A Trial of Clodronate-Liposomes As Anti-Macrophage Treatment in a Sheep Model of Arthritis

A trial of clodronate-liposomes as anti-macrophage treatment in a sheep model of arthritis

J. Highton1, D. Guévremont1, J. Thomson1, B. Carlisle2, I. Tucker3

Department of Medicine1, University of Otago Medical School; Department of Biochemistry2; School of Pharmacy3, University of Otago, Dunedin, New Zealand.

Abstract Objective Our previous research has concerned the role of macrophages in joint inflammation in rheumatoid arthritis. We have therefore been interested in liposomes containing clodronate as an anti- macrophage treatment for arthritis. We have used the antigen-induced arthritis model in sheep to evaluate the effect of clodronate liposomes. Methods Arthritis was induced in the right hock joint (day 0). We were able to demonstrate uptake of liposomes into macrophages within the inflamed joint lining. On day 7, sheep were given a single intra-articular injection of clodronate liposomes (group 1, n = 10) or saline liposomes (group 2, n = 10). A further 6 sheep (group 3) had no arthritis and no treatment. Results No difference in joint diameter was observed between the sheep in group 1 (clodronate) and group 2 (saline treated). Both groups had joint swelling which persisted until the end of the trial (day 20). Histologic scoring was also similar in group 1 and group 2 animals, and both were worse than group 3. Conclusion In vitro studies have shown that interaction of liposomes with neutrophils and monocytes stimulates a respiratory burst. Despite this possible pro-inflammatory effect we did not observe any increase in joint diameter following liposome injection. Thus we were unable to demonstrate a therapeutic effect of a single dose of clodronate liposomes in this large animal model of antigen-induced arthritis. Key words Clodronate-liposomes, treatment of arthritis, sheep model of arthritis.

Clinical and Experimental Rheumatology 1999; 17: 43-48. EDITORIALLiposome treatment of sheep arthritis / J. Highton et al. Hypothalamus-pituitary-adrenocortical and -gonadal axis in RA / M. Cutolo

J. Highton, MD FRACP, Associate Introduction ovalbumin in Freund’s complete adju- Professor; D. Guévremont, BSc, Junior Rheumatoid arthritis is a granulomatous vant and were then skin tested. The skin Research Fellow; J. Thomson, BSc, disease and activated macrophages are reaction was scored 24 and 48 hours af-

