Multiple Myeloma (MM), a type of monoclonal B-cell malignancy, is considered the second most common blood malignancy which accounts for approximately 13% of blood cancers in the US[1, 2]. The prognosis and patient overall survival have been greatly improved in the recent decade, owing to the development of a series of novel drugs and combination therapies. The immunomodulatory drugs (IMiDs), which include thalidomide, lenalidomide and pomalidomide, are widely used in different stages of MM progression and patients with different risk categories[3]. The second and third generation IMiDs, lenalidomide and pomalidomide, provide with improved safety and efficacy and are the standard of care drugs in patients with newly diagnosed MM as well as relapsed / refractory MM. However, MM progression is inevitable, and patients will develop resistance to IMiDs, with poor prognosis[3, 4].
The resistance mechanisms to IMiDs are still largely unknown, and those described so far mainly involve the direct binding target cereblon (CRBN) and its downstream signaling pathway, with involvement of two transcription factors Ikaros family zinc finger protein (IKZF1) and IKZF3[5-7]. The clinical response of IMiDs however is not crossing, which means patients may still response to a different IMiD after being treated with one[8, 9]. Despite similar structures, IMiDs also possess very different pharmacokinetic (PK) properties. For instance, lenalidomide is cleared primarily through renal excretion as parent drug with limited metabolism, while pomalidomide clearance occurs primarily though metabolism [10, 11]. Thalidomide is not a P-glycoprotein (P-gp) substrate[12], while both lenalidomide and pomalidomide are P-gp substrates[13-15]. Lenalidomide has lowest blood-brain barrier (BBB) penetration, while thalidomide has the highest BBB penetration[16, 17]. Collectively, these differences point to one or more yet unidentified transporters that are likely responsible for the observed differences in biodistribution among the IMiDs.
In this work, we build on prior clinical, in vitro, and in vivo data and conducted a series of in vitro assays to characterize the permeability difference among various MM cell lines, as well as several commonly used cell lines for transporter function determinations, such as HEK-293, HeLa, MDCKII, and Caco2. A general observed trend was highest uptake of lenalidomide compared to pomalidomide and thalidomide in most cell lines tested, and similar kinetics that pomalidomide intracellular level reached equilibrium much faster than lenalidomide and thalidomide. This was not the case in MDCKII and Caco2 cells, in which pomalidomide showed higher uptake at an earlier incubation time point, compared to lenalidomide and thalidomide. Particularly, as we observed different brain penetration in mice, which was unrelated to P-gp, we utilized a BBB cell model, the hCMEC/D3 cell[18], for further permeability characterization and transporter identification. An interesting phenomenon that high concentration of unlabeled IMiDs, particularly pomalidomide, could dramatically increase the apparent uptake of hot pomalidomide, and to some extent, lenalidomide and thalidomide was observed. This observation suggested this was due to a transporter mediated effect, though eventually we determined this to be caused by co-precipitation of radiolabeled IMiDs with unlabeled pomalidomide, which has relatively low solubility. Lastly, we performed pharmacokinetic analysis of data from a phase 1b clinical trial combining pomalidomide and AR-42, a novel histone deacetylase inhibitor previously found to restore IMiD sensitivity to MM cells[19]. We constructed population pharmacokinetic models for both drugs and examined the impact of missing covariates as well as including prior information on the model outcome. Both models fitted the limited dataset well and parameters estimated were close to published values.
In conclusion, the data presented herein remain supportive of transporter-mediated causes for the observed differences between the three IMiD drugs, though the identification of those transporters is still undergoing.