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Lenalidomide is a synthetic derivative of thalidomide exhibiting multiple immunomodulatory activities beneficial in the treatment of several hematological malignancies. Murine pharmacokinetic characterization necessary for translational and further preclinical investigations has not been published. Studies herein define mouse plasma pharmacokinetics and tissue distribution after intravenous (IV) bolus administration and bioavailability after oral and intraperitoneal delivery. Range finding studies used lenalidomide concentrations up to 15 mg/kg IV, 22.5 mg/kg intraperitoneal injections (IP), and 45 mg/kg oral gavage (PO). Pharmacokinetic studies evaluated doses of 0.5, 1.5, 5, and 10 mg/kg IV and 0.5 and 10 mg/kg doses for IP and oral routes. Liquid chromatography–tandem mass spectrometry was used to quantify lenalidomide in plasma, brain, lung, liver, heart, kidney, spleen, and muscle. Pharmacokinetic parameters were estimated using noncompartmental and compartmental methods. Doses of 15 mg/kg IV, 22.5 mg/kg IP, and 45 mg/kg PO lenalidomide caused no observable toxicity up to 24 h postdose. We observed dose-dependent kinetics over the evaluated dosing range. Administration of 0.5 and 10 mg/kg resulted in systemic bioavailability ranges of 90–105% and 60–75% via IP and oral routes, respectively. Lenalidomide was detectable in the brain only after IV dosing of 5 and 10 mg/kg. Dose-dependent distribution was also observed in some tissues. High oral bioavailability of lenalidomide in mice is consistent with oral bioavailability in humans. Atypical lenalidomide tissue distribution was observed in spleen and brain. The observed dose-dependent pharmacokinetics should be taken into consideration in translational and preclinical mouse studies.
Lenalidomide is a synthetic immunomodulatory drug (IMiD) analog of the anti-angiogenic drug, thalidomide. Although initially selected as a lead compound for potent inhibition of tumor necrosis factor-alpha (1), lenalidomide exhibits further activities including anti-angiogenic properties (2,3), increased erythropoiesis (4), direct induction of tumor apoptosis (5), modulation of pro-survival and inflammatory cytokines (6), an increase in immune effector cell response (5,7,8), and synergistic effects in combination therapies (9,10). With this extensive activity profile, exploration of drug efficacy across multiple diseases has been underway since the drug’s clinical release in the early 2000s (11). In the last decade, lenalidomide has met Food and Drug Administration approval for multiple myeloma (MM) and myelodysplastic syndrome (MDS). It also demonstrates clinical activity in systemic amyloidosis (12,13), chronic lymphocytic leukemia (CLL) (14–16), mantle cell lymphoma (17), myelofibrosis with myeloid metaplasia (18), and non-Hodgkin’s lymphoma (19,20) and continues to be investigated for treatment of other malignancies and hematological conditions. This widespread anticancer response is believed to result from a unique combination of the drug’s many activities. However, the mechanism responsible for induction of these events remains unclear.
Lenalidomide is orally administered with rapid absorption and bioavailability greater than 80% in healthy individuals (21). It is a small molecule (molecular weight=259) with moderate solubility at physiological pH where it is not ionized. In acidic conditions, such as in the stomach and proximal intestine, lenalidomide becomes protonated and its solubility increases greater than tenfold (22). While passive diffusion likely plays a role in its absorption, the high bioavailability and absorption of protonated lenalidomide may also be facilitated by transporter-mediated pathways. Approximately 90% of lenalidomide clearance occurs primarily by renal excretion, and a portion of this is believed to be transporter mediated (21,23). Renal reabsorption has not been directly studied for lenalidomide, and it is therefore unknown if this contributes to its overall disposition. Plasma protein binding in patients is between 35% and 45%, does not vary significantly in patients with renal impairment, and is constant over physiological concentrations (24). Hydroxylated and acetylated metabolites (21) are formed at a low abundance less than 5% of dose (21). Hydrolysis products of lenalidomide have also been observed (21,25); however, to our knowledge, their structures and activities have not been reported. The contributing effects of these properties on lenalidomide linearity and differential tissue distribution need to be further characterized.
