Comparison of the distribution of radioiodinated di- and tri-hydroxyphenylethylene estrogens in the immature female rat

NW/. Med. Biol. Vol. 22, No. 5, PP. 679-687, 1995

0%9-8051(95)00002-X

Copyright 0 1995 Elsevier Science l.td Printed in Great Britain. All rights reserved 0969-80511’95 $9.50 + 0.00

Comparison of the Distribution of Radioiodinated Di- and Tri-hydroxyphenylethylene Estrogens in the Immature Female Rat EUGENE

R. DESOMBRE

I,‘*, JAMES

PRIBISH3

and ALUN

HUGHES’

‘The Ben May Institute and *Cancer Research Center, The University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637 and 3Central Research and Development, The Dow Chemical Co., Midland, MI 48674, U.S.A. (Accepted 10 October 1994) The uptake, retention and tissue to blood ratios of two non-steroidal, ‘2SI-labeled iodoestrogens, an iodotrihydroxyphenylethylene, 2-iodo-1,2-bis(4-hydroxyphenyl)-2-(3-hydroxyphenyl)ethylene and an iododihydroxyphenylethylene, 2-iodo- 1,l -his (4-hydroxyphenyl)-2-phenylethylene, were compared after intraperitoneal injection in immature female rats. The iodotrihydroxyphenylethylene showed an unexpectedly prolonged specific retention in estrogen target tissue, lasting up to 72 h. It was rapidly cleared from blood and non-target tissues so that uterus or ovary to blood ratios of greater than 100 were seen at 2 and 3 days. This iodotrihydroxyphenylethylene may have clinical potential for estrogen receptor-containing cancers

Introduction There has been continuing interest in the use of radiolabeled estrogens for the diagnosis and therapy for estrogen receptor (ER)-containing cancers. Radioiodinated estrogens have shown promise for detecting ER-positive breast cancers (Pavlik et al., 1990; Ribeiro-Barras et al., 1992). Furthermore, there has been progress with the preparation of estrogens containing 18F for use in positron emission tomography, PET, for locating ER-positive lesions (Mintun et al., 1988; Van Brocklin et al., 1990). Radioiodinated ‘231-labeled estrogens not only have the potential ior diagnosis due to the 159 keV y-ray emission, but, by virtue of their emission of Auger electrons, have the potential for therapy as well. Although the effective range of the low-energy Auger electrons is short (Charlton, 1986; Kassis et al., 1987), there is extensive evidence that when incorporated into DNA such radiation is highly toxic (Bloomer and Adelstein, 1978). On the other hand, if the Auger cascade occurs predominantly in the cytoplasm or outside the cell, the cytotoxicity is reduced by several orders of magnitude or becomes insignificant (Bloomer and Adelstein, 1978; Hofer ef al., 1975). It is now well-established that ER is present in nuclei of target cells (King and Green, 1984) and, when bound ~----~. *Author

for correspondence

to estrogen, is localized at distinct estrogen response elements in the DNA (Carson-Jurica er al., 1990; DeSombre, 1993). Thus the delivery of an Auger electron-emitting estrogen to the DNA of a hormoncdependent cancer, mediated by ER, can render the radiation from such low-energy electrons very potent for damaging the DNA of the target cell, while sparing adjacent cells and tissues. We have recently reported (DeSombre et al., 1992) that “31-labeled estrogens can effect a dose-dependent, unlabeled estrogen-inhibitable radiotoxicity of ER-positive, but not ER-negative, cells in vitro. Others have even reported the radiotoxicity of the longer half-life nuelide ‘*‘I, in cell culture (Beckmann et al., 1993; Broizert et al., 1982; McLaughlin et al., 1989). However, since the biological half-life of most estrogens in target tissues in vir)o is generally less than 24 h, while the physical half-life of “‘1 is 60 days, only a small proportion of the incorporated very [“‘I]iodoestrogen would decay while associated with the DNA of the target cell. Even with the 13 h half-life ‘231,there is an obvious advantage to using iodoestrogens with as long a biological half-life as possible to optimize the effectiveness of the Auger decay of the iodoestrogen in target cells. To that end we have been investigating the distribution of a number of iodoestrogens in cells in culture and in tissues in animals (DeSombre et al., 1987, 1988; Gatley et (11.. 1991; Hughes et al., 1993) to evaluate 619

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their potential for use for radiotherapy. This report shows that an iodotrihydroxyphenylethylene has dramatically longer target tissue retention than its dihydroxyphenyl analog and possesses the longest biological half-life in ER-containing tissues of any estrogen reported in the immature rat.

