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Tocainide suppression of immune-complex-mediated dermal inflammation: Comparison with prostaglandin E1
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
39, 452-463 (1986)
Tocainide Suppression of Immune-Complex-Mediated Inflammation: Comparison with Prostaglandin RICHARD H. WHITE,
DICK L. ROBBINS,GARY ANDDALLAS M. HYDE
Dermal El
L. HENDERSON,
Departments of Medicine and Pharmacology, School of Medicine and Department School of Veterinav Medicine, University of California, Davis, California
of Anatomy, 95616
Local anesthetic agents have been shown to alter a variety of polymorphonuclear leukocyte (PMN) functions and may be useful as anti-inflammatory agents. We compared the anti-inflammatory effects of therapeutic doses of the recently released local anesthetic-antiarrythmic drug tocainide to pharmacologic doses of prostaglandin El (PGEl) on immune-complex-mediated dermal inflammation in female Sprague-Dawley rats. Intense dermal inflammation was produced using a classic reverse passive Arthus reaction, and the inhibition of PMN accumulation in the subdermis was quantitated in biopsy samples taken 2.5 hr after the reaction was initiated and the drug was given. Using a light microscope with a counting grid, biopsy sections were randomly sampled in a blinded fashion and an inflammation index equal to the ratio of PMNs to fibroblasts was determined for each animal. The mean inflammation index in 10 animals given 25 mg of tocainide (mean serum level = 14.6 kg/ml) was 9.3 t 1.2 ( f SEM), which was significantly less than the index of 17.7 + 2.5 in 10 control animals (P < 0.025). Similarly, the five animals that received either 500 or 250 ug of PGEl had a significantly reduced index, with the effect of 250 ug PGEl comparable to the effect of the tocainide. These findings suggest that therapeutic levels of tocainide reduce the accumulation of PMNs in immune-complex-mediated dermal inflammation; thus, local anesthetic agents may be useful in the treatment of certain inflammatory disorders. 0 1986 Academic Press. Inc.
INTRODUCTION
While there have been major advances in the treatment of large-vessel necrotizing vasculitis in the past 10 years (I), the treatment of small vessel or “hypersensitivity” vasculitides remains problematic (2). The pathogenesis of this group of disorders, which share in common the histologic finding of leukocytoclastic vasculitis (2, 3), appears to involve immune complex formation with a polymorphonuclear leukocyte (PMN)-mediated local inflammatory response in the perivenular and pericapillary bed (4, 5). Treatment is often unsatisfactory since conventional forms of therapy, such as high doses of glucocorticoids and immunosuppressive drugs, are usually ineffective (1, 2). One group of drugs that may hold some promise in the treatment of inflammatory disorders are the local anesthetics. It has been recognized for some time that local anesthetics (cationic tertiary amines) have potent effects on membrane-dependent responses to surface stimuli in a variety of cell types, including PMNs, macrophages, and platelets (6). There is evidence that these compounds prevent mobilization of Caz+ stores in cells (7), reduce the osmotic fragility of erythrocytes (8), inhibit exocytosis in several types of secretory cells (9), inhibit cell fusion (lo), and diminish intercellular and cell-substrate adhesion (11). Specific 452 0090-1229186 $1.50 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
TOCAINIDE
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effects on PMNs include decreased release of lysosomal enzymes on exposure to phagocytosable particles, and alteration of cell membrane structure, as determined by electron microscope (6). In addition, local anesthetics can alter the locomotion, spreading, and particle ingestion of phagocytic cells (6, 12). Two commonly used local anesthetics are lidocaine and tocainide. The latter is a recently released oral analog of lidocaine that has excellent bioavailability when given orally and a long serum half-life of lo-15 hr in humans (13). Stewart and her colleagues have shown that both lidocaine and tocainide inhibit leukocyte adherence to and migration through venous endothelium in dogs (14, 15). In humans, the constant intravenous infusion of lidocaine prevents the development of postoperative deep vein thrombosis (16), and this effect may be due to inhibition of leukocyte adhesion to the venous vascular endothelium (17). Prostaglandin El (PGEl) is an important mediator in both the immune and inflammatory responses (18, 19). The effects of PGEl on PMN function involve reduced lysosomal enzyme excretion induced by endocytosis (20), suppression of acute inflammation (21), and inhibition of immune-complex-mediated acute Arthus reactivity (22). The studies by Kunkel and his colleagues suggest that this latter effect is due, in part, to an alteration in PMN chemotaxis to complementderived chemotactic peptides (22). The effect of local anesthetics on experimentally induced immunologically mediated inflammation has not been well studied. The purpose of this study was to investigate the effect of intraperitoneally administered tocainide on immune-complex-mediated dermal vasculitis in rats using the classic reverse passive Arthus reaction (RPA). Results of this study indicate that therapeutic serum levels of tocainide reduce tissue inflammation and the magnitude of the effect is comparable to the anti-inflammatory effects of subcutaneously administered PGE 1. MATERIALS
AND
METHODS
Animals. Adult female Sprague-Dawley rats (Simonsens Breeding Laboratories) weighing 185-200 g (mean = 195 g) were used in all experiments. Reverse passive arthus reaction. Rabbit IgG containing precipitating antibody to bovine serum albumin (BSA) was obtained from Cappel Laboratories (Cochransville, Pa.). After shaving all hair from the anterior abdominal wall, 2.5 mg of antibody nitrogen in 50 ~1 of 0.15 M phosphate-buffered saline (PBS), pH 7.4, was injected intradermally in three separate locations spaced 2 cm apart on the right side of the abdomen. Parallel to each site on the left side of the abdomen, 50 ~1 of PBS was injected intradermally as a control. Fifteen to twenty minutes after administering antibody subcutaneously, a 0.5-cc bolus of a solution containing 250 mg nitrogen BSA was injected intravenously in a tail vein. Two hours and thirty minutes after administering the BSA, the animals were anesthetized with ether and the skin test sites were surgically excised and immediately placed in 10% formaldehyde. Tocainide administration. Powdered tocainide was kindly provided by Astra Pharmaceutical (Framingham, Mass.). Preliminary data suggested that intraperitoneal injection of 20 mg of tocainide into 190 to 200-g Sprague-Dawley rats resulted in low-therapeutic serum levels. In 10 experimental animals, a solution
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ET AL.
