Isotopic dilution GC-MS measurement of 4-Hydroxyandrostendione in postmenopausal breast cancer

Isotopic

dilution

GC-MS

measurement

of

4-hydroxyandrostenedione

in

postmenopausal

breast

cancer

G

Danza,

S

Dini,

G

Bartolucci,

G

Lentini,

A

_Becorpi,

G

B

_Massi,

A

Guarna

and M

Serio

Dipartimentodi_{FisiopatologiaClinica,}UnitàdiEndocrinologia,

DipartimentodiGinecologiaeOstetricia and DipartimentodiChimica

OrganicaUgo Schiff,Università diFirenze,Firenze,Italy

(Requestsforoffprintsshould be addressedtoMSerio,Dipartimentodi

FisiopatologiaClinica,Unità diEndocrinologia,Università diFirenze,Viale

G Pieraccini6,1-50134_{Firenze,Italy)}

Abstract

Using

a novel

_isotopic

dilution _gas

_{chromatography-mass}

_spectrometry

method,

the

plasma

kinetics of

_{4-hydroxyandrostenedione}

(4-OHA)aftera

_single

i.m.

_injection

of250or

500 mgof the

_drug

have been studied intwogroupsof

postmenopausal

womenaffected

by

advanced breast cancer.

Although

the meanvalues of

_CMax

_(the maximum

concentra-tion),

_t\m=1/2\

and AUC (thearea under thecurve)for the500 _mgdose_groupwere

higher,

the

differences were not

statistically significant,

possibly

because of the

_{high intersubject}

variability.

In

addition,

the 4-OHA levels in the breast

_adipose

tissue of the

_quadrant

where the

tumorwaslocatedweremeasured in anothergroupofwomenaffected

_by

breastcancer

who received a 250mg dose of

_drug

2, 7 and 15

_days

before

_surgical

intervention. The tissue concentrations of 4-OHAwere morethan five times

_higher

than the

_{corresponding}

plasma

concentrations;

inaddition

_they

correlated

_{significantly}

with the

_circulating

levels of the

_drug

and showedan

_{exponential disappearance}

_pattern.

Endocrine-Related Cancer(1996)3137-143

Introduction

4-Hydroxyandrostenedione

(4-OHA)

is a suicide

inhibitor of aromatase and is more

_potent

than

aminoglutethimide

invitro. 4-OHA has been demon¬ strated to be useful for the treatment of

postmenopausal

breast cancer

_{(Miller 1990).}

The

administration schedule

(one

injection

every 2

weeks)

was based on theobservation that

suppres-sion of

_plasma

_estrogens

_persists

for 14

_days.

The dose

_usually

_employed

is 250 or 500 mg every 2

weeks.

However,

the incidence ofside-effects with

pain

and inflammationatthe

_injection

site wasless

with the dose of 250 mg every 14

days,

and this schedule has been

_adopted

for

_parenteral

use

(Dowsett

etal.

_1989).

Arandomised trial in 40_post¬

menopausal

womenwith breastcancer

compared

the

weeks)

on

plasma

estradiol levels

showing

thesame

degree

of

_suppression

_(Dowsett

et al.

_1989).

However the clinical non-randomised studies

performed

with thetwo_{schedules gave}

contradictory

results. Inone clinical

study

_(Dowsett

etal.

₁₉₈₉₎

therewas nodifference in the responseratebetween thetwodose

levels,

while in another

_study

the

_objec¬

tive responses were 28% in the 250 mg group and

46%inthe 500_mggroup

_(Bajetta

etal.

_1994).

Recently,

Bulun &

_Simpson

(1994),

using

competitive polymerase

chain reaction

_following

reverse

transcription,

have demonstrated that the

highest expression

of P450aromatasein the stromal cell

_compartment

of

_adipose

tissue is in the

_quadrant

where thetumoris located. Since wehave

_recently

developed

an

isotopie

dilution gas

chromatography-mass

_spectrometry

_(GC-MS)

method for the determi¬

nation of 4-OHA in human

_plasma

_(Guarna

et al.

1989,

Danza etal.

1993)

we have set_up asimilar

method for themeasurementof this

_drug

in the breast

adipose

tissue of

_{postmenopausal}

womenaffected

by

breastcancer.

Thepresent

study

wastherefore

organised

to

re-investigate

the

_plasma

_{kinetics of 250 and 500 mg} doses of 4-OHA

and,

in

_addition,

_{in another group of}

patients

treated with an i.m.

injection

of 250 mg

4-OHA at different intervals before surgery, to

measure the concentration of 4-OHA in

adipose

breast tissue taken from the

_quadrant

where the

tumorwaslocated.

