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FIBRINOLYTIC EFFECTS OF DIGUANIDES PLUS ETHYLŒSTRENOL IN OCCLUSIVE VASCULAR DISEASE
1008
FIBRINOLYTIC EFFECTS OF DIGUANIDES PLUS ETHYLŒSTRENOL IN OCCLUSIVE VASCULAR DISEASE
G. R. FEARNLEY
R. CHAKRABARTI
M.D. Lond., F.R.C.P.
M.B. Calcutta
PHYSICIAN
ELIZABETH D. HOCKING M.B. Cantab., M.R.C.P.E. *
With the technical assistance
of
J. F. EVANS GLOUCESTERSHIRE ROYAL
GLOUCESTER
investigation lasting 33 months, the fibrinolytic effects of clofibrate plus androsterone (’ Atromid ’), phenformin, metformin, and combinations of each diguanide with the anabolic steroid, ethylœstrenol, were compared in two groups of patients with occlusive vascular disease. Atromid was relatively unsatisfactory as a fibrinolytic drug; phenformin and metformin increased fibrinolytic activity, but partial resistance developed to the fibrinolytic effect of both. The addition of ethylœstrenol overcame this resistance; metformin plus ethylœstrenol and phenformin plus ethylœstrenol given twice a day for 12 months produced a sustained increase of fibrinolytic activity in 80% and 89% of the patients treated. When the diguanides were withdrawn, ethylœstrenol alone failed to maintain the full fibrinolytic effect of combined treatment, but this was restored when metformin and phenformin were restituted. Both combinations of drugs reduced plasma-fibrinogen; serum-cholesterol was reduced by phenformin but not by metformin. The findings suggest that phenformin plus ethylœstrenol is suitable for trial as a prophylactic measure in patients with occlusive vascular disease. Summary
In
HOSPITAL,
an
Introduction fibrinolysis has been proposed as a prophylactic measure in patients with occlusive vascular disease (Fearnley 1965). The sulphonylureas (Fearnley,
PHARMACOLOGICAL
Chakrabarti, and Vincent 1960),
testosterone
(Fearnley
Chakrabarti 1962), corticosteroids (Chakrabarti, Fearnley and Hocking 1964), phenformin, oral anabolic steroids (Fearnley and Chakrabarti 1964), and metformin (Chakrabarti, Hocking and Fearnley 1965) increase the spontaneous fibrinolytic activity of blood. Resistance to the fibrinolytic effect of sulphonylureas and of oral anabolic steroids developed in many patients after a few weeks’ treatment, but phenformin produced a sustained increase of fibrinolysis when evaluated for 3 months (Fearnley and Chakrabarti 1964). In 1964 we began an investigation, which has lasted 33 months, with the initial object of comparing the effects of clofibrate plus androsterone (’ Atromid’), phenformin, and metformin on fibrinolysis, plasmafibrinogen, and serum-cholesterol in outpatients with arterial diseases. The results of the first year of this study have been described by Hocking, Chakrabarti, Evans, and Fearnley (1967): clofibrate plus androsterone was the least effective fibrinolytic drug; metformin and phenformin increased fibrinolytic activity, but after 3-4 months’ treatment partial resistance developed to the fibrinolytic effect of both drugs. This was disappointing, and it occurred to us that two fibrinolytic drugs might be more effective than one. Accordingly during the
and
*Present
appointment: medical assistant,
Manor Park
Patients and Methods
Design
SENIOR RESEARCH REGISTRAR
RESEARCH REGISTRAR
21 months of the trial we have evaluated the effect of the oral anabolic steroid ethyloestrenol in patients receiving phenformin or metformin.
remaining
Hospital, Bristol.
The design and conduct of the trial have been described elsewhere (Hocking, Chakrabarti, Evans, Fearnley 1967). In brief, three control readings of the dilute blood-clot-lysis time (B.L.T.), the euglobulin-lysis time (E.L.T.), plasma-fibrinogen, and serum-cholesterol were made on outpatients with occlusive vascular disease, after which the patients were divided into two groups. Group i (eighteen patients) received atromid 20-30 mg. per kg. body-weight daily in three divided doses for 4 months, after which placebo capsules were substituted for 2 months. They were then given metformin 1 g. twice daily for 1 month, and 0-5 g. twice daily for a further 5 months, (At this point two patients were unable to continue in the trial leaving sixteen, one of whom died suddenly a month later, The remaining fifteen patients continued the trial.) At month 12 ethylcestrenol was added, the patients receiving metformin 0-5 g. twice daily together with ethyloestrenol 4 mg. twice daily for the next 12 months. Metformin was then omitted, the patients continuing to take ethyloestrenol alone for 3 months. At the end of this time metformin 0-5 g. twice daily was restituted, the patients receiving combined therapy for a further 6 months. Group II (eighteen patients) received metformin 0-5 g. thrice daily for 4 months, placebo tablets for 2 months, phenformin capsules 50 mg. twice daily for 6 months, phenformin 50 mg. twice daily together with ethyloestrenol 4 mg. twice daily for 12 months, ethyloestrenol alone for 3 months, and phenformin 50 mg. twice daily together with ethyloestrenot 4 mg. twice daily for the final 6 months. Methods measured in duplicate by the method of Fearnley (1957) with reading of the end-point as modified by Fearnley (1964). Normal lysis-times by this method range from 11/2 to 7 hours. E.L.T. was measured in duplicate according to the method of von Kaulla (1963), modified as follows: a low-temperature technique was used from the time when the blood was obtained B.L.T. was
et
al.
