Ivan Spasojevic

The SLCO1B1*5 Genetic Variant Is Associated With Statin-Induced Side Effects

OBJECTIVES We sought to identify single nucleotide polymorphisms associated with mild statin-induced side effects. BACKGROUND Statin-induced side effects can interfere with therapy. Single nucleotide polymorphisms in cytochrome P450 enzymes impair statin metabolism; the reduced function SLCO1B1*5 allele impairs statin clearance and is associated with simvastatin-induced myopathy with creatine kinase (CK) elevation. METHODS The STRENGTH (Statin Response Examined by Genetic Haplotype Markers) study was a pharmacogenetics study of statin efficacy and safety. Subjects (n = 509) were randomized to atorvastatin 10 mg, simvastatin 20 mg, or pravastatin 10 mg followed by 80 mg, 80 mg, and 40 mg, respectively. We defined a composite adverse event (CAE) as discontinuation for any side effect, myalgia, or CK >3x upper limit of normal during follow-up. We sequenced CYP2D6, CYP2C8, CYP2C9, CYP3A4, and SLCO1B1 and tested 7 reduced function alleles for association with the CAE. RESULTS The CAE occurred in 99 subjects (54 discontinuations, 49 myalgias, and 9 CK elevations). Sex was associated with CAE (percent female in CAE vs. no CAE groups, 66% vs. 50%, p < 0.01). SLCO1B1*5 was associated with CAE (percent with > or = 1 allele in CAE vs. no CAE groups, 37% vs. 25%, p = 0.03) and those with CAE with no significant CK elevation (p < or = 0.03). Furthermore, there was evidence for a gene-dose effect (percent with CAE in those with 0, 1, or 2 alleles: 19%, 27%, and 50%, trend p = 0.01). Finally, the CAE risk appeared to be greatest in those carriers assigned to simvastatin. CONCLUSIONS SLCO1B1*5 genotype and female sex were associated mild statin-induced side effects. These findings expand the results of a recent genome-wide association study of statin myopathy with CK >3x normal to milder, statin-induced, muscle side effects.

Profiling the effects of isocitrate dehydrogenase 1 and 2 mutations on the cellular metabolome

Point mutations of the NADP+-dependent isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) occur early in the pathogenesis of gliomas. When mutated, IDH1 and IDH2 gain the ability to produce the metabolite (R)-2-hydroxyglutarate (2HG), but the downstream effects of mutant IDH1 and IDH2 proteins or of 2HG on cellular metabolism are unknown. We profiled >200 metabolites in human oligodendroglioma (HOG) cells to determine the effects of expression of IDH1 and IDH2 mutants. Levels of amino acids, glutathione metabolites, choline derivatives, and tricarboxylic acid (TCA) cycle intermediates were altered in mutant IDH1- and IDH2-expressing cells. These changes were similar to those identified after treatment of the cells with 2HG. Remarkably, N-acetyl-aspartyl-glutamate (NAAG), a common dipeptide in brain, was 50-fold reduced in cells expressing IDH1 mutants and 8.3-fold reduced in cells expressing IDH2 mutants. NAAG also was significantly lower in human glioma tissues containing IDH mutations than in gliomas without such mutations. These metabolic changes provide clues to the pathogenesis of tumors associated with IDH gene mutations.

Superoxide Dismutase Mimics: Chemistry, Pharmacology, and Therapeutic Potential

Oxidative stress has become widely viewed as an underlying condition in a number of diseases, such as ischemia-reperfusion disorders, central nervous system disorders, cardiovascular conditions, cancer, and diabetes. Thus, natural and synthetic antioxidants have been actively sought. Superoxide dismutase is a first line of defense against oxidative stress under physiological and pathological conditions. Therefore, the development of therapeutics aimed at mimicking superoxide dismutase was a natural maneuver. Metalloporphyrins, as well as Mn cyclic polyamines, Mn salen derivatives and nitroxides were all originally developed as SOD mimics. The same thermodynamic and electrostatic properties that make them potent SOD mimics may allow them to reduce other reactive species such as peroxynitrite, peroxynitrite-derived CO(3)(*-), peroxyl radical, and less efficiently H(2)O(2). By doing so SOD mimics can decrease both primary and secondary oxidative events, the latter arising from the inhibition of cellular transcriptional activity. To better judge the therapeutic potential and the advantage of one over the other type of compound, comparative studies of different classes of drugs in the same cellular and/or animal models are needed. We here provide a comprehensive overview of the chemical properties and some in vivo effects observed with various classes of compounds with a special emphasis on porphyrin-based compounds.

