Structure and pharmacokinetics/pharmacodynamics of the anticoagulant tetradecasaccharide oHG-14 as an intrinsic tenase inhibitor.

The intrinsic tenase complex (iXase) is an attractive antithrombotic target to treat or prevent pathological thrombosis with negligible bleeding risk. Fucosylated glycosaminoglycan (FG) is a promising anticoagulant by inhibiting iXase. A depolymerized FG (dHG-5) as an anticoagulant has been approved for clinical trials. Given that dHG-5 is a multi-component drug candidate consisting of a homologous series of oligosaccharides, it is difficult to predict a clear pharmacokinetics. Here, as a major oligosaccharide component, the tetradecasaccharide (oHG-14) was purified from dHG-5 and its structure was defined as L-Fuc3S4S-α(1,3)-L-Δ4,5GlcA-α(1,3)-{D-GalNAc4S6S-ß(1,4)-[L-Fuc3S4S-α(1,]3)-D-GlcA-ß(1,3)-}3-D-GalNAc4S6S-ß(1,4)-[L-Fuc3S4S-α(1,]3)-D-GlcA-ol. oHG-14 showed potent iXase inhibitory activity in vitro and antithrombotic effect in vivo comparable to dHG-5. After single subcutaneous administration of oHG-14 at 8, 14.4 and 32.4 mg/kg to rats, the absolute bioavailability was 71.6 %-80.9 % determined by the validated bioanalytical methods. The maximum concentration (Cmax) was 3.73, 8.07, and 11.95 µg/mL, respectively, and the time reaching Cmax (Tmax) was about 1 h. oHG-14 was mainly excreted by kidney as the parent compound with the elimination kinetics of first-order linear model. Anticoagulant activity of oHG-14 was positively correlated with its concentration in rat plasma. The pharmacokinetics/pharmacodynamics (PK/PD) of oHG-14 is similar to that of dHG-5. This study could provide supportive data for the clinical trial of dHG-5 and further development of pure oligosaccharide as an antithrombotic drug candidate.

Preparation, antioxidant and tyrosinase inhibitory activities of chitosan oligosaccharide-hydroxypyridinone conjugates.

Two novel chitosan oligosaccharide (COS)-hydroxypyridone (HPO) conjugates were prepared by reacting chitosan oligosaccharide with 2-chloromethyl-5-hydroxypyridone (HPO), which was synthesized by a series of reactions starting from kojic acid. The degree of substitution of COS-HPO2 reached 1.2, with a yield of 74.9%. The structure of the two conjugates (COS-HPO1 and COS-HPO2) was identified by NMR and FT-IR analysis. The two conjugates showed significantly higher free radical (DPPH•, ABTS+• and •OH) scavenging activity and reducing power than those of COS and HPO (p < 0.05). Both COS-HPO1 and COS-HPO2 possessed significantly stronger tyrosinase inhibitory activity than those of COS, with IC50 values of 0.67 and 0.28 mg/mL for monophenolase, 0.73 and 0.30 mg/mL for diphenolase, respectively. In addition, the conjugates were found to be non-toxic to RAW264.7 macrophages and MRC-5 human lung cells. This work proposes a facile method to enhance the oxidative and tyrosinase inhibitory properties of COS.

Protective Effect of Chitosan Oligosaccharide against Hydrogen Peroxide-Mediated Oxidative Damage and Cell Apoptosis via Activating Nrf2/ARE Signaling Pathway.

Chitosan oligosaccharide (COS), hydrolyzed and deacetylated from chitosan, has been reported to possess varieties of biological activities. Alzheimer's disease (AD) is a multifactorial progressive neurodegenerative disorder characterized by cognitive decline and memory loss, where oxidative stress was reported to be an overwhelming cause of the occurrence of AD. We have previously reported that COS could significantly decrease cell death, ROS generation, and lipid peroxidation, though the potential mechanism was yet to be determined. This study was designed to investigate the neuroprotective effect of COS against hydrogen peroxide (H2O2)-induced oxidative stress and apoptosis in neuronal SH-SY5Y cells. Our results indicated that COS could dose-dependently scavenge H2O2 in the cell-free systems. Accordingly, COS markedly decreased H2O2-induced cell apoptosis and intracellular ROS generation, while increased antioxidant capacity in SH-SY5Y cells. Further, COS significantly reduced the expression of Bax and upregulated Bcl-2. The mRNA and protein expression levels of nuclear Nrf2, heme oxygenase 1 (HO-1), and NAD(P)H: quinone oxidoreductase 1 (NQO1) were significantly increased upon COS treatment. Moreover, Nrf2-siRNA evidently reversed the promotive effect of COS on expression levels of HO-1 and NQO1, and ARE-driven transcriptional activity as determined by double-luciferase reporter gene assay. Besides, COS reversed H2O2-mediated increased phosphorylation of ERK1/2 and p38 MAPK. In conclusion, our findings indicate that COS could protect SH-SY5Y cells from oxidative damage and apoptosis via regulating Nrf2/ARE signaling pathway, which may provide new applications for the prevention and treatment of AD.

