Optimizing Early-stage Clinical Pharmacology Evaluation to Accelerate Clinical Development of Giredestrant in Advanced Breast Cancer.

Purpose: We describe the clinical pharmacology characterization of giredestrant in a first-in-human study.

Experimental Design: This phase Ia/Ib dose-escalation/-expansion study (NCT03332797) evaluated the safety, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of giredestrant in estrogen receptor-positive HER2-negative locally advanced/metastatic breast cancer. The single-agent dose-escalation stage evaluated giredestrant 10, 30, 90, or 250 mg once daily.

Volumetric absorptive microsampling-LC-MS/MS assays for quantitation of giredestrant in dried human whole blood.

Volumetric absorption microsampling devices offer minimally invasive and user-friendly collection of capillary blood in volumes as low as 10 μl. Herein we describe the assay validation for determination of the selective estrogen receptor degrader giredestrant (GDC-9545) in dried human whole blood collected using the Mitra and Tasso-M20 devices. Both LC-MS/MS assays met validation acceptance criteria for the linear range 1-1000 ng/ml giredestrant.

Exploring the potential of dried plasma collection cards for liquid chromatography coupled with tandem mass spectrometry quantitation of giredestrant in human plasma.

Microsampling technology for dried blood-derived samples provides an advantageous alternative to conventional venous blood for drug quantitation. Unlike conventional whole blood microsampling techniques, Noviplex is a novel, card-based technology for rapid dried plasma spot collection that retains the benefits of microsampling during collection and transportation, while avoiding the disadvantages of using whole blood samples. Giredestrant is a promising small-molecule therapeutic agent under development by Genentech to treat patients with estrogen receptor-positive breast cancer.

Pharmacokinetics of Ipatasertib in Subjects With Hepatic Impairment Using 2 Methods of Classification of Hepatic Function.

Ipatasertib is a highly selective small-molecule pan-Akt inhibitor in clinical development. Ipatasertib is predominantly eliminated by the liver, and therefore, the effect of hepatic impairment on ipatasertib pharmacokinetics (PK) was evaluated. In this phase 1 open-label, parallel group study, the PK of ipatasertib were evaluated in subjects with hepatic impairment based on both the Child-Pugh and the National Cancer Institute Organ Dysfunction Working Group classification for hepatic impairment.

A phase 1, randomized study to evaluate safety, tolerability, and pharmacokinetics of GDC-3280, a potential novel anti-fibrotic small molecule, in healthy subjects.

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease. Although anti-fibrotic treatments, such as pirfenidone, are available that reduce the rate of disease progression, these medications have limitations in tolerability, and IPF patients still have poor prognoses. GDC-3280, an orally available small molecule that was designed to improve upon pirfenidone's activity, has anti-fibrotic activity in animal models.

A Novel Depurination Methodology to Assess DNA Alkylation of Chloro-Bis-Seco-Cyclopropylbenzoindoles Allowed for Comparison of Minor-Groove Reactivity.

Duocarmycins [including cyclopropyl pyrroloindole (CPI) or cyclopropyl benzoindole (CBI)] are a class of DNA minor-groove alkylators and seco-CPI/CBIs are synthetic pro-forms that can spirocyclize to CPI/CBI. Bis-CPI/CBIs are potential drug candidates because of their enhanced cytotoxicity from DNA crosslinking, but it is difficult to analyze them for structure-activity correlation because of their DNA reactivity. To study their DNA alkylation, neutral thermal hydrolysis has been frequently applied to process depurination.

Determination of a novel antifibrotic small molecule GDC-3280 in human plasma and urine by liquid chromatography tandem mass spectrometry to support its first-in-human clinical trial.

A specific and robust LC-MS/MS method was developed and validated for the quantitative determination of GDC-3280 in human plasma and urine. The nonspecific binding associated with urine samples was overcome by the addition of CHAPS. The sample volume was 25 μL for either matrix, and supported liquid extraction was employed for analyte extraction.

