Mupirocin-Loaded Chitosan Microspheres Embedded in Piper betle Extract Containing Collagen Scaffold Accelerate Wound Healing Activity.

This study reports the formulation of mupirocin-loaded chitosan microspheres embedded in Piper betle extract containing collagen scaffold as combinational drug delivery for improved wound healing. Selection of chitosan type (molecular weight and degree of deacetylation) was carried out based on their antibacterial efficacy. The low molecular weight chitosan was selected owing to the highest antibacterial action against gram-positive as well as gram-negative bacteria. Low molecular weight chitosan-microspheres showed spherical shape with largely smooth surface morphology, 11.81% of mupirocin loading, and its controlled release profile. The XRD, DSC thermograms, and FT-IR spectral analysis revealed the mupirocin loaded in molecularly dispersed or in amorphous form, and having no chemical interactions with the chitosan matrix, respectively. The in vivo study indicates potential effect of the mupirocin, Piper betle, and chitosan in the collagen scaffold in the wound healing efficiency with approximately 90% wound healing observed at the end of 15 days of study for combinational drug-loaded chitosan microspheres-collagen scaffold-treated group. The histopathology examination further revealed tissue lined by stratified squamous epithelium, collagen deposition, fibroblastic proliferation, and absence of inflammation indicating relatively efficient wound healing once treated with combinational drug-loaded chitosan microspheres containing scaffold.

Synthesis and characterization of novel carboxymethyl Assam Bora rice starch for the controlled release of cationic anticancer drug based on electrostatic interactions.

Carboxymethyl Assam Bora rice starch (CM-ABRS) was chemically synthesized in non-aqueous medium with the optimum degree of substitution (DS) of 1.23, and physicochemically characterized by FT-IR, DSC, XRD, and SEM analysis. Comparative evaluation of CM-ABRS with native starch (ABRS) for powder flow characteristics, swelling index, apparent solubility, rheological properties, textural properties, and mucoadhesive studies were carried out. The aim of the current work was to investigate the potential of CM-ABRS as a novel carrier for the water-soluble chemotherapeutic, doxorubicin hydrochloride (DOX). Formation of drug/polymer complex (DOX-CM-ABRS) via electrostatic interaction has been evaluated for the controlled release of DOX in three different pH media (phosphate-buffered saline (PBS), pH 7.4, 6.8, and 5.5). In vitro drug release studies illustrated faster release of drug in PBS at pH 5.5 as compared to pH 6.8 and pH 7.4, respectively, indicating the importance of pH-sensitive drug release from the DOX-CM-ABRS complex in malignant tissues.

Intracellular Availability of pDNA and mRNA after Transfection: A Comparative Study among Polyplexes, Lipoplexes, and Lipopolyplexes.

Intracellular availability of nucleic acids from synthetic vectors is critical and directly influences the transfection efficiency (TE). Herein, we evaluated the TE of polymer- and lipid-based nanoplexes (polyplexes, lipoplexes and lipopolyplexes) of EGFP-encoding mRNA and pDNA. To determine the translation and transcription efficiency of each nucleic acid nanoplex, in vitro expression was measured in HEK293T7 cells that permit gene expression in the cytoplasmic region. Globally, mRNA transfection profile was well corroborative with cytoplasmic transfection of pT7-pDNA as well as with nuclear transfection of pCMV-DNA. Irrespective of the nucleic acid, high TE was observed with histidinylated l-polyethylenimine (His-lPEI) polyplexes and dioleyl succinyl paromomycin/O,O-dioleyl-N-histamine phosphoramidate (DOPS/MM27) lipoplexes. Moreover, His-lPEI polyplexes yielded higher in vitro expression of EGFP for pDNA than for mRNA. Furthermore, a significant enhancement in the TE in the presence of an excess of His-lPEI was observed indicating that this polymer promotes cytosolic delivery. Compared to other nanoplexes, His-lPEI polyplex showed high intracellular availability of DNA and mRNA along with low cytotoxicity, owing to its rapid (complete or partial) unpacking in the cytosol and/or endosomes. This study gives an insight that, whether with mRNA or pDNA, enhancing nanoplex unpacking in the endosomes and cytosol would improve the delivery of nucleic acid in the cytosol and particularly in the case of pDNA where a sufficient available amount of pDNA in the cytoplasm would definitely improve its transport toward the nucleus.

