A Simple, Sensitive, and Greener HPLC-DAD Method for the Simultaneous Analysis of Two Novel Orexin Receptor Antagonists.

The orexin receptor antagonist (ORA) is one of the new psychopharmacological agents used in the treatment of insomnia. There are currently no documented greener high-performance liquid chromatography-diode array detector (HPLC-DAD) methods for the analysis of ORA antagonists, lemborexant (LMB) and suvorexant (SUV) simultaneously. Therefore, in this study, a simple, sensitive, and greener HPLC-DAD method has been developed for the simultaneous quantitative analysis of LMB and SUV in bulk and laboratory-prepared mixture. The developed method was validated for numerous validation parameters and evaluated for greenness. The C18 Waters Spherisorb CN (4.6 × 250 mm2; 5 µm) column was used for the chromatographic separation. The mobile phase composition was ethanol: 10 mM KH2PO4 buffer in a ratio of (60:40 v/v). The DAD detection was performed at 253 nm using a Waters DAD detector. The greenness was evaluated using the analytical Eco-Scale (AES), ChlorTox, and analytical GREEnness (AGREE) techniques. The calibration curves showed excellent linearity for LMB and SUV between the concentration range of 125-5000 ng/mL and 250-10,000 ng/mL, respectively. In addition, the proposed HPLC-DAD method was accurate, precise, robust, highly sensitive, and greener. AES, ChlorTox, and AGREE scales were predicted by the HPLC-DAD method to be 91, 1.14 g, and 0.79, respectively, showing an excellent greenness profile. The greener HPLC-DAD method was successfully used to analyze both medicines quantitatively in bulk and laboratory-prepared synthetic mixtures. The findings of this study indicated that the proposed HPLC-DAD method may be consistently applied to evaluate LMB and SUV in bulk and dosage forms.

Molecular insights into anti-Protozoal action of natural compounds against Cryptosporidium parvum: a molecular simulation study.

The current study used the major target protein lactate dehydrogenase Cryptosporidium parvum to identify potential binders. Our approach was a comprehensive three-step screening of 2,569 natural compounds. First, we used molecular docking techniques, followed by an advanced DeepPurpose ML model for virtual screening. The final step involved meticulous re-docking and detailed interaction analysis. The known inhibitor FX11 was considered as a control that was used for comparative analysis. Our screening process led to the identification of three promising compounds: 5353794, 18475114, and 25229652. These compounds were chosen due to their exceptional ability to form hydrogen bonds and their high binding scores with the protein. Here, all three hits showed H-bonds with the functional residues (Asn122 and Thr231) of protein, while 25229652 also showed H-bond with the catalytic site residue (His177). RMSD behaviour reflected stable and consistent complex formation for all the compounds in their last 30 ns trajectories. Principal component analysis (PCA) and free energy landscape (FEL) showed a high frequency of favourable low free energy states. Using the MM/GBSA calculation, compounds 5353794 (ΔGTOTAL = -34.92 kcal/mol) and 18475114 (ΔGTOTAL = -34.66 kcal/mol) had the highest binding affinity with the protein however, 25229652 (ΔGTOTAL = -22.62 kcal/mol) had ΔGTOTAL comparable to the control FX11. These natural compounds not only show the potential for hindering C. parvum lactate dehydrogenase but also open new avenues in its drug development. Their strong binding properties and stable interactions mark them as the prime candidates for further research and experimental validation as anti-cryptosporidiosis agents.Communicated by Ramaswamy H. Sarma.

Topical Sustained-Release Dexamethasone-Loaded Chitosan Nanoparticles: Assessment of Drug Delivery Efficiency in a Rabbit Model of Endotoxin-Induced Uveitis.

Uveitis is an ocular illness that if not treated properly can lead to a total loss of vision. In this study, we evaluated the utility of HA-coated Dexamethasone-sodium-phosphate (DEX)-chitosan nanoparticles (CSNPs) coated with hyaluronic acid (HA) as a sustained ocular delivery vehicle for the treatment of endotoxin-induced-uveitis (EIU) in rabbits. The CSNPs were characterized for particle size, zeta potential, polydispersity, surface morphology, and physicochemical properties. Drug encapsulation, in vitro drug release, and transcorneal permeation were also evaluated. Finally, eye irritation, ocular pharmacokinetics, and pharmacodynamics were in vivo. The CSNPs ranged from 310.4 nm and 379.3 nm pre-(uncoated) and post-lyophilization (with HA-coated), respectively. The zeta potentials were +32 mV (uncoated) and -5 mV (HA-uncoated), while polydispersity was 0.178-0.427. Drug encapsulation and loading in the CSNPs were 73.56% and 6.94% (uncoated) and 71.07% and 5.54% (HA-coated), respectively. The in vitro DEX release over 12 h was 77.1% from the HA-coated and 74.2% from the uncoated NPs. The physicochemical properties of the CSNPs were stable over a 3-month period when stored at 25 °C. Around a 10-fold increased transcorneal-flux and permeability of DEX was found with HA-CSNPs compared to the DEX-aqueous solution (DEX-AqS), and the eye-irritation experiment indicated its ocular safety. After the ocular application of the CSNPs, DEX was detected in the aqueous humor (AH) till 24 h. The area under the concentrations curve (AUC0-24h) for DEX from the CSNPs was 1.87-fold (uncoated) and 2.36-fold (HA-coated) higher than DEX-AqS. The half-life (t1/2) of DEX from the uncoated and HA-coated NPs was 2.49-and 3.36-fold higher, and the ocular MRT0-inf was 2.47- and 3.15-fold greater, than that of DEX-AqS, respectively. The EIU rabbit model showed increased levels of MPO, TNF-α, and IL-6 in AH. Topical DEX-loaded CSNPs reduced MPO, TNF-α, and IL-6 levels as well as inhibited NF-κB expression. Our findings demonstrate that the DEX-CSNPs platform has improved the delivery properties and, hence, the promising anti-inflammatory effects on EIU in rabbits.

