[The use of the Epidemiology Cancer registry Baden-Württemberg in the follow-up of the EPIC cohort]. / Nutzung des Epidemiologischen Krebsregisters Baden-Württemberg bei der Nachbeobachtung der EPIC-Kohorte.

The European Prospective Investigation into Cancer and Nutrition (EPIC) is a prospective multicentre study that has been implemented to further the understanding of the association between diet and chronic diseases with emphasis on cancer. In Heidelberg from June 1994 until October 1998 about 25,500 subjects aged 35 to 65 years in women and 40 to 65 years in men were recruited. Apart from extensive questions about food intake, the participants were also asked to provide detailed information about their smoking habits, physical activity, subjective well-being, medical history and use of medications. As well as completing the questionnaire and a personal interview, the participants also gave blood samples and anthropometiric measures and the blood pressure were taken in standardized manner. The analyses of the EPIC study will depend on achieving a comprehensive record of all new cancer cases, and all deaths, together with the corresponding cause of death, within the study population. To date all self-reported incident cancer cases are verified by comparing them with pathology reports and hospital records. They are then coded according to the coding list for the International Classification of Diseases (ICD-O2) issued by the World Health Organisation (WHO). Since at begin of the investigation in the study region no cancer registration existed, the participants are followed -up by interval questioning ('active follow-up'). In order to integrate increasingly the data of the Cancer registry Baden-Württemberg (EKR-BW) attempts were made to explore record linkage systems. For this purpose, in the years 2000, 2002, 2003 record linkages between EPIC-Heidelberg and EKR-BW were performed. Procedures were evaluated for performing an anonymous linkage of the EPIC data with the data of the EKR-BW. After a pilot project on the feasibility of the linkage the program was evaluated on the EPIC data, record linkages are performed regularly. Different coding systems were applied. Simultaneously, the EPIC data about on cancer cases among the Heidelberg study participants are passed on to the Cancer Registry, thus contributing to improve completeness of the registry. So far the active follow-up can not be replaced by passive follow-up through record linkage with the cancer registry, but in the long-term it may be possible. Since the technical requirements are complied with, attempts should be made to improve the completeness of the epidemiological cancer registry Baden- Württemberg.

Effect of chemopreventive agents on separate stages of progression of benzo[alpha]pyrene induced lung tumors in A/J mice.

The effects of aerosol budesonide and dietary myo-inositol on progression of benzo[alpha]pyrene (B[alpha]P) induced carcinogenesis were studied in A/J mouse lung. First, we determined when to intervene in the carcinogenesis process by exposing several animals to B[alpha]P at 100 and 150 mg/kg of body wt. Groups of these animals were necropsied from 1 to 36 weeks post-carcinogen. The presence of different categories of lung tumors was noted over the 36 week time period. Hyperplasia first appeared approximately 6 weeks post-carcinogen followed by adenoma at 9 weeks, then by carcinoma at 26 weeks. From this temporal sequence we determined we could test for effects of preventive agents on progression to hyperplasia by intervening at 3 weeks, for effects on progression to adenoma by intervening at 6 weeks and for effects on progression to carcinoma by intervention at 12 weeks. Intervention at 3 weeks post-carcinogen with aerosolized budesonide delayed both hyperplasia and adenoma formation. Once hyperplasia appeared in budesonide treated animals, however, it increased at the same rate as in control animals, indicating a delay in progression. Progression from adenoma to carcinoma was reduced when budesonide was given 12 weeks post-carcinogen. Dietary myo-inositol failed to suppress progression from adenoma to carcinoma when started 12 weeks post-carcinogen. In summary, budesonide is a chemopreventive agent that has inhibitory effects on B[alpha]P induced carcinogenesis of the lung in A/J mice at all stages of progression from hyperplasia formation to cancer.

Analysis of the diffusion of bile salt/phospholipid micelles in rat intestinal mucin.

