Micellar electrokinetic capillary chromatographic determination of pirfenidone and 5-carboxy-pirfenidone by direct injection of plasma from patients receiving treatment for idiopathic pulmonary fibrosis (IPF)

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Micellar electrokinetic capillary chromatographic determination of pirfenidone and 5-carboxy-pirfenidone by direct injection of plasma from patients receiving treatment for idiopathic pulmonary fibrosis (IPF) Salvatore Sotgia ConceptualizationMethodologyFormal analysisWriting - Original Draft , Alessandro G Fois ResourcesConceptualization , Elisabetta Sotgiu Formal analysisData Curation , Angelo Zinellu ConceptualizationFunding acquisition , Panagiotis Paliogiannis Data Curation , Arduino A Mangoni , Ciriaco Carru ConceptualizationFunding acquisition PII: DOI: Reference:

S0026-265X(19)32062-4 https://doi.org/10.1016/j.microc.2019.104536 MICROC 104536

To appear in:

Microchemical Journal

Received date: Revised date: Accepted date:

4 August 2019 11 December 2019 12 December 2019

Please cite this article as: Salvatore Sotgia ConceptualizationMethodologyFormal analysisWriting - Original Draft , Alessandro G Fois ResourcesConceptualization , Elisabetta Sotgiu Formal analysisData Curation , Angelo Zinellu ConceptualizationFunding acquisition , Panagiotis Paliogiannis Data Curation , Arduino A Mangoni , Ciriaco Carru ConceptualizationFunding acquisition , Micellar electrokinetic capillary chromatographic determination of pirfenidone and 5-carboxy-pirfenidone by direct injection of plasma from patients receiving treatment for idiopathic pulmonary fibrosis (IPF), Microchemical Journal (2019), doi: https://doi.org/10.1016/j.microc.2019.104536

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Highlights 

To our knowledge, there is no literature about using capillary electrophoresis in its micellar electrokinetic capillary chromatographic variant for the measurement of pirfenidone and 5carboxy-pirfenidone;



Detection and measurement of the two analytes by direct injection of plasma samples without pre-treatments;



Detection and measurement of the two analytes in human plasma from patients under treatment for idiopathic pulmonary fibrosis;

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Micellar electrokinetic capillary chromatographic determination of pirfenidone and 5carboxy-pirfenidone by direct injection of plasma from patients receiving treatment for idiopathic pulmonary fibrosis (IPF) Salvatore Sotgia1, Alessandro G Fois2, Elisabetta Sotgiu1, Angelo Zinellu1, Panagiotis Paliogiannis1, Arduino A Mangoni3, Ciriaco Carru1,4

1

Department of Biomedical Sciences, School of Medicine, University of Sassari, Sassari, Italy

2

Department of Clinical and Experimental Medicine, School of Medicine, University of Sassari, Sassari,

Italy 3

Department of Clinical Pharmacology, College of Medicine and Public Health, Flinders University and

Flinders Medical Centre, Adelaide, Australia 4

Quality Control Unit, University Hospital of Sassari (AOU-SS), Sassari, Italy

*Correspondence: Salvatore Sotgia, e-mail: [email protected] Department of Biomedical Sciences, University of Sassari Viale San Pietro 43/B -I-07100 SASSARI - ITALY 2

Phone: +39 079 229775 - Fax: +39 079 228120 Abstract A micellar electrokinetic capillary chromatographic method, a variant of capillary electrophoresis, was developed for the detection and measurement of pirfenidone and 5-carboxy-pirfenidone by direct injection of plasma from patients under treatment for idiopathic pulmonary fibrosis. To achieve the best separation a 35 mmol/L N-lauroylsarcosine sodium salt surfactant added with a 16 mmol/L sodium 1-heptanesulfonate solution was used as a running buffer. Analytes were eluted in a fairly short time, 7.35 and 9.40 min for pirfenidone and 5-carboxy-pirfenidone , respectively, at 15 °C and with an applied voltage of 30 kV in an 80 cm, 75 µm i.d. uncoated fused-silica capillary. In the range of 6.25 to 200 µmol/L, calibration curves exhibited acceptable linearity with the coefficient of determination higher than 0.999. The recovery of analytes spiked in samples of plasma was found to be 95–109% and 93–106% for pirfenidone and 5-carboxy-pirfenidone, respectively. Intra- and inter-day precisions were around 5%. LOD and LOQ for pirfenidone and 5carboxy-pirfenidone were, respectively, 1.0 and 4.0 µmol/L. The method, applied to measure analytes in six idiopathic pulmonary fibrosis male patients, provided mean concentrations of 68.91±26.31 and 36.21±26.39 µmol/L for pirfenidone and 5-carboxy-pirfenidone , respectively.

