4-methylmorpholine 4-oxide, monohydrate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-04-11 to 2021-01-18

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Objective of study:

hepatic clearance 

metabolism

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The aim of the study was to investigate the metabolism of the test item NMMO in primary hepatocytes from rat and human by liquid chromatography-mass spectrometry. In a first pilot study (assay 1), 3 different test item concentrations (i.e. 30, 100 and 300 μM) were tested to select an appropriate condition for the main study part. However, NMMO was highly stable during 120 min of incubation with hepatocytes from both species and no difference was observed for the different test item concentrations. Therefore, the test concentration was further recuded to 0.3 and 3 μM in a second pilot study (assay 2). Also with this conditions, the test item was poorly metabolized with rat and human hepatocytes and the sponsor decided to cancel any further testing.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-methylmorpholine 4-oxide, monohydrate

EC Number:

231-391-8

EC Name:

4-methylmorpholine 4-oxide, monohydrate

Cas Number:

7529-22-8

Molecular formula:

C5H11NO2

IUPAC Name:

4-methyl-4λ⁵-morpholin-4-one

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identity: 4-methylmorpholine 4-oxide, monohydrate, CAS 7529-22-8 (NMMO)
- Lot/batch number of test material: 12.01.2018
- Purity: 50% aqueous solution

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Working solutions of test item were diluted from the stock solutions in water, to obtain working solutions of 100-fold higher strength than the final incubation concentrations and the respective final organic solvent content. In a second step, working solutions were further diluted in incubation medium to reach 10-fold concentrated starting solutions.

Radiolabelling:

no

Test animals

Species:

other: rat and human

Administration / exposure

Route of administration:

application in vitro

Vehicle:

water

Duration and frequency of treatment / exposure:

120 min

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

300 other: µM

Doses / concentrations 2

Dose / conc.:

100 other: µM

Doses / concentrations 3

Dose / conc.:

30 other: µM

Doses / concentrations 4

Dose / conc.:

3 other: µM

Doses / concentrations 5

Dose / conc.:

0.3 other: µM

Positive control reference chemical:

7-Ethoxycoumarin (Sigma-Aldrich, E1379, Lot: MKBN6201V)

Details on study design:

- Preparation of test solutions:
The concentration of the test item stock solution in water was 4880.75 mM, while the reference item stock solution contained 10 mM 7-ethoxycoumarin in acetonitrile. The concentration of the working solutions of the test item NMMO was 30, 10, 3, 0.3 and 0.03 mM in water, while the reference item working solution was 0.5 mM 7-ethoxycoumarin in ACN. By default, working solutions of test and reference items were diluted from the stock solutions in an appropriate solvent, to obtain working solutions of 100-fold higher strength than the final incubation concentrations and the respective final organic solvent content. In a second step, working solutions were further diluted in incubation medium to reach 10-fold concentrated starting solutions.

- Preparation of calibration standards of test and reference items:
By default, working solutions of the test and reference items were prepared for each calibration level by appropriate dilution of the corresponding stock solution with the corresponding solvent by serial dilution. Calibration solutions were processed for ACN/MeOH precipitation and quantitative bioanalysis.
Calibration standards were prepared by mixing 196 μl of incubation medium (WME supplemented with 25 mM HEPES and 2 mM L-glutamine) and the corresponding working solution (4 μl). The final standard solutions of the reference item contained 1% ACN. The standard solutions of the test item contained no organic solvent.

- Sample preparation (ACN precipitation):
As internal standards (ISTDs), compounds were chosen from the Pharmacelsus pool known to be suitable for ACN precipitation. Injection volumes of all measurements were 2 µl for NMMO samples and 0.5 µl (orbitrap) and 15 µl (triple quadrupole) for 7-ethoxycoumarin samples, respectively.
Metabolic stability samples from hepatocyte incubations were stopped by addition of 200 μl ACN/MeOH containing the internal standards (1 μM Diazepam, 1 μM Griseofulvin and 10 μM Diclofenac) to 200 μl sample or calibration standards, respectively. Samples were shaken vigorously (10 seconds) and spun down (4,800 x g, room temperature, 5 minutes). The resulting supernatant was transferred to auto sampler vials and subsequently subjected to LC-MS analysis.

- Liquid chromatography – mass spectrometry (LC-MS):
For quantitative analysis of test and reference items LC-MS systems Surveyor MS Plus HPLC (Thermo Electron) HPLC system connected to a TSQ Quantum Discovery Max (Thermo Electron) triple quadrupole mass spectrometer equipped with an electrospray (ESI) or APCI interface (Thermo Fisher Scientific, USA); connected to a PC running the standard software Xcalibur 2.0.7.
LC-HRMS: Accela U-HPLC pump and an Accela Open auto sampler (Thermo Fisher Scientific, USA) connected to an Q-Exactive mass spectrometer (Orbitrap); data handling with the standard software Chromeleon 7.2 SR5 MVf.