Effects of Apigenin on Pharmacokinetics of Dasatinib and Probable Interaction Mechanism.

Dasatinib (DAS), a narrow-therapeutic index drug, Bcr-Abl, and Src family kinases multitarget inhibitor have been approved for chronic myelogenous leukemia (CML) and Ph-positive acute lymphocytic leukemia (Ph+ ALL). Apigenin (APG) has a long history of human usage in food, herbs, health supplements, and traditional medicine, and it poses low risk of damage. The concomitant use of APG containing herbs/foods and traditional medicine may alter the pharmacokinetics of DAS, that probably lead to possible herb-drug interactions. The pharmacokinetic interaction of APG pretreatment with DAS in rat plasma following single and co-oral dosing was successfully deliberated using the UPLC-MS/MS method. The in vivo pharmacokinetics and protein expression of CYP3A2, Pgp-MDR1, and BCPR/ABCG2 demonstrate that APG pretreatment has potential to drastically changed the DAS pharmacokinetics where escalation in the Cmax, AUC(0-t), AUMC(0-inf_obs), T1/2, Tmax, and MRT and reduction in Kel, Vd, and Cl significantly in rats pretreated with APG 40 mg/kg, thus escalating systemic bioavailability and increasing the rate of absorption via modulation of CYP3A2, Pgp-MDR1, and BCPR/ABCG2 protein expression. Therefore, the concomitant consumption of APG containing food or traditional herb with DAS may cause serious life-threatening drug interactions and more systematic clinical study on herb-drug interactions is required, as well as adequate regulation in herbal safety and efficacy.

Topical Application of Linezolid-Loaded Chitosan Nanoparticles for the Treatment of Eye Infections.

Linezolid (LZ) loaded chitosan-nanoparticles (CSNPs) was developed by the ionic-gelation method using Tripolyphosphate-sodium as a crosslinker for topical application for the treatment of bacterial eye infections. Particles were characterized by Zeta-Sizer (Malvern Nano-series). TEM was used for structural morphology. Encapsulation and drug loading were estimated by measuring the unencapsulated drug. In-vitro drug release in STF (pH 7) was performed through a dialysis membrane. Storage stability of LZ-CSNPs was checked at 25 °C and 40 °C for six months. The antimicrobial potency of NPs was evaluated on different Gram-positive strains. Ocular irritation and pharmacokinetic studies were completed in rabbits. Ex-vivo transcorneal permeation of the drug was determined through the rabbit cornea. Ionic interaction among the oppositely charged functional groups of CS and TPP generated the CSNPs. The weight ratio at 3:1, wt/wt (CS/TPP) with 21.7 mg of LZ produced optimal NPs (213.7 nm with 0.387 of PDI and +23.1 mV of ZP) with 71% and 11.2% encapsulation and drug loading, respectively. Around 76.7% of LZ was released from LZ-AqS within 1 h, while 79.8% of LZ was released from CSNPs at 12 h and 90% at 24 h. The sustained drug release property of CSNPS was evaluated by applying kinetic models. The linearity in the release profile suggested that the release of LZ from CSNPs followed the Higuchi-Matrix model. LZ-CSNPs have shown 1.4 to 1.6-times improved antibacterial activity against the used bacterial strains. The LZ-CSNPs were "minimally-irritating" to rabbit eyes and exhibited 4.4-times increased transcorneal permeation of LZ than from LZ-AqS. Around 3-, 1.2- and 3.1-times improved Tmax, Cmax, and AUC0-24 h, respectively were found for LZ-CSNPs during the ocular pharmacokinetic study. AqS has shown 3.1-times faster clearance of LZ. Conclusively, LZ-CSNPs could offer a better alternative for the prolonged delivery of LZ for the treatment of bacterial infections in the eyes.

