Introduction
Rheumatoid arthritis (RA) is a systemic disorder of unknown etiology characterized by chronic inflammation and symmetric, progressive destruction of arthritic joints. The abundance and activation of macrophages (Mφ) in the inflamed synovial membrane significantly correlates with the severity of RA [
1,
2]. In addition, activation of the monocytic lineage extends to systemic parts of the mononuclear phagocyte system [
3‐
7]. Thus, selective counteraction of Mφ activation is a promising approach to diminish local and systemic inflammation or to prevent irreversible joint damage.
In addition to disease-modifying anti-rheumatic drugs (DMARDs) and to recently introduced biologicals (for example, antibodies against tumor necrosis factor- (TNF)-α or soluble TNF-α-receptors, [
8‐
14]), anti-inflammatory glucocorticoids are still frequently employed to bridge the gap before the onset of action of DMARDs [
15‐
17] and to improve the therapeutic control of RA. Due to their unequalled efficacy, bridging application and wide indication range (for example, renal failure, pregnancy), glucocorticoids remain valuable therapeutic tools. However, concerns about long-term side effects, such as Cushing syndrome or bone demineralization, strongly emphasize the need for safer treatment modalities. Specific targeting of glucocorticoids to phagocytic cells by liposomal encapsulation potentially increases drug efficacy and longevity while minimizing side-effects [
18,
19].
Previous studies have demonstrated good therapeutic efficacy of water-soluble prednisolone in neutral polyethylene glycol-modified (PEGylated) liposomes in animal models of arthritis and multiple sclerosis [
20‐
23]. However, evidence has emerged that repeated injections can result in the generation of anti-PEG antibodies, emphasizing the need for a PEG-free liposomal formulation [
24‐
27].
In this study, water-soluble dexamethasone phosphate (DxM-P) was encapsulated in a novel, non-PEGylated liposome formulation (Micromethason). All lipids in this formulation have market approval, thereby minimizing the risk of lipid-associated toxicity. The efficacy of liposomal DxM-P was evaluated in rat adjuvant arthritis (AA), a severe animal model characterized by histopathological similarities to RA, including both systemic and local features of inflammation [
28]. The effects of treatment with free DxM-P or different doses of liposomally-encapsulated DxM-P were also evaluated, in order to assess the increase of therapeutic potency by encapsulation. In addition, a pharmacokinetics and biodistribution study of DxM was carried out following administration of liposomal DxM-P or free DxM-P.
Discussion
Intravenous therapy with liposomal DxM-P in established AA (analogous to the clinical situation in RA) suppressed joint swelling and inflammation in a significant, dose-dependent, and long-lasting manner. Notably, liposomal DxM-P showed superior therapeutic efficacy in both early and advanced disease compared to matched doses of free DxM-P. Liposomal encapsulation, therefore, clearly potentiates the clinical efficacy of the well-established anti-rheumatic glucocorticoid (GC) DxM-P.
This superior efficacy is already achieved upon short-term treatment for three days, similar to successful, long-term suppression of human RA [
37] and rat antigen-induced arthritis (including an approximately 80% reduction of a flare-up induced seven days after the end of treatment) [
19]. The prolonged efficacy may represent a substantial advantage in the treatment of RA, with the potential to obtain therapeutic effects with lower and/or less frequent doses. Of interest, the AUC for arthritis score/paw volume was more markedly reduced by liposomal than by free DxM-P, suggesting a decrease of the
cumulative disease activity altogether.
Pharmacokinetic/biodistribution results showed a slower elimination of the liposomal drug from the circulation, confirming previous results [
38], and substantially higher levels and longer persistence of the liposomal form of the drug in synovial membrane, spleen and liver until at least 48 hours after the last injection. These depot effects likely contribute to the superior and prolonged clinical efficacy of the encapsulated formulation. Enhanced therapeutic efficacy extended until Day 9 post injection of the first dose (corresponding to Day 23 of AA; Figure
1), although no DxM was detectable in blood or synovial tissue at this time point. Typical GC-receptor mediated drug actions require 1 nM concentrations or less of DxM, a level that is below the quantification limit of 5 nM (2 ng/ml) of the LC-MS method employed. Residual liposomal drugs in circulation and/or tissues may thus contribute to the persistence of clinical effects [
19].
