Micronized Palmitoylethanolamide, Hempseed Oil, and Maritime Pine Bark Dry Extract (Pelvipea®) for Pelvic Pain: An In Vitro Study for Urothelial Inflammation Treatment.

Urothelial inflammation plays a key role in the pathogenesis of chronic pelvic pain due to its origin in the bladder. The aim of this study was to evaluate the efficacy of a patent-pending formulation (Pelvipea®) composed of micronized palmitoylethanolamide (PEA), hempseed oil, and maritime pine bark dry extract in reducing urothelial inflammation, as well as the effect of each ingredient individually, in order to define the synergistic effect of the three ingredients. An in vitro bladder urothelium model composed of the T24 cell line was exposed to a conditioned media obtained by treating macrophage-differentiated THP-1 cells with different concentrations of the functional ingredients and a mixture of them in the presence of the pro-inflammatory stimulus of Escherichia coli. Cells exposed only to the inflammatory stimulus in the absence of pre-treatment were considered as a positive control for inflammation. The impact of each functional ingredient and their mixture on inflammation was evaluated by the determination of transcription factor NF-kB and of pro-inflammatory cytokine expression. Statistical analysis was performed using the t-test, comparing the mixture and the single ingredients for every condition tested. All results were reported as fold change (mean ± standard deviation), the ratio between the values obtained from the respective treatments for inflammation control. The three functional ingredients did not induce negative effects on THP-1 cell vitality. The levels of NF-kB were reduced following treatment with hempseed oil, maritime pine bark dry extract, and the mixture at all tested concentrations, and with micronized PEA from 25 to 200 µg/mL. Treatment with the mixture resulted in the lowest expression levels of interleukins (IL)-1ß, IL-6, and IL-8 compared to the single functional ingredients at a concentration of 230 µg/mL, with values of 0.08 (±0.00), 0.01 (±0.00), and 0.32 (±0.01), respectively. The mixture of micronized PEA, hempseed oil, and maritime pine bark dry extract (Pelvipea®) at 230 µg/mL showed the best efficacy in urothelial IL-1ß, IL-6, and IL-8 reduction compared with the singular components. This formulation may represent a promising therapeutic option to relieve painful symptoms originating in the bladder. However, in vivo studies are needed to confirm these results.

Effects of short chain fructo-oligosaccharides on selected skin bacteria.

The human skin microbiota plays a key role in the maintenance of healthy skin, ensuring protection and biological barrier by competing with pathogens and by closely communicating with the immune system. The development of approaches which preserve or restore the skin microbiota represents a novel target for skincare applications. Prebiotics could be applied to balance almost any microbial community to achieve advantageous effects. However, information about their effectiveness as skin microbiota modulators is limited. The objective of the current study was to evaluate the effects of short chain fructo-oligosaccharides (scFOS) from sugar beet (DP 3-5), well-recognised prebiotics, on some representative bacterial strains of the skin microbiota. We measured the growth and competitive activity of these specific bacteria for the use of scFOS as energy source in minimal medium and in a reconstructed human epithelium (RHE) in vitro model. In minimal growth medium, scFOS promoted and sustained the growth of Staphylococcus epidermidis up to 24 h, considered a beneficial skin commensal bacterium, while inhibiting both Cutibacterium acnes and Staphylococcus aureus growth, regarded as opportunistic pathogens. S. epidermidis showed the highest colonization potential and 1% scFOS was effective in shifting the competition in favour of S. epidermidis with respect to C. acnes in the RHE model. This latter effect was observed following 24 h of exposure, suggesting a long-term effect of scFOS in a highly skin dynamic environment. Therefore, scFOS could be effectively implemented in skincare formulations for recovering skin microbiota homeostasis.

Intestinal Absorption Study of a Granular Form of Ferric Pyrophosphate.

