FORM 2
THE PATENTS ACT, 1970
(39 OF 1970)
THE PROVISIONAL SPECIFICATION
(See section 10)
1. "Sodium Hyaluronate Polymer of tailored molecular weight for treatment of osteoarthritis with minimized side effects".
2. CADILA PHARMACEUTICALS LTD., "CADILA CORPORATE CAMPUS", SARKHEJ-DHOLKA ROAD, BHAT, AHMEDABAD, 382210, GUJARAT, INDIA, AN INDIAN COMPANY.
3. THE FOLLOWING SPECIFICATION DESCRIBES AND ASCERTAINS THE NATURE OF THIS INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED.

Title : "Sodium Hyaluronate Polymer of tailored molecular weight for treatment of osteoarthritis with minimized side effects"

FIELD OF INVENTION
The present invention pertains to sodium hyaluronate (HA) polymer of tailored molecular weights for treatment of osteoarthritis with minimized side effects.

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BACKGROUND OF INVENTION:
This relates to the preparation of sodium hyaluronate (HA) polymer of tailored molecular weights for treatment of osteoarthritis. Sodium hyaluronate influence the invasion and activity of cells participating in the acute and chronic inflammatory processes.
Degradation of structures in articular cartilage is a typical characteristic of all diseases resulting in chronic destruction of joint structures. Examples of such disorders are rheumatoid arthritis, psoriatic arthritis and osteoarthritis. Also, acute inflammation of joints is often accompanied by destruction of the cartilage, although in most cases this will not develop into chronically destructive disease. However, the mechanism of cartilage destruction is not fully known.
Osteoarthritis and rheumatoid arthritis are representative of the disease accompanied by the destruction of the cartilage matrix. It is thought that the destruction of the matrix in these diseases is triggered by mechanical stresses with aging in case of osteoarthritis and by access proliferation of the surface layer cells of the synovial membrane, pannus formation and inflammatory cell infiltration in the case of rheumatoid arthritis.
sodium hyaluronate (HA) is obtained from diverse sources such as animal tissues, rooster comb, certain plants as well as by microbial fermentation of specific organisms, sodium hyaluronate is naturally occurring linear polysaccharide of repeating di-saccharide units composed of glucuronic acid and N-acetyl-glucosamine. It is viscoelastic and its water retention capacity changes with pressure.
Other applications of the sodium hyaluronate is with regard to chronic inflammation of tissues, wherein it improves the effect of anti inflammatory drug by decreasing its diffusion out of the articular space, sodium hyaluronate is also used to protect the articular cartilage surfaces from postoperative injuries.
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Anika Therapeutics (USA) markets a product (ORTHOVISC) which is 1. 5 % solution of sodium hyaluronate of molecular weights 1 to 2.9 million Daltons. The HA is extracted from rooster combs.
It is generally reported that about 47% of the treated patients developed pain and swelling in knee after treatment with natural sodium hyaluronate produced from rooster comb. (Ann Rheum Dis.1994; 53(8):529-34). Symptoms of Pseudogout arthritis and inflammation immediately following the treatment with natural sodium hyaluronate injection were also observed (Arthritis and rheumatism 1998, 41(5): 939-41). Natural sodium hyaluronate produced from rooster comb has a molecular weights 1 to 2.9 million Dalton.
Biomatrix /Genzyme/Wyeth(USA) markets chemically modified sodium hyaluronate as 0.8 % solution (SYNVISC) with the molecular weight of 6 million Daltons. LG Pharmaceuticals (Korea) markets its sodium hyaluronate (HYRUAN) as 1 % solution with microbial fermentation. In this product the active is obtained by microbial fermentation.
It is generally reported that chemically modified hyaluronate (of average molecular weight of 6 million Daltons) used for the treatments of osteoarthritis develop pain, swelling and effusion (Canadian Adverse Reaction Newsletter 2005, 15(2): 1-2). About 27% to 50% of patients developed local inflammatory reactions when treated with chemically modified preparation of
