Journal of In Silico & In Vitro Pharmacology

Keywords

Prucalopride; LC-MS/MS; Bioanalytical method; Serotonin receptor agonist

Abbreviation

GABA: Gamma-Aminobutyric Acid; HT: Hydroxytryptamine; hERG: human Ether-Ago-Go Related Gene; USFDA: United States Food and Drug Administration; CPU: Clinical Pharmacological Unit; CDSCO: Central Drugs Standard Control Organization; ISTD: Internal Standard; DMSO: Dimethyl Sulfoxide; LLOQ: Lower Limit of Quantification; LQC: Low-Quality Control; MQC: Middle-Quality Control; HQC: High-Quality Control; EMA: European Medicine Agency; API: Active Pharmaceutical Ingredient; LOD: Limit of Detection; ESI: Electrospray Ionization; ME: Matrix Effect; LLE: Liquid-Liquid Extraction; MTBE: Methyl Tert-Butyl Ether

Introduction

Serotonin receptors, activated by the neurotransmitter serotonin, are G-protein coupled receptors and ligand-gated ion channels found in the peripheral and central nervous systems [1]. But these receptors modulate releases of many neurotransmitters and hormones like GABA, dopamine, acetylcholine, epinephrine or norepinephrine, glutamate, and hormone-like oxytocin, prolactin, vasopressin, corticotrophin, and substances P [2,3]. All 5-HT receptors have excitatory activity except 5-HT1 and 5-HT5, which have inhibitory activity [4]. The pharmacological functions are anxiety, appetite, GI motility, learning, memory, mood respiration [5,6]. Constipation is a symptom which causes divided into congenital, primary (intrinsic problems of colonic or anorectal function), and secondary (related to organic disease, systemic disease, or medications) [7-11]. Prucalopride is a dihydro benzofuran carboxamide derivative from the benzofuran family that is a serotonin 5-HT4 receptor agonist and has enterokinetic properties that are it has potent prokinetic activity [12]. This drug changes the colonic motility pattern via serotonin 5-HT4 receptor stimulation and stimulates colonic mass movements, providing the main propulsive force for defecation [13-15]. Prucalopride is well absorbed in the GI tract. It reaches maximum plasma concentration around 3 hrs after initial administration, an overdose may cause severe diarrhea, and bioavailability is over 90% and not influenced by the ingestion of food. Still, plasma protein binding is 30% [16-20]. After reaching maximum plasma concentration, prucalopride is mainly excreted by the urine (84%) and remaining excreted by feces [21].

Ultra-high-performance mass spectrometry was used to measure prucalopride in human plasma after a literature analysis revealed one or two techniques. There was no mention of a precise bioanalytical approach, mass spectrometric fragmentation of the medication, or a complete process for validating the results.

As part of the bioequivalence study conducted by Estradiol Valerate (EV), we developed and validated a fast, rapid, sensitive, and specific LC-MS/MS (API-4000) method for quantifying prucalopride in Indian human plasma. This method was successfully applied to several pharmacokinetic studies on healthy human volunteers.

Materials and Methods

Chemical and reagents

Acetonitrile purchased from Merck (MERCK India Ltd., Mumbai), isopropyl alcohol, formic acid was AR grade, and solvents were HPLC grade. Milli-Q water was used in the study (Elix, Milli-Q A10 Academic, Bedford, MA, USA) until a resistivity of 18.2 MΩ was achieved. The blank human plasma with EDTA-K3 anticoagulant was collected from the Clinical Pharmacological Unit (CPU) of TAAB Biostudy Services, Kolkata, and stored at 20°C until analysis.

Ethical clearance

The protocol of prucalopride study and related documents like volunteers informed consent form, case record form, subject information sheet submitted to the HURIP Independent Bioethics committee, Kolkata, India (Central Drugs Standard Control Organization (CDSCO) registration: ECR/103/Indt/WB/2013/RR- 19 which is valid up to 21^st Nov, 2024) and the ethical clearance obtained before initiation of the study.

