Pharmacogenetics has arrived in clinical psychiatric practice with the FDA approval of the AmpliChip CYP450 Test that genotypes for two cytochrome P450 2D6 (CYP2D6) and 2C19 (CYP2C19) genes. Other pharmacogenetic tests, including those focused on pharmacodynamic genes, are far from ready for clinical application. CYP2D6 is important for the metabolism of many antidepressants and antipsychotics, and CY2C19 is important for some antidepressant metabolism. Poor metabolizers (PMs), lacking the enzyme, account for up to 7% of Caucasians for CYP2D6 and up to 25% of East Asians for CYP2C19. Patients having three or more active CYP2D6 alleles (up to 29% in North Africa and the Middle East), are called CYP2D6 ultrarapid metabolizers (UMs). CYP2D6 phenotypes (particularly PMs) are probably important in patients taking tricyclic antidepressants (TCAs), venlafaxine, typical antipsychotics, and risperidone. The CYP2C19 PM phenotype is probably important in patients taking TCAs and perhaps citalopram, escitalopram, and sertraline. On the basis of the literature and the authors’ clinical experience, the authors provide provisional recommendations for identifying and treating CYP2D6 PMs, CYP2C19 PMs, and CYP2D6 UMs. The next few years will determine whether CYP2D6 genotyping is beneficial for patients taking the new drugs aripiprazole, duloxetine, and atomoxetine. Practical recommendations for dealing with laboratories offering CYP2D6 and CYP2C29 genotyping are provided.

Previous studies have suggested elevated estrogen production in tumour-bearing breast quadrants as well as in breast cancers versus benign tissue. Using highly sensitive assays, we determined breast cancer tissue estrogen concentrations together with plasma and benign tissue estrogen concentrations in each quadrant obtained from mastectomy specimens (34 postmenopausal and 13 premenopausal women). We detected similar concentrations of each of the three major estrogens estradiol (E2), estrone (E1) and E1S in tumour-bearing versus non-tumour-bearing quadrants. Considering malignant tumours, intratumour E1 levels were reduced in cancer tissue obtained from pre- as well as postmenopausal women independent of tumour ER status (average ratio E1 cancer: benign tissue of 0.2 and 0.3, respectively; p < 0.001 for both groups), suggesting intratumour aromatization to be of minor importance. The most striking finding was a significant (4.1–8.6-fold) increased E2 concentration in ER positive tumours versus normal tissue (p < 0.05 and <0.001 for pre- and postmenopausal patients, respectively), contrasting low E2 concentrations in ER− tumours (p < 0.01 and <0.001 comparing E2 levels between ER+ and ER− tumours in pre- and postmenopausals, respectively). A possible explanation to our finding is increased ligand receptor binding capacity for E2 in receptor positive tumours but alternative factors influencing intratumour estrogen disposition cannot be excluded.

10532 Background: Aromatase inhibitors of the third generation like letrozole, anastrozole and exemestane are known to suppress plasma estrogens by 90–99% in postmenopausal women. However, little is known about their influence on tissue estrogen levels. Methods: We investigated the effect of neoadjuvant treatment with letrozole (2.5 mg o.d.) given for 16 weeks to patients with locally advanced breast cancers on tissue levels of estradiol (E2), estrone (E1) and estrone sulfate (E1S) measured with a previously published, highly sensitive, HPLC-RIA method (J. Steroid. Biochem. Mol. Biol. 72, 259–264, 2000). All patients had either estrogen receptor (ER) and/or progesterone receptor (PGR) positive tumors (at least one of the receptors expressed in > 50% of the tumor cells). Results: Mean tissue levels of E2, E1 and E1S were found to be 475.8, 272.4, and 160.5 fmol/g tissue at baseline (geometric mean values). Following 16 weeks of treatment with letrozole, these levels fell to 11.3, 18.2, and 16.0 fmol/g corresponding to a suppression of tissue levels of E2, E1 and E1S by 97.6%, 90.7%, and 90.1%, respectively. All plasma levels of E2, E1 and E1S were in the normal range expected for postmenopausal women prior to treatment with letrozole. The individual plasma estrogen fractions were found suppressed by 96.6%, 99.1% and 99.5%, respectively, during treatment with letrozole. Conclusions: Treatment with neoadjuvant letrozole 2.5 mg o.d. potently suppressed tissue estrogen levels in postmenopausal breast cancer patients by 90.1 to 97.6%, surpassing the results previously reported for anastrozole in a similar setting (Clin. Cancer Res. 7, 1230–1236, 2001). While a significant superiority of letrozole versus anastrozole concerning total body aromatase inhibition and plasma estrogen suppression has previously been recorded (J. Clin. Oncol. 20 (3), 751–757, 2002), we are now able to extend this observation to tissue estrogen levels as well. Letrozole (2.5 mg o.d.) seems superior to anastrozole 1 mg daily with respect to plasma as well as tissue estrogen suppression, advocating head to head comparison of these compounds in early breast cancer. [Table: see text]

