Pharmacodynamic and pharmacokinetic interaction of losartan with glimepiride-metformin combination in rats and rabbits.

OBJECTIVES: The presence of comorbidities such as cardiovascular disease, peripheral vascular disease, and chronic renal disease, or and the prevention of these ailments in diabetics, frequently demands multiple drug treatments, increasing the risk of drug-drug interactions (DDIs). The current study was focused on identifying possible DDIs on concomitant administration of losartan, a drug used to regulate hypertension along with a combination of glimepiride + metformin, widely used to treat diabetes mellitus. Possible pharmacodynamic and pharmacokinetic interactions were observed for, following single-dose as well as multiple-dose treatment protocols in normal and alloxan-induced diabetes in albino Wistar rats and rabbits.
MATERIALS AND METHODS: Blood samples from surviving rats/rabbits obtained through orbital venous sinus bleeding/marginal ear vein bleeding, respectively, at predetermined intervals and put through to biochemical estimations of sugar level in the blood by Glucose oxidase/peroxidase method; insulin levels in serum using the enzyme-linked immunosorbent assay and serum glimepiride levels using the high-performance liquid chromatography. RESULTS AND DISCUSSION: Losartan, when treated as a single drug, resulted in a slight lowering of blood glucose levels in normal rats, diabetic rats and normal rabbits. Hypoglycemic activity of a combination of glimepiride + metformin was enhanced when losartan was co-administered as a single dosage schedule as well as a multiple dose schedule as indicated by a reduced blood glucose level and enhanced levels of insulin in rats as well as in rabbits. Serum glimepiride levels were also higher and pharmacokinetic parameters of glimepiride including mean residence time, Cmax, T1/2, AUMC0-∞, AUMC0-t, and AUC0-∞, were significantly higher, whereas its clearance was decreased in the two regimens of losartan that was followed.
CONCLUSION: It can therefore be concluded, that in diabetics with hypertension as a comorbidity condition, co-administration of losartan with glimepiride + metformin should be avoided or the dosage of a combination of glimepiride + metformin needs to be tittered to avoid recurrence of hypoglycemic episodes.

Introduction

Diabetes, a group of metabolic disorders, is on a rapid rise worldwide and also in India since the past few decades. Recent epidemiological survey indicates that approximately 425 million adults at the age between 20 and 79 years are diabetes with the figures expected to rise exponentially to 629 million by 2045.[1] Etiopathogenic factors contributing to the severity and associated complications include lack of insulin production, its poor action and insulin resistance disturbing carbohydrate, protein and fat metabolism. Uncontrolled diabetes is associated with obesity, oxidative stress, vascular dysfunction, sympathetic excitation, activation of renin-angiotensin system, sodium retention that may ultimately lead to initiation and development of hypertension.[2] Chronic nature of the illness necessitates continues monitoring of glycemic levels and maintaining a tight control over it through treatment regimens along with precautionary measures aimed at avoiding hypoglycemia.

Diabetes with comorbidity hypertension needs treatment regimens aimed at controlling both hypertension and diabetes simultaneously.[3] Indiscriminate selection of antihypertensive to treat hypertension in diabetic patients can lead to serious drug interaction and needs careful consideration to avoid possible hypoglycemia or hyperglycemia induced by their use.

Glimepiride is frequently used in conjunction with metformin to achieve blood sugar control in diabetic individuals. Its metabolism is controlled by the cytochrome P450 subtype CYP2C9. Concomitant administration of drugs that influence inducers or inhibitors of CYP2C9 is known to affect its efficacy. Many drugs administered together have been found to potentiate or decrease the blood glucose-lowering effect of glimepiride and other sulfonylureas, resulting in higher or lower blood glucose levels. Combination with metformin has been approved to be safe and is devoid of serious drug interaction when used with glimepiride. Its elimination through tubular secretion without undergoing metabolic changes is a factor that favors its use in combinational therapy.[4]

