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PIMECROLIMUS: ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION IN HEALTHY VOLUNTEERS AFTER A SINGLE ORAL DOSE AND SUPPLEMENTARY INVESTIGATIONS IN VITRO

Novartis Pharma AG, Basel, Switzerland (M.Z., F.W., S.H., A.G.Z., U.G., J.-P.B., A.S., S.B., T.M.) and Novartis Institutes for BioMedical Research, Basel, Switzerland (G.Z.) and Vienna, Austria (M.A.G.)

(Received October 6, 2005; accepted February 1, 2006)

	Abstract

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The absorption and disposition of pimecrolimus, a calcineurin inhibitor developed for the treatment of inflammatory skin diseases, was investigated in four healthy volunteers after a single oral dose of 15 mg of [³H]pimecrolimus. Supplementary information was obtained from in vitro experiments. Pimecrolimus was rapidly absorbed. After t_max (1–3 h), its blood concentrations fell quickly to 3% of C_max at 24 h, followed by a slow terminal elimination phase (average t_1/2 62 h). Radioactivity in blood decreased more slowly (8% of C_max at 24 h). The tissue and blood cell distribution of pimecrolimus was high. The metabolism of pimecrolimus in vivo, which could be well reproduced in vitro (human liver microsomes), was highly complex and involved multiple oxidative O-demethylations and hydroxylations. In blood, pimecrolimus was the major radiolabeled component up to 24 h (49% of radioactivity area under the concentration-time curve_0–24 h), accompanied by a large number of minor metabolites. The average fecal excretion of radioactivity between 0 and 240 h amounted to 78% of dose and represented predominantly a complex mixture of metabolites. In urine, 0 to 240 h, only about 2.5% of the dose and no parent drug was excreted. Hence, pimecrolimus was eliminated almost exclusively by oxidative metabolism. The biotransformation of pimecrolimus was largely catalyzed by CYP3A4/5. Metabolite pools generated in vitro showed low activity in a calcineurin-dependent T-cell activation assay. Hence, metabolites do not seem to contribute significantly to the pharmacological activity of pimecrolimus.

View larger version (16K): [in this window] [in a new window]   FIG. 1. Chemical structure of pimecrolimus (SDZ ASM 981). Positions of tritium labeling are indicated by asterisks. In the in vitro blood distribution study [3H]pimecrolimus was labeled in position 32. In all other studies [3H]pimecrolimus was labeled in positions 5 and 6.

The oxidative metabolism of pimecrolimus is very complex, resulting in a plethora of minor metabolites, both in blood and excreta. All of them were present at low concentrations, were hardly separable by chromatography, and, thus, were hard to characterize in the sample matrices ex vivo. However, the same, very characteristic metabolite patterns were found in samples generated in vitro. The patterns resulted from oxidations by CYP3A at multiple positions of the pimecrolimus molecule, which appears to be a typical fate of bulky lipophilic CYP3A substrates. Thus, it is not surprising that the in vivo patterns could be reproduced in vitro using hepatic microsomes. Therefore, the analytically simpler in vitro samples were used for metabolite characterization, rather than the samples ex vivo. The same strategy had been used to compare the metabolism in humans and toxicological test species (Zollinger et al., 2002). The approach may be applicable to other developmental drugs when metabolism studies in vivo pose insurmountable analytical difficulties.

	Materials and Methods

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Chemicals. NADPH, NADP, DL-isocitrate, and isocitrate dehydrogenase were obtained from Sigma (St. Louis, MO), luciferin from Chemie Brunschwig (Basel, Switzerland), Rialuma liquid scintillation cocktail from Lumac (Groningen, The Netherlands), Pico-Fluor 40 and Permafluor E liquid scintillation cocktail from Packard (Meriden, CT), and Titrisol buffer concentrate from Merck (Darmstadt, Germany). All other reagents and solvents were of analytical grade and were readily available from commercial sources.

Study Drug. [³H]Pimecrolimus was synthesized by the Isotope Laboratory of Novartis, Basel, Switzerland (Moenius et al., 2001). In the human in vivo study, [³H]pimecrolimus with the label in position 5 and 6 on the piperidine ring (see Fig. 1) and a specific radioactivity of 607 MBq/mmol was used. The radiochemical purity was >98%. The in vitro assays were performed with different batches of [³H]pimecrolimus, again labeled in position 5 and 6. As an exception, the study on the distribution between blood cells and plasma was performed with [³H]pimecrolimus labeled in position 32 on the cyclohexyl ring. Unlabeled pimecrolimus was a product of Novartis.

Subjects and Design of the Human in Vivo Study. The clinical part of the study was performed at Simbec Research Ltd. (Merthyr Tydfil, UK) in accordance with Good Clinical Practice guidelines and the Declaration of Helsinki (1964 and subsequent revisions). All subjects had to give written informed consent before participation. The protocol was approved by an institutional review board and ethics committee and by the Administration of Radioactive Substances Advisory Committee, UK.

Four healthy male subjects were enrolled in this open-label, single oral dose, absorption, distribution, metabolism, and excretion study. The subjects were of good health as shown by past medical history, physical examination, electrocardiogram, laboratory tests, and urine analysis. Subjects of "poor metabolizer" genotype with regard to CYP2D6 were excluded (CYP2D6 was later found to be only marginally involved in the elimination of pimecrolimus). The subjects were nonsmokers, had no history of alcoholism or drug abuse, and did not take enzyme-inducing or -inhibiting drugs (e.g., phenobarbital, glutethimide, phenylbutazone, phenytoin, rifampin, isoniazid, dexamethasone, cimetidine, macrolides, ketoconazole, Ca-channel blockers) within 4 weeks before dosing. No medication other than the study drug was allowed from the initial day of screening (2–9 days before dosing) until the end of the evaluation period, with the exception of drugs needed to treat the subjects in the case of adverse events. The subjects had to fast for at least 10 h before dosing and to continue fasting for at least 4 h thereafter.

