Characterization of Mefenamic Acid-Guaiacol Ester: Stability and Transport Across Caco-2 Cell Monolayers

Purpose. Prodrug of non-steroidal anti-inflammatory drugs (NSAIDs) or NSAIDs linked with guaiacol have been reported to suppress gastrointestinal (GI) toxicity or induce GI protective effect. In this study, mefenamic-guaiacol ester was synthesized and its physicochemical properties, stability, and transport across Caco-2 monolayers were investigated. Methods. Synthesis of the ester was carried out using mefenamic acid, guaiacol, N, N-dimethylaminopyridine, and N, Ndicyclohexylcarbodiimide. The hydrolysis of the ester was investigated in aqueous buffer solutions pH 1-12 as well as in Caco-2 homogenate, human plasma, and porcine liver esterase. Caco-2 cell monolayers were utilized for transport studies. Due to the high lipophilicity of the ester with a calculated logP of 6.15, bovine serum albumin (BSA, 4%) was included in the receiver compartment to obtain a good in vitro-in vivo correlation. Permeation of the ester was assessed with or without the exposure of cells to PMSF, an inhibitor of esterase. Results. The ester was stable at a wide pH range from 1-10. However, it was hydrolyzed by enzymes from porcine liver esterase and Caco-2 homogenate. With the PMSF exposure on the apical (AP) side and in the presence of 4% BSA on the basolateral (BL) side, the transported amount of the ester from AP-to-BL direction was 14.63% after 3 hr with a lag time of 23 min. The Papp for the ester was 4.72 × 10^-6 cm s^-1. Conclusion. The results from hydrolysis studies indicate that this ester is a prodrug. The Papp value suggests the moderate absorption characteristic of the compound. The accumulation of this highly lipophilic ester in Caco-2 cells is reduced in the presence of BSA.     

Evaluation of Stereoselective Transdermal Transport and Concurrent Cutaneous Hydrolysis of Several Ester Prodrugs of Propranolol: Mechanism of Stereoselective Permeation

Purpose. The purpose of this study was to evaluate the stereoselective permeation and concurrent cutaneous hydrolysis of a series of ester prodrugs of propranolol (PL). Methods. In vitro studies were performed across full-thickness, stripped and diisopropylfluorophosphate (DFP) treated skins of hairless mouse with flow-through diffusion cells at 37°C. Results. The permeability coefficients (K _p), which were dependent on partition coefficients (PC), of all the prodrugs were markedly increased compared to the parent drug. In full-thickness skin, the (R) caproyl-PL (CR-PL) showed the highest K _p, which was about 52-fold greater than that of PL. Most of the more lipophilic prodrugs showed stereoselectivity in K _p (R > S). All the prodrugs underwent stereoselective hydrolysis (R > S) during penetration. The prodrugs which showed stereoselectivity in permeation were comparatively lipophilic and showed great differences in hydrolysis percentages between the enantiomers. Permeation studies with stripped skin revealed that prodrugs were more permeable across stratum corneum compared to PL, whereas reverse was happened across viable skin. Although CR-PL showed high stereoselectivity in permeation across full-thickness skin and underwent higher percent of concurrent stereoselective cutaneous hydrolysis, the prodrug showed no stereoselectivity in permeation across DFP, an esterase inhibitor, treated skin and the concurrent cutaneous hydrolysis was also stopped. Conclusions. Lipophilic prodrugs may readily pass the stratum corneum but may not be able to penetrate so easily through the deeper tissues. Unlike the (S) isomers, the (R) isomers of lipophilic prodrugs almost completely converted to propranolol in epidermis and can easily pass through the dermis layer, resulting in stereoselective penetration.     

