Murali Subramanian

High-speed simultaneous determination of nine antiepileptic drugs using liquid chromatography-mass spectrometry.

Therapeutic drug monitoring of antiepileptic drugs (AEDs) is important and widely practiced. However, simultaneous AED assays usually concentrate only on old or new AEDs. A new simultaneous assay was developed to monitor both older and newer AEDs in the same sample. This assay measures zonisamide (ZNS), lamotrigine (LTG), topiramate (TPM), phenobarbital (PB), phenytoin (PHT), carbamazepine (CBZ), carbamazepine-10,11-diol (CBZ-Diol), 10-hydroxycarbamazepine (MHD), and carbamazepine-10,11-epoxide (CBZ-E). Sample pretreatment consisted of a single solid-phase extraction (SPE) for all AEDs in a 100-μL plasma sample. HPLC separation was achieved on a Shimdazu Shimpack XR-ODS (4.6 id × 50 mm, 2.2-μm) column with a gradient mobile phase of acetate buffer, methanol, acetonitrile, and tetrahydrofuran. Four internal standards were used. Detection was achieved by atmospheric pressure chemical ionization mass spectrometry (APCI-MS) in selected-ion monitoring (SIM) mode with constant polarity switching. High recovery (88%-96%) was obtained for all compounds by SPE. Linearity was observed throughout an 80-fold concentration range, with correlation coefficient (r2) values higher than 0.99 for all AEDs. For the standards, the accuracy ranged from 89.3% to 111.8%. The within-run coefficient of variation (CVw) value was ≤9.7%, the between-run coefficient of variation (CVb) was ≤16.2%, and the total variability (CVt) was ≤16.8%. For the quality controls (QCs), accuracy ranged from 89.3% to 108.8%, CVw was ≤9.6%, CVb was ≤14.1%, and CVt was ≤15.1%. The correlation r2 values for comparison of this assay with existing validated assays in our laboratory (GC-MS or LC-MS) were 0.95, 0.91, 0.87, 0.95, and 0.95 for PHT, LTG, CBZ, CBZ-E, and CBZ-Diol, respectively.

CYP2C9 - CYP3A4 Protein-Protein Interactions: Role of the Hydrophobic N- Terminus

Cytochromes P450 (P450s) interact with redox transfer proteins, including P450 reductase (CPR) and cytochrome b5 (b5), all being membrane-bound. In multiple in vitro systems, P450-P450 interactions also have been observed, resulting in alterations in enzymatic activity. The current work investigated the effects and mechanisms of interaction between CYP2C9 and CYP3A4 in a reconstituted system. CYP2C9-mediated metabolism of S-naproxen and S-flurbiprofen was inhibited up to 80% by coincubation with CYP3A4, although Km values were unchanged. Increasing CYP3A4 concentrations increased the degree of inhibition, whereas increasing CPR concentrations resulted in less inhibition. Addition of b5 only marginally affected the magnitude of inhibition. In contrast, CYP2C9 did not alter the CYP3A4-mediated metabolism of testosterone. The potential role of the hydrophobic N terminus on these interactions was assessed by incubating truncated CYP2C9 with full-length CYP3A4, and vice versa. In both cases, the inhibition was fully abolished, indicating an important role for hydrophobic forces in CYP2C9-CYP3A4 interactions. Finally, a CYP2C9/CYP3A4 heteromer complex was isolated by coimmunoprecipitation techniques, confirming the physical interaction of the proteins. These results show that the N-terminal membrane binding domains of CYP2C9 and CYP3A4 are involved in heteromer complex formation and that at least one consequence is a reduction in CYP2C9 activity.

CYP2D6-CYP2C9 protein-protein interactions and isoform-selective effects on substrate binding and catalysis.