Materials

and Methods

All chemicals were of reagent grade or better. Acetonitrile (HPLC grade) was purchased from Baxter (McGaw Park, IL). lz51 (as Nalz51) was from Amersham (Arlington Heights, IL). Synthesis and speciJic activities c$[‘~~I] iodoestrogens The radioiodoestrogens were synthesized by the halodestannylation reaction (Hanson et al., 1982; Seitz et al., 1980), from the appropriate tributylstannyl derivatives as previously described in detail (DeSombre et al., 1992; Gatley et al., 1991; Hughes et al., 1993). The preparation of the l,l-bis(C hydroxyphenyl)-2-tributylstannyl-2-phenylethylene, was previously reported (Seevers et al., 1986; Shafii et al., 1993). The synthesis of l,l-bis(4-hydroxyphenyl)-2-(3-hydroxyphenyl)-2-tributylstannyl-ethylene will be reported separately. To prepare the radioiodinated estrogens, 100 pg of the appropriate tributylstannyl precursor in 100 PL ethanol was added to a small pear-shaped flask containing about 3.7 MBq (1 mCi) of Na12’I in 50 PL 0.1 M NaOH. Then 100 PL of 100: 1 glacial acetic acid: 30% hydrogen peroxide, previously incubated for 1 h at room temperature, was added and mixed and the reaction was allowed to proceed for 15 min at room temperature. The reaction mixture was then taken up in a gas-tight Hamilton syringe, the radioactivity assayed in a Capintec dose calibrator and injected onto a 4.6 x 100 mm, 10 p Econosil Cl8 reverse phase HPLC column and eluted with acetonitrile in water. For 2-iodo-l,l-b&(4-hydroxyphenyl)-2-phenylethylene (I-BHPE) a shallow gradient of 20-40% acetonitrile in water over 30 min was followed by a sharp gradient to 100% acetonitrile over the next 10min. I-BHPE eluted at about 33 min, generally cleanly separated from the reduced side product, BHPE, which eluted several minutes earlier. For 2-iodo- I,1 bis(4-hydroxyphenyl)-2-(3-hydroxyphenyl) ethylene (I-THPE) the shallow gradient of 20-40% acetonitrile over 30 min was followed by isocratic 40% acetonitrile for 15 min to elute the I-THPE. In each case the unreacted tin precursors were eluted in 100% acetonitrile. The peak fractions of the radioiodoestrogens were combined, evaporated to a small volume using a Buchi Rotovap (Brinkman Instruments), partitioned between water and ether, the water phase extracted with a second portion of ether and the ether extracts evaporated in the Rotovap to dryness. The radioactive iodoestrogens were taken up in absolute ethanol for assay, and diluted in 0.1% rat serum for injection.

