Supplementary Figure and Table Legends 1-4 from Combinatorial pharmacologic approaches target EZH2-mediated gene repression in breast cancer cells

Supplementary Table 3 from Combinatorial pharmacologic approaches target EZH2-mediated gene repression in breast cancer cells

DZNep is a potential epigenetic drug, and exerts potent anti-proliferative and pro-apoptotic effects on broad-spectrum carcinomas via disruption of the EZH2 pathway. Antitumor studies on DZNep have been stuck in the preclinical phase because of the lack of information about its integral pharmacokinetic (PK) properties. To circumvent this problem, we extensively investigated the disposition characteristics of the DZNep in rats. By incorporating the disposition data across species into a whole-body physiologically based pharmacokinetic (PBPK) models using the GastroPlus(TM) software, we simulated human PK properties of DZNep and determined whether DZNep could be developed for human cancer therapy. Firstly, DZNep was found to cause nephrotoxicity in a dose-dependent manner in rats and its safe dose was determined to be 10mg/kg. DZNep showed a short plasma elimination half-life (1.1h) in rats, a low protein binding in plasma (18.5%), a low partitioning to erythrocyte (0.78), and a low intrinsic hepatic clearance in rats and humans. There was extensive tissue distribution and predominant renal excretion (80.3%). The simulated rat PBPK model of DZNep was well-verified with satisfactory coefficients of determination for all the tested tissues (R(2)>0.781). The simulated human PBPK model successfully identified that intravenous administration of DZNep at appropriate dosing regimen could be further developed for human non-small cell lung carcinoma treatments. The present findings provide valuable information regarding experimental or in silico PK characteristics of DZNep in rats and humans, which is helpful to guide future studies of DZNep in both preclinical and clinical phases.

To establish using dried blood spot (DBS) as a surrogate to plasma for therapeutic drug monitoring (TDM) of carbamazepine (CBZ), we compared the population pharmacokinetic (PPK) estimates from concurrent DBS and plasma levels. The dose-concentration relationship, estimated parameter and variability were determined. A total of 98 observations from 97 people with epilepsy (PWE) were included in this study. Data was split into 3:1 ratio for the respective index group and validation group. Non-linear mixed effects regression with one compartment, first order absorption and elimination model was utilized. Covariates were screened for inclusion into final model via forward stepwise addition and backward elimination method. Predictive performances of the final models were assessed for bias and precision. The typical clearance for CBZ was estimated to be 5.85 and 5.68L/h from plasma and DBS concentrations, respectively. The final models for clearance estimates obtained from plasma concentrations (C-plasma) included total daily CBZ dose per unit weight (DD) and sex while from DBS concentrations (C-dbs) included only DD. The final models were both precise and non-bias. The developed PPK models had comparable estimates, errors and predictive performances. Our findings suggest that C-plasma and C-dbs could be used interchangeably for pharmacokinetic studies of CBZ.

BACKGROUND:A population pharmacokinetic model for phenytoin in Asian pediatric patients was developed to determine the influence of concurrent medications, patient demographics, and blood biochemistry on the pharmacokinetic profile of phenytoin. METHODS:Retrospective clinical data were obtained from 66 patients (age, 1-16 years) for the determination of pharmacokinetic parameters of phenytoin using WinNonmix. Data from 49 patients (74.2%) were allocated in the "index" group, and the other 17 patients (25.8%) in the "validation" group. Models were compared by final log likelihood, mean error as a measure of bias, and root-mean-squared error as a measure of precision. RESULTS:The Michaelis-Menten constant (km) and volume of distribution (V) were fixed at 9.08 mg/L and 1.23 L/kg, respectively. The saturated elimination rate (V × Vmax) of phenytoin was then found to be 0.525 mg/kg per hour (352.9 4 mg/d for a 28.0 kg individual). Patients' body surface area (in square meter) and catalytic activities of liver enzymes aspartate aminotransferase (U/L) and alkaline phosphatase (U/L) appeared to have significant correlation with Vmax, whereas coadministrating drugs with phenytoin did not yield any significant effect. The final model for the saturated elimination rate was (Equation is included in full-text article.) In validation of the final model, the mean error was found to be -0.805 (95% confidence interval, -3.67 to 2.06), and the root-mean-squared error was 7.92 (95% confidence interval, 3.41-12.43). CONCLUSIONS:The obtained results indicated the need to consider patients' body surface area and the catalytic activities of liver enzymes aspartate aminotransferase and alkaline phosphatase when dosing phenytoin. Based on the population pharmacokinetic parameters, a nomogram was subsequently developed for dose individualization of phenytoin in Asian pediatric patients.