Research Assistant; B. Carlisle, BSc Hons., a prominent feature of joint lesions and ter the antigen injection to assure their Junior Research Fellow; Ian Tucker, rheumatoid granulomas (nodules) (1). immune status. The protocol was approv- Professor, School of Pharmacy, We and others have demonstrated that ed by the Animal Ethics Committee. This work was supported by grants from the macrophages infiltrate the joint lining Health Research Council of New Zealand and and accumulate at the surface of the Trial of treatment with liposomes the Otago Medical Research Foundation. synovial lining where they drive inflam- Sheep were randomly allocated to three Please address correspondence and reprint matory processes resulting in joint de- groups. Group 1 (n = 10) and group 2 (n requests to: Dr. J. Highton, Department of struction (1, 2). Furthermore, the extent = 10) received an intra-articular injec- Medicine, University of Otago School of Medicine, P.O. Box 913, Dunedin, New of macrophage infiltration and lining tion of 5 mg ovalbumin in 0.5 ml saline Zealand. layer thickening correlates with joint in the right hock joint in order to induce Received on March 3, 1997; accepted damage (3). We have therefore been in- arthritis. Group 3 (n = 6) were normal in revised from on August 10, 1998. terested in means of targeting macro- non-arthritic untreated sheep. The left phages within the joint lining as a way hock joints of all sheep were left un- © Copyright CLINICAL AND of treating arthritis. The use of clodro- touched and were used as a further nega- EXPERIMENTAL RHEUMATOLOGY 1999. nate-containing liposomes appeared to tive control. Treatment was given in the offer a form of anti-macrophage therapy chronic phase of arthritis (day 7), in or- suitable for such a therapeutic trial. der to avoid the uptake of liposomes into The macrophage suicide technique, us- phagocytic cells within the acute joint ing clodronate encapsulated in liposomes effusions present at an early stage of the (4), was initially described as a way of arthritis (10). Group 1 (treatment group, studying the re-population of the liver clodronate-liposomes) received an intra- and spleen after macrophage depletion, articular injection of 0.5 ml of clodro- and to demonstrate the importance of nate-encapsulated liposomes (200 mg/ liver and spleen macrophages in the im- ml), in the right hock joint, while group mune response to particulate antigens 2 (control group) received 0.5 ml of sa- given by the intravenous route (5, 6). line liposomes. Group 3 (non-arthritic) These studies have shown that liposomes was left untreated. are selectively taken up by macrophages into phagosomes. Fusion of phagosomes Positive controls: treatment with intra- with lysosomes leads to the breakdown articular steroid preparations of liposomal membranes and to the re- A further 6 sheep with arthritis were lease of clodronate into the target cell. treated with steroid preparations. Ster- This results in the death of macrophages. oid group 1 received intra-articular in- By comparison with other bisphospho- jection of 0.35 ml of Betamethasone nates it was shown that clodronate is (Celestone chronodose, Schering most effective for the elimination of ma- Plough; 5.7 mg/ml) in the right hock crophages (7). joint. Steroid group 2 were treated with In order to apply this method in a thera- 0.5 ml microspheres impregnated with peutic trial, we thought it best to have a Dexamethasone (gift from Associate model of arthritis in animals big enough Professor Tony Whateley, University of to allow easy injection and accurate Strathclyde, UK; 4 mg/ml). In both cases measurement of joints. We therefore the dosage was 2 mg. Treatment was adapted the Dumonde Glynn model of given on day 7. arthritis (8) for use in sheep (9). Using this model, we evaluated the therapeu- Joint assessments tic potential of liposome-encapsulated The anterior-posterior (AP) diameter of clodronate as a drug delivery system both hock joints was measured in mm designed to eliminate macrophages in the with skin calipers using a standardised inflamed joint. technique. Triplicate measurements were made and results expressed as the mean Materials and methods of 3 measurements. Measurements were Induction of arthritis made 2 days apart for 20 days following Sheep were immunised 3 times at ap- the induction of arthritis. The results proximately 2-week intervals with 10 mg were averaged and expressed as a % dif-

