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. 2024 Apr;389(1):96–105. doi: 10.1124/jpet.123.002051

Pharmacokinetics of Panobinostat: Interspecies Difference in Metabolic Stability

Wenqiu Zhang 1,✉, Ju-Hee Oh 1, Wenjuan Zhang 1, Courtney C Aldrich 1, Rachael W Sirianni 1, William F Elmquist 1,✉
PMCID: PMC10949161  PMID: 38409112

Abstract

The deregulation of histone deacetylase (HDAC) expression is often seen in many cancers, and HDAC inhibitors have shown potency against a variety of cancer types. Panobinostat is a potent pan-HDAC inhibitor that has been tested in multiple studies for the treatment of brain tumors. There have been contrasting views surrounding its efficacy for the treatment of tumors in the central nervous system (CNS) following systemic administration when examined in different models or species. We conducted experiments using three different mouse strains or genotypes to have a more comprehensive understanding of the systemic as well as the CNS distributional kinetics of panobinostat. Our study found that panobinostat experienced rapid degradation in vitro in Friend leukemia virus strain B mouse matrices and a faster degradation rate was observed at 37°C compared with room temperature and 4°C, suggesting that the in vitro instability of panobinostat was due to enzymatic metabolism. Panobinostat also showed interstrain and interspecies differences in the in vitro plasma stability and was stable in human plasma. The objective of this study was to examine the in vitro metabolic stability of panobinostat in different matrices and assess the influence of that metabolic stability on the in vivo pharmacokinetics and CNS delivery of panobinostat. Importantly, the plasma stability in various mouse strains was not reflected in the in vivo systemic pharmacokinetic behavior of panobinostat. Several hypotheses arise from this finding, including: the binding of panobinostat to red blood cells, the existence of competing endogenous compounds to enzyme(s), the distribution into tissues with a lower level of enzymatic activity or the metabolism occurring in the plasma is a small fraction of the total metabolism in vivo.

SIGNIFICANCE STATEMENT

Panobinostat showed different in vitro degradation in plasma from different mouse strains and genotypes. However, despite the differences surrounding in vitro plasma stability, panobinostat showed similar in vivo pharmacokinetic behavior in different mouse models. This suggests that the interstrain difference in enzymatic activity did not affect the in vivo pharmacokinetic behavior of panobinostat and its central nervous system distribution in mice. This lack of translation between in vitro metabolism assays and in vivo disposition can confound drug development.

Introduction

Despite decades of research and an improved understanding of the genetic drivers, brain tumors are the leading cause of death from cancer in patients younger than 20 (Siegel et al., 2023). Glioblastoma multiforme (GBM), a highly aggressive brain tumor with dismal prognosis, is the most common malignant primary brain tumor (Ostrom et al., 2022). Somatic alterations in epigenetic regulators have been found in both adult and pediatric GBM patients (Schwartzentruber et al., 2012; Brennan et al., 2013) and the understanding of epigenetically different GBM subgroups is expected to help with the development of targeted therapeutics (Sturm et al., 2012). Epigenetic modifications have also been reported in other types of brain tumors, including the most common pediatric malignant brain tumor, medulloblastoma (Lindsey et al., 2005). The most lethal childhood brain tumor, diffuse intrinsic pontine gliomas (Morales La Madrid et al., 2015), also show these epigenetic modifications. Histone acetylation, regulated by histone acetyltransferases and histone deacetylases (HDACs), alters chromatin structure, and affects gene expression. Abnormal expression of HDACs have been found in various types of brain tumors (de Ruijter et al., 2003; Milde et al., 2010; Lee et al., 2015), and HDAC inhibitors have shown potency in multiple in vitro settings and preclinical studies of brain tumors (Bezecny, 2014; Grasso et al., 2015; Pak et al., 2019), leading to growing interest in applying HDAC inhibitors as therapeutic agents for brain tumors.

Many of the HDAC inhibitors are hydroxamic acid derivatives, and the plasma stability of hydroxamic acids has been investigated. Hermant et al. (2017) showed that hydroxamic acids are vulnerable to the metabolic activities of esterases in plasma. Carboxylic acids, common products from the hydrolysis of hydroxamic acids under physiologic conditions, are often less active than the parent compound and could be more limited in its blood–brain barrier permeability (Flipo et al., 2009). In vivo, metabolism in plasma can lead to rapid clearance, and hence a poor exposure that may then result in diminished efficacy (Gao and Hu, 2010). In vitro degradation in plasma specimens during sample storage and processing may cause inaccurate measurements in the bioanalysis and therefore lead to skewed results of pharmacokinetic studies. This can give rise to limitations in the development and use of the compound, therefore examination of the stability of these compounds in plasma is critically important.

Vorinostat, also known as suberoylanilide hydroxamic acid, was the first HDAC inhibitor approved by the Food and Drug Administration for the treatment of cutaneous T-cell lymphoma (Mann et al., 2007). Du et al. (2006) discovered that vorinostat was not stable in vitro in human plasma and suggested that human serum, in which vorinostat showed improved stability, be used as an alternative sample matrix to obtain accurate concentration measurements and pharmacokinetic and pharmacodynamic interpretations. Konsoula and Jung (2008) have studied the in vitro stability of vorinostat in plasma and found that the in vitro half-life of vorinostat was longest in mouse plasma (115 minutes), followed by rat and porcine plasma (86 and 87 minutes, respectively), and was shortest in human plasma (75 minutes). The difference in vitro metabolic stability profiles among species can affect the validity of the utilization of animal models in pre-clinical studies. In addition, vorinostat has a half-life of approximately 2 hours in patients following an oral dose (Kelly et al., 2005). The short half-life might be a reason for its lack of efficacy when targeting solid tumors (Chun, 2016).

These reports lead us to our interest in the in vitro stability of panobinostat, a pan-HDAC inhibitor showing in vitro potency and in vivo efficacy against multiple types of brain tumors (Grasso et al., 2015; Pei et al., 2016; Yao et al., 2017). Panobinostat has been reported to be extensively metabolized in patients with a median half-life of 30.7 hours (Clive et al., 2012). It has been discovered that panobinostat formed a carboxylic metabolite in mouse and rat plasma, but not human plasma (https://www.accessdata.fda.gov/drugsatfda_docs/nda/2015/205353Orig1s000PharmR.pdf). However, there are scarce data that show the in vitro stability of panobinostat in various biomatrices. Will the potential interspecies difference affect the translation from preclinical models to clinical studies for the treatment of CNS tumors? To begin to answer this, we investigated the in vitro metabolic stability of panobinostat in different matrices from various species and studied the in vivo pharmacokinetic behavior and CNS distribution of panobinostat in various mouse models. The results from this study highlight the importance of examining metabolic stability, and can inform the application of mouse models in the preclinical settings for panobinostat.

Materials and Methods

Chemicals and Reagents.

Panobinostat was ordered from Selleck Chemicals (Houston, TX). Panobinostat-d8 hydrochloride salt was obtained from Toronto Research Chemicals (Toronto, ON, Canada). Na-heparinized human, monkey, dog, and rat plasma were purchased from Innovative Research (Novi, MI). Na-heparin was purchased from Meitheal Pharmaceuticals (Chicago, IL). Recombinant mouse carboxylesterase 1c (CSB-YP338557MO) was obtained from Cusabio (Houston, TX). 4-Nitrophenyl acetate (4-NPA) was ordered from Sigma (St. Louis, MO). All other chemicals and reagents were high-performance liquid chromatography-grade and purchased from Thermo Fisher Scientific (Waltham, MA).

Animals.