Herbs are often administered in combination with therapeutic drugs, raising the potential of herb-drug interactions. An extensive review of the literature identified reported herb-drug interactions with clinical significance, many of which are from case reports and limited clinical observations.

Pharmacokinetic studies have become an integral part of modern drug development, but these studies are not regulatory needs for herbal remedies. This paper updates our current knowledge on the disposition pathways and pharmacokinetic properties of commonly used herbal medicines in humans. To retrieve relevant data, the authors have searched through computer-based literatures by full text search in Medline (via Pubmed), ScienceDirect, Current Contents Connect (ISI), Cochrance Library, CINAHL (EBSCO), CrossRef Search and Embase (all from inception to May 2010). Many herbal compounds undergo Phase I and/or Phase II metabolism in vivo, with cytochrome P450s (CYPs) and uridine diphosphate glucuronosyltransferases (UGTs) playing a major role. Some herbal ingredients are substrates of P-glycoprotein (P-gp) which is highly expressed in the intestine, liver, brain and kidney. As such, the activities of these drug metabolizing enzymes and drug transporters are determining factors for the in vivo bioavailability, disposition and distribution of herbal remedies. There are increasing pharmacokinetic studies of herbal remedies, but these studies are mainly focused on a small number of herbal remedies including St John's wort, milk thistle, sculcap, curcumin, echinacea, ginseng, ginkgo, and ginger. The pharmacokinetic data of a small number of purified herbal ingredients, including anthocyanins, berberine, catechins, curcumin, lutein and quercetin, are available. For the majority of herbal remedies used in folk medicines, data on their disposition and biological fate in humans are lacking or in paucity. For a herbal medicine, the pharmacological effect is achieved when the bioactive agents or the metabolites reach and sustain proper levels at their sites of action. Both the dose levels and fates of active components in the body govern their target-site concentrations after administration of an herbal remedy. In this regard, a safe and optimal use of herbal medicines requires a full understanding of their pharmacokinetic profiles. To optimize the use of herbal remedies, further clinical studies to explore their biological fate including the disposition pathways and kinetics in the human body are certainly needed.

3-Deazaneplanocin A (DZNep) is an attractive epigenetic anticancer agent through the inhibition of the cellular enhancer of zeste homolog 2 (EZH2) protein. The purpose of this study was to improve the pharmacokinetic characteristics of DZNep in vivo through developing a unilamellar pegylated liposomal formulation encapsulating DZNep (L-DZNep). A remote-loading method in the presence of phenylboronic acid (R-w-PBA) was developed to stably encapsulating DZNep inside liposomes (encapsulation efficiency = 50.7% at molar ratio of 1:10 of drug to lipids) through forming a transient PBA-DZNep complex. The pharmacokinetics of L-DZNep was investigated in Sprague-Dawley rats. In comparison with free drug, encapsulation of the DZNep in pegylated liposomes resulted in 99.3% reduction of the plasma clearance, whereas it increased the elimination half-life from 1.1 h to 8.0 h and the area under the plasma concentration curve by 138-fold. These findings demonstrate a novel approach (R-w-PBA method) through the development of L-DZNep, which may be extensively applied for the encapsulation of hydrophilic nucleoside analogs containing vicinal hydroxyl groups and protonable amino in the pegylated liposomes. Additionally, the pegylated liposomes could effectively prolong the retention of DZNep in the systemic circulation and therefore is highly likely to increase the DZNep's tumor localization. (C) 2011 Elsevier B.V. All rights reserved.

