Synthesis and Photochromism of Crowned Spirobenzothiapyran: Facilitated Photoisomerization by Cooperative Complexation of Crown Ether and Thiophenolate Moieties with Metal Ions.

Spirobenzothiapyrans bearing monoaza-12-crown-4, -15-crown-5, -18-crown-6, and oligooxyethylene moieties were synthesized, and their photochromism was examined in the presence of cations in acetonitrile. The cation complexation by their crown ether moieties cannot induce thermal isomerization to their corresponding colored merocyanine form, unlike the corresponding spirobenzopyran derivatives. The UV-light-induced isomerization was, however, facilitated by the cation complexation of the crown ether moieties and the affinity of the merocyanine thiophenolate anion to metal ions, especially in the presence of Li(+) and Ag(+). The presence of Ag(+) brought about the most remarkable effect in the facilitation of photoisomerization of the spirobenzothiapyrans and the thermal stability of the colored merocyanine form mainly due to the powerful interaction of the thiophenolate anion with the soft metal ion.

Administration-route-related difference in the micronucleus test with 7,12-dimethylbenz[a]anthracene.

The effect of route of administration on the outcome of the mouse micronucleus test was evaluated in 2 laboratories by administering a model chemical, 7,12-dimethylbenz[a]anthracene (DMBA) by intraperitoneal injection (i.p.) and oral gavage administration (p.o.) to males of 2 mouse strains, MS/Ae and CD-1. On the basis of a small-scale acute toxicity study and a pilot micronucleus test, a full-scale micronucleus test was performed with a 48-h sampling time at doses of 25, 50, 100, and 200 mg/kg by both administration routes in the 2 strains. At each dose level and in both strains, higher frequencies of micronucleated polychromatic erythrocytes (MNPCEs) were found after use of the i.p. route. In the MS/Ae strain, a linear, positive dose response was obtained by both routes. In the CD-1 strain, the maximum response was reached at 100 mg/kg and a downturn occurred at 200 mg/kg by both routes. The comparison of maximum responses indicated that MS/Ae was the higher responder for both routes of application. Although DMBA induced micronuclei more efficiently by the i.p. route than after oral administration on a mg/kg base, this route-related difference was reversed in both strains when the comparison was made on the basis of LD50 values and when the maximum responses were neglected.

Studies on eye irritation caused by chemicals in rabbits--II. Structure-activity relationships and in vitro approach to primary eye irritation of salicylates in rabbits.

Structure-activity relationships and in vitro evaluation of eye irritation potential of salicylates in rabbits were studied. The primary eye irritation potential of ten salicylates was evaluated according to Draize method. The effects of chemicals on model protein and lipid were investigated in vitro. The effects of chemicals on the protein could be detected by the production of aggregates of human serum gamma-globulin (HSG) and a good correlation was obtained between the ability of salicylates to produce aggregation of HSG and the potential of corneal irritation. The effects on the lipid could be detected by the adhesion potential of chemicals on lipid membrane and a linear correlation was not obtained between the adhesionary effects of salicylates on lipid membrane and the potential eye irritation. The corneal irritation and protein aggregation potential of salicylates were correlated with the acid dissociation constant more closely than octanol/water partition coefficient. The destruction of alpha-helix of proteins in corneal surface by salicylates were observed from the nondestructive structural analysis of corneal surface by Fourier Transform (FT)-IR spectroscopy. These results suggest that eye irritation caused by salicylates are mainly the results of denaturation of proteins in ocular tissue and that the effects on protein depend on the dissociation potential of molecules.

Studies on eye irritation caused by chemicals in rabbits--1. A quantitative structure-activity relationships approach to primary eye irritation of chemicals in rabbits.

