Cancer Chemotherapy and Pharmacology (2019) 84:447–452 https://doi.org/10.1007/s00280-019-03864-9 SHORT COMMUNICATION CNS penetration of the CDK4/6 inhibitor ribociclib in non‑tumor bearing mice and mice bearing pediatric brain tumors Yogesh T. Patel1,2 · Abigail Davis1 · Suzanne J. Baker3 · Olivia Campagne1 · Clinton F. Stewart1 Received: 22 January 2019 / Accepted: 3 May 2019 / Published online: 11 May 2019 © Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract Purpose Ribociclib, an orally bioavailable small-molecule CDK4/6 inhibitor is currently undergoing evaluation to treat pedi- atric central nervous system (CNS) tumors. However, it is crucial that it penetrates the brain and tumor. Thus, the objectives of the present study were to derive a clinically relevant mouse dosage for cerebral microdialysis studies, and to characterize ribociclib CNS penetration in non-tumor bearing mice and in mice bearing DIPGx7 (glioma) cortical allograft tumors. Methods A plasma pharmacokinetic study of ribociclib (100 mg/kg, orally) was performed in CD1 nude mice bearing glioma cortical allografts to obtain initial plasma pharmacokinetic parameters and to derive D-optimal plasma sampling time-points for microdialysis studies. Using a cerebral microdialysis technique, the extracellular fuid (ECF) disposition of ribociclib was evaluated after a single oral ribociclib dose (100 mg/kg) in non-tumor bearing mice and in mice bearing glioma cortical allografts. A one-compartment plasma model with absorption and ECF compartments were ft to plasma and ECF concentration–time data using a nonlinear mixed efects modeling approach (NONMEM 7.2). Results The mean unbound ribociclib plasma exposure (6812 ng/ml*h) was similar to that observed clinically at recom- mended dosages in adults. The median ribociclib ECF to plasma partition coefcient (Kp,uu) in non-tumor bearing and glioma mice was 0.10 and 0.07, respectively, and was not statistically diferent (t test, p = 0.19). Conclusions The CNS penetration observed was encouraging enough to move ribociclib forward with preclinical efcacy studies in models of pediatric brain tumors. Keywords Microdialysis · CDK4/6 · Pharmacokinetics · Ribociclib · Pediatrics Introduction some instances, chemotherapy is an important component of therapy. For children less than 3 years old, chemotherapy is Malignant central nervous system (CNS) tumors carry a preferentially given to avoid or delay irradiation of the devel- poor prognosis and are the most common solid tumors in oping brain [2]. Because drugs shown active to treat brain children [1]. Surgical resection and radiotherapy have been tumors in adults, such as temozolomide, have not shown the cornerstones of treatment for most brain tumors, but in efcacy in pediatric trials [3], new agents are needed to treat childhood brain tumors. Ribociclib is an orally bioavailable small-molecule inhib- Electronic supplementary material The online version of this itor of both cyclin-dependent kinases 4 and 6 (CDK4/6) article (https ://doi.org/10.1007/s0028 0-019-03864 -9) contains currently approved for the treatment of breast cancer [4]. supplementary material, which is available to authorized users. CDK4/6 regulate the mammalian cell-cycle progression * Clinton F. Stewart through G1 phase and initiation of S phase [5]. They respond [email protected] to mitogenic or pro-proliferative stimuli, complex with D-type cyclin, and phosphorylate the retinoblastoma (RB) 1 Department of Pharmaceutical Sciences, St. Jude Children’s tumor suppressor protein. This phosphorylation releases RB Research Hospital, Memphis, TN, USA from E2F transcription factors and enables E2F to transcribe 2 Present Address: Cognigen Corporation, Bufalo, NY, USA the genes that are required for G 1-S phase cell-cycle progres- 3 Department of Developmental Neurobiology, St. Jude sion and ultimately cell proliferation [5]. Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105-2794, USA Vol.:(0123456789)1 3 448 Cancer Chemotherapy and Pharmacology (2019) 84:447–452 Recent studies of difuse intrinsic pontine glioma (DIPG) Ribociclib protein binding in mouse plasma and pediatric non-brainstem high-grade glioma tissues have unraveled genetic alterations associated with tumor prolif- A plasma protein binding study of ribociclib was performed eration [6]. These studies reported that ~ 25–30% of these using a rapid equilibrium dialysis procedure. Ribociclib was tumors contain alterations in cell-cycle regulatory genes added to mouse plasma (BioIVT, Westbury, NY) to make such as amplifcation of D-type cyclins and CDK4/6, which fnal concentrations of 500, 1000, 2500, and 5000 ng/mL. are associated with abnormal proliferation [7]. Beside gli- 1 mL of spiked plasma was added to