While growth factors and hormones are known to influence aromatase expression in experimental systems, little is know about potential factors influencing peripheral aromatization in postmenopausal women. The fact that peripheral aromatase activity is higher in old compared to young women and the finding of relatively high tissue estradiol (E2) concentrations after the menopause suggests peripheral aromatization could be influenced by estrogen concentration. To test this hypothesis, we determined plasma hormone levels (n=9) and in vivo aromatization (n=3) in postmenopausal women suffering from advanced breast cancer before and during treatment (4 weeks) with diethylstilbestrol (DES) 5mg three times daily. Plasma levels of cortisol (C), corticosteroid-binding globulin (CBG), and sex hormone binding globulin (SHBG) were significantly increased in all patients (P<0.05 for all). While we found no change in total body aromatization and plasma estrone (E(1)) levels, estradiol (E(2)) and estrone sulfate (E(1)S) were suppressed by a mean of 48.8 and 68.2%, respectively (P=0.043 and 0.008). Surprisingly, plasma levels of androstenedione (A), testosterone (T), dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) were also suppressed by a mean in the range of 32.1 to 52.6% (P<0.05 for all androgens). In contrast, no change in plasma progesterone or 17alpha-hydroxyprogesterone was found. Thus, one possible explanation to our findings could be that DES administered in high doses reduces 17,20-lyase activity in the adrenal gland.

Endocrine dysfunctions have previously been reported in human immunodeficiency virus (HIV) infection. In this study we evaluated the relation of immunological parameters, virus load, clinical stage, and wasting to several parameters of the insulin-like growth factor (IGF) system in 76 patients with HIV infection, of whom 37 had developed acquired immune deficiency syndrome (AIDS). A subgroup of 26 untreated patients was followed during longitudinal testing, while the effects of antiretroviral therapy were evaluated in 34 patients (nucleoside analogs in 9, nucleoside analogs in combination with protease inhibitors in 25). Twenty healthy sex- and age-matched controls were analyzed for comparison. IGF-II was decreased (P = 0.03) and IGF-binding protein-2 (IGFBP-2) and IGFBP-3 protease activity were increased (P < 0.001) in AIDS patients compared with other HIV-infected individuals and controls. Plasma levels of IGFBP-2 and IGFBP-3 protease activity correlated positively to virus load (P < 0.001) and tumor necrosis factor-alpha (P < 0.025) and negatively to CD4(+) and CD8(+) cell counts (P < 0.001). AIDS patients with wasting (n = 13) had lower IGF-II levels (P = 0.001) and higher IGFBP-2 levels (P = 0.001) than other AIDS patients. Although no significant change in any of the IGF-parameters was observed in patients during antiretroviral therapy, patients with elevated IGFBP-3 protease activity before therapy (5 of 34) all had a decrease during treatment. During longitudinal testing in patients followed without antiretroviral therapy, disease progression was associated with increases in IGFBP-3 protease activity and IGFBP-2 levels. Our results reveal several alterations in the IGF system during HIV infection with decreased IGF-II levels, increased concentration of IGFBP-2, and an increased IGFBP-3 protease activity in advanced disease.

This study was designed to evaluate time and dose dependency of alterations in insulin-like growth factor (IGF)-I, free IGF-I and the functional status of IGF-binding protein (IGFBP)-3 in breast cancer patients during treatment with megestrol acetate (MA). In 16 patients receiving MA 160mg daily, total IGF-I levels increased gradually (significant after 3 days on treatment) by a maximum of 2.66-fold after 5–6 months on treatment. However, free (readily dissociable) IGF-I levels increased to a smaller extent (1.23–2.15-fold). This discrepancy may be due to an increase in intact IGFBP-3 determined by Western ligand blotting (WLB). Similar findings were observed in 12 patients treated with MA in escalating doses from 40–800mg daily. A dose-dependent increase in IGF-I was observed up to a dose level of 120mg daily. We conclude that treatment with MA caused a profound increase in plasma levels of total IGF-I accompanied by a moderate increase in free IGF-I. This may explain the anabolic effects of MA in patients suffering from cachexia, but refute the hypothesis that alterations in the IGF-system may contribute to the antitumour effects of MA in breast cancer patients.