Lenalidomide therapy is approved for daily oral administration. In adults, lenalidomide doses are not adjusted to body weight or body surface area, and there is no accumulation of drug after multiple, daily doses (11,22). In healthy subjects, lenalidomide has a 3–4 h half-life of elimination and typical maximum plasma concentration occurring 0.6–1.5 h postdose (24). Data are commonly described by a one-compartment model with mono-exponential decline of plasma concentrations (24,26). Pharmacokinetic analyses of orally administered lenalidomide mostly conclude plasma exposures are linear with increasing doses. One exception to this was a recent report of greater than proportional maximum observed plasma concentrations (Cmax) and concentration–time curve (AUC) in a phase 1 trial of lenalidomide dosed from 5 mg up to 40 mg daily in patients with refractory metastatic cancers (27). The authors evaluated a Michaelis–Menten function to describe this perceived nonlinearity, but ultimately utilized a dose-proportional model which fit the data equally well. In addition to humans (21,23,24,28), pharmacokinetic studies of lenalidomide have been reported in rats and monkeys (3,29).
Disease-specific toxicity profiles and potentially life-threatening adverse events associated with lenalidomide treatment are responsible for considerable diversity in starting doses. In the majority of indications, neutropenia and thrombocytopenia are the most frequent dose-limiting toxicities (11,30,31). However, increased severity of these adverse events limits the dose to 10 mg in MDS compared to the standard 25 mg dose in MM (11). In contrast to lower-grade myelosuppression, patients with MM are at greater risk for thromboembolytic events (32,33) as compared to MDS patients. The conventional 25 mg starting dose in clinical trials is also poorly tolerated in other hematological malignancies such as systemic amyloidosis (12) and CLL (34). Tumor flare reaction appears to be exclusive to CLL and remains poorly understood mechanistically. Typically associated with a cytokine release syndrome, the extreme severity of lenalidomide’s response in CLL is accountable for the significant limitations in starting dose for current clinical trials. In contrast, the maximum tolerable dose in patients with relapsed acute leukemias was found to be 50 mg (35) and in pediatric patients with primary tumors of the central nervous system (CNS) or with refractory solid tumors or MDS, maximum tolerated dose was not reached at normalized doses up to 70 or 116 mg/m2 (36). The variable and unknown mechanism of action and ill-defined sources of adverse effects in these patient populations reflect an unmet need for further translational research. Use of mouse models to evaluate lenalidomide efficacy and mechanisms underlying its activity and toxicity is highlighted in the recent literature (9,37–42). As the plasma concentration achieved in mice after lenalidomide dosing is unreported, it is unclear if the doses used in these previous studies produce systemic concentrations that are relevant to those observed in the clinic. In support of ongoing preclinical and translational evaluation of lenalidomide, we report a comprehensive pharmacokinetic characterization of lenalidomide in plasma and tissues of mice.
Stock lenalidomide powder was purified from commercial capsules in a method previously reported (34) and stored at room temperature with minimum light exposure. Dosing solutions were prepared by adding lenalidomide to the appropriate volume of sterile phosphate-buffered saline (PBS) containing 1% hydrochloric acid (HCl). Following complete drug dissolution, the pH of this preparation was adjusted to 7.0–7.6 using sodium hydroxide and sterile filtered using a 0.22 μm Steriflip filter (Millipore, Billerica, MA, USA) prior to dosing. Genistein (>98% HPLC grade) was obtained from Sigma (St. Louis, MO, USA). All other materials were purchased as analytical or cell culture grade from commercial sources.
Imprinting control region (ICR) mice 8–10 weeks of age were obtained from Harlan Laboratories (Indianapolis, IN, USA) and acclimated to the animal care facilities for a minimum of 48 h before study initiation. To minimize exogenous exposure, mice were housed in micro-insulator cages. Room temperature was regulated between 70°F and 72°F, and automatic 12-h light/dark cycles were maintained. Mice received food and water ad libitum, with the exception of orally administered mice which had food removed on the evening prior to dosing and withheld until 3 h post dosing. All animal care and experiments were approved and performed in compliance with the Institutional Animal Care and Use Committee guidelines.
Mice were administered sterile preparations of lenalidomide normalized to body weight. Intravenously (IV) dosed animals received drug by bolus tail vein injections, and extravascularly dosed mice received drug by bolus intraperitoneal injections (IP) or oral gavage (PO). Dosing solution, concentrations were adjusted so dose volumes ranged between approximately 100 and 150 μL for IV injections and between approximately 150 and 250 μL for IP and PO dosing in the pharmacokinetic study. However, for the range-finding study, increased dose volumes were used (up to 200 μL IV, 300 μL IP, and 600 μL PO, per approved animal use protocol) to explore elevated lenalidomide doses. The bolus injection rates for all IV, IP, or PO injections were less than 5 s. Concentrations of dosing solutions were verified by liquid chromatography–mass spectrometry.
