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282 Effects of nifedipine on histopathological changes of kidney in diabetic rat
s78
Poster Session P12. Drug toxicology
dose (20 mg/kg) did not increased general indomethacine (Imc) toxicity (mortality rate) and decreased Imc-analgesic effect (hote plate and acetic acid test). Ver protected against the hepatotoxic effect of Imc (risen glutamate-pyruvate transaminase activity in blood plasma was normalized). Although Ver alone decreased significantly the alkaline content in the blood plasma, when it was applied in combination with Imc this parameter was normalized. The bleeding time also was not affected by the combination of Ver+Imc. Strong ulceroprotective effect of Ver was established in Imc-treated animals. The possible mechanisms of ulceroprotective effect of Ver as a modulator of Ca2+ channels, as an antistressor agent or a stimulant of prostaglandin E-synthesis were discussed. 282
EFFECTS OF NIFEDIPINE ON HISTOPATHOLOGICAL CHANGES OF KIDNEY IN DIABETIC RAT
H. Mahdavinasab 1 , H. Mehrani 2 , H. Imani 1 , H. Sadrai 1 , H. Dashtnavard 1 , M. Mofid 1 , M. Ahmadian 1 . 1 Department of Anatomy, Faculty of Medicine, Baghyiatallah University, Tehran-Iran; 2 Department of Biochemistry, Faculty of Medicine, Baghyiatallah University, Tehran-Iran Diabetic nephropathy is the most common renal disease, which is complicated by another form of glumerular disease. The most characteristics lesion of diabetic glomerulonephropathy is nodular intercapillary glumerulosclerosis. In this study we designed three groups: 1) Diabetic group that diabetes was induced by sterptozotocin. 2) Diabetic + Nifedipine group that after 7 day of diabetes were orally treated daily with 40 mg/kg nifedipine for 4 month. 3) Control group were received water alone. For histopathological study all group rats were sacrificed and their kidneys were taken, fixed, dehydrated, embedded in paraffin, and sectioned at 5 micron from different area. The sections were then stained with H&E technique. The results of this study showed that internal diameter of collecting tubules were significantly decreased both in Diabetic and Diabetic + Nifedipine groups when compared to control group (respectively P<0.01 and P<0.05). Diffuse nodular glumerulosclerosis, acute tubular necrosis, intranuclear inclusions and some large and acellular nodules were also significantly increased in Diabetic and Diabetic + Nifedipine groups when compared to control group (respectively P<0.01 and P<0.05). In conclusion: 1) Diabetes could induce glumerulonephropathy in rats. 2) Nifedipine as a calcium blocker could reduce glumerulonephropathy in diabetic Rats. 283
EVALUATION OF ANTISECRETORY EFFECT OF GLYCERYL TRINITRATE IN RATS TREATED WITH CYCLOOXYGENASE NON-SELECTIVE AND SELECTIVE NSAIDs
´ c 3 , Z. Milovanovi´c 2 , D. Bokonji´c 2 . R. Velev 1 , S. Dobri´c 2 , V. Cupi´ 1 Faculty of Veterinary Medicine, Lazar Pop Traajkov 5–7, 1000 Skopje, Republic of Macedonia; 2 National Poison Control Center, Military Medical Academy, Belgrade, Yugoslavia and; 3 Faculty of Veterinary Medicine, Belgrade, Yugoslavia Indomethacin (IND) and nimesulide (NIM) are very potent nonsteroidal anti-inflammatory drugs (NSAIDs). The first one is cyclooxygenase (COX) non-selective NSAID, while the second belongs to the group of COX-2 selective inhibitors. Our earlier investigations showed that both drugs produced dose-related gastrotoxic effects after single administration, but they were significantly more pronounced in rats given IND than NIM. Recently it was demonstrated that nitric oxide (NO) may play an important role in gastric mucosal defense. The aim of this study was to evaluate the influence of glyceryl trinitrate (GTN), NO-generating compound, on secretion and total acid output of gastric juice in rats treated by IND and NIM. Adult male Wistar rats deprived of food for 24h and pylorus-ligated were used in the experiment. NSAIDs tested were given by gastric tube in a single dose of 25 mg/kg. GTN in gastroprotective dose of 6.25 mg/kg and 0.78 mg/kg was given p.o. or i.p. immediately after IND or NIM, respectively. In a separate group of experiments L-arginine (100 mg/kg i.p.), as an endogenous NO-donor, was given alone or concomitantly with L-NAME (1mg/kg i.p.), an inhibitor
