
==== Front
Eur J Drug Metab Pharmacokinet
Eur J Drug Metab Pharmacokinet
European Journal of Drug Metabolism and Pharmacokinetics
0378-7966
2107-0180
Springer International Publishing Cham

39105991
909
10.1007/s13318-024-00909-0
Original Research Article
The Effect of Concomitant Administration of Proton Pump Inhibitors on the Pharmacokinetics of CDK4/6 Inhibitors in Rats: Implications for the Evaluation of Hepatic and Transporter-Mediated Drug–Drug Interactions
Patil Prajakta Harish 1
Desai Mrunal 1
Birangal Sumit 1
Gurupur Gautham Shenoy 1
Rao Mahadev 2
Yadav Anandkumar 3
Kurawattimath Vishwanath 3
Chaudhari Avinash 3
Sharma Tarun 3
Pinjari Jakir 3
http://orcid.org/0000-0001-5956-7499
Channabasavaiah Jagadish Puralae jagadish.pc@manipal.edu

1
1 https://ror.org/02xzytt36 grid.411639.8 0000 0001 0571 5193 Department of Pharmaceutical Chemistry, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal, Karnataka 576104 India
2 https://ror.org/02xzytt36 grid.411639.8 0000 0001 0571 5193 Department of Pharmacy Practice, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal, Karnataka 576104 India
3 Drug Metabolism and Pharmacokinetics, SAI Life Sciences Ltd, Hyderabad, Telangana 500032 India
6 8 2024
6 8 2024
2024
49 5 631644
16 7 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License, which permits any non-commercial 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://creativecommons.org/licenses/by-nc/4.0/.
Background and Objectives

Numerous clinical concerns have been expressed regarding the potential worsening of cyclin-dependent kinase 4/6 inhibitor effects in breast cancer patients because of co-administration of proton pump inhibitors. Hence, this study evaluated the effects of proton pump inhibitors on the pharmacokinetics of palbociclib and ribociclib in terms of  cytochrome P450 (CYP) 3A4 and P-glycoprotein involvement.

Methods

The effects of omeprazole and rabeprazole on drug metabolism and efflux of these drugs were investigated using molecular docking, metabolic stability assay in rat liver microsomes, human recombinant CYP3A4 (rCYP3A4) enzymes, and Caco-2 cell monolayers, and in vivo pharmacokinetics with omeprazole and rabeprazole in (5 and 10 mg/kg) 30 min and 7 days before orally dosing palbociclib and ribociclib (10 mg/kg).

Results

Omeprazole and rabeprazole inhibited CYP3A4 enzyme activity in rCYP3A4 baculosomes with a 50–60% inhibition at 30 μM. Additionally, both omeprazole and rabeprazole (10 µm) significantly reduced the P-glycoprotein-mediated drug efflux of palbociclib and ribociclib. The 7-day pretreatment of omeprazole at a dose of 10 mg/kg resulted in 24% and 26% reductions in palbociclib's mean maximum plasma concentration) Cmax and area under the plasma concentration–time curve (AUC0-24 h), respectively. Palbociclib's pharmacokinetics were not significantly altered by the pretreatment with rabeprazole; however, ribociclib pharmacokinetics exhibited an 83.94% increase in AUC0-24 h.

Conclusion

The findings indicate that long-term treatment with therapeutic doses of both omeprazole and rabeprazole can alter the pharmacokinetics of palbociclib and ribociclib. The co-administration of rabeprazole may alter the pharmacokinetics of palbociclib and ribociclib via CYP enzyme and P-glycoprotein inhibition.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13318-024-00909-0.

http://dx.doi.org/10.13039/501100001411 Indian Council of Medical Research 2020-4462 Channabasavaiah Jagadish Puralae Manipal Academy of Higher Education, ManipalOpen access funding provided by Manipal Academy of Higher Education, Manipal

issue-copyright-statement© Springer Nature Switzerland AG 2024
==== Body
pmcKey Points

The net intestinal transport of palbociclib and ribociclib was significantly suppressed by the administration of verapamil, omeprazole, and rabeprazole.	
The absorption of palbociclib and ribociclib was significantly affected by the co-administration of omeprazole and rabeprazole in rats.	
The renal clearance of ribociclib was decreased by 2.14-fold in the presence of rabeprazole.	

Introduction

The pursuit of targeting the cyclin D-CDK4/6-Rb pathway has been a compelling endeavor. Palbociclib, ribociclib, and abemaciclib are the three cyclin-dependent kinases (CDK) 4/6 inhibitors that the USFDA has currently approved for the treatment of metastatic HR+/HER2– breast cancer [1, 2]. These drugs have been shown to improve progression-free survival (PFS), which can range from 24 to 28 months when combined with aromatase inhibitors [3, 4]. However, the notable adverse effects linked to this recently introduced category of therapies were significant and could pose challenges in the supportive care setting, particularly when used over an extended period. Gastrointestinal adverse effects, including nausea, vomiting, and diarrhea, are commonly experienced with CDK4/6 inhibitor therapy [5]. Proton pump inhibitors (PPIs), in particular acid-reducing agents, are frequently recommended medications for the goal of providing supportive care to cancer patients worldwide who exhibit symptoms of gastroesophageal reflux disease [6, 7]. The most often prescribed agent within this class of medications, PPIs, were found to be used by 33% of cancer patients in the United States, according to a retrospective review of healthcare databases with a population of more than 1.8 million people [8].

Palbociclib exhibits pH-dependent solubility, with the solubility rapidly decreasing to less than 0.5 mg/mL as the pH rises above 4.5. When multiple doses of rabeprazole were given alongside palbociclib, there was a significant decrease in the mean area under the plasma concentration–time curve (AUC0-24h) and maximum plasma concentration (Cmax). These reductions were observed to be, respectively, 62% and 80% under fasting conditions and 13% and 41% under fed conditions [9]. The impact of rabeprazole on palbociclib pharmacokinetics in fed conditions was considered insignificant from a clinical perspective. Based on clinical trial data and population pharmacokinetics, it was found that ribociclib absorption remained consistent regardless of stomach pH values resulting from food intake or concurrent use of PPIs [10]. However, contrary to the findings of the above study, no assessment of the impact of rabeprazole on clinical outcomes has been made in other reported clinical studies of palbociclib and ribociclib. The drug labels for palbociclib and ribociclib do not specify any restrictions regarding concurrent use of PPIs [11].