A quantitative structure-activity relationship (QSAR) approach has been applied to the analysis of the relation between the structural features of chemicals and the primary eye irritation in rabbits. One hundred thirty one heterogeneous chemicals including medicals, pesticides, detergents and organic solvents were used in this study. The eye irritation ratings were made in three classes on the basis of the recovery time of corneal and conjunctival damages. Thirty six descriptors were used to describe the molecules. To correlate eye irritation ratings with the descriptors, a QSAR model was formulated by the adaptive least-squares method. A three-class discrimination was made as follows; class I included 23 chemicals which induced the damages recovering within 24 hr, class II included 64 chemicals which induced the damages persisting for more than 24 hr but recovering within 21 days, class III included 44 chemicals which induced the damages not recovering within 21 days. The discriminant function included 18 descriptors. The accuracy in classifying the chemicals was 86.3% in the recognition and 74.0% in the leave-one-out prediction. These results suggest that QSAR analysis is valuable to predict the primary eye irritation of chemicals.

Studies on eye irritation caused by chemicals in rabbits--II. An in vitro testing method using rat red blood cells for the prediction of eye irritation potential of chemicals.

Rat red blood cells were used as an in vitro method to evaluate the eye irritation potential of chemicals in rabbits. The results using 116 chemicals of various categories including medicines, pesticides, detergents and solvents were analyzed for the prediction of possibility of eye irritation potentials. Eye irritation of chemicals was examined according to Draize method and chemicals were classified into three categories, (1) non or mild irritants, (2) moderate or severe irritants and (3) strong or corrosive irritants, based on the recovery of damages. The in vitro method consisted of two methods detecting the effects of chemicals mainly on protein and lipid in the membrane, which were evaluated by the induction of methemoglobin and hemolysis, respectively. Non- or mild irritants induced neither methemoglobin formation nor hemolysis. Most of moderate or severe irritants induced hemolysis, however, the potentials were low. Strong or corrosive irritants had high potentials for the induction of methemoglobin. The multivariate estimation by the above two in vitro data sets were 77.6% predictive of the in vivo classification.

[Single dose toxicity studies of calcipotriol (MC903) in rats and dogs].

A single dose toxicity of calcipotriol (MC903), an anti-psoriasic agent, administered subcutaneously (s.c.) and percutaneously (p.c.) was studied in Slc:SD rats (s.c. and p.c.) and beagle dogs (s.c.). The LD50 values of MC903 were as follows: rats, 2.19 mg/kg in males and 2.51 mg/kg in females by s.c., and more than 15 mg/kg in both sexes by p.c.; dogs, more than 1.5 mg/kg in males by p.c. No sexual difference was noted in LD50 values of rats. Death of rats was observed from 1 to 3 days after administration by both routes. Dead animals showed decreases in body weight and locomotor activity, reddish tear, abnormal gait and dirty hair by both routes. Furthermore, dead animals administered by s.c. showed salivation, nasal discharge, piloerection, ptosis, diarrhea, urorrhea, nasal and vaginal bleeding, subnormal temperature, loose stool, cyanosis, irregular and deep respirations, clonic and tonic convulsions. Survival of rats showed similar signs to those of dead animals except for nasal discharge, nasal and vaginal bleeding, cyanosis, agonal respiration and convulsion. Discoloration of the kidney, white patch of the heart and a dilatation of the stomach wall were observed on macroscopic examinations. No mortalities were observed in dogs which showed vomiting, conjunctival congestion, circumoral and auricular reddenings, periblepharal purplish reddening, decreases in locomotor activity and defecation, emaciation, eye discharge, skin desquamation of treated area and an increase in respiration. On macroscopic examination, desquamation of the skin, reddening of the circumoral mucosa, pale gray yellow striations in renal tubules of the cortex and discoloration of the thyroid were observed. Histopathological findings revealed epidermal thickening with parakeratosis, fibrocytes, hypertrophy and hypersecretion of the sebaceous and sweat glands, formation of epitheloid glanulomas and infiltration of neutrophils in the subcutaneous tissues. Furthermore, moderate calcium deposits in the renal tubules, fatty cells and slight calcium deposits in interstitial tissues of the thyroid, and a cystic nest of an ectopic intestinal epithelium between muscle layers of the duodenum were observed at the highest dose. On the basis of results obtained in the present study, rats administered MC903 by s.c. or p.c. died probably due to the circulatory and renal disturbance resulted from effects of this drug on the heart and kidney.

[A 4-week repeated percutaneous dose toxicity study of calcipotriol (MC903) followed by a 4-week recovery test in rats].