44 Hypothalamus-pituitary-adrenocortical and -gonadal axis in RA / M. Cutolo Liposome treatment of sheep arthritis / J.EDITORIAL Highton et al. ference [(right AP diameter - left AP di- Confocal Microscopy fluorescence was measured by FAC- ameter)/left AP diameter) x 100] in or- Sheep were injected in the right hock Scan™ analysis (12). der to adjust for size variation between joint with liposomes constituted with individual sheep. Measurements of joint BODIPY-phosphatidyl choline (100 µg, Statistical analysis diameter were made by an observer un- Molecular probe, Eugene, Oregon) seven Anova (Statview, single factor) were aware of which sheep were treated. days after the induction of arthritis so that performed to assess differences between uptake of liposomes could be traced by the AP diameter, and Student’s t-test for Histological and pathological analysis immunofluorescence. Sheep were killed the histological scores of the different The post mortem examinations and his- at 2, 6, and 24 hours after injection. Sec- treatment groups and in the coagulation tologic evaluations were carried out tions of synovial membrane (10 µm) test. We considered values of less than blind, i.e. without prior knowledge of were incubated with mouse anti-ovine 0.05 to be statistically significant. which group the animal belonged to. Post macrophage CD14 antibodies (Serotec, mortem synovial joint lining specimens UK), washed and then incubated with Results were taken from the right and left hock Texas Red goat anti-mouse IgG conju- Preliminary studies joints on the last day of the experiment gate (Molecular probe, Eugene, Oregon), Incubation of liposomes containing (day 20 post induction of arthritis). Fresh washed again and mounted with Prolong fluorescein acetate with whole hepar- synovial membrane was fixed in 10% mountant (Molecular probe, Eugene, inised blood followed by FACScan ana- buffered formalin and embedded in wax. Oregon). These double-labelled sections lysis confirmed the uptake of liposomes Sections (4 µm thick) were stained with were then examined by fluorescence mi- into monocytes. This was maximal at 4 haematoxylin and eosin. croscopy and confocal microscopy to hours. Incubation of clodronate lipo- For each sample, we assigned a total his- localise liposome uptake, and to see if somes with cultured macrophages re- tological score based on semi-quantita- joint macrophages were effectively tar- sulted in the depletion of cell numbers tive assessments of: (a) lining layer geted by liposomes. in comparison with saline liposomes, thickness (average of 3 areas, number of thus confirming the ability of clodronate cells thick); (b) degree of infiltration with Preparation of liposomes liposomes to kill macrophages as has mononuclear cells, where 1 = localised Clodronate was encapsulated within been described (13) (data not shown). and confined to sublining, 2 = perivascu- unilamellar vesicles using the thin film Cells loaded with the dye dihydrorho- lar and sublining, 3 = comprehensive hydration method (11). The lipid com- damine 123 (12) showed increased fluo- infiltration with inflammatory cells; (c) ponents, 40 mg total cholesterol, 80 mg rescence upon interaction with lipo- fibrin deposition, scored 1 = small local- egg phosphatidylcholine (both 99% pure somes, indicating the presence of a res- ised deposits, 2 = large localised depos- from Sigma, St Louis, USA) and 8 mg piratory burst (Fig. 1). This suggested its, 3 = widespread fibrin deposition; and phosphatidylserine (Sigma, St Louis, that liposomes could potentially have a (d) neutrophils present or not present. USA) were dissolved in chloroform (10 pro-inflammatory effect. Individual scores were added to give a ml) and dried to a thin film in a 500 ml Injection of Bodipy-labelled liposomes total histological score. round bottomed flask. Negatively into inflamed sheep hock joints and fluo- Each specimen was also used for immu- charged liposomes containing clodronate rescence microscopy carried out on nohistologic studies. These specimens were formed by the addition of clodro- synovial samples removed at 2, 6 and 24 were embedded in OCT compound nate (200 mg/ml) or normal saline fol- hours showed maximum uptake in the 6 (Miles Scientific, Naperville, IL), frozen lowed by hand shaking of the flask. The hour sample. Liposomes had dispersed in isopentane over liquid nitrogen, and preparation was left at room temperature by 24 hours. Figure 2 shows confocal mi- stored at -70°C. Frozen sections (8 µm for 2 hours and then probe sonicated for croscopic images from inflamed syno- thick) mounted on gelatin-coated slides 5 minutes at 4°C. The liposomes were vium from a sheep hock joint injected were fan-dried for 30 minutes and fixed washed 3 times with saline by centrifu- with Bodipy-liposomes 6 hours previ- in acetone at 4°C for 7 minutes. The sec- gation (4°C) and resuspended in a final ously. These experiments indicated that tions were incubated with mouse mono- volume of 2 ml for use. All glassware injected liposomes do gain access to clonal antibody to bovine CD5 (CC17, was sterilised and treated to remove en- synovial macrophages. Serotec, UK) and mouse anti-ovine mac- dotoxin and all solutions were filtered. rophage CD14 (VPM65, Serotec, UK) Treatment of arthritis with clodronate antibodies for 30 minutes at room tem- Dihydrorhodamine oxidative burst liposomes perature. After washing with PBS, sec- assay Injection of 5 mg ovalbumin into the tions were labelled with HRPO conju- Cell suspensions (500 µl monocytes) right hock joint of ovalbumin-sensitised gated antimouse antibody (DAKO Im- were incubated at 37°C for the required sheep resulted in acute swelling of the munoglobulins, Copenhagen), washed time with 50 µl Dihydrorhodamine (1 joint as shown by a 20 - 30% increase in again in PBS and developed with DAB mM), plus 150 µl negatively charged li- joint diameter (Table I). The most acute (diaminobenzidine, DAKO). Sections posomes; or 50 µl FMLP (formyl meth- swelling abated after 24 - 48 hours and were briefly counterstained with haemo- ionyl-leucyl-phenylalanine) diluted with the arthritis entered a more chronic toxylin. 100 µl saline; or 150 µl saline. Mean phase. Sheep in group 1 were treated

