Molecularly Imprinted Polymers Doped with Carbon Nanotube with Aid of Metal-Organic Gel for Drug Delivery Systems.

PURPOSE: The incidence of breast cancer worldwide has been on the rise since the late 1970s, and it has become a common tumor that threatens women's health. Aminoglutethimide (AG) is a common treatment of breast cancer. However, current treatments require frequent dosing that results in unstable plasma concentration and low bioavailability, risking serious adverse reactions. Our goal was to develop a molecularly imprinted polymer (MIP) based delivery system to control the release of AG and demonstrate the availability of this drug delivery system (DDS), which was doped with carbon nanotube with aid of metal-organic gel. METHODS: Preparation of MIP was optimized by key factors including composition of formula, ratio of monomers and drug loading concentration. RESULTS: By using multi-walled carbon nanotubes (MWCNT) and metal-organic gels (MOGs), MIP doubled the specific surface area, pore volume tripled and the IF was 1.6 times than the reference. Compared with commercial tablets, the relative bioavailability was 143.3% and a more stable release appeared. CONCLUSIONS: The results highlight the influence of MWCNT and MOGs on MIP, which has great potential as a DDS.

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.

Antidepressant-like effect of Ro5-4864, a peripheral-type benzodiazepine receptor ligand, in forced swimming test.

This study examined the effects of peripheral-type benzodiazepine receptors in the forced swimming test. PK 11195 (1-(2-chloro-phenyl)-N-methyl-N-(1-methylpropyl)-1-isoquinoline carboxamide) and Ro5-4864 (7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepine-2-one) were i.p. injected in mice, according to an acute (1 or 24 h) and a repeated (14 days) schedule. Pretreatment with the agonist, Ro5-4864, significantly reduced immobility time 1 h after treatment but not 24 h after it, whereas the antagonist, PK11195, did not interfere with the test parameters. Nevertheless, PK11195 pretreatment inhibited the Ro5-4864 antidepressant-like effect. Animals repeatedly treated with Ro5-4864 had a similar profile of action with no sign of motor impairment or locomotor activation as evaluated in the rota-rod and open-field tests, respectively. Aminoglutethimide pretreatment, which blocks the early step of steroid synthesis, inhibited the antidepressant-like effect of Ro5-4864. The present findings suggest an antidepressant-like profile for the benzodiazepine, Ro5-4864, that seems to involve steroid synthesis as underlying mechanism.

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.

Endocrine, pharmacokinetic and clinical studies of the aromatase inhibitor 3-ethyl-3-(4-pyridyl)piperidine-2,6-dione ('pyridoglutethimide') in postmenopausal breast cancer patients.

The aromatase inhibitor, 'pyridoglutethimide' (PyG), has been shown previously to suppress serum oestrogen levels in postmenopausal breast cancer patients and to achieve clinical responses at a dose of 500 mg twice daily (b.d.). This report gives the results of a detailed pharmacokinetic and endocrine study of PyG in ten patients. Four doses were tested at intervals of 2 weeks in the following order: 200 mg b.d., 400 mg b.d., 800 mg b.d., 1200 mg b.d. Concentration-time profiles of serum levels of PyG were curvilinear in all patients probably reflecting a saturation of metabolic enzymes. During repeat-dosing metabolism was enhanced approximately 2-fold. Plasma levels of oestradiol were significantly suppressed by the lowest dose of PyG. Although higher doses appeared to achieve greater suppression this was not statistically significant in this small group of patients. There were no significant effects at any dose on the serum levels of cortisol, aldosterone, luteinising hormone, follicle stimulating hormone, prolactin, sex hormone binding globulin or thyroid stimulating hormone. There was a dose-related increase in 17 alpha-hydroxyprogesterone levels and a dose-related decrease in levels of dehydroepiandrosterone sulphate (DHAS). The androgens DHA, testosterone and androstenedione also were significantly suppressed with at least one of the doses of PyG. Synacthen tests did not support these changes being a result of inhibition of 17,20 lyase. It is possible that they are due to enhanced clearance of DHAS. Two patients experienced no toxicity throughout the study, whilst a total of four patients were withdrawn because of side-effects: one at 400 mg b.d., two at 800 mg b.d., and one at 1200 mg b.d. The most frequent side-effects were nausea and lethargy. One patient showed an objective response to treatment.

