Cyclosporin Inhibits Hyperalgesia and Edema in Arthritic Rats: Role of the Central Nervous System

Home , Edema

BrazilianCyclosporin Journal inhibition of Medical of arthritic and Biological hyperalgesia Research and edema (1997) 30: 101-111 101 ISSN 0100-879X

Cyclosporin inhibits hyperalgesia and edema in arthritic rats: role of the central nervous system

J.N. Francischi1, 1Departamento de Farmacologia, Instituto de Ciências Biológicas, L.S.M. Pereira1 Universidade Federal de Minas Gerais, 31270-010 Belo Horizonte, MG, Brasil and M.S. Castro2 2Departamento de Farmacodinâmica, Faculdade de Farmácia, Universidade Federal de Minas Gerais, 30180-112 Belo Horizonte, MG, Brasil

Abstract

Correspondence Since arthritis induced by Mycobacterium products (adjuvant) in rats Key words J.N. Francischi is considered to be immunologically driven, the objective of the • Cyclosporin Departamento de Farmacologia present study was to determine if the immunosuppressor drug • Hyperalgesia Instituto de Ciências Biológicas cyclosporin could affect hindpaw edema and joint hyperalgesia simul- • Edema Universidade Federal de taneously. Female Holtzman rats (140-170 g) presented hyperalgesia • Adjuvant arthritis Minas Gerais Av. Antonio Carlos, 6627 and edema on the 8th and 12th day following adjuvant injection. Daily 31270-010 Belo Horizonte, MG systemic (oral or intramuscular) administration of cyclosporin (0.5- -1 -1 -1 -1 Brasil 5.0 mg kg day ) or dexamethasone (0.01-0.1 mg kg day ) for 15 Fax: 55 (031) 441-0835 days starting on day zero dose-dependently inhibited the hindpaw E-mail: [email protected] edema and hyperalgesia in arthritic rats. However, hyperalgesia but not edema could be detected two days after cyclosporin withdrawal. Research supported by We concluded that a) the continuous presence of cyclosporin is CNPq (No. 520449/94-0), FAPEMIG (No. CBS-1652/92) essential to reduce the development of joint hyperalgesia and that b) and PRPq, MG, Brasil. different mechanisms underlie the appearance of hyperalgesia and edema in this model. The intracerebroventricular (icv) administration of 5-50-fold smaller doses of cyclosporin (1.5-150 µg/day) or dexamethasone (15 µg/day) also reduced the arthritic hindpaw edema and Received May 14, 1996 hyperalgesia. Peripheral blood from animals injected with effective Accepted October 25, 1996 systemic cyclosporin doses showed detectable levels of the drug, whereas peripheral blood from those injected with icv cyclosporin did not, as measured by specific RIA. Our results indicate that cyclosporin administered by the central route is as effective as by the systemic route to reduce joint hyperalgesia and hindpaw edema in arthritic rats. The antiarthritic effect induced by low doses of cyclosporin in the central nervous system (CNS) could be explored to avoid its often associated systemic side effects during chronic therapy. However, the mechanism(s) involved in the antiarthritic response to cyclosporin in the CNS remain to be elucidated.

Braz J Med Biol Res 30(1) 1997 102 J.N. Francischi et al.

