WANG Pei, ZHAO Songbo, ZHONG Ling, HE Shupeng, ZHOU Yan

(1.The Second Clinical Medical College of Lanzhou University, Lanzhou,730030, China;2.College of Pharmacy, Lanzhou University, Lanzhou, 730000, China; 3.College of Animal Science and Technology, Gansu Agricultural University, Lanzhou,730070, China;4.Department of Pharmacy, Gansu College of Traditional Chinese Medicine, Lanzhou,730000, China)

[Abstract] Objective: To investigate the pharmacokinetics, plasma concentration and tissues (liver and kidney)distribution and urinary excretion of metformin, and expression of rat organic cation transporters (rOct2)and multidrug and toxin extrusion protein 1 (rMate1)in estrogen deficiency model of ovariectomized rats.Methods: Rats were randomly separated into two groups with 8 rats in each, ovariectomy group (Ovx)with bilateral ovaries removed and Sham group treated by the same surgical procedure but no ovary removed.Both groups were treated with metformin.Plasma, urine and tissue concentration of metformin was determined by HPLC.Plasma lactate (LCA)level was determined by biochemical analysis, while renal expression of rOct2 and rMate1 was measured by Western-blot.Results: The results showed that AUC0→t and tissue concentration of metformin in Ovx group were significantly higher than that in sham group (P<0.01).24 h cumulative urinary excretion of metformin and renal expression of rMate1 decreased while the expression of rOct2 increased significantly in Ovx group (P<0.01)when compared with sham group.The level of LCA in plasma significantly increased in Ovx group (P<0.05).Conclusion: The study provided evidences that decreased urinary excretion of metformin was associated with estrogen deficiency after ovariectomy in rats.

[Key words] metformin; ovariectomy; rat multidrug and toxin extrusion protein 1 (rMate1); rat organic cation transporters (rOct2); estrogen; urinary excretion

Menopause typically occurred in women in midlife during their late 40s or early 50s, which was signaling the end of the fertile phase of a woman’s life.It was commonly defined as the permanent cessation of the primary function of the ovaries[1-2].However, it was suggested that loss of ovarian function and subsequent estrogen deficiency may enhance many diseased risks.Obesity, metabolic syndrome and diabetes, cardiovascular disease, osteoporosis and osteoarthritis, cognitive decline, dementia and depression, and cancer are the major diseases concerned[3], and Type 2 Diabetes Mellitus (T2DM)was found to be the most commonly concerned disease[4].

Metformin was a widely used drug for treatment of T2DM.It ameliorated hyperglycemia without stimulating insulin secretion, promoting weight gain, nor causing hypoglycemia[5].Invivo, metformin was not metabolized by hepatic cytochrome P450 but excreted by urine (about 78.9%-99.9%).The renal clearance of metformin was approximately four or five fold higher than creatinine clearance, indicating substantial active tubular secretion[6-7].

Kidney played an important role in the urinary excretion of drugs and their metabolites via glomerular filtration and/or tubular secretion.Considerable researches has indicated that organic cation transporters 2 (Oct2/SLC22A2)expressed in the apical membranes of kidney, and multidrug and toxin extrusion 1 (Mate1/SLC47A1)expressed in the luminal membranes of renal proximal tubules were mainly responsible for vectorial secretion and urinary excretion of metformin[6, 8].Genetic polymorphisms ofOct2-808 G>T had a significant effect on metformin pharmacokinetics, yielding a higher peak concentration with a larger area under serum concentration-time curve[9].Meanwhile, rat multidrug and toxin extrusion protein 1 (rMate1/slc47a1)was found to mediate extrusion of organic cations from cells into tubular lumen by using transmembrane H+gradient as driving force, which was considered for being responsible for the final step of urinary excretion of cationic drugs.Tsuda et al.[10]found that renal clearance and renal secretory clearance of metformin in miceMate1-/-were approximately 18% and 14% of those in wild-type mice (Mate1+/+), respectively.Therefore, it was thought that rat organic cation transporters 2 (rOct2)andrMate1determine the pharmacokinetics of metformin.

