Second line endocrine treatment of postmenopausal advanced breast cancer. Preliminary endocrine results of a 5-ARM randomized phase II trial of medium vs low dose aminoglutethimide, with or without hydrocortisone vs hydrocortisone alone (EORTC 10861)

The supposed mechanism of action of aminoglutethimide (AG), medical adrenalectomy, has been challenged. AG is now considered to act as an inhibitor of the aromatization of mainly adrenal androgens to estrogens in peripheral tissues and/or breast cancer itself. To further establish the AG dose required to sufficiently reduce estrogen levels in plasma and the possible role of hydrocortisone (HC) in combination with AG or by itself, postmenopausal advanced breast cancer patients received AG low (125 mg bid) or medium (250 mg bid) dose alone or combined with HC (20 mg bid) or HC alone (20 mg bid). Preliminary hormonal data show a similar reduction of serum estrone and estrone sulphate by at least some 50% at 8 wk in all treatment groups. At 6 months these effects persist except for patients treated with HC alone. In the latter a normalization of estrone levels is observed with effective suppression of adrenal androgen precursors, suggesting increased aromatase activity with prolonged glucocorticoid treatment.     

Inability of gestational hormones to account for the inhibitory effects of pregnancy plasmas on lymphocyte responses in vitro.

Plasma from pregnant women has a marked inhibitory effect on lymphocyte responses in vitro. While much evidence suggests that this is due to an immunologic mechanism, an apparent lack of specificity and the known suppressive effects of several hormones on immune function has led to speculation that the inhibitory effects could be due to increased concentrations of gestational hormones. We have investigated the effects of a wide range of concentrations of estrone, estradiol, estriol, progesterone, human chorionic gonadotropin (HCG), and hydrocortisone on lymphocyte responses to mitogens and allogeneic cells. None of these hormones were capable of inhibiting lymphocyte DNA synthesis even at concentrations several times the maximum physiologic plasma levels occurring during pregnancy. Very high, supraphysiologic concentrations were found to be inhibitory. In investigating the mechanism of the hormonal inhibition we found that if they were removed from the media at various times after initiation of culture, the estradiol, HCG, and to a lesser extent the hydrocortisone effects were all reversible. Estradiol and HCG differed from hydrocortisone in that the former were inhibitory only when added at the initiation of culture, whereas hydrocortisone was inhibitory even when added 24 hr later. In summary, while extremely high concentrations of gestational hormones are inhibitory, the quantities which occur physiologically in gestational plasmas are not able to suppress lymphocyte responses and thus cannot account for their inhibitory effects.     

A new method for hydrolyzing sulfate and glucuronyl conjugates of steroids

A new method for hydrolyzing steroid conjugates (both sulfates and glucuronides conjugates) that is efficient, effective, and inexpensive is described. This method comprises incubation of the conjugates--after salting-out into ethyl acetate or elution from a C18 cartridge--with anhydrous methanolic hydrogen chloride (methanolysis) for 10 min. It has been successfully applied to our routine radioimmunoassay screening and GC/MS confirmation studies of steroids in prerace and postrace equine urine samples. Comparative GC/MS studies on entire (male horse) urine samples showed that methanolysis gave amounts of free steroids (estrone, estradiols, testosterone, estrenediols, nandrolone, androstanediols) at least as large as those obtained by solvolysis. Similar studies on urine samples from a gelding that had been administered nandrolone phenylpropionate showed that methanolysis gave larger amounts of free steroids (nandrolone, estranediols) than Helix pomatia enzymatic hydrolysis or solvolysis. Also, TLC studies on methanolysis of corticosteroid conjugates such as hydrocortisone 21-sulfate and hydrocortisone 21-phosphate showed that free corticosteroid was released in 5 min.     

