EA044994B1 - PARENTERAL COMPOSITION AND METHOD FOR ITS PREPARATION - Google Patents

Description

The present invention relates to a parenteral composition suitable for use in the treatment of joint disorders, a method of preparing said composition and the use of said composition for the treatment of joint disorders.

BACKGROUND OF THE INVENTION

Articular cartilage plays a key role in the movement of joints in humans and animals. It provides a smooth outer surface between adjacent bones, allowing joints to move almost without friction. The synovial fluid inside the joint cavity acts as a lubricant. In joints, articular cartilage distributes compressive stresses across the surfaces of the articular plates of the joint, thereby protecting the load-bearing bones from abrasion and wear. Articular cartilage is composed of chondrocytes embedded in an extracellular matrix containing proteoglycans, collagen, glycosaminoglycans, and low molecular weight (MW) glycoproteins. Chondrocytes are cartilaginous cells embedded in lacunae within the cartilage matrix. Proteoglycans are essential for maintaining the strength of cartilage tissue so that it can withstand compression. Collagen gives fabric tensile strength and shear resistance. In a healthy joint, the extracellular matrix is maintained by a balance between the synthesis and secretion of these macromolecules by chondrocytes and their subsequent degradation by proteolytic enzymes. Damage to articular surface cartilage can disrupt this balance so that degradation is not sufficiently compensated by the ability of chondrocytes to synthesize macromolecules necessary for cartilage tissue repair. Chronic imbalance between the synthesis and degradation of cartilage matrix macromolecules is associated with the development of osteoarthritis.

Osteoarthritis (OA) is a degenerative disease affecting the joints. In veterinary medicine, it is also known as Degenerative Joint Disease (DJD). Osteoarthritis is the most common form of arthritis and is the result of the progressive degeneration of cartilage that accompanies aging. Typically, OA appears after injury or chronic joint damage caused by other types of arthritis, such as rheumatoid arthritis. Additionally, OA can result from overuse of a specific joint. OA can be classified as:

primary, in which the root cause is not obvious; secondary, when there is a predisposing factor associated with it, such as injury, repetitive stress (occupation, sport), congenital anomalies, metabolic diseases (disorders) or other bone/joint disease (disorder); and erosive, which is characterized by periods of acute inflammation and progressive destruction of the joints, occurring more often in middle-aged women. OA is more common in the joints of the finger, hip, knee, spine, thumb and base of the hallux. Clinically, OA is characterized by joint pain, limitation of movement, crepitus (crackling) and unrelenting progressive disability. It may affect only one of these joints or all joints. Although most tissues in the body are capable of repairing themselves after an accident, the self-repair of cartilage is hampered by a limited blood supply and the lack of an effective cartilage replacement mechanism.

Historically, treatment of osteoarthritis and articular cartilage lesions has been limited to symptomatic pain therapy, joint load reduction, physical therapy, and orthopedic surgery. Most of these treatments are aimed at relieving symptoms rather than treating the underlying condition. Recently, research has focused on the development of chondroprotective treatments. Such methods provide long-term therapeutic treatment aimed at preserving or stimulating the formation of cartilage. Current treatment for arthritis includes first-line medications to control pain and inflammation, classified as non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, ibuprofen and naproxen. Secondary treatment includes corticosteroids that act as slow-acting anti-rheumatic drugs (SAARDs) or disease-modifying antirheumatic drugs (DMARDs), eg penicillamine, cyclophosphamide, gold salts, azathioprine and levamisole for rheumatoid arthritis.

Although NSAIDs are one of the main groups of drugs used in the treatment of OA, their side effects are often considered unfavorable in terms of risk/benefit ratio, especially in the elderly. Depending on individual circumstances, NSAIDs may cause gastrointestinal (GI) bleeding, ulceration, or perforation of the GI tract, while some cause bone marrow suppression or fluid retention and therefore may contribute to kidney failure . The significance of these effects is due to the fact that such treatment is often long-term. In addition, the use of medicinal plants for the treatment of various disorders (or conditions) in mammals is known.

Another approach that is used in the treatment of osteoarthritis and various forms of arthrosis is the introduction of drugs into the joint that improve the gliding of articular surfaces. The said treatment is carried out by infiltrating the joint with a therapeutic solution, usually containing

- 1 044994 hyaluronic acid. Although the above-mentioned drugs and treatments have achieved a certain degree of success in the preventive treatment of osteoarthritis, new therapeutic solutions are constantly being sought that can effectively reduce the progression of lesions and cartilage degradation in a mammal suffering from osteoarthritis. Therefore, it is an object of the present invention to provide a method for effectively treating arthritis, degenerative intervertebral disc diseases, rheumatoid arthritis and cartilage diseases.

The essence of the invention

The inventors of the present invention have unexpectedly discovered that certain substances, at certain concentrations, or mixtures thereof, have effective therapeutic effects in the treatment of disorders (or conditions) affecting articular cartilage in mammals. The figures appended to this specification and the experiments performed by the authors and described below show the effectiveness of the claimed substances and combinations.

Therefore, the object of the present invention is to provide a parenteral composition containing an extract or one or more fractions of Centella asiatica and/or an extract or one or more fractions of Echinacea purpurea, and at least one pharmaceutically acceptable carrier, therapeutic uses of the said composition and methods for preparing the same.

Detailed description of drawings

In fig. 1 shows, in various panels, the histological condition of rat joints treated with monoiodine acetate (MIA) and then with vehicle alone (designated MIA only), Centella extract, fraction soluble in 70% hydroalcoholic solution (MIA+Centella 14G1862), Echinacea purpurea extract, fraction soluble in 40 to 50% aqueous-alcoholic solution, (MIA+Echinacea 13C1608), and bovine synovial fluid (MIA+synovial fluid) during the period of 14, 30 and 60 days. Panel a shows a morphological score related to inflammatory infiltrate (a high score corresponds to a prominent inflammatory infiltrate), panel b shows a morphological score related to synovial hyperplasia, panel c shows a morphological score related to fibrin deposition, panel d shows a morphological score related to synovial vascularity, panel e shows a morphological score related to cartilage erosion, panel f shows a morphological score related to bone erosion, and panel g shows a morphological score related to space reduction ( gap) between the two ends of the joint (in this latter case, a high value corresponds to a reduced joint space). MIA-treated animals demonstrate, at the articular level, the presence of an inflammatory infiltrate (MIA; panel a) and morphological damage, manifested by the development of synovial hyperplasia (MIA; panel b) and increased vascularity (MIA; panel d), reaching a maximum level at 14 th day and showing a tendency to gradually decrease over several days. Cartilage and bone erosion (MIA; panels e-f), fibrin deposition (MIA; panel c), and joint space reduction (MIA; panel g) instead show a time-dependent increase until day 30. Treatment with extract (above fraction) from Centella and Echinacea effectively counteracts MIA-induced morphological damage, keeping most of the assessed parameters low (panels a-g), whereas synovial fluid does not appear to be effective in protecting the MIA-damaged joint (panels a-g).

In fig. Figure 2 shows the general histological picture: A Control; In MIA; D MIA+Centella 14G1862; EMIA+Echinacea 13C1608; F MIA+bovine synovial fluid. Images obtained from treated animals show fibrin deposition in the joint space (black arrows), cartilage erosion (black arrows), and bone erosion (asterisks). Calibration scale: 100 µm.

In fig. 3 shows, in various panels, the histological condition of rat joints treated with monoiodoacetate (MIA) and subsequently with vehicle alone (designated MIA only), Centella extract, EtOH 70% soluble fraction (MIA+Centella 14G1862), Centella extract, insoluble fraction, solubilized in EtOH 13% (MIA+ABO-AR-2016-391), Centella extract, filtered fraction -391, permeate (MIA+ABO-AR-2016-392), Centella extract, filtered fraction -391, retentate (M1A+ABO-AP-2016-393) and triamcinolone acetonide (MIA+Triam) after 14, 30 and 60 days.

Panel a presents a morphological score related to inflammatory infiltrate (a high score indicates a prominent inflammatory infiltrate), panel b shows a morphological score related to synovial hyperplasia, panel c shows a morphological score related to fibrin deposition, panel d shows a quantitative morphological index related to synovial vascularity, panel e presents a quantitative morphological index related to cartilage erosion, panel f presents a quantitative morphological index related to bone erosion, and panel g presents a quantitative morphological index related to space reduction ( gaps) between the two ends of the joint (in this latter case

- 2 044994 a high value corresponds to a reduced joint space). MIA-treated animals demonstrate, at the articular level, the presence of an inflammatory infiltrate (MIA; panel a) and morphological damage, manifested by the development of synovial hyperplasia (MIA; panel b) and increased vascularity (MIA; panel d), reaching a maximum level at 14 th day and showing a tendency to gradually decrease over several days. Cartilage and bone erosion (MIA; panels e-f), fibrin deposition (MIA; panel c), and joint space reduction (MIA; panel g) instead show a time-dependent increase up to day 30 (MIA; panel c, g) or up to day 60 (MIA; panels e-f). The figure shows how fractions from Centella 14G1862, ABO-AR-2016-391 and ABO-AR-2016-392 can protect the joint from MIA-induced damage across multiple parameters assessed. Instead, the ABO-AP-2016-393 fraction is not particularly effective (M1A+ABO-AP-2016-392; panels a-g), while the drug triamcinolone only partially reduces the damaging effects of MIA (MIA+Triam; panels a, b,c,e,f).

In fig. Figure 4 shows the general histological picture: A: MIA; B: MIA + Centella 14G1862; C: MIA + ABO-AR-2016-391; D: MIA + ABO-AR-2016-392; E: MIA+ABO-AR-2016-393; F: MIA + triamcinolone. Images of treated animals show fibrin deposition in the joint space (asterisk) and cartilage erosion (black arrows). Calibration scale: 100 µm.

In fig. 5 to 13 graphically depict the data depicted in tables 1 to 9. In the figures and text, MIX (co-extract) series 17H0246 corresponds to a mixture of Echinacea extract, a fraction soluble in an aqueous-alcoholic solution according to the present description, lyophilized, and extract of Centella asiatica, the hydroalcoholic soluble fraction as described herein, which was re-suspended with NaCl in water, incubated at 40 to 55°C for 4 to 8 hours, filtered and lyophilized.

Fig. 14 to 17: The mixture according to the invention was analyzed in separate experiments in relation to individual fractions of Centella and Eschacea. Behavioral test data were therefore expressed as % improvement compared to their positive control (MIA-treated animal + vehicle) to obtain a numerical value indicating the improvement of the animals after treatment. In this way, the effect of different treatments can be compared: the % value mentioned allows you to quantify the increased or decreased effectiveness of a sample compared to another sample. Figures 14 to 17 graphically present the results of these comparisons.

