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Histological changes in liver and jejunum induced by R103757, a new inhibitor of microsomal triglyceride transfer protein (MTP)
Thursday, 27 May 1999 Poster session: Lipid lowering drugs In contrast, in dogs exposure increased dose-proportionally. In rats, but not in dogs, females showed higher exposure than males. Male rats showed highest exposure to M2, whereas female rats and (pregnant) rabbits showed highest exposure to M l. in dogs, as in man, exposure was highest to M3. At the highest doses in all species the exposure to RI03757, MI and M2 (and M3 in rabbits) exceeded exposure in man (100 rag). At the mid-doses the same applies for R103757 and M2 in rabbits and Ml in female rats and rabbits. Possible species differences in plasma protein binding were not taken into account (remains to be determined). At 5 mg/kg/day, R103757 increased plasma total cholesterol in rats, did not affect plasma lipids in rabbits and decreased total cholesterol, phospholipids and triglycerides in plasma in dogs; in man a dose of 100 mg/day had a pronounced cholesterol lowering effect. The widely diverging pharmacodynamic effects in the species examined can probably only partly be explained by differences in R103757 pharmacokinetics. METABOLISM OF RI03757, A NEW MTP INHIBITOR, IN ANIMAL SPECIES AND IN MAN, IN VITRO AND IN VIVO
39
Table 1: Median percent change from baseline in lipid variables in 3 different phase i trials after 4 weeks of treatment with:
150 mg (solution)
100 mg (tablet)
200 mg (tablet)
300 mg (tablet)
ApoB
-33""
-21""
-26""
-36" °
HDL
+20
+5
+5
+13 -26" •
LDL
-36""
-25""
-26""
cholesterol
-29""
-13""
-23 ° °
-25""
ttiglycendes
-25""
-20
-29
-39""
Lp (a)
~1.3" °
-22
-50"
-17
( ' p < 0.05. " ' p < 0.01 statistically significantly different from baseline)
These phase I trials consistently show that RI03757 has the potential to reduce apolipoprotein B levels, total cholesterol, LDL cholesterol, triglycerides and Lp(a), and to increase HDL cholesterol in man. It is concluded that MTP inhibition may represent a valuable new approach for lipid lowering in patients with dyslipidemia.
W. Bode, M, Vermeir, J. Hendrickx, G, Mannens, R. Woestenborghs, K. Lavrijsen, W. Meuldermans. Department ofPharmacokinetics, Janssen Researvh Foundation, Beerse. Belgium
HISTOLOGICAL CHANGES IN LIVER AND JEJUNUM INDUCED BY R!03757, A NEW "INHIBITOR OF M1CROSOMAL TRIGLYCER1DE TRANSFER PROTEIN (MTP)
Objective: RI03757 is an inhibitor of microsomal triglyceride transfer protein (MTP), which is in development as a lipid-lowering drug. In order to validate the various animal models used in pharmacological and toxicity studies, in vitro metabolism of RI03757 was examined in liver preparations of mouse, rat, rabbit, dog and man, and compared with in uivo metabolism data. Methods: In vitro metabolism was studied using hepatocyte suspensions, hepatocyte primary cell cultures, hepatic microsomes and hepatic 12,000 x g supernatant prepared from livers of mouse, rat, rabbit, dog and man. The liver preparations were incubated with [14-C]-R103757, in which the label was located at the metabolically stable 2-position in the 1,3-dioxolane ring. The cytochrome P-450 (CYP) forms involved in the metabolism of R103757 were identified using human microsomes incubated with RI03757 and diagnostic CYP inhibitors. In oiuo metabolism was studied by determination of metabolites in plasma, obtained from toxicity studies in various animal species and from a pharmacokinetic study in human volunteers. Sample analysis was performed by HPLC with UV detection (and, if applicable, tandem radioactivity detection). Results and Conclusions: The main in oitro hepatic metabolic pathway in all species including man was oxidation, with the formation of metabolites MI, M2 and M3. In human liver, glucuronidation of R103757 was also a major pathway, also occurring to some extent in liver of all animal species. O-Dealkylation followed by glucuronidation was observed in all species; however, as O-dealkylation was also observed in boiled liver preparations, it may not be metabolic in nature. Inhibition of metabolism of RI03757 into MI, M2 and M3 in human microsomes was largest with troleandomycin and gestodene, two CYP3A4 diagnostic inhibitors. Inhibition with other diagnostic inhibitors was much more limited. For confirmation, R103757 was incubated with yeast microsomes containing heterologously expressed human liver CYP3A4 and rat reductase, resulting in the formation of all three metabolites MI, M2 and M3. The metabolites M l, M2 and M3 were also found at substantial levels in plasma of mice, rats, rabbits, dogs and man after oral dosing with R103757.
