Vol. I. I5Il—I5l5, December [995
`
`Clinical Cancer Research 1511
`
`In Vivo Measurement of Aromatase Inhibition by Letrozole
`
`(CGS 20267) in Postmenopausal Patients with
`
`Breast Cancer
`
`Mitchell Dowsett,1 Alison Jones,
`Stephen R. D. Johnston, Stephen Jacobs,
`Patrick Trunet, and Ian E. Smith
`Academic Department of Biochemistry [M. D.. S. R. D. J.. S. 1.] and
`Department of Medicine ll. E. 5.]. Royal Marsden National Health
`Service Trust. Fulham Road. London SW3 6]}. United Kingdom:
`Department of Medicine. Royal Free Hospital. London. United Kingdom
`IA. 1.]: and Ciba Pharmaceuticals. Basel. Switzerland [P. T.]
`
`ABSTRACT
`
`Thirteen postmenopausal women with advanced breast
`cancer were enrolled in an open randomized Phase I trial of
`a new p.o. active aromatase inhibitor, CGS 20267 (letrozole).
`The primary aim of the trial was to assess the impact of two
`doses of letrozole (0.5 and 2.5 mg/day) on the peripheral
`aromatization of androstenedione to estrone. An in viva
`
`isotopic technique was used to measure peripheral aroma-
`tization in each patient before treatment. The patients were
`then randomly assigned to one of the two doses, and mea-
`surements of aromatization were repeated after 6 weeks. At
`0.5 mg and 2.5 mg/day, letrozole inhibited aromatization by
`98.4% (97.3 to >99.1) and >98.9% (98.5 to >99.1; geomet-
`ric means and ranges), respectively. Plasma estrogen levels
`were also measured before and during treatment. At the dose
`of 0.5 mg/day estrone and estradiol levels fell by 82.0% and
`84.1% (geometric means). respectively. At the dose of 2.5 mg/
`day. the estrogens fell by 80.8% and 68.1%, respectively.
`There were no significant differences between the doses
`in aromatase inhibition. No formal statistical analysis was
`performed on the estrogen data. Letrozole is therefore a
`highly effective inhibitor of aromatase, causing near com-
`plete inhibition of the enzyme in peripheral tissues at the
`doses investigated. The falls in estrogen levels were greater
`than those seen with earlier generation aromatase inhibitors.
`
`INTRODUCTION
`Between one-third and a one-half of breast carcinomas are
`
`dependent on estrogen for their continued growth and develop-
`ment. As a result of this. pharmaceutical agents which deprive
`the tumor of estrogenic signals are widely used. The most
`frequently used agent tamoxifen is thought to exert the bulk of
`its therapeutic effectiveness as a result of antagonism of estro-
`gen (l). However. over the last 15 years,
`inhibitors of the
`enzyme of estrogen synthesis. aromatase. have also been found
`to be useful agents in breast cancer treatment. The first of these
`
`agents. AG.2 established the role of such inhibitors (2. 3). but its
`use is limited by its lack of specificity and its association with
`a number of toxic side effects (4). The only other aromatase
`inhibitor which is licensed is 4-hydroxyandrostenedione (form-
`estane: Lentaron). This is more specific than AG and has few
`systemic side effects (5). but has poor p.o. pharmacological
`activity and therefore has to be given by i.m. injection (6). The
`associated local side effects limit the dose of formestane to
`one which achieves between 80 and 90% inhibition of aro-
`matization (7).
`A number of nonsteroidal inhibitors are now completing
`Phase III studies in advanced breast cancer. In earlier studies on
`one of these. letrozole (CGS 20267). we demonstrated that it
`was a highly potent and effective suppressant of estrogen levels
`in postmenopausal volunteers (single dose) and in postmeno-
`pausal patients with advanced breast cancer (8. 9). A single dose
`of letrozole was able to suppress estrone and estradiol
`levels
`below the detection limit of sensitive assays in many patients. ln
`the majority of volunteers. estrogen levels did not return to
`baseline within 2 weeks of this single dosage. Letrozole had
`been shown to be highly selective in vitro. in rodents (l0). and
`in clinical studies (8. 9. ll).
`At the present time. there are no data directly demonstrat-
`ing the effectiveness of this compound on its target enzyme.
`This study set out to derive these data. to allow comparison of
`the drug's pharmacological effectiveness with that of other
`compounds under development. and to assess the effectiveness
`of two different doses which are currently being compared in
`clinical Phase III studies.
