JP6706632B2 - Composition for treating meniscus degeneration - Google Patents

Description

The present invention relates to a composition for treating meniscus degeneration, which has a long-lasting high efficacy.

Meniscus degeneration is a general term for degenerated, damaged, and/or ruptured medial and/or lateral menisci in the knee joint. Conventionally, it has been known that when a strong external force is suddenly applied to the meniscus during sports, etc., in recent years, with the advance of MRI imaging technology, even if a strong external force is not applied, it may be due to aging, etc. It has become clear that the meniscus gradually degenerates, which may cause osteoarthritis of the knee. In addition, it has been found that many adults have mild meniscal degeneration, may cause pain, and may be a reserve arm for knee osteoarthritis.

From this, it is strongly desired to develop a therapeutic drug that treats meniscal degeneration, that is, suppresses pain associated with meniscus degeneration, maintains its effect for a long time, and has a high effect of repairing the meniscus itself. ing.

The object of the present invention is to use hyaluronic acid and/or a salt thereof having high safety, but to obtain a significantly higher drug efficacy than the prior art against meniscus denaturation, and the drug efficacy lasts for a long period of time. It is intended to provide a composition for treating meniscus degeneration, which has a high effect of repairing itself.
The present inventors have obtained a high efficacy against long-term meniscal degeneration, including mild cases, which was recently found by using non-crosslinked and unmodified hyaluronic acid and/or its salt, and have a long efficacy. A study was conducted to sustain the period. When the efficacy of each composition was evaluated, it was surprisingly found that the composition containing non-crosslinked and unmodified hyaluronic acid and/or a salt thereof has a higher viscosity at a low shear rate than that of a conventional joint injection. It was found for the first time that a high efficacy against meniscal degeneration and a long-lasting effect were obtained. Further, it has been clarified that the higher the viscosity average molecular weight of the non-crosslinked and non-modified hyaluronic acid and/or its salt, the higher the efficacy even at a low concentration. Furthermore, not only the outer edge of the degenerated meniscus but also the inner edge, which is known to be difficult to be repaired, has no repair of blood vessels, and the repair effect was recognized. Further, since the non-crosslinked and unmodified hyaluronic acid and/or its salt are substances existing in the human body, side effects to humans are less likely to occur than the crosslinked or modified hyaluronic acid and/or its salt. Furthermore, it can be used even in patients who are allergic to cross-linking substances and modifiers.
Here, it is known from the common general technical knowledge of those skilled in the art that when the content of the active ingredient to be administered is the same in the injection, the drug effect does not change even if the amount of the solvent is changed. However, this time, a comparison was made between the present composition and a composition having a low viscosity by increasing the amount of the solvent, with the content of the non-crosslinked and unmodified hyaluronic acid and/or its salt being the active ingredient being constant. When it did, it became clear that the composition which made the viscosity low by increasing the amount of solvent becomes less effective than this composition surprisingly.
Further, as described above, it was found that the viscosity at low shear rate has a high correlation with the drug effect, but the viscosity at high shear rate has not been highly correlated with the drug effect. This cannot be inferred at all from the common general technical knowledge of those skilled in the art that the shear rate applied to the knee joint fluid during walking is very high.
Further, a therapeutic agent for osteoarthritis containing non-crosslinked and unmodified sodium hyaluronate as an active ingredient is said to have high viscoelasticity as one of its pharmacological actions, but the viscoelasticity of the composition and meniscus It was found that the correlation with drug efficacy against degeneration was not high.
Based on these findings, as a composition with dramatically improved therapeutic effect on meniscus degeneration, non-crosslinked and unmodified hyaluronic acid and/or its salt are contained at a concentration of 1.5 w/v% or more. Then, a composition for treating meniscus degeneration, which has a viscosity of 500 Pa·s or more at 25° C. and a shear rate of 0.1 s−1, was found, and the present invention was completed.

That is, according to the present invention, non-crosslinked and unmodified hyaluronic acid and/or a salt thereof is contained at a concentration of 1.5 w/v% or more, and the viscosity at 25°C and the shear rate of 0.1 s-1 is 500 Pa. A composition for treating meniscus degeneration, which is characterized by being s or more. Since it has a higher viscosity at a lower shear rate than the prior art, a high drug effect is obtained and the drug effect lasts for a long period of time. The meniscus is also repaired in cases where it is known that the degenerated part is a transverse fissure and reaches the inner edge of the meniscus, which is known to be difficult to repair.

3 shows a graph of changes in pain over time in Evaluation Example 1. The graph of the pain index in Evaluation Example 1 is shown. 9 shows a graph of changes in pain over time in Evaluation Example 2. The graph of the pain index in Evaluation Example 2 is shown. 9 shows a graph of changes in pain over time in Evaluation Example 3. The graph of the pain index in Evaluation Example 3 is shown. 9 is a graph showing changes over time in pain in Evaluation Example 4. The graph of the pain index in Evaluation Example 4 is shown. 7 shows a graph of changes in pain over time in Evaluation Example 5. The graph of the pain index in Evaluation Example 5 is shown. The representative photograph of the meniscus 30 days after surgery in Evaluation Example 6 is shown.

Hereinafter, preferred embodiments of the present invention will be described.

