Enhancement of the anti-herpetic effect of trichosanthin by acyclovir and interferon

Trichosanthin (TCS) is a type I ribosome-inactivating protein that has a wide range of pharmacological activities. The present study investigated the effectiveness of TCS on herpes simplex virus (HSV-1). The anti-viral activity and toxicity of TCS on Vero cells were measured. Results showed that the ED(50), TD(50) and the therapeutic indices were 38.5, 416.5 and 10.9 microg/ml, respectively. Anti-viral activity of TCS was substantially potentiated when it was used in conjunction with other anti-viral agents. The ED(50) of TCS was reduced 125-fold by acyclovir at a concentration of 0.001 microg/ml, which was practically devoid of significant anti-viral activity. Similarly, the ED(50) of TCS was reduced 100-fold by interferon-alpha2a at a concentration of 100 IU/ml. In conclusion, TCS is effective against HSV-1 and other anti-viral agents such as acyclovir or interferon can potentiate its action substantially.     

Comparison of physical dependence of ohmefentanyl stereoisomers in mice

Stereo-structural difference of ohmefentanyl stereoisomers on analgesic action and receptor affinity has been studied. To assess the difference of ohmefentanyl stereoisomers in physical dependence, the potency of physical dependence was quantified by estimating the ED50 value of ohmefentanyl stereoisomers in the naloxone-precipitated jumping test in mice. Morphine was used to assess the method and as a drug of comparison. The results indicate that the degree of physical dependence of morphine can been quantified by estimating the ED50 value of morphine withdrawal jumping induced by naloxone. A significant difference was observed in withdrawal jumping ED50 values among ohmefentanyl stereoisomers. Of these isomers, F9202 and F9204 had similarly potent analgesic action, but very significant difference in naloxone precipitated withdrawal response. Dependent potency index of F9204 was 618-fold weaker than that of F9202. It is concluded that a stereo-structural difference in physical dependence is found to exist among ohmefentanyl stereoisomers. Compound F9204 displayed a strong analgesic action and weak physical dependent potency.     

Glucose regulation of insulin receptor affinity in primary cultured adipocytes

Treatment of primary cultured adipocytes with 20 mM glucose resulted in a progressive increase in specific 125I-insulin binding that began almost immediately (no lag period) and culminated in a 60% increase by 24 h. This effect was dose-dependent (glucose ED50 of 4.6 mM) and mediated by an increase in insulin receptor affinity. Moreover, it appears that glucose modulates insulin receptor affinity through de novo protein synthesis rather than through covalent modification of receptors, since cycloheximide selectively inhibited the glucose-induced increase in insulin binding capacity (ED50 of 360 ng/ml) and restored receptor affinity to control values. Importantly, insulin sensitivity of the glucose transport system was increased by glucose treatment (63%) to an extent comparable with the enhancement in receptor affinity, thus indicating a functional coupling between insulin binding and insulin action. When the long term effects of insulin were assessed (24 h), we found that insulin treatment reduced 125I-insulin binding by greater than 60% by down-regulating the number of cell surface receptors in a dose-dependent manner (insulin ED50 of 7.4 ng/ml). On the basis of these studies, we conclude that 1) insulin binding is subject to dual regulation (glucose controls insulin action by enhancing receptor affinity, whereas insulin controls the number of cell surface receptors); and 2) glucose appears to modulate insulin receptor affinity through the rapid biosynthesis of an affinity regulatory protein.     

Synthesis of anticonvulsive AMPA antagonists: 4-oxo-10-substituted-imidaz

The overstimulation of excitatory amino acid receptors such as the glutamate AMPA receptor has been implicated in the physiopathogenesis of epilepsy as well as in acute and chronic neurodegenerative disorders. An original series of readily water soluble 4-oxo-10-substituted-imidazo[1,2-a]indeno[1,2-e]pyrazin-2-carboxylic acid derivatives was synthesized. The most potent derivative 6a exhibited nanomolar binding affinity (IC50 = 35nM) and antagonist activity (IC50 = 6nM) at ionotropic AMPA receptor. This compound also demonstrated potent anticonvulsant properties in MES in mice and rats with long durations of action with ED50 values in the 1-3 mg/kg dose range following ip and iv administration.     

