Anna Furimsky

Gene Expression Analysis Reveals New Possible Mechanisms of Vancomycin-Induced Nephrotoxicity and Identifies Gene Markers Candidates

Vancomycin, one of few effective treatments against methicillin-resistant Staphylococcus aureus, is nephrotoxic. The goals of this study were to (1) gain insights into molecular mechanisms of nephrotoxicity at the genomic level, (2) evaluate gene markers of vancomycin-induced kidney injury, and (3) compare gene expression responses after iv and ip administration. Groups of six female BALB/c mice were treated with seven daily iv or ip doses of vancomycin (50, 200, and 400 mg/kg) or saline, and sacrificed on day 8. Clinical chemistry and histopathology demonstrated kidney injury at 400 mg/kg only. Hierarchical clustering analysis revealed that kidney gene expression profiles of all mice treated at 400 mg/kg clustered with those of mice administered 200 mg/kg iv. Transcriptional profiling might thus be more sensitive than current clinical markers for detecting kidney damage, though the profiles can differ with the route of administration. Analysis of transcripts whose expression was changed by at least twofold compared with vehicle saline after high iv and ip doses of vancomycin suggested the possibility of oxidative stress and mitochondrial damage in vancomycin-induced toxicity. In addition, our data showed changes in expression of several transcripts from the complement and inflammatory pathways. Such expression changes were confirmed by relative real-time reverse transcription–polymerase chain reaction. Finally, our results further substantiate the use of gene markers of kidney toxicity such as KIM-1/Havcr1, as indicators of renal injury.

Glucuronidation of trans‐resveratrol by human liver and intestinal microsomes and UGT isoforms

Resveratrol (trans‐resveratrol, trans‐3,5,4′‐trihydroxystilbene) is a naturally occurring stilbene analogue found in high concentrations in red wine. There is considerable research interest to determine the therapeutic potential of resveratrol, as it has been shown to have tumour inhibitory and antioxidant properties. This study was performed to investigate the glucuronidation of resveratrol and possible drug interactions via glucuronidation. Two glucuronide conjugates, resveratrol 3‐O‐glucuronide and resveratrol 4′‐O‐glucuronide, were formed by human liver and intestinal microsomes. UGT1A1 and UGT1A9 were predominantly responsible for the formation of the 3‐O‐glucuronide (Km = 149 μm) and 4′‐O‐glucuronide (Km = 365 μm), respectively. The glucuronide conjugates were formed at higher levels (up to 10‐fold) by intestinal rather than liver microsomes. Resveratrol was co‐incubated with substrates of UGT1A1 (bilirubin and 7‐ethyl‐10‐hydroxycamptothecin (SN‐38)) and UGT1A9 (7‐hydroxytrifluoromethyl coumarin (7‐HFC)). No major changes were noted in bilirubin glucuronidation in the presence of resveratrol. Resveratrol significantly inhibited the glucuronidation of SN‐38 (Ki = 6.2 ± 2.1 μm) and 7‐HFC (Ki = 0.6 ± 0.2 μm). Hence, resveratrol has the potential to inhibit the glucuronidation of concomitantly administered therapeutic drugs or dietary components that are substrates of UGT1A1 and UGT1A9.

A parallel chiral-achiral liquid chromatographic method for the determination of the stereoisomers of ketamine and ketamine metabolites in the plasma and urine of patients with complex regional pain syndrome

A parallel chiral/achiral LC-MS/MS assay has been developed and validated to measure the plasma and urine concentrations of the enantiomers of ketamine, (R)- and (S)-Ket, in complex regional pain syndrome (CRPS) patients receiving a 5-day continuous infusion of a sub-anesthetic dose of (R,S)-Ket. The method was also validated for the determination of the enantiomers of the Ket metabolites norketamine, (R)- and (S)-norKet and dehydronorketamine, (R)- and (S)-DHNK, as well as the diastereomeric metabolites hydroxynorketamine, (2S,6S)-/(2R,6R)-HNK and two hydroxyketamines, (2S,6S)-HKet and (2S,6R)-Hket. In this method, (R,S)-Ket, (R,S)-norKet and (R,S)-DHNK and the diastereomeric hydroxyl-metabolites were separated and quantified using a C(18) stationary phase and the relative enantiomeric concentrations of (R,S)-Ket, (R,S)-norKet and (R,S)-DHNK were determined using an AGP-CSP. The analysis of the results of microsomal incubations of (R)- and (S)-Ket and a plasma and urine sample from a CRPS patient indicated the presence of 10 additional compounds and glucuronides. The data from the analysis of the patient sample also demonstrated that a series of HNK metabolites were the primary metabolites in plasma and (R)- and (S)-DHNK were the major metabolites found in urine. The results suggest that norKet is the initial, but not the primary metabolite and that downstream norKet metabolites play a role in (R,S)-Ket-related pain relief in CRPS patients.

