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Quantification of venadaparib, a novel PARP inhibitor, in the rat and dog plasma using liquid chromatography/tandem mass spectrometry

Quantification of venadaparib, a novel PARP inhibitor, in the rat and dog plasma using liquid... Venadaparib ( VEN), a next‑ generation inhibitor of poly (ADP‑ribose) polymerases, is under development for oral use in patients having cancers with deoxyribonucleic acid repair defects. The objective of this study was to develop and validate a sensitive and robust analytical method for quantifying VEN in a small volume of plasma samples from rats and dogs, and to assess the feasibility of the assay for application in pharmacokinetic/toxicokinetic studies. Plasma samples were subjected to deproteination, and an aliquot was injected into an LC–MS/MS system. VEN and imipra‑ mine were analyzed in the positive ion mode and quantified by monitoring the transition at m/z 407.2 → 70.0 for VEN and 281.2 → 86.1 for imipramine. The lower and upper limits of the assay were determined to be 1 and 1000 ng/mL, respectively, with acceptable linearity (r > 0.995). Validation parameters, such as accuracy, precision, dilution, recovery, matrix effect, and stability, were within acceptable ranges. This method was adequately applied to the characteriza‑ tion of pharmacokinetics of VEN in rats and dogs at the oral dose of 30 and 0.5 mg/kg, respectively. These findings suggest that the validated assay is applicable to the kinetic studies of VEN with a small volume of plasma samples from the animals. Keywords Method validation, Bioanalysis, Mass spectrometry, Pharmacokinetics, Poly (ADP‑ribose) polymerases inhibitor, Venadaparib Introduction Genomic instability, a characteristic feature of cancer *Correspondence: development, occurs due to defects in cellular responses Jong‑Hwa Lee to deoxyribonucleic acid (DNA) damage (Pilie et  al. jhl@kitox.re.kr SukJae ‑ Chung 2019). Although the damage is normally corrected by sukjae@snu.ac.kr DNA repair systems, some may still remain unrepaired/ Idience Co., Ltd, Seoul 06752, Republic of Korea misrepaired, leading to an enhanced risk of cancer devel- Research Laboratories, Ildong Pharmaceutical Co., Ltd, Hwaseong 18449, Republic of Korea opment (Alhmoud et  al. 2020; Hoeijmakers 2001). Poly National OncoVenture, National Cancer Center, Goyang 10408, Republic (ADP-ribose) polymerases (PARPs), essential enzymes of Korea in the repair of damage in single-strand DNA (Sugimura Bioanalytical and Pharmacokinetic Study Group, Korea Institute of Toxicology, Daejeon 34114, Republic of Korea and Miwa 1994), are responsible for recruiting proteins Human and Environment Toxicology, University of Science & to promote DNA repair/cell survival when cells are Technology, Daejeon 34113, Republic of Korea exposed to genotoxic insults. Therefore, inhibition of College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea PARPs can result in the accumulation of unrepaired dam- College of Pharmacy and Research Institute of Pharmaceutical Sciences, age in single-strand DNA and, ultimately, breaks in DNA Seoul National University, Seoul 08826, Republic of Korea double strands (Haber 1999; Ashworth 2008). When cells © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Lee et al. Journal of Analytical Science and Technology (2023) 14:8 Page 2 of 13 with genetic defects in the DNA repair system, notably in various cancer models at a tolerable dose (Lee et al. 2017, the homologous recombination pathway (e.g., breast can- 2018, 2023). Furthermore, VEN was found to be well tol- cer type 1 and 2 susceptibility proteins), are treated with erated in human subjects in a recently completed phase PARP inhibitors, breaks in DNA double strands would I clinical study in Korea (Identifier NCT03317743 2022; not be adequately repaired, viz. synthetic lethal con- Kim et  al. 2021), suggesting that the new investigational dition (Bryant et  al. 2005; Farmer et  al. 2005; Lord and drug is a next-generation PARP inhibitor for the man- Ashworth 2017). In this respect, PARP inhibition appears agement of advanced cancer patients with limited thera- to be a pharmaco-therapeutically attractive approach peutic options. The compound is currently under phase for targeting tumors. Currently, four PARP inhibitors, Ib/IIa studies in the USA, Korea, and China (Identifier olaparib, rucaparib, niraparib, and talazoparib, are com- NCT04174716 2022; Identifier NCT04725994 2022; Lee mercially available in the USA (AstraZeneca 2022; Clovis et al. 2021; Im et al. 2021). Oncology 2021; GlaxoSmithKline 2021; Pfizer 2021). PK and toxicokinetic (TK) studies are often neces- Venadaparib (VEN, Fig.  1A), a novel derivative of sary for the further development of new drugs (e.g., phthalazinone, selectively inhibits PARPs with a balanced drug–drug interaction and toxicology studies). For the trapping effect (Lee et  al. 2017, 2018, 2023). The com - case of PARP inhibitors, hematologic toxicities, includ- pound reportedly possesses potent in  vitro and in  vivo ing anemia, neutropenia, and thrombocytopenia, are antitumor effects with a wide therapeutic range, as evi - the common adverse events (AstraZeneca 2022; Clovis denced by the fact that the inhibition of tumor growth Oncology  2021; GlaxoSmithKline 2021; Pfizer 2021). It by VEN is greater than that by other PARP inhibitors in is expected that blood samples are collected for multiple Fig. 1 Structures and product ion scan spectra of A VEN and B IS (i.e., imipramine) L ee et al. Journal of Analytical Science and Technology (2023) 14:8 Page 3 of 13 purposes (e.g., TK, hematology, and clinical chemistry) solvent A (0.1% formic acid in water) and solvent B (0.1% in repeated dose toxicity studies for VEN. Therefore, it formic acid in methanol) and was delivered at a flow rate is important to keep the amount of blood sample to the of 0.6 mL/min. Compounds were separated by a gradient minimum in those studies: If the blood removal is above elution [i.e., (the percent of solvent composition in v/v%) 7.5% of the total blood volume, more than 1 week recov- 0–0.2  min, 25% B; 0.2–2.5  min, 25–40% B; 2.5–3.0  min, ery period is necessary (Diehl et al. 2001) for the animal. 40–80% B; 3.0–4.0  min, 80% B; 4.0–4.1  min, 80–98% B; In addition, reducing the blood sampling below 3% of the 4.1–4.5 min, 98% B; 4.5–4.6 min, 98–25% B; 4.6–5.5 min, total blood volume in a week allowed to reduce pain and 25% B]. In addition, the injection volume was set at 3 μL, impact on hematological parameters (Yokoyama et  al. and the temperature of the sample manager was main- 2020; Wang and Li 2021). tained at 10 °C. Considering these, we were interested in developing a sensitive bioanalytical assay that would involve minimal Mass spectrometry conditions volumes of blood samples. The objective of this study Mass spectrometric detection was performed using an was to develop and validate a sensitive and robust ana- API 5000 triple quadrupole mass spectrometer (Sciex, lytical methodology, consistent with the guidelines for Framingham, Massachusetts, USA) equipped with an assay validation recommended by the US Food and Drug electrospray ionization source operating in the positive Administration and the European Medicines Agency ion mode by optimization. The ions were simultaneously (Food and Drug Administration  2018; European Medi- monitored in the multiple reaction monitoring (MRM) cines Agency 2011), for quantifying VEN in plasma sam- mode at ion transitions of m/z 407.2 → 70.0 for VEN and ples from typical preclinical animal species, namely rats m/z 281.2 → 86.1 for the IS. The first and second quadru - and dogs. Accordingly, the desired sensitivity of the assay ples were set at unit mass resolution while maintaining was set at a lower limit of quantification (LLOQ) of 1 ng/ the dwell time at 70 ms. The optimized source tempera - mL using 20  μL of plasma samples for PK/TK studies. ture was 600  °C, and the ion spray voltage was 5,500  V. The findings represented herein indicate that the vali - The conditions for curtain gas, nebulizer gas, heater gas, dated analytical method is successfully developed with a and collision gas were 30, 50, 50, and 5 psi, respectively. limited volume of plasma samples from the animal mod- The analytical parameters for mass spectrometry for the els and that the assay is applied to PK/TK studies in the VEN and IS are summarized in Table  1. All data collec- animals. tion from the mass spectrometric detector, processing, and storage was performed using Analyst software (Ver- sion 1.6; Sciex, Framingham, Massachusetts, USA) run- Methods/experimental ning on a computer. Chemicals and reagents VEN (100.3% purity, hydrochloride salt form) was pro- Preparation of standard and quality control solutions vided by Ildong Pharmaceutical (Seoul, Korea), and imi- The primary stock solution of VEN was prepared in pramine (IS) was purchased from Cerilliant Corporation dimethyl sulfoxide at a concentration of 1  mg/mL. The [Dorset, UK]. LC–MS grade acetonitrile, methanol, for- VEN standard working solutions and quality control mic acid, and analytical reagent grade dimethyl sulfox- (QC) solutions were then prepared by successive dilu- ide were obtained from VWR International (Poole, UK) tion of the stock solution with methanol–water–formic and used without further purification. Deionized water acid (25:75:0.1, v/v/v). The primary standard solution of was purified using the Sartorius Arium Comfort system IS was obtained as a certified solution of concentration (Sartorius AG, Göttingen, Germany). Blank plasma and 1 mg/mL. The IS working solution was obtained by dilut - blood samples were obtained from Covance Laboratories ing the standard solution with methanol–water (50:50, Ltd. (Huntingdon, UK). v/v). An aliquot of the VEN standard solution was added to rats and dogs blank plasma, resulting in eight nonzero Liquid chromatographic conditions An Acquity LC system (Waters Corporation, Milford, Massachusetts, USA), consisting of a binary solvent man- Table 1 Analytical condition of mass spectrometry for VEN and ager with an in-built degasser, a sample manager with a IS sample organizer, and a column manager, was used in Analyte SRM transition Declustering Collision Cell exit this study. Chromatographic separation was achieved potential (V) energy potential with an InfinityLab Poroshell 120-EC C18 column (eV) (V) (3.0 mm × 50 mm, with a particle size of 2.7 μm, Agilent VEN 407.2 → 70.0 90 38 11 Technologies, Santa Clara, California, USA; temperature