In fig. 18 shows, in various panels, the histological condition of rat joints treated with monoiodoacetate (MIA) and then with vehicle alone (designated MIA alone), MIX (co-extract) Centella + Echinacea series 17H0246, Hyalgan, Jonexa and triamcinolone acetonide after 14. 30 and 60 days. Panel a presents a morphological score related to inflammatory infiltrate (a high score indicates a prominent inflammatory infiltrate), panel b shows a morphological score related to synovial hyperplasia, panel c shows a morphological score related to fibrin deposition, panel d shows a quantitative morphological index related to synovial vascularity, panel e presents a quantitative morphological index related to cartilage erosion, panel f presents a quantitative morphological index related to bone erosion, and panel g presents a quantitative morphological index related to space reduction ( gap) between the two ends of the joint (in this latter case, a high value corresponds to a reduced joint space). In fig. 19 shows the general histological picture: images typical of the histological picture detected after 30 days: A: control; In: MIA; C: MIA+MIX (joint extract) Centella+Echinacea lot 17Н0246; D: MIA+Jonexa; E: MIA+Hyalgan; F: MIA+triamcinolone acetonide. Images of treated animals show fibrin deposition in the joint space (black arrows) and erosion of the covering cartilage and subchondral bone (asterisks). Calibration scale: 100 µm.

The term co-extract series 17H0246 in the figure legends and tables refers to the fact that the Centella extract and the Echinacea extract are mixed and subjected to an additional extraction step in an aqueous solution prior to use in the final composition, as described below herein.

Detailed Description of the Invention

The present invention relates to a parenteral composition containing an extract of Centella asiatica, an extract of Echinacea purpurea and at least one pharmaceutically acceptable carrier.

For the purposes of the present invention, a parenteral composition means a composition that can be introduced into the body by piercing the covering tissues with a needle so that various cavities, organs or tissues can be penetrated. For the purposes of the present invention, the composition will in particular be an intra-articular composition, that is, a composition suitable for injection directly into a joint.

For the purposes of the present invention, the term extract means a preparation in liquid, solid or soft form from parts of fresh or dried plants, which can be obtained by suitable extraction methods, for example with suitable solvents and/or by soaking, percolation, filtration, as well as other suitable means.

As is known, methods of extraction from herbal medicinal products can contain multiple steps and, therefore, at the end or during the extraction, numerous different fractions can be obtained, thus the term extract in accordance with the invention is also intended to refer to individual fractions that can be obtained during extraction, or mixtures thereof. In any paragraph of the present description and claims, therefore, the term extract may be replaced by the term extract fraction or mixture of extract fractions.

In accordance with one embodiment of the present invention, a Centella asiatica extract can be, for example, obtained from crushed Centella leaves that are soaked in alcohol (65% to 75%) at a level of about 65%, about 70%, about 75% ( non-limiting example: ethanol: water 70:30 v/v). In one exemplary embodiment, Centella extract is prepared in a 1:8 ratio between Centella and 70% alcohol at a suitable extraction temperature of 40 to 60°C. Extraction usually continues for 6 to 8 hours, then the spent residue is separated from the solution by decanting and subsequent filtration (for example, on a paper filter).

In one embodiment, the extract (more specifically, the resulting hydroalcoholic fraction) is concentrated and dried by lyophilization, in accordance with techniques known to one skilled in the art.

In one embodiment of the present invention, therefore, the term extract corresponds to the fraction soluble in an aqueous-alcoholic solution as described above in the example.

In certain examples depicted herein representing embodiments of the present invention, the Centella extract, designated in the figures and tables as Centella 14G1862, is the aqueous-alcoholic soluble fraction of the aerial parts of the plant obtained as reported above.

According to another embodiment of the present invention, the fractions of Centella asiatica soluble in an aqueous-alcoholic solution may also be fractions obtained by subjecting the plant part of interest to primary extraction with a suitable alcohol at a concentration of about 96% (e.g. ethanol:water 96:4 v/v ./vol.) in a ratio between drug and 96% alcohol of about 1:8, at a suitable temperature in the range of 30 to 50°C for a period of 6 to 8 hours.

After the said period of time, the supernatant is removed and the remainder of the drug is further extracted with a suitable alcohol of about 13% concentration (e.g. ethanol:water 13:87 v/v) in a ratio of drug to 13% alcohol equal to 1:8, at a suitable temperature ranging from 30 to 50° C. for a period of 6 to 8 hours. Thereafter, the solid residue is discarded by decanting and filtering on paper, whereby a 13% alcoholic fraction is collected.

In one embodiment, the extract is represented by said 13% alcohol fraction, which is subjected to evaporation of the alcohol and subsequently filtered on paper to obtain a clear aqueous concentrate, which is freeze-dried in accordance with a method known to one skilled in the art (e.g., ABO fraction -AR-2016-391 in accordance with this description). In addition, a portion of the aqueous concentrate prepared as mentioned above may be filtered through a membrane of specified molecular pore size to obtain a permeate fraction and a retentate fraction. In one embodiment, said fractions are concentrated and dried by lyophilization, in accordance with techniques known to one skilled in the art. Examples of fractions according to this embodiment are the permeate fraction ABO-AR-2016-392 and the retentate fraction ABO-AR-2016-393, as reported herein.

According to the present description, the following membranes can be used: a filtration membrane with a defined molecular pore size, which has a MWCO (molecular weight cutoff) of 150 to 300 Da, or alternatively 700 Da, or alternatively 1000 Da, or alternatively 2500 Yes, or alternatively 5000 Yes, or alternatively 10000 Yes, or alternatively 20000 Yes, or alternatively 50000 Yes. Said membranes may be helical, made of polyamide, polysulfone, polyethersulfone or ceramic material.

In one embodiment of the invention, the Echinacea purpurea extract can be obtained by hydroalcoholic extraction from crushed leaves of the aerial parts of Echinacea, for example, in a suitable hydroalcoholic solution with an alcohol concentration of from 40 to 55%, for example, about 40%, about 45% , about 50%, about 55% (eg, but not limited to, ethanol:water 45:55 v/v).

In one exemplary embodiment, without intending to limit the present invention in any way, the Echinacea extract is prepared in a ratio of 1:8 to 1:10 between

- 4 044994

Echinacea and 45% ethanol, at a suitable extraction temperature of 40 to 60°C.

Typically, extraction can continue for 6 to 8 hours, then the spent residue is separated from the solution by decanting and subsequent filtration (for example, on a paper filter). In one embodiment, the resulting hydroalcoholic fraction is concentrated and dried by lyophilization, in accordance with techniques known to one skilled in the art.

In one embodiment of the present invention, the term extract therefore corresponds to the fraction soluble in an aqueous-alcoholic solution, as described in the example above.

In the examples depicted herein, the Echinacea extract, designated in the figures and tables as Echinacea 13C1608, is the hydroalcoholic soluble fraction obtained from the aerial parts of the plant (leaves, flowers), prepared as above.

What has been stated above with respect to extract, meaning the hydroalcoholic solution-soluble fraction from Echinacea, applies mutatis mutandis to the hydroalcoholic solution-soluble fraction from Centella according to the invention.

In one embodiment of the invention, the above components are in lyophilized form and are redissolved, with the addition of NaCl or other suitable salt, in a suitable concentration, in water; the resulting mixture is heated to a temperature in the range from 30 to 70°C, preferably from 40 to 50°C, for a certain period of time ranging from 4 to 8 hours. The mixture thus treated is decanted and/or filtered as already as above, and the supernatant or filtrate (depending on whether decantation or filtration was performed) is then lyophilized and solubilized, optionally immediately before use, in a pharmaceutically acceptable carrier.

As an example, individual extracts (also meaning one or more extract fractions, as mentioned above) from Centella and Echinacea, as described above, are mixed in a ratio of 7:3 to 9.5:0.5 parts by weight of said extracts in lyophilized form.

In one preferred embodiment, a ratio of about 8:2 or 9:1 is used, for example, therefore adding 9 parts by weight of lyophilized extract of Centella asiatica and 1 part by weight of lyophilized extract of Echinacea, NaCl (as needed to obtain 0.9% vol. /vol. solution), and everything is resuspended in water and processed as above.

The mixture, which is incubated at a temperature ranging from 30 to 70°C, preferably from 40 to 50°C, for a period of time ranging from 4 to 8 hours, can therefore be a mixture containing from 1 to 3% by weight of the extract, lyophilized as described above, from Centella asiatica, from 0.001 to 0.5% by weight of the extract, lyophilized as described above, from Echinacea purpurea, from 0.5 to 9% NaCl, water (preferably desalted or deionized) to reaching the final volume of 100 ml.

Therefore, after filtration and lyophilization, an extract, or more precisely a co-extract, is obtained, which can be mixed with one or more suitable pharmaceutically acceptable carriers to prepare the composition of the present invention.

This joint extract has the form of a powder, highly soluble in water.

In one embodiment of the present invention, a Centella asiatica extract in lyophilized form obtained by extraction in a 70% aqueous-alcoholic solution as described above, and a Centella asiatica extract in lyophilized form obtained by extraction in a 45% aqueous-alcoholic solution as described above , are mixed in a Centella.Echinacea ratio of about 9:1 and suspended in water in which NaCl is dissolved at a concentration ranging from 0.8 to 4%, preferably 1.5 to 2.5%, e.g. %, 1.6%, 1.7%, 1.8% 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4% or 2.5% .

The said mixture is then incubated at a temperature of 30 to 70°C, preferably 40 to 50°C, for a specified period of 4 to 8 hours and then filtered to remove suspended particles (undissolved particles), and the filtrate thus obtained , is lyophilized to obtain a co-extract in powder form, readily soluble in water.

Said co-extract is then dissolved in one or more pharmaceutically acceptable carriers to prepare the parenteral composition of the present invention.

An example of such a co-extract is that which is designated herein and in the figures as series 17H0246.

The term therapeutically active agent or therapeutic active component means that component or set of components that is responsible for improving the pathology or condition being treated.

According to another embodiment, in the parenteral composition of the invention, the therapeutically active principle is contained in an extract (or one or more fractions of an extract) from Centella asiatica, as defined above, in combination with an extract (or one or more fractions of an extract) from Echinacea purpurea as defined above.

The composition of the invention can also be defined based on the content of certain classes of substances; Below are the mass concentrations of some substances relative to the mass of the composition in lyophilized form.

In one preferred embodiment, the parenteral composition of the invention, as defined above, is also characterized in that said composition, in lyophilized form, comprises:

In addition, the parenteral composition of the invention can also be characterized by the fact that it contains, in lyophilized form: total phenols: from 0.08 to 2% w/w, preferably from 0.1 to 0.7%.

Again, the parenteral composition of the invention can also be characterized by what it contains, in lyophilized form: polysaccharides with a molecular weight MW>20,000 Dalton: 1 to 2% w/w, preferably 1.3 to 1.8% .

Finally, the parenteral composition of the invention can also be characterized by the fact that it includes, in lyophilized form: total tannins: from 0.002 to 1% w/w.

Therefore, the object of the invention is a parenteral composition comprising, in lyophilized form, from 0.08 to 2% wt./wt. Total phenols, from 0.002 to 1% w/w. total tannins, 1 to 2% w/w. polysaccharides with a molecular weight higher than 20,000 Da, and at least one pharmaceutically acceptable carrier, said phenols, tannins; polysaccharides; obtained from Centella asiatica and Echinacea purpurea.

For the purposes of this description, derived from means directly obtained, therefore, extracts (or one or more fractions) from or contained in extracts (or one or more fractions) from.

As used herein, a pharmaceutically acceptable carrier means a carrier that is commonly used in the pharmaceutical industry for the preparation of injection solutions.

