KR20160002256A - Pharmaceutical composition for treating osteoarthritis comprising cross-linked hyaluronan as an active ingredient and method for treating osteoarthritis in a patient - Google Patents

Abstract

The present invention relates to a composition for preventing and treating cartilage damage comprising crosslinked hyaluronic acid and / or a composition for preventing and treating arthritis. More particularly, the present invention relates to a composition for preventing and treating arthritis, which comprises crosslinked hyaluronic acid in an animal model of chronic osteoarthritis, And thus can be effectively used for prevention of cartilage damage and / or prevention and treatment of arthritis.

Description

[0001] The present invention relates to a pharmaceutical composition for treating osteoarthritis comprising a crosslinked 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 crosslinked hyaluronic acid as an active ingredient, and a method for treating osteoarthritis using the same.

Hyaluronic acid is a biosynthetic natural substance that exists in many skin areas such as animals. It is a constituent of matrix in articular cartilage. It is a kind of mucopolysaccharide that is involved in making proteoglycan. It is composed of N-linked glycosidic bonds acetyl-D-glucosamine and D-glucuronic acid. Hyaluronic acid is a hydrophilic substance because it has many hydroxyl groups (-OH), and acts as a moisturizing effect in the skin of animals and the like.

Osteoarthritis is a degenerative disease caused by abnormality of knee cartilage damage and joint lubrication, arthritis arising from degenerative changes in cartilage and surrounding bone in the lubricated joint. The cartilage is a slippery tissue that covers the tip of the bone in the joints. Healthy cartilage helps to absorb the impact of bone movement. In osteoarthritis, the upper layer of the cartilage is degraded and worn, allowing the bones below the cartilage to come into 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 osteoporosis. In addition, bone and cartilage debris can fall and float within the joint space, leading to greater pain and damage.

Currently, drug therapy, exercise therapy, or surgical therapy may be used to treat osteoarthritis. Drugs such as acetaminophen, nonsteroidal anti-inflammatory drugs (NSAIDs), and corticosteroids have been used as dual drug therapies. However, most drugs treat inflammation to alleviate pain, which does not inhibit the underlying cartilage degeneration, but rather accelerates cartilage damage, or it is pointed out that side effects such as cardiovascular, gastrointestinal, kidney, and liver problems.

It is known that hyaluronic acid is administered directly into the joints, which enables it to contain a large amount of water, thereby achieving a large volume, restoring viscosity and elasticity and preventing further damage to the joints.

Balazs et al. Show that synovial fluid contains 1.5-4 mg hyaluronic acid per ml and viscoelasticity exhibits 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.

Synvisc has been developed and marketed as a therapeutic concept that directly replenishes hyaluronic acid similar in nature to normal joint synovial fluid. It is mixed with 20% of cross-linked hyaluronic acid having a molecular weight of 6 MDa and 80% of uncrosslinked hyaluronic acid It is known that the initial effect is similar to that of normal joint synovial fluid, with viscoelasticity at 2.5 Hz, G '= 111 ± 13 Pa, G "= 25 ± 2 Pa as a gel fluid.

However, in the case of a hyaluronic acid composition having physiological properties similar to those of the joint synovial fluid, there is a problem that the content of hyaluronic acid and the viscoelasticity of the synovial fluid of the osteoarthritis rapidly decrease over time.

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

Another aspect provides a method for treating osteoarthritis in a subject, comprising administering to the subject 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 of such treatment.

As used herein, the term "treating or treating " means treating a disease or medical condition (e.g., edema of osteoarthritis and / or osteoporosis) in an individual such as a mammal (particularly a human) Includes:

(a) inhibiting the disease or medical condition, i.e. slowing or stopping the progression of the disease or medical condition in the individual; or

(b) alleviating the disease or medical condition in the individual.

The term "osteophytosis" refers to the formation of osteophytes, bony projections formed along joint margins. The osteophyte can be formed due to an increase in the surface area of the injured joint. Osteoporosis generally limits joint motion and usually causes pain.

