Patent Abstract:
methods and pharmaceutical combinations for inhibiting estrogen - dependent tumors via the co - administration of antiestrogen triphenylethylenes , including tamoxifen , and alkyl pcdfs .

Detailed Description:
the present invention provides a formulation for the inhibition of estrogen - dependent tumors comprising a 1 , 3 , 6 , 8 - substituted or 2 , 4 , 6 , 8 - substituted alkyl pcdf used in combination with a second compound of the triphenylethylene class . possible substituents include halogens such as bromine , chlorine , fluorine and / or linear or branched substituents such as alkyl groups of about one to about five carbons . the 2 , 4 , 6 or 8 and 1 , 3 , 6 or 8 positions may also be individually and independently occupied by a hydrogen instead of a substituent . suitable alkyl substituents include , but are not limited to , methyl , ethyl , propyl , isopropyl ( i - propyl ), n - butyl , sec - butyl , or tert - butyl groups . the pcdfs used in the present invention are described , for example , in u . s . pat . no . 5 , 516 , 790 , issued to stephen safe on may 14 , 1996 , which is hereby incorporated by reference herein in its entirety . the pcdfs may include , but are not limited to , those having the formula : ## str2 ## wherein r 1 , r 3 , r 6 and r 8 or r 2 , r 4 , r 6 and r 8 are individually and independently a hydrogen or a substituent selected from the group consisting of chlorine , fluorine and bromine , and a linear or branched alkyl group of one to four carbons , and wherein the compound has at least one alkyl substituent and at least two halogen substituents ; furthermore , the halogen may be chlorine , the alkyl substituent may be selected from the group consisting of methyl , ethyl and propyl ; r 6 may be an alkyl substituent and r 1 , r 3 , and r 8 may be selected from the group consisting of chlorine , fluorine and bromine ; further still r 8 may be an alkyl substituent and r 1 , r 3 , and r 6 may be selected from the group consisting of chlorine , fluorine and bromine , the alkly substituent may be methyl ; still further r 6 may be an alkyl and r 2 , r 4 , and r 8 may be selected from the group consisting of chlorine , fluorine and bromine ; and , for example , r 8 may be an alkyl substituent and r 2 , r 4 , and r 6 may be selected from the group consisting of chlorine , fluorine and bromine . examples of pcdfs may include , but are not limited to , in one embodiment , pcdfs possessing two lateral substituents (` lateral ` substituents may be on position 2 , 3 , 7 , or 8 of dibenzofuran ) is used in combination with tamoxifen . in a preferred embodiment , 6 - mcdf is used as a representative pcdf in combination with tamoxifen . in another preferred embodiment , 6 - i - propyl - 1 , 3 , 8 - tricdf is used in combination with tamoxifen . in yet another preferred embodiment , 8 - mcdf is used as a representative pcdf in combination with tamoxifen . the pcdf and the triphenylethylene class compounds are combined in a ratio by weight essentially 50 : 1 to 1 : 50 , preferably 25 : 1 to 1 : 25 , and more preferably 10 : 1 to 1 : 10 . most preferred ratios for alkyl pcdf : tamoxifen are 1 : 1 to 1 : 4 to 4 : 1 ( pcdf : tamoxifen ). the present invention also provides a method for the treatment of estrogen - dependent tumors comprising administering to a patient a therapeutically effective amount of a pcdf in combination with a triphenylethylene class compound ( see above ). the pcdf and the triphenylethylene class compounds are administered simultaneously or sequentially . simultaneous administration of