Pharmacokinetics and pharmacodynamics of the aromatase inhibitor 3-ethyl-3-(4-pyridyl)piperidine-2,6-dione in patients with postmenopausal breast cancer.

The pyridylglutarimide 3-ethyl-3-(4-pyridyl)-piperidine-2,6-dione (PyG) is a novel inhibitor of aromatase that was shown to cause effective suppression of plasma oestradiol levels in postmenopausal patients. In four patients receiving oral doses of PyG (500 mg) twice daily for 3-4 days, oestradiol levels fell to 31.1% +/- 6.3% of baseline values within 48 h and remained suppressed during treatment. Of a further six patients who received oral PyG (1 g) as a single dose, five had quantifiable oestradiol levels. Oestradiol suppression was sustained for 36 h and recovery correlated with a fall of PyG concentrations below a threshold value of ca. 2 micrograms/ml. The pharmacokinetics of PyG were non-linear and, when fitted to the integrated Michaelis-Menten equation, yielded good parameter estimates for Co (21.7 +/- 1.82 micrograms/ml), Km (2.66 +/- 0.68 micrograms/ml) and Vmax (0.86 +/- 0.06 micrograms ml-1 h-1). On subsequent repeated dosing with PyG, both the Km (4.31 +/- 0.48 micrograms/ml) and the Vmax (1.83 +/- 0.13 micrograms ml-1 h-1) values increased and recovery from oestradiol suppression was more rapid, indicating that PyG induces its own metabolism.

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.

Clinical development of aromatase inhibitors for the treatment of breast and prostate cancer.

Numerous aromatase inhibitors are under development for breast cancer treatment. The major aims are to obtain a drug which at its dose of maximum efficacy has no effect on other endocrine systems, has no clinical side-effects and is convenient to administer. During the early clinical stages of development detailed endocrine and pharmacokinetic analyses are a valuable aid in the establishment of a drug's selectivity and its optimum dose, route and frequency of administration. The optimal dose may be defined as the minimum that will achieve maximal and sustained suppression of aromatase activity. This has generally been measured indirectly by comparing the suppression of plasma oestrogen levels at a selection of dosages. This approach has major advantages in speeding dose selection for therapeutic clinical trials. However, it also has some disadvantages including the unproven assumption that clinical response has a direct relationship with the degree of oestrogen suppression. In addition there are technical difficulties of analysis, of wide variability in endocrine response between patients and of demonstrating oestrogen suppression to be equivalent between doses (necessary to show maximal suppression). The direct measurement of aromatase inhibition in vivo by isotopic infusion analysis provides support to these indirect estimates. Its value is shown by our recent results with CGS16949A. The additional value of collating pharmacokinetic and endocrine measurements is apparent from our investigations of 4-hydroxyandrostenedione (4-OHA) and pyridoglutethimide. A consideration of our experience with these inhibitors may be helpful in directing the development of future agents. Whilst the value of aromatase inhibition in breast cancer is established its value in prostatic cancer is in doubt: we have found that 4-OHA is only poorly efficacious in advanced prostatic cancer.

Tissue distribution and elimination of 14C-aminoglutethimide in the mouse.

Following oral administration of 14C-aminoglutethimide (Orimeten), male mice excreted 75% and female mice 59% of the administered dose during 24 h. After 72 h, elimination was essentially complete in male mice, whilst recovery from female mice was 74%. As assessed by quantitative assay and whole-body autoradiography, a wide distribution of radioactivity was found with high concentrations of radioactivity occurring only in the bile, gastro-intestinal tract and liver at early times. Residual (72 h) tissue levels of radioactivity were less than 1 microgram equiv. of 14C-aminoglutethimide/g tissue. Metabolite profiles in urine, tissues and faeces showed extensive metabolism of the drug in a pattern similar to that found in the rat.

