Annals of the Rheumatic Diseases 1993; 52: 817-822

Effects of the molecular weight of hyaluronic acidand its action mechanisms on experimental jointpain in rats

Sachiko Gotoh, Jun-Ichi Onaya, Mayumi Abe, Kyosuke Miyazaki, Akio Hamai,Katsuyuki Horie, Kiyochika Tokuyasu

Tokyo ResearchInstitute,SeikagakuCorporation,Tokyo, JapanS GotohJ-I OnayaM AbeK MiyazakiA HamaiK HorieK TokuyasuCorrespondence to:Sachiko Gotoh,Tokyo Research Institute,Seikagaku Corporation,1253 Tateno 3-chome,Higashiyamato-shi,Tokyo 207, Japan.Accepted for publication23 June 1993

AbstractObjectives-It has been shown previouslythat hyaluronic acid (HA) has an analgesicaction on bradykinin induced pain in theknee joints of rats. This study furtherclarifies the effects of the molecularweight ofHA and its mechanism of actionin the same model using HA of molecularweight 800 to 2-3 X 106 daltons and abradykinin antagonist.Methods-Bradykinin and the test HApreparations were given to rats by intra-articular injection, and the severity ofpain was evaluated by a change in thewalking behaviour.Results-HA with a molecular weightgreater than 40 kilodaltons producesanalgesic effects with a simultaneous orearlier injection. The ID50 values of HAwith molecular weight 40, 310, 860, and2300 kilodaltons were greater than 2 5, 0-6,0 07, and 0-06 mg/joint respectively. Theduration of the analgesic effect of 860and 2300 kilodalton HA was 72 hours at10 mg/ml, whereas that of 310 kilodaltonHA was short, being undetectable after24 hours. The analgesic action of HA of860 kilodaltons was not changed by pre-treatment with four saccharide HA andinhibited by pretreatment with HA largerthan six to eight saccharides, capable ofbinding to HA receptors. Further, HA didnot interfere with the analgesic action ofthe bradykinin antagonist, indicating thatHA does not directly bind with bradykininreceptors.Conclusions-HA with a molecular weightof greater than 40 kilodaltons producedan analgesic effect, and HA of 860 and2300 kilodaltons produced high and long-lasting analgesia. These effects of HAappear to be caused by the interactionbetween HA and HA receptors.

(Ann Rheum Dis 1993; 52: 817-822)

The main clinical symptom of osteoarthritis isjoint pain, which makes daily life unpleasant.The pain may also promote progress of thedisease process,'"3 and result in decreasedmobility of the joint due to lack of use. Conse-quently, reducing pain is important for themaintenance of joint activity and quality of life.

Hyaluronic acid (HA) is the main com-ponent of synovial fluid. High molecularweight HA with a random coil configurationhas viscoelasticity and occupies a large amountof hydrodynamic space in solution.4 Itsfunctions in the joint are considered to beas a lubricant,56 a protector of cartilage,7-9 andan effector of the metabolism of synovialtissue.'0

Intra-articular HA treatment of the knee ofpatients with osteoarthritis reduces painfulsymptoms and improves general activities ofdaily living and joint mobility."-"' The mech-anism of joint pain is not clear, so the role ofHA in pain suppression requires further study.In our previous study using the bradykinininduced joint pain model in rats, we showedthat HA acting in a joint can induceanalgesia. 14

In this study we investigated the effects ofthe molecular weight of HA on analgesia andfurther attempted to clarify its mode of actionusing HA of molecular weight 800 to 2-3 X 106daltons in the same model.

Materials and methodsMATERIALSHA preparations were extracted and highlypurified from rooster combs; their average mol-ecular weights were 6-8 (HA 6-8), 40 (HA 40),310 (HA 310), 860 (HA 860), and 2300kilodaltons (HA 2300). The molecular weightwas calculated from the intrinsic viscosityaccording to the formula of Laurent et al.'5 HAof four saccharide units (4S HA) and a mixtureof six and eight saccharide HA (8S HA) wereprepared from HA 6-8 as follows. HA 6-8 wasdigested by partially purified bovine testicularhyaluronidase (Seikagaku, Tokyo, Japan) at50°C for 96 hours. After digestion, degradedHA was fractionated by ethanol precipitation,followed by gel filtration chromatography withCellulofine-GCL 90 m (Seikagaku). Each oligo-saccharide fraction was desalted, lyophilisedand analysed by high performance liquidchromatography (Hitachi Model 638-30)according to the method of Yoshida et al."6Bradykinin and bradykinin specific inhibitor(bradykinin antagonist), Des-Arg9-(Leu8)-bradykinin, were purchased from the PeptideInstitute (Osaka, Japan). All preparations weredissolved in phosphate buffered saline (pH 7 4)or saline.

