Reaction mass of 9-icosyl-9-phosphabicyclo[3.3.1]nonane and 9-icosyl-9-phosphabicyclo[4.2.1]nonane

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Meets generally accepted scientific standards with acceptable restrictions

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1979

Materials and methods

Objective of study:

metabolism

Test guideline

Qualifier:

according to guideline

Guideline:

other: No data

Principles of method if other than guideline:

Six female rats were given single oral doses of [14C] RM-17 in corn oil.

Urine and faeces were collected daily. After radioassay of the individual samples the daily urine samples were combined and daily faecal samples were combined.

The metabolites of RM-17 in the urine and faeces were separated by chromatographic techniques. The major metabolites were isolated and quantified by physicochemical techniques.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 9-icosyl-9-phosphabicyclo[3.3.1]nonane and 9-icosyl-9-phosphabicyclo[4.2.1]nonane

EC Number:

915-206-4

Molecular formula:

C28H55P

IUPAC Name:

Reaction mass of 9-icosyl-9-phosphabicyclo[3.3.1]nonane and 9-icosyl-9-phosphabicyclo[4.2.1]nonane

Test material form:

not specified

Details on test material:

- Name of test material (as cited in study report): [14C] RM-17 (a mixture of 9-[1-14C]eicosyl-9-phosphabicyclo-[421] and [331] nonanes)
- Analytical purity: Non-radiolabelled RM-17 was analysed by glc and was shown to contain greater than 50% of the two oxides of RM-17. It was therefore purified by column chromatography on silica (30 cm x 2 cm) eluting with nitrogen bubbled hexane and collecting 20 ml fractions. The fractions were analysed by glc. Those containing RM-17 were combined and evaporated with a stream of nitrogen. A final analysis of the RM-17 showed that it was 64% RM-17 and 36% RM-17 oxides (present due to rapid oxidation of RM-17.
- Radiochemical purity (if radiolabelling): The compound was shown by glc analysis to contain 91% [14C] RM-17, 5% RM-17 oxides (III and IV) and 4% unknown.
- Other: [14C] RM-17 was diluted with the purified non-labelled RM-17 to a specific activity of 1.0 µCi.mg-1 (37 kBq.mg-1) to give a total weight of 225 mg of [14C] RM-17 (225 µCi, 8.33MBq). It was dissolved in corn oil (4.5 ml). This dose solution was radioassayed when the animals were treated in order to determine the radiochemical content of the solution an hence the dose to each animal.

Radiolabelling:

yes

Remarks:

14C

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Each rat was individually housed in a glass metabolism cage and given access to food and water ad libitum.

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

corn oil

Details on exposure:

The rats were given single oral doses of [14C] RM-17 (25.4 mg, equivalent to approximately 120 mg.kg-1 bodyweight; 24 µCi, 888 kBq) in corn oil (0.5 ml).

Duration and frequency of treatment / exposure:

The rats were given single oral doses.

Doses / concentrations

Remarks:

Doses / Concentrations:
[14C] RM-17 (25.4 mg, equivalent to approximately 120 mg.k-1 bodyweight; 24 µCi, 888 kBq) in corn oil (0.5 ml).

No. of animals per sex per dose / concentration:

6 female rats

Control animals:

not specified

Positive control reference chemical:

No data

Results and discussion

Preliminary studies:

Preliminary chromatography: A composite urine sample (0-2 day urine), containing 25% of the administered radiochemical was analysed directly by two dimensional tlc: first solvent a, second solvent b. The radio-components were quantified by exising the silica followed by liquid scintillation counting as described in the method section under thin-layer plates.

Main ADME results

Type:

metabolism

Results:

The major urinary metabolites (18% of dose in two days) was partially identified as an ω-carboxylic acid although its full structure was not determined.

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Isolation of the urinary metabolites of [14C] RM-17

Extraction of metabolites: The combined (0-2 day) urine sample was extracted with diethyl ether. Six percent of the urinary activity partitioned into the organic phase (Ue). The aqueous phase was taken to pH2 with HCl and extracted again with ether. No further radioactivity partitioned into the organic phase. The anlysis of Ue (solvent a) indicated that it contained only one radio-component.

Isolation of metabolites:
UeA (1% of dose) - This metabolite was isolated by extraction of the urine followed by preparative tlc in solvent c. The chromatographic characteristics of the metabolite indicated that it was slightly more polar than the two isomers of RM-17 oxide in solvents b, c and d. Analysis by mass spectroscopy indicated that at least two components were present and since the fraction represented less than 1% of the dose, no further work was performed.

Urine after extraction with diethyl ether Uw - This metabolite fraction contained the polar, non-extractable metabolites. Tlc analysis in solvent a indicated that it contained two major radio-components (B and D). Uw was purified by hplc as described in methods above.

