WO2016002983A1 - Pharmaceutical composition, for treating osteoarthritis, comprising cross-linked hyaluronic acid as active ingredient and osteoarthritis treatment method using same - Google Patents

Abstract

The present invention relates to a composition for inhibiting cartilage damage and/or for preventing and treating arthritis, the composition comprising cross-linked hyaluronic acid and, more specifically, to cross-linked hyaluronic acid which has excellent cartilage protective effect in an animal model of chronic osteoarthritis or an animal model of acute osteoarthritis and thereby can be utilized for inhibiting cartilage damage and/or preventing and treating arthritis.

Description

Pharmaceutical composition for treating osteoarthritis comprising cross-linked hyaluronan as an active ingredient and a method for treating osteoarthritis using the same

The present invention relates to a composition for inhibiting osteoarthritis comprising a crosslinked hyaluronic acid as an active ingredient and a method for treating osteoarthritis using the same.

Hyaluronic acid (hyaluronic acid) is a biosynthetic natural substance found in many skins such as animals. It is a component of the matrix in articular cartilage and is a type of mucopolysaccharide that is involved in making proteoglycans. It is linked to N- by glycosidic bonds. Glycoprotein complex of acetyl-D-glucosamine and D-glucuronic acid. Hyaluronic acid is a hydrophilic substance because it has a lot of hydroxyl groups (-OH), and plays a role of moisturizing in the skin of animals and the like.

Osteoarthritis is a degenerative disease caused by damage to knee cartilage and joint lubricating fluid. Osteoarthritis is osteoarthritis caused by degenerative changes in the cartilage and surrounding bone at the lubricated joint. Cartilage is a slippery tissue that covers the ends of bones in the joints. Healthy cartilage helps to absorb shocks caused by bone movement. In osteoarthritis, the upper layer of cartilage breaks down and wears, which allows bones under the cartilage to make direct contact. This direct contact results in pain, swelling, and loss of motion of the joints, which may lose their normal shape over time and lead to osteoproliferation. In addition, the debris of bone or cartilage can fall off and float in the joint space, resulting in greater pain and damage.

Currently, for the treatment of osteoarthritis, drug therapy, exercise therapy or surgery may be used. Among them, drugs such as acetaminophen, nonsteroidal anti-inflammatory drugs (NSAIDs), and corticosteroids (steroids) are used. However, most drugs are used to treat inflammation to relieve pain, and do not inhibit cartilage degeneration, but rather to accelerate cartilage damage or side effects such as cardiovascular, gastrointestinal tract, kidney and liver.

When hyaluronic acid is administered directly into the joint, it is possible to contain a large amount of water to achieve a large volume and to restore viscosity and elasticity to prevent further joint damage.

Balazs et al. Show that normal synovial fluid contains 1.5-4 mg of hyaluronic acid per ml and viscoelasticity exhibits properties of G '= 117 ± 13 Pa and G "= 45 ± 8 Pa at 2.5 Hz (Balazs, EA The physical properties of synovial fluid and the special role of hyaluronic acid.In disorders of the knee.63-75, 1974).

Synvisc has been developed and released under the concept of direct replenishment of hyaluronic acid with properties similar to those of normal joint synovial fluid, and is mixed with 20% cross-linked hyaluronic acid with a molecular weight of 6MDa and 80% uncrosslinked hyaluronic acid. It is known that the gel fulid has a viscoelasticity at 2.5 Hz and shows G '= 111 ± 13Pa and G ″ = 25 ± 2Pa, similar to normal joint synovial fluid and have good initial effect.

However, in the case of the hyaluronic acid composition having a physiological property similar to that of the joint synovial fluid, there is a problem in that the content and viscoelasticity of hyaluronic acid in the synovial fluid of osteoarthritis patients rapidly decreases with time.

One aspect provides a pharmaceutical composition for treating osteoarthritis comprising cross-linked hyaluronan as an active ingredient.

Another aspect provides a method for treating osteoarthritis of an individual comprising administering to the individual a therapeutically effective amount of hyaluronan.

As used herein, the term “therapeutically effective amount” means an amount sufficient to produce a therapeutic effect when administered to a subject in need thereof.

As used herein, the term “treating or treatment” means treating a disease or medical condition (eg, swelling and / or osteoproliferation of osteoarthritis) in an individual, such as a mammal (especially a human), which is Contains:

(a) inhibiting the disease or medical condition, ie, slowing or stopping the progression of the disease or medical condition in an individual; or

(b) alleviate a disease or medical condition in an individual.

The term “osteophytosis” refers to the formation of osteophytes, which are bony projections that form along joint margins. Osteoblasts can form due to an increase in the surface area of the damaged joint. Osteoprolifers generally limit joint movement and usually cause pain.

The term “subject” refers to a mammalian subject suffering from or likely to have osteoarthritis. The osteoarthritis may include osteoproliferation and / or edema. The mammal includes apes such as humans, pigs, cows, dogs, cats, horses, or sheep. The subject may be a mammal other than a human.

The term "about" may indicate varying by ± 10%, 5%, or 1%.