of NO-synthase, immediately after indomethacin. Four hours after administration of NSAIDs the animals were sacrificed, gastric juice was collected and its acidity was determined by titration with 0.1 N NaOH by using phenolphtaleine as an indicator. In rats given IND, but not NIM significant increase of gastric acidity was found, without significant changes in the volume of gastric juice. Treatment by GTN, regardless the route of administration normalised acidity of gastric juice without influence on its volume in rats treated with IND. L-arginine, like GTN normalised acidity of gastric juice in rats treated by IND, while L-NAME completely abolished this effect of L-arginine. Our results suggest that an increase in gastric acidity caused by IND could at least partly be responsible for its high gastrotoxic potential. Mechanism of antiulcer activity of GTN, among the other ones, could include NO-mediated processes influencing the gastric acidity. 284
BENZAMIDE-BASED TRICHOSTATIN A ANALOGUES ARE POTENT AND METABOLICALLY STABLE INHIBITORS OF HISTONE DEACETYLASE
G. Elaut 1 , G. Laus 2 , P. Papeleu 1 , V. Breckx 2 , J. Van Hemel 2 , M. Erra 3 , G. Brosch 3 , S. Snykers 1 , T. Vanhaecke 1 , D. Tourwé 2 , V. Rogiers 1 . Departments of Toxicology1 and Organic Chemistry2 , Vrije Universiteit Brussel, Brussels, Belgium, 3 Department of Microbiology, University of Innsbruck, Innsbruck, Austria Histone deacetylase (HDAC) inhibitors show great therapeutic potential for the treatment of diseases characterized by dedifferentiation and aberrant proliferation of mature cells, such as cancer. This stems from their ability to convert proliferating cells to a differentiated, non-proliferating phenotype through modulation of the eukaryotic chromatin structure, affecting DNA accessibility and gene expression. A number of hydroxamate-based HDAC inhibitors have been shown to inhibit tumor growth both in vitro and in vivo, and several of them are currently in clinical trial. Little is known, however, with respect to their pharmacokinetic and toxicologic properties, which are important determinants of their further success as a drug. In this context, the use of liver-derived in vitro models can provide valuable information on both biotransformation and drug-induced toxicity. They are of great value in the early discovery stage as well as throughout the rest of the drug development process. The natural compound, Trichostatin A (TSA), was the first specific hydroxamate-based HDAC inhibitor discovered, active in the nanomolar range. Previous in vitro studies performed in our lab showed a rapid and extensive phase I biotransformation of TSA in rat hepatocyte suspensions, implying a limited in vivo efficacy. In this study, we therefore focused on the synthesis of nine benzamide-containing structural analogues of TSA. HPLC-MS and tandem MS enabled us to separate and identify the analogues and their phase I metabolites. For the quantification of their inhibitory potencies towards hepatocyte HDAC, the removal of 3 H-acetate from prelabeled histones was measured. Acute cytotoxic effects caused by the mother compounds and their metabolites were evaluated by lactate dehydrogenase leakage in the incubation medium of rat hepatocytes. Our results show that benzamide-containing analogues of TSA represent a group of easily synthesizable, metabolically stable compounds that selectively inhibit HDAC in the (low) micromolar range and show little toxicity towards well-differentiated cells. 285
PL 14736: A 4-WEEK INTRAVENOUS TOXICITY STUY IN RATS FOLLOWED BY A 4-WEEK RECOVERY PERIOD AND A 4-WEEK INTRAVENOUS TOXICITY STUDY IN DOGS
M. Veljaˇca 1 , Ž. Krni´c 1 , Ž. Ferenˇci´c 1 , M. Kolega 1 . PLIVA Pharmaceutical Industry Inc., Zagreb, Croatia PL 14736 is a synthetic pentadecapeptide that shows protective and healing activity in trinitrobenzene sulphonic acid (TNBS) model of colitis in rats. It has been developed for the treatment of ulcerative colitis. Repeated dose toxicity studies were performed in rats (Sprague Dawley Crl:CD (SD) BR) and dogs (Beagle). Groups of male and female animals received single daily doses of 0, 1, 3 or 10 mg/kg/day
