The selective estrogen receptor modulator, raloxifene: a segment II/III delivery study in rats

Raloxifene is a nonsteroidal, selective estrogen receptor modulator developed by Eli Lilly and Company as a therapeutic agent for postmenopausal osteoporosis. Raloxifene was administered orally by gavage at doses of 0, 0.1, 1, or 10 mg/kg/d to female CD rats (25/group) on Gestation Day 6 (GD 6) through Postpartum Day 20 (PD 20). Females were allowed to deliver and maintain their progeny until PD 21. All dead pups and pups culled on PD 1 were given internal and external examinations. One pup/sex/litter was assigned to each of the following assessment groups: 1) the primary pair for the F1 generation study, in which survival, growth, development, behavior, indicators of sexual maturation, and reproductive performance were evaluated; 2) terminal necropsy evaluations at PD 21; 3) terminal necropsy evaluations at 60 d of age; and 4) assessments of immune function at 5 to 6 weeks of age. At termination on PD 21, 60, or approximately 140, a necropsy was performed; crown rump and tibia lengths were measured; pituitary weights were taken; and a portion of the anterior pituitary was retained for growth hormone, luteinizing hormone, and prolactin content determinations (control and 10-mg/kg groups only). The remainder of the pituitary and reproductive tissues were retained for histologic evaluations. Dose-related depressions in maternal body weight and food consumption occurred during gestation. Mean gestation length was increased at 1 and 10 mg/kg. Delayed, extended, and/or disrupted parturition occurred in dams given 10 mg/kg, which resulted in a high incidence of maternal morbidity and/or death, increased numbers of dead pups, and the survival of only 66% of live pups to PD 21. Progeny body weights were not decreased at birth, but were depressed progressively in a dose-related manner during the 3-week lactation period. Negative geotaxis and incisor eruption were apparently accelerated in the 1- and 10-mg/kg groups, but eye opening was delayed at 10 mg/kg. Postweaning activity levels, auditory startle, and passive avoidance performance were not affected in the raloxifene groups. Dose-related decreases in spleen cellularity and thymus weights occurred in both sexes, but immune system function, as measured by splenic natural killer cell activity and antibody response to sheep red blood cells, was not affected. Postweaning body weights and growth parameters, as well as pituitary hormone content, were affected in both an age- and sex-specific manner. Preputial separation was not affected, but vaginal patency occurred ca 2 d earlier than controls in females from the 10-mg/kg group. Estrous cycles of the F1 females were not affected during the first two weeks after vaginal opening, but were disrupted at 12 to 14 weeks of age in the 10-mg/kg group. These females showed poorer mating and fertility indices, and litter size was reduced in the two females that were pregnant. Histologically, reproductive organs were not affected in males at any age or in females at PD 21. At PD 60, vaginal mucification occurred in females from the 0.1- and 1-mg/kg groups. At PD 140, the only finding was a high rate of uterine hypoplasia in the 10-mg/kg group, and this finding occurred in the absence of any concomitant ovarian or vaginal changes. These reproductive and developmental findings are consistent with estrogen antagonist activity of raloxifene.     

Activation of the human estrogen receptor by estrogenic and antiestrogenic compounds in Saccharomyces cerevisiae: a positive selection system