The pump flow rate was set to 600 μl/min and the analytes were separated as follows: 7-Ethoxycoumarin using a Kinetex Phenyl-Hexyl analytical column, 2.6 μm, 50x2.1 mm (Phenomenex, Germany) or Poroshell 120, 2.7 μm, 100x3.0 mm (Agilent, Germany), and NMMO using a Poroshell EC-C18, 2.7 μm, 100x3 mm (Agilent, Germany) with a corresponding pre-column using the gradients as presented in Table 1 in Section "Any other information on materials and methods incl. tables".
For measurements of 7-Ethoxycoumarin applying the triple quadrupole technology, full scan mass spectra were acquired in the positive mode using syringe pump infusion to identify the protonated quasimolecular ions [M+H]+. Auto-tuning was carried out for maximising ion abundance followed by the identification of characteristic fragment ions using a generic parameter set: ESI ion-transfer-capillary temperature 350°C, capillary voltage 3.8 kV, collision gas 0.8 mbar argon, sheath gas, ion sweep gas and auxiliary gas pressure were 40, 2 and 10 arbitrary units. Ions with the highest S/N ratio were used to quantify the item in the selected reaction monitoring mode (SRM) and as qualifier, respectively.
For measurements of 7-Ethoxycoumarin and NMMO applying the OrbitrapTM technology with accurate mass (Q-Exactive), as MS tune file, a generic tune file was used and as a lock mass for internal mass calibration the [M+H]+ ion of the Diisooctyl phthalate (m/z 391.28429), which is ubiquitously present in the solvent system, was used. Full MS-SIM (m/z ranges are given in Table 2 in Section "Any other information on materials and methods incl. tables") analysis was applied with the mass resolution of the Orbitrap set as given in Table 2. Further analyser settings were as follows: max. trap injection time 250 ms, sheath gas 40, aux gas 10, sweep gas 2, capillary voltage 4 kV for the positive and 2.8 kV for the negative mode, capillary temperature 350°C, H-ESI heater temperature 350°C.

QUALITY CONTROL
- Bioanalysis:
For quantification the ISTD method was applied and the system calibrated using an appropriate regression as mathematical model. To improve the accuracy at low concentrations, a suitable weighting was applied. The limit of quantification (LOQ) was defined as the lowest standard concentration used for the corresponding calibration curve.
- Biological assays:
In order to demonstrate adequate enzyme activity of the cryopreserved hepatocytes during the metabolic stability assay, the positive control substrate 7-ethoxycoumarin (5 μM) was incubated for 0, 60 and 120 min and the depletion of the marker compound was monitored. Test items were classified based on their intrinsic clearance (Clint) expressed as μl/min/10E6 cells. High stability is assumed if the calculated Clint was determined to be ≤ 3.5 or 5.0 μl/min/106 cells with human or rat hepatocytes, respectively. Low metabolic stability is characterized by Clint values of ≥ 19.0 or 27.5 μl/min/10E6 cells with human or rat hepatocytes, respectively.

Details on dosing and sampling:

- Test concentration selection:
The selected test concentrations were 0.3, 3, 30, 100 and 300 μM NMMO in all test item incubations (in absence of any organic solvent) and 5 μM with a solvent content of 1% ACN in reference item incubations.
Working solutions were prepared as described above. Primary hepatocytes from rats (pooled, male) and humans (5-donor-pool) were thawed according to the instructions of the manufacturer. The incubation samples were composed of 0.2 x 106 cells/well in 225 μl incubation medium and 25 μl test item solution (3, 30, 300, 1000 or 3000 μM in incubation medium), resulting in a final concentration of 0.3, 3, 30, 100 and 300 μM without any organic solvent. 200 μl samples were taken from the suspension cultures after 0, 30, 60, 90 and 120 minutes of incubation and processed for LC-MS analysis, applying protein precipitation using 1 volume of ACN/MeOH (1:1, v/v). After centrifugation the supernatant was analyzed by LC-MS.

Positive control incubations were performed using 7-ethoxycoumarin as substrate. The incubation samples were composed of 0.2 x 106 cells/well in 225 μl incubation medium and 25 μl reference item solution (50 μM in incubation medium), resulting in a final concentration of 5 μM in presence of 1% ACN. Metabolic turnover rates were measured at 0, 60 and 120 minutes of incubation. Aliquotes were taken from the incubations for sample preparation and analysis. Hepatocyte enzyme activity was assessed in terms of 7-ethoxycoumarin turnover, i.e. loss of 7-ethoxycoumarin.
Negative controls were performed to observe non-metabolic degradation processes; i.e. test item concentrations remaining stable over the investigated time suggests that a decrease of the parent compound is mainly due to metabolism. Negative control incubations were performed in line with all experiments using incubation medium with test and reference item in absence of hepatocytes. Samples were taken from the incubations at 0 and 120 minutes and processed as described above.

Statistics:

The amount of compound in the samples was expressed as percentage of remaining compound compared to time point zero (=100%). These percentages were plotted against the corresponding time points. In vitro intrinsic clearance (CLint) and half-life (t1/2) estimates were determined using the rate of precursor disappearance and following formula, based on the well-stirred liver model.

