Efficient and highly selective covalent labeling of the estrogen receptor with [3H]tamoxifen aziridine.
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Animals↗
Biomedical subjects
J A Katzenellenbogen
Publications and source records attributed to J A Katzenellenbogen.
Factors affecting the target site uptake selectivity of estrogen radiopharmaceuticals: serum binding and endogenous estrogens.
The binding affinity of various substituted estrogens for human sex steroid binding protein (SBP) and rat alpha-fetoprotein (AFP) have been measured by hydroxylapatite adsorption (relative to estradiol = 100%). While 17 alpha-ethynyl and 11 beta-methoxy substituents reduce the affinity of estrogens for these serum binding proteins markedly, a 16 alpha-bromo or a 16 alpha-iodo substituent actually increases their affinity for AFP, though lowering it for SBP. As a consequence, the uterine uptake selectivity of 16 alpha [77Br]-bromoestradiol (relative affinity for AFP = 230%) and 16 alpha [125 I]-iodoestradiol (relative affinity for AFP = 180%) in young rats (day 19-23), when AFP levels are still substantial, is considerably less than in older animals (day 24-27). 11 beta-Methoxy-16 alpha [77Br]-bromoestradiol, which has lower affinity for AFP (5.1%), does not show this age-dependent uptake selectivity. In adult cycling female rats bearing dimethylbenz(a)anthracene(DMBA)-induced mammary tumors, there is a strong dependence of uterine and tumor uptake selectivity on the stage of the estrous cycle: uptake is maximal during diestrus and minimal during estrus. The effective use of estrogen radiopharmaceuticals as receptor-based imaging agents requires careful consideration of not only the binding affinity of the agent for the estrogen receptor, but also its interaction with non-receptor binding proteins. The modulation of receptor concentrations by endogenous ligands during endocrine cycles and physiological differences between animals will also affect markedly certain measures of the extent of receptor-mediated uptake by target sites.
Animals↗
Plasmin-activated prodrugs for cancer chemotherapy. 1. Synthesis and biological activity of peptidylacivicin and peptidylphenylenediamine mustard.
Many tumors contain elevated levels of plasminogen activator and thus produce elevated levels of the protease plasmin in the milieu of the tumor. We have hypothesized, therefore, that it should be possible to prepare peptidyl prodrug derivatives of anticancer drugs that would be locally activated by tumor-associated plasmin. As an initial test of this hypothesis, we synthesized the peptidyl prodrugs of the anticancer drugs (alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin, AT-125) and N,N-bis(2-chloroethyl)-p-phenylenediamine (phenylenediamine mustard) by mixed anhydride coupling of the parent drug with the protected tripeptide, Boc-D-Val-Leu-Lys(Boc)-OH, followed by deprotection with trifluoroacetic acid. The prodrugs showed an increased selective in vitro cytotoxicity for Rous sarcoma virus transformed chicken embryo fibroblasts (which produce elevated levels of plasminogen activator) compared to nontransformed fibroblasts (which produce low levels of plasminogen activator). In the presence of the plasmin inhibitor, p-nitrophenyl p'-guanidinobenzoate at 2 micrograms/mL, the selectivity of the phenylenediamine mustard prodrug was reduced, but there was no effect on the cytotoxicity of the free drug. Furthermore, the prodrug analogue D-valylleucyl-D-lysylphenylenediamine mustard (in which L-Lys has been replaced by D-Lys) was inactive. Finally, the prodrug derivative of acivicin did not display selective toxicity for transformed cells when the cells were cultured in plasminogen-free medium. These results suggest that plasmin hydrolysis is necessary for the activation of the prodrugs. The prodrugs were tested in vivo for antitumor activity. The prodrug of acivicin, like acivicin itself, was inactive against the B16 melanoma, a murine tumor that produces high levels of plasminogen activator. This prodrug was active against the M5076 carcinoma, a tumor that displays only moderate levels of plasminogen activator; however, despite the fact that the prodrug was 2- to 3-fold less toxic on a molar basis than acivicin, there was no evidence of an increased therapeutic index. The prodrug of phenylenediamine mustard was also slightly less toxic than the parent drug, but again there was no evidence for an improved therapeutic index against the B16 tumor.
