Determination of the new aromatase inhibitor exemestane in biological fluids by automated high-performance liquid chromatography followed by radioimmunoassay

I | N O F I I A N JOURNAL | P

PHARMACEUTICAL

ELSEVIER

European Journal of Pharmaceutical Sciences 4 (1996) 331-340

SClUCES

Determination of the new aromatase inhibitor exemestane in biological fluids by automated high-performance liquid chromatography followed by radioimmunoassay S. Persiani*, F. Broutin, P. Cicioni, P. Stefanini, M. Strolin Benedetti 1 Department of Pharmacokinetics and Metabolism, Pharmacia S.p.A., Nerviano (MI). Italy Received 24 September 1995; accepted 11 December 1995

Abstract A procedure for the determination of exemestane, a new aromatase inhibitor, in biological fluids is described in this paper. Exemestane is extracted from human plasma and urine by solid-phase and liquid-liquid extraction, respectively. The test compound is then isolated from endogenous steroids, its metabolites and/or degradation products by HPLC. The exemestane-containing fraction is collected and its exemestane content measured by radioimmunoassay (RIA). The automated HPLC system allowed a high specificity and reproducibility of retention times, and eliminated almost all manual operations. The RIA allowed the accurate and precise measurement of 12 pg of exemestane/ml in plasma (inter- and intra-assay R S D = I 7 . 7 and 13.4%, respectively) and 25 pg/ml in urine (inter- and intra-assay R S D = 14.5% and 8.7%, respectively). The recovery of the whole procedure was evaluated by comparison of the RIA calibration curve obtained in plasma or urine (after extraction and HPLC) with that obtained directly in RIA buffer (without extraction and HPLC). The calibration curves were practically superimposable, indicating that the recovery of the whole procedure was excellent. The method was validated in terms of reproducibility, recovery and precision in the range 10-500 pg of exemestane/ml of plasma and 20-1000 pg/ml of urine. Finally the plasma levels of exemestane in a postmenopausal healthy volunteer treated daily for 7 days with oral exemestane at a dose of 1 mg (the lowest dose administered in clinical trials) were monitored using the method here described. Exemestane was detectable in all plasma samples collected (up to 24 h after drug intake). Therefore the analytical method described here should be sufficiently sensitive and specific for the determination of exemestane in plasma and urine from clinical trials in which therapeutic doses of the drug (10-25 rag/day) are administered. Keywords: Exemestane; Aromatase inhibitor; Combination of HPLC and RIA

1. Introduction The aromatase system is an enzyme complex that converts androgens to estrogens (Ryan, 1959). The extraglandular conversion of androstenedione to estrone catalyzed by the aromatase enzyme is considered to be the most important source of circulating *Corresponding author. Present address: Department of Pharmacokinetics and Metabolism, Zambon Group S.p.A., Via Lillo del Duca 10, 20091 Bresso (MI), Italy. Tel.: (39-2) 6652-4359; Fax: (39-2) 6650-1492. ~Current address: Zambon Group S.p.A., Via Lillo del Duca 10, 20091 Bresso (MI), Italy. 0928-0987/96/$32.00 © 1996 Elsevier Science B.V. All fights reserved PII S0928-0987(96)00171-6

estrogens in the postmenopausal woman (Grodin et al., 1973). Since one-third of human breast tumors are hormone-dependent (Henderson and Canellos, 1980) and estrogens are the most important hormones involved in supporting the growth of these hormonedependent tumors (Segaloff, 1978; Kirschner, 1979), inhibition of estrogen biosynthesis by means of selective aromatase inhibitors in postmenopausal women is a potentially useful therapeutic option in hormone-sensitive breast cancer (Santen, 1987). Exemestane, 6-methylenandrosta- 1,4-diene-3,17dione (code name FCE 24304; Fig. I), is a new selective and irreversible aromatase inhibitor (Di

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O

O

menopausal healthy volunteer treated orally with repeated daily doses of 1 mg of the drug.

