Agents Actions, 35 (t992)
0065-4299/92/040273-07 $1.50+ 0.20/0
9 1992 Birkh~iuserVerlag, Basel
The influence of superoxide scavenging compound CTC 23 on type II collagen-induced arthritis in mice P.H. Wooley and J.D. Whalen Division of Rheumatology,Departmentsof Internal Medicine and Immunology,WayneState UniversityMedical School, Detroit, MI 48201, USA
Abstract
The anti-arthritic activity of the superoxide scavenging compound CTC 23 was tested in murine type II collagen-induced arthritis (CIA). CTC 23 demonstrated a dose-dependant prevention of the onset of CIA, with a significant reduction of disease incidence in mice receiving prophylactic administration of CTC 23 from 2 . 5 - 5 mg/kg/day. CTC 23 also delayed the development of the anti-collagen immune response, causing a significant decrease in antibody levels 14 days after immunization. CTC 23 reduced the arthritis score and number of involved limbs in mice with established CIA. The results suggest that CTC 23 acts as a disease modifying anti-arthritic compound to prevent the progression of the experimental arthritis.
Introduction
The physiological role of free oxygen radicals in the cellular damage that accompanies the inflammatory response is currently an active area of research [1, 2]. Scavengers of oxygen radicals have been indicated as anti-inflammatory agents, based on the findings that the enzyme superoxide dismutase (SOD) has a beneficial effect upon several experimental models of arthritis [3, 4], and has been used for the treatment of human arthritides [5-7]. However, the immunogenic nature of bovine SOD compromises its potential as a pharmacological agent, which has led to an vigorous search for compounds with oxygen anion scavenging activity. CTC 23 [8] (Figure 1) is a member of a novel class of organometallic compounds developed by Dr D. Gershon and Dr. Z. Dori (Departments of Biology and Chemistry, Technion - Israel Institute of Technology, Haifa), synthesized by procedures de-
scribed elsewhere [9] and initially selected for the ability to scavenge superoxide ions using the method of Crapo and McCord [10]. Recent findings have indicated that CTC 23 has anti-viral activity on HSV-l-induced ocular epithelial and stromal disease in the rabbits [11], and anti-inflammatory activity in classical assays including the mouse carrageenan paw edema model and rat adjuvant arthritis [12]. The novel nature of this compound and its activities prompted the current assessment in CIA, as this experimental model has the ability to select for atypical anti-arthritic agents. Murine CIA is an experimental model of arthritis with a number of pathological, immunological and genetic features in common with rheumatoid arthritis [13, 14]. Immunization of susceptible strains of mice with type II collagen, the major component of joint cartilage, causes a progressive, inflammatory arthritis in the majority of immunized animals. The histopathology of affected joints reveals synovitis, pannus formation, and
274
Agents Actions, 35 (1992)
+
"%
C
0
C
Compounds and administration
9' 21-
/
c
.
/IN
C--CH
\
Figure 1 The chemical structure of CTC 23.
erosion of cartilage and subchondral bone. Immunological findings include high antibody levels to type II collagen, and hypergammaglobulinemia. This model has been used for in vivo testing of potential anti-arthritic compounds, since its relative insensitivity to cyclo-oxygenase inhibitors may assist in the identification of anti-inflammatory compounds with a novel mode of action [15, 16]. The results of the current study suggest that CTC 23 may exert a disease modifying anti-arthritic effect on CIA.
CTC 23 was prepared daily by dissolving preweighed aliquots in sterile PBS. Compound or placebo (sterile PBS alone) was administered by daily intraperitoneal (i.p.) injection.
Prophylactic treatment. Fifty mice were assigned randomly to one of five treatment groups: (1) Saline control (2) CTC 23 at 1 mg/kg/day (3) CTC 23 at 2.5 mg/kg/day (4) CTC 23 at 5 mg/kg/day (5) CTC 23 at 5 mg/kg/quaque oltra die (QOD, every other day). Dosing was commenced 5 days prior to collagen immunization. Five days after the initial dosing, all mice were injected with 100 gg bovine type II collagen in Freund's complete adjuvant (FCA) intradermally at the base of the tail as described above. Therapeutic treatment. Mice were injected with 100 gg bovine type II collagen in Freund's complete adjuvant (FCA) intradermally at the base of the tail, and examined daily for the onset of disease. Immediately following arthritis onset, mice were assigned randomly to one of five treatment groups: (1) Saline control (2) CTC 23 at 1 mg/kg/ day (3) CTC 23 at 2.5 mg/kg/day (4) CTC 23 at 5 mg/kg/day (5) CTC 23 at 5 mg/kg/quaque oltra die (QOD), or every other day.
Materials and methods
Assessment of arthritis
Animals
Mice were monitored by daily examination for the onset of disease, and the onset dates recorded. Affected animals were clinically assessed three times per week for ten weeks after disease onset. Mice without signs of arthritis ten weeks after immunization were considered disease negative. Disease was evaluated using an established arthritis index system [13, 17]. Limbs were assigned a clinical score based on the index: 0 = normal appearance and flexion 1 = erythema and edema 2 = visible joint distortion 3 = ankylosis detectable on flexion Paw and ankle widths were measured using a constant tension caliper (Schnelltaster, GDR).
Female DBA/1 LacJ mice ( 7 - 8 weeks old) were obtained from Jackson Labs (Bar Harbor, Maine), and housed in the Department of Laboratory Animal Resources at Wayne State University, in accordance with N I H animal care guidelines. Mice were housed randomly at 15 per cage, and identified by ear punching. All procedures commenced following a 2 week quarantine period.
Induction of arthritis Bovine type II collagen was solubilized at 2 mg/ml in 0.01 M acetic acid overnight at 40 C, and emulsified with an equal volume of cold Freund's complete adjuvant (Difco Ra37), containing additional M. tuberculosis at 2.0 mg/ml/FCA. Mice were injected intradermally at a single site at the base of the tail with 100/,tl of cold emulsion as described elsewhere [17].
