Design, optimization, in vitro and in vivo evaluation of triamcinolone acetonide nanocrystals loaded in situ gel for topical ocular delivery.

Triamcinolone acetonide (TAA), a long-acting synthetic glucocorticoid, is commonly used for the management of posterior uveitis (PU) because of its anti-inflammatory and immunosuppressive characteristics. The commercially available formulation is in the suspension form advised for intravitreal injection, which has a number of serious problems. In the present research work, we prepared TAA nanocrystals (TAA-NCs) using the principles of design of experiments (DoE). The optimized TAA-NCs had a particle size of 243.0 ± 6.5 nm and a yield (%) of 89.4 ± 4.3%. The optimized TAA-NCs were suspended in a dual-responsive in situ gelling system, which has been previously reported by our team. The TAA-NCs loaded in situ gel (TAA-NC-ISG) formulations were evaluated for rheology, stability, in vitro and in vivo characteristics. The ocular pharmacokinetic investigations revealed that TAA-NCs loaded in situ gel achieved higher concentrations (Cmax of TAA-NC-ISG = 854.9 ng/mL) of the drug in vitreous humor and sustained (MRT0-∞ of TAA-NC-ISG = 11.2 h) the drug concentrations for longer duration compared to aqueous suspension of TAA-NCs (TAA-NC-Susp) and aqueous suspension of TAA with 20% hydroxypropyl ß-cyclodextrin(TAA-HP-ß-CD-Susp) reported in our previous work. This higher exposure of TAA by TAA-NC-ISG is due to the combined effect of the nanometric size of the TAA nanocrystals and the in situ gelling properties of the formulation.

Optimization of a HPLC-UV bioanalytical method using Box-Behnken design to determine the oral pharmacokinetics of neratinib maleate in Wistar rats.

The current research work reports the development and validation of a sensitive, robust and reproducible bioanalytical method for quantifying neratinib maleate in rat plasma. More than 85% of the drug was extracted from the plasma samples by protein precipitation. The method was optimized using Box-Behnken design, a response surface method. The effect of three critical factors, viz., the pH of the buffer (X1 ), the aqueous phase proportion in the mobile phase (X2 ) and the mobile phase flow rate (X3 ), was studied on two response variables, retention time (Y1 ) and United States Pharmacopoeia (USP) width (Y2 ). With the highest overall desirability function value of 0.943, the obtained optimized method conditions were: X1  = 2.4 ± 0.1; X2  = 66.7 ml, and X3  = 0.85 ml/min. Under the optimized conditions, the values of Y1 and Y2 for a sample containing 1 ppm of the drug were found to be 14.1 min and 0.50 ± 0.003, respectively. Single-dose intravenous bolus (7.5 mg/kg) and oral (15 mg/kg) pharmacokinetic studies were performed to determine the absolute bioavailability of the drug. The optimized bioanalytical method was sensitive enough to capture 95% of the drug eliminated from the body. The absolute oral bioavailability of the drug was 49.30%.

Design, Characterization and Pharmacokinetic-Pharmacodynamic Evaluation of Poloxamer and Kappa-Carrageenan-Based Dual-Responsive In Situ Gel of Nebivolol for Treatment of Open-Angle Glaucoma.

