Investigating the mechanism of enhanced cytotoxicity of HPMA copolymer-Dox-AGM in breast cancer cells.

Recently we have described an HPMA copolymer conjugate carrying both the aromatase inhibitor aminoglutethimide (AGM) and doxorubicin (Dox) as combination therapy. This showed markedly enhanced in vitro cytotoxicity compared to the HPMA copolymer-Dox (FCE28068), a conjugate that demonstrated activity in chemotherapy refractory breast cancer patients during early clinical trials. To better understand the superior activity of HPMA copolymer-Dox-AGM, here experiments were undertaken using MCF-7 and MCF-7ca (aromatase-transfected) breast cancer cell lines to: further probe the synergistic cytotoxic effects of AGM and Dox in free and conjugated form; to compare the endocytic properties of HPMA copolymer-Dox-AGM and HPMA copolymer-Dox (binding, rate and mechanism of cellular uptake); the rate of drug liberation by lysosomal thiol-dependant proteases (i.e. conjugate activation), and also, using immunocytochemistry, to compare their molecular mechanism of action. It was clearly shown that attachment of both drugs to the same polymer backbone was a requirement for enhanced cytotoxicity. FACS studies indicated both conjugates have a similar pattern of cell binding and endocytic uptake (at least partially via a cholesterol-dependent pathway), however, the pattern of enzyme-mediated drug liberation was distinctly different. Dox release from PK1 was linear with time, whereas the release of both Dox and AGM from HPMA copolymer-Dox-AGM was not, and the initial rate of AGM release was much faster than that seen for the anthracycline. Immunocytochemistry showed that both conjugates decreased the expression of ki67. However, this effect was more marked for HPMA copolymer-Dox-AGM and, moreover, only this conjugate decreased the expression of the anti-apoptotic protein bcl-2. In conclusion, the superior in vitro activity of HPMA copolymer-Dox-AGM cannot be attributed to differences in endocytic uptake, and it seems likely that the synergistic effect of Dox and AGM is due to the kinetics of intracellular drug liberation which leads to enhanced activity.

Stereoselective determination of pyridoglutethimide enantiomers in serum with a chiral cellulose-based high-performance liquid chromatographic column using solid phase extraction and UV detection.

A sensitive method for the separation and determination of R(+)- and S(-) enantiomers of pyridoglutehimide in serum by high performance liquid chromatography (HPLC) with UV detection was developed. The assay involves the use of a solid-phase extraction for serum sample clean-up prior to HPLC analysis using a C18 Bond-Elute column. Chromatographic resolution of the enantiomers was performed on a reversed-phase cellulose-based chiral column (Chiralcel OD-R, 250 x 4.6 mm I.D.) under isocratic conditions using a mobile phase of 25:75 v/v acetonitrile-0.3 M aqueous sodium perchlorate (pH 6.2 adjusted with perchloric acid) at a flow rate of 0.8 ml/min. Recoveries for R(+)- and S(-)-pyridoglutethimide enantiomers were in the range 86-91% at 300-900 ng/ml level. Intra-day and inter-day precision calculated as %R.S.D. were in the ranges of 2.9-3.9 and 1.5-4.7% for both enantiomers, respectively. Intra-day and inter-day accuracies calculated as percentage error were in the ranges of 1.9-3.3 and 1.5-3.9% for both enantiomers, respectively. Linear calibration curves in the concentration ranges of 100-1500 ng/ml for each enantiomer show correlation coefficient (r) of more than 0.9995. The limit of quantification (LOQ) of each enantiomer was 100 ng/ml using 1 ml of serum. The detection limit (LOD) for each enantiomer in serum using a UV detection set at 257 nm was 50 ng/ml (S/N = 2).

Synthesis and biochemical evaluation of novel inhibitors of aromatase (AR) using an enhanced representation of the active site of AR derived from the consideration of the reaction mechanism.

