Relationship between structure and permeability of dipeptide derivatives containing tryptophan and related compounds across human intestinal epithelial (Caco-2) cells

The permeability of dipeptide derivatives containing tryptophans and indole derivatives through Caco-2 cells was used as an in vitro intestinal absorption model in order to clarify structural factors which influence their intestinal epithelial permeation and metabolism. Most peptide derivatives were hydrolysed not only by the cytosolic enzymes in Caco-2 cells during permeation but also by enzymes released to the apical solution before cell permeation. The N-terminal blocked dipeptides were more resistant to hydrolases expressed in the Caco-2 cells and indole derivatives were not entirely degraded. Based on compound concentration dependency and comparison of permeability coefficients in apical-to-basolateral and basolateral-to-apical directions, the main absorption mechanism of compounds were determined. Compounds were then classified into three groups; (1) passively transported compounds, (2) actively transported compounds and (3) compounds excreted by P-glycoprotein.     

Microbial transport: Adaptations to natural environments

The cytoplasmic membrane of bacteria is the matrix for metabolic energy transducing processes such as proton motive force generation and solute transport. Passive permeation of protons across the cytoplasmic membrane is a crucial determinant in the proton motive generating capacity of the organisms. Adaptations of the membrane composition are needed to restrict the proton permeation rates especially at higher temperatures. Thermophilic bacteria cannot sufficiently restrict this proton permeation at their growth temperature and have to rely on the much␣lower permeation of Na + to generate a sodium motive force for driving metabolic energy-dependent membrane processes. Specific transport systems mediate passage across the membrane at physiological rates of all compounds needed for growth and metabolism and of all end products of metabolism. Some of transport systems, the secondary transporters, transduce one form of electrochemical energy into another form. These transporters can play crucial roles in the generation of metabolic energy. This is especially so in anaerobes such as Lactic Acid Bacteria which live under energy-limited conditions. Several transport systems are specifically aimed at the generation of metabolic energy during periods of energy-limitation. In their natural environment bacteria are also often exposed to cytotoxic compounds, including antibiotics. Many bacteria can respond to this live-threatening condition by overexpressing powerful drug-extruding multidrug resistance systems.     

Permeation of aromatic carboxylic acids across lipid bilayers: the pH-partition hypothesis revisited

According to the pH-partition hypothesis the charged species of organic compounds do not contribute to lipid bilayer permeation as they generally show negligible partitioning into n-octanol. With this assumption, membrane permeation is related to the molar fraction of the neutral species at a particular pH. A recently developed permeation assay permits us to directly determine pH-dependent permeation of aromatic carboxylic acids. Tb(3+)-loaded liposomes are incubated with aromatic carboxylic acids and upon excitation at the absorption wavelength of the acid, permeation kinetics can be measured as an increase in Tb(3+) luminescence. The anions of the tested acids permeated egg phosphatidylcholine membranes only 12 (2-hydroxynicotinic acid), 66 (salicylic acid), and 155 (dipicolinic acid) times slower than the net neutral species. The anions, therefore, controlled the total permeation already at 1-2 pH units above their pK(a). These results indicate that in contrast to the expectations of the pH-partition hypothesis, lipid bilayer permeation of an acidic compound can be completely controlled by the anion at physiological pH.     

Synthesis, stability and in vitro dermal evaluation of aminocarbonyloxymethyl esters as prodrugs of carboxylic acid agents

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.     

Synthetic ceramide analogues as skin permeation enhancers: structure-activity relationships

The study presents new information about the structure–activity relationships of the skin permeation enhancers. A series of ceramide analogues including eight different polar head groups and six different chain lengths was synthesised. The compounds were evaluated as permeation enhancers in vitro using porcine skin. The physico-chemical parameters of the tested compounds obtained by computer modelling were used to evaluate, by multiple linear regression, the enhancement ratios (ERs) of the compounds. The regression analysis suggests that the hydrogen bonding ability of the compounds is inversely related to the ER values and that the molecular size and lipophilicity must be well balanced. In the studied enhancers having the same chain length, the enhancement activity is dependent only on their permeability coefficients. This finding confirms the Warner's hypothesis that the polar head of an enhancer is responsible for the permeation and anchoring of the molecule into the stratum corneum lipids and that it does not influence the mechanism of action. For the specific action of enhancers, that is disordering of the intercellular lipid packing, the length of the hydrophobic chain(s) and not the lipophilicity is important. Furthermore, the examination of the FTIR spectra indicated that the most active substances possess the most ordered chains. The described relationships could bring more rational approaches in designing new potent enhancers for transdermal formulations.     