This study developed a dual-responsive in situ gel of nebivolol (NEB), a selective ß-adrenergic antagonist. The gel could achieve sustained concentrations in the aqueous humor to effectively treat glaucoma. The gel was prepared using a combination of poloxamers (Poloxamer-407 (P407) and Poloxamer-188 (P188)) and kappa-carrageenan (κCRG) as thermo-responsive and ion-sensitive polymers, respectively. Box-Behnken design (BBD) was used to optimize the effect of three critical formulation factors (concentration of P407, P188 and κCRG) on two critical response variables (sol-to-gel transition temperature of 33-35 °C and minimum solution state viscosity) of the in situ gel. A desirability function was employed to find the optimal concentrations of P407, P188 and κCRG that yielded a gel with the desired sol-to-gel transition temperature and solution state viscosity. An NEB-loaded gel was prepared using the optimized conditions and evaluated for in vitro drug release properties and ex vivo ocular irritation studies. Furthermore, ocular pharmacokinetic and pharmacodynamics studies were conducted in rabbits for the optimized formulation. The optimized NEB-loaded gel containing P407, P188 and κCRG had a sol-to-gel transition temperature of 34 °C and exhibited minimum viscosity (212 ± 2 cP at 25 °C). The optimized NEB-loaded gel sustained drug release with 86% drug release at the end of 24 h. The optimized formulation was well tolerated in the eye. Ocular pharmacokinetic studies revealed that the optimized in situ gel resulted in higher concentrations of NEB in aqueous humor compared to the NEB suspension. The aqueous humor Cmax of the optimized in situ gel (35.14 ± 2.25 ng/mL) was 1.2 fold higher than that of the NEB suspension (28.2 ± 3.1 ng/mL), while the AUC0-∞ of the optimized in situ gel (381.8 ± 18.32 ng/mL*h) was 2 fold higher than that of the NEB suspension (194.9 ± 12.17 ng/mL*h). The systemic exposure of NEB was significantly reduced for the optimized in situ gel, with a 2.7-fold reduction in the plasma Cmax and a 4.1-fold reduction in the plasma AUC0-∞ compared with the NEB suspension. The optimized gel produced a higher and sustained reduction in the intra-ocular pressure compared with the NEB suspension. The optimized gel was more effective in treating glaucoma than the NEB suspension due to its mucoadhesive properties, sustained drug release and reduced drug loss. Lower systemic exposure of the optimized gel indicates that the systemic side effects can be significantly reduced compared to the NEB suspension, particularly in the long-term management of glaucoma.

Optimization and in vivo evaluation of triamcinolone acetonide loaded in situ gel prepared using reacted tamarind seed xyloglucan and kappa-carrageenan for ocular delivery.

In the current work, triamcinolone acetonide (TAA) loaded dual responsive in situ gelling system was designed and optimized using reacted tamarind seed xyloglucan (RXG) (thermoresponsive) and kappa-Carrageenan (κ-CRG) (ion-sensitive) polymers. Tamarind seed xyloglucan (TSX) was subjected to purification followed by enzymatic treatment to produce RXG with ~40 % reduction in galactose content compared to TSX. RXG was characterized using size exclusion chromatography, Fourier transform infrared and proton nuclear magnetic resonance spectroscopy to confirm the ~40 % reduction in galactoside content compared to TSX. The proportions of RXG and κ-CRG in the in situ gels (TAA loaded RXG-κ-CRG) were optimized based on their rheological properties. The optimized in situ gel exhibited good flow properties at 25 °C, but transformed rapidly into a stronger gel in the presence of STF at 35 °C. The optimized formulation had strong mucoadhesion with good spreadability on the surface of excised goat cornea. The drug release followed zero-order kinetics from the optimized in situ gel. Ex vivo ocular toxicity studies indicate that the optimized formulation was well tolerated. The ocular pharmacokinetic studies in rabbits showed significantly higher and sustained vitreous humor exposure of TAA for optimized in situ gel compared to hydroxypropyl-ß-cyclodextrin based aqueous suspension of TAA.

Design and evaluation of rufinamide nanocrystals loaded thermoresponsive nasal in situ gelling system for improved drug distribution to brain.