Iron deficiency is one of the most prevalent nutritional disorders worldwide. The standard treatment involves iron supplementation, but this task is challenging because of poor solubility and organoleptic issues. Moreover, the need to increase iron bioavailability represents a challenge for treating iron-related disorders. In this study, gastroresistance and iron intestinal absorption of an innovative granular formulation composed of ferric pyrophosphate, modified starch and phospholipids branded as Ferro Fosfosoma® was investigated. Gastroresistant properties were studied using standard protocols, and a bioaccessible fraction was obtained by exposing a food supplement to in vitro digestion. This fraction was used for investigating iron absorption in Caco-2 and human follicle-associated intestinal epithelium (FAE) models. Ferro Fosfosoma® showed an improved resistance to gastric digestion and higher intestinal absorption than ferric pyrophosphate salt used as a control in both models. In the FAE model, Ferro Fosfosoma® induces larger iron absorption than in the Caco-2 monolayer, most likely due to the transcytosis ability of M cells. The larger iron absorption in the Ferro Fosfosoma®-treated FAE model corresponds to higher ferritin level, proving physiological iron handling that was once delivered by granular formulation. Finally, the formulation did not induce any alterations in viability and barrier integrity. To conclude, Ferro Fosfosoma® favors iron absorption and ferritin expression, while preserving any adverse effects.

(Z)-Endoxifen and Early Recurrence of Breast Cancer: An Explorative Analysis in a Prospective Brazilian Study.

Adherence to treatment and use of co-medication, but also molecular factors such as CYP2D6 genotype, affect tamoxifen metabolism, with consequences for early breast cancer prognosis. In a prospective study of 149 tamoxifen-treated early-stage breast cancer patients from Brazil followed up for 5 years, we investigated the association between the active tamoxifen metabolite (Z)-endoxifen at 3 months and event-free survival (EFS) adjusted for clinico-pathological factors. Twenty-five patients (16.8%) had recurred or died at a median follow-up of 52.3 months. When we applied a putative 15 nM threshold used in previous independent studies, (Z)-endoxifen levels below the threshold showed an association with shorter EFS in univariate analysis (p = 0.045) and after adjustment for stage (HR 2.52; 95% CI 1.13-5.65; p = 0.024). However, modeling of plasma concentrations with splines instead of dichotomization did not verify a significant association with EFS (univariate analysis: p = 0.158; adjusted for stage: p = 0.117). Hence, in our small exploratory study, the link between impaired tamoxifen metabolism and early breast cancer recurrence could not be unanimously demonstrated. This inconsistency justifies larger modeling studies backed up by mechanistic pharmacodynamic analyses to shed new light on this suspected association and the stipulation of an appropriate predictive (Z)-endoxifen threshold.

An investigation on [5 fluorouracil and epigallocatechin-3-gallate] complex activity on HT-29 cell death and its stability in gastrointestinal fluid.

Recently an enhancement of the sensitivity of colorectal cancer (CRC) cells by 5-fluorouracil (5FU) due to the concurrent treatment with epigallocatechin-3-gallate (EGCG) has been found. In the present paper, to investigate on this aspect, adenocarcinoma cells HT29 were treated with 5FU, EGCG and an equimolar mixture of 5FU and EGCG ([5FU+EGCG]) and cell viability was determined. While 5FU exhibits a clear activity, EGCG alone does not express any activity. However by treating the cells with [5FU+EGCG] a strong effect of EGCG is evidenced: the sensitivity of HT29 cells to 5FU was increased by 12-fold. A simulation of the behavior of [5FU+EGCG] in different compartments of the gastrointestinal digestion model was also performed. 5FU and EGCG solubilized into a mixture of digestive fluids analyzed by mass spectrometry did not lead to signals of 5FU, EGCG and the related complex, while by diluting the solution they become detectable. On the contrary, when 5FU and EGCG are submitted to the step-by-step digestion model procedure, the analysis did not show the presence of 5FU, EGCG and [5FU+EGCG]. This behaviour could be ascribed to the instability of these compounds due to the too severe digestion conditions and/or to the complexity of the matrix which could lead in ESI conditions to the suppression of the signals of the analytes of interest.

Capryloyl glycine and soy isoflavonoids in hypertrichosis: An experimental and placebo-controlled clinical study.

