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).

Pharmacokinetics of S- and R-enantiomers of aminoglutethimide following oral administration of racemic drug in breast cancer patients.

This study was undertaken to examine the pharmacokinetics of both enantiomers of AG--that is, (R-AG) and (S-AG) and respective acetyl metabolites, R-AcAG and S-AcAG--in breast cancer patients. Six patients received a single dose (500 mg) of the racemic drug, and serial plasma samples and urine were collected over a 48-hour period. R-AG, S-AG, R-AcAG, and S-AcAg were measured simultaneously by high-performance liquid chromatography using two serial chiral separation columns with ultraviolet detection. The plasma concentrations of R-AG were about 1.5 times higher than those of S-AG, and the data for both enantiomers exhibited the characteristics of the one-compartment open model. There were no significant differences between R- and S-AG in ka, tmax, V/F, and t1/2. The formation of R- and S-AcAG was rapid, and no correlation was found between the t1/2 values of the AG enantiomers with that of their acetylated metabolites. Overall, 41% of the dose was excreted in urine as AG (15% R-AG and 26% S-AG) and 5.1% as AcAG (2.9% R-AcAG and 2.2% S-AcAG). Renal clearance of S-AG was significantly greater (i.e., 2.3-fold) than that of R-AG and appears to be most likely the cause for the other pharmacokinetic differences observed. Both enantiomers had low renal extraction ratios, suggesting extensive tubular reabsorption of the compounds. However, based on the data obtained, it was concluded that the main factor contributing to the therapeutic effectiveness of racemic AG is the large potency difference between the R- and S- forms (R > S). The pharmacokinetic differences between R-AG and S-AG appear to contribute only marginally to the activity of this drug as an aromatase inhibitor.

Determination of aminoglutethimide enantiomers as dansyl derivative in human plasma by HPLC with fluorescence detection.

Determination of aminoglutethimide enantiomers as a dansyl derivative in plasma by HPLC has been achieved using cellulose tris(3,5-dimethylphenyl carbamate) chiral stationary phase known as Chiralcel OD, and a mobile phase consisted of ethanol-cyclohexane-methanol (95:5:2 v/v/v). The limit of detection for each enantiomer of aminoglutethimide using fluorescence detector was 20 ng ml-1.

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.

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.

Characterisation of metabolites of 3-ethyl-3-(4-pyridyl)-piperidine-2,6-dione, a potential breast cancer drug.

The identification of metabolites from the pyridylglutarimide 3-ethyl-3-(4-pyridyl)piperidine-2,6-dione (PG, Rogletimide) was achieved using liquid chromatography-mass spectrometry with a thermospray interface (LC-TSP-MS). The urinary metabolites include PG N-oxide, the products of 4- and 5-hydroxylation in the piperidine residue (4- and 5-hydroxy-PG) and a gamma-butyrolactone derived via terminal hydroxylation in the ethyl residue. In addition to the above metabolites, several products of glutarimide ring-opening could be detected in the plasma extracts after multiple-dose treatment. Thus LC-TSP-MS is potentially a simple and rapid technique in studies of drug metabolism for the important glutarimide class of drug.

Aminoglutethimide in advanced breast cancer: plasma levels and clinical results after low and high doses.

Drug plasma levels, metabolism data and clinical results were evaluated after the daily administration of either 500 or 1,000 mg aminoglutethimide (AG, Orimeten, Ciba-Geigy) plus hydrocortisone acetate (20 mg b. i. d.). A total of 34 patients with advanced breast cancer entered the study: 17 were given 1,000 mg/day and 17 received 500 mg/day for at least 3 months. A novel HPLC method was developed to determine the levels of AG and its known metabolites [N-acetyl-AG (NAG), formyl-AG, nitroglutethimide, hydroxy-AG] in the biological samples. AG plasma concentration was significantly higher during the 1,000-mg/day regimen. NAG was the only metabolite observed in plasma, always occurring at concentrations lower than those of the parent drug. The ratios between NAG and AG levels distinguish two statistically different groups of patients. Irrespective of the dose, a partial response was observed in 44% of the patients; no change in 32% of cases; and progressive disease had an incidence of 24%. The probability of response was not dependent on the drug AUC or on the NAG/AG ratio and did not significantly depend on previous hormone treatment. Neither the plasmatic level of the AG or metabolite concentrations nor the NAG/AG ratio seemed to affect the incidence of side effects.

