The lipid film was hydrated with DXM-P (25 mg/ml in PBS, pH 7.5), and the resulting vesicles were extruded through 400 nm membranes. persistent anti-inflammatory effect. Comparable clinical benefit was achieved with a single administration of 4 mg/kg liposomal DXM-P or daily administrations of 1 1.6 mg/kg free drug for at least 7 days. For the liposomal form, but not for the free form, we observed a limitation of the suppression of the HPA axis in time and an absence of the drug-induced TM6089 gluconeogenesis. == Conclusions == Liposomal DXM-P, but not free DXM-P, achieves therapeutic persistence in mouse collagen-induced arthritis, which results in drug-free periods of therapeutic benefit. The physical absence of drug after day 2 is associated with a reduction of the typical glucocorticoid side-effects profile. Liposomal DXM-P thereby has an improved therapeutic window. == Introduction == Glucocorticoids have long been used in the treatment of rheumatoid arthritis, and are an essential part of the first-line anti-inflammatory treatment. Dose escalation and long-term, chronic use of glucocorticoids lead to a number of well-characterized clinical side-effects, however, such as Cushing syndrome [1,2], diabetes [3], or bone demineralization [4], all of which are limiting their therapeutic use. Strategies for an improvement of the therapeutic index of glucocorticoids focus on the drug molecule itself, its specificity or metabolic conversion [5,6]. Others have investigated selective glucocorticoid receptor agonists to improve the ratio between the therapeutic effect and adverse reaction [7,8]. In addition, the targeted delivery of these drugs using liposomal formulations has been introduced, and a number of studies have demonstrated superior efficacy for water-soluble prednisolone in neutral, polyethylene glycol-modified (PEGylated) liposomes in rheumatoid arthritis animal models and multiple sclerosis [9-12]. A preliminary report on the first clinical use of these formulations confirmed potency and safety in patients [13]. PEGylation on small-sized liposomes is known to minimize uptake into phagocytic cells such as macrophages, which results in extended circulation times, but may be TM6089 counterindicated for the treatment of inflammatory disorders where macrophages are key producers of proinflammatory cytokines. PEGylation of liposomes has also been associated with the generation of anti-PEG antibodies [14]. We thus developed a non-PEGylated liposomal dexamethasone phosphate (DXM-P), a material that shows cellular uptake in monocytes and macrophages and is devoid of antibody formation even after TM6089 repeated administration (U Rauchhaus, unpublished results). The material showed a very high accumulation in spleen, whereas drug levels transported into the liver did not exceed peak plasma concentrations. A complete and persistent therapeutic benefit was observed after a single administration [15]. Given the specific distribution of liposomal DXM-P in combination with its therapeutic persistence, we here analyse the potential of this material for a separation of the therapeutic benefit from glucocorticoid-related side-effects. First, a single administration of liposomal DXM-P and daily injections with the free drug were adjusted for equal therapeutic efficacy in mouse collagen-induced arthritis. Second, we characterized the glucocorticoid-related side-effects in both therapeutic modalities. Eventually, the presence of liposomes and dexamethasone were monitored in plasma to correlate presence of the drug with the appearance of its side-effects. == Materials and methods == == Preparation of liposomal dexamethasone phosphate == Liposomes were prepared from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-(phosphor-rac-(1-glcerol)), sodium salt (DPPG), and cholesterol (50:10:40 mol%) using the lipid film extrusion method [16,17]. The lipid film was hydrated with DXM-P (25 mg/ml in PBS, pH 7.5), and the resulting vesicles were extruded through 400 nm membranes. Non-encapsulated DXM-P was removed by gel filtration. The particle Rab25 size (283 to 310 nm) and polydispersity (<0.3) were determined by dynamic light scattering. The drug/lipid ratio was 40 g/mol and the concentration was adjusted to 500 g DXM-P/ml. == Animal model of collagen-induced arthritis == All animal studies described here were approved by the Government Commission for Animal Protection. Arthritis was developed in male DBA/1 mice (age range, 7 to 10 weeks; Taconic Europe, Ry, Denmark) as previously described [18]. Mice were injected with 100 l type II bovine collagen emulsified in complete Freund's adjuvant (Sigma, Taufkirchen, Germany) on days X1 and X21, and disease progression was monitored in one joint per paw.