THE ORTHO EFFECT MAKES MANGANESE(III) MESO-TETRAKIS-(N-METHYLPYRIDINIUM-2-YL)PORPHYRIN A POWERFUL AND POTENTIALLY USEFUL SUPEROXIDE DISMUTASE MIMIC

The ortho, meta, andpara isomers of manganese(III) 5,10,15,20-tetrakis(N-methylpyridyl)porphyrin, MnTM-2-PyP5+, MnTM-3-PyP5+, and MnTM-4-PyP5+, respectively, were analyzed in terms of their superoxide dismutase (SOD) activity in vitro and in vivo. The impact of their interaction with DNA and RNA on the SOD activity in vivo and in vitro has also been analyzed. Differences in their behavior are due to the combined steric and electrostatic factors. In vitro catalytic activities are closely related to their redox potentials. The half-wave potentials (E½) are +0.220 mV, +0.052 mV, and +0.060 Vversus normal hydrogen electrode, whereas the rates of dismutation (k cat) are 6.0 × 107, 4.1 × 106, and 3.8 × 106 m −1 s−1 for theortho, meta, and para isomers, respectively. However, the in vitro activity is not a sufficient predictor of in vivo efficacy. The ortho andmeta isomers, although of significantly different in vitro SOD activities, have fairly close in vivo SOD efficacy due to their similarly weak interactions with DNA. In contrast, due to a higher degree of interaction with DNA, thepara isomer inhibited growth of SOD-deficientEscherichia coli.

A small molecular weight catalytic metalloporphyrin antioxidant with superoxide dismutase (SOD) mimetic properties protects lungs from radiation-induced injury.

Abstract Radiation therapy (RT) is an important therapeutic modality in the treatment of thoracic tumors. The maximum doses to these tumors are often limited by the radiation tolerance of lung tissues. Lung injury from ionizing radiation is believed to be a consequence of oxidative stress and a cascade of cytokine activity. Superoxide dismutase (SOD) is a key enzyme in cellular defenses against oxidative damage. The objective of this study was to determine whether the SOD mimetic AEOL 10113 [manganese (III) mesotetrakis (N-ethylpyridinium-2-yl) porphyrin (MnTE-2-PyP5+)] increases the tolerance of lung to ionizing radiation. AEOL 10113 was able to significantly reduce the severity of RT-induced lung injury. This was strongly supported with histopathology results and measurements of collagen deposition (hydroxyproline content). There was a significant reduction in the plasma level of the profibrogenic cytokine transforming growth factor-β (TGF-β) in the group of rats receiving RT + AEOL 10113. In conclusion, the novel SOD mimetic, AEOL 10113, demonstrates a significant protective effect from radiation-induced lung injury.

Antihelminth Compound Niclosamide Downregulates Wnt Signaling and Elicits Antitumor Responses in Tumors with Activating APC Mutations

Wnt/β-catenin pathway activation caused by adenomatous polyposis coli (APC) mutations occurs in approximately 80% of sporadic colorectal cancers (CRC). The antihelminth compound niclosamide downregulates components of the Wnt pathway, specifically Dishevelled-2 (Dvl2) expression, resulting in diminished downstream β-catenin signaling. In this study, we determined whether niclosamide could inhibit the Wnt/β-catenin pathway in human CRCs and whether its inhibition might elicit antitumor effects in the presence of APC mutations. We found that niclosamide inhibited Wnt/β-catenin pathway activation, downregulated Dvl2, decreased downstream β-catenin signaling, and exerted antiproliferative effects in human colon cancer cell lines and CRC cells isolated by surgical resection of metastatic disease, regardless of mutations in APC. In contrast, inhibition of NF-κB or mTOR did not exert similar antiproliferative effects in these CRC model systems. In mice implanted with human CRC xenografts, orally administered niclosamide was well tolerated, achieved plasma and tumor levels associated with biologic activity, and led to tumor control. Our findings support clinical explorations to reposition niclosamide for the treatment of CRC.

