PEGylated γ-tocotrienol isomer of vitamin E: Synthesis,characterization, in vitro cytotoxicity,and oral bioavailability

Vitamin E refers to a family of eight isomers divided into two subgroups, tocopherols and the therapeutically active tocotrienols (T₃). The PEGylated α-tocopherol isomer of vitamin E (vitamin E TPGS) has been extensively investigated for its solubilizing capacity as a nonionic surfactant in various drug delivery systems. Limited information, however, is available about the PEG conjugates of the tocotrienol isomers of vitamin E. In this study two PEGylated γ-T₃ variants with mPEG molecular weights of 350 (γ-T₃PGS 350) and 1000 (γ-T₃PGS 1000) were synthesized by a two-step reaction procedure and characterized by ¹H NMR, HPLC, and mass spectroscopy. The physical properties of their self-assemblies in water were characterized by zeta, CMC, and size analysis. Similar physical properties were found between the PEGylated T₃ and vitamin E TPGS. PEGylated T₃ were also found to retain the in vitro cytotoxic activity of the free T₃ against the MCF-7 and the triple-negative MDA-MB-231 breast cancer cells. PEGylated γ-T₃ also increased the oral bioavailability of γ-T₃ by threefolds when compared to the bioavailability of γ-T₃ formulated into a self-emulsified drug delivery system. No significant differences in biological activity were found between the PEG 350 and 100 conjugates. Results from this study suggest that PEGylation of γ-T₃ represents a viable platform for the oral and parenteral delivery of γ-T₃ for potential use in the prevention of breast cancer.     

Safety and pharmacokinetic studies of liposomal antioxidant formulations containing N-acetylcysteine, α-tocopherol or γ-tocopherol in beagle dogs

Abstract

The safety and pharmacokinetic profile of liposomal formulations containing combinations of the antioxidants α-tocopherol, γ-tocopherol or N-acetylcysteine in beagle dogs was examined. Each group consisted of beagle dogs of both genders with a control group receiving empty dipalmitoylphosphatidylcholine (DPPC) liposomes (330?mg/kg DPPC, EL), and test groups receiving liposomes prepared from DPPC lipids with (i) N-acetylcysteine (NAC) (60?mg/kg NAC [L-NAC]); (ii) NAC and α-tocopherol (αT) (60?mg/kg NAC and 25?mg/kg α-tocopherol [L-αT-NAC]) and (iii) NAC and γ-tocopherol (60?mg/kg NAC and 25?mg/kg γ-tocopherol (γT) [L-γT-NAC]). The dogs in the control group (EL) and three test groups exhibited no signs of toxicity during the dosing period or day 15 post treatment. Weight gain, feed consumption and clinical pathology findings (hematology, coagulation, clinical chemistry, urinalysis) were unremarkable in all dogs and in all groups. Results from the pharmacokinetic study revealed that the inclusion of tocopherols in the liposomal formulation significantly increased the area under the curve (AUC) and β-half life for NAC; the tocopherols had greater impact on the clearance of NAC, where reductions of central compartment clearance (CL) ranged from 56% to 60% and reductions of tissue clearance (CL₂) ranged from 73% to 77%. In conclusion, there was no treatment-related toxicity in dogs at the maximum feasible dose level by a single bolus intravenous administration while the addition of tocopherols to the liposomal formulation prolonged the circulation of NAC in plasma largely due to a decreased clearance of NAC.     

Preparation of meloxicam-β-cyclodextrin-polyethylene glycol 6000 ternary system: characterization, in vitro and in vivo bioavailability

Ternary complexes of meloxicam (ML), a poorly water-soluble anti-inflammatory drug, with β-cyclodextrin (βCD) and polyethylene glycol (PEG) 6000 were prepared from an equimolar (ML-βCD) and 10% of PEG. Characterization of the ternary complex was carried out by differential scanning calorimetry and X-ray diffractometry. The solubility of ML increased as a function of increasing the concentration of βCD and PEG 6000. Ternary system increased significantly ML solubility in water. Ternary complexes improved drug release compared with ML and ML-βCD. The oral bioavailability of ML-βCD-PEG was investigated by administration to rat and compared with ML and ML-βCD. The results confirmed that the oral bioavailability of ML was significantly improved by complexation with βCD in the presence of PEG.     

