Liposomal simvastatin sensitizes C26 murine colon carcinoma to the antitumor effects of liposomal 5-fluorouracil in vivo.

5-Fluorouracil-based therapy remains the main approach in colorectal cancer, even though there are still some drawbacks, such as chemoresistance. In this study we combined 5-fluorouracil encapsulated in long-circulating liposomes with simvastatin, also encapsulated in long-circulating liposomes, that was previously proved to exert antitumor actions on the same tumor model. The production of angiogenic/inflammatory proteins was assessed by protein array and the production of markers for tumor aggressiveness (Bcl-2, Bax, and nuclear factor [NF]-κB) were determined by western blot analysis. Intratumor oxidative stress was evaluated through measurement of malondialdehyde level by HPLC, and through spectrophotometric analysis of catalytic activity of catalase and of total antioxidant capacity. Immunohistochemical analysis of tumors for CD31 expression was assessed. Intratumor activity of MMP-2 by gelatin zymography was also carried out. Our results revealed that combined therapies based on liposomal formulations exerted enhanced antitumor activities compared with combined treatment with free drugs. Sequential treatment with liposomal simvastatin and liposomal 5-fluorouracil showed the strongest antitumor activity in C26 colon carcinoma in vivo, mainly through inhibition of tumor angiogenesis. Important markers for cancer progression (Bcl-2, Bax, NF-κB, and intratumor antioxidants) showed that liposomal simvastatin might sensitize C26 cells to liposomal 5-fluorouracil treatment in both regimens tested. The outcome of simultaneous treatment with liposomal formulations was superior to sequential treatment with both liposomal types as the invasive capacity of C26 tumors was strongly increased after the latest treatment. The antitumor efficacy of combined therapy in C26 colon carcinoma might be linked to the restorative effects on proteins balance involved in tumor angiogenesis.

Optimization of prednisolone-loaded long-circulating liposomes via application of Quality by Design (QbD) approach.

Quality by design principles (QbD) were used to assist the formulation of prednisolone-loaded long-circulating liposomes (LCL-PLP) in order to gain a more comprehensive understanding of the preparation process. This approach enables us to improve the final product quality in terms of liposomal drug concentration, encapsulation efficiency and size, and to minimize preparation variability. A 19-run D-optimal experimental design was used to study the impact of the highest risk factors on PLP liposomal concentration (Y1- µg/ml), encapsulation efficiency (Y2-%) and size (Y3-nm). Out of six investigated factors, four of them were identified as critical parameters affecting the studied responses. PLP molar concentration and the molar ratio of DPPC to MPEG-2000-DSPE had a positive impact on both Y1 and Y2, while the rotation speed at the formation of the lipid film had a negative impact. Y3 was highly influenced by prednisolone molar concentration and extrusion temperature. The accuracy and robustness of the model was further on confirmed. The developed model was used to optimize the formulation of LCL-PLP for efficient accumulation of the drug to tumor tissue. The cytotoxicity of the optimized LCL-PLP on C26 murine colon carcinoma cells was assessed. LCL-PLP exerted significant anti-angiogenic and anti-inflammatory effects on M2 macrophages, affecting indirectly the C26 colon carcinoma cell proliferation and development.

A step forward towards the development of stable freeze-dried liposomes: a quality by design approach (QbD).

This study highlights the advantages of using a Quality by Design (QbD) approach in order to gain a more comprehensive understanding of the freeze-drying process of pravastatin-loaded long-circulating liposomes (LCL-PRAV). Within the QbD paradigm, the present study aimed to establish the design space for the optimization of freeze-dried LCL-PRAV by means of Design of Experiment (DOE). The encapsulated solute retention (ESR), the average particle size, and zeta potential after freeze-drying, the residual moisture content, the macroscopic cake appearance, the glass transition temperature (Tg) of the freeze-dried cake, and the primary drying time were defined as critical quality attributes (CQAs) for the freeze-dried final product. Further on, the influence of lyoprotectant type, freezing rate, shelf temperature during primary drying, and the presence of an annealing step on the CQAs was investigated through a 21-run D-optimal experimental design. Three-dimensional response surfaces were generated to complete the statistical analysis and for a better understanding of the influence of variables and their interactions on the responses. The developed model was then used to build the design space for the freeze-dried liposomes, within which the product quality was assured and the process variability was minimized.

