The effect of freeze-drying on mucoadhesion and transport of acrylated chitosan nanoparticles.

Nanoparticle-based mucosal drug delivery is a promising method to increase the residence time of a drug in the mucosa. It is known that the stability of polysaccharide-based nanoparticles in aqueous solutions is limited, due to hydrolysis; hence the long-term stability of a formulation is usually improved by freeze-drying. The aim of this study was to investigate the effect of cryoprotection and freeze-drying on the physical and chemical properties of mucoadhesive acrylated chitosan (ACS) nanoparticles including the potential of these carriers to deliver drugs. The results showed that the most effective cryoprotection was achieved using sucrose. The incorporation of a hydrophilic macromolecular drug, dextran sulfate, increased the nanoparticle size and decreased the zeta potential for both fresh and freeze-dried nanoparticle formulations. In addition, the freeze-dried nanoparticles presented penetration across a mucus gel layer and the flow through technique revealed that short term mucoadhesive properties were not impaired. ACS nanoparticles were able to deliver a model drug across a mucin gel layer but could not improve drug penetration through the triple co-culture cell model that was used in order to mimic the small intestine epithelium.

Chitosan-dextran sulphate nanocapsule drug delivery system as an effective therapeutic against intraphagosomal pathogen Salmonella.

OBJECTIVES: The ability to target conventional drugs efficiently inside cells to kill intraphagosomal bacteria has been a major hurdle in treatment of infective diseases. We aimed to develop an efficient drug delivery system for combating infection caused by Salmonella, a well-known intracellular and intraphagosomal pathogen. Chitosan-dextran sulphate (CD) nanocapsules were assessed for their efficiency in delivering drugs against Salmonella. METHODS: The CD nanocapsules were prepared using the layer-by-layer method and loaded with ciprofloxacin or ceftriaxone. Antibiotic-loaded nanocapsules were analysed in vitro for their ability to enter epithelial and macrophage cells to kill Salmonella. In vivo pharmacokinetics and organ distribution studies were performed to check the efficiency of the delivery system. The in vivo antibacterial activity of free antibiotic and antibiotic loaded into nanocapsules was tested in a murine salmonellosis model. RESULTS: In vitro and in vivo experiments showed that this delivery system can be used effectively to clear Salmonella infection. CD nanocapsules were successfully employed for efficient targeting and killing of the intracellular pathogen at a dosage significantly lower than that of the free antibiotic. The increased retention time of ciprofloxacin in the blood and organs when it was delivered by CD nanocapsules compared with the conventional routes of administration may be the reason underlying the requirement for a reduced dosage and frequency of antibiotic administration. CONCLUSIONS: CD nanocapsules can be used as an efficient drug delivery system to treat intraphagosomal pathogens, especially Salmonella infection. This delivery system might be used effectively for other vacuolar pathogens including Mycobacteria, Brucella and Legionella.

Novel nanostructured lipid-dextran sulfate hybrid carriers overcome tumor multidrug resistance of mitoxantrone hydrochloride.

Novel nanostructured lipid-dextran sulfate hybrid carriers (NLDCs) were successfully developed for sustained delivery of water-soluble cationic mitoxantrone hydrochloride (MTO) and overcoming multidrug resistance. The introduction of negative polymer of dextran sulfate sodium significantly improved the encapsulation efficiency (97.4%) and sustained the release of MTO (86.9% at 72 hours). In vivo pharmacokinetics in rats after intravenous administration demonstrated that MTO-loaded NLDCs (MTO-NLDCs) had higher area under the curve and longer half-life than MTO solution (MTO-Sol). In the biodistribution study, NLDCs significantly improved the MTO levels in plasma, spleen, and brain, and decreased the distribution of MTO in heart and kidney. In comparison with MTO-Sol, MTO-NLDCs efficiently enhanced cytotoxicity through the higher accumulation of MTO in breast cancer resistance protein (BCRP)-overexpressing MCF-7/MX cells. MTO-NLDCs entered into the resistant cancer cells by the clathrin-mediated endocytosis pathway, which escaped the efflux induced by BCRP transporter and thereby overcame the multidrug resistance of MCF-7/MX cells. FROM THE CLINICAL EDITOR: In this study, novel nanostructured lipid-dextran sulfate hybrid carriers were synthesized and utilized for sustained delivery of mitoxantrone hydrochloride. The utilized methods successfully addressed multidrug resistance to this chemotherapy agent.

