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Toxicological information

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
Remarks:
Conducted on a read-across material.
Justification for type of information:
The toxicological profile of the test material would not be different than that of the read-across material.

Data source

Reference
Reference Type:
publication
Title:
THE METABOLISM AND EXCRETION OF BUPRENORPHINE IN HUMANS.
Author:
CONE EJ, GORODETZKY CW, YOUSEFNEJAD D, BUCHWALD WF, JOHNSON RE
Year:
1984
Bibliographic source:
Drug metabolism and Disposition 12 (5): 577 - 581.

Materials and methods

Objective of study:
excretion
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Following subcutaneous, sublingual, and oral test material administration to a single subject, total metabolite excretion in urine and faeces were assessed using Gas Chromatography.
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed

Test animals

Species:
other: Human
Strain:
other: Not applicable
Sex:
male
Details on test animals and environmental conditions:
The subjects were healthy male volunteers between the ages of 21 and 45 years who gave informed consent to participate in the study. All had histories of chronic opiate abuse but were nondependent at the time of the study. The subjects were housed on the research ward of the Addiction Research Center (Baltimore, MD). Each subject underwent extensive pretest history and physical, psychological, and laboratory examinations prior to the study. During their first 3 days on the ward, they were monitored for possible signs of opiate withdrawal and urine was screened for opiates and other drugs of abuse. During participation in the study subjects were monitored at all times by the ward nursing staff and a physician was available for potential emergencies

Administration / exposure

Route of administration:
other: subcutaneous, sublingual and oral administration
Vehicle:
other: Yes
Details on exposure:
The test material was administered in doses of 1 and 2 mg subcutaneously, 2 and 4 mg sublingually, and 20 and 40 mg orally.
To provide data for a valid bioassay, an additional dose of 10 mg orally and 1 mg sublingually was administered to each subject as the last drug treatment.
Drug was administered under double-dummy conditions, i.e. each drug treatment consisted of a sc injection and an oral or sublingual preparation.
Active drug was administered by only one route for each treatment.
Inactive vehicle by both routes served as the placebo treatment.
Subjects were randomly assigned to a particular order of drug treatment and double blind conditions were maintained throughout the study.
The subcutaneous injection volume was 2 mL; oral and sublingual medication was administered in liquid form, 50 mL as an oral drink and 0.5 mL placed under the tongue with an Eppendorf pipette for sublingual administration.
Duration and frequency of treatment / exposure:
Each subject receiving five different drug treatments (5 x 5 Latin square) administered at 4-day intervals.
Doses / concentrationsopen allclose all
Dose / conc.:
1 other: mg
Remarks:
subcutaneous
Dose / conc.:
2 other: mg
Remarks:
subcutaneous
Dose / conc.:
2 other: mg
Remarks:
Sublingual
Dose / conc.:
4 other: mg
Remarks:
Sublingual
Dose / conc.:
20 other: mg
Remarks:
Oral
Dose / conc.:
40 other: mg
Remarks:
Oral
Dose / conc.:
1 other: mg
Remarks:
An additional dose administered sublingually
Dose / conc.:
10 other: mg
Remarks:
An additional dose administered orally
Control animals:
no
Details on dosing and sampling:
Urine and faeces were collected from all subjects from time of admission to time of discharge from the research ward. Following drug administration at 0900, urine collection periods ended at 2, 4, 8, 12, 24, 36, 48, 60, 72, 84, and 96 hours. Urine samples for each subject were pooled within specified collection periods.
Faeces were collected ad libitum by having the subject defecate into a plastic bag lining a bed pan. All samples were labelled and frozen until time of assay.
Frozen faeces samples were weighed in a large beaker and homogenised in 1 L of methanol with stirring overnight. The supernatant was decanted and centrifuged for 15 mm at 1000g. Aliquots (1-5 mL) of the supernatant were removed and evaporated, and the residue was dissolved in water (1-5 mL). For hydrolysis of faeces and urine samples, aliquots (1 mL) were treated with 1 mL of 2 N sodium acetate buffer (pH 5.2), 30,000 units of glucuronidase, and 6,000 units of sulfatase (Endo Laboratories) and incubated at 37°C for 20 hours. Etorphine (200 ng) was added prior to hydrolysis for quantitative studies.
Extraction of untreated or hydrolysed faeces and urine samples was performed at pH 10 (1 mL of 10 N phosphate buffer) with 6 mL (x2) of heptane/ethyl acetate (1:5, v/v). The organic extracts were combined and extracted with 3 mL of 0.1 N H2SO4. The organic phase was discarded and the acid phase was washed with 6 mL of hexane. The pH of the aqueous phase was adjusted to pH 10 with sodium hydroxide solution, buffered with 10 N phosphate buffer, and extracted with 6 mL of heptane/ ethyl acetate (1:5. v/v). The organic extract was removed and evaporated to dryness under nitrogen at 40°C.
Extracts were derivatised with PFPA (20 µL) in 100 µL toluene at room temperature for 1 hour. The excess reagent was evaporated under nitrogen at room temperature. The residue was dissolved in ethyl acetate (100 µL) for analysis.

