Aortic Binding of AZD5248: Mechanistic Insight and Reactivity Assays To Support Lead Optimzation.

The oral dipeptidyl peptidase 1 (DPP1) inhibitor AZD5248 showed aortic binding in a rat quantitative whole-body autoradiography (QWBA) study, and its development was terminated prior to human dosing. A mechanistic hypothesis for this finding was established invoking reactivity with aldehydes involved in the cross-linking of elastin, a major component of aortic tissue. This was tested by developing a simple aldehyde chemical reactivity assay and a novel in vitro competitive covalent binding assay. Results obtained with AZD5248, literature compounds, and close analogues of AZD5248 support the mechanistic hypothesis and provide validation for the use of these assays in a two tier screening approach to support lead optimization. The strengths and limitations of these assays are discussed.

Cytomegalovirus infection and neonatal outcome in extremely preterm infants after freezing of maternal milk.

BACKGROUND: Cytomegalovirus (CMV) infection acquired from breast milk can cause serious illness in extremely preterm (EPT) infants (<28 weeks). Some neonatal centers freeze maternal milk (MM) to prevent CMV transmission; however, this practice is controversial. In this study, we assessed the CMV transmission rate and neonatal outcome in EPT infants after routine freezing of all MM. METHODS: EPT infants (n = 140) and their mothers were randomized to the intervention group (only freeze-thawed MM) or the control group (combined fresh and freeze-thawed MM). Freeze-thawed MM was frozen at -20°C for ≥3 days before thawing. Mothers had serological tests for CMV, and MM was analyzed for CMV by polymerase chain reaction and CMV culture. Infants underwent CMV screening with urine analysis by CMV-polymerase chain reaction and CMV culture until 12 weeks of age. RESULTS: Congenital CMV infection was detected in 2% of screened infants. The CMV transmission rate in infants fed with CMV-DNA positive milk was 8% (3 of 37) in the intervention group and 6% (2 of 33) in controls. All infants infected by CMV were asymptomatic. The final per-protocol analysis included 56 infants in the intervention group and 65 controls. Neonatal mortality was comparable between the groups (7% vs. 6%). Neonatal morbidity was similar, except for late onset Candida sepsis, which was more frequent in the controls (12% vs. 0%). CONCLUSIONS: Routine freezing of all MM did not affect the rate of CMV transmission but may help to prevent fungal sepsis in EPT infants. This observation merits further investigation.

Implementing Kangaroo mother care in a resource-limited setting in rural Bangladesh.

AIM: This study evaluated stable and unstable low birthweight infants admitted to a Kangaroo mother care (KMC) unit at a resource-limited rural hospital in Bangladesh. METHODS: This was a descriptive consecutive patient series study of 423 low birthweight neonates <2500 g enrolled from July 2007 to December 2010. KMC was initiated as soon as possible after birth, regardless of health, and we monitored skin-to-skin contact, weight gain, exclusive breastfeeding, length of hospital stay and death rates. RESULTS: Mean birthweight was 1796 g, and mean gestational age was 34.9 weeks. Mean (median, 90th percentile) time of skin-to-skin initiation for stable and unstable neonates was 1.1 h (0.3-2.5) and 1.7 h (0.3-3.0), respectively. Adjusted mean daily skin-to-skin contact duration was significantly higher for unstable infants. About 99% of neonates were exclusively breastfed. The death rate was 8.3% (stable 1.9%, unstable 19%) at discharge. Neonatal mortality rate was 90 per 1000 live births (stable: 23 per 1000; unstable: 203 per 1000). CONCLUSION: Skin-to-skin duration was higher for unstable than stable low birthweight infants, and exclusive breastfeeding was almost universal at discharge. KMC was suitable for unstable infants and may be successfully implemented in resource-limited hospitals.

Development and use of a quantum dot probe to track multiple yeast strains in mixed culture.

Saccharomyces cerevisiae strains vary in their ability to develop and enhance sensory attributes of alcoholic beverages and are often found growing in mixed strain fermentations; however, quantifying individual strains is challenging due to quantification inaccuracies, low marker longevity, and compromised kinetics. We developed a fluorescent probe, consisting of glutathione molecules conjugated to a quantum dot (QD). Two S. cerevisiae strains were incubated with different coloured probes (QD attached to glutathione molecules, QD-GSH), fermented at multiple ratios, and quantified using confocal microscopy. The QD method was compared with a culture method using microsatellite DNA analysis (MS method). Probes were taken up by an ADP1 encoded transporter, transferred from mother cell to daughter cell, detectable in strains throughout fermentation, and were non-toxic. This resulted in a new quantification method that was more accurate and efficient than the MS method.