`
`PATIENTS AND METHODS
`Treatment
`
`Twelve patients were to be randomly allocated to a daily
`p.o dose of 0.5 mg or 2.5 mg letrozolc for a treatment period of
`at least 6 weeks. One of the l2 patients was replaced because the
`tracer injection was given without [”Clestrone. All patients
`were postmenopausal or had received a bilateral ovariectomy
`(n = 2). Four patients had ceased menstruation for <5 years.
`and one patient had a radiation menopause.
`In these patients.
`menopausal
`status was confirmed by the measurement of
`plasma gonadotrophin levels. The median age of the patients
`was 64 (range. 44—76) years. and the median weight was 63.7
`(range. 43—79) kg. The median age for the patients treated with
`0.5 mg was 49.5 (range, 44—76) years. and the median age for
`the 2.5-mg group was 68 (range. 45—73) years. The median
`
`Received 4/20/95: revised 7/ 12/95: accepted 7/17/95.
`I To whom requests for reprints should be addressed.
`
`3 The abbreviations used are: AG. aminoglutehimide: CI. confidence
`interval.
`
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`

`1512 Aromatase Inhibition by Letrozole in Breast Cancer Patients
`
`weights for the two groups were 59.5 (range. 43—79) kg and 72.2
`(range, 56—76) kg, respectively.
`Patients had local regional recurrence or progressive met-
`astatic breast cancer that had been histologically or cytologically
`diagnosed. Patients who were estrogen receptor-negative were
`excluded from treatment. Five of the patients were estrogen
`receptor positive, and the other eight were unknown. All pa-
`tients had received previous therapy but had been off treatment
`for at least 4 weeks prior to the initiation of this study. Previous
`treatment with aromatase inhibitors was not allowed. Patients
`
`with rapidly progressive metastases, endocrine disorders, renal
`or hepatic dysfunction. or hematological disorders and concur-
`rent malignant disease were excluded. The measurement of
`peripheral aromatization was conducted in the 4 days prior to
`starting therapy (from days 1—4 of the trial) and after 5'/2 weeks
`of treatment (days 43—46 of the trial). Plasma was drawn for
`measurement of esu'one and estradiol on days 1, 4, 22. 43, and
`46 and for pharrnacokinetic measurement on days 22. 43,
`and 46.
`
`Clinical response was not a primary end point of this study
`but was recorded according to standard Union International
`Contre Cancer criteria.
`
`The protocol was approved by the Ethics Committee of the
`Royal Marsden Hospital, and each of the participants gave
`written informed consent.
`Injections of radioactive material
`were covered by a certificate for the "Administration of Radio-
`active Substances for Medicinal Purposes.“
`
`In Vivo Aromatization
`
`This methodology has been described in detail previously
`(12), but some minor modifications were introduced in this
`study to increase the sensitivity of the method. since the effi-
`ciency of inhibition of aromatization with letrozole was antici-
`pated to be high. In brief, patients were given injections of 500
`uCi
`[7—3H]androstenedione (Amersham International, United
`Kingdom) and 5 p.Ci [4-‘4C]estrone (New England Nuclear.
`United Kingdom) in a saline:ethanol mixture of 99:1. Aliquots
`of the isotopes in the injection mixture were taken for calcula-
`tion of the ratio of 3H:"‘C. All urine was collected for 72 h after
`injection, and the urine was then pooled, the volume was mea-
`sured. and it was stored at —20°C until analysis.
`Pre— and on—treatment samples were analyzed simulta-
`neously. Two-thirds of the urine volume was concentrated on an
`XAD Amberlite column (Sigma, United Kingdom). Free ste—
`roids were removed on a salt gradient column packed with
`DEAE Sephadex (Pharmacia, Uppsala, Sweden). The eluate was
`enzymatically hydrolyzed with B—glucuronidase for 48 h. and
`androgens were then removed using a phenolic extraction
`method as follows. The incubate was extracted with 3 X 30 ml
`
`ether for 5 min, and the aqueous phase was discarded. The
`pooled ether fractions were washed with 10 ml 8% NaHCO3 for
`5 min. The ether was then extracted with 3 X 20 ml 0.1 N NaOH.
