Assessment of Pharmacokinetic Interaction of Spirulina with Glitazone in a Type 2 Diabetes Rat Model

Abstract

The objective of the current study was to assess the possible pharmacokinetic interactions of spirulina with glitazones in an insulin resistance rat model. Wistar male albino rats were equally divided into five groups: insulin resistant rats+spirulina (500 mg/kg)+pioglitazone (10 mg/kg), insulin resistant rats+pioglitazone (10 mg/kg), insulin resistant rats+spirulina (500 mg/kg)+rosiglitazone (10 mg/kg), insulin resistant rats+rosiglitazone (10 mg/kg), and insulin resistant rats+spirulina (500 mg/kg). Described doses of pioglitazone, rosiglitazone, or spirulina were per orally administered and the plasma drug concentrations were determined. The pharmacokinetic parameters such as T_max, C_max, AUC_0-α, t_1/2, and Kel were determined by plotting the drug concentration as a function of time. The data observed in this acute study indicated that there was no statistically significant difference in any of the pharmacokinetic parameters (T_max, C_max, AUC_0-α, t_1/2, and Kel) of glitazones (pioglitazone, rosiglitazone) or spirulina, when they were coadministered. Given the promising results, this study concludes that the coadministration of spirulina does not influence the pharmacokinetics of glitazones in a type 2 diabetes rat model. Further chronic in vivo studies are recommended to assess the real time effect.

Introduction

Type 2 diabetes is a clinical syndrome characterized by inappropriate hyperglycemia caused by insufficient insulin sensitivity or production or a combination of these two factors. It is a major health problem affecting more than 200 million people worldwide and is projected to become one of the world's main disablers and killers within the next 25 years.¹ Unfortunately, the management of type 2 diabetes remains a global problem and the successful treatment remains elusive. Despite the presence of known antidiabetic medicines in the pharmaceutical market, remedies from medicinal plants are used with varying success by many diabetic patients.² More than 400 plants are used in different forms (crude or extracts) for their hypoglycemic effects in treating diabetes by patients and practitioners.³ In this context, it is likely that the adjuvant therapy using herbal formulations or indigenous plants in addition to the regular therapy could be more effective for the long-term management of type 2 diabetes. However, the combination therapy is possible only when there is no herb–drug interaction or adverse effect, and provides a safe and clinically effective therapy. Consequently, the research on the herb–drug interactions continue and are increasingly important for obtaining more specific information about the pharmacokinetics and the possible interaction between the herbs and drug molecules.⁴

Glitazones are one of the most important oral hypoglycemic agents, which increase the insulin sensitivity in diabetic patients; however, this type of therapy can lead to several side effects and systemic toxicity.⁵ Typically, oral hypoglycemic agents are high-affinity ligands and activators of the peroxisome proliferators activated receptor-gamma (PPAR-γ), and most of its effects are mediated through this transcription factor.^5
–7 The potential broad clinical application of glitazones, for treatment of a variety of pathologies, demands studies on the effects of these compounds on organs that express PPAR-γ. PPAR-γ is expressed in bone, particularly in mesenchymal stem cells, and earlier in vitro investigation furnished evidence that pioglitazone-activated PPAR-γ2 functions as a dominant negative regulator of osteoblast differentiation.⁸

Spirulina is receiving more attention from medical scientists as a nutraceutical and a source of potential pharmaceuticals. It is a blue-green algae (cyanobacterium) belonging to the family Osillatoriaceae. Spirulina fusiformis possess potent antiviral activity,⁹ anticancer effects,¹⁰ strengthens the immune system,^11,12 radioprotective properties,¹³ and metalloprotective effects.¹⁴ Spirulina is also rich in proteins and minerals, and has also attracted attention due to its ability to stimulate mineral absorption by its effects on intestinal microflora.¹⁵ This evidence suggested that spirulina acts on bone metabolism, although this has not yet been studied. Recently, we observed that the coadministration of spirulina reduces the risk of bone fracture and acts synergistically with glitazones (pioglitazone and rosiglitazone) in an insulin resistant rat model.^16,17 The objective of the current study was to investigate the possible alteration on the pharmacokinetics of two glitazones (pioglitazone and rosiglitazone) in the presence of spirulina in a rat model of type 2 diabetes.

