Pharmacokinetics of SDZ RAD and cyclosporin including their metabolites in seven kidney graft patients after the first dose of SDZ RAD

Abstract

Aims

The aim of the study was to investigate the pharmacokinetics and metabolism of the new immunosuppressant SDZ RAD during concomitant therapy with cyclosporin in stable renal transplant patients. Furthermore, we studied the influence of SDZ RAD on the pharmacokinetics of cyclosporin at steady state levels.

Methods

SDZ RAD was administered orally in different doses (0.25–15 mg day⁻¹) to seven patients, who were on standard cyclosporin-based immunosuppression. The blood concentrations of both drugs including their main groups of metabolites were measured simultaneously by LC/electrospray-mass spectrometry.

Results

The mean area under the blood concentration-time curve to 12 h (AUC(0,12 h)) was 4244 ± 1311 µg l⁻¹ h for cyclosporin before SDZ RAD treatment and 4683 ± 1174 µg l⁻¹ h (P = 0.106) on the day of SDZ RAD treatment (95% CI for difference -126, 1003). On both study days C_max, and t_max of cyclosporin were not significantly different. The metabolite pattern of cyclosporin did not change. The pharmacokinetic data of SDZ RAD dose-normalized to 1 mg SDZ RAD were as follows: AUC(0,24 h): 35.4 ± 13.1 µg l⁻¹ h, C_max: 7.9 ± 2.7 µg l⁻¹ and t_max: 1.5 ± 0.9 h. The metabolites of SDZ RAD found in blood were hydroxy-SDZ RAD, dihydroxy-SDZ RAD, demethyl-SDZ RAD, and a ring-opened form of SDZ RAD.

Conclusions

A single dose of SDZ RAD did not influence significantly the pharmacokinetics of cyclosporin. The most important metabolite of SDZ RAD was the hydroxy-SDZ RAD, its AUC(0,24 h) being nearly half that of the parent compound SDZ RAD.

Introduction

The new macrolide immunosuppressant SDZ RAD[40-O-(2-hydroxy)ethyl-rapamycin] has a molecular weight of 957.6 dalton (C₅₃H₈₃NO₁₄) and is currently under clinical investigation in stable renal transplant recipients. SDZ RAD has a mode of action different from that of cyclosporin or tacrolimus. In contrast to the latter drugs, which inhibit the expression of T-cell growth factors such as IL-2 [1–3], SDZ RAD inhibits in general growth factor driven cell proliferation, including that of T-cells and vascular smooth muscle cells [4]. SDZ RAD and cyclosporin show greater than additive immunosuppression and the drugs are intended to be given in combination [5]. The advantage of SDZ RAD seems to be that it is less nephrotoxic [unpublished data from Novartis] than the standard immunosuppressants cyclosporin and tacrolimus which are associated with nephrotoxicity [6–9].

Cyclosporin, tacrolimus and sirolimus (rapamycin) are metabolized mainly by cytochrome P450 3A4 [10–13]. Therefore it may be predicted that SDZ RAD is metabolized by the same enzymes. In in vitro studies liver microsomes generated at least seven SDZ RAD metabolites and we could identify three SDZ RAD metabolites resulting from hydroxylation, demethylation and degradation [14]. The immunosuppressive activity of the SDZ RAD metabolites is unknown.

Since h.p.l.c. with u.v. detection is not sensitive enough to assay concentrations lower than 2 µg l⁻¹ in patients' blood, we had developed an LC/electrospray-mass spectrometric method for simultaneous measurement of SDZ RAD, cyclosporin and their metabolites [15].

It was the aim of the study using a specific LC/electrospray-mass spectrometric method to analyse the pharmacokinetics of SDZ RAD, cyclosporin and their metabolites after the first oral SDZ RAD dose and to compare the pharmacokinetic data of cyclosporin under steady-state conditions and after a single dose of SDZ RAD.