Transcorneal Permeation of l- and d-Aspartate Ester Prodrugs of Acyclovir: Delineation of Passive Diffusion Versus Transporter Involvement

PURPOSE: The aim of this study was to evaluate the contribution of amino acid transporters in the transcorneal permeation of the aspartate (Asp) ester acyclovir (ACV) prodrug. METHODS: Physicochemical characterization, solubility and stability of acyclovir L: -aspartate (L: -Asp-ACV) and acyclovir D: -aspartate (D: -Asp-ACV) were studied. Transcorneal permeability was evaluated across excised rabbit cornea. RESULTS: Solubility of L: -Asp-ACV and D: -Asp-ACV were about twofold higher than that of ACV. The prodrugs demonstrated greater stability under acidic conditions. Calculated pK(a) and logP values for both prodrugs were identical. Transcorneal permeability of L: -Asp-ACV [Formula: see text] was fourfold higher than D: -Asp-ACV [Formula: see text] and ACV [Formula: see text]. ACV generation during the transport process was minimal. L: -Asp-ACV transport was sodium and energy dependent but was not inhibited by glutamic acid. Addition of BCH, a specific B(0,+) and L amino acid transporter inhibitor, decreased transcorneal L: -Asp-ACV permeability to [Formula: see text]. L: -Asp-ACV and D: -Asp-ACV did not demonstrate significant difference in stability in ocular tissue homogenates. CONCLUSION: The results demonstrate that enhanced transport of L: -Asp-ACV is as a result of corneal transporter involvement (probably amino acid transporter B(0,+)) and not as a result of changes in physicochemical properties due to prodrug derivatization (permeability of D: -Asp-ACV and ACV were not significantly different).     

Amino Acid Ester Prodrugs of Floxuridine: Synthesis and Effects of Structure,Stereochemistry, and Site of Esterification on the Rate of Hydrolysis

Purpose. To synthesize amino acid ester prodrugs of floxuridine (FUdR) and to investigate the effects of structure, stereochemistry, and site of esterification of promoiety on the rates of hydrolysis of these prodrugs in Caco-2 cell homogenates. Methods. Amino acid ester prodrugs of FUdR were synthesized using established procedures. The kinetics of hydrolysis of prodrugs was evaluated in human adenocarcinoma cell line (Caco-2) homogenates and pH 7.4 phosphate buffer. Results. 3-Monoester, 5-monoester, and 3,5-diester prodrugs of FUdR utilizing proline, L-valine, D-valine, L-phenylalanine, and D-phenylalanine as promoieties were synthesized and characterized. In Caco-2 cell homogenates, the L-amino acid ester prodrugs hydrolyzed 10 to 75 times faster than the corresponding D-amino acid ester prodrugs. Pro and Phe ester prodrugs hydrolyzed much faster (3- to 30-fold) than the corresponding Val ester prodrugs. Further, the 5-monoester prodrugs hydrolyzed significantly faster (3-fold) than the 3,5-diester prodrugs. Conclusions. Novel amino acid ester prodrugs of FUdR were successfully synthesized. The results presented here clearly demonstrate that the rate of FUdR prodrug activation in Caco-2 cell homogenates is affected by the structure, stereochemistry, and site of esterification of the promoiety. Finally, the 5-Val and 5-Phe monoesters exhibited desirable characteristics such as good solution stability and relatively fast enzymatic conversion rates.     

Glycosyl Derivatives of Dopamine and l-dopa as Anti-Parkinson Prodrugs: Synthesis,Pharmacological Activity and In Vitro Stability Studies

Novel glycosyl derivatives of dopamine and _l-dopa (I–IV) are synthesized in order to overcome the problem of blood–brain barrier low permeability of dopamine and of low bioavailability of its precursor _l-dopa.

Esters synthesized link dopamine and _l-dopa, by a succinyl linker, to C-3 position of glucose (I and III) and to C-6 of galactose (II and IV). The chemical and enzymatic stabilities of esters synthesized were evaluated in order to determine both their stability in aqueous medium and their feasibility in undergoing enzymatic cleavage by rat plasma to regenerate the original drug. Furthermore, we have shown the central effects of esters I–IV on classic dopaminergic models, such as morphine induced locomotion and reserpine-induced hypolocomotion.

From the result obtained compounds I–IV appeared moderately stable in a pH 7.4 buffered solution and in rat plasma. Furthermore, pharmacological studies showed that both dopamine derivatives (I and II) were equiactive in reversing reserpine-induced hypolocomotion in rats, and both were more active than _l-dopa or ester III and IV, while II and III were more potent in reducing morphine-induced locomotion than I and IV. The minimal vascular effects of these derivatives allow us to underline the possibility to use them in pathologies, such as Parkinson disease, characterised by an evident decreasing of dopamine concentration in the brain.     