the compounds is the preferred method of delivery . in the event of sequential delivery , however , it is preferred that the first administered compound still be bioactive at the time that the second compound is delivered . in one embodiment , both the pcdf and the triphenylethylene class compounds are delivered orally . they may also , for example , be administered intraperotineally . female virgin sprague - dawley rats were obtained from harlan ( houston ) and were allowed to acclimate for 10 days , allowed access to food and water ad libitum , and maintained on a 12 h light / dark schedule . mammary tumors were induced in 50 ± 3 day old - rats by administering a single gavage dose of 20 mg dmba in 0 . 5 ml corn oil . after 30 to 75 days , tumors could be detected by palpitation in the ductal tubes of the mammary glands . multiple tumors often developed on a single rat . when the tumor or the largest of the tumors reached a small size ( 50 - 100 mm 3 ), rats were treated daily by gavage with corn oil alone ( vehicle control ), 0 . 4 mg / kg 6 - mcdf , 0 . 4 mg / kg tamoxifen , or a cotreatment of 0 . 4 mg / kg 6 - mcdf plus 0 . 4 mg / kg tamoxifen for 20 days , and then euthanized on the 21 st day . tumor sizes were measured with calipers , and volumes were calculated by formula ( length × width × depth )/ 6π and are expressed as percent control . one week after their last injection , rats were euthanized by asphyxiation . all tumors were removed , weighed , and sectioned . one portion was frozen in liquid nitrogen for pcr analysis ; one portion was placed in 10 % formalin for histopathological analysis ; and the majority was immediately homogenized for the preparation of microsomal and cytosolic fractions . livers were perfused and weighed , and cytosol and microsomes were prepared as previously described . the results of this study are summarized in table 1 . at doses of 0 . 4 mg / kg / day , both tamoxifen and 6 - mcdf alone significantly inhibited tumor volume ( 58 . 5 and 66 . 4 % inhibition , respectively ). table 1______________________________________ % inhibitiontreatment tumor volume tumor weight______________________________________tamoxifen 58 . 5 * ns decrease 6 - mcdf 66 . 4 * ns decrease tamoxifen / 6 - mcdf 86 . 2 * 78 . 0 * ______________________________________ percent inhibition of tumor volume or tumor weight with tamoxifen alone ( 0 . 4 mg / kg / day ), 6mcdf alone ( 0 . 4 mg / kg / day ), and tamoxifen plus 6mcdf ( each 0 . 4 mg / kg / day ). * p & lt ; 0 . 05 compared to untreated control using anova and duncan &# 39 ; s statistical test for significance . both drugs alone also decreased tumor weight ; however , this inhibitory response was not significant . in contrast , there was a significant 86 . 2 and 78 . 0 % inhibition of both mammary tumor volume and tumor weight in animals cotreated with tamoxifen plus 6 - mcdf . the results illustrated in table 1 clearly show that tumor growth in animals cotreated with both drugs is essentially blocked . moreover , treatment with the compounds alone or in combination did not affect body or organ weights or cause any apparent histopathological changes in these tissues . the induction of cyp1a1 - dependent erod activity by toxic halogenated aromatics such as tcdd is one of the most sensitive indicators of exposure to these compounds and there is an excellent correlation between toxic versus erod induction potencies . alternate - substituted pcdfs are characterized by their low toxicity and low potency as inducers of hepatic erod activity . the results summarized in table 