Synthesis,in vivo and in silico analgesic and anti-inflammatory studies of α-D-ribofuranose derivatives

Five α-D-ribofuranose analogues (2, 3, 4, 5 and 6) were synthesized in good yields from 3-O-benzyl-4-C-(hydroxymethyl)-1, 2-O-isopropylidene-α-D-ribofuranose (1). The synthesized compounds were then subjected to analgesic, anti-inflammatory, antimicrobial and antioxidant assays. Compound 3 demonstrated 79.74% (P < 0.001) writhing inhibition and highest reaction time of 2.55 ± 0.13 min (P < 0.001) after 30 min of oral administration in peripheral and central analgesic assay, respectively, at 50 mg/kg dose. Compound 2 and 6 exhibited significant anti-inflammatory activity at 100 mg/kg dose with paw edema inhibition of 91.15% (P < 0.001) and 95.13% (P < 0.001), respectively, in 4th hour. The synthesized analogues did not show notable antioxidant and antibacterial properties. Molecular docking study revealed higher binding affinity of −8.1 kcal/mol and −8.9 kcal/mol of compound 3 towards cyclooxygenase-1 and phospholipase A_2, respectively, compared to −7.7 and −7.6 kcal/mol respectively for corresponding native ligands. Compound 2 demonstrated binding affinity of −9.1 kcal/mol towards interleukin-1 receptor-associated kinase-4 compared to −8.7 kcal/mol of the native ligand. The molecular properties related to drug likeness of compounds were found to be within acceptable range. Synthesized D-ribofuranose analogues demonstrated promising analgesic and anti-inflammatory activities and further development may lead to new potent analgesic and anti-inflammatory agents.     

Enhanced ocular bioavailability of fluconazole from niosomal gels and microemulsions: formulation,optimization, and in vitro–in vivo evaluation

Fungal keratitis may cause vision loss if it is not treated. Methods other than ocular delivery exhibited several limitations. No previous studies investigated and compared ocular bioavailability of fluconazole (FLZ) from niosomal gels and microemulsions. Niosomal gels of FLZ (0.3% w/w) based on Span^® 60 and cholesterol (CH) using 1% w/w carbopol^® 934 (CP) were evaluated. FLZ microemulsions (0.3% w/v) containing isopropyl myristate (IPM, as oil phase) and a 3:1 mixture of Tween^® 80 (as surfactant) and polyethylene glycol 400 (PEG 400, as cosurfactant) were characterized. Optimized formulations were compared for their ocular bioavailability in rabbit’s. Nanoscopic niosomes (63.67–117.13?nm) and microemulsions (57.05–59.93?nm) showed respective negative zeta potential ranges of ?45.37 to ?61.40 and ?20.50 to ?31.90?mV and sustained release up to 12?h. Entrapment efficiency (EE%) of niosomes ranged from 56.48% to 70.67%. Niosomal gels were more sustainable than niosomes and microemulsions. The most stable niosomal gel based on Span^® 60 and CH at a molar ratio of 5:5 and microemulsion containing 45% w/w IPM and 40% w/w of 3:1 Tween^® 80-PEG 400 mixture significantly (p?<?0.0001) enhanced FLZ ocular bioavailability compared with its solution. Niosomal gel showed higher bioavailability than microemulsion by ≈2-fold.     

Preparation of meloxicam–β-cyclodextrin–polyethylene glycol 6000 ternary system: characterization,in vitro and in vivo bioavailability

Ternary complexes of meloxicam (ML), a poorly water-soluble anti-inflammatory drug, with β-cyclodextrin (βCD) and polyethylene glycol (PEG) 6000 were prepared from an equimolar (ML–βCD) and 10% of PEG. Characterization of the ternary complex was carried out by differential scanning calorimetry and X-ray diffractometry. The solubility of ML increased as a function of increasing the concentration of βCD and PEG 6000. Ternary system increased significantly ML solubility in water. Ternary complexes improved drug release compared with ML and ML–βCD. The oral bioavailability of ML–βCD–PEG was investigated by administration to rat and compared with ML and ML–βCD. The results confirmed that the oral bioavailability of ML was significantly improved by complexation with βCD in the presence of PEG.     

