- Email: [email protected]
Sulphation of (−)-salbutamol in the human liver and duodenum: Interindividual variability
Sulphation of (-)-salbutamol in the human liver and duodenum: Interindividual variability G,M. Pacifici*, B, Giulianetti*, M. Quilici*, L. Giuliani §, R. Spisni §, M. Nervi §. Departments of Biomedicine* and Surgery §, University of Pisa, Pisa, Italy.
DRUG METABOLISM 1N THE RAT LIVER AFTER EXPOSURE TO A PYROGENIC INFLAMMATORY AGENT
Salbutamol (S) is administered by mouth to treat premature labour. S is a racemate and (-)-S retains most of the ~-adrenergic activity. Treatment with S is often associated with side effects which should reflect greater exposition to this drug. S undergoes presystemic conjugation with sulphate which limits his bioavailability to 50%. Variability in the amounts of S sulphated might cause variability in the bioavailability, and in turn, in the tissue concentrations of this drug. We thus ascertained whether the rate of (-)-S sulphation varies among subjects. As sulphotransferase is well expressed in the intestinal mucosa and in the liver, our study included human duodenum mucosa (HDM, 100 samples) and human liver (HL, 100 samples). There is a wide interindividual variability in the rate of (-)-S sulphation which varies over 4-fold within 4-2 S.D. units either in the HDM and HL. The rate of (-)-S sulphation is influenced neither by sex nor by ageing in both HDM and HE In HDM, the average rate (pmol/min/mg) of (-)-S sulphation is (329) 4 times greater than in HL (76.2), thus the intestine contributes with the liver to the presystemic sulphation of (-)-S. In the liver, the rate of (-)-S sulphation is positively skewed and the normal equivalent deviation is compatible with the presence of t w o subgroups in the population, The correlations with the two main forms of sulphotransferase have indicated that dopamine sulphotransferase should be responsible for sulphation of (-)-S in HDM whereas, in the liver, (-):S should be sulphated mainly by phenol sulphotransferase. In conclusion, the present results indicate that sulphation of (-)-S occurs in both HL and HDM and, in both tissues, it is subjected to a considerable variability.
Lipopolysaccharide (I.PS) (lyophilized powder of E. coil, serotype 055:B5, Sigma) is well known for inducing an acute inflammation. LPS w&g tested in rats (10mg/kg,, s.c., [or two days) for its possible inl]uence on hepatic drug metabolizing enzymes (DMEs). At constitutive levels, total glutafl~ione transfera~es (GSTs) and m'yl hydro-cm'bon hydroxylase (AItH) were not affected by LPS. On the contrary, the activities of ethoxyresorufin-O-deet[lylase (EROD) and two aldehyde dehydrogenase (ALDt{) isozymes (ALDH3c and ALDH3m) were diminished by LPS, when measured 12 and 24hr after treatment, in a way proportional to the time elapsed. In addition, 24hr after treatment, the liver/body weight ratio was increased, and no statistically significant changes were noticed in animal body tempera/ure. The cytosolic ALDH-isozyme (ALDtI3c) has been found before Io reflect the activity of Ihe subfamily CYP1AI of cytochrome P-450, as well as other enzymes functionally related with the aryt-hydrocarbon receptor (tile so-called "Ah-recel~tor enzyme baltery"), which is induced by 3 methylcholanthrenc (3MC) and other toxic pollutants (polycyclic or halogenated aromatic hydrocarbons). Co~adminislration of I.PS and 3MC (50mg/kg, s.c., once) resulted in a significant decrease of the induclion of the DMEs acliviqies, which are usually greatly enhanced after treatment with 3MC. These results indicate Ihal I.PS is aftectfi~glhe normal [unction mid the biochemicg alertness of the hepatocyles. It is not dear, however, il this is a direct toxic effect of the pyrogen or an indirect action mediated through other humoral or hormonal mechanisms.
P. Papp_~, P. Stephaeou, V.:Vasiliou m~d M. Marsclos Department of Pharmacology, Medical School, University of loannina, 45 l 10 Io~'mnina,Greece.
This work was supported by The European Community by contracts BMH1-CT94-1622 and BMH1-CT92-0097 and The Italian Ministry of University.
CYCLOSPORIN A METABOLISM IN H U M A N A N D RAT L U N G SLICES, A N D H U M A N B R O N C H I A L EPITHELIAL CELLS.