Introduction Material and Methods

The rat adjuvant arthritis model has been Animals widely used to study experimentally the pathophysiology of human rheumatoid ar- Female Holtzman rats (140-170 g) were thritis because of the similarities between used throughout this study. The animals were the two syndromes (1-5). This model has weighed every four days from day zero to the also proved to be a useful tool for investigat- end of the experiments, always before any ing new analgesic and anti-inflammatory experimental procedure. Unless otherwise compounds (6,7). stated, the animals were kept in cages (maxi- Experimental arthritis has been shown to mum of 6 animals per cage) with water and be immunologically driven (8,9), and immu- food ad libitum, a temperature of 26 ± 3oC, nosuppressive drugs such as cyclosporin and on a 12-h light-dark cycle. (CsA) and rapamycin are known to affect its development (10,11). The immunosuppres- Induction of adjuvant arthritis sive action of cyclosporin and rapamycin has been associated with their ability to re- Rats were injected subcutaneously with a duce the inflammatory hindpaw edema pres- single dose of 0.2 ml mineral oil-water emul- ent in arthritic animals and with their capac- sion (10:1, v/v) containing 400 µg of dried ity to interfere with normal T-cell functions. Mycobacterium butyricum into the dorsal Both drugs inhibit the expression of a dis- root of the tail under ether anesthesia. The crete set of lymphokines by promoting the time of adjuvant injection is referred to as binding of cytosolic proteins called cyclo- day zero. Control rats received a subcutane- philin and FK-binding protein (FKBP) to the ous injection of 0.2 ml of the oil-water emul- corresponding genes. Extensive reviews of sion without bacilli on the same day. the action of cyclosporin and rapamycin have been provided by many authors (11-13). Measurement of articular hyperalgesia and Corticosteroids exert their anti-inflam- hindpaw edema in rats matory and immunosuppressive effects pri- marily by interacting with cytosolic recep- The method for measuring hyperalgesia tors (14) different from those for cyclosporin has been previously described (16). Briefly, and rapamycin. In addition to regulating tar- the tendency of normal (naive), control and get gene expression, corticosteroids are also arthritic rats to vocalize following flexion of able to modify post-translational events and the tarsotibial joints of both hindpaws was second messenger systems including the ac- tested daily for 22 days starting from day

tivity of phospholipase A2 (15). zero. The results are reported as the mean Using different approaches, we and oth- number ± SEM of vocalizations obtained for ers have shown that arthritic rats develop five flexions per paw. Hindpaw volume (as hyperalgesia (16-18). The objective of the an indicator of edema) was measured daily present study was to determine whether the using an Ugo Basile hydroplethysmometer hyperalgesia of arthritic rats could be influ- (model 7150) after the test for hyperalgesia. enced by CsA administered either peripher- The volume (ml) of one hindpaw was essen- ally or into the central nervous system (CNS) tially the same as that of the contralateral of the animals. For comparison, dexametha- paw (data not shown) and is reported as the sone, a standard anti-inflammatory drug, was mean ± SEM. All measurements were ob- tested in parallel. tained at the same time of day.

Braz J Med Biol Res 30(1) 1997 Cyclosporin inhibition of arthritic hyperalgesia and edema 103

Schedules for systemic and CNS drug Radioimmunoassay for cyclosporin in blood administration samples