Specific activity determinations were carried out by comparing the specific binding of the [“‘Iliodoestrogens with that of [‘Hlestradiol to the estrogen receptor from a low salt rat uterine extract by sedimentation analysis as we have previously reported (Hughes et al., 1993). In this procedure the unlabeled estradiol (E2)-inhibited binding of the [‘*‘I]iodoestrogens to the 8S form of ER is compared to that of [3H]estradiol, of known specific activity, to the same extract. In this way the problem of quantitating the minute mass of iodoestrogen present is circumvented and, in addition, the result provides an “effective” specific activity. That is, if the preparation is contaminated by small amounts of non-radioactive estrogen, such as BHPE, which has a slightly higher affinity for ER than does I-BHPE itself (DeSombrc et al., 1988), this contaminant would compete for uptake by ER of target tissues as it does the binding to ER in the uterine extract, and thus reduce the effective specific activity of the iodoestrogen when used in rive. However contamination of the iodoestrogen with any other UV adsorbing material, which may reduce the apparent specific activity by conventional methods using UV to determine mass but does not bind to ER, would not affect the “effective” specific activity as described above. For this reason particular efforts are made to separate the reductive destannylation products, like BHPE (often in considerable excess over the iodoestrogen since the stannyl precursor is used in large excess to maximize the radiochemical yield) from the radioiodinated products. The radiochemical yields of final isolated iodoestrogens were in the range of 60-80%. The specific activities of the two [‘25I] iodoestrogens in the various experiments reported here were found to be between 1740-2200 Ci/mmol, so it appears that only minor amounts of such reduced precursors may have been present in some of the preparations. Experiments

in immature jkmale rats

Animal experiments were carried out in compliance with U.S. federal regulations concerned with the The raconduct of animal experimentation. dioiodoestrogens were prepared for injection in 1.OmL 0.1% rat serum in isotonic saline to minimize the adsorption of ligand to the syringe, alone or along with 5 pg unlabeled estradiol to inhibit the ER-mediated binding. In the several experiments from 500&700 kBq (- 15-20 PCi) were injected in each animal. From three to six 22-23-day-old SpragueDawley rats (Sasco) were included in each group. The amount injected in each animal was determined from assays of “‘1 in each syringe before and after injection using a Capintec dose calibrator. At various times after IP injection the rats were killed by decapitation, blood was collected directly into heparin-containing vials and 100 PL aliquots of each blood sample were taken for assay of radioiodine. The entire uterus, vagina, ovaries, adrenals and one kidney were removed intact, dissected free of connective tissues

Iodotriphenylethylene estrogens in and fat, blotted on filter paper, weighed and transferred to 12 x 75 mm tubes for counting in a Packard model 5 130 Auto-gamma Spectrometer. Portions (100-200 mg) of larger tissues (liver, muscle, brain) were similarly assayed. The pituitary was excised and transferred directly, without weighing, to the assay tube to eliminate the weighing error due to rapid dehydration of the tiny organ. The weight used for pituitary (2 mg) was based on an average of 100 combined pituitaries determined separately. For assay of thyroid radioactivity, the organ was excised along with adjacent tissues and the results were based on an average weight of 4mg/organ. Results were calculated as mean and standard deviations of the % injected dose per gram wet weight, as well as the ratios of radioactivity in various tissues to that found in blood.

Results We initially compared the uptake and retention of the two iodophenylethylene estrogens over the course of one day, comparing the tissue concentration of radioiodine in the tissues and blood of animals administered either one of the iodoestrogens alone, or in the presence of an excess of unlabeled estradiol to inhibit the specific, i.e. estrogen receptor-mediated, uptake. The route of administration in each case was IP to evaluate the ability of the iodotriphenylethylene estrogens to preferentially bind to peritoneal target tissues relative to distant ER-positive tissues (Hughes et al., 1993). These results, shown in Fig. 1, for the 1, 4 and 24 h time points confirmed the substantial specific uptake of I-BHPE (right panel) in the uterus, insofar as the tissue concentration of radioiodine was always substantially higher in animals where it was administered alone than in those where it was administered with the large dose of competing estrogen, estradiol. It can also be seen that there is estradiol-inhibitable binding in both the ovary and vagina at all three time points, with a lesser amount of specific binding in the pituitary (Hughes et al., 1993). As also seen in Fig. 1, there are substantial amounts of specific binding of I-THPE (left panel) in the uterus, ovary and vagina at 1, 4 and 24 h and generally no estradiol-inhibitable binding in the other tissues (adrenal, kidney, liver, muscle and brain). While the radioiodine content of the pituitary of immature female rats administered I-THPE alone tends to be higher than that of animals given I-THPE along with unlabeled estradiol, the differences were small and not statistically significant. Several important differences were found between the specific uptake of the I-THPE compared to I-BHPE. Most significant was the substantially better long-term retention. While the concentrations of radioiodine after administering the two different iodoestrogens was similar at both 1 and 4 h, significant differences were evident at 24 h. With I-BHPE only about 10% of the radioiodine found at 4 h was

