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Objectives: LK-157 is a novel 10-ethylidene tricyclic carbapenem that resembles the structure of the broad-spectrum antibiotic sanfetrinem and acts as a potent inactivator of beta-lactamases of classes A, C and D. LK-157 is a highly soluble but poorly permeable drug. Since most of the beta-lactams are poorly absorbed, ester prodrugs LK-159, LK-157E1 and LK-157E2 were designed to enhance membrane permeability. This study investigated the permeability of LK-157 and the three ester prodrugs across rat intestine in vitro. The morpholinoethyl ester of sanfetrinem was also investigated., Method: Permeability across rat jejunum was determined using EasyMount side-by-side diffusion chambers., Key Findings: The solubility and permeability of morpholinoethyl ester LK-157E2 were superior to those of LK-159 and LK-157E1. The morpholinoethyl ester of sanfetrinem LK-176E1 had the highest observed permeability coefficient and consequently the highest predicted absorption in humans., Conclusions: These results suggest that the morpholinoethyl esters of LK-157 and sanfetrinem could be further investigated to assess bioavailability in vivo.

It has been demonstrated that the absorption of EPA and DHA is significantly lower for ethyl esters than for the corresponding free fatty acids. Since these fatty acids exist in nature and are catabolized by beta-oxidation, we instead wanted to investigate the absorption, distribution and biological effects of a non-beta-oxidizable modified fatty acid. The modified fatty acid tetradecylthioacetic acid (TTA) and the ethyl ester of TTA (EtTTA) were administered to rats for 10 days, in doses corresponding to 150 mg TTA/kg BW/day. No significant differences were found between the accumulated amounts of TTA or its Delta9 desaturated metabolite in plasma, liver, heart and epididymal white adipose tissue between EtTTA and TTA treated rats. EtTTA and TTA increased the activities of carnitine palmitoyltransferase-II and fatty acyl-CoA oxidase in liver, with no differences between the two treatment groups, but did not affect these activities in heart. EtTTA and TTA treatment decreased the plasma levels of triacylglycerols, cholesterol and phospholipids to similar extents, but no significant effects were seen in hepatic and cardiac lipid levels. EtTTA and TTA had similar effects on the fatty acid composition in plasma, liver, heart and epididymal white adipose tissue. Based on changes in fatty acid indexes it seems that these drugs had comparable stimulating effects on stearoyl-CoA desaturase and Delta6 desaturase, and reduced the Delta5 desaturase activity in liver. From the presented results we conclude that the absorption and distribution of the ethyl ester and the free form of TTA are not significantly different, and that the two administered forms of TTA have similar effects on lipid metabolism in rats.

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 (12.1 +/- 1.48 x 10(-6) cm/s) was fourfold higher than D-Asp-ACV (3.12 +/- 0.36 x 10(-6) cm/s) and ACV (3.25 +/- 0.56 x 10(-6) cm/s). 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 2.66 +/- 0.21 x 10(-6) cm/s. 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).

The ethyl ester derivative of thymopentin was synthesized and characterized. The biological activities of thymopentin were evaluated by mouse spleen lymphocyte proliferation test and superoxide dismutase activity assay. Compared with thymopentin, the synthesized ethyl ester showed more potent immunoregulation activity in the MTT assay and anti-oxidation activity in the immune-suppressed rat model induced by hydrocortisone. The half life of the ester derivate in rat plasma determined by HPLC was slightly longer than that of thymopentin. The ester derivate gained advantages in activity and stability compared to thymopentin.

The novel methylphosphonic acid monoethylester (H(4)dotp(OEt)) has been synthesized and characterized and their complexes with Sm(III) and Ho(III) ions were studied. Dissociation constants of the ligand are lower than those of H(4)dota. The stability constants of the Ln(III)-H(4)dotp(OEt) complexes are surprisingly much lower that those of H(4)dota (H(4)dota=1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) probably due to a lower coordination ability of the phosphonate monoester groups. Acid-assisted decomplexation studies have shown that both complexes are less kinetically inert than the H(4)dota complexes, but still much more inert than complexes of open-chain ligands. Nevertheless, the synthesis of (153)Sm and (166)Ho complexes with this ligand led to stable complexes both in vitro and in vivo. A very low binding of these complexes to hydroxyapatite (HA) and calcified tissues was observed confirming the assumption that a fully ionized phosphonate group(s) is necessary for a strong bone affinity. Both complexes show similar behaviour in vivo and, in general, follow the biodistribution trend of the H(4)dota complexes with the same metals.

The objective of this study was to determine the in vitro transdermal permeation through human epidermis of zalcitabine, lamivudine and the synthesised N-acyl lamivudine esters, with and without the use of Pheroid as delivery system and to establish a correlation, if any, with selected physicochemical properties. Six N-acyl lamivudine esters were prepared by acylation of lamivudine with six different acid chlorides. The experimental aqueous solubility, log D and in vitro transdermal flux values were determined for these compounds. There was an inverse correlation between the aqueous solubility and the log D values. The median flux of zalcitabine (0.442 micromol/cm2 h) in PBS was lower than that of lamivudine (4.289 micromol/cm2 h), but in Pheroid, lamivudine (0.011 micromol/cm2 h) had a slightly lower median flux than zalcitabine (0.015 micromol/cm2 h). Entrapment of compounds in Pheroid was confirmed by confocal laser scanning microscopy.

The purpose of this study was to evaluate the physicochemical properties, skin permeation and accumulation profiles of model lipophilic ketorolac fatty ester (esters) prodrugs. Ketorolac linoleate (C18:2), oleate (C18:1) and stearate (C18:0) were evaluated for their solubility, capacity factor, enzymatic hydrolysis, chemical stability, and skin permeation and accumulation profiles using the combination of common permeation enhancing techniques such as the use of supersaturated solution of permeants in the enhancer vehicle, lipophilic receptor solution, enhancer pretreatment of skins, removal of stratum corneum and delipidization of skins etc. Esters were highly lipophilic, chemically stable for the duration of observation, enzymatically unstable in hairless mouse skin/liver homogenates and plasma, and impermeable into the receptor solution. Absence of skin permeation, relative enzymatic stability during permeation and chemical stability of these esters could delineate preliminary possibilities for designing safer topical agents without systemic absorption.

Purpose: Classic penetration enhancement/retardation methods for improved dermal drug delivery primarily focus on co-applied chemicals aided alterations in skin accumulation/permeation profile, and in many cases, this has been achieved by compromising the systemic absorption/toxicities of penetrant/enhancer/retarder. In this study, higher dermal accumulation without systemic absorption of ketorolac and its fatty esters (esters) will be achieved by synthesizing lipophilic fatty ester soft prodrugs of ketorolac., Methods: Ketorolac decenoate (C10:1), dodecenoate (C12:1) and palmitoleate (C16:1) were synthesized and evaluated for their lipophilicity, enzymatic hydrolysis, chemical stabilities, and skin permeation and accumulation profiles using the combination of common permeation enhancing techniques such as the use of lipophilic receptor solution, enhancer pretreatment of skins, removal of stratum corneum and delipidization of skins etc., Results: Esters were highly lipophilic, chemically stable, enzymatically unstable in hairless mouse skin/liver homogenates and impermeable into the receptor solution., Conclusion: Higher dermal accumulation, absence of skin permeation, relative enzymatic stability in whole skins during permeation study and the pharmaceutical stability of esters could delineate a preliminary possibility for designing safer dermal agents with minimum potential for systemic absorption without the co-application of permeation enhancers or retarders.