2. Materials and methods CH~ Exemestane

CH2 Labelled hapten (tritium is randomly distributed in the molecule)

Fig. 1. Structure of exemestane and of the radiolabelled hapten used in the RIA.

Salle et al., 1992) currently under investigation for the treatment of postmenopausal breast cancer (Evans et al., 1992). A single 5 mg oral dose of exemestane was found to cause a long-lasting suppression of plasma estrogens in postmenopausal women (Di Salle et al., 1994) and the lowest dose of exemestane that has been administered in a clinical trial is 0.5 mg (Evans et al., 1992). The available high-performance liquid chromatographic methods with UV (Breda et al., 1993) or mass spectrometric Allievi et al., 1995) detection, having a limit of quantitation of 10 and 1 ng/ml of plasma, respectively, therefore have insufficient sensitivity to monitor the drug in clinical trials. Thus a RIA for exemestane in which the test compound was extracted from human plasma and urine was developed. The antiserum cross-reacted with androstenedione and androsterone. Since these endogenous steroids are present in human plasma under physiological conditions at concentrations of about 700 and 800 pg/ml, respectively (Bblanger et al., 1990), their plasma concentrations are able to produce significant interference in the measurement of exemestane by RIA after extraction alone. Therefore, a purification step was applied to the plasma and urine extracts before the RIA. The available highperformance liquid chromatography procedure (Breda et al., 1993) with minor modifications was used on plasma and urine extracts to isolate exemestane from interfering androstenedione, and androsterone and from exemestane metabolites and/or degradation products. The present paper describes the combined H P L C RIA procedure for the determination of exemestane in human plasma and urine and its validation results. The procedure has been applied for the measurement of exemestane plasma levels in a post-

2.1. Chemicals

The following compounds were supplied by Pharmacia S.p.A., Chemistry Department (Nerviano, Italy): Exemestane (FCE 24304), FCE 27236, FCE 24140, FCE 25071, FCE 27560, FCE 27561, FCE 27562, FCE 27247, FCE 27472, FCE 27473, FCE 27353, FCE 27474, FCE 27278 and FCE 24204. Androstenedione, androsterone, dehydroepiandrosterone, progesterone, 17-OH-progesterone, pregnenolone, estrone, 17-estradiol, 3-androstane-diol, testosterone, 5-dihydrotestosterone and bovine serum albumin (BSA) were from Sigma (St. Louis, MO, USA). N-hydroxy-succinimide (NHS) was from Janssen Chemicals (Geel, Belgium). Dicyclohexylcarbodiimide (DCC) was from Fluka (Buchs, Switzerland). Freund's complete and incomplete adjuvants were from Difco (Detroit, MI, USA). Dextran TI0 and T70 were from Pharmacia (Uppsala, Sweden). Charcoal norit A was from Fisher Scientific (Pittsburgh, PA, USA). Ultima Gold liquid scintillation cocktail was from Canberra Packard (Meriden, CT, USA). All other chemicals were from Farmitalia Carlo Erba (Milan, Italy). The RIA buffer was prepared as follows: 13.61 g KH2PO 4, 30 mg dextran T10, 9 g NaC1 and 1 g/1 NaN 3 were dissolved in 1 1 of bidistilled water and 0.1 g/1 BSA was added. The pH was then adjusted to 7.2 with NaOH. The charcoal suspension was prepared from 2 g charcoal and 20 mg dextran T70 dissolved in 100 ml of RIA buffer, pH 7.2.