Consumption of a Single Dose of Prebiotic Galacto-Oligosaccharides Does Not Enhance Iron Absorption from Micronutrient Powders in Kenyan Infants: A Stable Iron Isotope Study.

BACKGROUND: Long-term feeding of prebiotic galacto-oligosaccharides (GOS) increases iron absorption in African infants, but the underlying mechanism and how long GOS need to be fed to infants to achieve an increase in absorption is uncertain. OBJECTIVES: In Kenyan infants, we tested whether the addition of GOS to a single test meal would affect iron absorption from a micronutrient powder (MNP) containing ferrous sulfate (FeSO4) and another MNP containing ferrous fumarate (FeFum) and sodium iron ethylenediaminetetraacetate (NaFeEDTA). METHODS: In a randomized-entry, prospective crossover study, iron deficient (87%) and anemic (70%) Kenyan infants (n  = 23; mean ± SD age, 9.9 ± 2.1 months) consumed 4 stable iron isotope-labeled maize porridge meals fortified with MNPs containing 5 mg iron as FeFum + NaFeEDTA, or FeSO4, either without or with 7.5 g GOS. The primary outcome, fractional iron absorption (FIA), was assessed by erythrocyte incorporation of isotopic labels. Data were analyzed using a 2-way repeated-measures ANOVA. RESULTS: There was no significant interaction between GOS and the iron compounds on FIA, and the addition of GOS did not have a significant effect on FIA. There was a statistically significant difference in FIA between the meals fortified with FeSO4 and with FeFum + NaFeEDTA (P  < 0.001).Given with GOS, FIA from FeSO4 was 40% higher than from FeFum + NaFeEDTA (P  < 0.001); given without GOS, it was 51% higher (P  < 0.01). CONCLUSIONS: The addition of GOS to a single iron-fortified maize porridge test meal in Kenyan infants did not significantly increase iron absorption, suggesting long-term feeding of GOS may be needed to enhance iron absorption at this age. This study was registered at clinicaltrials.gov as NCT02666417.

The role of Chito-oligosaccharide in regulating ovarian germ stem cells function and restoring ovarian function in chemotherapy mice.

In recent years, the discovery of ovarian germ stem cells (OGSCs) has provided a new research direction for the treatment of female infertility. The ovarian microenvironment affects the proliferation and differentiation of OGSCs, and immune cells and related cytokines are important components of the microenvironment. However, whether improving the ovarian microenvironment can regulate the proliferation of OGSCs and remodel ovarian function has not been reported. In this study, we chelated chito-oligosaccharide (COS) with fluorescein isothiocyanate (FITC) to track the distribution of COS in the body. COS was given to mice through the best route of administration, and the changes in ovarian and immune function were detected using assays of organ index, follicle counting, serum estrogen (E2) and anti-Mullerian hormone (AMH) levels, and the expression of IL-2 and TNF-α in the ovaries. We found that COS significantly increased the organ index of the ovary and immune organs, reduced the rate of follicular atresia, increased the levels of E2 and AMH hormones, and increased the protein expression of IL-2 and TNF-α in the ovary. Then, COS and OGSCs were co-cultured to observe the combination of COS and OGSCs, and measure the survival rate of OGSCs. With increasing time, the fluorescence intensity of cells gradually increased, and the cytokines IL-2 and TNF-α significantly promoted the proliferation of OGSCs. In conclusion, COS could significantly improve the ovarian and immune function of chemotherapy model mice, and improve the survival rate of OGSCs, which provided a preliminary blueprint for further exploring the mechanism of COS in protecting ovarian function.

Evaluation of Neutrophil Gelatinase-Associated Lipocalin as a Predictor of Glomerular Filtration Rate and Amikacin Clearance During Early Rat Endotoxemia: Comparison with Traditional Endogenous and Exogenous Biomarkers.