Release of vancomycin from multilayer coated absorbent gelatin sponges.

Wounds have the potential to become infected during any surgical procedure. Gelatin sponges that are commonly used to absorb blood during invasive surgeries would benefit tremendously if they released antibiotics. In this work, we have examined coating a commercial gelatin sponge with degradable polymer multilayer films containing vancomycin. The effect of the film on sponge absorption capabilities and the effect of the sponge on drug release kinetics were both examined. Application of vancomycin containing layer-by-layer assembled films to this highly porous substrate greatly increased drug loading up to approximately 880% compared to a flat substrate. Vancomycin drug release was extended out to 6 days compared to 2 days for film coated flat substrates. Additionally, the absorbent properties of the gelatin sponge were actually enhanced by up to 170% due to the presence of the vancomycin film coating. A comparison of film coated sponges with sponges soaked directly in vancomycin demonstrated the ability of the multilayer films to control drug release. Film released vancomycin was also found to remain highly therapeutic with unchanged antimicrobial properties compared to the neat drug, demonstrated by quantifying vancomycin activity against Staphylococcus aureus in vitro.

Oral administration of mesalazine protects against mucosal injury and permeation in dextran sulfate sodium-induced colitis in rats.

OBJECTIVE. Mesalazine, from which 5-aminosalicylic acid is released, is a therapeutic drug for inflammatory bowel disease. There has been no study concerning the effect of orally administered mesalazine on dextran sodium sulfate (DSS)-induced colitis in the rat model of ulcerative colitis. MATERIAL AND METHODS. Colitis was evaluated by means of the length of the colon, white blood cell count (WBC), tissue myeloperoxidase (MPO) activity, and histological inflammation scores. Colonic mucosal permeation was evaluated using Evans blue. The localization of a tight junction protein, occludin, was evaluated immunohistochemically and examined using confocal laser scanning microscopy. RESULTS. Mesalazine significantly improved changes in the length of the colon, tissue MPO activity, WBC, and the histological inflammation score as compared with DSS-induced colitis. Furthermore, the drug completely inhibited the increased permeation in DSS-induced colitis in rats. The immunofluorescence signals of occludin were disrupted and irregularly distributed in DSS-induced colitis, while the signals appeared as a typical reticular pattern but with reduced intensity by the administration of mesalazine, without any reduction in the protein content. In addition, the oral administration of mesalazine significantly improved mucosal permeation, thereby protecting the intestinal mucosa against injury in DSS-induced colitis in rats. CONCLUSIONS. These findings suggest that the recovery of mucosal impairment due to treatment with mesalazine may be associated with the protection of the tight junction protein occludin in DSS-induced colitis.

The renal clearance of dextran sulfate decreases in puromycin aminonucleoside-induced glomerulosclerosis: a puzzle observation.