BACKGROUND: The management of acquired hypertrichosis (HT) is based on the search of the causes and subsequent specific treatment. However, simultaneous hair removal is important. No single method for hair removal is appropriate for all patients and skin areas. Treatment options are actually limited and clinical results are often unsatisfactory. Ornithine decarboxylase 1 (ODC1), an enzyme present in hair follicles, is considered as a potential target to inhibit hair growth. Only eflornithine hydrochloride, an inhibitor of ODC1, showed to be partially effective in the management of acquired HT. AIMS: The aim of our study was to evaluate the potential inhibition of ODC1 activity by a cream containing 4% capryloyl glycine, an ODC1 inhibitor, and 1% glycine soy-fermented extract (soy isoflavonoids). Furthermore, we present the results of a placebo-controlled clinical study that evaluated the efficacy and tolerability of this cream. METHODS: The ODC1 activity was detected by measuring absorbance at 340 nm. In the presence of ODC1 inhibitors, absorbance decreases as a function of inhibition. Difluoromethylornithine (DFMO) was provided as an inhibitor control. ODC1 activity inhibition was expressed as percentage of control (untreated sample). All data were presented as mean ± standard deviation of three independent experiments. To determine if statistically significant differences between treatments were present, a t test analysis was performed. The differences between groups were considered significant at p < 0.05. Twelve Caucasian female adult patients, with HT located on the forearms, were enrolled. The study cream (product A) was applied twice/day for four months on the right forearm. A placebo cream (product B) was applied twice/day for four months on the left forearm. Clinical efficacy was evaluated by means of macrophotography. RESULTS: The cream significantly inhibited ODC1 activity (35.1 ± 0.5% inhibition, equivalent to a 64.9 ± 0.5% ODC1 activity). DFMO completely abolished the enzymatic activity (100 ± 5% inhibition, equivalent to 0 ± 5% ODC1 activity). All patients were considered evaluable. In 11 out of 12 patients (91.7%), who were treated with product A, an improvement was observed. No improvement was observed in patients treated with product B. The global assessment showed good efficacy in 7 patients (58.3%) and moderate efficacy in 5 patients (41.7%) treated with the product A. No efficacy was detected in patients treated with product B. CONCLUSIONS: The study cream showed to be effective in Caucasian, adult, female patients with hypertrichosis located on the forearms.

CD44 Targeting Mediated by Polymeric Nanoparticles and Combination of Chlorine TPCS2a-PDT and Docetaxel-Chemotherapy for Efficient Killing of Breast Differentiated and Stem Cancer Cells In Vitro.

The presence of rare but highly tumorigenic cancer stem cells (CSCs) within the tumors is recognized as one of the major reasons of failure of conventional chemotherapies, mainly attributed to the development of drug resistance and increasing metastatic potential. Here, we propose a therapeutic strategy based on the simultaneous delivery of docetaxel (DTX) and the photosensitizer meso-tetraphenyl chlorine disulfonate (TPCS2a) using hyaluronic acid (HA) coated polymeric nanoparticles (HA-NPs) for the targeting and killing of CD44 over-expressing breast cancer (BC) cells, both differentiated and CSCs (CD44high/CD24low population), thus combining chemotherapy and photodynamic therapy (PDT). Using the CD44high MDA-MB-231 and the CD44low MCF-7 cells, we demonstrated the occurrence of CD44-mediated uptake of HA-NPs both in monolayers and mammosphere cultures enriched in CSCs. Cell treatments showed that combination therapy using co-loaded NPs (HA@DTX/TPCS2a-NPs) had superior efficacy over monotherapies (HA@DTX-NPs or HA@TPCS2a-NPs) in reducing the self-renewal capacity, measured as mammosphere formation efficiency, and in eradicating the CSC population evaluated with aldehyde dehydrogenase activity assay and CD44/CD24 immunostaining. In summary, these in vitro studies demonstrated for the first time the potential of the combination of DTX-chemotherapy and TPCS2a-PDT for killing CSCs using properly designed NPs.

Keratin nanoparticles co-delivering Docetaxel and Chlorin e6 promote synergic interaction between chemo- and photo-dynamic therapies.