The term "subject" refers to a mammalian subject with or likely to be suffering from osteoarthritis. The osteoarthritis may include osteoporosis and / or edema. The mammal includes an ape, such as a human, a pig, a cow, a dog, a cat, a horse, or a sheep. The subject may be a mammal other than a human.

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

The term "hyaluronan" refers to hyaluronic acid, hyaluronate or "HA ", or a pharmaceutically acceptable salt thereof, having the formula of formula 1.

(One)

In formula (1), n is the number of repeating units. Hyaluronic acids of all origins are useful, including bacterial and avian origin. Useful hyaluronic acid may be present in an amount of from about 0.5 MDa to about 6.0 MDa, such as from about 1.5 MDa to about 6.0 MDa, from about 2.5 MDa to about 6.0 MDa, from about 3.5 MDa to about 6.0 MDa, from about 0.5 MDa to about 5.0 MDa, MDa to about 4.0 MDa, or from about 0.5 MDa to about 3.0 MDa.

The term " therapeutically acceptable salt "means a salt that is acceptable for administration to an individual, such as a mammal (e. G., A salt having acceptable mammalian safety for a given dosing schedule). 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, second manganese, first manganese, potassium, sodium and zinc.

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 bridging can be distinguished by intermolecular or intramolecular dehydration which results in a single polymer or a lactone, anhydride, or ester within two or more chains. Intramolecular crosslinking may also be included in the compositions referred to 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 form a covalent bond between two or more molecules. The cross-linking agent may be homobifunctional (i. E. Having two identical reactive ends) or heterobifunctional (i. E. Having two different reactive ends). As used herein, the crosslinking agent includes a functional group of hyaluronic acid and a complementary functional group so that the crosslinking reaction proceeds. The crosslinking agent may be selected from butanediol diglycidyl ether (BDDE), divinylsulfone (DVS), or 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) Lt; / RTI > In one embodiment, the crosslinking agent 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 chemical formula of Formula 2.

(2)

An example of a BDDE derivative of hyaluronic acid is a derivative of the formula 3.

(3)

As described above, BDDE derivatives of hyaluronic acid may be covalently bound to hyaluronic acid by reacting two epoxides at both ends of BDDE. For example, some BDDE derivatives of hyaluronic acid are covalently attached at both ends (i.e., cross-linked) between two distinct hyaluronic acid polymers whereas other BDDE derivatives are covalently attached at both ends in a single hyaluronic acid polymer Lt; / RTI > BDDE derivatives include BDDE derivatives covalently bonded at one or both ends to hydroxyl groups from one or more additional BDDE derivatives that are themselves covalently bonded to hyaluronic acid.

Also included are BDDE derivatives covalently bound to hyaluronic acid at only one terminus. For example, one of the epoxide rings may be opened by covalent attachment to a single stretch of hyaluronic acid polymer while the other epoxide ring may remain closed (i.e., unreacted) ). Such crosslinked hyaluronic acid-containing compositions also include those that do not affect the biocompatibility of the composition because the concentration of unreacted epoxide is too low. 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 ring may be open by hydrolysis.

The term "buffer" refers to a solution for stabilizing pH, which comprises a mixture of a weak acid and its base or weak base and its acid. 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 crosslinked hyaluronan as an active ingredient.

The pharmaceutical composition may be one 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 at least one 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 0.5 MDa to about 6.0 MDa, such as about 1.5 MDa to about 6.0 MDa, about 2.5 MDa to about 6.0 MDa, about 3.5 MDa to about 6.0 MDa From about 0.5 MDa to about 5.0 MDa, from about 0.5 MDa to about 4.0 MDa, or from about 0.5 MDa to about 3.0 MDa.

In the pharmaceutical composition, the weight content of the cross-linked hyaluronan per volume of final composition is from about 5 mg / ml to about 40 mg / ml, such as from about 10 mg / ml to about 40 mg / ml, From about 5 mg / ml to about 30 mg / ml, from about 5 mg / ml to about 20 mg / ml, or from about 5 mg / ml to about 10 mg / ml. The hyaluronic acid may be homogeneously distributed in the composition.

