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Increased fibrinolytic potential induced by gliclazide in types I and II diabetic patients
Increased Fibrinolytic Potential Induced by Gliclazide in Types I and II Diabetic Patients JORGEN
GRAM,
M.D.,
JORGEN
JESPERSEN,
M.D.,
DSC.
Esbjerg, Denmark
This study examined the effect of gliclazide on tissue-type plasminogen activator (t-PA)related fibrinolysis in 23 Type I diabetic patients without residual p-cell function and 17 Type II diabetic patients initially treated with tolbutamide. The Type I diabetic patients received gliclazide for a period of 6 months; the l)pe II diabetic patients were shifted from tolbutamide to gliclazide. In Type I diabetic patients, after 2-3 months of treatment with gliclazide, we observed a significant increase in plasma concentrations of total t-PA antigen that remained stable until discontinuation of the drug (p <0.0002), whereas the plasma concentrations of plasminogen activator inhibitor (PAI) did not change significantly during the study. Next, we investigated the possibility of gliclazide inducing t-PA-related fibrinolysis in a subset of Type II diabetics without detectable concentrations of t-PA during treatment with tolbutamide. The concentrations of active t-PA increased significantly 3 months after a change in treatment to gliclazide, and active t-PA again decreased in one patient to undetectable levels after 12 months with gliclazide. Moreover, the plasma concentrations of total t-PA antigen increased significantly (p CO.021 in this group of diabetic patients while PA1 remained unchanged. The changes in t-PA-related fibrinolysis could not be related in either Type I or Type II diabetics to changes in metabolic state evaluated by blood glucose, HbA1,, cholesterol, triglycerides, or apolipoproteins A and B. We conclude that gliclazide has the potential to exert extrametabolic non-insulin-mediated effects on t-PA-related fibrinolysis in diabetic patients.
From the Section of Coagulation and Fibrrnolysis, Department of Clinrcal Chemistry, and Department of Internal Medicine, Ribe County Hospital in Esbjerg; Section of Thrombosis Research, South Jutland University Centre, Esbjerg, DenRequests for reprints should be addressed to Jorgen Gram, M.D., Section of Coagulation and Fibrinolysis, Department of Clinical Chemistry, Ribe County Hospital in Esbjerg. DK-6700 Esbjerg, Denmark.
6A-62S
June 24,
1991
The American Journal of Medicine
A
ngiopathy with lesions of the small and large vessels is a consistent feature of late diabetes. An increased deposition of fibrin in the vascular system of diabetic patients may cause enhanced arteriosclerosis, impaired tissue repair, and dysfunction of organs [l-4]. It is of particular interest that attention has been drawn to fibrin deposition within the small vessels playing a pathogenetic role in the injury of the diabetic nerve and that a disturbed balance in the dynamics of deposition and resolution of fibrin possibly could explain the phasic variation of neuropathy [5]. Others have found evidence for some form of capillary diffusion barrier related to pericapillary fibrin caused in part by a reduced fibrinolytic response in diabetic subjects
Bl. It might therefore be important to enhance endogenous fibrinolysis in diabetic patients in an attempt to retard the onset of diabetic complications. First-generation sulfonylureas have been reported to have a fibrinolysis-enhancing effect that lasts for only 3-4 weeks [71. Recently, gliclazide, a secondgeneration sulfonylurea, has been reported to affect fibrinolysis more consistently [8-111. In this paper we report our observations on the effect of gliclazide on the extrinsic tissue-type plasminogen activator (t-PA&related fibrinolysis in Type I and Type II diabetic patients.
SUBJECTSAND METHODS Subjects Included in the study were 23 patients with Type I and 17 patients with Type II diabetes. The Type I diabetic patients had no significant residual p-cell function, verified by a lack of release of C-peptide after an intravenous injection of 1 mg glucagon (Novo Industry, Copenhagen, Denmark). These patients were given 160 mg (n = 11) or 240 mg (n = 12) gliclazide (Diamicron, Laboratoires Servier, Neuilly-sur-Seine, France) daily for a period of 6 months. Blood samples were obtained in the morning from fasting individuals immediately before administration; 1, 2, 4, and 6 months after administration; and 1 month after discontinuation of gliclazide. All patients were males with a median age of 47 years (range 23-58 years) and a median duration of diabetes of 15 years (range 6-27 years).