Moreover, a clinical study by Del Re et al. [12] revealed an interesting finding regarding the use of concomitant PPIs in chemotherapy with palbociclib. The study showed that patients who were taking PPIs experienced a significantly shorter PFS compared to patients who were only receiving palbociclib and endocrine therapy (14.0 months vs. 37.9 months, p < 0.0001). Patients were instructed to take a dose of lansoprazole (15 mg), esomeprazole (20 mg), omeprazole (10 mg), or pantoprazole (20 mg) [12]. However, from the study by Del Re et al. [12], it was evident that co-administration of PPIs did not compromise the efficacy of ribociclib in a real-life setting [13]. In a study by Çağlayan et al. [32], it was found that PPIs have a potential impact on PFS in PPI-treated patient populations which may be due to the effect of PPIs on the solubility of palbociclib and ribociclib. Nevertheless, in this study involving the simultaneous administration of PPIs, the hazard ratio of multivariate analysis for rabeprazole was lower. However, it should be noted that the study did not evaluate the impact of various PPIs on changes in the pharmacokinetics of both CDK4/6 [14]. According to a study by Eser et al. [15], it was observed that patients who were prescribed both palbociclib and ribociclib with PPIs exhibited a statistically significant reduction in PFS. The objective of this study was to examine the utilization of various doses of PPIs, namely lansoprazole 30 mg, esomeprazole 40 mg, omeprazole 40 mg, pantoprazole 40 mg, and rabeprazole 20 mg. However, the study did not assess the influence of different PPIs on the clinical outcomes of palbociclib and ribociclib [15].

Concerning the mechanism behind the negative effect of PPIs on PFS, the rise in gastric pH might have reduced the plasma concentrations of palbociclib and ribociclib, thus impacting the effectiveness of the treatment. Another appealing question to consider is whether the effects of PPIs on PFS observed in the reported studies may have been influenced by factors such as P-glycoprotein (P-gp) or CYP3A4/CYP2C19 inhibition. It is well-established that PPIs have a moderate inhibitory effect on P-gp, and it is worth noting that palbociclib/ribociclib are substrates of P-gp [16–18]. In addition, pantoprazole was found to impact the pharmacokinetics of tyrosine kinase inhibitors through its effects on breast cancer resistance protein and P-gp. It has been discovered that rabeprazole can impact the function of P-gp at concentrations that are relevant in the context of work. As a result, there is a decrease in palbociclib exposure, while ribociclib remains unaffected. Similarly, our previous research findings that have been published also demonstrate statistical variations in the incidence of CYP3A4- and P-gp-mediated interaction in the presence or absence of two PPIs, omeprazole and rabeprazole [19]. In addition, the effect of rabeprazole on palbociclib pharmacokinetics may have been underestimated due to the short duration of treatment. Short-term use of PPIs does not have a significant impact on the expression of CYP enzymes or transporters. Considering that palbociclib and ribociclib have been identified as substrates of CYP3A4 and P-gp enzymes, and PPIs such as omeprazole or rabeprazole have a moderate inhibitory effect on CYP3A4 or P-gp, it is important to investigate the potential pharmacokinetic drug–drug interaction (DDI) between these drugs and the extent to which it may occur. If the reported study had primarily focused on the inhibition of P-gp or CYP3A4 by PPIs, it would have resulted in a contrasting effect to the one caused by the rise in gastric pH. This would have led to higher exposure and potential signs of toxicity. Consequently, this investigation aimed to assess the influence of simultaneous PPI usage on the pharmacokinetics of palbociclib and ribociclib. The current investigation employed an in vitro–in vivo mechanistic risk assessment model to explore the potential involvement of CYP3A4 and P-gp in this interaction.

Materials and Methods

Chemicals and Reagents

Standards of palbociclib and ribociclib (purity > 99%) were generously gifted by MSN Laboratories, Hyderabad, India, while omeprazole and rabeprazole (purity > 99%) were acquired as gift samples from HETERO, India. Standards of terfenadine, verapamil (a reference CYP3A4 and P-gp inhibitor used as positive control), and glipizide (used as internal standard) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Human CYP3A4 baculosomes (P2377) produced from cDNA utilizing baculovirus-infected insect cells (1648816) were procured from Life Technologies (Thermo Fisher Scientific, MA, USA). Pooled rat liver microsomes (Cat No-452501) expressing CYP1A2, CYP2C9, CYP2D6, and CYP3A4 were procured from Corning, Discovery Labware (Woburn, MA, USA). The Caco-2 cell line (originating from a human colorectal carcinoma) was purchased from the European Collection of Authenticated Cell Cultures (ECACC Cat. no. 09042001). Phosphate-buffered saline (PBS), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (heat-inactivated, USA), and other reagents or solvents used were commercially available and of reagent grade. Millicell-24 Cell Culture Insert Plate, polycarbonate, 0.4 µm, was purchased from Nunc, Denmark. The Millicell–ERS instrument used for measuring the transepithelial electrical resistance (TEER) of the Caco-2 cell monolayers was obtained from Millipore (Bedford, MA, USA). Formulation excipients, Gelucire 44/14 (GATTEFOSSE, France), methyl cellulose (Colorcon Asia), Solutol HS-15, and Tween 80 (Sigma-Aldrich) were provided as gifts. Thermo Fisher Scientific supplied acetonitrile and formic acid suitable for liquid chromatography–mass spectrometry (LC/MS-MS). All other chemicals and solvents were of HPLC quality. Milli-Q was utilized to purify deionized water (Millipore, Billerica, MA).

