Targeted next-generation sequencing of EUS-guided through-the-needle-biopsy sampling from pancreatic cystic lesions.

BACKGROUND AND AIMS: Recent advances have introduced molecular subtyping of pancreatic cystic lesions (PCLs) as a possible amendment to the diagnostic algorithm. The study evaluated the feasibility and diagnostic accuracy of molecular analysis and subtyping of PCLs using the recently introduced EUS-guided through-the-needle-biopsy (TTNB) sampling. METHODS: We prospectively included 101 patients in the study who presented with PCLs >15 mm in the largest cross-section. EUS-guided TTNB samples were obtained by a micro-biopsy forceps introduced through a 19-gauge needle. The TTNB samples were analyzed by next-generation sequencing (NGS) for point mutations in tumor suppressors and oncogenes using a 51-gene customized hotspot panel. Sensitivity and specificity were calculated with the histologic diagnosis as reference. RESULTS: After initial microscopic evaluation of the samples, 91 patients had residual TTNB samples available for NGS. Of these, 49 harbored mutations, most frequently in KRAS and GNAS, reflecting an excess frequency of intraductal papillary mucinous neoplasms (IPMNs) in the study population. A sensitivity and specificity of 83.7% (95% confidence interval [CI], 70.3-92.7) and 81.8% (95% CI, 48.2-97.7), respectively, were demonstrated for the diagnosis of a mucinous cyst and 87.2% (95% CI, 74.2-95.2) and 84.6% (95% CI, 54.5-98.1) for the diagnosis of an IPMN. CONCLUSIONS: Thus, molecular analysis of TTNB samples by NGS has high sensitivity and specificity for diagnosing mucinous cysts and IPMNs. Although the procedure comes with a risk of adverse events of 9.9%, TTNB samples are a robust alternative to cyst fluid for a combined histologic and molecular diagnosis of PCLs. (Clinical trial registration number: NCT03578445.).

In Vitro-In Vivo Correlation of Buprenorphine Transdermal Systems Under Normal and Elevated Skin Temperature.

PURPOSE: Application of external heat using a heating pad over buprenorphine transdermal system, Butrans® has been shown to increase systemic levels of buprenorphine in human volunteers. The purpose of this study was to perform in vitro permeation studies at normal as well as elevated temperature conditions to evaluate the correlation of in vitro data with the existing in vivo data. METHODS: In vitro permeation tests (IVPT) were performed on human skin from four donors. The IVPT study design was harmonized to a previously published clinical study design and skin temperature was maintained at either 32 ± 1 °C or 42 ± 1 °C to mimic normal and elevated skin temperature conditions, respectively. RESULTS: IVPT studies on human skin were able to demonstrate heat induced enhancement in flux and cumulative amount of drug permeated from Butrans® which was reasonably consistent with the corresponding enhancement observed in vivo. Level A in vitro-in vivo correlation (IVIVC) was established using unit impulse response (UIR) based deconvolution method for both baseline and heat arms of the study. The percent prediction error (%PE) calculated for AUC and Cmax values was less than 20%. CONCLUSIONS: The studies indicated that IVPT studies performed under the same conditions as those of interest in vivo may be useful for comparative evaluation of the effect of external heat on transdermal delivery system (TDS). Further research may be warranted to evaluate factors, beyond cutaneous bioavailability (BA) assessed using an IVPT study, that can influence plasma exposure in vivo for a given drug product.

Investigator Impact on Reproducibility of Drug Bioavailability in Stratum Corneum Sampling by Tape Stripping.

PURPOSE: Skin sampling by tape stripping measures the local bioavailability of topical drug products in the stratum corneum (SC). The goal of the current study was to evaluate the impact of different investigators in studies that utilize a tape stripping protocol designed to minimize investigator variability. METHODS: Two open-label clinical studies compared two lidocaine patches and a diclofenac patch and solution in twelve healthy volunteers. The mass of drug was determined in SC samples collected on tape strips at three time points following product removal in duplicate by two investigators. Investigator results were compared with each other and with results for the diclofenac solution measured by another laboratory using a similar protocol. RESULTS: For drug mass, the geometric mean ratio comparing two investigators is within the acceptable bioequivalence interval for most measurement times and drug products. Drug uptake into the SC from the diclofenac solution was not statistically different from that determined in another laboratory. The average flux from the SC over the clearance intervals for the four drug products correspond well with flux measurements from in vitro permeation tests. CONCLUSIONS: Results from different investigators are reproducible within the limitations of measurement variability, which can be managed by increasing volunteer numbers.