Serological assessment Mice were anesthetized by the i.p. injection of Avertin (tribromoethanol, 5 mg/kg), and blood
275
Agents Actions, 35 (1992)
samples obtained by retro-orbital puncture. Bloods were centrifuged at 1500 rpm for 10 minutes, and sera separated. Sera were stored at - 8 0 ~ prior to assessment.
Anti-collagen antibody ELISA. A modified ELISA technique was used to detect antibody binding to collagen [18]. Micro-ELISA plates (Nunc Immuno plate II, Frankly, PA.), were coated with 100 gl of collagen solution at a concentration of 30 Ixg/ml in 0.4 M phosphate buffer, pH 7.6, by incubation overnight at 4~ Plates were washed 3 times with PBS containing 0.05% Tween (PBS-T) and blocked with 1% bovine serum albumin (BSA; Sigma, MO) for 1 hr or overnight at 4 ~ Subsequently, 100 gl of the serum to be tested was added diluted 1/200 in PBS/BSA-T, and incubation continued overnight. The amount of bound antibody was determined by incubation with goat-antimouse immunoglobulin covalently linked to alkaline phosphatase (Fisher-Biotech, NY). After washing 10 times, bound enzyme was quantified by the addition of p-nitrophenyt phosphate as the chromatogen substrate, and the resulting optical density determined using a UVmax spectrophotometer (Molecular Devices, CA) at 405 rim. A standard mouse anti-type II collagen antiserum was titered on each plate to ensure uniformity of the assay system. Negative (normal) control sera were also included on each plate. Immunoglobulin levels. Immunoglobulin levels were measured using an ELISA technique [18]. Micro-ELISA plates (Corning), were coated with 100 gl of a 1/500 dilution of rabbit anti-mouse Ig (Fisher-Biotech), in PBS, by incubation overnight at 4 ~ Plates were washed 3 times with PBS containing 0.05% Tween (PBS-T) and blocked with I% bovine serum albumin (BSA; Sigma, MO) for i hr or overnight at 4~ Subsequently, 100 gl of the serum to be tested was added diluted 1/2000 in PBS/BSA-T, and incubation continued overnight. The amount of bound immunoglobulin was determined by incubation with goat-anti-mouse immunoglobulin covalently linked to alkaline phosphatase (Fisher-Biotech, NY). After washing x 6, bound enzyme was quantified by the addition of p-nitrophenyl phosphate as the chromatogen substrate, and the resulting optical density determined using a UVmax spectrophotometer (Molecular Devices, CA) at 405 nm. A standard mouse sera (Miles, IN) containing known levels of the various
Ig classes was titered on the plate to provide a standard curve. Negative (normal) control sera were also included on each plate. The Ig levels in the test sera were determined by regression analysis from the standard curve, and expressed as mg/ dL.
Statistical analysis All data points were recorded using a microcomputer database and downloaded to the SPSS/PC statistical package for analysis. Group analysis for the incidence of arthritis used Chi squared 2 x 2 contingency analysis and ANOVA. Differences in disease onset points, arthritis index scores, peak swelling, and anti-collagen antibody measurements were tested for using Students T test or the Mann-Whitney U-test where appropriate. Results
Effect of prophylactic CTC 23 treatment on collagen arthritis The incidence of arthritis in mice treated with CTC 23 prophylactically is shown in Figure 2. CTC 23 reduced the incidence of disease in a dose dependant fashion, with a statistically significant reduction in CIA from 2.5 mg/mouse/day. The incidence of CIA in the CTC 23 group receiving 5 mg/ mouse/day group was highly statistically different (p < 0.005) from the incidence in the control group. A long-acting activity for CTC 23 was indicated in
100
I
75co 50;'-2 25
0 I Fi~llre 2
CONTROL lmg/kg
2.5mg/kg 5rng/kg 5mg/kg QOD
The incidence o f CIA in mice treated prophylactically with various doses o f C T C 23. G r o u p size varied f r o m ten to fifteen animals per group. A significant r e d u c t i o n in disease incidence was seen f r o m 2.5 m g / k g / d a y .
276
Agents Actions, 35 (1992)
100-
80-
- - CONTROL .....1.0 mg/kg/day 2.5 m g / k g / d o y ...... $,0 m g / k g / d o y
~
-
-
co
Uc
60-
~ 40w o_
20/" / '
/
O- .................... ~.../~i.J...-i20
50
40
/
,
~
,
50
60
70
80
Figure 3 DAYS AFTER IMMUNIZATION The onset of CIC in mice treated prophylactically with CTC 23, and immunized with bovine type II collagen at day 0.
mice receiving a similar dose QOD, since this group displayed a similar reduction in disease. The onset of arthritis in mice treated with CTC 23 prophylactically is shown in Figure 3. Although mice in the control group developed disease earlier (from Day 34) than the mice in the groups treated with CTC 23 there was no statistically significant differences in the onset of disease compared with control mice. Lower arthritis index scores were seen in the groups receiving CTC from 2 . 5 - 5 m g / k g / d a y compared with the control animals, but arthritis severity was not statistically different due to small numbers of arthritic mice in the treatment groups. No differences were observed between paw widths in treated and control groups, indicating that a similar inflammatory response occurred in individual affected paws due to the presence of CIA, despite the lower incidence of affected paws in the CTC 23 treated mice.
Effect of CTC 23 on established arthritis No statistical variations in the time of onset, initial disease parameters, or anticollagen antibodies were seen between the groups at the commencement of therapeutic treatment with CTC 23, and the treatment group size varied from six to eleven animals due to the random assignment of mice. The concluding mean arthritis index scores of mice treated with CTC 23 from the time of arthritis onset is shown in Figure 4. A significant reduction in the arthritis score was seen in mice receiving CTC 5.0 mg/kg/day (p = 0.03) and 5.0 mg/kg/QOD
Figure ~,
CONTROL lrng/kg
2,5mg/kg
5r'og/kg
5mg/kg QOD
The clinical arthritis score in mice treated therapeutically with CTC 23 from the onset of disease. The group size varied from six to eleven animals. A significant reduction in disease score was seen at 5 mg/kg/day and 5 mg/kg/QOD.