Molecular Docking and Simulation

The Schrödinger’s Maestro (version 11.8; Schrödinger, New York, NY, USA) was utilized to perform molecular docking and dynamics simulation investigations on a Linux Ubuntu 18.04.1 LTS console (Intel® Core™ i5-10600K Processor (10th Generation), 16 GB RAM, and NVIDIA® GeForce® 4-GB graphics card) on an HP workstation. The X-ray crystal structure 5X0R (resolution: 2.67 Å) in a complex with an antagonist and the SRC-1 coactivator peptide representing PXR LBD were downloaded from the protein data bank (https://www.rcsb.org/). The protein was prepared and processed with the protein preparation wizard using the Schrödinger Maestro module. The PXR protein chains A and C were deleted, whereas chains B and D were kept and optimized for the protonation and side-chain orientation of the binding site residues at pH 7.4. The grid box for docking was generated at the docked ligand of the PXR protein near the SRC-1 coactivator peptide. The structures of palbociclib, ribociclib, PPIs (4 compounds), and known inhibitors of PXR were prepared in Maestro using the Lig-prep module with the OPLS-2003 force field. The prepared ligands were docked into the generated grid using the induced fit docking (IFD) protocol, and the best docking pose out of 20 poses was kept for each ligand. The estimated energy differences of the optimized ligand–protein complex were calculated using the prime MMGBSA model in Kcal/mol. The top-ranked poses of palbociclib, ribociclib, and PPIs were selected for molecular dynamic simulation to determine the protein–ligand stability. Using the Desmond module's 100-ns simulation, the ligand–protein interaction, inhibitor–substrate stability, and ionic bonding interaction between the ligand and protein were better characterized.

In Vitro Metabolic Stability of CDK4/6 Inhibitors in the Presence of PPIs

The impact of omeprazole and rabeprazole on the CYP3A4-mediated metabolism of palbociclib and ribociclib was investigated in an in vitro metabolic stability experiment using RLM. A total of 100 µL of incubated mixture contained 50 mM PBS (pH 7.4), 1 mM NADPH, 0.25 mg/mL RLM, palbociclib, and ribociclib at 1-µm concentrations with and without omeprazole, rabeprazole (1 and 10 µm), and positive control inhibitor (Verapamil, 10 µM). The palbociclib, ribociclib, and positive control were spiked in RLM and pre-incubated at 37 °C for 5 min and the corresponding inhibitors were spiked to the accurate concentrations in 35 µL of PBS before being added to each well of a 96-well reaction plate. The mixture was then incubated at 37 °C for a further 60 min with the addition of NADPH to start the reaction. For a metabolic stability assay using human recombinant CYP3A4 isoforms, a total of 100 µL of incubated mixture contained 50 mM PBS (pH 7.4), 1 mM NADPH, 50 pmol/mL human rCYP3A4, palbociclib, ribociclib, and terfenadine (substrate of CYP3A4) at 1-μM concentrations with and without omeprazole, rabeprazole (30 μM), and ketoconazole (10 µm) with 0.1% DMSO. Further incubation was the same as followed for the RLM assay. Samples were taken out and quenched with 100 µL of ice-cold acetonitrile containing an internal standard at 0, 10, 15, 30, and 60 min. The sample analysis was performed using the developed LC–MS/MS method. Details are described in supplementary file 1.

Bidirectional Transport Studies of CDK4/6 Inhibitors in the Presence of PPIs

Cell Culture

The Caco-2 cells (ECACC Cat. no. 09042001; passage of 33–35) were seeded on cell insert plates (Millicell, Cat#PSHT010R5) at a density of 80,000 cells per well and the cells were maintained for 18–21 days in culture medium (DMEM (×1) from Gibco with 10% FBS, 0.1 mg/mL penicillin/streptomycin from Sigma) to enable differentiation. The culture medium was changed every alternate day. The TEER values for the Caco-2 cells were measured using a Millicell-ERS instrument, and monolayers exceeding TEER values of ≥ 500 Ω cm2 were selected for the experiment.

Bidirectional Permeability Study of CDK4/6 Inhibitors

An intermediate stock solution of palbociclib and ribociclib was prepared in DMSO at a concentration of 1 mM and spiked in the assay buffer to get a target concentration of 10 μM. The organic content of the final drug preparation was 1% v/v. On the day of the experiment, the cultured monolayer was washed twice with assay buffer. For the apical to basal experiment, aliquots of 0.4 mL donor solution (assay buffer, pH 7.4, containing the test compound with and without inhibitors, 10 µm) and 0.8 mL of receiver solutions (assay buffer, pH 7.4, only) were added to the apical and basolateral compartments, respectively. For the basal to apical experiment, aliquots of 0.8 mL donor solution (assay buffer, pH 7.4, containing the test compound with and without inhibitors) and 0.4 mL of receiver solutions (assay buffer, pH 7.4) were added to the basolateral and apical compartments, respectively and incubated at 37 °C for 120 min. Control studies with propranolol (high permeable), atenolol (low permeable), digoxin (high rEfflux–P-gp substrate), and digoxin + verapamil (P-gp inhibitor) in both directions (AP > BL and BL > AP) were performed in separate wells on the same/experiment day. A bidirectional study was performed with standard compounds to ascertain the apical to basal and basal to apical permeability. The study samples (collected from apical and basolateral compartments after 120 min incubation) were analyzed in the LC–MS/MS instrument. Both the apical to basolateral (Papp, AP-BL) and basolateral to apical (Papp, BL-AP) apparent permeability coefficients (Papp) of each compound were calculated using:1 Papp=VAArea ratio×time×drugacceptordruginitial donor

where VA is the volume in the acceptor well, the area ratio equals the surface area ratio of the membrane, time is the total transport time in seconds, and Papp is the apparent permeability.

In Vivo Pharmacokinetics Study of CDK4/6 Inhibitors in Presence of PPIs

Animals

The animal experimental protocol was approved by the Institutional Animal Ethics Committee of Sai Life Sciences Limited, Hyderabad, India, for the Study Protocol SAIDMPK/PK-21-11-1079 (Reference No-2121/PO/Rc/S/21/CPCSEA). All the experiments were carried out as per the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines (Government of India, New Delhi). An in vivo pharmacokinetic study was carried out using healthy adult female Wistar rats that were housed in standard laboratory conditions, such as temperature of 22 ± 2 °C, relative humidity of 50 ± 20%, light cycle of 12 h/12 h, fed with pellet diet, and water ad libitum.

