Development and Validation of Different Ultraviolet-Spectrophotometric Methods for the Estimation of Besifloxacin in Different Simulated Body Fluids.

In the present study a simple, accurate, precise, economical and specific UV-spectrophotometric method for estimation of besifloxacin in bulk and in different pharmaceutical formulation has been developed. The drug shows maximum λmax289 nm in distilled water, simulated tears and phosphate buffer saline. The linearity range of developed methods were in the range of 3-30 µg/ml of drug with a correlation coefficient (r(2)) 0.9992, 0.9989 and 0.9984 with respect to distilled water, simulated tears and phosphate buffer saline, respectively. Reproducibility by repeating methods as %RSD were found to be less than 2%. The limit of detection in different media was found to be 0.62, 0.72 and 0.88 µg/ml, respectively. The limit of quantification was found to be 1.88, 2.10, 2.60 µg/ml, respectively. The proposed method was validated statically according to International Conference on Harmonization guidelines with respect to specificity, linearity, range, accuracy, precision and robustness. The proposed methods of validation were found to be accurate and highly specific for the estimation of besifloxacin in different pharmaceutical formulations.

Synthesis and characterization of thermoresponsive copolymers for drug delivery.

We report the synthesis and characterization of two nontoxic, thermogelling drug delivery systems which are liquid at room temperatures but become a gel at physiological temperature (37°C) potentially leading to release of a drug molecule. We selected temperature as the stimulus for drug release as it is physiologically invariant. A free radical polymerization of N-isopropylacrylamide (NIPAM) and N-vinylpyrrolidone (VP) was carried out under nitrogen atmosphere in double-distilled water at two different temperatures (30°C and 70°C), and the copolymers obtained were characterized by various analytical techniques. The molar ratios of the two monomers were altered with increasing NIPAM content and their cloud point temperature or least critical solution temperature (LCST) was determined. The copolymer at 9:1 ratio of NIPAM to VP resulted in the formation of nanoparticle-based gel (NG1) at 30°C; however, at 70°C, a microgel (MG1) was formed. The LCST of the nanogel and microgel was 33.5-34°C and 36.5-37°C, respectively. Thus, both the copolymers are water soluble at room temperature, but distinct phases appear at physiological temperatures. We hypothesized that these copolymers on entrapment with a drug could be used for topical application to the skin or eye for controlled drug delivery applications. Toxicological studies revealed that the copolymers are nontoxic in HeLa cells. Finally, our experiments show that a model drug [bovine serum albumin (BSA)] is released at 37°C with zero-order kinetics and confirmed using multiple well-known mathematical models.

Evidence of carrier mediated transport of ascorbic acid through mammalian cornea.

The purpose of the present study was to evaluate the transport of ascorbic acid, a water soluble molecule, through a predominantly lipophilic cornea. Thus in-vitro permeation of ascorbic acid from aqueous drops through freshly excised mammalian cornea was studied. Aqueous isotonic ophthalmic solutions of ascorbic acid of different concentrations (0.125% w/v to 2% w/v) (pH 5.4) were made. Further 1.0% w/v or 0.5% w/v ascorbic acid solution containing NaCl or dextrose as tonicity modifiers or Na(+)K(+)-ATPase inhibitors were also made. Permeation characteristics of drug were evaluated by putting 1 ml formulation on freshly excised cornea fixed between donor and receptor compartments of an all-glass modified Franz diffusion cell and measuring the drug permeated in the receptor by spectrophotometry at 265 nm, after 120 min. Statistical analysis was done by one-way analysis of variance (ANOVA) followed by Dunnett's test or paired t-test. Increase in drug concentration in the formulation resulted in an increase in the quantity permeated but after a certain level increase in permeation with increase in concentration was minimal. Aqueous drops made isotonic with dextrose showed decreased permeation through paired cornea compared with aqueous drops made isotonic with NaCl from 1% w/v ascorbic acid solution suggesting likely involvement of Na(+) co-transporter but there was decreased permeation through 0.5% w/v ascorbic acid solution made isotonic with NaCl as compared to solution made isotonic with dextrose. Further aqueous drops containing Na(+)K(+)-ATPase inhibitor {MAG-Mono Ammonium Glycyrrhizinate (25 µmol)} showed decreased corneal permeation from 0.5% w/v ascorbic acid solution but there was not significant decrease from 1% ascorbic acid solution since MAG is a competitive inhibitor of ascorbic acid. Aqueous drops containing Na(+)K(+)-ATPase inhibitor {MAG (50 µmol) or Ouabain (1 mmol)} showed decreased corneal permeation of ascorbic acid compared with control from 1% ascorbic acid solution confirming the involvement of Na(+) co-transporter.