Implantation and persistence of yeast inoculum in Pinot noir fermentations at three Canadian wineries.

Inoculated fermentations are practiced in most wine regions of the world. This type of fermentation involves adding a commercial Saccharomyces cerevisiae strain as an inoculant. It is often assumed that the inoculant maintains dominance throughout the fermentation; however, sometimes commercial or indigenous yeasts, which were not intentionally added, end up as the dominant yeast in the winery fermentation. The aim of this study was to compare implantation/persistence of inoculants among three Canadian wineries (Quails' Gate, Cedar Creek, and Road 13 wineries). In 2010, three inoculated fermentation tanks at each of three wineries were sampled at four stages of fermentation (pre-inoculation, early, mid, and end). In addition, results from the end stage of fermentation, from two of the three wineries, were compared among different vintages (resulting in a 4-year comparison at Quails' Gate winery and a 2-year comparison at Cedar Creek winery). Strains of S. cerevisiae were discriminated by microsatellite analysis and identified using commercial microsatellite databases, whereas DNA sequencing was used to identify non-Saccharomyces. The percent implantation/persistence of the inoculum was significantly lower at Quails' Gate and Cedar Creek wineries as compared with the Road 13 winery in the 2010 vintage. Relatively low persistence of the inoculum at Quails' Gate winery was also found in the 2009 vintage, but low values were not found at Quails' Gate winery in 2011 and 2012 or at Cedar Creek winery in 2012. In all tanks having <80% relative abundance of the inoculant, the commercial strain (Lalvin ICV-D254®/Fermol® Premier Cru) was the dominant or co-dominant yeast. Our findings highlight year-to-year variation in inoculum implantation/persistence and the idea that unless strain typing of S. cerevisiae is conducted at the winery, there are no obvious fermentation factors that would indicate a relatively low inoculum implantation/persistence.

A correlation between the in vitro drug toxicity of drugs to cell lines that express human P450s and their propensity to cause liver injury in humans.

Drug toxicity to T-antigen-immortalized human liver epithelial (THLE) cells stably transfected with plasmid vectors that encoded human cytochrome P450s 1A2, 2C9, 2C19, 2D6, or 3A4, or an empty plasmid vector (THLE-Null), was investigated. An automated screening platform, which included 1% dimethyl sulfoxide (DMSO) vehicle, 2.7% bovine serum in the culture medium, and assessed 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium reduction, was used to evaluate the cytotoxicity of 103 drugs after 24h. Twenty-two drugs caused cytotoxicity to THLE-Null cells, with EC50 ≤ 200 µM; 21 of these drugs (95%) have been reported to cause human liver injury. Eleven drugs exhibited lower EC50 values in cells transfected with CYP3A4 (THLE-3A4 cells) than in THLE-Null cells; 10 of these drugs (91%) caused human liver injury. An additional 8 drugs, all of which caused human liver injury, exhibited potentiated THLE-3A4 cell toxicity when evaluated using a manual protocol that included 0.2% or 1% DMSO, but not bovine serum. Fourteen of the drugs that exhibited potentiated THLE-3A4 cell toxicity are known to be metabolized by P450s to reactive intermediates. These drugs included troglitazone, which was shown to undergo metabolic bioactivation and covalent binding to proteins in THLE-3A4 cells. A single drug (rimonabant) exhibited marked THLE cell toxicity but did not cause human liver injury; this drug had very low reported plasma exposure. These results indicate that evaluation of toxicity to THLE-Null and THLE-3A4 cell lines during drug discovery may aid selection of drugs with reduced propensity to cause drug-induced liver injury and that consideration of human exposure is required to enhance data interpretation.

In vitro exploration of potential mechanisms of toxicity of the human hepatotoxic drug fenclozic acid.

The carboxylic acid NSAID fenclozic acid exhibited an excellent preclinical safety profile and promising clinical efficacy, yet was withdrawn from clinical development in 1971 due to hepatotoxicity observed in clinical trials. A variety of modern in vitro approaches have been used to explore potential underlying mechanisms. Covalent binding studies were undertaken with [(14)C]-fenclozic acid to investigate the possible role of reactive metabolites. Time-dependent covalent binding to protein was observed in NADPH-supplemented liver microsomes, although no metabolites were detected in these incubations or in reactive metabolite trapping experiments. In human hepatocytes, covalent binding was observed at lower levels than in microsomes and a minor uncharacterizable metabolite was also observed. In addition, covalent binding was observed in incubations undertaken with dog and rat hepatocytes, where a taurine conjugate of the drug was detected. Although an acyl glucuronide metabolite was detected when liver microsomes from human, rat and dog were supplemented with UDPGA, there was no detectable UDPGA-dependent covalent binding. No effects were observed when fenclozic acid was assessed for P450-dependent and P450-independent cytotoxicity to THLE cell lines, time-dependent inhibition of five major human cytochrome P450 enzymes, inhibition of the biliary efflux transporters BSEP and MRP2 or mitochondrial toxicity to THLE or HepG2 cells. These data suggest that Phase 1 bioactivation plays a role in the hepatotoxicity of fenclozic acid and highlight the unique insight into mechanisms of human drug toxicity that can be provided by investigations of biotransformation and covalent binding to proteins.