In diabetics with hypertension as comorbidity, angiotensin-converting enzyme (ACE) inhibitors or angiotensin-II receptor blockers (ARB) are the most commonly employed antihypertensive drugs.[5] ARBs additionally have a favorable influence on insulin's actions and triglyceride (TG) metabolism that are impaired in type 2 diabetes.[6] Many compounds in this category affect glimepiride's effectiveness by stimulating or inhibiting CYP2C9, resulting in undesirable hypoglycemia or hyperglycemia. Losartan, an ARB is in use as an antihypertensive agent and gets metabolized by the isoenzymes of cytochrome P450 viz., (CYP) 3A4, 2C9 and 2C10. There is hardly any evidence indicating its ability to alter the CYP450 enzyme, especially those involved in the metabolism of glimepiride, and therefore, it is considered to be safe for concomitant administration with glimepiride in diabetic hypertensive patients. In addition to its antihypertensive action, losartan is known to reduce blood sugar level, and therefore, its concomitant administration with anti-diabetic agents can result in hyperglycemic or hypoglycemic states. With that background, the current study was conducted to assess the drug interaction between selected oral hypoglycemic agents (glimepiride and metformin) with an angiotensin II receptor antagonist, losartan using preclinical studies.

Materials and Methods

Drugs and chemicals

Apotex Research Pvt. Ltd., Bengaluru, provided Glimepride, Metformin, and Losartan as gift samples. All of the other chemicals employed in this experiment have been of the analytical grade.

Experimental animals

Sri. Venkateswara Enterprises, Bengaluru, India, provided albino wistar rats and rabbits. 25°C ± 2°C was the Standard laboratory temperature with 50% ± 15% relative humidity that was maintained to accustomed the experimental animals. All experimental animals were maintained at 12 h light/12 h dark cycle. They have been provided standard pellet diet (Hindustan Lever Ltd., Bangalore, India) as well as water ad libitum. Approval for the research was granted vide consent letter (No: VIPS/1442/15-16, Dated: 14.01.2016) by IAEC.

Selection of doses

Doses of drugs used in the experiment were arrived at by extrapolation of their oral dose in humans to (Losartan-25 mg; glimipiride-1 mg; Metformin-500 mg) to rats along with rabbits on the bases of body to surface area index. Human equivalent dose of Losartan used was 2.25 mg/kg bd. wt for rats and 1.75 mg/1.5kg bd. wt for rabbits. Equivalent dose of Glimiperide and metformin was 0.09 mg + 45 mg/kg bd. wt in rats and 0.07 mg + 35 mg/1.5 kg bd. wt. in rabbits correspondingly.[7]

Measurement of biochemical parameters

The blood samples of experimental animals were obtained by retro orbital puncture in case of rats and by puncturing of the left marginal ear vein in rabbits. They were transferred into clean disposable centrifuge tubes. Glucose oxidase/peroxidase method was used to analyze blood glucose level, enzyme-linked immunosorbent assay for insulin level, and high-performance liquid chromatography for the plasma concentration of the drugs.[8910]

Study in normal rats

Six albino rats of Wistar strain weighing between 250 and 300 g were administered glimepiride (0.09 mg/kg) along with metformin (45 mg/kg) p.o. A wash out period of 7 days was followed and the procedure was repeated with losartan 2.25 mg/kg body per orally. Following a washout period of an additional 7 days, the same animals have been administered Losartan, 2.25 mg/kg body weight and a combination of metformin (45 mg/kg) + glimepiride (0.09 mg/kg) per orally 30 min after the administration of losartan.

In multiple dose study, 2.25 mg/kg body weight losartan was administered 30 min prior to metformin (45 mg/kg) + glimepiride (0.09 mg/kg) for 7 constitutive days. Blood samples have been gathered from retro orbital vein at 0, 1, 2, 4, 6, 8, 10, 12, 14, and 16 h interval.

Study in diabetic induced rats

To induce diabetes, rats were injected with alloxan monohydrate in two category of doses such that 100 mg/kg body weight on the 1st day followed by 50 mg/kg body weight dose on the second.[11] Rats above 250 mg/dL have been considered to be diabetic with blood glucose levels. Groups of six diabetic-induced rats were used for the study. The study protocol followed was similar to the one followed in case of nondiabetic rats for single and multiple dose treatments.

Study in normal rabbits

Six albino rabbits weighing between 1.38 and 1.7 kg were administered with glimepiride + metformin (0.07 mg + 35 mg/kg) orally. The same group has been administered with 1.75 mg/kg of losartan in single and muliple dose treatments as conducted in rats at 0 (predose), 1, 2, 3, 4, 6, 8, 12, 18, and 24 h Blood samples were collected after drug administration by puncturing the left marginal ear vein and estimated for blood sugar, insulin, and serum glimepiride.

Statistical analysis

Blood glucose levels; serum insulin levels and plasma concentration of glimepiride was stated as mean ± standard error of the mean calculated utilizing individual values obtained within the groups. The significance of difference was determined using a “two-way ANOVA,” followed by a “Bonferroni post test.”

