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The intrinsic dissolution rate and solubility of carbamazepine was measured in aqueous solutions of sodium lauryl sulfate (SLS) prepared with two different grades of purity, 95 and 99%, and 95% SLS in 0.15 M NaCl to determine the effect of surface???active impurities and electrolytes. Four significant observations resulted from this work: (1) the equilibrium coefficients calculated from the solubility experiments in the 99% SLS, 95% SLS, and 95% with 0.15 M NaCl SLS solutions were 295, 265, and 233 L/mol, respectively; (2) the dissolution rate enhancement in the 99% SLS was 10% greater than that in the 95% SLS and 95% with 0.15 M NaCl solutions, which were not significantly different; (3) the diffusion coefficients of the drug???loaded micelles estimated from the dissolution experiments were 8.4 ?? 10 ???7 cm 2 /s for the 99% SLS, 9.5 ?? 10 ???7 cm 2 /s for the 95% SLS, and 1.2 ?? 10 ???6 cm 2 /s for the 95% with 0.15 M NaCl; and (4) the critical micelle concentrations for the 99% SLS, 95% SLS, and 95% SLS with 0.15M NaCl were 6.8, 4.2, and 0.35 mM, respectively. The results of this study clearly illustrate the sensitivity of the micelle to impurities and electrolytes with regard to size and loading capacity and the effect these changes have on the solubility and dissolution rate. Therefore, when using surfactants in dissolution media for in vitro testing of dosage forms, consideration must be given to the level of impurities present so that the results are consistent and reliable. Intrinsic dissolution rate, surface tension, or solubility measurements may be useful, convenient methods for identifying changes in the surfactant due to either degradation or lot???to???lot variability.

Stainless-steel templates of various thicknesses (75, 200, 800, and 1600??m) were used to apply propylene glycol/water gels containing methyl or propyl p -aminobenzoates to silicone rubber membranes, and drug delivery was studied with the use of the Bronaugh diffusion cell under conditions in which the drug was initially in thermodynamic equilibrium with respect to the application and membrane. Theoretical diffusion profiles were generated with the use of a model which assumes that diffusional gradients exist within the application. To use the model equation, previously derived for the initial condition in which the drug is in thermodynamic equilibrium with respect to the application and membrane, drug diffusivity in both the application and the membrane and the drug's membrane/ vehicle partition coefficient were independently determined. In general, agreement between experimental and theoretical results was within 25%.

There are presently no standards for in vitro research dealing with the release and delivery of drugs from semisolid dosage forms, largely because of inherent experimental difficulties. Among the problems, it has proven difficult to apply dosage forms to membranes mounted in in vitro diffusion cells in facsimile to the manner in which the dosage forms are applied clinically. In the present studies, methodology has been developed which allows films with thicknesses approaching clinical dimensions to be spread evenly over silicone rubber membranes. Using methyl p -aminobenzoate as a test permeant and gelled water and water/propylene glycol solvent systems as test vehicles, it has proven possible to spread films as thin as 75??m, yielding highly reproducible delivery profiles. Using this application technique, it has been shown how the diffusive clearance of drug from films of fixed composition placed over a resistant membrane is dependent on the thickness of application. For a given medium and thickness of application, when the vehicle composition is enriched in propylene glycol, partitioning into the membrane is suppressed, resulting in a lessening of the absolute rate of delivery and, consequently, a prolongation of the period over which drug is released. Increasing the membrane's resistance, i.e., increasing the membrane's thickness, likewise slows down the absolute delivery rate, extending the effective period of total clearance of drug from the applied film.

The effect of entrapped PEG-4000, inulin, and insulin on the in situ uptake from the rat jejunum of liposomes was determined. Each of these markers greatly reduced or completely eliminated jejunal uptake unless the liposomes were repeatedly???washed??? to remove marker from the external bilayer surface. Addition of free marker to a suspension of empty liposomes resulted in complete loss of liposomal uptake. It appears that???inert??? markers can profoundly affect the rate and extent of liposomal uptake from the rat jejunum and the preparative technique employed can greatly affect the behavior of the system.