Clinical impact of endoscopic ultrasound-guided through-the-needle microbiopsy in patients with pancreatic cysts.

BACKGROUND: The limited data on the utility of endoscopic ultrasound (EUS)-guided through-the-needle biopsies (TTNBs) in patients with pancreatic cystic lesions (PCLs) originate mainly from retrospective studies. Our aim was to determine the clinical impact of TTNBs, their added diagnostic value, and the adverse event rate in a prospective setting. METHODS: This was a prospective, single-center, open-label controlled study. Between February 2018 and August 2019, consecutive patients presenting with a PCL of 15 mm or more and referred for EUS were included. Primary outcome was a change in clinical management of PCLs following TTNB compared with cross-sectional imaging and cytology. Adverse events were defined according to the ASGE lexicon. RESULTS: 101 patients were included. TTNBs led to a change in clinical management in 11.9 % of cases (n = 12). Of these, 10 had serous cysts and surveillance was discontinued, while one of the remaining two cases underwent surgery following diagnosis of a mucinous cystic neoplasm. The diagnostic yield of TTNBs for a specific cyst diagnosis was higher compared with FNA cytology (69.3 % vs. 20.8 %, respectively; P < 0.001). The adverse event rate was 9.9 % (n = 10; 95 % confidence interval 5.4 % - 17.3 %), with the most common event being acute pancreatitis (n = 9). Four of the observed adverse events were severe, including one fatal outcome. CONCLUSIONS: TTNBs resulted in a change of clinical management in about one in every 10 patients; however, the associated adverse event risk was substantial. Further studies are warranted to elucidate in which subgroups of patients the clinical benefit outweighs the risks.

Cutaneous Pharmacokinetics of Acyclovir Cream 5% Products: Evaluating Bioequivalence with an In Vitro Permeation Test and an Adaptation of Scaled Average Bioequivalence.

PURPOSE: The in vitro permeation test (IVPT) with a new statistical approach was investigated to evaluate the utility of an IVPT methodology as a sensitive tool to support a demonstration of bioequivalence (BE) for topical dermatological drug products. METHODS: IVPT experiments were performed utilizing ex vivo human skin. The initial screening tests involved four differently formulated acyclovir 5% creams: the U.S. Zovirax® as the reference product and the U.K. Zovirax®, Aciclovir 1A Pharma® and Aciclostad® as test products. Subsequently, a pivotal BE study was conducted comparing the two Zovirax® creams. The resulting data was used to evaluate BE of test (T) versus reference (R), T versus T, and R versus R, with an adaption of scaled average BE approach to address high variability in IVPT data. RESULTS: More acyclovir permeated into and through the skin from the two Zovirax® creams compared to the two non-Zovirax® creams. The U.S. Zovirax® cream showed a significantly higher Jmax and total amount permeated over 48 h, compared to the U.K. Zovirax® cream. The statistical analysis indicated that the test and reference products were not bioequivalent, whereas each product tested against itself was shown to be bioequivalent. CONCLUSIONS: The current study demonstrated that the IVPT method, with an appropriate statistical analysis of the results, is a sensitive and discriminating test that can detect differences in the rate and extent of acyclovir bioavailability in the skin from differently formulated cream products.

Effect of Controlled Heat Application on Topical Diclofenac Formulations Evaluated by In Vitro Permeation Tests (IVPT) Using Porcine and Human Skin.

PURPOSE: Heat therapy is widely used for pain relief and may unintentionally be used in conjunction with pain relieving topical formulations. The purpose of this study was to evaluate the influence of heat on the permeation of diclofenac through porcine and human skin, comparing four marketed products. METHODS: In vitro permeation tests (IVPT) were performed on porcine skin from a single miniature pig and human skin from three donors. Skin temperature was maintained at either 32 ± 1°C or 42 ± 1°C to mimic normal and elevated skin temperature conditions, respectively. RESULTS: IVPT studies on porcine and human skin were able to demonstrate heat-induced enhancement in flux and cumulative amount of drug permeated from the four diclofenac products. The pivotal data showed the most significant heat-induced enhancement for the solution, followed by the patch and two gels in decreasing order of significance based on p values. Diclofenac solution showed the highest flux and cumulative amount permeated at both baseline and elevated skin temperature compared to the patch and gels. CONCLUSIONS: The studies demonstrated that exposure to heat can alter drug permeation from topical formulations, but the increased levels are not expected to lead to systemic concentrations that are of concern. Formulation design and excipients can influence drug permeation at elevated skin temperature.