Centrifree® ultrafltra- oma, D-type cyclins and CDK4/6 genes were also found to tion devices [molecular weight cut of: 10 K Daltons; Milli- be amplifed in other pediatric brain tumors highlighting the poreSigma, Darmstadt, Germany]. The plasma samples were clinical potential of CDK4/6 inhibitors such as ribociclib in centrifuged for 30 min at 3600 rpm at 37 °C. The fltrate was this therapeutic area [8]. saved and stored at − 80 °C until analysis. Fraction unbound If ribociclib has adequate CNS exposure (i.e., concentra- of ribociclib in plasma was calculated as ratio of unbound tion vs. time), it could be an important molecularly targeted ribociclib concentration in the fltrate to total ribociclib con- agent to treat childhood brain tumors. Thus, the objectives centration in the plasma sample. of the present study were to derive a clinically equivalent mouse dosage for the cerebral microdialysis studies, to Plasma pharmacokinetic study establish a limited sampling model (LSM) for plasma sam- ple collection during the cerebral microdialysis studies, and CD1 nude mice (n = 9) bearing SJ-DIPGx7 cortical allograft to characterize ribociclib CNS penetration in non-tumor tumors were dosed with 100 mg/kg ribociclib (oral; 10 mg/ bearing mice (i.e., normal blood–brain barrier) and in mice mL) formulated in 0.5% methyl cellulose. Multiple samples bearing DIPGx7 (glioma) cortical allograft tumors. were collected per mouse for up to 24 h post-dose using a population-based study design. Blood samples ( ~ 75 µL) Materials and methods were collected by retro-orbital eye bleed using heparin coated capillary tubes (Fisher Sci, Pittsburgh, PA), except Drugs and chemicals for the terminal sample, which was collected by cardiac stick with a 1 mL heparin pre-coated syringe. Immediately after collection, blood samples were centrifuged, plasma was sep- Ribociclib succinate (purity, > 99%) was provided by arated, and stored at − 80 °C until analysis with an LC–MS/ Novartis Oncology (East Hanover, NJ). Methyl cellu- MS method [9]. lose was purchased from Sigma-Aldrich (St. Louis, MO). Ribociclib was formulated at concentration of 10 mg/mL in 0.5% w/v methyl cellulose. Acetonitrile, ethyl acetate, Development of pharmacokinetic limited sampling and formic acid were purchased from Fisher Scientifc (Fair modeling for ribociclib Lawn, NJ, USA). All solvents used were HPLC grade. Water was purifed using Milli-Q Advantage A10 system (Milli- During microdialysis, only three plasma samples per mouse pore, Billerica, MA, USA). Mouse plasma (CD1 strain) in could be obtained due to blood sampling volume restric- sodium heparin was purchased from BioIVT (Westbury, NY, tions and plasma volume required for bioanalysis. Thus, USA). Ringer’s solution was purchased from Frey Scientifc a pharmacokinetic LSM was selected to provide the most (Nashua, NH, USA). statistically informative time-points for the plasma sample collection during microdialysis. The LSM was developed Animals and cell lines based on the pharmacokinetic parameters obtained from the plasma disposition study, using the D-optimality method Female CD1 nude mice (Charles River, Wilmington, MA, implemented in ADAPT 5 (BSMR, Los Angeles, CA, USA). USA) were kept under a controlled environment where temperature, humidity, and 12-h day and night cycles were In vitro probe recovery studies maintained artifcially. One cohort of mice were cortically 6 implanted with 1 × 10 cells of SJ-DIPGx7 (Line 7 passage To assess the dialyzability of ribociclib, the microdialysis 26 with luciferase-YFP marker). Line 7 was established from probe recovery was determined using in vitro recovery stud- a patient autopsy DIPG sample, and carries the H3.3K27M ies as previously described [10]. Specifcally, each probe mutation. All procedures were approved by the St. Jude used for the in vivo microdialysis study was placed in a Institutional Animal Care and Use Committee and met the beaker containing a ribociclib solution prepared in Ringer’s guidelines of the Association for Assessment and Accredita- solution at a concentration of 1 µg/mL and maintained at tion of Laboratory Animal Care (AAALAC). 1 3 Cancer Chemotherapy and Pharmacology (2019) 84:447–452 449 37 °C. The probe was perfused with blank Ringer’s solu- was estimated as a ratio of brain/tumor ECF AUC0–24 h and tion at a fow-rate of 0.5 µL/min. After equilibration for 1 h, plasma AUC0–24 h,u . three consecutive fractions each of 1 h interval were col- lected. Dialysate samples were stored at − 80 °C until fur- ther analysis using a mass spectrometry method developed Results in our laboratory validated for use with Ringer’s solution. Probe recovery was calculated using the ratio of ribociclib Ribociclib plasma protein binding concentration in the dialysate