Disposition of bazedoxifene in rats.

Bazedoxifene acetate (BZA), a novel selective estrogen receptor modulator, is currently being developed for the prevention and treatment of osteoporosis in post-menopausal women. In this study, the disposition of BZA was determined in the rat, a pharmacology and safety model. After a single 0.2 mg/kg intravenous (IV) administration to ovariectomized female rats, the plasma clearance (3.9 L/h/kg) and volume of distribution (16.8 L/kg) of BZA were high and the elimination half-life was short (3.8 h). The bioavailability was low (16%) after a 1 mg/kg oral dose of BZA. Radioactivity was rapidly absorbed (t(max) = 0.35 h), widely distributed into tissues and slowly eliminated (t(1/2) = 29 h) in female rats following a 1 mg/ kg oral dose of [(14)C]BZA. Following a 3 mg/kg oral dose to male rats, the tissue to plasma ratios of radioactivity ranged from 1 to 55 at 2 and 8 h post-dose in ascending order in heart, kidney, lung, and liver. BZA was extensively metabolized in both male and female rats. BZA-5-glucuronide was the major metabolite and BZA-4'-glucuronide was minor in plasma and tissues. Both glucuronides were major metabolites in bile. In vitro metabolite profiles were similar in rat liver and intestinal microsomes and they qualitatively correlated well with bile profiles. Feces was the major route of excretion (>97%) after the IV or oral dose. BZA was the predominant radioactive component in feces.

Distribution and metabolism of the retinoid, N-(4-methoxyphenyl)-all-trans-retinamide, the major metabolite of N-(4-hydroxyphenyl)-all-trans-retinamide, in female mice.

The metabolism and disposition of N-(4-methoxyphenyl)-all-trans-retinamide (MPR), the major metabolite of N-(4-hydroxyphenyl)-all-trans-retinamide (4-HPR), were investigated in female B6D2F1 (BDF) mice. Following a single oral dose of 10 mg/kg, MPR distributed to the serum, liver, mammary gland, urinary bladder, and skin. The highest levels of MPR were detected in the liver and mammary gland, and the largest values for AUC were in the mammary gland followed by the skin and liver. The t1/2 for MPR was 5.1 hr in liver, 5.6 hr in serum, 18.7 hr in urinary bladder, 23.1 hr in skin, and 26.6 hr in mammary gland. MPR and five metabolites were detected; levels varied between tissues. One metabolite was 4-HPR; the other four, which eluted at 7, 12, 13, and 18 min, remain unidentified. The major metabolite of MPR was the 18-min metabolite and comprised 17% of total retinoid in skin and 14% in mammary gland. 4-HPR was only a minor metabolite of MPR; 4-HPR was not detectable in serum or urinary bladder and accounted for less than 4% of total retinoid in the other tissues. In mice dosed with 10 mg/kg 4-HPR, the parent compound, MPR, a putative 4-HPR ester, and three of the MPR metabolites (7, 13, and 18 min) were found. These data suggest that the interconversion of 4-HPR and MPR greatly favors formation of MPR.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of the chemopreventive retinoid N-(4-hydroxyphenyl)retinamide by mammary gland in organ culture.

N-(4-Hydroxyphenyl)retinamide (4-HPR) is considered to be the most effective chemopreventive retinoid for chemically induced mammary carcinogenesis in rats. However, the mechanism of 4-HPR action in mammary cells is poorly understood. In the present study we examined the metabolism of 4-HPR in the mouse mammary gland in organ culture. Mammary glands excised from BALB/c mice were incubated with 4-HPR in the presence of insulin, prolactin and steroid hormones for 6 days. The glands were extracted with chloroform/methanol (2:1, v/v), and the metabolites were separated on a reversed-phase h.p.l.c. column. Three metabolites were separated in addition to 4-HPR; one of the metabolites, M2, was co-eluted with 13-cis-4-HPR, M3 was co-eluted with N-(4-methoxyphenyl)retinamide (4-MPR) and M1 remains unidentified. There appeared to be some hormonal regulation in the distribution of metabolites in the glands. Increased levels of 4-MPR and M1 were observed in insulin-plus-prolactin-treated glands as compared with the glands incubated with steroid hormones. Furthermore, it was observed that M1 isolated from the livers of 4-HPR-treated rats competed for the cellular retinoic acid-binding protein (CRABP) sites; however, 4-HPR did not bind to CRABP. These results indicate that mouse mammary gland can metabolize 4-HPR and that the metabolites which compete for CRABP sites may have physiological significance in the retinoid inhibition of mammary carcinogenesis.