Enhanced anti-tumor efficacy of paclitaxel with PEGylated lipidic nanocapsules in presence of curcumin and poloxamer: In vitro and in vivo studies.

Cancer chemotherapeutic drug containing PEGylated lipidic nanocapsules (D-LNCs) were formulated by the controlled addition of organic phase (combined solution of paclitaxel and curcumin in a mixture of oleic acid and MPEG2000-DSPE (90:2.5 molar ratio) in acetone) to the aqueous phase (consist of Poloxamer 407 as emulsifying agents and glycerol as a co-solvent) at a temperature of 55-60°C followed by evaporation of organic solvent. The obtained pre-colloidal dispersion of D-LNCs was processed through high pressure homogenization to get more uniformly and nano-sized particles. Effect of concentration of emulsifying agent and process variables of high pressure homogenization (pressure and number of cycles) on average particle size and entrapment efficiency was further investigated by constructing Box-Behnken experimental design to achieve the optimum manufacturing process. D-LNCs were characterized by dynamic light scattering, scanning and transmission electron microscopy, Fourier transform infrared spectroscopy, and differential scanning calorimetry. In vitro release studies showed a sustained release pattern of drug from the PEGylated D-LNCs, whereas in vivo pharmacokinetic studies after a single-dose intravenous (i.v.) administration of paclitaxel (15mg/kg) in Ehrlich ascites tumor (EAT)-bearing female Swiss albino mice showed a prolonged circulation time and slower elimination of paclitaxel from D-LNCs as compared with marketed formulation (Paclitec®). From the plasma concentration vs. time profile, i.v. bioavailability (AUC0-∞) of paclitaxel from D-LNCs was found to be increased approximately 2.91-fold (P<0.001) as compared to Paclitec®. In vitro cell viability assay against MCF-7 and MCF-7/ADR cell lines, in vivo biodistribution studies and tumor inhibition study in EAT-bearing mice, all together prove its significantly improved potency towards cancer therapy.

Collagen loaded nano-sized surfactant based dispersion for topical application: formulation development, characterization and safety study.

Collagen, a high molecular weight, hydrophilic and highly abundant protein is known to have anti-ageing, anti-wrinkle, anti-acne, anti-scar and wound healing properties. High molecular weight and hydrophilic nature hinder its effective topical delivery. So, the objective of present study was to develop effective topical nano-surfactant dispersion (NSD) for collagen delivery. NSD was prepared from sorbitan monostearate (Span60) and cholesterol using ethanol injection method followed by probe sonication. NSD was characterized for entrapment efficiency (%EE), size and size distribution (Z-avg and polydispersity index (PDI)), shape, zeta-potential (ζ), in vitro drug release, skin hydration and skin irritation test and histopathological examination. Optimized NSD (NSD3) had %EE, z-avg, PDI and ζ-potential of 77.56% ± 1.09%, 158.1 ± 2.31 nm, 0.211 and -17.2 ± 0.64 mV, respectively. In in vivo skin hydration test, NSD treatment showed nearly 2.5-fold and 3-fold increase in the thickness of stratum corneum (SC) as compared to the collagen gel treated and untreated skin, respectively. The mean scores of skin irritation test in two animal species, rats and rabbits, were found to be 1.42 ± 1.01 and 1.71 ± 0.29, respectively, indicating the non-irritant nature of collagen loaded NSD. Histopathology of the skin after application of developed NSD showed non-significant changes in skin anatomy indicating its safe nature.

Peptide spiders are emerging as novel therapeutic interventions for nucleic acid delivery.

