Scandinavian Journal of Rheumatology
ISSN: 0300-9742 (Print) 1502-7732 (Online) Journal homepage: http://www.tandfonline.com/loi/irhe20
Inhibition of Prostaglandin Biosynthesis by Tolfenamic Acid in Vitro Inge-Britt Lindén, J. Parantainen & H. Vapaatalo To cite this article: Inge-Britt Lindén, J. Parantainen & H. Vapaatalo (1976) Inhibition of Prostaglandin Biosynthesis by Tolfenamic Acid in Vitro, Scandinavian Journal of Rheumatology, 5:3, 129-132 To link to this article: http://dx.doi.org/10.3109/03009747609165450
Published online: 28 Aug 2009.
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Date: 26 December 2015, At: 07:59
Scand J Rheumatology 5: 129-132, 1976
INHIBITION OF PROSTAGLANDIN BIOSYNTHESIS BY TOLFENAMIC ACID IN VITRO
Downloaded by [University of California, San Diego] at 07:59 26 December 2015
Inge-Britt Linden, J. Parantainen and H. Vapaatalo From the Research Laboratories of Medica Ltd., P . 0 .Box 325, SF40101 Helsinki 10, and the Institute of Biomedical Sciences, University of Tampere, SF-33520 Tampere 52, Finland
Non-steroidal anti-inflammatory drugs are potent inhibitors of the biosynthesis of prostaglandins (PGs) both in vitro and in vivo, and this mechanism is also regarded as the basis for the therapeutic action of these drugs (2, 3, 16, 17). We have tested the activity of tolfenamic acid on the biosynthesis of PGs and compared it with that of acetylsalicylic acid and indomethacin. Rabbit kidney medulla was chosen as a source of the enzyme system (PG synthetase) because of the high Tolfenamic acid, N-(2-methyl-3-chlorphenyl)-an- enzyme activity in this tissue (1). Preliminary thranilic acid, is a new anthranilic acid deriva- results have been presented in part earlier (10). tive. Chemically it resembles mefenamic acid and flufenamic acid (Fig. 1). The anti-inflammatory and MATERIAL AND METHODS analgetic properties too have been found much A PG synthetase system was prepared from rabbit kidney the same in animal experiments (6). In clinical medulla as described by Bhattacherjee and Eakins (I). studies, tolfenamic acid appears to be comparable New Zealand rabbits of either sex, weighing 3.5-4.0kg, to other non-steroidal anti-inflammatory drugs (7). were killed by a blow on the head. The kidneys were Tolfenamic acid is well tolerated by patients (7, removed and the medullas excised from the cortex in 9, 12, 13, 14), and the clinical results in long- Krebs solution at 4°C. The tissue was homogenized with term treatment of rheumatoid arthritis are similar to the results obtained with indomethacin and phenylbutazone (12, 13, 14). Dysmenorrhea seems to be another promising indication for tolfenamic acid therapy (8).
ABSTRACT. The effect of tolfenamic acid on prostaglandin biosynthesis was investigated by .using rabbit kidney medulla microsomal fraction, and the results compared with those obtained with indomethacin and acetylsalicylic acid. Tolfenamic acid inhibited the conversion of arachidonic acid to prostaglandin & very effectively and in a dose-dependent manner. IC,, values were 0.64 p M for tolfenamic acid, 0.76 FM for indomethacin, and 7 mM for acetylsalicylic acid. The inhibition of prostaglandin biosynthesis may be related to the good anti-inflammatory action of tolfenamic acid. '
@COOH
3 5 CI
tolfenamlc acid
@$'COOH
:,:A
Q
O
O
H
c6)&
rnefenamlc aC id
flufenarnic acid
Fig. 1 . Formulas of the anthranilic acid derivatives tolfenamic acid, mefenamic acid, and flufenamic acid. 9-76 I864
Log molar conc.
