Oral administration of PEDV-dissolved Alg-CS gel induces high and sustained mucosal immunity in mice.

Porcine epidemic diarrhea (PED) is a serious disease in piglets that leads to high mortality. An effective measure that provides higher IgA levels in the intestine and milk is required to decrease losses. Porcine epidemic diarrhea virus (PEDV) was dissolved in calcium alginate (Alg) and combined with chitosan (CS) via electrostatic interactions between cationic chitosan and anionic alginate to create a porous gel (Alg-CS+PEDV). The gel was used to immunize mice orally or in combination with subcutaneous injections of inactivated PEDV vaccine. At 12 and 24 days after immunization, levels of IgA and IgG in Alg-CS+PEDV were higher than with normal PEDV oral administration. At 24 days after immunization, the concentration of IFN-γ in Alg-CS+PEDV was higher than with normal PEDV oral administration. Furthermore, oral administration combining subcutaneous immunization induced higher levels of IgG and IgA than oral administration alone. Our study provides a new method for the preparation and administration of oral vaccines to achieve enhanced mucosal immunity against PEDV.

Evaluation of the oral administration of α-l-guluronic acid on COX-1 and COX-2 gene expression profile in ankylosing spondylitis patients.

Ankylosing spondylitis (AS) is a chronic autoimmune arthritis disease with a genetic background, affecting the skeletal axis, sacroiliac, and peripheral joints. Nonsteroidal anti-inflammatory drugs (NSAIDs) are the first-line treatment for AS to alleviate the inflammation and pain. Despite the beneficial effect, their use is accompanied by a wide variety of possible side effects in the gastrointestinal and kidneys. The α-l-guluronic acid (G2013) is a new nonsteroidal anti-inflammatory patented (PCT/EP2017/067920) drug, which has shown its anti-inflammatory properties in the previous investigations. The present study revealed the oral administration effect of G2013 on COX-1 and COX-2 gene expression in AS patients. The blood samples of twelve 18-45 years old patients suffering AS and BASDAI >4, and BASFI >4, before and after 12 weeks of treatment with G2013 and 12 blood samples of healthy volunteers were collected and the effect of G2013 on the gene expression of COX-1 and COX-2 enzymes were assessed by Real-Time PCR. The results indicate that G2013 is able to reduce the gene expression level of COX-1 and COX-2 enzymes in treated AS patients compared to healthy control. Statistically significant differences were not observed between the treatment and the healthy control groups. According to the findings, G2013 might be categorized and introduced as a novel NSAID for the treatment of AS.

Effects of mannuronic acid (M2000) on gene expression profile of signal transducer and activator of transcription proteins (STATs) in rheumatoid arthritis patients.

Rheumatoid arthritis (RA), a form of inflammatory arthritis, is a chronic joint disease characterized by pain and inflammation that affects 0.5% to 1% of the population worldwide. The safety, efficacy, tolerability, and potency of ß-D-mannuronic acid (M2000) as a novel NSAID with immunosuppressive property has been reported by several in vitro studies, experimental models and clinical trials phase I/II and III in ankylosing spondylitis and rheumatoid arthritis (RA) patients This research is designed to study the therapeutic efficacy of oral administration of mannuronic acid in RA patients who had inadequate response to conventional drugs and to assess the effect of this drug on gene expression of the signal transducer and activator of transcription (STATs) protein (STAT1, STAT3, STAT4, and STAT6). The study has included 15 RA patients who had an insufficient response to the conventional therapy. The oral dose of mannuronic acid was 1000mg divided into two 500 mg doses per day for 3 months as an addition to conventional therapy. There were 15 healthy volunteer in the control group. Blood samples were collected from both groups, once from healthy controls and twice from RA patients before and after treatment by M2000. The peripheral blood mononuclear cells (PBMCs) were isolated to assess the gene expression level of STAT1, STAT3, STAT4, and STAT6 using the real-time PCR method. Results obtained in this study demonstrated a significant difference in the gene expression level of STAT1 between healthy controls and patients before treatment as well as a significant reduction in RA patients after treatment compared with the level before treatment. In addition, the gene expression level of STAT3 and STAT4 showed a significant reduction in RA patients after treatment compared to patients before treatment, while there was no significant difference between RA patients before treatment and the healthy control group for both molecules. On the other hand, there was no change in the gene expression level of STAT6 among all groups. The outcomes of this study confirmed that ß-D-mannuronic acid (M2000) has the ability to control the levels of STAT1, STAT3 and STAT4 in RA patients, and might be beneficial in the management and therapy of RA.