IS 281.2 → 86.1 90 23 14 maintained at 40 °C). The mobile phase was composed of Lee et al. Journal of Analytical Science and Technology (2023) 14:8 Page 4 of 13 Carryover calibration standards, with VEN at concentrations of 1, 2, The degree of carryover, that is, transfer of VEN from the 10, 50, 100, 500, 900, and 1,000  ng/mL. In our prelimi- preceding sample in a batch sequence, was assessed by nary studies, the addition of a small volume of organic injecting at least two blank matrix extracts sequentially solvent to plasma samples had no appreciable effect on analyzed immediately after the upper limit of quantifica - the outcome of the assay. Using a similar method, the tion (ULOQ) sample. The level of carryover was consid - LLOQ, low QC (LQC), mid-QC (MQC), and high QC ered acceptable if the response in the blank matrix extract (HQC) samples were prepared to obtain plasma with was less than or equal to 20% of the mean response from VEN at a concentration of 1, 3, 300, or 800  ng/mL of the LLOQ sample in the batch. plasma, respectively. All calibration and QC samples were prepared immediately and separately before analy- sis, except for the samples used in stability studies. Accuracy, precision, and dilution integrity Accuracy (relative error, RE) and within-run precision (coefficient of variation, CV) were assessed using six QC Sample preparation samples consisting of four concentrations of VEN (i.e., 1, An aliquot (20  μL) of plasma was transferred to a well 3, 300, and 800 ng/mL) in replicates (n = 6), together with and mixed with methanol–water (20 µL, 50:50, v/v) con- a set of calibration standards. Between-run precision was taining the IS (15  ng/mL). The resulting mixture was also determined by analyzing the four levels of QC sam- vortexed for 2  min at 1,000  rpm, and then acetonitrile ples on three separate occasions. The RE was determined (100 µL) was added. The mixture was vortexed for 5 min at each concentration for both within and between (over- at 1,000  rpm and centrifuged at 3,000 × g for 10  min at all) batches, and the accuracy was considered acceptable 4  °C. A 50  µL of the supernatant was gently mixed with if the value was within ± 15% (± 20% for LLOQ) for both 200  μL of solvent A and transferred to an analytical within and between batches. The CV was determined 96-well plate for LC–MS/MS analysis. at each concentration and the precision was considered adequate if the value was within ≤ 15% (≤ 20% for LLOQ) for both within and between batches. Method validation The effect of dilution of the test samples was also stud - The current analytical method was validated according ied: The samples were prepared at concentrations greater to the guidelines for the validation of bioanalytical meth- than the ULOQ (i.e., 16,000 ng/mL). A 20 μL of the sam- ods from the US Food and Drug Administration and the ple was diluted 20-fold with blank matrix, in replicates European Medicines Agency (Food and Drug Adminis- (n = 6). The concentration of VEN was determined in the tration 2018; European Medicines Agency 2011). diluted sample and the original concentration was calcu- lated and compared with the nominal VEN concentration in the original sample. Selectivity and specificity The selectivity of the assay was studied by analyzing blank plasma samples from six independent sources, for Recovery and matrix effect the presence of interfering peaks for VEN and the IS. The absolute recovery of samples was determined for The interfering peaks were considered absent when the analytes, that is, VEN and IS, by comparing the response response at the retention time of VEN in the plasma was from the injection of the extract obtained with plasma less than or equal to 20% of the response from the LLOQ added with the analytes to that from the injection of samples, or when the response of the IS in the control a mixture of the analytes added with the extract of was less than or equal to 5% of the response from the blank plasma. Thus, blank plasma was first extracted as LLOQ samples. described in Sect.  “Sample preparation,” and VEN was added to obtain LQC, MQC, and HQC concentration in replicates (n = 6) in the presence of a fixed concentration Linearity of the IS. Separately, blank plasma samples were added to Calibration curves were constructed based on the results VEN to obtain LQC, MQC, and HQC concentrations in of the analysis of the eight concentrations of VEN. Dupli- replicates (n = 6) at one fixed IS concentration. The sam - cate calibration samples in the plasma at VEN concentra- ples were extracted as described in Sect. “Sample prepa- tions of 1, 2, 10, 50, 100, 500, 900, and 1,000 ng/mL were ration.” The mean responses of the two sets of samples analyzed. One set of standards was analyzed at the start were then compared. of the batch, and one set of standards was analyzed at the The matrix factor was assessed using six independ - end of the batch. Fresh calibration standards were pre- ent sources of blank plasma from normal blood and two pared for each analytical batch during method validation. L ee et al. Journal of Analytical Science and Technology (2023) 14:8 Page 5 of 13 sources of blank plasma from hemolyzed blood. Thus, for at least 3  months at − 70  °C. Aliquots of the QC the blank matrix sample was first extracted in replicates, samples after the storage period were analyzed in six as described in Sect. “Sample preparation,” and then VEN replicates. The stability was considered adequate if the was added. For this study, VEN was set at LQC and HQC concentration of the samples was within ± 15% of the concentrations, with one fixed concentration of the IS nominal concentration. (i.e., post-extraction spiked sample). When necessary, the When necessary, the stability of the processed sample reference solution was prepared at LQC and HQC con- in the sample manager (10  °C) was studied using sam- centrations of VEN with one fixed concentration of the ples at LQC and HQC concentrations (3 and 800  ng/ IS. In this study, the matrix factor was obtained by com- mL; n = 3 for each concentration). The QC samples paring the response of the post-extraction spiked sample were injected twice, immediately after sample prepara- with that of the reference solution at the corresponding tion and again at least 24  h after the first injection. The concentrations. storage in the sample manager was considered adequate when CV and RE were ≤ 15% (≤ 20% for the LLOQ) and within ± 15% (± 20% for the LLOQ) for all QC concen- Stability trations, respectively. The stability of the analytes was assessed under various storage and handling conditions: Standard solutions were prepared to obtain VEN PK studies with VEN in rats and dogs at concentrations ranging from 0.2 to 1.0  mg/mL and To determine the applicability of the current assay in PK stored at 4  °C or allowed to stand at room temperature studies of VEN, VEN was dissolved in deionized water, for 24  h. Thereafter, the response of the stored samples and the aqueous solution was administered orally at a was compared with that of the freshly prepared standard dose of 30  mg/kg (as a free base; administration vol- solutions. ume of 10  mL/kg) to Wistar Han [Crl:WI(Han)] rats In whole blood sample stability, the stability of VEN (Charles River UK Ltd., Kent, UK), weighing 183–253  g was assessed at LQC and HQC concentrations (i.e., 3 (i.e., 7–8  weeks of age). In addition, VEN was adminis- and 800  ng/mL), representing 0  min, and after at least tered orally in a form of a gelatin capsule at the dose 60  min. The blood samples were prepared by dissolv - of 0.5  mg/kg (as free base) to Beagle dogs (Marshall ing VEN in pre-warmed (37 °C) whole blood. The 0 min BioResources, East Yorkshire, UK), weighing 6.4–9.5  kg plasma samples were then obtained by the centrifugation (i.e., 5–6  months of age). The study was conducted in of the blood samples at 2,500 × g for 10 min at 4 °C. The accordance with the applicable sections of the UK Ani remaining blood samples were stored for 60 min on wet mals (Scientific Procedures) Act 1986 and Amendment ice. The samples were then subjected to the centrifuga - Regulations 2012 (the Act). Blood samples (approxi- tion at 2,500 × g for 10 min at 4 °C to obtain plasma. The mately 100  μL) were collected in K EDTA tubes (Cov- stability of VEN in whole blood was considered adequate 2 ance, Huntingdon, UK) at pre-dosing, 0.167, 0.33, 0.5, if the mean concentration of samples at 60 min remained 1, 4, 8, and 12  h post-dose either via the jugular vein in within ± 15% of that of samples at time 0  min. The ade - rats or pre-dosing, 0.25, 0.5 1, 4, 8, and 12 h via the jugu- quacy was also determined if CV was less than or equal lar vein in dogs. The blood samples were centrifuged at to 15% for two concentrations of two time points. 2,500 × g at 4  °C for 10  min, and plasma samples were In plasma sample stability, the stability of VEN was collected and stored at − 70  °C prior to analysis. When evaluated at LQC and HQG concentrations (i.e., 3 and necessary, systemic PK parameters, e.g., area under the 800  ng/mL) under typical sample-handling conditions. curve from time zero to measurable concentration (AUC For the evaluation of short-term room temperature ), were calculated using the linear trapezoidal method stability, aliquots of QC samples were stored at room 0-t and the standard area extrapolation method (Gibaldi and temperature (i.e., 22 °C) over 20 h and a portion of the Perrier 1982). The maximum concentration (C ) and sample was analyzed in replicates (n = 6 for each con- max the time point at a maximum concentration ( T ) were centration). When necessary, freeze–thaw stability was max read directly from the concentration–time profile of VEN studied by repeating the freeze-and-thaw cycle. The in the plasma. In this study, the terminal phase half-life QC samples were stored at − 70 °C for at least 12 h and (T ) was calculated by dividing 0.693 by the slope of the thawed or allowed to stand for at least 2 h at room tem- 1/2 terminal log-linear portion of the concentration–time perature. The samples were then transferred back to the profile. PK parameters were calculated using Phoenix freezer and maintained for at least 12  h. The cycle of WinNonlin (version 8.1; Pharsight Corp. Mountain View, thawing and freezing was repeated, and six replicates California, USA) from either the mean (rats) or individ at each concentration were analyzed. In addition, long- ual concentration–time profiles (dogs). term stability was evaluated after