Inhibition of cell-surface neutral protease of Ehrlich ascites tumor cells by potassium p-(3,3-dimethyl-1-triazeno) benzoate

Ehrlich ascites tumour (EAT) cells possess a trypsin-like neutral protease on the cell surface. The antimetastatic triazene drug potassium p-(3,3-dimethyl-1-triazeno) benzoate (DM-COOK) inhibits this neutral protease, and also trypsin. Incubation of EAT cells with human erythrocytes (ratio of 1 to 5) at 37 degrees C for 18 h caused haemolysis of 28.8% of the erythrocytes. Addition of millimolar concentrations of DM-COOK to a fixed quantity (2.5 X 10(8)) of EAT cells caused a dose-related inhibition of haemolysis. DM-COOK was strongly bound to EAT cells and could not be removed by repeated washing.     

Response of selected fetal organs to antineoplastic agents

This study was designed to quantitate the effects of 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DIC) and 5-(3,3-bis(2-chlorethyl)-1-triazeno)-imidazole-4-carboxamide (BIC) on growth and selected components of rat fetal organs. Twelve-day pregnant rats were given single intraperitoneal injections of 600 mg/kg of DIC and 900 mg/kg of BIC and autopsied on day 21 of gestation. Fetal liver, brain, kidney, and placenta were removed, weighed, and assayed for total DNA, RNA, and protein. DIC significantly reduced weight, total DNA, RNA, and protein of all four fetal organs as compared to age-matched controls. The brain was most severely affected by this compound. BIC also significantly reduced weight, DNA, RNA, and protein of fetal brain, kidney, and placenta, but in fetal liver only weight and total protein were significantly depressed, while DNA and RNA remained essentially unchanged. The effect of BIC was maximal on the placenta.     

Immunologic cross-reactivity of antigen(s) induced by drug treatment in two leukemic sublines.

Previous studies have demonstrated that new antigenic specificities, not detectable on parental cells, can be induced by in vivo treatment of murine leukemic cells with anti-neoplastic agents. The immunologic properties of leukemic cells altered by treatment with 5-(3,3-dimethyl-1-triazeno) imidazole-4-carboxamide (DIC) were investigated further. Immunologic cross-reactivity between two DIC-treated leukemic sublines has been demonstrated by cell-mediated immunity in vitro and by active or adoptive immunity in vivo. Rabbit antiserum to DIC-treated sublines absorbed with the parental cells showed residual activity against the DIC-sublines that was specifically inhibited by further absorption with DIC-cells.     

Cellular and biochemical aspects of growth retardation in rat fetuses induced by maternal administration of selected anticancer agents.

Single ip injections of 600 mg/kg 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DIC) and 900 mg/kg 5-[3,3-bis(2-chlorethyl)-1-triazeno]-imidazole-4-carboxamide (BIC) were given to pregnant Wistar rats at day 12 and the animals were killed 4 h after injection and at days 13-17 of gestation. Fetal tissues were used to determine total DNA, RNA, and protein and the data used to derive cell number and cell weight, RNA, and protein/cell. Both compounds reduced total fetal body weight, DNA, RNA, and protein but reduction of RNA by BIC was not statistically significant. These effects were observed 4 h after injection, increased with age (days 13-17), and were 3-4 times greater for DIC than BIC. By using the value of 6.2 mumug DNA/cell, cell number and per-cell values for weight, RNA, and protein, and weight: DNA, RNA:DNA, and protein:DNA ratios were computed. The per-cell values and ratios in the DIC-exposed animals were 8-44% greater and in BIC-treated animals 0-11% greater than control animals of the same gestational age. Percentage of body water was the same in the experimental and control animals. The differences in DNA, RNA, and protein are believed to be related to drug-induced growth retardation incident to total fetal DNA reduction resulting in diminished cell number.     

