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EFFECT OF TOPICAL ADMINISTRATION OF ANTIINFLAMMATORY DRUGS TO RATS WITH ADJUVANT ARTHRITIS RICHARD D. HEILMAN and RICHARD R. R E 0 Oral or intramuscular administration of antiinflammatory agents produces numerous undesirable side effects. This work explores the hypothesis that topical administration of such agents directly to the site of inflammation would have beneficial antiinflammatory effects. Topical administration of steroidal and nonsteroidal antiinflammatory drugs to rats with adjuvant arthritis was as effective as oral or intramuscular administration. Peak blood levels of radioactivity following administration of equal doses of hydrocortisone3H were considerably lower after topical administration than after oral administration. Current steroidal and nonsteroidal antiinflammatory drugs provide symptomatic relief of arthritic disease and local inflammatory conditions. However the therapeutic effectiveness of the drugs is limited because severe side effects accompany their use. I n cases of arthritis or local inflammation limited to one or several discrete areas, administration of an antiinflammatory agent directly to the site of inflammation might be more advantageous than oral administration. It was hypothesized that direct From the Ortho Research Foundation, Raritan, New 08869. Richard D. Heilman, Ph.D.: Ortho Research Foundation; Richard R. Reo, B.S.: Ortho Rcsearch Foundation. Address reprint requests to Dr. Heilman. Submitted for publication October 31, 1974; accepted March 21, 1975.

administration would produce relatively high concentrations, on a mg:mg basis, of the antiinflammatory drug at the inflammation site as compared to oral administration. At the same time circulating levels of the drug would be expected to be relatively low after local administration, assuming that systemic absorption from the skin would not be as efficient as that from the gastrointestinal tract. A reduction in the level of circulating drug, with a concomitant high level of the drug at the site of inflammation, would be expected to produce an efficacious antiinflammatory effect with a reduction in drug-induced untoward effects. Intramuscular and intraarticular administration of antiinflammatory steroids is accepted, whereas indomethacin and phenylbutazone are administered orally. T h e efficient topical absorption of acetylsalicylic acid and hydrocortisone in a hydroalcoholic vehicle by rats with adjuvant arthritis has been demonstrated (1). T h e purpose of the present investigation is to support the concept of administering an antiinflammatory agent topically to the site of inflammation. T h e relative potency, efficacy, and blood concentration of several topically administered steroidal antiinflammatory agents to rats with adjuvant arthritis is demonstrated.

Jersey

Arthritis and Rheumatism, Vol. 18, No. 5 (September-October 1975)

MATERIALS AND METHODS Mycobacterial adjuvant arthritis and periarthritis were induced in rats as follows: Alale rats (Wistar-Lewis; Charles River Breeding Lab, Inc) weighing approximately

HEILMAN AND RE0

520

Table 1. Comparison of Antiinflammatory Efficacy of Administration Daily or at a 5-Day Interval of ORF 10703 to Rats with Adjuvant Arthritis Paw Volume, Mean (day 25)

* SE

Administered Daily (days 15 through 25)

Administered at a 5-Day Interval (days 15 and 20)

0.50 (oral) 1.00 (oral) 0.50 (topical) 1.00 (topical)

2.67 C 0.34 (3)*t 1.72 C 0.04 (5)t 2.40 C 0.08 (5)t 1.96 k 0.10 (5)t

3.64 f 0.44 (5) 2.48 iz 0.12 (5)t 2.94 C 0.18 (5)t 2.90 k 0.13 (4)t

Arthritic control (no treatment)

4.50 f 0.30 (4)

3.84 C 0.31 (5)

Total Dose (mg/kg)

Table 2. Comparison of Antiinflammatory Effects on the Adjuvant-injected Hind Paw of ORF 10703 Administered Orally or Topically to Rats

Dose (mg/kg/5 days)

0.075 0.150 0.300 0.600 1.250 2.500 Arthritic control (no treatment)

Paw Volume, Mean C SE (day 25) Oral

*

3.00 0.19 (5)* 3.00 k 0.18 (4) 2.98 f 0.10 (5)t 2.72 f 0.18 (5)t 2.58 f 0.15 (5)t 2.44 f 0.60 (5)t

Topical 2.05 f 0.35 (4)t 1.98 iz 0.13 (4)t

-

2.60 t 0.21 (5)t 1.64 zk 0.15 (5)t 2.14 f 0.15 (5)t

3.60 C 0.25 (5)

*Number of animals in parentheses. +Significantlydifferent from arthritic control (P < 0.05).