2.2. Antisera

The immunogen was made by conjugating an analog of exemestane (2-carboxy-6-methylenandrosta-1,4-diene-3,17-dione; FCE 27236) to bovine serum albumin (Fig. 2) via an active ester reaction using NHS. To prepare the active ester, FCE 27236 (0.5 mmol)

S. Persiani et al. / European Journal of Pharmaceutical Sciences 4 (1996) 3 3 1 - 3 4 0

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CH2 ~>

0

333

calculated that about 28 molecules of FCE 27236 were bound per molecule of BSA. The lyophilized immunogen (30 mg), dissolved in 6 ml of 0.9% NaC1 solution, was divided into 0.7 ml aliquots and stored at -20°C. At each immunization time, an aliquot was thawed, homogenized with 2.8 ml of 0.9% NaCI solution and 3.5 ml Freund's complete adjuvant and administered to five rabbits. Each animal received about 0.5 mg of the immunogen in 1 ml final solution at 20 different dorsal sites by intradermal and subcutaneous injection. Booster injections of the immunogen, emulsified with Freund's incomplete adjuvant, were given after 1, 5, 7, 14 and 19 months. Antibody titer was monitored 10 days after each booster.

O~ M IMUN~GEN CH2 Fig. 2. Synthesis of the immunogen, N-hydroxysuccinimide (NHS); dicyclohexylcarbodiimide (DCC): bovine serum albumin (,HN-BSA).

2.3. Antiserum characterization was dissolved in 25 ml of dry dioxane. To this solution 0.5 mmol of NHS and 0.5 mmol of DCC were added. This reaction mixture was stirred for 1 hour at 10°C and then for 5 h at room temperature, then a further 0.1 mmol of DCC and 0.1 mmol NHS were added. The reaction was monitored by thinlayer chromatography (mobile phase: dichloromethane-ethanol, 95:5). The reaction was complete after 36 h. After elimination of the white precipitate of dicyclohexylurea by paper filtration, the reaction mixture was evaporated, washed with ether and dried. In the second step of the synthesis, the active ester (0.15 mmol), dissolved in 13.2 ml dry dioxane, was slowly added (2 h) to a solution obtained by dissolving BSA (0.003 mmol) in 21 ml 0.1 M phosphate buffer (pH 8.8) at 4°C under stirring. The reaction mixture was left for 16 h at room temperature and then dialysed for three days against distilled water (3 l/day) to remove any trace of organic solvent, buffer and steroid not covalently bound to BSA. The number of exemestane molecules conjugated to BSA in the immunogen was measured at 247 nm (the maximum absorption wavelength of exemestane) by subtracting from the absorbance of the immunogen, the absorbance contribute by a BSA standard solution at the same molar concentration of the immunogen. With this procedure, and assuming that the conjugation does not affect the molar absorption coefficient of both FCE 27236 and BSA, it was

The affinity constant of the antiserum for exemestane was calculated by the Scatchard method (Scatchard, 1949). The cross-reactivity of the antiserum with several exemestane derivatives synthesized as possible metabolites (Buzzetti et al., 1993) and with endogenous steroids was tested. The exemestane derivatives were the following: FCE 24304, FCE 27236, FCE 24140, FCE 25071, FCE 27560, FCE 27561, FCE 27562, FCE 27247, FCE 27472, FCE 27473, FCE 27353, FCE 27474, FCE 27278 and FCE 24204. The endogenous steroids were the following: androstenedione, androsterone, dehydroepiandrosterone, progesterone, 17ce-OH-progesterone, pregnenolone, estrone, 17/3-estradiol, 3o~-androstane-diol, testosterone and 5c~-~hydrotestosterone. The structural formulae of the exemestane derivatives and of androstenedione and androsterone are shown in Fig. 3. The cross-reactivity of the antiserum with a given compound was defined as the amount of exemestane causing 50% binding expressed as a percentage of the amount of compound which produced the same binding. 2.4. Labelled hapten The labelled hapten ([-H]6-methylenandrosta-4ene-3,17-dione, [3H]FCE 24124, Fig. 1) was obtained by exchange procedure, using tritiated water, from Amersham (Amersham, UK). The specific activity of the radiolabelled hapten was 40 Ci/mmol and its radiochemical purity (measured by radio-HPLC) was

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O

ANDROSTENEDIONE ¢7.3%1

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ANDROSTERONE 13,2~ OH

OH

FCE 25071 CH2 10.3%) O

OH

FCE 27560 CH=OH (1,3-a~)

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FCE 27561 CI~OH (1.8-a%) O

o

FeE 27562 (1.a'~.,}

o

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FCE 27247 (0.011%)

FCE 27472 (0.28%} OH

FCE 27474 10.o3~)

O

FeE 27473 10.01~ 0

FCE 27278 ~1.5~)

OH

O

o

CH=OH

FCE 27353 (0.4%) 0

FCE 24204 (2.ev,}

Fig. 3. Structures of the endogenous steroids and of the exemestane analogs tested for cross-reactivity with the antiserum. Cross-reactivity percentages are shown in parentheses.

