Influence of trazodone on the pharmacodynamics and pharmacokinetics of pioglitazone.

BACKGROUND: These days, poly pharmacy is very common for the treatment of multiple diseases and majority of drugs were metabolized with CYP 450 enzymes. Diabetes mellitus is such a disorder, which requires continuous therapy for the control of blood glucose concentration. Depression was quite common in diabetic patients. Therefore, multiple drugs required to treat diabetes mellitus and depression. Simultaneous administration of these drugs leads to drug interaction. Pioglitazone and trazodone metabolized by CYP3A4 enzymes which may lead to potential drug interaction.
OBJECTIVES: This study aimed to find the influence of trazodone on the pharmacokinetics & pharmacodynamics of pioglitazone in normal & diabetic rats, also on rabbits and subsequently effectiveness and safety of the combination was evaluated. METHODS AND MATERIAL: Blood glucose concentration was determined by Glucose oxidase/peroxidase method in normal and diabetic rats. Diabetes was induced with Streptozotocin at a dose of 55 mg/kg body weight. Serum pioglitazone concentration was estimated by high performance liquid chromatography method for pharmacokinetic data. The values were expressed as Mean ± Standard Error Mean (SEM), GraphPad Prism 3.0 (San Diego, California, USA) software was used to express the data. Student's paired 't' test was used to determine the significance.
RESULTS: Pioglitazone produces hypoglycaemia in normal rats with a maximum decrease of 36.78 % ± 0.81 at 3 hours interval and anti-hyperglycaemic activity in diabetic rats with maximum reduction of 45.13 % ± 1.52 at 2 hours interval. Trazodone altered the pharmacokinetics of pioglitazone and improved the pioglitazone hypoglycaemic effect.
CONCLUSION: Trazodone apparently produced pharmacokinetic interaction with pioglitazone which might be by attenuating the metabolism of pioglitazone. Therefore, care should be taken in simultaneous therapy with pioglitazone and trazodone.

Introduction

Investigation of the mechanism of drug interaction (DI) is essential for certain drugs having less margin of therapeutic index and drugs used for chronic disorders. Hyperglycemia is associated with diabetes mellitus (DM) and requires continuous treatment.[12] Globally, the estimation of 14.3 crore people suffering from DM may increase double by the 2030 year.[3] In India, the incidence of DM is estimated to be 1%–5%.[4] Depression is two folds more in diabetic patients.[56] Nowadays, polypharmacy is more common to treat multiple disorders.[7] Therefore, the incidence of the combination of these two diseases is very high and requires treatment with many drugs to control hyperglycemia and depression. The majority of drugs are metabolized with cytochrome P450 (CYP450) enzymes.[28] Trazodone (TZ) is an extensively used drug for the treatment of depression and pioglitazone (PG) for the management of Type 2 DM. PG is known to act by stimulating the peroxisome proliferator-activated receptor γ, and TZ is a selective serotonin reuptake inhibitor. Both TZ and PG are metabolized by CYP3A4 enzymes.[89] Simultaneous administration of these two drugs may cause potential DI by altering the metabolism, absorption, distribution, and excretion of pioglitazone. Therefore, the chance of concurrent use of PG and TZ for treating DM and depression is more, and this research was performed to find the influence of TZ on pharmacokinetic (PK) parameters and the hypoglycemic effect of PG.

Materials and Methods

Ethical clearance

The study began after permission from Institutional Animal Ethics Committee (IAEC) (Reg.No: 878/Po/Re/s/05/CPCSEA/001/2017).

Procurement of chemicals

Gift samples of PG and TZ were obtained from DRL, Hyderabad. Streptozotocin (STZ), acetonitrile, methanol, dichloromethane, and glucose kits (ERBA) were purchased from Innovative and reliable chemicals and equipments, Guntur.

Selection of animals

Either sex of albino Wistar rats of 6–8 weeks age (220–280 g of weight), and albino rabbits of 3 months age (1.3–1.7 kg of weight), were selected for the study. Animals fed with pellet diet and ad libitum water. Eighteen hours of fasting were maintained before the experiment.

Dose calculation and suspension preparation of test compound

In medical practice, TZ 50 mg is given to humans orally as an antidepressant; rabbit dose is calculated as 3.5 mg (rabbit dose = human dose × 0.07), and rat dose is calculated as 0.9 mg (rat dose = human dose × 0.018).[810] However, PG dose for rabbit/rat trials was considered 10 mg/kg body weight, based on the effect of DRC on PG glucose levels in the blood.[11] Carboxymethylcellulose sodium 2% was used as a suspending agent to prepare oral TZ and PG suspension. Oral gavage was used to administer all the drugs to the different groups.

