Wound healing efficacy of a polymeric spray film solution containing Centella asiatica leaf extract on acute wounds.

OBJECTIVE: To study the clinical efficacy of a polymeric spray film containing Centella asiatica extract to heal acute wounds. METHOD: A polymeric spray film solution for wound healing was formulated using Centella asiatica extract, which contained triterpenes, including asiatic acid, madecassic acid, asiaticoside and madecassoside. The stability and physicochemical properties of the formulation were evaluated, and a multicentre, randomised, controlled trial was conducted to assess its clinical wound-healing efficacy. The Pressure Ulcer Scale for Healing (PUSH Tool) score was used to evaluate wound healing on days 0, 3, 5 and 7. RESULTS: The cohort consisted of 60 volunteers with clean-contaminated wounds (class 1), randomly assigned to the Control (n=30) and Testing (n=30) groups. The spray product contained asiatic acid, madecassic acid, asiaticoside and madecassoside at 0.20±0.02mg/ml, 0.16±0.01mg/ml, 0.32±0.03mg/ml and 0.10±0.00mg/ml, respectively. The pH value was 5.5±0.01, and the viscosity was 33±4cP. The product was stable for six months when stored at 30±2°C and at 40±2°C, in 75±5% relative humidity. The tested product significantly reduced the total PUSH and exudate scores, indicating that the polymeric spray film solution containing Centella asiatica improved wound healing. The average healing recovery times for the Testing and Control groups were 4.6±1.1 days and 4.87±1.0 days, respectively. CONCLUSION: In this study, Centella asiatica extract-containing polymeric spray film solution was beneficial as an acute wound medication, which could shorten healing time with no adverse effects.

Atmospheric and Room Temperature Plasma (ARTP) Mutagenesis Improved the Anti-MRSA Activity of Brevibacillus sp. SPR20.

Brevibacillus sp. SPR20 produced potentially antibacterial substances against methicillin-resistant Staphylococcus aureus (MRSA). The synthesis of these substances is controlled by their biosynthetic gene clusters. Several mutagenesis methods are used to overcome the restriction of gene regulations when genetic information is absent. Atmospheric and room temperature plasma (ARTP) is a powerful technique to initiate random mutagenesis for microbial strain improvement. This study utilized an argon-based ARTP to conduct the mutations on SPR20. The positive mutants of 40% occurred. The M27 mutant exhibited an increase in anti-MRSA activity when compared to the wild-type strain, with the MIC values of 250-500 and 500 µg/mL, respectively. M27 had genetic stability because it exhibited constant activity throughout fifteen generations. This mutant had similar morphology and antibiotic susceptibility to the wild type. Comparative proteomic analysis identified some specific proteins that were upregulated in M27. These proteins were involved in the metabolism of amino acids, cell structure and movement, and catalytic enzymes. These might result in the enhancement of the anti-MRSA activity of the ARTP-treated SPR20 mutant. This study supports the ARTP technology designed to increase the production of valuable antibacterial agents.

Silver Nanoparticles Conjugated with Colistin Enhanced the Antimicrobial Activity against Gram-Negative Bacteria.

Colistin is a potent peptide antibiotic that is effective against Gram-negative bacteria. However, nephrotoxicity limited its clinical use. Silver nanoparticles (AgNPs) have gained attention as a potential antimicrobial agent and nanodrug carrier. The conjugation of antibiotics and AgNPs has been found to increase the activity and decrease drug toxicity. In this study, colistin was conjugated with AgNPs (Col-AgNPs), which was confirmed by Fourier-transform infrared (FT-IR) and energy-dispersive X-ray (EDX) spectra. The optimized Col-AgNPs had the proper characteristics, including spherical shape, monodispersity, nanosized particle, high surface charge, and good stability. The powder X-ray diffraction (PXRD) pattern supported the crystallinity of Col-AgNPs and AgNPs. The drug loading of Col-AgNPs was 11.55 ± 0.93%. Col-AgNPs had higher activity against Gram-negative bacteria (Escherichia coli, Klebsiella pneumonia, and Pseudomonas aeruginosa) than AgNPs and colistin. The mechanism of actions of Col-AgNPs involved membrane disruption and genomic DNA damage. The Col-AgNPs and AgNPs were biocompatible with human red blood cells and renal cells at concentrations up to 16 µg/mL. Interestingly, Col-AgNPs exhibited higher cell survival than AgNPs and colistin at 32 µg/mL. Our results revealed that the Col-AgNPs could enhance the antimicrobial activity and cell biocompatibility more than colistin and AgNPs.