Cytogenetic effects of 1-p-(3-methyltriazeno)benzoic acid potassium salt on human lymphocytes in vitro

The triazene derivative 1-p-(3-methyltriazeno)benzoic acid potassium salt (MTBA) shows pharmacological properties similar to those of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC, trade name dacarbazine), which is known to induce antigenic modulation in tumor cells (xenogenization) and is currently used in cancer therapy. Mutagenic, teratogenic and cancerogenic properties of triazene derivatives have been demonstrated but there is no report on their possible clastogenicity. We describe here the in vitro cytogenetic effects of MTBA on human peripheral blood lymphocytes. The drug was tested at different culture times in a range of concentrations from 2 to 500 micrograms/ml. MTBA caused a dose-dependent increase in the frequency of chromosomal breaks. Different blood donors showed different sensitivity to the treatment. Cell proliferation, as evaluated by [3H]thymidine incorporation, was inhibited at the highest concentrations of the drug. These data might be relevant for comparison with in vivo effects of the drug in clinical trials and to investigate the possible relations between xenogenization induced by MTBA and its genetic and cytogenetic effects in human lymphocytes.     

Analgesic and anti-inflammatory activity of the proanthocyanidin shellegueain A from Polypodium feei METT.

Analgesic and antiinflammatory activity of proanthocyanidin isolated from Polypodium feei roots has been tested using acetic acid-induced writhing and carrageenan-induced paw edema methods, respectively. The compound at doses of 50 and 100 mg/kg significantly decreased writhing responses of mice induced by 0.7 % acetic acid along the 60 min test in a dose-dependent manner. The compound at a dose of 100 mg/kg gave the percent protection of 76.23 higher than that of acetylsalicylic acid (59.84 %) at a dose of 50 mg/kg. In the antiinflammatory test, this compound caused significant inhibition of the rats' plantar edema induced by 1 % of carrageenan, but this activity was observed only at a higher dose (200 mg/kg). These findings suggest that proanthocyanidin of P. feei roots might have analgesic and antiinflammatory activity, and its mechanism of action might be due to the inhibition of prostaglandin biosynthesis, because the proanthocyanidin fraction had an inhibitory effect on cyclooxygenase, but not on 5-lypoxygenase enzymes.     

Antigenic changes of a murine lymphoma by in vivo treatment with triazene derivatives

Summary Five aryltriazenes were studied for efficacy in mediating immunogenic changes of tumor cells by in vivo treatment of lymphoma-bearing mice. It was found that four analog compounds produced increase in cell immunogenicity similar to that described for 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC), one of the five being by contrast completely inactive. Moreover, the use of a drug-resistant lymphoma illustrates that cytotoxic activity is not mandatory for the appearance of the immunogenic changes.The results show that a drug-mediated increase of tumor immunogenicity (DMITI) can be induced by triazene derivatives not containing the imidazole moiety.     

Reversal of indomethacin-induced decreases in renal function by an isosterically-modified prostaglandin analog

A recently discovered isosterically-modified prostaglandin analog, 4-(3-[3-[2-(1-hydroxycyclohexyl)ethyl]-4-oxo-2-thiazolidinyl ] propyl) benzoic acid, was studied in conscious Na-deficient dogs to determine if this compound could reverse the deleterious renal effects induced by inhibition of renal cyclooxygenase. Indomethacin (2 mg/kg i.v.) reduced renal function significantly in all dogs studied: GFR decreased from 38 +/- 3 to 26 +/- 1 ml/min (P less than 0.01) and ERPF from 124 +/- 15 to 79 +/- 8 ml/min (P less than 0.01). On separate occasions, the six dogs used in this study were treated with a saline placebo intravenously or with the PG analog (0.1 mg/kg i.v.) 60 min after receiving indomethacin. After placebo treatments renal function remained suppressed for the duration of observation (2 hours). After treatment with PG analog, GFR was restored to pre-indomethacin levels within 1 hour (36 +/- 3 ml/min) and remained at this level or higher for the duration of the experiment. ERPF was restored to pre-indomethacin levels within 30 min of PG analog injection (140 +/- 7 ml/min) and subsequently rose ml/min) for the duration of the experiment. Urinary electrolyte excretion was suppressed by indomethacin and despite the large increase in ERPF, Na excretion was not augmented by PG analog. This study demonstrates that a synthetic, isosterically-modified prostaglandin analog can effectively reverse the hemodynamic effects of non-steroidal antiinflammatory drug treatment on renal function while not affecting renal Na excretion.     

The mechanism of cytoprotective effect of CHINOIN-127.