Pharmacokinetic Experimental Design

Pharmacokinetic studies were performed following oral administration of palbociclib and ribociclib (10 mg/kg) alone and in combination with omeprazole or rabeprazole concomitantly and pre-treatment for 7 days. Thirty Wistar female rats weighing 220–250 g (8–10 weeks old) were randomly divided into different study groups as represented in Table 1. The rats were fasted for 12 h before dosing, and food was withheld for another 3 h with water supplied ad libitum [20, 21]. Oral solutions of palbociclib (5% n-methyl pyrrolidine; NMP), 10% Gelucire 4/14, and 5% Solutol HS-15) and ribociclib (5%NMP and 95% normal saline), and oral suspensions of omeprazole and rabeprazole (0.5% Tween 80 and 0.5% w/v methylcellulose) were prepared as a pre-formulation for DDI study. A single dose of omeprazole or rabeprazole (Groups 2, 3, 7, and 8) was administered 0.5 h before palbociclib or ribociclib dosing. In the case of groups 4, 5, 9, and 10, omeprazole and rabeprazole were administered for 7 days at 10 mg/kg and 5 mg/kg doses (the animal equivalent doses were calculated from a human equivalent dose of marketed formulations), respectively, and, on the 8th day, palbociclib or ribociclib were administered after 0.5 h of omeprazole and rabeprazole administration at the same dose level to the respective groups. Blood samples were collected under light isoflurane anesthesia (Surgivet®) from the retro-orbital plexus from each group at pre-dose, and at 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h (approx. 100 µL of blood/ timepoint) after dosing of palbociclib and ribociclib. Immediately after blood collection, plasma was harvested by centrifugation at 4000 rpm, 10 min at 4 °C, and the samples were stored at − 80 ± 10 °C until bioanalysis. Table 1 Pharmacokinetic study design for in vivo interaction studies

Group No	Treatment (dosing arm)	
1	Palbociclib alone (10 mg/kg)	
2	Palbociclib (10 mg/kg) + omeprazole (10 mg/kg) concomitantly	
3	Palbociclib (10 mg/kg) + rabeprazole (5 mg/kg) concomitantly	
4	Palbociclib (10 mg/kg) + omeprazole (10 mg/kg) pre-treatment for 7 days	
5	Palbociclib (10 mg/kg) + rabeprazole (5 mg/kg) pre-treatment for 7 days	
6	Ribociclib alone (10 mg/kg)	
7	Ribociclib (10 mg/kg) + omeprazole (10 mg/kg) concomitantly	
8	Ribociclib (10 mg/kg) + rabeprazole (5 mg/kg) concomitantly	
9	Ribociclib (10 mg/kg) + omeprazole (10 mg/kg) pre-treatment for 7 days	
10	Ribociclib (10 mg/kg) + rabeprazole (5 mg/kg) pre-treatment for 7 days	

LC–MS/MS Bioanalysis

Waters ACQUITY UPLC H-Class paired with a triple quadrupole mass spectrometer, Q-trap 4000, with an electrospray ionization source (Applied Biosystems/MDS Sciex, Concord, Canada) comprised the UPLC-MS/MS system. A Waters BEH C18+, 50 × 2.1 mm, 1.7 µ column was used to analyze the palbociclib and ribociclib with the mobile phase of solvent A: 0.1% formic acid in acetonitrile, and solvent B: 0.1% formic acid in water. Linear gradient elution conditions were 98% B to 2% B in 0.3 min, 2% B from 0.5 min until the end of 1.30 min, and 98% B to re-equilibrium to 2.0 min with an injection volume of 2 μL, and 0.8 mL/min flow rate. Data acquisition was performed via a multiple reaction monitoring method for quantification for palbociclib (448.2/381.3), ribociclib (435.4/322.6), and glipizide (IS) (446.3/347.0) for the [M + H]-precursor ion to the selected product ion (m/z).The Applied Biosystems/MDS Sciex Analyst program (version 1.6.2) was used to conduct instrument monitoring, data acquisition, and data evaluation. Details of the mass parameters of all the analytes are listed in Supplementary File 1.

Data Analysis

Graph Pad Prism (version 10.0; GraphPad Software, San Diego, CA, USA) was used to plot the metabolic stability of palbociclib and ribociclib in the presence of omeprazole/rabeprazole, with the percent compound remaining on the Y axis and time on the X axis, and the slope (K) of the curve was determined. The intrinsic clearance values (in units of mL/min/mg) and in vitro t½ values were determined. The apparent permeability coefficients (Papp) of all tested compounds in both apical to basolateral (Papp, AP-BL) and basolateral to apical (Papp, BL-AP) directions were measured in triplicate. The non-compartmental-analysis tool of Phoenix WinNonlin® (version 8.0) was used to assess the pharmacokinetic parameters. Cmax and time for the peak plasma concentration (Tmax) were the observed values. The AUClast and AUC0-24 h were calculated from the linear trapezoidal rule. The terminal elimination rate constant, ke was determined by regression analysis of the linear terminal portion of the log plasma concentration–time curve. All mean values are presented with their standard deviation (mean ± SD). Data were analyzed for statistically significant differences using analysis of variance followed by the two-sided paired Student’s t test. Differences were considered to be significant at a level of p < 0.05.

Results

Molecular Docking and Simulations

The drug-binding domain and the ligand-binding domain (LBD) at the C-terminus, which contain the binding pocket, the activation function (AF-1) region, which regulates the receptor in a ligand-independent manner, and the activation function (AF-2) region are the major structural features of the PXR receptor [22]. As shown in Fig. 1, the largest hot spots are R (Right) and U (Up) which are primarily determined by the residues PHE288, TRP299, and TYR306 well occupied by palbociclib and ribociclib. Each drug resides inside a large hydrophobic pocket formed by three important side chains of PXR with a binding energy of − 92.03 and − 83.50 Kcal/mol, respectively. The hydrophobic interactions with PHE288, TRP299, and HID407 exhibit a high affinity for probe binding and permit substrate binding in multiple orientations. Additionally, palbociclib displays the polar interaction with SER247 that was absent in the PXR–ribociclib complex. The nearly spherical shape and predominantly hydrophobic nature of the binding site suggest that orientational specificity is limited. Among the four PPIs, omeprazole and rabeprazole were identified as strong binders of PXR with the lowest binding energy of − 86.00 and − 88.77 Kcal/mol, respectively. The common feature that was observed in the docking models of all the PPIs is that the sulfinyl moiety of all five compounds forms a hydrogen bond with either the SER247 or GLN285 side-chain of PXR. This hydrogen bond appears to act as a primary anchor for positioning the ligand in PXR LBD. As shown in Table 2, the binding of omeprazole, rabeprazole, lansoprazole, and pantoprazole involves residues from cluster R (GLN285, PHE288, TRP299, and TYR306), cluster U (GLU235, ILE236, LEU240, MET243, and SER247), and few amino acid residues from the cente cluster.Fig. 1 PXR protein showing PXR-LBD and AF-2 SRC-1 regions. Binding mode of 3D interaction scheme at the PXR LBD binding site near to AF-2 helix of a palbociclib, b ribociclib, c omeprazole, and d rabeprazole. Key amino acids found in the binding pocket are labeled (represented by thick gray tubes)