Keywords: capillary electrophoresis; micellar electrokinetic capillary chromatography; drug therapy monitoring; Introduction 3

Idiopathic pulmonary fibrosis (IPF) is a specific form of chronic fibrosing interstitial pneumonia of unknown etiology [1]. This rare parenchymal lung disease, occurring in 5 per 100,000 person-years [2], is characterized by thickening, stiffening, and scarring (fibrosis) of pulmonary tissue [3]. These alterations are considered the result of a chronic repair process secondary to epithelial cell damage in the small airways and alveoli [4]. Although the causes are unknown, older age, male sex, family history, and cigarette smoking have been recognized as risk factors [5]. In addition, occupational and environmental exposure to pollutants, specific medication or drugs, radiation therapy, and some connective tissue diseases have been identified as possible triggers of IPF development and/or worsening [6]. IPF progression is unpredictable and highly variable, with some patients exhibiting a rapid deterioration and others experiencing a relatively slow decline that can be, however, characterized by acute and often fatal respiratory failure [7]. Although recent data suggest a reduction in mortality risk [8], the five-year survival rate remains extremely low with a median survival after diagnosis of only 3–5 years [8,9]. Treatment with corticosteroids and immunosuppressants has proven to be ineffective in the stabilization of pulmonary function and improvement of survival [10]. Recently, however, a better management of the disease has been achieved with pirfenidone (PFD, 5-methyl-1-phenyl-2-(1H)-pyridone), a drug originally approved for the specific treatment of mild to moderate IPF [8]. PFD is a pleiotropic orally administered drug, with antifibrotic, anti-inflammatory and antioxidant effects [8], which has demonstrated efficacy in multiple fibrotic conditions, including those affecting the lung, kidney, and liver [11]. The primary clearance mechanism involves the oxidation of the methyl group on the pyridone ring to 5hydroxymethyl-pirfenidone, followed by the formation of the carboxylic acid, 5-carboxypirfenidone (5-PFD) [12]. Several high- and ultra-performance liquid chromatographic methods using ultraviolet and fluorescence detection or mass spectrometry have been developed for the assessment of PFD in plasma, serum, and urine of animals, healthy volunteers, and children [1214]. Different drawbacks, however, make these methods potentially not suitable for intensive practical use as they require expensive and/or time-consuming sample preparation (SPE, extraction 4

by halogenated solvents, evaporation under nitrogen), large sample volumes, gradient elution, or lengthy analysis. To our knowledge, no methods are available for analyzing PFD and 5-PFD in biological specimens or pharmaceutical preparation by capillary electrophoresis (CE). The latter has proven to be an attractive approach, orthogonal to HPLC, for the analysis of pharmaceutical compounds, providing fast, efficient, accurate, reproducible, and low-cost separations [15]. However, PFD behaves as a neutral compound in aqueous solution whereby separations by CE is hard to achieve [16]. In addition, although sample preparation is generally expensive, timeconsuming, poorly eco-friendly, a potential source of error and restricts the possibility to develop a high throughput method, it is a critical step for the robust measurement of the analyte. To overcome these analytical challenges, a variant of CE, the micellar electrokinetic capillary chromatography (MECK), was used in this study. MECK extended the potential of CE to the separation of uncharged analytes using surfactant micelles acting as a pseudo-stationary phase [17]. This allowed the differential partitioning of the analytes between micelles and the surrounding aqueous phase, resulting in the elution of the analytes at different velocities. MECK may be also a time- and costsaving alternative to usual sample preparation because it allows the direct injection of plasma. In principle, this analytical option is challenging in CE mainly due to the protein absorption to the chemically reactive uncoated fused-silica capillary wall. MECK overcomes this major drawback as the surfactant additives keep the proteins in solution and give them an electric charge that reduces the interaction with the inner fused-silica capillary wall [18]. Thus, this study describes the development and validation of a rapid MECK method for the measurement of PFD and of its primary metabolite 5-PFD in human plasma of patients under treatment for IPF.

