Formulation study of PLGA in situ films for topical delivery of salicylates.

A film-forming system (FFS) represents a convenient topical dosage form for drug delivery. In this study, a non-commercial poly(lactic-co-glycolic acid) (PLGA) was chosen to formulate an FFS containing salicylic acid (SA) and methyl salicylate (MS). This unique combination is advantageous from a therapeutic point of view, as it enabled modified salicylate release. It is beneficial from a technological perspective too, because it improved thermal, rheological, and adhesive properties of the in situ film. DSC revealed complete dissolution of SA and good miscibility of MS with the polymer. MS also ensures optimal viscoelastic and adhesive properties of the film, leading to prolonged and sustained drug release. The hydrolysis of MS to active SA was very slow at skin pH 5.5, but it apparently occurred at physiological pH 7.4. The film structure is homogeneous without cracks, unlike some commercial preparations. The dissolution study of salicylates revealed different courses in their release and the influence of MS concentration in the film. The formulated PLGA-based FFS containing 5 % SA and 10 % MS is promising for sustained and prolonged local delivery of salicylates, used mainly for keratolytic and anti-inflammatory actions and pain relief.

Cannabidiol disrupts tryptophan metabolism in the human term placenta.

The increasing use of cannabis during pregnancy raises concerns about its impact on fetal development. While cannabidiol (CBD) shows therapeutic promise, its effects during pregnancy remain uncertain. We investigated CBD's influence on tryptophan (TRP) metabolism in the human placenta. TRP is an essential amino acid that is metabolized via the serotonin and kynurenine (KYN) pathways, which are critical for fetal neurodevelopment. We used human term villous placental explants, an advanced ex vivo model, to study CBD's impact on key TRP metabolic enzymes. In addition, vesicles isolated from the microvillous membrane (MVM) of the human placenta were used to assess CBD's effect on placental serotonin uptake. Explants were exposed to CBD at therapeutic (0.1, 1, 2.5 µg/ml) and non-therapeutic (20 and 40 µg/ml) concentrations to determine its effects on the gene and protein expression of key enzymes in TRP metabolism and metabolite release. CBD upregulated TRP hydroxylase (TPH) and downregulated monoamine oxidase (MAO-A), resulting in reduced levels of 5-hydroxyindoleacetic acid (HIAA). It also downregulated serotonin transporter expression and inhibited serotonin transport across the MVM by up to 60% while simultaneously enhancing TRP metabolism via the kynurenine pathway by upregulating indoleamine-pyrrole 2,3-dioxygenase (IDO-1). Among kynurenine pathway enzymes, kynurenine 3 monooxygenase (KMO) was upregulated while kynurenine aminotransferase 1 (KAT-1) was downregulated; the former is associated with neurotoxic metabolite production, while the latter is linked to reduced neuroprotective metabolite levels. Overall, these results indicate that CBD modulates TRP catabolism in the human placenta, potentially disrupting the tightly regulated homeostasis of the serotonin and KYN pathways.

Pathological shifts in tryptophan metabolism in human term placenta exposed to LPS or poly I:C†.

Maternal immune activation during pregnancy is a risk factor for offspring neuropsychiatric disorders. Among the mechanistic pathways by which maternal inflammation can affect fetal brain development and programming, those involving tryptophan (TRP) metabolism have drawn attention because various TRP metabolites have neuroactive properties. This study evaluates the effect of bacterial (lipopolysaccharides/LPS) and viral (polyinosinic:polycytidylic acid/poly I:C) placental infection on TRP metabolism using an ex vivo model. Human placenta explants were exposed to LPS or poly I:C, and the release of TRP metabolites was analyzed together with the expression of related genes and proteins and the functional activity of key enzymes in TRP metabolism. The rate-limiting enzyme in the serotonin pathway, tryptophan hydroxylase, showed reduced expression and functional activity in explants exposed to LPS or poly I:C. Conversely, the rate-limiting enzyme in the kynurenine pathway, indoleamine dioxygenase, exhibited increased activity, gene, and protein expression, suggesting that placental infection mainly promotes TRP metabolism via the kynurenine (KYN) pathway. Furthermore, we observed that treatment with LPS or poly I:C increased activity in the kynurenine monooxygenase branch of the KYN pathway. We conclude that placental infection impairs TRP homeostasis, resulting in decreased production of serotonin and an imbalance in the ratio between quinolinic acid and kynurenic acid. This disrupted homeostasis may eventually expose the fetus to suboptimal/toxic levels of neuroactive molecules and impair fetal brain development.