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

The crosslinked hyaluronan can be prepared by incubating hyaluronic acid or a salt thereof in the presence of a crosslinking agent in a basic solution, for example, a basic aqueous solution. The hyaluronic acid or its salt may be hydrated before it is crosslinked, for example, before it is added to the aqueous solution. The hydration may be carried out 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 the hyaluronic acid or its salt in an amount of 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 / 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 aqueous solution may contain from about 0.1 mol% to about 50.0 mol%, for example, from about 1.0 mol% to about 50.0 mol%, from about 5.0 mol% to about 50.0 mol%, based on the disaccharide monomer, which is a repeating unit of hyaluronic acid About 50.0 mole percent to about 50.0 mole percent, about 20.0 mole percent to about 50.0 mole percent, about 30.0 mole percent to about 50.0 mole percent, about 40.0 mole percent to about 50.0 mole percent, about 5.0 mole percent to about 30.0 mole percent, From about 5.0 mol% to about 25.0 mol%, from about 5.0 mol% to about 20.0 mol%, from about 15.0 mol% to about 25.0 mol%, or from about 10.0 mol% to about 20.0 mol% of a crosslinking agent, BDDE can be bridged.

Hyaluronic acid disaccharide Conventional units consist of D-glucuronic acid and N-acetylglucosamine. The cross-link rate (%) is the ratio of total hyaluronic acid disaccharide units. The amount of covalently crosslinked agent per disaccharide unit, i.e., the degree of crosslinking, can be ascertained by known methods. For example, ion exchange chromatography, which is a method of performing reversible ion exchange between a stationary phase and a mobile phase using ion exchange chromatography and separating and analyzing using a difference in affinity for a fixed phase of a sample ion, chromatography, or NMR . The formula for the degree of crosslinking is as follows.

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

The crosslinked hyaluronan can be formed when hyaluronic acid or its salt is contacted with a crosslinking agent. As used herein, the crosslinking agent includes a functional group of hyaluronic acid and a complementary functional group so that the crosslinking reaction proceeds. The crosslinking agent may be selected from butanediol diglycidyl ether (BDDE), divinylsulfone (DVS), or 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) Lt; / RTI > In one embodiment, the crosslinking agent is BDDE. The crosslinked hyaluronan may be a BDDE derivative.

The pharmaceutical composition may further comprise suitable carriers, excipients and diluents conventionally 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 may be in the physiological pH range, for example, about 7. The pH may be adjusted by the addition of 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 the osmolarity and ion concentration.

The composition of the present invention can be formulated in the form of a liquid preparation or a sterile injectable solution. When the composition is formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like which are usually used may be used.

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art.

The composition may be administered once a day or divided into several doses. The administration of the composition can be carried out by confirming the damaged region through magnetic resonance imaging (MRI) or computed tomography (CT).

The crosslinked hyaluronan may have a storage (elastic) modulus G 'value of 250 to 600 when measured at 2.5 Hz. The G 'can be measured by a known method, for example, a frequency sweep test method according to a change in frequency. The viscoelasticity measurement method according to the frequency change measures the viscoelasticity of hyaluronic acid at the time of shear deformation and changes the viscosity from 0.1 to 1 Hz to measure the viscosity change according to the shear rate change. The viscoelasticity is measured, The result is 2.5 Hz.

In this specification, the storage (elastic) modulus (G ') and the loss (viscous) modulus (G ") depend on the rate of deformation (test frequency) The two moduli are a linear function of frequency and they have proven to be sensitive probes for the structure of polymer solutions or gels G 'and G " Both increase with frequency, but one increases faster than the other. At the point G '= G', this frequency is called the cross-over frequency fc. % Elasticity means the hardness of the gel, which is the degree of external force that can cause the gel to deform. Therefore, a high% elasticity means that the gel has a high cohesive force, meaning that the force that is so deformed acts to a great extent.