98%. The compound stored in ethanol was stable at - 2 0 ° C for at least 1 year.

The protocol of the total assay procedure outlined in Table 1 consists of three steps: extraction of steroids, automated HPLC and radioimmunoassay.

C18 cartridge (Lida) previously washed with 10 ml of ethanol and conditioned with 20 ml of water. After washing with 20 ml of water the absorbed compounds were eluted with 5 ml of ethyl acetate. The organic phase was then evaporated in the vacuum concentrator. A liquid-liquid extraction procedure was used for urine samples since the solid-phase extraction produced interference in the RIA due to endogenous compounds. Urine samples (0.5 ml) were extracted with 1.5 ml of diethyl-ether. The tubes were vortexed for 1 min, centrifuged at 1200×g for 5 min and stored at - 2 0 ° C in the dark. After 2 h the organic supernatants containing exemestane were separated from the frozen aqueous fractions and poured into separate tubes. The organic extracts were evaporated to dryness under a nitrogen gas stream. The dried extracts from plasma and urine samples were reconstituted with 250 /xl of a mixture of acetonitrile-water (34:66) and then subjected to HPLC.

2. 7. Extraction procedure

2.8. Chromatographic conditions

The following procedures were employed for the extraction of calibration, quality control and unknown plasma and urine samples (calibration and quality control samples were left to equilibrate at room temperature for 15 min before extraction): 1 ml of plasma was loaded onto an Extra-Sep RC

The chromatographic separation was a modification of a method previously described (Breda et al., 1993) and was the same for plasma and urine extracts. The column was a 150 mm x 4.6 mm I.D. C 18 Hypersil (Alltech), particle size 3 / z m , fitted with a guard column filled with Pellicular ODS media with

2.5. Equipment The Tricarb 1900 TR fl-counter and the Probe 1000 automated sample preparer were from Canberra Packard. The integrated HPLC system was a model 1050 from Hewlett Packard equipped with an UV detector operating at 247 nm, an autosampler (set for an injection volume of 225/xl) and a thermostat oven for the chromatographic column. The fraction collector, model Foxy Junior, was from ISCO. The vacuum concentrator equipped with a centrifuge, model AES 1000, was from Savant Instruments.

2.6. Methods"

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Table 1 Flow sheet of the complete exemestane assay in plasma and urine A.

Ai

Extraction Extraction of plasma samples 1 ml plasma sample (spiked or unknown) Transfer to Extra-Sep RC C18 cartridge Elute with 5 ml ethyl acetate Evaporate using vacuum concentrator Redissolve in 250 #1 of water-acetonitrile mixture (66:34)

A2

Extraction of urine samples 0.5 ml urine sample (spiked or unknown) Add 1.5 ml of ethyl-ether Vortex for 1 rain and store at - 2 0 ° C for 2 h in the dark Separate organic supernatant and evaporate under nitrogen gas stream Redissolve in 250 /~1 of water-acetonitrile mixture (66:34)

High performance liquid chromatography Transfer to autosampler Automatic injection (225 #1) and chromatography Automatic collection of the exemestane fraction (3 ml in I tube) Evaporate exemestane fraction using the vacuum concentrator

Radioimmunoassay Redissolve evaporated fraction in 350 #l of RIA buffer Add radiolabelled tracer, diluted antiserum (0.1 ml each) Incubate for 4 hr at 4°C Add charcoal suspension (0.1 ml) Separate supernatant containing the antibody-bound radioactivity by centrifugation Count radioactivity in supernatant by liquid scintillation spectrometry. Calculate results

a particle size of 30-38 /zm (Whatman, Maidstone, UK). The separation of exemestane from androstenedione and androsterone was performed at constant temperature (35°C) and flow rate (1 ml/min) using a 25 min isocratic step with acetonitrile-water (34:66). At the end of the isocratic step, a 7 min washing step with acetonitrile-water (80:20) and a 5 min stabilisation step with acetonitrile-water (34:66) was applied before the next injection.