A novel molecular modeling study, involving inhibitors bound to the iron of cytochrome P450 heme, is described for nonsteroidal inhibitors of aromatase (AR). Study of compounds such as aminoglutethimide (AG) suggests that it utilizes hydrogen bonding group(s) at the active site which would usually H-bond to the steroid C(17) carbonyl group. Interaction between AG's carbonyl groups and the area of the active site corresponding to the substrate C(3)==O group is not possible due to steric interaction. Possible reasons for the difference in activity of enantiomers of alternative inhibitors is also suggested, as well as the mode of action of the new AR inhibitor, Arimidex-whose inhibitory activity previously has not been rationalized. The present study proposes that it is able to use hydrogen bonding groups at the active site corresponding to the steroid C(17)==O and C(3)==O area, contradicting a previous study where it is postulated that azole-type compounds only use polar groups at the active site corresponding to the steroid D ring. Using the hypotheses of the modeling study, we designed and synthesized a number of novel (enantiomerically pure) inhibitors, which upon biochemical evaluation were found to be good inhibitors; the N-nonyl derivative of the S-enantiomer was found to possess 39% inhibition at 100 microM inhibitor concentration (using androstenedione as the substrate), under similar conditions, and AG possessed 20% inhibition.

[Aromatase inhibitors]. / Les anti-aromatases.

Aromatase inhibitors used in breast cancer, are drugs that inhibit the transformation of androstenedione and testosterone, respectively in estradiol and estrone. Two classes have been described: steroidal inhibitors which act competitively and irreversibly and non steroidal inhibitors which block the P 450 cytochrome. The first one is aminoglutethimide which has an adrenal effect on 11, 18 and 21 hydroxylase. Rogletimide, less powerful and less specific is a aminoglutethimide analogue. The response rates obtained with formestane is not different. The clinical development has been stopped due to a lack of specificity. Letrozole, vorozole, exemestane and anastrozole are more powerful and more specific. Letrozole and vorozole are at least as efficient and better tolerated than aminoglutéthimide. Anastrozole, letrozole and vorozole are at least as efficient as megestrol acetate and better tolerated in advanced breast cancer patients receiving a second line hormone therapy.

Use of aromatase inhibitors in postmenopausal women with advanced breast cancer.

Surgeons have been involved in the management of metastatic breast cancer since the technique of ovarian ablation was introduced in 1896. However, as newer hormonal and chemotherapeutic regimens were developed, drug therapy gradually replaced surgery as the preferred treatment for metastatic breast cancer. Thus, management of metastatic breast cancer has largely shifted from surgeons to medical oncologists. Advances in hormonal pharmacology have placed hormonal therapy alongside surgery and radiation therapy as a standard treatment option for women with advanced breast cancer. The purpose of this article is to update surgeons on the current use of hormonal agents for treatment of advanced breast cancer in postmenopausal women, and to review the aromatase inhibitors, a new line of hormonal agents for the treatment of advanced breast cancer.

Enantioselective separation of several piperidine-2, 6-diones on a covalently bonded cellulose 3,5-dimethylphenyl carbamate/10-undecenoate chiral selector.

A series of piperidine-2, 6-dione drugs were enantiomerically resolved on a covalently bonded cellulose 3,5-dimethylphenyl carbamate/10-undecenoate chiral stationary phase (CSP), under normal- or reversed-phase conditions. A single column that can be applied in both modes may be advantageous when considering the shorter setup time required and the solubility of the compound to be analysed since many samples possess different solubilities. The covalently bonded CSP was compared to a commercially available chiral adsorbent, Chiralcel OD, which was previously used in our laboratory for the enantiomeric resolution of the above-mentioned drug series. Chiralcel OD is used in the normal-phase mode and is more fragile than the column used in this study. The user is restricted to the range of solvents available as eluents. Hence, it was of interest to look at the possible advantages of using a chemically bonded phase that can be applied in dual mode.

Synthesis and biological evaluation of 3-(prop-2-enyl)- and 3-(prop-2-ynyl)pyrrolidine-2,5-dione derivatives as potential aromatase inhibitors.

3-(4'-Aminophenyl)pyrrolidine-2,5-dione (WSP3), a known reversible inhibitor of P450 aromatase, was modified using molecular graphics and our model of reversible inhibitor and substrate binding to resemble 10 beta-prop-2-ynylestr-4-ene-3,17-dione (PED), a mechanism-based inactivator of the enzyme. The analogues prepared were 3-substituted 3-(prop-2-enyl) or 3-(prop-2-ynyl) pyrrolidine-2,5-diones and their N-alkyl derivatives. The reported compounds demonstrated no irreversible (time-dependent) inhibition of the human placental P450 aromatase enzyme. However, some reversible activity was seen in several of the 3-(prop-2-ynyl) compounds.