The interaction of sodium taurocholate/egg phosphatidylcholine (TC/PC) micelles with mucin was determined to investigate the exclusion of lipids by mucus in the absorption process. The distribution of TC/PC was assessed at two intermicellar and three phospholipid concentrations with isolated, rat intestinal mucin (RIM) by dialysis. The diffusion coefficients were measured by NMR spectroscopy. At high [PC], RIM had lower [PC] relative to the control, while the concentration of TC was largely independent of mucin concentration. The PC diffusion coefficients were reduced in the presence of RIM. The magnetization decay of TC was compared with simulations to provide estimates of the monomeric diffusivity and exchange rate constant. The rate constants increased with increasing micelle concentration, and the free TC diffusion coefficient was reduced in the presence of mucin. Mucin has an exclusive effect on TC/PC mixed micelles that has been quantitatively determined through the use of diffusion measurements of dialyzed samples.

Interaction of bile salt and phospholipids with bovine submaxillary mucin.

PURPOSE: The purpose of this study was to determine the distribution and diffusion of sodium taurocholate-phospholipid micelles with mucin in order to provide the foundation for understanding the transport of ingested fat and poorly water-soluble drugs through the intestinal mucous layer. METHODS: Sodium taurocholate (NaTC) was dispersed with egg phosphatidylcholines (PC) to yield mixed micelles of a specific size and concentration. A preliminary study was conducted to determine the time required for equilibration of PC/TC micellar solutions with mucin. PC/TC micellar solutions were dialyzed against fixed and variable concentrations of bovine submaxillary mucin after which the concentration of PC and NaTC was measured by an assay for total phosphorus and by HPLC, respectively. In addition, a quantitative assay of TC and PC by NMR was developed and used to estimate the mobile fraction of lipids in the samples. Finally, pulsed-field gradient spin echo NMR self-diffusion measurements were made of the water, TC, and PC in the samples obtained from dialysis. RESULTS: TC/PC micellar solutions achieved equilibrium with mucin in 7 days. Mucin did not affect the equilibrium concentration of PC or TC, except at high concentrations of mucin (5%), and then the effect was small. NMR quantitation was valid for PC and TC systems containing small micelles, but deviated significantly with systems containing large micelles. Mucin decreased the diffusivity of water and the phospholipids, but the effect was relatively small. Mucin dramatically affected the mobility of TC, which prevented a straightforward interpretation of the calculated diffusion coefficients. CONCLUSIONS: Mucin has a minor effect on the equilibrium distribution of phospholipids and bile salts. However, lipids are readily accommodated by mucus, which can significantly increase the permeability of the mucous layer, particularly for poorly water-soluble drugs.

Ultrasonic nebulization system for respiratory drug delivery.

An ultrasonic spray system was tested for the production of aerosols for ultimate use in the respiratory delivery of drug to animals. A Sono-Tek ultrasonic spray system was mounted on top of a baffle to entrain aerosol particles within the carrier gas. Solvent was removed from the aerosol cloud by passing the droplets through drying columns composed of either charcoal or silica. The efficiency of removing ethanol and water were determined by measuring the outflow concentrations. Sodium fluorescein and sodium cromolyn dissolved in water were tested, and the effect of the liquid flow rate and drug concentration entering the atomizer on the output, and particle size distribution, were determined by the filter capture method, and by cascade impactor, respectively. Similar studies were conducted with budesonide and indomethacin dissolved in ethanol. The theoretical count median size distribution was calculated and compared with the experimental values calculated from the observed mass median aerodynamic diameter. The output rate expressed as the mass of aerosol collected in unit time increased nearly proportionately with the liquid flow rate (0.18-0.7 ml/min) and with the concentration of drug (0.19-12 mg/ml) entering the nebulizer. The mean particle size increased with solute concentration, but not by liquid flow rate. The calculated count median diameters were dependent on the type of solvent, but were independent of solute. At the high dose of cromolyn, there was very good agreement between the theoretical and observed. At lower doses, the observed size was larger than predicted, which was also true for the ethanol soluble solutes. The system has the potential of providing a wide range of dose levels for testing of drug delivery to animals including high doses with a controlled and relatively narrow particle size distribution.

