Shelley Orwick

Interaction of imatinib with human organic ion carriers

Purpose: The activity of imatinib in leukemia has recently been linked with expression of the organic cation transporter 1 (OCT1) gene SLC22A1. Here, we characterized the contribution of solute carriers to imatinib transport in an effort to further understand mechanisms involved in the intracellular uptake and retention (IUR) of the drug. Experimental Design: IUR of [3H]imatinib was studied in Xenopus laevis oocytes and HEK293 cells expressing OATP1A2, OATP1B1, OATP1B3, OCT1-3, OCTN1-2, or OAT1-3. Gene expression was determined in nine leukemia cell lines using the Affymetrix U133 array. Results: Imatinib was not found to be a substrate for OCT1 in oocytes (P = 0.21), whereas in HEK293 cells IUR was increased by only 1.20-fold relative to control cells (P = 0.002). Furthermore, in 74 cancer patients, the oral clearance of imatinib was not significantly altered in individuals carrying reduced-function variants in SLC22A1 (P = 0.99). Microarray analysis indicated that SLC22A1 was interrelated with gene expression of various transporters, including ABCB1, ABCC4, ABCG2 (negative), and OATP1A2 (positive). Imatinib was confirmed to be a substrate for the three efflux transporters (P < 0.05) as well as for OATP1A2 (P = 0.0001). Conclusions: This study suggests that SLC22A1 expression is a composite surrogate for expression of various transporters relevant to imatinib IUR. This observation provides a mechanistic explanation for previous studies that have linked SLC22A1 with the antitumor activity of imatinib. Because of its high expression in the intestine, ciliary body, gliomas, and leukemia cells, OATP1A2 may play a key role in imatinib pharmacokinetics-pharmacodynamics.

Pharmacogenetic pathway analysis of docetaxel elimination

The purpose of this study was to evaluate the affinity of docetaxel for 14 transporter proteins and assess the functional significance of 17 variants in five genes involved in drug elimination. Among the transfected models investigated, OATP1B3 (SLCO1B3) was identified as the most efficient influx transporter for docetaxel. None of the observed genotypes (SLCO1B3, ABCB1, and ABCC2) was related with docetaxel clearance in 92 white patients (P > 0.17). However, the simultaneous presence of the CYP3A4*1B and CYP3A5*1A alleles was associated with a 64% increase in docetaxel clearance (P = 0.0015), independent of both sex and CYP3A activity (as determined using the erythromycin breath test). This haplotype was also associated with increased midazolam clearance in another population (P = 0.0198). An analysis of the CYP3A locus among CEPH‐HapMap samples revealed that CYP3A4*1B is present exclusively among a subset of CYP3A5 expressors. Therefore, future studies should first stratify the population on the basis of CYP3A5 genotype and then compare CYP3A activity between individuals with and without the CYP3A4*1B allele.

Interaction of the Multikinase Inhibitors Sorafenib and Sunitinib with Solute Carriers and ATP-Binding Cassette Transporters

Purpose: To compare side-by-side the uptake of sorafenib and sunitinib in vitro by human uptake solute carriers of the SLC22A and SLCO families, the transport by and inhibition of efflux ATP-binding cassette (ABC) transporters, and the role of ABCB1 in the plasma pharmacokinetics and brain penetration of these agents. Experimental Design: Uptake of [3H]sorafenib or [3H]sunitinib was assessed in Xenopus laevis oocytes or mammalian cells transfected with cDNAs coding for human OATP1A2, OATP1B1, OATP1B3, OCT1, OAT2, OAT3, OCTN1, or OCTN2. Efflux and inhibition experiments were conducted in cells transfected with human ABCB1, ABCG2, ABCC2, or ABCC4. In vivo pharmacokinetic studies were done in knockout mice lacking Abcb1-type transporters. Results: Intracellular uptake was not appreciably affected by any of the studied solute carriers and was minute relative to the respective prototypical substrates. Sorafenib and sunitinib showed concentration-dependent (1 and 10 μmol/L), low to moderate affinity for ABCB1 but were not affected by the other ABC transporters. Both agents inhibited all tested ABC transporters. The absence of Abcb1 had no affect on plasma pharmacokinetics, but brain penetration was moderately increased by 1.9- and 2.9-fold for sorafenib and sunitinib, respectively, in knockout animals versus controls. Conclusions: Unlike other tyrosine kinase inhibitors, sorafenib and sunitinib do not appear to rely on active transport to enter the cell nor are they high-affinity substrates for ABC efflux transporters. Based on these characteristics, these two drugs may be less susceptible to transporter-mediated alterations in systemic exposure and transporter-related resistance mechanisms. (Clin Cancer Res 2009;15(19):6062–9)