Animals↗
Plasmin-activated prodrugs for cancer chemotherapy. 2. Synthesis and biological activity of peptidyl derivatives of doxorubicin.
We have synthesized peptidyl prodrugs of doxorubicin (Dox) designed to be selective substrates of plasmin. Such prodrugs might be locally activated by the elevated levels of plasmin produced near many solid tumors under the action of tumor-associated plasminogen activators. One such prodrug, 3'-(D-Val-Leu-Lys)-Dox, was obtained via a mixed-anhydride coupling with isobutyl chloroformate between the protected peptide Fmoc-D-Val-Leu-N epsilon-Fmoc-Lys-OH and doxorubicin, followed by removal of the Fmoc groups with anhydrous ammonia. Compared to doxorubicin, the prodrug showed about a 7-fold improved selective cytotoxicity against chicken embryo fibroblasts transformed with the Rous sarcoma virus (which produce high levels of plasminogen activator) compared to normal cells (which produce low levels of plasminogen activator). However, the prodrug was a very poor plasmin substrate, and although in vivo tests against the murine B16 melanoma showed that the prodrug was active, the maximum T/C obtained was less than that achieved by doxorubicin even at 25 times the molar concentration of prodrug. Qualitatively similar results were obtained for a far more hydrophobic prodrug, 3'-(Boc-Val-Leu-Lys)-Dox. These results demonstrate that peptidyl prodrugs of doxorubicin designed as plasmin substrates are more selective anticancer agents in vitro than doxorubicin itself but that the bulky anthracycline moiety probably prevents efficient plasmin-catalyzed conversion to the active parent drug, so that, in their present form, these drugs are not potent enough to allow a determination as to whether or not they are more selective in vivo.
Animals↗
New approach for visualizing estrogen receptors in target cells using inherently fluorescent ligands and image intensification.
Four fluorescent estrogen ligands were investigated as agents for visualization of estrogen receptors in cells: 2-(2,4-dihydroxyphenyl)-6-hydroxy-3-benzofurancarboxylic acid delta-lactone (coumestrol) and 9(11)-dehydro-12-oxoestradiol [12-oxo-1,3,5-(10),9(11)-estratetraene-3, 17 beta-diol] (12-oxoestradiol), which are inherently fluorescent compounds; and tamoxifen [Z)-1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenyl-1-butene) and 4-hydroxytamoxifen [Z)-1-[4-(2-dimethylaminoethoxy) phenyl]-1-(4-hydroxyphenyl)-2-phenyl-1-butene), which become maximally fluorescent only after ultraviolet irradiation. By conventional fluorescence techniques, these agents can be detected down to 10(-8) M in water, but only to 10(-6) to 10(-7) M in protein solutions; however, by photon-counting spectrofluorimetry, coumestrol and 12-oxoestradiol can be detected in protein solutions down to 5 X 10(-10) M. Three of these compounds have good affinity for the estrogen receptor: coumestrol (20%); 12-oxoestradiol (12%); and 4-hydroxytamoxifen (37%), relative to estradiol (100%). Under conditions where autoradiographic controls indicate that most of the estrogen receptor of MCF-7 human breast cancer cells is in the nucleus, we could demonstrate nuclear fluorescence using 10(-9) M concentrations of coumestrol, 12-oxoestradiol, and 4-hydroxytamoxifen. This nuclear fluorescence was abolished by a 200-fold excess of diethystilbestrol and could only be observed through a fluorescence microscope equipped with a microchannel image intensifier and a video camera detector that together provide a sensitivity enhancement of approximately 10(4). These studies indicate that the estrogen receptor in breast cancer cells can be visualized by fluorescence techniques, provided that the visualizing ligands have adequate affinity and specificity for the receptor and appropriate fluorescence characteristics, and provided that the fluorescence instrument has adequate sensitivity to observe fluorescence emission from cells treated with nM concentrations of the fluorescent agents.
Breast Neoplasms↗
Haloenol lactones: enzyme-activated irreversible inactivators for serine proteases. Inactivation of alpha-chymotrypsin.
