Clinical Studies of Tolazamide and Tolbutamide: Comparative Effectiveness of Control of Diabetes Mellitus

Tolazamide, a new oral hypoglycemic agent, was compared with tolbutamide, a related chemical compound, for stability of control of 12 patients suffering from maturity-onset diabetes mellitus. A short 12-week study was conducted which incorporated a cross-over design and the results were examined by variance analysis after dosage was individualized to the patient''s requirements. Greater stability of fasting blood sugar was found on tolazamide; patients also had less glycosuria and lower fasting blood sugar on tolazamide. Tolazamide appeared to be between five and six times as potent as tolbutamide, mg. for mg.No hepatic, renal, hematologic or symptomatic toxic reactions were observed during the total of 72 person-weeks of tolazamide therapy.     

Glucose and Insulin Secretory Response Patterns Following Diet and Tolazamide Therapy in Diabetes

Glucose and insulin secretory response patterns during glucose tolerance tests were determined in 28 maturity-onset diabetics, and the sequential effects of diet and a sulphonylurea, tolazamide, were assessed. Untreated diabetics showed hyperglycaemia, increased serum immunoreactive insulin response patterns, delayed insulin release, and relative insulin deficiency. Diet alone partially corrected the hyperglycaemia and serum immunoreactive insulin response but had no effect on the delayed insulin release or relative insulin deficiency. Tolazamide plus diet restored all values towards normal. The net effect of maintenance tolazamide therapy was to (1) restore the insulin secretory response pattern to normal, (2) reduce total pancreatic insulin output, and (3) improve the efficiency of insulin secretion. The results suggest that there is a rational basis for the use of sulphonylurea in all maturity-onset diabetics, including patients with mild carbohydrate intolerance and those who are apparently controlled by diet alone.     

Adjunctive use of tolazamide in newly-diagnosed diabetic children

Recent data suggests that one of the major actions of sulfonylureas is to potentiate the anabolic cellular effects of insulin. This is the first study to examine the use of sulfonylureas as adjunctive therapy in newly-diagnosed type I diabetic children. A random, prospective, double blind study over 15 months, stratified by age at diagnosis, was conducted. The treatment group (n = 13) received daily oral weight-adjusted tolazamide whereas the control group (n = 11) received placebo. Monthly comparison of the HbA1 values between groups revealed no statistical difference; likewise, the fasting serum C-peptide values were not dissimilar. The mean daily insulin dose per kilogram, however, was less in the tolazamide group (P less than 0.001). The data suggests that the addition of tolazamide may not be of therapeutic benefit in newly diagnosed juvenile diabetics, although insulin requirements may be reduced.     

The effect of various hypoglycaemic sulphonylureas on the cardiotoxicity of glycosides and arrhythmogenic activity due to myocardial ischaemia

The effects of different sulphonylureas on the electrical cardiac activity were studied in 145 rabbits and in 103 rats as well as in 278 digitalis-treated, non-smoker non-insulin-dependent diabetics on the same therapy at least during the previous three months. In rabbits and rats glibenclamide (0.0032-100 mumol. kg-1) decreased, while tolbutamide and carbutamide (0.008-1000 mumol. kg-1) increased strophantidin toxicity and myocardial ischaemia induced transitory ventricular fibrillation dose-dependently. The differences between the dose-response curve of glibenclamide and those of tolbutamide or carbutamide were significant. In digitalized non-insulin-dependent diabetics, multifocal ectopic ventricular beats could be observed in none among the 80 glibenclamide-treated diabetics, while in 12 cases of the 71 tolbutamide and in 10 cases of the 61 carbutamide treated diabetics. Two of the 66 non-insulin-dependent diabetics receiving only diet and 7 of the 278 age and sex matched, non-smoker, metabolically healthy patients had multifocal ectopic ventricular beats. No significant difference could be found between the therapeutical groups. It was concluded that instead of tolbutamide, glibenclamide must be preferred in digitalis-treated diabetics, when metabolic control is not satisfactorily achieved by diet and regime alone.     