ENANTIOSEPARATION
M.C. Spaans, R. Fisher, K. Brendel, and A. Vickers. Drug Safety, Sandoz Phanna Ltd., Basle Switzerland. Dept. Pharmac. and Tox., Univ. of Arizona.
OF
t~.~Sz6k6 a n d K. M a g y a r D e p a r t m e n t of P h a r m a c o d y n a m i c s , S e m m e l w e i s U n i v e r s i t y of M e d i c i n e , N a g y v & a d t6r 4. B u d a p e s t , 1089 Hungary
This study demonstrates the use of human and rat lung slices in comparison to liver for gaining insight of lung biotransformation of cyclosporin A (CSA), a promising asthmatic drug. Human lung slices (~ 400-500 gm do), rat lung and liver slices (~ 200 'lain dO) were prepared from 8 mm diameter cores using a Krumdieck Tissue slicer. The lungs were inflated with agarose (0.8-1%) which is soluble at 2530 °C. After a preincubation of 1.5 hr, medium containing 3H-CSA (3 blCi/ml, 0.1, 1 and 10 btM) was added for 24 hr. Lung slices from 3 humans metabolized CSA to the primary hydroxylated metabolites AM1, AM9, trace amounts of AM4N and the secundary metabolite AMIc. Rat lung and liver slices metabolized CSA to the primary hydroxylated metabolites AMI, AM9, AM4N, and the secondary metabolite AM19. Rat liver additionally formed AM19 and AM4N9. The metabolite profile of CSA in human bronchial epithelial cells was the same as in human lung slices but no AM4N was formed. The extent of total metabolism in human lung slice cultures for 24 hr was 0.5 pmol/mg slice protein for 0.1 hiM CSA and increased 10-fold for 1 gM and 75-fold for 10 ~M CSA, whereas in rat lung slices the extent was 316.5 pmol/mg slice protein for 0.1 btM CSA and increased 6-fold for 1 gM and 24-fold for 10 /tM CSA. In human bronchial epithelial cells the extent of total metabolism for 24 hr was 13.2 pmol/mg cell protein for 0.1 gM CSA and increased 18-fold for 1 btM and 76-fold for 10 oM CSA. Based on these initial data the order of extent of CSA metabolism was rat lung > rat liver ~- human liver > human bronchial epithelial cells > human lung for 0.1, 1 mid 10 I.tM CSA.
m 4 9
AND IDENTIFICATION BY C A P I L L A R Y
SELEGILINE METABOLITES ELECTROPHORESIS
Selegiline R ( R @ ) - d e p r e n y l ) is a selective i n h i b i t o r o f m o n o a m i n e o x i d a s e B e n z y m e a n d is u s e d i n the t r e a t m e n t of P a r k i n s o n ' s d i s e a s e . This c o m p o u n d , as well as its m a j o r m e t a b o l i t e s : a m p h e t a m i n e a n d m e t h a m p h e t a m i n e d i s p l a y e n a n t i o s e l e c t i v i t y in t h e i r p h a r m a c o l o g i c a l actions. The S(+) e n a n t i o m e r s of the m e t a b o l i t e s are m o r e effective p s y c h o s t i m u l a n t s , t h a n t h e R(-) isomers. O u r goal w a s to d e t e r m i n e the e n a n t i o m e r c o m p o s i t i o n of d e p r e n y l m e t a b o l i t e s e x c r e t e d in t h e u r i n e of rats t r e a t e d chronically with R(-)-deprenyl. A cyclodextrin-modified capillary electrophoresis method was developed. The chiral s e p a r a t i o n s w e r e p e r f o r m e d in a n acidic b u f f e r c o n t a i n i n g h e p t a k i s - ( 2 , 6 - d i - O - m e t h y l ) - [ 3 - c y c l o d e x t r i n as a chiral selector. M e t h a n o l a n d h y d r o x y p r o p y l m e t h y l cellulose w e r e also i n c l u d e d i n t h e s e p a r a t i o n buffer. R(-)a m p h e t a m i n e a n d R ( - ) - m e t h a m p h e t a m i n e w e r e f o u n d in the u r i n e of rats t r e a t e d c h r o n i c a l l y w i t h R ( - ) - d e p r e n y l . T h e f o r m a t i o n of S ( ÷ ) - e n a n t i o m e r s of t h e s e m e t a b o l i t e s in c o n c e n t r a t i o n a b o v e t h e d e t e c t i o n limit of the s e p a r a t i o n m e t h o d c o u l d be e x c l u d e d . The p e a k s of f o u r o t h e r m e t a b o l i t e s also a p p e a r e d in t h e u r i n e s a m p l e s , t h e i r identification needs further studies.