Comparison of permeation through phosphatidylcholine bilayers of N-dipicolinyl-alpha- and -beta-oligopeptides

Cell-membrane permeation of small therapeutic peptides and peptidomimetics is a fundamental issue in pharmaceutical research. Using a Tb(3+)-based permeation assay, we have examined the ability of alpha- and beta-peptides, bearing proteinogenic side chains and an N-terminal dipicolinic acid (DPA) monoamide group, to enter liposomes composed of egg phosphatidylcholine bilayers. A series of 12 DPA-peptides of increasing chain length was prepared and characterized by CD and NMR analysis. An interesting destabilizing effect of the N-terminal DPA group on the helical structure of a beta-hexapeptide was discovered. Significant differences in permeation were observed between the DPA-alpha- and the DPA-beta-peptides, with all beta-peptidic compounds permeating better than their alpha-analogs. Thus, beta-peptides have been shown to interact with lipid bilayers in a manner that is distinctly different from that of alpha-peptides. Together with the fact that beta-peptides are proteolytically stable in mammalian organisms, and that they fold to form helices and hairpin turns with short chain lengths, the new results further emphasize the biomedical potential of beta-peptides.     

Boronated derivatives of chlorin <Emphasis Type="Italic">e</Emphasis><Subscript>6</Subscript> and fluoride-containing porphyrins as penetrating anions: a study using bilayer lipid membranes

Boronated derivatives of porphyrins are studied extensively as promising compounds for boron-neutron capture therapy and photodynamic therapy. Understanding of the mechanism of their permeation across cell membranes is a key step in screening for the most efficient compounds. In the present work, we studied the ability of boronated derivatives of chlorin e ₆ and porphyrins, which are mono-, di-, and tetra-anions, to permeate through planar bilayer lipid membranes (BLM). The translocation rate constants through the hydrophobic part of the lipid bilayer were estimated for monocarborane and its conjugate with chlorin e ₆ by the method of electrical current relaxation. They were similar, 6.6 and 6.8 sec⁻¹, respectively. Conjugates of porphyrins carrying two and four carborane groups were shown to permeate efficiently through a BLM although they carry two charges and four charges, respectively. The rate of permeation of the tetraanion estimated by the BLM current had superlinear dependence on the BLM voltage. Because the resting potential of most mammalian cells is negative inside, it can be concluded that the presence of negatively-charged boronated groups in compounds should hinder the accumulation of the porphyrins in cells.     

Relationships between structure and high-throughput screening permeability of peptide derivatives and related compounds with artificial membranes: application to prediction of Caco-2 cell permeability

To evaluate absorption of compounds across the membrane via a transcellular route, the permeability of peptide derivatives and related compounds was measured by the parallel artificial membrane permeation assay (PAMPA). The permeability coefficients by PAMPA were analyzed quantitatively using classical QSAR and Volsurf approaches with the physicochemical parameters. The results from both approaches showed that hydrogen bonding ability of molecules in addition to hydrophobicity at a particular pH were significant in determining variations in PAMPA permeability coefficients. The relationship between Caco-2 cell permeability and artificial lipid membrane permeability was then determined. The compounds were sorted according to their absorption pathway in the plot of the Caco-2 cell and PAMPA permeability coefficients.     