until the clots had been formed. The euglobulin precipitate from 1 ml. plasma was suspended in 2 ml. saline solution and phosphate buffer, from which 1 ml. duplicates were clotted with 5 units of topical thrombin (Parke Davis). The buffer consisted of phosphate buffer (Fearnley et al. 1957) one part and physiological saline solution three parts. Normal lysistimes by this method range from 3/4 to 2 hours. Plasma-fibrinogen was estimated gravimetrically by the method of Fearnley and Chakrabarti (1966). Serum cholesterol was measured in an ’AutoAnalyser’ by the method of Zlatkis et al. (1953).
Results
Group
I
Fig. 1 shows the mean B.L.T., E.L.T., serum-cholesterol, plasma-fibrinogen of the fifteen patients. During treatment with atromid the B.L.T. was prolonged, but the E.L.T. was moderately reduced and lengthened during months 5 and 6 when no treatment was given. Metformin, 2 g. daily, gave a concordant reduction of the B.L.T. and E.L.T., but when after 1 month, the dosage was changed to 1 g. daily for 5 months both means returned towards their control levels. The addition of ethylcestrenol 8 mg. daily at month 12 resulted in a pronounced reduction of both lysis-times which fell in parallel and remained reduced throughout the 12 months during which metformin plus ethyloestrenol were given. When metformin was omitted and the patients continued on ethyloestrenol alone (months 24-27) there was a biphasic prolongation of both the B.L.T. and E.L.T., which were again reduced and
1009 TABLE I-MEAN AND
*
Female; all the remaining patients
were
male... =Unable
to
attend for
%
FALL IN E.L.T.
tests.
(HR.)
MI = Myocardial infarction.
AP =Angina pectoris.
IC=Intermittent
claudication.
when metformin was restituted for the last 6 months of the trial. The mean plasma-fibrinogen decreased at the 4th month of treatment with atromid, fluctuated during treatment with metformin, and was reduced by about 12% compared with the control levels during the 12 months’ treatment with metformin plus ethylcestrenol. When metformin was withdrawn for 3 months, the fibrinogen level rose sharply and decreased again when metformin was restituted. Serum-cholesterol was reduced by atromid, fell transiently during treatment with metformin, and
became elevated by about 15% compared with the control levels during the 12 months when metformin plus ethylcestrenol were given. It fell slightly during the 3 months when metformin was omitted, and rose again when treatment with metformin plus ethyloestrenol was resumed. Table i gives for each patient in group I the mean E.L.T. during the control period, the means of the last three E.L.T.s during the various treatments, and the differences between these means and those of the control period expressed as percentages of the control. (Except during treatment with atromid when they were discordant the B.L.T. and E.L.T. were closely correlated for each patient, and since the E.L.T. is in more general use and provides the more direct measure of plasminogen activator, only the data obtained with the E.L.T. are given.) A 25% or greater reduction of the E.L.T. taken as signifying an important increase of fibrinolytic activity was obtained with atromid in five, with metformin in six, with metformin plus ethylœstrenol in twelve, with ethyloestrenol alone in nine, and with metformin plus ethyloestrenol again in twelve of the fifteen patients. The results in individual patients taken in conjunction with the mean lysis-times shown in fig. 1 establish that metformin plus ethylcestrenol was superior to atromid and to metformin given alone, both in respect of duration of response and of the number of patients (80%) who responded. It is also clear that the fibrinolytic response of most patients to metformin plus ethylcestrenol was diminished during treatment with ethyloestrenol alone but was restored when both drugs were given again.
Group II
Fig. 2 shows the mean results obtained in the eighteen patients in group II during the control period and throughout the trial. Treatment with metformin 1-5 g. daily concordant reduction of the mean B.L.T. and E.L.T., but at the 4th month of treatment B.L.T. was prolonged, which suggests that resistance to the fibrinolytic effect of metformin was beginning to develop. Phenformin 100 mg. daily gave reductions of the B.L.T. and E.L.T. lasting 2 and 3 months, respectively, which thereafter rose to levels close to a 25% reduction compared with the control readings. The addition of ethylcestrenol, 8 mg. daily, at month 12 resulted in a pronounced reduction of both the B.L.T. and E.L.T. which fell in parallel and remained reduced throughout the 12 months during which phenformin plus ethylœstrenol were given. When phenformin was omitted, the patients to take ethylcestrenol alone (months 24-27), continuing there was a rebound prolongation of both the B.L.T. and gave
,
Fig. 1—Mean E.L.T., B.L.T., plasma-fibrinogen, and serumcholesterol of fifteen patients (group I) throughout the trial.
