Retinyl ethers as cancer chemopreventive agents. Suppression of mammary cancer.

It had been demonstrated previously that retinyl methyl ether (RME) can suppress carcinogen-induced mammary cancers in vivo. It had also been shown that RME is demethylated enzymatically to retinol and produces the toxic effects of retinol; however, a rationale was developed for further investigations of retinyl ethers and was the basis for the synthesis and biological evaluations of new retinyl ethers for the chemoprevention of mammary cancer, reported herein. Two of the new retinyl ethers, retinyl 3-methyl-2-butenyl ether (RMBE) and retinyl 2-propynyl ether (RPE), were evaluated for the suppression of mammary cancers in vivo. RMBE, RPE, RME, the 2,3,6-trimethyl-4-methoxyphenyl analogue of RME, and retinyl acetate (a positive control) were incorporated individually into the feed of rats that had been injected with N-methyl-N-nitrosourea to induce mammary cancers. Ninety-day tests of these compounds for suppression of mammary cancer showed that RPE has significant cancer chemopreventive activity, comparable to that of retinyl acetate in simultaneous tests. RMBE demonstrated borderline activity. Both RPE and RMBE were less toxic than retinyl acetate or RME and, in contrast to the other retinoids, did not cause accumulation of large amounts of retinyl palmitate in the liver. Further investigations of RPE showed that it accumulated in mammary tissue after a single oral dose was administered to female rats, reached maximum concentrations within 24 h, and was still present at 75-80% of maximum concentrations after 72 h. In ethanol at 25 degrees C, RPE slowly underwent intramolecular cyclization; small amounts of the cyclized product also appeared in mammary tissue of rats dosed with RPE. During the mammary cancer bioassay, however, RPE was essentially stable in the feed. Some of the new retinyl ethers, as well as RME, bind to cellular retinol-binding protein.

A possible suicide by diltiazem overdose.

This report describes a possible suicide by overdosage with diltiazem. The decreased, a 39-year-old male who had been under treatment for an unidentified heart condition, was discovered on the bathroom floor in his residence. Comprehensive drug screens performed on available postmortem specimens revealed the presence of 6.9 mg/L diltiazem, 0.182 g/100 mL ethanol, and a trace of propranolol in blood, and 4.7 mg/L diltiazem and 0.251 g/100 mL ethanol in urine.

Disposition of 2',3'-dideoxyadenosine and 2',3'-dideoxyinosine in mice.

Mice were dosed with [3H]2',3'-dideoxyadenosine ([3H]ddA) in three procedures: intravenously, intraperitoneally, and interperitoneally following a dose of 2'-deoxycoformycin (dCF). For mice dosed intravenously, the content of radioactivity in plasma and tissue samples were essentially constant after 30 min. Of the radioactivity in plasma and brain samples collected between 30 min and 24 hr, more than 94% was present as 3H2O, indicating that most of the tritium from [3H]ddA had exchanged with water. No intact ddA was detected, and the deamination product, 2',3'-dideoxyinosine (ddI), was present only transiently. In the urine, the major radioactive material was [3H]ddI. Also detected were 3H2O and small amounts of [3H]hypoxanthine and [3H]ddA. Following intraperitoneal doses to mice, levels of radioactivity in plasma, liver, and kidney increased to a maximum by 15-30 min after dosing but dropped to essentially constant levels thereafter, again indicating that the tritium had exchanged with water. At 5, 15, and 30 min after dosing, ddI was the major radioactive component in plasma. Only small amounts of ddA were present. When dCF was administered 24 hr prior to intraperitoneal [3H]ddA, levels of radioactivity in plasma, liver, and kidney reached a maximum of 30 to 60 min after dosing and decreased to essentially constant levels thereafter. The dCF transiently inhibited the deamination of ddA to ddI, since, in plasma, [3H]ddA was the main radioactive component at 5 and 15 min after dosing. Comparison of HPLC assays based on radioactivity detection and UV absorbance showed that they were equivalent for measuring ddA and ddI in samples derived from dosed mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Disposition of 2-mercaptobenzothiazole and 2-mercaptobenzothiazole disulfide in rats dosed intravenously, orally, and topically and in guinea pigs dosed topically.