Purpose . Topical delivery has been suggested to reduce systemic side effects while targeting cytokines for the treatment of certain skin conditions. Liposomes have been proposed as an enhancing agent for such a delivery. We have tested the potential of liposomes to augment the uptake of biologically active recombinant human interferon-gamma (rhIFN-??) into human skin lacking adnexa in an in vivo model.

The influence of formulation variables on the rheology of polyanionic formulations and the relationships between viscoelastic properties and mucociliary transport rate were investigated. Polymeric samples were oscillated from 0.001 to 5 Hz using either a "cone and plate??? or a "coaxial cylinder??? measuring system. The mucociliary transport rates of polymeric samples were determined and compared movement of charcoal powder on the frog palate. For the linear polymeric solutions, sodium carboxymethylcellulose and sodium alginate, the elastic modulus (G???) increased with increasing amplitudes during frequency scan. However, the G??? or viscous modulus (G???) of partially cross-linked polyacrylic acid (cPAA) samples did not change significantly under oscillation. Both G??? and G??? of cPAA samples were significantly influenced by the amount of salt present in the formulation. The rheology of 2% (w/w) cPAA in 90:10 (w/w) propylene glycol:alcohol changed from a viscous fluid to a coarse suspension after neutralization. The pH increased gradually when the nonaqueous formulation reacted with water and the maximum dynamic moduli were obtained after incorporating 20% (w/w) water in the formulation. A negative correlation was found between the G??? of linear polyanionic samples and the relative transport rate. However, the lowest mucociliary transport rate was observed when the loss tangent (G???/ G???) was around 0.4???0.5.

Expression plasmid DNA for the human interleukin-1 receptor antagonist (IL-1ra) protein was formulated with nonionic:cationic (NC) liposomes or phosphatidylcholine:cationic (PC) liposomes and applied to the auricular skin of hamsters in single- and multiple-dose protocols. Confocal microscopy identified delivery of plasmid DNA proximal to perifollicular cells, and successful transfection of perifollicular cells was identified by immunohistochemistry and ELISA. Skin treated for 3 days with the NC liposomes had statistically significant levels of transgenic IL-1ra present for 5 days post-treatment. Expression of transgenic IL-1ra was specific to areas of skin treated with NC liposomes but not PC liposomes. The results indicate that the NC liposomes can deliver expression plasmid DNA to perifollicular cells and mediate transient transfection in vivo.

The effect of particle size of liposomes on the deposition of drugs into the strata of skin was evaluated using hairless mice, hamster and pig skin. In vitro diffusion studies were performed in an attempt to find an optimum formulation for topical drug delivery as well as to try to explain the mechanism of topical drug delivery by liposomes. The results indicate that an optimum particle size for optimal drug delivery exists. The study proved that the follicular route play an important role in determining the kinetics of drug transfer from liposomes into the skin.

The viscoelastic propertied of model polymer, hydroxypropylmethylcellulose (HPMC), in ternary solvent mixtures and the variation of viscoelastic properties under dilution with water were investigated. HPMC was dispersed in mixtures of glycerol formal (GF) or ethyl alcohol, propylene glycol (PG), and water. Each polymeric sample was oscillated at different frequencies with fixed stress amplitude and the changes in elastic modulus, viscous modulus, and apparent viscosity were measured at 30[deg]C. The moisture absorption rate of HPMC in PG:GF solvent mixtures in a 94% relative humidified environment was also studied. A linear relationship between the elastic modulus or viscous modulus and polymer concentration was observed for the HPMC samples. The relative mucociliary transport rate tested on the non-depleted frog palate model revealed a curvilinear correlation with the loss tangent (a ratio of the viscous modulus to the elastic modulus) of the polymeric solution. Using GF as a solvent for HPMC resulted in a formation of a rigid gel with the highest elastic modulus and viscous modulus among solvents selected. A higher affinity for water was found for HPMC in the PG:GF 90:10 mixture compared with HPMC in the PG:GF 70:30 mixture. However, the elastic modulus and viscous modulus were much higher for HPMC in the PG:GF 70:30 mixture.