All subjects received a single oral 15-mg (11.2-MBq) dose of [³H]pimecrolimus, formulated as a 20% solid dispersion containing, in addition, hydroxypropyl methyl cellulose (70%) and poloxamer 188 (10%). The dose was administered as three capsules of 5 mg [³H]pimecrolimus, swallowed essentially simultaneously. The whole body radiation dose ("effective dose") was calculated to be below the radiation dose limit for the public in the United Kingdom (0.5 mSv per year).

Blood was collected at 1, 2, 3, 4, 6, 8, 12, 24, 32, 48, 72, 96, 120, 144, 192, and 240 h postdose into EDTA-coated polypropylene tubes by direct venipuncture or by an indwelling cannula inserted in a forearm vein. Volumes of 25 ml were taken up to 120 h, followed by 5-ml samples at later times. Complete urine was collected in 24-h intervals between dosing and 240 h postdose. Each subject voided his bladder before drug administration and at the end of each collection interval. During a collection interval, the urine was kept refrigerated at 4–8°C. Complete feces were collected after dose administration up to 240 h postdose. The samples were stored at –20°C until analysis.

Radioactivity was measured in blood, urine, urine distillates, and feces by liquid scintillation counting (LSC). Concentrations of parent drug were determined in blood by liquid chromatography-mass spectrometry (LC-MS). Metabolite patterns in blood, urine, and feces were determined by high-pressure liquid chromatography (HPLC) with radioactivity monitoring after appropriate sample preparation. Details of the procedures are given below.

Safety assessments included the monitoring and recording of all adverse events, regular checks of routine blood chemistry, hematology and urine values, electrocardiogram recordings, measurements of vital signs, and physical examinations.

Liquid Scintillation Counting of Human in Vivo Samples. Blood samples (duplicate 500-µl aliquots) were dried and combusted in a Canberra Packard (Schwadorf, Austria) model 307 sample oxidizer before LSC. Urine (duplicate 1-ml aliquots) and urine distillate samples (duplicate 300-µl aliquots of single distillates) were measured directly. Feces samples were homogenized in water. Aliquots of the homogenates (quadruplicate aliquots of approximately 0.7 g) were dried and combusted like the blood samples. The measurements in urine and urine distillates included volatile radioactivity (tritiated water); those in blood and feces did not because of drying. Pico-Fluor 40 and Permafluor E were used as liquid scintillation cocktails for the uncombusted and the combusted samples, respectively. The measurements were performed on a Wallac (Turku, Finland) 1409 liquid scintillation counter with counting times of 3 min (urine distillates) or 2 min (all other samples). Quench correction was performed by an external standard ratio method. To establish quench correction curves, sealed tritium standards (Packard) were used.

Determination of Parent Drug in Blood. Concentrations of pimecrolimus in individual blood samples were determined by LC-MS after extraction. The blood samples were diluted with 2 volumes of water before freezing to reduce the risk of coagulation. From each sample of diluted blood an aliquot of 1 ml was spiked with internal standard (21-propyl homolog of pimecrolimus; Novartis), alkalinized with 500 µl of Titrisol buffer concentrate, pH 10, and extracted with 5 ml of tert-butyl methyl ether (approximately 80% recovery). The organic extract was evaporated, reconstituted in 250 µl of water/methanol 20:80 (v/v), and 200 µl was injected. The chromatographic separation was performed at 75°C on a 250 x 4 mm Nucleosil 100 C18 AB column packed with 5-µm particles (Macherey-Nagel, Düren, Germany), preceded by an 8 x 4 mm precolumn of the same stationary phase. The components were eluted isocratically with 0.02 M aqueous ammonium acetate/methanol 15:85 (v/v) at a flow rate of 1 ml/min. Pimecrolimus (retention time approximately 4.4 min) and the internal standard (retention time approximately 5.2 min) were detected by mass spectrometry with negative ion atmospheric pressure chemical ionization and selected ion monitoring using an API 165 quadrupole mass spectrometer (PE Sciex, Foster City, CA). The [M – H]^– ions of pimecrolimus at m/z 808.6 and of the internal standard at m/z 822.6 were monitored. The limit of quantification was 0.11 pmol/ml (0.09 ng/ml), referring to undiluted blood.

Determination of Metabolite Patterns in Blood, Urine, and Feces. Blood samples were partially hemolyzed by dilution with 2 volumes of water immediately after sampling. They were analyzed individually. Each sample of diluted blood was extracted three times with methanol. The extracts were acidified with trifluoroacetic acid (TFA) to a pH of approximately 3.5 (pimecrolimus solutions are most stable at pH 2–3; Novartis Pharma AG, unpublished data), combined, and evaporated to dryness. The residue was reconstituted in three different solvents in the following way. First it was taken up in water/methanol 80:20 (v/v) by sonication, followed by centrifugation and separation of the supernatant (extract A). The remaining pellet was extracted two more times, once with water/methanol 60:40 (v/v) containing 0.012% (v/v) TFA (extract B) and once with water/acetonitrile 40:60 (v/v) containing 0.008% (v/v) TFA (extract C). The three extracts were analyzed in a single HPLC run after sequentially loading them onto the column in the order C-B-A. By this procedure, both polar and nonpolar components were dissolved and concentrated on the head of the HPLC column before starting the elution.

Urine was pooled over the collection interval of 0 to 168 h for each subject individually. Of each pool, 2.7 ml was diluted with 0.3 ml of acetonitrile and acidified with TFA to a pH of approximately 3. After centrifugation, an aliquot of the supernatant was injected onto the HPLC column. The contributions of tritiated water to the radioactivity in the urine pools were determined by measuring radioactivity in native and dried aliquots. Feces homogenates were pooled over the collection interval of 0 to 168 h for each subject individually and the samples were processed as described above for blood.

