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In vitro metabolism of THF-γ2 in human, Rat and mouse blood
THE DEXNORFENFLURAMINE-TO-DEXFENFLU RAMINE PLASMA RATIO IN NON-HUMAN PRIMATES A. BergamL C. Fracasso and S. Caccia Istituto di Ricerche Farmacologiche "Mario Negri", via Eritrea 62, 20157 Milan, Italy.
IN VITRO METABOLISM OF THF-72 IN HUMAN, RAT AND MOUSE BLOOD R.Battaglia ~, M. Martignoni ~, R. Forino', K.F. Tipton z, M. Strolin Benedetti ~ Pharmacia, Milan, Italy, 2 Dept of Biochemistry, Trinity Coilege, Dublin, Ireland
Dexfenfluramine (DF) causes in animals a dose-dependent decrease of serotoninergic markers but none of the sepcies so far investigated shows sufficient metabolic similarities with man to be a valid model for safety studies. The plasma kinetics of DF and its metabolite dexnorfenfluramine (DNF) were therefore studied in baboon, rhesus and cynomolgus monkeys given the drug orally (2 mg/kg) in order to investigate whether any of these primates was particularly similar to man. The drug was rapidly N-deethylated to DNF achieving comparatively low mean maximum plasma levels (Cmax) in all primates and rapidly disappearing thereafter with half-lives (tt/2) ranging from 2 to 6 h. Its metabolite reached higher mean Cma x and tl/2 were longer, varying from about 11 to 22 h. The metabolite-to-parent drug ratio (14-37), in terms of plasma area under the curve (AUC), greatly exceeded that in man (<1), being higher than in all species investigated so far. Plasma AUC of unchanged drug were lower and those of DNF higher in "all primates than in man given 20 mg DF. After correcting for dose differences, it appeared that the dosage in primates would need to be increased several-fold to achieve comparable DF AUC, producing DNF AUC several times those in man, whilst for comparable metabolite AUC the parent drug concentrations would be correspondingly too low. In view of the different interactions of DF and DNF with the serotoninergic system none of the these primates is therefore a suitable model for safety assessment in terms of exposure of the active moieties in comparison to man.
Thymic humoral factor '72 (THF-72, thymoctonan) is a synthetic octapeptide originally isolated from calf thymus, which is used as an immunomodulator. The compound has the amino acid sequence Leu-GluAsp-Gly-Pro-Lys-Phe-Leu. The metabolism of THF-,,/2 in hnman, rat and mouse blood was determined in vitro at 37°C for times ranging between 0 and 60 rain using THF-3,2 labelled by 3H in preline. Blood concentration of about 30 ng/ml of [~H]THF-72 was used, and the metabolite profile was determined by radio-TLC analysis after suitable extraction. THF-72 was rapidly metabolised in vitro at 37°C. However, in human and mouse blood THF-72 was still detectable after 30 min incubation, accounting for 3-4% of the extracted radioactivity. In rat blood THF-72 was still detectable after 20 min incubation (5%). Upon incubation in human and rat blood THF-72 afforded three major fragments, A, B and C, while in mouse blood fragment B was present only in small amounts. Fragment A appeared very quickly, after only 1 min incubation in all three species, roached a maximum after 10 min in human and rat blood and alter 15 min in mouse blood, then decreased. Fragment B was first detected after 5 min incubation and increased until 60 min in human blood. In rat blood fragment B reached a maximum at 20 min, and then decreased. In mouse blood fragment B appeared as a small peak at 5-10 min and remained virtually constant up to 60 min. Fragment C started to appear after 5 min and increased until 60 rain; at this time it was the most important fragment in human and rat blood. Two'or three minor metabolites were also present. Incubation of [3H]THF3,2 in human blood in the presence of various peptidase inhibitors showed that at least two different types of enzymes are involved in the metabolism in vitro of THF-3,2, i.e. a metallo-amino peptidase and a metalloendopeptidase.