Enteroendocrine K and L cells in healthy and type 2 diabetic individuals.

AIMS/HYPOTHESIS: Enteroendocrine K and L cells are pivotal in regulating appetite and glucose homeostasis. Knowledge of their distribution in humans is sparse and it is unknown whether alterations occur in type 2 diabetes. We aimed to evaluate the distribution of enteroendocrine K and L cells and relevant prohormone-processing enzymes (using immunohistochemical staining), and to evaluate the mRNA expression of the corresponding genes along the entire intestinal tract in individuals with type 2 diabetes and healthy participants. METHODS: In this cross-sectional study, 12 individuals with type 2 diabetes and 12 age- and BMI-matched healthy individuals underwent upper and lower double-balloon enteroscopy with mucosal biopsy retrieval from approximately every 30 cm of the small intestine and from seven specific anatomical locations in the large intestine. RESULTS: Significantly different densities for cells positive for chromogranin A (CgA), glucagon-like peptide-1, glucose-dependent insulinotropic polypeptide, peptide YY, prohormone convertase (PC) 1/3 and PC2 were observed along the intestinal tract. The expression of CHGA did not vary along the intestinal tract, but the mRNA expression of GCG, GIP, PYY, PCSK1 and PCSK2 differed along the intestinal tract. Lower counts of CgA-positive and PC1/3-positive cells, respectively, were observed in the small intestine of individuals with type 2 diabetes compared with healthy participants. In individuals with type 2 diabetes compared with healthy participants, the expression of GCG and PYY was greater in the colon, while the expression of GIP and PCSK1 was greater in the small intestine and colon, and the expression of PCSK2 was greater in the small intestine. CONCLUSIONS/INTERPRETATION: Our findings provide a detailed description of the distribution of enteroendocrine K and L cells and the expression of their products in the human intestinal tract and demonstrate significant differences between individuals with type 2 diabetes and healthy participants. TRIAL REGISTRATION: NCT03044860.

Dynamic contrast-enhanced EUS for quantification of tumor perfusion in colonic cancer: a prospective cohort study.

BACKGROUND AND AIMS: Dynamic contrast-enhanced EUS (CE-EUS) for quantification of perfusion in colonic tumors has not previously been reported in the literature. The aim of this study was to investigate correlations between perfusion parameters and vessel density assessed by immunohistochemical staining with antibodies toward CD31 and CD105. METHODS: We conducted a prospective clinical study of 28 patients with left-sided colonic adenocarcinoma who underwent CE-EUS and left-sided hemicolectomy within 2 weeks. CE-EUS recordings were analyzed in 2 regions of interest: the entire tumor and the most enhanced area. Immunohistochemical staining with CD31 and CD105 was performed on tumor tissue sections. The slides were manually scanned for highly vascularized areas, and counting of vessels was performed in hotspots within the tumor and invasive front. New vasculature was assessed by CD105. Associations between CE-EUS and CD31 and CD105 were investigated using Spearman correlation. RESULTS: We found significant P values for the correlation between CD31 and rise time (rho = .603 [95% confidence interval (95% CI), .238-.816]; P = .001) in tumor tissue and for the correlation between CD31 and rise time (rho = .50 [95% CI, .201-.695]; P = .008) and fall time (rho = .52 [95% CI, .204-.723]; P = .006) corresponding to the invasive front. We found no correlations between perfusion values evaluated by CE-EUS and CD105. CONCLUSIONS: Our results show a significant correlation for vessel density evaluated by CD31 and perfusion parameters evaluated by CE-EUS. This may be the first step toward using real-time CE-EUS for monitoring antiangiogenic therapies in colonic cancer. (Clinical trial registration number: NCT02324023.).

A novel endoscopic ultrasound-guided through-the-needle microbiopsy procedure improves diagnosis of pancreatic cystic lesions.

BACKGROUND: Pancreatic cystic lesions represent a diagnostic dilemma as some may harbor malignancy or have potential for malignant transformation. The aim of this study was to present our experience with a novel endoscopic ultrasound (EUS)-guided microbiopsy procedure enabling procurement of tissue from the wall of the cystic lesion. METHODS: We collected data from 31 consecutive patients with pancreatic cystic lesions who underwent an EUS-guided microbiopsy procedure at our institution. Records were retrospectively reviewed from a prospectively maintained database. RESULTS: The technical success was 87.1 %. Diagnostic yield of microbiopsies was 71.0 %. Microbiopsies offered sufficient tissue for morphological and immunohistochemical characterization of the lesions, as well as determination of grade of dysplasia. Furthermore, evaluation of microbiopsies changed the clinical management in six patients (19.4 %). Three nonsevere adverse events were observed (9.7 %): two cases of mild infection and one case of mild pancreatitis. All three patients recovered completely. CONCLUSIONS: EUS-guided microbiopsy procedure was technically feasible, with a high diagnostic yield. Further prospective studies are needed to confirm these promising results.