Investigation of a Potential Pharmacokinetic Interaction Between Nebivolol and Fluvoxamine in Healthy Volunteers.

PURPOSE: To investigate whether fluvoxamine coadministration can influence the pharmacokinetic properties of nebivolol and its active hydroxylated metabolite (4-OH-nebivolol) and to assess the consequences of this potential pharmacokinetic interaction upon nebivolol pharmacodynamics. METHODS: This open-label, non-randomized, sequential clinical trial consisted of two periods: Period 1 (Reference), during which each volunteer received a single dose of 5 mg nebivolol and Period 2 (Test), when a combination of 5 mg nebivolol and 100 mg fluvoxamine was given to all subjects, after a 6-days pretreatment regimen with fluvoxamine (50-100 mg/day). Non-compartmental analysis was used to determine the pharmacokinetic parameters of nebivolol and its active metabolite. The pharmacodynamic parameters (blood pressure and heart rate) were assessed at rest after each nebivolol intake, during both study periods. RESULTS: Fluvoxamine pretreatment increased Cmax and AUC0-∞  of nebivolol (Cmax: 1.67 ± 0.690  vs 2.20 ± 0.970  ng/mL; AUC0-∞: 12.1 ± 11.0  vs 19.3 ± 19.5  ng*h/mL ) and of its active metabolite (Cmax: 0.680  ± 0.220  vs 0.960 ± 0.290  ng/mL; AUC0-∞: 17.6 ±20.1  vs 25.5 ± 29.9  ng*h/mL). Apart from Cmax,AUC0-t and AUC0-∞, the other pharmacokinetic parameters (tmax, kel and t½) were not significantly different between study periods. As for the pharmacodynamic analysis, decreases in blood pressure and heart rate after nebivolol administration were similar with and without fluvoxamine concomitant intake. CONCLUSIONS: Due to enzymatic inhibition, fluvoxamine increases the exposure to nebivolol and its active hydroxylated metabolite in healthy volunteers. This did not influence the blood pressure and heart-rate lowering effects of the beta-blocker administered as single-dose. However, more detail studies involving actual patients are required to further investigate the clinical relevance of this drug interaction. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Defining the design space for freeze-dried orodispersible tablets with meloxicam.

OBJECTIVE: This work focused on simultaneously investigating formulation variables and freeze-drying parameters when preparing orodispersible tablets with meloxicam (Mel), by a Quality by Design (QbD) approach. MATERIALS AND METHODS: Methylcellulose (MC) was selected as a matrix forming agent and mannitol (Man) as cryoprotectant, both at two concentration levels. The freezing regime was also varied between fast and shelf-ramped, to find out how it affects the final products. The tablet formulations were characterized for their disintegration time, wetting properties, mechanical properties, morphology and in vitro dissolution. Response Surface Modeling completed the statistical analysis that assessed the effects of independent variables on the responses. RESULTS: All the responses showed good fitting to the chosen model. The increase in MC content determined a positive effect on disintegration time, wetting time, mechanical strength and a negative effect on Mel dissolution. High levels of Man-determined brittle products with low-absorption capacity and fast Mel dissolution. The freezing rate had an important effect on the structure of tablets: fast freezing determined slightly thicker pore walls with smooth surfaces, while shelf-ramped freezing led to a multiple-layer structure with increased hardness. Still, shelf-ramped freezing yielded higher Mel release, due to physical changes of the active substance during the freeze-drying process. CONCLUSION: From the generated design space, an optimal formulation was obtained and the results validated the experimental design. The QbD approach was an efficient manner of understanding formulation and process parameters at the freeze-dried orodispersible tablets preparation.

Optimizing long-circulating liposomes for delivery of simvastatin to C26 colon carcinoma cells.

