Synthesis and evaluation of etoposide and podophyllotoxin analogs against topoisomerase IIα and HCT-116 cells.

Etoposide is a widely-used anticancer agent that targets human type II topoisomerases. Evidence suggests that metabolism of etoposide in myeloid progenitor cells is associated with translocations involved in leukemia development. Previous studies suggest halogenation at the C-2' position of etoposide reduces metabolism. Halogens were introduced into the C-2' position by electrophilic aromatic halogenation onto etoposide (ETOP, 1), podophyllotoxin (PPT, 2), and 4-dimethylepipodophyllotoxin (DMEP, 3), and to bridge the gap of knowledge regarding the activity of these metabolically stable analogs. Five halogenated analogs (6-10) were synthesized. Analogs 8-10 displayed variable ability to inhibit DNA relaxation. Analog 9 was the only analog to show concentration-dependent enhancement of Top2-mediated DNA cleavage. Dose response assay results indicated that 8 and 10 were most effective at decreasing the viability of HCT-116 and A549 cancer cell lines in culture. Flow cytometry with 8 and 10 in HCT-116 cells provide evidence of sub-G1 cell populations indicative of apoptosis. Taken together, these results indicate C-2' halogenation of etoposide and its precursors, although metabolically stable, decreases overall activity relative to etoposide.

Impact of cytochrome P450 variation on meperidine N-demethylation to the neurotoxic metabolite normeperidine.

1. Meperidine is an opioid analgesic that undergoes N-demethylation to form the neurotoxic metabolite normeperidine. Previous studies indicate that meperidine N-demethylation is catalyzed by cytochrome P450 2B6 (CYP2B6), CYP3A4, and CYP2C19.2. The purpose of this study was to examine the relative P450 contributions to meperidine N-demethylation and to evaluate the effect of CYP2C19 polymorphism on normeperidine generation. Experiments were performed using recombinant P450 enzymes, selective chemical inhibitors, enzyme kinetic assays, and correlation analysis with individual CYP2C19-genotyped human liver microsomes.3. The catalytic efficiency (kcat/Km) for meperidine N-demethylation was similar between recombinant CYP2B6 and CYP2C19, but markedly lower by CYP3A4.4. In CYP2C19-genotyped human liver microsomes, normeperidine formation was significantly correlated with CYP2C19 activity (S-mephenytoin 4´-hydroxylation).5. CYP2C19 inhibitor (+)-N-3-benzylnirvanol and CYP3A inhibitor ketoconazole significantly reduced microsomal normeperidine generation by an individual donor with high CYP2C19 activity, whereas donors with lower CYP2C19 activity were sensitive to inhibition by ketoconazole but not benzylnirvanol.6. These findings demonstrate that the relative CYP3A4, CYP2B6, and CYP2C19 involvement in meperidine N-demethylation depends on the enzyme activities in individual human liver microsomal samples. CYP2C19 is likely an important contributor to normeperidine generation in individuals with high CYP2C19 activity, but additional factors influence inter-individual metabolite accumulation.

DARK Classics in Chemical Neuroscience: Fentanyl.

Fentanyl rose to prominence as an alternative analgesic to morphine nearly 50 years ago; today, fentanyl has re-emerged as a dangerous recreational substance. The increased potency and analgesic effect of fentanyl are advantageous in the treatment of pain but are also responsible for the rise in unintentional opioid overdose deaths. In response to this crisis, fentanyl, its analogues, and even precursors are under heightened regulatory scrutiny. Despite this controversial history, derivatization of fentanyl has resulted in numerous synthetic analogues that provide valuable insights into opioid receptor binding and signaling events. In this review, the impact of fentanyl on chemical neuroscience is shown through its synthesis and properties, manufacturing, metabolism, pharmacology, approved and off-label indications, adverse effects, and the responsibility it has in the opioid epidemic.

DARK Classics in Chemical Neuroscience: Morphine.