The Sensitivity of In Vitro Permeation Tests to Chemical Penetration Enhancer Concentration Changes in Fentanyl Transdermal Delivery Systems.

Chemical penetration enhancers (CPEs) are frequently incorporated into transdermal delivery systems (TDSs) to improve drug delivery and to reduce the required drug load in formulations. However, the minimum detectable effect of formulation changes to CPE-containing TDSs using in vitro permeation tests (IVPT), a widely used method to characterize permeation of topically applied drug products, remains unclear. The objective of the current exploratory study was to investigate the sensitivity of IVPT in assessing permeation changes with CPE concentration modifications and subsequently the feasibility of IVPT's use for support of quality control related to relative CPE concentration variation in a given formulation. A series of drug-in-adhesive (DIA) fentanyl TDSs with different amounts of CPEs were prepared, and IVPT studies utilizing porcine and human skin were performed. Although IVPT could discern TDSs with different amounts of CPE by significant differences in flux profiles, maximum flux (Jmax) values, and total permeation amounts, the magnitudes of the CPE increment needed to see such significant differences were very high (43-300%) indicating that IVPT may have limitations in detecting small changes in CPE amounts in some TDSs. Possible reasons for such limitations include formulation polymer and/or other excipients, type of CPE, variability associated with IVPT, skin type used, and disrupted stratum corneum (SC) barrier effects caused by CPEs.

On the Road to Development of an in Vitro Permeation Test (IVPT) Model to Compare Heat Effects on Transdermal Delivery Systems: Exploratory Studies with Nicotine and Fentanyl.

PURPOSE: At elevated temperatures, the rate of drug release and skin permeation from transdermal delivery systems (TDS) may be higher than at a normal skin temperature. The aim of this study was to compare the effect of heat on the transdermal delivery of two model drugs, nicotine and fentanyl, from matrix-type TDSs with different formulations, using in vitro permeation tests (IVPT). METHODS: IVPT experiments using pig skin were performed on two nicotine and three fentanyl TDSs. Both continuous and transient heat exposures were investigated by applying heat either for the maximum recommended TDS wear duration or for short duration. RESULTS: Continuous heat exposure for the two nicotine TDSs resulted in different effects, showing a prolonged heat effect for one product but not the other. The Jmax enhancement ratio due to the continuous heat effect was comparable between the two nicotine TDS, but significantly different (p < 0.05) among the three fentanyl TDSs. The Jmax enhancement ratios due to transient heat exposure were significantly different for the two nicotine TDSs, but not for the three fentanyl TDSs. Furthermore, the transient heat exposure affected the clearance of drug from the skin depot after TDS removal differently for two drugs, with fentanyl exhibiting a longer heat effect. CONCLUSIONS: This exploratory work suggests that an IVPT study may be able to discriminate differences in transdermal drug delivery when different TDS are exposed to elevated temperatures. However, the clinical significance of IVPT heat effects studies should be further explored by conducting in vivo clinical studies with similar study designs.

Development and validation of a high performance liquid chromatography quantification method of levo-tetrahydropalmatine and its metabolites in plasma and brain tissues: application to a pharmacokinetic study.

Levo-tetrahydropalmatine (l-THP) is an alkaloid isolated from Chinese medicinal herbs of the Corydalis and Stephania genera. It has been used in China for more than 40 years mainly as an analgesic with sedative/hypnotic effects. Despite its extensive use, its metabolism has not been quantitatively studied, nor there a sensitive reliable bioanalytical method for its quantification simultaneously with its metabolites. As such, the objective of this study was to develop and validate a sensitive and selective HPLC method for simultaneous quantification of l-THP and its desmethyl metabolites l-corydalmine (l-CD) and l-corypalmine (l-CP) in rat plasma and brain tissues. Rat plasma and brain samples were processed by liquid-liquid extraction using ethyl acetate. Chromatographic separation was achieved on a reversed-phase Symmetry® C18 column (4.6 × 150 mm, 5 µm) at 25°C. The mobile phase consisted of acetonitrile-methanol-10 mm ammonium phosphate (pH 3) (10:30:60, v/v) and was used at a flow rate of 0.8 mL/min. The column eluent was monitored at excitation and emission wavelengths of 230 and 315 nm, respectively. The calibration curves were linear over the concentration range of 1-10,000 ng/mL. The intra- and interday reproducibility studies demonstrated accuracy and precision within the acceptance criteria of bioanalytical guidelines. The validated HPLC method was successfully applied to analyze samples from a pharmacokinetic study of l-THP in rats. Taken together, the developed method can be applied for bioanalysis of l-THP and its metabolites in rodents and potentially can be transferred for bioanalysis of human samples.