Lead optimization of 3-carboxyl-4(1H)-quinolones to deliver orally bioavailable antimalarials.

Malaria is a protozoal parasitic disease that is widespread in tropical and subtropical regions of Africa, Asia, and the Americas and causes more than 800,000 deaths per year. The continuing emergence of multidrug-resistant Plasmodium falciparum drives the ongoing need for the development of new and effective antimalarial drugs. Our previous work has explored the preliminary structural optimization of 4(1H)-quinolone ester derivatives, a new series of antimalarials related to the endochins. Herein, we report the lead optimization of 4(1H)-quinolones with a focus on improving both antimalarial potency and bioavailability. These studies led to the development of orally efficacious antimalarials including quinolone analogue 20g, a promising candidate for further optimization.

Effect of resveratrol on 17β-estradiol sulfation by human hepatic and jejunal S9 and recombinant sulfotransferase 1E1

The purpose of this study was to investigate the sulfation of resveratrol (3,5,4′-trihydroxystilbene) and its potential to exhibit drug-drug interactions via sulfation. The possible interaction of resveratrol with 17β-estradiol (E2), a major estrogen hormone and prototypic substrate for sulfate conjugation, was studied. Resveratrol and E2 are both known to undergo sulfate conjugation catalyzed by human sulfotransferases (SULTs). Resveratrol is a phytoestrogen with mixed estrogen agonist/antagonist properties that is being developed as a chemopreventive agent. The sulfate conjugation of E2 and resveratrol were studied individually using S9 fractions from human liver and jejunum as well as recombinant human SULT isoforms. The sulfation of E2 (3–20 nM) was then investigated in the presence of various concentrations (0, 0.5, 1, and 2 μM) of resveratrol using the two S9 preparations as well as recombinant SULT1E1, the major isoform responsible for E2 sulfation. Resveratrol inhibited E2 sulfation with estimated Ki values of 1.1 μM (liver), 0.6 μM (jejunum), and 2.3 μM (SULT1E1), concentrations that could be pharmacologically relevant. The results suggest that these phytoestrogens can potentially alter the homeostasis of estrogen levels. These findings also imply that resveratrol may inhibit the metabolism of other estrogen analogs or therapeutic agents such as ethinylestradiol or dietary components that are also substrates for SULT1E1.

In Vitro Metabolism and Stability of the Actinide Chelating Agent 3,4,3‐LI(1,2‐HOPO)

The hydroxypyridinonate ligand 3,4,3-LI(1,2-HOPO) is currently under development for radionuclide chelation therapy. The preclinical characterization of this highly promising ligand comprised the evaluation of its in vitro properties, including microsomal, plasma, and gastrointestinal fluid stability, cytochrome P450 inhibition, plasma protein binding, and intestinal absorption using the Caco-2 cell line. When mixed with active human liver microsomes, no loss of parent compound was observed after 60 min, indicating compound stability in the presence of liver microsomal P450. At the tested concentrations, 3,4,3-LI(1,2-HOPO) did not significantly influence the activities of any of the cytochromal isoforms screened. Thus, 3,4,3-LI(1,2-HOPO) is unlikely to cause drug-drug interactions by inhibiting the metabolic clearance of coadministered drugs metabolized by these enzymes. Plasma protein-binding assays revealed that the compound is protein-bound in dogs and less extensively in rats and humans. In the plasma stability study, the compound was stable after 1 h at 37°C in mouse, rat, dog, and human plasma samples. Finally, a bidirectional permeability assay demonstrated that 3,4,3-LI(1,2-HOPO) is not permeable across the Caco-2 monolayer, highlighting the need to further evaluate the effects of various compounds with known permeability enhancement properties on the permeability of the ligand in future studies.

Lead Optimization of Antimalarial Propafenone Analogues

Previously reported studies identified analogues of propafenone that had potent antimalarial activity, reduced cardiac ion channel activity, and properties that suggested the potential for clinical development for malaria. Careful examination of the bioavailability, pharmacokinetics, toxicology, and efficacy of this series of compounds using rodent models revealed orally bioavailable compounds that are nontoxic and suppress parasitemia in vivo. Although these compounds possess potential for further preclinical development, they also carry some significant challenges.