4-
3-
b
z-
0
Figure
- -
,
,
CONTROL lmg/kg
,
2.5mg/kg
5mg/kg
5mg/kg QOD
The number of involved limbs in mice treated therapeutically with CTC 23 from the onset of disease. A significant reduction in the final number of involved limbs was seen at 5 mg/kg/day and 5 mg/kg/QOD.
(p=0.001) compared to the saline controls. Although less severe arthritis was seen in the groups receiving CTC l - 2 . 5 m g / k g / d a y , this was not statistically different from the severity seen in the control mice. The number of involved paws at the conclusion of the trial is shown in Fig. 5. Significantly less arthritic paws were seen in mice receiving CTC 23 at 5 mg/kg/day and 5 mg/kg/QOD compared to the control group. Although a lower number of involved limbs was seen in the groups receiving CTC 1-2.5 mg/kg/day, this was not statistically different from the number of involved limbs seen in the control mice. No significant differences were observed between maximum paw widths in treated and control
277
Agents Actions, 35 (1992)
2.000 9 0
1.500
E {u3 0
--
9
Saline
--
0
1.0mg/kg/day
Control
9 -A --
9 2.5mg/kg/day ~, 5 . O r n g / k g / d a y
[]
[]
--
5.Omg/kg/QOD
1.000
,-4 d 0.500
I [ I PREBLEED DAY 14 DAY 2 8 DAY 70 Anti-collagen antibody levels in mice treated prophylactically with various doses of CTC 23. A significant reduction in antibody level was seen at day 14 in mice treated with 2.5 mg/kg/day and 5.0 mg/kg/QOD.
0.000
Figure 6
groups, again indicating the development of a similar inflammatory response in individual affected paws. Antibody response to type H collagen The serum antibody response to type II collagen in mice treated prophylactically with CTC 23, measured at prebleed and days 14, 28, and 70 is shown in Figure 6. The antibody levels in mice treated with CTC 23 at 2.5 mg/kg/day and 5.0 mg/kg/ QOD were significantly lower than the saline control (p< 0.05) 14 days post immunization, and although this decrease was still apparent 28 days post immunization, the reduction was not statistically significant. These data suggest that CTC 23 delays the antibody response to type II collagen. CTC 23 caused no significant modulation of the circulating immunoglobulin levels at any of the doses or time points tested (Data not shown).
Discussion
CTC 23 exhibited an anti-arthritic effect in this study, both modulating the development of type II collagen-induced arthritis and modifying the dis-
ease in mice with established arthritis. Whereas steroid treatment has been shown to generate similar data, non-steroidal anti-inflammatory compounds and anti-arthritic agents have not been generally successful in this experimental disease model [J5, 16, 19, 20]. The precise sequence of immune and inflammatory events in the etiology of CIA has not been elucidated, although the generation of a vigorous immune response to collagen is a requisite for the development of CIA [21, 22]. Since CTC 23 reduced anti-collagen antibodies 14 days after immunization, it is likely that CTC 23 influenced the development of immunity to collagen, and this action may contribute to its antiarthritic activity. In adjuvant-induced arthritis, therapy with SOD was shown to have an effect both on disease parameters and peripheral blood immunoglobulin levels [23]. However, total immunoglobulin levels in our study were not effected by ~TC 23, suggesting that the active production of anti-collagen was preferentially influenced. It is currently unclear whether the anti-arthritic and anti-viral activity of CTC 23 are both dependant upon the superoxide scavenging properties of this compound. SOD has been shown to have an anti-arthritic effect in CIA [24], adjuvant-induced arthritis [3, 23, 25] and streptococcal cell wall-induced arthritis [4]. The plant phenol apocynin,
278
which is a strong inhibitor of neutrophil superoxide anion release in vitro, was also demonstrated to be active in the CIA model [26]. However, the exact mode of action of SOD therapy in arthritis remains to be elucidated, despite trials of bovine SOD in human disease [5-7]. Other synthetic organometallic compounds have been shown to possess antiarthritic activity in experimental models, notably spirogermanium [27] in the adjuvant arthritis model. It was hypothesized that organometallic compounds may activate suppressor cell, N K cell, and macrophage populations, and influence gamma interferon production [28], and similar activities have also been reported for the organic germanium compound Ge-132 [29]. The capacity of CTC-23 to influence cytokines and immune cell functions is currently under investigation to determine possible mechansims for antiarthritic activity. However, in vitro studies on the influence of superoxide anions upon production of an IL-l-like cytokine, and its down-regulation by SOD [30], provide evidence for one possible pathway for the suppressive activity of CTC 23 on the arthritogenic response to type II collagen. IL-I has been shown to provoke murine CIA [31, 32], while IL-1 receptor antagonist protein (IRAP) both reduces the antibody response and the development of arthritis in response to collagen [33]. However, further studies are required to determine the influence of CTC 23 on the cytokines involved in both arthritis and viral infections. Overall, CTC 23 was successful in the prevention and treatment of collagen-induced arthritis in mice. The drug exhibited dose-dependant behavior and may be long-acting in this effect, based on the observation that QOD dosing was equal or more efficacious than daily dosing. Although it remains to be established whether organometallic compounds with in vitro superoxide scavenging action are an appropriate treatment for chronic joint disease, this study indicates compounds such as CTC 23 represent a viable experimental approach to a novel arthritis therapy. Acknowledgements The authors wish to thank David Scheer (Scheer & Company, Inc.) and Dr Stephen Shapiro for their thoughtful contributions to the design and interpretation of the trial, and Chai-Tech Corporation for the generous support of this study. Received 21 May, 1991; accepted by I. G. Otterness, 8 August 1991
