Characterization of the mutational specificity of DNA cross-linking mutagens by the Lac+ reversion assay with Escherichia coli.

The mutational specificities of DNA cross-linking compounds such as cisplatin, transplatin, carboplatin, mitomycin C, psoralen, and 8-methoxypsoralen were investigated in lacZ reversion assay systems of Escherichia coli. Tester strains were constructed by introducing the six kinds of F' plasmids (lacI-, lacZ461, and proAB+), each of which carries a different base-substitution mutation within the lacZ gene. Each of the six possible base-substitution mutations was assayed by Lac+ reversion. Cisplatin induced G.C-->A.T transitions and G.C-->T.A transversions, with the former predominating. Transplatin induced A.T-->G.C transitions in addition to G.C-->A.T transitions and G.C-->T.A. Carboplatin weakly induced G.C-->A.T transitions. On the other hand, mitomycin C induced only G.C-->T.A transversions, while psoralen and 8-methoxypsoralen reactivated with near-UV irradiation induced A.T-->G.C transitions preferentially. The Lac(+) reversion system was very convenient for rapidly determining mutational spectra.

[Oral single-dose toxicity study of a new antineoplastic agent S-1, and its components, CDHP, and Oxo].

S-1, an antineoplastic formulation of a fluorinated pyrimidine derivative containing tegafur (FT), CDHP, and potassium oxonate (Oxo) in a molar ratio of 1:0.4:1, was recently developed by Taiho Pharmaceutical Co., Ltd., with the aim of prolonging the effective plasma concentration of 5-fluorouracil (5-FU) over that produced by FT alone and reducing its dose-limiting gastrointestinal toxicity. As a part of the S-1 toxicity study, the single-dose toxicity of S-1 as well as that of its components, CDHP and Oxo, was investigated in mice, rats, and dogs. The following results were obtained. 1. In mice and rats, excretion of diarrheal stools, salivation, and alopecia were observed after S-1 administration. In severe cases, the animals subsequently showed emaciation due to weight loss or suppressed weight gain, decreased spontaneous motor activity, an anemic appearance, bradypnea, prone position, and death. In the CDHP and Oxo treatment groups of rats, the only toxic signs were soft or diarrheal stools on the dosing day. 2. In dogs, vomiting and excretion of diarrheal, mucous, or soft stools was observed after S-1 administration. In the CDHP and Oxo treatment groups, excretion of soft and diarrheal stools and vomiting were observed relatively frequently from the dosing day until day 1. 3. In the pathological examination of the animals given S-1, mice and rats showed pulmonary congestion/edema, dark red discoloration of the mesenteric lymph nodes, atrophy of lymphatic tissues such as the thymus and lymph nodes, decreases of lymphocytes in the splenic white pulp and mesenteric lymph nodes, a decrease in bone marrow cells, congestion of the glandular stomach, and aggregates of bacteria in the lung, liver, or spleen. In dogs, abnormal changes were observed mainly in the lymphatic organs such as the thymus and lymph nodes. 4. The LD50 values of S-1 in terms of the amount FT they contained were estimated to be 549 mg/kg for mice(male), 441-551 mg/kg for rats (both sexes) and about 53 mg/kg for dogs (male). The LD50 values of CDHP and Oxo were 2000 mg/kg or higher for both rats (both sexes) and dogs (male). 5. Hematopoietic and lymphatic impairments, immunosuppression associated with respiratory were considered to be the cause of death from S-1. The toxicity of S-1 reflects the toxicity of 5-FU and was not found the different toxicity by the addition of CDHP and Oxo.

[A 13-week oral repeated dose toxicity study of a new antineoplastic agent S-1 in rats].