The FDA has approved many nucleic acid (NA)-based products. The presence of charges and biological barriers however affect stability and restrict widespread use. The electrostatic complexation of peptide with polyethylene glycol-nucleic acids (PEG-NAs) via nonreducible and reducible agents lead to three parts at one platform.. The reducible linkage made detachment of siRNA from PEG easy compared with a nonreducible linkage. A peptide spider produces a small hydrodynamic particle size, which can improve drug release and pharmacokinetics. Several examples of peptide spiders that enhance stability, protection and transfection efficiency are discussed. Moreover, this review also covers the challenges, future perspectives and unmet needs of peptide-PEG-NAs conjugates for NAs delivery.

Colorectal cancer targeted Irinotecan-Assam Bora rice starch based microspheres: a mechanistic, pharmacokinetic and biochemical investigation.

The purpose of this investigation was to evaluate the colon-targeted Irinotecan Hydrochloride (ITC-HCl) loaded microspheres by pharmacokinetic and biochemical studies. The microspheres were prepared by double emulsion solvent evaporation method with natural polymer Assam Bora rice starch. The microspheres were characterized for their micromeritics properties, incorporation efficiency, in vitro and in vivo drug release studies. The release study confirmed the insignificant release of ITC-HCl in physiological condition of stomach and small intestine and major drug release in the caecal content. In vivo release study of the optimized microsphere was compared with immediate release (IR) ITC-HCl. ITC-HCl was distributed predominantly in the upper GI tract from the IR, whereas ITC-HCl was distributed primarily to the lower part of GI tract from the microspheres formulation. Enhanced levels of liver enzymes were found in animals given IR ITC-HCl as well as augmented levels of serum albumin, creatinine, leucocytopenia and thrombocytopenia was also observed. In summary, Assam Bora rice starch microspheres exhibit slow and extended release of ITC-HCl over longer periods of time with reduced systemic side-effects.

Assam Bora rice starch based biocompatible mucoadhesive microsphere for targeted delivery of 5-fluorouracil in colorectal cancer.

The aim of this study was to develop novel colon targeted mucoadhesive microspheres (MAMs) for site specific delivery of 5-fluorouracil (5-FU) to colon without the drug being released in the stomach or small intestine. MAMs were prepared using Assam Bora rice starch, a natural mucoadhesive polymer, by a double emulsion solvent evaporation method. The microspheres were characterized for their shape, size, surface morphology, size distribution, incorporation efficiency, and in vitro and in vivo drug release studies. The release study confirmed the insignificant release of 5-FU in physiological condition of stomach and small intestine and major drug release in the cecal content. In vivo release study of the optimized MAMs was compared with immediate release (IR) 5-FU. 5-FU was distributed predominantly in the upper GI tract from the IR, whereas 5-FU was distributed primarily to the lower part of the GI tract from the MAM formulation. Enhanced levels of liver enzymes were found in animals given IR 5-FU as well as augmented levels of serum albumin, creatinine, leucocytopenia and thrombocytopenia was also observed. Thus to sum up, it can be appropriately established that the 5-FU release pattern from MAMs exhibits slow and extended release over longer periods of time with reduced systemic side effects.

Development and evaluation of nanosized niosomal dispersion for oral delivery of Ganciclovir.