The absorption of retinoic acids from the gastrointestinal tract is dependent upon chemical structure.

PURPOSE: The gastrointestinal permeability of a number of retinoic acids was determined in order to evaluate whether the gastrointestinal membrane was able to distinguish between retinoids in which the polyene chain was present in several different isomeric forms. In addition, the structure of the six-membered ring was varied in order to determine which portion of the molecule was most important for its recognition by the membrane. The role of bile salt micelle composition in the intestinal absorption of retinoids was also evaluated. METHODS: In situ perfused rat intestinal segment preparations (= 78) were used, and the retinoids were each perfused at a concentration of approximately 1 microg/ml in either simple micelles of sodium taurocholate (10 mM) or mixed micelles of sodium taurocholate/egg phosphatidylcholine (10 mM/10 mM). The flow rate of the perfusate was either 0.1 or 0.35 ml/min. RESULTS: For each retinoid, the mixed micelles were associated with a higher degree of retinoid uptake into the jejunal cells than were the simple micelles. In addition, the permeability was higher when the perfusate flow was greater, indicating that the aqueous boundary layer of the intestine contributes to the resistance to the disappearance of the retinoid from the intestinal lumen. Retinoid structure was also found to have a significant effect on the permeability in the mixed micelle systems at both low and high flow rates, but not with simple micelles. The structure of the six-membered ring was not a major determinant of the permeability. However, the permeability of the retinoids with the polyene chain in the 13-cis position was significantly greater than when the chain was all-trans or in the 9-cis position. CONCLUSIONS: The isomeric position of the polyene chain and the presence of phospholipid in the micellar vehicle have a significant influence on the membrane transport of the retinoic acids.

Note: dissolution of aerosol particles of budesonide in Survanta, a model lung surfactant.

The effect of a pulmonary surfactant extract from bovine lung, Survanta, on the dissolution rate of aerosol particles of budesonide was determined. Aerosol particles of budesonide were generated from an ethanol solution, dried, and collected by a cascade impactor for characterization or by a liquid impinger for dissolution experiments. Powder x-ray diffraction, differential scanning calorimetry, differential thermal analysis, and scanning electron microscopy were used to characterize the aerosol particles and starting material. No change in phase was detected, although the aerosol particles appeared to contain residual solvent. The dissolution rate of the aerosol particles in saline was low and variable. Survanta increased the extent of dissolution of budesonide in proportion to the added concentration, which was also verified by equilibrium solubilization studies. Survanta also increased rate of dissolution, in a manner similar to sodium dodecyl sulfate. Analysis of the concentration of budesonide following ultracentrifugation indicated that there is rapid equilibration of budesonide between the Survanta and aqueous phase. These results show that lung surfactant has the potential of enhancing the rate and extent of dissolution of drugs administered to the lung.

Distribution and diffusion of sodium taurocholate and egg phosphatidylcholine aggregates in rat intestinal mucin.