Results

Concomitant administration of losartan with glimepiride-metformin combination in diabetics with hypertension is common in clinical practice. Losartan's use as a safe drug for concomitant administration with glimepiride has been advocated due to a lack of evidence on its capacity to change the CYP450 enzyme, particularly those involved in the metabolism of glimepiride. The influence of inherent ability of losartan to lower glucose level and the pharmacodynamic or pharmacokinetics changes that it can bring about in the regulation of blood sugar level when administered along with antidiabetic drugs has not been studied. The present study has been designed for identifying the pharmacokinetic as well as pharmacodynamic interactions between losartan and a combination of glimepiride + metformin in normal and diabetic conditions, using animal models.

When normal or diabetic rats were given a combination of glimepiride and metformin, blood glucose levels decreased significantly in a dose-dependent way. When measured at the end of 4 h, a combination of 0.09 mg/kg of glimepiride and 45 mg/kg of metformin resulted in an ideal reduction in blood glucose of 33.60%. Losartan alone lowered blood glucose levels by 28.51% at 2.25 mg/kg body weight dose. Insulin levels were observed to be increased when blood glucose levels were reduced at the end of 6 h with losartan. Losartan enhanced the hypoglycemic activity produced by glimepiride + metformin with single dose to (43.59%) and to (43.56%) when administered in a component of multidose regimen spanning over 7 days [Figure 1]. Insulin levels measured at peak lowering in blood sugar levels were also determined to be altered in single and multiple dose regimens at 6 h [Table 1] indicates that in normal rats, there was an existence of pharmacodynamic interaction between losartan and the combination of glimepiride plus metformin.

Figure 1

Effect of single dose and multiple dose treatments of Losartan (2.25 mg/kg) with Glimepiride (0.09 mg/kg) + Metformin (45 mg/kg) combination on blood glucose reduction in normal rats

Table 1

“Mean serum glucose level (mg/dL) with mean serum insulin (µIU/mL) in glimepiride + metformin losartan alone and single and multiple dose treatments with glimepiride + metformin and losartan in normal rats; diabetic rats and normal rabbits

Groups	Time (h)	Mean serum glucose levels (mg/dL)	Serum insulin (µIU/mL)”
Glimepiride (0.09 mg/kg) + metformin (45 mg/kg) in normal rats	0.00	78.83±1.53	9.97±0.13
	4.00	50.50±2.05	10.03±0.15
Glimepiride (0.09 mg/kg) + metformin (45 mg/kg) in diabetic rats	0.00	262.33±6.59	7.34±0.24
	4.00	157.17±4.17	10.36±0.27
Glimepiride (0.07 mg/1.5 kg) + metformin (35 mg/1.5 kg) in normal rabbits	0.00	94.20±3.32	8.95±0.10
	4.00	59.00±2.45	10.20±0.15
Losartan (2.25 mg/kg) in normal rats	6.00	58.33±2.09	10.39±0.19
Losartan (2.25 mg/kg) in diabetic rats	6.00	183.00±3.20	8.96±0.17
Losartan (1.75 mg/1.5 kg) in normal rabbits	4.00	72.00±1.79	9.92±0.25
Glimepiride + metformin and losartan (SD) in normal rats	6.00	49.67±1.40	10.71±0.15
Glimepiride + metformin and losartan (SD) in diabetic rats	6.00	143.83±3.93	10.01±0.15
Glimepiride + metformin and losartan (SD) in normal rabbits	4.00	52.60±1.50	11.36±0.15
Glimepiride + metformin and losartan (MD) in normal rats	6.00	47.83±2.11	10.61±0.19
Glimepiride + metformin and losartan (MD) in diabetic rats	6.00	138.50±3.89	10.15±0.23
Glimepiride + metformin and losartan (MD) in normal rabbits	4.00	51.20±1.66	11.84±0.09

SD=Single dose, MD=Multiple dose

The combination of 0.09 mg/kg glimepiride and 45 mg/kg metformin resulted in a 40.09% reduction in blood glucose levels in diabetic rats at the end of 4 h. Similarly, losartan alone, at 2.25 mg/kg body weight dose, decreased blood glucose levels by 31.09% at the end of 6 h [Figure 2]. Insulin levels were found to be enhanced at 4th h as compared to 0 h at which the reduction of blood sugar level was found to be maximum following the administration of combination of glimepiride + metformin. Losartan when administered concomitantly reduced the blood sugar level to (44.61%) and (46.62%) when administered as a component of single dose regimen as well as component in a multidose regimen [Figure 2]. The insulin levels at peak reduction in the levels of blood glucose were also observed to be altered with both single and multiple dose treatments [Table 1] signifying an existence of pharmacodynamic interaction between losartan and glimepiride + metformin in diabetic rats.

