THE METABOLISM OF DEXAMETHASONE EFFECT OF PHENYTOIN J.
ENGLISH, J. CHAKRABORTY
and V.
IN THE RAT-
MARKS
Department of Biochemistry. University of Surrey, Guildford, Surrey, England (Rrceivecl24
May 1974)
SUMMARY
The metabolism and excretion pattern of tritium labelled dexamethasone was investigated in the male rat. Thirty-one per cent of the administered radioactivity was excreted in the urine in 4 days and 25% in the faeces. Most of the urinary excretion occurred within the first 24 h. The urinary radioactivity was largely present in the non-conjugated steroid fraction and accounted for 22.5% of the total dose administered. Unchanged dexamethasone accounted for 13.6%; 6-hydroxydexamethasone for 7.4% and 20-dihydrodexamethasone for 1.1%. Phenytoin pretreatment did not affect overall metabolism or excretion of [3H]-dexamethasone but significantly reduced its initial urinary excretion rate.
INTRODUCTION
zeneemethanol(8: 1, v/v). Merck silica gel plates (F, 54), thickness 0.25 mm, were used for thin layer chromatNumerous reports are available on the metabolism of ography (t.1.c.). the natural corticoids but rather less on their synthetic Forty-eight-day old male Wistar albino rats were analogues. Major differences are known to exist in given chromatographically pure [3H]-dexamethasone their rates and routes of metabolism, largely due to the orally at a dose of 1.14 nmol/kg body weight. In some presence of a A’-double bond and substituents in the experiments a large dose of native dexamethasone (64 C-6, C-9 and C-16 positions in the commercially pmol/kg) was given to rats in order to produce sufficient available corticosteroids [ 1,2]. amounts of metabolites for chemical characterization. following excretion of radioactivity The Phenytoin pretreated animals were given @3 mmol/kg administration of radiolabelled dexamethasone (9c~body weight of the drug twice a day for 3 days by intrafluoro-l1~,17r,2l-trihydroxy-16ix-methyl-l,4-pregnaperitoneal injection. The last dose of phenytoin was diene-3,20-dione) has been studied in man [3,4], but given 16 h before C3H]-dexamethasone administration. the nature of its metabolism and the chemistry of its Urine and faeces were collected from 0 to 6 h and metabolites remains unknown. It has been shown [4] from 6 to 24 h and thereafter at 24 h intervals for 4 that the anticonvulsant drug, phenytoin (5,5-diphenyldays. Urine was collected under toluene and stored hydantoin), may cause false positive results when dexafrozen until analysed. methasone is used in suppression tests for the diagAll radioactivity measurements were made using a nosis of Cushing’s syndrome and the suggestion has Packard Tri-Carb Liquid Scintillation Counter, Model been made that this might be due to accelerated con2425. The scintillation mixture (10 ml) contained 06% version of dexamethasone _to an inactive metabolite. butyl PBD [2-(4’-tert-butylphenyl)-5-4”-biphenylyl)The present work was undertaken to investigate the 1,3,4-oxadiazole] in sulphur-free toluene-Triton X-100 metabolism of dexamethasone in the rat, identify its (2: 1, v/v). 0.1 ml aliquots of urine were counted in urinary metabolites and to determine the influence of duplicate. phenytoin on its metabolism. Faeces were soaked overnight in 207; aqueous methanol, thoroughly mixed and extracted twice (2 vol.) with the same solvent. The extracts were pooled, conMATERIALS AND METHODS centrated and 1.0 ml aliquots counted for radioactivity. Dexamethasone, 6-hydroxybetamethasone acetate The efficiency of this method for counting faecal (9a-fluoro-6P,l la,l7p-trihydroxy-16B_methyl-1,4radioactivity was confirmed in some experiments using pregnadiene-3,20-dione-2 l-acetate) and phenytoin were the digestive procedure of Mahin and Dofberg [6]. gifts from Roussel Laboratories Ltd., Middlesex; Glaxo Tissue samples (300 mg) were digested with 60% methResearch Ltd., Middlesex, England and Parke Davis anolic KOH and extracted twice with ethyl acetate. and Co., Pontypool respectively. 20-DihydrodexaThe extracts were pooled, evaporated to dryness and methasone was prepared by sodium borohydride counted for radioactivity. Replicate determination of added radioactivity to reduction of dexamethasone. Acetylation, oxidation faeces (5 g; 10,000 d.p.m.) and tissue (200 mg muscle; and reduction of the steroids were carried out by stan500 d.p.m.) gave recoveries of 96% f 2.5 (counting effidard methods [S]. [1,2-3H]-Dexamethasone (S.A. ciency 22%) and 97% + 1.6 (counting efficiency 29%) 22 Ci/mmol) was purchased from the Radiochemical Centre, Amersham, England and stored at 4°C in benrespectively. 65