A 4-week repeated percutaneous dose toxicity of calcipotriol (MC903), an anti-psoriasic agent, followed by a recovery for 4 weeks was studied in Slc:SD rats at doses of 4, 20 and 100 micrograms/kg/day as low, mid and high dose levels. 1. One male and female at high dose died probably due to stress and circulatory failure. One female at mid dose died with clonic convulsion considered to be results in attached error of a neck collar. Survival of rats showed reddish tear, reddening and desquamation of the skin at application site, and vocalization at all groups including control. Furthermore, abnormal gait, dirty hair, emaciation and opacity of the eyeball surface in both sexes were observed at high dose. 2. A decreased body weight and a slight increased water consumption in both sexes, and a decreased food consumption in males were observed at high dose. 3. An increased incidence of corneal opacity was noted significantly in both sexes as compared with control at high dose. Urinalysis revealed an increased Ca excretion in both sexes at more than mid dose, and lower pH in females at mid dose and in both sexes at high dose, and a decreased urinary volume in males at high dose. The increases of neutrophil and serum beta-globulin ratios in females, and serum Ca level in both sexes were observed at high dose. The increased mineralization of the cornea in males at mid dose and in both sexes at high dose, and of the Kidney in males at high dose were observed. At the skin of application site, cellular infiltration in the epidermis and dermis in both sexes at more than mid dose was observed. Furthermore, hyperplasia of the squamous cell in females, and hyperkeratosis in the epidermis and hypertrophy of the sebaceous gland in both sexes were observed at high dose. 4. After a 4-week recovery period, the changes related with application disappeared except for opacity of the eyeball surface and cornea, and mineralization of organs. 5. On the basis of results obtained in the present study, it is considered that 4 micrograms/kg/day is the no-toxic dose of MC903 applied percutaneously in both sexes of rats.

[A 26-week repeated subcutaneous dose toxicity study of calcipotriol (MC903) followed by a 5-week recovery test in rats].

A 26-week repeated subcutaneous dose toxicity of calcipotriol (MC903), an anti-psoriasic agent, followed by a recovery for 5 weeks was studied in Slc:SD rats at doses of 0.4, 2 and 10 micrograms/kg/day as low, mid and high dose levels. 1. No mortality during the experimental period was observed in both sexes of all groups including control. An increased incidence of opacity of the eyeball surface in males was noted at high dose. There were no difference in body weight and food consumption between control. An increased water consumption in both sexes was observed at high dose. 2. An increased incidence of the corneal opacity was noted significantly at high dose in both sexes compared with that observed in control. Urinalysis revealed the increased excretions of Ca at more than mid dose, and Na, Cl and IP in males at high dose, and an decreased urinary volume in females and lower pH in both sexes at high dose. An increased serum Ca level in males at mid dose and in both sexes at high dose, and an elevated ALP activity in males at high dose were observed. The increased weights of the kidney in males at more than mid dose and adrenal gland in both sexes at high dose were observed. The increased incidence of mineralization of the cornea and kidney was noted significantly in males at more than mid dose as compared with control. Dilatation of endoplasmic reticulum of distal tubular cells of the kidney in both sexes was observed at high dose on electron microscopic examination. 3. After a 5-week recovery period, the changes related with the treatment of MC903 almost disappeared except for mineralizations of the cornea and kidney. 4. On the basis of results obtained in the present study, it is considered that 0.4 microgram/kg/day is the no-toxic dose of MC903 administered subcutaneously in both sexes of rats.

[A 26-week repeated percutaneous dose toxicity study of calcipotriol (MC903) in rats].

A 26-week repeated percutaneous dose toxicity of calcipotriol (MC903), an anti-psoriasic agent, was studied in Slc:SD rats at doses of 0.8, 4 and 20 micrograms/kg/day as low, mid and high dose levels. 1. No mortality were observed in both sexes of all groups including control. An increased water consumption was observed in females at mid dose and in both sexes at high dose. 2. An increased incidence of the corneal opacity in males at mid dose and in both sexes at high dose was noted significantly as compared with that observed in control. Urinalysis revealed a slight increased urinary volume, increased excretions of Ca and IP, and lower pH in both sexes at more than mid dose. Levels of the serum IP in females and Ca in both sexes were elevated at high dose. 3. The increased weights of the kidney in males and adrenal gland in females were observed at high dose. The kidney in females at mid dose and in both sexes at high dose showed a higher incidence of mineralization than in control. Furthermore, osteosclerosis of the sternum and femur in both sexes, and hyperkeratosis of the skin at application site in females at high dose were observed. Electron microscopic examination revealed no abnormality in the liver and kidney. 4. On the basis of results obtained in the present study, it is considered that 0.8 microgram/kg/day is the no-toxic dose of MC903 applied percutaneously in both sexes of rats.