According earlier, investigations nitrogen bridgehead compounds make a representative group of non-prostaglandin type gastroprotective agents. One member of this group is CHINOIN-127 (1,6-dimethyl-4-oxo-1, 6, 7, 8, 9, 9a-hexahydro-4H-pyrido-(1, 2a)-pyrimidine-3-carbox-amide). CHINOIN-127 is a potent non-narcotic analgesic and antiinflammatory agent and has a remarkable protective effect on indomethacin induced ulcer (ED50 = 25 mg/kg p.o.) and on acidified ethanol induced ulcer (ED50 = 26 mg/kg p.o.). In this study we examined the mechanism of action of cytoprotective effect of this drug and we made a comparison between the cytoprotective effect of 20% ethanol and 25 mg/kg CHINOIN-127. In the gastric mucosa of control rats we observed a balance between TxA2 and PGI2 (PGI2/TxA2 = 3.8) and between the cytoprotective prostaglandins (PGI2 and PGE2) and ulcerogen eicosanoids (TxA2 and leukotrienes) (PGI2 + PGE2/TxA2 + LTs = 3.9). 100% ethanol treatment causes disintegration of this balance, shifting the synthesis towards the ulcerogen eicosanoids production. CHINOIN-127 and 20% ethanol pretreatment improves the deranged balance between cytoprotective prostaglandins and ulcerogen eicosanoids. Our results demonstrate that CHINOIN-127 and 20% ethanol have a similar mechanism of cytoprotective action on ethanol induced ulcer in rats.     

Antiinflammatory efficacy of extracts of latex of Calotropis procera against different mediators of inflammation

The latex of the plant Calotropis procera has been reported to exhibit potent antiinflammatory activity against carrageenin and formalin that are known to release various mediators. In the present study, we have evaluated the efficacy of extracts prepared from the latex of C procera against inflammation induced by histamine, serotonin, compound 48/80, bradykinin (BK), and prostaglandin E2 (PGE2) in the rat paw oedema model. The paw oedema was induced by the subplantar injection of various inflammagens and oedema volume was recorded using a plethysmometer. The aqueous and methanol extracts of the dried latex (DL) and standard antiinflammatory drugs were administered orally 1 hour before inducing inflammation. The inhibitory effect of the extracts was also evaluated against cellular influx induced by carrageenin. The antiinflammatory effect of aqueous and methanolic extracts of DL was more pronounced than phenylbutazone (PBZ) against carrageenin while it was comparable to chlorpheniramine and PBZ against histamine and PGE2, respectively. Both extracts produced about 80%, 40%, and 30% inhibition of inflammation induced by BK, compound 48/80, and serotonin. The histological analysis revealed that the extracts were more potent than PBZ in inhibiting cellular infiltration and subcutaneous oedema induced by carrageenin. The extracts of DL exert their antiinflammatory effects mainly by inhibiting histamine and BK and partly by inhibiting PGE2.     

Aggravation of nonsteroidal antiinflammatory drug gastropathy by glucocorticoid deficiency or blockade of glucocorticoid receptors in rats

Our previous investigations suggest that the reduction of stress-induced corticosterone release, or inhibition of corticosterone actions, promotes stress-induced gastric erosions in rats. In this study the effect of glucocorticoid deficiency on susceptibility to gastric mucosal injury by nonsteroidal antiinflammatory drugs (NSAIDs) was evaluated in rats. Gastric erosions induced in male rats by indomethacin (25 mg/kg sc) or acidified aspirin (40 mM po) were studied one week after adrenalectomy with or without corticosterone replacement or after occupation of glucocorticoid receptors by the antagonist RU-38486 during the period of erosion formation. Corticosterone for replacement (4 mg/kg sc) was injected 15 min before the administration of indomethacin or acidified aspirin to adrenalectomized rats. The antagonist RU-38486 (10 mg/kg po) was administered twice, 20 min before and 60 min after NSAID administration. Plasma corticosterone levels were measured by fluorometry. Gastric erosions were quantitated by measuring the area of damage. Indomethacin or acidified aspirin induced both plasma corticosterone rise and gastric erosions. Adrenalectomy decreased both basal and NSAID-induced corticosterone levels and markedly promoted gastric erosion formation caused by the NSAID. An acute corticosterone replacement mimicking indomethacin-and aspirin-induced corticosterone rise prevented the effect of adrenalectomy on the gastric erosions. The administration of the glucocorticoid/progesterone antagonist RU-38486 significantly potentiated the formation of gastric erosions induced by indomethacin as well as aspirin. These observations suggest a gastroprotective action of glucocorticoids released in response to NSAID treatment against NSAID-induced injury.     