Entrapment of ketorolac tromethamine in polymeric vehicle for controlled drug delivery.

The most common method for applying a drug in to the eye is to formulate the drug in the form of an eye drop, but this method is not considered ideal for ocular delivery of drug because of poor bioavailability arising from precorneal loss processes, this loss of drug from the precorneal area is a net effect of drainage, tear secretion and noncorneal absorption. Following the above lead we tried to improve the ocular bioavailability by increasing the corneal contact time and the feasible way was to formulate a drug with mucoadhesive/viscosity imparting agents. The adhesive strength of various polymers on corneal surface was studied with the help of self modified Franz diffusion cell and freshly excised goat/bovine cornea. The polymers hydroxypropylmethylcellulose, carboxymethylcellulose sodium, Eudragit type E/RL/RS, Carbopol ETD 2020 and Carbopol 934 National Formulary were formulated with drug, ketorolac tromethamine. The adhesive strength of polymers on corneal surface and permeation characteristics of drug through cornea were investigated by using above said formulations. Eudragit type E/RL/RS did not show any improvement in mucoadhesion, but the formulations containing Carbopol ETD 2020 and Carbopol 934 national formulary showed good mucoadhesion on corneal surface in the concentration as low as 0.75%. The mucoadhesive strength was also evaluated using the combination of Carbopol acrylates/C 10-30 alkylacrylate with allylpentaerithrital and preservative benzalkonium chloride, which also resulted in good mucoadhesion with improved corneal permeation. Observations made in this study indicate the potentiality of the ophthalmic formulations containing mucoadhesive/viscosity imparting agents.

In vitro permeation characteristics of moxifloxacin from oil drops through excised goat, sheep, buffalo and rabbit corneas.

The objective of present investigation was to study the in vitro permeation characteristics of moxifloxacin from oil drops through freshly excised goat, sheep, buffalo and rabbit corneas. Moxifloxacin, 0.043 to 0.048% (w/v) ophthalmic solutions with or without (0.5% v/v) benzyl alcohol were made in arachis, castor, cottonseed, olive, soybean, sunflower and sesame oils. Permeation studies were conducted by putting 1 ml oil formulation on cornea (0.50 cm2) fixed between donor and receptor compartments of an all glass modified Franz diffusion cell and measuring the drug permeated in receptor (containing 10 ml bicarbonate ringer, pH 7.4 at 37 degrees C under stirring) by spectrophotometry at 291 nm, after 120 min. Post permeation corneal hydration was measured to assess corneal damage. The study was designed with paired corneas i.e. one cornea of an animal received formulation without benzyl alcohol while the contralateral cornea received formulation with benzyl alcohol. Moxifloxacin ophthalmic solution in castor oil showed maximum permeation with all the corneas. Addition of benzyl alcohol, a preservative, to oil drops reduced permeation of moxifloxacin from each oil drop, with corneas of all the species. Partition experiments with moxifloxacin oil drops and phosphate buffer (pH 7.4) indicated higher partitioning of drug in the oil phase, in presence of benzyl alcohol. Thus results of permeation are consistent with the partition characteristics of drug between oil and aqueous phase. Corneal hydration obtained with all the formulations was between 75 to 80% indicating no corneal damage.

Biomedical evaluation of polyvinyl alcohol-gelatin esterified hydrogel for wound dressing.

The wound is a biosynthetic environment in which numerous cellular processes are interlinked in the process of repair. Modern dressings are designed to facilitate wound healing rather than just to cover it. Hydrogel dressing can protect injured skin and keep it appropriately moist to speed the healing process by absorbing exudates while maintaining the products of tissue repair, including growth factor and lysosomes, in contact with the wound. The design and development of novel membrane of hydrogels prepared by esterification of polyvinyl alcohol with gelatin were attempted. Contact angle of goat blood was determined. The hydrogel was characterized by hemolysis test and water vapor transmission rate. Diffusion coefficient of salicylic acid (SA) and gatifloxacin, a fourth generation fluoroquinolone, through the membrane was determined. Both the drugs were used as model drug. Methyl tetrazolium dye assay of the membrane was done using L929 fibroblast cell line and mice splenocytes to establish the biocompatibility of the membrane. The equilibrium goat blood-in-air contact angles of measured ester films ranged from 56 to 60 degrees . The hydrogel was found to be hemocompatible and moisture retentive indicating its possible use in moist wound care. The diffusion coefficient of SA and gatifloxacin through the membrane was found to be 1.49 x 10(-5) and 3.97 x 10(-6) cm(2)/s respectively. The membrane was found to be compatible with the L929 fibroblast cell line and mice splenocytes.