Rufinamide (Rufi) is an antiepileptic drug used to manage Lennox-Gastaut Syndrome and partial seizures. The oral bioavailability of Rufi is less due to its poor solubility and low dissolution rate in the gastrointestinal fluids. This results in less amount of drug reaching the brain following the oral administration of drug. Oral formulations of Rufi are prescribed at a high dose and dosing frequency to increase its distribution to the brain. A Rufi loaded thermoresponsive nasal in situ gel which showed significantly high brain concentrations compared to aqueous suspension of Rufi administered through nasal route was developed by our research group and published. In the current work, we have formulated nanocrystals of Rufi and suspended them in a xyloglucan based thermoresponsive gel to improve the nose-to-brain distribution. The particle size, polydispersity index, and yield (%) of the optimized Rufi nanocrystals were 261.2 ± 2.1 nm, 0.28 ± 0.08, and 89.6 ± 2.0 respectively. The narrow PDI indicates that the manufacturing process is reproducible and reliable. Higher % yield suggested that the method of preparation is efficient. The sol-to-gel transition of in situ gel loaded with Rufi nanocrystals was at 32°C which suggested that the formulation transforms into gel at nasal epithelial temperatures. The nasal pharmacokinetic studies showed that Rufi nanocrystals loaded in situ gel produced higher concentration of the drug in brain (higher brain Cmax) and maintained the drug concentrations for longer duration (higher mean residence time) compared to aqueous suspension of Rufi nanocrystals as well aqueous suspension of Rufi and Rufi loaded in situ gel, reported previously. Nanometric size of the Rufi nanocrystals combined with the in situ gelling properties helped the optimized formulation achieve higher brain distribution and also sustain the drug concentrations in brain for longer duration compared to any of the formulations studied by our research group.

Dose identification of triamcinolone acetonide for noninvasive pre-corneal administration in the treatment of posterior uveitis using a rapid, sensitive HPLC method with photodiode-array detector.

Triamcinolone acetonide (TAA) is the drug of choice in the management of ocular inflammations due to its anti-inflammatory and immuno-suppressant activity. Available marketed formulations (Triesence, Trivaris, Kenalog) are in the suspension form recommended to be administered via intravitreal injection, which has many major complications. In the present study, we have designed and evaluated Hydroxypropyl-ß-cyclodextrin (HP-ß-CD),) based conventional formulations of TAA (aqueous suspensions) with different dose strengths to identify the dose strength required for achieving the effective concentrations in vitreous humor following pre-corneal administration of the formulations. Ocular pharmacokinetic studies of conventional formulations of triamcinolone acetonide (TAA) with different dose strengths (1 mg/30µL, 2 mg/30µL, 4 mg/30µL) were performed to identify the dose strength required to produce effective concentrations of TAA in the aqueous and vitreous humor. A rapid, sensitive, selective, accurate and precise bioanalytical method utilizing a small sampling volume (<45 µL) was developed and validated for quantification of TAA in the samples obtained from the ocular pharmacokinetic studies. Aqueous suspensions of TAA with 20% HP-ß-CD produced time course profiles in the aqueous humor at all the dose strengths. However, measurable concentrations and time course of TAA in vitreous humor were achieved only with 4 mg/30µL dose strength.

UGT1A1 and UGT1A3 activity and inhibition in human liver and intestinal microsomes and a recombinant UGT system under similar assay conditions using selective substrates and inhibitors.

In vitro enzyme kinetics and inhibition data was compared for UGT1A1 and UGT1A3 isoforms under similar assay conditions using human liver microsomes (HLM), human intestinal microsomes (HIM) and recombinant UGT (rUGT) enzyme systems.UGT1A1 catalysed ß-estradiol 3-ß-D-glucuronide formation showed allosteric sigmoidal kinetics in all enzyme systems; while UGT1A3 catalysed CDCA 24-acyl-ß-D-glucuronide formation exhibited Michaelis-Menten kinetics in HLM, substrate inhibition kinetics in HIM and rUGT systems. Corresponding Km or S50 concentrations of ß-estradiol and CDCA were employed in the respective UGT inhibition studies.Atazanavir inhibited the production of ß-estradiol 3-ß-D-glucuronide with IC50 values of 0.54 µM and 0.16 µM in HLM and rUGT1A1, respectively. But its inhibition potential was not observed in HIM, indicating potential cross-talk with other high-affinity intestinal UGT isozymes. On the other hand, zafirlukast, a pan UGT inhibitor, exhibited moderate inhibition in HIM with an IC50 value of 16.70 µM. Lithocholic acid, inhibited the production of CDCA 24-acyl-ß-D-glucuronide with IC50 values of 1.68, 1.84, and 12.42 µM in HLM, rUGT1A3, and HIM, respectively.These results indicated that HLM, HIM, and rUGTs may be used as complementary in vitro systems to evaluate hepatic and intestinal UGT mediated DDIs at the screening stage.