E F F E C T S O F H E P A T O T O X I C D R U G S O N r - O X I D A T I O N AS A S S E S S E D B Y 14C02 E L I M I N A T I O N IN T H E E X P I R E D A I R O F R A T S G I V E N 14C-PALMITIC ACID
PHARMACOKINETICS OF ISOSORBIDE DINITRATE A N D ITS T W O M O N O N I T R A T E METABOLITES IN MAN DURING AND AFTER 24HOUR I N T R A V E N O U S INFUSION. A. Bergami, R. Bernasconi S. Caccia, R. Urso, M. Sardina, R. Latini "Mario Negri" Institute, Milano and Italfarmaco Medical Department, Sesto S. Giovanni, Italy.
M.G. Castelli G. Salgarollo, M. Rocchetti, M. S t r o l i n Benedetti Pharmacia, V i a Bisceglie, 104, 20152 Milan, Italy. Several drugs or toxins w i t h a CO2H group in their structure, such as amineptine CA), v a l p r o i c a c i d (VA) and hypoglycin, h a v e b e e n shown to cause m i c r o v e s i c u l a r steatosis, likely as a result from the inhibition of the ~ - o x i d a t i o n of fatty acids. The objective o f this study was to develop a method to evaluate the interference of drugs w i t h the ~ - o x i d a t i o n of fatty acids as a means to assess their potential hepatotoxicity. A, V A and m i l a c e m i d e (M) were u s e d as p o s i t i v e controls, and p a l m i t i c a c i d (PA) as n e g a t i v e control. In the control group, 3 S p r a g u e - D a w l e y rats, fasted for 16 h, were o r a l l y g i v e n [U-14ClPA (I mg/kg). 14C02 e x h a l a t i o n was m e a s u r e d at time intervals up to 48 h post-dosing. In the treated groups (3 rats/group), A and PA (593 ~mol/kg), V A and M (1500 #mol/kg) were g i v e n o r a l l y 1 h prior to [U-14C]PA. The dose of the 3 drugs was chosen on the basis of the EDs0 value in a test considered as representative of the pharmacological activity. In the control group, the e x h a l a t i o n of l~C02, e x p r e s s e d as cumulative % of the a d m i n i s t e r e d radioactivity, was 0.2 (0.5 h), 0.6 (i h), 4.0 (2 h), 10.3 (3 h), 17.4 (4 h), 25.3 (6 h), 33.7 {8 h), 56.6 (24 h), 6 3 . 4 (32 h) and 69.7 (48 h). No decrease in the cumulative a m o u n t of 14CO2 exhaled was found after PA. C o m p a r e d to the control group, the cumulative e x h a l a t i o n of 14CO2 was ca 80% at 4 h in the V A - t r e a t e d group, ca 85% at 6 h in the M - t r e a t e d group, and ca 56% at 8 h in the A - t r e a t e d group, and r e a c h e d values similar to those of controls at the successive times. Thus, a d m i n i s t r a t i o n of a large amount of PA, w h i c h is c a t a b o l i z e d by ~-oxidation, d i d not affect the m e t a b o l i s m of a trace amount of [U-14C]PA. In contrast, VA, M and A s e e m to affect enzyme(s) involved in the m e t a b o l i s m of fatty acids. The r e l a t i o n s h i p b e t w e e n the inhibitory effect and the p o t e n t i a l h e p a t o t o x i c i t y of drugs w a r r a n t s further examination.
Isosorbide dinitrate (ISDN) is a drug widely used in angina pectoris, acute myocardial infarction and congestive heart failure. Although long-term intravenous infusions of ISDN are used in critically ill patiens, its pharmacokinetics has not been described in these conditions. We measured by capillary (30m x 0.32ram ID, AT50 column) gas-chromatography with electron capture detection ISDN and its two active metabolites, isosorbide-5-mononitrate (IS5MN) and isosorbide-2-mononitrate (IS2MN) in plasma Of 9 healthy Volunteers given an i.v. infusion of ISDN for 24 h, at doses of 1 to 6 ~tg*mi'n-I*kg-1. Plasma concentrations of ISDN and its two metabolites were simultaneously fitted to a linear 2compartimental model by SAAM program for Macintosh. Terminal half-lives were 1.12+0.16 h, 4.31+0.75 h and 1.76+0.30 h for ISDN, IS5MN and IS2MN, respectively. Rate constants for production of metabolites decreased with increasing rates of infusion. Steady state concentrations of ISDN increased more than proportionally with the increase in infusion rate: 20+4 ng*ml-I at 1 gg,min-l*kg-t, and 167+39 at 4-6 ~tg*min-l*kg-I. In conclusion, ISDN infused for 24 h shows evidences of nonlinear kinetics that might be attributed to saturation of metabolism of ISDN and/or to feed-back inhibition by one of its metabolites.