containing 50 mg/ml of tocainide was prepared in 0.15 Ad PBS and 0.5 cc (25 mg) was injected intraperitoneally immediately after intradermal injection of antibody and the PBS in controls. In order to determine peak serum tocainide levels, 1 cc of blood was drawn from the tail artery 20 min after administration of the drug. All animals were bled at the time they were sacrificed and tocainide levels measured using duplicate samples. PGEZ administration. A total of 10 animals was treated with ‘subcutaneously administered PGEl, which was provided by the Upjohn Company (Kalamazoo, Mich.). Five animals received 500 kg and five animals received 250 kg. All injections were made over the back immediately after antibody was administered. Controls. Ten animals served as positive controls and were treated with 0.5 cc of PBS injected intraperitoneally immediately after administration of antibody and 20 min before receiving 250 pg nitrogen of BSA. One animal served as a control for the effect of intradermally administered antibody. This animal was given three intradermal injections of antibody and three intradermal injections of PBS but was never treated with intravenous BSA. Measurement of serum tocainide levels. Serum tocainide levels were determined using a gas chromatograph method using thermionic-specific (nitrogenphosphorous-specific) detection. Sample preparation. Serum samples (0.5 ml) or fortified control samples were transferred to 60 x 125-mm Teflon-lined screw-cap culture tubes. The internal standard [2-amino-N-(2,4,6,-trimethylphenyl) propanamide, obtained from A. B. Haessle, Moelndal, Sweden] was added (0.1 ml of a solution containing 25 kg/ml), followed by 0.1 ml hydroxylamine, 0.3 ml 1 N NaOH, 1 ml saturated NaCl solution, and 6 ml methylene chloride. The mixture was shaken using a vortex mixer for 10 set and then a horizontal shaking table for 30 min. The phases were separated by centrifugation (1OOOg for 10 min) and the aqueous (top) layer was removed by vacuum aspiration and discarded. The organic layer was transferred to a test tube and evaporated at 44°C under a gentle stream of nitrogen. The sides of the test tubes were washed down with methanol and the solvent was evaporated again under nitrogen. Each sample was frozen until ready for analysis or reconstituted with 100 ~1 methanol. Two microliters of this solution was injected into the gas chromatograph. Gas chromatographic analysis. A Varian Model 1400 gas chromatograph equipped with a thermionic specific detector (TSD) was used. The column was a 36 x ‘/s in. glass column packed with 3% OV-17 on 100/120-mesh Gas Chrom Q. The carrier gas (nitrogen) was set at 30 ml/min, the air flow at 130 ml/min, and the hydrogen flow rate at approximately 4 ml/min (gauge reading of 3 psig). The column temperature was 2OO”C, the injector temperature was 285°C and the detector temperature was 300°C. The bias voltage was set at -4 V and the bead current adjusted to give a standing current of 70% full-scale deflection. Retention times for tocainide and the internal standard were 4.3 and 2.8 min, respectively. Calibration curves were constructed using pooled rat plasma fortified with tocainide to give concentrations of 0, 1, 5, 10, and 20 l&ml and with the internal standard at a fixed amount of 2 pg (0.1 ml of 25 pg/ml standard solution). The ratio of the area under the peak for tocainide to the area under the peak for the
TOCAINIDE
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internal standard was used for quantitation. Data points were fitted by linear regression. The curves were linear over the range of concentrations analyzed with correlation coefficients greater than 0.980. Histologic analysis. Formaldyde-fixed specimens were embedded in paraffin and 3 Frn-thick sections from the center of each specimen were stained with hematoxylin and eosin. Because of the variable degree of local edema in the samples, the degree of tissue inflammation in the subdermis was measured by determining the ratio of the number of PMNs in a field to the number of fibroblasts. This was accomplished using light microscopy and a square lattice grid (AIOO) at a final magnification of 400 x . All sampled fields were restricted to the subdermis as the reference area by orienting the counting grid in the subdermis parallel with the epidermis and muscularis layers. The initial field was randomly selected at the border of the area of inflammation and each subsequent field was located by using a micrometer to move the slide exactly 5 mm along the area of inflammation. Four fields adjacent to the epidermis were counted and four fields adjacent to the muscularis layer were counted in each specimen. All