storing the samples Lee et al. Journal of Analytical Science and Technology (2023) 14:8 Page 6 of 13 Accuracy, precision, and dilution integrity Results The accuracy, precision, and dilution integrity of the Mass spectrometry and chromatography assays are summarized in Table  2. The QC samples at The full scan mass spectra of VEN and the internal the LLOQ, LQC, MQC, and HQC were analyzed at standard (IS) indicate the presence of a prominent pro- three separate occasions, in six replicates for each occa- tonated molecule, [M + H] , at m/z 407.2 and 281.2, sion, together with a set of calibration standards. The respectively. Major fragment ions, formed by the cleav- within-run RE for rat and dog samples was 0.3–7.0%, age of amide bond, were readily evident at m/z 70.0 for and 1.7–6.0%, respectively; the CV for rat and dog VEN and 86.1 for the IS. Thus, the ion transitions of samples was 1.4–7.3%, and 1.7–4.2%, respectively. The m/z 407.2 → 70.0, and 281.2 → 86.1 were used for the between-run RE for rat and dog samples was 4.2–9.0%, subsequent monitoring of VEN and the IS, respectively and − 2.0 to 2.0% with the CV for rat and dog sam- (Fig.  1A and B). To achieve adequate chromatographic ples of − 1.7 to 5.0%, and 7.4–9.0%, respectively. These separation of VEN/IS peaks from interfering peaks results suggest that the assay is accurate and precise for (1.82, 3.26, and 3.35 min) in the rat plasma, the gradient the determination of VEN concentrations ranging from with 0.1% formic acid in methanol and 0.1% formic acid 1 to 1000  ng/mL in rat and dog plasma samples. To was used. For the case of the dog plasma, no apparent determine the adequacy of sample dilution for estimat- interfering peaks were evident with the elution condi- ing the concentration in samples exceeding the ULOQ, tion. Under these chromatographic conditions, the a set of plasma samples containing VEN at 16,000  ng/ retention time was 1.75  min for VEN and 3.45  min for mL was prepared and then diluted 20-fold with the the IS, and the analytical run was completed within corresponding blank plasma. The RE for the analyte in 5.5 min per sample (Fig. 2). These analytical conditions the plasma after the 20-fold dilution with blank rat and were used in subsequent experiments. dog samples was 0.0% and − 0.6% with at a CV of 3.3% and 2.4% in rat and dog plasma samples, respectively. Selectivity and specificity These observations indicate that samples at concentra - Representative LC–MS/MS chromatograms of the tions exceeding the ULOQ can be diluted with the cor- LLOQ at 1  ng/mL are shown in Fig.  2. The chromato - responding blank plasma to determine accurately and gram of six different lots of blank analyses indicates precisely the original concentration using the current that the analyte peaks are adequately separated from assay. interfering peaks, suggesting that the selectivity of the LC–MS/MS assay is adequate for the quantification of VEN in rat and dog plasma samples. Recovery and matrix effect The mean recovery of VEN in plasma samples for rats and Linearity dogs was within the range of 88.7–98.7% and 88.7–101%, The calibration curve was linear over the concentra - respectively, with six replicates at the LQC, MQC, and tion ranges of 1–1,000  ng/mL for VEN in plasma sam- 2 HQC levels. In addition, the mean recovery for IS ranged ples from rats and dogs. A 1/x -weighted least squares from 88.1 to 96.2% and from 96.1 to 102% for the rat and quadratic regression analysis of the data was used to dog samples, respectively, at a fixed IS concentration of calculate the slope, intercept, and coefficient of deter - 2 15  ng/mL in the presence of VEN at LQC, MQC, and mination (r ) for samples from the two species. The HQC concentrations in the plasma. Collectively, these typical equation of VEN calibration curve was y = 0. 2 2 observations indicate that the recovery of both VEN and 000000521x + 0.00691x − 0.000260 (r = 0.998) and 2 IS was consistent and reproducible in the concentration y = 0.00623x − 0.000106 (r = 0.998) for rats and dogs, range. The matrix effect and IS-normalized matrix effects respectively. Where y represents the ratio of the peak were also studied with six independent sources of matrix area of the VEN to that of IS, and x represents the and two sources of matrix obtained from hemolyzed plasma concentration of VEN. The mean coefficient blood. The matrix factor of VEN and the IS was in the of determination for the calibration curves for rat and range of 0.95–1.05 and 0.91–1.00, respectively. The CV dog plasma samples was 0.996 and 0.995, respectively. of the matrix factor was less than 15% regardless of the The LLOQ for VEN was determined to be at 1  ng/mL concentration or origin of the matrix. In addition, it was with acceptable an accuracy and precision (Sect. "Accu- readily evident that the IS-normalized matrix effect was racy, precision, and dilution integrity"), and the signal- consistent (Table 3): As a result, the matrix effect of VEN to-noise ratio was greater than 5 for the two species and IS appeared negligible in the current assay. Taken studied. together, these findings indicate that the current assay provides virtually complete recovery of the analytes with L ee et al. Journal of Analytical Science and Technology (2023) 14:8 Page 7 of 13 Fig. 2 MRM chromatograms for samples in the rat plasma. A Double blank; B VEN at LLOQ with IS; C VEN at the ULOQ with IS. MRM chromatograms for samples in the dog plasma. D Double blank; E VEN at LLOQ with IS; F VEN at the ULOQ with IS Lee et al. Journal of Analytical Science and Technology (2023) 14:8 Page 8 of 13 Fig. 2 continued L ee et al. Journal of Analytical Science and Technology (2023) 14:8 Page 9 of 13 Fig. 2 continued Lee et al. Journal of Analytical Science and Technology (2023) 14:8 Page 10 of 13 Table 2 Accuracy and precision of the assay for VEN in plasma samples from the rat and dog Table 3 Matrix effects for VEN and IS in plasma samples of the rat and dog Batch Theoretical concentration (ng/mL) Matrix factor IS-normalized 1 3 300 800 800 VEN ISMatrix effect Rat plasma Rat plasma (A) Within-run accuracy and precision LQC (3 ng/mL) Mean estimated concentration 1.07 3.01 301 802 800 1 A 0.77 0.83 0.92 RE, % 7.0 0.3 0.3 0.3 0.0 2 B 1.06 1.00 1.06 CV, % 7.3 2.4 1.4 2.9 3.3 C 0.97 1.03 0.95 (B) Between-run accuracy and precision D 0.99 1.07 0.92 Mean estimated concentration 1.05 3.09 295 802 E 0.99 0.99 1.00 RE, % 6.9 9.0 4.2 5.9 F 0.91 0.98 0.93 CV, % 5.0 3.0 − 1.7 0.3 A (Hemolyzed) 1.00 1.02 0.97 Dog plasma B (Hemolyzed) 1.05 0.99 1.06 (A) Within-run accuracy and precision Mean ± SD 0.97 ± 0.09 0.99 ± 0.07 0.98 ± 0.06 Mean estimated concentration 1.06 3.05 308 828 795 CV (%) 9.5 7.0 5.8 RE, % 6.0 1.7 2.7 3.5 − 0.6 HQC (800 ng/mL) CV, % 3.6 3.3 1.7 4.2 2.4 A 0.96 0.94 1.02 (B) Between-run accuracy and precision B 1.00 1.00 1.00 Mean estimated concentration 1.02 2.98 296 784 C 1.01 1.00 1.01 RE, % 2.0 − 0.7 − 1.3 − 2.0 D 1.00 0.96 1.03 CV, % 7.6 9.0 7.4 7.8 E 0.98 0.99 0.99 Accuracy (RE, %) = (calculated concentration − theoretical concentration)/ F 1.04 1.00 1.04 theoretical concentration × 100 A (Hemolyzed) 1.01 1.02 0.99 Precision (CV, %) = standard deviation of the concentration/mean B (Hemolyzed) 1.03 1.01 1.02 concentration × 100 Mean ± SD 1.00 ± 0.02 0.99 ± 0.03 1.01 ± 0.02 Analyzed after a 20-fold dilution with blank plasma (viz. 16,000 → 800 ng/mL) CV (%) 2.5 2.7 2.0 Dog plasma no appreciable matrix effect in plasma samples of the two LQC (3 ng/mL) species. A 0.82 0.79 1.05 B 0.96 0.97 0.99 C 0.88 0.93 0.94 Stability D 1.14 0.92 1.24 The stability of VEN was evaluated under various stor - E 1.09 0.98 1.11 age and handling conditions. In general, the compound F 0.93 0.90 1.04 was relatively stable and the assay satisfied the require - A (Hemolyzed) 0.80 0.80 0.99 ments set by the guidelines for assay validation under the B (Hemolyzed) 0.97 0.96 1.01 Mean ± SD 0.95 ± 0.12 0.91 ± 0.07 1.05 ± 0.09 conditions studied, regardless of the concentration and CV (%) 12.8 8.3 8.8 origin of the matrix. For example, the concentration of HQC (800 ng/mL) the compound and IS in a stock solution that had been A 1.03 0.93 1.11 stored for 24 h at room temperature was 97.3% and 98.6% B 1.03 0.97 1.06 of the initial concentration, respectively. In addition, the C 1.06 1.01 1.05 concentration after 1 month of storage under refrigerated D 1.11 1.03 1.08 conditions was 99.8% and 94.2% of the initial concentra- E 1.07 1.02 1.05 tion, respectively. Furthermore, the analyte in the rat and F 1.04 1.00 1.04 dog plasma was found to be stable in various conditions A (Hemolyzed) 1.03 1.01 1.05 [i.e., in room temperature for 22 and 24  h; after freeze– B (Hemolyzed) 1.04 1.01 1.03 thaw cycles, and at − 70  °C over 3  months (Table  4)]. In Mean ± SD 1.05 ± 0.03 1.00 ± 0.03 1.05 ± 0.03 this study, degradation was not apparent in the sam- CV (%) 2.7 3.3 2.6 ple manager of the LC–MS/MS system (i.e., operating Matrix factor = [peak area of analyte added post-extraction]/[peak area of at 10  °C) for 8  days (Table  4). VEN was stable in whole analyte standards] blood on ice for up to 1 h. Collectively, these observations L ee et al. Journal of Analytical Science and Technology (2023) 14:8 Page 11 of 13 Table 3 (continued) Normalized Matrix Factor = Matrix Factor for Analyte/Matrix Factor for IS CV (%) = standard deviation/mean × 100 Table 4 Summary of stability studies for VEN in QC samples Batch Theoretical concentration (ng/mL) Rat Dog 3 800 3 800 (A) Short-term stability at room temperature (22 °C, 22 h for rats, 24 h for dogs, n = 6) Mean estimated concentration 2.62 725 3.15 688 RE (%) − 12.7 − 9.4 5.0 − 14.0 CV (%) 5.7 5.6 7.5 3.3 (B) Freeze–thaw stability (3 cycles for rats, 4 cycles for dogs, n = 6) Mean estimated concentration 2.59 710 2.90 735 RE (%) − 13.7 − 11.3 − 3.5 − 8.1 CV (%) 5.9 7.6 8.1 2.9 (C) Long-term stability (107 days for rats, 87 days for dogs, n = 6) Mean estimated concentration 3.42 907 29.7 693 Fig. 3 Mean plasma concentration–time curves of VEN in A rats RE (%) 14.0 13.4 − 1.0 − 13.4 and B dogs that had received an oral administration of 30 mg/kg CV (%) 12.1 5.0 4.4 4.0 and 0.5 mg/kg of VEN, respectively (key: opened circles, male; closed circles, female). Data were represented as mean ± standard deviation (D) Processed sample stability (at 10 °C for 8 days, n = 3) of n = 3 (for rats) or n = 6 (for dogs) Mean estimated concentration 3.00 773 3.08 785 RE (%) 0.0 − 3.4 2.7 − 1.9 CV (%) 2.4 4.1 0.7 1.4 Table 5 Summary of systemic PK parameters following oral RE (%) = (calculated concentration − theoretical concentration)/theoretical administration of VEN to rats and dogs concentration × 100 1 2 Pharmacokinetic Rat Dog CV (%) = standard deviation of the concentration/mean concentration × 100 parameters Male Female Male Female Mean Mean ± SD suggest that VEN is stable under typical storage and han- dling conditions. Dose (mg/kg) 30.0 0.50 T (h) 0.333 1.00 0.583 ± 0.342 0.583 ± 0.342 max Applicability of the assay to pharmacokinetic studies C (ng/mL) 1,130 4160 120 ± 61 156 ± 54 max One of the objectives of this study was to determine T (h) 1.92 1.88 1.43 ± 0.64 1.72 ± 0.29 1/2 whether the current assay would be applicable to PK AUC (ng·h/mL) 5,310 12,900 243 ± 111 361 ± 88 0→t studies of VEN in rats and dogs. The temporal profiles AUC (ng·h/mL) 5,430 13,100 267 ± 169 369 ± 98 0→∞ of the plasma concentration of the PARP inhibitor after Three replicates: Each rat covered different regions in the time profile and, as oral administration to the rat and dog at a dose of 30 mg/ results, the calculation of the standard deviation (SD) was not possible kg and 0.5  mg/kg, respectively, are depicted in Fig.  3. In 2 Six replicates general, the concentration was readily detected up to AUC from time of dosing extrapolated to infinity 12  h after administration, suggesting that the sensitivity was adequate for the quantification of plasma concentra - to male dogs, female dogs showed 1.30- and 1.49-fold tions of the drug in PK studies with rats and dogs at the higher values for C and AUC , respectively.). max 0-t doses tested. PK parameters of the drug in rats and dogs, calculated using standard moment analysis, are listed in Table 5. Interestingly, a gender difference in VEN PK was Discussion noted in rats (i.e., compared to male rats, the C and Inhibition of PARP results in severe hematologic toxici- max AUC in female rats were 3.68- and 2.43-fold higher, ties that form neutropenia in laboratory animals at low 0-t respectively). However, in dogs, the gender difference dose levels in chronic studies. The fact that toxic doses was reduced for the key PK parameters (i.e., compared could be as low as one-tenth of the effective dose for Lee et al. Journal of Analytical Science and Technology (2023) 14:8 Page 12 of 13 PARP inhibitors is now well established (AstraZeneca kg was 120 and 156  ng/mL for male and female dogs, 2022; Clovis  Oncology 2021; GlaxoSmithKline 2021), respectively. In this study, we did not directly study the suggesting that an adequate dose of VEN may be lower mechanisms for the kinetic difference of VEN by gen - than the efficacy or PK study in certain toxicity studies. der. However, olaparib, a structural analog of VEN, was In addition, exploratory repeated dose PK studies may reported to have up to 14-fold higher exposure in female be conducted in animal models (e.g., the identification rats than in male rats, while the difference was markedly of drug–drug interaction mechanisms). Frequent blood reduced (approximately twofold) in dogs (Application sampling could exacerbate hematologic toxicity of PARP Number:206162Orig1s000 2014), suggesting a common inhibitors, a small sampling volume would be ideal for mechanism for the gender difference in the PK for the PARP inhibitors. Thus, a bioanalytical method which PARP inhibitors. This aspect of PARP inhibitors warrants enables the quantification of analyte using a small vol - additional studies. ume of plasma samples could be helpful in PK/TK stud- ies of VEN. In this study, we attempted to develop and Conclusions validate a sensitive and accurate LC–MS/MS method A sensitive and robust LC–MS/MS assay was developed for the quantification of VEN in the rat and dog plasma and validated for the quantification of VEN plasma sam - samples, and quantified VEN at concentrations as low ples from rats and dogs in terms of accuracy, precision, as 1  ng/mL using 20  µL plasma samples. Assuming lin- dilution, recovery, matrix effect, and stability, which ear PK for VEN in the animal models, the LLOQ, 1  ng/ were within acceptable ranges. The blood sampling can mL, represented less than 5% of the expected C after be reduced below 3% of the total blood volume in PK/ max oral administration of VEN at 1 and 0.1  mg/kg in rats TK studies in rats using this method. This method can and dogs, respectively. Taken together with the possibil- be applied to the chronic study of VEN to minimize the ity of low-dose studies of VEN in animals, this observa- hematological effect of blood sampling. Collectively, tion suggests that the current assay can be readily applied these observations indicate that the current assay can be to typical TK/PK studies of the inhibitor in two animal reliably applied to PK and TK studies of VEN in rats and models. dogs using the limited sample volume. Using gradient elution, interfering peaks originating from the plasma were reasonably separated from the ana- Abbreviations lytes, while limiting a run-time of approximately 5.5 min. AUC Area under the curve from time zero to measurable concentration 0→t The deuterated form of VEN is not commercially avail - AUC Ar ea under the curve from time zero to infinity 0→∞ AUMC Ar ea under the first moment curve from time zero to infinity 0→∞ able; hence, a series of compounds had to be screened for C Maximum concentration max an IS. In the preliminary screening study, imipramine was CV Coefficient of variation selected as the IS, considering its recovery and matrix HQC High quality control IS Internal standard effects with VEN and IS (Table  3). We found that the LC–MS/MS Liquid chromatography–tandem mass spectrometry analytes were almost completely extracted in the current LLOQ Lower limit of quantification assay as the recoveries for the rats and dogs, 88.7–98.7% LQC Low quality control MQC Mid‑quality control and 88.7–101%, respectively, for VEN; 88.1–96.2% and MRM Multiple reaction monitoring 96.1–102%, respectively, for IS. In addition, the recovery MRT Mean residence time values were consistent over the concentration range used PARP Poly (ADP‑ribose) polymerase) PK Pharmacokinetic in this study. The experimental data for selectivity, linear - QC Quality control ity, precision, accuracy, and stability were found accept- RE Relative error able according to the guidelines for bioanalytical method SD Standard deviation T Half‑life 1/2 validation from the US Food and Drug Administration T Time point at maximum concentration max and the European Medicines Agency. TK Toxicokinetic In this study, we found that the assay was applica- ULOQ Upper limit of quantification UK United Kingdom ble to PK studies for VEN in rats and dogs at oral doses US United States of 30  mg/kg and 0.5  mg/kg, respectively. Based on the VEN Venadaparib LLOQ of the current assay, we were able to monitor the Acknowledgements plasma concentration–time profile up to 12  h after oral Not applicable. administration, which accounted for over six times the T for VEN (T ≤ 2  h for VEN, Table  5). It was noted Author contributions 1/2 1/2 ML, EJ, and JL contributed to conceptualization; ML and EJ contributed to that gender differences in PK were found for VEN in the methodology; ML performed formal analysis, data curation, writing—original animal models: C at 30 mg/kg was 1130 and 4160 ng/ max draft preparation, and visualization; ML, J‑HL, and S‑ JC contributed to writ‑ mL for male and female rats, respectively; C at 0.5 mg/ ing—review and editing; SC, WSL, and S‑ JC supervised the study; NSB and SL max L ee et al. Journal of Analytical Science and Technology (2023) 14:8 Page 13 of 13 administered the project; Y‑ WP performed funding acquisition. All authors Identifier NCT03317743, Study to Assess the Safety and Tolerability of have read and agreed to the published version of the manuscript. NOV140101(IDX‑1197) in Patients With Advanced Solid Tumors. https:// clini caltr ials. gov/ ct2/ show/ NCT03 317743. Accessed on 12 Nov 2022. Funding Identifier NCT04174716, Basket Trial of IDX ‑1197, a PARP Inhibitor, in Patients This study was conducted with the National‑ OncoVenture supported by the With HRR Mutated Solid Tumors ( VASTUS). https:// clini caltr ials. gov/ ct2/ National Cancer Center, designated by the Ministry of Health and Welfare show/ NCT04 174716. Accessed on 12 Nov 2022. Korea (HI17C2196). Identifier NCT04725994, Study to Assess the Safety, Tolerability, and Efficacy of IDX‑1197 in Combination With XELOX or Irinotecan in Patients With Availability of data and materials Advanced Gastric Cancer. https:// clini caltr ials. gov/ ct2/ show/ NCT04 The data that support the findings of this study are available from the cor ‑ 725994. Accessed on 12 Nov 2022. responding author upon reasonable request. Im S‑A, Kim JE, Lee KS, Moon YW, Ahn HK, Lee KH, Ock C‑ Y, Roh EJ, Lee M, Hong MJ et al. Phase Ib study of venadaparib, a potent and selective PARP inhibitor, in homologous recombination repair (HRR) mutated breast Declarations cancer. In: Proceedings of the ESMO Congress, 2021. Kim YM, Bae K‑S, Baek NS, Kim K, Lee WS, Roh E‑ J, Ock C‑ Y, Kim S‑B. Abstract Competing interests 3107. First‑in‑human dose ‑finding study of venadaparib (IDX ‑1197), a The authors declare that they have no competing interests. potent and selective PARP inhibitor, in patients with advanced solid tumors. In: Proceedings of the ASCO Annual meeting, 2021. Lee M, Park J‑ T, Yang J‑h, Kim D, Je I‑ G, Lee YS, Jeong J, Song DK, Park S, Lee Received: 16 November 2022 Accepted: 18 January 2023 H‑S et al. Abstract A106: Development of IDX ‑1197, a novel, selective, and highly potent PARP inhibitor. 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Quantification of venadaparib, a novel PARP inhibitor, in the rat and dog plasma using liquid chromatography/tandem mass spectrometry

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Abstract

