Drug Metabolism Letters - Volume 8, Issue 1, 2014

At the outset, I would like to thank the present and previous authors for their quality and impactful contributions to Drug metabolism Letters. With the journal being open to a wide variety of areas in Biochemistry, Pharmacology and Pharmaceutical Sciences, the journal has consistently achieved a high Impact Factor in the last area, in the past four years. This is a true testament to the impactful science published and the caliber of scientists the journal attracts for submission of their research. We continue to strive to improve the quality of articles published, based on innovative, thought-provoking science. This has been streamlined, under the leadership of the Editor-in-Chief Chandra Prakash, by eminent Associate Editors, Editorial Board members, and Review Panel members (http://benthamscience.com/journal/editorial-board.php?journalID=dml#top). Behind this world class scientific team, working in the background, is the publisher’s staff, who work tirelessly and with much dedication to keep the manuscripts flowing to reviewers and authors so we can make timely decisions. My special thanks to the editorial office staff! The current special issue contains articles from the Editorial Board Members (EBM) as well as from select scientists, with the following highlights that the readers should find of great interest. Albert P. Li (EBM) and Kari E. Schlicht provide a new, composite Km estimation for the major CYPs using hepatocytes from 16 donors and isoform-selective substrates in a high-throughput fashion. The composite Km was derived for each pathway by normalization of individual activities to their respective Vmax. The composite Km values were similar to those in the literature generated using various hepatic sub-fractions. Using this more complete hepatic system, the authors claim that the composite Km values could be used to assess compounds’ impact on DDIs in humans. In a second article, Dr Li and Qian Yang devised a novel medium to overcome the issue of spontaneous decrease in CYP gene expression during hepatocytes incubation for days. The hepatocytes with the new medium were responsive to the CYP gene expression changes induced by proinflammatory IL6 cytokine, confirming the viability of the medium. Another example of leading science in the area of covalent binding of drugs to therapeutic target is presented by Mehran F. Moghaddam (EBM) et al. There is tremendous interest in the covalent modification of proteins leading to target silencing, as shown by the recent approval of drugs in both oncology as well as non-oncology areas. New criteria are proposed for a meaningful screening paradigm for such class of therapeutics. Mert Ulgen (EBM) and his colleagues have devised a simplified approach to simultaneously measure combo agents fluticasone and salmeterol present in a dry powder Seretide Diskus inhalers for asthma. One approach requires no purification steps. Natalia Penner, Xiaojing Liu, and Chandra Prakash (Editor-in-Chief) have designed a new trapping reagent to identify reactive species formation during metabolism. The reagent provides greater sensitivity and broader selectivity to detect reactive species, compared to commonly used trapping agents. Hiroshi Yamazaki (EBM) and his group carried out elegant docking simulations for CYP3A4 and CYP3A5 catalytic active sites with a series of substrates and showed that compounds with greater specificity for one of the CYP isoforms also docked closely to the heme of that CYP. These simulations partly explained the difference in Km values of the substrates for CYP3A4 and CYP3A5. Massimiliano Fonsi has emphasized the need for caution for in vitro-in vivo correlations (IVIVC) based on largely hepatic assessments. The article covers three cases where the IVIVC broke down, determined later to be due to involvement of extrahepatic metabolism. It also shows thorough mechanistic investigations undertaken to design corrective strategies that were eventually adopted for screening purposes. Demir et al. describe the purification of paraxonase (PON1) from sera of patients with Behcet’s disease and analysis of certain drugs, used in the treatment of this disease, as inhibitors of this enzyme. PON1 is one of the body’s defense mechanisms to prevent atherosclerosis. PON1 from healthy subjects was activated by the tested drugs, while that from patients was inhibited. Ramifications of these results to understanding the disease control process will require further studies.

A higher throughput platform was developed for the determination of KM values for isoformselective P450 substrates in human hepatocytes via incubation of the hepatocytes with substrates in 384- well plates and metabolite quantification by RapidFire™ mass spectrometry. Isoform-selective P450 substrates were incubated at 8 concentrations in triplicate with cryopreserved human hepatocytes from 16 donors. The metabolic pathways examined were the CYP1A2-catalyzed tacrine 1-hydroxylation, CYP2B6-catalyzed bupropion hydroxylation, CYP2C8-catalyzed amodiaquine N-deethylation, CYP2C9- catalyzed diclofenac 4'-hydroxylation, CYP2D6-catalyzed dextromethorphan O-demethylation, and CYP3A4-catalyzed midazolam 1'-hydroxylation. Typical saturation enzyme kinetics was observed for all the pathways evaluated. Individual differences in the apparent Vmax and KM values were observed among the human hepatocytes from each of the 16 individual donors, with no statistically significant gender- or age-associated differences. A “composite” KM value was calculated for each of the pathways via normalizing the individual activities to their respective Vmax values to develop “relative activities” followed by Michaelis-Menten analysis of the mean relative activities of the 16 donors at each of the 8 substrate concentrations. The resulting “composite” KM values for the P450 substrates may be used to guide in vitro P450 inhibition and induction studies and kinetic modeling of in vivo drug-drug interaction. Biography: Dr. Albert Li (Ph. D., Biomedical Sciences) is CEO and President of In Vitro ADMET Laboratories, Columbia, MD and Malden, MA. He is a pioneer in hepatocyte cryopreservation and application of hepatocytes in the assessment of ADMET drug properties. He has published over 160 scientific articles and edited 6 books.

