Rosuvastatin effects on the HDL proteome in hyperlipidemic patients.

The advancements in proteomics have provided a better understanding of the functionality of apolipoproteins and lipoprotein-associated proteins, with the HDL lipoprotein fraction being the most studied. The focus of this study was to evaluate the HDL proteome in dyslipidemic subjects without an established cardiovascular disease, as well as to test whether rosuvastatin treatment alters the HDL proteome. Patients with primary hypercholesterolemia or mixed dyslipidemia were assigned to 20 mg/day rosuvastatin and blood samples were drawn at study entry and after 12 weeks of treatment. A label-free LC-MS/MS protein profiling was conducted, coupled with bioinformatics analysis. Sixty-nine HDL proteins were identified, belonging to four main biological function clusters: lipid transport and metabolism; platelet activation, degranulation, and aggregation, wound response and wound healing; immune response; inflammatory and acute phase response. Five HDL proteins showed statistically significant differences in the abundance (Anova ≤ 0.05), before and after rosuvastatin treatment. Platelet factor 4 variant (PF4V1), Pregnancy-specific beta-1-glycoprotein 2 (PSG2), Profilin-1 (PFN1) and Keratin type II cytoskeletal 2 epidermal (KRT2) showed decreased expressions, while Integrin alpha-IIb (ITGA2B) showed an increased expression after treatment with rosuvastatin. The ELISA validation of PFN1 segregated the subjects into responders and non-responders, as PFN1 levels after rosuvastatin were shown to mostly depend on the subjects' inflammatory phenotype. Findings from this study introduce novel insights into the HDL proteome and statin pleiotropism.

Antioxidative Effects of Rosuvastatin in Low-to-Moderate Cardiovascular Risk Subjects.

Background: Although vast clinical evidence supports the oxidative CVD hypothesis, little is known on the effects of statins on LDL/HDL oxidative functionality. Therefore, the aim of this study was to evaluate the antioxidative effects of rosuvastatin by monitoring the susceptibility of LDL to oxidation and the antioxidative HDL potential in low-to-moderate CV risk subjects. Methods: 40 adult ambulatory patients (aged 53.8±10.9 years, 27 women and 13 men) were included in the study. Data was collected from patients' records, physical examination, and blood sampling. Subjects were prescribed rosuvastatin at 20mg/day. Traditional risk-factors/indicators, lipid parameters, inflammatory/immune markers, LDL susceptibility to oxidation and HDL antioxidative potential were monitored and statistically analyzed with t-test, Chi-square test, one-way ANOVA, Mann-Whitney, and Kruskal-Wallis tests. Multivariate logistic regression analyses were made. Results were considered significant when p≤0.05. Results: 67% of the patients showed lower susceptibility of LDL to oxidation after rosuvastatin treatment (p=0.03), with no significant effect on baseline LDL oxidation and lag time. All three LDL oxidative indices were seen to be dependent on the subjects' lipid profile, hemoglobin levels and the IL-1α and IL-8 pro-inflammatory marker levels. 53% of the patients showed higher HDL antioxidative capacity after treatment, but without statistical significance (p=0.07). Increased antioxidative potential of HDL with rosuvastatin treatment was more likely in males (OR=9.350; p=0.010), and subjects achieving lower post-treatment CV relative risk levels (higher CV risk reduction) (OR=0.338; p=0.027). Conclusions: This study suggests the need of a comprehensive approach when investigating oxidative stress and LDL/HDL functions, especially in low-to-moderate CVD risk subjects.

Anti-inflammatory and immunomodulatory effects of rosuvastatin in patients with low-to-moderate cardiovascular risk.

Statins have shown anti-inflammatory pleiotropic effects in subjects with/at risk of cardiovascular disease. The aim of this study was to evaluate the inflammatory/immunomodulatory properties of rosuvastatin in subjects at low-to-moderate cardiovascular risk. Data was collected from patients' records, physical examination and blood sampling. Subjects were assigned to rosuvastatin 20 mg per day. Rosuvastatin significantly decreased C-reactive protein (p = 0.045), and increased vascular endothelial growth factor (p = 0.004) and epidermal growth factor (p = 0.009). A multivariate analysis identified total cholesterol (p = 0.027) and vascular endothelial growth factor (p = 0.011) to be independently associated with rosuvastatin treatment. Given beneficial/harmful role of growth factors, vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF), in cardiovascular disease, one would suggest the need for routine monitoring of growth factor levels, especially in patients on long-term statin therapy.

