Abstract: Background and objective: Disturbance in glucose homeostasis is one of the serious outcomes of using fluoroquinolones in diabetes. The study aimed to assess the risk of severe hypoglycemia and oxidative stress among diabetic rats received ciprofloxacin or levofloxacin with glimepiride and to determine the involvement of calcium ion in their actions. Methodology: Male Wistar rats were classified into 5 groups. The 1st group served as control animals, meanwhile, the remaining rats were rendered diabetic by streptozotocin (STZ, 50 mg kg–1, i.p.). Group II received saline (diabetic control), groups III-V received glimepiride (0.5 mg kg–1, p.o.) alone or combined with ciprofloxacin (40 mg kg–1, i.p.) or levofloxacin (50 mg kg–1, i.p.) daily for 2 weeks. Blood and liver samples were collected for estimation of blood lactate dehydrogenase and superoxide dismutase activities and levels of glucose, insulin, lipid peroxides and reduced glutathione, as well as liver glycogen content. In an in vitro experiment, isolated islets of Langerhan’s were incubated with 100 μM of ciprofloxacin or levofloxacin, with or without 50 μM verapamil in presence of basal (3 mM) or stimulatory (16.7 mM) glucose concentration for 1 h to assess their effects on insulin release. Results: Glimepiride reduced diabetic-associated changes in carbohydrate metabolism and oxidative stress. fluoroquinolones augmented the effect of glimepiride on serum glucose and insulin levels as well as liver glycogen, meanwhile, they antagonized glimepiride effect on oxidative stress biomarkers. In addition, fluoroquinolones increased insulin release in in vitro study and their effects were antagonized by verapamil. Conclusion: Fluoroquinolones may augment the risk of glimepiride-induced hypoglycemia as they increased insulin release by a mechanism involve activation of calcium influx. In addition, they counteract the improvement of oxidative stress biomarkers caused by glimepiride, therefore, they may increase diabetic-associated changes of oxidative stress in diabetic patient.
INTRODUCTION
Fluoroquinolones antibacterial agents have a broad spectrum of activity against Gram-positive and Gram-negative bacteria and have been widely used for the treatment of infectious diseases1.
Disturbances in glucose homeostasis are increasingly recognized as one of the most relevant adverse effects of fluoroquinolones2. Their glycemic abnormalities are not fully illustrated where both hypoglycemic and hyperglycemic episodes were reported3-6. The hypoglycemic episodes were life-threatening and required hospitalization7.
Oxidative stress is currently suggested as mechanism underlying diabetes and diabetic complications8. Previous studies revealed that fluoroquinolones induced oxidative stress by producing Reactive Oxygen Species (ROS) that may play a role in their adverse effects9.
The objective of this study was to investigate the effect of concurrent administration of ciprofloxacin or levofloxacin "The most prescribed quinolones" with glimepride "A drug of sulphonylurea" on carbohydrate metabolism as well as oxidative stress in STZ-induced diabetic rats. Another goal of this study was to determine the effect of ciprofloxacin and levofloxacin on insulin release from rat isolated islets of langerhan’s, in addition to their effects in the presence of "Calcium channel blocker" verapamil to explore the importance of the role of calcium in their action.
MATERIALS AND METHODS
Animals: Male albino Wistar rats weighing 150-200 g were used. They were obtained from the animal house of Faculty of Veterinary Medicine, Cairo University and were housed for accommodation for at least 1 week in the laboratory room prior experiment under standard housing condition (room temperature 24-27°C and 60% humidity with alternating 12 h light and dark cycles). Animals were fed standard laboratory pellet with water ad libitum. All animals procedures were performed in accordance to Ethics Committee of Faculty of Pharmacy, Cairo University.
Materials: Glimepiride tablets and levofloxacin vials were purchased from Aventis Pharm (Egypt). Ciprofloxacin vial and verapamil ampoule were purchased from Amriya Pharmaceutical industries Co (Alexandria, Egypt). Streptozotocin (STZ), collagenase enzyme, Bovine Serum Albumin (BSA) and HEPES were purchased from Sigma-Aldrich Co (USA). Glimepiride tablets were suspended in 2% tween 80 and streptozotocin was dissolved in 0.1 M citrate buffer (pH 4.5).
Methods
Induction of diabetes: The STZ (50 mg kg–1) was i.p., injected into overnight fasted rats10. Diabetes was confirmed by the presence of glucosuria using glucotest strips for 7 days to ensure persistent diabetes. The equivalent volume of 0.1 M of citrate buffer was injected to control group (non-diabetic rats).
Experimental design: The rats were classified into 5 groups (n = 8). Group I received citrate buffer and served as normal control. The remaining rats were rendered diabetic by i.p., injection of STZ (50 mg kg–1) then treated as follows: Group II received normal saline and served as diabetic control, group III-V received glimepiride (0.5 mg kg–1, p.o.) alone or combined with ciprofloxacin (40 mg kg–1, i.p.) or levofloxacin (50 mg kg–1, i.p.) daily. Treatment with test agents continued for 2 weeks. At the end of treatment period, blood and liver samples were collected for determination of the chosen parameters.
