INTRODUCTION:

Patients with type 2 diabetes mellitus (T2DM) may have symptoms other than high blood glucose levels. Several comorbidities are present in the majority of patients, necessitating further pharmacological therapy¹. In recent years, class III antiarrhythmic medications, particularly amiodarone (a broad-spectrum antiarrhythmic agent), have acquired favour in clinical practise². In most cases, CYP-mediated interactions occur regardless of drug administration time; hence, timing the administration of implicated drugs is of limited use³. Glibenclamide is one of the most often prescribed sulphonylureas⁴. Sulfonylureas have long been recognised to boost insulin synthesis and glucose utilisation in tissues at the cellular level, lowering blood glucose levels⁵.

CYP3A4 is the enzyme that metabolises glibenclamide the most, followed by CYP2C9, CYP2C19, CYP3A7, and CYP3A5^6,7. Although there are more than 50 CYP450 enzymes, the CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5 enzymes are responsible for 90% of drug metabolism^8,9. Many medication interactions are caused by a change in CYP450 metabolism¹⁰. Drugs have a variety of interactions with the CYP450 system. Drugs may be processed by a single CYP450 enzyme (e.g., metoprolol by CYP2D6) or by numerous enzymes (e.g., warfarin [Coumadin] by CYP1A2, CYP2D6, and CYP3A4)¹¹.One or more CYP450 enzymes are inhibited by inhibitors. The degree to which an inhibitor influences a drug's metabolism is determined by factors such as the inhibitor's capacity to bind to the enzyme and its dosage¹². If a person is a poor metabolizer or has a CYP450 enzyme inhibitor added to therapy, standard therapeutic dosages may elicit undesirable effects associated to higher drug serum levels^13,14. Amiodarone is an antiarrhythmic medication having mostly Class III (Vaughan-Williams classification) effects¹⁵. Numerous cytochrome P450 pathways, including CYP 2C9 (which metabolises warfarin [Coumadin]), CYP 2D6 (which metabolises several beta-blockers and narcotics), and CYP 3A4 (which metabolises several beta-blockers and narcotics), limit amiodarone metabolism¹⁶.

The purpose of this study is to see if hypoglycemia drugs like Glibenclamide and anti-arrhythmic drugs like amiodarone interact with one another in healthy and diabetic rats. The basic limitations tested for the interaction between the drugs previously described are the influence of amiodarone on Glibenclamide at the start, duration, and peak of hypoglycemia. Glibenclamide and amiodarone might interact, but additional research is needed to see if this is true.

MATERIAL AND METHODS:

Animals:

For this study, we employed a total of 18 rats (both genders) procured from LACSMI BioFarms .

Method for oral administration:

A feeding needle and a 1 mL glass syringe are required for oral medicine administration, both of which may be acquired at space Labs in Nasik. Flexible polythene tubing was used to cover and dull the tip of the 18-gauge needle to protect it. A 1 ml tuberculin syringe was then linked to the needle. The rats had to be held in place with their heads slightly angled to the left, glycerin-soaked tubes inserted into their throats, and medications administered by gently pressing the plunger. This ensured that the appropriate dose was given.

Method for blood sampling:

To put the rats to sleep, they were placed in a sedative chamber and administered sedative ether. The rat was taken out of the anaesthetic chamber after a brief anaesthesia. The animal's tail vein is widened by squeezing it with xylene, then cutting off its tip and collecting blood in epindroff tubes with an anticoagulant solution (proportion of sodium fluoride to potassium oxalate, which is 1:3).

Estimation of blood glucose:

Trinder's GOD/POD technology¹⁷, created in 1964, is one of the company's most cutting-edge tactics. Easy, step-by-step, fast, trustworthy and acceptable: these are the qualities of this approach's procedure. Despite the higher operational time and expense, central laboratory glucose testing provides a more accurate means of patient diagnosis and therapy¹⁸. In this study, this strategy was employed as a consequence.

Effect of pre-treatment of Amiodarone on Glibenclamide hypoglycemic activity in healthy albino rats:

During the study's initial phase, healthy albino rats were demonstrated to exhibit hypoglycemic effects by glibenclamide. The hypoglycemic action of Glibenclamide 1.03 milligram/kg on the same animals was shown to be affected by amiodarone 50 milligram/kilogram every day for a week.

