Briefing on Investigational Chimeric Antigen Receptor T-Cell Immunotherapies in Pediatric Neoplasms.

The promising results seen in the treatment of refractory hematologic malignancies with tisagenlecleucel (Kymriah), the pioneering Chimeric Antigen Receptor (CAR) T-cell immunotherapy, has stimulated a fast growth in research and clinical testing of novel CAR-T constructs, targets, and various generations of CAR T-cells. Pharmacists may receive inquiries about active clinical trials or may be contributing to the care of patients participating in these studies. This briefing discusses the on-going clinical trials that explore novel CAR T-cell immunotherapies in pediatric refractory malignancies of hematologic and solid organ origins. It can be valuable in assisting practitioners in navigating the rapidly evolving field of CAR T-cell immunotherapies.

The Positive Association between Proton Pump Inhibitors and Clostridium Difficile Infection.

BACKGROUND: Proton pump inhibitors (PPIs) are among the most commonly used medications by patients due to its availability over the counter and frequent prescribing by physicians to treat and alleviate symptoms of gastroesophageal reflux disease. Recently, the FDA issued a warning with respect to the utilization of PPIs and risk of developing Clostridium difficile infections (CDI). The most commonly known medications to cause CDI are antibiotics. However, available studies suggest an association and increase in risk for CDI with PPI use as well. OBJECTIVE: The purpose of this research is to review and summarize data currently available on the association between PPIs and CDI. METHODS: To search for eligible studies, EBSCO engines were investigated using proton pump inhibitors or PPIs and Clostridium difficile or C. diff. as search terms. Meta analyses and systematic reviews published between 2000 and 2020 on adult patients were considered. RESULTS: Eight meta-analyses and systematic reviews met the inclusion criteria. They included studies conducted in the US, Europe, Asia and Canada on inpatient and outpatient adults. The final result for all 8 studies showed a statistically significant association between PPIs and CDI ranging from mild to high risk. CONCLUSION: Currently available data suggest a positive association between PPIs and CDI.

Team-Based Approach to Management of Hypertension Associated with Angiogenesis Inhibitors.

Angiogenesis inhibitors, also known as vascular endothelial growth factor (VEGF) or vascular signaling pathway (VSP) inhibitors, have improved care of neoplastic diseases over the past decade. However, cardiovascular toxicities associated with these agents, such as hypertension and less commonly left ventricular systolic dysfunction and heart failure, have often been a limiting factor for continued use. Balancing the benefits of these agents with the associated toxicities is critical to ensure these therapies do not negatively impact oncological outcomes. The care of cancer patients with cardiovascular risks is challenging due to the heterogeneity of cardiovascular complications, paucity of evidence-based guidelines, and lack of channels for collaboration among healthcare providers. Herein, we provide a team-based approach for treatment of angiogenesis inhibitor-induced hypertension along with recommendations on monitoring and appropriate selection of anti-hypertensive agents.

Chimeric antigen receptor T-cell therapy: Foundational science and clinical knowledge for pharmacy practice.

The Food and Drug Administration has recently approved two autologous chimeric antigen receptor T-cell immunotherapies tisagenlecleucel (Kymriah™) and axicabtagene ciloleucel (Yescarta™) for patients with advanced lymphocytic malignancies. Both immunotherapies target the CD19-positive B-cell neoplasms. Kymriah™ is indicated for the treatment of relapsed or refractory acute lymphoblastic leukemia and large B-cell lymphoma. Yescarta™ is indicated for lymphoma only. Although the new therapy offers a promise for patients with advanced disease, it is associated with adverse events including neurotoxicity and cytokine release syndrome, which can be fatal and may require a high level of multidisciplinary supportive care. Due to the risks, both Kymriah™ and Yescarta™ are subject to Risk Evaluation and Mitigation Strategy (REMS) protocols. As active members of multidisciplinary clinical teams, pharmacists are likely to be responsible for the execution of Kymriah™ and Yescarta™ REMS programs. This manuscript describes foundational science and clinical knowledge of chimeric antigen receptor T-cell immunotherapies, common therapy-specific toxicities and REMS requirements for Kymriah™ and Yescarta™ in relation to practice of pharmacy.

