European Journal of Clinical Pharmacology

Europ. J. clin. Pharmacol. 12, 291-296 (1977)

© by Springer-Verlag 1977

Double Blind Study of the Effect of Glafenine (Glifanan ®) on Oral Anticoagulant Therapy with Phenprocoumon (Marcumar®) J. K. Boeijinga and W. J. F. van der Vijgh Department of Internal Medicine, Academical Hospital, Vrije Universiteit, Amsterdam, The Netherlands

Summary. The interaction between phenprocoumon (Marcumar ®) and glafenine (Glifanan ®) was investigated in a double blind study of twenty patients receiving long term treatment with phenprocoumon. Thrombotesttime (TT) values had been stable for more than three months before the study. Patients taking glafenine showed a significant increase in TT during the second and third week of the trial (P < 0.05) compared with the placebo group. The increase in TT was not significant in the fourth week. The average concentrations of phenprocoumon were similar in both groups, which suggests that displacement of the drug from binding was not important. Concentrations of clotting factors II, VII and X showed a decrease in all patients at the time of the maximum TT values. A possible explanation for this interaction is discussed, but the mechanism remains uncertain.

Interaction between coumarin derivatives and other drugs is among the best studied of all interactions, probably because of the possible clinical consequences. A potentiating effect has been shown, e. g. for phenylbutazone (Sigg et al., 1956), oxyphenbutazone (Hobbs et al., 1965; Aggeler et al., 1967), paracetamol (Loeliger, 1973) and salicylic acid (Quick and Clesceri, 1969). However, the increased risk of bleeding in patients on coumarin therapy receiving aspirin is mainly the result of alteration in platelet function (Mustard and Packham, 1970) and a haemorrhagic effect on gastric mucosa (KochWeser and Sellers, 1971). An effect on the anticoagulant state could not be demonstrated for acetaminophen (Antlitz and Awalt, 1969), glafenine (no literature available except the preliminary study mentioned) and several other analgesics.

Key words: Interactions, glafenine, phenprocoumon, hypoprothrombinaemia, clotting factors.

Material and Methods

In a preliminary study (Boeijinga et al., 1974) the possibility of interaction between glafenine (Glifanan ®) and coumarin derivatives was suggested. Five out of seven patients in that study showed such an increase in hypoprothrombinaemia, that reduction in the dose of coumarin was necessary. A Thrombotesttime (TT; Owren, 1962; 1963) of two hundred seconds was taken as an arbitrary limit for the reduction. Because of the less stable hypoprothrombinaemia produced by acenocoumarol (Sintrom ®) than by phenprocoumon (Breed et al., 1969), it was decided to confine the study to phenprocoumon alone.

Twenty volunteers were selected from patients controlled by the Amsterdam Thrombosis Service. For admission to the study they had to meet the following criteria: - phenprocoumon was their oral anticoagulant. - haemostasis, measured by TT, should have been so stable that there had been no need to change the dose of coumarin for more than three months before commencement of the study. - further medication: only those drugs were permitted which were unlikely to influence haemostasis. No change in medication should have been made during the three month period preceding the study or during the study itself. Digitalis glycosides and diuretics proved to be the most frequently used drugs (Table 1 ) . - There was no history of serious diseases, e.g. infection, liver disease, or manifest heart failure (Weenink, 1971).

J. K. Boeijinga and W. J. F. van der Vijgh: Glafenine and Phenprocoumon

292 Table 1. Details of the subjects

Nr.

Sex

Age

Glafenine/ Placebo

01 02 03 04 05 06 07 08 09 10 11 12 13 14 15

M M M M M F M M M M M M M M M

68 72 40 64 53 52 72 67 63 60 64 65 69 72 56

G G G G G G G G G G P P P P P

16 17

M F

66 69

P P

18 19 20

M M F

58 79 71

P P P

Additional

digoxine, furosemiede digoxine, practolol digoxine, nitroglycerine digoxine digoxine digoxine, clofibrate digoxine digoxine, furosemide digoxine, practolol quinidine sulphate, alprenolol amiodaron HC1 L- a methyldopa digoxine, nitroglycerine quanidinesulpbate, amiloride HC1 digoxine digoxine, amiloride HC1

Table 2. Tablet weight and glafenine content in mg (glafenine

n = 10, placebo n 10)