fields counted were in the dermis in the area of greatest inflammation and edema. This sampling scheme was designed to adhere to area-weighted periodic sampling (23). Polymorphonuclear leukocytes in the lumen of capillaries and venules were not counted nor were vascular endothelial cells counted. All slides were coded and randomly mixed prior to being counted by a trained microscopist, who was unaware of the exact purpose of this experiment. An inflammation index equal to the total number of PMNs in each of eight fields divided by the total number of fibroblasts in each of the eight fields was calculated for each biopsy specimen. The inflammation index for each animal was calculated as the mean of inflammation index for each individual biopsy site. Statistical analysis. A two-tailed Student’s t test was used to analyze the data. A level of P < 0.05 was considered statistically significant. RESULTS
Control animals. Inflammation indices (no. PMN/no. fibroblast) in control and untreated animals are shown in Table 1. A total of 30 RPA sites were biopsied in 10 experimental positive control animals, and 26 of these sites were anatomically intact and oriented in a fashion to allow quantitation of the degree of inflammation. Seven of the animals had three biopsies, two of the animals had two biopsies, and one animal had one biopsy. The value for the inflammation index in the 10 control animals ranged from 10.1 to 30.8 with a mean of 17.7 ? 2.5 ( + SEM). The mean inflammation index of biopsy samples from PBS control sites (negative control) in both treated and untreated animals was uniformly low: the inflammation index ranging from 0.1 to 1.8 with a mean of 0.38 ? 0.01. The one animal treated with intradermal antibody but without intravenous BSA had an inflammation index of 1.3. Tocainide-treated group. In the 10 animals that received 25 mg of tocainide intraperitoneally, 30 sites were biopsied and of these 28 could be adequately analyzed. Eight animals had three biopsies and two animals had two biopsies. The inflammation indices in the tocainide-treated group ranged from 3.8 to 16.3 with a
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1
INFLAMMATION INDEX (No. PMNslNo. FIBROBLASTS 2 SEM) IN SUBDERMIS FOLLOWING A REVERSE PASSIVE ARTHUS REACTION IN RATS TREATED WITH T~CAINIDE (25 MG/ANIMAL) OR PGEl
(500 kg or 250 pg/animal) Experimental Animal 1 2 3 4 5 6 7 8 9
30.8 ZIZ11.8 32.1 k 20.2 13.3 f 4.4 10.1 k 7.9 14.3 2 3.8 15.2 i 9.1 13.8 + 8.1 15.6 2 9.0 21.9 f 7.5
10
10.4
Mean f SEM Note. uP < bP < =P < dP <
Positive control
17.7 2 2.5
Tocainide (25md 12.0 13.8 16.3 5.1 9.8 8.2 7.4 10.2 6.4 3.8 9.3
k 1.5 f 5.2 -c 3.1 2 1.9 -+ 2.7 k 2.3 k 2.6 2 1.6 f 0.3 ” 0.2 _’ 1.23”
group
PGEl (500 I%) 6.6 4.0 7.3 7.4 4.8
f + t f ?
2.2 1.4 3.3 1.1 2.3
6.0 f 0.67b
PGEl
(250I.4 11.8 11.9 8.3 2.0 9.3
+- 4.1 f 2.8 2 1.2 + 1.0
8.7 f 1.8’
Negative control 0.28 0.60 0.11 0.14 1.81
0.43 0.07 0.09 0.16 0.14 0.38 2 O.Old
The index for each animal represents the mean of one to three biopsy sites. 0.025. 0.01. 0.05. 0.001.
mean inflammation index of 9.3 + 1.2, which was significantly lower than the value of 17.7 _t 2.5 in the control group (P < 0.025). All of the animals treated with tocainide became drowsy but were able to crawl in the cage, and no seizure activity was noted. PGEI-treated group. Five animals were treated with 500 kg PGEl and 15 sites were biopsied, of which 14 sites could be analyzed histologically. Inflammation indices in this group ranged from 4.0 to 7.3 with a mean of 6.0 + 0.7. This was significantly lower than the inflammation index in the control group (P < 0.01) and also significantly lower than the index noted in the tocainide-treated group (P = 0.05).
Five animals were treated with 250 kg of PGEI, and 15 biopsy samples were obtained, of which 13 could be analyzed. The inflammation index for each animal ranged from 2.0 to 11.9 with a mean of 8.7 k 1.8. In comparison to control animals, the inflammation index was significantly lower (P < 0.05), but it was not significantly different from the tocainide-treated animals (NS). Within a few minutes after receiving the PGEI , all treated animals were extremely sedated and did not move. Serum tocainide levels. Serum tocainide levels measured in the 10 treated animals at the time they were sacrificed ranged from 5.6 to 19.4 t&ml, with a mean of 14.6 +- 4.0 (+ SD). Serum was obtained at the time of administration of BSA (20 min after injection of the tocainide) in the two animals; serum levels were 20.4 and 26.9 t&ml in these two animals. The serum level of tocainide in these two animals fell to 10.4 and 11.5 Fg/ml, respectively, 2% hr later at the time that the RPA sites were excised.