Encapsulation of Ganciclovir in lipophilic vesicular structure may be expected to enhance the oral absorption and prolong the existence of the drug in the systemic circulation. So the purpose of the present study was to improve the oral bioavailability of Ganciclovir by preparing nanosized niosomal dispersion. Niosomes were prepared from Span40, Span60, and Cholesterol in the molar ratio of 1:1, 2:1, 3:1, and 3:2 using reverse evaporation method. The developed niosomal dispersions were characterized for entrapment efficiency, size, shape, in vitro drug release, release kinetic study, and in vivo performance. Optimized formulation (NG8; Span60:Cholesterol 3:2 molar ratio) has shown a significantly high encapsulation of Ganciclovir (89±2.13%) with vesicle size of 144±3.47 nm (polydispersity index [PDI]=0.08). The in vitro release study signifies sustained release profile of niosomal dispersions. Release profile of prepared formulations have shown that more than 85.2±0.015% drug was released in 24 h with zero-order release kinetics. The results obtained also revealed that the types of surfactant and Cholesterol content ratio altered the entrapment efficiency, size, and drug release rate from niosomes. In vivo study on rats reveals five-time increment in bioavailability of Ganciclovir after oral administration of optimized formulation (NG8) as compared with tablet. The effective drug concentration (>0.69 µg/mL in plasma) was also maintained for at least 8 h on administration of the niosomal formulation. In conclusion, niosomes can be proposed as a potential oral delivery system for the effective delivery of Ganciclovir.

mRNA Lipoplexes with Cationic and Ionizable α-Amino-lipophosphonates: Membrane Fusion, Transfection, mRNA Translation and Conformation.

Cationic liposomes are attractive carriers for mRNA delivery. Here, mRNA lipoplexes (LX) were prepared with the cationic lipids α-aminolipophosphonate (3b) or imidazolium lipophosphoramidate (2) associated with various α-aminolipophosphonates co-lipids comprising protonable groups (imidazole or pyridine) and DOPE. Physicochemical parameters of liposomes and their membrane fusion activity were measured. LXs comprising either 3b- or 2- allowed transfection of ~25% and 40% of dendritic cells with low cytotoxicity, respectively; the efficiency increased up to 80% when 2 was combined with the imidazole-based co-lipid 1. The transfections were high with 3b/1, 3b/DOPE, 2/1 and 2/DOPE LXs. We observed that the transfection level was not well correlated with the acid-mediated membrane fusion activity of liposomes supposed to destabilize endosomes. The mRNA release from LXs and its translation capacity after release were studied for the most efficient LXs. The results showed that the more mRNA was condensed, the poorer the translation efficiency after release was. In contrast to DNA, circular dichroism performed on mRNA complexed with 2/DOPE revealed the presence of denatured mRNA in LXs explaining this lack of translation efficiency. This is an important parameter that should be stressed for the preparation of mRNA LXs with a conserved mRNA translation activity.

Stability-indicating ultra-performance liquid chromatography method for the estimation of thymoquinone and its application in biopharmaceutical studies.

Thymoquinone (THQ) is known for its neuroprotective and anti-convulsant properties in preclinical studies. We herewith describe a simple, rapid, selective, sensitive and stability-indicating UPLC method for the estimation of THQ and its application to biopharmaceutical studies such as in vitro release from nanoparticulate system and in vivo pharmacokinetic study. The method employed gradient elution using a Waters Acquity HSS-T3 C(18) (100 × 2.1 mm, 1.8 µm) UPLC column. The mobile phase consisted of water and acetonitrile, pumped at a flow rate of 0.5 mL/min. The injection volume was 5 µL and THQ was monitored at 294 nm wavelength with a total run time of 6 min. In solution as well as in plasma, the method was found to be linear (r ≥ 0.998), precise (CV ≤ 2.45%) and accurate (recovery ≥ 84.8%) in the selected concentration range of 0.1-0.8 µg/mL. Forced degradation studies revealed that THQ undergoes degradation under acidic, basic, oxidation and UV light stress conditions. However, the developed UPLC method could effectively resolve degradation product peaks from THQ. Further, no interference was found at the retention time of THQ from any plasma components, indicating selectivity of the developed method. For solutions, the limits of detection and quantitation of the method were found to be 0.001 and 0.0033 µg/mL, respectively; while in plasma they were 0.006 and 0.02 µg/mL, respectively. The validated method was successfully applied to quantify THQ in dissolution medium as well as oral in vivo pharmacokinetic study of THQ suspension and THQ- solid lipid nanoparticle (THQ-SLN) formulation. A 2-fold increase in the relative bioavailability was observed with the THQ-SLN compared with THQ. The results indicate that the SLN significantly increased plasma concentrations and retention within the systemic circulation.

