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B cell Chronic Lymphocytic Leukemia (CLL), the most common leukemia in the western world, is characterized by an accumulation of abnormal B cells in the blood and lymphoid organs. Broad immunosuppression is a serious complication for these patients, with infections and secondary cancers recognized as the leading causes of morbidity and mortality. Although the immune suppression is in part due to clonotypic CLL B cells crowding immune organs, immune responses may also be suppressed by the production of soluble mediators. We and others found that CLL cells produce interleukin-10 (IL-10), a cytokine that suppresses T cell effector function. We hypothesized that this IL-10 may reduce anti-tumor T cell responses. In our studies we use human cells as well as the Eμ-Tcl1 mouse model for U-CLL, in which the oncogene Tcl1 is expressed under the immunoglobulin VH promoter and μ-enhancer. Previously, we found that IL-10R−/− T cells control CLL growth better than WT T cells in the Eμ-Tcl1 adoptive transfer model. We also saw that CLL IL-10 production is dependent on BCR signaling and activation of the transcription factor Sp-1. IL-10 production can be blocked by mithramycin (MTM), an Sp-1 inhibitor. However, MTM is not used routinely as an anti-cancer agent due to its short half-life and associated toxicities, so we prepared and tested novel analogues of MTM to reduce these effects. One of these analogues, MTM23, shows promise as it has similar potency to MTM, but does not interfere with T cell gamma-interferon production or viability. This approach is novel as current therapies for CLL do not target IL-10, or address the immunosuppression seen in CLL patients. IL-10 blockade could therefore be a strategy for harnessing anti-tumor T cells to treat CLL in humans.

The narrow efficacy-toxicity window of anticancer agents necessitates understanding of factors contributing to their disposition. This is especially true for camptothecins as they exist in the lactone and carboxylate forms with each moiety differentially interacting with efflux or uptake transporters. Here we determined the disposition of the lactone and carboxylate forms of AR-67, a 3rd generation camptothecin analogue. Pharmacokinetic studies were conducted in rats given intravenous boluses of AR-67 lactone or carboxylate with or without pharmacologic inhibitor pretreatment (GF120918 or rifampin). Pharmacokinetic modeling was used to estimate clearances, while simulations assessed the impact of clearance changes on overall AR-67 exposure. Our modeling showed that carboxylate clearance was 3.5-fold higher than that of the lactone. GF120918 decreased lactone clearance only, but rifampin decreased both lactone and carboxylate clearances. Simulations showed that decreasing carboxylate clearance, which controls the overall drug disposition, leads to significant increase in AR-67 exposure. The apparent in vivo blood stability of AR-67 is partly dependent on the increased carboxylate clearance. This may have clinical implications for populations with single nucleotide polymorphisms that impair the function of uptake transporter genes (e.g., SLCO1B1), which are potentially responsible for AR-67 carboxylate clearance.

Introduction: Chromosomal translocations involving the ETS family of transcription factors are common in Ewing sarcoma, prostate cancer, and leukemia. These translocations lead in overexpression of aberrant ETS transcription factors, which drive tumorigenesis. Mithramycin (MTM) inhibits EWS-FLI1, the most common ETS-related transcription factor in Ewing sarcoma, presumably through interference at its DNA binding sites on promoter regions. However, MTM has a narrow therapeutic window marked by severe liver and hematologic toxicities. These are likely the result of interference with the ubiquitously expressed SP1 transcription factor. Here, we sought to develop analogues with specificity toward ETS transcription factors and reduced interaction with SP1. Methods: Using MTM-SA and semisynthetic approaches a series of analogues were obtained. To determine specificity toward cells expressing ETS-related chromosomal translocations, MTM-SA analogues were screened for growth inhibition in a panel of Ewing sarcoma cell lines (n=8), all expressing EWS-ETS translocations, and compared to a panel of non-Ewing cell lines (n=9) that do not express these translocations. Luciferase reporter constructs were developed to evaluate interference with EWS-FLI1 and SP1 regulated genes following MTM-SA analogue treatment. Select analogues were tested in vivo to identify the maximum tolerated dose and determine pharmacokinetics (PK). Results: In growth inhibition assays, several MTM-SA analogues resulted in > 10-fold specificity toward Ewing sarcoma cell lines vs. MTM. Luciferase reporter assays identified two analogues, MTM-SA-Tryptophan-A2 and MTM-SA-Tryptophan-A10, with 10-fold reduced inhibition of SP1 but similar inhibition of EWS-FLI1, as compared to MTM. Additionally, MTM-SA-Phe analogue has lower toxicity, despite longer plasma half-life. Conclusion: These data show that MTM-SA analogues are cytotoxic against tumor cell lines expressing aberrant ETS transcription factors. Further, their reduced interference with the ubiquitously expressed SP1 transcription factor, as well as their improved PK, may result in a wider therapeutic window. Citation Format: Markos Leggas, Joseph Eckenrode, Prithiba Mitra, Amit Jha, Shaimaa Salem, Abhisek Mandal, Jon Thorson, Jurgen Rohr. Mithramycin-SA analogues with reduced toxicity for the treatment of ETS transcription factor-driven tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B043.

Our preclinical and clinical data suggest that pretreatment with dexamethasone 4 days prior to chemotherapy increased the efficacy and decreased the toxicity of carboplatin and gemcitabine. To translate these findings to patients, we have undertaken a Phase 1/2 clinical trial. Thirty patients with advanced non-small cell lung cancer (NSCLC) received gemcitabine, 1,000 mg/m2 on days 1 and 8, and carboplatin, AUC 5.5 on day 1. Patients were randomized (1:2:2) to receive, no dexamethasone (cohort 1), or oral dexamethasone at 8 mg (cohort 2) or 16 mg (cohort 3) twice per day, 4 days before and of the day of chemotherapy. Dexamethasone was administered to patients in cohorts 2 and 3 during courses 2–4. In cohorts 1, 2, and 3, patients completing four planned courses of therapy were: 1/6, 6/12, 9/12. Partial responses (RECIST) were: 2/6, 6/12, and 7/12. Overall, dexamethasone significantly improved AGC and platelet nadirs and recovery times. There were no significant differences in non-hematologic toxicities between cohorts and no significant differences in pharmacokinetic parameters between course 1 and 2 in any cohort. These data support our previous preclinical and clinical observations that dexamethasone pre-treatment decreases hematopoietic toxicity and improves efficacy of this chemotherapeutic regimen in patients with metastatic non-small cell lung cancer and suggests that further randomized trials should be undertaken.

Purpose: To design nanoparticles containing accessible gadolinium atoms (Gd-NPs) as a contrast agent for magnetic resonance imaging of tumors. Methods: Nanoparticles containing phospholipid-chelates (phosphoethanolamine diethylenetriaminepentaacetate) and DSPE-PEG (MW5000) were prepared from Brij 78 and stearyl alcohol using the nanotemplate engineering approach. After addition of GdCl 3 , the presence of gadolinium on the surface of nanoparticles was quantified using inductively coupled plasma atomic emission spectroscopy. The in vitro relaxivities of the Gd-NPs in phosphate buffered saline were assessed at 4.7 T. The conditional binding constants of nanoparticle formulations were determined spectrophotometrically by competitive titration. Transmetallation kinetics of Gd 3+ from nanoparticles with Cu 2+ and Zn 2+ as the competing ions was measured in acetate buffer. The biodistribution profiles, pharmacokinetics, and contrast enhancement in tumor region was studied after administration of Gd-NPs to nude mice bearing A549 lung carcinoma xenografts. Results: Gd-NPs with an average diameter of 138 nm possessing surface chelating functions were prepared from GRAS (generally regarded as safe) materials. The longitudinal relaxivity (r 1 ) and transverse relaxivity (r 2 ) of Gd-NPs in 10% fetal bovine serum at 4.7 T were 7.1 (±0.2) and 13.0 (±0.7) 1/mM/s, respectively. These pegylated Gd-NPs had enhanced relaxivities and exhibited particle size stability, sufficient binding affinity, and kinetic inertness under physiologic conditions. The contrast enhancement in tumors was demonstrated 40, 120, and 360 minutes after intravenous injection of Gd-NPs at a dose of 0.1 mmol Gd/kg. The Gd plasma concentration of Gd-NPs over a period of 24 hours fit a two-compartmental model with Cl SYS = 0.89 mL/h and MRT = 5.93 h. The amount of Gd that accumulated in the tumor region was consistent with the estimated value obtained by T 1 measurements using MR imaging. Conclusion: Pegylated nanoparticles composed of biocompatible, biodegradable materials and possessing accessible Gd ions on their surface induce relaxivities in the bulk water signal and accumulated sufficiently in tumors, demonstrating their utility as potential magnetic resonance imaging tumor contrast enhancement agents.