One embodiment of the present invention contains non-crosslinked and unmodified hyaluronic acid and/or a salt thereof at a concentration of 1.5 w/v% or more, and has a viscosity of 500 Pa at 25°C and a shear rate of 0.1 s-1. -A composition for treating meniscus degeneration, which is characterized by being s or more. By injecting such a composition for treating meniscus degeneration into a joint cavity, a high drug effect can be obtained and the drug effect can be maintained for a long period of time as demonstrated in Examples described later. The meniscus is also repaired in cases where it is known that the degenerated part is a transverse fissure and reaches the inner edge of the meniscus, which is known to be difficult to repair. Another embodiment is a composition for treating internal degeneration of the meniscus, which comprises the above composition. There is also a therapeutic effect on the degeneration of the inner edge of the meniscus, which was conventionally considered to have no therapeutic effect by hyaluronic acid. In another embodiment, the viscosity average molecular weight of the non-crosslinked and unmodified hyaluronic acid and/or its salt is 1.5 million or more, and the concentration of this hyaluronic acid and/or its salt is 1.5 w/v% or more. And a viscosity of 500 Pa·s or more at 25° C. and a shear rate of 0.1 s−1, the composition for treating meniscus degeneration. Such a composition for treating meniscus degeneration can obtain a high drug effect at a relatively low concentration and can maintain the drug effect for a long period of time.

Here, "high drug efficacy" means that the drug efficacy is higher than that of sodium hyaluronate joint injections used in the prior art, for example, Alz. In addition, the term "sustaining the drug effect for a long period of time" means that the drug effect is exerted for a longer period of time after administration, as compared with the sodium hyaluronate joint injection used in the prior art.
The inner edge of the meniscus is the inner part of the meniscus, which occupies about 2/3 of the area and is not covered by blood vessels. The outer edge of the meniscus is the outer part of the meniscus, which occupies about one-third of the area through which blood vessels pass.
The transverse crack of the meniscus is a mode in which the meniscus is cut substantially linearly from the inner edge portion to the outer edge portion.
Meniscus repair refers to a state in which the fractured part of the meniscus is partially or wholly filled with the regenerated tissue. The repaired state of the meniscus may be visually observed by an endoscopic examination or the like, or may be discriminated from the morphological and/or qualitative evaluation result of the meniscus using an image diagnostic method such as MRI imaging. In visual observation and morphological evaluation, it is judged that the smaller the fractured part of the meniscus, the higher the repair effect. If the fracture near the inner edge of the meniscus has been repaired, it is judged that the repair effect is particularly high. In the qualitative evaluation, it is judged that the closer the collagen content, water content, proteoglycan content, etc. in the regenerated tissue are to a normal meniscus, the higher the repair effect is.
In this specification, the low shear rate is a shear rate of 0.1 s-1, and the high shear rate is a shear rate of 1000 s-1.

In the present specification, the meniscus degeneration means the remaining half moon, the disk-shaped outer meniscus, the disk-shaped inner meniscus, the disk-shaped meniscus, and the outer meniscus described in the injury/illness name master of the social insurance medical fee payment fund. Encapsulation, lateral meniscus incarnation, lateral meniscus disorder, lateral meniscus injury, lateral meniscus tear, lateral meniscus degeneration, lateral meniscus calcification of the knee, medial meniscus calcification of the knee, old lateral Meniscus injury, old medial meniscus injury, medial meniscus swelling, medial meniscus incarnation, medial meniscus disorder, medial meniscus damage, medial meniscus tear, medial meniscus degeneration, exfoliation external meniscus, exfoliation medial Including meniscus, detached meniscus, meniscus edema, meniscus disorder, meniscus injury, meniscus tear, meniscus bucket pattern tear, meniscus degeneration, meniscus rocking, knee meniscus ganglion, knee meniscus calcification be able to. Among these, there is a high possibility that surgery will not be necessary by administration of the present composition for treating meniscus degeneration, outer meniscus disorder, outer meniscus damage, outer meniscus tear, outer meniscus degeneration, old outer meniscus. Injury, old medial meniscus damage, medial meniscus failure, medial meniscus damage, medial meniscus tear, medial meniscus degeneration, meniscus failure, meniscus damage, meniscus tear, meniscus bucket pattern tear, meniscus It can be preferably used for plate modification and meniscus locking.

The meniscus degeneration is a disease in which the properties of the meniscus in the knee joint are different from those of a healthy person, and the meniscus is torn or deformed. It has been known for some time that the meniscus is physically damaged due to sports injuries, etc.However, due to recent improvements in MRI diagnostic technology, aging and repetitive stress may cause the meniscus to become meniscus even without being subjected to particularly strong external forces. It has been found that there are many cases where a part of the plate is torn or deformed.
On the other hand, osteoarthritis of the knee is a disease in which the elasticity of knee cartilage is reduced and the knees are worn away, resulting in deformation of the joint.
Therefore, since meniscus degeneration is a disease of the meniscus and osteoarthritis of the knee is a disease of the knee cartilage, the tissues causing the disease are clearly different.
Meniscal degeneration and knee osteoarthritis can be easily diagnosed using MRI. Meniscal degeneration can be diagnosed by MRI, which shows cracks and tears in the meniscus and degeneration inside the meniscus, and can be classified into grades 1 to 3 depending on the degree of degeneration. Furthermore, if T2 mapping is used, detection can be performed with higher sensitivity.
Osteoarthritis of the knee can be diagnosed by detecting wear or loss of cartilage by MRI imaging, or by declining cartilage collagen using the T2 mapping method. In the case of osteoarthritis of the knee, diagnosis based on Kellgren-Lawrence classification by simple X-ray is widely used rather than MRI, and grade 2 or more out of grade 0 to 4 is diagnosed as osteoarthritis of the knee. It is difficult to diagnose meniscal degeneration by plain X-ray.