One of the most important tasks in pharmaceutical technology is the optimization of drug release. The hot-melt technology is an important method with which to modify the bioavailability. Sugar esters (SEs) have a wide range of HLB values (1-16). Due to their low melting points, they are promising carriers for the melting method. The aims of the present work were to study the thermal properties (DSC) and the structures (XRPD) of SEs with low, medium or high HLB values, and to evaluate their applicability in the hot-melt technology. Relationships were found between the HLB value, the structure and the thermal behaviour. After melting and solidification, the SEs have partially amorphous layered structures which slowly crystallize in time; the original structure does not return for SEs with high, moderate, or low HLB values. These results demonstrate that changes in morphology must be considered during research and development. During the examination of meloxicam-SE melted products the SEs influenced the drug release, depending on their HLB values. In the cases of ibuprofen-SE melted products, the SEs did not influence the drug release. Here, a change in the drug distribution was the predominant effect, which was accompanied by movement in the SE structure.

Omega-3 fatty acids have a long history of use as dietary supplements and more recently for therapeutic applications as prescription pharmaceuticals. Achieving a high concentration is critical for developing convenient, practical therapeutic formulations. The objective of the study was to explore the uptake and effects of different concentrations of omega-3 acid ethyl esters. Three different omega-3 concentrations were investigated in a clinical study with 101 subjects. All participants were dosed for 14 days with 5.1g per day of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) ethyl esters provided in three concentrations: 62.5%, 80% and 85% of total fatty acids. Key endpoints of the study were serum phospholipids and standard fasting lipid panels at day 14. Although administered the same quantity of omega-3 fatty acids, the patients taking the more concentrated formulations had higher levels of EPA/DHA in serum phospholipids and greater reductions in serum triglyceride and VLDL cholesterol levels. Total and non-HDL cholesterol were significantly reduced from baseline with all three formulations. In conclusion the concentration of omega-3 fatty acids of the formulations studied had independent effects on the uptake and effect outcomes during short-term administration. Very high concentrations of omega-3 acid ethyl esters (80%) appear to have higher uptake and are more potent for reducing triglycerides (TGs) and VLDL-cholesterol than formulations with lower concentrations.

FRET-based fluorogenic substrates for lipases and esterases were prepared in four steps from commercially available building blocks. The substrates are pyrenebutyric acid monoesters of aliphatic 1,2-diols bearing a dinitrophenylamino group as a quencher. The most enzyme-reactive substrate is ester 2a. The substrates do not show any measurable background reaction in the absence of enzyme even at pH 11, but react fast and specifically with lipases and esterases. These substrates offer an unprecedented and practical solution to the long-standing problem of a simple yet efficient high-throughput screening tool for lipase activities under basic conditions.

In the aim to develop an efficient fluorinated probe for positron emission tomography (PET) exploration of the dopamine transporter (DAT), we studied several in vitro and in vivo characteristics of the phenyltropane derivative (E)-N-(4-fluorobut-2-enyl)-2beta-carbomethoxy-3beta-(4'-tolyl)nortropane (LBT-999). In vitro on rat striatal membrane, [(3)H]LBT-999 bound to a single site with a K(d) of 9 nM, B(max) of 17 pmol/mg protein, and a very high selectivity for the DAT [IC(50) for 1-{2-[bis-(4-fluorophenyl)-methoxy]ethyl}-4-(3-phenylpropyl)piperazine (GBR 12909) and (E)-N-(3-iodoprop-2-enyl)-2beta-carbomethoxy-3beta-(4'-methylphenyl)nortropane (PE2I): 2.4 and 18 nM, respectively; IC(50) for paroxetine, citalopram, N,N-dimethyl-2-(2-amino-4-methylphenyl thio)benzylamine, nisoxetine, and desipramine >1 muM]. In vitro on post-mortem human brain sections, LBT-999 bound with high intensity to the caudate-putamen, weakly to the thalamus, and not in the neocortex and cerebellum. This binding was totally abolished in the presence of PE2I. Ex vivo cerebral biodistribution of [(11)C]LBT-999 in rats showed striatum/cerebellum radioactivity ratios of 18 and 25 at 30 and 60 min postinjection, respectively. This accumulation was strongly prevented by preinjection of GBR 12909, whereas paroxetine and nisoxetine had no effect. An in vivo kinetic PET study in three baboons showed a fast and very high uptake in the striatum, with a plateau at 30 min postinjection and a maximal putamen/cerebellum ratio of 30. Taken together, these findings demonstrate that LBT-999 is a highly promising agent for in vivo exploration of the DAT. This probe is currently labeled with (18)F for further characterizations.

A rapid and simple high-performance liquid chromatographic (HPLC) method has been developed for the determination of p-coumaric acid in rat plasma and applied to a pharmacokinetic study in rats after administration of a prodrug, E-6-O-p-coumaroyl scandoside methyl ester, isolated from Hedyotis diffusa (Willd.). Sample preparation involved protein precipitation with acetonitrile. The supernatant was then injected onto a Diamonsil C(18) column (250 mm x 4.6mm i.d., 5 microm). The mobile phase consisted of acetonitrile-water (21:79, v/v) with 1% glacial acetic acid. The UV detector was set at 310 nm. The lower limit of quantification of p-coumaric acid in rat plasma was 0.02 microg/mL. The calibration curves were linear over the concentration range 0.02-5 microg/mL with correlations greater than 0.999. The assay procedure was applied to the study of the metabolite pharmacokinetics of E-6-O-p-coumaroyl scandoside methyl ester in rat.

The consumption of alcohol is known to have both positive and negative effects on the functioning of the cardiovascular system in general, and on platelet function in particular. Fatty acid ethyl esters (FAEEs) are non-oxidative metabolite of ethanol that may mediate the ethanol effect on platelet function leading to either bleeding or clotting. The aim of the current study was to investigate the synthesis, uptake, and hydrolysis of FAEEs by human platelets. Isolated platelets were incubated with ethanol for various times, and FAEE synthesis were measured by gas chromatography mass-spectrometry (GC-MS). In addition, platelets were incubated with (14)C-ethyl oleate, and FAEE uptake and hydrolysis were measured. There was significant synthesis of FAEEs by human platelets within 30 min of exposure to ethanol. The major FAEE species formed by human platelets exposed to ethanol were ethyl palmitate and ethyl stearate. FAEE uptake by human platelets showed maximum uptake by 60 s. The majority of FAEEs (50-80%) incorporated into platelets remained intact for up to 10 min. FAEE hydrolysis led to an increase in free fatty acids, with minimal subsequent esterification of the free fatty acids into phospholipids, triglycerides, and cholesterol esters. These studies show that FAEEs, non-oxidative metabolite of ethanol, can be incorporated into, synthesized, and hydrolyzed by human platelets.