Carboxylesterase (Ces) 1c knockout (B6.Cg-Ces1ctm1.1Loc/J, Ces1cKO) mice and wild-type C57BL6/J mice were sourced from The Jackson Laboratory (Bar Harbor, ME). Ces1cKO mice are homozygous knockout mice. Mice between 12 to 24 weeks old were used for in vitro studies and in vivo pharmacokinetic studies. Friend leukemia virus strain B (FVB) mice were sourced from Taconic Biosciences, Inc. (Germantown, NY) and mice between 8 to 16 weeks old were used for in vitro stability study. The colonies of Ces1cKO mice and FVB mice have been housed and maintained following an established breeding protocol in the Research Animal Resources facility located at University of Minnesota. Breeding and experiment protocols have been approved by University of Minnesota Institutional Animal Care and Use Committee and performed in accordance with the Guide for the Care and Use of Laboratory Animals established by the US National Institutes of Health.

In Vitro Stability Study.

The stability studies were initially conducted in FVB mouse matrices because we previously observed the degradation during the in vitro incubation of panobinostat in FVB mouse plasma (Zhang et al., 2023). The FVB strain is routinely used for the investigation of efflux transporters, and we have shown that p-glycoprotein influences the distribution of panobinostat to the CNS (Zhang et al., 2023). Plasma, brain, and spinal cord were harvested from FVB, Ces1cKO, and wild-type C57BL6/J mice. Blood was collected using preheparinized needles into tubes containing Na-heparin and centrifuged at 7,500 rpm at 4°C for 10 minutes to obtain plasma. For the comparison between blood and plasma, whole blood collected from multiple wild-type FVB mice was pooled, and half of the blood was centrifuged to obtain plasma. Whole blood was collected using syringe without anti-coagulant into covered tubes and left at room temperature for 30 minutes to allow the blood to clot, and serum was obtained by centrifugation at 12,000 rpm for 5 minutes at 4°C. Human brain tissue was obtained from our collaborators at Mayo Clinic (Rochester, MN). Plasma, serum, brain and spinal cord were kept at –20°C before use. Brain and spinal cord homogenates were prepared in three times volume of PBS (1×, pH 7.4) using a mechanical Omni THb homogenizer (Omni International Inc., Kennesaw, GA). On the day of usage, the pH of CNS tissue homogenates was adjusted to 7.4 at the temperature at which the study was intended to be conducted. Human, monkey (Rhesus), dog (Canine), and rat (Wistar) plasma was purchased from Innovative Research. Plasma and serum were used without dilution. Panobinostat was spiked into PBS or matrices to obtain a final concentration of 1 μm. The incubation concentration was determined based on our observations of in vivo panobinostat plasma concentrations. The drug-containing buffer and murine matrices were separated into three groups and each group was incubated at 37°C, room temperature (RT), and 4°C, respectively, with agitation at 150 rpm for the designated time points. Plasma from different species were spiked with panobinostat at 1 μm and incubated at 37°C with agitation. Each group was prepared in triplicate. Samples were collected at different time points over 24 hours (100 μl per replicate per time point), snap-frozen on dry ice, and kept at –80°C before analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Cleavage by the Recombinant Mouse Carboxylesterase 1c.

The enzymatic activity of the recombinant mouse carboxylesterase 1c (rmCes1c) was confirmed using 4-NPA (Sigma-Aldrich, Inc.) in Tris buffer (50 mM Tris, 150 mM NaCl, pH7.5). Once the degradation of the substrate 4-NPA was observed, the same batch of thawed rmCes1c was used for the study of panobinostat on the same day. The carboxylesterase concentration is approximately 80 μg/ml in mouse plasma (Li et al., 2005) and hence the rmCes1c was spiked into the Tris buffer, pooled human plasma, and pooled Ces1cKO mouse plasma to a final concentration of 80 μg/ml together with 1 μm panobinostat. Panobinostat was spiked to Tris buffer and FVB mouse plasma without rmCes1c as the negative and positive control, respectively. After 30 minutes, 1 hour, and 4 hours of incubation at 37°C, 50 μl of matrix was sampled from each group (n = 3) and added into 200 μl cold acetonitrile, followed by a 5-minute vortex and then 5-minute centrifugation at 14,000 rpm at 4°C. A total of 100 μl of the clear supernatant was transferred to a new tube and stored at –80°C until LC-MS/MS analysis.

Blood-to-Plasma Ratio.

Considering the in vitro stability of panobinostat, blood from Ces1cKO mice was used for the quantification of the blood-to-plasma ratio. Whole blood (∼6 ml) from Ces1cKO mice was collected on the day of experiment. Whole blood was pooled before 4 ml whole blood was centrifuged at 7,500 rpm for 10 minutes at 4°C to obtain control plasma. Whole blood and control plasma were prewarmed to 37°C. Panobinostat stock (30 μg/ml) was prepared in methanol (3% DMSO). Ten microliters of panobinostat stock was added into each 1.5 ml Eppendorf tube (n = 5). We observed that coincubation with 3.3% methanol (0.1% DMSO) caused hemolysis and led to reddish plasma, therefore, the aliquoted panobinostat stocks were dried using a SpeedVac (ISS110, Savant) before 300 μl pre-warmed whole blood or control plasma was added into each tube and mixed gently to reach a final concentration of 1000 ng/ml. Our preliminary study showed that a distributional equilibrium was reached after approximately 2 hours of incubation, therefore, panobinostat-spiked whole blood and control plasma samples were incubated at 37°C for 2 hours with mild agitation (50 rpm). After 2 hours, whole blood samples were centrifuged at 7,500 rpm for 10 minutes at 4°C to obtain plasma, and concentrations were measured and labeled as Cp. Concentrations in the control plasma were determined and labeled as Ccp. The blood-to-plasma ratio was calculated according to a previously reported method (Wen et al., 2010):

graphic file with name jpet.123.002051e1.jpg

Unbound Fractions in Mouse Plasma, Brain, and Spinal Cord.

The Centrifree ultrafiltration device (MilliporeSigma, Burlington, MA) was used to determine the unbound fractions of panobinostat in mouse plasma, brain and spinal cord. Each centrifugal unit has an Ultracel regenerated cellulose membrane with a 30 kDa molecular weight cut-off. Matrices from Ces1cKO mice were collected and kept at –20°C before use. Plasma was prewarmed to room temperature before panobinostat was added to reach a final concentration of 1 μm. The concentration was selected considering the range of plasma concentrations observed in our in vivo studies. Panobinostat-spiked plasma was aliquoted into five tubes and incubated at room temperature for 5 minutes with agitation (150 rpm). After incubation, a 100-μl plasma sample was collected from each replicate for the measurement of total concentrations (Cp,total). A 1-ml plasma sample was then added to the ultrafiltration device, followed by centrifugation at 3500 rpm for 5 minutes at room temperature (Clay Adams Triac centrifuge). The protein-free ultrafiltrate in the filtrate cup was collected for the determination of free concentrations (Cp,free). We have previously found that the nonspecific binding of panobinostat to the ultrafiltration device was negligible at the tested concentration (Zhang et al., 2023). The plasma unbound fraction (Fu,plasma) of panobinostat was determined using the following equation:

graphic file with name jpet.123.002051e2.jpg

Brain and spinal cord homogenates were prepared in three times volume of PBS using the THb homogenizer on an ice bath. Panobinostat was spiked into the CNS tissue homogenates (pH adjusted to 7.4 at room temperature before use) to a final concentration of 1 μm. After a 5-minute incubation at RT with agitation, 100 μl homogenates were collected for the measurement of total concentrations (Cb,total and Csc,total for brain and spinal cord, respectively) (n = 5). Supernatant was collected after the centrifugation of the homogenates at RT for 15 minutes at 12,000 rpm and added to the ultrafiltration device. Following 10-minute centrifugation at 3500 rpm at RT, the ultrafiltrate from each unit was collected for the measurement of the free concentrations in homogenates (Cb,free and Csc,free). The brain and spinal cord unbound fractions of panobinostat were determined following the reported equation (Kalvass and Maurer, 2002):

graphic file with name jpet.123.002051e3.jpg

where the Fu,diluted for the brain is the ratio of the Cb,free to the Cb,total (Csc,free to the Csc,total for the spinal cord), and D is the dilution factor (in this case D = 4).