SOD Therapeutics: Latest Insights into Their Structure-Activity Relationships and Impact on the Cellular Redox-Based Signaling Pathways

SIGNIFICANCE Superoxide dismutase (SOD) enzymes are indispensable and ubiquitous antioxidant defenses maintaining the steady-state levels of O2·(-); no wonder, thus, that their mimics are remarkably efficacious in essentially any animal model of oxidative stress injuries thus far explored. RECENT ADVANCES Structure-activity relationship (half-wave reduction potential [E1/2] versus log kcat), originally reported for Mn porphyrins (MnPs), is valid for any other class of SOD mimics, as it is dominated by the superoxide reduction and oxidation potential. The biocompatible E1/2 of ∼+300 mV versus normal hydrogen electrode (NHE) allows powerful SOD mimics as mild oxidants and antioxidants (alike O2·(-)) to readily traffic electrons among reactive species and signaling proteins, serving as fine mediators of redox-based signaling pathways. Based on similar thermodynamics, both SOD enzymes and their mimics undergo similar reactions, however, due to vastly different sterics, with different rate constants. CRITICAL ISSUES Although log kcat(O2·(-)) is a good measure of therapeutic potential of SOD mimics, discussions of their in vivo mechanisms of actions remain mostly of speculative character. Most recently, the therapeutic and mechanistic relevance of oxidation of ascorbate and glutathionylation and oxidation of protein thiols by MnP-based SOD mimics and subsequent inactivation of nuclear factor κB has been substantiated in rescuing normal and killing cancer cells. Interaction of MnPs with thiols seems to be, at least in part, involved in up-regulation of endogenous antioxidative defenses, leading to the healing of diseased cells. FUTURE DIRECTIONS Mechanistic explorations of single and combined therapeutic strategies, along with studies of bioavailability and translational aspects, will comprise future work in optimizing redox-active drugs.

Phase I Trial of Doxorubicin-Containing Low Temperature Sensitive Liposomes in Spontaneous Canine Tumors

Purpose: To determine the maximum tolerated dose, dose-limiting toxicities, and pharmacokinetic characteristics of doxorubicin encapsulated in a low temperature sensitive liposome (LTSL) when given concurrently with local hyperthermia to canine solid tumors. Experimental Design: Privately owned dogs with solid tumors (carcinomas or sarcomas) were treated. The tumors did not involve bone and were located at sites amenable to local hyperthermia. LTSL-doxorubicin was given (0.7-1.0 mg/kg i.v.) over 30 minutes during local tumor hyperthermia in a standard phase I dose escalation study. Three treatments, given 3 weeks apart, were scheduled. Toxicity was monitored for an additional month. Pharmacokinetics were evaluated during the first treatment cycle. Results: Twenty-one patients were enrolled: 18 with sarcomas and 3 with carcinomas. Grade 4 neutropenia and acute death secondary to liver failure, possibly drug related, were the dose-limiting toxicities. The maximum tolerated dose was 0.93 mg/kg. Other toxicities, with the possible exception of renal damage, were consistent with those observed following free doxorubicin administration. Of the 20 dogs that received ≥2 doses of LTSL-doxorubicin, 12 had stable disease, and 6 had a partial response to treatment. Pharmacokinetic variables were more similar to those of free doxorubicin than the marketed liposomal product. Tumor drug concentrations at a dose of 1.0 mg/kg averaged 9.12 ± 6.17 ng/mg tissue. Conclusion: LTSL-doxorubicin offers a novel approach to improving drug delivery to solid tumors. It was well tolerated and resulted in favorable response profiles in these patients. Additional evaluation in human patients is warranted.

Pure MnTBAP selectively scavenges peroxynitrite over superoxide: comparison of pure and commercial MnTBAP samples to MnTE-2-PyP in two models of oxidative stress injury, an SOD-specific Escherichia coli model and carrageenan-induced pleurisy.

