Filed of the Invention:

The present invention relates to Pentosan Polysulfate Sodium characterized by Capillary Electrophoresis, wherein the ascending portion exhibits a multitude of peaks (1-10) and descending portion exhibits no peaks. This invention further relates to area percent after the sulphate peak in ascending portion of the electropherogram is not less than (NLT) 30 % of total area percentage of all the peaks between 1.29 to 1.80 relative migration time (RMT).

Background of the Invention:

Pentosan Polysulfate Sodium [PPS] is a semi-synthetic polyanionic sulfated polysaccharide that is structurally similar to glycosaminoglycans (GAGs), PPS is based on a natural product Xylan, extracted from the bark of Beech wood tree. PPS chemically known as (I,4)-P-D-xylan-2,3-bis(hydrogen sulfate), sodium salt with a molecular weight of 4000-6000 Dalton is represented by the formula I. Pentosan Polysulfate Sodium salt is currently marketed under the trade name Elmiron® by Ortho McNeil in the form of oral 100 mg capsule for the treatment of interstitial cystitis.

US 2,689,848 discloses process for the preparation of Pentosan Polysulfate Sodium from Xylan. Xylan is a polysaccharide found in plant cell walls, is toxic and hence not suitable for pharmacological usage. Degree of polymerization is a measure of toxicity and hence is directly related to molecular size and configuration. Best therapeutic index measured in Zn viscosity values is found to be between 0.0138 and 0.0201 and sulfuric acid ester salt of Xylan is found to possess these values.

Xylan is first converted into sodium salt of sulfuric acid ester of Xylan by reacting with chlorosulphonic acid in pyridine at 80°C and followed by treating with sodium hydroxide to get Sulfonic acid salt of Xylan which is isolated in methanol. The sodium salt of sulfuric acid ester obtained is a polymer of high molecular weight and is subjected to oxidative depolymerisation using sulphuric acid and hydrogen peroxide, Depolymerized Sodium Xylan Polysulfate is subjected to dialysis (membrane filtration) in DM water. Later solution is concentrated by distillation to get residue. Residue is treated with small quantities of sodium acetate and isolated in alcohol: acetone (1:1) solvent mixture to get crude Pentosan Polysulfate sodium. It is purified in a mixture of water and acetone to give PPS. However, in this patent analytical method is not disclosed.

WO 2007/123800 discloses qualitative analysis of PPS by capillary electrophoresis wherein, substantially bell shaped curve in the electropherogram corresponding to the presence of PPS in a graph of change-in-absorption-versus-time. The bell shaped curve has a first half corresponding to an earlier absorption and a second half corresponding to a later absorption. Though this application explains CE absorption curve with a bell shape defining the area percent of Pentosan Polysulfate Sodium, it does not disclose any quantitative analysis for assessing the purity of each peak obtained in the electropherogram of Pentosan Polysulfate sodium.

WO 2008/107906 claims amorphous Pentosan Polysulfate sodium with molecular weight of range 3000 to 10000 Dalton, wherein PPS is subjected to crystallization in alcohol or
lyophilized to obtain amorphous PPS. This patent application also does not reveal any
analytical method for determining the quality control limits of PPS.

Journal of Pharmaceutical and Biomedical Analysis 31 (2003) 133-141 discloses the varied therapeutic uses of PPS. It has been investigated for its effectiveness against osteoarthritis, interstitial cystitis, and myocardial ischemia (obstruction of blood flow in the heart). Also PPS has been found to possess anticoagulant properties helpful in preventing brain paralysis and also a potent substituent for relatively costlier Heparin. Furthermore, PPS, along with other sulfated polyanions, have shown beneficial effects in treatment of Alzheimer's cerebral ischemia, HIV related encephalopathy or prion diseases (degenerative brain diseases), This journal discloses Capillary Electrophoresis method of analysis to characterize GAGs. However the disclosed analytical method does not mention the quantitative aspects of PPS.

Chromatographia 2003, 58, 343-348 discloses analytical method for PPS using Fourier Transform infrared spectroscopy, wherein the mid-IR region (IP region of the spectrum below 1800 cm'1) is found to give accurate peaks relating to PPS. This Journal article discloses comparison data of different samples of PPS by using mid-IR region. However even this article does not disclose any quantitative analytical method for PPS.

Journal of Urology Vol 175, 1143-1147 discloses the levels of PPS in urine which correspond to Low molecular weight fractions of Pentosan Polysulfate Sodium. High molecular weight fractions do not get absorbed by the gastrointestinal tract and hence not excreted out but instead get accumulated in the body. This difference is because commercial PPS is a heterogeneous mixture of various molecular sizes. Therefore the molecular size of Pentosan Polysulfate Sodium affects the urine excretion.