The term "hyaluronan" refers to hyaluronic acid, hyaluronate or "HA", or a pharmaceutically acceptable salt thereof, having the formula:

(1)

(One)

In Equation 1, n is the number of repeating units. Hyaluronic acid of all origins, including bacterial and algal origin, are useful. Useful hyaluronic acid is about 0.5MDa to about 6.0MDa, for example about 1.5MDa to about 6.0MDa, about 2.5MDa to about 6.0MDa, about 3.5MDa to about 6.0MDa, about 0.5MDa to about 5.0MDa, about 0.5 MDa to about 4.0MDa, or about 0.5MDa to about 3.0MDa.

The term “therapeutically acceptable salt” means salts that can be administered to an individual such as a mammal (eg, salts with acceptable mammalian safety for a given dosage regimen). The salts may be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids. Salts derived from such inorganic bases include ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganese, manganese, potassium, sodium, zinc and the like.

At least a portion of the hyaluronic acid referred to herein may be cross-linked. The term "cross-linked" refers to two or more polymer chains of hyaluronic acid covalently bonded through a cross-linking agent. Such crosslinking may be fractionated by intermolecular or intramolecular dehydration resulting in lactones, anhydrides, or esters in a single polymer or in two or more chains. Intramolecular crosslinking may also be included in the compositions mentioned herein. The term "cross-linked" also refers to hyaluronic acid covalently linked to a BDDE derivative.

The term "cross-linking agent" includes at least two reactive functional groups that create covalent bonds between two or more molecules. The crosslinking agent may be homobifunctional (ie have two identical reactive ends) or heterodifunctional (ie have two different reactive ends). As used herein, the crosslinking agent includes a functional group and a complementary functional group of hyaluronic acid to allow the crosslinking reaction to proceed. The crosslinking agent is butanediol diglycidyl ether (BDDE), divinyl sulfone (DVS), or 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), or May be a combination. In one embodiment, the crosslinker is BDDE.

The term "BDDE derivative" refers to the form of BDDE in which one or two epoxides of BDDE are reacted with hyaluronic acid. BDDE has the formula of formula (2).

(2)

(2)

One example of a BDDE derivative of hyaluronic acid is a derivative of formula 3.

(3)

(3)

As described above, the BDDE derivative of hyaluronic acid may be covalently bound to hyaluronic acid by reacting two epoxides at both ends of the BDDE. For example, some BDDE derivatives of hyaluronic acid are covalently bound (ie, crosslinked) at two ends between two separate hyaluronic acid polymers, while other BDDE derivatives are covalently linked at the end of a single hyaluronic acid polymer. It may be combined into. BDDE derivatives include BDDE derivatives covalently linked at one or both ends to hydroxyl groups from one or more additional BDDE derivatives which are covalently bound to hyaluronic acid per se.

Also included are BDDE derivatives covalently bound to hyaluronic acid at only one end. For example, one of the epoxide rings can be opened by covalent attachment to a single stretch of hyaluronic acid polymer while the other epoxide ring can be kept closed (ie, unreacted). ). Compositions comprising the crosslinked hyaluronic acid include those in which the concentration of unreacted epoxide is too low to affect the biocompatibility of the composition. In addition, one of the epoxide rings may be opened by covalent attachment to a single stretch of hyaluronic acid polymer while the other epoxide rings may include those opened by hydrolysis.

The term "buffer" refers to a solution for stabilizing pH, comprising a weak acid and its base or mixture of weak bases and their base acids. The buffer solution may include phosphate buffered saline (PBS), tris (hydroxymethyl) aminomethane (Tris), MOPS, and the like.

One aspect provides a pharmaceutical composition for treating osteoarthritis comprising cross-linked hyaluronan as an active ingredient.

The pharmaceutical composition may be for inhibiting cartilage damage. The pharmaceutical composition may also be for inhibiting one or more of osteophytosis, joint edema, and joint damage.

The osteoarthritis may include one or more symptoms of osteophytosis, joint edema, joint pain, tenderness, stiffness, locking and effusion.

In the pharmaceutical composition, the crosslinked hyaluronic acid has a molecular weight of about 0.5MDa to about 6.0MDa, for example, about 1.5MDa to about 6.0MDa, about 2.5MDa to about 6.0MDa, about 3.5MDa to about 6.0MDa , About 0.5MDa to about 5.0MDa, about 0.5MDa to about 4.0MDa, or about 0.5MDa to about 3.0MDa.

In the pharmaceutical composition, the weight content per volume of the final composition of the crosslinked hyaluronan is from about 5 mg / ml to about 40 mg / ml, for example from about 10 mg / ml to about 40 mg / ml, from about 20 mg / ml to about 40 mg / ml, about 5 mg / ml to about 30 mg / ml, about 5 mg / ml to about 20 mg / ml, or about 5 mg / ml to about 10 mg / ml. The hyaluronic acid may be to be distributed homogeneously in the composition.

The crosslinked hyaluronic acid may be a BDDE derivative of hyaluronic acid. The crosslinked hyaluronan is from about 0.1 mol% to about 50.0 mol% relative to the repeating disaccharide monomer, which is covalently bonded at two ends between two separate hyaluronic acid polymers, for example, About 1.0 mole% to about 50.0 mole%, about 5.0 mole% to about 50.0 mole%, about 10.0 mole% to about 50.0 mole%, about 20.0 mole% to about 50.0 mole%, about 30.0 mole% to about 50.0 mole%, About 40.0 mol% to about 50.0 mol%, about 5.0 mol% to about 30.0 mol%, about 5.0 mol% to about 25.0 mol%, about 5.0 mol% to about 20.0 mol%, about 15.0 mol% to about 25.0 mol%, Or from about 10.0 mol% to about 20.0 mol% BDDE.