Two haloenol lactones have been shown to inactivate alpha-chymotrypsin by an enzyme-mediated process. According to the postulated mechanism, acylation of the active site serine by the haloenol lactone reveals an alpha-haloketone, which alkylates the enzyme at the active site and renders the enzyme inactive. The inactivation has been shown to require enzymatic activation of the inhibitor to its reactive form to take place within the acyl-enzyme intermediate and to be irreversible.
Benzene Derivatives↗
Methodology for the synthesis and specific activity determination of 16 alpha-[77Br]-bromoestradiol-17 beta and 16 alpha-[77Br]-11 beta-methoxyestradiol-17 beta, two estrogen receptor-binding radiopharmaceuticals.
16 alpha-[77 Br]-Bromoestradiol-17 beta (3a) has been synthesized from estrone enol diacetate (1) by bromination with Na77Br and hydrogen peroxide-acetic acid, followed by reduction with lithium aluminum hydride to give a mixture of 16 alpha-[77 Br]-bromoestradiol-17 beta (3a) and 16 alpha-[77Br]-bromoestradiol-17 alpha (3b) from which the desired epimer (3a) can be obtained in 50% overall radiochemical yield (from Na77Br) by HPLC. Analogous procedures can be used in the preparation of 16 alpha-[77Br]-bromo-11 beta-methoxyestradiol-17 beta. (4a) The effective specific activities of these radiopharmaceuticals, determined by binding to the uterine estrogen receptor, are 900-1500 Ci/mmol. Both have affinity and good binding selectivity for the estrogen receptor and are useful as imaging agents for mammary tumors.
Animals↗
Tamoxifen antiestrogens. A comparison of the activity, pharmacokinetics, and metabolic activation of the cis and trans isomers of tamoxifen.
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Animals↗
Antiestrogen basicity--activity relationships: a comparison of the estrogen receptor binding and antiuterotrophic potencies of several analogues of (Z)-1,2-diphenyl-1-[4-[2-(dimethylamino)ethoxy]phenyl]-1-butene (tamoxifen, Nolvadex) having altered basicity.
A series of N-substituted (Z)-1,2-diphenyl-1-[4-(2-aminoethoxy)phenyl]-1-butenes, analogues of the antiestrogen tamoxifen (Nolvadex), in which the side-chain basicity is varied over a wide range, has been prepared to probe the importance of basicity in evoking estrogen antagonism. All of the compounds, except the pyrrole analogue 14, were found to possess significant antiestrogenic activity in the rat, as measured by their ability to inhibit estrogen-induced uterine growth. This implies that in the tamoxifen series the level of side-chain basicity, at least in the Lowry-Brønsted sense, is not a determining factor in the estrogen antagonist potencies of these compounds.
Animals↗
Nonsteroidal estrogens: synthesis and estrogen receptor binding affinity of derivatives of (3R*,4S*)-3,4-bis(4-hydroxyphenyl)hexane (hexestrol) and (2R*,3S*)-2,3-bis(4-hydroxyphenyl)pentane (norhexestrol) functionalized on the side chain.
A series of nonsteroidal, side-chain functionalized estrogens based on (3R*,4S*)-3,4-bis(4-hydroxyphenyl)hexane (hexestrol) and (2R*,3S*)-2,3-bis(4-hydroxyphenyl)pentane (norhexestrol) has been prepared; these include amide, diazo ketone, ester, alcohol, ketone, fluoro, bromo, iodo, and saturated hydrocarbon derivatives. Analysis of the binding affinity of these compounds to the uterine estrogen receptor, measured by competitive binding assay, reveals trends that can be related to the steric size, the hydrophobicity, and the hydrogen bond accepting character of the side-chain substituents. Comparison of binding affinities between norhexestrol and hexestrol derivatives indicates that, in general, the norhexestrols show significantly higher receptor binding affinities, making this series of compounds ideally suited as functional probes for the estrogen receptor.
Animals↗
(2R*,3S*)-1-[125I]Iodo-2,3-bis(4-hydroxyphenyl)pentane ([125I]iodonorhexestrol) and (2R*,3S*)-1-[77Br]Bromo-2,3-bis(4-hydroxyphenyl)pentane ([77Br]bromonorhexestrol), two gamma-emitting estrogens that show receptor-mediated uptake by target tissues in vivo.