To determine the metabolic disposition of [14C]-2-mercaptobenzothiazole (MBT) and [14C]-2-mercaptobenzothiazole disulfide (MBTS), male and female rats were dosed topically. Topical doses were 36.1 micrograms/animal for [14C]MBT and 33.6 micrograms/animal for [14C]MBTS. Although more MBT passed through the skin than MBTS and although, relative to rats, guinea pigs absorbed a greater percentage of the dose (33.4% compared to 16.1-17.5% of the MBT and 12.2% compared to 5.94-7.87% for MBTS), the disposition of radioactivity derived from the two compounds was similar. Washing of the skin removed more of the radioactivity from guinea pigs than from rats. For both sexes of rats dosed intravenously with [14C]MBT (0.602 mg/kg) or [14C]MBTS (0.571 mg/kg), disposition of the compounds was similar. In 72 h, 90.9-101% of the dose appeared in the urine and 3.79-15.1% in the feces. At this time, a small portion of the administered radioactivity (1.52-1.96% of the dose) remained associated with erythrocytes. Oral dosing of rats for 14 d with unlabeled MBT (0.510 mg/kg.d) prior to a single dose of [14C]MBT (0.503 mg/kg) or with unlabeled MBTS (0.521 mg/kg.d) prior to a single dose of [14C]MBTS (0.730 mg/kg). For both sexes, disposition of the compounds was similar. At 96 h after dosing, a small portion of the administered radioactivity (1.20-1.69% of the dose) remained associated with erythrocytes, most of which was bound to the membranes. For both compounds and sexes, 60.8-101% of the radioactivity administered appeared in the urine and 3.46-9.99% in the feces in 96 h. At the time, only trace amounts of radioactivity remained in tissues other than blood. Of these tissues, thyroid contained the highest concentration. In the urine, there was a detectable MBT or MBTS, but there were two metabolites, one of which was identified as a thioglucuronide derivative of MBT. The other was possibly a sulfonic acid derivative of MBT. In conclusion, there were similarities in absorption, distribution, and metabolism of [14C]MBT and [14C]MBTS in rats and in guinea pigs, indicating that [14C]MBTS was readily converted to [14C]MBT.

Disposition of 2-(2-quinolyl)-1,3-indandione (D. C. yellow #11) in rats dosed orally or intravenously.

The disposition of 2-(2-quinolyl)-1,3-indandione (D. C. yellow #11, DCY) in male Fischer rats dosed intravenously or by feeding was determined. For rats given [14C]DCY in the feed (0.00044-0.41% of the diet), recovery of radioactivity during the 24-h dosing period and the 72-h period thereafter ranged from 89.1 to 93.9% for feces and from 4.98 to 6.25 for urine. Tissues contained only trace amounts. Following intravenous dosing with [14C]DCY (0.93 mg/kg), radioactivity distributed readily into most tissues; maximum amounts were present at 5 min, the earliest time of assay. Maximum amounts of radioactivity in fat, skin, and gut tissue, however, were present at 30 min after dosing. These three tissues also had relatively long alpha phases for the elimination of radioactivity. In 24 h after intravenous dosing, rats excreted 81.1% of the dose in the feces and 16.0% of the dose in the urine. For rats fitted with biliary cannulas, 54.5% of the dose, all of which was metabolites of [14C]DCY, was recovered in the bile in 4 h. Associated with the rapid and extensive biliary excretion of metabolites of intravenously administered [14C]DCY was the appearance of large amounts of radioactivity in the feces and also, at intermediate time points, in the liver, gut contents, and gut tissue. In conclusion, rats rapidly distribute, metabolize, and excrete [14C]DCY.

The pharmacokinetics of all-trans-retinoic acid and N-(2-hydroxyethyl)retinamide in mice as determined with a sensitive and convenient procedure. Solid-phase extraction and reverse-phase high performance liquid chromatography.

An improved method for the analysis of retinoids in mouse plasma allows quantification of all-trans-retinoic acid (RA) and N-(2-hydroxyethyl)retinamide (NHERA) in amounts as low as 2 ng/ml (7 X 10(-9) M), the approximate endogenous concentration of RA. The procedure, involving solid-phase extraction and reverse-phase HPLC, can be used for rapid processing of large numbers of samples. With this procedure, we have determined the terminal half-life for RA (0.5 hr) and have confirmed the half-life for NHERA (3.6 hr) in the plasma of mice dosed orally. We have also noted that the serum content of retinol is temporarily reduced following dosing with RA, but not with NHERA.

Disposition of decabromobiphenyl ether in rats dosed intravenously or by feeding.