Characterization of the sulfonylurea receptor on beta cell membranes

Specific, high affinity sulfonylurea receptors were characterized on membranes of an insulin-secreting hamster beta cell line (HIT cells). Saturable binding of the sulfonylurea, [3H]glyburide, was linear up to 0.8 mg/ml membrane protein. Scatchard analysis of equilibrium binding data at room temperature indicated the presence of a single class of saturable, high affinity binding sites with a Kd of 0.76 +/- 0.04 nM and a Bmax of 1.09 +/- 0.13 pmol/mg protein, n = 9. The insulin secretory potency of glyburide, glipizide, tolbutamide, tolazamide, and carboxytolbutamide was compared to the ability of these ligands to displace [3H]glyburide from the sulfonylurea receptor. Tolbutamide, tolazamide, and glipizide demonstrated reasonable agreement with ED50 values of 15 microM, 3 microM, and 30 nM and Ki values of 25.3 microM, 7.2 microM, and 45 nM, respectively. The inactive tolbutamide metabolite, carboxytolbutamide, at the highest concentration tested, only partially displaced [3H]glyburide from the receptor and was a very poor secretagogue. At 37 degrees C the affinity of [3H]glyburide binding, Kd = 2.0 nM, was similar to the ED50 of 5.5 nM when the free glyburide concentrations were corrected for binding of the drug to albumin. These studies suggest that sulfonylureas initiate their biologic effect through a high affinity, specific interaction with sulfonylurea receptors on the beta cell membrane.     

A Comparison of the Effects of Tolazamide and Tolbutamide Upon Blood Glucose and Serum Insulin and Lipid Levels in Diabetic Subjects

The effects of tolazamide (300 mg. daily), a new oral hypoglycemic sulfonylurea, and tolbutamide on the blood glucose, serum insulin, cholesterol and triglyceride levels were compared in 14 subjects with maturity-onset diabetes of varying severity. The mean effects of the two drugs in the pharmacologically equivalent doses were the same. In particular, the mean reduction of the blood glucose level was 19% and of the serum triglyceride level was 17% with both agents. However, an individual subject might respond to one agent and not to the other; neither the blood glucose nor the serum lipid response could be predicted from the pretreatment blood glucose level or the per cent of ideal body weight.     

Effect of physiological concentrations of insulin and antidiabetic drugs on RNA release from isolated liver nuclei

The addition of 10(-11) M insulin to a cell-free system from rat liver promotes the release of messengerlike RNA from isolated prelabeled nuclei. The stimulation was similar whether the nuclei were preincubated with insulin, or if insulin was added directly to the cell-free system with or without a protease inhibitor. Dot blot hybridization using cloned cDNA for alpha 2u-globulin mRNA showed that this was one of the messages whose release was enhanced by insulin. Nuclei isolated from rats treated with either of the antidiabetics tolbutamide or tolazamide showed no increase in RNA release in the presence of insulin over the concentration range 10(-5) - 10(-14) M. Furthermore, these nuclei did not release detectable levels of alpha 2u-globulin mRNA.     

Sulfonylurea binding to adipocyte membranes and potentiation of insulin-stimulated hexose transport

We have previously shown that the sulfonylureas increase insulin-stimulated glucose transport in adipocytes mainly by enhancing the insulin-induced recruitment of glucose transporter from its intracellular storage pool to the plasma membrane (Jacobs, D. B., and Jung, C. Y. (1985) J. Biol. Chem. 260, 2593-2596). In order to determine if this sulfonylurea effect is mediated by a specific membrane-associated sulfonylurea-binding protein, in the present report we measured exact dose dependence of the transport enhancement activities of different sulfonylureas in adipocytes in primary culture and equilibrium binding affinities of these agents to various adipocyte membrane fractions. Glycuride was found to increase the insulin-stimulated, 3-O-methyl-D-glucose equilibrium exchange in cultured rat adipocytes by up to 60% with little effect in the absence of insulin. The effect developed gradually reaching the maximum level at 24 h of incubation. The effect was concentration dependent showing a simple, one-to-one stoichiometry and an apparent activation constant (Ka) of approximately 1 microM. Glypizide, tolazamide, and tolbutamide also enhanced the insulin-stimulated hexose transport by up to 60%, but with Ka of approximately 2, 11, and 25 microM, respectively. HB-699 and ciglitazone, non-sulfonylureas, were without effect under the same condition. In equilibrium binding experiments, [3H]glyburide was found to bind to adipocyte membranes at two or more protein-specific, saturable sites, with similar apparent dissociation constants (KD) ranging 1-3 microM. These protein-specific glyburide bindings were displaced not only by tolazamide and tolbutamide, but also by ciglitazone and HB-699, with indicated KD of 11-16, 80-85, 20-25, and 85-95 microM, respectively. However, with the plasma membrane fraction, the displacements by ciglitazone and HB-699 were partial and did not exceed 56-61% at maximum. Based on these findings, we propose that there is a sulfonylurea-specific-binding protein in the plasma membrane of adipocytes, and that this sulfonylurea-binding protein may play a key role in the enhancement of insulin-stimulated hexose transport by sulfonylureas, probably via potentiation of the insulin-induced recruitment of glucose transporter.     