Fig. 2 . Dose-response curves for tolfenamic acid, indomethacin, and acetylsalicylic acid in the inhibition of the PG-synthetase system of rabbit kidney medulla. S w n d J Rhrumutologv 5
130
I.-B. Linden et al. 1 V
TOLFENAMIC
ACID
.o9%
/
2000
15001
2500-
ACID
2000-
1500-
1000-
Downloaded by [University of California, San Diego] at 07:59 26 December 2015
ACETYLSALICYLIC
i"/
+99
1 V
25001
INDOMETHACIN
2000-
1500-
1000-
Fig. 3 . Lineweaver-Burk plots calculated from inhibition of the PG-synthetase system by tolfenamic acid, indomethacin, and acetylsalicylic acid.
a Sorvall homogenizer in 100 mM Ssrensen phosphate buffer, pH 7.4 (2000 rpm for 1 min at +4"C). The homogenate was centrifuged for 10 min at 10000 g (+4"C) and the supernatant for 60 min at 80000 g (+4"C). The remaining microsomal pellet was lyophilized, and stored at -20°C. The amount of protein was deter-
Table 1. Relative potency of tolfenamic acid, indomethacin, and acetylsalicylic acid on PG biosynthesis ICSO Tolfenamic acid Indomethacin Acetylsalicylic acid Scand J Rheumcitoloyy 5
0.64pM 0.76 pM 7.0mM
Relative potency 1.2 1 O.OOO1
mined according to the method of Lowry et al. ( I I). When used, the powder was dissolved in 100 mM phosphate buffer, pH 7.4, which was used throughout the experiment. The reaction system (2 ml) consisted of PG synthetase (1 mg proteinlml), arachidonic acid as substrate (20 pg/ ml), and the co-factors glutathione (100 pg/ml) and hydroquinone (20 &ml; all final concentrations). The substances were dissolved in phosphate buffer, pH 7.4, and the test drugs added in different concentrations. The reaction systems were saturated with oxygen and incubated at 37°C for 20 min. The reaction was terminated by heating the tubes in boiling water for 45 sec, and the precipitate was separated by centrifugation. The formation of PGs was assayed for PGE, on hamster stomach strip (15) by superfusion technique (18). The purity of the end product was controlled by thin-layer radiochromatography (scanning by liquid scintillator counter Wallac Decem-NTL 314).
Tolfenamic acid and PG biosynthesis
131
The following drugs were used: Tolfenamic acid (Medica Ltd., Clotam@,lic. A/S GEA), indomethacin (ICFI), acetylsalicylic acid (RhBne-Poulenc), arachidonic acid 99% (Sigma Chemical Co.), IJ4C arachidonic acid 58 mCi/mmol (The Radiochemical Centre, Amersham), reduced glutathione (Merck,), hydroquinone (Fluka AG), and PGG, PGA, and PGF:o standards (Chinoin and Upjohn Co.). Lineweaver-Burk plot was used in the enzyme kinetic studies. The regression lines and IC5,, values were obtained mathematically.
The plasma levels of tolfenamic acid in patients on a dose regimen of 3x 100 mg/day exceed the concentrations needed for 50% inhibition of PG biosynthesis in the present study. This favours the opinion that the effect of tolfenamic acid in vivo can be mediated by the same mechanism as has formerly been suggested for the action of other fenamates (4) and of indomethacin (16).
RESULTS
The authors wish to thank Dr Pike (Upjohn Co.) and Dr Virig (Chinoin) for the generous gift of prostaglandin standards.
Downloaded by [University of California, San Diego] at 07:59 26 December 2015
ACKNOWLEDGEMENTS The basal formation of PGs in our test system was about 100-150 ng/mg protein. When substrate and co-factors were added, 1800-2200 ng of PGs was synthesized. Thin-layer chromatography with I-l4C arachidonic acid revealed that the main constituent formed during conversion was P G b . The dose-response curves for the drugs tested (Fig. 2) show that tolfenamic acid and indomethacin were active within the concentration range to 10-5M, while acetylsalicylic acid was to needed in much higher concentrations ( 5x IO+!M). The relative potencies of the agents are seen in Table I. The IC50 values for tolfenamic acid and indomethacin were of the same magnitude (0.64 p M and 0.76 pM, respectively), and for acetylsalicylic acid much higher (7.0 mM). Kinetic studies revealed that the type of inhibition for all the test substances is apparently competitive (Fig. 3). DISCUSSION The effect of tolfenamic acid on PG biosynthesis has not been tested before. Its inhibitory potency in the present study was about the same as that of indomethacin. This is in good agreement with the results obtained with other fenamates, of which meclofenamic acid is somewhat more effective than indomethacin, and mefenamic acid slightly less so. The inhibitory activity of indomethacin (IC50 0.76 p M ) in our test system was somewhat stronger than in two other studies with rabbit kidney PG synthetase, where the IC5,, values were 1.50 pM (1) and 3.7 p M (9, respectively. Acetylsalicylic acid was a rather weak inhibitor in the present work, but even this ICs0 value (7.0 mM) is about the same magnitude as in another study (5) with microsomal enzyme preparation of rabbit kidney medulla (IC502.8 mM).