The impact of molecular tumor profiling on the design strategies for targeting myeloid leukemia and EGFR/CD44-positive solid tumors.

Nanomedicine has emerged as a novel cancer treatment and diagnostic modality, whose design constantly evolves towards increasing the safety and efficacy of the chemotherapeutic and diagnostic protocols. Molecular diagnostics, which create a great amount of data related to the unique molecular signatures of each tumor subtype, have emerged as an important tool for detailed profiling of tumors. They provide an opportunity to develop targeting agents for early detection and diagnosis, and to select the most effective combinatorial treatment options. Alongside, the design of the nanoscale carriers needs to cope with novel trends of molecular screening. Also, multiple targeting ligands needed for robust and specific interactions with the targeted cell populations have to be introduced, which should result in substantial improvements in safety and efficacy of the cancer treatment. This article will focus on novel design strategies for nanoscale drug delivery systems, based on the unique molecular signatures of myeloid leukemia and EGFR/CD44-positive solid tumors, and the impact of novel discoveries in molecular tumor profiles on future chemotherapeutic protocols.

Association of TNF-a (rs361525 and rs1800629) with susceptibility to cervical intraepithelial lesion and cervical carcinoma in women from Republic of North Macedonia.

Cervical cancer (CCa) is one of the most common malign diseases in women associated with human papillomavirus (HPV). The virus is an initiating factor, but not sufficient for the development of cervical intraepithelial lesions (CIN) and CCa. The disease might be a result of the influence of host's genetic factors and polymorphisms in inflammatory-related genes that modify the immune response to HPV and attribute to cancer susceptibility. We carried out a study to determine the association between TNF-a-238G/A and TNF-a-308 G/T polymorphisms with HPV-positive CIN and CCa in women living in the Republic of North Macedonia. Using multiplex SNaPshot analysis for single nucleotide polymorphisms (SNPs), we analysed the genotype and allele distributions of TNF-a-238G/A and TNF-a-308 G/T in 134 cases (HPV-positive and histologically confirmed CIN and CCa) and in 113 controls (cytological and HPV-negative women). For further analysis, the case group was stratified in three subgroups (all cases: CINs+ CCa- group; CIN2+ -group and CIN1- group). Data analysed using the odds ratio (OR) and chi-square test showed the frequency of AA genotypes and A alleles are not significantly higher in cases compared to the controls for both SNPs: AA of TNF-a-238 (0.7% versus 0%) and TNF-a-308 (1.5% versus 0.9%) as well as A allelic frequency (3.0% versus 1.7%) and (13.1% versus 10.6), respectively. The comparison of the case's subgroups with the control group did not show a statistically significant difference. Compared to controls, TNF-a-238G/A and TNF-a-308 G/T are not associated with the risk of HPV associated CIN or CCa in the studied women.

AKR1D1*36 C>T (rs1872930) allelic variant is associated with variability of the CYP2C9 genotype predicted pharmacokinetics of ibuprofen enantiomers - a pilot study in healthy volunteers.

The relative contribution of CYP2C9 allelic variants to the pharmacokinetics (PK) of ibuprofen (IBP) enantiomers has been studied extensively, but the potential clinical benefit of pharmacogenetically guided IBP treatment is not evident yet. The role of AKR1D1*36C>T (rs 1872930) allelic variant in interindividual variability of CYP450 mediated drug metabolism was recently elucidated. A total of 27 healthy male subjects, volunteers in IBP single-dose two-way cross-over bioequivalence studies were genotyped for CYP2C9*2, CYP2C9*3 and AKR1D1*36 polymorphisms. The correlation between CYP2C9 and AKR1D1 genetic profile and the PK parameters for S-(+) and R-(-)-IBP was evaluated. Remarkable changes in the PK values pointing to reduced CYP2C9 enzyme activity were detected only in the CYP2C9*2 allelic variant carriers. Statistically significant association between the AKR1D1*36 allele and the increased IBP metabolism (low AUC0-t and 0-∞, high Cltot and short tmax values for both enantiomers) was observed in subjects carrying the CYP2C9 *1/*3 or CYP2C9*1/*1 genotype. The clinical value of concomitant CYP2C9 and AKR1D1 genotyping has to be further verified.