a
1010 was reduced slightly with strikingly (by about 15%) with phenformin and with phenformin plus ethyloestrenol. It rose somewhat while phenformin was withdrawn and fell again when phenformin was resumed. Table 11 gives for each patient in group n the mean E.L.T. during the control period and the means of the last three E.L.T.s during treatment with metformin, phenformin, phenformin plus ethyloestrenol, ethylcestrenol alone, and phenformin plus ethyloestrenol again, together with the percentage reduction of lysis-time obtained with these drugs. Again taking a 25% or greater reduction of lysis-time as important, it can be seen that this was obtained with metformin in fourteen, with phenformin in eleven, with phenformin plus ethylcestrenol in sixteen, with ethyloestrenol alone in eleven, and with phenformin plus ethyloestrenol again in seventeen of the eighteen patients. (Had metformin been given for longer than 4 months there is little doubt that developing resistance, shown by prolongation of the B.L.T. [Fig. 2], would have reduced the number of those who gave a sustained fibrinolytic response to this drug, as happened in group I.) These results, taken in conjunction with the mean lysis-times shown in fig. 2, establish that phenformin plus ethyloestrenol was superior to metformin and phenformin alone, both in respect of duration of response and of the number of patients (89%) who responded. The fibrinolytic response of most patients to phenformin plus ethyloestrenol was diminished during treatment with ethyloestrenol alone, but was restored when both drugs were given again. Tables i and 11 show not only that more patients responded but also that the magnitude of response was greater with combined therapy than with any of the drugs given singly. In group i, a 50% or greater reduction of the E.L.T. was obtained by no patient on atromid, by three on metformin, by seven on metformin plus ethyloestrenol) by two on ethyloestrenol alone and by six on metformin plus ethyloestrenol given again. Similarly, in group n, two patients gave a 50% response or greater to metformin, one to phenformin, eight to phenformin plus ethyloestrenol, none to ethyloestrenol alone, and nine to phenformin plus ethyloestrenol given again. Half the patients who responded to metformin with ethyloestrenol and to phenformin with ethyloestrenol, therefore, had a 50% or greater increase of fibrinolytic activity. It is noteworthy that fibrinolytic responses to combined
The
mean
serum-cholesterol
metformin, and
Fig. 2-Mean cholesterol of
E.L.T., B.L.T., plasma-fibrinogen, and serum eighteen patients (group II) throughout the trial.
which were again reduced when phenformin was restituted for the last 6 months of the trial. The mean plasma-fibrinogen did not change during treatment with metformin, was reduced during the first 2 months’ treatment with phenformin, and fluctuated during the next 4 months as partial resistance to the fibrinolytic effect of this drug developed. During the 12 months’ treatment with phenformin plus ethyloestrenol, the plasma-fibrinogen dropped to a level commensurate with a 25% reduction compared with the control readings. When phenformin was withdrawn the plasma-fibrinogen level increased in parallel with the B.L.T. and E.L.T., and decreased again when phenformin was restituted. E.L.T.
TABLE II-MEAN AND
the remaining claudication.
Female; all
patients
were
mate...=Unabte
to
%
attend for
FALL IN E.L.T.
tests.
more
(HR.)
MI=Myocardial infarction AP= Angina pectoris. tC= Intermittent
1011
therapy were not confined in either group to those whose fibrinolytic activity was low (E.L.T. more than 2 hours)
diguanides in this respect. Fibrinolysis was enhanced not only in patients whose fibrinolytic activity before treat-
before treatment, but were also obtained in most of the patients whose activity was initially within the normal range. In group l, patients 1-7 had low fibrinolytic activity, and of these six had a fibrinolytic response; patients 8-15 had normal activity, and six of these responded. In group II, patients 1-10 had low activity and all responded; patients 11-18 had normal activity, and six of these responded.