Materials and methods Chemicals Sodium hydroxide (NaOH), DMSO, sodium dodecyl sulfate (SDS), sodium cholate hydrate (SCH), N-lauroylsarcosine sodium salt (LLS), and sodium 1-heptanesulfonate (NaHpSo) were purchased 5

from Sigma Aldrich (Milan, Italy). Pirfenidone (PFD) and 5-carboxy-pirfenidone (5-PFD) were obtained from DBA Italia (Milan, Italy), a licensed dealer of Toronto Research Chemicals (Toronto, Canada). High-purity water, obtained from a Millipore Milli-Q system, was used throughout the experiments (Merck Millipore, Italy). Solutions NaOH was prepared in Milli-Q water as 1 mmol/L solution. PFD and 5-PFD were prepared in DMSO as 5 mmol/L stock solutions. The latter were further diluted with Milli-Q water to obtain working solutions of calibration standards at different concentrations. Calibration standards for 5PFD and PFD were prepared in Milli-Q water at concentrations of 200, 100, 50, 25, 12.5, and 6.25 µmol/L. Background electrolyte (BGE) was prepared daily as an aqueous 35 mmol/L LLS buffer containing 16 mmol/L NaHpSo resulting in a solution at a pH of 8. Apparatus and CE conditions All experiments were performed on an Agilent 7100 capillary electrophoresis system equipped with a diode array detector. Agilent ChemStation software (Revision C.01.03) was used for instrument control and data analysis (Agilent Technologies, Milan, Italy). The uncoated fused-silica capillary (Agilent Technologies, Milan, Italy) was of 75 µm i.d. and had a total length of 80 cm. The capillary was daily flushed, at the beginning of each experimental session, with 1 mmol/L sodium hydroxide for 10 min, followed by Milli-Q water for 5 min and finally with BGE for 5 min. Between consecutive analyses, the inlet and outlet vials were emptied and filled with new BGE by the automatic replenishment system, then the capillary was rinsed with 1 mmol/L NaOH solution for 50 sec and with BGE for 50 sec. The operating conditions consisted of a temperature of 15 °C, applied voltage of 30 kV, injection pressure of 50 mbar, injection time of 10 s, and detection wavelengths of 235, 255, 304, and 312 nm with the latter used for the quantitative analysis of both analytes and to plot the presented electropherograms. Participants to study and samples collection

6

Six IPF male patients (age 71±3 years) treated for one year with a total daily PFD dose of 2,403 mg, administered three times a day, were randomly selected for this study. After informed written consent was given, fasting blood samples were obtained in the morning by 09:00 am, after an approximately ten-hour overnight fast. Blood was collected by venipuncture in 5.4 mg K3EDTA vacutainer tubes. Without delay, blood was centrifuged at 4°C and 3000 x g for 10 min to separate plasma, which was rapidly stored in 150-µL aliquots at -80 °C until use. The study was performed following the principles outlined in the Declaration of Helsinki and all procedures were approved by the local ethics committee (Local Health Authority of Sassari (Italy), prot. 2175/CE, 21/04/2015). Sample treatment A 50 µL-volume of plasma was three-fold diluted with Milli-Q water and mixed thoroughly by vigorous vortex-mixing. Tubes were then centrifuged at 17,000 x g for 5 min at room temperature. Finally, 100 µL of the pale yellow-colored supernatant was recovered for the analysis.