Methods

Patients and blood sample collection

Seven Caucasian stable kidney transplant recipients (1 female/6 male) under primary therapy with cyclosporin (administered every 12 h; trough levels between 80 and 150 µg l⁻¹) and prednisone (dose < 15 mg day⁻¹) participated in a phase I study with SDZ RAD. These patients were part of an European multicentre study. The patients were instructed to take the drugs exactly at 08.00 h and 20.00 h 3 days before and during the study. Compliance was checked by a questionaire. In addition to their basic immunosuppression patients received between 0.25 mg day⁻¹ and 15 mg day⁻¹ of SDZ RAD for only one day in a single dose. SDZ RAD was administered as capsules. Patients were excluded who had to take other drugs known either to inhibit or induce cytochrome P450 3A [16] with the exceptions of methylprednisolone and prednisone. The mean age of the patients was 43 years (range 25–61 years). The patients had a mean weight of 78 kg (range 52–102 kg) and the mean time after transplantation was 6.6 ± 4.2 years (range 1.25 years to 14.5 years). The study was approved by the local ethics committee, and all patients gave their written informed consent according to the revised Declaration of Helsinki.

Blood samples were taken into EDTA containing tubes on the day before and on the day of SDZ RAD administration and were immediately frozen at −20 °C until measurement. The samples were collected immediately before (0 time) and 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 8, 10, and 12 h after cyclosporin and SDZ RAD intake.

In all patients clinical chemistry and haematological parameters on the day before SDZ RAD administration were measured. The mean value of gamma glutamyl transferase (γ-GT) was slightly increased (mean = 27 U l⁻¹; normal: up to 18 U l⁻¹). All the other liver function tests [serum bilirubin, activities of alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), cholinesterase (CHE), glutamate dehydrogenase (GLDH)] were normal. Additionally glucose, cholesterol, triglycerides, leucocytes, haematocrit and thrombocytes were in normal ranges. The mean renal function tests were abnormal: serum creatinine averaged 125 µmol l⁻¹ (normal 50–80 µmol l⁻¹) and urea 10 mmol l⁻¹ (normal 3.3–6.7 mmol l⁻¹). The mean value of haemoglobin was decreased: 11.3 g dl⁻¹ (normal 12–16 g dl⁻¹). The serum parameters were measured daily using standard methods (Institut für Klinische Chemie, Medizinische Hochschule Hannover, Hannover, Germany).

Method of SDZ RAD, cyclosporin and their metabolites

The h.p.l.c./electrospray-mass spectrometric technique used has been previously described [15]. Blood samples were deproteinized by methanol/0.4 mol l⁻¹ zinc sulphate solution (80 : 20, v/v). After centrifugation (4900 rev min⁻¹ for 6 min) supernatants were injected into the h.p.l.c. system (HP 1090 series II liquid chromatograph; Hewlett-Packard, Waldbronn, Germany) and transferred to an extraction column (C-18 column). The compounds were enriched on the extraction column, while other compounds in the sample were transferred through the extraction column into the waste. Then the switching valve changed position and the extract was eluted in the back-flush mode and directly transferred onto the analytical column (250 × 2 mm Hypersil ODS column, particle size 5 µm). The compounds were separated on the analytical column under isocratic conditions (0.2 ml min⁻¹ of methanol/water 90/10; v/v) with a column temperature of 35 °C, and were then injected into the electrospray-mass spectrometer.

The h.p.l.c. system was linked to an API-electrospray cabinet HP59987A which was connected to an HP5989B mass spectrometer engine (Hewlett-Packard, Waldbronn, Germany). Data were recorded and analysed by HP LC/MS ChemStation consisting of the HP G1034C MS ChemStation and the G1047A LC/MS software (Hewlett Packard).