tsko

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still present in the uterus at 24 h, while with I-THPE the amount of radioiodine in the uterus at 24 h was about 70% of the concentration at 1 h. It therefore appeared that the target tissue retention of I-THPE may be substantially better than that of I-BHPE. Secondly, the level of non-competable radioiodine (i.e. that when unlabeled E2 was co-injected) in the peritoneal target tissues after administration of ITHPE seemed to be significantly higher than that seen after I-BHPE, especially comparing the 1 and 4 h results. Since the I-THPE seemed to be specifically retained in the ER target tissues longer, it was of interest to see just how long specific binding was evident in ER target tissues with this iodoestrogen. Therefore a similar experiment was carried out to confirm the retention patterns for the first day, but also to study the long-term retention of I-THPE in ER target tissues. As shown in Fig. 2, the substantial uterine retention of I-THPE continues even up to 72 h. In fact, the half maximal level of uterine I-THPE is not reached until after 2 days. On the other hand, confirming the results of the earlier experiment, most of the uterine I-BHPE is lost during the first day. Also confirming the results of our earlier experiment, the level of radioiodine present in the uteri of animals administered I-THPE along with 5 pg of unlabeled estradiol was found to be appreciably higher than the radioiodine in uteri of animals given I-BHPE and cold E2. In fact, this unexpectedly elevated level of apparently non-specific binding of I-THPE continues up to 72 h. Also shown in Fig. 2 are the radioiodine concentrations in other ER-containing target tissues of animals given I-BHPE or I-THPE, showing a similar pattern of specific, long-term retention of I-THPE in the ovary (Fig. 2B) and, while lower in magnitude, also in the vagina (Fig. 2C). It is also of interest that the uptake of radioiodine by the pituitary (Fig. 2D) following I-THPE is lower than that after I-BHPE. However, the retention pattern of I-THPE, seen in the other target tissues, is also apparent in the pituitary, where despite the quite low maximum uptake compared with I-BHPE the time to reach the half-maximal level of I-THPE is more than 1 day. The radioiodine in the pituitary is rapidly lost from animals given I-BHPE so that by 30 h the level is lower from I-BHPE than I-THPE. When the two iodoestrogens were administered with unlabeled E2 to the animals, the level of radioiodine in the pituitaries were comparable however. In contrast to the significantly longer retention of I-THPE compared to I-BHPE in ER target tissues. no such difference in radioiodine concentration is seen in non-target tissues, Fig. 3. Rather there seemed to be a tendency, at least at the 2 h time point, for the radioiodine levels to be higher in non-target tissues after the I-BHPE, probably due to the higher 2 h blood level (Fig. 3A). In the non-target tissues no significant differences in the patterns of the

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1 hour time point

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Fig. 1. Distribution of radioactivity in various tissues of the immature female rat following intraperitoneal administration of radioiodotriphenylethylenes. The radioiodoestrogens were administered alone or along with 5 kg of unlabeled estradiol and at 1 h (top), 4 h (middle) or 24 h (bottom) later the animals were sacrificed and blood and the indicated tissues collected, weighed and assayed for radioactivity. The results are presented as % injected dose per gram wet weight of tissue (or per mL of blood) *SD for 2-[‘2SI]iodo-l,l-bis(4-hydroxyphenyl)-2-(3-hydroxyphenyl)-ethylene, I-THPE, left panel, alone ( unlabeled estradiol (0) and 2-[‘251]iodo-1 ,I-bis(4-hydroxyphenyl)-2-phenylethylene, I-BHPE; right panel, alone (a) or along with unlabeled estradiol (0).