Calculations of 13 C NMR chemical shifts and F-C coupling constants of ciprofloxacin.

Ciprofloxacin is a widely used fluoroquinolone antibiotic. In this work, a comprehensive evaluation of MP2 and DFT with different functionals and basis sets was carried out to select the most suitable level of theory for the study of the NMR properties of ciprofloxacin. Their relative predictive capabilities were evaluated comparing the theoretically predicted and experimental spectral data. Our computational results indicated that in contrast to the solid state, the molecule of ciprofloxacin does not exist as a zwitterion in gaseous state. The results of the calculations of the chemical shifts most close to the experimental were obtained with B3LYP/aug-cc-pVDZ. The F-C coupling constants were calculated systematically with different DFT methods and several basis sets. In general, the calculations of the coupling constants with the BHandH computational method including the applied in this work 6-311++G**, EPRII, and EPRIII basis sets showed a good reproducibility of the experimental values of the coupling constants.

Rational development of nanomedicines for molecular targeting in periodontal disease.

Recent advances in understanding the etiology and pathogenesis of periodontal disease and polymicrobial synergy in the dysbiotic oral microbial community endorsed novel therapeutic targets and assured further improvement in periodontal disease treatment. Moreover, understanding of the events at the molecular level inspired the researchers to alleviate the stress from the disease by applying the bottom-up approach and delivering the drugs at the site of action, using nanoscale medicines. This review is focused on promising strategies for rational design of nanopaharmaceuticals for periodontal disease treatment based on novel therapeutic targets and the potential of advanced concepts for inflammation cascade targeting. Due to their size, nanomedicines are capable to interact with the elements of the immune system through cell receptor binding and to subsequently influence specific intracellular signaling pathways activation. They might also interfere with different signaling molecules continuously involved in the disease progression, in order to abolish cell activation and block the production of proinflammatory substances. Different biomacromolecules can be trafficked to the site of action using nanomedicines for gene targeting: i) decoy oligodeoxynucleotide (ODN) for suppression of NF-κB transcription activity, ii) DNA therapeutics for modulation of cell inflammatory response and iii) siRNA for cytokine production silencing. However, despite the potential of the nanotechnology for improvement of periodontal disease treatment, the translation of nano-drug delivery systems to clinical therapy is hindered by the lack of standard procedures for proper safety and efficacy profile evaluation.

Design and biological response of doxycycline loaded chitosan microparticles for periodontal disease treatment.

The aim of this study was to develop chitosan (CS) microparticulated mucoadhesive drug delivery system (DDS) with improved therapeutic performance and biological responce. Ionotropic gelation/spray drying process was used for preparation of doxycycline hyclate (DOXY) loaded low and medium molecular weight (LMw and MMw) CS/sodium tripolyphosphate microparticles (CS/TPP MPs), further coated with ethyl cellulose (EC) using coacervation/solvent displacement technique. The relevant physico-chemical and biopharmaceutical properties were optimized using experimental design approach. Both coated and uncoated CS/TPP MPs showed high mucoadhesive potential and did not affect the viability of the tested epithelial cell line. The MPs induced slow and gradual apoptotic response in murine macrophage cell line RAW 264.7 and the observed effect depended upon formulation type and MP concentration. Biological effect of the CS-based MPs observed in our experiments point to synergism of the biological response of the carrier with the anti-inflammatory effect of DOXY.

Lactobacillus casei Encapsulated in Soy Protein Isolate and Alginate Microparticles Prepared by Spray Drying.

This article presents a novel formulation for preparation of Lactobacillus casei 01 encapsulated in soy protein isolate and alginate microparticles using spray drying method. A response surface methodology was used to optimise the formulation and the central composite face-centered design was applied to study the effects of critical material attributes and process parameters on viability of the probiotic after microencapsulation and in simulated gastrointestinal conditions. Spherical microparticles were produced in high yield (64%), narrow size distribution (d50=9.7 µm, span=0.47) and favourable mucoadhesive properties, with viability of the probiotic of 11.67, 10.05, 9.47 and 9.20 log CFU/g after microencapsulation, 3 h in simulated gastric and intestinal conditions and four-month cold storage, respectively. Fourier-transform infrared spectroscopy confirmed the probiotic stability after microencapsulation, while differential scanning calorimetry and thermogravimetry pointed to high thermal stability of the soy protein isolate-alginate microparticles with encapsulated probiotic. These favourable properties of the probiotic microparticles make them suitable for incorporation into functional food or pharmaceutical products.

