Influence of microencapsulation method and peptide loading on formulation of poly(lactide-co-glycolide) insulin nanoparticles.

Insulin stability during microencapsulation and subsequent release is essential for retaining its biological activity. Therefore we investigated a novel solid/oil/water anhydrous encapsulation method with a combination of stabilizers for maintaining the integrity of insulin during formulation and delivery. Two methods were used for preparation of nanoparticles, namely water/oil/water solvent evaporation and s/o/w anhydrous encapsulation to study the influence of the microencapsulation method on nanoparticle characteristics such as size and morphology, drug content, encapsulation efficiency, and in vitro and in vivo release profile. Poly (lactic-co-glycolic) acid (PLGA) with co-polymer ratio 50:50 was selected to prepare drug-loaded nanoparticles. When nanoparticles were prepared by solvent evaporation higher encapsulation efficiencies could be obtained, e.g. 74 +/- 13 with 5% target loading, whereas with 12% target loading, encapsulation efficiency was 27 +/- 8.6. The s/o/w method has a direct influence on the evaluation parameters where very poor encapsulation efficiencies 11 +/- 6.8 (max) were observed. The presence of stabilizers in the nanoparticles resulted in an increase in particle size but a reduction of encapsulation efficiency. Insulin release rate was comparatively higher for the batches prepared by the w/o/w method containing stabilizers than the s/o/w method. Also the presence of stabilizers resulted in sustained release of insulin resulting in prolonged reduction of blood glucose levels in streptozotocin induced diabetic rats. From the in vitro and in vivo studies, it can be concluded that careful selection of processing conditions and combination of stabilizers also result in beneficial effects without compromising the advantages of these delivery systems.

Development and validation of a reversed-phase HPLC method for the determination of ezetimibe in pharmaceutical dosage forms.

Ezetimibe belongs to a group of selective and very effective 2-azetidione cholesterol absorption inhibitors that act on the level of cholesterol entry into enterocytes. A rapid, specific reversed-phase HPLC method has been developed for assaying ezetimibe in pharmaceutical dosage forms. The assay involved an isocratic elution of ezetimibe in a Kromasil 100 C18 column using a mobile phase composition of water (pH 6.8, 0.05%, w/v 1-heptane sulfonic acid) and acetonitrile (30:70, v/v). The flow rate was 0.5 ml/min and the analyte monitored at 232 nm. The assay method was found to be linear from 0.5 to 50 microg/ml. All the validation parameters were within the acceptance range. The developed method was successfully applied to estimate the amount of ezetimibe in tablets.

Biodisposition of PEG-coated lipid microspheres of indomethacin in arthritic rats.

Conventional lipid microspheres (LM) were prepared using soybean oil and lipid at a 5.5:1 weight ratio with lipid phase consisting of PC (phosphatidyl choline):CH (cholesterol) (1:0.5) by molar ratio. The average diameter of the particles was 150 nm. Long-circulating microspheres (S-LM) were also prepared similarly but the lipid phase consisted of PC:CH:DSPE-PEG (phosphatidyl choline:cholesterol:distearoyl phosphatidyl ethanolamine-polyethylene glycol) 1:0.5:0.16 by molar ratio. A comparative biodistribution study was conducted between free indomethacin and lipo-indomethacin (LM and S-LM) in the arthritic rats by administering the formulations at a dose equivalent to 12 mg of indomethacin/kg. It was observed that the free drug as well as the encapsulated drug followed biphasic clearance from the blood. Pharmacokinetic parameters, such as AUC(0-t), terminal half-life, MRT increased significantly when the drug was used in encapsulated form (p < 0.05). Clearance of the drug was reduced 1.4 times with the conventional lipid microspheres and was reduced three-fold when encapsulated in polyethylene glycol-coated lipid microspheres. The overall drug targeting efficiency (T(e)) with the PEG-coated lipid microspheres was 7.5-fold higher than the conventional lipid microspheres. The high accumulation of the drug in arthritic paw with S-LM system may be accounted for by the reduced uptake by RES cells, and thereby, availability for extravascularization in the inflammatory tissues.

Beta-cyclodextrin complexes of celecoxib: molecular-modeling, characterization, and dissolution studies.

