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Radioimmunoassay for dexamethasone 17,21-dipropionate and its metabolites in plasma and urine after topical application
RADIOIM~UNOASSAY FOR DEXAMETHASONE 17,210DIPROPIONATE AND ITS METABOLITES IN PLASMA AND URINE AFTER TOPICAL APPLICATION Yukitaka Miyachi,a Masaru Ishihara,b Satoshi Kurihara,b Masahiko Yoshida,c Hirotoshi Masuda,c Masahito Komuro,c Kazuki Taira,c and Yasuro Kawaguchic ‘The First D,epartment of Internal Medicine and bDepartment of Dermatology, Toho University School of Medicine, Otaku, Tokyo 143; ‘Research Institute, Taiho Pharmaceutical Co. Ltd., Tokushima 771-01; Japan Correspondingauthor: Yukitaka Miyachi Received January 2‘1987 RevisedMarch 17,1988
ABSTRACT A sensitive radioimmunoassay for dexamethasone 17,21-dipropionate and its four metabolites in human plasma and urine has been developed using single anti-dexamethasone antiserum. The
antiserum was obtained by immunizing rabbits with dexamethasone3-oxime-bovine serum albumin conjugate. All of the endogenous steroids tested cross-reacted less than 0.07%. Before radioimmunoassay, dexamethasone 17,21-dipropionate and dexamethasone 17-propionate were hydrolyzed to dexamethasone, and &OH-dexamethasone 17-propionate was hydrolyzed to 68-OH-dexamethasone in 3% a~onia/methanol at 5 C for 16 h. A standard curve was established with a useful range between 0.005 and 2 ng in the case of dexamethasone, between 0.05 and 5 ng in the case of 6BOH-dexamethasone. Measurement of plasma concentrations and percent urinary excretion of the metabolites in healthy men was performed following occlusive dressing of dexamethasone 17,21dipropionate cream and ointment. The main metabolites in plasma were dexamethasone 17-propionate and dexamethasone, which increased gradually and reached maximum levels (160-200 pg/mL) at 24-32 h after application. The major metabolites observed in urine were 68-OH-dexamethasone 17-propionate and 6B-OH-dexamethasone. Total percentage of their urinary excretions within 72 h after application amounted to 0.28-0.50% of the dose administered. INTRODUCTION The measure
method not
of
steroid radioimmunoassay has been
only endogenous steroids (l-3) but
STEROIDS52/1-2
July-August 1988(137-153)
also
used
to
synthetic
137
138
Miyachi et al: RIA FOR DEXAMETHASONE
DERIVATIVES
steroids, such as betamethasone (4), prednisolone (5), dexamethasane(6)
and
methylprednisolone (7).
These steroid
drugs
are
mainly used orally and their plasma concentrations are relatively high.
In this report,
a sensitive radioimmunoassay using single
anti-dexamethasone antiserum of
dexamethasone
for determining low
17,21-dipropionate
(dexamethasone 17-propionate,
and its
concentrations
four
metabolites
dexamethasone, 6B-OH-dexamethasone
17-propionate, and 6B-OH-dexamethasone) in plasma
or urine after
topical application of dexamethasone 17,21_dipropionate
ointment
and cream is described. MATERIALS AND METHODS Chemicals [1,2,4-3H1 Dexamethasone with a specific activity of 42 Ci/ mmol was purchased from Amersham Co.(Amersham Buckinghamshire, England). Its purity was more than 95% as determined by high performance liquid chromatography (HPLC). Steroids and other chemicals used were as follows: dexamethasone 17,21_dipropionate, dexamethasone 17,21_dipropionate ointment and cream, dexamethasone 17-propionate, 6B-OH-dexamethasone 17-propionate, 68-OHdexamethasone (Taiho Pharmaceutical Co.,Tokushima, Japan), dexamethasone (Roussel Uclaf Co.,Paris, France), prednisolone, cortisol , progesterone, testosterone and estradiol (Nakarai Chemicals, LTD.,Kyoto, Japan), charcoal (Norit SX plus, Wako Pure Chemical Industries, LTD.,Osaka, Japan), dextran-T70 (Pharmacia Fine Chemicals AB,Uppsala, Sweden). and Enzymes used were as follows: B-glucuronidase TypelX arylsulfatase TypeIV (Sigma Chemical Co.,St. Louis, USA). All solvents purchased commercially were reagent grade and distilled when necessary.
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Miyachi et al: RIA FOR DEXAMETHASONE
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Antigen Preparation Dexamethasone 21-hemisuccinate (dexamethasone-21-HS) and dexamethasone-3-(O-carboxylmethyl)oxime(dexamethasone-3-oxime) were synthesized and conjugated to bovine serum albumin(BSA) by the method described by Erlanger et al(8). Based on spectrophotomerit measurements, dexamethasone-21-HS-BSA and dexamethasone-3oxime-BSA contained 16 and 6 dexamethasone residues per albumin molecule, respectively. Immunization New Zealand white rabbits (male , 3-4 kg) were immunized intradermally at multiple sites with 1 mg of dexamethasone-8SA conjugate in saline emulsified with complete Freund's adjuvant, with booster injections being given at two week intervals. Blood was collected once in 2 weeks from the marginal ear vein. Serum was harvested and frozen at -80 C in small aliquots for future use. All assays were performed with antiserum obtained 2 months after immunization. Sample Preparation One milliter of plasma sample was diluted P-fold with water and extracted twice with 15 mL of dichloromethane. The combined dichloromethane extract was washed using 5 mL of 0.1 M NaHCO -Na2 CO solution (pH 10) and 5 mL of 0.02 N HCl (pH 2) and e aaporazed. The residue redissolved in methanol(100 pL) was injected in an HPLC apparatus. One-half milliliter of urine sample was treated with 1200 units of B-glucuronidase and 6 units of arylsulfatase previously prepared in 1.5 mL of 10 mM acetate buffer(pH 5.0) at 37 C for 16 The mixture was extracted twice with 15 mL of dichlorome:hane /ethylacetate (2:l v/v). Subsequent experiments were carried out in the same manner as plasma. HPLC HPLC conditions used in this study were as follows: apparatus, Waters Model ALC/GPC 204; column, Zorbax SIL(4.6 x 250 mm,Du Pont Co.,Wilmington, USA); mobile phase, dichloromethane/methanol (95.5:4.5); flow rate, 1.0 mL/min.