Mean tablet weight SD Extreme values Glafenine content SD Extreme values

Placebo

Glafenine

391.7 4.9 385.6-400.4

353.3 3.3 347.2-358.5 196 3.8 192-202

- u r i n a l y s i s , haematology and blood chemistry screening, including urea, creatinine, uric acid, liver function tests, protein spectrum, L.E. phenomenon and A. N. F. were all normal. The volunteers were told that the possible influence of a simple analgesic, which had been widely used for many years without any side effects having been reported, on their anticoagulant therapy would be investigated. Written consent was obtained from all patients. Volunteers were randomised with the aid of a table of random permutation into glafenine and placebo groups each of ten subjects. All participants were seen twice a week and they could always reach the investigator by phone, which resulted in very good cooperation. Possible side effects were noted on a blank sheet to avoid suggesting any particular symptom. These data, none of

which affected the anticoagulant state, are to be published elsewhere, together with data from a subsequent study of the possible gastrointestinal toxicity of glafenine. The trial lasted for exactly four weeks. The patients were asked to take one tablet of glafenine 200 mg or placebo at 9.00, 16.00 and 23.00 h. The above mentioned laboratory tests were repeated on the last day of the trial and after four weeks. During the trial and for the following four weeks the TT was measured on the same day each week, in fresh blood obtained by venepuncture; to each 5 ml blood 20% sodium citrate 0.1 ml was added and mixed by shaking. The TT was done with a Lode coagulatometer ~ by technicians who knew that an interaction study was going on, but who were ignorant of the phase of the investigation at any given moment. The glafenine and placebo tablets were specially made for the study, charge numbers 4.04.427 and 4.04.428, respectively2. Ten glafenine tablets were analysed according to the prescribed methods of Roussel Ltd.: each tablet was shaken with 100 ml HC1 (0.1 N), for 30 rain, in a reaction vessel. The solution obtained was filtered and the first 20 ml discarded. The next 0.5 ml filtrate was diluted to 100 ml with HC1 (0.1 N). The absorption (A) of this solution was measured spectrophotometrically at 343 nm in a 1 cm cuvette (mg glafenine per tablet = A × 200.000/490). The concentration of phenprocoumon in plasma samples was determined according to Seiler and Duckert (1968). Blood clotting factors II (prothrombin), VII (proconvertin) and X (Stuart Prower) were determined by the one step method, using artificial or congenital deficient plasma, according to Biggs (1972).

Results

Details of the patients data are shown in Table 1. The indication for anticoagulant therapy was a previous coronary infarotion in all but three subjects, in whom it was impending infarction (nos. 06 and 11) or atrial fibrillation (no. 08). Perhaps because of the intensive control, the trial drop out percentage was zero. Neither the coumarin dose nor that of any other drug was changed during the full trial period, except in patient number 03, whose phenprocoumon medication had to be stopped for one day when a TT of

1 Lode Ltd., Groningen, Netherlands 2 Roussel Laboratories Ltd., Paris, France

293

J. K. Boeijinga and W. J. F. van der Vijgh: Glafenine and Phenprocoumon Table 3. Measured TT (Thrombotesttimes) in seconds before, during and after the trial period Nr.

- 9

- 6

- 3

0

1

2

3

4

5

6

8

01 02 03 04 05 06 07 08 09 10

168 150 146 142 126 I08 116 I20 115 174

133 135 94 148 123 113 118 104 I41 154

114 163 95 129 106 124 122 134 148 142

135 170 146 154 150 116 136 112 132 146

117 160 184 153 174 141 139 102 155 150

t39 164 190 180 167 150 138 131 196 188

130 157 239" 165 145 188 159 146 160 143

162 153 181 147 132 107 I57 127 164 153

142 142 168 147 124 136 158 159 146 158

135 I49 157 131 129 137 157 123 175 t43

137 131 165 151 116 104 122 1t8 152 151

11 12 13 14 15 16 17 t8 19 20

126 118 140 115 108 177 140 139 94 121

146 140 131 129 120 121 106 163 122 126

156 142 111 122 99 109 116 120 143 118

138 186 179 142 149 146 141 88 125 153

125 139 123 98 130 144 96 107 106 131

142 153 122 123 123 126 89 132 117 146

111 142 128 135 106 121 88 128 132 158

97 138 122 156 119 140 88 114 109 135

127 129 122 151 142 145 93 142 122 135

117 128 126 154 136 164 104 127 118 156

141 125 113 150 142 169 122 125 121 125

T1" 0 - I 0 TTll-20

136 128

126 130

128 124

140 145

147 130

164 t27

163 125

148 122

148 131

143 133

135 133

before GiN

trig

time in weeks

Glafenine

Placebo

after trial

phenprocoumon dose reduction

E E c,

•

Glafenine

~o

x

Placebo

-

o

Glafenine

-

A

Placebo

g-g

oh c 4: 0 13. ,,~

O O

+

2

group

(TT) [phenprocoumon]4

Patient 0 6 excluded

~8 ~g

I--

r0

O

-9

-6

-3

0

1

2

3

4

5

6

8 Time

in w e e k s

i

t70

/\

150

j~J

130

iX

--2.4C t10 - - 2 . 3 C --

2.2C

90 - - 2 . 1 0

I

)

