- Email: [email protected]
Formation of 13,14-dihydro-prostaglandin E1 during intravenous infusions of prostaglandin E1 in patients with peripheral arterial occulsive disease
PROSTAGLANDINS
FORMATION OF 13,14-DIHYDRO-PROSTAGLANDIN E 1 DURING INTRAVENOUS INFUSIONS OF PROSTAGLANDIN E ~ IN PATIENTS WITH PERIPHERAL ARTERIAL OCCLUSIVE]~ISEASE B.A. Peskar , ~.H. Hesse I, ~. Rogatti 2, C. Die~m 3, G. Rudofsky ~, H. Schweer J and H.W. Seyberth ~
1Department of P h a ~ c o l o g y and Toxicology, Ruhr University, ~-4630 Bochum, FRG, Schwarz Pharma AG, D-4019 Monheim, FRG, ~Department of Internal M~dicine, University of Heidelberg, D-6900 Heidelberg, FRG, Department of Intern81 Medicine, University of Essen, D-4300 Essen, FRG, and Children s Hospital, University of Marburg, D-3550 Marburg, FRG ABSTRACT Formation of 13,14-dihydro-prostaglandin (PG) E1 during intravenous infusions of PGEI in patients with peripheral a r t e r i a l occlusive disease was investigated. Using both high performance l i q u i d chromatography ( h . p . l . c . ) combined with radioimmunoassay and gas chromatograp h y / t r i p l e stage quadrupole mass spectrometry (GC/MS/MS) basal levels of 13,14-dihydro-PGE1 were found to be close to or below the detection limits of the assay methods. Levels of the PGEI metabolite increased s i g n i f i c a n t l y during the infusion periods and decreased a f t e r t h e i r end. Since 13,14-dihydro-PGE1, in contrast to i t s precursors 15-keto-PGE1 and 15-keto-13,14-dihydro-PGE1, is b i o l o g i c a l l y active, i t s formation could contribute to the beneficial effects of PGEI administered intravenously in patients with peripheral a r t e r i a l occlusive disease. INTRODUCTION For more than a decade intravenous PGEI infusions have been used in the treatment of peripheral a r t e r i a l occlusive disease (1,2). The beneficial e f f e c t of PGEI administered intravenously is remarkable, since a major portion of c i r c u l a t i n g PGEI is rapidly metabolized during passage through the human lung (3). I n i t i a l metabolism of PGEI occurs via the 15-hydroxy-PG-dehydrogenase/a13-reductase pathway (4) resulting in formation of 15-keto-13,14-dihydro-PGE1 as the major c i r c u l a t i n g metaboliteo This compound lacks almost completely biological a c t i v i t y (5). I t was, therefore, of interest to investigate the possible formation of 13,14-dihydro-PGE1, a b i o l o g i c a l l y active metabolite (5,5), during intravenous infusions of PGEI. METHODS PGEI (Prostavasin R, Schwarz Pharma AG, Monheim, FRG) was infused intravenously either at 80 wg/60 min/patient (n=6, age 21-79, 4 male, 2 female) or at 60 pg/30 min/patient (n=4, age 60-70, 2 male, 2 female). In the f i r s t group of patients blood was taken before and 40 min after the start as well as at the end (60 min) and 30 min a f t e r the end of the infusion period. In the second group of patients blood was taken before and at the end of the infusion period (30 mtn) as well as 60 min l a t e r . Blood (20 ml) was collected into syringes con-
MARCH 1991 VOL. 41 NO. 3
225
PROSTAGLANDINS t a i n i n g sodium EDTA as anticoagulant and indomethacin as cyclooxygenase i n h i b i t o r ( f i n a l concentrations 5.4 and 0.1 mM, r e s p e c t i v e l y ) . Plasma was separated by immediate c e n t r i f u g a t i o n (1500 x g, 4oc, 15 min) and analyzed f o r 13,14-dihydro-PGE1 using h . p . l . c . (7) combined with radioimmunoassay of eluted f r a c t i o n s f o r the f i r s t group of 6 p a t i e n t s . The anti-PGE1 antiserum (8) used f o r the radioimmunoassay e x h i b i t s 100 % r e l a t i v e cross-reaction with 13,14-dihydro-PGE1. Blood from the second group of 4 patients was analyzed by GC/MS/MS (to be published) using a p u r i f i c a t i o n procedure described previously (9). Standard amounts of 13,14-dihydro-PGE1 (100 pg - I ng) added to human whole blood were shown to be stable f o r at least 2 - 3 hours. The r e s u l t s were calculated as means + S.E.M. S t a t i s t i c a l was performed by use of Student's t - t e s t f o r paired values.