`and the ether was discarded. Hydrochloric acid was added to the
`NaOH to reduce the pH to between 2 and 4, and the solution was
`extracted with 2 X 40 ml ether. The ether extract was washed
`
`with 10 ml 8% NaHCO3 and with 10 ml distilled water. The
`ether was dried down and was then available for further purifi-
`cation by column chromatography.
`
`The phenolic extract was further purified on a DEAE
`Sephadex column and a QAE Sephadex (Pharmacia) column
`prior to separation of the individual estrogens by HPLC. This
`was conducted on a Hypersil ODS S-um (Chrompack) 4.6 X
`250—mm reverse phase column and a mobile phase of 0.05 M
`acetonitrilezphosphate buffer (38:62), pH 3. This technique has
`been shown to derive radiochemically pure estrogens (12). The
`fractions from the HPLC column were counted for 20 min on a
`
`TriCarb 1900 CA liquid scintillation analyzer. The 3H:"‘C ratio
`for each of the estrogens was calculated, and the rate of aroma-
`tization was determined by comparison to the 3LH:”C in the
`injection mixture. The percentage of inhibition of aromatization
`on treatment was derived by comparison of the on-treatment to
`the pretreatment level.
`The sensitivity of the modified methodology for measuring
`aromatase activity and its inhibition was determined by assess-
`ing the 95% counting error of zero 3H counts in the presence of
`the mean number of '4C counts found in the estrogen fractions.
`By expressing the upper limit of the 95% confidence limits for
`3H in terms of aromatase activity. the upper limit of detection of
`aromatase inhibition was calculated as 99.1%.
`
`Plasma Estrogen Analyses
`The RIA for estradiol has been described in detail else-
`
`where (13). This assay has a detection level of 3 pmol/liter.
`The RIA for estrone was conducted as described by Trunet
`er al. (14). The sensitivity limit of this assay was 10 pmol/liter.
`
`Statistical Methodology
`General. The trial was designed assuming a type I error
`rate of 10% (i.e.. on = 0.10). An 80% confidence interval was
`calculated for the variables described below, since the trial was
`
`designed on the basis of a one-tailed 90% test assuming that the
`higher dose would show a greater percentage of aromatase
`inhibition, Such a confidence interval reflects the design if only
`one limit is inspected. Geometric means rather than standard
`arithmetic means are quoted if a logarithmic transformation to
`the data was performed for analysis purposes and back-trans—
`formed for presentation purposes.
`In Vivo Inhibition of Aromatization. Percentage of aro-
`matization was calculated for both pretreatment and on-treat—
`ment values for estrone and estriol individually and for their
`total. The percentage of inhibition was calculated for each as
`described above. Logarithmic transformations were made to the
`values calculated for percentage of inhibition. A t
`test
`for
`independent samples was performed on these to compare the
`values for the two dose groups. The geometric means for each
`dose group were calculated. The ratio for the geometric means
`and respective 80% confidence intervals were calculated as
`estimates of the difference between the dose groups in terms of
`percentage of inhibition. In addition. a paired t test was per—
`formed on the percentage of aromatization within each dose
`group to investigate whether a significant change occurred in
`their absolute values over time. Summary statistics and esti-
`mates for the changes in absolute values over time with the
`respective 80% confidence intervals were again calculated.
`
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`

`Clinical Cancer Research 1513
`
`Individual values of the aromatization rate for patients
`Table 1
`at the two doses of letrozole and the percentage of inhibition
`of aromatization on-treatment
`AromatiIation
`Dose
`
`Patient
`(mg)
`Pretreatment
`On-treatment
`‘7r Inhibition
`2
`0.5
`1.547
`0.042
`973
`6
`1.691
`0030
`98.2
`7
`1.416
`0.01 1
`>99]
`'0
`11‘7“
`“-022
`>991
`U
`1‘3‘8
`0‘0“
`98'4
`1
`L2”
`0019
`984
`5
`L406
`0'0“)
`98'6
`8
`2.362
`0009
`>991
`13
`2,560
`0.006
`>99]
`
`13
`2.988
`0.018
`>991
`
`3-5
`
`RESULTS
`
`Two of the patients were inevaluable for in viva aromati-
`zation. As stated above. one of the patients did not begin
`treatment after recruitment and was replaced by another ran-
`domized patient. One patient took the medication before the first
`tracer injection, which invalidated the pretreatment aromatiza-
`tion measurement.