UPLC-MS/MS Based Identification and Quantification of a Novel Dual Orexin Receptor Antagonist in Plasma Samples by Validated SWGTOX Guidelines.

Lemborexant (LEM) is a novel dual orexin receptor antagonist (DORA), recently approved for the treatment of insomnia. As with other DORAs, LEM has potential of abuse and therefore placed in Schedule IV class by the United States Drug Enforcement Administration (USDEA). In this study, a sensitive and accurate UPLC-MS/MS assay was developed for the quantification of LEM in human plasma sample using losartan as an internal standard (IS). The chromatographic separation was performed by using gradient elution of mobile phase, comprising of 10 mM ammonium acetate and acetonitrile with a flow rate of 0.3 mL/min. An Acquity UPLC BEH C18 (1.7 µm, 2.1 × 50 mm) column was used for separation of LEM and IS by maintaining the oven temperature of 40 °C. The electrospray ionization in positive mode was used for sample ionization. The precursor to product ion transition of 411.12 > 175.09 (qualifier) and 411.1 > 287.14 (quantifier) was used for detection and quantification of LEM, respectively, in multiple reaction monitoring mode. Being a drug of abuse, the assay was validated according to "Scientific Working Group for Toxicology" (SWGTOX) guidelines, including limit of detection (LOD), limit of quantification (LOQ), precision and bias, calibration model, interferences, carry-over effects, matrix effects, and stability parameters. The LOD and LOQ of the assay were 0.35 and 1.0 ng/mL, respectively. The linear range was between 1-300 ng/mL with correlation coefficient of ≥0.995. The method was also cross validated in rat plasma samples with acceptable ranges of precision and accuracy before its application for pharmacokinetic study in rats.

Mesoporous Silica Nanoparticles Coated with Carboxymethyl Chitosan for 5-Fluorouracil Ocular Delivery: Characterization, In Vitro and In Vivo Studies.

This study investigates the development of topically applied non-invasive amino-functionalized silica nanoparticles (AMSN) and O-Carboxymethyl chitosan-coated AMSN (AMSN-CMC) for ocular delivery of 5-Fluorouracil (5-FU). Particle characterization was performed by the DLS technique (Zeta-Sizer), and structural morphology was examined by SEM and TEM. The drug encapsulation and loading were determined by the indirect method using HPLC. Physicochemical characterizations were performed by NMR, TGA, FTIR, and PXRD. In vitro release was conducted through a dialysis membrane in PBS (pH 7.4) using modified Vertical Franz diffusion cells. The mucoadhesion ability of the prepared nanoparticles was tested using the particle method by evaluating the change in zeta potential. The transcorneal permeabilities of 5-FU from AMNS-FU and AMSN-CMC-FU gel formulations were estimated through excised goat cornea and compared to that of 5-FU gel formulation. Eye irritation and ocular pharmacokinetic studies from gel formulations were evaluated in rabbit eyes. The optimum formulation of AMSN-CMC-FU was found to be nanoparticles with a particle size of 249.4 nm with a polydispersity of 0.429, encapsulation efficiency of 25.8 ± 5.8%, and drug loading capacity of 5.2 ± 1.2%. NMR spectra confirmed the coating of AMSN with the CMC layer. In addition, TGA, FTIR, and PXRD confirmed the drug loading inside the AMSN-CMC. Release profiles showed 100% of the drug was released from the 5-FU gel within 4 h, while AMSN-FU gel released 20.8% of the drug and AMSN-CMC-FU gel released around 55.6% after 4 h. AMSN-CMC-FU initially exhibited a 2.45-fold increase in transcorneal flux and apparent permeation of 5-FU compared to 5-FU gel, indicating a better corneal permeation. Higher bioavailability of AMSN-FU and AMSN-CMC-FU gel formulations was found compared to 5-FU gel in the ocular pharmacokinetic study with superior pharmacokinetics parameters of AMSN-CMC-FU gel. AMSN-CMC-FU showed 1.52- and 6.14-fold higher AUC0-inf in comparison to AMSN-FU and 5-FU gel, respectively. AMSN-CMC-FU gel and AMSN-FU gel were "minimally irritating" to rabbit eyes but showed minimal eye irritation potency in comparison to the 5 FU gel. Thus, the 5-FU loaded in AMSN-CMC gel could be used as a topical formulation for the treatment of ocular cancer.

Nanocrystallization Improves the Solubilization and Cytotoxic Effect of a Poly (ADP-Ribose)-Polymerase-I Inhibitor.