In an independent arthritis study, administration of one dose of liposomal DxM-P (1 mg/kg) and three doses of free DxM-P (3 × 1 mg/kg) showed comparable therapeutic efficacy. In addition, long-term suppression of AA by three medium doses of liposomal DxM-P (3 × 0.1 mg/kg) was basically equivalent to that of three high doses of free DxM-P (3 × 1 mg/kg). Furthermore, blood DxM concentrations 24 hours after a single dose of liposomal administration (1 mg/kg) were 17-fold and 5-fold higher, respectively, than 24 hours after a single or triple administration of free drug (one or three doses of 1 mg/kg). Thus, the present results indicate that liposomal encapsulation allows a dose reduction by a factor of at least 3, if not 10. Also, the difference between the clinical efficacy of matched doses of liposomal and free DxM-P was augmented with rising DxM-P concentrations, suggesting that potentiation by liposomal encapsulation may be enhanced by a higher dose of DxM-P.
Dose reductions via encapsulation have been recently described in AA with prednisolone-containing PEGylated-liposomes [
22,
23]. Avnir
et al. [
23] achieved an effective suppression of arthritis by administering two or three doses of 10 mg/kg methylpredisolone either in early AA (Days 10 and 14, or Days 10, 14 and 18) or in established AA (Days 19 and 23) using PEGylated, remote-loaded liposomes. The latter results are very similar to those of the present study, in particular when considering the differences between methylprednisolone and DxM in terms of glucocorticoid equivalent. To our knowledge, however, this study provides the first evidence of a prolonged and nearly 100% remission in severe established AA with DxM-P in non-PEGylated liposomes.
The long-term benefit of short-term therapy with liposomal DxM-P was histologically confirmed by significant reduction of inflammation, as well as bone and cartilage destruction. At this relatively early stage of disease (Day 21), the histological effects of free DxM-P were similar to those of liposomal DxM-P, and did not yet reflect the clinical rebound observed with free DxM-P after Day 19. Interestingly, only liposomal DxM-P accumulated in the inflamed synovial membrane, matching the targeting of the inflamed joint achieved by other novel liposome formulations [
23]. Based on the known direct and indirect contribution of synovial Mφ to cartilage and bone destruction
via TNF-α, IL-1β, or IL-6 [
2,
3,
7,
39], the present histological findings suggest that both Mφ activation and secretion of pro-inflammatory mediators are inhibited by effectively targeting these cells with liposomal DxM-P. Local efficacy of liposomal DxM-P extended to periarticular tissue (data not shown) and pop LN, in which liposomal (but not free) DxM-P reduced the number of lymphocytes presumably involved in regional perpetuation of inflammation [
40].
The present study demonstrates for the first time that, in contrast to free DxM-P, i.v.-injected liposomal DxM-P showed systemic anti-inflammatory effects by profoundly decreasing ESR and total WBC during acute and chronic AA. Although leukocytosis in PBS-treated AA was already paralleled by an increase of the relative percentage of PMN and a reduction of the lymphocyte percentage, both free DxM-P and liposomal DxM-P caused a further significant decrease of circulating lymphocytes. Lymphocytes were reduced to levels below 50% and 75% of those in healthy controls by both free and liposomal DxM-P, respectively, suggesting that lymphopenia may be the result of direct DxM effects in addition to indirect effects via Mϕ inactivation.
Free GCs, in particular DxM-P, are known to diminish the number of circulating lymphocytes via two mechanisms [
41]: 1) redistribution to spleen, lymph nodes, bone marrow, and perivascular compartments; 2) induction of cell apoptosis. In the present study, however, significantly lower numbers of lymphocytes in lymphoid organs were observed after therapy with liposomal DxM-P, suggesting that redistribution to spleen and LN either did not occur or was balanced by increased local lymphocyte apoptosis. Since liposomes are hardly taken up by lymphocytes
in vitro ([
42] and own results, data not shown), direct induction of lymphocyte apoptosis by liposomal DxM-P is unlikely. Thus, the specific contribution of the prolonged accumulation of liposomal DxM in the spleen to systemic lymphocyte redistribution remains to be analyzed.