OBJECTIVE: To develop a slow-release carboplatin formulation for intratumoral administration to cats. DESIGN: Preliminary study to analyze pharmacokinetic effects of purified sesame oil in the carboplatin formulation for intratumoral administration, and a second study to evaluate the efficacy and toxicosis of intratumoral administration of carboplatin in purified sesame oil. ANIMALS: 23 cats with squamous cell carcinomas of the nasal plane. PROCEDURE: Eight cats with advanced-stage tumors were submitted to intratumoral administration of 100 mg of carboplatin/m2 of body surface area, with or without purified sesame oil, using a two-period, cross-over design. Fifteen additional cats were treated by intratumoral administration of carboplatin in purified sesame oil. Four weekly intratumoral chemotherapy injections of carboplatin in purified sesame oil at a dosage of 1.5 mg/cm3 of tissue were given. RESULTS: Purified sesame oil in the formulation significantly reduced systemic exposure to carboplatin and drug leakage from the sites of injection. Cumulative effects of repeated intratumoral administrations on plasma concentrations of carboplatin were not observed. Systemic toxicosis was not observed, and local toxicosis was minimal. Healing of ulcerated lesions was not compromised. Rates of complete clinical tumor clearance and complete response were 67 and 73.3%, respectively. Product-limit estimates of mean progression-free survival times was 16 +/- 3.3 months. The 1-year progression-free survival rate was 55.1 +/- 13%. Local recurrence was observed in 7 cats; 4 had marginal tumor recurrence, and 3 had in-field and marginal tumor recurrence. CONCLUSIONS: Intratumoral carboplatin chemotherapy is safe and effective for cats with squamous cell carcinoma of the nasal plane. Future studies to improve treatment efficacy could include evaluation of increased dose-intensity as well as combination of this modality with radiotherapy. CLINICAL RELEVANCE: Intratumoral administration of carboplatin in a water-sesame-oil emulsion was found to be a practical and effective new treatment for facial squamous cell carcinomas in cats.     

An acidic amino acid in the N-methyl-D-aspartate receptor that is important for spermine stimulation

The polyamine spermine has multiple effects on N-methyl-D-aspartate (NMDA) receptors, including "glycine-independent" stimulation, which is seen in the presence of saturating concentrations of glycine; "glycine-dependent" stimulation, which is due to an increase in the affinity of the receptor for glycine; and voltage-dependent block. These effects may involve three separate polyamine binding sites on the receptor. To identify amino acid residues that are important for spermine binding, we used site-directed mutagenesis to alter amino acids in and around a region of the NR1 subunit of the NMDA receptor that shows homology with PotD, a polyamine binding protein from Escherichia coli. Mutated subunits, expressed in heteromeric and homomeric NMDA receptors, were studied by voltage-clamp recording in Xenopus oocytes. Mutation of two acidic residues (E339-E342) to neutral amino acids reduced or abolished glycine-independent stimulation by spermine without affecting glycine-dependent stimulation or voltage-dependent block by spermine. Mutation of these residues also had modest effects on sensitivity to protons and to ifenprodil but did not alter sensitivity to glutamate and glycine or to voltage-dependent block by Mg2+. Residue E342 in NR1 appears to be critical for glycine-independent spermine stimulation. Mutations at equivalent positions in NR2A(E352Q) or NR2B(E353Q) had no effect on sensitivity to spermine, pH, or ifenprodil. Residue E342 in NR1 may form part of a discrete spermine binding site on the NMDA receptor or be involved in the mechanism of modulation by polyamines. This residue may also be involved in modulation by protons and ifenprodil.     

The interaction of the general anesthetic etomidate with the gamma-aminobutyric acid type A receptor is influenced by a single amino acid

The gamma-aminobutyric acid type A (GABAA) receptor is a transmitter-gated ion channel mediating the majority of fast inhibitory synaptic transmission within the brain. The receptor is a pentameric assembly of subunits drawn from multiple classes (alpha1-6, beta1-3, gamma1-3, delta1, and epsilon1). Positive allosteric modulation of GABAA receptor activity by general anesthetics represents one logical mechanism for central nervous system depression. The ability of the intravenous general anesthetic etomidate to modulate and activate GABAA receptors is uniquely dependent upon the beta subunit subtype present within the receptor. Receptors containing beta2- or beta3-, but not beta1 subunits, are highly sensitive to the agent. Here, chimeric beta1/beta2 subunits coexpressed in Xenopus laevis oocytes with human alpha6 and gamma2 subunits identified a region distal to the extracellular N-terminal domain as a determinant of the selectivity of etomidate. The mutation of an amino acid (Asn-289) present within the channel domain of the beta3 subunit to Ser (the homologous residue in beta1), strongly suppressed the GABA-modulatory and GABA-mimetic effects of etomidate. The replacement of the beta1 subunit Ser-290 by Asn produced the converse effect. When applied intracellularly to mouse L(tk-) cells stably expressing the alpha6beta3gamma2 subunit combination, etomidate was inert. Hence, the effects of a clinically utilized general anesthetic upon a physiologically relevant target protein are dramatically influenced by a single amino acid. Together with the lack of effect of intracellular etomidate, the data argue against a unitary, lipid-based theory of anesthesia.     