Venadaparib ( VEN), a next‑ generation inhibitor of poly (ADP‑ribose) polymerases, is under development for oral use in patients having cancers with deoxyribonucleic acid repair defects. The objective of this study was to develop and validate a sensitive and robust analytical method for quantifying VEN in a small volume of plasma samples from rats and dogs, and to assess the feasibility of the assay for application in pharmacokinetic/toxicokinetic studies. Plasma samples were subjected to deproteination, and an aliquot was injected into an LC–MS/MS system. VEN and imipra‑ mine were analyzed in the positive ion mode and quantified by monitoring the transition at m/z 407.2 → 70.0 for VEN and 281.2 → 86.1 for imipramine. The lower and upper limits of the assay were determined to be 1 and 1000 ng/mL, respectively, with acceptable linearity (r > 0.995). Validation parameters, such as accuracy, precision, dilution, recovery, matrix effect, and stability, were within acceptable ranges. This method was adequately applied to the characteriza‑ tion of pharmacokinetics of VEN in rats and dogs at the oral dose of 30 and 0.5 mg/kg, respectively. These findings suggest that the validated assay is applicable to the kinetic studies of VEN with a small volume of plasma samples from the animals. Keywords Method validation, Bioanalysis, Mass spectrometry, Pharmacokinetics, Poly (ADP‑ribose) polymerases inhibitor, Venadaparib Introduction Genomic instability, a characteristic feature of cancer *Correspondence: development, occurs due to defects in cellular responses Jong‑Hwa Lee to deoxyribonucleic acid (DNA) damage (Pilie et  al. jhl@kitox.re.kr SukJae ‑ Chung 2019). Although the damage is normally corrected by sukjae@snu.ac.kr DNA repair systems, some may still remain unrepaired/ Idience Co., Ltd, Seoul 06752, Republic of Korea misrepaired, leading to an enhanced risk of cancer devel- Research Laboratories, Ildong Pharmaceutical Co., Ltd, Hwaseong 18449, Republic of Korea opment (Alhmoud et  al. 2020; Hoeijmakers 2001). Poly National OncoVenture, National Cancer Center, Goyang 10408, Republic (ADP-ribose) polymerases (PARPs), essential enzymes of Korea in the repair of damage in single-strand DNA (Sugimura Bioanalytical and Pharmacokinetic Study Group, Korea Institute of Toxicology, Daejeon 34114, Republic of Korea and Miwa 1994), are responsible for recruiting proteins Human and Environment Toxicology, University of Science & to promote DNA repair/cell survival when cells are Technology, Daejeon 34113, Republic of Korea exposed to genotoxic insults. Therefore, inhibition of College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea PARPs can result in the accumulation of unrepaired dam- College of Pharmacy and Research Institute of Pharmaceutical Sciences, age in single-strand DNA and, ultimately, breaks in DNA Seoul National University, Seoul 08826, Republic of Korea double strands (Haber 1999; Ashworth 2008). When cells © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Lee et al. Journal of Analytical Science and Technology (2023) 14:8 Page 2 of 13 with genetic defects in the DNA repair system, notably in various cancer models at a tolerable dose (Lee et al. 2017, the homologous recombination pathway (e.g., breast can- 2018, 2023). Furthermore, VEN was found to be well tol- cer type 1 and 2 susceptibility proteins), are treated with erated in human subjects in a recently completed phase PARP inhibitors, breaks in DNA double strands would I clinical study in Korea (Identifier NCT03317743 2022; not be adequately repaired, viz. synthetic lethal con- Kim et  al. 2021), suggesting that the new investigational dition (Bryant et  al. 2005; Farmer et  al. 2005; Lord and drug is a next-generation PARP inhibitor for the man- Ashworth 2017). In this respect, PARP inhibition appears agement of advanced cancer patients with limited thera- to be a pharmaco-therapeutically attractive approach peutic options. The compound is currently under phase for targeting tumors. Currently, four PARP inhibitors, Ib/IIa studies in the USA, Korea, and China (Identifier olaparib, rucaparib, niraparib, and talazoparib, are com- NCT04174716 2022; Identifier NCT04725994 2022; Lee mercially available in the USA (AstraZeneca 2022; Clovis et al. 2021; Im et al. 2021). Oncology 2021; GlaxoSmithKline 2021; Pfizer 2021). PK and toxicokinetic (TK) studies are often neces- Venadaparib (VEN, Fig.  1A), a novel derivative of sary for the further development of new drugs (e.g., phthalazinone, selectively inhibits PARPs with a balanced drug–drug interaction and toxicology studies). For the trapping effect (Lee et  al. 2017, 2018, 2023). The com - case of PARP inhibitors, hematologic toxicities, includ- pound reportedly possesses potent in  vitro and in  vivo ing anemia, neutropenia, and thrombocytopenia, are antitumor effects with a wide therapeutic range, as evi - the common adverse events (AstraZeneca 2022; Clovis denced by the fact that the inhibition of tumor growth Oncology  2021; GlaxoSmithKline 2021; Pfizer 2021). It by VEN is greater than that by other PARP inhibitors in is expected that blood samples are collected for multiple Fig. 1 Structures and product ion scan spectra of A VEN and B IS (i.e., imipramine) L ee et al. Journal of Analytical Science and Technology (2023) 14:8 Page 3 of 13 purposes (e.g., TK, hematology, and clinical chemistry) solvent A (0.1% formic acid in water) and solvent B (0.1% in repeated dose toxicity studies for VEN. Therefore, it formic acid in methanol) and was delivered at a flow rate is important to keep the amount of blood sample to the of 0.6 mL/min. Compounds were separated by a gradient minimum in those studies: If the blood removal is above elution [i.e., (the percent of solvent composition in v/v%) 7.5% of the total blood volume, more than 1 week recov- 0–0.2  min, 25% B; 0.2–2.5  min, 25–40% B; 2.5–3.0  min, ery period is necessary (Diehl et al. 2001) for the animal. 40–80% B; 3.0–4.0  min, 80% B; 4.0–4.1  min, 80–98% B; In addition, reducing the blood sampling below 3% of the 4.1–4.5 min, 98% B; 4.5–4.6 min, 98–25% B; 4.6–5.5 min, total blood volume in a week allowed to reduce pain and 25% B]. In addition, the injection volume was set at 3 μL, impact on hematological parameters (Yokoyama et  al. and the temperature of the sample manager was main- 2020; Wang and Li 2021). tained at 10 °C. Considering these, we were interested in developing a sensitive bioanalytical assay that would involve minimal Mass spectrometry conditions volumes of blood samples. The objective of this study Mass spectrometric detection was performed using an was to develop and validate a sensitive and robust ana- API 5000 triple quadrupole mass spectrometer (Sciex, lytical methodology, consistent with the guidelines for Framingham, Massachusetts, USA) equipped with an assay validation recommended by the US Food and Drug electrospray ionization source operating in the positive Administration and the European Medicines Agency ion mode by optimization. The ions were simultaneously (Food and Drug Administration  2018; European Medi- monitored in the multiple reaction monitoring (MRM) cines Agency 2011), for quantifying VEN in plasma sam- mode at ion transitions of m/z 407.2 → 70.0 for VEN and ples from typical preclinical animal species, namely rats m/z 281.2 → 86.1 for the IS. The first and second quadru - and dogs. Accordingly, the desired sensitivity of the assay ples were set at unit mass resolution while maintaining was set at a lower limit of quantification (LLOQ) of 1 ng/ the dwell time at 70 ms. The optimized source tempera - mL using 20  μL of plasma samples for PK/TK studies. ture was 600  °C, and the ion spray voltage was 5,500  V. The findings represented herein indicate that the vali - The conditions for curtain gas, nebulizer gas, heater gas, dated analytical method is successfully developed with a and collision gas were 30, 50, 50, and 5 psi, respectively. limited volume of plasma samples from the animal mod- The analytical parameters for mass spectrometry for the els and that the assay is applied to PK/TK studies in the VEN and IS are summarized in Table  1. All data collec- animals. tion from the mass spectrometric detector, processing, and storage was performed using Analyst software (Ver- sion 1.6; Sciex, Framingham, Massachusetts, USA) run- Methods/experimental ning on a computer. Chemicals and reagents VEN (100.3% purity, hydrochloride salt form) was pro- Preparation of standard and quality control solutions vided by Ildong Pharmaceutical (Seoul, Korea), and imi- The primary stock solution of VEN was prepared in pramine (IS) was purchased from Cerilliant Corporation dimethyl sulfoxide at a concentration of 1  mg/mL. The [Dorset, UK]. LC–MS grade acetonitrile, methanol, for- VEN standard working solutions and quality control mic acid, and analytical reagent grade dimethyl sulfox- (QC) solutions were then prepared by successive dilu- ide were obtained from VWR International (Poole, UK) tion of the stock solution with methanol–water–formic and used without further purification. Deionized water acid (25:75:0.1, v/v/v). The primary standard solution of was purified using the Sartorius Arium Comfort system IS was obtained as a certified solution of concentration (Sartorius AG, Göttingen, Germany). Blank plasma and 1 mg/mL. The IS working solution was obtained by dilut - blood samples were obtained from Covance Laboratories ing the standard solution with methanol–water (50:50, Ltd. (Huntingdon, UK). v/v). An aliquot of the VEN standard solution was added to rats and dogs blank plasma, resulting in eight nonzero Liquid chromatographic conditions An Acquity LC system (Waters Corporation, Milford, Massachusetts, USA), consisting of a binary solvent man- Table 1 Analytical condition of mass spectrometry for VEN and ager with an in-built degasser, a sample manager with a IS sample organizer, and a column manager, was used in Analyte SRM transition Declustering Collision Cell exit this study. Chromatographic separation was achieved potential (V) energy potential with an InfinityLab Poroshell 120-EC C18 column (eV) (V) (3.0 mm × 50 mm, with a particle size of 2.7 μm, Agilent VEN 407.2 → 70.0 90 38 11 Technologies, Santa Clara, California, USA; temperature IS 281.2 → 86.1 90 23 14 maintained at 40 °C). The mobile phase was composed of Lee et al. Journal of Analytical Science and Technology (2023) 14:8 Page 4 of 13 Carryover calibration standards, with VEN at concentrations of 1, 2, The degree of carryover, that is, transfer of VEN from the 10, 50, 100, 500, 900, and 1,000  ng/mL. In our prelimi- preceding sample in a batch sequence, was assessed by nary studies, the addition of a small volume of organic injecting at least two blank matrix extracts sequentially solvent to plasma samples had no appreciable effect on analyzed immediately after the upper limit of quantifica - the outcome of the assay. Using a similar method, the tion (ULOQ) sample. The level of carryover was consid - LLOQ, low QC (LQC), mid-QC (MQC), and high QC ered acceptable if the response in the blank matrix extract (HQC) samples were prepared to obtain plasma with was less than or equal to 20% of the mean response from VEN at a concentration of 1, 3, 