In the present specification, the non-crosslinked and unmodified hyaluronic acid and/or its salt may be hyaluronic acid that has not been crosslinked with a crosslinker or modified with a modifier, or sodium hyaluronate. Hyaluronates such as potassium hyaluronate, zinc hyaluronate, calcium hyaluronate, magnesium hyaluronate, and ammonium hyaluronate may be used. Among these, sodium hyaluronate is preferable from the viewpoint that a desired viscosity can be obtained and a sufficient therapeutic effect can be expected. The chemical name of sodium hyaluronate is, for example, [→3)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-β-D-sodium glucopyranosyluronate-(1→]n(IUPAC) The source of the non-crosslinked and unmodified hyaluronic acid and/or its salt can be used regardless of its origin, whether it is extracted from animal tissue or produced by fermentation. From the viewpoint of little concern about side effects, those produced by the fermentation method are preferred.When produced by the fermentation method, the strain to be used is a non-cross-linked and unmodified hyaluronic acid such as Streptococcus sp. A microorganism having the ability to produce a salt thereof, or Streptococcus equi FM-100 described in JP-A-63-123392 (Microtechnical Laboratory No. 9027), and Streptococcus described in JP-A-2-234689. A mutant strain capable of stably producing a non-crosslinked and unmodified hyaluronic acid and/or a salt thereof in a high yield, such as Equi FM-300 (Microtechnology Research Institute No. 2319), is preferable. What has been cultured and purified is used.

Meniscus degeneration treatment composition used herein, meniscus inner edge degeneration treatment composition, pain suppressing composition associated with meniscus degeneration, pain suppressing composition associated with meniscus inner edge degeneration, meniscus degeneration The meniscus repairing composition in the above, and the meniscus inner edge repairing composition in the modification of the inner edge of the meniscus are commercialized as a pharmaceutical product in addition to non-crosslinked and unmodified hyaluronic acid and/or its salt and water. It may contain various active ingredients and additives required in. For example, active ingredients include local anesthetics (lidocaine, xylocaine, etc.), nonsteroidal anti-inflammatory drugs (indomethacin, diclofenac, etc.), steroidal anti-inflammatory drugs (prednisolone, dexamethasone, etc.), antibiotics (cefmetazole, erythromycin, etc.), It may include muscle relaxants, contrast agents, antibody drugs, nucleic acid drugs and the like. As additives, isotonic agents (sodium chloride, potassium chloride, etc.), pH adjusters (sodium hydrogen phosphate hydrate, sodium dihydrogen phosphate, acetic acid, sodium acetate, boric acid, sodium borate, etc.), Stabilizers (L-methionine, sodium citrate, xylitol, zinc chloride, ethanol, glycerin, etc.) can be included, but from the viewpoint of toxicity and stability, boric acid, boric acid such as sodium borate and borates. It is preferable to contain sodium chloride, sodium hydrogen phosphate hydrate, sodium dihydrogen phosphate, and L-methionine without the acid salt component. The composition may be in a solution state or a suspension state, but a solution state is preferable from the viewpoint of easy inspection. Various commonly known methods can be used for the preparation of the present composition. For example, after mixing and dissolving components (water, active ingredients, additives) other than the non-crosslinked and unmodified hyaluronic acid and/or its salt, the non-crosslinked and unmodified hyaluronic acid and/or its salt are mixed. In addition, after mixing and dissolving, a container such as a vial or a syringe can be filled. To obtain a sterile composition, a method of performing all operations aseptically, a method of sterilizing by filtration, a method of sterilizing by heating after mixing and dissolving, or after filling can be used.

The non-crosslinked and unmodified hyaluronic acid and/or salt thereof according to one of the embodiments may have a concentration of 1.5 w/v% or more and a concentration that does not impair workability. Although it may be a value, the concentration of the non-crosslinked and unmodified hyaluronic acid and/or its salt is 3.0 w/v% or less from the viewpoint of production cost reduction and workability. The range of the concentration includes a range between the upper limit value or the lower limit value and a numerical value included therebetween. For example, the concentration ranges are 1.5 w/v%, 1.6 w/v%, 1.7 w/v%, 1.8 w/v%, 1.9 w/v%, 2.0 w/v%, 2.1 w/v%, 2.2 w/v%, 2.3 w/v%, 2.4 w/v%, 2.5 w/v%, 2.6 w/v%, 2.7 w/v%, 2 It may be a range between two selected from the group consisting of 0.8 w/v%, 2.9 w/v%, 3.0 w/v%.

The viscosity of the non-crosslinked and unmodified hyaluronic acid and/or its salt according to one of the embodiments at a shear rate of 0.1 s-1 may be any value as long as it is 500 Pa·s or more. In order to maintain the temperature for a longer period of time, the pressure is preferably 550 Pa·s or more, more preferably 600 Pa·s or more, and further preferably 700 Pa·s or more. Further, from the viewpoint of reducing the production cost and the upper limit of the solubility of hyaluronic acid and/or its salt, the viscosity of the non-crosslinked and unmodified hyaluronic acid and/or its salt at a shear rate of 0.1 s-1 is 4,000 Pa·s. Or less, preferably 3000 Pa·s or less, more preferably 2000 Pa·s or less, still more preferably 1000 Pa·s or less. The range of the viscosity includes a range between the upper limit value or the lower limit value and a numerical value included therebetween. For example, the viscosity range is 500 Pa·s, 550 Pa·s, 600 Pa·s, 650 Pa·s, 700 Pa·s, 800 Pa·s, 900 Pa·s, 1000 Pa·s, 1100 Pa·s, 1200 Pa·s, 1300 Pa·s, 1300 Pa·s. s, 1400Pa·s, 1500Pa·s, 2000Pa·s, 2300Pa·s, 2500Pa·s, 3000Pa·s, 3500Pa·s, 4000Pa·s may be a range between two selected from the group consisting of: ..