*Number of animals in parentheses. *Significantly different from arthritic control (P < 0.05).

130 to 150 g were given a single intradermal-subcutaneous injection of 0.1 ml of a white mineral oil suspension of heat-killed Mycobacterium tuberculosis (concentration: 3.5 mg/ml) into the subplantar area of the left hind paw. Before the injection the suspension was sonified and autoclaved a t 230°C for 10 minutes. O n day 15, 14 days after the injection of adjuvant, the rats were anesthetized with ether, weighed, and their hind paws measured by a mercury displacement method. T h e rats were then placed in groups of 5 so that mean valves of left hind paw mercury displacement did not vary significantly among the groups. Groups of less than 5 animals i n the results section indicate deaths before day 25. T h e body weight and hind paw mercury displacement were measured o n days 20 and 25 of the study. T h e volume displaced by the hind paws is expressed i n all tables as cc of water displaced. Statistical analyses were performed using Student's t test for group data according to Snedecor (2). Drugs administered orally were suspended in water 1% Tween 80 with sonification. O R F 10703 (6a,9-difluoro-l18,16a, 17,20,tetrahydroxypregna-l,4-diene-3,20-dione3 ethylenedithioketal 16, 17 acetonide), hydrocortisone, indomethacin, and phenylbutazone were soluble in Gantrez@ ES-435 (GAF Corporation). Gantrez is the monobutyl ester of poly (methyl vinyl ether/maleic acid). A 50% solution i n isopropyl alcohol was used. Drugs were administered topically to the left hind paw in Gantrez with a syringe and a blunt-ended needle. Concentrations of drugs were prepared so that Gantrez was applied at a dose of 0.1 ml/lOO g body weight. Rats i n the following studies were treated with drugs twice, o n days 15 and 20, ie, once every 5 days beginning o n day 15. For comparison, i n one study rats were treated daily from day 15 through day 24. In the radioactivity study the rats received only a single dose o n day 15 of hydrocortisone-3H added to 10 mg/kg cold hydrocortisone. Blood samples i n the radioactivity study were collected from the tail into 280 81 Micro-Natelson heparinized capillary tubes. They were weighed, dried i n a n oven a t 50"C, burned i n a Packard Tritium Oxidizer, mixed with

15 ml of Bray's cocktail, and counted for radioactivity. Antiinflammatory efficacy, ie inhibition of paw edema, was not measured in the radioactivity study.

+

RESULTS Effect of Daily versus I n t e r m i t t e n t A d m i n i s t r a tion. A comparison was m a d e of t h e a n t i i n f l a m m a t o r y effects of ORF 10703 a d m i n i s t e r e d daily for 10 consecutive d a y s ( s t a r t i n g on day 15), w i t h a d m i n i s t r a t i o n on d a y s 15 and 20. Paw v o l u m e s in b o t h g r o u p s were m e a s u r e d on d a y s 20 and 25. The results, s u m m a r i z e d for day 25 in Table 1, i n d i c a t e t h a t daily a d m i n i s t r a t i o n was more effective t h a n a d m i n i s t r a t i o n o n c e every 5 days. However b o t h m e t h o d s of a d m i n i s t r a t i o n p r o d u c e d significant a n t i i n f l a m m a t o r y effects. Comparison of t h e P o t e n c y of a Steroid Administered by Various R o u t e s . T h e results of a c o m p a r i s o n s t u d y in w h i c h ORF 10703 was a d m i n i s t e r e d orally a n d topically t o r a t s w i t h a d j u v a n t a r t h r i t i s a r e s u m m a r i z e d in T a b l e 2. ORF 10703 was more p o t e n t w h e n a d m i n i s t e r e d topically. ORF 10703 p r o d u c e d a near 100% m a x i m u m i n h i b i t i o n of inflammation. OKF 10703 was a d m i n i s t e r e d topically in Gantrezm or i n t r a m u s c u l a r l y in saline. The results, s u m m a r i z e d in Table 3, s h o w t h a t ORF 10703 was more p o t e n t when delivered topically t h a n w h e n administered intramuscularly. T a b l e 4 s u m m a r i z e s t h e effects of topical a n d i n t r a m u s c u l a r a d m i n i s t r a t i o n on t h e c o n t r a l a t e r a l ( n o n a d j u v a n t injected) hind paw. W h e r e a s a l t e r n a t e flanks were used for t h e i n t r a m u s c u l a r injections, topical a d m i n i s t r a t i o n was always t o t h e a d j u v a n t i n j e c t e d (left) hind paw. I n f l a m m a t i o n i n t h e r i g h t hind paw was significantly i n h i b i t e d by i n t r a m u s c u l a r