As the major psychoactive agent in opium and direct precursor for heroin, morphine is a historically critical molecule in chemical neuroscience. A structurally complex phenanthrene alkaloid produced by Papaver somniferum, morphine has fascinated chemists seeking to disentangle pharmacologically beneficial analgesic effects from addiction, tolerance, and dependence liabilities. In this review, we will detail the history of morphine, from the first extraction and isolation by Sertürner in 1804 to the illicit use of morphine and proliferation of opioid use and abuse disorders currently ravaging the United States. Morphine is a molecule of great cultural relevance, as the agent that single-handedly transformed our understanding of pharmacognosy, receptor dynamics, and substance abuse and dependence disorders.

HU-331 and Oxidized Cannabidiol Act as Inhibitors of Human Topoisomerase IIα and ß.

Topoisomerase II is a critical enzyme in replication, transcription, and the regulation of chromatin topology. Several anticancer agents target topoisomerases in order to disrupt cell growth. Cannabidiol is a major non-euphoriant, pharmacologically active component of cannabis. Previously, we examined the cannabidiol derivative HU-331 in order to characterize the mechanism of the compound against topoisomerase IIα. In this current work, we explore whether cannabidiol (CBD) impacts topoisomerase II activity, and we additionally examine the activity of these compounds against topoisomerase IIß. CBD does not appear to strongly inhibit DNA relaxation and is not a poison of topoisomerase II DNA cleavage. However, oxidation of CBD allows this compound to inhibit DNA relaxation by topoisomerase IIα and ß without poisoning DNA cleavage. Additionally, we found that oxidized CBD, similar to HU-331, inhibits ATP hydrolysis and can result in inactivation of topoisomerase IIα and ß. We also determined that oxidized CBD and HU-331 are both able to stabilize the N-terminal clamp of topoisomerase II. Taken together, we conclude that while CBD does not have significant activity against topoisomerase II, both oxidized CBD and HU-331 are active against both isoforms of topoisomerase II. We hypothesize that oxidized CBD and HU-331 act against the enzyme through interaction with the N-terminal ATPase domain. According to the model we propose, topoisomerase II inactivation may result from a decrease in the ability of the enzyme to bind to DNA when the compound is bound to the N-terminus.

Drugs of Abuse and Addiction: An integrated approach to teaching.

BACKGROUND AND PURPOSE: To describe the design, implementation, and student perceptions of a Drugs of Abuse and Addiction elective course utilizing an integrated teaching model. EDUCATIONAL ACTIVITY AND SETTING: Third-year pharmacy students enrolled in the two credit hour elective. Teaching methodology included didactic lecture, journal club, simulated addiction assignment with reflection, debates, external speakers, site visit to a residential drug court program and research paper with presentation. FINDINGS: A course objective survey was administered upon course completion. All students strongly agreed that having science- and clinical-based faculty members develop and deliver course content was beneficial. Additionally, all students agree to strongly agree that their research project helped them integrate and comprehend the science and practice surrounding drugs of abuse and addiction. DISCUSSION AND SUMMARY: Students enjoyed an integrated teaching approach and multiple teaching methodologies leading to increased engagement and enhancement of student learning. Course enrollment was beneficial for personalized learning, but limited student perspective.

Two-Mechanism Model for the Interaction of Etoposide Quinone with Topoisomerase IIα.

Topoisomerase II is an essential nuclear enzyme involved in regulating DNA topology to facilitate replication and cell division. Disruption of topoisomerase II function by chemotherapeutic agents is in use as an effective strategy to fight cancer. Etoposide is an anticancer therapeutic that disrupts the catalytic cycle of topoisomerase II and stabilizes enzyme-bound DNA strand breaks. Etoposide is metabolized into several species including active quinone and catechol metabolites. Our previous studies have explored some of the details of how these compounds act against topoisomerase II. In our present study, we extend those analyses by examining several effects of etoposide quinone on topoisomerase IIα including whether the quinone impacts ATP hydrolysis, DNA ligation, cleavage complex persistence, and enzyme/DNA binding. Our results demonstrate that the quinone inhibits relaxation at 100-fold lower levels of drug when compared to that of etoposide. Further, the quinone inhibits ATP hydrolysis by topoisomerase IIα. The quinone does appear to stabilize single-strand breaks similar to etoposide suggesting a traditional poisoning mechanism. However, there is minimal difference in cleavage complex persistence in the presence of etoposide or etoposide quinone. In contrast to etoposide, we find that etoposide quinone blocks enzyme/DNA binding, which provides an explanation for previous data showing the ability of the quinone to inactivate the enzyme over time. Finally, etoposide quinone is able to stabilize the N-terminal protein clamp implying an interaction between the compound and this portion of the enzyme. Taken together, the evidence supports a two-mechanism model for the effect of the quinone on topoisomerase II: (1) interfacial poison and (2) covalent poison that interacts with the N-terminal clamp and impacts the binding of DNA.