Intestinal transport and metabolism of glucose-conjugated kyotorphin and cyclic kyotorphin: metabolic degradation is crucial to intestinal absorption of peptide drugs

Intestinal transport and metabolism of modified kyotorphin (KTP) were studied in rats. Modified KTPs studied were C-terminally modified KTP with p-aminophenyl-beta-D-glucoside (KTP-pAPbeta glc), N-terminally modified KTP-pAPbeta glc with t-butyloxycarbonyl group (Boc-KTP-pAPbeta glc) and the N- and C-terminally modified KTP by cyclization (cyclic KTP). KTP-pAPbeta glc was metabolized at a similar rate to that of KTP, and did not appear on the serosal side. Although Boc-KTP-pAPbeta glc was also metabolized, it was more stable than KTP and appeared on the serosal side. Cyclic KTP was also quite stable and appeared on the serosal side. The modified KTPs were evaluated kinetically for absorption consisting of membrane transport and metabolism. Absorption clearance (CL(abs)) of cyclic KTP, Boc-KTP-pAPbeta glc and Boc-KTP was higher than that of KTP (0.247 microl/min/cm) (Mizuma et al., Biochim. Biophys. Acta 1335 (1997) 111-119), which is the theoretical maximum by complete inhibition of peptidase activity, indicating that derivatization of KTP increases the membrane permeability. Furthermore, the data clearly showed that the greater the metabolic clearance (CL(met)) of KTP and the KTP derivatives, the lower the absorption clearance (CL(abs)). These results and further simulation study led to the conclusion that metabolic degradation in the intestinal tissues is more critical than membrane permeability (transport) for oral delivery of peptide drugs. Based on the stability of cyclic KTP in serum, this appears to be a good candidate analgesic peptide drug.     

Evaluation of gene panel mRNAs in urine samples of kidney transplant recipients as a non-invasive tool of graft function

Non-invasive monitoring may be useful after kidney transplantation (KT), particularly for predicting acute rejection (AR). It is less clear whether chronic allograft nephropathy (CAN) is also associated with changes in urine cells. To identify non-invasive markers of allograft function in kidney transplant patients (KTP), mRNA levels of AGT, TGF-beta1, EGFR, IFN-gamma, TSP-1, and IL-10 in urine (Ur) samples were studied using QRT-PCR. Ninety-five KTP and 111 Ur samples were evaluated. Patients (Pts) were divided as, within six months (N = 31), and with more than six months post-KT (N = 64). KTP with more than six months post-KT were classified as KTP with stable kidney function (SKF) (N = 32), KTP with SKF (creatinine < 2 mg/dL) and proteinuria > 500 mg/24 h (N = 18), and KTP with biopsy proven CAN (N = 14). F-test was used to test for equality of variances between groups. IL-10 mRNA was decreased in Ur samples from KTP with less than six months post-KT (P = 0.005). For KTR groups with more than six months post-KT, AGT and EGFR mRNA were statistically different among KTP with SKF, KTP with SKF and proteinuria, and CAN Pts (P = 0.003, and P = 0.01), with KTP with SKF having higher mean expression. TSP-1 mRNA levels also were significantly different among these three groups (P = 0.04), with higher expression observed in CAN Pts. Using the random forest algorithm, AGT, EGFR, and TGF-beta1 were identified as predictors of CAN, SKF, SKF with proteinuria. A characteristic pattern of mRNA levels in the different KTP groups was observed indicating that the mRNA levels in Ur cells might reflect allograft function.     

Antisera reactive directly to estrone sulfate.

Derivatives of estrone were prepared and linked to bovine serum albumin or its methyl-esterified form to produce immunogens which were effective in raising antisera to estrone sulfate. The most effective was estrone-3-methylphosphonothioate, electrostatically complexed with methylated bovine serum albumin. The ionically combined hapten functioned as an antigenic determinant as do covalently bound haptens when administered to sheep in emulsions with Freund's complete adjuvant. Estrone-3-phosphate covalently or electrostatically linked to bovine serum albumin also produced antisera reactive to estrone sulfate. Estrone sulfate itself, after electrostatically complexing to methylated bovine serum albumin and administration with Freund's complete adjuvant to sheep, was ineffective in producing antisera. The sera which had workable titres to estrone sulfate showed considerable cross-reaction with free estrone but was otherwise highly specific with little or no reaction with other steroid sulfates, glucosiduronates or other free steroids. Radioimmunoassay curves using [6,7-3H]-estrone sulfate were highly sensitive and were effective in the range of 5-250 pg estrone sulfate.     