Analysis of the intralesional adriamycin-induced regression of primary and metastatic growth of line-10 guinea-pig hepatoma

Summary A single intralesional injection of 2.4 mg adriamycin/kg into 7-day-old intradermal strain-2 guinea-pig hepatoma, line-10, caused regression of the tumor and prevented the growth of regional lymph node metastasis. Cured animals were resistant to challenge with the same tumor. Intratumoral injection of 20 mg DTIC/kg was not effective in causing tumor regression or preventing growth of regional lymph node metastasis. Comparative histological examinations performed on the tumor site and the draining lymph node showed that both chemotherapeutic drugs caused extensive necrosis at the injection site and that within 7 days only adriamycin eradicated tumor cells from the draining lymph nodes.The number of host lymphocytes and monocytes in the adriamycin-treated tumor sites was less than that seen in the saline- or DTIC-injected animals at all time intervals examined. Minimal peripheral effects, as measured by total and differential analysis of the blood, were noted in drug-treated normal and tumor-bearing animals. In addition, the concentration of drug used did not interfere with the development of immunity.The results suggest that the effect(s) of intralesional adriamycin treatment is probably caused by a combination of cytotoxic and cytostatic actions of the drug and the development of tumor-specific immunity. Abbreviations used: DTIC, 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboximide; BCNU, 1,3-bis-(2-chloroethyl)-1-nitrosourea; SDA, superficial distal axillary lymph node     

Effect of indomethacin, tiaprofenic acid and dicrofenac on rat gastric mucosal damage and content of prostacyclin and prostaglandin E2

Gastric ulcerogenicity and depletion of endogenous prostaglandins (PGs) content induced by tiaprofenic acid, dicrofenac and indomethacin were examined using the same antiinflammatory effective doses. Male Wistar rats were given each of these drugs intragastrically 24, 18, and 3 hrs before sacrifice in the following doses (mg/kg): indomethacin (0.8, 4 and 20); tiaprofenic acid (1.2, 6 and 30); dicrofenac (0.8, 4 and 20). Endogenous prostacyclin (PGI2) and PGE2 in fundic mucosa were determined by radioimmunoassay. The three compounds produced fundic mucosal lesions in a dose-dependent manner. However, tiaprofenic acid and dicrofenac were both less potent than indomethacin in producing gastric mucosal lesions at similar antiinflammatory doses. Mucosal PGE2 content was abolished by the three compounds in the following doses (mg/kg): indomethacin (4 and 20); tiaprofenic acid (6 and 30); dicrofenac (20). Mucosal PGI2 was maintained around 50% of the control value in rats given tiaprofenic acid in a dose of 6 mg/kg or dicrofenac in a dose of 4 mg/kg, while indomethacin in a dose of 4 mg/kg markedly reduced mucosal PGI2 to 17% of the control value. In larger doses, tiaprofenic acid and dicrofenac were also significantly less potent in reducing mucosal PGI2 than indomethacin. These results suggest that the difference in ulcerogenicity between indomethacin and the other two compounds was closely related to their potency in decreasing PGI2 in the gastric (fundic) mucosa.     

Broad-Spectrum Antimicrobial Activity of a New Triazenoimidazole

Methyl-5(or 4)-(3,3-dimethyl-1-triazeno)-imidazole-4(or 5)-carboxylate was shown to have in vitro antimicrobial activity against gram-positive and gram-negative bacteria, yeasts, filamentous fungi, and algae. Preliminary studies with mice, experimentally infected with Staphylococcus aureus, have shown that this new antimicrobial agent has in vivo chemotherapeutic activity comparable to that observed with penicillin.     

