WO2005034945A1

WO2005034945A1 - Use of norphenazone and coenzyme q for curing arthrose, arthritis and osteoarthritis

Info

Publication number: WO2005034945A1
Application number: PCT/CH2004/000021
Authority: WO
Inventors: Thierry Schwitzguebel
Original assignee: Thierry Schwitzguebel
Priority date: 2003-10-11
Filing date: 2004-01-16
Publication date: 2005-04-21

Abstract

The invention relates to a pharmaceutical preparation containing norphenazone (3-methyl-1-phenyl-2-pyrazolin-5-one) or the pharmaceutically acceptable salts thereof or coenzyme Qn (N ranging from 6 to 10) or the mixture of the components thereof which is very efficient for therapeutically curing arthrose, arthritis and osteoarthritis and for prophylactics during interventions on articulations. The inventive preparation is particularly useful for any surgical intervention on articulations, in particular for arthroscopies and for above-mentioned diseases. Said preparation reduces the Chondrocytes destruction in affected tissues and the degradation of a polymeric matrix encompassing the Chondrocytes of the related articulation cartilage.

Description

title

USE OF NORPHENAZONE AND COENZYME Q FOR THE TREATMENT OF ARTHROSIS, ARTHRITIS AND OSTEO ARTHRITIS

Description

The subject of the present invention is the use of a pharmaceutical preparation whose active substances make it possible to preserve the chondrocytes and the polymeric matrix surrounding them when the joints are affected.

The subject of the invention is more specifically the use of substances chosen alone or in admixture, such as Norphenazone (3-methyl-1-phenyl-2-pyrazolin-5-one), norphenazone salts which are acceptable for pharmaceutical use. , Coenzyme Q _N OR N represents an integer between 6 and 10.

The invention more specifically relates to the prophylactic treatment of joints during surgical procedures, for example arthroscopy, etc. and the therapeutic treatment of osteoarthritis, arthritis and osteoarthritis.

Mammalian joints have reduced mechanical resistance to movement and elasticity to compressive loads due to the presence of cartilage covering the contacting bone surfaces. Cartilage is a non-vascularized tissue, where homeostasis is essentially directed by diffusion.

Some medical procedures, some trauma and some diseases have the consequence of damaging the cartilage of the joints. This covers in particular arthroscopies and other surgical procedures that cut or scratch the surface of the cartilage, occasional or repetitive trauma of the cartilage surface by mechanical load, osteoarthritis, chronic, degenerative and non-inflammatory joint disease , arthritis caused by trauma, and osteoarthritis. These conditions are associated with destructive cartilage damage and joint pain and cracking. These destructive alterations are accompanied by the death of the cartilage cells, the chondrocytes, as well as the decrease of the volume and the loss of elasticity of the polymeric matrix surrounding these chondrocytes. This reduction in elasticity is particularly observed during the loss of Glucosaminoglycans, which are the main constituents of the elastic polymeric network of cartilage. There is currently no effective pharmaceutical composition recognized for the prophylactic or therapeutic treatment of cartilaginous tissues of the joints related to the conditions described above. (Martindale Ref .: The Extra Pharmacopeia). The treatments currently offered merely reduce the pain observed by various means in order to maintain the mobility of the affected joint, or to replace certain parts of the joint with prostheses. The present invention describes an innovative method of preserving cartilage tissue and its functionality in mammalian joints in the conditions described above.

The use of Norphenazone, or pharmaceutically acceptable salts thereof, is known as an antipyretic agent (GB Patent 1072262) or an anti-inflammatory agent (US Patent 4579844; JP63132833) under general conditions of vascularized tissue. These conditions, as well as the effects observed, can not be applied to the cartilage of the joints. The application of Norphenazone in ophthalmic treatments is described (EP0633025), it corresponds to very particular oxidative and photo oxidative phenomena of the constituents of the eye and its pathologies.

