Clark Williard

Sirolimus PK trial: A pharmacokinetic study of the sirolimus-eluting Bx Velocity stent in patients with de novo coronary lesions

This study was conducted to assess the systemic drug release and distribution of sirolimus‐eluting stents. Early results with sirolimus‐eluting stents have demonstrated a favorable outcome for reducing restenosis post coronary intervention. However, the clinical systemic pharmacokinetics of sirolimus released from these stents has not been investigated. Sirolimus‐eluting stents (150–178 mcg/18 mm stent) were implanted in 19 patients with coronary artery disease using standard techniques. Blood samples were obtained at multiple times to determine the kinetics of sirolimus release and elimination. Non‐compartmental analysis showed that the maximum blood concentration of sirolimus occurred between 3 and 4 hr after implantation, with a peak concentration of 0.57 ± 0.12 ng/mL (mean ± SD) and 1.05 ± 0.39 ng/mL in patients receiving one or two stents, respectively. Terminal‐phase elimination half‐life was independent of the number of stents and averaged at 213 hr, a value longer than that seen in patients following oral dosing. The apparent clearance was 1.46 ± 0.45 L/hr with an apparent volume of distribution in the terminal phase of 407 ± 111 L (data for both stent doses pooled). Minimal measurable blood levels were detectable at 7 days. Peak whole blood level following sirolimus stent implantation in humans is proportional to the number of stents implanted. The prolonged terminal half‐life may reflect kinetics of blood clearance combined with continued drug elution and secondary local tissue release.

TSPYL5 SNPs: association with plasma estradiol concentrations and aromatase expression.

We performed a discovery genome-wide association study to identify genetic factors associated with variation in plasma estradiol (E2) concentrations using DNA from 772 postmenopausal women with estrogen receptor (ER)-positive breast cancer prior to the initiation of aromatase inhibitor therapy. Association analyses showed that the single nucleotide polymorphisms (SNP) (rs1864729) with the lowest P value (P = 3.49E-08), mapped to chromosome 8 near TSPYL5. We also identified 17 imputed SNPs in or near TSPYL5 with P values < 5E-08, one of which, rs2583506, created a functional estrogen response element. We then used a panel of lymphoblastoid cell lines (LCLs) stably transfected with ERα with known genome-wide SNP genotypes to demonstrate that TSPYL5 expression increased after E2 exposure of cells heterozygous for variant TSPYL5 SNP genotypes, but not in those homozygous for wild-type alleles. TSPYL5 knockdown decreased, and overexpression increased aromatase (CYP19A1) expression in MCF-7 cells, LCLs, and adipocytes through the skin/adipose (I.4) promoter. Chromatin immunoprecipitation assay showed that TSPYL5 bound to the CYP19A1 I.4 promoter. A putative TSPYL5 binding motif was identified in 43 genes, and TSPYL5 appeared to function as a transcription factor for most of those genes. In summary, genome-wide significant SNPs in TSPYL5 were associated with elevated plasma E2 in postmenopausal breast cancer patients. SNP rs2583506 created a functional estrogen response element, and LCLs with variant SNP genotypes displayed increased E2-dependent TSPYL5 expression. TSPYL5 induced CYP19A1 expression and that of many other genes. These studies have revealed a novel mechanism for regulating aromatase expression and plasma E2 concentrations in postmenopausal women with ER(+) breast cancer.

Estrogens and their precursors in postmenopausal women with early breast cancer receiving anastrozole.

PURPOSE We determined hormone concentrations (estradiol [E2], estrone [E1], estrone conjugates [E1-C], androstenedione [A], testosterone [T]) before and on anastrozole therapy where we also determined plasma concentrations of anastrozole and its metabolites. EXPERIMENTAL Postmenopausal women who were to receive adjuvant anastrozole for resected early breast cancer were studied. Pretreatment, blood samples were obtained for the acquisition of DNA and for plasma hormone measurements (E2, E1, E1-C, A, and T). A second blood draw was obtained at least 4 weeks after starting anastrozole for hormone, anastrozole and metabolite measurements. For hormone assays, a validated bioanalytical method using gas chromatography negative ionization tandem mass spectrometry was used. Anastrozole and metabolite assays involved extraction of plasma followed by LC/MS/MS assays. RESULTS 649 patients were evaluable. Pretreatment and during anastrozole, there was large inter-individual variability in E2, E1, and E1-C as well as anastrozole and anastrozole metabolite concentrations. E2 and E1 concentrations were below the lower limits of quantitation in 79% and 70%, respectively, of patients on anastrozole therapy, but those with reliable concentrations had a broad range (0.627-234.0 pg/mL, 1.562-183.2 pg/mL, respectively). Considering E2, 8.9% had the same or higher concentration relative to baseline while on anastrozole, documented by the presence of drug. CONCLUSIONS We demonstrated large inter-individual variability in anastrozole and anastrozole metabolite concentrations as well as E1, E2, E1-C, A, and T concentrations before and while on anastrozole. These findings suggest that the standard 1mg daily dose of anastrozole is not optimal for a substantial proportion of women with breast cancer.

