Preparation and purification of cationic solid lipid nanospheres--effects on particle size, physical stability and cell toxicity.

Cationic solid lipid nanospheres (SLN) were prepared by the microemulsion technique with polysorbate 80 (Tween 80) and butanol as surfactants. The SLN (diameter 100-500 nm, zetapotential around +15 mV) consisted mainly of stearylamine (SA) and different triglycerides. Three different purification methods, ultrafiltration, ultracentrifugation and dialysis, were investigated and compared with the cellular toxicity and physical stability of the dispersions. The cell toxicity was dependent on both the SLN composition and the purification method. Dialysis was found to easily and efficiently remove excessive surfactant determined by dynamic light scattering (DLS), leading to reduced cell toxicity and increased physical stability of the SLN on storage. The cationic SLN might constitute a promising DNA delivery system.

Physical stability of redispersible dry emulsions containing amorphous sucrose.

The objective of the present study was to estimate the stability of redispersible dry emulsions containing amorphous sucrose. Dry emulsions were prepared by spray drying liquid o/w-emulsions in a laboratory spray dryer. The effect of hydroxypropyl methylcellulose (HPMC) on the glass transition temperature T(g) of spray dried sucrose-HPMC mixtures, relative to the T(g) of amorphous sucrose, was investigated. For the sucrose-HPMC mixtures the values of T(g) followed the ideal Gordon-Taylor equation up to 30% HPMC. For dry emulsions containing 40% HPMC, 30% lipid and 30% sucrose, the T(g) was increased by 12 degrees C relative to the T(g) of amorphous sucrose. The stability of the dry emulsions was investigated by a conventional stability study and by an enthalpy relaxation study. The measured enthalpy recovery of amorphous sucrose below T(g) was used to calculate molecular relaxation time parameters based on the Williams-Watts equation. The molecular mobility of amorphous sucrose at temperatures 50 degrees C below T(g) was low and negligible with respect to the shelf life stability. It was concluded that the dry emulsions are physically stable with respect to the lifetime of a pharmaceutical product when stored in dry condition and at temperatures up to 28 degrees C.

Comparison of the lymphatic transport of a lipophilic drug from vehicles containing alpha-tocopherol and/or triglycerides in rats.

The applicability of alpha-tocopherol as a lymphotropic carrier for a highly lipophilic drug has been evaluated. Transport to the intestinal lymph of the highly lipophilic model drug, Lu28-179, in rats after administration to the stomach in an alpha-tocopherol emulsion was compared with lymphatic transport after administration of a sesame oil emulsion and an alpha-tocopherol/sesame oil emulsion. Lymphatic transport of the triglycerides and of alpha-tocopherol was determined. A conscious rat model was used, and the mesenteric lymph was collected. There was no significant difference between the cumulative masses of triglyceride from the two emulsions containing triglyceride 24 h after administration. Administration of an alpha-tocopherol emulsion seemed to induce mobilization of endogenous triglyceride. The lymphatic transport of alpha-tocopherol was less than 1 mg 24 h after administration of both emulsions containing alpha-tocopherol. The absorption of Lu28-179 from the alpha-tocopherol emulsion was very low, with a lymphatic recovery of 0.05%. When administered in an alpha-tocopherol/sesame oil emulsion, the recovery of Lu28-179 increased sevenfold to 0.35%. However, after administration of Lu28-179 in a sesame oil emulsion, the lymphatic recovery increased a further 13-fold to 4.5%. In conclusion, the study showed that alpha-tocopherol did not promote lymphatic absorption of Lu28-179 and thus was not a good lymphotropic carrier, as compared with sesame oil. Alpha-tocopherol in combination with sesame oil was not a good lymphotropic carrier either. The non-absorbed alpha-tocopherol fraction in the intestine might be able to prevent the absorption of Lu28-179.

Comparison of total oral bioavailability and the lymphatic transport of halofantrine from three different unsaturated triglycerides in lymph-cannulated conscious rats.

The lymphatic transport and the portal absorption of the lipophilic drug halofantrine were investigated in a conscious rat model. The rats were dosed with 0.1 g with triolein, trilinolein or trilinolenin containing 2 mg halofantrine. Following oral administration of the triglycerides, the mesenteric lymph and plasma samples were collected. The lymphatic transport for halofantrine was 11.1+/-1.2 after administration of trilinolein, 9.0+/-3.5 for trilinolenin and 8.6+/-2.2 for triolein and the total amount of halofantrine transported in the lymph was linear proportional with the amount of triglyceride in the lymph. The absorption of halofantrine directly into the blood showed a trend towards a higher AUC for trilinolien and trilinolenin compared to triolein, but no statistical difference could be found. The statistically analysis of the mean total bioavailability therefore shows that the absorption of halofantrine was largely independent on triglyceride unsaturation.