Serum and urinary estrone sulfate during the menstrual cycle, measured by a direct radioimmunoassay, and fate of exogenously injected estrone sulfate

Serum and early-morning urinary levels of estrone sulfate during the menstrual cycle were measured by a direct radioimmunoassay without hydrolysis. These levels were high and showed prominent peaks [serum, 2.67 +/- 0.37 ng/ml (mean +/- SE); urine, 5.82 +/- 2.3 micrograms/l] around the day of the preovulatory estradiol-17 beta peak, and increased again during the luteal phase. Following intravenous injection of estrone sulfate, serum estrone sulfate, estrone and estradiol-17 beta were measured. The conversion of estrone sulfate to estrone and/or estradiol-17 beta was very small during their transit in the general circulation.     

A Novel Sesquiterpene,Tuberonone, from Solanum tuberosum L.

The effects of the ovarian hormones progesterone and estrone on the conversion of tryptophan to nicotinamide in rats were investigated. Female rats were fed for 35 days with a 20% casein diet, or with a 20% casein diet containing 0.1% progesterone, or 0.001% estrone, or 0.1% progestrone and 0.001% estrone. The conversion ratio of tryptophan to nicotinamide on the last day of the experiment was 2% in the groups fed with the 20% casein diet and the diet containing 0.1% progesterone, but around 1.2% in the group fed with 0.001% estrone, and 0.7% in the group fed with 0.1% progesterone and 0.001% estrone. These results demonstrated that administration of ovarian hormones significantly decreased the conversion of tryptophan to nicotinamide.     

Side-effects of analgesic kyotorphin derivatives: advantages over clinical opioid drugs

The adverse side-effects associated with opioid administration restrain their use as analgesic drugs and call for new solutions to treat pain. Two kyotorphin derivatives, kyotorphin-amide (KTP–NH₂) and ibuprofen–KTP–NH₂ (IbKTP–NH₂) are promising alternatives to opioids: they trigger analgesia via an indirect opioid mechanism and are highly effective in several pain models following systemic delivery. In vivo side-effects of KTP–NH₂ and IbKTP–NH₂ are, however, unknown and were evaluated in the present study using male adult Wistar rats. For comparison purposes, morphine and tramadol, two clinically relevant opioids, were also studied. Results showed that KTP-derivatives do not cause constipation after systemic administration, in contrast to morphine. Also, no alterations were observed in blood pressure or in food and water intake, which were only affected by tramadol. A reduction in micturition was detected after KTP–NH₂ or tramadol administrations. A moderate locomotion decline was detected after IbKTP–NH₂-treatment. The side-effect profile of KTP–NH₂ and IbKTP–NH₂ support the existence of opioid-based mechanisms in their analgesic actions. The conjugation of a strong analgesic activity with the absence of the major side-effects associated to opioids highlights the potential of both KTP–NH₂ and IbKTP–NH₂ as advantageous alternatives over current opioids.     

Effects of L-thyrosyl - L-arginine (kyotorphin) on the behavior of rats and goldfish

The effects of kyotorphin (KTP), a dipeptide (L-Tyr-L-Arg), on the level of sensory attention to stimuli of different modalities in rats and the exploratory behavior in goldfish were investigated. In both cases KTP was found to suppress the exploratory activity. When 5-HTP, a precursor of serotonin synthesis, is activated the inhibitory effects of KTP increased. It is assumed that the regulatory effect of KTP on the exploratory behavior of animals is mediated by the monoaminergic (neurotransmitting) brain systems, as distinct from its analgetic effects, which are mediated by the opioid brain systems.     

Steroid levels after intramuscular injection of radioactive estradiol-17beta, estrone, progesterone and testosterone in the bovine