Celecoxib, a specific inhibitor of cycloxygenase-2 (COX-2) is a poorly water-soluble nonsteroidal anti-inflammatory drug with relatively low bioavailability. The effect of beta-cyclodextrin on the aqueous solubility and dissolution rate of celecoxib was investigated. The possibility of molecular arrangement of inclusion complexes of celecoxib and beta-cyclodextrin were studied using molecular modeling and structural designing. The results offer a better correlation in terms of orientation of celecoxib inside the cyclodextrin cavity. Phase-solubility profile indicated that the solubility of celecoxib was significantly increased in the presence of beta-cyclodextrin and was classified as A(L)-type, indicating the 1:1 stoichiometric inclusion complexes. Solid complexes prepared by freeze drying, evaporation, and kneading methods were characterized using differential scanning calorimetry, powder x-ray diffractometry, and scanning electron microscopy. In vitro studies showed that the solubility and dissolution rate of celecoxib were significantly improved by complexation with beta-cyclodextrin with respect to the drug alone. In contrast, freeze-dried complexes showed higher dissolution rate than the other complexes.

Enhancement of dissolution and oral bioavailability of gliquidone with hydroxy propyl-beta-cyclodextrin.

The virtual insolubility of gliquidone in water results in poor wettability and dissolution characteristics, which may lead to a variation in bioavailability. To improve these characteristics of gliquidone, binary systems with hydroxypropyl-beta-cyclodextrin (HP-beta-CD) were prepared by classical methods such as physical mixing, kneading, co-evaporation and co-lyophilization. The solid state interaction between the drug and HP-beta-CD was assessed by evaluating the binary systems with X-ray diffraction, differential scanning calorimetry and IR- spectroscopy. The results establish the molecular encapsulation and amorphization of gliquidone. The phase solubility profile of gliquidone in aqueous HP-beta-CD vehicle resulted in an A(L) type curve with a stability constant of 1625 M(-1). The dissolution rate of binary systems was greater than that of pure drug and was significantly higher in the case of co-lyophilized and co-evaporated systems. Upon oral administration, [AUC]-alpha was significantly higher in case of co-lyophilized (2 times) and co-evaporated systems (1.5 times) compared to pure drug suspension while other binary systems showed only a marginal improvement. The study ascertained the utility of HP-beta-CD in enhancing the oral bioavailability of gliquidone, and points towards a strong influence of the preparation method on the physicochemical properties.

Effect of pH and complexation on transdermal permeation of gliquidone.

Gliquidone, a second generation sulfonylurea has been investigated for transdermal delivery. The poor aqueous solubility of the drug prompted the use of hydroxypropyl-beta-cyclodextrin (HP-beta-CD), a cyclic oligosaccharide, which is known to facilitate transdermal permeation of many drugs by enhancing the solubility and thus improving the diffusible species of the drug molecules at the skin-vehicle interface. In order to optimize the transdermal delivery of gliquidone, the effect of pH along with complexation on the solubility and permeation has been investigated. The solubility profiles of the drug, on increasing the concentration of HP-beta-CD were of Higuchi's AL type at the three pH values evaluated. However, the solubilization slope of the drug at pH 7.0 was 22 times that at pH 3.0 as a result of greater intrinsic solubility of the ionized form of the drug at pH 7.0. Transdermal flux of gliquidone at pH 7.0 was significantly greater than the flux at pH 3.0 in the presence of 15% w/v HP-beta-CD, attributable to the better solubility of the drug at pH 7.0 in the presence of HP-beta-CD. The effect of increasing concentrations of HP-beta-CD investigated at variable drug loading in the donor phase at pH 7.4 endorsed the earlier observations from studies on other drugs, that the drug has to be present at saturation in HP-beta-CD aqueous vehicle to achieve an optimized flux. While at saturation, the steady state flux of gliquidone from the aqueous HP-beta-CD (25% w/v) vehicle was enhanced 31 times compared to pure drug suspension at pH 7.4, unsaturation in the donor phase resulted in the decreased flux of gliquidone. It was concluded from the present study that enhanced transdermal flux of gliquidone can be achieved by adjusting the pH and the concentration of HP-beta-CD to achieve a better solubility of the drug.