Mode of antitumor action of recombinant human tumor necrosis factor on the sarcoma Meth A transplanted in the mouse

The mode of antitumor action of rHu-TNF was elucidated in BALB/c mice bearing Meth A fibrosarcoma 7 days after transplantation with respect to time course, dose-response relationships and selectivity of the effects. The maximal cytotoxic effect on tumor cells revealed by inhibition of DNA synthesis and maximal lesional effect on tumor vasculature revealed by change in blood pool-size in the tissue were detected at 30 min and 1 h after administration of rHu-TNF, respectively. The dose-response relationship between cytotoxic and tumoricidal effects of rHu-TNF was irrespective of administration route. ED50s of these antitumor effects after i.v. administration of rHu-TNF were about 50 times as high as ED50s after i.t. administration. ED50 of i.t. given rHu-TNF for vascular effect was about 20 times as high as that for cytotoxicity while ED50 of i.v. rHu-TNF for vascular effect was only 2-3 times as high as that for cytotoxicity. The whole body autoradiographies with [125I]HSA given i.v. to see the blood influx into tumor tissue and [14C]thymidine given i.v. to see DNA synthesis in the whole body after administration of rHu-TNF revealed that the distribution of radioactivity was markedly changed in the tumor alone without any detectable change in other whole body tissues. In conclusion, the in vivo antitumor effect of rHu-TNF given i.t. or i.v. appears to be exerted through the direct action on Meth A sarcoma rather than indirectly on tumor vasculature. Under present conditions, the effect of rHu-TNF in the whole body tissues seems rather selective on cells and vasculature of the tumor.     

The effect of a novel CCK-antagonist (lorglumide) on human and guinea pig gallbladder strips: a tensiometric study

The effect of a novel CCK-antagonist (lorglumide, CR 1409) was evaluated by "in vitro" tensiometric studies on 16 human (gallstone patients) and 12 guinea pig gallbladder smooth muscle strips. In the animal experiments, increasing doses of lorglumide (0.2-6.5 uM) caused a rightward shift of the dose-response curves of CCK-OP, with an increase of the ED50 from 8.2 nM +/- 1.62 SEM, n = 12; to 100 nM +/- 12, n = 4) without affecting the maximal effect (Emax). Schild plot gave an affinity constant of 7.19. In human gallbladders, the effect of lorglumide was also present (ED50 increased from 47 nM +/- 8 SEM, n = 16; to 300 nM +/- 10 SEM, n = 4) coexisting with a large inter-sample variation for CCK-OP ED50s and maximal contractions, most likely due to the histological changes of the wall in chronic cholecystitis. The affinity constant was similar to that found in animal experiments. We confirm the studies previously reported in animals on the existence of a competitive antagonism of lorglumide on CCK gallbladder receptors. Moreover, our results on gallbladders from gallstone patients show that lorglumide is a highly effective antagonist of CCK-induced contractions despite the presence of chronic cholecystitis. Our study might help for a better comprehension of the role of these new anti-CCK drugs in the treatment of biliary pain.     

Antagonism of peptidoleukotrienes and inhibition of systemic anaphylaxis by RG 12525 in guinea pigs

RG 12525 was determined to be a specific, competitive and orally effective antagonist of the peptidoleukotrienes, LTC4, LTD4 and LTE4, in several assays utilizing guinea pigs. In vitro, RG 12525 competitively inhibited 3H-LTD4 binding to lung membranes (Ki = 3.0 +/- 0.3 nM) and competitively antagonized the spasmogenic activity of LTC4, LTD4 and LTE4 on lung strips (KB values = 3 nM) with greater than 8000 fold selectivity. In vivo, RG 12525 orally inhibited LTD4 induced wheal formation (ED50 = 5 mg/kg with a t1/2 = 10 hrs at 9 mg/kg), LTD4 induced bronchoconstriction (ED50 = 0.6 mg/kg), and anaphylactic death (ED50 = 2.2 mg/kg with a t1/2 = 7 hrs at 10 mg/kg) and antigen induced bronchoconstriction (ED50 = 0.6 mg/kg). RG 12525 represents a significant improvement in receptor affinity and oral efficacy and thus, is a valuable pharmacological tool to evaluate peptidoleukotrienes in allergic diseases.     