300, or 800  ng/mL of the LLOQ sample in the batch. plasma, respectively. All calibration and QC samples were prepared immediately and separately before analy- sis, except for the samples used in stability studies. Accuracy, precision, and dilution integrity Accuracy (relative error, RE) and within-run precision (coefficient of variation, CV) were assessed using six QC Sample preparation samples consisting of four concentrations of VEN (i.e., 1, An aliquot (20  μL) of plasma was transferred to a well 3, 300, and 800 ng/mL) in replicates (n = 6), together with and mixed with methanol–water (20 µL, 50:50, v/v) con- a set of calibration standards. Between-run precision was taining the IS (15  ng/mL). The resulting mixture was also determined by analyzing the four levels of QC sam- vortexed for 2  min at 1,000  rpm, and then acetonitrile ples on three separate occasions. The RE was determined (100 µL) was added. The mixture was vortexed for 5 min at each concentration for both within and between (over- at 1,000  rpm and centrifuged at 3,000 × g for 10  min at all) batches, and the accuracy was considered acceptable 4  °C. A 50  µL of the supernatant was gently mixed with if the value was within ± 15% (± 20% for LLOQ) for both 200  μL of solvent A and transferred to an analytical within and between batches. The CV was determined 96-well plate for LC–MS/MS analysis. at each concentration and the precision was considered adequate if the value was within ≤ 15% (≤ 20% for LLOQ) for both within and between batches. Method validation The effect of dilution of the test samples was also stud - The current analytical method was validated according ied: The samples were prepared at concentrations greater to the guidelines for the validation of bioanalytical meth- than the ULOQ (i.e., 16,000 ng/mL). A 20 μL of the sam- ods from the US Food and Drug Administration and the ple was diluted 20-fold with blank matrix, in replicates European Medicines Agency (Food and Drug Adminis- (n = 6). The concentration of VEN was determined in the tration 2018; European Medicines Agency 2011). diluted sample and the original concentration was calcu- lated and compared with the nominal VEN concentration in the original sample. Selectivity and specificity The selectivity of the assay was studied by analyzing blank plasma samples from six independent sources, for Recovery and matrix effect the presence of interfering peaks for VEN and the IS. The absolute recovery of samples was determined for The interfering peaks were considered absent when the analytes, that is, VEN and IS, by comparing the response response at the retention time of VEN in the plasma was from the injection of the extract obtained with plasma less than or equal to 20% of the response from the LLOQ added with the analytes to that from the injection of samples, or when the response of the IS in the control a mixture of the analytes added with the extract of was less than or equal to 5% of the response from the blank plasma. Thus, blank plasma was first extracted as LLOQ samples. described in Sect.  “Sample preparation,” and VEN was added to obtain LQC, MQC, and HQC concentration in replicates (n = 6) in the presence of a fixed concentration Linearity of the IS. Separately, blank plasma samples were added to Calibration curves were constructed based on the results VEN to obtain LQC, MQC, and HQC concentrations in of the analysis of the eight concentrations of VEN. Dupli- replicates (n = 6) at one fixed IS concentration. The sam - cate calibration samples in the plasma at VEN concentra- ples were extracted as described in Sect. “Sample prepa- tions of 1, 2, 10, 50, 100, 500, 900, and 1,000 ng/mL were ration.” The mean responses of the two sets of samples analyzed. One set of standards was analyzed at the start were then compared. of the batch, and one set of standards was analyzed at the The matrix factor was assessed using six independ - end of the batch. Fresh calibration standards were pre- ent sources of blank plasma from normal blood and two pared for each analytical batch during method validation. L ee et al. Journal of Analytical Science and Technology (2023) 14:8 Page 5 of 13 sources of blank plasma from hemolyzed blood. Thus, for at least 3  months at − 70  °C. Aliquots of the QC the blank matrix sample was first extracted in replicates, samples after the storage period were analyzed in six as described in Sect. “Sample preparation,” and then VEN replicates. The stability was considered adequate if the was added. For this study, VEN was set at LQC and HQC concentration of the samples was within ± 15% of the concentrations, with one fixed concentration of the IS nominal concentration. (i.e., post-extraction spiked sample). When necessary, the When necessary, the stability of the processed sample reference solution was prepared at LQC and HQC con- in the sample manager (10  °C) was studied using sam- centrations of VEN with one fixed concentration of the ples at LQC and HQC concentrations (3 and 800  ng/ IS. In this study, the matrix factor was obtained by com- mL; n = 3 for each concentration). The QC samples paring the response of the post-extraction spiked sample were injected twice, immediately after sample prepara- with that of the reference solution at the corresponding tion and again at least 24  h after the first injection. The concentrations. storage in the sample manager was considered adequate when CV and RE were ≤ 15% (≤ 20% for the LLOQ) and within ± 15% (± 20% for the LLOQ) for all QC concen- Stability trations, respectively. The stability of the analytes was assessed under various storage and handling conditions: Standard solutions were prepared to obtain VEN PK studies with VEN in rats and dogs at concentrations ranging from 0.2 to 1.0  mg/mL and To determine the applicability of the current assay in PK stored at 4  °C or allowed to stand at room temperature studies of VEN, VEN was dissolved in deionized water, for 24  h. Thereafter, the response of the stored samples and the aqueous solution was administered orally at a was compared with that of the freshly prepared standard dose of 30  mg/kg (as a free base; administration vol- solutions. ume of 10  mL/kg) to Wistar Han [Crl:WI(Han)] rats In whole blood sample stability, the stability of VEN (Charles River UK Ltd., Kent, UK), weighing 183–253  g was assessed at LQC and HQC concentrations (i.e., 3 (i.e., 7–8  weeks of age). In addition, VEN was adminis- and 800  ng/mL), representing 0  min, and after at least tered orally in a form of a gelatin capsule at the dose 60  min. The blood samples were prepared by dissolv - of 0.5  mg/kg (as free base) to Beagle dogs (Marshall ing VEN in pre-warmed (37 °C) whole blood. The 0 min BioResources, East Yorkshire, UK), weighing 6.4–9.5  kg plasma samples were then obtained by the centrifugation (i.e., 5–6  months of age). The study was conducted in of the blood samples at 2,500 × g for 10 min at 4 °C. The accordance with the applicable sections of the UK Ani remaining blood samples were stored for 60 min on wet mals (Scientific Procedures) Act 1986 and Amendment ice. The samples were then subjected to the centrifuga - Regulations 2012 (the Act). Blood samples (approxi- tion at 2,500 × g for 10 min at 4 °C to obtain plasma. The mately 100  μL) were collected in K EDTA tubes (Cov- stability of VEN in whole blood was considered adequate 2 ance, Huntingdon, UK) at pre-dosing, 0.167, 0.33, 0.5, if the mean concentration of samples at 60 min remained 1, 4, 8, and 12  h post-dose either via the jugular vein in within ± 15% of that of samples at time 0  min. The ade - rats or pre-dosing, 0.25, 0.5 1, 4, 8, and 12 h via the jugu- quacy was also determined if CV was less than or equal lar vein in dogs. The blood samples were centrifuged at to 15% for two concentrations of two time points. 2,500 × g at 4  °C for 10  min, and plasma samples were In plasma sample stability, the stability of VEN was collected and stored at − 70  °C prior to analysis. When evaluated at LQC and HQG concentrations (i.e., 3 and necessary, systemic PK parameters, e.g., area under the 800  ng/mL) under typical sample-handling conditions. curve from time zero to measurable concentration (AUC For the evaluation of short-term room temperature ), were calculated using the linear trapezoidal method stability, aliquots of QC samples were stored at room 0-t and the standard area extrapolation method (Gibaldi and temperature (i.e., 22 °C) over 20 h and a portion of the Perrier 1982). The maximum concentration (C ) and sample was analyzed in replicates (n = 6 for each con- max the time point at a maximum concentration ( T ) were centration). When necessary, freeze–thaw stability was max read directly from the concentration–time profile of VEN studied by repeating the freeze-and-thaw cycle. The in the plasma. In this study, the terminal phase half-life QC samples were stored at − 70 °C for at least 12 h and (T ) was calculated by dividing 0.693 by the slope of the thawed or allowed to stand for at least 2 h at room tem- 1/2 terminal log-linear portion of the concentration–time perature. The samples were then transferred back to the profile. PK parameters were calculated using Phoenix freezer and maintained for at least 12  h. The cycle of WinNonlin (version 8.1; Pharsight Corp. Mountain View, thawing and freezing was repeated, and six replicates California, USA) from either the mean (rats) or individ at each concentration were analyzed. In addition, long- ual concentration–time profiles (dogs). term stability was evaluated after storing the samples Lee et al. Journal of Analytical Science and Technology (2023) 14:8 Page 6 of 13 Accuracy, precision, and dilution integrity Results The accuracy, precision, and dilution integrity of the Mass spectrometry and chromatography assays are summarized in Table  2. The QC samples at The full scan mass spectra of VEN and the internal the LLOQ, LQC, MQC, and HQC were analyzed at standard (IS) indicate the presence of a prominent pro- three separate occasions, in six replicates for each occa- tonated molecule, [M + H] , at m/z 407.2 and 281.2, sion, together with a set of calibration standards. The respectively. Major fragment ions, formed by the cleav- within-run RE for rat and dog samples was 0.3–7.0%, age of amide bond, were readily evident at m/z 70.0 for and 1.7–6.0%, respectively; the CV for rat and dog VEN and 86.1 for the IS. Thus, the ion transitions of samples was 1.4–7.3%, and 1.7–4.2%, respectively. The m/z 407.2 → 70.0, and 281.2 → 86.1 were used for the