Preparation of Alendronate Liposomes for Enhanced Stability and Bioactivity: In Vitro and In Vivo Characterization

Liposomes containing bisphosphonates have been shown to deplete circulating monocytes and reduce experimental restenosis. However, acceptable shelf life was not achieved, and the disruption extent and rate of the vesicles in the circulation has not been examined. Designing an optimal liposomal formulation in general, and for an anti-inflammatory effect in particular, requires careful consideration of the factors that contribute to their in vitro stability and integrity in the blood after injection. An improved liposomal alendronate formulation was prepared by a modified thin lipid film hydration technique followed by extrusion, resulting in relatively smaller size vesicles, narrow size distribution, and low drug to lipid ratio in comparison to the reverse phase evaporation method. In order to rule out premature leakage of the drug, the integrity of the vesicles was examined by means of size-exclusion chromatography in vitro and in vivo, with subsequent analysis of size, drug (fractions of encapsulated and free) and lipid concentrations. Vesicles were found to be stable in serum, with 15 +/- 3% leakage of the drug after 10 min in rabbit's circulation, and intact liposomes were detected in the circulation 24 h following administration. It is concluded that the new formulation results in increased stability (2.5 years) as determined by the insignificant changes in vesicle size, drug leakage, lipid and drug stability, in vitro bioactivity (macrophages inhibition), as well as in vivo in depleting circulating monocytes and inhibition of restenosis in rabbits. Our in vitro stability results regarding dilution in serum paralleled in vivo data. Thus, in vitro assessment may provide a valuable tool in assessing in vivo integrity of liposomal formulations.     

Synthesis,Characterization, and In Vitro Evaluation of New Ibuprofen Polymeric Prodrugs Based on 2-Hydroxypropyl Methacrylate

The present research work describes the synthesis and evaluation of new acrylic-type polymeric systems having degradable ester bonds linked to ibuprofen as materials for drug delivery. Ibuprofen was linked to 2-hydroxy-propyl methacrylate by an activated ester methodology in a one-pot procedure with a high yield. The resulting material was copolymerized with either 2-hydroxyethyl methacrylate or methyl methacrylate (in 1:3 mole ratios) by the free radical polymerization method, utilizing azoisobutyronitrile at 65–70 °C. The characterization of the resulting products by FTIR, ¹H NMR, ¹³C NMR, DSC, and elemental analysis confirmed their synthesis successfully. Ibuprofen release from the obtained polymers was preliminarily evaluated at different buffered solutions (pH 1, 7.4, and 10) into dialysis bags to show the capacity of prodrugs to release the drug under hydrolytic conditions. Detection of hydrolysis by UV spectroscopy at selected intervals showed that the drug can be released by selective hydrolysis of the ester bond at the side of the drug moiety. The release profiles indicated that the hydrolytic behavior of polymers is strongly based on the polymer hydrophilicity and the pH value of the hydrolysis solution. The results suggest that these polymers could be useful in controlled release systems.     

Cyclic Analogs of Chemotactic Formylpeptides,I.: Synthesis and Biological Activity of For-Cys-Leu-Phe-Cys-OMe

By using the side chain to side chain mode of cyclization the 14-membered disulfide cyclopeptide ( 5 ) has been synthesized as conformationally constrained analog of the prototypical chemotactic tripeptide For-Met-Leu-Phe-OMe. Compound 5 is inactive towards human neutrophils. The lack of biological activity of cyclotetrapeptide 5 is discussed taking into account the backbone folding and the side chain topography.     

In Vitro Biological Control of Aspergillus flavus by Hanseniaspora opuntiae L479 and Hanseniaspora uvarum L793, Producers of Antifungal Volatile Organic Compounds

Aspergillus flavus is a toxigenic fungal colonizer of fruits and cereals and may produce one of the most important mycotoxins from a food safety perspective, aflatoxins. Therefore, its growth and mycotoxin production should be effectively avoided to protect consumers’ health. Among the safe and green antifungal strategies that can be applied in the field, biocontrol is a recent and emerging strategy that needs to be explored. Yeasts are normally good biocontrol candidates to minimize mold-related hazards and their modes of action are numerous, one of them being the production of volatile organic compounds (VOCs). To this end, the influence of VOCs produced by Hanseniaspora opuntiae L479 and Hanseniaspora uvarum L793 on growth, expression of the regulatory gene of the aflatoxin pathway (aflR) and mycotoxin production by A. flavus for 21 days was assessed. The results showed that both yeasts, despite producing different kinds of VOCs, had a similar effect on inhibiting growth, mycotoxin biosynthetic gene expression and phenotypic toxin production overall at the mid-incubation period when their synthesis was the greatest. Based on the results, both yeast strains, H. opuntiae L479 and H. uvarum L793, are potentially suitable as a biopreservative agents for inhibiting the growth of A. flavus and reducing aflatoxin accumulation.     