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

In one embodiment of the present invention, in order to investigate the effect of the crosslinked hyaluronic acid-containing composition on osteoarthritis including cartilage damage and osteoporosis, a rat known to induce a lesion similar to degenerative joint disease in humans (Sprague-Dawley rats and Wistar rats) were used to induce chronic osteoarthritis through right fusiform knee anterior cruciate ligament incision, followed by hyaluronic acid. As a result, it was observed that the test group to which hyaluronic acid was administered had less cartilage damage and osteoproteger than the negative control group and the positive control group to which the phosphate buffered saline was administered.

Also, in one embodiment of the present invention, in order to investigate the effect of the crosslinked hyaluronic acid-containing composition on osteoarthritis including cartilage damage and osteoporosis, a diagnostic (osteoproliferative formation, proteoglycan destruction, etc.) similar to human arthritis and Hyaluronic acid was administered after acute induction of osteoarthritis using a MIA (Monosodium iodoacetate) chemical model (Wistar species rats) known to exhibit histopathological findings. As a result, it was observed that the test group to which hyaluronic acid was administered had less cartilage damage and osteoproteger than the negative control group and the positive control group to which the phosphate buffered saline was administered. Therefore, it can be seen that the composition of the present invention has an inhibitory effect on osteoarthritis.

The composition 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 in a subject, comprising administering to the subject a therapeutically effective amount of hyaluronan.

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

The administration may be by local administration to joints where osteoarthritis is present. Such administration may be by intra-articular administration, for example, to joints. The joint may be a knee, a finger, a toe, a hip, a spine, a neck, or a shoulder joint. Such administration may be by administering to the joints of an individual, for example, medial, lateral or both sites of the knee. The administration may be by injection into the joint cavity.

For such administration, the dosage varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art.

The "therapeutically effective amount" means an amount sufficient to produce a therapeutic effect when administered to a subject in need of osteoarthritis treatment. The therapeutically effective amount is in the range of 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, 1000 mg to 1500 mg, 1000 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 a pharmaceutical composition as described above.

The subject refers to a mammalian subject who is suffering from osteoarthritis or is likely to be sick. The osteoarthritis may include osteoporosis and / or edema. The mammal includes an ape, such as a human, a pig, a cow, a dog, a cat, a horse, or a sheep. The subject may be a mammal other than a human.

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

According to another aspect, a method for treating osteoarthritis of an individual can effectively treat osteoarthritis of an individual.

Fig. 1 shows the results of the saprinin-O staining of the untreated group, negative control group, positive control group and test group according to Example 1 (OARSI, post-operative 13 weeks, and 40 magnification).
FIG. 2 is a graphical representation of the degree of cartilage damage in the untreated group, the negative control group, the positive control group and the test group according to Example 2 (6 weeks after administration of MIA).
FIG. 3 shows visual observation of cartilage damage in the untreated group, the negative control group and the test group according to Example 3 (9 weeks after administration of MIA).

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited by the following examples.

Materials and methods

The following materials and methods were used in the following examples.

1. Bridge: Bridged  Production of hyaluronic acid

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

2. Cleaning

The crosslinked-hyaluronic acid gel was dialyzed to remove sodium hydroxide and any unreacted BDDE. It was confirmed that the LC concentration was controlled to be below the detection limit of 2 ppm.

3. Formulating

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

The resulting crosslinked hyaluronic acid-containing aqueous composition is a pharmaceutical composition obtained by crosslinking as described above, using 0.5 to 6 MDa of natural hyaluronic acid and 5 to 30 mol% of BDDE as a hyaluronic acid disaccharide monomer.