The recovery of radioactivity after sample processing and HPLC was around 100% for blood and urine. For feces, the recovery of radioactivity was 74 to 83% after sample preparation and approximately 91% (of the amount injected onto the column) after HPLC. The stability of pimecrolimus during blood and feces sample processing and HPLC analysis was investigated using blank samples spiked with [³H]pimecrolimus. No degradation of pimecrolimus was detected. The stability of the numerous metabolites in blood and feces during sample processing and HPLC was not investigated. However, due to their structural similarity to the parent drug (mainly products of O-demethylations, hydroxylations, and oxidative dechlorination with intact macrocycle; see below), they can be assumed to be stable as well.

The chromatography was performed on an HP 1090 liquid chromatograph (Hewlett-Packard, Waldbronn, Germany). Radioactivity was monitored by LSC either off-line after collection of 0.5-min fractions (blood extracts and urine samples) or on-line using a Berthold (Wildbad, Germany) LB 507A radioactivity monitor. For the latter purpose, 3 ml/min Rialuma liquid scintillation cocktail was added to the column effluent. The separations were performed on a 250 x 4.6 mm Nucleosil 100 C18 AB column (5-µm particles), preceded by an 8 x 4 mm precolumn of the same stationary phase. The column temperature was 60°C. The components were eluted with a gradient of 0.02% (v/v) aqueous TFA (mobile phase A) versus acetonitrile (mobile phase B). The proportion of the mobile phase B was kept at 10% until 5 min after injection and was then increased linearly to 100% at 140 min, where it was kept for another 10 min. The total flow rate was 1 ml/min.

Blood concentrations and percentages of dose in the excreta were estimated for pimecrolimus and its metabolites from the radiochromatograms based on the relative peak areas and the concentrations or amounts of radioactivity in the original biological samples, after subtraction of the losses during sample preparation and chromatography.

Pharmacokinetic Calculations. Pharmacokinetic parameters in vivo were calculated using noncompartmental methods. They are given as mean ± S.D. except for t_max, for which medians are given. Area under the concentrationtime curve (AUC) values were calculated by the linear trapezoidal method and extrapolated to infinity according to AUC_t- = C_t · t_1/2/ln 2. The t_1/2 values were calculated by log-linear regression of plasma concentrations during the terminal elimination phase.

The amount of total tritiated water formed in the body was estimated from the radioactivity concentrations in the distillates of the urine samples collected between 72 and 240 h postdose. The concentrations were extrapolated back to time 0 assuming a half-life of 9.5 days (Richmond et al., 1962). The extrapolated concentrations were multiplied by the amounts of total body water, taken as 55% of the individual body weights (Richmond et al., 1962) to obtain the total amounts of tritiated water formed, which were expressed as percentage of dose.

Distribution of Pimecrolimus between Blood Cells and Plasma in Vitro. Pooled fresh heparinized blood from three human donors was spiked with [³H]pimecrolimus at five concentrations in the range of 1 ng/ml to 1 µg/ml and incubated in triplicates at 37°C for 30 min. Thereafter, two aliquots were removed and the remaining blood was centrifuged to obtain plasma. Radioactivity concentrations in all whole blood and plasma samples were determined by LSC. The stability of [³H]pimecrolimus during the incubation was confirmed by HPLC analysis with radioactivity monitoring of blood extracts. No degradation was found during 1.5 h at 37°C.

Metabolism of [³H]Pimecrolimus by Human Liver Microsomes. A commercial pool of liver microsomes from 10 individuals (BD Gentest, Woburn, MA; Catalog No. H161, lot 7) with a total cytochrome P450 (P450) content of 0.42 nmol/mg protein was used. Microsomal incubations were performed at 37°C in 0.1 M phosphate buffer, pH 7.4, containing 0.2 mM NADPH and an NADPH regenerating system consisting of (final concentrations) 1 mM NADP, 5 mM DL-isocitrate, 1 U/ml isocitrate dehydrogenase, and 5 mM MgCl₂. [³H] Pimecrolimus was added as a solution in acetonitrile. The concentration of acetonitrile in the final incubate was less than or equal to 0.7% (v/v). [³H]Pimecrolimus was incubated at 1 µM initial substrate concentration with 50 µg microsomal protein/ml or at 40 µM initial substrate concentration with 4 mg microsomal protein/ml for different times. In all incubates the concentration of radioactivity was 0.22 MBq/ml. The enzymatic reactions were stopped by adding 1 volume of ice-cooled acetonitrile, followed by cooling on ice for 5 min and ultracentrifugation. Aliquots of the supernatants of the 1 and 40 µM[³H]pimecrolimus incubates were analyzed by HPLC with radioactivity detection using the chromatographic conditions described above for the in vivo samples. Moreover, aliquots of the 40 µM [³H]pimecrolimus incubates were investigated by LC-MS with parallel radioactivity monitoring as described below for characterizing metabolite structures. The chemical stability of [³H]pimecrolimus (1 µM) under the incubation conditions was checked by a control incubation for 120 min (longest incubation time used in the study) in the absence of microsomes. The degradation amounted to less than 2%.