Coenzyme Q is known as a cytochrome C inhibitor, it is also known for its antioxidant properties in hydrophobic media or highly vascularized tissues. These conditions do not apply in the case of an essentially aqueous and non-vascularized cartilaginous tissue, the living cells of which are inside a matrix impeπnable to the product. This product is also described as an ophthalmic adjunct during surgery (patent EP1231909), which by the nature of the organ in question can not be applied to the cartilage of the joints. Dispersion methods for coenzyme Q are described (patent WO9835660), but they can not be applied because they introduce harmful fatty acids to the cartilaginous tissues.

Unexpectedly, it has been observed that pharmaceutical preparations containing one or more of the following constituents, Norphenazone, a pharmaceutically acceptable salt of Norphenazone, Coenzyme Q _N (N between 6 and 10), could reduce the amount of chondrocytes. as well as the disintegration of the polymer matrix of the articular cartilage during traumas or affections described previously.

The amount of Norphenazone or the pharmaceutically acceptable salt in the preparation found in the present invention is such that the concentration of the active substance in contact with the cartilage surface in the joint is between 1 * 10 ^-7 and 1 * 10. ^"2 Molar. Optimally this concentration is between 1 * 10 ^" and 1 * 10 ^" Molar. The amount of Coenzyme QN (N between 6 and 10) in the preparation discovered in the present invention is such that the concentration of the active substance in contact with the surface cartilage in the joint is between 1 * 10 ^" and 1 * 10 ^" Molar. Optimally this concentration is between 1 * 10 ^-6 and 1 * 10 ^-4 molar. Coenzyme Q _N , used in this invention, is previously dissolved in an oil, a fat or a pharmaceutically acceptable hydrophobic substance, the essential characteristics of which are the minimum content of unsaturated fatty acids, and in particular the absence of fatty acid such as arachidonic acid, lonolenic acid, linoleic acid. The amount of the hydrophobic phase used in the preparation is between 50 and 0.01% by weight of the preparation, and optimally between 10 and 0.1% by weight of the preparation. The aqueous and hydrophobic phases are emulsified using, for this purpose, any phanaceutically acceptable emulsifier in proportions corresponding to the state of the art. If necessary, adequate amounts of ionizing agent, pH regulator, thickener, or preservative are included in the preparation. Adequate treatment is applied to the preparation so that it can be injected into the joint of which the cartilaginous tissue is concerned by the present invention. In an alternative manner, any agent peπnettant a control of the dissolution after the injection, such as by the creation of inclusions based on cyclodextrin, or liposomes, will be added to the preparation. Alternatively the control of the amount of preparation in contact with the cartilage surface can be adjusted by the use of a solid element implanted near the cartilage. The pharmaceutical preparation thus presented is useful as a prophylactic or therapeutic drug for the various lesions of the cartilage.

Example 1

An In Vitro model is used to demonstrate the degrading effect of a mechanical injury on cartilage chondrocytes. This model is described in the following references: Matrix and cell injury due to sub-impact loading of adult bovine articular cartilage explants: effects of strain rate and peak stress; T. Quinn; Journal of Orthopedic Research 19 (2001) - Cartilage injury by radiation compression near the gel diffusion rate; V. Morel, EPFL (Switzerland);

The pieces of bovine cartilage are obtained by cylindrical biopsy of the head of the animal 48 hours after his death. The elements tested measures 4mm in height from the outer surface of the cartilage to the inner side of the exponent subchondrial bone. The bone part of the element is cylindrical with a diameter of 4mm, while the size of the attached cartilage is a cylinder of 2.7mm in diameter.

The explant cartilages are individually placed in 1 ml wells in a culture medium. The culture medium is renewed every 24 hours, and the wells containing the cartilages are stored in an incubator at 37 degrees Celsius, with an enriched atmosphere of 4.8% volume of CO.