Experimental Procedure:

Healthy Albino Rats:

The experiment utili LACSMI BioFarms LACSMI BioFarms sed a total of 6 albino rats, both sexes, weighing between 150 and 180 grammes. They'd been labelled to make tracing easier. According to industry regulations, the animals were housed in colony cages. The meal was taken away 18 hours before the start time on the day before the experiment. Unrestricted access to water, on the other hand, was given. Fasting was required for the length of the research. Blood was drawn from the tail vein (0.5 ml each rat) for testing to ensure that the baseline glucose level was met.The animals were then given Glibenclamide suspension 1.03 milligram/kilogram orally in the first portion of the trial. Blood was collected from a tail vein and evaluated for blood glucose levels using the GOD/POD technique at 0.5, 1, 2, 4, 6, 8, 12, 18, and 24 hours. The same animals were employed in the second half of the investigation, and Amiodarone in a 2 percent gum acacia solution was administered for 7 days. The rats fasted for 18 hours on the seventh day after receiving Amiodarone. Fasting continued until all of the findings were received. On the eighth day, one hour after receiving amiodarone 50 milligram/kilogram, the same animals were given Glibenclamide 1.03 milligram/kilogram.Following that, blood samples (0.5 ml) were collected at various time intervals and analysed using the GOD/POD method to determine glucose concentration. At time "t," this condition was utilised to determine how rapidly blood glucose levels fell.

A - B

% Blood sugar reductionat time ‘t’ ---------------- x100

Where A = Pre-drug administration initial blood glucose content.

B = After drug administration, levels of blood glucose at the time"t".

Diabetic Rat:

When Amiodarone is utilised in rats, the efficiency of anti-diabetic medications in pathological circumstances like diabetes mellitus is uncertain. In order to demonstrate this viewpoint, diabetic rats will be employed as test subjects in the current study. Glibenclamide 1.03 milligram/kg was found to have a substantial influence on peak hypoglycemia when given before Amiodarone (50 milligram/kg).

Experimental diabetes induction methods:

Induction of diabetes:

To induce diabetes, albino rats of both sexes were employed. The intraperitoneal approach was used to deliver 100 milligrammes per kilogramme of freshly synthesised alloxan monohydrate, followed by 50 milligrammes per kilogramme of body weight the next day. To prevent hypoglycemia from arising straight soon, 10 percent dextrose was given. The study employed fasting glucose levels of rats more than 250 mg/dL to measure glucose and conduct the investigation.

Experimental procedure:

Alloxan was given to diabetic male and female rats for 48 hours. Six animals were chosen using a random selection method. The rats were housed in large colony cages under regular husbandry settings. The animals had to fast for 18 hours before to the experiment. The rats had unrestricted access to water during this time. There was no food provided for the duration of the trial. Blood samples were used to calculate fasting blood glucose levels. During the first portion of the investigation, rats were given oral doses of Glibenclamide 1.03 milligram/kilogram suspension. GOD/POD methods were used to monitor blood glucose levels after samples were obtained on a regular basis.The same rats were utilised in the second half of the trial, and all of the rats were given amiodarone 50 milligram/kilogram orally seven days in a row. After taking amiodarone for 6 hours, the rats were hungry for 18 hours on the 7th day. On the seventh day, following 60 minutes of Amiodarone 50 milligram/kilogram oral delivery, Glibenclamide 1.03 milligram/kilogram was administered orally. The GOD/POD approach was used to analyse blood samples obtained at intervals of 0, 0.5, 1, 2, 4, 6, 8, 12, 18, and 24 hours. Blood glucose levels were measured in milligrammes per deciliter (mg/dL). Glibenclamide's anti-diabetic effects and the percent of blood glucose decrease at various time intervals before and after treatment with Amiodarone were assessed, the results were compared.

A - B

% Blood sugar reductionat time ‘t’ = ---------- x100

Where A = Pre-drug administration initial blood glucose content.

B = After drug administration, levels of blood glucose at the time"t".

RESULTS:

The following parameters were used to evaluate glibenclamide-induced hypoglycemia: onset of hypoglycemia (time required to reduce blood glucose levels by 15% -20%), duration of hypoglycemia (time required to maintain blood glucose levels by 15% - 20% for an extended period of time), and peak hypoglycemia.