Table 2 Molecular interaction of proton pump inhibitors at the binding domain of PXR protein by induced fit docking

Sample	Title	Docking score	MMGBSA DG Bind	H bond	Pi–pi stacking	Pi–cation	
1	Palbociclib	− 12.76	− 92.03	SER274, PHE281	PHE429	PHE288, TRP299	
2	Ribociclib	− 10.69	− 83.50	 -	HID407	PHE288, TRP299	
3	Omeprazole	− 9.14	− 86.00	SER247, GLN285	PHE281, PHE429	TRP299	
4	Rabeprazole	− 10.19	− 88.77	SER247, GLN285	PHE288, TRP299	PHE288, TRP299	
5	Pantoprazole	− 10.03	− 73.70	GLN285	PHE281, PHE288, TRP299	 -	
6	Lansoprazole	− 13.23	− 77.28	GLN285	PHE288, TRP299, HID407	 -	
7	Ketoconazole	− 12.04	− 92.63	LYS277, GLN285	PHE288, TRP299, TYR306	 -	
8	Laniquidar	− 12.14	− 126.81	 -	PHE251, PHE288, TRP299	 -	
9	Tariquidar	− 11.80	− 137.37	SER247, GLN285, HID407	PHE288, TRP299	 -	
10	Zosuquidar	− 13.707	− 119.44	SER247, HID407	PHE288, TRP299, PHE429	PHE281	

However, PPIs like omeprazole and rabeprazole reside close to the hot spot cluster L (PHE281, HID407, LEU411, PHE420, PHE425, and PHE429). The most important property of the next hot spot region, L, is that it is in contact with four residues that are on helix 10, close to the activation function (AF-2) helix which is identified as the antagonist binding region. The binding model is anticipated to be similar to those of the PXR–ketoconazole interactions where the pyridine ring and benzimidazole of structural moiety were found to interact with amino acids such as PHE288, TRP299, and HID407 with a hydrophobic bond (Fig. 1). The polar interaction and hydrophobic interactions identified in IFD were retained in molecular dynamic simulation by the omeprazole–PXR and rabeprazole–PXR complexe, which were found to be stable throughout 100 ns of simulation with protein and ligand RMSD values of 2.1 Å and 2.4 Å for omeprazole and 2 Å and 4.8 Å for rabeprazole. Figure 2 shows the binding free energy (MMGBSA dG bind) of the compound PXR protein's palbociclib, ribociclib, omeprazole, and rabeprazole for every 10th frame up to the final frame (1000). Except for a few splurges, the dG bind remained between 70 and 95 Kcal/mol throughout the exercise. This protein–ligand interaction profile discovered by MD is consistent with the theories that were put forth in response to the findings of the induced-fit docking study [23–25].Fig. 2 MMGBSA dG bind versus frame plots of palbociclib, ribociclib, omeprazole, and rabeprazole PXR LBD binding interactions after dynamics simulations

In Vitro Metabolic Stability of CDK4/6 Inhibitors in Presence of PPIs

To better explain the experimental model being used to evaluate the CYP3A4 metabolism, the inhibitory effects of omeprazole and rabeprazole on CYP3A4-mediated phase I metabolism of palbociclib and ribociclib were examined in RLM (at 1 and 10 µm) and human rCYP3A4 (at 30 µM). Palbociclib and ribociclib exhibited metabolic half-lives of 90.44 and 49.78 min, respectively, with palbociclib having moderate and ribociclib with high in vitro metabolic clearance in RLM. The concentrations of inhibitors (omeprazole and rabeprazole) in in vitro assay were chosen based on the Cmax of PPIs for the highest strength. The metabolisms of both palbociclib and ribociclib in RLM incubates were found to be inhibited by omeprazole and rabeprazole at a concentration of 10 µM. The inhibitory effect of both inhibitors at a concentration of 1 µm was found to be ineffective in terms of inhibiting CYP3A4-mediated metabolism of palbociclib and ribociclib (Fig. 3). The enzymatic instability in RLM was investigated using verapamil as a positive control in combination with the test drugs. During the 60-min incubation phase, the positive control verapamil stayed at 18%. The impact of ribociclib stability on microsomal intrinsic clearance (CLint) was affected by rabeprazole and omeprazole, leading to reductions of 61.45% and 48.95%, respectively (Table 3). The metabolic stability data of palbociclib and ribociclib, both individually and in the presence of 30 µm omeprazole or rabeprazole in rCYP3A4, are presented in Table S5. The in vitro hepatic intrinsic clearance (CLint) values for palbociclib and ribociclib in human recombinant CYP3A4 incubates were 30.60 and 82.19 mL/min/mg of protein, respectively. The statistical analysis revealed that the observed differences were statistically significant (p < 0.05). Figure S3 displays representative plots illustrating the depletion of individual test compounds over time in recombinant CYP3A4 incubations with terfenadine as a positive control. The metabolic rate of palbociclib in human recombinant CYP3A4 incubations can reach a maximum of 54% with the metabolic rate influenced by the presence of 30 µm of rabeprazole, followed by omeprazole. Ribociclib was found to undergo metabolism up to 60% in recombinant CYP3A4 incubates. The impact of ribociclib stability on hepatic intrinsic clearance (CLint) was affected by rabeprazole and omeprazole, leading to reductions of 3-fold and 5-fold, respectively.Fig. 3 Residuals metabolic activity of a palbociclib and b ribociclib in the presence and absence of omeprazole and rabeprazole at different concentrations in rat liver microsomes at *p < 0.05 or **p < 0.01 compared with the control group