The MS was run in the selected ion monitoring (SIM) mode and focused on the sodium adduct ions [M + Na]⁺ of the following masses: 966.6 atomic mass unit (amu) (demethyl-SDZ RAD), 980.6 amu (SDZ RAD), 996.6 amu (hydroxy-SDZ RAD), 998.6 amu (ring-opened form of SDZ RAD), 1012.6 amu (dihydroxy-SDZ RAD), 1224.9 amu (cyclosporin), 1240.9 amu (hydroxy-cyclosporin) and 1256.9 amu (dihydroxy-cyclosporin).

For quality control during the study two precision and calibration control samples each were run for every 10 study samples. The calibration curve in whole blood comprised nine data points at concentrations of 0/0, 1/20, 3/60, 9/180, 15/300, 25/500, 35/700, 50/1000, 75/1500 µg l⁻¹ with n = 5 for each concentration of SDZ RAD/cyclosporin. The lower limit of quantification was 0.4 µg l⁻¹ for SDZ RAD, cyclosporin and their metabolites. The calibration curves were linear from 0.4 to 100 µg l⁻¹ for SDZ RAD (r² = 0.99) and 10–1300 µg l⁻¹ for cyclosporin (r² = 0.99). Recoveries of SDZ RAD and cyclosporin from blood were 96.0 ± 6% (mean ± s.d.) and 94 ± 5% (mean ± s.d.), respectively. The interassay coefficients of variation were 9.2% at 3 µg l⁻¹ and 8.8% at 50 µg l⁻¹ for SDZ RAD and 9.8% at 60 µg l⁻¹ and 9.6% at 700 µg l⁻¹ for cyclosporin (n = 15).

Pharmacokinetic analysis

Compartment-independent pharmacokinetic parameters of SDZ RAD, hydroxy-SDZ RAD, dihydroxy-SDZ RAD, ring-opened form of SDZ RAD, demethyl-SDZ RAD, and of cyclosporin, hydroxy-cyclosporin, and dihydroxy-cyclosporin were evaluated using the TopFit version 2.0 software [17]. The maximal concentration (C_max), the corresponding time (t_max), the clearance and area under the curve (AUC(0,12 h) or AUC(0,24 h)) were determined. Since SDZ RAD exhibited dose proportional increases in C_max and AUC (r² = 0.96 and r² = 0.92, respectively), the mean values of SDZ RAD including its metabolites were given in dose-normalized units.

Statistical analysis

For statistical analysis Student's t-test for paired data was used to compare baseline with day 1 data. Differences between the pre and post-treatment values were given as means and 95% confidence intervals (CI). We used ‘SPSS for Windows 9.0; SPSS Inc., Chicago, IL’;

P-values of < 0.05 were considered significant. Figure 2 was designed with ‘GraphPad Prism, Version 2.0’.

Figure 2.

Cyclosporin clearance as a function of SDZ RAD dose in seven patients. The regression line and a confidence interval for the regression slope as shown. CSA = cyclosporin.

Results

Pharmacokinetics of cyclosporin and its metabolites in the steady-state

All seven patients were treated orally with cyclosporin (baseline) so that cyclosporin trough levels were between 80 and 150 µg l⁻¹. The pharmacokinetic profiles of cyclosporin and its metabolites of all patients are shown in Figure 1a–c. The main groups of cyclosporin metabolites were hydroxy-cyclosporin and dihydroxy-cyclosporin. During the first 2.5 h the concentration of cyclosporin was higher than that of hydroxy-cyclosporin, but thereafter the concentration of hydroxy-cyclosporin increased while the concentration of cyclosporin rapidly decreased. The pharmacokinetic parameters (t_max, C_max, and AUC) of cyclosporin and its metabolites are shown in Table 1. During baseline therapy the mean AUC(0,12 h) value was 4244 ± 1311 µg l⁻¹ h. Mean C_max was 1069 ± 291 µg l⁻¹ which was reached at a time of 1.3 ± 0.3 h.