1 show that tamoxifen , 6 - mcdf and tamoxifen plus 6 - mcdf did not induce hapatic erod activity and this is consistent with the low toxicity of 6 - mcdf . these results clearly demonstrate that 6 - mcdf enhances the antitumorigenic activity of tamoxifen in the dmba - induced rat mammary tumor model indicating that combined treatment with tamoxifen plus alkyl pcdfs is a new and more effacious endocrine therapy for treatment of mammary cancer . example 2 , summarized in table 2 and fig1 shows the effects of 6 - mcdf and tamoxifen , alone and in combination in the dmba - induced rat mammary tumor model . methods : exact aged virgin female sprague - dawley rats were dosed on day 55 in the afternoon with 20 mg / rat 7 , 12 - dimethylbenz [ a ] anthacene in a volume of 0 . 5 ml / rat corn oil ( rats weigh approximately 165 g , resulting in 120 mg / kg dmba and 3 ml / kg corn oil ). rats were treated by gavage daily with corn oil ( vehicle ), 0 . 4 mg / kg 6 - mcdf , 0 . 4 mg / kg tamoxifen , or a cotreatment of 0 . 4 mg / kg 6 - mcdf plus 0 . 4 mg / kg tamoxifen for 20 days , and then euthanized on the 21 st day . table 2__________________________________________________________________________effects of 6 - mcdf and tamoxifen , alone and in combination , in the dmba - induced rat mammary tumor model control tamoxifen 6 - mcdf tam ± mcdf__________________________________________________________________________number of 13 13 11 10 animals final tumor 2734 ± 956 1135 ± 587 * 918 ± 305 * 376 ± 111 ** volume ( mm . sup . 3 ) final tumor 100 ± 31 . 5 41 . 5 ± 20 * 33 . 6 ± 11 . 2 * 18 . 8 ± 4 . 1 ** volume (% control ) tumor 3 . 82 ± 1 . 18 2 , 35 ± 1 . 16 1 . 68 ± 0 . 53 0 . 84 ± 0 . 27 * weight ( g ) tumor wt 100 ± 27 . 7 61 . 5 ± 30 . 4 43 . 9 ± 12 . 8 22 . 0 ± 6 . 3 * (% control ) erod 118 ± 40 169 ± 74 140 ± 32 92 ± 21 activity ( pmol / mg / min ) final body 250 ± 5 243 ± 4 247 ± 4 244 ± 4 weight ( g ) liver weight 3 . 13 ± 0 . 12 3 . 64 ± 0 . 18 3 . 49 ± 0 . 16 3 . 32 ± 0 . 19 (% bw ) uterine wt . 0 . 212 ± 0 . 017 0 . 142 ± 0 . 006 * 0 . 230 ± 0 . 037 0 . 146 ± 0 . 10 (% bw ) heart weight 0 . 374 ± 0 . 005 0 . 420 ± 0 . 018 0 . 405 ± 0 . 10 0 . 395 ± 011 (% bw ) spleen wt . 0 . 257 ± . 022 0 . 236 ± 0 . 019 0 . 255 ± 0 . 017 0 . 213 ± 0 . 008 (% bw ) kidney 0 . 349 ± 0 . 007 0 . 386 ± 0 . 012 0 . 361 ± 0 . 016 0 . 397 ± 0 . 027 weight (% bw ) __________________________________________________________________________ * p & lt ; 0 . 05 ** p & lt ; 0 . 01 statistical analysis was done using anova , and significance was determine using the duncan new multiple range test . fig1 shows tumor volume and the effect of treatment on mammary tumors of 0 . 4 mg / kg / day tamoxifen , mcdf , or co - treatment . example 3 shows bone effects of daily doses of tamoxifen and 6 - mcdf in ovariectomized rats . the ovariectomized rat is used as a model for osteoporosis . current studies measure bone histomorphometry ( evans et al . ( 1996 ) or bone mineral density with x - ray abosrptiometry ( ezawa ( 1995 ) as well as bone length , wet weight and dry weight ( takahashi et al . ( 1996 ). in 1987 it was reported that tamoxifen acted as an estrogen agonist in bone tissue of ovariectomized rats ( turner et al . ( 1987 ), although a study indicates that tamoxifen has no effect in cycling rats ( takahashi et al . ( 1996 ). the femurs of rats treated with tamoxifen , mcdf , or tamoxifen + mcdf were analyzed to determine the tissue - specific antiestrogenicity of mcdf . methods : rats were ovariectomized at 97 days of age . after approximately 3 weeks , rats were orally closed for 20 days with corn oil ( vehicle ), 0 . 