Actinomadura melliaura ATCC 39691, a strain isolated from a soil sample collected in Bristol Cove, California, is a known producer of the disaccharide-substituted AT2433 indolocarbazoles (6-9). Reinvestigation of this strain using new media conditions led to40-fold improvement in the production of previously reported AT2433 metabolites and the isolation and structure elucidation of the four new analogues, AT2433-A3, A4, A5, and B3 (1-4). The availability of this broader set of compounds enabled a subsequent small antibacterial/fungal/cancer SAR study that revealed disaccharyl substitution, N-6 methylation, and C-11 chlorination as key modulators of bioactivity. The slightly improved anticancer potency of the newly reported N-6-desmethyl 1 (compared to 6) contrasts extensive SAR of monoglycosylated rebeccamycin-type topoisomerase I inhibitors where N-6 alkylation has contributed to improved potency and ADME. Complete 2D NMR assignments for the known metabolite BMY-41219 (5) and (13)C NMR spectroscopic data for the known analogue AT2433-B1 (7) are also provided for the first time.

Aging occurs over time with gradual and progressive loss of physiological function. Strategies to reduce the rate of functional loss and mitigate the subsequent onset of deadly age-related diseases are being sought. We demonstrated previously that a combination of rapamycin and myriocin reduces age-related functional loss in the Baker’s yeast Saccharomyces cerevisiae and produces a synergistic increase in lifespan. Here we show that the same drug combination also produces a synergistic increase in the lifespan of the fission yeast Schizosaccharomyces pombe and does so by controlling signal transduction pathways conserved across a wide evolutionary time span ranging from yeasts to mammals. Pathways include the target of rapamycin complex 1 (TORC1) protein kinase, the protein kinase A (PKA) and a stress response pathway, which in fission yeasts contains the Sty1 protein kinase, an ortholog of the mammalian p38 MAP kinase, a type of Stress Activated Protein Kinase (SAPK). These results along with previous studies in S. cerevisiae support the premise that the combination of rapamycin and myriocin enhances lifespan by regulating signaling pathways that couple nutrient and environmental conditions to cellular processes that fine-tune growth and stress protection in ways that foster long term survival. The molecular mechanisms for fine-tuning are probably species-specific, but since they are driven by conserved nutrient and stress sensing pathways, the drug combination may enhance survival in other organisms.

Previous studies suggest an interdependent relationship between liver and intestine for cholesterol elimination from the body. We hypothesized that a combination of ursodiol (Urso) and ezetimibe (EZ) could increase biliary secretion and reduce cholesterol reabsorption, respectively, to promote cholesterol excretion. Treatment with Urso increased hepatic ABCG5 ABCG8 (G5G8) protein and both biliary and fecal sterols in a dose-dependent manner. To determine whether the drug combination (Urso-EZ) further increased cholesterol excretion, mice were treated with Urso alone or in combination with two doses of EZ. EZ produced an additive and dose-dependent increase in fecal neutral sterol (FNS) elimination in the presence of Urso. Finally, we sequentially treated wide-type and G5G8-deficient mice with Urso and Urso-EZ to determine the extent to which these effects were G5G8 dependent. Although biliary and FNS were invariably lower in G5G8 KO mice, the relative increase in FNS following treatment with Urso alone or the Urso-EZ combination was not affected by genotype. In conclusion, Urso increases G5G8, biliary cholesterol secretion, and FNS and acts additively with EZ to promote fecal sterol excretion. However, the stimulatory effect of these agents was not G5G8 dependent.

OBJECTIVE:The study goal was to determine whether clonidine treatment of neonatal abstinence syndrome (NAS) would result in a better neurobehavioral performance compared with morphine.METHODS:This pilot study prospectively enrolled infants ≥35 weeks’ gestational age admitted for treatment of NAS. After informed consent was obtained, infants were randomized to receive morphine (0.4 mg/kg per day) or clonidine (5 μg/kg per day) divided into 8 doses. A 25% dose escalation every 24 hours was possible per protocol (maximum of 1 mg/kg per day for morphine and 12 μg/kg per day for clonidine). After control of symptoms, the dose was tapered by 10% every other day. Clinical staff monitored infants by using Finnegan scoring. Masked research staff administered the NICU Network Neurobehavioral Scale (NNNS) at 1 week and at 2 to 4 weeks after initiation of treatment and the Bayley Scales III, and Preschool Language Scale IV, at 1-year adjusted age. Analyses included descriptive statistics, repeated measures analysis of variance, and Wilcoxon tests.RESULTS:Infants treated with morphine (n = 15) versus clonidine (n = 16) did not differ in birth weight or age at treatment. Treatment duration was significantly longer for morphine (median 39 days) than for clonidine (median 28 days; P = .02). NNNS summary scores improved significantly with clonidine but not with morphine. On subsequent assessment, those receiving clonidine had lower height of arousal and excitability (P < .05). One-year motor, cognitive, and language scores did not differ between groups.CONCLUSIONS:Clonidine may be a favorable alternative to morphine as a single-drug therapy for NAS. A multicenter randomized trial is warranted.

The 4-anilinoquinazoline (4-AQ) derivative gefitinib (Iressa) is an oral epidermal growth factor receptor tyrosine kinase inhibitor. Oral administration of 4-AQ molecules, such as gefitinib, inhibits ATP-binding cassette (ABC) transporter–mediated drug efflux and strongly increases the apparent bioavailability of coadministered drug molecules that are transporter substrates. Based on in vitro studies investigating 4-AQ interactions with several transporters, these effects have primarily been attributed to the inhibition of breast cancer resistance protein (BCRP; ABCG2). Although 4-AQ shows in vitro inhibition of P-glycoprotein [multidrug resistance protein (MDR1); ABCB1], the in vivo effect on this and other transporters is not known. In our studies, pretreatment of Abcg2−/− and Mdr1(a/b)−/− mice with gefitinib increased oral absorption and decreased systemic clearance of topotecan, a model substrate, indicating that additional transporters were inhibited. These results were extended to human orthologues using engineered cell lines to show that gefitinib inhibited the efflux of BCRP and MDR1 substrates and restored vincristine sensitivity in MDR1-expressing cells. Although gefitinib inhibited BCRP more potently than MDR1 (10-fold), the inhibition of both transporters occurred at clinically relevant concentrations (e.g., 1-5 μmol/L). These studies illustrate the broad implications for the therapeutic combination of gefitinib or other 4-AQ molecules with agents that are BCRP and MDR1 substrates. 4-AQ molecules may offer a means to increase the low and variable oral drug absorption of transporter substrates while decreasing interpatient variability and reversing tumor drug resistance. (Cancer Res 2006; 66(9): 4802-7)

Most reverse transcriptase and protease inhibitors used in highly active antiretroviral therapy for treating human immunodeficiency virus (HIV) infections exhibit poor penetration into the brain, raising the concern that the brain may be a sanctuary site for the development of resistant HIV variants. This study explores the relationship between the dose and plasma and brain concentrations of zosuquidar and the effect of this selective P-glycoprotein inhibitor on central nervous system penetration of the HIV protease inhibitor nelfinavir maintained at steady state by intravenous infusions in rats. Nelfinavir was infused (10 mg/kg/h) for up to 10 h with or without concurrent administration of an intravenous bolus dose of 2, 6, or 20 mg/kg zosuquidar given at 4 h. Brain tissue and plasma were analyzed for both drug concentrations. Brain tissue/plasma nelfinavir concentration ratios (uncorrected for the vascular contribution) increased nonlinearly with zosuquidar dose from 0.06 +/- 0.03 in the absence of zosuquidar and 0.09 +/- 0.02 between 2 and 6 h after 2 mg/kg zosuquidar to 0.85 +/- 0.19 after 6 mg/kg and 1.58 +/- 0.67 after 20 mg/kg zosuquidar. Zosuquidar brain tissue/plasma concentration ratios exhibited a similar abrupt increase from 2.8 +/- 0.3 after a 2 mg/kg dose to approximately 15 after the 6 and 20 mg/kg doses. The apparent threshold in the plasma concentration of zosuquidar necessary to produce significant enhancement in brain uptake of nelfinavir appears to be close to the plasma concentrations associated with the maximum tolerated dose reported in the literature after repeated dosing of zosuquidar in patients.