The disposition of 14C-labeled decabromobiphenyl ether (DBBE) in male Fischer rats dosed by feeding (0.025-5.0% of the diet) or intravenously (1 mg/kg) was determined. For rats dosed by feeding, intestinal absorption of DBBE was evident in that the intact compound was present in extracts of liver. For these rats, the size of the liver increased with increasing concentration of DBBE in the diet. Liver contained a maximum of 0.449% of the administered radioactivity at 24 h after feeding rats a diet containing 0.0277% [14C]DBBE; no other organ or tissue contained more than 0.26%. The total amount of radioactivity found in tissues was less than 1% of the dose. Of the radioactivity recovered in the feeding experiments, more than 99% was in the feces and gut contents at 72 h; a maximum of 0.012% of the dose was in the urine. In the feces of rats fed [14C]DBBE, there were three metabolites, which together comprised 1.5-27.9% of the radioactivity. Since absorption was minimal, most of the metabolism of [14C]DBBE apparently took place in the gastrointestinal tract. The metabolites increased in percent of total radioactivity with the content of DBBE in the diet, an indication that enzyme induction in intestinal bacteria may have occurred at the higher doses. More extensive metabolism of [14C]DBBE occurred after intravenous administration; only 37% of the radioactivity in the feces was unchanged DBBE. At 72 h after dosing, fecal excretion accounted for 70% of the dose; only 0.129% appeared in the urine. Muscle retained 12.9% and skin 7.25% of the radioactivity administered. In 4 h, rats with biliary cannulas excreted in the bile 7.17% of the intravenously administered radioactivity; less than 1% was excreted as intact DBBE. Biliary excretion was apparently the major route for elimination of the intravenously administered compound. The rapid excretion and extensive metabolism of DBBE, relative to other polyhalogenated compounds, are advantageous properties that may allow it to be used in place of structurally similar compounds presently employed in industrial applications.

Disposition of sucrose octa-isobutyrate in rats, dogs and monkeys.

The disposition of 200 mg/kg of 14C-labelled sucrose octa-isobutyrate (14C-SOIB), a component of sucrose acetate isobutyrate (SAIB), a beverage emulsion stabilizer, was studied in rats, dogs and monkeys. After oral administration of 14C-SOIB to three rats, 3-15% of the dose was excreted as volatile products, 1-2% appeared in urine and 78-93% was recovered in faeces. In dogs, recoveries of radiolabel in CO2, urine and faeces were approximately 1%, less than 2% and 77-94%, respectively. Monkeys excreted the majority of the dose in faeces; less than 2% of the administered radioactivity was eliminated in either CO2 or urine. The biliary excretion of radiolabel from 14C-SOIB was negligible in rats and monkeys; however, in dogs, 3-10% of the dose was excreted into bile. It was demonstrated by chromatographic analyses of faeces that 14C-SOIB was more extensively hydrolysed in the gastro-intestinal tract of rats and dogs than in monkeys. The results indicate that after oral administration, rats and dogs absorb SOIB following hydrolysis of the sugar ester in the gut. The proportion of the dose that is absorbed by the rat is oxidized to CO2. In the dog, little of the absorbed product is oxidized; rather, it is circulated through an enterohepatic pathway. In contrast, in the monkey, SOIB is not detectably hydrolysed in the gut or absorbed. These findings show that there is a species difference in the disposition of SOIB; the most salient findings relate to a difference in the disposition of SOIB in the dog compared with the rat.

Disposition of 2-hydroxy-4-methoxybenzophenone in rats dosed orally, intravenously, or topically.

Administration to rats of oral doses of [14C]-2-hydroxy-4-methoxybenzophenone (HMB) in the range of 3.01-2570 mg/kg revealed that a dose-dependent elimination process was operative at the highest dose. Urinary excretion (63.9-72.9% of the dose in 72 h) was the major route for elimination of radioactivity. An intravenous dose (4.63 mg/kg) distributed rapidly throughout the body of rats and appeared in the urine in an amount (67.4%) similar to those for the oral doses. Rats absorbed large portions of doses of [14C]HMB administered topically, either as an ethanolic solution (50, 200, or 800 micrograms/rat) or formulated in a lotion (50 micrograms/rat). For rats with biliary cannulas, 36.6% of the radioactivity of an intravenous dose (4.46 mg/kg) appeared in the bile in 4 h; the initial half-life for biliary elimination was 40 min. In the bile, at least five radioactive components, none of which was intact HMB, were present. The two major components were glucuronides of HMB and demethylated HMB, and a third was probably a sulfate ester of hydroxylated HMB. In urine, there were nine radioactive components, two of which were unchanged HMB and its glucuronide.

Pharmacokinetic and metabolism studies of two novel 1-deaza-7,8-dihydropteridines in mice.