52 1

ANTIINFLAMMATORY DRUGS I N RATS

Table 3. Comparison of Antiinflanittiatory Effects on the Adjuvant-injected Hind Paw of ORF 10703 Adtninistered Intramuscularly or Topically to Rats Paw Volume, Mean f SE (day 25) Dose (mg/kg/5 days) 0.075 0.150 0.300 0.600 1.250 2.500 5.000 Arthritic control (no treatment)

Intramuscular

Topical

2.86 k 0.33 (!I)* 2.12 t 0.14 (5)t 2.18 i 0.17 (5)t 2.00 t 0.15 (5)t 1.70 t 0.04 (5)f 1.85 t 0.14 (4)t

2.3.5 f 0.13 (4)t 2.38 f 0.17 (5)t 2.02 k 0.07 (5)t 1.72 f 0.05 (5)t 1.54 f 0.07 (5)t

-

-

1.45 f 0.09 (4)t

Table 4. Comparison of Antii,if/attriiiatory Effects on the h'otiadjuvrint-iirjecte~Hind Paw of ORF 10703 Administered Intrattrusrularly or Topirrtl1.y to Rats Noninjectctl Paw Volume, Ifcan f SE (day 25) Dose (mg/kg/5 days)

Intramuscular 2.24 C 0.25 (5)* 1.78 +- 0.10 ( 5 ) 1.60 0.08 (5)t 1.40 t 0.07 (5)t 1.34 f 0.04 (5)t 1.38 f 0.03 (4)t

0.075 0.150

*

0.300 0.600 1.250 2.500 5.000

-

Arthritic control (no treatment)

2.72 k 0.09 (5)

Topical 1.88 f 0.19 (4) I .YO f 0.23 (5) 1.46 f 0.04 (5)t 1.22 f 0.04 (5)t 1.26 t 0.04 (5)f

-

1.10 & 0.06 (4)t

2.04 k 0.09 (5)

*Number of animals in parentheses. +Significantly different from arthritic control (P < 0.05).

*Number of animals in parentheses. tsignificantly different from arthritic control ( P < 0.05).

and topical administration of ORF 10703, but at higher doses than those required to significantly inhibit left hind paw edema (compare Tables 3 and 4). Studies with Hydrocortisone-3H. Hydrocortisone-3H was administered orally, topically, and intramuscularly to determine the peak plasma level of radioactivity and the time of the peak plasma level. T h e results of the experiment are summarized in Table 5. Peak blood levels of radioactivity were reached within 1 hour following oral administration of 25 or 125 pC of hydrocorti~one-~H. Intramuscular administration of the drug produced earlier blood radioactivity peaks (15 minutes) whereas topical administration produced blood radioactivity peaks at 15 to 24 hours. Oral administration produced the greatest relative blood level of radioactivity. Intramuscular administration produced about 447$ of the oral peak, and topical administration in Gantrezm produced about 8 to 17y0 of the oral peak. Comparison of the Potency a n d Efficacy of Indomethacin and Phenylbutazone Administered by

Various Routes. T h e results of the comparison of oral and topical administration of indomethacin and phenylbutazone to rats with adjuvant arthritis are summarized in Tables 6 and 7. Indomethacin was less potent when administered topically than when administered orally. By either route of administration an antiinHammatory effect with a maximum inhibition of inflammation of about 65u/, was produced. Plienylbutazone was less potent than indomethacin. A maximum of 56% inhibition of inflammation was produced by phenylbutazone when administered orally. Phenylbutazone did not produce a dose response curve by the topical route. However phenylbutazone administered topically significantly inhibited inflammation by about 30y0 at the doses employed. Indomethacin was lethal at the highest dose level.