The culture medium has the following composition: 500 ml Dubellco's MEM media culture (Biochrom KG) - 5 ml Non essential amino acids mixture (Biochrom KG) 50 ml Fetal Bovine Serum (Cambrex) - 0.4 mM L-Proline (Sigma) - 5 Antibiotics, anti-fungal media (Penicillin G 100 000 U / ml, Streptomycin sulphate 10 000 μg / ml, Amphotericin B 25 μg / ml)

For the evaluation of the pharmaceutical preparation described in this invention, an amount of 6 mg of Norphenazone corresponding to a concentration of 6.1 * 10 ^-5 molar of the substance in the culture medium is directly dissolved therein.

The cartilage culture program after the death of the animal is as follows: - Day 2 biopsy of the cartilages with subchondrial bone. - Day 7 compression of the cartilages - Day 7 put in culture medium containing the pharmaceutical preparation. - Day 11 measurement of viability of chondrocytes (destructive measurement) - Day 18 measurement of viability of chondrocytes.

The compression is performed using a plane support of the sample whose displacement against a fixed cylindrical plunger of diameter much greater than that of the sample is controlled by a motor. The force exerted during the compression is measured by a sensor on the plunger. The move is saved at the same time. The assembly used contains the following elements: - Displacement controller (PM500-C from Newport Instruments) - Engine PM500-1 A.100 (Newport Instruments) - Stress recorder (Model 31, Sensotec) The stress conditions chosen are a constant strain of 7% per second of the thickness of the cartilage until a maximum force of 14 MPa is obtained. The constraint is then immediately released.

The measurement of viability of the chondrocytes is carried out by confocal cellular imaging of a vertical slice of the cylinder of the biopsy element. Dead chondrocytes are differentiated from living chondrocytes by selective staining with two distinct and specific fluorophores for the purpose of the measurement. The images obtained are processed using a computer program for detecting relative surfaces containing only dead chondrocytes, and those containing only living chondrocytes. These surfaces are expressed as a percentage of the total area of the biopsy cartilage. The results are statistically treated for their variance using ANOVA multiple regression model. The differences are then tested in pairs using the Tu Key method (Biostatistics, L.D. Fisher). The following equipment is used: Fiuorophores: Calcein AM / Ethidium homodimer-1 (Live / Dead Viability / Cytotoxicity kit Molecular Probe Inc.) Confocal microscope: Carl Zeiss LSM410 with Argon Laser excitation source

The results obtained are presented in Table 1.

The difference between these results has a degree of confidence greater than 99% (Tukey test).

Example 2

An In Vitro model is used to demonstrate the degrading effect of a mechanical injury on the polymeric matrix of cartilage. This model is described in the following references: Matrix and cell injury due to sub-impact loading of adult bovine articular cartilage explants: effects of strain rate and peak stress; T. Quinn; Journal of orthopedic research 19 (2001) Cartilage injury by ramp compression near the gel diffusion rate; V. Morel;

The samples are prepared according to the same procedure as in Example 1. The culture medium, as well as the conditions are the same as in Example 1, likewise for the program of the experiment and the mechanical conditions of the trauma applied. .

For the evaluation of the pharmaceutical preparation described in this invention, an amount of 33.6 mg of Coenzyme Q10 corresponding to a concentration of 6.95 * 10 ^-5 molar of the substance in the culture medium is first dissolved in a 11.2 g of rapeseed oil corresponding to 2% by weight of the final culture medium, 2.8 g of 0.5% by weight relative to the propylene glycol culture medium is added to the oil. before being mechanically altered with the culture medium described in Example 1. During the course of the study, the culture media are changed every 24 hours and their glucosaminoglycan content (called GAG in the rest of the document) are analyzed The method of analysis of GAG is by colorimetry following the methods described in: - RW Farndale, DJ Buttle, AJ Barrett, Improved quantification and discrimination of sulphated glycosaminoglycans by use of dimethyl blue, Biochemica and Bi ophysica acta, 883, 1986, p 173-177. - RW Farndale, CA Sayers, AJ Barrett, A Direct Spectrophotometric Microassay for Sulphated Glycosaminoglycans in Cartilage Cultures, Connective Tissue Research, Vol. 9, 1982, p 247-248.