Eight 2 year old Hereford cows from days 8 to 12 of the estrous cycle were injected intramuscularly with 5 ml of corn oil containing 5 mg of estradiol-17beta (two cows), estrone (two cows), progesterone (two cows) or testosterone (two cows). Each cow treated with estradiol received 494 microc of estradiol-17beta-6, 7 H3 and each cow treated with estrone received 492 microc of estrone-6, 7 H3. Each cow treated with progesterone or testosterone received 400 muc of H3 compound labeled in the 7 position. Total urine was collected by urethral catheterization of the cows treated with estrogens. Blood samples for plasma and serum were collected via jugular cannulae. Blood and urine samples from estrogen-treated cows were collected hourly for the first 24 hr, at 2 hr intervals for the next 26 hr, at 4 hr intervals for the next 12 hr and at 12 hr intervals until background was reached. Blood samples were collected hourly from 1 to 8 hr after injection from progesterone or testosterone-treated cows. Plasma and serum levels of radioactive estradiol-17beta, estrone, progesterone and testosterone were similar. Blood levels of radioactivity peaked at 2 hr post-injection in cows receiving estradiol-17beta and at 3 hr in cows receiving estrone. Blood levels of labeled estradiol-17beta and estrone were nondetectable by 54 hr and 83 hr, respectively. Peak urinary excretion of radioactivity was reached at 7 hr for estradiol-17beta and at 14 hr for estrone and nondetectable levels were reached by 95 hr for estradiol-17beta and 14 hr for estrone. At these times, 15.5% of the total dose of radioactive estradiol-17beta and 17.5% of the injected estrone had been excreted in the urine. Peak blood and urinary excretion levels were reached earlier for radioactive estradiol-17beta than for estrone, and excretion of estradiol-17beta was completed more rapidly. No difference was found in plasma and serum levels for any steroid studies; thus, endogenous steroid titers in blood plasma and serum are not different in the cow.     

Role of GSH in estrone sulfate binding and translocation by the multidrug resistance protein 1 (MRP1/ABCC1)

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent efflux pump that can confer resistance to multiple anticancer drugs and transport conjugated organic anions. Unusually, transport of several MRP1 substrates requires glutathione (GSH). For example, estrone sulfate transport by MRP1 is stimulated by GSH, vincristine is co-transported with GSH, or GSH can be transported alone. In the present study, radioligand binding assays were developed to investigate the mechanistic details of GSH-stimulated transport of estrone sulfate by MRP1. We have established that estrone sulfate binding to MRP1 requires GSH, or its non-reducing analogue S-methyl GSH (S-mGSH), and further that the affinity (Kd) of MRP1 for estrone sulfate is 2.5-fold higher in the presence of S-mGSH than GSH itself. Association kinetics show that GSH binds to MRP1 first, and we propose that GSH binding induces a conformational change, which makes the estrone sulfate binding site accessible. Binding of non-hydrolyzable ATP analogues to MRP1 decreases the affinity for estrone sulfate. However, GSH (or S-mGSH) is still required for estrone sulfate binding, and the affinity for GSH is unchanged. Estrone sulfate affinity remains low following hydrolysis of ATP. The affinity for GSH also appears to decrease in the post-hydrolytic state. Our results indicate ATP binding is sufficient for reconfiguration of the estrone sulfate binding site to lower affinity and argue for the presence of a modulatory GSH binding site not associated with transport of this tripeptide. A model for the mechanism of GSH-stimulated estrone sulfate transport is proposed.     

Studies on nuclear binding of dehydroepiandrosterone in hepatocytes.

Experiments were conducted to determine if dehydroepiandrosterone (DHEA) specifically binds to liver nuclei or interferes in the binding of other steroid hormones. Hepatocytes were isolated from obese, female Zucker rats that were treated with and without 0.6% DHEA in their diets for 2 weeks. The hepatocytes were incubated with either [3H]DHEA, [3H]estrone, or [3H]corticosterone. The nuclei isolated from the incubated cells from either control or DHEA-treated rats had no binding with [3H]DHEA, but had the expected binding with [3H]estrone and [3H]corticosterone. Furthermore, increasing concentrations of cold, unlabeled DHEA in the incubation media with [3H]estrone or [3H]corticosterone failed to have any effect on the binding of either of these steroid hormones to the nuclei. These results suggest that DHEA treatment does not exert its effects on cellular metabolism through a receptor-mediated mechanism like other steroid hormones or by interfering in the expression of steroid hormones such as estrone and corticosterone.     