Correlation of the intrinsic clearance of donepezil (Aricept®) between in vivo and in vitro studies in rat,dog and human

1. Donepezil hydrochloride (Aricept®) is used for the treatment of Alzheimer's disease. Here the correlation of the intrinsic clearance (Cl_int) of donepezil between the in vivo and in vitro states was studied in rat, dog and human. 2. In an experiment with ¹⁴C-donepezil and human microsomes the routes of metabolism were identified as N-dealkylation and O-demethylation, and no unknown metabolites were detected. 3. The Cl_int of donepezil in the male rat, female rat, dog and human liver microsomes were 33.7, 13.4, 37.0 and 6.35 μl/min/mg microsomal protein respectively, and sex difference in rat and interspecies difference in the estimated Cl_int were found. 4. After a single intravenous administration to the male rat, female rat and dog, total plasma clearance (Cl^P_total) was 78.6, 29.5 and 88.3 ml/min/kg respectively, and a sex difference was observed in rat. 5. After a single oral administration to the male rat, dog and healthy volunteer, Cl^P_total/F was 140, 105 and 2.35 ml/min/kg respectively, and remarkable differences were observed between animals and man. 6. The contribution of renal clearance to blood clearance (Cl_r) was low in all species. The predicted in vitro hepatic clearance (Cl_h-pre) was in the rank order: male rat (15.91 ml/min/kg) &gt; dog (7.96) &gt; female rat (7.67) &gt; human (1.04). Although Cl_h-pre was underestimated, Cl_h-pre was significantly correlated with that of ClBtotal in the different animal species and in man, indicating that the in vitro-in vivo ranking order was conserved.     

Combination of α-glucosidase inhibitor and ribavirin for the treatment of dengue virus infection in vitro and in vivo

Cellular α-glucosidases I and II are enzymes that sequentially trim the three terminal glucoses in the N-linked oligosaccharides of viral envelope glycoproteins. This process is essential for the proper folding of viral glycoproteins and subsequent assembly of many enveloped viruses, including dengue virus (DENV). Imino sugars are substrate mimics of α-glucosidases I and II. In this report, we show that two oxygenated alkyl imino sugar derivatives, CM-9-78 and CM-10-18, are potent inhibitors of both α-glucosidases I and II in vitro and in treated animals, and efficiently inhibit DENV infection of cultured human cells. Pharmacokinetic studies reveal that both compounds are well tolerated at doses up to 100mg/kg in rats and have favorable pharmacokinetic properties and bioavailability in mice. Moreover, we showed that oral administration of either CM-9-78 or CM-10-18 reduces the peak viremia of DENV in mice. Interestingly, while treatment of DENV infected mice with ribavirin alone did not reduce the viremia, combination therapy of ribavirin with sub-effective dose of CM-10-18 demonstrated a significantly enhanced antiviral activity, as indicated by a profound reduction of the viremia. Our findings thus suggest that combination therapy of two broad-spectrum antiviral agents may provide a practically useful approach for the treatment of DENV infection.     

Orthostatic hypotensive effect of antipsychotic drugs in Wistar rats by in vivo and in vitro studies of α1-adrenoceptor function

Rationale It has been suggested that the increase in serotonin transmission induced by indirect agonists such as fenfluramine and fluoxetine attenuates cue-elicited reinstatement of cocaine-seeking in rats through a 5-HT_2C receptor-dependent mechanism. Objective We investigated whether Ro 60-0175, a nonselective 5-HT_2B–2C agonist, influences cue-elicited reinstatement of cocaine-seeking behavior. We evaluated the 5-HT_2C receptor’s role in Ro 60-0175 by studying its interaction with SB-242,084, a selective 5-HT_2C antagonist. The study also explored whether Ro 60-0175 influences cue-elicited seeking behavior associated with sucrose, a highly palatable nutritive reinforcer. Materials and methods Different groups of free-feeding rats were trained to associate discriminative stimuli (S^Ds) with the availability of cocaine or a sucrose pellet or no-reward in two-lever operant cages. Cocaine and sucrose pellets were available under an FR1 schedule of reinforcement, and each reinforcer was followed by a 20-s timeout signaled by a cue light coming above the active lever. After extinction of reinforced responding in the absence of cue, the reinforcer-associated stimuli were reintroduced in reinstatement sessions in which reinforcers were withheld. Results Ro 60-0175, at IP doses from 0.1 to 1 mg/kg, dose-dependently reduced cocaine-seeking behavior, while 1 mg/kg had no such effect for the sucrose pellet. Pretreatment with 1 mg/kg SC SB-242,084 completely prevented the effect on cocaine-seeking behavior. Conclusions These findings, provided they can be extrapolated to abstinent human addicts, suggest therapeutic potential for the selective 5-HT_2C agonist in preventing cue-controlled cocaine-seeking and relapse. An erratum to this article can be found at     