Steroidogenesis in Fundulus heteroclitus. I. Production of 17 alpha-hydroxy,20 beta-dihydroprogesterone, testosterone, and 17 beta-estradiol by prematurational follicles in vitro.

In order to understand better the mechanism of gonadotropin action on steroidogenesis in prematurational follicles of Fundulus heteroclitus, follicle synthesis of 17 alpha-hydroxy, 20 beta-dihydroprogesterone (17 alpha-OH,20 beta-DHP), testosterone (T), and 17 beta-estradiol (E2) from a variety of precursors and the maturational response of oocytes were simultaneously followed in vitro. The addition of 25-hydroxycholesterol, pregnenolone, or progesterone to unstimulated follicles increased media 17 alpha-OH,20 beta-DHP, T, and E2, as well as oocyte germinal vesicle breakdown (GVBD) in a dose-dependent manner. Inhibition of cholesterol side-chain cleavage by aminoglutethimide blocked 25-hydroxycholesterol-promoted steroid accumulation and GVBD, indicating that 25-hydroxycholesterol does not directly induce GVBD, but rather is metabolized in the follicle to an active steroid (presumably 17 alpha-OH,20 beta-DHP). Likewise, trilostane, an inhibitor of delta 5-3 beta-hydroxysteroid dehydrogenase, blocked pregnenolone action. Both inhibitors also completely abolished steroid accumulation and GVBD promoted by a F. heteroclitus pituitary extract (FPE), but not GVBD induced by exogenous 17 alpha-OH,20 beta-DHP. FPE also significantly depressed T but enhanced E2 production from exogenous precursors. We have concluded from these observations that (1) cholesterol side-chain cleavage and pregnenolone conversion to progesterone are essential for gonadotropin-promoted follicle steroid production and the resulting reinitiation of meiosis by the oocyte, (2) the enzymes necessary for the conversion of cholesterol to 17 alpha-OH,20 beta-DHP, T, and E2 are present in the unstimulated, prematurational follicle, and (3) gonadotropin initiates steroidogenesis by acting at a step prior to the conversion of cholesterol to pregnenolone; it also appears to enhance aromatase activity.

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.

Mechanisms of action of aminoglutethimide as endocrine therapy of breast cancer.

During the last decade aminoglutethimide has been recognised as a valuable alternative in endocrine therapy for advanced breast cancer. Although some side effects do occur, most often these are initial effects which subside within a few weeks, and cessation of therapy is not usually indicated. Aminoglutethimide was originally introduced as an inhibitor of steroidogenesis in the adrenal cortex. It was soon recognised, however, that inhibition of the non-glandular aromatase, blocking the conversion of androgenic prohormones to oestrogens, was more important, resulting in decreased blood levels of oestrogens. In this review the role of aromatase inhibition as the only important aspect of the mechanism of action of aminoglutethimide is challenged. Evidence has accumulated during the last few years that aminoglutethimide is a most potent inducer of microsomal enzymes. In addition to the pharmacological implications this has (suggesting important interactions), it also points to the possibility that levels of oestrogens are decreased due to accelerated metabolism of these hormones. Based on new experimental data, and also clinical work with alternative aromatase inhibitors, it appears that the antitumour activity of aminoglutethimide may be due to both aromatase inhibition and accelerated metabolism of oestrogens. This seriously challenges the importance of aromatase inhibition alone as a strategy in endocrine therapy of breast cancer, and furthermore suggests that accelerated metabolism of key hormones is an alternative strategy to be explored.

Metabolism profiles and excretion of 14C-aminoglutethimide in several animal species and man.