Hybrid molecules of estrone: new compounds with potential antibacterial, antifungal, and antiproliferative activities

New hybrid molecules of estrone were synthesized as compounds indicating promising biological activity (antibacterial, antimycobacterial, antifungal, and antiproliferative). The prepared molecules contained various heterocyclic units (pyridine, benzylsulfanyl derivatives of pyridine or derivatives of tetrazole) linked to estrone by n-heptyl bridges. The compounds with charge on molecule (the hybrid pyridinium or benzylsulfanylpyridinium salts) exhibited significant biological activity (antibacterial, antimycobacterial, antifungal, and antiproliferative). On the other hand, the compounds not in the form of salts (omega-(1-phenyl-5-tetrazolylthio)heptylethers of estrone) were inactive. The antimycobacterial activities of three different series of tetrazole derivatives (i.e., the hybrid molecules with estrone, tetrazole-5-thiols, and 5-benzylsulfanyl-1-phenyltetrazoles) with the same substituents on phenyl ring were compared. Amongst them, the 5-benzylsulfanyl-1-phenyltetrazoles were the most potent.     

Alterations in the activities of subcellular fractions marker enzymes in rat liver and brain by hydrocortisone and corticosterone treatment

The effect of subcutaneous injection of hydrocortisone and corticosterone on the activity values of some subcellular fractions marker enzymes from rat liver and brain was investigated and compared with controls (without treatment with hormones). The following enzymes were studied (subcellular fraction are shown between parentheses): N-acetyl-beta-D-glucosaminidase and beta-glucuronidase (lysosomes); succinate dehydrogenase = SDH (mitochondria); glucose-6-phosphatase (endoplasmic reticulum); 5'-nucleotidase and Na+-K+-Mg2+ ATPase (plasma membrane). The specific activity of lysosomal enzymes from liver showed no change when rats were injected either with hydrocortisone or corticosterone. The same enzymes from brain showed significant increases in their activities with both hydrocortisone or corticosterone except beta-glucuronidase; this enzyme gave activity values remaining between the control levels, after treatment with corticosterone. The activity of mitochondrial SDH was increased after corticosterone injection either in liver or brain. After hydrocortisone injection, its activity rises significantly in brain (72%), but it falls in liver compared to the control values. Glucose-6-phosphatase behaves similarly in brain or liver fractions; its activity increases always after corticosterone treatment and decreases by hydrocortisone. The plasma membrane marker enzymes did not change practically in brain fractions, excepted Na+-K+-Mg2+ ATPase which tends to rise its activity after hydrocortisone injection. In liver fractions, both 5'-nucleotidase and Na+-K+-Mg2+ ATPase activities increase either by corticosterone or hydrocortisone treatment, except 5'-nucleotidase which specific activity decreases in liver after hydrocortisone treatment.     

Effects and interactions of epidermal growth factor, insulin, hydrocortisone, and estradiol on the cloning of human tumor cells

We investigated the effects and interactions of epidermal growth factor (EGF), insulin, hydrocortisone, and estradiol on the growth of 18 freshly obtained human tumors in our human tumor stem cell assay (HTSCA) cultured at a reduced serum concentration (8.5% ml). All possible combinations of these four supplement factors were added to the assay to determine the ability of each component to enhance colony formation. We found that hydrocortisone was the most effective single supplement in stimulating colony growth in the HTSCA. Supplementation with insulin, estradiol, or both had some growth-promoting effect but not as great as hydrocortisone. Moreover, the addition of insulin, estradiol, or both often demonstrated a negative interaction with hydrocortisone. EGF supplementation alone; in dual combination with insulin, estradiol, or hydrocortisone; or in combination with estradiol and insulin in the assay did not significantly increase colony formation. However, EGF added to the cultures containing hydrocortisone with insulin and/or estradiol significantly increased colony formation and reversed the negative effect of insulin and estradiol on hydrocortisone activity. Thus, under conditions of our assay, the most effective combination in promoting colony growth contained all four factors.     

Tissue-specific concentration changes of estrone and estradiol during spontaneous and ACTH-induced parturition in sheep