In any embodiment of the invention, the extracts used may be the above-mentioned extracts or processings of the same (eg, joint extracts as described above).

In one embodiment, the composition of the invention may consist of an extract (or one or more extract fractions, such as any of those described herein) from Centella asiatica, an extract (or one or more extract fractions, such as, for example, any of those described here) from Echinacea purpurea, and at least one pharmaceutically acceptable carrier, the above extracts being in accordance with any of the embodiments described above. In the formulation of the composition of the invention, the therapeutically active principle, as defined herein, may be provided in lyophilized form, with a separately provided carrier, for resuspension before use, or may be provided already dissolved in a suitable carrier.

All uses reported herein also apply to the embodiment defined by the classes of substances defined above.

In formulating the composition in any of the embodiments according to the present description, one skilled in the art may conveniently select carriers and/or auxiliaries from among those generally known in the art.

As used herein and in the practice of the present invention, the term pharmaceutically acceptable carriers is understood in a broad sense and includes solvents, emulsifiers, buffers, pH adjusters, antioxidants, preservatives, osmotic agents.

In addition to water, for injectable preparations, buffers selected, for example, from citric, phosphoric and acetic acid and their sodium salts can be used; pH adjusters such as hydrochloric acid and sodium hydroxide, isotonic agents such as sodium chloride, sugars such as, for example, glucose, mannitol, dextrose; and, in some cases, antioxidants, which may be selected, for example, from sodium bisulfite, sodium metabisulfite or sodium salts of ethylenediaminetetraacetic acid.

You can resort to the use of preservatives such as benzalkonium chloride, benzethonium chloride, methyl p-hydroxybenzoate, benzyl alcohol.

Emulsifiers can be used, such as, for example, lecithins or ethoxylated fatty acid derivatives.

For purposes of illustration, and not intended to be limiting of the invention, in implementing the composition according to the present description, solvents can be selected from one or more of: water, polyvinylpyrrolidone, buffered and unbuffered isotonic solutions; emulsifiers can be selected from lecithins, ethoxylated fatty acid derivatives; buffering agents can be selected from citric acid and its sodium salts, phosphoric acid and its sodium salts, acetic acid and its sodium salts; pH correctors can be selected from hydrochloric acid and sodium hydroxide; antioxidants can be selected from sodium bisulfite, sodium metabisulfite, ethylenediaminetetraacetic acid and its sodium salts; preservatives may be selected from benzyl alcohol, methyl p-6044994 hydroxybenzoate, benzalkonium chloride, benzethonium chloride, and osmotic agents may be selected from sodium chloride, sugars, mannitol, dextrose, glucose.

Methods for preparing these sterile preparations are those typically used in the art. After preparing a solution of extracts and excipients by using temperature to accelerate solubilization, the solution thus obtained can be sterilized by sterilizing filtration or resorting to steam sterilization. In some cases, gamma rays may be used for terminal sterilization.

As described herein and as demonstrated by the examples, a parenteral composition in accordance with any of the embodiments set forth in the present description and claims is suitable for use as a drug.

In particular, a parenteral composition in accordance with any of the embodiments set forth in the present description and claims is particularly suitable for use in the treatment of disorders that affect articular cartilage in mammals.

In an illustrative and non-limiting form, mentioned disorders affecting articular cartilage are selected from arthritis, osteoarthritis, joint inflammation, cartilage damage caused by trauma, spinal disc degeneration, rheumatoid arthritis. The parenteral composition according to any of the embodiments set forth in the present specification and claims may be used to treat mammals in general, and therefore would be a veterinary composition, or may be used to treat human patients, and therefore would be will constitute a pharmaceutical composition or medical device in accordance with applicable law. It is also an object of the present invention to provide a therapeutic method for the treatment of disorders affecting articular cartilage as described above, wherein the composition is administered to the patient by infiltration/injection into the area of interest, and thereby infiltration/injection into the joint. The patient who, as already defined above, exhibits disorders affecting articular cartilage may be a human or even an animal, such as a mammal.

Any embodiment described above relates to the therapeutic treatment of the present invention.

It is also an object of the present invention to provide a method for preparing a composition according to any of the described embodiments, wherein the therapeutically active substances as defined above, in dry form, preferably in lyophilized form, are mixed and subjected to a subsequent extraction step with NaCl and water, then decanted or filtered, lyophilize and resuspend in the vehicle as defined above.

Any method generally known to one skilled in the art can be used to prepare the composition according to the invention. According to an embodiment, which is to be understood as merely exemplary and non-limiting, the method of preparing the composition of the invention can be carried out as follows.

1. Extracts obtained from aqueous-alcoholic extraction, lyophilized, are dissolved in water with 0.8 to 4% NaCl, heating the mixture at a temperature of 30 to 80 ° C, preferably from 30 to 70 ° C, from 40 to 50 ° C for a period of time ranging from 4 to 8 hours to accelerate the dissolution of the extract,

2. The mixtures obtained in the previous step, after cooling, are filtered to eliminate any undissolved extract particles and then dried, preferably by lyophilization, and resuspended in a suitable pharmaceutically acceptable carrier.

3. At this stage, if necessary, oily substances and emulsifiers can be added if surface-activated systems are to be obtained.

4. To the mixture of extracts (or fractions of extracts, such as any of those described herein) are added other excipients, soluble in water, such as buffers, isotonic agents.

5. Measure and properly correct, if necessary, the pH of the prepared solution.

6. The solution thus obtained can be sterilized by sterilizing filtration or moist heat (steam) in an autoclave.

Therefore, it is also an object of the invention to provide a method for preparing a parenteral composition as defined above and as defined in the claims, in which

a) the leaves of Centella asiatica are subjected to one or more extraction steps with hydroalcoholic mixtures, and the extract (or fraction(s)/extract, such as, for example, any of those described herein) thus obtained is subjected to lyophilization;

b) the aerial parts of Echinacea purpurea are subjected to one or more extraction steps with water-alcohol mixtures, and the extract (or fraction(s)/extract, such as, for example, any of those described herein) thus obtained is lyophilized;

c) the lyophilized extracts obtained in a) and b), in any order, are mixed in an extract ratio (or fraction(s)/extract, such as, for example, any of those described herein).

- 7 044994 herein) from Centella asiatica: extract (or fraction(s)/extract, such as, for example, any of those described herein) from Echinacea purpurea, ranging from 7:3 to 0.5:9.5;

d) the extracts (or extract fractions, such as, for example, any of those described herein) thus mixed are resuspended in water with the addition of 0.8 to 4% sodium chloride;

e) the mixture thus obtained is brought to a temperature in the range of 30 to 70° C. for a period of 4 to 8 hours and then subjected to filtration or decantation;

e) the filtrate or supernatant obtained in e) is lyophilized;

g) the lyophilisate obtained in step f) is resuspended in one or more pharmaceutically acceptable carriers.

According to the invention, said extract in step a) may be a portion of the extract that is soluble in an aqueous-alcoholic solution where the alcohol concentration is between 65% and 75% v/v, a fraction of the extract that is insoluble in EtOH at a concentration of 90 to 96% v/v and soluble in EtOH at a concentration of 10 to 20% v/v, or a mixture thereof, as described above.

In addition, said extract in step b) may be a fraction soluble in an aqueous-alcoholic solution, where the concentration of said alcohol is from 40 to 50% v/v.

It is also an object of the invention to provide a composition prepared in the manner described above, for all uses and in all forms provided herein for the parenteral composition of the invention.

The individual extracts (or fractions, such as, for example, any of those described herein) in steps a) and b) can be prepared in accordance with any of the embodiments exemplified herein and any other suitable conventional method, known to one skilled in the art.

The method as described above makes it possible to prepare a parenteral composition containing from 0.08 to 2.0% wt./wt. total phenols, from 0.002 to 1 wt./wt. total tannins, from 1% to 2% w/w. polysaccharides with a molecular weight above 20,000 Da; and at least one pharmaceutically acceptable carrier, wherein said phenols, tannins, polysaccharides are derived from Centella asiatica and Echinacea purpurea, according to the present description.

Illustrative examples of some embodiments of the present invention follow.

The examples mentioned are in no way to be construed as limiting the embodiments of the present invention, but are intended only to illustrate possible compositions practiced according to the directions given herein and having the therapeutic features described.

Examples of compositions.

Composition 1.

Lyophilized Centella-Echinacea joint extract (9:1) - NaCl* 10 mg.

Water for injectable preparations (f.i.p.) in a quantity sufficient to obtain (q.s.) 1 ml.

Composition 2.

Lyophilized Centella-Echinacea joint extract (8:2) - NaCl* 10 mg.

Water f.i.p. q.s. up to 1 ml

Composition 3.

Lyophilized Centella-Echinacea co-extract (9:1) -NaCl* 30 mg.

Tetrasodium salt of ethylenediaminetetraacetic acid 1 mg.

Sodium hydroxide 0.1 mg.

Benzalkonium chloride 0.2 mg.

Composition 4.

Lyophilized Centella-Echinacea co-extract (8:2) -NaCl* 30 mg.

Tetrasodium salt of ethylenediaminetetraacetic acid 1 mg.

Sodium hydroxide 0.1 mg.

Benzalkonium chloride 0.2 mg.

Composition 5.

Lyophilized Centella-Echinacea co-extract (9:1) - NaCl* 50 mg.

Acetic acid 1 mg.

Sodium acetate 2 mg.

Water f.i.p. q.s. up to 1 ml.

Composition 6.

Lyophilized Centella-Echinacea joint extract (8:2) - NaCl* 50 mg.

Acetic acid 1 mg.

Sodium acetate 2 mg.

Water f.i.p. q.s. up to 1 ml.

Composition 7.

Lyophilized Centella-Echinacea co-extract (9:1) - NaCl* 7 mg.

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Monobasic sodium phosphate 1 mg.

Dibasic sodium phosphate 2 mg.

Water f.i.p. q.s. up to 1 ml.

Composition 8.

Lyophilized Centella-Echinacea co-extract (8:2) - NaCl* 7 mg.

Monobasic sodium phosphate 1 mg.

Dibasic sodium phosphate 2 mg.

Water f.i.p. q.s. up to 1 ml.

Composition 9.

Lyophilized Centella-Echinacea co-extract (9:1) - NaCl* 70 mg.

Benzalkonium chloride 1 mg.

Water f.i.p. q.s. up to 1 ml.

Composition 10.

Lyophilized Centella-Echinacea joint extract (8:2) - NaCl* 70 mg.

Benzalkonium chloride 1 mg.

Water f.i.p. q.s. up to 1 ml.

*NaCl q.s. for preparing an isotonic solution.

Composition 11.

Lyophilized Centella extract 1 mg.

Lyophilized Echinacea extract 0.1 mg.

NaCl 0.009 mg.

Water f.i.p. q.s. up to 1 ml.

Composition 12.

Lyophilized Centella extract 25 mg.

Lyophilized Echinacea extract 3 mg.

NaCl 0.018 mg.

Water f.i.p. q.s. up to 1 ml.

Composition 13.

Lyophilized Centella extract 50 mg.

Lyophilized Echinacea extract 7 mg NaCl 0.009 mg.

Water f.i.p. q.s. up to 1 ml.