hyaluronic acid (J Rheumatol 1995; 22(7): 1311-4. Clin Exp Rheumatol 2002; 20:445-54).
Chemically modified hyaluronate produced antibodies and granulomatous inflammation in animal models (Toxicologic pathol, 2003; 31(3):321-25). Increase in incidence of severe inflammatory reactions in 20% patients, to chemically modified hyaluronate, on repeat treatment (Journal of Bone Joint Surg. 2002; 84A; 1619-1623. Clin orthop 2004, 419:130-7).
Formulations available in the market such as those mentioned above and others not described here suffer from side effects such as:
There is a long standing need in the industry for preparations of sodium hyaluronate that reduce the problems of pain, swelling and inflammatory reactions with minimum side effects.
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SUMMARY OF INVENTION
The main object of the invention is to provide preparation of sodium hyaluronate polymer as injectable solution for treatment of osteoarthritis with reduced side effects.
Another object of the invention is to provide hydrolyzed sodium hyaluronate of tailored molecular weights from natural sodium hyaluronate.
It is yet another object of the invention is to provide sodium hyaluronate of specified visco-elastic properties.
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DETAILED DESCRIPTION OF INVENTIONS
In accordance with this invention the process of obtaining sodium hyaluronate of specified molecular weights comprises:
A. Extraction of bulk natural sodium hyaluronate from rooster combs.
B. Processing of the extracted bulk sodium hyaluronate to obtain HA tailored molecular
weights.
(A) Extraction of bulk sodium hyaluronate (HA) comprises steps of:
i. Collection and storage of rooster combs;
ii. Cleaning and treatment of tissue;
iii. Extraction of sodium hyaluronate from tissues of rooster combs;
iv. Removal of impurities;
v. Sterilization of sodium hyaluronate by membrane filtration;
vi. Isolation of sterilized natural sodium hyaluronate;
Rooster combs from slaughterhouse and poultries stored at 0° C to -5° C are transported to the facility and preserved at -70 . The rooster combs are cleaned followed by washing with alcohol and cutting into small pieces of HA is extracted with water for about 60 - 70 hours at 20° C to 30 C. The aqueous extract is treated with organic solvents 2 to 4 times at pH of 4 to 8; preferably with chloroform wherein the aqueous extract: solvent ratio at least 1:1. The aqueous sodium hyaluronate extract is sterile filtered. Sodium hyaluronate is precipitated preferably with alcohol or acetone using aqueous solution : solvent ratio of at least 1:2 v/v. The precipitate is further washed with solvent and dried. With a 1% solution of the HA in phosphate buffer saline (pH 7.0 - 7.6) has viscosity of more than 50,000 cPs and average molecular weight of 1.6 to 2 million Daltons.
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(B) Processing of the bulk sodium hyaluronate to obtain tailored molecular weights :
Bulk sodium hyaluronate produced in step (a) is dissolved in phosphate saline buffer and is treated with steam under preset conditions of temperature, pressure and time to obtain the hydrolyzed sodium hyaluronate polymer of tailored molecular weight between 0.6 - 1.45 million Dalton.
Bulk sodium hyaluronate (HA) so produced is dissolved in phosphate buffer saline by stirring at 2-15 °C at 7.0 - 7.6 pH. The solution is then subjected to steam hydrolysis at 100 to 130°C for 10 to 90 minutes at 0.3 to 1.8 atmospheres.
Example 1
Effect of duration of steam hydrolysis
A 1% solution of sodium hyaluronate of average molecular weight of about 1.6 to 2 million Dalton (prepared by the process described above) in phosphate buffer saline having pH of about 7.2 to 7.4 is exposed to steam under pressure to obtain partially de-polymerized material of molecular weight 0.8 to 1.3 million Dalton. The viscosity of a 1% aqueous solution is about 10,000 to 30,000 cPs. The results of steam hydrolysis at about 121°C at various times is depicted in Table 1. TABLE 1: Effect of duration of steam hydrolysis on molecular weight of HA.