2.9. Determination of the time interval for collection of the exemestane fraction To determine the time interval during which the collection of the fraction corresponding to exemestane should be carried out, a standard solution containing UV-detectable amounts of androstenedione and exemestane (1 /.~g each in a volume of 5 ml) was prepared in acetonitrile:water (34:66). A 250 ~1 aliquot (50 ng) of this solution was used to reconstitute the dried extract from 1 ml of blank plasma (or 0.5 ml of blank human urine) obtained using the extraction procedures described above. Exemestane and androstenedione retention times were monitored before each calibration and unknown

samples batch. The HPLC fraction corresponding to exemestane was then collected automatically using the fraction collector, evaporated using the vacuum concentrator, and the exemestane concentration measured by RIA.

2.10. Radioimmunoassay The following procedure was used for the characterization of the antisera and for all the RIA determinations. Approximately 20 pg of labelled hapten ( 100 Bq in 0.1 ml) freshly diluted in RIA buffer without BSA, from a working ethanol solution stored at -20°C was added to each tube. The diluted antisera (0.1 ml), the labelled hapten (0.1 ml), and exemestane at different concentrations (0.1 ml) were distributed by the Probe 1000 adjusting the final incubation volume to 0.55 ml with RIA buffer. The tubes were then incubated at 4°C for 4 h. The separation of free from antiserumbound hapten was performed at 4°C by adding 0.1 ml of charcoal suspension then incubated for 15 min and centrifuged for 20 min at 1200×g. The supernatant containing the antibody-bound hapten (0.5 ml) was then transferred into counting vials by the Probe

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1000, and counted for 10 min by liquid scintillation spectrometry. The antibody bound fraction of radioactivity was expressed as percentage B/B o, the amount bound (B) relative to the amount bound in the absence of the analyte (Bo). The non-specific binding (NSB) was measured by incubating the radiolabelled hapten for 4 h at 4°C in RIA buffer with no antiserum. The logit of the percentage bound was plotted against the log of exemestane concentration (range 10-500 pg/ml for plasma and 20-1000 pg/ml for urine) and computer-fitted to a straight line. Quality control and unknown sample concentrations were calculated from the standard curve. 2.11. Assay validation Two parameters were used to define the suitability of the chromatographic system (United States Pharmacopoeia XXII, 1990). The efficiency of the chromatographic column, evaluated as the number of theoretical plates of the column, was calculated from the equation N = 16(tR/ W) 2, where t R is the retention time (mm) of exemestane and W is the baseline peak width (mm). The resolution factor (R) between the peaks of exemestane and androstenedione was calculated from the equation R = 2 (t2--tl)/(Wm + w 2 ) , where tl and t 2 refer to the respective retention times, and wl and w: are their baseline widths measured in min, as shown in Fig. 4. To determine the precision and recovery of the whole procedure, three quality control samples containing exemestane at concentrations of 12.5, 50 and 450 pg/ml of plasma (or 25, 100 and 900 pg/ml of urine) were prepared as previously described in triplicate, extracted, subjected to HPLC and the fraction corresponding to exemestane analysed by RIA, on six different working days over a period of one month. The intra- and inter-day relative standard deviation (RSD, %) of the three repeated measurements were used as an index of precision. The percentage of recovered exemestane/amount of compound added was used as an index of recovery (Table 2). The sensitivity limit of the method was defined as the concentration obtained from the standard curve (in plasma or urine) at three times the intra-assay S.D. of the Bo value ([Bo-3SD]/Bo%), whereas the

z 6

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;o

,'s

2'o

I

2's

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Fig. 4. Typical HPLC chromatogram of exemestane and androstenedione in the presence of the extract from 1 ml of human plasma. W, and W 2 were used to calculate the resolution factor (see text for details). The collection of the exemestane fraction started at time A and ended at time B.