Figure 1.

a) Mean (± s.d.) blood concentrations of cyclosporin, b) hydroxy-cyclosporin and c) dihydroxy-cyclosporin in seven stable kidney graft patients the day before (cyclosporin only, •) and on the day (cyclosporin and SDZ RAD, □) of SDZ RAD administration measured by LC/electrospray-mass spectrometry.

Table 1.

Pharmacokinetic parameters (mean ± s.d.) of cyclosporin and its metabolites in the steady-state (baseline) and of cyclosporin and SDZ RAD including their metabolites during combination therapy (day 1). Based upon the assumption that there is a dose linearity the pharmacokinetic data of SDZ RAD including its metabolites were given as dose normalized values. The blood concentrations were measured by LC/electrospray-mass spectrometry.

	Cmax (µg l−1)	tmax(h)	AUC(0,12 h)(µg l−1h)
Baseline
Cyclosporin	1069 ± 291	1.3 ± 0.3	4244 ± 1311
Hydroxy-cyclosporin	638 ± 79	2.2 ± 0.4	4906 ± 779
Dihydroxy-cyclosporin	106 ± 14	3.4 ± 0.9	785 ± 84
Day 1
Cyclosporin	1231 ± 345	1.6 ± 0.6	4683 ± 1174
Hydroxy-cyclosporin	737 ± 9.9	2.4 ± 0.6	5559 ± 923
Dihydroxy-cyclosporin	128 ± 60	3.2 ± 0.6	997 ± 220
			AUC(0,24 h)
Day 1			(µg l−1h)
SDZ RAD	7.9 ± 2.7	1.5 ± 0.9	35.4 ± 13.1
Hydroxy-SDZ RAD	2.8 ± 2.8	1.7 ± 0.9	16.0 ± 6.5
Dihydroxy-SDZ RAD	1.4 ± 1.0	1.6 ± 0.2	8.5 ± 5.7
Demethyl-SDZ RAD	1.6 ± 1.3	2.0 ± 1.0	10.7 ± 15.8
Ring-opened form of SDZ RAD	0.5 ± 0.4	1.2 ± 0.9	2.3 ± 2.1

t_max = time to maximal serum concentration, C_max = maximum serum concentration, AUC = area under the concentration time curves.

Influence of a single dose of SDZ RAD on the pharmacokinetics of cyclosporin

In addition to cyclosporin the patients received a single, oral dose of various concentrations of SDZ RAD (0.25 mg to 15 mg) for 1 day (day 1). The concentration-time curves of the mean ±s.d. concentrations of cyclosporin and its metabolites at baseline and day 1 are shown in Figure 1a,b,c. The pattern of the metabolites of cyclosporin did not change in the presence of SDZ RAD (Figure 1b,c). C_max of cyclosporin amounted to 1231 ± 345 µg l⁻¹ at t_max of 1.6 ± 0.6 h (Table 1). The mean AUC(0,12 h) was 4683 ± 1174 µg l⁻¹ h. SDZ RAD, cyclosporin and most of the metabolites reached C_max at about the same time (approximately 1.5 h). We could show that the mean C_max values of baseline and day 1 (1069 µg l⁻¹vs 1231 µg l⁻¹) were not significantly different (mean difference 162 µg l⁻¹; 95% CI −136–459 µg l⁻¹; P = 0.233). The difference between the mean cyclosporin AUC(0, 12 h) values alone and in the presence of SDZ RAD (4244 µg l⁻¹ h vs 4683 µg l⁻¹ h) did not reach significance (mean difference 439 µg l⁻¹ h; 95% CI −126–1003 µg l⁻¹; P = 0.106). In the presence of SDZ RAD the metabolite pattern of cyclosporin did not show significant changes. The AUC(0,12 h) (mean difference 653 µg l⁻¹ h; 95% confidence interval −189–1164 µg l⁻¹ h; P = 0.123) and C_max (mean difference 99 µg l⁻¹; 95% CI −38–134 µg l⁻¹; P = 0.060) of hydroxy-cyclosporin and AUC(0,12 h) (mean difference 212 µg l⁻¹; 95% CI −7.5–359 µg l⁻¹; P = 0.057) and C_max (mean difference 22 µg l⁻¹; 95% CI −38–65 µg l⁻¹; P = 0.528) of dihydroxy-cyclosporin did not change significantly in the presence of SDZ RAD. Cyclosporin clearance did not change significantly (P = 0.778) with increasing SDZ RAD doses (Figure 2).