Investigation of in vivo toxicity of hydroxylamine sulfate and the efficiency of intoxication treatment by α-tocopherol acetate and methylene blue

ObjectivesInvestigation of hydroxylamine sulfate toxicity mechanism in vivo and estimation of α-tocopherol acetate and methylene blue efficiency in poisoning treatments.MethodsIn vivo experiments were conducted on 102 Wistar Han rats. The experiments investigated the hematotoxic and oxidative stress effects of hydroxylamine sulfate in acute and subacute toxicity treatment of animals. Electron Spin Resonance was used for quantitative determination of blood and liver tissue parameters alterations after intoxication. The osmotic fragility of erythrocytes, lipid peroxidation intensity and level of SH-groups in liver of rats were determined by established biochemical assays.ResultsHydroxylamine sulfate cause an acute hematotoxicity and oxidative stress in vivo as demonstrated by the appearance of free oxidized iron in blood, reduced glutathione content and increased lipid peroxidation in liver. The experimental studies showed the formation of Hb–NO, MetHb in erythrocytes and as well of stable complex of reduced iron (Fe²⁺) with hydroxylamine sulfate. Methylene blue treatment does not reduce the Hb–NO or MetHb levels in intoxicated animals while administration of α-tocopherol acetate reduces substantially lipid peroxidation.ConclusionsOxidative stress is a key mechanism of acute hematotoxicity caused by hydroxylamine sulfate. Methylene blue is not suitable antidote in case of hydroxylamine intoxication.     

Generic sustained release tablets of trimetazidine hydrochloride: Preparation and in vitro–in vivo correlation studies

The aim of the current work was to develop generic sustained-release tablets containing 35 mg trimetazidine dihydrochloride and to establish an in vitro–in vivo correlation that could predict the bioavailability. The marketed sustained release tablet (Vastarel MR) used as reference, a sustained-release matrix tablet was prepared using hydroxypropyl methylcellulose (HPMC) as matrix by wet granulation and the in vitro dissolution profiles of the self-made tablets were determined in four different dissolution media (0.1 M HCl, pH 4.5 PBS, pH 6.8 PBS and water). A higher similarity between prepared tablets and Vastarel MR was established, with similarity factor (f₂) ranging from 60 to 75 in the four media. The in vivo pharmacokinetics was studied in six healthy beagles. Compared with Vastarel MR, the C_max of self-made tablets was slightly decreased, while the T_max and MRT_0–t were slightly prolonged, but with no significant difference (P > 0.05). The average of relative bioavailability (F) was 102.52% based on AUC_0–t. For log-transformed AUC_0–t and C_max, the upper confidence limit on the appropriate criterion is <0, indicating these two formulations were population bioequivalent. The in vivo–in vitro correlation coefficient obtained from point-to-point analysis of self-made tablets was 0.9720. In conclusion, the prepared tablets were bioequivalent to the marketed tablets, according to both the in vitro release rate and extent of absorption, and a good in vivo–in vitro correlation was established for the self-made tablets that indicated in vitro dissolution tests could be used as a surrogate for bioavailability studies.     

Different starting times of α-tocopherol and γ-tocotrienol supplementation and tumor marker enzyme activities in-the rat chemically induced with cancer

• 1.1. α-Tocopherol (α-T) and γ-tocotrienol (γ-T) were supplemented continuously for 8 weeks in the diets of normal rats and rats chemically induced with cancer using diethylnitrosamine (DEN), 2-acetylaminofluorene (AAF) and partial hepatectomy. Hepatocarcinogenesis was followed by determining the plasma γ-glutamyl-transpeptidase (GGT) and alkaline phosphatase (ALP) activities as well as placental glutathione S-transferase (PGST) and GGT activities histochemically, at 4-week intervals.
• 2.2. Male Rattus norvegicus were supplemented α-T and γ-T at two different doses of 30 and 300 mg/kg diet. The supplementation was started at three different times: simultaneously with DEN administration; 4 weeks; and 8 weeks after DEN administration.
• 3.3. Elevation of plasma GGT activities and formation of PGST and GGT positive foci were attenuated significantly (P< 0.05) when α-T and γ-T were supplemented simultaneously with cancer induction. Supplementation begun 4 and 8 weeks after cancer induction did not affect plasma enzyme activities and formation of enzyme-positive foci.
• 4.4. α-T was more effective than γ-T, and a lower dose of 30 mg/kg was found to be more effective in reducing the severity of hepatocarcinogenesis.