The mechanism of potentiation of the antitumor effect of 5-fluorouracil by methionine-free intravenous amino acid solution (AO-90) in rats

AO-90, a methionine-free intravenous amino acid solution (7.43%) showed to potentiate the antitumor effect of 5-fluorouracil (5-FU) when concomitantly used as the nitrogen source in total parenteral nutrition (TPN) in Yoshida sarcoma (YS)-bearing rats. In the present experiment, this potentiation mechanism was studied by determining the serum methionine level and tumor methylenetetrahydrofolate (CH2FH4) content in YS-bearing Donryu rats given AO-90 (nitrogen 0.73g/kg on the 1st day and 1.46g/kg for the remaining 6 days) by TPN for 1 week. The rats were subcutaneously inoculated with 10(4) YS cells in the dorsum 3 days before the start of TPN. Inhibition of thymidylate synthase activity in tumor tissue after dosing of AO-90 (nitrogen 0.68g/kg on the 1st day and 1.36 g/kg for the remaining 6 days) by TPN along with daily intraperitoneal dosing of 5-FU (10 mg/kg) was also evaluated with the inoculation of 10(6) tumor cells. The results were compared with those in tumor-bearing rats given TPN with a commercially available amino acid solution containing methionine. On day 5 of TPN, the tumor-bearing rats given AO-90 showed a significantly lower serum methionine level than the control rats: 101 +/- 11 mumol/l versus 29 +/- 14 mumol/l (p < 0.01); and a higher CH2FH4 content in tumor: 7.0 +/- 2.8 pmol/g protein versus 23.7 +/- 16.6 pmol/g protein (p < 0.05). Thymidylate synthase inhibition was 81.2 +/- 5.1% in the AO-90 group and 30.9 +/- 26.3% in the control group (p < 0.01). The results of the present study suggest that AO-90 potentiate the antitumor effect of 5-FU by biochemical modulation. AO-90 concomitantly given with 5-FU for 7 days was effective not only in the allogeneic tumor model, but also in WKAH and SHR rats previously inoculated with 10(6) of syngeneic KDH-8 hepatoma cells and SST-2 adenocarcinoma cells, respectively. Weight of SST-2 adenocarcinoma in SHR rats after the TPN period was significantly smaller in the AO-90 group than in the control rats given methionine-containing TPN and 5-FU: 2.66 +/- 0.91 versus 5.12 +/- 2.11 (p < 0.05).     

Epidermal growth factor (EGF) increases the renal amino acid transport capacity in amino acid loaded rats

In anaesthetized adult female rats, the influence of epidermal growth factor (EGF) on renal amino acid handling was investigated in glutamine, arginine (both 50 mg/100 g b.wt. per hour), or alanine (90 mg/100 g b.wt. per hour) loaded animals. Continuous infusions of the three amino acids were followed by an increase in the fractional excretion (FE) of the administered amino acids as well as of the other endogenous amino acids. Under load conditions (alanine, arginine or glutamine), EGF pretreatment (8 micrograms/100 g b.wt. subcutaneously for 8 days, twice daily 8 a.m. and 4 p.m.) was followed by a stimulation of renal amino acid reabsorption. The increase in the fractional excretion of the administered amino acids was significantly lower than in non-EGF-treated rats. These changes in amino acid transport were connected with a significant reduction of GFR after EGF pretreatment (0.96 +/- 0.10 vs. 0.62 +/- 0.07 ml/min x 100 g b.wt.) and a distinct increase in sodium excretion (2.98 +/- 0.55 vs. 4.97 +/- 0.71 muval/100 g b.wt. x 20 min). After loading with p-aminohippurate (PAH; 200 mg/100 g b.wt.), PAH excretion in EGF rats was increased by about 20%, whereas urinary protein excretion was lower in EGF pretreated rats (control: 0.45 +/- 0.04 vs. EGF: 0.18 +/- 0.03 mg/100 g b.wt. x 20 min). The PAH load reduced amino acid reabsorption as a sign of overloading of renal tubular transport capacity, but in EGF pretreated animals the amino acid excretion was only slightly increased under these conditions. Furthermore, EGF pretreatment depressed normal kidney weight gain significantly (874 +/- 18 vs. 775 +/- 32 mg/100 g b.wt.). EGF can improve the renal tubular transport capacity, but, compared to well-known stimulators of renal transport like dexamethasone or triiodothyronine, its effect is only of a moderate degree.     

