Markos Leggas

Mrp4 Confers Resistance to Topotecan and Protects the Brain from Chemotherapy

ABSTRACT 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.

Gefitinib enhances the antitumor activity and oral bioavailability of irinotecan in mice.

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.

Gefitinib Modulates the Function of Multiple ATP-Binding Cassette Transporters In vivo

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 micromol/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.

Synthesis and evaluation of a series of benzothiophene acrylonitrile analogs as anticancer agents

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.

DEPENDENCE OF NELFINAVIR BRAIN UPTAKE ON DOSE AND TISSUE CONCENTRATIONS OF THE SELECTIVE P-GLYCOPROTEIN INHIBITOR ZOSUQUIDAR IN RATS

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 ∼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.

Nanoparticles Containing Anti-inflammatory Agents as Chemotherapy Adjuvants II: Role of Plasma Esterases in Drug Release

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.

Nanotemplate-engineered nanoparticles containing gadolinium for magnetic resonance imaging of tumors.

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 GdCl3, 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 Gd3+ from nanoparticles with Cu2+ and Zn2+ 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 (r1) and transverse relaxivity (r2) 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 Clsys = 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 T1 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.

Morphine Versus Clonidine for Neonatal Abstinence Syndrome

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.

A Phase I Study of 7-t-Butyldimethylsilyl-10-Hydroxycamptothecin in Adult Patients with Refractory or Metastatic Solid Malignancies

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

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 ATP-binding cassette transporter family G member 2 (Abcg2) knockout model to study nitrofurantoin transfer into milk.

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 μg/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 (p < 0.05), 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; p < 0.05). 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 ABCG2s role in drug transfer into milk during breastfeeding.