BACKGROUND AND OBJECTIVES: Renal elimination of amikacin and other aminoglycosides is slowed down in sepsis-induced acute kidney injury increasing the risk of adverse effects. Since neutrophil gelatinase-associated lipocalin (NGAL) and aminoglycosides share the mechanisms for renal excretion, the predictive power of NGAL was examined towards the changes in amikacin pharmacokinetics during early endotoxemia in anesthetized Wistar rats. METHODS: Endogenous biomarkers of inflammation and acute kidney injury were assessed including NGAL in saline-injected controls and two groups of rats challenged with an intravenous injection of bacterial lipopolysaccharide (5 mg/kg)-a fluid-resuscitated group (LPS) and a fluid-resuscitated group infused intravenously with 8 µg/kg/h terlipressin (LPS-T). Sinistrin and amikacin were infused to measure glomerular filtration rate (GFR) and amikacin clearance (CLam). The investigations included blood gas analysis, chemistry and hematology tests and assessment of urine output, creatinine clearance (CLcr) and sinistrin clearance (CLsini). RESULTS: Within 3 h of injection, systemic and renal inflammatory responses were induced by lipopolysaccharide. Gene and protein expression of NGAL was increased in the kidneys and the concentrations of NGAL in the plasma (pNGAL) and urine rose 4- to 38-fold (P < 0.01). The decreases in CLam and the GFR markers (CLcr, CLsini) were proportional, reflecting the extent to which endotoxemia impaired the major elimination mechanism for the drug. Terlipressin attenuated lipopolysaccharide-induced renal dysfunction (urine output, CLcr, CLsini) and accelerated CLam. The pNGAL showed a strong association with the CLsini (rs = - 0.77, P < 0.0005). Concerning prediction of CLam, pNGAL was comparable to CLcr (mean error - 24%) and inferior to CLsini (mean error - 6.4%), while the measurement of NGAL in urine gave unsatisfactory results. CONCLUSIONS: During early endotoxemia in the rat, pNGAL has a moderate predictive ability towards CLam. Clinical studies should verify whether pNGAL can support individualized dosing of aminoglycosides to septic patients.

Chemosensitizing indomethacin-conjugated chitosan oligosaccharide nanoparticles for tumor-targeted drug delivery.

A chitosan oligosaccharide (CSO)-indomethacin (IDM) conjugate (CI) was synthesized to fabricate chemosensitizing nanoparticles (NPs) for tumor-targeted drug delivery. IDM was conjugated to a CSO backbone via amide bond formation, of which successful synthesis was confirmed by proton-nuclear magnetic resonance analyses. Doxorubicin (DOX)-loaded CI (CI10/DOX; CI:DOX=10:1 [w/w]) NPs with <75nm of mean diameter, polydispersity index of ∼0.2, and positive zeta potential were prepared. The release of DOX from the NPs was enhanced at acidic pH (pH 5.5 and 6.8) compared to physiological pH (pH 7.4). The release of IDM increased in the presence of A549 cell lysates. In A549 cells (human lung carcinoma cells), more efficient cellular uptake of CI10/DOX NPs than that of free DOX was observed by using confocal laser scanning microscopy and flow cytometry. The in vitro cytotoxicity of CI10/DOX NPs in A549 cells was higher than those of free DOX and CI NPs with free DOX groups. In vivo pharmacokinetic studies after intravenous administration in rats showed significantly lower clearance of DOX from NPs compared with the free DOX group. Tumor targetability of the developed CI NPs was also verified by a real-time optical imaging study. In summary, the chemosensitizing CI/DOX NP with enhanced anticancer activity, prolonged blood circulation, and passive tumor targeting can be a promising anticancer drug delivery system for tumor-targeted therapy. STATEMENT OF SIGNIFICANCE: Chemosensitizing nanoparticles (NPs) based on amphiphilic chitosan oligosaccharide-indomethacin (CSO-IDM; CI) conjugate were developed for tumor-targeted delivery of doxorubicin (DOX). IDM was introduced to the CSO backbone as a hydrophobic residue to synthesize an amphiphilic conjugate and a chemosenstizer of DOX for improving antitumor efficacies. IDM, conjugated to CSO, may inhibit the efflux of cellular uptaken DOX via multidrug resistance-associated protein (MRP) and subsequently augment the anti-proliferation potentials of DOX in A549 cells (MRP-expressed human lung cancer cells). Chemosensitizing properties of developed CI NPs were assessed in cell culture models and the tumor targetability of CI/DOX NPs was demonstrated in A549 tumor-xenografted mouse model by a real-time optical imaging. Developed CI NPs can be used as a multifunctional nanosystem for the therapy of MRP-expressed cancers.