limit of quantitation was set at the lowest measurable concentration with an inter-assay RSD lower than 20%. 2.12. Plasma samples from a healthy postmenopausal volunteer Blood samples were obtained from a 68-year-old postmenopausal healthy volunteer weighing 60 kg and 164 cm tall. The volunteer was treated orally for 7 days with 1 m g / d a y of exemestane as sugar-coated tablet at the Aster Clinical Research Centre (Paris, France). The Institutional Ethics Committee approved the study and the volunteer gave written, informed consent. On the seventh day of treatment, blood samples (6 ml) were collected, in heparinized tubes, immediately before drug intake (time 0) and 0.5, 1, 1.5, 2, 4, 6, 8, 12 and 24 h after treatment. The collected blood samples were centrifuged at 1200×g for 10 rain; the separated plasma samples were stored at - 2 0 ° C until analysis using the HPLC-RIA procedure here described.

3. Results

3.1. Extraction The mean recovery_+SD of the extraction procedure, calculated using five plasma samples (1 ml)

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Table 2 Precision and recovery of the radioimmunoassay for the determination of exemestane in human plasma and urine after extraction and HPLC Amount added (pg/ml)

Mean amount found (pg/ml)

Inter-assay recovery (%)

Inter-assay RSD (%)

Intra-assay RSD (%)

Plasma

12.5 (n= 17) 50 ( n = l S ) 450 (n = 17 )

12.6 52.4 479.6

100.8 (n=17) 104.8 (n=18) 106.6 (n - 17 )

17.7 (n= 17) 10.9 (n=18) 16.2 (n = t 7 )

13.4 (n=3) 5.8 (n=3) l 5.4 (n = 3 )

Urine

25 ( n - 1 8 ) 100 ( n = l S ) 900 (n=17)

24.0 103.3 936.7

96.0 (n=18) 103.3 (n=18) 104.1 ( n - 1 8 )

14.5 (n 18) 9.2 ( n - 1 8 ) 16.2 (n=18)

8.7 (n=3) 4.9 (n=3) 11.5 (n 3)

containing about 7 pg of the tritium labelled hapten ([3H]6-methylenandrosta-4-ene-3,17-dione; see Fig. 1 for its structural formula), was 89.1___3.9% (RSD= 4.4%). The recovery of the extraction procedure in urine, calculated as previously described, was 94.9___4.1 (RSD=4.3%). 3.2. HPLC

Fig. 4 shows a typical chromatogram of exemestane and androstenedione in the presence of the extract from 1 ml of human plasma (see the method section for details of the sample preparation). Under the chromatographic conditions described above, exemestane had a retention time of about 23 min, and was separated from androstenedione by a 3-min interval. Androsterone was eluted during the washing step (water-acetonitrile 20:80; data not shown). Therefore androstenedione was used to evaluate the suitability of the chromatographic system. To ensure that controlled conditions are maintained, the number of theoretical plates (N) must be at least 4000 for exemestane whereas the resolution factor (R) must be at least 2.3. Based on these results the collection of the exemestane fraction started 1 minute before the retention time of the drug (time A) and ended 2 min after (time B; see Fig. 4). The chromatograms obtained in the presence of the extract from 0.5 ml of human urine were similar to those obtained in the presence of human plasma (data not shown), therefore the time interval for the collection of the exemestane fraction was identical for plasma and urine samples. 3.3. RIA

Four rabbits raised antisera against exemestane.