Mannuronic Acid in Low-Risk and Intermediate-1-Risk Myelodysplastic Syndromes.

The discovery of hematologic improvement and bone marrow modification by the drug ß-D mannuronic acid (M2000) during treatment of rheumatoid arthritis in phase 1/2/3 clinical trials prompted us to design a new trial to target hematologic deficits in myelodysplastic syndromes (MDS). In this open-label, randomized phase 2 clinical trial, the potential effect and tolerability of drug M2000 was assessed in patients with low- and intermediate-1-risk MDS. The primary efficacy end point was hematologic improvement after 12 weeks of ß-D-mannuronic acid therapy. Among 34 enrolled patients, half received their conventional therapy plus ß-D-mannuronic acid, and the other half received only conventional drugs. In the conventional + ß-D mannuronic acid treatment group, hematologic improvement and development of transfusion independence and/or reduction in transfusion requirements were seen in 12 patients (92.3%) and 1 patient (7.7%), respectively. Moreover, 5 patients (38.5%), 2 patients (15.4%), and 1 patient (7.7%) in the ß-D-mannuronic acid-treated group showed hematologic improvement of the major parameters of erythroid, neutrophil, and platelet responses, respectively, based on the International Working Group criteria), whereas in the conventional treatment group as control, no hematologic improvements including erythroid, neutrophil, and platelet response was seen. In this trial, the addition of ß-D mannuronic acid to conventional treatment showed promising results in MDS patients with low and intermediate-1 risk with effects on hematologic improvements without significant adverse effect.

Pivotal cytokines and their transcription factors are the targets of guluronic acid (G2013) for inhibiting the immunopathogenesis process of multiple sclerosis.

The α-L-guluronic acid (G2013), is a novel immunosuppressive drug (PCT/EP2017/067920). One of the most popular ideas in designing drugs for multiple sclerosis (MS) is to restrict the main inflammation-related lymphocytes and cytokines. The foremost problems with conventional drugs are their side effects and low efficacy. In order to rectify these problems, we examined the effect of two doses of G2013 on the gene expression of IFN-γ, IL-17, IL-22, T-bet, RORC, and AHR, in MS patients PBMCs. RNA was extracted from peripheral blood mononuclear cell (PBMC) of 12 relapsing-remitting MS patients and 12 healthy volunteers and the effect of two doses of G2013 on the gene expression of IFN-γ, IL-17, IL-22, T-bet, RORC, and AHR, were assessed by real-time PCR. Overall, the results show that G2013 is able to significantly reduce the gene expression of IL-22, AHR, RORC, and T-bet. Collectively, G2013 might be considered and studied as a new drug of possible use to MS patients due to its immunosuppressive property on some of the main inflammatory cytokine and transcription factors.

The Situation of Chemokine Ligands and Receptors Gene Expression, Following the Oral Administration of Drug Mannuronic Acid in Rheumatoid Arthritis Patients.