The viscosity can be measured under the following conditions using a commercially available rheometer.
Rheometer: rotary type (for example, MCR300 manufactured by Anton Paar)
・Measuring jig: Cone plate type jig (for example, CP50-1 manufactured by Anton Paar)
・Measuring temperature: 25℃
・Shear rate: 0.1 (1/s)

The viscosity average molecular weight of the non-crosslinked and unmodified hyaluronic acid and/or its salt described in one of the embodiments may be any value as long as it is 1,500,000 or more, but a high drug effect can be obtained even at a relatively low concentration. Therefore, it is preferably 1.6 million or more, more preferably 1.7 million or more, and further preferably 1.9 million or more. Further, from the viewpoint of reducing the production cost and the upper limit of the solubility of the uncrosslinked and unmodified hyaluronic acid and/or its salt, the viscosity average molecular weight of the uncrosslinked and unmodified hyaluronic acid and/or its salt is 3.5 million. It is preferably not more than 2.5 million, more preferably not more than 2.5 million, still more preferably not more than 2.2 million. The range of the viscosity average molecular weight includes a range between the upper limit value or the lower limit value and a numerical value included therebetween. For example, the range of the viscosity average molecular weight is 1.5 million, 1.6 million, 1.7 million, 1.8 million, 1.9 million, 2 million, 2.1 million, 2.2 million, 2.3 million, 2.4 million, 2.5 million, 2.7 million, 3 million, 340. The range may be between two selected from the group consisting of 10,000,000 and 3.5,000,000.

The viscosity average molecular weight is based on the 16th revised Japanese Pharmacopoeia General Test Method Viscosity Measurement Method 1 using the value of intrinsic viscosity obtained by measuring with an Ubbelohde viscometer at a measurement temperature of 30°C. It can be determined by the Laurent formula.
The viscosity-average molecular weight = ((intrinsic viscosity ^{(dL / g)) × 10} 5/36) 1 / 0.78

The composition can be applied where meniscus degeneration occurs anywhere in the meniscus. Specifically, in the inner meniscus and/or the outer meniscus, degeneration inside the meniscus, degeneration and/or tearing of only the inner edge portion, degeneration and/or tearing of only the outer edge portion, both inner edge portion and outer edge portion Examples include denaturation and/or tearing. Among these, particularly preferable is application to degeneration and/or tearing of only the inner edge portion, or degeneration and/or tearing including both the inner edge portion and the outer edge portion, which are difficult to repair with existing formulations.
In addition, the present composition can be applied regardless of the mode of meniscus modification. Specific examples include degeneration that does not cause rupture, transverse rupture, longitudinal rupture, beak rupture, horizontal rupture, and bucket pattern rupture in the inner meniscus and/or the outer meniscus. Of these, particularly preferable is application to transverse fissures, which are difficult to repair with existing preparations.

Meniscal degeneration can be diagnosed by medical examination, physical examination, MRI imaging, endoscopy, and the like. In MRI imaging, T2-weighted images, T1-weighted images, T2 mapping, T1ρ mapping, etc. are used, and by examining the presence or absence of degeneration or rupture of the meniscus, rupture state, collagen sequence, water content, proteoglycan content, etc., The morphological and qualitative evaluation of meniscus is possible.

Since this composition has high safety, it can be applied even when a disease other than meniscus degeneration is co-occurring. However, in the case of osteoarthritis of the knee, it may be difficult to determine the therapeutic effect of meniscal degeneration, so in the Kellgren-Lawrence classification of plain X-ray used for the diagnosis of osteoarthritis, It is preferably applied in the case of Grade 0 or Grade 1. Furthermore, it is more preferable to apply it in Grade 0, that is, in the case where osteoarthritis of the knee is not complicated.
The composition has clear causes such as sports trauma, secondary meniscus degeneration, and can be applied to any of the primary meniscus degeneration that occurs with aging without a clear cause, but in particular, , Can be preferably applied to primary meniscus degeneration.

The techniques for prescribing and administering a composition for treating meniscus to a joint are the same as those for general joint injections. For example, the latest version and the latest supplement of the Japanese Pharmacopoeia, “REMINGTON'S PHARMACEUTICAL” SCIENCES" (Maack Publishing Co., Easton, PA).

When the composition for treating meniscus is injected into a joint, the content of the non-crosslinked and unmodified hyaluronic acid and/or its salt as an active ingredient is the same as that of non-crosslinked and non-modified hyaluronic acid and/or its salt. Can be contained in an amount effective to achieve the intended purpose. A “therapeutically effective amount” or “pharmacologically effective amount” is well recognized by those skilled in the art and refers to an amount of an agent effective to produce a pharmacological result. Determination of a therapeutically effective amount is well known to those of ordinary skill in the art.

A therapeutically effective amount refers to that amount of drug which, upon administration, reduces the condition of the disease. Therapeutic efficacy and toxicity can be determined by standard pharmaceutical procedures in cell culture or experimental animals. The dosage lies preferably within a range of circulating concentrations that include the LD50 with little or no toxicity. This dose will vary within this range depending upon the dosage form used, the sensitivity of the patient, and the route of administration. As an example, the dose of the complex is appropriately selected depending on age and other conditions of the patient, the type of disease, the type of complex used, and the like.

The present invention will be described below with reference to examples, but the present invention is not limited thereto. Hyaluronic acid and/or its salts used in the examples are uncrosslinked and unmodified unless otherwise stated.

(Sample preparation example 1)
Sodium hyaluronate having a viscosity average molecular weight of 1.9 million (manufactured by Denka Co., Ltd.) was sampled in a dry weight of 20.0 g, and aseptically placed in a sterilized 2 L cylindrical plastic bottle (manufactured by Corning). To this, 1 L of phosphate buffered saline (pH 7.2) prepared aseptically was added and shaken. This was dissolved by stirring at room temperature for 1 day to give “2.0 w/v%-Mη1.9 million sample”. The dry weight of sodium hyaluronate was calculated from the wet weight and the weight loss value obtained in the section on weight loss of purified sodium hyaluronate in each article of the 16th edition of the Japanese Pharmacopoeia.

(Sample preparation example 2)
The same procedure as in Sample Preparation Example 1 was conducted except that 25.0 g of a dry weight of sodium hyaluronate having a viscosity average molecular weight of 1.9 million (manufactured by Denka Co., Ltd.) was collected, and a "2.5 w/v%-Mη 1.9 million sample" was obtained. ..