It is presently unclear whether the therapeutic efficacy of liposomal DxM-P is due to sustained immunosuppression. On one hand, the DTH to pathogenetic Mb was significantly increased on Day 21 after liposomal DxM-P treatment when compared to AA/PBS, which speaks against immunosuppression. This may indicate a shift of the Th1/Th2 balance [
40,
43] in favor of a protective T cell reactivity, for example by modulating the Mφ cytokine pattern [
44] and/or by inducing a
regulatory Th1 type DTH [
45]. On the other hand, liposomal (but also free) DxM-P completely reverted the pathologically increased levels of specific serum anti-Mb IgG after Day 21; thus, the therapeutic effect of liposomal DxM-P may at least be partially due to, or associated with, effects on humoral immunity. However, since total IgG was not evaluated, it remains unclear whether this represents broad suppression of the humoral immune response or rather selective inhibition of disease-triggered immunity. The delay of the effects on the specific IgG levels compared to those on the cell-mediated immunity is likely due to the long half-life of the circulating IgG.
Mφ were clearly addressed by the liposomal DxM-P, as shown by a significant, persistent inhibition of the secretion of pro-inflammatory cytokines in peritoneal Mφ (until five days after the end of therapy). At this point in time, liposomal DxM-P had little effect on TNF-α secretion by peritoneal Mφ, suggesting that, if these results are transferable to the arthritic joints [
40], clinical improvement may not be primarily mediated by local reduction of TNF-α, but rather by reduction of IL-1β and IL-6.
As described above, clinical efficacy of liposomal DxM-P may be related to its prolonged circulation in the blood and its enhanced accumulation in the affected joint; however, marked accumulation in other immunologically active organs (spleen and liver) is also likely to influence AA on a local and systemic level. A similar marked accumulation was observed in the spleen of AIA rats following injection of our liposomal DxM-P [
19]; thus, the spleen may represent a key efficacy target of these novel liposomes, in line with the known contribution of the spleen to inflammation severity in acute AA [
40]. On the other hand, liver acumulation may reduce the number of activated macrophages in the liver and thereby suppress the subsequent local production of pro-inflammatory cytokines such as IL-1β [
46]. However, in addition to local and systemic depot effects of liposomal DxM, also non-genomic effects of high DxM peak levels may contribute to its therapeutic efficacy [
38].
The administration of three doses of 1 mg/kg liposomal DxM-P transiently reduced the body weight. This is a known, rat-specific side-effect of glucocorticoids (observed at doses above 0.05 mg/kg DxM), mediated by decreased ribosomal amino acid incorporation into muscle cells [
47], and possibly related to low plasma levels of released DxM-P [
20]. Thus, lymphopenia (see above) and body weight reduction appeared to be the only evident side-effects of treatment. Of note, higher drug adsorption of liposomal DXM-P in tissues and organs is not associated with stronger GC side effects. Instead, triggering of the HPA axis, gluconeogenesis or lymphocyte alterations are markedly reduced in comparison with the effects of free DxM-P [
38].
Drug-free liposomes showed a modest pro-inflammatory effect in acute and chronic AA (especially after Day 26). PEGylated liposome formulations are known to induce allergic reactions [
26,
48], possibly as a result of complement activation [
49]. However, liposome size (especially above 90 nm) can also play a key role in complement activation [
50], potentially explaining the slightly increased lymphocyte number in pop LN observed with the non-PEGylated PBS-liposomes in our study (mean size 300 nm). Pro-inflammatory effects in the arthritic joint, in turn, may be due to increased liposome uptake by activated Mφ, possibly triggered by lysosomal degradation of DPPC to lyso-phosphatidylcholine, which significantly increases Mφ ingestion [
51]. Recent improvements such as the increase of the drug-to-lipid ratio may allow diminishing the lipid load [
23].
Competing interests
Steffen Panzner is founder and shareholder of Novosom AG. Novosom AG holds patent WO/2004/047792: Liposomal glucocorticoids, which is granted or pending in several jurisdictions. RB and RWK have each received a one-time compensation of <10,000 Euros as inventors.
Authors' contributions
RA carried out the study, performed the statistical analysis and drafted the manuscript. AF, MC and FP participated in the design, coordination and execution of the in vivo studies. DP carried out the dose-response analysis and MG evaluated the histological slides. APS, TK, and CT designed, executed and described the pharmacokinetics/biodistribution experiments. RB coordinated dose response analysis, histological studies, and immunoglobulin ELISAs. SP and UR manufactured the liposomes and participated in the drafting of the manuscript. RWK initiated the study, participated in its design and coordination, and was the main contributor to the manuscript. All authors read and approved the final manuscript.