The renal rOct2 expression in males was much higher than that in females[11].So Urakami et al.[12]hypothesized that rOct2 expression might be under the control of sex hormones, and they further found that testosterone upregulated renal rOct2 expression and estradiol moderately downregulated rOct2.Although estrogen status could affect the expression of Octs and Mate1 and be related to the renal clearance of metformin, few studies had evaluated the pharmacokinetics of metformin in case of estrogen deficiency.In view of the important role of metformin in the menopause women with T2DM, the potential effect of estrogen deficiency on urinary excretion of metformin was of great importance for clinical treatment.The present study investigated the pharmacokinetics, plasma concentration and tissue (liver and kidney)distribution, urinary excretion and expression of rOct2 and rMate1 by designing model of estrogen deficiency in ovariectomized rats.

1 MATERIALS AND METHODS

1.1 Reagents, chemicals and instruments

Metformin (97% purity)was purchased from Sigma-Aldrich (St Louis, MO, USA).Norethindrone (internal standard)was purchased from Nation Institute for Control of pharmaceutical and Biological Products (Beijing, China).Methanol was of chromatographic grade (Fisher Scientific, NJ, USA).All other reagents and solvents were of analytical grade and were commercially available.Biochemical auto analyzer (OLYMPLLS AU2700, Olympus Co., Tokyo, Japan).

1.2 Animals

Five months old Virgin Wistar female rats(n=48)weighing 220-250 g (Experimental Animal Center of Lanzhou University, Gansu, China，SYXK Gan 2014-0006)were used in this study.The rats were housed in a room that provided alternating 12 h of light and 12 h of darkness with the room temperature at 23±1 ℃ and humidity at 55±5%.All experiments were approved by the Ethical Committee for Animal Experiments of Lanzhou University (LDYYLL2018-136)and carried out in accordance with the guidelines of Lanzhou University Ethics Review Committee.

1.3 Experimental design

1.3.1 Surgical procedure

Rats were anesthetized by intraperitoneal injection of 7% (m/v)chloral hydrate (350 mg/kg).Dorsal bilateral incisions were made aseptically midway between lower ribs and crest of ileum.In Ovx group, both ovaries of rats were isolated and removed.The rats in Sham group was treated by the same surgical procedure, except that both ovaries were not removed.The incision has closed with suture clips and rats were allowed to recover before being returned to the rat facility.Then rats were fed for one month to stabilize hormonal level before subsequent studies.

1.3.2 Investigate the pharmacokinetic profile of metformin via HPLC

Rats were randomly divided into two groups, ovariectomy group (Ovx)(n=8)and Sham group (n=8).Before the experiments, rats were fasted for 12 h with free access to water.Under anesthesia with ether, a polyethylene tube (0.28 mm, i.d.,0.61 mm, o.d.)was inserted into the right femoral artery of rat.Rats in both Ovx and Sham group were orally and intravenous treated with metformin at 100 mg/kg (oral)and 25 mg/kg (i.v.)after consciousness.Serial blood samples (0.25 mL)were collected through the right femoral artery with heparinized syringes at 0, 10, 20, 40, 60, 90, 120, 180, 240, 360, 480, 600 and 720 min in oral group, while at 2, 5, 10, 20, 30, 60, 90, 120, 240, and 480 min in i.v.group postdose.Plasma concentration of metformin was determined by HPLC, and the pharmacokinetic parameters were calculated using DAS2.0 program.

1.3.3 Investigate the plasma concentration, tissue (liver and kidney)distribution and urinary excretion of metformin via HPLC

Rats were divided into two groups, Ovx group (n=8)and Sham group (n=8), both of which were orally treated with metformin at 100 mg/kg.Blood samples from orbital and dissected liver and kidney were collected at 2 h postdose.Urine samples were collected during the interval of 0-2, 2-4, 4-6, 6-8, 8-10, 10-12, and 12-24 h postdose metformin.Tissue samples were weighed and homogenized in saline (250 mg/mL), and urine samples were diluted 10-fold with blank urine.Tissue homogenate, urine diluent and blood sample were centrifuged at 18 000 g for 10 min.The supernatant (100 μL)were added with 100 μL of the internal standard solution (48 μg/mL norethindrone)and 300 μL methanol for deproteinization.The treated samples were measured for metformin concentration by HPLC method as described by Ma et al.[13].