ment was
in the low range, but also in those in whom it within normal limits. Reduction of the E.L.T. indicates that the drugs raise the level of plasminogen activator; whether they do this by increasing the rate of formation of activator or by decreasing its rate of removal by an organ such as the liver is unknown. Although both combinations of drugs had a satisfactory fibrinolytic effect, only phenformin reduced serumcholesterol and this drug gave a somewhat greater reducSide-effects Besides the patients reported here we have given tion of plasma-fibrinogen than did metformin. Since phenformin plus ethyloestrenol to another one hundred phenformin plus ethyloestrenol also reduces platelet and eighteen patients with occlusive vascular disease, and stickiness (Chakrabarti and Fearnley 1967)-and we do metformin plus ethyloestrenol to another thirty patients. not yet know whether metformin plus ethylcestrenol will do so--on the present evidence phenformin would seem No serious toxicity has been encountered with either to have advantages over metformin. The diguanides cause gastrointestinal combination. The favourable influence of phenformin plus ethylaesymptoms in about 15% of patients; anorexia, nausea, strenol on four factors believed to be of importance in and vomiting with phenformin and diarrhoea with metforrriin. The symptoms are mild in more than half occlusive vascular disease-i.e., fibrinolytic activity, the patients who experience them and subside after a few plasma-fibrinogen, serum-cholesterol, and platelet stickiness-makes this combination of drugs suitable for trial weeks’ treatment; in about 5% of patients they necessitate treatment. Patients unable to tolerate stopping phen- in the prophylaxis of venous and arterial occlusions. The formin capsules, however, may be able to take metformin diguanide may also be expected to correct the impaired cartablets without ill-effect, and vice versa. bohydrate tolerance found by Wahlberg (1962) and others Both phenformin and metformin cause loss of weight to be a feature of many patients with ischaemic disease. While no firm conclusions can be drawn from an in about half the patients treated. This is usually of the uncontrolled trial, the moderate death-rate and low rate order of 6-10 lb. (3-5 kg.), and after this has been lost, of reinfarction the weight generally stabilises at the new level. Greater among our patients encourage the hope that a combination of fibrinolytic drugs may have prophyand continuing weight loss is uncommon, but may very lactic value in occlusive vascular disease. Twice-daily rarely necessitate stopping treatment. Ethyloestrenol is well tolerated by most patients, though dosage helps to ensure that the drugs are taken regularly, occasionally it causes headache. It may raise the blood- an important consideration in the long-term treatment of those patients who are untroubled by symptoms. It pressure in hypertensive patients, and can exacerbate must be emphasised that while the effect of phenformin or in who have had are on the symptoms patients recently left of ventricular while these can plus ethyloestrenol on fibrinolysis is usually apparent failure; verge having or controlled with diuretic often be prevented tablets within a month of starting treatment, in some patients such patients must be watched carefully and told to the maximal effect is not obtained until the drugs have been given for 2 months. Furthermore, reduction of report any undue dyspnoea. platelet stickiness was not observed until the 3rd month of Outcome in nearly half the patients who responded in During the 21 months of combined therapy, including treatment this respect (Chakrabarti and Fearnley 1967). It the months when ethyloestrenol was given alone, there follows that should phenformin plus ethyloestrenol were two deaths and one reinfarction, all of which occurred exercise a protective influence in occlusive vascular conin group i. The first death was that of a man, not included in the data of table i who died suddenly after ditions, their full effects will not become available in many patients until treatment has been given for up to 3 months. the first month of treatment with metformin plus This work was supported by grants from the South Western ethyloestrenol. Necropsy was not done. The second Regional Hospital Board, the British Heart Foundation, the Nuffield death was that of patient no. 15 in tablet who died Foundation, Bayer Products Ltd., and Organon Laboratories, Ltd. suddenly during month 33 at the end of the trial. No We thank the two pharmaceutical companies for supplies of phenformin and ethyloestrenol, and Rona Laboratories for supplies of necropsy was done. The sole reinfarction was in patient metformin; Dr. C. C. Downie, of Imperial Chemical Industries for no. 8 (table l) during month 29. He made a good recovery supplies of atromid and for undertaking the determination of serumand continued in the trial. cholesterol during this investigation; and patients for continuing The total number of man-months of combined therapy cooperation throughout a long trial. in groups i and n was 694, giving a mortality-rate of Requests for reprints should be addressed to G. R. F. REFERENCES 0-3 per 100 man-months. Twenty-two of the patients had Chakrabarti, R., Fearnley, G. R. (1967) Lancet, ii, 1012. survived one or more myocardial infarctions and the total Hocking, E. D. (1964) Br. med. J. i, 534. number of man-months of combined therapy for these Hocking, E. D., Fearnley, G. R. (1965) Lancet, ii, 256. G. R. (1964) J. clin. Path. 17, 307. patients was 462, giving a reinfarction-rate of 0-2 per Fearnley, Balmforth, G., Fearnley, E. B. (1957) Clin. Sci. 16, 645. 100 man-months. (1965) Fibrinolysis, p. 82, London. was
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—
Discussion
The problem of resistance which
1
develops sooner or given by mouth has been overcome by giving two such drugs together. Both phenformin and metformin combined with ethyloestrenol gave a substantial and sustained increase of fibrinolytic activity, there being little to choose between the two
later when fibrinolytic drugs
B
I I
are
Chakrabarti, R., Vincent, C. T. (1960) Lancet, ii, 622. (1962) ibid. ii, 128. (1964) J. clin. Path. 17, 328. (1966) Lancet, ii, 757. Hocking, E. D., Chakrabarti, R., Evans, J., Fearnley, G. R. (1967) J. Atheroscler. Res. 7, 121. von Kaulla, K. N. (1963) Chemistry of Thrombolysis: Human Fibrinolytic Enzymes; p. 79. Illinois. Wahlberg, F. (1962) Acta. med. scand. 171, 1. Zlatkis, A., Zak, B., Boyle, G. J. (1953) J. Lab. clin. Med 41, 486. —
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FIBRINOLYTIC EFFECTS OF DIGUANIDES PLUS ETHYLŒSTRENOL IN OCCLUSIVE VASCULAR DISEASE
G. R. FEARNLEY
R. CHAKRABARTI
M.D. Lond., F.R.C.P.