Figure 2

“Effect of single and multiple dose treatments of Losartan (2.25 mg/kg) with Glimepiride (0.09 mg/kg) + Metformin (45 mg/kg) combination on blood glucose reduction in diabetic rats”

In experiments conducted on normal rabbits, combination of glimepiride (0.07 mg/1.5 kg) + metformin (35 mg/1.5 kg) produced an optimal decrease in blood glucose level by 37.41% at the end of 4th h. Similarly, losartan at 1.75 mg/1.5 kg dose decreased blood glucose level by 25.80% at the end of the 4 h. Losartan in co-administered with a combination of glimepiride + metformin significantly enhanced the hypoglycemic activity of glimepiride + metformin in single (45.18%) as well as multiple dose regimens (48.32%) [Figure 3].

Figure 3

Effect of single and multiple dose treatments of Losartan (1.75 mg/1.5 kg) on Glimepiride (0.07 mg/1.5 kg) + Metformin (35 mg/1.5 kg) combination on blood glucose reduction in normal rabbits

Following the administration of combination of glimepiride + metformin, insulin levels were found to be enhanced at 4th h as compared to 0 h. Highest reduction in blood sugar level was also found to occur at the 4th h. Serum levels of glimepiride have been observed to be improved appreciably with single as well as multiple dose treatments of losartan from 1 h to 24 h postdrug administration [Tables 2 and 3]. A significant increase in the pharmacokinetic parameters of glimepiride such as AUC0-∞, AUMC 0-t, AUMC 0-∞, T1/2, Cmax and mean residence time was observed, whereas clearance (Cl) was significantly decreased in experiments involving single as well as multiple dose treatment with losartan coadministered with glimepiride + metformin. The increase in serum glimepiride + metformin levels and pharmacokinetic parameters suggests that losartan and glimepiride + metformin have a pharmacokinetic interaction.

Table 2

Serum glimepiride levels in normal rabbits treated losartan single and multiple dose treatments

Time (h)	Serum glimepiride levels (µg/ml)
	Glimepiride	Glimepiride + losartan (SD)	Glimepiride + losartan (MD)
0	0.00±0.00	0.00±0.00ns	0.00±0.00ns
1	1.46±0.18	3.21±0.07**	5.22±0.18***
2	5.43±0.23	8.13±0.15***	10.65±0.23***
4	8.14±0.15	12.14±0.23***	15.83±0.29***
6	6.57±0.19	9.59±0.38***	12.83±0.23***
8	5.22±0.14	7.97±0.21***	10.22±0.22***
12	3.25±0.15	6.02±0.05***	8.09±0.16***
18	1.40±0.15	3.16±0.10***	5.57±0.15***
24	0.63±0.07	1.32±0.10***	3.83±0.16***

nsP>0.05, P<0.05, **P<0.01, ***P<0.001 significance followed by two-way ANOVA followed by Bonferroni posttest when compared with Glimepiride (0.035 mg/1.5 kg). SD=Single dose, MD=Multiple dose, ns=Not significant

Table 3

Pharmacokinetic parameters of serum glimepiride alone and combination with losartan in single and multiple dose treatments

Pharmacokinetic parameter	Mean pharmacokinetic parameters (mean±SEM)
	Glimepiride	Glimepiride + losartan (SD)	Glimepiride + losartan (MD)
AUC0-24 (µg/ml/h)	125.31±4.47	185.82±2.49*	187.97±1.85*
AUC0-α (µg/ml/h)	136.29±6.69	247.66±4.71*	247.51±5.85*
AUMC0-24 (µg/ml/h*h)	1032.17±110.86	2541.22±67.56***	2519.96±66.29*
AUMC0-α (µg/ml/h*h)	1400.09±197.62	5035.71±328.02*	4909.26±301.35*
Ka (h-1)	1.15±0.00	1.15±0.00ns	1.15±0.00ns
T1/2 (h)	6.26±0.39	11.14±0.51*	11.03±0.45*
VdSS (ml/kg)	1568.60±70.49	1777.41±103.25ns	1779.58±45.08ns
Cl (ml/h/kg)	172.79±8.03	95.04±2.17*	94.50±2.32*
Tmax (h)	4.00±0.00	4.00±0.00ns	4.00±0.00ns
Cmax (µg/ml)	11.83±0.24	14.17±0.18*	14.11±0.36*
MRT (h)	10.10±0.88	20.25±0.99*	19.77±0.81*

nsP>0.05, *P<0.05 significance followed by unpaired t-test. SEM=Standard error of the mean, SD=Single dose, MD=Multiple dose, ns=Not significant, AUC=Area under the concentration time-curves, AUMC=Area under first moment curve, MRT=Mean residence time