between-run RE for rat and dog samples was 4.2–9.0%, subsequent monitoring of VEN and the IS, respectively and − 2.0 to 2.0% with the CV for rat and dog sam- (Fig.  1A and B). To achieve adequate chromatographic ples of − 1.7 to 5.0%, and 7.4–9.0%, respectively. These separation of VEN/IS peaks from interfering peaks results suggest that the assay is accurate and precise for (1.82, 3.26, and 3.35 min) in the rat plasma, the gradient the determination of VEN concentrations ranging from with 0.1% formic acid in methanol and 0.1% formic acid 1 to 1000  ng/mL in rat and dog plasma samples. To was used. For the case of the dog plasma, no apparent determine the adequacy of sample dilution for estimat- interfering peaks were evident with the elution condi- ing the concentration in samples exceeding the ULOQ, tion. Under these chromatographic conditions, the a set of plasma samples containing VEN at 16,000  ng/ retention time was 1.75  min for VEN and 3.45  min for mL was prepared and then diluted 20-fold with the the IS, and the analytical run was completed within corresponding blank plasma. The RE for the analyte in 5.5 min per sample (Fig. 2). These analytical conditions the plasma after the 20-fold dilution with blank rat and were used in subsequent experiments. dog samples was 0.0% and − 0.6% with at a CV of 3.3% and 2.4% in rat and dog plasma samples, respectively. Selectivity and specificity These observations indicate that samples at concentra - Representative LC–MS/MS chromatograms of the tions exceeding the ULOQ can be diluted with the cor- LLOQ at 1  ng/mL are shown in Fig.  2. The chromato - responding blank plasma to determine accurately and gram of six different lots of blank analyses indicates precisely the original concentration using the current that the analyte peaks are adequately separated from assay. interfering peaks, suggesting that the selectivity of the LC–MS/MS assay is adequate for the quantification of VEN in rat and dog plasma samples. Recovery and matrix effect The mean recovery of VEN in plasma samples for rats and Linearity dogs was within the range of 88.7–98.7% and 88.7–101%, The calibration curve was linear over the concentra - respectively, with six replicates at the LQC, MQC, and tion ranges of 1–1,000  ng/mL for VEN in plasma sam- 2 HQC levels. In addition, the mean recovery for IS ranged ples from rats and dogs. A 1/x -weighted least squares from 88.1 to 96.2% and from 96.1 to 102% for the rat and quadratic regression analysis of the data was used to dog samples, respectively, at a fixed IS concentration of calculate the slope, intercept, and coefficient of deter - 2 15  ng/mL in the presence of VEN at LQC, MQC, and mination (r ) for samples from the two species. The HQC concentrations in the plasma. Collectively, these typical equation of VEN calibration curve was y = 0. 2 2 observations indicate that the recovery of both VEN and 000000521x + 0.00691x − 0.000260 (r = 0.998) and 2 IS was consistent and reproducible in the concentration y = 0.00623x − 0.000106 (r = 0.998) for rats and dogs, range. The matrix effect and IS-normalized matrix effects respectively. Where y represents the ratio of the peak were also studied with six independent sources of matrix area of the VEN to that of IS, and x represents the and two sources of matrix obtained from hemolyzed plasma concentration of VEN. The mean coefficient blood. The matrix factor of VEN and the IS was in the of determination for the calibration curves for rat and range of 0.95–1.05 and 0.91–1.00, respectively. The CV dog plasma samples was 0.996 and 0.995, respectively. of the matrix factor was less than 15% regardless of the The LLOQ for VEN was determined to be at 1  ng/mL concentration or origin of the matrix. In addition, it was with acceptable an accuracy and precision (Sect. "Accu- readily evident that the IS-normalized matrix effect was racy, precision, and dilution integrity"), and the signal- consistent (Table 3): As a result, the matrix effect of VEN to-noise ratio was greater than 5 for the two species and IS appeared negligible in the current assay. Taken studied. together, these findings indicate that the current assay provides virtually complete recovery of the analytes with L ee et al. Journal of Analytical Science and Technology (2023) 14:8 Page 7 of 13 Fig. 2 MRM chromatograms for samples in the rat plasma. A Double blank; B VEN at LLOQ with IS; C VEN at the ULOQ with IS. MRM chromatograms for samples in the dog plasma. D Double blank; E VEN at LLOQ with IS; F VEN at the ULOQ with IS Lee et al. Journal of Analytical Science and Technology (2023) 14:8 Page 8 of 13 Fig. 2 continued L ee et al. Journal of Analytical Science and Technology (2023) 14:8 Page 9 of 13 Fig. 2 continued Lee et al. Journal of Analytical Science and Technology (2023) 14:8 Page 10 of 13 Table 2 Accuracy and precision of the assay for VEN in plasma samples from the rat and dog Table 3 Matrix effects for VEN and IS in plasma samples of the rat and dog Batch Theoretical concentration (ng/mL) Matrix factor IS-normalized 1 3 300 800 800 VEN ISMatrix effect Rat plasma Rat plasma (A) Within-run accuracy and precision LQC (3 ng/mL) Mean estimated concentration 1.07 3.01 301 802 800 1 A 0.77 0.83 0.92 RE, % 7.0 0.3 0.3 0.3 0.0 2 B 1.06 1.00 1.06 CV, % 7.3 2.4 1.4 2.9 3.3 C 0.97 1.03 0.95 (B) Between-run accuracy and precision D 0.99 1.07 0.92 Mean estimated concentration 1.05 3.09 295 802 E 0.99 0.99 1.00 RE, % 6.9 9.0 4.2 5.9 F 0.91 0.98 0.93 CV, % 5.0 3.0 − 1.7 0.3 A (Hemolyzed) 1.00 1.02 0.97 Dog plasma B (Hemolyzed) 1.05 0.99 1.06 (A) Within-run accuracy and precision Mean ± SD 0.97 ± 0.09 0.99 ± 0.07 0.98 ± 0.06 Mean estimated concentration 1.06 3.05 308 828 795 CV (%) 9.5 7.0 5.8 RE, % 6.0 1.7 2.7 3.5 − 0.6 HQC (800 ng/mL) CV, % 3.6 3.3 1.7 4.2 2.4 A 0.96 0.94 1.02 (B) Between-run accuracy and precision B 1.00 1.00 1.00 Mean estimated concentration 1.02 2.98 296 784 C 1.01 1.00 1.01 RE, % 2.0 − 0.7 − 1.3 − 2.0 D 1.00 0.96 1.03 CV, % 7.6 9.0 7.4 7.8 E 0.98 0.99 0.99 Accuracy (RE, %) = (calculated concentration − theoretical concentration)/ F 1.04 1.00 1.04 theoretical concentration × 100 A (Hemolyzed) 1.01 1.02 0.99 Precision (CV, %) = standard deviation of the concentration/mean B (Hemolyzed) 1.03 1.01 1.02 concentration × 100 Mean ± SD 1.00 ± 0.02 0.99 ± 0.03 1.01 ± 0.02 Analyzed after a 20-fold dilution with blank plasma (viz. 16,000 → 800 ng/mL) CV (%) 2.5 2.7 2.0 Dog plasma no appreciable matrix effect in plasma samples of the two LQC (3 ng/mL) species. A 0.82 0.79 1.05 B 0.96 0.97 0.99 C 0.88 0.93 0.94 Stability D 1.14 0.92 1.24 The stability of VEN was evaluated under various stor - E 1.09 0.98 1.11 age and handling conditions. In general, the compound F 0.93 0.90 1.04 was relatively stable and the assay satisfied the require - A (Hemolyzed) 0.80 0.80 0.99 ments set by the guidelines for assay validation under the B (Hemolyzed) 0.97 0.96 1.01 Mean ± SD 0.95 ± 0.12 0.91 ± 0.07 1.05 ± 0.09 conditions studied, regardless of the concentration and CV (%) 12.8 8.3 8.8 origin of the matrix. For example, the concentration of HQC (800 ng/mL) the compound and IS in a stock solution that had been A 1.03 0.93 1.11 stored for 24 h at room temperature was 97.3% and 98.6% B 1.03 0.97 1.06 of the initial concentration, respectively. In addition, the C 1.06 1.01 1.05 concentration after 1 month of storage under refrigerated D 1.11 1.03 1.08 conditions was 99.8% and 94.2% of the initial concentra- E 1.07 1.02 1.05 tion, respectively. Furthermore, the analyte in the rat and F 1.04 1.00 1.04 dog plasma was found to be stable in various conditions A (Hemolyzed) 1.03 1.01 1.05 [i.e., in room temperature for 22 and 24  h; after freeze– B (Hemolyzed) 1.04 1.01 1.03 thaw cycles, and at − 70  °C over 3  months (Table  4)]. In Mean ± SD 1.05 ± 0.03 1.00 ± 0.03 1.05 ± 0.03 this study, degradation was not apparent in the sam- CV (%) 2.7 3.3 2.6 ple manager of the LC–MS/MS system (i.e., operating Matrix factor = [peak area of analyte added post-extraction]/[peak area of at 10  °C) for 8  days (Table  4). VEN was stable in whole analyte standards] blood on ice for up to 1 h. Collectively, these observations L ee et al. Journal of Analytical Science and Technology (2023) 14:8 Page 11 of 13 Table 3 (continued) Normalized Matrix Factor = Matrix Factor for Analyte/Matrix Factor for IS CV (%) = standard deviation/mean × 100 Table 4 Summary of stability studies for VEN in QC samples Batch Theoretical concentration (ng/mL) Rat Dog 3 800 3 800 (A) Short-term stability at room temperature (22 °C, 22 h for rats, 24 h for dogs, n = 6) Mean estimated concentration 2.62 725 3.15 688 RE (%) − 12.7 − 9.4 5.0 − 14.0 CV (%) 5.7 5.6 7.5 3.3 (B) Freeze–thaw stability (3 cycles for rats, 4 cycles for dogs, n = 6) Mean estimated concentration 2.59 710 2.90 735 RE (%) − 13.7 − 11.3 − 3.5 − 8.1 CV (%) 5.9 7.6 8.1 2.9 (C) Long-term stability (107 days for rats, 87 days for dogs, n = 6) Mean estimated concentration 3.42 907 29.7 693 Fig. 3 Mean plasma concentration–time curves of VEN in A rats RE (%) 14.0 13.4 − 1.0 − 13.4 and B dogs that had received an oral administration of 30 mg/kg CV (%) 12.1 5.0 4.4 4.0 and 0.5 mg/kg of VEN, respectively (key: opened circles, male; closed circles, female). Data were represented as mean ± standard deviation (D) Processed sample stability (at 10 °C for 8 days, n = 3) of n = 3 (for rats) or n = 6 (for dogs) Mean estimated concentration 3.00 773 3.08 785 RE (%) 0.0 − 3.4 2.7 − 1.9 CV (%) 2.4 4.1 0.7 1.4 Table 5 Summary of systemic PK parameters following oral RE (%) = (calculated concentration − theoretical concentration)/theoretical administration of VEN to rats and dogs concentration × 100 1 2 Pharmacokinetic Rat Dog CV (%) = standard deviation of the concentration/mean concentration × 100 parameters Male Female Male Female Mean Mean ± SD suggest that VEN is stable under typical storage and han- dling conditions. Dose (mg/kg) 30.0 0.50 T (h) 0.333 1.00 0.583 ± 0.342 0.583 ± 0.342 max Applicability of the assay to pharmacokinetic studies C (ng/mL) 1,130 4160 120 ± 61 156 ± 54 max One of the objectives of this study was to determine T (h) 1.92 1.88 1.43 ± 0.64 1.72 ± 0.29 1/2 whether the current assay would be applicable to PK AUC (ng·h/mL) 5,310 12,900 243 ± 111 361 ± 88 0→t studies of VEN in rats and dogs. The temporal profiles AUC (ng·h/mL) 5,430 13,100 267 ± 169 369 ± 98 0→∞ of the plasma concentration of the PARP inhibitor after Three replicates: Each rat covered different regions in the time profile and, as oral administration to the rat