Purification and Characterization of Novel Anti-MRSA Peptides Produced by Brevibacillus sp. SPR-20.

Methicillin-resistant Staphylococcus aureus (MRSA) is listed as a high-priority pathogen because its infection is associated with a high mortality rate. It is urgent to search for new agents to treat such an infection. Our previous study isolated a soil bacterium (Brevibacillus sp. SPR-20), showing the highest antimicrobial activity against S. aureus TISTR 517 and MRSA strains. The present study aimed to purify and characterize anti-MRSA substances produced by SPR-20. The result showed that five active substances (P1-P5) were found, and they were identified by LC-MS/MS that provided the peptide sequences of 14-15 residues. Circular dichroism showed that all peptides contained ß-strand and disordered conformations as the major secondary structures. Only P1-P4 adopted more α-helix conformations when incubated with 50 mM SDS. These anti-MRSA peptides could inhibit S. aureus and MRSA in concentrations of 2-32 µg/mL. P1 (NH2-VVVNVLVKVLPPPVV-COOH) had the highest activity and was identified as a novel antimicrobial peptide (AMP). The stability study revealed that P1 was stable in response to temperature, proteolytic enzymes, surfactant, and pH. The electron micrograph showed that P1 induced bacterial membrane damage when treated at 1× MIC in the first hour of incubation. The killing kinetics of P1 was dependent on concentration and time. Mechanisms of P1 on tested pathogens involved membrane permeability, leakage of genetic material, and cell lysis. The P1 peptide at a concentration up to 32 µg/mL showed hemolysis of less than 10%, supporting its safety for human erythrocytes. This study provides promising anti-MRSA peptides that might be developed for effective antibiotics in the post-antibiotic era.

Enhanced dissolution of sildenafil citrate as dry foam tablets.

Dry foam formulation technology is alternative approach to enhance dissolution of the drug. Sildenafil citrate was suspended in sodium dodecyl sulfate solution and adding a mixture of maltodextrin and mannitol as diluent to form a paste. Sildenafil citrate paste was passed through a nozzle spray bottle to obtain smooth foam. The homogeneous foam was dried in a vacuum oven and sieved to obtain dry foam granules. The granules were mixed with croscarmellose sodium, magnesium stearate and compressed into tablet. All formulations were evaluated for their physicochemical properties and dissolution profiles. All the tested excipients were compatible with sildenafil citrate by both differential scanning calorimetry (DSC) and infrared (IR) analysis. There are no X-ray diffraction (XRD) peaks representing crystals of sildenafil citrate observed form dry foam formulations. The hardness of tablets was about 5 kg, friability test <1% with a disintegration time <5 min. The sildenafil citrate dry foam tablet had higher dissolution rate in 0.1 N HCl in comparison with commercial sildenafil citrate tablet, sildenafil citrate prepared by direct compression and wet granulation method. Sildenafil citrate dry foam tablet with the high-level composition of surfactant, water and diluent showed enhanced dissolution rate than that of the lower-level composition of these excipients. This formulation was stable under accelerated conditions for at least 6 months.

Unveiling a New Antimicrobial Peptide with Efficacy against P. aeruginosa and K. pneumoniae from Mangrove-Derived Paenibacillus thiaminolyticus NNS5-6 and Genomic Analysis.