M.B. Calcutta
PHYSICIAN
ELIZABETH D. HOCKING M.B. Cantab., M.R.C.P.E. *
With the technical assistance
of
J. F. EVANS GLOUCESTERSHIRE ROYAL
GLOUCESTER
investigation lasting 33 months, the fibrinolytic effects of clofibrate plus androsterone (’ Atromid ’), phenformin, metformin, and combinations of each diguanide with the anabolic steroid, ethylœstrenol, were compared in two groups of patients with occlusive vascular disease. Atromid was relatively unsatisfactory as a fibrinolytic drug; phenformin and metformin increased fibrinolytic activity, but partial resistance developed to the fibrinolytic effect of both. The addition of ethylœstrenol overcame this resistance; metformin plus ethylœstrenol and phenformin plus ethylœstrenol given twice a day for 12 months produced a sustained increase of fibrinolytic activity in 80% and 89% of the patients treated. When the diguanides were withdrawn, ethylœstrenol alone failed to maintain the full fibrinolytic effect of combined treatment, but this was restored when metformin and phenformin were restituted. Both combinations of drugs reduced plasma-fibrinogen; serum-cholesterol was reduced by phenformin but not by metformin. The findings suggest that phenformin plus ethylœstrenol is suitable for trial as a prophylactic measure in patients with occlusive vascular disease. Summary
In
HOSPITAL,
an
Introduction fibrinolysis has been proposed as a prophylactic measure in patients with occlusive vascular disease (Fearnley 1965). The sulphonylureas (Fearnley,
PHARMACOLOGICAL
Chakrabarti, and Vincent 1960),
testosterone
(Fearnley
Chakrabarti 1962), corticosteroids (Chakrabarti, Fearnley and Hocking 1964), phenformin, oral anabolic steroids (Fearnley and Chakrabarti 1964), and metformin (Chakrabarti, Hocking and Fearnley 1965) increase the spontaneous fibrinolytic activity of blood. Resistance to the fibrinolytic effect of sulphonylureas and of oral anabolic steroids developed in many patients after a few weeks’ treatment, but phenformin produced a sustained increase of fibrinolysis when evaluated for 3 months (Fearnley and Chakrabarti 1964). In 1964 we began an investigation, which has lasted 33 months, with the initial object of comparing the effects of clofibrate plus androsterone (’ Atromid’), phenformin, and metformin on fibrinolysis, plasmafibrinogen, and serum-cholesterol in outpatients with arterial diseases. The results of the first year of this study have been described by Hocking, Chakrabarti, Evans, and Fearnley (1967): clofibrate plus androsterone was the least effective fibrinolytic drug; metformin and phenformin increased fibrinolytic activity, but after 3-4 months’ treatment partial resistance developed to the fibrinolytic effect of both drugs. This was disappointing, and it occurred to us that two fibrinolytic drugs might be more effective than one. Accordingly during the
and
*Present
appointment: medical assistant,
Manor Park
Patients and Methods
Design
SENIOR RESEARCH REGISTRAR
RESEARCH REGISTRAR
21 months of the trial we have evaluated the effect of the oral anabolic steroid ethyloestrenol in patients receiving phenformin or metformin.
remaining
Hospital, Bristol.
The design and conduct of the trial have been described elsewhere (Hocking, Chakrabarti, Evans, Fearnley 1967). In brief, three control readings of the dilute blood-clot-lysis time (B.L.T.), the euglobulin-lysis time (E.L.T.), plasma-fibrinogen, and serum-cholesterol were made on outpatients with occlusive vascular disease, after which the patients were divided into two groups. Group i (eighteen patients) received atromid 20-30 mg. per kg. body-weight daily in three divided doses for 4 months, after which placebo capsules were substituted for 2 months. They were then given metformin 1 g. twice daily for 1 month, and 0-5 g. twice daily for a further 5 months, (At this point two patients were unable to continue in the trial leaving sixteen, one of whom died suddenly a month later, The remaining fifteen patients continued the trial.) At month 12 ethylcestrenol was added, the patients receiving metformin 0-5 g. twice daily together with ethyloestrenol 4 mg. twice daily for the next 12 months. Metformin was then omitted, the patients continuing to take ethyloestrenol alone for 3 months. At the end of this time metformin 0-5 g. twice daily was restituted, the patients receiving combined therapy for a further 6 months. Group II (eighteen patients) received metformin 0-5 g. thrice daily for 4 months, placebo tablets for 2 months, phenformin capsules 50 mg. twice daily for 6 months, phenformin 50 mg. twice daily together with ethyloestrenol 4 mg. twice daily for 12 months, ethyloestrenol alone for 3 months, and phenformin 50 mg. twice daily together with ethyloestrenot 4 mg. twice daily for the final 6 months. Methods measured in duplicate by the method of Fearnley (1957) with reading of the end-point as modified by Fearnley (1964). Normal lysis-times by this method range from 11/2 to 7 hours. E.L.T. was measured in duplicate according to the method of von Kaulla (1963), modified as follows: a low-temperature technique was used from the time when the blood was obtained B.L.T. was
et
al.