Comparison of the lymphatic transport of halofantrine administered in disperse systems containing three different unsaturated fatty acids.

PURPOSE: To compare the influence of the degree of fatty acid unsaturation (oleic [C18:], linoleic [C18:2], or linolenic acid [C18:3]), with the intestinal lymphatic transport of halofantrine free base from disperse systems in anesthetized rats. METHODS: The mesenteric lymph duct was cannulated in anesthetized rats. Lipid vehicle containing halofantrine was administered by intraduodenal infusion. The concentration of halofantrine in blood and lymph samples was analyzed. RESULTS: The rank order of the lymphatic transport of halofantrine was C18:2 > C18:1 > C18:3. Comparison of the area under the curve (AUC) from the three fatty acids showed no statistically significant differences between the AUCs from the lymph cannulated rats. In terms of rank order effects, the plasma concentrations of halofantrine were highest for the rats dosed C18:2 followed by C18:3 and C18:1. CONCLUSIONS: Using C18:2 as a vehicle increased the lymphatic transport of halofantrine 16.6-fold over that observed for the system containing C18:3. The extent of lymphatic transport for the C18:1 system did not differ from the other two formulations, but the combined lymph and plasma data indicated that the C18:2 was the most suitable lipid vehicle for the oral delivery of halofantrine.

A dynamic in vitro lipolysis model. II: Evaluation of the model.

A lipolysis model was characterised and evaluated by investigating the composition of the aqueous phase and the concentration of probucol and danazol in the aqueous phase. Effects of bile salt levels at 5, 10, 20, and 30 mM were investigated. Samples were taken at 0%, 50%, 75% and 95% hydrolysis of the triglycerides, and the aqueous phases were isolated by ultra-centrifugation, whereby the concentrations of bile salts, fatty acids, mono-, di-, triglycerides, and drug substances were measured. At high Ca(2+)-concentrations, bile salts were believed to precipitate with Ca(2+). The concentration of lipolytic products (fatty acids + monoglycerides) was dependent on the bile salt concentration. The ratio between lipolytic product and bile salts was 1.55+/-0.09 (S.D.). This ratio is equivalent to mixed bile salt micelles and vesicles in equilibrium. The aqueous solubility of probucol and danazol was increased in the presence of bile salts. The concentration of danazol in the aqueous phase was dependent on the solubilisation capacity of the aqueous phase. In the case of probucol, the concentration in the aqueous phase was dependent on the partition of probucol between the aqueous phase and the remaining triglyceride phase. This difference between danazol and probucol was attributed to the effect of different lipophilicity.

A dynamic in vitro lipolysis model. I. Controlling the rate of lipolysis by continuous addition of calcium.

Lipolysis by pancreatic lipase was investigated with the aim to establish an in vitro lipolysis model, which can be used to investigate the dissolution of poorly soluble lipophilic drug substances at controlled hydrolysis rates. The effects of three experimental parameters -- the concentrations of bile salts and Ca(2+) and the lipase activity -- were investigated. The effect on the rate of hydrolysis of emulsified soybean oil was investigated in experiments in a pH-stat at pH 6.5 and 37 degrees C. The free fatty acids produced by the hydrolysis were titrated at pH 6.5. It was shown that all three investigated parameters influence the initial rate of hydrolysis, whereas only the lipase activity and the concentration of Ca(2+) affect the subsequent stages. It was also shown that the rate of lipolysis can be controlled by the rate of adding Ca(2+). Thus, it is possible to design an in vitro model using readily available and inexpensive materials in which the hydrolysis rate can be controlled by the continuous addition of Ca(2+).

The effect of alpha-tocopherol on the in vitro solubilisation of lipophilic drugs.

alpha-Tocopherol is an excellent solvent for many poorly soluble drugs. The aim of this work was to study whether or not the presence of alpha-tocopherol has an influence on the solubilisation of poorly soluble drugs in simulated intestinal fluids (SIF). The solubilizing capacity of mixed micelles containing alpha-tocopherol towards three lipophilic drugs was investigated. The solubilisation of alpha-tocopherol in an aqueous micellar phase was increased by the addition of monoglycerides (MG) and free fatty acids (FFA), preferably of medium chain length, as compared to a simple bile salt solution. The addition of alpha-tocopherol to mixed micellar solutions seems to have an effect on the solubilizing capacity, which can be correlated to the partition coefficient of the drug to be solubilised. A positive effect on the solubilisation of griseofulvin and felodipine was found. For a highly lipophilic drug (Lu28-179), a positive effect on solubilisation was observed only in media containing MG and FFA of medium chain length. Generally, alpha-tocopherol cannot be considered an important factor for the solubilisation of highly lipophilic drugs in SIF. The presence of lipolytic digestion products (LDP) of the proper chain length in relation to the drug to be solubilised is much more important.

Preparation of redispersible dry emulsions by spray drying.