Fig. 1. Average TT values in the glafenine and placebo groups, nine weeks before, during and for four weeks after the trial. Average phenprocoumon concentrations are shown during the trial period and four weeks afterwards (see text)

J. K. Boeijinga and W. J. F. van der Vijgh: Glafenine and Phenprocoumon

294

Table 4. Measured phenprocoumon concentrations in ~tg/ml plasma before, during and after the trial period Nr.

0

I

2

3

4

8

01 02 03 04 05 06 07 08 09 i0

1.79 1,88 2.86 1.89 1.71 4.14 2.31 2.29 2.72 2.36

1.72 1.79 2.86 1.85 1.81 4.53 2.34 2.16 2.81 2.18

1.67 1.62 2.86 1.82 1.91 4.47 2.34 2.20 2.91 1,99

1.72 1.78 2.77 1.65 1.84 4.45 2.28 2.25 2.74 2.27

1.72 1.73 2.59 1.66 1.87 4.74 2,15 2.40 2.86 2,16

1.50 1.62 2.59 1.63 1.76 4.40 2.04 2.09 2.77 2.27

11 12 13 14 15 16 17 18 t9 20

1.45 3.17 1.09 1.36 1.85 2.78 2.14 1.65 2.90 2.89

1.37 2.95 1.04 1.11 1.80 3.01 2.16 1.72 3.01 2.67

1.48 3.14 0.98 1.24 1.99 2.93 1.75 2.26 2.90 2.38

1.58 2.76 0.85 1.07 2.05 2.98 2,11 2.20 2.63 2.63

1.54 3.21 0.84 1.23 2.06 2.81 2.15 2.42 3.13 2.52

1.50 2.63 0.69 1.06 2.47 2.63 2.15 2.05 3.17 2.52

2.38 2.19 a

2.40 2.38 2.17 ~ 2.15"

2.37 2.14 a

2.39 2.27 2.13 a 2.03 a

2.13

2.08

2.09

2.19

time in weeks

Glafenine

Placebo

conc.

01-10 cone.

11-20

2.10

before trial trial

2.09

after trial

a patient 06 excluded

Table 5. Blood clotting factors in patients on glafenine (% of average normal value), a before trial, b at the time of maximum TT II Nr.

a

01 02 03 04 05 06 07 08 09 10

VII b

X

a

b

a

b

12.5 4.0 7.6 7.6 7.0 1.4 9.5 4.5 16.0 × 22.0 5.3 9.5 7.6 18.0 10.5 10.5 4.0 9,5 6.2

15.0 20.0 12.5 20.0 20.0 26.0 29.0 26.0 26.0 23.0

6.6 16.5 5.0 20.0 × 6.3 15.0 12.5 13.5 21.0

11.0 4.7 6.0 9.6 5.6 12.0 5.4 12.0 10.0 8.4

6.8 3.6 3.0 5.3 X 3.1 5.6 5.6 3.0 4.1

12.4

21,9 6.0

8,6 12.7

Discussion

average % control value 4.5

239 sec was found. However, it was recommenced on the following day in the same dose as before. During the trial no patient suffered from infectious disease and none had increased heart failure as judged by the degree of effort induced dyspnoea, nocturia or peripheral oedema. The results of urinalysis, haematology and blood chemistry screening after the trial did not reveal any noticeable changes from the pre-existing, normal values. According to the manufacturer's specifications, the tablet weight should have been in the range 332.5 to 367.5 rag, and the glafenine content between 190 and 210 rag. These requirements were met by the tables used, as shown in Table 2. The placebo tablets could not be distinguished from the glafenine tablets in appearance, colour or taste. The TT values are shown in Table 3 and Figure 1. Gtafenine, given in the normal therapeutical dose of 200 mg t.i.d., induced a statistically significant lengthening of TT in patients taking phenprocoumon. A two-sided Wilcoxon's two sample test with a = 0.05 as the level of significance was applied to the TT values for the second and third weeks: tail probability P < 0.05 for both weeks. The plasma phenprocoumon concentrations (Table 4 and Fig. 1) were stable and no significant differences between the two groups were seen when values from patient 06, who could be considered as an outlier, were omitted. The elevated values in this patient, who took one tablet (3 mg) of phenprocoumon daily, remain unexplained. The assays of dotting factors II, VII and X are shown in Table 5. In almost all cases values at the time of maximum TF were dearly lower than basal levels.

average % at the time of max. TI?