Simvastatin (SIM) is a lipophilic statin that has potential benefits for prevention and treatment of several types of malignancies. However, its low water solubility and the toxicity associated with administration of high doses recommend it for encapsulation in carriers able to deliver the therapeutic dose in the tumor. In this work, liposomes with long-circulating properties were proposed as delivery systems for SIM. The objective of this study was to optimize the formulation of SIM-loaded long-circulating liposomes (LCL-SIM) by using D-optimal experimental design. The influence of phospholipids concentration, phospholipids to cholesterol molar ratio and SIM concentration was studied on SIM liposomal concentration, encapsulation efficiency and liposomal size. The optimized formulation had liposomal SIM concentration 6238 µg/ml, EE % of 83.4% and vesicle size of 190.5 nm. Additionally we evaluated the in vitro cytotoxicity of the optimized liposomal SIM (LCL-SIM-OPT) on C26 murine colon carcinoma cells cultivated in monoculture as well as in co-culture with murine peritoneal macrophages at a cell density ratio that provides an approximation of physiological conditions of colon carcinoma development in vivo. Our preliminary studies suggested that LCL-SIM-OPT exerted cytotoxicity on C26 cells probably via enhancement of oxidative stress in co-culture environment.

Testing the disintegration and texture-related palatability predictions for orodispersible tablets using an instrumental tool coupled with multivariate analysis: Focus on process variables and analysis settings.

Orodispersible tablets (ODTs) represent a growing category of dosage forms intended to increase the treatment acceptability for special groups of patients. ODTs are designed to rapidly disintegrate in the oral cavity and to be administered without water. In addition, ODTs are easy to manufacture using standard excipients and pharmaceutical equipment. This study adds to previously published research that developed an instrumental tool to predict oral disintegration and texture-related palatability of ODTs with different formulations. The current study aimed to challenge the predictive capacity of the models under variable process conditions. The studied process parameters with potential impact on the pharmaceutical properties, texture profiles, and palatability were the compression pressure, punch shape and diameter. Subsequently, for all the placebo and drug-loaded ODTs, the in vivo disintegration time and texture-related palatability were determined with healthy volunteers. Previously developed regression models were applied to predict the formulation's disintegration time and texture-related palatability characteristics of ODTs obtained under different experimental conditions. The influence of process variables on the predictive performance of the models was estimated by calculating the residuals as the difference between the predicted and observed values for the investigated response. Increasing the speed of the analyser`s probe from 0.01 mm/s to 0.02 mm/s led to an improved differentiation of the texture profiles. The in vivo disintegration time and texture-related palatability scores were only influenced by the mechanical resistance and the tablet shape. Lower score was observed for the larger diameter tablets (10 mm). Overall, the prediction of the disintegration time at 0.02 mm/s was more accurate, except for stronger tablets. The best prediction of texture-related palatability was achieved for the 10 mm tablets, tested at 0.01 mm/s speed. The same model achieved good predictions of the oral disintegration time for all API-loaded formulations, which confirmed the ability of the texture analysis to capture process-related variability. Drug loading decreased the predictive capacity of the texture-related palatability because of the taste effect.

In Vivo Study of the Effects of Propranolol, Timolol, and Minoxidil on Burn Wound Healing in Wistar Rats.

Propranolol, timolol, and minoxidil have all shown benefits in treatment of burn injury and other skin wounds. The study evaluated their effects on full-thickness thermal skin burns in a Wistar rat model. Performed on 50 female rats; two dorsal skin burns were created on each animal. On the next day, the rats were divided into 5 groups (n = 10); each has received a specific treatment daily for 14 days: group I-topical vehicle (control), group II-topical silver sulfadiazine (SSD), group III-oral propranolol (5.5 mg) associated with topical vehicle, group IV-topical timolol 1% cream, and group V-topical minoxidil 5% cream. Wound contraction rates, malondialdehyde (MDA), glutathione (GSH, GSSG), and catalase activity in skin and/or serum were evaluated, and histopathological analyses were performed. Propranolol did not show advantages in necrosis prevention and wound contraction and healing, and did not reduce oxidative stress. It impaired keratinocyte migration, and promoted ulceration, chronic inflammation, and fibrosis, yet reducing the necrotic zone. Timolol prevented necrosis and promoted contraction and healing, increased antioxidant capacity and promoted keratinocyte migration and neo capillarization in comparison to the other treatments. Minoxidil reduced necrosis and enhanced contraction, resulting in positive outcomes after 1 week of treatment regarding local antioxidant defense, keratinocyte migration, neo capillarization, chronic inflammation, and fibrosis rates. However, after 2 weeks, it resulted in contrasting outcomes. In conclusion, topical timolol promoted wound contraction and healing, reducing local oxidative stress and improving keratinocyte migration, bringing arguments for potential benefits in skin epithelization.