analysis
RESULTS The r e s u l t s are shown in Table I . While preinfusion plasma l e v e l s of 13,14-dihydro-PGE1 were close to or below the detection l i m i t s of Table I Levels of 13,14-dihydro-PGE1 (pg/ml) in patients with peripheral a r t e r i a l occlusive disease before, during and a f t e r the end of i n t r a venous infusions of PGEI. The f i r s t group of patients (n=6) received infusions of 80 pg PGEI f o r 60 min; t h e i r plasma was analyzed by h.p.l.c./radioimmunoassay. The second group of 4 patients received infusions of 60 pg PGEI f o r 30 min; t h e i r plasma was analyzed by GC/MS/MS. Results represent means + S.E.M. Plasma 13,14-dihydro-PGE1 (pg/ml) Time
h.p.l.c./radioimmunoassay
GC/MS/MS
(min)
(n=6)
(n=4)
0
5+2
30
n.d.
40
19 + 6 *
60
22 + 5 **
90
13 + 5 +
0.7+0.1 13
+ 1 *** n.d n.d.
4
+ 0.2 +++
n . d . : not determined; * p < ~@~5, ** p < 0.01, * * * p < 0.001 as compared to 0 min; p < 0.05, p < 0.001 as compared to end of i n f u sion period (60 or 30 min, r e s p e c t i v e l y ) . the assay methods, concentrations at the end of the infusion periods (60 and 30 min, r e s p e c t i v e l y ) had s i g n i f i c a n t l y increased to 22 + 5 pg/ml (n=6, p < 0.01) and to 13 + I pg/ml (n=4, p < 0.001), r e s p e c t i v e l y . T h i r t y min a f t e r the en~ of the infusion period l e v e l s of 13,14-dihydro-PGE1 had s i g n i f i c a n t l y (p < 0.05) decreased to 13 + 5 pg/ml in those 6 patients that had received 8o ~g PGEI f o r 60 A n . S i m i l a r l y a s i g n i f i c a n t (p < 0.001) decrease in the metabolite l e v e l s
226
MARCH 1991 VOL. 41 NO. 3
PROSTAGLANDINS to 4 + 0.2 pg/ml was observed I hour after the end of the infusion period- in those 4 patients that had received 60 pg PGEI for 30 min (Table I ) . DISCUSSION The results c l e a r l y demonstrate for the f i r s t time formation of 13,14-dihydro-PGE1 during intravenous administration of PGEI to man. On the other hand, formation of t h i s metabolite in v i t r o (5,10,11) as well as in the rat in vivo (12) has long been known to occur. In man, the analogous metabolite 13,14-dihydro-PGF2~ has been found to occur in plasma a f t e r intravenous administration of PGF2~ (13,14). Similar to PGEI, 13,14-dthydro-PGE1 has been shown to lower blood pressure (5) and to i n h i b i t p l a t e l e t aggregation (6). Although the mechanism of beneficial action of PGEI in peripheral a r t e r i a l occlusive disease remains unknown and the concentrations of 13,14-dihydro-PGE1 formed during the infusions of PGEI are rather low, the b i o l o g i c a l l y active metabolite could contribute to the therapeutic effects of intravenous infusions of PGEI observed in such patients (1,2). ACKNOWLEDGEMENTS We thank Mrs. K. Rezny for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (Pe 256/4-I and Se 263/9). REFERENCESS I.
Carlsson, L.A. and A.G. Olsson. Intravenous prostaglandin severe peripheral vascular disease. Lancet i i : 810, 1976.
2.
Sinzlnger, H., I. V i r g o l i n i and J. O'Grady. Clinical t r i a l s of PGEI, PGI2 and mimetics in patients with peripheral vascular disease. In: Prostaglandins in Clinical Research, (K. SchrOr and H. Sinzinger, eds.) Alan Liss Inc., New York, 1989, p. 85.
3.
Golub M, P. Zia, M. Matsuno and R. Horton. Metabolism of prostaglandins AI and El in man. J. Clin. Invest. 56: 1404, 1975.
4.
Hamberg, M. and B. Samuelsson. On the metabolism of prostaglandins El and E2 in man. J. Biol. Chem. 246: 6713, 1971.
5.
Angg~rd E. The biological a c t i v i t i e s of three metabolites prostaglandin El. Acta physiol, scand. 66: 509, 1966.