Effects of pretreatment with the retinoid N-(4-hydroxyphenyl)-all-trans-retinamide and phenobarbital on the disposition and metabolism of N-(4-hydroxyphenyl)-all-trans-retinamide in mice.

The effects of pretreatment with N-(4-hydroxyphenyl)-all-trans-retinamide (4-HPR) and phenobarbital (PB) on the distribution and metabolism of 4-HPR, and on the levels of hepatic cytochromes, were investigated in female BDF mice. Pretreatment of mice for 3 days with 10 mg/kg 4-HPR had no effect on the disposition of 4-HPR in the serum, liver, mammary gland, or urinary bladder. 4-HPR pretreatment also had no effect on the pharmacokinetics of any of its metabolites in the liver, or on the levels of hepatic cytochromes P450 or b5. By contrast, pretreatment of mice for 3 days with 80 mg/kg PB had a significant effect on the disposition of 4-HPR in all the tissues examined; the areas under the concentration-time curves for PB-pretreated mice were half those for vehicle-pretreated mice. PB pretreatment also significantly reduced the levels of four metabolites of 4-HPR in the liver and increased the levels of hepatic cytochromes P450 and b5. These data suggest that prior or concomitant administration of drugs that induce the mixed function oxidase system could result in changes in retinoid disposition and metabolism; such changes may alter retinoid chemopreventive activity.

Genetic variability in deacetylation of 2-acetylaminofluorene and N-hydroxy-2-acetylaminofluorene in inbred strains of mice.

Genetic variability in 2-acetylaminofluorene (AAF) and N-hydroxy-2-acetylaminofluorene (N-OH-AAF) deacetylase activities was examined in 19 inbred strains of mice. AAF deacetylase activities ranged from 0.60 to 1.33 nmol/min/mg protein, and there was an approximately 2.5-fold difference in AAF deacetylase activity between the fastest (C57BL/6J) and slowest (RIIIS/J) mouse strains. N-OH-AAF deacetylase activities ranged from 3.28 to 13.24 nmol/min/mg protein, and the difference between the fastest (AU/SsJ) and slowest (RIIIS/J) strains was 4-fold. N-OH-AAF deacetylase activity was higher (5-13 times) than AAF deacetylase activity in all strains examined. Thus, there are genetic differences in AAF and N-OH-AAF deacetylase activities; these differences may play an important role in individual susceptibility to the mutagenic and carcinogenic effects of the aromatic amides.

Comparative activity of dietary or topical exposure to three retinoids in the promotion of skin tumor induction in mice.

The activity of dietary and topical administration of three retinoids, all-trans-retinoic acid, 13-cis-retinoic acid, and N-(4-hydroxyphenyl)retinamide (4-HPR), as promoters of skin tumor induction in SENCAR mice was studied. When administered as dietary supplements at their maximum tolerated dose levels, all three retinoids promoted tumorigenesis in mice initiated with a single topical dose of 5 micrograms 7,12-dimethylbenz(a)anthracene. Maximal promoting activity was observed with dietary 13-cis-retinoic acid; dietary 4-HPR was significantly less active than was either isomer of retinoic acid. When administered via topical application, all-trans- and 13-cis-retinoic acids both promoted skin tumor induction; 4-HPR did not. HPLC analysis of skin samples from mice receiving dietary 4-HPR showed the parent compound and six metabolites; these metabolites were not found in the skin of mice receiving topical 4-HPR exposure, although 4-HPR itself was present. These data indicate that skin tumor promotion can be induced by systemic administration as well as topical application of the all-trans- and 13-cis-retinoic acids. Substitution of a 4-hydroxyphenylamide terminal group results in a significant reduction in promoting activity. 4-HPR appears to require metabolic activation for tumor promoting activity; this metabolism does not occur in the skin following topical application, but is observed following systemic exposure.

Potentiation of vitamin A hepatotoxicity by butylated hydroxytoluene.