Chemical Profile and Biological Effects of an Herbal Mixture for the Development of an Oil-in-Water Cream.

Three individual hydroalcoholic extracts derived from Hamamelis virginiana leaves, Krameria lappacea root, Salix alba bark, and the resulting herbal mixture (HM) were assessed for the phytochemical profile as well as for antibacterial and cytotoxic potential. The chemical composition of the individual extracts and of their mixture was analyzed by chromatographical (LC-MS) and spectrophotometrical methods. The antimicrobial properties were evaluated by using the agar-well diffusion and the broth microdilution assays, whereas the potential cytotoxicity was investigated on human keratinocyte cell line by MTT method and apoptosis test. The HM composition revealed important amounts of valuable polyphenolic compounds provided from the individual extracts, having synergistic biological effects. All tested extracts displayed in vitro antimicrobial properties, with a significantly higher efficacy noticed for the HM when tested against Staphylococcus aureus. Moreover, none of the tested extracts was responsible for in vitro cytotoxicity against the human keratinocytes in the selected concentration range. Furthermore, the HM was included in an oil-in-water cream for the nonpharmacological treatment of seborrheic dermatitis, developed and optimized by using a QbD approach. A D-optimal experimental plan with four factors that varied on two levels was used to investigate the effect of the quantitative variation of the formulation factors (emulsifier, co-emulsifier, thickening agent, oily phase ratio) on the characteristics of the cream in terms of firmness, consistency, adhesiveness, stringiness, spreadability, and viscosity. Based on the experimental results, an optimal formulation containing 2.5% emulsifier and 20% oily phase was prepared and analyzed. The obtained results showed appropriate quality characteristics of this novel cream, which may be used in the future to manage the associated symptoms of seborrheic dermatitis.

Effect of fluvoxamine on the pharmokinetics of zolpidem: a two-treatment period study in healthy volunteers.

1. Our objective was to evaluate a possible pharmacokinetic interaction between zolpidem and fluvoxamine in healthy volunteers. 2. The study consisted of two periods: Period 1 (reference), when each volunteer received a single dose of 5 mg zolpidem; and Period 2 (test), when each volunteer received a single dose of 5 mg zolpidem and 100 mg fluvoxamine. Between the two periods, the subjects were treated for 6 days with a single daily dose of 100 mg fluvoxamine. 3. Pharmacokinetic parameters of zolpidem given in each treatment period were calculated using non-compartmental analysis and the data from two periods were compared to determine statistically significant differences. 4. In the two periods of treatments, the mean peak plasma concentrations (C(max)) were 56.4 ± 25.6 ng/mL (zolpidem alone) and 67.3 ± 25.8 ng/mL (zolpidem after pretreatment with fluvoxamine). The t(max), times taken to reach C(max), were 0.83 ± 0.44 and 1.26 ± 0.74 h, respectively, and the total areas under the curve (AUC(0-∞)) were 200.9 ± 116.8 and 512.0 ± 354.6 ng h/mL, respectively. The half-life of zolpidem was 2.24 ± 0.81 h when given alone and 4.99 ± 2.92 h after pretreatment with fluvoxamine. 5. Fluvoxamine interacts with zolpidem in healthy volunteers and increases its exposure by approximately 150%. The experimental data showed the pharmacokinetic interaction between zolpidem and fluvoxamine, and suggest that the observed interaction might be clinically significant, but its relevance has to be confirmed.

Characterization and In Vitro Biocompatibility of Two New Bioglasses for Application in Dental Medicine-A Preliminary Study.

