Determination of ciclosporin A and its six main metabolites in isolated T-lymphocytes and whole blood using liquid chromatography–tandem mass spectrometry

doi:10.1016/j.jchromb.2007.01.039

Journal of Chromatography B

Volume 852, Issues 1–2, 1 June 2007, Pages 345-352

https://doi.org/10.1016/j.jchromb.2007.01.039 Get rights and content

Abstract

A specific and sensitive method for determination of intracellular ciclosporin A (CsA) and its six main metabolites AM1, AM9, AM1c, AM1c9, AM19 and AM4N, in isolated T-lymphocytes and whole blood is described. T-lymphocytes were separated from whole blood using Prepacyte^®. The analytes were extracted and purified from isolated lymphocytes and whole blood by protein precipitation followed by solid-phase extraction (SPE). The analytes and the internal standard, ciclosporin C (CsC), were separated on a reversed phase C8 column (30 mm × 2.1 mm, 3 μm) with a 10 mm × 2 mm, 5 μm Drop-In Guard Cartridge, using gradient elution chromatography and tandem ion trap mass spectrometry detection. The method has been validated in accordance with FDA guidelines and showed linear range from 0.25 to 500 ng/mL for CsA, 0.5 to 500 ng/mL for AM1, AM9 and AM19, 1 to 500 ng/mL for AM4N, AM1c and AM1c9 in intracellular matrix, and 2.5 to 3000 ng/mL for all analytes in whole blood. The applicability of the method is shown on patient samples.

Introduction

Ciclosporin A (CsA) is a cornerstone of the immunosuppressive therapy in solid organ transplant recipients and has resulted in increased survival rates of both patients and grafts since its introduction in the market in the early 80s [1], [2]. In 2003, 77% of all kidney transplant recipients in Norway were treated with CsA [3]. However, CsA treatment is associated with serious side-effects such as increased blood pressure, development of diabetes, dyslipidemia, intermittent renal hypoperfusion and chronic nephrotoxicity [4], [5], [6]. The pharmacokinetics of CsA exhibits extensive inter- and intrapatient variation, and CsA has a narrow therapeutic window with serious consequences when over- and underdosed. Due to this, routine therapeutic drug monitoring of CsA is necessary.

All routine monitoring is optimally performed in whole blood due to the distribution of CsA into erythrocytes, which is temperature and concentration dependent [7], [8]. The immunosuppressive site of action for CsA is inhibition of the intracellular phosphatase calcineurin in T-lymphocytes [9].

CsA is both a substrate and an inhibitor of the transmembrane transporter P-glycoprotein (P-gp), which is expressed in the T-lymphocytes and transports CsA out of the cells [10], [11]. The expression of P-gp will therefore influence the intracellular concentration of CsA and affect its pharmacodynamic effect. An up-regulation of P-gp following transplantation has been shown in lymphocytes from renal transplanted patients [12]. Measurement of intracellular CsA concentration in T-lymphocytes could therefore give more relevant information with regards to efficacy of CsA as compared to whole blood concentrations. Previous work has shown interesting results on the correlation between clinical effect and CsA lymphocyte binding [13], [14]. However, the lymphocyte separation technique referred to in those studies (Fiqoll centrifugation) is relatively unspecific and does not allow selective isolation of T-lymphocytes [15].

The cytochrome P-450 3A (CYP 3A) subfamily is responsible for metabolizing CsA to more than 30 metabolites [16]. Generally, the metabolites are considered to have both less immunosuppressive effect and less toxic effect than CsA [17], [18]. However, some data indicate that the secondary metabolites AM19 and AM1c9 are more nephrotoxic than CsA and other metabolites [19], [20]. Studies also indicate higher formation of the secondary metabolites AM19 and AM1c9 in patients with functional CYP3A5 enzymes [21].

The method described in this article is a validated, sensitive analytical tool capable of measuring CsA and six of its main metabolites AM19, AM1c9, AM1, AM9, AM1c and AM4N, intracellularly in T-lymphocytes as well as in whole blood from solid organ transplant recipients. This is the first report of a sensitive analysis of CsA and its main metabolites suitable for routine intralymphocyte measurements. The method will be used to monitor solid organ transplant recipients prospectively after transplantation to elucidate the understanding of the mechanism behind both the lack of immunosuppressive effect during acute rejection episodes and the cause of the nephrotoxic adverse event of CsA.