Structural Characterization of in Vitro Metabolites by LC-MS. The chromatographic part of the instrumentation consisted of two Kontron (Milan, Italy) 420 pumps, a manual Rheodyne (Cotati, CA) 7725i injector, and an SPH 99 column oven from Spark Holland (Emmen, The Netherlands). After the column, the flow was split into two parts. Between 70 and 95% of the flow was combined with Rialuma (3 ml/min) and directed into a Berthold LB 507B radioactivity monitor. The remaining 5 to 30% of the effluent was passed into the electrospray LC-MS interface (model API 2; Finnigan MAT, San Jose, CA) of a Finnigan MAT TSQ7000 tandem quadrupole mass spectrometer. The HPLC column, the column temperature, the mobile phase gradient, and the total flow rate were the same as described above in the context of the metabolite patterns in vivo. In front of the electrospray interface, a flow of 1 µl/min aqueous 50 mM lithium acetate was added by a syringe pump to generate M + Li⁺ ions. The electrospray interface was operated with nitrogen as sheath gas (80 psi), nitrogen as auxiliary gas (10 flowmeter units), and methanol/water 90:10 (v/v) as sheath liquid (20 µl/min). The spray capillary was set to 4.5 kV. The transfer capillary was heated to 290 to 350°C. Single-stage positive ion mass spectra were recorded at unit mass resolution by using the first quadrupole as mass analyzer. The range of m/z 200 to 1100 was scanned in 2 s. The upfront collision offset was set to 0 V. Thermal excitation in the heated capillary of the LC-MS interface was sufficient to generate fragment ions.

For determining the number of exchangeable hydrogens of the metabolites, LC-MS runs were performed as described above except that D₂O was used instead of H₂O in the mobile phase A and as solvent for lithium acetate. No sheath liquid was used in these experiments.

In Vitro Characterization of Isoenzymes Responsible for Pimecrolimus Metabolism; Enzyme Kinetics. Microsomes prepared from recombinant human B-lymphoblastoid cell lines heterologously expressing human P450 isoenzymes (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP4A11) or expressing P450 reductase only (control) were obtained from BD Gentest. [³H]Pimecrolimus was incubated with these microsomes for 20 min at an initial substrate concentration of 1 µM under conditions recommended by the supplier. The incubation volumes were 200 µl. The amount of recombinant P450 added (12.5, 21.5, 5.3, 19.4, 13.5, 12.3, 6.4, 8.3, 29.1, and 17.0 pmol of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP4A11, respectively) was representative of either the highest P450 concentration or the highest P450 marker activity found for the respective isoform in a panel of liver microsomes from 12 human individuals (BD Gentest, Catalog No. H003, H006, H023, H030, H042, H043, H056, H066, H070, H089, H093, and H112).

For determining enzyme kinetic parameters (K_m, V_max) as well as for investigating the effect of the CYP3A inhibitor ketoconazole or of a specific anti-CYP3A4/5 monoclonal antibody (BD Gentest, Catalog No. A334, lot 2) on the metabolism of [³H]pimecrolimus, the compound was incubated for 10 min with pooled human liver microsomes from six individual donors (BD Gentest, Catalog No. H161, lot 3) at 0.1 mg/ml microsomal protein. The extent of biotransformation of [³H]pimecrolimus was shown, before, to increase linearly with incubation time and microsomal protein concentration up to 15 min and 0.5 mg/ml, respectively. For investigating the enzyme kinetics, initial substrate concentrations of 0.1 to 8 µM were used. For the inhibition experiments, initial substrate concentrations of 1 µM were chosen. The ketoconazole concentrations were between 0.1 and 2 µM. For the antibody inhibition experiments, human liver microsomes (pool of six individuals) were pretreated with increasing amounts of monoclonal antibody according to the recommendations of the supplier (BD Gentest). Immunoinhibited human liver microsomes were incubated at a final microsomal protein concentration of 0.1 mg/ml for 10 min with either 1 µM [³H]pimecrolimus or 50 µM midazolam.

All enzymatic reactions were stopped by the addition of ice-cooled aceto-nitrile, followed by centrifugation. The supernatants were analyzed by HPLC with radioactivity monitoring to determine the rate of disappearance of [³H]pimecrolimus or with UV detection to determine the rate of formation of hydroxy-midazolam.

Determination of Pharmacological Activity of Metabolite Pools by Interleukin-2 Reporter Gene Assay. [³H]Pimecrolimus (10 µM initial concentration; 0.22 MBq/ml) was incubated with human liver microsomes (1 mg microsomal protein/ml) for 5, 15, or 60 min. The microsomes and the concentrations of NADPH and of the components of the NADPH regenerating system were the same as described above for the in vitro metabolism investigations. Two pools of metabolites were isolated from each of the three incubates by preparative HPLC: a metabolite fraction 1 (highly polar metabolites) eluting between 0 and approximately 10 min and a metabolite fraction 2 (moderately polar and nonpolar metabolites but no parent drug) eluting between approximately 10 and 93 min under the chromatographic conditions of the metabolite patterns (cf. Results). Fractions 1 and 2 were evaporated and redissolved in water (fractions 1) or water/ethanol 1:1 (v/v) (fractions 2). The amounts of total [³H]pimecrolimus metabolites in the isolated fractions were determined from the total radioactivity, measured by LSC, and the specific radioactivity of the parent drug in the microsomal incubates. Small aliquots of fractions 2 were analyzed by HPLC to confirm the metabolite patterns and the absence of unchanged [³H]pimecrolimus (except for insignificant traces).

For determining the effects of the solvents and of pimecrolimus-unrelated components from the incubation medium on the interleukin-2 (IL-2) reporter gene assay, incubations of human liver microsomes without pimecrolimus were performed, which were processed identically to the pimecrolimus incubates described above. Fraction 1 and fraction 2 samples of the control incubates did not show any significant activity in the IL-2 reporter gene assay at dilutions of 1:50 and 1:200, respectively (data not shown). Furthermore, when these samples were spiked with 1 nM pimecrolimus, the pimecrolimus activity was not affected at dilutions of 1:50 of fraction 1 and 1:500 of fraction 2 (data not shown). Thus, the fraction 1 and fraction 2 samples containing pimecrolimus metabolite pools were diluted at least 1:50 and 1:500, respectively.