The crosslinked hyaluronic acid can be prepared by incubating hyaluronic acid or a salt thereof in a basic solution, for example a basic aqueous solution, in the presence of a crosslinking agent. The hyaluronic acid or salts thereof may be hydrated before crosslinking, for example prior to addition to the aqueous solution. The hydration may be performed for 1 minute to 12 hours, for example, 1 to 60 minutes, 1 hour to 12 hours, 1 hour to 4 hours, 1 hour to 3 hours, or 1 hour to 2 hours.

The aqueous solution may contain about 5 mg / ml to about 40 mg / ml, for example, about 10 mg / ml to about 40 mg / ml, about 20 mg / ml to about 40 mg / ml, about 5 mg / ml to about 30 mg / ml, about 5 mg / ml to about 20 mg / ml, or about 5 mg / ml to about 10 mg / ml may be included in the final composition.

The aqueous solution is about 0.1 mol% to about 50.0 mol%, for example, about 1.0 mol% to about 50.0 mol%, about 5.0 mol% to about 50.0, based on the disaccharide monomer which is a repeating unit of hyaluronic acid. Mole%, about 10.0 mole% to about 50.0 mole%, about 20.0 mole% to about 50.0 mole%, about 30.0 mole% to about 50.0 mole%, about 40.0 mole% to about 50.0 mole%, about 5.0 mole% to about 30.0 Mole%, about 5.0 mole% to about 25.0 mole%, about 5.0 mole% to about 20.0 mole%, about 15.0 mole% to about 25.0 mole%, or about 10.0 mole% to about 20.0 mole% of a crosslinking agent, for example, Can be crosslinked with BDDE.

The existing units of hyaluronic acid disaccharide consist of D-glucuronic acid and N-acetylglucosamine, and the cross-link rate (%) is the ratio of total hyaluronic acid disaccharide units. The amount of covalently bound crosslinking agent per disaccharide unit, ie the degree of crosslinking, can be ascertained by known methods. For example, ion exchange chromatography (ion exchange) is a method in which reversible ion exchange between a stationary phase and a mobile phase is carried out using ion exchange chromatography to separate and analyze sample ions using affinity differences to the stationary phase. chromatography) or NMR . The formula of crosslinking degree is as follows.

Cross-linke rate (%) = ∑ (peak area x No. of HA dimer x cross-linked HA dimer ratio) x 100 / ∑ (peak area x No. of HA dimer units)

The crosslinked hyaluronic acid can be formed when hyaluronic acid or a salt thereof is contacted with a crosslinking agent. As used herein, the crosslinking agent includes a functional group and a complementary functional group of hyaluronic acid to allow the crosslinking reaction to proceed. The crosslinking agent is butanediol diglycidyl ether (BDDE), divinyl sulfone (DVS), or 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), or May be a combination. In one embodiment, the crosslinker is BDDE. The crosslinked hyaluronan may be a BDDE derivative.

The pharmaceutical composition may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. The carrier may comprise, for example, water or a buffer. The buffer may be such that the pH of the solution hardly changes with the addition of the components of the composition. The pharmaceutical composition may be an aqueous buffered composition. The pH of the aqueous buffered composition can be in a physiological pH range, for example about 7. The pH can be adjusted by adding an appropriate amount of a suitable base such as Na ₂ CO ₃ or NaOH. In one embodiment the aqueous buffered composition comprises phosphate buffered saline (PBS). In another embodiment the aqueous buffered composition comprises Tris (hydroxymethyl) aminomethane (Tris) having the formula (HOCH ₂ ) ₃ CNH ₂ . In some embodiments additional solutes, such as sodium chloride, calcium chloride, and / or potassium chloride, are added to adjust osmolarity and ion concentration.

The composition of the present invention can be formulated in the form of a liquid or sterile injectable solution. In formulating the composition, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants that are commonly used may be used.

Preferred dosages of the compositions of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route and duration of administration, and may be appropriately selected by those skilled in the art.

Administration of the composition may be administered once a day, may be divided several times. Administration of the composition may be carried out by identifying the site of injury through magnetic resonance imaging (MRI) or computed tomography (CT).

When the cross-linked hyaluronic acid is measured at 2.5 Hz, the storage (elastic) modulus G 'value may be 250 to 600. The G 'may be measured by a known method, for example, a frequency sweep test method according to a frequency change. Viscoelasticity measurement according to the frequency change measures the viscoelasticity of hyaluronic acid during shear deformation and changes the frequency from 0.1 to 1Hz to measure the viscoelasticity according to the shear rate change and measures the viscoelasticity and corresponds to the load on the joint The result value is 2.5 Hz.