Systemic administration. Various doses of Blood (1-2 ml) was collected into EDTA cyclosporin (0.5, 2.5 and 5.0 mg kg-1 day-1) tubes by cardiac puncture from ketamine- or dexamethasone (0.01 and 0.1 mg kg-1 day-1) xylazine-anesthetized arthritic rats, which diluted in sterile saline were administered by were already receiving cyclosporin, on the gavage (1 ml/100 g animal) or intramuscu- 8th or 12th day of treatment. Blood samples larly (im, 0.1 ml/100 g animal) from day zero were also collected from two other groups of to the 14th day after induction of arthritis. animals (2 rats/group) which had received All drugs were administered 2 h after the 15 or 150 µg/day CsA, respectively, 4 h determination of hindpaw hyperalgesia and before. The blood samples were stored at edema, except on day zero, when the drugs -15oC until assayed. Whole blood is the pre- were administered 2 h before induction of ferred sample for assay (21). The whole arthritis. Control arthritic rats received blood samples were assayed by radioimmu- the same volume of vehicle (diluted cremo- noassay (RIA) according to manufacturer phor El or saline) administered by the same instructions (Sandimmun kit, Sandoz, Santo route. Amaro, SP), using monoclonal antibodies CNS administration. The animals were specific for the form of cyclosporin injected. prepared for intracerebroventricular (icv) The ratio of RIA and high-performance liq- injection under ketamine-xylazine anesthe- uid chromatography (HPLC) in whole blood sia as used by Lemaire et al. (19). Metal was not significantly different from one (22). cannulae were adapted from hypodermic The limit of determination of the assay was needles (7-gauge) and fixed in a hole previ- 25 ng/ml. Briefly, aliquots of 50 µl of whole ously made in the exposed skull of each blood from all samples (standard, control, anesthetized animal. The stereotaxic coordi- and unknown) were pretreated with 950 µl nates used for cannulation were: anteropos- methanol. Aliquots of the methanolic super- terior, -1.4 mm, laterolateral, -1.5 mm and natant (50 µl) were prediluted with buffer vertical, 3.1 to 3.5 mm below the skull sur- (500 µl) and incubated with [3H]-CsA and face (20). The animals were housed in sepa- the specific monoclonal antibody for 2 h at rate cages (one animal per cage) and arthritis 4oC. Bound and free CsA were separated at was induced 4-5 days following the implan- 4oC after the addition of a charcoal suspen- tation of the cannulae, when the animals sion and centrifugation. Bound activity was started to put on weight. Single doses of CsA measured in the supernatant by liquid scin- (1.5, 15 or 150 µg/day) or dexamethasone tillation counting (RackBeta, Beckman, (15 µg/day) diluted in sterile saline were Irvine, CA). cautiously injected into awake animals over a period of 15 days (final volume, 10 µl). Materials The injections were performed using the free extremity of the fixed metal cannulae and Mycobacterium butyricum powder was occurred during a 30-sec period. Control purchased from Difco (lot 43887JC, Detroit, arthritic rats received an icv injection of 10 MO). The following solutions were used: µl of diluted cremophor El (cyclosporin ve- ketamine (Ketalar, Parke-Davies, Aché- hicle) or physiological saline instead of CsA Laboratórios, SP), xylazine (Rompun, Bayer, or dexamethasone, respectively. RS) and dexamethasone (Decadron, MSD,

Braz J Med Biol Res 30(1) 1997 104 J.N. Francischi et al.

Prodome Química Farmacêutica, SP). Re- considered to indicate statistical significance. agents for RIA and cremophor El were sup- plied by Sigma (St. Louis, MO) and Results cyclosporin (Sandimmun) and Sandimmun kit were a generous gift from Mr. Álvaro De To establish the initial experimental con- Paoli from Sandoz, Santo Amaro, SP, Brazil. ditions, 400 µg of M. butyricum (adjuvant) was injected into the dorsal root of the tail of Statistics ether-anesthetized rats. This dose induced a significant hyperalgesia in the hindpaw joints The results were analyzed by the one- by the 8th day after injection. The number of tailed Student t-test for single comparisons vocalizations ranged from 0.2 ± 0.08 to a and one-tailed ANOVA for multiple com- maximum value of one (1.0) per five flex- parisons with a control. P less than 0.05 was ions of the paws on the 12th day of observation as compared with naive (non-injected)

1.20 or control animals (P<0.05; Figure 1A). Hy- A peralgesia remained elevated up to the 21st * * * * * * * 1.00 * day of observation (Figure 1A) by which time the incidence of paw ankylosis was * 0.80 high (35%) and precluded prolongation of

0.60 the experiments (data not shown). The level of edema was significant by the 12th day Hyperalgesia 0.40 * after injection and resulted in a more than (vocalization/5 flexions) two-fold increase in the volume of both 0.20 hindpaws by the 21st day of observation, as

0.00 illustrated for one of the hindpaws in Figure 0 2 4 6 8 1012141618 2022 1B. A dose of 200 µg of M. butyricum did not induce significant hyperalgesia or edema 6.00 (data not shown). B * In order to determine the effect of 5.20 cyclosporin in this model of arthritis and * compare it with dexamethasone, various 4.40 doses of both drugs were administered sys- * * temically to adjuvant-induced arthritic rats.