HU-331 is a catalytic inhibitor of topoisomerase IIα.

Topoisomerases are essential enzymes that are involved in DNA metabolism. Topoisomerase II generates transient DNA strand breaks that are stabilized by anticancer drugs, such as doxorubicin, causing an accumulation of DNA damage. However, doxorubicin causes cardiac toxicity and, like etoposide and other topoisomerase II-targeted agents, can induce DNA damage, resulting in secondary cancers. The cannabinoid quinone HU-331 has been identified as a potential anticancer drug that demonstrates more potency in cancer cells with less off-target toxicity than that of doxorubicin. Reports indicate that HU-331 does not promote cell death via apoptosis, cell cycle arrest, caspase activation, or DNA strand breaks. However, the precise mechanism of action is poorly understood. We employed biochemical assays to study the mechanism of action of HU-331 against purified topoisomerase IIα. These assays examined DNA binding, cleavage, ligation, relaxation, and ATPase activities of topoisomerase IIα. Our results demonstrate that HU-331 inhibits topoisomerase IIα-mediated DNA relaxation at micromolar levels. We find that HU-331 does not induce DNA strand breaks in vitro. When added prior to the DNA substrate, HU-331 blocks DNA cleavage and relaxation activities of topoisomerase IIα in a redox-sensitive manner. The action of HU-331 can be blocked, but not reversed, by the presence of dithiothreitol. Our results also show that HU-331 inhibits the ATPase activity of topoisomerase IIα using a noncompetitive mechanism. Preliminary binding studies also indicate that HU-331 decreases the ability of topoisomerase IIα to bind DNA. In summary, HU-331 inhibits relaxation activity without poisoning DNA cleavage. This action is sensitive to reducing agents and appears to involve noncompetitive inhibition of the ATPase activity and possibly inhibition of DNA binding. These studies provide a promising foundation for the exploration of HU-331 as a catalytic inhibitor of topoisomerase IIα.

Catalytic core of human topoisomerase IIα: insights into enzyme-DNA interactions and drug mechanism.

Coordination between the N-terminal gate and the catalytic core of topoisomerase II allows the proper capture, cleavage, and transport of DNA during the catalytic cycle. Because the activities of these domains are tightly linked, it has been difficult to discern their individual contributions to enzyme-DNA interactions and drug mechanism. To further address the roles of these domains, we analyzed the activity of the catalytic core of human topoisomerase IIα. The catalytic core and the wild-type enzyme both maintained higher levels of cleavage with negatively (as compared to positively) supercoiled plasmid, indicating that the ability to distinguish supercoil handedness is embedded within the catalytic core. However, the catalytic core alone displayed little ability to cleave DNA substrates that did not intrinsically provide the enzyme with a transport segment (i.e., substrates that did not contain crossovers). Finally, in contrast to interfacial topoisomerase II poisons, covalent poisons did not enhance DNA cleavage mediated by the catalytic core. This distinction allowed us to further characterize the mechanism of etoposide quinone, a drug metabolite that functions primarily as a covalent poison. Etoposide quinone retained some ability to enhance DNA cleavage mediated by the catalytic core, indicating that it still can function as an interfacial poison. These results further define the distinct contributions of the N-terminal gate and the catalytic core to topoisomerase II function. The catalytic core senses the handedness of DNA supercoils during cleavage, while the N-terminal gate is critical for capturing the transport segment and for the activity of covalent poisons.