Drug information and study design

It is a randomized, open-label, balanced, laboratory blind, two treatment, two-period, two sequences, single-dose, truncated, two way, crossover, bioequivalence study of Prucalopride 2 mg tablet as a test with Pruvict 2 (Prucalopride 2 mg) tablet of Torrent pharmaceutical Ltd as a reference product, in healthy adult human subjects under fasting condition. The volunteers have received either test or reference product with 240 ml drinking water based on the randomization code in each clinical period as specified in Table 1.

Test product Prucalopride 2 mg tablets were compared to a reference product under fasting conditions with the primary goal being to compare the rate and amount of absorption. Prucalopride 2 mg tablet will be administered orally to healthy adult human volunteers under fasting conditions with the secondary goal of monitoring safety and acceptability. Total 21 blood sampling time point were 0 hour (before drug administration) and 0.50 hours, 1.00 hours, 1.33 hours, 1.67 hours, 2.00 hours, 2.33 hours, 2.67 hours, 3.00 hours, 3.33 hours, 3.67 hours, 4.00 hours, 4.50 hours, 5.00 hours, 6.00 hours, 8.00 hours, 10.00 hours, 12.00 hours, 24.00 hours, 48.00 hours, 72.00 hours, which were collected from cubital vein and separated plasma by centrifugation and stored in -20˚C. 5 ml of blood taken at each time point. The demographic data of each volunteer is specified in Table 2.

Bio-analytical method development by LC-MS/ MS (API-4000)

Prucalopride is a dihydro benzofuran carboxamide derivative from the benzofurane family, with chemical name 4-Amino-5-chloro- 2methoxybenzoic acid, 3-dihydro-N-[1-(3-methoxypropyl)-4- piperidinyl]-7-benzofurancarboxamide group and represented in Figure 1 [22]. It acts as an agonist of the 5-HT4 receptor. In addition, it acts as a prokinetic drug that stimulates these receptors and colonic mass movements, providing the primary propulsive force for defecation [23].

Figure 1. Chemical structure of Prucalopride.

The exact mass of prucalopride is 367.16 (molecular weight 367.88) and the PKa value of prucalopride is 14.64 (conjugate base) and 8.98 (strongest basic) for benzofuran and piperidine heterocyclic structure. Benzofuran is a fragile basic compound depending on its PKa value -2.9 that is strongly conjugate basic and used as psychoactive substances which interacted with 5-HT receptors and activated 5-HT4 receptors. Piperidine is an azacycloalkane that is cyclohexane. A more substantial base replaces one carbon because the lone pair of electrons in piperidine is in an sp3 hybrid orbital and PKa value 11.28, which is used as an analgesic in prucalopride. So drug prucalopride itself a basic [24,25].

Due to lower protein binding, optimum conditions of plasma extraction and method development are required for a different type of pH value of the analyte. Therefore, propranolol is used as an Internal Standard (IS) for quantitation positive polarity to achieve adequate response for their simultaneous analysis. Moreover, the positive ionization mode is selective and highly sensitive for compounds with low electron affinity. Thus positive ionization mode was selected to fragment the analyte and IS to obtain intense and consistent product ions.

The protonated precursor ions (M+H)⁺ at 368.1 m/z (highest peak), 370.1 (2^nd peak), were observed in Q1 MS in which selected parent ion 368.1 for prucalopride and characteristic product ions or fragment ions found in Q3 MS were at m/z 196.0, 278.8, 156.1. However, the most stable and consistent fragment ion selected was m/z 196.0 (M-C₁₉H₂₀N_2O+H)⁺ for releasing nonanohydrazide and the product ion chemical name 2-[(4-chlorophenyl) methylideneamino]acetate, which monoisotopic mass 196.0 and displayed in Figure 2.

Figure 2. Parent (Q1) and product ion (Q3) scan of Prucalopride.