Validation of a method for quantifying enzalutamide and its major metabolites in human plasma by LC–MS/MS

BACKGROUND Enzalutamide is an androgen receptor inhibitor that targets multiple steps in the androgen receptor signaling pathway. Oral enzalutamide was recently approved by the US FDA and health authorities in other regions for the treatment of patients with metastatic castration-resistant prostate cancer who previously received docetaxel. The objective of this study was to validate a method for quantification of enzalutamide and its two major metabolites in human plasma. RESULTS The analytes were extracted from plasma by an LLE procedure, separated by reversed phase HPLC and detected by MS/MS in positive mode ESI. The quantitation range was 0.0200-50.0 µg/ml. CONCLUSION The method proved to be rapid and simple, and met FDA validation criteria.

Safety and Pharmacokinetics of Sirolimus-eluting Stents in the Canine Cerebral Vasculature: 180 Day Assessment

OBJECTIVE:We evaluated local and systemic pharmacokinetics and pharmacodynamics of sirolimus-eluting stents (SES) in canine cerebral vessels. METHODS:SES (1.5 × 8 mm, 79 &mgr;g/479 &mgr;g sirolimus) and control stents (1.5 × 8 mm stainless steel with or without polymer) were implanted in canine basilar and ventral spinal arteries. Animals were sacrificed for local pharmacokinetic (36 animals at 1, 3, 8, 30, 90, 180 days) and pharmacodynamic (60 animals at 3, 30, 90, 180 days) assessment. RESULTS:Postrecovery adverse clinical events were not serious, requiring no unscheduled treatment. Histologically, brain and spinal cord sections revealed scattered microinfarcts and minimal gliosis consistent with postprocedure changes in all four stent-treatment groups. All stented vessels at all time points demonstrated good luminal patency with low injury and inflammation scores and no thrombosis of either stented or branch arteries. Endothelialization was complete in all stent groups by 30 days. Intimal smooth muscle cell scores were reduced in both SES groups at 30, 90, and 180 days. Systemic sirolimus levels peaked between 1 and 7 hours postimplant (maximum concentration, 1.2 ± 1.47, 79 &mgr;g; 4.5 ± 1.23 ng/ml, 479 &mgr;g), then declined rapidly to 1 ng/ml or less by 96 hours. Peak local tissue sirolimus levels were 41.5 ng/mg (79 &mgr;g) and 65 ng/mg (479 &mgr;g). CONCLUSION:SES in canine cerebral vessels were associated with good luminal patency to 180 days, with complete endothelialization and no evidence of acute thrombosis. This model has shown that SES deployed within the brain do not cause neurotoxicity during a 180-day time course, even when exaggerated doses are used. The findings support the contention that SES are safe to use and maintain patency in cerebral vessels.

The 10th GCC Closed Forum: rejected data, GCP in bioanalysis, extract stability, BAV, processed batch acceptance, matrix stability, critical reagents, ELN and data integrity and counteracting fraud

The 10th Global CRO Council (GCC) Closed Forum was held in Orlando, FL, USA on 18 April 2016. In attendance were decision makers from international CRO member companies offering bioanalytical services. The objective of this meeting was for GCC members to meet and discuss scientific and regulatory issues specific to bioanalysis. The issues discussed at this closed forum included reporting data from failed method validation runs, GCP for clinical sample bioanalysis, extracted sample stability, biomarker assay validation, processed batch acceptance criteria, electronic laboratory notebooks and data integrity, Health Canadas Notice regarding replicates in matrix stability evaluations, critical reagents and regulatory approaches to counteract fraud. In order to obtain the pharma perspectives on some of these topics, the first joint CRO-Pharma Scientific Interchange Meeting was held on 12 November 2016, in Denver, Colorado, USA. The five topics discussed at this Interchange meeting were reporting data from failed method validation runs, GCP for clinical sample bioanalysis, extracted sample stability, processed batch acceptance criteria and electronic laboratory notebooks and data integrity. The conclusions from the discussions of these topics at both meetings are included in this report.

The importance of chromatographic resolution when analyzing steroid biomarkers.