Neutralization of EP217609, a new dual-action FIIa/FXa anticoagulant, by its specific antidote avidin: a phase I study.

INTRODUCTION: EP217609 is a representative of a new class of synthetic parenteral anticoagulants with a dual mechanism of action. It combines in a single molecule a direct thrombin inhibitor and an indirect factor Xa inhibitor. EP217609 can be neutralized by a specific antidote avidin, which binds to the biotin moiety of EP217609. PURPOSE: The primary objective was to assess the neutralization of EP217609 by avidin in healthy subjects. Secondary objectives were to define the optimal avidin monomer/EP217609 molar ratio to achieve an adequate neutralization of EP217609 and to assess the safety and tolerability of EP217609 and avidin. METHODS: Healthy subjects (n = 36) were randomized to a 3 by 3 replicated Latin square design between 3 EP217609 doses (4, 8, 12 mg) and 3 avidin monomer/EP217609 molar ratios (1:1; 2:1; 3:1). EP217609 was administered as a single intravenous bolus, and avidin as a 30-min intravenous infusion, starting 90 min after EP217609 administration. RESULTS: Overall, EP217609 and avidin were well tolerated. One subject experienced a benign and transient typical pseudo-allergic reaction. The administration of EP217609 resulted in dose-dependent increases in pharmacodynamic markers. Avidin triggered a rapid and irreversible neutralization of EP217609 without rebound effect. Adequate neutralization of the anticoagulant activity was achieved with both 2:1 and 3:1 avidin monomer/EP217609 molar ratios. All safety parameters did not show any treatment-emergent clinically relevant changes or abnormalities in any dose group. CONCLUSIONS: These results will allow further investigation in patients requiring a neutralizable anticoagulant as those undergoing cardiac surgery. STUDY REGISTRATION: EudraCT number 2010-020216-10.

Chitosan oligosaccharide: Biological activities and potential therapeutic applications.

Chitosan oligosaccharide (COS) is an oligomer of ß-(1➔4)-linked d-glucosamine. COS can be prepared from the deacetylation and hydrolysis of chitin, which is commonly found in the exoskeletons of arthropods and insects and the cell walls of fungi. COS is water soluble, non-cytotoxic, readily absorbed through the intestine and mainly excreted in the urine. Of particular importance, COS and its derivatives have been demonstrated to possess several biological activities including anti-inflammation, immunostimulation, anti-tumor, anti-obesity, anti-hypertension, anti-Alzheimer's disease, tissue regeneration promotion, drug and DNA delivery enhancement, anti-microbial, anti-oxidation and calcium-absorption enhancement. The mechanisms of actions of COS have been found to involve the modulation of several important pathways including the suppression of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPK) and the activation of AMP-activated protein kinase (AMPK). This review summarizes the current knowledge of the preparation methods, pharmacokinetic profiles, biological activities, potential therapeutic applications and safety profiles of COS and its derivatives. In addition, future research directions are discussed.

Thermoresponsive nanospheres with independent dual drug release profiles for the treatment of osteoarthritis.

UNLABELLED: Dual drug delivery of drugs with different therapeutic effects in a single system is an effective way to treat a disease. One of the main challenges in dual drug delivery is to control the release behavior of each drug independently. In this study, we devised thermo-responsive polymeric nanospheres that can provide simultaneous and independent dual drug delivery in the response to temperature change. The nanospheres based on chitosan oligosaccharide conjugated pluronic F127 grafting carboxyl group were synthesized to deliver kartogenin (KGN) and diclofenac (DCF) in a single system. To achieve the dual drug release, KGN was covalently cross-linked to the outer part of the nanosphere, and DCF was loaded into the inner core of the nanosphere. The nanospheres demonstrated immediate release of DCF and sustained release of KGN, which were independently controlled by temperature change. The nanospheres treated with cold temperature effectively suppressed lipopolysaccharide-induced inflammation in chondrocytes and macrophage-like cells. The nanospheres also induced chondrogenic differentiation of mesenchymal stem cells, which was further enhanced by cold shock treatment. Bioluminescence of the fluorescence-labeled nanospheres was significantly increased after cold treatment in vivo. The nanospheres suppressed the progression of osteoarthritis in treated rats, which was further enhanced by cold treatment. The nanospheres also reduced cyclooxygenase-2 expression in the serum and synovial membrane of treated rats, which were further decreased with cold treatment. These results suggest that the thermo-responsive nanospheres provide dual-function therapeutics possessing anti-inflammatory and chondroprotective effects which can be enhanced by cold treatment. STATEMENT OF SIGNIFICANCE: We developed thermo-responsive nanospheres that can provide a useful dual-function of suppressing the inflammation and promoting chondrogenesis in the treatment of osteoarthritis. For a dual delivery system to be effective, the release behavior of each drug should be independently controlled to optimize their desired therapeutic effects. We employed rapid release of diclofenac for acute anti-inflammatory effects, and sustained release of kartogenin, a newly found molecule, for chondrogenic effects in this polymeric nanospheres. This nanosphere demonstrated immediate release of diclofenac and sustained release of kartogenin, which were independently controlled by temperature change. The effectiveness of this system to subside inflammation and regenerate cartilage in osteoarthritis was successful demonstrated through in vitro and in vivo experiments in this study. We think that this study will add a new concept to current body of knowledge in the field of drug delivery and treatment of osteoarthritis.