Supercritical fluid chromatography-tandem mass spectrometry for high throughput bioanalysis of small molecules in drug discovery.

Liquid chromatography tandem mass spectrometry (LC-MS/MS) has been a golden standard for high throughput small molecule bioanalysis in drug discovery for decades. Supercritical fluid chromatography (SFC) has caught more attention in recent decade due to its advantages of greener mobile phase, lower backpressure and higher separation power. For the first time, we evaluated supercritical fluid chromatography tandem mass spectrometry (SFC-MS/MS) as a high throughput technique for bioanalysis of small molecule drug candidates and compared it to reversed phase LC-MS/MS.

Strategy for CYP3A Induction Risk Assessment from Preclinical Signal to Human: a Case Example of a Late-Stage Discovery Compound.

Purpose: The exposure of G2917 decreased by four-fold at oral doses of 100 mg/kg twice daily for seven days in cynomolgus monkeys. Additional investigative work was conducted to understand: (1) the causes for the significant reduction in G2917 exposure in monkeys; (2) the extrapolation of in vitro induction data to in vivo findings in monkeys, and (3) the relevance of this pre-clinical finding to humans at the projected human efficacious dose.

Methods: Pharmacokinetic and induction potency (in vitro and in vivo) of G2917 in monkeys, and the in vitro human induction potency were studied.

A Pharmacokinetic Bioequivalence Study Comparing Pirfenidone Tablet and Capsule Dosage Forms in Healthy Adult Volunteers.

Introduction: Pirfenidone film-coated tablets were developed to offer an alternative to the marketed capsule formulation. This study assessed the bioequivalence of the tablet and capsule formulations under fed and fasted states.

Methods: A Phase I, open-label, randomized, four-treatment-period, four-sequence, crossover pharmacokinetics study (NCT02525484) was conducted.

Replication studies of carboxymethylated DNA lesions in human cells.

Metabolic activation of some N-nitroso compounds (NOCs), an important class of DNA damaging agents, can induce the carboxymethylation of nucleobases in DNA. Very little was previously known about how the carboxymethylated DNA lesions perturb DNA replication in human cells. Here, we investigated the effects of five carboxymethylated DNA lesions, i.

Development of a multi-matrix LC-MS/MS method for urea quantitation and its application in human respiratory disease studies.

In human respiratory disease studies, liquid samples such as nasal secretion (NS), lung epithelial lining fluid (ELF), or upper airway mucosal lining fluid (MLF) are frequently collected, but their volumes often remain unknown. The lack of volume information makes it hard to estimate the actual concentration of recovered active pharmaceutical ingredient or biomarkers. Urea has been proposed to serve as a sample volume marker because it can freely diffuse through most body compartments and is less affected by disease states.

Quantification of Azaserine-Induced Carboxymethylated and Methylated DNA Lesions in Cells by Nanoflow Liquid Chromatography-Nanoelectrospray Ionization Tandem Mass Spectrometry Coupled with the Stable Isotope-Dilution Method.

Humans are exposed to N-nitroso compounds through environmental exposure and endogenous metabolism. Some N-nitroso compounds can be metabolically activated to yield diazoacetate, which is known to induce DNA carboxymethylation. DNA lesion measurement remains one of the core tasks in toxicology and in evaluating human health risks associated with carcinogen exposure.

Roles of Aag, Alkbh2, and Alkbh3 in the Repair of Carboxymethylated and Ethylated Thymidine Lesions.

Environmental and endogenous genotoxic agents can result in a variety of alkylated and carboxymethylated DNA lesions, including N3-ethylthymidine (N3-EtdT), O(2)-EtdT, and O(4)-EtdT as well as N3-carboxymethylthymidine (N3-CMdT) and O(4)-CMdT. By using nonreplicative double-stranded vectors harboring a site-specifically incorporated DNA lesion, we assessed the potential roles of alkyladenine DNA glycosylase (Aag); alkylation repair protein B homologue 2 (Alkbh2); or Alkbh3 in modulating the effects of N3-EtdT, O(2)-EtdT, O(4)-EtdT, N3-CMdT, or O(4)-CMdT on DNA transcription in mammalian cells. We found that the depletion of Aag did not significantly change the transcriptional inhibitory or mutagenic properties of all five examined lesions, suggesting a negligible role of Aag in the repair of these DNA adducts in mammalian cells.