Characterization of a human placental clearance system to regulate serotonin levels in the fetoplacental unit.

BACKGROUND: Serotonin (5-HT) is a biogenic monoamine with diverse functions in multiple human organs and tissues. During pregnancy, tightly regulated levels of 5-HT in the fetoplacental unit are critical for proper placental functions, fetal development, and programming. Despite being a non-neuronal organ, the placenta expresses a suite of homeostatic proteins, membrane transporters and metabolizing enzymes, to regulate monoamine levels. We hypothesized that placental 5-HT clearance is important for maintaining 5-HT levels in the fetoplacental unit. We therefore investigated placental 5-HT uptake from the umbilical circulation at physiological and supraphysiological levels as well as placental metabolism of 5-HT to 5-hydroxyindoleacetic acid (5-HIAA) and 5-HIAA efflux from trophoblast cells. METHODS: We employed a systematic approach using advanced organ-, tissue-, and cellular-level models of the human placenta to investigate the transport and metabolism of 5-HT in the fetoplacental unit. Human placentas from uncomplicated term pregnancies were used for perfusion studies, culturing explants, and isolating primary trophoblast cells. RESULTS: Using the dually perfused placenta, we observed a high and concentration-dependent placental extraction of 5-HT from the fetal circulation. Subsequently, within the placenta, 5-HT was metabolized to 5-hydroxyindoleacetic acid (5-HIAA), which was then unidirectionally excreted to the maternal circulation. In the explant cultures and primary trophoblast cells, we show concentration- and inhibitor-dependent 5-HT uptake and metabolism and subsequent 5-HIAA release into the media. Droplet digital PCR revealed that the dominant gene in all models was MAO-A, supporting the crucial role of 5-HT metabolism in placental 5-HT clearance. CONCLUSIONS: Taken together, we present transcriptional and functional evidence that the human placenta has an efficient 5-HT clearance system involving (1) removal of 5-HT from the fetal circulation by OCT3, (2) metabolism to 5-HIAA by MAO-A, and (3) selective 5-HIAA excretion to the maternal circulation via the MRP2 transporter. This synchronized mechanism is critical for regulating 5-HT in the fetoplacental unit; however, it can be compromised by external insults such as antidepressant drugs.

Dynamics of Tryptophan Metabolic Pathways in Human Placenta and Placental-Derived Cells: Effect of Gestation Age and Trophoblast Differentiation.

L-Tryptophan is an essential amino acid and a precursor of several physiologically active metabolites. In the placenta, the serotonin and kynurenine metabolic pathways of tryptophan metabolism have been identified, giving rise to various molecules of neuroactive or immunoprotective properties, such as serotonin, melatonin, kynurenine, kynurenic acid, or quinolinic acid. Current literature suggests that optimal levels of these molecules in the fetoplacental unit are crucial for proper placenta functions, fetal development and programming. Placenta is a unique endocrine organ that, being equipped with a battery of biotransformation enzymes and transporters, precisely orchestrates homeostasis of tryptophan metabolic pathways. However, because pregnancy is a dynamic process and placental/fetal needs are continuously changing throughout gestation, placenta must adapt to these changes and ensure proper communication in the feto-placental unit. Therefore, in this study we investigated alterations of placental tryptophan metabolic pathways throughout gestation. Quantitative polymerase chain reaction (PCR) analysis of 21 selected genes was carried out in first trimester (n = 13) and term (n = 32) placentas. Heatmap analysis with hierarchical clustering revealed differential gene expression of serotonin and kynurenine pathways across gestation. Subsequently, digital droplet PCR, Western blot, and functional analyses of the rate-limiting enzymes suggest preferential serotonin synthesis early in pregnancy with a switch to kynurenine production toward term. Correspondingly, increased function and/or protein expression of serotonin degrading enzyme and transporters at term indicates efficient placental uptake and metabolic degradation of serotonin. Lastly, gene expression analysis in choriocarcinoma-derived cell lines (BeWo, BeWo b30, JEG-3) revealed dissimilar expression patterns and divergent effect of syncytialization compared to primary trophoblast cells isolated from human term placentas; these findings show that the commonly used in vitro placental models are not suitable to study placental handling of tryptophan. Altogether, our data provide the first comprehensive evidence of changes in placental homeostasis of tryptophan and its metabolites as a function of gestational age, which is critical for proper placental function and fetal development.