Development of stable dry emulsions being able to reform the original o/w-emulsion by reconstitution in water is presented. Dry emulsions were prepared by spray drying liquid o/w-emulsions in a laboratory spray dryer. Three hydroxypropylmethylcellulose (HPMC) types were applied as solid carrier and emulsifier. The lipid phase was fractionated coconut oil. The ratio of solid carrier to lipid phase influenced the reconstitution properties. It was possible to prepare redispersible dry emulsions of a lipid content up to 40% dry powder mass. The different HPMC types had no noticeable effect on the reconstitution properties, but too viscous liquid o/w-emulsions were difficult to atomise. The type of rotary atomizer, or the rate of rotation did not affect the technical properties of the dry emulsions containing 40% lipid. It was concluded that low viscosity HPMC was a useful solid carrier. The dry emulsions remained physically stable for at least 6 months.

Technical optimisation of redispersible dry emulsions.

Preparation of dry emulsions suitable for tablet processing was examined in this study. Liquid o/w-emulsions were spray dried in a laboratory spray dryer applying hydroxypropylmethylcellulose (HPMC) as a solid carrier and emulsifier. As the lipid phase, fractionated coconut oil was used. The ability of various excipients to increase the density of dry emulsions was investigated. Adding sucrose to the formulation, redispersible dry emulsions with higher density were obtained. The type of rotary atomizer did not affect the dry emulsions containing sucrose nor the rate of rotation of the atomizer applied in the spray drying process. By wet granulation, using ethanol as a binder, free-flowing and compactable dry emulsions were obtained and simultaneously the reconstitution properties were preserved. It was concluded that dry emulsions could be optimised for tablet processing by wet granulation. Tablets having a lipid content up to 20% had proper tablet properties.

Direct estimation of the in vivo dissolution of spironolactone, in two particle size ranges, using the single-pass perfusion technique (Loc-I-Gut) in humans.

AIM: The objective of this in vivo dissolution study was to investigate the usefulness of the Loc-I-Gut technique for differentiating between the in vivo dissolution rate of two particle sizes of spironolactone, and to compare these in vivo results with corresponding in vitro data. METHODS: The study included six volunteers, and consisted of three sequential parts (I, II, III). In parts I and III the in vivo dissolution was measured directly by perfusing a semi-open segment in the proximal jejunum. In part II, a solution of spironolactone was administered orally, and the plasma concentration time profile was followed for 48 h. The in vitro dissolution was measured using flow-through cells and different dissolution media simulating human gastrointestinal fluids. RESULTS: A difference in in vivo dissolution rate of the two different particle sizes was observed, based on perfusion data. This difference was not pronounced in the relative bioavailability of spironolactone administered in two different particle sizes. The relative bioavailability was dependent on the bile acid concentration in vivo. In vitro, dissolution rate of the smaller particles was improved at fasted state bile acid concentrations, while the larger particles were only significantly affected at fed state bile acid concentrations. CONCLUSION: In vivo dissolution studies discriminated between the dissolution rate of the two different particle sizes of spironolactone, based on the perfusate samples. The lack of difference in relative bioavailability, might be explained by the insufficient wash-out of particles after ending the perfusion, reabsorption of surface active ingredients along the GI tract, relatively small difference in particle size and the large inter- and intra-individual differences in pharmacokinetic variables.

Dissolution of hydrocortisone in human and simulated intestinal fluids.

PURPOSE: To compare solubility and dissolution rate of hydrocortisone in aspirated human intestinal fluids (HIFs) with simulated intestinal fluids (SIFs) and buffer. METHODS: Solubility and flux from a rotating disk of hydrocortisone were measured. The bile salt content, pH and osmotic pressure were determined in HIFs. RESULTS: In fasted state the solubility of hydrocortisone was higher in HIFs than in the buffer and SIFs. The flux of hydrocortisone in HIFs was similar to the flux in the buffer but lower than the flux in SIFs at fasted state. Addition of intestinal surfactants in SIFs increased solubility and flux at both fasted and fed state. The increase in solubility was caused by micelle formation in SIFs. The increase in flux may partly be explained by increased solubility. The bile salt content of the HIFs did not correlate with the solubility or the flux but pH in the HIFs seems to have some effect on the components of the HIFs resulting in increased solubility. CONCLUSIONS: It is possible to perform comparable dissolution tests in HIFs and SIFs. The lack of correlation between the results in HIFs and the bile salt content may be explained by the relatively low lipophilicity of the model drug.

Growth mechanisms in melt agglomeration with a low viscosity binder.