Stealth liposomes form an important subset of liposomes, demonstrating prolonged circulation half-life and improved safety in vivo. Caelyx® (liposomal doxorubicin; Merck & Co., Whitehouse Station, New Jersey, USA) is a successful example of the application of stealth liposomes in anticancer treatment. However, multidrug resistance (MDR) to chemotherapy still remains a critical problem, accounting for more than 90% of treatment failure in patients with advanced cancer. To circumvent MDR, fluoxetine and doxorubicin were tested in combination for synergistic activity in MCF-7 (human breast carcinoma) and MCF-7/adr (doxorubicin-resistant human breast carcinoma) cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell-viability assay. Coencapsulation of doxorubicin and fluoxetine, using an ammonium sulphate gradient, was investigated, and a factorial experiment was designed to determine the optimal drug-to-lipid (D/L) ratio for coencapsulation. Drug release from Dox-Flu-SL (stealth liposome coencapsulating doxorubicin and fluoxetine) under both in vitro and in vivo conditions was determined. In MCF-7 cells, synergism was demonstrated at specific doxorubicin:fluoxetine ratios of between 0.09 and 0.5 (molar ratio), while MCF/7/adr cells demonstrated synergism across all drug ratios. Coencapsulation of doxorubicin and fluoxetine (Dox-Flu-SL) was successfully achieved (optimal doxorubicin:fluoxetine:lipid molar ratio of 0.02:0.05:1), obtaining a mean concentration of 257 ± 12.1 and 513 ± 29.3 µM for doxorubicin and fluoxetine, respectively. Most important, Dox-Flu-SL demonstrated drug release in synergistic ratios in cell-culture media, accounting for the improved cytotoxicity of Dox-Flu-SL over liposomal doxorubicin (LD) in both MCF-7 and MCF-7/adr cells. Pharmacokinetic studies also revealed that Dox-Flu-SL effectively prolonged drug-circulation time and reduced tissue biodistribution. Dox-Flu-SL presents a promising anticancer formulation, capable of effective reversal of drug resistance, and may constitute a novel approach for cancer therapy.

Herbal medicines, an important group of multicomponent therapeutics, are widely and increasignly used worldwide. Despite the popularitiy of herbal medicines, the clinical evidence that support the use of most herbal medicines is weak. Pharmacokinetic and absorption, distribution, metabolism and excretion (ADME) studies have been integrated into modern drug development, but ADME studies are generally not needed for herbal remedy discovery and development. For the majority of herbal medicines, data on their ADME and pharmacokinetic properties in humans are lacking or scant. An extensive literature search indicates that there are limited data on ADME properties of herbal medicines in humans. Many herbal compounds undergo Phase I and/or Phase II metabolism in vivo, with cytochrome P450s (CYPs) and uridine diphosphate glucuronosyltransferases (UGTs) playing a major role. Some herbal ingredients are substrates of P-glycoprotein (P-gp/MDR1/ABCB1) which is highly expressed in the intestine, liver, brain and kidney. As such, the activities of these drug metabolizing enzymes and drug transporters are critical determining factors for the in vivo ADME processes of herbal remedies. There are increasing ADME studies of herbal remedies, but these studies are mainly focused on a small number of herbal medicines including St John's wort, milk thistle, curcumin, echinacea, ginseng, ginkgo, and ginger. For an herbal medicine, the pharmacological activity is gained when the active agents or the active metabolites reach and sustain proper levels at their sites of action. Both the dose levels and ADME processes of active herbal components in the body govern their target-site concentrations and thus the therapeutic responses. In this regard, a safe and optimal use of herbal medicines requires a full understanding of their ADME profiles. To optimize the use of herbal remedies, further studies to explore their ADME properties in humans are certainly warranted.