Insulin and Non-esterified Fatty Acid Metabolism in Asymptomatic Diabetics and Atherosclerotic Subjects

Glucose, insulin and non-esterified fatty acid (NEFA) metabolism was studied in 18 patients (mean age 49) with ischemic heart disease (IHD) who did not have any concurrent disorder known to affect glucose tolerance.Significant hyperglycemia and hyperinsulinemia were observed in the IHD patients after oral glucose. The serum NEFA declined to a lower level in IHD patients than in normal subjects who received glucose.In response to hypoglycemia following the oral administration of sodium tolbutamide the serum NEFA in IHD patients rose to a higher level in the rebound phase than in normal subjects. This rise was preceded by a sharp decline in the concentration of circulating insulin.In 72% of the patients (IHD sub-group) the blood glucose values after oral glucose satisfied the criteria for the diagnosis of diabetes mellitus. The metabolic changes following oral glucose in the IHD sub-group and in asymptomatic diabetics (AD), free of clinical atherosclerosis and with similar impairment in glucose tolerance, were compared. Despite insignificantly lower insulin concentrations, the AD showed a significantly lesser fall in circulating NEFA than did the patients in the IHD sub-group. After oral sodium tolbutamide the IHD sub-group patients showed a greater insulin response and a greater rebound increase in circulating NEFA than did the AD.These differences in response to oral glucose and to sodium tolbutamide suggest that the pathogenesis of the impaired glucose tolerance in IHD may be different from that responsible for abnormal carbohydrate tolerance in asymptomatic diabetics without evident atherosclerosis. The abnormalities demonstrated in glucose, insulin and NEFA metabolism may play a role in the genesis of the hyperlipoproteinemia and atherosclerosis of IHD. One possible mechanism leading to hyperlipoproteinemia in ischemic heart disease compatible with the data is discussed.     

Screening,library-assisted identification and validated quantification of oral antidiabetics of the sulfonylurea-type in plasma by atmospheric pressure chemical ionization liquid chromatography-mass spectrometry

An atmospheric pressure chemical ionization liquid chromatographic-mass spectrometric (APCI-LC-MS) LC-MS assay is presented for fast and reliable screening and identification as well as precise and sensitive quantification of oral antidiabetics of the sulfonylurea-type (OADs) in plasma. It allowed the specific diagnosis of an overdose situation or a Munchausen syndrome caused by ingestion of OADs. After liquid-liquid extraction, the OADs glibenclamide, glibornuride, gliclazide, glimepiride, glipizide, gliquidone, glisoxepide, tolazamide and tolbutamide were separated using fast gradient elution. After screening and identification in the scan mode using our new LC-MS library, the OADs were quantified in the selected-ion mode. The quantification assay was validated according to the criteria established by the Journal of Chromatography B. All validation data were inside the required limits. The assay is part of a general LC-MS procedure for fast screening, identification and quantification of different toxicologically relevant compounds in plasma and has proven to be appropriate for OADs.     

Inhibitory effect of nicotine and its metabolites on tolbutamide hydroxylation in rat liver microsomes

A simple HPLC/fluorescence method to detect hydroxytolbutamide (a major metabolite of the anti-diabetic drug tolbutamide) has been developed. The effects of nicotine and some of its metabolites on tolbutamide hydroxylation is described. An extraction procedure with diethyl ether was followed by isocratic HPLC analysis of tolbutamide hydroxylation with a binary mobile phase composed of 10 mM monobasic sodium phosphate in methanol (45:55, v/v, apparent pH 2.28). A detection limit of sub-nanogram amounts (0.353 ng) of hydroxytolbutamide was obtained with fluorescence detection at 226 nm for excitation and 318 nm for emission. Overall precision values for hydroxytolbutamide was determined with coefficients of variation of 1.4–4.6% when nanogram levels of the metabolite were analyzed. Differential inhibitory responses were demonstrated for tolbutamide hydroxylation to nicotine and its metabolites. Tolbutamide hydroxylation was apparently inhibited by cotinine and relatively less inhibited by nicotine. Nornicotine, however, caused very little inhibition of tolbutamide hydroxylation. The implication is that nornicotine may not share similar affinity for the substrate binding site for tolbutamide. The results also suggest that heavy smokers may experience reduction in tolbutamide metabolism. The assay system itself will be useful for future studies of tolbutamide, and possibly related sulfonylureas.     