139
140
Miyachietal:RiAFORDEXAMETHASONE
DERIVATIVES
The retention times of each steroids were determined by ultraviolet absorption at 280 nm and each fraction,approximately 1.5 ml, was taken to dryness under N gas at 40 C. The retention times of dexamethason$ 17,21-dipropionate and its metabolites in these conditions were as follows: dexamethasone 17,21_dipropionate, 4.5820.04 min; dexamethasone 17-propionate, 6.72+0.01 min; dexamethasone, 9.12kO.01 min; 68-OH-dexamethasone 17-propionate, 12.12+0.01 min; and 68-OH-dexamethasone, 15.39+0.11 min. Hydrolysis -of Ester Metabolites Ester metabolites, dexamethasone 17,21-dipropionate, dexamethasone 17-propionate, and 68-OH-dexamethasone l7-propionate, were dissolved in methanol saturated with N2 gas, treated with 3% ammonia/methanol(l:lO v/v), hydrolyzed at 5 C for 16 h, and then taken to dryness under N2 gas at room temperature. The hydrolysis of dexamethasone 17,21-dipropionate to dexamethasone was 95% and that of dexamethasone 17-propionate to dexamethasone was 101%. Radioimmunoassay All samples were assayed in duplicate. Equivalent extract of plasma 100 pL, equivalent extract of urine 50 $L, or standards containing 0.005-2.0 ng of dexamethasone and 0.05-5.0 ng of 68OH-dexamethasone in ch~oroform/methano~(l:l v/v) were pipetted into assay tubes, and taken to dryness under3N2 gas at 40 C. Each tube received 5000 dpm [1,2,4- H fdexamethasone in 0.5 mL diluent, followed by 1:5000 diluted antiserum(0.5 mL) raised against dexamethasone-3-oxime-BSA, giving a final dilution The assay tubes were incubated of l:lO,OOO in the assay tube. for 16 h at 4 C. Following incubation, 0.2 mL of the dextran-coated charcoal solution, which consisted of 0.5% charcoal and 0.5% dextran-T70 in 0.9% saline, was added to each tube and then the tubes were The supernatant was decanted into counting vials centrifuged. measured in a liquid scintillation counter and radioactivity (Mark-IB ,Tracor Analytic, Grove Village Illinois, USA) using Amersham Co.,~ersham Buckinghamshire, scintillator (ACS-II , The sample values were calculated by weighted fourEngland). parameter logistic curve-fitting method according to D. Rodbard Diluent for all components of the assay system was et at(g). borate-saline prepared by adding bovine gamma globulin to buffer{50 mM boric acid - 1 mM borax in 0.9% sodium chloride solution; pH 7.6) to a concentration of 1 mg/mL (3).
Miyachietal:
RIAFORDEXAMETHASONE
DERlVATiVES
141
Human Experiment Written informed consent was obtained from ten male adult volunteers. Five healthy men (24-40 years) were studied in the case of ointment application and five other healthy men (26-34 years) were studied in the case of cream application. Five grams of dexamethasone 17,21_dipropionate at a concentration of 0.1% in ointment or cream was applied to the back skin and covered with an occlusive plastic dressing at 9:OO AR. The dressing was left in place for 24 h. Blood was collected at 0, 4, 8, 12, 24, 48, Urine and 72 h and plasma was obtained after centrifugation. was collected for 24 h before application and for 3 days at intervals of 24 h after application, and then urinary volume was measured. Each sample was stored frozen until analysis. RESULTS Specificity Shown
in
Table 1 are experimental values for
cross-reactivity dexamethasone)
(measured of
at 50%
displacement
the
of
percent
t1,2,4-3Hl
various steroids for antisera raised
against
dexamethasone-21-HS-BSA and dexamethasone-3-oxime-BSA. Antiserum reactivity with ever,
to
dexamethasone-21-HS-BSA
with cortisol and
relatively high
both dexamethasone 17-propionate and antiserum
cross-reactivity
showed
1.1% cross-
cross-reactivity
betamethasone.
How-
to dexamethasone-3-oxime-BSA showed only
0.07%
with
cortisol and
moderate
cross-reactivity
(13.5%) with 6e-OH-dexamethasone , suggesting high specificity for dexamethasone.
The dexamethasone-3-oxime-BSA antiserum was used
for further studies.
1.
of
Dexamethasone
Comparison
0.04
0.2
Prednisolone
0.03 c 0.0004 < 0.0004
Progesterone
Testosterone
Estradiot
< 0.0005
0.03
0.2
1.1
5.5
87.2
1.2
0.9
46.7
a.9
100
Anti-Dexamethasone21-HS-BSA
aDetermined at 50% displacement,
0.07
Cortisol
Endogenous
6.2
Betamethasone
13.5
0.6
Dexa~thasone-17-propionate
6B-OH-dexamethasone 17-propionate
100
against
Cross-Reactivity (%)"
0.02
6f3-OH-dexamethasone
Antisera
Conjugates
of
Anti-Dexamethasone3-oxime-BSA
Albumin
Specificity
Oexamethasone-17,21-dipropionate
Dexamethasone
Synthetic
Steroids
Different
Table
Miyachi et al: RIA FOR DEXAMETHASONE
DERIVATIVES
143
Standard Curve A typical standard curve by the logistic method is shown Figure used,
1. the
When
5000 dpm (0.03 ng) of
was
useful range of dexamethasone was established between
0.005 and 2 ng,
and that of 6B-OH-dexamethasone between 0.05 and
Sensitivity of dexamethasone and
5 ng.
