Combined anti-inflammatory agent

A pharmaceutical composition for treating inflammatory diseases, comprising (A) an effective amount of hyaluronic acid or its salt, and (B) an effective amount of an anti-inflammatory agent. The composition exhibits a synergistic therapeutic effect on inflammations and is useful for treating inflammatory diseases, particularly diseases of joint with inflammation.

BACKGROUND OF THE INVENTION 
The present invention relates to a combined medicine for the purpose of 
treating inflammatory diseases and more particularly to a combined 
medicine useful for treating diseases of a joint with inflammation. 
It has been known that hyaluronic acid or its salt is effective to some 
kinds of arthropathies in clinical and fundamental tests. The theoretical 
bases are as follows: (1) Hyaluronic acid is one of the main components of 
joint liquid. In the case of rheumatoid arthritis and osreoarthritis which 
are included in arthropathy, the hyaluronic acid contained in the joint 
liquid has a reduced molecular weight and a reduced concentration. (2) As 
the main pharmacological actions of hyaluronic acid, there are exemplified 
an action of covering the surface of a cartilage, an inhibitory action on 
the liberation of proteoglycan, which action is exhibited by the 
hyaluronic acid migrated into a cartilage matrix, and an improving action 
on the spinnability of the joint liquid. 
However, most of the reported clinical cases wherein hyaluronic acid was 
applied are osteoarthritis and rheumatoid arthritis and its applicable 
range is relatively narrow. Further, although symptoms such as pain and 
stiffness become in serious problem in the treatment of joint diseases, 
hyaluronic acid does not possess any direct activity of improving such 
symptoms. 
Anti-inflammatory agents are widely used in the clinical treatment of 
arthropathy. The reason therefor is presumed that many kinds of 
arthropathies involves inflammation. The reason why nonsteroidal 
anti-inflammatory agents are especially widely used is presumed that they 
have strong analgesic activity. 
However, when anti-inflammatory agents are administered in a usual clinical 
method such as oral administration, administration using suppository, 
subcutaneous or intramuscular administration, side effects such as the 
inflammation and ulcer of digestive system, and diarrhea tend to develop 
because the drug reaches its effective concentration not only at a part to 
be treated but also in tissues of the whole body including blood. Further 
the development of the side effects is promoted due to the fact that a 
large amount of dose is required for the treatment because of the 
distribution of the drug over the whole body and the fact that the 
administration period is prolonged because most arthropathies are chronic. 
For the reasons, a sufficient amount of the drug required for the 
treatment cannot be administered or the administration is obliged to be 
interrupted, which results in failure of a suitable treatment. 
Consequently, there are many cases wherein the condition of the disease is 
worsened. 
It is an object of the present invention to provide an 
inflammation-treating agent, in particular, which is capable of curing 
inflammation in joint diseases and possesses the activity of improving 
symptoms such as pain and stiffness. 
Another object of the invention is to provide an efficient 
inflammation-treating agent, in particular, which does not develop any 
side effect even when a sufficient amount thereof required for the 
treatment of joint diseases is administered. 
These and other objects of the present invention will become apparent from 
the description hereinafter. 
SUMMARY OF THE INVENTION 
The present invention provides a pharmaceutical composition for treating 
inflammatory diseases, comprising (A) an effective amount of hyaluronic 
acid or its salt, and (B) an effective amount of an anti-inflammatory 
agent.

DETAILED DESCRIPTION 
A combined agent of hyaluronic acid or its salt with an anti-inflammatory 
agent in accordance with the present invention is an excellent agent for 
treating joint diseases, which develops the respective merits of both 
drugs and supresses the respective demerits of both drugs. 
Hyaluronic acid and its salts possess anti-inflammatory activity. Examples 
of the salt of hyaluronic acid include sodium salt, potassium salt, 
ammonium salt and salts with lower alkyl amines (C.sub.1 to C.sub.5) The 
sodium salt is preferred. 