The aim of this study was to determine the impact of the administration route and cigarette smoking on plasma oestrogen levels during oral and parenteral oestrogen replacement therapy (ERT). Fourteen healthy postmenopausal women (six smokers and eight non-smokers) were recruited for a prospective, randomised, crossover study at a private outpatient medical centre in Oslo, Norway. All patients were randomised to receive cyclic therapy with oestradiol and norethisterone orally or by the transdermal route each for a 6-month period. Plasma levels of oestrone (Oe(1)), oestradiol (Oe(2)) and oestrone sulphate (Oe(1)S) were determined using highly sensitive RIA methods before and during hormone replacement therapy given by the oral and transdermal route. Comparing smokers and non-smokers, plasma levels of Oe(1), Oe(2) and Oe(1)S were all found to be 40-70% lower in smokers compared with non-smokers when ERT was given orally (Oe(1)S, P<0.05; Oe(1) and Oe(2), P<0.01 for both). Oe(2) given orally caused a higher Oe(1)S/Oe(2) ratio but also a higher Oe(1)/Oe(2) ratio compared with parenteral therapy in smokers (40.2 versus 7(.)0, P<0.01; and 3.2 versus 0.8, P<0.05 respectively). No significant differences in these parameters in the different test-situations were seen in non-smokers. Except for a lower level of Oe(1)S in smokers (non-significant), no difference in plasma oestrogen levels between smokers and non-smokers was observed during parenteral therapy. In conclusion, cigarette smoking has been shown to have major impact on plasma oestrogen levels during oral but not during parenteral Oe(2) replacement.

The influence of the novel antiprogestin onapristone on the serum insulin-like growth factor (IGF) system was studied in a group of 13 postmenopausal women with metastatic breast cancer. Blood samples were obtained before treatment and subsequently after 1, 2 and 3 months on therapy. IGF-I, IGF-II and IGF-binding protein (IGFBP)-2 were measured by radioimmunoassay (RIA). In addition, the IGFBP profile was evaluated by Western ligand blotting (WLB), and IGFBP-3 fragmentation determined by immunoblotting. A moderate (29%) but significant increase in IGF-I was observed after 3 months on treatment (p<0.05). IGFBP-2 showed a significant, progressive increase during treatment when evaluated both by WLB (44% increase over baseline at 3 months) and by RIA (33% increase over baseline at 3 months). There was a non-significant trend towards an initial decrease in IGFBP-3 fragmentation. No significant alterations were observed in IGF-II or any of the binding proteins (except IGFBP-2) determined by Western ligand blotting. Due to the observation that onapristone treatment caused a moderate suppression of serum cortisol and androstenedione, we postulate the observed increase in IGF-I to be due to a slight glucocorticoid agonistic effect of the drug. On the contrary, the increase in IGFBP-2 may be related to disease progression as has been observed in patients suffering from prostatic cancer.

The aromatase inhibitor aminoglutethimide (AG) is widely used in the treatment of advanced breast cancer in postmenopausal women. Apart from the inhibition of estrogen synthesis, previous studies by our group have shown that AG selectively enhances plasma clearance of the estrogen conjugate estrone sulphate (E1S). In the present study we used a novel, highly sensitive radioimmunoassay to measure plasma E1S during treatment with AG. Treatment with AG decreased plasma levels of E1S from a mean pretreatment value of 372.4 to 50.6 pmol/l (mean suppression to 14.5% of pretreatment values) whereas plasma levels of E1 and E2 were suppressed to 40.7 and 32.8% of pretreatment values, respectively. Dehydroepiandrosterone sulphate levels decreased from a mean value of 0.8 to 0.5 μmol/l (mean suppression to 59.6% of pretreatment values), whereas the ratios of E1SE1 and DHEASDHEA decreased to 30.8% (P < 0.001) and 55.5% (P < 0.005) of pretreatment values, respectively. In conclusion, we found that AG suppressed plasma levels of E1S more extensively compared to previous studies. The simultaneous suppression of the DHEASDHEA ratio suggests that AG may influence the disposition of steroid sulphates in general.