REFERENCES I . Bhattacherjee, P. & Eakins, K. E.: Inhibition of the prostaglandin synthetase system in ocular tissues by indomethacin. Br J Pharmac 50: 227, 1974. 2. Ferreira, S. H., Moncada, S. & Vane, J. R.: Indomethacin and aspirin abolish prostaglandin release from the spleen. Nature, New Bio1231: 237, 1971. 3. Ferreira, S . H. & Vane, J. R.: New aspects of the mode of action of nonsteroid anti-inflammatory drugs. Ann Rev Pharmac 14: 57, 1974. 4. Flower, R., Gryglewski, R., Herbaczynska-Cedro, K. & Vane, J. R.: The effects of anti-inflammatory drugs on a cell-free prostaglandin synthetase system from dog spleen. Nature, New Bio1238: 104, 1972. 5. Flower, R. J. & Vane, J. R.: Some pharmacologic and biochemical aspects of prostaglandin biosynthesis and its inhibition. I n : Prostaglandin Synthetase Inhibitors (ed. H. J. Robinson & J. R. Vane), p. 9. Raven Press, New York, 1974. 6. A/S GEA Pharmacological Dept.: Unpublished results. 7. Kajander, A., Laine, V. & Gothoni, G.: Effect of tolfenamic acid in rheumatoid arthritis. Scand J Rheum I: 91, 1972. 8. Kauppila, A.: Personal communication. 9. Kauppila, A., Vapaatalo, H. & Taskinen, P. J.: A double-blind clinical study on long-term use of oxyphenbutazone and tolfenamic acid in connection with telecobalt therapy. Arzneim.-Forsch. 25: 1082, 1975. 10. Linden, I.-B., Parantainen J., Karppanen, H. & Vapaatalo, H.: Inhibitory effects of tolfenamic acid, indomethacin and acetylsalicylic acid on prostaglandin synthetase and phosphodiesterase in vitro. Sixth International Congress of Pharmacology. Helsinki, Finland, July 20-25, 1975. Abstract 1448, 1975. 11. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J.: Protein measurement with the fohn phenol reagent. J Biol Chem 193: 265, 1951. 12. Martio, J., Kajander, A., Mutru, 0. & Gothoni, G.: Long-term treatment with tolfenamic acid in inflammatory rheumatoid diseases. Scand J Rheum 4: Suppl. 8, Abstract 136, 1975.
132
I.-B. Lindkn et al.
Downloaded by [University of California, San Diego] at 07:59 26 December 2015
13. Oka, M. & Anttinen, J.: Clinical trial with tolfenamic acid in rheumatoid arthritis. To be published. 14. Ruotsi, A., Oka, M. & Rekonen, A,: Tolfenamic acid in rheumatoid arthritis. Scandinavian Congress of Rheumatologists, Abstract 12, 1972. 15. Ubatuba, F. B.: The use of hamster stomach strip in vitro as an assay preparation for prostaglandins. Br J Pharmac49: 662, 1973. 16. Vane, J. R.: Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature, New Biol231: 232, 1971. 17. Vane, J. R.: Mode of action of aspirin and similar compounds. In: Prostaglandin Synthetase Inhibitors
(ed. H. J. Robinson & J. R. Vane), p. 155. Raven Press, New York. 1974. 18. Vapaatalo, H., Lindtn, I.-B. & Parantainen, J.: A sensitive biological assay for PGE and acetylcholine. J Pharm Pharmac 28:188, 1976. Submitted f o r publication February 5 , 1976
J. Parantainen Medica Ltd., Library P. 0. Box 325 SF-00101 Helsinki 10 Finland
ISSN: 0300-9742 (Print) 1502-7732 (Online) Journal homepage: http://www.tandfonline.com/loi/irhe20
Inhibition of Prostaglandin Biosynthesis by Tolfenamic Acid in Vitro Inge-Britt Lindén, J. Parantainen & H. Vapaatalo To cite this article: Inge-Britt Lindén, J. Parantainen & H. Vapaatalo (1976) Inhibition of Prostaglandin Biosynthesis by Tolfenamic Acid in Vitro, Scandinavian Journal of Rheumatology, 5:3, 129-132 To link to this article: http://dx.doi.org/10.3109/03009747609165450
Published online: 28 Aug 2009.