In the present specification, the storage (elastic) modulus (G ') and the loss (viscosity) modulus (G ") can be recovered by each material as the rate of deformation (test frequency) changes. Elastic response) or flowable (viscosity). Both modulus is a linear function of frequency. It has been demonstrated that they can be sensitive probes of the structure of polymer solutions or gels. G 'and G " Both increase with increasing frequency, but one increases faster than the other. At the G '= G' point, this frequency is called the cross-over frequency (fc). % Elasticity refers to the hardness of the gel and is the degree of external force that can lead to the deformation of the gel. Therefore, high% elasticity means that the force to be deformed to the gel should act largely, indicating that the gel has a high cohesive force.

The pharmaceutical composition may be for administration into a joint of a patient. The joint may be a knee, hip, shoulder, spine, neck, finger or toe joint. Such "intra-articular" administration includes "intra-articular" administration. In addition, the pharmaceutical composition may be used for the purpose of administration into the joint of the patient. The pharmaceutical composition may be used for topical administration to a joint injury site of a patient. For example, the pharmaceutical composition may be in the form or use for administration to the joint of the individual.

In one embodiment of the present invention to investigate the effect of the cross-linked hyaluronic acid-containing composition on osteoarthritis including cartilage damage and osteoproliferative rats known to cause lesions similar to degenerative joint disease (degenerative joint disease) in humans Animal models of (Sprague-Dawley rat and Wistar rat) were used to induce chronic osteoarthritis through anterior cruciate ligament incision in the right hind knee and then administered hyaluronic acid. As a result, it was observed that the test group administered with hyaluronic acid had less cartilage damage and osteoblasts than the negative control group and the positive control group administered with phosphate buffered saline.

In addition, in one embodiment of the present invention in order to investigate the effect of the cross-linked hyaluronic acid-containing composition on osteoarthritis including cartilage damage and osteoproliferative diagnosis (osteoprogenitor formation, proteoglycan loss, etc.) and Hyaluronic acid was administered after acute induction of osteoarthritis using a MIA (Monosodium iodoacetate) chemical model (using Wistar species rats), which is known to show histopathological findings. As a result, it was observed that the test group administered with hyaluronic acid had less cartilage damage and osteoblasts than the negative control group and the positive control group administered with phosphate buffered saline. Therefore, it can be seen that the composition of the present invention has an inhibitory effect on osteoarthritis.

The compositions of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and / or biological response modifiers to inhibit osteoarthritis.

Another aspect provides a method for treating osteoarthritis of an individual comprising administering to the individual a therapeutically effective amount of hyaluronan.

The method may be for suppressing cartilage damage. In addition, the method may be to inhibit one or more of osteophytosis, joint edema, and joint damage.

The administration may be locally administered to the joint site with osteoarthritis. The administration may be in intra-articular, for example, intra-articular cavity. The joint may be a knee, finger, toe, hip, spine, neck, or shoulder joint. The administration can be to the subject's joint, eg, the medial, lateral or both sites of the knee. The administration may be by injection into the joint cavity.

In the above administration, the dosage depends on the condition and weight of the patient, the severity of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art.

By "therapeutically effective amount" is meant an amount sufficient to produce a therapeutic effect when administered to a subject in need thereof. The therapeutically effective amount is from 0.0001 mg to 1500 mg, 0.001 mg to 1500 mg, 0.01 mg to 1500 mg, 0.1 mg to 1500 mg, 1 mg to 1500 mg, 5 mg to 1500 mg, 10 mg to 1500 mg, 50 mg to 1500 mg, 100 mg to 1500 mg, 300 mg to 70 kg body weight. 1500 mg, 500 mg to 1500 mg, 800 mg to 1500 mg, 1000 mg to 1500 mg, 1200 mg to 1500 mg, 0.0001 mg to 1200 mg, 0.001 mg to 1200 mg, 0.01 mg to 1200 mg, 0.1 mg to 1200 mg, 1 mg to 1000 mg, 5 mg to 800 mg, 10 mg to 700 mg, 50 mg to 600 mg, 100 mg to 500 mg, or 10 mg to 150 mg.

The administration may be in the form of the pharmaceutical composition described above.

The subject represents a mammalian subject suffering from, or highly likely to have osteoarthritis. The osteoarthritis may include osteoproliferation and / or edema. The mammal includes apes such as humans, pigs, cows, dogs, cats, horses, or sheep. The subject may be a mammal other than a human.

According to the pharmaceutical composition for treating osteoarthritis according to one aspect, it can be used to effectively treat osteoarthritis.

According to a method for treating osteoarthritis of an individual according to another aspect, the osteoarthritis of the individual can be effectively treated.

Figure 1 shows the safranin-O tissue staining results of the untreated group, negative control group, positive control group and test group according to Example 1 (OARSI, 13 weeks after surgery, and 40 times magnification).

Figure 2 shows the results of visual observation of the degree of cartilage damage in the untreated group, negative control group, positive control group and test group according to Example 2 (6 weeks after MIA administration).

Figure 3 shows the results of visual observation of cartilage damage of the untreated group, negative control group and test group according to Example 3 (9 weeks after MIA administration).

Hereinafter, the present invention will be described in more detail through examples according to the present invention, but the scope of the present invention is not limited to the examples given below.

Materials and methods

Unless stated otherwise, the following materials and methods were used in the following examples.