Gas Chromatography.
Assays were performed on a Perkin-Elmer Sigma 2 gas chromatograph equipped with a 63Ni electron capture detector. The glass column (2 mm i.d. x 1.84 m) was packed with 3 % OV-17 on Gas-Chrom Q (100-120 mesh). The conditions were: Injector, 275°C: column, 260°C, detector, 320°C carrier flow (nitrogen), 20 mL min-1.

Daily standard curves were constructed for the test material and metabolite 1 by the peak height ratio method. The curves were prepared from the analyses of control urine (or faeces) containing etorphine (250 ng) and test material, metabolite 1, and metabolite 2 added in equal concentrations in ranges of 10-100, 25-250, or 500-2000 ng/mL. Samples for standard curves were processed and analysed in the same manner as drug specimens. Responses were linear over the concentrations measured with correlation coefficients (r) ≥ 0.98. The lower limits of the assay were ~10 ng/mL for the test material and ~5 ng/mL for metabolite 1. Samples were assayed in duplicate and the mean values are reported.

Gas Chromatography-Mass Spectrometry.
Urinary extracts were derivatised with PFPA and analysed with a Finnigan model 4021 GC-MS operating in the negative or positive chemical ionization mode: A glass column (2 mm id. x 30 cm) packed with 3% OV-210 on Gas-Chrom Q ( 100- 120 mesh) was coupled to the mass spectrometer by a glass-lined stainless steel tube. The injector, column, interface oven, and source were maintained at 245, 240, 250, and 250 °C respectively. Methane was used as carrier and reagent gas at a flow rate of 12 mL/mm. The electron energy was 80 eV. Total ion scans of extracted standards and samples were collected over the range of 250-800 amu.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on excretion:
Urinary Excretion:
The pattern of excretion of metabolites in urine was determined for an individual subject who had received two doses of the test material by the oral, subcutaneous, and sublingual routes of administration. The samples were analysed by gas chromatography for free and total (following Glusulase hydrolysis) parent and metabolite content. The amount of conjugated metabolite was determined as the difference between the free and total drug concentration. The limits of detectability of the assay for the test material and metabolite 1 from urine were approximately 10 and 5 ng/mL, respectively.
Total overall recoveries of metabolites from urine were consistently low, ranging from 1.9-14.3% of the administered dose; this is excluding the 2-mg subcutaneous dose which had metabolite overlap from the preceding dose as evidenced by the presence of metabolite in the control (zero hour) sample. Overall recoveries from urine for other subjects were also within this range.
The absence of free test material in urine was consistent across all doses, routes, and subjects. The pattern of excretion of metabolites for the human subject varied somewhat with the route of administration. Generally, the highest concentration of metabolites by the subcutaneous and oral routes were found in the first 24-hour period with the amount of conjugated metabolite 1 predominating over that of free metabolite 1 or conjugated test material by a factor of 2 or more.
Excretion of metabolites following sublingual drug administration presented a slightly different pattern in that larger amounts of conjugated test material were excreted (8.2% and 6.5%), matching or exceeding the overall amounts of conjugated metabolite 1 excreted in urine. Also, peak excretion of conjugated test material occurred during a somewhat later time interval (24-48 hour) particularly for the 4 mg sublingual dose.
The pattern of urinary excretion of metabolites following a 40 mg oral dose for one subject was quite similar to the first subject, although the overall recovery of 6% was somewhat less. Conjugated metabolite 1 (4.2%) was the predominant metabolite followed by free metabolite 1 (1%) and conjugated test material (0.8%).