Volume 90 (suppl 6A)
SYMPOSIUM ON GLICIAZIDE
All patients were characterized by background retinopathy. The Type II diabetic patients, initially treated with tolbutamide, were changed to equipotent doses of gliclazide, i.e., 13 patients consuming a daily dose of 1.5 g tolbutamide were changed to 240 mg gliclazide and four patients consuming a daily dose of 1.0 g tolbutamide were changed to 160 mg gliclazide. Blood samples from these patients were obtained in the morning from fasting individuals at four periods: during treatment with tolbutamide; immediately before change to gliclazide treatment; after 3 months of treatment with gliclazide; after 12 months of treatment with gliclazide. All patients had diabetes of greater than 5 years’ duration. Their mean age was 61 years (range 58-76 years), mean weight 78.3 kg (range 59-105 kg), and mean height 176 cm (range 158-188 cm). The blood sampling from both groups of patients and the preparation and handling of plasma specimens followed standardized procedures [lo]. Analytic Methods For the evaluation of the glycemic control, the patients’ serum glucose was determined by a dehydrogenase method on a Technicon RA-1000 analyzer (Technicon Instruments Corporation, NY), and hemoglobin Ai, (HbA,,) by isoelectric focusing [ 111. Apolipoproteins A and B in serum on Mpartigen plates (Behringwerke, Marburg, Germany), and triglyceride and total cholesterol in serum were measured by enzymatic methods using commercially available reagents (Boehringer Mannheim, Germany). The following variables were determined for the evaluation of the extrinsic t-PA-related fibrinolysis: the concentration of active (free) t-PA by a parabolic kinetic method [12], the concentrations of total t-PA antigen by an enzyme-linked immunosorbent assay using commercially available reagents (Biopool5-t-PA, Biopool, Umea, Sweden), and the plasminogen activator inhibitor (PAI) by spectrophotometric determination of residual t-PA after addition of purified two-chain human melanoma t-PA to plasma [13]. Statistical Analysis Possible systematic fluctuations during the whole study period were evaluated by a Friedman chisquared test. Comparisons between paired observations were evaluated by a paired t-test.
RESULTS During the study period, the insulin demand of the group of Type I diabetic patients did not change significantly; neither did the patients’ weight or
I GRAM and JESPERSEN
8-
6-
3 4 Months
5
6
Figure 1. Median concentratrons and the quartiles of total tissue-type plasminogen activator (t-PA) antigen (0) expressed as nanograms per milliliter and plasminogen activator inhibitor (PAI) activity(r) expressed as international units per milliliter in 23 Type I diabetic patients given a daily dose of gliclazrde of 160 mg (n = 11) or 240 mg (n = 12). The treatment period is indicated by the horizontal open bar.
their metabolic regulation as evaluated by fasting glucose, HbAi,, apolipoproteins A and B, triglycerides, and total cholesterol [141. Similarly, the Type II diabetic patients had unchanged average weight during the study. The average values in fasting blood glucose, HbA,,, apolipoproteins A and B, and triglycerides also remained constant [15]. However, the mean serum concentrations of cholesterol showed a continuous decrease from 5.90 mmol/L during treatment with tolbutamide to 5.41 mmol/L after 12 months of treatment with gliclazide. This fall was statistically significant (p <0.002). The plasma concentration of total t-PA antigen increased significantly (p < 0.0002) during the administration of gliclazide to the group of Type I diabetic patients without residual P-cell function (Figure 1). This effect was delayed because the increase in t-PA antigen was constant during the first 2 months and increased significantly between 2 and 3 months of treatment. One month after gliclazide was discontinued, the t-PA antigen concentrations were back to baseline level. In contrast, the plasma concentrations of the main t-PA inhibitor, PAI, remained constant during the whole study period (Figure 1). These findings demonstrate that
June 24, 1991
The American Journal of Medicine
Volume 90 (suppl 6A)
6A63S
SYMPOSIUMON GUClAZlDE/GRAM and JESPERSEN
At-PA
At-PA
mlU/ml
mlU/ml
120
-
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-
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80
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40
-
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.
-120
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-43*---
-4
months At-PA 120 -
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-120
months At-PA mllJ/ml
mpJ/ml
-80
<
-
;:
4
-
-80
-
-
-120
-
-43*---
Figure 3. Individual changes in concentrations of active tissue-type plasminogen activator (At-
-4 months
June 24,
1991 The American Journal af Medicine
12 *-months
gliclazide has the potential to increase the concentrations of t-PA in the blood of Type I diabetics, but without affecting the inhibition of t-PA. The next step was to study the effect of gliclazide on the t-PA-related fibrinolysis in the patients for whom the drug is primarily intended, i.e., Type II diabetics. The particular purpose was to study whether an abnormally low blood concentration of
6Ad4s
12 I)---
Figure 2. Individual changes in concentrations of active tissue-type plasminogen aetivator (AtPA) in lOType II diabetic patients after 3 months (left panel) and after 12 months (rigw panel) of treatment with gliclazide. The 10 patients had no detectable active CPA before they were shifted from tolbutamide to glickszide, determined on @9 occasions with an interval of 3 months.