BACKGROUND: Regarding the leukocytes infiltration into the synovium of Rheumatoid Arthritis (RA) patients is mostly mediated by chemokine ligands and receptors, and following the efficient and motivating results of international Phase III clinical trial of ß-D-Mannuronic acid (M2000) patented EP067919 (2017), as a novel anti-inflammatory drug, in patients with RA, the present research was designed. OBJECTIVES: This study aimed to assess the oral administration effects of this new drug on gene expression of some chemokine receptors and ligands, including CXCR4, CXCR3, CCR2, CCR5 and CCL2/MCP-1 in PBMCs of patients with active form of RA. METHODS: Twelve patients suffering from RA, with inadequate response to conventional drugs were selected (Clinical trial identifier IRCT2017100213739N10) and 1000mg/day of M2000 was orally administrated to them for 12 weeks. The mRNA expression of target molecules was then evaluated in PBMCs of the patients before and after treatment with M2000 using real-time PCR and was compared to healthy controls. Patents related to this study were also reviewed. RESULTS: The results showed that M2000 was able to significantly down-regulate the mRNA expression of CXCR4, CCR2 and CCL2/MCP-1 in the PBMCs of the RA patients. It should be noted that the gene expression situation of the target molecules was in coordinate with the clinical and paraclinical assessments in the patients. CONCLUSION: Taken together, the results of this investigation revealed the part of molecular and immunological mechanisms of drug Mannuronic acid (M2000) in the treatment of RA, based on chemokine ligands and receptors mediated processes.

Effects of guluronic acid, as a new NSAID with immunomodulatory properties on IL-17, RORγt, IL-4 and GATA-3 gene expression in rheumatoid arthritis patients.

Aim: Rheumatoid arthritis (RA) is a prevalent inflammatory, autoimmune diseases characterized by inflammation and destruction of joints. Disease-modifying anti-rheumatic drugs (DMARDs) and biological drugs can have modulatory interference in disease process. In this study, the effect of Guluronic Acid (G2013) as a novel non-steroidal anti-inflammatory drug (NSAID) with immunomodulatory effects was evaluated on IL-17, RORγt, IL-4 and GATA-3 gene expression in RA patients.Methods: Fourteen patients with RA who had an inadequate response to conventional treatments were included in this clinical trial. During this trial, patients were permitted to continue the conventional therapy excluding NSAIDs. G2013 was administered orally at dose of 500 mg twice daily for 12 weeks. The peripheral blood mononuclear cells (PBMCs) were collected before and after treatment to evaluate the gene expression levels of IL-4, GATA-3, IL-17 and RORγt.Results: Primary and secondary efficacy endpoints and Disease Activity Score (DAS) 28 showed an improvement after 12 weeks of treatment. G2013 has a potent efficacy on gene expression of these molecules, so that it could decrease IL-17 and RORγt levels and increase IL-4 and GATA-3 levels after 12 weeks of treatment. Reduction of IL-17 was statistically non-significant whereas for its transcription factor (RORγt) was statistically significant. Moreover, the gene expression results were in accordance with the clinical and preclinical assessments.Conclusion: G2013 as a natural novel drug showed a significant increase on IL-4 and GATA-3 and a significant decrease on RORγt gene expression after 12 weeks oral administration of this drug in RA patients. (Clinical trial identifier: IRCT2016092813739N5).

Colon-targeted dexamethasone microcrystals with pH-sensitive chitosan/alginate/Eudragit S multilayers for the treatment of inflammatory bowel disease.

Oral colon-targeted drug delivery has gained popularity as an effective strategy for treatment of inflammatory bowel disease (IBD). In this study, we prepared colon-targeted dexamethasone microcrystals (DXMCs) coated with multilayers of chitosan oligosaccharide (CH), alginate (AG), and finally Eudragit S 100 (ES) (ES1AG4CH5-DXMCs) using a layer-by-layer (LBL) coating technique. Particle size, surface charge, in vitro drug release, and in vivo anti-inflammatory activity of ES1AG4CH5-DXMCs were evaluated. ES1AG4CH5-DXMCs had an average particle size of 2.34 ±â€¯0.19 µm and a negative surface charge of - 48 ±â€¯9 mV. ES1AG4CH5-DXMCs demonstrated pH-dependent dexamethasone release, avoiding initial burst drug release in acidic pH conditions of the stomach and small intestine, and providing subsequent sustained drug release in the colonic pH. Importantly, ES1AG4CH5-DXMCs exhibited a significant therapeutic activity in a mouse model of colitis compared to other DXMCs. Overall, the LBL-coated DXMCs presented here could be a promising colon-targeted therapy for IBD.