Section snippets

Chemicals and reagents

CsA, Ciclosporin C (CsC) and the metabolites AM1, AM9 and AM1c were provided by Novartis (Basel, Switzerland). The metabolites AM4N, AM19 and AM1c9 were kindly provided by Dr. U. Christians (University of Colorado Health Sciences Centre, Denver, USA). Verapamil hydrochloride was purchased from Sigma Chemical, St. Louis, USA. The standard solutions of the metabolites were made by dissolving 5 mg of pure metabolites in 5 mL of methanol. The lymphocyte isolation medium Prepacyte^® and erythrocyte

MS–MS conditions

To optimize detection, the chromatogram was divided in three segments and each segment consisted of two or more scan events (Fig. 2C and Table 1). All analytes were detected with unique scan event, except AM1 and AM9 as these two analytes produced identical daughter ions after fragmentation. Optimal sensitivity for the analytes was obtained by multiple reaction monitoring. The sum of signals of the daughter ions listed in Table 1 was used for quantification.

Optimal SPE conditions

Fig. 3A shows a plot of analyte

Conclusion

A unique specific method has been developed for the measurement of CsA and its six main metabolites intracellularly in T-lymphocytes as well as in whole blood. The method is highly specific towards CsA and its metabolites, and shows no interference with other drugs. Determinations of lymphocyte-associated concentration in previous work have shown interesting results when correlating the lymphocyte concentration to clinical effect. However, this previously used method utilizes a less specific