Synbiotic loaded chitosan-Ca-alginate microparticles reduces inflammation in the TNBS model of rat colitis.

New therapeutic strategies against inflammatory bowel disease (IBD) consider the usage of probiotics, prebiotics and synbiotics as beneficial for the intestinal microbial balance. Limitations of such an approach are addressed into difference in survival, persistence, colonization and variable effects among different probiotic strains, lack in understanding of probiotic mechanisms of action, as well the complex etiology of IBD. The anti-inflammatory activity of Lactobacillus casei 01 (L. casei 01) was assessed in trinitrobenzenesulphonic (TNBS) acid model of rat colitis when the probiotic was used alone and/or in combination with oligofructose-enriched inulin (Synergy 1), and as synbiotic (L. casei 01+Synergy 1) loaded chitosan-Ca-alginate microparticles; all suspended in ayran. The results from the probiotic/synbiotic treatments (8.5-8.9log CFU g-1L. casei 01 and 1.5% Synergy 1) have shown reduction in the colonic damage and increased lactobacilli counts in feces. Lactobacilli translocation to sterile extra-intestinal organs demonstrated acceptable safety of the probiotic strain used. The best effect at reducing inflammation and lesions associated with a significant decline in myeloperoxidase (MPO) activity was observed in rats that received synbiotic microparticles. This finding suggests colon targeted delivery of the probiotics/synbiotics, as an advantageous approach in prevention and treatment of IBD.

Original research paper. Hydrazinyldiene-chroman-2,4-diones in inducing growth arrest and apoptosis in breast cancer cells: Synergism with doxorubicin and correlation with physicochemical properties.

This study evaluates the effects of previously synthesized hydrazinyldiene-chroman-2,4-diones on cell proliferation and apoptosis, cell cycle distribution and migration capacity of MCF-7 breast cancer cells in synergy with doxorubicin. Physicochemical properties of the synthesized compounds were correlated with their structure and activity. Significant cell viability decrease in comparison with the effect of doxorubicin alone and the reference 4-hydroxycoumarin was observed when combination treatment comprising doxorubicin and the title compounds was applied. Synergistic effect with doxorubicin was also observed in down-regulation of phospho-Thr308Akt levels, confirming reduced proliferation and increased apoptosis. Combined treatment increased the percentage of cells arrested at the G2/M stage. Additive inhibition of cell migration was also observed, pointing to the possibility of reducing the risk of metastases. With their solubility profile and log D7.4, all the synthesized compounds follow Lipinski's rule of five for good permeability (absorption) potential.

Secondary Hodgkin Lymphoma and Myelodysplastic Syndrome (MDS) After Paclitaxel-Carboplatin Treatment in a Patient with Small Cell Lung Cancer.

Herein synchronous occurrence of Hodgkin lymphoma and secondary myelodysplastic syndrome in a 60 year old male patient with small cell lung cancer treated with combined chemotherapy (carboplatin and paclitaxel) and radiotherapy is presented. The objective of this report is to stress the importance of documenting and monitoring adverse drug reactions that arise from chemotherapy. After four years of treatment with the combined chemotherapy, the patient presented inguinal lymphadenopathy and enlarged lymph nodes and histopathology rapport was suggestive for plasmacytoid variant of Castleman disease. Three years later, biopsy of lymph node was performed and diagnosis of Hodgkin lymphoma - mixed cellularity has been established. Molecular analyses revealed presence of dominant monoclonal population of the immunoglobulin genes in the oligo/monoclonal background. Bone marrow biopsy findings suggested secondary myelodysplasia and revealed signs of hematopoietic cells dismaturation with signs of megaloblastic maturation of the erytropoetic lineage, appearance of ALIP (abnormal localization of immature precursors) in the myeloid lineage and dysplastic megakaryocytes. In addition, an increased level of polyclonal plasmacytes (lambda vs kappa was 60%:40%) was found. Hodgkin lymphoma and MDS occurring after 4 years of carboplatin/paclitaxel therapy might be contributed to the accumulation of alkylator-related DNA damage. This emphasize the need of outlining a monitoring plan regarding development of secondary leukemia and other malignant hematological proliferations should be outlined in the protocols.