13Hldexamethasone
in
68-OH-dexamethasone was
0.005 and 0.05 ng, respectively. Blank and Precision -For
volunteers not receiving dexamethasone
17,21-dipropio-
nate, blank values of dexamethasone in plasma and urine were less than 0.03 and 0.06 ng,
respectively.
Those of 6B-OH-dexametha-
sone in urine were less than 0.15 ng. For assay For
the plasma dexamethasone assay,
coefficient of variation was 7.9 and
the urinary dexamethasone assay,
8.9%,
the intra- and
respectively.
7.2%,
inter-
respectively.
these values were 6.3
and
For the urinary 6B-OH-dexamethasone assay,
these values were 9.4 and 8.8%, respectively. Recovery The recovery of dexamethasone 17,21_dipropionate, dexamethasone
17-propionate,
and dexamethasone was more than
dichloromethane extraction. 17-propionate
The
90%
after
recovery of 66-OH-dexamethasone
and 6#3-OH-dexamethasonewere more than 95 and
65%
Figure
1.
0.01
Dexamethasone Dexamethasone propionate, were assayed
0.005
Dexanethasone 17-proplonote
0.35
0.1
0.2
0.5
I
68-OH-dexmethasone
2
5
and 6B-OH-dexamethasone standard 17,21-dipropionate, dexamethasone and hf3-OH-dexamethasone 17-propionate after hydrolysis.
0.02
Dexanethasone
10
w/tube
curve. 17-
f
Miyachi et al: RIA FOR DEXAMETHASONE
after
the
DERIVATIVES
dichloromethane/ethylacetate
respectively.
(2:l
145
v/v)
extraction,
The recovery of dexamethasone after HPLC was about
80%. Human Experiment Plasma levels of dexamethasone 17,21_dipropionate, dexamethasone 17-propionate, of
and dexamethasone after topical application
dexamethasone 17,21_dipropionate ointment or cream to healthy
volunteers
are shown in Figure 2.
The concentrations of
dexa-
methasone 17,21_dipropionate were less than 100 pg/mL during test period. gradually tion.
the
The levels of dexamethasone 17-propionate increased
and reached maxima within 24 h after topical
Maximum
applica-
concentrations of dexamethasone 17-propionate in
groups treated with dexamethasone 17,21_dipropionate ointment and cream were 152k45 and 166+15 pg/mL, respectively. The levels of dexamethasone in groups treated with
dexamethasone
17,21_dipropionate ointment
and cream reached maxima (209t60 and
202k59 pg/mL, respectively)
at 32 h
and then decreased gradual-
ly. Urinary
excretions of dexamethasone 17,21_dipropionate
and
its metabolites after topical application of dexamethasone 17,21dipropionate ointment or cream to healthy volunteers are shown in Figure
3.
Total percentage of dexamethasone 17,21_dipropionate
and its metabolites excreted in the urine within 72 h amounted to
146
Miyachi et al: RIA FOR DEXAMETHASONE
(A) Ointment
DERIVATIVES
(B) Cream
Dexamethasane 17,21-dipropionate
Oexamethasone 17,21-dipropionate
200 150
0
4812
242832
48
T
72
ipplicatiok
Dexamethasone
t
<
Application
Application T
0
4812
242832
7
48
72
0
4812 ‘
Application
242832
48
72
f
Application
Time (hf Figure
2.
levels of dexamethasone 17,21-dipropionate and its metabolites after single topical application (ODT) of dexamethasone 17,21-dipropionate ointment (A) or cream (B) for 24 h in healthy men. Values are means and SE for 5 volunteers. Plasma
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Miyachi
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al:
RIA
FOR
DEXAMETHASONE
I 0 k3 m S
147
DERIVATIVES
Dexmthasone 17,21-dlpmionote Dexmthasone 17-propionflte Dexmethasone BE-OH-dexmthosone 17-proplonate Be-OH-dexanethosone
(A) Ointment
-
s
”
c ?1
0 - 24
24 -
48
48 -
72
h
6 9 ADLJllcotlon
:
Y
z
I?
3
0.10
0.05
0
0 - 24
24 - 48
48 - 72
h
< * Awllcatlon
Figure
3. Urinary excretion of dexamethasone 17,21-dipropionate and its metabolites after single topical application (ODT) of dexamethasone 17,21_dipropionate ointment (A) or cream (B) for 24 h in healthy men. Values are means and SE for 5 volunteers.
148
Miyachi et al: RIA FOR DEXAMETHASONE
0
pddt
0
14 12
DERIVATIVES
T
Oint~nt
Cream
10
8
~ we
72
24
h
Application Figure
4.
Plasma levels of cortisol after single topical tion (ODT) of dexamethasone 17,21-dipropionate Values are or cream for 24 h in healthy men. SE for 5 volunteers.
STEROIDS 5211-2
applicaointment means and
July-August
1988
Miyachi
et al: RIA FOR DEXAMETHASONE
0.28kO.038
of
dose
in
the
17,21_dipropionate ointment treated
149
DERIVATIVES
group
treated
with
dexamethasone
and 0.5OkO.O9% of dose
in the group The
with dexamethasone 17,21-dipropionate cream.
metabolites in
the urine were 6s-OH-dexamethasone
main
17-propionate
and 68-OH-dexamethasone. In accord with low absorption, level
was
no change of plasma cortisol
observed after a single application of
dexamethasone
17,21_dipropionate ointment or cream (Fig. 4).
DISCUSSION A
sensitive
propionate
and
radioimmunoassay for
dexamethasone
its four metabolites in human plasma
using single anti-dexamethasone antiserum is reported.
I7,21-diand
The anti-
serum raised against dexamethasone-3-oxime-BSA reacts with methasone
and
dexamethasone. with
also
shows
13.5% cross-reactivity
This antiserum shows
cortisol,indicating
steroid is negligible.
urine
with
dexa6&OH-
only 0.07% cross-reactivity
that interference by
this
endogenous
The cross-reactivity of the ester metabo-
lites, dexamethasone 17,21-dipropionate, dexamethasone 17-propionate, The
and 6@-OH-dexamethasone 17-propionate, was relatively low.
propionic acid moiety at the 17 or 21 position of dexametha-
sone may disturb the binding to the antibody.