Pharmacokinetics and CNS Distribution of Panobinostat following a Single Intravenous Bolus Dose in Ces1cKO and Wild-Type C57BL6/J Mice.

The dosing solution of panobinostat was formulated in water with 0.8% DMSO, 0.8% Tween80, and 19.2% PEG300 at a final drug concentration of 2 mg/ml on the day of use. Mice received a bolus intravenous dose of panobinostat at 10 mg/kg (5 μl volume/g body weight) via injection into the tail vein. Adult mice were weighed on the day of experiment before dosing to be given an accurate dose. Samples were collected from 10 minutes to 24 hours post-dose. At each time point, mice were euthanized using a carbon dioxide chamber. Blood, brain, and spinal cord were sampled immediately on euthanasia. Blood samples were collected via cardiac puncture and kept in an ice bath before being centrifuged at 7,500 rpm for 10 minutes at 4°C for plasma separation. CNS tissues were rinsed with saline, and the blood and meninges on the tissue surface were carefully removed using Kimwipes. Plasma and CNS tissues were fast-frozen using dry ice on collection and kept at –80°C until LC-MS/MS analysis. An equal number of male and female adult mice were allocated to each time point group (n = 4 per group), and no sex difference has been observed.

LC-MS/MS Analysis.

Panobinostat concentrations were measured using LC-MS/MS. CNS tissue homogenates were prepared in three times volume of 5% bovine serum albumin (w/v). Brains were homogenized by the THb homogenizer and spinal cords were homogenized using a handheld cordless motor (Pellet Pestles, DWK Life Sciences, LLC., Millville, NJ). CNS tissue homogenates and plasma were extracted via liquid–liquid extraction. A pH 12 buffer, containing 100 μm sodium hydroxide and 48 μm sodium bicarbonate, was prepared at room temperature. The master stock of panobinostat at 1 mg/ml was prepared in DMSO and diluted using methanol to a series of substocks. The master stocks of panobinostat for standards and quality controls (QCs) were prepared from independent weighings. For each batch of analysis, standards and QCs were processed in parallel with samples. Substocks of panobinostat were added into Eppendorf tubes and dried using the SpeedVac for the preparation of standards and QCs. An equal amount (10 ng) of panobinostat-d8 (in methanol) was added to standards, QCs, and samples as the internal standard. Panobinostat and its internal standard were partitioned by adding one time volume of pH 12 buffer and five times volume of ethyl acetate, followed by vortexing for 5 minutes. After the centrifugation at 14,000 rpm for 5 minutes at 4°C, the supernatant was then transferred to a new tube and evaporated under a gentle nitrogen gas flow. An 82:18 water (0.1% formic acid):acetonitrile (0.1% formic acid) (v/v) solution was used for reconstitution. A reversed-phase liquid chromatography method was set up using an YMC-PACK ODS-AM column (35 × 2.0 mm, S-3 μm, 12 nm; YMC, Inc.). A gradient separation method running on an UltiMate 3000 LC system (Thermo Fisher Scientific, Waltham, MA) coupled with a TSQ Vantage triple quadrupole MS system (Thermo Finnigan LLC, San Jose, CA) was used for all the measurements. The phase A consisted of water with 0.1% formic acid, and the phase B consisted of acetonitrile with 0.1% formic acid. The flow rate of the mobile phase was 0.5 ml/min. The composition of the mobile phase was started at 82% of phase A and converted to 10% of phase A at 3 minutes to wash the column for 3 minutes and altered back to 82% of phase A. The mass-to-charge transition ratios for panobinostat and panobinostat-d8 under positive electrospray ionization mode were 350.27 → 158.08 and 358.32 → 164.1, respectively. The electrospray ion source parameters were as follows: spray voltage 3500 V, collision energy 23 V, vaporizer temperature 430°C, and capillary temperature 300°C. For all matrices, the limit of quantification was 10 ng/ml and standard calibration curves were linear within the range of 10–1000 ng/ml (r2 >0.99 with weighting factor 1Y2). The acceptance criteria for accuracy and precision (CV <15%) were met for all batches of samples.

Pharmacokinetic Data Analysis.

Panobinostat concentrations in the plasma and spinal cord were obtained directly from LC-MS/MS measurements. The concentrations of panobinostat in the brain were corrected for residual blood following the Oh method (Oh et al., 2022). The correction was performed by subtracting the panobinostat amount in the vascular blood from the total panobinostat amount measured in the brain homogenates. The concentration-time profiles of panobinostat in the plasma, brain, and spinal cord were analyzed using non-compartmental analysis performed by Phoenix WinNonlin 8.3 (Certara USA Inc., Princeton, NJ) to estimate pharmacokinetic parameters. Areas under the curve (AUCs) were calculated by linear trapezoidal integration method until the last measured time point to measure the exposure following dosing. The AUC from the last measured time point extrapolated to infinity was determined by dividing the concentration at the last time point by the first-order rate constant associated with the terminal portion of the concentration–time curve.

The standard deviations around the averages of area under the concentration-time curve from time zero to infinity (AUCinf) were calculated using the reported methods (Bailer, 1988; Yuan, 1993). A tissue-to-plasma partition coefficient (Kp) was determined by the ratio of AUCinf in tissues (AUCinf,brain/spinal cord) to the AUC in plasma (AUCinf,plasma). The standard errors around the means of Kp were determined according to the principle of propagation of error (You et al., 2013). The unbound tissue-to-plasma partition coefficient (Kp,uu) is calculated as follows:

graphic file with name jpet.123.002051e4.jpg

Statistical Analysis.

All experimental data are presented as mean ± standard deviation except for the Kps that are reported as mean ± standard error. For in vitro stability studies, the degradation rate constants for panobinostat were determined using plots of the logarithm of the percentage of panobinostat remaining versus incubation time. All statistical tests were conducted using GraphPad Prism 9.4.0 (La Jolla, CA), and differences were considered statistically significant when P < 0.05. Unpaired two-tailed t tests (not assuming equal standard deviations between two groups) and one-way ANOVA followed by a Tukey post-hoc test were performed when there were two groups and more than two groups for comparison, respectively.

Results

In Vitro Stability of Panobinostat in FVB Matrices.

Panobinostat was evaluated for its in vitro stability in PBS and FVB mouse specimens including plasma, brain, and spinal cord, at different temperatures. Panobinostat was stable in PBS and unstable in mouse matrices at all three temperatures tested (Fig. 1). The slopes were determined by performing linear regression analysis for the logarithm percent of drug remaining-time profiles. The corresponding in vitro half-lives are listed in Table 1. Panobinostat showed robust in vitro degradation in the FVB mouse plasma, while being least stable when incubated at 37°C, and more stable at RT (P < 0.0001) and 4°C (P < 0.0001). Panobinostat was unstable to some degree in all three FVB matrices investigated. At 37°C, the degradation of panobinostat in the brain was faster than in the spinal cord (P < 0.05) and slower than in the plasma (P < 0.0001). To summarize, panobinostat was stable in the buffer and unstable in FVB mouse matrices with temperature-dependent degradation rates. These results suggested that the in vitro degradation of panobinostat was due to enzyme-mediated metabolism.