Macrocyclic diterpenes from Euphorbia nivulia.

The latex of Euphorbia nivulia afforded two ingol diterpenes 3,12-diacetyl-8-benzoylingol (4) and 3,12-diacetyl-7-benzoyl-8-nicotinylingol (5) along with three known ingol diterpenes 1, 2,and 3, and two known triterpenes cycloart-25-en-3beta-ol and cyclonivulinol. Their structures have been assigned on the basis of their structural data as well as their acetylated products. The diterpenes 1-5 were tested for the LPS induced PGE(2) inhibition activity.

An immunosuppressive tryptophan-derived alkaloid from Lepidagathis cristata.

An immunosuppressive, tryptophan-derived alkaloid cristatin A (1), and two known compounds, cycloartenol and stigmasta-5,11(12)-diene-3 beta-ol, were isolated from the whole plant Lepidagathis cristata Willd. The structures of the isolates were established by interpretation of their spectral data.

A new triterpenoid from the fern Adiantum lunulatum and evaluation of antibacterial activity.

A new triterpenoid, 22,29xi-epoxy-30-norhopane-13beta-ol (1) was isolated together with six known compounds viz., fern-9(11)-en-6alpha-ol. fern-9(11)-ene, fern-9(11)-en-25-oic acid, fern-9(11)-en-28-ol, filicenol-B, adiantone and oxidation product of fern-9(11)-en-6alpha-ol obtained as 6-oxofern-9(11)-ene from the whole plant of Adiantum lunulatum, and their structures were elucidated by means of spectroscopic analysis and antibacterial evaluation of these compounds were conducted.

Drug release kinetics from polymeric films containing propranolol hydrochloride for transdermal use.

Polymeric films containing propranolol hydrochloride (PPN) were formulated and evaluated with a view to select a suitable formulation for the development of transdermal drug delivery systems. Films containing different ratios of ethyl cellulose (EC), poly(vinylpyrrolidone) (PVP), and PPN were prepared by mercury substrate method. In vitro drug release and skin permeation studies were conducted using paddle over disk and modified Franz diffusion cell, respectively. The drug release profiles from the polymeric film indicated that the drug content in the film decreased at an apparent first-order rate, whereas the quantity of drug release was proportional to the square root of time. The release rate of PPN increased linearly with increasing drug concentration and PVP fraction in the film, but was found to be independent of film thickness. The increase in release rate may be due to leaching of hydrophilic fraction of the film former, which resulted in the formation of pores. It was also observed that the release of drug from the films followed the diffusion-controlled model at low drug concentration. A burst effect was observed initially, however, at high drug loading level, which may be due to rapid dissolution of the surface drug followed by the diffusion of the drug through the polymer network in the film. The in vitro skin permeation profiles displayed increased flux values with increase of initial drug concentration in the film, and also with the PVP content. From this study, it is concluded that the films composed of EC/PVP/PPN, 9:1:3, 8:2:2, and 8:2:3, should be selected for the development of transdermal drug delivery systems using a suitable adhesive layer and backing membrane for potential therapeutic applications.

Synthesis and conformational studies of peptidomimetics containing furanoid sugar amino acids and a sugar diacid.