Design and evaluation of thermo-responsive nasal in situ gelling system dispersed with piribedil loaded lecithin-chitosan hybrid nanoparticles for improved brain availability.

Piribedil (PBD) is a compound that has shown efficacy in clinical trials to treat motor and non-motor symptoms of Parkinson's disease. However, drug delivery issues like low oral bioavailability, high dosing frequency (3-5 tablets/day), gastrointestinal side-effects reduced the clinical use of PBD. In this work, we have developed lecithin-chitosan hybrid nanoparticles (PBD-LCNs) to improve the direct nose to brain uptake of PBD. PBD-LCNs were optimized using hybrid design approach based on DoE. The mean particle size and drug loading of PBD-LCNs were 147 nm, and 12%, respectively. The PBD-LCNs showed good stability and were found to be nearly spherical in shape. Further, the optimized LCNs were loaded in methylcellulose thermo-responsive in situ gel (PBD-LCN-ISG) to overcome rapid mucociliary clearance upon intranasal administration. Plasma and brain pharmacokinetic studies in rats showed that PBD-LCN-ISG increased the relative bioavailability of PBD in brain (AUCbrain) by about 6.4-folds and reduced the (Cmax)plasma by 3.7-folds when compared to plain intranasal suspension of PBD (PBD-Susp). Further, PBD-Susp showed limited direct nose to brain uptake with DTP values less than 0, while the optimized PBD-LCNs showed DTP value of 56% indicating efficient direct nose to brain uptake. Overall, the development of nanoformulations significantly improved the direct nose to brain uptake of PBD.

Design, optimization and pharmacokinetic evaluation of Piribedil loaded solid lipid nanoparticles dispersed in nasal in situ gelling system for effective management of Parkinson's disease.

Piribedil (PBD) is an anti-Parkinson's drug that gained interest recently due to its unique pharmacological profile. But its clinical use is severely limited by drug delivery issues like high dosing frequency (up to 5 tablets/day), low oral bioavailability (<10%), severe GI side-effects, etc. In this work, we have developed solid lipid nanoparticles (PBD-SLNs) to access the nose to brain pathways for direct uptake of PBD. PBD-SLNs were optimized using design of experiments approach to a mean particle size of 358 nm, and drug loading of 15%. The optimized PBD-SLNs were found to be nearly spherical in shape and showed good stability. Further, the SLNs were loaded in thermoresponsive Methyl Cellulose in situ gel (PBD-SLN-ISG) to delay mucociliary clearance upon intranasal administration in rats. Intranasal administration at the olfactory region was achieved with a cannula-microtip setup. In vivo pharmacokinetic studies showed that PBD-SLN-ISG increased the PBD (AUC)brain by about 4-folds and reduced the (Cmax)plasma by 2.3-folds when compared to plain intranasal suspension of PBD (PBD-Susp). Further, PBD-Susp showed limited direct nose to brain uptake with direct transport percentage (DTP) values less than 0, while the optimized PBD-SLN-ISG showed DTP value of 27% indicating efficient direct nose to brain uptake.

Rufinamide-Loaded Chitosan Nanoparticles in Xyloglucan-Based Thermoresponsive In Situ Gel for Direct Nose to Brain Delivery.