TOCAINIDE
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Histopatholo~y. Examples of representative tissue sections from control animals, tocainide-treated animals, and PGEl-treated animals are shown in Fig. 1. Sections from positive control animals universally showed vessels full of PMNs, extensive interstitial accumulation of PMNs, and edema. Tocainide- and PGEItreated animals also showed small vessels full of PMNs (Fig. 2) and some interstitial edema; however, the extent of PMN accumulation in the dermal and muscularis was markedly reduced. DISCUSSION
The results of this study demonstrated that tocainide, when given in quantities that approximate the upper limit of the therapeutic range recommended in humans (15 &ml), significantly suppressed acute dermal inflammation induced experimentally in rats using the reverse passive Arthus reaction. The magnitude of this anti-inflammatory effect appears comparable to the effect of 250 pg of subcutaneously administered of PGE 1, a prostaglandin that has been shown to have a potent anti-inflammatory action (22). High doses of PGEl (500 kg/animal) exerted a small but statistically significant greater suppressive effect than the tocainide. Higher doses of tocainide were not studied since we were interested only in the anti-inflammatory effect of doses of tocainide that would approximate the therapeutic range in humans. Mechanisms responsible for the acute inflammatory response that develops in the RPA reaction have been well studied (24). Antigen-antibody complexes formed at the site where antibody is injected intradermally activate the complement system, generating numerous biologically active polypeptides, including C5a, which is highly chemotactic for PMNs (25, 26). Complement depletion or removal of PMNs using specific antisera or cytotoxic drugs ameliorates tissue injury (25). Since the experimental work of Stewart et al. suggested that tocainide may alter PMN migration through vascular endothelium (14, 15), we chose to measure the anti-inflammatory properties of tocainide by directly counting the number of PMNs in the dermal interstitium relative to the number of fibroblasts. Measurement of the relative uptake of radioactively labeled BSA in RPA biopsy sites has been used by others as an index of complement-mediated vascular injury (22). However, since some complement-derived peptides directly increase vascular permeability, this latter assay technique does not specifically assess the effect of the drug on PMN migration. The morphologic features of the dermal inflammation in the treated animals support the hypothesis that both tocainide and PGEl do not inhibit chemoattraction of PMNs to the region of immune-complexmediated complement activation but rather inhibit the migration of PMNs FIG. 1. Reverse passive Arthus Positive control biopsy showing (arrows) full of PMNs. (B) Biopsy (C) Biopsy from a PGEI-treated overall degree of edema formation imals (10% formaldehyde fixed, x
reaction in control, tocainide-treated. and PGEl-treated rats. (A) intense inflammation with edema in the subdermis and venules from a tocainide-treated animal showing less intense inflammation. animal (500 Fgl. (D) Biopsy from a negative control animal. The appeared comparable in control, tocainide- and PGEl-treated an100).
458
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through the vascular endothelium. In the animals that received tocainide, small venules lined with PMNs were noted in most tissue sections. The major difference in appearance between control animals and treated animals was a signiticantly lower relative number of PMNs to fibroblasts in the interstitium. Whether tocainide also impairs PMN ingestion and digestion of immune complexes in viva was not studied. The rationale for using a drug that may alter migration of PMNs through vascular endothelium to treat inflammatory disorders is not without precedent. One of the numerous effects of glucocorticoids is to inhibit the normal migration of PMNs from the intravascular space to the interstitium (27). Given the great difficulty that clinicians have in treating small vessel leukocytoclastic vasculitis, any well-tolerated drug that reduces dermal inflammation would be of great value. In light of the findings in this study, further experimental work to define the efficacy of tocainide in inhibiting immune-complex-mediated vascular inflammation appears warranted. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. IO. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