The IL-2 reporter gene assay was performed as described previously (Zenke et al., 2001). In brief, the luciferase gene was placed under the transcriptional control of the human IL-2 promoter and stably transfected into the human T-cell line Jurkat. IL-2 promoter-driven luciferase expression was quantified by measuring the bioluminescence upon reaction with its substrate luciferin. Its production correlated with IL-2 expression and was thus a direct measure of IL-2 transcription. Cells were stimulated with 20 ng/ml phorbol myristate acetate and 1 µg/ml phytohemagglutinin in the presence of serial dilutions of test samples in duplicates and incubated 5.5 h at 37°C. Serial dilutions (0.01–10 nM in duplicates) of unlabeled pimecrolimus (1 mM stock solution in ethanol) were performed on each assay plate, and the resulting dose-response curves were used to quantify the pimecrolimus-related pharmacological activity of the test samples. In two independent experiments, serial dilutions of the pimecrolimus metabolite pools were tested. The IC₅₀ values of pimecrolimus were between 0.6 and 1.0 nM. Pimecrolimus did not show any activity at 0.12 to 0.37 nM concentrations.

View larger version (17K): [in this window] [in a new window]   FIG. 2. Time profiles of total radiolabeled components (radioactivity) and pimecrolimus in blood of four healthy male subjects treated with a single oral dose of 15 mg of [3H]pimecrolimus.

	Results

Top Abstract Materials and Methods Results Discussion References

Blood Concentrations of Radioactivity and Pimecrolimus; Pharmacokinetic Parameters in Vivo. Both the total radiolabeled components (radioactivity) and pimecrolimus reached their maximum blood concentrations already at 1 h (median value) after the oral dose of 15 mg of [³H]pimecrolimus. After t_max, the average blood concentrations of pimecrolimus decreased rapidly to 11% of C_max at 8 h and 3% at 24 h. The average blood concentrations of radioactivity decreased more slowly to 8% of C_max at 24 h (Fig. 2; Table 1). The terminal half-life of radioactivity in blood could only roughly be estimated (approximately 145 h) and was clearly longer than that of the parent drug (62 h on average). The AUC_0- of radioactivity in blood was approximately 4 times higher than that of the parent drug.

View larger version (39K): [in this window] [in a new window]   FIG. 3. Representative metabolite patterns in vivo (A–D) after a single oral dose of 15 mg of [3H]pimecrolimus administered to a healthy male subject and in vitro (E–H) after incubation of [3H]pimecrolimus (1 µM initial concentration) with human liver microsomes (pool of 10 individuals; 50 µg/ml microsomal protein).

The small amounts of radioactivity excreted in urine (2.3 ± 0.5% of dose between 0 and 168 h; mean ± S.D.) consisted mainly of highly polar, chromatographically unretained material, a minor part of which (0.5 ± 0.2% of dose in urine collected between 0 and 168 h) was due to tritiated water (example of a radiochromatogram in Fig. 3C). No parent drug was detected in urine.

The metabolite patterns in feces were extremely complex, consisting mainly of a very broad and complex peak of unresolved metabolites centered at 55 min but extending from 20 to 90 min retention time (example in Fig. 3D). Minor amounts of parent drug were observed in the feces of three of the four subjects, and minor front-peaks were found in the feces chromatograms of all four subjects.

Only 0.8 ± 0.5% of the administered radioactivity (mean ± S.D.) was converted to tritiated water in total. The low extent of tritiated water formation demonstrated that the ³H-label was placed in a metabolically stable position.

Due to the low amounts of pimecrolimus metabolites in blood and urine, and the complexity of the radiochromatograms of the feces extracts, no attempt was made to directly characterize the structures of the metabolites in vivo. Instead, material from incubates of [³H]pimecrolimus with human liver microsomes was used to obtain structural information on the biotransformation products (see paragraph on metabolite structures).

Excretion of Radioactivity in Urine and Feces. Radioactivity was excreted predominantly with the feces (78 ± 14% of dose between 0 and 240 h; mean ± S.D.). Only 2.5 ± 0.6% of the administered radioactivity appeared in urine between 0 and 240 h (Table 3). Most of the excretion occurred between 0 and 96 h postdose (on average, 72% of dose in feces, 2.0% in urine). The recovery of radioactivity up to 240 h varied considerably among the subjects, ranging from 63 to 94% of dose. The reason for the low recovery, mainly in one subject, remains unknown but may be due to losses during feces collection.

Blood Distribution of Pimecrolimus in Vitro. The distribution of pimecrolimus between human plasma and blood cells in vitro was found to be concentration-dependent (Table 4). At low blood concentrations (1–34 ng/ml), only approximately 12% of the compound was localized in plasma. At higher concentrations, the percentage in plasma increased to approximately 70% at 1 µg/ml.

Metabolite Patterns in Incubates of [³H]Pimecrolimus with Human Liver Microsomes. The biotransformation of pimecrolimus was further investigated in vitro using human liver microsomes. At 1 µM initial substrate concentration and a concentration of microsomal protein of 50 µg/ml, the metabolite patterns shown in Fig. 3, E to H, were obtained. The patterns after short incubation times (5–10 min) closely resembled those obtained in blood in vivo between 1 and 8 h postdose. With increasing incubation times, the complexity of the patterns and the average polarity of the metabolites gradually increased until patterns like those in the feces (Fig. 3D) were obtained. Hence, metabolite patterns very similar to those found in vivo could be generated in vitro. Therefore, the structural characterization of metabolites was limited to those formed in vitro, which were much better amenable to LC-MS analysis than those in the in vivo samples due to higher concentrations and a less complex background of drug-unrelated components. The similarity of the metabolism of pimecrolimus in vitro and in vivo was further supported by data from the rat (not shown), which metabolized pimecrolimus by pathways similar to those in humans. After an oral dose of 100 mg/kg pimecrolimus, major metabolites (including Dx_1) in rat blood could be characterized mass spectrometrically and compared in detail with those formed by rat liver microsomes. The data showed a close correspondence of the in vivo and in vitro metabolism.