Agents Actions, 35 (1992)
References [1] N. A. Roberts, Free radicals immunoglabulins and complement as mediators of inflammation. Baillieres Clinical Rheumatology 2, 211-232 (1988). [2] B. Haliwe11, J. R. Hoult and D. R. Blake, Oxidants, inflammation, andanti-inflammatory drugs. Faseb Journal 2, 28672873 (1988). [3] A. Vaille, G. Jadot and E. Elizagaray, Anti-inflammatory activity of various superoxide dismutases on polyarthritis in the Lewis rat. Biochemical Pharmacology 39, 247-255 (1990). [4] W. E. Linhart, G. Steinwender, W. Weybora, S. Zadravec and H. Esterbauer, Influence of superoxide disrnutase on staphylococcal arthritis - A histological and biochemical investigation using an experimental animal model Agents Actions 29, 259-265 (1990). [5] D. Dejica, G. Szegli, A. Herold, N. Bucurenci and E. Mazilu, Local treatment with Epurox (superoxide dismutase and catalase of human origin) in the chronic inflammatory and degenerative rheumatism. Archives Roumaines De Patholofie Experimentale Et De Microbiologie 45, 299-310 (1986). [6] K. Lund-Oleson and K. B. Menander-Huber, Orgotein: a new anti-inflammatory metalloprotein drug: preliminary evaluation of clinical efficacy and safety in degenerative joint disease. Curt Ther Res 16, 706-717 (1974). [7] K. B. Menander-Huber, Orgotein in the treatment of rheumatoid arthritis. Eur J Rheumatol Inflamm 4, 201-2t 1 (1981). [8] U. S. Patent #4866054. [9] G. Costa, G. Mestroni, G. Tauzder and L. Stefani, Organometallic derivatives of cobalt chelates of Bis(acetylacetone) ethylendiamine. J. Organometal. Chem. 6, 181-187 (1966). [10] J. D. Crapo, J. M. McCord and I. Fridovich, Preparation and assay of superoxide dismutases. Methods in Enzymology 53, 382-393 (1978). [11] E. C. Dunkel, P. A. Geary, J. Brooks and D. PavanLangston, CTC 23 efficacy in vitro and on HSV-l-induced ocular epithelial and stromal disease in the rabbti. Anti-Viral Research, 1, 135, (1990). [12] D. Gershon, Z. Dori, Anti-inflammatory and anti-arthritic activity of metal compounds with superoxide scavenging ability. Technical Report, Chai-Tech Corp., Greenvale, NY 11548, (1986). [13] P. H. Wooley, H. S. Luthra, J. M. Stuart and C. S. David, Type H collagen-induced arthritis in mice. L Major histocompatibility complex (I region) linkage and antibody correlates. Journal of Experimental Medicine 154, 688-700 (1981). [14] J. S. Courtenay, M. J. DaUman, A. D. Dayan, A. Martin and B. Mosedale, Immunisation against heterologous type H collagen induces arthritis in mice. Nature 283, 666 668 (1980). [15] K. Phadke, R. L. Fouts, J. E. Parrish and L. D. Butler, Evaluation of the effects o f various anti-arthritic durgs on type H collagen-induced mouse arthritis model. Immunopharmacology 10, 51-60 (1985). [16] G.W. Cannon, S. McCall, B. C. Cole, M. M. Griffiths, L. A. Radov and J. R. Ward, Effects ofindomethaein, cyclosporin, cyclophosphamide, and placebo on collagen-induced arthritis of mice. Agents Actions 29, 315-323 (1990). [17] P. H. Wooley, Collagen-inducedarthritis in the mouse. Methods in Enzymology 162, 361-373 (1988). [18] P. H. Wooley, J. R. Seibold, J. D. Whalen and J. M. Chapdelaine, Pristane-induced arthritis. The immunological and ge-
Agents Actions, 35 (1992) netic features of a n experimental murine model of autoimmune disease. Arthritis & Rheumatism 32, 1022-1030 (1989). [19] J. M. Stuart, L. K. Myers, A. S. Townes and A. H. Kang, Effect of cyclophosphamide, hydrocortisone and levamisole on collagen-induced arthritis in rats. Arthritis & Rheumatism 24, 790-794 (1981). [20] R. B. Gilbertsen, Effects ofpentostatin (2'deoxycoformycin), an inhibitor of adenosine deaminase, on type 11 collageninduced arthritis in rats. Journal of Immunopharmacology 7, 3252-341 (1985). [21] J. M. Stuart, M. A. Cremer, A. H. Kang and A. S. Townes, Collagen-induced arthritis in rats. Evaluation of early immunologic events. Arthritis & Rheumatism 22, 1344-1351 (1979). [22] D. E. Trentham, A. S. Townes, A. H. Kang and J. R. David, Humoral and cellular sensitivity to collagen in type H collagen- induced arthritis in rats. Journal of Clinical Investigation 61, 89-96 (1978). [23] G. Jadot, A. M. Michelson and K. Puget, Anti-inflammatory activity of superoxide dismutases: studies on adjuvant induced polyarthritis in rats. Free Radical Research Communications 2, 27-42 (1986). [24] C. Beauchamp, R. B. Gilbertson, J. Rantone and D. P. Menapace, Fed. Proc. 42, 6354 (1983). (Abstract) [25] M. Castell, J. J. Moreno, J. C. Oliva, J. Queralt and M. C. Castellote, Effect of indomethacin, benoxaprofen and superoxide dismutase on serum sulfhydryl levels in adjuvant arthritis. Farmaco-Edizione Pratica 42, 233-Edizione P (1987). [26] B. A. Hart, J. M. Simons, S. Knaan-Shanzer, N. P. Bakker and R. P. Labadie, Antiarthritic activity of the newly developed neutrophil oxidative burst antagonist apocynin. Free Radical Biology & Medicine 9, 127-131 (1990).