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Table I Changes in walking behaviour caused by intra-articular injection of bradykinin

Walking behaviouir Grade Indication

Normal No change in walking

Lameness Lam-I Lameness alone for 20 seconds or lessLam-II Lameness alone for more than 20 seconds

Pulling Pul-I Only transient pulling up of the injected leg (five seconds or less)Pul-II Pulling up of the injected leg, followed by lameness

Three legs Thr-I Transient (for five seconds or less) walking on three legs, followed by lamenessThr-II Walking on three legs for more than five seconds, followed by lameness

INDUCTION OF KNEE PAIN AND EVALUATIONSYSTEMMale SD rats, bred by Japan SLC (Shizuoka,Japan), aged 5-6 weeks, were used. Theanimals were previously placed for more thanfive minutes in long steel cages, allowing clearobservation of their feet, and acclimatised tothe new environment. Each injection wascarried out with 0 05 ml/joint a time. A controlgroup was set up in every experiment to checkthe reproducibility. Tests were performed bythree workers; one operator prepared andinjected the test solutions and the rest of theworkers observed the animals for two minutesand evaluated the pain reaction blindly. Intra-articularly injected bradykinin causes shortknee pain reactions from five to 10 seconds toabout two minutes after the injection. A lowdose of bradykinin causes transient lameness,which is extended by slightly increasing theamount of bradykinin. If the dose is increasedfurther, pain symptoms include pulling up ofthe injected leg, and subsequently walking onthe three uninjected legs. The last two werefollowed by lameness in most instances. Thechanges were differentiated and evaluated asshown in tables 1 and 2.

S'FATISTICSResults are shown as the mean (SE) of severalexperiments. Differences among the groupswere evaluated by one way analysis of variancefollowed by Newman-Keuls's test for multiplecomparisons.

ResultsBRADYKININ INDUCED KNEE JOINT PAIN

RESPONSE AND ITlS REPRODUCIBILITY

Bradykinin caused dose dependent painreactions in rats when 0 05 ml was injected ata concenltration of 0-56-45 pLg/ml (fig 1). Theanalgesic score was 3 at a bradykinin concen-tration of 5 pLg/ml. A score of 3 means that therats pulled up the injected leg, followed bylameness, and the rating of score 3 is easy.Unless noted otherwise, 5 pLg/ml bradykininwas used to induce pain in the subsequent

Table 2 Behaviour grading cnteria for evaluating painreaction

Grade of Walking Evaluationalgesia behaviour score

None Normal walking 0Slight Lam-I IMild Lam-II or Pul-I 2Moderate Pul-II or Thr-I 3Severe Thr-II 4

4

0Ca)0

al)

3

2

0.1 1 10Concentration of bradykinin (kg/ml)

100

Figure I Dose-response curve ofpainl response induced by,izltra-articular injection of bradykinin 0 05 nizl in rats.Results are shozwn as niean (SE) values (n = 6-38).

examinations. There was no difference inthe pain level among experimental days when5 p.g/ml bradykinin or bradykinin 30 minutesafter a saline injection was given (fig 2).

EFFECT1 S OF MOLECULAR WEIGH'T OF HA

INJECTED WITH BRADYKININ ON PAIN RESPONSEHA preparations of different molecular weightsof various concentrations were injected withbradykinin simultaneously. HA 40, 310, 860,and 2300 inhibited the pain at concentrationsof -50, ¢10, >-2-5, and -1 25 mg/mlrespectively. HA 6-8 had no effect, even at100 mg/ml (fig 3).The percentage inhibition was obtained with

respect to the control. The ID50 values of HA40, 310, 860, and 2300 were >50, 12, 1 4, and1-2 mg/ml as concentration, and >2-5, 0-6,0 07, and 0-06 mg/joint as dose respectively(fig 4). The ID50 value decreased withincreased molecular weight of HA, but nodifference between the ID50 values of HA 860and HA 2300 was noted.

DURATI ON OF ANALGESIA

A pain reaction was induced 24, 48, 72, and96 hours after administration of HA 310, 860,and 2300. HA 860 and 2300 inhibited pain at:5 mg/ml after 24 hours, at 10 mg/ml after 48and 72 hours, and had no effect after 96 hours.HA 310 had no effect at any of these times(fig 5).

EFFECTIS OF HA OLIGOSACCHARIDE ON

ANALGESIA OF HA 860This test was conducted to examine whetherthe analgesic action of HA is produced by theinteraction ofHA with HA receptors. It is well

n

818

Effects of hyaluronic acid on joint pain

6IEI~

4 1A

o

C.)

