Title of Invention

A PHARMACEUTICAL COMPOSITION FOR THE HEALING OF CELLS

Abstract

ABSTRACT IN/PCT/2002/01652/CHE A pharmaceutical composition for the healing of cells The present invention relates to a pharmaceutical composition for use in the healing of cells comprising from 0.0001 percentage weight for volume (%w/v) to 2.0 %w/v of an alginic acid or a salt of alginic acid having a molecular weight in the range of from above 250,000 to 1,000,000, provided that if the composition additionally contains an alginic acid or an alginic acid salt having a molecular weight in the range of from 20,000 to 250,000 it is present in an amount of less than 1% w/w.

Full Text

This invention relates to a pharmaceutical composition for the treatment and/or prophylaxis of mucosal damage in a mammal. In particular, this invention relates to such pharmaceutical compositions including water soluble salts of alginic acid. The gastrointestinal (GI) tract in mammals consists of the oral cavity, the oesophagus, the stomach and the small and large intestine. The tract is lined by a layer of cells known as the mucosa, being stratified squamous non-keratinised epithelium in the case of the oral cavity and the oesophagus and columnar epithelium in the case of the remainder. This mucosa suffers a number of assaults from time to time, including: - mechanical damage and/or hot/cold stress from food and drink; - exposure to contents from the stomach and duodenum (reflux); and - systemically and/or topically induced mucosal damage such as that caused by the action of prostaglandin inhibitors (aspirin) or indomethacin. All of the above assaults (but particularly reflux) can result in local injury to the mucosa which itself can lead to oesophagitis (mucosal inflammation) and in severe or chronic instances, Barrett's oesophagus (in which a lower portion of the oesophagus becomes lined with columnar epithelium) may develop. The upper GI tract has many defence mechanisms which act to counter the assaults mentioned above. These include: - the secretion of mucus and bicarbonate which is generally considered to have both a protective and a neutralising function; - the secretion of saliva which acts to lubricate the oesophagus and raise the pH in the oral cavity and oesophagus; - the lower oesophageal or cardiac sphincter which acts to confine gastric content to the stomach; and - peristalsis or clearance whereby, through the action of swallowing, the bolus is moved through the oesophagus to the stomach by peristaltic waves. All too often, however, these defences prove inadequate and damage to the mucosa or cellular lining of the oral cavity, oesophagus and/or stomach occurs. All of this damage is characterised by injury to the upper cell layers in the mucosa. GB-A-2324725 discloses a pharmaceutical composition suitable for forming a mucoadhesive lining in the gastrointestinal tract which comprises an alginate or alginic acid having a mannuronic acid to guluronic acid residue ratio (M/G) of at least 1. The compositions disclosed comprise a combination of a low viscosity low molecular weight alginate and a high viscosity high molecular weight alginate. GB-A-232 4724 discloses a pharmaceutical composition comprising high concentrations (e.g. 8 to 15% w/w) of sodium alginate having an M/G ratio of at least 0.6:1. EP-A-0059221 discloses a composition for the protection of the gastrointestinal tract which comprises a water soluble salt of alginic acid. The water soluble salt has a molecular weight of 60,000 to 250,000. WO 91/11205 discloses a method of treating wounds comprising applying to a wound a biopolymer composition comprising at least 70% molar p-D-mannuronate. The specification proposes that the alginate biopolymers may be made into fibres and spun, woven, mixed or otherwise incorporated into wound dressings. For internal wounds such as ulcers the biopolymer is provided as a solution that will coat the walls of the gastrointestinal tract. Accordingly a need exists for a pharmaceutical composition for use in the healing of mucosal damage in a mammal. A further need exists for the manufacture of a pharmaceutical composition which provides for the healing of mucosal damage in a mammal. According to a first aspect of the present invention there is provided a pharmaceutical composition for use in the healing of cells comprising from 0.0001 percentage weight for volume (%w/v) to 2.0 %w/v of an alginic acid or a salt of alginic acid having a molecular weight in the range of from above 250,000 to 1000,000, provided that if the composition additionally contains an alginic acid or a salt of alginic acid