Novel LRRK2 GTP-binding inhibitors reduced degeneration in Parkinson's disease cell and mouse models.

Mutations in the leucine-rich repeat kinase-2 (LRRK2) gene cause autosomal-dominant Parkinson's disease (PD) and contribute to sporadic PD. LRRK2 contains Guanosine-5'-triphosphate (GTP) binding, GTPase and kinase activities that have been implicated in the neuronal degeneration of PD pathogenesis, making LRRK2, a potential drug target. To date, there is no disease-modifying drug to slow the neuronal degeneration of PD and no published LRRK2 GTP domain inhibitor. Here, the biological functions of two novel GTP-binding inhibitors of LRRK2 were examined in PD cell and mouse models. Through a combination of computer-aided drug design (CADD) and LRRK2 bio-functional screens, two novel compounds, 68: and 70: , were shown to reduce LRRK2 GTP binding and to inhibit LRRK2 kinase activity in vitro and in cultured cell assays. Moreover, these two compounds attenuated neuronal degeneration in human SH-SY5Y neuroblastoma cells and mouse primary neurons expressing mutant LRRK2 variants. Although both compounds inhibited LRRK2 kinase activity and reduced neuronal degeneration, solubility problems with 70: prevented further testing in mice. Thus, only 68: was tested in a LRRK2-based lipopolysaccharide (LPS)-induced pre-inflammatory mouse model. 68: reduced LRRK2 GTP-binding activity and kinase activity in brains of LRRK2 transgenic mice after intraperitoneal injection. Moreover, LPS induced LRRK2 upregulation and microglia activation in mouse brains. These findings suggest that disruption of GTP binding to LRRK2 represents a potential novel therapeutic approach for PD intervention and that these novel GTP-binding inhibitors provide both tools and lead compounds for future drug development.

Pharmacokinetics, microbial response, and pulmonary outcomes of multidose intravenous azithromycin in preterm infants at risk for Ureaplasma respiratory colonization.

The study objectives were to refine the population pharmacokinetics (PK) model, determine microbial clearance, and assess short-term pulmonary outcomes of multiple-dose azithromycin treatment in preterm infants at risk for Ureaplasma respiratory colonization. Fifteen subjects (7 of whom were Ureaplasma positive) received intravenous azithromycin at 20 mg/kg of body weight every 24 h for 3 doses. Azithromycin concentrations were determined in plasma samples obtained up to 168 h post-first dose by using a validated liquid chromatography-tandem mass spectrometry method. Respiratory samples were obtained predose and at three time points post-last dose for Ureaplasma culture, PCR, antibiotic susceptibility testing, and cytokine concentration determinations. Pharmacokinetic data from these 15 subjects as well as 25 additional subjects (who received either a single 10-mg/kg dose [n = 12] or a single 20-mg/kg dose [n = 13]) were analyzed by using a nonlinear mixed-effect population modeling (NONMEM) approach. Pulmonary outcomes were assessed at 36 weeks post-menstrual age and 6 months adjusted age. A 2-compartment model with all PK parameters allometrically scaled on body weight best described the azithromycin pharmacokinetics in preterm neonates. The population pharmacokinetics parameter estimates for clearance, central volume of distribution, intercompartmental clearance, and peripheral volume of distribution were 0.15 liters/h · kg(0.75), 1.88 liters · kg, 1.79 liters/h · kg(0.75), and 13 liters · kg, respectively. The estimated area under the concentration-time curve over 24 h (AUC24)/MIC90 value was ∼ 4 h. All posttreatment cultures were negative, and there were no drug-related adverse events. One Ureaplasma-positive infant died at 4 months of age, but no survivors were hospitalized for respiratory etiologies during the first 6 months (adjusted age). Thus, a 3-day course of 20 mg/kg/day intravenous azithromycin shows preliminary efficacy in eradicating Ureaplasma spp. from the preterm respiratory tract.

