Impact of Leucine and Magnesium Stearate on the Physicochemical Properties and Aerosolization Behavior of Wet Milled Inhalable Ibuprofen Microparticles for Developing Dry Powder Inhaler Formulation.

This study investigated the development and characterization of leucine and magnesium stearate (MgSt) embedded wet milled inhalable ibuprofen (IBF) dry powder inhaler (DPI) formulations. IBF microparticles were prepared by a wet milling homogenization process and were characterized by SEM, FTIR, DSC, XRD and TGA. Using a Twin-Stage Impinger (TSI), the in vitro aerosolization of the formulations with and without carrier lactose was studied at a flow rate of 60± 5 L/min and the IBF was determined using a validated HPLC method. The flow properties were determined by the Carr's Index (CI), Hausner Ratio (HR) and Angle of Repose (AR) of the milled IBF with 4-6.25% leucine and leucine containing formulations showed higher flow property than those of formulations without leucine. The fine particle fraction (FPF) of IBF from the prepared formulations was significantly (p = 0.000278) higher (37.1 ± 3.8%) compared to the original drug (FPF 3.7 ± 0.9%) owing to the presence of leucine, which enhanced the aerosolization of the milled IBF particles. Using quantitative phase analysis, the XPRD data revealed the crystallinity and accurate weight percentages of the milled IBF in the formulations. FTIR revealed no changes of the structural integrity of the milled IBF in presence of leucine or MgSt. The presence of 2.5% MgSt in the selected formulations produced the highest solubility (252.8 ± 0.6 µg/mL) of IBF compared to that of unmilled IBF (147.4 ± 1.6 µg/mL). The drug dissolution from all formulations containing 4-6.25% leucine showed 12.2-18.6% drug release in 2.5 min; however, 100% IBF dissolution occurred in 2 h whereas around 50% original and dry milled IBF dissolved in 2 h. The results indicated the successful preparation of inhalable IBF microparticles by the wet milling method and the developed DPI formulations with enhanced aerosolization and solubility due to the presence of leucine may be considered as future IBF formulations for inhalation.

Robust Wet Milling Technique for Producing Micronized Ibuprofen Particles with Improved Solubility and Dissolution.

This study investigated a systematic approach for producing ibuprofen (IBF) particles with leucine by wet milling. Using a high shear homogenizer, the particles size of the IBF was reduced. Prepared IBF microparticles were freeze-dried and characterized by using Mastersizer, SEM, DSC, XRD, ATR-FTIR, and TGA. The drug saturation solubility and in-vitro dissolution performance were carried out in phosphate buffer solution (PBS, pH 7.4) at 37°C temperature and IBF were determined using a validated HPLC method. The wet-milled method reduced the particle size from 71.3 to 1.7 µm. The minimum particle size of IBF was obtained in 0.05% Tween 80 solution homogenized at 17,000 rpm for 15 min. The saturated solubility (168.7 µg/mL) of the micronized IBF particles with leucine showed higher compared to that of the original IBF (147.4 µg/mL) in PBS solution. The prepared IBF particles containing 2.5-6.25% leucine showed significantly higher IBF release (100%) compared to that of original drug particles (55.9%) in 120 min. The excipient leucine played a major role in enhancing the solubility and dissolution profile of the prepared IBF particles probably by the formation of hydrogen bonding. The developed wet milling was an efficient and robust technique for reducing the particle size of IBF and could be a useful method for manufacturing drug particles with enhanced solubility and dissolution.

Rapid Assay for the Therapeutic Drug Monitoring of Edoxaban.

Edoxaban is a direct oral anticoagulant (DOAC) that has been recently indicated for the treatment of pulmonary embolism (PE) in SARS-CoV-2 infections. Due to its pharmacokinetic variability and a narrow therapeutic index, the safe administration of the drug requires its therapeutic drug monitoring (TDM) in patients receiving the treatment. In this work, we present a label-free method for the TDM of edoxaban by surface enhanced Raman spectroscopy (SERS). The new method utilises the thiol chemistry of the drug to chemisorb its molecules onto a highly sensitive SERS substrate. This leads to the formation of efficient hotspots and a strong signal enhancement of the drug Raman bands, thus negating the need for a Raman reporter for its SERS quantification. The standard samples were run with a concentration range of 1.4 × 10-4 M to 10-12 M using a mobile phase comprising of methanol/acetonitrile (85:15 v/v) at 291 nm followed by the good linearity of R2 = 0.997. The lowest limit of quantification (LOQ) by the SERS method was experimentally determined to be 10-12 M, whereas LOQ for HPLC-UV was 4.5 × 10-7 M, respectively. The new method was used directly and in a simple HPLC-SERS assembly to detect the drug in aqueous solutions and in spiked human blood plasma down to 1 pM. Therefore, the SERS method has strong potential for the rapid screening of the drug at pathology labs and points of care.