The disposition of two 1-deaza-7,8-dihydropteridines, NSC 269416 and NSC 350386, was studied in mice dosed iv. After dosing with 25 mg/kg of NSC 269416, serum levels fell in two phases with half-lives of 3.1 and 32 min. Highest levels were in liver, kidney, spleen, lungs, and small intestines; little compound was detected in brain, muscle, or fat. Although no metabolites were detected in serum or tissues, four metabolites, but no parent compound, were present in urine. After administration of 7 mg/kg of NSC 350386, serum levels fell with apparent half-lives of 4.3 (alpha-phase) and 49 min (beta-phase). At most times of analysis, liver, kidney, spleen, lung, brain, fat, and small intestines had similar concentrations of the compound. In 24 hr, less than 5% of the dose was excreted into urine unchanged. Analyses of collected urinary products indicated that, in vivo, NSC 350386 was metabolically hydroxylated and conjugated with glucuronic acid and also cleaved, a process involving removal of the ethoxycarbonyl group.

Effect of long-term administration of retinoids on rats exposed transplacentally to ethylnitrosourea.

A neurogenic cancer model, involving transplacental administration of ethylnitrosourea (ENU) to Sprague-Dawley rats, was employed to evaluate the efficacy of retinyl acetate, 13-cis-retinoic acid, and all-trans-retinoic acid in prevention of nervous system tumors in the offspring. Supplementation of the diet with either of these retinoids did not alter the incidence, number, or latency period of the induced neurogenic tumors. Long-term administration of high doses of 13-cis-retinoic acid (240 mg/kg of diet) or all-trans-retinoic acid (65 mg/kg of diet) produced lethal toxicity in this strain of rats, possibly due to interference with vitamin K absorption and the resulting internal hemorrhages associated with hypoprothrombinemia. Prolonged feeding of retinyl acetate increased the retinyl palmitate level in the liver. The concentration reached was not dose-dependent; a maximum level (approximately 10-fold that of controls) was observed after six months of feeding. An unexpected observation was the decrease in liver retinyl palmitate concentration in the livers of rats fed 13-cis-or all-transretinoic acid.

Disposition of 9-aminoacridine in rats dosed orally or intravenously and in monkeys dosed topically.

Following administration of [14C]-labeled 9-aminoacridine ([14C]9AA) hydrochloride either orally or intravenously to rats, the excretion of radioactivity was similar, with 20-26% of the dose appearing in the urine and 57-68% in the feces. The pattern of tissue distribution was also similar for the two routes. This information suggests that absorption of the oral doses was extensive and that, for both routes of administration, biliary excretion accounted for most of the radioactivity in the feces. Biliary excretion of radioactivity derived from [14C]9AA was confirmed in an experiment involving rats with inserted biliary cannulas. For these rats, 49.5% of the dose administered appeared in the bile in 4 h. The major urinary and biliary metabolite of [14C]9AA of rats was identified as an O-beta-glucuronide of hydroxylated 9AA. Absorption of 9AA through the skin could not be conclusively demonstrated. For monkeys dosed topically with [14C]9AA, only small amounts of radioactivity (a total of less than 0.8% of the dose) appeared in the urine and various tissues in 24 h.

Effects of phenobarbital, 3-methylcholanthrene, and retinoid pretreatment on disposition of orally administered retinoids in mice.

Following oral administration of doses of 10 mg/kg, the concentrations of all-trans-retinoic acid (RA), N-(2-hydroxyethyl)retinamide (N-HOERA), and 13-cis-retinoic acid (13-cis-RA) were measured in serum and tissues of mice with and without pretreatment with phenobarbital (PB), 3-methylcholanthrene (3MC), or the respective retinoid. For RA, the areas under the concentration-time curves (AUC values) for serum and tissues, relative to controls, were reduced, on the average, by 54 or 37% on pretreatment with RA or PB, respectively. Pretreatment with 3MC did not affect disposition of RA. The AUC values for N-HOERA were reduced, on the average, by 39 or 30% following pretreatment with PB or 3MC, respectively, but only 13% by the retinoid. For 13-cis-RA, the AUC values were reduced, on the average, by 56 or 37% following pretreatment with PB or 3MC, respectively; pretreatment with the retinoid had no appreciable effect. Thus, the disposition of orally administered retinoids appears to be affected by several inducible metabolic processes. Furthermore, comparison of data on the tissue distribution of retinoids with in vivo results demonstrates a general correlation between distribution and anticarcinogenic activity.

Disposition of 2-mercaptobenzimidazole in rats dosed orally or intravenously.