For the internal standard, the protonated precursor ions [M+H]⁺ at m/z 260.1 (highest peak) were observed in Q1 MS for propranolol, and characteristic product ions or fragment ions found in Q3 MS were m/z 116.0, 182.9. However, the most stable and consistent fragment ion selected was m/z 116.0[(M-C₁₀H₈O+H)⁺] forreleasingnaphthalen-2-ol and the product ion chemical name hexanamide monoisotopic mass 116.0. The chromatographic elution of the analytes on a Phenomenex Kinetex 5μ C18 100A 50 × 3 mm column was initiated as a rapid, susceptible, and rugged bioanalytical method covering the dynamic linear range. Mobile phase selection was necessary for the analysis of the drug depending on its pka value. Thus, the pH of the mobile phase, buffer concentration, and choice and proportion of diluents were necessary for chromatographic resolution with adequate response to achieve the desired sensitivity. Optimized instrumental (mass) parameters for prucalopride and IS are illustrated in Table 3.

Initially, acetonitrile/methanol with one mM ammonium acetate buffer (pH 6.5) gave a response for prucalopride. However, the response was not reproducible. The signal of the lower limit of quantification concentration was changed when buffer concentration increased from 1 mM to 10 mM. Further, the chromatography was better with a higher response using an acetonitrile buffer than a methanol-buffer combination. At pH above 5.0 that is basic, the resolution of prucalopride was affected, which further deteriorated with an increase in PH. Thus, to achieve more excellent reproducibility and better chromatography, low pH buffers were tried.

The lower drug concentration’s reproducibility and peak shape were better in 1.0% formic acid, but the inadequate signalnoise ratio. So as a final solvent was used, 1% formic Acid was in Milli Q water and mixed with 10 mM ammonium acetate as an aqueous solvent having apparent pH 1.20 and 1% formic acid in acetonitrile as an organic solvent at a flow rate of 0.5000 ml/ min then a superior signal to noise ratio (≥ 22), and baseline resolution achieved. So the selected mobile phase was 1% formic acid in Milli Q water and mixed with 10 Mm ammonium acetate as an aqueous solvent, and 1% formic acid in acetonitrile as an organic solvent drug was totally basic, so for better ionization and more negligible matrix effect, the acidic solvent was necessary. For analysis of prucalopride, the bioanalytical method was performed by gradient technique in which 90% aqueous solvent was used for 0.01 min to 0.50 min. From 0.50 min to 3.50 min of total run time, 10% aqueous solvent was used, and from 3.50 min to rest of the entire run time (7.00 min) for washing purposes used 90% aqueous solvent was. The gradient curve is illustrated in Figure 3. For better intensity and a narrower peak, the acidic aqueous solvent runs for a long time, and in this timing, the analyte and IS eluted. The chromatographic elution time for prucalopride and IS (propranolol) was found at 1.76 and 1.82 min respectively, in a total run time of 7.00 min. The representative MRM chromatograms were showed in Figures 4-9.

Figure 3. Gradient curve of method development.

Figure 4. Blank sample chromatogram of prucalopride.

Figure 5. Blank IS sample chromatogram of Prucalopride.

Figure 6. LLOQ sample chromatogram of Prucalopride.

Figure 7. LOC sample chromatogram of Prucalopride.

Figure 8. MQC sample chromatogram of Prucalopride.

Figure 9. HQC sample chromatogram of prucalopride.