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Early reports with intracellular cyclosporine quantification highlighted that using laboratory techniques able to improve the efficacy of lymphocyte isolation can increase the prediction ratio of intracellular measurements of immunosuppressive drugs for acute rejection episodes. Falck et al. used an antibody-dependent homocytophilic cell agglutination technique to obtain an enriched suspension in peripheral blood T lymphocyte [14]. Cyclosporine levels in these T lymphocyte enriched suspensions were significantly lower among kidney transplant recipients with allograft rejection [12].
Monitoring tacrolimus (Tac) exposure in cell matrices enriched with lymphocytes can improve Tac therapeutic drug monitoring (TDM) in solid organ transplant recipients. An UPLC–MS/MS based assay for Tac quantification in peripheral blood T CD4+ and B CD19+ lymphocytes was developed. Peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation and highly purified (purity >90%) T CD4+ and B CD19+ cell suspensions were acquired by magnetic negative selection from whole blood of 6 healthy volunteers. The purity of lymphocyte suspensions was checked by flow cytometry. Tac extraction was performed in a liquid–liquid zinc sulfate, methanol and acetonitrile based medium. Ascomycin was used as internal standard. The equipment used was a Waters^® Acquity™ UPLC system (Waters Corporation, Milford, MA, USA). The chromatographic run was performed on a Waters^® MassTrak TDM C18 (2.1 × 10 mm) column (Waters Corporation, Milford, MA, USA). at a flow rate of 0.4 mL/min. The instrument was set in electrospray positive ionization mode. The method was validated according to Clinical Laboratory Standard Institute (CLSI) guidelines and showed a high sensitivity and specificity over a range of 0–5.2 ng/mL in PBMC, 0–5.0 ng/mL in T CD4+ Lymphocytes and 0–5.3 ng/mL in B CD19+ lymphocytes. Precision was appropriate with CV of intra-assay quantifications ranging from 4.9 to 7.4%, and of inter-assay quantifications from 7.2 to 13.9%. Limit of detection and quantification were 0.100 and 0.115 ng/mL in PBMC, 0.058 and 0.109 ng/mL in T CD4+ and 0.017 and 0.150 ng/mL in B CD19+ cells. Matrix effect was not significant among all the studied matrices. Samples showed stability for Tac quantification over a period of 90 days either at room temperature or at −30 °C storage conditions. The method was applied to clinical samples of 20 kidney transplant recipients. Concentrations ranged from 2.200 to 11.900 ng/mL in whole blood, from 0.005 to 0.570 ng/10⁶ cells in PBMC, from 0.081 to 1.432 ng/10⁶ cells in T CD4+, and from 0.197 to 1.564 ng/10⁶ cells in B CD19+ cell matrices. The method has potential applicability for Tac TDM in solid organ transplant recipients.
Liquid chromatography tandem mass spectrometry for therapeutic drug monitoring of immunosuppressive drugs: Achievements, lessons and open issues
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Currently therapeutic drug monitoring (TDM) of immunosuppressive drugs is one of the best established fields of application of TDM as a tool dedicated to therapy guidance and patient care improvement. LC-MS/MS techniques were introduced to this field about 20 years ago, and enabled a huge progress towards providing more adequate overall quality and turnaround time for TDM services. However, numerous LC-MS/MS difficulties have also been recognized and approaches to deal with them have been developed. Moreover, some further issues must be resolved to realize the full potential of this technique. This article aims to provide an update of the LC-MS/MS literature with a focus on new developments and applications that have been reported during the last five years. In addition, it summarizes important lessons and defines current needs in the context of TDM services, which must be addressed in the near future.
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Unfortunately, the results are not yet available. Falck validated an LC⿿MS/MS assay to determine CsA and six metabolites in T-lymphocytes and whole blood with LOQ of 0.25 ng/106 cells and 2.5 ng/mL, respectively [53]. This method has been used for three prospective studies [54⿿56].
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Hydroxylated, Hydroxymethylated, Dihydroxylated, and Trihydroxylated Cyclosporine Metabolites Can Be Nephrotoxic in Kidney Transplant Recipients
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Cyclosporine (CsA) is an immunosuppressive agent whose use is associated with adverse effects, including nephrotoxicity. There are reports indicating that some CsA metabolites may contribute to these effects. This study was aimed at evaluation of CsA metabolites and correlating them with kidney function.
In 62 kidney transplant recipients (41.9% women; overall mean age, 48.44 ± 11.75 years), concentrations of CsA and 4 groups of metabolites were assessed: hydroxylated (HCsA), hydroxymethylated (HMCsA), dihydroxylated (DHCsA), and trihydroxylated (THCsA). The results were normalized with the use of the metabolite-to-parent drug ratio, and results were linked with estimated glomerular filtration rate (eGFR) at 3 months before (−3M), point zero (0M), and after 3 (+3M) and 12 (+12M) months.
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Although therapeutic drug monitoring has been standard of care in immunosuppressive therapy for quite a while, acute rejections remain a significant problem, particularly immediately following transplantation and despite trough whole blood concentrations within the therapeutic range. The widely used calcineurin inhibitors exert their effect inside the lymphocyte, where they bind to specific proteins, thus interfering with T-cell activation. This makes the intracellular compartment of peripheral blood mononuclear cells an attractive target for concentration monitoring of calcineurin inhibitors. A number of papers have shown that there is no or only a weak correlation between intracellular concentrations of calcineurin inhibitors and their whole blood trough concentrations, which are currently regularly monitored. However, it has also been reported that such measurements that have become feasible through advances in analytics, such as the widespread introduction of liquid chromatography combined with tandem mass spectrometry, have the potential to better predict clinical outcomes than is possible with conventional whole blood monitoring. However, these results are still early, and more work needs to be done in this field.
Semi-synthesis of cyclosporins
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Conversion of the newly formed thioamides to thioimidates can be followed by hydrolysis to bring about ring cleavage between residues 4 and 5, and residues 7 and 8 [57,206]. In an effort to mimic the human metabolic pathway [207] of CsA using microorganisms, Traber and coworkers found that the actinomycte bacteria Sebekia benihana selectively converted CsA to [4′-HOMeLeu]4CsA (35% yield), with smaller amounts of its N-demethylated analog [4′-HOLeu]4CsA (4.5%) as well as the bishydroxylated [4′-HOMeLeu]4[4′-HOMeLeu]6CsA (8.6%) also isolated [208]. The finding that this hydroxylated product retains cyclophilin binding activity but has significantly reduced immunosuppressive activity has led to optimization of this reaction on an industrial scale.
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This review systematically collates the synthetic chemistry performed at each of the eleven amino acids, and provides examples of the utility of such transformations. The various modifications of CsA are traced from early, modest chemistry performed at the unique Bmt residue, through the remarkable use of a polyanion enolate that can be stereoselectively manipulated, and onto application of more recently developed olefin metathesis chemistry to prepare new CsA derivatives with unexpected biological activity.
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Determination of ciclosporin A and its six main metabolites in isolated T-lymphocytes and whole blood using liquid chromatography–tandem mass spectrometry

Abstract

Introduction

Section snippets

Chemicals and reagents

MS–MS conditions

Optimal SPE conditions

Conclusion

Cell

J. Biol. Chem.

Transplant. Proc.

Transplant. Proc.

Pharmacol. Ther.

Transplant. Proc.

Biochem. Pharmacol.

J. Immunol. Methods

Drugs