Shared Decision-Making and Women's Adherence to Breast and Cervical Cancer Screenings.

We examined the effect of shared decision-making (SDM) on women's adherence to breast and cervical cancer screenings and estimated the prevalence and adherence rate of screenings. The study used a descriptive cross-sectional design using the 2017 Health Information National Trends Survey (HINTS) data collected by the National Cancer Institute. Adherence was defined based on the guidelines from the American Cancer Society and the composite measure of shared decision-making was constructed using three items in the data. Multivariable logistic regression was performed to examine the association between the SDM and adherence, controlling for cancer beliefs and socio-demographic variables. The analysis included 742 responses. Weighted to represent the U.S. population, 68.1% adhered to both breast and cervical cancer screening guidelines. The composite measure of SDM was reliable (α = 0.85), and a higher SDM score was associated with women's screening adherence (b = 0.17; p = 0.009). There were still women who did not receive cancer screenings as recommended. The results suggest that the use of the SDM approach for healthcare professionals' communication with patients can improve screening adherence.

Acute treatment with doxorubicin affects glutamate neurotransmission in the mouse frontal cortex and hippocampus.

Doxorubicin (DOX) is a potent chemotherapeutic agent known to cause acute and long-term cognitive impairments in cancer patients. Cognitive function is presumed to be primarily mediated by neuronal circuitry in the frontal cortex (FC) and hippocampus, where glutamate is the primary excitatory neurotransmitter. Mice treated with DOX (25mg/kg i.p.) were subjected to in vivo recordings under urethane anesthesia at 24h post-DOX injection or 5 consecutive days of cognitive testing (Morris Water Maze; MWM). Using novel glutamate-selective microelectrode arrays, amperometric recordings measured parameters of extracellular glutamate clearance and potassium-evoked release of glutamate within the medial FC and dentate gyrus (DG) of the hippocampus. By 24h post-DOX injection, glutamate uptake was 45% slower in the FC in comparison to saline-treated mice. In the DG, glutamate took 48% longer to clear than saline-treated mice. Glutamate overflow in the FC was similar between treatment groups, however, it was significantly increased in the DG of DOX treated mice. MWM data indicated that a single dose of DOX impaired swim speed without impacting total length traveled. These data indicate that systemic DOX treatment changes glutamate neurotransmission in key nuclei associated with cognitive function within 24h, without a lasting impact on spatial learning and memory. Understanding the functional effects of DOX on glutamate neurotransmission may help us understand and prevent some of the debilitating side effects of chemotherapeutic treatment in cancer survivors.

A Team-based Assignment to Integrate Basic Science and Pharmacotherapeutic Principles for Anticancer Agents.

Objective. To implement and evaluate an active-learning, team-based assignment centered on anticancer agents for the integration of basic science and pharmacotherapeutic principles. Methods. Student teams were assigned a specific anticancer agent and were expected to answer a series of questions on the written section of the assignment, followed by a presentation to the class. Each assignment was assessed using a grading rubric that was mapped to the 2013 CAPE educational outcomes. Student perceptions of the assignment were assessed using a short survey. Results. Student cohort performance on the assignment was in the B range (83%) with a mean of 33.2 out of 40. Using the grading rubric, the 12 student cohorts performed particularly well under professionalism (Domain 4.4) that focuses on personal and professional development from CAPE 2013 with means >4 on a 1-5 scale. Student impressions of the assignment suggested that students believed the assignment had a positive effect on their learning and should be continued. Conclusion. The assignment provided a focused review of basic science and pharmacotherapeutic principles and enabled integration of concepts relating to the therapeutic application of anticancer agents, and management of anticancer agent mediated adverse effects. The assignment could contribute toward preparing students for the evolving role of the pharmacist in the management of cancer.

Drug interactions with aprepitant or fosaprepitant: Review of literature and implications for clinical practice.