The syntheses of a series of novel ester-linked derivatives of the icosahedral [closo-B12(OH)12]2- boron cluster (closomer esters) are described using several synthetic methods. The reaction of bis(tetrabutylammonium)-closo-dodecahydroxy-dodecaborate, [NBu4]2 1, with carboxylic acid chlorides and anhydrides, vinyl esters with a Y5(OiPr)13O catalyst and 1,1'-carbonyldiimidazole-activated carboxylic acids yields the corresponding dianionic dodeca-ester closomers. The method using 1,1'-carbonyldiimidazole-activated carboxylic acids may be employed as a general synthetic strategy. The use of elevated reaction temperatures, achievable under pressure, to expedite syntheses is described. An attractive methodology using immobilized scavenger reagents for the expeditious purification of the closomer esters was employed. The developed methodology is compatible with a variety of peripheral functional groups attached to the termini of densely packed, carboxylate ester-linked radial arms bonded to the icosahedral borane surface. A closomer ester having twelve terminal amino groups was prepared, and without isolation, fully acetylated in good yield.

The purpose of this study was to investigate the possibility of parenteral delivery of poorly water-soluble lipophilic drugs using a phospholipid-based microemulsion system. Ibuprofen eugenol ester (IEE), a highly lipophilic compound, was synthesized from ibuprofen and eugenol, and isolated as an amorphous whitish solid with a melting point at 40.2+/-0.1 degrees C, which structure was confirmed by IR, 1H-NMR and MS spectra. A pharmaceutically acceptable microemulsion system using Miglyol 812, soybean phosphatidylcholine (SbPC) and poly (ethylene glycol) (660)-12-hydroxystearate (Solutol HS-15), and PEG400 and ethanol as oil phase, surfactants and cosurfactants, respectively, was presented and characterized in terms of stability, droplet size distribution (DSD) and their solubilization capacity of IEE. The solubility of IEE in the optimized microemulsion formulation consisting of 6.4% ibuprofen eugenol, 9.6% Miglyol 812, 6% SbPC, 6% HS-15, 8.4% PEG400, 3.6% ethanol and 60% distilled water (w/w) was about 21,000 times higher than that in water. The ibuprofen blood concentration after intravenous administration of microemulsions was determined and compared with that of ibuprofen solution. It was concluded that the presented microemulsion system might be a promising intravenous dosage form of poorly water-soluble lipophilic drugs.

The glucoside and mannoside derivatives of the NSAIDs flurbiprofen, ibuprofen, ketoprofen and naproxen were synthesised and their penetration through human skin was determined. Experimental transdermal flux data showed that the parent NSAIDs penetrated human skin to a much higher extent than the glycosides.

Aim: To reconstruct of a tissue engineering skin in vitro for the study of the use of drug percutaneous penetration and metabolism., Methods: Dermal fibroblasts were embedded in collagen type I. HaCaT cells were seeded on the top of the gel. The skin was generated through air-liquid interface culture. Effects of various culture media on tissues morphology were investigated. Sections of the cultured skin were stained with hematoxylin and eosin and examined under microscope. Permeation and metabolism of ketoprofen and its isopropyl ester through the cultured skin were investigated., Results: HaCaT cells initially developed a multilayer epithelium at the air-liquid interface, but it showed a parakeratotic stratum corneum. Vitamin C enhanced cell proliferation obviously. Vitamin D3 promoted cell differentiation. And estradiol showed little effect on the tissue engineering skin. Ketoprofen isopropyl ester was hydrolyzed into ketoprofen when penetrated through the cultured skin, which resembled in the skin cell homogenates metabolism., Conclusion: Cultured at the air-liquid interface, HaCaT cells developed a parakeratotic mutilayer epithelium. Enzyme activity was reserved. This cultured skin could serve as an appropriate model for drug percutaneous metabolism and skin irritation.

Ketorolac is a potent nonsteroidal anti-inflammatory drug. Recently, a novel ester of ketorolac, ketorolac pentyl ester, was synthesized. When prepared in injectable oil, the new agent demonstrated a long duration of action. Ketorolac pentyl ester was synthesized using a prodrug design by esterification of ketorolac, and appeared to be a prodrug of ketorolac in vivo, which needed to be confirmed. The aim of the present study was to establish the prodrug's pharmacokinetics in vivo, and to confirm whether or not ketorolac pentyl ester was a prodrug of ketorolac. Pharmacokinetic profiles of intravenous ketorolac and its pentyl ester on an equal-molar basis in six rabbits were evaluated. A high-performance liquid chromatographic method was used to determine the plasma concentrations of ketorolac and its pentyl ester. We found that the plasma concentrations of ketorolac pentyl ester declined rapidly after injection and so did the conversion of ketorolac pentyl ester to ketorolac. Also, the conversion of ketorolac was proved complete when compared with intravenous ketorolac under an equi-molar basis. In conclusion, this in vivo pharmacokinetic study confirmed that keterolac pentyl ester was a prodrug of keterolac.

The utility of polyamines as vectors for the intracellular transport of iron chelators is further described. Consistent with earlier results with polyamine analogues, these studies underscore the importance of charge in the design of polyamine-vectored chelators. Four polyamine conjugates are synthesized, two of terephthalic acid [N(1)-(4-carboxy)benzoylspermine (7) and its methyl ester (6)] and two of (S)-2-(2,4-dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylic acid [(S)-4'-(HO)-DADFT] [(S)-4,5-dihydro-2-[2-hydroxy-4-(12-amino-5,9-diazadodecyl-oxy)phenyl]-4-methyl-4-thiazolecarboxylic acid (10) and its ethyl ester (9)]. These four molecules were evaluated in murine leukemia L1210 cells for their impact on cell proliferation (48- and 96-h IC(50) values), their ability to compete with spermidine for the polyamine transport apparatus (K(i)), and their intracellular accumulation. The data revealed that when neutral molecules (cargo fragments) were fixed to the polyamine vector, the conjugates competed well with spermidine for transport and were accumulated intracellularly to millimolar levels. However, this was not the case when the cargo fragments were negatively charged. Metabolic studies of the polyamine-vectored (S)-4'-(HO)-DADFTs in rodents indicated that not only did the expected deaminopropylation step occur, but also a surprisingly high level of oxidative deamination at the terminal primary nitrogens took place. Finally, the iron-clearing efficiency of the (S)-4'-(HO)-DADFT conjugates was determined in a bile-duct-cannulated rodent model. Attaching the ligand to a polyamine vector had a profound effect on increasing the iron-clearing efficiency of this chelator relative to its parent drug.