PURPOSE: The permeability of rat intestinal mucin (RIM) to sodium taurocholate/egg phosphatidylcholine (TC/PC)-mixed micelles has been investigated. METHODS: The time dependence for the equilibration of TC/PC-mixed lipid micelles with isolated RIM was determined. Thereafter the distribution of TC/PC-mixed lipid micelles was assessed at low and high PC and intermicellar concentrations (IMC) and with different RIM concentrations. The equilibrium distribution of PC and TC was determined by analysis for phosphorus and by high-performance liquid chromatography, respectively, as well as by nuclear magnetic resonance spectroscopy. In addition, the diffusion coefficients of water, PC, and TC were measured by pulsed field gradient nuclear magnetic resonance spectroscopy. Two model solutes, phenylmethyltrimethylsilane (PTMS) and tetramethylsilyl-tetradeutero-proprionic acid (TSP), were added to the high PC, low IMC samples, and the diffusion coefficients were determined. RESULTS: The time to reach equilibrium was 2 days for a system with a high intermicellar concentration of sodium taurocholate. At low PC concentrations, RIM had slightly higher PC concentrations relative to the control. In contrast, at high PC concentrations, RIM samples had lower PC concentrations. The concentration of TC was largely independent of mucin concentration. The water diffusivity was reduced proportionately to the concentration of RIM, and analysis indicated that about 150 g of water moved as a kinetic unit with each gram of mucin. The diffusion coefficients of PC were also reduced with increasing RIM concentration. The magnetization decay of TC did not always follow a monoexponential decay, reflecting the simultaneous diffusion and exchange among sites imparting different relaxation behavior on the TC. Magnetization decay curves were simulated and the diffusivity of TC in mucin was estimated. The diffusion coefficient of TSP was 10 times larger than that of PTMS in the presence of micelles and mucin. CONCLUSIONS: RIM is highly hydrated, and dilute solutions have a minor exclusive effect on the high concentration of PC/TC micelles. At low concentrations of PC, there appears to be preferential association of the PC with the RIM. The permeability of mucin to solutes in the presence of bile salt mixed micelles critically depends on the degree of association of the solute with the micelle.

Drug solubilization in lung surfactant.

The relative affinity of glucocorticosteroids for lung surfactant was determined for the purpose of identifying chemopreventive agents with a high therapeutic index for lung cancer. The aqueous solubility and the extent of solubilization in Survanta, a native extract of bovine lung, of budesonide, triamcinolone acetonide, dexamethasone, and flunisolide were determined as a function of temperature by a dialysis technique. The aqueous solubilites at 37 degrees C were 19.6, 35.8, 104 and 120 microg/ml for the above listed compounds, respectively. The temperature dependence of the solubilities was modest consistent with the hydrophobic properties of the steroids. The amount of drug in solution was significantly enhanced in the presence of Survanta with solubilization ratios of 0. 019, 0.023, 0.014, and 0.02 microg drug dissolved per microg of Survanta phospholipid, respectively. In addition, the extent of solubilization also generally increased with temperature, although the phase transition of the surfactant lipid appeared to complicate the functional relation between temperature and solubilization. These results show that there is enhanced solubilization of glucocortosteroids by lung surfactant which is secreted by the cancer susceptible type II cells of the lung.

Hydraulic high-pressure nebulization of solutions and dispersions for respiratory drug delivery.

The purpose of this investigation was to assess hydraulic high-pressure nebulization as a means for respiratory drug delivery. A hydraulic high-pressure nebulizer was designed and constructed. In a design study, the output efficiency and the aerosol particle size were determined for the nebulizer as a function of nozzle diameter (5, 10, and 20 microns), gas flow rate (2 and 8 l/min), applied hydraulic pressure (2200 and 4000 psig), and distance between the nozzle orifice and impaction surface (0.25-4 cm) with an aqueous solution of fluorescein. The output efficiency was also measured with an ethanol solution and an aqueous phospholipid dispersion of liposomes. For the design study, each factor had an effect. The efficiency tended to increase with a decrease in the nozzle diameter, although the differences between the 5- and 10-micron nozzle were more sensitive to the air flow rate and nozzle-to-impaction-surface distance. Greater efficiencies were always obtained at the higher ancillary air flow rates. Operating the nebulizer at different pressures caused a change in the functional relationship between the efficiency and the nozzle-to-impaction-surface distance. For the 5-micron nozzle at high pressure, efficiency fell with increasing nozzle-to-impaction-surface distance, whereas for the data obtained with the 20-micron nozzle, the efficiency increased with nozzle-to-impaction-surface distance, with lower efficiencies obtained at the higher pressures. For the remaining observations made with the 5- and 10-micron nozzles, the efficiency as a function of nozzle-to-impaction-surface distance appeared to be variable. For the 5- and 10-micron size nozzle, there was no significant effect of the air flow rate, pressure, or nozzle-to-impaction-surface distance on the mass median aerodynamic diameter and geometric standard deviation. For the 20-micron size nozzle, the particles were not completely dried. Ethanol solutions gave somewhat higher efficiencies, whereas the phospholipid dispersion gave efficiencies comparable to the aqueous solutions nebulized under similar conditions. The efficiency of the hydraulic high-pressure nebulizer appears to be correlated with the calculated properties of the liquid jet. For respiratory drug delivery, the hydraulic high-pressure nebulizer provides reasonably high outputs of respirable particles independent of time from a single pass of liquid through the nebulizer.