Inhibition of Intestinal Metabolism of the Antiviral Ester Prodrug bis(POC)-PMPA by Nature-Identical Fruit Extracts as a Strategy to Enhance Its Oral Absorption: An In Vitro Study

Purpose. To explore the usefulness of fruit extracts as enhancers of the oral absorption of esterase-sensitive prodrugs. Methods. Inhibition of esterase-mediated degradation by nature-identical fruit extracts was evaluated using 1) p-nitrophenylacetate (model substrate for esterase-activity) in rat intestinal homogenates and 2) bis(isopropyloxycarbonyloxymethyl)-(R)-9-[(2-phosphonomethoxy) propyl]adenine [bis(POC)-PMPA] (esterase-sensitive prodrug of the antiviral agent PMPA) in Caco-2 cell homogenates and in intestinal homogenates from rat, pig and man. Subsequently, transport of the ester prodrug was studied across Caco-2 monolayers in the presence or absence of fruit extracts. Results. In homogenates from rat ileum, the esterase activity could be reduced significantly by the inclusion of fruit extracts (1%): the initial enzymatic degradation of p-nitrophenylacetate was inhibited by 77% (strawberry), 16% (passion fruit) and 57% (banana). A similar inhibition of bis(POC)-PMPA metabolism by fruit extracts was observed in intestinal homogenates from several species and in homogenates from Caco-2 cells. Transport of total PMPA across Caco-2 monolayers was enhanced 3-fold by co-incubation with strawberry extract (1%). The fraction of intact prodrug appearing in the acceptor compartment increased from virtually zero to 67%. Conclusions. The results suggest that co-incubation with nature-identical fruit extracts might be useful as a strategy to enhance the transepithelial transport of esterase-sensitive prodrugs through inhibition of intracellular metabolism of the prodrug.     

Transdermal Drug Transport and Metabolism. I. Comparison of In Vitro and In Vivo Results

Using excised human skin and tissue grafted to athymic mice, the in vitro and in vivo delivery and metabolism of a salicylate diester were compared. Concentration profiles of this drug and its metabolites were obtained for the outer several hundred microns of the skin. These results show significant differences in the extent of enzymatic cleavage and distribution of metabolites between in vitro and in vivo studies. Furthermore, these data suggest that in vitro results may overestimate metabolism because of increased enzymatic activity and/or decreased capillary removal.     

Vitamin K Prodrugs: 1. Synthesis of Amino Acid Esters of Menahydroquinone-4 and Enzymatic Reconversion to an Active Form

The efficacy and toxicity of vitamin K depends on the pathway and the extent of enzymatic reductive activation to vitamin K hydroquinone, which is an essential cofactor for the synthesis of clotting factors. Parenteral use of vitamin K is impaired by its water insolubility. With the aim to improve delivery problems associated with menahydroquinone-4 (MKH, 2), an active form of menaquinone-4, N,N-dimethylglycine esters of 2 (1-mono, 4-mono, and 1,4-bis) were synthesized and assessed as potential water-soluble prodrugs for parenteral use. The esters can deliver the hydroquinone to its active site without a quinone reductive activation step. The hydrochloride salts of the esters were found to be quite soluble in water. The hydrolysis of the esters in 20% rat liver homogenate 9000 × g supernatant, rat plasma and phosphate buffer, pH 7.4, at 37°C was kinetically studied in the presence and absence of an esterase inhibitor. The hydrolysis was catalyzed by esterases located in the rat liver and rat plasma and quantitatively yielded 2. These results suggest that esterification of 2 with N,N-dimethylglycine is a promising way for obtaining water-soluble prodrug forms of 2. Based on the high susceptibility to liver esterase, the esters are potential prodrugs for achieving the site-specific delivery of 2.     

