Influence of electric pulse characteristics on the cellular internalization of chemotherapeutic drugs and cell survival fraction in electroporated and vasoconstricted cancer tissues using boundary element techniques.

Electroporation has emerged as a suitable technique to induce the pore formation in the cell membrane of cancer tissues, facilitating the cellular internalization of chemotherapeutic drugs. An adequate selection of the electric pulse characteristics is crucial to guarantee the efficiency of this technique, minimizing the adverse effects. In the present work, the dual reciprocity boundary element method (DR-BEM) is applied for the simulation of drug transport in the extracellular and intracellular space of cancer tissues subjected to the application of controlled electric pulses, using a continuum tumour cord approach, and considering both the electro-permeabilization and vasoconstriction phenomena. The developed DR-BEM algorithm is validated with numerical and experimental results previously published, obtaining a satisfactory accuracy and convergence. Using the DR-BEM code, a study about the influence of the magnitude of electric field (E) and pulse spacing (dpulses) on the time behavior and spatial distribution of the internalized drug, as well as on the cell survival fraction, is carried out. In general, the change of drug concentration, drug exposure and cell survival fraction with the parameters E and dpulses is ruled by two important factors: the balance between the electro-permeabilization and vasoconstriction phenomena, and the relative importance of the sources of cell death (electric pulses and drug cytotoxicity); these two factors, in turn, significantly depend on the reversible and irreversible thresholds considered for the electric field.

Yeast Double Transporter Gene Deletion Library for Identification of Xenobiotic Carriers in Low or High Throughput.

The routes of uptake and efflux should be considered when developing new drugs so that they can effectively address their intracellular targets. As a general rule, drugs appear to enter cells via protein carriers that normally carry nutrients or metabolites. A previously developed pipeline that searched for drug transporters using Saccharomyces cerevisiae mutants carrying single-gene deletions identified import routes for most compounds tested. However, due to the redundancy of transporter functions, we propose that this methodology can be improved by utilizing double mutant strains in both low- and high-throughput screens. We constructed a library of over 14,000 strains harboring double deletions of genes encoding 122 nonessential plasma membrane transporters and performed low- and high-throughput screens identifying possible drug import routes for 23 compounds. In addition, the high-throughput assay enabled the identification of putative efflux routes for 21 compounds. Focusing on azole antifungals, we were able to identify the involvement of the myo-inositol transporter, Itr1p, in the uptake of these molecules and to confirm the role of Pdr5p in their export. IMPORTANCE Our library of double transporter deletion strains is a powerful tool for rapid identification of potential drug import and export routes, which can aid in determining the chemical groups necessary for transport via specific carriers. This information may be translated into a better design of drugs for optimal absorption by target tissues and the development of drugs whose utility is less likely to be compromised by the selection of resistant mutants.

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.

Crude plant extract versus single compounds for vitiligo treatment: Ex vivo intestinal permeability assessment on Brosimum gaudichaudii Trécul.

Roots´ bark extract of Brosimum gaudichaudii Trécul (EBGT) is traditionally used for photochemotherapy of vitiligo due to the presence of furanocoumarins psoralen (PSO) and 5-methoxypsoralen (5-MOP) as major compounds. Though plant extracts may provide additional highly permeable psoralens-like substances which may act synergically on vitiligo's therapy. Thus, the aim of this work was to develop an LC-MS/MS method for screening new highly permeable furanocoumarins from B. gaudichaudii and to compare biomarkers permeability and solubility provided as single compounds or as crude extract, according to BCS. An optimized LC-MS/MS method showed twelve permeable and bioactive compounds, among which 9 furanocoumarins, 2 pyranocoumarins and 1 dihydrocinnamic acid derivative were detected in EBGT samples. Solubility of PSO and 5-MOP was found to be, respectively, six- and eleven-fold higher in crude extract than as pure compounds. Permeability (Papp) of PSO and 5-MOP in EBGT were higher than metoprolol, the low/high BCS permeability class boundary reference compound. Hence, both biomarkers were considered as highly permeable (BCS2) compounds. Their permeability were concentration-dependent displaying values from 30.26 ± 5.13-8.21 ± 2.16 × 10-6 cm/s and 10.72 ± 1.73-6.07 ± 1.27 × 10-6 cm/s, respectively, over a wide range (2.3-200.0 mg mL-1). Thus, a carrier-mediated absorption process is suggested as the main mechanism. Accordingly, all additional permeated coumarins, identified by LC-MS/MS, showed to be at comparable amount of biomarkers in the permeated samples inferring similar high permeability rate. Moreover, biomarkers and other highly absorbable and bioactive linear furanocoumarins from EBGT may be used for vitiligo´s photochemotherapy. Taken together, these findings bring additional evidences for using crude plant extract when aiming synergistic effects of bioactive compounds on melanogenic therapies.