Equation 1: t1/2= ln2/-k
t1/2 = half life [min]
k = slope from the linear regression of log [test compound] versus time plot [1/min]

Equation 2: CL int=(-k) * V * fu
CLint = in vitro intrinsic clearance [μl/min/106 cells]
t1/2 = half life [min]
k = slope from the linear regression of log [test compound] versus time plot [1/min]
V = ratio of incubation volume and cell number
fu = unbound fraction in the blood

As fu is not known for the tested compound, the calculation was performed with fu =1.
CLint was used to calculate in vivo intrinsic clearance (CLint in vivo) on the basis of Equation 3. Scaling parameters are given in Table 3 in Section "Any other information on materials and methods incl. tables".

Equation 3: CL int in vivo = CLint* w liver *cd
CLint in vivo = in vivo intrinsic clearance [ml/min/kg]
CLint = in vitro intrinsic clearance [ml/min/106 cells]
w liver = liver weight [g/kg]
cd = liver cell density [10E6 hepatocytes / g liver]

Hepatic clearance (CLhep) was calculated as follows:

Equation 4: CLhep= CLint in vivo*Q/CLint in vivo+Q
CLhep= hepatic clearance [ml/min/kg]
CLint in vivo = in vivo intrinsic clearance [ml/min/kg]
Q = blood flow [ml/min/kg]

Results and discussion

Main ADME results

Main ADME results 1

Type:

metabolism

Results:

NMMO was highly stable during 120 min of incubation with pimary hepatocytes from rat and human, resulting in 98.9 to 108.4% or 102.6 to 112.8% remaining parental compound for rat and human hepatocytes, respectively.

Main ADME results 2

Type:

metabolism

Results:

Positive control 7-ethoxycoumarin showed half-lives of 13.3 and 19.3 min, (CLint in vitro 65.2 and 44.9 μl/min/10E6cells) in human hepatocytes and half-lives of 81.4 and 79.9 minutes (CLint in vitro 10.7 and 10.9 μl/min/10E6 cells) in rat hepatocytes.

Any other information on results incl. tables

Table 4: Stability of test item (0.3, 3, 30, 100 and 300 μM) with cryopreserved primary rat hepatocytes and William’s Medium E (n=1)

Time [± 1 min]	Rat hepatocytes (male), % remaining compound
	0.3 μM	3 μM	30 μM	100 μM	300 μM
0	100.0	100.0	100.0	100.0	100.0
30	109.7	102.8	106.8	108.8	105.0
60	110.0	96.4	103.4	105.0	103.2
90	104.3	106.7	110.7	110.5	108.1
120	108.4	104.4	106.3	108.1	98.9

Table 5: Stability of test item (0.3, 3, 30, 100 and 300 μM) with cryopreserved primary human hepatocytes and William’s Medium E (n=1)

Time [± 1 min]	Human hepatocytes (5-donors, mixed gender), % remaining compound
	0.3 μM	3 μM	30 μM	100 μM	300 μM
0	100.0	100.0	100.0	100.0	100.0
30	104.2	93.6	108.8	106.2	100.0
60	101.7	95.7	112.6	105.7	108.0
90	107.2	96.2	108.2	115.0	103.3
120	105.9	102.6	110.9	112.8	109.4

Applicant's summary and conclusion

Conclusions:

The test item 4-methylmorpholine 4-oxide, monohydrate (NMMO) was tested for its metabolic stability at different concentrations (i.e. 0.3, 3, 30, 100 and 300 μM) with primary hepatocytes from rat and human. Independent on the test concentration, NMMO was highly stable within 120 min of incubation, resulting in 98.9 to 108.4% or 102.6 to 112.8% remaining parental compound for rat and human hepatocytes, respectively.

Executive summary:

In a non-guideline metabolic stability study, the test item 4-methylmorpholine 4-oxide, monohydrate (NMMO), was incubated with primary hepatocytes from male rats and primary human hepatocytes from 5 mixed gender donors in vitro at concentrations of 0.3, 3, 30, 100 and 300 μM. Samples were taken after 0, 30, 60, 90 and 120 min and analysed by liquid chromatography-mass spectrometry (LC-MS).

Independent of the test concentration, NMMO was highly stable within 120 min of incubation, resulting in 98.9 to 108.4% or 102.6 to 112.8% remaining parental compound for rat and human hepatocytes, respectively. Due to the high stabililty, clearance and half-life calculation was not applicable. Based on these results, the hepatocyte-specific metabolism of NMMO is predicted to be low, inter-species differences between rat and human were not observed.

Incubation of hepatocytes from all test species with the positive control 7-ethoxycoumarin confirmed the high metabolic activity of the primary cell model and were in accordance with historical laboratory control data.

This metabolism study in rat and human hepatocytes is classified acceptable for the analysis of metabolic stability of NMMO.