Lenalidomide was incompletely soluble at 3.5 mg/mL and above in PBS containing 1% HCl, as visible particulates remained after thorough mixing. We therefore selected 3 mg/mL as the maximum dosing solution concentration (with no visible particulates). Single, individual mice were initially dosed with 3, 10, or 15 mg/kg IV; 4.5, 15, or 22.5 mg/kg IP; and 9, 30, or 45 mg/kg PO. Additional mice (n=4) were then evaluated at the maximum dose achievable by volume and solubility of lenalidomide in the dosing solution. All mice were monitored closely for 1 h and re-evaluated for toxicities 3, 6, and 24 h postdose.
Mice were euthanized by carbon dioxide asphyxiation followed by exsanguination at time points 2 min to 8 h postdose (n=4 or 5 mice per time point). Blood was collected by cardiac puncture into lithium heparinized plasma separator tubes (BD Microtainer, Becton, Dickson and Company, Franklin Lakes, NJ, USA) and immediately centrifuged at room temperature for 1.5 min at 4,200 RCF. Plasma was immediately removed and placed on dry ice prior to storage at −80°C until analysis. Liver, lung, heart, spleen, kidney, hind limb muscle, and brain were individually wrapped in foil and flash frozen in liquid nitrogen. Solid tissues were individually pulverized via mortar and pestle on dry ice, weighed in aliquots, and stored at −80°C until processing. Short-term stability of lenalidomide was not determined in the various sample types collected, although as IMiDs are reported relatively unstable in aqueous media (43–45), sample handling at room temperature was minimized to the 10 min collection period prior to freezing.
Sample processing and quantitative analysis using liquid chromatography–tandem mass spectrometry (LC-MS/MS) is described elsewhere (manuscript submitted). Briefly, frozen samples were removed from storage and warmed to room temperature prior to processing. Standard curves were prepared in blank mouse plasma, neat solution (100% acetonitrile) or homogenized tissues. After brief vortex mixing and centrifugation, plasma aliquots were taken for processing and quantification. Solid tissue homogenates were incubated in an equal volume of water for 20 min prior to extraction. Drug was extracted from plasma and tissues via protein precipitation with 4°C acetonitrile containing 200 nM genistein as internal standard. Supernatant was removed and evaporated under vacuum for 6 h. Samples were reconstituted in HPLC grade water containing 0.1% formic acid and transferred to autosampler vials. Extracted impurities were separated by an Extended C-18 column (Agilent, 50×0.5 mm, 3 μm particle size) using an ultra high performance Accela liquid chromatography system (Thermo, Waltham, MA, USA) and a flow gradient of water and acetonitrile both containing 0.1% formic acid. Analytes were ionized with atmospheric pressure chemical ionization, and fragment ions were detected on a TSQ Quantum Discovery Max triple quadrupole mass spectrometer system (Thermo).
Pharmacokinetic (PK) parameter estimates from each dose/route were generated from quantifiable plasma and tissue data using WinNonlin Professional version 5.2.1 (Pharsight Corporation, Mountain View, CA, USA) and NONMEM, v. 7.1.2 (Icon, Hanover, MD, USA). Concentration measurements below the lower limit of quantification (e.g., 0.3 nM in plasma) were excluded from analysis. Non-compartmental analysis was performed for each dose level in WinNonlin using the mean time and concentration data at each nominal time point. A linear up/log down approach was used for AUC calculation, and uniform weighting was used for terminal phase regression. Compartmental model fitting of data from each dose/route was completed in WinNonlin by evaluating one-, two- and three-compartment models with uniform, 1/Y, 1/Y2, 1/Yhat, and 1/Yhat2 weighting of naïve pooled data. Final model selection was based on visual fit, weighted residuals, individual parameter coefficients of variation, and Akaike’s information criterion. Simultaneous fitting of IV and PO plasma data for the 0.5 and 10 mg/kg doses was performed using two- and three-compartment models, respectively, in NONMEM and WinNonlin. The NONMEM data file was constructed with unique individual identification numbers assigned to each plasma concentration–time observation. Base simultaneous models with a dosing compartment, a central compartment, and either one or two peripheral compartments were coded with differential equations using the ADVAN 13 subroutine in NONMEM. For these single-response population data, between-subject variability was fixed to zero, and residual unexplained variability (RUV) parameters were estimated for both IV and PO data using proportional error models.