Single dose pharmacokinetics of aminoglutethimide by a rapid SIM methodology.

The kinetics of unchanged aminoglutethimide and of its major metabolite N-acetylaminoglutethimide were investigated in healthy volunteers with a new multiple selected ion monitoring (SIM) technique. This method allows a rapid detection of both the unchanged drug and of its metabolite with a single injection and after minimal handling of the samples. This rapid method provided similar kinetic findings, compared to those described with the more time consuming high pressure liquid chromatographic (HPLC) procedure. Moreover, the SIM method allowed the detection of the N-acetylamino metabolite in plasma at longer time intervals vs. the HPLC method. Some typical features of the kinetic behavior (e.g., a discontinuity in the plasma die-away curve for both unchanged drug and metabolite), attributable to partial liver extraction, could also be more clearly observed with the new procedure. This new, rapid technique confirms that aminoglutethimide and N-acetylaminoglutethimide have very similar plasma die-away curves in subjects with a normal conjugating capacity, and that kinetic patterns or individual blood levels can be readily obtained by SIM with minimal acquisition of supplementary equipment.

Alterations in the metabolism of oestrogens during treatment with aminoglutethimide in breast cancer patients. Preliminary findings.

In this small study, the effect of aminoglutethimide on the disposition of oestrogens in women with advanced breast cancer was investigated using bolus injections of 4-[14C]-oestradiol and 6,7-[3H]-oestrone sulphate, alone or in combination. No alterations in oestrogen disposition were seen after short term (6 hours) aminoglutethimide administration. During long term (3 weeks to 8 months) aminoglutethimide treatment mean 4-[14C]-oestradiol clearance was not changed. 14C-Oestrone sulphate AUC was reduced by 43% at a low dose of aminoglutethimide (125 mg twice daily) and by 65% at a high dose (250 mg 4 times daily) with hydrocortisone acetate 25 mg twice daily. The oestrone sulphate terminal elimination rate constant (lambda z) was concurrently increased (mean of 46 and 79%, respectively, with the 2 dosage regimens). A possible increase in oestrone sulphate clearance during long term treatment was tested for by injecting 6,7-[3H]-oestrone sulphate. These studies revealed a marked increase (mean 104%) in oestrone sulphate clearance in patients receiving the high dose aminoglutethimide schedule. Following injection of 4-[14C]-oestradiol plus 6,7-[3H]-oestrone sulphate, the fraction of 4-[14C]-oestradiol metabolised to oestrone sulphate was found to be reduced in all patients (mean 13%). A mean increase of 80% in the urinary excretion of 14C-oestriol was observed after 4-[14C]-oestradiol administration. Our results, although preliminary, suggest that aminoglutethimide is a potent inducer of aminoglutethimide metabolism, thereby producing a significant reduction in plasma bioavailability of oestrone sulphate. These effects may have a role in the action of aminoglutethimide, a finding which warrants further investigation.

Interactions of aminoglutethimide with analgesics using antinociceptive tests in mice.

The action of aminoglutethimide in alleviating bone pain in women with metastatic breast cancer may be due to either an inherent analgesic effect or an interaction with other analgesic drugs. These possibilities have been investigated in mice by conventional antinociceptive tests. In the abdominal constriction test, aminoglutethimide alone had a dose-related antinociceptive activity. A low dose (which had no pharmacological activity) when co-administered with an effective sub-maximal dose of the analgesic, potentiated the effects of the non-steroidal anti-inflammatory drugs (NSAIDs) tested. In the tail immersion test, aminoglutethimide was inactive and did not enhance the antinociceptive activity of the centrally acting analgesics. As cytochrome P450-dependent routes which are inhibited by aminoglutethimide are not involved in the metabolism of the NSAIDs studied, an interaction at the drug metabolism level cannot explain these results. The NSAID-like activity of aminoglutethimide provides some evidence that the drug's mode of action may involve more than the suppression of oestrogen biosynthesis.

Aminoglutethimide as an inducer of microsomal enzymes. Part 2: Endocrine aspects.

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.