Volume 90 (suppl
PA) in seven Type II diabetic pabents alter 3 months (left panel) and after 12 months (right panel) of treatment with gliclazide. The seven patients had marked concentrations of active t-PA (average, 66 mlU/ml) before they were shifted from tolbutamide to gliclazide.
active (free) t-PA in individual patients could be increased by a change in treatment from tolbutamide to gliclazide. During the treatment with tolbutamide, 19 patients had undetectable concentrations of active t-PA. After 3 months of treatment with glielazide, these Id patients had raised concentrations of active t-PA (Figure 2, left panel); and 12 months after
64
SYMpOSlUM
change in treatment 9 of the 10 patients still had raised concentrations of active t-PA (Figure 2, right panel). The seven patients with detectable concentrations of active t-PA during treatment with tolbutamide (average 55 mIU/L) did not show a trend toward change during the study (Figure 3). The concentrations of total t-PA, i.e., t-PA antigen, increased significantly after 3 months of treatment with gliclazide (p ~0.05) in the subset of patients who had undetectable active t-PA during treatment with tolbutamide, and the trend toward higher concentrations of total t-PA antigen (0.05 < p ~0.10) remained unchanged after 12 months of treatment with gliclazide (Table I). In this group of patients the concentrations of active PA1 remained unchanged during the study (Table I). In the other subset of seven patients who had marked concentrations of total t-PA during treatment with tolbutamide, neither the concentrations of total t-PA nor the concentration of active PA1 changed significantly during the study period. In order to determine if there was a covariance between the increase in concentrations of total t-PA and the decrease in cholesterol, we correlated the paired observations of cholesterol and t-PA antigen in the 10 Type II diabetic patients with no detectable concentration of active t-PA during treatment with tolbutamide. This correlation was not significant.
CQMMENTS Studies on the effect of oral antidiabetic drugs on the fibrinolytic system of diabetic patients raise methodologic problems, because the drug per se, as well as possible drug-induced changes of the metar bolic state of the patients, might affect plasminogen activation [16-H]. In our endeavor to obtain the simplest metabolic model, we initiated our studies on the effect of glielazide on the fibrinolytic system of diabetic patients by the administration of the drug to Type I diabetics without residual P-cell function. Because of the large interindividual variance of tibrinolytic variables in diabetic subjects, we designed our study so that each patient served as his own control. During the period of administration of 160 mg or 240 mg gliclazide to the Type I diabetic patients, we observed a small but statistically significant increase in the blood concentration of total t-PA antigen, while the inhibition of t-PA remained unchanged (Figure 1). The effect was retarded until 2-3 months after gliclazide initiation, indicating an increased de novo synthesis of t-PA, which is produced by endothelial cells [191, rather than an immediate release from an extravascular
June 24,
ON GLICLAZIDE I GRAM and JESPERSEN
TABLE I Mean (SD) Concentrations in Plasma of Total t-PA (t-PA antigen) and Free Active PAI t.PfMie Sampling Periods During tolbutamide During gliclazide (3 months) During gliclazide (12 months)
13.2 (2.3)
15.013.7)* 14.7(6.3)
PAI Activity (It-l/ml) 43.7 (37.3) 46.2 (36.2) 43.5 (23.2)
I
I
Mean concentrations were determined in 10 type II diabetic patients with Initially no detectable active (free) t-PA levels during treatment with tolbutamide and after 3 and 12 months of treatment with gliclazide. t-PA = tissue type plasminogen activator: PAI = plasmlnogen activator inhibltor. *p ~0.05, different from tolbutamlde.
t-PA pool. This conclusion is supported by a recent study on the effect of sulfonylureas on the synthesis and secretion of plasminogen activators from bovine aortic endothelial cells [20]. On an insulin-free medium, it was demonstrated that glipizide, another second-generation sulfonylurea, increased fivefold the concentration of total t-PA antigen in the growth media, and that the increased release of t-PA involved an enhanced mRNA and protein synthesis. After gliclazide had been discontinued in our patients, the concentration of total t-PA antigen decreased to baseline levels (Figure 1). Since the metabolic state of the patients did not change significantly during the study period, these results suggest that gliclazide exerts extrametabolic noninsulin-mediated effects on the t-PA-related fibrinolytic system of Type I diabetic patients. In the second study we wished to elucidate whether gliclazide can enhance t-PA-related fibrinolysis in Type II diabetics and particularly whether the drug can increase the concentrations of active t-PA in a subset of patients with undetectable levels of t-PA. This might be of interest because angiopathy is an obligate feature of late diabetes [l-4] and because an abnormally low activity of t-PA has been reported to increase the risk of myocardial infarction in nondiabetic subjects with arteriosclerosis [21-241. All 10 Type II diabetic patients with undetectable active t-PA during treatment with tolbutamide showed increased concentrations of active t-PA 3 months after the treatment change from tolbutamide to gliclazide, while the concentration in one patient decreased to undetectable levels after 12 months of treatment with gliclazide (Figure 2). The increase in concentrations of active t-PA in this subset of Type II diabetic patients paralleled an increase in concentrations of total t-PA antigen, whereas PA1 remained constant (Table I). These changes are probably not related to a changed metabolic state during the study, as described previously [15]. In the other subset of Type II diabetic patients with marked concentrations of active t-PA during treatment with tolbutamide, we
1991 The American Journal of Medicine
Volume 90 (suppl 6A)
6A-65S
SYMPOSIUM
ON GLICIAZIDE
/ GRAM and JESPERSEN
did not observe a change in concentrations of active t-PA, total t-PA antigen, or PA1 during the study. Combining our results on Type I and Type II diabetic patients, we have obtained suggestive evidence that gliclazide (or some of its metabolites) has a non-insulin-mediated potential to increase the blood concentrations of total t-PA antigen without affecting PA1 levels. It should be noted that t-PA is produced by the endothelial cells [19], but whether the changes in t-PA-related fibrinolysis reported here reflect an improved function of endothelial cells or a change in removal mechanisms in the diabetic patients should be examined in further detail in prospective clinical trials.
