In-depth structural characterization of pentosan polysulfate sodium complex drug using orthogonal analytical tools.

Rapid advances have been made in developing analytical technologies for characterization of highly heterogeneous active ingredients of complex drugs, such as pentosan polysulfate (PPS), active ingredient of the drug Elmiron®, approved by the Food and Drug Administration and marketed in the United States to treat interstitial cystitis. PPS sulfated polysaccharides comprise of a repeat unit of ß(1-4)-D-xylopyranoses randomly substituted by 4-O-methyl-glucopyranosyluronic acid. To define the critical quality attributes (CQAs) of such a complex drug, it is critical to develop an approach that integrates data from orthogonal analytical methodologies. Here, we developed an approach integrating diverse analytical tools including gel permeation chromatography, LC/ESI-MS and NMR to measure CQAs of PPS. The proposed mathematical framework integrates the data from these diverse analytical methods as function of PPS chain length and building blocks. Our approach would facilitate in establishing a scientific foundation for comparative characterization of drug products with complex active ingredients.

Bottom-up and top-down profiling of pentosan polysulfate.

Pentosan polysulfate (PPS) is a semi-synthetic glycosaminoglycan (GAG) mimetic. PPS, synthesized through the chemical sulfonation of a plant-derived ß-(1 → 4)-xylan, is the active pharmaceutical ingredient of the drug Elmiron™ used to treat interstitial cystitis. Unlike natural GAGs that can be enzymatically broken down into oligosaccharides for analysis, PPS is an unnatural polyanionic polysaccharide and is not amenable to such an analytical approach. Instead reactive oxygen species were used for the controlled depolymerization of PPS and the resulting oligosaccharide fragments were then analyzed by liquid chromatography-mass spectrometry (LC-MS) to obtain bottom-up information on its composition. Because PPS has an average molecular weight ranging from 4000 to 6000 Da, similar to that of low molecular weight heparin, this suggested that it might be possible to use LC-MS on its intact chains and perform top-down analysis. The bottom-up and top-down analysis of PPS provides the first detailed compositional and structural information on PPS. Finally, we examined whether PPS would interfere with polysaccharide lyases and hydrolases, used in the analysis of natural GAGs such as chondroitin sulfates, heparan sulfate, and keratan sulfates. We found that PPS did not interfere with GAG analysis, suggesting that a combination of chemical and enzymatic treatment could be used to analyze samples containing both natural GAGs and PPS.

Plasma and synovial fluid concentrations of calcium pentosan polysulphate achieved in the horse following intramuscular injection.

Results from in vitro studies have indicated that calcium pentosan polysulphate (CaPPS) may be of therapeutic value in osteoarthritis (OA) in the horse. However, no controlled clinical trials using this drug in equine OA have yet been reported. If CaPPS is to be developed for such use, the relationship between the proposed i.m. dose of CaPPS to be used and the concentrations of drug attained in plasma and synovial fluid of the target joint should first be established. An investigation was undertaken to determine these concentrations after a single 2 mg/kg i.m. injection of CaPPS. Blood and synovial fluid samples were taken from 6 healthy, sound horses following i.m. CaPPS administration. Concentrations of CaPPS measured in the synovial fluid were, on the basis of published studies, sufficient to elicit a potential therapeutic effect on synoviocyte metabolism, and possibly also to stimulate proteoglycan synthesis and reduce matrix metalloproteinase activities in articular cartilage. It would therefore seem justified to investigate further the therapeutic effect of CaPPS in OA in the horse.

Improvements in pentosan polysulfate sodium quality assurance using fingerprint electropherograms.

Complex samples from polymer production, plant extracts or biotechnology mixtures can be characterized by fingerprints. Currently, the standard approach for sample characterization employs near-infrared (NIR) spectroscopy fingerprinting. Up to now, however, fingerprints obtained by chromatography or electrophoresis could only be visually evaluated. This type of inspection is very labor-intensive and difficult to validate. In order to transfer the use of fingerprints from spectroscopy to electrophoresis, spectra-like properties must be obtained through a complete alignment of the electropherograms. This has been achieved by interpolation and wavelet filtering of the baseline signal in the present work. The resulting data have been classified by several algorithms. The methods under survey include self-organizing maps (SOMs), artificial neural networks (ANNs), soft independent modeling of class analogy (SIMCA) and k-nearest neighbors (KNNs). In order to test the performance of this combined approach in practice, it was applied to the quality assurance of pentosan polysulfate (PPS). A recently developed capillary electrophoresis (CE) method using indirect UV detection was employed in these studies [1]. All algorithms were well capable of classifying the examined PPS test batches. Even minor variations in the PPS composition, not perceptible by visual inspection, could be automatically detected. The whole method has been validated by classifying various (n = 400) unknown PPS quality assurance samples, which have been correctly identified without exception.

Quality control of pentosane polysulfate by capillary zone electrophoresis using indirect detection.

Pentosane polysulfate sodium salt (PPS) is a mixture of multiply charged anionic polysaccharides, used for urological treatment. Several constituents of the polysaccharide can be characterized by a highly reproducible fingerprint. In comparison with earlier approaches the separation efficiency has been further improved using an anionic benzene-1,2,4-tricarboxylic acid buffer (8.7 mmol l-1, pH = 4.9) with indirect UV detection (lambda = 217 nm) and a special capillary pretreatment (1 M NaOH for 10 h at 25 degrees C applying -20 kV). The method has been optimized with regard to buffer concentration and pH. The robustness was tested on several capillaries. PPS was separated from all major synthetic impurities such a sulfate, chloride and acetate. Twelve PPS batches from two manufactures were measured and compared.

A monoclonal antibody that recognizes 2,3-, 2,6-, and 4,6-disulphate ester ring substitution in pyranose-containing polysaccharides. Its production, characterization and application for the quantitation of pentosan polysulphate, dextran sulphate, glycosaminoglycan polysulphate and chondroitin sulphate E.

A method is described for the preparation of a monoclonal antibody (MAb) which binds specifically to polysaccharides which contain 2,3-, 2,6- and 4,6-disulphate ester pyranose ring substitution. Such molecules include the semisynthetic heparinoids, pentosan polysulphate (PPS), dextran sulphate (DS) and glycosaminoglycan polysulphates (GAGPS), as well as the naturally occurring polysulphated polysaccharides, chondroitin sulphate E, and the 2,6-disulphated galactoses of carrageenans. The antibody (MAb 5-B-10) did not significantly cross-react with other sulphated polysaccharides such as heparin, heparan sulphate, the chondroitin sulphates, A, B, C or D, or keratan sulphate. No cross-reactivity was found with non-sulphated polysaccharides or polyanions including hyaluronic acid, xylan, or DNA. MAb 5-B-10 was characterized as IgM and kappa light chains, and was to develop an amplified enzyme-linked immunosorbent inhibition assay (ELISIA) to detect and quantitate some of the polysulphated polysaccharides in biological fluids. Using this assay, the lower limits of detection of these compounds in serum were in the order of 50 ng/ml; however 50% inhibition was obtained between 200-500 ng/ml. The intra- and inter-assay coefficients of variation for PPS were 4.2 +/- 2.8 and 16.7 +/- 13.8% respectively. The MAb 5-B-10 and the ELISIA were used to determine the levels of PPS in plasma of three healthy non-fasted volunteers for up to 120 min post-intravenous infusion (1.0 mg/kg). The results obtained compared favourably with kinetic data reported by others using a competitive binding assay method for this drug.