Profiling of Tryptophan Metabolic Pathways in the Rat Fetoplacental Unit During Gestation.

Placental homeostasis of tryptophan is essential for fetal development and programming. The two main metabolic pathways (serotonin and kynurenine) produce bioactive metabolites with immunosuppressive, neurotoxic, or neuroprotective properties and their concentrations in the fetoplacental unit must be tightly regulated throughout gestation. Here, we investigated the expression/function of key enzymes/transporters involved in tryptophan pathways during mid-to-late gestation in rat placenta and fetal organs. Quantitative PCR and heatmap analysis revealed the differential expression of several genes involved in serotonin and kynurenine pathways. To identify the flux of substrates through these pathways, Droplet Digital PCR, western blot, and functional analyses were carried out for the rate-limiting enzymes and transporters. Our findings show that placental tryptophan metabolism to serotonin is crucial in mid-gestation, with a subsequent switch to fetal serotonin synthesis. Concurrently, at term, the close interplay between transporters and metabolizing enzymes of both placenta and fetal organs orchestrates serotonin homeostasis and prevents hyper/hypo-serotonemia. On the other hand, the placental production of kynurenine increases during pregnancy, with a low contribution of fetal organs throughout gestation. Any external insult to this tightly regulated harmony of transporters and enzymes within the fetoplacental unit may affect optimal in utero conditions and have a negative impact on fetal programming.

Serotonin homeostasis in the materno-foetal interface at term: Role of transporters (SERT/SLC6A4 and OCT3/SLC22A3) and monoamine oxidase A (MAO-A) in uptake and degradation of serotonin by human and rat term placenta.

AIM: Serotonin is crucial for proper foetal development, and the placenta has been described as a 'donor' of serotonin for the embryo/foetus. However, in later stages of gestation the foetus produces its own serotonin from maternally-derived tryptophan and placental supply is no longer needed. We propose a novel model of serotonin homeostasis in the term placenta with special focus on the protective role of organic cation transporter 3 (OCT3/SLC22A3). METHODS: Dually perfused rat term placenta was employed to quantify serotonin/tryptophan transport and metabolism. Placental membrane vesicles isolated from human term placenta were used to characterize serotonin transporters on both sides of the syncytiotrophoblast. RESULTS: We obtained the first evidence that serotonin is massively taken up from the foetal circulation by OCT3. This uptake is concentration-dependent and inhibitable by OCT3 blockers of endogenous (glucocorticoids) or exogenous (pharmaceuticals) origin. Population analyses in rat placenta revealed that foetal sex influences placental extraction of serotonin from foetal circulation. Negligible foetal serotonin levels were detected in maternal-to-foetal serotonin/tryptophan transport and metabolic studies. CONCLUSION: We demonstrate that OCT3, localized on the foetus-facing membrane of syncytiotrophoblast, is an essential component of foeto-placental homeostasis of serotonin. Together with serotonin degrading enzyme, monoamine oxidase-A, this offers a protective mechanism against local vasoconstriction effects of serotonin in the placenta. However, this system may be compromised by OCT3 inhibitory molecules, such as glucocorticoids or antidepressants. Our findings open new avenues to explore previously unsuspected/unexplained complications during pregnancy including prenatal glucocorticoid excess and pharmacotherapeutic risks of treating pregnant women with OCT3 inhibitors.