In the preliminary study (Boeijinga et al., 1974) prolongation of the TT to more than 200 sec was found in five out of seven patients; a similar large increase was found in only one patient in the present study. Yet, as a whole, the TF prolongation proved to be statistically significant in the second and third week, but not in the fourth week. This cannot be readity interpreted. The occurrence of this phenomenon in individual patients was unpredictable. The data obtained point to the need for frequent measurement of TT from the second week of combined use onward, at least at weekly intervals. From comparison of Tables 3 and 4 it is evident that there

J. K. Boeijinga and W. J. F. van der Vijgh: Gtafenine and Phenprocoumon

was no correlation between TT elevation and phenprocoumon concentration. If a competitive effect on protein binding had had a significant role, an increase in the free phenprocoumon concentration in plasma would be expected, resulting in a decrease in the total plasma concentration of phenprocoumon, but this was not found. Comparison of the percentages of factors II, VII and X before the trial and at the time of the maximum individual TT showed marked reduction in all of them in accordance with the TT value. The inhibition of clotting factor synthesis depends directly either on the concentration of free coumarin (Nagaskima et al., 1969; O'Reilly and Aggeler, 1970) or on a coumarin-like effect of any other compound. The action of vitamin K 1 on the biosynthesis of prothrombin has been studied intensively; several hypotheses have been put forward, but recent experimental data strongly suggest that vitamin K t has a promoting or completing effect on clotting factor precursors (Johnson et al., 1972; Malhotra, 1972; Prydz and Gaudernack, 1971; Reekers et al., 1973; Schade and Janowski, 1976; Shah and Suttie, 1971), related to the Protein Induced by Vitamin K Absence or Antagonists (PIVKA) (Hemker et al., 1968; 1970). The reduced concentrations of factors II, VII and X, together with the unchanged phenprocoumon concentration may suggest that glafenine could have had a coumarin-like inhibitory effect on the completing activity of vitamin K 1 or on precursors related to the enzyme systems involved.

Acknowledgements. The authors are indebted to the staff of the Amsterdam Thrombosis Service (Head: Dr.T.B. Gan) for their outstanding cooperation. We wish to thank Prof. C. L. Riimke and Ir. P. D. Bezemer (Department of Medical Statistics, Vrije Universiteit, Amsterdam) for statistical advice, Prof. E. A. Loeliger (Department of Haematology, Rijksuniversiteit Leiden) for the clotting factor analysis, and Dr. J. D. Wiener for his critical comments. References 1. Aggeler, P.M., O'Reilly, R.A., Leong, I.: Potentiation of anticoagulant effect of warfarin by phenylbutazone. New Engl. J. Med. 276, 496-501 (1967) 2. Antlitz, A.M., Awalt, L.F.: A double blind study of acetaminophen used in conjunction with oral anticoagulant therapy. Curr. Ther. Res. 11, 360-361 (1969) 3. Biggs, R.: Human blood coagulation, haemostasis and thrombosis Chapter III. Oxford: Blackwell 1972