1. Crosslinking: Preparation of Crosslinked Hyaluronic Acid

Butanediol diglycidyl ether (BDDE) to completely dissolve the hyaluronic acid with a molecular weight of about 0.5 to 6 MDa in a NaOH solution at a concentration of 14 (w / w)% and then crosslink by reaction with a hydroxyl group Was added. BDDE was added at 5-30% by weight of repeat units of HA. The mixed solution was reacted at 35 ° C. for 20 hours for complete crosslinking of the HA crosslinked product. Thereafter, the prepared HA hydrogel was sealed with a pre-washed dialysis membrane, and dialyzed in 0.01 M PBS (phosphate buffered saline, pH 7.4) to remove residual BDDE.

2. washing

The cross-linked hyaluronic acid gel was dialyzed to remove any unreacted BDDE with sodium hydroxide. LC analysis showed that the detection limit was controlled below 2 ppm.

3. Formulating

The completely dried crosslinked hyaluronic acid was formulated as a final composition at a final concentration of 10 mg / ml to 50 mg / ml HA. The formulation was formulated in 10 mM PBS at pH 7.4. As a control, an uncrosslinked natural hyaluronic acid solution and SYNVISC-ONE ^™ as a positive control were prepared or purchased.

The prepared crosslinked hyaluronic acid-containing aqueous composition is a pharmaceutical composition obtained by crosslinking as described above using 0.5 to 6MDa natural hyaluronic acid and BDDE in an amount of 5 to 30 mol% based on the disaccharide of the hyaluronic acid.

Example 1 Confirmation of Osteoarthritis Inhibitory Effect of Hyaluronic Acid in Surgical Rat Osteoarthritis Model (chronic Arthritis Animal Model)

Anterior cruciate ligament dissection creates instability similar to osteoarthritis naturally occurring in humans due to trauma and is suitable for the slow progression of osteoarthritis from a etiological point of view. Lesions occur on the medial tibial plateau and medial femoral condyle, where the weight-bearing effects are the major factors of joint movement, and the meniscus degeneration of the meniscus between the two bone surfaces. The main diagnostic findings are histological degeneration of articular cartilage and subchondral plate, large amounts of osteophyte, and loss of proteoglycan.

The animal model of this experiment was male SD (Sprague-Dawley) rats. The group consisted of untreated group (Sham, arthroplasty, no substance administration), negative control (50uL phosphate buffered saline solution, PBS), Positive control group (SYNVISC-ONE ^TM 100uL, administered three times at weekly intervals), test group (administration of 100 μL of hyaluronic acid example after surgery), four groups in total, and administered in the knee joint cavity of 5-7 animals per group. . SYNVISC-ONE ^TM (hylan GF 20) (Genzyme Biosurgery, USA) is a viscoelastic high molecular weight fluid (elasoviscous high molecular weight) comprising hylan A and hylan B (average molecular weight 6 MDa) polymers produced from chicken combs. fluid). hylan is a derivative of hyaluronan (sodium hyaluronate). hylan GF 20 is unique in that it is chemically crosslinked. Hyaluronic acid is a long chain polymer containing repeating disaccharide units of Na-glucuronate-N-acetylglucosamine. SYNVISC-ONE ^™ has elasticity (111 ± 13 Pa and viscosity (loss modulus G ”) 25 ± 2 Pa at 2.5 Hz storage module G '. The elasticity and viscosity of knee synovial fluid from 18 to 27 years old are comparable. method), G '= 117 ± 13 Pa, G ″ = 45 ± 8 Pa at 2.5 Hz.

Rheological properties of the positive control composition and the test group composition (2M2020) were measured by viscoelasticity measurement according to frequency change. Viscoelasticity measurement according to the frequency change measures the viscoelasticity of hyaluronic acid during shear deformation and changes the frequency from 0.1 to 1Hz to measure the viscoelasticity according to the shear rate change and measures the viscoelasticity and corresponds to the load on the joint Take a result of 2.5 Hz. The measurement temperature was 25 ° C., the measurement geometry was 40 mm, and the gap was 1000 mm.

Table 1 shows the measured rheological properties. In Table 1,% elasticity represents G'x100 / (G '+ G ").

Table 1 Composition Rheological properties G '(Pa) G "(Pa) % Elasticity SYNVISC-ONE ^TM 100 18 84.7 Test group 1 332 53 86

Prior to intraarticular administration of the test substance, the anterior cruciate ligament, which plays the largest role in joint stability, is incision alone (right Fuji knee), followed by treadmill 20 times at a rate of 20 m / min 3 times per week. Exercise for 11 minutes and under these conditions Chronic osteoarthritis was induced by the forced exercise of the liver to increase joint wear caused by physical joint instability.

The effectiveness of hyaluronic acid arthritis treatment using surgical model was measured by the vernier calipers measuring the width of the knee joint at the surgical site (test site) and non-surgical site (non-administration site) in the same individual. The degree of joint swelling was evaluated, and tissues were extracted and stained (Safranin-O / Fast green) at about 13 and 20 weeks after surgery to determine the condition of abrasion and osteoarthritis of osteoarthritis Research Society International (OARSI). It was evaluated by histopathological evaluation as presented in.