REFERENCES 1. Wilkens HJ, Back N. Fibrinolysis and risk factors of atherosclerotic disease, with special emphasis on diabetes mellitus. Circ Shock 1978; 5: 125-43. 2. Fuller JH, Keen H, Jarret RI, et al. Haemostatic variables associated with diabetes and its complications. Br Med J 1979; 2: 964-8. 3. Colwell JA, Winocour PD, Lopes-Virella M, Halushka PV. New concepts about the pathogenesis of atherosclerosis in diabetes mellitus. Am J Med 1983: 75 (Suppl5B): 67-80. 4. Stofar MW. Atherosclerosis in diabetes: the role of hyperinsulinemia. Metabolism 1988; 37 (Suppl 1): l-9. 5. Timperley WR, Ward JD, Preston FE, et al. Clinical and histalogical studies in diabetic neuropathy. A reassessment of vascular factors in relation to intravascular coagulation. Diabetologia 1976; 12: 237-43. 6. Haitas B, Barnes AJ, Cederholm-Williams SA, et al. Abnormal endothelial release of fibrinolytic activity and fibronectin in diabetic microangiopathy. Diabetologia 1984; 27: 493-6. 7. Feamley GR, Chakrabarti R. Vincent CT. Effect of sulphonylureas on fibrinolysis. Lancet 1960; 2: 622-5. 8. Almer L-O. Vascular fibnnolytic activity in long term treatment with second generation sulfonylurea compounds. Acta Endocrinol (Copenh) 1980; 239 (Suppl): 53-5. 9. Almer L-O. Effect of chlorpropamide and gliclazide on plasminogen activator activity in vascular walls of patients with maturity onset diabetes. Thromb Res 1984;
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June 24, 1991
The American
Journal of Medicine
35: 173-8. 10. Jespersen J, Knudsen LH, Sidelmann J. The use of evacuated glass tubes for collection of blood samples for fibrinolytic assays. Thromb Res 1982; 25: 173-6. 11. Poulsen JH, Jespersen J. A comparison of the determination of glycosylated hemoglobin by isoelectric focusing and cation-exchange chromatography on mmicolumns. Stand J Clin Lab Invest 1986; 46: 259-63. 12. Verheijen JH, Muellaart E, Chang GTG, Kluft C. A simple sensitive spectrophotometric assay for extrinsic (tissue-type) plasminogen activator appltcable to measurement In plasma. Thromb Haemost 1982; 48: 266-9. 13. Gram J, Jespersen J. A simplified esbmation of tissue plasminogen acbvator (t-PA) inhibition in human plasma. Fibrinolysis 1987; 1: 33-7. 14. Gram J, Jespersen J, Kold AA. Effects of an oral antidiabetic drug on the fibrlnolytic system of blood in insulin-treated diabetic patients. Metabolism 1988; 37: 937-43. 15. Gram J, Kold AA, Jespersen J. Rise of plasma t-PA fibrinolytic activity in a group of maturity onset diabetic patients shifted from a first generation (tolbutamrde) to a second sulphonylurea (gliclazide). J Intern Med 1989; 225: 241-7. 16. Brownlee M, Vlassara H, Ceramr A. Nonenzymatic glycosylahon reduces the susceptibility of fibrin to degradation by plasmin. Diabetes 1983; 32: 600-4. 17. Geiger M, Binder BR. Plasminogen activation in diabetes mellitus. J Biol Chem 1984; 259: 2676-81. 18. Auwerx J, Bouillon R, Collen D, Geboers J. Tissue-type plasminogen activator antigen and plasminogen activator inhibitor in diabetes mellitus. Atherosclerosis 1988; 8: 68-72. 19. Rijken DC, Wijngaards G, Welbergen J. Relationship between tissue plasminogen activator and the activators in blood and vascular wall. Thromb Res 1980; 18: 815-30. 20. Kuo B-S, Korner G, Bjornsson TD. Effects of sulfonylureas on the synthesis and secretion of plasminogen activator from bovine aortic endothelial cells. J Clin Invest 1988; 81: 730-7. 21. Gram J, Jespersen J. A selective depression of tissue plasminogen activator (t-PA) activity in euglobulins characterises a risk group among survivors of acute myocardial infarction, Thromb Haemost 1987; 57: 137-9. 22. Gram J, Jespersen J, Kluft C, Rijken D. On the usefulness of fibrinolysis variables in the characterization of a risk group for myocardial infarction. Acta Med Stand 1987; 221: 149-63. 23. Hamsten A, de Faire U, Wellelius G, et al. Plasminogen activator inhibitor in plasma: a risk factor for recurrent myocardial infarction. Lancet 1987; 2: 3-8. 24. Aznar J, Estelles A, Tormo G, et al. Plasminogen activator inhibitor activity and other fibrinolytic variables in patients with coronary artery disease. Br Heart J 1988; 59 535-41.