Aqueous pathways dominate permeation of solutes across Pisum sativum seed coats and mediate solute transport via diffusion and bulk flow of water

The permeability of seed coats to solutes either of biological or anthropogenic origin plays a major role in germination, seedling growth and seed treatment by pesticides. An experimental set‐up was designed for investigating the mechanisms of seed coat permeation, which allows steady‐state experiments with isolated seed coats of Pisum sativum. Permeances were measured for a set of organic model compounds with different physicochemical properties and sizes. The results show that narrow aqueous pathways dominate the diffusion of solutes across pea seed coats, as indicated by a correlation of permeances with the molecular sizes of the compounds instead of their lipophilicity. Further indicators for an aqueous pathway are small size selectivity and a small effect of temperature on permeation. The application of an osmotic water potential gradient across isolated seed coats leads to an increase in solute transfer, indicating that the aqueous pathways form a water‐filled continuum across the seed coat allowing the bulk flow of water. Thus, the uptake of organic solutes across pea testae has two components: (1) by diffusion and (2) by bulk water inflow, which, however, is relevant only during imbibition.     

Novel oxoisoaporphine-based inhibitors of acetyl- and butyrylcholinesterase and acetylcholinesterase-induced beta-amyloid aggregation

A series of novel oxoisoaporphine-based inhibitors (10-aminoalkylamino-1-azabenzanthrone Ar-NH(CH(2))(n)NR(1)R(2)) of acetylcholinesterase (AChE) has been designed, synthesized, and tested for their ability to inhibit AChE, butyrylcholinesterase (BChE) and AChE-induced β-amyloid (Aβ) aggregation. The synthetic compounds exhibited high AChE inhibitory activity with IC(50) values in the submicromolar range in most cases. Non-competitive binding mode was found for these derivatives by the graphical analysis of steady-state inhibition data. Moreover, all compounds exhibit significant inhibitory activity on AChE-induced Aβ aggregation with inhibitory potency from 54.5% to 93.5%. Finally, six out of twelve synthetic compounds were predicted to be able to cross the blood-brain barrier (BBB) to reach their targets in the central nervous system (CNS) according to a parallel artificial membrane permeation assay for BBB. The result encourages us to study this class of compounds thoroughly and systematically.     

Synthesis and evaluation of the physicochemical properties of esterase-sensitive cyclic prodrugs of opioid peptides using coumarinic acid and phenylpropionic acid linkers.

In an attempt to improve the membrane permeabilities of opioid peptides, we have synthesized cyclic prodrugs of [Leu5]-enkephalin and DADLE using a coumarinic acid or a phenylpropionic acid linker. The synthesis of the coumarinic acid- and phenylpropionic acid-based cyclic prodrugs followed similar strategies. Key intermediates were the compounds with the C-terminal amino acids of opioid peptides (L-Leu, [Leu5]-enkephalin; D-Leu, DADLE) attached to the phenol hydroxyl group and the remaining amino acids of the peptide linked via the N-terminal amino acid (L-Tyr) attached to the carboxylic acid groups of the prodrug moieties (coumarinic acid or propionic acid). Cyclization of these linear precursors gave the cyclic prodrugs in 30-50% yields. These cyclic prodrugs exhibited excellent transcellular permeation characteristics across Caco-2 cell monolayers, an in vitro model of the intestinal mucosa. To correlate the cellular permeabilities of these cyclic prodrugs with their physicochemical properties, we calculated their Stokes-Einstein molecular radii from their diffusion coefficients which were determined by NMR and we determined their membrane interaction potentials using immobilized artificial membrane (IAM) column chromatography. The cyclic prodrugs exhibited molecular radii similar to those of the parent compounds, [Leu5]-enkephalin and DADLE. However, these cyclic prodrugs were shown to have much higher membrane interaction potentials than their corresponding opioid peptides. Therefore, the enhanced cellular permeation of the cyclic prodrugs is apparently due to the alteration of their lipophilicity and hydrogen bonding potential, but not their molecular sizes.     