Hence the polydisperse nature of Pentosan Polysulfate Sodium necessitates in the art for an effective reproducible method of quantitative analytical procedure that detects variations in active pharmaceutical ingredient relating to size, degree of sulfation. Also there exists a need in the art for reproducible quality control method to assess different batches of Pentosan Polysulfate Sodium provided by potential suppliers thereby purity of Pentosan Polysulfate sodium can be optimized.

Surprisingly, the present inventors have found a quantitative analytical procedure wherein PPS purity is optimized and peaks corresponding to Pentosan Polysulfate Sodium are characterized. This analytical method will help to obtain Pentosan Polysulfate Sodium with minor variations in molecular size thereby reducing the toxic effects due to polydisperse nature of Pentosan Polysulfate Sodium as described above.

Object of the Invention:

The main object of the present invention is to provide Pentosan Polysulfate Sodium characterized by Capillary Electrophoresis, wherein the ascending portion exhibits a multitude of peaks (1-10) and descending portion exhibits no peaks.

Another object of the present invention is to provide Pentosan Polysulfate Sodium characterized by Capillary Electrophoresis, wherein area percent after the sulphate peaks in ascending portion of the electropherogram is not less than (NLT) 30 % of total area percentage of all the peaks between 129 to 1.80 relative migration time (RMT),

Yet another object of the present invention is to provide Pentosan Polysulfate Sodium characterized by Capillary Electrophoresis, wherein the lower part of the ascending portion exhibits not more than 2 peaks between 1.0 to 1.23 relative migration time (RMT).

Summary of the Invention:

The present invention provides stable method of analysis for active pharmaceutical ingredient Pentosan Polysulfate Sodium containing substantially homogenous molecular weights.

Pentosan Polysulfate Sodium samples were analysed by capillary electrophoresis (CE). Capillary Zone Electrophoresis may be conducted in a free solution. Separation of components during capillary zone electrophoresis may be based on differences in various components' charge to mass ratio. A homogenous buffer solution may be used. A constant electric field may be applied. The process of capillary zone electrophoresis may depend on pH. Buffer additives may include any of the following organic salts, organic solvents, urea, sulfonic acids, cationic surfactants, cellulose derivatives, amines, organic acids and organic polymers. The analytic capability of CE on a PPS active ingredient in accordance with various embodiments of the invention allows quantification of sulfate in the presence or absence of a known amount of sulfite; quantification of total Pentosan levels, detection of oligosaccharides.

The main aspect of the present invention is to provide Pentosan Polysulfate Sodium characterized by Capillary Electrophoresis, wherein the ascending portion exhibits a multitude of peaks (1-10) and descending portion exhibits no peaks. The ascending portion contains peaks which correspond to chlorides, sulfates and also non light absorbing Pentosan Polysulfate Sodium molecules. The descending portion does not show any peaks and if present correspond to non-Pentosan Polysulfate Sodium peaks.

Another aspect of the present invention is to provide Pentosan Polysulfate Sodium characterized by Capillary Electrophoresis, wherein area percentage of first four peaks after sulphate peaks in ascending portion of the electropherogram is not less than (NLT) 30 % of total area percentage of all the peaks between 1.29 to 1.80 relative migration time (RMT).

Yet another aspect of the present invention is to provide Pentosan Polysulfate Sodium characterized by Capillary Electrophoresis, wherein the lower part of the ascending portion exhibits not more than 2 peaks between 1.0 to 1,23 relative migration time (RMT). Peaks in the lower part eluted small anions chlorides and sulfates.

Brief Description of the Drawings:

Figure 1: shows an electropherogram of PPS. Detailed Description of the Invention:
Various embodiments of the present invention are directed to quantitative analysis of Pentosan Polysulfate Sodium. The active ingredient PPS is used as an anticoagulant to treat osteoarthritis, interstitial cystitis, transmissible spongiform encephalopathy. Phase one clinical trials have been undertaken to evaluate the antiviral and anti tumor effects of PPS. Pentosan Polysulfate Sodium due to its varied pharmacological activities demands an effective quantitative analytical method to differentiate fine structural details of polysaccharides and should exhibit uniformity in all the production batches. The natural product Xylan can vary from batch to batch and hence may give rise to structural differences in the product obtained from such batches. Therefore there exists a need for an efficient qualitative analytical method for optimizing the purity of Pentosan