Volume 9Q (suppl 6A)
GRAM,
M.D.,
JORGEN
JESPERSEN,
M.D.,
DSC.
Esbjerg, Denmark
This study examined the effect of gliclazide on tissue-type plasminogen activator (t-PA)related fibrinolysis in 23 Type I diabetic patients without residual p-cell function and 17 Type II diabetic patients initially treated with tolbutamide. The Type I diabetic patients received gliclazide for a period of 6 months; the l)pe II diabetic patients were shifted from tolbutamide to gliclazide. In Type I diabetic patients, after 2-3 months of treatment with gliclazide, we observed a significant increase in plasma concentrations of total t-PA antigen that remained stable until discontinuation of the drug (p <0.0002), whereas the plasma concentrations of plasminogen activator inhibitor (PAI) did not change significantly during the study. Next, we investigated the possibility of gliclazide inducing t-PA-related fibrinolysis in a subset of Type II diabetics without detectable concentrations of t-PA during treatment with tolbutamide. The concentrations of active t-PA increased significantly 3 months after a change in treatment to gliclazide, and active t-PA again decreased in one patient to undetectable levels after 12 months with gliclazide. Moreover, the plasma concentrations of total t-PA antigen increased significantly (p CO.021 in this group of diabetic patients while PA1 remained unchanged. The changes in t-PA-related fibrinolysis could not be related in either Type I or Type II diabetics to changes in metabolic state evaluated by blood glucose, HbA1,, cholesterol, triglycerides, or apolipoproteins A and B. We conclude that gliclazide has the potential to exert extrametabolic non-insulin-mediated effects on t-PA-related fibrinolysis in diabetic patients.
From the Section of Coagulation and Fibrrnolysis, Department of Clinrcal Chemistry, and Department of Internal Medicine, Ribe County Hospital in Esbjerg; Section of Thrombosis Research, South Jutland University Centre, Esbjerg, DenRequests for reprints should be addressed to Jorgen Gram, M.D., Section of Coagulation and Fibrinolysis, Department of Clinical Chemistry, Ribe County Hospital in Esbjerg. DK-6700 Esbjerg, Denmark.
6A-62S
June 24,
1991
The American Journal of Medicine
A
ngiopathy with lesions of the small and large vessels is a consistent feature of late diabetes. An increased deposition of fibrin in the vascular system of diabetic patients may cause enhanced arteriosclerosis, impaired tissue repair, and dysfunction of organs [l-4]. It is of particular interest that attention has been drawn to fibrin deposition within the small vessels playing a pathogenetic role in the injury of the diabetic nerve and that a disturbed balance in the dynamics of deposition and resolution of fibrin possibly could explain the phasic variation of neuropathy [5]. Others have found evidence for some form of capillary diffusion barrier related to pericapillary fibrin caused in part by a reduced fibrinolytic response in diabetic subjects
Bl. It might therefore be important to enhance endogenous fibrinolysis in diabetic patients in an attempt to retard the onset of diabetic complications. First-generation sulfonylureas have been reported to have a fibrinolysis-enhancing effect that lasts for only 3-4 weeks [71. Recently, gliclazide, a secondgeneration sulfonylurea, has been reported to affect fibrinolysis more consistently [8-111. In this paper we report our observations on the effect of gliclazide on the extrinsic tissue-type plasminogen activator (t-PA&related fibrinolysis in Type I and Type II diabetic patients.