Faecal Excretion:
The excretion of the test material and metabolites in faeces for two individual human subjects was shown, with samples obtained following 10, 20, and 40 mg oral doses of test material at time periods of 2-7 days following drug administration. Free and conjugated test material were present in all samples, with free test material exceeding that of the conjugated metabolite. Metabolite 1 was present in all but one sample tested with free metabolite content exceeding the conjugated form. Overall per cent dose recoveries for the two subjects based on the prior dosage ranged from 0.2-11.5% with the largest amount being excreted in the 3-6 day period following drug administration. A similar pattern of faecal excretion was found for other subjects following sublingual dosing. For these subjects, test material and conjugate 1 were excreted in nearly equivalent amounts. Drug concentrations in faeces generally peaked during the 4th day following drug administration.

Metabolite characterisation studies

Metabolites identified:
yes
Remarks:
norbuprenorphine (metabolite 1)
Details on metabolites:
Identification of metabolite 1 as a metabolite of the test material in urine and faeces:
Urinary and faecal extracts were examined by thin layer chromatography and GC-MS for the presence of metabolites. Only one metabolite was detected by these methods. The urine was collected 4-8 hours following a 40 mg dose of the test material. The retention time of the major component was identical to that of standard metabolite (derivatised with PFPA). The partial negative chemical ionization spectra clearly established the identity of the metabolite. This metabolite was detected in the urine and faeces of subjects receiving the test material by the oral, sublingual, and subcutaneous routes of administration.

Any other information on results incl. tables

Discussion

Substantial amounts of free test material were found in faecal samples following oral and sublingual dosing at times when there was very little conjugated test material being excreted in urine. Also, the ratio of total test material to metabolite 1 in faecal samples was quite high when compared to that found in urine. This is likely due to a substantial first pass effect by the liver favouring the formation, conjugation, and urinary excretion of metabolite 1.

Applicant's summary and conclusion

Conclusions:
Following subcutaneous, sublingual, and oral test material administration to a single subject, total metabolite excretion in urine was 2, 13.4, and 12.1%, respectively.
No free parent drug was detected in urine. The amount of metabolite 1 excreted in urine generally exceeded that of conjugated test material.
In contrast, free and conjugated test material equalled or greatly exceeded total metabolite 1 content in faecal samples following oral or sublingual administration. The greatest amount of drug and metabolite eliminated in faeces occurred at 4 to 6 days following test material administration at times when there was very Iittle urinary excretion of conjugated test material. This latter evidence indicates an enterohepatic circulation of the test material in humans.
Executive summary:

Following subcutaneous, sublingual, and oral test material administration to a single subject, total metabolite excretion in urine and faeces were assessed using Gas Chromatography.

Total metabolite excretion in urine was 2, 13.4, and 12.1%, respectively.

No free parent drug was detected in urine. The amount of metabolite 1excreted in urine generally exceeded that of conjugated test material.

In contrast, free and conjugated test material equalled or greatly exceeded total metabolite 1 content in faecal samples following oral or sublingual administration. The greatest amount of drug and metabolite eliminated in faeces occurred at 4 to 6 days following test material administration at times when there was very little urinary excretion of conjugated test material. This latter evidence indicates an enterohepatic circulation of the test material in humans.