Materials And Methods

Animals

Adult male albino Wistar rats weighing about 180–200 g were used with the approval of the Institute Animal Ethics Committee (MMCP/IEC/10/01). The animals were housed in a controlled environment: temperature (24–28°C), relative humidity (60–70%), and a 12-h light–12-h dark cycle. The animals were fed with a standard pellet diet (Lipton Bangaluru India, Ltd., India) and water ad libitum.

Spirulina fusiformis in the form of powder was obtained from RECON Ltd. (Bangaluru, India). It was suspended in vehicle (olive oil) and was administered orally using oral gavage.

Drugs and chemicals

Dexamethasone sodium phosphate was obtained as a gift from M/s. Strides Arcolabs (Bangaluru, India) and pioglitazone maleate and rosiglitazone maleate from Torrent Pharmaceutical Ltd. (Ahmedabad, India). Methanol–acetonitrile used was of high-performance liquid chromatography (HPLC) grade and was supplied by Rankem Ltd. (New Delhi, India). Dimethyl sulfoxide and dichloromethane used were of analytical grade and were purchased from Qualikem Fine Chem. Pvt. Ltd. (New Delhi, India). Glacial acetic acid and ammonium acetate used were of analytical grade and were purchased from Central Drug House (New Delhi, India).

Dexamethasone-induced insulin resistance model

Animals were divided into the following five groups as described below.

Group 1: insulin resistant rats+spirulina (500 mg/kg b.w./day per oral)+pioglitazone (10 mg/kg b.w./day per oral) (IR+S+P).

Group 2: insulin resistant rats+pioglitazone (10 mg/kg b.w./day per oral) (IR+P).

Group 3: insulin resistant rats+spirulina (500 mg/kg b.w./day per oral)+rosiglitazone (10 mg/kg b.w./day per oral) (IR+S+R).

Group 4: insulin resistant rats+rosiglitazone (10 mg/kg b.w./day per oral) (IR+R).

Group 5: insulin resistant rats+spirulina (500 mg/kg b.w./day per oral) (IR+S).

In vivo studies

The pharmacokinetic studies were carried out by peroral administration of specific doses of pioglitazone, rosiglitazone, and spirulina. To perform the pharmacokinetic analysis, serial blood samples were collected from retro-orbital plexus at different time intervals (0.5, 1.5, 3, 5, and 7.5 h), following the administration of pioglitazone, rosiglitazone, or spirulina. The blood sample was centrifuged at 8000 g for 10 min for plasma separation. About 250 μL of plasma was subjected to protein precipitation with equal volumes of acetonitrile and was centrifuged at 8000 g for 10 min. The supernatant was filtered and injected into HPLC.

The area under the curve (AUC_0-α) is a measure of the bioavailability of a drug. The maximum serum concentration (C _max) and the time to reach maximum concentration (T _max) indicate the rate of delivery of drug into the systemic circulation. AUC_0-α was calculated using the trapezoid rule and C _max, T _max were determined from the concentration–time curves. The mean pharmacokinetic parameters of different groups recorded in Tables 1 –3 were calculated from the plasma concentration in rats.

Table 1.

Mean Pharmacokinetic Parameters of Pioglitazone in Plasma Following Oral Administration of Pioglitazone with Spirulina and of Control for a Period of 8 h in Adult Male Albino Wistar Rats

Parameter	Pioglitazone+spirulina	Control	P value
T _max (h)	1.5	1.5
C _max (μg/mL)	19.42±4.06	21.26±6.42	.5661
AUC_0-α (μg·h/mL)	97.83±26.55	104.03±31.17	.7184
t_1/2 (h)	4.60±0.85	4.44±1.19	.794
Kel	0.15±0.04	0.16±0.05	.710

n=6. Dosages: pioglitazone 10 mg/kg + spirulina 500 mg/kg; control, pioglitazone 10 mg/kg.