Adenosine modulation of fat cell responsiveness to insulin and oxytocin

We have investigated the effects of adenosine on the stimulation of glucose oxidation and lipogenesis by oxytocin and insulin in rat epididymal adipocytes. The addition of adenosine deaminase (1 U/ml) to the assay medium reduced the maximal oxytocin response (glucose oxidation and lipogenesis) to between 25 and 50% of the maximum response in control cells. The maximal response to insulin was not appreciably affected under these conditions. The addition of adenosine (10 or 30 microM) increased the cell sensitivity to oxytocin by elevating the maximum rate of oxytocin-stimulated glucose metabolism. Adenosine also increased the cell sensitivity to insulin by decreasing its ED50. A change in ED50, however, was observed only when control or adenosine-treated cells were compared to adenosine deaminase-treated cells; but not when control and adenosine-treated cells were compared. On its own, adenosine also caused an appreciable increase in both glucose oxidation and lipogenesis (ED50 approximately equal to 3 microM adenosine). The difference in the effect of adenosine on oxytocin action, compared with the effect on insulin action, points to differences in the mechanisms by which insulin and oxytocin stimulate glucose metabolism in adipocytes.     

A new simple mouse model for the in vivo evaluation of cholecystokinin (CCK) antagonists: comparative potencies and durations of action of nonpeptide antagonists

A new simple mouse assay for the in vivo evaluation of CCK antagonists which is based upon visual determination of the gastric emptying of a charcoal meal is described. CCK-8 (24 micrograms/kg s.c.) but not various other peptide and nonpeptide agents effectively inhibited gastric emptying in this test system. The effect of CCK-8 was antagonized by established peripheral CCK antagonists but not representative agents of various other pharmacological classes. The rank order of potency of the CCK antagonists were: L-364,718 (ED50 = 0.01 mg/kg, i.v.; 0.04 mg/kg, p.o.) greater than Compound 16 (ED50 = 1.5 mg/kg, i.v.; 2.0 mg/kg p.o.) greater than asperlicin (ED50 = 14.8 mg/kg i.v.) greater than proglumide (ED50 = 184 mg/kg i.v.; 890 mg/kg, p.o.). Duration of action studies based upon ED50 values determined at various time intervals after oral administration showed that L-364,718 and proglumide are considerably longer acting than Compound 16. Asperlicin (ED50 greater than 300 mg/kg, p.o.) was ineffective as a CCK antagonist when administered orally. These data provide the first direct comparisons of the in vivo potencies of current CCK antagonists and demonstrate the utility of a new simple mouse assay for the in vivo characterization of peripheral CCK antagonists.     

Anorectic activities of serotonin uptake inhibitors: correlation with their potencies at inhibiting serotonin uptake in vivo and 3H-mazindol binding in vitro

The mechanism of anorectic action of several serotonin uptake inhibitors was investigated by comparing their anorectic potencies with several biochemical and pharmacological properties and in reference to the novel compound SL 81.0385. The anorectic effect of the potent serotonin uptake inhibitor SL 81.0385 (ED50 = 4 mg/kg, i.p.) was potentiated by pretreatment with 5-hydroxytryptophan and blocked by the serotonin receptor antagonist metergoline. A good correlation (r = 0.98, p less than 0.01) was obtained between the ED50 values of anorectic action and the ED50 values of serotonin uptake inhibition in vivo (but not in vitro) for several specific serotonin uptake inhibitors. Most of the drugs tested displaced [3H]-mazindol from its binding to the anorectic recognition site in the hypothalamus, except the pro-drug zimelidine which was inactive (IC50 greater than 100 microM). Excluding zimelidine, a good correlation (r = 0.835, p less than 0.01) was obtained between the affinities of these drugs for [3H]-mazindol binding and their anorectic action indicating that their anorectic activity may be associated with an effect mediated through this site. Taken together these results suggest that the anorectic action of serotonin uptake inhibitors is directly associated to their ability to inhibit serotonin uptake and thus increasing the synaptic levels of serotonin. The interactions of these drugs with the anorectic recognition site labelled with [3H]-mazindol is discussed in connection with the serotonergic regulation of carbohydrate intake.     

The saxitoxin/tetrodotoxin binding site on cloned rat brain IIa Na channels is in the transmembrane electric field.