Novel nanoemulsion gel containing triple natural bio-actives combination of curcumin, thymoquinone, and resveratrol improves psoriasis therapy: in vitro and in vivo studies.

Curcumin, resveratrol, and thymoquinone are the potential natural bio-actives reported with good anti-psoriatic activity. However, poor aqueous solubility and limited skin permeation of these natural bio-actives hinder their effective delivery and potential therapeutic outcome. In this regard, current research work focuses on the design and optimization of nanoemulsion (NE) gel formulation for the concurrent delivery of these three drugs. The NE system is consisting of oleic acid as oil phase, Tween 20 as surfactant, and PEG 200 as co-surfactant. The optimized formulation exhibited the droplet size 76.20 ± 1.67 nm, PDI of 0.12 ± 0.05, RI of 1.403 ± 0.007, and viscosity of 137.9 ± 4.07 mp. Carbopol 940 (0.5% w/v) was used as the gelling agent to prepare the NE gel which exhibited a good texture profile. The optimized formulation exhibited a higher % of growth inhibition on A-431 cells and demonstrated good anti-angiogenic activity in the HET-CAM test. Finally, in vivo studies in Balb/c mice model showed improved anti-psoriatic conditions which indicated that the triple natural bio-actives combination in nanoemulgel formulation is effective in the management of psoriasis.

Lipid/polymer-based nanocomplexes in nucleic acid delivery as cancer vaccines.

Cancer vaccines consist of nucleic acid derivatives such as plasmid DNA, small interfering RNA and mRNA, and can be customized according to the patient's needs. Nanomedicines have proven to be exceptionally good as miniaturized drug carriers, and thus they offer great advantages for delivering cancer vaccines. This review provides an overview of the literature on cancer vaccines, from their inception to current developments in the field.

Quantitative analysis of safranal in saffron extract and nanoparticle formulation by a validated high-performance thin-layer chromatographic method.

INTRODUCTION: Safranal is an effective anticonvulsant shown to act as an agonist at GABA(A) receptors. Nose to brain delivery via nanoparticle formulation might improve its brain delivery. A selective and sensitive analytical method is required for evaluation of safranal-based novel drug delivery systems. OBJECTIVE: To develop and validate a high-performance thin-layer chromatographic (HPTLC) method for the quantitative analysis of safranal as bulk, in saffron extract and in developed safranal-loaded nanoparticle formulation. METHODOLOGY: Chromatographic separation was achieved on silica gel pre-coated TLC aluminium plates 60F-254, using n-hexane:ethyl acetate (9 : 1, v/v) as the mobile phase. Quantitative analysis was carried out by densitometry at a wavelength of 310 nm. The method was validated and applied to detect related impurities, to analyse safranal in saffron extract and to evaluate safranal-loaded nanoparticles. RESULTS: Compact spots of safranal were observed at R(f) value 0.51 +/- 0.02. The method was linear (r = 0.9991) between 0.5 and 5.0 ng/spot. The intra- and inter-day precisions were 1.08-2.17 and 1. 86-3.47%, respectively. The limit of detection was 50 ng/spot and the limit of quantification was 150 ng/spot. The method proved to be accurate (recovery 97.4-102.0%) and was selective for safranal. Evaluation of safranal-loaded nanoparticle formulation demonstrated drug loading of 23.0%, encapsulation efficiency of 42.0% and sustained drug release following biphasic pattern. CONCLUSION: The present method is useful for the quantitative and qualitative analysis of safranal and safranal-loaded nanoparticle formulation. It provides significant advantages in terms of greater specificity and rapid analysis.

Co-encapsulation of docetaxel and thymoquinone in mPEG-DSPE-vitamin E TPGS-lipid nanocapsules for breast cancer therapy: Formulation optimization and implications on cellular and in vivo toxicity.