In a preferable embodiment of the present invention, hyaluronic acid or its 
salt is used in the form of a solution wherein it is dissolved in water or 
an aqueous solvent in such a concentration that the solution shows 
spinnability. An aqueous solution of hyaluronic acid or its salt which 
shows suitable spinnability has a viscosity of about 500 to 2,000 cps at 
300.degree. C. In the case of sodium hyaluronate having a molecular weight 
of 8 .times. 10.sup.5, a concentration of not less than 0.5% (w/v %, 
hereinafter the same), preferably 0.8 to 1.2%, is required to obtain such 
a suitable spinnability. A lower concentration (lower than 0.5%) is 
adoptable with increasing molecular weight of sodium hyaluronate and a 
higher concentration (more than 0.5%) is required with decreasing 
molecular weight of sodium hyaluronate. 
Hyaluronic acid or its salt having a molecular weight within a wide range 
can be used in the present invention. From the viewpoints of the 
anti-inflammatory activity and spinnability, the preferred molecular 
weight ranges from 4 .times. 10.sup.5 to 3 .times. 10.sup.6. When the 
molecular weight is less than the above range, the anti-inflammatory 
activity is poor and a suitable spinnability is not obtainable. When the 
molecular weight is more than the above range, the viscosity of the 
resulting solution extremely increases and consequently the administration 
by injection is difficult, which results in the impossibility of practical 
application to the treatment of arthropathy. 
In a preferable embodiment of the present invention, the anti-inflammatory 
agent, which is preferably in the form of finely divided particles, is 
dissolved or suspended into a solution of hyaluronic acid or its salt in 
water or an aqueous solvent. The resulting solution or suspension is 
preferably adjusted so that the pH value is from 6.0 to 7.0 and the ratio 
of its osmotic pressure to that of a 0.9% physiological saline solution is 
from 0.8 to 1.2, yielding a preparation suitable for administration in an 
articular cavity. Examples of the aqueous solvent include physiological 
saline solutions, 3 to 5% glucose solutions and 3 to 5% xylitol solutions 
and phosphate buffer solutions. 
Both steroidal anti-inflammatory agents including prednisolone and 
nonsteroidal anti-inflammatory agents can be used as an anti-inflammatory 
agent. The nonsteroidal anti-inflammatory agents are preferred. 
Preferable examples of the nonsteroidal anti-inflammatory agent are as 
follows: 
I Carboxylic acid anti-inflammatory agent 
1. Salicylic acid anti-inflammatory agent 
Salicylic acid 
Aspirin 
2. Anthranilic acid anti-inflammatory agent 
Mefenamic acid 
II Acetic acid anti-inflammatory agent 
1. Phenylacetic acid anti-inflammatory agent 
Diclofenac 
Alclofenac 
2. Indole anti-inflammatory agent 
Indometacin 
3. Heteroarylacetic acid anti-inflammatory agent 
Tolmethin 
III Propionic acid anti-inflammatory agent 
1. Phenyl anti-inflammatory agent 
Ibuprofen 
2. Naphthalene anti-inflammatory agent 
Naproxen 
3. Tricyclic anti-inflammatory agent 
Pranoprofen 
IV Pyrazolone anti-inflammatory agent 
Phenylbutazone 
V Benzthiazine anti-inflammatory agent 
Piroxicam 
The above-mentioned anti-inflammatory agents include their salts, if any. 
These anti-inflammatory agents may be used singly or in admixtures 
thereof. 
The preferred anti-inflammatory agents are nonsteroidal acid 
anti-inflammatory agents represented by the following structure formulas 
(I) and (II): 
##STR1## 
wherein R.sup.1 is 
##STR2## 
and R.sup.2 is --H or --Ch.sub.3. 
##STR3## 
wherein R.sup.3 is --COOH or --CH.sub.2 COOH, R.sup.4 is --H or --Cl, 
R.sup.5 is --Cl or --CH.sub.3, and R.sup.6 is --H or --CH.sub.3. 
The more preferred anti-inflammatory agents are shown in Table 1. 