Minoxidil dissolved in propylene glycol/ethanol/water (20:60:20; v/v) was more efficiently delivered into hairless mouse skin from a 0.02% solution than from a 2% formulation during in vitro mass balance experiments. Evaporation of the vehicle after application of the 2% minoxidil solution led to compositional changes so drastic that saturation is attained quickly. The shifting compositions of the vehicle were experimentally profiled and the drug's solubility was measured in media reflecting these changes over time. The original 2% formulation has excess capacity to dissolve minoxidil but, following its application, the solution rapidly saturates through evaporation, reaching saturation within 30 minutes. While the 2% formulation definitely functions at a proportionally higher thermodynamic activity than the 0.02% formulation at early times, most of this thermodynamic advantage is lost abruptly upon precipitation of the drug. The driving force following drug precipitation only reflects the dissolved minoxidil, and thus the relative delivery rate of the 2% formulation eventually drops to only several multiples of the delivery rate of the 0.02% solution which never quite attains saturation.

The extent of uptake of gamma interferon ([gamma]-IFN) in various strata of hairless mouse, human and hamster skin upon application of a liposomal formulation and an aqueous solution were determined by in vitro diffusion cell experiments. For each of the animal species studied, 70-80% of the liposomally entrapped IFN was deposited onto or penetrated into the skin as determined 24 h after in vitro application. However, a significant fraction of this total amount ([approximate] 0.25-0.30) is either adsorbed to or associated with the stratum corneum. The drug content found in the deeper skin strata, where the receptor sites reside, suggests that drug deposition is strongly influenced by the skin species tested. The percent of applied drug found in this strata 24 h after application followed the order: hamster (6.1)>= human (0.9)> hairless mouse (0.3), although the amounts of drug in the total skin of each species tested were approximately the same. This indicates that the deposition of drug into the living epidermis and/or dermis cannot be predicted by determination of the amount of drug in the total skin. The amounts in the deeper skin strata were also in the order of increasing number of follicles/hair in the skin species, suggesting that the transfollicular route is an important pathway for liposomal topical therapeutics.

The effectiveness of three adhesive tapes, 3M's No. 810, No. 845 and No. 850, in stripping stratum corneum from hairless mouse skin, was compared by measuring transdermal water loss (TEWL) and distribution profiles of a marker, minoxidil, from a solution formulation. While No. 850 tape failed to increase TEWL perceptively even after 24 strippings, both No. 810 and 845 tapes effectively removed the whole stratum corneum, at least as reflected in TEWL measurements. TEWL specifically increased incrementally from somewhere between 4 and 8 g/m2 per h to 120 g/m2 per h. From both TEWL measurements and distribution profiles of minoxidil in skin 2 h post-application of its formulation, it appears that the 3M No. 845 tape more efficiently removes stratum corneum than 3M No. 810 tape. The difference between No. 810 and No. 845 tapes in stripping formulation-treated skin held in a diffusion cell was application-time dependent, since the No. 845 tape harvested no more minoxidil than the No. 810 tape at 12 and 24 h following application of the formulation.