45 EDITORIALLiposome treatment of sheep arthritis / J. Highton et al. Hypothalamus-pituitary-adrenocortical and -gonadal axis in RA / M. Cutolo

somes could also be compared with positive controls treated with steroid preparations which were more effective (p < 0.01, ANOVA, Table I). Post mortem synovial specimens were assessed for thickening of the synovial lining, the intensity of the inflammatory cellular infiltrate and the extent of fibrin deposition (Fig. 3). There was no significant difference in these individual parameters or in the overall total histologic score (mean ± SEM, clodronate- liposomes 9.8 ± 0.5, control saline liposomes 6.7 ± 0.2, p = 0.6, t-test). Results for both types of liposomes were also significantly worse than for the non-arthritic group, (total score 1.8 ± 0.1, t-test p < 0.001 for both group 1 and 2). His- Fig. 1. Figures represent net mean fluorescence ± s.d. of monocytes loaded with dihydrorhodamine and tological scores for steroid-treated joints exposed to liposomes (measured by FacscanTM analysis). Net figures for fluorescence intensity were were not statistically superior to the calculated as the test mean minus the mean without stimulation. The bacterial peptide FMLP (formyl methionyl-leucyl-phenylalanine) was used as a positive control. Results shown are the mean of 3 clodronate-liposome-treated joints (beta- experiments ± s.d. SMFI = specific mean fluorescence intensity. Monocyte FMLP and liposomes results methasone (n = 3) total score 6.3 ± 1.1, were not significantly different (ANOVA, p > 0.05). The neutrophil FMLP and liposomes results (not t-test, p = 0.08; dexamethasone (n = 3), shown) were similar and showed a significant difference only at 0.3 hours (ANOVA, p = 0.002). total score 5.0 ± 0.6, t-test, p = 0.16). during this phase at day 7 by injection swollen in comparison to non-arthritic Discussion of 0.5 ml clodronate liposomes into ar- sheep when the experiment was termi- The prevailing view of rheumatoid arthritic joints. Comparison with group 2 nated at day 20: group 1, 16.3 ± 0.1, thritis is that it is a T-lymphocyte driven controls injected with saline liposomes group 2, 9.9 ± 0.1, group 3, -0.7 ± 0.6 (p disease. Despite this the evidence for the showed no significant difference be- < 0.01, t-test, for both group 1 and group involvement of T-lymphocytes is mainly tween the two groups (p = 0.9, ANOVA) 2 vs group 3). The results for sheep in indirect, and therapies directed against (Table I). Joints in both groups were still group 1 treated with clodronate lipo- T lymphocytes have met with limited success (14). The principal alternative view is that rheumatoid arthritis is mainly a disease of disordered macrophage function (15). In both models, the activated synovial macrophage is the cell which orchestrates those inflammatory events which culminate in joint destruc- tion (1, 16). Macrophage production of pro-inflammatory cytokines such as IL- 1 and TNF are critical events, and inhibition of such macrophage-produced cytokines is therapeutic (17). The use of antibodies to TNF has been shown to be effective in a double-blind clinical trial (18). This suggests that a therapeutic attack targeted directly at macrophages should be an effective treatment for inflammatory joint diseases such as rheumatoid arthritis. Conse- quently we undertook a trial of clodronate liposomes as anti-macrophage treat- Fig. 2. Confocal microscopic images of inflamed synovial lining from one sheep hock joint injected ment in an animal model of arthritis. The with Bodipy-liposomes 6 hours previously. Red fluorescence shows macrophages labelled with antibody sheep was chosen as it provided a model to sheep CD14 and texas-red; green fluorescence identifies cells that have taken up Bodipy-liposomes; with joints of sufficient size to be read- yellow (green plus red) cells are macrophages that have taken up Bodipy-liposomes. ily injected and measured.