150
Miyachietat:
In this study,
RIAFORDEXAMETHASONE
DERIVATIVES
the method of radioimmunoassay combined with
chemical pretreatment was developed.
The
pretreatment,
which
included the organic extraction, HPLC fractionation, and hydrolysis
of
useful
the 17- or 21-propionate of
the ester
metabolites,
for determination of these metabolites with
high
is
sensi-
tivity and accuracy. The
plasma
'levelsand urinary excretions of
17,21-dipropionate and its metabolites were low, only
dexamethasone suggesting that
small amounts of dexamethasone 17,21-dipropionate
sorbed gradually through the transdermal system. low
absorption,
application
of
plasma
are
In accord with
cortisol did not change after a
dexamethasone
17,21-dipropionate
ab-
single
ointment
or
cream. The
value
of topical application of
dipropionate in the treatment
dexamethasone
17,21-
of various skin diseases has
been
well established (lO,ll), and the present study clearly indicates that
this type of steroid therapy has few possibilities of
sys-
temic side-effects. The
low
plasma levels of dexamethasone
17,21-dipropionate
and its metabolites remained for a long time,
showing that dexa-
methasone corneum
17,21-dipropionate
which accumulated in
the
may be absorbed continuously even after removal
stratum of
the
dressing.
S~EROlDS52/1-2
July-August1988
Miyachi et al: RIA FOR DEXAMETHASONE
The
amounts
of
DERIVATIVES
151
dexamethasone 17,21_dipropionate
and
its
metabolites excreted in the urine were lower in the dexamethasone 17,21_dipropionate treated
group,
ointment-treated group
than
in
the
cream-
suggesting that the absorption rate of the cream
is higher than that of the ointment. The compared
plasma dexamethasone 17,21_dipropionate level was with the plasma dexamethasone 17-propionate
methasone levels. to
and
low, dexa-
Dexamethasone 17,21_dipropionate is converted
dexamethasone via dexamethasone 17-propionate by enzymic
drolysis,
with esterase distributed in the skin,
hy-
plasma, liver,
or other tissues at relatively high speed (12). The present study of clinical pharmacology using a sensitive and nate
specific radioimmunoassay for dexamethasone and
17,21-dipropio-
its metabolites can be useful to make the best use
of
steroid therapy using a topical application. TRIVIAL AND IUPAC NAMES Steroid Dexamethasone 17,21-dipropionate: 9a-fluoro-116,17,21trihydroxy-16a-methyl-pregna-1,4-diene-3,2O-dione-l7,21dipropionate Metabolites Dexamethasone 17-propionate: 9a-fluoro-116,17,21-trihydroxy16a-methyl-pregna-l,4-diene-3,20-dione-17-propionate Dexamethasone: 9a-fluoro-118,17,21-trihydroxy-16a-methylpregna-1,4-diene-3,20-dione 6B-OH-dexamethasone 17-propionate: 9a-fluoro-6B,ll6,17,21tetrahydroxy-16a-methyl-pregna-1,4-diene-3,2O-dione-l7propionate 6f3-OH-dexamethasone:9a-fluoro-6B,llg,l7,21-tetrahydroxy16a-methyl-pregna-1,4-diene-3,20-dione
Miyachi et ai: RIA FOR D~~AMETHASQN~
152
DERIVATIVES
Endogenous Cortisol: 11~,17~,2l-trihy~roxy~4-pregnene-3,ZO-diane Progesterone: 4-pregnene-3~20"djone Testosterone: 178-hydroxy-4-androstene-3-one Estradiol: 1,3,5,(10)-estratriene-3,17B-diol Synthetic Eetamethasone: 9~-fluoro-ll~,l7,Zl-trihydroxy-l68-m~thy~preg~a-1~4-d~ene-3,20-dione Prednisolone: 11~ ,17~21-tribydroxy-pregna-~~4-diene-3,20~ dione Methylprednisolone: 11~,17,21-trihydroxy-6~-methyl pregna1,4-diene 3,20-dione REFERENCES
2. 2. 3. 4.
5, 6. 7. 8. 9, 10,
Abraham GE (1969). Solid-phase radioimmunoassay of estradiol-17B. J CLIN ENDOCRINOL METAB 29:866-870. Furuyama S, Mayes DM, and Nugent CA(197v. Radioimmunoassay for plasma testosterone. STEROIDS 16:415-428, Ruder HJ, Guy RL, and Lipsett ME(l972f. ~radioimmunoassay for cortisof in plasma and urine. J CLfN E~DOCR~NOL METAE 35:219-224, Miyach?f, Yotsumoto H, Kano T, Mizuchi A, Muto T, and Yanagibashi K(1979). Blood levels of synthetic glucocorticoids after administration by various routes. J ENDOCRINOL 82:149-157. zlburn WA and Buller RH(l973). Radioimmunoassay for prednisolone. STEROIDS 21~833-846. Meikle AW, Lagerquist LK and Tyler F~(l973). A plasma dexamethasone radioimmunoassay. STEROIDS 2:193-202. Colburn WA and Buller RW(1973). Radioimmunoassay for methylprednisolone (MEDRQL). STEROIDS 22:687-698. Erlanger BF, Borek F, Beiser SM, and Lzberman S(1957). Steroid-protein conjugates. J BIOL CHEM 228~713-727. Rodbard D and Hutt DM(l974~. S~posium ~rad~o~mmunoassay and related procedure in medicine, Int Atomic Energy Agency, tiienna,Austria 165. Ishihara M and Ito M(1984). Studies on the vasoconstrictor activity of Dexamethasone 17,21-dipropionate, a new synthetic topical corticosteroid(1). Yakuri to Chiryo -12:1259-1268.
STEROlDS
52/-l-2
July-August 1988
Miyachietal:RIAFORDEXAMETHASONE
11.
12.