Purpose: Physicochemical characterization and in vitro human skin diffusion studies of branched-chain ester and carbonate prodrugs of naltrexone (NTX) were compared and contrasted with straight-chain ester and carbonate NTX prodrugs., Methods: Human skin permeation rates, thermal parameters, solubilities in mineral oil and buffer, and stabilities in buffer and plasma were determined. Partition coefficients between stratum corneum and vehicle were determined for straight- and branched-chain esters with the same number of carbon atoms., Results: Branched prodrugs had lower melting points, lower buffer solubilities, and higher mineral oil solubilities than NTX. The transdermal flux values from all of these branched prodrugs were significantly lower than flux values from the straight-chain ester and the methyl carbonate prodrugs. Straight-chain prodrugs had higher partition coefficient values and higher calculated thermodynamic activities than their branched-chain counterparts. The prodrug hydrolysis to NTX in buffer and plasma was slower for prodrugs with increased branching., Conclusions: Branched-chain prodrugs with bulky moieties had smaller stratum corneum-vehicle partition coefficients and lower thermodynamic activities that resulted in smaller transdermal flux values than straight-chain prodrugs.

We have evaluated the capacity of the cell-binding heptapeptide SIGYPLP to enhance transgene expression using non-viral and viral gene delivery vectors. Targeted polyplex based vectors showed good levels of DNA uptake in freshly isolated human umbilical vein endothelial cells (HUVECs) compared to untargeted controls, whilst displaying only modest increases in reporter gene activity. The targeted polyplexes showed reduced levels of DNA uptake in cells of a none endothelial origin although they mediated higher levels of transgene expression. The enhanced efficiency of transgene expression may relate to the more rapid rate of cell division. However, since in vivo application of polyplexes is compromised by instability to serum proteins, serum-resistant polyplexes (surface modified with multivalent reactive hydrophilic polymers based on poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA)) were also evaluated for their ability to mediate transgene expression. Surface modification of polyplexes with pHPMA ablates non-specific cell entry, reducing levels of transgene expression, whilst the incorporation of the SIGYPLP peptide into the hydrophilic polymer resulted in restored transgene expression in all formulations tested. The technology of surface modification using pHPMA can also be applied in the context of viruses, masking receptor-binding epitopes and enabling the linkage of novel cell targeting ligands, enabling construction of a virus with receptor-specific infectivity. Retargeting of adenovirus based vectors using the same polymer-peptide construct enhanced levels of transgene expression in HUVECs to greater than 15 times that observed using parental (unmodified) virus, whilst restoring levels of transgene expression in non-endothelial cell lines tested. The use of constructs based on conjugates between hydrophilic polymers and small receptor-binding oligopeptides as agents for retargeting viral or non-viral vectors to cellular receptors represents a simple alternative to the use of antibodies as targeting ligands for cell specific gene delivery.

A series of novel corticosteroid derivatives featuring C-17 furoate ester functionality have been synthesised. Profiling in vitro and in vivo has resulted in the identification of a compound with a longer duration of action and a lower oral side effect profile in rodents compared to budesonide.

Oil-based depot formulations may constitute a future delivery method for small peptides. Thus, a requirement is attainment of sufficient oil solubility for such active compounds. A model dipeptide (Gly-Phe) has been converted into lipophilic prodrugs by esterification at the C-terminal carboxylic acid group. The decomposition kinetics of octyl ester of Gly-Phe (IV) has been investigated at pH 7.4 (37 degrees C) and IV was shown to degrade by first-order kinetics via two parallel pathways (1) intramolecular aminolysis resulting in formation of a 2,5-diketopiperazine and (2) hydrolysis of the ester bond producing the dipeptide. The cyclisation reaction was dominating in the decomposition of methyl (II) butyl (III) octyl (IV) decyl (V) and dodecyl (VI) esters of Gly-Phe at pH 7.4. However, this degradation pathway was almost negligible for pH below 6. During degradation of the dipeptide esters in 80% human plasma pH 7.4 (37 degrees C) a minimal amount of cyclo(-Gly-Phe) was formed. A faster degradation of the esters in 80% human plasma pH 7.4 compared to those in aqueous solution pH 7.4 was suggested to be due to fast cleavage of the peptide bond. Low oil solubilities for Gly-Phe and the hydrochlorides of the dipeptide esters III and VI were observed. Although the solubility of Gly-Phe in oil solutions was enhanced by hydrophobic ion pairing with sodium decyl sulfonate the oil solubility was still less than 1 mg Gly-Phe/ml. By addition of a solubiliser, 10% N,N-dimethylacetamide (DMA), to Viscoleo the solubility of the HIP complexes increased significantly. The present study indicates that sufficient oil solubility might only be obtained for relatively small peptides by using the prodrug approach in combination with solubility enhancing organic solvents like DMA.

Purpose: The purpose of this study was to analyze the transport of amino acid esters and the amino-acid-based prodrug valganciclovir by the Na(+)/Cl(-)-coupled amino acid transporter ATB(0,+)., Methods: The interaction of amino acid esters and valganciclovir with the cloned rat ATB(0,+) was evaluated in a mammalian cell expression system and in the Xenopus oocyte expression system., Results: In mammalian cells, expression of ATB(0,+) induced glycine uptake. This uptake was inhibited by valine and its methyl, butyl, and benzyl esters. The benzyl esters of other neutral amino acids were also effective inhibitors. Valganciclovir, the valyl ester of ganciclovir, was also found to inhibit ATB(0,+)-mediated glycine uptake competitively. Exposure of ATB(0,+)-expressing oocytes to glycine induced inward currents. Exposure to different valyl esters (methyl, butyl, and benzyl), benzyl esters of various neutral amino acids, and valganciclovir also induced inward currents in these oocytes. The current induced by valganciclovir was saturable with a K0.5 value of 3.1+/-0.7 mM and was obligatorily dependent on Na+ and Cl-. The Na+:Cl-:valganciclovir stoichiometry was 2 or 3:1:1., Conclusions: Amino acid esters and the amino-acid-based prodrug valganciclovir are transported by ATB(0,+). This shows that ATB(0,+) can serve as an effective delivery system for amino acid-based prodrugs.