TABLE 1.

In vitro half-lives of panobinostat in different matrices (n = 3, mean ± S.D.).

Matrix Half-life (h)
FVB Mice Ces1cKO Mice Wild-Type C57BL6/J Mice Rat Human Monkey Dog
Plasma 1.1 ± 0.0 (stable) 130.5 ± 25.7 34.3 ± 2.5 (stable) (stable) (stable)
Brain 11.8 ± 0.3 12.4 ± 0.2 10.5 ± 0.1 N.D. 44.6 ± 3.1 N.D. N.D.
Spinal cord 32.8 ± 1.8 22.4 ± 1.3 31.3 ± 1.7 N.D. N.D. N.D. N.D.
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N.D., not determined.

Fig. 1.

Fig. 1.

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In vitro stability of panobinostat in the (A) PBS, (B) FVB mouse plasma, (C) FVB mouse brain, and (D) FVB mouse spinal cord at 37°C RT and 4°C (n = 3, mean ± S.D.).

In Vitro Stability of Panobinostat in Matrices from Different Species.

Panobinostat has been reported to be converted to its carboxylic acid metabolite when incubated at 37°C in rodent plasma, but not in human, monkey, and dog plasma (https://www.accessdata.fda.gov/drugsatfda_docs/nda/2015/205353Orig1s000PharmR.pdf). Therefore, we investigated the in vitro degradation rates of panobinostat in plasma from multiple species (Fig. 2A). The anticoagulant for all the plasma matrices was sodium heparin. Results showed that panobinostat was unstable in rat plasma and least stable in FVB mouse plasma. In contrast, panobinostat was stable in human, monkey, and dog plasma. These inter-species differences around in vitro stability in plasma might influence the in vivo pharmacokinetic behavior of panobinostat. To examine whether there is a difference in panobinostat degradation between human and mouse brain, we measured the stability of panobinostat in the human brain at 37°C under the same experimental conditions (Fig. 2B). A higher percentage of panobinostat remained after a 24-hour incubation at 37°C in human brain compared with FVB mouse brain (P < 0.0001). The influence of residual blood in murine CNS tissues on its in vitro stability was determined by measuring the percentage of panobinostat remaining in the homogenates prepared from saline-perfused CNS tissues (Supplemental Method 1). The results showed that although the residual blood induced to some extent compound degradation, panobinostat was unstable in the saline-perfused brain and spinal cord, indicating that factors within the mouse brain and spinal cord were the predominant reasons for the in vitro degradation of panobinostat in CNS tissues (Supplemental Fig. 1).

Fig. 2.

Fig. 2.

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In vitro stability of panobinostat in (A) the plasma from different species and (B) mouse and human brain at 37°C (n = 3, mean ± S.D.). Data for the FVB mouse plasma are the same as those in Figure 1.

4-NPA is a prototypical substrate of esterases that is used for the measurement of enzymatic activities by monitoring the formation of 4-nitrophenoxide at 410 nm (Anderson et al., 1994). The hydrolytic activities of plasma from different species and mouse strains and genotypes were measured using the hydrolysis of 4-NPA (Supplemental Method 2). Plasma from wild-type FVB and wild-type C57BL6/J mice showed the highest hydrolytic activities against 4-NPA (Supplemental Fig. 2 and Supplemental Table 1). On the contrary, plasma from Ces1cKO mice showed similar hydrolytic activities as human, monkey, and dog plasma. The in vitro plasma stability of panobinostat was different among species and the interspecies differences were in line with the expression of carboxylesterase in plasma among species. It has been reported that there is no expression of carboxylesterase in human, monkey, and dog plasma (Li et al., 2005), and carboxylesterase 1c (Ces1c) is the isoform of carboxylesterase that is present in mouse plasma (Di, 2019). Therefore, we proposed that the in vitro degradation of panobinostat in mouse plasma might be the result of carboxylesterase-mediated metabolism. The recombinant mouse Ces1c enzyme and Ces1cKO mice were used to study the potential role of Ces1c.

In Vitro Stability of Panobinostat in Matrices from Different Mouse Strains.

It has been reported that there is no detectable carboxylesterase activity in the plasma but normal carboxylesterase activities in tissues of Ces1cKO mice (Duysen et al., 2011). Panobinostat showed varied stability in plasma from different mouse strains and genotypes. Panobinostat was significantly more stable in wild-type C57BL6/J plasma than in FVB plasma (P < 0.001) (Fig. 3A). There was also difference between the in vitro stability of panobinostat in Ces1cKO and wild-type C57BL6/J plasma (P < 0.01). Whether the Ces1c was responsible for the in vitro plasma degradation of panobinostat needs further investigation. Panobinostat was also unstable in the brain and spinal cord from both Ces1cKO and wild-type C57BL6/J mice (Fig. 3). The in vitro stability of panobinostat in Ces1cKO brain was similar to that in the wild-type C57BL6/J brain (P = 0.3515), indicating that the absence of Ces1c did not influence the degradation of panobinostat in the mouse brain. This also suggested that contributors for the panobinostat degradation in mouse plasma and CNS tissues might be different.

Fig. 3.

Fig. 3.

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In vitro stability of panobinostat in the plasma, brain, and spinal cord from (A) the wild-type (WT) C57BL6/J mice and (B) Ces1cKO mice at 37°C (n = 3, mean ± S.D.).

Cleavage by the Recombinant Mouse Carboxylesterase 1c.

Recombinant mouse Ces1c (rmCes1c) was used to assess the role of carboxylesterase in the in vitro degradation of panobinostat. The hydrolytic activity of rmCes1c was confirmed using 4-NPA in the Tris buffer (Supplemental Fig. 3). The addition of rmCes1c into panobinostat-spiked Tris buffer at the same pH and temperature did not induce the degradation of panobinostat (Fig. 4), indicating that there might be cofactors that participated in the plasma degradation of panobinostat. To address this, rmCes1c was added into human or Ces1cKO plasma, in which panobinostat was stable, at the reported in vivo concentration of Ces1c in mice (80 μg/ml) (Li et al., 2005). Results showed that panobinostat was stable with the presence of rmCes1c in both human and Ces1cKO plasma, suggesting that the in vitro degradation of panobinostat observed in wild-type mouse plasma was Ces1c-independent.

Fig. 4.

Fig. 4.

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The percentage of panobinostat remaining after incubation with or without recombinant mouse Ces1c in vitro in buffer and different matrices at 37°C.

Blood-to-Plasma Ratio and Unbound Fractions.

The blood-to-plasma ratio of panobinostat was 2.17 ± 0.19 (n = 5) as determined in fresh blood and plasma collected from Ces1cKO mice. The standard deviation was calculated via the propagation of error for divisions (You et al., 2013). The average value 2.17 was used for the correction of the contribution of residual blood to the panobinostat brain concentrations.

Considering the in vitro instability of panobinostat, rapid ultrafiltration was performed to determine the free fraction in various matrices. A 5-minute incubation at room temperature was applied with the assumption that the drug-binding site reaction occurs rapidly on the addition of the compound into matrices. Mouse matrices and ultrafiltration devices were prewarmed to room temperature before the experiment. The unbound percent of panobinostat were 12.39 ± 0.56 in Ces1cKO plasma, 4.16 ± 0.44 in Ces1cKO brain, and 4.90 ± 0.37 in Ces1cKO spinal cord. The unbound fraction of panobinostat in the plasma was higher than that in the brain (P < 0.0001) and spinal cord (P < 0.0001). Panobinostat showed higher binding in the brain than in the spinal cord (P < 0.05). The average unbound fractions were used to calculate the unbound tissue-to-plasma partition coefficient (Kp,uu) of panobinostat using eq. 4.

