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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: acceptable, well-documented publication which meets basic scientific principles, tested substance was methylamine hydrochloride.

Data source

Reference
Reference Type:
publication
Title:
Further studies on the metabolism of methylamine by semicarbazide-sensitive amine oxidase activities in human plasma, umbilical artery and rat aorta
Author:
Lyles, G.A., Holt, A. and Marshall, C.M.S.
Year:
1990
Bibliographic source:
J.Phar. Pharmacol. 1990, 42: 322-338

Materials and methods

Objective of study:
metabolism
Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
no
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
[14C]methylamine hydrochloride (55 µCi/µmol) and [7-14C]benzylamine hydrochloride (51 µCi/µmol). Unlabelled amine was added to give 100 mM stock solutions at final specific activities of [ (MA) and 0-5 µCi /µmol (BZ) which were stored frozen between experiments.
BZ served as a competitive inhibitor of an enzym, which metabolizes both BZ and MA.
Radiolabelling:
yes

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
The animals were obtained from the Departmental breeding colony, Animal Services Unit, University of Dundee.
The animals served only as a source of aorta. The metabolism of MA was studies in the homogenates of the aorta.

Human plasma and umbilical artery were also used for studying of the MA metabolism. Human umbilical cords were supplied by the Maternity Unit, Ninewells Hospital, usually from deliveries involving Caesarean sections.

For studies on human plasma, blood (approx. 20 mL) was cotlected by antecubital venepuncture from four healthy male volunteers (age range 22-38 years) into glass tubes without anticoagulant, allowed to clot and then centrifuged (1700 g for 10 min). The serum was removed, divided into several smaller portions and stored as above for subsequent use in experiments.

Administration / exposure

Route of administration:
other: added to blood vessels homogenates
Vehicle:
not specified
Duration and frequency of treatment / exposure:
Colorimetric amine oxidase assay: incubations for 30 min of [14C]MA hydrochloride with the homogenates prepared above.
Radiochemical amine oxidase assay: In some inhibitor studies, samples were preincubated with inhibitors for 20 min at 37°C before further ice-cooling and addition of substrate. Samples were then incubated at 37°C for either 30 min (umbilical artery), 60 min (aorta) or 120 min (plasma).
Doses / concentrations
Remarks:
Doses / Concentrations:
Radiochemical amine oxidase assay: Assays, in triplicates, (25 µL enzyme source, 25 µL distilled water (or aqueous inhibitor solution, in inhibitor studies) and 50 µL appropriate [l4C]MA concentration (prepared in 0-2 M potassium phosphate buffer pH 7-8)).
Dialysis studies: 50 µL 2mM of [14C]MA
No. of animals per sex per dose:
not applicable
Positive control:
Assays performed without competitive inhibitors of MA metabolism.
Competitive inhibitors used: clorgyline and semicarbazide
Details on study design:
The following tests were performed:
1. Comparison of MA metabolism determined by colorimetric and radiochemical assay.
2. Effects of clorgyline and semicarbazide upon MA metabolism in rat aorta, human umbilical artery and plasma.
3. The determination of kinetic constants for MA metabolism in rat aorta, human umbilical artery and plasma.
4. Inhibition of [14C]MA metabolism in human umbilical artery and plasma by unlabelled BZ.
5. Kinetic constants for (14C]BZ metabolism in human umbilical artery and product formation from mixtures of[14C]BZ and [14C]MA.
6. Effects of β-aminopropionitrile (BAPN) upon MA metabolism in human umbilical artery
Details on dosing and sampling:
1. Comparison of MA metabolism determined by colorimetric and radiochemical assay: Three homogenates from different umbilical arteries were assayed concurrently by the two methods at a final MA concentration of 1 mM.
2. Effects of clorgyline and semicarbazide upon MA metabolism in rat aorta, human umbilical artery and plasma: Tissue homogenates or plasma samples were preincubated with inhibitor concentrations from 0.1 µM to 1 mM, and remaining enzyme activity was determined with 1 mM [14C]MA as substrate in the ion exchange assay, and compared with activity in control samples preincubated without inhibitor.
3. Kinetic constants for MA metabolism in rat aorta, human umbilical artery and plasma: The metabolism of [14C]MA was measured at final assay concentrations of 0.05-1 mM in rat aorta, and 0.1-2 mM in human umbilical artery homogenates.
4. Inhibition of [14C]MA metabolism in human umbilical artery and plasma by unlabelled BZ: Assays of [14C]MA metabolism (0.2-2 mM in umbilical artery, 0.1-1 mM in plasma) were carried out in the presence of unlabelled BZ at final assay concentrations of 100, 200 or 400 µM.
5. Kinetic constants for (14C]BZ metabolism in human umbilical artery and product formation from mixtures of [14C]BZ and [14C]MA: the [14C] metabolite formation from 200 µM [14C]BZ and 800 µM [14C]MA, these substrates being used both individually and also as a mixture with each homogenate tested.
6. Effects of β-aminopropionitrile (BAPN) upon MA metabolism in human umbilical artery: the effects of 100, 200 and 500 µM BAPN upon the deamination of 0-2-2 mM MA.
Statistics:
not reported

Results and discussion

Preliminary studies:
listed in the original paper
Main ADME results
Type:
metabolism
Results:
MA is a substrate for the semicarbazide-sensitive amine oxidase (SSAO)

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
Vt was determined. Vt=product formation from a mixture of the two substrates (A and B). Specific inhibition of MMA-metabolism by semicarbazide. These results suggest that MA is predominantly, if not exclusively, a substrate for the soluble amine oxidase in human plasma.