The cross-reactivity of these antisera with androstenedione was similar; therefore, the one with the highest titer was chosen for the development of the RIA. In the assay conditions previously described, the antiserum was used diluted 1:3000. The affinity constant of the antiserum for exemestane was 3" 10~° 1/mol. The cross-reactivity of the chosen antiserum with several endogenous steroids and with exemestane derivatives synthesized as potential metabolites is shown in parentheses in Fig. 3. Androstenedione and androsterone were the only endogenous steroids tested with significant cross-reactivity (7 and 3%, respectively) Fig. 5 shows the mean standard curve obtained by averaging the results of six different standard curves obtained on six different days using 1 ml of plasma (A) or 0.5 ml of urine (B) spiked with exemestane at different concentrations and processed using the whole HPLC/RIA procedure. In the presence of human plasma the percentage of specific binding (Bo) averaged 39.4___3.2% (mean___SD; range 35.444.0%), the non-specific binding (NSB) averaged 2.9___0.4% (mean__+SD; range 2.4-3.5%). The mean standard curve obtained was defined by the equation: logit(B/B o, in % ) = - 2 . 6 1 1 log(exemestane concentration in pg/ml)+4.154 (r>0.999). In the presence of human urine the percentage of specific binding ( / 3 o ) averaged 35.2___4.2% (mean_SD; range 30.3-39.6%), the non-specific binding (NSB) averaged 2.6___0.5% (mean+_SD; range 2.2-3.5%). The mean standard curve obtained was defined by the equation: logit(B/B,, in % ) = 2.380 log(exemestane concentration in pg/0.5ml)+ 3.616 (r>0.999). To evaluate the recovery of the whole procedure and the ability of the HPLC purification to remove all interfering endogenous compounds, the mean standard curve in plasma after HPLC/RIA was compared

S, Persiani et al. / European Journal of Pharmaceutical Sciences 4 (1996) 3 3 1 - 3 4 0

338

I~ en

90-

90

70-

70

50-

50

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2'0

s6o

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'

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EXEMESTANE (pglml)

90-

Fig. 6. Mean RIA calibration curves obtained after extraction and HPLC of lml of human plasma ( 0 ) or directly in RIA buffer without extraction and HPLC (ll),

7050-

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"~ 30-

3.4. Recovery, precision and limit of quantitation of the whole procedure

10521

1'0

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5b0

EXEMESTANE (I~I0.5 ml)

Fig. 5. Mean standard curve obtained by averaging 6 standard curves from 6 different days after extraction and HPLC of 1 ml of human plasma (A) or 0.5 ml of human urine (B).

with the mean standard curve obtained directly in RIA buffer without extraction and HPLC (mean of six standard curves obtained on six different days). The concentrations of the mean standard curve samples after HPLC/RIA were corrected to take into consideration that only 225 out of 250 /zl of the reconstituted extract were injected into the HPLC (Fig. 6). As can be seen the two standard curves were practically superimposable. The non-specific binding (NSB) values in RIA buffer ranged from 1.7 to 3.0% ( m e a n _ S D = 2.3__.0.5%) and the specific binding (Bo) values ranged from 35.4 to 52.5% (mean_ SD= 43.0__.6.1%). The equation defining the mean standard curve in RIA buffer was the following: logit (B/B o, in % ) = - 2 . 5 9 0 log(exemestane concentration in pg/tube)+3.971. The recovery experiments described above gave identical results when 0.5 ml of urine were used in place of 1 ml of plasma (data not shown).

The recovery of the method in plasma ranged from 100.8 to 106.6%; the inter-assay precision (expressed as RSD) ranged from 10.9 to 17.7%, and the intraassay RSD ranged from 5.8 to 15.4%. In urine the recovery ranged from 96.0 to 104.1%; the inter-assay precision ranged from 9.2 to 16.2% and the intraassay precision ranged from 4.9 to 11.5% The sensitivity limit of the assay was 4 and 8 pg of exemestane/ml of plasma and urine, respectively. The limit of quantitation was set at 12.5 and 25 pg/ml in plasma and urine, respectively.