Ihe concentration ofcompounds I. II and III in the orlginal sample. The second method of quontitation LIXCI N;IS ;I reverse isotope dilution proccdurc [7]. Knou II amounts. 300 /lg. of the steroids under c\amination were added to the original cthht acctatc extract and thoroughly mixed. The steroid components \+crc then scparatcd and purified bq t.t.c. and their specific activity determined. Steroid mass mcasurcmcnts wcrc made using the Porter -Silber reaction [S] and radioactivity was determined by liquid scintillation counting. As preliminary cxperimcnts demonstrated goodagreement between the two methods only the lirst was used routinely in subsequent expcrimcnts. Fraction III was puritied by thin layer chromatography in systems A & B before isotope dilution study. 20-Dihydrodexamethasone (30 mg) was added to the sample and repeatedly crystallized from acttone heptanc.
The tolucne overlying the urine was cvaporatcd undcr nifrogcn and the urine adjusted to pH 7. Unconjugated steroids wcrc extracted twice \vith ethyl acetate (2 vol.). The cutracts Lvcrc pooled. \vashed once with 0.1 N NaOH (@I vol.) t\vicc with distilled water and (b I ml ;diquots of the ethyl-acetate layer taken for tritium counting. The rest was c\aporated to dryness, under reduced pressure. at 45 C‘ and the rcsiduc was dissolved in 0.1 ml cthyt acetate and applied. as a streak. to tl1c t.l.c. plakx R,. \:~ILICSof the unconjugated steroids and their ace1yla tion. oxidation and reduction products were dctermined in each of the following three solvent systems: cthql acetate methylene chloride ethanol (Al (5535: IO by vol.): (B) chloroform methylrne chloride ethanol (80: IO: IO by vat): and (C) bcnzenc methanol
(X0:
20
c
v).
Tests \vcrc run in parallel with authentic reference compounds. as far as was possible. Bccuasc no “authcntic” samptc of 6-hydrox~dcxametli~~sol7c was availahlc its I (l/i-isomer. 6-hydroxy-bctatncthasonc was used instead as reference compound to metabolite II. This would stem to be justified as in our c.t.c. studies \+c ohscrvcd that in all sotvcnt systems (A. B & C) dexamethasone and bctamethasonc gave identical R,. vatue~ as fret compounds and their acetytation and oxidation products. Steroids were located on the t.1.c. pIale under U.V. light. Variations in R,. values. between runs, \zcrc corrected bq rcfercnce to a cortisol market-. (‘hromatograms wcrc scamxd for radioactivity bq means of ;I Dunnschicht-Scanner II (LB 2723 Berthold). and the three radioactive bands corresponding to three dcxamcthasone mctabolitcs scraped off the plntcs. The scrapings wcrc elutcd 3 times with 3 ml methanol prior to counting in the Packard scintillation counter. The varmus components (I, II and I II) of the mlconjugated steroid fraction were mcasurcd by two diffcrent methods. In the first. the amount of radioactivity associated with each compound after t.1.c. separation in sqstcm A was dctcrmined. Previous tests had shown that only YO”,,of the radioactivity applied to the plate could be recovered by elution after t.1.c. development. This \V;IS atto\+ed for in all suhscquent calculations of
Table I. C tlmt~li~~ivc
cucrction
of rnd~oac~iv~t~
Radioactivity remaining in the urine after ethyl acetate cxtraclion was assumed to reside wholly in the conjugated steroid fraction. The propriety of this assumption is open to question but since the total radioactivity involved was small it is doubtful that this introduced any significant error.
