Inhibition of phorbol ester-induced tumor promotion in mice by vitamin A analog and anti-inflammatory steroid.

The effects of a vitamin A analog, TMMP ethyl retinoate [or ethyl-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl-trans-2,4,6,8-nonatetraenoate] (abbreviated Ro 10-9359), and an anti-inflammatory steroid, fluocinolone acetonide (or 6 alpha, 9 alpha-difluoro-11 beta, 16 alpha, 17,21-tetrahydroxypregna-1,4-diene-3,20-dione cyclic 16,17-acetal) (abbreviated FA), given alone or together were studied in a two-stage carcinogensis system. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was used as the tumor promoter in a 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mouse skin system. Two stocks of female mice, CD-1 and Sencar, which differ in their degrees of sensitivity to skin carcinogenesis, were used. A dose-dependent inhibition of carcinogenic expression, as determined by a decreased number of papillomas per animal, was observed in each mouse stock with the use of both FA and Ro 10-9359 when given alone. When FA and Ro 10-9359 were given together, an enhanced effect on the lowering of tumor incidence was noted. FA effectively inhibited tumor formation in the sensitive mouse stock even when the steroid was given 1 day prior to TPA treatment under conditions of unusually high doses of initiator (DMBA) and/or promoter (TPA). These results suggest that both anti-inflammatory steroids and retinoids inhibit tumor promotion and can be effectively used as a combination regimen for increased chemopreventive response.

Inhibition of murine tumor growth by an interferon-inducing imidazoquinolinamine.

The low-molecular-weight imidazoquinolinamine derivative, 1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine (imiquimod, previously described as R-837), induced alpha-interferon (IFN-alpha) in mice. IFN induction was identified at oral doses as low as 3 mg/kg. The 10% lethal dose for daily treatment with imiquimod was 200 mg/kg. Oral treatment with 30 mg/kg imiquimod once every three days significantly inhibited MC-26 colon carcinoma. Delay of treatment from day 1 to day 5, when tumors were easily palpable, did not reduce benefits. Ten daily treatments were slightly more effective than five. However, delivery of the same total dose of imiquimod either once every day for 20 days, once every 4 days, once every 7 days, or once every 10 days inhibited tumor growth to the same level. The antitumor effects of imiquimod were significantly abrogated by an antiserum to murine IFN-alpha, suggesting that the antitumor effect was to a substantial extent mediated by IFN induction. Imiquimod also significantly reduced the number of lung colonies in mice inoculated i.v. with MC-26 tumor cells. Combination of treatment with imiquimod and cyclophosphamide was significantly (P less than 0.01) better than treatment with either drug alone. Combination treatment with cyclophosphamide led to cures in some of the mice inoculated either s.c. or i.v. with MC-26 cells. Treatment with imiquimod also inhibited the growth of RIF-1 sarcoma and Lewis lung carcinoma but was ineffective for P388 leukemia. Imiquimod is an oral IFN-alpha inducer with antitumor effectiveness for transplantable murine tumors.

Cytokine induction in mice by the immunomodulator imiquimod.

Imiquimod has been identified as a potent antiviral and antitumor agent in animal models. The biological activity associated with imiquimod has been attributed to its induction of interferon (IFN)-alpha. The present studies evaluated imiquimod administered orally for its ability to stimulate production of IFN and other cytokines in mice. The cytokine profile induced by imiquimod was compared with other known immunomodulators. Imiquimod was found to stimulate increased serum IFN in mice. Daily dosing of imiquimod for five consecutive days led to diminished production of IFN in mice as measured after the final dose. Elevated levels of serum tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 but not IL-1 alpha were found in serum from mice treated with imiquimod. Imiquimod produced significantly higher levels of IFN but lower levels of TNF and IL-6 and IL-1 alpha than lipopolysaccharide. Polyinosinic acid:polycytidylic acid induced significantly higher amounts of IFN but lower levels of TNF and IL-6 than imiquimod. Imiquimod stimulated significantly higher levels of IFN when compared with 2-amino-5-bromo-6-phenyl-4(3H)-pyrimidinone (ABPP) and similar levels of IFN when compared with tilorone. Neither ABPP nor tilorone induced TNF or IL-6. Finally, imiquimod stimulated TNF, IFN, and IL-6 production in cultures of mouse spleen and bone marrow cells. These studies demonstrate that imiquimod induces not only IFN but other cytokines as well, all of which may contribute to its biological activity.

Inhibitors of polyamine biosynthesis. 3. (+/-)-5-Amino-2-hydrazine-2-methylpentanoic acid, an inhibitor of ornithine decarboxylase.