BACKGROUND: Puromycin aminonucleoside-induced nephrosis is characterized by increased renal excretion of plasma proteins. We employed this experimental model to study the urinary clearance of dextran sulfate. METHODS: The dextran sulfate eliminated by the urine was determined using a metachromatic assay. Polysaccharide fragments were analyzed by chromatographic and electrophoretic procedures. Disaccharide composition of the glomerular heparan sulfate was assessed using digestion with specific lyases. RESULTS: In normal rats dextran sulfate is partially degraded to lower molecular weight fragments and only then eliminated by the urine. Surprisingly, in puromycin aminonucleoside-induced glomerulosclerosis the molecular size of the fragments of dextran sulfate found in the urine is the same or even lower than in control animals in spite of the marked proteinuria. Furthermore, urinary excretion of dextran sulfate decreases in the experimentally induced nephrosis. This observation cannot be totally attributed to a reduced number of physiologically active nephrons since the glomerular filtration rate decreases approximately 32% after puromycin aminonucleoside administration while the urinary excretion of 8 kDa-dextran sulfate decreases 3-fold. The glomerular heparan sulfate shows reduced sulfation when compared with normal animals. Possibly puromycin aminonucleoside decreases the activity of kidney endoglycosidases, which reduce the molecular size of the sulfated polysaccharide, leading to a decrease in its renal clearance. Reduced sulfation of the glomerular heparan sulfate in the puromycin aminonucleoside-induced nephrosis does not alter the size of the dextran sulfate eliminated by the kidney, as suggested for protein. CONCLUSIONS: Each pathological process induces a particular modification in the kidney, which in turn can affect the renal selectivity to specific macromolecules in different ways.

Reduced urinary excretion of sulfated polysaccharides in diabetic rats.

The aim of the present study was to further understand the changes in renal filtration that occur in the early stages of diabetes mellitus. Diabetes was induced in male Wistar rats by a single injection of streptozotocin. Glycemia, body weight, 24-h urine volume and urinary excretion of creatinine, protein and glycosaminoglycans were measured 10 and 30 days after diabetes induction. All the diabetic animals used in the present study were hyperglycemic, did not gain weight, and presented proteinuria and creatinine hyperfiltration. In contrast, the glycosaminoglycan excretion decreased. Dextran sulfates of different molecular weights (6.0 to 11.5 kDa) were administered to the diabetic rats, and to age-matched, sham-treated controls. Most of the dextran sulfate was excreted during the first 24 h, and the amounts excreted in the urine were inversely proportional to the dextran sulfate molecular weight for all groups. Nevertheless, diabetic rats excreted less and accumulated more dextran sulfate in kidney and liver, as compared to controls. These differences, which were observed only for the dextran sulfates of higher molecular weights (>7 kDa), increased with the duration of diabetes. Our findings suggest differential renal processing mechanisms for proteins and sulfated polysaccharides, with the possible involvement of kidney cells.

Hemin potentiates nitric oxide-mediated nitrosation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) to 2-nitrosoamino-3-methylimidazo[4,5-f]quinoline.

Heme has been reported to be an important contributor to endogenous N-nitrosation within the colon and to the enhanced incidence of colon cancer observed with increased intake of red meat. This study uses the heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) as a target to evaluate hemin potentiation of nitric oxide (NO)-mediated nitrosation. Formation of 14C-2-nitrosoamino-3-methylimidazo[4,5-f]quinoline (N-NO-IQ) was monitored by HPLC following incubation of 10 microM IQ with the NO donor spermine NONOate (1.2 microM NO/min) at pH 7.4 in the presence or absence of hemin. N-NO-IQ formation due to autoxidation of NO was at the limit of detection (0.1 microM) and increased 22-fold in the presence of 10 microM hemin and an in situ system for generating H2O2 (glucose oxidase/glucose). A linear increase in N-NO-IQ formation was observed from 1 to 10 microM hemin. Significant nitrosamine formation occurred at fluxes of NO and H2O2 as low as 0.024 and 0.25 microM/min, respectively. Potentiation by hemin was not affected by a 400-fold excess flux of H2O2 over NO or a 4.8-fold excess flux of NO over H2O2. Reactive nitrogen species produced by hemin potentiation had a 46-fold greater affinity for IQ than those produced by autoxidation. Azide inhibited autoxidation, suggesting involvement of the nitrosonium ion, NO+. Hemin potentiation was inhibited by NADH, but not azide, suggesting oxidative nitrosylation with NO2* or a NO2*-like species. IQ and 2,3-diaminonaphthylene were much better targets for nitrosation than the secondary amine morpholine. Apc(min) mice with dextran sulfate sodium-induced colitis demonstrated increased levels of urinary nitrite and nitrate consistent with increased expression of iNOS and NO synthesis. As reported previously, identical conditions increased fecal N-nitroso compounds. Thus, hemin potentiation of NO-mediated nitrosation of heterocyclic amines provides a testable mechanism by which red meat consumption can generate N-nitroso compounds and initiate colon cancer under inflammatory conditions, such as colitis.