DERIVATIVES
Takeda K, Kukita Y, Asada Y, Shimano S, Watanabe Y, and Miyachi Y(1987). Studies on clinical efficacy and percutaneous systemic and/or topical effects of a new synthetic corticoid. 17th World Congress of Dermatology, Berlin 325. Shindo T, Tsukamoto Y, Yasuda Y, Marunaka T, Umeno Y, Minami Y, and Tanaka M(1984). Studies on the metabolic fate of Dexamethasone 17,21-dipropionate (THS-101)(S). Oyo Yakuri -27:1057-1072.
153
ABSTRACT A sensitive radioimmunoassay for dexamethasone 17,21-dipropionate and its four metabolites in human plasma and urine has been developed using single anti-dexamethasone antiserum. The
antiserum was obtained by immunizing rabbits with dexamethasone3-oxime-bovine serum albumin conjugate. All of the endogenous steroids tested cross-reacted less than 0.07%. Before radioimmunoassay, dexamethasone 17,21-dipropionate and dexamethasone 17-propionate were hydrolyzed to dexamethasone, and &OH-dexamethasone 17-propionate was hydrolyzed to 68-OH-dexamethasone in 3% a~onia/methanol at 5 C for 16 h. A standard curve was established with a useful range between 0.005 and 2 ng in the case of dexamethasone, between 0.05 and 5 ng in the case of 6BOH-dexamethasone. Measurement of plasma concentrations and percent urinary excretion of the metabolites in healthy men was performed following occlusive dressing of dexamethasone 17,21dipropionate cream and ointment. The main metabolites in plasma were dexamethasone 17-propionate and dexamethasone, which increased gradually and reached maximum levels (160-200 pg/mL) at 24-32 h after application. The major metabolites observed in urine were 68-OH-dexamethasone 17-propionate and 6B-OH-dexamethasone. Total percentage of their urinary excretions within 72 h after application amounted to 0.28-0.50% of the dose administered. INTRODUCTION The measure
method not
of
steroid radioimmunoassay has been
only endogenous steroids (l-3) but
STEROIDS52/1-2
July-August 1988(137-153)
also
used
to
synthetic
137
138
Miyachi et al: RIA FOR DEXAMETHASONE
DERIVATIVES
steroids, such as betamethasone (4), prednisolone (5), dexamethasane(6)
and
methylprednisolone (7).
These steroid
drugs
are
mainly used orally and their plasma concentrations are relatively high.
In this report,
a sensitive radioimmunoassay using single
anti-dexamethasone antiserum of
dexamethasone
for determining low
17,21-dipropionate
(dexamethasone 17-propionate,
and its
concentrations
four
metabolites
dexamethasone, 6B-OH-dexamethasone
17-propionate, and 6B-OH-dexamethasone) in plasma
or urine after
topical application of dexamethasone 17,21_dipropionate
ointment
and cream is described. MATERIALS AND METHODS Chemicals [1,2,4-3H1 Dexamethasone with a specific activity of 42 Ci/ mmol was purchased from Amersham Co.(Amersham Buckinghamshire, England). Its purity was more than 95% as determined by high performance liquid chromatography (HPLC). Steroids and other chemicals used were as follows: dexamethasone 17,21_dipropionate, dexamethasone 17,21_dipropionate ointment and cream, dexamethasone 17-propionate, 6B-OH-dexamethasone 17-propionate, 68-OHdexamethasone (Taiho Pharmaceutical Co.,Tokushima, Japan), dexamethasone (Roussel Uclaf Co.,Paris, France), prednisolone, cortisol , progesterone, testosterone and estradiol (Nakarai Chemicals, LTD.,Kyoto, Japan), charcoal (Norit SX plus, Wako Pure Chemical Industries, LTD.,Osaka, Japan), dextran-T70 (Pharmacia Fine Chemicals AB,Uppsala, Sweden). and Enzymes used were as follows: B-glucuronidase TypelX arylsulfatase TypeIV (Sigma Chemical Co.,St. Louis, USA). All solvents purchased commercially were reagent grade and distilled when necessary.
STEROIDS
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1988
Miyachi et al: RIA FOR DEXAMETHASONE
DERIVATIVES
Antigen Preparation Dexamethasone 21-hemisuccinate (dexamethasone-21-HS) and dexamethasone-3-(O-carboxylmethyl)oxime(dexamethasone-3-oxime) were synthesized and conjugated to bovine serum albumin(BSA) by the method described by Erlanger et al(8). Based on spectrophotomerit measurements, dexamethasone-21-HS-BSA and dexamethasone-3oxime-BSA contained 16 and 6 dexamethasone residues per albumin molecule, respectively. Immunization New Zealand white rabbits (male , 3-4 kg) were immunized intradermally at multiple sites with 1 mg of dexamethasone-8SA conjugate in saline emulsified with complete Freund's adjuvant, with booster injections being given at two week intervals. Blood was collected once in 2 weeks from the marginal ear vein. Serum was harvested and frozen at -80 C in small aliquots for future use. All assays were performed with antiserum obtained 2 months after immunization. Sample Preparation One milliter of plasma sample was diluted P-fold with water and extracted twice with 15 mL of dichloromethane. The combined dichloromethane extract was washed using 5 mL of 0.1 M NaHCO -Na2 CO solution (pH 10) and 5 mL of 0.02 N HCl (pH 2) and e aaporazed. The residue redissolved in methanol(100 pL) was injected in an HPLC apparatus. One-half milliliter of urine sample was treated with 1200 units of B-glucuronidase and 6 units of arylsulfatase previously prepared in 1.5 mL of 10 mM acetate buffer(pH 5.0) at 37 C for 16 The mixture was extracted twice with 15 mL of dichlorome:hane /ethylacetate (2:l v/v). Subsequent experiments were carried out in the same manner as plasma. HPLC HPLC conditions used in this study were as follows: apparatus, Waters Model ALC/GPC 204; column, Zorbax SIL(4.6 x 250 mm,Du Pont Co.,Wilmington, USA); mobile phase, dichloromethane/methanol (95.5:4.5); flow rate, 1.0 mL/min.