This study focused on the discovery of the antimicrobial peptide (AMP) derived from mangrove bacteria. The most promising isolate, NNS5-6, showed the closest taxonomic relation to Paenibacillus thiaminolyticus, with the highest similarity of 74.9%. The AMP produced by Paenibacillus thiaminolyticus NNS5-6 exhibited antibacterial activity against various Gram-negative pathogens, especially Pseudomonas aeruginosa and Klebsiella pneumoniae. The peptide sequence consisted of 13 amino acids and was elucidated as Val-Lys-Gly-Asp-Gly-Gly-Pro-Gly-Thr-Val-Tyr-Thr-Met. The AMP mainly exhibited random coil and antiparallel beta-sheet structures. The stability study indicated that this AMP was tolerant of various conditions, including proteolytic enzymes, pH (1.2-14), surfactants, and temperatures up to 40 °C for 12 h. The AMP demonstrated 4 µg/mL of MIC and 4-8 µg/mL of MBC against both pathogens. Time-kill kinetics showed that the AMP acted in a time- and concentration-dependent manner. A cell permeability assay and scanning electron microscopy revealed that the AMP exerted the mode of action by disrupting bacterial membranes. Additionally, nineteen biosynthetic gene clusters of secondary metabolites were identified in the genome. NNS5-6 was susceptible to various commonly used antibiotics supporting the primary safety requirement. The findings of this research could pave the way for new therapeutic approaches in combating antibiotic-resistant pathogens.

In Vitro Evaluation of Colistin Conjugated with Chitosan-Capped Gold Nanoparticles as a Possible Formulation Applied in a Metered-Dose Inhaler.

Inhaled colistin is used to treat pneumonia and respiratory infections through nebulization or dry powder inhalers. Nevertheless, the development of a metered-dose inhaler (MDI) for colistin, which could enhance patient convenience and treatment efficacy, has not yet been developed. Colistin is known for its ability to induce cellular toxicity. Gold nanoparticles (AuNPs) can potentially mitigate colistin toxicity. Therefore, this study aimed to evaluate the antimicrobial effectiveness of colistin conjugated with chitosan-capped gold nanoparticles (Col-CS-AuNPs) and their potential formulation for use with MDIs to deliver the aerosol directly to the deep lung. Fourier-transform infrared spectroscopy, nuclear magnetic resonance, and elemental analysis were used to characterize the synthesized Col-CS-AuNPs. Drug release profiles fitted with the most suitable release kinetic model were evaluated. An MDI formulation containing 100 µg of colistin per puff was prepared. The aerosol properties used to determine the MDI performance included the fine particle fraction, mass median aerodynamic diameter, and geometric standard deviation, which were evaluated using the Andersen Cascade Impactor. The delivered dose uniformity was also determined. The antimicrobial efficacy of the Col-CS-AuNP formulation in the MDI was assessed. The chitosan-capped gold nanoparticles (CS-AuNPs) and Col-CS-AuNPs had particle sizes of 44.34 ± 1.02 and 174.50 ± 4.46 nm, respectively. CS-AuNPs effectively entrapped 76.4% of colistin. Col-CS-AuNPs exhibited an initial burst release of up to 60% colistin within the first 6 h. The release mechanism was accurately described by the Korsmeyer-Peppas model, with an R2 > 0.95. The aerosol properties of the Col-CS-AuNP formulation in the MDI revealed a high fine particle fraction of 61.08%, mass median aerodynamic diameter of 2.34 µm, and geometric standard deviation of 0.21, with a delivered dose uniformity within 75-125% of the labeled claim. The Col-CS-AuNP MDI formulation completely killed Escherichia coli at 5× and 10× minimum inhibitory concentrations after 6 and 12 h of incubation, respectively. The toxicity of CS-AuNP and Col-CS-AuNP MDI formulations in upper and lower respiratory tract cell lines was lower than that of free colistin. The stability of the Col-CS-AuNP MDI formulation was maintained for at least 3 months. The Col-CS-AuNP MDI formulation effectively eradicated bacteria over a 12-h period, showing promise for advancing lung infection treatments.