In 2004, the US FDA approved Rufinamide, an anti-epileptic drug under the brand name Banzel®. In 2015, Banzel® received approval for its use in pediatric patients (ages 1-4 years). Rufinamide shows low oral bioavailability due to a low dissolution rate resulting in less of the drug reaching the brain. This has led to the high dose and dosing frequency of Rufinamide. In this work, using the principle of design of experiments (DoE), we have formulated Rufinamide-loaded chitosan nanoparticles and suspended them in a solution of a thermoresponsive polymer-tamarind seed xyloglucan to form a nasal in situ gel for direct nose to brain delivery of Rufinamide. The nanoparticles were characterized for particle size, entrapment efficiency, zeta potential, and physical stability. The in situ gel formulations were characterized for rheological properties, stability, and in vivo plasma and brain pharmacokinetics. Pharmacokinetic parameters were computed for aqueous suspension of nanoparticles and in situ gelling formulation for nanoparticles and compared with the pharmacokinetic parameters of an aqueous suspension of plain Rufinamide. The percentage of direct transport efficiency (% DTE) and direct transport percentage (%DTP) values were calculated for all the formulations. The optimized nanoparticle formulation showed a size of 180 ± 1.5 nm, a zeta potential of 38.3 ± 1.5 mV, entrapment efficiency of 75 ± 2.0%, and drug loading of 11 ± 0.3%. The in situ gelling formulation of nanoparticles showed a solution to the gel transition temperature of 32°C. The %DTE values for aqueous suspension of nanoparticles and in situ gelling formulation for nanoparticles were 988.5 and 1177.3 and the %DTP values were 86.06 and 91.5 respectively.

Cilnidipine loaded poly (ε-caprolactone) nanoparticles for enhanced oral delivery: optimization using DoE, physical characterization, pharmacokinetic, and pharmacodynamic evaluation.

Cilnidipine (CND), an anti-hypertensive drug, possesses low oral bioavailability due to its poor aqueous solubility, low dissolution rate, and high gut wall metabolism. In the present study, an attempt has been made to prepare CND loaded polycaprolactone based nanoparticles (CND-PCL-NPs) by nanoprecipitation method applying the concepts of Design of Experiments. Critical factors affecting particle size and loading efficiency (LE%) were assessed by a hybrid design approach, comprising of Mini Run Resolution IV design followed by Box-Behnken design. Particle size, PDI, zeta potential and LE% of optimized formulations of CND-PCL-NPs were 220.3 ± 2.6 nm, 0.25 ± 0.1, -19.5 ± 0.9 mV, and 46.4 ± 1.8%, respectively. No significant changes were observed in the physical stability of nanoparticles when stored at 25 °C/60% RH over a period of 3 months. Oral pharmacokinetic studies revealed that Fabs of CND-PCL-NPs (0.55) were significantly higher than the CND suspension (0.26). Pharmacodynamic studies have revealed that the mean percent reduction in systolic blood pressure (% ΔSBP) was significantly higher in the case of CND-PCL-NPs (42%) as compared to CND suspension (24%). Optimized CND-PCL-NPs offer great potential in providing higher and sustained antihypertensive effect compared to conventional formulations of CND.

Piribedil loaded thermo-responsive nasal in situ gelling system for enhanced delivery to the brain: formulation optimization, physical characterization, and in vitro and in vivo evaluation.

Methyl cellulose (MC) based nasal in situ gels were developed to enhance the brain delivery of piribedil (PBD), an anti-Parkinson's drug. Different grades of MC and several solutes (NaCl, KCl, Na.Citrate, STPP, PEG-6000, sucrose, etc.) were screened to formulate thermo-responsive nasal in situ gelling systems. Formulations were evaluated for their sol-gel transition temperature and time, rheological behaviour, in vitro drug release, mucociliary clearance (MCC), ex vivo nasal toxicity, and in vivo brain availability studies in Wistar rats. Intranasal (i.n.) administration was carried out using a cannula-microtip setup to deliver PBD at the olfactory region of the nose. The concentration and viscosity grade of MC and also the concentration and type of solute used were found to affect the rheological behaviour of the formulations. Among the solutes tested, NaCl was found to be effective for formulating MC in situ gels. The developed in situ gels significantly delayed the MCC of PBD from the site of administration when compared with conventional suspension (p < 0.05). Further, formulations with higher gel strength showed lower in vitro drug release rate and longer intranasal residence (delayed MCC) (p < 0.05). The absolute brain availability (brain AUC0-t) of PBD increased to 35.92% with i.n. delivery when compared to 4.71% with oral administration. Overall, it can be concluded that intranasal delivery of PBD is advantageous when compared to the currently practiced oral therapy. Graphical abstract.

