Biomimetic Artificial Membranes Research Articles

Preformulation Studies of Novel Menthol Prodrugs with Antiparasitic Activity: Chemical Stability, In Silico, and In Vitro Permeability Assays

Based on the demonstrated and reported trypanocidal, leishmanicidal, and antiplasmodial activities of two menthol prodrugs, it was decided to proceed with preformulation studies, which are of key relevance in the drug discovery process. The aim of this study is to examine the stability and permeability of two new menthol prodrugs with antiparasitic activity. To determine the stability of menthol and its prodrugs, the corresponding studies were carried out in buffered solutions at pH values of 1.2, 5.8, and 7.4 at 37 °C. In silico permeability studies were performed using the free PerMM software and then in vitro permeability studies were performed using a biomimetic artificial membrane (BAM). Permeability studies conducted in silico predicted that both menthol and its prodrugs would pass through biological membranes via flip-flop movement. This prediction was subsequently confirmed by in vitro BAM permeability studies, where it was observed that the menthol prodrugs (1c and 1g) exhibited the highest Papp (apparent permeability) value compared to the parent compound. The study reveals that menthol prodrugs exhibit stability at a pH of 5.8 and possess sufficient in vitro permeability values as preformulation parameters.

Study and development of microemulsion formulations to increase the permeability of acyclovir

The purpose of this study was to improve the permeability of acyclovir (ACV) by developing a water-in-oil microemulsion, since it is an important factor that influences the oral absorption and, consequently, the oral bioavailability of drugs. Pseudoternary phase diagrams (PTPD) were constructed to identify the regions for obtaining microemulsions (ME). From the region of fluid transparent systems, five ME were chosen to evaluate the effect on ACV permeability by using two-compartment horizontal Franz diffusion cells, using a bio-mimetic artificial membrane, which simulates the typical lipophilic characteristics of the biological membranes. The characterization of ME was carried out by PTPD, conductivity determination and Nuclear Magnetic Resonance studies. The formulation that showed the higher incorporation of ACV (81.57 mg/ml) consisted of 13.3% Polysorbate 80, 13.3% ethanol, 6.6% ginger oil and 66% water. An increase in ACV permeability was observed, reaching an apparent permeability coefficient of 1.99.10−6 cm/s, corresponding to a fraction of human absorbed dose of 99.97%, according to the correlation previously established by our research group. These results indicated the ability of ME to improve the permeability of ACV, representing a system with the potential to improve the efficacy of ACV in oral administrated pharmaceutical formulation.

HPV Lesions and Other Issues in the Oral Cavity Treatment and Removal without Pain.

Due to different oral and dental conditions, oral mucosa lesions such as those caused by the human papilloma virus and temporomandibular joint pathologies often have to be treated by surgical, ablative or extractive procedures. The treatment and control of pain and inflammation during these procedures is essential to guarantee the patient’s well-being. For the foregoing reason, a hydrogel based on sodium alginate and hyaluronic acid containing 2% of ketorolac tromethamine has been developed. We characterized it physically, mechanically and morphologically. The rheological results suggest that the formulation can be easily and gently applied. Ex vivo permeation studies show that Ketorolac Tromethamine is able to penetrate through the buccal and sublingual mucosae, in addition to being retained in the mucosae’s structure. Through an in vitro test, we were able to evaluate the role that saliva plays in the bioavailability of the drug, observing that more than half of the applied dose is eliminated in an hour. The histological and cytotoxic studies performed on pigs in vivo showed the excellent safety profile of the formulation, as well as its high tolerability. In parallel, a biomimetic artificial membrane (PermeaPad®) was evaluated, and it showed a high degree of correlation with the oral and sublingual mucosa.

Biomimetic Artificial Membrane Permeability Assay over Franz Cell Apparatus Using BCS Model Drugs.

A major parameter controlling the extent and rate of oral drug absorption is permeability through the lipid bilayer of intestinal epithelial cells. Here, a biomimetic artificial membrane permeability assay (Franz–PAMPA Pampa) was validated using a Franz cells apparatus. Both high and low permeability drugs (metoprolol and mannitol, respectively) were used as external standards. Biomimetic properties of Franz–PAMPA were also characterized by electron paramagnetic resonance spectroscopy (EPR). Moreover, the permeation profile for eight Biopharmaceutic Classification System (BCS) model drugs cited in the FDA guidance and another six drugs (acyclovir, cimetidine, diclofenac, ibuprofen, piroxicam, and trimethoprim) were measured across Franz–PAMPA. Apparent permeability (Papp) Franz–PAMPA values were correlated with fraction of dose absorbed in humans (Fa%) from the literature. Papp in Caco-2 cells and Corti artificial membrane were likewise compared to Fa% to assess Franz–PAMPA performance. Mannitol and metoprolol Papp values across Franz–PAMPA were lower (3.20 × 10−7 and 1.61 × 10−5 cm/s, respectively) than those obtained across non-impregnated membrane (2.27 × 10−5 and 2.55 × 10−5 cm/s, respectively), confirming lipidic barrier resistivity. Performance of the Franz cell permeation apparatus using an artificial membrane showed acceptable log-linear correlation (R2 = 0.664) with Fa%, as seen for Papp in Caco-2 cells (R2 = 0.805). Data support the validation of the Franz–PAMPA method for use during the drug discovery process.