Pharmacokinetics and CNS Distribution of Panobinostat following a Single Intravenous Bolus Dose in Ces1cKO and Wild-Type C57BL6/J Mice.

The accuracy and precision of the LC-MS/MS assay and extraction efficiency of the liquid-liquid extractions were examined to validate the LC-MS/MS assay (Supplemental Method 3; Supplemental Tables 2 and 3). We observed that the addition of bovine serum albumin can help improve the in vitro stability of panobinostat in CNS tissues (Supplemental Fig. 4). Therefore, the brain and spinal cord specimens obtained from in vivo pharmacokinetic studies were homogenized in three volumes of 5% bovine serum albumin (w/v in distilled water). We have also observed that panobinostat was equally stable in serum as in plasma (Fig. 5A). However, panobinostat was unstable in blood, with a similar degradation rate as in plasma (Fig. 5B).

Fig. 5.

Fig. 5.

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In vitro stability of panobinostat in the (A) plasma and serum from wild-type FVB mice and (B) whole blood and plasma from wild-type FVB mice (n = 3, mean ± S.D.).

The plasma concentration-time profiles of panobinostat in Ces1cKO and wild-type C57BL6/J mice are depicted in Fig. 6A and compared with previous observations in wild-type FVB mice (Zhang et al., 2023). Despite differences around the in vitro plasma stability, the super-imposable plasma concentration-time profiles indicated essentially identical systemic exposures of panobinostat following an intravenous bolus dose of 10 mg/kg in adult Ces1cKO, wild-type C57BL6/J, and wild-type FVB mice. The distributional kinetics of panobinostat into CNS tissues was similar among Ces1cKO, wild-type C57BL6/J and wild-type FVB mice as well (Fig. 6, B and C). The brain-to-plasma and spinal cord-to-plasma concentration ratios reached a plateau after approximately 8 hours in all tested mouse models (Fig. 7). The unbound brain-to-plasma partition coefficients (Kp,uu,brain) and unbound spinal cord-to-plasma partition coefficients (Kp,uu,spinal cord) were similar between Ces1cKO and wild-types C57BL6/J mice (Table 2), indicating that the CNS distribution of panobinostat was similar between these two genotypes. The Kp,uu,brain and Kp,uu,spinal cord observed in wild-type FVB mice were 0.32 and 0.21, respectively (Zhang et al., 2023). The CNS distributional kinetics of panobinostat was similar among these three different mouse models. Despite vastly different in vitro plasma stability, the systemic pharmacokinetic behavior, as well as CNS distribution, of panobinostat was not different among the three mouse models.

TABLE 2.

Pharmacokinetic parameters of panobinostat in Ces1cKO and wild-type C57BL6/J mice following the administration of a single intravenous dose of 10 mg/kg.

Parameter Unit Ces1cKO Wild-type C57BL6/J
Plasma Brain Spinal Cord Plasma Brain Spinal Cord
t1/2 h 4.9 15.8 17.4 8.1 17.1 16.9
CL l/h/kg 6.6 — — 5.6 — —
Vss l/kg 14.1 — — 18.9 — —
AUC0-∞ ng*h/ml 1506 ± 75 1379 ± 27 704 ± 19 1785 ± 96 1568 ± 54 943 ± 49
Kp — 0.92 ± 0.05 0.47 ± 0.03 — 0.88 ± 0.06 0.53 ± 0.04
Kp,uu — 0.31 0.19 — 0.30 0.21
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*Standard error of Kp is calculated via propagation of error (You et al., 2013).

Fig. 6.

Fig. 6.

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Concentration-time profiles of panobinostat in (A) plasma, (B) brain, and (C) spinal cord in wild-type FVB, Ces1cKO, and wild-type C57BL6/J mice following a 10 mg/kg intravenous bolus dose (n = 4, mean ± S.D.). The data for panobinostat in wild-type FVB mice are from previous study (Zhang et al., 2023).

Fig. 7.

Fig. 7.

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(A) Brain-to-plasma and (B) spinal cord-to-plasma concentration ratios of panobinostat following a 10 mg/kg intravenous dose in wild-type FVB, Ces1cKO, and wild-type C57BL6/J mice (n = 4, mean ± S.D.). The data for panobinostat in wild-type FVB mice are from a previous study (Zhang et al., 2023).

Discussion

The stability of some synthetic hydroxamates, but not HDAC inhibitors like panobinostat, has been examined in rodent plasma (Hermant et al., 2017). The in vitro degradation of compounds in plasma and tissue specimens obtained from pharmacokinetic studies could cause imprecise measurement of concentration, which may lead to inaccurate interpretation of the pharmacokinetic parameters and metrics. Li et al. (2019) minimized the impact of esterase-mediated degradation of simvastatin during blood sampling in rat by adding an inhibitor and obtained higher systemic exposure and lower clearance of simvastatin compared with calculations from no–inhibitor-treated samples. Therefore, after finding such degradation of panobinostat in mouse matrices, precautions were taken to improve the accuracy of drug concentration measurements; yielding a more accurate determination of the in vivo kinetics. Panobinostat samples collected during our studies were snap-frozen on dry ice and kept at –80°C until LC-MS/MS analysis to minimize the influence of the in vitro degradation on the measurements. The use of a deuterium internal standard and preparation of the standards and QCs in parallel with unknowns helped improve the precision and accuracy of the quantitative LC-MS/MS bioanalysis.

In addition, the determination of some pharmacokinetic parameters can be challenging for unstable compounds. For instance, we found that the mass recovery of panobinostat after a 6-hour incubation with FVB plasma in the donor chamber at 37°C with agitation (600 rpm) in a rapid equilibrium dialysis (RED) device was approximately 50%. The degradation of panobinostat was preventing the dialysis system from reaching the distributional equilibrium between the donor (plasma) and receiver (PBS) chambers. To overcome this issue in RED, Eng et al. (2010) used an irreversible carboxylesterase inhibitor, bis-para-nitrophenylphosphate (BNPP), to stabilize a hydrolytically unstable amide derivative. Although our data indicated that a common esterase in rodent plasma, Ces1c, was not causing the degradation of panobinostat, the structure of panobinostat suggests that it may still be vulnerable to esterase activities. Therefore, we tested several inhibitors and found that multiple esterase inhibitors (Hermant et al., 2017), including BNPP, 5,5-dithio-bis(2-nitrobenzoic acid), phenylmethylsulfonyl fluoride, profenamine, ethylenediaminetetraacetic acid, and an inhibitor cocktail (cOmplete ULTRA tablets inhibiting serine proteases and cysteine proteases, Roche), did not improve the in vitro plasma stability of panobinostat (data not shown). Therefore, assuming that the binding reaction reached a rapid equilibrium on the addition of panobinostat, we performed ultrafiltration at room temperature instead of using RED at 37°C to determine unbound fractions of panobinostat in FVB mouse matrices and significantly shortened the incubation time for ultrafiltration (Zhang et al., 2023). Considering that panobinostat was unstable during the incubation in the Ces1cKO brain and spinal cord homogenates, ultrafiltration was used for the measurement of unbound fractions of panobinostat in matrices from Ces1cKO mice.