Rationally designed combination nano-therapy approaches have emerged as a promising strategy for resistant breast cancer treatment. This research reports the combination of Docetaxel (DTX) and Thymoquinone (THQ) co-encapsulated within long circulating sub-100 nm mPEG-DSPE-Vitamin E TPGS-Lipid nanocapsules (DxTq-LNCs). DxTq-LNCs with sufficient drug loading exhibited controlled drug release, enhanced protein binding resistance (confirming its long circulation in physiological environment and suitability for iv application) and retained the antioxidant effects of THQ. DxTq-LNCs were further subjected to cytotoxicity analysis against human breast cancer cells (MCF-7 & MDA-MB-231). The presence of multidrug resistance (MDR) reversal agents; Vitamin E TPGS and THQ, along with the nanoencapsulation, re-sensitized the resistant triple negative breast cancer (TNBC) cells to the anticancer effects of DTX. Greater inhibition of cell migration indicated improved anti-metastatic effects. Drastic changes in cellular morphology indicated by nuclear fragmentation (the hall marks of apoptosis), were observed upon DxTq-LNCs treatment to the breast cancer cells. In vivo toxicity studies indicated no substantial blood biochemical and histological changes with near normal appearance of kidney and liver tissue sections upon DxTq-LNCs treatment in contrast to free drug that showed parenchymal degeneration, areas of interstitial haemorrhage, glomerular atrophy and other histological changes, indicating hepato- and nephro-protective potential of DxTq-LNCs. Furthermore, enhanced antitumor efficacy was observed with DxTq-LNCs treatment to mice bearing ehrlich ascites carcinoma. Thus, nanocapsules presents a simple yet effective approach for successful combination chemotherapy with reduced unwanted toxicity.

Emerging advances in synthetic cancer nano-vaccines: opportunities and challenges.

Introduction: Cancer immunotherapy is a fast-growing field that has achieved tremendous progress in recent years. It is one of the most potent tools that can activate the immune system against cancer. Nevertheless, the development of safe and effective vaccines to overcome emerging new disease remains challenging since several emerging antigens are poorly immunogenic. Nanotechnology has provided a realistic resolution for the drawback of traditional cancer immunotherapy. Area covered: This review discusses different cancer immunotherapy approaches focusing on recent advancements in nanomedicine-based cancer immunotherapy. The literature review method includes inclusion and exclusion criteria to categorize important articles. The literature survey was carried out using PubMed, Google Scholar, Scopus, and the Saudi digital library. Expert opinion: In the last two decades, the development and application of nanoparticles incorporating antigen/adjuvant in cancer immunotherapy have experienced rapid growth. Soon, progressively multifaceted nanovaccines presenting different antigens and co-delivered with antigens will be clinically translated. Better understanding and improved knowledge of nanomedicines-based delivery approaches and immunostimulatory action, and in-vivo biodistribution would inevitably facilitate the altruistic design of cancer nanovaccine for humankind.

Omega-3 fatty acids as adjunctive therapeutics: prospective of nanoparticles in its formulation development.

Omega-3 polyunsaturated fatty acids (ω-3-PUFAs) are dietary components that have been extensively recognized for their therapeutic value and have shown diverse therapeutic effects including anti-inflammatory, antiarrhythmic, antithrombotic, immunomodulatory and antineoplastic activities. Most of the ω-3-PUFAs are obtained through diet or supplements because the body does not synthesize them. The high instability of ω-3-PUFAs to oxidative deterioration, lower bioavailability at the target tissues and reduced bioactivity of ω-3-PUFAs is an impediment for achieving their therapeutic potential. The present review provides an overview of potential therapeutic activities of ω-3-PUFAs and different novel technical approaches based on nanotechnology, which have been emphasized to overcome instability problems as well as enhance the bioactivity of ω-3-PUFAs. Future prospects related to this area of research are also provided.