6.
Westwick, J. The e f f e c t of pulmonary metabolites of prostaglandins El, E2 and F2~ on ADP-induced aggregation of human and rabbit p l a t e l e t s . Br. J. Pharmacol. 58: 297P, 1976.
7.
Hesse, W.H., H. Schweer, H.W. Seyberth and B.A. Peskar. Separation and determination of prostaglandin EI metabolites by high-
MARCH 1991 VOL. 41 NO. 3
El in
of
227
PROSTAGLANDINS
performance l i q u i d chromatography. J. Chromatogro, in press 8.
Diekmann, J.M., A. Jobke, B.A. Peskar and G. Hertting. Angiotensin ll-induced contractions of rabbit splenic capsular strips and release of prostaglandins. Use of radioimmunoassays for prostaglandins El and E2. Naunyn-Schmiedeberg's Arch. Pharmacol. 297: 177, 1977.
9.
Schweer, H., C.O. Meese, O. FOrst, P.G. KOhl and H.W. Seyberth. Tandem mass spectrometric determination of 11-dehydro-thromboxane B2, and index metabolite of thromboxane A2 in plasma and urine. Anal. Biochem. 164: 156, 1987.
10.
~ngg~rd E. and B. Samuelsson. Prostaglandins and related factors. 28. Metabolism of prostaglandin El in guinea pig lung: the structures of two metabolites. J. Biol. Chem, 239: 4097, 1964.
11.
Angg~rd E. and C. Larsson. The sequence of the early steps in the metabolism of prostaglandin El. Eur. J. Pharmacol. 14: 66, 1971.
12.
Samuelsson, B. Prostaglandins and related factors. 27. Synthesis of t r i t i u m - l a b e l e d prostaglandin El and studies on its d i s t r i bution and excretion in the rat. J. Biol. Chem. 239, 4091, 1964.
13.
GranstrOm. E. On the metabolism of prostaglandin F2~ in female subjects. Structures of two metabolites in blood. Eur. J. Biochem. 27: 462, 1972.
14.
Green, K. Quantitative mass spectrometric analysis of prostaglandins. Adv. Biosci. 9: 91, 1973.
Editor:
228
P.W. R a m w e l l
Received:
10-17-90
Accepted:
12-7-90
MARCH 1991 VOL. 41 NO. 3
FORMATION OF 13,14-DIHYDRO-PROSTAGLANDIN E 1 DURING INTRAVENOUS INFUSIONS OF PROSTAGLANDIN E ~ IN PATIENTS WITH PERIPHERAL ARTERIAL OCCLUSIVE]~ISEASE B.A. Peskar , ~.H. Hesse I, ~. Rogatti 2, C. Die~m 3, G. Rudofsky ~, H. Schweer J and H.W. Seyberth ~
1Department of P h a ~ c o l o g y and Toxicology, Ruhr University, ~-4630 Bochum, FRG, Schwarz Pharma AG, D-4019 Monheim, FRG, ~Department of Internal M~dicine, University of Heidelberg, D-6900 Heidelberg, FRG, Department of Intern81 Medicine, University of Essen, D-4300 Essen, FRG, and Children s Hospital, University of Marburg, D-3550 Marburg, FRG ABSTRACT Formation of 13,14-dihydro-prostaglandin (PG) E1 during intravenous infusions of PGEI in patients with peripheral a r t e r i a l occlusive disease was investigated. Using both high performance l i q u i d chromatography ( h . p . l . c . ) combined with radioimmunoassay and gas chromatograp h y / t r i p l e stage quadrupole mass spectrometry (GC/MS/MS) basal levels of 13,14-dihydro-PGE1 were found to be close to or below the detection limits of the assay methods. Levels of the PGEI metabolite increased s i g n i f i c a n t l y during the infusion periods and decreased a f t e r t h e i r end. Since 13,14-dihydro-PGE1, in contrast to i t s precursors 15-keto-PGE1 and 15-keto-13,14-dihydro-PGE1, is b i o l o g i c a l l y active, i t s formation could contribute to the beneficial effects of PGEI administered intravenously in patients