Purpose Aprepitant and its parenteral formulation fosaprepitant are widely used for the prevention of chemotherapy-induced nausea and vomiting. Aprepitant exerts modest inhibitory effect on CYP3A4 and modest inductive effect on CYP2C9 substrates such as some antineoplastics and multiple other medications. This article is aimed to provide pharmacists and other healthcare professionals with an updated summary of drug-drug interactions of aprepitant/fosaprepitant and implications for clinical practice. Method We reviewed publications reporting drug-drug interactions between aprepitant/fosaprepitant and other medications. Results Coadministration of aprepitant with antineoplastics or opiods may result in significant elevations in the serum levels of the agents metabolized via CYP3A4, with the best documentation for cyclophosphamide, ifosfamide, erlotinib and oxycodone. These alterations did not translate into adverse outcomes and/or necessitate dosing adjustments. The levels of warfarin were significantly decreased by aprepitant requiring prolonged monitoring after discontinuation of aprepitant. Among direct oral anticoagulants, a theoretical interaction between aprepitant and rivaroxaban or apixaban exists. Interactions between aprepitant and quetiapine or diltiazem or sirolimus required dose reductions to avoid adverse outcomes. The intravenous route had a weaker inhibitory effect on CYP3A4 than the oral pathway. Conclusion The evidence on drug interactions of aprepitant with other medications is limited, and the impact on therapeutic outcomes remains to be determined. The intravenous regimen may be a preferred option. As utilization of aprepitant is expanding, practitioners and patients need to be educated about the potential for drug interactions and a need for careful monitoring of patients concurrently receiving aprepitant and CYP2C9 or CYP3A4 substrates, especially those with a narrow therapeutic window.

Elevated glutathione is not sufficient to protect against doxorubicin-induced nuclear damage in heart in multidrug resistance-associated protein 1 (Mrp1/Abcc1) null mice.

Cardiotoxicity is a major dose-limiting adverse effect of doxorubicin (DOX), mediated in part by overproduction of reactive oxygen species and oxidative stress. Abcc1 (Mrp1) mediates the efflux of reduced and oxidized glutathione (GSH, GSSG) and is also a major transporter that effluxes the GSH conjugate of 4-hydroxy-2-nonenal (HNE; GS-HNE), a toxic product of lipid peroxidation formed during oxidative stress. To assess the role of Mrp1 in protecting the heart from DOX-induced cardiac injury, wild-type (WT) and Mrp1 null (Mrp1(-/-)) C57BL/6 littermate mice were administered DOX (15 mg/kg) or saline (7.5 ml/kg) i.v., and heart ventricles were examined at 72 hours. Morphometric analysis by electron microscopy revealed extensive injuries in cytosol, mitochondria, and nuclei of DOX-treated mice in both genotypes. Significantly more severely injured nuclei were observed in Mrp1(-/-) versus WT mice (P = 0.031). GSH and the GSH/GSSG ratio were significantly increased in treatment-naïve Mrp1(-/-) versus WT mice; GSH remained significantly higher in Mrp1(-/-) versus WT mice after saline and DOX treatment, with no changes in GSSG or GSH/GSSG. GS-HNE, measured by mass spectrometry, was lower in the hearts of treatment-naïve Mrp1(-/-) versus WT mice (P < 0.05). DOX treatment decreased GS-HNE in WT but not Mrp1(-/-) mice, so that GS-HNE was modestly but significantly higher in Mrp1(-/-) versus WT hearts after DOX. Expression of enzymes mediating GSH synthesis and antioxidant proteins did not differ between genotypes. Thus, despite elevated GSH levels in Mrp1(-/-) hearts, DOX induced significantly more injury in the nuclei of Mrp1(-/-) versus WT hearts.

The metalloporphyrin antioxidant, MnTE-2-PyP, inhibits Th2 cell immune responses in an asthma model.