Edema (ml) 3.60 * Administration of cyclosporin (0.5-5.0 mg kg-1 day-1) or dexamethasone (0.01-0.1 mg * 2.80 kg-1 day-1) by the oral or im route dose- dependently reduced both the joint hyperal- 2.00 0246810121418202216 gesia and hindpaw edema of arthritic rats Time (days) (Figure 2A-D). An im 5 mg kg-1 day-1 dose of cyclosporin completely blocked both the Figure 1 - Development of hyperalgesia (A) and edema (B) in arthritic rats. Arthritis was development of hyperalgesia and edema (Fig- induced by the subcutaneous injection of 0.2 ml of an emulsion containing 400 µg Myco- bacterium butyricum ( ) into the tail base of female rats on day zero. The reponses of ure 2B and D, respectively) throughout the £ control ( ) and normal ( ) rats are also shown. The daily hyperalgesia measurements are 15 days of administration. A significant re- · ! reported as the number of vocalizations upon 5 flexions of the tibiotarsal joints of the duction in hyperalgesia was also observed animals (mean ± SEM, N = 4-6). Hindpaw edema was measured using an Ugo Basile with the lowest oral dose of cyclosporin (0.5 hydroplethysmometer (model 7150). Hindpaw volumes are reported as the mean increase -1 -1 in volume (ml) ± SEM. *P<0.05 compared to naive or vehicle-treated animals (ANOVA t- mgkg day ; Figure 2A), although the same test). dose did not affect hindpaw edema to a

Braz J Med Biol Res 30(1) 1997 Cyclosporin inhibition of arthritic hyperalgesia and edema 105 similar extent (Figure 2C). Dexamethasone tribution of the central nervous system to (0.1 mg kg-1 day-1) administered either orally this “analgesic” effect of the drug. Icv ad- or im was virtually identical to cyclosporin ministration of cyclosporin (1.5-150 µg/day) (5 mg kg-1 day-1) in reducing joint hyperalge- dose-dependently reduced joint hyperalgesia and hindpaw edema in arthritic rats sia and hindpaw edema in arthritic rats (Fig- throughout the period of drug administration ure 3). As with the systemic response this (Figure 2A-D). A lower oral dose of dexa- reduction was observed throughout the pe- methasone (0.01 mg kg-1 day-1) was less ef- riod of drug administration, and was most fective in inhibiting the development of both complete at the highest dose of cyclosporin arthritic indices (Table 1). However, follow- used (150 µg/day, Figure 3A and B). In ing cessation of drug treatment, recurrence addition, as in the case of systemic treat- of hyperalgesia in the arthritic rats was more ment, a significant level of hyperalgesia, but rapid for 0.1 mg kg-1 day-1 dexamethasone no edema, could be detected after terminat- than for 5 mg kg-1 day-1 cyclosporin (Figure ing treatment with this dose of cyclosporin 2A and B). There was no recurrence of (150 µg/day, Figures 3A and 3B). A shorter hindpaw edema at these doses after oral or period of treatment (8 days starting from day im cyclosporin and dexamethasone adminis- zero) with 15 mg of cyclosporin/day admin- tration (Figure 2C and D). istered icv was also effective in inhibiting Since cyclosporin had inhibited joint hy- the hyperalgesia and edema in adjuvant-in- peralgesia, we decided to determine the con- jected rats (Table 2). In this case, hyperalge-

1.20 1.20 A - oral B - im 1.00 1.00

0.80 0.80 * * ** 0.60 * 0.60 * * * Hyperalgesia

Hyperalgesia * 0.40 * * 0.40 * (vocalization/5 flexions) (vocalization/5 flexions) 0.20 * * * 0.20 * * * * * * * * * * * * * * * 0.00 * * 0.00 0 2 4 6 8 1012141618 2022 0 2 4 6 8 1012141618 2022