Mixing 20 microliter of culture medium and 200 microliter of methylene blue solution gives a mixture absorption variation at 530 nm as a function of the GAG content of the setting. The recorded values are converted to GAG content by comparison with a calibration line constructed using known concentrations of 10 to 100 micrograms per ml of Chondrocyte Sulfate (Sigma). At the end of the culture period, the measured cartilage is totally digested to determine its total GAG content. This value is used to express the daily GAG cartilage losses as a percentage of it. For the measurement of the total content of GAG cartilages, they are digested in 1 ml of 60 Deg Papagen solution. C for 16 hours. The Papain solution has the following structure: - 75 ml of PB S containing 0.0075 mg of NaN ₃ - 65 mg of cysteine HC1 - 375 microliter of suspension of Papain 25 mg / ml (Sigma, P-3125) The solution obtained after digestion is diluted 50 times in the same culture medium as that used during the study to perform the colorimetric measurement. A 20 microliter uptake is mixed with 200 microliters of methylene blue solution as before. The presentation of the results obtained for the pharmaceutical composition of this invention is made in graphical form by representing the average values obtained and their standard deviation.

Chart 1

The above examples confirm that the pharmaceutical preparation disclosed in the present invention containing one or a mixture of the constituents selected from Norphenazone, the pharmaceutically acceptable salts of Norphenazone and Coenzyme QN (N between 6 and 10) are effective. as prophylactic and therapeutic treatment of trauma and cartilage disorders, such as, for example, surgical procedures, single or repeated mechanical stress, osteoarthritis, arthritis or osteoarthritis.

Claims

1. A pharmaceutical preparation containing a beneficial amount of Norphenazone or its pharmaceutically acceptable salts, as well as the excipients, diluents, emulsifiers, and additives phaπnaceutically acceptable for its formulation, useful for the prophylactic or therapeutic treatment of the damage of the cartilage of the joints of mammals .

2. A pharmaceutical preparation containing a beneficial amount of Coenzyme Q _N (N between 6 and 10), as well as excipients, diluents, emulsifiers, and pharmaceutically acceptable additives for its formulation, useful for the prophylactic or therapeutic treatment of cartilage damage joints of mammals.

3. A phaπnaceutical preparation containing a beneficial quantity of the mixture, of the active substances, Coenzyme Q _N (N between 6 and 10) and of Norphenazone or its pharmaceutically acceptable salts, as well as the excipients, diluents, emulsifiers, and pharmaceutically acceptable additives for its formulation, useful for the prophylactic or therapeutic treatment of cartilage damage in mammalian joints.

4. A pharmaceutical preparation according to claims 1 to 3, used when the damage to the cartilage is caused by a surgical operation, for example an arthroscopy.

5. A phaπnaceutical preparation according to claims 1 to 3, used when the damage to the cartilage is caused by a single or repeated mechanical trauma.

6. A phaπnaceutical preparation according to claims 1 to 3, used when the damage to the cartilage is caused by osteoarthritis, arthritis or osteoarthritis.

7. A method for the prophylactic or therapeutic treatment of the cartilage disorders linked to claims 4 to 6 in mammals by the local administration of a pharmaceutical preparation containing a beneficial amount of the active substances claimed in points 1 to 3.

8. A pharmaceutical preparation described in claims 1 to 3, characterized by an emulsion form of oil, of fat, or of a hydrophobic substance pharmaceutically acceptable in water, the specificity of the lipid phase of which is characterized by the absence of the following fatty acids: arachidonic, linoleic or linolenic.

9. A pharmaceutical preparation described in claims 1 to 3 and 8, sterilized to be able to be applied locally by injection according to usual pharmaceutical practices.

10. A phamiaceutical preparation as described in claims 1 to 3 and 8 to 9, characterized by a treatment which allows to obtain inclusions or liposomes according to the usual phanaceutical practices.

11. A pharmaceutical preparation prepared according to claims 8 to 10 characterized by its incorporation in an implant intended to be placed in the vicinity of the damaged cartilage.