Example  1: Surgical Rat  Confirmation of osteoarthritis inhibitory effect of hyaluronic acid in osteoarthritis model (chronic arthritis animal model)

Anterior cruciate ligament surgery produces instability similar to naturally occurring osteoarthritis in humans by trauma and is suitable for osteoarthritis studies that are slow in pathologic aspects. Lesions occur on the medial tibial plateau and medial femoral condyle, where weight bearing is the main cause of joint weight loss, and degeneration of the meniscus is progressed between these two bone surfaces. Major diagnostic features include histologic degeneration of the articular cartilage and subchondral plate, large amounts of osteophyte, proteoglycan loss.

The animal models of this experiment were male Sprague-Dawley (SD) sprague-dawley rats, and the groups were divided into two groups: no treatment (sham, articular incision, no substance administration), negative control (postoperative phosphate buffered saline, The mice were divided into 4 groups: a positive control (SYNVISC-ONE ^TM 100uL, a total of 3 doses administered at intervals of 1 week) and a test group (100 ul of a hyaluronic acid example after surgery), and 5 to 7 mice were injected into the knee joint . The SYNVISC-ONE ^™ (Hylan GF 20) (Genzyme Biosurgery, USA) is an elasoviscous high molecular weight fluid containing 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 hyaluronan is chemically cross-linked. Hyaluronan is a long-chain polymer containing repeating units of two units of Na-glucuronate-N-acetylglucosamine. The SYNVISC-ONE ^TM has elasticity (111 ± 13 Pa and viscosity (loss modulus G ") 25 ± 2 Pa at 2.5 Hz for storage module G. The elasticity and viscosity of knee synovials in people aged 18-27 are comparable method), G '= 117 ± 13 Pa and G "= 45 ± 8 Pa at 2.5 Hz.

The rheological properties of the positive control composition and the test group composition (2M2020) were determined by viscoelastic measurement according to frequency change. The viscoelasticity measurement method according to the frequency change measures the viscoelasticity of hyaluronic acid at the time of shear deformation and changes the viscosity from 0.1 to 1 Hz to measure the viscosity change according to the shear rate change. The viscoelasticity is measured, Takes the result value of 2.5Hz. The measurement temperature was 25 占 폚, the measurement geometry was 40 mm, and the interval was 1000 mm.

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

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

The anterior cruciate ligament, which plays a major role in the joint stability, was isolated (right fusiform knee) prior to the intra-articular administration of the test substance, and then the treadmill was used at a rate of 20 m / min three times per week Minimal exercise was performed. Under these conditions, 11 weeks Induced joint osteoarthritis by increasing joint wear caused by physical joint instability.

The efficacy of the hyaluronic acid arthritis treatment using a surgical model was evaluated by measuring the width of the knee joints of the surgical site side (test substance administration site) and the non-administration site side (non-administration site) in the same individual with vernier calipers, The osteoarthritis of the osteoarthritis was evaluated by OARSI (Osteoarthritis Research Society International) through osteoarthritis and osteoporosis using Safranin-O / Fast green. The histopathologic evaluation method was used.

According to the OARSI method, a score of 0 to 6.5 according to the degree of damage of the cartilage, a score of 0 to 4 according to the area occupied by the lesion, . Table 2 shows the OA cartilage histopathology grade evaluation - an advanced grade evaluation standard.

* Rating (Key Features) Secondary rating (optional) Relevant Criteria (Organizational Response) Grade 0: Intact surface and cartilage (no damage) No secondary grade Intact and intact cartilage Grade 1: Full surface 1.0 Cell Integrity
1.5 cell death Substrate: Whole surface layer, edema and / or fibrillation
Cells: proliferation (cluster), hypertrophy Class 2: Discontinuous Surface (Rough) 2.0 Texture of the surface layer
2.5 Surface wear with surface layer substrate degradation With the above content,
+ Discontinuous surface layer
± Decreased staining of the upper third of the cartilage (middle layer) (sapranin O or toluidine blue)
± irregularity of cartilaginous pillars Class 3: Vertical cracks / crevices 3.0 Simple cracks / crevices
3.5 Cracked / Composite Niches With the above content,
± Staining reduction in 2/3 lower (deep layer) of cartilage (sopranin O or toluidine blue) Grade 4: Erosion 4.0 Surface Layer Separation
4.5 Middle layer recess Cartilage matrix loss, cartilage matrix cyst formation Grade 5: Peeling 5.0 Exact bone surface
5.5 Restoration or recovery tissue presence Restored tissue, including joints with hardened bone or fibrous cartilage Grade 6: Transformation 6.0 Peripheral osteoid proliferation
6.5 Appearance of peripheral and central osteophytes in joints Bone remodeling,
Deformation of articular surface contour, including microcracking and restoration