The joint extract according to the examples shown above is prepared from the extract of Centella asiatica in lyophilized form obtained by extraction in a 70% aqueous-alcoholic solution as described above, and the extract of Centella asiatica in lyophilized form obtained by extraction in a 45% aqueous solution. alcohol solution as described above, which are mixed in a Centella: Echinacea ratio of about 8:2 or 9:1, and suspended in water, NaCl being dissolved in a concentration of from 0.8 to 4%, preferably from 1.5 up to 2.5%, for example, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2, 3%, 2.4% or 2.5%. Said mixture is then incubated at a temperature ranging from 30 to 70°C, preferably from 40 to 50°C, for a period of 4 to 8 hours and then filtered to remove suspended particles (undissolved particles), and so The resulting filtrate is lyophilized to obtain a co-extract in the form of a powder, easily soluble in water.

Said co-extract is then dissolved in one or more pharmaceutically acceptable carriers to prepare the parenteral composition of the present invention.

The Centella extract according to the examples depicted above may be an extract obtained by hydroalcoholic extraction. The extract may, for example, be prepared from crushed Centella leaves that are steeped in alcohol (65% to 75%) at a level of about 65%, about 70%, about 75% (non-limiting example: ethanol:water 70:30 vol. /about.). In one exemplary embodiment, Centella extract can be prepared with a 1:8 ratio of Centella to 70% alcohol, at a suitable extraction temperature of 40 to 60°C. Typically, extraction can continue for 6 to 8 hours, then the spent residue is separated from the solution by decantation and subsequent filtration. The filtrate is then lyophilized and ready for use.

The extract from Echinacea purpurea according to the examples shown above may be an extract obtained by hydroalcoholic extraction. The extract may, for example, be prepared by aqueous-alcoholic extraction from ground aerial parts of Echinacea, for example in a suitable hydroalcoholic solution with an alcohol concentration of from 40 to 55%, for example about 40%, about 45%, about 50 %, about 55% (for example, without limitation, ethanol: water 45:55 v/v).

In one exemplary embodiment, it can be prepared in a 1:10 ratio of Echinacea and 45% ethanol, at a suitable extraction temperature of 40 to 60°C. Typically, extraction can continue for 6 to 8 hours, then the spent residue is separated from the solution by decantation and subsequent filtration. Then the filtrate is lyophilized and ready for use.

In each of the above compositions, the soluble fractions in the aqueous-alcoholic solution provided herein, for example, in the ratios mentioned in the examples, description and claims, can be advantageously used as extracts.

The following data was obtained by the authors of the present invention and demonstrates the effectiveness of various embodiments of the composition of the present invention.

Experimental analyzes performed.

Animals.

Male Sprague-Dawley rats weighing approximately 200 grams were provided from Harlan Company, Italy. Four animals per cage were housed in cages of the following dimensions: 26x41 cm, at the Center for Stabulation of Laboratory Animals (Ce.S.A.L. - Centro per la Stabulazione Animali da Laboratorio) at the University of Florence under a 12-hour light/dark cycle, with water and food. optional. The rats were fed a standard diet and kept at a temperature of 23±1°C. The experimental protocol was approved by the local Committee for the control of experimenting on laboratory animals. All experiments were carried out in accordance with the European Communities Council Directives of 22 September 2010 (directive 2010/63/EU) on the treatment of animals used for experimental purposes and

The National Institute of Health Guide for the Care and Use of Laboratory Animals, adopting the guidelines of the International Association for the Study of Pain and in accordance with the guidelines ARRIVE. All necessary measures have been taken to minimize suffering and reduce the number of animals used. Moniodoacetate-Induced Model of Osteoarthritis Pain Joint damage was induced by individual intra-articular injection, into the paw of a pre-anesthetized rat, of 2 mg sodium monoacetate (MIA) (volume: 25 μl) as described by Guingamp et al., Arthritis Rheum., 40:16701679, 1997. The use of the mentioned dosage is additionally based on experiments carried out in the laboratory of the inventors, from which it became known that doses of 0.5 and 1 mg cause hyperplasia, which disappears within 2 weeks, while doses of 4 mg cause an inflammatory condition that lasts longer than the first week. As reported by Janusz et al., Osteoarthritis Cartilage, 9: 751-760, 2002 and Fernihoughetal., Pain 112:83-93, 2004, a dose of 2 mg causes death of chondrocytes and severe hyperalgesia and allodynia. MIA is inserted into the joint after the paw is flexed at an angle of 90°, using a 26Gx3/8 needle. Control rats received an intra-articular injection of saline (25 μl).

Preparation and administration of substances.

The experiments described below were performed by injecting µL of control (saline) into the joint;

extract of lyophilized Echinacea purpurea, a fraction soluble in an aqueous-alcoholic solution with a concentration of 40 to 50%, designated in the figures as 13C1608: 2-10-20 mg/ml in PBS (phosphate-buffered saline solution);

extract of lyophilized Centella asiatica, the fraction soluble in an aqueous-alcoholic solution with a concentration of 65 to 75%, designated in the figures as 14G1862: 0.2-1-2 mg/ml in PBS;

bovine synovial fluid: as is;

Hyalgan (low molecular weight sodium salt of hyaluronic acid (MW-500 to 730 kDa)): as is;

Jonexa (a mixture consisting of hylastan gel, that is, hyaluronic acid cross-linked with divinyl sulfone, and liquid sodium hyaluronate in a gel-liquid ratio of 80:20): as is;

extract of Centella asiatica, the fraction soluble in aqueous-alcoholic solution with a concentration of 65 to 75%, designated in the figures as 14G1862: 2 mg/ml in PBS extract of lyophilized Centella asiatica, fraction soluble in aqueous-alcoholic solution, designated in the figures as ABO-AR-2016391: 2 mg/ml in PBS; extract of lyophilized Centella asiatica, the fraction soluble in aqueous alcohol solution corresponding to the permeate fraction of ABO-AR-2016-391 after filtration, designated in the figures as ABO-AR-2016-392: 2 mg/ml in PBS; extract of lyophilized Centella asiatica, fraction soluble in aqueous-alcoholic solution corresponding to the retentate fraction ABOAR-2016-391 after filtration, designated in the figures as ABO-AR-2016-393: 2 mg/ml in PBS;

triamcinolone acetonide: 100 μg/20 ml MIX (joint extract) Centella + Echinacea series 17H0246, as described above: 11 mg/ml in water f.p.i.

Extracts, also referred to as specific fractions obtained by extraction method, from Echinacea and Centella were obtained by hydroalcoholic extraction as described above in the detailed part of the present invention and in the examples depicted below.

Injected material: 20 μl of each sample to be analyzed was injected into the rat tibiotarsal joint (using a 26Gx3/8 needle) on day 7 from injury induction by MIA.

Behavioral assessments using paw pressure tests, Von Frey, disability,

- 10 044994 balance beam, Rotarod and ANIMEX, and histological analyzes of articular and periarticular tissue were performed on days 14, 30 and 60 from the beginning of the experiment.

Solubilization and introduction of substances.

The mixture of plant extracts was solubilized according to the following protocol: 5.5 g of the lyophilized mixture was added to Becker containing 450 g in water and left under stirring for 15 minutes; After this, the liquid mass was adjusted to 500 g. For each injection, 30 g of the solution was collected and filtered in series with filters with pore sizes of 0.45 μm and 0.22 μm. During each filtration, the first 3 to 4 g of solution was discarded. 20 μl of the mixture was injected into the tibiotarsal joint of the rat (using a 26Gx3/8 needle) after paw flexion at 90°, on the 8th day from induction of articular damage by MIA. Other substances were also administered by injection of 20 μl of solution (Hyalgan 10 mg/ml; triamcinolone acetonide 100 μg/20 ml).

Paw pressure test.

An analgesiometer (Ugo Basile, Varese, Italy) was used. Pressure increasing at a constant rate (32 g/s) was applied to the animal's hind paw using a blunt conical element. The rat's nociceptive threshold has been expressed as the force to which the animal responds by withdrawing its paw or making sounds (Leighton et al., 1988).

Von Frey test.

The animals were placed in a plexiglass cage measuring 20x20x15 cm, the floor of which was formed by a metal mesh, and were allowed to get used to the environment for 15 minutes. An electronic Von Frey apparatus (Ugo Basile, Italy), with a built-in flexible metal probe, with which the flat surface of the rat's front paw was stimulated, was placed under a metal mesh. The applied pressure was increased gradually to a maximum of 50 g, with an accuracy of 0.2 g. The paw sensitivity threshold was defined as the minimum pressure required to obtain a clear and immediate paw withdrawal reflex. Movement-related voluntary movements were not considered an answer. The stimulus was applied five times to each forepaw, and each value reported as threshold was the average of 10 consecutive measurements (Sakurai et al. Pain 147: 165-174, 2009).

Disability test.

The test is based on assessing the pressure exerted by a rat under normal posture conditions on each of the hind legs (Hay et al. Neurosci. 78: 843-850, 1997). The rats were placed in a small Plexiglas box with their hind paws resting on two sensors that recorded the mass that the rat distributed to each of its two paws. If neuropathy or inflammation occurs on one paw, the rat will rest less weight on said paw compared to the other. Balance beam test A 1 meter long wooden beam with a 1 cm square cross-section was placed at a height of about 40 cm. A black plastic box measuring 15 cm x 15 cm x 8 cm was attached to the end of the beam. The animal was placed on a beam and its locomotor abilities were assessed, assigning a higher score, from 0 to 4, depending on the severity of the motor deficits (0, correct gait; 1, clings with four paws; 2, glides with one paw; 3, glides with two paws; 4, falls in <60 s) (Song et al., 2006).

Animex.

The device allows you to record the spontaneous activity of animals through the use of electromagnetic fields and resonant circuits. A cage containing animals is located above the instrument; animals, moving from one side of the cage to the other, modify the magnetic fields created by the device on its surface. Variations are recorded using a numerical counter.

Histology.

Tissue samples were fixed in 4% formalin in phosphate buffer for 24 hours.

Subsequently, the samples were decalcified for 28 days in a suitable decalcifying solution (1.6 M formic acid, 0.76 M sodium formate). At the end of decalcification, the samples were dehydrated by passages in alcohol with increasing titer, and then embedded in paraffin. From samples thus processed, 7-μm-thick sections were obtained and stained with hematoxylineosin.

The samples were observed under an optical microscope in transmitted light to assess the morphological parameters of the object of analysis:

inflammatory infiltrate, synovial hyperplasia, fibrin deposition in the joint space, synovial vascularization, cartilage erosion, bone erosion, joint space width.

Each morphological parameter was assessed with a specially assigned quantitative indicator (0: absent, 1: little, 2: moderate, 3: severe). The evaluation was performed by three separate operators who were unaware of the origin of the slides. A fourth operator collected the data and obtained the final averages. At least three slides were evaluated for each joint, and at least 6 animals were evaluated for each group. Results are expressed as mean ± standard error.

Statistical analysis.

All experimental results are expressed as mean±SEM. One-way analysis of variance (ANOVA) was performed, followed by a Bonferroni test to test significance between the two means. Analysis of variance and Bonferroni test were performed using Origin Pro 9.1 statistical software. Statistically significant values of P < 0.05 or P < 0.01 were considered.

Results.