Materials and Methods

Patients

For the

_{plasma pharmacokinetic study, eight}

_post¬

menopausal

women with advanced breast cancer

received 250 or 500_mg 4-OHA. The

_mean(±S.D.)

age and

body weight

of the first groupwere

_57(±6.4)

years and

63.5(±7.3)

kg respectively,

those of the second group were

64.7(±11.5)

_{years and}

63(±8.7)

kg respectively.

Blood

_samples

were

collected before and

4, 24, 48, 72,

96

192,

240 and 336 h after the

_injection

of the

_{drug. Immediately}

after

collection,

the

_samples

were

centrifuged

and

plasma

wasstoredat-20 °C until

_analysis.

For the tissue

_{pharmacokinetic study, eight}

patients

with breastcancer

_(see

Table 2 for clinical

details),

undergoing

breast surgery,weretreated with anintramuscular

injection

of 250 mg 4-OHA at

2,

7

or15

days

before the

operation.

Tissue

samples

were

obtained at

_operation

and

_immediately

frozen at

-80°C. Blood

_samples

weretaken before the

injec¬

tion of the

_drug

and

_just

before the

_operation;

samples

were

centrifuged

and

plasma

stored at

-20 °C until

_analysis.

This

_protocol

has been

_{approved by}

the ethical committee of USL

10D, Florence,

Italy,

and written informedconsentwasobtained from the

patients.

Materials

4-OHAwas akind

gift

ofDr ABrodie

(University

of

Maryland,

Baltimore, MD,

USA).

7,7'-[2H]2-4-Hydroxyandrostenedione

(D2-4-OHA)

was

synthe-sised in our laboratories as

previously

described

(Guama

etal.

_1989).

Thesolvents used for the

_puri¬

fication

_procedure

were

analytical

grade

and were

purchased

from J Baker

_{(Phillisburg,}

NJ,

USA).

Extrelut 3 columns were

purchased

from Merck

(Darmstadt,

Germany)

and the

_{Chromatographie}

stationary phase

Bio-beads SX3 was obtained from

Bio-Rad

_(Richmond,

CA,

USA).

The HPLC instru¬

mentwas aPerkin-Elmer LC series 3B.AllGC-MS

analyses

were

performed using

a Hewlett-Packard

(Palo

Alto, CA,

USA)

GC-MS _systemwitha5890

seriesIIgas

Chromatograph equipped

witha5971A mass

_spectrometry

detector anda7673A automatic

injector.

The GC

_capillary

column usedwasanHP1

(12

m 0.2mm 0.33

_pm)

_{(Hewlett-Packard).}

Internal standardand stock and

_working

solutions

D2-4-OHA, synthesised

as

_previously

described

(Guama

etal.

_1989),

wasused as internal standard

(IS).

Working

solutionsof 4-OHA and

_D2-4-OHA

in ethanolwere

prepared

_by

dilutionof stock solutions

(1

mg/ml

in

_ethanol).

Analysis

of

_plasma

and

_adipose

tissue

samples

Plasma

_samples

were

_purified

and

_analysed

as

previ¬

ously

described

_(Danza

etal.

1993).

For the

analysis

of

_adipose

tissue

_samples

anew methodwasestab¬

lished. The extraction of 4-OHA was

performed

according

tothe

_{following procedure:}

atissue

_sample

of_{0.5-1.0 g}was minced with scissors and 20 ngIS were added

₍₂₀

_{µ 1 of} 1

_ng/pl

_solution).

Minced tissues were extracted with 3x5 ml methanol: dichloromethane

_{(90:10, v/v);}

the extracts were

cyclohexane:dichloromethane (85:15, v/v)

were

added and the solutionsweredriedonshortcolumns of

_anhydrous

_Na2S04.

The vials and the

_Na2S04

columns were then washed with 2 3 ml

cyclohexane:dichloromethane

(85:15).