`
`Of the 11 evaluable patients. 6 received 0.5 mg and 5
`received 2.5 mg.
`Aromatase Inhibition. Table 1 shows the individual val-
`
`ues for aromatization before and on—treatment along with the
`measurement of the percentage of inhibition. Pretreatment levels
`of aromatization varied between 1.2 and 3.0%. In two patients
`on 0.5 mg and three patients on 2.5 mg. the residual amount of
`aromatization on treatment was below the detection limit of the
`
`assay. All patients on the lower dose showed >97% inhibition
`and on the higher dose >98%. There were no statistically
`significant differences between the doses.
`Plasma Estrogens. There was a marked and highly sig-
`nificant suppression of plasma estrone and estradiol levels by
`both doses of letrozole (Table 2 and Fig. 1). The pretreatment
`levels of both estrogens were lower for the 0.5-mg group. All
`values of estrone for both doses at day 43 and 46 were below the
`detection limit (10 pmol/liter). and the majority of the on-
`treatment values for estradiol were also close to the detection
`
`to
`is therefore difficult
`It
`limit of that assay (3 pmol/liter).
`express these data as a true percentage of pretreatment values.
`For statistical purposes, concentrations below the detection limit
`were given a value 1 decimal point below the respective detec-
`
`tion limit (e.g.. <10 was given a value of 9.99). Using these
`values by week 6. the mean suppression of estrone was 82.0%
`(80% Cl, 79.6—84.l%) and 80.8% (80% CI. 78.3—83.1%) and
`of estradiol was 84.1% (80% CI, 78.3—88.3%) and 68.1% (80%
`.
`CL 54-41—77-799? at 1h": 0-5- and 2.5-mg.doses. respectively.
`There was no evrdence 1n the data suggesting any difference in
`the suppresston of estrone and estradiol. Because so few sam-
`ples were available and valid for analysis by week 6. no formal
`comparative statistical analysis was performed.
`Clinical Response and Tolerability. Assessment of
`clinical response was not a primary aim of this study but was
`corded t the t'me of the second in'ect' n ('
`6 w ek
`ft r
`re
`‘
`a
`1
`.
`j
`10 La.
`e
`s a e
`starting treatment). At thls stage. one of the patients was unas-
`sessable. Of the other 12. 4 of the patients were responders (one
`complete and three partial). Four of the patients were charac-
`terized as no change and four as having progressive disease.
`The drug was well tolerated. with few adverse experiences
`being considered to be associated with the drug. One patient
`showed an increase in hot flushes which was considered to be
`
`possibly related to drug usage. No patient required discontinu-
`ation of treatment for anything other than progressive disease.
`There were no clinically significant changes in hematological or
`biochemical laboratory investigations.
`
`DISCUSSION
`
`Over recent years, numerous aromatase inhibitors have
`entered into early clinical trials. The interest in aromatase in-
`hibitors for the treatment of estrogen-dependent diseases. or
`potentially the manipulation of estrogen-dependent physiologi-
`cal processes. was instigated by the effectiveness of AG (3).
`Two different types of inhibitor have since been developed: (a)
`steroidal substrate analogues such as 4-hydroxyandrostenedione
`(forrnestane) and exemestane and (b) nonsteroidal
`inhibitors
`which bind directly to the cytochrome P450 prosthetic group of
`aromatase such as rogletimide (15—17) and fadrozole hydrochlo-
`ride (14. 18. 19). Most recently, a group of triazole inhibitors
`have been studied clinically. including letrozole. vorozole. and
`arimidex (8. 9. 11. 20—22).
`All three of these inhibitors appear to suppress estrogen
`levels close to or below the detection limit of sensitive estrogen
`assays. but even with the most sensitive assays it has not been
`possible to demonstrate >80—90% suppression.