Fauci, A. S., Haynes. B. F., and Katz, P., Ann. Intern. Med. 87, 660, 1978. Sams, W. M., Thorne. E. G., and Small, P., et rtl., Arch. Dermuto/. 112, 219. 1976. Millikon, L. E.. and Duvall, J. M., C~fis 21, 817, 1978. Mackel. S. E.. Tapperino, G., Brumfield. H., er ~1.. J. C/in. Inwst. 67, 1652, 1979. Kauffmann. R. H., and Valentija, R. M.. Net/l. J. Med. 25, 193, 1982. Goldstein, I. M., Lind, S.. Hoffstein, S., and Weissman, G.. J. Eq. Med. 146, 483, 1977. Feinstein, M. D.. Fiekers, J.. and Fraser /. Phnr-t77ucol. Exp. Tlzer. 197, 215. 1976. Seeman, P., Phurnzucol. Rev. 24, 583, 1972. Kazimierczak, W., Peret, M., and Maslinski, C.. Biochenr. Phrmuco/. 25, 1747. 1976. Papadimitriou, J. M., and Storcina, D.. Ex~. Cell. Res. 91, 233. 1975. Rabinovitch, M., and De Stefano, M., J. Cell Physiol. 68, 327. 1972. Cullen. B. F.. and Haschke, R. H.. Anesthesiology 40, 142, 1974. Kutalek, S. P.. Morganroth. J.. and Horowitz, L. N.. Ann. Intern. Med. 103. 387. 1985. Stewart, G. H., Ritchie. W. G. M., and Lynch, P. R., Amer. J. Puthol. 74, 507. 1974. Stewart, G. H., Knight. L. C.. Arbogart. B. W., and Stern, H. S.. Lab. Inws~. 42, 307, 1980. Cooke. E. D., Bowcock. S. A.. Lloyd, M. J., and Pilcher, M. F.. Lancer 1, 797, 1977. Giddon. D. B., and Lindhe. J.. Amer. J. Purhol., 68, 327. 1972. Robinson, D. R.. 3Ot3): December 1983. Zurier. R. B., and Sayadoff, D. M.. and Torrey. S. B.. Arfhrifis Rlleurn. 20, 723, 1977. Zurier, R. B., Weissmann, G.. and Hoffstein, S., et (I/., J. Clir7. Itnwt. 53, 297. 1971. Bonta, 1. L., Parnham, M. J.. and Van Vliet, L., Ann. Rhelltn. Dis. 37, 212, 1978. Kunkel, S. L., Thrall. R. S.. Kunkel, R. G., McCormick. J. R.. Ward, P. A., and Zurier, R. B.. J. C/in. Invest, 64, 1525. 1979. Cruz-Orive, L. M.. and Weibel, E. R.. J. Micros<,. 122, 235, 1981. Cochrane, C. G., and Dixon, F. J., “Immune Complex Injury in Immunologic Diseases. (M. Sampter, Ed.), p. 210. Little. Brown, Boston, 1979. Kniker. W. T., and Cochrane. C. G., J. Exp. Med. 122, 83, 1965. Cochrane, C. G.. and Aikin. B. S., J. E.up. Med. 124, 733, 1966. Fauci, A. S., Dale. D. C., and Balow, J. E.. Ann. Intern. Med. 84, 304, 1976.
Received October 30, 1985; accepted with revision January 18, 1986
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
39, 452-463 (1986)
Tocainide Suppression of Immune-Complex-Mediated Inflammation: Comparison with Prostaglandin RICHARD H. WHITE,
DICK L. ROBBINS,GARY ANDDALLAS M. HYDE
Dermal El
L. HENDERSON,
Departments of Medicine and Pharmacology, School of Medicine and Department School of Veterinav Medicine, University of California, Davis, California
of Anatomy, 95616
Local anesthetic agents have been shown to alter a variety of polymorphonuclear leukocyte (PMN) functions and may be useful as anti-inflammatory agents. We compared the anti-inflammatory effects of therapeutic doses of the recently released local anesthetic-antiarrythmic drug tocainide to pharmacologic doses of prostaglandin El (PGEl) on immune-complex-mediated dermal inflammation in female Sprague-Dawley rats. Intense dermal inflammation was produced using a classic reverse passive Arthus reaction, and the inhibition of PMN accumulation in the subdermis was quantitated in biopsy samples taken 2.5 hr after the reaction was initiated and the drug was given. Using a light microscope with a counting grid, biopsy sections were randomly sampled in a blinded fashion and an inflammation index equal to the ratio of PMNs to fibroblasts was determined for each animal. The mean inflammation index in 10 animals given 25 mg of tocainide (mean serum level = 14.6 kg/ml) was 9.3 t 1.2 ( f SEM), which was significantly less than the index of 17.7 + 2.5 in 10 control animals (P < 0.025). Similarly, the five animals that received either 500 or 250 ug of PGEl had a significantly reduced index, with the effect of 250 ug PGEl comparable to the effect of the tocainide. These findings suggest that therapeutic levels of tocainide reduce the accumulation of PMNs in immune-complex-mediated dermal inflammation; thus, local anesthetic agents may be useful in the treatment of certain inflammatory disorders. 0 1986 Academic Press. Inc.
INTRODUCTION
While there have been major advances in the treatment of large-vessel necrotizing vasculitis in the past 10 years (I), the treatment of small vessel or “hypersensitivity” vasculitides remains problematic (2). The pathogenesis of this group of disorders, which share in common the histologic finding of leukocytoclastic vasculitis (2, 3), appears to involve immune complex formation with a polymorphonuclear leukocyte (PMN)-mediated local inflammatory response in the perivenular and pericapillary bed (4, 5). Treatment is often unsatisfactory since conventional forms of therapy, such as high doses of glucocorticoids and immunosuppressive drugs, are usually ineffective (1, 2). One group of drugs that may hold some promise in the treatment of inflammatory disorders are the local anesthetics. It has been recognized for some time that local anesthetics (cationic tertiary amines) have potent effects on membrane-dependent responses to surface stimuli in a variety of cell types, including PMNs, macrophages, and platelets (6). There is evidence that these compounds prevent mobilization of Caz+ stores in cells (7), reduce the osmotic fragility of erythrocytes (8), inhibit exocytosis in several types of secretory cells (9), inhibit cell fusion (lo), and diminish intercellular and cell-substrate adhesion (11). Specific 452 0090-1229186 $1.50 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
TOCAINIDE
INHIBITION
OF INFLAMMATION
453
