Synthesis and Evaluation of 2‘-Substituted 4-(4‘-Carboxy- or 4‘-carboxymethylbenzylidene)-<i>N</i>-acylpiperidines: Highly Potent and in Vivo Active Steroid 5α-Reductase Type 2 Inhibitors

Sixteen compounds derived from N-acyl-4-benzylidenepiperidine-4‘-carboxylic acids were synthesized and evaluated for inhibition of rat and human steroid 5α-reductase isozymes types 1 and 2. In the dicyclohexylacetyl series, fluorination in the 2-position of the benzene nucleus (15), exchange of the carboxy group by a carboxymethyl moiety (20), and combination of both structural modifications (25) led to highly active inhibitors of the human type 2 isozyme (IC50 values:  15, 11 nM; 20, 6 nM; 25, 7 nM; finasteride, 5 nM). In vivo all compounds tested markedly reduced the prostate weights in castrated testosterone-treated rats. Oral activity was shown for compound 7. From the finding that compound 15 is active in the rat, although it is a rather poor inhibitor of the rat enzyme and is a strong inhibitor of the human enzyme, it is concluded that it should be highly potent in men

LC–MS/MS Bioanalysis of Radioligand Therapeutic Drug Candidate for Preclinical Toxicokinetic Assessment

Radioligand therapy (RLT) has gained significant momentum in recent years in the diagnosis, treatment, and monitoring of cancers. In preclinical development, the safety profile of RLT drug candidate(s) is investigated at relatively low dose levels using the cold (non-radioactive, e.g., 175Lu) ligand as a surrogate of the hot (radioactive, e.g., 177Lu) one in the “ligand-linker-chelator” complex. The formulation of the test article used in preclinical safety studies contains a mixture of free ligand (i.e., ligand-linker-chelator without metal) and cold ligand (i.e., ligand-linker-chelator with non-radioactive metal) in a similar molar ratio as seen under the manufacturing conditions for the RLT drug for clinical use, where only a fraction of free ligand molecules chelate the radioactive metal to form a hot ligand. In this very first report of LC–MS/MS bioanalysis of RLT molecules in support of a regulated preclinical safety assessment study, a highly selective and sensitive LC–MS/MS bioanalytical method was developed for the simultaneous determination of free ligand (NVS001) and cold ligand (175Lu-NVS001) in rat and dog plasma. Several unexpected technical challenges in relation to LC–MS/MS of RLT molecules were successfully addressed. The challenges include poor assay sensitivity of the free ligand NVS001, formation of the free ligand (NVS001) with endogenous metal (e.g., potassium), Ga loss from the Ga-chelated internal standard during sample extraction and analysis, “instability” of the analytes at low concentrations, and inconsistent IS response in the extracted plasma samples. The methods were validated according to the current regulatory requirements in a dynamic range of 0.5–250 ng/mL for both the free and cold ligands using a 25 μL sample volume. The validated method was successfully implemented in sample analysis in support of regulated safety studies, with very good results from incurred sample reanalysis. The current LC–MS/MS workflow can be expanded to quantitative analysis of other RLTs in support of preclinical RLT drug development

New Microfluidic-Based Sampling Procedure for Overcoming the Hematocrit Problem Associated with Dried Blood Spot Analysis

Hematocrit (Hct) is one of the most critical issues associated with the bioanalytical methods used for dried blood spot (DBS) sample analysis. Because Hct determines the viscosity of blood, it may affect the spreading of blood onto the filter paper. Hence, accurate quantitative data can only be obtained if the size of the paper filter extracted contains a fixed blood volume. We describe for the first time a microfluidic-based sampling procedure to enable accurate blood volume collection on commercially available DBS cards. The system allows the collection of a controlled volume of blood (e.g., 5 or 10 μL) within several seconds. Reproducibility of the sampling volume was examined in vivo on capillary blood by quantifying caffeine and paraxanthine on 5 different extracted DBS spots at two different time points and in vitro with a test compound, Mavoglurant, on 10 different spots at two Hct levels. Entire spots were extracted. In addition, the accuracy and precision (<i>n</i> = 3) data for the Mavoglurant quantitation in blood with Hct levels between 26% and 62% were evaluated. The interspot precision data were below 9.0%, which was equivalent to that of a manually spotted volume with a pipet. No Hct effect was observed in the quantitative results obtained for Hct levels from 26% to 62%. These data indicate that our microfluidic-based sampling procedure is accurate and precise and that the analysis of Mavoglurant is not affected by the Hct values. This provides a simple procedure for DBS sampling with a fixed volume of capillary blood, which could eliminate the recurrent Hct issue linked to DBS sample analysis

Association of replication origins with chromatin states and timing.