Improved chemotherapeutic efficacy against resistant human breast cancer cells with co-delivery of Docetaxel and Thymoquinone by Chitosan grafted lipid nanocapsules: Formulation optimization, in vitro and in vivo studies.

In recent years, multi-targeted chemotherapeutic combinations have received considerable attention in solid tumor chemotherapy. Here, we optimized low-molecular-weight chitosan (CS)-grafted lipid nanocapsules (LNCs, referred to as CLNCs) for the co-delivery of docetaxel (DTX) and thymoquinone (THQ) to treat drug-resistant breast cancer. We first screened size reduction techniques (homogenization vs ultrasonication), and then the 33-Box-Behnken design was employed to determine optimal conditions of the final LNCs with the desired quality attributes. Uncoated LNCs had a particle size of 141.7 ± 2.8 nm (Polydispersity index, PdI: 0.17 ± 0.02) with entrapment efficiency (%EE) of 66.1 ± 3.5 % and 85.3 ± 3.1 % for DTX and THQ, respectively. The CS functionalization of LNCs improved the uptake and endosomal escape effect, and led to a significantly higher cytotoxicity against MCF-7 and triple-negative (MDA-MB-231) breast cancer cells. Furthermore, an enhanced antiangiogenic effect was observed with DTX- and THQ-carrying CLNCs in the Chick embryo chorioallantoic membrane (CAM) assay.

Therapeutic Nanoemulsion: Concept to Delivery.

Nanoemulsions (NEs) or nanometric-scaled emulsions are transparent or translucent, optically isotropic and kinetically stable heterogeneous system of two different immiscible liquids namely, water and oil stabilized with an amphiphilic surfactant having droplet size ranges up to 100 nm. They offer a variety of potential interests for certain applications: improved deep-rooted stability; excellent optical clarity; and, enhanced bioavailability due to its nanoscale of particles. Though there is still comparatively narrow insight apropos design, development, and optimization of NEs, which mainly stems from the fact that conventional characteristics of emulsion development and stabilization only partly apply to NEs. The contemporary article focuses on the nanoemulsion dosage form journey from concept to key application in drug delivery. In addition, industrial scalability of the nanoemulsion, as well as its presence in commercial and clinical practice, are also addressed.

Systematic development of a bioanalytical UPLC-MS/MS method for estimation of risperidone and its active metabolite in long-acting microsphere formulation in rat plasma.

A systematic approach to develop a UPLC-MS/MS method was applied for quantifying of risperidone (RISP), its active metabolite, 9-hydroxy risperidone (9-OH-RISP) and internal standard (propranolol) in rat plasma. Liquid-liquid extraction was performed using methyl tert-butyl ether for quantification of drug and its active metabolite by MS detection in the positive ion mode. Acquity UPLC system with BEH C18 (2.1 mm × 100 mm, particle size 1.7 µm) column was used along with acetonitrile (0.1% formic acid)-2 mM (milli mole) ammonium acetate in isocratic condition was used as the mobile phase. Detection was performed by multiple reactions monitoring with precursor-to-product ion transitions with m/z 411.2 â†’ 191.0 for RISP, m/z 427.2 â†’ 207.0 for 9-OH-RISP and m/z 260.1 â†’ 116.0 for IS. The method was validated as per the FDA guidance on bioanalytical method validation. Linearity (r2 = 0.999) was observed in the drug concentration ranging between 0.1 and 50 ng mL-1, while all other parameters were found to be within the acceptable ranges. Method robustness was optimized by Box-Behnken design to monitor the influential variables to achieve maximal recovery of the analytes in the rat plasma. Pharmacokinetic evaluation of the analytes from long-acting microparticles in rat plasma showed two peaks indicating an initial burst effect within 24 h of administration followed by controlled drug release pattern upto 45 days, while marketed formulation (Risperdal Consta®) showed no plasma concentration during the lag-time of 21 days followed by maximal drug absorption between 28 and 40 days.