and dog at a dose of 30 mg/ results, the calculation of the standard deviation (SD) was not possible kg and 0.5  mg/kg, respectively, are depicted in Fig.  3. In 2 Six replicates general, the concentration was readily detected up to AUC from time of dosing extrapolated to infinity 12  h after administration, suggesting that the sensitivity was adequate for the quantification of plasma concentra - to male dogs, female dogs showed 1.30- and 1.49-fold tions of the drug in PK studies with rats and dogs at the higher values for C and AUC , respectively.). max 0-t doses tested. PK parameters of the drug in rats and dogs, calculated using standard moment analysis, are listed in Table 5. Interestingly, a gender difference in VEN PK was Discussion noted in rats (i.e., compared to male rats, the C and Inhibition of PARP results in severe hematologic toxici- max AUC in female rats were 3.68- and 2.43-fold higher, ties that form neutropenia in laboratory animals at low 0-t respectively). However, in dogs, the gender difference dose levels in chronic studies. The fact that toxic doses was reduced for the key PK parameters (i.e., compared could be as low as one-tenth of the effective dose for Lee et al. Journal of Analytical Science and Technology (2023) 14:8 Page 12 of 13 PARP inhibitors is now well established (AstraZeneca kg was 120 and 156  ng/mL for male and female dogs, 2022; Clovis  Oncology 2021; GlaxoSmithKline 2021), respectively. In this study, we did not directly study the suggesting that an adequate dose of VEN may be lower mechanisms for the kinetic difference of VEN by gen - than the efficacy or PK study in certain toxicity studies. der. However, olaparib, a structural analog of VEN, was In addition, exploratory repeated dose PK studies may reported to have up to 14-fold higher exposure in female be conducted in animal models (e.g., the identification rats than in male rats, while the difference was markedly of drug–drug interaction mechanisms). Frequent blood reduced (approximately twofold) in dogs (Application sampling could exacerbate hematologic toxicity of PARP Number:206162Orig1s000 2014), suggesting a common inhibitors, a small sampling volume would be ideal for mechanism for the gender difference in the PK for the PARP inhibitors. Thus, a bioanalytical method which PARP inhibitors. This aspect of PARP inhibitors warrants enables the quantification of analyte using a small vol - additional studies. ume of plasma samples could be helpful in PK/TK stud- ies of VEN. In this study, we attempted to develop and Conclusions validate a sensitive and accurate LC–MS/MS method A sensitive and robust LC–MS/MS assay was developed for the quantification of VEN in the rat and dog plasma and validated for the quantification of VEN plasma sam - samples, and quantified VEN at concentrations as low ples from rats and dogs in terms of accuracy, precision, as 1  ng/mL using 20  µL plasma samples. Assuming lin- dilution, recovery, matrix effect, and stability, which ear PK for VEN in the animal models, the LLOQ, 1  ng/ were within acceptable ranges. The blood sampling can mL, represented less than 5% of the expected C after be reduced below 3% of the total blood volume in PK/ max oral administration of VEN at 1 and 0.1  mg/kg in rats TK studies in rats using this method. This method can and dogs, respectively. Taken together with the possibil- be applied to the chronic study of VEN to minimize the ity of low-dose studies of VEN in animals, this observa- hematological effect of blood sampling. Collectively, tion suggests that the current assay can be readily applied these observations indicate that the current assay can be to typical TK/PK studies of the inhibitor in two animal reliably applied to PK and TK studies of VEN in rats and models. dogs using the limited sample volume. Using gradient elution, interfering peaks originating from the plasma were reasonably separated from the ana- Abbreviations lytes, while limiting a run-time of approximately 5.5 min. AUC Area under the curve from time zero to measurable concentration 0→t The deuterated form of VEN is not commercially avail - AUC Ar ea under the curve from time zero to infinity 0→∞ AUMC Ar ea under the first moment curve from time zero to infinity 0→∞ able; hence, a series of compounds had to be screened for C Maximum concentration max an IS. In the preliminary screening study, imipramine was CV Coefficient of variation selected as the IS, considering its recovery and matrix HQC High quality control IS Internal standard effects with VEN and IS (Table  3). We found that the LC–MS/MS Liquid chromatography–tandem mass spectrometry analytes were almost completely extracted in the current LLOQ Lower limit of quantification assay as the recoveries for the rats and dogs, 88.7–98.7% LQC Low quality control MQC Mid‑quality control and 88.7–101%, respectively, for VEN; 88.1–96.2% and MRM Multiple reaction monitoring 96.1–102%, respectively, for IS. In addition, the recovery MRT Mean residence time values were consistent over the concentration range used PARP Poly (ADP‑ribose) polymerase) PK Pharmacokinetic in this study. The experimental data for selectivity, linear - QC Quality control ity, precision, accuracy, and stability were found accept- RE Relative error able according to the guidelines for bioanalytical method SD Standard deviation T Half‑life 1/2 validation from the US Food and Drug Administration T Time point at maximum concentration max and the European Medicines Agency. TK Toxicokinetic In this study, we found that the assay was applica- ULOQ Upper limit of quantification UK United Kingdom ble to PK studies for VEN in rats and dogs at oral doses US United States of 30  mg/kg and 0.5  mg/kg, respectively. Based on the VEN Venadaparib LLOQ of the current assay, we were able to monitor the Acknowledgements plasma concentration–time profile up to 12  h after oral Not applicable. administration, which accounted for over six times the T for VEN (T ≤ 2  h for VEN, Table  5). It was noted Author contributions 1/2 1/2 ML, EJ, and JL contributed to conceptualization; ML and EJ contributed to that gender differences in PK were found for VEN in the methodology; ML performed formal analysis, data curation, writing—original animal models: C at 30 mg/kg was 1130 and 4160 ng/ max draft preparation, and visualization; ML, J‑HL, and S‑ JC contributed to writ‑ mL for male and female rats, respectively; C at 0.5 mg/ ing—review and editing; SC, WSL, and S‑ JC supervised the study; NSB and SL max L ee et al. Journal of Analytical Science and Technology (2023) 14:8 Page 13 of 13 administered the project; Y‑ WP performed funding acquisition. All authors Identifier NCT03317743, Study to Assess the Safety and Tolerability of have read and agreed to the published version of the manuscript. NOV140101(IDX‑1197) in Patients With Advanced Solid Tumors. https:// clini caltr ials. gov/ ct2/ show/ NCT03 317743. Accessed on 12 Nov 2022. Funding Identifier NCT04174716, Basket Trial of IDX ‑1197, a PARP Inhibitor, in Patients This study was conducted with the National‑ OncoVenture supported by the With HRR Mutated Solid Tumors ( VASTUS). https:// clini caltr ials. gov/ ct2/ National Cancer Center, designated by the Ministry of Health and Welfare show/ NCT04 174716. Accessed on 12 Nov 2022. Korea (HI17C2196). Identifier NCT04725994, Study to Assess the Safety, Tolerability, and Efficacy of IDX‑1197 in Combination With XELOX or Irinotecan in Patients With Availability of data and materials Advanced Gastric Cancer. https:// clini caltr ials. gov/ ct2/ show/ NCT04 The data that support the findings of this study are available from the cor ‑ 725994. Accessed on 12 Nov 2022. responding author upon reasonable request. Im S‑A, Kim JE, Lee KS, Moon YW, Ahn HK, Lee KH, Ock C‑ Y, Roh EJ, Lee M, Hong MJ et al. Phase Ib study of venadaparib, a potent and selective PARP inhibitor, in homologous recombination repair (HRR) mutated breast Declarations cancer. In: Proceedings of the ESMO Congress, 2021. Kim YM, Bae K‑S, Baek NS, Kim K, Lee WS, Roh E‑ J, Ock C‑ Y, Kim S‑B. Abstract Competing interests 3107. First‑in‑human dose ‑finding study of venadaparib (IDX ‑1197), a The authors declare that they have no competing interests. potent and selective PARP inhibitor, in patients with advanced solid tumors. In: Proceedings of the ASCO Annual meeting, 2021. Lee M, Park J‑ T, Yang J‑h, Kim D, Je I‑ G, Lee YS, Jeong J, Song DK, Park S, Lee Received: 16 November 2022 Accepted: 18 January 2023 H‑S et al. Abstract A106: Development of IDX ‑1197, a novel, selective, and highly potent PARP inhibitor. 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Journal

Journal of Analytical Science & Technology – Springer Journals

Published: Feb 1, 2023

Keywords: Method validation; Bioanalysis; Mass spectrometry; Pharmacokinetics; Poly (ADP-ribose) polymerases inhibitor, Venadaparib

References

  • DNA damage/repair management in cancers
    Alhmoud, JF; Woolley, JF; Al Moustafa, AE; Malki, MI
  • A synthetic lethal therapeutic approach: poly(ADP) ribose polymerase inhibitors for the treatment of cancers deficient in DNA double-strand break repair
    Ashworth, A
  • Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase
    Bryant, HE; Schultz, N; Thomas, HD; Parker, KM; Flower, D; Lopez, E; Kyle, S; Meuth, M; Curtin, NJ; Helleday, T
  • A good practice guide to the administration of substances and removal of blood, including routes and volumes
    Diehl, KH; Hull, R; Morton, D; Pfister, R; Rabemampianina, Y; Smith, D; Vidal, JM; van de Vorstenbosch, C
  • Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy
    Farmer, H; McCabe, N; Lord, CJ; Tutt, AN; Johnson, DA; Richardson, TB; Santarosa, M; Dillon, KJ; Hickson, I; Knights, C
  • DNA recombination: the replication connection
    Haber, JE
  • Genome maintenance mechanisms for preventing cancer
    Hoeijmakers, JH
  • PARP inhibitors: synthetic lethality in the clinic
    Lord, CJ; Ashworth, A
  • State-of-the-art strategies for targeting the DNA damage response in cancer
    Pilie, PG; Tang, C; Mills, GB; Yap, TA
  • Poly(ADP-ribose): historical perspective
    Sugimura, T; Miwa, M
  • Haematologic toxicities with PARP inhibitors in cancer patients: an up-to-date meta-analysis of 29 randomized controlled trials
    Wang, C; Li, J
  • Lack of toxicological influences by microsampling (50 microL) from jugular vein of rats in a collaborative 28-day study
    Yokoyama, H; Hattori, N; Ohtsuka, H; Murata, E; Kobayashi, A; Muta, K; Takumi, A; Kitaura, H; Jinno, F; Iwai, A