1,4-dioxacyclohexadecane-5,16-dione

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Objective of study:

metabolism

Test guideline

Qualifier:

no guideline followed

GLP compliance:

no

Test material

Test material information

1

Reference substance name:

1,4-dioxacyclohexadecane-5,16-dione

EC Number:

259-423-6

EC Name:

1,4-dioxacyclohexadecane-5,16-dione

Cas Number:

54982-83-1

Molecular formula:

C14H24O4

IUPAC Name:

1,4-dioxacyclohexadecane-5,16-dione

Test material form:

liquid

Radiolabelling:

no

Administration / exposure

Details on study design:

Blank Wistar Han mix-gender rat plasma with K2 EDTA as anticoagulant was obtained from BioIVT (Burgess Hill, West Sussex, UK). Blank matrices were stored in the ultralow freezer (≤ -75°C).

The LC-MS/MS conditions, preparation procedure of the solutions and sample processing method

Selectivity: The selectivity of the analytical method was evaluated by analyzing blank matrix. The chromatograms of the blank samples showed no interfering components at the retention time of the analyte (RT~1.05 min). The analytical method was found to be selective for the analyte in rat plasma.

Plasma Stability Evaluation:
- Experiment 1: Rat K2EDTA plasma will be spiked with analyte solution at 10 and 50 µg/mL and gently mixed at approximately 37°C. The spiked plasma samples will be aliquoted at multiple time points of 2, 5, 10, 20, 60, and 120 minutes and processed according to the established bioanalytical procedure. The experiment will be performed in triplicate.
- Experiment 2: Rat K2EDTA plasma will be spiked with analyte solution at 500 µg/mL and kept on ice. The spiked plasma samples will be aliquoted at multiple time points between 0 and at least 300 seconds and processed according to the established bioanalytical procedure. There will be at least 10 time points for aliquoting. The experiment will be performed in triplicate.
- Experiment 3: Rat K2EDTA plasma will be spiked with analyte solution at 100 µg/mL and gently mixed at approximately 37°C. The spiked plasma samples will be aliquoted at multiple time points of 0, 0.5, 1, 2, 4, and 6 hours and processed according to the established bioanalytical procedure. The experiment will be performed in triplicate.

Results and discussion

Main ADME results

Main ADME results 1

Type:

metabolism

Results:

Half-life Zenolide: <1% remaining after 1 minute at 0°C

Main ADME results 2

Type:

metabolism

Results:

Half-life First metabolite of Zenolide: 20 minutes at 37°C

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Experiment 1: In the preliminary results regarding the stability of Zenolide it was not possible to identify Zenolide when spiked to rat plasma at room temperature at concentration from 1-50 µg/mL as initially planned by the original study plan. It was however possible to qualify Zenolide in already extracted (plasma protein precipitated by acetonitrile that most probably is inhibiting the enzymes responsible for degradation of Zenolide) rat plasma. Zenolide was not present when spiked to plasma and consecutively processed, which indicated its instability. Thus two different experiments were set (see below).

Experiment 2: Rat K2EDTA plasma was spiked with analyte solution at 500 μg/mL and kept on ice. This experiment proved clearance of Zenolide when spiked to rat plasma and formation of assumed Zenolide metabolite (as this compound is not present in the blank matrix sample and it has the same MW as Zenolide but different retention time). The spiked plasma samples were aliquoted at multiple time points between 0 and at least 300 seconds and processed according to the established bioanalytical procedure. The chromatograms showed fast interconversion of Zenolide to its metabolite. Statistics of this experiment indicates very good reproducibility and reliability for the method and experimental design. Very fast decrease for the Zenolide concentration when spiked to plasma at 500 µg/mL on ice (peak at 1.06 min RT) was observed, and increase of the Zenolides assumed metabolite (peak at 0.74min RT). See table 1 in "Any other information on results" and the attached figure in which the intraconversion of Zenolide (red dots) to its metabolite (blue dots) is presented.

Experiment 3: This experiment was designed to evaluate clearance of the assumed metabolite of Zenolide. Rat K2 EDTA plasma was spiked with analyte solution at 100 μg/mL concentration levels and gently mixed at approximately 37°C. The spiked plasma samples were aliquoted at time points of 0, 0.5, 1, 2, 4 and 6 hours and processed according to the established bioanalytical procedure. This experiment shows clearance of the assumed Zenolides metabolite at the time slightly after 2 hours and indicates that the metabolite originates from 100% conversion of Zenolide. See table 1 in "Any other information on results" and see the attached figure in which the clearance of the metabolite of Zenolide is presented.

Any other information on results incl. tables

Table 1: Results of experiment 2: Peak area instrument response of zenolide (RT=1.06) and its metabolite (RT=0.75). Sample name annotation “t=” seconds.

	Metabolite	Zenolide	Metabolite	Zenolide	Metabolite	Zenolide
	Average response		St dev		RSD
	Metabolite	Zenolide	Metabolite	Zenolide	Metabolite	Zenolide
Plasma-Stability-H t=0	60920	172140	8677	12197	14%	7%
Plasma-Stability-H t=5	78795	152937	9766	6553	12%	4%
Plasma-Stability-H t=10	100795	136760	9816	7077	10%	5%
Plasma-Stability-H t=15	131005	115252	17489	2997	13%	3%
Plasma-Stability-H t=20	150556	91169	9070	2909	6%	3%
Plasma-Stability-H t=30	212060	52915	20055	10398	9%	20%
Plasma-Stability-H t=45	287044	10954	13415	4103	5%	37%
Plasma-Stability-H t=60	306718	2534	10181	652	3%	26%
Plasma-Stability-H t=120	315732	902	7588	121	2%	13%
Plasma-Stability-H t=300	315807	869	4933	113	2%	13%

Table 2: Results of experiment 3: Peak area instrument response of the metabolite of Zenolide (RT=0.75). Sample name annotation “t=” minutes.

	Average peak area	St dev	RSD%
Plasma-Stability-L t=0	56619	3612	6%
Plasma-Stability-L t=30	20670	2192	11%
Plasma-Stability-L t=60	6310	20	0%
Plasma-Stability-L t=120	676	64	10%
Plasma-Stability-L t=240	0	0
Plasma-Stability-L t=360	0	0

Applicant's summary and conclusion

Conclusions:

The stability of the test substance in rat K2EDTA plasma was assessed using a bioanalytical method based on liquid chromatography with tandem mass spectrometric detection (LC-MS/MS). It has been shown that zenolide is quickly degrading (in matter of seconds at 0°C) and converted to its metabolite when spiked to rat plasma. In an additional experiment the degradation of this metabolite was also followed. This metabolite has an estimated half-life of 20 minutes at 37°C.

Executive summary:

Stability of Zenolide was evaluated in rat plasma in two separate experiments in triplicate using LC-MS/MS.In the first preliminary experiment, rat plasma was spiked with 1-50 µg/ml Zenolide and incubated at 37°C and no Zenolide could be detected. Thereafter, Zenolide was also incubated with denaturated proteins (thus without carboxyl esterases breaking down the d-ester bond) and Zenolide could be detected. Further work was carried out to see if the metabolisation of Zenolide could be slowed down. In the next experiment Zenolide was incubated at 500 µg/ml on ice (0°C) and there it could be seen that Zenolide disappeared within 1 minute (<1% remaining) in plasma. In this test a metabolite appeared on the LC-MS detector. To further follow the pathway of this intermediate metabolite, another test was carried out.In this third experiment 100 µg/ml Zenolide was incubated in rat plasma at 37°C. Here it was shown that this intermediate metabolite disappeared with a half-life of 20 minutes.