Cytochrome P450 (CYP) 3A4 is the most abundant hepatic and intestinal phase I enzyme that metabolizes approximately 50% marketed drugs. A number of important drugs have been identified as substrates, inducers and/or inhibitors of CYP3A4. The ability of drugs to act as inducers, inhibitors, or substrates for CYP3A is predictive of whether concurrent administration of these compounds with a known CYP3A substrate might lead to altered drug disposition, efficacy or toxicity. The substrates of CYP3A4 considerably overlap with those of P-glycoprotein (P-gp). To date, the identified clinically important CYP3A4 inhibitors mainly include macrolide antibiotics (e.g., clarithromycin, and erythromycin), anti-HIV agents (e.g., ritonavir and delavirdine), antidepressants (e.g. fluoxetine and fluvoxamine), calcium channel blockers (e.g., verapamil and diltiazem), steroids and their modulators (e.g., gestodene and mifepristone), and several herbal and dietary components. Many of these drugs are also mechanism-based inhibitors of CYP3A4. A small number of drugs are identified as inducers of CYP3A4. The inhibition or induction of CYP3A4 by drugs often causes unfavorable and long-lasting drug-drug interactions and probabaly fatal toxicity, depending on many factors associated with the enzyme, drugs and the patients. Clinicians are encouraged to have a sound knowledge on drugs that behave as substrates, inhibitors or inducers of CYP3A4, and take proper cautions and close monitoring for possible drug interactions when using drugs that are CYP3A4 inhibitors or inducers.

A sensitive and selective LC–MS/MS based bioanalytical method was developed and validated for the quantification of 3-deazaneplanocin A (DZNep), a novel epigenetic anti-tumor drug candidate, in Sprague–Dawley (SD) rat biosamples (plasma, urine, feces and tissue samples). The method comprises a phenylboronic acid (PBA)-containing solid phase extraction procedure, serving for binding and clean-up of DZNep in rat biosamples spiked with tubercidin (as internal standard). The analytes were separated on an Agilent hydrophilic interaction chromatography (HILIC) column. LC–MS/MS in positive ion mode was used to perform multiple reaction monitoring at m/z of 263/135 and 267/135 for DZNep and tubercidin, respectively. The limit of quantification (LOQ) of DZNep in rat biosamples was 20ng/mL. The data of intra-day and inter-day accuracy were within 15% of nominal concentration while the precision (relative standard deviation) less than 10% for all biosamples. The extraction recoveries for DZNep and tubercidin were consistent and reproducible (around 80%) and the matrix effects were negligible (around 10% suppression) in all biosamples. This method was demonstrated to be applicable for pharmacokinetic studies of DZNep in SD rats.

Human CYP1A2 is one of the major CYPs in human liver and metabolizes a variety of clinically important drugs (e.g., clozapine, tacrine, tizanidine, and theophylline), a number of procarcinogens (e.g. benzo[a]pyrene and aflatoxin B1), and several important endogenous compounds (e.g. steroids and arachidonic acids). Like many of other CYPs, CYP1A2 is subject to induction and inhibition by a number of compounds, which may provide an explanation for some drug interactions observed in clinical practice. A large interindividual variability in the expression and activity of CYP1A2 and elimination of drugs that are mainly metabolized by CYP1A2 has been observed, which is largely caused by genetic (e.g., SNPs) and epigenetic (e.g., DNA methylation) and environmental factors (e.g., smoking and comedication). CYP1A2 is primarily regulated by the aromatic hydrocarbon receptor (AhR) and CYP1A2 is induced through AhR-mediated transactivation following ligand binding and nuclear translocation. To date, more than 15 variant alleles and a series of subvariants of the CYP1A2 gene have been identified and some of they have been associated with altered drug clearance and response to drug therapy. For example, lack of response to clozapine therapy due to low plasma drug levels has been reported in smokers harboring the −163A/A genotype; there is an association between CYP1A2*1F (−163C>A) allele and the risk for leflunomide-induced host toxicity. The *1F allele is associated with increased enzyme inducibility whereas *1C causes reduced inducibility. Further studies are warranted to explore the clinical and toxicological significance of altered CYP1A2 expression and activity caused by genetic, epigenetic, and environmental factors.