Results and discussion To develop a MECK method for the plasma measurement of PFD and 5-PFD, different anionic surfactants such as SDS, SCH, and LLS were trialed in this study. Without the addition of other additives, rough tests were performed for all surfactants around their corresponding critical micellar concentration (CMC), 9 mmol/L for SDS, and 15 mmol/L for SCH and LLS, as well as to the native pH of the solutions. In these conditions, PFD and 5-PFD eluted at a lower speed than the electroosmotic flow (EOF) only when SDS was used as surfactant, with the retention time of 5-PFD slightly greater than PFD (fig. 1a). On the contrary, either with SCH or LLS, only 5-PFD eluted after EOF while PFD eluted with or near the EOF (fig. 1b and c). With SDS, the resolution between the analytes was good as well as the symmetry degree of the peaks, 0.95 and 1.04 for PFD and 5PFD, respectively. With the other surfactants, peaks shape and resolution were just as good, notwithstanding the co-elution of PFD with EOF. As shown in figure 2 , regardless of the 7

surfactants used the quality of the electropherograms following the direct injection of plasma did not allow the easy identification of the analytes and their quantification. To this regard, as displayed in figure 3 (supporting materials), UV-spectrum of PFD shows maximum adsorption at 312 nm and a shoulder at 235 nm. Similarly, 5-PFD shows adsorption around 300 nm and a maximum and specific wavelength at 255 nm, potentially useful to distinguish it from PFD. In the attempt to improve the electropherograms after the direct injection of plasma, surfactant concentrations were double those of CMCs. No improvements were obtained by doubling CMC of SHC and SDS, as PFD either co-eluted with 5-PFD (fig. 4a) or continued to elute with EOF (fig. 4b). Despite an improvement in resolution, a further rise in the concentration of SHC and SDS resulted in the loss of symmetry of the peaks, increased retention times as well as of the current with frequent voltage drops. On the contrary, as displayed in figure 4c, the use of LLS at twice CMC resulted in an enhanced separation, as PFD eluted after EOF and an increase of the resolution between analytes also occurred. The electropherogram of the plasma sample was good as well, despite some degree of peaks asymmetry, 1.38 and 0.92 for PFD and 5-PFD, respectively (fig. 5a). To further improve the peaks shape and overall separation, some tentative combinations of LLS either with SHC and/or SDS were tried without, however, achieving any noticeable results. Improved results were found bringing the concentration of LLS to 35 mmol/L and adding NaHpSo, a well-known alkylsulfonic compound used as ion-pairing reagent in HPLC and CE analysis of peptides. NaHpSo has a CMC value of 302 mmol/L but, in this study, it was used below CMC at a concentration of 16 mmol/L. Higher concentrations of NaHpSo, in fact, did not bring any improvement and resulted in an excessive increase of the current followed by voltage drops. Conversely, below the concentration of 16 mmol/L, the effect of the addition of NaHpSo to LLS was negligible. As shown in figure 5b, at the optimized 16 mmol/L NaHpSo concentration, PFD and 5-PFD eluted at 7.35 and 9.40 min, respectively, with a sharp improvement in symmetry of peaks compared to LLS alone: 1.03 vs. 1.38 for PFD and 1.06 vs. 0.92 for 5-PFD, respectively. Thus, the optimum electrophoretic conditions that allowed a minimal analysis time, symmetric peaks shape, stable electrical current, repeatability 8