Olaparib (OLA) is an anticancer agent that acts by inhibiting the poly (ADP-ribose)-polymerase-I (PARP-I). Due to its low solubility and low permeability, it has been placed as a BCS Class-IV drug and hence its clinical use is limited. In this study, we develop the nanocrystals of OLA as a way to improve its solubility and other performances. The OLA-NCs were prepared by antisolvent precipitation method through homogenization and probe sonication technique using a novel amphiphilic polymeric stabilizer (Soluplus®). Particle characterization resulted approximately 103.13 nm, polydispersity-index was 0.104 with positive zeta-potential of +8.67 mV. The crystal morphology by SEM of OLA-NCs (with and without mannitol) exhibited nano-crystalline prism-like structures as compared to the elongated OLA-pure. The DSC, XRD and FTIR were performed to check the interaction of Soluplus, mannitol and OLA did not exhibit any physical interaction among the OLA, Soluplus® and mannitol that is indicated by the presence of parent wave number peak. Two-fold increased solubility of OLA was found in PBS with Soluplus® from the NCs (69.3 ± 6.2 µgmL−1) as compared to pure drug (35.6 ± 7.2 µgmL−1). In vitro release of drug from OLA-NCs was higher (78.2%) at 12 h at pH 6.8 and relatively lower (53.1%) at pH 1.2. In vitro cellular cytotoxicity and anticancer effects were examined on MCF-7 cells. OLA-NCs were found effectively potent to MCF-7 cells compared with OLA-pure with approximately less than half IC50 value during MTT assay. Estimation of p53, Caspase-3 and Caspase-9 in MCF-7 cells indicated that OLA-NCs have significantly (p < 0.05) increased their expressions. After single oral dose in rats, 12 h plasma drug concentration-time profile indicated approximately 2.06-, 2.29-, 2−25- and 2.62-folds increased Cmax, AUC0-12 h, AUC0-∞ and AUMC0-∞, respectively, from the NCs as compared to OLA-pure. Storage stability indicated that the OLA-NCs was physically and chemically stable at 4 °C, 25 °C and 40 °C up to 6-months. Overall, OLA-NCs were deliberated; its potential feasibility to overwhelm the formulation challenges related to poorly soluble drugs and its future clinical applications.

Identification of Doxorubicin as Repurposing Inhibitory Drug for MERS-CoV PLpro.

Middle East respiratory syndrome coronavirus (MERS-CoV), belonging to the betacoronavirus genus can cause severe respiratory illnesses, accompanied by pneumonia, multiorgan failure, and ultimately death. CoVs have the ability to transgress species barriers and spread swiftly into new host species, with human-to-human transmission causing epidemic diseases. Despite the severe public health threat of MERS-CoV, there are currently no vaccines or drugs available for its treatment. MERS-CoV papain-like protease (PLpro) is a key enzyme that plays an important role in its replication. In the present study, we evaluated the inhibitory activities of doxorubicin (DOX) against the recombinant MERS-CoV PLpro by employing protease inhibition assays. Hydrolysis of fluorogenic peptide from the Z-RLRGG-AMC-peptide bond in the presence of DOX showed an IC50 value of 1.67 µM at 30 min. Subsequently, we confirmed the interaction between DOX and MERS-CoV PLpro by thermal shift assay (TSA), and DOX increased ΔTm by ~20 °C, clearly indicating a coherent interaction between the MERS-CoV PL protease and DOX. The binding site of DOX on MERS-CoV PLpro was assessed using docking techniques and molecular dynamic (MD) simulations. DOX bound to the thumb region of the catalytic domain of the MERS-CoV PLpro. MD simulation results showed flexible BL2 loops, as well as other potential residues, such as R231, R233, and G276 of MERS-CoV PLpro. Development of drug repurposing is a remarkable opportunity to quickly examine the efficacy of different aspects of treating various diseases. Protease inhibitors have been found to be effective against MERS-CoV to date, and numerous candidates are currently undergoing clinical trials to prove this. Our effort follows a in similar direction.

Apigenin Loaded Lipoid-PLGA-TPGS Nanoparticles for Colon Cancer Therapy: Characterization, Sustained Release, Cytotoxicity, and Apoptosis Pathways.

Colon cancer (CC) is one of major causes of mortality and affects the socio-economic status world-wide. Therefore, developing a novel and efficient delivery system is needed for CC management. Thus, in the present study, lipid polymer hybrid nanoparticles of apigenin (LPHyNPs) was prepared and characterized on various parameters such as particle size (234.80 ± 12.28 nm), PDI (0.11 ± 0.04), zeta potential (−5.15 ± 0.70 mV), EE (55.18 ± 3.61%), etc. Additionally, the DSC, XRD, and FT-IR analysis determined drug entrapment and affinity with the selected excipient, demonstrating a promising drug affinity with the lipid polymer. Morphological analysis via SEM and TEM exhibited spherical NPs with a dark color core, which indicated drug entrapment inside the core. In vitro release study showed significant (p < 0.05) sustained release of AGN from LPHyNPs than AGN suspension. Further, the therapeutic efficacy in terms of apoptosis and cell cycle arrest of developed LPHyNPs against CC was estimated by performing flow cytometry and comparing its effectiveness with blank LPHyNPs and AGN suspension, which exhibited remarkable outcomes in favor of LPHyNPs. Moreover, the mechanism behind the anticancer attribute was further explored by estimating gene expression of various signaling molecules such as Bcl-2, BAX, NF-κB, and mTOR that were involved in carcinogenic pathways, which indicated significant (p < 0.05) results for LPHyNPs. Moreover, to strengthen the anticancer potential of LPHyNPs against chemoresistance, the expression of JNK and MDR-1 genes was estimated. Outcomes showed that their expression level reduced appreciably when compared to blank LPHyNPs and AGN suspension. Hence, it can be concluded that developed LPHyNPs could be an efficient therapeutic system for managing CC.