The solutions

were

evaporated

to

_dryness

under

_nitrogen;

the residues were redissolved with 0.5 ml

cyclohexane:dichloromethane

(85:15)

and the

resultant solutions were

injected

into a

semiprep¬

arative

_{low-pressure gel permeation}

column

(Bio-Beads

_SX3),

_gelified

with

_cyclohexane:

dichloromethane

_(85:15),

connected to the HPLC instruments. The columnwaselutedataflowrateof

1 ml/min and the fractions

_containing

4-OHA and

D2-4-OHA

(23-33 min)

were

collected,

evaporated

to

_dryness

under

_nitrogen

and derivatised for 1 h at

144 192

Time

_(h)

336

Time

_(h)

Figure 1 Plasma

pharmacokinetic

profilesof 4-OHA afterasinglei.m.

injection

of(a)

Table 1 Meanvalues of_plasma kineticparametersfor thetwodosesof 4-OHA(250and 500mg)

Parameter 4-OHA(250mg)4-OHA(500mg)

C^x

(ng/ml)

10.78 17.9

tMax(h) 48 48

tK(h)

99.0 173.3

AUCq.336

(ng

h/ml) 1279 2075.8

C336

(ng/ml)

1.011 3.05*

*P=0.006comparedwith result for 250 mg4-OHA.

room

_temperature

with 25

_pi

bis-trimethylsilyl-trifluoroacetamide to obtain the

_{4-O-trimethylsilyl}

(4-O-TMS)

derivative. The

_sample

solutions were

evaporated

to

_dryness

under

_nitrogen,

dissolved in 30

_{pi n-heptane}

and 3

_pi

were

autoinjected

into the

GC-MS instrument.

The mean

analytical

recovery of4-OHA from

adipose

tissue,

calculated as

previously

described

(Guarna

etal.

_1995),

was63%.

The

_selectivity

of the method wasevaluated

_by

analysing

blank

_samples

and 4-OHA-free tissue

samples.

The calibrationcurve wasmadewithseven

points

in_{the range 0-70ng with}20_ngIS.Peakarea

ratios of 4-OHA and IS were

plotted

versus4-OHA

concentration and a

good linearity

was obtained

(r=0.997)

The

_precision

and accuracy of the methodwere

determined

_{by replicate}

_interday

_{analyses (n=3)}

of tissue

_{samples (0.5 g)}

ata4-OHA concentration of

20

_ng/g.

Precision,

evaluated

_by

the coefficient of

variation,

was

13%,

and accuracy, calculatedas mean

measured concentration versus nominal concentra¬

tion,

was92%.

The

_sensitivity

limit of the instrumentwaseval¬

uated

_{by injection}

of

_decreasing

amountsof 4-OHA and

_calculating

the

_signal-noise

ratio

_(S/N);

_{10 pg of}

injected

4-OHA gave S/N=50. The

sensitivity

limit of the assaywasevaluated

analysing

tissue

samples

(1

g)

at

_decreasing

concentrations of 4-OHA and

calculating

S/N;

samples

at500

_pg/g

gave S/N=30.4

when 3

_pi

ofthe 30

_pi

final volumewere

injected.

Analyses

were

performed using

an HP1

(12

m 0.2mm 0.33

_pm)

GC

capillary

column

with a100%

methylsilicone phase.

The

_temperature

program was: 70 °C for 1

min,

then an increase of

40 °C/min to 220

°C,

220 °C for 0.5

min,

then an

increase of 3 °C/min to 300 °C. The transfer line

temperature

was_{280 °C. The carrier gas}washelium withaninlet pressure of 35 kPa.Inthese

conditions,

the retention times of 4-OHA and

_D2-4-OHA

were

14.14 min and 14.12 min

_{respectively. Injections}

were

performed

in the

unsplit

mode witha

purge-off

time of1 minandan

injector

_temperature

of 280 °C.

Selected ion

_{monitoring analyses}

were made

acquiring

the ions at 359.2 and 361.2m/z corre¬

sponding

to the M-15

_fragment

of the 4-O-TMS derivatives of 4-OHA and

_D2-4-OHA

_{respectively.}

Plasma and tissue

_{pharmacokinetic profiles}

Plasma

_{pharmacokinetic}

_parameters for the two

doses of 4-OHA

₍₂₅₀

and500

_mg)

werecalculated asfollows:

CMax

wasthe maximum concentration of

Table2_Comparisonbetweenadiposebreast tissue and_plasma4-OHAconcentrationsat_{different intervals}

afterasingle250 mginjectionof thedrug.Theage,bodyweight, heightandTNM_{stagingsystemaccording}to

the UICC classificationarealso_reportedfor individual_patients

Patient Age Body Height TNM _{Days Breast Plasma}

(years) weight (cm) after _adipose4-OHA 4-OHA

(kg) injection (ng/g) (ng/ml) 72 64 63 62 56 64 49 71 68 54 59 84 65 65 55 85 163 165 158 160 155 155 158 160

TlN0M0

,

T2N0M0

T4dN1Mx

T2N2M-|

TiN0M0

2 2 2 2 2 7 15 15 134.6 104.7 57.4 77.4 97.8 20.2 2.82 1.26 21.4 21.4 10.7 23.9 9.46 4.7 0.08 0.00