TABLE 1 
__________________________________________________________________________ 
R.sup.1 R.sup.2 
R.sup.3 R.sup.4 
R.sup.5 
R.sup.6 
__________________________________________________________________________ 
##STR4## CH.sub.3 
CH.sub.2 COOH 
Cl Cl H 
##STR5## H COOH H CH.sub.3 
CH.sub.3 
__________________________________________________________________________ 
In the present invention, the ratio of hyaluronic acid or its salt to the 
anti-inflammatory agent can vary over a wide range. For the purpose of 
obtaining a good synergistic effect, the ratio of hyaluronic acid or its 
salt to the anti-inflammatory agent ranges preferably from 1:0.03 to 2 (by 
weight), more preferably from 1:0.1 to 1. When the proportion of 
hyaluronic acid or its salt is lower than the above range, the 
anti-inflammatory agent is not sufficiently retained by an aqueous 
solution of hyaluronic acid or its salt. When the proportion of hyaluronic 
acid or its salt is higher than the above range, the anti-inflammatory 
activity is lowered. 
The combined agent of the present invention may contain other medicaments 
such as adrenocortical hormones, local anesthetic agents and antibiotics. 
Further, it may contain various additives including stabilizing agents, 
for example, antioxidants such as sodium sulfite and sodium 
hydrogen-sulfite; buffers such as citrates and phosphates; solubilizers or 
solubilizing agents such as alcohols, polyethylene glycols; and 
preservatives such as benzoic acid and salicylic acid. 
The combined agent of the present invention can be applicable to the 
treatment of a variety of arthropathies such as osteoarthrisis, rheumatoid 
arthritis and periarthritis; and gout, and to treatments after operation 
of joints and eyes. 
In the treatment of arthropathy, the combined agent of the present 
invention is preferably administered into an articular cavity in a dose of 
27.5 to 50 mg/one time per adult (based on the total amount of both 
drugs). More concretely, for example, 2.5 ml ampuls are prepared, each 
containing 25 mg of sodium hyaluronate and a given amount of an 
anti-inflammatory agent (e.g. 25 mg of diclofenac, ibuprofen or 
phenylbutazone, 7.5 mg of indometacin, or 375 mg of sodium salicylate) in 
an isotonic phosphate buffer solution as an aqueous solvent. The 
preparation is administered into an articular cavity in a dose of one 
ampul once per 7 to 10 days. In such a manner, the administration is 
continuously conducted 4 to 5 times while varying the dose if necessary. 
The combined agent of hyaluronic acid or its salt and an anti-inflammatory 
agent in accordance with the present invention is able to exhibit the 
effects mentioned below. 
The combined agent of the present invention has a wide application as an 
arthropathy-treating agent because it is composed of two kinds of 
arthropathy-treating agents different in mechanism of action from each 
other. The combined agent of the present invention has a strong 
therapeutic effect due to a synergistic effect of the combination of the 
two kinds of the components. 
The combined agent of the present invention can be administered directly to 
an affected part to be treated so that the concentration of the drugs 
becomes higher at the affected part to be treated and lower in tissues, 
including the tissues of digestive system, other than the tissue to which 
the instant agent is administered. Thus a strong therapeutic effect is 
obtained at the affected part and side effects such as ulcer and 
inflammation of digestive system and diarrhea hardly develop. The 
interruption of the administration and the extreme reduction of the dose 
due to the side effects can be avoided. Consequently a sufficient medical 
treatment is made possible. 
Moreover, in the case of the instant combined agent of hyaluronic acid or 
its salt with an anti-inflammatory agent, the anti-inflammatory agent 
dissolved in the aqueous solvent is retained in a hydrated hyaluronic acid 
or its salt for a long time and released gradually therefrom. The 
effective concentration of the drugs can be retained in the tissue to 
which the instant combined agent is administered and the action of the 
drugs continues. Consequently, it is sufficient to administer the instant 
combined agent about once per a week. 
The present invention is more specifically described and explained by means 
of the following Examples. It is to be understood that the present 
invention is not limited to the Examples, and various change and 
modifications may be made in the invention without departing from the 
spirit and scope thereof. 
TEST EXAMPLE 1 
The inhibitory effect on carrageenan-induced edema was investigated as to 
combined agents of sodium hyaluronate (molecular weight: 8 
.times.10.sup.5) and various anti-inflammatory agents. 