Synthesis, carbonic anhydrase enzyme inhibition evaluations, and anticancer studies of sulfonamide based thiadiazole derivatives.

The sulfonamide-based thiadiazole derivatives (STDs) with different hydrophobic/hydrophilic substitutions were synthesized to investigate their potentials in carbonic anhydrase inhibition (CAI). The CAI activity of the STDs (4a-4h) and the mechanism of the inhibition kinetics were determined. STD 4f contained both methoxy and Cl groups at benzene ring in STD 4f showed the lowest IC50 value. The molecular docking study confirmed that STDs bind strongly with the active sites of the target protein PDBID 1V9E. With the help of Lineweaver-Burk plots, inhibition kinetics of PDBIR 1V9E protein with STDs were determined. Cytotoxicity was checked against human keratinocyte cell lines and the anticancer properties were determined against MCF-7 cell lines. The electrochemical method was used to investigate the binding study with DNA and CA enzymes. Anticancer studies showed that STDs have weak bonding ability to DNA and strong binding ability with CA. It is concluded that anticancer activity is through CAI rather than by DNA binding.

Inhaled Edoxaban dry powder inhaler formulations: Development, characterization and their effects on the coagulopathy associated with COVID-19 infection.

Herein, we demonstrated the development and characterization of a dry powder inhaler (DPI) formulation of edoxaban (EDX); and investigated the in-vitro anticoagulation effect for the management of pulmonary or cerebral coagulopathy associated with COVID-19 infection. The formulations were prepared by mixing the inhalable micronized drug with a large carrier lactose and dispersibility enhancers, leucine, and magnesium stearate. The drug-excipient interaction was studied using X-Ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) methods. The drug and excipients showed no physical inter particulate interaction. The in-vitro drug aerosolization from the developed formulation was determined by a Twin Stage Impinger (TSI) at a flow rate of 60 ± 5 L /min. The amount of drug deposition was quantified by an established HPLC-UV method. The fine particle fraction (FPF) of EDX API from drug alone formulation was 7%, whereas the formulations with excipients increased dramatically to almost 7-folds up to 47%. The developed DPI formulation of EDX showed a promising in-vitro anticoagulation effect at a very low concentration. This novel DPI formulation of EDX could be a potential and effective inhalation therapy for managing pulmonary venous thromboembolism (VTE) associated with COVID-19 infection. Further studies are warranted to investigate the toxicity and clinical application of the inhaled EDX DPI formulation.

Puerarin dry powder inhaler formulations for pulmonary delivery: Development and characterization.

This study aims at developing and characterizing the puerarin dry powder inhaler (DPI) formulations for pulmonary delivery. The inhalable particles size (<2 µm) was accomplished by micronization and its morphology was examined by scanning electron microscopy (SEM). The puerarin-excipient interaction in powder mixtures was analyzed by using Fourier transform infrared spectroscopy (FTIR), Raman confocal microscopy, X-Ray powder Diffraction (XRD), and differential scanning calorimetry (DSC) methods. Using a Twin stage impinger (TSI), the in-vitro aerosolization of the powder formulations was carried out at a flow rate of 60 L/min and the drug was quantified by employing a validated HPLC method. No significant interactions between the drug and the excipients were observed in the powder formulations. The fine particle fraction (FPF) of the drug alone was 4.2% which has increased five to six-fold for the formulations with aerosolization enhancers. Formulation containing lactose as large carriers produced 32.7% FPF, which further increased with the addition of dispersibility enhancers, leucine and magnesium stearate (40.8% and 41.2%, respectively). The Raman and FTIR techniques are very useful tool for understanding structural integrity and stability of the puerarin in the powder formulations. The puerarin was found to be compatible with the excipients used and the developed DPI formulation may be considered as an efficient formulation for pulmonary delivery for the management of various diseases at a very low dose.