(Sample preparation example 3)
The same procedure as in Sample Preparation Example 1 was conducted except that 30.0 g of a dry weight of sodium hyaluronate having a viscosity average molecular weight of 1.9 million (manufactured by Denka Co., Ltd.) was collected, and a “3.0 w/v%-Mη1.9 million sample” was obtained. ..

(Sample preparation example 4)
The same procedure as in Sample Preparation Example 1 was carried out except that 10.0 g of a dry weight of sodium hyaluronate having a viscosity average molecular weight of 800,000 (manufactured by Denka Co., Ltd.) was collected, and a “1.0 w/v%-Mη800,000 sample” was obtained. ..

(Sample preparation example 5)
The same procedure as in Sample Preparation Example 1 was carried out except that 20.0 g of sodium hyaluronate having a viscosity average molecular weight of 800,000 (manufactured by Denka Co., Ltd.) was collected as a dry weight to give “2.0 w/v%-Mη800,000 sample”. ..

(Sample Preparation Example 6)
The same procedure as in Sample Preparation Example 1 was carried out except that 30.0 g of a dry weight of sodium hyaluronate having a viscosity average molecular weight of 800,000 (manufactured by Denka Co., Ltd.) was collected to give a “3.0 w/v%-Mη800,000 sample”. ..

(Sample Preparation Example 7)
The same procedure as in Sample Preparation Example 1 was carried out except that 10.0 g of a dry weight of sodium hyaluronate having a viscosity average molecular weight of 3.4 million (manufactured by Denka Co., Ltd.) was collected, and a “1.0 w/v%-Mη3.4 million sample” was obtained. ..

For the following, commercial products were used as samples.
Sodium hyaluronate joint injection "Svenir" (trade name, manufactured by Chugai Pharmaceutical Co., Ltd.)

(Sample Preparation Example 8)
Sodium hyaluronate having a viscosity average molecular weight of 1.5 million (manufactured by Denka Co., Ltd.) was collected in a dry weight of 2.2 g, and put into a sterilized 200 mL plastic bottle (manufactured by Corning) aseptically. The same procedure as in Sample Preparation Example 1 was carried out except that 100 mL of phosphate buffered saline (pH 7.2) prepared aseptically was added thereto to give "2.2 w/v%-M η 1.5 million sample". ..

(Sample Preparation Example 9)
"2.2 w/v%-Mη 1.7 million sample" was performed in the same manner as in Sample Preparation Example 8 except that 2.2 g of a dry weight of sodium hyaluronate having a viscosity average molecular weight of 1.7 million (manufactured by Denka Corporation) was collected. ..

(Sample Preparation Example 10)
The 2.0 w/v%-Mη 1.9 million sample was diluted 1.053 times with phosphate buffered saline (pH 7.2) to give "1.9 w/v%-Mη 1.9 million sample".

(Sample Preparation Example 11)
The same procedure as in Sample Preparation Example 8 was carried out except that 1.8 g of sodium hyaluronate having a viscosity average molecular weight of 2.2 million (manufactured by Denka Co., Ltd.) was collected as a dry weight to give "1.8 w/v%-Mη 2.2 million sample". ..

(Sample Preparation Example 12)
"1.6 w/v%-Mη 2.5 million sample" was performed in the same manner as in Sample Preparation Example 8 except that 1.6 g of a dry weight of sodium hyaluronate having a viscosity average molecular weight of 2.5 million (manufactured by Denka Corporation) was collected. ..

(Sample Preparation Example 13)
The same procedure as in Sample Preparation Example 8 was conducted except that 2.0 g of a dry weight of sodium hyaluronate having a viscosity average molecular weight of 3.4 million (manufactured by Denka Co., Ltd.) was sampled to give “2.0 w/v%-Mη3.4 million sample”. ..

(Sample Preparation Example 14)
A 2.0 w/v%-Mη 3.4 million sample was diluted 1.333 times with a phosphate buffered saline (pH 7.2) to give "1.5 w/v%-Mη 3.4 million sample".

(Sample Preparation Example 15)
An attempt was made to prepare 4.0 w/v%-Mη 1.9 million sample in the same manner as in Sample Preparation Example 8 except that 4.0 g of a dry weight of sodium hyaluronate having a viscosity average molecular weight of 1.9 million (manufactured by Denka Corporation) was collected. However, it could not be dissolved because the solubility was exceeded.

(Sample Preparation Example 16)
An attempt was made to prepare a 3.0 w/v%-Mη 3.4 million sample in the same manner as in Sample Preparation Example 8 except that 3.0 g of a dry weight of sodium hyaluronate having a viscosity average molecular weight of 3.4 million (manufactured by Denka Corporation) was collected. However, it could not be dissolved because the solubility was exceeded.

<Viscosity measurement of sodium hyaluronate solution>
MCR300 (trade name, manufactured by Anton Paar) was used as a rheometer which is a viscosity/viscoelasticity measuring device for each of Sample Preparation Examples 1 to 16 and each sample of a commercial product. As the cone plate, CP50-1 (cone angle 1.009°, D=49.938 mm) was used, the measurement temperature was 25° C., the shear rate was 0.01 to 1000 (1/s) (the logarithm of the shear rate was a constant interval). Increase), the number of measurement points: 51, the sample amount: 0.6 mL, and the measurement time: 10 seconds/point. Of the obtained data, the shear rate of 0.1 (1/s) is the closest to 0.1 (1/s) and is based on the two data points that sandwich 0.1 (1/s). The value of the viscosity at a shear rate of 0.1 (1/s) was calculated by the interpolation method. Regarding the shear rate of 1000 (1/s), the viscosity value at the shear rate of 999.9 or 1000 (1/s) was used as it was. Table 1 shows the measurement results of the viscosity.
The sodium hyaluronate concentrations having a viscosity at a low shear rate of more than 500 Pa·s are 1.5 w/v% and 1.6 w/v when the viscosity average molecular weights are 3.4 million, 2.5 million, 2.2 million, 1.9 million and 1.5 million, respectively. It was v%, 1.8 w/v%, 1.9 w/v%, 2.2 w/v%. As described above, it can be seen that the use of high molecular weight sodium hyaluronate makes it possible to obtain a composition having a lower concentration and a higher viscosity.