Cytochrome P450 (P450) protein-protein interactions have been observed with various in vitro systems. It is interesting to note that these interactions seem to be isoform-dependent, with some combinations producing no effect and others producing increased or decreased catalytic activity. With some exceptions, most of the work to date has involved P450s from rabbit, rat, and other animal species, with few studies including human P450s. In the studies presented herein, the interactions of two key drug-metabolizing enzymes, CYP2C9 and CYP2D6, were analyzed in a purified, reconstituted enzyme system for changes in both substrate-binding affinity and rates of catalysis. In addition, an extensive study was conducted as to the “order of mixing” for the reconstituted enzyme system and the impact on the observations. CYP2D6 coincubation inhibited CYP2C9-mediated (S)-flurbiprofen metabolism in a protein concentration-dependent manner. Vmax values were reduced by up to 50%, but no appreciable effect on Km was observed. Spectral binding studies revealed a 20-fold increase in the KS of CYP2C9 toward (S)-flurbiprofen in the presence of CYP2D6. CYP2C9 coincubation had no effect on CYP2D6-mediated dextromethorphan O-demethylation. The order of combination of the proteins (CYP2C9, CYP2D6, and cytochrome P450 reductase) influenced the magnitude of catalysis inhibition as well as the ability of increased cytochrome P450 reductase to attenuate the change in activity. A simple model, congruent with current results and those of others, is proposed to explain oligomer formation. In summary, CYP2C9-CYP2D6 interactions can alter catalytic activity and, thus, influence in vitro-in vivo correlation predictions.

Electrocatalytic Drug Metabolism by CYP2C9 Bonded to A Self-Assembled Monolayer-Modified Electrode

Cytochrome P450 (P450) enzymes typically require the presence of at least cytochrome P450 reductase (CPR) and NADPH to carry out the metabolism of xenobiotics. To address whether the need for redox transfer proteins and the NADPH cofactor protein could be obviated, CYP2C9 was bonded to a gold electrode through an 11-mercaptoundecanoic acid and octanethiol self-assembled monolayer (SAM) through which a current could be applied. Cyclic voltammetry demonstrated direct electrochemistry of the CYP2C9 enzyme bonded to the electrode and fast electron transfer between the heme iron and the gold electrode. To determine whether this system could metabolize warfarin analogous to microsomal or expressed enzyme systems containing CYP2C9, warfarin was incubated with the CYP2C9-SAM-gold electrode and a controlled potential was applied. The expected 7-hydroxywarfarin metabolite was observed, analogous to expressed CYP2C9 systems, wherein this is the predominant metabolite. Current-concentration data generated with increasing concentrations of warfarin were used to determine the Michaelis-Menten constant (Km) for the hydroxylation of warfarin (3 μM), which is in good agreement with previous literature regarding Km values for this reaction. In summary, the CYP2C9-SAM-gold electrode system was able to carry out the metabolism of warfarin only after application of an electrical potential, but in the absence of either CPR or NADPH. Furthermore, this system may provide a unique platform for both studying P450 enzyme electrochemistry and as a bioreactor to produce metabolites without the need for expensive redox transfer proteins and cofactors.

A Systematic Evaluation of Solubility Enhancing Excipients to Enable the Generation of Permeability Data for Poorly Soluble Compounds in Caco-2 Model

The study presented here identified and utilized a panel of solubility enhancing excipients to enable the generation of flux data in the Human colon carcinoma (Caco-2) system for compounds with poor solubility. Solubility enhancing excipients Dimethyl acetamide (DMA) 1 % v/v, polyethylene glycol (PEG) 400 1% v/v, povidone 1% w/v, poloxamer 188 2.5% w/v and bovine serum albumin (BSA) 4% w/v did not compromise Caco-2 monolayer integrity as assessed by trans-epithelial resistance measurement (TEER) and Lucifer yellow (LY) permeation. Further, these excipients did not affect P-glycoprotein (P-gp) mediated bidirectional transport of digoxin, permeabilities of high (propranolol) or low permeability (atenolol) compounds, and were found to be inert to Breast cancer resistant protein (BCRP) mediated transport of cladribine. This approach was validated further using poorly soluble tool compounds, atazanavir (poloxamer 188 2.5% w/v) and cyclosporine A (BSA 4% w/v) and also applied to new chemical entity (NCE) BMS-A in BSA 4% w/v, for which Caco-2 data could not be generated using the traditional methodology due to poor solubility (<1 µM) in conventional Hanks balanced salt solution (HBSS). Poloxamer 188 2.5% w/v increased solubility of atazanavir by >8 fold whereas BSA 4% w/v increased the solubility of cyclosporine A and BMS-A by >2-4 fold thereby enabling permeability as well as efflux liability estimation in the Caco-2 model with reasonable recovery values. To conclude, addition of excipients such as poloxamer 188 2.5% w/v and BSA 4% w/v to HBSS leads to a significant improvement in the solubility of the poorly soluble compounds resulting in enhanced recoveries without modulating transporter-mediated efflux, expanding the applicability of Caco-2 assays to poorly soluble compounds.