In vitro and in vivo evaluation of mucoadhesive microspheres consisting of dextran derivatives and cellulose acetate butyrate.

The objective of this study was to evaluate mucoadhesive properties and gastrointestinal transit of microspheres made of oppositely charged dextran derivatives and cellulose acetate butyrate (CAB). The microspheres were prepared by emulsion solvent evaporation method. A reference microsphere was made of lactose instead of dextran derivatives. Microspheres with a diameter of 425-710 microm were examined for in vitro mucoadhesion by the everted sac method. The results indicated that the percentage of adherence to the rat small intestine was affected by the amount of dextran derivatives in the microspheres. After 1.5h, the adhering percent of the reference microspheres and the microspheres containing 50% of dextran derivatives were 34 and 74%, respectively. Then gastrointestinal transit after oral administration to rats was evaluated by counting the microspheres remaining in the stomach and small intestine. The microspheres containing 40% of dextran derivatives adhered to the stomach rather than the small intestine. Mathematical analysis revealed that the time required for 50% of microspheres to leave the stomach was 1.42h, three times longer than the reference. These findings suggest that the microsphere is a promising device as a multiple-unit mucoadhesive system.

Electrically enhanced transdermal delivery of a macromolecule.

The purpose of this study was to establish the delivery parameters for the enhanced transdermal delivery of dextran sulfate (MW 5000 Da). Full-thickness pig skin or epidermis separated from human cadaver skin was used. Silver-silver chloride electrodes were used to deliver the current (0.5 mA cm-2). For electroporation experiments, one or more pulses were given using an exponential decay pulse generator. The correct polarity for iontophoresis and pulsing was first established as cathode in the donor. The amount of drug delivered increased with increasing donor concentration up to a point, but not any further. The amount delivered also increased with pulse voltage, the delivery being twice as much as with iontophoresis alone (144.5+/-10.35 microg cm(-2)), when 6 pulses of 500 V were applied at time zero before iontophoresis (276+/-45.2 microg cm(-2)). It was observed that the amount delivered was a function of increasing pulse length when the apparent charge delivered was kept constant. Transport through pig skin (107.4+/-24.4 microg cm(-2)) was found to be comparable with that through human epidermis (84.9+/-18.4 microg cm(-2)). In conclusion, we have demonstrated the transdermal delivery of a 5000 Da molecular weight dextran sulfate using iontophoresis. It was also seen that iontophoretic delivery could be enhanced by simultaneous electroporation.

A model for dextran sodium sulfate (DSS)-induced mouse colitis: bacterial degradation of DSS does not occur after incubation with mouse cecal contents.

Germ-free (GF) mice are highly susceptible to dextran sodium sulfate (DSS)-induced colitis in comparison to conventionalized (CVz) mice. It is hypothesized that degradation of DSS by intestinal microflora is involved in the susceptibility to DSS-induced colitis of GF mice. This study evaluates the ability of bacteria in mouse cecal contents (CC) to degrade DSS in vitro, and provides confirmatory evidence that DSS was not degraded when incubated with CC. Our results suggest that intestinal microflora do not contribute directly to the difference in susceptibility of GF mice to DSS-induced colitis through degradation.

Correlation of rectum-associated lymph nodules with the development of experimentally induced acute colonic inflammation in rats.