MnTE-2-PyP, a superoxide dismutase mimetic, inhibited OVA-induced airway inflammation in mice suggesting an effect on Th2 responsiveness. Thus, we hypothesized that MnTE-2-PyP may alter dendritic cell-Th2 interactions. Bone marrow derived dendritic cells (DC) and OVA(323-339)-specific Th2 cells were cultured separately in the presence or absence of MnTE-2-PyP for 3 days prior to the co-culturing of the two cell types in the presence of an OVA(323-339) peptide and in some cases stimulated with CD3/CD28. MnTE-2-PyP-pretreated DC inhibited IL-4, IL-5 and IFNγ production and inhibited Th2 cell proliferation in the DC-Th2 co-culturing system in the presence of the OVA(323-339) peptide. Similar results were obtained using the CD3/CD28 cell-activation system; the addition of MnTE-2-PyP inhibited Th2 cell proliferation. MnTE-2-PyP suppressed CD25 expression on OVA-specific Th2 cells, which implied that MnTE-2-PyP can inhibit the activation of Th2 cells. MnTE-2-PyP also down-regulated co-stimulatory molecules: CD40, CD80 and CD86 on immature DC. Our studies suggest that the major mechanism by which MnTE-2-PyP inhibits airway inflammation is by acting on the DC and suppressing Th2 cell proliferation and activation.

The G671V variant of MRP1/ABCC1 links doxorubicin-induced acute cardiac toxicity to disposition of the glutathione conjugate of 4-hydroxy-2-trans-nonenal.

OBJECTIVE: Doxorubicin-induced acute cardiotoxicity is associated with the Gly671Val (G671V; rs45511401) variant of multidrug resistance-associated protein 1 (MRP1). Doxorubicin redox cycling causes lipid peroxidation and generation of the reactive electrophile, 4-hydroxy-2-trans-nonenal (HNE). Glutathione forms conjugates with HNE, yielding an MRP1 substrate, GS-HNE, whose intracellular accumulation can cause toxicity. METHODS: We established stable HEK293 cell lines overexpressing wild-type MRP1 (HEKMRP1), G671V (HEKG671V), and R433S (HEKR433S), a variant not associated with doxorubicin-induced cardiotoxicity and investigated the sensitivity of HEKG671V cells to doxorubicin and transport capacity of G671V toward GS-HNE. RESULTS: In ATP-dependent transport studies using plasma membrane-derived vesicles, the Vmax (pmol/min/mg) for GS-HNE transport was the lowest for G671V (69±4) and the highest for R433S (972±213) compared with wild-type MRP1 (416±22), whereas the Km values were 2.8±0.4, 6.0 or more, and 1.7±0.2 µmol/l, respectively. In cells, the doxorubicin IC50 (48 h) was not different in HEKMRP1 (463 nmol/l) versus HEKR433S (645 nmol/l), but this parameter was significantly lower in HEKG671V (181 nmol/l). HEKG671V retained significantly (approximately 20%) more, whereas HEKR433S retained significantly less intracellular doxorubicin than HEKMRP1. Similarly, HEKG671V cells treated with 1.5 µmol/l of doxorubicin for 24 h retained significantly more GS-HNE. In cells treated with 0.5 µmol/l of doxorubicin for 48 , glutathione and glutathione disulfide levels and the glutathione/glutathione disulfide ratio were significantly decreased in HEKG671V versus HEKMRP1; these values were similar in HEKR433S versus HEKMRP1. CONCLUSION: These data suggest that decreased MRP1-dependent GS-HNE efflux contributes to increased doxorubicin toxicity in HEKG671V and potentially in individuals carrying the G671V variant.

p53 Regulates oxidative stress-mediated retrograde signaling: a novel mechanism for chemotherapy-induced cardiac injury.