As a single agent the ERBB1 inhibitor, gefitinib (Iressa; ZD1839) showed minimal activity against a panel of 10 pediatric tumor xenografts that do not express the ERBB1 receptor. However, combined with irinotecan (CPT-11), significantly greater than additive activity was observed in four of eight models (P < 0.05), and the combination showed enhanced activity against three additional tumor lines. Breast cancer resistance protein (ABCG2), a transporter that confers resistance to SN-38 (the active metabolite of irinotecan), was readily detected in six of nine xenograft models examined by immunohistochemistry. In vitro gefitinib potently reversed resistance to SN-38 only in a cell line that overexpressed functional ABCG2. However, overexpression of ABCG2 did not decrease accumulation nor increase the rate of efflux of [14C]gefitinib. On the basis of these results and the distribution of Abcg2 in mouse tissues, we assessed the ability of gefitinib to modulate irinotecan pharmacokinetics. Oral gefitinib coadministration resulted in no change in clearance of intravenously administered irinotecan. However, gefitinib treatment dramatically increased the oral bioavailability of irinotecan after simultaneous oral administration. It is concluded that gefitinib may modulate SN-38 activity at the cellular level to reverse tumor resistance mediated by ABCG2 through inhibiting drug efflux and may be used potentially in humans to modulate the oral bioavailability of a poorly absorbed camptothecin such as irinotecan.

The ABC transporter, Mrp4, transports the sulfated steroid DHEA-s, and sulfated bile acids interact with Mrp4 with high affinity. Hepatic Mrp4 levels are low, but increase under cholestatic conditions. We therefore inferred that up-regulation of Mrp4 during cholestasis is a compensatory mechanism to protect the liver from accumulation of hydrophobic bile acids. We determined that the nuclear receptor CAR is required to coordinately up-regulate hepatic expression of Mrp4 and an enzyme known to sulfate hydroxy-bile acids and steroids, Sult2a1. CAR activators increased Mrp4 and Sult2a1 expression in primary human hepatocytes and HepG2, a human liver cell line. Sult2a1 was down-regulated in Mrp4-null mice, further indicating an inter-relation between Mrp4 and Sult2a1 gene expression. Based on the hydrophilic nature of sulfated bile acids and the Mrp4 capability to transport sulfated steroids, our findings suggest that Mrp4 and Sult2a1 participate in an integrated pathway mediating elimination of sulfated steroid and bile acid metabolites from the liver.

Objective. To determine whether admissions data alone adequately predicts student success in the first-year doctor of pharmacy (PharmD) curriculum or whether academic monitoring and intervention has greater value toward successful completion of first-year coursework. Methods. A systematic evaluation of the literature assessing student success was performed to ascertain historical evidence of student success metrics. We then retrospectively analyzed internal admissions data and first-year outcomes for our pharmacy classes of 2016-2019 using available data. We conducted an interim evaluation of voluntary academic monitoring and mentoring with the hypothesis that admission data alone cannot predict student success in early foundational coursework, and intentional intervention might improve success. Results. Pre-pharmacy grade point average (GPA), science GPA, Pharmacy College Admission Test (PCAT) score, and prior degree status each retain some predictive value regarding success, and combinations of these factors may improve the ability to predict student success in early foundational coursework. There remains a significant, and perhaps insurmountable, gap in identifying quantitative metrics that forecast student success. Although admission data can stratify incoming students based on predicted academic ability, early monitoring and intervention provide an actionable means for enhancing student success in first-year coursework. Conclusion. Quantitative academic measures, such as PCAT scores and GPA, historically have demonstrated limited value in predicting student success. While these measures allow stratification of predicted academic performance among incoming students, monitoring of first-year, institution-specific data, such as midterm grades, can direct intentional intervention and remediation strategies that may provide more benefit to ensure students succeed.

The breast cancer resistance protein (BCRP; ABCG2) is an ATP-dependent efflux multidrug transporter that belongs to the G family of half-transporters that consist of six transmembrane-spanning domains and must homodimerize to form the active membrane transporter. It is expressed in the apical plasma membrane domain of the small intestine, endothelium, and liver, where it has been shown to play an important role in limiting drug absorption and distribution and in enhancing drug clearance, respectively. BCRP is also expressed in the apical membrane of mammary alveolar epithelia, where it mediates efflux of substrates into milk, and in the placental syncytiotro-phoblasts, where it reduces fetal exposure to these substrates. BCRP substrates include numerous drugs (topotecan, nitrofurantoin, cimetidine) as well as food carcinogens (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) and the vitamins riboflavin and folic acid. BCRP expression is regulated by a number of nuclear transcription factors, including the peroxisome proliferator-activated receptor-gamma and Hif-1. This issue of Molecular Pharmacology includes a study (p. 845) now conclusively demonstrating that progesterone acts via the progesterone A and B receptors to regulate BCRP expression in a placental cell line.

A new library of small molecules with structural features resembling combretastatin analogs was synthesized and evaluated for anticancer activity against a panel of 60 human cancer cell lines. Three novel acrylonitrile analogs (5, 6 and 13) caused a significant reduction in cell growth in almost all the cell lines examined, with GI50 values generally in the range 10-100 nM. Based on the structural characteristics of similar drugs, we hypothesized that the cytotoxic activity was likely due to interaction with tubulin. Furthermore, these compounds appeared to overcome cell-associated P-glycoprotein (P-gp)-mediated resistance, since they were equipotent in inhibiting OVCAR8 and NCI/ADR-Res cell growth. Given that antitubulin drugs are among the most effective agents for the treatment of advanced prostate cancer we sought to validate the results from the 60 cell panel by studying the representative analog 6 utilizing prostate cancer cell lines, as well as exploring the molecular mechanism of the cytotoxic action of this analog.

Disease incidence rises rapidly with age and increases both human suffering and economic hardship while shortening life. Advances in understanding the signaling pathways and cellular processes that influence aging, support the possibility of reducing the incidence of age-related diseases and increasing lifespan by pharmacological intervention. Here, we demonstrate a novel pharmacological strategy that both reduces signs of aging in the budding yeast Saccharomyces cerevisiae and generates a synergistic increase in lifespan. By combining a low dose of rapamycin, to reduce activity of the target of rapamycin complex 1 (TORC1) protein kinase, and myriocin, to reduce sphingolipid synthesis, we show enhancement of autophagy, genomic stability, mitochondrial function, and AMP kinase pathway activity. These processes are controlled by evolutionarily conserved signal transduction pathways that are vital for maintaining a healthy state and promoting a long life. Thus, our data show that it ought to be possible to find pharmacological approaches to generate a synergistic reduction in the incidence of human age-related diseases to improve health quality in the elderly and enhance lifespan.

AR-67 (7-t-butyldimethylsilyl-10-hydroxycamptothecin) is a lipophilic camptothecin analog, currently under early stage clinical trials. Transporters are known to have an impact on the disposition of camptothecins and on the response to chemotherapeutics in general due to their expression in tumor tissues. Therefore, we investigated the interplay between the breast cancer resistance protein (BCRP), multidrug resistant protein 1 (MDR1), and organic anion-transporting polypeptide (OATP) 1B1/1B3 transporters and AR-67 and their impact on the toxicity profile of AR-67. Using cell lines expressing the aforementioned transporters, we showed that the lipophilic AR-67 lactone form is a substrate for efflux transporters BCRP and MDR1. Additionally, OATP1B1 and OATP1B3 facilitated the uptake of AR-67 carboxylate in SLCO1B1- and SLCO1B3-transfected cell systems compared with the mock-transfected ones. Notably, both BCRP and MDR1 conferred resistance to AR-67 lactone. Prompted by recent studies showing increased OATP1B3 expression in certain cancer types, we investigated the effect of OATP1B3 expression on cell viability after exposure to AR-67 carboxylate. OATP1B3-expressing cells had increased carboxylate uptake as compared with mock-transfected cells but were not sensitized because the intracellular amount of lactone was 50-fold higher than that of carboxylate and comparable between OATP1B3-expressing and OATP1B3-nonexpressing cells. In conclusion, BCRP- and MDR1-mediated efflux of AR-67 lactone confers resistance to AR-67, but OATP1B3-mediated uptake of the AR-67 carboxylate does not sensitize OATP1B3-expressing tumor cells.