Wister male rats weighing 240 to 260 g (6 weeks old) were preliminarily 
bred for not less than 1 week. Eight rats were used in one group. Each 
test agent shown in Table 2 was dissolved or suspended in an isotonic 
phosphate buffer solution (pH 7.0) to give a 1% solution or suspension 
(hereinafter referred to as "1% solution"). The solution was administered 
subcutaneously into the right foot pad of each rat. Six hours after the 
administration, a 1% solution of carrageenan was administered 
subcutaneously as an irritating agent into the right foot pad of the rat 
in a dose of 0.1 ml/animal. The volume of the right foot pad was measured 
before and 4 hours after the administration of carrageenan. The rate of 
edema inhibition (hereinafter referred to as "inhibitory rate") by each 
test agent was calculated from the obtained measurements and thus the 
inhibitory effect on edema was evaluated. The results are shown in Table 
2. 
Table 2 reveals that all anti-inflammatory agents tested, when being used 
in combination with sodium hyaluronate, showed strong inhibitory effect on 
edema in comparison with either each anti-inflammatory agent alone or 
sodium hyaluronate alone. Among the anti-inflammatory agents tested, 
diclofenac, ibuprofen and indometacin, particularly, showed great 
synergistic effect in combination with sodium hyaluronate. 
The measurement of the volume was carried out according to the method of 
Fujimura et al. (see Iyakuhin Kaihatsu Kisokoza, Vol. 6, "Yakubutsu no 
Hyoka (1)" editted by Tsuda and Nogami, p 239-282, Kabushiki Kaisha 
Chizin-sha, 1971). 
The rate of inhibition (%) of the edema of the foot pad of each rat was 
calculated according to the following formula (III). 
##EQU1## 
MCEV: Average swollen rate of the foot pad 4 hours after the 
administration of carrageenan in the control group 
MTEV: Average swollen rate of the foot pad 4 hours after the administration 
of carrageenan in the drug-given group 
##EQU2## 
CEV: Volume of foot pad of each rat before the administration of 
carrageenan 
TEV: Volume of foot pad of each rat 4 hours after the administration of 
carrageenan 
With respect to each agent, the evaluation of the inhibitory effect on 
edema was carried out according to the following criteria: Synergistic 
effect was observed: 
______________________________________ 
Very great HA % + DG % .ltoreq. ED % 
Great TD % .ltoreq. ED % &lt; HA % + DG % 
Small MD % &lt; ED % &lt; TD % 
______________________________________ 
No synergistic effect was observed: 
EQU ED % .ltoreq. MD % 
HA % : Inhibitory rate by the administration of sodium hyaluronate alone 
DG % : Inhibitory rate by the administration of the anti-inflammatory agent 
alone 
ED % : Inhibitory rate by the administration of the combined agent 
MD % : {The greater one between HA % and DG %} .times. 1.2 
TD % : HA % + DG % (1--HA %/100) 
Besides, the stomach and the small intestine were autopsyed under 
anesthesia with ether 6 hours after the administration of carrageenan and 
no abnormal symptom was observed in the drug-given group. 
TABLE 2 
__________________________________________________________________________ 
Inhibitory 
HA % 
Dose rate + Synergistic 
Test agent (mg/kg) 
(%) TD % 
DG % 
effect 
__________________________________________________________________________ 
Sodium hyaluronate (HA) 
4.0 23.5 
Diclofenac sodium 
4.0 16.9 
HA + Diclofenac sodium 
4.0 + 4.0 
62.9 36.4 
40.4 
very great 
Ibuprofen 4.0 18.2 
HA + Ibuprofen 
4.0 + 4.0 
47.6 37.4 
41.7 
very great 
Sodium salicylate 
50.0 46.6 
HA + Sodium salicylate 
4.0 + 50.0 
57.0 59.1 
70.1 
small 
Indometacin 1.2 12.9 
HA + Indometacin 
4.0 + 1.2 
66.2 33.4 
36.4 
very great 
Phenylbutazon 
4.0 46.1 
HA + Phenylbutazon 
4.0 + 4.0 
59.3 58.8 
69.6 
great 
Piroxicam 4.0 34.6 
HA + Piroxicam 
4.0 + 4.0 
47.1 50.0 
58.1 
small 
Prednisolone 0.5 21.7 
HA + Prednisolone 
4.0 + 0.5 
37.0 40.1 
45.2 
small 
__________________________________________________________________________ 
Note: 
MD % is smaller than ED % with all agents tested. 
