Radioimmunoassay for fluoxymesterone (halotestin®

43 RADIOIMMUNOASSAY

FOR FLUOXYMESTERONE

(HALOTESTI8)

W. A. Colburn Control Received:

Analytical Research and Development The Upjohn Company Kalamazoo, Michigan 49001

1014174

ABSTRACT A specific, sensitive, precise and accurate radioimmunoassay has 9u-fluoro-116,17B-dihydroxy-17been developed for fluoxymesterone, methyl-4-androsten-3-one (Halotestinq. The method is capable of detecting 25 picograms of drug in 0.1 ml of unextracted serum. The primary antibody was prepared against fluoxymesterone 3-[O-(carboxymethoxime)] (CMO) bovine serum albumin. The specificity of the assay is greatly influenced by the hydroxyl group at position 11 and the methyl group at position 17. Physiological levels of endogenous steroids did not cross-react significantly with the primary antibody. Blood levels of fluoxymesterone were determined in both human subjects and male beagle dogs after oral administration of Halotestin. Fluoxymesterone, terone agent

has proven in

replace

rat

an orally to be a potent

and man.

testicular inoperable

dose

from

The

lack

It

in

breast

2-50

has inhibited described

used

for

therapeutic *See appendix

WRegistered

Volwne

of

the

represents levels for

of

25, Nwnber

1

more

of testos-

and anabolic

than

a decade

to

and employed

in

the

The recommended

female.

study

of the

radioimmunoassay of drug the

first

names

used

of The Upjohn

S

and reliable

was developed

fluoxymesterone

trivial

trademark

the here

the measurement

radioimmunoassay

in

sensitive,

radioimmunoassay been

hormone for

the male cancer

of a specific,

the advent

used

analog

the palliation

mg/day.

fluoxymesterone* Since

synthetic

androgenic

has been

hormones

of advanced ranges

active

TIIROXD6

drug

method

capable

body this

for

serum.

this

technique in

in

in

to fill

products in

assay

(l),

serum

The

void. it

(2-10).

has This

of quantitating

fluids. manuscript.

Company.

January,

1975

S

44

TIIROXDE

METHODS AND MATERIALS derivative of Antigen Preparation - The 3-(O-(carboxymethoxime)] fluoxymesterone was conjugated to bovine serum albumin (BSA) at the The carboxymethoxime (CMO) was derived by reacting a 3-position. mixture of 2.6 g fluoxymesterone, 1.7 g of aminooxyacetic acid hemihydrochloride, 0.98 g KOH, and 12.5 ml H20 in 125 ml of 95% ethanol in a steam bath for three hours. The solution was cooled in ice, then diluted 1:lO in H20 and brought to pH 10.5 with KOH. After extracting this mixture twice with 300 ml portions of ethyl the aqueous phase was acidified to pH 3.0 with concentrated acetate, HCl. The solution was cooled in an ice bath before collecting the precipitate by filtration and air drying. Melting point, mass, infrared, ultraviolet, NMR spectral and elemental analyses were performed on the final product. The 3.-[O-(carboxymethoxime)] derivative of fluoxymesterone was conjugated to bovine serum albumin (BSA) by the carbonyldiimidazole One hundred twenty milligrams (0.3 mmole) of the reaction (11). steroid and 60 mg (0.3 mmole) carbonyldiimidazole were dissolved in 1 ml of dimethylformamide (DMF) under reduced pressure. One hundred eighty milligrams of BSA (0.003 mmole) were dissolved in 1.5 ml of H20. The DMF solution was added dropwise to the aqueous solution while mixing. The mixture was adjusted to pH 9.0 with O.lM NaOH before DMF was added to clear the solution. Only part of the mixture was solubilized by this method. After dialysis, the cloudy solution was filtered. The filtrate was air dried and the soluble portion was freeze dried. Ultraviolet characterization analysis was carried out to determine the success of conjugation. Antibody Preparation - The fluoxymesterone 3-BSA hapten was prepared in a 1 mg per ml concentration in phosphate buffer (0.2M, pH 6.5) then emulsified with an equal volume of Freund's Adjuvant. Four New Zealand white rabbits were injected in multiple flank areas weekly for one month with monthly booster injections for three months. Blood was collected from the ear vein or artery (12). Antibody titres were approximately 1:lOO at the end of four months. In an attempt to raise the titres, two rabbits were injected once by the intravenous route with 100 ug of hapten in 1 ml of phosphate buffer. Titres continued to increase until on the 90th day a titre of 1:5000 was established. At a 1:5000 dilution, the antibody bound 75% of the radioactive fluoxymesterone. After establishing dually pooled. performed after to the specificity

a suitable titre, each rabbit's serum was indiviTitration and characterization of the pools was several bleedings. The best pool was chosen according it offered.