Effects of Rosuvastatin Versus Atorvastatin, Alone or in Combination, on Lipoprotein (a).

BACKGROUND: There are little evidences about the therapeutic efficacy of different lipid-lowering agents in the reduction of elevated lipoprotein(a) [Lp(a)]. OBJECTIVE: testing the effect of different lipid-lowering agents on elevated Lp(a). METHODS: prospective interventional study performed in patients with CAD, or high CAD risk, with Lp(a), >50 mg/dL. Lp(a), total cholesterol (C), HDL-C, LDL-C, triglycerides (TGs), apolipoprotein (Apo) A1, Apo B, enzymes of myocyte and hepatic injury were comparatively analyzed between 4 lipid-lowering strategies: rosuvastatin (R group) 40 mg, atorvastatin (A group) 80 mg, atorvastatin 40 mg add-on micronized fenofibrate (A+F group), and atorvastatin 40 mg add-on 1 g extended-release niacin (A+ERN group). Comparison was made for their therapeutic efficacy on Lp(a), and safety. RESULTS: 87 patients with mean Lp(a) 94.6 ± 39.6 mg/dL were analyzed. Groups: 25 patients in the R, 22 in the A, 20 in the A+F and 20 in A+ERN group. Significant reduction in all lipid fractions in all treatment groups was reported after 6 months. The average reduction of Lp(a) was 15.9 ± 21.0 mg/dL, with: 18.2 ± 24.8 (P = 0.001) in the R group, 17.3 ± 10.4 (P = 0.001) in A+F, 19.5 ± 10.9 (P = 0.001) in A+ERN and the lowest in the A group (11.24 ± 22.91, P = 0.032). No adverse effects were observed in any of the treatment groups. CONCLUSIONS: When compared with atorvastatin, it seems that rosuvastatin can achieve more significant decrease of Lp(a).The efficacy of the second one can be increased by adding fibrate or ERN.

Implementation of quality by design principles in the development of microsponges as drug delivery carriers: Identification and optimization of critical factors using multivariate statistical analyses and design of experiments studies.

Microsponges drug delivery system (MDDC) was prepared by double emulsion-solvent-diffusion technique using rotor-stator homogenization. Quality by design (QbD) concept was implemented for the development of MDDC with potential to be incorporated into semisolid dosage form (gel). Quality target product profile (QTPP) and critical quality attributes (CQA) were defined and identified, accordingly. Critical material attributes (CMA) and Critical process parameters (CPP) were identified using quality risk management (QRM) tool, failure mode, effects and criticality analysis (FMECA). CMA and CPP were identified based on results obtained from principal component analysis (PCA-X&Y) and partial least squares (PLS) statistical analysis along with literature data, product and process knowledge and understanding. FMECA identified amount of ethylcellulose, chitosan, acetone, dichloromethane, span 80, tween 80 and water ratio in primary/multiple emulsions as CMA and rotation speed and stirrer type used for organic solvent removal as CPP. The relationship between identified CPP and particle size as CQA was described in the design space using design of experiments - one-factor response surface method. Obtained results from statistically designed experiments enabled establishment of mathematical models and equations that were used for detailed characterization of influence of identified CPP upon MDDC particle size and particle size distribution and their subsequent optimization.

Potential antiproliferative effect of isoxazolo- and thiazolo coumarin derivatives on breast cancer mediated bone and lung metastases.

The study highlights the current progress in the development of coumarin scaffolds for drug discovery as novel anticancer agents in metastatic breast cancer. Eight compounds, combining the coumarin core and five membered heterocycles (isoxazoles and thiazoles) in hydrazinyldiene- -chroman-2,4-diones, were characterized in terms of a potential antiproliferative effect on bone (SCP1833) and lung (SCP4175) metastatic breast cancer cell lines using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Cell viability was evaluated after 48 and 72 h of treatment and the 50 % inhibitory concentrations were determined. The results demonstrated dose- and time-dependent activity, with the most potent molecules having a thiazole moiety, without or with additional methyl group(s) attached to the carbon(s) at position(s) 5 and/or 4 in the thiazole ring. These molecules possessed significantly higher potency against both test cell lines compared to 4-hydroxycoumarin.

Formulation and characterization of ORMOSIL particles loaded with budesonide for local colonic delivery.