Development and validation of a chiral high-performance liquid chromatography assay for rogletimide and rogletimide-N-oxide isomers in plasma.

The purpose of the present study was to develop and validate a stereo-specific high-performance liquid chromatography (HPLC) assay for rogletimide (Rog) and rogletimide-N-oxide (Nox) isomers in plasma. The assay was performed with a chiral cellulose-[4-methylbenzoate]ester column (Chiracel OJ). Optimal separation was achieved isocratically with a mobile phase consisting of n-hexane/anhydrous ethanol (65/35, v/v) at a flow rate of 0.9 ml/min, with the column being thermostated at +35 degrees C (UV detection at 257 nm). Under these conditions, retention times were approximately 17, 28, 31 and 76 min for R-Rog, S-Rog, R-Nox and S-Nox, respectively. S-aminoglutethimide (S-Ag) served as the internal standard (retention time 70 min). An extraction procedure from plasma samples was developed on Bond Elut RP8 500-mg cartridges; conditioning was performed with 5 ml methanol and 5 ml water, after which 1 ml plasma that had previously been spiked with 5 microM S-Ag was applied. Washing was done with 6 ml water and elution, with 4 ml methanol. After evaporation to dryness, residues were dissolved in 400 microliters anhydrous ethanol and 12-48 microliters was injected onto the HPLC system. Blank plasma from healthy donors showed the random presence of a small interference eluting at the retention time of R-Rog, precluding the accurate quantification of R-Rog concentrations below 2.5 microM. Reproducibility assays demonstrated the need to use an internal standard. Taking into account the internal standard, at 2.5 microM the intra- and inter-assay coefficients of variation were 10.5% and 21.0% for R-Rog 5.5% and 8.7% for S-Rog, 7.6% and 20.8% for R-Nox and 11.7% and 6.4% for S-Nox, respectively. The detection limit was 2.5 microM for R-Rog, 0.5 microM for S-Rog, 0.25 microM for R-Nox and 0.5 microM for S-Nox. Linearity was satisfactory at concentrations ranging from 2.5 to 10 microM for R-Rog, from 0.5 to 10 microM for S-Rog, from 0.25 to 2.5 microM for R-Nox and from 0.50 to 2.5 microM for S-Nox. This assay was applied to plasma obtained from rog-letimide-treated breast cancer patients receiving conventional oral doses and demonstrated its feasibility with regard to sensitivity. The preliminary pharmacokinetic results reported herein suggest for the first time that both the R-Rog and S-Rog isomers are metabolized into rogletimide-N-oxide.

Liquid chromatographic separation and measurement of optical isomers of aminoglutethimide and its acetyl metabolite in plasma, saliva, and urine.

An accurate and specific liquid chromatographic method for the separation and analysis of the R(+) and S(-) enantiomers of both aminoglutethimide (AG) and its acetylated metabolite (AcAG) in plasma, saliva, and urine is described. The separation was achieved by use of two serial Chiralcel OD columns [cellulose tris(3,5-dimethylphenyl carbamate)] with a mixture of hexane/isopropanol/methanol (65:17.5:17.5, per volume) as a mobile phase. The flow rate was 0.7 ml/min, and the compounds were detected in the effluent spectrophotometrically at 245 nm. The plasma, saliva, or urine sample (300 microliters) was extracted with dichloromethane after the addition of an equal volume of acetate buffer (pH 5.6) to the sample. The extraction recovery of the R(+) and S(-) enantiomers of AG and AcAG from plasma, saliva, and urine at different concentrations under these conditions was > 80.9%. No interference from any endogenous substance or concomitantly used drug was observed. The ratio of the peak area of R(+) and S(-) enantiomers of both AG and AcAG/internal standard was linearly (r > or = 0.995) related to concentration in the range 0.83-40.0 micrograms/ml, and the coefficient of variation (CV) at different concentrations was consistently < or = 13%. We are presently employing this method to study the pharmacokinetics of each of these enantiomers in breast cancer patients.

Effect of dexamethasone and cytochrome P450 inhibitors on the formation of 7 alpha-hydroxydehydroepiandrosterone by human adipose stromal cells.