Purpose: To investigate the in vitro trans-esterification of 1-[2(R)-(2-amino-2-methylpropionylamino)-3-(1H-indol-3-yl)propionyl]-3(S)-benzyl-piperidine-3-carboxylic acid ethyl ester (compound A) and to determine the effects of ethanol on its in vivo pharmacokinetics in male Sprague-Dawley rats., Methods: The effects of deuterated [d5]ethanol on the hydrolysis and trans-esterification of compound A in rat plasma and rat liver microsomes in the presence or absence of bis(p-nitrophenyl) phosphate (BNPP), a carboxylesterase inhibitor, were investigated. Following an oral pretreatment with deuterated ethanol in conjunction with an intravenous dose of compound A to rats, the pharmacokinetics of compound A and deuterated compound A were evaluated., Results: It was observed that the amount of deuterated compound A generated increased with increasing amounts of deuterated ethanol in incubates, whereas the amount of hydrolyzed product (compound B) decreased. BNPP inhibited both the hydrolysis and the trans-esterification of compound A. Furthermore, the pharmacokinetics of compound A in rats receiving ethanol was altered, such that the plasma clearance decreased by 1.5-fold and the elimination rate constant decreased by 2-fold. Deuterated compound A was determined, confirming that trans-esterification proceeded in vivo; approximately one third of the intravenous dose of compound A underwent trans-esterification., Conclusions: In the presence of ethanol, compound A underwent trans-esterification catalyzed by carboxylesterases. Ethanol pretreatment resulted in a decrease in the in vivo clearance of compound A mainly due to trans-esterification with ethanol.

alpha-Lipoic acid [5-[1,2]-dithiolan-3-yl-pentanoic acid (LA)] is a natural antioxidant and cofactor of several enzymes. It increases the glucose transport activity in skeletal muscles and adipocytes in a non-insulin dependent manner. Therefore, LA is widely used in Type 2 diabetic patients as an oral auxiliary drug. However, large doses of LA (0.8-1.8 gr/day p.o.) are required due to its unfavorable pharmacokinetic parameters. In order to improve these parameters, we synthesized ester and amide LA derivates. Two of these newly synthesized compounds, 5-[1,2]-dithiolan-3-yl-pentanoic acid 3-(5-[1,2]dithiolan-3yl-pentanoylamino)-propyl]-amide (AN-7) and 5-[1,2]-dithiolan-3-yl-pentanoic acid 3-(5-[1,2]-dithiolan-3yl-pentanoyloxy)-propyl ester (AN-8) augmented the rate glucose transport in myotubes in culture in the absence or presence of insulin. Their potency was 12-fold higher than that of the parent compound; their maximal stimulatory effect was 1.5-fold higher than that of LA. When tested in vivo in streptozotocin-diabetic C57/Black mice, AN-7 (10 mg/kg/day for 2 weeks, s.c.) reduced blood glucose level by 39% while a higher dose of LA (50 mg/kg/day for 2 weeks, s.c.) lowered it by 30%. These results indicate that AN-7 is more potent than LA in augmenting glucose transport in skeletal muscles and reducing blood glucose in diabetic animals.

A series of N-fluoroethylpiperidinyl (1), N-fluoroethylpiperidinemethyl (2) and N-fluoroethylpyrrolidinyl (3) esters were synthesized and examined as new (18)F-labeled radiotracers for measuring brain cholinesterase activity. The fluoroethyl group, instead of methyl group, results in slower in vitro enzymatic cleavage rates and higher selectivity for AChE. Based on metabolism in mouse blood and PET time-activity curves in rats, two radiotracers were identified as potential candidates for further in vivo evaluation in higher species.

Although 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) has proven to be clinically beneficial for the treatment of certain cancers, including a variety of skin cancers, optimal tissue localisation still remains a problem. An approach to improve the bioavailability of protoporphyrin IX (PpIX) is the use of ALA derivatives instead of ALA. In this work, we employed a subcutaneous murine mammary adenocarcinoma to study the tissue distribution pattern of the ALA hexyl ester (He-ALA) in comparison with ALA after their topical application in different vehicles. He-ALA induced porphyrin synthesis in the skin overlying the tumour (SOT), but it did not reach the tumour tissue as efficiently. Only 5 h after He-ALA lotion application, tumour porphyrin levels surpassed control values. He-ALA delivered in cream induced a substantially lower porphyrin synthesis in SOT, reinforcing the importance of the vehicle in the use of topical PDT. Porphyrin levels in internal organs remained almost within control values when He-ALA was employed. The addition of DMSO to ALA formulation slightly increased tumour and SOT porphyrin biosynthesis, but it did not when added to He-ALA lotion.

The association of the alpha(v)beta(3) integrin with tumor metastasis and tumor related angiogenesis has been suggested. Therefore, by imaging the alpha(v)beta(3) receptor with PET, information concerning the tumor status could be obtained. Cyclic peptides including the RGD sequence, were radiolabeled by direct electrophilic fluorination with [(18)F]AcOF. In tumor-bearing mice, the labeled peptides accumulated at the tumor with a high tumor to blood ratio. These findings suggest that an assessment of tumor characteristics may be obtained by using these (18)F-labeled peptides., (Copyright 2002 Elsevier Science Inc.)

A series of novel, highly potent alpha(v)beta(3) receptor antagonists with favorable pharmacokinetic profiles has been identified. In this series of antagonists, 2-aryl beta-amino acids function as potent aspartic acid replacements.

Ganciclovir (GCV), a promising antiviral compound, has poor ocular bioavailability as a result of its relatively low partition coefficient. In this study, lipophilic ester prodrugs of GCV were synthesized in an effort to improve its uptake into ocular tissues. In vitro permeability studies were conducted on isolated rabbit corneal membranes using aliphatic mono-acyl ester prodrugs of GCV to determine the effect of lipophilicity and corneal hydrolysis rate on transcorneal diffusion. The GCV prodrugs showed a progressive decrease in solubility and a corresponding increase in Log P values as the chain length was ascended. Permeation studies using freshly isolated rabbit corneas showed that all prodrugs permeated as intact prodrug as well as hydrolyzed GCV. Corneal permeability coefficients increased with increasing lipophilicity for mono-ester prodrugs having more than three carbon atoms in the side chain. The permeability of GCV increased about 6-fold in ascending from the parent drug-ganciclovir (3.82 +/- 0.19 x 10(-6) cm sec(-1)) to its valerate ester prodrug (23.70 +/- 1.36 x 10(-6) cm sec(-1)). Among the prodrugs studied, the valerate ester showed the highest permeability and holds the most potential for development. Overall prodrug permeability correlated linearly with increased susceptibility of the GCV esters to undergo hydrolysis in the cornea. The present work indicates that the ideal prodrug is one that not only possesses enhanced partitioning characteristics, but also high enzyme susceptibility. Concentration of active GCV penetrating the corneal epithelium was substantially increased through the bio-reversible ester prodrug strategy.

Synthetic iron chelators based on the natural siderophore ferrichrome have previously been shown to bind Fe(III) with high affinity (pKf > 27) and have shown no toxicity to mammalian cell cultures in vitro. A new class of lipophilic ferrichrome analogues carrying acetoxymethyl ester moieties has been synthesized. We have shown that these molecules penetrate rapidly through cell membranes and turn highly hydrophilic while inside the cells, upon esterase mediated hydrolysis of the lipophilic termini. The intracellular retention was visualized by labeling these analogues with a fluorescent naphthalic diimide probe. The prohydrophilic iron chelators have been shown to inhibit the metal-catalyzed intracellular formation of reactive oxygen species with high effectivity, and preliminary results suggest these molecules to be potent antimalarial agents.