Abemaciclib-loaded ethylcellulose based nanosponges for sustained cytotoxicity against MCF-7 and MDA-MB-231 human breast cancer cells lines.

Abemaciclib (AC) is a novel, orally available drug molecule approved for the treatment of breast cancer. Due to its low bioavailability, its administration frequency is two to three times a day that can decrease patient compliance. Sustained release formulation are needed for prolong the action and to reduce the adverse effects. The aim of current study was to develop sustained release NSs of AC. Nanosponges (NSs) was prepared by emulsion-solvent diffusion method using ethyl-cellulose (EC) and Kolliphor P-188 (KP-188) as sustained-release polymer and surfactant, respectively. Effects of varying surfactant concentration and drug: polymer proportions on the particle size (PS), polydispersity index (PDI), zeta potential (ζP), entrapment efficiency (%EE), and drug loading (%DL) were investigated. The results of AC loaded NSs (ACN1-ACN5) exhibited PS (366.3-842.2 nm), PDI (0.448-0.853), ζP (-8.21 to -19.7 mV), %EE (48.45-79.36%) and %DL (7.69-19.17%), respectively. Moreover, ACN2 showed sustained release of Abemaciclib (77.12 ± 2.54%) in 24 h Higuchi matrix as best fit kinetics model. MTT assay signified ACN2 as potentials cytotoxic nanocarrier against MCF-7 and MDA-MB-231 human breast cancer cells. Further, ACN2 displayed drug release property without variation in the % release after exposing the product at 25 °C, 5 °C, and 45 °C storage conditions for six months. This investigation proved that the developed NSs would be an efficient carrier to sustain the release of AC in order to improve efficacy against breast cancer.

Fabrication and Characterization of Tedizolid Phosphate Nanocrystals for Topical Ocular Application: Improved Solubilization and In Vitro Drug Release.

Positively charged NCs of TZP (0.1%, w/v) for ocular use were prepared by the antisolvent precipitation method. TZP is a novel 5-Hydroxymethyl-Oxazolidinone class of antibiotic and is effective against many drug-resistant bacterial infections. Even the phosphate salt of this drug is poorly soluble, therefore the NCs were prepared for its better solubility and ocular availability. P188 was found better stabilizer than PVA for TZP-NCs. Characterization of the NCs including the particle-size, PDI, and ZP by Zeta-sizer, while morphology by SEM indicated that the preparation technique was successful to get the optimal sized (151.6 nm) TZP-NCs with good crystalline morphology. Mannitol (1%, w/v) prevented the crystal growth and provided good stabilization to NC1 during freeze-drying. FTIR spectroscopy confirmed the nano-crystallization did not alter the basic molecular structure of TZP. DSC and XRD studies indicated the reduced crystallinity of TZP-NC1, which potentiated its solubility. An increased solubility of TZP-NC1 (25.9 µgmL-1) as compared to pure TZP (18.4 µgmL-1) in STF with SLS. Addition of stearylamine (0.2%, w/v) and BKC (0.01%, w/v) have provided cationic (+29.4 mV) TZP-NCs. Redispersion of freeze-dried NCs in dextrose (5%, w/v) resulted in a clear transparent aqueous suspension of NC1 with osmolarity (298 mOsm·L-1) and viscosity (21.1 cps at 35 °C). Mannitol (cryoprotectant) during freeze-drying could also provide isotonicity to the nano-suspension at redispersion in dextrose solution. In vitro release in STF with SLS has shown relatively higher (78.8%) release of TZP from NC1 as compared to the conventional TZP-AqS (43.4%) at 12 h. TZP-NC1 was physically and chemically stable at three temperatures for 180 days. The above findings suggested that TZP-NC1 would be a promising alternative for ocular delivery of TZP with relatively improved performance.

Boosting the Anticancer Activity of Sunitinib Malate in Breast Cancer through Lipid Polymer Hybrid Nanoparticles Approach.