A Novel Bacitracin-like Peptide from Mangrove-Isolated Bacillus paralicheniformis NNS4-3 against MRSA and Its Genomic Insights.

The global rise of antimicrobial resistance (AMR) presents a critical challenge necessitating the discovery of novel antimicrobial agents. Mangrove microbes are valuable sources of new antimicrobial compounds. This study reports the discovery of a potent antimicrobial peptide (AMP) from Bacillus paralicheniformis NNS4-3, isolated from mangrove sediment, exhibiting significant activity against methicillin-resistant Staphylococcus aureus (MRSA). The AMP demonstrated a minimum inhibitory concentration ranging from 1 to 16 µg/mL in the tested bacteria and exhibited bactericidal effects at higher concentrations. Structural analysis revealed a bacitracin-like configuration and the peptide acted by disrupting bacterial membranes in a time- and concentration-dependent manner. The AMP maintained stability under heat, proteolytic enzymes, surfactants, and varying pH treatments. The ten biosynthetic gene clusters (BGCs) of secondary metabolites were found in the genome. Detailed sequence comparison of the predicted bacitracin BGC indicated distinct DNA sequences compared to previously reported strains. Although the antibiotic resistance genes were found, this strain was susceptible to antibiotics. Our findings demonstrated the potential of Bacillus paralicheniformis NNS4-3 and its AMP as a promising agent in combating AMR. The genetic information could be pivotal for future applications in the healthcare industry, emphasizing the need for continued exploration of marine microbial diversity in drug discovery.

BrSPR-20-P1 peptide isolated from Brevibacillus sp. developed into liposomal hydrogel as a potential topical antimicrobial agent.

A novel BrSPR-20-P1 antimicrobial peptide (P1-AMP; NH2-VVVNVLVKVLPPPVV-COOH) isolated from Brevibacillus sp. SPR-20 was encapsulated in a liposome containing varying proportions of l-α-phosphatidylcholine (PC) and cholesterol (CH). P1-AMP liposomes were incorporated into a chitosan hydrogel to achieve a peptide concentration of 0.02%. P1-AMP has been tested for its antibacterial and in vitro wound healing activities. The physicochemical characteristics of liposomes and hydrogel were investigated, including in vitro drug release, permeability, cell toxicity, antimicrobial activities, and stability studies. P1-AMP showed higher antimicrobial and wound-healing activities than the negative control. A toxicity test of P1-AMP in keratinocyte cell lines revealed cell viability of 100% at a concentration range of 1.96-1000 µg mL-1. The empty liposomes exhibited an average particle size ranging from 324.5 ± 8.6 to 1823.7 ± 288.2 nm. The size range of P1-AMP liposomes was 378.6 ± 14.0 to 2363.0 ± 255.6 nm. The zeta potential of the blank liposome ranged from -40.43 ± 2.51 to -60.17 ± 0.93 mV and it decreased to -57.33 ± 0.72 to -70.33 ± 0.15 mV of the liposome loaded with peptide. SEM images showed liposomes were ovoid spheres with smooth surfaces. The chosen formulation, composed of PC to CH in an 18 : 1 ratio (formulation F3), had the highest entrapment effectiveness with small particle size and possessed an acceptable zeta potential. The developed P1-AMP liposome-loaded hydrogels exhibited a yellowish-clear appearance with a viscosity of 758.0 ± 149.8 cPs. The P1-AMP was rapidly released from the P1-AMP-loaded liposome hydrogel formulation. The P1-AMP-loaded liposome showed high permeability compared to P1-AMP alone or P1-AMP in hydrogel without the incorporation of liposomes. The minimum inhibitory concentration (MIC) against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) of P1-AMP-loaded liposome hydrogel was 2 µg mL-1, equivalent to P1-AMP. It completely killed S. aureus at 10× and 5× MIC after 6 and 12 h of incubation, respectively. The formulation did not induce cytotoxicity to the tested keratinocyte cell and remained stable for at least 6 months under the studied conditions.