A major challenge for the evaluation of cytokine-induced down regulation of CYP gene expression in primary cultured hepatocytes is the spontaneous decrease in expression of the genes with culture duration. Based on our recent discovery that hepatocytes cultured for 7 days in a novel medium, Li’s Differentiation Maintenance Medium (LDMM), would retain gene expression for markers of differentiation and most CYP isoforms at levels similar to those of the first day of culture, we examined the effects of the prototypical pro-inflammatory cytokine IL-6 in the "LDMM-stabilized (LS)" human hepatocyte model. The LS-human hepatocyte cultures were found to be responsive to IL-6 induction of the inflammatory gene marker, C-reactive protein (CRP), suggesting the expression of IL-6 receptors and the subsequent signaling pathways. Results from two independent laboratories with human hepatocytes from three donors demonstrated dose-dependent down regulation of the gene expression of several CYPs, i.e. 1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4. The results suggest that the LS-human hepatocytes may represent a physiologically relevant experimental model for mechanistic investigation of the down-regulatory effects of inflammatory cytokines. Biography:Dr. Albert Li (Ph. D., Biomedical Sciences) is CEO and President of In Vitro ADMET Laboratories, Columbia, MD and Malden, MA. He is a pioneer in hepatocyte cryopreservation and application of hepatocytes in the assessment of ADMET drug properties. He has published over 160 scientific articles and edited 6 books.

The in vitro and in vivo preclinical ADME properties of 10 clinically late stage or marketed covalent inhibitors were evaluated in order to define advancement criteria for discovery of future drugs in this arena. Our studies revealed the following: After incubating with S9 fractions for 30 minutes, the rat and human in vitro stability for these compounds ranged from 1% to 100%. The blood stability ranged from 30% to 100%. There was a broad range of CYP inhibition with prevalence for time-dependent inhibition of at least one enzyme. The Caco-2 permeability (A→B) ranged from negligible (0.6 x 10-6 cm/s) to highly permeable (31 x 10-6 cm/s) and the efflux ratio also varied widely (0.2-30). Most of the compounds were highly protein bound in both rat and human with binding ≥ 90%. Rat plasma clearance for the 10 compounds ranged from slow (11 mL/min/kg) to very rapid (350 mL/min/kg). The Vss ranged from low (0.67 L/kg) to very high (115 L/kg). MRT’s also ranged from short (0.5 hr) to long (7.4 hr). The oral exposures also showed a very broad range with Cmax’s ranging from 0.01-77 μM and exposure levels ranging from 0.03-106 μM.hr. In conclusion, the wide range in in vitro and in vivo ADME data makes these particular ADME assays non-discriminatory in the selection of promising compounds. In our opinion, non-traditional assays such as target mass modification, target confirmation by amino acid sequencing, cellular target occupancy, and target turnover rate data in combination with the pharmacokinetic profiles are the critical considerations for progression of irreversible compounds in early discovery. Biography: Mehran Moghaddam, PhD, is the head of Discovery DMPK at Celgene, a global biopharmaceutical company with products in oncology and immune-inflammatory related diseases. He has 18 years of industrial experience with DuPont, Pfizer, and Celgene. His expertise includes animal experimentation, pharmacokinetics, metabolism, metabolite/catabolite identification, and bioanalysis of small and macro-molecules.

In the present study, two new methods were developed for the quantitative determination of active components of Seretide®, commercially available pharmaceutical preparation in the diskus form. One of these methods was based on derivative spectrophotometry and used a zero-crossing technique. The determinations of fluticasone propionate and salmeterol xinafoate were performed by first order derivatisation at 216.5 nm and second order derivatisation at 250 nm, respectively. The concentration ranges were 5.0-32.5 μg/mL for fluticasone propionate and 2-12 μg/mL for salmeterol xinafoate. The second method developed also included high performance liquid chromatography. In this method, a methanol-water mobile phase mixture (95:5, v/v) and a C18 chromasil column as a stationary phase were used. The wavelength of the diode array UV detector was 260 nm; the flow rate was 1 mL/min. The concentration ranges were 2-16 μg/mL for fluticasone propionate and 1-8 μg/mL for salmeterol xinafoate. The results for both methods from diskus are in the pharmacopea limits. For the statistical determination of these results, these two methods were compared with t-test for the means and with F-test for the standard deviations. Biography: Dr. Mert Ulgen is a Full Professor of Pharmaceutical Chemistry and he is currently the Vice Director of the Institute of Health Sciences and also of the Vocational School of Health Sciences at Acıbadem University, Istanbul, Turkey. He also maintains directorship of Continuous Education Center at the same University. He received his M.Sc. degre in Pharmaceutical Chemistry in 1987 (Marmara University, Turkey) and Ph.D. degree in Drug Metabolism in 1992 (King’s College London). He has a number of publications on in vitro metabolism of drugs and model compounds. He is currently supervising a M.Sc. program, “Management in Drug Industry” at Acıbadem University (For further details please visit www.acibadem.edu.tr, http://www.acibadem.edu.tr.