The interaction between the natural vitamin A ester retinyl acetate (RA) and the phenolic antioxidant butylated hydroxytoluene (BHT) in the induction of biliary hyperplasia and hepatic fibrosis in female Sprague-Dawley rats was characterized. Using a 3 X 3 matrix design, rats were fed diets supplemented with (per kilogram diet) 0, 125, or 250 mg RA and/or 0, 2500, or 5000 mg BHT. The 125-mg dose of RA induced no gross hepatotoxicity, while the 250-mg dose of RA induced a low incidence of hepatic fibrosis in rats examined after 120 and 180 days of exposure. Exposure to BHT alone induced hepatocellular hypertrophy and dose-related increases in liver weight, but no hepatocellular pathology. Simultaneous administration of RA plus BHT resulted in significant increases in the incidence of biliary hyperplasia and hepatic fibrosis compared to that induced by RA alone. BHT reduced total hepatic vitamin A content at all RA dose levels. Thus, mechanisms other than increases in liver vitamin A levels must underlie the potentiation by BHT of RA hepatotoxicity.

N-(4-hydroxyphenyl)-all-trans-retinamide pharmacokinetics in female rats and mice.

The distribution of N-(4-hydroxyphenyl)-all-trans-retinamide (4-HPR) and its metabolites was investigated in the liver, serum, mammary gland, and urinary bladder of female rats and mice. Following an iv dose of 5 mg/kg to rats, 4-HPR distributed to all tissues examined with the highest levels reached in the liver. The distribution period was completed in about 4 hr and was followed by first order elimination kinetics. The t1/2 for 4-HPR elimination from the liver was 9.4 hr, from the serum was 12.0 hr (not significantly different from liver), from the mammary gland was 43.6 hr, and from the urinary bladder was 9.3 hr. A 5-day ip dosing study (5 mg/kg/day of 4-HPR) in both rats and mice revealed that 4-HPR distributed to all tissues examined with the highest levels reached in the urinary bladder. 4-HPR and four metabolites were detected in the tissues. One coeluted with a cis isomer of 4-HPR (M2), another with N-(4-methoxyphenyl)-all-trans-retinamide (4-MPR) (M3), a third appeared to be a 4-HPR-ester (M4), and the fourth remains unidentified (M1). However, the amount of each metabolite varied between tissues and between species. The concentration of 4-HPR was significantly 2-4 times lower and the percentage of M3 (4-MPR) was 3 times higher in the mouse tissues than in the corresponding tissues of the rat. M2 (cis-4-HPR) and M4 (4-HPR-ester) were present in rat liver but not in mouse liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective high-efficiency cross-linking of mammalian ribosomal proteins with cleavable thiol-directed heterobifunctional reagents: identification and binding directions of major protein complexes.

Rat liver and mouse ascitic tumour ribosomal proteins are cross-linked selectively in good yield with the newly developed cleavable heterobifunctional reagents 2-(4-hydroxy-2-maleimidophenylazo)benzoic acid N-hydroxysuccinimide ester (reagent A) and 4-(4-hydroxy-3-maleimidophenylazo)[carboxyl-14C]benzoic acid N-hydroxysuccinimide ester (reagent B). The primary function of the reagents, an N-aroylated maleimide, binds quantitatively at low pH to accessible cysteine groups. After eliminating the free reagent, the pH is increased to make the secondary function, a juxtanuclear aroyl ester, reactive against neighboring amino groups, essentially lysine. The spacer, 4-phenylazophenol, is readily cleaved by reduction with dithionite. The ranges of cross-linking of the two reagents are approx. 8 and 12 A, respectively. Using the radiolabelled reagent B the secondarily attached protein (and its contact sequence) is made recognizable even in trace amounts. The order of binding of the interacting proteins is thereby established. The two reagents produce similar, but not identical, patterns of selective cross-linking. The following protein complexes are readily observed after conventional staining. With reagent A: S8-S11, L4-L14, L4-L18, L6-L29 and L21-L18a. With the radioactive, longer-range reagent B: L4 ---- L13a, L4 ---- L18, L4 ---- L18a, L4 ---- L26, L6 ---- L29, L14 ---- L13a, L21 ---- L18a and L27 ---- L30 (arrows indicating the direction of binding). Ternary and quaternary complexes are also obtained, especially of the large protein L4. With both reagents a protein designated L6' is cross-linked to L23. The predominant cross-linked complexes can be obtained on a preparative scale for isolation and characterization of contact sequences by optional fragmentation and fractionation methods.