In the current study, lipid-polymer hybrid nanoparticles (LPHNPs) fabricated with lipoid-90H and chitosan, sunitinib malate (SM), an anticancer drug was loaded using lecithin as a stabilizer by employing emulsion solvent evaporation technique. Four formulations (SLPN1-SLPN4) were developed by varying the concentration of chitosan polymer. Based on particle characterization, SLPN4 was optimized with size (439 ± 5.8 nm), PDI (0.269), ZP (+34 ± 5.3 mV), and EE (83.03 ± 4.9%). Further, the optimized formulation was characterized by FTIR, DSC, XRD, SEM, and in vitro release studies. In-vitro release of the drug from SPN4 was found to be 84.11 ± 2.54% as compared with pure drug SM 24.13 ± 2.67%; in 48 h, release kinetics followed the Korsmeyer-Peppas model with Fickian release mechanism. The SLPN4 exhibited a potent cytotoxicity against MCF-7 breast cancer, as evident by caspase 3, 9, and p53 activities. According to the findings, SM-loaded LPHNPs might be a promising therapy option for breast cancer.

Development and Characterization of PEGylated Fatty Acid-Block-Poly(ε-caprolactone) Novel Block Copolymers and Their Self-Assembled Nanostructures for Ocular Delivery of Cyclosporine A.

Low aqueous solubility and membrane permeability of some drugs are considered major limitations for their use in clinical practice. Polymeric micelles are one of the potential nano-drug delivery systems that were found to ameliorate the low aqueous solubility of hydrophobic drugs. The main objective of this study was to develop and characterize a novel copolymer based on poly (ethylene glycol) stearate (Myrj™)-block-poly(ε-caprolactone) (Myrj-b-PCL) and evaluate its potential as a nanosystem for ocular delivery of cyclosporine A (CyA). Myrj-b-PCL copolymer with various PCL/Myrj ratios were synthesized via ring-opening bulk polymerization of ε-caprolactone using Myrj (Myrj S40 or Myrj S100), as initiators and stannous octoate as a catalyst. The synthesized copolymers were characterized using 1H NMR, GPC, FTIR, XRD, and DSC. The co-solvent evaporation method was used to prepare CyA-loaded Myrj-b-PCL micelles. The prepared micelles were characterized for their size, polydispersity, and CMC using the dynamic light scattering (DLS) technique. The results from the spectroscopic and thermal analyses confirmed the successful synthesis of the copolymers. Transmission electron microscopy (TEM) images of the prepared micelles showed spherical shapes with diameters in the nano range (<200 nm). Ex vivo corneal permeation study showed sustained release of CyA from the developed Myrj S100-b-PCL micelles. In vivo ocular irritation study (Draize test) showed that CyA-loaded Myrj S100-b-PCL88 was well tolerated in the rabbit eye. Our results point to a great potential of Myrj S100-b-PCL as an ocular drug delivery system.

Development of Apremilast Nanoemulsion-Loaded Chitosan Gels: In Vitro Evaluations and Anti-Inflammatory and Wound Healing Studies on a Rat Model.

Apremilast (APL) has profound anti-inflammatory and wound healing activity, alongside other dermal care. This study aims to develop APL-loaded NEs (ANE1-ANE5) using eucalyptus oil (EO) as the oil and Tween-80 and transcutol-HP (THP) as a surfactant and co-surfactant, respectively. The prepared NEs were then evaluated based on mean droplet size (12.63 ± 1.2 nm), PDI (0.269 ± 0.012), ZP (-23.00 ± 5.86), RI (1.315 ± 0.02), and %T (99.89 ± 0.38) and ANE4 was optimized. Further, optimized NEs (ANE4) were incorporated into chitosan gel (2%, w/v). The developed ANE4-loaded chitosan gel was then evaluated for pH, spreadability, in vitro diffusion, and wound healing and anti-inflammatory studies. Moreover, in vivo studies denoted improved anti-inflammatory and wound healing activity and represented a decrease in wound size percentage (99.68 ± 0.345%) for the APNE2 gel test compared to a negative control (86.48 ± 0.87%) and standard control (92.82 ± 0.34%). Thus, the formulation of ANE4-loaded chitosan gels is an efficient topical treatment strategy for inflammatory and wound healing conditions.

Effect of Solvents, Stabilizers and the Concentration of Stabilizers on the Physical Properties of Poly(d,l-lactide-co-glycolide) Nanoparticles: Encapsulation, In Vitro Release of Indomethacin and Cytotoxicity against HepG2-Cell.