The rat brain IIa (BrIIa) Na channel alpha-subunit and the brain beta 1 subunit were coexpressed in Xenopus oocytes, and peak whole-oocyte Na current (INa) was measured at a test potential of -10 mV. Hyperpolarization of the holding potential resulted in an increased affinity of STX and TTX rested-state block of BrIIa Na channels. The apparent half-block concentration (ED50) for STX of BrIIa current decreased with hyperpolarizing holding potentials (Vhold). At Vhold of -100 mV, the ED50 was 2.1 +/- 0.4 nM, and the affinity increased to a ED50 of 1.2 +/- 0.2 nM with Vhold of -140 mV. In the absence of toxin, the peak current amplitude was the same for all potentials negative to -90 mV, demonstrating that all of the channels were in a closed conformation and maximally available to open in this range of holding potentials. The Woodhull model (1973) was used to describe the increase of the STX ED50 as a function of holding potential. The equivalent electrical distance of block (delta) by STX was 0.18 from the extracellular milieu when the valence of STX was fixed to +2. Analysis of the holding potential dependence of TTX block yielded a similar delta when the valence of TTX was fixed to +1. We conclude that the guanidinium toxin site is located partially within the transmembrane electric field. Previous site-directed mutagenesis studies demonstrated that an isoform-specific phenylalanine in the BrIIa channel is critical for high affinity toxin block.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anticonvulsant evaluation and mechanism of action of benzylamino enaminones

The mechanism of anticonvulsant action was evaluated for the benzylamino enaminones. The most potent enaminone in this series was the unsubstituted benzylamine analog (30; methyl 4-benzylamino-6-methyl-2-oxocyclohex-3-en-1-oate) which had an oral effective dose (ED50) in rats of 27 mg/kg against maximal electroshock seizures, and a concentration 10-fold less than this dose depressed excitatory synaptic transmission, and action potential firing in the rat brain in vitro.     

Diltiazem potentiates the negative inotropic action of nimodipine in heart

In Langendorff perfused rat hearts, nimodipine enhances coronary flow and inhibits contractility. The binding of [3H]nimodipine (160 Ci/mmol) to sarcolemma isolated from dog heart revealed a KD of 0.2 nM. d-cis-Diltiazem, but not 1-cis-diltiazem, a less active stereoisomer, stimulated [3H]nimodipine (0.17 nM) binding to sarcolemmal membranes (ED50 for diltiazem = 1.1 microM). In the presence of 10 microM d-cis-diltiazem, [3H]nimodipine binding sites were doubled, but there was no change in the apparent affinity. Perfused rat hearts were treated with 250 nM d-cis-diltiazem. The negative inotropic response to nimodipine was dramatically potentiated (I50, from 1.1 to 0.033 microM). The pharmacological and binding effects were observed only at 37 degrees C. It is possible that diltiazem in some way converts low affinity to high affinity sites.     

Cardioselective profile of AF-DX 116, a muscarine M2 receptor antagonist

AF-DX 116 (see chemical name below) is a competitive antagonist of muscarine receptors in peripheral organs. In contrast to pirenzepine, its behaviour in functional experiments indicates selectivity for the M2 muscarinic subtype. In pithed rats AF-DX 116 inhibits vagally-induced bradycardia, an M2 response, (ED50 32 micrograms/kg i.v.) in preference to the M1-mediated pressor response to McN-A-343 (ED50 211 micrograms/kg i.v.). AF-DX 116 further discriminates among M2 receptors, showing a high affinity for the cardiac muscarine receptors. In isolated preparations, AF-DX 116 has a tenfold higher affinity for the muscarine receptors of the heart (pA2 7.33) than for those in smooth muscles (pA2 6.39-6.44). The same profile appears from animal studies, where the compound is a more potent antagonist of either endogenously or exogenously activated cardiac muscarine responses as compared to vascular, smooth muscle or secretory responses. In general, the ratios of potencies (ED50) observed in cardiac vs. other muscarine mediated functions ranged between 30 and 50. Atropine showed no discrimination, inhibiting all muscarine responses in the same range of doses. In the conscious dog intravenous AF-DX 116 increased basal heart rate, and completely reversed the reflex bradycardia induced by clonidine. Tachycardia was dose-related (ED50 79 micrograms/kg i.v.), and occurred independently of background sympathetic tone. AF-DX 116 clearly distinguishes between M1- and M2-mediated responses; it also emphasizes the long-recognized heterogeneity among the peripheral M2 subtypes. AF-DX 116, for its pronounced cardioselectivity, may have a therapeutic potential in the treatment of sinus bradycardia.     