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IN VITRO METABOLISM OF THF-72 IN HUMAN, RAT AND MOUSE BLOOD R.Battaglia ~, M. Martignoni ~, R. Forino', K.F. Tipton z, M. Strolin Benedetti ~ Pharmacia, Milan, Italy, 2 Dept of Biochemistry, Trinity Coilege, Dublin, Ireland
Dexfenfluramine (DF) causes in animals a dose-dependent decrease of serotoninergic markers but none of the sepcies so far investigated shows sufficient metabolic similarities with man to be a valid model for safety studies. The plasma kinetics of DF and its metabolite dexnorfenfluramine (DNF) were therefore studied in baboon, rhesus and cynomolgus monkeys given the drug orally (2 mg/kg) in order to investigate whether any of these primates was particularly similar to man. The drug was rapidly N-deethylated to DNF achieving comparatively low mean maximum plasma levels (Cmax) in all primates and rapidly disappearing thereafter with half-lives (tt/2) ranging from 2 to 6 h. Its metabolite reached higher mean Cma x and tl/2 were longer, varying from about 11 to 22 h. The metabolite-to-parent drug ratio (14-37), in terms of plasma area under the curve (AUC), greatly exceeded that in man (<1), being higher than in all species investigated so far. Plasma AUC of unchanged drug were lower and those of DNF higher in "all primates than in man given 20 mg DF. After correcting for dose differences, it appeared that the dosage in primates would need to be increased several-fold to achieve comparable DF AUC, producing DNF AUC several times those in man, whilst for comparable metabolite AUC the parent drug concentrations would be correspondingly too low. In view of the different interactions of DF and DNF with the serotoninergic system none of the these primates is therefore a suitable model for safety assessment in terms of exposure of the active moieties in comparison to man.
Thymic humoral factor '72 (THF-72, thymoctonan) is a synthetic octapeptide originally isolated from calf thymus, which is used as an immunomodulator. The compound has the amino acid sequence Leu-GluAsp-Gly-Pro-Lys-Phe-Leu. The metabolism of THF-,,/2 in hnman, rat and mouse blood was determined in vitro at 37°C for times ranging between 0 and 60 rain using THF-3,2 labelled by 3H in preline. Blood concentration of about 30 ng/ml of [~H]THF-72 was used, and the metabolite profile was determined by radio-TLC analysis after suitable extraction. THF-72 was rapidly metabolised in vitro at 37°C. However, in human and mouse blood THF-72 was still detectable after 30 min incubation, accounting for 3-4% of the extracted radioactivity. In rat blood THF-72 was still detectable after 20 min incubation (5%). Upon incubation in human and rat blood THF-72 afforded three major fragments, A, B and C, while in mouse blood fragment B was present only in small amounts. Fragment A appeared very quickly, after only 1 min incubation in all three species, roached a maximum after 10 min in human and rat blood and alter 15 min in mouse blood, then decreased. Fragment B was first detected after 5 min incubation and increased until 60 min in human blood. In rat blood fragment B reached a maximum at 20 min, and then decreased. In mouse blood fragment B appeared as a small peak at 5-10 min and remained virtually constant up to 60 min. Fragment C started to appear after 5 min and increased until 60 rain; at this time it was the most important fragment in human and rat blood. Two'or three minor metabolites were also present. Incubation of [3H]THF3,2 in human blood in the presence of various peptidase inhibitors showed that at least two different types of enzymes are involved in the metabolism in vitro of THF-3,2, i.e. a metallo-amino peptidase and a metalloendopeptidase.
E F F E C T S O F H E P A T O T O X I C D R U G S O N r - O X I D A T I O N AS A S S E S S E D B Y 14C02 E L I M I N A T I O N IN T H E E X P I R E D A I R O F R A T S G I V E N 14C-PALMITIC ACID
PHARMACOKINETICS OF ISOSORBIDE DINITRATE A N D ITS T W O M O N O N I T R A T E METABOLITES IN MAN DURING AND AFTER 24HOUR I N T R A V E N O U S INFUSION. A. Bergami, R. Bernasconi S. Caccia, R. Urso, M. Sardina, R. Latini "Mario Negri" Institute, Milano and Italfarmaco Medical Department, Sesto S. Giovanni, Italy.