Simulation of the influence of voltage level and pulse spacing on the efficiency, aggressiveness and uniformity of the electroporation process in tissues using meshless techniques.

Electroporation is a widely used method consisting of application of high-voltage, short-duration electric pulses to increase cell membrane permeability, allowing cellular internalization of medications. In this work, the influence of two primary parameters, voltage level (V) and pulse spacing (N), on electroporation efficiency, uniformity and aggressiveness, as quantified by the total mass transport to viable cells, intracellular concentration gradients and an aggressiveness factor introduced here, is studied by means of numerical simulations of drug transport in electroporated tissues. The global method of approximate particular solutions (Global MAPS) is used to solve the governing equations, together with domain scaling, singular value decomposition and smoothing algorithms, to address the ill-conditioning of the final system and suppress small scale oscillations. The accuracy of Global MAPS is evaluated by comparing the initial extracellular concentration, Ce , and final intracellular concentration, Ci , with previous finite volume method results, obtaining similar behavior of Ce and Ci along the tissue domain, with some differences for Ci in high-gradient zones. According to the Global MAPS results, the influence of V and N on Ci is only significant over a certain range, within which the largest drug transport to viable cells occurs. In general, both electroporation efficiency and aggressiveness change in nonuniform manner with V and decrease with N, whereas the electroporation uniformity decreases as V increases and N decreases. The contour plots obtained here can be considered useful tools to compare electroporation-based treatments in terms of their efficiency, aggressiveness and uniformity, assisting in the selection of a suitable treatment plan for cancer.

Evaluation of potential herbal-drug interactions of a standardized propolis extract (EPP-AF®) using an in vivo cocktail approach.

ETHNOPHARMACOLOGICAL RELEVANCE: Propolis has been employed extensively in many cultures since ancient times as antiseptic, wound healing, anti-pyretic and others due to its biological and pharmacological properties, such as immunomodulatory, antitumor, anti-inflammatory, antioxidant, antibacterial, antiviral, antifungal, antiparasite activities. But despite its broad and traditional use, there is little knowledge about its potential interaction with prescription drugs. AIM OF THE STUDY: The main objective of this work was to study the potential herbal-drug interactions (HDIs) of EPP-AF® using an in vivo assay with a cocktail approach. MATERIALS AND METHODS: Subtherapeutic doses of caffeine, losartan, omeprazole, metoprolol, midazolam and fexofenadine were used. Sixteen healthy adult volunteers were investigated before and after exposure to orally administered 125 mg/8 h (375 mg/day) EPP-AF® for 15 days. Pharmacokinetic parameters were calculated based on plasma concentration versus time (AUC) curves. RESULTS: After exposure to EPP-AF®, it was observed decrease in the AUC0-∞ of fexofenadine, caffeine and losartan of approximately 18% (62.20 × 51.00 h.ng/mL), 8% (1085 × 999 h.ng/mL) and 13% (9.01 × 7.86 h.ng/mL), respectively, with all 90% CIs within the equivalence range of 0.80-1.25. On the other hand, omeprazole and midazolam exhibited an increase in AUC0-∞ of, respectively, approximately 18% (18.90 × 22.30 h.ng/mL) and 14% (1.25 × 1.43 h.ng/mL), with the upper bounds of 90% CIs slightly above 1.25. Changes in pharmacokinetics of metoprolol or its metabolite α-hydroxymetoprolol were not statistically significant and their 90% CIs were within the equivalence range of 0.80-1.25. CONCLUSIONS: In conclusion, our study shows that EPP-AF® does not clinically change CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A activities, once, despite statistical significant, the magnitude of the changes in AUC values after EPP-AF® were all below 20% and therefore may be considered safe regarding potential interactions involving these enzymes. Besides, to the best of our knowledge this is the first study to assess potential HDIs with propolis.