Submit your article to this journal
Article views: 4
View related articles
Citing articles: 7 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=irhe20 Download by: [University of California, San Diego]
Date: 26 December 2015, At: 07:59
Scand J Rheumatology 5: 129-132, 1976
INHIBITION OF PROSTAGLANDIN BIOSYNTHESIS BY TOLFENAMIC ACID IN VITRO
Downloaded by [University of California, San Diego] at 07:59 26 December 2015
Inge-Britt Linden, J. Parantainen and H. Vapaatalo From the Research Laboratories of Medica Ltd., P . 0 .Box 325, SF40101 Helsinki 10, and the Institute of Biomedical Sciences, University of Tampere, SF-33520 Tampere 52, Finland
Non-steroidal anti-inflammatory drugs are potent inhibitors of the biosynthesis of prostaglandins (PGs) both in vitro and in vivo, and this mechanism is also regarded as the basis for the therapeutic action of these drugs (2, 3, 16, 17). We have tested the activity of tolfenamic acid on the biosynthesis of PGs and compared it with that of acetylsalicylic acid and indomethacin. Rabbit kidney medulla was chosen as a source of the enzyme system (PG synthetase) because of the high Tolfenamic acid, N-(2-methyl-3-chlorphenyl)-an- enzyme activity in this tissue (1). Preliminary thranilic acid, is a new anthranilic acid deriva- results have been presented in part earlier (10). tive. Chemically it resembles mefenamic acid and flufenamic acid (Fig. 1). The anti-inflammatory and MATERIAL AND METHODS analgetic properties too have been found much A PG synthetase system was prepared from rabbit kidney the same in animal experiments (6). In clinical medulla as described by Bhattacherjee and Eakins (I). studies, tolfenamic acid appears to be comparable New Zealand rabbits of either sex, weighing 3.5-4.0kg, to other non-steroidal anti-inflammatory drugs (7). were killed by a blow on the head. The kidneys were Tolfenamic acid is well tolerated by patients (7, removed and the medullas excised from the cortex in 9, 12, 13, 14), and the clinical results in long- Krebs solution at 4°C. The tissue was homogenized with term treatment of rheumatoid arthritis are similar to the results obtained with indomethacin and phenylbutazone (12, 13, 14). Dysmenorrhea seems to be another promising indication for tolfenamic acid therapy (8).
ABSTRACT. The effect of tolfenamic acid on prostaglandin biosynthesis was investigated by .using rabbit kidney medulla microsomal fraction, and the results compared with those obtained with indomethacin and acetylsalicylic acid. Tolfenamic acid inhibited the conversion of arachidonic acid to prostaglandin & very effectively and in a dose-dependent manner. IC,, values were 0.64 p M for tolfenamic acid, 0.76 FM for indomethacin, and 7 mM for acetylsalicylic acid. The inhibition of prostaglandin biosynthesis may be related to the good anti-inflammatory action of tolfenamic acid. '
@COOH
3 5 CI
tolfenamlc acid
@$'COOH
:,:A
Q
O
O
H
c6)&
rnefenamlc aC id
flufenarnic acid
Fig. 1 . Formulas of the anthranilic acid derivatives tolfenamic acid, mefenamic acid, and flufenamic acid. 9-76 I864
Log molar conc.