Amiodarone Pre-Treatment Effect on Glibenclamide Hypoglycemic Effect in Healthy Albino Rats:

The onset of hypoglycemia was significantly altered by pre-treatment with amiodarone (50 milligram/kilogram) in this trial (17.47±0.18% before treatment to 31.05±0.31% increase after treatment at 1^sthr), peak hypoglycemia was markedly increased (42.16±0.46% before treatment to 59.00±0.15% after treatment at 6^thhour. Meanwhile, the duration of hypoglycemia was 11.78±0.39% before therapy and 23.87±0.57% after therapy at 24-hour treatment.

Table no. 1 provide the findings, which are schematically shown in figure no. 1.

Table 1: Percentage blood glucose level reduction in healthy albino rats before and after treatment with Glibenclamide (1.03 milligram/kilogram) and Amiodarone (50 milligram/kilogram).

Time in (h)	Glibenclamide (G) blood glucose levels (mg/dl)	Amiodarone (A) blood glucose levels (mg/dl)	Glibenclamide (G) + Amiodarone (A) blood glucose levels (mg/dl)
Time in (h)	Mean ± SEM	Mean ± SEM^**	Mean ± SEM^*
0	1.72±0.14	0.55±0.07	5.14±0.39
½	7.83±0.22	1.34±0.11	23.00±0.22
1	17.47±0.18	2.54±0.15	31.05±0.31
2	23.85±0.39	3.11±0.11	41.43±0.30
4	32.97±0.27	4.34±0.17	49.30±0.19
6	42.16±0.46	5.52±0.14	59.00±0.15
8	34.32±0.55	6.43±0.06	48.13±0.58
12	28.32±0.43	4.97±0.17	39.91±0.46
18	20.79±0.55	3.98±0.16	32.54±0.58
24	11.78±0.39	3.05±0.17	23.87±0.57

G+ A: Glibenclamide+Amiodarone; SEM: Standard error of the mean

Mean±SEM; *** Significant at P<0.001; ** Significant at P<0.01; * Significant at P< 0.05 compared to glibenclamide control

Figure 1: Percentage Blood glucose reduction with Glibenclamide (1.03mg/kg) in healthy Albino rats before and after Amiodarone treatment (50 mg/kg)

Effect of Amiodarone pre-treatment on Glibenclamide's anti-diabetic effects in diabetic rats:

In prior investigations, pre-treatment with Amiodarone improved the hypoglycemic effect of Glibenclamide in healthy albino rats. The effect of Amiodarone pre-treatment on the same drugs was investigated in albino rats that had been intentionally made to acquire diabetes as a consequence of this study. In this study, pre-treatment with Amiodarone 50 milligram/kilogram significantly exacerbated the onset of hypoglycemia. Peak hypoglycemia was significantly raised (before treatment 16.77±0.23 percent and grew to 29.72±0. 63 percent after treatment at 1st hour), and peak hypoglycemia was significantly increased (before treatment 42.600±38% and rose to 58.610±52 percent after treatment at 6th hour). Meanwhile, the duration of hypoglycemia was 11.680±38% before treatment and improved to 23.190±29% after 24 hours of treatment. The results are presented in tables 2 and visually in figure 2.

Table 2: Percentage blood glucose levels in diabetic albino rats before and after treatment with Glibenclamide (1.03 milligram/kilogram) and Amiodarone (50 milligram/kilogram).

Time in hr	Glibenclamide (G) blood glucose levels (mg/dl)	Amiodarone (A) blood glucose levels (mg/dl)	Glibenclamide (G) + Amiodarone (A) blood glucose levels (mg/dl)
Time in hr	Mean ± SEM	Mean ± SEM^**	Mean ± SEM^*
0	1.75±0.09	0.52±0.05	5.46±0.31
½	7.14±0.27	1.26±0.05	21.98±0.72
1	16.77±0.23	2.29±0.06	29.72±0.63
2	22.89±0.22	3.63±0.05	40.48±0.58
4	32.57±0.26	4.61±0.06	49.05±0.33
6	42.60±0.38	5.89±0.10	58.61±0.52
8	34.65±0.51	6.66±0.07	47.43±0.46
12	28.90±0.37	6.08±0.05	39.52±0.55
18	20.83±0.36	4.72±0.11	31.65±0.40
24	11.68±0.38	3.43±0.06	23.19±0.29