Synthetic macromolecules such as copolymers of N-(2-hydroxypropyl)methacrylamide (pHPMA) are potential carriers for the delivery of drugs owing to their ability to passively accumulate in solid tumours [enhanced permeation and retention (EPR) effect]. To gain further knowledge about the biodistribution and the cellular localisation, poly(HPMA) was prepared for labelling by introducing biotin molecules. Biotinylated pHPMA (5 mol%) was intravenously injected into tumour-bearing rats and the accumulation of biotin-pHPMA was visualised using a streptavidin-alkaline phosphatase technique at day 7 post injection. In spite of the high solubility of pHPMA copolymers and the lack of attachment to cell structures, the biotinylated polymer could be easily detected in tissues fixed in 10% paraformaldehyde-phosphate buffer at 4 degrees C for 48 h. While biotin-pHPMA could be detected intracytoplasmically in liver and spleen, a predominantly interstitial localisation was observed within the anaplastic prostate carcinoma (Dunning R3327-AT1). How biotin as a label influences the biodistribution of poly(HPMA) was assessed by scintigraphy, autoradiography and histology comparing homopolymer poly(HPMA) with biotin-pHPMA. The organ distribution patterns of the two polymers correlated well, except with respect to kidney. It is assumed that the accumulation of biotin-pHPMA in the distal tubuli is due to a biotin transporter in the brush border membrane. The technique presented is useful for a more comprehensive understanding of the biodistribution of soluble macromolecules.

Properties of the intestinal digestion of the dietary phytosterols, cholesterol and cholestanol, and the mechanisms by which phytosterols inhibit the intestinal absorption of cholesterol in healthy human subjects are poorly known. We have studied the hydrolysis of dietary plant sterol and stanol esters and their subsequent micellar solubilization by determining their concentrations in micellar and oil phases of the jejunal contents. Two liquid formulas with low (formula 1) and high (formula 2) plant stanol concentrations were infused via a nasogastric tube to the descending duodenum of 8 healthy human subjects, and intestinal contents were sampled for gas-liquid chromatographic sterol analysis 60 cm more distally. During the duodenal transit, phytosterol esters were hydrolyzed. This was especially profound for sitostanol, as its esterified fraction per milligram of sitosterol decreased 80% (P < 0.001) in formula 1 and 61% (P < 0.001) in formula 2. Contrary to that, esterified fraction of cholesterol per milligram of sitosterol was increased fourfold (P < 0.001) in formula 1 and almost sixfold (P < 0.001) in formula 2, whereas that of cholestanol remained unchanged. Percentages of esterified sterols and stanols in total intestinal fluid samples were higher after the administration of formula 2 than of formula 1. Esterified cholesterol and stanols accumulated in the oil phase, and free stanols replaced cholesterol in the micellar phase. At high intestinal plant stanol concentrations, cholesterol looses its micellar solubility possibly by replacement of its free fraction in the micellar phase by hydrolyzed plant stanols, which leads to a decreased intestinal absorption of cholesterol.

In vitro studies have demonstrated that water-soluble, nontoxic phosphoramidates of azidothymidine (zidovudine [AZT]) have significant and specific anti-human immunodeficiency virus and anticancer activity. Although polar, these compounds are internalized and processed to the corresponding nucleoside monophosphates. Eight methyl amide and methyl ester phosphoramidate monoesters composed of D- or L-phenylalanine or tryptophan and AZT were synthesized. The plasma stability and protein binding studies were carried out in vitro. Then in vivo pharmacokinetic evaluations of six of the compounds were conducted. Sprague-Dawley rats received each compound by intravenous bolus dose, and serial blood and urine samples were collected. AZT and phosphoramidate concentrations in plasma and urine were quantitated by high-performance liquid chromatography with UV or fluorescence detection. Pharmacokinetic parameters were calculated by standard noncompartmental means. The plasma half-lives of the phosphoramidates were 10- to 20-fold longer than the half-life of AZT. Although the renal clearances of the phosphoramidates were similar to AZT, their total body clearances were significantly greater than that of AZT. The 3- to 15-fold-larger volume of distribution (Vss) for the phosphoramidates relative to AZT appeared to be dependent on the stereochemistry of the amino acid, with the largest values being associated with the L-amino acids. The increased Vss indicates a much greater tissue distribution of the phosphoramidate prodrugs than of AZT. Amino acid phosphoramidate monoesters of AZT have improved pharmacokinetic properties over AZT and significant potential as in vivo pronucleotides.

Aminocarbonyloxymethyl esters based on (S)-amino acid carriers were synthesised and evaluated as potential prodrugs of carboxylic acid agents. In addition, the compounds were evaluated as topical prodrugs with the aim of improving the dermal delivery of two non-steroidal anti-inflammatory agents: naproxen and flufenamic acid. The lipophilicities of these compounds were determined and their hydrolyses in aqueous solutions and in human plasma were examined. Compounds containing a secondary carbamate group were hydrolysed at pH 7.4 by two different routes: (i) direct nucleophilic attack at the ester carbonyl carbon leading to the release of the parent carboxylic acid and (ii) intramolecular rearrangement involving an O-->N acyl migration, leading to the formation of the corresponding amide. The rearrangement pathway is highly dependent on the size of the carboxylic acid and amino acid substituents, being eliminated when the amino acid is valine or leucine. In contrast, compounds decomposed in plasma exclusively through ester hydrolysis, most releasing the parent carboxylic acid quantitatively with half-lives shorter than 5 min. The permeation of selected prodrugs across excised postmortem human skin was studied in vitro. All prodrugs evaluated exhibited a lower flux than the corresponding parent carboxylic acid. The poor skin permeation observed for compounds is most probably due to their low aqueous solubility and high partition coefficient.