The untranslated first exon ''exon 0S'' of the rat estrogen receptor (ER) gene

Occasionally, ipsilateral ischemia develops following the groin insertion of an intra-aortic balloon catheter. Various treatment options have evolved, and include replacing the catheter in the opposite groin, removing it completely, or performing a femorofemoral bypass to deliver blood flow below the catheter. Outlined in this paper is a simple method to restore blood flow to a threatened limb, during femoral artery exploration, in the presence of an intra-aortic balloon. This method is also appropriate for optimal positioning of the balloon catheter prior to femorofemoral bypass.     

Characterization of a gene that is inversely correlated with estrogen receptor expression (ICERE-1) in breast carcinomas

The concurrent external validity of the diagnosis of cocaine abuse, based on the DSM criteria, is shown to be established to an unexpectedly high degree, based on a method of determining the association between a summary score derived by quantifying the DSM criteria and another summary score derived from weighting several measures of frequency of use of cocaine and a measure of its mode of use. The degree of validity was cross-validated by performing the same analysis on two study samples: one of inpatients (N = 179) and one of urban-community African-Americans (N = 204).     

Human free secretory component is composed of the first 585 amino acid residues of the polymeric immunoglobulin receptor

The main objective of this work was to unequivocally determine the C-terminal sequence of human milk free secretory component (SC). It was found to end at arginine-585, i.e. 33 amino acids downstream from the major heterogeneous C-terminal residue previously identified for colostrum SC. In contrast, our data showed that the C-terminal end of SC was found to be homogeneous. Conflicting assignments, Asp/Gln, a missing Asn-211, Asp/Asn, Glu/Gln were corrected and found to agree with the cDNA sequence. An Ala/Val substitution at position 562 (domain VI) was identified. Its genetic significance is uncertain at present.     

Ontogeny of estrogen receptor messenger ribonucleic acid expression in the postnatal rat uterus

During the first 2 wk of postnatal life, the rodent uterus undergoes a period of marked growth and differentiation. To further examine the role of the estrogen receptor (ER) in the mediation of uterine development, we analyzed the ontogeny of ER mRNA expression in the postnatal rat uterus using in situ hybridization. ER mRNA was present in the uterine stroma on the day of birth and progressively increased in abundance during the first 2 wk of postnatal life. In contrast, ER mRNA was not detectable in the luminal epithelium at birth and did not become abundant in this region until postnatal day (P) 7. ER mRNA abundance increased in the luminal epithelium and in the invaginating and fully formed glandular epithelium during the second week of life. At P21 ER mRNA was more abundant in the glandular epithelium than in any other uterine cell type. These results are consistent with, and extend the findings of, previous studies using uterine homogenate binding assays and immunohistochemistry to define ER ontogeny in rodents. Delineation of the temporal and cell-type specific pattern of ER mRNA ontogeny in the postnatal rat uterus furthers our understanding of the molecular basis of both endogenous and exogenous estrogen effects on uterine growth and development.     

The interaction of human estrogen receptor with DNA is modulated by receptor-associated proteins