Iodotriphenylethylene estrogens in rko Uterus

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Hours Hours Fig. 2. Radioiodine concentrations in estrogen target tissues as a function of time following IP administration of radioiodotriphenylethylenes in ho. The percent injected dose per gram wet weight + SD for 2-[‘ZSI]iodo-1,l -his (4-hydroxyphenyl)-2-(3-hydroxyphenyl)-ethylene, I-THPE, alone (a), or along with 5 pg estradiol (E2), (m) and for 2-[‘2SI]iodo-l,l-bi~(4-hydroxyphenyl)-2-phenylethylene, I-BHPE, alone (0) or given with 5 pg estradiol (0) are plotted according to the time following administration for the uterus (A), ovary (B), vagina (C) and pituitary (D).

radioiodine levels were found between I-BHPE and I-THPE administered alone or with excess unlabeled E2. As seen in Fig. 3, the radioiodine concentrations of blood, muscle, liver, kidney and brain are very low by 30 h after administration of either iodoestrogen. It appears that the level of radioiodine in the brain (Fig. 3C) may be higher after administration of I-BHPE (with or without unlabeled E2) than after I-THPE, but these levels are quite low and by 30 h all return to background. Judging by the level of radioiodine in the thyroid, there does not appear to be a major differences in deiodination of the I-BHPE and I-THPE. While the highest thyroid content is the 2 h level after I-BHPE alone, by 7 h no differences are evident. The prolonged retention of I-THPE, compared to I-BHPE is especially evident when one looks at the ratios of radioactivity in the various tissues to that in blood (Fig. 4). While the ratios are relatively low for the first two time points (2 and 7 h) the tissue to blood ratio for both the uterus and ovary increase dramatically at the longer time points for animals given I-THPE, while they remain fairly constant for animals given I-BHPE. Ratios of 400 and 150 were found for these two tissues at the 48 h time point, reflecting the excellent retention by the target tissues while the ligand is cleared from the blood and other tissues. It is of interest that quite large ratios are also seen for these tissues in the animals given excess

unlabeled E2 along with the I-THPE. On the other hand, no such differences in the tissue to blood ratios are seen for the liver. In that tissue the ratios are somewhat above unity at the 2 hr time point, but remain quite close to one at 7 h and beyond.

Discussion A number of radiohalogenated estrogens have been studied with respect to their potential for imaging and therapy for ER-positive cancers. For both applications it is imperative that the potential haloestrogen show substantial uptake, and particularly long-term retention, by ER-target tissues. For therapy it is especially important that the radiohaloligand be retained a relatively long time in the target tissues to increase the probability that the potentially cytotoxic decay occurs while the radiohaloestrogen is still present in the nucleus of the target tissue. For imaging it is less critical that the retention in the tissues of interest be long, but as a practical consideration it is important to have time for the non-receptor-mediated uptake of other tissues and blood to clear, to provide a low background for imaging. Previous reports on haloestrogens with the best estrogen target tissue retention have involved steroidal estrogens. However, to our knowledge no other haloestrogen has previously been reported to have a biologic half-life of as much as 2 days in the rat uterus as has

Eugene R. DeSombre e/ crl

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been reported here. 17a-iodovinyl- I I fi-ethylestradiol (Hanson ~‘t (II., 1993) appeared to have a half-life of just slightly more than 24 h after IV administration. lha-iodo-1 Ifi-methoxyestradiol, also administered IV, showed excellent uterine uptake (Zielinski c’t al., 1989), but by 24 h the uterine content of radioiodine was less than 25% of the maximum seen at 1 h (Zielinski et al., 1986). 17r-iodovinyl-I lpmethoxyestradiol (I-VME2), essentially the iodo derivative of the much studied estrogen, moxestrol (the

on -c d -0 B s 4 .9 te

estrogen used extensively in contraceptives), also has excellent affinity for ER (Ah et ul., 1991). It showed significantly better uterine uptake and selectivity (assessed as uterus to blood ratio) than either 17xiodovinylestradiol or 16a-iodocstradiol (Hanson and Franke, 1984). Comparison of the E and Z isomers of I-VME2 indicated a higher affinity of the Z isomei for ER, but interestingly, the E isomer showed higher uterine uptake in rats (Ah er al.. 1991) and mice (Foulon et a/., 1992). However, the I-VME2 was also