A biocompatible, biodegradable and FDA-approved polymer [Poly lactic-co-glycolic acid (PLGA)] was used to prepare the nanoparticles (NPs) to observe the effect of solvents, stabilizers and their concentrations on the physical properties of the PLGA-NPs, following the encapsulation and in vitro release of Indomethacin (IND). PLGA-NPs were prepared by the single-emulsion solvent evaporation technique using dichloromethane (DCM)/chloroform as the organic phase with Polyvinyl-alcohol (PVA)/Polyvinylpyrrolidone (PVP) as stabilizers to encapsulate IND. The effects of different proportions of PVA/PVP with DCM/chloroform on the physiochemical properties (particle size, the polydispersity index, the zeta potential by Malvern Zetasizer and morphology by SEM) of the NPs were investigated. DSC was used to check the physical state, the possible complexation of PLGA with stabilizer(s) and the crystallinity of the encapsulated drug. Stabilizers at all concentrations produced spherical, regular-shaped, smooth-surfaced discrete NPs. Average size of 273.2-563.9 nm was obtained when PVA (stabilizer) with DCM, whereas it ranged from 317.6 to 588.1 nm with chloroform. The particle size was 273.2-563.9 nm when PVP was the stabilizer with DCM, while it was 381.4-466.6 nm with chloroform. The zeta potentials of PVA-stabilized NPs were low and negative (-0.62 mV) while they were comparatively higher and positive for PVP-stabilized NPs (+17.73 mV). Finally, drug-loaded optimal NPs were composed of PLGA (40 mg) and IND (4 mg) in 1 mL DCM/chloroform with PVA/PVP (1-3%), which resulted in sufficient encapsulation (54.94-74.86%) and drug loading (4.99-6.81%). No endothermic peak of PVA/PVP appeared in the optimized formulation, which indicated the amorphous state of IND in the core of the PLGA-NPs. The in vitro release study indicated a sustained release of IND (32.83-52.16%) from the PLGA-NPs till 72 h and primarily followed the Higuchi matrix release kinetics followed by Korsmeyer-Peppas models. The cell proliferation assay clearly established that the organic solvents used to prepare PLGA-NPs had evaporated. The PLGA-NPs did not show any particular toxicity in the HepG2 cells within the dose range of IND (250-500 µg/mL) and at an equivalent concentration of PLGA-NPs (3571.4-7142.7 µg/mL). The cytotoxicity of the hepatotoxic drug (IND) was reduced by its encapsulation into PLGA-NPs. The outcomes of this investigation could be implemented to prepare PLGA-NPs of acceptable properties for the encapsulation of low/high molecular weight drugs. It would be useful for further in vitro and in vivo applications to use this delivery system.

Development and Evaluation of Chitosan Nanoparticles for Ocular Delivery of Tedizolid Phosphate.

This study investigates the development of topically applied non-invasive chitosan-nanoparticles (CSNPs) for ocular delivery of tedizolid phosphate (TZP) for the treatment of MRSA-related ocular and orbital infections. An ionic-gelation method was used to prepare TZP-encapsulated CSNPs using tripolyphosphate-sodium (TPP) as cross-linker. Particle characterization was performed by the DLS technique (Zeta-Sizer), structural morphology was observed by SEM. The drug encapsulation and loading were determined by the indirect method. In-vitro release was conducted through dialysis bags in simulated tear fluid (pH 7) with 0.25% Tween-80. Physicochemical characterizations were performed for ocular suitability of CSNPS. An antimicrobial assay was conducted on different strains of Gram-positive bacteria. Eye-irritation from CSNPs was checked in rabbits. Transcorneal flux and apparent permeability of TZP from CSNPs was estimated through excised rabbit cornea. Ionic interaction between the anionic and cationic functional groups of TPP and CS, respectively, resulted in the formation of CSNPs at varying weight ratios of CS/TPP with magnetic stirring (700 rpm) for 4 h. The CS/TPP weight ratio of 3.11:1 with 10 mg of TZP resulted in optimal-sized CSNPs (129.13 nm) with high encapsulation (82%) and better drug loading (7%). Release profiles indicated 82% of the drug was released from the TZP aqueous suspension (TZP-AqS) within 1 h, while it took 12 h from F2 to release 78% of the drug. Sustained release of TZP from F2 was confirmed by applying different release kinetics models. Linearity in the profile (suggested by Higuchi's model) indicated the sustained release property CSNPs. F2 has shown significantly increased (p < 0.05) antibacterial activity against some Gram-positive strains including one MRSA strain (SA-6538). F2 exhibited a 2.4-fold increased transcorneal flux and apparent permeation of TZP as compared to TZP-AqS, indicating the better corneal retention. No sign or symptoms of discomfort in the rabbits' eyes were noted during the irritation test with F2 and blank CSNPs, indicating the non-irritant property of the TZP-CSNPs. Thus, the TZP-loaded CSNPs have strong potential for topical use in the treatment of ocular MRSA infections and related inflammatory conditions.

Eudragit-Coated Sporopollenin Exine Microcapsules (SEMC) of Phoenix dactylifera L. of 5-Fluorouracil for Colon-Specific Drug Delivery.