In our study, panobinostat showed various in vitro stabilities in plasma from different species. It was stable in human plasma while being unstable in FVB mouse plasma. This interspecies difference in stability can influence the translation from preclinical animal models to human pharmacokinetics and thus, confound drug development. The extrapolation of clearance and the ensuant systemic exposure of a therapeutic agent would be difficult if there were interspecies differences in plasma and tissue metabolism. A preclinical species, one in which the drug candidate shows significantly different clearance from humans, may not be a feasible model for the prediction of the pharmacokinetic behavior and metabolic profiles in humans. Dorywalska et al. (2015) found that the antibody-drug linker they used was cleaved much faster in mouse plasma than in primate plasma. They injected multiple antibody-drug conjugates that showed different mouse plasma linker stabilities into tumor-bearing mice and observed differential growth inhibition effects as a result of varying degrees of payload loss resulting in different antibody-drug conjugate exposure. Their results demonstrate that this type of interspecies difference should be clarified when conducting preclinical efficacy and safety studies in rodents. Ubink et al. (2018) found that the antibody-drug conjugate SYD985 was unstable in mouse plasma, yet stable in human and monkey plasma, because of the presence and absence of carboxylesterase 1c. They observed higher plasma exposure of SYD985 in Ces1cKO (−/−) mice than in Ces1c wild-type (+/+) mice following an intravenous bolus dose and improved efficacy of SYD985 in Ces1cKO tumor-bearing mice. In the case of SYD985, the instability observed in vitro had an impact on its in vivo behavior so that Ces1c knockout mice more reliably predicted the pharmacokinetics of SYD985 in humans than using the Ces1c wild-type mice. Therefore, we investigated the potential impact of the in vitro instability of panobinostat in the rodent on its in vivo systemic pharmacokinetics and CNS distributional kinetics. Super-imposable plasma concentration–time profiles of panobinostat in FVB, Ces1cKO, and wild-type C57BL6/J mice indicated that the systemic exposure of panobinostat, when samples are properly processed, was not influenced by vastly different in vitro plasma stabilities. Moreover, the CNS distribution of panobinostat was not affected based on our observations of panobinostat CNS tissue concentrations and unbound brain/plasma partition coefficients (Kp,uus) of panobinostat in CNS tissues.

Stability studies with panobinostat in FVB and C57BL6/J mouse matrices indicated that there was a strain difference in what was responsible for its in vitro plasma degradation (Supplemental Fig. 5). Even though in the three mouse models (i.e., FVB, C57BL6/J, and Ces1cKO) panobinostat showed significantly different in vitro plasma stability, the in vivo pharmacokinetic behavior of panobinostat, as determined by the superimposable plasma–concentration time profiles (Fig. 5), was not different among the three strains. This was true for both the systemic exposure and the CNS distribution. We propose several hypotheses to address this lack of in vitro–in vivo correlation. Given that panobinostat showed an average blood-to-plasma ratio of 2.17, our first hypothesis is that the partitioning of panobinostat into red blood cells is competing with its interaction with the soluble plasma enzyme(s). We measured the in vitro stability of panobinostat in the fresh whole blood and plasma from wild-type FVB mice, and results show that panobinostat had similar stability in the whole blood compared with plasma (Fig. 5B). This suggests that the partitioning into red blood cells is not improving the stability of panobinostat to a significant extent. In line with this same mechanism, the competitive inhibition of panobinostat binding to plasma enzyme(s) can come from endogenous substrates that may be renewed in vivo, but not under in vitro conditions. Hence, panobinostat might be more vulnerable to enzymatic activities in vitro in the absence of competing endogenous substrates. This mechanism has been seen for CYP3A4-mediated zonisamide metabolism that was observed to be inhibited in vitro by adding androstenedione, an endogenous androgen, into human liver microsomes (Nakamura et al., 2002). The relatively large volume of distribution (∼15 l/kg) indicates that panobinostat largely distributes outside the site of measurement (i.e., the plasma) into tissues. With that in mind, our third hypothesis is that panobinostat distribution into tissues that have diminished enzymatic activity versus plasma can also lead to in vivo protection from plasma esterase activity. These organs would act as reservoirs to protect panobinostat from robust enzymatic degradation in the plasma. To test this, we collected saline-perfused tissues from FVB wild-type and Ces1cKO mice (Supplemental Method 1) and performed in vitro stability studies of panobinostat in perfused-tissue homogenates. In FVB wild-type mouse matrices, panobinostat had varying degrees of stability in all the tissues tested, but was most unstable in the plasma (Supplemental Fig. 6A). This suggests that the distribution of panobinostat into other tissues apart from the plasma might be “protecting” it so that in FVB wild-type mice panobinostat showed longer in vivo plasma half-life than in vitro plasma half-life. When we compared the wild-type FVB with Ces1cKO mouse matrices, the interstrain difference in stability was only observed in plasma but in no other tissues (Supplemental Fig. 6). Clarifying the interstrain differences in plasma stability is of interest for specimen handling in future studies examining the pharmacokinetics of compounds susceptible to esterase-mediated metabolism. It has been reported that panobinostat is cleared through metabolism via both CYP- and non-CYP–mediated pathways (Clive et al., 2012). If the metabolism occurring in the plasma is a small portion of the overall metabolism of panobinostat, the removal of the metabolism in plasma, as would be seen in the Ces1cKO mouse, may not produce observable changes to the in vivo pharmacokinetic behavior of panobinostat.

Even though the use of recombinant Ces1c enzyme and carboxylesterase-specific inhibitor, BNPP, indicated that the in vitro degradation of panobinostat was Ces1c-independent, we have observed different stabilities of panobinostat between Ces1cKO and wild-type C57BL6/J mouse plasma (Supplemental Fig. 6). The knockout of the Ces1c gene might introduce other genetic changes to the C57BL6/J mice and hence improve the stability of panobinostat in Ces1cKO plasma. The situation when the expression of an enzyme has been altered when another gene was knocked out has been reported before. Tang et al. (2014) found that the expression of Ces1c was upregulated in the plasma of triple-knockout (TKO) (lacking both p-glycoprotein and breast cancer resistance protein, on an FVB background) mice compared with the wild-type FVB (Tang et al., 2014). Their results indicated that the binding interaction between everolimus and plasma Ces1c was preventing the compound from degradation by another unidentified plasma protein and led to higher systemic exposure and lower clearance in the knockout mice compared with wild-type mice. It has been noted that Ces1 family knockout mice have more body fat and altered lipid metabolism (Gan et al., 2023). Given the non-Ces1c change in systemic metabolism in Ces1cKO mice, it is clearly possible that Ces1cKO mice have lower expression of the degrading enzyme(s) or higher expression of the competing endogenous compound(s) so that panobinostat was stabilized in Ces1cKO plasma compared with wild-type C57BL6/J plasma. In addition, we tested the in vitro stability of panobinostat in plasma from wild-type FVB and TKO mice and results show that panobinostat had similar degradation rates in these two matrices (Supplemental Fig. 7). Considering a higher level of Ces1c in TKO mouse plasma as reported by Tang et al. (2014), this similar stability of panobinostat in wild-type and TKO mouse plasma further indicates that the plasma degradation of panobinostat was Ces1c-independent. Given the degradation of panobinostat in CNS tissues, an esterase like paraoxonase-2 is not likely involved because it is expressed in both human brain and plasma (Manco et al., 2021). A potential contributing factor to the enzymatic metabolism of panobinostat in the brain may be Ces1d and Ces5a; carboxylesterases that have been reported to be expressed in appreciable levels in the mouse brain (Jones et al., 2013).

The investigation of the in vitro plasma stability of panobinostat is applicable to similar compounds. Several other HDAC inhibitors that have shown in vitro potency and in vivo efficacy in mouse models against brain tumors are hydroxamate-based, such as vorinostat (Yin et al., 2007; Galanis et al., 2009) and quisinostat (Pak et al., 2019). The hydroxamic acid group in their structures is also likely to be susceptible to similar enzymatic reactions. The current study of panobinostat plasma stability points to the need for compounds susceptible to plasma esterase activity to be: 1) tested for that enzymatic activity, 2) protected in sampling procedures, 3) examined for strain differences, and 4) recognized that in vitro–in vivo metabolic liabilities may differ. Importantly, this study shows that the lack of in vitro plasma stability does not necessarily translate into the lack of in vivo stability, and, hence, lack of effect.