[An antigenicity study of calcipotriol (MC903)].

Antigenicity of calcipotriol (MC903), an anti-psoriasic agent, was investigated in mice and guinea-pigs. 1. In mice, MC903 administered alone or with an adjuvant (Alum) did not result in the production of MC903-specific IgE antibody. 2. In guinea-pigs sensitized with MC903 alone or plus an adjuvant (FCA), systemic anaphylaxis was not induced by challenging with MC903. IgG1 antibody in MC903-sensitized guinea-pigs was not detected by the 4-hr PCA test. 3. Ovalbumin induced the production of ovalbumin-specific IgE antibody in mice, and ovalbumin-specific IgG1 antibody in guinea-pigs. Intense systemic anaphylaxis in the ovalbumin-sensitized guinea-pigs was induced by challenging with ovalbumin. 4. Results obtained in the present study suggest that MC903 does not induce the production of IgE antibody in mouse, and IgG1 antibody in guinea-pig, and in MC903-sensitized guinea-pig systemic anaphylaxis is not induced by challenging with MC903.

[A mutagenicity study of calcipotriol (MC903)].

Calcipotriol (MC903), an anti-psoriasic agent, was examined for mutagenicity in the reverse mutation test and the chromosomal aberration test in vitro, and the micronucleus test in Slc:ddY mice. 1. In the reverse mutation test using Salmonella typhimurium (TA100, TA1535, TA98, TA1537) and Escherichia coli (WP2uvrA), MC903 did not significantly increase revertant colonies in any of the test strains with or without metabolic system. 2. In the chromosomal aberration test with a Chinese hamster fibroblast cell line (CHL), MC903 did not significantly increase aberrant cells in the direct method or in the activation method. 3. In the micronucleus test with male mice, MC903 did not significantly increase in the number of polychromatic erythrocytes with micronuclei in the bone marrow. These results suggest that MC903 has no mutagenic as well as clastogenic effects under the present experimental condition.

[Dermatotoxicity studies of calcipotriol (MC903) ointment--primary skin irritation, skin sensitization, phototoxicity and skin photosensitization].

MC903 ointment was studied on primary skin irritation in rabbits and skin sensitization, skin photosensitization and phototoxicity in guinea-pigs. 1. MC903 ointment, ointment base and deteriorated MC903 ointment induced erythematous changes in rabbit skin. These erythematous changes were not so serious, and they were classified as a mild irritant. 2. MC903 ointment was almost negative in skin sensitization study. 3. MC903 ointment has no skin phototoxicity in guinea-pigs. 4. MC903 ointment has no skin photosensitizing activity in guinea-pigs.

Effects of the tumor inhibitor IKP-104, a 4(1H)-pyridinone derivative, on cytoskeletal microtubules of cultured tumor cells.

The effects of IKP-104, a 4(1H)-pyridinone derivative, on the mitotic profile and cytoskeletal microtubule dynamics of cultured B16 melanoma cells were examined in order to investigate the mechanism of its antitumor activity. The exposure to IKP-104 caused accumulation of cells in abnormal metaphase with chromosomes scattered within the cytoplasm and induced polyploid and multinucleate cells as detected by differential staining microscopy with brilliant blue R and safranin O. An immunofluorescence study with monoclonal anti-alpha-tubulin antibody revealed that IKP-104 diminished cytoskeletal microtubules of both interphase and mitotic cells, resulting in induction of a few fragments resembling "microtubular bundles" induced by vinblastine (VLB). These results indicated that IKP-104 arrests cells in the mitotic phase by inhibition of polymerization and induction of depolymerization of cytoskeletal microtubules, similarly to VLB.