Anti-methicillin-resistant Staphylococcus aureus and antibiofilm activity of new peptides produced by a Brevibacillus strain.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is listed as a highly prioritized pathogen by the World Health Organization (WHO) to search for effective antimicrobial agents. Previously, we isolated a soil Brevibacillus sp. strain SPR19 from a botanical garden, which showed anti-MRSA activity. However, the active substances were still unknown. Methods: The cell-free supernatant of this bacterium was subjected to salt precipitation, cation exchange, and reversed-phase chromatography. The antimicrobial activity of pure substances was determined by broth microdilution assay. The peptide sequences and secondary structures were characterized by tandem mass spectroscopy and circular dichroism (CD), respectively. The most active anti-MRSA peptide underwent a stability study, and its mechanism was determined through scanning electron microscopy, cell permeability assay, time-killing kinetics, and biofilm inhibition and eradication. Hemolysis was used to evaluate the peptide toxicity. Results: The pure substances (BrSPR19-P1 to BrSPR19-P5) were identified as new peptides. Their minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) against S. aureus and MRSA isolates ranged from 2.00 to 32.00 and 2.00 to 64.00 µg/mL, respectively. The sequence analysis of anti-MRSA peptides revealed a length ranging from 12 to 16 residues accompanied by an amphipathic structure. The physicochemical properties of peptides were predicted such as pI (4.25 to 10.18), net charge at pH 7.4 (-3 to +4), and hydrophobicity (0.12 to 0.96). The CD spectra revealed that all peptides in the water mainly contained random coil structures. The increased proportion of α-helix structure was observed in P2-P5 when incubated with SDS. P2 (NH2-MFLVVKVLKYVV-COOH) showed the highest antimicrobial activity and high stability under stressed conditions such as temperatures up to 100 °C, solution of pH 3 to 10, and proteolytic enzymes. P2 disrupted the cell membrane and caused bacteriolysis, in which its action was dependent on the incubation time and peptide concentration. Antibiofilm activity of P2 was determined by which the half-maximal inhibition of biofilm formation was observed at 2.92 and 4.84 µg/mL for S. aureus TISTR 517 and MRSA isolate 2468, respectively. Biofilm eradication of tested pathogens was found at the P2 concentration of 128 µg/mL. Furthermore, P2 hemolytic activity was less than 10% at concentrations up to 64 µg/mL, which reflected the hemolysis index thresholds of 32. Conclusion: Five novel anti-MRSA peptides were identified from SPR19. P2 was the most active peptide and was demonstrated to cause membrane disruption and cell lysis. The P2 activity was dependent on the peptide concentration and exposure time. This peptide had antibiofilm activity against tested pathogens and was compatible with human erythrocytes, supporting its potential use as an anti-MRSA agent in this post-antibiotic era.

Identification and Characterization of a Potential Antimicrobial Peptide Isolated from Soil Brevibacillus sp. WUL10 and Its Activity against MRSA Pathogens.

Methicillin-resistant Staphylococcus aureus (MRSA) is a severe threat to public health globally. The development of novel agents has encountered the repeated mechanism of drug resistance. This study aimed to investigate an anti-MRSA substance isolated from a promising soil bacterium. The result showed that an isolate (WUL10) was in the Brevibacillus genus. The minimum inhibitory concentration (MIC) of the purified substance was 1 µg/mL against S. aureus TISTR 517 and MRSA strains. This substance showed the bactericidal effect at the concentration of 1-2 µg/mL against these bacterial indicators. The activity of the substance retained more than 95% when encountering high temperatures and a wide range of pH, but it was sensitive to proteolytic enzymes and SDS. It was identified as a novel antimicrobial peptide (KVLVKYLGGLLKLAALMV-COOH) with the predicted structure of α-helix. The substance could rupture the cell wall of the tested pathogen. MIC and MBC of the synthesized peptide were 16 and 64 µg/mL, respectively. The difference in the activity between the isolated and synthetic peptides might be from the synergistic effects of other AMPs in the purified substance. This novel AMP would provide an advantage for further development of anti-MRSA substances to manage the situation of antibiotic resistance.