Evaluation of the effectiveness of samples of the joint extract of Centella + Echinacea (series 17H0246), and Hyalgan, Jonexa, triamcinolone acetonide.

The purpose of the present study was to evaluate the antihyperalgesic and antiallodynic efficacy of a mixture consisting of the above-mentioned extracts as a co-extract as described herein (batch 17H0246) in the same pain model. In addition, the effectiveness of the above mixture was compared with reference substances that are commonly used intra-articularly in clinical practice for the treatment of joint pain, namely the hyaluronic acid preparations Jonexa and Hyalgan and the corticosteroid triamcinolone acetonide. Histological assessments followed behavioral assessments performed on days 14, 30 and 60.

Treatment groups:

carrier + carrier,

MIA + carrier

MIA + mixture Centella/Echinacea series 17H0246 (20 µl),

MIA + Hyalgan (20 µl),

MIA + Jonexa (20 µl),

MIA + triamcinolone acetonide (100 µg/20 µl).

The osteoarthritis (OA) model used in this study was made by single i.a. (intra-articular) injection of MIA (2 mg/25 µl). On days +8 from the moment of damage by the algogenic agent, when OA pathology had already begun to manifest itself, 20 μl of each sample (MIX (joint extract) series 17H0246; Hyalgan, 10 mg/ml; Jonexa; triamcinolone acetonide) were injected intra-articularly. Behavioral assessments were performed + 14 + 30 and + 60 days from the start of the experiment, that is, from the moment of MIA injection, as well as histological assessments on the rat joint. The tests carried out allowed us to evaluate the antihyperalgesic effect (paw pressure test) of the samples, the effect they have on the posture of the animals and on the weight burdening the contralateral paw relative to the ipsilateral paw (disability test), coordination and motor resources (balance test on a beam) and spontaneous activity (Animex test).

In table 1 presents data on the antihyperalgesic effect of substances measured through a painful mechanical stimulus + 14 + 30 and + 60 days after induction of damage. Intra-articular injection of MIA reduced the algic threshold of the rat ipsilateral paw in a statistically significant manner relative to the control group (vehicle + vehicle) until day 60; the peak pain response was observed 2 weeks after injury (Table 1). A single intra-articular injection of a mixture of plant extracts (series 17N0246) appears to be effective in reducing joint pain starting on the 7th day after its administration (14th day from the MIA injury). This effect is enhanced, culminating on day 30 with the reversal of algic states, as the values recorded on the ipsilateral paw are comparable to those recorded in the control group. The effect persists even on the 60th day. Hyalgan appears to be active on the 14th and 30th days, although it shows lower potency than the herbal mixture and does not culminate in a change in disease state. Jonexa is active only on the 30th day, while triamcinolone acetonide significantly reduces the pain response in the joints at any time, however, it exhibits less antihyperalgesic effectiveness compared to the plant mixture (Table 1).

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Table 1

Evaluation of analgesic effects due to intra-articular injection of samples

Table 1. Paw pressure test Weight (g) Day 14 Day 30 Day 60 Treatment ipsilateral contralateral ipsilateral contralateral ipsilateral contralateral carrier + carrier 66.7 ± 1.7 68.3 ± 1.7 67.3 ± 0.9 66.4 ± 1.5 65.3 ±0.8 66.2 ±0.8 MIA + carrier 41.7 ± 1.7** 61.7 ±1.7 49.3 ± 1.7** 69.2 ± 0.8 55.5 ± 1.4* 66.7 ± 1.7 MIA + MIX (joint extract) Centella + Echinacea series 17Н0246 57.2 ± 1.1 ^LL 65.6 ± 1.0 65.0 ± 1.5 ^LL 70.0 ± 1.4 65.0 ± 1.4 ^LL 67.5 ± 0.8 MIA + Hyalgan 52.6 ± 0.9 ^ll 62.8 ± 1.7 58.3 ± 0.8 ^l 68.4 ± 1.4 59.2 ± 1.6 63.7 ± 1.6 MIA + Jonexa 46.7 ± 1.5 66.7 ± 0.8 56.1 ± 0.5 ^l 66.7 ± 0.8 58.4 ±2.0 66.8 ±0.9 MIA+ triamcinolone acetonide 51.9 ± 1.3 ^LL 64.5 ± 0.9 57.4 ± 0.4 ^l 68.4 ±1.6 63.0 ± 1.5 ^L 64.9 ±2.1

Monoarthritis was induced by injection of 2 mg/25 μl monoiodoacetate (MIA, Sigma-Aldrich) into the tibiotarsal joint. On day 8 after MIA administration, 20 μl of a mixture of Centella and Echinacea extracts, Hyalgan, Jonexa (as is) and triamcinolone acetonide (100 μg) was injected into the tibiotarsal joint of a rat. **P<0.01 vs vehicle + vehicle; ^L P < 0.05 and ^ll P < 0.01 vs. MIA + vehicle The disability test shows greater effectiveness of treatment with the mixture, compared with other substances, in reducing the postural change caused by the administration of MIA, which leads to the animals carrying more weight degrees on the contralateral paw relative to the ipsilateral paw (Table 2). MIX (combined extract) Centella + Echinacea batch 17H0246 actually returns the above-mentioned change to normal in all time periods considered (14, 30 and 60 days), reaching control values two months from the start of the experiment. Triamcinolone acetonide reduced, also in a statistically significant manner, the changes caused by MIA at all time periods examined, although with less effectiveness compared to MIX (co-extract) Centella + Echinacea lot 17H0246. As shown in the paw pressure test, Hyalgan is active on days 14 and 30, then loses effectiveness, whereas Jonexa only reaches statistical significance on day 30 (Table 2).

table 2

Table 2. Disability Test D Mass (g) Difference in points (contralateral - ipsilateral paw) Treatments Day 14 Day 30 Day 60 carrier + carrier -0.8 ± 1.2 6.7 ± 4.9 3.6 + 7.8 MIA + carrier 73.0+13.0** 49.8 + 2.8** 36.2 + 3.2** MIA + MIX (joint extract) Centella + Echinacea batch 17Н0246 30.4+ 1.9 ^LL 16.7 +6.4 ^LL 3.4±2.1 ^ll. MIA + Hyalgan 42.8 ± 7.5 ^LL 23.8 ± 2.5 ^LL 28.9 + 5.8

- 13 044994

MIA + Jonexa 56.9 ± 18.1 32.0 ±6.3 32.4 ±6.1 MIA+ triamcinolone acetonide 45.7 ± 3.9 ^LL 25.4 ± 4.3 ^LL 20.9 ± 5.4 ^L

Monoarthritis was induced by injecting 2 mg/25 μl monoiodoacetate (MIA, Sigma-Aldrich) into the tibiotarsal joint. On day 8 after MIA injection, 20 μl of a mixture of Centella+Echinacea, Hyalgan, Jonexa and triamcinolone acetonide (100 μg) was injected into the tibiotarsal joint of the rat. **P<0.01 vs vehicle + vehicle; ^l P < 0.05 and ^ll P < 0.01 versus MIA+vehicle.

The balance beam test (Table 3) was performed to assess the animal's motor abilities when placed on a wooden beam raised above the ground. The score assigned to each group ranges from 0 to 4 (0 for correct gait, 1 if grasping with four paws, 2 if sliding with one paw, 3 if sliding with two paws, 4 if falling in <60 s). Animals treated with MI A (MIA + vehicle) have great difficulty placing the ipsilateral paw on the beam and for this reason slip when walking on it. Repeating this experiment at different times from the administration of MIA shows how the effect of the said drug causes motor impairment, which is maximum in the first 14 days, later the effect tends to spontaneously decrease over time. A single administration of MIX (co-extract) Centella + Echinacea lot 17H0246 results in the score assigned to these animals being halved relative to the MIA + vehicle group in all cases considered, even if statistical significance is only achieved on day 14, also as for Hyalgan and triamcinolone acetonide. Jonexa appears to be ineffective (Table 3).

Table 3

Table 3. Balance test on beam Limit: normal (0) to pathological (4) values Treatments Day 14 Day 30 Day 60 carrier + carrier 0.3 ± 0.2 0.2 ± 0.3 0.2 ± 0.3 MIA + carrier 3.2 ± 0.4** 1.5 ±0.2* 1.3 ±0.2* MIA + MIX (joint extract) CentellaL Echinacea batch 17Н0246 1.3 ± 0.2 ^LL 0.5 ± 0.2 0.7 ±0.2 MIA + Hyalgan 1.5 ± 0.3 ^ll 1.0 ±0.1 1.0 ±0.2 MIA + Jonexa 2.3 ± 0.4 1.0 ±0.2 1.0 ±0.2 MIA+ triamcinolone acetonide 1.8±0.2 ^l 1.3 ± 0.2 0.8 ± 0.2

Monoarthritis was induced by injecting 2 mg/25 μl monoiodoacetate (MIA, Sigma-Aldrich) into the tibiotarsal joint. On day 8 after MIA injection, 20 μl of a mixture of Centella+Echinacea, Hyalgan, Jonexa and triamcinolone acetonide (100 μg) was injected into the tibiotarsal joint of the rat.

**P<0.01 vs vehicle+vehicle; lp<0.05 and lp<0.01 versus MIA + vehicle Finally, the animal’s spontaneous mobility was assessed using the Animex test (Table 4). In this case, the number of spontaneous movements of a rat once placed in a plexiglass cage was recorded. The results obtained are similar to those recorded in previous tests, the decrease in spontaneous movements of the animal that is recorded in the MIA + vehicle group is significantly reduced

- 14 044994 by treating with MIX (combined extract) Centella + Echinacea lot 17Н0246 in all time periods considered. Although this also occurs with triamcinolone acetonide, its effectiveness is lower than that of the herbal mixture. Hyalgan reached statistical significance on days 14 and 30, while Jonexa only reached statistical significance on day 30 (Table 4). Briefly, intra-articular treatment with a mixture of plant extracts (MIX (combined extracts) Centella + Echinacea batch 17H0246) was more effective in reducing joint pain after injection of MIA into an experimental animal, compared with injection of Hyalgan, Jonexa and triamcinolone acetonide. In addition to greater effectiveness, the antihyperalgesic profile of this treatment lengthens over time until the algic pathology is completely changed.

Table 4

Table 4. Animex test Number of movements Treatments Day 14 Day 30 Day 60 carrier + carrier 736.4 ± 45.9 698.3 ±35.9 720.6 ± 52.3 MIA + carrier 298.7 ± 60.4** 455.0 ± 26.7** 510.7 ± 42.0* MIA + MIX (JOINT EXTRACT) Centella+Echinacea batch 17Н0246 547.3 ± 50.8 ^LL 664.3 ± 39.8 ^LL 704.9 ± 53.1 ^l MIA + Hyalgan 517.0 ± 35.6 ^LL 549.6 ± 45.1 ^l 599.9 ± 54.3 MIA + Jonexa 358.1 ± 42.8 568.1 ± 38.7 ^L 604.3 ± 39.7 MIA + triamcinolone acetonide 487.3 ± 57.Z ^l 583.3 ± 45.2 ^L 688.9 ± 54.2 ^L

Monoarthritis was induced by injecting 2 mg/25 μl monoiodoacetate (MIA, Sigma-Aldrich) into the tibiotarsal joint. On day 8 after MIA injection, 20 μl of a mixture of Centella+Echinacea, Hyalgan, Jonexa and triamcinolone acetonide (100 μg) was injected into the tibiotarsal joint of the rat. **P<0.01 vs vehicle + vehicle; ^L P < 0.05 and ^ll P < 0.01 versus MIA + vehicle.