139
140
Miyachietal:RiAFORDEXAMETHASONE
DERIVATIVES
The retention times of each steroids were determined by ultraviolet absorption at 280 nm and each fraction,approximately 1.5 ml, was taken to dryness under N gas at 40 C. The retention times of dexamethason$ 17,21-dipropionate and its metabolites in these conditions were as follows: dexamethasone 17,21_dipropionate, 4.5820.04 min; dexamethasone 17-propionate, 6.72+0.01 min; dexamethasone, 9.12kO.01 min; 68-OH-dexamethasone 17-propionate, 12.12+0.01 min; and 68-OH-dexamethasone, 15.39+0.11 min. Hydrolysis -of Ester Metabolites Ester metabolites, dexamethasone 17,21-dipropionate, dexamethasone 17-propionate, and 68-OH-dexamethasone l7-propionate, were dissolved in methanol saturated with N2 gas, treated with 3% ammonia/methanol(l:lO v/v), hydrolyzed at 5 C for 16 h, and then taken to dryness under N2 gas at room temperature. The hydrolysis of dexamethasone 17,21-dipropionate to dexamethasone was 95% and that of dexamethasone 17-propionate to dexamethasone was 101%. Radioimmunoassay All samples were assayed in duplicate. Equivalent extract of plasma 100 pL, equivalent extract of urine 50 $L, or standards containing 0.005-2.0 ng of dexamethasone and 0.05-5.0 ng of 68OH-dexamethasone in ch~oroform/methano~(l:l v/v) were pipetted into assay tubes, and taken to dryness under3N2 gas at 40 C. Each tube received 5000 dpm [1,2,4- H fdexamethasone in 0.5 mL diluent, followed by 1:5000 diluted antiserum(0.5 mL) raised against dexamethasone-3-oxime-BSA, giving a final dilution The assay tubes were incubated of l:lO,OOO in the assay tube. for 16 h at 4 C. Following incubation, 0.2 mL of the dextran-coated charcoal solution, which consisted of 0.5% charcoal and 0.5% dextran-T70 in 0.9% saline, was added to each tube and then the tubes were The supernatant was decanted into counting vials centrifuged. measured in a liquid scintillation counter and radioactivity (Mark-IB ,Tracor Analytic, Grove Village Illinois, USA) using Amersham Co.,~ersham Buckinghamshire, scintillator (ACS-II , The sample values were calculated by weighted fourEngland). parameter logistic curve-fitting method according to D. Rodbard Diluent for all components of the assay system was et at(g). borate-saline prepared by adding bovine gamma globulin to buffer{50 mM boric acid - 1 mM borax in 0.9% sodium chloride solution; pH 7.6) to a concentration of 1 mg/mL (3).
Miyachietal:
RIAFORDEXAMETHASONE
DERlVATiVES
141
Human Experiment Written informed consent was obtained from ten male adult volunteers. Five healthy men (24-40 years) were studied in the case of ointment application and five other healthy men (26-34 years) were studied in the case of cream application. Five grams of dexamethasone 17,21_dipropionate at a concentration of 0.1% in ointment or cream was applied to the back skin and covered with an occlusive plastic dressing at 9:OO AR. The dressing was left in place for 24 h. Blood was collected at 0, 4, 8, 12, 24, 48, Urine and 72 h and plasma was obtained after centrifugation. was collected for 24 h before application and for 3 days at intervals of 24 h after application, and then urinary volume was measured. Each sample was stored frozen until analysis. RESULTS Specificity Shown
in
Table 1 are experimental values for
cross-reactivity dexamethasone)
(measured of
at 50%
displacement
the
of
percent
t1,2,4-3Hl
various steroids for antisera raised
against
dexamethasone-21-HS-BSA and dexamethasone-3-oxime-BSA. Antiserum reactivity with ever,
to
dexamethasone-21-HS-BSA
with cortisol and
relatively high
both dexamethasone 17-propionate and antiserum
cross-reactivity
showed
1.1% cross-
cross-reactivity
betamethasone.
How-
to dexamethasone-3-oxime-BSA showed only
0.07%
with
cortisol and
moderate
cross-reactivity
(13.5%) with 6e-OH-dexamethasone , suggesting high specificity for dexamethasone.
The dexamethasone-3-oxime-BSA antiserum was used
for further studies.
1.
of
Dexamethasone
Comparison
0.04
0.2
Prednisolone
0.03 c 0.0004 < 0.0004
Progesterone
Testosterone
Estradiot
< 0.0005
0.03
0.2
1.1
5.5
87.2
1.2
0.9
46.7
a.9
100
Anti-Dexamethasone21-HS-BSA
aDetermined at 50% displacement,
0.07
Cortisol
Endogenous
6.2
Betamethasone
13.5
0.6
Dexa~thasone-17-propionate
6B-OH-dexamethasone 17-propionate
100
against
Cross-Reactivity (%)"
0.02
6f3-OH-dexamethasone
Antisera
Conjugates
of
Anti-Dexamethasone3-oxime-BSA
Albumin
Specificity
Oexamethasone-17,21-dipropionate
Dexamethasone
Synthetic
Steroids
Different
Table
Miyachi et al: RIA FOR DEXAMETHASONE
DERIVATIVES
143
Standard Curve A typical standard curve by the logistic method is shown Figure used,
1. the
When
5000 dpm (0.03 ng) of
was
useful range of dexamethasone was established between
0.005 and 2 ng,
and that of 6B-OH-dexamethasone between 0.05 and
Sensitivity of dexamethasone and
5 ng.
13Hldexamethasone
in
68-OH-dexamethasone was
0.005 and 0.05 ng, respectively. Blank and Precision -For
volunteers not receiving dexamethasone
17,21-dipropio-
nate, blank values of dexamethasone in plasma and urine were less than 0.03 and 0.06 ng,
respectively.
Those of 6B-OH-dexametha-
sone in urine were less than 0.15 ng. For assay For
the plasma dexamethasone assay,
coefficient of variation was 7.9 and
the urinary dexamethasone assay,
8.9%,
the intra- and
respectively.