1. Following administration of a single oral dose of 14C-aminoglutethimide to rats, guinea-pigs, rabbits and man, greater than 89% of the dose was excreted in urine and faeces within 72 h; dogs eliminated only 51% in this time. 2. Extensive metabolism occurred in all species, with N-acetylaminoglutethimide being the major metabolite except for dog and man. In the latter two species unchanged drug was the main product excreted. 3. A metabolite, 3-(4-acetamidophenyl)-3-(2-carboxamidoethyl)tetrahydrofuran-2-one, not previously found in human urine, was identified. 4. Chronic administration of aminoglutethimide to rats produced no detectable change in the excretory or metabolite patterns of the drug. However chronic administration of phenobarbitone decreased the urinary excretion of 14C over a 72 h period. 5. Residual (72 h) tissue levels of 14C were less than 1 microgram equivalent of 14C-aminoglutethimide/g tissue in the rat, guinea-pig and rabbit. Dog tissues retained a considerable quantity of 14C at this time.

Clinical pharmacology of aminoglutethimide in patients with metastatic breast cancer.

The pharmacology of aminoglutethimide (AG) was studied in two subsequent trials without hydrocortisone supplementation. A total of 79 patients with metastatic breast cancer entered the study, and their plasma and urine samples were analyzed by high-performance liquid chromatography (HPLC). Thirty evaluable patients with a median age of 57 years (range, 37-79) were treated with the standard dose of 1000 mg/day, and 37 evaluable patients with a median age of 59 years (range, 35-79) received 500 mg/day. The median follow-up in the two groups was 5 months (range, 1-16) and 4 months (range, 1-21), respectively. After the first oral dose of 500 mg, peak plasma concentrations of AG were observed 1-4 h after administration in 15 patients. The elimination half-life was 10.1 +/- 1.7 h (mean +/- SD) after initial dosage; it decreased significantly to 6.9 +/- 1.2 h after 8 weeks of treatment. The area under the curve of AG concentrations was 92.5 +/- 14.2 micrograms/ml x h. The total clearance rate was 5.5 +/- 0.9 1/h and the volume of distribution was 80 +/- 111. About 23% of the drug was excreted unchanged in the urine. The major metabolite, N-acetyl-AG (AAG), had the same half-life as AG. A comparison on day 7 of treatment revealed that doses of 1000 and 500 mg yielded AG plasma concentrations of 9.0 +/- 1.2 and 4.5 +/- 0.5 micrograms/ml, respectively. After 1 month of treatment, however, AG plasma levels of 6-7 and 4-5 micrograms/ml were observed, respectively. A 50% reduction of dose, therefore, resulted in only 30% lower AG levels during continuous treatment. Apparently, the induction of metabolism is of greater importance in standard-dose than in lower dose treatment. The plasma concentrations of AG did not bear a relationship to the clinical response.

Aminoglutethimide bioavailability, pharmacokinetics, and binding to blood constituents.

The bioavailability of aminoglutethimide tablets was examined using a spectrophotometric assay. For six subjects receiving 500 mg of aminoglutethimide as an oral solution, the average peak concentration was 6.2 micrograms/ml with a median time of 0.8 hr. The corresponding average peak concentration for tablet administration was 5.9 micrograms/ml with a median time of 1.5 hr. Average values for the area under the curve (AUC) extrapolated to infinity were 89.0 and 96.8 micrograms hr/ml for the solution and tablets, respectively. The tablets had a 9% larger mean for the AUC than the solution and a 5% lower value for the mean maximum concentration. The bioavailability of the tablets is considered equal to that of oral solution. Data for individual concentration versus time curves were treated by nonlinear least-squares curve fitting. A two-compartment model with first-order absorption gave an acceptable fit for most data sets, but the individual absorption rate coefficients were not reliably determined. Values were estimated for plasma clearance, renal clearance, and volume of distribution. The distribution of aminoglutethimide between plasma and cells of human blood was examined in vitro; the drug concentration in cells was 1.4-1.7 times the concentration in plasma. The binding of aminoglutethimide to plasma proteins of human blood was measured by equilibrium dialysis at starting concentrations of 5 and 10 micrograms/ml. The binding ranged from 21.3 to 25.0% without concentration dependence.