279 [27] A. M. Badger, D. A. Schwartz, D. H. Picker, J. W. Dorman, F. C. Bradley, E. N. Cheeseman, M. J. DiMArtino, N. Hanna and C. K. Mirabelli, Antiarthritic and supressor cell inducing activity of azaspiranes: structure-function relationships o f a novel class of immunodulatory agents. J. Med. Chem. 33, 2963-2970 (1990). [28] S. Goodman, Therapeutic effects of organic germanium. Medical Hypothesis 26, 207-215 (1988). [29] H. Aso, E Suzuki, T. Yamaguchi, T. Ebina and N. Ishida, Induction of interferon and activation of N K cells and macrophages in mice by oral administration of Ge-132, an organic germanium compound. Gan To Kagaku Ryoho 9, 1976 1980 (1982). [30] T. Kasama, K. Kobayashi, T. Fukushima, M. Tabata, I. Ohno, M. Negishi, H. Ide, T. Takahashi and Y. Niwa, Production of interleukin l-like factor from human peripheral blood monocytes and polymorphonuclear leukocytes by superoxide anion: the role of interleukin 1 and reactive oxygen species in inflamed sites. Clinical Immunology & Immunopathology 53, 439 448 (1989). [31] J. T. Hom, A. M. Bendele and D. G. Carlson, In vivo administratgon with 1L-1 accelerates the development of collagen-induced arthritis in mice. J. Immunol. 141, 834-841 (1988). [32] L. M. Killar and C. J. Dunn, lnterleukin-1 potentiates the development of collagen-induced arthritis in mice. Clinical Science 76~ 535-538 (1989). [33] P. H. Wooley, N. D. Staite, 3. D. Whalen, D. L. Chapman, A. E. Berger, D. G. Aspar and K. A. Richard, The effect of interleukin-1 receptor antagonist protein on type II collagen and antigen-induced arthritis in mice. Arthritis & Rheumatism 33, $20 (1990). (Abstract)
0065-4299/92/040273-07 $1.50+ 0.20/0
9 1992 Birkh~iuserVerlag, Basel
The influence of superoxide scavenging compound CTC 23 on type II collagen-induced arthritis in mice P.H. Wooley and J.D. Whalen Division of Rheumatology,Departmentsof Internal Medicine and Immunology,WayneState UniversityMedical School, Detroit, MI 48201, USA
Abstract
The anti-arthritic activity of the superoxide scavenging compound CTC 23 was tested in murine type II collagen-induced arthritis (CIA). CTC 23 demonstrated a dose-dependant prevention of the onset of CIA, with a significant reduction of disease incidence in mice receiving prophylactic administration of CTC 23 from 2 . 5 - 5 mg/kg/day. CTC 23 also delayed the development of the anti-collagen immune response, causing a significant decrease in antibody levels 14 days after immunization. CTC 23 reduced the arthritis score and number of involved limbs in mice with established CIA. The results suggest that CTC 23 acts as a disease modifying anti-arthritic compound to prevent the progression of the experimental arthritis.
Introduction
The physiological role of free oxygen radicals in the cellular damage that accompanies the inflammatory response is currently an active area of research [1, 2]. Scavengers of oxygen radicals have been indicated as anti-inflammatory agents, based on the findings that the enzyme superoxide dismutase (SOD) has a beneficial effect upon several experimental models of arthritis [3, 4], and has been used for the treatment of human arthritides [5-7]. However, the immunogenic nature of bovine SOD compromises its potential as a pharmacological agent, which has led to an vigorous search for compounds with oxygen anion scavenging activity. CTC 23 [8] (Figure 1) is a member of a novel class of organometallic compounds developed by Dr D. Gershon and Dr. Z. Dori (Departments of Biology and Chemistry, Technion - Israel Institute of Technology, Haifa), synthesized by procedures de-
scribed elsewhere [9] and initially selected for the ability to scavenge superoxide ions using the method of Crapo and McCord [10]. Recent findings have indicated that CTC 23 has anti-viral activity on HSV-l-induced ocular epithelial and stromal disease in the rabbits [11], and anti-inflammatory activity in classical assays including the mouse carrageenan paw edema model and rat adjuvant arthritis [12]. The novel nature of this compound and its activities prompted the current assessment in CIA, as this experimental model has the ability to select for atypical anti-arthritic agents. Murine CIA is an experimental model of arthritis with a number of pathological, immunological and genetic features in common with rheumatoid arthritis [13, 14]. Immunization of susceptible strains of mice with type II collagen, the major component of joint cartilage, causes a progressive, inflammatory arthritis in the majority of immunized animals. The histopathology of affected joints reveals synovitis, pannus formation, and
274
Agents Actions, 35 (1992)
+
"%
C
0
C
Compounds and administration
9' 21-
/
c
.
/IN
C--CH
\
Figure 1 The chemical structure of CTC 23.
erosion of cartilage and subchondral bone. Immunological findings include high antibody levels to type II collagen, and hypergammaglobulinemia. This model has been used for in vivo testing of potential anti-arthritic compounds, since its relative insensitivity to cyclo-oxygenase inhibitors may assist in the identification of anti-inflammatory compounds with a novel mode of action [15, 16]. The results of the current study suggest that CTC 23 may exert a disease modifying anti-arthritic effect on CIA.
CTC 23 was prepared daily by dissolving preweighed aliquots in sterile PBS. Compound or placebo (sterile PBS alone) was administered by daily intraperitoneal (i.p.) injection.
Prophylactic treatment. Fifty mice were assigned randomly to one of five treatment groups: (1) Saline control (2) CTC 23 at 1 mg/kg/day (3) CTC 23 at 2.5 mg/kg/day (4) CTC 23 at 5 mg/kg/day (5) CTC 23 at 5 mg/kg/quaque oltra die (QOD, every other day). Dosing was commenced 5 days prior to collagen immunization. Five days after the initial dosing, all mice were injected with 100 gg bovine type II collagen in Freund's complete adjuvant (FCA) intradermally at the base of the tail as described above. Therapeutic treatment. Mice were injected with 100 gg bovine type II collagen in Freund's complete adjuvant (FCA) intradermally at the base of the tail, and examined daily for the onset of disease. Immediately following arthritis onset, mice were assigned randomly to one of five treatment groups: (1) Saline control (2) CTC 23 at 1 mg/kg/ day (3) CTC 23 at 2.5 mg/kg/day (4) CTC 23 at 5 mg/kg/day (5) CTC 23 at 5 mg/kg/quaque oltra die (QOD), or every other day.
Materials and methods
Assessment of arthritis
Animals
Mice were monitored by daily examination for the onset of disease, and the onset dates recorded. Affected animals were clinically assessed three times per week for ten weeks after disease onset. Mice without signs of arthritis ten weeks after immunization were considered disease negative. Disease was evaluated using an established arthritis index system [13, 17]. Limbs were assigned a clinical score based on the index: 0 = normal appearance and flexion 1 = erythema and edema 2 = visible joint distortion 3 = ankylosis detectable on flexion Paw and ankle widths were measured using a constant tension caliper (Schnelltaster, GDR).