G+ A: Glibenclamide+Amiodarone; SEM: Standard error of the mean

Mean±SEM; *** Significant at P<0.001; ** Significant at P<0.01; * Significant at P< 0.05 compared to glibenclamide control

Figure 2: Percentage Blood glucose reduction with Glibenclamide (1.03mg/kg) in healthy Albino rats before and after Amiodarone treatment (50 mg/kg)

DISCUSSION:

Anti-arrhythmic medicines are typically used in conjunction with oral anti-diabetes medications since patients with diabetes are more likely to have cardiovascular issues¹⁹. According to published data, when amiodarone was given to rats repeatedly, it caused an increase in the number of inactive cytochrome P-450-Fe (ll) metabolite complexes^20,21. According to published data, when amiodarone was given to rats repeatedly, it caused an increase in the number of inactive cytochrome P-450-Fe (ll) metabolite complexes²². In certain individuals taking sulphonylurea treatment, toxic responses (such as allergies, hyponatraemia, elevated liver enzyme activity, and hepatocellular or cholestatic jaundice) have been reported in addition to hypoglycemia²³. Patients with T2DM are predisposed to drug-drug interactions because to the intricacy of their treatment²⁴.

When Amiodarone and oral antidiabetic medicines are taken together under healthy circumstances, we found that DDI occurs. But in pathophysiological illnesses like diabetes, the link remained unclear. Consequently, in the second stage of our investigation, Alloxan-induced diabetic rats were used and Glibenclamide was administered to diabetic animals and the onset, duration, and peak antidiabetic activity were evaluated in hypoglycemic animals. Amiodarone (50 milligram/kilogram for one week) substantially increased the development of hypoglycaemia in the same rats as did oral antidiabetic drug Glibenclamide (1.03 milligram/kilogram) in the same animals. (from16.77±0.23% to 29.72±0.63% at 1 hour) and the peak hypoglycaemia was considerably increased (from 42.60±0.38% to 58.61±0.52%, at 6^th hr) and duration of hypoglycemia was also increased from 11.68±0.38% before treatment to 23.19±0.29% after treatment, induced by Glibenclamide (Table 2, Figure 2)

Diabetic patients are likely to suffer from cardiovascular disorders and hence most often anti-arrhythmic are co-administered along with oral anti-diabetic drugs. Use of sulphonylureas carries a high risk of hypoglycaemia even when low doses are used^25-28. Use of sulphonylureas carries a high risk of hypoglycaemia even when low doses are used. Diabetic patients are likely to suffer from cardiovascular disorders and hence most often anti-arrhythmic are co-administered along with oral anti-diabetic drugs^29-34. Amiodarone had no impact on blood glucose levels in healthy albino or diabetic rats. Diabetic patients are likely to suffer from cardiovascular disorders and hence most often anti-arrhythmic are co-administered along with oral anti-diabetic drugs^35-36.

CONCLUSION:

Amiodarone had no impact on blood glucose levels in healthy albino or diabetic rats. According to these findings, Amiodarone has no hypoglycemia impact, indicating that glibenclamide and Amiodarone have a pharmacokinetic type of drug interaction. The onset of hypoglycemia with glibenclamide was increased in healthy albino rats treated with Amiodarone for one week, although peak hypoglycemia and hypoglycemic duration were both enhanced. Furthermore, in diabetic rats, one week of amiodarone pre-treatment increased hypoglycemia's onset while increasing its peak and duration when combined with glibenclamide.

The inhibition of CYP3A4 and CYP2C8 isoenzyme pathways by amiodarone may allow the hypoglycemic effects of oral antidiabetic medications to be amplified. This data suggests that when Glibenclamide and Amiodarone are taken simultaneously, blood sugar monitoring is necessary. When Amiodarone is used with oral antidiabetic medicines, the dosage and frequency of these medications should be changed as well.

ACKNOWLEDGEMENT:

The authors are thankful to DCS’s A.R.A. College of Pharmacy, India for providing an experimental facility.

CONFLICTS OF INTEREST:

The authors declare no conflict of interest.

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Received on 04.03.2022 Modified on 10.06.2022

Research J. Pharm. and Tech 2023; 16(3):1134-1138.

DOI: 10.52711/0974-360X.2023.00189