(0a)

2 -

1 -

1 2 3 4 5 6 1 2 3 4 5 6Exoerimental number

Figure 2 Reproducibility of bradykinin induced knee pain response. (A) Bradykinin injection of5 Ag/ml (n = 4-8);(B) Bradykinin of 5 ,ug/ml at 30 minutes after saline injection (n = 3-10).

~L T

T

10 25 50 5 10 20

HA-40 HA-31 0

II

0 63 1 25 2 5 5 10

HA-860

0o63125 2 5 5 10 mg/mi

HA-2300

Figure 3 Effects of hyaluronic acid (HA) with different molecular weights on bradykinin inducedjoint pain in rats.Each HA was injected into a joint simultaneously with bradykinin (n = 6-31). *p < 0-05; **p < 0 01. Molecular weightofHA-6-8, HA-40, HA-310, HA-860, and HA-2300 are 6-8, 40, 310, 860, and 2300 kilodaltons.

known that four saccharide HA does not bind

to the HA receptors on the cell surface, andHA with six or eight saccharide units is theminimum size for binding.'7 18 Thus weexamined whether the analgesic effect ofHA 860 was suppressed by pretreatment with

100 r

80F

0

.0

._

0..... .4

60

40 [

20

n01 1 TO 100Concentration of hyaluronic acid (mg/ml)

Figure 4 Dose-response curves of hyaluronic acid (HA)with different molecular weights. Each percentage inhibitionofpain response was obtained with respect to the controlusing the data offig 3. (L) HA-6-8; (A) HA-40;(A) HA-310; (-) HA-860; and (0) HA-2300.The abbreviations are the same as those in fig 3.

4S HA, 8S HA, and HA 68. The pain reactionwas induced by the injection of bradykininalone and by the simultaneous injection ofbradykinin and HA 860 of 10 mg/ml 30minutes after administration of 4S HA, 8S HA,and HA 6&8. HA 860 inhibited the pain inknees with preinjected saline, 4S HA of10 mg/ml, and 8SHA of 2-5 mg/ml, but didnot inhibit it in knees injected with 10 mg/ml8S HA or ¢2-5 mg/ml HA 68. The resultsindicated that pretreatment with 8S HA andHA 6-8 suppressed the analgesic effect ofHA 860, and HA 6-8 greatly decreased it,whereas 4S HA did not (fig 6). These findingssuggest that the analgesia of HA is producedby the interaction between HA and HAreceptors.

EFFECT OF LOW MOLECULAR WEIGHT HA ONANALGESIA OF BRADYKININ ANTAGONISTTo examine whether HA binds directly tobradykinin receptors and competes againstbradykinin antagonist activity, a test wasconducted with the combined use of HA.

4

3

a)oC.)U,)0U)a)CD

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10 100

HA-6.8

0

Cont

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819

2

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Gotoh, Onaya, Abe, et al

-

Figure 5 Time courses of analgesic effects of hyaluronic acid (HA) with different molecular weights. HA was administeredintra-articularly 24, 48, 72, and 96 hours before the bradykinin injection. (A) 24 hours (n = 6-28); (B) 48 hours(n = 6-21); (C) 72 hours (n = 6-27); and (D) 96 hours (n = 7-19). *p < 0-05; **p < 0-01. The abbreviations are thesame as those infig 3.

Bradykinin antagonist at 2-5 and 1O,ug/mlinhibited pain, depending on the dose, wheninjected simultaneously with 5 ,ug/ml brady-kinin (fig 7). As the bradykinin antagonist hasa short life in vivo, bradykinin was injected fiveminutes after administration of the bradykinin

t 2

Figure 6 Antagonistic effects ofhyaluronic acid (HA) oligosaccharide on analgesia ofHA with high molecular weight. Saline and each oligosaccharide were administeredintra-articularly 30 minutes before bradykinin or injection of a combination of bradykininand high molecular weight HA (n = 6-36). **p < 0 01. (4S-HA) four saccharide HA,(8S-HA) mixture ofsix and eight saccharide HA. The abbreviations are the same as thosein fig 3.

Figure 7 Antagonistic effect of Des-ArgY-(Leu)-bradykinin(bradykinin antagonist) on bradykinin induced painresponse. Drug was injected simultaneously with bradykinin(n = 7-38). **p < 0-01. Concentration of bradykinin,S wug/ml.

antagonist. This short interval betweeninjections enhanced the pain. Thus in this testa bradykinin concentration of 2-5 pLg/ml was

chosen to obtain an analgesic score of 3 in thecontrol group for easy rating. HA 6-8 was

selected as it binds to HA receptors but showsno analgesia, and will suppress bradykininantagonist activity if it binds to bradykinin

2

.