UDP glucuronosyltransferases (UGTs) are an important class of Phase II enzymes involved in the metabolism and detoxification of numerous xenobiotics including therapeutic drugs and endogenous compounds (e.g. bilirubin). To date, there are 21 human UGT genes identified, and most of them contain single-nucleotide polymorphisms (SNPs). Non-synonymous SNPs (nsSNPs) of the human UGT genes may cause absent or reduced enzyme activity and polymorphisms of UGT have been found to be closely related to altered drug clearance and/or drug response, hyperbilirubinemia, Gilbert’s syndrome, and Crigler-Najjar syndrome. However, it is unlikely to study the functional impact of all identified nsSNPs in humans using laboratory approach due to its giant number. We have investigated the potential for bioinformatics approach for the prediction of phenotype based on known nsSNPs. We have identified a total of 248 nsSNPs from human UGT genes. The two algorithms tools, sorting intolerant from tolerant (SIFT) and polymorphism phenotyping (PolyPhen), were used to predict the impact of these nsSNPs on protein function. SIFT classified 35.5% of the UGT nsSNPs as “deleterious”; while PolyPhen identified 46.0% of the UGT nsSNPs as “potentially damaging” and “damaging”. The results from the two algorithms were highly associated. Among 63 functionally characterized nsSNPs in the UGTs, 24 showed altered enzyme expression/activities and 45 were associated with disease susceptibility. SIFT and Polyphen had a correct prediction rate of 57.1% and 66.7%, respectively. These findings demonstrate the potential use of bioinformatics techniques to predict genotype–phenotype relationships which may constitute the basis for future functional studies.

ObjectivesThe effects of the flavonoid rutin on the anticoagulant activity of oral warfarin and the protein binding and pharmacokinetics of its enantiomers were investigated in rats.

Abstract Polycomb protein EZH2-mediated gene silencing is implicated in breast tumorigenesis through methylation of histone H3 on Lysine 27 (H3K27). We have previously shown that S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A can modulate histone methylation and disrupt EZH2 complex. Here, we used 3-deazaneplanocin A, together with other chromatin remodeling agents, as well as RNA interference–mediated EZH2 depletion, to probe the role of EZH2 in coordination with other epigenetic components in gene regulation in breast cancer cells. Through genome-wide gene expression analysis, coupled with extensive chromatin immunoprecipitation analysis of histone modifications, we have identified a variety of gene sets that are regulated either by EZH2 alone or through the coordinated action of EZH2 with HDAC and/or DNA methylation. We further found that tumor antigen GAGEs were regulated by distinct epigenetic mechanisms in a cell context–dependent manner, possibly reflecting mechanistic heterogeneity in breast cancer. Intriguingly, we found that EZH2 regulates a remarkable cohort of genes whose functions are highly enriched in immunoresponse and autocrine inflammation network, and that their transcriptional activation upon EZH2 perturbation is cancer specific, revealing a potential novel role of EZH2 in regulating cancer immunity. These findings show the complexity and diversity of epigenetic regulation in human cancer and underscore the importance for developing combinatorial pharmacologic approaches for effective epigenetic gene reactivation. [Mol Cancer Ther 2009;8(12):3191–202]

CYP1A2 is one of the major CYPs in human liver ( approximately 13%) and metabolises a variety of clinically important drugs, such as clozapine, lidocaine, theophylline, tacrine, and leflunomide. CYP1A2 is one of the major enzymes that bioactivate a number of procarcinogens and thus induction of CYP1A2 may increase the carcinogenicity of these compounds. This enzyme also metabolizes several important endogenous compounds including steroids, retinols, melatonin, uroporphyrinogen and arachidonic acid. In the recently published crystal structure of CYP1A2 in complex with alpha-naphthoflavone, its compact active site is closed without clear solvent or substrate access channels. Not surprisingly, CYP1A2 has a relatively small volume of the active site cavity of 375 A(3), which is 44.2% larger than that of CYP2A6 (260 A(3)), but much smaller than that of CYP3A4 (1385 A(3)) and 2C8 (1438 A(3)). Generally, CYP1A2 substrates contain planar ring that can fit the narrow and planar active site of the enzyme. Like many of other CYPs, CYP1A2 is subject to induction and inhibition by a number of compounds. Similar to CYP1A1 and 1B1, CYP1A2 is primarily regulated by the aromatic hydrocarbon receptor (AhR), a ligand-activated transcription factor and a basic helix-loop-helix protein belonging to the Per-Arnt-Sim family of transcription factors. Knockout of Cyp1a2 in mice has provided a very useful tool for the functional investigation of this gene. Further studies are needed to explore the clinical and toxicological significance of CYP1A2.