The cumulative excretion data for radioactivit) present in the urine and facces, estimated at various time intervals over a period of 4 daqs following oral administration of [“HI-dcxamethasone to 4X-day malt rats. arc shown in Table I. The major part of the urinary excretion occurred within the first 24 h and that of faeces within 48 h. After 4 days. 31.4 i_ S.E.M. I._?“ 55”,, could he found in the urine bind faeces in 4 days. Our more recent studies (unpublished results) show that after a larger dose. tho total amount ofradioactivityexcretcd in the urine and f%~es over the same period went up to X5 9S”,, of the dose. More detailed investi~~~tions on rhe interrclatiorishir between the size of [“HI-desameth~tsoric dose and its rate of clearance from the body with particular rcfcrencc to residual radioactivity in all tissues arc in progress.
REFEREUCES
I. Chen P. S. Jr. and Borrevang P.: In Ifat~dhoo~ of’E.x/wiwlttuI Pharwcoloy~ (Edited hy F. A. Smith). SpringcrVerlag, Berlin, Vol. XX. Part 2 (1970) pp. 193 252. 2. Fotherhy K. and James F.: In .3Jt~1rws in Stewili Bio~i~rrnistr~ arrtl ~~~~~~/t~~~~~~~u~~~ (Edited by M. H. Briggs and G. A. Christie). Academic Press, London. Vol. 3 (1972) pp. 67- 165. 3 Jubiz W.. Meikle A. W.. Levinson R. A.. Mizutani S.. West C. D. and Tyler F. H.: jv;(,~\, Enyl J. bfctl. 283 (1970) 11 14. 4 Haque N.. Thrasher K.. Wcrk E. ti. Jr.. Knowles H. c‘. Jr. and Shotiton L. J.: J.
ENGLISH, J. CHAKRABORTY
and V.
IN THE RAT-
MARKS
Department of Biochemistry. University of Surrey, Guildford, Surrey, England (Rrceivecl24
May 1974)
SUMMARY
The metabolism and excretion pattern of tritium labelled dexamethasone was investigated in the male rat. Thirty-one per cent of the administered radioactivity was excreted in the urine in 4 days and 25% in the faeces. Most of the urinary excretion occurred within the first 24 h. The urinary radioactivity was largely present in the non-conjugated steroid fraction and accounted for 22.5% of the total dose administered. Unchanged dexamethasone accounted for 13.6%; 6-hydroxydexamethasone for 7.4% and 20-dihydrodexamethasone for 1.1%. Phenytoin pretreatment did not affect overall metabolism or excretion of [3H]-dexamethasone but significantly reduced its initial urinary excretion rate.