UwB (4% of dose) - This metabolite was isolated by hplc. It was a polar material which, on analysis, had a Rf=O in neutral organic solvents (e.g. b). It was subjected to enzyme hydrolysis by ß-glucuronidase and sulphatase. However, no less polar aglycone was formed by this procedure. UwB was treated with diazomethane in ether. The reaction was analysed by tlc (solvent a); UwB was quantitatively converted to a marginally less polar component (UwB-Me) (solvent a). This methylated derivative was analysed by mass spectroscopy, and was shown to consist of more than one component and no structure analysis could be performed on the data obtained.

UwD (18% of dose) - This metabolite was isolated by hplc. It had similar chromatographic properties to UwB as described above. It was incubated with ß-glucuronidase and sulphatase but no aglycone was formed. It was quantitatively methylated by diazomethane in ether to form the methylated derivative UwD-Me. This derivative was analysed by mass spectroscopy (electron impact, chemical ionisation, field desorption) but inconsistent results were obtained and no structure analysis could be performed on the data. Proton nmr studies on UwD-Me showed that the terminal methyl group (chemical shift = 0.87δ) on the C-20 side-chain in the parent molecule was missing and that a methyl ester (3.70 δ) and methylene group adjacent to a carbonyl group (2.55 δ) were now present. This data confirms that the terminal methyl group has been oxidised to a carboxylic acid and subsequently derivatised by the diazomethane. The rest of the nmr spectrum showed similar, but not identical, structural units to RM-17 oxide. The 13C nmr spectrum proved the presence of carbonyl (178.3 δ) and methyl ester (54.4 δ) groups in the molecule, but no other information could be obtained from the spectrum.

UwD-Me and UwD were refluxed with 4N sodium hydroxide and 6N hydrochloric acid for 4 h respectively. On tlc analysis, no hydrolysis products were present. The only radio-component observed was UwD.

UwD-Me was analysed by ion-exchange chromatography. UwD-Me was placed on a column of carboxymethyl cellulose (H+ form) and eluted with water. None of the radioactivity was retained by the ion-exchange column. A similar experiment was performed with CG120 resin (Amberlite sulphonic acid resin, H+ form); all the radioactivity was retained by the column and could be eluted with 1M hydrochloric acid.

UwD-Me was analysed by Hve at pH2 and at pH9.5. The compound migrated to the same extent as the electro-osmotic marker at both pH values indicating it to be a neutral molecule at these two pH values.

Components UwA, C, E, F and G individually represented 1% of the dose or less and were not analysed further.

Isolation of the faecal metabolites of [14C] RM-17

Extraction of metabolites: The 0-2 day faeces containing 62% of the administered dose were extracted by stirring at room temperature with ethanol. Most of the faecal radio-activity (93%) extracted into the ethanol. The extract was analysed by two-dimensional tlc: first solvent d, second solvent c. The radio-components were quantified by excising the silica followed by liquid scintillation counting as described in the tlc plates section above.

Isolation of metabolites:
FeA2 (6% of dose) - This fraction was isolated from Fe by preparative tlc using solvents d, e and f. The isolated metabolite was analysed by chemical ionisation mass spectroscopy. Examination of the mass spectra obtained showed that two compounds were present in FeA2. The molecular weights of the two components were 455 and 429. The higher molecular weight compound corresponds to a hydroxylated RM-17 oxide molecule. The structure of the second component could not be established from the mass spectral data.

FeD (38% of dose) - This metabolite was isolated from Fe by preparative tlc in solvent d. The material was analysed by two-dimensional tlc (solvents c then b then g) followed by autoradiography; it co-chromatographed with an authentic standard of the more polar isomers of RM-17 oxide. The mass spectrum of FeD was also identical to that of the authentic more polar isomer of RM-17 oxide.

FeE (11% of dose) - This fraction was purified from Fe by preparative tlc in solvent a. This fraction co-chromatographed on tlc analysis (solvents a and b) with the urinary metabolite UwD. FeE was esterified by diazomethane in ether. The esterified material was analysed by tlc in solvents a and b. 74% of the radiolabelled compound was converted to a slightly less polar compound (FeE-Me), 13% remained unchanged and 13% was shown to co-chromatograph with the more polar isomer of RM-17 oxide by two-dimensional tlc using solvent d then c. On hve analysis at pH6.1 and 9.5 FeE migrated appreciably towards the cathode (as did the dichlorobenzoic acid marker) indicating a negatively charged species. At all three pH values (pH2, 6.1, 9.5) FeE-Me migrated towards the anode to the same extent as the electro-osmotic marker indicating that FeE-Me was a neutral compound. FeE-Me had identical electrophoretic properties to UwD-Me.

Fractions FeAl, B and C individually represent less than 1% of the administered dose and were not analysed further.

Applicant's summary and conclusion

Conclusions:

The major metabolite of RM-17 was excreted in the faeces and was identified as RM-17 oxide. The major urinary metabolite (18% of dose in two days) was partially identified as ω-carboxylic acid although its full structure was not determined.