Alteration of epidermal growth factor receptor activity by mutation of its primary carboxyl-terminal site of tyrosine self-phosphorylation

The epidermal growth factor (EGF) receptor, which exhibits intrinsic protein tyrosine kinase activity, undergoes a rapid, intramolecular self-phosphorylation reaction following EGF activation. The primary sites of tyrosine self-phosphorylation in vivo are located in the extreme carboxyl-terminal region of the molecule, principally Tyr-1173. To test the biological and biochemical consequences of this EGF receptor self-phosphorylation, we made the mutation Tyr----Phe-1173. Membranes containing the mutated receptor exhibited an ED50 for EGF activation of tyrosine kinase activity equivalent to control receptor at both high and low substrate levels, but exhibited reduced basal and EGF-stimulated tyrosine kinase activity at low, non-saturating substrate levels. The Tyr----Phe-1173 mutant possessed high affinity EGF binding and could still self-phosphorylate other tyrosine sites in an intramolecular fashion with a low Km for ATP (200 nM), suggesting that this alteration did not grossly change receptor structure. When EGF-dependent growth of Chinese hamster ovary cells expressing comparable levels of control or mutant EGF receptor was measured, the ability of the mutant receptor to mediate cell growth in response to EGF was reduced by approximately 50%, yet both receptors exhibited a similar affinity and ED50 for EGF. These results support the concept that this self-phosphorylation site can act as a competitive/alternate substrate for the EGF receptor, and that this region of the molecule is important in modulating its maximal biological activity.     

Pharmacological characterization of an Endogenous Negative Inotrophic Factor (ENIF) from porcine heart

Recently we have been successful in isolating an endogenous negative inotropic factor (ENIF) from porcine left ventricular tissue. In this study, we have characterized its pharmacological properties. The results of the study demonstrated that ENIF produces a concentration-dependent negative inotropic response on both guinea pig left atria and right ventricular trabeculae. The maximal reduction in contractile force produced by 300 ul of ENIF (5 ml bath) on atria and trabeculae were 90.0 ± 0.8% and 77.5 ± 6%. Atria, however, was significantly more sensitive to ENIF than trabeculae. The ED 50 of ENIF for atria was found to be 38 ul as opposed to ED 50 of 100 ul of ENIF for trabeculae.Acetylcholine (ACh), a muscarinic receptor agonist, decreased the contractile force of guinea pig atria in a dose-dependent manner with a maximal decline in the contractile force of 90%. However, none of the concentration of ACh used affected the contractile function of the trabeculae. Atropine (1 uM) completely blocked the negative inotropic response on atria of all the doses of ACh used. The same dose of atropine, however, was unable to influence the negative inotropic effect of any of the doses of ENIF used on either the atria or trabeculae preparations in our study. The maximal decline in the contractile force of atria was e.g. 94 and 95% in the presence and absence of atropine respectively. These data demonstrate that the myocardial negative inotropic effect of ENIF is not mediated via the cholinegic receptor mechanism.     

Assessment of delta-opioid receptor activation by a series of peptides in cultured cells.

This study was designed to investigate the relative ability of a series of cyclic opioid peptides to initiate the first activation steps following their binding of delta-opioid receptors. The extent of stimulation of low Km guanosine-triphosphatase (GTPase activity) and inhibition of hormonally-stimulated cAMP accumulation in the NG108-15 (neuroblastoma-glioma) hybrid cell line were determined and compared for six closely related peptides. In addition, their binding affinity was assessed by competition with 3H-[D-Pen2D-Pen5]-enkephalin (3H-DPDPE) in membranes from these cells. All peptides tested elicited comparable maximal effects for both functional responses. Different potencies in stimulating the low Km GTPase was observed at sub-maximal agonist concentrations, although the shallow dose-response behavior did not allow accurate determination of ED50s. Estimation of ED50s for inhibition of cAMP accumulation could be made by curve fitting and were similar for four of these peptides, while DCDPE and 3R-methylDCDPE, the highest affinity analogs, were considerably more potent. In general, the observed differences in hormonal activity somewhat parallel the rank order of binding affinities, but no strict relationship was found between receptor binding and activation.     

