Topoisomerase II inhibitors fail to induce chromosome-type aberrations in etoposide-resistant cells: evidence for essential contribution of the cleavable complex formation to the induction of chromosome-type aberrations.

The induction of chromosome-type aberrations is mediated by stabilization of the DNA-topoisomerase II (topo-II) complex, the cleavable complex (CC), induced by topo-II inhibitors. In the present study, in order to confirm the critical contribution of topo-II in producing chromosome-type aberrations, the inducibility of chromosome-type aberrations by topo-II inhibitors was compared between human epidermoid cancer KB cells and their mutant cells (KB/ VP-2 cells) which were resistant to etoposide (VP-16) and which have reduced levels of topo-II and its gene expression. KB/VP-2 cells were resistant to the cytotoxicity of topo-II inhibitors and most resistant to etoposide. In KB cells treated with etoposide which had accumulated CC, chromosome-type aberrations but not chromatid-type aberrations were efficiently induced, as has already been reported in Chinese hamster fibroblasts. In contrast, in KB/VP-2 cells with no accumulation of CC, etoposide induced mainly chromatid-type aberrations, with a few chromosome-type aberrations. Unlike etoposide, however, adriamycin, which was known to accumulate CC in Chinese hamster fibroblastic cells, neither induced chromosome-type aberrations nor accumulated CC in either KB or KB/VP-2 cells. No difference in cell incorporation of [3H]etoposide between the two cell lines was observed. These findings indicate that CC formation contributes to the induction of chromosome-type aberrations, although the reason why adriamycin could not accumulate CC in KB cells is not clear. This may suggest a mechanism for resistance to topo-II inhibitors in cancer chemotherapy.

Carbon tetrachloride does not induce micronucleus in either mouse bone marrow or peripheral blood.

We performed mouse bone marrow and peripheral blood micronucleus tests on carbon tetrachloride (CCl4). In the bone marrow assay, bone marrow cells were collected once after 24 h and twice, with a 24-h interval at a dose of 500, 1000 and 2000 mg/kg. In the peripheral blood assay, blood samples were collected 0, 24, 48 and 72 h after a single intraperitoneal injection at a dose of 1000, 2000 and 3000 mg/kg. As a result, micronucleated polychromatic erythrocytes (MNPCEs) were observed neither in the bone marrow assay nor the peripheral blood assay. We concluded that CCl4 does not induce chromosomal aberrations in the mouse bone marrow cells under these experimental conditions.

Bone disorder in cardiomyopathic hamsters.

Bones of cardiomyopathic hamsters (UM-X7.1 Syrian hamsters), at 5, 10 and 20 weeks of age, were compared chemically and histomorphologically with those of normal Syrian hamsters. Femurs of UM-X7.1 hamsters were significantly shorter than those of normal hamsters, and the mean dry weight, mean volume, mean ash weight per unit bone volume and mean ash as a percentage of dry weight of femurs were all significantly less in UM-X7.1 hamsters. The bone disorder preceded the myocardial calcium precipitation and myocardial hypertrophy in the cardiomyopathic hamsters. In addition, the percentage of cortical area measured on the cross-section of tibia and the appositional rate of bone minerals, determined by a tetracycline labelling technique, were also lower in the UM-X7.1 hamsters. These findings suggest that the bone disorder was associated with decreased bone formation in the UM-X7.1 Syrian hamsters.

Studies on topical antiinflammatory agents. I. Synthesis and vasoconstrictive activity of corticosteroid 17-succinyl esters.

A series of 17-succinyl derivatives of four corticosteroids was prepared. They were tested for vasoconstrictive activity in humans, using 9 alpha-fluoro-11 beta, 21-dihydroxy-16 beta-methyl-17 alpha-valeryloxy-1,4-pregnadiene-3,20-dione (betamethasone 17-valerate, BV) as a standard. The activities of the 21-chloro 17-methylsuccinate compounds (6A, 6C and 6D) were greater than that of BV. A structure-activity relationship study showed that the activities of the 21-chloro 17-methylsuccinates were more potent than those of the corresponding 21-esters.

Comparative toxicity study of hydrocortisone 17-butyrate 21-propionate (HBP) ointment and other topical corticosteroids in rats.