The combination of chemotherapy and photodynamic therapy (PDT) is considered a valuable strategy for increasing therapeutic response in cancer treatment, and the re-formulation of pharmaceuticals in biocompatible nanoparticles (NPs) is particularly appealing for the possibility of co-loading drugs exerting cytotoxicity by different mechanisms, with the aim to produce synergic effects. We report the in-water synthesis of a novel keratin-based nanoformulation for the co-delivery of the antimitotic Docetaxel (DTX) and the photosensitizer Chlorin e6 (Ce6). The drug-induced aggregation method allowed the formation of monodisperse NPs (DTX/Ce6-KNPs) with an average diameter of 133 nm and loaded with a drug ratio of 1:1.8 of Ce6 vs DTX. The efficacy of DTX/Ce6-KNPs was investigated in vitro in monolayers and spheroids of DTX-sensitive HeLa (HeLa-P) and DTX-resistant HeLa (HeLa-R) cells. In monolayers, the cytotoxic effects of DTX/Ce6-KNPs toward HeLa-P cells were comparable to those induced by free DTX + Ce6, while in HeLa-R cells the drug co-loading in KNPs produced synergic interaction between chemotherapy and PDT. Moreover, as respect to monotherapies, DTX/Ce6-KNPs induced stronger cytotoxicity to both HeLa-P and HeLa-R multicellular spheroids and reduced their volumes up to 50%. Overall, the results suggest that KNPs are very promising systems for the co-delivery of chemotherapeutics and PSs, favoring synergic interactions between PDT and chemotherapy.

Co-delivery of Docetaxel and Disulfonate Tetraphenyl Chlorin in One Nanoparticle Produces Strong Synergism between Chemo- and Photodynamic Therapy in Drug-Sensitive and -Resistant Cancer Cells.

Cancer therapies based on the combinations of different drugs and/or treatment modalities are emerging as important strategies for increasing efficacy and cure, decreasing unwanted toxicity, and overcoming drug resistance, provided that optimized drug concentration ratios are delivered into the target tissue. To these purposes, delivery systems such as nanoparticles (NPs) offer the unique opportunity to finely tune the drug loading and the release rate of drug combinations in the target tissues. Here, we propose double-layered polymeric NPs for the delivery of the chemotherapeutic docetaxel (DTX) and the photosensitizer disulfonate tetraphenyl chlorin (TPCS2a) coated with hyaluronic acid (HA), which allows cell targeting via CD44 receptors. The simultaneous delivery of the two drugs aims at killing DTX-sensitive (HeLa-P, MDA-MB-231) and DTX-resistant (HeLa-R) cancer cells by combining chemotherapy and photodynamic therapy (PDT). Using the Chou and Talalay method that analyses drug interactions and calculates combination index (CI) using the median-effect principle, we compared the efficiency of DTX chemotherapy combined with TPCS2a-PDT for drugs delivered in the standard solvents, coloaded in the same NP (DTX/TPCS2a-NP) or loaded in separate NPs (DTX-NPs + TPCS2a-NPs). Along with the drug interaction studies, we gained insight into cell death mechanisms after combo-therapy and into the extent of TPCS2a intracellular uptake and localization. In all cell lines considered, the analysis of the viability data revealed synergistic drug/treatment interaction especially when DTX and TPCS2a were delivered to cells coloaded in the same NPs despite the reduced PS uptake measured in the presence of the delivery systems. In fact, while the combinations of the free drugs or drugs in separate NPs gave slight synergism (CI < 1) only at doses killing more than 50% of the cells, the combination of drugs in one NPs gave high synergism also at doses killing 10-20% of the cells. Furthermore, the DTX dose in the combination DTX/TPCS2a-NPs could be reduced by ∼2.6- and 10.7-fold in HeLa-P and MDA-MB-231, respectively. Importantly, drug codelivery in NPs was very efficient in inducing cell mortality also in DTX resistant HeLa-R cells overexpressing P-glycoprotein 1 in which the dose of the chemotherapeutic can be reduced by more than 100 times using DTX/TPCS2a-NPs. Overall, our data demonstrate that the protocol for the preparation of HA-targeted double layer polymeric NPs allows to control the concentration ratio of coloaded drugs and the delivery of the transported drugs for obtaining a highly synergistic interaction combining DTX-chemotherapy and TPCS2a-PDT.

Adenocarcinoma de Células Claras na era pós Dietilestilbestrol / Cervical clear Cell Adenocarcinoma in the era after Diethylstilbestrol

Carcinoma de células claras (CCC) em colo uterino é raro. A colpocitologia pode estar normal. Cirurgia radical é indicada, podendo associar-se a quimioterapia (QT) e radioterapia (RT) adjuvantes. Relatamos CCC de colo uterino e metástase recidivada em mulher jovem. Paciente possuía estadiamento IA1 e não havia tido contato com dietilestilbestrol. Foi submetida à histerectomia radical e tumor era moderadamente diferenciado. Em 3 anos, surgiram metástases pulmonares, todas ressecadas e conduzidas com QT adjuvante. Tamanho tumoral aumentado, grau histológico indiferenciado e presença de metástases são fatores prognósticos ruins, o que pode explicar a evolução desfavorável da paciente. Conquanto seja de patologia rara, é imprescindível que o CCC faça parte do diagnóstico diferencial de tumores de colo uterino, mesmo que colpocitologia normal