Table 3 Apparent intrinsic microsomal clearance of palbociclib and ribociclib (CL’int) in RLM

Compound	CL’int (mL/min/mg of protein)	
Palbociclib	30.89	
Palbociclib + rabeprazole	15.91	
Palbociclib + omeprazole	9.39	
Ribociclib	47.85	
Ribociclib + rabeprazole	19.06	
Ribociclib + omeprazole	25.26	
Statistical analysis was performed using a one-sample t test at *p < 0.05 compared with palbociclib and ribociclib alone and with treatment with rabeprazole and omeprazole at 10 µm at n = 2

Bidirectional Transport Studies of CDK4/6 Inhibitors in the Presence of PPIs

The bi-directional transport experimental Papp (AP-BL) and Papp (BL-AP) assays were performed in Caco-2 cell monolayers to investigate the ability of PPIs to modulate the transport/efflux of palbociclib and ribociclib. The basic transport characteristics of palbociclib or ribociclib (10 µm) in the Caco-2 cell system are shown in Fig. 4, with or without the presence of the P-gp inhibitor verapamil, and PPIs. The calculation of efflux ratio based on the observed Papp (AP-BL) and Papp (BL-AP) values for palbociclib and ribociclib individually validates that they are substrates of P-gp, in particular ribociclib, and exhibit high efflux ratios of 3.50 and 7.46, respectively. As illustrated in Fig. 4, statistically significant inhibition was observed at concentrations of 10 µm for omeprazole and rabeprazole (with inhibition to palbociclib BL-AP 26.75 and 38.96% transport, respectively). However, in the case of the results of the in vitro DDIs study between ribociclib and PPIs, omeprazole decreased BL-AP transport to the greatest extent (by 75.02%) compared to rabeprazole (by 61.45%). The net intestinal transport of palbociclib and ribociclib was significantly suppressed by the inhibition of BL-AP transport, as well as the administration of verapamil, omeprazole, and rabeprazole. This was observed through a decrease in efflux ratio compared to the control.Fig. 4 Apical to basolateral (AP-BL) and basolateral to apical (BL-AP) flux of a palbociclib and b ribociclib across Caco-2 cell mono-layer expressed as a percentage of control in the presence of 10 µm of the respective PPIs. *Data are represented as mean ± SD, n = 2. The concentrations of test compounds in the basolateral-to-apical (B–A) and apical-to-basolateral (A–B) direction across a Caco-2 cell monolayer at the end of 120 min in the presence and absence of inhibitors. A probability of less than 0.05 (p < 0.05) was statistically significant performed by paired t tests

In Vivo Pharmacokinetics Study of CDK4/6 Inhibitors in Presence of PPIs

Mean plasma concentration versus time profiles of palbociclib and ribociclib following an oral dose of 10 mg/kg alone (control) and after co-administration with PPIs (omeprazole and rabeprazole) as single-dose and 7-day pre-treatment are presented in Figs. 5 and 6. The quantitative values obtained for the pharmacokinetic parameters of palbociclib and ribociclib following a single dose of omeprazole and rabeprazole align with the findings of our previous pH-dependent micro-dissolution analysis in biorelevant media [26, 27]. The overall absorption of palbociclib and ribociclib was significantly affected by the co-administration of omeprazole and rabeprazole. The 30-min prior and pre-treatment for 7 days with the omeprazole to palbociclib group significantly lowers the Cmax of palbociclib, compared to only palbociclib. The AUC0-24h of palbociclib upon pre-treatment with omeprazole was lowered but, subsequently, in vivo CLobs was not changed in a significant manner. Changes in pharmacokinetic parameters of palbociclib with treatment with rabeprazole (Groups 3–5) in comparison with a control group (Group 1) were not statistically significant. Unexpectedly, rabeprazole concomitant and pre-treatment for 7 days did not significantly alter mean palbociclib plasma pharmacokinetics (p < 0.05), with a slight increase in Cmax (Table 4). Following oral administration, ribociclib showed slow absorption (Tmax 3.3 h) followed by distribution and disposition in rats. Relative to the degree of systemic exposure, AUC0-24h, and in vivo CLobs, statistically significant differences were not detected for the rabeprazole (5 mg/kg)-treated group. The administration of 5 mg/kg rabeprazole significantly increased the AUC0-24h of ribociclib by 1.72-fold. Consequently, the renal clearance of ribociclib was decreased by 2.14-fold in the presence of rabeprazole. Oral administration of omeprazole (10 mg/kg) did not affect the pharmacokinetics of ribociclib except for systemic exposure, which was lowered in the presence of omeprazole single- and 7-day pre-treatment (Table 5).Fig. 5 Plasma concentrations-time profiles (mean ± SD) of palbociclib (dose: 10 mg/kg) in female Wistar rats following a a single per oral (PO) dose with and without single and b pre-treatment (7 days) dose of omeprazole and rabeprazole

Fig. 6 Plasma concentration–time profiles (mean ± SD) of ribociclib (dose: 10 mg/kg) in female Wistar rats following a a single per oral (PO) dose with and without single and b pre-treatment (7 days) dose of omeprazole and rabeprazole

Table 4 Pharmacokinetic parameters estimated by non-compartmental analysis of the plasma concentration–time profiles of palbociclib (10 mg/kg) in female Wistar rat following oral administration with and without single oral and pre-treatment (7 days) with omeprazole and rabeprazole

Parameters	Palbociclib	Palbociclib + omeprazole (single dose)	Palbociclib + omeprazole (7 days pretreated)	Palbociclib + rabeprazole (single dose)	Palbociclib + rabeprazole (7 days pretreated)	
Cmax (ng/mL)	313.24 ± 19.16	298.62 ± 16.72*	242.85 ± 35.10*	355.98 ± 14.44	298.53 ± 72.08	
AUClast (h·ng/mL)	3563.56 ± 10.99	3015.47 ± 3.10	2628 ± 12.12*	3294.29 ± 11.52	3014.08 ± 17.01	
Tmax (h)	4 ± 0.00	6 ± 0.00	3.33 ± 1.15	4 ± 0.00	4 ± 0.00	
Half-life (h)	5.08 ± 0.51	3.1 ± 0.26	5.13 ± 0.39	3.52 ± 0.01	3.65 ± 0.46	
Cl_F_obs (mL/h/kg)	2700.84 ± 72.10	3285.4 ± 94.48*	3668.27 ± 13.01*	2997.32 ± 12.71	3319.85 ± 17.00	
Data are expressed as the mean values ± standard deviation (SD), n = 3