To assess pharmacokinetic dose proportionality, IV and PO data was combined, and the effect of dose was evaluated as a continuous covariate in NONMEM. This mixed effects approach was chosen instead of the standard comparison of PK parameter and standard error estimates between dose groups because it allowed for evaluation of objective function value as well as reduction in residual error to determine if dose had a significant impact on model fits and PK parameter estimates. The dose covariate was evaluated on each model parameter using the relationship in equation 1, where the dose impact on CL is shown as an example,
where CLi is lenalidomide clearance for individual mouse i, TVCL is the typical or population value of clearance, Dosei is the dose for individual i, and θ1 is the estimated exponential representing the influence of dose on the PK parameter, CL. Base and covariate models were evaluated by the log-likelihood ratio test, visual inspection of fit and residual plots, standard errors of estimate, and reduction in RUV. A minimum reduction in the objective function value (OFV) of 3.84 was used as a cutoff to indicate the dose covariate had a significant impact on PK with α=0.05.
Doses administered to mice were limited by solubility of lenalidomide in the PBS dosing solution. Maximum achievable doses of up to 15, 22.5, and 45 mg/kg (n=4 each) were given through IV, IP, and PO routes, respectively. IV administration was fatal in one mouse immediately following the 15 mg/kg bolus injection. Cause of death was not determined, and no other fatalities or observable toxicities were noted at maximum doses. Due to solubility limitations which prevented further dose escalation, a maximum tolerated dose was not established.
As the range finding study resulted in mortality in one mouse at the 15 mg/kg maximum dose in IV dose toxicity studies, upper-limit doses of 10 mg/kg were utilized in pharmacokinetic studies regardless of administration route. Dose proportionality was assessed following IV bolus doses of 0.5, 1.5, 5 and 10 mg/kg body weight. Observed plasma concentration versus time plots are shown in Fig. 1, and noncompartmental observed Cmax and AUCs are listed in Table I. Cmax at 2 min postdose ranged from 0.28 to 17.75 μg/mL across the four evaluated doses, and noncompartmental AUCs ranged from 7.8 to 226 μg×min×mL−1 over the chosen dose range.
For data modeling, all final compartmental best fit IV models employed a 1/Yhat weighting scheme (Fig. 1). Following IV doses of 1.5 mg/kg and greater, lenalidomide concentrations decreased triexponentially, while the 0.5 mg/kg concentration–time profile produced a biphasic decline. Table I lists compartmental estimated PK parameters. Plasma concentrations and AUC appeared to saturate at the higher doses. The data indicate an approximate 17-fold increase in AUC while the dose increased threefold from 0.5 to 1.5 mg/kg. Following doses of 1.5 mg/kg and greater, the increase in plasma AUC is less than proportional, with an average fold increase of 0.51 per unit fold increase in dose. It should be noted that, while the three-compartment models provided the overall best fits to the IV data, uncertainty in parameter estimation is significant (Table I). Three-compartment α-phase kinetics (and γ-phase kinetics for the 1.5 mg/kg dose group) was estimated with sparse data. Given the high uncertainty in α-phase kinetics across the dose groups, IV Cmax estimates should be interpreted with caution.
Bioavailability was assessed for IP and PO administration routes following doses of 0.5 and 10 mg/kg. Concentration–time plots and resulting compartmental fit models are shown in Fig. 2. Maximum observed concentrations following 0.5 and 10 mg/kg were 0.246 and 8.343 μg/mL for IP administration and 0.092 and 2.447 μg/mL for PO administration (Table I). Observed time of maximum concentration (Tmax) was 10 min for both IP doses and 20 and 40 min for 0.5 and 10 mg/kg oral doses, respectively. IP dosing achieved a Cmax 2.7- to 4.2-fold greater than oral administration, whereas orally dosed mice sustained drug concentrations above 1.00 μg/mL 1.5 h post a 10 mg/kg dose compared to 0.39 μg/mL for mice receiving IP doses.
IP and oral compartmental data were weighted as 1/Y. Apparent absorption rate constants (Ka) were greater for IP than PO routes, with a fold difference of 7.3 and 2.9 for 0.5 and 10 mg/kg, respectively (Table I). Concentration–time data indicated monophasic and biphasic decline following PO and IP administration, respectively. Compartmental estimates for Tmax were 10 and 27 min, respectively, regardless of dose. Systemic bioavailability determined from AUC estimates were 69% and 75% for PO doses versus 90% and 105% for IP doses of 0.5 and 10 mg/kg, respectively.