Fig. 2 . Dose-response curves for tolfenamic acid, indomethacin, and acetylsalicylic acid in the inhibition of the PG-synthetase system of rabbit kidney medulla. S w n d J Rhrumutologv 5
130
I.-B. Linden et al. 1 V
TOLFENAMIC
ACID
.o9%
/
2000
15001
2500-
ACID
2000-
1500-
1000-
Downloaded by [University of California, San Diego] at 07:59 26 December 2015
ACETYLSALICYLIC
i"/
+99
1 V
25001
INDOMETHACIN
2000-
1500-
1000-
Fig. 3 . Lineweaver-Burk plots calculated from inhibition of the PG-synthetase system by tolfenamic acid, indomethacin, and acetylsalicylic acid.
a Sorvall homogenizer in 100 mM Ssrensen phosphate buffer, pH 7.4 (2000 rpm for 1 min at +4"C). The homogenate was centrifuged for 10 min at 10000 g (+4"C) and the supernatant for 60 min at 80000 g (+4"C). The remaining microsomal pellet was lyophilized, and stored at -20°C. The amount of protein was deter-
Table 1. Relative potency of tolfenamic acid, indomethacin, and acetylsalicylic acid on PG biosynthesis ICSO Tolfenamic acid Indomethacin Acetylsalicylic acid Scand J Rheumcitoloyy 5
0.64pM 0.76 pM 7.0mM
Relative potency 1.2 1 O.OOO1
mined according to the method of Lowry et al. ( I I). When used, the powder was dissolved in 100 mM phosphate buffer, pH 7.4, which was used throughout the experiment. The reaction system (2 ml) consisted of PG synthetase (1 mg proteinlml), arachidonic acid as substrate (20 pg/ ml), and the co-factors glutathione (100 pg/ml) and hydroquinone (20 &ml; all final concentrations). The substances were dissolved in phosphate buffer, pH 7.4, and the test drugs added in different concentrations. The reaction systems were saturated with oxygen and incubated at 37°C for 20 min. The reaction was terminated by heating the tubes in boiling water for 45 sec, and the precipitate was separated by centrifugation. The formation of PGs was assayed for PGE, on hamster stomach strip (15) by superfusion technique (18). The purity of the end product was controlled by thin-layer radiochromatography (scanning by liquid scintillator counter Wallac Decem-NTL 314).
Tolfenamic acid and PG biosynthesis
131
The following drugs were used: Tolfenamic acid (Medica Ltd., Clotam@,lic. A/S GEA), indomethacin (ICFI), acetylsalicylic acid (RhBne-Poulenc), arachidonic acid 99% (Sigma Chemical Co.), IJ4C arachidonic acid 58 mCi/mmol (The Radiochemical Centre, Amersham), reduced glutathione (Merck,), hydroquinone (Fluka AG), and PGG, PGA, and PGF:o standards (Chinoin and Upjohn Co.). Lineweaver-Burk plot was used in the enzyme kinetic studies. The regression lines and IC5,, values were obtained mathematically.
The plasma levels of tolfenamic acid in patients on a dose regimen of 3x 100 mg/day exceed the concentrations needed for 50% inhibition of PG biosynthesis in the present study. This favours the opinion that the effect of tolfenamic acid in vivo can be mediated by the same mechanism as has formerly been suggested for the action of other fenamates (4) and of indomethacin (16).
RESULTS
The authors wish to thank Dr Pike (Upjohn Co.) and Dr Virig (Chinoin) for the generous gift of prostaglandin standards.
Downloaded by [University of California, San Diego] at 07:59 26 December 2015
ACKNOWLEDGEMENTS The basal formation of PGs in our test system was about 100-150 ng/mg protein. When substrate and co-factors were added, 1800-2200 ng of PGs was synthesized. Thin-layer chromatography with I-l4C arachidonic acid revealed that the main constituent formed during conversion was P G b . The dose-response curves for the drugs tested (Fig. 2) show that tolfenamic acid and indomethacin were active within the concentration range to 10-5M, while acetylsalicylic acid was to needed in much higher concentrations ( 5x IO+!M). The relative potencies of the agents are seen in Table I. The IC50 values for tolfenamic acid and indomethacin were of the same magnitude (0.64 p M and 0.76 pM, respectively), and for acetylsalicylic acid much higher (7.0 mM). Kinetic studies revealed that the type of inhibition for all the test substances is apparently competitive (Fig. 3). DISCUSSION The effect of tolfenamic acid on PG biosynthesis has not been tested before. Its inhibitory potency in the present study was about the same as that of indomethacin. This is in good agreement with the results obtained with other fenamates, of which meclofenamic acid is somewhat more effective than indomethacin, and mefenamic acid slightly less so. The inhibitory activity of indomethacin (IC50 0.76 p M ) in our test system was somewhat stronger than in two other studies with rabbit kidney PG synthetase, where the IC5,, values were 1.50 pM (1) and 3.7 p M (9, respectively. Acetylsalicylic acid was a rather weak inhibitor in the present work, but even this ICs0 value (7.0 mM) is about the same magnitude as in another study (5) with microsomal enzyme preparation of rabbit kidney medulla (IC502.8 mM).