with peripheral a r t e r i a l occlusive disease. INTRODUCTION For more than a decade intravenous PGEI infusions have been used in the treatment of peripheral a r t e r i a l occlusive disease (1,2). The beneficial e f f e c t of PGEI administered intravenously is remarkable, since a major portion of c i r c u l a t i n g PGEI is rapidly metabolized during passage through the human lung (3). I n i t i a l metabolism of PGEI occurs via the 15-hydroxy-PG-dehydrogenase/a13-reductase pathway (4) resulting in formation of 15-keto-13,14-dihydro-PGE1 as the major c i r c u l a t i n g metaboliteo This compound lacks almost completely biological a c t i v i t y (5). I t was, therefore, of interest to investigate the possible formation of 13,14-dihydro-PGE1, a b i o l o g i c a l l y active metabolite (5,5), during intravenous infusions of PGEI. METHODS PGEI (Prostavasin R, Schwarz Pharma AG, Monheim, FRG) was infused intravenously either at 80 wg/60 min/patient (n=6, age 21-79, 4 male, 2 female) or at 60 pg/30 min/patient (n=4, age 60-70, 2 male, 2 female). In the f i r s t group of patients blood was taken before and 40 min after the start as well as at the end (60 min) and 30 min a f t e r the end of the infusion period. In the second group of patients blood was taken before and at the end of the infusion period (30 mtn) as well as 60 min l a t e r . Blood (20 ml) was collected into syringes con-
MARCH 1991 VOL. 41 NO. 3
225
PROSTAGLANDINS t a i n i n g sodium EDTA as anticoagulant and indomethacin as cyclooxygenase i n h i b i t o r ( f i n a l concentrations 5.4 and 0.1 mM, r e s p e c t i v e l y ) . Plasma was separated by immediate c e n t r i f u g a t i o n (1500 x g, 4oc, 15 min) and analyzed f o r 13,14-dihydro-PGE1 using h . p . l . c . (7) combined with radioimmunoassay of eluted f r a c t i o n s f o r the f i r s t group of 6 p a t i e n t s . The anti-PGE1 antiserum (8) used f o r the radioimmunoassay e x h i b i t s 100 % r e l a t i v e cross-reaction with 13,14-dihydro-PGE1. Blood from the second group of 4 patients was analyzed by GC/MS/MS (to be published) using a p u r i f i c a t i o n procedure described previously (9). Standard amounts of 13,14-dihydro-PGE1 (100 pg - I ng) added to human whole blood were shown to be stable f o r at least 2 - 3 hours. The r e s u l t s were calculated as means + S.E.M. S t a t i s t i c a l was performed by use of Student's t - t e s t f o r paired values.
analysis
RESULTS The r e s u l t s are shown in Table I . While preinfusion plasma l e v e l s of 13,14-dihydro-PGE1 were close to or below the detection l i m i t s of Table I Levels of 13,14-dihydro-PGE1 (pg/ml) in patients with peripheral a r t e r i a l occlusive disease before, during and a f t e r the end of i n t r a venous infusions of PGEI. The f i r s t group of patients (n=6) received infusions of 80 pg PGEI f o r 60 min; t h e i r plasma was analyzed by h.p.l.c./radioimmunoassay. The second group of 4 patients received infusions of 60 pg PGEI f o r 30 min; t h e i r plasma was analyzed by GC/MS/MS. Results represent means + S.E.M. Plasma 13,14-dihydro-PGE1 (pg/ml) Time
h.p.l.c./radioimmunoassay
GC/MS/MS
(min)
(n=6)
(n=4)
0
5+2
30
n.d.
40
19 + 6 *
60
22 + 5 **
90
13 + 5 +
0.7+0.1 13
+ 1 *** n.d n.d.