TEST EXAMPLE 2 
The inhibitory effect on carrageenan-induced edema was investigated as to 
combined agents of various grades of sodium hyaluronates having different 
molecular weights with diclofenac sodium. 
Wister male rats weighing 230 to 265 g (6 weeks old) were preliminarily 
bred for not less than 1 week. Eight rats were used in one group. A 1% 
solution of each test agent shown in Table 3 was administered 
subcutaneously into the right foot pad of each rat. Six hours after the 
administration, a 1% solution of carrageenan was administered 
subcutaneously as an irritating agent into the right foot pad of the rat 
in a dose of 0.1 ml/animal. The volume of the right foot pad was measured 
before and 4 hours after the administration of carrageenan. Then the 
inhibitory effect on edema was evaluated in the same way as in Test 
Example 1. The results are shown in Table 3. 
Table 3 reveals that sodium hyaluronates having a molecular weight within a 
wide range showed synergistic effect in combination with diclofenac 
sodium. However, sodium hyaluronate having too small molecular weight 
showed a smaller inhibitory effect itself and therefore the inhibitory 
effect of the combined agent thereof was also smaller. Accordingly the 
preferable molecular weight of sodium hyaluronate is not less than 4 
.times. 10.sup.5 . 
TABLE 3 
______________________________________ 
Molecular Inhibitory 
weight Dose rate 
Test agent of HA (mg/kg) (%) 
______________________________________ 
Sodium hyaluronate (HA) 
28 .times. 10.sup.4 
4.0 -36.4 
HA + Diclofenac 28 .times. 10.sup.4 
4.0 + 4.0 
19.6 
HA 58 .times. 10.sup.4 
4.0 17.9 
HA + Diclofenac 58 .times. 10.sup.4 
4.0 + 4.0 
38.0 
HA 80 .times. 10.sup.4 
4.0 29.9 
HA + Diclofenac 80 .times. 10.sup.4 
4.0 + 4.0 
49.6 
HA 210 .times. 10.sup.4 
4.0 43.2 
HA + Diclofenac 210 .times. 10.sup.4 
4.0 + 4.0 
52.5 
Diclofenac -- 4.0 16.9 
______________________________________ 
TEST EXAMPLE 3 
The inhibitory effect on carrageenan-induced edema was investigated with 
respect to combined agents of sodium hyaluronate (molecular weight: 8 
.times. 10.sup.5) and diclofenac sodium wherein the ratio of diclofenac 
sodium to sodium hyaluronate varied. 
Wister male rats weighing 235 to 260 g (6 weeks old) were preliminarily 
bred for not less than 1 week. Eight rats were used in one group. A 1% 
solution of each test agent shown in Table 4 was administered 
subcutaneously into the right foot pad of each rat. Six hours after the 
administration, a 1% solution of carrageenan was administered 
subcutaneously as an irritating agent into the right foot pad of the rat 
in a dose of 0.1 ml/animal. The volume of the right foot pad was measured 
before and 4 hours after the administration of carrageenen. Then, the 
inhibitory effect on edema was evaluated in the same way as in Test 
Example 1. The results are shown in Table 4. 
As is clear from Table 4, when the ratio of diclofenac sodium to sodium 
hyaluronate is approximately equal, the preferable results can be 
expected. 
TABLE 4 
______________________________________ 
Dose Inhibitory rate 
Test agent (mg/kg) (%) 
______________________________________ 
Sodium hyaluronate 
4 25.9 
(HA) 
HA + Diclofenac 4 + 2 29.8 
HA + Diclofenac 4 + 4 58.2 
HA + Diclofenac 4 + 8 63.9 
______________________________________ 
TEST EXAMPLE 4 
The inhibitory effect on carrageenan-induced edema was investigated with 
respect to combined agents of sodium hyaluronate (molecular weight: 8 
.times. 10.sup.5) and various acid anti-inflammatory agents. 