DISCUSSION Adjuvant arthritis in rats has been shown to bear a resemblance in many respects to human arthri-

Table 5. Comparison of the Eoect of Route of Administration on Blood Levels of Radioactivity (Hydrocortisone-JH)in Rats with Adjuvant Arthritis

Group Oral (25 pC) Topical (25 pC) Intrasmuscular (25 p C ) Oral (125 pC) Topical (125 pC)

*Animal number.

Mean Peak Blood Level f SE Relative Blood Level DPM/ml Blood of Radioactivity 172,773 t 18,429 14,387 t 364 76,670 t 7,537 817,712 -C 92,649 137,636 i 14,899

1.o 0.08 0.44 4.73 0.80

T i m e of Peak Blood Radioactivity/Rat (hours)

(I)* 7

7

(3)* 1 15

% %

%

%

1

15

15

1 15

%

(2)*

%

HEILMAN AND R E 0

522

Table 6. Comparison of Oral and Topical Antiinflammatory Eflects of Indomethacin on the Adjuvant-injected Hind Paw in Rats Injected Paw Volume, Mean f SE (day 25) Dose (mg/kg/S days) 5 10 20 40 Arthritic control (no treatment)

Oral 2.82 f 0.14 (5)*t 2.43 f 0.09 (3)t 2.35 +- 0.25 (2)t

-

Topical 3.22 & 0.39 2.80 f 0.19 (5)t 2.50 0.19 (4)t 2.50 (1)

*

4.26 & 0.19 (5)

*Number of animals in parentheses. ?Significantly different from arthritic control (P < 0.05).

tis (3). Tlie rat adjuvant polyarthritis model is currently accepted and preferred by many investigators as the best available animal model of rheumatoid arthritis (4-6). Adjuvant arthritis in rats therefore seemed to be a suitable model for the study of the feasibility of topical administration of antiinflammatory drugs to provide efficacious symptomatic relief of arthritic or local inflammatory conditions. ORF 10703, the steroidal antiinflammatory agent used in this study, was shown in previous unpublished studies to be similar to fluocinolone acetonide (SynalarB) in animal studies. Tlie results clearly demonstrate the effectiveness of topical administration of steroidal and nonsteroidal antiinflammatory drugs to the rat. I n fact the potency of a steroidal compound, O R F 10703, seems to he enhanced by topical administration. Tlie antiinflammatory effects o n tlie hind paw opposite to that treated topically with O R F 10703 suggest that a) the drug is absorbed in sufficient quantity to produce a systemic effect, or b) treatment of tlie adjuvantinjected paw in rat adjuvant arthritis relieves symptoms manifested in places otlier than tlie primary site. T h e first alternative is supported by the results with radioactive hydrocortisone. I n addition to the fact that the drug is absorbed systemically, the liytlrocortisone-{H studies indicate that a) systemic absorption following topical administration is slower than oral absorption, and b) the peak blood level of tlie drug is lower with topical than with oral administration. Although the total area under the radioactive curve5 was not compared, the differences in absorption antl peak blood levels suggest that following topical administration a sustained but relatively low level of circulating drug may accompany an efficacious antiinflammatory effect.

Table 7. Comparison of Oral and Topical Antiinflammatory Eflects of Phenylbutazone on the Adjuvant-injected Hind Paw in Rats Injected Paw Volume, Mean f SE (day 25) Dose (mg/kg/5 days) 125 250 500 1000 Arthritic control (no treatment)

Oral 2.94 f 0.21 (5)*t 3.23 f 0.24 (4)t 2.45 k 0.14 (4)t -

Topical 3.13 & 0.18 (4)t 3.30 & 0.30 (2)t 2.96 f 0.09 (5)t 3.23 f 0.54 (4)$

4.26 f 0.19 (5)

*Number of animals in parentheses. +Significantly different from arthritic control (P < 0.05). $P < 0.20.