Acknowledgments

The authors would like to thank Yingchun Zhao, Analytical Biochemistry, Masonic Cancer Center, University of Minnesota, for his help with the development of the LC-MS/MS methods. The authors are grateful to Tetsuya Terasaki, University of Eastern Finland, for his contribution to the discussion.

Data Availability

The authors declare that all the data supporting the findings of this study are available within the paper and its Supplemental Material.

Abbreviations

AUC

area under the curve

BNPP

bis-para-nitrophenylphosphate

Ces

carboxylesterase

CNS

central nervous system

4-NPA

4-nitrophenyl acetate

GBM

glioblastoma multiforme

HDAC

histone deacetylase

Kp

tissue-to-plasma partition coefficient

Kp,uu

unbound tissue-to-plasma partition coefficient

LC-MS/MS

liquid chromatography-tandem mass spectrometry

QCs

quality controls

RED

rapid equilibrium dialysis

RT

room temperature

TKO

triple-knockout

Authorship Contributions

Participated in research design: Wenqiu Zhang, Oh, Sirianni, Elmquist.

Conducted experiments: Wenqiu Zhang, Wenjuan Zhang.

Performed data analysis: Wenqiu Zhang, Oh, Elmquist.

Wrote or contributed to the writing of the manuscript: Wenqiu Zhang, Oh, Aldrich, Sirianni, Elmquist.

Footnotes

This work was supported by National Institutes of Health National Institute of Child Health and Human Development, National Institute for Neurologic Disease and Stroke [Grants R01HD099543, R01NS116657, and R01NS111292], National Cancer Institute [Grant U19CA264362], and National Brain Tumor Society [Grant AWD 21-004061].

No author has an actual or perceived conflict of interest with the contents of this article.

Inline graphicThis article has supplemental material available at jpet.aspetjournals.org.