Self-assembled lecithin-chitosan nanoparticles improve the oral bioavailability and alter the pharmacokinetics of raloxifene.

In this study, we report the development and evaluation of soy lecithin-chitosan hybrid nanoparticles to improve the oral bioavailability of raloxifene hydrochloride. The nanoparticles were formed by interaction of negatively charged soy lecithin with positively charged chitosan. The ratio of soy lecithin to chitosan was critical for the charge, and hence the size of the nanoparticles. The optimal soy lecithin to chitosan ratio was 20:1 to obtain nanoparticles with particle size of 208 ± 3 nm, a ζ-potential of 36 ± 2 mV and an entrapment efficiency of 73 ± 3%. The nanoparticles were also characterized by differential scanning calorimetry and FT-IR spectrophotometer. In-vitro drug release was assessed using dialysis bag method in pH 7.4 buffer. The drug loaded nanoparticles did not cause significant reduction in the cell viability at low doses. Pharmacokinetic studies in female Wistar rats showed significant improvement (~4.2 folds) in the oral bioavailability of the drug when loaded into nanoparticles. Further, the modified everted gut sac study showed that these nanoparticles are taken up by active endocytic processes in the intestine. The ex-vivo mucoadhesion studies proved that the nanoparticles get bound to the mucus layer of the intestine, which in turn correlates with reduced excretion of the drug in faeces. In conclusion, the proposed nanoparticles appear promising for effective oral delivery of poorly bioavailable drugs like raloxifene hydrochloride.

Oral Bioavailability Enhancement of Raloxifene with Nanostructured Lipid Carriers.

Raloxifene hydrochloride (RLX) shows poor bioavailability (<2%), high inter-patient variability and extensive gut metabolism (>90%). The objective of this study was to develop nanostructured lipid carriers (NLCs) for RLX to enhance its bioavailability. The NLC formulations were produced with glyceryl tribehenate and oleic acid. The particle characteristics, entrapment efficiency (EE), differential scanning calorimetry (DSC), in vitro drug release, oral bioavailability (in rats) and stability studies were performed. The optimized nanoparticles were 120 ± 3 nm in size with positive zeta potential (14.4 ± 0.5 mV); % EE was over 90% with the drug loading of 5%. The RLX exists in an amorphous form in the lipid matrix. The optimized RLX-NLC formulation showed sustained release in vitro. The RLX-NLC significantly (p < 0.05) enhanced oral bioavailability 3.19-fold as compared to RLX-free suspension in female Wistar rats. The RLX-NLC can potentially enhance the oral bioavailability of RLX. It can also improve the storage stability.

Pharmacodynamic, pharmacokinetic and physical characterization of cilnidipine loaded solid lipid nanoparticles for oral delivery optimized using the principles of design of experiments.

Cilnidipine (CND), an anti-hypertensive drug, is known to have low oral bioavailability due to its poor aqueous solubility, low dissolution rate and high gut wall metabolism. In the present study, CND loaded compritol based nanoparticles (CND-CMP-NPs) were prepared by emulsification-solvent evaporation method applying the concepts of design of experiments. Critical factors affecting particle size and loading efficiency (LE%) were assessed by hybrid design approach, comprising of Mini Run Resolution IV design followed by Box-Behnken design. Particle size, PDI, zeta potential and LE% of optimized formulations of CND-CMP-NPs were 207.1 ± 2.9 nm, 0.27 ± 0.1, -22.2 ± 1.9 mV and 15.9 ± 1.3% respectively. No significant changes were observed in physical stability of NPs when stored at 25 °C/60% RH over a period of three months. Pharmacokinetic studies revealed that Fabs of CND-CMP-NPs (0.66) was significantly higher than the free CND (0.27). The Cmax and AUC0-∞ of CND-CMP-NPs (572.4 ± 25.3 ng/mL and 5588.6 ± 229.5 ng/mL × h) were significantly higher (Pcal < 0.0001) as compared to free CND (363.6 ± 23.5 ng/mL and 2316.1 ± 163.6 ng/mL × h). MRT of CND-CMP-NPs (9.8 ± 0.9 h) was significantly higher (Pcal < 0.0001) as compared to free CND (5.7 ± 0.5 h). Pharmacodynamic studies showed a maximum of 38% decrease in systolic blood pressure with more than 20% drop in systolic blood pressure sustained for a total duration of 64 h in the case of CND-CMP-NPs as compared to free CND. CND-CMP-NPs not only provide higher and sustained plasma levels of CND but also higher and sustained antihypertensive therapy as compared to free CND.