Plasma extraction procedure of prucalopride

The liquid-liquid extraction technique performed plasma extraction. 400 μl volume of plasma sample transferred to a 15 ml plastic Carson tube. Then 100 μl of internal standard propranolol (25 μg) spiked for getting 5 μg/ml of standard internal concentration in the final prepared sample and added 500 μl n-Hexane then vortex 1 min added 1.0 ml of Diethyl Ether and vortex for 1 min then 3 ml TBME added to the sample tubes. After that, the sample was vortex mixed for 5 min and then centrifuged at 5000 r.p.m for 10 min. Then obtained supernatant organic layer 3.5 ml was transferred to a 15 ml plastic Carson tube and evaporated to dryness at 400°C under a stream of nitrogen. Then the dried extract was reconstituted in 200 μl of diluents (mobile phase) acetonitrile: water (50:50) and vortex for 2 min, then taken into autosampler vial, and 10 μl aliquot injected into chromatographic system.

Stock solution and calibration standards preparation

Preparation of prucalopride and propranolol stock solution (W/V): Weighted about 1.0 mg of prucalopride and propanolol separately and dissolved in 1.0 ml DMSO in a separate tube then mixed well by a vortex. So this each of the two stocked solution concentrations was 1mg/ml and stored in the refrigerator at 2˚C-8˚C. This stock solution was used to prepare an intermediate concentrated solution of prucalopride and internal standard, respectively.

Preparation of calibration concentrations in plasma: To prepare calibration concentrations in plasma of prucalopride, use a liquidliquid extraction procedure to extract prucalopride from human plasma. So from prepared stock solutions of prucalopride and IS (propranolol) diluted with ACN: water 50: 50 (v/v) and prepared intermediate concentrations. The intermediate each of the corresponding concentrated solutions 500 μl of Prucalopride transfer into 500 μl blank human plasma to achieve calibration concentrated points 0.25 ng/ml, 0.50 ng/ml, 1.00 ng/ml, 2.00 ng/ ml, 4.00 ng/ml, 8.00 ng/ml, 16.00 ng/ml including LLOQ 0.25 ng/ ml, LQC 0.75 ng/ml, MQC 6.0 ng/ml, HQC 12.0 ng/ml.

Method validation: The method validation performed as per guidelines for bioavailability and bioequivalence studies published by Central Drugs Standard Control Organization (CDSCO) and EMA [26].

Results and Discussion

Method validation

Specificity, selectivity, and linearity: The prepared plasma calibration concentrations of prucalopride were 0.25 ng/ml, 0.50 ng/ml, 1.00 ng/ml, 2.00 ng/ml, 4.00 ng/ml, 8.00 ng/ml and 16.00 ng/ml and LLOQ was 0.25 ng/ml, LQC was 0.75 ng/ml, MQC and HQC were 6 ng/ml and 12 ng/ml respectively. The concentrations range of the proposed assay was linear, and it was 0.25 ng/ml to 16 ng/ml in plasma. The calibration curve of linearity was showed in Figure 10, which codes no LIN-2. The Limit of Detection (LOD) of lower concentrations of prucalopride in plasma was 0.10 ng/ ml. The mean regression value of three days linearity was 0.9985, and the %of CV was 0.16. Pre-study linearity of detector response with statistics represented in Table 4 and Table 5.

Figure 10. Plasma calibration curve of prucalopride.

Precision and accuracy: In the case of between-run precision and accuracy, the percent of coefficient of variation range was 3.825 to 5.516 for precision, and the percent of absolute bias was 101.33% for LLOQ, 102.58% for LQC, 99.99% for MQC, and 96.14% for HQC for accuracy In the case of within-run precision and accuracy, the percent of coefficient of variation range was 2.645 to 3.683 for precision, and percent of absolute bias range was 94.92 to 100.00 for LLOQ to HQC range accuracy. The obtained result of precision and accuracy illustrated in Table 6.

Stability: The result of the stability study illustrated in Table 7.