having a molecular weight in the range of from 20,000 to 250,000 it is present in an amount of less than 1% w/w. All references to molecular weight are weight average molecular weights (MW). The applicants have surprisingly found that the amount of an alginic acid or an alginic acid salt as hereinbefore described, being significantly less than that taught to be effective in the prior art, is nonetheless effective in the healing of cells in a mammal, particularly mucosal cells. In terms of the present invention, the term 'mucosa' is intended to encompass the tissue layer found lining the GI tract of a mammal. As such, it is composed of a layer of epithelium containing numerous mucous glands and an underlying layer of areolar and lymphoid tissue. The present invention takes cognisance of the fact that the healing of cells contained in the mucosa is evidenced by the cells displaying one or more characteristics selected from: - endocytosis and/or ruffling; - a decrease in cell-cell adhesion; - an increase in cell motility, for example in wound restitution; and - an epidermal growth factor (EGF) like response, for example, stimulation of the epidermal growth factor receptors (EGFr). The present invention therefore provides a pharmaceutical composition as hereinbefore described for use in promoting any one or more of - endocytosis and/or ruffling; - a decrease in cell-cell adhesion; - an increase in cell motility; and - a stimulation of the epidermal growth factor receptors (EGFr) in a cell, preferably a mucosal cell. The applicants have surprisingly discovered that high molecular weight alginic acids and alginic acid salts, particularly the water soluble salts, are more effective than low molecular weight alginic acids or alginic acid salts in promoting endocytic or/ruffling activity in cells and/or decreasing cell adhesion and/or increasing cell motility and/or mimicking the effect of epidermal growth factor by stimulating the EGFr on cells, In addition, the applicants have surprisingly discovered that, although lower molecular weight water soluble salts of alginic acid are not substantially effective in having a healing effect on cells, higher concentrations of these lower molecular weight salts do have this effect. The alginic acids or alginic acid salts for use in the present invention are those having a molecular weight of from 250,000 to 1,000,000, more preferably 300,000 to 400,000. Water soluble salts are preferred. Examples of suitable grades of such water soluble salts of alginic acid are the sodium salts Protanal SF/LF, SF60L and H120L, all supplied by FMC Biopolymer a.s. Another alginate which is particularly preferred is known as Poly M. Alginic acid and alginates are linear hetero-polysaccharides comprising units of p-D-mannuronic acid (denominating M units) and oc-L-mannuronic acid (denoting G units). Alginates, such as H120L, may be exposed to epimerases which cause the epimerisation of some M units to G units. For example HI20L may be epimerised to H120LA which is an alternase with alternating G units or H120B which is a blockase with blocks of G units, respectively. Poly M may comprise 95 to 100% M units with an estimated molecular weight of up to 1,000,000. Poly M may have a slight degree of epimerisation which accounts for it having a less than 100% mannuronate content. Poly M may be degraded, for example by heating, to have a molecular weight of about 400,000. The M/G ratio of the alginic acid or alginic acid salts used in the present invention is preferably at least 1 and may be at least 10 or above. Relatively small amounts of the alginic acid or alginic acid salts are required in the present invention such that preferred concentrations are from 0.02 to 1.8 %w/v, more preferably 0.05 to 1.5 %w/v. However, in combination with the above, a lower molecular weight alginic acid or alginic acid salt may be used, but at a relatively higher concentration. Thus, an alginic acid or alginic acid salt having a molecular weight of from 20,000 to 250,000 may be used at concentrations of from 2.0 to 10.0%w/v, preferably 2.5 to 8.0 %w/v. Examples of suitable grades of the lower molecular weight water soluble salts of alginic acid are the sodium salts Protanal LFR5/60, SF120, LFIOL and LF120 supplied by FMC Biopolymer a.s. The mucosal damage may be systemically and/or topically induced, for example, by the consumption of prostaglandin inhibitors, for example acetyl salicyclic acid (aspirin) or indomethacin coupled to exposure to gastric contents, or the damage may be mechanically induced. The damage may also be induced by the action of gastric contents on the