Control of pepsin secretion by regulatory peptides in the rat stomach: comparison with acid secretion.

Previous studies of the control of pepsin secretion by neurohumoral agents showed some discrepancies between in vitro (isolated cells) and in vivo experiments. In the present work, the effects on pepsin secretion of CCK, pentagastrin, secretin, VIP, neurotensin, histamine, and methacholine were reinvestigated in conscious gastric fistula rats, in comparison to acid secretion. ED50's and doses inducing maximal responses were measured to directly compare the potency and efficacy of these substances. Methacholine was the most efficient (maximal response = 4.5 x basal level, ED50 = 1.3 mumol/kg.h) and CCK the most potent (ED50 = 1.9 nmol/kg.h) stimulant, whereas secretin was a potent (ED50 regulators of pepsin secretion in the rat. Pentagastrin and histamine did not stimulate pepsin output, as found by others with isolated chief cells in vitro. Neurotensin and large doses of VIP marginally inhibited pepsin secretion.     

Amiloride potentiates atrial natriuretic factor inhibitory action by increasing receptor binding in bovine adrenal zona glomerulosa

The interaction of atrial natriuretic factor (ANF) with the diuretic amiloride was studied in bovine adrenal zona glomerulosa. Amiloride enhances 2 to 3-fold high affinity binding of [125I] ANF to zona glomerulosa membrane receptor with an ED50 of 10 microM. This effect is due to a recruitement of high affinity receptor sites and to an increase of their affinity from a Kd of 23 to 8 pM. This enhancing effect is almost equipotently elicited by guanabenz, while clonidine is 20-fold less potent and arginine is inactive. ATP reduces by 30 to 50% [125I] ANF binding with an IC50 of 50 microM. Amiloride and ATP opposite effects on [125I] ANF binding are mutually competitive. Low concentrations of amiloride (less than 100 microM) potentiate the inhibitory effect of ANF in hormone-stimulated steroid secretion with a 3-fold decrease in ANF IC50 at 10 microM amiloride. Higher concentrations of amiloride (greater than 100 microM) directly inhibit aldosterone secretion with an IC50 of 500 microM and a maximum of 80 to 100% reversal of stimulation by various secretagogues. These results indicate that amiloride synergistically potentiates ANF inhibitory action by altering ANF receptor binding properties. They also suggest a role for sodium transport and for phosphorylation-dephosphorylation mechanisms in the mode of action of ANF.     

Exemestane (FCE 24304), a new steroidal aromatase inhibitor.

Exemestane (FCE 24304; 6-methylenandrosta-1,4-diene-3,17-dione) is a novel orally active irreversible aromatase inhibitor. Its in vitro and in vivo pharmacological properties have been compared to 4-hydroxyandrostenedione (4-OHA). In preincubation studies with human placental aromatase, exemestane, like 4-OHA, showed enzyme inactivating properties with a similar affinity (Ki 26 vs 29 nM) and a lower rate of inactivation (t1/2 13.9 vs 2.1 min). Conversely, when tested in pregnant mares' serum gonadotropin-treated rats, exemestane was more potent in reducing microsomal ovarian aromatase activity than 4-OHA, after both subcutaneous (ED50 1.8 vs 3.1 mg/kg) and oral dosing (ED50 3.7 vs greater than 100 mg/kg). No interference of exemestane on desmolase or 5 alpha-reductase activity was found. The compound did not show any relevant binding affinity to steroidal receptors, but slight binding to the androgen receptor (approximately 0.2% of dihydrotestosterone), like 4-OHA. In the first phase I trial, healthy postmenopausal volunteers were given single oral doses of exemestane, ranging from 0.5 to 800 mg, and plasma [estrone (E1), estradiol (E2) and estrone sulphate (E1S)] and urinary estrogens (E1 and E2) were measured up to 5-8 days. The minimal effective dose in decreasing estrogens was 5 mg. At 25 mg the maximal suppression was observed at day 3: plasma estrogens fell to 35 (E1), 39 (E2) and 28% (E1S), and urinary estrogens fell to 20 (E1) and 25% (E2) of basal values, these effects still persisting on day 5. No effects on plasma levels of cortisol, aldosterone, 17-hydroxyprogesterone, DHEAS, LH and FSH, and no significant adverse events were observed up to the highest tested dose of 800 mg exemestane.