To obtain sufficient amounts of metabolites for LC-MS analysis, [³H]pimecrolimus was incubated additionally at an initial substrate concentration of 40 µM and with 4 mg microsomal protein/ml for 2 and 60 min. The metabolite patterns were almost identical with those obtained at 1 µM initial substrate concentration and 50 µg microsomal protein/ml, suggesting that, both qualitatively and quantitatively, the same metabolites were formed under the two incubation conditions despite potential enzyme saturation at 40 µM substrate concentration.

Metabolite Structures. The chemical structures of pimecrolimus metabolites formed in human liver microsomal incubations (described above) were investigated by LC-MS with electrospray ionization in the positive ion mode. Lithium acetate was added postcolumn to form M + Li⁺ ions. In the absence of lithium acetate, pimecrolimus formed a mixture of adduct ions, including M + Na⁺, whereas essentially no M + H⁺ ions were observed (data not shown). The mass spectrum of pimecrolimus itself (Fig. 4) showed the M + Li⁺ ion at m/z 816 together with a number of fragment ions of which fragment A, resulting from a cleavage of the macrocyclic ring at the C24,C25-bond and the O,C26-bond, predominated. The cleavages were induced by thermal activation in the heated capillary between the ion source and the mass analyzer. Very similar fragmentations were obtained by the more conventional collisional activation method (data not shown). Metabolites of pimecrolimus also formed M + Li⁺ and fragment A-type ions, which allowed to localize metabolic changes within fragment A (substructure x) or outside fragment A (substructure y; cf. Fig. 4). Cleavage products in addition to fragment A were observed in a few of the mass spectra of the metabolites. They were in agreement with the proposed partial structures described below. Further support of these structures was obtained by mass spectrometric H/D exchange experiments, which revealed the number of heteroatom-bound hydrogens.

View larger version (18K): [in this window] [in a new window]   FIG. 4. Electrospray mass spectrum of pimecrolimus (main tautomer) in the presence of lithium acetate, positive ion mode. Instrumental settings were as described under Materials and Methods.

The results of the mass spectrometric characterization of the metabolites in a 2-min incubate of [³H]pimecrolimus with human liver microsomes is shown in Fig. 5 in the form of a radiochromatogram labeled with metabolite names that reflect the type and number of metabolic changes. The legend of Fig. 5 explains the nomenclature.

View larger version (23K): [in this window] [in a new window]   FIG. 5. Metabolite pattern of [3H]pimecrolimus in an incubate with human liver microsomes. The initial substrate concentration was 40 µM, and pooled microsomes were from 10 individual livers, 4 mg microsomal protein/ml, 2 min incubation time. The information on the metabolite structures obtained by LC-MS is given in the form of metabolite names consisting of reaction codes, substructure codes, and isomer numbers. Reaction codes: D, O-demethylation; K, oxidative dechlorination; O, hydroxylation. Substructure codes: x, reaction within substructure x; y, reaction within substructure y (see Fig. 4). Isomer numbers: numbering of isomers or tautomers (irrespective of substructure codes); discontinuous numbers due to additional isomers detected in incubates with animal liver microsomes (not shown). Example: 2DxxOx_7, two O-demethylations (2D), both in substructure x; in addition, one hydroxylation (O) in substructure x; isomer (or tautomer) number 7.

The mass spectral data on the metabolites in the 60-min incubate with human liver microsomes (data not shown) suggested that the additional biodegradation products formed at this longer incubation time were simply products of consecutive biotransformations of the types found already in the 2-min incubate. Additional types of metabolic reactions were of negligible importance. The observed biotransformations, including possible tautomerizations (see Discussion), are summarized in Fig. 6.

View larger version (24K): [in this window] [in a new window]   FIG. 6. Overview of metabolic pathways and tautomerizations of pimecrolimus in humans.

Enzyme Kinetics and P450 Isoforms Responsible for Pimecrolimus Metabolism in Humans. The rate of metabolism of pimecrolimus by human liver microsomes followed apparent single-enzyme Michaelis-Menten kinetics with K_m and V_max values of 0.7 µM and 394 (pmol/min)/mg protein, respectively. From a panel of 10 P450 isoenzymes tested, CYP3A4 dominated against all other isoforms with regard to the turnover of [³H]pimecrolimus under the experimental conditions used (Fig. 7). CYP2A6, CYP2C19, CYP2D6, and CYP2E1 also metabolized pimecrolimus, although at a relatively low rate, whereas no measurable biotransformation occurred with CYP1A2, CYP2B6, CYP2C8, CYP2C9, and CYP4A11. In line with this result, ketoconazole, a well established high-affinity inhibitor probe against CYP3A isoforms in vitro (Baldwin et al., 1995), strongly inhibited the metabolism of pimecrolimus (1 µM) by human liver microsomes with an IC₅₀ of 0.25 µM. Moreover, a monoclonal antibody inhibitory to CYP3A4/5 inhibited the metabolism of pimecrolimus (1 µM) and the hydroxylation of midazolam (mediated predominantly by CYP3A4/5; Kronbach et al., 1989) to a similar extent. These data suggest that CYP3A4 and possibly CYP3A5 play a major role in the biotransformation of pimecrolimus in humans.