Comparative study of cilnidipine loaded PLGA nanoparticles: process optimization by DoE, physico-chemical characterization and in vivo evaluation.

Cilnidipine (CND) is known to have low oral bioavailability due to its poor aqueous solubility, low dissolution rate, and high gut wall metabolism. In the present study, CND-loaded PLGA nanoparticles (CND-PLGA-NPs) were prepared with two different grades of PLGA (50:50 and 75:25) by design of experiment. Critical factors affecting particle size and entrapment efficiency (EE%) were assessed by mixed design approach, comprising of Plackett-Burman design followed by rotatable central composite design. Particle size, PDI, zeta potential, and EE% of optimized formulations of CND-PLGA(50:50)-NPs and CND-PLGA(75:25)-NPs were 211.6 ± 1.8 nm, 0.21 ± 0.05, - 15.1 ± 1.6 mV, and 85.9 ± 1.5% and 243.5 ± 2.4 nm, 0.23 ± 0.06, -19.6 ± 1.3 mV, and 92.0 ± 1.2% respectively. No significant changes were observed in physical stability of NPs when stored at 25 °C/60% RH over a period of 3 months. Pharmacokinetic studies revealed that Fabs of CND-PLGA(50:50)-NPs (1.15) and CND-PLGA(75:25)-NPs (2.23) were significantly higher than the free CND (0.26). The Cmax and AUC0-∞ of CND-PLGA(50:50)-NPs (787.42 ± 27.38 ng/mL and 9339.37 ± 252.38 ng/ml × h) and CND-PLGA(75:25)-NPs (803.49 ± 19.63 ng/mL and 18,153.34 ± 543.05 ng/ml × h) were significantly higher (p Ë‚ 0.0001) compared with free CND (367.69 ± 47.22 ng/mL and 2107.95 ± 136.40 ng/ml × h). MRTOral of CND-PLGA(50:50)-NPs (33.36 ± 0.48 h) and CND-PLGA(75:25)-NPs (48.37 ± 0.61 h) were significantly higher (p ˂ 0.0001) compared with free CND (4.69 ± 0.58 h). CND-PLGA-NPs can provide higher and sustained plasma drug levels of CND and be effective in antihypertensive therapy. Graphical abstract.

Development and validation of a bio-analytical method for simultaneous quantification of nebivolol and labetalol in aqueous humor and plasma using LC-MS/MS and its application to ocular pharmacokinetic studies.

Beta blockers is the class of choice of drugs in treatment of open angle Glaucoma. However, many of these drugs suffer from systemic side effects due to their absorption into systemic circulation via nasolachrymal duct. To evaluate the safety and efficacy of nebivolol and labetalol for the treatment of open angle glaucoma, it is important to have a bioanalytical method for measuring the drug concentrations both in aqueous humor and plasma. A simple, sensitive and high throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with protein precipitation technique was developed for simultaneous quantification of nebivolol and labetalol using nebivolol-d4 and metoprolol, respectively, as internal standards in aqueous humor and plasma. Nebivolol and labetalol were monitored in electrospray positive ionization (ESI) mode at transition 406.2/151.1 and 329.2/162.0, respectively. Mobile phase comprised of mixture of aqueous buffer (solvent A) and organic phase (solvent B) (mixture of A:B in the ratio of 30:70, v/v). The aqueous buffer was 5 mM ammonium acetate buffer adjusted to pH 3.5 ±â€¯0.05 with formic acid while the organic phase was a mixture of methanol and acetonitrile in the ratio of 25:75, v/v. Chromatographic separation was achieved using reverse phase Zorbax SB-C18 column (4.6 × 100 mm, 3.5 µm). Method was linear in both the matrices in the concentration range of 0.43-750 ng/mL for nebivolol and 0.39-668 ng/mL for labetalol with r2 > 0.99. Accuracy values, expressed in terms of bias (%), for nebivolol in aqueous humor and plasma were ≤9.6% and ≤11.4% and for labetalol were ≤8.6% and ≤5.9%, respectively. Inter-day and intra-day precision values, expressed in terms of RSD (%), for both the drugs were within 11.4%. No interference was obtained due to matrix components. Mean recovery (%) values in aqueous humor and plasma were 72.4% and 73.0% for nebivolol and 56.7% and 54.4% for labetalol, respectively. No significant degradation was observed in both the drugs in both the matrices when stored at -20 °C for 1 month. Aqueous humor and plasma samples of nebivolol and labetalol on bench top were stable for 18 h and 8 h, respectively. The developed method was applied for determining pharmacokinetic parameters of both drugs in aqueous humor following single dose ocular administration in rabbits.