oesophageal mucosa ('a little acid in the wrong place') through reflux of the luminal contents. The mucosal damage may be damage to any part of the epithelium of the gastrointestinal tract, including but not limited to the oral cavity, the oesophagus and the stomach. Previously, one approach to the problem of reflux has been to administer a preparation which on contact with gastric acid generates a carbonated gelatinous foam or raft which floats on the stomach contents. When reflux occurs it is this raft which precedes the stomach contents into the oesophagus, thus protecting the mucosa from further irritation. Kmswn preparations of this type include solid preparations in the form of powder or tablets containing a water soluble salt of alginic acid, sodium bicarbonate and antacid materials or liquid preparations containing sodium alginate, sodium bicarbonate and calcium carbonate marketed under the name GAVISCON (RTM Reckitt & Colman Products Ltd). Therefore, in one embodiment of the present invention, the compositions as hereinbefore described may be liquid, or become liquid upon vigorous shaking. Alternatively, the compositions may be presented as chewable tablets, granules, powders, soft gels and reconstitutable liquids. The inventors have found that through the use of the the alginic acid or alginic acid salts as hereinbefore described, a pharmaceutical composition may be arrived at which not only serves to physically prevent damage to the mucosa (as per GAVISCON (RTM) ) but also has a healing effect on existing damage to the mucosa. In the broadest sense, however, the invention includes a pharmaceutical composition including what has heretobefore been considered sub-clinical doses of selected forms of alginic acid or alginic acid salt to exert a healing effect on the mucosa at a cellular level rather than presenting a physical barrier to reflux and/or having a coating effect (not unlike a conventional plaster dressing). The water soluble salts of alginic acid may be sodium, potassium and/or magnesium salts. Preferably however, the water soluble salt is sodium alginate. (The term alginate' is intended to be used interchangeably with the term 'alginic acid'.) Preferably, high viscosity grade sodium alginate is used to prepare the compositions as hereinbefore described. These are grades of sodium alginate for which the viscosity of a 1% weight/volume aqueous solution, when determined on a Brookfield RVT viscometer using spindle number 3 at 20 r.p.m. at 20'C, falls within the range 200 - 1500 mPa.s. An example of a suitable commercial grade of sodium alginate is Protanal H120L as supplied by FMC Biopolymer a.s. The compositions as hereinbefore described may further comprise preservatives to prevent contamination and subsequent deterioration by micro-organisms. Examples of suitable preservatives are methyl, ethyl, propyl and butyl para-hydroxybenzoates and their salts, which are preferably used in combination e.g. methyl and propyl or ethyl and butyl. Preferred concentrations for the preservatives are 0.01 to 0.5% w/v. The compositions as hereinbefore described may also include one or more of the following ingredients: thickeners, suspending agents, tabletting agents, glidants, diluents, colouring, sweetening, flavouring or pH adjusting ingredients. According to a second aspect of the present invention there is provided a pharmaceutical composition comprising from 0.0001 percentage weight for volume {%w/v) to 2.0 %w/v of alginic acid or a salt of alginic acid having a molecular weight in the range of from above 250,000 to 1,000,000 for use in the healing of cells in a mammal, as evidenced by the promotion of one or more of endocytosis and/or ruffling; a decrease in cell-cell adhesion; an increase in cell motility; and a stimulation of the EGF receptors (EGFr) in a cell, provided that if the composition additionally contains an alginic acid or a salt of alginic acid having a molecular weight in the range of from 20,000 to 250,000 it is contained in an amount of less than 1% w/v. According to a further aspect of the present invention there is provided the use of alginic acid or an alginic acid salt in the manufacture of a pharmaceutical composition for the healing of cells in a mammal, as evidenced by the promotion of one or more of endocytosis and/or ruffling; a decrease in cell-cell adhesion; an increase in cell motility; and a stimulation of the EGF receptors (EGFr) in a cell, the composition comprising from 0.0001 percentage weight for volume (%w/v) to 2.0 %w/v of an alginic acid or a salt of alginic acid having a molecular weight in the range of from 250,000 