To evaluate the pharmacokinetics of simvastatin at the maximum tolerated dose (MTD) of 7.5 mg/kg, twice daily, in the context of a pilot trial enrolling patients with recurrent and refractory chronic lymphocytic leukemia. Patients received simvastatin orally at MTD for 7 days during a 21-day cycle for 6 cycles. Blood samples were collected during cycle 1. Simvastatin lactone and carboxylate concentrations were measured in plasma and peripheral blood mononuclear cells (PBMCs) using a validated HPLC–MS/MS assay. Patients accrued to this study showed high variability in their exposure to simvastatin. Exposure was dose proportional (AUC and C max) as compared to those receiving standard hyperlipidemia therapy. Peak plasma concentrations ranged from 0.08 to 2.2 and from 0.03 to 0.6 μM for simvastatin lactone and carboxylate, respectively. Our study shows that when simvastatin is administered at its MTD, only low micro-molar concentrations are achieved in plasma and PBMCs, which is consistent with the results observed in previous studies with lovastatin, but far lower than the concentrations required for anticancer effects in vitro. However, whether simvastatin at its MTD can confer therapeutic benefits to patients still remains to be determined.

The angucycline group of antibiotics is one of the largest groups of polycyclic aromatic polyketides, rich in chemical scaffolds and biological activities, predominantly anticancer and antibacterial.1–3 Saquayamycins,4–8 urdamycins,9–17 and landomycins,18–24 are well known angucycline antitumor antibiotics. The structures of both saquayamycins and urdamycins contain the same aquayamycin (17)25–27 as aglycone with the C-glycosidic sugar D-olivose attached at C-9 of the angucycline chromophore. So far, seven saquayamycin analogues were reported from Streptomyces spp. Saquayamycins differ from urdamycins by their saccharide patterns, which are attached at C-9 and C-3 positions in the saquayamycins, but at C-9- and C-12b-positions in urdamycins. Saquayamycins A-D (7–10) were first isolated from Streptomyces nodosus MH190-16F3 and were reported as platelet aggregation inhibitors.4 Saquayamycins A-B (7–8) contain three different O-glycosidically linked deoxysugars, L-rhodinose, L-aculose, and L-cinerulose (only in saquayamycin B). Saquayamycin A (7) was reported to be unstable to acid, and even contact with silica gel led to its conversion to saquayamycin B (8). Saquayamycins A1 (11), B1 (6), and C1 (12) were generated by partial acid hydrolysis of saquayamycins A-C (7–9), respectively.4 The two isomers, saquayamycins E (13) and F (14) were produced by Actinomyces strain MK290-AF1 and reported to inhibit the FPTase from bovine brain with IC50 values of 1.8 and 2.0 μM, respectively.5 They differ slightly from the saquayamycins A (7) and C (9) with respect to their sugar moieties. The saquayamycin analogue A-7884 (16) was isolated from the Streptomyces sp. #AM1699; it has a trisaccharide side chain connected at C-9, which contains an L-rhodinose sugar moiety between the first sugar, a C-glycosidic bound D-olivose, and L-aculose of saquayamycin A1 (11).28 Compound 16 showed inhibitory activity in the inducible nitric oxide synthase (iNOS) assay with IC50 values of 43.5 μM, better than saquayamycin A1 (11; IC50 values of 101.2 μM).28 Recently, the largest saquayamycin analogue, saquayamycin Z (15), was reported from Micromonospora sp. strain Tu6368.6 Saquayamycin Z (15) contains tetra- and pentasaccharide side chains linked at C-3- and C-9-positions of the benz[a]anthracene core. The tetrasaccharide side chain of saquayamycin Z (15) is striking due to the presence of an L-oliose, which is not a usual sugar constituent of the angucyclines.6, 29 Very recently, Ren et al., reported three novel members of the angucycline family named N05WA963 A, B and D, with the same aglycones as in the moromycins A (18) and B (19), except for an additional methoxy group attached at C-5.30 N05WA963 A, B and D were reported to have antiproliferative effects on a panel of cancer cell lines such as SW620 (colon cancer), K-562 (chronic myelogeneous leukemia), MDA-MB-231 (estrogen receptor negative breast cancer), YES-4 (esophageal cancer), T-98 and U251SP (both glioblastomae). To study the structure-activity relationship (SAR) of this saquayamycin group of antibiotics, we looked for further saquayamycin analogues produced by Streptomyces sp. K40-1, the strain earlier reported as the producer of moromycins A-B (18–19).7 The saccharide attachments in moromycins A (18) and B (19) are like those of saquayamycins B (8) and B1 (6), respectively, however, their tetracyclic angucyclinone core has an aromatic ring B, and thus no angular hydroxy groups at C-4a and C-12b positions. We found five new metabolites, designated as saquayamycins G-K (1–5), produced by repeated fermentations of the same strain, along with saquayamycin B1 (6), which was previously not described as a natural product, and known saquayamycins. Two of the new angucyclines, saquayamycins H (2) and I (3), bear the unusual aminosugar rednose, which was found for the first time in an angucycline compound. Aminosugar-containing angucyclines are very rare, and previously only three examples had been reported, namely the marmycins A and B,31, 32 and mayamycin.33 The marmycins contain an unusual branched and doubly (C- and N)-linked aminosugar, 3-epi-, 4-epi-vancosamine, and mayamycin contains a unique C-glycosidically linked aminosugar, N-demethylangolosamine, attached at the C-5-position of the benz[a]anthracenone core.

We have previously shown that the bacterial enzyme thiaminase 1 has antitumor activity. In an attempt to make thiaminase I a more effective pharmaceutical agent, we have modified it by adding polyethylene glycol (PEG) chains of various lengths. We were surprised to find that 5k-PEGylation eliminated thiaminase cytotoxic activity in all cell lines tested. Both native thiaminase and 5k-PEGylated thiaminase efficiently depleted thiamine from cell culture medium, and both could use intracellular phosphorylated thiamine as substrates. However, native enzyme more effectively depleted thiamine and thiamine diphosphate in RS4 leukemia cell cytosol, and native thiaminase depressed cellular respiration, whereas PEGylated thiaminase did not. Despite the lack of in vitro cytotoxicity, PEGylation markedly increased the in vivo toxicity of the enzyme. Pharmacokinetic studies revealed that the half-life of native thiaminase was 1.5 h compared with 34.4 h for the 5k-PEGylated enzyme. Serum thiamine levels were depleted by both native and 5k-PEGylated enzyme. Despite superior pharmacokinetics, 5k-PEGylated thiaminase showed no antitumor effect against an RS4 leukemia xenograft, in contrast to native thiaminase, which showed antitumor activity. PEGylation of thiaminase I has demonstrated that depression of mitochondrial function contributes, at least in part, to its anticancer activity. PEGylation also enhances plasma retention time, which increased its vivo toxicity and decreased its activity against a leukemia xenograft, the opposite of the desired effects. These studies suggest that the mechanism of anticancer cytotoxicity of thiaminase requires acute depression of cellular respiration, whereas systemic toxicity is related to the duration of extracellular thiamine depletion.

The in vitro and in vivo characterization of an optimized formulation of nanoparticles (NPs) loaded with a high content of dexamethasone palmitate (DEX-P), a chemotherapeutic adjuvant that decreases interstitial fluid pressure in tumors, and (111) In, a signaling agent, is described. These NPs are uniform in size and composition. Single photon emission computed tomography imaging demonstrates significant tumor uptake of (111) In-labeled DEX-P NPs in tumor-bearing mice. As with many nanoparticle-based drug delivery systems, significant liver accumulation is observed. Assessment of liver histology and blood tests show no apparent hepatic or renal toxicity of the DEX-P NPs. Conversion of DEX-P to DEX occurs when DEX-P NPs are incubated with mouse plasma, human tumor homogenate and ascites from tumor bearing mice, but not with human plasma. This conversion is slower in plasma from Es1(e) ((-/-)) /SCID mice, a potential alternative animal model that better mimics humans; however, plasma from these mice are not completely devoid of esterase activity. The difference between blood and tumor esterase activity in humans facilitates the delivery of DEX-P NPs to tumors and the release of dexamethasone by an esterase trigger.

7-tert-Butyldimethylsilyl-10-hydroxycamptothecin (AR-67; also known as DB-67) is a novel lipophilic camptothecin analog in early-phase anticancer clinical trials. In support of these studies, we evaluated the metabolism of AR-67 in vitro and identified potential metabolites in patient samples. The lactone form of AR-67 was found to be preferentially metabolized over AR-67 carboxylate in human microsomes. Subsequently, the lactone form was tested as a substrate in a panel of CYP450 and UDP-glucuronosyltransferase (UGT) enzymes known to metabolize the majority of clinically approved molecules. AR-67 was metabolized by CYP3A5, CYP3A4, CYP1A1, and CYP1A2, in order of activity. Extrahepatic UGT1A8 and UGT1A7 possessed at least 6-fold higher metabolizing activity than UGT1A1 and other UGT enzymes tested. CYP1A1 and UGT1A7 displayed Michaelis-Menten kinetics, whereas CYP3A4, CYP3A5, and UGT1A8 displayed kinetics consistent with substrate inhibition. Chromatographic analysis of representative patient plasma and urine samples demonstrated the presence of AR-67 glucuronides and oxidized products in the urine but only in very minimal amounts. We conclude that limited in vivo metabolism of AR-67 by UGT1A1 may partly explain the absence of AR-67 glucuronides in plasma and hypothesize that UGT1A8- and CYP3A-mediated biotransformation within the gastrointestinal epithelium may provide protective mechanisms against AR-67 gastrointestinal toxicity.