The OARSI evaluation method gives a score of 0 to 6.5 points according to the degree of cartilage damage, a score of 0 to 4 points according to the area occupied by the lesion, and the maximum number of points is 26 by multiplying them. Can be given. Table 2 shows OA Cartilage Histopathology Grading—Advanced Grading Criteria.

TABLE 2 Grade (Key Features) Secondary grade (optional) Related Criteria (Tissue Response) Grade 0: intact surface and cartilage (no damage) No secondary rating Intact and intact cartilage Class 1: Intact Surface 1.0 Cell Integrity 1.5 Cell Death Substrate: Intact surface layer, edema and / or fibrosis: Proliferation (cluster), hypertrophy Class 2: Discontinuous Surface (Rough) 2.0 Fibrosis of the Surface Layer 2.5 Surface Abrasion with Substrate Reduction of the Surface Layer Including the above, + discontinuous surface layer ± reduction of the staining of the upper 1/3 of the cartilage (middle layer) (safranin O or toluidine blue) ± irregularity of the cartilage column Grade 3: vertical cracks / gaps 3.0 Simple cracks / gaps 3.5 Cracks / compound gaps Including the above, ± 2/3 lower (deep) staining of cartilage (safranin O or toluidine blue) Grade 4: Miran 4.0 Surface layer separation 4.5 Interlayer depression Loss of cartilage stroma, cyst formation in cartilage stroma Grade 5: Peel 5.0 Intact bone surface 5.5 Restoration or repair tissue Repaired tissue with articular surface including hardened bone or fibrocartilage Grade 6: variant 6.0 Peripheral Osteoarthritis Appearance 6.5 Peripheral and Central Osteoarthritis Appearance Deformation of the joint profile, including bone remodeling, microcracks and repair

Grade: cartilage damage progress

Table 3 shows OA cartilage histopathology-stage criteria.

TABLE 3 *step % Lesion (surface, area, volume) Step 0 No osteoarthritis lesion Step 1 <10% Step 2 10-25% Step 3 25-50% Step 4 > 50%

* Step: lesion size

Table 4 shows the results of joint edema measurement (6 weeks, 13 weeks, and 20 weeks after surgery).

Table 4 Classification (mean ± standard error) Knee Joint Width Difference (Right-Left, mm) 6 Weeks 13 Weeks 20 Weeks Sham (n = 7) 0.15 ± 0.13 0.09 ± 0.10 - Negative Control (PBS) (n = 7) 1.37 ± 0.25 2.31 ± 0.37 1.78 ± 0.19 Positive control group (Synvisc-one ^TM ) (n = 7) 0.94 ± 0.21 1.46 ± 0.27 0.88 ± 0.12 ^* Test group 1 (n = 7) 1.21 ± 0.18 1.39 ± 0.22 ^a 1.20 ± 0.11 ^*

^* P <0.05 (student's t-test, compared with PBS group)

^a P = 0.052 (student's t-test, compared with PBS group)

Table 5 shows histopathological evaluation results (OARSI, 13 weeks after surgery).

Table 5 Classification (mean ± standard error) MFC MTP 13 Weeks 13 Weeks Sham (n = 7) 1.16 ± 0.19 3.48 ± 0.24 Negative Control (PBS) (n = 7) 20.83 ± 0.97 15.57 ± 1.07 Positive control group (Synvisc-one ^TM ) (n = 7) 14.68 ± 1.32 ^** 12.31 ± 0.97 ^* Test group 1 (n = 7) 13.59 ± 1.83 ^** 9.98 ± 0.13 ^**

^* P <0.05 (student's t-test, compared with PBS group)

^** P <0.01 (student's t-test, compared with PBS group)

MFC: medial femoral condyle

MTP: medial tibial plateau

Table 6 shows the histopathological evaluation results (OARSI, 20 weeks after surgery).

Table 6 Classification (mean ± standard error) MFC MTP 20 Weeks 20 Weeks Negative Control (PBS) (n = 5) 19.91 ± 1.62 18.10 ± 1.52 Positive control group (Synvisc-one ^TM ) (n = 6) 17.70 ± 1.46 14.34 ± 1.30 Test group 1 (n = 6) 18.16 ± 2.81 12.69 ± 1.33 ^*

^* P <0.05 (student's t-test, compared with PBS group)

^** P <0.01 (student's t-test, compared with PBS group)

MFC: medial femoral condyle

MTP: medial tibial plateau

As shown in Table 4, Table 5, Table 6 and Figure 1, hyaluronic acid was found to inhibit osteoarthritis including joint edema and inhibit osteoarthritis of chronically induced rats.

1 shows safranin-O tissue staining results of the untreated group, the negative control group, the positive control group, and the test group according to Example 1 (OARSI, 13 weeks after surgery, and 40 magnifications). 1 shows histopathological lesions of the knee joint surface at post-13 parking. That is, the untreated group, the negative control group, the test group, and the positive control group show the cut surface of the knee joint. Two staining photographs are included for each group. In Fig. 1, the arrow head represents the joint surface discontinuity, del represents the surface layer separation of the joint surface, fc represents the fibrous joint formation, and of represents the osteoplasia.