<Viscoelasticity measurement of sodium hyaluronate solution>
For each of the sample preparation examples 1 to 7 and each of the commercially available products, the same rheometer and cone plate as those used for viscosity measurement were used, measurement temperature: 37° C., strain: 5% (constant), frequency: 10 to 0.01 (Hz). (The logarithm of the frequency decreases so that the intervals are constant), the number of measurement points: 31, the sample amount: 0.6 mL, the measurement time: automatic (output when the value stabilizes). The data is plotted on a graph with frequency on the horizontal axis and storage elastic modulus G'and loss elastic modulus G" on the vertical axis, and the cross point where the curve plotting G'and the curve plotting G" intersect is obtained. It was Table 2 shows the results obtained by the interpolation method for the frequency at this cross point and the G'value (=G" value).

<Measurement of viscosity average molecular weight of sodium hyaluronate>
For each of Sample Preparation Examples 1 to 16 and each of the commercially available products, the flow-down time was 0.2 mol/when measured with an Ubbelohde viscometer in accordance with the section of the 16th Revised Japanese Pharmacopoeia, Pharmaceutical Articles, Purified Sodium Hyaluronate Diluted with 0.2 mol/L sodium chloride test solution so that it becomes 2.0 to 2.4 times as much as L sodium chloride test solution, and further dilute this solution in 4 steps, and then the 16th revision Japanese Pharmacopoeia general test method Viscosity measurement method According to the first method, the flow-down time of each sample was measured with an Ubbelohde viscometer to calculate the intrinsic viscosity. From the value of the intrinsic viscosity, the viscosity average molecular weight was calculated by the following formula. Table 3 shows the measurement results of the viscosity average molecular weight.
Viscosity average molecular weight=((intrinsic viscosity (dL/g))×10 ⁵ /36) ^1/0.78

(Evaluation example 1)
<Comparison of Svenyl, 2.0 w/v%-Mη 1.9 million sample, 2.0 w/v%-Mη 800,000 sample>
The effects of intra-articular injection of sodium hyaluronate on pain in meniscal degeneration were investigated using an experimental model of meniscus degeneration due to partial rupture of the meniscus in the knee joint of rabbits.

As animals, 5 Kbl:JW (SPF) rabbits and males aged 10 to 11 weeks were used per group. Every day from 1 to 8 days after the arrival of the animals, the animals were put into a main body container (holder) of a small animal analgesic evaluation apparatus Incapacitance Tester (manufactured by Linton Instrument) and left stationary for 5 seconds.

Animals are individually housed in a bracket-type metal wire mesh floor cage (350W x 500D x 350H mm) mounted on a movable rack, and the temperature is 23 ± 3°C, the humidity is 55 ± 20%, and the ventilation rate is 12 to 18 times/hour. The animals were bred under the environment of illumination time 7:00 to 19:00 (light 12 hours, dark 12 hours). As the feed, a solid feed RC4 (made by Oriental Yeast Co., Ltd.) for laboratory animals was fed as a restricted feed of 100 g/day by a stainless steel feeder, and drinking water was freely given by a polypropylene water bottle (stainless steel pipe). . The individual identification of the animal was made by writing the individual identification number on the auricle with magic ink, and the cage was provided with a card having the individual identification number.

<Preparation of experimental meniscus degeneration model (partial fracture of meniscus)>
The day of partial meniscus rupture surgery was defined as day 0 after surgery. An experimental model of meniscal degeneration was prepared using 11-12 week old animals.

The left knee joint of the rabbit was hair-removed under combined anesthesia (auricular vein injection) of ketamine hydrochloride (500 mg for ketaral intramuscular injection, manufactured by Sankyo Ale Pharmaceutical Co., Ltd.) and seractal (xylazine 2% injection, manufactured by Bayer). Fixed in the dorsal position on the Kitajima type fixator (Natsume Seisakusho). Aseptically, an incision of about 2 cm was made in the skin just below the lateral side of the patella to expose the lateral collateral ligament, and then the ligament was excised. Furthermore, the lateral meniscus was exposed by excising the tendon at the origin of the popliteal muscle. The incision was made in such a manner that the center of the inner edge of the meniscus was ruptured, and the incision was extended in a substantially straight line up to the outer edge of the meniscus. Then, the subcutaneous muscle layer and the skin were each sutured by nodules.

<Animal selection and grouping>
All groups were divided into groups on the 4th day after the operation (the day of the onset of pain). On the day of grouping, the body weight and weight distribution of both hind paws of all the cases were measured. The left hind paw weight distribution ratio ((left load/total load on both sides)×100(%)) was calculated from the measured hind paw weight distribution. Based on the left hind paw weight distribution ratio, the individual values were selected in the order of closeness to the average value. Selected animals were assigned to each group using a stratified, continuous randomization method with a left hind paw weight distribution ratio. After confirming that the average value of the left hind paw weight distribution ratio was the same in each group and there was no difference between the groups, the average value of body weight also showed the same value in each group, and the difference between the groups was I confirmed that there is no.