C _max, maximum concentration; T _max, time of maximum concentration; Kel, elimination rate constant; AUC_0-α, area under the serum concentration–time curve; t_1/2, elimination half-life.

Table 2.

Mean Pharmacokinetic Parameters of Rosiglitazone in Plasma Following Oral Administration of Rosiglitazone with Spirulina and of Control for a Period of 8 h in Adult Male Albino Wistar Rats

Parameter	Rosiglitazone+spirulina	Control	P value
T _max (h)	2.0	2.0
C _max (μg/mL)	13.16±2.97	12.31±3.61	.665
AUC_0-α (μg·h/mL)	92.15±16.55	86.21±18.43	.570
t_1/2 (h)	4.88±1.18	4.21±1.79	.461
Kel	0.14±0.05	0.16±0.04	.461

n=6. Dosages: rosiglitazone 10 mg/kg + spirulina 500 mg/kg; control, rosiglitazone 10 mg/kg.

Table 3.

Mean Pharmacokinetic Parameters of Spirulina in Plasma Following Oral Administration of Spirulina with Pioglitazone or Rosiglitazone and of Control for a Period of 8 h in Adult Male Albino Wistar Rats

Parameter	Rosiglitazone+spirulina	Pioglitazone+spirulina	Control	P value
T _max (h)	3.0	3.0	3.0
C _max (μg/mL)	1417.29±602.45	1407.05±671.21	1205.52±418.26	.7747
AUC_0-α (μg·h/mL)	11,699.23±1094.35	11,185.20±992.24	10,245.20±995.56	.0755
t_1/2 (h)	10.58±2.06	9.63±1.99	11.70±1.72	.4618
Kel	0.07±0.03	0.07±0.02	0.06±0.03	.765

n=6. Dosages: spirulina 500 mg/kg + pioglitazone 10 mg/kg; spirulina 500 mg/kg + rosiglitazone 10 mg/kg; control, spirulina 500 mg/kg.

HPLC analysis

The amount of glitazones and beta carotene in the plasma samples were quantified by HPLC (Cyberlab, LC P100) using a reverse phase RP-18 analytical column (4.6mm×250 mm, Shiseido, 5 μm). Briefly, for pioglitazone, the mobile phase consists of acetonitrile and ammonium acetate (pH 3, 20 mM; pH was adjusted with glacial acetic acid) in proportion of 60:40 (v/v). The flow rate was 1.0 mL/min and the effluent was monitored at 268 nm.¹⁸ The retention time of pioglitazone was found to be 5.5±0.2 min. In the case of rosiglitazone, the mobile phase consists of ammonium acetate (10 mM, pH 5.2) and acetonitrile (56.5:43.5, v/v) with a flow rate of 1 mL/min. The effluent was monitored using a fluorescence detector with an excitation wavelength of 247 nm and emission wavelength of 367 nm.¹⁹ The retention time of rosiglitazone was found to be 7.8±0.2 min. For beta carotene in spirulina, the mixture of acetonitrile:methanol (containing 0.1 M ammonium acetate:dichloromethane) in the proportion of 71:22:7 (v/v) was used. The flow rate was 0.5 mL/min and the effluent was monitored at 450 nm.²⁰ The retention time of beta carotene was found to be 5.3±0.3 min.

Pharmacokinetic analysis

The pharmacokinetic parameters were calculated using a noncompartmental pharmacokinetic model. Pharmacokinetic variables of interest included the area under the concentration–time curve from time 0 to ∞ (AUC_∞), peak concentration (C _max), terminal elimination rate constant (Kel), half-life of the terminal phase (t_1/2), and time to peak concentration (T _max). A linear-up/log-down method of estimation was used for the calculation of AUC_∞ and was obtained by adding C _last/Kel to AUC_0–t. The terminal elimination rate constant (Kel) was determined from the slope of the terminal exponential phase of the logarithmic plasma concentration–time curve. The elimination half-life (t_1/2) was calculated using 0.693/Kel.