Comparative systemic and topical toxicity in male rats treated on the dorsal skin for 14 consecutive days with a volume of 0.15 g/100 g (body weight) of 0.1% hydrocortisone 17-butyrate 21-propionate (HBP) ointment, 0.05% clobetasol propionate (CP) ointment, 0.1% predonisolone 17-valerate 21-acetate (PVA) ointment and 0.1% diflucortolone valerate (DV) ointment was studied. In all the treated groups body weight gain was suppressed, serum concentration of total cholesterol and triglycerides increased and the lymphatic tissues and skin were atrophic. The DV and CP groups had adrenal atrophy and renal lesions, and the DV group also had gastric and hepatic lesions. The systemic effect of HBP ointment was weaker than that of the other drugs (DV greater than CP much greater than PVA greater than HBP). All the drugs significantly reduced the skin fold thickness in treated areas throughout the application period. The dermal atrophic effect of HBP ointment was also relatively weaker than that of the other drugs. From the above evidence, it was concluded that HBP ointment was less toxic than the other topical corticosteroids.

Effect of 2'-carboxymethoxy-4,4'-bis(3-methyl-2-butenyloxy) chalcone (sofalcone) on chronic gastric ulcers in rats.

The anti-ulcer effect of sofalcone, an isoprenyl chalcone derivative, on acetic acid-induced gastric ulcers in rats was studied histologically and histochemically. After administrations of sofalcone at 50 and 200 mg/kg twice daily for 10 days, contraction of the ulcer, mucosal regeneration, accelerated development of the collagen fibers in the granulation tissue at the base of the ulcer, and increase of acid mucopolysaccharides, an alcian blue stain-positive substance covering the regenerated mucosa, were noted. The healing effect of sofalcone was balanced in mucosal regeneration and connective tissue proliferation (formation of the collagen fibers). Sofalcone of 50 mg/kg showed a greater healing effect than gefarnate at the same dose and had a similar healing effect as L-glutamine at 200 mg/kg.

[The analgesic and antipyretic effects of a non-steroidal anti-inflammatory drug, oxaprozin, in experimental animals].

The analgesic and antipyretic effects of oxaprozin were investigated in comparison with those of indomethacin, ibuprofen, phenylbutazone and aspirin. On the various writhing tests in mice, the analgesic effect of oxaprozin was about 2 to 9 times more potent than those of ibuprofen, phenylbutazone and aspirin. On the other hand, the analgesic and antipyretic effects of oxaprozin in rats were roughly equivalent to those of aspirin, but less effective than those of the other drugs tested. On the urate synovitis test in dogs, only oxaprozin showed a prophylactic effect. Therefore, The effect of oxaprozin in mice and dogs was more potent than ibuprofen, phenylbutazone and aspirin. The metabolic rate of oxaprozin in rats is 3.5 and 7.2 times more rapid than in mice and dogs, respectively, and its blood level in rats is low. Moreover, the biological half-life of oxaprozin is 39 to 43 hr and 49 to 69 hr in dogs and humans, respectively. From these results, it is suggested that oxaprozin is more potent than ibuprofen, phenylbutazone and aspirin, and in clinical use, it is a long acting anti-inflammatory drug.

Studies on the alpha-glucoside hydrolase inhibitor, adiposin. IV. Effect of adiposin on intestinal digestion of carbohydrates in experimental animals.

Adiposins showed a potent inhibitory activity against alpha-glucoside hydrolases such as salivary and pancreatic alpha-amylases, and intestinal disaccharidases in vivo. They suppressed the increase of the blood glucose level and the secretion of insulin in mice and rats which both had been fasted and then forced-fed on cooked corn starch, sucrose or maltose. The suppression of the increase in the body weight gains, and of the secretion of insulin and triglyceride in blood were observed in the experimental animals which were given diet containing adiposins. Hematological and histopathological examinations of animals treated with adiposins did not reveal any remarkable changes after a 3 months toxicity test. Adiposins did not show any intravenous acute toxicity in mice at dose levels (p.o.) less than 10 g/kg of body weight.

[Pharmacological study on hydrocortisone 17-butyrate 21-propionate (author's transl)].