Ureidopyrazine Derivatives: Synthesis and Biological Evaluation as Anti-Infectives and Abiotic Elicitors.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) has become a frequently deadly infection due to increasing antimicrobial resistance. This serious issue has driven efforts worldwide to discover new drugs effective against Mtb. One research area is the synthesis and evaluation of pyrazinamide derivatives as potential anti-TB drugs. In this paper we report the synthesis and biological evaluations of a series of ureidopyrazines. Compounds were synthesized by reacting alkyl/aryl isocyanates with aminopyrazine or with propyl 5-aminopyrazine-2-carboxylate. Reactions were performed in pressurized vials using a CEM Discover microwave reactor with a focused field. Purity and chemical structures of products were assessed, and the final compounds were tested in vitro for their antimycobacterial, antibacterial, and antifungal activities. Propyl 5-(3-phenylureido)pyrazine-2-carboxylate (compound 4, MICMtb = 1.56 µg/mL, 5.19 µM) and propyl 5-(3-(4-methoxyphenyl)ureido)pyrazine-2-carboxylate (compound 6, MICMtb = 6.25 µg/mL, 18.91 µM) had high antimycobacterial activity against Mtb H37Rv with no in vitro cytotoxicity on HepG2 cell line. Therefore 4 and 6 are suitable for further structural modifications that might improve their biological activity and physicochemical properties. Based on the structural similarity to 1-(2-chloropyridin-4-yl)-3-phenylurea, a known plant growth regulator, two selected compounds were evaluated for similar activity as abiotic elicitors.

Mucoadhesive plasticized system of branched poly(lactic-co-glycolic acid) with aciclovir.

Commercially available antibacterial semisolid preparations intended for topical application provide only short-term drug release. A sustained kinetics is possible by exploitation of a biodegradable polymer carrier. The purpose of this work is to formulate a mucoadhesive system with aciclovir (ACV) based on a solid molecular dispersion of this drug in poly(lactic-co-glycolic acid) branched on tripenterythritol (PLGA/T). The ACV incorporation into PLGA/T was carried out either by solvent method, or melting method, or plasticization method using various plasticizers. The drug-polymer miscibility, plasticizer efficiency and content of residual solvent were found out employing DSC. Viscosity was measured at the shear rate range from 0.10 to 10.00 s(-1) at three temperatures and data were analyzed by Newtonian model. The mucoadhesive properties were ascertained in the tensile test on a mucin substrate. The amount of ACV released was carried out in a wash-off dissolution test. The DSC results indicate a transformation of crystalline form of ACV into an amorphous dissolved in branched polyester carrier, and absence of methyl formate residuals in formulation. All the tested plasticizers are efficient at Tg depression and viscosity decrease. The non-conventional ethyl pyruvate possessing supportive anti-inflammatory activity was evaluated as the most suitable plasticizer. The ACV release was strongly dependent on the ethyl pyruvate concentration and lasted from 1 to 10 days. The formulated PLGA/T system with ACV exhibits increased adhesion to mucosal hydrophilic surfaces and prolonged ACV release controllable by degradation process and viscosity parameters.

Study of hydrophobic properties of biologically active open analogues of flavonoids.

Hydrophobicity can either be determined experimentally or predicted by means of commercially available programs. In the studies concerning biological activities of pyrazine analogues of chalcones, 3-(2-hydroxyphenyl)-1-(pyrazin-2-yl)prop-2-en-1-ones were more potent than the corresponding 3-(4-hydroxyphenyl)-1-(pyrazin-2-yl)prop-2-en-1-ones. As the difference in lipophilicity may be a factor responsible for the difference in the potency, R(M) values of the compounds were determined by RP-TLC and compared with logP values calculated by various commercially available programs. Important discrepancies were found between experimental and computational lipophilicity data. Therefore, we have tried to find a reliable method for calculating R(M) values from in silico derived molecular parameters. The R(M) values obtained with the chromatographic system consisting of Silufol UV 254 plates impregnated with silicon oil as the stationary phase and acetone-citrate buffer (pH=3) 50:50 (v/v) as the mobile phase correlated well with van der Waals volumes (V(W)) and hydration energies [Formula: see text] derived of molecular models calculated on RHF/AM1 level.