7.2%,
inter-
respectively.
these values were 6.3
and
For the urinary 6B-OH-dexamethasone assay,
these values were 9.4 and 8.8%, respectively. Recovery The recovery of dexamethasone 17,21_dipropionate, dexamethasone
17-propionate,
and dexamethasone was more than
dichloromethane extraction. 17-propionate
The
90%
after
recovery of 66-OH-dexamethasone
and 6#3-OH-dexamethasonewere more than 95 and
65%
Figure
1.
0.01
Dexamethasone Dexamethasone propionate, were assayed
0.005
Dexanethasone 17-proplonote
0.35
0.1
0.2
0.5
I
68-OH-dexmethasone
2
5
and 6B-OH-dexamethasone standard 17,21-dipropionate, dexamethasone and hf3-OH-dexamethasone 17-propionate after hydrolysis.
0.02
Dexanethasone
10
w/tube
curve. 17-
f
Miyachi et al: RIA FOR DEXAMETHASONE
after
the
DERIVATIVES
dichloromethane/ethylacetate
respectively.
(2:l
145
v/v)
extraction,
The recovery of dexamethasone after HPLC was about
80%. Human Experiment Plasma levels of dexamethasone 17,21_dipropionate, dexamethasone 17-propionate, of
and dexamethasone after topical application
dexamethasone 17,21_dipropionate ointment or cream to healthy
volunteers
are shown in Figure 2.
The concentrations of
dexa-
methasone 17,21_dipropionate were less than 100 pg/mL during test period. gradually tion.
the
The levels of dexamethasone 17-propionate increased
and reached maxima within 24 h after topical
Maximum
applica-
concentrations of dexamethasone 17-propionate in
groups treated with dexamethasone 17,21_dipropionate ointment and cream were 152k45 and 166+15 pg/mL, respectively. The levels of dexamethasone in groups treated with
dexamethasone
17,21_dipropionate ointment
and cream reached maxima (209t60 and
202k59 pg/mL, respectively)
at 32 h
and then decreased gradual-
ly. Urinary
excretions of dexamethasone 17,21_dipropionate
and
its metabolites after topical application of dexamethasone 17,21dipropionate ointment or cream to healthy volunteers are shown in Figure
3.
Total percentage of dexamethasone 17,21_dipropionate
and its metabolites excreted in the urine within 72 h amounted to
146
Miyachi et al: RIA FOR DEXAMETHASONE
(A) Ointment
DERIVATIVES
(B) Cream
Dexamethasane 17,21-dipropionate
Oexamethasone 17,21-dipropionate
200 150
0
4812
242832
48
T
72
ipplicatiok
Dexamethasone
t
<
Application
Application T
0
4812
242832
7
48
72
0
4812 ‘
Application
242832
48
72
f
Application
Time (hf Figure
2.
levels of dexamethasone 17,21-dipropionate and its metabolites after single topical application (ODT) of dexamethasone 17,21-dipropionate ointment (A) or cream (B) for 24 h in healthy men. Values are means and SE for 5 volunteers. Plasma
STEROIDS
52/l-2
Juiy-August 1988
Miyachi
et
al:
RIA
FOR
DEXAMETHASONE
I 0 k3 m S
147
DERIVATIVES
Dexmthasone 17,21-dlpmionote Dexmthasone 17-propionflte Dexmethasone BE-OH-dexmthosone 17-proplonate Be-OH-dexanethosone
(A) Ointment
-
s
”
c ?1
0 - 24
24 -
48
48 -
72
h
6 9 ADLJllcotlon
:
Y
z
I?
3
0.10
0.05
0
0 - 24
24 - 48
48 - 72
h
< * Awllcatlon
Figure
3. Urinary excretion of dexamethasone 17,21-dipropionate and its metabolites after single topical application (ODT) of dexamethasone 17,21_dipropionate ointment (A) or cream (B) for 24 h in healthy men. Values are means and SE for 5 volunteers.
148
Miyachi et al: RIA FOR DEXAMETHASONE
0
pddt
0
14 12
DERIVATIVES
T
Oint~nt
Cream
10
8
~ we
72
24
h
Application Figure
4.
Plasma levels of cortisol after single topical tion (ODT) of dexamethasone 17,21-dipropionate Values are or cream for 24 h in healthy men. SE for 5 volunteers.
STEROIDS 5211-2
applicaointment means and
July-August
1988
Miyachi
et al: RIA FOR DEXAMETHASONE
0.28kO.038
of
dose
in
the
17,21_dipropionate ointment treated
149
DERIVATIVES
group
treated
with
dexamethasone
and 0.5OkO.O9% of dose
in the group The
with dexamethasone 17,21-dipropionate cream.
metabolites in
the urine were 6s-OH-dexamethasone
main
17-propionate
and 68-OH-dexamethasone. In accord with low absorption, level
was
no change of plasma cortisol
observed after a single application of
dexamethasone
17,21_dipropionate ointment or cream (Fig. 4).
DISCUSSION A
sensitive
propionate
and
radioimmunoassay for
dexamethasone
its four metabolites in human plasma
using single anti-dexamethasone antiserum is reported.
I7,21-diand
The anti-
serum raised against dexamethasone-3-oxime-BSA reacts with methasone
and
dexamethasone. with
also
shows
13.5% cross-reactivity
This antiserum shows
cortisol,indicating
steroid is negligible.
urine
with
dexa6&OH-
only 0.07% cross-reactivity
that interference by
this
endogenous
The cross-reactivity of the ester metabo-
lites, dexamethasone 17,21-dipropionate, dexamethasone 17-propionate, The
and 6@-OH-dexamethasone 17-propionate, was relatively low.
propionic acid moiety at the 17 or 21 position of dexametha-
sone may disturb the binding to the antibody.
150
Miyachietat:
In this study,
RIAFORDEXAMETHASONE
DERIVATIVES
the method of radioimmunoassay combined with
chemical pretreatment was developed.