4 mg / kg / day tamoxifen , 0 . 8 mg / kg / day 6 - mcdf , or cotreatment of 0 . 4 mg / kg tamoxifen plus 0 . 8 mg / kg 6 - mcdf daily . rats were euthanized on day 21 . after uteri were excised and processed , both left and right femurs were excised and the length measured with calipers . because there was no cheap protocol in the literature for loosening all the muscle from the bone , a 4 hour wash with pbs , ph 2 . 0 , was used , and softened the connective tissue without visibly affecting the bone . wet weight was measured after residual connective tissue was stripped away . dry weight was measured after the bones were dried for 12 ours at 130 ° c . table 3__________________________________________________________________________bone effects of daily doses of tamoxifen ( 0 . 4 mg / kg ) and 6 - mcdf ( 0 . 8 mg / kg ) in the ovariectomized sprague - dawley rat control tamoxifen 6 - mcdf tam + mcdf__________________________________________________________________________wet bone 0 . 347 ± 0 . 009 0 . 403 ± 0 . 007 * 0 . 341 ± 0 . 008 0 . 412 ± 0 . 020 * weight (% body weight ) dry bone 0 . 180 ± 0 . 005 0 . 216 ± 0 . 005 * 0 . 183 ± 0 . 003 0 . 235 ± 0 . 010 * weight (% body weight ) bone length 12 . 1 ± 0 . 4 14 . 2 ± 0 . 3 * 12 . 17 ± 0 . 3 15 . 0 ± 0 . 9 * index ( mm / body weight × 100 ) __________________________________________________________________________ values expressed as means ± standard errors . * significantly different ( p & lt ; 0 . 05 ) from control treatment . example 4 shows uterine effects of daily doses of tamoxifen and 6 - mcdf in ovariectomized rats . methods : rats were ovariectomized at 97 days of age , and treatments began at 117 days of age . rats were orally dosed for 20 days with corn oil ( vehicle ), 0 . 4 mg / kg / day tamoxifen , 0 . 4 mg / kg / day 6 - mcdf , or a co - treatment of 0 . 4 mg / kg / day tamoxifen plus 0 . 4 mg / kg / day 6 - mcdf , with 5 rats in each treatment group . rats were euthanized on day 21 . 6 - mcdf caused a slight but significant increase in splenic wet weight . table 4__________________________________________________________________________uterine effects of daily doses of tamoxifen ( 0 . 4 mg / kg ) and 6 - mcdf ( 0 . 4 mg / kg ) in the ovariectomized sprague - dawley rate control tamoxifen mcdf tam + mcdf__________________________________________________________________________uterine wet 0 . 045 ± 0 . 002 0 . 085 ± 0 . 004 * 0 . 060 ± 0 . 006 0 . 078 ± 0 . 004 * weight (% body wt ) uterine wet 0 . 131 ± 0 . 006 0 . 211 ± 0 . 011 * 0 . 180 ± 0 . 019 * 0 . 195 ± 0 . 012 * weight ( g ) uterine 1 . 00 ± 0 . 06 17 . 5 ± 0 . 60 ** 1 . 05 ± 0 . 02 11 . 48 ± 0 . 24 ** peroxidase assay ( abs / mg protein ) ( fold increase over control ) uterine 1 . 00 ± 0 . 06 28 . 41 ± 0 . 73 ** 1 . 56 ± 0 . 03 22 . 06 ± 0 . 46 ** peroxidase assay ( abs / uterus ) ( fold increase over control ) uterine 1 . 00 ± 0 . 06 17 . 7 ± 0 . 45 ** 1 . 14 ± 0 . 02 14 . 84 ± 0 . 31 ** peroxidase assay ( abs / g uterus ) fold increase over control ) uterine 308 ± 142 674 ± 52 * 338 ± 31 677 ± 108 * cytosolic pr levels ( fmol / mg protein ) uterine 1100 ± 508 4501 ± 350 * 1925 ± 177 3685 ± 122 * cytosolic pr levels ( fmol / uterus ) uterine 8368 ± 3868 21301 ± 1655 * 10693 ± 986 18863 ± 625 * cytosolic pr levels ( fmol / g uterus ) __________________________________________________________________________ * significantly different ( p & lt ; 0 . 05 ) from control . ** significantly different ( p & lt ; 0 . 