Chemoprevention of pulmonary carcinogenesis by brief exposures to aerosolized budesonide or beclomethasone dipropionate and by the combination of aerosolized budesonide and dietary myo-inositol.

This investigation is part of an effort to develop chemoprevention for carcinogenesis of the lung. It focuses on the efficacy of low doses of synthetic glucocorticoids administered either as single agents or in combination with a second compound, myo-inositol. Glucocorticoids are potent inhibitors of carcinogenesis. The use of low doses is important to avoid potential side-effects. The synthetic glucocorticoid budesonide, administered by aerosol for 20 s three times a week, was studied to determine its effects on benzo[a]pyrene-induced pulmonary adenoma formation in female A/J mice. Two dose levels were employed, 10 and 25 microg/kg body wt. The lower dose produced a 34% reduction in lung tumor formation and the higher dose level a 60% reduction in lung tumors. In additional groups of mice, the effects of 0.3% myo-inositol added to the diet was found to reduce pulmonary tumor formation by 53%. The two agents given in combination resulted in a greater inhibition of lung tumor formation than either by itself. Budesonide at 10 microg/kg body wt plus 0.3% myo-inositol reduced the number of tumors by 60% and budesonide at 25 microg/kg body wt plus 0.3% myo-inositol reduced lung tumor formation by 79%. To determine whether a glucocorticoid other than budesonide would have inhibitory effects in this experimental model, beclomethasone dipropionate administered by aerosol for 20 s three times a week was studied as a single agent and showed almost identical inhibitory properties to budesonide. The doses of the glucocorticoids calculated on a daily basis are within the range of those used widely for control of chronic allergic respiratory diseases in the human. The capacity of low doses of inhaled glucocorticoids to prevent pulmonary neoplasia and the enhancement of this preventive effect by myo-inositol, an essentially non-toxic compound, are findings that should encourage further work to evaluate the applicability of these agents to the prevention of neoplasia of the lung in the human.

Analysis of a diffusion dryer for the respiratory delivery of poorly water soluble drugs.

PURPOSE: The purpose of this study was to analyze a diffusion dryer as a means to remove organic solvents from aerosol particles of poorly water soluble drugs. METHODS: Aerosols of methanol, ethanol, and ethyl acetate were generated with an ultrasonic nebulizer, and inflow to outflow concentration ratio of vapor in a annular charcoal column was determined as a function of time by gas chromotography at two to four different airflow rates. In addition, the particle transmission efficiency was determined with an ethanol solution of the test compound, budesonide. The results were analyzed with equations originally developed for assessing the loss of drug from intravenous tubing along with independent measures of the adsorption isotherm of the vapors onto charcoal. RESULTS: Aerosol production was relatively constant with time, and the transmission of solid particles through the column occurred with efficiency nearing 100%. The inlet to outlet vapor concentration ratio was adequately described by a model of three resistances in series composed of the inner tube, the screen mesh, and the charcoal bed. CONCLUSIONS: The diffusion dryer was found to be satisfactory for the removal of methanol, ethanol, and ethyl acetate and the efficiency may be assessed from the adsorption isotherms on charcoal and the geometry of the dryer.