Histological evaluation of the tibiotarsal joint of animals treated with a combined extract of Centella + Echinacea (lot 17H0246), Hyalgan, Jonexa, triamcinolone acetonide.

MIA: In general, the analyzed animals showed a decrease in the joint space, independent of observation time, and deposition of fibrin filaments in the joint space, increasing 30 and 60 days after treatment. There is a time-dependent trend toward worsening erosion phenomena on the covering cartilage and subchondral bone. The phenomena of synovial hyperplasia and vascularization are detected. Moreover, the presence of an inflammatory infiltrate is clearly observed, showing a decrease after 60 days from inoculation (Figures 18 A-G and 19). MIA+MIX (combined extract) Centella+Echinacea lot 17H0246: treatment with MIX (joint extract) Centella+Echinacea lot 17H0246 changes the analyzed morphological parameters. Regarding the inflammatory infiltrate, the mentioned treatment significantly reduces it after 30 and 60 days. At the synovial level, this reverses the damage caused by MIA treatment. Erosion of covering cartilage and subchondral bone tissue in animals treated with MIX (combined extract) Centella + Echinacea lot 17H0246 disappears after 30 and 60 days. Fibrin deposition in the joint space is absent after 30 and 60 days; likewise, joint space dimensions returned to values consistent with those found in control animals (Figures 18 A-G and 19).

MIA+Jonexa: Jonexa treatment did not significantly improve the analyzed morphological parameters compared to the group of animals treated with MIA alone. A slight improvement in joint space width is clearly observed (Figs. 18 A-G and 19).

MIA+Hyalgan: No significant improvement was noted after treatment with Hyalgan. A slight improvement in joint space width is clearly observed (Figs. 18 A-G and 19).

MIA+triamcinolone acetonide: treatment with triamcinolone acetonide can eliminate the presence of

- 15 044994 inflammatory infiltrate in all analyzed time intervals. A similar result was noted for erosion phenomena affecting articular cartilage and subchondral bone. Obvious effects can be clearly observed on the joint space after 30 and 60 days. More moderate effects can be clearly observed in hyperplastic and vascular phenomena on the synovium and in relation to fibrin deposition in the joint space (Figures 18 A-G and 19).

Evaluation of the effectiveness of extracts of Echinacea (13C1608) and Centella (14G1862) (fractions soluble in aqueous-alcoholic solution) compared with bovine synovial fluid. 20 μl of each substance was injected into the rat tibiotarsal joint with the pathology of osteoarthritis having already begun (8 days after induction or joint damage caused by intra-articular injection of MIA). Behavioral assessments were performed on days 14, 30, and 60 after lesion induction. The effectiveness of Centella (fraction 14G1862) and Echinacea (fraction 13C1608) extracts and the antihyperalgesic effect of bovine synovial fluid were assessed using the paw pressure test (measuring the animal's pain threshold using a painful mechanical stimulus) and the von Frey test (measuring the animal's pain threshold using a painless mechanical stimulus). stimulus). Differences in weight bearing on the animal's hind paws were assessed using a disability test (an animal with unilateral pain tends to place more weight on the contralateral paw compared to the ipsilateral paw).

In table Figure 5 shows the effect of various substances on the pain threshold assessed on the ipsilateral paw (paw pressure test). In the group of animals that were injected with MIA (MIA+vehicle), there is a decrease in the pain threshold compared to the group of control animals (vehicle+vehicle), which is maximum on the 14th day from the induction of damage (43.7 ± 1.7 and 64 ,2±1.5, respectively). This difference in the two experimental groups remains significant even on the 30th and 60th days, although a spontaneous decrease in pain pathology is noted within several days.

A single intra-articular injection of a fraction soluble in a 45% aqueous-alcoholic solution of Echinacea (20 mg/ml) appears to be effective in reducing joint pain starting on the 6th day after its administration (14th day from the injury caused by MIA) . The effect of complete reversal of osteoarthritis pathology is achieved on the 60th day, since the values recorded on the ipsilateral paw in the control group and in the group of animals treated with MIA + Echinacea fraction (20 mg/ml) are comparable. The lower dose of the Echinacea fraction (2 mg/ml) tested is not active, while the 10 mg/ml dose shows a significant effect only on day 14.

The Centella fraction, soluble in a 70% aqueous-alcoholic solution, is effective only at the maximum analyzed dose (2 mg/ml). The antihyperalgesic effect is detectable as early as 14 days from MIA injury and remains significant, reaching full recovery from injury at 30 and 60 days.

A single intra-articular injection of bovine synovial fluid induces an effect of reducing osteoarthritis symptoms, which is statistically significant on the 14th day; subsequent estimates do not reach the mentioned value. In table Figure 6 shows the antiallodynic effect achieved at different times after a single tibiotarsal administration of various products in animals subjected to non-painful mechanical stimulation (von Frey test). Animals treated with MIA exhibited half the load on the ipsilateral paw compared to controls. The allodynic effect caused by MIA injection persists throughout the experiment (60 days), however, a stronger effect appears 14 days after its administration. The Echinacea and Centella fractions show a comparable pattern throughout the experiment. For both drugs, the antiallodynic effect is recorded already on day 14; the mentioned effect gradually increases over several days and ends with a change in pain symptoms on the 60th day after joint damage. Regarding synovial fluid, the data obtained are comparable to those obtained from the paw pressure test.

The disability test shows higher efficacy of Centella (2 mg/ml) and Echinacea (20 mg/ml) fractions compared to synovial fluid injection. The injection of two plant fractions entails a halving of the difference in the load weight on the contralateral paw of the animal compared to the ipsilateral paw (Table 7).

This effect turns out to be statistically significant in all measurements performed (14, 30 and 60 days). Both fractions, once tested at lower doses, were not effective.

Tests to determine locomotor ability, motor coordination abilities (bar balance and Rotarod test, respectively) and spontaneous locomotor activity (Animex test) demonstrated that animals that were damaged by MIA and subsequently treated with Echinacea and Centella fractions retained good locomotor abilities and motor coordination (data not shown).

Briefly, Echinacea (20 mg/ml) and Centella (2 mg/ml) fractions appear to be more active in reducing joint pain after MIA administration in experimental animals compared to synovial fluid injection. In particular, two extracts exhibit an antihyperalgesic profile that remains constant over time following their single intra-articular administration.

Table 5. Paw pressure test Weight (g) Day 14 Day 30 Day 60 Treatments Ipsila teral naya contralate ralnaya ipsilatera lana contrala teral ipsilater alnaya contrala teral carrier + carrier 64.2 ± 1.5 62.1 ±0.9 65.8 ± 1.1 62.1 ± 1.2 63.4 ±0.8 63.7 ± 1.5 MIA + carrier 43.3 ± 1.7** 65.0 ±0.3 51.3 ± 1.2** 62.5 ± 1.4 57.5 ± 1.4* 65.8 ±0.8 MIA + Echinacea extract, fraction 13С1608 2 mg/ml 46.8 ± 1.6 60.5 ± 1.2 50.6 ± 2.5 63.4 ± 0.7 61.4 ±2.5 63.7 ± 1.4 MIA + Echinacea extract, fraction 13С1608 10 mg/ml 52.1 ± 0.4 ^L 64.4 ± 0.9 49.7 ± 0.9 62.9 ± 0.5 59.8 ±2.6 60.7 ±2.8 MIA + Echinacea extract, fraction 20 mg/ml 54.2 ± 2.2 ^LL 63.3 ±0.8 58.3 ±0.8 ^l 61.7 ±0.8 63.3 ± 1.7 65.8 ±0.8 MIA + Centella extract, fraction 14G1862 0.2 mg/ml 44.7 ± 1.6 66.1 ±2.3 52.7 ± 0.8 61.6 ± 1.4 61.6 ±0.7 64.3 ± 1.5 MIA + Centella Extract, 47.6 ± 2.1 62.9 ± 0.9 52.9 ± 1.3 64.8 ± 2.3 63.4 ± 1.1 61.9 ±2.5

fraction 14G1862 1 mg/ml MIA ± Centella extract, fraction 14G1862 2 mg/ml 59.2 ± 3.0 ^LL 64.2 ± 0.8 60.0 ± 1.4 ^LL 63.3 ± 0.8 63.3 ± 1.7 64.2 ± 0.8 MIA ± Synovial fluid 53.3 ± 1.7 ^LL 62.5 ± 1.4 55.8 ±0.8 61.7 ± 1.7 59.2 ±3.0 63.3 ±0.8

Monoarthritis was induced by injection of 2 mg/25 μl monoiodoacetate (MIA, Sigma-Aldrich) into the tibiotarsal joint. On day 8 post-administration, 20 μl of each plant fraction or compound was injected into the tibiotarsal joint of the rat. Echinacea and Centella fractions were dissolved in PBS. *P<0.05 and **P<0.01 versus vehicle + vehicle; ^l P < 0.05 and ^ll P < 0.01 versus MIA+vehicle.

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Table 6. Von Frey test Retraction threshold (g) Day 14 Day 30 Day 60 Treatments ipsila teral may contrala teral ipsilater alnaya contralate ralnaya ipsilater alnaya contralate ralnaya carrier + carrier 23.7 ± 0.7 22.5 ± 1.4 25.6 ± 1.4 23.7 ±2.8 24.5 ± 1.3 24.6 ±2.1 MIA + carrier 11.0 ± 0.3** 22.7 ± 0.4 14.1 ± 12** 23.8 ±0.8 16.5 ± 1.0** 23.4 ± 1.7 MIA + Echinacea extract, fraction 13С1608 2 mg/ml 12.3 ± 1.5 23.4 ±0.8 14.9 ± 2.0 20.1 ±2.3 18.7 ± 1.1 22.9 ± 0.7 MIA + Echinacea extract, fraction 13С1608 10 mg/ml 13.6 ± 2.1 21.6 ±2.4 15.7 ±0.8 25.0 ± 1.1 21.3±0.4 ^l 23.0 ±2.3 MIA+ 16.0 ± 0.8 ^LL 24.0 ± 0.6 19.5 ± 1.7 25.2 ±0.8 22.4 ± 0.8 ^liters 24.7 ± 0.5

Echinacea extract, fraction 13С1608 20 mg/ml М1А± Centella extract, fraction 14G1862 0.2 mg/ml 11.9± 0.8 23.7 ± 1.6 16.4 ± 1.5 23.7 ± 1.6 19.6 ±2.1 22.6 ± 1.3 М1А± Centella extract, fraction 14G1862 1 mg/ml 12.6 ± 1.6 20.8 ± 0.9 16.7 ± 0.6 20.8 ± 0.9 18.4 ± 1.9 21.8 ±0.9 MIA ± Centella extract, fraction 14G1862 2 mg/ml 15.9 ± 0.5 ^LL 22.8 ± 0.6 18.7±0.9 ^l 25.1 ±0.8 22.3 ± 1.4 ^LL 24.7 ± 1.7 M1A± Synovial fluid 14.9 ± 1.0 ^ll 23.4 ±0.3 18.2±0.8 ^l 24.1 ±0.3 19.9 ± 1.1 24.5 ± 0.5

Monoarthritis was induced by injection of 2 mg/25 μl monoiodoacetate (MIA, Sigma-Aldrich) into the tibiotarsal joint. On day 8 post-administration, 20 μl of each plant fraction or compound was injected into the tibiotarsal joint of the rat. Echinacea and Centella fractions were dissolved in PBS. **P<0.01 vs vehicle + vehicle; ^l P < 0.05 and ^ll P < 0.01 versus MIA+vehicle.