200-1

4-OHA

_{(ng/ml plasma)}

Figure2 Correlation betweenadiposebreast tissue andplasmaconcentrations of 4-OHA afterasingle_{250 mg}injection (y=0.75+4.91_x,r=0.96).

the

_{pharmacokinetic}

_profile

and

_tMax

wasthetimeat

which themaximum concentrationwasreached. The

ti/2

wascalculatedas

_{t[/2=0.693/ATe/}

where Kel is the

eliminationconstant.The

_{AUC0_336}

_(area

under the

curve)

wascalculated with the

_trapezoidal

mie.

_C336

wasthe4-OHAconcentrationat336hafter the

_drug

injection.

All the_parametersfor thetwodoseswere

compared

with Student'sr-test.

For the tissue

_{pharmacokinetic}

_profile

_only

the

elimination

_phase

wasvaluable and the AUCwasnot

determined.

Results

The

_plasma

kinetics of4-OHA measured after the

injection

of thetwo doses of4-OHA are shown in

Fig.

\a and b and the mean values of the relevant

parameters

are

reported

in Table 1. In

_spite

of the

large intersubject

variability,

the

_highest

concentra¬

tionwas obtainedat48 h with both doses.For that

reason, 2

days

before

surgical

intervention was

chosenasthe first

point

forthe tissue

study. Although

the

_CMax,

_t1/2

and AUC obtained with the500_mg

dose were

higher

than those obtained with the

250 mg

dose,

the results were not

_{significantly}

different

_(P>0.05)

when

_{compared by}

Student's

t-test.

_Only

the levels measured at 336h

₍₁₄

_days)

were

significantly higher

_(3.05

versus1.011

_ng/ml

of

plasma,

/>=0.006)

forthe 500 mg dose. The tissue concentrations of4-OHAwerefive times

higher

than

the

_{corresponding}

_plasma

concentrations

_{(Table 2);}

they

correlated

_{significantly}

with

_circulating

levels of the

_drug

_(r=0.96)

_(Fig.

₂₎

andshowedan_exponen¬

tial

_{disappearance}

_pattern

_(Fig.

_3).

The

_CMax

in breast

_adipose

tissue and the _apparent

_ti/2

were

respectively

94.4

_ng/g

and57.8 h.

Discussion

The

_plasma

kinetics of 4-OHA aftera

single injection

of 250or_{500 mgof}the

_drug

have also beenstudied

by

Dowsettetal.

₍₁₉₈₉₎

_using

anRIA. Their results are in _agreement with those of the _present

_study

showing

amaximal concentrationof about 10

_ng/ml

for the _{250 mg dose and about 18}

_ng/ml

for the 500 mg dose on thesecond

day

after

injection.

The

concentration of 4-OHA in breast

_adipose

tissue from the

_quadrant

wherethetumorislocatedhasnot

V. íoo-o

-I

c O 336 Time

_(h)

Figure3_Exponentialreduction of the concentration of4-OHAin_adiposebreasttissue afterasingle_{250 mg}

injection

of the

drug.

been measured before.

However,

Reedetal.

₍₁₉₉₁₎

measured 4-OHA levels in normal andtumorbreast

tissue.

_They

foundthat theconcentration of

_drug

in breast tumor tissue was similar to thatin

_plasma

while that in normal breast tissuewas lower.

_They

found alsoa

significant

correlation between

plasma

and tissue concentrations of the

_drug.

Inthe_present

study

the

_circulating

levels of 4-OHA still correlated with the tissue levels but the concentration in

_adipose

tissue was five times

higher

than that in

plasma.

In

breast

_adipose

tissue the concentration of 4-OHA neededtoreduce thearomatase

_activity

to50% has been calculated to be 30

_ng/g

_(Perei

etal.

_1981),

which is threefold lowerthan that which we meas¬

uredonthe second

day.

On the basis of the

_apparent

tissue half-life we cancalculate that 4-OHA levels

which are able to reduce the aromatase

activity

to

50%

_persist

in breast

_adipose

tissue for 6

_days.

In

conclusion,

the

_ability

tomeasureconcentra¬

tions of4-OHA in both

_plasma

and breast

_adipose

tissue allowsustoobtainnewinformation

regarding

the

_disposition

of this

_potent

and useful

_drug.

Acknowledgements

Thiswork was

supported

in_part

by Consiglio

Nazi¬

onale delle Ricerche

_progetto

ACRO

_(96.36.49).

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