Polysulfate Sodium

The main embodiment of the present invention relates to optimization of Pentosan Polysulfate Sodium characterized by capillary electrophoresis. Electrophoresis is an electro-analytical technique used for the separation of large molecules from small molecules. The differential movement of molecules through a capillary under the influence of an electric field is called capillary electrophoresis. The main components of capillary electrophoresis are

a) Source vial, destination vial and capillary tube filled with electrolytic buffer solution.

b) Capillary is introduced into the sample vial and then returned to source vial.

c) Migration of analytes initiated by electric field (E) applied b/w source &
destination vial.

d) All ions (+)ve or (-)ve are pulled through capillary in same direction by electroosmotic flow (EOF).

e) Separated analytes get detected near the outlet end of the tube which is sent to data output device & displayed as electropherogram as peaks with different migration times.

The detectors can be either UV-Vis absorbance; Florescence detection or CE can be coupled with MS or surface Enhanced Raman Spectroscopy (SERS).

PPS is a semi-synthetic polysulfated oligosaccharide comprising a mixture of multiply charged anionic polysaccharides. After injection of a sample into the capillary tube, the right end of the tube and an electrode (cathode) are placed in a buffer reservoir, while the left end of the tube and another electrode (anode) are simultaneously placed in another buffer reservoir. A voltage is applied between the electrode and cathode. The bulk fluid in the capillary flows with the positively charged, hydrated cations in a process called electro-osmosis or electro osmotic flow (EOF). Other positively charged analytes move in the same direction. Negatively charged, anionic molecules move in the opposite direction towards the cathode. If EOF exceeds electrophoretic mobility for a group of anions, the anions will move in the other direction. The polarity of the electrodes may be reversed to ensure that the negatively charged anions in the sample pass the detector.

PPS has anionic ends and is non-light absorbing. As the PPS sample and buffer move through the capillary, the similarly charged, non-light absorbing sample displaces the light absorbing buffer, creating a zone of reduced buffer concentration. The sample passes the detector window, wherein the detector will detect a reduction in light absorption EOF of buffer solution & electrophoretic mobilities of different anions will enable different components of PPS to travel the capillary tube at different rates which are seen at the detector. Therefore small and multiple charged anions move quickly and are detected at early times. Homogeneity (units of similar characteristics) of PPS may be detected by calculating relative size of small oligosaccharide peaks as % of total PPS peaks in electrophoregram.

Relative Migration Time [RMT] is calculated by time required for migration by Pentosan peaks in relation to sulfate peak.

In another embodiment, the present invention provides a pharmaceutical composition that includes a therapeutically effective amount of pure Pentosan Polysulfate and its salts prepared according to the processes of the present invention and one or more pharmaceutically acceptable carriers, excipients or diluents.

Accordingly, the pharmaceutical composition comprising pure Pentosan Polysulfate or their pharmaceutically acceptable salts along with one or more pharmaceutically acceptable carriers of this invention may further be formulated as: solid oral dosage forms such as, but not limited to, powders, granules, pellets, tablets, and capsules; liquid oral dosage forms such as but not limited to syrups, suspensions, dispersions, and emulsions, and injectable preparations such as but not limited to solutions, dispersions, and freeze dried compositions. Formulations may be in the form of immediate release, delayed release or modified release. The compositions may be prepared by direct blending, dry granulation, or wet granulation or by extrusion and spheronization. Compositions may be presented as uncoated, film coated, sugar coated, powder coated, enteric coated or modified release coated. Compositions of the present invention may further comprise one or more pharmaceutically acceptable excipients.

Instrumentation:

The analysis was performed on the Agilent CE instrument, equipped with a UV diode-array detector. A polyimide coated fused silica capillary (Agilent Technologies, Vic, Austrialia) (ID=50 micro mts) with an effective length of 48 cm was used. The wavelength of UV detector was set at 350 nm (reference wavelength set at 240 nm), Chemstation installed on a personal computer allowed for instrumental operation and integration strategies. MINITAB statistical software was used to achieve surface response methodology information.

Sample Preparation

Background electrolyte: Benzene- 1,2,4-tricarboxyIic acid (8.75 mmol/lt; pH-4.9); Milli-Q water. Approximately 42 ml of freshly prepared 0.1 M NaOH was added to obtain a pH of 4.9. This solution was then made to 200 ml with Milli-Q water. The final concentration of 8.75 mmol"1 was obtained for the BTC solution.

Sample: 18.5 milligram of PPS was made upto 10 ml with Milli-Q water. The final concentration of- mg ml'1 was obtained.