Insulin loaded eudragit L100 microspheres for oral delivery: preliminary in vitro studies.

Eudragit L100 microspheres were prepared using water-in-oil-in water (w/o/w) emulsion-solvent evaporation with polysorbate 20 as dispersing agent in the internal aqueous phase, and PVA/PVP as stabilizer in the external aqueous phase. Smaller internal and external aqueous phases provided higher drug encapsulation. The PVA-stabilized microspheres having maximum drug encapsulation (84.5 2.8%) released 7% insulin at pH 1.0 in 2 h. In phosphate buffer (pH 7.4), microspheres showed an initial burst release of 21% in 1 h with additional 35% release in the next 5 h. The smaller the volumes of internal and external aqueous phases, the lower the initial burst release. The release of drug from microspheres followed Higuchi kinetics. Scanning electron microscopy of PVA stabilized microspheres demonstrated spherical particles with smooth surface and laser diffractometry revealed a mean particle size (V(m)) of 59.11 30 m.

Polyvinyl alcohol--gelatin patches of salicylic acid: preparation, characterization and drug release studies.

Polyvinyl alcohol-gelatin patches were developed and salicylic acid was incorporated at different stages of preparation of the patches. The patches were characterized by Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), differential scanning calorimetry, tensile strength test, and scanning electron microscopy. The release patterns of the drug from the patches were also studied. The FTIR spectra of the blank patch indicated complete esterification of the free carboxylic groups of gelatin. The XRD studies indicated a crystalline form of the drug entrapped in the patches. Release of the drug from the patches followed Higuchian/Fickian kinetics indicating a diffusion-controlled release process.

Development of carboxymethyl cellulose acrylate for various biomedical applications.

The purpose of this work is to prepare a pH-sensitive hydrogel membrane of sodium carboxymethyl cellulose acrylate for drug delivery and other biomedical applications. The hydrogel was made by esterification of sodium carboxymethyl cellulose (SCMC) and acryloyl chloride (ACl). The esterified product was characterized by FTIR spectroscopy and XRD. Swelling, hemocompatibility, water vapor transmission rate, contact angle and diffusional studies were also done. Biocompatibility of the membrane was established by quantification of cell growth of L929 cells and mice splenocytes. The FTIR spectrum of the hydrogel suggested the formation of ester bonds between the hydroxyl groups of sodium carboxymethyl cellulose and the carbonyl group of acryloyl chloride. Water vapor transmission rate, hemocompatibility, contact angle and swelling studies indicated that the hydrogel can be tried as a wound dressing material. The hydrogel showed pH-dependent swelling behavior arising from the acidic pendant group in the polymer network. The permeability of the hydrogel membrane produced, as shown by salicylic acid diffusion, increased in response to an increase in pH of the external medium. The hydrogel membrane was permeable to salicylic acid at pH 7.2 but not at pH 2.0 (0.01N HCl). The effect of changes of pH on the hydrogel's permeability was found to be reversible. The hydrogel membrane was found to be compatible with the L929 mice fibroblast cell line and mice splenocytes. The esterified product of SCMC and ACl swells on increase of pH indicating its possible use in a pH-sensitive drug delivery system and as a wound dressing material.

Characterization of prepared corn starch-based hydrogel membranes.

The design and development of novel hydrogel membranes prepared by crosslinking polyvinyl alcohol with heat-treated cornstarch (CS) and CS suspensions was attempted. Degradation studies of the hydrogel membranes were conducted using á -amylase. The mechanical property of the hydrogel membranes was characterized by tensile tests. The swelling behavior of the membranes was also evaluated. Methyl tetrazolium assay of the membranes was performed using mice splenocytes and the L929 cell line to establish cytocompatibility. The membranes had sufficient strength and water holding capacity. The membrane was found to be biocompatible with L929 fibroblast cells and mice splenocytes.