6.00 6.00 C - oral D - im 5.20 5.20

4.40 4.40

3.60 3.60 Edema (ml) * Edema (ml)

2.80 2.80 * * * * * * * * * * * 2.00 2.00 0369121518 21 0 246 81012 14 16 18 20 22 Time (days) Time (days)

Figure 2 - Dose-response curves of cyclosporin administered im and orally on hyperalgesia (A and B) and edema (C and D) presented by arthritic rats. Cyclosporin (ª = 0.5; = 2.5, = 5.0 mg kg-1 day-1, N = 6-9) was administered from day zero to the 14th day of arthritis induction orally (A and C) or · ! intramuscularly (B and D). The daily hyperalgesia measurements are reported as the number of vocalizations upon 5 flexions of the tibiotarsal joints of the animals (mean ± SEM, N = 4-6). Hindpaw volumes are reported as the mean volume increase (ml) ± SEM. For comparison, the results of hyperalgesia and edema presented by rats receiving 0.1 mg kg-1 day-1 dexamethasone ( , N=6) and control arthritic rats ( ) are also shown. *P<0.05 £ compared to control arthritic rats (ANOVA t-test). @

Braz J Med Biol Res 30(1) 1997 106 J.N. Francischi et al.

Table 1 - Dose-dependent reduction of joint hyperalgesia and hindpaw edema presented by arthritic rats treated with dexamethasone by the oral route.

Dexamethasone or saline (control) was administered once a day orally (by gavage) to rats from day zero to the 14th day of arthritis induction (N = 6-11 per group). Hyperalgesia: number of vocalizations upon 5 flexions of the hindpaw tarsotibial joints of the animals (mean ± SEM). The hindpaw edema was measured by plethysmometry and is reported as volume increase in ml (mean ± SEM). *P<0.05 compared to paired control (ANOVA t-test).

Group Day after induction of arthritis

Hyperalgesia Edema

0 8th 12th 0 8th 12th

Control 0.20 ± 0.04 0.26 ± 0.07 0.98 ± 0.07 2.11 ± 0.02 2.11 ± 0.01 2.43 ± 0.05 Dexamethasone 0.01 mg kg-1 day-1 0.25 ± 0.08 0.11 ± 0.06 0.43 ± 0.20* 2.12 ± 0.01 2.07 ± 0.02 2.17 ± 0.02* 0.1 mg kg-1 day-1 0.28 ± 0.08 0.06 ± 0.03* 0.06 ± 0.03* 2.08 ± 0.02 2.05 ± 0.02 2.03 ± 0.01*

Figure 3 - Intracerebroventricular administration 1.20 of cyclosporin dose-dependently reduced the A hyperalgesia (A) and edema (B) presented by 1.00 arthritic rats. Cyclosporin ( = 1.5, · = 15, ª = 150 µg/day in a 10 µl volume)! was administered 0.80 by the intracerebroventricular route from day zero to the 14th day of arthritis induction. Con- * trol arthritic rats ( ) received 10 µl cremophor El 0.60 £ * * (vehicle) diluted in sterile physiological saline * * * * using the same schedule. The daily hyperalgesia Hyperalgesia * 0.40 * * * measurements are shown as the number of

(vocalization/5 flexions) * * vocalizations upon 5 flexions of the tibiotarsal 0.20 * * joints of the animals (mean ± SEM, N = 4-7). * * Hindpaw volumes are presented as the mean ± * 0.00 SEM (in ml). For comparison, the results of hy- 0 2 4 6 8 10 12 14 1618 20 22 peralgesia and edema presented by rats treated with 15 µg/day dexamethasone ( , N = 4) are also shown. *P<0.05 compared@ to animals treated with vehicle (ANOVA t-test). 5.00 B

4.20

3.40 * * * Edema (ml) *

2.60 * * * * *

1.80 0 2 4 6 8 10 12 14 1618 20 22 Time (days)