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Table 7. Disability Test A Masses (g) (contralateral - ipsilateral paw) Treatments Day 14 Day 30 Day 60 carrier + carrier 2.7 ±3.8 10.5 ± 6.7 -5.9 ± 4.2 MIA + carrier 55.4 ± 1.9** 38.9 ± 1.5** 17.3 ± 1.4** MIA + Echinacea extract, fraction 13С1608 2 mg/ml 44.9 ±3.7 42.5 ± 4.6 13.1 ±3.6 MIA + Echinacea extract, fraction 13С1608 10 mg/ml 39.6 ± 10.9 31.4 ±5.8 11.8 ±3.9 MIA + Echinacea extract, fraction 13С1608 20 mg/ml 29.7 ± 2.7 ^LL 28.0 ± 0.9 ^ll 7.3 ± 0.2 ^ll MIA + Centella extract, fraction 14G1862 0.2 mg/ml 48.6 ±9.4 33.6 ± 1.9 14.8 ± 2.4 MIA + Centella extract, fraction 14G1862 1 mg/ml 39.3 ± 9.8 35.0 ±5.6 12.3 ± 3.5 MIA + Centella extract, fraction 14G1862 2 mg/ml 19.8 ± 7.4 ^LL 12.8 ± 4.1 ^liters 8.1 ± 1.2 ^L MIA + Synovial fluid 34.7 ± 2.0 ^liters 29.4 ± 0.9 ^ll 10.8 ± 0.9 ^l

Monoarthritis was induced by injection of 2 mg/25 μl monoiodoacetate (MIA, Sigma-Aldrich) into the tibiotarsal joint. On day 8 post-administration, 20 μl of each plant fraction or compound was injected into the tibiotarsal joint of the rat. Echinacea and Centella fractions were dissolved in PBS. **P<0.01 vs vehicle + vehicle; ^l P < 0.05 and ^ll P < 0.01 versus MIA+vehicle.

Histological evaluation of the tibiotarsal joint of animals treated with extracts of Echinacea (fraction 13C1608) and Centella (fraction 14G1862), bovine synovial fluid MIA: in general, the analyzed animals showed a decrease in the joint space, independent of the observation time, and deposition of fibrin filaments in the joint cracks, increasing 30 and 60 days after treatment. In general, cartilage and bone erosion show an increase over time. Synovial hyperplasia and vascularization are present and improve with treatment over time. Moreover, the presence of a moderate inflammatory infiltrate is clearly observed, the nature of which does not depend on time (Fig. 1 and 2).

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MIA+Centella (14G1862) (2 mg/ml): Treatment with Centella 14G1862 generally reduced all identified morphological changes in a statistically significant manner, primarily at 30 and 60 days.

Minor fibrin deposits or small areas of bone erosion remain (Figures 1 and 2).

MIA+Echinacea purpurea (13C1608) (20 mg/ml): Treatment with Echinacea 13C1608 generally reduced all identified morphological changes in a statistically significant manner, primarily at 30 and 60 days. Minor fibrin deposits or small areas of bone erosion remain (Figures 1 and 2).

MIA+Bovine Synovial Fluid: Treatment with bovine synovial fluid does not appear to alter morphological parameters relative to the changes described for MIA-treated animals (Figures 1 and 2).

Evaluation of the effectiveness of various fractions from Centella asiatica, soluble in aqueous-alcoholic solutions.

Using the same animal model of MIA-induced osteoarthritis, the efficacy of intra-articular administration of three Centella fractions (ABO-AR-2016-391, ABO-AR-2016-392 and ABO-AR2016-393) and, finally, the effect generated by intra-articular treatment was assessed with triamcinolone acetonide. Behavioral assessments were performed 14, 30, and 60 days after injury induction, while intra-articular administration of substances was performed on days 8 after MIA administration (when osteoarthritis pathology had already begun).

In table Figure 8 shows the effect of various treatment methods on the threshold of pain perception by a painful mechanical stimulus (paw pressure test). The group of animals that were injected with MIA (MIA + vehicle) shows a statistically significant decrease in the pain threshold in relation to the control group, which is maximum on the 14th day from the induction of damage (41.5 ± 1.9 g, and 65.0 ± 1 .6 g, respectively). This difference between the two experimental groups remains significant even on the 30th day, and then decreases after 60 days, when spontaneous recovery from osteoarthritis pathology occurs (Table 8). A single intra-articular injection of a 70% EtOH soluble fraction from Centella (14G1862) reduces joint pain starting on day 6 of administration (day 14 of MIA-induced injury). An almost complete return to the initial level of joint pain is achieved on the 30th day, when the values recorded on the ipsilateral paw in the control group and in the group of animals receiving MIA + Centella extract, fraction 14G1862, become comparable (65.2 ± 0.9 g and 60.0±1.4 g, respectively). Centella fractions ABO-AR-2016-391 and ABO-AR-2016-392, prepared as described in Preparation Example 4 below, became significantly more active at 14 and 30 days, demonstrating lower potency at day 14 compared to treatments with Centella extract, fraction 14G1862, since they only partially reduce MIA-induced hyperalgesia. Centella extract, fraction ABO-AP-2016-393, reached statistical significance only on day 30 (58.3 ± 1.7 g), and it also showed efficacy comparable to that of Centella extract, fraction (14G1862). The efficacy of the ABO-AR-2016-391 and ABO-AR2016-392 fractions was comparable to that induced by intra-articular treatment with triamcinolone acetonide (52.8 ± 1.1 g and 56.7 ± 2.5 g, on days 14 and 30, respectively) (Table 8).

Table 8. Paw Pressure Test Weight (g) Day 14 Day 30 Day 60 Treatments ipsilateral I contralateral ipsilateral contralateral ipsilateral contralateral I carrier + carrier 65.0 ± 1.6 65.0 ±2.5 65.2 ± 0.9 66.3 ± 1.3 62.5 ±2.5 65.0 ±0.6 MIA + carrier 41.5 ± 1.9** 65.0 ±2.9 48.0 ± 1.8** 65.8 ±3.0 59.2 ± 0.8 63.3 ± 1.7 MIA + Centella extract, fraction 14G1862 59.2 ± 3.0 ^LL 64.2 ± 0.8 60.0 ± 1.4 ^LL 63.3 ± 0.8 63.3 ± 1.7 64.2 ± 0.8 MIA + Centella extract, fraction ABO-AR2016-391 50.4 ± 2.2 ^L 64.4 ± 0.9 57.5 ± 1.4 ^L 65.8 ±0.8 56.7 ±0.8 70.0 ± 2.9 MIA + Centella extract, fraction 51.2 ± 1.3 ^LL 63.3 ±0.8 56.7 ±3.3 63.4 ± 1.4 61.3 ± 1.3 65.8 ±0.8

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ABO-AR2016-392 MIA + Centella extract, fraction ABO-AR2016-393 48.8 ± 2.2 66.1 ±2.3 58.3 ± 1.7 ^LL 64.2 ± 2.2 63.3 ± 1 0.7 72.5 ± 1.4 MIA + Triamcinolone Acetonide 52.8 ± 1.1 65.3 ± 1.0 56.7 ± 2.5 ^LL 65.8 ±0.5 61.3 ± 1 0.6 66.4 ± 0.7

Monoarthritis was induced by injection of 2 mg/25 μl monoiodoacetate (MIA, Sigma-Aldrich) into the tibiotarsal joint. On day 8 post-administration, 20 μl of each plant fraction (2 mg/ml) or triamcinolone acetonide (100 μg) was injected into the tibiotarsal joint of the rat. Centella fractions were dissolved in PBS.

**P<0.01 vs vehicle + vehicle; ^l P < 0.05 and ^ll P < 0.01 versus MIA + vehicle The disability test shows an effectiveness higher than that of Centella fractions 14G1862, ABO-AR-2016-392 relative to the introduction of fractions ABO-AR-2016-391, ABO- AR-2016-393 and in relation to drug treatment based on the drug cortisone. Injection of fractions 14G1862, ABO-AR-2016-392 entails a halving of the weight that animals carry on the contralateral paw relative to the ipsilateral paw (Table 9). This effect is statistically significant when measured both after 14 and 30 days. ABO-AR-2016-391 induced a significant effect at 14 days (30.9±5.0 g) against MIA-induced damage, while the ABO-AR-2016-393 fraction reached statistical significance at day 30 (10. 6±0.8 g). Treatment with triamcinolone acetonide was effective on both days 14 and 30, but, as mentioned above, with less efficiency compared to Centella 14G1862 and ABO-AR-2016-392 fractions (Table 9).

Table 9. Disability Test Δ Weight (g) (paw contralateral - ipsilateral) Treatments Day 14 Day 30 Day 60 carrier + carrier -9.8 ± 2.5 3.5 ± 5.2 9.7 ± 5.2 MIA + carrier 56.8 ±7.1** 31.5 ± 1.5* 10.4 ± 2.7 MIA + Extract Centella faction 14G1862 19.8±7.4 ^AA 12.8 ± 4.1 ^AA 8.1 ± 1.2 MIA + Extract Centella faction ABO-AR-2016-391 30.9 ± 5.0 ^l 24.1 ±3.8 6.3 ± 4.5

MIA ± Centella extract, fraction AR2016-392 20.5 ± 3.1 ^liters 6.3±3.4 ^AL -2.6 ±8.8 MIA ± Extract Centella faction ABO-AR-2016-393 41.6 ±2.8 10.6 ± 0.8 ^AA 5.6 ±4.4 MIA ± Triamcinolone acetonide 28.7 ± 3.2 ^A 15.6 ± 5.4 ^L 5.6 ±4.4

Monoarthritis was induced by injection of 2 mg/25 μl monoiodoacetate (MIA, Sigma-Aldrich) into the tibiotarsal joint. On day 8 post-administration, 20 μl of each plant fraction (2 mg/ml) or triamcinolone acetonide (100 μg) was injected into the tibiotarsal joint of the rat. Centella fractions were dissolved in PBS.

**P<0.01 vs vehicle + vehicle; llr<0.05 and llr<0.01 vs. MIA + carrier Ac assessment test

- 21 044994 spontaneous movement activity (Animex test) allowed us to demonstrate that animals treated with Centella extract retained good movement abilities (data not shown). In this test, the most effective extracts are extracts from Centella, fractions 14G1862 and ABOAR-2016-392, as they increase in a statistically significant way the number of spontaneous movements of the animal, both 14 days and 30 days after injury (data not shown). Briefly, the Centella extracts that have shown greater efficacy in reducing hyperalgesia and motor impairment following MIA administration are the Centella fraction 14G1862 and the ABO-AR-2016392 fraction. In particular, they exhibited an analgesic profile that remained constant over time after their individual intra-articular administration.