S-1, an antineoplastic formulation of a fluorinated pyrimidine derivative containing tegafur (FT), CDHP, and potassium oxonate (Oxo) in a molar ratio of 1:0.4:1, was recently developed by Taiho Pharmaceutical Co., Ltd., with the aim of prolonging the effective plasma concentration of 5-fluorouracil (5-FU) over that produced by FT alone and reducing its dose-limiting gastrointestinal toxicity. As a part of the S-1 toxicity study, a 13-week oral repeated dose toxicity study and a recovery study using male and female rats was conducted. Doses of S-1 were adjusted to deliver 1.5, 5, and 15 mg/kg/day as doses of FT, and FT was given at 15 mg/kg/day. The following results were obtained. 1. In clinical observation, edema of the limbs and face or swelling of the auricle of the ear and an anemic appearance were observed in both sexes in the 15 mg/kg/day group as dose of FT. Subsequently, males in this group developed severe anemia, decreased spontaneous motor activity, emaciation, and subnormal skin temperature, and many males died. In the survivors, keratosis of the palm, sole, or tail was observed, with necrosis and loss of the tail tip in the severe cases. 2. Body weight gain was suppressed from about week 2 of treatment in both sexes in the 15 mg/kg/day group as dose of FT, and there was almost no weight gain after week 4-5. Food consumption was consistently less than the control value for males in the 15 mg/kg/day group as dose of FT throughout the treatment period. 3. No marked changes were observed in water intake and on opthalmologic examination. 4. In the fecal test for occult blood, a positive tendency was observed in both sexes in the 15 mg/kg/day group as dose of FT. 5. Urinalysis disclosed a slight increase in protein and decrease in sodium, potassium, and chloride in males, and an increase in protein in females in the 15 mg/kg/day group as dose of FT. 6. Hematologically, both sexes in the 15 mg/kg/day group as dose of FT showed decreases in red blood cell count, hemoglobin, and hematocrit, and increases in platelet count and fibrinogen, with a slight decrease in white blood cell count in males. 7. In the blood biochemical test, abnormal findings included increases in total cholesterol and free cholesterol, and decreases in non-esterified fatty acid and albumin in both sexes in the 15 mg/kg/day group as dose of FT. 8. In organ weight measurement, abnormal changes included a decrease in thymus weight in both sexes in the 5 mg/kg/day or higher dosage groups and a decrease in the testis weight in males and an increase in the liver weight in females in the 15 mg/kg/day group as dose of FT. 9. Histopathologically, both sexes in the 15 mg/kg/day group as dose of FT showed a decrease in the red pulp of the bone marrow, atrophy of the thymus, white pulp of the spleen, and testes. degeneration of the renal tubules, and ulcerative changes of the skin or oral mucosa. 10. The findings were unremarkable in the FT group. 11. During the recovery study, all the toxic effects tended to reverse. 12. The NOAEL of S-1 was estimated to be 1.5 mg/kg/day as dose of FT for both sexes.

[A 26-week oral repeated dose toxicity study of a new antineoplastic agent S-1 in rats].

S-1 was administered to male and female rats by gavage for 26 weeks at 0, 1, 5, and 10 mg/kg/day followed by 5-week recovery period for the control, 5, and 10 mg/kg/day groups. Treatment at 5 or 10 mg/kg/day in both sexes produced keratosis of the tail, palm or sole. Weight gain and average food consumption were lowered by the treatment. Urine showed increases in protein and epithelial or white blood cells and a decrease in specific gravity. The blood showed decreases in red blood cell count, hemoglobin, and hematocrit as well as increases in MCH, platelet count, fibrinogen, and MCV. A/G ratio, albumin, and chloride were decreased while total cholesterol, free cholesterol, triglycerides, and phospholipids were increased. Histopathologically, treatment-related changes at 5 and 10 mg/kg/day were observed mainly in the lymphoid tissues and kidneys. Those changes included atrophy in the lymphoid tissues and chronic nephropathy-like changes in the kidneys. Other changes in the 10 mg/ kg/day group, included acanthosis and/or inflammation in the epidermis of the tail, sole, or palm, degeneration and disarrangement of ameloblasts, and atrophy of the testes. In a recovery study, although some changes in the sole, palm, or tail, and the kidneys remained, they were less extensive than they had been at the end of the treatment period. Based upon these observations, the non-toxic dose level was estimated to be 1 mg/kg/day (2.3 mg/kg/day, as the summed doses of tegafur, CDHP, and Oxo) in both sexes.

Negative inotropic action of denbufylline through interfering with the calcium channel independently of its PDE IV inhibitory activity in guinea pig ventricle papillary muscles.

The inotropic actions of xanthine derivatives with long alkyl chains were investigated in guinea pig ventricular papillary muscle. A potent and nonselective phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine, elicited a positive inotropy and inhibited the negative inotropic effects of calcium channel inhibitors, as did a selective PDE III inhibitor, amrinone, and these effects were canceled by a protein kinase inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89). However, 1,3-di-n-butyl-7-(2'oxopropyl)xanthine (denbufylline) and 1-n-butyl-3-n-propylxanthine (XT-044), which have potent and selective PDE IV-inhibitory activities, showed negative inotropic actions that became more potent in the presence of H-89. Denbufylline abolished the late restoration phase induced by ryanodine. This xanthine derivative attenuated the effects of both the calcium channel acting agents Bay K 8644 and verapamil, without interaction with caffeine and dihydropyridine calcium channel inhibitors, and denbufylline had little direct influence on the specific binding of [(3)H]azidopine and [(3)H]desmethoxyverapamil to cardiac membranes. A nonxanthine PDE IV inhibitor, Ro 20-1724, did not affect the inotropic actions of calcium channel inhibitors. The attenuation by denbufylline or XT-044 of the negative inotropic action of verapamil was not influenced by treatment with H-89. These results suggest that in the ventricular papillary muscle, these xanthine derivatives elicit negative inotropy by acting on a verapamil-sensitive site of the calcium channel without involving their PDE-inhibitory activity.