<p>Origins associated with a given chromatin state (in percent) correspond to origins that overlap with published chromatin state coordinates <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004282#pgen.1004282-Ernst1" target="_blank">[22]</a>, from the UCSC Genome Browser with chromHMM 1: active promoter, chromHMM 2: weak promoter, chromHMM 3: inactive/poised promoter, chromHMM 4: strong enhancer, chromHMM 5: strong enhancer, chromHMM 6: weak/poised enhancer, chromHMM 7: weak/poised enhancer, chromHMM 8: insulator, chromHMM 9: transcriptional transition, chromHMM 10: transcriptional elongation, chromHMM 11: weak transcribed, chromHMM 12: Polycomb-repressed, chromHMM 13: heterochromatin; low signal, chromHMM 14: repetitive/copy number variation, chromHMM 15: repetitive/copy number variation. The expected (Exp.) percentage and significance of association ( for significant enrichment, with and for significant depletion) were calculated with random genomic segments sampled from mappable regions (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004282#s4" target="_blank">Materials and Methods</a>).</p

Spatial interaction of K562 replication origins with chromatin marks in discriminant analysis.

<p>A: boxplot of the 3 discriminant axis (DA) coordinates of origins according to timing categories (from early to late origins, as explained in the Methods Section). B,C: correlation circles of the three discriminant axis, with distances to chromatin marks. Correlations between discriminant axes and distances to chromatin marks are given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004282#pgen-1004282-t007" target="_blank">Table 7</a>. High correlation means that the variables highly contribute to the creation of the axis.</p

Association of replication origins with chromatin marks.

<p>Chromatin mark coordinates were downloaded from the UCSC genome browser (ChIP-Seq peaks), and an origin of replication is associated with a given mark if the origin and the peak overlap. Expected (Exp.) percentage and significance of association ( for significant enrichment, with and for significant depletion) were computed with random genomic segments sampled from mappable regions (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004282#s4" target="_blank">Materials and Methods</a>).</p

Comparison of SNS-scan origins and SNS-SoleS origins.

<p>Overlap between SNS-Scan origins and SNS-SoleS origins <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004282#pgen.1004282-Besnard1" target="_blank">[10]</a>. % Overlap (SoleS in scan) corresponds to the number of scan origins that overlap with SoleS origins divided by the total number of scan origins. Expectations are assessed by randomly sampling genomic intervals on the mappable fraction of the human genome (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004282#s4" target="_blank">Materials and Methods</a>). SNS-SoleS were clustered so that origins less than 12 kb apart were clustered into one single (larger) origin.</p

Effect of histone marks associations on origins efficiency, length and density (K562).

<p>A: Percentage of origins associated with chromatin marks according to timing categories (from early to late origins, as explained in the Methods Section) and CGI association. B: Variations of origin efficiency (as defined by the number of reads divided by the length of the origin) with replication timing, mark associations, and association with CGIs. C: Variations of origin length in kb with replication timing, mark associations, and association with CGIs. D: Variations of origins density (as defined by the number of origins per Megabase) with replication timing, mark associations, and association with CGIs. Values correspond to mean values with 95% confidence intervals. A comparison of the solid and dashed lines highlights the effect of associations of marks. OpenMarks indicates origins associated with either H2AZ, H3K9ac or H3K4me3.</p

Global view of origins datasets and detections characteristics.

<p>Global view of origins datasets and detections characteristics.</p

Association of replication origins with CGIs and TSSs, as a function of replication timing category.

<p>Number and percentage of origins in each timing category (from early to late). Origins classified as CGI (or TSS) correspond to origins that strictly overlap with a CGI (or TSS). The positions of CGIs and TSSs were taken from the UCSC Genome Browser annotation. Expected (Exp.) percentage and significance of association ( for significant enrichment, with ) are computed with random genomic segments sampled from mappable regions (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004282#s4" target="_blank">Materials and Methods</a>).</p