Relationship with histological evaluations of the tibiotarsal joint of animals treated with different fractions of Centella and triamcinolone acetonide MIA: In general, the animals analyzed showed a decrease in the joint space, independent of observation time, and deposition of fibrin filaments in the joint space, increasing after 30 and 60 days after treatment. In general, erosion of cartilage and bone tissue increases over time. Synovial hyperplasia and vascularization are present and decrease with time after treatment. In addition, the presence of a moderate inflammatory infiltrate is clearly visible, the nature of which does not depend on time (Fig. 3 and 4).

MIA+Centella 14G1862: Treatment with Centella 14G1862 generally reduced all identified morphological changes in a statistically significant manner, primarily at 30 and 60 days. Minor fibrin deposits or small areas of bone erosion remain (Figs. 3 and 4).

MIA+ABO-AR-2016-391: Treatment with ABO-AR-2016-391 improves in a statistically significant way some of the parameters considered (bone and cartilage erosion and joint space reduction). The fraction shows effectiveness in relation to other parameters after 30 and 60 days of treatment (Figures 5 and 6).

M1A+ABO-AR-2016-392: treatment with ABO-AR-2016-392 shows effectiveness after 30 and 60 days for all parameters analyzed, with the exception that in the presence of an inflammatory infiltrate, the fraction shows activity only after 60 days. With regard to cartilage and bone erosion and joint space reduction, the fraction appears to be significantly effective at all three experimental time periods (Figures 3 and 4).

M1A+ABO-AR-2016-393: treatment with ABO-AR-2016-393 shows an effect on all analyzed morphological parameters, with the exception that in the presence of an inflammatory infiltrate, the fraction has no effect. The improvement in morphological parameters induced by ABO-AR-2016-393 is, however, lower than that recorded with the other two fractions (Figs. 3 and 4).

MIA+Triamcinolone: Treatment with triamcinolone shows significant effectiveness in reducing inflammatory infiltration and erosion of both cartilage and bone tissue. With regard to synovial hyperplasia, fibrin deposition, synovial vascularization and joint space reduction, it shows reduced effectiveness after 15 and 30 days of treatment and more noticeable effectiveness after 60 days of treatment (Figs. 3 and 4).

Examples

Examples of the preparation of therapeutically active substances according to the invention are shown below. One skilled in the art may use other similar procedures or make modifications to the procedures presented below to obtain extracts or fractions suitable for practicing the invention based on knowledge generally known in the art.

1. Example of preparation of extract of fraction 14G1862 from Centella asiatica.

The dried and crushed leaves of Centella asiatica are subjected to extraction with 70% ethanol (ethanol:water 70:30 v/v), in a static extractor by percolation method, with the plant immersed in the solvent and the solvent is re-fed from bottom to top by a mechanical pump.

The drug-solvent ratio (DSR) is 1:8, the extraction temperature is 50°C, the extraction duration is from 6 to 8 hours. From the resulting mixture, the insoluble residue is separated from the soluble fraction by static decantation and subsequently filtration at paper filter. The resulting solution is concentrated by evaporating the ethanol using a vacuum concentration system. When the concentration ratio reaches 10:1, concentration stops and drying is completed by lyophilization. Lyophilized material constitutes the fraction of interest (DER 4-10:1). The protocol described herein is applicable, mutatis mutandis, to formulations with other alcohol contents as reported in the detailed description.

2. An example of the preparation of an extract of fraction 13C1608 from Echinacea purpurea Dried and crushed leaves of Echinacea purpurea are subjected to extraction with 45% ethanol (ethanol: water 45:55 v/v), in a static extractor by percolation, with the plant immersed in a solvent and solvent recirculated from bottom to top by a mechanical pump.

The drug-solvent ratio (DSR) is 1:8, the extraction temperature is 50°C, the extraction duration is from 6 to 8 hours. From the resulting mixture, insoluble

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Claims (11)

The washable residue is separated from the soluble fraction by static decantation and subsequently filtration on a paper filter. The resulting solution is concentrated by evaporating the ethanol using a vacuum concentration system. When the concentration ratio reaches 10:1, concentration stops and drying is completed by lyophilization. Lyophilized material constitutes the fraction of interest (DER 4-10:1). The protocol described in this document applies, mutatis mutandis, to other alcohol contents reported in the detailed description. 3. An example of the preparation of Centella asiatica fractions ABO-AR-2016-391, ABO-AR-2016-392, ABOAR-2016-393. Centella leaves were fragmented and subjected to two extraction steps in ethanol at decreasing concentrations; performing the first step with 96% ethanol, the plant parts treated with 96% EtOH were then subjected to the second step with 13% ethanol. The extraction conditions of each extraction step were characterized by a time duration of 8 hours, a drug-solvent ratio (DSR) of 1:8, and an extraction temperature of 40°C. The first alcoholic fraction obtained from the 96% ethanol step was discarded. The second alcoholic fraction obtained from the 13% ethanol step was concentrated by evaporating the ethanol using a vacuum distillation system according to a standard protocol, resulting in a concentrated aqueous extract after removing the ethanol. The resulting concentrated aqueous extract was filtered on a plate filter with a paper filter having a pore size cut-off of 25 micrometers. A portion of this concentrate was freeze-dried to provide the corresponding lyophilized fraction (ABO-AR-2016-391) (DER 5-10:1). The filtered concentrated aqueous extract obtained as described above was subjected to a 150 to 300 Da membrane filtration step according to standard procedures, providing a permeate fraction and a retentate fraction. The above fractions were freeze-dried, providing the corresponding lyophilized fractions: permeate (DER 10-30:1) (ABO-AR-2016-392) and retentate (DER 5-20:1) (ABO-AR2016-393). The protocol described in this document applies, mutatis mutandis, to other membranes provided in the detailed description. 4. An example of preparing a composition according to the invention. 1. The lyophilized extracts are solubilized in NaCl water by heating the mixture at a temperature ranging from 30 to 80°C, preferably 30 to 70°C, 40 to 50°C for a period of 4 to 8 hours in order to to promote solubilization of the extract. 2. The mixture obtained in the previous step, after cooling, was filtered to eliminate any undissolved extract particles and then dried, preferably by lyophilization, and resuspended in a suitable pharmaceutically acceptable vehicle. 3. At this stage, if necessary, oily substances and emulsifiers can be added if surface-activated systems are to be obtained. 4. Other water-soluble auxiliary substances, such as buffers, isotonic agents, were added to the extract mixture. 5. The pH of the prepared solution is measured and adjusted if necessary. 6. The solution thus obtained was sterilized by sterilizing filtration or moist heat (steam) in an autoclave. CLAIM 1. A method of preparing a parenteral composition containing an extract of Centella asiatica, an extract of Echinacea purpurea and at least one pharmaceutically acceptable carrier, in which a) the leaves of Centella asiatica are subjected to one or more stages of extraction with water-alcohol mixtures, and the extract thus obtained is subjected to lyophilization; b) the aerial parts of Echinacea purpurea are subjected to one or more extraction steps with water-alcohol mixtures and the extract thus obtained is lyophilized; c) the lyophilized extracts obtained in stages a) and b) are mixed in any order in the ratio of Centella asiatica extract: Echinacea purpurea extract in the range from 8:2 to 0.5:9.5; d) the extracts thus mixed are resuspended in water with the addition of 0.8 to 4% sodium chloride; e) the mixture thus obtained is brought to a temperature in the range of 30 to 70°C for a period of time in the range of 4 to 8 hours and then subjected to filtration or decantation; f) the filtrate or supernatant obtained in step e) is lyophilized; g) the lyophilized co-extract obtained in step f) is resuspended in one or more pharmaceutically acceptable carriers. 2. The method according to claim 1, in which the specified extract at stage a) is a fraction of the extract - 23 044994 that, soluble in aqueous-alcoholic solution, in which alcohol is from 65% to 75% v/v, extract fraction, insoluble in from 90 to 96% v/v. EtOH and soluble in 10 to 20% v/v. EtOH, or mixtures thereof, and/or wherein said extract in step b) is a fraction of the extract soluble in an aqueous-alcoholic solution in which said alcohol constitutes from 40% to 50% v/v. 3. A parenteral composition obtained by the method according to any one of claims 1 or 2, wherein the parenteral composition contains an extract of Centella asiatica and an extract of Echinacea purpurea, and at least one pharmaceutically acceptable carrier, wherein the therapeutically active principle is contained in said Centella asiatica extract in combination with said extract of Echinacea purpurea, wherein said extract of Centella asiatica is an extract from the leaves of said plant, and wherein said extract of Echinacea purpurea is an extract of the aerial parts of said plant, and wherein said extract of Centella asiatica and said extract of Echinacea purpurea are a lyophilized extract in an aqueous-alcohol solution. 4. Parenteral composition according to claim 3, characterized in that it includes, in lyophilized form, total phenols: from 0.08 to 2% w/w, and/or polysaccharides with a molecular weight above 20,000 Da: from 1 to 2% w/w, and/or total tannins: 0.002 to 1% w/w. 5. Parenteral composition according to claim 4, containing from 0.08 to 2% wt./wt. total phenols, from 0.002 to 1% wt./wt. total tannins, from 1 to 2% w/w. polysaccharides with a molecular weight greater than 20,000 Da, being in lyophilized form, and at least one pharmaceutically acceptable carrier, wherein said phenols, tannins, polysaccharides are derived from Centella asiatica and Echinacea purpurea. 6. Parenteral composition according to any one of claims 3 to 5, in which the pharmaceutically acceptable carrier is selected from solvents, emulsifiers, buffering agents, pH regulators, antioxidants, preservatives, osmotic agents. 7. The parenteral composition according to claim 6, wherein said solvent is selected from one or more of: water, polyvinylpyrrolidone, isotonic saline solutions; and/or said emulsifiers are selected from one or more of: lecithins, ethoxylated fatty acid derivatives; and/or said buffering agents are selected from one or more of: citric acid and its sodium salts, phosphoric acid and its sodium salts, acetic acid and its sodium salts; and/or said pH adjusters are selected from one or more of: hydrochloric acid and sodium hydroxide; and/or said antioxidants are selected from one or more of: sodium bisulfite, sodium metabisulfite, ethylenediaminetetraacetic acid and its sodium salts; and/or said preservatives are selected from one or more of: benzyl alcohol, methyl phydroxybenzoate, benzalkonium chloride, benzethonium chloride: and/or said osmotic agents are selected from one or more of: sodium chloride, sugars, mannitol, dextrose, glucose. 8. Use of a parenteral composition according to any one of claims 3 to 7 for the treatment of disorders affecting articular cartilage in vertebrate animals, including humans. 9. Use according to claim 8, wherein the disorders affecting articular cartilage are selected from arthritis, osteoarthritis, joint inflammation, cartilage damage caused by trauma, spinal disc degeneration, rheumatoid arthritis. 10. A method for treating disorders affecting articular cartilage, comprising administering to the patient a parenteral composition according to any one of claims 3 to 7. 11. The method according to claim 10, wherein said composition is administered by infiltration/articular injection. -