Effect of Complexes and Microemulsions on the Permeability of Drugs: Determination Using a New Biomimetic Artificial Membrane.

The aim of this work was to predict the permeability of two model drugs, sulfamerazine (SMR) and indomethacin (INM), and to determine the effect on their apparent permeabilities by complexation with cyclodextrins and/or meglumine or incorporation in microemulsions. Permeation experiments were performed using two-chamber diffusion cells with a new composition of bio-mimetic membrane composed of 80% of Lipoid® S100 and 20% of cholesterol in n-octanol 10% w/w solution, at 37 ± 0.5°C and 14,000rpm. The predictive capacity of the permeability of passive diffusion absorbed compounds was evaluated using 20 drug standards and showed an exponential correlation between the apparent permeability coefficients (Papp) and the fraction absorbed percentages in humans (Fa%), with an R2 value of 0.67942 and a constant value of - 4.1 ± 0.8. SMR and INM were classified as Class II and I, respectively, according to the Biopharmaceutical Classification System. These drugs were complexed and incorporated in microemulsions. The Fa% from all the drug products was higher than 90%. SMR in the complexes and both drugs in microemulsions were classified as highly soluble. Thus, SMR and INM incorporated in these pharmaceutical products could be classified as Class I.

Droplet Interface Bilayer as Cell Membrane Mimics: Water Permeability Studies

The process of water permeation across lipid membranes has significant implications for cellular physiology and homeostasis, and its study may lead to a greater understanding of the relationship between the structure of lipid bilayer and the role that lipid structure plays in water permeation. We have created a biomimetic artificial membrane, through contact of water droplets in an oil solution containing lipids. Using optical microscopy, we have measured water transport between droplets, as water moves from one droplet to another due to concentration difference. This was assessed as a function of lipid content, structure, and additives, such as cholesterol, which is an essential component of cell membrane. Our results show that cholesterol can increase the activation energy of water permeability several-fold, depending upon the structure of the lipid that makes up the bilayer, thus shedding light on how this singular sterol is vital to control of water movement. We demonstrate that the droplet interface bilayer can be employed as a convenient model membrane to rapidly explore subtle structural effects on bilayer water permeability.

Paraben Transport and Metabolism in the Biomimetic Artificial Membrane Permeability Assay (BAMPA) and 3-Day and 21-Day Caco-2 Cell Systems

Noncellular and cellular in vitro models for predicting intestinal absorption were used to investigate the transport and metabolism of parabens. The biomimetic artificial membrane permeability assay (BAMPA) membrane was constructed by impregnating a lipid solution on a hydrophobic filter. Caco-2 cells at passage numbers 65 to 80 were cultured in either the accelerated 3-day Biocoat system or the standard 21-day Transwell cell culture system. Paraben transport across the BAMPA system showed a parabolic relationship. The lowest log P (p-hydroxybenzoic acid) and highest log P compounds (heptyl and octyl parabens) had apparent permeabilities (Papp) less than 1.0 x 10(-6) cm/s and Papp was maximal at approximately 8.5 x 10(-6)cm/s for the intermediate log P (ethylparaben) compound. With the Biocoat, a similar parabolic relationship was found. In the 21-day Caco-2 cells, the parabens were metabolized by esterases at to p-hydroxybenzoic acid. In conclusion, the in vitro models added complementary insight into the absorption process, such as the transport route, intrinsic permeability, and extent of metabolism of the parabens. This study indicated that presystemic metabolism of orally ingested parabens to the p-hydroxybenzoic acid in the intestine may limit systemic exposure to alkyl-paraben esters in vivo.

Comparison of in Vitro Models for the Prediction of Compound Absorption across the Human Intestinal Mucosa

Several in vitro assays have been developed to evaluate the gastrointestinal absorption of compounds. Our aim was to compare 3 of these methods: 1) the bio-mimetic artificial membrane permeability assay (BAMPA) method, which offers a high-throughput, noncellular approach to the measurement of passive transport; 2) the traditional Caco-2 cell assay, the use of which as a high-throughput tool is limited by the long cell differentiation time (21 days); and 3) The BioCoat high-throughput screening Caco-2 Assay System, which reduces Caco-2 cell differentiation to 3 days. The transport of known compounds (such as cephalexin, propranolol, or chlorothiazide) was studied at pH 7.4 and 6.5 in BAMPA and both Caco-2 cell models. Permeability data obtained was correlated to known values of human absorption. Best correlations (r = 0.9) were obtained at pH 6.5 for BAMPA and at pH 7.4 for the Caco-2 cells grown for 21 days. The Caco-2 BioCoat HTS Caco-2 Assay System does not seem to be adequate for the prediction of absorption. The overall results indicate that BAMPA and the 21-day Caco-2 system can be complementary for an accurate prediction of human intestinal absorption.