The analyses of endogenous substances as biomarkers presents challenges that are distinctly different from the analyses of drugs or other xenobiotic substances. This is particularly true for estrogens. When no matrix is available which does not contain some level of the biomarker of interest, specificity cannot be demonstrated. Therefore it cannot be known whether the analyte signal includes a response from another substance. This uncertainty is increased by the fact that biomarkers are often created as part of a complex biosynthetic process that also creates a large number of substances with very similar structures and sometimes the same mass. Because of this, the two most powerful selectivity tools in the analysis of drugs, mass selective detection and MS/MS, are often rendered ineffective. The only remaining selectivity tool is chromatography and as will be demonstrated these separations can be very challenging. Failure to achieve specificity is perhaps the leading cause for inaccuracy of biomarker data and inter-laboratory variability.

Bioanalytical method transfer considerations of chromatographic-based assays

Bioanalysis is an important part of the modern drug development process. The business practice of outsourcing and transferring bioanalytical methods from laboratory to laboratory has increasingly become a crucial strategy for successful and efficient delivery of therapies to the market. This chapter discusses important considerations when transferring various types of chromatographic-based assays in todays pharmaceutical research and development environment.

Abstract C114: Androstenedione levels in postmenopausal women with resected early-stage breast cancer are associated with SNPs in CYP11B1 and CYP11B2 identified by a genome-wide association study (GWAS).

Introduction: Androstenedione is a precursor for estrogen biosynthesis catalyzed by aromatase. Plasma androstenedione concentrations in postmenopausal women with early stage breast cancer display large interindividual variation (Ingle et al., Cancer Res. 70:3278–86, 2010). We performed a GWAS to identify single nucleotide polymorphisms (SNPs) associated with plasma levels of androstenedione in 776 postmenopausal women with resected early stage breast cancer. Methods: Plasma androstenedione concentrations were measured by GC-MS/MS. GWAS was performed using Illumina Human610-Quad BeadChips. The GWAS results were controlled for population stratification (PS) by Eigenstrat analyses. Plasma androstenedione concentrations were associated with genotype by using a quasi-likelihood model with adjustment for PS, BMI, age and other clinical variables associated with androstenedione concentrations (p Results: 563,945 SNPs were analyzed, followed by imputation. The genotyped SNP with the lowest p value, rs4736317 (p=4.61E-06), was on chromosome 8 near the CYP11B1 and CYP11B2 genes. This SNP had a multiplicative effect of 1.128 (95% Cl: 1.07, 1.19) and a MAF of 0.40. After imputation, 20 SNPs with apparent p Conclusion: We have identified a series of SNPs in a region of chromosome 8 in and around CYP11B1 and CYP11B2 that are associated with elevated plasma androstenedione concentrations in postmenopausal women with resected early stage breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C114.

Genes regulating estradiol and estrone-conjugate levels in postmenopausal women with resected early-stage breast cancer detected by a genome-wide association study (GWAS).

1001 Background: There is large inter-individual variation in baseline estrone (E1), estradiol (E2), E1-conjugate (E1-C), androstenedione (A), and testosterone (T) levels in postmenopausal women with resected early stage breast cancer (Ingle et al., Ca Res 70:3278-86, 2010). We hypothesized that genetic variation can explain some of this variation in baseline (before anastrozole) hormone levels and performed a GWAS. METHODS Baseline plasma was obtained for E1, E2, E1-C, A, and T assays by GC-MS-MS. Genotypes were determined with the Illumina Human610-Quad BeadChip. Eigenstrat analyses were performed to control for population stratification (PS). Analysis of each baseline hormone level was assessed with each genotype using a quasi-likelihood model because of the skewness in baseline hormone levels, with adjustment for PS as well as body mass index, age and any additional clinical variables associated with baseline hormone levels (p<0.01). SNPs were imputed within 200 Kb on either side of regions containing SNPs with p < 3E-05 using HapMap. RESULTS 563,945 SNPs were used in the analyses. SNP genotypes were modeled in terms of the number of minor alleles (0, 1 or 2), with SNPs achieving genome-wide significance after Bonferroni correction (p<5E-08) identified for both E2 and E1-C. For E2 (n=757 patients), the genotyped SNP (rs1864729) with the lowest p-value (p=1.13E-08) was on chromosome (chr) 8 and had a multiplicative effect (ME: estimated fold change per minor allele) of 1.62 (95% CI: 1.38, 1.91), with the closest gene being TSPYL5 (Testis-specific Y-encoded-like protein 5), which we showed to be regulated by E2. There were 8 imputed SNPs near rs1864729 with lower p-values (1.03E-08-3.85E-09). For E1-C (n=752 patients), one nonsynonymous SNP (rs4149056) on chr 12 in the SLCO1B1 gene (which encodes an E1-C transporter) was significant (p=5E-08) with ME 1.41 (95% CI: 1.25, 1.60). CONCLUSIONS This GWAS revealed novel loci associated with the baseline levels of E2 and E1-C in postmenopausal women that may be of importance for understanding the estrogen physiology of postmenopausal women and their response to aromatase inhibitor therapy.