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Hours Hours Fig. 3. Radioiodine concentrations in estrogen non-target tissues as a function of time following IP administration of radioiodotriphenylethylenes in vim. The percent injected dose per gram wet weight (mL of blood) + SD for 2-[‘25I]-iodo-l,l-bi~(4-hydroxyphenyl)-2-(3-hydroxyphenyl)-ethylene, I-THPE, alone (lJ), or alone with 5pg estradiol (E2), (m) and for 2-[‘251]iodo-l,l-bi~(4-hydroxyphenyl)-2phenylethylene, I-BHPE, along (0) or given with 5 pg estradiol (a) are plotted according to the time following administration for the blood (A), muscle (B), brain/cerebrum (C), liver (D), kidney (E) and thyroid (F).

72

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tissues seems to be even more dramatic for the I-THPE, reported here. Significantly less I-THPE + E2 than I-BHPE reaches the pituitary. However, with the enhanced retention of I-THPE seen in general in . IBHPE + E2 ER target tissues, the integrated dose-time function for I-BHPE and I-THPE in the pituitary are quite similar. Katzenellenbogen and co-workers have prepared and studied other non-steroidal haloestrogens, such 0 12 24 36 48 60 12 as iodohexestrol (Goswami et al., 1980) and bromo Hours and iodonorhexestrol (Landvatter et al., 1982). Although all of these nonsteroidal haloestrogens Ovary T showed excellent affinity for ER (Goswami et al., 1980; Landvatter and Katzenellenbogen, 1982) and 150 uptake by the immature rat uterus in Guo, the halonorhexestrols possessed significant metabolic or 100 solvolytic lability in tlitro and in uivo, ascribed to their p-hydroxyphenylalkyl halide structure (Landvatter 50 et al., 1982). Earlier studies had shown that radioiodohexestrols in which the iodine was present in 0 12 24 36 48 60 72 the aromatic ring of hexestrol (Komai er a/., 1977) bound to ER but showed only modest uptake by the Hours immature rat uterus in vim. Liver Because of its unusually long target tissue retention, the I-THPE shows very high target tissue-toblood or target tissue-to-non-target tissue ratios. especially after 7 h (Fig. 4). Although few studies reported in the literature have followed haloestrogens up to 3 days following administration, we are not aware of tissue to blood ratios in the range of 400, seen for the uterus 48 h after I-THPE administration. 0 12 24 36 48 60 72 I-VME2, which was studied up to 48 h, has shown Hours high tissue to blood ratios, although the various Fig. 4. Tissue to blood ratios of radioactivity in selected reports in the literature are not completely consistent. tissues as a function of time after IP administration of Zielinski et al. reported that its highest tissue to blood radioiodotriphenylethylenes in oivo. The ratios of the con- ratio (i.e. 40) was found at 1 h, but at 48 h, the ratio centrations of radioactivity in tissue (% ID/g) to that in the blood (%ID/mL of blood) and the SD of the ratios are was still in the range of 30 (Zielinski et al., 1986). plotted as a function of time for the uterus (top), ovary Others have reported uterus to blood ratios of 30-60 (middle) and liver (bottom). The symbols and lines for the in rats (Ali et al., 1991) and mice (Foulon et al., 1992) various injectants are the same as indicated in the legends during the first 5 h after IV administration. Jagoda for Figs 2 and 3. et al. reported a uterus to plasma ratio of 100 at 6 h after administration of I-VME2 to immature rats cleared relatively fast from the uterus in that the 5 h (Jagoda et al., 1984) but did not report results at later uterine concentration of radioiodine after giving the time points. It should be recognized that the mode of adminisE isomer was only half of that at the 2 h peak. Our earlier comparison of I-BHPE with the E tration may have affected the high target tissue to blood ratios we see for I-THPE. Indeed it was our isomer of I-VME2 (Hughes et al., 1993) showed that in most of the ER target tissues the retention of the hope that IP administration would allow the hgand to reach the peritoneal target tissues before being I-VME2 was better than that seen for I-BHPE. The diluted appreciably as would be expected to occur on difference in tissue concentration was especially eviIV administration. Nonetheless, in this study the dent in the pituitary, where after IP administration tissue to blood ratio for the vagina, which our the iodotriphenylethylene estrogen showed very much lower levels of ligand. This characteristic is previous studies (DeSombre et al., 1990; Hughes et al., 1993) had indicated obtains the l&and largely potentially valuable when considering iodoestrogens from the blood, rather than directly on IP adminisfor therapy of tumors located in the peritoneal cavity (e.g. ovarian cancer), as the ratio of I-BHPE in the tration, is more than 30 at the 48 h time point. ln view of the generally significantly lower uptake of estroER+ peritoneal tissues to that in pituitary and gens by the vagina compared with the uterus when peripheral target tissues was higher than that for I-VME2. This preferential localization of an ligand is given IV, it appears the longer target tissue retention of the I-THPE would provide high tissue to iodoestrogen in peritoneal versus peripheral target Uterus