<Sample administration>
Once after grouping, 0.1 ml/kg of Svenir, 2.0 w/v%-Mη 1.9 million samples and 2.0 w/v%-Mη 800,000 samples, respectively, as a sample, was applied to the surgical side (left side) knee joint cavity. A 1 ml syringe (Terumo syringe 1 ml for tuberculin, Terumo Corporation) and a 23G injection needle (Terumo injection needle 23G, Terumo Corporation) were used for administration. The administration liquid volume was calculated individually by converting the liquid volume based on the body weight measured on the administration day.

<Method of measuring pain suppressing effect>
An analgesic evaluation device for small animals, Incapacitance Tester (manufactured by Linton Instrument, UK), was used to measure the weight distribution of both hind paws. This device uses the dual-channel sensor pad installed on the bottom of the container to accurately detect the weight distribution to the left and right legs of the animal in the main container, and accurately detect the left and right weights in grams. Were averaged at the set time. The body container used was for rabbits. The measurement setting time was 5 seconds when the animal was stationary.

The animal was moved into the main body container (holder) for rabbits, and the measurement was performed in the static state of the animal (first time), and then the animal was taken out of the holder and re-inserted to measure in the static state (second time). The measurement was repeated up to 10 times. For each of the hind paw weight distributions measured 10 times, the left hind paw weight distribution ratio (%) was calculated from the left and right weights (loads) by the following formula.
Left hind foot weight distribution ratio (%)={left load (g)/(right load (g)+left load (g))×100}

The average value of the left hind paw weight distribution ratio (%) calculated 10 times was defined as the left hind paw weight distribution ratio (%) per measurement. When the left hind paw weight distribution ratio is 50% or less, the higher the left hind paw weight distribution ratio is, the smaller the pain is, that is, the higher the drug efficacy is, and the left hind paw weight distribution is longer than 14 days after the operation. When the ratio remains high, it can be said that the drug effect lasts for a long time. Further, the area [area under the time curve of the decrease width (pain) of the left hind paw weight distribution ratio] calculated by the following calculation formula is defined as a pain index (IP: Index of Pain). The pain index (IP _1-Xd ) in the entire administration period ( _{1 to} X days after surgery) was calculated by the trapezoidal method. The smaller the pain index number, the higher the medicinal effect.
_{IP 1-Xd = Σ (n} = 1- (X-1)) [{( distribution ratio after 50 ^* -n ^{date) + (50 * - (n} + x) days distribution ratio after)} × x / 2]
*: The left hind paw weight distribution ratio (normal value) of individuals without pain is 50 (%).
n: number of postoperative days, x: number of days between n days and the next measurement day. As a result, as shown in FIGS. 1 and 2, 2.0 w/v%-Mη1.9 million samples were svenyl or 2.0 w/v. It was found that the drug effect was clearly higher than that of the %-Mη 800,000 sample, and that it was long-lasting.

(Evaluation example 2)
<Comparison of Svenyl, 2.0 w/v%-Mη 1.9 million sample, 2.5 w/v%-Mη 1.9 million sample, 3.0 w/v%-Mη 1.9 million sample>
Evaluations were made in the same manner as in Evaluation Example 1 except that Svenyl, 2.0 w/v%-Mη 1.9 million samples, 2.5 w/v%-Mη 1.9 million samples and 3.0 w/v%-Mη 1.9 million samples were used as samples. .. As a result, as shown in FIGS. 3 and 4, the 2.0 w/v%-Mη 1.9 million sample, the 2.5 w/v%-Mη 1.9 million sample, and the 3.0 w/v%-Mη 1.9 million sample were all obtained from Svenile. It was found that the drug was clearly effective and lasted for a long time.

(Evaluation example 3)
<Comparison of Svenyl, 1.0 w/v%-Mη 800,000 sample, 2.0 w/v%-Mη 800,000 sample, 3.0 w/v%-Mη 800,000 sample>
Evaluation was performed in the same manner as in Evaluation Example 1 except that Svenyl, 1.0 w/v%-Mη 800,000 sample, 2.0 w/v%-Mη 800,000 sample, and 3.0 w/v%-Mη 800,000 sample were used as samples. .. As a result, as shown in FIGS. 5 and 6, the efficacy of the 1.0 w/v%-Mη 800,000 sample, the 2.0 w/v%-Mη 800,000 sample, and the 3.0 w/v%-Mη 800,000 sample were all Svenyl. It was found that the drug efficacy was the same as that of Eq. Since the 1.0 w/v%-Mη 800,000 sample has the same sodium hyaluronate concentration and viscosity average molecular weight as that of Alz, in Alz used in the prior art and Svenyl which is a high molecular weight sodium hyaluronate joint injection, Regarding the pain-suppressing effect associated with meniscal degeneration, it can be said that the efficacy and duration of efficacy are the same.

(Evaluation example 4)
<Comparison of Svenyl, 2.0 w/v%-Mη 800,000 sample, 2.0 w/v%-Mη 1.9 million sample, 1.0 w/v%-Mη 3.4 million sample>
Evaluation was performed in the same manner as in Evaluation Example 1 except that Svenyl, 2.0 w/v%-Mη 800,000 sample, 2.0 w/v%-Mη 1.9 million sample and 1.0 w/v%-Mη 3.4 million sample were used as samples. .. As a result, as shown in FIGS. 7 and 8, it was found that the 2.0 w/v%-Mη 1.9 million sample had obviously higher drug efficacy and the duration of drug efficacy than the other samples. The efficacy and duration of efficacy were similar between Svenir, 2.0 w/v%-Mη 800,000 sample, and 1.0 w/v%-Mη 3.4 million sample.