Osteotropic Drug Delivery System (ODDS) Based on Bisphosphonic Prodrug. I: Synthesis and in Vivo Characterization of Osteotropic Carboxyfluorescein

Abstract

An osteotropic drug delivery system (ODDS) based on a bisphosphonic prodrug was designed as a novel method for site-specific and controlled delivery of drugs to the bone. Due to the chemical adsorption of bisphosphonic promoiety to the mineral component, hydroxyapatite, a bisphosphonic prodrug is predominantly taken up into the bone. To verify the concept, bisphosphonic promoiety was chemically introduced into 6-carboxyfluorescein (CF) as a model compound and the disposition after intravenous injection was studied in rats. The bisphosphonic prodrug of CF, disodium (fluorescein-6-carbonyloxy) acetoaminomethylene bisphosphonate (CF-BP) was highly taken up to the skeleton (62.1% of dose) and the remainder was excreted into the urine (35.9% of dose). Subsequently, regeneration of CF by hydrolysis of CF-BP in the bone was observed. The microscopic observation showed that CF-BP was buried into the bone with a calcification of the bone. According to the remodeling of the bone, bisphosphonic prodrug buried was supposed to be released in the vicinity of the osteoclast or resorption surface of the bone. Thus, it is suggested that ODDS has a potential to achieve osteoclast-speciftc or resorption surface-specific targeting of the drugs.     

In situ Gel of Metoprolol Tartrate: Physicochemical Characterization,In vitro Diffusion and Histological Studies

The purpose of the present investigation was to prepare an intranasal in situ gel with increased nasal residence time in order to improve bioavailability of metoprolol tartrate. The in situ gel systems containing carbopol, hydroxypropyl methylcellulose K4M and K15M in different concentrations were prepared. The samples were characterized for viscosity, rheological behavior, gelation behavior, gel strength, and mucoadhesion. The formulations F10 (0.4% w/v carbopol, 1% w/v hydroxylpropyl methylcellulose K15M) and F13 (0.3% w/v carbopol, 1% w/v hydroxypropyl methylcellulose K15M) showed gel strength of 40.33±0.47 and 43.00±1.41, respectively, and mucoadhesion strength 31.48±0.14×10³ and 32.12±0.05×10³ dyne/cm², respectively. In vitro release profiles showed initial burst followed by slow release. F10 and F13 released 88.08±0.98 and 91.18±1.09% drug in 8 h. R² value for F10 (0.9953) and F13 (0.9942) was maximum for Higuchi, showing mixed order kinetics while n value obtained on treatment with Korsemayer Pappas equation were near to 0.5, suggesting release by fickian diffusion mechanism. The nasal permeability of formulations F10 and F13 were found to be 0.057 and 0.063 cm/s, respectively. Histopathological examination revealed slight degeneration of nasal epithelium with increased vascularity by F10 but no inflammation by formulation F13. Thus, a pH triggered in situ gel system containing low concentration (0.3% w/v) of carbopol demonstrated sustained release of metoprolol tartrate without any destructive effect on the mucosa.     

lontophoretic Delivery of Apomorphine I: In Vitro Optimization and Validation

Purpose. To investigate the feasibility of transdermal iontophoretic delivery of apomorphine in patients with Parkinson's disease, transdermal transport rates were optimized and validated across human stratum corneum and freshly dermatomed human skin in vitro. Methods. In all experiments R-apomorphine hydrochloride was applied in the anodal compartment. The effect on the flux of the following parameters was studied, using a flow through transport cell: current density, pH, concentration, ionic strength, osmolarity, buffer strength, temperature and skin type. Results. Transdermal transport of apomorphine was directly controlled by the presence or absence of current. Passive delivery was minimal and no depot effect was observed. A linear relationship was found between current density and steady-state flux. At room temperature the lag time was 30 to 40 minutes. A maximal steady-state flux was obtained when the donor concentration approached maximum solubility. By increasing the temperature of the acceptor chamber to 37°C, the steady-state flux was increased by a factor of 2.3 and the lag time decreased to ± 3 minutes. No effect of osmolarity and buffer strength, and only a small effect of ionic strength and pH on the transport rate were observed. The flux through dermatomed human skin was decreased compared to stratum corneum. This effect was shown not to be caused by skin metabolism. Conclusions. The results obtained in vitroindicate that the iontophoretic delivery of apomorphine can be controlled and manipulated accurately by the applied current. The in vitro flux furthermore depends on the donor composition, temperature and skin type. Under optimized conditions, transport rates resulting in therapeutically effective plasma concentrations are feasible, assuming a one to one in vitro/in vivo correlation.     