Preferential tumor accumulation and desirable interstitial penetration of poly(lactic-co-glycolic acid) nanoparticles with dual coating of chitosan oligosaccharide and polyethylene glycol-poly(D,L-lactic acid).

Despite advances in polymeric nanoparticles (NPs) as effective delivery systems for anticancer drugs, rapid clearance from blood and poor penetration capacity in heterogeneous tumors still remain to be addressed. Here, a dual coating of poly (ethylene glycol)-poly (d,l-lactic acid) (PEG-PDLLA) and water-soluble chitosan oligosaccharide (CO) was used to develop PLGA-based NPs (PCPNPs) with colloidal stability for delivery of paclitaxel (PTX). The PCPNPs were prepared by a modified nanoprecipitation process and exhibited homogeneous size of 165.5nm, and slight positive charge (+3.54mV). The single PEG-PDLLA-coated PLGA NPs (PPNPs) with negative charge (-13.42mV) were prepared as control. Human breast cancer MDA-MB-231 cell and mice MDA-MB-231 xenograft model were used for in vitro and in vivo evaluation. Compared to Taxol®, both PCPNPs and PPNPs increased the intracellular uptake and exerted stronger inhibitory effect on tumor cells in vitro, especially for PCPNPs. Particularly, due to the near neutral surface charge and shielding by the dual coating, the blank cationic NP presented low cytotoxicity. With the synergistic action of PEG-PDLLA and CO, PCPNPs not only strongly inhibited macrophage uptake and extended the blood circulation time, but also improved the selective accumulation and interstitial penetration capacity to/in tumor site. Consequently, a significantly enhanced antitumor efficacy was observed for the cationic PCPNPs. Our findings suggest that, the dual PEG-PDLLA/CO coating can effective improve the tumor accumulation and interstitial penetration of NPs and, therefore may have great potential for tumor treatment. STATEMENT OF SIGNIFICANCE: Rapid clearance from blood and poor penetration capacity in heterogeneous tumors represent great challenge for polymeric nanoparticles (NPs) as effective delivery systems for anticancer drugs. This study provides a promising cationic nanoparticle (PCPNPs) with dual coating of chitosan oligosaccharide (CO) and PEG-PDLLA to address the above problem. The PCPNPs prepared with 165.5nm and slight positive charge (+3.54mV) showed an improved accumulation and interstitial penetration capacity to/in tumor site, and thus led to an enhanced antitumor efficacy. This is the first time to report the cooperative effect of PEG-PDLLA and CO on PLGA NPs in this field. This work can arouse broad interests among researchers in the fields of nanomedicine, nanotechnology, and drug delivery system.

Stachyose increases absorption and hepatoprotective effect of tea polyphenols in high fructose-fed mice.

SCOPE: The consumption of tea polyphenols (TP) and stachyose contributes to preventive effects on hepatic injury. This study examined the effects of stachyose on absorption and hepatoprotective effects of TP in mice. METHODS AND RESULTS: GC-MS measurement showed that stachyose significantly increased serum total phenolic, ECG and EGCG contents in mice. The mice fed with high fructose (HF)-diet for 56 days exhibited oxidative stress observed by an increase in hepatic MDA levels and decreases in GSH-Px and SOD activities. Serum TC, TG, LDL-C and CRP levels, and ALT and AST activities were increased, while HDL-C concentrations were decreased following HF diet. Co-supplementation of stachyose and TP more effectively improved all parameters mentioned above when compared to administration of stachyose or TP alone. Histological observations of hepatic tissues also confirmed the beneficial effects of co-administration of stachyose and TP. CONCLUSION: Our results suggest that stachyose enhances absorption and hepatoprotective effects of TP, and combined ingestion of stachyose and TP is a novel strategy for alleviating HF diet-induced hepatic injury.