In this study, 5-fluorouracil (5-FU)-loaded pollens of Phoenix dactylifera and their coating with ERS was done and evaluated for the colon-targeted delivery of 5-FU to treat colon cancer. Sporopollenin exine microcapsules (SEMC) from the pollens of Phoenix dactylifera were extracted by the reflux method and 5-FU into SEMC was encapsulated by the vacuum-assisted loading method. 5-FU loaded SEMC was coated with Eudragit® RS-100 (ERS) by the organic solvent-evaporation technique under vacuum to avoid the discharge of 5-FU in the stomach and small intestine. Morphological and physicochemical characterization of drug-loaded SEMC (coated/uncoated) was performed by scanning electron microscopy (SEM), FTIR, XRD, and DSC. The encapsulation and drug loading were determined by the direct method, and an in vitro release study was performed in simulated gastric and intestinal fluids (SGF/SIF). The colon-specific delivery of 5-FU from the SEMC was assessed in terms of pharmacokinetics and gastrointestinal tract distribution after oral administration in rats. The successful encapsulation and loading of 5-FU into SEMC by a vacuum-assisted loading technique and its coating with ERS by a solvent-evaporation technique were achieved. SEM images of uncoated SEMC have shown porous structures, and coating with ERS reserved their morphology with a smooth surface and discrete microstructures and the 5% w/v ERS acetone solution. ERS-coated SEMC sustained the release of 5-FU until 24 h in SIF, while it was up to 12 h only from uncoated SEMC. The maximum plasma concentration (Cmax) of 5-FU from uncoated SEMC was 102.82 µg/mL after 1 h, indicating a rapid release of 5-FU in the upper gastrointestinal tract. This concentration decreased quickly with a half-life of 4 h, AUC0-t was 264.1 µg/mL.h, and MRT0-inf was 5.2 h. The Cmax of 5-FU from ERS-coated SEMC was 19.47 µg/mL at 16 h. The Cmax of 5-FU in small intestines was 406.2 µg/g at 1 h from uncoated SEMC and 1271.5 µg/g at 12 h from coated SEMC. Conclusively, a 249.9-fold higher relative bioavailability of 5-FU was achieved with the ERS-coated SEMC in colon tissues than that from uncoated SEMC.

Formulation and in vitro evaluation of topical nanosponge-based gel containing butenafine for the treatment of fungal skin infection.

In the current study, four formulae (BNS1-BNS4) of butenafine (BTF) loaded nanosponges (NS) were fabricated by solvent emulsification technology, using different concentration of ethyl cellulose (EC) and polyvinyl alcohol (PVA) as a rate retarding polymer and surfactant, respectively. Prepared NS were characterized for particle size (PS), polydispersity index (PDI), zeta potential (ZP), entrapment efficiency (EE) and drug loading (DL). Nanocarrier BNS3 was optimized based on the particle characterizations and drug encapsulation. It was further evaluated for physicochemical characterizations; FTIR, DSC, XRD and SEM. Selected NS BNS3 composed of BTF (100 mg), EC (200 mg) and 0.3% of PVA showed, PS (543 ± 0.67 nm), PDI (0.330 ± 0.02), ZP (-33.8 ± 0.89 mV), %EE (71.3 ± 0.34%) and %DL (22.8 ± 0.67%), respectively. Fabricated NS also revealed; polymer-drug compatibility, drug-encapsulation, non-crystalline state of the drug in the spherical NS as per the physicochemical evaluations. Optimized NS (BNS3) with equivalent amount of (1%, w/w or w/v) BTF was incorporated into the (1%, w/w or w/v) carbopol gel. BTF loaded NS based gel was then evaluated for viscosity, spreadability, flux, drug diffusion, antifungal, stability and skin irritation studies. BNS3 based topical gels exhibited a flux rate of 0.18 (mg/cm2.h), drug diffusion of 89.90 ± 0.87% in 24 h with Higuchi model following anomalous non-Fickian drug release. The BNS3 based-gel could be effective against pathogenic fungal strains.

UPLC-MS/MS assay of Tedizolid in rabbit aqueous humor: Application to ocular pharmacokinetic study.

Tedizolid phosphate (TZP) a prodrug of Tedizolid (TDZ) is a novel oxazolidinone antibiotic, used for the treatment of acute bacterial skin, skin structure infections and other serious gram positive and MRSA infections. In the present study, a sensitive UPLC-MS/MS analytical method was developed and validated for the quantification of TDZ in rabbit's aqueous humor (AqH) by using linezolid as internal standard (IS). Both TDZ and IS were separated on an Acquity™ HILIC column using an isocratic mobile phase comprising of acetonitrile: 20 mM ammonium acetate (85:15, v/v), eluted at 0.3mLmin-1 flow rate with total run time of 3 min. The AqH samples were processed by protein precipitation method by using acetonitrile as precipitating agent. TDZ and IS were detected in positive mode using electrospray ionization source. The precursor to product ion transitions at m/z 371.15 to 343.17 for TDZ and m/z 338.18 to 296.22 for IS were used for the quantification in multiple reaction monitoring mode. The calibration curve was linear in the concentration range of 4.98-1000ngmL-1 and the lower limit of detection was 1.97ngmL-1 only. The method was validated following US-FDA-guidelines and the results of validation parameter were found within the set limits. The developed UPLC-MS/MS method was fast, sensitive and reliable for the quantification of TDZ in the rabbit AqH and was successfully employed for ocular pharmacokinetic study of TDZ in AqH after topical ocular application of TZP-containing formulations in rabbit eyes.