in retention times and peaks integration were obtained with an aqueous 35 mmol/L (CMC x 2.33) LLS buffer containing 16 mmol/L NaHpSo, resulting in a solution at a pH of 8. In such conditions, without the automatic replenishment system of BGE, analytes showed a %RSDs of the migration times, obtained after the injection of the same plasma sample 20 times in a row, of 1.52 and 1.32% for PFD and 5-PFD, respectively (Table 1). Analytes in the samples were identified by comparing their retention times to those of authentic standards and with a blank plasma sample (figure 6a). The linearity of the method, evaluated by the regression analysis of standards over the concentration range of the calibration curve, 6.25 to 200 µmol/L for both analytes, was satisfactory in the assessed range with a coefficient of determination of R^2 ≥ 0.999. No difference was observed by preparing the calibration curve in MilliQ water compared with drug-free plasma spiked with the analytes. Precision, assessed as intra-day precision and as inter-day precision, was quantified in 3 individually prepared replicates of three samples in duplicate within a single batch (intra-day) and quantifying three plasma samples in duplicate on 3 separate occasions over a period of 2 weeks (inter-day). Intra-day precision, expressed as %RSD of the measurements, was 3.23 and 3.95 for PFD and 5-PFD, respectively, while %RSDs of the inter-day precision were 4.53 and 5.22 for PFD and 5-PFD, respectively. The accuracy, expressed as percent recovery, was determined by recovery experiments by spiking three samples at three different concentrations, 25, 50, and 100 µmol/L, for both analytes. Recoveries were close to 100% at each concentration for all analytes: 98±2.4, 109±1.8, and 95±1.5% for PFD and 93±2.5, 106±2.7, 104±1.1% for 5-PFD, respectively. The limit of detection (LOD) and the limit of quantification (LOQ) were assessed on spiked drug-free plasma samples. Thus, LOD, i.e., the concentration of analyte that would result in a peak intensity equal to 3 times the noise level (S/N = 3), was 1.0 µmol/L for both analytes (figure 6b). LOQ, i.e., the concentration of analyte that would result in a peak intensity equal to 10 times the noise level (S/N 10), was 4.0 µmol/L for both analytes. As reported in table 2, the application of the established method to the plasma measurement of PFD and 5-PFD in six male IPF patients under treatment with PFD from a year provided mean concentrations of 68.91±26.31 µmol/L (12.76±4.87 mg/L) 9

and 36.21±26.39 µmol/L (7.79±5.68 mg/L) for PFD and 5-PFD, respectively. The inter-individual variability of around 38% might suggest a different metabolic rate or unsatisfactory compliance among patients. Conclusion The first CE method for the simultaneous determination of PFD and 5-PFD in human plasma was developed and applied to the measurement of the drug and of its metabolite in IPF patients. Given that a free solution CE system failed to achieve a complete separation, a MECK strategy was used for this purpose. This strategy also allowed the direct injection of plasma, avoiding the rate-limiting sample pre-treatment and thus meeting the requirements of a high throughput analysis. Compared with published HPLC assays, the developed MECK method is inexpensive and effortless, has an acceptable sensitivity and a short analysis time which makes it an attractive approach for the fast and reproducible quantitative determination of PFD and 5-PFD in large clinical trials and for the therapeutic drug monitoring in IPF patients.

Acknowledgements Arduino A. Mangoni contributed to this study during a Visiting Professorship at the University of Sassari.

Compliance with ethical standards This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

CRediT author statement Salvatore Sotgia: Conceptualization, Methodology, Formal analysis, Writing - Original Draft Alessandro G Fois: Resources, Conceptualization 10

Elisabetta Sotgiu: Formal analysis, Data Curation Angelo Zinellu: Conceptualization, Funding acquisition Panagiotis Paliogiannis: Data Curation Arduino A Mangoni: Conceptualization Ciriaco Carru: Conceptualization, Funding acquisition