Behaviour of small solutes and large drugs in a lipid bilayer from computer simulations

To reach their biological target, drugs have to cross cell membranes, and understanding passive membrane permeation is therefore crucial for rational drug design. Molecular dynamics simulations offer a powerful way of studying permeation at the single molecule level. Starting from a computer model proven to be able to reproduce the physical properties of a biological membrane, the behaviour of small solutes and large drugs in a lipid bilayer has been studied. Analysis of dihedral angles shows that a few nano seconds are sufficient for the simulations to converge towards common values for those angles, even if the starting structures belong to different conformations. Results clearly show that, despite their difference in size, small solutes and large drugs tend to lie parallel to the bilayer normal and that, when moving from water solution into biomembranes, permeants lose degrees of freedom. This explains the experimental observation that partitioning and permeation are highly affected by entropic effects and are size-dependent. Tilted orientations, however, occur when they make possible the formation of hydrogen bonds. This helps to understand the reason why hydrogen bonding possibilities are an important parameter in cruder approaches which predict drug absorption after administration. Interestingly, hydration is found to occur even in the membrane core, which is usually considered an almost hydrophobic region. Simulations suggest the possibility for highly polar compounds like acetic acid to cross biological membranes while hydrated. These simulations prove useful for drug design in rationalising experimental observations and predicting solute behaviour in biomembranes.     

Possible mechanism of uptake for several compounds in ionized form through human erythrocyte membrane

The mechanism underlying uptake of certain compounds in ionized form across human red blood cell membrane was examined. The ionized forms of salicylate, 5-methoxysalicylate and phenylalanylphenylalanine were significantly taken up into the interior space of human red blood cells instead of remaining in the membrane. Inhibition of the uptake of these compounds by 4,4′-diisothiocyano-2,2′-disulfonate stilbene and phlorizin indicates that their permeation of the erythrocyte membrane may involve the membrane protein fraction. Chelation at the protein site does not appear to occur. Instead, an amino group in the protein structure may mediate the transport of these ionized compounds.     

Interaction of insecticides with lipid membranes.

The permeability of liposome membranes is increased by organophosphorus and organochlorinated insecticides at concentrations of 10(-5)--10(-4) M. The order of effectiveness is similar to the toxicity of the compounds to mammals, and is the following for permeation of non-electrolytes and for valinomycin-induced permeation of K+: parathion greater than 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT) approximately aldrin greater than malathion greater than lindane. The degree of effectiveness for X-537A-induced permeation of Ca2+ was the following: aldrin greater than or equal to DDT greater than parathion greater than malathion greater than lindane. The organophosphorus compound, ethyl azinphos (10(-4) M), dramatically increases the permeability of liposome membranes to all the tested substances, probably as a consequence of surfactant effects. Some organochlorinated insecticides appear to react with cation ionophores and modulate their motion across lipid membranes. It is suggested that the insecticides may exert some of their toxic actions by modifying certain mechanisms in the cell membrane.     

Ionic strength has a greater effect than does transmembrane electric potential difference on permeation of tryptamine and indoleacetic acid across Caco-2 cells

The effects of transmembrane electric potential difference and ionic strength on the permeation of tryptamine and indoleacetic acid across a Caco-2 cell monolayer were examined. A decrease in the transmembrane electric potential difference caused by the addition of potassium ion to the transport buffer had no effect on the permeation rate of either compound. On the other hand, an increase in ionic strength resulted in a decrease in the permeation rate of tryptamine and an increase in the permeation rate of indoleacetic acid. The changes in the permeation rate with changes in the ionic strength were correlated with the membrane surface potential monitored by 1-anilino-8-naphthalenesulfonic acid (ANS), a fluorescent probe. We tested these effects using several other cationic and anionic compounds. These effects of ionic strength were found to be common to all drugs tested. The compound that showed a relatively lower permeation rate was given relatively stronger effect. The possibility of overestimation or underestimation caused by these effects should be considered when the permeation of an ionic compound is evaluated using a cell monolayer system.     

Ionic strength has a greater effect than does transmembrane electric potential difference on permeation of tryptamine and indoleacetic acid across Caco-2 cells

The effects of transmembrane electric potential difference and ionic strength on the permeation of tryptamine and indoleacetic acid across a Caco-2 cell monolayer were examined. A decrease in the transmembrane electric potential difference caused by the addition of potassium ion to the transport buffer had no effect on the permeation rate of either compound. On the other hand, an increase in ionic strength resulted in a decrease in the permeation rate of tryptamine and an increase in the permeation rate of indoleacetic acid. The changes in the permeation rate with changes in the ionic strength were correlated with the membrane surface potential monitored by 1-anilino-8-naphthalenesulfonic acid (ANS), a fluorescent probe. We tested these effects using several other cationic and anionic compounds. These effects of ionic strength were found to be common to all drugs tested. The compound that showed a relatively lower permeation rate was given relatively stronger effect. The possibility of overestimation or underestimation caused by these effects should be considered when the permeation of an ionic compound is evaluated using a cell monolayer system.     