The RING heterodimer BRCA1-BARD1 is a ubiquitin ligase inactivated by the platinum-based anticancer drugs.

The breast cancer susceptibility protein 1 (BRCA1) participates in the maintenance of cells genomic integrity through DNA repair, cell cycle checkpoint, protein ubiquitination, and transcriptional regulation. The N-terminus of BRCA1 contains a RING domain that preferentially forms a heterodimeric complex with BARD1. The BRCA1-BARD1 RING complex has an E3 ubiquitin ligase activity that plays an essential role in response to DNA damage. Preclinical and clinical studies have recently revealed that structural changes to the heterodimer result in alterations to the BRCA1-mediated DNA repair pathways in cancer cells, and lead to hypersensitivity to several chemotherapeutic agents. It is of interest to approach the BRCA1 RING domain as a potentially molecular target for platinum-based drugs for cancer therapy. A previous study has shown that the anticancer drug cisplatin formed intramolecular and intermolecular BRCA1 adducts in which His117 was the primary platinum-binding site, and conferred conformational changes and induced thermostability. Here, we have studied the functional consequence of the in vitro platination of the BRCA1 RING domain by a number of platinum complexes. The BRCA1 ubiquitin ligase activity was inhibited by transplatin > cisplatin > oxaliplatin > carboplatin in that order. The consequences of the binding of the platinum complexes on the reactivity of the BRCA1 were also discussed. The data raised the possibility of selectively targeting the BRCA1 DNA repair for cancer therapy.

Substitution of aspartic acid with glutamic acid at position 67 of the BRCA1 RING domain retains ubiquitin ligase activity and zinc(II) binding with a reduced transition temperature.

Breast cancer susceptibility protein 1 (BRCA1) participates in genomic integrity maintenance through DNA repair, cell cycle checkpoint, protein ubiquitination, and transcriptional regulation. The N-terminus of BRCA1 contains a RING domain which forms two Zn(2+) binding sites in an interleaved fashion. A number of deleterious BRCA1 missense mutations, which predispose an individual to a subset of hereditary breast and ovarian cancers, have been identified in the RING domain. Disruption of Zn(2+) binding sites and protein structure results in the inactivation of BRCA1 tumor suppression function. An unprecedented D67E BRCA1 mutation, identified in Thai familial breast cancer patients, is located in the vicinity of Zn(2+) binding site II, and its pathogenic significance remains elusive. The present study revealed that the D67E BRCA1 RING protein assumes a preformed structure in the absence of Zn(2+). The Zn(2+)-bound mutant protein was more folded, resulting in enhanced proteolytic resistance and dimerization. This indicated that the mutation retained Zn(2+) binding, and barely perturbed the native global structure of the BRCA1 RING domain. The complex between D67E BRCA1 and BARD1 RING domains exhibited a substantial ubiquitin ligase activity compared with a defective complex containing the C61G BRCA1 mutation. However, the D67E mutation was slightly less stable toward thermal denaturation. This implies that the D67E mutation might be a neutral or mild cancer-risk modifier of other defective mechanisms underlying BRCA1-mutation-related breast cancer.

Preparation and physicochemical characterization of sildenafil cocrystals.