`It had previ-
`ously been argued (23) that the use of assays based on direct
`measurement of aromatase activity by isotopic analysis is a
`more precise measurement for comparisons between drugs or
`
`Table 2 Geometric mean levels and 80% Cls (range) of estrone and estradiol at each time point and each dose of letrozole
`Estrone
`Estradiol
`
`
`
`0.5 mg
`2.5 mg
`0.5 mg
`2.5 mg
`
`7 Treatment
`Day_
`Mean
`Range
`Mean
`Range
`Mean
`Range
`Mean
`Range
`Pre-
`1
`40.9
`30—59
`60.3
`35—84
`19.4
`12—32
`32.5
`15—52
`4
`44.1
`33—82
`67.8
`54—90
`21.3
`13—41
`23.2
`16—37
`
`On-
`
`22
`43
`46
`
`11.4
`<10.0
`<10.0
`
`<10—17
`<10.()
`<10.0
`
`< 10.0
`<10.0
`<10.0
`
`<10.()
`< | 0.0
`<10.0
`
`46
`4.0
`3.7
`
`<3.0—11
`3.4—4.7
`<3.0—4.3
`
`3.9
`5.2
`5.9
`
`3.6—4.6
`4.9—5.6
`3.7-10
`
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`

`1514 Aromatase Inhibition by Letrozole in Breast Cancer Patients
`
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` Fig] Geometric mean levels of estrone
`
`and estradiol at each time point and each
`dose of letrozole.
`
`dosages of the same drug. As such, we have previously dem-
`onstrated that forrnestane at its conventional dose of 250 mg
`inhibits aromatase activity between 80 and 85% (7). and fadro—
`zole at doses of l or 2 mg twice a day inhibits aromatase by 82.4
`or 92.6%, respectively (19). This technique also demonstrated
`that the use of p.o. 4-hydroxyandrostenedione or roglethimide is
`associated with substantially lower degrees of aromatase inhi-
`bition than this (7, 16).
`Given the degree of suppression of estrogen observed with
`the triazole compounds (8, 9, 20), it was anticipated that letro-
`zole would be more effective than any of the agents measured so
`far. and as a result of this the aromatase assay was sensitized and
`subjected to a formal analysis of sensitivity (99.1% inhibition).
`It is clear that letrozole achieves aromatase inhibition greater
`than any of the inhibitors that we have previously analyzed:
`inhibition was virtually absolute at both doses and there was no
`significant difference between the two doses.
`The pretreatment levels of aromatase ranged between 1.2
`and 3%. and these are consistent with previous measurements in
`postmenopausal women (7. l6, 19, 24). It is notable that the age
`and weight of the patients on 0.5 mg/day was lower than those
`on 2.5 mg, and this is consistent with the higher levels of
`aromatization and plasma estrogen in the 2.5-mg group at base-
`line. It seems unlikely that this would have significantly affected
`the results on percentage of inhibition, since statistically signif-
`icant reduction in the percentage of aromatization was seen over
`time with each dose.
`
`The inhibition of aromatase by vorozole. another triazole
`inhibitor, at doses of 1.0, 2.5. and 5.0 mg/day has been reported
`to be approximately 93—94% (25). It seems likely. however. that
`the sensitivity level of that analysis was insufficient to distin—
`guish between the doses or to allow measurement to the degree
`of sensitivity that has been achieved in the current study. As
`such. comparison of letrozole with the other triazole compounds
`in their pharmacological effectiveness awaits the application of
`more sensitive assays to patients treated with these other com-
`pounds.
`Although clinical efficacy and tolerability were not pri-
`mary end points for this study. it was notable that 4 of the 12
`patients studied showed an objective response within 6 weeks of
`starting treatment. Given that at least 3 months of treatment is
`
`generally given prior to estimates being made of the clinical
`response, continued follow-up may have revealed further objec-
`tive responders from among the patients with stable disease at 6
`weeks. Our previous data on response with this agent also
`demonstrated a 33% response rate (9). Additionally. we previ-
`ously found that tolerability of letrozole is excellent, and our
`experience in the present study confirmed this assessment.
`Letrozole is a well-tolerated and clinically effective com-
`pound which achieves a greater degree of aromatase inhibition
`in viva than has been previously reported for other compounds.
`The two doses of 0.5 and 2.5 mg appear to inhibit aromatization
`in viPO to a similar degree. There is therefore no reason to expect
`a higher degree of antitumor efficacy at the higher dose. The
`current ongoing clinical Phase III
`trials will demonstrate
`whether this is the case.
`
`ACKNOWLEDGMENTS
`We thank Michele Lunn for her valued statistical advice.
`
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`

`Clinical Cancer Research 1515
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`In vivo measurement of aromatase inhibition by letrozole (CGS
`20267) in postmenopausal patients with breast cancer.
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`M Dowsett, A Jones, 8 R Johnston, et al.
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`Clin Cancer Res 1995;1 :1511-1515.
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