Pharmacological study of Stachytarpheta cayennensis Vahl in rodents

Freeze-dried aqueous extracts (AEs, 0.1–1 g/kg body wt., p.o.) obtained from entire or selected parts of Stachytarpheta cayennensis were tested for their effects on gastric secretion, gastric motility, inflammation and pain in rodents, with the purpose of validating the plant's ethnomedical uses. The AE-Total, AE-Flowers and AE-Leaves but not AE-Stems inhibited the gastric acid secretion in pylorus-ligated rats with varying potency. Purification of AEs yielded the semipurifed fractions EtFs rich in iridoids. All the EtFs with exception of EtF-Stems inhibited gastric acid secretion of pylorus ligated mice. While AE-Total stimulated the intestinal transit of mice by 43%, AE-Leaves delayed it by 38%. These effects on intestinal transit were not observed when the EtFs were tested. Only AE-Leaves and AE-Flowers altered the gastric emptying of semisolids, increasing it by 45% and 69%, respectively. These results indicate that the compounds related to inhibition of gastric acid secretion and gastrointestinal motility are different. The AE-Total reduced abdominal writhing induced by acetic acid potently (ED₅₀ VALUE=700 mg/kg, p.o.) without altering the writhes induced by acetylcholine. Attempts to identify the mechanism of analgesia were unsuccessful since the AE-Total did not show analgesic effects when tested in different models of pain such as formalin and capsaicin or the tail-flick test. Pretreatment of animals with AE-Total did not show antiinflammatory activity in any of the acute (paw edema induced by carrageenin, dextran or histamine, pleurisy induced by carrageenin and capsaicin-induced mouse ear edema) or chronic (air pouch) models used. No toxic signs were observed after administration of the different extracts up to 2 g/kg body wt., p.o.

Collectively, the results confirmed folk information indicating presence of analgesic, mild laxative and potent inhibition of gastric secretion activities in the aqueous extracts of S. cayennensis. The results do not, however confirm the folk use of the plant as an antiinflammatory medicine.     

Chemical xenogenization of murine lymphoma cells with triazene derivatives: Immunotoxicological studies

Summary Equitoxic doses of 5-(3-3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC) and aryl-triazene derivatives (compounds all capable of inducing a marked increase in murine tumor cell immunogenicity) were studied for their effects on the host immune system. At different times after drug exposure the animals were tested for allograft responses, competence in producing lymphocytes active in lethal graft-versus-host disease, delayed-type hypersensitivity, humoral antibody production, and mitogen responsiveness. While some of the aryl-triazenes tested (DM-COOK DM-NO₂) showed a pattern of immunodepression similar to that of DTIC, others were less (MIC, MM-COOK, MM-Cl) or far less (DM-Cl, MM-NO₂) active than DTIC in impairing host immunocompetence, although all retained or even augmented their ability to induce chemical xenogenization.This work was supported by Progetto Finalizzato Controllo della Crescita Neoplastica contracts no. 83.00815.96 and no. 83.00838.96 (CNR, Rome, Italy)     

The role of alpha-adrenergic receptors in the vasoconstrictor response induced by indomethacin in the kidney.

The effect of indomethacin, an inhibitor of prostaglandin (PG) synthesis, was studied on the renal circulation, Na+ and water excretion in anaesthesized dogs during alpha-receptor inhibition. Indomethacin decreased cortical blood flow (CBFcontr, 454 +/- 142; CBFindo, 332 +/- 51 ml per min per 100 g; p less than 0.02) as well as medullary blood flow (OMBFcontr, 339 +/- 95; OMBFindo, 183 +/- 46 ml per min per 100 g; p less than 0.001), salt and water excretion, further it caused a shift in the intrarenal blood flow distribution toward the cortex. Alpha-blockade prevented the indomethacin-induced vasoconstriction in the cortex (CBF alpha inhibition + indo, 455 +/- 76 ml per min per 100 g) but not in the medullar (OMBF alpha inhibition + indo, 259 +/- 102 ml per min per 100 g, p less than 0.05). Alpha-blockade failed to prevent the indomethacin-induced antidiuresis, antinatriuresis and the intrarenal blood flow redistribution. GFR remained unaffected in all three series of studies. Our experimental findings are in line with the presumption that alpha-receptors are involved in the renal circulatory changes caused by indomethacin, probably as a result of an enhanced NE release during the inhibition of PG production. A NE--PG feed back mechanism is suggested in the regulation of renal circulation. The reduction of salt and water output induced by indomethacin appears to be independent of the alterations in renal haemodynamics, and seems rather to be the result of enhanced Na+ reabsorption, predominantly at the distal segment of the nephron, in the absence of PG, and/or a direct action of indomethacin.     

