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Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
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:
publication
Title:
Tritium-labelled dihydromorphine: Its metabolic fate and excretion in the rat.
Author:
Hug CC and Mellett LB
Year:
1965
Bibliographic source:
The Journal of Pharmacology and Experimental Therapeutics 149 (3) 446 – 453

Materials and methods

Objective of study:
excretion
metabolism
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The investigation was undertaken to determine the metabolism and excretion of the tritium-labelled test material by rats. The test material was injected subcutaneously at 5 mg/kg. Animals were housed in metabolism cages and urine and faeces collected. Ninety two hours after injection the rats were sacrificed and the intestinal contents were removed and added to the cage collected faeces.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Specific details on test material used for the study:
RADIOLABELLING INFORMATION
Tritium-labelled test material was synthesised and purified. The radioactive drug was diluted with non-radioactive test material to provide a specific radioactivity of 70 µc/mg.
Radiolabelling:
yes

Test animals

Species:
rat
Strain:
other: Holtzman
Details on species / strain selection:
None specified
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: Male rats weighing 350 – 390 g and female rats weighing 230 – 270 g.
- Housing: Each group of animals was placed in an individual metabolism cage.
- Diet: Fasted during the first 24 hours, ad libitum thereafter.
- Water: Ad libitum

Administration / exposure

Route of administration:
subcutaneous
Vehicle:
not specified
Details on exposure:
Each animal was injected subcutaneously with tritium-labelled test material at a dose of 5 mg/kg (calculated as free base).
Duration and frequency of treatment / exposure:
Single dose
Doses / concentrations
Dose / conc.:
5 other: mg/kg (calculated as free base)
No. of animals per sex per dose:
Six male rats were divided into two groups of three, and eight female rats were separated into two groups of four.
Control animals:
yes, concurrent no treatment
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY
- Tissues and body fluids sampled: Urine, faeces. Ninety two hours after injection, the rats were sacrificed and the intestinal contents were removed at sacrifice and added to the cage-collected faeces.
- Time and frequency of sampling: Urine was collected beneath the cage under toluene for three time periods: 0 to 24, 24 to 48 and 48 to 92 hours. Faeces were accumulated for the full 92 hours on a screen beneath the cage floor. Faeces were combined for the two groups of the same sex.
Cage-collected urine was diluted with water in proportion to the anticipated amount of radioactivity in the sample. Wet-weighed faeces were homogenised in 0.5 N hydrochloric acid in a Waring Blender. Two- or 4-mL aliquots of the dilution or homogenate were analysed. A double purification procedure was necessary to eliminate quenching materials from the final extract. This was accomplished by extracting the original sample aliquot buffered at pH into 10 mL of ethylene dichloride containing 30 % n-amyl alcohol. An 8 mL aliquot of organic solvent extract was transferred to a glass-stoppered centrifuge tube containing 5 mL of 0.5 N HCl and the tube was shaken for 20 minutes. The mixture was centrifuged and a 4 mL aliquot of the aqueous layer (upper) was transferred to a clean centrifuge tube, neutralised and buffered at pH 9, and extracted with 10 mL of ethylene dichloride containing 30 % n-amyl alcohol as before. Eight mL of this final extract were placed in a scintillation-grade counting vial and evaporated to dryness at 65 °C. The sample was prepared for counting as described.
Total tritium-labelled test material (i.e. free plus conjugated) was determined by autoclaving liquid samples with 1/10th volume conc. HCl as described previously and subsequently analysing for tritium-labelled test material using the double purification procedure.
Recoveries of total amounts of tritium-labelled test material ranging from 12 to 4800 ng added to 1:5 aqueous dilutions of urine were 100.5 ± 31 (SD) %. Additions of 500 ng quantities of tritium-labelled test material to acid homogenates of faeces were recovered to the extent of 89.9 ± 1.4 (SD) %. This recovery figure was used to correct final values for analysis of tritium-labelled test material in faeces to 100 %. As little as 0.1 ng of tritium-labelled test material with a specific radioactivity of 0.2 curies per millimole could be determined using the double purification procedure with accuracy of ± 20 %; 0.4 ng was determined with an accuracy of ± 4.5 %. The above recovery figures have been corrected for aliquot volumes transferred at each step of the double purification procedure.
In order to evaluate the specificity of the method, aliquots of non-autoclaved and acid autoclaved urine from rats given the tritium-labelled test material were extracted as above. The concentrated extracts were analysed by paper chromatography using the technique described below. In each case there was a single area of radioactivity which corresponded in Rf to that of non-radioactive test material applied to the same chromatograms.
Since tritium-labelled test material might be expected to be N-demethylated to dihydronormorphine-H3 in vivo, it was necessary to estimate to what degree the metabolite would interfere in the analysis for the tritium-labelled test material. Analysis of aqueous solutions of dihydronormorphine using the double purification procedure for tritium-labelled test material showed that only 14 % of the dihydronormorphine could be recovered. Thus, for example if up to 20 % of the radioactivity in a sample was due to dihydronormorphine-H3, it would introduce an error of less than 3.5 % in the analysis for tritium-labelled test material.


METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: Urine, faeces. Ninety two hours after injection, the rats were sacrificed and the intestinal contents were removed at sacrifice and added to the cage-collected faeces.
- Time and frequency of sampling: Faeces were accumulated for the full 92 hours on a screen beneath the cage floor.
- Number of animals: Faeces were combined for the two groups of the same sex.
- Method type for identification: Paper chromatographic methods for tritium-labelled test material:
Descending paper chromatography was done using non-buffered and buffered Whatman No. 1 or No. 3 chromatography paper. Buffered paper was prepared by dipping the paper in an aqueous solution of potassium phosphate (0.2 M) of the appropriate pH (6 – 10) and allowing it to dry in room air for 1 to 2 hours before applying solutions to be chromatographed. Buffered chromatograms were developed in tert.-amyl alcohol, n-butanol, n-butyl ether, water (80:7:13 v/v) which is designated as the “buffered solvent system”. Non-buffered chromatograms were developed in n-butanol, glacial acetic acid, water (4:1:2 v/v) which is referred to as the “non-buffered solvent system”. Occasionally paper was buffered with 0.2 M borate buffer instead of potassium phosphate and the chromatogram was developed in tert.-amyl alcohol saturated with the buffer (“borate-buffered system”).
Non-radioactive test material and dihydronormorphine were applied routinely to each chromatogram to serve as controls on technique. These compounds were localised on dry chromatograms by visualisation under UV light and by spraying with iodoplatinate reagent and / or with a freshly prepared solution of Diazoblue-B. Rf values were calculated using the midpoint of the coloured spot.
Scanning chromatograms for radioactivity involved cutting out the path of migration and dividing it lengthwise into 1 cm sections. Each section was placed in a scintillation-grade glass vial and 4 mL of eluting solvent were added. One of three eluting solvents was used: Absolute methanol; methanol containing 10 % concentrated hydrochloric acid; or aqueous 0.25 N hydrochloric acid. After 1 to 2 hours the section of paper was pressed against the vial to remove as much elute as possible and then discarded. The elute was evaporated at 60 °C. The residue was dissolved in 0.2 mL absolute methanol and 5 mL of phosphor solution were added. The radioactivity was determined in an automatic liquid scintillation spectrometer. The average counts per minute (cpm) determined from two or more counting periods were plotted against centimetres of migration from the origin. The Rf value for an area of radioactivity was calculated using the centimetre-area containing the highest number of absolute cpm (i.e. total cpm minus background cpm).


TREATMENT FOR CLEAVAGE OF CONJUGATES:
Faeces: Non-autoclaved and autoclaved portions of acid homogenates of faeces from male rats (groups I and II) and from female rats (groups III and IV) were neutralized to pH 7 and centrifuged. Aliquots of the supernatants were chromatographed directly on non-buffered and buffered paper (pH 7, 8, 9, 10).
Urine:
1. To a 56 mL portion of male rat urine (15 % of the total collection for group 1) 100 ng of non-radioactive DNM were added and the solution was autoclaved in 1.2 N hydrochloric acid for 30 minutes Throughout the entire procedure, two controls, one consisting of tritium-labelled test material and non-radioactive DNM in aqueous solution, and the other of tritium-labelled test material and DNM in urine from non-injected male rats were handled in a fashion identical to that for urine from tritium-labelled test material-injected rats.
The pH of the autoclaved urine was adjusted to approximately 10 and then buffered at pH 10.35 with 40 % potassium phosphate. The total volume was divided into suitable aliquots and each aliquot was extracted three times. Less than 1 % of the total radioactivity remained in the aqueous phase.
The coloured extracts were pooled and concentrated to dryness at 60 °C. The brown residue was dissolved in methanol and chromatographed using the non-buffered system. Scanning a strip of the large chromatogram showed that a single, sharp peak of radioactivity migrated coincidentally with non-radioactive, standard test material, Rf 0.58.
The radioactive area (Rf 0.58) from the remainder of the chromatogram was eluted with 3- to 300-mL portions of methanol and the eluates were pooled and evaporated to dryness at 60 °C. Suitable aliquots of a methanol solution of the residue were chromatographed at various pH’s using buffered systems. A portion of each chromatogram was scanned for radioactivity. Attempts to elute and re-chromatograph the radioactive material corresponding to DNM on pH 8 buffered paper were inconclusive probably as a result of decomposition of the material during manipulation.
2. Portions of urine from male rats (group I) and from female rats (group III) were neutralized to pH 7 or acid-autoclaved and then neutralized before they were applied to non-buffered and buffered papers for chromatography.
3. Aliquots of urine from each of the four groups of animals were diluted 1:10 in 0.2 M sodium acetate (pH 6.9). Duplicate 2-mL volumes of each dilution were incubated with 25 mg (approximately 872 Fishman units) of β-glucuronidase for 24 hours at 37 °C. Similar volumes of each dilution were: (a) incubated without enzyme, (b) acid-autoclaved, (c) and left untreated. All samples were analysed for free tritium-labelled test material.