SUBJECTSAND METHODS Subjects Included in the study were 23 patients with Type I and 17 patients with Type II diabetes. The Type I diabetic patients had no significant residual p-cell function, verified by a lack of release of C-peptide after an intravenous injection of 1 mg glucagon (Novo Industry, Copenhagen, Denmark). These patients were given 160 mg (n = 11) or 240 mg (n = 12) gliclazide (Diamicron, Laboratoires Servier, Neuilly-sur-Seine, France) daily for a period of 6 months. Blood samples were obtained in the morning from fasting individuals immediately before administration; 1, 2, 4, and 6 months after administration; and 1 month after discontinuation of gliclazide. All patients were males with a median age of 47 years (range 23-58 years) and a median duration of diabetes of 15 years (range 6-27 years).
Volume 90 (suppl 6A)
SYMPOSIUM ON GLICIAZIDE
All patients were characterized by background retinopathy. The Type II diabetic patients, initially treated with tolbutamide, were changed to equipotent doses of gliclazide, i.e., 13 patients consuming a daily dose of 1.5 g tolbutamide were changed to 240 mg gliclazide and four patients consuming a daily dose of 1.0 g tolbutamide were changed to 160 mg gliclazide. Blood samples from these patients were obtained in the morning from fasting individuals at four periods: during treatment with tolbutamide; immediately before change to gliclazide treatment; after 3 months of treatment with gliclazide; after 12 months of treatment with gliclazide. All patients had diabetes of greater than 5 years’ duration. Their mean age was 61 years (range 58-76 years), mean weight 78.3 kg (range 59-105 kg), and mean height 176 cm (range 158-188 cm). The blood sampling from both groups of patients and the preparation and handling of plasma specimens followed standardized procedures [lo]. Analytic Methods For the evaluation of the glycemic control, the patients’ serum glucose was determined by a dehydrogenase method on a Technicon RA-1000 analyzer (Technicon Instruments Corporation, NY), and hemoglobin Ai, (HbA,,) by isoelectric focusing [ 111. Apolipoproteins A and B in serum on Mpartigen plates (Behringwerke, Marburg, Germany), and triglyceride and total cholesterol in serum were measured by enzymatic methods using commercially available reagents (Boehringer Mannheim, Germany). The following variables were determined for the evaluation of the extrinsic t-PA-related fibrinolysis: the concentration of active (free) t-PA by a parabolic kinetic method [12], the concentrations of total t-PA antigen by an enzyme-linked immunosorbent assay using commercially available reagents (Biopool5-t-PA, Biopool, Umea, Sweden), and the plasminogen activator inhibitor (PAI) by spectrophotometric determination of residual t-PA after addition of purified two-chain human melanoma t-PA to plasma [13]. Statistical Analysis Possible systematic fluctuations during the whole study period were evaluated by a Friedman chisquared test. Comparisons between paired observations were evaluated by a paired t-test.
RESULTS During the study period, the insulin demand of the group of Type I diabetic patients did not change significantly; neither did the patients’ weight or
I GRAM and JESPERSEN
8-
6-
3 4 Months
5
6
Figure 1. Median concentratrons and the quartiles of total tissue-type plasminogen activator (t-PA) antigen (0) expressed as nanograms per milliliter and plasminogen activator inhibitor (PAI) activity(r) expressed as international units per milliliter in 23 Type I diabetic patients given a daily dose of gliclazrde of 160 mg (n = 11) or 240 mg (n = 12). The treatment period is indicated by the horizontal open bar.
their metabolic regulation as evaluated by fasting glucose, HbAi,, apolipoproteins A and B, triglycerides, and total cholesterol [141. Similarly, the Type II diabetic patients had unchanged average weight during the study. The average values in fasting blood glucose, HbA,,, apolipoproteins A and B, and triglycerides also remained constant [15]. However, the mean serum concentrations of cholesterol showed a continuous decrease from 5.90 mmol/L during treatment with tolbutamide to 5.41 mmol/L after 12 months of treatment with gliclazide. This fall was statistically significant (p <0.002). The plasma concentration of total t-PA antigen increased significantly (p < 0.0002) during the administration of gliclazide to the group of Type I diabetic patients without residual P-cell function (Figure 1). This effect was delayed because the increase in t-PA antigen was constant during the first 2 months and increased significantly between 2 and 3 months of treatment. One month after gliclazide was discontinued, the t-PA antigen concentrations were back to baseline level. In contrast, the plasma concentrations of the main t-PA inhibitor, PAI, remained constant during the whole study period (Figure 1). These findings demonstrate that
June 24, 1991
The American Journal of Medicine
Volume 90 (suppl 6A)
6A63S
SYMPOSIUMON GUClAZlDE/GRAM and JESPERSEN
At-PA
At-PA
mlU/ml
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-80
<
-
;:
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Figure 3. Individual changes in concentrations of active tissue-type plasminogen activator (At-
-4 months
June 24,
1991 The American Journal af Medicine
12 *-months
gliclazide has the potential to increase the concentrations of t-PA in the blood of Type I diabetics, but without affecting the inhibition of t-PA. The next step was to study the effect of gliclazide on the t-PA-related fibrinolysis in the patients for whom the drug is primarily intended, i.e., Type II diabetics. The particular purpose was to study whether an abnormally low blood concentration of
6Ad4s
12 I)---
Figure 2. Individual changes in concentrations of active tissue-type plasminogen aetivator (AtPA) in lOType II diabetic patients after 3 months (left panel) and after 12 months (rigw panel) of treatment with gliclazide. The 10 patients had no detectable active CPA before they were shifted from tolbutamide to glickszide, determined on @9 occasions with an interval of 3 months.