Permeation characteristics of a hydrophilic basic compound across a bio-mimetic artificial membrane

In the present study, the permeation characteristics of a hydrophilic basic compound (HBC) in a bio-mimetic parallel artificial membrane permeability assay (bio-mimetic PAMPA) were investigated in detail. The bio-mimetic PAMPA membrane was constructed on a hydrophobic filter by impregnating a lipid solution consisting of phosphatidylcholine (0.8%, w/w), phosphatidylethanolamine (0.8%, w/w), phosphatidylserine (0.2%, w/w), phosphatidylinositol (0.2%, w/w), cholesterol (1.0%, w/w), and 1,7-octadiene (97.0%, w/w). The pH–permeability curve (pH 3–10), the effect of lipid composition, concentration dependency (0.02–2.00 mM), and inhibition by other cationic compounds, were investigated for several HBCs. Ketoprofen and methylchlorpromazine were also employed as an acidic and a quaternary ammonium compound, respectively. At pH 3–6, the permeability of timolol, a HBC, was higher than expected from the pH-partition hypothesis, especially in the PI-containing membrane, whereas the pH–permeability curve of ketoprofen followed the pH-partition hypothesis. Permeation of HBC was saturable and inhibited by basic and quaternary ammonium compounds. Similar results were also found for methylchlorpromazine. The permeation characteristics of HBC observed in the present study are not usually expected in a passive permeation process across an artificial membrane. The participation of facilitated permeation of cationic species was suggested, in addition to a simple passive diffusion of un-dissociated species. Ion pair transport was suggested as a possible permeation mechanism of cationic species. However, further investigation is necessary to clarify the reason for the permeation characteristics of HBC.

Prediction of human intestinal permeability using artificial membrane permeability

The purpose of the present study was to examine a correlation between the human intestinal permeability ( P eff) and the bio-mimetic artificial membrane permeability corrected by the paracellular pathway model based on the Renkin function ( P PAMPA-PP-RF) and to construct a prediction scheme. The effect of the unstirred water layer was incorporated to the prediction scheme. Eighteen P eff values of passively absorbed drugs were employed for the analysis. The correlation coefficient (CC) between the predicted and observed log P eff was 0.91. P eff of furosemide, hydrochlorothiazide and creatinine were underestimated by P PAMPA-PP-RF. When these compounds were excluded, CC was 0.97. Without the correction for the paracellular pathway, P eff of small, cationic and hydrophilic compounds were underestimated. Therefore, P PAMPA-PP-RF was found to be an adequate in vitro surrogate for P eff.

Prediction of passive intestinal absorption using bio-mimetic artificial membrane permeation assay and the paracellular pathway model

The purpose of this study was to construct and examine the prediction model for total passive permeation through the intestinal membrane. The paracellular pathway prediction model based on Renkin function (PP-RF) was combined with a bio-mimetic artificial membrane permeation assay (BAMPA), which is an in vitro method to predict transcellular pathway permeation, to construct the prediction model (BAMPA-PP-RF model). The parameters of the BAMPA-PP-RF model, e.g. apparent pore radius and potential drop of the paracellular pathway, were calculated from BAMPA permeability, the dissociation constant, the molecular radius and the fraction of a dose absorbed in humans consisting of 80 structurally diverse compounds. The apparent pore radius and the apparent potential drop obtained in this study were 5.61–5.65 Å and 75–86 mV, respectively, and these were in accordance with the previously reported values. The mean square root error of the BAMPA-PP-RF model was 13–14%. The BAMPA-PP-RF model was shown to be able to predict the total passive permeability more adequately than BAMPA alone.

Optimized conditions of bio-mimetic artificial membrane permeation assay

Effects of pH and co-solvents on the bio-mimetic artificial membrane permeation assay were investigated to determine the optimal conditions for the prediction of oral absorption. The permeability ( P am) of 33 structurally diverse drugs to the PC/PE/PS/PI/CHO/1,7-octadiene membrane system (bio-mimetic lipid (BML) membrane) was measured at pH 5.5, 6.5, and 7.4. The pH dependence of P am was in accordance with the pH partition theory. The better prediction of oral absorption (fraction of a dose absorbed) was shown under the pH 5.5 condition ( r=0.866, n=25) and/or pH 6.5 ( r=0.865, n=28), rather than pH 7.4 ( r=0.767, n=24). Then, the appropriate conditions for determining the permeability of poorly soluble compounds were examined. Dimethysulfoxide (DMSO), ethanol (EtOH) and polyoxyethyleneglycol 400 (PEG 400) were added up to 30% to the transport medium as solubilizers. DMSO, EtOH and PEG 400 decreased P am of hydrocortisone and propranolol. For example, DMSO (30%) decreased P am of hydrocortisone by 60% and by 70% in the case of propranolol. DMSO and PEG 400 also decreased P am of ketoprofen. In contrast, EtOH produced an opposite effect on permeability, i.e. an increased P am of ketoprofen. Therefore, the high concentration of these co-solvents could lead to the under- or overestimation of drug permeability.