View larger version (15K): [in this window] [in a new window]   FIG. 7. Turnover of pimecrolimus by recombinant microsomes. [3H]Pimecrolimus (1 µM initial substrate concentration) was incubated with microsomes prepared from recombinant human B-lymphoblastoid cell lines heterologously expressing human P450 isoenzymes. The amounts of recombinant P450 in the incubates (12.5, 21.5, 5.3, 19.4, 13.5, 12.3, 6.4, 8.3, 29.1, and 17.0 pmol of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP4A11, respectively) were representative of the highest P450 concentration or marker activity found for the respective isoform in a panel of liver microsomes from 12 human individuals. Given are the amounts of [3H]pimecrolimus metabolized during the 20-min incubation time. Control, microsomes from recombinant cells containing P450 reductase only.

Pharmacological Activity of Metabolites. Pools of metabolites of pimecrolimus, isolated from incubates with human liver microsomes, were investigated for immunosuppressive activity using an IL-2 reporter gene assay. To cover the full range of biodegradation products, microsomal incubations were performed for 5, 15, and 60 min. The metabolite pattern in the 5-min incubate resembled that in Fig. 3E (and that in blood in Fig. 3A), that in the 15-min incubate resembled the pattern in Fig. 3G, and that in the 60-min incubate resembled the pattern in Fig. 3H, although without any parent drug left. The metabolites in the three incubates were isolated by HPLC in the form of two pools. Fraction 1 contained only highly polar metabolites (corresponding to those eluting between 0 and approximately 10 min in Fig. 3); fraction 2 contained the moderately polar and nonpolar metabolites (corresponding to those eluting between about 10 and 93 min in Fig. 3), but no parent drug.

The fractions 1 were devoid of immunosuppressive activity. The fractions 2 showed moderate to low activities, decreasing with increasing incubation time, i.e., with increasing polarity of the metabolites (Table 5). The highest concentration-normalized activity was found in fraction 2 of the 5-min incubate, amounting to 9% of the concentration-normalized activity of pimecrolimus.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In the present study, the absorption and disposition of pimecrolimus were investigated in healthy volunteers after a single oral dose of 15 mg. Supplementary information on the blood distribution, the biotransformation, including a characterization of the responsible enzymes, as well as the pharmacological activity of the metabolites was obtained from in vitro investigations.

Pimecrolimus was rapidly absorbed. Maximum blood concentrations of radioactivity and parent drug were reached at 1 h after dosing in three subjects and at 2 to 3 h in one subject. The small amounts of parent drug detected in feces (0.7 ± 0.9% of dose) suggest a nearly complete absorption. Even though some of the unabsorbed pimecrolimus may have undergone degradation by the intestinal microflora, this does not seem to be a significant process. Metabolism by intestinal microorganisms is expected to occur predominantly by reduction and hydrolysis (Scheline, 1973; Smith, 1978), whereas under in vitro conditions, producing metabolite patterns very similar to those in feces (Fig. 3), only products of oxidative biotransformation were identified. The absolute oral bioavailability of pimecrolimus could not be determined from the data of the present study.

At t_max of radioactivity, pimecrolimus accounted for approximately 70% of the radioactivity in blood. Thereafter, the parent drug concentrations decreased rapidly, and faster than those of the radioactivity, reflecting the increasing presence of metabolites in the circulation, predominantly highly polar ones. The terminal half-lives of parent drug (average t_1/2 62 h) and radioactivity (t_1/2 approximately 145 h) in blood were rather long.

The extent of tissue distribution of pimecrolimus was probably high inasmuch as the terminal volume of distribution divided by the bioavailability amounted to 6700 liters, on average. Within the blood compartment, the distribution of pimecrolimus between blood cells and plasma was concentration-dependent (in vitro data). At the blood concentrations found after a 15-mg oral dose, the compound resided predominantly in the blood cells (approximately 12% in plasma). However, at higher blood concentrations, the fraction in plasma increased.

The metabolism of pimecrolimus, both in vivo and in vitro, was found to be highly complex due to numerous parallel and consecutive biotransformations. The occurrence of multiple relatively stable tautomers of the metabolites further increased the complexity of the chromatograms (see below). The complexity was most obvious from the metabolite patterns in feces, showing a broad peak of chromatographically unresolved components. However, in blood, also, the number of metabolites (all minor) was too high for complete chromatographic separation. Therefore, no attempt was made to directly determine the structures of the metabolites in vivo. Instead, in vitro metabolites, formed in incubates with human liver microsomes, were investigated. The substitution of in vitro for in vivo metabolites for structural characterization is justified both by the similarity of the metabolite patterns in the two human systems (Fig. 3) and by an in vitro-in vivo comparison of the pimecrolimus metabolism in the rat based on retention times as well as mass spectra, as mentioned under Results.

For a large number of in vitro metabolites, LC-MS provided structural information (mainly partial structures) and hence information on the types and number of metabolic reactions. The metabolites of the first generation formed by human liver microsomes were mainly products of (oxidative) O-demethylations, including the most abundant metabolite Dx_1, which had undergone O-demethylation at position 13 or 15. The two possibilities could not be differentiated. It is notable that four metabolites with code Dx (Dx_1, Dx_2, Dx_3, and Dx_5), which had undergone a single O-demethylation in substructure x (see Fig. 4 and legend of Fig. 5), were detected, even though there are only two O-methyl groups in this substructure. This can be explained with the existence of relatively stable, slowly interconverting tautomers, appearing as separate chromatographic peaks, as observed for the parent drug. The phenomenon of stable tautomers has been described before for structurally related macrolides (Hughes et al., 1992; Namiki et al., 1993). Demethylations in substructure x generate hydroxy groups capable of forming additional hemiacetal functions with the tricarbonyl system, thereby increasing the number of possible tautomers, compared with those of pimecrolimus. In contrast, only one product of an O-demethylation in substructure y (Dy_6; demethylated at position 31) was observed. As additional metabolic reactions, hydroxylations and, to a minor extent, oxidative dechlorination were observed. Secondand higher-generation metabolites seemed to be formed mainly by consecutive O-demethylations and hydroxylations. Interestingly, no products of an opening of the macrocycle were found among the metabolites characterized by LC-MS. Even though a lactone hydrolysis might have been followed by a dehydration, resulting in a zero net change in molecular weight, such a sequence of biotransformations would have resulted in an additional exchangeable hydrogen, which would have been noted in the mass spectrometric H/D exchange experiments. This is in contrast to the behavior of the related macrolides rapamycin and SDZ RAD 001, which undergo prominent cleavage at the lactone group (Streit et al., 1996; Vidal et al., 1998). The front-peak, observed both in the in vitro and in vivo metabolite patterns, seems to be due to one or several highly polar metabolites of pimecrolimus which, because of their polarity, seem to contain only a small structural fragment of the parent drug molecule, including the labeled piperidine ring. Even though these components might have accounted for a considerable portion of the radioactivity in blood at late times after dosing, their quantitative importance, expressed in percentage of dose, was small (less than 4%), as judged from the metabolite patterns in the excreta.