7 alpha-Hydroxydehydroepiandrosterone (7 alpha-OHDHA) is a major metabolite of dehydroepiandrosterone (DHA) using adipose stromal cells. To gain a better understanding of the factors regulating DHA metabolism, we examined the effect of dexamethasone and cytochrome P450 inhibitors on the formation of 7 alpha-OHDHA. Dexamethasone (10(-9) to 10(-7) M) stimulated 7 alpha-OHDHA formation in a dose-dependent manner with a 2- to 5-fold stimulation at 10(-7) M. The dexamethasone stimulated 7 alpha-OHDHA formation was inhibited by RU486 in a dose-dependent manner with suppression to basal levels at 10(-6) M. Progesterone (10(-7) M) had no effect on 7 alpha-OHDHA formation suggesting that the dexamethasone stimulation was acting through the glucocorticoid receptor. Conversion of DHA to 7 alpha-OHDHA was inhibited by ketoconazole and metyrapone. An inhibition of 70-80% was obtained with ketoconazole and 25-60% with metyrapone at concentrations of 10(-5) M. Aminoglutethimide phosphate was less effective than either ketoconazole or metyrapone in inhibiting 7 alpha-OHDHA formation with < 30% inhibition at 10(-5) M. These studies indicate that 7-hydroxylation provides an alternative pathway for the metabolism of DHA in peripheral tissues. This pathway, which is regulated by glucocorticoids, may influence the amount of DHA available for conversion to androstenedione and its subsequent aromatization to estrone. The biological role of the 7-oxygenated metabolites and their effects on other steroidogenic pathways have not been established.

In vitro inhibition of aromatase by the enantiomers of aminoglutethimide and analogs.

The in vitro aromatase activity in microsomal fractions from rat ovary and its inhibition by enantiomers of aminoglutethimide (AG), rogletimide (RG), and cyclohexylaminoglutethimide (ChAG) were studied by analysing the [3H]H2O released when [1 beta-3H]androstenedione was converted to estrone. Maximum velocity (Vmax) and the Michaelis-Menten constant (Km) of the microsomal aromatase enzyme were 17.40 +/- 0.45 pmol/ml/mg protein/min and 1.02 +/- 0.06 microM, respectively. The IC50s for the enantiomers were similar for (+)-R-AG and (-)-R-ChAG (0.86 +/- 0.06 and 0.89 +/- 0.15 microM, respectively. (+)S-ChAG was most potent with IC50 of 0.075 +/- 0.003 microM. The IC50s for (-)-S-AG, (+)-R-RG, and (-)-S-RG were in the same range (23.15 +/- 2.74, 24.58 +/- 2.46, and 24.43 +/- 2.20 microM, respectively.

Aromatase inhibitors--mechanisms for non-steroidal inhibitors.

The conversion of androgens to estrogens occurs in a variety of cells and tissues, such as ovarian granulosa and testicular cells, placenta, adipose tissue, and various sites of the brain. The extragonadal synthesis of estrogens has great pathophysiological importance. Estrogens produced by, for example, adipose tissue have a role in the pathogenesis of certain forms of breast cancer and endometrial adenocarcinoma. The biosynthesis of estrogens is catalyzed by the aromatase, an enzyme localized in the endoplasmic reticulum that consists of two components: a cytochrome P450 (P450 Arom, P450 19 product of the CYP19 gene) and the NADPH cytochrome P450 reductase. The alignment of the amino acid sequences of human P450 19 with other mammalian P450s shows little sequence similarity, which indicates not only that P450 19 is a unique form of the P450 superfamily but also that the aromatase may be a good target for the development of selective P450 inhibitors. Aminoglutethimide (AG) is the pioneer drug of the reversible competitive nonsteroidal aromatase inhibitors. Since AG is a nonspecific aromatase inhibitor and presents some problems with tolerability, a number of structural analogues have been synthesized. For example, rogletimide is slightly less potent than AG but has the advantage of not inhibiting the cholesterol side-chain cleavage and is devoid of sedative action. Elongation of the ethyl substituent of AG and rogletimide leads to an increase in aromatase inhibition. Further studies led to the discovery of a new generation of much more potent aromatase inhibitors. An example is fadrozole. However, although fadrozole is a poor inhibitor of the cholesterol side-chain cleavage, it suppresses aldosterone release by ACTH-stimulated human adrenocortical cells. More selective aromatase inhibitors are the triazole derivatives. Examples are CGS 20267, CGS 47645, R 76 713, and ICI D1033. R 76 713's aromatase inhibitory effect is largely due to its (+)-S-enantiomer, vorozole. Computer modeling studies of the interaction of vorozole with part of the "I-helix" of P450 19 suggest that the chlorine-substituted phenyl ring of vorozole interacts with the gamma-carbonyl group of Glu-302. Thr-310, which corresponds to the highly conserved Thr-252 in P450 101, interacts with vorozole's triazole ring, and the 1-methyl-benzotriazole moiety binds near Asp-309.