Opioids and efflux transporters. Part 4: influence of N-substitution on P-glycoprotein substrate activity of noroxymorphone analogues.

The efflux transporter protein P-glycoprotein (P-gp) is capable of affecting the central distribution of diverse neurotherapeutics, including opioid analgesics, through their active removal from the brain. P-gp located at the blood brain barrier has been implicated in the development of tolerance to opioids and demonstrated to be up-regulated in rats tolerant to morphine and oxycodone. We have previously examined the influence of hydrogen-bonding oxo-substitutents on the P-gp-mediated efflux of 4,5-epoxymorphinan analgesics, as well as that of N-substituted analogues of meperidine. Structure-activity relationships (SAR) governing N-substituent effects on opioid efficacy is well-established, however the influence of such structural modifications on P-gp-mediated efflux is unknown. Here, we present SAR describing P-gp recognition of a short series of N-modified 4,5-epoxymorphinans. Oxymorphone, naloxone, naltrexone, and nalmexone all failed to demonstrate P-gp substrate activity, indicating these opioid scaffolds contain structural features that preclude recognition by the transporter. These results are examined using mathematical molecular modeling and discussed in comparison to other opioid scaffolds bearing similar N-substituents.

Etoposide quinone is a covalent poison of human topoisomerase IIß.

Etoposide is a topoisomerase II poison that is utilized to treat a broad spectrum of human cancers. Despite its wide clinical use, 2-3% of patients treated with etoposide eventually develop treatment-related acute myeloid leukemias (t-AMLs) characterized by rearrangements of the MLL gene. The molecular basis underlying the development of these t-AMLs is not well understood; however, previous studies have implicated etoposide metabolites (i.e., etoposide quinone) and topoisomerase IIß in the leukemogenic process. Although interactions between etoposide quinone and topoisomerase IIα have been characterized, the effects of the drug metabolite on the activity of human topoisomerase IIß have not been reported. Thus, we examined the ability of etoposide quinone to poison human topoisomerase IIß. The quinone induced ~4 times more enzyme-mediated DNA cleavage than did the parent drug. Furthermore, the potency of etoposide quinone was ~2 times greater against topoisomerase IIß than it was against topoisomerase IIα, and the drug reacted ~2-4 times faster with the ß isoform. Etoposide quinone induced a higher ratio of double- to single-stranded breaks than etoposide, and its activity was less dependent on ATP. Whereas etoposide acts as an interfacial topoisomerase II poison, etoposide quinone displayed all of the hallmarks of a covalent poison: the activity of the metabolite was abolished by reducing agents, and the compound inactivated topoisomerase IIß when it was incubated with the enzyme prior to the addition of DNA. These results are consistent with the hypothesis that etoposide quinone contributes to etoposide-related leukemogenesis through an interaction with topoisomerase IIß.

Etoposide catechol is an oxidizable topoisomerase II poison.

Topoisomerase II regulates DNA topology by generating transient double-stranded breaks. The anticancer drug etoposide targets topoisomerase II and is associated with the formation of secondary leukemias in patients. The quinone and catechol metabolites of etoposide may contribute to strand breaks that trigger leukemic translocations. To further analyze the characteristics of etoposide metabolites, we extend our previous analysis of etoposide quinone to the catechol. We demonstrate that the catechol is ∼2-3-fold more potent than etoposide and under oxidative reaction conditions induces high levels of double-stranded DNA cleavage. These results support a role for etoposide catechol in contributing to therapy-induced DNA damage.

Etoposide quinone is a redox-dependent topoisomerase II poison.