Effect of short-term clofibrate on glucose metabolism and insulin secretion in patients with mild maturity-onset diabetes mellitus

The effect of 1 week clofibrate administration on glucose and insulin responses to oral glucose and to intravenous tolbutamide was evaluated in 21 patients with mild maturity-onset diabetes (fasting plasma glucose 108-152 mg/100 ml). After treatment, oral glucose tolerance and hypoglycaemic effect of tolbutamide were significantly improved; plasma insulin response was reduced after glucose and unmodified after tolbutamide; fasting plasma glucose was also significantly reduced. These findings did not correlate with the observed fall in serum lipids. Short-term clofibrate improves glucose metabolism in mild diabetes irrespective of its effects on lipid metabolism. It is suggested that the drug's action may be mediated by reduced insulin resistance.     

Proliferation of C6 glioma cells is blunted by the increase in gap junction communication caused by tolbutamide.

We have previously reported that tolbutamide prevents the inhibition of gap junction communication in astrocytes. Here, we show that tolbutamide increases gap junction communication and connexin 43 expression in poorly coupled C6 glioma cells. The increase in communication is concurrent with the inhibition of the rate of proliferation due to a block of the progression of C6 glioma cells through the S phase of the cell cycle. The effects of tolbutamide were quantitatively similar to that found after the elevation of intracellular cAMP. Furthermore, the effects of tolbutamide and cAMP were additive. The possible beneficial effect of tolbutamide on gene therapy for gliomas is discussed.     

Demonstration of C-peptide immunoreactivity in various body fluids and clinical evaluation of the determination of urinary C-peptide immunoreactivity.

C-peptide immunoreactivity (CPR) was demonstrated not only in plasma, but in urine, ascites, cerebrospinal fluid and pleural effusion. The concentration of CPR in urine was very high compared with that in the other body fluids and was easy to assay. CPR in urine after glucose administration or tolbutamide injection increased parallel to the change of CPR in plasma and also to that of IRI in normals or diabetics without renal disturbances. The result suggest that the determination of CPR in urine before and after stimulation of insulin secretion could serve as a simple indicator of insulin secretory function of pancreas.     

Effectiveness of hypoglycemic agents in guinea pigs exposed to mixed gamma-neutron radiations

The effect of 200, 1000, and 5000 rads of mixed gamma-neutron radiations on total blood reducing sugar and blood glucose levels in guinea pigs was investigated 2 and 24 hours, and 9, 22, and 60 days postirradiation. In addition, the effectiveness of insulin and tolbutamide in these animals was evaluated before and after irradiation. Glucose increased to a lesser degree and later than did the nonglucose fraction of the blood sugar. Insulin and tolbutamide were at least as effective in irradiated animals as in unirradiated ones except after 5000 rads, when tolbutamide was significantly less effective. These results suggest that: (1) insufficient insulin is released by the pancreas in response to elevated blood sugar levels following irradiation; (2) the pancreas does produce insulin at these times and is able to release it in response to tolbutamide; and (3) a decrease in insulin production occurs following supralethal doses of radiation.     

Successful use of Oral Diazoxide in the Treatment of Severe Toxaemia of Pregnancy

Treatment with oral diazoxide for periods of 4 to 10 weeks in hospital allowed adequate control of hypertension with continuation of fetal growth in four patients with severe toxaemia. Two were insulin-dependent diabetics and one of these suffered from renal failure. In all patients albuminuria diminished markedly, while the fluid retention was controlled with frusemide. Three pregnancies were successfully terminated by lower segment caesarean section and in the remaining case vaginal delivery resulted in the birth of a healthy baby. Control of blood sugars presented no problems in the diabetic patients. Only minimal adjustments of insulin dosage were required. One of the non-diabetic patients developed mild hyperglycaemia which responded to tolbutamide. The babies, ranging in age from 5 to 12 months, have continued to thrive.     