Bioactive glasses (BGs), also known as bioglasses, are very attractive and versatile materials that are increasingly being used in dentistry. For this study, two new bioglasses-one with boron (BG1) and another with boron and vanadium (BG2)-were synthesized, characterized, and tested on human dysplastic keratinocytes. The in vitro biological properties were evaluated through pH and zeta potential measurement, weight loss, Ca2+ ions released after immersion in phosphate-buffered saline (PBS), and scanning electron microscopy (SEM) coupled with energy dispersive spectroscopy (EDS) analysis. Furthermore, biocompatibility was evaluated through quantification of lactate dehydrogenase activity, oxidative stress, transcription factors, and DNA lesions. The results indicate that both BGs presented the same behavior in simulated fluids, characterized by high degradation, fast release of calcium and boron in the environment (especially from BG1), and increased pH and zeta potential. Both BGs reacted with the fluid, particularly BG2, with irregular deposits covering the glass surface. In vitro studies demonstrated that normal doses of the BGs were not cytotoxic to DOK, while high doses reduced cell viability. Both BGs induced oxidative stress and cell membrane damage and enhanced NFkB activation, especially BG1. The BGs down-regulated the expression of NFkB and diminished the DNA damage, suggesting the protective effects of the BGs on cell death and efficacy of DNA repair mechanisms.

Design, in vitro bioactivity and in vivo influence on oxidative stress and matrix metalloproteinases of bioglasses in experimental skin wound.

BACKGROUND: The bioactive glasses (BGs) are very attractive materials increasingly used in healing skin lesions due to their antibacterial effect and stimulation of collagen deposition and angiogenesis. In this study, three specimens of bioactive glasses (BG1, BG2 and BG3) have been synthesized and characterized. METHODS: In order to evaluate their in vitro bioactivity, the pH measurements, zeta potential and the concentration of Ca2+ and fluor ions released after immersion in phosphate buffered saline (PBS) followed by scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy, inductively coupled plasma optical emission spectrometry (ICP-OES) and for BG1 and BG3, X-ray powder diffraction analysis, were performed. X-ray photoelectron spectroscopy (XPS) was also used for detection of different ions in the solid bioglasses before immersion in PBS. The impact of BG1 and BG3 on skin healing mechanisms was evaluated by oxidative stress and matrix metalloproteases (MMP)-2 and -9 and by histopathological analysis. RESULTS: The results have shown that all the BGs tested are characterized by a very high degradation rate and a very fast Ca2+, fluor and boron releases and displayed changed surface morphology at SEM, after 7 and 14 days of immersion in PBS. In addition, BG1 and BG3 reduced in vivo the lipid peroxidation, increased the nitric oxide, especially at 14 days and improved superoxide dismutase activity, mainly in BG1 treated animals. In parallel, both BG1 and BG3, diminished MMP-9 at 14 days and increased the proportion of normal collagen in the bed of the wound, particularly BG3. CONCLUSION: These results suggested that due to the antioxidant and anti-inflammatory properties of components released from BGs and regulatory properties on MMPs activities, BGs can exert beneficial effects in wound healing.

Influence of palm oil on doxorubicin induced cytotoxicity in normal and tumor cell cultures.

The vitamin E of palm oil, unlike most other vegetal fats, consists largely of tocotrienols (TT), products previously reported as having antioxidant and tumor-inhibitory properties. A tocotrienols containing palm oil, in the form of liposomes entrapping dosages of 0.5-0.05 microgTT/mL, was studied in combined treatments with doxorubicin (30 min before drug administration). The IC(50) values of doxorubicin, at 24 h, showed that its cytotoxic effects were decreased by palm oil, in a dose effect relationship (p < 0.01, ANOVA), in both normal (Hfl-1, Huvec) and tumor (HepG2, Mls) cells. These results demonstrated an unselective protective activity of tocotrienols, in vitro, on some normal and tumor cultured cells treated with doxorubicin.

Statins in risk-reduction and treatment of cancer.

Statins, which are competitive inhibitors of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, reduce cholesterol blood levels and the risk of developing cardiovascular diseases and their related complications. In addition to this main activity, statins show pleiotropic effects such as antioxidant, anti-inflammatory and antiproliferative properties, with applications in many pathologies. Based on their antiproliferative properties, in vitro and in vivo studies have investigated their effects on various types of cancer (i.e., breast cancer, prostate cancer, colorectal cancer, ovarian cancer, lung cancer) with different genetic and molecular characteristics. Many positive results were obtained, but they were highly dependent on the physiochemical properties of the statins, their dose and treatment period. Combined therapies of statins and cytotoxic drugs have also been tested, and synergistic or additive effects were observed. Moreover, observational studies performed on patients who used statins for different pathologies, revealed that statins reduced the risk of developing various cancers, and improved the outcomes for cancer patients. Currently, there are many ongoing clinical trials aimed at exploring the potential of statins to lower the mortality and the disease-recurrence risk. All these results are the foundation of new treatment directions in cancer therapy.