820

Effects of hyaluronic acid on joint pain

4

3

(1)

0

U)

C.)

a)C)

2

1 st treatment

Bradykinin antagonist

HA-6 8

2nd treatment

Bradykinin +

+ +

+ +

+

+

Figure 8 Effect oflow molecular weight hyaluronic acid (HA) on analgesic effect ofbradykinin antagonist. Drugs were given intra-articularlyfive minutes before bradykinininjection (n = 7-38). *p < 0 05; **p < 0 01. Concentrations of bradykinin, bradykininantagonist and low molecular weight HA (HA-6-8) were 2-5, 10, and 10 mg/mlrespectively. The abbreviations are the same as those in figs 3 and 7.

receptors. A pain reaction was induced byinjecting 2-5 ,ug/ml bradykinin five minutesafter administration of bradykinin antagonist(10 ,ug/ml), HA 6 8 (10 mg/ml), and a mixtureof the two. Bradykinin antagonist and themixture with HA 6-8 inhibited the painequally, but HA 6*8 did not. This indicates thatHA had no effect on the interaction betweenbradykinin and bradykinin receptors (fig 8).

DiscussionAlthough many investigations have suggestedthat HA plays an important part in variousbiological activities, the precise function ofHAin these events is not yet clear. Nevertheless, itis likely that the influence of HA depends on

(a) its remarkable hydrodynamic propertiesand (b) its interaction with the cell surface.'9We hypothesised that information on the

effects of the molecular weight ofHA might bethe key for revealing the mechanisms of theanalgesia of HA. In this study using HA witha molecular weight of 800 to 2.3 x 106 daltons,the results indicated that HA with a molecularweight range of 40 to 860 kilodaltons producedan analgesic action dependent on molecularweight and concentration. HA 860 andHA 2300 showed almost the same ID50 valueat 1-4 and 1-2 mg/ml (0 07 and 0-06 mg/joint)on simultaneous injection with bradykinin, andthe same duration of analgesia (72 hours).HA 310 showed a short term effect, suggestingthat a relatively high molecular weight HA isneeded to produce longlasting analgesia. HA 860and HA 2300 show almost the same activity,however, in spite of the fact that HA 2300 has

a higher viscosity. A previous study suggestedthat the analgesic effect ofHA was obtained bycovering the tissue and cell surface painreceptors, and not by viscosity."4 From thesefindings, we have concluded that the analgesiceffect of HA is due to its affinity to cells ratherthan to the direct hydrodynamic physicalproperties and viscosity corresponding tomolecular weight.The interaction of HA with HA binding

receptors on the cell surface is considered to bean important factor in determining cellbehaviour. Underhill and Toole"7 and Laurentet al'8 investigated the effect of the molecularweight of HA on the binding ability to thereceptor in cultured cells in vitro and foundthat binding of HA to its receptors occurredwhen the saccharide number of HA was largerthan six or eight. The larger the molecule ofHA, the higher the affinity; when the hydro-dynamic size of exogenous HA was very large,the number of binding molecules that couldoccupy the available cell surface receptors wasreduced.

This led us to investigate the role of HAreceptors on the cell surface in the analgesicaction of HA. Among 4S HA, 8S HA, andHA 6-8, 8S HA and HA 6-8, capable ofbinding to receptors, suppressed the biologicalactivity ofHA 860, and the degree of inhibitiondepended on the concentration and saccharidechain length of HA. On the other hand, theaction of the bradykinin antagonist was notaffected by HA 6-8, indicating that HAmolecules do not appear to bind directly tobradykinin receptors. Consequently, the effectof HA appears to be brought on by the inter-action between HA and HA receptors, and notby the interaction between HA and bradykininreceptors.We had previously proposed another possible

explanation for the analgesic action. HA has anexpanded random coil configuration whichcauses entanglement between neighbouringmolecules. We had thought that HA in thesynovial fluid may trap bradykinin in itsmolecule by the ion bonds formed between thecarboxyl base in HA with the negative chargeand bradykinin with the cation charge,followed by maintaining an entangled coilconfiguration. This is not feasible, however,because analgesia by HA 860 no longeroccurred after HA oligosaccharide pretreat-ment, as shown in fig 5, and when thebradykinin solution was dialysed with the filtermembrane, the dialysis rate of bradykinin wasnot changed by adding HA in solution (datanot shown).The results of this study indicate that (a) HA

with a molecular weight greater than 40 kilo-daltons produces analgesic effect, (b) high andlonglasting analgesia is induced by HA withmolecular weights of 860 and 2300 kilodaltonsequally, and (c) that analgesia was inhibited byoligosaccharides of HA. Moreover, HA did notdirectly bind to bradykinin receptors, indicatingthat analgesia ofHA appears to be brought onby the interaction of HA and HA receptors onor surrounding the free nerve endings thatdetect pain in the joint tissue.

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