Development of novel gastroretentive salbutamol sulfate-loaded sodium alginate-pectin bubble beads prepared by co-axial needle air-injection method and in vivo clinical evaluation by ultrasound studies.

In the present study, the salbutamol sulfate-loaded sodium alginate-pectin (SS-loaded SA-PEC) bubble beads have been optimized and evaluated for drug loading, in vitro drug release, in vivo floating behavior in the stomach, etc. Nine batches (F1-F9) of bubble beads with different SA and PEC contents were prepared by novel co-axial needle air-injection method and related to their percent drug loading efficiency (%DLE) and percent drug release at 4 h (%R4h) as response factors. The multivariate analysis has shown the effect of SA/PEC ratio, total polymer content, as well as their interaction on %DLE and %R4h. In the quantitative modeling, the satisfactory adjustment of the linear models (along with interaction terms) with the experimental data for both %DLE and %R4h has confirmed the findings of the multivariate analysis. The optimized SS-loaded SA-PEC bubble beads based on 2D (contours), 3D, desirability, and overlay plots has exhibited %DLE of 87.35 ±â€¯2.48% (n = 3 and error = 2.94%) and %R4h of 85.79 ±â€¯2.98% (n = 3 and error = 0.25%). The in vitro drug release studies have shown almost complete (≥85%) SS release from all the batches within 4-6 h in simulated gastric fluid (SGF) pH 1.2. The in vivo clinical findings by ultrasound studies have shown excellent floatation (>6 h) behavior of bubble beads in the upper gastrointestinal tract (GIT) and efficient stomach-specific gastroretention.

Pea-protein alginate encapsulation adversely affects development of clinical signs of Citrobacter rodentium-induced colitis in mice treated with probiotics.

The efficacy of two strains of Lactobacillus probiotics (Lactobacillus rhamnosus R0011 and Lactobacillus helveticus R0052) immobilized in microcapsules composed of pea protein isolate (PPI) and alginate microcapsules was assessed using a mouse model of Citrobacter rodentium-induced colitis. Accordingly, 4-week-old mice were fed diets supplemented with freeze-dried probiotics (group P), probiotic-containing microcapsules (group PE) (lyophilized PPI-alginate microcapsules containing probiotics), or PPI-alginate microcapsules containing no probiotics (group E). Half of the mice (controls, groups P, PE, and E) received C. rodentium by gavage 2 weeks after initiation of feeding. Daily monitoring of disease symptoms (abnormal behavior, diarrhea, etc.) and body weights was undertaken. Histopathological changes in colonic and cecal tissues, cytokine expression levels, and pathogen and probiotic densities in feces were examined, and the microbial communities of the distal colon mucosa were characterized by 16S rRNA sequencing. Infection with C. rodentium led to marked progression of infectious colitis, as revealed by symptomatic and histopathological data, changes in cytokine expression, and alteration of composition of mucosal communities. Probiotics led to changes in most of the disease markers but did not have a significant impact on cytokine profiles in infected animals. On the basis of cytokine expression analyses and histopathological data, it was evident that encapsulation materials (pea protein and calcium alginate) contributed to inflammation and worsened a set of symptoms in the cecum. These results suggest that even though food ingredients may be generally recognized as safe, they may in fact contribute to the development of an inflammatory response in certain animal disease models.

Design and characterization of emulsified spray dried alginate microparticles as a carrier for the dually acting drug roflumilast.