In this study, hybrid silica xerogel particles were developed as carriers of budesonide (BDS) for efficient local treatment of inflammatory bowel diseases (IBD). Organically modified silica particles (ORMOSILs) were prepared by co-condensation of 3-aminopropyltriethoxysilane (APTES) and tetraethyl orthosilicate (TEOS) by an ambient temperature acid catalysed sol-gel process followed by spray-drying. Formulation for preparation of BDS-loaded particles was optimized and their physicochemical parameters and drug release profiles were evaluated in vitro. Optimal formulation had a small particle size (mean diameter of 1.45±0.02µm) with unimodal narrow size distribution and high encapsulation efficiency (98.0 ± 1.85%). Due to the positive surface charge originated from amino group of APTES, ORMOSILs showed excessive mucoadhesiveness in comparison to native TEOS particles. The drug release decreased with increasing pH from 2.0 to 7.4. In order to avoid undesirable erroneous performance in the upper GI tract, particles were additionally coated with Eudragit(®) FS 30D, as a barrier to the drug release at pH range from 2.0 to 7.0. After Eudragit(®) FS 30D coating, the release of BDS in acidic media was sustained, while no significant differences in drug release were observed at pH 7.4. In conclusion, pH-responsive ORMOSILs showed great potential for efficient BDS delivery to the colon region.

Optimization of the formulation for preparing Lactobacillus casei loaded whey protein-Ca-alginate microparticles using full-factorial design.

CONTEXT: This article presents specific approach for microencapsulation of Lactobacillus casei using emulsion method followed by additional coating with whey protein. METHODS: Experimental design was employed using polynomial regression model at 2nd level with three independent variables, concentrations of alginate, whey protein and CaCl2. Physicochemical, biopharmaceutical and biological properties were investigated. RESULTS: In 11 series generated, negatively charged microparticles were obtained, with size 6.99-9.88 µm, Ca-content 0.29-0.47 mg per 10 mg microparticles, and viability of the probiotic 9.30-10.87 log10CFU/g. The viability after 24 hours in simulated gastrointestinal conditions was between 3.60 and 8.32 log10CFU/g. DISCUSSION: Optimal formulation of the microparticles that ensures survival of the probiotic and achieves controlled delivery was determined: 2.5% (w/w) alginate, 3% (w/w) CaCl2 and 3% (w/w) whey protein. CONCLUSION: The advantageous properties of the L. casei-loaded microparticles make them suitable for incorporation in functional food and/or pharmaceutical products.

Poly(lactide-co-glycolide) microparticles as systems for controlled release of proteins -- preparation and characterization.

Poly(DL-lactide-co-glycolide) (PDLLGA) and poly(L-lactide-co-glycolide) (PLLGA) copolymers were prepared by bulk ring opening polymerization of lactide and glycolide and characterized by GPC, FTIR, 1H NMR and DSC. Copolymers with different molar masses at a constant lactide/glycolide ratio were used for preparation of bovine serum albumin (BSA)-loaded microparticles by the double emulsion w/o/w method. The influence of the copolymer molar mass and composition on the microparticle morphology, size, yield, degradation rate, BSA-loading efficiency and BSA release profile were studied. For microparticles prepared from PDLLGA copolymers, a biphasic profile for BSA release was found and for those made from PLLGA copolymers the release profile was typically triphasic; both of them were characterized by high initial burst release. Possible reasons for such behavior are discussed.

Biodistribution of 131I-BSA loaded gelatin microspheres after peroral application to BALB/c mice--particle size study.

Biodistribution studies of radiolabelled 131I-BSA loaded gelatin microspheres were carried out on BALB/c mice after peroral administration. To two groups, radiolabelled 131I-BSA gelatin microspheres of different particle size, 1.2 +/- 1.1 microm and 7.0 +/- 1.2 microm, were administered orally. To the control group, a solution of 131I-BSA was administered orally as well. Biodistribution was followed periodically within 15 days as the percent of total radioactivity present in the stomach and small intestine with Peyer's patches and mesentery, in colon with Peyer's patches, appendix and mesentery, in liver, spleen, blood, kidney, lungs and heart. Preliminary in vitro biodegradation and drug release studies confirmed the potential of gelatin microspheres to protect the antigen of interest from enzymatic degradation in the gut, and to release it in a controlled manner. The biodistribution data confirmed that particle uptake into Peyer's patches and passage to the liver and spleen via the mesentery lymph supply and nodes increased with decreasing particle size.