Assay Procedure - A double antibody immunoprecipitation procedure is used to quantitate the drug in serum. The primary antiserum is rabbit antifluoxymesterone 3-(CMO) BSA. Both labeled (6,7-3H-fluoxymesterone (32.7 Ci/mmole) and unlabeled drug are allowed to compete for a limited number of binding sites. The assay sensitivity varied directly with the antiserum dilution. With the addition of the goat anti-rabbit gamma globulin second anticomplex becomes insoluble. After precipitation, body, the antibody aliquots of the supernatant containing the unbound tritiated fluoxyare placed into counting vials, mixed with the counting mesterone solution, and counted to determine the amount of unlabeled drug in Quantitation is accomplished by reading values from a each tube. standard curve of known concentrations of fluoxymesterone. Standard curves, as well as 100% control, baseline control, and reference are assayed in parallel with each group of unknown samples. tubes, Analytical 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Procedure

-

Label 10 x 75 mm disposable culture tubes for controls, standards and unknowns. Add 0.5 ml Phosphate buffer (O.ZM, pH 6.5) to 100% control tubes. Add 0.1 ml Phosphate buffer to control and unknown tubes. Add 0.1 ml of control serum to control and standard tubes. Add 0.5 ml of anti-fluoxymesterone antiserum (1:5000) to standard and unknown tubes. Add 0.1 ml Phosphate buffer containing standard dilutions to standard tubes. Add 0.1 ml of sample to unknown tubes. Add 0.2 ml 3H-fluoxymesterone solution (4,000 cpm), shake and allow to equilibrate for two hours at room temperature. Add 0.1 ml anti-rabbit gamma globulin (second antibody) to the tube, shake and allow to equilibrate at 4'C overnight. Centrifuge at 2,000 rpm for 20 minutes and count 0.6 ml of the supernatant in 15 ml of Bio-Solv counting solution using a suitable liquid scintillation counter. Plot standards on semi-log paper (log concentration vs. percent free radioactivity) and interpolate results from the curve.

Assay Characteriziation - The precision, accuracy, and sensitivity the method were determined by repetitive duplicate assays of known In an effort to determine the relative specificity unknown samples. many structurally similar synthetic, potential the primary antibody, metabolic and endogenous steroids were allowed to react with the fluoxymesterone-3-BSA antiserum.

of and of

In vivo Studies - Two human subjects were given 50 mg of fluoxymesThe first subject terone in the form of Halotestin (5 x 10 mg). Sharp was administered 1.0 mg of dexamethasone (Decadron 62, Merck, The second and Dohme) eight hours prior to treatment with Halotestin. subject was not pretreated. Serum samples were withdrawn at 0, 1, 3, The pretreatment with dexamethasone 5, and 8 hours after treatment.

S

46 was used n) establish

the

TXBROXD~

effect

of adrenal

suppression

on the

assay.

Six male beagle dogs were administered either 2, 5, or 10 mg of fluoxymesterone in the form of Halotestin. Serum was withdrawn at 0, l/2, 1, 2, 3, 5, 8, and 12 hours after administration in an attempt to establish a potential dose response. RESULTS Following produced

the

(Figure

described

procedure,

the

fluoxymesterone

1).

3-CM0 was OH

OH 'CH2C02H

3

+ OH

+NH,OCH,:-OH L

L

l/2

Chemical tant

analysis

derivative W:

I.R.:

HCl

1235,

335 (l.OO), S 1.03

2.10-2.40

All structure.