Roflumilast is a selective inhibitor of phosphodiesterase-4 isoenzyme in lung cells. Having psychiatric adverse reactions when administered orally affects negatively the patients' adherence to the drug. This work aimed to prepare emulsified spray dried alginate microparticles for the pulmonary delivery of roflumilast. Sodium alginate was used as microparticle-forming material, isopropyl myristate as an oil, Tween®80 as surfactant and calcium beta-glycerophosphate as cross-linking agent to enhance the mechanical properties of the particles. The prepared particles were evaluated for their encapsulation efficiency, particle size and in-vitro drug release. From the studied carriers, beta-cyclodextrin (CD) was the best regarding giving formulation with smaller particle size and more sustained drug release. The inhalation profile of CD-based microparticles was investigated using Anderson cascade impactor. The aerosolization profile of CD-based microparticles suggested their efficiency to deliver the drug deep in the lung. The CD-based microparticles possessed more inhibitory effects on the viability of A549 cells and on the pro-inflammatory cytokines (TNF-α, IL-6 and IL-10) compared to the pure drug. Hence, CD-based microparticles could regulate the tumorigenesis besides tumor-associated inflammation. Finally, CD-based microparticles showed more sustained bronchodilatation properties in healthy human volunteers when compared to Ventolin®HFA. CD-based microparticles proved to be a promising carrier for inhaled roflumilast in human.

Nasal delivery of chitosan/alginate nanoparticle encapsulated bee (Apis mellifera) venom promotes antibody production and viral clearance during porcine reproductive and respiratory syndrome virus infection by modulating T cell related responses.

In this study, we administered specially developed chitosan/alginate nanoparticle encapsulated BV (CH/AL-BV) which has slow-releasing properties and mucosal adhesiveness to pig via nasal route and evaluate whether it can facilitate systemic immune response and improve clearance of porcine reproductive and respiratory syndrome virus (PRRSV). The CH/AL-BV-administered group with PRRSV vaccination showed significantly enhanced Th1-related responses including a high population of CD4+ T lymphocyte and cytokine mRNA levels including interferon-gamma (IFN-γ) and interleukin (IL)-12 and increased PRRSV-specific IgG levels. In the PRRSV challenge experiment, the CH/AL-BV group showed a significant decrease of viral burden in the sera and tissues (lung and bronchial lymph node) and mild interstitial pneumonia signs on both lung gross examination and microscopic evaluation with high levels of PRRSV-specific IgG and viral neutralizing antibody. CH/AL-BV also effectively induced not only Th1-related immune responses including increase in portion of CD4+ T lymphocyte, cytokines (IFN-γ and IL-12), and transcriptional factors (STAT4 and T-bet), but also stimulated IFN-γ-secreting cell families such as CD4+ T lymphocytes and Th/memory cells. Interestingly, the CH/AL-BV group showed decrease in PRRSV-specific immune-suppressive actions, including the T regulatory cell population and its related cytokines (IL-10 and TGF-ß) and transcriptional factors (STAT5 and Foxp3). Therefore, nasal-delivered CH/AL-BV may effectively induce non-specific immune stimulating actions, particularly those related to Th1 responses and viral clearance activities against PRRSV infection. Based on these results, CH/AL-BV could be a promising strategy for overcoming the disadvantages of classical PRRSV vaccination and can be applied as a preventive agent against PRRSV and other viral diseases, particularly those with immune-suppressive characteristics.

Incorporation of beads into oral films for buccal and oral delivery of bioactive molecules.