4
+ 0.2 +++
n . d . : not determined; * p < ~@~5, ** p < 0.01, * * * p < 0.001 as compared to 0 min; p < 0.05, p < 0.001 as compared to end of i n f u sion period (60 or 30 min, r e s p e c t i v e l y ) . the assay methods, concentrations at the end of the infusion periods (60 and 30 min, r e s p e c t i v e l y ) had s i g n i f i c a n t l y increased to 22 + 5 pg/ml (n=6, p < 0.01) and to 13 + I pg/ml (n=4, p < 0.001), r e s p e c t i v e l y . T h i r t y min a f t e r the en~ of the infusion period l e v e l s of 13,14-dihydro-PGE1 had s i g n i f i c a n t l y (p < 0.05) decreased to 13 + 5 pg/ml in those 6 patients that had received 8o ~g PGEI f o r 60 A n . S i m i l a r l y a s i g n i f i c a n t (p < 0.001) decrease in the metabolite l e v e l s
226
MARCH 1991 VOL. 41 NO. 3
PROSTAGLANDINS to 4 + 0.2 pg/ml was observed I hour after the end of the infusion period- in those 4 patients that had received 60 pg PGEI for 30 min (Table I ) . DISCUSSION The results c l e a r l y demonstrate for the f i r s t time formation of 13,14-dihydro-PGE1 during intravenous administration of PGEI to man. On the other hand, formation of t h i s metabolite in v i t r o (5,10,11) as well as in the rat in vivo (12) has long been known to occur. In man, the analogous metabolite 13,14-dihydro-PGF2~ has been found to occur in plasma a f t e r intravenous administration of PGF2~ (13,14). Similar to PGEI, 13,14-dthydro-PGE1 has been shown to lower blood pressure (5) and to i n h i b i t p l a t e l e t aggregation (6). Although the mechanism of beneficial action of PGEI in peripheral a r t e r i a l occlusive disease remains unknown and the concentrations of 13,14-dihydro-PGE1 formed during the infusions of PGEI are rather low, the b i o l o g i c a l l y active metabolite could contribute to the therapeutic effects of intravenous infusions of PGEI observed in such patients (1,2). ACKNOWLEDGEMENTS We thank Mrs. K. Rezny for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (Pe 256/4-I and Se 263/9). REFERENCESS I.
Carlsson, L.A. and A.G. Olsson. Intravenous prostaglandin severe peripheral vascular disease. Lancet i i : 810, 1976.
2.
Sinzlnger, H., I. V i r g o l i n i and J. O'Grady. Clinical t r i a l s of PGEI, PGI2 and mimetics in patients with peripheral vascular disease. In: Prostaglandins in Clinical Research, (K. SchrOr and H. Sinzinger, eds.) Alan Liss Inc., New York, 1989, p. 85.
3.
Golub M, P. Zia, M. Matsuno and R. Horton. Metabolism of prostaglandins AI and El in man. J. Clin. Invest. 56: 1404, 1975.
4.
Hamberg, M. and B. Samuelsson. On the metabolism of prostaglandins El and E2 in man. J. Biol. Chem. 246: 6713, 1971.
5.
Angg~rd E. The biological a c t i v i t i e s of three metabolites prostaglandin El. Acta physiol, scand. 66: 509, 1966.
6.
Westwick, J. The e f f e c t of pulmonary metabolites of prostaglandins El, E2 and F2~ on ADP-induced aggregation of human and rabbit p l a t e l e t s . Br. J. Pharmacol. 58: 297P, 1976.
7.
Hesse, W.H., H. Schweer, H.W. Seyberth and B.A. Peskar. Separation and determination of prostaglandin EI metabolites by high-
MARCH 1991 VOL. 41 NO. 3
El in
of
227
PROSTAGLANDINS
performance l i q u i d chromatography. J. Chromatogro, in press 8.
Diekmann, J.M., A. Jobke, B.A. Peskar and G. Hertting. Angiotensin ll-induced contractions of rabbit splenic capsular strips and release of prostaglandins. Use of radioimmunoassays for prostaglandins El and E2. Naunyn-Schmiedeberg's Arch. Pharmacol. 297: 177, 1977.
9.
Schweer, H., C.O. Meese, O. FOrst, P.G. KOhl and H.W. Seyberth. Tandem mass spectrometric determination of 11-dehydro-thromboxane B2, and index metabolite of thromboxane A2 in plasma and urine. Anal. Biochem. 164: 156, 1987.
10.
~ngg~rd E. and B. Samuelsson. Prostaglandins and related factors. 28. Metabolism of prostaglandin El in guinea pig lung: the structures of two metabolites. J. Biol. Chem, 239: 4097, 1964.
11.
Angg~rd E. and C. Larsson. The sequence of the early steps in the metabolism of prostaglandin El. Eur. J. Pharmacol. 14: 66, 1971.
12.
Samuelsson, B. Prostaglandins and related factors. 27. Synthesis of t r i t i u m - l a b e l e d prostaglandin El and studies on its d i s t r i bution and excretion in the rat. J. Biol. Chem. 239, 4091, 1964.
13.
GranstrOm. E. On the metabolism of prostaglandin F2~ in female subjects. Structures of two metabolites in blood. Eur. J. Biochem. 27: 462, 1972.
14.
Green, K. Quantitative mass spectrometric analysis of prostaglandins. Adv. Biosci. 9: 91, 1973.
Editor:
228
P.W. R a m w e l l
Received:
10-17-90
Accepted:
12-7-90
MARCH 1991 VOL. 41 NO. 3