Volume 90 (suppl
PA) in seven Type II diabetic pabents alter 3 months (left panel) and after 12 months (right panel) of treatment with gliclazide. The seven patients had marked concentrations of active t-PA (average, 66 mlU/ml) before they were shifted from tolbutamide to gliclazide.
active (free) t-PA in individual patients could be increased by a change in treatment from tolbutamide to gliclazide. During the treatment with tolbutamide, 19 patients had undetectable concentrations of active t-PA. After 3 months of treatment with glielazide, these Id patients had raised concentrations of active t-PA (Figure 2, left panel); and 12 months after
64
SYMpOSlUM
change in treatment 9 of the 10 patients still had raised concentrations of active t-PA (Figure 2, right panel). The seven patients with detectable concentrations of active t-PA during treatment with tolbutamide (average 55 mIU/L) did not show a trend toward change during the study (Figure 3). The concentrations of total t-PA, i.e., t-PA antigen, increased significantly after 3 months of treatment with gliclazide (p ~0.05) in the subset of patients who had undetectable active t-PA during treatment with tolbutamide, and the trend toward higher concentrations of total t-PA antigen (0.05 < p ~0.10) remained unchanged after 12 months of treatment with gliclazide (Table I). In this group of patients the concentrations of active PA1 remained unchanged during the study (Table I). In the other subset of seven patients who had marked concentrations of total t-PA during treatment with tolbutamide, neither the concentrations of total t-PA nor the concentration of active PA1 changed significantly during the study period. In order to determine if there was a covariance between the increase in concentrations of total t-PA and the decrease in cholesterol, we correlated the paired observations of cholesterol and t-PA antigen in the 10 Type II diabetic patients with no detectable concentration of active t-PA during treatment with tolbutamide. This correlation was not significant.
CQMMENTS Studies on the effect of oral antidiabetic drugs on the fibrinolytic system of diabetic patients raise methodologic problems, because the drug per se, as well as possible drug-induced changes of the metar bolic state of the patients, might affect plasminogen activation [16-H]. In our endeavor to obtain the simplest metabolic model, we initiated our studies on the effect of glielazide on the fibrinolytic system of diabetic patients by the administration of the drug to Type I diabetics without residual P-cell function. Because of the large interindividual variance of tibrinolytic variables in diabetic subjects, we designed our study so that each patient served as his own control. During the period of administration of 160 mg or 240 mg gliclazide to the Type I diabetic patients, we observed a small but statistically significant increase in the blood concentration of total t-PA antigen, while the inhibition of t-PA remained unchanged (Figure 1). The effect was retarded until 2-3 months after gliclazide initiation, indicating an increased de novo synthesis of t-PA, which is produced by endothelial cells [191, rather than an immediate release from an extravascular
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ON GLICLAZIDE I GRAM and JESPERSEN
TABLE I Mean (SD) Concentrations in Plasma of Total t-PA (t-PA antigen) and Free Active PAI t.PfMie Sampling Periods During tolbutamide During gliclazide (3 months) During gliclazide (12 months)
13.2 (2.3)
15.013.7)* 14.7(6.3)
PAI Activity (It-l/ml) 43.7 (37.3) 46.2 (36.2) 43.5 (23.2)
I
I
Mean concentrations were determined in 10 type II diabetic patients with Initially no detectable active (free) t-PA levels during treatment with tolbutamide and after 3 and 12 months of treatment with gliclazide. t-PA = tissue type plasminogen activator: PAI = plasmlnogen activator inhibltor. *p ~0.05, different from tolbutamlde.