Gold-Deposited Nickel Foam as Recyclable Plasmonic Sensor for Therapeutic Drug Monitoring in Blood by Surface-Enhanced Raman Spectroscopy.

A sensitive and recyclable plasmonic nickel foam sensor has been developed for surface-enhanced Raman spectroscopy (SERS). A simple electrochemical method was used to deposit flower-shaped gold nanostructures onto nickel foam substrate. The high packing of the gold nanoflowers onto the nickel foam led to a high enhancement factor (EF) of 1.6 × 1011. The new SERS sensor was utilized for the direct determination of the broad-spectrum ß-lactam carbapenem antibiotic meropenem in human blood plasma down to one pM. The sensor was also used in High Performance Liquid Chromatography (HPLC)-SERS assembly to provide fingerprint identification of meropenem in human blood plasma. Moreover, the SERS measurements were reproducible in aqueous solution and human blood plasma (RSD = 5.5%) and (RSD = 2.86%), respectively at 200 µg/mL (n = 3), and successfully recycled using a simple method, and hence, used for the repeated determination of the drug by SERS. Therefore, the new sensor has a strong potential to be applied for the therapeutic drug monitoring of meropenem at points of care and intensive care units.

Development and characterization of meropenem dry powder inhaler formulation for pulmonary drug delivery.

Meropenem (MPN), a broad spectrum ß-lactam antibiotic, has been increasingly used in the treatment of moderate to severe bacterial infections. However, due to its short plasma half-life and chemical instability in solution form, it has been challenging to use in the intravenous formulation. This study aims to develop and characterize MPN dry powder inhaler (DPI) formulation for pulmonary delivery. The inhalable MPN particles (1-5 µm) were prepared by micronization. Lactose, L-leucine and magnesium stearate (MgSt) were used in the powder formulation as carriers and dispersibility enhancers. The formulations were characterized by Scanning electron microscopy (SEM), Transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), Raman confocal microscopy, X-Ray powder diffraction analysis (PXRD), and differential scanning calorimetry (DSC) methods. The concentration of MPN was determined by using a validated HPLC method. The Fine Particle Fraction (FPF) of meropenem from powder mixtures was determined by a Twin Stage Impinger (TSI) at a flow rate of 60 L/min. The FPF of the original MPN was 1.91% which was significantly increased to 37.5% for the formulations with excipients. No physical interactions between the drug and the excipients observed. This study revealed the potential of a stable meropenem DPI formulation for pulmonary delivery.

Utilizing the thiol chemistry of biomolecules for the rapid determination of anti-TNF-α drug in blood.

The detection of anti-TNF-α drugs require rapid, selective and sensitive biosensors that can be easily utilised at the point of care. Herein, we demonstrate a new biosensing approach that employs target-specific nanomaterial and label free surface-enhanced Raman spectroscopy (SERS) for the selective extraction and rapid determination of Adalimumab (ADB) in human blood plasma. The new method utilises the tumour necrosis factor (TNF-α) for the fabrication of a target-specific nanomaterial for extraction of ADB. The method also uses the thiol chemistry of the purified antibody drug for its chemisorption onto a gold-coated copper oxide substrate. A handheld Raman spectrophotometer is used for the determination of ADB by label free SERS. The limits of quantification (LOQ) and detection (LOD) of the purified and reduced drug by SERS were 0.10 fM and 0.03 fM respectively. ELISA was used for the cross validation of the SERS quantification of ADB where a 98.8% agreement was found between the two methods. Many proteins have disulfide bonds in their molecular structure. Therefore, the demonstrated biosensing approach can be extended for the rapid screening of other proteins and antibody drugs by developing target-specific extractor nanomaterial and utilizing the disulfide bond structure of the purified biomolecules for their label free SERS detection.

Preconcentration and SERS-based determination of infliximab in blood by using a TNF-α-modified gold-coated copper oxide nanomaterial.