REFERENCES I . Bhattacherjee, P. & Eakins, K. E.: Inhibition of the prostaglandin synthetase system in ocular tissues by indomethacin. Br J Pharmac 50: 227, 1974. 2. Ferreira, S. H., Moncada, S. & Vane, J. R.: Indomethacin and aspirin abolish prostaglandin release from the spleen. Nature, New Bio1231: 237, 1971. 3. Ferreira, S . H. & Vane, J. R.: New aspects of the mode of action of nonsteroid anti-inflammatory drugs. Ann Rev Pharmac 14: 57, 1974. 4. Flower, R., Gryglewski, R., Herbaczynska-Cedro, K. & Vane, J. R.: The effects of anti-inflammatory drugs on a cell-free prostaglandin synthetase system from dog spleen. Nature, New Bio1238: 104, 1972. 5. Flower, R. J. & Vane, J. R.: Some pharmacologic and biochemical aspects of prostaglandin biosynthesis and its inhibition. I n : Prostaglandin Synthetase Inhibitors (ed. H. J. Robinson & J. R. Vane), p. 9. Raven Press, New York, 1974. 6. A/S GEA Pharmacological Dept.: Unpublished results. 7. Kajander, A., Laine, V. & Gothoni, G.: Effect of tolfenamic acid in rheumatoid arthritis. Scand J Rheum I: 91, 1972. 8. Kauppila, A.: Personal communication. 9. Kauppila, A., Vapaatalo, H. & Taskinen, P. J.: A double-blind clinical study on long-term use of oxyphenbutazone and tolfenamic acid in connection with telecobalt therapy. Arzneim.-Forsch. 25: 1082, 1975. 10. Linden, I.-B., Parantainen J., Karppanen, H. & Vapaatalo, H.: Inhibitory effects of tolfenamic acid, indomethacin and acetylsalicylic acid on prostaglandin synthetase and phosphodiesterase in vitro. Sixth International Congress of Pharmacology. Helsinki, Finland, July 20-25, 1975. Abstract 1448, 1975. 11. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J.: Protein measurement with the fohn phenol reagent. J Biol Chem 193: 265, 1951. 12. Martio, J., Kajander, A., Mutru, 0. & Gothoni, G.: Long-term treatment with tolfenamic acid in inflammatory rheumatoid diseases. Scand J Rheum 4: Suppl. 8, Abstract 136, 1975.
132
I.-B. Lindkn et al.
Downloaded by [University of California, San Diego] at 07:59 26 December 2015
13. Oka, M. & Anttinen, J.: Clinical trial with tolfenamic acid in rheumatoid arthritis. To be published. 14. Ruotsi, A., Oka, M. & Rekonen, A,: Tolfenamic acid in rheumatoid arthritis. Scandinavian Congress of Rheumatologists, Abstract 12, 1972. 15. Ubatuba, F. B.: The use of hamster stomach strip in vitro as an assay preparation for prostaglandins. Br J Pharmac49: 662, 1973. 16. Vane, J. R.: Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature, New Biol231: 232, 1971. 17. Vane, J. R.: Mode of action of aspirin and similar compounds. In: Prostaglandin Synthetase Inhibitors
(ed. H. J. Robinson & J. R. Vane), p. 155. Raven Press, New York. 1974. 18. Vapaatalo, H., Lindtn, I.-B. & Parantainen, J.: A sensitive biological assay for PGE and acetylcholine. J Pharm Pharmac 28:188, 1976. Submitted f o r publication February 5 , 1976
J. Parantainen Medica Ltd., Library P. 0. Box 325 SF-00101 Helsinki 10 Finland