until the clots had been formed. The euglobulin precipitate from 1 ml. plasma was suspended in 2 ml. saline solution and phosphate buffer, from which 1 ml. duplicates were clotted with 5 units of topical thrombin (Parke Davis). The buffer consisted of phosphate buffer (Fearnley et al. 1957) one part and physiological saline solution three parts. Normal lysistimes by this method range from 3/4 to 2 hours. Plasma-fibrinogen was estimated gravimetrically by the method of Fearnley and Chakrabarti (1966). Serum cholesterol was measured in an ’AutoAnalyser’ by the method of Zlatkis et al. (1953).
Results
Group
I
Fig. 1 shows the mean B.L.T., E.L.T., serum-cholesterol, plasma-fibrinogen of the fifteen patients. During treatment with atromid the B.L.T. was prolonged, but the E.L.T. was moderately reduced and lengthened during months 5 and 6 when no treatment was given. Metformin, 2 g. daily, gave a concordant reduction of the B.L.T. and E.L.T., but when after 1 month, the dosage was changed to 1 g. daily for 5 months both means returned towards their control levels. The addition of ethylcestrenol 8 mg. daily at month 12 resulted in a pronounced reduction of both lysis-times which fell in parallel and remained reduced throughout the 12 months during which metformin plus ethyloestrenol were given. When metformin was omitted and the patients continued on ethyloestrenol alone (months 24-27) there was a biphasic prolongation of both the B.L.T. and E.L.T., which were again reduced and
1009 TABLE I-MEAN AND
*
Female; all the remaining patients
were
male... =Unable
to
attend for
%
FALL IN E.L.T.
tests.
(HR.)
MI = Myocardial infarction.
AP =Angina pectoris.
IC=Intermittent
claudication.
when metformin was restituted for the last 6 months of the trial. The mean plasma-fibrinogen decreased at the 4th month of treatment with atromid, fluctuated during treatment with metformin, and was reduced by about 12% compared with the control levels during the 12 months’ treatment with metformin plus ethylcestrenol. When metformin was withdrawn for 3 months, the fibrinogen level rose sharply and decreased again when metformin was restituted. Serum-cholesterol was reduced by atromid, fell transiently during treatment with metformin, and
became elevated by about 15% compared with the control levels during the 12 months when metformin plus ethylcestrenol were given. It fell slightly during the 3 months when metformin was omitted, and rose again when treatment with metformin plus ethyloestrenol was resumed. Table i gives for each patient in group I the mean E.L.T. during the control period, the means of the last three E.L.T.s during the various treatments, and the differences between these means and those of the control period expressed as percentages of the control. (Except during treatment with atromid when they were discordant the B.L.T. and E.L.T. were closely correlated for each patient, and since the E.L.T. is in more general use and provides the more direct measure of plasminogen activator, only the data obtained with the E.L.T. are given.) A 25% or greater reduction of the E.L.T. taken as signifying an important increase of fibrinolytic activity was obtained with atromid in five, with metformin in six, with metformin plus ethylœstrenol in twelve, with ethyloestrenol alone in nine, and with metformin plus ethyloestrenol again in twelve of the fifteen patients. The results in individual patients taken in conjunction with the mean lysis-times shown in fig. 1 establish that metformin plus ethylcestrenol was superior to atromid and to metformin given alone, both in respect of duration of response and of the number of patients (80%) who responded. It is also clear that the fibrinolytic response of most patients to metformin plus ethylcestrenol was diminished during treatment with ethyloestrenol alone but was restored when both drugs were given again.
Group II
Fig. 2 shows the mean results obtained in the eighteen patients in group II during the control period and throughout the trial. Treatment with metformin 1-5 g. daily concordant reduction of the mean B.L.T. and E.L.T., but at the 4th month of treatment B.L.T. was prolonged, which suggests that resistance to the fibrinolytic effect of metformin was beginning to develop. Phenformin 100 mg. daily gave reductions of the B.L.T. and E.L.T. lasting 2 and 3 months, respectively, which thereafter rose to levels close to a 25% reduction compared with the control readings. The addition of ethylcestrenol, 8 mg. daily, at month 12 resulted in a pronounced reduction of both the B.L.T. and E.L.T. which fell in parallel and remained reduced throughout the 12 months during which phenformin plus ethylœstrenol were given. When phenformin was omitted, the patients to take ethylcestrenol alone (months 24-27), continuing there was a rebound prolongation of both the B.L.T. and gave
,
Fig. 1—Mean E.L.T., B.L.T., plasma-fibrinogen, and serumcholesterol of fifteen patients (group I) throughout the trial.