The topical and systemic anti-inflammatory activities of hydrocortisone 17-butyrate 21-propionate (HBP) were studied. The systemic anti-inflammatory activities of HBP and reference steroids were examined for their effects on dinitrochlorobenzene dermatitis, carrageenin edema, cotton pellet granuloma and adjuvant arthritis in rats and by the delayed allergic edema test in mice. The topical anti-inflammatory activities of these steroids were examined for their effects on croton oil dermatitis, croton oil ear edema, carrageenin edema and cotton pellet granuloma in rats. Furthermore, effects of these steroids on liver glycogen deposition in mice, thymolysis, and decrease of serum corticosterone level in rats were examined. Systemically administered HBP was less potent than betamethasone 17-valerate (BV), but was almost equal to hydrocortisone 17-butyrate (HB) in anti-inflammatory activity, and its effects on liver glycogen deposition, thymolysis, and the decrease of serum corticosterone level. However, the topical anti-inflammatory activity of HBP was more potent than that of BV and HB, although in the same experiment, thymolytic activity of HBP was less potent than that of BV, but was almost equal to HB. The inhibitory effect of HBP on hypotonic induced hemolysis was weaker than that of BV, but was stronger than that of HB in vitro. The affinity of HBP was higher than that of BV and HB to polymorphonuclear leucocytes used as the inflammatory cells in vitro. On the other hand no marked difference was observed in the affinity to erythrocytes used as the non-inflammatory cells in vitro. These results suggest that HBP is a useful drug which has superior topical anti-inflammatory activity, but has a weak systemic effect.

[Studies of D-penicillamine (5): effects on rat adjuvant arthritis (author's transl)].

D-Penicillamine (D-PA), 80-100 mg/kg/day enhanced the early phase of adjuvant arthritis (AA) in rats when it was administered orally for about 4 weeks after the adjuvant injection day, whereas dexamethasone (1 mg/kg/day, p.o.) and chloroquine diphosphate (25 mg/kg/day, p.o.) inhibited AA in the same dosing regimen. On the other hand, subcutaneous injection of sodium aurothiomalate (12.5 mg/kg/day) enhanced AA initially, but inhibited it later. The enhancing effect of D-PA on the early phase of AA was observed also at doses of 50 mg/kg/day and 200 mg/kg/day, but, in the case of 200 mg/kg/day, inhibited the later phase of AA. When the administration of D-PA was started before the adjuvant injection, it showed a tendency to suppress AA on proportion to the dosing period. The effect of D-PA, however, was not observed in the model when the drug administration was started after the establishment of arthritis. The co-administration pyridoxine HCl did not influence the effect of D-PA on AA. A good correlation was not obtained between the inflammatory score and the PPD induced skin reaction, serum metals level and histopathological changes of lymph node in the AA rats treated with D-PA. Thus the effect of D-PA on AA was related to dose, timing and duration of the administration. It was suggested that the enhancing and inhibitory effects of D-PA on AA were not based on vitamin B6, depletion, but rather were caused by inhibition of T1 and T2 lymphocytes which may be regulating this arthritis process.

Effects of cathepsin B, H, and D in pectoral muscle of dystrophic chickens (line 413) of in vivo administration of E-64-c (N-[N-(L-3-transcarboxyoxirane-2-carbonyl)-L-leucyl]-3-methyl-butylamine).

The total activities of cathepsin B and cathepsin H in pectoral muscle of dystrophic chickens (Line 413) were about two times higher than in control chickens (Line 412), and cathepsin D activity was about 3 times higher in this muscle in the dystrophic chickens. When E-64-c, a synthesized potent thiol inhibitor was injected subcutaneously, in various doses, daily for 80 days into dystrophic chickens (L 413), the activities of cathepsin B and cathepsin H were reduced to the levels in control chickens (Line 412), but cathepsin D activity, which is insensitive to E-64-c in vitro, was not changed.

[Studies on toxicity of hydrocortisone 17-butyrate 21-propionate -1. Acute toxicity of hydrocortisone 17-butyrate 21-propionate and its analogues in mice, rats and dogs (author's transl)].

The acute toxicity of hydrocortisone 17-butyrate 21-propionate (HBP), hydrocortisone 17-butyrate (HB17) and hydrocortisone 21-butyrate (HB21) were investigated by three administration routes (s.c., i. p. and p. o.) in mice, rats and dogs. In the case of HBP, LD50 by oral administration was the highest, and followed by subcutaneous and intraperitoneal administration in mice and rats. And LD50 of HB17 and HB21 were not different from HBP in mice by subcutaneous administration. The depression of spontaneous movement and respiratory rate, ptosis, larcrymation and the collapse were commonly observed in all drugs, and it was independent of administration routes. The autopsy revealed the atrophy of thymus, spleen and adrenal glands, the supprative nodules of heart and liver and the ulcers of alimentary tract in mice and rats. But the changes observed in mice and rats were recognized when 1000 mg/kg of HBP was administered to dogs subcutaneously. Many of these changes were common to glucocorticoids, and the LD50 of HBP was rather high compared with other synthetic steroids; therefore, HBP was among less toxic steroid.