The present study demonstrates that in vitro comparisons of delivery vehicles are reliable as long as one performs the studies on skin samples taken from the same cadaver section of skin. The in vitro rates of delivery of minoxidil and hydrocortisone from an ointment and a propylene glycol/ethanol/water vehicle through skin sections taken from the same and different cadaver abdomens are specifically compared. When tested on skins from 8 human cadavers, the fluxes of minoxidil suspended in the ointment ranged from 0.37 x 10-5 mg/cm2/h to 9.2 x 10-5 mg/cm2/h, a 25-fold spread, and the fluxes of minoxidil from the propylene glycol/ethanol/water vehicle ranged from 1.25 x 10-5 mg/cm2/h to 18.7 x 10-5 mg/cm2/h, a 15-fold spread. However, when the ratios of fluxes between these two formulations were calculated for individual subjects, the values ranged from 1.3 to 3.5 (only a 3-fold spread). Four human cadaver sections were used to evaluate the delivery of hydrocortisone. The flux values ranged from 696 to 1335 cpm/cm2/h for the ointment and 946 to 1291 cpm/cm2/h for the propylene glycol/ethanol/water vehicle (up to a 2-fold spread). When the ratios of fluxes between these two formulations were calculated for each individual subject, the values ranged from 0.96 to 1.39. The results with these two compounds suggest that even in the unavoidable presence of large inter-subject variations in the cadaver skin, the influence of vehicle on drug delivery performance can be determined. In addition to these findings concerning intra-subject variability, it was observed that the flux of minoxidil from the ointment attains a steady-state after an initial lag period of between 6 and 8 h but steady delivery is not apparent for the minoxidil solution, suggesting that for the solution formulation the thermodynamic activity of. the drug changes markedly as the experiment proceeds. No comparable curvature is seen for the hydrocortisone solution presumably because its thermodynamic activity

Freeze-fracture has been used to examine the effects of gentamicin on membrane structure in liposomes of different anionic phospholipids combined with a neutral phospholipid, phosphatidylcholine. The molar ratios of neutral: anionic lipid were 1:1 (high anionic lipid ratio) and 4:1 (low anionic lipid) and the liposomes were incubated with 0.1 mM (low) and 1 mM (high) gentamicin. With the anionic phospholipid phosphatidylinositol bisphosphate, an identifiable disruption of the membrane bilayer was observed as well as aggregation of liposomes leading to membrane fusion. These effects occurred both at low gentamicin concentration and low anionic lipid content of the liposomes; these responses were not inhibited by 1 mM Ca2+. With the other anionic lipids tested (phosphatidylserine, phosphatidylinositol and phosphatidylinositol monophosphate), only aggregation and fusion of liposomes was observed and this effect only occurred at high gentamicin concentration and high anionic lipid content. Further, 1 mM Ca2+ inhibited the responses of these other anionic lipids to gentamicin. The results demonstrate the unique character of the interaction between gentamicin and phosphatidylinositol bisphosphate and provide further support for the hypothesis that a specific binding to this lipid is a key step in the ototoxic action of aminoglycoside antibiotics. They also suggest that such an interaction in vivo might cause alterations to the structure and properties of cell membranes in the inner ear.

An in vitro technique has been developed for evaluating the delivery performance of topical semi-solid formulations. A thin and uniform layer of formulation was applied in facsimile to actual usage conditions by troweling the vehicle across a thin. circular copper template (200 [mu]m in thickness). Approximately 30-45 mg of an oil-in-water cream, a water-in-oil cream or an ointment, each containing a range of concentrations of minoxidil, were applied over human cadaver skin within a defined circular area of 1.54 cm2. The rates of permeation of minoxidil from these formulations were determined by finite dose diffusion experiments. For formulations containing 2% minoxidil, the flux from the w/o cream tested was about 4 times higher than fluxes from the o/w cream and the ointment. Even though all w/o formulations were initially saturated with drug, the flux of minoxidil from these creams increased as the concentration of minoxidil was increased from 0.5% to 2%. In contrast, the delivery rates from the o/w cream and the ointment did not appear to be dependent on the minoxidil concentration applied (0.5-2%). Under the operative experimental conditions, the percent coefficients of variation of flux of minoxidil from these formulations were less than 20%. To achieve this low level of variability, the skin samples were all obtained from the same cadaver abdomen. If one assumes that the efficacy of a particular formulation is dependent on the ability of the drug to be released from the vehicle and diffuse through the skin, the studies show that the nature of the vehicle can profoundly affect delivery even when excess solid drug is present. They also indicate that reliable in vitro comparisons of drug delivery are possible as long as one performs the studies on skin samples taken from the same section of skin.