Diabetes induction

Rats were injected with nicotinamide 100 mg/kg, intraperitoneally. Fifteen minutes later, STZ was administered intravenously at a dose of 55 mg/kg body weight. Animals were given 10% glucose to combat the early phase of hypoglycemia.[1112] The fasting blood glucose levels that were conducted on the rats after 72 h of STZ treatment, confirmed the induction of DM. Only those rats that had blood glucose concentration of 0.2 g/100 mL and above only considered for the study as diabetic rat (DR).[1314] Blood was collected at regular intervals of 0, 1, 2, 3, 4, 6, 8, 10, and 12 h from each rat through the retro-orbital plexus and analyzed for blood glucose using a semiauto analyzer (ERBA).[114]

Study of pharmacodynamic interaction in normal and diabetic rats

PG was administered orally to a group of six normal rats (NRs) following TZ administration after 7 days washout period. Later, the combination of PG and TZ was administered to the same group, maintaining the washout period. Later to the single-dose (SD) study, the same group continued with TZ administration for the coming 8 days. After the fasting period, on the 9th day, the rats were given the combined treatment of PG and TZ. Similar treatment was given to STZ-nicotinamide-induced DRs in SD and multiple-dose (MD) studies. Blood samples were withdrawn in rats from the retro-orbital plexus at defined breaks of 0, 1, 2, 3, 4, 6, 8, 10, and 12 h intervals. Glucose concentration in blood was found by glucose oxidase (GOD)/peroxidase (POD) technique.

Study of pharmacokinetic interaction in rabbits

PG was administered orally to a group of five rabbits, and the same group was treated orally with TZ. Afterwords, the combination of TZ and PG was adminiistered with a gap of washout period. Later to the SD study, the same group continued with the administration of TZ for the coming 8 days. After the fasting period, on the 9thday, the rats were given the combined treatment of PG and TZ. Blood samples were withdrawn in rabbits from the ear marginal vein at defined breaks of 0, 1, 2, 4, 6, 8, 12, 18, and 24 h intervals.[11] Serum PG concentrations were estimated using high-performance liquid chromatography (HPLC).[15] Serum PG was estimated by HPLC in rabbits, and glucose concentration in blood was found by GOD/POD technique in rabbits and rats. PG and PK parameters were determined from the concentration–time profile using software (WinNonlin 5.0.1 version).

Data and statistical analysis

The values were expressed as mean ± standard error of the mean; GraphPad Prism 3.0 (San Diego, California, USA) was used to express the data. Paired t-test was used to determine the significance.

Results

Study of pharmacodynamic interaction in normal rats and diabetic rats

PG produces hypoglycemia in NR with a maximum decrease of 36.78% ± 0.81 at 3-h interval [Table 1] and anti-hyperglycemic effect in DR with a maximum decrease of 45.13% ± 1.52% at 2-h interval [Table 2]. TZ alone not produced a significant difference in glucose concentrations of blood in both NR and DR. Combination of PG with TZ shows a significant decrease of glucose concentrations in blood at a defined interval of 1, 2, 3, 4, 6, 8, 10, 12 h when compared with PG control in both normal and DRs in both SD and MD study [Tables 1 and 2].

Table 1

Effect of single-dose and multiple-dose treatments of trazodone on pioglitazone blood glucose levels in normal rats

Time (h)	Percentage of blood glucose reduction, mean±SEM
	PG only	TZ only	PG + TZ SD	PG + TZ MD
0	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00
1	27.15±0.76	4.85±0.86	45.57±1.52*	51.57±0.97*
2	30.88±0.86	5.27±0.45	50.67±1.10*	50.67±1.10*
3	36.78±0.81	5.39±1.71	52.52±1.37*	46.21±0.97*
4	27.45±1.03	7.15±1.09	48.32±1.46*	44.75±1.06*
6	21.10±0.46	8.11±0.85	46.63±2.17*	42.62±0.49*
8	16.64±0.77	12.39±1.54	37.44±1.63*	38.3±1.68*
10	15.82±0.65	15.55±0.93	31.33±1.34*	31.38±1.34*
12	12.24±0.73	16.91±1.58	28.59±0.94*	27.68±0.72*

*P<0.05 is considered statistically significant compared to PG control. TZ=Trazodone, PG=Pioglitazone, SD=Single dose, MD=Multiple dose, SEM=Standard error of the mean

Table 2

Effect of single-dose and multiple-dose treatments of trazodone on pioglitazone blood glucose levels in diabetic rats

Time (h)	Percentage of blood glucose reduction, mean±SEM
	PG only	TZ only	PG + TZ SD	PG + TZ MD
0	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00
1	40.81±1.86	2.18±0.55	53.76±0.49*	52.89±0.52*
2	45.13±1.52	4.29±1.69	57.44±0.63*	55.36±0.36*
3	36.06±1.49	6.22±0.79	51.93±0.47*	49.28±0.47*
4	27.85±1.72	10.07±1.4	48.16±0.76*	48.28±0.48*
6	26.99±1.79	12.87±1.49	45.11±0.54*	46.62±1.54*
8	23.89±1.51	14.93±0.55	39.71±1.11*	44.94±0.74*
10	21.25±1.75	18.53±1.39	30.54±1.09*	40.45±0.63*
12	20.49±0.97	17.21±1.30	29.09±0.59*	27.29±0.92*