Antibacterial activity of Ocimum sanctum L. fixed oil.

Ocimum sanctum fixed oil showed good antibacterial activity against Staphylococcus aureus, Bacillus pumilus and Pseudomonas aeruginosa, where S. aureus was the most sensitive organism. Sesame and soyabean oils also showed moderate activity against S. aureus. Higher content of linolenic acid in O. sanctum fixed oil could contribute towards its antibacterial activity. The antibacterial activity combined with anti-inflammatory and analgesic activities of the oil, could make it useful in inflammatory disorder resulting from staphylococcal infection.

Effect of preservative, antioxidant and viscolizing agents on in vitro transcorneal permeation of ketorolac tromethamine.

The influence of formulation additives, e.g. preservative, antioxidant and viscolizing agents on in vitro transcorneal permeation of ketorolac tromethamine from 0.5%(w/v) aqueous drop was studied using goat cornea. Permeation characteristics of drug, from selected formulations, through excised rabbit cornea were also evaluated. Aqueous solution of ketorolac tromethamine (0.5% w/v), pH 6.5 or 7.0 having ionic strength 0.2, was prepared. To this solution perservatives either alone or in combination with other additives were added to have drops of various composition. Permeation studies with goat cornea showed maximum permeation of ketorolac tromethamine from formulation containing benzalkonium chloride and disodium edetate. Increase in viscosity of drop resulted in decreased permeation of drug. Formulation containing benzalkonium chloride and disodium edetate also increased permeation of drug through rabbit cornea. Cumulative permeation of drug through rabbit cornea was found to be 2.3-2.4 fold higher than that observed with goat cornea.

Effect of Ocimum sanctum fixed oil on blood pressure, blood clotting time and pentobarbitone-induced sleeping time.

Ocimum sanctum fixed oil produced hypotensive effect in anaesthetised dog, which seems to be due to its peripheral vasodilatory action. The oil increased blood-clotting time and percentage increase was comparable to aspirin and could be due to inhibition of platelet aggregation. The oil also increased pentobarbitone-induced sleeping time in rats indicating probable inhibitory effect of oil towards cytochromic enzyme responsible for hepatic metabolism of pentobarbitone.

Permeation through cornea.

The permeability of the cornea to drugs is clinically important because it is the major factor determining the efficacy of topically applied ophthalmic preparations. With this perspective, the present article gives a brief update and overview of corneal structure and proposed mechanisms of permeation. Physiological, physicochemical and formulation factors affecting drug permeation through cornea are highlighted. Influence of ocular penetration enhancers on drug permeation is also discussed.

Ketorolac entrapped in polymeric micelles: preparation, characterisation and ocular anti-inflammatory studies.

Polymeric micelles made of copolymer of N-isopropylacrylamide (NIPAAM), vinyl pyrrolidone (VP) and acrylic acid (AA) having cross-linkage with N,N'-methylene bis-acrylamide (MBA) were used as host carrier in which up to 30%w/w ketorolac (free acid) was entrapped to make the formulation. The lyophilised powder was used for physical characterisation. The drug entrapment was found to be about 80% and the formulation was stable for 8-10 days at room temperature. The smaller the amount of ketorolac dissolved into the micelles, the longer was the formulation shelf life. The size of the particles as measured by dynamic light scattering was found to be around 35 nm diameter at 25 degrees C. TEM picture showed spherical particles. The structure of the polymer and its morphology were characterised by FTIR, NMR and XRD measurements. IR data indicated weak interaction between polymer and ketorolac in the encapsulated system. NMR spectra indicated rigid polymer backbone with intermittent iso-propyl group in the chain. XRD spectra showed significant loss of crystallinity of the drug while being entrapped in the polymeric micelles. The release of drug in aqueous buffer (pH 7.2) from the polymeric micelles at 25 degrees C were 20 and 60% after 2 and 8 h respectively and is temperature and pH dependent. In vitro corneal permeation studies through excised rabbit cornea indicated two fold increase in ocular availability with no corneal damage compared to an aqueous suspension containing same amount of drug as in nanoparticles. The formulation showed significant inhibition of lid closure up to 3 h and PMN migration up to 5 h compared to the suspension containing non-entrapped drug, which did not show any significant effect.

Evaluation of the gastric antiulcer activity of fixed oil of Ocimum sanctum (Holy Basil).