Statistical analysis

The results were analyzed statistically by using either paired or unpaired Student's t-tests to determine the level of significance. The statistical significances between means were analyzed using one-way analysis of variance followed by the Tukey's multiple comparison test. A P<.05 was considered as statistically significant.

Results And Discussion

A recent report by the World Health Organization indicates that noncommunicable diseases, comprising cardiovascular diseases, cancers, and diabetes, are the leading causes of death globally, killing more people each year than all other causes combined.²¹ Unfortunately, the worldwide prevalence of diabetes is significantly high and controlling the incidence remains a challenge. Recent discoveries may not translate quickly enough into effective, affordable and safe medical products for patients, to counteract this situation. On the other hand, the existing oral hypoglycemic agents are relatively effective, but do not offer permanent cure for this disorder. Alternatively, a combination therapy has been attempted to treat diabetes, but was limited by the potential side effects of these drugs during long-term treatment. For instance, glitazone administration (10 mg/kg) results in significant bone loss due to sensitized PPAR-γ2 isoforms, which is critical for the regulation of osteoblast and adipocyte differentiation. In addition, the long-term usage of pioglitazone in diabetic patients showed the risk of incidental cancer²² although there is no clear evidence to prove the association between use of pioglitazone and risk of cancer.²³

In the insulin resistant rat model (dexamethasone induced experimental type 2 diabetes),²⁴ change of insulin action seems more important than the alteration of insulin secretion. Defects in the insulin-signaling pathway, owing to mutations in the insulin receptor gene, the presence of antibodies to the insulin receptor, or to insulin itself are some of the factors responsible for insulin resistance. Dexamethasone causes insulin resistance as measured by several markers, including a decrease in insulin-stimulated glucose uptake and glucose oxidation. Whereas the precise cause or causes are not yet known, it is accepted that dexamethasone affects insulin signaling at several levels.²⁵ In addition to this, insulin resistance is also major risk factor for bone fractures and poor skeletal health.²⁶

To overcome these side effects, some physicians preferred alternative therapy, either from herbal formulations, nutraceuticals, or indigenous plants, as adjunct therapies in the long-term management of type 2 diabetes. This factor has prompted us to assess the effect of coadministration of spirulina along with glitazones to treat type 2 diabetes and osteoporosis, and has been reported recently.^16,17 Excited by the promising results, an acute study was carried out to investigate the possible pharmacokinetic interaction of spirulina with glitazones in a type 2 diabetes rat model. A fixed low dose of glitazones (10 mg/kg) and a higher dose of spirulina (500 mg/kg) were administered to assess the maximum possible effect spirulina on the pharmacokinetics of glitazones.

The pharmacokinetic data such as T _max, C _max, AUC_0-α, t_1/2, and Kel were determined following the oral administration of glitazones and spirulina to different groups. Figure 1 represents the plasma profile of pioglitazone following oral administration of pioglitazone (10 mg/kg) with spirulina (500 mg/kg) and of control (pioglitazone dose of 10 mg/kg) for a period of 8 h in adult male albino Wistar rats. It is apparent from Figure 1 that the pharmacokinetic profiles of both groups were comparable, indicating no difference statistically in the pioglitazone profile due to coadministration of spirulina. Interestingly, the profile also signifies that the coadministration of spirulina with pioglitazone has no influence on the absorption or elimination of the drug (Fig. 1). Furthermore, the observed pharmacokinetic parameters such as T _max, C _max, AUC_0-α, t_1/2, and Kel were also found to be comparable in both the cases (Table 1). Moreover, the pharmacokinetic data also indicate that the pioglitazone is rapidly absorbed following oral administration and possesses a short biological half-life (∼4.5 h). Indeed, these results suggest that the coadministration of spirulina has no effect on the overall pharmacokinetics of pioglitazone.

FIG. 1.

Plasma drug profiles obtained after oral administration of pioglitazone (dose of 10 mg/kg) with spirulina (dose of 500 mg/kg) and of control (pioglitazone dose of 10 mg/kg) for a period of 8 h in adult male albino Wistar rats. The data represent mean±SD of six determinations.