(+/-)-5-Amino-2-hydrazino-2-methylpentanoic acid [alpha-hydrazino-alpha-methyl-(+/-)-ornithine] was obtained from 1-phthalimidopentan-4-one by treatment with hydrazine and KCN followed by acid hydrolysis. The title compound was found in vitro to be a potent competitive inhibitor of ornithine decarboxylase obtained from the prostate glands of rats. This inhibition was abolished at high concentrations of pyridoxal phosphate. The title compound also blocked the increase in putrescine levels normally observed in bovine lymphocytes transformed by conconavalin A.

Potential inhibitors of polyamine biosynthesis. 2. alpha-Alkyl- and benzyl-(+/-)-ornithine.

Alpha-Methyl-(+/-)-ornithine hydrochloride was not a substrate for ornithine decarboxylase from rat prostate glands. It produced equal inhibition of ornithine decarboxylase obtained from rat prostate glands, spleens of mice inoculated with L1210 leukemic cells, and regenerating rat liver indicating its lack of selectivity for any of these tissues. In these three tissues the inhibition was competitive with L-ornithine. A number of alpha-alkyl- and alpha-aralkyl-substituted analogs of (+/-)-ornithine were synthesized and evaluated in vitro as inhibitors of the enzyme L-ornithine decarboxylase obtained from prostate glands of rats. These compounds were obtained by the reaction of alkyl iodide or benzyl bromide with the anion obtained by treatment of 3-(benzalimino)piperidin-2-one with sodium hydride. The following alpha-substituted analogs of (+/-)-ornithine were obtained: ethyl, n-propyl, n-butyl, n-hexyl, n-octyl, and benzyl. The synthesized compounds were found to be much less active than alpha-methyl-(+/-)-ornithine as competitive inhibitors of ornithine decarboxylase in vitro. The most active compound in the series was alpha-n-octyl-(+/-)-ornithine which was 60-fold less active than alpha-methyl-(+/-)-ornithine and the least active analog was alpha-n-butyl-(+/-)-ornithine which was 270-fold less active than the alpha-methyl-(+/-)-ornithine.

The effects of weak or non-carcinogenic polycyclic hydrocarbons on 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene skin tumor-initiation.

Benzo[e]pyrene (B[e]P) inhibited 7,12-dimethylbenz[a]anthracene (DMBA) skin tumor-initiation in mice by 84%, whereas pyrene and fluoranthene inhibited DMBA initiation by 50 and 34%, respectively. However, B[e]P, pyrene and fluoranthene had either no significant effect or a slight enhancing effect on benzo[a]pyrene (B[a]P) skin tumor-initiation. In addition, B[e]P had essentially no effect on the initiating ability of (+/-)B[a]P-7 beta,8 alpha-diol-9 alpha,10 alpha-epoxide. As a tumor-initiator, B[e]P was found to have very weak activity at a 252 microgram/level (0.4 papillomas/mouse at 40 weeks) and no activity at 100 microgram. When given at a dose of 100 microgram twice weekly, B[e]P induced 2.1 papillomas/mouse at 30 weeks, and 25% of the mice had carcinomas at 40 weeks. However, B[e]P carcinogenic activity is weak when compared to B[a]P, which can induce a comparable tumor response at a dose of 5 microgram twice weekly. When B[e]P was tested as a tumor promoter at a dose of 100 microgram twice weekly after DMBA initiation, it induced 4.5 papillomas/mouse at 30 weeks and a 45% carcinoma incidence at 40 weeks, which was approximately twice as effective as B[e]P alone. The data show that B[e]P is a very weak tumor initiator, a weak complete carcinogen, a moderate tumor promoter, possibly a weak co-tumor-initiator when given with B[a]P, and a potent anit-tumor-initiator when given with DMBA. The anti-tumor initiating and co-tumor-initiating effects of B[e]P appear to be related to its ability to modify the conversion of the tumor initiator into an electrophilic intermediate(s) which are capable of covalently binding to DNA. In addition, B[e]P induced epidermal cellular proliferation which may be related to its promoting ability.

Oral flecainide pharmacokinetics in patients with impaired renal function.