Characterization of Recombinantly Expressed Rat and Monkey Intestinal Alkaline Phosphatases: In Vitro Studies and In Vivo Correlations

Intestinal alkaline phosphatases (IALPs) are widely expressed in the brush border of epithelial cells of the intestinal mucosa. Although their physiologic role is unclear, they are very significant when it comes to the release of bioactive parent from orally dosed phosphate prodrugs. Such prodrugs can be resistant to cleavage by IALP, or alternatively undergo rapid cleavage leading to the release and precipitation of the less soluble parent. Because purified IALPs from preclinical species are not commercially available, and species differences have not been investigated to date, an effort was made to recombinantly express, purify, and characterize rat and cynomolgus monkey IALP (rIALP). Specifically, recombinant IALP (rIALP)-catalyzed cleavage of five prodrugs (fosphenytoin, clindamycin phosphate, dexamethasone phosphate, ritonavir phosphate, and ritonavir oxymethyl phosphate) was tested in vitro and parent exposure was assessed in vivo (rat only) following an oral dose of each prodrug. It was determined that the rate of phosphate cleavage in vitro varied widely; direct phosphates were more resistant to bioconversion, whereas faster conversion was observed with oxymethyl-linked prodrugs. Overall, the rat rIALP-derived data were qualitatively consistent with in vivo data; prodrugs that were readily cleaved in vitro rendered higher parent drug exposure in vivo. Of the five prodrugs tested, one (ritonavir phosphate) showed no conversion in vitro and no in vivo parent exposure. Finally, the apparent Km values obtained for fosphenytoin and clindamycin phosphate in vitro suggest that IALP is not likely to be saturated at therapeutic doses.

A Novel Liquid Chromatography Tandem Mass Spectrometry Method for the Estimation of Bilirubin Glucuronides and its Application to In Vitro Enzyme Assays

BACKGROUND Bilirubin is a toxic waste product of metabolism, eliminated mainly through UGT1A1 mediated conjugation to mono- and di-glucuronides. Due to the potentially low Km value of bilirubin glucuronidation, the quantitative sensitivity obtained with most UV/visible light detection methods are not sufficient to accurately calculate UGT1A1 enzyme kinetics at low bilirubin concentrations. In addition, bilirubin, as well as its metabolites, are unstable during sample preparation and bioanalysis. This necessitates the need for a rapid, sensitive and robust assay to measure bilirubin glucuronides. METHODS A robust LC-MS/MS method was developed to measure low levels of bilirubin glucuronides accurately from in vitro incubations, as well as stabilizing the analytes during sample preparation and analysis. The metabolites were quantified using a qualitative/quantitative approach utilizing UV to MS correction, thereby eliminating the need for synthetic standards. RESULTS The method was sensitive enough to quantify mono- and di-glucuronides as low as 3 nM from in vitro incubations, and kinetic data was determined for total glucuronide formation. The Km and Vmax values for total bilirubin glucuronide formations were determined to be 0.05 ± 0.01 μM and 181.9 ± 5.3 pmol/min/mg-protein, respectively, in human recombinant UGT1A1, and 0.23 ± 0.05 μM and 875 ± 45 pmol/min/mg protein in human liver microsomes (HLM). CONCLUSION We have developed a sensitive LC-MS/MS based method for the quantitation of bilirubin and its glucuronides from in vitro incubations. This method was successfully utilized to determine bilirubin glucuronidation kinetics in HLM and human rUGT1A1.