ABC Transporters in Extrahepatic Tissues: Pharmacological Regulation in Heart and Intestine.

ATP binding cassette (ABC) transporters are transmembrane proteins expressed in secretory epithelia like the liver, kidneys and intestine, in the epithelia exhibiting barrier function such as the blood-brain barrier and placenta, and to a much lesser extent, in tissues like reproductive organs, lungs, heart and pancreas, among others. They regulate internal distribution of endogenous metabolites and xenobiotics including drugs of therapeutic use and also participate in their elimination from the body. We here describe the function and regulation of ABC transporters in the heart and small intestine, as examples of extrahepatic tissues, in which ABC proteins play clearly different roles. In the heart, they are involved in tissue pathogenesis as well as in protecting this organ against toxic compounds and druginduced oxidative stress. The small intestine is highly exposed to therapeutic drugs taken orally and, consequently, ABC transporters localized on its surface strongly influence drug absorption and pharmacokinetics. Examples of the ABC proteins currently described are Multidrug Resistance-associated Proteins 1 and 2 (MRP1 and 2) for heart and small intestine, respectively, and P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) for both organs.

Delivering Resveratrol on the Buccal Mucosa Using Mucoadhesive Tablets: A Potential Treatment Strategy for Inflammatory Oral Lesions.

BACKGROUND: Resveratrol is a polyphenol that has gained momentum in therapeutics in the last few years. OBJECTIVE: In this study, we hypothesised that resveratrol could act prophylactically and/or treat inflammatory lesions of the oral cavity after being delivered by two different formulations of buccal mucoadhesive tablets (F1 and F2, which differed in terms of viscosity agents used). METHODS: This hypothesis was assessed through permeation studies, to verify diffusion of the drug through the buccal mucosa using a porcine model to predict human in vivo behaviour. RESULTS: F2 (tablet with less viscosity agent) presented better permeation than F1, but the amount of drug that crossed the mucosa was still low compared to the amount retained within it (35.90 µg found in the receptor medium and 15.63 mg quantified within the mucosa). CONCLUSION: This accounted for a local effect rather than a systemic one, which is desirable for local processes, such as oral mucositis, lichen planus, erythema multiforme, nicotinic stomatitis, recurrent aphthous stomatitis, among others. In this sense, resveratrol-loaded mucoadhesive tablets appear to be a prominent alternative to prevent and/or cure inflammatory lesions of the oral cavity.

Modulation of expression and activity of intestinal multidrug resistance-associated protein 2 by xenobiotics.

The multidrug resistance-associated protein 2 (MRP2/ABCC2) is a transporter that belongs to the ATP-binding cassette (ABC) superfamily. In the intestine, it is localized to the apical membrane of the enterocyte and plays a key role in limiting the absorption of xenobiotics incorporated orally. MRP2 may also play a role in systemic clearance of xenobiotics available from the serosal side of the intestine. MRP2 transports a wide range of substrates, mainly organic anions conjugated with glucuronic acid, glutathione and sulfate and its expression can be modulated by xenobiotics at transcriptional- and post-transcriptional levels. Transcriptional regulation is usually mediated by a group of nuclear receptors. The pregnane X receptor (PXR) is a major member of this group. Relevant drugs described to up-regulate intestinal MRP2 via PXR are rifampicin, spironolactone and carbamazepine, among others. The constitutive androstane receptor (CAR, NR1I3) was also reported to modulate MRP2 expression, phenobarbital being a typical activator. Dietary compounds, including micronutrients and other natural products, are also capable of regulating intestinal MRP2 expression transcriptionally. We have given them particular attention since the composition of the food ingested daily is not necessarily supervised and may result in interactions with therapeutic drugs. Post-transcriptional regulation of MRP2 activity by xenobiotics, e.g. as a consequence of inhibitory actions, is also described in this review. Unfortunately, only few studies report on drug-drug or nutrient-drug interactions as a consequence of modulation of intestinal MRP2 activity by xenobiotics. Future clinical studies are expected to identify additional interactions resulting in changes in efficacy or safety of therapeutic drugs.