Effects of silver and gold nanoparticles phytosynthesized with Cornus mas extract on oral dysplastic human cells.

Background: Oral cancer is highly aggressive due to difficult diagnosis, therapy resistance and increasing frequency; thus finding prevention therapies is very important. Aim: This study evaluates the use of gold and silver nanoparticles (NPs), phyto-synthesized with Cornus mas extract against oral dysplastic lesions. Methods: NPs were characterized by UV-Vis, Fourier-transform infrared spectroscopy, transmission electron microscopy, x-ray diffraction and laser Doppler microelectrophoresis. Biological testing employed two human oral cell lines: gingival fibroblasts and dysplastic keratinocytes and evaluated viability, cell death mechanisms and cellular uptake. Results: NPs induced selective toxic effects against dysplastic cells. p53/BAX/BCL2 activation and PI3K/AKT inhibition led to cell death through necrosis and apoptosis. NPs also induced antioxidant and anti-inflammatory effects. Conclusion: NPs of gold and silver showed promising beneficial effects in the therapy of oral dysplasia.

Solid Lipid Nanoparticles: Vital Characteristics and Prospective Applications in Cancer Treatment.

Cancer nanotechnology is a new field of interdisciplinary research cutting across biology, chemistry, engineering, and medicine, aiming to lead to major advances in cancer treatment. Over the past several years, solid lipid nanoparticles (SLNs) have attracted the interest of researchers due to their ability to overcome the limitations of classic chemotherapeutics. We reviewed the most recent data on the therapeutic use of SLNs in oncology, presenting their main advantages and disadvantages, along with various production methods and different routes of administration. In accordance with these aspects, the long-term physical stability, the controlled release of the loaded drugs, and the efficient targeted delivery of drugs as methods of surpassing the pharmaceutical limitations of anticancer drugs, natural products and gene therapy have been discussed. In addition, we have also emphasized briefly the crosstalk between SLNs and the new trend in oncology, immunotherapy, as future possible antineoplastic treatment, especially in melanoma. This review highlights the potential of SLNs in providing very positive perspectives for future cancer treatment by improving the efficiency of present chemotherapy and reducing its side effects. SLNs allow targeted delivery of anticancer drugs and could improve the efficiency of current chemotherapy in neoplasia.

Formulation Optimization of Freeze-Dried Long-Circulating Liposomes and In-Line Monitoring of the Freeze-Drying Process Using an NIR Spectroscopy Tool.

The effect of lyoprotectant type and concentration on the stability of freeze-dried prednisolone sodium phosphate-loaded long-circulating liposomes was investigated. Trehalose at a 5:1 carbohydrate to lipid molar ratio proved to be superior in maintaining the structural integrity and the permeability properties of the liposome bilayers, assuring the desired characteristics of the final product: a cake with a porous structure and easy to reconstitute, a similar size to the liposomes before freeze-drying, a high percent of encapsulated drug, and a low residual moisture content. Further on, the study demonstrated the possibility of near-infrared spectroscopy to provide valuable insights for detecting critical changes in acyl chain packing of the liposome bilayer. By visualizing the spectra after principal component analysis, one can predict if any harm has occurred to liposome integrity during the process. Moreover, near-infrared spectroscopy enabled us to determine the end points of primary and secondary drying without disturbing the normal freeze-drying procedure, which allowed us to gain a better understanding of the process and to improve process efficiency by optimizing the primary and secondary drying time.

In Vivo Double Targeting of C26 Colon Carcinoma Cells and Microenvironmental Protumor Processes Using Liposomal Simvastatin.