The rate of release from multilamellar liposomes of the fluoroscent probe carboxyfluorescein was determined as a measure of membrane permeability. Liposomes of phosphatidylcholine and different anionic phospholipids were incubated with low (1 [mu]M) and high (3 mM) concentrations of calcium in the absence or presence of aminoglycoside antibiotics. The leakage of carboxyfluorescein into the medium was not caused by liposomal fusion as no vesicle fusion was observed in experiments with terbium and dipicolinic acid-loaded liposomes. The basal rate of carboxyfluorescein release (in the absence or presence of 1 [mu]M calcium) from all types of liposomes ranged from 0.1 to 0.3% of trapped carboxyfluorescein per hour. The presence of 3 mM calcium caused the greatest increase in the rate of carboxyfluorescein release (about 9-fold) in liposomes containing phosphatidylinositol 4,5-bisphosphate (PIP2) whereas liposomes containing the other anionic phospholipids (phosphatidylserine, phosphatidylinositol and phosphatidylinositol 4-phosphate) showed an approximate 5-fold increase. In the presence of 1 [mu]M calcium, the aminoglycosides neomycin and gentamicin also increased the rate of carboxyfluorescein release, with PIP2-containing liposomes showing a 3-5-times greater response than the other liposomes, releasing up to 4.6% of trapped carboxyfluorescein per hour. This drug-induced release was dose-dependent and antagonized by calcium. In the presence of 3 mM calcium, 0.1 mM gentamicin or neomycin were ineffective while the drug at 1 mM affected carboxyfluorescein release from PIP2-liposomes only. The aminoglycoside antibiotics, neomycin, gentamicin, tobramycin, kanamycin, amikacin, netilmicin, as well as neamine and spectinomycin (all at 0.1 mM) showed a graded effect on the rate of carboxyfluorescein release from PIP2-containing vesicles in the presence of 0.1 mM calcium. The magnitude of the effect correlated well with the ototoxicity of the drugs previously determined direc

Carboxyfluorescein is the most commonly used probe to measure the rate of release of vesicle contents. The validity of the data obtained by this method depends on obtaining an end point based on the complete release of the dye on treatment of the liposomes with a detergent, usually Triton X-100. However, Triton does not completely release entrapped carboxyfluorescein from multilamellar liposomes and the amount and rate of release of marker upon detergent treatment is a function of lipid composition of the liposome, Triton concentration and temperature and duration of detergent incubation. The fluorescence `end point' for distearoyl--[alpha]-phosphatidylcholine/cholesterol (2:1, mol%) multilamellar liposomes treated with 0.5% Triton at 22[deg]C (a condition often used) is only about one-fifth the value for liposomes treated with 5% Triton at 72[deg]C. The conditions of treatment appear to affect the release of carboxyfluorescein from the lipid of the partially or completely disrupted liposome and the subsequent partitioning of the free dye into the aqueous phase. This effect can lead to serious errors in the interpretation of multilamellar liposome stability data. However, Triton allows complete release of entrapped dye from small unilamellar vesicles under all conditions tested.