A similar effect was observed in the case of rosiglitazone. Figure 2 shows the plasma profile of rosiglitazone following oral administration of rosiglitazone (10 mg/kg) along with spirulina (500 mg/kg) and control (rosiglitazone dose of 10 mg/kg). All pharmacokinetic parameters were found to be statistically insignificant, under the current experimental conditions (Table 2). Thus, these results further substantiated the data observed in the case of pioglitazone, indicating no specific pharmacokinetic interactions between glitazones and spirulina in type 2 diabetes rats.

FIG. 2.

Plasma drug profiles obtained after oral administration of rosiglitazone (dose of 10 mg/kg) with spirulina (dose of 500 mg/kg) and of control (rosiglitazone dose of 10 mg/kg) for a period of 8 h in adult male albino Wistar rats. The data represent mean±SD of six determinations.

We also assessed the pharmacokinetic data of spirulina by measuring the beta carotene (major constituent in spirulina) content in plasma.²⁷ The sample withdrawn at time zero served as the baseline value. The pharmacokinetic parameters of spirulina (measured as beta carotene) in plasma following oral administration of spirulina (500 mg/kg) along with pioglitazone (10 mg/kg) or rosiglitazone (10 mg/kg) and spirulina (500 mg/kg, control) are presented in Figure 3. It was apparent that the profiles were comparable and the pharmacokinetics of spirulina was not influenced by the administration of glitazones (pioglitazone and rosiglitazone), under the experimental conditions. It was also evident from the figure that the spirulina constituent (beta carotene) was rapidly absorbed (T _max=3 h) from the gastrointestinal tract, probably by the enterocytes and incorporated into chylomicrons and released into the circulation.²⁸ The different pharmacokinetic parameters measured are summarized in Table 3. It can be seen from Table 3 that the pharmacokinetic parameters of spirulina are not affected by coadministration with glitazones. Furthermore, the half-life (∼10 h) observed also suggested that the beta carotene was gradually metabolized in the body.

FIG. 3.

Plasma drug profiles obtained after oral administration of spirulina (dose of 500 mg/kg) with pioglitazone (dose of 10 mg/kg) or rosiglitazone (dose of 10 mg/kg) and of control (spirulina dose of 500 mg/kg) for a period of 8 h in adult male albino Wistar rats. The data represent mean±SD of six determinations.

The literature indicates that several factors directly or indirectly influence the CYP-mediated metabolism and are likely to be potential for drug interaction either as a result of induction or inhibition of the CYP enzyme. Some of the researchers reported the induction of the CYP enzyme with spirulina^29,30 and similar results were also observed when curcumin was coadministered with pioglitazone.³¹ However, another study showed the inhibition of the CYP enzyme with spirulina.^10,32 However, the feasibility of any such interactions is not ruled out in the current study, although no pharmacokinetic interactions between the glitazones and spirulina were observed.

In conclusion, an acute in vivo study was carried out to assess the possible alteration on the pharmacokinetics of two glitazones (pioglitazone and rosiglitazone) in the presence of spirulina in a type 2 diabetes rat model. The observed data suggest that there were no significant pharmacokinetic interactions between the glitazones and spirulina. Further studies are recommended to assess the dose-dependent pharmacokinetic interaction of glitazones and spirulina in a type 2 diabetes rat model. Moreover, the possible interaction and toxicity of glitazones with spirulina also need to be assessed in chronic treatments to substantiate the current findings.

Footnotes

Acknowledgments

The authors are grateful to the management for offering the requisite technical help to accomplish this study. A word of gratitude to Recon Limited, Bangalore, India, for providing Spirulina fusiformis and to Torrent Pharmaceutical Ltd., Ahmedabad, India, for pioglitazone malleate gratis samples. The authors gratefully acknowledge their colleagues for encouraging and providing the necessary research facilities to conduct this study. There was no funding from any outside agency.

Author Disclosure Statement

No competing financial interests exist.

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