Braz J Med Biol Res 30(1) 1997 Cyclosporin inhibition of arthritic hyperalgesia and edema 107 sia relapse occurred the second day after the µg) of cyclosporin did not contain detectable end of treatment (data not shown). As ob- levels of the drug. served for cyclosporin, a similar dose of 15 An invariable sign of arthritis develop- µg/day dexamethasone administered icv sig- ment in induced rats was the stabilization or nificantly reduced the hindpaw hyperalgesia a net decrease of their weight about the 8th (Figure 3A; P<0.05, ANOVA) and com- day of induction (Figure 4). In contrast, ar- pletely blocked the hindpaw edema of arthritic rats treated orally with 5 mg cyclo- thritic rats (Figure 3B). However, one of the sporin kg-1 day-1 had the same profile of 5 animals was found dead on the last day of weight gain as control (Figure 4) or normal treatment (14th). The hyperalgesia and edema animals (data not shown). Different from observed in arthritic animals injected icv cyclosporin, however, rats given 0.1 mg of with either saline or vehicle were not signif- dexamethasone kg-1 day-1 by the oral route icantly different from each other. significantly lost weight between the 4th and Since the antiarthritic effect of the low the 12th day of arthritis. This response was doses of cyclosporin administered icv could not observed when dexamethasone was ad- have derived from a peripheral action, we ministered im or icv (data not shown). No collected systemic blood samples from those other behavioral or neurological sign of tox- animals in an attempt to detect cyclosporin. icity was observed in cyclosporin- or dexa- Table 3 summarizes the CsA concentrations methasone-treated animals. obtained by specific RIA in whole blood samples. The time course of CsA concentra- Discussion tion for the oral route during the treatment with a 5-mg/kg dose showed an increase The rat adjuvant arthritis model repro- from the 8th to the 12th day of administra- duces many of the signs and symptoms of tion, whereas samples from icv-treated ani- human rheumatoid arthritis (1-4). Drugs mals did not contain any detectable amount which reduce or block the development of of the drug (Table 3). In addition, peripheral hindpaw edema in arthritic rats have been blood collected at the 12th day of treatment useful in increasing our understanding of the from animals which had been treated 4 h pathophysiology of rheumatoid arthritis and before with effective icv doses (15 or 150 in the detection of anti-inflammatory, immu-

Table 2 - Effect of the duration of cyclosporin administration by the intracerebroventricular route on joint hyperalgesia and hindpaw edema in arthritic rats.

Cyclosporin was administered at a daily dose of 15 µg/animal in a volume of 10 µl. Control animals were injected with the same volume of vehicle (N = 5-7). Hyperalgesia: number of vocalizations upon 5 flexions of the hindpaw tarsotibial joints of the animals (mean ± SEM). Hindpaw edema was measured by plethysmometry and is reported as volume increase in ml (mean ± SEM). *P<0.05 compared to paired control (ANOVA t-test).

Duration of Day after induction of arthritis treatment Hyperalgesia Edema

8th 12th 15th 8th 12th 15th

Control 0.56 ± 0.04 1.0 1.0 2.07 ± 0.01 2.42 ± 0.04 4.09 ± 0.04

15 days 0.02 ± 0.01* 0.25 ± 0.05* 0.44 ± 0.13* 2.01 ± 0.12 2.08 ± 0.08* 2.88 ± 0.20*

8 days 0.04 ± 0.02* 0.82 ± 0.08 1.0 2.02 ± 0.02 2.22 ± 0.05* 4.10 ± 0.12

Braz J Med Biol Res 30(1) 1997 108 J.N. Francischi et al.