Female DBA/1 LacJ mice ( 7 - 8 weeks old) were obtained from Jackson Labs (Bar Harbor, Maine), and housed in the Department of Laboratory Animal Resources at Wayne State University, in accordance with N I H animal care guidelines. Mice were housed randomly at 15 per cage, and identified by ear punching. All procedures commenced following a 2 week quarantine period.
Induction of arthritis Bovine type II collagen was solubilized at 2 mg/ml in 0.01 M acetic acid overnight at 40 C, and emulsified with an equal volume of cold Freund's complete adjuvant (Difco Ra37), containing additional M. tuberculosis at 2.0 mg/ml/FCA. Mice were injected intradermally at a single site at the base of the tail with 100/,tl of cold emulsion as described elsewhere [17].
Serological assessment Mice were anesthetized by the i.p. injection of Avertin (tribromoethanol, 5 mg/kg), and blood
275
Agents Actions, 35 (1992)
samples obtained by retro-orbital puncture. Bloods were centrifuged at 1500 rpm for 10 minutes, and sera separated. Sera were stored at - 8 0 ~ prior to assessment.
Anti-collagen antibody ELISA. A modified ELISA technique was used to detect antibody binding to collagen [18]. Micro-ELISA plates (Nunc Immuno plate II, Frankly, PA.), were coated with 100 gl of collagen solution at a concentration of 30 Ixg/ml in 0.4 M phosphate buffer, pH 7.6, by incubation overnight at 4~ Plates were washed 3 times with PBS containing 0.05% Tween (PBS-T) and blocked with 1% bovine serum albumin (BSA; Sigma, MO) for 1 hr or overnight at 4 ~ Subsequently, 100 gl of the serum to be tested was added diluted 1/200 in PBS/BSA-T, and incubation continued overnight. The amount of bound antibody was determined by incubation with goat-antimouse immunoglobulin covalently linked to alkaline phosphatase (Fisher-Biotech, NY). After washing 10 times, bound enzyme was quantified by the addition of p-nitrophenyt phosphate as the chromatogen substrate, and the resulting optical density determined using a UVmax spectrophotometer (Molecular Devices, CA) at 405 rim. A standard mouse anti-type II collagen antiserum was titered on each plate to ensure uniformity of the assay system. Negative (normal) control sera were also included on each plate. Immunoglobulin levels. Immunoglobulin levels were measured using an ELISA technique [18]. Micro-ELISA plates (Corning), were coated with 100 gl of a 1/500 dilution of rabbit anti-mouse Ig (Fisher-Biotech), in PBS, by incubation overnight at 4 ~ Plates were washed 3 times with PBS containing 0.05% Tween (PBS-T) and blocked with I% bovine serum albumin (BSA; Sigma, MO) for i hr or overnight at 4~ Subsequently, 100 gl of the serum to be tested was added diluted 1/2000 in PBS/BSA-T, and incubation continued overnight. The amount of bound immunoglobulin was determined by incubation with goat-anti-mouse immunoglobulin covalently linked to alkaline phosphatase (Fisher-Biotech, NY). After washing x 6, bound enzyme was quantified by the addition of p-nitrophenyl phosphate as the chromatogen substrate, and the resulting optical density determined using a UVmax spectrophotometer (Molecular Devices, CA) at 405 nm. A standard mouse sera (Miles, IN) containing known levels of the various
Ig classes was titered on the plate to provide a standard curve. Negative (normal) control sera were also included on each plate. The Ig levels in the test sera were determined by regression analysis from the standard curve, and expressed as mg/ dL.
Statistical analysis All data points were recorded using a microcomputer database and downloaded to the SPSS/PC statistical package for analysis. Group analysis for the incidence of arthritis used Chi squared 2 x 2 contingency analysis and ANOVA. Differences in disease onset points, arthritis index scores, peak swelling, and anti-collagen antibody measurements were tested for using Students T test or the Mann-Whitney U-test where appropriate. Results
Effect of prophylactic CTC 23 treatment on collagen arthritis The incidence of arthritis in mice treated with CTC 23 prophylactically is shown in Figure 2. CTC 23 reduced the incidence of disease in a dose dependant fashion, with a statistically significant reduction in CIA from 2.5 mg/mouse/day. The incidence of CIA in the CTC 23 group receiving 5 mg/ mouse/day group was highly statistically different (p < 0.005) from the incidence in the control group. A long-acting activity for CTC 23 was indicated in
100
I
75co 50;'-2 25
0 I Fi~llre 2
CONTROL lmg/kg
2.5mg/kg 5rng/kg 5mg/kg QOD
The incidence o f CIA in mice treated prophylactically with various doses o f C T C 23. G r o u p size varied f r o m ten to fifteen animals per group. A significant r e d u c t i o n in disease incidence was seen f r o m 2.5 m g / k g / d a y .
276
Agents Actions, 35 (1992)
100-
80-
- - CONTROL .....1.0 mg/kg/day 2.5 m g / k g / d o y ...... $,0 m g / k g / d o y
~
-
-
co
Uc
60-
~ 40w o_
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/
O- .................... ~.../~i.J...-i20
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40
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60
70
80
Figure 3 DAYS AFTER IMMUNIZATION The onset of CIC in mice treated prophylactically with CTC 23, and immunized with bovine type II collagen at day 0.
mice receiving a similar dose QOD, since this group displayed a similar reduction in disease. The onset of arthritis in mice treated with CTC 23 prophylactically is shown in Figure 3. Although mice in the control group developed disease earlier (from Day 34) than the mice in the groups treated with CTC 23 there was no statistically significant differences in the onset of disease compared with control mice. Lower arthritis index scores were seen in the groups receiving CTC from 2 . 5 - 5 m g / k g / d a y compared with the control animals, but arthritis severity was not statistically different due to small numbers of arthritic mice in the treatment groups. No differences were observed between paw widths in treated and control groups, indicating that a similar inflammatory response occurred in individual affected paws due to the presence of CIA, despite the lower incidence of affected paws in the CTC 23 treated mice.