• Short term stability: The stability samples of LQC, MQC and HQC analyzed after keeping the samples in the refrigerator at 2˚C-8˚C for 24 hours and compared these samples with fresh samples of the same concentration. The result of short term stability of prucalopride obtained from 93.53%- 101.70%

• Freeze-thaw stability: The stability samples of LQC, MQC and HQC analyzed after keeping the samples in the refrigerator at 2˚C-8˚C for three cycles and compared these samples with fresh samples of the same concentration. The result of freeze-thaw stability of prucalopride obtained from 96.52%-101.71%

• Auto-sampler stability: The stability samples of LQC, MQC and HQC analyzed after keeping the samples in an auto sample at 15˚C and compared with fresh samples of the same concentration. The result of autosampler stability of prucalopride obtained from 94.36%-98.82%

• Benchtop stability: The stability samples of LQC, MQC and HQC analyzed after keeping the samples on the sample preparation bench at laboratory environmental room temperature and compared these samples with fresh samples of the same concentration. The result of benchtop stability of prucalopride obtained from 95.68%-96.23%

• Long term stability: The stability samples of LQC, MQC and HQC analyzed after keeping the samples in the refrigerator at 2˚C-8˚C for seven days and compared these samples with fresh samples of the same concentration. The result of long term stability of prucalopride obtained from 95.52%- 105.23%

Matrix effect: The matrix effect of mass analysis of the analyte is the suppression of ionization of the analyte and IS in mass spectrometry. This parameter calculates by the peak area of the analyte and IS of the plasma extracted samples of LQC, MQC and HQC concentrations comparing with the unextracted samples of the same concentrations. The matrix factor of prucalopride was 0.92-0.95, and matrix effect ranges were 92.31%-94.82%, and for IS matrix factor was 0.95 and matrix effect was 94.79%-95.18%. The matrix effect of prucalopride illustrated in Table 8.

Recovery: Recovery of mass analysis of the analyte and IS calculated by the peak area of the analyte and IS of the plasma extracted samples of LQC, MQC, and HQC concentrations comparing with the unextracted samples of the same concentrations. The recovery of prucalopride was 100.43%- 111.83%, and for IS, it was 95.66%-106.02%. The recovery result of prucalopride is illustrated in Table 9 and Table 10.

Result of comparative pharmacokinetic study of prucalopride in human volunteers

From the analysis of unknown concentrations of prucalopride 2 mg in human plasma by LCMS/MS, itobserved that after administration of reference film-coated tablet of prucalopride 2 mg as a single dose in the fasting state reached maximum plasma concentration 4.98 ng/ml ± 0.95 ng/ml (Cmax) at the time of 2.71 hours ± 0.33 hours. (Tmax) and on the other hand, after administration of test preparation of film-coated prucalopride 2 mg in same volunteers, it observed that the maximum plasma concentration of prucalopride 2 mg reached 5.01 ng/ml ± 1.05 ng/ml (Cmax) at the time of 2.64 hours ± 0.28 hours (Tmax). The area under the curve of plasma concentration time of reference preparation was 30.34 ng.h/ml ± 8.07 ng.h/ml (AUC_0-t) and 31.96 ng.h/ml ± 8.09 ng.h/ml (AUC_0-∞), but in test preparation, it was 30.44 ng.h/ml ± 9.78 ng.h/ml (AUC_0-t) and 31.82 ng.h/ml ± 10.10 ng.h/ml (AUC_0-∞) shown in Figure 11. The elimination constant (Kel) value in reference preparation was 0.057 hour ± 0.014 hour. 1 hour and 0.058 hour ± 0.011 hour. (1 hour for test preparation). The elimination half life (T1/T2) of reference preparation was 12.84 hour ± 2.78 hour and 12.37 hour ± 2.28 hour for test preparation. From the above comparative pharmacokinetic study of prucalopride 2 mg, the relative bioavailability of the prucalopride test sample was 100.35% compared to the reference tablet prucalopride. The comparative pharmacokinetic parameters illustrated in Table 11.

Figure 11. Mean pharmacokinetics plasma concentration time profile of prucalopride.

Result of statistical analysis

Statistical ANOVA test using SAS 9.1.3 Version-Grizzles Model by PROC GLM was applied to calculate Ln Cmax, AUC_0-t and Ln AUC_0-t values.