To better define the role of accessory protein as mediators of estrogen receptor (ER) function, we have used immuno-, steroid-, and site-specific DNA-affinity chromatography to identify and characterize proteins that associate with ER in extracts from ER-expressing Chinese hamster ovary (CHO-ER) cells. Two associated proteins [70 and 55 kilodaltons (kDa)] were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis silver stain analysis of all three column eluates. Two additional proteins (45 and 48 kDa) were preferentially retained by the ER-specific DNA affinity column. The 70-kDa protein was subsequently identified by Western blot analysis as a heat shock protein (hsp70). The 55-kDa protein was identified by N-terminal microsequencing as a member of the protein disulfide isomerase family. The 48- and 45-kDa proteins remain unidentified. To determine the possible differential effects of estrogen agonists and antagonists on human (h) ER interaction with these proteins, CHO-ER cells were labeled with [35S]methionine in the presence of estradiol, 4-hydroxytamoxifen (OH-Tam; partial agonist/antagonist), or ICI 182,780 (complete antagonist). None of these ligands altered the pattern of associated proteins when hER complexes were isolated by adsorption to the vitellogenin A2 estrogen response element (ERE). However, when hER was isolated by immunoadsorption, a reduction in the level of associated hsp70 was observed following treatment with estradiol or OH-Tam, compared to no treatment or treatment with ICI 182,780. By gel retardation analysis, maximal interaction of affinity-purified hER with ERE occurred in the presence of all four associated proteins. Removal of the 48- and 45-kDa proteins and/or hsp70 resulted in a decrease in hER/ERE association, which could be restored by the addition of purified hsp70 and/or a mixture of the 48- and 45-kDa proteins. The increased stability of the restored complex was due primarily to an increase in the association rate of hER with ERE. These results suggest that accessory proteins may be required for maximal ER interaction with EREs and that estrogens and estrogen antagonists may promote differential retention of hsp70 in the presence or absence of a specific ERE.     

Binding of the estrogen receptor to DNA. The role of waters

Molecular dynamics simulations are carried out to investigate the binding of the estrogen receptor, a member of the nuclear hormone receptor family, to specific and non-specific DNA. Two systems have been simulated, each based on the crystallographic structure of a complex of a dimer of the estrogen receptor DNA binding domain with DNA. One structure includes the dimer and a consensus segment of DNA, ds(CCAGGTCACAGTGACCTGG); the other structure includes the dimer and a nonconsensus segment of DNA, ds(CCAGAACACAGTGACCTGG). The simulations involve an atomic model of the protein-DNA complex, counterions, and a sphere of explicit water with a radius of 45 A. The molecular dynamics package NAMD was used to obtain 100 ps of dynamics for each system with complete long-range electrostatic interactions. Analysis of the simulations revealed differences in the protein-DNA interactions for consensus and nonconsensus sequences, a bending and unwinding of the DNA, a slight rearrangement of several amino acid side chains, and inclusion of water molecules at the protein-DNA interface region. Our results indicate that binding specificity and stability is conferred by a network of direct and water mediated protein-DNA hydrogen bonds. For the consensus sequence, the network involves three water molecules, residues Glu-25, Lys-28, Lys-32, Arg-33, and bases of the DNA. The binding differs for the nonconsensus DNA sequence in which case the fluctuating network of hydrogen bonds allows water molecules to enter the protein-DNA interface. We conclude that water plays a role in furnishing DNA binding specificity to nuclear hormone receptors.     

Identification of amino acid residues contributing to the pore of a P2X receptor

P2X receptors are ion channels opened by extracellular ATP. The seven subunits currently known are encoded by different genes. It is thought that each subunit has two transmembrane domains, a large extracellular loop, and intracellular N- and C-termini, a topology which is fundamentally different from that of other ligand-gated channels such as nicotinic acetylcholine or glutamate receptors. We used the substituted cysteine accessibility method to identify parts of the molecule that form the ionic pore of the P2X2 receptor. Amino acids preceding and throughout the second hydrophobic domain (316-354) were mutated individually to cysteine, and the DNAs were expressed in HEK293 cells. For three of the 38 residues (I328C, N333C, T336C), currents evoked by ATP were inhibited by extracellular application of methanethiosulfonates of either charge (ethyltrimethylammonium, ethylsulfonate) suggesting that they lie in the outer vestibule of the pore. For two further substitutions (L338C, D349C) only the smaller ethylamine derivative inhibited the current. L338C was accessible to cysteine modification whether or not the channel was opened by ATP, but D349C was inhibited only when ATP was concurrently applied. The results indicate that part of the pore of the P2X receptor is formed by the second hydrophobic domain, and that L338 and D349 are on either side of the channel 'gate'.     