The side effects of cancer therapy on normal tissues limit the success of therapy. Generation of reactive oxygen species (ROS) has been implicated for numerous chemotherapeutic agents including doxorubicin (DOX), a potent cancer chemotherapeutic drug. The production of ROS by DOX has been linked to DNA damage, nuclear translocation of p53, and mitochondrial injury; however, the causal relationship and molecular mechanisms underlying these events are unknown. The present study used wild-type (WT) and p53 homozygous knock-out (p53(-/-)) mice to investigate the role of p53 in the crosstalk between mitochondria and nucleus. Injecting mice with DOX (20 mg/kg) causes oxidative stress in cardiac tissue as demonstrated by immunogold analysis of the levels of 4-hydroxy-2'-nonenal (4HNE)-adducted protein, a lipid peroxidation product bound to proteins. 4HNE levels increased in both nuclei and mitochondria of WT DOX-treated mice but only in nuclei of DOX-treated p53((-/-)) mice, implicating a critical role for p53 in causing DOX-induced oxidative stress in mitochondria. The stress-activated protein c-Jun amino-terminal kinase (JNKs) was activated in response to increased 4HNE in WT mice but not p53((-/-)) mice receiving DOX treatment, as determined by co-immunoprecipitation of HNE and pJNK. The activation of JNK in DOX treated WT mice was accompanied by Bcl-2 dissociation from Beclin in mitochondria and induction of type II cell death (autophagic cell death), as evidenced by an increase in LC3-I/LC-3-II ratio and γ-H2AX, a biomarker for DNA damage. The absence of p53 significantly reduces mitochondrial injury, assessed by quantitative morphology, and decline in cardiac function, assessed by left ventricular ejection fraction and fraction shortening. These results demonstrate that p53 plays a critical role in DOX-induced cardiac toxicity, in part, by the induction of oxidative stress mediated retrograde signaling.

2-Mercaptoethane sulfonate prevents doxorubicin-induced plasma protein oxidation and TNF-α release: implications for the reactive oxygen species-mediated mechanisms of chemobrain.

Doxorubicin (DOX), an anthracycline used to treat a variety of cancers, is known to generate intracellular reactive oxygen species. Moreover, many patients who have undergone chemotherapy complain of cognitive dysfunction often lasting years after cessation of the chemotherapy. Previously, we reported that intraperitoneal administration of DOX led to elevated TNF-α and oxidative stress in the plasma and brain of mice. However, the mechanisms involved in nontargeted tissue damage remain unknown. In this study, we measured plasma oxidative stress and cytokine levels in patients treated with DOX. We observed increased plasma protein carbonylation and elevation of TNF-α 6 h after DOX administration in the context of multiagent chemotherapy regimens. Importantly, patients not treated coincidentally with 2-mercaptoethane sulfonate (MESNA) showed statistically significantly increased plasma protein-bound 4-hydroxynonenal, whereas those who had been coincidentally treated with MESNA as part of their multiagent chemotherapy regimen did not, suggesting that concomitant administration of the antioxidant MESNA with DOX prevents intravascular oxidative stress. We demonstrate in a murine model that MESNA suppressed DOX-induced increased plasma oxidative stress indexed by protein carbonyls and protein-bound HNE, and also suppressed DOX-induced increased peripheral TNF-α levels. A direct interaction between DOX and MESNA was demonstrated by MESNA suppression of DOX-induced DCF fluorescence. Using redox proteomics, we identified apolipoprotein A1 (APOA1) in both patients and mice after DOX administration as having increased specific carbonyl levels. Macrophage stimulation studies showed that oxidized APOA1 increased TNF-α levels and augmented TNF-α release by lipopolysaccharide, effects that were prevented by MESNA. This study is the first to demonstrate that DOX oxidizes plasma APOA1, that oxidized APOA1 enhances macrophage TNF-α release and thus could contribute to potential subsequent TNF-α-mediated toxicity, and that MESNA interacts with DOX to block this mechanism and suggests that MESNA could reduce systemic side effects of DOX.

Differential gene expression in liver and small intestine from lactating rats compared to age-matched virgin controls detects increased mRNA of cholesterol biosynthetic genes.