effects on PMNs include decreased release of lysosomal enzymes on exposure to phagocytosable particles, and alteration of cell membrane structure, as determined by electron microscope (6). In addition, local anesthetics can alter the locomotion, spreading, and particle ingestion of phagocytic cells (6, 12). Two commonly used local anesthetics are lidocaine and tocainide. The latter is a recently released oral analog of lidocaine that has excellent bioavailability when given orally and a long serum half-life of lo-15 hr in humans (13). Stewart and her colleagues have shown that both lidocaine and tocainide inhibit leukocyte adherence to and migration through venous endothelium in dogs (14, 15). In humans, the constant intravenous infusion of lidocaine prevents the development of postoperative deep vein thrombosis (16), and this effect may be due to inhibition of leukocyte adhesion to the venous vascular endothelium (17). Prostaglandin El (PGEl) is an important mediator in both the immune and inflammatory responses (18, 19). The effects of PGEl on PMN function involve reduced lysosomal enzyme excretion induced by endocytosis (20), suppression of acute inflammation (21), and inhibition of immune-complex-mediated acute Arthus reactivity (22). The studies by Kunkel and his colleagues suggest that this latter effect is due, in part, to an alteration in PMN chemotaxis to complementderived chemotactic peptides (22). The effect of local anesthetics on experimentally induced immunologically mediated inflammation has not been well studied. The purpose of this study was to investigate the effect of intraperitoneally administered tocainide on immune-complex-mediated dermal vasculitis in rats using the classic reverse passive Arthus reaction (RPA). Results of this study indicate that therapeutic serum levels of tocainide reduce tissue inflammation and the magnitude of the effect is comparable to the anti-inflammatory effects of subcutaneously administered PGE 1. MATERIALS
AND
METHODS
Animals. Adult female Sprague-Dawley rats (Simonsens Breeding Laboratories) weighing 185-200 g (mean = 195 g) were used in all experiments. Reverse passive arthus reaction. Rabbit IgG containing precipitating antibody to bovine serum albumin (BSA) was obtained from Cappel Laboratories (Cochransville, Pa.). After shaving all hair from the anterior abdominal wall, 2.5 mg of antibody nitrogen in 50 ~1 of 0.15 M phosphate-buffered saline (PBS), pH 7.4, was injected intradermally in three separate locations spaced 2 cm apart on the right side of the abdomen. Parallel to each site on the left side of the abdomen, 50 ~1 of PBS was injected intradermally as a control. Fifteen to twenty minutes after administering antibody subcutaneously, a 0.5-cc bolus of a solution containing 250 mg nitrogen BSA was injected intravenously in a tail vein. Two hours and thirty minutes after administering the BSA, the animals were anesthetized with ether and the skin test sites were surgically excised and immediately placed in 10% formaldehyde. Tocainide administration. Powdered tocainide was kindly provided by Astra Pharmaceutical (Framingham, Mass.). Preliminary data suggested that intraperitoneal injection of 20 mg of tocainide into 190 to 200-g Sprague-Dawley rats resulted in low-therapeutic serum levels. In 10 experimental animals, a solution
454
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ET AL.
containing 50 mg/ml of tocainide was prepared in 0.15 Ad PBS and 0.5 cc (25 mg) was injected intraperitoneally immediately after intradermal injection of antibody and the PBS in controls. In order to determine peak serum tocainide levels, 1 cc of blood was drawn from the tail artery 20 min after administration of the drug. All animals were bled at the time they were sacrificed and tocainide levels measured using duplicate samples. PGEZ administration. A total of 10 animals was treated with ‘subcutaneously administered PGEl, which was provided by the Upjohn Company (Kalamazoo, Mich.). Five animals received 500 kg and five animals received 250 kg. All injections were made over the back immediately after antibody was administered. Controls. Ten animals served as positive controls and were treated with 0.5 cc of PBS injected intraperitoneally immediately after administration of antibody and 20 min before receiving 250 pg nitrogen of BSA. One animal served as a control for the effect of intradermally administered antibody. This animal was given three intradermal injections of antibody and three intradermal injections of PBS but was never treated with intravenous BSA. Measurement of serum tocainide levels. Serum tocainide levels were determined using a gas chromatograph method using thermionic-specific (nitrogenphosphorous-specific) detection. Sample preparation. Serum samples (0.5 ml) or fortified control samples were transferred to 60 x 125-mm Teflon-lined screw-cap culture tubes. The internal standard [2-amino-N-(2,4,6,-trimethylphenyl) propanamide, obtained from A. B. Haessle, Moelndal, Sweden] was added (0.1 ml of a solution containing 25 kg/ml), followed by 0.1 ml hydroxylamine, 0.3 ml 1 N NaOH, 1 ml saturated NaCl solution, and 6 ml methylene chloride. The mixture was shaken using a vortex mixer for 10 set and then a horizontal shaking table for 30 min. The phases were separated by centrifugation (1OOOg for 10 min) and the aqueous (top) layer was removed by vacuum aspiration and discarded. The organic layer was transferred to a test tube and evaporated at 44°C under a gentle stream of nitrogen. The sides of the test tubes were washed down with methanol and the