Results and discussion

Main ADME results
Type:
excretion
Results:
Males excreted 84.1 % of the injected dose in urine and faeces in 92 hours following injection; females excreted 86.2 %.

Toxicokinetic / pharmacokinetic studies

Details on excretion:
Urinary and faecal excretion of free and conjugated tritium-labelled test material.
Rats of either sex excreted about the same percentage of the dose as unchanged (free) tritium-labelled test material in urine. Female rats excreted about twice as much conjugated tritium-labelled test material; the difference is significant (P < .05). Analysis of faeces revealed that male rats excreted approximately 1.5 times as much free tritium-labelled test material as the females; the difference cannot be evaluated statistically because of the pooling of the faeces for each sex. Both sexes excreted small amounts of conjugated tritium-labelled test material in faeces. The combined total excretion of tritium-labelled test material in urine and faeces in the 92-hour period following injection was almost identical for the two sexes: Males, 84.1 % of the injected dose; females, 86.2 %.

Identification of tritium-labelled test material in faeces.
Non-autoclaved and acid-autoclaved aliquots of the supernatant from faecal homogenates were analysed chromatographically. In all instances there was a small, but definite amount of radioactivity associated with the migration of standard DNM. Approximately 7.5 % of the total tritium in male rat faeces was present as tritium-labelled test material.
No radioactivity was present at the Rf of DNM in control chromatograms of non-radioactive faeces to which tritium-labelled test material was added in vitro. Whenever tritium-labelled test material was mixed with urine or faeces in vitro or in vitro and then chromatographed, a small amount of radioactivity remained at the origin on buffered chromatograms. It would appear that the radioactivity retained at the origin of buffered paper represented an artefact of manipulation inasmuch as there was a single area of radioactivity on non-buffered chromatograms, which were able to separate all of the expected in vitro metabolites of tritium-labelled test material except the N-demethylated form.

Metabolite characterisation studies

Metabolites identified:
yes
Remarks:
N-demethylated metabolite
Details on metabolites:
Identification of tritium-labelled test material and its metabolites in, urine: The radioactive area designated by Rf 0.58 was eluted and portions of the eluate were chromatographed on buffered papers. In all cases the largest portion of tritium radioactivity migrated with standard test material. A small amount of radioactivity migrated with DNM on pH 8 buffered paper but not on papers buffered at a higher pH.
Radioactivity in male rat urine chromatographed directly (i.e., without extraction) on pH 8 buffered paper separated into two areas, one of which corresponded to tritium-labelled test material. After autoclaving the urine, almost all of the radioactivity migrated with standard test material. No radioactivity was detected at the Rf of tritium-labelled test material.
Non-autoclaved male rat urine produced two, possibly three, areas of radioactivity on non-buffered chromatograms. A single radioactive peak was present at the Rf of standard test material when acid-autoclaved urine was chromatographed similarly
Results similar to those just described for direct chromatography of male rat urine were obtained when female rat urine was chromatographed before and after acid-autoclaving. Free and conjugated tritium-labelled test material was identified but no suggestion of the N-demethylated metabolite was evident.
β-Glucuronidase hydrolysed one-third (males) to one-half (females) of the total conjugated drug in rat urine. The possibility of urinary constituents interfering with optimal β-glucuronidase activity was not ruled out as an explanation for the incomplete hydrolysis of conjugated tritium-labelled test material. However, the results were reproduced in repeated incubations of different dilutions of urine suggesting that urinary constituents were not preventing enzymatic hydrolysis.

Identification of metabolites in faeces.
The faeces from female rats contained only a trace of the N-demethylated drug (1.8 % of the radioactivity in the homogenate).

Applicant's summary and conclusion

Conclusions:
Following subcutaneous injection of tritium-labelled test material in rats, males excreted 84.1 % of the injected dose in urine and faeces in 92 hours following injection; females excreted 86.2 %.
Executive summary:

The investigation was undertaken to determine the metabolism and excretion of tritium-labelled test material by rats. The test material was injected subcutaneously at 5 mg/kg. Animals were housed individually in metabolism cages and urine and faeces collected. Ninety two hours after injection the rats were sacrificed and the intestinal contents were removed and added to the cage collected faeces.

Males excreted 84.1 % of the injected dose in urine and faeces in 92 hours following injection; females excreted 86.2 %.

Free and conjugated tritium-labelled test material was identified in urine but no suggestion of the N-demethylated metabolite was evident.

Faeces from female rats contained only a trace of the N-demethylated drug (1.8 % of the radioactivity in the homogenate).