Effect of CTC 23 on established arthritis No statistical variations in the time of onset, initial disease parameters, or anticollagen antibodies were seen between the groups at the commencement of therapeutic treatment with CTC 23, and the treatment group size varied from six to eleven animals due to the random assignment of mice. The concluding mean arthritis index scores of mice treated with CTC 23 from the time of arthritis onset is shown in Figure 4. A significant reduction in the arthritis score was seen in mice receiving CTC 5.0 mg/kg/day (p = 0.03) and 5.0 mg/kg/QOD
Figure ~,
CONTROL lrng/kg
2,5mg/kg
5r'og/kg
5mg/kg QOD
The clinical arthritis score in mice treated therapeutically with CTC 23 from the onset of disease. The group size varied from six to eleven animals. A significant reduction in disease score was seen at 5 mg/kg/day and 5 mg/kg/QOD.
4-
3-
b
z-
0
Figure
- -
,
,
CONTROL lmg/kg
,
2.5mg/kg
5mg/kg
5mg/kg QOD
The number of involved limbs in mice treated therapeutically with CTC 23 from the onset of disease. A significant reduction in the final number of involved limbs was seen at 5 mg/kg/day and 5 mg/kg/QOD.
(p=0.001) compared to the saline controls. Although less severe arthritis was seen in the groups receiving CTC l - 2 . 5 m g / k g / d a y , this was not statistically different from the severity seen in the control mice. The number of involved paws at the conclusion of the trial is shown in Fig. 5. Significantly less arthritic paws were seen in mice receiving CTC 23 at 5 mg/kg/day and 5 mg/kg/QOD compared to the control group. Although a lower number of involved limbs was seen in the groups receiving CTC 1-2.5 mg/kg/day, this was not statistically different from the number of involved limbs seen in the control mice. No significant differences were observed between maximum paw widths in treated and control
277
Agents Actions, 35 (1992)
2.000 9 0
1.500
E {u3 0
--
9
Saline
--
0
1.0mg/kg/day
Control
9 -A --
9 2.5mg/kg/day ~, 5 . O r n g / k g / d a y
[]
[]
--
5.Omg/kg/QOD
1.000
,-4 d 0.500
I [ I PREBLEED DAY 14 DAY 2 8 DAY 70 Anti-collagen antibody levels in mice treated prophylactically with various doses of CTC 23. A significant reduction in antibody level was seen at day 14 in mice treated with 2.5 mg/kg/day and 5.0 mg/kg/QOD.
0.000
Figure 6
groups, again indicating the development of a similar inflammatory response in individual affected paws. Antibody response to type H collagen The serum antibody response to type II collagen in mice treated prophylactically with CTC 23, measured at prebleed and days 14, 28, and 70 is shown in Figure 6. The antibody levels in mice treated with CTC 23 at 2.5 mg/kg/day and 5.0 mg/kg/ QOD were significantly lower than the saline control (p< 0.05) 14 days post immunization, and although this decrease was still apparent 28 days post immunization, the reduction was not statistically significant. These data suggest that CTC 23 delays the antibody response to type II collagen. CTC 23 caused no significant modulation of the circulating immunoglobulin levels at any of the doses or time points tested (Data not shown).
Discussion
CTC 23 exhibited an anti-arthritic effect in this study, both modulating the development of type II collagen-induced arthritis and modifying the dis-
ease in mice with established arthritis. Whereas steroid treatment has been shown to generate similar data, non-steroidal anti-inflammatory compounds and anti-arthritic agents have not been generally successful in this experimental disease model [J5, 16, 19, 20]. The precise sequence of immune and inflammatory events in the etiology of CIA has not been elucidated, although the generation of a vigorous immune response to collagen is a requisite for the development of CIA [21, 22]. Since CTC 23 reduced anti-collagen antibodies 14 days after immunization, it is likely that CTC 23 influenced the development of immunity to collagen, and this action may contribute to its antiarthritic activity. In adjuvant-induced arthritis, therapy with SOD was shown to have an effect both on disease parameters and peripheral blood immunoglobulin levels [23]. However, total immunoglobulin levels in our study were not effected by ~TC 23, suggesting that the active production of anti-collagen was preferentially influenced. It is currently unclear whether the anti-arthritic and anti-viral activity of CTC 23 are both dependant upon the superoxide scavenging properties of this compound. SOD has been shown to have an anti-arthritic effect in CIA [24], adjuvant-induced arthritis [3, 23, 25] and streptococcal cell wall-induced arthritis [4]. The plant phenol apocynin,
278
which is a strong inhibitor of neutrophil superoxide anion release in vitro, was also demonstrated to be active in the CIA model [26]. However, the exact mode of action of SOD therapy in arthritis remains to be elucidated, despite trials of bovine SOD in human disease [5-7]. Other synthetic organometallic compounds have been shown to possess antiarthritic activity in experimental models, notably spirogermanium [27] in the adjuvant arthritis model. It was hypothesized that organometallic compounds may activate suppressor cell, N K cell, and macrophage populations, and influence gamma interferon production [28], and similar activities have also been reported for the organic germanium compound Ge-132 [29]. The capacity of CTC-23 to influence cytokines and immune cell functions is currently under investigation to determine possible mechansims for antiarthritic activity. However, in vitro studies on the influence of superoxide anions upon production of an IL-l-like cytokine, and its down-regulation by SOD [30], provide evidence for one possible pathway for the suppressive activity of CTC 23 on the arthritogenic response to type II collagen. IL-I has been shown to provoke murine CIA [31, 32], while IL-1 receptor antagonist protein (IRAP) both reduces the antibody response and the development of arthritis in response to collagen [33]. However, further studies are required to determine the influence of CTC 23 on the cytokines involved in both arthritis and viral infections. Overall, CTC 23 was successful in the prevention and treatment of collagen-induced arthritis in mice. The drug exhibited dose-dependant behavior and may be long-acting in this effect, based on the observation that QOD dosing was equal or more efficacious than daily dosing. Although it remains to be established whether organometallic compounds with in vitro superoxide scavenging action are an appropriate treatment for chronic joint disease, this study indicates compounds such as CTC 23 represent a viable experimental approach to a novel arthritis therapy. Acknowledgements The authors wish to thank David Scheer (Scheer & Company, Inc.) and Dr Stephen Shapiro for their thoughtful contributions to the design and interpretation of the trial, and Chai-Tech Corporation for the generous support of this study. Received 21 May, 1991; accepted by I. G. Otterness, 8 August 1991
Agents Actions, 35 (1992)
References [1] N. A. Roberts, Free radicals immunoglabulins and complement as mediators of inflammation. Baillieres Clinical Rheumatology 2, 211-232 (1988). [2] B. Haliwe11, J. R. Hoult and D. R. Blake, Oxidants, inflammation, andanti-inflammatory drugs. Faseb Journal 2, 28672873 (1988). [3] A. Vaille, G. Jadot and E. Elizagaray, Anti-inflammatory activity of various superoxide dismutases on polyarthritis in the Lewis rat. Biochemical Pharmacology 39, 247-255 (1990). [4] W. E. Linhart, G. Steinwender, W. Weybora, S. Zadravec and H. Esterbauer, Influence of superoxide disrnutase on staphylococcal arthritis - A histological and biochemical investigation using an experimental animal model Agents Actions 29, 259-265 (1990). [5] D. Dejica, G. Szegli, A. Herold, N. Bucurenci and E. Mazilu, Local treatment with Epurox (superoxide dismutase and catalase of human origin) in the chronic inflammatory and degenerative rheumatism. Archives Roumaines De Patholofie Experimentale Et De Microbiologie 45, 299-310 (1986). [6] K. Lund-Oleson and K. B. Menander-Huber, Orgotein: a new anti-inflammatory metalloprotein drug: preliminary evaluation of clinical efficacy and safety in degenerative joint disease. Curt Ther Res 16, 706-717 (1974). [7] K. B. Menander-Huber, Orgotein in the treatment of rheumatoid arthritis. Eur J Rheumatol Inflamm 4, 201-2t 1 (1981). [8] U. S. Patent #4866054. [9] G. Costa, G. Mestroni, G. Tauzder and L. Stefani, Organometallic derivatives of cobalt chelates of Bis(acetylacetone) ethylendiamine. J. Organometal. Chem. 6, 181-187 (1966). [10] J. D. Crapo, J. M. McCord and I. Fridovich, Preparation and assay of superoxide dismutases. Methods in Enzymology 53, 382-393 (1978). [11] E. C. Dunkel, P. A. Geary, J. Brooks and D. PavanLangston, CTC 23 efficacy in vitro and on HSV-l-induced ocular epithelial and stromal disease in the rabbti. Anti-Viral Research, 1, 135, (1990). [12] D. Gershon, Z. Dori, Anti-inflammatory and anti-arthritic activity of metal compounds with superoxide scavenging ability. Technical Report, Chai-Tech Corp., Greenvale, NY 11548, (1986). [13] P. H. Wooley, H. S. Luthra, J. M. Stuart and C. S. David, Type H collagen-induced arthritis in mice. L Major histocompatibility complex (I region) linkage and antibody correlates. Journal of Experimental Medicine 154, 688-700 (1981). [14] J. S. Courtenay, M. J. DaUman, A. D. Dayan, A. Martin and B. Mosedale, Immunisation against heterologous type H collagen induces arthritis in mice. Nature 283, 666 668 (1980). [15] K. Phadke, R. L. Fouts, J. E. Parrish and L. D. Butler, Evaluation of the effects o f various anti-arthritic durgs on type H collagen-induced mouse arthritis model. Immunopharmacology 10, 51-60 (1985). [16] G.W. Cannon, S. McCall, B. C. Cole, M. M. Griffiths, L. A. Radov and J. R. Ward, Effects ofindomethaein, cyclosporin, cyclophosphamide, and placebo on collagen-induced arthritis of mice. Agents Actions 29, 315-323 (1990). [17] P. H. Wooley, Collagen-inducedarthritis in the mouse. Methods in Enzymology 162, 361-373 (1988). [18] P. H. Wooley, J. R. Seibold, J. D. Whalen and J. M. Chapdelaine, Pristane-induced arthritis. The immunological and ge-
Agents Actions, 35 (1992) netic features of a n experimental murine model of autoimmune disease. Arthritis & Rheumatism 32, 1022-1030 (1989). [19] J. M. Stuart, L. K. Myers, A. S. Townes and A. H. Kang, Effect of cyclophosphamide, hydrocortisone and levamisole on collagen-induced arthritis in rats. Arthritis & Rheumatism 24, 790-794 (1981). [20] R. B. Gilbertsen, Effects ofpentostatin (2'deoxycoformycin), an inhibitor of adenosine deaminase, on type 11 collageninduced arthritis in rats. Journal of Immunopharmacology 7, 3252-341 (1985). [21] J. M. Stuart, M. A. Cremer, A. H. Kang and A. S. Townes, Collagen-induced arthritis in rats. Evaluation of early immunologic events. Arthritis & Rheumatism 22, 1344-1351 (1979). [22] D. E. Trentham, A. S. Townes, A. H. Kang and J. R. David, Humoral and cellular sensitivity to collagen in type H collagen- induced arthritis in rats. Journal of Clinical Investigation 61, 89-96 (1978). [23] G. Jadot, A. M. Michelson and K. Puget, Anti-inflammatory activity of superoxide dismutases: studies on adjuvant induced polyarthritis in rats. Free Radical Research Communications 2, 27-42 (1986). [24] C. Beauchamp, R. B. Gilbertson, J. Rantone and D. P. Menapace, Fed. Proc. 42, 6354 (1983). (Abstract) [25] M. Castell, J. J. Moreno, J. C. Oliva, J. Queralt and M. C. Castellote, Effect of indomethacin, benoxaprofen and superoxide dismutase on serum sulfhydryl levels in adjuvant arthritis. Farmaco-Edizione Pratica 42, 233-Edizione P (1987). [26] B. A. Hart, J. M. Simons, S. Knaan-Shanzer, N. P. Bakker and R. P. Labadie, Antiarthritic activity of the newly developed neutrophil oxidative burst antagonist apocynin. Free Radical Biology & Medicine 9, 127-131 (1990).
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