Any other information on results incl. tables

1.Comparison of MA metabolism determined by colorimetric and radiochemical assay:

Mean (± S.E.) specific enzyme activities for the group were 281 ± 32 nmol H2O2 (mg protein)/1 h for the colorimetric assay and 366 ± 31 nmol MA metabolized (mg protein)/1 h for the radiochemical assay.

2.Effects of clorgyline and semicarbazide upon MA metabolism in rat aorta, human umbilical artery and plasma:

The deamination of both MA and BZ was largely resistant to inhibition by clorgyline concentrations up to 1 mM, whereas similar concentrations of semicarbazide produced a progressive degree of inhibition which resulted in virtually complete inhibition of MA metabolism, and around 80% inhibition of BZ metabolism at 1 mM semicarbazide. These results suggest that MA is predominantly, if not exclusively, a substrate for the soluble amine oxidase in human plasma.

3. Kinetic constants for MA metabolism in rat aorta, human umbilical artery and plasma

Mean values (± S.E.) from four different samples were: Km(µM) of 516 ± 74 (MA) and 225 ± 36 (BZ): Vmax (nmol (mL serum)/1 h) of 48 ± 5 (MA) and 28 ± 3(BZ).

4. Inhibition of [14C]MA metabolism in human umbilical artery and plasma by unlabelled BZ

BZ was found to be a competitive inhibitor of MA metabolism. Mean values (± S.E.) for Ki (µM) from experiments with different samples of each enzyme source were 220 ±10 (umbilical artery, n=3) and 172 ± 27 (plasma, n=4).

5. Kinetic constants for (14C]BZ metabolism in human umbilical artery and product formation from mixtures of[14C]BZ and [14C]MA

200 µM [14C]BZ and 800 µM [14C]MA were cocultivated with homogenates of human umbilical artery. The substrate concentrations were chosen to approximate closely to their Km values-found in the earlier work reported in the original paper. If a single enzyme metabolizes two different substrates at the same catalytic site, the product formation calculated (for detailed calculations see original paper) should be comparable with that experimentally determined. Table 1 shows the results obtained in this experiment:

Table 1. Comparison of predicted and actual metabolite formation in mixtures of [14C]BZ and [14C]MA.

Expt. no.

vA

vB

Predicted vT

Actual vT

Ratio (Actual vT/ Predicted vT)

d min-1

d min-1

d min-1

d min-1

 

1

623

3526

2832

2743

0.97

2

577

3460

2757

2822

1.02

3

858

5401

4274

4759

1.11

4

721

4481

3552

3681

1.04

 

 

 

 

Mean ratio= 1.04 ± 0.03

vAand vBrepresent [14C]metabolite formation (in d min-1) from 200 µMBZ and 800 µMMA, respectively, in assays containing the appropriate amine individually. The predicted metabolite formation (vT) from a mixture of the two amines if metabolized by the same enzyme was determined as described in the text, and compared with actual experimentally determined values. Experiments were carried out on 4 different arteries, assays on each artery involving concurrent determinations (in triplicate) of BZ and MA metabolism alone, and in a mixture.

 

6. Effects of β-aminopropionitrile (BAPN) upon MA metabolism in human umbilical artery

BAPN was found to be a competitive inhibitor of MA metabolism

Applicant's summary and conclusion

Conclusions:
SSAO metabolizes MA.
The vasculature and plasma contain semicarbazide-sensitive amine oxidase (SSAO) which metabolizes MA
Executive summary:

Lyles et al. investigated in 1990 the metabolism of methylamine hydrochloride in vitro. An ion exchange radiochemical assay has been developed to study the deamination of [14C]methylamine (MA) in homogenates of rat aorta and human umbilical artery, as well as in samples of human plasma. MA metabolism was found to be inhibited almost completely by 1 mM semicarbazide, but virtually unaffected by 0-1 mM clorgyline, suggesting that MA is a substrate for the semicarbazide-sensitive amine oxidase (SSAO) activities which also metabolize benzylamine (BZ) in these sources. Mean Km values for MA metabolism by aorta, umbilical artery and plasma were 182, 832 and 516 µM, respectively, with corresponding Vmax values in aorta and umbilical artery of 100 and 590 nmol /(mg prot.) h, and in plasma of 48 nmol /(mL serum) h. Kinetic constants determined for [14C]BZ metabolism in plasma (by an organic solvent extraction assay) and in umbilical artery (by the ion exchange assay) yielded mean Km values of 225 µM (plasma), 222 µM (umbilical artery), and Vmax values of 28 nmol (mL serum)/ 1 h (plasma) and 377 nmol /(mg prot.) h (umbilical artery). The deamination of [14C]MA was inhibited competitively by unlabelled BZ, with Ki values in umbilical artery and plasma of 220 and 172 µM, respectively. Also, metabolite formation from mixtures of [14C]BZ (200 µM) and [14C]MA (800 µM) was extremely close to that predicted for a single enzyme capable of metabolizing two alternative substrates in a competitive fashion. β-Aminopropionitrile was found to be a reversible, competitive inhibitor (Ki of 165 µM) of [14C]MA metabolism in umbilical artery, inhibitory properties characteristic of those found previously for the effects of β-aminopropionitrile upon BZ-metaboIizing SSAO activities in other tissues.