a
1010 was reduced slightly with strikingly (by about 15%) with phenformin and with phenformin plus ethyloestrenol. It rose somewhat while phenformin was withdrawn and fell again when phenformin was resumed. Table 11 gives for each patient in group n the mean E.L.T. during the control period and the means of the last three E.L.T.s during treatment with metformin, phenformin, phenformin plus ethyloestrenol, ethylcestrenol alone, and phenformin plus ethyloestrenol again, together with the percentage reduction of lysis-time obtained with these drugs. Again taking a 25% or greater reduction of lysis-time as important, it can be seen that this was obtained with metformin in fourteen, with phenformin in eleven, with phenformin plus ethylcestrenol in sixteen, with ethyloestrenol alone in eleven, and with phenformin plus ethyloestrenol again in seventeen of the eighteen patients. (Had metformin been given for longer than 4 months there is little doubt that developing resistance, shown by prolongation of the B.L.T. [Fig. 2], would have reduced the number of those who gave a sustained fibrinolytic response to this drug, as happened in group I.) These results, taken in conjunction with the mean lysis-times shown in fig. 2, establish that phenformin plus ethyloestrenol was superior to metformin and phenformin alone, both in respect of duration of response and of the number of patients (89%) who responded. The fibrinolytic response of most patients to phenformin plus ethyloestrenol was diminished during treatment with ethyloestrenol alone, but was restored when both drugs were given again. Tables i and 11 show not only that more patients responded but also that the magnitude of response was greater with combined therapy than with any of the drugs given singly. In group i, a 50% or greater reduction of the E.L.T. was obtained by no patient on atromid, by three on metformin, by seven on metformin plus ethyloestrenol) by two on ethyloestrenol alone and by six on metformin plus ethyloestrenol given again. Similarly, in group n, two patients gave a 50% response or greater to metformin, one to phenformin, eight to phenformin plus ethyloestrenol, none to ethyloestrenol alone, and nine to phenformin plus ethyloestrenol given again. Half the patients who responded to metformin with ethyloestrenol and to phenformin with ethyloestrenol, therefore, had a 50% or greater increase of fibrinolytic activity. It is noteworthy that fibrinolytic responses to combined
The
mean
serum-cholesterol
metformin, and
Fig. 2-Mean cholesterol of
E.L.T., B.L.T., plasma-fibrinogen, and serum eighteen patients (group II) throughout the trial.
which were again reduced when phenformin was restituted for the last 6 months of the trial. The mean plasma-fibrinogen did not change during treatment with metformin, was reduced during the first 2 months’ treatment with phenformin, and fluctuated during the next 4 months as partial resistance to the fibrinolytic effect of this drug developed. During the 12 months’ treatment with phenformin plus ethyloestrenol, the plasma-fibrinogen dropped to a level commensurate with a 25% reduction compared with the control readings. When phenformin was withdrawn the plasma-fibrinogen level increased in parallel with the B.L.T. and E.L.T., and decreased again when phenformin was restituted. E.L.T.
TABLE II-MEAN AND
the remaining claudication.
Female; all
patients
were
mate...=Unabte
to
%
attend for
FALL IN E.L.T.
tests.
more
(HR.)
MI=Myocardial infarction AP= Angina pectoris. tC= Intermittent
1011
therapy were not confined in either group to those whose fibrinolytic activity was low (E.L.T. more than 2 hours)
diguanides in this respect. Fibrinolysis was enhanced not only in patients whose fibrinolytic activity before treat-
before treatment, but were also obtained in most of the patients whose activity was initially within the normal range. In group l, patients 1-7 had low fibrinolytic activity, and of these six had a fibrinolytic response; patients 8-15 had normal activity, and six of these responded. In group II, patients 1-10 had low activity and all responded; patients 11-18 had normal activity, and six of these responded.