[Influence of hydrocortisone 17-butyrate 21-propionate on the visual and the auditory organs (author's transl)].

The visual toxicity and the ototoxicity of hydrocortisone 17-butyrate 21-propionate (HBP), a newly synthesized anti-inflammatory steroid, was investigated using rats and dogs. (1) Electroretinogram (ERG) and visually evoked potential (VEP) in rats were not changed when HBP was administered intravenously and intraperitoneally, even at the semilethal doses. Consequently, it was suggested that HBP had no effect on the visual nervous system. (2) Similar to other corticosteroids, the intra ocular pressure rose in the dogs received HBP. Nevertheless, these dogs showed neither the remarkable changes in ERG, in the fundus of the eye, in the histological examination, nor the turbidity of the cornea and the lens. Based on these facts, it was reasonable to think that the the rise in the intra ocular pressure caused by HBP was not so severe that induce the secondary influence to other visual functions. (3) The rats received HBP did not show any changes in the auditory function by the audiometry. As the result, HBP was thought to be one of the rather safety corticosteroids concerning the visual toxicity and the ototoxicity.

[Studies on toxicity of hydrocortisone 17-butyrate 21-propionate -2. Subacute toxicity in rats by subcutaneous administration (author's transl)].

Subacute toxicity of hydrocortisone 17-butyrate 21-propionate (HBP), a new synthetic corticosteroid, was studied in rats, using betamethasone 17-valerate (BV) and hydrocortisone 17-butyrate (HB) as the reference drugs. HBP was subcutaneously injected to rats at the daily doses of 0.08, 0.4, 2.0, 10 and 50 mg/kg for 30 days. BV and HB were also administered at the daily doses of 0.08, 0.4 and 2.0 mg/kg. The recovery test was performed for 4 weeks after administration of HBP, BV and HB. The suppression of body weight gain by HBP was observed at the doses more than 0.08 mg/kg in male and more than 2.0 mg/kg in female rats. In addition, at the doses more than 0.4 mg/kg of HBP induced the dose-dependent symptoms such as decrease in the number of circulating white blood cells, lymphocyte counts and S-ALP level, increase in total cholesterol, GOT and GPT level of serum, and regressive changes in adrenals, lymphatic and hematopoietic tissues. There were fatal cases in rats given 50 mg/kg of HBP. These changes are considered to be common phenomena to other corticosteroids, and less toxic in female than male rats. Changes of symptoms caused by the administration of HBP 2.0 mg/kg were almost recovered after withdrawal. The toxicities of three corticosteroids were in the order of BV greater than HB greater than or equal to HBP in strength. As the result, maximum non-toxic dose of HBP was estimated to be 0.08 mg/kg in female and lower than that in male rats.

[Studies on antigenicity, phototoxicity and other specific toxicities of hydrocortisone 17-butyrate 21-propionate (HBP) (author's transl)].

The present experiments were undertaken to determine the antigenicity and other toxicities of HBP; such as phototoxicity, photosensitivity, ulcerogenicity, adrogenic-myotropic, estrogenic and progestational activities, and mutagenicity. No antibody formation and delayed type skin reaction of HBP were seen in rabbits. Active systemic anaphylaxis was not observed in guinea pigs challenged by HBP. In the phototoxicity and photosensitivity test, 0.1% HBP ointment, 0.1% HBP cream and 10% HBP acetone solution did not show any skin reaction with or without irradiation of ultraviolet light. Repeated subcutaneous administration of HBP irritated the gastric and intestinal mucosa dose dependently in rats as hydrocortisone 17-butyrate and betamethasone 17-valerate (BV). HBP had neither androgenic-myotropic nor estrogenic activity, but antiestrogenic activity was observed. The progestational activity of HBP in immature rabbit pretreated with estrone was less potent than BV. In the mutagenicity test os HBP investigated by the reverse mutation according to the method by Ames, no significant increase in the number of revertants was observed in the presence or absence of S9 mixture.

[Studies on toxicity of hydrocortisone 17-butyrate 21-propionate -3. Subacute toxicity in rats by percutaneous administration (author's transl)].