to 1,000,000 and optionally from 2.0 %w/v to 10.0 %w/v of an alginic acid or a salt of alginic acid having a molecular weight of from 20,000 to 250,000. The treatment may comprise administering to a mammal in need thereof, a therapeutically effective amount of the composition. A therapeutically effective amount of the composition may present from 0.1 to 50 mg of the alginic acid salt per kilogram of the mammal, preferably from 1 to 10 mg/kg. Such dosages may be repeated every 4 to 24 hours. According to a further aspect to the present invention there is provided a process for the preparation of pharmaceutical composition comprising from 0.0001 percentage weight for volume (%w/v) to 2.0 %w/v of alginic acid or a salt of alginic acid having a molecular weight of from above 250,000 to 1,000,000 and a pharmaceutically acceptable carrier, the process including the steps of adding the alginic acid or alginic acid salt to the carrier and mixing. According to a further aspect to the present invention there is provided a method for the healing of cells in a mammal, the method comprising the steps of administering to a mammal in need thereof a pharmaceutically effective amount of a pharmaceutical composition comprising from 0.0001 percentage weight for volume (%w/v) to 2.0 %w/v of alginic acid or a salt of alginic acid having a molecular weight in the range of from 250,000 to 1,000,000 and optionally from 2.0 %w/v to 10.0 %w/v of alginic acid or a salt of alginic acid having a molecular weight in the range of from 20,000 to 250,000. The pharmaceutical composition which is administered to a mammal promotes any one or more of: - endocytosis and/or ruffling; - a decrease in cell-cell adhesion; - an increase in cell motility; and - a stimulation of the epidermal growth factor receptors (EGFr) in a cell, preferably a mucosal cell. The present invention is further described with reference to the Figures of the accompanying drawings, in which: Figure lA illustrates the results of the effect of certain alginates (2mg/ml) in promoting endocytosis in squamosa carcinoma cells (0E21); and Figure IB illustrates the results of the effect of certain alginates {2mg/ml) in promoting endocytosis in adenocarcinoma cells (OE33) . Figure 2 illustrates the migration dose response of OE33 cell lines in different concentrations of H120L and LFR5/60. It can clearly be seen from this illustration that lower molecular weight alginates such as LFR5/60 do not have an appreciable effect on cell migration at low concentrations whereas higher molecular weight alginates such as H120L do have an effect at these lower concentrations. Figure 3 illustrates the endocytosis dose response of 0E21 cell lines in response to differing concentrations of H120L. It can be seen from this illustration that there is a maximum effect at 5mg/ml H120L. The invention will now be described with reference to the following examples. Example 1 - Indomethacin-induced mucosal damage in rats Alginates Protanal H120L, LFR5/60 and LFIOL (supplied by FMC Biopolymer a.s.) were used in the study. The different samples of alginate (4mg/ml in H2O containing 2% w/v hydroxypropylmethyl cellulose (HPMC)) were administered by gastric gavage to rats (2ml per rat) . 2% w/v HPMC in H2O was administered by gastric gavage as the negative control. SOpg/kg Epidermal Growth Factor (EGF) in H2O containing 2% w/v HPMC was administered by gastric gavage as the positive control. After 30 minutes all rats were subcutaneously injected with 20mg/] Three hours later the rats were sacrificed, their stomachs removed and fixed in formalin overnight. Damage to the gastric mucosa was assessed macroscopically by opening the stomachs and pinning them out/ then placing a transparent sheet with a 1mm grid placed on top and thereafter assessing the total area of macroscopic damage. The results were as follows: Table 1: Results of macroscopic gastric mucosal damage in rats. From these results it can be seen that alginate H120L significantly outperforms in ameliorating gastric mucosal damage in rats having had gastric mucosal damage systemically induced by indomethacin. Example 2 - Indomethacin-induced mucosal damage in rats (second study) The same protocol as for Example 1 was used but the following alginate/control solutions were administered to the rats (all weights per volume): 2% HPMC 2% HPMC + 50vjg/kg EGF 50yg/kg EGF 4 mg/ml H120L + 2% HPMC 4 mg/ml H120L 4 mg/ml H120L(a) (autoclaved) Table 2: Results of macroscopic gastric mucosal damage in rats. These results show the effect of H120L is similar to the effects of EGF in protecting GI mucosa in rats. The HPMC