46 Hypothalamus-pituitary-adrenocortical and -gonadal axis in RA / M. Cutolo Liposome treatment of sheep arthritis / J.EDITORIAL Highton et al. Our data from this trial has demonstrated that clodronate-liposomes, as an anti- macrophage treatment, did not show a significant therapeutic effect in antigen- induced arthritis in sheep when injected intra-articularly on a single occasion. Van Lent et al. (10) reported that a single dose of clodronate-liposomes given by intra-articular injection was effective in mice with antigen-induced arthritis. Efficacy for the depletion of synovial macrophages and therapy was depend- ent upon injecting clodronate liposomes in the chronic phase of arthritis. Clodronate liposomes were given to sheep in this study after the initial acute phase of inflammation and consumption of liposomes by the acute inflammatory infiltrate of neutrophils is therefore an unlikely explanation for the lack of ther- Fig. 3. Histological scores. Individual measures and total scores for sheep injected with clodronate- apeutic effect in this study. Other authors liposomes (group 1, treatment n = 10), saline-liposomes (group 2, control n = 10) or untreated (no have demonstrated efficacy when clo- liposomes, group 3, n = 6), where *** is p < 0.001 and **p < 0.01, and 1 indicates group 1 vs group 3, dronate liposomes were given intrave- and 2 indicates group 2 vs group 3. nously to rats with adjuvant arthritis (19), but not when clodronate liposomes were administered intra-articularly to Table I. Mean difference in anterior-posterior (AP) hock joint diameter (%) in sheep with rats with antigen-induced arthritis, simi- antigen-induced arthritis (day 0) who on day 7 were given either a single intra-articular injection of clodronate liposomes (group 1), saline liposomes (group 2) or no treatment. lar to our results. These results suggest that successful treatment with clodronate Positive controls liposomes might be influenced by the Clodronate-lip. Control Betamethasone Dexamethasone Control animal species and type of arthritis in- Days Group 1 Group 2 Steroid 1 Steroid 2 Group 3 duced. We confirmed that the clodronate lipo- -2 -0.1 ± 0.1 0.6 ± 0.1 -1.7 ± 0.5 -1.3 ± 0.5 2.0 ± 0.3 somes we manufactured were capable of -1 0.7 ± 0.2 0.1 ± 0.1 -0.67 ± 0.7 -1.3 ± 0.5 -1.2 ± 0.3 eliminating macrophages in vitro. We 1 19.3 ± 0.6 25.1 ± 0.6 25.7 ± 1.0 31.7 ± 2.5 0.7 ± 0.3 were also able to demonstrate that such 3 20.1 ± 0.1 22.5 ± 0.5 20.7 ± 1.8 19.0 ± 3.3 0.5 ± 0.2 liposomes rendered fluorescent with BO- 5 18.4 ± 0.2 21.9 ± 0.9 21.0 ± 8.9 18.7 ± 3.0 0.0 ± 0.2 DIPY were readily taken up by macrophages throughout the inflamed synovi- 8 22.4 ± 0.3 19.4 ± 0.7 13.7 ± 6.7 11.3 ± 2.5 0.0 ± 0.3 al lining of sheep with antigen-induced 10 18.5 ± 0.1 17.5 ± 0.9 8.0 ± 7.2 9.7 ± 2.4 0.7 ± 0.3 arthritis. Other authors have demon- 12 17.6 ± 0.1 16.1 ± 0.7 8.7 ± 3.9 9.3 ± 1.9 -0.7 ± 0.4 strated the ability of such liposomes to 14 16.0 ± 0.3 14.6 ± 0.8 9.3 ± 2.3 6.7 ± 0.7 -0.5 ± 0.3 deplete synovial macrophages (21, 22). 16 12.8 ± 0.5 13.4 ± 0.9 8.0 ± 1.5 5.0 ± 0.3 -0.8 ± 0.2 Thus, failure to access synovial macro- 18 14.6 ± 0.3 10.3 ± 0.6 7.3 ± 1.7 5.0 ± 0.6 0.0 ± 0.4 phages is also an unlikely explanation 20 16.3 ± 01 9.9 ± 0.7 7.0 ± 2.0 6.3 ± 0.5 -0.7 ± 0.6 of the lack of therapeutic effect demonstrated in this trial. Figures represent the mean difference in AP diameter between left (normal) and right (arthritic) hock Trials conducted to date in animals with joints, expressed as a % ± SEM. Lines at day 0 and day 7 indicate the induction of arthritis and the therapeutic joint injection respectively. There was no significant difference between group 1 (clodronate antigen-induced arthritis have showed treated arthritic sheep, n = 10) and group 2 (saline treated arthritic sheep, n = 10), p = 0.9, ANOVA. At the efficacy in mice (10), but not in rats (19) end of the experiment (day 20), both groups were still worse than the non-arthritic controls (n = 6) in or sheep with the same type of arthritis. group 3 (p < 0.01 respectively, Student’s t-test). Therefore, there could be species-spe- Both of the steroid-treated groups were significantly better than group 1 treated with clodronate liposomes (p < 0.01, ANOVA). Steroid group 1 sheep (n = 3) were treated with a standard intra-articular steroid cific effects as well as effects from the preparation, celestone chronodose (betamethasone, Schering Plough). Steroid group 2 sheep (n = 3) model of arthritis chosen, e.g. adjuvant were treated with microspheres impregnated with dexamethasone. In both cases the dosage was 2 mg. arthritis versus antigen-induced arthritis. Dexamethasone microspheres were a gift from Associate Professor Tony Whateley, University of Strath- Our data is derived from relatively few clyde, U.K. animals. However, they are large animals