--
DRUG METABOLISM 1N THE RAT LIVER AFTER EXPOSURE TO A PYROGENIC INFLAMMATORY AGENT
Salbutamol (S) is administered by mouth to treat premature labour. S is a racemate and (-)-S retains most of the ~-adrenergic activity. Treatment with S is often associated with side effects which should reflect greater exposition to this drug. S undergoes presystemic conjugation with sulphate which limits his bioavailability to 50%. Variability in the amounts of S sulphated might cause variability in the bioavailability, and in turn, in the tissue concentrations of this drug. We thus ascertained whether the rate of (-)-S sulphation varies among subjects. As sulphotransferase is well expressed in the intestinal mucosa and in the liver, our study included human duodenum mucosa (HDM, 100 samples) and human liver (HL, 100 samples). There is a wide interindividual variability in the rate of (-)-S sulphation which varies over 4-fold within 4-2 S.D. units either in the HDM and HL. The rate of (-)-S sulphation is influenced neither by sex nor by ageing in both HDM and HE In HDM, the average rate (pmol/min/mg) of (-)-S sulphation is (329) 4 times greater than in HL (76.2), thus the intestine contributes with the liver to the presystemic sulphation of (-)-S. In the liver, the rate of (-)-S sulphation is positively skewed and the normal equivalent deviation is compatible with the presence of t w o subgroups in the population, The correlations with the two main forms of sulphotransferase have indicated that dopamine sulphotransferase should be responsible for sulphation of (-)-S in HDM whereas, in the liver, (-):S should be sulphated mainly by phenol sulphotransferase. In conclusion, the present results indicate that sulphation of (-)-S occurs in both HL and HDM and, in both tissues, it is subjected to a considerable variability.
Lipopolysaccharide (I.PS) (lyophilized powder of E. coil, serotype 055:B5, Sigma) is well known for inducing an acute inflammation. LPS w&g tested in rats (10mg/kg,, s.c., [or two days) for its possible inl]uence on hepatic drug metabolizing enzymes (DMEs). At constitutive levels, total glutafl~ione transfera~es (GSTs) and m'yl hydro-cm'bon hydroxylase (AItH) were not affected by LPS. On the contrary, the activities of ethoxyresorufin-O-deet[lylase (EROD) and two aldehyde dehydrogenase (ALDt{) isozymes (ALDH3c and ALDH3m) were diminished by LPS, when measured 12 and 24hr after treatment, in a way proportional to the time elapsed. In addition, 24hr after treatment, the liver/body weight ratio was increased, and no statistically significant changes were noticed in animal body tempera/ure. The cytosolic ALDH-isozyme (ALDtI3c) has been found before Io reflect the activity of Ihe subfamily CYP1AI of cytochrome P-450, as well as other enzymes functionally related with the aryt-hydrocarbon receptor (tile so-called "Ah-recel~tor enzyme baltery"), which is induced by 3 methylcholanthrenc (3MC) and other toxic pollutants (polycyclic or halogenated aromatic hydrocarbons). Co~adminislration of I.PS and 3MC (50mg/kg, s.c., once) resulted in a significant decrease of the induclion of the DMEs acliviqies, which are usually greatly enhanced after treatment with 3MC. These results indicate Ihal I.PS is aftectfi~glhe normal [unction mid the biochemicg alertness of the hepatocyles. It is not dear, however, il this is a direct toxic effect of the pyrogen or an indirect action mediated through other humoral or hormonal mechanisms.
P. Papp_~, P. Stephaeou, V.:Vasiliou m~d M. Marsclos Department of Pharmacology, Medical School, University of loannina, 45 l 10 Io~'mnina,Greece.
This work was supported by The European Community by contracts BMH1-CT94-1622 and BMH1-CT92-0097 and The Italian Ministry of University.
CYCLOSPORIN A METABOLISM IN H U M A N A N D RAT L U N G SLICES, A N D H U M A N B R O N C H I A L EPITHELIAL CELLS.
ENANTIOSEPARATION
M.C. Spaans, R. Fisher, K. Brendel, and A. Vickers. Drug Safety, Sandoz Phanna Ltd., Basle Switzerland. Dept. Pharmac. and Tox., Univ. of Arizona.