As shown in FIG. 1, in the tissue photograph, the red stained portion is a normal cartilage, based on the untreated group. When comparing the test group and the negative control group, the cartilage surface damage (grade 5 ~ 5.5) of the medial femoral joint (MFC) of the negative control group was prominent, the repair tissue (fibrocartilage) was confirmed, and the staining was also light. . The medial tibial surface (MTP) is observed to have a detachment of the surface layer (grade 4) and osteoblasts (grade 6). In contrast, MFC and MTP in the test group show discontinuous joints (grades 2 to 2.5) as major lesions. The area of lesion showed 4 levels of MFC and MTP of negative control group, compared with 3 levels of MFC of test group and 4 levels of MTP.

Example 2: Confirmation of Osteoarthritis Inhibitory Effect According to Molecular Weight of Hyaluronic Acid in Chemical Rat Osteoarthritis Model (Acute Arthritis Animal Model)

Monosodium iodoacetate (MIA) results in inhibition of the action of glyceraldehyde-3-phosphate (G3P) in chondrocytes, activation of degenerate marker matrix metalloproteinase (MMP), and inhibition of proteoglycan synthesis. It is an experimental model that causes arthritis similar to degenerative arthritis by causing necrosis of chondrocytes.

Arthritis using MIA is also known to show similar diagnostic (osteoprogenitor formation, proteoglycan loss) and histopathological findings similar to human arthritis.

In this experimental animal model, male Wistar rats were used in the control group (Control), negative control group (MIA administration, 50uL phosphate buffered saline, PBS), positive control group (Synvisc-one ^TM 100uL, at 1 week intervals). Total 3 administrations), and cross-linked test groups 2 to 3 (1 administration of 100 uL of HA after surgery), 6 groups in total, and 6 mice per group were administered intraarticularly.

Table 7 shows the rheological properties measured for the positive control or test group compositions.

TABLE 7 Composition Rheological properties G '(Pa) G "(Pa) % Elasticity Test group 2 379 124 75.4 Test group 3 153 35 81.5

0.5 mg / 50 uL MIA was administered in the right posterior knee joint cavity of anesthetized rats to induce acute osteoarthritis. After administration, test substance or phosphate buffered saline was administered to the same site and incapacitance test was performed to evaluate pain relief and joint mobility improvement. Table 8 shows the incapacitance test.

Table 8 Day 0 Day 7 Day 14 Day 21 Control 50 49.70 ± 0.18 50.13 ± 0.20 50.18 ± 0.20 PBS 10.31 14.31 ± 2.03 35.29 ± 1.29 36.62 ± 4.63 SYNVIS-Con e 13.085 13.08 ± 1.68 32.51 ± 5.81 35.91 ± 0.47 Test group 2 11.346 28.61 ± 4.45 43.37 ± 1.42 41.26 ± 3.12 Test group 3 14.977 31.31 ± 1.78 39.73 ± 2.51 38.28 ± 3.32

Figure 2 shows the results of visual observation of the degree of cartilage damage in the untreated group, negative control group, positive control group and test group according to Example 2 (6 weeks after MIA administration).

As shown in Table 8 and Figure 2, it was confirmed that improving the viscoelasticity of HA through crosslinking effectively inhibits osteoarthritis as compared with the positive control (synvisc one) similar to the viscoelasticity of the joint synovial fluid.

Example 3 Confirmation of Osteoarthritis Inhibitory Effect According to Hyaluronic Acid Content and Crosslinking Degree in Chemical Rat Osteoarthritis Model (Acute Arthritis Animal Model)

In this experimental animal model, male Wistar rats were used in the control group (Control), negative control group (50uL phosphate buffered saline after PIA), test groups 4 and 5 (uncrosslinked hyaluronic acid) Content of 10 to 50 mg / ml), and test groups 6 to 9 having improved viscoelasticity through cross-linking were prepared, totaling 8 groups, and 6 mice per group were administered intraarticularly.

Table 9 shows the rheological properties measured for the test group compositions.

Table 9 Composition Rheological properties G '(Pa) G "(Pa) % Elasticity Test group 4 31 25 55.1 Test group 5 212 114 65.1 Test group 6 18 12 60.9 Test group 7 90 45 66.6 Test group 8 102 27 79.0 Test group 9 347 54 86.5

0.5 mg / 50 uL MIA was administered in the right posterior knee joint cavity of anesthetized rats to induce acute osteoarthritis. After administration, test substance or phosphate buffered saline was administered to the same site and incapacitance test was performed to evaluate pain relief and joint mobility improvement. Table 10 shows the incapacitance test.