solvent was evaporated again under nitrogen. Each sample was frozen until ready for analysis or reconstituted with 100 ~1 methanol. Two microliters of this solution was injected into the gas chromatograph. Gas chromatographic analysis. A Varian Model 1400 gas chromatograph equipped with a thermionic specific detector (TSD) was used. The column was a 36 x ‘/s in. glass column packed with 3% OV-17 on 100/120-mesh Gas Chrom Q. The carrier gas (nitrogen) was set at 30 ml/min, the air flow at 130 ml/min, and the hydrogen flow rate at approximately 4 ml/min (gauge reading of 3 psig). The column temperature was 2OO”C, the injector temperature was 285°C and the detector temperature was 300°C. The bias voltage was set at -4 V and the bead current adjusted to give a standing current of 70% full-scale deflection. Retention times for tocainide and the internal standard were 4.3 and 2.8 min, respectively. Calibration curves were constructed using pooled rat plasma fortified with tocainide to give concentrations of 0, 1, 5, 10, and 20 l&ml and with the internal standard at a fixed amount of 2 pg (0.1 ml of 25 pg/ml standard solution). The ratio of the area under the peak for tocainide to the area under the peak for the
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internal standard was used for quantitation. Data points were fitted by linear regression. The curves were linear over the range of concentrations analyzed with correlation coefficients greater than 0.980. Histologic analysis. Formaldyde-fixed specimens were embedded in paraffin and 3 Frn-thick sections from the center of each specimen were stained with hematoxylin and eosin. Because of the variable degree of local edema in the samples, the degree of tissue inflammation in the subdermis was measured by determining the ratio of the number of PMNs in a field to the number of fibroblasts. This was accomplished using light microscopy and a square lattice grid (AIOO) at a final magnification of 400 x . All sampled fields were restricted to the subdermis as the reference area by orienting the counting grid in the subdermis parallel with the epidermis and muscularis layers. The initial field was randomly selected at the border of the area of inflammation and each subsequent field was located by using a micrometer to move the slide exactly 5 mm along the area of inflammation. Four fields adjacent to the epidermis were counted and four fields adjacent to the muscularis layer were counted in each specimen. All fields counted were in the dermis in the area of greatest inflammation and edema. This sampling scheme was designed to adhere to area-weighted periodic sampling (23). Polymorphonuclear leukocytes in the lumen of capillaries and venules were not counted nor were vascular endothelial cells counted. All slides were coded and randomly mixed prior to being counted by a trained microscopist, who was unaware of the exact purpose of this experiment. An inflammation index equal to the total number of PMNs in each of eight fields divided by the total number of fibroblasts in each of the eight fields was calculated for each biopsy specimen. The inflammation index for each animal was calculated as the mean of inflammation index for each individual biopsy site. Statistical analysis. A two-tailed Student’s t test was used to analyze the data. A level of P < 0.05 was considered statistically significant. RESULTS
Control animals. Inflammation indices (no. PMN/no. fibroblast) in control and untreated animals are shown in Table 1. A total of 30 RPA sites were biopsied in 10 experimental positive control animals, and 26 of these sites were anatomically intact and oriented in a fashion to allow quantitation of the degree of inflammation. Seven of the animals had three biopsies, two of the animals had two biopsies, and one animal had one biopsy. The value for the inflammation index in the 10 control animals ranged from 10.1 to 30.8 with a mean of 17.7 ? 2.5 ( + SEM). The mean inflammation index of biopsy samples from PBS control sites (negative control) in both treated and untreated animals was uniformly low: the inflammation index ranging from 0.1 to 1.8 with a mean of 0.38 ? 0.01. The one animal treated with intradermal antibody but without intravenous BSA had an inflammation index of 1.3. Tocainide-treated group. In the 10 animals that received 25 mg of tocainide intraperitoneally, 30 sites were biopsied and of these 28 could be adequately analyzed. Eight animals had three biopsies and two animals had two biopsies. The inflammation indices in the tocainide-treated group ranged from 3.8 to 16.3 with a
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1
INFLAMMATION INDEX (No. PMNslNo. FIBROBLASTS 2 SEM) IN SUBDERMIS FOLLOWING A REVERSE PASSIVE ARTHUS REACTION IN RATS TREATED WITH T~CAINIDE (25 MG/ANIMAL) OR PGEl
(500 kg or 250 pg/animal) Experimental Animal 1 2 3 4 5 6 7 8 9
30.8 ZIZ11.8 32.1 k 20.2 13.3 f 4.4 10.1 k 7.9 14.3 2 3.8 15.2 i 9.1 13.8 + 8.1 15.6 2 9.0 21.9 f 7.5
10
10.4
Mean f SEM Note. uP < bP < =P < dP <
Positive control
17.7 2 2.5
Tocainide (25md 12.0 13.8 16.3 5.1 9.8 8.2 7.4 10.2 6.4 3.8 9.3
k 1.5 f 5.2 -c 3.1 2 1.9 -+ 2.7 k 2.3 k 2.6 2 1.6 f 0.3 ” 0.2 _’ 1.23”
group
PGEl (500 I%) 6.6 4.0 7.3 7.4 4.8
f + t f ?