nomodulatory and analgesic effects of drugs tected in the blood and joint cavities of ar- (6,18,23-25). thritic persons as well as in adjuvant-in- In the present study, we have shown that jected rats (27-29). IL-1 induces T-lympho- the development of joint hyperalgesia (by cyte activation and proliferation through the the 8th day) precedes the development of release of another cytokine, IL-2 (27,30), hindpaw edema (12th day) following the which, when injected intra-articularly into induction of arthritis. The development of the rat knee joint, is also able to induce this hyperalgesia was paralleled by a weight mechanical hyperalgesia (31). The disease- loss compared to control animals and may induced release of IL-1 may thus activate reflect the increasing difficulty of the ani- immunocompetent cells with a consequent mals in reaching their food. However, a de- release of IL-2 into the joints, thereby ex- creased appetite could also contribute to the plaining the articular hyperalgesia observed weight loss observed since this phenomenon in arthritic rats in this study. The presence of is an established feature of the active im- IL-1 itself in the knee joint could also ac- mune phase of arthritis in humans (26). Thus, count for some of the hyperalgesia detected hyperalgesia, weight loss and decreased ap- since this cytokine is able to induce hyperal- petite may indicate involvement of the CNS gesia in the rat paw (32) and in the rat knee in the pathophysiology of the experimental joint (29). disease in a manner similar to that observed In accordance with the above hypothesis, for the human condition. immunosuppressive drugs such as cyclo- The involvement of the immune system sporin, which inhibit the release of various in both human and experimental arthritis is cytokines including IL-2 by activated lym- well known. Interleukin 1 (IL-1), one of the phocytes (33), should be able to inhibit the prominent cytokines involved in the early development of hyperalgesia in arthritic rats. activation of T-lymphocytes, has been de- Indeed, cyclosporin was reported to allevi- ate the pain associated with arthritis in human trials (34,35). In the present study, we Table 3 - Cyclosporin (CsA) concentration in whole blood detected by specific radioimmunoassay after oral and intracerebroventricular adminis- demonstrated that the daily oral or im admin- tration to rats. istration of cyclosporin blocked the development of hyperalgesia and edema in arthritic Aliquots of 50 µl whole blood from all samples (internal standard-added, oral rats in a dose-dependent manner. While the and icv) were pretreated with 950 µl methanol. Aliquots of the methanolic supernatant (50 µl) were prediluted with buffer (500 µl) and incubated with anti-edematogenic effect remained blocked [3H]-CsA and the specific monoclonal antibody for 2 h at 4oC. Bound and throughout the period of observation (22 o free CsA were separated at 4 C after the addition of a charcoal suspension days), even after treatment was stopped, the and centrifugation. Bound activity was measured in the supernatant by liquid scintillation counting (RackBeta, Beckaman). Data are reported as analgesic effect of oral cyclosporin was mean ± SD for N animals in each group. Measurements were made on the clearly reversed within two days after drug 8th and 12th days following oral or icv administration, and in additional withdrawal. These results suggest that a) groups on the 12th day (15 and 150 µg/day), 4 h after icv cyclosporin administration. N.D. = Not detectable. continuous presence of cyclosporin is essential in order to reduce the development of Route and dose CsA concentration in whole blood (ng/ml) joint hyperalgesia and that b) different mecha-

8th day 12th day 12th day nisms underlie the appearance of joint hyperalgesia and hindpaw edema in this model. Oral Cyclosporin, an undecapeptide with a -1 -1 5 mg kg day 170 ± 28 (N = 5) 640 ± 161 (N = 4) - pronounced hydrophobic character (36), is icv considered not to cross the blood-brain bar- 15 µg/day N.D. (N = 6) N.D. (N = 6) N.D. (N = 2) 150 µg/day N.D. (N = 5) N.D. (N = 3) N.D. (N = 2) rier (BBB) to any significant extent following systemic administration in man and mice

Braz J Med Biol Res 30(1) 1997 Cyclosporin inhibition of arthritic hyperalgesia and edema 109

