Non-Linear Pharmacokinetics of Oral Roscovitine (Seliciclib) in Cystic Fibrosis Patients Chronically Infected with Pseudomonas aeruginosa: A Study on Population Pharmacokinetics with Monte Carlo Simulations

Roscovitine (Seliciclib), a new protein kinase inhibitor, was administered orally to adult patients with cystic fibrosis for the first time in the ROSCO-CF trial, a dose-escalation, phase IIa, randomized, controlled trial. Extensive pharmacokinetic sampling was performed up to 12 h after the first oral dose. Roscovitine and its main metabolite M3 were quantified by liquid chromatography coupled with tandem mass spectrometry. The pharmacokinetics analyses were performed by non-linear mixed effects modelling. Monte Carlo simulations were performed to assess the impact of dose on the pharmacokinetics of oral roscovitine. Twenty-three patients received oral doses ranging from 200 to 800 mg of roscovitine and 138 data points were available for both roscovitine and M3 concentrations. The pharmacokinetics was best described by a two-compartment parent-metabolite model, with a complex saturable absorption process modelled as the sum of Gaussian inverse density functions. The Monte Carlo simulations showed a dose-dependent and saturable first-pass effect leading to pre-systemic formation of M3. The treatment with proton-pump inhibitors reduced the rate of absorption of oral roscovitine. The pharmacokinetics of oral roscovitine in adult patients with cystic fibrosis was non-linear and showed significant inter-individual variability. A repeat-dose study will be required to assess the inter-occasional variability of its pharmacokinetics.

The solutions were labeled and stored according to the procedures in force at the Test Facility.

Assay method
Calibration standards and QC samples were prepared from two different stock solutions.

Preparation of (R)-Roscovitine stock solution
An amount of (R)-Roscovitine, batch No. N0-MRT0-200-3-16 (5.05 ± 0.02 mg), determined taking into account the total correction factor (1.01), was accurately weighed and dissolved into a 5 mL class A volumetric flask by hand shaking in acetonitrile. If necessary stock solutions was sonicated. The volume was adjusted to 5 mL with acetonitrile giving a parent stock solution at 1000 ng/µL.
The stock solution was stored in amber-15mL flask at 5±4°C and stable for at least 26 days.

Preparation of M3 stock solution
An amount of M3, batch No. N0-M3 (5.25±0.02 mg), determined taking into account the total correction factor (1.05), was accurately weighed and dissolved into a 5 mL class A volumetric flask by hand shaking in acetonitrile. If necessary stock solutions was sonicated. The volume was adjusted to 5 mL with acetonitrile giving a parent stock solution at 1000 ng/µL.
The stock solution was stored in an amber-15mL flask at 5±4°C and stable for at least 26 days.

Preparation of the standard working solutions
The standard working solutions were prepared daily from stock solutions. An amount of (R)-Roscovitine-D6, batch No. N0-RoscoD6 (5.15 ±0.01mg), determined taking into account the total correction factor (1.03), was accurately weighed and dissolved into a 5.00 mL class A volumetric flask by hand shaking in acetonitrile. The volume was adjusted to 5.00 mL with acetonitrile giving a parent stock solution at 1000 ng/µL.

Concentration
The stock solution was stored in an amber-15mL flask at 5±4°C and stable for at least 26 days. An amount of M3-D6, batch No. N0-M3D6 (5.30 ±0.02mg), determined taking into account the total correction factor (1.06), was accurately weighed and dissolved into a 5.00 mL class A volumetric flask by hand shaking in acetonitrile. The volume was adjusted to 5.00 mL with acetonitrile giving a parent stock solution at 1000 ng/µL.
The stock solution was stored in an amber-15mL flask tube at 5±4°C and stable for at least 26 days.

Preparation of internal standard working solution
The stock solutions were diluted daily in acetonitrile in order to obtain a final concentration at 1.00 ng/µL.

Preparation of pH3 buffer
pH 3 buffer was prepared by dissolving around 385 mg of ammonium acetate and diluting 9.28 mL of acetic acid (>99%) with bidistilled water in a 1 L class A volumetric flask. The pH was measured at each buffer preparation.

Calibration standards
A hundred µL of blank matrix were spiked with 5.00 µL of working solution. This preparation, made daily, is detailed in the following

Sample preparation
Calibration standards and QC samples were prepared daily. Samples were left to thaw at room temperature for approximately 30 minutes. They were shaken and if necessary, they were centrifuged during three minutes at 4000 rpm (2890g) before sampling.
A portion of each sample (100 µL) was transferred into a microtube. Ten 10.0 µL of the internal standard working solution were added. Samples were then diluted to obtain a constant volume: 5.00 µL of acetonitrile were added to samples and blank matrix spiked with IS, 15.0 µL of acetonitrile were added to blank sample. Samples were then prepared by deproteinization. 500 µL of acetonitrile were added. The tubes were shaken and were centrifuged at 13000 rpm (16000 g) for ten minutes at 4°C. The supernatant was transferred into a glass tube and evaporated under a stream of pure nitrogen at approximately 40°C during 15 minutes. The residue was dissolved in 400 µL of injection solvent and transferred into polypropylene vials. Before injection, vials were centrifuged 3 min at 4000 rpm (2890g).
Chromatography conditions

Mass spectrometry conditions
The compounds were ionized using turbo V ionization source and detected using the multiple reaction monitoring (MRM) scan type.
Nitrogen was used as nebulizer gas and for collisionally activated dissociation in the collision cell. Resolution Q1: unit Resolution Q3: unit The first presented MS/MS transitions for analyte and IS were used for the quantification.

Data acquisition and calculations
HPLC system, data acquisition, data representation and post-acquisition quantitative analysis were carried out with the LC-MS/MS system manager and integration software of AB Sciex: Analyst TM.1.6