Aromatase inhibition is a well-defined treatment option for postmenopausal breast cancer. Although several aromatase inhibitors such as aminoglutethimide, formestane, fadrozole have been found to inhibit in vivo aromatization by >85%, previous studies reported plasma estrogen levels to be sustained at approximately 20–50% of their control level during treatment with these drugs. The discrepancy could be due to lack of sensitivity or non-specific crossreactions in the radioimmunoassay (RIA) methods. Mean plasma levels of estrone (E1) and estradiol (E2) in postmenopausal women are approximately 80 and 20 pmol/l, respectively; on the contrary, mean plasma levels of the estrogen conjugate estrone sulphate (E1S) are approximately 4–500 pmol/l. Most RIA methods for plasma E2 and E1 measurements have sensitivity limits in the range of 2–3 and 7–10 pmol/l, respectively; accordingly, the suppression of plasma estrogens by more than 80–90% will produce hormone values below the sensitivity limit of the method in many patients. Recently, we developed a new method to determine plasma E1S. This assay has a sensitivity limit of 2.7 pmol/l. In theory, this method may allow the determination of plasma E1S levels suppressed to less than 2% of control values in the majority of patients. Using this method, we found different aromatase inhibitors such as formestane, aminoglutethimide, formestane and aminoglutethimide administered in concert or anastrozole to suppress plasma E1S levels down to 24, 13, 7 and 4%, respectively. The suppression of plasma E1S evaluated with this method thus approaches the percentage aromatase inhibition measured with tracer studies.

A major obstacle to the understanding of the mechanisms of action of aromatase inhibitors in breast cancer is the observation that plasma estrogens are sustained at about 30–50% of their control levels despite 85–95% inhibition of the conversion of tracer androstenedione (A) to estrone (E1). The discrepancy could be due to lack of sensitivity of current RIAs. Due to low levels of plasma estradiol (E2) (mean about 20 pM) and E1 (mean about 75 pM) in postmenopausal women, it is difficult to develop RIA methods with the sensitivity required to detect > 90% suppression from baseline. In contrast, the plasma level of the estrogen conjugate estrone sulphate (E1S) is substantially higher (mean level about 400 pM). This paper describes a new assay to measure plasma E1S in the low range aiming to detect > 95% suppression of E1S from baseline values in patients treated with aromatase inhibitors. E1S was separated from unconjugated estrogens, hydrolysed and purified as unconjugated E1. E1 was subsequently reduced to E2, purified, and measured by a highly sensitive RIA using oestradiol-6-(O-car☐ymethyl) oximino-(2-[125I]iodohistamine as ligand. The sensitivity limit of the method was 2.7 pM. Patients on treatment with the aromatase inhibitors formestane or aminoglutethimide or both drugs in concert were found to have plasma levels of E1S ranging from 3 to 274 pM with a mean suppression of 78, 86 and 95%, respectively, compared to baseline, a lower suppression than that reported in previous trials with these drugs.

Droloxifene (3-hydroxytamoxifen) is a novel antiestrogen currently undergoing clinical investigations for treatment of breast cancer patients. We measured plasma levels of sex hormone binding globulin (SHBG) and the gonadotrophins (LH and FSH) at baseline and after 3 months on treatment in a group of fourteen postmenopausal women treated with droloxifene 40 mg daily. Plasma levels of estrone (E1), estradiol (E2) and estrone sulphate (E1S) were measured in a subgroup of eight patients. Plasma SHBG increased during treatment with droloxifene by a mean value of 16.6% (P < 0.05), while plasma levels of LH and FSH decreased by a mean value of 15.7% (n.s.) and 18.1% (P < 0.05), respectively. Plasma levels of E2 and E1 fell slightly (mean decrease 19.4 and 16.7% respectively, n.s.). On the contrary, plasma levels of E1S increased by a mean value of 23.5% (P = 0.068). The ratio of E1S to E1 and E1S to E2 increased by a mean value of 48.3% (P < 0.025) and 53.2% (P < 0.025), respectively. The effect of droloxifene 40 mg daily on plasma levels of SHBG resembles what is seen during treatment with tamoxifen but occurs to a smaller extent. Contrary to tamoxifen, droloxifene caused a minor suppression of plasma LH levels, suggesting droloxifene to have less estrogen agonistic effects on the pituitary.

Estrone sulphate (E1S) may be an important estrogen source in breast cancers, particularly in postmenopausal women. Recent studies have shown that tamoxifen inhibits the uptake and metabolism of E1S to estradiol (E2) in cell cultures. To evaluate a possible influence of tamoxifen on E1S dispositionin vivo, we measured plasma levels of E1S together with unconjugated estrogens (E1 and E2), androgens (T, A, DHEA and DHEAS), SHBG, FSH and LH in 32 postmenopausal breast cancer patients before and during tamoxifen treatment. In a subgroup of 10 patients, we measured 24 h urinary excretion of estrogen metabolites to evaluate the influence of tamoxifen treatment on estrogen metabolism and total estrogen production. Tamoxifen increased plasma levels of E1 S (mean increase of 18.1%, P < 0.05) and the ratio of E1/SE1 (mean increase of 25.7%, P < 0.01) and E1/SE2 (mean increase of 34.7%, P < 0.0005). No significant change in plasma E1 was seen, but plasma E2 was reduced (mean reduction of 12.1%, P < 0.005). The fall in plasma E2 was probably secondary to a fall in plasma T (mean reduction of 11.9%, P < 0.05) due to a reduced ovarian excretion of this androgen. The mechanism may be a reduced gonadotrophin stimulation of the ovary, as plasma FSH and LH fell by mean values of 45.5 and 48.1%, respectively (P < 0.0001 for both). The increase in plasma E1S was accompanied by a reduced ratio of 2OHE1/E1 in urine (mean reduction of 38.2%, P < 0.025) indicating reduced 2-hydroxylation. Possible mechanisms for these alterations are discussed.