Purpose: 7-t-Butyldimethylsilyl-10-hydroxycamptothecin (AR-67) is a novel third generation camptothecin selected for development based on the blood stability of its pharmacologically active lactone form and its high potency in preclinical models. Here, we report the initial phase I experience with i.v. AR-67 in adults with refractory solid tumors. Experimental Design and Methods: AR-67 was infused over 1 hour daily five times, every 21 days, using an accelerated titration trial design. Plasma was collected on the 1st and 4th day of cycle 1 to determine pharmacokinetic parameters. Results: Twenty-six patients were treated at nine dosage levels (1.2-12.4 mg/m2/d). Dose-limiting toxicities were observed in five patients and consisted of grade 4 febrile neutropenia, grade 3 fatigue, and grade 4 thrombocytopenia. Common toxicities included leukopenia (23%), thrombocytopenia (15.4%), fatigue (15.4%), neutropenia (11.5%), and anemia (11.5%). No diarrhea was observed. The maximum tolerated dosage was 7.5 mg/m2/d. The lactone form was the predominant species in plasma (>87% of area under the plasma concentration-time curve) at all dosages. No drug accumulation was observed on day 4. Clearance was constant with increasing dosage and hematologic toxicities correlated with exposure (P < 0.001). A prolonged partial response was observed in one subject with non–small cell lung cancer. Stable disease was noted in patients with small cell lung cancer, non–small cell lung cancer, and duodenal cancer. Conclusions: AR-67 is a novel, blood-stable camptothecin with a predictable toxicity profile and linear pharmacokinetics. The recommended phase II dosage is 7.5 mg/m2/d five times every 21 days. Clin Cancer Res; 16(2); 673–80

Solute transporters that are selectively expressed on tumor cell membranes could be targeted with small molecule toxins that are selective substrates for these transporters. HeLa cells transfected to express the solute transporter OATP1B1 are exquisitely sensitive in vitro to microcystin LR (MCLR) and its analogs, and undergo rapid morphologic changes after exposure to MCLR. Immunoblot analyses revealed HSP27 phosphorylation increased prior to the rapid MCLR-induced morphologic changes. However, transfection of OATP1B1-expressing cells with HSP27 dominant negative mutants did not reverse MCLR toxicity. Although the MAP kinase p38 inhibitor SB202190 partially reversed MCLR cytotoxicity, the control molecule, SB202474, had similar effects. Unexpectedly, both SB202190 and SB202474 inhibited OATP1B1 uptake activity, indicating an alternative explanation for cytotoxicity reversal that did not involve p38 MAP kinase. Similarly, although the potassium chloride co-transporter (KCC) inhibitor (dihydro-indenyl)oxyalkanoic acid (DIOA), and the anti-oxidant, N-acetyl cysteine (NAC) both reversed MCLR cytotoxicity, both were also found to be unexpected OATP1B1 transport inhibitors. Therefore, the mechanism of MCLR-induced cytotoxicity is obscured by the inhibition of OATP1B1 uptake activity by MAP kinase inhibitors, DIOA, and NAC. Finally, growth of OATP1B1-expressing HeLa xenografts was inhibited by MCLR, suggesting that MCLR structural analogs selected for a broader therapeutic index could target OATP-expressing tumors.

Purpose: To compare the expression of pregnane xenobiotic receptor and certain multi-drug resistance proteins in retinoblastoma. Methods: Using tissue microarray analyses, we studied 62 pathology specimens for expression of pregnane xenobiotic receptor, multi-drug resistance 1/P glycoprotein, multi-drug resistance proteins 1, 2, and 4, and breast cancer resistant protein. Results: Comparing tumors treated with primary enucleation with tumors treated with chemotherapy and/or radiation showed no significant differences in the expression of multi-drug resistance proteins or pregnane xenobiotic receptor. Pregnane xenobiotic receptor was correlated with multi-drug resistance protein 2 expression (p < 0.001). Conclusion: Our results indicate selection, rather than induction, of chemoresistant cells as a cause for treatment failure in managing retinoblastoma with primary systemic chemotherapy.

The objectives of these studies were to investigate and compare solid lipid nanoparticles (SLNs) of two anthracyclines, idarubicin (IDA) and doxorubicin (DOX), against Pgp-mediated multiple drug resistance (MDR.) in-vitro and in-vivo using different human and murine cancer cell models. IDA and DOX SLNs were developed from warm microemulsion precursors comprising emulsifying wax as the oil phase, and polyoxyl 20-stearyl ether (Brij 78) and D-alpha-tocopheryl polyethylene glycol succinate (Vitamin E TPGS) as the surfactants. Anionic ion-pairing agents, sodium taurodeoxycholate (STDC) and sodium tetradecyl sulfate (STS), were used to neutralize the charges of the cationic anthracyclines and enhance entrapment of the drugs in the SLN. The in-vitro cytotoxicity results showed that the IC50 value of DOX NPs was 9-fold lower than that of free DOX solution in resistant P388/ADR cell line. In contrast, free IDA had comparable IC50 values as IDA NPs in Pgp-overexpressing P388/ADR and HCT-15 cells. In the in-vivo P388/ADR leukemia mouse model, the median survival time of DOX NPs was significantly greater than that of free DOX, and controls. In contrast, free IDA was equally as effective as IDA NPs in P388 and Pgp-overexpressing HCT-15 mouse tumor models. The cell uptake of IDA formulated as free IDA and IDA NPs was comparable in Pgp-overexpressing cells. In conclusion, DOX NPs could overcome Pgp-mediated MDR both in-vitro in P388/ADR leukemia cells and in-vivo in the murine leukemia mouse model. The present study suggests that our SLNs may offer potential to deliver anticancer drugs for the treatment of Pgp-mediated MDR in leukemia; however, selection of target drug may be very important.

Publisher Summary This chapter provides a context for understanding drug distribution into tissues and addresses those factors that play a major role in determining the rate and extent of drug distribution. Transporters can greatly facilitate the passage of a drug from one side of a biological membrane to the other. The orientation of transporters is either apical or basolateral and these terms also are referred to as luminal or abluminal, respectively. Drug movement across barrier-forming biological membranes can generally be accomplished by three mechanisms. Carrier-mediated transport is the most prevalent mechanism because it affords greater control as to the type and quantity of molecules that can be translocated. Passive diffusion is a major process by which drugs cross cell membranes. The cell bilayer, which is permeable to lipophilic molecules and favors their partitioning into the membrane, represents an important first step in flux across the membrane. Most drugs leave the site of absorption and distribute to sites of action and elimination by traveling in the blood. Distribution concepts need to consider the limitations of this physiological reality.

The pre-administration of the anti-inflammatory drugs dexamethasone (DEX) and cortisone acetate reduces toxicity and enhances efficacy of anticancer agents in murine models and in human clinical trials (1–5). We previously reported on the formulation of the lipophilic dexamethasone palmitate ester (DEX-P) in nanoparticles (NPs) employing a microemulsion template engineering technique to achieve tumor-specific delivery of dexamethasone (6). The nanoparticles exhibited significantly enhanced stealth properties as indicated by reduced macrophage uptake and decreased adsorption of opsonin proteins in in vitro assays (6). Unexpectedly, preliminary biodistribution studies of NPs containing [3H]-DEX-P in tumor-bearing mice showed that the radiolabel was cleared from the circulation rapidly and exhibited high liver uptake. Our previous in vitro release studies demonstrated that rapid release of the radiolabel from the NPs was observed when 10% mouse plasma was used as the medium, while nominal release was observed in phosphate-buffered saline (PBS) buffer (6). Esterolysis of NP-associated DEX-P was presumed to be the main cause for the rapid drug release in plasma, as most of the released radioactivity was in the form of DEX and not DEX-P. High degradation rates of ester prodrugs in rodent plasma has been attributed to increased esterase activity, while only minimal degradation in human plasma has been observed (7–9). Based on our observation of the release of [3H]-DEX from NPs in mouse plasma, we studied the release of DEX from nanoparticles in various plasma sources as a guide for the design of future in vivo experiments.