Transcriptional inhibition and mutagenesis induced by N-nitroso compound-derived carboxymethylated thymidine adducts in DNA.

N-nitroso compounds represent a common type of environmental and endogenous DNA-damaging agents. After metabolic activation, many N-nitroso compounds are converted into a diazoacetate intermediate that can react with nucleobases to give carboxymethylated DNA adducts such as N3-carboxymethylthymidine (N3-CMdT) and O(4)-carboxymethylthymidine (O(4)-CMdT). In this study, we constructed non-replicative plasmids carrying a single N3-CMdT or O(4)-CMdT, site-specifically positioned in the transcribed strand, to investigate how these lesions compromise the flow of genetic information during transcription.

Translesion synthesis of 8,5'-cyclopurine-2'-deoxynucleosides by DNA polymerases η, ι, and ζ.

Reactive oxygen species can give rise to a battery of DNA damage products including the 8,5'-cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine (cdG) tandem lesions. The 8,5'-cyclopurine-2'-deoxynucleosides are quite stable lesions and are valid and reliable markers of oxidative DNA damage. However, it remains unclear how these lesions compromise DNA replication in mammalian cells.

In-vitro replication studies on O(2)-methylthymidine and O(4)-methylthymidine.

O(2)- and O(4)-methylthymidine (O(2)-MdT and O(4)-MdT) can be induced in tissues of laboratory animals exposed with N-methyl-N-nitrosourea, a known carcinogen. These two O-methylated DNA adducts have been shown to be poorly repaired and may contribute to the mutations arising from exposure to DNA methylating agents. Here, in vitro replication studies with duplex DNA substrates containing site-specifically incorporated O(2)-MdT and O(4)-MdT showed that both lesions blocked DNA synthesis mediated by three different DNA polymerases, including the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I (Kf(-)), human DNA polymerase κ (pol κ), and Saccharomyces cerevisiae DNA polymerase η (pol η).

A quantitative assay for assessing the effects of DNA lesions on transcription.

Most mammalian cells in nature are quiescent but actively transcribing mRNA for normal physiological processes; thus, it is important to investigate how endogenous and exogenous DNA damage compromises transcription in cells. Here we describe a new competitive transcription and adduct bypass (CTAB) assay to determine the effects of DNA lesions on the fidelity and efficiency of transcription. Using this strategy, we demonstrate that the oxidatively induced lesions 8,5'-cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine (cdG) and the methylglyoxal-induced lesion N(2)-(1-carboxyethyl)-2'-deoxyguanosine (N(2)-CEdG) strongly inhibited transcription in vitro and in mammalian cells.

Accurate and efficient bypass of 8,5'-cyclopurine-2'-deoxynucleosides by human and yeast DNA polymerase η.

Reactive oxygen species (ROS), which can be produced during normal aerobic metabolism, can induce the formation of tandem DNA lesions, including 8,5'-cyclo-2'-deoxyadenosine (cyclo-dA) and 8,5'-cyclo-2'-deoxyguanosine (cyclo-dG). Previous studies have shown that cyclo-dA and cyclo-dG accumulate in cells and can block mammalian RNA polymerase II and replicative DNA polymerases. Here, we used primer extension and steady-state kinetic assays to examine the efficiency and fidelity for polymerase η to insert nucleotides opposite, and extend primer past, these cyclopurine lesions.

In vitro replication studies of carboxymethylated DNA lesions with Saccharomyces cerevisiae polymerase η.