BACKGROUND AND AIMS: The mechanisms of inflammation in ulcerative colitis occurring initially in the rectum and then extending throughout the colon are still unknown. The aim of this study is to investigate the correlation of rectum-associated lymph nodules (RALN) and the development of a dextran sulfate sodium-induced experimentally acute colonic inflammation in rats. METHODS: We immunohistochemically analyzed the lymph nodules in the rectal region of rats using monoclonal antibodies to specific markers. RESULTS: Rectum-associated lymph nodules resembled the Peyer's patches, where CD4+, CD8+ lymphocytes and Mar 1+ macrophages were observed. After the administration of dextran-sulfate sodium (DSS), RALN showed hypertrophy with an increase in the number of CD4+, CD8+, and interleukin (IL)-2R+ lymphocytes in the periphery, as well as Mar 1+ macrophages in the entire region. Concurrently, platelet/endothelin cell adhesion molecule-1 (PECAM-1+) blood vessels, including high-endothelial venules (HEV), were increased in number in the interfollicular areas. Furthermore, a number of small lymph nodules varying in size were observed in the upper region of the rectum. Some of them were initially confined to the lamina propria mucosae, and became large enough to extend deeper into the tela submucosa. These DSS-induced lymph nodules contained large numbers of CD4+ lymphocytes, IL-2R+ lymphocytes, and Mar 1+ macrophages. CONCLUSIONS: The pathological changes of ulcerative colitis were well correlated with the development of both RALN and DSS-induced lymph nodules. The immunological reaction that occurred in DSS-induced lymph nodules is significantly associated with the expansion of colitis from the lower region of rectum to the upper region of rectum and colon.

Correlation of transarterial transport of various dextrans with their physicochemical properties.

Local vascular drug delivery provides elevated concentrations of drug in the target tissue while minimizing systemic side effects. To better characterize local pharmacokinetics we examined the arterial transport of locally applied dextran and dextran derivatives in vivo. Using a two-compartment pharmacokinetic model to correct the measured transmural flux of these compounds for systemic redistribution and elimination as delivered from a photopolymerizable hydrogel surrounding rat carotid arteries, we found that the diffusivities and the transendothelial permeabilities were strongly dependent on molecular weight and charge. For neutral dextrans, the effective diffusive resistance in the media increased with molecular weight approximately 4.1-fold between the molecular weights of 10 and 282 kDa. Similarly, endothelial resistance increased 28-fold over the same molecular weight range. The effective medial diffusive resistance was unaffected by cationic charge as such molecules moved identically to neutral compounds, but increased approximately 40% when dextrans were negatively charged. Transendothelial resistance was 20-fold lower for the cationic dextrans, and 11-fold higher for the anionic dextrans, when both were compared to neutral counterparts. These results suggest that, while low molecular weight drugs will rapidly traverse the arterial wall with the endothelium posing a minimal barrier, the reverse is true for high molecular weight agents. With these data, the deposition and distribution of locally released vasotherapeutic compounds might be predicted based upon chemical properties, such as molecular weight and charge.

Surface hydrophobicity of the rat colonic mucosa is a defensive barrier against macromolecules and toxins.

BACKGROUND: Mucosal surface hydrophobicity is a key factor of the gastric acid defence barrier. In the colon, surface hydrophobicity is high but its biological function remains unexplored. AIMS: To investigate the functional changes of the barrier due to removal of the surface active phospholipid layer by a detergent, or to reinforcement of the surface active phospholipid by local application of a suspension of lipids. METHODS: Surface hydrophobicity (contact angle measurement), colonic permeability (lumen to blood clearance of mannitol and dextran), and mucosal resistance against luminal aggression (distal colitis induced by dextran sodium sulphate, DSS) were investigated in three study groups: (a) rats pretreated with a detergent (Brij 35) known to remove surfactant lipids; (b) rats pretreated with a suspension of surface active lipids (tripalmitin and dipalmitoyl-phosphatidylcholine); and (c) control rats pretreated with the corresponding vehicles. RESULTS: In controls, surface hydrophobicity was low on the caecal mucosa and high in colon and rectum. Detergent treatment reduced surface hydrophobicity, and increased colonic permeability to mannitol and dextran. Conversely, treatment with lipids increased surface hydrophobicity, and reduced colonic permeability. Administration of DSS induced a progressive loss of colonic surface hydrophobicity, and an increase in permeability to mannitol and dextran. Detergent treatment increased susceptibility to epithelial damage and mucosal inflammation by DSS. Treatment with lipids reduced susceptibility to DSS colitis. CONCLUSION: Colonic surface hydrophobicity modulates permeability to hydrophilic molecules and protects against toxins.