The association of alginate beads and guar-gum films in a single delivery system was idealized to promote a more effective buccal and oral delivery of bioactive molecules. A response surface method (experimental design approach) was performed to obtain optimal formulations of alginate beads to be incorporated into guar gum oral films as combined buccal and oral delivery systems for caffeine delivery. The combined formulation was further characterized regarding physicochemical properties, drug release, cell viability and buccal permeability. Beads average size, determined by dynamic light scattering (DLS), was of 3.37 ±â€¯6.36 µm. Film thickness was set to 62 µm. Scanning electron microscopy micrographs revealed that beads were evenly distributed onto the film matrix and beads size was in accordance to data obtained from DLS analysis. Evaluation of Fourier-transform infrared spectra did not indicate the formation of new covalent bonds between the matrix of guar-gum films, alginate beads and caffeine. In vitro release assays by dialysis membrane allowed understanding that the combination of guar-gum films and alginate beads assure a slower release of caffeine when compared with the delivery profile of free caffeine from alginate beads or guar-gum films alone. MTT assay, performed on human buccal carcinoma TR146 cell line, allowed concluding that neither guar-gum film, alginate beads nor guar-gum film incorporated into alginate beads significantly compromised cell viability after 12 h of exposure. As demonstrated by in vitro permeability assay using TR146 human buccal carcinoma cell lines, combination of guar-gum films and alginate beads also promoted a slower release and, thus, lower apparent permeability (1.15E-05 ±â€¯3.50E-06) than for caffeine solution (2.68E-05 ±â€¯7.30E-06), guar-gum film (3.12E-05 ±â€¯4.70E-06) or alginate beads (2.01E-05 ±â€¯3.90E-06). The conjugation of alginate beads within an orodispersible film matrix represents an effective oral/buccal delivery system that induces a controlled release along with an enhanced intimate contact with cell layers that may promote higher in vivo bioavailability of carried drugs.

The impact of polymers on 3D microstructure and controlled release of sildenafil citrate from hydrophilic matrices.

Sildenafil citrate has short biological half-life in humans. Thus, matrix tablets of controlled release were designed and prepared by compaction on the basis of hydrophilic polymers, i.e. HPMC, sodium alginate, carbomer, poloxamer and their mixtures. The impact of these polymers on sildenafil release in vitro and its pharmacokinetics in vivo was evaluated. Since drug release rate from hydrophilic matrices can be govern by the porosity of the matrix, the microstructure of tablets was studied using X-ray microcomputed tomography. 3D network of either open (percolating) or closed (non-percolating) pores was reconstructed. The tortuosity and the diameter of both kinds of pores were determined. Their spatial distribution within the matrix was analyzed in linear and radial direction. Polymer-dependent characteristics of the open pores (Ø > 2 µm) architecture was shown. The release profiles of sildenafil from matrix tablets fitted to Korsmeyer-Peppas model (r2: 0.9331-0.9993) with either Fickian diffusion or anomalous transport involved. Mean dissolution time (MDT) from tablets made of HPMC, carbomer or a mixture of HPMC and sodium alginate (2:1) was ca. 100 min, which was more than twelve times longer as compared to matrices prepared of silicified microcrystalline cellulose (MDT = 8 min). MDT correlated with the number of the open pores (Pearson's r = 0.94). Sustained release of sildenafil from ground carbomer tablets reflected in the slow absorption of the drug (tmax = 5.0 ±â€¯1.2 h) in vivo and the relative bioavailability of 151%. Interestingly, the relative bioavailability of sildenafil from binary matrices composed of HPMC and sodium alginate (2:1) was almost four times higher than that of sildenafil alone.

A phase I/II randomized, controlled, clinical trial for assessment of the efficacy and safety of ß-D-mannuronic acid in rheumatoid arthritis patients.