The results of Cmax, AUC_0-t, AUC_0-∞, Tmax, Kel, T1/2 of ANOVA for untransformed and logtransformed data of Cmax show that the parameters like Period and Treatment are not statistically significant at 5% level in both untransformed and log-transformed data. Still, subject and sequence are statistically significant at 5% in both untransformed and log-transformed data i.e. p<0.05.

The 90% confidence interval for the ratio (Test/Reference) of geometric means, based on the log-transformed data for Cmax, was found to be 97.12%-103.98% relative to Test Preparation with Reference Preparation, for AUC_0-t found to be 97.95%- 101.04% relative to Test Preparation with Reference Preparation, for AUC_0-inf found to be 97.53%-101.02% close to Test Preparation with Reference Preparation.

The 95% confidence interval for the ratio (Test/Reference) of geometric means, based on the log-transformed data for Cmax, was found to be 97.63%-101.36% relative to Test Preparation with Reference Preparation, for AUC_0-t found to be 97.63%- 101.36% relative to test preparation with reference preparation, for AUC_0-inf found to be 97.16%-101.38% close to test preparation with reference preparation.

Application of paired t-test that the Cmax (untransformed data) for test preparation and reference preparation is not statistically significant at 5% level and p-value=0.874. Both the trials are similar effectson the body. The summarised statistical analysis report shown in Table 12.

Dependent variable: Cmax in Tables 13-16.

Dependent variable: AUC0-t in Tables 17-20.

Dependent variable : AUC0-∞ in Tables 21-24.

Dependent variable: Ln Cmax in Tables 25-28.

Dependent variable: Ln AUC0-t in Tables 29-32.

Dependent variable : Ln AUC0-∞ Tables 33-36.

Dependent variable : Tmax Tables 37-40.

Dependent variable : Kel in Tables 41-44.

Dependent variable : T1/2 in Tables 45-51.

Overall Conclusion

According to the criteria, it was sensitive, selective, had minimal ion suppression, was highly recoverable, and was repeatable. Aside from that, this technique successfully utilised to quantify prucalopride in unidentified volunteer plasma samples and an in-vivo pharmacokinetic and bioequivalence investigation. There were just a few articles on bioanalytical technique development, based on the literature review. LC-MS/MS API-4000 was utilised in this work to establish bioanalytical methods to quantify prucalopride in human plasma.

Conflict of Interest

None

Acknowledgement

The author is thankful to M/S, TAAB Biostudy Services, Kolkata-700032, India, for providing human plasma and delivering the necessary instrumental facilities. In addition, the authors acknowledge theDepartment of Pharmaceutical Technology, Jadavpur University, Kolkata, India, Calcutta School of Tropical Medicine, Kolkata 700073, India.National Institute of Technology, Tiruchirappalli, Government of India, India, for supporting this analysis.

Ethical Considerations

The protocol of prucalopride study and related documents like volunteers informed consent form, case record form, subject information sheet was submitted to the HURIP Independent Bioethics committee, Kolkata, India (Central Drugs Standard Control Organization (CDSCO) registration: ECR/103/Indt/ WB/2013/RR-19 which is valid up to 21-Nov, 2024) and the ethical clearance obtained before initiation of the study.

Safety Assessment

Clinical examinations were done at the time of screening, checkin and checked out from the clinical facility in each period. All vital signs like blood pressure, pulse ratemeasured, and wellbeing enquiry performed before check-in, before dosing, at 3 hours, 6 hours, 9 hours and 12 hours post-dose ( ± 60 minute) and check Fout of each period whenever felt necessary by the investigators or medical doctors Body temperature was monitored during the pre-dose and at the time of check out. In addition to the above well being, the assessment performed during each ambulatory sample collection. Clinical laboratory tests performed during screening and at the end of the study. The subjects did not report any adverse events and toxic effects at the time of the survey of prucalopride.

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