Background/aims: Prolonged skin occlusion increases stratum corneum water content and often increases skin permeability and irritant dermatitis. As skin wetness from wearing diapers is considered an important factor favouring the onset of diaper dermatitis, optimal diapering might decrease skin hyperhydration and dermatitis. Our aim is to define the quantitative relationship between nicotinate ester (a model penetrant) skin permeability and hydration, as measured by water evaporation rate (WER), decay curves (at individual time points) and WER-area under the curve (WER-AUC); and also to determine the level of skin hydration and skin permeability to nicotinates following a diapering simulation., Methods/results: Nine healthy Caucasian adult women were enrolled after a prescreening procedure (time to peak redness response to nicotinate); each received three wet occlusive patches for different exposure times (10 min, 30 min, and 3h) and two wet model diapers (3 and 8 h). Prior to patching or diapering of forearms, basal values of WER, skin blood flow volume (BFV), capacitance (Cap) and redness (a*) were measured on premarked sites (a, b, c and d). Immediately, following occlusive patch or diaper removal, 20 microL of each nicotinate (methyl and hexyl nicotinate) was applied to its respective site (a or b). The WER and Cap readings were recorded at designated sites (c and d) with the following intervals after nicotinate applications: 0, 5,10,15 and 20 min. The a* and BFV measurements were made on each nicotinate challenged site (a and b) with the following intervals after nicotinate applications: 5, 10, 15, 20, 30, 40, and 60 min., Results: WER-AUC and thus, skin hyperhydration, increased with occlusive patch and diaper exposure time, but there was no statistical difference between 3 and 8h diaper sites. All patched sites had significantly (P<0.05) increased hydration in comparison to control sites (undiapered or unpatched skin). Cap increased with occlusion time with patches, but not with diapers. The degree and time-course of redness from nicotinates did not vary with extent of skin hydration, but was significantly increased compared to non-hydrated skin. BFV-AUC did not show a significant increase between diapers at 3 and 8h sites; the BFV-AUC values varied on the patched sites, but some were significantly (P<0.05) higher than control site., Conclusion: Wet patches and diapers increased skin hyperhydration proportional to exposure time. Permeation of nicotinates was increased for hydrated skin vs. control, even after only 10 min of patch exposure. For these model permeants, we found no evidence of increased permeation rates with increased hyperhydration, once a relatively low threshold of hyperhydration was achieved (e.g. that reached after a 10 min wet patch). The data showed no meaningful differences in permeation following either diapering simulation and also suggested that the WER-AUC method was superior to capacitance for measuring the absolute extent of hyperhydration. We believe this is a suitable model for evaluating the quality of diaper product performance, as well as in pharmacologic assays of occlusive therapy.

Measurement of Carboxylesterase (CES) Activities (Masakiyo Hosokawa, Chiba University, Chiba, Japan and Tetsuo Satoh, Biomedical Research Institute, Chiba, Japan). Mammalian carboxyesterase (CES) efficiently catalyzes the hydrolysis of a variety of ester- and amide-containing chemicals. CES is involved in detoxification or metabolic activation of various drugs, environmental toxicants, and carcinogens. This unit contains three protocols for measuring CES activities.

Five new oligoethylene ester derivatives (9-13) of 5-iodo-2'-deoxyuridine (IDU) were synthesized and assayed to determine their lipophilicity by both experimental lipophilicity indices (log K') and calculated partition coefficients (CLOGP). In vitro experiments were carried out to evaluate the chemical and enzymatic stability and fluxes through excised human skin of these new IDU derivatives. Esters 9-13 showed increased lipophilicity compared with the parent drug (IDU), had good stability in phosphate buffer (pH 7.4), and were readily hydrolyzed by porcine esterase. No correlation between lipophilicity and skin permeation fluxes of synthesized esters 9-13 was observed. Results from in vitro percutaneous absorption studies showed that, among all esters synthesized, only esters 9 and 10 significantly increased the cumulative amount of IDU that penetrated through excised human skin compared with the parent drug (IDU)., (Copyright 2002 Wiley-Liss, Inc.)

With the aim at improving the transport of the current HIV protease inhibitors across the intestinal and blood brain barriers and their penetration into the central nervous system, the synthesis of various acyl and carbamatoyl glucose-containing prodrugs derived from saquinavir, indinavir and nelfinavir, their in vitro stability with respect to hydrolysis, and their anti-HIV activity have been investigated. D-Glucose, which is actively transported across these barriers, was connected through its 3-hydroxyl to these antiproteases via a linker. The liberation of the active free drug during the incubation time of the prodrugs with the cells was found to be crucial for HIV inhibition. The labile ester linking of the glucose-containing moiety to the peptidomimetic hydroxyl of saquinavir or to the indinavir C-8 hydroxyl, which is not part of the transition state isostere, is not an obstacle for anti-HIV activity. This is not the case for its stable carbamate linking to the peptidomimetic hydroxyl of saquinavir, indinavir and nelfinavir. The chemical stability with respect to hydrolysis of some of the saquinavir and indinavir prodrugs reported here, the liberation rate of the active free drug and the HIV inhibitory potency are acceptable for an in vivo use of these prodrugs. These glucose-linked ester and carbamate prodrugs display a promising therapeutic potential provided that their bioavailability, penetration into the HIV sanctuaries, and/or the liberation of the active free drug from the carbamate prodrugs are improved. Furthermore, no cytotoxicity was detected for the prodrugs for concentrations as high as 10 or even 100 microM, thus indicating an encouraging therapeutic index.

Entacapone is a new inhibitor of catechol-O-methyltransferase (COMT) that is used as an adjunct to L-dopa therapy in the treatment of Parkinson's disease. The bioavailability of orally administered entacapone is, however, relatively low (29-46%). In this study we have prepared more lipophilic acyl and acyloxyacyl esters, an acyloxy alkyl ether and an alkyloxycarbonyl ester of entacapone, and we have evaluated them as potential prodrugs to enhance the oral bioavailability of entacapone. All the derivatives fulfilled prodrug criteria and released entacapone in human serum in-vitro. The oral bioavailability of monopivaloyl (1a) and dipivaloyl (1b) esters of entacapone were investigated further in rats. The lipophilicity of 1b was high (log Papp 4.0 at pH 7.4) but its oral bioavailability was low (F = 0.6%), most probably due to its low aqueous solubility. The monopivaloyl ester of entacapone (1a) had a higher lipophilicity (log Papp 0.80) than entacapone (log Papp 0.18) at pH 7.4 while maintaining an aqueous solubility equal to entacapone. However, oral bioavailability was not increased when compared with the parent drug entacapone (F = 7.0% and 10.4%, respectively).

The class B, type I scavenger receptor (SR-BI) mediates the selective uptake of high density lipoprotein (HDL) cholesteryl esters and the efflux of free cholesterol. SR-BI is predominantly associated with caveolae in Chinese hamster ovary cells. The caveola protein, caveolin-1, binds to cholesterol and is involved in intracellular cholesterol trafficking. We previously demonstrated a correlative increase in caveolin-1 expression and the selective uptake of HDL cholesteryl esters in phorbol ester-induced differentiated THP-1 cells. The goal of the present study was to determine if the expression of caveolin-1 is the causative factor in increasing selective cholesteryl ester uptake in macrophages. To test this, we established RAW and J-774 cell lines that stably expressed caveolin-1. Transfection with caveolin-1 cDNA did not alter the amount of 125I-labeled HDL that associated with the cells, although selective uptake of HDL [3H]cholesteryl ether was decreased by approximately 50%. The amount of [3H]cholesterol effluxed to HDL was not affected by caveolin-1. To directly address whether caveolin-1 inhibits SR-BI-dependent selective cholesteryl ester uptake, we overexpressed caveolin-1 by adenoviral vector gene transfer in Chinese hamster ovary cells stably transfected with SR-BI. Caveolin-1 inhibited the selective uptake of HDL [3H]cholesteryl ether by 50-60% of control values without altering the extent of cell associated HDL. We next used blocking antibodies to CD36 and SR-BI to demonstrate that the increase in selective [3H]cholesteryl ether uptake previously seen in differentiated THP-1 cells was independent of SR-BI. Finally, we used beta-cyclodextrin and caveolin overexpression to demonstrate that caveolae depleted of cholesterol facilitate SR-BI-dependent selective cholesteryl ester uptake and caveolae containing excess cholesterol inhibit uptake. We conclude that caveolin-1 is a novel negative regulator of SR-BI-dependent selective cholesteryl ester uptake.