Table 10 control PBS Test group 4 Test group 5 Test group 6 Test group 7 Test group 8 Test group 9 Day 0 50.37 14.97 14.35 14.25 13.07 13.1 12.8 13.08 Day 7 50.27 ± 0.24 23.53 ± 5.44 25.62 ± 1.05 39.34 ± 4.23 42.86 ± 5.20 35.01 ± 7.22 28.81 ± 8.19 34.03 ± 6.29 Day 14 49.94 ± 0.30 23.83 ± 2.62 36.75 ± 3.59 32.22 ± 5.61 32.21 ± 5.69 29.93 ± 2.40 31.09 ± 2.64 42.58 ± 2.26 Day 21 50.51 ± 0.42 32.40 ± 1.15 39.96 ± 3.01 43.06 ± 2.34 43.78 ± 4.38 43.72 ± 3.33 37.18 ± 4.25 47.19 ± 1.63 Day 28 49.98 ± 0.21 28.97 ± 1.62 36.96 ± 1.47 35.92 ± 2.80 39.38 ± 1.81 41.00 ± 2.82 30.39 ± 2.11 43.70 ± 1.38 Day 35 50.22 ± 0.11 32.11 ± 2.84 39.85 ± 1.87 33.79 ± 2.37 41.34 ± 3.13 42.91 ± 1.88 35.07 ± 3.07 43.08 ± 1.80 Day 38 50.49 ± 0.22 30.28 ± 1.18 37.20 ± 1.43 37.70 ± 1.51 40.99 ± 0.69 43.34 ± 1.71 40.28 ± 0.84 42.84 ± 1.59

Figure 3 shows the results of visual observation of cartilage damage of the untreated group, negative control group and test group according to Example 3 (9 weeks after MIA administration).

As shown in Table 10 and Figure 3, when the hyaluronic acid is not cross-linked it was confirmed that the initial effect is faster than the other group, but in the long run it was confirmed that the cross-linked group is more excellent in pain relief effect. In addition, the crosslinking test group with improved viscoelasticity was found to be more effective in cartilage protection, pain relief and motility than the test group similar to the viscoelasticity of joint synovial fluid.

Example 4: Confirmation of effect according to molecular weight and degree of crosslinking of hyaluronic acid according to visual observation

Visual observation of cartilage damage is generally assessed by the femoral condyle effect (FCE) method, which is graded according to the following three criteria and is determined within the range of 0 to 18, the sum of the values according to each evaluation. High scores show negative patterns and severe tissue damage, while low scores show positive patterns and tissue damages near normal.

In this experimental animal model, male Wistar rats were used as a control group; These improved test groups 10-11 were prepared, totaling 5 groups and administered 6 intra-articularly in each group.

Table 11 shows the rheological properties measured for the test group compositions.

Table 11 Composition Rheological properties G '(Pa) G "(Pa) % Elasticity Test group 10 162 43 79.0 Test group 11 359 58 86.1

Table 12 shows the criteria for measuring the depth of cartilage erosion on medial femoral condyle and lateral femoral condyle with a score of 0-3.

Table 12 Surface area erosion Surface erosion rate 0 No erosion One 10% erosion 2 11-25% erosion 3 26-50% erosion 4 51-75% erosion 5 76-100% erosion

Table 13 shows the criteria for measuring the depth of cartilage erosion on medial femoral condyle and lateral femoral condyle with a score of 0-3.

Table 13 Grade of erosion Erosion depth 0 none 0.5 Almost unrecognizable One Slightly 2 Noticeably 3 Quite a lot

Table 14 shows the criteria for measuring the degree of cracking of the cartilage surface of the medial femoral condyle and the lateral femoral condyle with a score of 0-2.

Table 14 ranking Presence of cracks 0 none One 1 crack is present 2 There are two cracks

Table 15 shows visual observation results measured according to FCE.

Table 15 Total score Medial Outside corrosion(%) Erosion depth Cracked area No treatment 0 0 0 0 0 0 PBS treatment group 11.3 5.6 5.4 7.1 3.9 0.3 Synvisc-one 11.2 5.5 5.4 7.2 3.7 0.4 Test group 1 13.5 6.2 6.4 8.1 4.5 1.0 Test group 11 10.4 5.1 4.9 6.5 3.4 0.4

As shown in Table 15, compared with the positive control group, test group 11 administered with cross-linked hyaluronic acid showed less cartilage damage according to FCE in the MIA animal model.

Claims (15)

A pharmaceutical composition for treating osteoarthritis, comprising a crosslinked hyaluronan. The pharmaceutical composition of claim 1, for inhibiting cartilage damage. The pharmaceutical composition of claim 1, for inhibiting one or more of osteophytosis, joint edema, and joint injury. The pharmaceutical composition of claim 1, wherein the G 'value is 250 to 600 when the pharmaceutical composition is measured at 2.5 Hz. The pharmaceutical composition of claim 1, wherein the hyaluronan has a molecular weight of 0.5MDa to 6MDa. The pharmaceutical composition of claim 1, wherein the weight content per volume of the hyaluronan composition is 10 mg / ml to 50 mg / ml. The pharmaceutical composition of claim 1, for administration into a joint of an individual. The pharmaceutical composition of claim 8, wherein the joint is a knee, finger, toe, hip, spine, neck, or shoulder. A method for treating osteoarthritis in an individual, comprising administering to the individual a therapeutically effective amount of hyaluronan. The method of claim 9, wherein said administering is in an articular. The method of claim 9, for inhibiting cartilage damage. The method of claim 9, which inhibits one or more of osteophytosis, joint edema, and joint injury. The method of claim 9, wherein the administration is administration of a pharmaceutical composition having a G ′ value of 250 to 600 when measured at 2.5 Hz. The method of claim 9, wherein the hyaluronan has a molecular weight of 0.5MDa to 6MDa. The method of claim 10, wherein the administering is administering a pharmaceutical composition having a weight content of 10 mg / ml to 50 mg / ml by volume of the composition of hyaluronan.

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