3.5. Plasma levels of exemestane in a healthy postmenopausal volunteer Plasma concentrations of exemestane at steady state in a healthy postmenopausal volunteer measured at day 7 after 6 consecutive daily administrations of the drug at the dose of 1 mg are shown in Fig. 7. Exemestane concentrations in plasma ranged from 35 pg/ml (immediately before the seventh drug administration) to 710 pg/ml (1.5 h after drug adminstration).

4. D i s c u s s i o n

The specificity of the antiserum against the available exemestane derivatives was satisfactory.

S. Persiani et al. / European Journal of Pharmaceutical Sciences 4 (1996) 331-340

700 1 600

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LU Z

400

O9 ILl

300

UJ X LU

200 1000~ 0

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5

10

15

20

q

25

TIME (h) Fig. 7. Plasma levels of exemestane at steady state in a healthy postmenopausal volunteer receiving daily doses of 1 mg of the drug for 7 days.

From the results of the cross-reaction experiments it can be deduced that exemestane derivatives modified in the 17 position were characterized by a larger reduction of immunoreactivity than derivatives modified in the 6 position (see the respective cross-reaction percentages of FCE 25071 and of FCE 24204). This was expected considering the structure of the immunogen used to raise the antiserum in which the 17 position of the steroid skeleton is farther removed from the carrier protein compared to the 6 position and therefore more exposed to immunological recognition (Fig. 2). The mean standard curves in the presence of the extracts of 1 ml of plasma after HPLC and in RIA buffer without HPLC were almost superimposable, indicating that the interference given by plasma constituent(s) (androstenedione and/or androsterone) was eliminated by the HPLC prepurification of the sample (Fig. 6) and that the recovery of the method proved excellent notwithstanding the several steps involved in the procedure. This is due to the fact that, except for the solid-phase extraction, the procedure was performed with the aid of automated instruments. Future automation of the solid-phase extraction procedure is planned. Using the procedure described here it was possible to analyse about 70 plasma samples and 100 urine

339

samples per working week. The time includes one day for the extraction and evaporation, three days for the HPLC (run overnight) with relative fraction evaporation and about one day for the RIA. It is noteworthy that in separate experiments, the results of which are not shown, the analytical method described here was able to selectively and accurately measure exemestane in plasma in the presence of 10 ng/250 /_tl of androstenedione. This androstenedione concentration correponds to more than ten times that found in plasma under physiological conditions (Bblanger et al., 1990). Moreover, when applied to several baseline plasma and urine samples t¥om healthy postmenopausal volunteers the procedure never gave false positive results, further indicating that the HPLC purification was able to eliminate all the interferences given by endogenous plasma and urine constituents (data not shown). The difference in the quantitation limit of the method in plasma (12.5 pg/ml) and urine (25 pg/ml) was due only to the different volume extracted (1 ml for plasma and 0.5 ml for urine). The HPLC procedure was able to separate exemestane from all its available derivatives synthesized as possible metabolites (data not shown). This should allow the collection of other fraction(s) corresponding to the retention time of identified exemestane metabolite(s) that could be analysed using a different radioimmunoassay. For this purpose an antiserum against an identified exemestane metabolite, FCE 25071 (Cocchiara et al., 1994), is being developed. Finally the method here described has been applied for the determination of exemestane in plasma of a healthy postmenopausal volunteer receiving daily doses of exemestane of 1 mg. The drug appeared to be absorbed rapidly, since the maximum plasma concentration occurred at about 1 h after dosing. Even at the dose level of 1 mg, i.e., a dose 10-25times lower than the one chosen for phase II and III clinical trials, the sensitivity of the method was sufficient to monitor the drug in plasma up to the last time of blood collection (24 h after the daily drug intake). In conclusion the described procedure for the determination of exemestane in human plasma proved useful for the determination of the test compound in plasma samples from a clinical trial in which a low

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dose of exemestane was administered. The procedure is currently being applied for the determination of exemestane in plasma samples from several clinical trials and its reproducibility and robustness have been confirmed.

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