Cervical clear cell adenocarcinoma is rare. Colpocytology may be normal. Radical surgery is indicated, and may be associated with chemotherapy (QT) and radiotherapy (RT) adjuvants. We report a patient with CCC of uterine cervix and recurrent metastasis in young women. Patient had IA1 staging and had no contact with diethylstilbestrol. It was subjected to radical hysterectomy and tumor was moderately differentiated. At 3 years, pulmonary metastases appeared, all resected and conducted with adjuvant QT. Increased tumor size, undifferentiated histological grade and presence of metastases are poor prognostic factors, which may explain the unfavorable evolution of the patient. Although it is rare, it is essential to be part of the differential diagnosis of uterine cervix tumors, even if normal colpocytology

Pluronic® P123/F127 mixed micelles delivering sorafenib and its combination with verteporfin in cancer cells.

Here, we developed Pluronic® P123/F127 (poloxamer) mixed micelles for the intravenous delivery of the anticancer drug sorafenib (SRB) or its combination with verteporfin (VP), a photosensitizer for photodynamic therapy that should complement well the cytotoxicity profile of the chemotherapeutic. SRB loading inside the core of micelles was governed by the drug:poloxamer weight ratio, while in the case of the SRB-VP combination, a mutual interference between the two drugs occurred and only specific ratios could ensure maximum loading efficiency. Coentrapment of SRB did not alter the photophysical properties of VP, confirming that SRB did not participate in any bimolecular process with the photosensitizer. Fluorescence resonance energy-transfer measurement of micelles in serum protein-containing cell-culture medium demonstrated the excellent stability of the system in physiologically relevant conditions. These results were in line with the results of the release study showing a release rate of both drugs in the presence of proteins slower than in phosphate buffer. SRB release was sustained, while VP remained substantially entrapped in the micelle core. Cytotoxicity studies in MDA-MB231 cells revealed that at 24 hours, SRB-loaded micelles were more active than free SRB only at very low SRB concentrations, while at 24+24 hours a prolonged cytotoxic effect of SRB-loaded micelles was observed, very likely mediated by the block in the S phase of the cell cycle. The combination of SRB with VP under light exposure was less cytotoxic than both the free combination and VP-loaded micelles + SRB-loaded micelles combination. This behavior was clearly explainable in terms of micelle uptake and intracellular localization. Besides the clear advantage of delivering SRB in poloxamer micelles, our results provide a clear example that each photochemotherapeutic combination needs detailed investigations on their particular interaction, and no generalization on enhanced cytotoxic effects should be derived a priori.

Uptake and photo-toxicity of Foscan®, Foslip® and Fospeg® in multicellular tumor spheroids.

In cancer photodynamic therapy (PDT), an efficient and homogeneous intratumoral accumulation of the photosensitizer (PS) is required to induce cell damages in the entire tumor mass after light activation. Thus, in this study we investigated penetration ability and photodynamic efficiency of meta-tetra(hydroxyphenyl)chlorin (m-THPC) in standard formulation (Foscan®) and in its non PEGylated and PEGylated liposomal formulations, Foslip® and Fospeg®, in HeLa multicellular spheroids, as in vitro avascular models of solid tumors. Confocal microscopy studies demonstrated that m-THPC fluorescence was confined in the external cell layers of spheroids with a slightly higher accumulation of Foslip® and Fospeg® with respect to Foscan®. Irradiation with red light, following 24h incubation of spheroids with the m-THPC formulations, caused however photodamages also in cells located in the central part of spheroids, as documented by transmission electron microscopy analyses. Overall, the photodynamic effects of the three m-THPC formulations on HeLa cell spheroids were comparable in terms of cell viability measured with the MTS assay. It is however worth noting that the delivery of m-THPC by liposomes significantly abolished its cytotoxicity in the dark, slightly improved the cellular uptake and, following PDT, promoted cell loss and spheroid disassembling to a higher extent when compared to Foscan®.