Selective high-efficiency cross-linking of mammalian ribosomal proteins with cleavable thiol-directed heterobifunctional reagents: separation and identification of contact sequences of neighboring proteins after CNBr fragmentation.

Mammalian ribosomal proteins were cross-linked in situ with the primarily cysteine-selective heterobifunctional reagents N-succinimidyl 2-(4-hydroxy-2-maleimidophenylazo)benzoate (reagent A, maximum range approx. 8 A) and N-succinimidyl 4-(4-hydroxy-3-maleimidophenylazo)[carboxyl-14C]benzoate (reagent B, maximum range approx. 12 A). With reagent B the secondarily attached (N-aryolated) protein becomes labelled specifically at the receptor amino group (lysine). The cross-linked proteins were fragmented with CNBr in attempts to isolate and identify sequences involved in the next-neighbor contacts. Two experimental schemes were adopted. Heavy complexes containing the large protein L4 cross-linked to protein L14 and/or L18 were isolated and treated with CNBr. The split products were submitted to diagonal electrophoresis for separation and identification of the two pairs of contact fragments. Proteins cross-linked with the radiolabelled reagent B were submitted to diagonal electrophoresis. The labelled receptor proteins were excised and treated with CNBr. Fragments carrying the contact sequences were separated by gradient gel electrophoresis and identified by autoradiography. By use of these methods CNBr fragments were isolated containing one or the dual contact sites of the following binary protein complexes: L4-L14, L4-L18, L4-L13a/L18a, L6'-L23, L6-L29, L7-L29, L14-L13a, L21-L18a, and L27-L30 (asterisks indicate the labelled receptor proteins). By varying the site of labelling of the heterobifunctional reagents and the methods of protein fragmentation a complete analysis of the contact sequences of these proteins should be possible.

A class of cleavable heterobifunctional reagents for thiol-directed high-efficiency protein crosslinking: synthesis and application to the analysis of protein contact sites in mammalian ribosomes.

The synthesis of a new class of cleavable crosslinking reagents is described. The primary function, a ring-substituted maleimide, binds selectively and very efficiently at low pH to cysteine-containing protein sequences. At increased pH the secondary function, an N-hydroxysuccinimide ester of a ring-attached carboxyl group, becomes reactive against adjacent amino groups. The spacer, azobenzene, is readily cleaved by reduction with dithionite provided that a hydroxyl group is included in the ring system. By altering the relative positions of the reactive groups the range of crosslinking can be varied within approximately 8-12 A. After degradation of the crosslinked proteins by optional methods the contact sequences are readily identified by diagonal electrophoresis. By radiolabeling the carboxyl group of the reagent the order of binding of the proteins can be established, and the secondarily attached protein and its contact sequences can be recognized even in trace amounts. The usefulness of the reagents is illustrated by the selective, high-efficiency crosslinking of mammalian ribosomal proteins and the identification of their contact fragments as obtained by CNBr degradation.

Genetic differences in 2-aminofluorene pharmacokinetics between intact C57BL/6J and A/J mice.

Studies of genetically determined differences in arylamine acetylation with the model carcinogen 2-aminofluorene in C57BL/6J and A/J inbred mouse strains showed that individual differences in the pharmacokinetics of 2-aminofluorene were dependent on differences in 2-aminofluorene N-acetyltransferase activity in liver and blood. Elimination rates of 2-aminofluorene from blood of mice administered a single ip dose of 30, 50, or 100 mg/kg of 2-aminofluorene were dose-dependent in both strains. At a dose of 100 mg/kg, the average rate of 2-aminofluorene elimination was approximately three times faster in rapid acetylator (C57BL/6J) mice than in slow acetylator (A/J) mice (0.36 +/- 0.02 hr-1 vs. 0.12 +/- 0.02 hr-1), and that in B6AF1 mice was intermediate (0.24 +/- 0.02 hr-1) to the parental lines. These results support previous observations that acetylation of arylamines is controlled by intermediate dominant inheritance of two major alleles at a single locus. Comparison of the average rate of elimination of 2-aminofluorene from blood of the congenic mouse line, A.B6-NATr, (0.27 +/- 0.05 hr-1) which has the rapid acetylator allele placed on the A/J background provided evidence that modifying genes of the A/J mouse significantly reduced the effect of the rapid acetylator allele on the rate of 2-aminofluorene elimination. A trend was observed toward a greater apparent volume of distribution for 2-aminofluorene in A/J than in C57BL/6J mice.