05 ) from control . example 5 shows the effect on body and organ weight of treatment with tamoxifen , mcdf , and co - treatment . methods : the procedure for preparing the dosing solutions is as follows : mcdf requires gentle heating to dissolve , and tamoxifen requires that it be dissolved in ethanol ( 5 % of final volume ) which is then added to corn oil . the ethanol is evaporated off with a gentle stream of air . first , the corn oil for the tamoxifen dose was heated in a boiling water bath , and allowed to cool . then , mcdf for both mcdf alone and tam ± mcdf was weighed and dissolved in appropriate amounts of corn oil with heating . for the control vehicle and the mcdf vehicle ethanol was added ( 5 % v / v ) and then evaporated away . tamoxifen for both the tamoxifen treatment and the tam ± mcdf was dissolved in ethanol . an aliquot from each was then added to the appropriate corn oil vehicle ( the preheated corn oil for tamoxifen , and the corn oil containing mcdf for tam + mcdf ), and the ethanol was evaporated away . table 5__________________________________________________________________________effect of treatment on body and organ weights control tamoxifen mcdf tam + mcdf__________________________________________________________________________rat weight at start 273 . 1 ± 8 . 1 275 . 7 ± 7 . 6 274 . 7 ± 7 . 0 278 . 3 ± 7 . 1 of treatments ( g ) rat weight prior 293 . 2 ± 10 . 9 249 . 8 ± 7 . 6 * 298 . 3 ± 5 . 0 250 . 0 ± 4 . 5 * to euthanasia ( g ) spleen wet 0 . 247 ± 0 . 007 0 . 252 ± . 008 0 . 298 ± 0 . 015 * 0 . 253 ± 0 . 005 weight (% body wt ) spleen wet 0 . 72o ± 0 . 012 0 . 631 ± 0 . 037 0 . 887 ± 0 . 043 * 0 . 632 ± 0 . 011 weight ( g ) liver weight (% 4 . 06 ± 0 . 23 4 . 26 ± 0 . 26 4 . 37 ± 0 . 26 4 . 03 ± 0 . 09 body wt ) liver weight ( g ) 11 . 88 ± 0 . 78 10 . 70 ± 0 . 92 13 . 04 ± 0 . 87 10 . 06 ± 0 . 24 heart weight (% 0 . 319 ± 0 . 006 0 . 346 ± 0 . 005 0 . 344 ± 0 . 014 0 . 354 ± 0 . 012 body wt ) heart weight ( g ) 0 . 935 ± 0 . 039 0 . 864 ± 0 . 031 1 . 024 ± 0 . 037 0 . 863 ± 0 . 013 kidney weight (% 0 . 318 ± 0 . 010 0 . 334 ± 0 . 008 0 . 332 ± . 004 0 . 323 ± 0 . 005 body wt ) kidney weight ( g ) 0 . 933 ± 0 . 038 0 . 837 ± 0 . 045 0 . 991 ± 0 . 026 0 . 807 ± 0 . 016__________________________________________________________________________ example 6 ( tables 6 and 7 and fig2 and 3 ) show uterine effects of daily doses of tamoxifen and 6 - mcdf in ovariectomized rats . for table 6 and fig2 rats were ovariectomized at 97 days of age . after approximately 3 weeks , rats were orally dosed for 20 days with corn oil ( vehicle ), 0 . 4 mg / kg / day tamoxifen , 0 . 8 mg / kg / day 6 - mcdf , or a cotreatment of 0 . 4 mg / kg tamoxifen plus 0 . 8 mg / kg 6 - mcdf daily . rats were euthanized on day 21 . table 6__________________________________________________________________________uterine effects of daily doses of tamoxifen ( 0 . 4 mg / kg ) and 6 - mcdf ( 0 . 8 mg / kg ) in the ovariectomized sprague - dawley rat effects of treatment on uterine assaysassay control tamoxifen 6 - mcdf tam + mcdf__________________________________________________________________________ # rats / treatment group 4 5 5 6 uterine wet weight ( g ) 0 . 171 ± 0 . 15 0 . 208 ± 0 . 009 * 0 . 131 ± 0 . 004 *† 0 . 197 ± 0 . 009 uterine wet weight 0 . 0583 ± 0 . 0055 0 . 0857 ± 0 . 0036 * 0 . 0454 ± 0 . 018 ±† 0 . 0848 ± 0 . 0062 * (% body weight ) uterine peroxidase 1 . 00 ± 0 . 01 26 . 76 ± 0 . 22 * 0 . 99 ± 0 . 01 . dagger . 20 . 87 ± 0 . 36 * activity ( absorbance / mg protein )( fold increase over control ) uterine peroxidase 1 . 00 ± 0 . 01 38 . 66 ± 0 . 32 * 0 . 57 ± 0 . 01 *. dagge r . 