Furanoid sugar amino acids (1) were synthesized and used as dipeptide isosteres to induce interesting turn structures in small linear peptides. They belong to a new variety of designed hybrid structures that carry both amino and carboxyl groups on rigid furanose sugar rings. Four such molecules, 6-amino-2,5-anhydro-6-deoxy-D-gluconic acid (3, Gaa) and its mannonic (4, Maa), idonic (5, Iaa), and a 3,4-dideoxyidonic (6, ddIaa) congeners were synthesized. The synthesis followed a novel reaction path in which an intramolecular 5-exo S(N)2 opening of the hexose-derived terminal aziridine ring in 2 by the gamma-benzyloxy oxygen with concomitant debenzylation occurred during pyridinium dichromate oxidation of the primary delta-hydroxyl group to carboxyl function, leading to the formation of furanoid sugar amino acid frameworks in a single step. Incorporation of these furanoid sugar amino acids into Leu-enkephalin replacing its Gly-Gly portion gave analogues 8-11. Detailed structural analysis of these molecules by circular dichroism (CD) and various NMR techniques in combination with constrained molecular dynamics (MD) simulations revealed that two of these analogues, 8a and 10a, have folded conformations composed of an unusual nine-membered pseudo beta-turn-like structure with a strong intramolecular H-bond between LeuNH --> sugarC3-OH. This, in turn, brings the two aromatic rings of Tyr and Phe in close proximity, a prerequisite for biological activities of opioid peptides. The analgesic activities of 8a,b determined by mouse hot-plate and tail-clip methods were similar to that of Leu-enkephalin methyl ester. The syn disposition of the beta-hydroxycarboxyl motif on the sugar rings appears to be the driving force to nucleate the observed turn structures in some of these molecules (8 and 10). Repetition of the motif on both sides of a furanose ring resulted in a novel molecular design of sugar diacid, 2,5-anhydro-D-idaric acid (7, Idac). Bidirectional elongation of the diacid moieties of 7 with identical peptide strands led to the formation of a C2-symmetric reverse-turn mimetic 12 which displayed a very ordered structure consisting of identical intramolecular H-bonds at two ends between LeuNH --> sugar-OH, the same as in 8 and 10.

Biodistribution of liposomes of terbutaline sulfate in guinea pigs.

A series of liposomes was prepared with various lipid (egg phosphatidyl choline [egg PC], phosphatidyl glycerol [PG], dipalmitoyl phosphatidyl choline [DPPC], distearoyl phosphatidyl choline [DSPC], dipalmitoyl phosphatidyl glycerol [DPPG], phosphatidyl ethanolamine [PE], cholesterol [CH], and stearylamine [SA]) compositions, such as egg PC:PG:CH (55:5:40), DPPC:PG:CH (55:5:40), DSPC:DSPG:CH (55:5:40) egg PC:SA:CH (55:5:40), DSPE:DSPG:CH (55:5:40) in molar ratio. Liposomal formulations were administered to guinea pigs intravenously; 3 hr after the treatment, serum samples and various organs (e.g., liver, spleen, lung) were removed and analyzed for drug concentration by a high-performance liquid chromatographic (HPLC) method. Based on the above study, a liposomal preparation with better lung specificity was selected, and the time profile of these liposomes was determined in guinea pigs. Three hours postadministration, a significant difference in blood levels was observed between free terbutaline sulfate and the various liposomal formulations. Localization of the drug in the lungs increased considerably when encapsulated drug was used, and the highest percentage localization was observed with DSPC:DSPG:CH (55:5:40) liposomes. The percentage recovery of the drug in the lungs with egg PC:CH:SA (55:40:5) liposomes did not change significantly when compared with egg PC:CH:PG (55:40:5) liposomes. To establish the time course of disposition of the liposomes, DSPC:DSPG:CH (55:5:40) liposomes were selected. Terminal half-life t1/2 of the drug in blood with free drug solution was about 12 hr, whereas with liposomes, a twofold increase in t1/2 was observed. The disposition data indicated that the clearance of the drug was delayed by 1.5 times when incorporated into liposomes.

Long-circulating liposomes of indomethacin in arthritic rats--a biodisposition study.

To improve the targeting efficiency of liposomes of indomethacin to the arthritic joints, circulation half-life of the liposomes was increased by grafting amphipathic polyethylene glycol-2000 to the bilayer surface. A comparative biodistribution study was performed between the conventional liposomes (PC:CH:PE--1:0.5:0.16) and long-circulating liposomes (PC:CH:PE-PEG--1:0.5:0.16) in arthritic rats. Pharmacokinetics of the drug changed significantly when administered in liposomal form. Pharmacokinetic parameters of the drug such as AUC0-t (trapezoidal), clearance and t1/2 (elimination half-life) changed significantly (p < 0.05) when encapsulated in liposomes. Significant difference in pharmacokinetics was observed in AUC0-t and clearance between the conventional liposomes and long-circulating liposomes. The increased AUC0-t and reduced clearance of the drug with long-circulating liposomes, increased the availability of the drug by reducing RES uptake, in turn localization in arthritic paw tissue was also increased. A concentration of 0.33 microgram of indomethacin/g of the tissue was achieved with S-liposomes after 24 h whereas it was only 0.26 microgram of drug/g of the tissue with conventional liposomes. From the study, in may be concluded that the targeting efficiency of the long-circulating liposomes was about four times more than the conventional liposomes.