Analysis of the solubilization of steroids by bile salt micelles.

Bile salt mixed micelles play an important role in the emulsification, solubilization, and absorption of cholesterol, fats, and lipid-soluble vitamins. Studies have also revealed the importance of wetting and solubilization of drugs by the bile salts; however, the capacity and specificity of bile salt simple and mixed micelles for solubilization are still undefined. Thus, in this study the aqueous solubility and the extent of solubilization in bile salt micellar solutions of a series of steroids were determined. The melting point and enthalpy of fusion were measured to determine the ideal mole fraction solubility of each steroid. From the ideal mole fraction solubility and the observed solubility of each steroid, the activity coefficient of each steroid in solution was calculated with respect to the supercooled pure liquid as the standard state. In aqueous solution, the activity coefficients decreased with increasing number of hydroxyl groups on the steroids. This trend also occurred in the bile salt solutions, although the values of the activity coefficients were smaller by about three orders of magnitude. The stereochemical position of the hydroxyl groups influenced the rank order of the activity coefficients in the aqueous and micellar solutions. Incorporation of a fluoro or methyl group resulted in an increase in the activity coefficient, whereas aromatization of the A ring of the steroid frame markedly decreased the activity coefficient. The results for the aqueous solubility are consistent with the expected interaction of the functional groups with the polar environment. The relatively small activity coefficients observed with the micellar solutions result from the nonpolar environment of the aggregates. However, the similarity of the relationship between the activity coefficient and the number of hydroxyl groups in the aqueous and micellar solutions indicates the importance of polar interactions for solubilization. These results may provide new insight into the solubilization of steroids by bile salt micellar solutions and may provide a basis for predicting solubilization of other compounds by bile salt aggregates.

Chemoprevention of pulmonary carcinogenesis by aerosolized budesonide in female A/J mice.

This investigation is part of a continuing effort to develop effective chemoprevention for carcinogenesis of the lung. The present study explores the use of aerosol administrations for this purpose. The agent selected for initial study was the synthetic glucocorticoid budesonide. This selection was based on previous work in which budesonide added to the diet was found to inhibit pulmonary adenoma formation in female A/J mice. However, high dose levels were required, i.e., of the order of 300 microg/kg, of body weight [L. W. Wattenberg and R. D. Estensen, Carcinogenesis (Lond.), 18: 2015-2017, 1997]. For aerosol administration of budesonide, a nose-only technique has been developed that entails nebulization of the compound dissolved in ethanol and subsequent stripping off of the solvent (less than 3 microl ethanol/liter of air remaining at the site of inhalation). The budesonide particles produced by the apparatus had a mass median aerodynamic diameter of less than 1 microm. An experiment has been carried out in which the inhibitory effects of aerosolized budesonide, given for 1 min six times a week, were studied. Concentrations of budesonide of 26, 81, and 148 microg/liter of air (calculated doses of 23, 72, and 126 microg/kg of body weight) were used. The aerosols were started 1 week after three oral administrations of benzo(a)pyrene (2 mg/20 g of body weight) to female A/J mice. All three doses of budesonide resulted in more than 80% inhibition of pulmonary tumor formation compared to the aerosol control and 90% or greater compared to mice not exposed to aerosol. The difference in inhibition is due to the aerosol procedure itself, which produces a reduction in tumor formation. A decrease in splenic weight (evidence of a systemic effect) occurred at all doses of budesonide. To the best of our knowledge, this is the first published effort at the use of aerosol administration to prevent neoplasia of the respiratory tract. The results of the present study show that administration of a potential chemopreventive agent by aerosol at a low dose can inhibit the occurrence of pulmonary carcinogenesis in female A/J mice.

Ionization and solubilization of 4 alkyl benzoic acids and 4 alkyl anilines in sodium taurodeoxycholate solutions.