The
pretreatment,
which
included the organic extraction, HPLC fractionation, and hydrolysis
of
useful
the 17- or 21-propionate of
the ester
metabolites,
for determination of these metabolites with
high
is
sensi-
tivity and accuracy. The
plasma
'levelsand urinary excretions of
17,21-dipropionate and its metabolites were low, only
dexamethasone suggesting that
small amounts of dexamethasone 17,21-dipropionate
sorbed gradually through the transdermal system. low
absorption,
application
of
plasma
are
In accord with
cortisol did not change after a
dexamethasone
17,21-dipropionate
ab-
single
ointment
or
cream. The
value
of topical application of
dipropionate in the treatment
dexamethasone
17,21-
of various skin diseases has
been
well established (lO,ll), and the present study clearly indicates that
this type of steroid therapy has few possibilities of
sys-
temic side-effects. The
low
plasma levels of dexamethasone
17,21-dipropionate
and its metabolites remained for a long time,
showing that dexa-
methasone corneum
17,21-dipropionate
which accumulated in
the
may be absorbed continuously even after removal
stratum of
the
dressing.
S~EROlDS52/1-2
July-August1988
Miyachi et al: RIA FOR DEXAMETHASONE
The
amounts
of
DERIVATIVES
151
dexamethasone 17,21_dipropionate
and
its
metabolites excreted in the urine were lower in the dexamethasone 17,21_dipropionate treated
group,
ointment-treated group
than
in
the
cream-
suggesting that the absorption rate of the cream
is higher than that of the ointment. The compared
plasma dexamethasone 17,21_dipropionate level was with the plasma dexamethasone 17-propionate
methasone levels. to
and
low, dexa-
Dexamethasone 17,21_dipropionate is converted
dexamethasone via dexamethasone 17-propionate by enzymic
drolysis,
with esterase distributed in the skin,
hy-
plasma, liver,
or other tissues at relatively high speed (12). The present study of clinical pharmacology using a sensitive and nate
specific radioimmunoassay for dexamethasone and
17,21-dipropio-
its metabolites can be useful to make the best use
of
steroid therapy using a topical application. TRIVIAL AND IUPAC NAMES Steroid Dexamethasone 17,21-dipropionate: 9a-fluoro-116,17,21trihydroxy-16a-methyl-pregna-1,4-diene-3,2O-dione-l7,21dipropionate Metabolites Dexamethasone 17-propionate: 9a-fluoro-116,17,21-trihydroxy16a-methyl-pregna-l,4-diene-3,20-dione-17-propionate Dexamethasone: 9a-fluoro-118,17,21-trihydroxy-16a-methylpregna-1,4-diene-3,20-dione 6B-OH-dexamethasone 17-propionate: 9a-fluoro-6B,ll6,17,21tetrahydroxy-16a-methyl-pregna-1,4-diene-3,2O-dione-l7propionate 6f3-OH-dexamethasone:9a-fluoro-6B,llg,l7,21-tetrahydroxy16a-methyl-pregna-1,4-diene-3,20-dione
Miyachi et ai: RIA FOR D~~AMETHASQN~
152
DERIVATIVES
Endogenous Cortisol: 11~,17~,2l-trihy~roxy~4-pregnene-3,ZO-diane Progesterone: 4-pregnene-3~20"djone Testosterone: 178-hydroxy-4-androstene-3-one Estradiol: 1,3,5,(10)-estratriene-3,17B-diol Synthetic Eetamethasone: 9~-fluoro-ll~,l7,Zl-trihydroxy-l68-m~thy~preg~a-1~4-d~ene-3,20-dione Prednisolone: 11~ ,17~21-tribydroxy-pregna-~~4-diene-3,20~ dione Methylprednisolone: 11~,17,21-trihydroxy-6~-methyl pregna1,4-diene 3,20-dione REFERENCES
2. 2. 3. 4.
5, 6. 7. 8. 9, 10,
Abraham GE (1969). Solid-phase radioimmunoassay of estradiol-17B. J CLIN ENDOCRINOL METAB 29:866-870. Furuyama S, Mayes DM, and Nugent CA(197v. Radioimmunoassay for plasma testosterone. STEROIDS 16:415-428, Ruder HJ, Guy RL, and Lipsett ME(l972f. ~radioimmunoassay for cortisof in plasma and urine. J CLfN E~DOCR~NOL METAE 35:219-224, Miyach?f, Yotsumoto H, Kano T, Mizuchi A, Muto T, and Yanagibashi K(1979). Blood levels of synthetic glucocorticoids after administration by various routes. J ENDOCRINOL 82:149-157. zlburn WA and Buller RH(l973). Radioimmunoassay for prednisolone. STEROIDS 21~833-846. Meikle AW, Lagerquist LK and Tyler F~(l973). A plasma dexamethasone radioimmunoassay. STEROIDS 2:193-202. Colburn WA and Buller RW(1973). Radioimmunoassay for methylprednisolone (MEDRQL). STEROIDS 22:687-698. Erlanger BF, Borek F, Beiser SM, and Lzberman S(1957). Steroid-protein conjugates. J BIOL CHEM 228~713-727. Rodbard D and Hutt DM(l974~. S~posium ~rad~o~mmunoassay and related procedure in medicine, Int Atomic Energy Agency, tiienna,Austria 165. Ishihara M and Ito M(1984). Studies on the vasoconstrictor activity of Dexamethasone 17,21-dipropionate, a new synthetic topical corticosteroid(1). Yakuri to Chiryo -12:1259-1268.
STEROlDS
52/-l-2
July-August 1988
Miyachietal:RIAFORDEXAMETHASONE
11.
12.
DERIVATIVES
Takeda K, Kukita Y, Asada Y, Shimano S, Watanabe Y, and Miyachi Y(1987). Studies on clinical efficacy and percutaneous systemic and/or topical effects of a new synthetic corticoid. 17th World Congress of Dermatology, Berlin 325. Shindo T, Tsukamoto Y, Yasuda Y, Marunaka T, Umeno Y, Minami Y, and Tanaka M(1984). Studies on the metabolic fate of Dexamethasone 17,21-dipropionate (THS-101)(S). Oyo Yakuri -27:1057-1072.
153