Subacute toxicity of hydrocortisone 17-butyrate 21-propionate (HBP) was studied in rats. HBP was percutaneously given to rats with 0.1% and 0.5% creame (0.1% HBP-C, 0.5% HBP-C) and ointment (0.1% HBP-O, 0.5% HBP-O) at the daily dose level of 150 mg per 100 g body weight for 1 month. Rats receiving HBP-C and HBP-O showed some dose-dependent symptoms such as the suppression of body weight gain, emaciation, decrease in the number of white blood cells, hemoglobin, hematocrit, and serum total cholesterol level, regressive changes in adrenals, skin, lymphatic and hematopoietic tissues, which are known as toxic effects of synthetic corticosteroids. These symptoms were comparatively high toxic in male rats and in cream groups, and almost disappeared in rats elapsed recovery time of 1 month after withdrawal of HBP.

[Studies on toxicity of hydrocortisone 17-butyrate 21-propionate -4. Chronic toxicity in rats by subcutaneous administration (author's transl)].

Chronic toxicity of hydrocortisone 17-butyrate 21-propionate (HBP), a new synthetic corticosteroid, was studied in rats. HBP was subcutaneously injected to rats as the daily doses of 0.001, 0.01, 0.01, 1.0 and 3.0 mg/kg for 6 months, and the following recovery test was carried out for 4 weeks. Hydrocortisone 17-butyrate (HB) and betamethasone 17-valerate (BV) were used as the reference drugs at the doses of 0.1, 1.0 and 3.0 mg/kg. The suppression of body weight gain by the administration of HBP was observed at the doses more than 1.0 mg/kg in male and more than 0.1 mg/kg in female, and the dead animals were sent at the highest dose of HB and BV. Mainly at the doses more than 0.1 mg/kg HBP induced the dose-dependent symptoms such as decrease in the number of white blood cells and total protein level in serum, and increase in total cholesterol, GOT and GPT level in serum, and atrophic changes of adrenals, lymphatic tissues, skin and subsexual organs. No usual abnormality was recognized at the doses less than 0.01 mg/kg of HBP. These symptoms were more toxic in male, and the strength of toxicity was in the order of BV greater than HB greater than HBP. Many of these findings have known as common effects of corticosteroids. The changes observed in this study were almost recovered after withdrawal of HBP at the doses less than 0.1 mg/kg. As the result, it was suggested that the maximum non-toxic dose of HBP was 0.001 mg/kg.

[Studies of toxicity of hydrocortisone 17-butyrate 21-propionate -5. Chronic toxicity in rats by percutaneous administration (author's transl)].

Chronic toxicity of a new synthetic corticosteroid, hydrocortisone 17-butyrate 21-propionate (HBP), was investigated in rats of both sexes. HBP was percutaneously given to rats with 0.1%, 0.5% cream and ointment at the daily dose level of 150 mg per 100 g body weight for 6 months. For the comparison, the percutaneous toxicity with 0.12% betamethasone 17-valerate (BV) cream and ointment, and 0.1% hydrocortisone 17-butyrate (HB) cream and ointment at the daily dose level of 150 mg per 100 g body weight were studied. Rats receiving HBP showed the dose-dependent changes such as the suppression of body weight gain and food intake, emaciation, decrease in the number of white blood cells and lymphocytes, total protein, increase in the number of red cells, hematocrit, hemoglobin, blood sugar and total cholesterol, regressive changes in adrenal cortex, lymphatic and hematopoietic tissues and skin, and gastric erosion, which have been well known as toxic effects of synthetic corticosteroids. These findings were comparatively high toxic in male, and almost disappeared in rats elapsed recovery time of month after withdrawal of HBP. The toxicities of HBP, BV and HB were qualitatively same. However, the grade of effects of HBP toxicity was similar to that of HB, weaker than of BV.

[Studies on the physical dependence liability of chlorphenesin carbamate (author's transl)].

Physical dependence liability of chlorphenesin carbamate (CPC) was studied in parallel with phenobarbital-Na (PB). Beagle dogs were used and the overall duration of the experiment was 85 days, i.e. the first dosing period was 42 dyas (6 weeks) in which drugs were repeatedly administered orally once daily, followed by a withdrawal period (7 days), the second dosing period was continued from the 50th-78th day in which the form and schedule of drug administration was as in the first dosing period. The last 79th to 85th days were used for substitution experiments. In both dosing periods, PB but not CPC showed signs of tolerance formation. Severe withdrawal syndrome was observed in PB administered dogs whereas there were no changes of behavior observed in CPC-dogs by withdrawal and substitution procedures, respectively. CPC apparently does not have a physical dependence liability.