PURPOSE: The aqueous solubility and the extent of solubilization and ionization constant in sodium taurodeoxycholate (NaTDC) solutions of a series of benzoic acid and aniline derivatives were measured as a basis to characterize and thereby help predict the nature of the interaction of drugs with bile aggregates. METHODS: The aqueous solubility and the solubilization of two series of compounds, 4-alkyl benzoic acids and 4-alkyl anilines, was measured as a function of NaTDC in 0 and 150 mM NaCl. The ionization constants were determined in water and in 50 mM NaTDC at sodium chloride concentration of 0, 75 and 150 mM by spectrophotometric titration. The diffusion coefficients of NaTDC and the solutes were measured by pulsed-field gradient spin echo NMR spectroscopy. RESULTS: The aqueous solubilities decreased with increasing alkyl chain length in both series, and the aniline derivatives had larger solubilities than the benzoic acid derivatives. The number of moles of solute solubilized per mole of bile salt ranged from 0.17 to 0.31 for the benzoic acid derivatives and from 1.3 to 3.0 for the aniline derivatives. The pKa values of the benzoic acid derivatives in the presence of NaTDC were higher relative to the controls, and the difference in the pKa (delta pKa,obs) increased with increasing chain length. With the aniline derivatives, the pKa values were also shifted to higher values in NaTDC relative to the control but only in the absence of salt. The presence of the solute caused a decrease in the diffusion coefficient of NaTDC, and the diffusion coefficients of the solutes decreased with increasing alkyl chain length. With the hexyl derivative, the diffusion coefficient of the solute was smaller than the diffusion coefficient of the bile salt. The chemical shift of the protons attached to carbon 18 and 19 of the salt were decreased to a greater extent in the presence of the solutes than the protons attached to carbon 26. CONCLUSION: Both the solubilization and ionization behavior of solutes were affected by the presence of bile salt aggregates. The surface potential and effective polarity of NaTDC aggregates were found to be dependent on the alkyl chain length for these two homologous series of solutes. The solubilization ratio was largely independent of alkyl chain length, but the unitary partition coefficient was dependent on both alkyl chain length as well as ionization state. The derivatives reduced the diffusivity of the micelles suggesting the formation of larger size aggregates and the solutes (hexyl derivatives) appear to favor association with the larger sized aggregates. The phenyl ring of the solutes appears to be oriented parallel to the plane of the steroid frame with preferential positioning near the hydrophobic rings.

Human stratum corneum lipids have a distorted orthorhombic packing at the surface of cohesive failure.

The cohesive strength of the stratum corneum is determined by its unique molecular composition and structural architecture. Whereas the structure responsible for corneocyte cohesion has been visualized at the microscopic level, the structure of the intercellular domain has not been characterized at the molecular level. In this report, new insight into the molecular architecture of the stratum corneum has been provided by atomic force microscopy and x-ray photoelectron spectroscopy. The surface layer of human stratum corneum was stripped, yielding the characteristic polygonal corneocytes shown by scanning electron microscopy as well as low resolution atomic force microscopy. With atomic force microscopy, the resolution was increased to allow imaging of the molecular architecture of the stratum corneum. With the high resolution image, a repetitive pattern characteristic of lipids in an ordered state was visualized. The lattice appeared to be orthorhombic where the lattice distances were about 5.5 and 9 angstroms, and the lattice angle was close to 90 degrees. The atomic composition of the superficial layers was 82% carbon, 16.5% oxygen, and 1.4% nitrogen as determined by x-ray photoelectron spectroscopy. The high nitrogen content compared to the calculated stratum corneum lipid composition and measured model lipid composition suggests that proteins were detected. In summary, although proteins are present, the fracture plane of the stratum corneum is largely composed of lipids that appear to have a distorted orthorhombic packing.

Solubilization of retinoids by bile salt/phospholipid aggregates.