1.3.4 Investigate the plasma level of LCA via biochemical auto analyzer

The sample collected from 1.3.3 were measured for level of LCA by using biochemical auto analyzer, and the assay procedures were carried out according to the manufacturer’s protocol.

1.3.5 Investigate the expression of rOct2 and rMate1 in kidney via Western blot

Rats were divided into two groups, Ovx group (n=8)and Sham group(n=8), both of which were orally treated with metformin at 100 mg/kg.The expression of rOct2 and rMate1 in kidney were investigated via western blot.The antibodies used in the experiment included rabbit polyclonal antibody to rOct2 (1∶1 000, Abcam Inc., MA, USA), rabbit polyclonal antibody to rMate1 (1∶200, Santa Cruz Biotechnology, CA, USA), rabbit polyclonal to GAPDH (1∶3 000, Abcam Inc., MA, USA), and secondary antibody (1∶500, Abcam Inc., MA, USA).The scanned digital images were quantified using NIH ImageJ software.Data were expressed as the ratio of densitometric level of rOct2 or rMate1 and GAPDH protein.Specific experiment process was conducted according to the technique described by Ma et al.[13]

1.4 Statistical analysis

2 RESULTS

2.1 Pharmacokinetics of metformin in rats

The mean plasma concentration verus time curves of metformin after oral or intravenous administration of metformin in each group were shown in Fig.1, and the relevant pharmacokinetic parameters were listed in Table 1.After oral administration, the plasma concentration of metformin in Ovx group were significantly higher than those in Sham group at 20, 40, 60, 90, 120, 180, 240, 360 min (P<0.01, Fig.1A).Same phenomenon was observed after i.v.injection at 20, 30, 60, 90 and 120 min (P<0.05, Fig.1B).As shown in Table 1,t1/2β,ρmax and AUC0→tof Ovx group were significantly higher than Sham group, while CL/F and Ke of Ovx group were significantly lower than those of Sham group after oral and i.v.administration of metformin respectively(P<0.01).

1)Compared with Sham group，P<0.05; 2)Compared with Sham group，P<0.01.

Fig.1 Mean plasma concentration verus time curves of metformin after oral (A)or intravenous (B)administration

The subpart of Fig.1B was presented to show that plasma concentrations of metformin in Ovx group was significantly higher than those in Sham group at 20, 30, 60, 90 and 120 min.As shown in Table 1, the parameterst1/2βand AUC0→tof Ovx group were significantly increased compared to Sham group, and the CL and Ke of Ovx group were significantly lower than Sham group respectively(P<0.01).

2.2 Urinary excretion and plasma concentration, tissue distribution of metformin in rats

The results of urinary excretion and plasma concentration, tissue distribution of metformin in rats were shown in Fig.2 and Fig.3.As shown in Fig.2, cumulative urinary excretion of metformin during interval of 2-4, 4-6, 6-8, 8-10, 10-12, and 12-24 h postdose was significantly decreased in Ovx group (P<0.01).As shown in Fig.3, compared with Sham group, concentrations of metformin in plasma, liver and kidney were all significantly increased in Ovx group, especially in plasma and kidney(P<0.01).

2.3 Renal protein expression change of rOct2 and rMate1 in rats

To find the molecular mechanism of ovariectomy induced decrease of renal elimination of metformin, this study investigated renal protein expressions of rOct2 and rMate1 via western blot.As shown in Fig.4, compared with Sham group, the expression of rMate1 was decreased and that of rOct2 was increased significantly in Ovx group (P<0.01).

grouporalt1/2β/minVdF/(L·kg-1)CLF/(mL·min-1)AUC0-t/(μg·min·mL-1)Ka/min-1Ke/min-1ρmax/(μg·mL-1)tmax/minSham group246±215.3±0.620.2±4.94 568±2740.034±0.0120.006±0.00115.15±1.290±10.1Ovx group292±19 1)4.9±0.49.7±2.1 1)5 895±254 1)0.031±0.0140.003±0.001 1)19.25±1.7 1)88±9.7groupi.v.t1/2β/minVd/(L·kg-1)CL/(mL·min-1)AUC0-t(μg·min·mL-1)Ke/min-1Sham group54±60.51±0.0811.21±2.941 745±1290.016±0.004Ovx group105±101)0.48±0.067.47±1.87 1)2 354±136 1)0.011±0.003 1)

1)Compared with Sham group，P<0.01.