Method Development:

CE Method: All solutions were filtered through a 0.45 micro mt syringe filter. The sample was introduced at the cathode hydrodynarnically at an optimal setting of 40 s at 50 mbar. The optimal voltage applied was -20 kV. The temperature for the optimum analysis was held at 25°C. Before its first use, the capillary was flushed with freshly prepared 1.0M NaOH for 1 h. Equilibrium was performed for 1 h by flushing with BGE at 25°C. This has the effect of protonating the silanol sited on the capillary surface. The capillary was rinsed between each injection for 5 min with BGE. After each sequence or where the capillary was not used for extended periods, the capillary was rinsed with Milli-Q water for 30 min and dried by flushing with air for 30 min.

Pentosan Polysulfate Sodium samples were run using the above conditions and electropherogram for various samples were obtained. Buffer additives may include any of the following organic salts, organic solvents, urea, sulfonic acids, cationic surfactants, cellulose derivatives, amines, organic acids and organic polymers. The analytic capability of CE on a PPS active ingredient in accordance with various embodiments of the invention allows quantification of sulfate in the presence or absence of a known amount of sulfite; quantification of total Pentosan Polysulfate Sodium levels, detection of oligosaccharides.

An electropherogram for the active ingredient Pentosan Polysulfate Sodium is prepared using capillary electrophoresis in a manner that satisfies a peak resolution standard, the electropherogram comprising a change-in-absorption-versus-time graph. Figure I depicts eieclropherogram of Pentosan Polysulfate Sodium, wherein the ascending portion exhibits a multitude of peaks (i-10) and descending portion exhibits no peaks. The main peak generally appears in the middle of the electropherogram. The lower part of the ascending portion exhibits not more than 2 peaks with in 1.23 relative migration time (RMT). First two peaks of the ascending portion relate to chlorides and sulfates which account to less than 5 % of total area attributable to Pentosan Polysulfate Sodium. These results can estimate the degree of sulfonation in PPS sample. The later part of the ascending portion relates to non light absorbing Pentosan Polysulfate Sodium molecules which are detected earlier in time. Because they are of low molecular weight, they can travel more quickly through the capillary and are therefore detected much early. Area percentage of first four peaks in ascending portion of the electropherogram is not less than (NLT) 30 % of total area percentage of all the peaks between 1.29 to 1.80 relative migration time (RMT).

The descending portion exhibits no peaks corresponding to PPS. Any peak beyond 12 inin of migration time can be attributable to non Pentosan Polysulfate Sodium peaks. Hence presence of peaks beyond 12 min of migration time infers the presence of related substances. This can act as check point in the reaction monitoring. Reaction can be terminated immediately when peaks at 17 min begin to appear. This checkpoint will help us to obtain Pentosan with the desired molecular weight and purity thereby reducing the toxic effects due to the presence of more polymeric units.

We Claim:

1. A method of quantification of Pentosan Polysulfate sodium characterized by Capillary Electrophoresis.

2. The method according to claim 1, wherein Pentosan Polysulfate Sodium is characterized by Capillary Electrophoresis whose ascending portion exhibits a multitude of peaks (1-10) and descending portion exhibits no peaks.

3. The method according to claim 1, wherein Pentosan Polysulfate Sodium is characterized by Capillary Electrophoresis with an area percent without the sulphate peaks in ascending portion of the electropherogram not less than (NLT) 30 % of total area percentage of all the peaks between 1.29 to 1.80 relative migration time (RMT).

4. The method according to claim 1, wherein Pentosan Polysulfate Sodium is characterized by Capillary Electrophoresis with the lower part of the ascending portion exhibits not more than 2 peaks between 1.0 to 1.23 relative migration time (RMT).

5. The method according to claim 1, wherein Pentosan Polysulfate Sodium is characterized by Capillary Electrophoresis with the descending portion exhibits no peaks corresponding to Pentosan Polysulfate Sodium.

6. Pentosan Polysulfate Sodium characterized by Capillary Electrophoresis, wherein the descending portion of the electropherogram does not show any peaks.

7. The method according to claim 1, wherein Pentosan Polysulfate Sodium is characterized by electropherogram as depicted in figure 1.

8. A process for the preparation of Pentosan Polysulfate Sodium, wherein the reaction is terminated when peaks at 17 min in the CE electrop hero gram begin to appear.

9. Pentosan Polysulfate Sodium prepared as per process of claim 8.

10. A pharmaceutical composition comprising Pentosan Polysulfate Sodium, wherein Pentosan Polysulfate Sodium is prepared according to claim 8.