Compartmental Modeling of Transdermal Iontophoretic Transport: I. In Vitro Model Derivation and Application

PURPOSE: The objective of this study was to develop a family of compartmental models to describe in a strictly quantitative manner the transdermal iontophoretic transport of drugs in vitro. METHODS: Two structurally different compartmental models describing the in vitro transport during iontophoresis and one compartmental model describing the in vitro transport in post-iontophoretic period are proposed. These models are based on the mass transfer from the donor compartment to the acceptor compartment via the skin as an intermediate compartment. In these models, transdermal iontophoretic transport is characterized by 5 parameters: 1) kinetic lag time (tL), 2) steady-state flux during iontophoresis (Jss), 3) skin release rate constant (K(R)), 4) the first-order rate constant of the iontophoretic driving force from the skin to the acceptor compartment (I1), and 5) passive flux in the post-iontophoretic period (Jpas). The developed models were applied to data on the iontophoretic transport in human stratum corneum in vitro of R-apomorphine after pretreatment with phosphate buffered saline pH 7.4 (PBS) and after pretreatment with surfactant (SFC), as well as the iontophoretic transport of 0.5 mg ml(-1) rotigotine at pH 5 (RTG). RESULTS: All of the proposed models could be fitted to the transport data of PBS, SFC, and RTG groups both during the iontophoresis and in the post-iontophoretic period. The incorporation of parameter I1 failed to improve the fitting performance of the model. This might indicate a negligible contribution of iontophoretic driving force to the mass transfer in the direction from the skin to the acceptor compartment, although it plays an important role in loading the skin with the drug. The estimated values of Jss of PBS, SFC, and RTG were identical (p > 0.05) to the values obtained with the diffusion lag time method. Moreover, time required to achieve steady-state flux can be estimated based on the parameter tL and the reciprocal value of parameter K(R). In addition, accumulation of drug molecules in the skin is reflected in a reduction of the value of the K(R) parameter. CONCLUSIONS: The developed in vitro models demonstrated their strength and consistency to describe the drug transport during and post-iontophoresis.     

Design and Evaluation of an Emulsion Vehicle for Paclitaxel. I. Physicochemical Properties and Plasma Stability

PURPOSE: The current formulation of paclitaxel contains ethanol and Cremophor EL and has been reported to cause serious adverse reactions. The purpose of the present work was to develop an improved emulsion vehicle for paclitaxel and to study the physicochemical properties of such a system. METHODS: Emulsions were prepared by either microfluidization or sonication method and the droplet size characterized by dynamic light scattering and light microscopy. RESULTS: Stable emulsions could be made using mixtures of lecithin/sodium deoxycholate as the emulsifiers. The formulation was further improved by using a combination of free acid and the sodium salt. Paclitaxel could be loaded into the emulsions at 2.5 mg/ml without the formation of drug crystals. While these emulsions were stable on storage, they flocculated when mixed with plasma. Steric stabilization of the emulsion droplets with poloxamer 188 increased the stability of the emulsions in plasma but promoted the crystallization of paclitaxel. The crystallization tendency could be reduced by using PEG5000PE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[poly (ethylene glycol) 5000]), a less water-soluble stabilizer. CONCLUSIONS: Emulsions with good stability characteristics containing 2.5 mg/ml paclitaxel could be made using bile salt/acid and lecithin, and the excellent stability of these emulsions in plasma was achieved by steric stabilization using PEG5000PE.     