Wister male rats weighing 240 to 255 (6 weeks old) were preliminarily bred 
for not less than 1 week. Eight rats were used in one group. A 1% solution 
of each test agent shown in Table 5 was administered subcutaneously into 
the right foot pad of each rat. Six hours after the administration, a 1% 
solution of carrageenen was administered subcutaneously as an irritating 
agent into the right foot pad of each rat in a dose of 0.1 ml/animal. The 
volume of the right foot pad was measured before and 4 hours after the 
administration of carrageenen. Then, the inhibitory effect on edema was 
evaluated in the same way as in Test Example 1. The results are shown in 
Table 5. 
Table 5 reveals that all anti-inflammatory agents tested, when being used 
in combination with sodium hyaluronate, showed strong inhibitory effect on 
edema in comparison with either each anti-inflammatory agent alone or 
sodium hyaluronate alone. 
TABLE 5 
______________________________________ 
Inhibi- 
tory HA % Syner- 
Dose rate + gistic 
Test agent (mg/kg) (%) TD % DG % effect 
______________________________________ 
Sodium hyaluro- 
4 23.9 
nate (HA) 
Aspirin 4 14.6 
HA + Aspirin 
4 + 4 23.6 35.0 38.5 small 
Mefenamic acid 
4 34.2 
HA + Mefenamic 
4 + 4 44.6 49.9 58.1 small 
acid 
Alclofenac 4 22.9 
HA + Alclofenac 
4 + 4 40.4 41.3 46.8 small 
Tolmetin 4 25.1 
HA + Tolmetin 
4 + 4 42.5 43.0 49.0 small 
Pranoprofen 4 39.0 
HA + Pranoprofen 
4 + 4 46.8 53.6 62.9 small 
______________________________________ 
TEST EXAMPLE 5 
After preliminary breeding of mail ICR mice 5 weeks old weighing 23 to 28 g 
(5 mice per one group) for one week, a 1% solution of each test agent 
shown in Table 6 wa subcutaneously administered to the mice. The number of 
dead mice was counted 72 hours after the administration. The results are 
shown in Table 6. 
The results of Table 6 reveal that the inflammation-testing agents of the 
present invention have no toxicity. 
The doses of the test agent used in this test were about ten times those 
used in Test Example 1. 
TABLE 6 
______________________________________ 
Test agent Dose Number of dead mice 
______________________________________ 
Sodium hyaluronate (HA) 
40 0 
HA + Diclofenac sodium 
+40 0 
HA + Sodium salicylate 
+500 0 
HA + Ibuprofen +40 0 
HA + Indometacin 
+12 0 
HA + Phenylbutazon 
+40 0 
HA + Piroxicam +40 0 
HA + Prednisolone 
+5 0 
______________________________________ 
Note: 
The molecular weight of sodium hyaluronate is 8 .times. 10.sup.5. The 
doses in Table 6 mean the amounts (mg) administered per kg body weight. 
The dose values, to which the mark "+" is attached, as to the combined 
agents means the amounts of antiinflammatory agent added to 40 mg/kg of 
sodium hyaluronate (HA). 
TEST EXAMPLE 6 
Wister male rats weighing 240 to 260 g (6 weeks old) were preliminarily 
bred for not less than 1 week. Eight rats were used in one group. A 1% 
solution of each test agent shown in Table 7 was administered 
subcutaneously into the right foot pad of each rat. Sixteen hours after 
the administration, a 1% solution of carrageenan was administered 
subcutaneously as an irritating agent into the right foot pad of each rat 
in a dose of 0.1 ml/animal. The volume of the right foot pad was measured 
before and 4 hours after the administration of carrageenen. Then, the 
inhibitory effect on edema was evaluated in the same way as in Test 
Example 1. The results are shown in Table 7. 