A xanthine derivative denbufylline inhibits negative inotropic response to verapamil in guinea pig ventricular papillary muscles, independent of its phosphodiesterase inhibitory activity.

A phosphodiesterase (PDE) III inhibitor, amrinone, inhibited both the negative inotropic actions of verapamil and nicardipine in guinea pig ventricular papillary muscle; this effect was canceled by the protein kinase A inhibitor H-89. The PDE IV inhibitor 1,3-di-n-butyl-7-(2'-oxopropyl)xanthine (denbufylline), which elicited a negative inotropic action by itself, attenuated the action of verapamil up to 10 microM, without any interaction with nicardipine. The attenuation by denbufylline was not influenced by H-89. This suggests that in the ventricular papillary muscle, denbufylline acts on some verapamil-sensitive site(s) in the membrane and interferes with the calcium channel function without involvement of its PDE inhibitory activity.

Structure-activity relationships of alkylxanthines: alkyl chain elongation at the N1- or N7-position decreases cardiotonic activity in the isolated guinea pig heart.

Relationships between the alkyl substitutions (C1-C6) and cardiac inotropic activities of xanthine derivatives were studied in isolated guinea pig heart muscles. Most of the alkylxanthines exhibited positive inotropic activity on the left atrium, which was increased with an elongation of alkyl chain at the N3-position but decreased by substitution of a long alkyl group at the N1- or N7-position of the xanthine skeleton. Although positive inotropic activity in the right ventricular papillary muscle was also increased by longer alkyl groups at the N3-position, the inotropic activity became negative with an increment in alkyl chain length at the N1- or N7-position. The positive inotropic activity of alkylxanthines was correlated with their inhibitory activity on the phosphodiesterase (PDE) III isoenzyme. Adenosine A1 antagonism and PDE IV inhibitory activity were also partly associated with the inotropic activity because H-89, an inhibitor of cyclic AMP-dependent protein kinase, diminished the positive inotropic action and potentiated the negative inotropic action. These results indicate that the positive inotropic activity of alkylxanthines becomes weak with elongation of alkyl chains at the N1- and N7-positions; In particular, xanthines having two long alkyl chains show a negative inotropic activity on the right ventricular papillary muscle, an effect that could not be elucidated from their cyclic AMP-dependent action.

Effects of alkyl substituents of xanthine on phosphodiesterase isoenzymes.

The structure-activity relationships of a series of alkylxanthine derivatives were investigated. The partition coefficient of alkylxanthines enlarged with an elongation of the alkyl chain at the 1-, 3-, or 7-position of xanthine. There was a mild correlation between the apparent partition coefficient and the tracheal relaxant activity or the inhibitory activity on phosphodiesterase (PDE) IV isoenzyme, while the tracheal relaxant activity closely correlated with the PDE IV inhibitory activity. Regarding substituents at different positions, the alkylation at the 3-position increased the inhibitory activity on every PDE isoenzyme. The alkylation at the 1-position potentiated the inhibitory activity on PDE IV with the alkyl chain length, but decreased the activities on other PDE isoenzymes. The alkylation at the 7-position was characteristic in its decrease in inhibitory activity on PDE III. These results suggested that the potency of the inhibitory activity of xanthine derivatives on PDE isoenzymes is not dependent simply upon their hydrophobicity but upon change in the affinity for the active sites on PDE isoenzymes by the introduction of the alkyl group at particular positions of the xanthine skeleton.

Selective tracheal relaxation and phosphodiesterase-IV inhibition by xanthine derivatives.

The effects of substitutions in the xanthine nucleus on tracheal relaxant activity, atrium chronotropic activity, adenosine A1 affinity, and inhibitory activities on cyclic AMP-phosphodiesterase isoenzymes in guinea pigs were studied. Substitution with a long alkyl chain at the N1-position of xanthine nucleus increased the tracheal relaxant activity without leading to positive chronotropic action, and long alkyl chains at the N3-position increased both activities. N7-substitutions with n-propyl and 2'-oxopropyl groups, such as in denbufylline, increased bronchoselectivity. N7-substitution decreased the adenosine A1 affinity, but substitution at either the N1- or N3-position increased it. The bronchorelaxant activity of xanthine derivatives was closely correlated with their inhibition of phosphodiesterase-IV, but not with their adenosine A1 affinity; the positive chronotropic effects were related to their inhibition of phosphodiesterase-III. This study confirms that the bronchorelaxation of xanthine derivatives is mediated by inhibition of the isoenzyme phosphodiesterase-IV. The results of structure-activity analysis suggest that substitutions at the N1- and N7-positions should be tried in the development of xanthine derivatives that are selective bronchodilators and phosphodiesterase-IV inhibitors.