When antiinflammatory drugs are administered orally or intramuscularly, the time of the peak blood level of tlie drug is significant because it indicates the time that maximum quantities of the drug are circulating throughout the body. Such circulation distributes the drug to the site of inflammation. Following topical administration of an antiinflammatory drug directly to tlie site of inflammation, the time of the peak blood level of the drug may have a different meaning. Distribution of the drug through the systemic circulation following topical administration may in effect reduce the level of the drug at the inflamed site. Following oral or intramuscular administration a rapid dilution by distribution may be produced. Topical administration may produce relatively high concentrations of antiinflammatory drug at the site of inflammation for comparably long periods of time before the drug is diluted by absorption and distribution throughout the systemic circulation. T h e reason for the unexpected effectiveness of topical administration in this model is not known. However it may result from a “tissue depot” effect of the drug that enables it to be locally absorbed into the vascular system in the immediate area of inflammation. This absorption may provide relatively concentrated distribution of tlie drug (undiluted by tissue absorption throughout the body and without possible gastrointestinal antl liver metabolism) to the affected joint. In addition a “sustained” release of the drug into the systemic circulation may expose the affected joint to tlie drug for a relatively prolonged period. I n order to optimize the effectiveness of topical administration of antiinflammatory drugs, an ap-

A N T I I N F L A M M A T O R Y DRUGS I N R A T S

propriate vehicle must be coml)ined with the various chemical and physical characteristics of the drug to be employed i n order to provide maximum therapeutic results. Obviously the so1ul)ility of tlie drug in tlie vehicle, tlie extent of absorption through tlie skin, and the rate of absorption tlirough the connective tissue and into the systemic circulation are important. T h e importance of the proper vehicle-drug combination has been demonstrated with the topical use of antiinflammatory steroids for the therapy of dermatosis (7-1 1). Although Gantrezn was a highly compatible vehicle for the administration of the tliHuorinated steroid, OKF 10703, to rats, it is possible that a more appropriate vehicle coirltl have been selected for use with indomethacin and phenylbutazone. T h e antiinHammatory effectiveness of tlie latter nonsteroidal compounds could possibly have been improved by a better choice of vehicle.

ACKNOWLEDGMENTS T h e authors wish to thank J. Bisaha for her assistance with the manuscript, Art Costaris for his assistance with the radioactivity studies, a n d Geigy Chemical Corporatioil and Merck Sharp and Dohme for their gifts of phenylbutazone and indomethacin. O K F 10703 was synthesized at Ortho Pharmaceutical Corporation by J. A. Settepani and K. Mallory.

REFERENCES 1. Walz D T , Dolan MM, DiMartino hIJ, et al: Effects of topical hydrocortisone and acetylsalicylic acid o n the

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primary lesion of adjuvant-induced arthritis. Proc SOC Exp Riol hlctl 125:1466-1469, 1971 2. Snetlecor G W : Statistical hlethods. Fifth edition. Cedar Falls, Iowa State College Press, 1956 3. Pearson Chl: Experimental joint disease. Observations on adjuv~int-inducetlarthritis. J Chronic Dis 16:863874, 1963 4. Newl)ould BB: Chemotherapy of arthritis induced in rats by mycobacterial adjuvant. Br J Pharmacol 21: 127-136, 1963 5. Graeme hlL, Fabry E, Sigg EB: Mycobacterial atljuvant periarthritis in rodents a nd its modification by antiinHammatory arid analgesic drugs. J Pharmacol Exp T h e r 153:373-380, 1966 (i.Sofia KD, Vassar HB, Nalepa SD: Correlations between pithological changes in the hind paws of rats with adjuvant arthritis and their response to antiinHammatory arid analgesic drugs. Eur J Pharmacol 24: 108-1 12, 1973 7. Sarkany I, Hadgraft JW, Caron GA, et al: T h e role of vehicles in the percutaneous absorption of cortiocosteroids: an experimental and clinical study. Br J Dermatol 77:569-575, 1965 8. hIcKenzie AW: Percutaneous absorption of steroids. Arch Dermatol 86:611-614, 1962 9. Reid J. Brookes DB: Topical corticosteroids-an experimental evaluation of the vasoconstrictor test as ari index of anti-inHammatory activity. Br J Dermatol 80: 328-336, 1968 10. Feldmann KJ, Maibach HI: Percutaneous penetration of steroids in man. J Invest Dermatol 52:89-94, 1969 11. Cramer MB, Cates LA: Effects of dimethyl sulfoxide a nd trimethylphosphine oxide o n percutaneous absorption of corticosteroids in the rat. J Pharm Sci 63:793794, 1974