(11,37). When cyclosporin was injected into 220 the CNS of adjuvant-induced rats, it was able to produce the same antiarthritic effects 204 observed when systemically administered. Since cyclosporin does not cross the BBB, 188 one must conclude that this antiarthritic effect was exerted directly through one or more preexisting sites in the CNS which Weight (g) 172 were reached upon injection. This conclu- sion is supported by the 5- to 50-fold lower 156 * * dose of cyclosporin required to produce this * 140 antiarthritic effect compared to the doses 0 3 6 9 12 15 18 21 normally administered systemically to ani- Time (days) mals or humans. The antiarthritic response observed with a shorter schedule of cyclo- Figure 4 - Changes in the weight (g) of arthritic rats treated systemically with cyclosporin sporin or a low dose of dexamethasone in- and dexamethasone. The animals were weighed every four days from day zero to the 21st day after induction of arthritis. Cyclosporin (ª, 5 mg kg-1 day-1, N = 6) or dexamethasone ( , jected by the same route in arthritic rats gives 0.1 mg kg-1 day-1, N = 6) was administered orally up to the 14th day of arthritis induction.@ further support to this view. However, dexa- The weight of rats injected with mineral oil without adjuvant ( , N = 4) and of control ! arthritic rats ( ) is also shown. *P<0.05 compared to control arthritic rats (ANOVA t-test). methasone centrally or systemically admin- £ istered has shown to be unexpectedly more toxic than cyclosporin administered by the acute icv administration of effective (antiar- same route in arthritic rats, despite the cen- thritic) doses. Consistent with an antiarthritic tral clinical side effects reported during con- effect induced by cyclosporin in the CNS is ventional therapy with cyclosporin (38). the fact that neurons are a rich source of Dexamethasone-treated animals, especially cyclophilin (30), the cytosolic receptor for those orally treated, showed a pronounced cyclosporin, whose interaction could ulti- weight loss, indicating a probable drug-in- mately lead to inhibition of IL-2 production duced damaging action on the gastrointesti- by them. As yet, however, no definite link nal tract. In addition, the icv administration between the cellular action of cyclosporin at of an effective anti-arthritic dose of dexa- the neuronal cyclophilin level and its antiar- methasone resulted in the death of one ani- thritic effect in the CNS has been estab- mal. Together, the present data support the lished. Other cell lines such as the special- view that dexamethasone is more toxic to the ized macrophages present in the microglia animals than cyclosporin. could eventually contribute to the observed The concept of ‘immunological privi- cyclosporin-induced antiarthritic effect in the lege’ established for the CNS (39) has been rat CNS. challenged (40). In addition, a highly regu- In summary, the results of the present lated communication between the brain and study show that joint hyperalgesia precedes the immune system via the cervical lymphat- the development of hindpaw edema in ar- ics and BBB has been reported (41). In the thritic rats. Daily systemic (oral or intramus- present study we provide evidence showing cular) administration of cyclosporin dose- that the central administration of effective dependently reduced both hyperalgesia and low doses of cyclosporin did not reach the edema. Unexpectedly, a 5 to 50-fold lower periphery, since no detectable levels dose of cyclosporin administered icv also (<0.001%) were demonstrable in the sys- reduced the hyperalgesia and edema, thus temic blood collected from the animals fol- demonstrating for the first time a central lowing 8 or 12 days of treatment, or after antiarthritic effect for this drug. Although

Braz J Med Biol Res 30(1) 1997 110 J.N. Francischi et al.

the precise central site (or sites) for the ob- Acknowledgments served action was not determined, we suggest that the development of new formula- The authors gratefully acknowledge tions for delivery of cyclosporin to the CNS Dalton L. Ferreira-Alves (MD) and Maria of rodents may provide a similar antiarthritic Ap. K.F. Tatsuo (PhD) for their advice dur- effect. This would be of great benefit for the ing the study. We also wish to thank Mr. long term immunosuppression of selected Webster G.P. Dos Reis for excellent techni- cases of human rheumatoid arthritis and re- cal assistance. lated conditions, since it would avoid the side effects frequently associated with systemic cyclosporin therapy.

References

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