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blood ratios by other routes of administration as well, which will be tested in subsequent studies. It is not entirely clear at this time what the biologic basis is for the extremely long target tissue retention of I-THPE. Preliminary studies suggest that this iodoestrogen has a high affinity for ER, higher than that of I-BHPE. However, another difference between I-THPE and I-BHPE seen in these studies is the ability of a fairly high dose of unlabeled E2 to inhibit the ER target tissue uptake. While 5 pg of E2 is generally considered to be sufficient to completely block the specific uptake of other estrogens by ER target tissues, when given concomitantly, this dose reduced the initial uterine uptake of I-THPE by only about 60% (1 h) while reducing the I-BHPE uterine uptake by over 80%. And at later time points this higher level of apparently non-specific binding of I-THPE continued while it was not seen for either I-BHPE or I-VME2 (Hughes et al., 1993). There are two possible explanations. The first is that actually the affinity of the I-THPE for ER is significantly higher than that of E2. Our preliminary evidence suggests that the affinity of I-THPE may indeed be higher than that of E2 at 2” so that at 37’ this difference could be greater. Secondly, it is possible that there are non-receptor binding sites in the peritoneal cavity, not competed by unlabeled E2, which soak up large amounts of I-THPE allowing it to diffuse continuously to the receptor locally. In fact, assay of peritoneal fat at several times during these studies suggests that fat may take up appreciable amounts of I-THPE. If this were the case, it could be especially useful for IP therapy with I-THPE as it could provide a continuing supply of the ligand possibly safe from the metabolism which most likely occurs with blood borne ligand when it reaches the liver. Thus the fat might act as a depot for the ligand that would be able to diffuse into the peritoneal fluid as its concentration in the fluid decreases. We feel that both of these explanations may be operative. Clearly, even though lower levels of I-THPE reach both the vagina and pituitary, probably via the circulation, both of these tissues also show a prolonged retention, suggestive of a high affinity association of ligand to the ER positive tissue, not seen in tissues lacking ER. Clearly, by 30 h only insignificant levels of radioiodine are found in the blood, and most likely only a minute proportion of this radioiodine is I-THPE, whereas the vagina and pituitary still show specific binding after I-THPE, but not I-BHPE, at 30 h or longer. And it is possible that the high level of apparent non-specific binding found particularly in the peritoneal ER-target tissues, could be due to depot storage of I-THPE, but not unlabeled E2, in the peritoneal cavity, allowing the I-THPE to later diffuse to the target tissues to occupy available receptor sites. Although more studies with this interesting new iodoestrogen will be necessary to clarify the biologic basis for its enhanced retention in ER target tissues

in experimental animals, this estrogen shows propcrties in z+o which may be useful for imaging or therapy of ER-positive cancers in patients. Acknowiedgemem~We gratefully acknowledge the excellent technical assistance provided by B. Teastcr. R. Dizon and V. Chomhirun. This research was supported by the Department of Energy (ER 61655). The Dow Chemical Company and the Boothroyd Foundation.

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