Pharmacokinetics of SDZ RAD and its metabolites after a single dose

Four main types of metabolites: hydroxy-SDZ RAD, dihydroxy-SDZ RAD, demethyl-SDZ RAD and a ring-opened form of SDZ RAD were found in patients' blood (Figure 3). The pharmacokinetic data of SDZ RAD including its metabolites were given as dose-normalized to 1 mg. Hydroxy-SDZ RAD was the most important metabolite, its AUC(0,24 h) was nearly half of that of the parent compound SDZ RAD (16.0 ± 6.5 µg l⁻¹ h vs 35.4 ± 13.1 µg l⁻¹ h). Pharmacokinetic data including that of the other SDZ RAD metabolites are shown in Table 1. SDZ RAD reached after 1.5 ± 0.9 h (t_max) the C_max of 7.9 ± 2.7 µg l⁻¹. C_max of hydroxy-SDZ RAD was 2.8 ± 2.8 µg l⁻¹ and appeared after 1.7 ± 0.9 h.

Figure 3.

Mean dose normalized blood concentrations of SDZ RAD and its metabolites(• SDZ RAD, □ hydroxy SDZ RAD, dihydroxy SDZ RAD, x ring opened SDZ RAD, * demethyl SDZ RAD) in seven stable kidney graft patients on the day of SDZ RAD administration measured by LC/ESI-MS.

Discussion

This report has shown that in stable renal transplant recipients SDZ RAD is metabolized and that a single dose of SDZ RAD does not affect the pharmacokinetics of cyclosporin. The main metabolite of SDZ RAD appearing in blood is a monohydroxylated form. Dihydroxy and demethyl SDZ RAD and a ring-opened form were detectable in much lower concentrations. At least the first three metabolites suggest that the cytochrome P450 system is involved. In analogy to what is known from related compounds such as tacrolimus and sirolimus [10–13] it can be hypothesized that the subfamily CYP 3A plays a major role in the metabolism of SDZ RAD.

This in turn would raise the concern that there might be pharmacokinetic interactions between SDZ RAD and other drugs metabolized by CYP 3A, e.g. cyclosporin. Our pharmacokinetic data of cyclosporin before administration of SDZ RAD are in good agreement with the previously published data [18–20]. The present investigation excluded a significant influence of SDZ RAD in a single dose either on the time-concentration relationship or the metabolism of cyclosporin under steady state conditions. Further studies are needed to investigate the interactions of SDZ RAD and cyclosporin under long-term administration.

Irrespective of SDZ RAD dosage all patients had the same metabolite pattern (data not shown). Compared with blood concentrations of cyclosporin metabolites, which reached higher trough values than the parent compound [21], the SDZ RAD metabolite fraction in blood was relatively small. Nevertheless there is need to investigate the properties of the metabolites in further studies.

In our study we presented pharmacokinetic data and the metabolite pattern of SDZ RAD after oral intake in humans. SDZ RAD is metabolized to at least four types of metabolites. We showed that hydroxy-SDZ RAD is the main metabolite of SDZ RAD with an AUC(0,24 h) nearly half of that of the parent compound. Therefore it is important to investigate whether hydroxy-SDZ RAD or one of the main metabolites has immunosuppressive or toxic effects. Additionally, the pharmacokinetics of cyclosporin and its metabolites were not significantly influenced by a single dose of SDZ RAD.