Crenolanib is active against models of drug-resistant FLT3-ITD-positive acute myeloid leukemia

FLT3 kinase internal tandem duplication (ITD) mutations are common in acute myeloid leukemia (AML) and are associated with poor clinical outcomes. Although initial responses to FLT3 tyrosine kinase inhibitors (TKIs) are observed in FLT3-ITD-positive patients, subsequent relapse often occurs upon acquisition of secondary FLT3 kinase domain (KD) mutations, primarily at residues D835 and F691. Using biochemical assays, we determined that crenolanib, a novel TKI, demonstrates type I properties and is active against FLT3 containing ITD and/or D835- or F691-activating mutations. Potent activity was observed in FLT3-ITD-positive AML cell lines. Crenolanib delayed the outgrowth of MV4-11 cells in a xenograft mouse model, whereas in combination with the type II TKI sorafenib, a significant decrease in leukemic burden (P < .001) and prolonged survival (P < .01) was observed compared with either type I or II TKI alone. Crenolanib was active against Ba/F3 cells harboring FLT3-ITD and secondary KD mutations and sorafenib-resistant MOLM-13 cells containing FLT3-ITD/D835Y both in vitro and in vivo. In addition, crenolanib inhibited drug-resistant AML primary blasts with FLT3-ITD and D835H/Y mutations. These preclinical data demonstrate that crenolanib is effective against FLT3-ITD containing secondary KD mutations, suggesting that crenolanib may be a useful therapeutic agent for TKI-naive and drug-resistant FLT3-ITD-positive AML.

Contribution of OATP1B1 and OATP1B3 to the Disposition of Sorafenib and Sorafenib-Glucuronide

Purpose: Many tyrosine kinase inhibitors (TKI) undergo extensive hepatic metabolism, but mechanisms of their hepatocellular uptake remain poorly understood. We hypothesized that liver uptake of TKIs is mediated by the solute carriers OATP1B1 and OATP1B3. Experimental Design: Transport of crizotinib, dasatinib, gefitinib, imatinib, nilotinib, pazopanib, sorafenib, sunitinib, vandetanib, and vemurafenib was studied in vitro using artificial membranes (PAMPA) and HEK293 cell lines stably transfected with OATP1B1, OATP1B3, or the ortholog mouse transporter, Oatp1b2. Pharmacokinetic studies were conducted with Oatp1b2-knockout mice and humanized OATP1B1- or OATP1B3-transgenic mice. Results: All 10 TKIs were identified as substrates of OATP1B1, OATP1B3, or both. Transport of sorafenib was investigated further, as its diffusion was particularly low in the PAMPA assay (<4%) than other TKIs that were transported by both OATP1B1 and OATP1B3. Whereas Oatp1b2 deficiency in vivo had minimal influence on parent and active metabolite N-oxide drug exposure, plasma levels of the glucuronic acid metabolite of sorafenib (sorafenib-glucuronide) were increased more than 8-fold in Oatp1b2-knockout mice. This finding was unrelated to possible changes in intrinsic metabolic capacity for sorafenib-glucuronide formation in hepatic or intestinal microsomes ex vivo. Ensuing experiments revealed that sorafenib-glucuronide was itself a transported substrate of Oatp1b2 (17.5-fold vs. control), OATP1B1 (10.6-fold), and OATP1B3 (6.4-fold), and introduction of the human transporters in Oatp1b2-knockout mice provided partial restoration of function. Conclusions: These findings signify a unique role for OATP1B1 and OATP1B3 in the elimination of sorafenib-glucuronide and suggest a role for these transporters in the in vivo handling of glucuronic acid conjugates of drugs. Clin Cancer Res; 19(6); 1458–66. ©2013 AACR.

