Separating therapeutic efficacy from glucocorticoid side-effects in rodent arthritis using novel, liposomal delivery of dexamethasone phosphate: long-term suppression of arthritis facilitates interval treatment

Abstract

Introduction: Glucocorticoids have extensively been used in the treatment of rheumatoid arthritis and other inflammatory diseases. However, their side-effects remain the major limitation in clinical use and an improved therapeutic index is needed.

Methods: Therapeutic efficacy and persistence of free and liposomal dexamethasone phosphate (DXM-P) were determined in mouse collagen-induced arthritis. For regimens with equal therapeutic benefit, the side-effect profiles were analysed over time with respect to collagen breakdown, suppression of the hypothalamus-pituitary-adrenal (HPA) axis, changes in blood glucose levels and the haematological profile. In addition, the presence of drug was monitored in plasma.

Results: Liposomal DXM-P, but not free drug, resulted in a persistent anti-inflammatory effect. Comparable clinical benefit was achieved with a single administration of 4 mg/kg liposomal DXM-P or daily administrations of 1.6 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 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.

Figure 1

Study design. Treatment protocol for administration of free dexamethasone phosphate (DXM-P) and liposomal DXM-P. Blood samples for assessment of the side-effect profile were collected 2 and 7 days after last treatment. An additional blood sample was collected from mice treated with free DXM-P 2 days after start of the treatment. CFA, complete Freund's adjuvant; i.v., intravenous.

Figure 2

Therapeutic efficacy and persistence of liposomal dexamethasone phosphate. (a) Joint swelling was assessed by clinical scoring after a single injection of liposomal dexamethasone phosphate (DXM-P) (0.4 mg/kg, 1.6 mg/kg or 4 mg/kg) or after seven daily injections of free DXM-P (0.4 and 1.6 mg/kg). A single dose of free DXM-P at 1.6 mg/kg is also shown for comparison. Saline injections were used as controls. n = 12 for all groups; means ± standard error of the mean. (b) Urine pyridinoline levels 2 and 7 days after cessation of the treatment. *P ≤ 0.05 Mann-Whitney U test versus saline-treated inflamed animals. n = 6 for all groups; means ± standard error of the mean.

Figure 3

Impact of liposomal and free dexamethasone phosphate on corticosterone. Corticosterone levels in the blood of (a) arthritic animals or (b) healthy animals were measured following treatment with liposomal dexamethasone phosphate (DXM-P), free DXM-P or saline. **P ≤ 0.01 Mann-Whitney U test versus saline. n ≥ 6 for all groups; mean ± standard error of the mean.

Figure 4

Effects of liposomal and free dexamethasone phosphate on blood glucose, liver enzymes and haematology. (a) Glucose levels as a percentage of the initial blood value in arthritic animals following treatment with liposomal dexamethasone phosphate (DXM-P), free DXM-P, or saline. (b) Values of liver enzymes alkaline phosphatase (AP), alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) after treatment with liposomal DXM-P, free DXM-P, or saline. (c) Percentage of polymorphonuclear neutrophilic leukocytes (PMNs) and lymphocytes in the blood of arthritic animals following treatment with liposomal DXM-P, free DXM-P, or saline. *P ≤ 0.05 Mann-Whitney U test versus saline-treated inflamed animals. n = 6 for all groups; mean ± standard error of the mean.

Figure 5

Pharmacokinetics of liposomal and free dexamethasone phosphate in rats. (a) A dose of 4 mg/kg liposomal dexamethasone phosphate (DXM-P) (open squares) or free DXM-P (diamonds) was injected intravenously into rats, and the appearance of the converted dexamethasone (DXM) was monitored in plasma. In a separate experiment, radiolabelled liposomal DXM-P (triangles) was injected into rats and their pharmacokinetics was followed using ¹⁴C-1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). (b) Integrity of liposomal DXM-P. Radiolabelled liposomal DXM-P was injected as in (a) and the additional aqueous phase marker ³H-inuline was analysed. The resulting ³H/¹⁴C ratio was plotted with respect to the initial ratio. The constant ratio indicates high carrier integrity in the circulation. Error bars indicate standard deviations.