Location of the sulfhydryl groups involved in disulfide interaction between the neighboring proteins L6 and L29 in mammalian ribosomes. S-cleavage of the cyanylated proteins in polyacrylamide gels after separation by dodecylsulfate gel electrophoresis.

Proteins L6 and L29 occupy closely adjacent sites in mammalian 60-S ribosomal subparticles and are easily cross-linked by intermolecular disulfide bond formation. For locating the interacting thiols within the polypeptide chains the dissociated proteins L6 and L29 obtained from the isolated disulfide complex were subjected to S-cleavage following [14C]cyanylation of the two cysteine residues. Four split products of the [14C]cyanylated proteins were isolated by dodecylsulfate gel electrophoresis. Two of these could be identified by autoradiography as the selectively labeled C-terminal fragments. For unequivocal assignment of the fragments to the parent proteins, a simple and generally applicable method of cleaving cyanylated proteins in polyacrylamide gel for subsequent diagonal analysis was developed. The experiments indicated that the sulfhydryl group of L6 interacting with L29 is located at a distance of approximately 80 amino acid residues from the N-terminus. In the intact ribosome this sequence contains a clostripain-sensitive and trypsin-sensitive portion of the protein more or less exposed at the ribosomal surface. In the case of protein L29, the interacting sulfhydryl group was located at a distance of approximately 40 amino acid residues from the C-terminal.

Isolation of the contact regions of the neighboring mammalian ribosomal proteins L6 and L29. Cyanogen bromide cleavage of the disulfide complex after preparative electrophoresis in non-oxidative polyacrylamide gels.

Proteins L6 and L29 in the 60-S subparticle of mammalian ribosomes interact in situ under gentle conditions by intermolecular disulfide bond formation. For identifying the contact regions of the two proteins the disulfide complex was isolated from whole ribosomes by preparative polyacrylamide gel electrophoresis and subjected to cyanogen bromide cleavage. For effective cleavage non-oxidizing conditions had to be maintained throughout the preparation. A simple and generally applicable method of high-efficiency gel pre-electrophoresis with anionic and cationic thiols was developed. Under these conditions reversibly cross-linked CNBr fragments of L6 and L29 could be isolated in high yield (Mr approx. 13 000 and 7000, respectively). After [14C]carboxymethylation of the reduced disulfide links smaller contact sequences were obtained by pepsin digestion and characterized by two-dimensional peptide mapping. These smaller contact peptides were contained within the corresponding CNBr fragments. Both contact peptides were hydrophilic and relatively basic.

Analysis of the membrane-associated poly(A)+RNA in the cytoplasm of dormant Artemia cysts by DNA excess hybridization. Evidence for a nuclear origin.

Encysted embryos of Artemia contain latent mRNA, to a large extent associated with a fraction of cytoplasmic membranes. The membranes, purified by EDTA treatment and banding in a sucrose gradient at 1.17 g/cm3, include endoplasmic vesicles and mitochondria. The origin of the membrane-associated poly(A)+RNA was therefore investigated. In gel electrophoresis poly(A)+RNA from the purified membranes of dormant cysts forms two distinct bands at approx. 3 . 10(5) and 5 . 10(5) Da. Later during development the lighter component decreases. Nuclei from dormant cysts are devoid of poly(A)+RNA, while nuclei from developing embryos (50% emergence) contain a predominant poly(A)+RNA component of approx. 5 . 10(5) Da. 125I-labelled preparations of nuclear DNA and of nuclear and membrane-associated poly(A)+RNA were used in reassociation and hybridization experiments with excess nuclear DNA. Poly(A)+RNA from the membranes of dormant cysts hybridized to nuclear DNA to the same extent as the nuclear poly(A)+RNA from developing embryos. The hybridization of labelled, nuclear poly(A)+RNA to nuclear DNA was strongly inhibited by unlabelled membrane RNA from either dormant cysts or developing embryos. It is concluded that the stored, membrane-associated poly(A)+RNA in dormant cysts is essentially of nuclear origin. The 5 . 10(5)-Da component is largely homologous with the corresponding component of nuclear poly(A)+RNA at later stages.