* Rating: Progression of cartilage damage

Table 3 shows OA cartilage histopathology-stage evaluation criteria.

*step % Lesion% (surface, area, volume) Step 0 Osteoarthritis no lesion Step 1 <10% Step 2 10 to 25% Step 3 25 to 50% Step 4 > 50%

* Step: lesion size

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

division
(Mean ± standard error) Knee joint width difference (right-left, mm) 6 weeks 13 weeks 20 weeks No treatment group (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 A positive control (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 the histopathological evaluation results (OARSI, 13 weeks post-surgery).

Classification (mean ± standard error) MFC MTP 13 weeks 13 weeks No treatment group (Sham) (n = 7) 1.16 ± 0.19 3.48 ± 0.24 Negative control (PBS) (n = 7) 20.83 ± 0.97 15.57 ± 1.07 A positive control (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 histopathological evaluation results (OARSI, 20 weeks post-surgery).

Classification (mean ± standard error) MFC MTP 20 weeks 20 weeks Negative control (PBS) (n = 5) 19.91 ± 1.62 18.10 ± 1.52 A positive control (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 Tables 4, 5, 6 and 1, hyaluronic acid inhibited the osteoarthritis of chronic induced rats and inhibited osteoarthritis including joint edema.

Fig. 1 shows the results of the saprinin-O staining of the untreated group, negative control group, positive control group and test group according to Example 1 (OARSI, post-operative 13 weeks, and 40 magnification). Fig. 1 shows a histopathological lesion of the knee joint surface at the 13th postoperative day. That is, the untreated group, the negative control group, the test group, and the positive control group show cut surfaces of the knee joint. Include 2 dye photos per group. In Fig. 1, the arrowhead indicates the discontinuity of the joint surface, del indicates the surface layer separation of the joint surface, fc indicates the fibrous joint formation, and of indicates the osteophyte.

As shown in Fig. 1, in the tissue photograph, the reddish-stained portion is normal cartilage when viewed from 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, and the restoration tissue (fibroplastic cartilage) was confirmed and the staining was also light . In the medial tibial surface (MTP), separation of the surface layer (grade 4) and osteophyte (grade 6) are observed. In contrast, MFC and MTP of the test group showed discontinuous articular surface (grade 2 ~ 2.5) as the main lesion. The area of the lesion showed four levels of MFC and MTP in the negative control group, whereas the MFC in the test group showed three levels and the MTP showed four levels.

Example  2: Chemical Rat  Determination of osteoarthritis inhibitory effect according to molecular weight of hyaluronic acid in osteoarthritis model (acute arthritis animal model)

Monosodium iodoacetate (MIA) inhibits the action of glyceraldehyde-3-phosphate (G3P) in cartilage cells, activates matrix metalloproteinase (MMP), a degenerative marker, and inhibits proteoglycan synthesis. , Which causes necrosis of cartilage cells and causes arthritis similar to degenerative arthritis.

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

In this experiment, male Wistar rats were used. Control group, control group (50uL phosphate buffered saline, PBS), positive control group (Synvisc-one ^TM 100uL, Total of 3 doses), and cross-linked test groups 2 to 3 (total of 100 UL of HA after surgery). Six groups of 6 mice per group were administered intraarticularly.