Etoposide is a topoisomerase II poison that is used to treat a variety of human cancers. Unfortunately, 2-3% of patients treated with etoposide develop treatment-related leukemias characterized by 11q23 chromosomal rearrangements. The molecular basis for etoposide-induced leukemogenesis is not understood but is associated with enzyme-mediated DNA cleavage. Etoposide is metabolized by CYP3A4 to etoposide catechol, which can be further oxidized to etoposide quinone. A CYP3A4 variant is associated with a lower risk of etoposide-related leukemias, suggesting that etoposide metabolites may be involved in leukemogenesis. Although etoposide acts at the enzyme-DNA interface, several quinones poison topoisomerase II via redox-dependent protein adduction. The effects of etoposide quinone on topoisomerase IIα-mediated DNA cleavage have been examined previously. Although findings suggest that the activity of the quinone is slightly greater than that of etoposide, these studies were carried out in the presence of significant levels of reducing agents (which should reduce etoposide quinone to the catechol). Therefore, we examined the ability of etoposide quinone to poison human topoisomerase IIα in the absence of reducing agents. Under these conditions, etoposide quinone was ∼5-fold more active than etoposide at inducing enzyme-mediated DNA cleavage. Consistent with other redox-dependent poisons, etoposide quinone inactivated topoisomerase IIα when incubated with the protein prior to DNA and lost activity in the presence of dithiothreitol. Unlike etoposide, the quinone metabolite did not require ATP for maximal activity and induced a high ratio of double-stranded DNA breaks. Our results support the hypothesis that etoposide quinone contributes to etoposide-related leukemogenesis.

Opioid analgesics and P-glycoprotein efflux transporters: a potential systems-level contribution to analgesic tolerance.

Chronic clinical pain remains poorly treated. Despite attempts to develop novel analgesic agents, opioids remain the standard analgesics of choice in the clinical management of chronic and severe pain. However, mu opioid analgesics have undesired side effects including, but not limited to, respiratory depression, physical dependence and tolerance. A growing body of evidence suggests that P-glycoprotein (P-gp), an efflux transporter, may contribute a systems-level approach to the development of opioid tolerance. Herein, we describe current in vitro and in vivo methodology available to analyze interactions between opioids and P-gp and critically analyze P-gp data associated with six commonly used mu opioids to include morphine, methadone, loperamide, meperidine, oxycodone, and fentanyl. Recent studies focused on the development of opioids lacking P-gp substrate activity are explored, concentrating on structure-activity relationships to develop an optimal opioid analgesic lacking this systems-level contribution to tolerance development. Continued work in this area will potentially allow for delineation of the mechanism responsible for opioid-related P-gp up-regulation and provide further support for evidence based medicine supporting clinical opioid rotation.

ACS chemical neuroscience molecule spotlight on Qnexa.

Qnexa (VI-0521) is an investigational fixed-dose combination drug of phentermine and topiramate currently in Phase III clinical trials for the treatment of obesity. Vivus, Inc. has demonstrated efficacy of their product and are currently addressing FDA safety concerns with the possibility of a New Drug Application (NDA) resubmission.

ACS chemical neuroscience molecule spotlight on contrave.

Contrave is an investigational fixed-dose combination drug of naltrexone and bupropion currently in Phase III clinical trials for the treatment of obesity. Orexigen Therapeutics, Inc. has demonstrated efficacy of their product and is currently addressing FDA safety concerns and deciding future actions.

Behavioral effects of gamma-hydroxybutyrate, its precursor gamma-butyrolactone, and GABA(B) receptor agonists: time course and differential antagonism by the GABA(B) receptor antagonist 3-aminopropyl(diethoxymethyl)phosphinic acid (CGP35348).

Gamma-hydroxybutyrate (GHB) is used therapeutically and recreationally. The mechanism by which GHB produces its therapeutic and recreational effects is not entirely clear, although GABA(B) receptors seem to play an important role. This role could be complex, because there are indications that different GABA(B) receptor mechanisms mediate the effects of GHB and the prototypical GABA(B) receptor agonist baclofen. To further explore possible differences in underlying GABA(B) receptor mechanisms, the present study examined the effects of GHB and baclofen on operant responding and their antagonism by the GABA(B) receptor antagonist 3-aminopropyl(diethoxymethyl)phosphinic acid (CGP35348). Pigeons were trained to peck a key for access to food during response periods that started at different times after the beginning of the session. In these pigeons, GHB, its precursor gamma-butyrolactone (GBL), and the GABA(B) receptor agonists baclofen and 3-aminopropyl(methyl)phosphinic acid hydrochloride (SKF97541) decreased the rate of responding in a dose- and time-dependent manner. CGP35348 shifted the dose-response curve of each agonist to the right, but the magnitude of the shift differed among the agonists. Schild analysis yielded a pA(2) value of CGP35348 to antagonize GHB and GBL [i.e., 3.9 (3.7-4.2)] that was different (P = 0.0011) from the pA(2) value to antagonize baclofen and SKF97541 [i.e., 4.5 (4.4-4.7)]. This finding is further evidence that the GABA(B) receptor mechanisms mediating the effects of GHB and prototypical GABA(B) receptor agonists are not identical. A better understanding of the similarities and differences between these mechanisms, and their involvement in the therapeutic effects of GHB and baclofen, could lead to more effective medications with fewer adverse effects.