The pharmacokinetics of flecainide acetate were studied in 20 patients with varying degrees of renal impairment following a single oral dose. The patients were divided into two groups, on the basis of renal creatinine clearance (CLCR), for statistical and kinetic analysis. Patients with a CLCR between 4 and 41 mL/min/m2 were designated group 1 and those below 4 mL/min/m2 or unmeasurable because of lack of urine output were designated group 2. In both groups peak plasma flecainide concentrations, time to peak concentrations, and apparent volume of distribution (Vd) were similar to those reported in healthy subjects with normal renal function. The mean flecainide plasma elimination half-lives from both groups 1 and 2 were longer than those previously reported by several investigators in normal subjects. Nine patients in group 1 and seven patients in group 2 had half-lives within the range reported in healthy subjects. Therefore, CLCR alone is not a good predictor of plasma elimination half-life following a single oral dose of flecainide. Although renal clearance of flecainide is significantly reduced in end-stage renal disease (ESRD), total plasma clearance of flecainide (CLflec) was not reduced to the same degree, although there was a significant, modest correlation with CLCR. Less than 1% of the administered oral dose of flecainide was removed during hemodialysis. The relationship between dosage and plasma elimination half-life in patients with ESRD needs further study to evaluate possible dose-dependent kinetics.

The effect of flecainide acetate, a new antiarrhythmic, on plasma digoxin levels.

The possible effect of oral flecainide acetate on steady-state digoxin levels was assessed in 15 healthy men. Each volunteer received digoxin 0.25 mg daily (8 AM) for 22 consecutive days and flecainide 200 mg bid (8 AM and 8 PM) on days 11 through 15. Plasma digoxin and flecainide levels were measured by radioimmunoassay and gas-liquid chromatography methods, respectively. Flecainide levels were within the range associated with suppression of premature ventricular contractions in patients. Mean plasma digoxin levels just before the 8 AM dose were 0.46 ng/mL on days 9 and 10 (baseline), 0.57 ng/mL (P less than .05) on day 13, and 0.49 ng/mL (not significant [NS]) on day 15. Compared with a mean six-hour postdose baseline digoxin level of 0.58 ng/mL, postdose levels were 0.62 ng/mL (NS) and 0.65 ng/mL (P less than .05) on days 13 and 15, respectively. On an average for each subject, predose and six-hour postdose digoxin levels increased by 24 +/- 35% and 13 +/- 19%, respectively, during coadministration. The changes in electrocardiographic intervals and vital signs that occurred during concomitant drug administration were not clinically significant although a slight prolongation of the PR interval was noted in some subjects. Unless plasma digoxin levels are in the upper end of the therapeutic range, changes in magnitude as observed in this study should be clinically inconsequential for most patients.

Increased production of TNF-alpha and decreased levels of dienoic eicosanoids, IL-6 and IL-10 in mice fed menhaden oil and juniper oil diets in response to an intraperitoneal lethal dose of LPS.

Eicosapentaenoic acid (EPA) and the non-methylene interrupted fatty acids (NMIFA) displace arachidonic acid (AA: 20:4omega6 -5,8,11,14) in the membrane phospholipids. Unlike EPA (20:5omega3 -5,8,11,14,17), the NMIFA (20:3omega6 -5,11,14 and 20:4omega3 -5,11,14,17) lacking the delta-8 double bond are not substrates for the formation of eicosanoids. For 20 days, the mice were fed diets containing 5wt% dietary fats from various sources. The magnitudes in the production of eicosanoids and cytokines produced in response to an intraperitoneal injection of endotoxin in mice fed menhaden fish oil (MO) diets enriched with EPA were compared with those maintained on juniper oil (JO) containing NMIFA or on safflower oil (SO), a major source of the AA precursor, linoleic acid. The levels of PGE2, 6-keto-PGF1alpha and TXB2 were markedly lower (P < 0.01) in animals fed either MO or JO diets compared to the controls. The plasma levels of tumor necrosis factor (TNF)-alpha were significantly higher (P < 0.05) with a concomitant decrease of interleukin (IL)-6 and of IL-10 in mice fed MO or JO diets (P < 0.01) compared to those fed SO diet. These data suggest that the effects of consuming NMIFA of JO despite their inability to form eicosanoids are similar to those of feeding EPA which forms biologically active alternate metabolites.

Effect of weak-, non-, and co-carcinogenic chemicals on 2-acetylaminofluorene-induced mutation in Salmonella typhimurium.

The effects of various weak, non-, and co-carcinogenic chemicals on 2-acetylaminofluorene-induced mutation in Salmonella typhimurium were studied. We found that a single co-mutagen could provide for either enhanced or decreased mutagenesis. A differential effect on mutagenic expression was dependent upon: (1) the type of inducer of S-9 (supernatant from liver homogenate centrifuged at 9000 x g) liver enzymes; (2) the amount of S-9 enzyme preparation employed; and (3) the combined dose of mutagen plus co-mutagen studied. No effect was observed in the absence of S-9. Our data suggest that in S. typhimurium a primary factor in the alteration of mutagenesis by a combination of chemicals is changes in metabolism of the principal mutagen.

