Clinical efficacy of lenalidomide in multiple malignancies results from its many activities spanning from anti-angiogenic and hematological responses to enhanced erythropoiesis. Extensive clinical evaluations are underway, but dose-dependent and disease-specific toxicities remain a challenge. Further translational research in animal models is required to enhance our understanding of lenalidomide mechanisms of action and toxicity. With limited published pharmacokinetic data in animals, we sought to provide a thorough characterization of lenalidomide pharmacokinetics in mice to guide ongoing and future lenalidomide investigations in murine disease models.
In the present study, we characterized lenalidomide pharmacokinetics in ICR mice. Prior to conducting the pharmacokinetic studies, we explored toxicity of lenalidomide doses up to 15, 22.5, and 45 mg/kg via
IV, IP, and PO routes of administration. Limited by solubility in our PBS dosing vehicle, these maximum achievable lenalidomide doses were well tolerated with the exception of one mouse death (of four total dosed) at the 15 mg/kg IV dose. As cause of death was not determined, it is unclear if this was due to drug exposure, possible precipitation of lenalidomide in blood, or other drug-related causes. Notably, no other toxicities were observed in the study at IV doses of 15 mg/kg (n
3) or 10 mg/kg (n
45) or at any other dose level through IV, IP, and PO routes.
Low doses in mice result in similar plasma concentrations, high oral bioavailability, and monoexponential decline as seen in humans. With doses of 0.5–10 mg/kg, systemic bioavailability of lenalidomide ranged from 69% to 75% after oral administration and 90% to 105% after IP administration using compartmentally estimated AUC values. When comparing mouse pharmacokinetic data to that in humans, 1.5 and 5 mg/kg IV doses in mice produce noncompartmental AUCs of 2,130 and 3,200 h
ng/mL (128 and 192 min
μg/mL), which are most similar to observed lenalidomide plasma exposures following doses of approximately 25 mg in adults (examples, mean AUCs of 2,154 h
) or 2,415 h
)) or 15–30 mg/m2
in children (examples, mean AUCs ranging from 2,740 to 3,850 h
). Interestingly, the 1.5 and 5 mg/kg doses in mice translate to human equivalent doses of approximately 7 and 23 mg in adults or 4.5 and 15 mg/m2
in children, based on allometric scaling factors (49
). Notably, these doses are low relative to the observed data in clinical reports. Future doses used for translational studies in mice should consider this data while choosing clinically relevant doses.
Our data suggests lenalidomide has absorption rate limited and dose-dependent (or nonlinear) kinetics in ICR mice over a dosing range of 0.5–10 mg/kg. This nonlinearity is observed as a greater than proportional increase in plasma AUC and Cmax
between the 0.5 and 1.5 mg/kg doses, followed by a less than proportional increase in AUC and Cmax
between the 1.5, 5, and 10 mg/kg doses. These data indicate at least two distinct mechanisms contribute to the observed nonlinear pharmacokinetic behavior. Greater than proportional increases in AUC are consistent with saturated clearance mechanisms or altered distribution between two dose levels. Consistent with previous suggestions that active transport contributes to renal clearance of lenalidomide (21
), our group previously presented data suggesting P-glycoprotein is a potential mechanism for this apparent saturation in renal excretion (23
). Although additional transporters of lenalidomide have not yet been identified, a second contributing mechanism may be a saturation of active renal reabsorption. The likelihood of saturated reabsorption is uncertain and would depend on abundance of specific transporters and their affinities for lenalidomide.
Tissue distribution displayed in Fig. demonstrates a collective trend for the tissue AUC/plasma AUC ratio which is high for the 0.5 mg/kg dose, decreases for the 1.5 mg/kg dose, then increases for the 5 and 10 mg/kg doses. This indicates a greater portion of the low dose is distributed into the tissue volume as compared to the higher doses. Conceivably tissue binding is saturated at the 1.5 mg/kg dose, and a greater portion of lenalidomide is retained in plasma than at the 0.5 mg/kg dose. However as dose increases further, plasma protein binding may also become saturated thus increasing clearance at higher doses. In addition, solubility cannot be overlooked as a potential cause for the observed nonlinearity at higher doses. A 3 mg/mL concentration was selected for the maximum IV dosing solutions, which was just below the 3.5 mg/mL concentration with visible, undissolved particulates. These concentrated dosing solutions may have partially precipitated before or immediately upon injection into mice (fatality in one of the four mice dosed with 15 mg/kg IV lenalidomide may be evidence for the later). Although concentrations of dosing solutions were verified using LC-MS/MS, this required dilution to enable measured concentrations to fall within linear ranges of our assay, and may therefore not accurately represent the more concentrated dosing solutions. Furthermore, our observed limitations in solubility prevented an assessment of linearity and drug exposures with doses of 50 mg/kg (9
) and 100 mg/kg (51
), which were used by other groups previously in preclinical murine studies.
Lenalidomide was distributed to all analyzed tissues, although levels were undetectable in brain following 0.5 and 1.5 mg/kg doses. Tissue-to-plasma ratios calculated at each dose further demonstrate the nonlinearity observed in plasma. As an example, quantification of lenalidomide in eight tissues from IV dosed mice shows dissimilar kinetics in spleen and brain (Figs. and ). Lenalidomide distribution to spleen includes an absorption phase with a Tmax at 10 min, regardless of dose. Furthermore, spleen demonstrated the highest relative exposure when tissue AUCs were normalized to blood perfusion rate (Fig. ). This apparent accumulation may be explained by the presence of uptake transporters or high affinity binding to biomolecules expressed specifically in the spleen (either intra- or extra-cellular). To our knowledge, these possibilities have not been reported previously and should be further explored. This preferential distribution to spleen may be relevant for lenalidomide efficacy and toxicity mechanisms in lymphoid organs.
Lenalidomide distribution to brain was detectable only at doses of 5 mg/kg and above. With an assay lower limit of quantification of 0.3 ng/g in brain tissue, the data suggest relatively poor penetration into brain. This is in contrast to its analog, thalidomide (logP
), which has considerable brain penetration (54
). Although lenalidomide structure and lipophilicity (logP
) are similar to thalidomide, recent evidence indicates lenalidomide is a substrate of the efflux transporter, P-glycoprotein (23
). This active transporter mediates xenobiotic removal from the brain as part of the blood brain barrier (56
) which would reduce overall lenalidomide uptake in the CNS. A recent publication exploring distribution of lenalidomide in nonhuman primates determined 11% exposure in cerebrospinal fluid relative to plasma following a dose of 20 mg (29
). Similarly, we report lenalidomide brain exposures following 5 and 10 mg/kg in mice are approximately 0.9% and 2.3% of that observed in plasma. These results may explain why clinical neurotoxicities are minimal and reported in few patients relative to thalidomide (57
). While studies are being pursued to evaluate lenalidomide efficacy in cancers of the CNS and other neurological disorders, the relatively low lenalidomide CNS penetration may become an important factor.
This work provides data for plasma pharmacokinetics and tissue distribution of lenalidomide in mice at clinically relevant doses. Intraperitoneal and oral administration routes demonstrate good bioavailability in mice. These studies can be used to estimate concentrations achieved at relevant sites of drug action when lenalidomide efficacy studies are performed in murine models. As presented above, transporter-mediated disposition and potential intra- or extra-cellular preferential binding of lenalidomide offer possible explanations for the observed nonlinearity. Considerations of nonlinearity and selective tissue distribution reported herein may aid in selection of doses with greater clinical relevance to better enable interspecies extrapolation of results in continued clinical and translational development of lenalidomide.