(Evaluation example 5)
<Comparison according to the amount of Svenyl administered>
Evaluation was performed in the same manner as in Evaluation Example 1 except that svenyl was used as a sample and the administration liquid amount was shaken at 0.1, 0.2 and 0.3 ml/kg. These samples have the same sodium hyaluronate content as those obtained when svenyl, 2.0 w/v%-Mη 1.9 million samples and 3.0 w/v%-Mη 1.9 million samples were administered at 0.1 ml/kg, respectively. .. As a result, as shown in FIGS. 9 and 10, all the drug effects were equivalent to the administration of 0.1 ml/kg of svenyl. That is, in the case of a composition having a large amount of solvent and a low viscosity at a low shear rate even if the content of sodium hyaluronate is the same as that of the 2.0 w/v%-Mη 1.9 million sample and the 3.0 w/v%-Mη 1.9 million sample. , It turns out that it is not possible to obtain a higher medicinal effect than Svenir.

The results of Evaluation Examples 1 to 4 are summarized in Table 4. When the pain index is equivalent to 1.0 w/v%-Mη 800,000 sample corresponding to Alz or Svenir, x, when it is clearly higher than these, ○, when there is no data-, the viscosity is high even if the concentration is high. It can be seen that even a high average molecular weight is not sufficient to obtain a high drug effect.

Table 3 is summarized in Tables 5 and 6 using the viscosities at low and high shear rates as indexes. As a result, under the conditions of a shear rate of 0.1 s-1 and a low shear rate, when the viscosity was 500 Pa·s or more, the pain index was clearly lower than that of Alz equivalent 1.0 w/v%-Mη 800,000 sample or Svenir. It was found that, when the viscosity is less than 500 Pa·s, the pain index becomes equivalent to 1.0 w/v%-Mη800,000 sample or Svenir corresponding to Alz. Under the conditions of a shear rate of 1000 s-1 and a high shear rate, the efficacy of the 3.0 w/v%-Mη 800,000 sample was not higher than that of the 2.5 w/v%-Mη 1.9 million sample despite the higher viscosity. .. From this, it was revealed that the correlation between the viscosity under the high shear rate condition and the drug efficacy was not high.

Of the viscoelasticity, Table 3 is re-organized into Tables 7 and 8 using the cross-point frequency and G'(=G") value as indices. As a result, both the frequency and G'value show efficacy. It was found that the correlation was not high.

(Evaluation example 6)
<Confirmation of meniscus repair effect>
As a sample, using 2.0 w/v%-Mη 1.9 million sample of Svenir, meniscus partial rupture operation was performed in the same manner as in Evaluation Example 1 for 3 rabbits per group, and a sample of 0.1 ml/kg was evaluated. The same procedure as in Example 1 was administered once every 5 days and 5 times. On the 30th postoperative day, the femur of the left leg and the meniscus were separated, and the repaired condition of the lateral meniscus was visually observed. As a result, as shown in Table 9 and FIG. 11, in the 2.0 w/v%-Mη 1.9 million sample, the repair from the outer edge to the inner edge of the meniscus was visually confirmed in all three cases, but in Svenir, the outer edge of the meniscus was confirmed. The part was partially repaired or no repair was observed.

Claims (18)

Non-crosslinked and unmodified hyaluronic acid and/or its salt are contained at a concentration of 1.5 w/v% or more, and the viscosity at 25° C. and a shear rate of 0.1 s ⁻¹ is 500 Pa·s or more. A composition for treating meniscus degeneration. The composition for treating meniscus degeneration according to claim 1, wherein the non-crosslinked and unmodified hyaluronic acid and/or its salt has a viscosity average molecular weight of 1.5 million or more. The composition for treating meniscus degeneration according to claim 1 or 2, wherein the meniscus degeneration does not accompany knee osteoarthritis. The meniscus degeneration therapeutic composition according to claims 1 to 3, wherein the meniscus degeneration has a tear at the inner edge of the meniscus. The meniscus degeneration therapeutic composition according to claims 1 to 4, wherein the meniscus degeneration is transverse meniscus tear. A composition for treating internal degeneration of the meniscus, which comprises the composition according to claim 1. Non-crosslinked and unmodified hyaluronic acid and/or its salt are contained at a concentration of 1.5 w/v% or more, and the viscosity at 25° C. and a shear rate of 0.1 s ⁻¹ is 500 Pa·s or more. The composition for suppressing pain associated with meniscal degeneration, comprising: The composition for suppressing pain associated with meniscus degeneration according to claim 7, wherein the non-crosslinked and unmodified hyaluronic acid and/or its salt has a viscosity average molecular weight of 1,500,000 or more. The composition for suppressing pain associated with meniscus degeneration according to claim 7 or 8, wherein the meniscus degeneration does not accompany knee osteoarthritis. The composition for suppressing pain associated with meniscus degeneration according to claim 7, wherein the meniscus degeneration has a tear at the inner edge of the meniscus. The composition for suppressing pain associated with meniscal degeneration according to claim 7, wherein the meniscus degeneration is transverse meniscus tear. A composition for suppressing pain associated with degeneration of the inner edge of the meniscus, which comprises the composition according to claim 7. Non-crosslinked and unmodified hyaluronic acid and/or its salt are contained at a concentration of 1.5 w/v% or more, and the viscosity at 25° C. and a shear rate of 0.1 s ⁻¹ is 500 Pa·s or more. A composition for meniscus repair in meniscus degeneration. The composition for meniscus repair in meniscus modification according to claim 13, wherein the non-crosslinked and unmodified hyaluronic acid and/or its salt has a viscosity average molecular weight of 1.5 million or more. The composition for meniscus repair in meniscus degeneration according to claim 13 or 14, wherein the meniscus degeneration does not accompany knee osteoarthritis. The meniscus repair composition in meniscus degeneration according to claim 13 to 15, wherein the meniscus degeneration has a tear at the inner edge of the meniscus. The meniscus repair composition in meniscus degeneration according to claims 13 to 16, wherein the meniscus degeneration is a transverse meniscus tear. A composition for repairing the inner edge of the meniscus in the inner edge modification of the meniscus, which comprises the composition according to claim 13.