Single-Pass Intestinal Perfusion (SPIP) and prediction of fraction absorbed and permeability in humans: A study with antiretroviral drugs.

In recent years, the prediction of oral drug absorption in humans has been a challenge for researchers and many techniques for permeability studies have been developed for several purposes, including biowaiver processes. The Single-Pass Intestinal Perfusion (SPIP) method performed in rats can provide permeability results closest to in vivo condition. The purpose of the present study was to evaluate the intestinal permeability of the antiretroviral drugs lamivudine, stavudine and zidovudine using the SPIP method in rats and to predict their permeability (Peff,humans) and fraction absorbed (Fa) in humans. Metoprolol and fluorescein were used as marker compounds of high and low permeability, respectively. The effective permeability (Peff) results showed that stavudine and zidovudine have high permeability characteristics while lamivudine presented the lowest result. From Peff values obtained in rats, the Peff,humans and Fa were calculated. The use of SPIP in rats and calculations for absorption prediction in humans may indicate the transport mechanisms and/or pre-systemic metabolism involved on permeation processes of drugs, since this model is the closest to in vivo conditions.

Functional Validation of ABCA3 as a Miltefosine Transporter in Human Macrophages: IMPACT ON INTRACELLULAR SURVIVAL OF LEISHMANIA (VIANNIA) PANAMENSIS.

Within its mammalian host, Leishmania resides and replicates as an intracellular parasite. The direct activity of antileishmanials must therefore depend on intracellular drug transport, metabolism, and accumulation within the host cell. In this study, we explored the role of human macrophage transporters in the intracellular accumulation and antileishmanial activity of miltefosine (MLF), the only oral drug available for the treatment of visceral and cutaneous leishmaniasis (CL). Membrane transporter gene expression in primary human macrophages infected in vitro with Leishmania Viannia panamensis and exposed to MLF showed modulation of ABC and solute liquid carrier transporters gene transcripts. Among these, ABCA3, a lipid transporter, was significantly induced after exposure to MLF, and this induction was confirmed in primary macrophages from CL patients. Functional validation of MLF as a substrate for ABCA3 was performed by shRNA gene knockdown (KD) in THP-1 monocytes. Intracellular accumulation of radiolabeled MLF was significantly higher in ABCA3(KD) macrophages. ABCA3(KD) resulted in increased cytotoxicity induced by MLF exposure. ABCA3 gene expression inversely correlated with intracellular MLF content in primary macrophages from CL patients. ABCA3(KD) reduced parasite survival during macrophage infection with an L. V. panamensis strain exhibiting low in vitro susceptibility to MLF. Confocal microscopy showed ABCA3 to be located in the cell membrane of resting macrophages and in intracellular compartments in L. V. panamensis-infected cells. These results provide evidence of ABCA3 as an MLF efflux transporter in human macrophages and support its role in the direct antileishmanial effect of this alkylphosphocholine drug.

Modeling of Progesterone Release from Poly(3-Hydroxybutyrate) (PHB) Membranes.