Non-specific Digestion Artifacts of Bovine Trypsin Exemplified with Surrogate Peptides for Endogenous Protein Quantitation

In bottom-up MS-based proteomic workflows, peptides are used as surrogates of proteins either for identification or quantification. Trypsin is the most preferred and widely used protease to generate these surrogate peptides for peptide based quantitative targeted proteomics. During one of such surrogate based protein quantitations, one of the experiments to understand the stability of the selected surrogate peptides during the process of digestion, yielded very low recovery (2%) compared to an equimolar amount of the pure peptide. This prompted us to investigate the reasons using high-resolution mass spectrometry (Q-Exactive plus) with data dependent MS2 acquisition (ddMS2) and parallel reaction monitoring (PRM) experiments. Investigations revealed non-specific cleavage of the peptide bond predominantly after phenylalanine (F) and tyrosine (Y) amino acid residues of the surrogate peptide standard by trypsin. PRM data showed high abundance of these truncated peptides compared to full length peptide, thus explaining the low recovery of peptide standards during the digestion process. Further investigations suggested a possible role of pseudo-trypsin (with chymotrypsin like specificity), which is likely formed from autolysis of trypsin during the course of digestion. Sequence grade trypsin (TPCK treated and reductively methylated) did not show any such non-specific cleavage of the peptides. Our findings, therefore, demonstrate the importance of choosing the appropriate trypsin for protein quantitation experiments. The propensity of trypsin that can form pseudotrypsin can produce non-specific digestion artifacts that can under predict the protein concentrations in surrogate peptide based quantification methods.

Metabolite Identification, Reaction Phenotyping, and Retrospective Drug-Drug Interaction Predictions of 17-Deacetylnorgestimate, the Active Component of the Oral Contraceptive Norgestimate

Ortho Tri-Cyclen, a two-drug cocktail comprised of ethinylestradiol and norgestimate (13-ethyl-17-acetoxy-18, 19-dinor-17α-pregn-4-en-20yn-3 oxime), is commonly prescribed to avert unwanted pregnancies in women of reproductive age. In vivo, norgestimate undergoes extensive and rapid deacetylation to produce 17-deacetylnorgestimate (NGMN), an active circulating metabolite that likely contributes significantly to norgestimate efficacy. Despite being of primary significance, the metabolism and reaction phenotyping of NGMN have not been previously reported. Hence, detailed biotransformation and reaction phenotyping studies of NGMN with recombinant cytochrome P450 (P450), recombinant uridine 5′-diphospho-glucuronosyltransferases, and human liver microsomes in the presence and absence of selective P450 inhibitors were conducted. It was found that CYP3A4 plays a key role in NGMN metabolism with a fraction metabolized (fm) of 0.57. CYP2B6 and to an even lesser extent CYP2C9 were also observed to catalyze NGMN metabolism. Using this CYP3A4 fm value, the predicted plasma concentration versus time area under the curve (AUC) change in NGMN using a basic/mechanistic static model was found to be within 1.3-fold of the reported NGMN AUC changes for four modulators of CYP3A4. In addition to NGMN, we have also elucidated the biotransformation of norgestrel (NG), a downstream norgestimate and NGMN metabolite, and found that CYP3A4 and UGT1A1 have a major contribution to the elimination of NG with a combined fm value of 1. The data presented in this paper will lead to better understanding and management of NGMN-based drug-drug interactions when norgestimate is coadministered with CYP3A4 modulators.

Absolute quantification of imipramine and its metabolites in vivo utilizing calibrators from radiolabeled in vitro incubations.

BACKGROUND We have demonstrated the use of a single-point calibration approach, derived from in vitro metabolite identification studies utilizing radiolabeled imipramine, that allows for the quantitation of metabolites from in vivo studies in the absence of metabolite synthetic standards. RESULTS From the in vivo study of imipramine in rats, the concentration of parent and metabolites were determined using the single-point calibration approach. Sixty seven percent of the dosed imipramine was recovered within 24 h, with 95 and 5% of drug-related material detected in feces and urine, respectively. CONCLUSION Using a novel single-point calibration approach from radiolabeled in vitro studies, we quantified metabolites in vivo and determined various disposition pathways.