Poster Session P12. Drug toxicology
dose (20 mg/kg) did not increased general indomethacine (Imc) toxicity (mortality rate) and decreased Imc-analgesic effect (hote plate and acetic acid test). Ver protected against the hepatotoxic effect of Imc (risen glutamate-pyruvate transaminase activity in blood plasma was normalized). Although Ver alone decreased significantly the alkaline content in the blood plasma, when it was applied in combination with Imc this parameter was normalized. The bleeding time also was not affected by the combination of Ver+Imc. Strong ulceroprotective effect of Ver was established in Imc-treated animals. The possible mechanisms of ulceroprotective effect of Ver as a modulator of Ca2+ channels, as an antistressor agent or a stimulant of prostaglandin E-synthesis were discussed. 282
EFFECTS OF NIFEDIPINE ON HISTOPATHOLOGICAL CHANGES OF KIDNEY IN DIABETIC RAT
H. Mahdavinasab 1 , H. Mehrani 2 , H. Imani 1 , H. Sadrai 1 , H. Dashtnavard 1 , M. Mofid 1 , M. Ahmadian 1 . 1 Department of Anatomy, Faculty of Medicine, Baghyiatallah University, Tehran-Iran; 2 Department of Biochemistry, Faculty of Medicine, Baghyiatallah University, Tehran-Iran Diabetic nephropathy is the most common renal disease, which is complicated by another form of glumerular disease. The most characteristics lesion of diabetic glomerulonephropathy is nodular intercapillary glumerulosclerosis. In this study we designed three groups: 1) Diabetic group that diabetes was induced by sterptozotocin. 2) Diabetic + Nifedipine group that after 7 day of diabetes were orally treated daily with 40 mg/kg nifedipine for 4 month. 3) Control group were received water alone. For histopathological study all group rats were sacrificed and their kidneys were taken, fixed, dehydrated, embedded in paraffin, and sectioned at 5 micron from different area. The sections were then stained with H&E technique. The results of this study showed that internal diameter of collecting tubules were significantly decreased both in Diabetic and Diabetic + Nifedipine groups when compared to control group (respectively P<0.01 and P<0.05). Diffuse nodular glumerulosclerosis, acute tubular necrosis, intranuclear inclusions and some large and acellular nodules were also significantly increased in Diabetic and Diabetic + Nifedipine groups when compared to control group (respectively P<0.01 and P<0.05). In conclusion: 1) Diabetes could induce glumerulonephropathy in rats. 2) Nifedipine as a calcium blocker could reduce glumerulonephropathy in diabetic Rats. 283
EVALUATION OF ANTISECRETORY EFFECT OF GLYCERYL TRINITRATE IN RATS TREATED WITH CYCLOOXYGENASE NON-SELECTIVE AND SELECTIVE NSAIDs
´ c 3 , Z. Milovanovi´c 2 , D. Bokonji´c 2 . R. Velev 1 , S. Dobri´c 2 , V. Cupi´ 1 Faculty of Veterinary Medicine, Lazar Pop Traajkov 5–7, 1000 Skopje, Republic of Macedonia; 2 National Poison Control Center, Military Medical Academy, Belgrade, Yugoslavia and; 3 Faculty of Veterinary Medicine, Belgrade, Yugoslavia Indomethacin (IND) and nimesulide (NIM) are very potent nonsteroidal anti-inflammatory drugs (NSAIDs). The first one is cyclooxygenase (COX) non-selective NSAID, while the second belongs to the group of COX-2 selective inhibitors. Our earlier investigations showed that both drugs produced dose-related gastrotoxic effects after single administration, but they were significantly more pronounced in rats given IND than NIM. Recently it was demonstrated that nitric oxide (NO) may play an important role in gastric mucosal defense. The aim of this study was to evaluate the influence of glyceryl trinitrate (GTN), NO-generating compound, on secretion and total acid output of gastric juice in rats treated by IND and NIM. Adult male Wistar rats deprived of food for 24h and pylorus-ligated were used in the experiment. NSAIDs tested were given by gastric tube in a single dose of 25 mg/kg. GTN in gastroprotective dose of 6.25 mg/kg and 0.78 mg/kg was given p.o. or i.p. immediately after IND or NIM, respectively. In a separate group of experiments L-arginine (100 mg/kg i.p.), as an endogenous NO-donor, was given alone or concomitantly with L-NAME (1mg/kg i.p.), an inhibitor