Aminoglycoside interactions with various phospholipids were measured in three model systems and compared with the ototoxicities of the drugs: (a) competition for [14C]neomycin binding; (b) competition for 45Ca2+ binding; and (c) effect on surface pressure of monomolecular lipid films. The efficacies of the antibiotics in displacing neomycin from phosphatidylserine, phosphatidylinositol or phosphatidylinositol bisphosphate were netilmicin> neomycin [ges] gentamicin; the efficacies in displacing calcium from phosphatidylinositol, phosphatidylinositol phosphate or phosphatidylinositol bisphosphate were netilmicin> gentamicin> neomycin> kanamycin> spectinomycin. Neither measure correlated well with the ototoxicities of the drugs which were quantitated at equimolar drug concentrations in cochlear perfusions: neomycin> gentamicin>= tobramycin> netilmicin>= amikacin. When monomolecular films of phosphatidylcholine with phosphatidylserine, cardiolipin, phosphatidylinositol, or phosphatidylinositol phosphate or bisphosphate were challenged with neomycin, the phosphatidylinositol bisphosphate film showed a unique dose-dependent increase in surface pressure while the others showed a decrease or no significant effect. The abilities of aminoglycosides to increase the surface pressure of a film of phosphatidycholine : phosphatidylinositol bisphosphate (1:1 molar ratio) in the presence of 3 mM CaCl2 correlated well with their toxicities. Non-ototoxic cations increased the film pressure or left it unaffected. The results confirm the unique interactions between aminoglycosides and phosphatidylinositol bisphosphate as a possible basis of a mechanism of toxicity and development of a drug-screening system.

The transport of sterols incorporated into the lecithin bilayer of small unilamellar liposomes through a model membrane was studied. A two-chamber diffusion cell containing liposomes with incorporated [4-14C]cholesterol or [beta]-[4-14C]sitosterol in the donor chamber and liposomes with unlabeled cholesterol in the receiver chamber was used. The permeability coefficients of the sterols through silastic rubber membranes which served as a model membrane were measured. The permeability for cholesterol incorporated into liposomes in a phosphatidyl choline/cholesterol molar ratio of 1 : 1, produced by sonication for 1 h, and subsequent centrifugation at 100000 x g for 1 h, was 1.6 [middle dot] 10-8 cm sec-1. Dilution of the liposome suspension did not change the permeability coefficient significantly. The permeability coefficient of sitosterol incorporated into liposomes was about 4-times smaller than that of cholesterol. These results suggest that the sterols were delivered to the silastic membrane by the intact liposomes and that free solute was not involved in the transport to the membrane to a significant degree. The large differences in the permeability coefficients between cholesterol and sitosterol indicate that an aqueous interfacial barrier was crossed by the sterol during the delivery to the membrane.

The ototoxicities of eight aminoglycoside antibiotics and fragments were measured quantitatively by cochlear perfusion in the guinea pig. Perilymphatic spaces were perfused for 1 hr with `artificial perilymph' containing 10 mM drug, during which time continuous measurements of cochlear microphonic potentials were made. Kanamycin B and neomycin B caused the most rapid decline of cochlear microphonic potentials, followed by gentamicin C1a ~- ribostamicin> kanamycin A ~ G-418. Neamine and methylneobiosamine did not show significant effects. The same drugs were tested for their interaction with monomolecular films of polyphosphoinositides, and relative binding constants were determined. Neomycin B and kanamycin B had the highest affinities to the lipids, followed by the other drugs in the order as seen for toxicity. The correlation between the in situ and in vitro actions of the drugs was r = 0.9. These results support the hypothesis that binding to polyphosphoinositides plays an important role in the decrease of the cochlear microphonic potentials. Furthermore, the good correlation between the drug actions in the two test systems suggests that an in vitro assay may be possible for the assessment of aminoglycoside ototoxicity.

Neomycin and related aminoglycosidic antibiotics displace calcium from synaptosomes of guinea pig cerebral cortex and from preparations of phosphatidylinositol diphosphate. At low drug concentrations, inhibition of synaptosomal calcium binding is competitive (Ki = 3[middle dot]10-5M), at high concentrations it is non-competitive (Ki = 4[middle dot]10-4M). Monomolecular films of phosphatidylinositol diphosphate are contracted by low concentrations of neomycin in the subphase, and are expanded at high concentrations. This expansion persists even at the collapse pressure indicating a strong interaction between the drug and the lipid.