To identify the oral Ka, IV and PO data were simultaneously fit to a multicompartment model using NONMEM. A simultaneous, two-compartment base model was applied to data at the 0.5 mg/kg, while 10 mg/kg simultaneous data were modeled as three-compartment. Final model parameters and standard errors of estimate (expressed as CV%) are presented in Table II. Simultaneous fitting of IV and PO data indicated an absorption rate similar to the elimination rate estimate (Ke) at the 0.5 mg/kg dose. In contrast, Ka was much lower than Ke at the 10 mg/kg dose level in the evaluated models. Oral bioavailability estimates from these models were 65% and 87%, respectively. Notably, these population estimates are consistent with compartmental estimates determined with the naïve pooled approach in WinNonlin. Parameter estimates from these simultaneous models in NONMEM were confirmed using WinNonlin simultaneous user defined models (data not shown). Collectively, results from these analyses suggest lenalidomide exhibits absorption rate limited pharmacokinetic behavior when administered orally in mice. Therefore, the naïve pooled WinNonlin estimates for oral and intraperitoneal absorption and elimination rate constants are reported with Ke>Ka in Table I.
Dose linearity was assessed in NONMEM by combining IV and PO data to a two- and a three-compartment model. When dose was added as a covariate to each PK model parameter, OFV was decreased most significantly for dose as a covariate on Q2 (−7.51) in the two-compartment model and on Q3 (−37.245) in the three-compartment model. RUV also decreased 2% for the IV data in the two-compartment model and 1% and 7%, respectively, for the IV and PO data in the three-compartment model. Combined with the observed nonlinearity in drug exposure (Fig. 3a), results from these analyses provide further support that lenalidomide pharmacokinetic behavior is dose-dependent (or nonlinear) in ICR mice across the dose range evaluated.
Lenalidomide concentrations were quantified in multiple tissues collected from mice dosed with 0.5, 1.5, 5, and 10 mg/kg IV lenalidomide. The resulting concentration–time profiles from heart, lung, liver, muscle, spleen, brain, and kidney tissues are displayed in Fig. 4. Initial concentrations expressed by 2 min time points in order from most prominent highest to lowest concentration were lung, liver, heart, and spleen. Following a dose of 10 mg/kg, the observed 2-min concentrations were 15, 5, 2.4, and 1.3 μg/g, respectively. At 45 min post the 10 mg/kg dose, the highest concentrations (268–612 ng/g) were observed in lung, liver, and spleen. Less than 15 ng/g remained in each tissue after 8 h.
The proportional distribution of lenalidomide was varied across each tissue type. Additionally, the proportion of lenalidomide distributed to a single tissue was variable over the dose range evaluated. These apparent tissue nonlinearities are observed when tissue noncompartmental AUC’s are plotted against a linear dosing scale and when lenalidomide distribution to tissues is expressed as the ratio of tissue exposure/plasma exposure (Fig. 3). The proportion of lenalidomide distributed to tissues was frequently lowest within the 1.5 mg/kg dose. Tissues with the greatest lenalidomide exposures were lung, spleen and liver. Lenalidomide had the highest affinity or retention in spleen, as displayed in Fig. 3d when AUC is normalized to organ perfusion rate (46,47). This is consistent with our observation of an absorption phase in spleen (Fig. 4). Lung, liver, and muscle also displayed high lenalidomide concentrations after normalization to perfusion rate. Of note, an absorption phase was also observed in muscle; however, only at the 10 mg/kg dose level (Fig. 4). Lenalidomide could not be detected in brain tissues following 0.5 and 1.5 mg/kg doses but was quantifiable at low concentrations following 5 and 10 mg/kg injections.
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×ng/mL (11) or 2,415 h×ng/mL (35)) or 15–30 mg/m2 in children (examples, mean AUCs ranging from 2,740 to 3,850 h×ng/mL; 36,48). 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 (21,24), 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. 3 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 (9,50) and 100 mg/kg (51,52), 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. 3 and and4).4). 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. 3). 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=0.3; 53), which has considerable brain penetration (54,55). Although lenalidomide structure and lipophilicity (logP=−0.5; 53) 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 (57,58). 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.
This work was supported by an Eli Lilly Graduate Fellowship (DMR), a Leukemia and Lymphoma Society SCOR grant (JCB, AJJ, and MP), the D. Warren Brown Foundation (JCB), and NIH grants 1 P50 CA140158 (JCB and AJJ), K12CA133250 (AJJ), and 5KL2RR025754.
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