Infliximab (INF) is a chimeric monoclonal immunoglobulin acting against tumor necrosis factor-alpha (TNF-α). The drug is used for the treatment of chronic autoimmune and inflammatory diseases. A target-specific nanomaterial is presented for the extraction of INF from human plasma along with a label-free surface enhanced Raman spectroscopy (SERS) method for its determination using a handheld device. A gold-coated copper oxide chip was functionalized with TNF-α and used to extract the drug from plasma. INF was recovered from the extractor by lowering the pH value to 2.5. The disulfide bond structure of the drug was then reduced and used for its oriented chemisorption onto a gold-coated copper oxide substrate for SERS measurements using the INF-specific band at 936 cm-1. The working range of the SERS method was between 10-7 and 10-14 M of reduced INF. The relative standard deviation (RSD), between three different measurements was 4.2% (intra-day) and 7.1% (inter-day). The quantification and detection limits of the assay (LOQ, LOD) were 0.01 pM and 1.4 fM respectively. The SERS detection was cross-validated against ELISA where 99% agreement was found between the two methods. Graphical abstractSchematic representation of the determination of Infliximab (INF) in blood. A gold coated copper oxide chip was functionalised with tumor necrosis factor (TNF-α) and used to extract INF from blood plasma. The captured INF was released, reduced, chemisorbed onto a second gold-coated copper oxide substrate and screened by surface-enhanced Raman spectroscopy (SERS) using a handheld device.

Phytotherapeutics: The Emerging Role of Intestinal and Hepatocellular Transporters in Drug Interactions with Botanical Supplements.

In herbalism, botanical supplements are commonly believed to be safe remedies, however, botanical supplements and dietary ingredients interact with transport and metabolic processes, affecting drug disposition. Although a large number of studies have described that botanical supplements interfere with drug metabolism, the mode of their interaction with drug transport processes is not well described. Such interactions may result in serious undesired effects and changed drug efficacy, therefore, some studies on interaction between botanical supplement ingredients and drug transporters such as P-gp and OATPs are described here, suggesting that the interaction between botanical supplements and the drug transporters is clinically significant.

FOXO Transcriptional Factors and Long-Term Living.

Several pathologies such as neurodegeneration and cancer are associated with aging, which is affected by many genetic and environmental factors. Healthy aging conceives human longevity, possibly due to carrying the defensive genes. For instance, FOXO (forkhead box O) genes determine human longevity. FOXO transcription factors are involved in the regulation of longevity phenomenon via insulin and insulin-like growth factor signaling. Only one FOXO gene (FOXO DAF-16) exists in invertebrates, while four FOXO genes, that is, FOXO1, FOXO3, FOXO4, and FOXO6 are found in mammals. These four transcription factors are involved in the multiple cellular pathways, which regulate growth, stress resistance, metabolism, cellular differentiation, and apoptosis in mammals. However, the accurate mode of longevity by FOXO factors is unclear until now. This article describes briefly the existing knowledge that is related to the role of FOXO factors in human longevity.

5'-NUCLEOTIDASES OF NAJA NAJA KARACHIENSIS SNAKE VENOM: THEIR DETERMINATION, TOXICITIES AND REMEDIAL APPROACH BY NATURAL INHIBITORS (MEDICINAL PLANTS).

Present study was carried out regarding enzymatic assay for 5'-nucleotidase enzymes present in snake venom Naja naja karachiensis and to evaluate twenty eight medicinal plants as their antidotes. Elevated enzymatic activities i.e., 119, 183, 262 and 335 U/mL were observed in 10, 20, 30 and 40 µg of crude venom, respectively, in dose dependent manner. Among various plant extracts only two (Bauhinia vaiiegate L. and Citms linion (L.) Burm. f.) were found 94% effective at 160 µg to neutralize 112 U/mL activities (p 0.5) while reference standard was proved 93.2% useful at 80 pg to halt 111 U/mL activities. Cedrus deodara G. Don, Enicostemna hyssopifolium (Willd.) Verdoom, Terminalia arjuma Wight & Am. and Zingiber officinalis Rosc. (at 160 µg) were found ≥90% effective (0.5 ≥ p ≥ 0.1) while Citrulus colocynthis, Fogonia cretica L., Rhazya stticta Dcne and Stenolobiun stans (L.) D. Don (at 320 µg) were proved 90% effective (0.05 ≥ p ≥ 0.02). The remaining plant extracts were observed abortive (p ≥ 0.001) in neutralization of 5'-nucleotidases enzymatic actions. This study emphasizes further characterization of active plant extracts to further explore the antivenom influences of these herbal remedies against deleterious effects produced by 5'-nucleotidase enzymes after snake bite envenomation.