Two gamma-emitting estrogen analogues, (2R*,3S*)-1-[125I]iodo-2,3-bis(4-hydroxyphenyl)pentane ([125I]iodonorhexestrol) and (2R*,3S*)-1-[77Br]bromo-2,3-bis(4-hydroxyphenyl]pentane ([77Br]bromonorhexestrol), have been prepared by halide ion displacement on a labile trifluoromethanesulfonate derivative of a suitably protected precursor, followed by mild acid deprotection. Although halide displacement on a more stable tristrifluoromethanesulfonate derivative was successful, the basic conditions required for deprotection of this precursor resulted in destruction of the products by a base-induced spiroelimination reaction. In immature female rats, both of these halonorhexestrols demonstrated preferential uptake by the uterus that could be blocked selectively by coadministration of a large dose of unlabeled estradiol. In a double label comparison with 16 alpha-[125I]iodo-17 beta-estradiol the uterine uptake of [77Br]bromonorhexestrol was notably less selective. Stability studies in vitro and in vitro have indicated that both iodo- and bromonorhexestrol are quite labile, and this lability compromises the selectivity of their uptake by estrogen target tissues in vivo. p-Hydroxyphenethyl halides are known to be unusually prone to a base-catalyzed solvolysis, via cyclization of the phenolate to a spirocyclohexadienone intermediate. This unusual solvolytic mechanism may contribute to the lability of these halonorhexestrols in vivo.
Animals↗
16 alpha-[77Br]bromo-11 beta-methoxyestradiol-17 beta: a gamma-emitting estrogen imaging agent with high uptake and retention by target organs.
16 alpha[77Br]Bromo-11 beta-methoxyestradiol-17 beta [MBE(Br-77)], a compound with high affinity for the estrogen receptor and with low nonspecific binding, has been prepared with an effective specific activity of 770--1450 Ci per mmole at the time of synthesis. In immature female rats, this compound is taken up selectively by the uterus and is retained for prolonged periods. This is presumably due to the binding of this compound to the estrogen receptor, as uterine uptake is blocked selectively by coadministration of an excess of unlabeled estradiol, and administration of a chase dose of unlabeled estradiol results in a rapid decrease in activity in the uterus. In double-label experiments with 16 alpha[125I]estradiol and MBE(Br-77), the two compounds showed equally selective uterine uptake at 1 hr, but the bromine-labeled compound became increasingly more selective at 3 and 6 hr. MBE(Br-77) may prove to be a more favorable agent for imaging human breast tumors than our previously described compound, 16 alpha-[77Br]bromoestradiol-17 beta.
Animals↗
In vivo comparison of 16 alpha[77Br]bromoestradiol-17 beta and 16 alpha-[125I]iodoestradiol-17 beta.
An in vivo comparison of two estrogen-receptor-binding radiopharmaceuticals of high specific activity, 16 alpha-[77Br]bromoestradiol-17 beta and 16 alpha-[125I]iodoestradiol-17 beta, has been carried out in immature female rats. The iodoestradiol has slightly higher uterine uptake at 1 hr after injection, whereas the brominated analog has significantly enhanced uptake at later times. The similar behavior of the two compounds in vivo suggests that estradiol labeled with I-123, Br-77, or Br-75 could be used interchangeably for the imaging of breast tumors containing estrogen receptors. In addition, coinjection of 16 alpha-[125I]iodoestradiol as an internal standard has been shown to be useful for comparison of different radiohalogenated estrogens.
Animals↗
16 alpha-[77Br]bromoestradiol: dosimetry and preliminary clinical studies.
An estrogen-receptor-binding radiopharmaceutical, 16 alpha-[77Br]bromoestradiol-17 beta, has been used successfully at high specific activity to image carcinogen-induced mammary tumors in rats and in preliminary studies to image breast tumors in patients. The biodistribution of the labeled estrogen in rats and its clearance in a monkey were used to estimate the radiation absorbed doses to a human resulting from administration of the radiopharmaceutical. Preliminary imaging studies in patients with mammary carcinoma show promising results and warrant further development of radiolabeled estrogens, particularly those carrying positron emitters that could permit positron emission tomography--for example Br-75 or F-18.
Animals↗
Interaction of a high affinity anti-estrogen (alpha-[4-pyrrolidinoethoxy]phenyl-4-hydroxy-alpha'-nitrostilbene, CI628M) with uterine estrogen receptors.