Quantitative contrast-enhanced harmonic EUS in differential diagnosis of focal pancreatic masses (with videos).

BACKGROUND: The role of EUS with contrast agents can be expanded through the use of time-intensity curve (TIC) analysis and computer-aided interpretation. OBJECTIVE: To validate the use of parameters derived from TIC analysis in an artificial neural network (ANN) classification model designed to diagnose pancreatic carcinoma (PC) and chronic pancreatitis (CP). SETTING: Prospective, multicenter, observational trial-endoscopy units from Romania, Denmark, Germany, and Spain. PATIENTS: A total of 167 consecutive patients with PC or CP. INTERVENTIONS: Contrast-enhanced harmonic EUS (CEH-EUS) and EUS-guided FNA (EUS-FNA), TIC analysis, and ANN processing. MAIN OUTCOME MEASUREMENTS: Sensitivity, specificity, positive and negative predictive values (PPV, NPV) for EUS-FNA, CEH-EUS, and the ANN. RESULTS: After excluding all of the recordings that did not meet the technical and procedural criteria, 112 cases of PC and 55 cases of CP were included. EUS-FNA was performed in 129 patients, and the diagnosis was confirmed by surgery (n = 15) or follow-up (n = 23) in the remaining cases. Its sensitivity and specificity were 84.82% and 100%, respectively, whereas the PPV and NPV were 100% and 76.63%, respectively. The sensitivity of real-time quantitative assessment of CEH-EUS was 87.5%, specificity 92.72%, PPV 96.07%, and NPV 78.46%. Peak enhancement, wash-in area under the curve, wash-in rate, and the wash-in perfusion index were significantly different between the groups. No significant differences were found between rise time, mean transit time, and time to peak. For the ANN, sensitivity was 94.64%, specificity 94.44%, PPV 97.24%, and NPV 89.47%. LIMITATIONS: Only PC and CP lesions were included. CONCLUSION: Parameters obtained through TIC analysis can differentiate between PC and CP cases and can be used in an automated computer-aided diagnostic system with good diagnostic results. ( CLINICAL TRIAL REGISTRATION NUMBER: NCT01315548.).

Transporter-Mediated Disposition of Opioids: Implications for Clinical Drug Interactions.

Opioid-related deaths, abuse, and drug interactions are growing epidemic problems that have medical, social, and economic implications. Drug transporters play a major role in the disposition of many drugs, including opioids; hence they can modulate their pharmacokinetics, pharmacodynamics and their associated drug-drug interactions (DDIs). Our understanding of the interaction of transporters with many therapeutic agents is improving; however, investigating such interactions with opioids is progressing relatively slowly despite the alarming number of opioids-mediated DDIs that may be related to transporters. This review presents a comprehensive report of the current literature relating to opioids and their drug transporter interactions. Additionally, it highlights the emergence of transporters that are yet to be fully identified but may play prominent roles in the disposition of opioids, the growing interest in transporter genomics for opioids, and the potential implications of opioid-drug transporter interactions for cancer treatments. A better understanding of drug transporters interactions with opioids will provide greater insight into potential clinical DDIs and could help improve opioids safety and efficacy.

Curcumin and dimethoxycurcumin induced epigenetic changes in leukemia cells.

PURPOSE: Curcumin is an ideal chemopreventive and antitumor agent characterized by poor bioavailability and low stability. The development of synthetic structural analogues like dimethoxycurcumin (DMC) could overcome these drawbacks. In this study we compared the cytotoxicity, metabolism and the epigenetic changes induced by both drugs in leukemia cells. METHODS: Apoptosis and cell cycle analysis were analyzed by flow cytometry. Real-time PCR was used for gene expression analysis. DNA methylation was analyzed by DNA pyrosequencing. The metabolic stability was determined using human pooled liver microsomes. Chromatin Immunoprecipitation was used to quantify histone methylation. RESULTS: Clinically relevant concentration of curcumin and DMC were not cytotoxic to leukemia cells and induced G2/M cell cycle arrest. DMC was more metabolically stable than curcumin. Curcumin and DMC were devoid of DNA hypomethylating activity. DMC induced the expression of promoter methylated genes without reversing DNA methylation and increased H3K36me3 mark near the promoter region of hypermethylated genes. CONCLUSION: DMC is a more stable analogue of curcumin that can induce epigenetic changes not induced by curcumin. DMC induced the expression of promoter methylated genes. The combination of DMC with DNA methyltransferase inhibitors could harness their combined induced epigenetic changes for optimal re-expression of epigenetically silenced genes.

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.