Toxic Oil Syndrome (TOS) was an epidemic disease related to the consumption of rapeseed oil denatured with aniline that made its sudden appearance in Spain in 1981. The fatty acid esters of 3-(N-phenylamino)-1,2-propanediol (PAP), which is a chemical class of by-products resulting from the reaction of aniline with oil components, have shown a strong association with TOS-related oils. These compounds also show some structural similarities to platelet-activating factor (PAF). In search of a toxic agent that could explain the widespread systemic effects observed in TOS patients, we investigated the intestinal absorption and biotransformation of the different PAP esters found in TOS-related oil samples and the possible pathophysiological effect of these mediators and their metabolic products if acting as PAF analogs. Results indicate that PAP esters are absorbed in the gastrointestinal tract and are distributed and stored in different organs, particularly in the liver and brown adipose tissue. PAP in these organs showed different patterns of fatty acids, indicating the ability of the gastrointestinal tract to modify the fatty acid composition of the parent PAP. Thus, the fatty acid profile of the PAP esters found in intestine appears to be related to the type of oil used as vehicle. Some of these PAP esters, when a long acyl chain was present in the sn-1 position of the molecule, showed an inhibitory effect on the PAF synthesis. This is an important observation in line with the systemic nature of the disease.

Diferulic acids are potent antioxidants and are abundant structural components of plant cell walls, especially in cereal brans. As such, they are part of many human and animal diets and may contribute to the beneficial effect of cereal brans on health. However, these phenolics are ester-linked to cell wall polysaccharides and cannot be absorbed in this form. This study provides the first evidence that diferulic acids can be absorbed via the gastrointestinal tract. The 5-5-, 8-O-4-, and 8-5-diferulic acids were identified in the plasma of rats after oral dosing with a mixture of the three acids in oil. Our study also reveals that human and rat colonic microflora contain esterase activity able to release 5-5-, 8-O-4-, and 8-5-diferulic acids from model compounds and dietary cereal brans, hence providing a mechanism for release of dietary diferulates prior to absorption of the free acids. In addition, cell-free extracts from human and rat small intestine mucosa exhibited esterase activity towards diferulate esters. Hence, we have shown that esterified diferulates can be released from cereal brans by intestinal enzymes, and that free diferulic acids can be absorbed and enter the circulatory system. Our results suggest that the phenolic antioxidant diferulic acids are bioavailable.

Readily hydrolysable basic and dibasic esters of ampicillin were synthesised by alkylation of the carboxylate function of ampicillin to obtain prodrugs that may accumulate in cells and allow for an intracellular delivery of ampicillian (Fan et al., Bioorg. Med. Chem. Lett. 1997, 7, 3107). We found that the beta-lactam ring cleavage and the hydrolysis of the ester function were competitive reactions. The prerequisite for biological activity of compounds of this type is therefore that ester hydrolysis proceeds faster than ring opening. Some synthesised compounds show promise as prodrugs since they displayed a reasonable stability and regenerate large quantities of bioactive ampicillin in broth.

The headspace concentrations of three esters above solutions containing emulsified lipids were more resistant to dilution by a stream of gas than those above water alone. The effect was greatest for ethyl octanoate, and least for ethyl butyrate, with ethyl hexanoate showing intermediate behavior. This correlated with their solubility in the lipid fraction of the emulsion. Headspace analysis (comparing the emulsion with water) underestimated the release of the esters during consumption. The ratios observed between water and emulsion systems were different for the maximum breath concentration compared with headspace analysis. The emulsion appears to have acted as a reservoir for volatile release, counteracting the effects of sample dilution by saliva.

6(A)-O-(n-Butanoyl)-beta-cyclodextrin was prepared and its hydrolysis behavior in aqueous solutions and in rat intestinal fluids was investigated. Furthermore, the enzymatic hydrolyses of the n-butyric acid-beta-cyclodextrin conjugate using alpha-amylase and esterase were studied to gain insight into the release behavior of n-butyric acid from the conjugate. The hydrolysis of the conjugate proceeded according to a first-order kinetics in aqueous solution, and gave a V-shaped pH profile, indicating a specific acid-base-catalyzed hydrolysis at acidic and neutral-alkaline regions, respectively. The half-lives (t(1/2)) of the conjugate at pH 4.4, 6.8, and 7.4 at 37 degrees C were approximately 580, 43, and 6 days, respectively, indicating that the conjugate is stable in aqueous solution. No appreciable release of n-butyric acid from the conjugate was observed in the stomach and small intestinal contents of rats, or in the small and large intestinal homogenates of rats. On the other hand, a fast disappearance of the conjugate and an appearance of n-butyric acid were observed in the cecal and colonic contents of rats. The t(1/2) values of the disappearance were approximately 4, 1, and 6 h in 10 and 15% cecal contents and 10% colonic contents, respectively, and the appearance of n-butyric acid after 6 h was approximately 10% in the 15% cecal contents. Aspergillus oryzae alpha-amylase hydrolyzed the conjugate to small saccharide conjugates, such as the triose and maltose conjugates, but there was no appreciable release of n-butyric acid. The conjugate was less susceptible to carboxylic esterase (from porcine live), thus releasing no appreciable amounts of n-butyric acid. On the other hand, a fast release of n-butyric acid was observed when the esterase was employed after amylase hydrolysis, suggesting that two types of enzymes, sugar-degrading and ester-hydrolyzing enzymes, are necessary for the release of n-butyric acid from the conjugate in large intestinal contents., (Copyright 2000 Wiley-Liss Inc.)

Twelve medium to long chain fatty acid Esters (Esters), the total number of carbon atoms of which ranged from 17 to 34, were used to study the effect of the vehicle on the permeation of ketoprofen, and the effect was compared with the case of indomethacin. The solubility of ketoprofen was higher in Esters with a smaller number of carbon atoms. The permeation rate of ketoprofen from the Ester suspension through excised hairless rat skin was proportional to its solubility in the suspension, which was the same in the case of indomethacin. The diffusion constant and partition coefficient were calculated using the computer program MULTI(FILT). The diffusion constant decreased with increasing number of carbon atoms, and the partition coefficient was increased with increasing number of carbon atoms, in both cases of ketoprofen and indomethacin. Esters also penetrated the skin with the concentration of about 10 mg/g, independent of the number of carbon atoms. The Esters in the skin increase the diffusion rate of the drugs, especially in the case of Esters with a small number of carbon atoms. Also the drug solubility in the skin was improved, although the effect was similar for the range of Esters investigated in the present study. Then the permeation rate of ketoprofen and indomethacin increased.