47 EDITORIALLiposome treatment of sheep arthritis / J. Highton et al. Hypothalamus-pituitary-adrenocortical and -gonadal axis in RA / M. Cutolo whose joints are easily observed and 5. VAN ROOIJEN N, VAN DEN ENDE M, DIJK- macrophage ‘suicide’ technique. J Immunol measured and the complete lack of any STRA CD: Depletion and repopulation of ma- Methods 1990; 134: 153-61. crophages in spleen and liver of rat after in- 14. FOX DA: The role of T cells in the immuno- apparent therapeutic effect suggests that travenous treatment with liposome-encapsu- pathogenesis of rheumatoid arthritis. Arthri- larger numbers of animals would not lated dichloromethylene disphosphonate. Cell tis Rheum 1997; 40: 598-609. have resulted in a statistically significant Tissue Res 1990; 260: 215-22. 15. FIRESTEIN GS, ZVAIFLER NJ: How important outcome. It is also possible that a single 6. DELEMARRE FGA, KORS N, VAN ROOIJEN N: are T cells in chronic rheumatoid synovitis ? Elimination of spleen and lymph node ma- Arthritis Rheum 1990; 33: 768-73. dose of clodronate liposomes was insuf- crophages and its difference in the effect on 16. ZVAIFLER NJ: Macrophages and the synovial ficient, and that multiple doses would be the immune response to particular antigens. lining. Scand J Rheumatol 1995 (Suppl. 101): effective, for example, against the rapid Immunobiology 1990; 182: 70-78. 67-75. recruitment of fresh macrophages into 7. VAN ROOIJEN N, KORS N: Effects of intrac- 17. AREND WP, DAYER J-M: Inhibition of the pro- ellular disphosphonates on cells of the mono- duction and effects of interleukin-1 and tumor the joint lesion. In summary, although nuclear phagocyte system: In vivo effects of necrosis factor a in rheumatoid arthritis. Ar- this trial did not show a therapeutic ef- liposome-encapsulated disphosphonates on thritis Rheum 1995; 38: 151-160. fect, the concept of anti-macrophage different macrophage subpopulations in the 18. 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