Pharmacokinetics and metabolism of (R,R)-methoxyfenoterol in rat

(R,R)-fenoterol (Fen), a β2-adrenoceptor agonist, is under clinical investigation in the treatment of congestive heart disease. The pharmacokinetics and metabolism of the 4-methoxyphenyl derivative of (R,R)-Fen, (R,R)-MFen, have been determined following intravenous and oral administration to the rat and compared with corresponding results obtained with (R,R)-Fen. Results from the study suggest that (R,R)-MFen can offer pharmacokinetic and metabolic advantages in comparison to an earlier (R,R)-Fen. The oral administration revealed that the net exposure of (R,R)-MFen was about three-fold higher than that of (R,R)-Fen (7.2 versus 2.3 min × nmol ml−1), while intravenous administration proved that the clearance was significantly reduced, 48 versus 146 ml min−1 kg−1, the T1/2 was significantly longer, 152.9 versus 108.9 min, and the area under the curve (AUC) was significantly increased, 300 versus 119 min × nmol ml−1. (R,R)-MFen was primarily cleared by glucuronidation associated with significant presystemic glucuronidation of the compound. After intravenous and oral administration of (R,R)-MFen, (R,R)-Fen and (R,R)-Fen-G were detected in the urine samples indicating that (R,R)-MFen was O-demethylated and subsequently conjugated to (R,R)-Fen-G. The total (R,R)-Fen and (R,R)-Fen-G as a percentage of the dose after intravenous administration was 3.6%, while after oral administration was 0.3%, indicating that only a small fraction of the drug escaped presystemic glucuronidation and was available for O-demethylation. The glucuronidation pattern was confirmed by the results from in vitro studies where incubation of (R,R)-MFen with rat hepatocytes produced (R,R)-MFen-G, (R,R)-Fen and (R,R)-Fen-G, while incubation with rat intestinal microsomes only resulted in the formation of (R,R)-MFen-G.

Pharmacokinetics and Metabolism of 4R-Cembranoid

4R-cembranoid (4R) is a natural cyclic diterpenoid found in tobacco leaves that displays neuroprotective activity. 4R protects against NMDA, paraoxon (POX), and diisopropylfluorophosphate (DFP) damage in rat hippocampal slices and against DFP in rats in vivo. The purpose of this study was to examine the metabolism and pharmacokinetics of 4R as part of its preclinical development as a neuroprotective drug. 10 µM 4R was found to be very stable in plasma for up to 1 hr incubation. 4R metabolism in human microsomes was faster than in the rat. Ten metabolites of 4R were detected in the microsomal samples; 6 dihydroxy and 4 monohydroxy forms of 4R. Male rats received a single dose of 4R at 6 mg/kg i.v., i.m., or s.c. The i.v. group had the highest plasma concentration of 1017 ng/mL. The t1/2 was 36 min and reached the brain within 10 min. The brain peak concentration was 6516 ng/g. The peak plasma concentration in the i.m. group was 163 ng/mL compared to 138 ng/mL in the s.c. group. The t1/2 of 4R after i.m. and s.c. administration was approximately 1.5 hr. The brain peak concentration was 329 ng/g in the i.m. group and 323 ng/g for the s.c. group. The brain to plasma ratio in the i.v. group was 6.4, reached 10 min after dose, whereas in the i.m. and s.c. groups was 2.49 and 2.48, respectively, at 90 min after dose. Our data show that 4R crosses the BBB and concentrates in the brain where it exerts its neuroprotective effect.

Structure-based lead optimization to improve antiviral potency and ADMET properties of phenyl-1H-pyrrole-carboxamide entry inhibitors targeted to HIV-1 gp120

We are continuing our concerted effort to optimize our first lead entry antagonist, NBD-11021, which targets the Phe43 cavity of the HIV-1 envelope glycoprotein gp120, to improve antiviral potency and ADMET properties. In this report, we present a structure-based approach that helped us to generate working hypotheses to modify further a recently reported advanced lead entry antagonist, NBD-14107, which showed significant improvement in antiviral potency when tested in a single-cycle assay against a large panel of Env-pseudotyped viruses. We report here the synthesis of twenty-nine new compounds and evaluation of their antiviral activity in a single-cycle and multi-cycle assay to derive a comprehensive structure-activity relationship (SAR). We have selected three inhibitors with the high selectivity index for testing against a large panel of 55 Env-pseudotyped viruses representing a diverse set of clinical isolates of different subtypes. The antiviral activity of one of these potent inhibitors, 55 (NBD-14189), against some clinical isolates was as low as 63 nM. We determined the sensitivity of CD4-binding site mutated-pseudoviruses to these inhibitors to confirm that they target HIV-1 gp120. Furthermore, we assessed their ADMET properties and compared them to the clinical candidate attachment inhibitor, BMS-626529. The ADMET data indicate that some of these new inhibitors have comparable ADMET properties to BMS-626529 and can be optimized further to potential clinical candidates.