Intrinsic prostacyclin contributes to exudation induced by bradykinin or carrageenin: a study on the paw edema induced in IP-receptor-deficient mice

To prove that prostaglandin I2 (PGI2) is a major prostaglandin involved in bradykinin-induced exudation, we examined carrageenin- or bradykinin-induced paw edema in prostacyclin receptor-deficient mice (IPKO). Paw volume of wild-type mice (IPWT) increased gradually 5-6 hr after the carrageenin injection in a similar manner as in ICR mice, but the swelling in IPKO mice was significantly smaller (about 60% of the IPWT volume). Indomethacin, at 10 mg/kg, suppressed the swelling of the IPWT paw to the level of the non-pretreated IPKO, which was not affected by indomethacin, confirming the previous result that PGI2 is a major prostaglandin involved in the swelling. The paw edema of IPWT and IPKO was significantly attenuated by the nonpeptide bradykinin B2-receptor antagonist FR173657, at 30 mg/kg, to the same level of swelling, indicating kinin involvement. Injection of bradykinin (1.2 nmole) into the paw caused rapid edema, which peaked around 15 min in both mice. However, the edema induced in IPKO was smaller and almost at the same level as that elicited in the indomethacin-treated IPWT, suggesting that edema induced by bradykinin includes the intrinsic effect of PGI2. Concomitant injection of carbacyclin with bradykinin caused enhancement of edema in IPWT mice but not in IPKO mice, indicating that intrinsic PGI2 could cause enhancement of bradykinin- or even carrageenin-induced edema formation. These results clearly demonstrate that bradykinin released by carrageenin may be a key mediator to induce PGI2 formation, and both autacoids work together to induce enhanced inflammatory exudation.     

Metallothionein induction by nonsteroidal antiinflammatory drugs

In the present study we report on the effects of commonly used nonsteroidal antiinflammatory drugs on metallothionein (MT) and MT-I mRNA levels. A single dose of chloroquine (100 mg/kg), diclofenac (100 mg/kg), indomethacin (10 mg/kg), or piroxicam (100 mg/kg) was administered ip to C57B1 mice. After 18 h, MT levels were determined with a Cd-saturation radioassay. MT-I mRNA levels were measured by Northern Blot analyses using a probe containing the mouse MT-I gene. All drugs tested caused an increase in the MT content of the liver but not of the kidneys and lung. The lowest and highest effects were observed with chloroquine (8 times the control value) and diclofenac (18 times), respectively. In accordance with the stimulation of MT synthesis, increased accumulation of hepatic MT-I mRNA could be demonstrated. These results indicate that elevated MT levels may contribute to the effectiveness of nonsteroidal antiinflammatory drugs in the treatment of rheumatoid arthritis (RA).     

Effects of anti-inflammatory drugs on fever and neutrophilia induced by Clostridium difficile toxin B

This study investigated the ability of Clostridium difficile toxin B, isolated from the VPI 10463 strain, to induce fever and neutrophilia in rats. Intravenous injection of toxin B (0.005-0.5 mug/kg) evoked a dose-dependent increase in body temperature. The febrile response to 0.5 mug/kg of the toxin started in 2.5 h, peaked at 5 h, and subsided fully within 24 h. Toxin B also induced a dosedependent neutrophilia. Pretreatment with indomethacin (2 mg/kg, i.p.) did not affect the neutrophilia induced by toxin B, but significantly reduced the febrile response measured 4 to 8 h after toxin B injection. Dexamethasone (0.5 mg/ kg) also markedly diminished the febrile response induced by toxin B. These results show that Clostridium difficile toxin B induced a febrile response susceptible to inhibition by dexamethasone and indomethacin. Furthermore, they suggest that prostaglandins are not involved in the neutrophilia caused by this toxin.