Dissection of the molecular mechanism of action of GW5638, a novel estrogen receptor ligand, provides insights into the role of estrogen receptor in bone

The estrogen receptor (ER) mixed agonists tamoxifen and raloxifene have been shown to protect against bone loss in ovariectomized rats. However, the mechanism by which these compounds manifest their activity in bone is unknown. We have used a series of in vitro screens to select for compounds that are mechanistically distinct from tamoxifen and raloxifene in an effort to define the properties of an ER modulator required for bone protection. Using this approach, we identified a novel high affinity ER antagonist, GW5638, which when assayed in vitro functions as an ER antagonist, inhibiting the agonist activity of estrogen, tamoxifen, and raloxifene and reversing the "inverse agonist" activity of the pure antiestrogen ICI182,780. Thus, GW5638 appears to function as an antagonist in these in vitro systems, although in a manner distinct from other known ER modulators. Predictably, therefore, GW5638 alone displays minimal uterotropic activity in ovariectomized rats, but will inhibit the agonist activity of estradiol in this environment. Unexpectedly, however, this compound functions as a full ER agonist in bone and the cardiovascular system. These data suggest that the mechanism by which ER operates in different cells is not identical, and that classical agonist activity is not required for the bone protective activity of ER modulators.     

The cytoplasmic fragment of the aspartate receptor displays globally dynamic behavior

A number of cloned soluble fragments if the bacterial chemotaxis transmembrane receptors retain partial function. Prior studies of a fragment corresponding to the cytoplasmic domain (c-fragment) of the Escherichia coli aspartate receptor have correlated the signaling state of mutant receptors with the oligomerization state of the c-fragments: equilibria of smooth-swimming mutants are shifted toward oligomeric states; tumble mutants are shifted toward monomeric states [Long, D. G., & Weis, R. M. (1992) Biochemistry 31, 9904-9911]. We have applied several experimental probes of local and global structural flexibility to two signaling states, the wild-type (monomeric) and S461L smooth mutant (predominantly dimeric) c-fragments. Featureless near-UV CD spectra are observed, which indicate that the single Trp residue is in a symmetric environment (most likely averaged by fluctuations) and suggest that the C-termini of both proteins are highly mobile. Both proteins undergo extremely rapid proteolysis and enhance ANS fluorescence, which indicates that many sites are accessible to trypsin cleavage and hydrophobic sites are accessible to ANS binding. The global nature of the flexibility is demonstrated by 1H NMR studies. Lack of chemical shift dispersion suggests that fluctuations average the environments of side chains and backbone protons. Rapid exchange of 99% of the observable amide protons suggests that these fluctuations give high solvent accessibility to nearly the entire backbone. This evidence indicates that both monomeric and dimeric c-fragments are globally flexible proteins, with properties similar to "molten-globule" states. The significance of this flexibility depends on whether it is retained in functioning receptors: the c-fragment structure may lack important tertiary contacts, protein-protein interactions, or topological constraints needed to stabilize a nondynamic native structure, or the cytoplasmic domain of the native receptor may retain flexibility which may be modulated in the mechanism of transmembrane signaling.     

The hepato-renal syndrome: renal amino acid transport in bile duct ligated rats (DL)--influence of treatment with triiodothyronine or dexamethasone on renal amino acid handling in amino acid loaded rats

The influence of triiodothyronine or dexamethasone on renal amino acid handling was investigated in anaesthetized, bile duct-ligated (DL) adult female rats. 3 days after DL, glomerular filtration rate (GFR) was unchanged whereas urine flow was decreased. Plasma concentrations of 5 out of 16 amino acids were significantly enhanced after DL. On the other hand, the fractional excretion (FE) of 11 out of 16 amino acids was significantly reduced as a sign of improved reabsorption capacity. Bolus injections of leucine (20 mg/100 g b.wt.), glutamine (45 mg/100 g b.wt.), or taurine (45 mg/100 g b.wt.) were followed by a temporary increase in the FE of the administered amino acids as well of the endogenous amino acids which were not administered. This phenomenon was more pronounced in DL than in control rats. Under load conditions, dexamethasone (60 microg/100 g b.wt.) or triiodothyronine (20 microg/100 g b.wt.) treatment for 3 days, i.p. once daily, was followed by a stimulation of renal amino acid reabsorption in DL rats. The increase in fractional amino acid excretion after amino acid load was significantly lower than in untreated rats. This effect was also more pronounced in DL rats.