The polar metabolite of the anti-estrogen alpha-[4-pyrrolidinoethoxy]phenyl-4-methoxy-alpha'-nitrostilbene (CI628), that is selectively accumulated by the nuclear estrogen receptor in vivo, is identified as the corresponding free phenol (alpha-[4-pyrrolidinoethoxyl]phenyl-4-hydroxy-alpha'-nitrostilbene (CI628M) and has been prepared in high specific activity tritium-labeled form to study its interaction with the rat uterine estrogen receptor. CI628M has an affinity for the cytosol receptor that is comparable to that of estradiol (Kd (CI628M) = 0.16 nM; Kd (estradiol) = 0.28 nM), and the binding of these two compounds is mutually competitive. Dissociation of the CI628M complex with the estrogen receptor is somewhat slower than that of the estradiol complex, and prewarming the complexes at 28 degrees C causes a characteristic decrease in the rate of dissociation. On low salt sucrose gradients, the CI628M and estradiol cytoplasmic receptor complexes both sediment as 8S species; their sedimentation profiles on high salt gradients are somewhat different, however. While the estradiol receptor complex shows mainly a 4.6S species that shifts to a 5.8S species upon warming, the unwarmed CI628M complex is predominantly 5.8S and becomes somewhat more disperse upon warming. Under high salt conditions, there is a progressive shift of the 4.6S to the 5.8S species as cytosol concentration increases; the CI628M complex shows a greater tendency to form the more rapidly sedimenting form at all cytosol concentrations. After in vivo injection of [3H]CI628M, the uterine nuclear receptor sediments as a characteristic 5S species. Cytosol receptor complexes with estradiol and CI628M, maintained at 0 degrees C, show very little binding to ATP-Sepharose, while after warming, 50-60% of both complexes are bound by the resin. Both complexes also show similar elution profiles from DNA-cellulose columns; however, the extent of binding of the [3H]CI628M receptor complex to the column is consistently only 40-50% that of the estradiol receptor complex. These results indicate that the properties of the uterine estrogen receptor complexed with estradiol and with the high affinity anti-estrogen CI628M are very similar. The differences observed in the behavior of the CI628M cytosol receptor complex on high salt sucrose gradients and on DNA-cellulose columns, though quantitative rather than qualitative, may be indicative of differences in receptor properties that are important to the antagonist activity of this compound.
Animals↗
Chemically reactive estrogens: synthesis and estrogen receptor interactions of hexestrol ether derivatives and 4-substituted deoxyhexestrol derivatives bearing alkylating functions.
A series of chemically reactive derivatives of the nonsteroidal estrogen hexestrol have been synthesized as potential affinity labels for the estrogen receptor or as cytotoxic agents with selective activity against receptor-containing cells. These compounds are hexestrol ethers with halo ketone, halohydrin, or epoxide functions or 4-substituted deoxyhexestrols with halo ketone, benzyl halide, nitro, azide, sulfonyl fluoride, or sulfonyl azide groups. The alkylating activity of the electrophilic derivatives was measured using the colorimetric reagent nitrobenzylpyridine, the bromo derivatives being considerably more reactive than the chloro ones. Their reversible binding to the lamb uterine estrogen receptor was measured by competitive binding assays, and their irreversible reaction with receptor was measured by exchange assays that determine the rate and extent of receptor inactivation. In general, monoetherification of hexestrol or substitution of deoxyhexestrol produces compounds with relatively low affinity for the estrogen receptor (0.3-10% that of estradiol). Most of the electrophilic derivatives are rapid and effective inactivators of receptor (24-70% inactivation within 0.5-5 h at 25 degrees C). Of the photosensitive derivatives, 4-azidodeoxyhexestrol appears to be the most efficient receptor inactivator (49%). The high reactivity of these compounds toward the estrogen receptor and the lack of interference by their reaction with other cellular nucleophiles suggest that these compounds may be useful as affinity-labeling agents or as selective cytotoxic agents in intact systems.
Affinity Labels↗
A novel connector linkage applicable in prodrug design.
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Chemical Phenomena↗
Biological potency and uterine estrogen receptor interactions of the metabolites of the antiestrogens CI628 and U23,469.
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Animals↗