Activity of the Multikinase Inhibitor Sorafenib in Combination With Cytarabine in Acute Myeloid Leukemia

BACKGROUND Acute myeloid leukemia (AML) is a genetically heterogeneous cancer that frequently exhibits aberrant kinase signaling. We investigated a treatment strategy combining sorafenib, a multikinase inhibitor with limited single-agent activity in AML, and cytarabine, a key component of AML chemotherapy. METHODS Using 10 human AML cell lines, we determined the effects of sorafenib (10 μM) on antileukemic activity by measuring cell viability, proliferation, ERK1/2 signaling, and apoptosis. We also investigated the effects of sorafenib treatment on the accumulation of cytarabine and phosphorylated metabolites in vitro. A human equivalent dose of sorafenib in nontumor-bearing NOD-SCID-IL2Rγ(null) mice was determined by pharmacokinetic studies using high performance liquid chromatography with tandem mass spectrometric detection, and steady-state concentrations were estimated by the fit of a one-compartment pharmacokinetic model to concentration-time data. The antitumor activity of sorafenib alone (60 mg/kg) twice daily, cytarabine alone (6.25 mg/kg administered intraperitoneally), or sorafenib once or twice daily plus cytarabine was evaluated in NOD-SCID-IL2Rγ(null) mice bearing AML xenografts. RESULTS Sorafenib at 10 μM inhibited cell viability, proliferation and ERK1/2 signaling, and induced apoptosis in all cell lines studied. Sorafenib also increased the cellular accumulation of cytarabine and metabolites resulting in additive to synergistic antileukemic activity. A dose of 60 mg/kg in mice produced a human equivalent sorafenib steady-state plasma exposure of 10 μM. The more dose-intensive twice-daily sorafenib plus cytarabine (n = 15) statistically significantly prolonged median survival in an AML xenograft model compared with sorafenib once daily plus cytarabine (n = 12), cytarabine alone (n = 26), or controls (n = 27) (sorafenib twice daily plus cytarabine, median survival = 46 days; sorafenib once daily plus cytarabine, median survival = 40 days; cytarabine alone, median survival = 36 days; control, median survival = 19 days; P < .001 for combination twice daily vs all other treatments listed). CONCLUSIONS Sorafenib in combination with cytarabine resulted in strong anti-AML activity in vitro and in vivo. These results warrant clinical evaluation of sorafenib with cytarabine-based regimens in molecularly heterogeneous AML.

Emergence of Polyclonal FLT3 Tyrosine Kinase Domain Mutations during Sequential Therapy with Sorafenib and Sunitinib in FLT3-ITD–Positive Acute Myeloid Leukemia

Purpose: To evaluate the clinical activity of sequential therapy with sorafenib and sunitinib in FMS-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD)-positive acute myelogenous leukemia (AML) and monitor the emergence of secondary FLT3 tyrosine kinase domain (TKD) mutations during treatment. Experimental Design: Six children with relapsed/refractory AML were treated with sorafenib in combination with clofarabine and cytarabine, followed by single-agent sorafenib if not a candidate for transplantation. Sunitinib was initiated after sorafenib relapse. Bone marrow samples were obtained for assessment of FLT3 TKD mutations by deep amplicon sequencing. The phase of secondary mutations with ITD alleles was assessed by cloning and sequencing of FLT3 exons 14 through 20. Identified mutations were modeled in Ba/F3 cells, and the effect of kinase inhibitors on FLT3 signaling and cell viability was assessed. Results: Four patients achieved complete remission, but 3 receiving maintenance therapy with sorafenib relapsed after 14 to 37 weeks. Sunitinib reduced circulating blasts in two patients and marrow blasts in one. Two patients did not respond to sorafenib combination therapy or sunitinib. FLT3 mutations at residues D835 and F691 were observed in sorafenib resistance samples on both ITD-positive and -negative alleles. Deep sequencing revealed low-level mutations and their evolution during sorafenib treatment. Sunitinib suppressed leukemic clones with D835H and F691L mutations, but not D835Y. Cells expressing sorafenib-resistant FLT3 mutations were sensitive to sunitinib in vitro. Conclusions: Sunitinib has activity in patients that are resistant to sorafenib and harbor secondary FLT3 TKD mutations. The use of sensitive methods to monitor FLT3 mutations during therapy may allow individualized treatment with the currently available kinase inhibitors. Clin Cancer Res; 19(20); 5758–68. ©2013 AACR.