Time (min) Expt. 1 Expt. 2 Expt. 3
Molecular weight in million Dalton
0 2.0 1.7 1.66
20 1.71 1.12 1.1
40 1.36 1.03 1.0
60 1.03 0.64 0.6
The molecular weight decreases as the time of hydrolysis increases.
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Example 2
Viscosity change of processed HA
The samples as prepared in Example 1, were collected after 20 minutes, 40 minutes, and 60 minutes of steam hydrolysis by precipitating the HA for the reaction mixture by alcohol or acetone (1 volume of solution to 2 volumes of alcohol or acetone). Vacuum drying produced dry powder of sodium hyaluronate.
The bulk sodium hyaluronate (without treatment) and the de-polymerized sodium hyaluronate contained about 10 to 20 % of moisture after drying to constant weight.
Viscosity of the materials as 1% (w/v) solution determined using a Brookfield Viscometer at 25°C at shear rate of 0.08 is presented in Table 2.
TABLE 2: Effect of steam hydrolysis with time on viscosity of sodium hyaluronate solutions.

Time (min) Expt.1 Expt.2 Expt.3
Viscosity of 1% solution in centipoise
0 (before treatment) 200000 100000 90000
20 55556 22222 22000
40 19444 12778 12222
60 9444 2222 1667
The duration of treatment had effect on the viscosity. The initial molecular weight of the HA also had considerable impact on the viscocity change with the duration of treatment.
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Example 3
Effect of initial concentration of bulk HA on product formation
In order to ascertain the effect of initial concentration of sodium hyaluronate on steam hydrolysis, 0.5 %, 1 % and 2 % solution of sodium hyaluronate in phosphate buffer were subjected to steam hydrolysis as described in example 1. The results are summarized in Table 3.
TABLE 3: The effect of initial concentration of sodium hyaluronate on steam hydrolysis Mol. Wt. = Molecular weight in million Dalton.
Vise. = Viscosity in centipoises (cPs) (measured with Brookfield Viscometer at 0.08 shear rate and at 25° C) NM = Not measurable

Minutes 0 (Before treatment) 20 40 60
% Solution Vise. Mol. Wt Vise. Mol. Wt Vise. Mol. Wt Vise. Mol. Wt
0.5 555.6 1.63 NM 1.35 NM 1.11 NM 0.9
1 100000 1.63 27778 1.3 13333 1.17 2778 0.8
2 556000 1.63 280000 1.45 102000 1.15 31667 0.91
It is observed from Table 3 that 1 % solution subjected to hydrolysis for 20 to 40 minutes
provided materials of the Viscosity between 10,000 to 30,000 cPs and average molecular weight between 0.8 to 1.3 million Dalton.
Example 4
Effect of time and temperature of steam hydrolysis on viscosity and molecular weight
Experiments of molecular size reduction of a 1 % solution of sodium hyaluronate were
conducted at 100° C, 110° C and 121° C for 20, 40 and 60 minutes. The results are presented in Table 4.
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TABLE 4: Effect of steam hydrolysis of a 1 % solution of sodium hyaluronate for different time periods.

1% Solution 121 UC 110 °C 100 UC
Vise. Mol. Wt Vise. Mol. Wt Vise. Mol. Wt
0 100000 1.63 100000 1.63 100000 1.63
20 27778 1.3 68333 1.57 77222 1.6
40 13333 1.17 66111 1.43 67222 1.47
60 2778 0.8 55556 1.39 57223 1.4
Mol. Wt. = Molecular weight in million Daltons.
Vise. = Viscosity in centipoises (measured with Brookfield Viscometer at 0.08 shear rate at 25° C)
Table 4 indicates that steam hydrolysis of 1 % solution of sodium hyaluronate in phosphate buffer produces the hydrolysed product of 5,000 to 30,000 cPs and average molecular weight of 0.6 million Dalton to 1.45 million Daltons, at 121° C between the period of 20 to 40 minutes.
Example 5
Product efficacy studies in osteoarthritis
The pre-filled syringes produced as 1 % solution of hydrolysed sodium hyaluronate in phosphate buffer saline having viscosity of 10,000 to 30,000 cPs and average molecular weight between 0.8 million Dalton to 1.3 million Dalton were injected in human subjects suffering from osteoarthritis, after fulfilling the statutory requirements. The Table 5 shows the effect of hydrolysed product on 10 osteoarthritis patients.
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Table 5: The therapeutic effect of hydrolyzed sodium hyaluronate solution

Patient Age Sex Knee injected OsteoarthritisCondition(Stage) Pain at rest and on movement at joint (Based on VAS )
Before therapy At the end of study
Patient 1 55 F Right III Severe pain No pain
Patient 2 53 F Left III Severe pain No pain
Patient 3 48 F Left III Severe pain No pain
Patient 4 33 F Left III Severe pain No pain
Patient 5 49 F Left III Severe pain No pain
Patient 6 39 F Right III Severe pain No pain
Patient 7 42 M Left III Severe pain No pain
Patient 8 48 M Left III Severe pain No pain
Patient 9 72 M Right III Severe pain No pain
Patient 10 48 F Left III Severe pain No pain
# VAS: Visual Analog Scale
It is observed from Table 5 that the formulation has good rejuvenating effects on all the recipients. Moreover, none of the patient had any inflammatory reaction.
From the above examples it was observed that 1% solution of sodium hyaluronate polymer of viscosity about 10,000 to 30,000 cPs and average molecular weight about 0.8 million Dalton to 1.3 million Daltons is best suited for the treatment osteoarthritis with minimized side effects.
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Use of solutions of HA of molecular weight between 0.8 to 1.3 million Dalton as 0.8 - 2.5 %
solution showed considerably lower local inflammatory reaction when used in human subjects in knee joints.
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