Lactose monohydrate was melt agglomerated in an 8-l high shear mixer using stearic acid as meltable binder. The impeller speed was varied at six levels, and the temperature of the heating jacket was varied at three levels. The agglomerate growth mechanisms were found to be different from those observed in previous melt agglomeration experiments. This is attributed to the low viscosity of the molten stearic acid. A higher impeller speed and a higher jacket temperature resulted in an agglomerate growth that was markedly affected by comminution and accordingly gave rise to agglomerates of a wider size distribution and a higher porosity. A lower impeller speed and a lower jacket temperature resulted in smoother and more spherical agglomerates.

Electrostatic charging during a melt agglomeration process.

Lactose monohydrate was melt agglomerated in an 8-l high shear mixer using stearic acid, polyethylene glycol (PEG) 3000, and a mixture of stearic acid and glycerol monostearate as meltable binders. Electrostatic charging during processing at relative air humidities of 35 and 75%, respectively, was estimated from the voltage of a monitoring probe inserted into the mixer. Stearic acid produced a high level of electrostatic charges, whereas PEG 3000 gave rise to a markedly lower level. Addition of glycerol monostearate to the stearic acid reduced the level of electrostatic charges. A correlation was found between the resistivity of the binder and the level of electrostatic charges in the material. With the stearic acid and the binder mixture, the level of electrostatic charges was higher at a low air humidity. The amount of adhesion to the bowl was found to depend on the level of electrostatic charges.

Evaluation of melt agglomeration properties of polyethylene glycols using a mixer torque rheometer.

Lactose was melt agglomerated in a mixer torque rheometer with polyethylene glycol (PEG) 2000, 3000, 6000, 8000, 10 000, or 20 000 as meltable binder. A longer massing time caused an increase in mean torque until a maximum value after which the torque decreased. A smaller particle size of the PEG gave rise to a faster initial rise in mean torque. The higher viscosity of the PEG 20 000 resulted in a higher mean torque, whereas no clear difference in mean torque was obtained with the other PEGs. The binder concentration could be varied within a rather wide range without causing overwetting, the range being wider with PEG 3000 than with PEG 20 000. The mean torque values obtained were found to be related to the liquid saturation of the agglomerates. The reproducibility of the experiments was found to be very dependent on the experimental conditions, the highest binder viscosities and binder concentrations giving rise to a poor reproducibility. The results were compared with a few melt agglomeration experiments with PEG 3000 in a high shear mixer. The mixer torque rheometer was found not to be suitable for predicting melt agglomeration properties in the high shear mixer because of a marked difference in the shear forces in the two mixers.

A comparison between direct determination of in vivo dissolution and the deconvolution technique in humans.

AIM: The primary objective of this study was to investigate the in vivo dissolution of carbamazepine in humans and to compare it with the dissolution estimated by deconvolution of plasma concentrations as well as the in vitro dissolution. METHODS: The in vivo study included six healthy volunteers, and consisted of two sequential parts. In part 1 the dissolution was measured by perfusing a semi-open segment in the proximal jejunum in humans. In part 2 the volunteers were given a solution of carbamazepine orally. In both parts of the study, plasma samples were collected up to 48 h after administration of the dose. The in vitro dissolution was measured in a flow-through cell using dissolution medium with and without the addition of bile acids (3 mM). RESULTS: The direct measured in vivo dissolution profile of carbamazepine and the deconvoluted profile were found to be similar. The two dissolution profiles of carbamazepine obtained in vitro were statistically lower than the two in vivo dissolution profiles. The higher in vivo dissolution rate is probably due to efficient sink conditions as a consequence of the high permeability of carbamazepine and more pronounced intestinal motility. CONCLUSION: The jejunal perfusion system was successfully used for in vivo dissolution measurements of carbamazepine and agreed with the deconvoluted plasma profile regarding rate and extent of dissolution. Single-pass perfusion is therefore a meaningful tool for further studies of in vivo dissolution.

Quantitative analysis of povidone (PVP) in drug-PVP matrix using multicomponent analysis.

A method for the quantification of povidone (PVP), in solid dispersions and physical mixtures of the polymer and a very slightly soluble drug substance, has been developed by multicomponent analysis using the concepts of chemometrics. Because the UV-absorbance spectra of PVP is completely overlapped by the UV-absorbance spectra of the drug substance, a direct spectrophotometric method of PVP is impossible. However, UV-spectrophotometric data were analyzed by the Quant + Perkin Elmer software for quantitative multicomponent analysis using chemometrics, and by the optimal method developed using a solvent of pH 7.4, a fast, reliable, and precise detection of PVP was obtained when the content of PVP in the powder sample exceeded 20% (m/m). Two methods were developed by the calibration procedure, using buffers of pH 7.4, respectively pH 8.5. By applying a solvent of pH 8.5, more sample could be taken into use because of the enhanced solubility of the drug substance, and hence it was believed that as more PVP was taken into use, a better prediction of PVP would be obtained. However, as more drug substance was taken into use the UV-absorbance spectrum of PVP was even more overlapped, and an inferior prediction was obtained.