BACKGROUND: Lactation increases energy demands four- to five-fold, leading to a two- to three-fold increase in food consumption, requiring a proportional adjustment in the ability of the lactating dam to absorb nutrients and to synthesize critical biomolecules, such as cholesterol, to meet the dietary needs of both the offspring and the dam. The size and hydrophobicity of the bile acid pool increases during lactation, implying an increased absorption and disposition of lipids, sterols, nutrients, and xenobiotics. In order to investigate changes at the transcriptomics level, we utilized an exon array and calculated expression levels to investigate changes in gene expression in the liver, duodenum, jejunum, and ileum of lactating dams when compared against age-matched virgin controls. RESULTS: A two-way mixed models ANOVA was applied to detect differentially expressed genes. Significance calls were defined as a p < 0.05 for the overall physiologic state effect (lactation vs. control), and a within tissue pairwise comparison of p < 0.01. The proportion of false positives, an estimate of the ratio of false positives in the list of differentially expressed genes, was calculated for each tissue. The number of differentially expressed genes was 420 in the liver, 337 in the duodenum, 402 in the jejunum, and 523 in the ileum. The list of differentially expressed genes was in turn analyzed by Ingenuity Pathways Analysis (IPA) to detect biological pathways that were overrepresented. In all tissues, sterol regulatory element binding protein (Srebp)-regulated genes involved in cholesterol synthesis showed increased mRNA expression, with the fewest changes detected in the jejunum. We detected increased Scap mRNA in the liver only, suggesting an explanation for the difference in response to lactation between the liver and small intestine. Expression of Cyp7a1, which catalyzes the rate limiting step in the bile acid biosynthetic pathway, was also significantly increased in liver. In addition, decreased levels of mRNA associated with T-cell signaling were found in the jejunum and ileum. Several members of the Solute Carrier (SLC) and Adenosine Triphosphate Binding Cassette (ABC) superfamilies of membrane transporters were found to be differentially expressed; these genes may play a role in differences in nutrient and xenobiotic absorption and disposition. mRNA expression of SLC39a4_predicted, a zinc transporter, was increased in all tissues, suggesting that it is involved in increased zinc uptake during lactation. Microarray data are available through GEO under GSE19175. CONCLUSIONS: We detected differential expression of mRNA from several pathways in lactating dams, including upregulation of the cholesterol biosynthetic pathway in liver and intestine, consistent with Srebp activation. Differential T-Cell signaling in the two most distal regions of the small intestine (ileum and jejunum) was also noted, as well as differential expression of transporters that likely play a key role in nutrient uptake.

Hepatobiliary disposition of 3alpha,6alpha,7alpha,12alpha-tetrahydroxy-cholanoyl taurine: a substrate for multiple canalicular transporters.

Tetrahydroxy bile acids become major biliary bile acids in Bsep(-/-) mice and Fxr(-/-) mice fed cholic acid; we characterized disposition of these novel bile acids that also occur in patients with cholestasis. We investigated mouse Mrp2 (mMrp2) and P-glycoprotein [(P-gp) mMdr1a]-mediated transport of a tetrahydroxy bile acid, 6α-OH-taurocholic acid (6α-OH-TC), and its biliary excretion in wild-type and Mrp2(-/-) mice in the presence or absence of 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), a P-gp and breast cancer resistance protein inhibitor. 6α-OH-TC was rapidly excreted into bile of wild-type mice (78% recovery); coinfusion of GF120918 had no significant effect. In Mrp2(-/-) mice, biliary excretion was decreased (52% recovery) and coinfusion of GF120918 further decreased these values (34% recovery). In wild-type, but not Mrp2(-/-), mice, 6α-OH-TC increased bile flow 2.5-fold. Membrane vesicle transport studies of 6α-OH-TC (0.05-0.75 mM) yielded saturation kinetics with a higher apparent affinity for mMrp2 (K(m) = 0.13 mM) than for mMdr1a (K(m) = 0.33 mM); mBsep transported 6α-OH-TC with positive cooperativity (Hill slope = 2.1). Human multidrug resistance-associated protein (MRP) 2 and P-gp also transported 6α-OH-TC but with positive cooperativity (Hill slope = 3.6 and 1.6, respectively). After intraileal administration, the time course of 6α-OH-TC biliary recovery was similar to that of coinfused taurocholate, implying that 6α-OH-TC can undergo enterohepatic cycling. Thus, Mrp2 plays a key role in 6α-OH-TC biliary excretion, whereas P-glycoprotein plays a secondary role; Bsep likely mediates excretion of 6α-OH-TC in the absence of Mrp2 and P-gp. In Bsep(-/-) mice, efficient synthesis of tetrahydroxy bile acids that are Mrp2 and P-gp substrates can explain the noncholestatic phenotype.