Sildenafil is a specific inhibitor of the phosphodiesterase type 5 (PDE-5) enzyme that protects cyclic guanosine monophosphate from breakdown by PDE-5. It is a biopharmaceutical categorization system Class II medication with low bioavailability because it is almost insoluble in water. The objectives of this study were to prepare sildenafil cocrystals with co-former molecules including aspirin (acetylsalicylic acid [ASA]), fumaric acid (FMA), and benzoic acid (BZA) to improve the water solubility of sildenafil. The cocrystals were prepared by antisolvent addition (AA) and slow solvent evaporation (SE) methods. The stoichiometric ratios of sildenafil and co-former molecules were varied. The obtained crystals were characterized by stereomicroscope, Fourier transformed infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR), and powder X-ray diffraction (PXRD). The water solubility of sildenafil cocrystals was compared with sildenafil base. In the AA method, the crystals only form in sildenafil-ASA reaction. These crystals were not cocrystals between sildenafil and ASA because they were formed to new substances that were confirmed by single-crystal X-ray diffraction. In the SE method, the cocrystals were successfully prepared in the reaction of sildenafil with ASA, FMA, and BZA which use acetone or ethyl acetate as a solvent. The obtained crystals are irregular shapes and their FT-IR, NMR, and PXRD results exhibited the characteristics of sildenafil and its co-former. The stoichiometric ratios of sildenafil and co-formers after cocrystallization were different from an initial of crystallization. The sildenafil cocrystals with ASA, FMA, and BZA by SE method had higher water solubility than sildenafil base. The sildenafil-FMA cocrystals had the highest water solubility and increased up to five times when compared with sildenafil base.

Cisplatin affects the conformation of apo form, not holo form, of BRCA1 RING finger domain and confers thermal stability.

The breast cancer suppressor protein 1 (BRCA1) has been shown to participate in genomic integrity maintenance. Preclinical and clinical studies have recently revealed that the inactivation of BRCA1 in cancer cells leads to chemosensitivity. Approaching the BRCA1 RING protein as a potentially molecular target for a platinum-based drug might be of interest in cancer therapy. In the present study, the in vitro platination of the BRCA1 RING protein by the anticancer drug cisplatin was observed. The protein contained a preformed structure in the apo form with structural changes and resistance to limited proteolysis after Zn2+ binding. SDS-PAGE and mass-spectrometric analyses revealed that cisplatin preferentially formed monofunctional and bifunctional BRCA1 adducts. Tandem mass spectrometry (MS/MS) of the 656.29(2+) ion indicated that the ion arose from [Pt(NH3)2(OH)]+ bound to the BRCA1 peptide (111)ENNSPEHLK(119). The product-ion spectrum revealed the Pt-binding site on His117. Circular dichroism showed that the apo form, not holo form, of BRCA1 underwent more folded structural rearrangement upon cisplatin binding. Cisplatin-bound protein exhibited an enhanced thermostability by 13 degrees , resulting from the favorably intermolecular cross-links driven by the free energy. Our findings demonstrated the first conformational and thermal evidences for a direct binding of cisplatin to the BRCA1 RING domain and could raise a possibility of selectively targeted treatment of cancer with less toxicity or improved response to conventional regimens.

Development of a Sildenafil Citrate Microemulsion-Loaded Hydrogel as a Potential System for Drug Delivery to the Penis and Its Cellular Metabolic Mechanism.

Sildenafil citrate is used to treat mild to moderate erectile dysfunction and premature ejaculation. However, it has low oral bioavailability, numerous adverse effects, and delayed onset of action. These problems may be resolved by transdermal delivery to the penis. Hence, sildenafil citrate was formulated as a microemulsion system using isopropyl myristate, Tween 80, PEG400, and water (30:20:40:10). The hydrogel used in the microemulsion was 2% w/w poloxamer 188. The sildenafil microemulsion-loaded hydrogels were characterised for their appearance, particle size, pH, spreadability, swelling index, viscosity, sildenafil drug content, membrane permeation, epithelial cell cytotoxicity, and in vitro drug metabolism. The optimised formulated microemulsion showed the lowest droplet size and highest solubility of sildenafil citrate. The in vitro skin permeation of the sildenafil citrate microemulsion-loaded hydrogel was significantly higher than that of the sildenafil suspension, with a 1.97-fold enhancement ratio. The formulated microemulsion exhibited a 100% cell viability, indicating its safety for skin epithelial cells. The major metabolic pathway of sildenafil citrate loaded in the microemulsion formulation was hydroxylation. Furthermore, loading sildenafil in the microemulsion reduced the drug metabolite by approximately 50% compared to the sildenafil in aqueous suspension. The sildenafil citrate-loaded isopropyl myristate-based microemulsion hydrogels were physically and chemically stable over 6 months of storage. The sildenafil citrate microemulsion-loaded hydrogel showed in vitro results suitable for used as a transdermal drug delivery system.