As is clear from the results of Table 7, the combined agents composed of 
indometacin or diclofenac and sodium hyaluronate showed prolonged 
anti-inflammatory effects even in a low dose which does not cause any side 
effect. The effects were greater than that in the case of administering 
sodium hyaluronate alone or that in the case of administering each 
anti-inflammatory agent alone. Then it would be understood that the 
synergistic effect of the combined agent of the present invention is very 
great. 
TABLE 7 
______________________________________ 
Inhibi- 
tory HA % Syner- 
Dose rate + gistic 
Test agent (mg/kg) (%) TD % DG % effect 
______________________________________ 
Sodium hyaluro- 
4.0 30.0 
nate (HA) 
Diclofenac 3.0 9.7 
HA + Diclofenac 
4.0 + 3.0 
46.3 36.8 39.7 Very 
great 
Indometacin 3.0 20 
HA + Indometacin 
4.0 + 3.0 
52.0 44.0 50.0 Very 
great 
______________________________________ 
TEST EXAMPLE 7 
Rabbits (New Zealand White) weighing about 2 kg were preliminarily bred for 
not less than one week and healthy ones were selected (3 rabbits per 
group). The joint of right knee of each rabbit was fixed by applying a 
splint thereto and further completely fixed not so as to move by putting 
it in plaster. Then the rabbits were bred normally for one month. 
During the normal breading, a 1% solution of each test agent shown in Table 
8 was administered using an injection needle of gauge No. 27 into the 
articular cavity of each rabbit except those of the control group in a 
dose of 0.3 ml/kg body weight once per three days. 
One day after the last administration, the splint and the plaster were 
removed and the movable range of the joint was measured with a protractor 
under anesthesia with Nembutal (trademark). The rate of inhibition of the 
damage to the movable range of joint with respect to each test agent was 
calculated from the obtained measurements and the curative effect was 
evaluated. The results are shown in Table 8. 
In the case of the rabbits of the control group, the adhesion and 
deformation of the bones were caused in one month normal breeding and a 
gait disturbance was observed even after removal of the splint and the 
plaster because the movable range of joint was rendered narrow. 
In the case of the rabbits of the drug-given groups, the following is 
apparent from Table 8. The administration of either diclofenac alone or 
sodium hyaluronate alone exhibited some curative effect, but the 
administration of the combined agent of both drugs exhibited an 
outstanding curative effect That is, the movable range of joint was 
rendered wider than that of the single drug-given rabbits and the gait 
disturbance was also outstandingly remedied. 
The rate of inhibition of the damage to the movable range of joint was 
calculated according to the following formula (IV), and the evaluation of 
synergistic effect was conducted under the same condition as in Test 
Example 1. 
##EQU3## 
MCJV: Average movable range of joint with respect to the rabbits of the 
drug-given group 
MTJV: Average movable range of joint with respect to the rabbits of the 
drug-given group 
TABLE 8 
______________________________________ 
Inhibi- 
tory HA % Syner- 
Dose rate + gistic 
Test agent (mg/kg) (%) TD % DG % effect 
______________________________________ 
Sodium hyaluro- 
3.0 24.5 
nate (HA) 
Diclofenac 3.0 17.3 
HA + Diclofenac 
3.0 + 3.0 
38.1 36.6 41.8 Great 
______________________________________ 
Molecular weight of sodium hyaluronate: 8 .times. 10.sup.5 
PREATION EXAMPLE 1 
A combined agent having the following formula was prepared. 
______________________________________ 
Sodium hyaluronate 25 mg 
Diclofenac sodium 5 mg 
4% solution of glucose or 
2.5 ml 
4% solution of xylitol 
______________________________________ 
PREPATATION EXAMPLE 2 
Combined agents having following formulas were prepared. 
______________________________________ 
Sodium hyaluronate 25 mg 
Diclofenac sodium 5 to 25 mg 
Sodium hydrogensulfite 1 to 25 mg 
4% solution of glucose or 
2.5 ml 
4% solution of xylitol 
______________________________________ 
In addition to the ingredients used in the Examples, other ingredients can 
be used in the Examples as set forth in the specification to obtain 
substantially the same results.