Genetic knockout mice studies suggested ATP-binding cassette transporter family G member 2 (ABCG2)/Abcg2 translocates nitrofurantoin at the mammary-blood barrier, resulting in drug accumulation in milk. The purpose of this study was to establish the role of Abcg2 in nitrofurantoin accumulation in rat milk using N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918) as a "chemical knockout" equivalent. The inhibitory effect of GF120918 was verified in Madin-Darby canine kidney II cells stably expressing rat Abcg2 with Hoechst 33342 and nitrofurantoin flux in Transwells. Nitrofurantoin was infused (0.5 mg/h) in the absence and presence of GF120918 (10 mg/kg in dimethyl sulfoxide) to Sprague-Dawley lactating female rats using a balanced crossover design. Administration of GF120918 increased nitrofurantoin concentration in serum (from 443 +/- 51 to 650 +/- 120 ng/ml) and decreased concentration in milk (from 18.1 +/- 0.9 to 1.9 +/- 1.2 microg/ml), resulting in corresponding mean values for milk to serum concentration ratio (M/S) of 41.4 +/- 19.1 versus 3.04 +/- 2.27 in the absence and presence of GF120918 (p0.05), respectively. There was a decrease in systemic clearance with GF120918 (2.8 +/- 0.5 l/h/kg) compared with vehicle controls (4.1 +/- 0.5 l/h/kg; p0.05). Western blot analysis revealed good expression of Abcg2 and no P-glycoprotein (P-gp) expression in mammary gland, whereas immunohistochemistry confirmed the apical expression of Abcg2 in lactating mammary gland epithelia. Nitrofurantoin active transport into rat milk can be inhibited by GF120918 resulting in a 10-fold lower M/S. Although GF120918 inhibits both Abcg2 and P-gp, the high expression of Abcg2 and the absence of detectable P-gp expression in lactating mammary gland validate an important role for Abcg2 in nitrofurantoin accumulation in rat milk. GF120918 is particularly useful as a rat chemical knockout model to establish ABCG2's role in drug transfer into milk during breastfeeding.

The breast cancer resistance protein (BCRP; ABCG2) is an ATP-dependent efflux multidrug transporter that belongs to the G family of half-transporters that consist of six transmembrane-spanning domains and must homodimerize to form the active membrane transporter. It is expressed in the apical plasma membrane domain of the small intestine, endothelium, and liver, where it has been shown to play an important role in limiting drug absorption and distribution and in enhancing drug clearance, respectively. BCRP is also expressed in the apical membrane of mammary alveolar epithelia, where it mediates efflux of substrates into milk, and in the placental syncytiotro-phoblasts, where it reduces fetal exposure to these substrates. BCRP substrates include numerous drugs (topotecan, nitrofurantoin, cimetidine) as well as food carcinogens (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) and the vitamins riboflavin and folic acid. BCRP expression is regulated by a number of nuclear transcription factors, including the peroxisome proliferator-activated receptor-gamma and Hif-1. This issue of Molecular Pharmacology includes a study (p. 845) now conclusively demonstrating that progesterone acts via the progesterone A and B receptors to regulate BCRP expression in a placental cell line.

The pre-administration of dexamethasone (DEX) has previously been shown to enhance the anti-tumor efficacy of chemotherapeutic agents. The delivery of anti-inflammatory agents specifically to tumors via nanoparticle carriers is expected to promote the effectiveness of chemotherapeutic agents while avoiding systemic toxicities. The process for preparing solid lipid nanoparticles containing anti-inflammatory agents using the nanotemplate engineering method was optimized. Due to the solubilization of DEX in the bulk aqueous phase, its more lipophilic palmitate ester was synthesized and incorporated in nanoparticles that included a pegylating agent, PEG6000 mono-stearate, as part of the formulation. The stealth properties of these nanoparticles were demonstrated to be enhanced compared to latex particles by measuring the adsorption of radioiodinated IgG (185 microg vs. 6.7 microg IgG/mg NP). In addition, the uptake of (14)C-labeled nanoparticles by murine macrophages was shown to decrease from 36.6% to 14.7% of the nanoparticles/mg cell protein as the amount of pegylating agent in the formulation increased from 0 to 4 mg/mL. The high loading values and low burst effect observed for these DEX palmitate-containing nanoparticles in addition to their stealth properties are expected to allow for the delivery of sufficient amounts of DEX to tumors to enhance the uptake of chemotherapeutic agents.

The use of probe substrates and combinations of ATP-binding cassette (ABC) transporter knockout (KO) animals may facilitate the identification of common substrates between apparently unrelated ABC transporters. An unexpectedly low concentration of the purine nucleotide analogue, 9-(2-(phosphonomethoxy)ethyl)-adenine (PMEA), and up-regulation of Abcg2 in some tissues of the Mrp4 KO mouse prompted us to evaluate the possibility that Abcg2 might transport purine-derived drugs. Abcg2 transported and conferred resistance to PMEA. Moreover, a specific Abcg2 inhibitor, fumitremorgin C, both increased PMEA accumulation and reversed Abcg2-mediated PMEA resistance. We developed Mrp4 and Abcg2 double KO mice and used both single KOs of Abcg2 and Mrp4 mice to assess the role of these transporters in vivo. Abcg2 contributed to PMEA accumulation in a variety of tissues, but in some tissues, this contribution was only revealed by the concurrent absence of Mrp4. Abcg2 also transported and conferred resistance to additional purine analogues, such as the antineoplastic, 2-chloro-2′-deoxyadenosine (cladribine) and puromycin, a protein synthesis inhibitor that is often used as a dominant selectable marker. Purine analogues interact with ABCG2 by a site distinct from the prazosin binding site as shown by their inability to displace the substrate analogue and photoaffinity tag [125I]iodoarylazidoprazosin. These studies show that Abcg2, like Mrp4, transports and confers resistance to purine nucleoside analogues and suggest that these two transporters work in parallel to affect drug cytotoxicity and tissue distribution. This new knowledge will facilitate an understanding of how Abcg2 and Mrp4, separately and in combination, protect against purine analogue host toxicity as well as resistance to chemotherapy. [Cancer Res 2007;67(14):6965–72]

Introduction: EWS-FLI1 and related chromosomal translocations are prevalent in Ewing sarcoma and play a major role in modulating oncogenic transcription. Development of drugs that affect EWS-FLI1 oncoprotein function may lead to successful treatment for these patients. Mithramycin (MTM) was shown to inhibit transcriptional targets of EWS-FLI1, but it has a narrow therapeutic window attributed to its nonspecific toxicities. To overcome this, semisynthetic methods were developed to generate MTM analogs with unique pharmacologic properties. Mechanistic and pharmacologic studies are presented here. Methods: Studies were conducted using MTM and lead analogs (mithramycin-SK (MTM-SK), mithramycin-SA-tryptophan (MTM-SA-Trp), and mithramycin-SA-phenylalanine (MTM-SA-Phe)). EWS-FLI1 promoter occupancy was investigated using chromatin immunoprecipitation real-time PCR (ChIP-RTPCR). The effect of drug treatment on expression of genes controlled by EWS-FLI1 was evaluated by quantitative real-time PCR (qRT-PCR). The effect of treatment on cell cycle distribution was also compared among analogs. In vitro efficacy was evaluated by estimating GI50 parameters (72-hr). In addition, the maximum tolerated dose (MTD) and the effect of treatment on plasma total-calcium were used to assess relative toxicity in mice. Results: EWS-FLI1 promoter occupancy upstream from Nr0b1, Tgfbr2, and Rcor1 genes was evaluated in Ewing sarcoma cells (TC32) by ChIP-RTPCR. MTM and MTM-SA-Trp analog destabilized FLI1 binding to all three promoters and MTM-SA-Trp was shown to be the most destabilizing. Comparatively, MTM-SK appears to mostly stabilize FLI1. Additionally, qRTPCR showed that MTM and its analogs efficiently down-regulated mRNA expression in a dose-dependent manner (rank-order of efficiency: MTM-SA-Trp>MTM = MTM-SK). These data were in accord with the in vitro cytotoxicity data that show MTM-SA-Trp has relatively higher potency (lower GI50) among Ewing cell lines (n = 8) as compared to other analogs. Furthermore, the effect of drug treatment appears to lead to differences in cell-cycle progression. MTM and MTM-SK treated TC32 cells were primarily in G1/G2 phase, whereas MTM-SA-Trp treated cells showed increased S-phase accumulation. Compared to MTM, mice tolerated significantly higher single doses of the MTM analogs. Repeated dosing showed similar results except that MTM-SA-Trp was tolerated at lower doses. MTM treatment caused an acute drop in total-calcium, which also occurred with MTM-SA-Trp and MTM-SA-Phe analogs three days later within the two-week treatment. Comparatively, MTM-SK caused an increase in total-calcium. In all cases, total-calcium concentrations returned to baseline within two weeks following treatment. Conclusion: The work presented here demonstrates the ability to design more specific and less toxic analogs of MTM. Development of such analogs could lead to successful treatments of Ewing sarcoma. Citation Format: Joseph Eckenrode, Jamie Horn, Jhong-Min Chen, Jurgen Rohr, Markos Leggas. Mithramycin analogs with reduced toxicity for EWS-FLI1 targeting. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2628. doi:10.1158/1538-7445.AM2015-2628