DNA-loaded chitosan oligosaccharide nanoparticles with enhanced permeability across Calu-3 cells.

Chitosan oligosaccharide (oligoCS) is a low molecular weight chitosan and its potential for DNA delivery is described here. DNA-loaded oligoCS nanoparticles were prepared by ionic gelation using thiamine pyrophosphate (TPP) as cross-linker. The nanoparticles with oligoCS:DNA: TPP weight ratio of 50:1:25 were approximately 170 nm in diameter with a zeta potential of +40 mV, and were used in the permeability study. The cytotoxicity of oligoCS solutions and nanoparticles was evaluated by MTT assay. The concentrations that exhibited minimal cytotoxicity were employed to investigate their effect on trans-epithelial electrical resistance (TEER) and cellular uptake across the Calu-3 cell layer which was used as a nasal epithelial model. OligoCS nanoparticles were able to cause a significant and reversible decrease in TEER and promote efficient cellular uptake. In addition, the oligoCS nanoparticles were able to enhance paracellular permeability to a greater extent than oligoCS solutions at an equivalent concentration. However, the oligoCS nanoparticles were too large to cross the cell layers through the paracellular route. The transcellular pathway appeared to be the major mechanism of the transportation of oligoCS nanoparticles across the cell layers. OligoCS nanoparticles also allowed efficient DNA incorporation, thereby providing the possibility of controlled nucleic acids release and absorption across epithelial surface.

Transcutaneous assessment of renal function in conscious rats with a device for measuring FITC-sinistrin disappearance curves.

Determination of the urinary or plasma clearance of exogenous renal markers, such as inulin or iohexol, is considered to be the gold standard for glomerular filtration rate (GFR) measurement. Here, we describe a technique allowing determination of renal function based on transcutaneously measured elimination kinetics of fluorescein isothiocyanate (FITC)-sinistrin, the FITC-labeled active pharmaceutical ingredient of a commercially available marker of GFR. A low cost device transcutaneously excites FITC-sinistrin at 480 nm and detects the emitted light through the skin at 520 nm. A radio-frequency transmission allows remote monitoring and real-time analysis of FITC-sinistrin excretion as a marker of renal function. Due to miniaturization, the whole device fits on the back of freely moving rats, and requires neither blood sampling nor laboratory assays. As proof of principle, comparative measurements of transcutaneous and plasma elimination kinetics of FITC-sinistrin were compared in freely moving healthy rats, rats showing reduced kidney function due to unilateral nephrectomy and PKD/Mhm rats with cystic kidney disease. Results show highly comparable elimination half-lives and GFR values in all animal groups. Bland-Altman analysis of enzymatically compared with transcutaneously measured GFR found a mean difference (bias) of 0.01 and a -0.30 to 0.33 ml/min per 100 g body weight with 95% limit of agreement. Thus, with this device, renal function can be reliably measured in freely moving rats eliminating the need for and influence of anesthesia on renal function.

PCP copolymers grafted with RGD enhance the rates of RGD-PCP micelles internalized into cells.

RGD-PCP copolymers were fabricated by grafting Arg-Gly-Asp (RGD) peptide to poly(epsilon-caprolactone)-b-chitooligosaccharide-b-poly(ethylene glycol) (PCP) copolymers and the rate of internalization of RGD-PCP micelles by PC 12 cells were examined. Increasing intensity of the absorbance of amine groups in FT-IR spectra of RGD-PCP copolymers compared with those of PCP copolymers indicated the presence of RGD in new copolymers. Moreover, the grafting efficiency and molar ratio of RGD peptides to PCP copolymers were 88.2% and 0.45, respectively, analysed with HPLC. The RGD-PCP copolymers self-assemble to micelles at the critical micelle concentration (CMC) of 0.018 wt% (178 mg L(-1)) and with a mean diameter of 90 nm using a dynamic light-scattering (DLS) analyser. Interestingly, the internalization of DPH-loaded RGD-PCP micelles into PC 12 cells is much faster (e.g. within 5 min) than that of PCP micelles. The new RGD-PCP micelles may potentially be used in cellular drug delivery.

Synthesis and biological evaluation of polysulfated oligosaccharide glycosides as inhibitors of angiogenesis and tumor growth.