Summary OBJECTIVE Oestrogens, androgens and anti‐endocrine drugs such as tamoxifen and aminoglutethimide influence plasma Insulin‐like growth factor‐I (IGF‐I). IGF‐I, in turn, has been found to stimulate the peripheral aromatase in vitro. The aim of this study was to examine relations between sex hormones, IGF‐I and insulin‐like growth factor binding protein‐1 (IGFBP‐1) In post‐menopausal women with breast cancer. DESIGN To measure plasma sex steroids, sex hormone binding globulin (SHBG), IGF‐I, IGFBP‐1, Insulin and urinary oestrogen metabolites In post‐menopausal women with breast cancer not receiving any endocrine therapy. PATIENTS Thirty‐two patients had fasting blood samples obtained between 0800 and 1000h. A sub‐group of 10 patients had 24‐hour urine oestrogen metabolites determined. MEASUREMENTS Plasma steroids and proteins were measured by radioImmunoassays. Urinary oestrogens were measured by GC‐MS. RESULTS SHBG correlated negatively with plasma androstenedione ( P < 0·001), insulin ( P < 0·001), IGF‐I, height and plasma oestrone sulphate ( P < 0·025 for all), but positively with plasma IGFBP‐1 ( P < 0·025). IGFBP‐1 correlated negatively with IGF‐I ( P < 0·001) and the testosterone/SHBG ratio ( P < 0·05). Neither IGF‐I nor IGFBP‐1 correlated with any of the plasma or urinary sex hormones or with the oestrone/androstenedione and oestradiol/testosterone ratios. Multivariate analysis revealed plasma SHBG to correlate positively with IGFBP‐1 ( P = 0·029) and negatively with Insulin ( P = 0·031). Plasma IGFBP‐1 correlated negatively with IGF‐I ( P < 0·0001) but not with insulin. CONCLUSION Our results do not suggest any influence of plasma sex steroids in physiological concentrations on IGF‐I or IGFBP‐1 in post‐menopausal breast cancer patients, nor do they indicate IGF‐I at physiological concentrations Influences the ratios between plasma oestrogens and their androgen precursors.

The clinical and biochemical effects of combined treatment with the two aromatase inhibitors aminoglutethimide and 4-hydroxyandrostenedione were evaluated in 10 patients suffering from advanced breast cancer. All patients had become resistant to treatment with one of the drugs before having combined treatment. Seven patients progressing on 4-hydroxyandrostenedione who had aminoglutethimide added to their treatment and achieved a further suppression of plasma oestradiol by a mean of 40.0% (p<0.05). Plasma oestrone was suppressed by a mean of 40.6% (p<0.025) and plasma oestrone sulphate was suppressed by a mean of 63.6% (p<0.025). Two of the patients, neither of whom had responded to 4-hydroxyandrostenedione alone, experienced objective tumour regression when aminoglutethimide was given in concert. Three patients progressing on aminoglutethimide who had 4-hydroxyandrostenedione added showed no further suppression of their plasma oestrogen levels, and no tumour regression was observed. These findings suggest a dose-response relationship between plasma oestrogen suppression at low postmenopausal levels and objective tumour response in breast cancer.

Using a [4-14C]estradiol bolus injection technique possible effects of AG administration on estrogen metabolism were studied in seven patients. No alterations in steroid disposition were seen after a short term AG treatment. Chronic AG administration produced no change in mean estradiol clearance value. Chronic AG treatment as a low- or high-dose drug schedule produced a consistent reduction in estrone sulfate production rate (mean reductions 24 and 42%) as well as an increased estrone sulfate elimination rate constant (mean increase 30 and 69%). Urinary estriol secretion rate was consistently increased during chronic treatment (mean increase 95%) with no difference between low- and high dose drug schedules. These findings are consistent with AG being a potent enzyme inducer stimulating estrogen metabolism, and this mechanism may be an important part of AG mechanism of action by reducing estrone sulfate bioavailability.