The very small amounts of unchanged parent drug observed in the excreta (0.7% of dose on average) suggest that pimecrolimus is eliminated from the human body predominantly by oxidative metabolism. Even if biliary excretion of unchanged pimecrolimus, followed by degradation in the intestinal lumen, cannot be excluded, it seems unlikely that this was a major process, as discussed above in the context of absorption. Moreover, bile of rats treated intravenously with [³H]pimecrolimus did not contain any unchanged parent drug (data not shown). In the human subjects, radiolabeled material was excreted almost exclusively via the feces, indicating a predominance of biliary over renal excretion of pimecrolimus metabolites. A direct secretion of pimecrolimus metabolites through the intestinal wall into the intestinal lumen, as an alternative to biliary excretion, cannot be excluded but seems unlikely since, in bile duct-cannulated rats treated intravenously with [³H]pimecrolimus, 93% of the administered radio-activity was excreted in the bile, whereas only 2.4% appeared in the feces (data not shown).

The oxidative steps of pimecrolimus biotransformation are likely to be catalyzed by cytochrome P450 enzymes. Incubations with a panel of heterologously expressed single P450 isoenzymes and experiments with P450 isoenzyme-specific inhibitors revealed that CYP3A4/5 is mainly responsible for the metabolism of pimecrolimus. The broad catalytic selectivity of CYP3A4 (Guengerich, 1999) and its ability to oxidize substrates in multiple positions (Smith and Jones, 1992) may at least partially explain the complexity of the metabolism of pimecrolimus. In addition to CYP3A4/5, a few other P450 isoforms were shown to be able to metabolize [³H]pimecrolimus to some extent. The increase in exposure that an inhibition of the CYP3A pathway in vivo may provoke is therefore likely to be limited by the existence of alternative enzymes capable of metabolizing pimecrolimus. Because the metabolism of pimecrolimus by CYP3A5 was not specifically investigated, its contribution, relative to that of CYP3A4, remains unknown.

The pharmacological activity of metabolite pools was assessed by an IL-2 reporter gene assay, a very sensitive in vitro test that is relevant for the principal mode of action of pimecrolimus. Using this assay, only low activity was observed with pools of moderately polar to nonpolar metabolites, whereas pools of highly polar metabolites (front-peaks) showed no measurable activity at all. The biotransformation of pimecrolimus thus seems to be associated, in general, with a loss of pharmacological activity, an effect which increases with the degree of hydrophilicity of the metabolites. Therefore, and because of their low blood concentrations, the metabolites may contribute only to a small extent, if at all, to the therapeutic effect of orally administered pimecrolimus in patients with inflammatory skin diseases.

In conclusion, pimecrolimus is rapidly absorbed in fasting humans after a single oral dose of 15 mg. After t_max, the blood concentrations of pimecrolimus decrease rapidly, followed by a long terminal elimination phase. The compound seems to be extensively distributed into tissues. Its distribution into blood cells is high and concentration-dependent. Numerous parallel and consecutive biotransformations, together with the occurrence of multiple relatively stable tautomers, leads to complex metabolite patterns in blood and feces which could be reproduced in incubates with human liver microsomes. The main metabolic pathways are O-demethylations. CYP3A4 and possibly CYP3A5 provide the most important contribution(s) to the biotransformation of pimecrolimus. The elimination of pimecrolimus occurs almost exclusively by oxidative metabolism. The metabolites are excreted mainly via the bile into the feces. In urine, little drug-related material and no unchanged pimecrolimus is excreted. The metabolites are unlikely to contribute significantly to the pharmacological activity of the compound.

	Acknowledgments

 
We thank Dr. Jeffrey T. Smith from Simbec Research Ltd. (Principal Investigator), Dr. Pascale Burtin (Clinical Pharmacology Physician), and Dr. Jean-Baptiste Lecaillon (Bioanalytics and Pharmacokinetics Scientist) for their support of the human absorption, distribution, metabolism, and excretion study. The work by Bertrand Birlinger (in vitro enzyme identification), Regina Klünder (in vitro blood distribution), and Ulrike Strittmatter (IL-2 reporter gene assay) is gratefully acknowledged.

	Footnotes

 
Part of this work has been presented at the 50th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, FL, June 2–6, 2002.

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.105.007732.

ABBREVIATIONS: LSC, liquid scintillation counting; AUC, area under the concentration-time curve; P450, cytochrome P450; HPLC, highpressure liquid chromatography; IL-2, interleukin-2; LC-MS, liquid chromatography-mass spectrometry; TFA, trifluoroacetic acid.

Address correspondence to: Dr. Markus Zollinger, Novartis Pharma AG, WKL-135.2.21, P.O. box, CH-4002 Basel, Switzerland. E-mail: markus.zollinger{at}novartis.com

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