OF
t~.~Sz6k6 a n d K. M a g y a r D e p a r t m e n t of P h a r m a c o d y n a m i c s , S e m m e l w e i s U n i v e r s i t y of M e d i c i n e , N a g y v & a d t6r 4. B u d a p e s t , 1089 Hungary
This study demonstrates the use of human and rat lung slices in comparison to liver for gaining insight of lung biotransformation of cyclosporin A (CSA), a promising asthmatic drug. Human lung slices (~ 400-500 gm do), rat lung and liver slices (~ 200 'lain dO) were prepared from 8 mm diameter cores using a Krumdieck Tissue slicer. The lungs were inflated with agarose (0.8-1%) which is soluble at 2530 °C. After a preincubation of 1.5 hr, medium containing 3H-CSA (3 blCi/ml, 0.1, 1 and 10 btM) was added for 24 hr. Lung slices from 3 humans metabolized CSA to the primary hydroxylated metabolites AM1, AM9, trace amounts of AM4N and the secundary metabolite AMIc. Rat lung and liver slices metabolized CSA to the primary hydroxylated metabolites AMI, AM9, AM4N, and the secondary metabolite AM19. Rat liver additionally formed AM19 and AM4N9. The metabolite profile of CSA in human bronchial epithelial cells was the same as in human lung slices but no AM4N was formed. The extent of total metabolism in human lung slice cultures for 24 hr was 0.5 pmol/mg slice protein for 0.1 hiM CSA and increased 10-fold for 1 gM and 75-fold for 10 ~M CSA, whereas in rat lung slices the extent was 316.5 pmol/mg slice protein for 0.1 btM CSA and increased 6-fold for 1 gM and 24-fold for 10 /tM CSA. In human bronchial epithelial cells the extent of total metabolism for 24 hr was 13.2 pmol/mg cell protein for 0.1 gM CSA and increased 18-fold for 1 btM and 76-fold for 10 oM CSA. Based on these initial data the order of extent of CSA metabolism was rat lung > rat liver ~- human liver > human bronchial epithelial cells > human lung for 0.1, 1 mid 10 I.tM CSA.
m 4 9
AND IDENTIFICATION BY C A P I L L A R Y
SELEGILINE METABOLITES ELECTROPHORESIS
Selegiline R ( R @ ) - d e p r e n y l ) is a selective i n h i b i t o r o f m o n o a m i n e o x i d a s e B e n z y m e a n d is u s e d i n the t r e a t m e n t of P a r k i n s o n ' s d i s e a s e . This c o m p o u n d , as well as its m a j o r m e t a b o l i t e s : a m p h e t a m i n e a n d m e t h a m p h e t a m i n e d i s p l a y e n a n t i o s e l e c t i v i t y in t h e i r p h a r m a c o l o g i c a l actions. The S(+) e n a n t i o m e r s of the m e t a b o l i t e s are m o r e effective p s y c h o s t i m u l a n t s , t h a n t h e R(-) isomers. O u r goal w a s to d e t e r m i n e the e n a n t i o m e r c o m p o s i t i o n of d e p r e n y l m e t a b o l i t e s e x c r e t e d in t h e u r i n e of rats t r e a t e d chronically with R(-)-deprenyl. A cyclodextrin-modified capillary electrophoresis method was developed. The chiral s e p a r a t i o n s w e r e p e r f o r m e d in a n acidic b u f f e r c o n t a i n i n g h e p t a k i s - ( 2 , 6 - d i - O - m e t h y l ) - [ 3 - c y c l o d e x t r i n as a chiral selector. M e t h a n o l a n d h y d r o x y p r o p y l m e t h y l cellulose w e r e also i n c l u d e d i n t h e s e p a r a t i o n buffer. R(-)a m p h e t a m i n e a n d R ( - ) - m e t h a m p h e t a m i n e w e r e f o u n d in the u r i n e of rats t r e a t e d c h r o n i c a l l y w i t h R ( - ) - d e p r e n y l . T h e f o r m a t i o n of S ( ÷ ) - e n a n t i o m e r s of t h e s e m e t a b o l i t e s in c o n c e n t r a t i o n a b o v e t h e d e t e c t i o n limit of the s e p a r a t i o n m e t h o d c o u l d be e x c l u d e d . The p e a k s of f o u r o t h e r m e t a b o l i t e s also a p p e a r e d in t h e u r i n e s a m p l e s , t h e i r identification needs further studies.
--