2.2 1.4 3.3 1.1 2.3
6.0 f 0.67b
PGEl
(250I.4 11.8 11.9 8.3 2.0 9.3
+- 4.1 f 2.8 2 1.2 + 1.0
8.7 f 1.8’
Negative control 0.28 0.60 0.11 0.14 1.81
0.43 0.07 0.09 0.16 0.14 0.38 2 O.Old
The index for each animal represents the mean of one to three biopsy sites. 0.025. 0.01. 0.05. 0.001.
mean inflammation index of 9.3 + 1.2, which was significantly lower than the value of 17.7 _t 2.5 in the control group (P < 0.025). All of the animals treated with tocainide became drowsy but were able to crawl in the cage, and no seizure activity was noted. PGEI-treated group. Five animals were treated with 500 kg PGEl and 15 sites were biopsied, of which 14 sites could be analyzed histologically. Inflammation indices in this group ranged from 4.0 to 7.3 with a mean of 6.0 + 0.7. This was significantly lower than the inflammation index in the control group (P < 0.01) and also significantly lower than the index noted in the tocainide-treated group (P = 0.05).
Five animals were treated with 250 kg of PGEI, and 15 biopsy samples were obtained, of which 13 could be analyzed. The inflammation index for each animal ranged from 2.0 to 11.9 with a mean of 8.7 k 1.8. In comparison to control animals, the inflammation index was significantly lower (P < 0.05), but it was not significantly different from the tocainide-treated animals (NS). Within a few minutes after receiving the PGEI , all treated animals were extremely sedated and did not move. Serum tocainide levels. Serum tocainide levels measured in the 10 treated animals at the time they were sacrificed ranged from 5.6 to 19.4 t&ml, with a mean of 14.6 +- 4.0 (+ SD). Serum was obtained at the time of administration of BSA (20 min after injection of the tocainide) in the two animals; serum levels were 20.4 and 26.9 t&ml in these two animals. The serum level of tocainide in these two animals fell to 10.4 and 11.5 Fg/ml, respectively, 2% hr later at the time that the RPA sites were excised.
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Histopatholo~y. Examples of representative tissue sections from control animals, tocainide-treated animals, and PGEl-treated animals are shown in Fig. 1. Sections from positive control animals universally showed vessels full of PMNs, extensive interstitial accumulation of PMNs, and edema. Tocainide- and PGEItreated animals also showed small vessels full of PMNs (Fig. 2) and some interstitial edema; however, the extent of PMN accumulation in the dermal and muscularis was markedly reduced. DISCUSSION
The results of this study demonstrated that tocainide, when given in quantities that approximate the upper limit of the therapeutic range recommended in humans (15 &ml), significantly suppressed acute dermal inflammation induced experimentally in rats using the reverse passive Arthus reaction. The magnitude of this anti-inflammatory effect appears comparable to the effect of 250 pg of subcutaneously administered of PGE 1, a prostaglandin that has been shown to have a potent anti-inflammatory action (22). High doses of PGEl (500 kg/animal) exerted a small but statistically significant greater suppressive effect than the tocainide. Higher doses of tocainide were not studied since we were interested only in the anti-inflammatory effect of doses of tocainide that would approximate the therapeutic range in humans. Mechanisms responsible for the acute inflammatory response that develops in the RPA reaction have been well studied (24). Antigen-antibody complexes formed at the site where antibody is injected intradermally activate the complement system, generating numerous biologically active polypeptides, including C5a, which is highly chemotactic for PMNs (25, 26). Complement depletion or removal of PMNs using specific antisera or cytotoxic drugs ameliorates tissue injury (25). Since the experimental work of Stewart et al. suggested that tocainide may alter PMN migration through vascular endothelium (14, 15), we chose to measure the anti-inflammatory properties of tocainide by directly counting the number of PMNs in the dermal interstitium relative to the number of fibroblasts. Measurement of the relative uptake of radioactively labeled BSA in RPA biopsy sites has been used by others as an index of complement-mediated vascular injury (22). However, since some complement-derived peptides directly increase vascular permeability, this latter assay technique does not specifically assess the effect of the drug on PMN migration. The morphologic features of the dermal inflammation in the treated animals support the hypothesis that both tocainide and PGEl do not inhibit chemoattraction of PMNs to the region of immune-complexmediated complement activation but rather inhibit the migration of PMNs FIG. 1. Reverse passive Arthus Positive control biopsy showing (arrows) full of PMNs. (B) Biopsy (C) Biopsy from a PGEI-treated overall degree of edema formation imals (10% formaldehyde fixed, x
reaction in control, tocainide-treated. and PGEl-treated rats. (A) intense inflammation with edema in the subdermis and venules from a tocainide-treated animal showing less intense inflammation. animal (500 Fgl. (D) Biopsy from a negative control animal. The appeared comparable in control, tocainide- and PGEl-treated an100).
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through the vascular endothelium. In the animals that received tocainide, small venules lined with PMNs were noted in most tissue sections. The major difference in appearance between control animals and treated animals was a signiticantly lower relative number of PMNs to fibroblasts in the interstitium. Whether tocainide also impairs PMN ingestion and digestion of immune complexes in viva was not studied. The rationale for using a drug that may alter migration of PMNs through vascular endothelium to treat inflammatory disorders is not without precedent. One of the numerous effects of glucocorticoids is to inhibit the normal migration of PMNs from the intravascular space to the interstitium (27). Given the great difficulty that clinicians have in treating small vessel leukocytoclastic vasculitis, any well-tolerated drug that reduces dermal inflammation would be of great value. In light of the findings in this study, further experimental work to define the efficacy of tocainide in inhibiting immune-complex-mediated vascular inflammation appears warranted. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. IO. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
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Received October 30, 1985; accepted with revision January 18, 1986