The novel cysteine-cholamine (Cys-chol) trapping reagent was synthesized by coupling N-(tertbutoxycarbonyl)- S-trityl-L-cysteine with cholamine in the presence of HBTU (O-(benzotriazol-1-yl)- N,N,N',N'-tetramethyluronium hexafluorophosphate), and then deprotecting by trifluoroacetic acid. Cys-chol reagent enhanced the sensitivity of reactive metabolite screening 4 to 20 times without introducing additional sample preparation or derivatization steps. Retention of Cys-chol conjugates on reversed-phase column is higher than for respective GSH conjugates which helps in reduction of background interference. The use of Cys-chol trapping reagent can potentially improve sensitivity and specificity of routine reactive metabolite screening assay in drug discovery.

To investigate the reported differences in catalytic activities of cytochromes P450 (CYP or P450) 3A4 and 3A5, molecular docking studies were conducted of the interactions of 13 compounds with active sites of the reported three-dimensional structure of CYP3A4 and the modeled structure of CYP3A5. The primary sequence of human CYP3A5 was aligned with that of human CYP3A4 (1TQN) using threedimensional modeling software. Docking simulations were carried out after the energy of the CYP3A4 and CYP3A5 structures had been minimized. Kinetic parameters for the substrate oxidations were taken from the literature. Among the substrates that are preferably metabolized by CYP3A4, including carebastine, itraconazole, haloperidol, and fluvastatin, the former three compounds were found to closely dock to the heme region of CYP3A4 but not to that of CYP3A5. The ligand–CYP3A5 interaction energies (U values) for vincristine, R- and S-verapamil, and β-endosulfan were considerably lower than the corresponding ligand–CYP3A4 interaction energies; these substrates also had lower reported Michaelis constants (km) for CYP3A5 than for CYP3A4. Despite higher CYP3A5 km values for α-endosulfan and estradiol, the CYP3A5 U value for estradiol was lower than that for CYP3A4. No marked differences of U values between CYP3A4 and CYP3A5 were observed for midazolam and triazolam, and comparable km values for the oxidations of these two substrates have been reported for CYP3A4 and CYP3A5. Molecular docking simulation could partly explain the differences of the affinities (km) of the substrates for CYP3A4 and CYP3A5 based on the accessibility of substrates to the heme moiety of CYP3A molecules. Biography: Hiroshi Yamazaki has been Professor of Laboratory of Drug Metabolism and Pharmacokinetics in Showa Pharmaceutical University, Tokyo, Japan since 2005. A major area of his interest is polymorphic cytochrome P450 and flavincontaining monooxygenase involved in drug metabolism. He is recognized as a ‘Highly Cited’ researcher in Pharmacology by ISI/Thomson Reuters.

As the liver is generally considered the organ most involved in metabolic transformations, metabolism in other organs is often overlooked and in vitro screening systems largely adopted in drug discovery are generally based on liver tissue fractions. First pharmacokinetics of new chemical entities (NCEs) are initially based on preclinical species; rat is used in the majority of the cases to assess early in vitro-in vivo correlation (IVIVC). It is important, in this perspective, to address as early as possible the relevant differences between rat and human and the limits using pharmacokinetic studies in this species as a model for the human PK. In this paper the author reports at least three clear examples in drug discovery where the use of hepatic in-vitro systems resulted in a very poor IVIVC due to relevant extrahepatic metabolism in rats.

In this study, serum samples from 50 patients with the diagnosis of Behcet’s disease and 20 healthy volunteers were analyzed. The study consists of three parts. In the first part, paraoxonase (PON) activities were determined in the serum samples of 50 patients with Behcet’s disease and 20 healthy people. In the second part, equal volumes of serum samples from 50 patients were pooled and PON enzymes were purified by using Sepharose-4B-L-tyrosine tyrosine-1-naphtylamine affinity column. Optimum temperature, optimum pH, Vmax and Km values of the pure enzymes were determined. The same purification procedure was also performed in the serum samples of 20 healthy people. Electrophoretic mobility was observed (via SDS-PAGE) in the PON enzymes that were purified from the serum samples of patients with Behcet’s disease and healthy people. In the third part, in vitro effects of drugs containing azathioprine, methylprednisolone and colchicine that have already been used for the treatment of Behcet’s disease were tested on the PON enzymes of the patients with Behcet’s disease and control group. IC50 values and Ki constant values were measured and inhibition types were determined for the drugs containing azathioprine, methylprednisolone and colchicine that have already been used for the treatment of the Behcet’s disease and demonstrate in vitro inhibition effects.