U Lammens, H. Borghys, P. Roevens, J. Vandenberghe. Janssen Research Foundation. Turnhoutseweg 30, B-2340 Beerse. Belgium
CLINICAL EXPERIENCE WITH RI03757, A POTENT INHIBITOR OF MICROSOMAL TRIGLYCERIDE TRANSFER PROTEIN (MTP) A.G. Dupont, J. De Cree, R. Vermeulen, W. Salvo, E Deridder. danssen Research Foundation. Turnhoutseweg 30, B-2340 Beerse. Belgium R103757, a novel MTP-inhibitor, has been investigated in phase 1 trials in healthy volunteers. In a placebo-controlled repeated dose-escalation trial with once daily administration of R103757 during 11 days, (25 mg/day up to 150 mg/day given as an oral solution), 50 mg once daily significantly reduced apolipoprotein B (-14%) and LDL cholesterol (-21%); with the 75 mg dose and higher, there was also a statistically significant reduction in total cholesterol, and a trend to a reduction in triglycerides and lipoprotein a [Lp(a)]. Further double-blind placebo-controlled phase I trials in healthy volunteers with single daily oral administration of 150 mg (as cyclodextrin containing oral solution), and of 100 rag, 200 mg, 300 mg daily (as a tablet formulation) for 4 weeks, confirmed the lipid modifying effects. (Table 1). There were no consistent effects on apolipoprotein AI levels.
R103757 is an MTP inhibitor currently in development as a lipid-lowering compound. MTP, required for triglyceride-rich lipoprotein assembly, is localized in the endoplasmic reticulum o f hepatocytes and enterocytes. The pharmacological target organs of an MTP inhibitor, the liver and jejunum, were examined histologically in dogs treated with RI03757. Normolipidemic dogs were dosed orally at various doses ranging from 1.25 mg/kg/day to 5 mg/kg/day for 2 weeks up to 3 months. Studies also included control dogs dosed with the vehicle. Plasma triglyceride, phospholipid and cholesterol levels were measured. Liver and jejunum of vehicledosed dogs and R I03757-dosed dogs were examined histologicaly at the light microscopic level (haematoxylin-eosin (H.E.) stained paraffin sections and oil red O-stained frozen sections) and at the electron microscopic level. The lipid content of intestinal mucosa and liver was determined biochemicaly. Oral dosing with RI03757 resulted in a time- and dose-dependent decrease in plasma cholesterol, phospholipids and triglycerides, demonstrating the pharmacological effectiveness of RI03757 in the dog. In the liver of RI03757-dosed dogs, an increase in small lipid droplets within hepatocytes was observed on oil red O-stained frozen sections. In the jejunum of RI03757-dosed dogs, swelling and vacuolation of enterocytes was visible on H.E.-stained sections. On oil red O-stained frozen sections, the vacuolation ofenterocytes corresponded to an increased presence o f lipid droplets. Biochemical determination of the lipid content demonstrated that the histologicaly observed oil red O-positive droplets in liver and intestinal mucosa consist mainly of triglycerides. No degenerative changes were observed at the light microscopic level, neither in the liver nor in the jejunum. Electron microscopic examination of liver and jejunum o f R103757-dosed dogs confirmed the light microscopic observations. An increase in small lipid droplets within hepatocytes and enterocytes was the only ultrastructural change. Ultrastructural changes o f cell organelles were absent. Results o f histological examination indicated that the changes reach a maximum rather than a stage of continous progression and that the liver changes are completely reversible after 2 weeks of drug withdrawal. The RI03757-induced increase in lipid droplets in dog hepatocytes and enterocytes, is completely in line with the primary pharmacological effect o f the compound: inhibition of the assembly of triglyceride-rich lipoproteins due to inhibition of MTP. Similar changes are observed in patients with abetalipoproteinaemia, a disease caused by deficiency of MTP. THE EFFICACY OF SIMVASTATIN IN PATIENTS WITH HYPERTRIGLYCERIDEMIA J. isaacsohn 1, D. Hunninghake, H.A. Schrott, C.A. Dujovne, R. Knopp, S.R. Weiss, H. Bays, J.R. Crouse, M.H. Davidson, E Samuel, L.M. Keilson, J. McKenney, S.G. Korenman, A.S. Dobs, P.M. LaskatT.ewski, M. Liu, D. Plotkin, Y.B. Mitchel. t Metabolic and dtherosclerosis Research Center, Cincinnati, Ohio; Merck Research Labs, Rahway, NJ, USA Simvastatin (S) is effective in patients with hyperehotesterolemia and combined hyperlipidemia, however few studies have evaluated patients whose