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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[7] G. Sgalla, A. Biffi, L. Richeldi, Idiopathic pulmonary fibrosis: Diagnosis, epidemiology and natural history. Respirology 21 (2016) 427–437 [8] S.E. Torrisi, M. Pavone, A. Vancheri, C. Vancheri, When to start and when to stop antifibrotic therapies, Eur. Respir. Rev. 26 (2017) 170053 [9] E.F. Fabrellas, R.P. Sánchez, C.S. Abad, G.J. Samper, Prognosis and follow-up of idiopathic pulmonary fibrosis, Med. Sci. (Basel). 6 (2018) 51 [10] S.A. Papiris, E.D. Manali, L. Kolilekas, K. Kagouridis, C. Triantafillidou, I. Tsangaris, C. Roussos, Clinical review: Idiopathic pulmonary fibrosis acute exacerbations - unravelling Ariadne's thread, Crit. Care. 14 (2010) 246 [11] N.Y Huang, L. Ding, J. Wang, Q.Y. Zhang, X. Liu, H.D. Lin, W.Y. Hua, Pharmacokinetics, safety and tolerability of pirfenidone and its major metabolite after single and multiple oral doses in healthy Chinese subjects under fed conditions. Drug Res. 63 (2013) 388–395 [12] S.N. Giri, Q.J. Wang, Y. Xie, J. Lango, D. Morin, S.B. Margolin, A.R. Buckpitt, Pharmacokinetics and metabolism of a novel antifibrotic drug pirfenidone, in mice following intravenous administration Biopharm. Drug Dispos. 23 (2002) 203–211. [13] Y.G. Wen, X. Liu, X.L. He, D.W. Shang, X.J. Ni, M. Zhang, Z.Z Wang, J.Q. Hu, C. Qiu, Simultaneous determination of pirfenidone and its metabolite in human plasma by liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study. J. Anal. Toxicol. 38 (2014) 645-652 [14] M.L. Bruss, S.B. Margolin, S.N. Giri, Pharmacokinetics of orally administered pirfenidone in male and female beagles. J. Vet. Pharmacol. Ther. 27 (2004) 361–367 [15] W. Thormann, C.X. Zhang, A. Schmutz, Capillary electrophoresis for drug analysis in body fluids, Ther. Drug Monit. 18 (1996) 506-520 [16] Committee for Medicinal Products for Human Use (CHMP), Esbriet, Procedure No. EMEA/H/C/002154 (2010)

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[17] G. Hancu, B. Simon, A. Rusu, E. Mircia, Á. Gyéresi, Principles of Micellar Electrokinetic Capillary Chromatography Applied in Pharmaceutical Analysis, Adv. Pharm. Bull. 3 (2013) 1–8 [18] D.K. Lloyd, Capillary electrophoretic analyses of drugs in body fluids: sample pretreatment and methods for direct injection of biofluids, J. Chromatogr. A. 735 (1996) 29-42

Legends

Analyte

PFD

5-PFD

Linear range (µmol/L)

6.25 – 200

6.25 – 200

LOD (µmol/L)

LOQ (µmol/L)

1.0

1.0

4.0

4.0

Intra-day Precision (%RSD)

3.23

3.95

Inter-day Precision (%RSD)

4.53

5.22

Migration time (%RSD)

1.52

1.32

Recovery (%±SD) Level 1 25 µM Level 2 50 µM Level 3 100 µM Level 1 25 µM Level 2 50 µM Level 3 100 µM

98±2.4 109±1.8 95±1.5 93±2.5 106±2.7 104±1.1

Table 1. Summary of validation parameters of assay

Patient

PFD µmol/L±SD

5-PFD µmol/L±SD

#1

61.48±1.74

15.95±0.23

#2

25.82±1.82

19.01±1.61

#3

97.14±1.37

15.34±0.43

#4

58.51±1.65

48.01±2.72

#5

77.83±0.66

35.80±0.51

#6

92.68±2.62

83.16±2.35

13

Table 2. Concentrations of PFD and 5-PFD measured in six IPF patients treated for one year with 2,403 mg/day of PFD. Values are the mean of two measurements performed within a single batch

14

15

Figure 1. Electropherograms of a 100 µmol/L mixture of PFD and 5-PFD obtained using a) SDS, b) SCH, and c) LLS as surfactants at their CMC and native pHs

16

17

Figure 2. Electropherograms of a real plasma sample obtained using a) SDS, b) SCH, and c) LLS as surfactants at their CMC and native pHs

18

19

Figure 3. UV-spectrum of PFD (dot line) and 5-PFD (solid line). The wavelength at 312 nm was used for quantitative analysis

20

Figure 4. Electropherograms of a 100 µmol/L mixture of PFD and 5-PFD obtained using a) SDS, b) SCH, and c) LLS as surfactants at their doubled CMC and native pHs 21

22

Figure 5. Electropherograms of a real plasma sample obtained using a) LLS at its doubled CMC and b) LLS at a concentration of 35 µmol/L (CMC x 2.3) added with a 16 mmol/L NaHpSo solution

23

Figure 6. Electropherograms of a) a drug-free plasma sample and b) of a drug-free plasma sample spiked at LOD concentration of 1.0 µmol/L

24