A method for the quantitation of DB-67 ((20S)-10-hydroxy-7-tert-butyldimethylsilylcamptothecin) lactone and carboxylate in mouse plasma has been developed, validated, and applied in pharmacokinetic studies. The analytes were separated by reversed-phase chromatography with fluorescence detection. Validation demonstrated the selectivity and specificity for the carboxylate and lactone, with linearity between 1-300ng/mL and 2.5-300ng/mL for the carboxylate and lactone, respectively (accuracy 90-110% of theory and coefficient of variation < or =5.7%). Carboxylate to lactone conversion was

The number of new cases of primary malignant brain tumors diagnosed in 2003 more than double the number of diagnosed cases of Hodgkin’s disease, approximately 13,100 deaths were estimated to have occurred as a result of primary cancer of the central nervous system (CNS). Metastases to the brain from a systemic primary cancer are even more common. One estimate suggests that more than 100,000 patients per year die with symptomatic intracranial metastases. In addition, if benign cases are considered, approximately 36,000 brain tumors would have been diagnosed in 2002. The age- and sex-adjusted incidence of primary tumors of the CNS is estimated to be approximately 19 per 100,000 persons per year (11.8 per 100,000 for symptomatic tumors and 7.3 per 100,000 for asymptomatic tumors) (1).

The role of the multidrug resistance protein MRP4/ABCC4 in vivo remains undefined. To explore this role, we generated Mrp4-deficient mice. Unexpectedly, these mice showed enhanced accumulation of the anticancer agent topotecan in brain tissue and cerebrospinal fluid (CSF). Further studies demonstrated that topotecan was an Mrp4 substrate and that cells overexpressing Mrp4 were resistant to its cytotoxic effects. We then used new antibodies to discover that Mrp4 is unique among the anionic ATP-dependent transporters in its dual localization at the basolateral membrane of the choroid plexus epithelium and in the apical membrane of the endothelial cells of the brain capillaries. Microdialysis sampling of ventricular CSF demonstrated that localization of Mrp4 at the choroid epithelium is integral to its function in limiting drug penetration into the CSF. The topotecan resistance of cells overexpressing Mrp4 and the polarized expression of Mrp4 in the choroid plexus and brain capillary endothelial cells indicate that Mrp4 has a dual role in protecting the brain from cytotoxins and suggest that the therapeutic efficacy of central nervous system-directed drugs that are Mrp4 substrates may be improved by developing Mrp4 inhibitors.

We developed a chromatography method to measure lactone and carboxylate forms of topotecan (TPT) in mouse cerebrospinal fluid (CSF) using microdialysis sampling. The chromatography method utilized a microbore (0.8 mm) column. Analytes, which eluted in less than 5 min, were detected with a fluorescence detector. The calibration range was 0.25-100.0 ng/mL for both forms. The within-day and between-day precision wasor =16% for 0.8 ng/mL andor =8.0% for 3, 12, and 80 ng/mL. Accuracy was +/-15% (0.8 and 3 ng/mL) and +/-10% (12 and 80 ng/mL). TPT lactone hydrolyzes to the carboxylate during sampling, so we developed an equation and parameters to describe the TPT lactone hydrolysis in artificial CSF (aCSF). After TPT administration, CSF dialysate samples (2 microL) were analyzed for lactone and carboxylate using online injection. The hydrolysis of each dialysate sample was then estimated and a correction applied. We conclude that this HPLC method coupled with online microdialysis sampling allows for the rapid measurement of both TPT forms in small volumes of murine CSF dialysate. The system allows for the determination of TPT pharmacokinetics in murine CSF and provides a tool to extend pharmacological studies in this brain compartment.

Irofulven is a novel, small molecular weight semisynthetic compound, derived from a family of mushroom toxins known as illudins. This DNA alkylating agent has a chemical structure unlike any other chemotherapeutic agent in clinical use. The molecule is currently being studied in several Phase I, II, and III trials. The objectives of this study were to evaluate the antitumor activity of Irofulven in a panel of 20 pediatric solid tumor xenografts and to relate the Irofulven systemic exposure, defined as area under the concentration time curve, to the antitumor dose associated with tumor regression in the tumor models. Irofulven was administered i.v. daily for 5 days with courses repeated every 21 days for a total of three cycles. The minimum effective dose of Irofulven causing objective regression (or =50% volume regression) of advanced tumors was determined for each of 19 of 20 independently derived tumor models (12 brain tumors, 4 neuroblastomas, and 4 rhabdomyosarcomas). At the maximum tolerated dose for three cycles of treatment (4.6 mg/kg/day) objective regressions were determined in 14 of 18 tumor lines (78%). However, the dose-response relationship was acute. At 2 mg/kg only 3 of 15 tumors tested demonstrated objective regressions, and in 3 additional tumors volume regressions were not achieved at a higher dose level (3 mg/kg), hence were not additionally tested. After administering the maximum tolerated dose (tolerated for one or two cycles of treatment) of Irofulven, 7 mg/kg, to mice bearing sensitive and resistant human tumors plasma concentration-time profiles were determined. Tumors were highly sensitive to Irofulven, but the systemic exposure required for a significant rate of objective response in this panel of tumors is in excess of that achievable in patients at tolerable doses, using this schedule of drug administration.

Statins are established as safe and effective anti-hypercholesterolemia drugs. Preclinical evidence shows that statins exhibit antitumor activity against different tumors by reducing prenylation of small GTPase molecules, which play a critical role in intracellular cancer cell signaling. However, these effects have only been noted at higher concentrations, which cannot be achieved with the typical dosing. In a Phase I trial, simvastatin was well tolerated by patients with relapsed or refractory myeloma or lymphomas at amaximum tolerated dose (MTD) of 7.5mg/kg twice daily. However, the pharmacokinetic (PK) was not defined and it is unknown if simvastatin plasma concentrations can reach the levels necessary for the antitumor activity observed in vitro. Here we evaluated the PK, safety and efficacy of high dose simvastatin in the context of a pilot trial enrolling patients with recurrent and refractory CLL. Methods: Patients received simvastatin orally at 7.5mg/kg twice a day for 7 days during a 21 day cycle for 6 cycles. During study treatment, patients underwent weekly or bi-weekly evaluations that included physical examination, complete blood counts and comprehensive chemistry profiles. Toxicities were graded according to the National Cancer Institute Common Toxicity Criteria (version 3.0). The NCI CLL revised guidelines for diagnosis and treatment were utilized to determine the level of clinical response. Blood samples were collected during cycle 1 at predose, 15 min and 1,2,3,6,8, and 12 hr and at predose on day-7. Simvastatin lactone and acid concentrations were measured in plasma and peripheral blood mononuclear cells (PBMCs) using a validated HPLC-MS/MS assay. Biochemical assays assessing the apoptotic effects of simvastatin were also performed in CLL cell samples (the predose and day-7) obtained from the treated patients. Results: Patients were accrued in this pilot trial. High dose simvastatin was well tolerated and there were no signs of serious adverse effects. Simvastatin exposure was dose proportional (AUC and Cmax) as compared to the doses used to treat hyperlipidemia. Exposure was lower than that required for in vitro cytotoxicity against immortalized cells. Simvastatin exposure in both plasma and PBMCs was variable with an AUC12hr (%CV) of 800.6 hr*ng/ml (88.6%) and 11.03 hr*ng/mg (119.4%), respectively. Following 7 days exposure to high dose simvastatin, CLL cells had increased the expression of cleaved PARP, an apoptotic marker. Conclusion: Preliminary results show that high dose simvastatin is well tolerated with no signs of serious side effects. Although plasma concentrations were lower than that required for in-vitro cytotoxicity, the simvastatin concentrations achieved in the clinic induced apoptosis in patient CLL cells. Further studies in CLL patients are warranted to demonstrate its efficacy alone or in combination therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3778. doi:1538-7445.AM2012-3778