PURPOSE: The capacity and specificity of bile salt (BS)/ phosphatidylcholine (PC) mixed lipid aggregated systems in solubilizing four structurally related retinoids, etretinate, motretinid, fenretinide and N-ethyl retinamide, were determined. METHODS: Excess solid drug was dispersed into sodium taurocholate (NaTC)/egg PC systems at lipid ratios of 10:0, 10:2 and 10 mM:10 mM in isotonic HEPES buffer, pH 6.5. A sensitive HPLC method was used to quantify the amount solubilized. The melting point and associated enthalpy change as well as the aqueous solubilities were also measured. RESULTS: The retinoids had aqueous solubilities of less than 25 nM. The predicted aqueous solubility was less than 0.01 nM. The amount of retinoid in 10 mM NaTC was increased from three to four orders of magnitude relative to the aqueous solubility. Further increases in the amount solubilized were observed in the 10:10 mixed micelle dispersion. Fenretinide and N-ethyl retinamide were particularly well solubilized by BS and BS/PC aggregated systems which may be related to the presence of a cyclohexenyl ring. CONCLUSIONS: The discrepancy between the observed and predicted aqueous solubility may be due to self-association of the retinoids. Micellar/aqueous distribution ratios appear to be dominated by the hydrophobic effect, although specific interactions also are important. In considering intestinal absorption, the large increase in solubilization with BS/PC micelles would be capable of dramatically increasing the bioavailability in spite of the smaller effective diffusivity of the solubilized retinoid.

Diffusivity of bile salt/phospholipid aggregates in mucin.

PURPOSE: The objective of this study is to evaluate the effect of the mucous layer on the transport of the drug-solubilizing bile salt/phosphatidylcholine (BS/PC) aggregates. METHODS: The self-diffusion coefficient of BS/PC aggregates in bovine submaxillary mucin (BSM) was measured by Fourier-transform pulsed-field gradient spin-echo (FT-PGSE) 1H NMR spectroscopy. RESULTS: In spite of the complexity of the mixture, the FT-PGSE technique allowed the unambiguous determination of the diffusivity of PC and 1H2HO (HDO, natural abundance in D2O). With a series of BS/PC total lipid concentrations ranging from 1 to 7 g/dl, a progressive decrease in the effective diffusivity of HDO was observed with an increase in the both the BSM and total lipid concentration. The effective diffusivity of PC decreased with increasing lipid concentrations in the presence of mucin, while in the controls it increased. After correcting the effective diffusivity of PC for the obstruction effect of mucin, the size of the BS/PC mixed micelle was assessed. It appears that PC associates with BSM resulting in a decrease in the available PC for micellization. This reduces the average size of the mixed micelle within the mucous layer. CONCLUSIONS: The aggregation state of BS/PC micelle is altered by the presence of mucin which would have a direct impact on the transport of dietary lipid and solubilized drug through the aqueous boundary layer of the intestinal tract.

Retinoid absorption from simple and mixed micelles in the rat intestine.

The absorption of three retinoid analogs etretinate (ET), acitretin (ETA), and motretinid (MOE) from two distinct micellar systems was studied in the rat intestine. Each of the three drugs was loaded into simple micelles consisting of 10 mM sodium taurocholate (NaTC) and mixed micelles consisting of 10 mM egg phosphatidylcholine (PC) and 10 mM NaTC. Following perfusion through the jejunum segments, both the fraction of drug disappearing from the segment and the permeability of the drug from the lumen into the gut wall (Peff) was greater with the mixed micelles as compared to the simple micelles. Perfusion flow rate had an influence on the Peff for ET and ETA. Similar trends as for the jejunum were seen in the ileum perfusions. The simultaneous uptake of PC and NaTC during the retinoid perfusions was monitored. There appeared to be a correlation between the Peff values for PC and that of the retinoids. The viability of the in-situ perfusion system was confirmed histologically. There is evidence to indicate that the permeability of the intestine is sensitive to subtle differences in the chemical structure of the retinoids.