Preparation and pharmacodynamic evaluation of liposomes of indomethacin.

The side effects of indomethacin, such as ulceration of the kidney and central nervous system (CNS) toxicity, limit its use as a drug for rheumatoid arthritis. Encapsulation of this drug in liposomes may reduce the toxic effects. The aim of this study was to determine the factors influencing encapsulation of indomethacin in liposomes and to determine anti-inflammatory potential of liposomal indomethacin. A series of liposomal formulations of indomethacin were prepared using various phospholipids. The effects of method of preparation, lipid composition, charge, and cholesterol (CH) on encapsulation of indomethacin in liposomes were investigated. A significant variation in encapsulation of the drug in liposomes was observed when prepared by different methods. With all the methods of preparation tried, the favorable lipid composition for high encapsulation of this drug was egg phosphatidyl choline:CH: stearlyamine (PC:CH:SA) at a 1:0.5:0.1 molar ratio. Inclusion of cholesterol did not affect the encapsulation efficiency of the drug in liposomes. The drug release profile from the liposomes was biphasic, and the highest percentage drug release was observed with large unilamellar vesicles (LUVs) (100 nm). Inclusion of stearylamine (PC:CH:SA 1:0.5:0.1) and phosphatidyl glycerol (PG) (PC:CH:PG 1:0.5:0.2) in the liposomes reduced the release of the drug in comparison to the neutral liposomes (PC:CH 1:1). The slow release of the drug from stearylamine-containing liposomes may be explained by the electrostatic interaction between the acid moiety of the drug and the amine moiety of the lipid. It is assumed that the possible hydrogen bonding between--OH groups of phosphatidyl glycerol and the--COOH group of the drug might be the reason for the slow release of the drug from PC:CH:PG (1:0.5:0.2) containing liposomes. Pharmacodynamic evaluation of the liposomes was performed by carrageenan-induced rat paw edema (acute) and adjuvant arthritis (chronic) models. The anti-inflammatory activity was increased from the first to fifth hour PC:CH:PG (1:0.5:0.2) and PC:CH:SA (1:0.5:0.1) liposomes showed the highest percentage inhibition of edema. In both these models, anti-inflammatory activity of liposomal indomethacin was significantly higher than that of free indomethacin (p < .01). The ulcer index of the free drug was about three times more than the encapsulated drug when administered at the same dose intraperitoneally to arthritic rats consecutively for 21 days.

Analgesic, antiinflammatory and hypoglycaemic activities of Sida cordifolia.

Sida cordifolia extracts of the aerial and root parts showed good analgesic, antiinflammatory and hypoglycaemic activities. The ethyl acetate (EA) extract of root (SCR-E) showed comparable antiinflammatory activity with indomethacin and possessed significantly higher activity when compared with that of the methanol extract of the root part (SCR-M). The ethyl acetate extract of both root and aerial parts of Sida cordifolia (SCR-E and SCA-E) showed very good central and peripheral analgesic activities at a dose of 600 mg/kg. The methanol extract of root (SCR-M) was found to possess significant hypoglycaemic activity.

Pharmacodynamic and pharmacokinetic evaluation of lipid microspheres of indomethacin.

Lipid microspheres (LM) of indomethacin were prepared using phosphatidyl choline and soya bean oil. LM of required size (< 1 micron) were obtained by intermittent microscopic observation while homogenization. Anti-inflammatory activity of lipo-indomethacin was compared with free indomethacin by carrageenan induced rat paw edema model. It was found that at 30% edema inhibitory dose, lipo-indomethacin was about 1.5 times more potent than free indomethacin, indicating possible localization of LM at the inflammatory site. Biodistribution studies were performed in rats at the dose of 12 mg/kg. After 2 h of the treatment, concentration of the drug was 0.31 microgram/g of inflammatory tissue with lipo-indomethacin, whereas, only 0.05 microgram/g of the tissue was found with free indomethacin. Maximum difference in drug concentrations between the above two formulations was observed in lungs. LM were found not to cross the BBB as drug concentration in the brain after lipo-indomethacin treatment was below the detectable level. Thus, the pharmacokinetic data gave a quantitative evidence for high anti-inflammatory potential of lipo-indomethacin.