ment was
in the low range, but also in those in whom it within normal limits. Reduction of the E.L.T. indicates that the drugs raise the level of plasminogen activator; whether they do this by increasing the rate of formation of activator or by decreasing its rate of removal by an organ such as the liver is unknown. Although both combinations of drugs had a satisfactory fibrinolytic effect, only phenformin reduced serumcholesterol and this drug gave a somewhat greater reducSide-effects Besides the patients reported here we have given tion of plasma-fibrinogen than did metformin. Since phenformin plus ethyloestrenol to another one hundred phenformin plus ethyloestrenol also reduces platelet and eighteen patients with occlusive vascular disease, and stickiness (Chakrabarti and Fearnley 1967)-and we do metformin plus ethyloestrenol to another thirty patients. not yet know whether metformin plus ethylcestrenol will do so--on the present evidence phenformin would seem No serious toxicity has been encountered with either to have advantages over metformin. The diguanides cause gastrointestinal combination. The favourable influence of phenformin plus ethylaesymptoms in about 15% of patients; anorexia, nausea, strenol on four factors believed to be of importance in and vomiting with phenformin and diarrhoea with metforrriin. The symptoms are mild in more than half occlusive vascular disease-i.e., fibrinolytic activity, the patients who experience them and subside after a few plasma-fibrinogen, serum-cholesterol, and platelet stickiness-makes this combination of drugs suitable for trial weeks’ treatment; in about 5% of patients they necessitate treatment. Patients unable to tolerate stopping phen- in the prophylaxis of venous and arterial occlusions. The formin capsules, however, may be able to take metformin diguanide may also be expected to correct the impaired cartablets without ill-effect, and vice versa. bohydrate tolerance found by Wahlberg (1962) and others Both phenformin and metformin cause loss of weight to be a feature of many patients with ischaemic disease. While no firm conclusions can be drawn from an in about half the patients treated. This is usually of the uncontrolled trial, the moderate death-rate and low rate order of 6-10 lb. (3-5 kg.), and after this has been lost, of reinfarction the weight generally stabilises at the new level. Greater among our patients encourage the hope that a combination of fibrinolytic drugs may have prophyand continuing weight loss is uncommon, but may very lactic value in occlusive vascular disease. Twice-daily rarely necessitate stopping treatment. Ethyloestrenol is well tolerated by most patients, though dosage helps to ensure that the drugs are taken regularly, occasionally it causes headache. It may raise the blood- an important consideration in the long-term treatment of those patients who are untroubled by symptoms. It pressure in hypertensive patients, and can exacerbate must be emphasised that while the effect of phenformin or in who have had are on the symptoms patients recently left of ventricular while these can plus ethyloestrenol on fibrinolysis is usually apparent failure; verge having or controlled with diuretic often be prevented tablets within a month of starting treatment, in some patients such patients must be watched carefully and told to the maximal effect is not obtained until the drugs have been given for 2 months. Furthermore, reduction of report any undue dyspnoea. platelet stickiness was not observed until the 3rd month of Outcome in nearly half the patients who responded in During the 21 months of combined therapy, including treatment this respect (Chakrabarti and Fearnley 1967). It the months when ethyloestrenol was given alone, there follows that should phenformin plus ethyloestrenol were two deaths and one reinfarction, all of which occurred exercise a protective influence in occlusive vascular conin group i. The first death was that of a man, not included in the data of table i who died suddenly after ditions, their full effects will not become available in many patients until treatment has been given for up to 3 months. the first month of treatment with metformin plus This work was supported by grants from the South Western ethyloestrenol. Necropsy was not done. The second Regional Hospital Board, the British Heart Foundation, the Nuffield death was that of patient no. 15 in tablet who died Foundation, Bayer Products Ltd., and Organon Laboratories, Ltd. suddenly during month 33 at the end of the trial. No We thank the two pharmaceutical companies for supplies of phenformin and ethyloestrenol, and Rona Laboratories for supplies of necropsy was done. The sole reinfarction was in patient metformin; Dr. C. C. Downie, of Imperial Chemical Industries for no. 8 (table l) during month 29. He made a good recovery supplies of atromid and for undertaking the determination of serumand continued in the trial. cholesterol during this investigation; and patients for continuing The total number of man-months of combined therapy cooperation throughout a long trial. in groups i and n was 694, giving a mortality-rate of Requests for reprints should be addressed to G. R. F. REFERENCES 0-3 per 100 man-months. Twenty-two of the patients had Chakrabarti, R., Fearnley, G. R. (1967) Lancet, ii, 1012. survived one or more myocardial infarctions and the total Hocking, E. D. (1964) Br. med. J. i, 534. number of man-months of combined therapy for these Hocking, E. D., Fearnley, G. R. (1965) Lancet, ii, 256. G. R. (1964) J. clin. Path. 17, 307. patients was 462, giving a reinfarction-rate of 0-2 per Fearnley, Balmforth, G., Fearnley, E. B. (1957) Clin. Sci. 16, 645. 100 man-months. (1965) Fibrinolysis, p. 82, London. was
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Discussion
The problem of resistance which
1
develops sooner or given by mouth has been overcome by giving two such drugs together. Both phenformin and metformin combined with ethyloestrenol gave a substantial and sustained increase of fibrinolytic activity, there being little to choose between the two
later when fibrinolytic drugs
B
I I
are
Chakrabarti, R., Vincent, C. T. (1960) Lancet, ii, 622. (1962) ibid. ii, 128. (1964) J. clin. Path. 17, 328. (1966) Lancet, ii, 757. Hocking, E. D., Chakrabarti, R., Evans, J., Fearnley, G. R. (1967) J. Atheroscler. Res. 7, 121. von Kaulla, K. N. (1963) Chemistry of Thrombolysis: Human Fibrinolytic Enzymes; p. 79. Illinois. Wahlberg, F. (1962) Acta. med. scand. 171, 1. Zlatkis, A., Zak, B., Boyle, G. J. (1953) J. Lab. clin. Med 41, 486. —
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