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

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

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

Day  0 Day  7 Day  14 Day  21 Control group 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

FIG. 2 is a graphical representation of the degree of cartilage damage in the untreated group, the negative control group, the positive control group and the test group according to Example 2 (6 weeks after administration of MIA).

As shown in Table 8 and FIG. 2, it was confirmed that improving viscoelasticity of HA by cross-linking effectively inhibited osteoarthritis as compared with a positive control (synvisc one) similar to viscoelasticity of synovial fluid.

Example 3 : Chemical Rat  In the osteoarthritis model (acute arthritis animal model), the content of hyaluronic acid In the christian  Of Osteoarthritis

In this experiment, male Wistar rats were used. Control group, control group (50uL phosphate-buffered saline, PBS), test group 4 and 5 (final of non-crosslinked hyaluronic acid) And 10 to 50 mg / ml), and test groups 6 to 9 having improved viscoelasticity were prepared through bridge cleavage, to give a total of 8 groups, and 6 mice per group were administered intraarticularly.

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

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

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

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 占 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

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

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

Example  4: molecular weight of hyaluronic acid by visual observation and In the christian  Confirm the effect

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. The higher the score, the more negative the pattern and the more severe the tissue damage. On the other hand, the lower the score, the better the positive aspect and the tissue damage.

The experimental animals were divided into two groups using male Wistar rats. Control group, control group (50 uL phosphate-buffered saline, PBS), positive control group (Synvisc one), and cross- These improved test groups 10 to 11 were prepared and total of 5 groups were administered, and 6 mice per group were administered intra-articularly.

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

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 for medial femoral condyle and lateral femoral condyle with a score of 0-3.

Surface area erosion Surface erosion rate 0 No erosion One Less than 10% erosion 2 11 to 25% erosion 3 26 to 50% erosion 4 51 ~ 75% erosion 5 76 ~ 100% erosion

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

Depth of erosion Erosion depth 0 none 0.5 Hardly noticeable One Has a little 2 Stand out 3 There are quite a lot

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

Rating Presence of cracks 0 none One There is a single cleavage site 2 There are two cracks

Table 15 shows the visual observation results measured according to FCE.

Total score Medial Outside corrosion(%) Erosion depth Cleavage site Untreated group 0 0 0 0 0 0 PBS treated 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, test group 11 in which cross-linked hyaluronic acid was administered compared with the positive control group showed less degree of cartilage damage according to FCE in the MIA animal model.

Claims (15)

A pharmaceutical composition for treating osteoarthritis comprising crosslinked hyaluronan as an active ingredient. The pharmaceutical composition according to claim 1, for inhibiting cartilage damage. The pharmaceutical composition according to claim 1, for inhibiting osteophytosis, joint edema, and joint damage. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition has a G 'value of 250 to 600 when measured at 2.5 Hz. The pharmaceutical composition according to claim 1, wherein the hyaluronan has a molecular weight of 0.5 MDa to 6 MDa. The pharmaceutical composition according to claim 1, wherein the weight of the hyaluronan composition is from 10 mg / ml to 50 mg / ml. The pharmaceutical composition according to claim 1, for administration into the joint of an individual. 9. The pharmaceutical composition of claim 8, wherein the joint is a knee, a finger, a toe, a hip, a spine, a neck, or a shoulder. A method for treating osteoarthritis in a subject, comprising administering to the subject a therapeutically effective amount of hyaluronan. The method according to claim 9, wherein said administration is administered within the joints. The method according to claim 9, for inhibiting cartilage damage. 10. The method of claim 9, wherein the at least one of osteophytosis, joint edema, and joint damage is inhibited. The method according to claim 9, wherein said administration is administering a pharmaceutical composition having a G 'value of from 250 to 600 when measured at 2.5 Hz. The method of claim 9, wherein the molecular weight of the hyaluronan is from 0.5 MDa to 6 MDa. 11. The method according to claim 10, wherein the administration comprises administering a pharmaceutical composition wherein the weight content of hyaluronan is from 10 mg / ml to 50 mg / ml.

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