Bioactivation of the cannabinoid receptor antagonist rimonabant to a cytotoxic iminium ion metabolite.

The cannabinoid type 1 receptor (CB1r) antagonist rimonabant was approved in 2006 for the treatment of obesity but was withdrawn in 2008 due to serious drug-related psychiatric disorders. In vitro metabolism studies with rimonabant have revealed high levels of reactive metabolite formation, which resulted in irreversible time-dependent P450 3A4 inhibition and in covalent binding to microsomal proteins. In the present study, an in vitro approach has been used to explore whether metabolic bioactivation of rimonabant might result in cell toxicity. A panel of SV40-T-antigen-immortalized human liver derived (THLE) cells that had been transfected with vectors encoding various human cytochrome P450 enzymes (THLE-1A2, 2C9, 2C19, 2D6, and 3A4) or with an empty vector (THLE-Null) were exposed to rimonabant. Cell toxicity and covalent binding to cellular proteins were evaluated, as was metabolite formation. Rimonabant exhibited markedly potentiated dose and time dependent cytotoxicity to THLE-3A4 cells, compared to that of all other THLE cell lines. This was accompanied by high levels of covalent binding of [(14)C]-rimonabant to THLE-3A4 cell proteins (1433 pmol drug equivalents/mg protein) and the formation of several metabolites that were not generated by THLE-Null cells. These included N-aminopiperidine (NAP) and an iminium ion species. However, no toxicity was observed when THLE cells were incubated with NAP. Glutathione depletion did not alter the observed potent cell cytotoxicity of rimonabant to THLE-3A4 cells. Preincubation of THLE-3A4 cells with the cytochrome P450 3A4 inhibitor ritonavir blocked the selective toxicity of rimonabant to these cells. In addition, ritonavir pretreatment blocked the metabolism of the compound in the cells and thereby significantly decreased the covalent binding of [(14)C]-rimonabant to THLE-3A4 cell proteins. We conclude that the potent toxicity of rimonabant in THLE-3A4 cells occurs by a mechanistic sequence, which is initiated by cytochrome P450 3A4 mediated formation of a highly cytotoxic reactive iminium ion metabolite that binds covalently to cellular proteins.

Tryptophan 697 modulates hydride and interflavin electron transfer in human methionine synthase reductase.

Human methionine synthase reductase (MSR), a diflavin oxidoreductase, plays a vital role in methionine and folate metabolism by sustaining methionine synthase (MS) activity. MSR catalyzes the oxidation of NADPH and shuttles electrons via its FAD and FMN cofactors to inactive MS-cob(II)alamin. A conserved aromatic residue (Trp697) positioned next to the FAD isoalloxazine ring controls nicotinamide binding and catalysis in related flavoproteins. We created four MSR mutants (W697S, W697H, S698Δ, and S698A) and studied their associated kinetic behavior. Multiwavelength stopped-flow analysis reveals that NADPH reduction of the C-terminal Ser698 mutants occurs in three resolvable kinetic steps encompassing transfer of a hydride ion to FAD, semiquinone formation (indicating FAD to FMN electron transfer), and slow flavin reduction by a second molecule of NADPH. Corresponding experiments with the W697 mutants show a two-step flavin reduction without an observable semiquinone intermediate, indicating that W697 supports FAD to FMN electron transfer. Accelerated rates of FAD reduction, steady-state cytochrome c(3+) turnover, and uncoupled NADPH oxidation in the S698Δ and W697H mutants may be attributed to a decrease in the energy barrier for displacement of W697 by NADPH. Binding of NADP(+), but not 2',5'-ADP, is tighter for all mutants than for native MSR. The combined studies demonstrate that while W697 attenuates hydride transfer, it ensures coenzyme selectivity and accelerates FAD to FMN electron transfer. Moreover, analysis of analogous cytochrome P450 reductase (CPR) variants points to key differences in the driving force for flavin reduction and suggests that the conserved FAD stacking tryptophan residue in CPR also promotes interflavin electron transfer.