Changes in estrogen production are considered important in the sequence of events leading to parturition. We sought tissue-specific changes in the concentration of unconjugated estrone (E1) and estradiol (E2) in intrauterine fetal (amnion, chorion) and maternal (endometrium, myometrium) tissues during normal pregnancy, labour, and ACTH-induced labour in sheep. The mean concentrations of E1 and E2 in the fetal membranes were higher than in endometrium and myometrium. In amnion there were no consistent changes in estrone concentrations with gestation, although estradiol concentrations increased between day 130 and term. In the endometrium there were increases in both estrone and estradiol between day 100 and term, whereas in the myometrium increases in the concentrations of E1 and E2 occurred between days 130-135 and term. Animals showing a labourlike pattern of uterine contractions after intrafetal ACTH administration did not show significant differences in estrone or estradiol concentrations in amnion, chorion, or endometrium compared with saline-infused controls. However, there was a progressive increase in the concentration of estrone and estradiol in the myometrium during ACTH-induced labour. We conclude that changes in the concentrations of estrone and estradiol in intrauterine tissues vary between the tissues studied and the two estrogens. In general, estrogen concentrations increased towards term, but this trend was more marked in the maternal than fetal tissues. The changes in estrone concentrations in myometrium, but not in the other tissues, were replicated during ACTH-induced labour. Our results would be compatible with the suggestion that tissue-specific changes in estrogen concentrations may contribute to the local intrauterine steroid milieu during pregnancy and at term.     

Activation of human B lymphocytes. IV. Regulatory effects of corticosteroids on the triggering signal in the plaque-forming cell response of human peripheral blood B lymphocytes to polyclonal activation.

In vitro hydrocortisone in physiologic and pharmacologically attainable concentrations caused a marked enhancement of the PWM-induced PFC response of normal human peripheral blood B lymphocytes. This effect was seen only when hydrocortisone was added within the first 24 hr of culture and only when hydrocortisone and PWM were present together in cultures. Only suprapharmacologic concentrations of hydrocortisone (10(-3) M) were capable of suppressing early B cell activation. Late stages of antibody production and secretion were resistant to suppression by even these extraordinarily high concentrations. Hydrocortisone did not replace the T cell requirement of PWM-induced PFC responses. A single dose of in vivo hydrocortisone (400 mg) to normal adult volunteers did not produce this enhancing effect when PFC responses were measured in vitro in the absence of hydrocortisone. The data strongly suggest that the enhancing effect of hydrocortisone was due not to elimination of naturally occurring suppressor cells, but to a modulation of the triggering signal either directly on the B cell itself or via the balance of positive and negative T cell regulation of B cell activation.     

Estrogen interconversions in the induced cycle in female rhesus monkeys

To determine the extractions and interconversions of estrone and estradiol across and within the uterus, [3H]estradiol and [14C]estrone were infused at a constant rate in six ovariectomized female rhesus (Macaca mulatta) monkeys. Studies were done on Days 9, 14, and 23 of artificial menstrual cycles induced by the timed insertion and removal of Silastic capsules of estradiol and progesterone. Measurements of estrogen radioactivity were made from peripheral arterial blood and uterine venous blood as well as from endometrial biopsy samples. A significant increase occurred in the conversion of estradiol to estrone measured within the uterus on Day 23 compared to Days 9 and 14. The conversion of estrone to estradiol, measured within the uterus, fell progressively from Day 9 to Day 23, but this decrease was not significant. The extractions and interconversions across the uterus, and the overall interconversions of estrone and estradiol were not significantly different on Days 9, 14, or 23 of the cycle. Thus, we have been able to confirm in vivo the increase in the activity of the 17 beta-hydroxysteroid dehydrogenase, the enzyme responsible for estradiol to estrone interconversions, shown earlier by studies done in vitro. However, the increase in 17 beta-hydroxysteroid activity in the uterus is not reflected in the overall interconversions of estrone and estradiol as reflected by measurements in peripheral arterial blood.     

Expanding the role of photoselective vaporization of the prostate

The use of the potassium-titanyl-phosphate (KTP) laser for the ablative treatment of lower urinary tract symptoms secondary to benign prostatic hyperplasia (BPH) has gained wide acceptance in the urologic community. The efficacy and safety of photoselective vaporization of the prostate using 60-W or 80-W KTP have been demonstrated in multiple trials, with significant impact on special high-risk surgical populations (ie, patients with large prostates and anticoagulated patients with multiple comorbidities) with symptomatic BPH. The high-power KTP laser technique has also shown encouraging results in the management of urethral strictures. With catheter removal, improvement in voiding may not immediately occur; however, with the efficient vaporization and limited coagulation necrosis that are routinely noted with high-power KTP applications, improvement may occur in as early as a few days to 1 week. Because of the superior surgical hemostasis associated with laser prostatectomy, no restrictions on physical activity are required after the procedure, even in the immediate postoperative period.