INTRODUCTION
zeneemethanol(8: 1, v/v). Merck silica gel plates (F, 54), thickness 0.25 mm, were used for thin layer chromatNumerous reports are available on the metabolism of ography (t.1.c.). the natural corticoids but rather less on their synthetic Forty-eight-day old male Wistar albino rats were analogues. Major differences are known to exist in given chromatographically pure [3H]-dexamethasone their rates and routes of metabolism, largely due to the orally at a dose of 1.14 nmol/kg body weight. In some presence of a A’-double bond and substituents in the experiments a large dose of native dexamethasone (64 C-6, C-9 and C-16 positions in the commercially pmol/kg) was given to rats in order to produce sufficient available corticosteroids [ 1,2]. amounts of metabolites for chemical characterization. following excretion of radioactivity The Phenytoin pretreated animals were given @3 mmol/kg administration of radiolabelled dexamethasone (9c~body weight of the drug twice a day for 3 days by intrafluoro-l1~,17r,2l-trihydroxy-16ix-methyl-l,4-pregnaperitoneal injection. The last dose of phenytoin was diene-3,20-dione) has been studied in man [3,4], but given 16 h before C3H]-dexamethasone administration. the nature of its metabolism and the chemistry of its Urine and faeces were collected from 0 to 6 h and metabolites remains unknown. It has been shown [4] from 6 to 24 h and thereafter at 24 h intervals for 4 that the anticonvulsant drug, phenytoin (5,5-diphenyldays. Urine was collected under toluene and stored hydantoin), may cause false positive results when dexafrozen until analysed. methasone is used in suppression tests for the diagAll radioactivity measurements were made using a nosis of Cushing’s syndrome and the suggestion has Packard Tri-Carb Liquid Scintillation Counter, Model been made that this might be due to accelerated con2425. The scintillation mixture (10 ml) contained 06% version of dexamethasone _to an inactive metabolite. butyl PBD [2-(4’-tert-butylphenyl)-5-4”-biphenylyl)The present work was undertaken to investigate the 1,3,4-oxadiazole] in sulphur-free toluene-Triton X-100 metabolism of dexamethasone in the rat, identify its (2: 1, v/v). 0.1 ml aliquots of urine were counted in urinary metabolites and to determine the influence of duplicate. phenytoin on its metabolism. Faeces were soaked overnight in 207; aqueous methanol, thoroughly mixed and extracted twice (2 vol.) with the same solvent. The extracts were pooled, conMATERIALS AND METHODS centrated and 1.0 ml aliquots counted for radioactivity. Dexamethasone, 6-hydroxybetamethasone acetate The efficiency of this method for counting faecal (9a-fluoro-6P,l la,l7p-trihydroxy-16B_methyl-1,4radioactivity was confirmed in some experiments using pregnadiene-3,20-dione-2 l-acetate) and phenytoin were the digestive procedure of Mahin and Dofberg [6]. gifts from Roussel Laboratories Ltd., Middlesex; Glaxo Tissue samples (300 mg) were digested with 60% methResearch Ltd., Middlesex, England and Parke Davis anolic KOH and extracted twice with ethyl acetate. and Co., Pontypool respectively. 20-DihydrodexaThe extracts were pooled, evaporated to dryness and methasone was prepared by sodium borohydride counted for radioactivity. Replicate determination of added radioactivity to reduction of dexamethasone. Acetylation, oxidation faeces (5 g; 10,000 d.p.m.) and tissue (200 mg muscle; and reduction of the steroids were carried out by stan500 d.p.m.) gave recoveries of 96% f 2.5 (counting effidard methods [S]. [1,2-3H]-Dexamethasone (S.A. ciency 22%) and 97% + 1.6 (counting efficiency 29%) 22 Ci/mmol) was purchased from the Radiochemical Centre, Amersham, England and stored at 4°C in benrespectively. 65
Ihe concentration ofcompounds I. II and III in the orlginal sample. The second method of quontitation LIXCI N;IS ;I reverse isotope dilution proccdurc [7]. Knou II amounts. 300 /lg. of the steroids under c\amination were added to the original cthht acctatc extract and thoroughly mixed. The steroid components \+crc then scparatcd and purified bq t.t.c. and their specific activity determined. Steroid mass mcasurcmcnts wcrc made using the Porter -Silber reaction [S] and radioactivity was determined by liquid scintillation counting. As preliminary cxperimcnts demonstrated goodagreement between the two methods only the lirst was used routinely in subsequent expcrimcnts. Fraction III was puritied by thin layer chromatography in systems A & B before isotope dilution study. 20-Dihydrodexamethasone (30 mg) was added to the sample and repeatedly crystallized from acttone heptanc.