Comparison of antitumor effects of multitargeted tyrosine kinase inhibitors in acute myelogenous leukemia

We compared the antitumor activities of the multitargeted tyrosine kinase inhibitors imatinib, sorafenib, and sunitinib to determine which inhibitor is best suited to be used for the treatment of acute myelogenous leukemia (AML). In nine human AML cell lines, sorafenib and sunitinib were more potent inhibitors of cellular proliferation than imatinib (IC50, 0.27 to >40, 0.002-9.1, and 0.007-13 μmol/L for imatinib, sorafenib, and sunitinib, respectively). Sorafenib and sunitinib were potent inhibitors of cells with fms-like tyrosine kinase 3 internal tandem duplication (IC50, 2 and 7 nmol/L) and c-KIT N822K mutations (IC50, 23 and 40 nmol/L). In four cell lines (MV4-11, Kasumi-1, KG-1, and U937) that spanned a range of drug sensitivities, sorafenib and sunitinib had similar activity in apoptosis and cell cycle assays, except that sunitinib did not promote apoptosis in U937 cells. Both drugs inhibited mitogen-activated protein kinase signaling but had no effect on AKT signaling in most of the cell lines tested. Sorafenib was substantially more bound than sunitinib in human plasma (unbound fraction, 0.59% versus 8.4%) and cell culture medium (unbound fraction, 1.3% versus 39%), indicating that sorafenib was more potent than sunitinib and that unbound sorafenib concentrations with activity against most AML cell lines are achievable in vivo. There was more intracellular accumulation of sorafenib than of sunitinib and imatinib in AML cells. Between 1 and 10 μmol/L, sorafenib inhibited the proliferation of six of nine primary AML blast samples by ≥50%. Our results highlight the pharmacologic features of sorafenib that may provide it an advantage in the treatment of AML. [Mol Cancer Ther 2008;7(5):1110–20]

Effect of ABCC2 (MRP2) Transport Function on Erythromycin Metabolism

The macrolide antiobiotic erythromycin undergoes extensive hepatic metabolism and is commonly used as a probe for cytochrome P450 (CYP) 3A4 activity. By means of a transporter screen, erythromycin was identified as a substrate for the transporter ABCC2 (MRP2) and its murine ortholog, Abcc2. Because these proteins are highly expressed on the biliary surface of hepatocytes, we hypothesized that impaired Abcc2 function may influence the rate of hepatobiliary excretion and thereby enhance erythromycin metabolism. Using Abcc2 knockout mice, we found that Abcc2 deficiency was associated with a significant increase in erythromycin metabolism, whereas murine Cyp3a protein expression and microsomal Cyp3a activity were not affected. Next, in a cohort of 108 human subjects, we observed that homozygosity for a common reduced‐function variant in ABCC2 (rs717620) was also linked to an increase in erythromycin metabolism but was not correlated with the clearance of midazolam. These results suggest that impaired ABCC2 function can alter erythromycin metabolism, independent of changes in CYP3A4 activity.

Cisplatin-Induced Downregulation of OCTN2 Affects Carnitine Wasting

Purpose: Carnitine is an essential cofactor for mitochondrial fatty acid oxidation that is actively reabsorbed by the luminal transporter Octn2 (Slc22a5). Because the nephrotoxic agent cisplatin causes urinary loss of carnitine in humans, we hypothesized that cisplatin may affect Octn2 function. Experimental Design: Excretion of carnitine and acetylcarnitine was measured in urine collected from mice with or without cisplatin administration. The transport of carnitine was assessed in cells that were transfected with OCT1 or OCT2. The effect of cisplatin treatment on gene expression was analyzed using a mouse GeneChip array and validated using quantitative reverse transcriptase-PCR. Results: In wild-type mice, urinary carnitine excretion at baseline was ∼3-fold higher than in mice lacking the basolateral cisplatin transporters Oct1 and Oct2 [Oct1/2(−/−) mice], indicating that carnitine itself undergoes basolateral uptake into the kidney. Transport of carnitine by OCT2, but not OCT1, was confirmed in transfected cells. We also found that cisplatin caused an increase in the urinary excretion of carnitine and acetylcarnitine in wild-type mice but not in Oct1/2(−/−) mice, suggesting that tubular transport of cisplatin is a prerequisite for this phenomenon. Cisplatin did not directly inhibit the transport of carnitine by Octn2 but downregulated multiple target genes of the transcription factor peroxisome proliferator activated receptor α, including Slc22a5, in the kidney of wild-type mice that were absent in Oct1/2(−/−) mice. Conclusion: Our study shows a pivotal role of Oct1 and Oct2 in cisplatin-related disturbances in carnitine homeostasis. We postulate that this phenomenon is triggered by deactivation of peroxisome proliferator activated receptor α and leads to deregulation of carnitine-shuttle genes. Clin Cancer Res; 16(19); 4789–99. ©2010 AACR.