Induction of interferon and other cytokines by imiquimod and its hydroxylated metabolite R-842 in human blood cells in vitro.

The low-molecular-weight immunomodulator drug candidate, imiquimod (R-837), and its hydroxylated metabolite R-842, induce interferon-alpha (IFN-alpha) in human blood cells in vitro when tested in concentrations of 0.5 microgram/ml or more. The amounts of IFN-alpha found increased with time from 2-6 h of incubation up to 24-48 h, and were dependent on cell number and drug concentration. These two chemicals yielded more IFN-alpha in human peripheral blood mononuclear cell (PBMC) cultures than other known inducers tested in parallel. They also induced detectable amounts of interleukin (IL)-1, IL-6, IL-8, and tumor necrosis factor-alpha in human PBMC cultures in vitro.

An acute oral gavage study of 3beta-acetoxyandrost- 5-ene-7,17-dione (7-oxo-DHEA-acetate) in rats.

The present study was done to assess the tolerance of rats for 3-acetoxyandrost-5-ene-7,17-dione (7-oxo-DHEA-acetate, 7-ODA) when administered as a single oral gavage dose. Five groups of Sprague-Dawley rats (Crl:CD (SD) BR VAF/Plus) (five/sex/group) were treated with 7-ODA at a dose level of 0 (control), 250, 500, 1000, or 2,000 mg/kg of body weight in a dose volume of 10 ml/kg. Food and water were provided ad libitum. All animals survived in good health to the scheduled sacrifice on Day 15. The single oral administration of 7-ODA had no apparent effects on body weight. Food consumption was significantly higher for all female treated groups during week two; however, the statistically significant differences were not considered to be of clinical consequence. Treatment caused no apparent changes of gross or microscopic anatomical structures of nine different organs. This study demonstrated that the no-observable adverse effect level for a single oral dose of 7-ODA in male and female rats was 2,000 mg/kg.

An escalating dose oral gavage study of 3beta-acetoxyandrost-5-ene-7, 17-dione (7-oxo-DHEA-acetate) in rhesus monkeys.

To test the effects of 7-oxo-dehydroepiandrosterone-3 acetate (hereafter 7-ODA) in Rhesus macaques the steroid was administered by oral gavage to two male and two female monkeys. Dose levels of 250, 500, and 1,000 mg/kg body weight (BW)/day were administered on days 1, 3, and 5 respectively, and 1,000 mg/kg on days 7 through 11. Each group received the dose in a volume of 10 ml/kg BW. All animals survived to the scheduled sacrifice on day 12. No adverse clinical effects of 7-ODA were observed at the 250 or 500 mg/kg doses. Females vomited on non-treatment days and all animals vomited on some days after being given the 1000 mg/kg dose. Excessive salivation was observed before or immediately after dosing on days 9 through 11. Appearance, behavior and body weights were not altered by the treatments. Visual examination of all body cavities, and macroscopic and microscopic examination of 42 different organs and tissues found no lesions or abnormalities.

Assay of thiamine in foods, using manual and semiautomated fluorometric and microbiological methods.

Three methods for the determination of thiamine in foods were evaluated for accuracy, recovery, and precision: a manual fluorescent method, a semiautomated fluorescent method, and a Lactobacillus viridescens microbiological assay. Thiamine in the samples was destroyed with clam tissue thiaminase; a known amount of thiamine hydrochloride was then added to the extract; and the thiamine recovery was determined. For 14 commerically processed food products analyzed by the manual and semiautomated methods, the mean per cent recovery values and standard deviations were 91.2 +/- 8.92 and 99.3 +/- 3.13%, respectively. Eight of these products were analyzed by all 3 methods. The mean per cent recoveries and standard deviations for these 8 samples were 90.7 +/- 8.97, 101 +/- 2.52, and 99.9 +/- 1.03%, respectively, for the manual, semiautomated, and microbiological methods. The microbiological method with L. viridescens gave the best results for the products tested. The concentration of vitamin which can be measured is such that samples of low label declaration present no problems. The semiautomated method offers a rapid and accurate method of thiamine assay. The chemical reactions are identical to those of the official method. The major difference between the methods is in the sample cleanup. It is postulated that the low recovery observed for the manual method is due to incomplete elution of thiamine in the column purification step.