A series of polysulfated penta- and tetrasaccharide glycosides containing alpha(1-->3)/alpha(1-->2)-linked mannose residues were synthesized as heparan sulfate (HS) mimetics and evaluated for their ability to inhibit angiogenesis. The compounds bound tightly to angiogenic growth factors (FGF-1, FGF-2, and VEGF) and strongly inhibited heparanase activity. In addition, the compounds exhibited potent activity in cell-based and ex vivo assays indicative of angiogenesis, with tetrasaccharides exhibiting activity comparable to that of pentasaccharides. Selected compounds also showed good antitumor activity in vivo in a mouse melanoma (solid tumor) model resistant to the phase III HS mimetic 1 (muparfostat, formerly known as PI-88). The lipophilic modifications also resulted in reduced anticoagulant activity, a common side effect of HS mimetics, and conferred a reasonable pharmacokinetic profile in the rat, as exemplified by the sulfated octyl tetrasaccharide 5. The data support the further investigation of this class of compounds as potential antiangiogenic, anticancer therapeutics.

Population pharmacokinetic model of PI-88, a heparanase inhibitor.

PURPOSE: The aim of this study was to investigate typical population pharmacokinetic (PK) parameters, potential covariates, and interindividual and residual variabilities of PI-88, a heparanase endoglycosidase enzyme inhibitor being developed for the treatment of cancer. METHODS: A population PK model of PI-88 was developed and evaluated using nonlinear mixed effects modeling (NONMEM). Plasma concentration versus time data was obtained from a total of 76 subjects that participated in phase I trials of PI-88 delivered subcutaneously (SC) at doses ranging from 80 to 315 mg. Overall, the PK effects of 12 clinical covariates were evaluated, including weight, age, creatinine clearance, body surface area, body mass index, sex, cancer (vs. healthy subject), docetaxel coadministration, prior chemotherapy, prior investigational therapy, prior radiotherapy and prior surgery. RESULTS: Population PK analysis of the data-set showed that apparent clearance (CL/F) and apparent volume of distribution (V/F) of PI-88 were positively correlated with body surface area and the absorption rate constant (KA) was positively correlated with body mass index. In addition, CL/F was found to be significantly lower in patients with malignancies versus healthy subjects. By incorporating these covariates into the PK parameter equations, the interindividual variability of CL/F was reduced from 30.6 to 20.2% (decrease of 34%), V/F was reduced from 31.4 to 20.7% (decrease of 34.1%) and KA was reduced from 52.6 to 46.2% (decrease of 12.2%). CONCLUSIONS: This population PK model indicates that the PK variability of PI-88 can be significantly reduced by taking BSA into account when dosing this drug SC.

Avaliação da pectina fosfatada aplicada na formação de filmes isolados: material candidato a novos sistemas para liberação modificada de fármacos / Phosphated pectin application in the development of films for drug delivery systems: evaluation of permeability properties and swelling

Pectina fosfatada (Pect-TMFT) juntamente com o α-Gluco-oligossacarídeo (Bioecolians®) foram incorporados à dispersão aquosa de polimetacrilato (Eudragit® RS 30 D) para obtenção de filmes isolados pelo processo "casting" (50 ºC) em placa de Teflon®. A Pect-TMFT e o Bioecolians® foram adicionados à dispersão de Eudragit® RS 30 D nas concentrações: 90: 05: 05, 80: 10: 10 e 70: 20: 10 (4 por cento p/v). Citrato de trietila (TEC) foi utilizado como plastificante (20 por cento da massa do polimetacrilato). As dispersões propostas apresentaram habilidades filmogênicas. Os filmes isolados foram caracterizados pela determinação da permeabilidade ao vapor d'água (TVA), pelo índice de intumescimento (Ii por cento) em fluidos de simulação gástrica (FSG) e intestinal (FSI) e por microscopia eletrônica de varredura (MEV). O aumento do polissacarídeo modificado e do Bioecolians® nos filmes favoreceram a permeabilidade ao vapor d'água e o grau de hidratação quando em FSI (pH= 6,8). Dessa forma, o filme 70: 20: 10, poderá impedir a liberação prematura do fármaco em regiões superiores do TGI quando aplicado como revestimento de sistemas sólidos orais. Além disso, a presença da Pect-TMFT e do Bioecolians®, poderá favorecer a degradação específica da película por enzimas produzidas pela microflora colônica, possibilitando uma cinética de liberação modificada de fármacos apesar das variações inter-individuais de pH que possam existir.