295

4. Boeijinga, J.K., Gan, Tjong Bing, Van der Meer, J.: De invloed van glafenine (Glifanan®) op antistollingsbehandeling met coumarinederivaten. Ned. Tijdschr. Geneesk. 118, 1895-1898 (1974) 5. Breed, W.P.M., Van Hooff, J.P., Haanen, C.: A comparative study concerning the stability of the anticoagulant effect of acenocoumarol and phenproeoumon. Acta med. scand. 186, 283-288 (1969) 6. Coon, W.W., Willis, P.W. III: Some aspects of the pharmacology of oral anticoagulants. Clin. Pharmacol. Ther. 2, 312-336 (1970) 7. Hemker, H.C., Veltkamp, J.J., Loeliger, E.A.: Kinetic aspects of the interaction of bloodctotting enzymes. III. Demonstration of the existence of an inhibitor of prothrombin conversion in vitamin K deficiency. Thromb. Diath. Haemorrh. 19, 346-350 (1968) 8. Hemker, H.C., Miiller, A.D.: Kinetic aspects of the interaction of bloodclotting enzymes VI. Localization of the site of blood coagulation inhibitor by the protein induced by vitamin K absence (PIVKA). Thromb. Diath. Haemorrh. 20, 78-83 (1968) 9. Hemker, H.C., Miiller A.D., Loeliger, E.A.: Two types of prothrombin in vitamin K deficiency. Throm. Diath. Haemorrh. 23, 633-637 (1970) 10. Hobbs, C.B., Miller, A.L., Thornley, J.H.: Potentiation of anticoagulant therapy by oxyphenylbutazone: (a probable case). Postgrad. med. J. 41, 563-565 (1965) 11. Johnson, H.V., Boyd, C., Matinovic, J., Valkowich, G., Connor Johnson, B.: A new blood protein which increases with vitamin K deficiency. Biochim. Biophys. Acta 148, 431--442 (I972) 12. Koch-Weser, J., Sellers, E. M.: Drug interactions with coumafin anticoagulants. New Engl. J. Med. 285, (I) 487-498, (II) 547-558 (1971) 13. Loeliger, E.A.: De invloed van pharmaca op de behandeling met orale antistollingsmiddelen. Ned. Tijdschr. Geneesk. 117, 1396-1399 (1973) 14. Malhotra, O.P.: Atypical prothrombins induced by dicoumarol. Nature [New Biol.] 239, 59-60 (1972) 15. Mustard, J.F., Packham, M.A.: Factors influencing platelet function: adhesion, release and aggregation. Pharmacol. Rev. 22, 97-187 (1970) 16. Nagashina, R., O'Reilly, R.A., Levy, G.: Kinetics of pharmacologic effects in man: the anticoagulant action of warfarin. Clin. Pharmacol. Ther. 10, 22-35 (1969) 17. O'Reilly, R.A., Aggeler, P.M.: Determinants of the response to oral anticoagulant drugs in man. Pharmacol. Rev. 22, 35-96 (1970) 18. O'Reilly, R.A., Levy, G.: Kinetics of the anticoagulant effect of bishydroxycoumarin in man. Clin. PhannacoI. Ther. 11, 378-384 (1970) 19. Owren, P.A.: Critical studies of tests for control of anticoagulant therapy. Thromb. Diath. Haemorrh. Suppl. 1, 294306(1962) 20. Owren, P.A.: Control of anticoagulant therapy. The use ot new tests. Arch. intern. Med. 3, 248-258 (1963) 21. Prydz, H., Gaudernack, G.: Studies on the biosynthesis of factor VII (Proconvertin). The mode of action of warfarin. Biochim. Biophys. Acta 230, 373-380 (1971) 22. Quick, A.J., Clesceri, L.: Influence of acetylsalicylic acid and salicylamide on the coagulation of blood. J. Pharmacol. exp. Ther. 128, 95-98 (1960) 23. Reekers, P.M.M., Lindhout, M.J., Kop-Klaassen, B H. M., Hemker, H.C.: Demonstration of three anomalous plasma proteins induced by a vitamin K antagonist. Biochim. Biophys. Acta 317, 559-562 (1973)

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J.K. Boeijinga and W. J. F. van der Vijgh: Glafenine and Phenprocoumon

24. Schade, R., Janowski, K.: Der Wirkungsmechanismus von Vitamin K und seine Beeinflussung durch indirekte Antikoagulantien. Pharmazie 31, 585-589 (1976) 25. Seiler, K., Duckert, F.: Properties of 3-(1-phenyl-propyl)-4 oxycoumarin (Marcoumar ®) in the plasma when tested in normal cases and under influence of drugs. Thromb. Diath. Haemorrh. 19, 389-396 (1968) 26. Shah, D.V., Suttie, J.W.: Mechanism of action of vitamin K: evidence for the conversion of a precursor protein to prothrombin in the rat. Proc. Nat. Acad. Sci. USA 68, 1653-1657 (1971) 27. Sigg, A., Pestalozzi, H., Clauss, A., Koller, F.: Verst~irkung

der Antikoagulantienwirkung durch Butazolidin. Schweiz. Med. Wschr. 86, 1194-1195 (1956) 28. Weenink, A.: Profylaxe en therapie met anticoagulantia. Toepassing in kliniek en praktijk, alsmede controle. Propharma N.V. - Haarlem, Netherlands. P. 5-207 (1971) Received: January 17, 1977, in revised form: June 6, 1977, accepted: June 8, 1977 Dr. J. K. Boeijinga Internist Hermelijnedreef 33 Oss/Nederland