H, 7.88; of the

data

2630,

1105,

2560

(Acid

1.49m

Analytical:

The NMR, however,

the

resul-

Found:

M.S.:

ppm (m,sh),

indicates

consistent that

1630

M/3 = M+ 409 NMR: DMSO-dG,

1.75-2.10

5.59 for

C, 64.84; are

180-185OC; (C = 0).

31 (0.71);

Calculated

accumulated

1730

905 (C-o/N-O);

ppm (m, 0-CH2-C02),

N, 3.42,

that

M.P.:

OH),

43 (0.78),

J=3Hz),

4.47

was reasonable

3-CMO.

1030,

44 (0.85),

ppm (d,

ppm (m),

syn to C02HCH20N). C, 64.52;

it

247 nm, E max 19,150;

(OH) * 2740,

(C = N/ C = C), (0.07),

that

was fluoxymesterone

X max (ethanol),

3490;

60MHZ;

revealed

ppm (m);

ppm (m, vinylic C22H32FN05

H, 7.78; with there

are

(409.50):

N, 3.53. the

H,

assigned two

isomers

of the

derivative.

The proposed

fluoxymesterone Conjugation

was achieved

The resulting method

conjugate

repetitive while

the never

reached

(Table

13.9

2).

while

the

assay

varied

of

metabolites

primary

with

antibody

from

The potential

reacted

adding

is

that

5a-

The addition reduces is is

in

probability

not

detected

llS-hydroxyl

and the from

by the

(Table

three

similar was

group.

metabolites

of

fluoxymesterone

only

two cross-

metabolites

of

and 56-dihydrotestosterone

group

plasma levels

in plasma

antiserum,

curve

17-methyl the

Sa-dihydro

to testosterone

group

as well

specificity

known

tested,

of a 5u and 56 to a carbonyl

steroids

that

to form

of 5a-

level.

by comparing

with

groups two

0.05

standard

metabolites were

comparison

at the

llB-hydroxy-17-methyltes-

chosen

to fluoxymesterone

converted

zero

was determined

and 5S-dihydrofluoxymesterone

of the

the

was possible;

from

group

functional

of to

1 ml of serum

in

indicating

potential

dilution was able

of l:lO,OOO

significantly

were

These

low

same relationship

form

the six

the

assay

at a dilution

fluoxymesterone

llB-hydroxyl

The presence

very

implying

group

the

significantly.

plasma

drug

cross-reacted

metabolites

fluoxymesterone.

quantities

day coefficient

with

1-dehydrofluoxymesterone,

steroids:

Of the

human

day variation

and endogenous

the

and 17-methyltestosterone

testosterone

known

the

while

antibody

synthetic

synthetic

molecule.

directly

different

the

of similar

mainly

of by

using

The within

of 1:5000,

of 250 pg of

tosterone derived

determined

the between

of serum,

250 pg was significantly

The primary

were

by assaying

9.6%,

500 pg/ml

The specificity as potential

assay

was determined

At a dilution

detection

curves

the

was determined

of

detect

consistent

moles

1).

antibody.

consistently

thirty-eight

to human serum.

exceeded

to the

of BSA. Precision

The sensitivity primary

standard

for

reaction.

by UV according

Approximately

assays. added

of variation

i.e.,

reasonable

carbonyldiimidazole

analyzed

of

accuracy

fluoxymesterone

the

was deemed

the

and accuracy

duplicate

serum,

the

was again

--et al. (13). coupled per mole

were

The precision

of

using

of Gross

steroid

structure

3-CMO.

to the

the

would

after

drug until

Since probability

the

(14), bear

the

administration.

testosterone

reduction (15).

in

levels

nucleus the keto

hydroxy metabolic

of interference

S

48 from

these

metabolites

None of the ble

degree

with

circulating oral

is

decreased

endogenous

steroids

the

primary

steroids

occurred average

days.

tested

antiserum.