The influence of a graded dose schedule of aminoglutethimide on the disposition of the optical enantiomers of warfarin in patients with breast cancer.

The pharmacokinetics of the optical enantiomers of warfarin (R-warfarin and S-warfarin) were investigated in patients treated for breast cancer with aminoglutethimide (AG). The patients received 125 mg AG b.i.d. (i.e., low-dosage regimen); 250 mg AG q.i.d. together with cortisone acetate (i.e. high-dosage regimen); or an escalating dose schedule was followed (i.e. low-dosage regimen followed by high-dosage regimen). The pharmacokinetics for R-warfarin and S-warfarin were determined before initiation of AG treatment and again after 2, 4, or 8 weeks of continuous AG treatment. The plasma clearance for both enantiomers showed a moderate increase (mean 41.2%) in patients receiving the low AG dose, whereas in patients treated according to the high-dosage regimen a marked increase (mean 90.8%) was observed. There was a corresponding reduction in warfarin half-life, and no alteration in distribution volume. These effects on the warfarin pharmacokinetics appeared after 14 days of AG treatment, and after this time point there was no further increase in warfarin clearance. Notably, the effect of AG on warfarin kinetics was the same for both enantiomeric forms of warfarin. These data show that there is a dose-response relationship between AG dose and induction of warfarin metabolism.

Mitochondrial cholesterol availability during gonadotropin-induced Leydig cell desensitization.

In order to define the early lesion (before pregnenolone formation) of the androgen biosynthetic pathway induced by human CG (hCG) or LH in the Leydig cell, we initially have optimized the use of aminoglutethimide to obtain maximal and sustained inhibition of steroidogenesis in vivo and in vitro. Aminoglutethimide inhibited Leydig cell steroidogenesis in vitro at a dose of 100 micrograms/ml. The minimal serum concentration of aminoglutethimide necessary for maximal inhibition of testosterone in vivo was also 100 micrograms/ml (1 h after the ip injection of 20 mg aminoglutethimide). However, testosterone levels were normal 12 h later, coincident with a marked fall in the serum aminoglutethimide levels. The t 1/2 of the circulating aminoglutethimide was 5 +/- 0.7 h on the first day of treatment but was reduced to 3.0 +/- 0.4 and 2.25 +/- 0.35 h at 2 and 3 days of treatment. At the dose eliciting maximal and sustained steroid inhibition (60 mg/day) aminoglutethimide was able to prevent the estradiol-dependent late steroidogenic lesion (after pregnenolone formation) induced by 1 microgram hCG, with no effect on the early lesion (before pregnenolone formation) caused by 10 micrograms hCG. The aminoglutethimide-induced in vivo accumulation of cholesterol in the inner mitochondrial membrane (by 50%) was associated with an increase in the production of testosterone and pregnenolone by the Leydig cell when subsequently incubated in vitro. Similar increases in the steroidogenic capacity were observed after initial exposure of Leydig cells to aminoglutethimide in vitro, even after acid wash to remove the surface-bound endogenous LH. The steroidogenic cholesterol was also increased in desensitized Leydig cells (by 50-70%); however, the conversion of cholesterol to pregnenolone was substantially blocked in animals with the early lesion. Our findings define the requirement of increasing high levels of aminoglutethimide to inhibit cholesterol metabolism and provide a dose schedule suitable for studies on cholesterol availability and inhibition of steroidogenesis in the rat. These results support our proposal that the early lesion observed in desensitized Leydig cells is due to inhibition of the side-chain cleavage activity rather than to a decrease in the amount of metabolically available cholesterol.

Observations on the pharmacokinetics of low dose aminoglutethimide in patients with advanced breast cancer.

Serum aminoglutethimide (AG) and N-acetylaminoglutethimide (NAG) concentrations were measured by high pressure liquid chromatography (HPLC) in 24 postmenopausal women with advanced breast cancer receiving increasing doses of oral AG. Patients received 62.5 mg b.d., 125 mg b.d., 250 mg b.d., and 500 mg b.d. of AG alone, and 500 mg b.d. of AG combined with hydrocortisone (HC) 20 mg b.d. Dose was increased at monthly intervals. Each dose increment was accompanied by a significant rise in serum AG and NAG levels (P less than 0.05). The addition of HC to the dose of 500 mg b.d. of AG did not alter serum AG or NAG concentrations significantly. Although serum AG and NAG levels appeared to increase linearly with dose, serum NAG increased significantly more slowly, leading to a fall in the NAG:AG ratio during therapy. The NAG:AG ratio appeared to stabilise only after about 6 months of treatment.