BACKGROUND: Following the potent efficacy of ß-D-mannuronic acid (M2000) in phase I/II trial in ankylosing spondylitis patients, the present clinical trial was conducted to evaluate the efficacy, safety, and tolerability of this novel drug in rheumatoid arthritis (RA) patients who had inadequate response to conventional therapy. METHOD: The study was a 12-week randomized, controlled, phase I/II clinical trial with two treatment arms: M2000 and conventional treatment. Patients who had RA according to the modified American College of Rheumatology (ACR) criteria, with active disease at baseline also inadequate response to conventional therapy, were enrolled in this study. M2000 was administrated at a dose of two capsules (500 mg) per day orally during a period of 12 weeks. The primary endpoint was the proportion of patients fulfilling the ACR 20% improvement criteria after 12 weeks of M2000 therapy. Moreover, the patients were also followed up for safety. RESULTS: There were no statistically significant differences between treatment and conventional groups at baseline characteristics. The ACR20 response rate was significantly higher among M2000-treated patients than conventional-treated control, so that 74% of patients in treatment group showed an ACR20 response after 12 weeks of M2000 therapy (74 versus 16%; P = 0.011). 10% of M2000-treated patients and 57.1% of conventional-treated patient's adverse events occurred during this study. CONCLUSION: Treatment with M2000 in combination with conventional therapy showed a significantly superior efficacy along with a high safety profile compared to conventional-treated patients. Thereby, M2000 might be suggested as a suitable option in the treatment of RA.

A novel dressing for the combined delivery of platelet lysate and vancomycin hydrochloride to chronic skin ulcers: Hyaluronic acid particles in alginate matrices.

The aim of the present work was to develop a medication allowing for the combined delivery of platelet lysate (PL) and an anti-infective model drug, vancomycin hydrochloride (VCM), to chronic skin ulcers. A simple method was set up for the preparation of hyaluronic acid (HA) core-shell particles, loaded with PL and coated with calcium alginate, embedded in a VCM containing alginate matrix. Two different CaCl2 concentrations were investigated to allow for HA/PL core-shell particle formation. The resulting dressings were characterized for mechanical and hydration properties and tested in vitro (on fibroblasts) and ex-vivo (on skin biopsies) for biological activity. They were found of sufficient mechanical strength to withstand packaging and handling stress and able to absorb a high amount of wound exudate and to form a protective gel on the lesion area. The CaCl2 concentration used for shell formation did not affect VCM release from the alginate matrix, but strongly modified the release of PGFAB (chosen as representative of growth factors present in PL) from HA particles. In vitro and ex vivo tests provided sufficient proof of concept of the ability of dressings to improve skin ulcers healing.

Bone regeneration in osteoporosis by delivery BMP-2 and PRGF from tetronic-alginate composite thermogel.

As the life expectancy of the world population increases, osteoporotic (OP) fracture risk increase. Therefore in the present study a novel injectable thermo-responsive hydrogel loaded with microspheres of 17ß-estradiol, microspheres of bone morphogenetic protein-2 (BMP-2) and plasma rich in growth factors (PRGF) was applied locally to regenerate a calvaria critical bone defect in OP female rats. Three systems were characterized: Tetronic® 1307 (T-1307) reinforced with alginate (T-A), T-A with PRGF and T-A-PRGF with microspheres. The addition of the microspheres increased the viscosity but the temperature for the maximum viscosity did not change (22-24 °C). The drugs were released during 6 weeks in one fast phase (three days) followed by a long slow phase. In vivo evaluation was made in non-OP and OP rats treated with T-A, T-A with microspheres of 17ß-estradiol (T-A-ßE), T-A-ßE prepared with PRGF (T-A-PRGF-ßE), T-A-ßE with microspheres of BMP-2 (T-A-ßE-BMP-2) and the combination of the three (T-A-PRGF-ßE-BMP). After 12 weeks, histological and histomorphometric analyzes showed a synergic effect due to the addition of BMP-2 to the T-A-ßE formulation. The PRGF did not increased the bone repair. The new bone filling the OP defect was less mineralized than in the non-OP groups.

Injectable collagen/RGD systems for bone tissue engineering applications.