In silico Prediction of Human Oral Absorption Based on QSAR Analyses of PAMPA Permeability

The parallel artificial membrane permeation assay (PAMPA) was developed as a model for the prediction of transcellular permeation in the process of drug absorption. Our research group has measured the PAMPA permeability of peptide‐related compounds, diverse drugs, and agrochemicals. This work led to a classical quantitative structure–activity relationship (QSAR) equation for PAMPA permeability coefficients of structurally diverse compounds based on simple physicochemical parameters such as lipophilicity at a particular pH (log P_oct and |pK_a?pH|), H‐bond acceptor ability (SA_HA), and H‐bond donor ability (SA_HD). Since the PAMPA permeability of lipophilic compounds decreased with their apparent lipophilicity due to the unstirred water layer (UWL) barrier on membrane surfaces and to membrane retention, a bilinear QSAR model was introduced to explain the permeability of a broader set of compounds using the same physicochemical parameters as those used for the linear model. We also compared PAMPA and Caco‐2 cell permeability coefficients of compounds transported by various absorption mechanisms. The compounds were classified according to their absorption pathway (passively transported compounds, actively transported compounds, and compounds excreted by efflux systems) in the plot of Caco‐2 vs. PAMPA permeability. Finally, based on the QSAR analyses of PAMPA permeability, an in silico prediction model of human oral absorption for possibly transported compounds was proposed, and the usefulness of the model was examined.     

Ions and blockers in potassium channels: insights from free energy simulations

Potassium ion channels enable efficient and selective permeation of K+ ions across nonpolar biological membranes. Here we review the results of recent free energy calculations related to the permeation of monovalent cations through K+ channels and to the channel inhibition by blocker compounds. In particular, the progress in computational studies of the bacterial KcsA channel is discussed.     

Macrocyclic BACE-1 inhibitors acutely reduce Aβ in brain after po application

A series of macrocyclic peptidic BACE-1 inhibitors was designed. While potency on BACE-1 was rather high, the first set of compounds showed poor brain permeation and high efflux in the MDRI–MDCK assay. The replacement of the secondary benzylamino group with a phenylcyclopropylamino group maintained potency on BACE-1, while P-glycoprotein-mediated efflux was significantly reduced and brain permeation improved. Several compounds from this series demonstrated acute reduction of Aβ in human APP-wildtype transgenic (APP51/16) mice after oral administration.     

Reconstructed epidermis and full-thickness skin for absorption testing: influence of the vehicles used on steroid permeation

A protocol for percutaneous absorption studies has been validated, based on the use of reconstructed human epidermis (RHE) and aqueous solutions of test substances. However, it is often the case that it is more-complex formulations of drugs or chemicals which will make contact with the skin surface. To investigate whether RHE and the reconstructed full-thickness skin model (FT-model) can be used to predict uptake from formulations, we compared the permeation of hydrocortisone and testosterone when applied in emulsion form and as a solution containing the penetration enhancer, ethanol. Human and pig skin and a non-cornified alveolar model served as references. The results were compared with steroid release from the formulations. The permeation rates of the steroids were ranked as: alveolar model > RHE > FT-model, pig skin > human skin. In accordance with the rapid hydrocortisone release from the formulations, the permeation rates of this steroid exceeded those of testosterone. Only minor differences were observed when comparing the testosterone formulations, in terms of release and permeation. However, the ranking of the permeation of the hydrocortisone formulations was: solution > w/o emulsion > o/w emulsion, which permitted the elucidation of penetration enhancing effects, which is not possible with drug release studies. Differences in penetration were most obvious with native skin and reconstructed tissues, which exhibited a well-developed penetration barrier. In conclusion, RHE and skin preparations may be useful in the development of topical dermatics, and in the framework of hazard analysis of toxic compounds and their various formulations.