Follicular growth rate and lack of influence of oestradiol valerate, testosterone propionate and aminoglutethimide phosphate on ovarian follicular development in the musk shrew, Suncus murinus L.

Growth rate of follicles and effects of administration of oestradiol valerate (EV), testosterone propionate (TP) and aminoglutethemide phosphate (AGP) on the development of ovarian follicles of the adult musk shrew were investigated. The follicular growth rate was estimated by mitotic index before and 2 hr after colchicine treatment to the shrews. Mitotic index of granulosa cells increased linearly with the increase in the follicular diameter. The calculated mean duration of mitosis was 0.48 hr. The mean time taken for follicle to pass from primary follicle stage to preovulatory stage was estimated to be 12 days. Treatment of both the steroids EV and TP, failed to produce any noticeable effect on the atretic follicle of the musk shrew. The results indicate that neither EV or TP, nor the potent steroid inhibitor AGP had any influence on the growth of large follicles in this primitive eutherian mammal.

Competition for electron transfer between cytochromes P450scc and P45011 beta in rat adrenal mitochondria.

Rat adrenal mitochondria contain approximately equal levels of P450scc and P45011 beta, each reduced by NADPH through adrenodoxin reductase (ADX-reductase) and adrenodoxin (ADX). Constitutive cholesterol side-chain cleavage (SCC) can be increased over 20-fold through a combination of hormonal activation and inhibition of cholesterol metabolism in vivo prior to isolation of the mitochondria. This stimulation, which results from accumulated reactive cholesterol, does not significantly affect either the dependence of activities on the concentration of isocitrate (IC) and succinate (SU) or the ratio of maximum activities [3:1] supported by these reductants. Thus, the rate of cholesterol SCC is determined independently by electron transfer and the amount of reactive cholesterol. Hydroxylation of deoxycorticosterone (11 beta and 18 positions) required much higher levels of each reductant, indicating less effective reductant transfer to P45011 beta. Reactions at P450scc and P45011 beta, mediated by IC, are enhanced by low concentrations of various dicarboxylates anions (fumarate, SU). The actions of SU dehydrogenase inhibitors and the activity of fumarate, a poor direct reductant, suggest that higher production of NADPH results from malate-enhanced uptake of isocitrate. Only synergistic combinations of reductants are sufficient to sustain maximum rates of 11-deoxycorticosterone (DOC) metabolism, whereas IC is fully effective for P450scc. Increased reaction at P450scc (cholesterol loading or addition of 20 alpha-hydroxycholesterol) decreased simultaneous DOC metabolism at P45011 beta in inverse proportion to the estimated intramitochondrial generation of NADPH (1 mM or 10 mM SU > 1 mM IC > 10 mM IC). These decreases were reversed by inhibition of P450scc. Crossover inhibition caused by maximum DOC metabolism was less pronounced. EGTA/albumin treatment, which enhanced activities at both P450scc and P45011 beta, presumably via increased NADPH, diminished this cross-competition. The differential dependence on reductants and the characteristics of crossover competition are consistent with a roughly three-fold more favorable partitioning of electron transfer to P450scc, possibly caused by preferential interaction of reduced adrenodoxin with P450scc.

Separation of the enantiomers of some racemic nonsteroidal aromatase inhibitors and barbiturates by capillary electrophoresis.

