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Reference
Endpoint:
basic toxicokinetics, other
Remarks:
In Vitro ADME Assessment
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Objective of study:
absorption
distribution
excretion
metabolism
toxicokinetics
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Version / remarks:
The work is exemplified in OECD 417 (22nd July 2010) Guideline for the
testing of chemicals, Toxicokinetics, Supplemental approaches, use of in vitro information
(paragraphs 59-61), and use of toxicokinetic modelling (paragraph 65).
Principles of method if other than guideline:
Assess the in vivo pharmacokinetic behaviour of the test item via an in vitro to in vivo extrapolation (IVIVE) to help put into context the findings of toxicology studies carried out. In vitro microsomal metabolic stability, plasma protein binding and Caco2 permeability were
determined by Cyprotex Discovery Ltd., Macclesfield, UK and the data used in the physiologically based pharmacokinetic (PBPK) model, Cloe PKTM, to simulate the predicted in vivo exposure in male and female rat at 5, 550 and 2000 mg/kg.
GLP compliance:
no
Radiolabelling:
no
Species:
other: Pooled male Wistar rat and pooled female Wistar rat liver microsomes
Strain:
Wistar
Sex:
male/female
Route of administration:
other: incubation
Vehicle:
unchanged (no vehicle)
Details on exposure:
All incubations were performed in 96 well plates on a TAP baseplate robotic platform.
Microsomes (final protein concentration 0.5 mg/mL), 0.1 M phosphate buffer pH 7.4 and CA4920 (final substrate concentration 3 μM; final DMSO concentration 0.25%) were preincubated at 37°C prior to the addition of NADPH (final concentration 1 mM) to initiate the reaction. The final incubation volume was 50 μL. A control incubation was included where 0.1 M phosphate buffer pH 7.4 was added instead of NADPH (minus NADPH). Two control compounds with predefined acceptance criteria (diazepam and diphenhydramine) were also included. All incubations were performed singularly for the test item and control compounds.

Duration and frequency of treatment / exposure:
Each compound was incubated for 0, 5, 15, 30 and 45 minutes. The control (minus NADPH) was incubated for 45 minutes only. The reactions were stopped by the removal of 20 μL into 60 μL methanol containing internal standard (metoprolol) at the appropriate time points. The incubation plates were centrifuged at 2500 rpm for 20 min at 4 °C to precipitate the protein and the supernatants analysed for test compound concentration by LC-MS/MS.
No. of animals per sex per dose:
not applicable
Positive control:
not applicable
Details on study design:
Microsomal intrinsic clearance:
Pooled male Wistar rat and pooled female Wistar rat liver microsomes were purchased from In Vitro technologies Inc, Belgium.
All incubations were performed in 96 well plates on a TAP baseplate robotic platform. Microsomes (final protein concentration 0.5 mg/mL), 0.1 M phosphate buffer pH 7.4 and the test item (final substrate concentration 3 μM; final DMSO concentration 0.25%) were preincubated at 37°C prior to the addition of NADPH (final concentration 1 mM) to initiate the reaction. The final incubation volume was 50 μL. A control incubation was included where 0.1 M phosphate buffer pH 7.4 was added instead of NADPH (minus NADPH). Two control compounds with predefined acceptance criteria (diazepam and diphenhydramine) were also included. All incubations were performed singularly for CA4920 and control compounds. Each compound was incubated for 0, 5, 15, 30 and 45 minutes. The control (minus NADPH) was incubated for 45 minutes only. The reactions were stopped by the removal of 20 μL into 60 μL methanol containing internal standard (metoprolol) at the app opriate time points. The incubation plates were centrifuged at 2500 rpm for 20 min at 4 °C to precipitate the protein and the supernatants analysed for test compound concentration by LC-MS/MS.
Plasma protein binding
Separate solutions of the test item (3 μM, 0.5 % final DMSO concentration) were prepared in phosphate buffered saline (pH 7.4) and 50 % rat plasma (v/v in buffer). The experiment was performed using equilibrium dialysis with the two compartments separated by a semi-permeable membrane. The buffer solution was added to one side of the membrane and the plasma solution to the other side. All incubations were performed in a 96-well plate on a TAP baseplate robotic platform. After equilibration overnight at 37 °C, samples were taken from both sides of the membrane. Analytical standards were prepared in plasma (0.6 – 6 μM) and buffer (0.06 - 4.5 μM) and incubated at 37 °C overnight. CA4920 incubations were performed in duplicate. Warfarin was included as a control compound with predefined acceptance criteria and was performed in duplicate. Protein was precipitated in samples and standards by addition of methanol containing internal standard (metoprolol). Samples were centrifuged (2500 rpm at 4 ºC for 30 minutes) and diluted with water for analysis by LC-MS/MS. Recovery was calculated by ((final conc buffer+final conc plasma)/(initial conc buffer + initial conc plasma))*100.
Caco2 permeability
Caco-2 cells obtained from the American Tissue Culture Collection (ATCC) were used between passage numbers 40 - 60. Cells were seeded on to Millipore Multiscreen Caco-2 plates at 1 x 105 cells/cm2. They were cultured for 20 days in Dubelccos modified essential media (DMEM) and media was changed every two or three days. On day 20 the permeability study was performed. Hanks Balanced Salt Solution (HBSS) pH 7.4 buffer with 1% BSA, 25 mM HEPES and 4.45 mM glucose at 37 °C was used as the medium in the permeability study. Incubations were carried out in an atmosphere of 5% CO2 with a relative humidity of 95% at 37 °C. All incubations were performed in a CO2 incubator with dosing and sampling performed in 96- well plates on a TAP baseplate robotic platform.
On day 20, the monolayers were prepared by rinsing both basolateral and apical surfaces twice with HBSS at 37 oC. Cells were then incubated with HBSS i both apical and basolateral compartments for 40 minutes to stabilise physiological parameters. HBSS was then removed from the apical compartment and replaced with the test item dosing solutions. The solutions were made by diluting 10 mM DMSO concentrates with HBSS to give a final test item concentration of 5 μM (final DMSO concentration 1%). The fluorescent integrity marker lucifer yellow was also included in the dosing solution. The apical compartment inserts were then placed into ‘companion’ plates containing fresh HBSS. At 120 minutes the companion plate was removed and apical and basolateral samples diluted for analysis by LC-MS/MS (Section 3.3). The test item permeability was assessed in duplicate. Atenolol, propranolol and talinolol were run as controls with predefined acceptance criteria in duplicate. Analytical standards were made from dosing solutions. Standards (1 – 1250 nM) and samples are diluted with methanol containing internal standard (metoprolol) and water for analysis by LC-MS/MS.