t-PA pool. This conclusion is supported by a recent study on the effect of sulfonylureas on the synthesis and secretion of plasminogen activators from bovine aortic endothelial cells [20]. On an insulin-free medium, it was demonstrated that glipizide, another second-generation sulfonylurea, increased fivefold the concentration of total t-PA antigen in the growth media, and that the increased release of t-PA involved an enhanced mRNA and protein synthesis. After gliclazide had been discontinued in our patients, the concentration of total t-PA antigen decreased to baseline levels (Figure 1). Since the metabolic state of the patients did not change significantly during the study period, these results suggest that gliclazide exerts extrametabolic noninsulin-mediated effects on the t-PA-related fibrinolytic system of Type I diabetic patients. In the second study we wished to elucidate whether gliclazide can enhance t-PA-related fibrinolysis in Type II diabetics and particularly whether the drug can increase the concentrations of active t-PA in a subset of patients with undetectable levels of t-PA. This might be of interest because angiopathy is an obligate feature of late diabetes [l-4] and because an abnormally low activity of t-PA has been reported to increase the risk of myocardial infarction in nondiabetic subjects with arteriosclerosis [21-241. All 10 Type II diabetic patients with undetectable active t-PA during treatment with tolbutamide showed increased concentrations of active t-PA 3 months after the treatment change from tolbutamide to gliclazide, while the concentration in one patient decreased to undetectable levels after 12 months of treatment with gliclazide (Figure 2). The increase in concentrations of active t-PA in this subset of Type II diabetic patients paralleled an increase in concentrations of total t-PA antigen, whereas PA1 remained constant (Table I). These changes are probably not related to a changed metabolic state during the study, as described previously [15]. In the other subset of Type II diabetic patients with marked concentrations of active t-PA during treatment with tolbutamide, we
1991 The American Journal of Medicine
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did not observe a change in concentrations of active t-PA, total t-PA antigen, or PA1 during the study. Combining our results on Type I and Type II diabetic patients, we have obtained suggestive evidence that gliclazide (or some of its metabolites) has a non-insulin-mediated potential to increase the blood concentrations of total t-PA antigen without affecting PA1 levels. It should be noted that t-PA is produced by the endothelial cells [19], but whether the changes in t-PA-related fibrinolysis reported here reflect an improved function of endothelial cells or a change in removal mechanisms in the diabetic patients should be examined in further detail in prospective clinical trials.
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35: 173-8. 10. Jespersen J, Knudsen LH, Sidelmann J. The use of evacuated glass tubes for collection of blood samples for fibrinolytic assays. Thromb Res 1982; 25: 173-6. 11. Poulsen JH, Jespersen J. A comparison of the determination of glycosylated hemoglobin by isoelectric focusing and cation-exchange chromatography on mmicolumns. Stand J Clin Lab Invest 1986; 46: 259-63. 12. Verheijen JH, Muellaart E, Chang GTG, Kluft C. A simple sensitive spectrophotometric assay for extrinsic (tissue-type) plasminogen activator appltcable to measurement In plasma. Thromb Haemost 1982; 48: 266-9. 13. Gram J, Jespersen J. A simplified esbmation of tissue plasminogen acbvator (t-PA) inhibition in human plasma. Fibrinolysis 1987; 1: 33-7. 14. Gram J, Jespersen J, Kold AA. Effects of an oral antidiabetic drug on the fibrlnolytic system of blood in insulin-treated diabetic patients. Metabolism 1988; 37: 937-43. 15. Gram J, Kold AA, Jespersen J. Rise of plasma t-PA fibrinolytic activity in a group of maturity onset diabetic patients shifted from a first generation (tolbutamrde) to a second sulphonylurea (gliclazide). J Intern Med 1989; 225: 241-7. 16. Brownlee M, Vlassara H, Ceramr A. Nonenzymatic glycosylahon reduces the susceptibility of fibrin to degradation by plasmin. Diabetes 1983; 32: 600-4. 17. Geiger M, Binder BR. Plasminogen activation in diabetes mellitus. J Biol Chem 1984; 259: 2676-81. 18. Auwerx J, Bouillon R, Collen D, Geboers J. Tissue-type plasminogen activator antigen and plasminogen activator inhibitor in diabetes mellitus. Atherosclerosis 1988; 8: 68-72. 19. Rijken DC, Wijngaards G, Welbergen J. Relationship between tissue plasminogen activator and the activators in blood and vascular wall. Thromb Res 1980; 18: 815-30. 20. Kuo B-S, Korner G, Bjornsson TD. Effects of sulfonylureas on the synthesis and secretion of plasminogen activator from bovine aortic endothelial cells. J Clin Invest 1988; 81: 730-7. 21. Gram J, Jespersen J. A selective depression of tissue plasminogen activator (t-PA) activity in euglobulins characterises a risk group among survivors of acute myocardial infarction, Thromb Haemost 1987; 57: 137-9. 22. Gram J, Jespersen J, Kluft C, Rijken D. On the usefulness of fibrinolysis variables in the characterization of a risk group for myocardial infarction. Acta Med Stand 1987; 221: 149-63. 23. Hamsten A, de Faire U, Wellelius G, et al. Plasminogen activator inhibitor in plasma: a risk factor for recurrent myocardial infarction. Lancet 1987; 2: 3-8. 24. Aznar J, Estelles A, Tormo G, et al. Plasminogen activator inhibitor activity and other fibrinolytic variables in patients with coronary artery disease. Br Heart J 1988; 59 535-41.
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