Purpose: Besides cholesterol lowering effects, simvastatin (SIM) at very high doses possesses antitumor actions. Moreover our previous studies demonstrated that tumor-targeted delivery of SIM by using long-circulating liposomes (LCL) improved the therapeutic index of this drug in murine melanoma-bearing mice. To evaluate whether this finding can be exploited for future therapy of colorectal cancer the antitumor activity and the underlying mechanisms of long-circulating liposomal simvastatin (LCL-SIM) efficacy for inhibition of C26 murine colon carcinoma growth in vivo were investigated. Materials and Methods: To find LCL-SIM dose with the highest therapeutic index, dose-response relationship and side effects of different LCL-SIM doses were assessed in C26 colon carcinoma-bearing mice. The underlying mechanisms of LCL-SIM versus free SIM treatments were investigated with regard to their actions on C26 cell proliferation and apoptosis (via tumor tissues immunostaining for PCNA and Bax markers), tumor inflammation (via western blot analysis of NF-κΒ production), angiogenesis (using an angiogenic protein array), and oxidative stress (by HPLC assessment of malondialdehyde). Results: Our findings suggest that LCL-SIM antitumor activity on C26 colon carcinoma is a result of the tumor-targeting property of the liposome formulation, as free SIM treatment was ineffective. Moreover, LCL-SIM exerted significant antiproliferative and pro-apoptotic actions on C26 cells, notable suppressive effects on two main supportive processes for tumor development, inflammation and angiogenesis, and only slight anti-oxidant actions. Conclusion: Our data proved that LCL-SIM antitumor activity in C26 colon carcinoma was based on cytotoxic effects on these cancer cells and suppressive actions on tumor angiogenesis and inflammation.

A quality by design (QbD) study on enoxaparin sodium loaded polymeric microspheres for colon-specific delivery.

The aim of this study was to apply quality by design (QbD) for pharmaceutical development of enoxaparin sodium microspheres for colon-specific delivery. The Process Parameters (CPPs) and Critical Quality Attributes (CQAs) were identified. A central composite experimental design was used in order to develop the design space of microspheres for colon-specific delivery that have the desired Quality Target Product Profile (QTPP). The CPPs studied were Eudragit® FS-30D/Eudragit® RS-PO ratio, poly(vinyl alcohol) (PVA) concentration and sodium chloride (NaCl) concentration. The encapsulation efficiency increased with NaCl concentration increase, the percentages of enoxaparin sodium reaching 94% for some formulations. Increasing the ratio Eudragit® FS-30D/Eudragit® RS-PO ensured a relatively complete release of enoxaparin sodium in the environment simulating the colonic pH. Based on these results, the optimum conditions were decided and the optimum formulation was prepared. The results obtained for the latter in terms of in vitro enoxaparin sodium release were good, the microparticles releasing only 9.42% enoxaparin sodium in acidic environment and 15.16% in the medium which simulated duodenal pH, but allowing the release of up to 89.24% in the medium which simulated colonic pH. The in vitro release profile of enoxaparin sodium was close to the ideal one, therefore the system was successfully designed using QbD approach.

Liposomal prednisolone phosphate potentiates the antitumor activity of liposomal 5-fluorouracil in C26 murine colon carcinoma in vivo.

The antitumor efficacy of 5-fluorouracil (5-FU) in advanced colorectal cancer (CRC) is hindered not only by the low therapeutic index, but also by tumor cell resistance to this cytotoxic drug. Therefore, to enhance the 5-FU antitumor activity, the present research used a novel tumor-targeted therapy based on the co-administration of 5-FU encapsulated in long-circulating liposomes (LCL-5-FU) together with liposomal prednisolone phosphate (LCL-PLP), a formulation with known anti-angiogenic actions on C26 murine colon carcinoma cells. Thus, we assessed the in vivo effects of the combined liposomal drug therapy on C26 carcinoma growth as well as on the production of molecular markers with key roles in tumor development such as angiogenic, inflammatory, and oxidative stress molecules. To get further insight into the polarization state of tumor microenvironment after the treatment, we determined the IL-10/IL-12p70 ratio in tumors. Our results showed that combined liposomal drug therapy inhibited almost totally tumor growth and was superior as antitumor activity to both single liposomal drug therapies tested. The antitumor efficacy of the combined therapy was mainly related to the anti-angiogenic and anti-inflammatory actions on C26 carcinoma milieu, being favored by its controlling effect on intratumor oxidative stress and the skewing of polarization of tumor microenvironmental cells toward their antineoplastic phenotypes. Thus, our study unveils a promising treatment strategy for CRC that should be furthermore considered.