Pentosan polysulfate increases affinity between ADAMTS-5 and TIMP-3 through formation of an electrostatically driven trimolecular complex.

The semi-synthetic sulfated polysaccharide PPS (pentosan polysulfate) increases affinity between the aggrecan-degrading ADAMTSs (adamalysins with thrombospondin motifs) and their endogenous inhibitor, TIMP (tissue inhibitor of metalloproteinases)-3. In the present study we demonstrate that PPS mediates the formation of a high-affinity trimolecular complex with ADAMTS-5 and TIMP-3. A TIMP-3 mutant that lacks extracellular-matrix-binding ability was insensitive to this affinity increase, and truncated forms of ADAMTS-5 that lack the Sp (spacer) domain had reduced PPS-binding ability and sensitivity to the affinity increase. PPS molecules composed of 11 or more saccharide units were 100-fold more effective than those of eight saccharide units, indicating the involvement of extended or multiple protein-interaction sites. The formation of a high-affinity trimolecular complex was completely abolished in the presence of 0.4 M NaCl. These results suggest that PPS enhances the affinity between ADAMTS-5 and TIMP-3 by forming electrostatically driven trimolecular complexes under physiological conditions.

Optimisation for the separation of the oligosaccharide, sodium Pentosan Polysulfate by reverse polarity capillary zone electrophoresis using a central composite design.

The separation by reverse polarity capillary zone electrophoresis of the therapeutically developed sodium salt of Pentosan Polysulfate was optimised through the analysis of response surface methodologies, modeled using a central composite design. The optimisation investigated injection pressure, injection time and voltage and the effect of the conditions on retention times, peak areas, separation efficiency and the method sensitivity. The overall goal was to develop the most sensitive results with no decrease in separation efficiency. The following results were obtained: (1) retention times generally decreased as injection pressure, injection time and voltage increased, injection time having the least effect; (2) as expected peak areas increased as injection pressure and injection time increased but decreased as voltage increased; (3) separation efficiencies generally increased as injection pressure and injection time decreased, with voltage having almost no effect. For the optimum condition, the sample was introduced at the inlet vial at the cathode hydrodynamically, at optimal setting of 44 s at 35 mbar. The optimal voltage was -20 kV. In comparison with other methods, the optimum showed increased sensitivity, resolution and separation efficiency. Repeatability studies were performed on the optimum parameter conditions. Relative standard deviation values obtained were between 0.9 and 5.4%.

The production of antibodies to pentosanpolysulfate (ELMIRON, SP-54).

Pentosanpolysulfate (PPS) represents the product obtained after sulfation of xylan and is composed of beta 1----4-D-xylopyranose residues sulfated at C2 and C3. Studies have shown that this compound can often be effective in relieving the symptoms of interstitial cystitis (IC). In order to elucidate the mode of action of PPS in IC, a sensitive and reliable assay was needed. To this end we prepared an immunogenic form of PPS by coupling it to methylated bovine serum albumin (MBSA). This complex was used to immunize NZW rabbits (1 mg, IM). Four of five animals responded with anti-PPS antibodies, three of which had high titer (greater than 1/2000) as measured by an enzyme-linked immunosorbent assay (ELISA). All sera were routinely absorbed with an MBSA-Sepharose immunoadsorbent to remove anti-MBSA antibodies. ELISA inhibition tests were used to determine the sensitivity and specificity of the sera. At least 50 ng/ml of PPS could be routinely detected by this assay. A number of naturally occurring proteoglycans, polysaccharides, monosaccharides and disaccharides were examined for reactivity with the antibodies but only heparin was an effective inhibitor. Absorption with heparin immunoadsorbents reduced, but did not eliminate, the ability of heparin to inhibit anti-PPS binding. This activity could be destroyed by treatment with heparinase without affecting PPS inhibition. Normal urine did not affect the ELISA or ELISA inhibition tests and thus allowed the determination of PPS levels in IC patient urines. Initial analysis of seven IC patients receiving oral PPS revealed urine concentration of 0.8-16.0 micrograms/ml. No inhibition could be detected in pre-treatment urine samples.