71st EAS Congress and Satellite Symposia
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39
Table 1: Median percent change from baseline in lipid variables in 3 different phase i trials after 4 weeks of treatment with:
150 mg (solution)
100 mg (tablet)
200 mg (tablet)
300 mg (tablet)
ApoB
-33""
-21""
-26""
-36" °
HDL
+20
+5
+5
+13 -26" •
LDL
-36""
-25""
-26""
cholesterol
-29""
-13""
-23 ° °
-25""
ttiglycendes
-25""
-20
-29
-39""
Lp (a)
~1.3" °
-22
-50"
-17
( ' p < 0.05. " ' p < 0.01 statistically significantly different from baseline)
These phase I trials consistently show that RI03757 has the potential to reduce apolipoprotein B levels, total cholesterol, LDL cholesterol, triglycerides and Lp(a), and to increase HDL cholesterol in man. It is concluded that MTP inhibition may represent a valuable new approach for lipid lowering in patients with dyslipidemia.
W. Bode, M, Vermeir, J. Hendrickx, G, Mannens, R. Woestenborghs, K. Lavrijsen, W. Meuldermans. Department ofPharmacokinetics, Janssen Researvh Foundation, Beerse. Belgium
HISTOLOGICAL CHANGES IN LIVER AND JEJUNUM INDUCED BY R!03757, A NEW "INHIBITOR OF M1CROSOMAL TRIGLYCER1DE TRANSFER PROTEIN (MTP)
Objective: RI03757 is an inhibitor of microsomal triglyceride transfer protein (MTP), which is in development as a lipid-lowering drug. In order to validate the various animal models used in pharmacological and toxicity studies, in vitro metabolism of RI03757 was examined in liver preparations of mouse, rat, rabbit, dog and man, and compared with in uivo metabolism data. Methods: In vitro metabolism was studied using hepatocyte suspensions, hepatocyte primary cell cultures, hepatic microsomes and hepatic 12,000 x g supernatant prepared from livers of mouse, rat, rabbit, dog and man. The liver preparations were incubated with [14-C]-R103757, in which the label was located at the metabolically stable 2-position in the 1,3-dioxolane ring. The cytochrome P-450 (CYP) forms involved in the metabolism of R103757 were identified using human microsomes incubated with RI03757 and diagnostic CYP inhibitors. In oiuo metabolism was studied by determination of metabolites in plasma, obtained from toxicity studies in various animal species and from a pharmacokinetic study in human volunteers. Sample analysis was performed by HPLC with UV detection (and, if applicable, tandem radioactivity detection). Results and Conclusions: The main in oitro hepatic metabolic pathway in all species including man was oxidation, with the formation of metabolites MI, M2 and M3. In human liver, glucuronidation of R103757 was also a major pathway, also occurring to some extent in liver of all animal species. O-Dealkylation followed by glucuronidation was observed in all species; however, as O-dealkylation was also observed in boiled liver preparations, it may not be metabolic in nature. Inhibition of metabolism of RI03757 into MI, M2 and M3 in human microsomes was largest with troleandomycin and gestodene, two CYP3A4 diagnostic inhibitors. Inhibition with other diagnostic inhibitors was much more limited. For confirmation, R103757 was incubated with yeast microsomes containing heterologously expressed human liver CYP3A4 and rat reductase, resulting in the formation of all three metabolites MI, M2 and M3. The metabolites M l, M2 and M3 were also found at substantial levels in plasma of mice, rats, rabbits, dogs and man after oral dosing with R103757.