M.G. Castelli G. Salgarollo, M. Rocchetti, M. S t r o l i n Benedetti Pharmacia, V i a Bisceglie, 104, 20152 Milan, Italy. Several drugs or toxins w i t h a CO2H group in their structure, such as amineptine CA), v a l p r o i c a c i d (VA) and hypoglycin, h a v e b e e n shown to cause m i c r o v e s i c u l a r steatosis, likely as a result from the inhibition of the ~ - o x i d a t i o n of fatty acids. The objective o f this study was to develop a method to evaluate the interference of drugs w i t h the ~ - o x i d a t i o n of fatty acids as a means to assess their potential hepatotoxicity. A, V A and m i l a c e m i d e (M) were u s e d as p o s i t i v e controls, and p a l m i t i c a c i d (PA) as n e g a t i v e control. In the control group, 3 S p r a g u e - D a w l e y rats, fasted for 16 h, were o r a l l y g i v e n [U-14ClPA (I mg/kg). 14C02 e x h a l a t i o n was m e a s u r e d at time intervals up to 48 h post-dosing. In the treated groups (3 rats/group), A and PA (593 ~mol/kg), V A and M (1500 #mol/kg) were g i v e n o r a l l y 1 h prior to [U-14C]PA. The dose of the 3 drugs was chosen on the basis of the EDs0 value in a test considered as representative of the pharmacological activity. In the control group, the e x h a l a t i o n of l~C02, e x p r e s s e d as cumulative % of the a d m i n i s t e r e d radioactivity, was 0.2 (0.5 h), 0.6 (i h), 4.0 (2 h), 10.3 (3 h), 17.4 (4 h), 25.3 (6 h), 33.7 {8 h), 56.6 (24 h), 6 3 . 4 (32 h) and 69.7 (48 h). No decrease in the cumulative a m o u n t of 14CO2 exhaled was found after PA. C o m p a r e d to the control group, the cumulative e x h a l a t i o n of 14CO2 was ca 80% at 4 h in the V A - t r e a t e d group, ca 85% at 6 h in the M - t r e a t e d group, and ca 56% at 8 h in the A - t r e a t e d group, and r e a c h e d values similar to those of controls at the successive times. Thus, a d m i n i s t r a t i o n of a large amount of PA, w h i c h is c a t a b o l i z e d by ~-oxidation, d i d not affect the m e t a b o l i s m of a trace amount of [U-14C]PA. In contrast, VA, M and A s e e m to affect enzyme(s) involved in the m e t a b o l i s m of fatty acids. The r e l a t i o n s h i p b e t w e e n the inhibitory effect and the p o t e n t i a l h e p a t o t o x i c i t y of drugs w a r r a n t s further examination.
Isosorbide dinitrate (ISDN) is a drug widely used in angina pectoris, acute myocardial infarction and congestive heart failure. Although long-term intravenous infusions of ISDN are used in critically ill patiens, its pharmacokinetics has not been described in these conditions. We measured by capillary (30m x 0.32ram ID, AT50 column) gas-chromatography with electron capture detection ISDN and its two active metabolites, isosorbide-5-mononitrate (IS5MN) and isosorbide-2-mononitrate (IS2MN) in plasma Of 9 healthy Volunteers given an i.v. infusion of ISDN for 24 h, at doses of 1 to 6 ~tg*mi'n-I*kg-1. Plasma concentrations of ISDN and its two metabolites were simultaneously fitted to a linear 2compartimental model by SAAM program for Macintosh. Terminal half-lives were 1.12+0.16 h, 4.31+0.75 h and 1.76+0.30 h for ISDN, IS5MN and IS2MN, respectively. Rate constants for production of metabolites decreased with increasing rates of infusion. Steady state concentrations of ISDN increased more than proportionally with the increase in infusion rate: 20+4 ng*ml-I at 1 gg,min-l*kg-t, and 167+39 at 4-6 ~tg*min-l*kg-I. In conclusion, ISDN infused for 24 h shows evidences of nonlinear kinetics that might be attributed to saturation of metabolism of ISDN and/or to feed-back inhibition by one of its metabolites.
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