Pentosan polysulphate: activation of heparin cofactor II or antithrombin III according to molecular weight fractionation.

The relative potency of pentosan polysulphate in activation of heparin cofactor II/thrombin interaction has been compared to heparin and dermatan sulphate and found to be within the same order. A skewed distribution of molecular weight forms was observed upon gel filtration of pentosan polysulphate with an average molecular weight of 4500 daltons. Two peaks of activity were observed in activation of heparin cofactor II. The greatest activity was observed in high molecular weight fractions (5-fold greater than that of average molecular weight) and a concentration-dependent profile indicated a template mechanism of action. A lower peak of activity was observed at average molecular weight and the effect of increasing concentrations of this material on activity indicated a mechanism involving binding to proteinase inhibitor or proteinase alone. Potentiation of antithrombin III/thrombin interaction was observed only in fractions greater than the average molecular weight. Concentration-dependent profiles indicated binding to antithrombin III and thrombin was a requisite of activation. A fraction of low molecular weight showed no property of activation of antithrombin III or heparin cofactor II/thrombin interaction. Three fractions of high, average and low molecular weights tested in clotting assays showed relative potencies corresponding to those observed in the purified systems.

Metabolism of sodium pentosan polysulphate in man--catabolism of iodinated derivatives.

An iodinated derivative of the heparin analogue SP54 has been prepared and used in conjunction with unlabelled SP54 to study the catabolism and organ distribution of this potential antithrombotic agent in healthy human volunteers. As observed previously with 125I-heparin, we found that the 125I-SP54 was rapidly cleared from the circulation, returning later in a desulphated form. Organ distribution studies with 123I-SP54 suggested that the liver and spleen were major sites of desulphation. Gel filtration and Polybrene binding showed the presence of sulphated macromolecular SP54 and desulphated macromolecular and depolymerised SP54 in post-injection urines. No depolymerised material was present in plasma suggesting depolymerisation occurs in the kidney.

Isolation and anticoagulant properties of a new sulfated xylan: comparison with heparin and a sodium pentosan polysulfate (SP-54).

Larchwood xylan was purified by diaminoethylaminoethyl (DEAE) cellulose chromatography, sulfated by heating with chlorosulfonic acid -pyridine complex and the sulfated polysaccharide was isolated by epichlorohydrin triethanolamine (ECTEOLA) cellulose chromatography as the sodium salt. It's molecular weight was approximately 25000 and the specific rotation was [alpha]D 20-70 degrees. Electrophoretic analysis of the sulfated xylan along with heparin and SP-54 using lithium acetate-agarose technique showed that the charge density of the xylan sulfate was similar to heparin and it moved as a single component in contrast to commercial heparin which resolved into two while SP-54 moved at the same rate as the marker dye. Anticoagulant properties of the sulfated xylan were compared with heparin and SP-54 by studying its effect on the activated partial thromboplastin time (APTT), prothrombin time (PT) and the thrombin time (TT) using pooled normal human plasma. The sulfated xylan was more effective than SP-54 in delaying coagulation by all the three measurements while it was less active than heparin in inhibiting APTT and PT.

The effect of a sulfated polysaccharide on antithrombin III.

A major naturally occurring inhibitor of blood is AT-III. We have investigated the potentiation of AT-III inhibition of factors Xa, IIa, and plasmin by the heparinoid substance SP-54. Both coagulation and amidolytic methods were used. SP-54 potentiated AT-III inhibition of factors Xa and IIa in the absence of heparin. When heparin was present, potentiation of inhibition of factor Xa usually occurred, but not of factor IIa. SP-54 also potentiated the AT-III inhibition of plasmin action. Laurell immunoelectrophoresis showed no changes in AT-III in the presence of SP-54. In view of the recent importance placed on the role of AT-III and factor Xa in thrombogenesis, an oral agent which potentiates AT-III activity can have important implications for thrombotic therapy.