Imbalance crosslink density and polymer concentration gradient is formed within the traditional alginate hydrogel using calcium chloride as a crosslinking agent in external gelation for instantaneously process. In this studying, type I collagen (Col I) blended calcium salt form of poly(γ-glutamic acid) (γCaPGA) was mixing with RGD-modified alginate with convenient gelation process and suitable for practical use. The hydrophilicity of the resulting hydrogels was evaluated through swelling tests, water retention capacity tests, and water vapor permeation tests. Mineralization was qualitatively evaluated by alizarin red dyeing at day 14, verifying the deposition of calcium. The in vitro osteogenic differentiation is monitored by determining the early and late osteocalcin (OCN) and osteopontin (OPN) markers with MG63 cells. Obtained results demonstrated that no extremely changes in mechanical properties. After 14 days of culture, hydrogels significantly stimulated OCN/OPN gene expressions and MG63 cell proliferation. Unusually, γCaPGA with RGD-modified alginate appeared better calcium deposition in 14 days than the other. However, addition of Col I can counterpoise RGD effect in blood coagulation and platelet adhesion made the hydrogel more flexibility and selectively in use. This studying provided that non-covalently crosslinked hydrogel by γCaPGA with alginate can be upgrading by RGD and Col I in water uptake capability, obviously effective for MG63 cells and are remarkably biocompatible and exhibited no cytotoxicity. Moreover, results also displayed the injectable process without complicated procedure, have high cost/performance ratio and have great potential for bone regeneration.

Oral administration effects of ß-d-mannuronic acid (M2000) on Th17 and regulatory T cells in patients with ankylosing spondylitis.

BACKGROUND: To explore the effects of ß-d-mannuronic acid (M2000) on levels of Th17, regulatory T (Treg) cells and their related cytokines in patients with ankylosing spondylitis (AS). METHODS: 30 AS patients and 15 age and sex-matched healthy individuals were enrolled in this study. The frequencies of Th17 and Treg cells and serum levels of related cytokines were measured by flow cytometry analysis and ELISA respectively, before (baseline) and 3 months after M2000 therapy. RESULTS: Significantly higher baseline Th17 cells and serum IL-17, TNF-α and IL-6 were observed in AS patients than in normal controls, whereas baseline levels of Treg cells and serum IL-10 were not significantly different between AS patients and healthy controls. After M2000 therapy, frequencies of Th17 and serum levels of IL-17 and IL-6 significantly decreased in AS patients. The frequencies of Treg cells and serum level of IL-10 were not significantly changed, in comparison to before therapy. Moreover, the correlation analysis showed that frequencies of Th17 and levels of IL-17, TNF-α and IL-6 were positively correlated with Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and Bath Ankylosing Spondylitis Functional Index (BASFI) scores, whereas Treg cells were revealed to be negatively correlated with BASDAI and BASFI scores. CONCLUSIONS: It can be concluded that the oral administration of M2000 as a novel NSAID with the immunosuppressive property that down-regulates Th17 and Th17-related cytokines and facilitates the correction of the Th17/Treg imbalance can be effective in the process of AS treatment.

Alginate as a potential diphase solid dispersion carrier with enhanced drug dissolution and improved storage stability.

The objective of this study was to explore the feasibility of using alginate as a promising diphase solid dispersion carrier to enhance dissolution rate of BCS II drugs with improved stability. Taking lovastatin and indomethacin as model drugs, solvent evaporation method was used to prepare solid dispersions. The drug/polymer compatibility was predicted by Hansen solubility parameter and the drug/polymer ratio was screened based on dissolution study, drug existing state in solid dispersion was characterized by DSC and XRPD. Accelerated stability of the solid dispersion was assessed and compared with that of HPMCAS based system. Phase behavior of the solid dispersion before and after stability study was characterized using polar microscope and Raman mapping. It was found that the optimal drug/alginate ratio was drug dependent and drug existing state was related to drug/alginate miscibility. Stability studies revealed that alginate improved the stability of solid dispersions regardless of drug existing state and a better stability was obtained compared to HPMCAS based system. Raman mapping and SEM study revealed that micro phase separation of solid dispersion was the main reason for the slight decrease in drug dissolution after accelerating experiment. In conclusion, alginate can be used as a promising diphase solid dispersion carrier with significantly improved dissolution rate and storage stability.