Averages

day variations between serum

further.

were

concentration

were

calculated

1 and 3 hours

Therefore,

after

Table

4 ngfml 12 nglml 32 rig/ml

Assay 2 (n = 10)

4 rig/ml 12 rig/ml 32 rig/ml

Combined Assays (n = 20)

4 nglml 12 nglml 32 rig/ml

interference samples assayed

established (Figure

2).

and Accuracy

Peak

serum

The peak

Coef. of Variation 8.8 5.4 3.4

3.6 12.9 30.5

0.3 0.6 2.5

9.6 4.6 8.2

4.1 13.2 31.5

0.6 0.7 2.1

13.9 5.4 6.7

2 Data*

Steroids

% Cross-Reactivity

Halotestin (Fluoxymesterone) 9a-Fluoro-118, 17B-dihydroxy-17-methyl-4androsten-3-one

100%

Metabolites

9a-Fluoro-17B-hydroxy-17-methyl-4-androstene3,11-dione 9a-Fluoro-llB-17B-dihydroxy-l7-methyl-5Bandrostan-3-one 9a-Fluoro-17B-hydroxy-l7-methyl-58-androstane3,11-dione 9a-Fluoro-17-methyl-5B-androstane-3a,llB,l7S-triol 9a-Fluoro-114, 17@dihydroxy-17-methyl-5a-androstan3-one 9a-Fluoro-17-methyl-5a-androstane-3B, llB,

3.7% 8% 0.4% 7% 176-trio1

in

day and

1 hour.

Dev.

after

times

0.4 0.7 1.1

Cross-Reactivity

Potential

nine

Data

Std.

4.5 13.4 32.4

Table

from

obtained

and within

administration. at

to any apprecia-

1

Mean Assay 1 (n = 10)

were

was 335 ngfml

Precision

cross-reacted

The serum

of 50 mg of Halotestin

different

between

even

was negligible.

administration

seven

TBBOXD=

21% 12.7%

levels of the

(Table

2, Cont'd)

Synthetic

% Cross-Reactivity

go-Fluoro-116, 17S-dihydroxy-17-methyl-1,4-androstadien-3-one llg, 17S-dihydroxy-17-methyl-4-androsten-3-one 9a-Fluoro-116, 17R-dihydroxy-4-androstene-3-one 9a-Fluoro-llR-hydroxy-4-androstene-3,17-dione 4-Androstene-3,11,17-trione 17S-hydroxy-17-methyl-4-androsten-3-one llg, 17B-Dihydroxy-4-androstene-3-one

75% 33% 9.6% a% <1x 12.0% 4.3%

Endogenous US, 17~1, 21-trihydroxy-4-pregnene-3,20-dione 17f3-hydroxy-4-androsten-3-one llR-hydroxy-4-pregnene-3,20-dione Cholest-5-ene-38-01 *Cross reactivity fluoxymesterone.

determined

at 50% displacement

Figure 2 Serum Concentration in Man

Halotestin

1

1.9% 3.2%
2

3

4 Time

5 (Hours)

of

labeled

Time

Curve

6

7

8

Peak between

serum

concentrations

one and two hours

age administered had detectable

(2, levels

of fluoxymesterone after

serum

concentrations

were

for

2, 5 and 10 mg dosages,

after

120,

195,

respectively Table

0

l/2 1 2 3 5 a 12

2.0 mg Dose Mean + S.D. 0 6 113 120 92 44 ia 10

(Table

Time 10 mg A 5 w3 2 mg

l

at

3 and Figure

3

0 20 163 195 155 47 20 13 Figure

of

in

Dog

10.0 mg Dose Mean + S.D.

0 1 13 75 75 27 20 13

0 37 315 505 395 192 57 la

0 37 a5 25 35 48 11 16

Halotestin Time

Serum-Concentration Curve in Dog

3

(Hours) Halotestin

orally

Halotestin

orally

Halotestin

orally

the six

The peaks

and 505 rigs/ml

5.0 mg Dose Mean + S.D.

0 1 23 5 22 27 9 3

of

Five

dosing.

Mean Fluoxymesterone Serum Concentrations (Two Dogs/Treatment) Hour

occurred

irrespective

Halotestin).

30 minutes

average the

administration

5 or 10 mg of C.T. within

in dogs

dosdogs

of the

two hours 3).