Dextran sulfate inhibits IFN-gamma-induced Jak-Stat pathway in human vascular endothelial cells.

Human vascular endothelial cells can be induced by IFN-gamma to express class II MHC proteins. Previously, dextran sulfate was shown to selectively inhibit expression of class II MHC by preventing transcription of the gene encoding CIITA, a transactivator protein required for IFN-gamma-inducible expression of class II genes. In this study we characterized the effects of dextran sulfate on the intracellular events occurring prior to CIITA activation. Immunoprecipitation and Western blot analyses indicated that IFN-gamma-induced phosphorylation of Stat1 and Jak2 was blocked by dextran sulfate. In addition, electron micrographs showing the large accumulation of dextran sulfate particles in the cytoplasms of endothelial cells demonstrated that Stat and Jak proteins may directly interact with dextran sulfate. Binding of radiolabeled IFN-gamma to cells indicated that dextran sulfate may also modulate IFN-gamma interactions with the cell surface. Thus, dextran sulfate is capable of interfering with the IFN-gamma-induced expression of class II MHC genes at multiple sites.

Orally administered dextran sulfate is absorbed in HIV-positive individuals.

Preliminary in vivo studies suggested that oral dextran sulfate was poorly absorbed, but investigations were limited by inadequate methods for measuring the drug in the body. To determine absorption in HIV-positive subjects, hydrogenated dextran sulfate, average molecular weight 8000 (Usherdex 8), was orally administered in a short-term (single dose, 4 g/day for 5 days, 7 subjects) and in a long-term study (1 g, 4 times per day for 29 to 335 days, 8 subjects), which was a continuation of the short-term study with the inclusion of an additional subject. When an agarose gel electrophoresis technique with toluidine blue staining was used, the drug was recovered from plasma (67%, peak 2.2 microg/mL) and circulating peripheral blood lymphocyte (PBL) samples (50%, peak 333 microg/L blood) obtained at 5 and 15 minutes and 1, 3, 6, and 24 hours after the first day's dose and from plasma (56%) and PBL samples (38%) obtained 5 minutes after administration on 4 subsequent days in the short-term study. In the long-term study, the drug was found in plasma (67%, peak 2.4 microg/mL) and PBL samples (25%, peak 126 microg/L blood) obtained at monthly visits within 4 hours of the last dose. The drug was found in all urine samples from all subjects in both studies (short-term study, 24-hour samples up to 4 days after the final dose; long-term study, monthly samples within 4 hours of the last dose). In the long-term study, bone marrow preparations from 3 subjects showed metachromatic inclusions present in reticular cells when the cells were stained with toluidine blue, indicating the presence of sulfated polyanions. A significant rise in activated partial thromboplastin time and a drop in platelet count (P < .025) were demonstrated, with thrombocytopenia developing in 3 patients. Mild-to-moderate gastrointestinal disturbances were experienced by 6 subjects in the short-term study and by all subjects in the long-term study. One subject experienced mild central nervous system symptoms in the short-term study. These results indicate that dextran sulfate is absorbed after oral administration; therefore, further studies on its efficacy, particularly in the early stages of the disease, along with additional observations on its toxicity, are warranted.

Tissue distribution of dextran sulfate sodium (DSS) in the acute phase of murine DSS-induced colitis.

In the present study, we examined histochemically the tissue distribution of dextran sulfate sodium (DSS) in the acute phase of murine colitis induced by administering DSS in the drinking water. DSS was mainly observed in the Kupffer cells of the liver, in the macrophages of the mesenteric lymph node (MLN) and in the lamina propria of the large intestine after administration of DSS. We followed the time course of DSS distribution and found that DSS, which was considered as a large and negatively charged molecule that can not easily cross membranes, was distributed in the liver, the MLN, and the large intestine 1 day after the start of administration of DSS.