Cloe PKTM - PK simulation
Cloe PKTM (version 2.1.6) is a software system encompassing a collection of physiologically based pharmacokinetic (PBPK) prediction models. It predicts the likely exposure of compounds in the rat following intravenous or oral administration. It uses basic compound information to generate plasma and tissue concentration-time profiles together with pharmacokinetic summary parameters which are derived from these data (reference 1, 2, 3).
For the purposes of simulating systemic exposure of the test item, microsomal intrinsic clearance, fup and Caco-2 permeability were derived as described above. Physical chemistry parameters (log P, pKa and water solubility) were determined by Syngenta and provided to Cyprotex. Intravenous and oral doses of 5, 550 and 2000 mg/kg were simulated. These doses were selected for consistency with the toxicology studies being performed with this compound.
Preliminary studies:
not applicable
Type:
absorption
Results:
In vitro data indicates high permeability across the intestinal wall would be expected. Test item would be expected to be similar to the control compound propranolol which has a predicted in vivo absorption in humans of 90%
Type:
distribution
Results:
The free fraction in plasma was approximately 3% in both male and female rats and the predicted volume of distribution at steady-state was 2 L/kg indicating that the test item would be expected to distribute into tissues.
Type:
metabolism
Results:
Major clearance mechanism for the test item in the model is via metabolism.
Type:
excretion
Results:
predicted a low percentage renal excretion of test item (0.002 - 0.044% in males, 0.011 - 0.176% in females) being higher in females due to a lower fraction metabolised.
Details on absorption:
In vitro data indicates high permeability across the intestinal wall would be expected. The test item would be expected to be similar to the control compound propranolol which has a predicted in vivo absorption in humans of 90%. In combination with a water solubility of 0.0016 mg/mL and Log P of 4.1 the Cloe PKTM model predicted a high fraction absorbed across the gastrointestinal tract following a dose of 5 mg/kg of 98% decreasing to 18% at 550 mg/kg and 7% at 2000 mg/kg.
Details on distribution in tissues:
The free fraction in plasma was approximately 3% in both male and female rats and the predicted volume of distribution at steady-state was 2 L/kg indicating that the test item would be expected to distribute into tissues.
Predicted tissue distribution was similar at all doses in males and females. Tissues showing higher predicted concentrations than plasma were adipose (135 - 225-fold), skin (~2.4-fold) brain, gonads, intestines, pancreas and stomach (~1.4 fold). Liver had a liver:plasma concentration ratio of 0.17 in males and 0.35 in females. All other tissues had concentrations below to that in plasma. The predicted blood to plasma ratio was 0.7 suggesting there would be very some difference in plasma and blood concentrations.
Note: these ratios were predicted ratios calculated at the end of the time course (i.e. 43 h following 5 mg/kg, 168 h at 550 mg/kg and 168 h at 2000 mg/kg).
Details on excretion:
Cloe PKTM predicted a low percentage renal excretion of test item (0.002 - 0.044% in males, 0.011 - 0.176% in females) being higher in females due to a lower fraction metabolised. The intrinsic clearance of the test item in male microsomes was high with a major proportion being attributed to CYP-mediated metabolism.
Therefore, the major clearance mechanism for the test item in the model is via metabolism. Following a dose of 5 mg/kg approximately 100% metabolism would be anticipated over the time course of exposure in both males and females. The percentage metabolism decreases at higher doses due to the reduced % absorbed at these doses.
Metabolites identified:
not measured
Bioaccessibility testing results:
Predicted oral bioavailability was 1 - 22% in male rats (high to low dose) despite a high fraction absorbed at the low dose. A high metabolic clearance resulted in predicted first-pass
metabolism, thereby reducing the systemic exposure to the test item.
Predicted oral bioavailability in female rats was 4 - 59% (high to low dose) being higher than males as metabolic intrinsic clearance was lower in females.
Conclusions:
Based upon an in vitro to in vivo extrapolation utilising recognised, established and validated in vitro systems carried out by Cyprotex (Macclesfield, UK) together with their commercially accepted physiologically based pharmacokinetic model (Cloe PKTM), the test item, when administered in vivo to male and female rats, is predicted to have a high fraction absorbed across the gastrointestinal tract following a dose of 5 mg/kg (approx. 100%) reducing to 7% following a dose of 2000 mg/kg. However, due to a hepatic first pass effect following oral administration this translates to a systemic oral bioavailability of 1 - 22% in male rats over this dose range. Due to metabolic intrinsic clearance being lower in female compared to male rats oral bioavailability in female rats was predicted to range from 4 - 59%.
In general in vivo, the test item would be expected to be a low clearance compound that distributed into tissues with a mean residence time in the body of around 3 hours.
Executive summary:

The study was designed to assess the in vivo pharmacokinetic behaviour of the test item via an in vitro to in vivo extrapolation (IVIVE) to help put into context the findings of toxicology studies carried out separately to this study.

In vitro microsomal metabolic stability, plasma protein binding and Caco2 permeability were determined by Cyprotex Discovery Ltd., Macclesfield, UK and the data used in the physiologically based pharmacokinetic (PBPK) model, Cloe PKTM, to simulate the predicted in vivo exposure in male and female rat at 5, 550 and 2000 mg/kg. Physical chemistry data including solubility, logP and pKa was provided by Syngenta for the purposes of modelling and simulation.

The in vitro CLint of the test item in male Wistar rat microsomes was 247 ± 3.78 μL/min/mg protein. This was characterised by >99% depletion over 45 minutes. The in vitro CLint in female Wistar rat microsomes was 26.1 ± 2.69 μL/min/mg protein characterised by 44% depletion over 45 minutes. The fraction of test item unbound in plasma (fup) was 0.0292 ± 0.0000612 in male plasma and in female plasma was 0.030 ± 0.0143. The recovery of test item in the incubations was 80 and 75%, respectively. The apparent permeability (Papp) of the test item across Caco-2 cell monolayers was 29.6 x 10-6 cms-1 ± 2.06 x 10-6. The recovery of test item from the incubation media was 84%.

The predicted median pharmacokinetic parameters for the test item using Cloe PKTM simulating oral administration at doses of 5, 550 and 2000 mg/kg in male and female rats was also determined:

The test item, when administered in vivo to male and female rats, is predicted to have a high fraction absorbed across the gastrointestinal tract following a dose of 5 mg/kg (approx. 98%) reducing to 7% following a dose of 2000 mg/kg

Description of key information

Based upon an in vitro to in vivo extrapolation utilising recognised, established and validated in vitro systems carried out by Cyprotex Discovery Ltd. together with their commercially accepted physiologically based pharmacokinetic model (Cloe PKTM), the test item, when administered in vivo to male and female rats, is predicted to have a high fraction absorbed across the gastrointestinal tract following a dose of 5 mg/kg (approx. 98%) reducing to 7% following a dose of 2000 mg/kg. However, due to a hepatic first pass effect following oral administration this translates to a systemic oral bioavailability of 22 - 1% in male rats over this dose range. Due to metabolic intrinsic clearance being lower in female compared to male rats oral bioavailability in female rats was predicted to range from 59 – 4%. In general in vivo the test item would be expected to be a low clearance compound that distributed into tissues with a mean residence time in the body of around 3 hours.

Key value for chemical safety assessment

Bioaccumulation potential:
low bioaccumulation potential
Absorption rate - oral (%):
60

Additional information