Formulation and in vitro evaluation of polymeric films of diltiazem hydrochloride and indomethacin for transdermal administration.

Ethylcellulose-polyvinyl pyrrolidone films containing diltiazem hydrochloride and indomethacin were evaluated for their potential drug delivery at a controlled rate, using rat skin, to select a suitable formulation for the development of transdermal drug delivery systems. The influence of film composition, initial drug concentration, and film thickness on the in vitro drug release rate as well as drug permeation through rat abdominal skin were studied. Drug release studies were carried out employing the paddle over disk method and drug permeation through full thickness of the rat abdominal skin was tested using a modified Franz diffusion cell fastened with O-ring. The drug content of the film decreased at an apparent first-order rate, whereas the quantity of drug released was proportional to the square root of time. The release rates of both drugs increased linearly with increasing drug concentration and polyvinyl pyrrolidone fraction in the film, but was found to be independent of film thickness. The increase in release rate may be due to leaching of hydrophilic fraction of the film former which resulted in the formation of pores. It was also observed that the release of drugs from the films followed a diffusion-controlled model at low drug concentrations. A burst effect was observed initially, however, at high drug loading levels. This may be due to rapid dissolution of the surface drug followed by the diffusion of drug through the polymer network in the film. The in vitro skin permeation profiles showed increased flux values with increase of initial drug concentration in the film and also with the concentration of polyvinyl pyrrolidone. From this study, it is concluded that the films composed of ethylcellulose:polyvinyl pyrrolidone:diltiazem hydrochloride (8:2:2) and ethylcellulose:polyvinyl pyrrolidone:indomethacin (8:2:3) should be selected for the development of transdermal drug delivery systems, using a suitable adhesive layer and backing membrane, for potential therapeutic use.

Permeability studies of cellulose acetate free films for transdermal use: influence of plasticizers.

Permeability of cellulose acetate (CA) free films casted from chloroform solution containing different plasticizers were studied with a view to developing a suitable rate controlling membrane for transdermal use. Dibutyl phthalate (DBP), polyethylene glycol 600 (PEG 600) and propylene glycol (PG) were used as plasticizers at a concentration of 40% w/w of dry polymer weight. The free films were prepared by mercury substrate method and evaluated for uniformity of thickness, tensile strength and percentage elongation and water vapour transmission. Permeability characteristics of free films were studied using the drugs such as diltiazem hydrochloride (DLT) and indomethacin (Ind). The variation in film thickness was less than 2% which ensured uniformity. Tensile strength of films plasticized with DBP is more compared with other plasticized films. Water vapour transmission and drug diffusion through the free films followed zero order kinetics and decreased with increasing the film thickness. The films plasticized with PEG600 showed higher permeability for both drugs compared with other films. The order of decrease of permeability of plasticized films with plasticizers is PEG600 > PG > DBP. Diffusion of drugs through the free films of CA was extended over a longer period of time at a controlled rate and thus, these can be used as rate controlling membranes for the development of a transdermal drug delivery system.

Comparative pharmacokinetic evaluation of compressed suppositories of diclofenac sodium in humans.

Diclofenac sodium (CAS 15307-79-6) suppositories were formulated using polyethylene glycol 4000 as the base by dispersing the drug in the molten base, congealing the mass, followed by pulverization, sieving and subsequent compression of the resultant granules. These suppositories were evaluated with respect to their pharmacokinetic behaviour in 12 healthy, male human volunteers. The results were compared with those obtained after oral administration of a commercial enteric coated tablet. Bioequivalence between rectal suppositories and commercial tablets was observed with respect to AUC0-infinity and Cmax. However, tmax differed significantly (p < 0.05) in case of rectal administration (0.625 +/- 0.065 h) compared to oral tablet (1.58 +/- 0.06 h). The relative rectal bioavailability was 107.19 +/- 3.2.