Background: A new library of small molecules with structural features resembling combretastatin analogs was synthesized and two analogues were chosen by the NCI for screening. Both agents exhibited activity in all NCI-60 panel cell lines with GI50 values Methods: Biochemical assays to assess the interaction with tubulin were carried out using purified tubulin. Immunofluorescence staining for beta-tubulin was also evaluated in PC3 cells following drug treatment. Vinblastine and docetaxel were used to demonstrate tubulin destabilizing and stabilizing modes of action, respectively. Cytotoxicity assays (72 hour GI50) were carried out in PC3, DU145, LNCaP and C4/2 prostate cancer cells and in PC3-DR cells (docetaxel resistant PC3 cells). To assess the effect of P-gp, we used NCI/ADR-Res ovarian cancer cells and L-MDR1 porcine epithelial cells and their non-expressing parent lines. Assays were performed in 96-well plates using AlamarBlue cell viability dye. Cell-cycle analysis was performed using propidium iodide staining and flow activated cell sorting. Western blot analyses were used to assess the effect of drug treatment on apoptotic pathway and on cell cycle proteins. Results: The GI50 in all cell lines was in the 1–10 nM range with no apparent resistance in P-gp expressing cells or in docetaxel resistant PC3-DR cells. The lead combretastatin analog showed a concentration dependent antitubulin interaction with a mode of action similar to vinblastine. This suggests that these compounds act by interfering with the microtubule polymerization process. Immunofluorescence staining for beta-tubulin showed diffuse staining patterns with drug increased concentration suggesting the lack of microtubule formation. Cell-cycle analysis demonstrated a G2/M cell cycle accumulation after 24-hr treatment with drug. Interestingly, there was no drug dependent effect on the number of cells in the sub-G1 fraction suggesting that the mode of cell death may be different than apoptosis. This was corroborated with the lack of caspase activation as well as the lack of cleaved-PARP. A concentration dependent p-Bcl2 was noted, however, suggesting that the drug interferes with the anti-apoptotic role of Bcl-2. Nuclear staining with Hoechst 33342 and DAPI demonstrated the presence of multinucleated cells suggesting that mitotic catastrophe could be the primary mode of cell death. This was further corroborated by the concentration dependent centrosome overduplication and presence of multiple spindle poles. Conclusion: These exquisitely potent antitubulin agents appear to overcome multiple modes of drug resistance including P-glycoprotein, p-53 deregulation, and Bcl-2. Further studies are ongoing to verify the exact molecular mechanism mediating cell death and to determine the in-vivo activity of these novel compounds. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C216.

Background: Tipifarnib, a farnesyl transferase inhibitor, was initially designed to prevent Ras farnesylation and its subsequent membrane localization, which is necessary for Ras mediated signaling. Although, the clinical effectiveness of tipifarnib as a single agent in solid tumors was limited, it was modest in hematologic malignancies. Alternative prenylation by geranylgeranyl transferase has been postulated as an escape mechanism by which Ras evades the effect of tipifarnib. Simvastatin, an anti-hyperlipidemic drug that inhibits 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase, has been shown in several studies to induce apoptosis in cancer cells through blockade of the geranylgeranylation pathway of small GTPases. Thus, we hypothesized that simvastatin can augment the cytotoxic effect of tipifarnib by blocking the alternative Ras prenylation pathway. Our studies were carried out in a panel of leukemia cell lines. Methods: Studies were carried out using five cell lines of varied leukemic origin including HL60, K562, Molt4, Jurkat and HSB2. Cells were exposed to increasing drug concentrations for 72 hours either as a single or combination treatment with simvastatin and tipifarnib. Cell viability was measured using AlamarBlue (Invitrogen) and the combination index values were calculated based on the Chou-Talalay equations. Apoptosis was evaluated using Annexin V assay. Biochemical assays assessing the expression of caspases, c-PARP and Ras, in both membrane and cytosolic fractions, were also used. Results: The combination of tipifarnib with simvastatin was shown to induce a synergistic cytotoxic effect in all cell lines tested. Notably, our results showed that coadministarion of simvastatin and tipifarnib resulted in the induction of apoptosis through the activation of several apoptotic markers including Caspase 3, 7 and 9 as well as the upregulation of cleaved PARP. These effects were accompanied by disruption of Ras membrane localization and its sequestration into the cytosol. Conclusions: These findings demonstrate that combining simvastatin with tipifarnib effectively disrupts Ras prenylation and induces apoptosis in human leukemia cell lines. These effects were observed with simvastatin and tipifarnib concentrations that can be achieved in the clinic. Thus, the effectiveness of this combination should be explored further in future clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A78.

BACKGROUND: The overall survival and disease-free rates for Acute Myelogenous Leukemia (AML) patients treated with the gold-standard therapy comprised of Cytarabine (Ara-C) and Daunorubicin (DNR) remain significantly low. Camptothecins are potent anticancer drugs that confer their cytotoxic effect by interfering with Topoisomerase-I (Top1). Semi-synthetic camptothecin analogues have been used in the past to treat AML patients with relatively low response rates. Here, we examined the anti-leukemia activity of AR-67, a novel camptothecin analogue, alone or in combination with standard agents for the treatment of AML. METHODS: The anti-leukemia activity of AR-67, TPT, Ara-C and DNR was studied in HL60, KG1 and K562 cells and in primary leukemia cell cultures after exposure to increasing drug concentrations for 48 hours. Cell proliferation was determined using a colorimetric assay (MTT) assay and IC50s were obtained using nonlinear-regression analysis. Drug combination treatments were assessed for synergy. The expression of Top1, phosphohistone (γ-H2AX), Bcl-2 and c-PARP were evaluated using biochemical assays. RESULTS: The potency of AR-67 was in the nM range in the leukemic cell lines. Consistent with the lower proliferation rates of primary cells grown in culture, their IC50 values were variable and in the µM range. IC50s estimated for TPT were higher than the respective ones for AR-67. The IC50 values for Ara-C and DNR were in the μM range for the HL60, K562 and primary cells, and higher than the ones for AR-67. However, the KG-1 cell line appeared to be more sensitive to the AML gold standard therapy, but not as sensitive as to AR-67. AR-67 treatment decreased Top1 expression, increased γ-H2AX expression in a concentration-dependent manner suggesting removal of DNA-Top1 complexes and formation of double strand DNA breaks. Induction of apoptosis was manifested by decreased Bcl-2 and increased c-PARP expression suggesting a caspace-3 mediated effect. AR-67 combination treatments that included DNR and Ara-C showed synergy in HL60 cells. Additionally, pretreatment with Ara-C revealed synergy between AR-67 and DNR in both the KG1 and K562 cells, accompanied by increased γ-H2AX and c-PARP expression. CONCLUSIONS: Our results indicate AR-67 as a potent anticancer agent that induces DNA damage and apoptosis. Using in vitro leukemia models we have shown that AR-67 is a potentially efficacious agent when combined with standard agents for the treatment of AML and may benefit AML patients resistant to Ara-C. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3547. doi:10.1158/1538-7445.AM2011-3547

PURPOSE. To compare the expression of multidrug-resistant proteins in retinoblastoma tumors among eyes treated with primary enucleation. METHODS. A group of 18 patients with unilateral retinoblastoma with advanced intraocular disease was selected for the study. All patients had undergone primary enucleation. A histologic specimen from each patient was retrieved from the pathology archives and a tissue gene microarray was constructed (0.6 X 3-4 mm). Standard immunohistochemical techniques were used to study the tissue microarrays for the expression of the ATP-binding cassette (ABC) transporters: breast cancer resistance protein (BCRP; ABCG2), multidrug-resistant protein 1/P-glycoprotein (MDR1/Pgp; ABCB1), multidrug-resistant-associated protein 1 (MRP1; ABCC1), MRP2 (ABCC2), and MRP4 (ABCC4). RESULTS. Of the 18 specimens retrieved, 16 had adequate tissue for study. MRP1 was expressed in 8 (50%) of 16 tumors, and MRP2 was expressed in 5 (31%) of 16 tumors. MDR1/Pgp was found in 2 (12%) of 16 retinoblastomas. MRP4 and BCRP were not detected in any of the tumors studied. CONCLUSIONS. The results show that multiple ABC transporters were present in a cohort of sporadic patients with unilateral retinoblastoma who underwent primary enucleation. Studies are planned of the expression of ABC transporters in eyes treated by chemotherapy and/or radiation as a comparison with this group.