solution has no effect and the effect of H120L is significantly reduced when the structure is broken down by autoclaving {H120L(a)). Example 3 - Cell Migration A cell culture insert crucible having an B\im. millipore filter is suspended in each of the following test substances: H120L (Img/ml); epidermal growth factor (EGF) (10" M); OE33 oesophageal EGFr negative cells were added to the crucible and left for three hours. After this time the number of cells having moved through the filter into the test substance was estimated using bound dye technique (toluidine blue) via colorimetric assay. The results are as follows: Table 3: Cell Migration Assay These results indicate that H120L has an increased effect on cell migration over and above the known effect of epidermal growth factor. Thus H120L has be expected to have a correspondingly negative affect on cell-cell adhesion and a positive effect on wound restitution. Example 4 Using the technique as described in Example 3, the following test substances (all at Img/ml): Epidermal growth factor (EGF) H120L LFR 5/60 Poly M Poly M (degraded) H120L-A Table 4: Cell Migration Assays, Example 5 - Endocytosis A cell line of HCT116 cells was seeded out onto wells the night before the experiment. The cells were incubated for one hour with the alginic acid samples at 0.2mg/ml and 2mg/ml. Molecular probes (Fluorospheres (RTM) ) in the form of polystyrene microspheres were used as a fluorescent marker and the fluorescence in the cells was analysed by flow cytometry. In addition to the above, porcine oesophagus biopsies were incubated in Ussing chambers and analysed as above. The results are as follows (corrected for a normal value of 1.0) : Table 5: Endocytosis Assay These results clearly indicate that alginates have a positive effect in promoting endocytosis in GI cells in a mammal. Example 6 Cell lines of 0E21 squamosa carcinoma cells and of DE33 adenocarcinoma cells were seeded out into wells two days before the experiment. The cells were incubated for one hour with various alginate sample at concentrations of 2mg/ml and epidermal growth factor at a concentration of lOmg/ml. Molecular probes (Fluorospheres (RTM)) in the form of polystyrene microspheres were used as a fluorescent marker and the fluorescence in the cells was analysed by flow cytometry. The following alginates were tested in this experiment. H120L Poly M The results are shown in Figures lA and IB. These results indicate a significant increase in endocytosis in the presence of these alginates. Example 7 - Binding to Epidermal Growth "Factor Raceptors Oesophageal squamous carcinoma cells (Cell line OE33) were incubated for one hour with different alginate grades being SF60L, H120L, SF/LF and LFR5/60. The alginate was then removed and epidermal growth factor added. Fluorescent epidermal growth factor and CT-b FITC or transferring were added to the cell culture dish at the same time and incubated for one hour at 37°C with 5% CO2. Confocal microscopy and flow cytometry were used to analyse the results. Observations indicated that when the cells were incubated with the alginates, there was no evidence present of epidermal growth factor binding to epidermal growth factor receptors. This indicates that alginates seem to mimic the effect of epidermal growth factor on a cell in that the alginates seem to bind to the epidermal growth factor receptors on the cell membrane. Example 8 - Aqueous Liquid Compositions I. A composition containing Sodium Alginate H120L 0.4 %w/v Sodium Ethyl parahydroxybenzoate 2.0 %w/v Sodium Butyl parahydroxybenzoate 0.2 %w/v Sweetener q.s. Flavour q.s. Deionised water to 100% is made up as follows: 1. Dissolve the preservatives flavour and sweetener. 2. Add the alginate and stir to dissolve. 3. Make up to volume. II. As above but with the addition of 2 %w/v Hydroxy propyl methyl cellulose (2% solution with a viscosity of lOOmPas" at 25*0) added at stage 1. III. Buffered Solution Sodium Alginate H12 0L 0.6 %w/v Hydroxy Propyl Methyl Cellulose 2.0 %w/v Monopotassium Phosphate 0.02 %w/v Dipotassium Phosphate 0.04 %w/v Sodium Ethyl parahydroxybenzoate 2.0 %w/v Sodium Butyl parahydroxybenzoate 0.2 %w/v Sweetener q.s. Flavour q.s. Deionised water to 100% IV. With antacid component As III above plus Sodium bicarbonate and Calcium carbonate, both 2.0 %w/v Example 9 - Bioadhesive Liquids I mg/lOml Monopotassium phosphate 20.00 Dipotassium phosphate 40.00 Sodium bicarbonate 168.00 Ethyl paraben 20.00 Sodium butyl paraben 2.22 Sodium saccharin 10.00 Xanthan gum 4 9.00 Calcium carbonate 80.00 Locust bean gum 21.00 Sodium alginate H120L 4.00 Flavour 7.00 Deionised water to 10ml II mg/lOmI Monopotassium phosphate 20.00 Dipotassium phosphate 40.00 Sodium bicarbonate ]68.00 Ethyl paraben 20.00 Sodium butyl paraben 2.22 Sodium saccharin 10.00 Xanthan gum 49.00 Calcium carbonate 80.00 Locust bean gum 21.00 Sodium alginate H120L 4.00 Sodium Alginate LFR5/60 550.00 Flavour 7.00 Deionised water to 10ml Example 10 - Powders Dry powder mixture in sachet for dissolving in water prior to administration Sodium Alginate H120L 0.2g Sucrose 4 . 