     

In vitro studies-how good are they at replacing in vivo studies for measurement of skin absorption?

Measures of percutaneous penetration are required for risk assessment of exposure of man to chemicals. In vitro approaches and QSAR predictions can be used and reduce the use of in vivo animal experiments. The OECD Guidelines on in vitro dermal absorption studies were recently accepted but progress was hampered by a lack of direct in vitro/in vivo comparisons in humans or in rodents. Either flow through diffusion or static cell systems with full thickness, dermatomed skin or membranes can be used. In a study of the robustness of in vitro techniques, inter-skin variability was greater than inter-laboratory or between cell variability. Recent studies with a number of chemicals have shown a reasonably good prediction but the difference between in vitro and in vivo results was greater for lipophilic molecules as lipophilic molecules which were retained in the stratum corneum. The experimental flux obtained in vitro using conditions that reflect the potential occupational exposure may be the most appropriate figure for risk assessment purposes. A database of in vitro and in vivo dermal penetration has been established. Dermal absorption data using infinite doses has been combined in a number of databases used for predictive QSAR modelling approaches to dermal absorption. However, absorption values derived from QSAR may over estimate the actual absorption from a finite dose.     

Soluplus® as an effective absorption enhancer of poorly soluble drugs in vitro and in vivo

As many new active pharmaceutical ingredients are poorly water soluble, solubility enhancers are one possibility to overcome the hurdles of drug dissolution and absorption in oral drug delivery. In the present work a novel solubility enhancing excipient (Soluplus®) was tested for its capability to improve intestinal drug absorption. BCS class II compounds danazol, fenofibrate and itraconazole were tested both in vivo in beagle dogs and in vitro in transport experiments across Caco-2 cell monolayers. Each drug was applied as pure crystalline substance, in a physical mixture with Soluplus®, and as solid solution of the drug in the excipient. In the animal studies a many fold increase in plasma AUC was observed for the solid solutions of drug in Soluplus® compared to the respective pure drug. An effect of Soluplus® in a physical mixture with the drug could be detected for fenofibrate. In vitro transport studies confirm the strong effect of Soluplus® on the absorption behavior of the three tested drugs. Furthermore, the increase of drug flux across Caco-2 monolayer is correlating to the increase in plasma AUC and C_max in vivo. For these poorly soluble substances Soluplus® has a strong potential to improve oral bioavailability. The applicability of Caco-2 monolayers as tool for predicting the in vivo transport behavior of the model drugs in combination with a solubility enhancing excipient was shown. Also the improvement of a solid dispersion compared to physical mixtures of the drugs and the excipient was correctly reflected by Caco-2 experiments. In the case of fenofibrate the possible improvement by a physical mixture was demonstrated, underscoring the value of the used tool as alternative to animal studies.     

Nanoparticle-mediated delivery of oligonucleotides to the blood–brain barrier: in vitro and in situ brain perfusion studies on the uptake mechanisms

Abstract

Except for the few exceptions where topical administration is feasible, progress towards broad clinical application of nucleic acid therapeutics requires development of effective systemic delivery strategies. The central nervous system represents a particularly difficult organ for systemic delivery due to the blood–brain barrier. We previously reported a nanoparticulate delivery system for targeted brain delivery of oligonucleotides upon systemic administration, i.e. liposome-encapsulated polyethylenimine/oligonucleotides polyplexes. In this study, cellular uptake and intracellular trafficking of the nanoparticles were further investigated using in situ brain perfusion technique followed by colocalization and fluorescence resonance energy transfer techniques. The brain endothelial uptake and possibly parenchymal accumulation were readily visualized upon administration via internal carotid artery perfusion. The nanoparticles were colocalized with early-endosome antigen, which confirms the brain endothelial uptake through transferrin receptor-mediated endocytosis. Fluorescence resonance energy transfer analysis also suggested the nanoparticles entered the brain endothelial cells while maintaining their integrity. Together, the enhanced brain uptake, as claimed previously, of the antibody-targeted nanoparticles was clearly confirmed with more convincing evidences. In addition, the experimental techniques described here should be applicable to the studies involving nanoparticle-mediated brain delivery of nucleic acid therapeutics.     