The disposition of [14C]-labeled 2-mercaptobenzimidazole (MBI) in male Fischer-344 rats dosed orally (49 or 0.5 mg/kg) or intravenously (0.5 mg/kg) was determined. Absorption of the oral dose was evident, since, in 72 h, most of the radioactivity administered by either route appeared in the urine. Smaller amounts appeared in the feces. In 4 h, 12% of the radioactivity from an intravenous dose of 0.5 mg/kg was excreted in the bile of rats with biliary cannulas. For rats dosed intravenously, the half-life for disappearance of unchanged MBI from plasma was 125 min. In contrast, the terminal half-life for loss of radioactivity from blood was 83 h. The concentration of total radioactivity was higher in liver and kidney tissue than in blood. One of the major urinary metabolites was identified as benzimidazole, and a minor component was tentatively identified as unchanged MBI. Neither of these could be detected in bile.

Disposition of 13-cis-retinoic acid and N-(2-hydroxyethyl)retinamide in mice after oral doses.

The concentrations of 13-cis-retinoic acid and N-(2-hydroxyethyl)retinamide in serum and tissues from mice were measured after oral dosing with 10 mg/kg. Serum levels of the drugs reached their maxima within 15-30 min and then declined in an exponential fashion with t1/2 values of 19 min and 2.9 hr, respectively. Tissue levels of 13-cis-retinoic acid reached their maxima within 15-30 min, then declined exponentially with t1/2 values of 11-19 min. Tissue levels of N-(2-hydroxyethyl)retinamide reached their maxima within 30 min to 2 hr, then declined exponentially with t1/2 values of 2.1-4.7 hr. Longer t1/2 values were observed for both 13-cis-retinoic acid and N-(2-hydroxyethyl)retinamide in the bladder (51 min and 7.3 hr, respectively). Only small amounts of the unchanged retinoids were observed in bile and feces, and none was found in urine. Polar materials, however, were present in bile, feces, and urine.

Disposition of all-trans-retinoic acid in mice following oral doses.

The concentrations of all-trans-retinoic acid in serum and tissues from mice were measured following oral dosing with 10 mg/kg. Serum levels of the drug reached a maximum within 45 min, then declined after 2 hr in a linear fashion. Tissue levels reached a maximum within 30 to 120 min, then declined after 3 hr in an exponential fashion with t 1/2 values of 25 to 68 min. The longest t 1/2 value was observed in brain. Only small amounts of unchanged all-trans-retinoic acid were observed in bile and feces and none was found in urine. Polar material, however, was present in bile, feces, and urine.

Lipid activation of undecaprenol kinase from Lactobacillus plantarum.

Extraction of membranes of Lactobacillus plantarum with Triton X-100/glycerol solubilized up to 80% of the undecaprenol kinase activity. Fractionation of the extract by gel chromatography separated endogenous phospholipid from the enzyme but simultaneously inactivated the enzyme. The kinase was reactivated by reconstitution with various synthetic phosphatidylcholines and purified L. plantarum phospholipids. Ditetradecanoylphosphatidylcholine and lysylphosphatidylglycerol were the best activators. Furthermore, the optimal environment for enzyme stimulation was provided by different defined molar ratios of Triton X-100/phospholipid. The ratios for the phospholipids tested ranged from 1.25 to 6.3. Similar substrate specificity and kinetic constants were observed for both the solubilized and reconstituted enzymes suggesting that no fundamental changes in the enzyme activity occurred during the delipidation-reconstitution process.

Characterization of undecaprenol kinase from Lactobacillus plantarum.

A membrane-bound undecaprenol kinase from Lactobacillus has been identified by observing the ATP-dependent phosphorylation of [14C]undercaprenol. The product of this reaction was shown to be [14C]undecaprenyl monophosphate by comparison of its chromatographic mobilities with authentic undecaprenyl monophosphate. It was shown that 32P from [gamma-32P]ATP was incorporated into undecaprenyl monophosphate. The kinase was partially solubilized by a variety of methods utilizing Triton X-100. Both the membrane-associated and solubilized enzymes required Mg2+, Triton X-100 and dimethylsulfoxide for activity. The enzyme preferentially phosphorylated the C34, C50 AND C 55 polyprenols. Geranylgeraniol (C20) and dolichol (C100), however, were utilized only 6% and 13% as well as undecaprenol, respectively. Despite the 8-fold difference in apparent V values, the apparent Km values for dolichol and undecaprenol were both 14 microM. The apparent Km for the nucleotide cosubstrate, ATP, was 2 mM. No other nucleoside triphosphate could substitute for ATP.