Isolation of islet: Islets were isolated from pancreas of fed male Wistar rats (250-350 g) by collagenase digestion technique11, followed by preincubation for 30 min in Krebs-Ringer bicarbonate buffer gassed with O2/CO2 (19:1) supplemented with 3 mM glucose and BSA12 5 mg mL–1.
For the measurement of insulin release, groups of 3 islets were incubated at 37°C for 1 h in 0.5 mL of solutions gassed with O2/CO2 (19:1) containing (mM): NaCl 120.0, KCl 4.8, CaCl2 2.0, KH2PO4 1.2, MgSO4 1.2, NaHCO3 20.0, HEPES 10.0 and BSA 5 mg mL–1 under the indicated conditions. The pH of the solution was adjusted to 7.4 with 0.1 N NaOH. Islets were incubated with 100 μM of ciprofloxacin or levofloxacin at 3 m M glucose or 16.7 mM glucose with or without 50 μM verapamil for 1 h and insulin content in the medium was determined.
Biochemical estimations: The serum level of glucose was determined by the glucose oxidase method according to the method described by Trinder13 using glucose kit (Spinereact, Spain). Serum insulin level was determined by enzyme immunoassay using insulin kit (Alpco-diagnostic, USA) according to the method described by Kullin et al.14. Liver glycogen content was estimated according to the method described by Kemp and van Heijningen15.
Serum lactate dehydrogenase (LDH) and blood SOD activities were estimated using commercial reagent kits according to the methods described by Friedman and Young16 and Nishikimi et al.17, respectively. Blood glutathione (GSH) level was estimated using Ellman’s reagent according to the method described by Beutler et al.18, meanwhile serum malondialdehyde (MDA) level was estimated according to the method of Satoh19.
Statistical analysis: Data were expressed as Mean±Standard Error (SE) and statistical significance was analyzed by one way ANOVA followed by least significant difference (LSD) test. The p-values of <0.05 were considered as statistically significant.
RESULTS
Effect of administration of glimepiride and ciprofloxacin or levofloxacin on serum glucose, serum insulin and liver glycogen content in diabetic rats: The STZ (50 mg kg–1, i.p.) significantly increased serum glucose level to 362.99% and decreased serum insulin level and liver glycogen content to 36.01 and 37.00%, respectively as compared to the normal control value. Administration of glimepiride (0.5 mg kg–1, p.o.) significantly decreased serum glucose level to 52.22%, meanwhile, it increased serum insulin level and liver glycogen content to 174.61 and 152.36%, respectively as compared to diabetic control value.
Concurrent administration of ciprofloxacin (40 mg kg–1, i.p.) or levofloxacin (50 mg kg–1, i.p.) with glimepiride significantly decreased serum glucose level to 29.57 and 31.95%, respectively as compared to diabetic control value and 56.62 and 61.18%, respectively as compared to glimepiride treated group. In the same context, combination of ciprofloxacin or levofloxacin with glimepiride increased serum insulin level to 226.47 and 221.96%, respectively as compared to diabetic control value and 129.71 and 127.12%, respectively as compared to glimepiride treated group. The same combination regimens increased liver glycogen content to 254.72 and 233.96%, respectively as compared to diabetic control value and 167.18 and 153.56%, respectively as compared to glimepiride treated group (Table 1).
Effect of administration of glimepiride and ciprofloxacin or levofloxacin on serum LDH, serum MDA, blood GSH and blood SOD in diabetic rats: The STZ significantly increased serum LDH activity and MDA level to 229.75 and 177.64%, respectively and decreased blood GSH level and SOD activity to 57.28 and 54.67%, respectively as compared to normal control group. Administration of glimepiride significantly decreased serum LDH activity and serum MDA level to 70.20 and 70.41%, respectively and raised blood GSH level and SOD activity to 146.55 and 156.63%, respectively as compared to diabetic control. Concurrent administration of ciprofloxacin or levofloxacin with glimepiride significantly raised serum LDH activity to 137.97 and 144.54%, increased serum MDA level to 176.81 and 150.00%, decreased blood GSH level to 58.47 and 60.19% and decreased blood SOD activity to 76.02 and 72.39%, respectively as compared to glimepiride treated group (Table 2).
Effect of ciprofloxacin, levofloxacin and verapamil on basal (3 mM) and stimulated (16.7 mM) glucose on insulin secretion from isolated pancreatic islets of male rats: In the presence of 3 mM glucose, the mean normal control value of basal insulin released from 3 islets for 1 h was 2.05±0.22 ng h–1 per islet. When verapamil in concentration of 50 μM was added, basal insulin secretion was significantly reduced to 4.39% of the control value. Addition of ciprofloxacin or levofloxacin in concentrations of 100 μM significantly increased basal insulin secretion to 152.20 and 169.76%, respectively as compared to control value. Combination of ciprofloxacin or levofloxacin with verapamil significantly decreased basal insulin secretion to 8.78 and 10.73%, respectively as compared to control value (Fig. 1).