of NO-synthase, immediately after indomethacin. Four hours after administration of NSAIDs the animals were sacrificed, gastric juice was collected and its acidity was determined by titration with 0.1 N NaOH by using phenolphtaleine as an indicator. In rats given IND, but not NIM significant increase of gastric acidity was found, without significant changes in the volume of gastric juice. Treatment by GTN, regardless the route of administration normalised acidity of gastric juice without influence on its volume in rats treated with IND. L-arginine, like GTN normalised acidity of gastric juice in rats treated by IND, while L-NAME completely abolished this effect of L-arginine. Our results suggest that an increase in gastric acidity caused by IND could at least partly be responsible for its high gastrotoxic potential. Mechanism of antiulcer activity of GTN, among the other ones, could include NO-mediated processes influencing the gastric acidity. 284
BENZAMIDE-BASED TRICHOSTATIN A ANALOGUES ARE POTENT AND METABOLICALLY STABLE INHIBITORS OF HISTONE DEACETYLASE
G. Elaut 1 , G. Laus 2 , P. Papeleu 1 , V. Breckx 2 , J. Van Hemel 2 , M. Erra 3 , G. Brosch 3 , S. Snykers 1 , T. Vanhaecke 1 , D. Tourwé 2 , V. Rogiers 1 . Departments of Toxicology1 and Organic Chemistry2 , Vrije Universiteit Brussel, Brussels, Belgium, 3 Department of Microbiology, University of Innsbruck, Innsbruck, Austria Histone deacetylase (HDAC) inhibitors show great therapeutic potential for the treatment of diseases characterized by dedifferentiation and aberrant proliferation of mature cells, such as cancer. This stems from their ability to convert proliferating cells to a differentiated, non-proliferating phenotype through modulation of the eukaryotic chromatin structure, affecting DNA accessibility and gene expression. A number of hydroxamate-based HDAC inhibitors have been shown to inhibit tumor growth both in vitro and in vivo, and several of them are currently in clinical trial. Little is known, however, with respect to their pharmacokinetic and toxicologic properties, which are important determinants of their further success as a drug. In this context, the use of liver-derived in vitro models can provide valuable information on both biotransformation and drug-induced toxicity. They are of great value in the early discovery stage as well as throughout the rest of the drug development process. The natural compound, Trichostatin A (TSA), was the first specific hydroxamate-based HDAC inhibitor discovered, active in the nanomolar range. Previous in vitro studies performed in our lab showed a rapid and extensive phase I biotransformation of TSA in rat hepatocyte suspensions, implying a limited in vivo efficacy. In this study, we therefore focused on the synthesis of nine benzamide-containing structural analogues of TSA. HPLC-MS and tandem MS enabled us to separate and identify the analogues and their phase I metabolites. For the quantification of their inhibitory potencies towards hepatocyte HDAC, the removal of 3 H-acetate from prelabeled histones was measured. Acute cytotoxic effects caused by the mother compounds and their metabolites were evaluated by lactate dehydrogenase leakage in the incubation medium of rat hepatocytes. Our results show that benzamide-containing analogues of TSA represent a group of easily synthesizable, metabolically stable compounds that selectively inhibit HDAC in the (low) micromolar range and show little toxicity towards well-differentiated cells. 285
PL 14736: A 4-WEEK INTRAVENOUS TOXICITY STUY IN RATS FOLLOWED BY A 4-WEEK RECOVERY PERIOD AND A 4-WEEK INTRAVENOUS TOXICITY STUDY IN DOGS
M. Veljaˇca 1 , Ž. Krni´c 1 , Ž. Ferenˇci´c 1 , M. Kolega 1 . PLIVA Pharmaceutical Industry Inc., Zagreb, Croatia PL 14736 is a synthetic pentadecapeptide that shows protective and healing activity in trinitrobenzene sulphonic acid (TNBS) model of colitis in rats. It has been developed for the treatment of ulcerative colitis. Repeated dose toxicity studies were performed in rats (Sprague Dawley Crl:CD (SD) BR) and dogs (Beagle). Groups of male and female animals received single daily doses of 0, 1, 3 or 10 mg/kg/day