Poly(3-hydroxybutyrate) (PHB) biodegradable polymeric membranes were evaluated as platform for progesterone (Prg)-controlled release. In the design of new drug delivery systems, it is important to understand the mass transport mechanism involved, as well as predict the process kinetics. Drug release experiments were conducted and the experimental results were evaluated using engineering approaches that were extrapolated to the pharmaceutical field by our research group. Membranes were loaded with different Prg concentrations and characterized by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and Fourier transform infrared spectroscopy (FTIR). SEM images showed that membranes have a dense structure before and after the progesterone addition. DSC and FTIR allowed determining the influence of the therapeutic agent in the membrane properties. The in vitro experiments were performed using two different techniques: (A) returning the sample to the receptor solution (constant volume of the delivery medium) and (B) extracting total volume of the receptor solution. In this work, we present a simple and accurate "lumped" second-order kinetic model. This lumped model considers the different mass transport steps involved in drug release systems. The model fits very well the experimental data using any of the two experimental procedures, in the range 0 ≤ t ≤ ∞ or 0 ≤ M t ≤ M ∞. The drug release analysis using our proposed approaches is relevant for establishing in vitro-in vivo correlations in future tests in animals.

New structure-activity relationships of chalcone inhibitors of breast cancer resistance protein: polyspecificity toward inhibition and critical substitutions against cytotoxicity.

Adenosine triphosphate-binding cassette subfamily G member 2 (ABCG2) plays a major role in cancer cell multidrug resistance, which contributes to low efficacy of chemotherapy. Chalcones were recently found to be potent and specific inhibitors, but unfortunately display a significant cytotoxicity. A cellular screening against ABCG2-mediated mitoxantrone efflux was performed here by flow cytometry on 54 chalcone derivatives from three different series with a wide panel of substituents. The identified leads, with submicromolar IC50 (half maximal inhibitory concentration) values, showed that the previously identified 2'-OH-4',6'-dimethoxyphenyl, as A-ring, could be efficiently replaced by a 2'-naphthyl group, or a 3',4'-methylenedioxyphenyl with lower affinity. Such a structural variability indicates 3polyspecificity of the multidrug transporter for inhibitors. At least two methoxyl groups were necessary on B-ring for optimal inhibition, but substitution at positions 3, 4, and 5 induced cytotoxicity. The presence of a large O-benzyl substituent at position 4 and a 2'-naphthyl as A-ring markedly decreased the cytotoxicity, giving a high therapeutic ratio, which constitutes a critical requirement for future in-vivo assays in animal models.

Evaluation of antimicrobial activity and cell viability of Aloe vera sponges

Background: Aloe vera L., member of the Liliaceae family, has been shown to stimulate cell proliferation and contribute to healing and angiogenesis, has anti-bacterial, anti-fungal and anti-inflammatory activity. In addition, Aloe vera can be used as a support for drug transport. Our objective is to evaluate antimicrobial activity and cytotoxicity of sponges of Aloe vera L. for use as a carrying support of drugs. Results: In this work, sponge of free Aloe vera (AV) loaded with amoxicillin (AMX) or nystatin (NYS) at 1% w/w, were prepared and physico-chemically characterized via X-ray diffraction, Fourier Transform Infrared Spectroscopy and thermal analysis. Antimicrobial potency of AV sponge alone, loaded with AMX or NYS, against strains of Streptococcus mutans, Staphylococcus aureus, Aggregatibacter actinomycetemcomitans, Enterococcus faecalis and Candida albicans was determined. Osteoblasts and human gingival fibroblasts were cultivated on AV, Aloe vera loaded with amoxicillin (AV/AMX) and Aloe vera loaded with nystatin (AV/NYS) and cellular viability was assessed. The physico-chemical characterization performed suggested that the loaded drugs were dispersed in the sponge and those interactions between the AV sponge and the loaded drugs were weak. Furthermore, AV loaded with AMX or NYS demonstrated antimicrobial potency and osteoblasts and fibroblasts were viable after 24 hrs on free AV, and AV loaded with AMX or NYS. Conclusions: Our results indicate that sponges of free AV, loaded with AMX or NYS, are biocompatible and exhibit antimicrobial activity.