Cmax maximum plasma concentration, AUC area under the curve from the time of dosing to the time of the last measurable (positive) concentration, Tmax time for the peak plasma concentration, CL_F_obs total body clearance for extravascular administration

*p < 0.05 considered to be statistically significantly different from palbociclib control by statistical analysis performed by one-way ANOVA followed by Turkey post hoc test for multiple comparisons

Table 5 Pharmacokinetic parameters estimated by non-compartmental analysis of the plasma concentration-time profiles of ribociclib (10 mg/kg) in female Wistar rat following oral administration with and without single oral and pre-treatment (7 days) with omeprazole and rabeprazole

Parameters	Ribociclib	Ribociclib + omeprazole (single dose)	Ribociclib + omeprazole (7 days pretreated)	Ribociclib + rabeprazole (single dose)	Ribociclib + rabeprazole (7 days pretreated)	
Cmax (ng/mL)	162.06 ± 49.47	133.52 ± 2.86*	184.22 ± 55.43	143.38 ± 6.07	278.03 ± 24.15*	
AUClast (h·ng/mL)	1294.36 ± 36.42	1084.1 ± 13.03	1211.33 ± 13.74	992.53 ± 13.58	2380.95 ± 16.60*	
Tmax (h)	3.33 ± 1.15	4 ± 0.00	2.67 ± 1.15	4 ± 0.00	3.33 ± 1.15	
Half-life (h)	3.74 ± 0.44	4 ± 0.04	4.18 ± 1.07	3.28 ± 0.11	3.87 ± 0.75	
Cl_F_obs (mL/h/kg)	7955.26 ± 24.01	9036.46 ± 9.99*	8177.72 ± 12.01	9545.78 ± 63.21	4147.1 ± 26.61*	
Data are expressed as the mean values ± standard deviation (SD), n = 3

Cmax maximum plasma concentration, AUC area under the curve from the time of dosing to the time of the last measurable (positive) concentration, Tmax time for the peak plasma concentration, CL_F_obs total body clearance for extravascular administration

*p < 0.05 considered to be statistically significantly different from ribociclib control by statistical analysis performed by one-way ANOVA followed by Turkey post hoc test for multiple comparison

Discussion

Inadvertent prescribing practices, including those involving PPIs, can carelessly exacerbate the potential risk of pharmacokinetic DDIs, which may impede the effectiveness of a selected treatment approach. Activation of PXR has been linked to chemoresistance; therefore, performed in silico methods to identify the potential to activate PXR given the diverse nature of PPIs and CDK inhibitors in multiple orientations that can bind to the large and flexible PXR LBD [23]. Among the PPIs, the binding energy was calculated after induced-fit docking in which rabeprazole (BE = − 88.77), omeprazole (BE = − 86.00), pantoprazole (BE = − 73.70), lansoprazole (BE = − 77.28), palbociclib (BE = − 92.03), ribociclib (BE = − 83.50), and ketoconazole (BE = − 92.63) exhibited highest net binding scores confirming its substrate activity towards PXR (Table 2). Similar to the manner in which ketoconazole binds, rabeprazole occupies the AF-2 region and competes with the PXR coactivator for binding, disabling this crucial protein–protein interaction required for PXR activation [28].

In the in vitro experiment, the compounds omeprazole and rabeprazole at a concentration of 10 µm, along with ketoconazole at a concentration of 10 µm, demonstrated significant inhibition of 60–70% on the phase I hydroxylation of palbociclib and ribociclib in RLM. The metabolic stability of palbociclib and ribociclib was conducted in the presence of omeprazole and rabeprazole (1 and 10 µm) using RLM and demonstrated a dose-dependent inhibition of CYP3A4 enzymes. The co-incubation of omeprazole and rabeprazole in human rCYP3A4 resulted in a substantial increase in the in vitro half-life (t½) of palbociclib, with omeprazole increasing it by 3.08 times and rabeprazole increasing it by 10.01 times. Additionally, the in vitro drug clearance in human rCYP3A4 was reduced as a result of this co-incubation with inhibitors. In a manner like rabeprazole, the in vitro half-life of ribociclib was observed to increase by a factor of 3.5 in the presence of omeprazole. Additionally, the in vitro drug clearance was found to decrease by 77.98%. The findings appear to be consistent with our previous results of metabolic stability assay in HLM [19].

The findings from the in vivo pharmacokinetic drug–drug interaction study indicate that the administration of omeprazole, whether as a single dose or as a pre-treatment for a duration of 7 days, had a major impact on the pharmacokinetics of palbociclib in rats. The pre-treatment of omeprazole led to a decrease in the Cmax and AUC0-24h of palbociclib by 24% and 26%, respectively. Additionally, there was a 36% increase in total body clearance, and Tmax shifted from 4 to 6 h. However, it was evident from studies that the solubility of palbociclib will decrease as a result of the higher gastric pH levels caused by the administration of PPIs [27]. Therefore, it is anticipated that the dissolution rate of palbociclib will decrease as a result of the elevation in gastric pH induced by omeprazole, thereby affecting the absorption of palbociclib. This phenomenon may potentially lead to a decrease in the Cmax and a delay in the Tmax of palbociclib in rats. Furthermore, it has been observed that the administration of rabeprazole, whether as a single dose 30 min prior or pre-treatment for a duration of 7 days, did not result in any significant alterations in the pharmacokinetic characteristics of palbociclib, as depicted in Fig. 5a and b. The co-administration of rabeprazole results in a modest reduction in the Cmax of palbociclib. In both dosing regimens involving rabeprazole, it was observed that the total body clearance (in vivo CLobs) of palbociclib remained unchanged. However, it is important to consider the potential for rabeprazole to inhibit CYP3A4 or P-gp, as this may impact the overall clearance of palbociclib from the body.