Mrp1 localization and function in cardiac mitochondria after doxorubicin.

Multidrug resistance-associated protein 1 (Mrp1; Abcc1) is expressed in sarcolemma of murine heart, where it probably protects the cardiomyocyte by mediating efflux of endo- and xenobiotics. We used doxorubicin (DOX), a chemotherapeutic drug known to induce oxidative stress and thereby cardiac injury, as a model cardiotoxic compound and observed changes in the Mrp1 expression pattern in cardiac tissue of DOX-versus saline-treated mice. Confocal immunofluorescent and immunogold electron microscopy, together with subcellular fractionation followed by immunoblot analyses and transport measurements, localized functional Mrp1 to mitochondria after DOX. Expressions of Mrp1 in heart homogenate, sarcolemma, and submitochondrial particles (SMP) were increased 1.6-, 2-, and 3-fold, respectively, at 24 h after DOX. Mitochondrial Mrp1 expression was markedly increased 72 h after DOX, whereas transport of Mrp1 substrates in SMP was maximal at 24 h. ATP-dependent transport in SMP occurred into an osmotically sensitive space and was inhibited by the anti-MRP1 antibody QCRL3. Adduction of a 190-kDa protein with the reactive lipid peroxidation product 4-hydroxy-2-nonenal (HNE) was detected in SMP and was maximal at 72 h after DOX; immunoprecipitation confirmed Mrp1-HNE adduction. In vitro, HNE (10 muM) inhibited mitochondrial respiration and transport activity in SMP, suggesting that Mrp1 is adversely affected by oxidative stress. These data demonstrate that after DOX, functional Mrp1 is detected in mitochondria in addition to that in sarcolemma; however, adduction with HNE inhibits Mrp1 activity. Mrp1 may serve to protect the heart by mediating the efflux of toxic products of oxidative stress from mitochondria and cardiomyocytes.

Collateral damage in cancer chemotherapy: oxidative stress in nontargeted tissues.

Injury to nontargeted tissues in chemotherapy often complicates cancer treatment by limiting therapeutic dosages of anticancer drugs and by impairing the quality of life of patients during and after treatment. Oxidative stress, directly or indirectly caused by chemotherapeutics as exemplified by doxorubicin, is one of the underlying mechanisms of the toxicity of anticancer drugs in noncancerous tissues, including the heart and brain. A comprehensive understanding of the mechanisms of oxidative injury to normal tissue will be essential for the improvement of strategies to prevent or attenuate the toxicity of chemotherapeutic agents without compromising their chemotherapeutic value.

Increase in Mrp1 expression and 4-hydroxy-2-nonenal adduction in heart tissue of Adriamycin-treated C57BL/6 mice.