1)Compare with Sham group，P<0.01.

Fig.2 Cumulative urinary excretion curves of metformin in rats

1)Compared with Sham group，P<0.05; 2)Compared with Sham group，P<0.01.

Fig.3 Plasma concentration, tissue distribution of metformin in rats

1)Compared with Sham group，P<0.01.

Fig.4 Renal protein expression change of rMate1(A)and rOct2(B)in rats

2.4 Plasma LCA in rats

As shown in Fig.5, compared with Sham group, level of LCA in plasma was significantly increased in Ovx group (P<0.05), which indicated that ovariectomy could affect metabolism of LCA and result in accumulation of LCA.

1)Compared with Sham group，P<0.05

3 DISCUSSION

The epidemiological studies had shown that the prevalence of T2DM in the menopause women was higher than general women, which was likely to be connected with the low level of estrogen[14].Metformin, a prescribed first line antidiabetic drug, played an important role in patients with T2DM, including menopause women.Lactic acidosis was a fatal adverse effect of biguanide agents including metformin.Previous study showed that there were some adverse reactions when metformin accumulates in body, such as lactic acidosis[15].It was reported that metformin induced lactic acidosis was associated with an elevation in plasma concentrations of metformin in patients with renal failure[7].Study also showed that liver was of great importance on the lactic acidosis caused by metformin[22].As shown in our results, both plasma concentration of LCA and plasma, liver concentration of metformin was significantly higher in Ovx group than in Sham group, which indicated that the increased plasma concentration of LCA might be related with the increase of liver concentration and accumulation of metformin.Therefore, it is important to make it clear the metabolism and excretion of metformin in absence of estrogen.

In this work, ovariectomy rats were used as estrogen deficiency model.It discovered that plasma concentration of metformin increased in rats of estrogen deficiency Ovx group.According to the pharmacokinetic results,t1/2βand AUC0→tincreased while CL and Ke decreased significantly in Ovx group in case of oral and i.v.administration.The results indicated that estrogen deficiency may account for the decreased metabolism of metformin.Eyal et al.[16-17]reported that renal clearance of metformin increased significantly in mid (723±243 mL/min,P<0.01)and late pregnancy (625±130 mL/min,P<0.01)compared with postpartum (477±132 mL/min), which could support the idea that decreased metabolism of metformin was related with estrogen deficiency.

It was well known that rOct2 and rMate1 played crucial roles in renal elimination of metformin[21], thus this study investigated the expressions of rOct2 and rMate1 via Western blot.As shown in the results, compared with Sham group, reduction of rMate1 and elevation of rOct2 in Ovx group were noteworthy.The reduction of outputted transporter rMate1 decreased renal excretion of metformin, while elevation of rOct2 resulted in accumulation of metformin in renal tubular epithelial cells.It agreed with the higher metformin deposition in kidney in Ovx group than in Sham group.Also, cumulative urinary excretion decreased by 25% in Ovx group, compared with Sham group.The results indicated that estrogen deficiency may inhibit urinary excretion of metformin via downregulating renal expression of rMate1 as well as upregulating rOct2.The research of Meetam et al.[18]also showed an increase in number of Octs in Ovx mice, which was consistent with our results.

Kidney elimination of drug also was affected by renal blood flow and glomerular filtration, but elimination of metformin in kidney was finished via active tubular secretion (approximately 80%)[19].In consequence, to some extent, the effect of glomerular filtration on elimination of metformin could be ignored.With regard to renal blood flow, study reported that ovariectomy had no effect on it[20].

All in a word, estrogen deficiency might mainly account for the declined elimination of metformin observed in this study, which thus caused change in pharmacokinetic behavior of metformin in Ovx rats.The results indicated that it was important to focus more on the treatment of metformin in menopause patients with T2DM due to the increasing prevalence of T2DM in menopause women.