In Vitro Drug Absorption Models. I. Brush Border Membrane Vesicles,Isolated Mucosal Cells and Everted Intestinal Rings: Characterization and Salicylate Accumulation

Brush border membrane vesicles, isolated mucosal cells and everted rings from rat intestine were compared for their suitability for drug uptake studies. Vesicles from brush border membranes were judged to be metabolically and morphologically functional on the basis of biochemical and microscopic criteria. With the use of a collagenase-vascular-perfusion method, populations of villus, mid villus and crypt cells were separated. An alternative approach that is based on an EDTA-dissociation procedure afforded fractions enriched in villus and crypt cells. Although several enzymatic and metabolic activities of these two cell preparations were comparable, cell viability based on the Trypan Blue dye exclusion test, ultrastructural appearance and glucose uptake more closely conformed to in vivo values for cells isolated according to the EDTA-dissociation method. These cells were chosen as a model for drug transport investigation. The morphological and functional integrity of everted rings was verified by histological examination, extracellular space estimation and assessment of glucose transport ability. Sodium salicylate uptake studies using brush border membrane vesicles and isolated mucosal cells were highly variable, whereas everted segments exhibited good reproducibility in uptake experiments. Time dependence of salicylate uptake was demonstrated with membrane vesicles and everted rings. Time dependence was not observed in mucosal cell uptake studies, probably because of the time required to separate the cells from the incubation solution. Based on ease of preparation, technical aspects of in vitro incubation and reproducibility of results, everted intestinal rings were considered to be a good potential model for in vivo drug absorption. Brush border membrane vesicles were generally regarded as unacceptable because of variations after storage and between experiments. Isolated cells offered certain advantages, but the utility of cells as an in vitro model remains equivocal.     

Physicochemical Properties of Salts of p-Aminosalicylic Acid. I. Correlation of Crystal Structure and Hydrate Stability

The potassium (K), sodium (NA), calcium (CA), and magnesium (MG) salts of p-aminosalicylic acid were obtained, and their thermal behavior was characterized by means of differential scanning calorimetry (DSC) and thermogravimetric analysis (TG). Their crystal and molecular structures were determined by single-crystal X-ray diffraction after powder patterns had shown them to be nonisomorphous. Different degrees of hydration were observed for the solid salts, and an assessment of hydrate stability to dehydration was made from thermogravimetric studies. The onset temperature of dehydration (T _t) of each salt varied within the series and exhibited correlation with X-ray determined structure. The observed onset of dehydration of MG and CA was higher than that of NA and is consistent with stronger ion-dipole interactions for the divalent salts. Crystallographic determination of the bond lengths between the metal ion and the water oxygens were 2.4 and 2.9 for NA, between 2.0 and 2.1 for MG, and 2.4 for CA. The open nature and presence of a channel feature in the structure of the sodium salt may have facilitated escape of water molecules from the crystal. Particle presentation (e.g., size, crystallinity) was also shown to affect dehydration behavior.     

Development of Anti-Influenza Virus Drugs I: Improvement of Oral Absorption and In Vivo Anti-Influenza Activity of Stachyflin and Its Derivatives

Purpose. Stachyflin and its derivatives which are active against the influenza virus in vitro, were studied to improve their reduced in vivo activity after oral administration by chemical modification and some vehicles. Methods. The solubility was examined for different vehicles. The improvement of gastrointestinal absorption was evaluated by the plasma concentration after oral administration to mice or the in situ loop method with rats. The in vivo anti-influenza activity was examined using mice infected with the influenza virus and evaluated based on the virus titer in the lung by TCID₅₀. Results. PEG 400 showed the highest solubility of Stachyflin and its derivative among the vehicles studied. While no viral inhibition was found in the lung after oral administration of 0.5% HPMC suspension of Stachyflin, in vivo anti-influenza virus activity was found with the PEG 400 solution. The absorption of Stachyflin by PEG 400 showed about a fifty-fold increase in AUC compared with that of 0.5% HPMC suspension. Improving the oral absorption of Stachyflin led to an increase in the in vivo anti-influenza virus activity. When the Stachyflin derivative in PEG 4000 was administered orally, there was more enhancement of the oral absorption than with PEG 400. When the aqueous solution of the phosphate ester prodrugs of Stachyflin and its derivative was administered orally, the absorption of the parent compound was improved and in vivo anti-influenza virus activity was found. Conclusions. When Stachyflin and its derivatives were administered orally to mice with a solution in PEG and an aqueous solution of their phosphate ester, their oral absorption was improved and in vivo anti-influenza virus activity was observed.