High-performance liquid chromatographic assay for simultaneous estimation of aminoglutethimide and acetylaminoglutethimide in biological fluids.

A simple rapid high-performance liquid chromatographic assay for simultaneous estimation of aminoglutethimide and its acetylated metabolite acetylamidoglutethimide in plasma, saliva, and urine is described. This assay is suitable for pharmacokinetic studies in normal subjects and patients receiving other medication in addition to aminoglutethimide.

Influence of ACTH on aminoglutethimide induced reduction of plasma steroids in postmenopausal breast cancer.

In postmenopausal women estrogens are mainly produced by aromatase mediated conversion of adrenal 4-ene-steroids in peripheral tissue, a process which is inhibited by aminoglutethimide (AG). To assess possible fluctuations in adrenocortical steroid secretion and the impact on plasma estrogen levels the effect of physiological ACTH doses was studied in 24 postmenopausal women with advanced breast cancer treated with AG 4 X 250 mg and hydrocortisone 2 X 10 + 1 X 20 mg daily. Synthetic 1-24 ACTH (Synacthen) 0.5 mg was injected i.m. at 8.30 a.m. (t0), 10 h after the last AG + hydrocortisone dose. A definite decrease of t0 levels of the 5-ene-steroids dehydroepiandrosterone and its sulphate, and androstenediol was found at 1 month, with no further decrease at 2 months. 5-Ene-steroids responded decreasingly to ACTH under treatment. The 4-ene-steroids progesterone, androstenedione and testosterone, before and after ACTH were not suppressed by 1 or 2 months of treatment. Basal (t0) cortisol remained normal. Cortisol response to ACTH (delta max) was drastically diminished, but still present. Plasma estrogens were decreased. Estradiol (E2) at t0, being low from the start, fell by 50%. Estrone (E1) at t0 dropped to 30%. ACTH had measurable influence on E2, but did cause a transient increase of E1 (mean delta max 40% of baseline value) under treatment. The following conclusions are drawn: Treatment with AG + hydrocortisone for postmenopausal breast cancer reduces plasma estrogens, particularly E1. Plasma E1 reduction is not stable as demonstrated by the response to a physiological ACTH dose. The responses of 4-ene-steroids to ACTH are not affected by treatment, except for the response of cortisol which is significantly diminished.

Gas--liquid chromatographic assay of aminoglutethimide and a high-performance liquid chromatographic assay for its acetyl metabolite in biological fluids.

A rapid, sensitive and selective gas--liquid chromatographic assay for aminoglutethimide is described. The same extraction procedure may be employed prior to a high-performance liquid chromatographic assay for acetamidoglutethimide which is also detailed. Both assays are suitable for the study of the pharmacokinetics of aminoglutethimide and acetamidoglutethimide in biological fluids in man.

Determination of aminoglutethimide and N-acetylaminoglutethimide in human plasma by reversed-phase liquid chromatography.

A liquid chromatography method for the determination of aminoglutethimide and N-acetylaminoglutethimide in human plasma is described. The assay involved precipitation of the plasma proteins using a mixture of acetonitrile and perchloric acid, without an extraction procedure. The supernatant was subjected to chromatography on a 3-micrometers ODS Hypersil column eluted isocratically with 11% acetonitrile in 100 mM ammonium formate buffer, pH 3.5. The absorbance was routinely recorded at 242 nm. The standard curves were linear in the range of 0.1-100 micrograms/ml, and the lower detection limit was approximately 0.1 microgram/ml for aminoglutethimide and its plasma metabolite N-acetylaminoglutethimide. The precision of the method, given as the coefficient of variation, was 3.9%. With this method, it was determined that aminoglutethimide and N-acetylaminoglutethimide were present in the plasma of patients receiving single-dose or continuous treatment with aminoglutethimide for breast cancer. No N-formylaminoglutethimide or nitroglutethimide could be demonstrated in the plasma from these patients. Interference from several drugs commonly given to patients with breast cancer was ruled out.