The in vivo interaction profile of palbociclib when co-administered with omeprazole and rabeprazole exhibits a notable disparity compared to the in vitro findings of CYP3A4 or P-gp interactions. Based on a subsequent enzymology investigation, it has been determined that omeprazole and its metabolites demonstrate a moderate level of inhibition towards CYP3A4 and P-gp [29–31]. In the presence of omeprazole and rabeprazole, the efflux ratio of palbociclib was decreased by a factor of 1.8 and 2.8, respectively. In contrast to ribociclib, palbociclib exhibits moderate metabolism and demonstrates low substrate specificity towards P-gp, as evidenced by the in vitro findings. In comparison to omeprazole, rabeprazole exhibits a greater capacity to inhibit CYP3A4 and P-gp. This observation is also evident from in vivo studies conducted on rats, where palbociclib–rabeprazole demonstrated a notable interaction. The overall pharmacokinetic profile remains unaltered as a result of the inhibition of CYP3A4 and P-gp by rabeprazole, which counteracts the impact on solubility caused by PPIs at the absorption level. The changes induced on palbociclib pharmacokinetics by rabeprazole in the fed condition were considered not clinically relevant, and no restriction for the concomitant use of PPIs are reported in the palbociclib label. The results of the current investigation also align with the reported interaction of palbociclib with rabeprazole. Whereas in the reported clinical studies by Çağlayan, Del Re, and Eser, omeprazole (20mg) was administered concomitantly and the PPI-treated population of patients treated with palbociclib had a detrimental effect on PFS [12, 15, 32].

The 30-min prior dosing of omeprazole and rabeprazole had a significant impact on the pharmacokinetics of ribociclib. The administration of rabeprazole for 7 days before treatment with ribociclib led to a significant elevation in Cmax and AUC0-24h, with increases of 83.94% and 71.55%, respectively. The research indicates that the rabeprazole pre-treatment had a statistically significant impact (p < 0.05) on decreasing the complete body clearance (in vivo CLobs) of ribociclib. The observed effect can be ascribed to the potential enhancement of intestinal absorption resulting from the inhibition of P-gp and CYP3A4 enzymes. Based on the Caco-2 data, it can be inferred that ribociclib exhibits the characteristics of being a substrate for efflux transporters. Furthermore, it was observed that the absorption of ribociclib was significantly enhanced by inhibiting efflux transport with rabeprazole, leading to a notable 2.75-fold increase. The Cmax of ribociclib in rats that received pre-treatment with omeprazole in the 7-day treated group did not exhibit any alterations when compared to the single-dose regimens, as illustrated in Fig. 6b. On the other hand, omeprazole demonstrated a 2.23-fold decrease in P-gp-mediated efflux of ribociclib.

The potential interaction between ribociclib and drugs that elevate gastric pH has not been assessed in a clinical trial. Several studies, including simulations using physiologically based pharmacokinetic models, non-compartmental pharmacokinetic analysis, and population pharmacokinetic studies, have not indicated any changes in the absorption of ribociclib when co-administered with PPIs. The modifications in metabolism and permeability induced by omeprazole or rabeprazole primarily led to enhancements in the pharmacokinetic characteristics of ribociclib. Based on the results obtained, it is suggested that rabeprazole may potentially interact with ribociclib. This interaction could occur by inhibiting its hepatic metabolism mediated by CYP3A4 or its transport mediated by P-gp at the intestinal mucosal surface. Consequently, this interaction may result in reduced systemic clearance and/or increased absorption of ribociclib based on the dosing regimen of rabeprazole. The effects of omeprazole and rabeprazole on the intestinal absorption and metabolism of palbociclib and ribociclib have been investigated through both in vitro and in vivo studies. The findings indicate that individuals who use PPIs may face potential changes in the peak pharmacodynamic properties of palbociclib and ribociclib due to alterations in the concentrations of these components. Consequently, this could impact the therapeutic advantages associated with palbociclib and ribociclib.

Conclusion

The effect of concomitant administration of PPIs (omeprazole or rabeprazole) with palbociclib and ribociclib has been comprehensively evaluated by in vivo and in vitro studies in rats. The results obtained from the in vitro experiments are not completely consistent with the results of the in vivo experiments in both single and concomitant dosing, which showed that the most probable DDI between omeprazole and palbociclib/ribociclib could occur at the level of absorption due to the pH effect. The study well predicted the effect of concomitant use of rabeprazole on the pharmacokinetics of CDK4/6 inhibitors, which was due to metabolic and P-gp mediation where a small but significant increase in the AUC0-24h of oral ribociclib took place after long-term treatment with therapeutic doses of rabeprazole. The observed interaction with omeprazole or rabeprazole is likely to be of clinical relevance when the relatively low therapeutic indices of palbociclib and ribociclib are considered.

Supplementary Information

Below is the link to the electronic supplementary material.Supplementary file1 (PDF 159 KB)

Funding

Open access funding provided by Manipal Academy of Higher Education, Manipal.

Declarartions

Acknowledgements

The authors would like to thank Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal, as well as SAI Life Sciences Ltd., Hyderabad, India, for providing the necessary resources and facilities for this study.

Author Contributions

Conception, design, investigation, and data curation of the study: P.P., J.P.C.; Project administration and Funding acquisition: J.P.C.; Experimentation: P.P., M.D., S.B., A.Y., A.C.; Analysis/interpretation of data: P.P., M.D., G.S., M.R., V.KM., T.S., J.P., J.P.C.; Drafting the manuscript: P.P.; Critical reviewing of the manuscript: M.R., V.KM., J.P.C.; Approval of the final manuscript: P.P., G.S., M.R., V.KM., T.S., J.P., J.P.C.

Funding

The research was funded by the Indian Council of Medical Research (ICMR) (Ad-hoc Research Grant no. 2020-4462).

Data Availability

The datasets generated during and/or analysed during the current study will be available from the corresponding author upon reasonable request.

Code availability

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Conflict of interest

The author(s) declare that there are no conflicts of interest.

Ethics Approval

The experimental protocol (2121/PO/Rc/S/21/CPCSEA dated 10th March 2021 for project proposal no. FB-21-019) was approved by the Institutional Animal Ethical Committee of SAI Life Sciences, Hyderabad, India. The studies were carried out in conformity with the guidelines for the use and care of laboratory animals provided by CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals), Government of India, and confirms that the authors complied with the ARRIVE guidelines.
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