Oral administration of orinase (tolbutamide); clinical observations of effect on nondiabetic and diabetic humans

Ingestion of tolbutamide (Orinase(R)) by nondiabetic humans brought about a maximum reduction in blood sugar within one to two hours. In diabetic persons taking large doses of insulin, or who needed insulin for control, hyperglycemia, ketosis, and increased excretion of glucose in the urine developed when tolbutamide was substituted for insulin or was used before insulin therapy was begun. The only serious toxic manifestation observed was a skin rash in two patients. Successful control of diabetes with tolbutamide was limited to cases in which the disease was of mild, stable type and the patient was 40 or more years of age, of normal weight, and with a previous insulin requirement of 5 to 30 units per day. It was of benefit in 43.5 per cent of all diabetic patients in the series studied and in about 75 per cent of the group that might be referred to as selected. The duration of the disease and the duration of insulin therapy were unimportant in predicting effectiveness for tolbutamide therapy.     

Regulation of muscle fructose 2, 6-bisphosphate levels by sulfonylureas

We examined the effect of sulfonylureas on the level of fructose 2, 6-bisphosphate (F-2, 6-P2) in muscle using a mouse hindlimb flow-through perfusion system. The F-2, 6-P2 level in muscle was increased by adding glibenclamide or tolbutamide in a dose-dependent manner. The stimulatory potency of each drug on F-2, 6-P2 formation was parallel to its hypoglycemic potency. Sulfonylurea stimulation of muscle F-2, 6-P2 formation is thought to be an important extrapancreatic action improving the deranged carbohydrate metabolism in diabetics.     

Effect of tolbutamide on fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase in rat liver

The effects of tolbutamide on the activities of fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase were examined using rat hepatocytes. Tolbutamide stimulated fructose-6-phosphate,2-kinase activity and inhibited fructose-2,6-bisphosphatase activity, resulting in an increase of fructose-2,6-bisphosphate level. Changes in the activities of the enzyme by tolbutamide were due to variation in the Km value, but not dependent on alteration of Vmax. Glucagon inhibition of fructose-2,6-bisphosphate formation resulting from an inactivation of fructose-6-phosphate,2-kinase and an activation of fructose-2,6-bisphosphatase was released by tolbutamide. Tolbutamide stimulation of fructose-2,6-bisphosphate formation through regulation of fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase may produce enhancement of glycolysis and inhibition of gluconeogenesis in the liver.     

Effect of tolbutamide and glyburide on pyruvate kinase flux in isolated rat hepatocytes.

The effects of two representative sulfonylureas, tolbutamide and glyburide, on pyruvate kinase (PK) flux were examined in fasted rat hepatocytes. PK flux was estimated by trapping 14C from NaH14CO3 in a 2 mM lactate pool, accounting for any incomplete trapping by parallel incubations with L-[1-14C]alanine. Glyburide (20 microM) and tolbutamide (1 mM) decreased glucose formation by 34.9% and 54.8%, respectively, from 2 mM lactate. This decrease in glucose formation was associated with a proportional decrease in pyruvate carboxylase (PCOX) flux (32.7% and 50.5%, respectively). Under these conditions, no net change in PK flux was observed. When hepatocytes were preincubated with lactate and/or sulfonylurea addition for 30 min prior to radiolabeling with NaH14CO3, the metabolic state of the cells changed markedly. Glyburide produced a 34.6% decrease in glucose formation and a 31.3% decrease in PCOX flux, but no change in PK flux. In contrast, tolbutamide decreased glucose formation by 12.5% and increased PK flux by 53.2%, but no change in PCOX flux was observed. Such an increase in PK flux may be linked to tolbutamide-mediated increases in fructose-1,6-bisphosphate (F16P) via fructose-2,6-bisphosphate (F26P). These findings demonstrate that tolbutamide and glyburide decrease hepatic glucose production through various alterations in carbohydrate metabolism, depending upon the metabolic state of the cell. In addition, F26P may play a larger role in the hypoglycemic mechanism of action of tolbutamide than glyburide, since pyruvate carboxylase accounted for most of the decrease in glucose formation observed with glyburide and because preincubation with tolbutamide resulted in an activation of PK.