High-performance capillary electrophoresis (HPCE) and micellar electrokinetic capillary chromatography (MECC) were applied to the resolution of racemic nonsteroidal antiaromatase drugs and intermediates. Successful results were obtained in both modes using alpha-cyclodextrin (alpha-CD), beta-cyclodextrin (beta-CD), gamma-cyclodextrin (gamma-CD), or 2,6-di-O-methyl-beta-cyclodextrin (DM-beta-CD) as chiral selectors. Depending on the structure of the solute, one of the cyclodextrins was generally better suited for resolution of the racemate. The basic solutes were analyzed under HPCE conditions, whereas the nonionizable compounds such as glutethimide (Doriden) were analyzed in MECC mode. For the azole-type antiaromatase Fadrozole, both HPCE and MECC modes could be used to achieve the separation of the enantiomers. The influence of experimental factors such as pH, the presence of organic modifier, temperature, the micelle concentration, and the concentration of the chiral selector is also discussed on the basis of the results obtained with some chiral barbiturates. The possibility of analyzing the enantiomers directly in plasma samples was also demonstrated.

The influence of aminoglutethimide and its analogue rogletimide on peripheral aromatisation in breast cancer.

The influence of the prototype aromatase inhibitor Aminoglutethimide (AG) and its analogue Rogletimide (RG) on peripheral aromatisation were investigated in 13 postmenopausal women with advanced breast cancer. Seven patients received AG 1,000 mg daily plus Hydrocortisone (HC) cover and six received RG as dose escalation of 200 mg bd, 400 mg bd and 800 mg bd. In vivo aromatase inhibition was investigated using the double bolus injection technique with [4-14C] oestrone ([4-14C]E1) and [6,7-3H] androstenedione ([6,7-3H]4A) followed by a 96 h urine collection. The labelled urinary oestrogens were separated and purified by chromatography and HPLC. Plasma oestradiol (E2) was also measured. AG mean aromatase inhibition was 90.6% +/- 1.8 s.e.m. and E2 suppression 75.7% +/- 7.3 s.e.m. RG mean aromatase inhibition was 50.6% +/- 9.8 s.e.m. at 200 mg bd, 63.5% +/- 5.7 s.e.m. at 400 mg bd and 73.8% +/- 5.8 s.e.m. at 800 mg bd. E2 suppression was 30.7% +/- 9.5 s.e.m., 40.2% +/- 10.3 s.e.m. and 57.6% +/- 9.2 s.e.m. respectively. These results confirm the efficacy of AG as an aromatase inhibitor. RG produced dose dependent E2 suppression and aromatase inhibition, but even at the maximum tolerated dose of 800 mg bd had sub-optimal aromatase inhibition and oestradiol suppression compared with AG.

Conformational analysis of the aromatase inhibitor 3-ethyl-3-(4-pyridyl)piperidine-2,6-dione (rogletimide) and discovery of potent 5-alkyl derivatives.

Analysis of the proton NMR spectra of 3-ethyl-3-(4-pyridyl)piperidine-2,6-dione (rogletimide, 1) shows that it exists in solution with the aromatic ring in an axial position; the same conformation was found for aminoglutethimide. Excess lithium diisopropylamide treatment of 1 formed a dianion which methylated at C-5. The major product with the methyl group trans to the pyridyl ring retained this ring in an axial position and had higher aromatase inhibitory potency than 1. The minor diastereoisomer with an equatorial pyridyl ring had low potency. Upon elongating the alkyl chain, particularly high inhibitory activity was found for the major product isomer having a C-5 octyl, coinciding with the high activity in C-3 and N-1 octyl derivatives of 1, but there was only a small difference in the activity between the enantiomers of 5-octyl-1 and activity was reduced rather than increased when octyl also replaced ethyl at C-3. The results partially support a previously described model comparing binding of androstenedione to aromatase in as much as an axial pyridyl ring is needed to mimic the axial C-19 methyl group of the steroid and bind to the heme component of the enzyme, but for the derivatives bearing a C-5 octyl, the function of the glutarimide ring seems to be simply as a spacer between the hydrophobic chain and the pyridyl ring.

On-line continuous-flow dialysis thermospray tandem mass spectrometry for quantitative screening of drugs in plasma: rogletimide.

The application of a continuous-flow dialysis system, consisting of a membrane dialyser and a trace enrichment column, in on-line combination with tandem mass spectrometry via a thermospray interface is described. The method is applied to the quantitation of drugs in complex biological matrices containing macromolecular interferences. The potential of the method is demonstrated by the quantitative analysis of the anti-cancer drug rogletimide in the plasma of patients after treatment.