MnTBAP is often referred to as an SOD mimic in numerous models of oxidative stress. We have recently reported that pure MnTBAP does not dismute superoxide, but commercial or poorly purified samples are able to perform O2.- dismutation with low-to-moderate efficacy via non-innocent Mn-containing impurities. Herein, we show that neither commercial nor pure MnTBAP could substitute for SOD enzyme in a SOD-deficient Escherichia coli model, whereas MnTE-2-PyP-treated SOD-deficient E. coli grew as well as a wild-type strain. This SOD-specific system indicates that MnTBAP does not act as an SOD mimic in vivo. In another model, carrageenan-induced pleurisy in mice, inflammation was evidenced by increased pleural fluid exudate and neutrophil infiltration and activation: these events were blocked by 0.3 mg/kg MnTE-2-PyP and, to a slightly lesser extent, by 10 mg/kg of either MnTBAP. Also, 3-nitrotyrosine formation, an indication of peroxynitrite existence in vivo, was blocked by both compounds; again MnTE-2-PyP was 33-fold more effective. Pleurisy model data indicate that MnTBAP exerts some protective actions in common with MnTE-2-PyP, which are not O2.- related and can be fully rationalized if one considers that the common biological role shared by MnTBAP and MnTE-2-PyP is related to their reduction of peroxynitrite and carbonate radical, the latter arising from ONOOCO2 adduct. The log kcat (O2.-) value for MnTBAP is estimated to be about 3.16, which is approximately 5 and approximately 6 orders of magnitude smaller than the SOD activities of the potent SOD mimic MnTE-2-PyP and Cu,Zn-SOD, respectively. This very low value indicates that MnTBAP is too inefficient at dismuting superoxide to be of any biological impact, which was confirmed in the SOD-deficient E. coli model. The peroxynitrite scavenging ability of MnTBAP, however, is only approximately 2.5 orders of magnitude smaller than that of MnTE-2-PyP and is not significantly affected by the presence of the SOD-active impurities in the commercial MnTBAP sample (log k red (ONOO-) = 5.06 for pure and 4.97 for commercial sample). The reduction of carbonate radical is equally fast with MnTBAP and MnTE-2-PyP. The dose of MnTBAP required to yield oxidative stress protection and block nitrotyrosine formation in the pleurisy model is > 1.5 orders of magnitude higher than that of MnTE-2-PyP, which could be related to the lower ability of MnTBAP to scavenge peroxynitrite. The slightly better protection observed with the commercial MnTBAP sample (relative to the pure MnTBAP) could arise from its impurities, which, by scavenging O2.-, reduce consequently the overall peroxynitrite and secondary ROS/RNS levels. These observations have profound biological repercussions as they may suggest that the effect of MnTBAP observed in numerous studies may conceivably relate to peroxynitrite scavenging. Moreover, provided that pure MnTBAP is unable to dismute superoxide at any significant extent, but is able to partially scavenge peroxynitrite and carbonate radical, this compound may prove valuable in distinguishing ONOO-/CO3.- from O2.- pathways.

Urinary Biomarkers of Oxidative Status

Oxidative damage produced by reactive oxygen species (ROS) has been implicated in the etiology and pathology of many health conditions, including a large number of chronic diseases. Urinary biomarkers of oxidative status present a great opportunity to study redox balance in human populations. With urinary biomarkers, specimen collection is non-invasive and the organic/metal content is low, which minimizes the artifactual formation of oxidative damage to molecules in specimens. Also, urinary levels of the biomarkers present intergraded indices of redox balance over a longer period of time compared to blood levels. This review summarizes the criteria for evaluation of biomarkers applicable to epidemiological studies and evaluation of several classes of biomarkers that are formed non-enzymatically: oxidative damage to lipids, proteins, DNA, and allantoin, an oxidative product of uric acid. The review considers formation, metabolism, and exertion of each biomarker, available data on validation in animal and clinical models of oxidative stress, analytical approaches, and their intra- and inter-individual variation. The recommended biomarkers for monitoring oxidative status over time are F₂-isoprostanes and 8-oxodG. For inter-individual comparisons, F₂-isoprostanes are recommended, whereas urinary 8-oxodG levels may be confounded by differences in the DNA repair capacity. Promising urinary biomarkers include allantoin, acrolein-lysine, and dityrosine.