Humans are exposed to N-nitroso compounds (NOCs) both endogenously and exogenously from a number of environmental sources, and NOCs are both mutagenic and carcinogenic. After metabolic activation, some NOCs can induce carboxymethylation of nucleobases through a diazoacetate intermediate, which could give rise to p53 mutations similar to those seen in human gastrointestinal cancers. It was previously found that the growth of polymerase η-deficient human cells was inhibited by treatment with azaserine, a DNA carboxymethylation agent, suggesting the importance of this polymerase in bypassing the azaserine-induced carboxymethylated DNA lesions.

High-throughput analysis of the mutagenic and cytotoxic properties of DNA lesions by next-generation sequencing.

Human cells are constantly exposed to environmental and endogenous agents which can induce damage to DNA. Understanding the implications of these DNA modifications in the etiology of human diseases requires the examination about how these DNA lesions block DNA replication and induce mutations in cells. All previously reported shuttle vector-based methods for investigating the cytotoxic and mutagenic properties of DNA lesions in cells have low-throughput, where plasmids containing individual lesions are transfected into cells one lesion at a time and the products from the replication of individual lesions are analyzed separately.

Synthesis and characterization of oligodeoxyribonucleotides containing a site-specifically incorporated N6-carboxymethyl-2'-deoxyadenosine or N4-carboxymethyl-2'-deoxycytidine.

Humans are exposed to both endogenous and exogenous N-nitroso compounds (NOCs), and many NOCs can be metabolically activated to generate a highly reactive species, diazoacetate, which is capable of inducing carboxymethylation of nucleobases in DNA. Here we report, for the first time, the chemical syntheses of authentic N(6)-carboxymethyl-2'-deoxyadenosine (N(6)-CMdA) and N(4)-carboxymethyl-2'-deoxycytidine (N(4)-CMdC), liquid chromatography-ESI tandem MS confirmation of their formation in calf thymus DNA upon diazoacetate exposure, and the preparation of oligodeoxyribonucleotides containing a site-specifically incorporated N(6)-CMdA or N(4)-CMdC. Additionally, thermodynamic studies showed that the substitutions of a dA with N(6)-CMdA and dC with N(4)-CMdC in a 12-mer duplex increased Gibbs free energy for duplex formation at 25°C by 5.

Chemical synthesis of oligodeoxyribonucleotides containing N3- and O4-carboxymethylthymidine and their formation in DNA.

Humans are exposed to N-nitroso compounds from both endogenous and exogenous sources. Many N-nitroso compounds can be metabolically activated to give diazoacetate, which can result in the carboxymethylation of DNA. The remarkable similarity in p53 mutations found in human gastrointestinal tumors and in shuttle vector studies, where the human p53 gene-containing vector was treated with diazoacetate and propagated in yeast cells, suggests that diazoacetate might be an important etiological agent for human gastrointestinal tumors.

Thermodynamics and kinetics of the hydride-transfer cycles for 1-aryl-1,4-dihydronicotinamide and its 1,2-dihydroisomer.

Five 1-(p-substituted phenyl)-1,4-dihydronicotinamides (GPNAH-1,4-H(2)) and five 1-(p-substituted phenyl)-1,2-dihydronicotinamides (GPNAH-1,2-H(2)) were synthesized, which were used to mimic NAD(P)H coenzyme and its 1,2-dihydroisomer reductions, respectively. When the 1,4-dihydropyridine (GPNAH-1,4-H(2)) and the 1,2-dihydroisomer (GPNAH-1,2-H(2)) were treated with p-trifluoromethylbenzylidenemalononitrile (S) as a hydride acceptor, both reactions gave the same products: pyridinium derivative (GPNA(+)) and carbanion SH(-) by a hydride one-step transfer. Thermodynamic analysis on the two reactions shows that the hydride transfer from the 1,2-dihydropyridine is much more favorable than the hydride transfer from the corresponding 1,4-dihydroisomer, but the kinetic examination displays that the former reaction is remarkably slower than the latter reaction, which is mainly due to much more negative activation entropy for the former reaction.