Novel Liposomal Rolipram Formulation for Clinical Application to Reduce Emesis

Abstract

Introduction: The phosphodiesterase 4 (PDE4) inhibitor, rolipram, has beneficial effects on tissue inflammation, injury and fibrosis, including in the liver. Since rolipram elicits significant CNS side-effects in humans (ie, nausea and emesis), our group developed a fusogenic lipid vesicle (FLV) drug delivery system that targets the liver to avoid adverse events. We evaluated whether this novel liposomal rolipram formulation reduces emesis.

Methods: C57Bl/6J male mice were used to compare the effect of three doses of free and FLV-delivered (FLVs-Rol) rolipram in a behavioral correlate model of rolipram-induced emesis. Tissue rolipram and rolipram metabolite levels were measured using LC-MS/MS. The effect of FLVs-Rol on brain and liver PDE4 activities was evaluated.

Results: Low and moderate doses of free rolipram significantly reduced anesthesia duration, while the same doses of FLVs-Rol had no effect. However, the onset and duration of adverse effects (shortening of anesthesia period) elicited by a high dose of rolipram was not ameliorated by FLVs-Rol. Post-mortem analysis of brain and liver tissues demonstrated that FLVs affected the rate of rolipram uptake by liver and brain. Lastly, administration of a moderate dose of FLVs-Rol attenuated endotoxin induced PDE4 activity in the liver with negligible effect on the brain.

Discussion: The findings that the low and moderate doses of FLVs-Rol did not shorten the anesthesia duration time suggest that FLV delivery prevented critical levels of drug from crossing the blood-brain barrier (BBB) to elicit CNS side-effects. However, the inability of high dose FLVs-Rol to prevent CNS side-effects indicates that there was sufficient unencapsulated rolipram to cross the BBB and shorten anesthesia duration. Notably, a moderate dose of FLVs-Rol was able to decrease PDE4 activity in the liver without affecting the brain. Taken together, FLVs-Rol has a strong potential for clinical application for the treatment of liver disease without side effects.

Keywords: PDE4; liposomes; liver; rolipram; side-effects.

Figure 1

Effects of fusogenic lipid vesicles-rolipram (FLVs-Rol) and non-encapsulated rolipram on xylazine/ketamine anesthesia duration time. (A) Comparison of baseline vs anesthesia duration following administration of 1.6 (n=5), 3.3 (n=6) and 6.6 (n=6) mg/kg of i.v. rolipram. (B) Comparison of baseline vs anesthesia duration time following administration of i.v. FLVs-Rol (1.6 (n=6), 3.3 (n=6) and 6.6 mg/kg (n=6) of rolipram in 12.5 mg/mL of FLV lipid). Each mouse served as its own baseline control and anesthesia duration was measured by the return of the righting reflex. Values represent mean±SD; *P-value ≤ 0.05; **P-value ≤ 0.01.

Figure 2

Safety pharmacology studies comparing the effect of i.v. administration of a high dose of free rolipram (6.6 mg/kg) or fusogenic lipid vesicles-rolipram (FLVs-Rol) followed by anesthesia induction at 35-, 60-, 80-, 100-, and 120-min post-dosing. (A) Time course of onset and duration of CNS side-effects following a bolus infusion of rolipram (n=6 per time point). (B) Time course of onset and duration of CNS side-effects following a bolus infusion of FLVs-Rol (n=6 per time point except for 100 min where n=5). Each mouse served as its own baseline control and anesthesia duration was measured by the return of the righting reflex. Values represent mean±SD; *P-value ≤ 0.05; ** P-value ≤0.01.

Figure 3

Comparison of liver-to-brain ratios of rolipram, hydroxy-pyrrolidinone rolipram (HPR), decyclopentylated rolipram (DR) and hydroxy-cyclopentyl rolipram (HCR). Tissues from mice in the 3.3 mg/kg of rolipram dose response study groups were harvested at the end of experiments after receiving i.v. free drug (n=4) or fusogenic lipid vesicles-rolipram (FLVs-Rol) (n=4). Rx = treatment; Values represent mean±SD; *P-value ≤ 0.05.

Figure 4

FLVs-Rol inhibits lipopolysaccharide (LPS)-inducible PDE4 activity in the liver but not in the brain. Mice were administered FLVs-Rol, at a dose of 3.3. mg/kg for 24 hours followed by 5 mg/kg LPS i.p. for 3 hours. PDE4 activities were measure in (A) liver and (B) brain tissues of control mice (n=6, PBS vehicle control), mice treated with LPS alone (n=9) and mice pretreated with FLVs-Rol followed by LPS treatment (n=9). PDE4 activity changes are presented as fold change over untreated (UT). Data are presented as individual values with mean±SD. ** P-value ≤0.01; *** P-value ≤0.001.