*P<0.05 is considered statistically significant when compared with PG control. TZ=Trazodone, PG=Pioglitazone, SD=Single dose, MD=Multiple dose, SEM=Standard error of the mean

Pharmacokinetic interaction study in rabbits

Mean serum concentration versus time curve of PG alone, a combination of PG with TZ in SD and MD studies was shown in Figure 1. The peak serum concentration of PG was observed in the combination of PG with TZ in SD and MD studies. PK parameters of PG alone, and a combination of PG and TZ, in both SD and MD studies, are expressed in Table 3. PK parameters such as the area under the curve (AUC0-t), area under the first moment curve (AUMC0-t), AUC0-∞, AUMC0-∞, Cmax, and mean residence time shows a significant difference in the combination of PG and TZ in both SD and MD studies.

Figure 1

Mean serum PG concentration of PG alone and combination with TZ in SD and MD treatments in rabbits. PG: Pioglitazone, TZ: Trazodone, SD: Single dose, MD: Multiple dose

Table 3

Pharmacokinetic parameters of pioglitazone before and after administration of trazodone

PK parameters	PG	PG + TZ SD	PG + TZ MD
AUC0-t (ng/ml/h)	3195±33.01	4144±62.59*	3845±31.48*
AUMC0-t (ng/ml/h×h)	28790±253.6	35960±303.3*	33340513.7*
AUC0-∞	3785±51.61	4586±47.63*	4786±25.44*
AUMC0-∞	28970±264.2	23480±243.9*	24510±130.2*
Cmax (ng/ml/)	2192±8.728	3973±46.44*	3940±23.17*
Tmax (h)	2	2	2
Abs t1/2 (h)	1.05	1.05	1.05
MRT (h)	11.67±0.34	21.04±0.59*	21.01±0.28*

*P<0.05 is considered statistically significant compared to PG control. TZ=Trazodone, PG=Pioglitazone, SD=Single dose, MD=Multiple dose, PK=Pharmacokinetic, AUC=Area under the curve, AUMC=Area under the first moment curve, MRT=Mean residence time

High-performance liquid chromatography Method

PG and PK were studied using a simple, sensitive HPLC method in a rabbit model. The HPLC method was developed to measure the PG concentration in serum. Using HPLC, PG can be measured even in 20 ng/mL quantities in serum. Metformin was used as the internal standard, PG, and blank chromatograms are shown in Figures 2 and 3. The retention time of PG was found to be 6.81–7.52 min.

Figure 2

Blank rabbit serum chromatogram

Figure 3

Pioglitazone chromatogram and metformin (IS). IS: Internal standard

Discussion

In clinical practice, DIs are seen generally and the mechanism of interaction mechanism is evaluated generally in animals. Thus, the influence of TZ on the PK and pharmacodynamics of PG is studied by us in rats and rabbits. NR model helps to identify rapidly the DI and DR model validated the identical reaction.[12] Rabbit model was used to authenticate the existence of the DI. The impact of TZ MD, on PG action, was also studied for how TZ influences the prolonged treatment with it, as both are used for a longer period. The study revealed that TZ alone did not produce any significant effect on normal and DR blood glucose concentrations. Fascinatingly, the PG hypoglycemic activity was notably enhanced by TZ, following SD and MD treatment in rat and rabbit models, which established the existence of DI between PG and TZ. Since TZ did not change blood glucose concentration on its own, it is clear that the rise in the action of PG on blood glucose may be due to raised blood PG levels in the presence of TZ, as established by the PK DI study in rabbits. There was a notable increase in serum concentration of PG levels and an alteration in PK parameters of PG with SD and MD treatments of TZ. The rise in AUC and AUMC indicates enhanced availability of PG in the existence of TZ. The altered T1/2 indicates a change either in excretion or the metabolism process.[2] TZ and PG are biotransformed by the CYP450 system predominantly with CYP3A4 and there is an additional chance of TZ for modification of the metabolism of PG.[9] Raise in Cmax and AUC might be due to augmentation of absorption of PG in the existence of TZ, a well-known p-glycoprotein inhibitor.[1]

Conclusion

The DI appears to be PK and pharmacodynamic at metabolic and absorption levels. Since DI was observed in two different species of rat and rabbit, it is prone to occur in human beings also and leads to improved PG concentrations in plasma, which may require dose adjustment. Hence, safety measures should be taken when a combination of TZ and PG is prescribed to treat depression and diabetes, respectively.

Financial support and sponsorship

This work was funded by the UGC-SERO, Hyderabad, India.

Conflicts of interest

There are no conflicts of interest.