Phosphated pectin (Pect-TMFT) together with α-gluco-oligossacaride (Bioecolians®) were incorporated into aqueous dispersion of polymethacrylate (Eudragit® RS 30 D) to obtain free films by the casting process (50ºC) in Teflon® plate. Pect-TMFT and Bioecolians® were added into dispersions of Eudragit ® RS 30 D at different rates: 90:05:05, 80:10:10 and 70:20:10 (4 percent p/v). Triethyl citrate (TEC) was used as plasticizer (20 percent of mass of the polymethacrylate). The proposed dispersions showed film formation ability. The free films were characterized by the determination of water vapour transmission (WVT), by the swelling index (Ii percent) in fluids of gastric simulation (FGS) and intestinal (FIS) and by scanning electron microscopy (SEM). The increase of modified polysaccharide and Bioecolians® in the films favored their permeability to the water vapour and their hydration degree when in FIS (pH = 6.8). In that way, the obtained film in the concentration 70:20:10, can prevent the premature release of the drug in the up GIT when applied to develop oral solid systems coating. Besides, the presence of Pect-TMFT and Bioecolians® can contribute to the specific membrane degradation by colonic microflora enzymes, making possible a modified release kinetics of drugs even with the existence of inter-individual variations of pH.

A phase I pharmacological and biological study of PI-88 and docetaxel in patients with advanced malignancies.

PURPOSE: This study evaluated the safety, toxicity, pharmacological properties and biological activity of PI-88, a heparanase endoglycosidase enzyme inhibitor, with fixed weekly docetaxel in patients with advanced solid malignancies. EXPERIMENTAL DESIGN: This was a phase I study to determine the maximal-tolerated dose of escalating doses of PI-88 administered subcutaneously for 4 days per week, along with docetaxel 30 mg/m(2) given on days 1, 8, 15 of a 28-day schedule. RESULTS: Sixteen patients received a total of 42 courses of therapy. No dose-limiting toxicities were observed despite escalation to the highest planned dose level of PI-88 (250 mg/day). Frequent minor toxicities included fatigue (38%), dysgeusia (28.5%), thrombocytopenia (12%), diarrhea (14%), nausea (12%), and emesis (10%) in the 42 courses. No significant bleeding complications were observed. One patient developed a positive anti-heparin antibody test/serotonin releasing assay with positive anti-platelet factor 4/PI-88 antibodies and grade 1 thrombocytopenia in cycle 5, and was withdrawn from the study without any sequelae. PI-88 plasma concentrations (mirrored by APTT) and urinary elimination were linear and dose-proportional. Docetaxel did not alter the pharmacokinetic (PK) profile of PI-88, nor did PI-88 affect docetaxel PK. No significant relationship was determined between plasma or urine FGF-2, or plasma VEGF levels and PI-88 dose/response. Although no objective responses were observed; 9 of the 15 evaluable patients had stable disease for greater than two cycles of therapy. CONCLUSION: PI-88 administered at 250 mg/day for 4 days each week for 3 weeks with docetaxel 30 mg/m(2) on days 1, 8 and 15, every 28 days, was determined to be the recommended dose level for phase II evaluation. This combination was well tolerated without severe toxicities or PK interactions.

Cystatin C does not detect acute changes in glomerular filtration rate in early diabetic nephropathy.

BACKGROUND: The measurement of renal functional reserve (acute change in glomerular filtration rate [GFR] after protein load) allows the detection of sub-clinical renal dysfunction and has prognostic implications in diabetes. Our aim was to test cystatin C as an index of GFR and renal functional reserve. METHODS: GFR was measured by C(Sinistrin) at baseline and after protein load in 28 diabetic patients with serum creatinine <1.2 mg/dL. The C(Sinistrin) was compared with cystatin C, serum creatinine, creatinine clearance, and Cockcroft-Gault formula. RESULTS: Baseline C(Sinistrin) ranged from 67-172 mL/min. Regression analysis showed an overall low relationship between C(Sinistrin) and the indirect markers of GFR. The highest correlation with C(Sinistrin) was obtained for cystatin C clearance (R(2) = 0.58, r = 0.76, p < 0.001), the 1/serum cystatin C (R(2) = 0.58, r = 0.76, p < 0.001), and serum cystatin C (R(2) = 0.52, r = 0.72, p < 0.001). Renal functional reserve was preserved in 6 of 28 patients. There was no significant change in cystatin C in response to protein load. CONCLUSION: Wide variation in baseline GFR emphasizes the need for the early detection of renal dysfunction. Cystatin C correlated best with C(Sinistrin) at baseline, but did not detect renal functional reserve.