Formulation Development of Albendazole-Loaded Self-Microemulsifying Chewable Tablets to Enhance Dissolution and Bioavailability.

Albendazole is an anthelmintic agent with poor solubility and absorption. We developed a chewable tablet (200 mg drug equivalent), containing a self-microemulsifying drug delivery system (SMEDDS), with oral disintegrating properties. The emulsion was developed using sesame and soybean oils along with surfactant/co-surfactants, and the tablets were prepared by wet granulation using superdisintegrants and adsorbents. Infra-red (IR) spectral studies revealed no interaction between the drug and excipients, and all physical and chemical parameters were within acceptable limits. Stability studies for the formulation indicated no significant change over time. An in vitro release study indicated 100% drug release within 30 min, and in vivo plasma concentrations indicated that the area under the curve (AUC) of albendazole in rats administered SMEDDS chewable tablets was significantly higher than in those administered commercial tablets or powder (p-value < 0.05). The systemic bioavailability of albendazole achieved through the SMEDDS tablets was 1.3 times higher than that achieved by the administration of comparable quantities of albendazole commercial tablets. This was due to the higher dissolution of albendazole SMEDDS in the chewable tablets. We conclude that the SMEDDS chewable formulation can be used to improve the dissolution and systemic availability of poorly water-soluble drugs.

Physicochemical and Microbiological Stability of the Extemporaneous Sildenafil Citrate Oral Suspension.

Sildenafil is a potent and selective phosphodiesterase-5 inhibitor that is effectively used in the treatment of pulmonary arterial hypertension. In several countries, hospital pharmacists prepare the drug in an extemporaneous liquid preparation as there are no liquid formulations available for pediatric and adult uses. The purpose of this study was to evaluate the stability of an extemporaneous sildenafil citrate oral suspension for 90 days, according to the ASEAN guideline on stability studies of drug products. The results showed that the preparation was a white suspension with a sweet taste. It was a viscous and weakly acidic mixture with pseudoplastic behavior. The drug content was in the range between 99.23% and 102.23%, and the microbial examination met the general requirements throughout the study period. Therefore, the extemporaneously compounded sildenafil suspensions were physically, chemically, and microbiologically stable for at least 90 days when stored at 30° and 40°C. Furthermore, the in-use stability study showed that the preparations had acceptable attributes at least 14 days after the first-time use. This might provide an alternative option when the commercial suspension is unavailable.

In Vitro Enhanced Sensitivity to Cisplatin in D67Y BRCA1 RING Domain Protein.

BRCA1 is a tumor suppressor protein involved in maintaining genomic integrity through multiple functions in DNA damage repair, transcriptional regulation, cell cycle checkpoint, and protein ubiquitination. The BRCA1-BARD1 RING complex has an E3 ubiquitin ligase function that plays essential roles in response to DNA damage repair. BRCA1-associated cancers have been shown to confer a hypersensitivity to chemotherapeutic agents. Here, we have studied the functional consequence of the in vitro E3 ubiquitin ligase activity and cisplatin sensitivity of the missense mutation D67Y BRCA1 RING domain. The D67Y BRCA1 RING domain protein exhibited the reduced ubiquitination function, and was more susceptible to the drug than the D67E or wild-type BRCA1 RING domain protein. This evidence emphasized the potential of using the BRCA1 dysfunction as an important determinant of chemotherapy responses in breast cancer.