The inclusion of fluoxetine into γ-cyclodextrin increases its bioavailability: behavioural, electrophysiological and pharmacokinetic studies

The inclusion of a drug into cyclodextrin generally results in the modification of its physical and chemical properties and sometimes can increase its oral bioavailability. The aim of this study was to compare the effects of the fluoxetine HCl/gamma-cyclodextrin complex to that of traditional fluoxetine HCl. In the forced swimming test in mice, fluoxetine HCl/gamma-cyclodextrin was more effective than fluoxetine HCl, the ED30s being, respectively, 9.5 and 16.9 mg/kg PO. Both compounds (10 mg/kg PO) were able to reduce the firing rate of dorsal raphe neurons in the rat. However, between-groups comparisons showed no significant differences between fluoxetine HCl treated animals and the vehicle group, while fluoxetine HCl/gamma-cyclodextrin appeared significantly more effective than vehicle from minute 25 of the measurement period. In healthy volunteers, the relative oral bioavailability, calculated as the ratio AUC 0-infinity fluoxetine HCl/gamma-cyclodextrin on AUC 0-infinity fluoxetine HCl (20 mg PO), was equal to 249.9%. The three experiments taken together suggest that the complexation of fluoxetine HCl into gamma-cyclodextrin increases its pharmacological efficacy in animals, this effect being related to an enhancement of its oral bioavailability as demonstrated in human healthy subjects.     

Inactivation of hepatic enzymes by inhalant nitrite—In vivo and in vitro studies

We examined the effects of acute isobutyl nitrite (ISBN) exposure on the activity of several hepatic enzymes. Two strains of adult male mice (Balb/c and C57BL/6) were exposed to 900 ppm ISBN or ambient air for 45 minutes. The enzyme activity of hepatic cytochrome P450 (CYP)-mediated deethylation, glutathione S-transferase (GST), and carboxylesterase (CBE) was monitored through the substrates 3-cyano-7-ethoxycoumarin (CEC), 1-chloro-2,4-dinitrobenzene, and p-nitrophenyl acetate, respectively. Acute ISBN exposure led to a significant reduction in hepatic CYP-mediated CEC deethylation, GST, and CBE activity in Balb/c mice (of 81.5%, 74.7%, and 25.2%, respectively, vs control mice, each at P < .05) when livers were harvested immediately after inhalant exposure. The corresponding decreases in C57BL/6 mice were smaller (with reductions of 21.8%, 18.8%, and 13.3%, respectively, each at P < .05). This enzyme activity, tested in C57BL/6 mice only, returned to control values after a 24-hour period of nonexposure. Follow-up mechanistic investigations using rat liver GST indicated that ISBN-mediated enzyme inactivation was not caused by its metabolites: inorganic nitrite ion (NO2-) or nitric oxide. This inactivation could be prevented, but not reversed, by added glutathione, suggesting irreversible protein oxidation. Using different NO donors as comparative agents, we found that GST inactivation by ISBN was not associated with protein S-nitrosylation or disulfide formation, but with tyrosine nitration. Inhalant nitrite exposure, therefore, led to a significant reduction in hepatic enzyme activity in mice, possibly through tyrosine nitration of hepatic proteins. This effect raises the possibility of drug-drug metabolic interactions from inhalant nitrite abuse. However, determining the applicability of these findings to humans will require further study.     

Novel in situ gelling ocular inserts for voriconazole-loaded niosomes: design,in vitro characterisation and in vivo evaluation of the ocular irritation and drug pharmacokinetics

This work aimed to develop voriconazole in situ gelling ocular inserts loaded with niosomal suspension. Niosomes and mixed niosomes were prepared using span 40 and span 60 with pluronic L64 and pluronic F127. The entrapment efficiency percentages (EE%), mean vesicle size, polydispersity index (PI), zeta potential and in vitro drug release of these niosomes were evaluated. F3-mixed niosomes prepared with span 60 and pluronic L64 was selected, due to its highest EE; optimum vesicle size with smallest PdI and suitable release pattern of the drug (63% after 8?h). In situ ocular inserts were prepared using sodium carboxymethylcellulose (CMC Na) and sodium alginate (ALG) and characterised for surface morphology, surface pH, water uptake, mucoadhesion and in vitro release. ALG in situ ocular insert (S₂) was selected for further in vivo evaluation of the ocular irritation and drug pharmacokinetics in the aqueous humour of rabbit's eyes. S₂ in situ gelling ocular insert was non-irritant and showed significantly (p?<?0.01) higher C_max, delayed T_max and increased bioavailability.