Discussion

Elderly patients suffer from chronic diseases are often exposed to the practice of polypharmacy.[12] Such strategies amplify the chances of drug-drug interactions (DDIs) proportionally. The consequences such interactions enhances the risk associated with drugs having a narrow therapeutic index requiring changes in order, timing, and dosage of the drugs to be administered.[13] Majority of the DDIs contribute to the development of adverse drug reactions (ADR) and its contribution toward the same has been reported to be in a 20%–40%.[14] Clinical relevant drug interactions are found to be around 6% in patients with 2 to 4 medications, 50% with 5 medications, and 100% with more than 10 medications at once. Other factors contributed to DDIs are age, sex, comorbidities and genetics, and simultaneous usage of various treatment options. Pharmacokinetic and pharmacodynamic interactions play a role in drug-drug and drug herb potential interactions.

CYP enzymes play a key role in many drug interactions. CYP450 isoenzymes are primely involved in the modulation of patient response to the drugs in chronic inflammatory diseases such as type 2 diabetes mellitus (T2DM).[15] This factor has an impact on drug metabolizing enzymes to be consider in the management of T2DM and also to consider in clinically proven antidiabetic drugs and in multiple drug regimens aimed at reducing ADR and to optimize drug therapy. Combined use of glimepiride with insulin or with metformin may enhance the drug-induced hypoglycemia in patients. When a persistent hypoglycemia situation cannot be treated with a single medication in conjunction with diet and exercise, a combination of metformin or insulin with glimepiride must be prescribed.[16] ARBs are the recommended antihypertensive agents in diabetic hypertensive patients. However, it was not explore whether the ARBs have shown to produce positive result on insulin action and metabolism of TGs, both of which are impaired in type 2 diabetes mellitus. The commonly prescribe ARBs in diabetic hypertensives are losartan that block the effect of angiotensin mediated vasoconstriction and aldosterone secretion by acting on AT1 receptor.[17] Losartan is a CYP2C9 substrate and its metabolism is induced by CYP2C10, 3A4.[18] Losartan acts by mediating the improvement of insulin sensitivity might be due to enhanced nonoxidative glucose disposal and also increased flow of blood and its circulation in patients of diabetic hypertension.[19]

In both single along with multiple dose regimens, losartan increased the hypoglycemic of glimepiride + metformin activity; this could be attributed to a combination hypoglycemic activity (Pharmacodynamic effect) or to competitive inhibition of glimepiride metabolism by CYP2C9 and CYP3A4. Losartan pretreatment increases the glimepiride hypoglycemic effect by increasing insulin sensitivity as well as progressing insulin homeostasis, which could be attributable to CYP2C9 suppression.[20] Furthermore, oral administration of losartan to diabetic induced rats improved insulin sensitivity, resulting in lower fasting and fed glucose levels.[21]

Hypoglycemic losartan activity may be due both to insulin resistance and reduced hepatic glucose consumption in both normal and diabetic rats as previously established.[22] According to Chu et al.'s findings, AT1 receptor antagonists improve the glucose tolerance and β-cells function in juvenile type 2 diabetic mice.[23] Furthermore, oral losartan therapy to diabetic rats has been found for increasing insulin sensitivity, resulting in lower fasting along with fed glucose concentrations.[21] Studies showed that Losartan in people with type 2 diabetes and nephropathy, enhances insulin sensitivity, improves β-cell response to glucose, as well as augment glucose homeostasis.[24]

Conclusion

Losartan along with glimepiride and metformin combination was significantly associated with lowering the glucose level in both nondiabetic and diabetic rats and rabbits. The results obtained from the current study indicated that the losartan increases the hypoglycemia produced by glimepiride + metformin by both pharmacokinetic and pharmacodynamic interactions. As a consequence, clinicians must proceed cautiously while prescribing this combination to avoid severe hypoglycemia.[25]

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.