The fixed oil of Ocimum sanctum L. (Labiatae) was found to possess significant antiulcer activity against aspirin-, indomethacin-, alcohol-, histamine-, reserpine-, serotonin- and stress-induced ulceration in experimental animal models. Significant inhibition was also observed in gastric secretion and aspirin-induced gastric ulceration in pylorus ligated rats. The lipoxygenase inhibitory, histamine antagonistic and antisecretory effects of the oil could probably have contributed towards antiulcer activity. O. sanctum fixed oil may be considered to be a drug of natural origin which possesses both anti-inflammatory and antiulcer activity.

Effect of Ocimum sanctum fixed oil on vascular permeability and leucocytes migration.

Ocimum sanctum fixed oil significantly inhibited the rise in protein concentration and dye leakage in peritoneal fluid in experimentally induced peritoneal inflammation in mice. In carrageenan-induced pleurisy in rats, the fixed oil showed significant inhibition of leucocytes migration in the pleural exudate. The results suggest that the fixed oil can inhibit enhancement of the vascular/capillary permability and leucocyte migration following inflammatory stimulus.

Effect of concentration, pH, and preservative on in vitro transcorneal permeation of ibuprofen and flurbiprofen from non-buffered aqueous drops.

Influence of drug concentration, pH of aqueous drops and some commonly used preservatives on in vitro transcorneal permeation of ibuprofen and flurbiprofen were investigated using goat cornea. Increase in drug concentration in the drops made in normal saline resulted in increase in quantity permeated but decrease in cumulative percent permeation of both drugs. Permeation of each drug from 0.5% drops was maximum at acidic pH (6.4) and decreased with increase in pH of the drops. Normal saline, as a vehicle, favoured permeation of each drug, hence retained in the formulation. Benzalkonium chloride and chlorobutanol enhanced cumulative percent permeation of ibuprofen while benzalkonium chloride and phenyl mercuric nitrate increased permeation of flurbiprofen. Benzalkonium chloride being incompatible with 0.5% drops (pH 6.4) of either drug, chlorobutanol appears suitable for ibuprofen drops and phenyl mercuric nitrate for flurbiprofen drops.

Evaluation of antiinflammatory activity of fatty acids of Ocimum sanctum fixed oil.

Ocimum sanctum fixed oil and linolenic acid were found to possess significant antiinflammatory activity against PGE2, leukotriene and arachidonic acid-induced paw edema. Plant lipids like linseed oil and soyabean oil containing linolenic acid when tested along with O. sanctum fixed oil, also showed significant inhibition of carrageenan-induced paw edema. The results suggest that linolenic acid present in O. sanctum fixed oil has the capacity to block both the cyclooxygenase and lipoxygenase pathways of arachidonate metabolism and could be responsible for the antiinflammatory activity of the oil.

In vitro transcorneal permeation of ketorolac tromethamine from buffered and unbuffered aqueous ocular drops.

In vitro transcorneal permeation of ketorolac tromethamine from 0.5% w/v solutions containing equimolar (0.02 M) concentrations of citrate (pH 6.5), phosphate (pH 6.5 and 7), citrate-phosphate (pH 7) and borate (pH 7) buffers was studied using goat cornea. Cumulative % permeation was maximum with phosphate buffered drops of pH 6.5. The effect of pH and ionic strength on permeation of ketorolac tromethamine from buffered (phosphate) drops was next investigated. Cumulative % permeation of ketorolac tromethamine from buffered drops was pH dependent being maximum at pH 4.5. Adjustment of ionic strength of drops to 0.2 resulted in decreased permeation of drug. Permeation of ketorolac tromethamine from unbuffered drops of varying pH and ionic strength 0.2 was also pH dependent and was maximum at pH 4.5. Buffered drops of pH between 4.5-5.5, ionic strength 0.2, provided better permeation of drug compared to unbuffered drops of same pH and ionic strength. Above pH 6.5 unbuffered drops showed better permeation than buffered drops. Increase in molarity of phosphate buffer (pH 4.5) used in making drops, between 0 to 0.15 M increased permeation. Aqueous drops of ketorolac tromethamine formulated in 0.15 M phosphate buffer of pH 4.5 and ionic strength 0.2 showed maximum cumulative % permeation in vitro. Considering lacrimation induced drug loss in vivo, by buffer of high concentration, ketorolac tromethamine drops formulated in buffer of low molarity, pH 4.5 and ionic strength 0.2 appear suitable.