19 . 55 ± 0 . 33 *† activity ( absorbance / uterus ) ( fold increase over control ) uterine peroxidase 1 . 00 ± 0 . 01 31 . 94 ± 0 . 26 * 0 . 88 ± 0 . 01 * 19 . 69 ± 0 . 34 *† activity ( absorbance / g uterus ) ( fold increase over control ) uterine progesterone 198 ± 15 790 ± 21 * 152 . 9 ± 35 † 375 ± 61 *† receptor ( fmol / mg protein ) uterine progesterone 765 ± 69 9062 ± 244 * 441 . 5 ± 100 † 1875 ± 306 *† receptor ( fmol / uterus ) uterine progesterone 4462 ± 343 43666 ± 1175 * 3985 ± 907 . dagger . 11403 ± 1852 *† receptor ( fmol / g uterus ) __________________________________________________________________________ values expressed as means ± standard deviations . + significantly different ( p & lt ; 0 . 05 ) from control treatment . † significantly different ( p & lt ; 0 . 05 ) from tamoxifen treatment . fig2 a , 2b and 2c show uterine peroxidase activity ( nuclear extracts ) with treatment with tamoxifen , mcdf and co - treatment . for table 7 and fig3 and 3b , exact aged virgin female sprague - dawley rats were dosed on day 55 in the afternoon with 20 mg / rat 7 , 12 - dimethylbenz [ a ] anthacene in a volume of 0 . 5 ml / rat corn oil ( rats weigh approximately 165 g , resulting in 120 mg / kg dmba and 3 ml / kg corn oil ). rats were treated by gavage daily with corn oil ( vehicle ), 0 . 4 mg / kg 6 - mcdf , 0 . 4 mg / kg tamoxifen , or a cotreatment of 0 . 4 mg / kg 6 - mcdf plus 0 . 4 mg / kg tamoxifen for 20 days , and then euthanized on the 21 st day . table 7__________________________________________________________________________effects of treatment on body and organ weights control tamoxifen 6 - mcdf tam ± mcdf__________________________________________________________________________rat weight at 255 ± 3 261 ± 9 257 ± 4 244 ± 16 start of treatments ( g ) rat weight 295 ± 11 242 ± 6 * 289 ± 7 † 236 ± 11 * prior to euthanasia ( g ) liver weight 3 . 95 ± 0 . 16 4 . 03 ± 0 . 15 3 . 78 ± 0 . 11 4 . 07 ± 0 . 20 (% body weight ) heart weight 0 . 360 ± 0 . 010 0 . 357 ± 0 . 023 0 . 356 ± 0 . 012 0 . 374 ± 0 . 23 (% body weight ) spleen weight 0 . 289 ± 0 . 018 0 . 254 ± 0 . 018 0 . 265 ± 0 . 014 0 . 250 ± 0 . 004 (% body weight ) kidney weight 0 . 334 ± 0 . 010 0 . 369 ± 0 . 017 0 . 352 ± 0 . 015 0 . 357 ± 0 . 013 (% body weight ) __________________________________________________________________________ values expressed as means ± standard error . * significantly different ( p & lt ; 0 . 05 ) from control treatment . † significantly different ( p & lt ; 0 . 05 ) from tamoxifen treatment . fig3 a and 3b show uterine weights with treatment with tamoxifen , mcdf and co - treatment . 1 . b . astroff et al ., 6 - methyl - 1 , 3 , 8 - trichlorodibenzofuran as a 2 , 3 , 7 , 8 - tetrachlorodibenzo - p - dioxin antagonist inhibition of the induction of rat cytochrome p - 450 isozymes and related monoxygenase activites , mol . pharmacol . 33 ( 1988 ) 231 - 236 ; 2 . b . astroff et al ., comparative antiestrongenic activities of 2 , 3 , 7 , 8 - tetrachlorodibenzo - p - dioxin and 6 - methyl - 1 , 3 , 8 - trichlorodibenozofuran in the female rat , itoxicol . appl . pharmacol . 95 ( 1988 ) 435 - 443 ; 3 . b . astroff et al ., 6 - substituted - 1 , 3 , 8 - trichlorodibenzofurans as 2 , 3 , 7 , 8 - tetrachlorodibenzo - p - dioxin antagonists in the rat : structure - activity relationships , toxicology , 59 ( 1989 ) 285 - 296 ; 4 . b . astroff et al ., 6 - alkyl - 1 , 3 , 8 - trichlorodibenzofurans as antiestrogens in female sprague - dawley rats , toxicology 69 ( 1991 ) 187 - 197 ; 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