Design of experiments-based RP - HPLC bioanalytical method development for estimation of Rufinamide in rat plasma and brain and its application in pharmacokinetic study.

This study reports a fully validated HPLC-UV (High Performance Liquid Chromatography- Ultra Violet) method for quantitative estimation of Rufinamide (RUFI), an antiepileptic drug, in rat plasma and brain matrices. A response surface methodology based Box Behnken experimental design, using the principles of Design of Experiments (DoE), was employed to optimize critical chromatographic conditions viz. pH and proportion of the buffer and wavelength of detection, for achieving good sensitivity (peak area) and specificity (number of theoretical plates). A desirability function was employed to identify the optimized conditions, which gave a highest value of 0.971. The optimized chromatographic conditions were: pH of the buffer: 4.7, wavelength of detection: 215 nm and proportion of buffer in mobile phase: 84.7% v/v for responses: 124839.6 mV ∗ min as the peak area (1 µg/mL) and 20,000 as the theoretical plate number for the same. A simple protein precipitation method, using methanol, was employed to extract RUFI from the biological matrices. Piribedil was used as the internal standard (IS). At the optimized conditions, the LOQ values of RUFI in plasma and brain were found to be 13.84 ng/mL and 105.24 ng/g respectively. The developed method was validated as per ICH guidelines and its applicability in analysing RUFI in rat plasma and brain matrices was demonstrated by i.v. administration in rats. The AUC0-t in plasma was found to be 91.9% of AUC0-∞ indicating that the method is very sensitive and can capture almost the entire plasma time course of RUFI, at a dose of 0.5 mg/kg.

Development and validation of rapid and sensitive LC methods with PDA and fluorescence detection for determination of piribedil in rat plasma and brain tissues and their pharmacokinetic application.

Simple, selective and sensitive high-performance liquid chromatographic (HPLC) bioanalytical methods using fluorescence (FL) and photodiode array (PDA) detectors were developed and validated for determination of piribedil (PBD), an anti-Parkinson's drug, in rat plasma and brain samples, with telmisartan as internal standard (IS). Protein precipitation technique was used to extract PBD from both biological matrices. Chromatographic separation was achieved on a Phenomenex Kinetex C18 end-capped column (250 × 4.6 mm, 5 µm), with 38:62 v/v acetonitrile and ammonium acetate buffer (pH 5.0) as mobile phase at 1.0 mL/min flow rate. Linear response in the concentration ranges 5-300 and 150-3000 ng/mL in plasma, and 15-900 and 450-9000 ng/g in brain tissue were achieved in FL and PDA detectors, respectively. The chromatograms were extracted at 239 nm in case of PDA detection and at excitation wavelength of 239 nm and emission wavelength of 385 nm in case of FL detection. FL detection was found to be more sensitive compared with PDA detection. The developed methods were successfully employed in determining the plasma time course, brain distribution and the pharmacokinetic parameters of PBD following intravenous bolus administration of the drug in male Wistar rats.