In the presence of glucose (16.7 mM), insulin release was significantly increased to 7.65±0.32 ng h–1 per islet. Verapamil in concentration of 50 μM significantly decreased glucose (16.7 mM) stimulated insulin secretion to 9.15% of the control value. Ciprofloxacin and levofloxacin in concentrations of 100 μM significantly increased glucose (16.7 mM) stimulated insulin secretion to 194.38 and 164.31%, respectively as compared to control value.
Combination of ciprofloxacin or levofloxacin with verapamil significantly decreased glucose (16.7 mM) stimulated insulin secretion to 17.90 and 16.08%, respectively as compared to control value (Fig. 2).
DISCUSSION
Hypoglycemia is one of the serious adverse effects induced by fluoroquinolones20, particularly in diabetic patient21,22. The previous data reported that levofloxacin at (100 mg kg–1) decreased the serum glucose concentration23. In the same context, gatifloxacin both in vivo and in vitro augmented insulin release24,25.
In present study, concurrent administration of ciprofloxacin (40 mg kg–1, i.p.) or levofloxacin (50 mg kg–1, i.p.) with glimepiride (0.5 mg kg–1, p.o.) significantly decreased serum glucose level and raised serum insulin level and liver glycogen content as compared to diabetic control and glimepiride alone (Table 1). Thus, ciprofloxacin and levofloxacin may potentially enhance the effect of glimepiride and induce hypoglycemia in diabetic patient.
Administration of glimepiride (0.5 mg kg–1) significantly decreased serum LDH activity and MDA levels and increased blood GSH level and SOD activity (Table 2) as compared to diabetic control. These data find support in the study of Rabbani et al.26 and Amin et al.27 who suggested that glimepiride may effectively inhibit the development of the oxidative stress in diabetes. Meanwhile, Talla and Veerareddy28 reported that ciprofloxacin and levofloxacin induce reactive oxygen species and cause cell damage28.
In the present study, concurrent administration of ciprofloxacin or levofloxacin with glimepiride significantly raised serum LDH activity and MDA level and decreased blood GSH level and SOD activity as compared to glimepiride alone (Table 2). The obtained data indicate that ciprofloxacin or levofloxacin with glimepiride raise oxidative stress in diabetic rats and antagonize the effect of glimepiride. Thus, they may increase diabetic-associated changes of oxidative stress in diabetic patient, which it was possible to be improved in case of glimepiride alone.
In the presence o f a basal glucose concentration (3 mM), addition of ciprofloxacin or levofloxacin in concentration of 100 μm significantly initiated insulin secretion from isolated pancreatic islets as compared to control value (Fig. 1). These observations is in some differences to data of Ghaly et al.2 who had found none of the fluoroquinolones stimulated insulin secretion in the presence of a basal glucose concentration (5 mM), but raising the glucose concentration from 5-10 mM revealed the insulinotropic effect. The difference in results may be arised from using of NMRI mice in the study of Ghaly et al.2 while, used Wistar rats.
In the presence of (16.7 mM) glucose, addition of ciprofloxacin or levofloxacin in concentration of 100 μm significantly augmented insulin secretion from isolated pancreatic islets as compared to control value (Fig. 2)12,25.
The obtained data indicate that ciprofloxacin and levofloxacin are initiators themselves to insulin secretion where they stimulate insulin secretion in basal glucose concentration also they have a stimulatory effect as they enhance glucose induced insulin secretion in stimulatory glucose concentration.
The present study showed that verapamil abolished insulin release caused by ciprofloxacin or levofloxacin from pancreatic islets in both basal and stimulatory concentration of glucose as also reported previously12.
This may be refers to that ciprofloxacin and levofloxacin increase insulin release via opening of ca2+ channel, so ca2+ influx was important for the action of ciprofloxacin and levofloxacin. This find supports in the study of Ghaly et al.2 who found that in presence of 10 mM glucose, fluoroquinolones, which enhanced insulin secretion markedly elevated calcium concentration in B-cell from SUR1Ko islets. Also in the presence of 5 mM glucose, gatifloxacin and moxifloxacin at 500 μM elevated calcium. These findings indicated that the hypoglycemia (the insulinotropic effect) of ciprofloxacin and levofloxacin may be mediated via a pancreatic effect.
Novelty of this study, concurrent administration of glimepiride with ciprofloxacin or levofloxacin may potentially increase the risk of glimepiride-induced hypoglycemia in diabetic patient, as well as, they can antagonize the improvement of oxidative stress biomarkers caused by glimepiride. Therefore, they may increase diabetic-associated changes of oxidative stress in diabetic patient.
CONCLUSION
Ciprofloxacin and levofloxacin have insulinotropic effect and may induce hypoglycemia, these findings were confirmed by in vitro study, that ciprofloxacin and levofloxacin augmented insulin release of pancreatic islets and this effect could be mediated via calcium ions.
Fluoroquinolones augmented the effect of glimepiride on serum glucose and insulin levels, as well as liver glycogen, meanwhile, they antagonized glimepiride effects on oxidative stress biomarkers.
Clinicians should be cognizant of the potential adverse effect of life threatening hypoglycemia as well as increased diabetic oxidative stress in patients with diabetes who receive ciprofloxacin or levofloxacin therapy with glimepiride.