Evaluation of the P-glycoprotein (Abcb1) affinity status of a series of morphine analogs: comparative study with meperidine analogs to identify opioids with minimal P-glycoprotein interactions.

One of the major shortcomings of many commonly used opioids is the fact that they are P-gp substrates, which represents a major obstacle towards effective pain management. P-gp can affect opioids' oral absorption, CNS accumulation, systemic clearance, antinociceptive activity, and tolerance development to their analgesic effects. Moreover, P-gp can be the locus of drug-drug interactions between opioids and other concomitantly administered drugs that are P-gp substrates/inhibitors. The objective of this study was to identify opioids that are non-P-gp substrates to overcome some of the mentioned shortcomings. We evaluated the P-gp affinity status (substrate, non-substrate, or inhibitor) of a series of morphine analogs (10 opioid agonist and 2 opioid antagonists) and compared them to previously reported meperidine analogs. The fold stimulation of the morphine analogs ranged from 1.01 to 1.54 while for the meperidine analogs the fold stimulation ranged from 1.10 to 3.66. From each series (morphine and meperidine analogs) we selected potential candidate opioids that are non-P-gp substrates and conducted in vivo assessments of their antinociceptive effects using P-gp knockout and P-gp competent mice. 6-Desoxymorphine, meperidine and N-phenylbutyl normeperidine did not significantly (p>0.05) stimulate the basal P-gp ATPase activity, where, the fold stimulations of the basal P-gp ATPase activity were 1.01+/-0.11, 1.51+/-0.29 and 1.10+/-0.23, respectively. Evaluation of the influence of P-gp ablation on their antinociceptive effects indicated that P-gp did not significantly (p>0.05) affect their antinociceptive effects. Among the evaluated opioids in vivo, 6-desoxymorphine showed high potency and induced no apparent toxicity upon low- and high-dose administration. 6-Desoxymorphine is therefore an ideal lead compound to create a library of opioids that have negligible P-gp affinity for better management of pain.

Opioids and efflux transporters. Part 3: P-glycoprotein substrate activity of 3-hydroxyl addition to meperidine analogs.

Numerous studies have shown that many clinically employed opioid analgesics are substrates for P-glycoprotein (P-gp), suggesting that up-regulation of P-gp may contribute to the development of central tolerance to opioids. The studies herein focus on the development of SAR for P-gp substrate activity in the meperidine series of opioids. Addition of a 3-OH to meperidine and the ketone analog of meperidine yielding bemidone and ketobemidone, respectively, significantly increased P-gp substrate affinity. The results of this study have implications in the development of novel analgesics to be utilized as tools to study the contribution of P-gp on the development of central tolerance to opioids.

Opioids and efflux transporters. Part 2: P-glycoprotein substrate activity of 3- and 6-substituted morphine analogs.

Continuing our studies investigating opioids with reduced P-glycoprotein (P-gp) substrate activity, a series of known 3- and 6-hydroxy, -methoxy, and -desoxymorphine analogs was synthesized and analyzed for P-gp substrate activity and opioid binding affinity. 6-Desoxymorphine ( 7) showed high affinity for opioid receptors and did not induce P-gp-mediated ATP hydrolysis. Additionally, 7 demonstrated morphine-like antinociceptive potency in mice, indicating this compound as an ideal lead to further evaluate the role of P-gp in opioid analgesic tolerance development.