S

51

TIIEOIDE

DISCUSSION The radioimmunoassay assay

for

the

described of

measurement

here

is

capable

not

exceed

fluoxymesterone

variation

did

coefficient

in

The specificity at position

exceeds

tobe

accurately data

the the

serum within are

The assay samples.

mainly

in

this

from

at position form

parent

the methyl

11 on the of

potential

the

llB-hydroxyl

metabolite,

group the

concentrations. is

within

oral

the

first

of drugs

will

of

are

administration three

makes possible

levels it

levels

assay

quickly in

the

peri-

and maximum

hours. the

fluoxymesterone

be possible

group

D and C

fluoxymesterone

after

day day

molecule.

fluoxymesterone

presented tool,

the

that

Detectable

observed

The between

only

30 minutes

method

day and between

reflect

of circulating With

drug

derived

greatest

quantities

indicate

and metabolized.

measurement

for

oxidized

The assay

levels.

specific is

will

levels

The within respectively.

10% at low

and accurate

and nanogram

13.9%,

relatively

Since

fluids.

serum.

group

Man and animal absorbed

and

antibody

be anticipated

of the

9.6

hydroxyl

would

drug

peripheral

precise

biological picogram

the

respectively.

described

in

17 and the

rings,

specific,

of detecting

are of

a sensitive,

of drugs

of variation

The antibodies

pheral

is

precise

and accurate

in numerous

to determine

availability

serum. ACKNOWLEDGMENT

The author gratefully acknowledges interpretation of chemical analyses, Lewis, The Upjohn Company.

the assistance of R.K. Lustgarten in and the statistical work of A.R.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

Yale, R.S., and S.A. Berson, J CLIN INVEST 2, 1157 (1960). Landon, J., and F.C. Greenwood, LANCET February, 273 (1968). Oliver, G.C. et al., J CLIN INVEST 47, 1035 (1968). Cornette, J.C., K.T. Kirton, and G.W. Duncan, J CLIN ENDOCR METAB 3, 459 (1971). Colburn, W.A. and R.H. Buller, STEROIDS 1, 833 (1973). Colburn, W.A. and R.H. Buller, STEROIDS 22, 687 (1973). Spector, S. andE.J. Flynn, SCIENCE 173, 1036 (1971). Royer, M.E., H. Ko, A. Campbell, H.C. Murray, J.S. Evans and D. G. Kaiser, STEROIDS -23 (1974).

S

52 9. 10. 11. 12. 13. 14. 15.

TIIROXDE

Ruder, H.J., R.L. Guy, and M.R. Lipsett, J CLIN ENDOCR METAB 35, 219 (1972). Albano, J.D.M., R.P. Elkins, G. Martz, and R.C. Turner, ACTA ENDOCR lo, 487 (1972). Axen, U.F., PROSTAGLANDINS 5, 45 (1974). Niswender, G.D., L.E. Reichirt, Jr., A.R. Midgley, Jr., and A.V. Nalbanov, ENDOCR 84, 1166 (1969). Gross, S.J., Campbell, D.M., and H-H. Westall, IMMDNO CHEM 1, 55 (1968). Ito, T., and R. Horton, J CLIN ENDOCR 1, 362 (1970). Dorfman, R.I., and F. Ungar, METABOLISM OF STEROID HORMONES, Academic Press, New York, 407-408 (1965). APPENDIX

Trivial

names used

in

this

manuscript:

9a-fluoro-118, 178-dihydroxy-17-methyl-4-androstenFluoxymesterone: 3-one Cortisol: 118, 17, 21-trihydroxy-4-pregnene-3,20-dione Testosterone - 178-hydroxy-4-androsten-3-one 5a-Dihydrotestosterone - 178-hydroxy-5a-androstan-3-one 58-Dihydrotestosterone - 178-hydroxy-58-androstan-3-one 5a-Dihydrofluoxymesterone - 9a-fluoro-118, 178-dihydroxy-17-methyl5a-androstan-3-one 58-Dihydrofluoxymesterone - 9a-fluoro-118, 178-dihydroxy-17-methyl58-androstan-3-one 1-Dehydrofluoxymesterone - 9a-fluoro-118, 178-dihydroxy-17-methyl1,4-androstan-diene-3-one 118-Hydroxy-17-methyltestosterone - 118, 178-dihydroxy-17-methyl-4androsten-3-one 17-Methyltestosterone - 178-hydroxy-17-methyl-4-androsten-3-one