U Lammens, H. Borghys, P. Roevens, J. Vandenberghe. Janssen Research Foundation. Turnhoutseweg 30, B-2340 Beerse. Belgium
CLINICAL EXPERIENCE WITH RI03757, A POTENT INHIBITOR OF MICROSOMAL TRIGLYCERIDE TRANSFER PROTEIN (MTP) A.G. Dupont, J. De Cree, R. Vermeulen, W. Salvo, E Deridder. danssen Research Foundation. Turnhoutseweg 30, B-2340 Beerse. Belgium R103757, a novel MTP-inhibitor, has been investigated in phase 1 trials in healthy volunteers. In a placebo-controlled repeated dose-escalation trial with once daily administration of R103757 during 11 days, (25 mg/day up to 150 mg/day given as an oral solution), 50 mg once daily significantly reduced apolipoprotein B (-14%) and LDL cholesterol (-21%); with the 75 mg dose and higher, there was also a statistically significant reduction in total cholesterol, and a trend to a reduction in triglycerides and lipoprotein a [Lp(a)]. Further double-blind placebo-controlled phase I trials in healthy volunteers with single daily oral administration of 150 mg (as cyclodextrin containing oral solution), and of 100 rag, 200 mg, 300 mg daily (as a tablet formulation) for 4 weeks, confirmed the lipid modifying effects. (Table 1). There were no consistent effects on apolipoprotein AI levels.
R103757 is an MTP inhibitor currently in development as a lipid-lowering compound. MTP, required for triglyceride-rich lipoprotein assembly, is localized in the endoplasmic reticulum o f hepatocytes and enterocytes. The pharmacological target organs of an MTP inhibitor, the liver and jejunum, were examined histologically in dogs treated with RI03757. Normolipidemic dogs were dosed orally at various doses ranging from 1.25 mg/kg/day to 5 mg/kg/day for 2 weeks up to 3 months. Studies also included control dogs dosed with the vehicle. Plasma triglyceride, phospholipid and cholesterol levels were measured. Liver and jejunum of vehicledosed dogs and R I03757-dosed dogs were examined histologicaly at the light microscopic level (haematoxylin-eosin (H.E.) stained paraffin sections and oil red O-stained frozen sections) and at the electron microscopic level. The lipid content of intestinal mucosa and liver was determined biochemicaly. Oral dosing with RI03757 resulted in a time- and dose-dependent decrease in plasma cholesterol, phospholipids and triglycerides, demonstrating the pharmacological effectiveness of RI03757 in the dog. In the liver of RI03757-dosed dogs, an increase in small lipid droplets within hepatocytes was observed on oil red O-stained frozen sections. In the jejunum of RI03757-dosed dogs, swelling and vacuolation of enterocytes was visible on H.E.-stained sections. On oil red O-stained frozen sections, the vacuolation ofenterocytes corresponded to an increased presence o f lipid droplets. Biochemical determination of the lipid content demonstrated that the histologicaly observed oil red O-positive droplets in liver and intestinal mucosa consist mainly of triglycerides. No degenerative changes were observed at the light microscopic level, neither in the liver nor in the jejunum. Electron microscopic examination of liver and jejunum o f R103757-dosed dogs confirmed the light microscopic observations. An increase in small lipid droplets within hepatocytes and enterocytes was the only ultrastructural change. Ultrastructural changes o f cell organelles were absent. Results o f histological examination indicated that the changes reach a maximum rather than a stage of continous progression and that the liver changes are completely reversible after 2 weeks of drug withdrawal. The RI03757-induced increase in lipid droplets in dog hepatocytes and enterocytes, is completely in line with the primary pharmacological effect o f the compound: inhibition of the assembly of triglyceride-rich lipoproteins due to inhibition of MTP. Similar changes are observed in patients with abetalipoproteinaemia, a disease caused by deficiency of MTP. THE EFFICACY OF SIMVASTATIN IN PATIENTS WITH HYPERTRIGLYCERIDEMIA J. isaacsohn 1, D. Hunninghake, H.A. Schrott, C.A. Dujovne, R. Knopp, S.R. Weiss, H. Bays, J.R. Crouse, M.H. Davidson, E Samuel, L.M. Keilson, J. McKenney, S.G. Korenman, A.S. Dobs, P.M. LaskatT.ewski, M. Liu, D. Plotkin, Y.B. Mitchel. t Metabolic and dtherosclerosis Research Center, Cincinnati, Ohio; Merck Research Labs, Rahway, NJ, USA Simvastatin (S) is effective in patients with hyperehotesterolemia and combined hyperlipidemia, however few studies have evaluated patients whose
71st EAS Congress and Satellite Symposia
i
C G C