8g Flavour 0. Ig Sweeteners O.lg per sachet Dry blend and fill into sachets. Reconstitute by pouring sachet contents into 100ml water and stirring to dissolve. Example 11 - Chewable tablet 1. Alginate H120L 5mg 2. Xylitol lOOOmg 3. Mannitol 1350mg 4. Povidone K30 50mg 5. Flavour 25mg 6. Magnesium stearate 25mg Dry blend 1, 2 and 3. Granulate using a solution of 4 in isopropanol, dry at 50C. Pass through a 1000micron mesh. Add 5 and 6 to the granules, mix for 3 minutes and press into tablets. On chewing, combination with approximately lOmls of saliva gives a 0.05 %w/v active alginate solution. Example 12 - Oral Gel Alginate H120L 0.4 %w/v Hydroxy Propyl Methyl Cellulose 10.0 %w/v Sodium Ethyl parahydroxybenzoate 0.2 %w/v Sodium Butyl parahydroxybenzoate 0.2 %w/v Flavour q.s. Sweetener q.s. Water to 100% WE CLAIM; 1. A pharmaceutical composition for the healing of cells comprising from 0.0001 percentage weight for volume (%w/v) to 2.0 %w/v of an alginic acid or a salt of alginic acid having a molecular weight in the range of from above 250,000 to 1,000,000, provided that if the composition additionally contains an alginic acid or an alginic acid salt having a molecular weight in the range of from 20,000 to 250,000 it is present in an amount of less than 1 % w/w. 2. The composition as claimed in claim 1 wherein the composition comprises from 0.02 to 1.8 %w/v of an alginic acid or an alginic acid salt having a molecular weight of300,000 to 400,000. 3. The composition as claimed in claim 1 or claim 2 wherein the alginic acid or alginic acid or alginic acid salt has a mannuronic acid residue to guluronic acid residue ratio (M/G)ofatleast 1. 4. The composition as claimed in claim 3 wherein the M/G ratio is at least 10. 5. The composition as claimed in any one of the preceding claims wherein the composition is presented as a liquid, chewable tablet, granule, powder or soft gel. 6. The composition as claimed in any one of the preceding claims wherein the salt of alginic acid is a water soluble salt which is selected from sodium, potassium and magnesium salts. 7. The composition as claimed in any one of the preceding claim comprising any one or more of thickeners, suspending agents, tabletting agents, glidants, diluents, colouring, sweetening, flavouring, pH adjusting ingredients and preservatives selected from methyl, ethyl, propyl and butyl para-hydroxybenzoates and their salts. 8. A pharmaceutical composition comprising from 0.0001 percentage weight for volume (%w/v) to 2.0 %w/v of alginic acid or a salt of alginic acid having a molecular weight in the range of from above 250,000 to 1,000,000 for the healing of cells in a mammal, as evidenced by the promotion of one or more of endocytosis and/or ruffling; a decrease in cell-cell adhesion; an increase in cell motility; and a stimulation of the epidermal growth factor receptors (EGFr) in a cell, provided that if the composition additionally contains an alginic acid or an alginic acid salt having a molecular weight in the range of from 20,000 to 250,000 it is contained in an amount of less than 1% w/v. 9. A process for the preparation of a pharmaceutical composition comprising from 0.0001 percentage weight for volume (%w/v) to 2.0 %w/v of alginic acid or a salt of alginic acid having a molecular weight of from above 250,000 to 1,000,000 and a pharmaceutically acceptable carrier, the process including the steps of adding the alginic acid or a salt of alginic acid to the carrier and mixing.

Documents:

in-pct-2002-1652-che abstract-duplicate.pdf

in-pct-2002-1652-che abstract.pdf

in-pct-2002-1652-che claims-duplicate.pdf

in-pct-2002-1652-che claims.pdf

in-pct-2002-1652-che correspondence-others.pdf

in-pct-2002-1652-che correspondence-po.pdf

in-pct-2002-1652-che description (complete)-duplicate.pdf

in-pct-2002-1652-che description (complete).pdf

in-pct-2002-1652-che drawings-duplicate.pdf

in-pct-2002-1652-che drawings.pdf

in-pct-2002-1652-che form-1.pdf

in-pct-2002-1652-che form-19.pdf

in-pct-2002-1652-che form-26.pdf

in-pct-2002-1652-che form-3.pdf

in-pct-2002-1652-che form-5.pdf

in-pct-2002-1652-che pct.pdf

in-pct-2002-1652-che petition.pdf