Multidrug resistance-associated protein 1 (MRP1) mediates the ATP-dependent efflux of endobiotics and xenobiotics, including estradiol 17-(beta-d-glucuronide), leukotriene C(4), and the reduced glutathione conjugate of 4-hydroxy-2-nonenal (HNE), a highly reactive product of lipid peroxidation. Adriamycin is an effective cancer chemotherapeutic drug whose use is limited by cardiotoxicity. Adriamycin induces oxidative stress and production of HNE in cardiac tissue, which may contribute to cardiomyopathy. We investigated the role of Mrp1 in Adriamycin-induced oxidative stress in cardiac tissue. Mice were treated with Adriamycin (20 mg/kg, i.p.), and heart homogenate and sarcolemma membranes were assayed for Mrp1 expression and ATP-dependent transport activity. Expression of Mrp1 was increased at 6 and 24 hours after Adriamycin treatment compared with saline treatment. HNE-adducted proteins were significantly increased (P < 0.001) in the homogenates at 6 hours after Adriamycin treatment and accumulated further with time; HNE adduction of a 190-kDa protein was evident 3 days after Adriamycin treatment. Mrp1 was localized predominately in sarcolemma as shown by confocal and Western blot analysis. Sarcolemma membrane vesicles transported leukotriene C(4) with a K(m) and V(max) of 51.8 nmol/L and 94.1 pmol/min/mg, respectively, and MK571 (10 micromol/L) inhibited the transport activity by 65%. Exposure of HEK(Mrp1) membranes to HNE (10 micromol/L) significantly decreased the V(max) for estradiol 17-(beta-d-glucuronide) transport by 50%. These results show that expression of Mrp1 in the mouse heart is localized predominantly in sarcolemma. Adriamycin treatment increased Mrp1 expression and HNE adduction of Mrp1. Cardiac Mrp1 may play a role in protecting the heart from Adriamycin-induced cardiomyopathy by effluxing HNE conjugates.

Inhaled dexamethasone differentially attenuates disease relapse and established allergic asthma in mice.

Inhaled glucocorticoids are effective in patients with chronic allergic asthma. We examined the effects of inhaled glucocorticoids on relapse (allergen challenge after disease remission) and established/overt allergic asthma (repeated allergen challenge in weekly intervals) in mice to establish a reference standard for novel treatments. BALB/c mice were treated before relapse or during overt disease with 1 h of nebulized PBS or 10 mg% dexamethasone twice daily for 5 days. Dexamethasone eliminated airway hyperresponsiveness before relapse and during overt disease. They more efficiently reduced airway inflammation, mucus production, and OVA-specific IgG1 and IgE during relapse compared to overt disease. However, during overt disease, parenchymal inflammatory infiltrates were more effectively eliminated compared to relapse, suggesting that activated infiltrating leukocytes have increased sensitivity to steroids. These data demonstrate that inhaled corticosteroids attenuate relapse and overt disease differentially and suggest that both airway and parenchymal inflammation need to be evaluated for treatment efficacy.

Repeated aerosol allergen exposure suppresses inflammation in B-cell-deficient mice with established allergic asthma.

BACKGROUND: Repeated allergen administration is a well-established therapeutic strategy for desensitizing patients with allergic disease. Similarly, repeated inhalation of antigen by mice with established allergen-induced asthma suppresses allergic inflammation. The mechanisms underlying antigen-dependent suppression of allergic immune responses remain unknown. In previous studies, we found that repeated aerosol antigen challenges in sensitized mice reduced eosinophils while increasing plasma cells and antibody in the lungs. We sought to test whether plasma cells and antibody played a role in suppression of allergic disease. METHODS: We primed wild-type and B-cell-deficient (microMT) mice with 25 microg ovalbumin (OVA) precipitated in alum on days 0 and 5, nebulized weekly with 1% OVA, 1 h, twice daily, for up to 6 weeks, and assessed lung inflammation, mucus hypersecretion, and IgE/IgG1. RESULTS: Kinetic studies revealed that initial aerosol exposure induced high numbers of eosinophils, lymphocytes, and macrophages within lung infiltrates and increased mucus production in wild-type mice. After 3-4 weeks of antigen exposure, eosinophils diminished while lymphocytes, plasma cells, and macrophages and mucus hypersecretion increased. However, by 6 weeks, lung inflammation and mucus hypersecretion were dramatically reduced. In contrast, repeated aerosol challenges maintained OVA-specific IgG1 and IgE production. Repeated aerosol antigen challenges in microMT mice resulted in reduced lung inflammation and mucus hypersecretion and the development of smooth muscle hypertrophy of the pulmonary microvasculature. CONCLUSIONS: B cells and antibody do not appear to play a role in antigen-dependent suppression of allergic responses in mice.