Anomalous decrease in dextran sulfate clearance in the diabetic rat kidney.

The anomalous increase in charge selectivity as previously observed with reduced dextran sulfate clearances in diabetic rats (L. D. Michels, M. Davidman, and W. F. Keane. Kidney Int. 21: 699-705, 1982) was confirmed in 4-wk streptozotocin (STZ) diabetic Sprague-Dawley rats using the isolated perfused kidney technique. The apparent charge selectivity in both control and diabetic rats could be abolished by increasing the dextran sulfate concentration to 200 micrograms/ml in the perfusate. This was demonstrated by a high rate of processing of dextran sulfate (approximately 1,700 ng.min-1.kidney-1) by glomeruli in both control and diabetic kidneys and by the fact that charge interaction could not explain the concentration dependence. The amount of urinary desulfation of dextran sulfate was also found to be significantly less in the diabetic kidney as was glomerular sulfatase activity compared with controls. Dextran sulfate glomerular processing is therefore altered in the STZ diabetic rat kidney but could be rationalized in terms of previous models of endothelial cell receptor-mediated uptake of dextran sulfate. The results are consistent with recent work demonstrating that there is little or no electrostatic charge interaction operating on dextran sulfate or other negatively charged molecules at the glomerular capillary wall.

Circulating hyaluronan, chondroitin sulphate and dextran sulphate bind to a liver receptor that does not recognize heparin.

Chondroitin sulphate, injected intravenously into rats and given prior to intravenous 125I-labelled hyaluronan with a mean Mw of about 400 kDa, was shown to inhibit the rapid receptor-mediated uptake of hyaluronan by the liver. The labelled hyaluronan that remained in the circulation was shown, by size exclusion chromatography of serum and urine, to be rapidly degraded down to fragments of lower Mw and filtered out into the urine and tissues. When the uptake of 125I-hyaluronan was inhibited by unlabelled hyaluronan, only very low degradation and urinary excretion were found. Liver uptake could also be inhibited by dextran sulphate but not by heparin. Unlabelled hyaluronan could inhibit the liver uptake of labelled chondroitin sulphate but not labelled heparin. Unlabelled chondroitin sulphate and dextran sulphate inhibited cell association of labelled hyaluronan to liver endothelial cells in culture more effectively than unlabelled hyaluronan. Our data show that the liver hyaluronan receptors also recognize and effectively bind chondroitin sulphate and dextran sulphate but not heparin and that a hyaluronan-specific saturable degradative mechanism exists in the circulation. Such a mechanism could explain why hyaluronan in the general circulation has a much lower Mw than the hyaluronan in lymph. The results also indicate that increased hyaluronan levels in serum, and increased urinary excretion of hyaluronan, may be secondary to increased outflow of chondroitin sulphate from the tissues during some pathological conditions.

Urinary excretion of sulfated polysaccharides administered to Wistar rats suggests a renal permselectivity to these polymers based on molecular size.

Sulfated polysaccharides were administered to Wistar rats and their elimination from the blood as well as their urinary excretion were evaluated. Sulfated polysaccharides with differences in molecular mass, charge density and molecular structure were obtained from algae, marine invertebrates and vertebrates. A simple methodology based on the metachromatic property of these polysaccharides with 1,9-dimethylmethylene blue was used to estimate their concentration in urine and blood. Renal permselectivity to these macromolecules was based on molecular size, but the upper limit of molecular mass for excretion of a sulfated polysaccharide in urine varies among polymers with different structures. For dextran sulfates the upper limit is approximately 8 kDa. Chondroitin 4- and 6-sulfates were excreted as fragments of approximately 30 kDa, which is smaller than the injected polysaccharide. This suggests that they were degraded enzymatically in vivo. Large synthetic polymers (dextran sulfate > 8 kDa) were not excreted in urine, but slowly disappeared from the blood. Evaluation of their tissue distribution after intravenous administration indicated that these molecules are preferentially accumulated in the kidney.