The tolucne overlying the urine was cvaporatcd undcr nifrogcn and the urine adjusted to pH 7. Unconjugated steroids wcrc extracted twice \vith ethyl acetate (2 vol.). The cutracts Lvcrc pooled. \vashed once with 0.1 N NaOH (@I vol.) t\vicc with distilled water and (b I ml ;diquots of the ethyl-acetate layer taken for tritium counting. The rest was c\aporated to dryness, under reduced pressure. at 45 C‘ and the rcsiduc was dissolved in 0.1 ml cthyt acetate and applied. as a streak. to tl1c t.l.c. plakx R,. \:~ILICSof the unconjugated steroids and their ace1yla tion. oxidation and reduction products were dctermined in each of the following three solvent systems: cthql acetate methylene chloride ethanol (Al (5535: IO by vol.): (B) chloroform methylrne chloride ethanol (80: IO: IO by vat): and (C) bcnzenc methanol
(X0:
20
c
v).
Tests \vcrc run in parallel with authentic reference compounds. as far as was possible. Bccuasc no “authcntic” samptc of 6-hydrox~dcxametli~~sol7c was availahlc its I (l/i-isomer. 6-hydroxy-bctatncthasonc was used instead as reference compound to metabolite II. This would stem to be justified as in our c.t.c. studies \+c ohscrvcd that in all sotvcnt systems (A. B & C) dexamethasone and bctamethasonc gave identical R,. vatue~ as fret compounds and their acetytation and oxidation products. Steroids were located on the t.1.c. pIale under U.V. light. Variations in R,. values. between runs, \zcrc corrected bq rcfercnce to a cortisol market-. (‘hromatograms wcrc scamxd for radioactivity bq means of ;I Dunnschicht-Scanner II (LB 2723 Berthold). and the three radioactive bands corresponding to three dcxamcthasone mctabolitcs scraped off the plntcs. The scrapings wcrc elutcd 3 times with 3 ml methanol prior to counting in the Packard scintillation counter. The varmus components (I, II and I II) of the mlconjugated steroid fraction were mcasurcd by two diffcrent methods. In the first. the amount of radioactivity associated with each compound after t.1.c. separation in sqstcm A was dctcrmined. Previous tests had shown that only YO”,,of the radioactivity applied to the plate could be recovered by elution after t.1.c. development. This \V;IS atto\+ed for in all suhscquent calculations of
Table I. C tlmt~li~~ivc
cucrction
of rnd~oac~iv~t~
Radioactivity remaining in the urine after ethyl acetate cxtraclion was assumed to reside wholly in the conjugated steroid fraction. The propriety of this assumption is open to question but since the total radioactivity involved was small it is doubtful that this introduced any significant error.
The cumulative excretion data for radioactivit) present in the urine and facces, estimated at various time intervals over a period of 4 daqs following oral administration of [“HI-dcxamethasone to 4X-day malt rats. arc shown in Table I. The major part of the urinary excretion occurred within the first 24 h and that of faeces within 48 h. After 4 days. 31.4 i_ S.E.M. I._?“ 55”,, could he found in the urine bind faeces in 4 days. Our more recent studies (unpublished results) show that after a larger dose. tho total amount ofradioactivityexcretcd in the urine and f%~es over the same period went up to X5 9S”,, of the dose. More detailed investi~~~tions on rhe interrclatiorishir between the size of [“HI-desameth~tsoric dose and its rate of clearance from the body with particular rcfcrencc to residual radioactivity in all tissues arc in progress.
REFEREUCES
I. Chen P. S. Jr. and Borrevang P.: In Ifat~dhoo~ of’E.x/wiwlttuI Pharwcoloy~ (Edited hy F. A. Smith). SpringcrVerlag, Berlin, Vol. XX. Part 2 (1970) pp. 193 252. 2. Fotherhy K. and James F.: In .3Jt~1rws in Stewili Bio~i~rrnistr~ arrtl ~~~~~~/t~~~~~~~u~~~ (Edited by M. H. Briggs and G. A. Christie). Academic Press, London. Vol. 3 (1972) pp. 67- 165. 3 Jubiz W.. Meikle A. W.. Levinson R. A.. Mizutani S.. West C. D. and Tyler F. H.: jv;(,~\, Enyl J. bfctl. 283 (1970) 11 14. 4 Haque N.. Thrasher K.. Wcrk E. ti. Jr.. Knowles H. c‘. Jr. and Shotiton L. J.: J.
