depending on the prior evaluation, DES and benzoic acid (BA) formed the MCC. PLM and SEM results showed that crystal habit of combination between DES and BA differed from the constituent components. Moreover, the diffractogram pattern of DES-BA was distinct from the constituent components. The DSC thermogram showed a new peak which was distinct from both constituent components. The FTIR study proved a new spectrum. All characterizations indicated that a new solid crystal was formed, ensuring the MCC formation. In addition, DES-BA MCC had both chemical and physical stabilities for a period of 4 months.
RESUMEN
We report the first multicomponent crystal of desloratadine, an important anti-histamine drug, with a pharmaceutically acceptable coformer of benzoic acid. The single crystal structure analysis revealed that this novel multicomponent crystal is categorized as salt due to the proton transfer from benzoic acid to the desloratadine molecule. By forming the salt multicomponent crystal, we demonstrated that the tabletability and plasticity of the multicomponent crystal was improved from the parent drug. In addition, neither capping nor lamination tendency was observed in the desloratadine-benzoic acid multicomponent crystal. The existence of a layered structure and slip planes are proposed to be associated with this improvement. The desloratadine-benzoate in this case shows an improved solubility in water and HCl 0.1N media and a better dissolution profile in water. However, the dissolution rate in HCl 0.1N media was found to be essentially indifference.
Asunto(s)
Ácido Benzoico/química , Antagonistas de los Receptores Histamínicos H1 no Sedantes/química , Loratadina/análogos & derivados , Modelos Químicos , Cristalización , Composición de Medicamentos , Elasticidad , Enlace de Hidrógeno , Loratadina/química , Estructura Molecular , Solubilidad , Comprimidos , Resistencia a la TracciónRESUMEN
We report novel pharmaceutical cocrystal of a popular antipyretic drug paracetamol (PCA) with coformer 5-nitroisophhthalic acid (5NIP) to improve its tabletability. The cocrystal (PCA-5NIP at molar ratio of 1:1) was synthesized by solvent evaporation technique using methanol as solvent. The physicochemical properties of cocrystal were characterized by powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), thermogravimetry analysis (TGA), fourier transform infrared spectroscopy (FTIR), hot stage polarized microscopy (HSPM) and scanning electron microscopy (SEM). Stability of the cocrystal was assessed by storing them at 40°C/75% RH for one month. Compared to PCA, the cocrystal displayed superior tableting performance. PCA-5NIP cocrystal showed a similar dissolution profile as compared to PCA and exhibited good stability. This study showed the utility of PCA-5NIP cocrystal for improving mechanical properties of PCA.