References

  1. Anderson J, Byrne T, Woelfel KJ, Meany JE, Spyridis GT, Pocker Y (1994) The hydrolysis of p-nitrophenyl acetate: a versatile reaction to study enzyme kinetics. J Chem Educ 71:715. [Google Scholar]
  2. Bailer AJ (1988) Testing for the equality of area under the curves when using destructive measurement techniques. J Pharmacokinet Biopharm 16:303–309. [DOI] [PubMed] [Google Scholar]
  3. Bezecny P (2014) Histone deacetylase inhibitors in glioblastoma: pre-clinical and clinical experience. Med Oncol 31:985. [DOI] [PubMed] [Google Scholar]
  4. Brennan CW, Verhaak RGW, McKenna A, Campos B, Noushmehr H, Salama SR, Zheng S, Chakravarty D, Sanborn JZ, Berman SH, et al. ; TCGA Research Network (2013) The somatic genomic landscape of glioblastoma. Cell 155:462–477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chun P (2016) Histone deacetylase inhibitors in medical therapeutics, in Medical Epigenetics (Tollefsbol TM ed) pp 633–655, Academic Press, Boston. [Google Scholar]
  6. Clive S, Woo MM, Nydam T, Kelly L, Squier M, Kagan M (2012) Characterizing the disposition, metabolism, and excretion of an orally active pan-deacetylase inhibitor, panobinostat, via trace radiolabeled 14C material in advanced cancer patients. Cancer Chemother Pharmacol 70:513–522. [DOI] [PubMed] [Google Scholar]
  7. de Ruijter AJM, van Gennip AH, Caron HN, Kemp S, van Kuilenburg ABP (2003) Histone deacetylases (HDACs): characterization of the classical HDAC family. Biochem J 370:737–749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Di L (2019) The impact of carboxylesterases in drug metabolism and pharmacokinetics. Curr Drug Metab 20:91–102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dorywalska M, Strop P, Melton-Witt JA, Hasa-Moreno A, Farias SE, Galindo Casas M, Delaria K, Lui V, Poulsen K, Loo C, et al. (2015) Effect of attachment site on stability of cleavable antibody drug conjugates. Bioconjug Chem 26:650–659. [DOI] [PubMed] [Google Scholar]
  10. Du L, Musson DG, Wang AQ (2006) Stability studies of vorinostat and its two metabolites in human plasma, serum and urine. J Pharm Biomed Anal 42:556–564. [DOI] [PubMed] [Google Scholar]
  11. Duysen EG, Koentgen F, Williams GR, Timperley CM, Schopfer LM, Cerasoli DM, Lockridge O (2011) Production of ES1 plasma carboxylesterase knockout mice for toxicity studies. Chem Res Toxicol 24:1891–1898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Eng H, Niosi M, McDonald TS, Wolford A, Chen Y, Simila STM, Bauman JN, Warmus J, Kalgutkar AS (2010) Utility of the carboxylesterase inhibitor bis-para-nitrophenylphosphate (BNPP) in the plasma unbound fraction determination for a hydrolytically unstable amide derivative and agonist of the TGR5 receptor. Xenobiotica 40:369–380. [DOI] [PubMed] [Google Scholar]
  13. Flipo M, Charton J, Hocine A, Dassonneville S, Deprez B, Deprez-Poulain R (2009) Hydroxamates: relationships between structure and plasma stability. J Med Chem 52:6790–6802. [DOI] [PubMed] [Google Scholar]
  14. Galanis E, Jaeckle KA, Maurer MJ, Reid JM, Ames MM, Hardwick JS, Reilly JF, Loboda A, Nebozhyn M, Fantin VR, et al. (2009) Phase II trial of vorinostat in recurrent glioblastoma multiforme: a North Central Cancer Treatment Group study. J Clin Oncol 27:2052–2058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gan C, Wang J, Martínez-Chávez A, Hillebrand M, de Vries N, Beukers J, Wagenaar E, Wang Y, Lebre MC, Rosing H, et al. (2023) Carboxylesterase 1 family knockout alters drug disposition and lipid metabolism. Acta Pharm Sin B 13:618–631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gao S, Hu M (2010) Bioavailability challenges associated with development of anti-cancer phenolics. Mini Rev Med Chem 10:550–567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Grasso CS, Tang Y, Truffaux N, Berlow NE, Liu L, Debily MA, Quist MJ, Davis LE, Huang EC, Woo PJ, et al. (2015) Functionally defined therapeutic targets in diffuse intrinsic pontine glioma. Nat Med 21:555–559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hermant P, Bosc D, Piveteau C, Gealageas R, Lam B, Ronco C, Roignant M, Tolojanahary H, Jean L, Renard P-Y, et al. (2017) Controlling plasma stability of hydroxamic acids: a MedChem toolbox. J Med Chem 60:9067–9089. [DOI] [PubMed] [Google Scholar]
  19. Jones RD, Taylor AM, Tong EY, Repa JJ (2013) Carboxylesterases are uniquely expressed among tissues and regulated by nuclear hormone receptors in the mouse. Drug Metab Dispos 41:40–49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kalvass JC, Maurer TS (2002) Influence of nonspecific brain and plasma binding on CNS exposure: implications for rational drug discovery. Biopharm Drug Dispos 23:327–338. [DOI] [PubMed] [Google Scholar]
  21. Kelly WK, O’Connor OA, Krug LM, Chiao JH, Heaney M, Curley T, MacGregore-Cortelli B, Tong W, Secrist JP, Schwartz L, et al. (2005) Phase I study of an oral histone deacetylase inhibitor, suberoylanilide hydroxamic acid, in patients with advanced cancer. J Clin Oncol 23:3923–3931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Konsoula R, Jung M (2008) In vitro plasma stability, permeability and solubility of mercaptoacetamide histone deacetylase inhibitors. Int J Pharm 361:19–25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lee P, Murphy B, Miller R, Menon V, Banik NL, Giglio P, Lindhorst SM, Varma AK, Vandergrift WA 3rd, Patel SJ, et al. (2015) Mechanisms and clinical significance of histone deacetylase inhibitors: epigenetic glioblastoma therapy. Anticancer Res 35:615–625. [PMC free article] [PubMed] [Google Scholar]
  24. Li B, Sedlacek M, Manoharan I, Boopathy R, Duysen EG, Masson P, Lockridge O (2005) Butyrylcholinesterase, paraoxonase, and albumin esterase, but not carboxylesterase, are present in human plasma. Biochem Pharmacol 70:1673–1684. [DOI] [PubMed] [Google Scholar]
  25. Li Z, Zhang J, Zhang Y, Zuo Z (2019) Role of esterase mediated hydrolysis of simvastatin in human and rat blood and its impact on pharmacokinetic profiles of simvastatin and its active metabolite in rat. J Pharm Biomed Anal 168:13–22. [DOI] [PubMed] [Google Scholar]
  26. Lindsey JC, Anderton JA, Lusher ME, Clifford SC (2005) Epigenetic events in medulloblastoma development. Neurosurg Focus 19:E10. [DOI] [PubMed] [Google Scholar]
  27. Manco G, Porzio E, Carusone TM(2021) Human paraoxonase-2 (PON2): protein functions and modulation. Antioxidants (Basel) 10:256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mann BS, Johnson JR, Cohen MH, Justice R, Pazdur R (2007) FDA approval summary: vorinostat for treatment of advanced primary cutaneous T-cell lymphoma. Oncologist 12:1247–1252. [DOI] [PubMed] [Google Scholar]
  29. Milde T, Oehme I, Korshunov A, Kopp-Schneider A, Remke M, Northcott P, Deubzer HE, Lodrini M, Taylor MD, von Deimling A, et al. (2010) HDAC5 and HDAC9 in medulloblastoma: novel markers for risk stratification and role in tumor cell growth. Clin Cancer Res 16:3240–3252. [DOI] [PubMed] [Google Scholar]
  30. Morales La Madrid A, Hashizume R, Kieran MW (2015) Future clinical trials in DIPG: bringing epigenetics to the clinic. Front Oncol 5:148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nakamura H, Nakasa H, Ishii I, Ariyoshi N, Igarashi T, Ohmori S, Kitada M (2002) Effects of endogenous steroids on CYP3A4-mediated drug metabolism by human liver microsomes. Drug Metab Dispos 30:534–540. [DOI] [PubMed] [Google Scholar]
  32. Oh J-H, Power EA, Zhang W, Daniels DJ, Elmquist WF (2022) Murine CNS and bone marrow distribution of the aurora A kinase inhibitor alisertib: pharmacokinetics and exposure at the sites of efficacy and toxicity. J Pharmacol Exp Ther 383:44–55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ostrom QT, Price M, Neff C, Cioffi G, Waite KA, Kruchko C, Barnholtz-Sloan JS (2022) CBTRUS statistical report: primary brain and other central nervous system tumors diagnosed in the United States in 2015-2019. Neuro Oncol 24 (Suppl 5):v1–v95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pak E, MacKenzie EL, Zhao X, Pazyra-Murphy MF, Park PMC, Wu L, Shaw DL, Addleson EC, Cayer SS, Lopez BG-C, et al. (2019) A large-scale drug screen identifies selective inhibitors of class I HDACs as a potential therapeutic option for SHH medulloblastoma. Neuro Oncol 21:1150–1163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pei Y, Liu KW, Wang J, Garancher A, Tao R, Esparza LA, Maier DL, Udaka YT, Murad N, Morrissy S, et al. (2016) HDAC and PI3K antagonists cooperate to inhibit growth of MYC-driven medulloblastoma. Cancer Cell 29:311–323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schwartzentruber J, Korshunov A, Liu X-Y, Jones DTW, Pfaff E, Jacob K, Sturm D, Fontebasso AM, Quang D-AK, Tönjes M, et al. (2012) Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma. Nature 482:226–231. [DOI] [PubMed] [Google Scholar]
  37. Siegel RL, Miller KD, Wagle NS, Jemal A (2023) Cancer statistics, 2023. CA Cancer J Clin 73:17–48. [DOI] [PubMed] [Google Scholar]
  38. Sturm D, Witt H, Hovestadt V, Khuong-Quang D-A, Jones DTW, Konermann C, Pfaff E, Tönjes M, Sill M, Bender S, et al. (2012) Hotspot mutations in H3F3A and IDH1 define distinct epigenetic and biological subgroups of glioblastoma. Cancer Cell 22:425–437. [DOI] [PubMed] [Google Scholar]
  39. Tang SC, Sparidans RW, Cheung KL, Fukami T, Durmus S, Wagenaar E, Yokoi T, van Vlijmen BJM, Beijnen JH, Schinkel AH (2014) P-glycoprotein, CYP3A, and plasma carboxylesterase determine brain and blood disposition of the mTOR Inhibitor everolimus (Afinitor) in mice. Clin Cancer Res 20:3133–3145. [DOI] [PubMed] [Google Scholar]
  40. Ubink R, Dirksen EHC, Rouwette M, Bos ES, Janssen I, Egging DF, Loosveld EM, van Achterberg TA, Berentsen K, van der Lee MMC, et al. (2018) Unraveling the interaction between carboxylesterase 1c and the antibody-drug conjugate SYD985: improved translational PK/PD by using Ces1c knockout mice. Mol Cancer Ther 17:2389–2398. [DOI] [PubMed] [Google Scholar]
  41. Wen Z, Huang Y, Behler N, Bambal R, Bhoopathy S, Owen A (2010) Determination of red blood cell partitioning and whole blood to plasma ratio using human, rat, and mouse blood: Methods, model-compounds, and specific differences. Poster W4305 at the AAPS 2010 Annual Meeting.
  42. Yao Z-G, Li W-H, Hua F, Cheng H-X, Zhao M-Q, Sun X-C, Qin Y-J, Li J-M (2017) LBH589 inhibits glioblastoma growth and angiogenesis through suppression of HIF-1α expression. J Neuropathol Exp Neurol 76:1000–1007. [DOI] [PubMed] [Google Scholar]
  43. Yin D, Ong JM, Hu J, Desmond JC, Kawamata N, Konda BM, Black KL, Koeffler HP (2007) Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor: effects on gene expression and growth of glioma cells in vitro and in vivo. Clin Cancer Res 13:1045–1052. [DOI] [PubMed] [Google Scholar]
  44. You L, Qian J, Wu X, Sun X, Su M, Di B, Du Y, Mao B (2013) Propagation of error in ocular pharmacokinetic parameters estimate of azithromycin in rabbits. J Pharm Sci 102:2371–2379. [DOI] [PubMed] [Google Scholar]
  45. Yuan J (1993) Estimation of variance for AUC in animal studies. J Pharm Sci 82:761–763. [DOI] [PubMed] [Google Scholar]
  46. Zhang W, Oh J-H, Zhang Wenjuan, Rathi S, Larson JD, Wechsler-Reya RJ, Sirianni RW, Elmquist WF (2023) CNS distribution of panobinostat in preclinical models to guide dosing for pediatric brain tumors. J Pharmacol Exp Ther DOI: 10.1124/jpet.123.001826 [published ahead of print]. [DOI] [PMC free article] [PubMed] [Google Scholar]

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